[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33042008</article-id><article-id pub-id-type=\"pmc\">PMC7523498</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00629</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Mini Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Studying Brown Adipose Tissue in a Human <italic>in vitro</italic> Context</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Samuelson</surname><given-names>Isabella</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/991096/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Vidal-Puig</surname><given-names>Antonio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/862343/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Metabolic Research Laboratories, University of Cambridge</institution>, <addr-line>Cambridge</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Cellular Genetics, Wellcome Sanger Institute (WT)</institution>, <addr-line>Hinxton</addr-line>, <country>United Kingdom</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Jiri Jiracek, Academy of Sciences of the Czech Republic (ASCR), Czechia</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: P. Trayhurn, University of Liverpool, United Kingdom; Alexander W. Fischer, Harvard University, United States</p></fn><corresp id=\"c001\">*Correspondence: Isabella Samuelson <email>iks25@cam.ac.uk</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Experimental Endocrinology, a section of the journal Frontiers in Endocrinology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>629</elocation-id><history><date date-type=\"received\"><day>29</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>04</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Samuelson and Vidal-Puig.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Samuelson and Vidal-Puig</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>New treatments for obesity and associated metabolic disease are increasingly warranted with the growth of the obesity pandemic. Brown adipose tissue (BAT) may represent a promising therapeutic target to treat obesity, as this tissue has been shown to regulate energy expenditure through non-shivering thermogenesis. Three different strategies could be employed for therapeutic targeting of human thermogenic adipocytes: increasing BAT mass through stimulation of BAT progenitors, increasing BAT function through regulatory pathways, and increasing WAT browning through promotion of beige adipocyte formation. However, these strategies require deeper understanding of human brown and beige adipocytes. While murine studies have greatly increased our understanding of BAT, it is becoming clear that human BAT does not exactly resemble that of the mouse, highlighting the need for human <italic>in vitro</italic> models of brown adipocytes. Several different human brown adipocyte models will be discussed here, along with the potential to improve brown adipocyte culture through recreation of the BAT microenvironment.</p></abstract><kwd-group><kwd>brown adipose tissue (BAT)</kwd><kwd>3D culture</kwd><kwd>HPSC model</kwd><kwd>obesity</kwd><kwd>thermogenic adipocyte differentiation</kwd><kwd>brown adipogenesis</kwd><kwd>human adipocytes</kwd><kwd>beige adipocyte</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Medical Research Council<named-content content-type=\"fundref-id\">10.13039/501100000265</named-content></funding-source><award-id rid=\"cn001\">4050281695</award-id></award-group><award-group><funding-source id=\"cn002\">British Heart Foundation<named-content content-type=\"fundref-id\">10.13039/501100000274</named-content></funding-source><award-id rid=\"cn002\">RG/12/13/29853</award-id></award-group><award-group><funding-source id=\"cn003\">European Research Council<named-content content-type=\"fundref-id\">10.13039/501100000781</named-content></funding-source><award-id rid=\"cn003\">669879</award-id></award-group><award-group><funding-source id=\"cn004\">Wellcome Trust<named-content content-type=\"fundref-id\">10.13039/100004440</named-content></funding-source><award-id rid=\"cn004\">109153/Z/15/Z</award-id></award-group></funding-group><counts><fig-count count=\"1\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"83\"/><page-count count=\"8\"/><word-count count=\"6322\"/></counts></article-meta></front><body><sec id=\"s1\"><title>The Obesity Pandemic</title><p>Obesity, defined as excessive fat accumulation, has reached pandemic proportions. Among adults, more than 650 million were obese in 2016 (BMI ≥ 30), with over 340 million obese or overweight (BMI ≥ 25) children and adolescents (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Obesity is associated with the metabolic syndrome, which comprises a cluster of metabolic abnormalities including insulin resistance, obesity, dyslipidemia, and hypertension, which are risk factors for cardiovascular disease and diabetes (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). Furthermore, obesity is a risk factor for cancer and musculoskeletal disorders, including osteoarthritis, among other diseases (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Thus, obesity places a substantial burden on society and healthcare systems and despite extensive research into the mechanisms controlling energy balance, body weight, and appetite, safe and effective measures to treat and prevent obesity are lacking.</p></sec><sec id=\"s2\"><title>Brown Adipose Tissue</title><p>Brown adipose tissue (BAT) has become a focus area for weight loss therapies. This tissue, evolved to help maintain core body temperature, is able to burn nutrients as heat, thereby increasing total energy expenditure (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). This process is known as non-shivering thermogenesis and is mediated by the mitochondrial uncoupling protein1 (UCP1), and BAT activation leads to increased energy expenditure and decreased metabolic efficiency.</p><p>BAT has been extensively studied in the mouse, and these studies have revealed an important role for BAT in the control of energy balance and whole-body metabolism. BAT activation has, for instance, been shown to positively influence plasma triglyceride and cholesterol levels, atherosclerosis development, glucose tolerance, insulin sensitivity, and hepatic steatosis, in addition to promoting weight loss (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>).</p></sec><sec id=\"s3\"><title>Beige Adipose Tissue</title><p>Another type of adipose tissue (AT), which has received considerable attention for the treatment of obesity, is beige AT, which arises from white adipose tissue (WAT) browning. WAT browning is characterized by the presence of multilocular, brown adipocyte (BA)-marker expressing adipocytes within WAT—so-called beige or brite adipocytes. Beige adipocytes can appear in WAT, mainly subcutaneous (sc)WAT, after cold exposure, hypercaloric diet, β-adrenergic stimulation, and exercise (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>), and are believed to influence metabolism similarly to BAT (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Although beige adipocytes show high expression of UCP1 (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>), they are believed to originate from different lineages than BAs (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>), although this is still debated.</p></sec><sec id=\"s4\"><title>BAT in Humans—Beige or Brown?</title><p>In humans, BAT mass and prevalence have been found to correlate negatively with BMI, diabetes status, and glucose plasma levels (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>), and cold-induced BAT activation also had beneficial metabolic effects and induced weight loss (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B17\" ref-type=\"bibr\">17</xref>), suggesting that human and murine BAT may have similar metabolic roles. It has been estimated that adult lean men may have around 330 ml of cold-inducible BAT, with lower volumes (around 130 ml) found in obese men (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Furthermore, seasonal changes in WAT thermogenic gene expression have been described (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>), suggesting that human WAT has the capacity for browning, although the impact of browning on whole-body metabolism is unclear.</p><p>Based on this ability of BAT to increase energy expenditure and improve metabolic function, BAT seems a likely target for the development of obesity therapeutics. Importantly, the efficacy of these therapeutics depends on whether activation of BAT in adults is able to significantly influence energy expenditure. Cold exposure in adults is able to increase energy expenditure varying from 20 kcal/day (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>) to more than 100 kcal/day (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>), and this can be increased further through chronic cold adaptation or increased BAT mass (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>).</p><p>A growing body of research is focusing on identifying genes and molecules able to influence BAT biology; however, one question is how well the findings in mice translate to humans. First, there is debate around whether human BAT resembles murine brown or beige AT more closely. Analyses of human neck fat have revealed that on a molecular level, deep neck fat mostly resembles that of murine canonical BAT, whereas the superficial neck fat may be more white or beige (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). In a study characterizing beige adipocytes, Wu et al. (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>) generated murine brown and beige adipocyte gene expression signatures. Comparing these signatures with human supraclavicular BAT, they found that human BAT is closer to murine beige than brown AT. A similar study comparing murine AT gene signatures with human BAT from multiple anatomical locations came to the same conclusion (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). In contrast, a recent study suggests that when mice are physiologically humanized, meaning housed at thermoneutrality, fed a “Western diet,” and examined at 9–11 months old, their BAT becomes comparable with human supraclavicular BAT (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>), although this conclusion has raised some discussion (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). This debate may likely be solved as the composition of human and murine brown and beige AT is better characterized with the help of more sophisticated techniques such as single-cell RNA sequencing. Owing to discrepancies between mRNA and protein expression levels of certain genes, including <italic>UCP1</italic> (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>), purely transcriptomic analysis of different BA populations may not be adequate to fully characterize their identity and thermogenic potential. To this end, protein analysis and functional characterization, including β-adrenergic response and bioenergetics, are required.</p><p>Second, whereas treatment with β<sub>3</sub>-adrenergic receptor (β<sub>3</sub>AR) agonists in mice is able to selectively stimulate BAT and protect against diet-induced obesity, these effects are not seen in humans at doses low enough to prevent cardiovascular side effects (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>). Interestingly, a recent study found that the β<sub>1</sub>AR may be the predominant βAR in human BAT (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>), highlighting another confounding issue when translating murine BAT studies to human biology. Other attempts to selectively activate thermogenesis through sympathomimetic drugs or mitochondrial uncouplers have not been successful (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Thus, to achieve selective and safe activation of BAT in humans, we may need to look beyond adrenergic stimulation or uncoupling mechanisms and instead focus on alternative pathways to target BAT biology. Specifically, rather than focusing exclusively on increasing the activation of existing BAT, other strategies could include increasing BAT mass through differentiation of progenitors, or increasing thermogenic activity in WAT through WAT browning.</p></sec><sec id=\"s5\"><title>Strategies for the Targeting of Human BAT</title><p>Increased energy expenditure in humans through BAT thermogenesis could be achieved by (a) increasing functional BAT mass, (b) increasing BAT activity, and (c) increasing WAT browning.</p><sec><title>Increased BAT Mass/Recruitment</title><p>Increased BAT mass could be achieved through the recruitment of BA progenitors. As mentioned earlier, decreased BAT mass is found in obese individuals—the very individuals the BAT therapeutics are intended for—and thus even if a method to selectively activate BAT was found, this may be of limited use to obese recipients. However, BAT activity can be enhanced through cold acclimation even in obese individuals (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>), suggesting the presence of BA precursors, the differentiation of which may be promoted given appropriate stimulation. The development of targeted approaches to induce differentiation of BA progenitors requires in-depth knowledge of the developmental mechanisms of human BAT, which currently does not exist. Furthermore, newly differentiated BAs would still require activation through sympathetic or alternative pathways.</p></sec><sec><title>Increased BAT Activity</title><p>Increased BAT activity, as discussed earlier, has been challenging to achieve without cardiovascular side effects. However, several molecules have been identified as possible therapeutic targets for the activation of BAT, reviewed in (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). First, members of the bone morphogenetic protein (BMP) family have been reported to play roles in BAT or WAT browning, mainly using murine models. These include, among others, BMP4, which may promote browning of WAT (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>), BMP7, which can promote BAT development and activation (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>) as well as white adipocyte (WA) browning (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>), and BMP8B, which was found to increase the adrenergic response of BAT and induce scWAT browning (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>, <xref rid=\"B35\" ref-type=\"bibr\">35</xref>). In addition, factors released from heart, muscle, liver, and immune cells have been shown to regulate thermogenesis (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Although these studies reveal several candidate targets, most of them have been conducted in murine models or <italic>in vitro</italic>, and tangible therapeutics are still far from a reality.</p></sec><sec><title>Increased WAT Browning</title><p>Finally, increased WAT browning is a strategy which may have substantial potential given the high abundance of WAT compared with BAT. However, as for BAs, the safe activation of beige adipocytes likely requires identification of specific molecular pathways to induce WAT browning that do not rely directly on adrenergic or uncoupling actions. These include fibroblast growth factor 21 (FGF21), which may regulate browning of scWAT through activation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>), as well as through UCP1-independent mechanisms (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). Another potential molecule is irisin, which may activate UCP1 in scWAT via p38-MAPK/ERK pathways (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>), although the levels of circulating irisin and its metabolic effects in humans are debated (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>, <xref rid=\"B40\" ref-type=\"bibr\">40</xref>). In addition, more studies on human WAT browning are necessary to determine whether this process is able to influence whole-body energy expenditure in a notable manner.</p><p>The three strategies presented earlier are all limited by the lack of knowledge regarding human thermogenic adipocyte development and function. This lack of knowledge stems principally from the lack of robust <italic>in vitro</italic> models of human BAs.</p></sec></sec><sec id=\"s6\"><title><italic>In vitro</italic> Models of Human BAT</title><p>Models of human BAs have been developed by several different groups and from starting material such as primary BAT, fibroblasts, muscle cells, adult stem cells, and pluripotent stem cells, among others. Some of these models are discussed later and represented in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Schematic of <italic>in vitro</italic> human BA models. Human BA models have been generated from infant BAT (1), deep neck and supraclavicular BAT (2), dermal fibroblasts (3), MSCs from AT and bone marrow (4), skeletal muscle progenitor cells (5), and hPSCs (6). Standard adipogenic cocktails or variations were used for the differentiations unless otherwise indicated. SVF, stromal vascular fraction; BAs, brown adipocytes; MSCs, mesenchymal stem cells; h(i)PSCs, human (induced) pluripotent stem cells; BAPs, BA progenitors; EB, embryoid body; RA, retinoic acid.</p></caption><graphic xlink:href=\"fendo-11-00629-g0001\"/></fig><sec><title>Primary Cell-Derived BA Models</title><p>The study of human primary BAT is limited by the scarceness of the disperse tissue and the need for invasive procedures to obtain biopsies. Nonetheless, several <italic>in vitro</italic> BA models derived from primary human BAT do exist. If one does have access to primary BAT, preadipocytes from non-viable human fetal interscapular BAT can be cultured and differentiated <italic>in vitro</italic> (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>); however, these are not stable cell lines and are thus not a viable long-term solution for the study of human BAT. To this end, immortalized human BA lines may be of more use. One of these is the PAZ6 cell line from immortalized infant BAT vascular stromal cells (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). These cells readily differentiate to BAs and respond to β-adrenergic stimulation; however, the differentiation potential of these cells declines with increasing passage number, significantly limiting their experimental value. Similarly, subcloned immortalized BA-like cell lines have been generated from adult supraclavicular AT stromal vascular fraction (SVF) (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>) and neck AT SVF (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>). In addition, a more heterogeneous BA-like cell model was created from adult deep neck AT SVF without subcloning (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>). These BA cell models demonstrated expression of <italic>UCP1</italic>, lipolytic response to cAMP (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>), and increased oxygen consumption in response to β-adrenergic stimulation (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>, <xref rid=\"B45\" ref-type=\"bibr\">45</xref>), and two of the lines were maintained for at least 20 passages (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>, <xref rid=\"B45\" ref-type=\"bibr\">45</xref>). These cell lines thus represent useful models for the study of human BAT. However, their generation does require access to primary tissue, and they, therefore, come from limited starting material, which means that these cell lines will deplete eventually.</p><p>Rather than using primary BAT, BA-like cells can be directly generated from human dermal fibroblasts, eliminating the need for invasive biopsies. One study demonstrated that overexpression of CCAAT/enhancer binding protein beta (<italic>CEBPB</italic>) and avian myelocytomatosis viral oncogene homolog (<italic>C-MYC</italic>), coupled with an adipogenic cocktail, induced conversion of human fibroblasts to BA-like cells (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>). Another study demonstrated the direct conversion of primary fibroblasts into BA-like cells with only chemical compounds, without using gene transfer (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>). These compounds included rosiglitazone, which is an agonist of peroxisome proliferator-activated receptor gamma (PPARγ), and known to promote browning (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>).</p><p>Alternatively, BA-like cells were shown to be differentiated from fetal and adult muscle progenitor cells expressing CD34, a marker of hematopoietic progenitors as well as vascular endothelial progenitors (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>), thus supporting the common lineage of skeletal muscle and BAT. This technique does require invasive biopsies; however, muscle tissue may be more accessible than BAT.</p><p>In addition to primary tissue access, a limitation of the aforementioned models is the inherent risk of cell depletion. Thus, to circumvent this limitation, cell lines can be generated from human stem cells.</p></sec><sec><title>Generation of Adipocytes From Stem Cells</title><sec><title>Multipotent Stem Cell–Derived BAs</title><p>Multipotent mesenchymal stem cells (MSCs) are adult stem cells able to differentiate into a range of mesodermal cell types including adipocytes, and can be isolated from several tissues such as bone marrow, blood, and AT (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). MSCs isolated from human AT (termed human multipotent adipose-derived stem (hMADS) cells) could be differentiated to BA-like cells when stimulated with a PPARγ agonist on top of an adipogenic cocktail (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>), and with the addition of BMP7 (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>). Similarly, bone marrow–derived MSCs may also be able to differentiate to BA-like cells when overexpressing <italic>PPARGC1A</italic> (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). Thus, MSCs are a useful tool for the generation of BA-like cells, and the self-renewal ability of MSCs gives unlimited starting material, at least theoretically. As an alternative to MSCs, human pluripotent stem cells (hPSCs) have been used for the generation of BA cell models.</p></sec><sec><title>Pluripotent Stem Cell–Derived BAs</title><p>hPSCs are advantageous for the generation of cell models for several reasons, including that they are relatively easy to genetically engineer. Because of their pluripotency and the fact that protocols exist to differentiate hPSCs into a wide range of cell types (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>), it is possible to generate isogenic disease models of cells from all three embryonic germ layers. Furthermore, the use of human induced (hi)PSCs, which are reprogrammed from human somatic cells (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>), enables the generation of disease models with patient-specific genetic backgrounds.</p><p>Some studies have demonstrated the generation of adipocytes from hPSCs. Mature white and brown adipocytes were generated from hPSCs through transfection with lentiviral constructs to overexpress <italic>PPARG2</italic> or <italic>PPARG2, CEBPB</italic>, and <italic>PRDM16</italic>, respectively (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>). In this study, hPSCs were first differentiated into MSCs using embryoid bodies (EBs), after which transgene expression was induced and an adipogenic cocktail was given. A similar method was used to generate BAs from hiPSCs through transduction of <italic>PRDM16</italic> (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>). Here, EBs were generated in the presence of retinoic acid (RA), to obtain more myoblast-like cells (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>), accounting for the reduced transgene requirement. Overexpression of transgenes in hPSCs is an efficient method for the generation and study of mature cell types (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>–<xref rid=\"B60\" ref-type=\"bibr\">60</xref>), although it can be argued that this method of differentiation may not follow natural developmental steps and may instead shunt some of these pathways. Thus, this technique may not be optimal for the generation of a human BA model for the study of BA development.</p><p>Another group demonstrated the differentiation of BA progenitors from hiPSCs through EB formation and adipogenic cocktails (<xref rid=\"B61\" ref-type=\"bibr\">61</xref>). The differentiation efficiency was reported to be low but could be improved through overexpression of paired box 3 (<italic>PAX3</italic>), a marker of BA progenitors (<xref rid=\"B61\" ref-type=\"bibr\">61</xref>). A similar method was used to generate BA progenitors from hiPSCs without gene transfer, although this study reports higher differentiation yield, possibly due to inhibition of TGFβ signaling (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>). TGFβ signaling has been shown to induce the conversion of endothelial cells to a more mesenchymal phenotype (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>), and these findings, therefore, suggest an important role of endothelial cells in the adipogenic niche. The generation of BA-like cells from hPSCs using EB formation and a hemopoietin cocktail has also been reported (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>), which may suggest that the developmental origin of BAs is more diverse than previously believed. Finally, BA-like cells were generated from hiPSCs in cytokine-free medium through the formation of spheroids (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>), although the molecular mechanisms of this differentiation method remain unclear.</p><p>Although hPSCs can, in theory, give rise to any cell type of the human body, their immature/embryonic state means that it can be difficult to generate mature cell types from hPSCs without forward programming. This issue does not arise when using BA models derived from primary adult AT. As an alternative, hPSC-derived BAs can be matured <italic>in vivo</italic> by transplantation into mice, which is also a method of functional validation of the generated BAs (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>, <xref rid=\"B64\" ref-type=\"bibr\">64</xref>, <xref rid=\"B65\" ref-type=\"bibr\">65</xref>). Currently, transplantation <italic>in vivo</italic> may be the best technique for recapitulation of the physiological BAT microenvironment; however, advances are also being made toward recreating BAT <italic>in vitro</italic> using 3D cell cultures.</p></sec></sec></sec><sec id=\"s7\"><title>Recapitulating the BA Microenvironment <italic>in vitro</italic></title><p>Long-term culture of adipocytes <italic>in vitro</italic> can be difficult because of the buoyancy of mature, lipid-laden adipocytes, which makes their attachment to 2D cell culture plates difficult (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>). Furthermore, true understanding of BAT biology requires recapitulation of the <italic>in vivo</italic> BA microenvironment—the BA niche. <italic>In vivo</italic>, BAT is a highly vascularized and innervated tissue, containing not only BAs but also endothelial cells, fibroblasts, progenitor cells, and immune cells (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>, <xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Furthermore, the extracellular matrix (ECM) of the AT is vital as a structural scaffold and to facilitate adipocyte differentiation, AT expansion, mechanotransduction, and biomolecule signaling (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>–<xref rid=\"B69\" ref-type=\"bibr\">69</xref>).</p><sec><title>Culturing BAs in 3D</title><p>A first step toward recapitulating BAT <italic>in vitro</italic> may be to use 3D cultures, which can provide mechanical support, allow physiological cell organization, and prevent loss of mature adipocytes (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). 3D culture techniques are successful for WA culture (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>), allowing <italic>in vitro</italic> culture of unilocular primary mature human WAs (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>, <xref rid=\"B73\" ref-type=\"bibr\">73</xref>). Unilocular WAs can also develop <italic>in vitro</italic> when differentiated in spheroids (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>–<xref rid=\"B76\" ref-type=\"bibr\">76</xref>).</p><p>Fewer 3D culture systems have been tested on BAs, potentially because the focus is still on the generation of useful BA cell models rather than their further development. Using hanging drop spheroid formation followed by culture in ultra-low attachment plates, Klingelhutz et al. (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>) demonstrated improved differentiation of murine BAT SVF compared with 2D. Alginate microstrands have also been used for the differentiation of BAs from murine embryonic stem cells and brown preadipocytes (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>). Using 3D printed hyaluronic acid/gelatin gels, Kuss et al. (<xref rid=\"B78\" ref-type=\"bibr\">78</xref>) showed that whereas immortalized human WA progenitors prefer soft gels, immortalized human BA progenitors prefer stiffer gels, suggesting that developments within WA 3D culture methods cannot necessarily be directly translated to BA cultures.</p></sec><sec><title>ECM and Vascularization</title><p>The next step toward recapitulating BAT <italic>in vitro</italic> is the addition of ECM and supporting cell types. It is possible to generate 3D scaffolds from ECM proteins such as collagens and glycoproteins (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>), or to incorporate ECM components into synthetic hydrogels (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>, <xref rid=\"B79\" ref-type=\"bibr\">79</xref>). However, to recreate the human BAT ECM <italic>in vitro</italic>, its composition must first be fully characterized <italic>in vivo</italic>, in both physiological and pathophysiological states.</p><p>For the generation of functional models of AT, vascularization is an important parameter, which can also aid engraftment <italic>in vivo</italic> during transplantation studies, as shown (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>). Angiogenic signals, including VEGF (vascular endothelial growth factor), play important roles not only in BAT function but also in the development of BAs (<xref rid=\"B80\" ref-type=\"bibr\">80</xref>–<xref rid=\"B83\" ref-type=\"bibr\">83</xref>). For instance, VEGFA is able to trigger local angiogenesis in BAT, leading to brown adipogenesis and upregulation of <italic>Ucp1</italic> and <italic>Pgc1</italic>α (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>). Vascularized (white) adipocyte spheroids can be generated using AT SVF, which contains endothelial cells as well as preadipocytes, or by coculturing adipocytes with endothelial cells (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>, <xref rid=\"B75\" ref-type=\"bibr\">75</xref>). For the generation of vascularized human BA models, it may be advantageous to focus on coculture methods, to avoid the need for primary tissue. Similar challenges exist regarding the integration of immune cells into the BA cultures.</p></sec></sec><sec sec-type=\"discussion\" id=\"s8\"><title>Discussion</title><p>Our understanding of BAT in mice and humans has increased significantly over the last decade, and with this understanding, we are moving closer to the development of therapeutics targeting BAT for obesity treatment. One of the main limitations in the field is the limited understanding of human BAT, including its development and molecular signature. These questions are steadily being tackled through in-depth characterization of primary human BAT and the development of human <italic>in vitro</italic> BA models. Human BA models can be generated from immortalization of primary human BAs, from multipotent stem cells or hPSCs, among others, and each of these methods has distinct advantages and limitations. For instance, <italic>in vitro</italic> models generated from adult BAT may represent the most mature BA models, whereas hPSC-derived BAs may be less mature unless forward programming is employed. In contrast, hPSCs are useful for disease modeling involving genetic engineering or patient-specific genetic backgrounds. Without a defined molecular signature for human BAT, lacking due to the heterogeneity and disperse anatomical distribution of BAT depots, it may, however, be challenging to determine which <italic>in vitro</italic> model is the best. Furthermore, as BAT does not exclusively consist of BAs, true recapitulation of this tissue involves modeling of the BAT microenvironment, including physiological BA organization in 3D, crosstalk with supporting cells such as immune cells, vascularization, and an ECM to facilitate BA differentiation and cell–cell signaling.</p><p>The road toward the development of obesity therapeutics targeting human BAT thus has plenty of challenges. However, with continued development of BA models and characterization of the <italic>in vivo</italic> depots, these challenges may soon be overcome.</p></sec><sec id=\"s9\"><title>Author Contributions</title><p>IS and AV-P wrote the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s10\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The authors gratefully acknowledge support from the Medical Research Council (PO 4050281695), the British Heart Foundation (RG/12/13/29853), the European Research Council (669879), and Wellcome (109153/Z/15/Z).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>World Health Organization</collab></person-group>\n<source>Obesity and Overweight</source>. (<year>2020</year>). 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Mem Inst Oswaldo Cruz</journal-id><journal-id journal-id-type=\"iso-abbrev\">Mem. Inst. Oswaldo Cruz</journal-id><journal-id journal-id-type=\"publisher-id\">mioc</journal-id><journal-title-group><journal-title>Memórias do Instituto Oswaldo Cruz</journal-title></journal-title-group><issn pub-type=\"ppub\">0074-0276</issn><issn pub-type=\"epub\">1678-8060</issn><publisher><publisher-name>Instituto Oswaldo Cruz, Ministério da Saúde</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32997000</article-id><article-id pub-id-type=\"pmc\">PMC7523502</article-id><article-id pub-id-type=\"doi\">10.1590/0074-02760200006</article-id><article-id pub-id-type=\"other\">00339</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Molecular and serological characterization of occult hepatitis B among blood donors in Maputo, Mozambique</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-1848-4627</contrib-id><name><surname>Mabunda</surname><given-names>Nédio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"corresp\" rid=\"c\">\n<sup>+</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zicai</surname><given-names>Ana Flora</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ismael</surname><given-names>Nalia</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Vubil</surname><given-names>Adolfo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mello</surname><given-names>Francisco</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Blackard</surname><given-names>Jason T</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lago</surname><given-names>Barbara</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Duarte</surname><given-names>Vanessa</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Moraes</surname><given-names>Milton</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lewis</surname><given-names>Lia</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Jani</surname><given-names>Ilesh</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<label>1</label>Instituto Nacional de Saúde, Maputo, Mozambique</aff><aff id=\"aff2\">\n<label>2</label>Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Hepatites Virais, Rio de Janeiro, RJ, Brasil</aff><aff id=\"aff3\">\n<label>3</label>Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Hanseníase, Rio de Janeiro, RJ, Brasil</aff><aff id=\"aff4\">\n<label>4</label>University of Cincinnati College of Medicine, Division of Digestive Diseases, United States of America</aff><author-notes><corresp id=\"c\">+ Corresponding author: <email>nediojonas@gmail.com</email></corresp><fn fn-type=\"con\" id=\"fn2\"><p>NM, IJ and MM - Conceptualization; NM, AFZ, NI, AV, FM, BL and VD - data curation; NM, NI, FM, JTB and BL - formal analysis; NM and IJ - funding acquisition; NM, AFZ, NI, AV, FM, BL and VD - investigation; NM, NI, FM, BL, VD and JTB - methodology; MM and IJ - supervision; NM, AFZ, NI and AV - writing - original draft; JTB, MM, LL and IJ - writing - review and editing. The authors have declared no conflicts of interest.</p></fn></author-notes><pub-date pub-type=\"epub\"><day>23</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>115</volume><elocation-id>e200006</elocation-id><history><date date-type=\"received\"><day>08</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>8</month><year>2020</year></date></history><permissions><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License</license-p></license></permissions><abstract><sec><title>BACKGROUND</title><p> Occult hepatitis B virus (HBV) - characterized by the absence of detectable HBsAg in the presence of HBV DNA - represents a potential threat for blood safety.</p></sec><sec><title>OBJECTIVES</title><p> This study was conducted with the aim to investigate the serological and molecular characterization of occult HBV infection (OBI) among blood donors in Mozambique.</p></sec><sec><title>METHODS</title><p> 1,502 blood donors were tested for HBsAg. All HBsAg-negative individuals were tested for HBV DNA. Antibodies against HBV core, surface and HBe antigen (anti-HBc, anti-HBs, HBeAg) were measured in HBV DNA positive individuals.</p></sec><sec><title>FINDINGS</title><p> 1435 serum samples were HBsAg negative and 16 positive for HBV DNA, 14 confirmed to have OBI, corresponding to a frequency of 0.98%. Of the 14 OBI infections identified, 13/14 (92.8%) were positive for anti-HBc, 4/14 (28.5%) for anti-HBs, and no samples were reactive for HBeAg. Of the 14 OBI cases, nine samples (64.2%) were sequenced for the S/P region. Eight samples (88.9%) belonged to genotype A1 and one (11.1%) to genotype E. One escape mutation (T123A) associated with OBI and various amino acid substitutions for genotype A1 and E were observed.</p></sec><sec><title>MAIN CONCLUSIONS</title><p> Our results show the importance of using nucleic acid amplification test to detect occult hepatitis B infection in blood donors in Mozambique.</p></sec></abstract><kwd-group><title>Key words:</title><kwd>occult hepatitis B</kwd><kwd>blood donors</kwd><kwd>Mozambique</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"2\"/><ref-count count=\"30\"/></counts></article-meta></front><body><p>Significant efforts have been made to ensure safe blood for transfusion. However, several sub-Saharan African countries including Mozambique still face several challenges such as the lack of nucleic acid amplification tests (NAT).<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref> One of the main agents transmitted by blood transfusion is hepatitis B virus (HBV) that can lead to cirrhosis and hepatocellular carcinoma (HCC). Chronic HBV infection is defined by the presence of hepatitis B surface antigen (HBsAg), the main diagnostic marker for routine screening of donated blood, for at least six months in the serum.<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref> Widespread HBsAg screening using sensitive assays has substantially reduced the incidence of transfusion-transmitted HBV. However, it has been demonstrated that individuals without detectable HBsAg can transmit HBV.<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref> Transmission of HBV from HBsAg seronegative blood donors can occur (i) when the individual is in the window period viral exposure but before the detection of serological markers of HBV infection, or (ii) when individuals harbor HBV DNA at low levels in the absence of detectable HBsAg, a condition known as occult HBV infection (OBI).<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>\n</p><p>In developing countries, current HBV testing algorithms often rely on HBsAg detection alone or in combination with antibodies against viral antigens, and OBI cases are frequently undiagnosed.<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref> The presence of anti-HBc may serve as a marker of HBV infection in settings where molecular tests are unavailable. However, anti-HBc alone is not sufficient to identify OBI.<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref> Several mechanisms may explain the presence of HBV DNA in the absence of HBsAg, including: (i) low levels of HBV replication and HBsAg expression due to strong humoral and cellular immune response against HBsAg, (ii) mutations within the S gene that suppresses the synthesis of HBsAg or alter antigenic epitopes, (iii) the occurrence of HBsAg/anti-HBs immunocomplexes that inhibit HBsAg detection, (iv) the presence of virus reservoirs in peripheral mononuclear cells (monocytes and lymphocytes), and, (v) coinfections such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV) that can interfere with HBV replication.<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n</p><p>Based on the HBsAg positivity in the general population, Mozambique is classified as a country with high endemicity. The prevalence of HBsAg in Mozambique among blood donors is approximately 10.6%.<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref> Recently, Viegas et al.,<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref> reported a higher prevalence of 12.2% in 18-24 year-olds in the capital of Maputo. Carimo et al.,<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref> reported an 8.3% frequency of OBI among patients who were anti-HBc positive. The aim of this study was to characterize serological markers (anti-HBc, anti-HBs and HBeAg), viral genotypes, and mutations associated with OBI among blood donors.</p><sec sec-type=\"materials|methods\"><title>MATERIALS AND METHODS</title><p>\n<italic>Study design and population</italic> - A cross-sectional study was performed between November 2014 and October 2015 at the Blood Bank of Hospital Central de Maputo. Blood donors that fulfilled the national blood service eligibility criteria for blood donation were included. Exclusion criteria include the donor’s general health and the risk of having a blood transfusion transmission disease as reported in full by Stokx et al.<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref> All study participants (1502 blood donors) provided written informed consent. Demographic information was obtained from all consenting blood donors using a structured questionnaire. For each participant, 9 mL of whole blood was collected into 3 mL vacutainers with K3EDTA (Becton Dickinson, Franklin Lakes, NJ, USA) and plasma sample were obtained from blood donors. The study was approved by the National Health Bioethics Committee in Mozambique (number 263/CNBS/2014).</p><p>\n<italic>Serological assays</italic> - All plasma samples were screened for HBsAg, anti-HCV, and HIV antibodies/antigens. HBV screening was performed using the Advanced Quality HBsAg ELISA Test Kit (InTec Products, INC, China), and only reactive samples were confirmed using Advanced Quality HBsAg Rapid Test (InTec Products, INC, China). HCV screening was performed using the ADVANCED Test ELISA anti-HCV (InTec Products, INC, China) and confirmed with the ADVANCED QUALITY Rapid Anti-HCV (InTec Products, INC, China). HIV screening was conducted using a qualitative enzyme immunoassay GENSCREEN PLUS HIV Ag-Ab (Bio-Rad, Marnes-l Coquette, France) and only reactive samples were re-tested using the UNIGOLD (Trinity Biotech Plc, Bray, Co. Wicklow, Ireland) rapid test. All samples were considered negative if non-reactive in the first test, and positive if reactive for both the first and second tests. Sample was considered indeterminate if the result was discordant for both tests and was not included in the next step.</p><p>\n<italic>DNA quantification and detection</italic> - HBV DNA was quantified for all HBsAg-negative blood donors in test pools of six plasma samples using the COBAS AmpliPrep/COBAS TaqMan HBV Test, v2.0 (Roche Diagnostics, Germany) with a detection limit of 20 IU/mL according to the manufacturer’s instructions. For test pools with detectable viral load, samples were individually retested using the same kit. For the individual testing 1,100 µL of plasma was used and 184 µL per sample for test pool testing. Samples with detectable HBV DNA were subsequently tested for anti-HBc and anti-HBs using the Bioelisa Kits (Biokit, Barcelona, Spain), and HBeAg levels using the Liaison Kit (DiaSorin, Sallugia, Italy) following the manufacturer’s instructions for individual blood donors.</p><p>\n<italic>HBV amplification and sequencing</italic> - HBV DNA was extracted from 200 µL of plasma using the High Pure Viral Nucleic Acid Kit (Roche Applied Science, Mannheim, Germany). The following primers were used to amplify the S and P regions: first round primers S1 (position 124-143) and 4R (position 1120-1100), and second round primers 1F (position180-203) and 4R and 1F (position180-203). The thermal cycler for the first and second cycle was programmed as follows: 30 cycles with initial denaturation at 94ºC for 1 min, 94ºC for 15 s, 56ºC for 30 s, 68ºC for 1 min and 15 s, and a final extension at 68ºC for 10 min. The amplified product was subjected to electrophoresis on a 2% agarose gel supplemented with ethidium bromide stain and visualized by UV light. High Pure Product Purification Kit (Roche, Germany) was used to purify DNA according to manufacturer’s instruction. The S/P PCR products of ~900 bases were sequenced using an automated DNA sequence 3500 Genetic Analyzer (Foster City, CA, USA) as described by Mallory et al.<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref>\n</p><p>All laboratory activities were carried out following good laboratory practices (include routine daily practices, for example, decontamination of safety cabinets, inclusion of controls in all procedures).</p><p>\n<italic>Statistical analysis</italic> - Fisher’s exact test was used for group-to-group differences in gender, age, and donor type. Rank sum test was used to analyze HBV DNA levels. For all analyses, p-value < 0.05 was considered statistically significant.</p><p>\n<italic>GenBank accession numbers</italic> - Sequences were assigned the GenBank accession numbers MF615980-MF615989.</p><p>\n<italic>Phylogenetic analysis</italic> - Nucleotide alignments were performed with Clustal X 2.1<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref> and additional phylogenetic inference was performed using a Bayesian Markov Chain Monte Carlo (MCMC) approach as implemented in the Bayesian Evolutionary Analysis by Sampling Trees (BEAST) version 1.8.4 program<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref> with an uncorrelated log-normal relaxed molecular clock, general time-reversible model, and nucleotide site heterogeneity estimated using a gamma distribution. The MCMC analysis was run for a chain length of 100,000,000, and results were visualized to confirm adequate chain convergence with Tracer version 1.6. The effective sample size (ESS) was calculated for each parameter, and all ESS values were > 500 indicating sufficient sampling. The maximum clade credibility tree was selected from the posterior tree distribution after a 10% burn-in using Tree Annotator version 1.8.4 and visualized in FigTree version 1.4.3 as described previously.<xref rid=\"B16\" ref-type=\"bibr\">\n<sup>16</sup>\n</xref> Potential recombination was evaluated using the Jumping Profile Hidden Markov Model available at http://jphmm.gobics.de/submission_hbv.</p><p>\n<italic>Mutations and amino acid substitution analysis</italic> - To identify mutations associated with occult HBV infection, a consensus sequence of HBV/A1 and HBV/E was generated from 120 and 223 GenBank references, respectively. An alignment of nucleotide sequences containing the consensus sequence and the Mozambican OBI sequences was generated and the amino acids visualized to identify substitutions occurring in the S gene ORF.</p></sec><sec sec-type=\"results\"><title>RESULTS</title><p>A total 1,502 blood donors were screened for HBsAg, of which 1,435 (95.5%) were negative and 67 (4.5%) positive. From the 1,435 HBsAg negative samples, 16 (1.1%) had detectable HBV DNA. Two of 16 were negative for all serological markers (seronegative OBI or window period). Of the 14 (0.98%) occult HBV infections, 10 (71.4%) samples were positive for anti-HBc only, 14 (21.4%) were positive for anti-HBc and anti-HBs, and 1 (7.1%) was positive for anti-HBs only. The HBV DNA of OBI cases was significantly lower when compared to that of HBsAg-positive cases (<xref rid=\"t1\" ref-type=\"table\">Table I</xref>). Of the 14 OBI cases, nine samples (64.3%) could be polymerase chain reaction (PCR) amplified and sequenced, including four with an HBV viral load of < 20 IU/mL. HBV DNA levels for the remaining 5 ranged from 20 to 1326 IU/mL (<xref rid=\"t2\" ref-type=\"table\">Table II</xref>). No OBI case tested positive for HCV or HIV co-infection.</p><p>\n<table-wrap id=\"t1\" orientation=\"portrait\" position=\"float\"><label>TABLE I</label><caption><title>Characteristics of occult hepatitis B and HBsAg positive blood donors</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">HBsAg positive 67 (4.5)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Occult HBV (n = 14) 14 (0.98)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">p-value</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age (years); mean (SD)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30.6 (8.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33.6 (10.7)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.725</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Gender</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male; n (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53 (97.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12 (85.7)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.248</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Female; n (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (20.9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (14.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" colspan=\"4\" rowspan=\"1\">Donor type</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Replacement; n(%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">60 (89.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (92.9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Voluntary; n(%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (10.4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (7.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Median plasma HBV DNA, IU/mL (IQR)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1138 (149.5-7829)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23 (20-293)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">< 0.001</td></tr></tbody></table></table-wrap>\n</p><p>\n<table-wrap id=\"t2\" orientation=\"portrait\" position=\"float\"><label>TABLE II</label><caption><title>Molecular and serological characteristics of occult HBV infection (OBI) cases in blood donors at the blood bank of Hospital Central de Maputo</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"3\"/><col span=\"2\"/><col span=\"2\"/></colgroup><tbody><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">Code</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">Serological markers</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">Virological characteristics</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">Genetic variation in HBV MHR domain</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Anti-HBc</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Anti-HBs</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">HBeAg</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Viral load (IU/mL)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Genotypes and sub-genotypes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Substitution in MHR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Escape mutations</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">385</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">< 20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A (A1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">V190A, A194V, L209V, V224A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">532</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">< 20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A (A1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A194V</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">590</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1014</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A (A1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A194V</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T123A</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">800</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A (A1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A194V, S204G, L216F, F220L</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">885</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1326</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A (A1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A159V, A194V</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1498</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">503</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">E</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">P56L, T57I, E164G, S174N, L216*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1551</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">349</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A (A1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">L21S, R24K/R, L49R, P70LP, F134I, V184AV, A194V, S210I/T, L216*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1626</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">< 20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A (A1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">W36L, A194V</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1637</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">< 20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A (A1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A194V</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">805</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">806</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">< 20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">942</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">< 20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1629</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">< 20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1688</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">+</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">125</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN2\"><p>HBV: hepatitis B virus; MHR: major hydrophilic region. *The sign is a stop codon which means the termination of the translation process which includes an amino acid change at position 216 in the wild type virus to a stop codon.</p></fn></table-wrap-foot></table-wrap>\n</p><p>Eight OBI sequences clustered with genotype A1 references, while one grouped with genotype E (<xref ref-type=\"fig\" rid=\"f1\">Fig. 1</xref>). One escape mutation (T123A) associated with OBI in one sample was observed. Various amino acid substitutions for genotype A1 and E were observed in the major hydrophilic region (MHR). In addition, polymorphism I195V within the S gene for all the samples was also detected in OBI individuals belonging to genotype A1 (<xref rid=\"t2\" ref-type=\"table\">Table II</xref>, <xref ref-type=\"fig\" rid=\"f2\">Figs 2</xref>-<xref ref-type=\"fig\" rid=\"f3\">3</xref>).</p><p>\n<fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1:</label><caption><title>Bayesian phylogenetic analysis of partial S gene sequences from Mozambique (indicated by a 4-digit identifier) compared to reference sequences (indicated by their accession number, subgenotype, and country of origin). Hepatitis B virus (HBV) genotype A sequences (n = 8) are shown in red, while genotype E sequences (n = 1) are shown in blue.</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200006-gf1\"/></fig>\n</p><p>\n<fig id=\"f2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2:</label><caption><title>amino acid alignment of the S gene (aa 1-227) from occult hepatitis B virus (HBV)-infected individuals from Mozambique. The Major Hydrophilic Region (aa 99-169) is underlined (99-169) and the “a” determinant epitope region is represented in the box.</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200006-gf2\"/></fig>\n</p><p>\n<fig id=\"f3\" orientation=\"portrait\" position=\"float\"><label>Fig. 3:</label><caption><title>amino acid alignment of the S gene (aa 1-227) from occult hepatitis B virus (HBV)-infected individuals with genotype E from Mozambique. The Major Hydrophilic Region (aa 99-169) is underlined (99-169) and the “a” determinant epitope region is represented in the box.</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200006-gf3\"/></fig>\n</p></sec><sec sec-type=\"discussion\"><title>DISCUSSION</title><p>The present study is the first to identify and characterize OBI cases among blood donors in Mozambique, and highlights the presence of HBV DNA despite undetectable HBsAg and the absence of other serological markers. Fourteen OBI cases were identified among 1,435 HBsAg-negative blood donors an overall prevalence of 0.98%.</p><p>Studies conducted in Colombia and Nigeria found a prevalence of 1.98% and 17% among blood donors, respectively. Both studies tested for HBV DNA amongst all HBsAg-negative and anti-HBc positive samples.<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref> One the other hand, study that included blood donors from South Africa, Egypt, The Mediterranean, Northern and Central Europe, Southeast Asia and Oceania showed OBI rates ranging from 1:3,900 to 1:59,000.<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref> Although OBI data in sub-Saharan African is limited, other studies in Botswana and South Africa have identified OBI in 6.6% to 15% in HIV-infected patients.<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref> In this study, no hepatitis C or HIV co-infections were identified.</p><p>Recently, a study conducted in HIV patients naive to antiretroviral therapy in Mozambique reported an OBI prevalence of 8.2%.<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref> The difference in the prevalence reported in our study and that of Carimo et al. may be attributed to (i) differences in the study population group (HIV patients are at higher risk of OBI due to immunodeficiency), (ii) the inclusion criteria utilized by Carimo et al. that may lead to over or underestimation of OBI cases. With this criteria, only patients with anti-HBc alone were submitted to PCR assay and a portion of HBsAg gene was amplified and (iii) the pooling method used to determine HBV DNA might explain the low frequency of OBI observed in the current study.</p><p>Significantly lower levels of HBV DNA were detected in OBI samples, with the exception of two samples (BSM 885 and 590). This low level HBV DNA is in accordance with previous studies, indicating strong suppression by the immune response together with histological derangements occurring during acute or chronic HBV infection.<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref> In contrast, the presence of high HBV DNA in two samples may be associated with recent infection increasing the risk of infectivity in the transfused blood. Nevertheless, in agreement with other studies, other factors not observed in this study such as host factors and epigenetic modifications may account for the occult HBV phenotype observed in these individuals.<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref> In fact, <italic>in vitro</italic> studies have demonstrated that OBI strains are competent for replication.<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref>\n</p><p>Of the 14 identified OBI cases, nine were sequenced of which eight belonged to sub-genotype A1. Seven sequences were similar to South African sequences, one to a Tanzania sequence, and one to genotype E (<xref ref-type=\"fig\" rid=\"f1\">Fig. 1</xref>). These findings may be explained by the geographic proximity of Mozambique to South Africa and Tanzania. The present study revealed no difference in HBV genotypes observed between the occult HBV population and the HBV population co-infected with HIV previously observed in sub-Saharan countries where sub-genotype A1 predominates. Indeed, it has been proposed that genotype A originated in Africa with sub-genotype A1 from sub-Saharan Africa and India.<xref rid=\"B25\" ref-type=\"bibr\">\n<sup>25</sup>\n</xref> Genotype E is most prevalent in West Africa but have been reported sporadically in other African countries.<xref rid=\"B26\" ref-type=\"bibr\">\n<sup>26</sup>\n</xref> The introduction of genotype E is probably due to increased population migration occurring in recent years between Mozambique and West African countries. However, full genome sequencing and further phylogenetic analyses will be necessary to validate this hypothesis.</p><p>Understanding the immunologic and molecular characteristics that play a role in OBI cases determination is important and must be investigated further. Various studies propose the alteration of HBsAg antigenicity, inhibiting anti-HBs production associated with escape mutants in OBI cases within the PreS/S region. Changes in the epitope, such as a single mutation in the “a” determinant (amino acid 124-147), inhibit HBsAg secretion. On the other hand, amino acid substitutions in the reverse transcriptase (RT) domain can also result in low levels of HBV DNA and HBsAg synthesis influencing occult infection.<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref> In our study, amino acid substitutions were found within the MHR region in all samples. Substitutions V190 and F134I in genotype A samples have been reported in some studies, however their contribution to occult hepatitis has not yet been described.<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B28\" ref-type=\"bibr\">\n<sup>28</sup>\n</xref> Further studies are needed to determine effects of these and other variation we found in this study on HBsAg and HBV. In particular, T123A escape mutation located in the MHR of the S gene was distinctly present in one patient (sample BSM590). This finding is in concordance with several studies reporting the occurrence of T123A mutation in OBI individuals, changing its immunogenicity and making HBsAg unrecognizable by available commercial kits. The ability of different commercial tests to detect HBsAg in sample with escape mutations varies, reason why testing the same sample with different commercial tests is recommended.<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B29\" ref-type=\"bibr\">\n<sup>29</sup>\n</xref>\n</p><p>Furthermore, the A194V polymorphism observed among genotype A1 sequences in this study may represent a molecular signature for the HBV Mozambican infected population; however, further studies must be conducted to evaluate this hypothesis.</p><p>With regards to additional analysis for sample BSM1498 belonging to genotype E, amino acid substitution E164G was observed within the MHR. Interestingly, amino acid substitution E164G was also observed in a study conducted among Nigerian blood donor samples.<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>\n</p><p>In the present study, individuals positive for anti-HBc, anti-HBs and HBV DNA were observed, representing a viral persistency with low viral load as observed in other studies as well.<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref> A plausible explanation for this may be related to poor neutralization by the anti-HBs antibody that leads to the loss of recognition allowing the mutant virus to escape to neutralization in the presence of the protective antibody. Notably, HBeAg detection was not observed in the present study suggesting the absence of wild type variants previously described in HBV HBeAg positive individuals. Most individuals positive for anti-HBc only had a viral load below the detection limit in concordance with the theory by Prati et al.<xref rid=\"B30\" ref-type=\"bibr\">\n<sup>30</sup>\n</xref> which states that anti-HBc positivity alone contributes to low viral replication in OBI individuals.</p><p>Limitations of the current study include the lower limit of detection of the HBV DNA assay (20 UI/mL). Second, only participants with the eligibility for blood donation according to Stokx et al.<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref> were included in this study fact that may have influenced the with the overall low frequency of OBI infection detected in our study. Nevertheless, the detection of 16 out of 1,435 samples screened indicates that molecular approaches should utilized for routine blood screening to avoid HBV transmission.</p><p>\n<italic>In conclusion</italic> - This study is the first to demonstrate that OBI is prevalent among healthy asymptomatic blood donors in Mozambique. The presence of 0.98% detectable HBV DNA among negative HBsAg individuals suggests that asymptomatic blood donors with HBV infection will not be identified when screened with commonly available serological tests at the blood banks in Mozambique, increasing the risk of HBV transmission. The introduction of routine molecular screening tests for blood donation may prevent the transmission of HBV in the window period and occult hepatitis B. Mutations detected within the S gene and MHR region may have accounted for the occult nature of HBV infection in these blood donors observed in Mozambique.</p></sec></body><back><ack><title>ACKNOWLEDGEMENTS</title><p>To all the blood donors who voluntarily participated in the study, the team at the blood bank of Maputo Central Hospital, especial Sandra Oficiano, and the Molecular Virology Laboratory of the Instituto Nacional de Saude of Mozambique.</p></ack><fn-group><fn fn-type=\"financial-disclosure\" id=\"fn1\"><p>Financial support: This study was supported by The National Research Fund of Mozambique (FNI).</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id=\"B1\"><label>1</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>WHO - World Health Organization</collab></person-group><source>Global status report on blood safety and availability 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Mem Inst Oswaldo Cruz</journal-id><journal-id journal-id-type=\"iso-abbrev\">Mem. Inst. Oswaldo Cruz</journal-id><journal-id journal-id-type=\"publisher-id\">mioc</journal-id><journal-title-group><journal-title>Memórias do Instituto Oswaldo Cruz</journal-title></journal-title-group><issn pub-type=\"ppub\">0074-0276</issn><issn pub-type=\"epub\">1678-8060</issn><publisher><publisher-name>Instituto Oswaldo Cruz, Ministério da Saúde</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32997001</article-id><article-id pub-id-type=\"pmc\">PMC7523504</article-id><article-id pub-id-type=\"doi\">10.1590/0074-02760200310</article-id><article-id pub-id-type=\"other\">00421</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Short Communication</subject></subj-group></article-categories><title-group><article-title>Genomic and phylogenetic characterisation of an imported case of SARS-CoV-2 in Amazonas State, Brazil</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>do Nascimento</surname><given-names>Valdinete Alves</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Corado</surname><given-names>André de Lima Guerra</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>do Nascimento</surname><given-names>Fernanda Oliveira</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>da Costa</surname><given-names>Ágatha Kelly Araújo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Duarte</surname><given-names>Debora Camila Gomes</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Luz</surname><given-names>Sérgio Luiz Bessa</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gonçalves</surname><given-names>Luciana Mara Fé</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>de Jesus</surname><given-names>Michele Silva</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>da Costa</surname><given-names>Cristiano Fernandes</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Delatorre</surname><given-names>Edson</given-names></name><xref ref-type=\"aff\" rid=\"aff5\">\n<sup>5</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-2888-1060</contrib-id><name><surname>Naveca</surname><given-names>Felipe Gomes</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff6\">\n<sup>6</sup>\n</xref><xref ref-type=\"corresp\" rid=\"c\">\n<sup>+</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<label>1</label>Fundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Manaus, AM, Brasil</aff><aff id=\"aff2\">\n<label>2</label>Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Programa de Pós-Graduação em Biologia Celular e Molecular, Rio de Janeiro, RJ, Brasil</aff><aff id=\"aff3\">\n<label>3</label>Fundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Programa de Pós-Graduação em Biologia da Interação Patógeno-Hospedeiro, Manaus, AM, Brasil</aff><aff id=\"aff4\">\n<label>4</label>Fundação de Vigilância em Saúde do Amazonas, Manaus, AM, Brasil</aff><aff id=\"aff5\">\n<label>5</label>Universidade Federal do Espírito Santo, Centro de Ciências Exatas, Naturais e da Saúde, Departamento de Biologia, Vitória, ES, Brasil</aff><aff id=\"aff6\">\n<label>6</label>Rede Genômica de Vigilância em Saúde do Estado do Amazonas, Manaus, AM, Brasil</aff><author-notes><corresp id=\"c\">+ Corresponding author: <email>felipe.naveca@fiocruz.br</email></corresp><fn fn-type=\"con\" id=\"fn2\"><p>VAN, SLBL, CFC and FGN conceived the study; FGN designed the study protocol; VAN, ALGC, FON, AKAC, DCGD, LMFG, MSJ and FGN performed the molecular tests; VAN, ED and FGN performed the analysis and interpretation of these data; FON and MSJ collected biological sample; VAN, ALGC, AKAC, FON, DCGD, LMFG, ED and FGN wrote the manuscript; SLBL, CFC, ED and FGN critically revised the manuscript for intellectual content; FGN financed the study. All authors read and approved the final manuscript. . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p></fn></author-notes><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>115</volume><elocation-id>e200310</elocation-id><history><date date-type=\"received\"><day>14</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>8</month><year>2020</year></date></history><permissions><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License</license-p></license></permissions><abstract><p>A new coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] is currently causing a life-threatening pandemic. In this study, we report the complete genome sequencing and genetic characterisation of a SARS-CoV-2 detected in Manaus, Amazonas, Brazil, and the protocol we designed to generate high-quality SARS-CoV-2 full genome data. The isolate was obtained from an asymptomatic carrier returning from Madrid, Spain. Nucleotide sequence analysis showed a total of nine mutations in comparison with the original human case in Wuhan, China, and support this case as belonging to the recently proposed lineage A.2. Phylogeographic analysis further confirmed the likely European origin of this case. To our knowledge, this is the first SARS-CoV-2 genome obtained from the North Brazilian Region. We believe that the information generated in this study may contribute to the ongoing efforts toward the SARS-CoV-2 emergence.</p></abstract><kwd-group><title>Key words:</title><kwd>coronavirus</kwd><kwd>SARS-CoV-2</kwd><kwd>COVID-19</kwd><kwd>Brazil</kwd><kwd>Amazon Region</kwd><kwd>genome</kwd></kwd-group><counts><fig-count count=\"2\"/><table-count count=\"1\"/><ref-count count=\"29\"/></counts></article-meta></front><body><p>The coronavirus disease 2019 (COVID-19) is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that was identified for the first time in patients with pneumonia in Wuhan, China.<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref> Since its discovery at the end of 2019, SARS-CoV-2 transmission has been documented as being person-to-person, causing from an asymptomatic status, that may also generate transmission clusters, to a symptomatic disease with the typical symptoms manifested as fever, dry cough, myalgia, fatigue, dyspnea, diarrhea, and nausea.<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref> In the majority of the patients, the clinical outcome is a mild disease, although Wu and McGoogan described Chinese patients that developed a severe outcome (16%) or a critical condition (4%). Severe or critical outcomes usually occur in patients with comorbidities, and the disease can progress, presenting arrhythmia and shock, that could evolve to death.<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n</p><p>The SARS-CoV-2 belongs to family <italic>Coronaviridae</italic>, genus <italic>Betacoronavirus</italic>. In 2002 and 2012, two outbreaks occurred caused by new coronaviruses, SARS-CoV and MERS-CoV, with lethality ranging from nine to 33%, respectively.<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref> The coronaviruses are enveloped viruses, with 80 to 120 nm in diameter. Three proteins compose the virion surface: spike (S), membrane (M), and small membrane protein (E), giving the virus a crown-like visual on electron micrograph.<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref> This viral family has one of the largest RNA genome of all other RNA viruses, with a single non-segmented positive-stranded RNA of approximately 30 kb.<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref>\n</p><p>In Brazil, the first recorded case of SARS-CoV-2 infection occurred in the end of February, in the city of São Paulo, followed by cases in the Northeast (Bahia), Central-West (Brasília) and South (Rio Grande do Sul) regions.<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref> The northern of Brazil was the last region of the country to detected SARS-CoV-2 in its population. In March 13th, the first case in the State of Amazonas was detected in a woman that traveled to England and return to the Amazonas capital, Manaus.<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref> After three days, the second case of SARS-CoV-2 was detected in Manaus and characterised in the present study.</p><p>One 56-years-old man returning from Madrid, Spain, arrived asymptomatic in Manaus, Amazonas State, Brazil on 15-Mar-2020. At that time, a massive outbreak of COVID-19 was already established in several European countries, suggesting that travelers should be kept in quarantine at arrival. Even without the symptoms of a respiratory infection, nasal and oropharyngeal swabs were collected for SARS-CoV-2 testing as a routine established for respiratory virus surveillance at Instituto Leônidas e Maria Deane (ILMD) - Fiocruz, Amazonas State, Brazil, since 2019. The swabs were combined and submitted to total nucleic acid extraction with a commercial kit (Biogene, Recife, PE, Brazil) and immediately evaluated using the reverse transcription real-time polymerase chain reaction (RT-qPCR) protocol developed by the US Centers for Disease Control and Prevention (CDC/USA) (On 15-Mar-2020, CDC updated the RT-qPCR protocol removing the N3 target). This RT-qPCR assay employs different primers and probes sets aiming three regions of the SARS-CoV-2 nucleocapsid (N) gene (https://www.fda.gov/media/134922/download), and the human RNase P as an internal control.</p><p>The analysed sample tested positive for SARS-CoV-2 with Cts values of 14.43 (N1), 15.39 (N2) and15.33 (N3). Thus, we immediately generated cDNA with random primers and Superscript IV reverse transcriptase (ThermoFisher Scientific, Waltham, MA, United States). We had previously designed a PCR scheme to amplify the entire genome of the SARS-CoV-2 based on an alignment of all complete genome sequences available on GenBank at 03/03/2020. Conserved regions were chosen for primer design with Primer3 v2.3.7. embedded in Geneious Software 10.2.6,<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref> spanning around 2.2Kb and with an overlap region between 131 and 225 bp. This PCR scheme resulted in 15 amplicons with further details presented in the <inline-supplementary-material xlink:href=\"1678-8060-mioc-115-e200310-s1.pdf\" id=\"d38e422\" content-type=\"local-data\">Supplementary data</inline-supplementary-material> (Table I).</p><p>The SARS-CoV-2 whole genome was amplified with Platinum SuperFi II Green PCR master mix (ThermoFisher Scientific) using the 15 primers sets in individual reactions. Each amplicon was then visualised as a unique and intense DNA fragment on agarose gel electrophoresis stained with GelRed (Biotium, Hayward, CA, United States). The PCR amplicons were precipitated with molecular biology grade Polietilenoglicol (PEG) 8,000 (Promega, Madison, WI, United States) and then resuspended in nuclease-free water. After one-hour incubation at 37ºC, all amplicons were quantified in ng/μL using Qubit 2.0 and the dsDNA HS assay kit (Thermo Fisher Scientific). Finally, the number of DNA copies in each purified amplicon was estimated with ENDMEMO (http://www.endmemo.com/bio/dnacopynum.php), normalised, and pooled. A single library was constructed using the Nextera DNA Flex Library Prep and clustered with MiSeq Reagent Micro Kit v2 (300-cycles), following the manufacturer’s protocols. Nucleotide sequencing was performed in the MiSeq platform (Illumina, San Diego, CA, United States), installed at ILMD, in a paired-end run (2x150 cycles).</p><p>A total of 10,946,898 reads were trimmed for quality and adapters using BBDUK v37.25, embedded in Geneious software. Thus, 8,362,418 reads were mapped to the SARS-CoV-2 NCBI Reference Sequence NC_045512.2 using Geneious map-to-reference tool. The BR_AM_ILMD_20140001 final consensus genome sequence contains 29,789 nucleotides, with no gaps, a Q40 score of 100%, with no undetermined “N” bases and high average coverage (> 34,000X). To avoid any primers bias, we removed both primer binding sites at the 5′ and 3′ ends. Thus, our final sequence represents the positions between nucleotides 47 and 29,835 or 99.6% of the NCBI RefSeq previously mentioned.</p><p>We aligned the BR_AM_ILMD_20140001 genomic sequence with the SARS-CoV-2 NCBI Reference Sequence NC_045512 using MAFFT v7.388<xref rid=\"B16\" ref-type=\"bibr\">\n<sup>16</sup>\n</xref> to investigate any mutations throughout genome. A total of nine mutations were observed at nucleotide positions 8,782 (C to T); 9,477 (T to A); 12,781 (C to T); 14,805 (C to T); 25,979 (G to T); 26,642 (C to T); 28,144 (T to C); 28,657 (C to T) and 28,863 (C to T), with four of these leading to residues substitution in the deduced protein sequences (<xref rid=\"t\" ref-type=\"table\">Table</xref>).</p><p>\n<table-wrap id=\"t\" orientation=\"portrait\" position=\"float\"><label>TABLE</label><caption><title>Differences observed between sample BR_AM_ILMD_20140001 and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prototype</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Genome position</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8782</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9477</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12781</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14805</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25979</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26642</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28144</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28657</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28863</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NC_045512</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">G</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">BR_AM_ILMD</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">C</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T</td></tr><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">Codon position</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">AG<bold>C</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">TTT</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">TA<bold>C</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">TA<bold>C</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GGA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GCC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">TTA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GA<bold>C</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T<bold>C</bold>A</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">AG<bold>T</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T<bold>A</bold>T</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">TA<bold>T</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">TA<bold>T</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GTA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GCT</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">T<bold>C</bold>A</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GA<bold>T</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">TTA</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Mutation type</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">s</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">s</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">s</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">s</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">s</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">ns</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Protein</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">nsp4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">nsp4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">nsp9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">RdRp</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">ORF3a ptn</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">M glycoptn</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">ORF8 ptn</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">npptn</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">npptn</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Residue</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ser2839</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Phe3071Tyr</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Tyr4172</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Tyr4847</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Gly196Val</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ala40</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Leu84Ser</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Asp128</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Ser197Leu</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN\"><p>Nucleotides substituted in each codon are represented in bold. s: silent mutation; ns: non-silent mutation; nsp: non-structural protein; RdRp: RNA-dependent RNA-polymerase; ORF3a ptn: ORF3a protein; M glycoptn: M glycoprotein; ORF8 ptn: ORF8 protein; npptn: nucleocapsid phosphoprotein.</p></fn></table-wrap-foot></table-wrap>\n</p><p>To put the BR_AM_ILMD_20140001 genome in a global context, we aligned the new genome to a pool of all SARS-CoV-2 genomes with at least 25,000 nucleotides available at GISAID database<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref> on March 31st, 2020 using MAFFT. We adopted a subsampling strategy to reduce computation time, selecting subsets of 15-20 sequences retaining the most viral diversity from each country using the CD-HIT program.<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref> This subsampling approach resulted in a final sequence dataset with 490 sequences from 53 countries [<inline-supplementary-material xlink:href=\"1678-8060-mioc-115-e200310-s1.pdf\" id=\"d38e679\" content-type=\"local-data\">Supplementary data</inline-supplementary-material> (Table II)].</p><p>Complete coding sequences (CDS) were subjected to maximum likelihood (ML) phylogenetic reconstruction with PhyML v3.0,<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref> under the HKY+Γ4 nucleotide substitution model. According to the ML phylogenetic tree, the BR_AM_ILMD_20140001 sequence belonged to the lineage A<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref> (<xref ref-type=\"fig\" rid=\"f1\">Fig. 1</xref>A) clustering within a monophyletic cluster (aLRT = 1.00) comprising sequences from Spain, Chile, France, Greece, Georgia, Netherlands, Senegal (<xref ref-type=\"fig\" rid=\"f1\">Fig. 1</xref>B). The pangolin web application (pangolin.cog-uk.io) further assigned the sequences from this cluster to the A.2 lineage (<xref ref-type=\"fig\" rid=\"f1\">Fig. 1</xref>B).</p><p>\n<fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1:</label><caption><title>maximum-likelihood (ML) phylogeny of subsampled severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes. (A) ML tree rooted on the branch separating lineages A and B sequences colored following the legend. (B) A close view of the lineage A highlighting the A.2 lineage (light blue box) that comprises the BR_AM_ILMD_20140001 strain (indicated with an arrow). The nodes representing the most recent common ancestor (MRCA) of the lineage A.2 is indicated with a red diamond. In both trees, tips representing the Spanish strains inside lineage A.2 are coloured red and the scale bar represents nucleotide substitutions per site.</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200310-gf1\"/></fig>\n</p><p>After temporal validation with Tempest<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref> (<xref ref-type=\"fig\" rid=\"f2\">Fig. 2</xref>A), we conducted a Bayesian discrete spatiotemporal analysis with all lineage A sequences (<xref ref-type=\"fig\" rid=\"f2\">Fig. 2</xref>) using the BEAST v1.10 package,<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref> applying the strict molecular clock and the parametric exponential coalescent models. The strict molecular clock model was selected over the relaxed uncorrelated one,<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref> after marginal likelihood estimation using path sampling and stepping-stone sampling methods<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref> [<inline-supplementary-material xlink:href=\"1678-8060-mioc-115-e200310-s1.pdf\" id=\"d38e749\" content-type=\"local-data\">Supplementary data</inline-supplementary-material> (Table III)].The analysis under the uncorrelated relaxed molecular clock model, however, resulted in estimates similar to those of the strict clock model (data not shown). We estimated that the most recent common ancestor (MRCA) of lineage A.2 originated in Spain (posterior state probability, PSP = 0.99) in the beginning of February 2020 (95% highest posterior density, HPD = 2nd - 22nd Feb 2020). The phylogeographic analysis also pointed out that the BR_AM_ILMD_20140001 was most probably introduced from Spain (PSP = 0.43) (<xref ref-type=\"fig\" rid=\"f2\">Fig. 2</xref>B).</p><p>\n<fig id=\"f2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2:</label><caption><title>phylogeography of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage A. (A) Temporal signal analysis correlating the sampling date of each sequence and its genetic distance from the root of a maximum likelihood phylogeny. (B) Time-scaled Bayesian phylogeographic maximum clade credibility (MCC) tree of the SARS-CoV-2 complete coding sequences (CDS) classified as lineage A. The branch’s colors represent the most probable location of their descendent nodes (diamonds) as indicated at the legend. Branch support are indicated only at key nodes posterior (PP) and posterior state probability (PSP). The lineage A.2 is highlighted with a light blue box. All horizontal branch lengths are drawn to a scale of years.</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200310-gf2\"/></fig>\n</p><p>Like other viral infections, the infection by SARS-CoV-2 can be asymptomatic and this fact has been reported in different studies.<xref rid=\"B25\" ref-type=\"bibr\">\n<sup>25</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B26\" ref-type=\"bibr\">\n<sup>26</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref> It is noteworthy that according to the definition of suspected case adopted in Brazil at the time that we investigated this asymptomatic carrier, he would not be included in SARS-CoV-2 testing, despite returning from an area with active transmission like Madrid, Spain.<xref rid=\"B28\" ref-type=\"bibr\">\n<sup>28</sup>\n</xref> Until 24-Mar-2020, when we deposited the BR_AM_ILMD_20140001 genome information at https://www.gisaid.org, there were only 17 Brazilian SARS-CoV-2 complete genome sequences, and the sequence reported in the present work is the first complete genome from the northern Brazilian region.</p><p>Several laboratories over the world are now sequencing thousands of SARS-CoV-2 genomes, which is undoubtedly the most notable effort of viral sequencing in human history. Like any other virus, the new coronavirus is continuously evolving as more hosts, humans or animals, are getting infected. Thus, it is of paramount importance to generate and share high-quality full viral genomes from different regions over the world to better understand the SARS-CoV-2 evolution. The information related to the viral evolution is not only necessary for molecular epidemiology studies, but also to monitor if the newly identified mutations are linked to different clinical presentations or may drive into false-negative results when performing nucleic acid amplification assays, like real-time PCR.</p><p>Therefore, in this work, we aimed to describe and characterise the complete genome of the SARS-CoV-2 obtained from an asymptomatic carrier returning from Madrid, Spain. To achieve this goal, we decided to use a nucleotide sequencing strategy where firstly all the 15 amplicons, encompassing the entire SARS-CoV-2 genome, were confirmed by agarose gel electrophoresis. Subsequently, each amplicon was quantified and normalised in order to prevent that one region could be overrepresented during sequencing. In order to make our approach more straightforward, we decided to evaluate if longer amplicons could be generated in a very similar way. We were successful in generating amplicons around 6 Kb, with a minimum overlap of 131 bp, reducing the number of PCR reactions to 5 instead of 15 (data not showed). The conditions to amplify the 6 Kb amplicons followed the manufacturer’s recommendations, with the primers pairs details presented in the <inline-supplementary-material xlink:href=\"1678-8060-mioc-115-e200310-s1.pdf\" id=\"d38e802\" content-type=\"local-data\">Supplementary data</inline-supplementary-material> (Table I) of this manuscript. Of note, we observed that only samples with high viral load (e.g. Ct lower than 21) were fully amplified using the 6Kb protocol. We believe that this approach may be exciting not only to reduce the current protocol costs, but also for those interested in using long reads sequencing technologies like PacBio SMS and nanopore.</p><p>Recently, Rambaut and colleagues proposed a rational and dynamic virus classification for SARS-CoV-2 genomes based on a phylogenetic framework.<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref> Using this approach authors identified at the root of the phylogeny of SARS-CoV-2 two lineages that were simply denoted as lineages A and B. In our analysis, while all Brazilian SARS-CoV-2 sequences belonged to the lineage B, mostly from the B.1 lineage, the BR_AM_ILMD_20140001 genome clustered within the lineage A.2, indicating that BR_AM_ILMD_20140001 strain belongs to a distinct transmission cluster than the other full Brazilian genomes reported until March 31st, 2020. Originally, the lineage B.1 predominated in Europe and North America,<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref> and currently, constitutes the most prevalent SARS-CoV-2 variant circulating in Brazil.<xref rid=\"B29\" ref-type=\"bibr\">\n<sup>29</sup>\n</xref> The lineage A.2 constitutes a predominantly Spanish lineage, found in at least 25 countries<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref> and our phylogeographic analysis corroborates its origins and reinforces the importation scenario from Spain to Brazil.</p><p>In this study, we report and characterise the first SARS-CoV-2 genome obtained from an infected subject in the Brazilian North Region. Since this case was an asymptomatic carrier, it is not easy to suggest when infection has occurred. However, our phylogeographic analysis strongly indicates this individual was infected in Spain. Finally, we would like to emphasise that more fully genomes studies of the SARS-CoV-2 are necessary to better understand the evolution of this emerging life-threatening virus and the information of the nucleotide sequence described here may contribute to future molecular epidemiological studies in Brazil. In this sense, the protocol that we described in the present study may be useful to aid other researchers to generate other high-quality SARS-CoV-2 genomes.</p><p>\n<italic>Nucleotide sequence accession number</italic> - The complete genome sequence of the BR_AM_ILMD_20140001 isolate is available in GISAID since March 24, 2020, under the ID number EPI_ISL_417034.</p></body><back><ack><title>ACKNOWLEDGEMENTS</title><p>To the following authors from the originating laboratories responsible for obtaining the specimens and the submitting laboratories where genetic sequence data were generated and shared via the GISAID Initiative, on which this research is based. All GISAID accession numbers and data contributors are described in the <inline-supplementary-material xlink:href=\"1678-8060-mioc-115-e200310-s2.xlsx\" id=\"d38e854\" content-type=\"local-data\">Supplementary data</inline-supplementary-material> (Table IV). The authors thank the Program for Technological Development in Tools for Health - FIOCRUZ - for use of nucleotide sequencing facilities at ILMD - Fiocruz Amazônia.</p></ack><fn-group><fn fn-type=\"financial-disclosure\" id=\"fn1\"><p>Financial support: CNPq, CAPES, MS-DECIT, FIOCRUZ, FAPEAM - REGESAM. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Mem Inst Oswaldo Cruz</journal-id><journal-id journal-id-type=\"iso-abbrev\">Mem. Inst. Oswaldo Cruz</journal-id><journal-id journal-id-type=\"publisher-id\">mioc</journal-id><journal-title-group><journal-title>Memórias do Instituto Oswaldo Cruz</journal-title></journal-title-group><issn pub-type=\"ppub\">0074-0276</issn><issn pub-type=\"epub\">1678-8060</issn><publisher><publisher-name>Instituto Oswaldo Cruz, Ministério da Saúde</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32997002</article-id><article-id pub-id-type=\"pmc\">PMC7523505</article-id><article-id pub-id-type=\"doi\">10.1590/0074-02760200349</article-id><article-id pub-id-type=\"other\">00340</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Effect of <italic>Cinnamomum verum</italic> leaf essential oil on virulence factors of <italic>Candida</italic> species and determination of the <italic>in-vivo</italic> toxicity with <italic>Galleria mellonella</italic> model</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0003-3304-9110</contrib-id><name><surname>Wijesinghe</surname><given-names>Gayan Kanchana</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"corresp\" rid=\"c\">\n<sup>+</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Maia</surname><given-names>Flávia Camila</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>de Oliveira</surname><given-names>Thaís Rossini</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>de Feiria</surname><given-names>Simone N Busato</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Joia</surname><given-names>Felipe</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Barbosa</surname><given-names>Janaina Priscila</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Boni</surname><given-names>Giovana Cláudia</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sardi</surname><given-names>Janaina de Cássia Orlandi</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rosalen</surname><given-names>Pedro Luiz</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Höfling</surname><given-names>José Francisco</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<label>1</label>Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba, Área de Microbiologia e Imunologia, Departamento de Diagnóstico Oral, Campinas SP, Brasil</aff><aff id=\"aff2\">\n<label>2</label>Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba, Área de Farmacologia, Anestesiologia e Terapêutica, Departamento de Ciências Fisiológicas, Campinas, SP, Brasil</aff><author-notes><corresp id=\"c\">+ Corresponding author: <email>gkwijesinghe1989@gmail.com</email></corresp><fn fn-type=\"con\" id=\"fn2\"><p>GK - Conception or design of the work, data collection, data analysis and interpretation, drafting the article, critical revision of the article, final approval of the version to be published; FC - data collection, final approval of the version to be published; TR and SN - conception or design of the work, data collection, data analysis and interpretation, final approval of the version to be published; FJ, JP, GC, JD and PL - data collection, final approval of the version to be published; JF - conception or design of the work, data collection, data analysis and interpretation, drafting the article, critical revision of the article, final approval of the version to be published. The authors declare no conflict of interest.</p></fn></author-notes><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>115</volume><elocation-id>e200349</elocation-id><history><date date-type=\"received\"><day>02</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>8</month><year>2020</year></date></history><permissions><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License</license-p></license></permissions><abstract><sec><title>BACKGROUND</title><p> Essential oils (EO) extracted from <italic>Cinnamomum verum</italic> has been used as an antimicrobial agents for centuries. The effects of <italic>C. verum</italic> leaf oil against virulence of microorganisms is not well studied yet.</p></sec><sec><title>OBJECTIVES</title><p> This study evaluates the effect of <italic>C. verum</italic> leaf oil against three virulence factors of <italic>Candida albicans</italic>, <italic>C. tropicalis</italic> and <italic>C. dubliniensis</italic> and its <italic>in-vivo</italic> toxicity.</p></sec><sec><title>METHODS</title><p> Chemical composition of EO was determined using gas chromatography-mass spectrometry (GC-MS). Minimum inhibitory concentration (MIC) was determined using clinical and laboratory standards institute (CLSI) M27-A3 broth microdilution. Effect of EO on initial adhesion was quantified using XTT assay after allowing <italic>Candida</italic> cells to adhere to the polystyrene surface for 2 h. Biofilm formation of <italic>Candida</italic> in the presence of EO was quantified using XTT viability assay. Efficacy on reduction of germ tube formation was evaluated using standard protocol. Visualisation of biofilm formation and progression under the EO treatment were done using scanning electron microscope (SEM) and Time lapses microscope respectively. <italic>In-vivo</italic> toxicity of EO was determined using <italic>Galleria mellonella</italic> larvae. Chlorhexidine digluconate: positive control.</p></sec><sec><title>RESULTS</title><p> Eugenol was the main compound of EO. MIC was 1.0 mg/mL. 50% reduction in initial adhesion was achieved by <italic>C. albicans</italic>, <italic>C. tropicalis</italic> and <italic>C. dubliniensis</italic> with 1.0, > 2.0 and 0.34 mg/mL respectively. 0.5 and 1.0 mg/mL significantly inhibit the germ tube formation. MBIC<sub>50</sub> for forming biofilms were ≤ 0.35 mg/mL. 1.0 mg/mL prevent biofilm progression of <italic>Candida</italic>. SEM images exhibited cell wall damages, cellular shrinkages and decreased hyphal formation. No lethal effect was noted with <italic>in-vivo</italic> experiment model at any concentration tested.</p></sec><sec><title>CONCLUSION</title><p>\n<italic>C. verum</italic> leaf oil acts against virulence factors of <italic>Candida</italic> and does not show any toxicity.</p></sec></abstract><kwd-group><title>Key words:</title><kwd>Cinnamomum verum</kwd><kwd>essential oil</kwd><kwd><italic>Candida</italic> spp.</kwd><kwd>biofilms</kwd><kwd>anti-virulence</kwd><kwd>Galleria mellonella</kwd></kwd-group><counts><fig-count count=\"9\"/><table-count count=\"5\"/><ref-count count=\"42\"/></counts></article-meta></front><body><p>\n<italic>Candida</italic> spp. is the most common causative agent of opportunistic fungal infections, leading to a range of serious life-threatening invasive to non-life-threatening mucocutaneous diseases including skin and ear infections, genitourinary candidiasis, nosocomial pneumonias, medical device associated infections and Candidaemias.<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>\n</p><p>Some <italic>Candida</italic> species show commensalism and colonise the skin and mucosal surfaces of the human body. Severely ill or patients with compromised immunity are more prone to develop both superficial and life-threatening systemic <italic>Candida</italic> infections.<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref> Predisposing factors for the infections are: extremes of age, hormonal changes, nutritional deficiency, HIV incidence, frequent antimicrobial exposure, chemotherapeutic treatments, carbohydrate-rich (glucose) diets, use of prostheses and low immunity.<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref> as host factors. Based on the available data, the total occurrence of <italic>Candida</italic> infections is around 1.2-25 cases per 100 000 population or 0.19-2.5 per 1000 hospital admissions all over the world. 13% of these were reported with patients with underlying predisposing factor like compromised immunity etc.<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>\n</p><p>Among all <italic>Candida</italic> species, <italic>Candida albicans</italic> is the most frequent etiological agent in cases of fungal infections, being associated in up to 50% of cases of this disease.<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref> This species has the capacity to adapt and proliferate easily in the environment of the human body, in several places. This ability to survive in different tissues of the human body is related to its capacity for morphological transition between hyphal form and yeast form.<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref> According to Mayer et al., hyphae and yeast, are involved in the infection process, and the yeast, being smaller, is able to disseminate, while the hyphae invades the host tissue by penetrating the epithelium with the help of proteinases and phospholipases enzymes, escaping from phagocytic cells.<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B16\" ref-type=\"bibr\">\n<sup>16</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref> Another factor associated with virulence of <italic>Candida</italic> spp. is the ability to form biofilms, promoting cell adhesion and formation of the extracellular matrix of the biofilm, as well as the synthesis of proteins that favor its further adhesion. The biofilm structure increases the resistance to antimicrobial agents, due to the difficulty of penetration into the extracellular matrix, and the action of the immune system, besides allowing the increase of the gene expression of mechanisms of resistance to antifungals as the efflux pumps.<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>\n</p><p>Same as many bacterial and fungal species, antimicrobial resistance is an emerging problem with the <italic>Candida</italic> spp. Centre for Disease Control and Prevention (CDC) reports that, about 7% and 1.6% of all systemic infection causing <italic>Candida</italic> isolates from year 2012-2016 demonstrated a resistance to the first line antifungal drug, fluconazole and to echinocandins.<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref> Hence, new therapeutic options are needed to effectively address the emerging issue of developing antimicrobial resistance, in addition to overcoming the toxicities and drug interactions that are associated with available antifungal agents.<sup>20</sup> Plant derived natural therapeutics are becoming more popular among the vast majority of the modern world due to its low toxicity, easy accessibility, cost efficacy as well as the high therapeutic potency.<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref> In this aspect, <italic>Cinnamomum verum</italic>/ True Cinnamon, has been shown to be of great economic and pharmaceutical-medicinal interest.<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>\n</p><p>Extracted phytochemicals from various parts of the cinnamon tree (bark, leaves, fruits and flowers etc.) have been used as a culinary spice and medicinal plant since ancient times. It has also been used as an ingredient in many ancient Asian (especially in India, China and Sri Lanka) therapeutic preparations.<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref> In traditional folk medicine, cinnamon is used for the treatment of many disease conditions including eye inflammations, vaginitis, rheumatism, and neuralgia as well as wounds and toothaches apart from the use as an antimicrobial and anti-parasitic agent. Cinnamon is also useful for treatment of bronchitis, itching, and urinary tract and digestive tract related diseases.<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref>\n</p><p>Although, many <italic>in-vitro</italic> microbiological studies with <italic>C. verum</italic> show antimicrobial activities related to many microbial species in the health care area, including those that exhibit multiple antimicrobial resistance such as <italic>Acinetobacter</italic> spp., <italic>Bacillus</italic> spp., <italic>Enterobacter</italic> spp., <italic>Enterococcus faecalis</italic>, <italic>Escherichia coli</italic>, <italic>Mycobacterium tuberculosis</italic>, <italic>Klebsiella pneumoniae</italic> and <italic>Haemophilus influenzae</italic> etc.,<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref> the anti-<italic>Candida</italic> activity, specially, effect on virulence of <italic>Candida</italic> spp. is still less comprehensive.</p><p>The present study aimed to evaluate the effect of <italic>C. verum</italic> leaf essential oils (EO) on the initial adhesion, germ tube formation and biofilm formation of <italic>Candida</italic> spp., and determine its toxicity <italic>in-vivo</italic> using <italic>Galleria mellonella</italic> larvae model.</p><sec sec-type=\"materials|methods\"><title>MATERIALS AND METHODS</title><p>\n<italic>Fungal strains</italic> - Three <italic>Candida</italic> type strains, <italic>C. albicans</italic> (ATCC MYA-2876), <italic>C. tropicalis</italic> (ATCC 750) and <italic>C. dubliniensis</italic> (ATCC MYA-646) obtained from Microbiology and Immunology Area, Faculty of Dentistry in Piracicaba, UNICAMP, Brazil, were used in this study. The standard strains of <italic>Candida</italic> spp. stocks were maintained in 80% grycerol in ultrafreezer at -80ºC. To reactivate stock organisms, they were subcultured in freshly prepared Saboraud Dextrose Agar (SDA, OXOID) culture medium, incubated aerobically at 37ºC for 24 h.</p><p>The standard inocula were prepared by adjusting the turbidity in accordance with McFarland scale (0.5), which was equivalent to the absorbance of 0.08-0.10 (600 nm) corresponding to 5 x 10<sup>6</sup> colony forming unit (CFU)/mL.</p><p>\n<italic>Essential oil</italic> - The EO of <italic>C. verum</italic> leaves was purchased from Romik Lanka Marketing Services, Moratuwa, Sri Lanka (WCC/3569). The <italic>C. verum</italic> EO was diluted to 32 mg/mL in Tween 80 (0.05%) solution and Roswell Park Memorial Institute (RPMI) buffered with MOPS [3-(N-morpholino) propane sulfonic acid] followed by sonication, 1 cycle of 20 seconds.</p><p>\n<italic>Chemical analysis of the EO</italic> - The chemical analysis of <italic>C. verum</italic> EO was performed using HP-6890 (Agilent, USA) gas chromatograph coupled with HP-5975 (Agilent, USA) selective mass detector; HP-5MS Capillary Column (30 m x 0.25 mm x 0.25 µm); temperatures: injector (220ºC), detector (250ºC), column (60ºC), 3ºC/min, 240ºC; flow rate of carrier gas (highly dried He) of 1.0 mL/min<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>. 1.0 µL of oil was injected in split mode and the ionisation source was 70 eV. The relative abundance of the components were calculated using the following equation.<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref>\n</p><p>Percentage abundance (%) = (Area of the component/total area of the chromatogram) × 100</p><p>The database for identifying the analytes was NIST-11 (National Institute of Standards and Technology) mass spectral database and NIST mass spectral search program (Version 2.0 g).</p><p>\n<italic>Determination of minimum inhibitory concentration (MIC)</italic> - MIC was determined using the clinical and laboratory standards institute (CLSI) M27-A3 broth microdilution method with modification.<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref> Briefly, 100 µL of the dilution of the EO in RPMI 1640 with MOPS was mixed with 100 µL of the prepared standard fungal cell suspensions (1 × 10<sup>6</sup> cells/mL) in a 96 well sterile flat bottomed polystyrene microtiter plate followed by aerobic incubation at 37ºC for 24 h. After incubation, plates were visually observed for the presence and absence of growth (turbidity of the suspensions). Minimum concentration of the treatment without any turbidity was considered as MIC point.</p><p>Growth control: 100 µL of buffered RPMI 1640 instead of EO + standard cell suspension. Positive control: 120 mg/mL chlorhexidine digluconate was tested.</p><p>\n<italic>Effect on initial adhesion</italic> - Effect of <italic>C. verum</italic> leaf oil on <italic>C. albicans</italic> (ATCC MYA-2876), <italic>C. dubliniensis</italic> (ATCC MYA-646) and <italic>C. tropicalis</italic> (ATCC 750) adhesion to a sterile polystyrene surface was studied by using previously published methodology by Raut et al.<xref rid=\"B25\" ref-type=\"bibr\">\n<sup>25</sup>\n</xref>\n</p><p>Few colonies of 24 h old fresh <italic>Candida</italic> cultures on sabouraud dextrose agar (SDA) were inoculated into 50 mL of YPD broth in labeled sterile culture media bottle and incubated at 35-37ºC for 18-24 h in a shaking incubator at 30 rpm (Nova Instruments, Brazil).</p><p>After incubation, cells were harvested and washed twice with sterile phosphate buffered saline (PBS) and the inoculum was adjusted to 1.0 x 10<sup>7</sup> cells/mL in RPMI 1640 medium after counting cells using Neubauer improved counting chamber.<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref>\n</p><p>Ninety-six well sterile flat bottomed microtiter plates were seeded with these suspensions (100 µL/well) and allowed to adhere to polystyrene surface of plates for 2 h at 37ºC in a shaker incubator (75 rpm, Nova Instruments, Brazil), in the presence of different concentrations of <italic>C. verum</italic> leaf oil ranging from 2 mg/mL to 0.0039 mg/mL (100 µL/well). Wells without oil were kept as growth control. RPMI buffered with MOPS+0.05% Tween 80 was added to negative control wells. 120 mg/mL chlorhexidine digluconate in RPMI 1640 buffered with MOPS (Sigma-Aldrich, USA) was used as positive control.</p><p>After 2 h incubation, wells were washed with 200 µL sterile normal saline in order to remove non-adherent cells. Attached cells in each well were quantified using XTT metabolic assay. Briefly, 80 μL of XTT [2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide, Sigma-Aldrich] and menadione (Sigma-Aldrich) solution (4 µL of menadione in 10 mL XTT) was added to each well and plates were then covered with a piece of aluminium paper to protect from light and incubated for 2 h at 37<sup>º</sup>C. Then, the absorbance of resulting solution was measured at 490 nm using microtiter plate reader /Versa MAX, molecular Devices, USA.<xref rid=\"B26\" ref-type=\"bibr\">\n<sup>26</sup>\n</xref>\n</p><p>Percentage reduction of adhered cells was calculated by comparing the mean XTT activity of the test groups and negative control group.<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref>\n</p><p>\n<italic>Effect on germ tube formation</italic> - The formation of germ tubes from blastoconidia is the first step in the development of true hyphae and the ability to switch between morphological forms has been suggested as a potential virulence factor. Compared to the unicellular blastoconidial form, hyphae have an increased ability to adhere to and penetrate host tissues.</p><p>An experiment described by Hammer et al.<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref> was carried out to examine the effects of <italic>C. verum</italic> leaf oil on the morphological transition (germ tube/hyphal formation) from blastoconidia to hypha of <italic>C. albicans</italic> (ATCC MYA-2876) and <italic>C. dubliniensis</italic> (ATCC MYA-646).</p><p>Three concentrations (1/4 MIC, 1/2 MIC and MIC) of <italic>C. verum</italic> leaf oil and chlorhexidine digluconate were prepared in foetal bovine serum (FBS) (Sigma-Aldrich) in 1 mL volumes in sterile glass Bijou bottles at twice the desired final concentration as follows: 0.25, 0.5, 1.0 and 0 mg/mL.</p><p>Equal volumes (1 mL) of the prepared cell suspensions in yeast extract-peptone-dextrose (YPD) medium as explained previously were added to each <italic>C. verum</italic> leaf oil treatment and mixed thoroughly. All treatments were then incubated aerobically at 37ºC, without shaking, and were sampled at 0 h, 2 h, 4 h and 6 h after mixing thoroughly.</p><p>10 μL of prepared suspensions were loaded to Neubauer improved counting chamber at each time point and the number of germinated cells (Cells with germ tubes) in 500 <italic>Candida</italic> cells was counted and the percentage of germ tube formation was calculated by dividing the number of germinated cells by five.</p><p>\n<italic>Effect on biofilm development in the presence of (EO)</italic> - 100 μL of standard inoculum of each organism was inoculated to a 96-well sterile flat bottomed microplate plate, followed by aerobic incubation for 120 minutes under agitation (75 rpm at 37ºC)( Nova Instruments, Brazil). The plate was then washed once with sterile PBS and 100 μL of the each dilution of the essential oil was added separately to the treatment wells. 100 μL of RPMI 1640 buffered with MOPS was added to the growth control wells and buffered RPMI 1640 with 0.05% Tween 80 was added to negative control wells instead of oil.<xref rid=\"B28\" ref-type=\"bibr\">\n<sup>28</sup>\n</xref> Chlorhexidine digluconate was used as positive control. The plate was then aerobically incubated for 24 h at 37ºC.</p><p>Biofilm biomass was quantified after 24 h incubation using XTT metabolic assay explained previously.</p><p>\n<italic>Minimum toxic concentration for forming biofilms (MTC)</italic> - MTC determines the minimum concentration of <italic>C. verum</italic> leaf oil requires to kill the forming biofilms of <italic>Candida</italic> spp. completely. The CFU assay was used to detect the MTC of the oil on forming biofilms. After the 24 h treatment with different concentrations of <italic>C. verum</italic> leaf oil, the mass of the adhered biofilm was scraped using a sterile cell scraper and thoroughly homogenised in 100 µL of sterile normal saline and then the suspension was serially diluted (10<sup>-1</sup> to 10<sup>-6</sup>). Subsequently, an aliquot of 100 µL was plated on SDA medium. Agar plates were incubated at 37ºC for 24 h. After incubation, the colony forming units were counted and the results were expressed in CFU/mL.<xref rid=\"B29\" ref-type=\"bibr\">\n<sup>29</sup>\n</xref>\n</p><p>\n<italic>Scanning electron microscopy (SEM) of forming biofilms in the presence of C. verum leaf oil</italic> - Ultra structural properties of biofilms formed with the presence of different concentrations of <italic>C. verum</italic> oil was evaluated using SEM.<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref>\n</p><p>Sterile cover slips were placed in 12-well cell culture cluster separately. Coverslips were immersed in 1 mL standardised cell suspension (1 x 10<sup>7</sup> cells/mL) to ensure uniform <italic>Candida</italic> adhesion and incubated for 120 min at 37ºC in a shaker incubator (Nova Instruments, Brazil). Then the cover slips were moved carefully using a sterile forceps and gently immersed in new 12-well plate containing 0.5 mg/mL, 1.0 mg/mL and 2 mg/mL <italic>C. verum</italic> oil in RPMI 1640 (1 mL/well). Coverslips were incubated aerobically for 24 h at 37ºC. After 24 h incubation, the cover slips with formed biofilms in the presence of EO dilutions were subsequently washed three times with sterile PBS. Then they were transferred to a new 24-well cell culture cluster containing 2.5% glutaraldehyde at 4ºC. After fixing with glutraldehyde for 60 min, samples were dehydrated in a series of ethanol solutions (70% for 10 min, 80% for 10 min, 95% for 10 min and 10 min in absolute ethanol), and air-dried overnight in an incubator prior to sputter coating with gold. Chlorhexidine digluconate (0.25 mg/mL and 0.125 mg/mL) and Fluconazole (0.008 mg/mL) were used as positive controls to compare the post-exposure biofilm architecture with common antifungals and <italic>C. verum</italic> leaf oil treatment.</p><p>\n<italic>Biofilm progression analysis (Time Lapse Microscopy)</italic> - Progression of <italic>Candida in-vitro</italic> biofilms in the presence of <italic>C. verum</italic> leaf oil was evaluated using Time Lapse Microscopy. For analysis of biofilm progression in the presence of <italic>C. verum</italic> leaf oil, <italic>C. albicans</italic> (ATCC MYA-2876); <italic>C. dubliniensis</italic> (ATCC MYA-646) and <italic>C. tropicalis</italic> (ATCC 750) were adhered on to the polystyrene surface of 24-well cell culture cluster and treated with three concentrations (0.5 mg/mL, 1.0 mg/mL and 2.0 mg/mL) of <italic>C. verum</italic> leaf oil as explained previously. During the 24 h incubation at 37ºC after adding the treatment, images of forming biofilms were captured at time 0, 4 h, 8 h, 12 h, 16 h, 20 h and 24 h using time-lapse microscope<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref> (ZEISS ApoTome.2).</p><p>\n<italic>In-vivo toxicity of C. verum essential oil on G. mellonella larvae</italic> - The <italic>in-vivo</italic> toxicity experiment was performed on <italic>G. mellonella</italic> larvae as described previously by Wijesinghe (2019).<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref> Study groups and control group (n = 10) of larvae (RJ Mous Livebait, The Netherlands) were selected and selected larvae were kept in a Petri dishes (10/petri dish). Larvae with colour changes in their bodies including dark pigmentations or apparent melanisation, and larvae outside the body weight of 0.2- 0.3 g were excluded. Dilutions of EO of <italic>C. verum</italic> leaf were prepared in sterile PBS (Concentrations ranging from 0.5 to 1000 mg/mL).</p><p>Ten (10) µL of EO dilutions were injected in to the haemocele through the last left pro-leg of larvae using a 1 mL syringe. The prick area was decontaminated with 70% ethanol prior to administration of EO. Larvae containing dishes were then covered with a lid and kept in an aerobic incubator at 37ºC. 10 µL Sterile PBS with 0.05% Tween 80 was administered to negative control group (n = 10).</p><p>Viability of larvae was monitored by visual inspection of the body appearance (brown-dark brown colour) and by lack of body movement. The experiment was repeated two times.</p><p>\n<italic>Statistical analysis</italic> - The statistical analysis was carried out by using the software Statistical Package for Social Sciences (SPSS) version 16. Multiple means of more than three data sets were compared using one way analysis of variance (ANOVA) and two way ANOVA. The level of significance was taken at 5% (p < 0.05).</p></sec><sec sec-type=\"results\"><title>RESULTS</title><p>\n<italic>Chemical composition</italic> - Identified and characterised chemical constituents and their relative abundance were represented in <xref rid=\"t1\" ref-type=\"table\">Table I</xref>. The most abundant active compound was Eugenol (77.22%). Benzyl benzoate (4.53%), Trans caryophyllene (3.39%), Acetyle eugenol (2.75%) and Linalool (2.11%) were found as minor components with a > 95% similarity between the spectra (NIST) (<xref rid=\"t1\" ref-type=\"table\">Table I</xref>).</p><p>\n<table-wrap id=\"t1\" orientation=\"portrait\" position=\"float\"><label>TABLE I</label><caption><title>Chemical constituents and relative abundunce of <italic>Cinnamomum verum</italic> leaf essential oil (EO) detected by gas chromatography-mass spectrometry (GC-MS)</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Retention time (min)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Compound</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">% Abundance</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">21.41</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Eugenol</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">77.22</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">36.65</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Benzyl Benzoate</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.53</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">23.67</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Trans caryophyllene</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.39</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">28.03</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Acetyl eugenol</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.75</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">10.56</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Linalool</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.11</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">17.45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Trans-Cinnamaldehyde</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.69</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">24.71</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Acetic acid cinnamyl ester</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.49</td></tr></tbody></table></table-wrap>\n</p><p>\n<italic>MIC</italic> - <xref rid=\"t2\" ref-type=\"table\">Table II</xref> represents the MIC values corresponding to the <italic>C. verum</italic> EO and the positive control chlorhexidine digluconate. Both <italic>C. verum</italic> and chlorhexidine digluconate exhibited a similar efficacy on planktonic <italic>C. albicans</italic> (ATCC MYA-2876) and <italic>C. dubliniensis</italic> (ATCC MYA-646). <italic>C tropicalis</italic> (ATCC 750) was more susceptible for chlorhexidine digluconate compared to <italic>C. verum</italic> leaf oil.</p><p>\n<table-wrap id=\"t2\" orientation=\"portrait\" position=\"float\"><label>TABLE II</label><caption><title>Minimum inhibitory concentration (MIC) of <italic>Candida</italic> spp. Experiment was made in triplicates with three individual experiments. Chlorhexidine digluconate was used as positive control</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"2\"/><col span=\"1\"/><col span=\"1\"/></colgroup><tbody><tr><td align=\"left\" colspan=\"2\" rowspan=\"1\">Organism </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Cinnamomum verum</italic> oil mg/mL</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Chlorhexidine digluconate mg/mL</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>C. albicans</italic> (ATCC 5314)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>C. tropicalis</italic> (ATCC 750)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>C. dubliniensis</italic> (ATCC MYA-646)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.0</td></tr></tbody></table></table-wrap>\n</p><p>\n<italic>Effect on initial adhesion</italic> - The results of the microtiter plate XTT method in order to determine the effect of <italic>C. verum</italic> leaf oil on <italic>Candida</italic> adhesion on to a polystyrene surfaces was showed in <xref ref-type=\"fig\" rid=\"f1\">Fig. 1</xref>.</p><p>\n<fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1:</label><caption><title>percentage reduction in XTT metabolic activity of <italic>Candida albicans</italic> (ATCC MYA-2876), <italic>C. tropicalis</italic> (ATCC 750) and <italic>C. dubliniensis</italic> (ATCC MYA-646) in the presence of different concentrations of <italic>Cinnamomum verum</italic> (CV) leaf oil and chlorhexidine digluconate (CHL). All error bars represent the ± 2 standard deviations (SD).</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200349-gf1\"/></fig>\n</p><p>According to obtained data, <italic>C. verum</italic> leaf oil effectively reduce the adhesion of all test strains on to polystyrene surface compared to negative control. <italic>C. albicans</italic> achieved 50% reduction in adhesion with 1.0 mg/mL concentration, whereas, <italic>C. tropicalis</italic> and <italic>C. dubliniensis</italic> showed 50% reduction in adhesion with > 2.0 mg/mL and 0.34 mg/mL concentrations of oil respectively. Chlorhexidine digluconate exhibited 50% reduction in adhesion of <italic>C. albicans</italic>, <italic>C. tropicalis</italic> and <italic>C. dubliniensis</italic> with the concentrations of 1.06 mg/mL, 0.72 mg/mL and 1.32 mg/mL respectively (<xref rid=\"t3\" ref-type=\"table\">Table III</xref>).</p><p>\n<table-wrap id=\"t3\" orientation=\"portrait\" position=\"float\"><label>TABLE III</label><caption><title>Minimum concentrations of <italic>Cinnamomum verum</italic> leaf essential oil (EO) and chlorhexidine digluconate (CHL) required to reduce the adhesion of <italic>Candida albicans</italic> (ATCC MYA-2876), <italic>C. tropicalis</italic> (ATCC 750) and <italic>C. dubliniensis</italic> (ATCC MYA-646) by 50%</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"2\"/><col span=\"2\"/><col span=\"2\"/></colgroup><tbody><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<italic>C. albicans</italic>\n</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<italic>C. tropicalis</italic>\n</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<italic>C. dubliniensis</italic>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>C. verum</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CHL</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>C. verum</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CHL</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>C. verum</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CHL</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Concentration (mg/mL)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.06</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">> 2.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.72</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.32</td></tr></tbody></table></table-wrap>\n</p><p>\n<italic>Effect of C. verum leaf oil on germ tube formation</italic> - <xref ref-type=\"fig\" rid=\"f2\">Fig. 2</xref> shows the percentage of germ tube forming cells in the presence of 0.25, 0.5 and 1.0 mg/mL of <italic>C. verum</italic> leaf oil and chlorhexidine digluconate throughout 6 h experiment period.</p><p>\n<fig id=\"f2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2:</label><caption><title>percentage germ tube formation of (A) <italic>Candida albicans</italic> (ATCC MYA-2876) and (B) <italic>C. dubliniensis</italic> (ATCC MYA-646) with 0.25, 0.5 and 1.0 mg/mL <italic>Cinnamomum verum</italic> leaf oil and chlorhexidine digluconate within 6 h test period with 2 h intervals. 0 mg/mL concentration indicates the negative control. All error bars represent the ± 2 standard deviations (SD).</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200349-gf2\"/></fig>\n</p><p>According to obtained data, both <italic>C. verum</italic> leaf oil and chlorhexidine digluconate significantly reduced the germ tube formation of <italic>C. albicans</italic> and <italic>C. dubliniensis</italic> (p < 0.05) with all tested concentrations. Both <italic>C. verum</italic> leaf oil and chlorhexidine digluconate at 1.0 mg/mL (MIC) concentration completely inhibited the germ tube formation of both <italic>Candida</italic> species throughout the experiment period (6 h).</p><p>\n<italic>Minimum biofilm inhibitory concentration of forming biofilms</italic> - In this experiment, minimum concentration of <italic>C. verum</italic> leaf oil required to inhibit the biofilm formation by 50% (compared to negative control) was determined. Biomass/viability of formed biofilms in the presence of essential oil was determined using XTT viability assay. <xref ref-type=\"fig\" rid=\"f3\">Fig. 3</xref> showed the percentage reduction of XTT metabolic activity of forming biofilms compared to negative control.</p><p>\n<fig id=\"f3\" orientation=\"portrait\" position=\"float\"><label>Fig. 3:</label><caption><title>percentage reduction in XTT metabolic activity of <italic>Candida albicans</italic> (ATCC MYA-2876), <italic>C. tropicalis</italic> (ATCC 750) and <italic>C. dubliniensis</italic> (ATCC MYA-646) forming biofilms with the presence of different concentrations of <italic>Cinnamomum verum</italic> (CV) leaf oil and chlorhexidine digluconate (CHL). All error bars represent the ± 2 standard deviations (SD). All error bars represent the ± 2 standard deviations (SD).</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200349-gf3\"/></fig>\n</p><p>According to obtained data, concentrations required to reduce the biofilm formation by 50% as follows (<xref rid=\"t4\" ref-type=\"table\">Table IV</xref>).</p><p>\n<table-wrap id=\"t4\" orientation=\"portrait\" position=\"float\"><label>TABLE IV</label><caption><title>Minimum biofilm inhibitory concentrations (MBIC<sub>50</sub>) for forming biofilms of <italic>Candida albicans</italic> (ATCC MYA-2876), <italic>C. tropicalis</italic> (ATCC 750) and <italic>C. dubliniensis</italic> (ATCC MYA-646)</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"2\"/><col span=\"2\"/><col span=\"2\"/></colgroup><tbody><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">\n</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<italic>C. albicans</italic>\n</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<italic>C. tropicalis</italic>\n</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<italic>C. dubliniensis</italic>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Cinnamomum verum</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CHL</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>C. verum</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CHL</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>C. verum</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">CHL</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">MBIC<sub>50</sub> (mg/mL)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.35</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.025</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN4\"><p>CHL: chlorhexidine digluconate.</p></fn></table-wrap-foot></table-wrap>\n</p><p>\n<italic>MTC</italic> - Minimum concentration of <italic>C. verum</italic> leaf oil required to prevent the biofilm formation completely by killing <italic>Candida</italic> cells was determined using CFU assay. <xref ref-type=\"fig\" rid=\"f4\">Fig. 4</xref> represents the viability of forming biofilms with the presence of different concentrations of <italic>C. verum</italic> leaf oil and chlorhexidine digluconate determined by CFU assay on forming biofilms. <xref rid=\"t5\" ref-type=\"table\">Table V</xref> represents the minimum concentration of treatments required to prevent the biofilm formation of test strains completely.</p><p>1.0 mg/mL <italic>C. verum</italic> kill the forming biofilms of all test strains, <italic>C. albicans</italic> (ATCC MYA-2876), <italic>C. tropicalis</italic> (ATCC 750) and <italic>C. dubliniensis</italic> (ATCC MYA-646). Chlorhexidine digluconate kills forming biofilms of <italic>Candida</italic> species with low concentrations compared to <italic>C. verum</italic> leaf oil. For <italic>C. albicans</italic>, the killing concentration with chlorhexidine digluconate was 0.25 mg/mL (Four times low concentration compared to that value of <italic>C. verum</italic> leaf oil) and for <italic>C. tropicalis</italic> and <italic>C. dubliniensis</italic>, killing concentration was 0.125 mg/mL (Eight times less concentration compared to that value of <italic>C. verum</italic>).</p><p>\n<fig id=\"f4\" orientation=\"portrait\" position=\"float\"><label>Fig. 4:</label><caption><title>Log colony forming unit (CFU) values of forming <italic>Candida albicans</italic> (CA) (ATCC MYA-2876), <italic>C. tropicalis</italic> (CT) (ATCC 750) and <italic>C. dubliniensis</italic> (CD) (ATCC MYA-646) biofilms in the presence of different concentrations of (A) <italic>Cinnamomum verum</italic> (CV) leaf oil and (B) chlorhexidine digluconate (CHL). All error bars represent the ± 2 standard deviations (SD).</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200349-gf4\"/></fig>\n</p><p>\n<table-wrap id=\"t5\" orientation=\"portrait\" position=\"float\"><label>TABLE V</label><caption><title>Concentrations of <italic>Cinnamomum verum</italic> leaf oil and chlorhexidine digluconate that kill the forming biofilms (MTC) of <italic>Candida albicans</italic> (ATCC MYA-2876), <italic>C. tropicalis</italic> (ATCC 750) and <italic>C. dubliniensis</italic> (ATCC MYA-646) completely</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"2\"/></colgroup><tbody><tr><td align=\"left\" rowspan=\"2\" colspan=\"1\">Test strain</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">Concentration requires to kill the forming biofilms (mg/mL)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>C. verum</italic> oil</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Chlorhexidine digluconate</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>C. albicans</italic> (ATCC MYA-2876)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>C. tropicalis</italic> (ATCC 750)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.125</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>C. dubliniensis</italic> (ATCC MYA-646)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.125</td></tr></tbody></table></table-wrap>\n</p><p>\n<italic>SEM of forming biofilms</italic> - Ultrastructure of forming biofilms of <italic>C. albicans</italic> (ATCC MYA-2876), <italic>C. tropicalis</italic> (ATCC 750) and <italic>C. dubliniensis</italic> (ATCC MYA-646) in the presence of 0.5, 1.0 and 2.0 mg/mL <italic>C. verum</italic> leaf oil, chlorhexidine digluconate and Fluconazole was qualitatively evaluated by SEM (<xref ref-type=\"fig\" rid=\"f5\">Figs 5</xref>, <xref ref-type=\"fig\" rid=\"f6\">6</xref>, <xref ref-type=\"fig\" rid=\"f7\">7</xref>).</p><p>\n<italic>Cinnamomum verum</italic> leaf oil caused the <italic>Candida</italic> cell shrinkage by damaging the cell wall and cause leakage of intracellular materials. These effects are concentration dependent. Maximum cell damage was observed with 2 mg/mL <italic>C. verum</italic> leaf oil. And <italic>C. verum</italic> leaf oil reduced the biofilm development and biofilm cell density.</p><p>\n<fig id=\"f5\" orientation=\"portrait\" position=\"float\"><label>Fig. 5:</label><caption><title>scanning electron microscopy (SEM) images of <italic>Candida albicans</italic> (ATCC MYA-2876) forming biofilms in the presence of 0.5 mg/mL <italic>Cinnamomum verum</italic> leaf oil (A and D), 1.0 mg/mL <italic>C. verum</italic> leaf oil (B and E), 2 mg/mL <italic>C. verum</italic> leaf oil (C and F), 0.25 mg/mL chlorhexidine digluconate (G and J) and 0.008 mg/mL Fluconazole (H and K). I and L: negative control. Red circles: cell wall deformities with treatments.</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200349-gf5\"/></fig>\n</p><p>\n<fig id=\"f6\" orientation=\"portrait\" position=\"float\"><label>Fig. 6:</label><caption><title>scanning electron microscopy (SEM) images of <italic>Candida tropicalis</italic> (ATCC 750) forming biofilms in the presence of 0.5 mg/mL <italic>Cinnamomum verum</italic> leaf oil (A and D), 1.0 mg/mL <italic>C. verum</italic> leaf oil (B and E), 2.0 mg/mL <italic>C. verum</italic> leaf oil (C and F), 0.125 mg/mL chlorhexidine digluconate (G and J) and 0.008 mg/mL Fluconazole (H and K) I and L: negative control. Red circle: cell wall deformities with treatments. Red solid arrow: leakages of intracellular components.</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200349-gf6\"/></fig>\n</p><p>\n<fig id=\"f7\" orientation=\"portrait\" position=\"float\"><label>Fig. 7:</label><caption><title>scanning electron microscopy (SEM) images of <italic>Candida dubliniensis</italic> (ATCC MYA-646) forming biofilms in the presence of 0.5 mg/mL <italic>Cinnamomum verum</italic> leaf oil (A and D), 1.0 mg/mL <italic>C. verum</italic> leaf oil (B and E), 2mg/mL <italic>C. verum</italic> leaf oil (C and F), 0.125 mg/mL chlorhexidine digluconate (G and J) and 0.008 mg/mL Fluconazole (H and K) I and L: negative control. Red circle: cell wall deformities with treatments.</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200349-gf7\"/></fig>\n</p><p>\n<italic>Biofilm progression analysis using time lapses microscope</italic> - <xref ref-type=\"fig\" rid=\"f8\">Fig. 8</xref>A-C indicate the Time lapses microscopic images of forming biofilms of <italic>C. albicans</italic> (ATCC MYA-2876); <italic>C. dubliniensis</italic> (ATCC MYA-646) and <italic>C. tropicalis</italic> (ATCC 750) without essential oil treatment, 0.5 mg/mL, 1.0 mg/mL and 2.0 mg/mL <italic>C. verum</italic> leaf oil treatment for 24 h time period.</p><p>The progression of the untreated (negative) control biofilm demonstrates excessive biofilm developement throughout the observation period. 0.5 mg/mL concentration of <italic>C. verum</italic> leaf caused retardation of biofilm development of <italic>Candida</italic> spp. 1.0 mg/mL and 2.0 mg/mL <italic>C. verum</italic> leaf oil completely inhibited <italic>C. tropicalis</italic> and <italic>C. dubliniensis</italic> cell proliferation and biofilm development from 0 h.</p><p>With 1.0 mg/mL <italic>C. verum</italic> leaf oil, <italic>C. albicans</italic> exhibited slight cell proliferation up to 8 h, while 2.0 mg/mL completely inhibited biofilm development.</p><p>\n<fig id=\"f8\" orientation=\"portrait\" position=\"float\"><label>Fig. 8:</label><caption><title>(A) Time Lapses images of developing biofilms of <italic>Candida albicans</italic> (ATCC MYA-2876). Each column represents the concentration of <italic>Cinnamomum verum</italic> leaf oil treatment. (B) Time Lapses images of developing biofilms of <italic>C. tropicalis</italic> (ATCC 750). Each column represents the concentration of <italic>C. verum</italic> leaf oil treatment. (C) Time Lapses images of developing biofilms of <italic>C. dubliniensis</italic> (ATCC MYA-646). Each column represents the concentration of <italic>C. verum</italic> leaf oil treatment.</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200349-gf8\"/></fig>\n</p><p>\n<italic>In-vivo toxicity of C. verum leaf oil (G. mellonella larvae)</italic> - <italic>In-vivo</italic> toxicity of <italic>C. verum</italic> leaf EO was evaluated by using <italic>in-vivo G. mellonella</italic> larvae model after treatment with different concentrations of EO. 100% survival of the treated larvae was observed at all concentrations tested (0.5, 1.0, 2.0, 16.0, 32.0, 64.0, 128.0, 250.0, 500.0, 1000.0 mg/mL) throughout whole experiment period (5 d) which indicates the non-toxicity of <italic>C. verum</italic> leaf EO on experimental model (<xref ref-type=\"fig\" rid=\"f9\">Fig. 9</xref>).</p><p>\n<fig id=\"f9\" orientation=\"portrait\" position=\"float\"><label>Fig. 9:</label><caption><title>survival rate of <italic>Galleria mellonella</italic> larvae after administration of <italic>Cinnamomum verum</italic> leaf oil over five days experiment period. Control curve was obtained by administrating sterile phosphate buffered saline (PBS) in to larvae.</title></caption><graphic xlink:href=\"1678-8060-mioc-115-e200349-gf9\"/></fig>\n</p></sec><sec sec-type=\"discussion\"><title>DISCUSSION</title><p>\n<italic>Candida</italic> spp. displays many virulent factors that contribute to successfully colonise in host tissues. Adhesion to host surfaces (which is the initial step of biofilm formation), germ tube formation, biofilm formation, secretion of proteinases and other hydrolytic enzymes and aldehyde production are some of them.<xref rid=\"B30\" ref-type=\"bibr\">\n<sup>30</sup>\n</xref> Phytomedicinal therapeutics is becoming more popular in modern word because of the high occurrence of the antimicrobial resistance and increased virulence of microbial pathogens.<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref> On the other hand, easy access, lack of toxicity to the host, high efficacy and cost effectiveness contribute the widespread use of antimicrobial natural products as therapeutic alternatives.<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref> This study was conducted to evaluate the efficacy of such plant derived antimicrobial agent, <italic>C. verum</italic> leaf EO on controlling the virulence mechanisms of the most common human fungal pathogen, <italic>Candida</italic> spp. Further the toxicity on <italic>in-vivo</italic> laboratory experiment model, <italic>G. melonella</italic> larvae was also evaluated.</p><p>Adhesion to the host surfaces or abiotic surfaces is the very initial step of colonisation of the microbial pathogens. 1.0 mg/mL concentration of EO reduced adhesion of <italic>C. albicans</italic> by 50%, whereas, > 2.0 mg/mL and 0.34 mg/mL concentrations of oil reduced <italic>C. tropicalis</italic> and <italic>C. dubliniensis</italic> adhesion by 50% respectively. Reference antifungal chlorhexidine digluconate exhibited 50% of reduction in adhesion to <italic>C. albicans</italic>, <italic>C. tropicalis</italic> and <italic>C. dubliniensis</italic> with the concentrations of 1.06 mg/mL, 0.72 mg/mL and 1.32 mg/mL. Importantly, 50% reduction of adhesion of <italic>C. albicans</italic> and <italic>C. dubliniensis</italic> was achieved with lower concentrations of <italic>C. verum</italic> leaf oil compared to chlorhexidine digluconate control which indicates the higher efficacy of <italic>C. verum</italic> EO in anti-adhesion of those two strains. According to data obtained, 2.0 mg/mL chlorhexidine digluconate completely inhibit the adhesion of all test strains, but the observation may be due to killing of organisms completely during test period (2 h). Since adhesion on to the host surfaces is one of the major virulent factor and initial step of biofilm formation, the anti-adhesion properties of <italic>C. verum</italic> EO on <italic>Candida</italic> spp. contributes to its’ aptness as an antimicrobial agent.</p><p>Germ tube formation is another major virulent factor of <italic>C. albicans</italic> and <italic>C. dubliniensis</italic> that enables the absorption of nutrients from host tissues by invading them. Effect of <italic>C. verum</italic> at sub-MIC (0.25 and 0.5 mg/mL), both <italic>C. verum</italic> leaf oil and chlorhexidine digluconate significantly reduce the germ tube formation of both <italic>C. albicans</italic> and <italic>C. dubliniensis</italic> whereas MIC (1.0 mg/mL) completely inhibit the germ tube formation of test strains. With time (for <italic>C. albicans</italic>, after 2 h and for <italic>C. dubliniensis</italic>, after 4 h) the germ tube formation in all groups including control group gradually decreased since produced germ tubes transformed to pseudo hyphae and hyphae. There are no published scientific evidences available on effect of <italic>C. verum</italic> leaf EO on adhesion and germ tube formation of <italic>Candida</italic> spp. Current study was designed to find the efficacy of Cinnamon leaf oil on controlling virulent factors of <italic>Candida</italic> spp. in order to fill this gap of available data. Adhesion and germ tube formation are key attributes of infection causing ability of <italic>Candida</italic> and therefore antimicrobial substances which interfere with these factors are considered as potential anti-<italic>Candida</italic> agents. Hence <italic>C. verum</italic> leaf EO can be developed as a therapeutic alternative against <italic>Candida</italic> infections.</p><p>Biofilm is a surface attached microbial communities embedded in an extracellular matrix derived from cells and environment.<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref> Biofilms are more resistant to physical and chemical stresses compared to their planktonic counterpart as biofilms exhibit various mechanisms to neutralise those external stresses. Extracellular biofilm matrix acts as a physical or chemical barrier for diffusion of antimicrobial agents into the biofilm. Further, with the limited availability of nutrients, the biofilm community shifts towards slow or no growth status from exponential growth. All above factors and induction of a biofilm phenotype and quorum sensing contribute to high resistance to antimicrobial agents of a biofilm.<xref rid=\"B31\" ref-type=\"bibr\">\n<sup>31</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B32\" ref-type=\"bibr\">\n<sup>32</sup>\n</xref> Since the resistance of biofilms to available antimicrobial strategies is becoming more widespread, more studies should be carried out in order to invent novel, non-toxic, inexpensive and effective treatment options specially by controlling the virulence of the pathogen effectively. In the current study, efficacy of <italic>C. verum</italic> leaf EO as a potential phytomedicinal agent was evaluated on forming <italic>Candida</italic> biofilms. <italic>C. verum</italic> EO exhibited a potential antibiofilm effect on forming biofilms of <italic>C. albicans</italic> (ATCC MYA-2876), <italic>C. tropicalis</italic> (ATCC 750) and <italic>C. dubliniensis</italic> (ATCC MYA-646) test strains. Concentrations required to reduce the biofilm development by 50% (Compared to negative control) were 0.15, 0.35 and 0.2 mg/mL for three test strains respectively. And the MTC (concentrations of EO required to completely cease the biofilm development) was 1.0 mg/mL for all test strains. Importantly, these MTC values are lower than MIC and MFC values of test strains which indicates the high potency of killing of forming biofilms of <italic>C. verum</italic> EO though biofilms are considered as more resistant microbial communities than its planktonic counterpart. These data suggest the potential action of <italic>C. verum</italic> oil on interrupting the normal process of formation of <italic>Candida</italic> biofilms.</p><p>Biofilm progression under the chemical stress of <italic>C. verum</italic> leaf oil was visualised with time lapses microscope and results obtained from this experiment confirm above observation. 2 × MTC (2 mg/mL) of <italic>C. verum</italic> EO completely inhibited biofilm development of all test strains from time 0 h whereas slight cellular proliferation and biofilm development was observed in progressing <italic>C. albicans</italic> with MTC (1 mg/mL) from 0h to 8 h. After 8 h, no visible biofilm development was noted in <italic>C. albicans</italic> developing biofilms. <italic>C. tropicalis</italic> and <italic>C. dubliniensis</italic> biofilm development was completely inhibited at 0 h by MTC. Sub-MTC (0.5 mg/mL) causes retardation of biofilm development of all tested <italic>Candida</italic> strains. These results indicate the possible effective use of <italic>C. verum</italic> leaf EO as a biofilm preventive strategy. Further, data from SEM and time lapses microscopy reveal that the effect of EO on the progression of <italic>Candida</italic> biofilm is dose dependent.</p><p>SEM images were taken to understand the structure of the <italic>Candida</italic> biofilms developed with the presence of <italic>C. verum</italic> leaf EO. All biofilms of test strains exhibited reduced cell density, cell wall damages, cell wall deformities and leakages of intracellular materials with treatment of cinnamon leaf oil. Importantly, SEM images confirm the dose dependent nature of effects of <italic>C. verum</italic> leaf essential oil. Fluconazole is the known antifungal agent which belongs to azole group and inhibits synthesis of fungal sterol, ergosterol by preventing the conversion of lanosterol to ergosterol.<xref rid=\"B33\" ref-type=\"bibr\">\n<sup>33</sup>\n</xref> On the other hand chlorhexidine digluconate is a biguanide which is used as an antibacterial mouth rinse. It alters the morphology cells and damages the cell wall of microorganisms and releases intracellular components. It has been suggested as a well-known therapeutic antifungal agent for oral candidiasis.<xref rid=\"B34\" ref-type=\"bibr\">\n<sup>34</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B35\" ref-type=\"bibr\">\n<sup>35</sup>\n</xref> Since both antimicrobial agents have an effect on <italic>Candida</italic> cell wall, ultrastructure of forming <italic>Candida</italic> biofilms with 0.25 mg/mL chlorhexidine digluconate (for <italic>C. albicans</italic>), 0.125 mg/mL chlorhexidine digluconate (for <italic>C. tropicalis</italic> and <italic>C. dubliniensis</italic>) and 0.008 mg/mL Fluconazole (maximum recommended <italic>in vitro</italic> assay concentration, CLSI) were also visualised. Similar observations were obtained with the exposure to MTC of chlorhexidine digluconate and 0.008 mg/mL Fluconazole. The intensity of the post-exposure response of Fluconazole was minimal due to low concentration.</p><p>When considering chlorhexidine digluconate, it exhibited the similar effect as <italic>C. verum</italic> leaf oil (damaging cell walls of <italic>Candida</italic> cells and cause cytoplasmic leakages). Further, according to SEM observations, chlorhexidine digluconate decreased pseudo hyphae formation of <italic>C. tropicalis</italic> compared to negative control. Extracellular polymeric matrix that is characteristic of biofilms was not observed in any of the biofilms analysed using SEM since pre-treatments and electron beam of the equipment can cause the matrix destruction.</p><p>Though, the effect of <italic>C. verum</italic> oil on <italic>Candida</italic> biofilm formation is not well studied, few studies were conducted by various scientists to evaluate the efficacy of Cinnamon oil on virulence and biofilm depletion of different bacterial species. Kalia et al.<xref rid=\"B36\" ref-type=\"bibr\">\n<sup>36</sup>\n</xref> in 2015 studied the anti-quorum sensing activity of cinnamon oil against <italic>Pseudomonas aeruginosa</italic> by measuring the inhibition of biofilm formation and other quorum sensing (QS) associated virulence factors such as proteolytic enzyme production and swarming activity and effective biofilm reduction and antagonist effect on QS. Further, Erfan and Marouf in 2019 observed a downregulation of virulence genes of respiratory bacterial pathogens (<italic>Staphylococcus aureus</italic>, <italic>E. coli</italic>, <italic>Avibacterium paragallinarum</italic> and <italic>Pasteurella multocida</italic> etc.) by cinnamon oil. They suggested the possible use of cinnamon oil for the control of antibiotic-resistant bacterial infections instead of the routine antibiotics.<xref rid=\"B37\" ref-type=\"bibr\">\n<sup>37</sup>\n</xref> Another two studies conducted by Zhang et al. in 2020 and Kim et al. in 2014<xref rid=\"B38\" ref-type=\"bibr\">\n<sup>38</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B39\" ref-type=\"bibr\">\n<sup>39</sup>\n</xref> identified few anti-virulence mechanisms of <italic>C. verum</italic> bark oil including the inhibition of microbial toxins, interrupting the biofilm development and quorum sensing of pathogenic bacterial species. But the anti-virulence properties of <italic>C. verum</italic> leaf oil on fungal virulence was not widely studied.</p><p>Toxicological assessment of a natural product prior to its clinical application is an important aspect when the invention of a novel herbal therapeutic agent is concerned. The current study determines the <italic>in-vivo</italic> toxicity of <italic>C. verum</italic> EO using the <italic>G. mellonella</italic> experiment model. 100% survival was observed with <italic>G. mellonella</italic> larvae with all test concentrations ranging from 0.5 to 1000 mg/mL EO. This indicates the non-toxicity of <italic>C. verum</italic> leaf oil on experiment model. Since 1000 mg/mL is the neat concentration (undiluted) of <italic>C. verum</italic> leaf EO all dilutions including neat solution can be considered as non-toxic. Even though previous <italic>in-vivo</italic> studies conducted on animal models have also highlighted lack of significant toxicity with a significantly high therapeutic range of concentration of cinnamon bark oil,<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref> the <italic>in-vivo</italic> toxicity of cinnamon leaf oil on laboratory animal models was not well studied.</p><p>The results of the chemical analysis of the <italic>C. verum</italic> leaf EO agree with the findings of previous studies, which indicate that eugenol is its most abundant chemical component (77.22%) whereas the other chemical compounds appear in smaller concentrations.<xref rid=\"B40\" ref-type=\"bibr\">\n<sup>40</sup>\n</xref>\n</p><p>The high occurrence and mortality rates associated with Candidiasis emphasise the urgent necessity for the introduction of new therapeutic approaches to treat candidiasis. Various studies conducted by multiple research groups has provided important knowledge about the pathogenesis of candidiasis,<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B41\" ref-type=\"bibr\">\n<sup>41</sup>\n</xref> and this knowledge should represent a starting point for the transformation of their findings into treatment strategies that can directly benefit patients suffering from <italic>Candida</italic> and other infections. More effective and earliest option among them is the invention of novel anti-virulence approaches for the control and treatment of <italic>Candida</italic> infections.<xref rid=\"B42\" ref-type=\"bibr\">\n<sup>42</sup>\n</xref> Because germ tube formation, adhesion on to host surfaces and biofilm formation constitute the major virulence factors during candidiasis, they represent a clinically unexploited target for the innovation of such alternative anti-virulence strategies. In this study, felicity of a natural product, <italic>C. verum</italic> leaf EO as a non-toxic, anti-virulence strategy against <italic>Candida</italic> spp. was identified using both <italic>in vitro</italic> and <italic>in vivo</italic> experiment series.</p><p>Though a number of <italic>in vitro</italic> and <italic>in-vivo</italic> studies have used plant derived natural products for the treatment of <italic>Candida</italic> infections, the exact mechanism of action of the antifungal effect or the effect on Candidal physiology of those treatments were not clearly understood. The <italic>C. verum</italic> leaf EO used in the present study is a herbal product with potential for non-toxic therapeutic application in the treatment of candidiasis by interfering the virulence of <italic>Candida</italic>. However, further experiments including <italic>in-vivo</italic> toxicity investigations and clinical trials as well as molecular studies on gene regulation are recommended to develop an antifungal agent based on <italic>C. verum</italic> leaf EO.</p><p>In conclusion, this study provides evidence for the high efficacy of <italic>C. verum</italic> leaf EO as an anti-virulence approach against three key virulence factors of <italic>Candida</italic> spp., adhesion, germ tube formation and biofilm formation. The EO extracted from <italic>C. verum</italic> leaves has eugenol as its major active component and is non-toxic against <italic>G. mellonella</italic> larvae.</p></sec></body><back><ack><title>ACKNOWLEDGEMENTS</title><p>To all academic and nonacademic staff members of the area of Microbiology and Immunology, Department of Oral Diagnosis, Piracicaba Dental School, University of Campinas, Brazil, for their support.</p></ack><fn-group><fn fn-type=\"financial-disclosure\" id=\"fn1\"><p>Financial support: CNPq (Grant No. 132718/2018-9).</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id=\"B1\"><label>1</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kojic</surname><given-names>EM</given-names></name><name><surname>Darouiche</surname><given-names>RO</given-names></name></person-group><article-title>Candida infections of medical devices</article-title><source>Clin Microbiol 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Pharmacol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Pharmacol.</journal-id><journal-title-group><journal-title>Frontiers in Pharmacology</journal-title></journal-title-group><issn pub-type=\"epub\">1663-9812</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33041812</article-id><article-id pub-id-type=\"pmc\">PMC7523507</article-id><article-id pub-id-type=\"doi\">10.3389/fphar.2020.571266</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Pharmacology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>CU06-1004 Alleviates Experimental Colitis by Modulating Colonic Vessel Dysfunction</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Kim</surname><given-names>Ye-Seul</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Haiying</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Lee</surname><given-names>Sunghye</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Park</surname><given-names>Songyi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1082777\"/></contrib><contrib contrib-type=\"author\"><name><surname>Noh</surname><given-names>Minyoung</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Kim</surname><given-names>Young-Myeong</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Kwon</surname><given-names>Young-Guen</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1001321\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University</institution>, <addr-line>Seoul</addr-line>, <country>South Korea</country></aff><aff id=\"aff2\"><sup>2</sup><institution>R&D Department, Curacle Co. Ltd</institution>, <addr-line>Seongnam-si</addr-line>, <country>South Korea</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Vascular System Research Center, Kangwon National University</institution>, <addr-line>Chuncheon</addr-line>, <country>South Korea</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Stefano Fiorucci, University of Perugia, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Irina Leonardi, Cornell University, United States; Shinichi Kato, Kyoto Pharmaceutical University, Japan</p></fn><corresp id=\"fn001\">*Correspondence: Young-Guen Kwon, <email xlink:href=\"mailto:ygkwon@yonsei.ac.kr\" xlink:type=\"simple\">ygkwon@yonsei.ac.kr</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p>This article was submitted to Gastrointestinal and Hepatic Pharmacology, a section of the journal Frontiers in Pharmacology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>571266</elocation-id><history><date date-type=\"received\"><day>10</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Kim, Zhang, Lee, Park, Noh, Kim and Kwon</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Kim, Zhang, Lee, Park, Noh, Kim and Kwon</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Inflammatory bowel disease is an autoimmune disease that causes chronic inflammation of the gastrointestinal tract. Endothelial dysfunction, defined by a reduced endothelial barrier and an increase in the expression of adhesion molecules, is part of the pathology of inflammatory bowel disease. In this study, we assessed the therapeutic effect of CU06-1004, an endothelial dysfunction blocker that reduces vascular hyperpermeability and inflammation in a mouse model of colitis. Acute colitis was induced in mice using 3% (w/v) dextran sodium sulfate added to their drinking water for 7 days. Twenty-four hours after the addition of dextran sodium sulfate, either mesalazine or CU06-1004 was administered orally each day. Administration of CU06-1004 significantly reduced the clinical manifestations (weight loss, diarrhea, and bloody stool) and histological changes (epithelium loss, inflammatory cell infiltration, and crypt destruction) induced by dextran sodium sulfate. Proinflammatory cytokines were also reduced, indicating that inflammation was ameliorated. From a vascular perspective, CU06-1004 reduced interrupted and tortuous vessels, enhanced junction protein expression, and reduced inflammatory adhesion molecules, indicating a broad improvement of endothelial dysfunction. Endothelial protection induced epithelial barrier restoration and decreased epithelial inflammation. Blocking endothelial dysfunction with CU06-1004 significantly ameliorated the progression of inflammatory bowel disease. Therefore, CU06-1004 may represent a potential therapeutic agent for the treatment of inflammatory bowel disease as well as other inflammatory diseases.</p></abstract><kwd-group><kwd>CU06-1004</kwd><kwd>inflammatory bowel disease</kwd><kwd>ulcerative colitis</kwd><kwd>endothelium</kwd><kwd>endothelial dysfunction</kwd><kwd>colonic vessel</kwd><kwd>epithelium</kwd><kwd>epithelial barrier</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Ministry of Education<named-content content-type=\"fundref-id\">10.13039/501100002701</named-content></funding-source><award-id rid=\"cn001\">2019R1A2C3007142</award-id></award-group><award-group><funding-source id=\"cn002\">Ministry of Science and Technology<named-content content-type=\"fundref-id\">10.13039/100007225</named-content></funding-source><award-id rid=\"cn002\">NRF-2015M3A9B6066835</award-id></award-group></funding-group><counts><fig-count count=\"6\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"43\"/><page-count count=\"12\"/><word-count count=\"5658\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Inflammatory bowel disease (IBD) is a chronic gastrointestinal disorder associated with changes in mucosal immunity caused by a loss of intestinal homeostasis. It is characterized by abdominal pain, weight loss, diarrhea, and bloody stool (<xref rid=\"B19\" ref-type=\"bibr\">Khor et al., 2011</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Grinspan and Kornbluth, 2015</xref>). The dysregulated intestinal inflammation associated with IBD can also induce complications such as fistulas, stenosis, and abscesses (<xref rid=\"B8\" ref-type=\"bibr\">Blumberg, 2009</xref>). Furthermore, IBD patients have a higher risk of developing colorectal cancer and colitis-associated cancer (<xref rid=\"B26\" ref-type=\"bibr\">Mattar et al., 2011</xref>).</p><p>IBD consists of Crohn’s disease (CD) and ulcerative colitis (UC). These diseases have different but overlapping features. CD affects the entire gastrointestinal tract, but the terminal ileum and colon are most commonly affected. Moreover, it is characterized by an upregulation of interferon-γ, which has a pivotal role in Th1-type responses. On the other hand, UC inflammation occurs typically in the colon and rectum, and is associated with a Th2-type response featuring interleukin-4 and interleukin-13 secretions and activated natural killer T (NK-T) cells. NK-T cells are highly cytotoxic to epithelial cells, aggravating UC, by reducing the epithelial barrier (<xref rid=\"B9\" ref-type=\"bibr\">Brown and Mayer, 2007</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Dharmani and Chadee, 2008</xref>). CD and UC also have common features: imbalances between proinflammatory and antiinflammatory cytokines. Proinflammatory mediators such as interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) are found at high levels in the colonic mucosa. These mediators are produced by innate immune cells such as macrophages and induce cytotoxicity, apoptosis, and an acute-phase response (<xref rid=\"B31\" ref-type=\"bibr\">Papadakis and Targan, 2000</xref>). However, the antiinflammatory cytokine levels of both interleukin-4 and interleukin-10 are decreased. Interestingly, IBD patients (both CD and UC) present with lower intra-luminal pH due to inflammation compared with healthy people. Leukocyte infiltration of the gut and the concomitant increase in metabolic by-product levels makes the gut more acidic by impairing the cellular waste-removal process (<xref rid=\"B22\" ref-type=\"bibr\">Lardner, 2001</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Nugent et al., 2001</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Barkas et al., 2013</xref>).</p><p>In the intestine, blood vessels have many roles, such as supporting oxygen and nutrient exchange, maintaining osmotic balance, and allowing leukocyte migration in the extracellular compartment. During IBD, these blood vessels undergo fast and remarkable changes by a process known as immune-derived angiogenesis. Due to this angiogenesis, the total vessel surface area increases, which provides an opportunity for immune cell extravasation. In addition, endothelial dysfunction includes increased vascular permeability increases, and upregulation of adhesion molecules occurs not only in these new blood vessels in IBD patients, but also in their existing intestinal vasculature. This increased vascular permeability reduces overall barrier function and allows for luminal bacteria-leukocyte interactions, which cause intestinal hypoxia and increased expression of free radicals and inflammatory cytokines, and lead to intestinal epithelial cell damage. Given that the upregulation of these adhesion molecules is linked to disease severity (<xref rid=\"B12\" ref-type=\"bibr\">Cromer et al., 2011</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Alkim et al., 2015</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Gravina et al., 2018</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Sun et al., 2018</xref>), regulating these intestinal blood vessel changes is likely to be important for the prevention and treatment of IBD.</p><p>No studies have examined the endothelial dysfunction blocker, CU06-1004, for therapeutic efficacy in the inflammatory diseases such as IBD. Therefore, we assessed its therapeutic potential using a dextran sodium sulfate (DSS)-induced colitis model in the mouse. We previously demonstrated that CU06-1004 enhances endothelial cell survival and integrity <italic>via</italic> the upregulation of cyclic adenosine monophosphate levels and Rac signal activation that leads to cortical actin ring formation. CU06-1004 blocks endothelial hyperpermeability and the loss of junction proteins induced by multiple factors such as vascular endothelial growth factor, IL-1β, and histamine. It also inhibits the expression of inflammatory adhesion molecules such as ICAM-1 and VCAM-1 by nuclear factor-κB inhibition. In other pathologies such as cancer, diabetic retinopathy, and mouse models of cerebral ischemia, the therapeutic influence of CU06-1004 has been <italic>via</italic> the inhibition of vascular hyperpermeability and inflammation (<xref rid=\"B25\" ref-type=\"bibr\">Maharjan et al., 2013</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Agrawal et al., 2014</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Lee et al., 2014</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Batbold et al., 2016</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Zhang et al., 2017</xref>). Here, we demonstrate that oral administration of CU06-1004 ameliorated the macroscopic pathology of the colon as well as histological changes and inflammation. These therapeutic effects correlate with reductions in endothelial abnormalities and inflammatory cytokine expression, and result in the preservation of the epithelial barrier, which blocks further progression of IBD.</p></sec><sec sec-type=\"materials|methods\" id=\"s2\"><title>Materials and Methods</title><sec id=\"s2_1\"><title>Test Drug</title><p>CU06-1004, previously described as sac-1004, was synthesized as described previously.19 Briefly, CU06-1004 was tetrahydropyran-deprotected and subsequently glycosylated with 4, 6-di-O-acetyl-2,3-didieoxyhex-2-enopyran in the presence of acid. Mesalazine was purchased from Sigma-Aldrich, St. Louis, MO, USA. CU06-1004 and mesalazine were dissolved in olive oil. Since CU06-1004 was observed to be most effective at a dose of 10 mg/kg, that was the dose used in all experiments (<xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Figure S1</bold></xref> in <xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Supplementary Material</bold></xref>).</p></sec><sec id=\"s2_2\"><title>Mice</title><p>Male C57BL/6J mice (8–10 weeks) were obtained from DBL (Eumsung, Korea) and acclimated for one week under controlled conditions (12 h/12 h dark-light cycle, 22°C ± 1°C, 50%–60% humidity). All mice had access to sterilized tap water and standard mouse chow <italic>ad libitum</italic>. Mice maintained under pathogen-free conditions at Yonsei University. All animal experiments were conducted under the institutional guidelines established for the Animal Core Facility at Yonsei University of Medicine with approval of the institutional care and use committee.</p></sec><sec id=\"s2_3\"><title>DSS-Induced Colitis</title><p>Mice were randomly assigned to four groups numbering 4–6 mice: control, DSS, DSS+mesalazine, and DSS+1004. The control group received distilled water (no colitis induction). Mice in the other groups received a 3% aqueous solution (w/v) of DSS (M.W. = 36,000–50,000 Da) (MP Biochemicals, Solon, OH, USA) <italic>ad libitum</italic> for 7 days, replaced every other day (<xref rid=\"B20\" ref-type=\"bibr\">Kim et al., 2012</xref>). Control- and DSS-group mice received vehicle (olive oil) by oral gavage 24 h after DSS was provided. The DSS+1004 group received oral 1,004 (10 mg/kg body weight, dissolved in olive oil), and the DSS+mesalazine group received oral mesalazine (Sigma-Aldrich; 100 mg/kg body weight, dissolved in olive oil). Each experiment was repeated four times.</p></sec><sec id=\"s2_4\"><title>Assessment of Disease Severity</title><p>pt?>In each repeat, mice were examined daily for body weight, stool consistency, and fecal blood. These three factors were used to determine the additive disease activity index (DAI) score. Scores were determined by: (1) weight loss (0 = <1%, 1 = 1%–5%, 2 = 5%–10%, 3 = 10%–20%, 4 = >20%); (2) stool consistency (0 = normal, 2 = loose stool, 4 = diarrhea); and (3) fecal blood (0 = no blood, 2 = red, 4 = black) (<xref rid=\"B11\" ref-type=\"bibr\">Cooper et al., 1993</xref>). On day 7, mice were euthanized, and both colon length and weight were measured to calculate the edema rate (length/weight ratio). Data were expressed as the sum of mice across the four repeat experiments (weight loss was calculated relative to day 0 weight for each mouse. Stool consistency and fecal blood scores were examined quantitatively, not as relative measures. Therefore, all repeats of experiments were pooled and presented together). And the presented image was chosen in the same experiment, and has close to the median value.</p></sec><sec id=\"s2_5\"><title>Histopathological Assessment</title><p>After each mouse euthanized, its colon was removed and washed with cold phosphate-buffered saline (PBS). The distal colon was then fixed for 48 h in cold 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned at 4–6 μm. Sections were stained with hematoxylin and eosin (H&E) and observed under a phase-contrast microscope (100× magnification, Nikon, Japan). Epithelial loss, crypt disruption, and inflammatory cell infiltration were each graded separately: (0 = no change, 1 = localized and mild, 2 = localized and moderate, 3 = extensive and moderate, and 4 = extensive and severe). The three grades were then combined to obtain a total histological score (<xref rid=\"B28\" ref-type=\"bibr\">Nishiyama et al., 2012</xref>). Staining was repeated for each experimental repeat. Data were expressed as the sum of mice across the four repeat experiments. Images were chosen from an experiment that was close to the median value for that group.</p></sec><sec id=\"s2_6\"><title>Immunofluorescence Analysis</title><p>Distal colon sections (from the same sample used for H&E staining) were deparaffinized in xylene and then rehydrated with an alcohol gradient. For antigen retrieval (heat-induced epitope retrieval), samples were heated in 10 mM sodium citrate buffer using a microwave oven and then allowed to cool to room temperature. Sections were treated with 3% hydrogen peroxide to block endogenous peroxidase activity and permeabilized with 0.4% Triton X-100 in PBS. Sections were blocked with Protein Block solution (Dako, Santa Cruz, CA, USA) for 1 h and incubated with the following primary antibodies overnight at 4°C: anti-goat CD31 (R&D Systems, 1:200), anti-rabbit ZO-1 (Invitrogen, 1:100), anti-rabbit occludin (Invitrogen, 1:200), anti-rabbit claudin-5 (Invitrogen, 1:200), anti-rabbit VE-cadherin (Invitrogen, 1:200). Sections then incubated with appropriate secondary antibodies labeled with Alexa Fluor 488 or Alexa Fluor 594. Before imaging, nuclei stained using 4′,6-diamidino-2-phenylindole (DAPI). Images captured using a confocal microscope (Carl Zeiss). Staining was repeated for each experimental repeat. Data were expressed for single repeat of experiments. Images were chosen from an experiment that was close to the median value for that group.</p></sec><sec id=\"s2_7\"><title>Colon Whole-Mount Immunostaining</title><p>Colon whole-mount processing was performed as previously described (<xref rid=\"B6\" ref-type=\"bibr\">Bernier-Latmani and Petrova, 2016</xref>). Taking 7 days to complete. Briefly, an anesthetized mouse was perfused with 1× PBS and 1% (w/v) PFA by cardiac puncture. The colon was harvested, cut longitudinally, washed with ice-cold 1× PBS, and pinned onto a silicon plate lumen-side up. Colon samples were then post-fixed in 4% PFA overnight. On day 2, samples were washed with ice-cold PBS, incubated with 10% sucrose solution for 3 h at 4°C on an orbital shaker, and then incubated overnight in a 20% sucrose-glycerol solution at 4°C on an orbital shaker. On day 3, after washing with ice-cold PBS, the colon was cut into fragments measuring 0.5–1 cm in length and immersed in a blocking solution for 2 h at 4°C on an orbital shaker. Primary antibody treatment; CD31 (BD bioscience, 1:500) in blocking solution was performed overnight at 4°C on an orbital shaker. On day 4, segments were washed with ice-cold 0.3% PBS-Tween (PBS-T) and then incubated with secondary antibody (Alexa Fluor 594) in blocking solution overnight at 4°C on an orbital shaker. On day 5, segments were washed in ice-cold 0.3% PBS-T and then immersed in 4% PFA for two days at 4°C on an orbital shaker. On day 7, after washing with ice-cold PBS, segments were treated with FocusClear solution (CelExplorer labs, HSINCHU, TAIWAN) for 20 min. The FocusClear solution was then removed, and the segments were mounted with ProLong Gold (Life Technologies, CA, USA). Z-stack images were acquired using a confocal microscope (Carl Zeiss). Whole-mount staining was done in one of the four experimental repeats. Images were chosen that reflect the median value for each group.</p></sec><sec id=\"s2_8\"><title>RNA Isolation, Purification, and Quantitative Real-Time Polymerase Chain Reaction</title><p>Total RNA was extracted from colon samples using Easy-Blue reagent (Intron, Sungnam, Korea) according to the manufacturer’s instructions. RNA was purified by lithium chloride and sodium acetate precipitation to remove any remaining DSS (a possible polymerase inhibitor). The RNA solution was mixed with 0.1 volume of 8 M LiCl (Sigma-Aldrich), incubated on ice for 2 h, and then centrifuged at 14,000<italic>g</italic> for 30 min, at 4°C. The supernatant discarded and the pellet dissolved in diethyl pyrocarbonate-treated water. This procedure repeated once and then 0.1 volume of 3 M sodium acetate (Sigma-Aldrich) and two volumes of pre-chilled 100% ethanol were added. After a 30-min incubation, the RNA solution was centrifuged at 14,000<italic>g</italic> for 10 min at 4°C. The RNA pellet was then washed with pre-chilled 70% ethanol. The supernatant discarded, and the dried RNA pellet was dissolved in a volume smaller (or equal to) the initial volume (<xref rid=\"B39\" ref-type=\"bibr\">Viennois et al., 2018</xref>). RNA concentrations were determined using a Nanodrop instrument (Thermo Fisher Scientific). Reverse transcription of RNA (2 μg) into cDNA performed using reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) performed using a SYBR Green master mix (Thermoscientific, Waltham, MA, USA) with an initial denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, 70°C for 30 s, and 60°C for 15 s. Primer sequences are presented in the following <xref rid=\"T1\" ref-type=\"table\"><bold>Table 1</bold></xref>. mRNA expression levels for each target gene were normalized to glyceraldehyde 3-phosphate dehydrogenase levels. Relative expression was determined using the 2<sup>-ΔΔCt</sup> (fold change) method based on control mouse RNA as a reference. qRT-PCR was repeated twice in each of the four experimental repeats.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Primer list.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">gene</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Forward</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Reverse</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-1B</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TGGACCTTCCAGGATGAGGACA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GTTCATCTCGGAGCCTGTAGTG</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TNF-a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GGTGCCTATGTCTCAGCCTCTT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GCCATAGAACTGATGAGAGGGAG</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TACCACTTCACAAGTCGGAGGC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CTGCAAGTGCATCATCGTTGTTC</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">E-selectin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GGACACCACAAATCCCAGTCTG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TCGCAGGAGAACTCACAACTGG</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">P-selectin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AAGATGCCTGGCTACTGGACAC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAAGAGGCTGAACGCAGGTCAT</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VCAM1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GCTATGAGGATGGAAGACTCTGG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ACTTGTGCAGCCACCTGAGATC</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ICAM1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AAACCAGACCCTGGAACTGCAC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GCCTGGCATTTCAGAGTCTGCT</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CXCL12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GGAGGATAGATGTGCTCTGGAAC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AGTGAGGATGGAGACCGTGGTG</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CXCR4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GACTGGCATAGTCGGCAATGGA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAAAGAGGAGGTCAGCCACTGA</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HO-1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CACTCTGGAGATGACACCTGAG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> GTGTTCCTCTGTCAGCATCACC</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">iNOS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GAGACAGGGAAGTCTGAAGCAC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CCAGCAGTAGTTGCTCCTCTTC</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FLT1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TGGATGAGCAGTGTGAACGGCT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GCCAAATGCAGAGGCTTGAACG</td></tr></tbody></table></table-wrap></sec><sec id=\"s2_9\"><title>Evan’s Blue Assay</title><p>After the DAI and drug supply (at day 7), 0.5% w/v Evan’s blue was administered intravenously. Two hours later, the mice were euthanized and perfused with sterile PBS. For this experiment, we administered 4 μl of 0.5% w/v Evan’s blue per 1g body weight. After sacrifice, the colon was harvested, weighed, and dye was extracted with formamide (4 μl formamide per 1 mg of colon tissue) for 48 h at 60 . Then, we measured absorbance at 620 nm. These data were presented as the fold change in OD620 relative to the control group.</p></sec><sec id=\"s2_10\"><title>Statistical Analyses</title><p>All results are presented as means ± standard errors of the mean (SEM). An analysis of variance was used for comparisons between groups. The difference of the means among groups was statistically analyzed by one-way or two-way analysis of variance (ANOVA) with followed by turkey’s test. A P-value of <0.05 was considered statistically significant. Analyses performed using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA).</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec id=\"s3_1\"><title>CU06-1004 Reduced the Severity of DSS-Induced Colitis</title><p>The DSS mouse model was used to induce severe mucosal inflammation and colitis, a model for UC. Its pathophysiological and morphological characteristics are considered similar to UC (<xref rid=\"B30\" ref-type=\"bibr\">Okayasu et al., 1990</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Whittem et al., 2010</xref>). Mesalazine (5-aminosalicylic acid; 5-ASA), a Food and Drug Administration (FDA)-approved drug to treat UC, was used as a positive control treatment. The experimental scheme was shown in <xref ref-type=\"fig\" rid=\"f1\"><bold>Figure 1A</bold></xref>. The DAI, an indicator of UC severity in mice, was used for the daily assessment of body weight, stool consistency, and fecal blood. Body weight reductions in all DSS-treated groups began on day 4, with the other factors indicating severity on day 3. In the CU06-1004-treated group, this body weight reduction significantly prevented from day 4. Weight loss also blocked in the mesalazine-treated group (<xref ref-type=\"fig\" rid=\"f1\"><bold>Figure 1B</bold></xref>). Changes in stool consistency and fecal blood were also ameliorated in both of these groups (<xref ref-type=\"fig\" rid=\"f1\"><bold>Figures 1C, D</bold></xref>), leading to significantly decreased DAI scores in both CU06-1004- and mesalazine-treated mice (<xref ref-type=\"fig\" rid=\"f1\"><bold>Figure 1E</bold></xref>). Colon shortening is another indicator of colorectal inflammation, and the average colon length of the control group was 7.6 cm, but the average length in DSS-groups reduced to 5.43 cm. Average colon lengths from the mesalazine- and CU06-1004-treated groups were 5.93 cm and 6.14 cm, respectively (<xref ref-type=\"fig\" rid=\"f1\"><bold>Figures 1F, G</bold></xref>). An additional indicator of inflammation, the colon weight-length ratio (mg/cm), decreased by approximately 10% and 18% in the drug-treated groups (<xref ref-type=\"fig\" rid=\"f1\"><bold>Figure 1H</bold></xref>).</p><fig id=\"f1\" position=\"float\"><label>Figure 1</label><caption><p>CU06-1004 treatment attenuated the clinical features of dextran sodium sulfate (DSS)-induced colitis in mice. Mice divided into four groups; control (ctrl.), vehicle (DSS), Mesalazine (DSS+M), and CU06-1004 (DSS+1004). The control (normal) group received water and the other groups received Dextran Sodium Sulfate. Mice were administered 3% DSS <italic>ad libitum </italic>for 7 days, and vehicle or Mesalazine or CU06-1004 treated 24 h after DSS administration started. Mesalazine (an FDA-approved TNFα inhibitor) administered at 100 mg/kg per day, and CU06-1004 at 10 mg/kg, for 6 days <bold>(A)</bold>. Body weight change (%) <bold>(B)</bold>, diarrhea <bold>(C)</bold>, and fecal blood scores <bold>(D)</bold> were determined each day, and their sums determined a <italic>disease activity index</italic> (DAI) score <bold>(E)</bold>. Representative images of removed mouse colon <bold>(F)</bold>, colon length measurement <bold>(G)</bold>, and weight-length ratios <bold>(H)</bold>. <sup>###</sup>P < 0.001 versus the control group. *P < 0.05, **P < 0.01, ***P < 0.001 versus the DSS group. (n=18-20 per group).</p></caption><graphic xlink:href=\"fphar-11-571266-g001\"/></fig></sec><sec id=\"s3_2\"><title>CU06-1004 Treatment Attenuated Histological Damage and Leukocyte Infiltration</title><p>DSS treatment destroyed intestinal epithelial cells and crypt structure and induced inflammatory cell infiltration. Histological analyses of the distal colon performed using H&E-stained sections. Control-group mice exhibited an intact epithelial cell layer and full-crypt structure without any leukocyte infiltration. In contrast, DSS-administered mice showed epithelial cell and crypt damage with high levels of leukocyte infiltration. Tissues from both mesalazine- and CU06-1004-treated mice showed preserved colon histology, with reduced inflammation, compared with the DSS-only group (<xref ref-type=\"fig\" rid=\"f2\"><bold>Figures 2A, B</bold></xref>). The histological scores also showed that DSS induced histological defects and that these reduced by mesalazine and CU06-1004 treatments (<xref ref-type=\"fig\" rid=\"f2\"><bold>Figure 2C</bold></xref>).</p><fig id=\"f2\" position=\"float\"><label>Figure 2</label><caption><p>CU06-1004 prevented tissue damage and the infiltration of inflammatory cells into the colon. Colons were obtained 7 days after dextran sodium sulfate (DSS) administration and then sectioned and stained with H&E. Magnification 100×, scale bar: 20μm <bold>(A)</bold>. Histopathological scores were then assessed <bold>(B, C)</bold>. <sup>###</sup>P < 0.001 versus the control group. *P < 0.05, ***P < 0.001 versus the DSS group. (n=18–20 per group).</p></caption><graphic xlink:href=\"fphar-11-571266-g002\"/></fig></sec><sec id=\"s3_3\"><title>CU06-1004 Decreased the Level of Proinflammatory Cytokines in the Colitis-Induced Colon</title><p>Persistent inflammation is important for the development of UC. IL-1β, IL-6, and TNF-α all play critical roles in IBD progression. Therefore, we investigated the effects of CU06-1004 on the expression of these proinflammatory cytokines. Compared to control-group mice, the levels of IL-1β, IL-6, and TNF-α increased in DSS-group mice. However, mesalazine- and CU06-1004-group mice showed significantly reduced cytokine levels (<xref ref-type=\"fig\" rid=\"f3\"><bold>Figures 3A–C</bold></xref>). These results suggest that CU06-1004 has an antiinflammatory effect to prevent DSS-induced colitis.</p><fig id=\"f3\" position=\"float\"><label>Figure 3</label><caption><p>The effect of CU06-1004 on the expression of inflammatory biomarkers. Quantitative real-time polymerase chain reaction (qRT-PCR) performed using gut homogenates to detect proinflammatory cytokine expression levels. IL-1β <bold>(A)</bold>, IL-6 <bold>(B)</bold>, and TNF-α <bold>(C)</bold> ###P < 0.001, versus the control group. *P < 0.05, **P < 0.01, ***P < 0.001 versus the DSS group. (n=4–5 per group).</p></caption><graphic xlink:href=\"fphar-11-571266-g003\"/></fig></sec><sec id=\"s3_4\"><title>CU06-1004 Maintained Blood Vessel Integrity in the Colon</title><p>During the development of UC, blood vessels in the colon lose their normal integrity and appearance. During colitis, there is loss of vascular integrity. Indeed, the increase in colonic vascular permeability precedes the clinical symptoms and accelerates the development of colitis (<xref rid=\"B37\" ref-type=\"bibr\">Tolstanova et al., 2012</xref>). Colonic vessels become interrupted, tortuous, and have decreased diameters during colitis (<xref rid=\"B10\" ref-type=\"bibr\">Chidlow et al., 2007</xref>). While the DSS-administered group had increased leakage of vascular Evan’s blue, the CU06-1004-treated group exhibited markedly decreased residual Evan’s blue, suggesting that CU06-1004 reduced colonic vascular permeability (<xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Figure S2</bold></xref> in <xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Supplementary Material</bold></xref>). Blood vessels in the DSS and mesalazine-administered group colonic vessel were interrupted (<xref ref-type=\"fig\" rid=\"f4\"><bold>Figure 4A</bold></xref>, white arrow), and were tortuous (yellow asterisk). However, blood vessels in CU06-1004-treated mice were less interrupted and less tortuous than the other group (<xref ref-type=\"fig\" rid=\"f4\"><bold>Figure 4A</bold></xref>). The CU06-1004-treated group also had larger vessel diameters than the other group of mice (<xref ref-type=\"fig\" rid=\"f4\"><bold>Figure 4B</bold></xref>). During UC, vascular permeability in the colon increases (i.e., a loss of integrity), causing bleeding and inflammation (<xref rid=\"B33\" ref-type=\"bibr\">Saijo et al., 2015</xref>), and tight junctions play a pivotal role in maintaining vascular integrity (<xref rid=\"B16\" ref-type=\"bibr\">Erickson et al., 2007</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Edelblum and Turner, 2009</xref>). As CU06-1004 has been reported to block such leakage, we investigated whether its influence on these junctions. In the colon, tight junction proteins are expressed not only in the endothelium but also in the epithelium. As our focus was vascular, colon samples were double-stained for tight junction proteins and an endothelial cell marker (CD31) to exclude the epithelium. Occludin (OCLN), Zonula occludens-1 (ZO-1), and claudin-5 (CLDN-5) reported representing tight junction proteins that maintain vascular junctions. Expressions of OCLN, ZO-1, and CLDN-5 in DSS-group mice decreased. The colonic vessel junctions in the mesalazine group were slightly increased (no significance), and there was no apparent difference compared to DSS-treated mice. However, tight junction coverage was enhanced by approximately 53% in CU06-1004-treated mice versus DSS mice (<xref ref-type=\"fig\" rid=\"f4\"><bold>Figures 4C–F</bold></xref>). This strengthening of the vascular junctions also caused a reduction in hypoxia (<xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Figure S3A</bold></xref> in <xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Supplementary Material</bold></xref>) and pathological changes (<xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Figures S3B, C</bold></xref> in <xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Supplementary Material</bold></xref>), which may also prevent an increase in colitis severity.</p><fig id=\"f4\" position=\"float\"><label>Figure 4</label><caption><p>CU06-1004 attenuated vascular abnormalities and barrier destruction. Whole-colon immunofluorescence staining for CD31 (red), with an interrupted vessel (white arrow), and tortuous vessel (yellow asterisk) Magnification 200×, scale bar: 50 μm <bold>(A)</bold>. Vessel diameters measured by counting the number of pixels on vascular sections viewed by confocal microscopy (n=4 per group) <bold>(B)</bold>. Immunofluorescence staining of colon sections for CD31 (red) and the tight junction proteins (green) occludin <bold>(C)</bold>, Zonula occludens-1 <bold>(D)</bold>, and claudin-5 <bold>(E)</bold>. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue nuclei). CD31 and tight junction co-expression were quantified by optical density <bold>(F)</bold>. (n=4–5 per group). The coexpression of junction proteins and vascular markers represent the colon’s vascular barrier. Magnification 1,000×, scale bar: 10 μm, <sup>##</sup>P < 0.01, <sup>###</sup>P < 0.001 versus the control group. *P < 0.05, **P < 0.01, ***P < 0.001 versus the dextran sodium sulfate (DSS) group.</p></caption><graphic xlink:href=\"fphar-11-571266-g004\"/></fig></sec><sec id=\"s3_5\"><title>The CU06-1004 Antiinflammatory Effect Is Mediated by Inhibition of DSS-Induced Adhesion Molecules in the Colon</title><p>Leukocyte rolling, adhesion, and transendothelial migration from the bloodstream into the tissues of the colon are critical steps in the development of the IBD inflammatory response. In the DSS group, pronounced increases in colonic E-selectin (<xref ref-type=\"fig\" rid=\"f5\"><bold>Figure 5A</bold></xref>), P-selectin (<xref ref-type=\"fig\" rid=\"f5\"><bold>Figure 5B</bold></xref>), VCAM-1 (<xref ref-type=\"fig\" rid=\"f5\"><bold>Figure 5C</bold></xref>), and ICAM-1(<xref ref-type=\"fig\" rid=\"f5\"><bold>Figure 5D</bold></xref>) expressions were seen. However, the upregulation of RNA for these adhesion molecules was markedly inhibited in CU06-1004-treated mice (<xref ref-type=\"fig\" rid=\"f5\"><bold>Figure 5</bold></xref>). Therefore, the reduced inflammation in CU06-1004-treated colonic tissue (<xref ref-type=\"fig\" rid=\"f2\"><bold>Figure 2B</bold></xref>) is likely mediated by a decrease in leukocyte transendothelial infiltration of the colon <italic>via</italic> downregulation of adhesion molecules (<xref ref-type=\"fig\" rid=\"f5\"><bold>Figure 5</bold></xref>). Because mesalazine is an antiinflammatory and immunosuppressive drug, the expression of adhesion molecules in mesalazine-treated mice decreased even more than in the CU06-1004 treated groups, but it may cause severe side effects.</p><fig id=\"f5\" position=\"float\"><label>Figure 5</label><caption><p>Downregulation of adhesion molecule expression in CU06-1004-treated mice. Quantitative real-time polymerase chain reaction (RT-PCR) was performed using gut homogenates to detect adhesion molecule expression levels. E-selectin <bold>(A)</bold>, P-selectin <bold>(B)</bold>, VCAM-1 <bold>(C)</bold>, and ICAM-1 <bold>(D)</bold>. <sup>#</sup>P < 0.05, <sup>##</sup>P < 0.01, <sup>##</sup>P < 0.01, versus the control group. *P < 0.05, **P < 0.01, **P < 0.01, ***P < 0.001 versus the dextran sodium sulfate (DSS) group. (n=4–5 per group).</p></caption><graphic xlink:href=\"fphar-11-571266-g005\"/></fig></sec><sec id=\"s3_6\"><title>CU06-1004 Attenuated Epithelial Inflammation and Loss of Epithelial Barrier Integrity</title><p>Destruction of the gut epithelium occurs afterward endothelium injury and contributes to IBD clinical manifestation (<xref rid=\"B37\" ref-type=\"bibr\">Tolstanova et al., 2012</xref>). In the early phase of DSS-induced colitis (days 2-3) there was no damage to the colonic epithelium (<xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Figures S4D</bold></xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>S5D</bold></xref> in <xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Supplementary Material</bold></xref>), but the vessels did lose their junctions (<xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Figures S4G–J</bold></xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>S5 G–J</bold></xref> in <xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Supplementary Material</bold></xref>). This suggests that vessel destruction precedes epithelial injury. Indeed, inhibition of vascular dysfunction (e.g. through treatment with CU06-1004) prevents epithelial injury (as observed from immunohistochemistry), which relieved clinical symptoms. Epithelial tight junctions and adherens junctions showed strong expression in the control-group mice, but DSS-group mice showed weakened expression. However, colon samples from CU06-1004-treated mice showed more regular and stronger expression (<xref ref-type=\"fig\" rid=\"f6\"><bold>Figures 6A, B</bold></xref>), indicating restoration of epithelial barrier integrity with CU06-1004 treatment. SDF-1, secreted by damaged colonic epithelial cells, facilitates leukocyte infiltration, and increases severity during the development of UC (<xref rid=\"B14\" ref-type=\"bibr\">Dotan et al., 2010</xref>). Both SDF-1 and its main receptor, CXCR4, are known to increase during UC development. Colonic expression of both SDF-1 and CXCR4 derived by epithelium (<xref ref-type=\"fig\" rid=\"f6\"><bold>Figures 6C, D</bold></xref>) and decreased in CU06-1004-treated mice compared with DSS mice (<xref ref-type=\"fig\" rid=\"f6\"><bold>Figures 6C–F</bold></xref>). Therefore, CU06-1004 induces vessel protection, which inhibits epithelial damage and has a therapeutic benefit in colitis.</p><fig id=\"f6\" position=\"float\"><label>Figure 6</label><caption><p>CU06-1004 reduced epithelial barrier destruction and epithelial inflammation. Epithelial junctions (occluding, Zonula occludens-1, and E-cadherin expressions) were upregulated in the CU06-1004-treated group. Magnification 200×, scale bar: 50 μm <bold>(A)</bold>. Epithelial junctions were quantified by optical density <bold>(B)</bold>. Levels of chemokine SDF-1 and its receptor CXCR4 were evaluated using immune-histochemistry <bold>(C, D)</bold> and RT-PCR on RNA from gut homogenates (n=4–5 per group) <bold>(E, F)</bold>. <sup>##</sup>P < 0.01, <sup>###</sup>P < 0.001 versus the control group. *P < 0.05, **P < 0.01, ***P < 0.001 versus the dextran sodium sulfate (DSS) group.</p></caption><graphic xlink:href=\"fphar-11-571266-g006\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Various factors can cause IBD, including environmental, genetic, and lifestyle factors, and infection (<xref rid=\"B38\" ref-type=\"bibr\">Vedamurthy and Ananthakrishnan, 2019</xref>). Many studies have investigated IBD pathogenesis, but its exact etiology remains unclear. Colonic blood vessel dysfunction is one factor that often influences the development of IBD (<xref rid=\"B12\" ref-type=\"bibr\">Cromer et al., 2011</xref>). Therefore, blood vessel protection is a promising strategy to block IBD development. The DSS mouse model often used in the process of drug discovery because of its ease of use and high success rate for disease induction. DSS administration causes colonic inflammation, ulceration, crypt dilation, edema, and mixed immune cell infiltration — all similar characteristics of UC (<xref rid=\"B7\" ref-type=\"bibr\">Blumberg et al., 1999</xref>). Here we report that CU06-1004 has a tissue-protection effect on DSS-induced UC by attenuating endothelial dysfunction and inflammation.</p><p>The gut changes induced by DSS occurred in an early phase (days 2–3) and a late phase (days 4–5). In the early phase, DSS injured endothelial cells in the deep layer of the lamina propria directly or indirectly and induced endothelial dysfunction. Endothelial dysfunction characterized by reduced barrier function, upregulation of cellular adhesion molecules, and increased leukocyte diapedesis (<xref rid=\"B33\" ref-type=\"bibr\">Saijo et al., 2015</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Sun et al., 2018</xref>). Colon blood vessels become twisted, dilated, and irregularly distributed, and vessel diameters reduced (<xref rid=\"B10\" ref-type=\"bibr\">Chidlow et al., 2007</xref>). Our results show that CU06-1004 increased junction protein expression levels, which, in turn, blocked DSS-induced increases in vascular permeability, and prevented further development of UC. Furthermore, immunostaining of colon whole mounts showed strengthened and less tortuous colonic blood vessels in CU06-1004-treated mice. CU06-1004 had reported protecting the endothelium from the junction-destroying influence of multiple cytokines, so we hypothesized that CU06-1004 would also prevent cytokine vasodilation effects (many studies have shown cytokine level increases in IBD (<xref rid=\"B12\" ref-type=\"bibr\">Cromer et al., 2011</xref>)), even though it did not reduce some cytokine level directly (data not shown).</p><p>Increased expression of adhesion molecules is another characteristic of endothelial dysfunction. It leads to abnormal leukocyte extravasation from the blood into colonic tissue, contributes to the devastation of the epithelium, and plays a crucial role in the pathogenesis of colitis. Leukocytes infiltrate the region in three steps; rolling, activation, and adhesion. During leukocyte-endothelium interactions, various adhesive and migratory molecular events occur, with selectin and cell adhesion molecules (CAMs) having the most relevance. The initial process for leukocyte recruitment is selectin-mediated low-affinity and transient tethering and rolling of leukocytes, followed by integrin-dependent firm adhesion (<xref rid=\"B24\" ref-type=\"bibr\">Ley et al., 2007</xref>). Our results indicate that CU06-1004 significantly suppressed selectin and CAM expression, and reduced leukocyte extravasation by preventing both the rolling and the adhesion process. Therefore, reduced adhesion and leukocyte infiltration could feedback into the inflammatory process and attenuate it. A reduction in the inflammatory response, seen as a decrease in the proinflammatory cytokines IL-1β, IL-6, and TNF-α, also observed in the CU06-1004-treated mice intestine. These cytokines amplify inflammatory cascades and result in intestinal tissue damage in UC patients. The treatment of UC often involves the downregulation of TNF-α using antibodies (e.g., infliximab and adalimumab) (<xref rid=\"B3\" ref-type=\"bibr\">Argollo and Danese, 2019</xref>). IL-1β is known to stimulate diarrhea, immune cell infiltration, and intestinal necrosis, and IL-6 reduction had shown to slow down the development of UC and colitis-associated colon cancer (<xref rid=\"B31\" ref-type=\"bibr\">Papadakis and Targan, 2000</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Perrier and Rutgeerts, 2011</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Moriasi et al., 2012</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Song et al., 2013</xref>). Although an antibody-based drug can target its corresponding cytokine, CU06-1004 inhibits the effects of multiple cytokines. Therefore, the overall effect of CU06-1004 is to reduce inflammation by reducing the expression of endothelial adhesion molecules and inhibiting multiple cytokines.</p><p>In the late phase of UC, the destruction of the mucosal epithelium occurs and leads to crypt pathology disorders (<xref rid=\"B33\" ref-type=\"bibr\">Saijo et al., 2015</xref>). As a result of vascular protection, this late-phase feature of epithelial injury also decreased in CU06-1004-treated mice. Improved epithelial integrity and decreased epithelial inflammation was verified by assessing junction protein expression and SDF-1 expression (reported to be upregulated in injured epithelium, particularly in colitis (<xref rid=\"B43\" ref-type=\"bibr\">Zimmerman et al., 2008</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Dotan et al., 2010</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Kulkarni et al., 2017</xref>)). Reduced epithelial injury also reduced the primary clinical manifestations of fecal blood, weight loss, and loose stools. These three features were verified each day and represented by the DAI. Upon analysis of the colon at the early time points, after mice had been administered DSS for 3 days, there were no significant changes in colonic epithelium, but the mice presented with vascular junction loss and the beginnings of inflammatory cell infiltration due to increased vascular permeability. However, CU06-1004 treated mice had stronger junction protein expression and decreased immune cell infiltration than the DSS-alone group (<xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Figures S5D–J</bold></xref> in <xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Supplementary Material</bold></xref>). Therefore, CU06-1004 induces vessel protection before epithelial damage can occur, providing therapeutic benefits to protect against colitis. This is an important insight into the mechanism by which CU06-1004 modulates colonic blood vessels and further protects colonic tissue.</p><p>Due to its unknown etiology, there is no precise treatment for IBD, and the application of most drugs depends on disease severity (timing of the disease) and specific location. Typical treatments usually consist of supportive medications and immunosuppressive drugs, which have many undesirable side effects and complications (<xref rid=\"B41\" ref-type=\"bibr\">Wilhelm and Bryan, 2017</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Seyedian et al., 2019</xref>). Therefore, the development of better and different therapeutic approaches that have practical effects and minimal or no side effects is very important. CU06-1004 treatment after 7 days of DSS administration led to milder disease severity, as reflected by improvements in body weight (increased) and diarrhea (less severe) relative to the DSS group (<xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Figure S6</bold></xref> in <xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Supplementary Material</bold></xref>). Further, the colonic vessel integrity was increased in the CU06-1004 mice relative to the DSS-treated group (<xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Figure S7</bold></xref> in <xref ref-type=\"supplementary-material\" rid=\"SM1\"><bold>Supplementary Material</bold></xref>). This may suggested that CU06-1004 also has therapeutic effects with protective effects on against colitis by improving vascular integrity. Moreover, since CU06-1004 regulates vessels, and not the immune system directly, there may be fewer side effects than other immunomodulatory drugs. Consequently, CU06-1004 may be beneficial for the treatment of IBD both during and after disease progression in safe.</p><p>In conclusion, the results demonstrate that oral administration of CU06-1004 has a therapeutic influence on DSS-induced UC. CU06-1004 treatment reduced vascular permeability by upregulating junction protein expression, reduced leukocyte extravasation by downregulating adhesion molecule expression, decreased proinflammatory cytokine expression, and increased vessel normality in the colon. Furthermore, epithelial integrity, which has a pivotal role in colitis, was improved indirectly following reduced endothelial dysfunction. Moreover, CU06-1004 not only has protective effects, but also clinical effects, aiding the recovery of severely damaged colon tissue. These findings suggest that colonic vessel integrity is vital to colitis inhibition and recovery. Therefore, the endothelial dysfunction blocker CU06-1004 may provide a novel protective and treatment strategy for the pharmacological treatment of IBD. And this suggests an important insight into therapeutic access of IBD, not directly impede the immune system.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The studies involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Yonsei University, Seoul, Korea.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>Y-SK and HZ and carried out the experimental work. Y-SK wrote the manuscript. HZ proofread the manuscript. SL contributed revision experiment. MN and SP contributed data analysis. Y-MK and Y-GK supervised and edited the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec sec-type=\"funding-information\" id=\"s8\"><title>Funding</title><p>This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology [grant number 2019R1A2C3007142], and the Bio & Medical Technology Development Program of the National Research Foundation of Korea [NRF-2015M3A9B6066835].</p></sec><sec id=\"s9\"><title>Conflict of Interest</title><p>Author HZ was employed by the company Curacle Co. Ltd.</p><p>The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><sec sec-type=\"supplementary-material\" id=\"s10\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fphar.2020.571266/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fphar.2020.571266/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"DataSheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name><surname>Agrawal</surname><given-names>V.</given-names></name><name><surname>Maharjan</surname><given-names>S.</given-names></name><name><surname>Kim</surname><given-names>K.</given-names></name><name><surname>Kim</surname><given-names>N. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33042009</article-id><article-id pub-id-type=\"pmc\">PMC7523508</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00630</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Pancreatic Alpha-Cells Contribute Together With Beta-Cells to CXCL10 Expression in Type 1 Diabetes</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Nigi</surname><given-names>Laura</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/884923/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Brusco</surname><given-names>Noemi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1032828/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Grieco</surname><given-names>Giuseppina E.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/477193/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Licata</surname><given-names>Giada</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1066453/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Krogvold</surname><given-names>Lars</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1021561/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Marselli</surname><given-names>Lorella</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/141032/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Gysemans</surname><given-names>Conny</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/486796/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Overbergh</surname><given-names>Lut</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1052477/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Marchetti</surname><given-names>Piero</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/140807/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Mathieu</surname><given-names>Chantal</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/511444/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Dahl Jørgensen</surname><given-names>Knut</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Sebastiani</surname><given-names>Guido</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/476480/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Dotta</surname><given-names>Francesco</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff8\"><sup>8</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/851302/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Diabetes Unit, Department of Medicine, Surgery and Neurosciences, University of Siena</institution>, <addr-line>Siena</addr-line>, <country>Italy</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Fondazione Umberto Di Mario, c/o Toscana Life Sciences</institution>, <addr-line>Siena</addr-line>, <country>Italy</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Faculty of Odontology, University of Oslo</institution>, <addr-line>Oslo</addr-line>, <country>Norway</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Faculty of Medicine, University of Oslo</institution>, <addr-line>Oslo</addr-line>, <country>Norway</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Department of Clinical and Experimental Medicine, University of Pisa</institution>, <addr-line>Pisa</addr-line>, <country>Italy</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Clinical and Experimental Endocrinology (CEE), Katholieke Universiteit Leuven (KU LEUVEN)</institution>, <addr-line>Leuven</addr-line>, <country>Belgium</country></aff><aff id=\"aff7\"><sup>7</sup><institution>Division of Paediatric and Adolescent Medicine, Oslo University Hospital</institution>, <addr-line>Oslo</addr-line>, <country>Norway</country></aff><aff id=\"aff8\"><sup>8</sup><institution>Tuscany Centre for Precision Medicine (CReMeP)</institution>, <addr-line>Siena</addr-line>, <country>Italy</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Roberto Mallone, Institut National de la Santé et de la Recherche Médicale (INSERM), France</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Malin Flodström-Tullberg, Karolinska Institutet (KI), Sweden; Helen Thomas, University of Melbourne, Australia</p></fn><corresp id=\"c001\">*Correspondence: Francesco Dotta <email>francesco.dotta@unisi.it</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Diabetes: Molecular Mechanisms, a section of the journal Frontiers in Endocrinology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors share first authorship</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>630</elocation-id><history><date date-type=\"received\"><day>04</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>04</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Nigi, Brusco, Grieco, Licata, Krogvold, Marselli, Gysemans, Overbergh, Marchetti, Mathieu, Dahl Jørgensen, Sebastiani and Dotta.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Nigi, Brusco, Grieco, Licata, Krogvold, Marselli, Gysemans, Overbergh, Marchetti, Mathieu, Dahl Jørgensen, Sebastiani and Dotta</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>C-X-C Motif Chemokine Ligand 10 (CXCL10) is a pro-inflammatory chemokine specifically recognized by the ligand receptor CXCR3 which is mostly expressed in T-lymphocytes. Although CXCL10 expression and secretion have been widely associated to pancreatic islets both in non-obese diabetic (NOD) mice and in human type 1 diabetic (T1D) donors, the specific expression pattern among pancreatic endocrine cell subtypes has not been clarified yet. Therefore, the purpose of this study was to shed light on the pancreatic islet expression of CXCL10 in NOD, in C57Bl/6J and in NOD-SCID mice as well as in human T1D pancreata from new-onset T1D patients (DiViD study) compared to non-diabetic multiorgan donors from the INNODIA European Network for Pancreatic Organ Donors with Diabetes (EUnPOD). CXCL10 was expressed in pancreatic islets of normoglycaemic and new-onset diabetic NOD mice but not in C57Bl/6J and NOD-SCID mice. CXCL10 expression was increased in pancreatic islets of new-onset diabetic NOD mice compared to normoglycaemic NOD mice. In NOD mice, CXCL10 colocalized both with insulin and glucagon. Interestingly, CXCL10-glucagon colocalization rate was significantly increased in diabetic vs. normoglycaemic NOD mouse islets, indicating an increased expression of CXCL10 also in alpha-cells. CXCL10 was expressed in pancreatic islets of T1D patients but not in non-diabetic donors. The analysis of the expression pattern of CXCL10 in human T1D pancreata from DiViD study, revealed an increased colocalization rate with glucagon compared to insulin. Of note, CXCL10 was also expressed in alpha-cells residing in insulin-deficient islets (IDI), suggesting that CXCL10 expression in alpha cells is not driven by residual beta-cells and therefore may represent an independent phenomenon. In conclusion, we show that in T1D CXCL10 is expressed by alpha-cells both in NOD mice and in T1D patients, thus pointing to an additional novel role for alpha-cells in T1D pathogenesis and progression.</p></abstract><kwd-group><kwd>type 1 diabetes</kwd><kwd>pancreas</kwd><kwd>alpha-cells</kwd><kwd>chemokines</kwd><kwd>CXCL10</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Innovative Medicines Initiative<named-content content-type=\"fundref-id\">10.13039/501100010767</named-content></funding-source><award-id rid=\"cn001\">115797-INNODIA</award-id></award-group></funding-group><counts><fig-count count=\"5\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"58\"/><page-count count=\"13\"/><word-count count=\"8458\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Type 1 diabetes (T1D) is an autoimmune disease, characterized by a progressive destruction of pancreatic insulin-producing beta-cells driven by autoreactive T-lymphocytes (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>) and leading to chronic hyperglycemia and to the development of chronic complications (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>).</p><p>C-X-C Motif Chemokine Ligand 10 (CXCL10) is a pro-inflammatory chemokine secreted by a wide spectrum of cells. It is involved in multiple mechanisms and reported to have pleiotropic effects, including immune cell migration and attraction to inflammation sites, angiogenesis, and cancer cell growth (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>). CXCL10 is produced by pancreatic islet cells upon inflammatory stress (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>) and is specifically recognized by C-X-C Motif Chemokine Receptor 3 (CXCR3) which is expressed by activated T-lymphocytes and other immune cells (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Several reports demonstrated that CXCL10 plays an important role in the natural history of T1D mainly through the attraction of autoreactive T-lymphocytes to the islets, thus leading to the subsequent destruction of pancreatic beta-cells (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Of note, although still debated, CXCL10 has been proposed as a possible therapeutic target, supported by several studies showing the beneficial effects of CXCL10 inhibition (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>).</p><p>In animal models, transgenic overexpression of CXCL10 in beta-cells, coupled to the induction of T1D through lymphocytic choriomeningitis virus (LCMV) infection, accelerated the autoimmune process by enhancing the migration of antigen-specific lymphocytes (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>). Of note, neutralization of CXCL10 reduced the occurrence of the disease by affecting lymphocyte migration to the pancreatic islets and by enhancing beta-cell proliferation (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>–<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Similar results, although more controversial, were reported in CXCR3-deficient mouse models or upon antagonistic blockade of CXCR3, leading to delayed insulitis and diabetes onset (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Therefore, CXCL10:CXCR3-based pancreatic immune cell trafficking has been reported as an important component in the natural history of T1D.</p><p>In man, increased CXCL10 levels have been detected in serum of T1D patients compared to non-diabetic subjects and to type 2 diabetic (T2D) individuals, thus indicating a relationship between this phenomenon and autoimmune diabetes (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>–<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). Of note, CXCL10 levels were positively correlated with the numbers of autoreactive CD4 T cells and negatively associated with T1D duration and age at disease onset, suggesting important implications for CXCL10 in autoimmune diabetes progression and severity (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>).</p><p>Using immunohistochemistry and immunofluorescence, we and others have previously demonstrated that CXCL10 expression is increased in pancreatic islets of T1D vs. non-diabetic donors (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>–<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). However, the exact CXCL10 expression pattern in pancreatic islet endocrine cell subsets has not been addressed.</p><p>Here, by using immunohistochemical fluorescence and confocal imaging, we aimed at elucidating the CXCL10 expression pattern among pancreatic islet endocrine cells in T1D through the analysis of pancreas samples from NOD, C57Bl/6J and NOD-SCID mice as well as from new-onset T1D patients (DiViD study) and non-diabetic multiorgan donors from the INNODIA European Network for Pancreatic Organ Donors with Diabetes (EUnPOD).</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Animals</title><p>C57Bl/6J, NOD-SCID, and NOD mice were housed and inbred in the animal facility of Katholieke Universiteit Leuven (KU Leuven, Leuven, Belgium) as previously described (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). All animal procedures were performed in accordance with the NIH guidelines for the care and use of laboratory animals and protocols were approved by the Ethics Committees of the KU Leuven. NOD female mice used in this study were screened for the onset of diabetes by evaluating urine glucose levels (Diastix Reagent Strips; Bayer, Leverkusen, Germany) and venous blood glucose levels (Accu-Chek; Roche Diagnostics, Vilvoorde, Belgium). Mice were diagnosed as diabetic when they had glycosuria and two consecutive blood glucose measurements exceeding 200 mg/mL. Pancreatic sections used for histological analysis were collected from 8-week-old C57Bl/6J (<italic>n</italic> = 4), 15- to 20-week-old C57Bl/6J (<italic>n</italic> = 3), 2- to 3-week-old NOD (Non-Obese Diabetic)-SCID (severe combined immunodeficient) (<italic>n</italic> = 4), 20-week-old NOD-SCID (<italic>n</italic> = 3), 20- to 22-week-old normoglycaemic NOD (<italic>n</italic> = 4) and 12- to 21-week-old new-onset diabetic NOD mice (<italic>n</italic> = 4).</p></sec><sec><title>Human Donors</title><p>Human pancreatic sections analyzed in this study were collected from two different cohorts of subjects.</p><sec><title>INNODIA EUnPOD Cohort</title><p>Following acquisition of informed research consent, pancreata (<italic>n</italic> = 3) were obtained from brain-dead multiorgan donors within the European Network for Pancreatic Organ Donors with Diabetes (EUnPOD) (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>), a project launched in the context of the INNODIA consortium (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.innodia.eu\">www.innodia.eu</ext-link>). Whole pancreata were processed following standardized procedures at University of Pisa. Formalin fixed paraffin embedded (FFPE) pancreatic tissue sections were obtained from <italic>n</italic> = 3 non-diabetic and islet-autoantibodies negative donors.</p></sec><sec><title>DiViD Cohort</title><p>Following the acquisition of appropriate consents, 6 new-onset T1D patients underwent pancreatic biopsy by adopting laparoscopic pancreatic tail resection, in the context of the Diabetes Virus Detection (DiViD) study (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). The pancreatic tissue was processed for multiple purposes including FFPE processing (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). From each patient included in the DiViD study, we analyzed two pancreatic sections from separate parts of the pancreas tail for CXCL10-INS-GCG staining and one pancreatic section for CXCL10-INS-CD45 staining. Collection of pancreatic tissue in the DiViD study was approved by the Norwegian Governments Regional Ethics Committee. Written informed consent was obtained from all individuals with type 1 diabetes after they had received oral and written information from the diabetologist and the surgeon separately (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). INNODIA EUnPOD multiorgan donors' pancreata were obtained with approval of the local Ethics Committee at the University of Pisa.</p><p>Clinical characteristics of EUnPOD donors and of DiViD T1D subjects are reported in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Demographics and main clinical parameters of T1D and non-diabetic donors.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Case ID</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Gender</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Age</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Disease duration (weeks)</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Cause of death</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>IA</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>GADA</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>IA−2A</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>ZnT8A</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Non-diabetic (EUnPOD)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20171031</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">54</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cardiovascular disease</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20171114</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cardiovascular disease</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20171118</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Trauma</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Type 1 diabetic (DiViD)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DiViD-1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DiViD-2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DiViD-3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DiViD-4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DiViD-5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DiViD-6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">n/a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">pos</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">neg</td></tr></tbody></table></table-wrap></sec></sec><sec><title>Immunofluorescence Analysis of Mouse and Human Pancreatic Sections</title><p>Formalin-Fixed Paraffin Embedded (FFPE) pancreatic sections obtained from mouse and human pancreata were analyzed using single or triple immunofluorescence and confocal imaging analysis in order to simultaneously evaluate the expression of CXCL10, insulin, glucagon or CXCL10, insulin, and CD45.</p><sec><title>Mouse Pancreatic Sections</title><p>After deparaffinization and rehydration through alcohol series, pancreatic sections (5 μm thickness) were incubated with Tris-buffered saline (TBS) supplemented with 3% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) to reduce non-specific reactions. Antigen retrieval was performed using 10 mM citrate buffer pH 6.0 in microwave (600 W) for 10 min. Sections were incubated with polyclonal rabbit anti-murine CXCL10 [dilution 1:25, cat. 500-P129, Peprotech, Rocky Hill, NJ, USA; purified by affinity chromatography employing immobilized mCXCL10 matrix from sera of rabbits pre-immunized with highly pure (>98%) recombinant mCXCL10] (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>), polyclonal guinea pig anti-insulin (dilution 1:500, cat. A0564, Dako-Agilent Technologies, Santa Clara, CA, USA) and monoclonal mouse anti-glucagon (dilution 1:300, cat. MAB1249 clone 181402, R&D Systems, Minneapolis, MN, USA) as primary antibodies; subsequently with goat anti-guinea pig Alexa-Fluor 488 conjugate (dilution 1:500, cat. A11073, Molecular Probe, Thermofisher, Waltham, MA, USA), goat anti-rabbit Alexa-Fluor 594 conjugate (dilution 1:500, cat. A11037, Molecular Probe, Thermofisher), goat anti-mouse 647 conjugate (dilution 1:500, cat. A21236, Molecular Probe, Thermofisher) as secondary antibodies. DNA was counterstained with DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride, dilution 1:3,000, cat. D8517, Sigma Aldrich). Sections were mounted with VECTASHIELD (cat. H-1000, Vector Laboratories, Burlingame, CA, USA) antifade medium and analyzed immediately or stored at 4°C until ready for confocal image analysis. Potential non-specific binding of goat anti-mouse-647 conjugate secondary antibody to mouse endogenous immunoglobulins was evaluated through a negative control staining with secondary antibody (without mouse anti-glucagon primary antibody), demonstrating the specificity of the reaction (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 1</xref>).</p></sec><sec><title>Human Pancreatic Sections</title><p>FFPE Pancreatic sections obtained from DiViD and EUnPOD collections were analyzed by triple immunofluorescence using different combinations of antibodies. After deparaffinization and rehydration through alcohol series, 5 μm thick sections were incubated with methanol supplemented with 3% H<sub>2</sub>O<sub>2</sub> to block endogenous peroxidases and with phosphate-buffered saline (PBS) supplemented with 3% BSA to reduce non-specific reactions. Antigen retrieval was performed with 10 mM citrate buffer pH 6.0 for 10 min. Sections were incubated with polyclonal guinea pig anti-insulin (dilution 1:500, cat. A0564, Dako), monoclonal mouse anti-glucagon (dilution 1:300, cat. MAB1249, clone #181402, R&D Systems), polyclonal rabbit anti-human CXCL10 [dilution 1:300, cat. 500-P93, Peprotech; specific antibody was purified by affinity chromatography employing immobilized hCXCL10 matrix from sera of rabbits pre-immunized with highly pure (>98%) <italic>Escherichia coli</italic> recombinant hCXCL10] (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>–<xref rid=\"B32\" ref-type=\"bibr\">32</xref>) and/or monoclonal mouse anti-human CD45 (pre-diluted, cat. IR751, clone #2B11 + PD7/26, Dako) as primary antibodies. Subsequently, the following secondary antibodies were adopted: goat anti-guinea pig Alexa-Fluor 488 conjugate (dilution 1:500, cat. A11073, Molecular Probes, Thermofisher), goat anti-mouse Alexa-Fluor 647 conjugate (dilution 1:500, cat. A21236, Molecular Probes, Thermofisher), swine anti-rabbit HRP (dilution 1:100, cat. P0217, Dako), goat anti-guinea pig Alexa-Fluor 647 conjugate (dilution 1:500, cat. A21450, Molecular Probes, Thermofisher), goat anti-mouse Alexa-Fluor 488 conjugate (dilution 1:500, cat. A21236, Molecular Probes, Thermofisher). TSA Fluorescein system (dilution 1:50, cat. NEL742001KT, Perkin Elmer) was used to amplify CXCL10 signal. DNA was counterstained with DAPI. Sections were finally mounted with VECTASHIELD Antifade Medium (Vector Laboratories). Antibodies details and main reagents used in the study are reported in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Table 1</xref>.</p></sec></sec><sec><title>Image Acquisition and Analysis</title><p>Images were acquired using Leica TCS SP5 confocal laser scanning microscope system (Leica Microsystems, Wetzlar, Germany). Images were acquired as a single stack focal plane or in <italic>z</italic>-stack mode capturing multiple focal planes (<italic>n</italic> = 5) for each identified islet. Sections were scanned and images acquired at 40 × or 63 × magnification. The same confocal microscope setting parameters (laser power, photomultiplier voltage gain and offset values, pinhole value) were applied to all stained sections before image acquisition in order to uniformly collect detected signal related to each channel.</p><p>The analysis of CXCL10, insulin, and glucagon positive signal were performed using Volocity 6.3 software (Perkin Elmer, Waltham, MA, USA). A background threshold filter was uniformly applied to all processed images before the evaluation of specific parameters. A 3D model reconstruction adopting voxels quantification method or pixels, was used to compute single channel signals and to evaluate single channel volumes or the percentage of colocalization coefficient M<sub>1</sub> (Mander's coefficient) (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>) between CXCL10 and insulin and between CXCL10 and glucagon. Colocalization Coefficient M<sub>1</sub> considers the percentage of pixels (or voxels in case of volume) of a given channel which overlaps to total pixels (or voxels) related to the other channel. Of note, Mander's coefficient is independent of absolute signal as it measures the fraction of one protein that colocalizes with a second protein where <italic>M</italic> represents the fraction (reported in percentage) of colocalizing pixels channel-1/channel-2 on total channel-1 pixels.</p><p>Colocalization coefficient M was analyzed for each identified mouse and human islet. In mouse islets, the following parameters were collected for each islet: islet/endocrine volume, insulin volume, glucagon volume, and CXCL10 volume.</p></sec><sec><title>Statistics</title><p>Results were expressed as mean ± SD. Statistical analyses were performed using Graph Pad Prism 8 software. Comparisons between two groups were carried out using Mann–Whitney <italic>U</italic> test (for non-parametric data) or Wilcoxon matched signed rank test. Multiple comparisons were analyzed using ordinary one-way ANOVA. Differences were considered significant with <italic>p</italic>-values <0.05.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>CXCL10 Expression in Pancreas of NOD Mice</title><p>CXCL10 expression was not detectable in pancreatic samples from 8-week-old and 20-week-old control C57Bl/6J mice, and absent or barely visible in pancreatic islets of 3-week-old and 20-week-old NOD-SCID mice, which showed no sign of immune cell infiltration or islet inflammation (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 2</xref>).</p><p>In NOD mice pancreata, CXCL10 expression was not observed in exocrine tissue. In pancreatic islets of 20-22-week-old NOD normoglycaemic mice, CXCL10 expression was weak and localized in few cells within islet parenchyma (<xref ref-type=\"fig\" rid=\"F1\">Figure 1a</xref>, panels A,B). In contrast, in new-onset diabetic NOD mice (12- to 21-week-old) the expression of CXCL10 was higher compared to NOD normoglycaemic mice, as shown by confocal z-stack imaging analysis of pancreatic islets (<xref ref-type=\"fig\" rid=\"F1\">Figure 1a</xref>, panels C,D), revealing an absolute increase of CXCL10 positive volume (<xref ref-type=\"fig\" rid=\"F1\">Figure 1b</xref>), as well as increased CXCL10 signal normalized per total islet volume (<xref ref-type=\"fig\" rid=\"F1\">Figure 1c</xref>). Of note, CXCL10 volume normalization based on beta-cell content, similarly showed an increase of CXCL10 positive signal in pancreatic islets of new-onset diabetic NOD mice (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 3a</xref>). Collectively, these results corroborated previous findings regarding the increase of CXCL10 in pancreatic islets of NOD mice in autoimmune diabetes.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>CXCL10 is increased in pancreatic islets of new-onset diabetic NOD mice. <bold>(a)</bold> Immunofluorescence staining of CXCL10 in pancreatic tissue sections of <italic>n</italic> = 4 normoglycaemic NOD mice and in <italic>n</italic> = 4 new-onset diabetic NOD mice. Representative images of normoglycaemic (panels A,B) and new-onset diabetic NOD mice (panels C,D) are reported. CXCL10 is reported in red; nuclei in white/gray. Scale bar is 50 μm. Analysis of CXCL10 total voxels absolute volume <bold>(b)</bold> and normalized per total islet volume <bold>(c)</bold>, in pancreatic islets of <italic>n</italic> = 4 normoglycaemic and <italic>n</italic> = 4 NOD new-onset diabetic NOD mice. A total of <italic>n</italic> = 27 and <italic>n</italic> = 25 pancreatic islets were individually analyzed in normoglycaemic and new-onset diabetic mice, respectively; individual values for each islet are reported in μm<sup>3</sup>\n<bold>(b)</bold> or as a volumetric ratio <bold>(c)</bold>. Exact <italic>p</italic>-values were analyzed using non-parametric Mann–Whitney <italic>U</italic> test (<italic>p</italic> < 0.05).</p></caption><graphic xlink:href=\"fendo-11-00630-g0001\"/></fig></sec><sec><title>CXCL10 Is Expressed in Beta- and in Alpha-Cells of NOD Mice</title><p>In order to verify whether the increase of CXCL10 expression observed in islets of new-onset diabetic NOD mice was exclusively dependent on its hyperexpression in beta-cells, we performed triple immunofluorescence staining for CXCL10, insulin and glucagon in pancreas sections of normoglycaemic and new-onset diabetic NOD mice. Interestingly, CXCL10 was expressed both in beta- and in alpha-cells (<xref ref-type=\"fig\" rid=\"F2\">Figure 2a</xref>), suggesting that its expression is not an exclusive feature of beta-cells. Of note, in NOD normoglycaemic mice, colocalization analysis between CXCL10-insulin and CXCL10-glucagon revealed that the proportion of beta- and alpha-cells positive for CXCL10 was similar [CXCL10-INS 24.3 ± 15.3% vs. CXCL10-GCG 18.7 ± 15.2% (mean ± SD), respectively] (<xref ref-type=\"fig\" rid=\"F2\">Figure 2b</xref>). In new-onset diabetic NOD mice, CXCL10-glucagon colocalization was significantly higher compared to CXCL10-insulin [40.6 ± 15.7% vs. 21.3 ± 16.0% (mean ± SD) <italic>p</italic> < 0.0001] (<xref ref-type=\"fig\" rid=\"F2\">Figures 2b,c</xref>). Moreover, CXCL10-insulin colocalization did not differ between normoglycaemic and new-onset diabetic NOD mice [24.3 ± 15.3% vs. 21.3 ± 16.0% (mean ± SD)], suggesting that alpha-cells significantly contribute to the increase of CXCL10 in pancreatic islets of new-onset diabetic NOD mice. Importantly, such increase was not dependent on changes in islets volume (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 3b</xref>).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>CXCL10 is increased in alpha-cells of new-onset diabetic NOD mice. <bold>(a)</bold> Representative images of triple immunofluorescence reporting the expression of insulin (INS, green), glucagon (GCG, blue), and CXCL10 (red) in pancreatic islets of normoglycaemic (panels A–E) and of new-onset diabetic NOD mice (panels F–J). In panels E,K, zoom-in of pancreatic islets are shown. Colocalization between insulin and CXCL10 is shown in yellow and indicated by yellow arrows; colocalization between glucagon and CXCL10 is reported in magenta and indicated by red arrows. Scale bar in panels D,I = 50 μm. Scale bar panels E,J = 20 μm. <bold>(b)</bold> Colocalization rate are reported as the results of Manders's coefficient evaluation between CXCL10 and insulin (CXCL10-INS) (green dots) and CXCL10 and glucagon (CXCL10-GCG) (blue dots) in individual pancreatic islets of <italic>n</italic> = 4 normoglycaemic NOD mice and <italic>n</italic> = 4 new-onset diabetic NOD mice. Each dot represents an individual islet. A total of <italic>n</italic> = 27 pancreatic islets of normoglycaemic and new-onset diabetic NOD mice are reported. Values are reported as the percentage of CXCL10 signal overlapping with total INS or GCG signal. Exact <italic>p</italic>-values were analyzed using multiple comparison ordinary one-way ANOVA test (<italic>p</italic> < 0.05). Dotted lines represent mean ± SD. <bold>(c)</bold> Colocalization plots of CXCL10-Insulin (left) and CXCL10-glucagon (right) of a new-onset diabetic NOD mouse pancreatic islet. Positive pixels for CXCL10 (red), insulin (green), and glucagon (blue), alongside with colocalizing pixels (CXCL10-insulin: yellow; CXCL10-glucagon: magenta), are reported in the plots. Significant colocalizing pixels are within the area delimited by white lines, representing background and threshold levels relative to each channel. Each pixel is reported as a gray-scale RGB intensity value (0–255).</p></caption><graphic xlink:href=\"fendo-11-00630-g0002\"/></fig></sec><sec><title>Four Different Islet Subsets Can Be Identified in Pancreata of New-Onset T1D Patients Based on CXCL10 Expression Pattern</title><p>Results obtained in NOD mice demonstrated a peculiar CXCL10 expression pattern, pointing out to a significant involvement of alpha-cells in chemokine secretion in T1D. These findings prompted us to investigate its distribution in pancreatic islets of T1D donors.</p><p>Firstly, we further confirmed that CXCL10 was absent in pancreatic islets of non-diabetic donors. We analyzed pancreatic sections derived from 3 EUnPOD-INNODIA non-diabetic multiorgan donor (subjects characteristics reported in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>) using triple immunofluorescence analysis for CXCL10, insulin and glucagon. Results demonstrated that CXCL10 was not expressed in non-diabetic pancreatic islets (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 4</xref>). Then, we analyzed CXCL10 expression pattern distribution in pancreatic sections of 6 new-onset (≤9 weeks from diagnosis) T1D subjects from DiViD study (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><p>CXCL10 expression was not observed in exocrine tissue nor in CD45<sup>+</sup> cells surrounding or infiltrating pancreatic islets or scattered in acinar tissue (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 5</xref>). In line with previous reports, we confirmed that CXCL10 was expressed in pancreatic islets but not in exocrine/acinar tissue. Of note, a heterogeneous pattern of CXCL10 expression among pancreatic islets of T1D DiViD cases was clearly observed. Indeed, based on CXCL10 positivity and on the presence or absence of insulin [insulin-containing islets (ICIs) and insulin-deficient islets (IDIs)], four different islet subsets can be readily distinguished: ICIs with CXCL10 expression (ICI-CXCL10<sup>pos</sup>) and ICIs without any sign of CXCL10 positivity (ICI-CXCL10<sup>neg</sup>); IDIs containing CXCL10 positive cells (IDI-CXCL10<sup>pos</sup>) and IDIs without any positivity for the chemokine (IDI-CXCL10<sup>neg</sup>) (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>CXCL10 is expressed in ICIs and IDIs of new-onset diabetic individuals and distinguished four pancreatic islet subsets. Triple immunofluorescence analysis of insulin (INS, green), glucagon (GCG, blue), and CXCL10 (red) in pancreatic sections of new-onset T1D DiViD cases. Representative pancreatic islet 40 × confocal microscope images are shown for each channel, alongside with digital zoom-in for each set of panel. <bold>(A–E)</bold> ICI showing positivity for CXCL10. <bold>(F–J)</bold> ICI without CXCL10 positivity. <bold>(K–O)</bold> IDI showing CXCL10 positivity. <bold>(P–T)</bold> IDI without positivity for CXCL10. Scale bar =100 μm. Scale bar zoom-in = 40 μm.</p></caption><graphic xlink:href=\"fendo-11-00630-g0003\"/></fig><p>For each T1D DiViD case, we analyzed two non-consecutive sections derived from two different paraffin blocks of the pancreas tail. Overall, using fluorescent confocal microscopy, we manually screened a total of 1,148 pancreatic islets from 6 new-onset T1D DiViD subjects. Of 1,148 islets, 343 were ICIs and 805 were IDIs, in line to what has been previously observed from multiple histological analyses of the same DiViD cases, showing a higher number of IDIs vs. ICIs. Of 343 ICIs, the majority (<italic>n</italic> = 332, 96.7% of the total ICIs) were positive for CXCL10, while 11 (3.3%) were negative for the chemokine. Interestingly, among 805 IDIs, 514 (63.8%) contained CXCL10 positive cells, while 291 (36.1%) were negative (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary File 1</xref>).</p><p>A case-by-case analysis showed a heterogeneous CXCL10 staining pattern and distribution of islet subsets, confirming a substantial heterogeneity among T1D individuals (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref> and <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). In all cases, regardless of the section analyzed, almost all ICIs were positive for CXCL10.</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Distribution of islet subsets in T1D DiViD individuals based on CXCL10 expression and ICI/IDI classification. Histological evaluation of islet subsets distribution in each of the six recent-onset DiViD individuals, based on the analysis of two non-consecutive pancreatic sections/case. Distribution of islets is reported as percentage value of total islets identified per section.</p></caption><graphic xlink:href=\"fendo-11-00630-g0004\"/></fig><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Table reporting the percentage values and the absolute number (in parentheses) of ICIs and IDIs positive or negative for CXCL10 in two non-consecutive pancreatic sections derived from two different formalin-fixed paraffin-embedded pancreatic tissue histological blocks of the same DiViD case.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Section #</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>ICI CXCL10<sup>POS</sup> % (absolute)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>ICI CXCL10<sup>NEG</sup> % (absolute)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>IDI CXCL10<sup>POS</sup> % (absolute)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>IDI CXCL10<sup>NEG</sup> % (absolute)</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Case 1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.9 (3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">90.2 (46)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.9 (2)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.0 (5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">81.7 (67)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.2 (10)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Case 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.2 (22)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.5 (3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">42.9 (36)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.4 (23)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44.9 (53)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.8 (1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">47.4 (56)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.77 (8)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Case 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40.6 (41)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.9 (1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5 (5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53.5 (54)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76.6 (69)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.5 (5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.0 (11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.5 (5)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Case 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3 (15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.2 (1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.2 (1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62.2 (28)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.9 (23)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">58.0 (61)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20 (21)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Case 5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.6 (16)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67.8 (143)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.6 (52)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.7(13)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">64.7 (66)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22.5 (23)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Case 6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.4 (1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.7 (11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">82.9 (58)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Section #2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">81.6 (71)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.3 (9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.0 (7)</td></tr></tbody></table><table-wrap-foot><p><italic>See <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary File 1</xref> for an extended version of this table</italic>.</p></table-wrap-foot></table-wrap><p>In both sections analyzed, Case-1, Case-2, and Case-5 showed a consistent and significant higher proportion of IDI-CXCL10<sup>pos</sup> compared to IDI-CXCL10<sup>neg</sup> (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Of note, in these cases, IDIs represent the major source of CXCL10, being higher compared to ICI-CXCL10<sup>pos</sup> (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>, <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>, and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary File 1</xref>).</p><p>In contrast, in Case-3, Case-4, and Case-6, we found a striking heterogeneity between the two sections analyzed, mainly due to the different rate of IDIs positive for CXCL10 (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Notable, in Case-6, the high heterogeneity observed in terms of ICIs and IDIs presence (section#1: 1.5% ICIs vs. 98.5% IDIs; section#2: 81.6% ICIs vs. 18.4% IDIs) between the two sections is paralleled by strong differences in CXCL10 islets positivity (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>, <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>, and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary File 1</xref>) being more frequent in section#2 within ICIs (100% of ICIs CXCL10<sup>pos</sup>) compared to IDIs in section#1 (15.7% of IDIs CXCL10<sup>pos</sup>).</p></sec><sec><title>Alpha-Cells Contribute to CXCL10 Expression in Pancreatic Islets of New-Onset T1D Patients</title><p>The relevant presence of IDIs showing positivity for CXCL10 strongly suggests that also in human context, CXCL10 expression is not exclusively expressed by beta-cells. Indeed, triple immunofluorescence staining aimed at detecting insulin, glucagon, and CXCL10 expression in pancreatic sections of 6 new-onset T1D subjects from DiViD study, demonstrated that: (a) in ICIs, both beta- and alpha-cells stained positive for CXCL10 (<xref ref-type=\"fig\" rid=\"F5\">Figure 5a</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 6</xref>); (b) in IDIs, CXCL10 was expressed only in alpha-cells, since the (whole) signal of the chemokine perfectly overlapped with glucagon (<xref ref-type=\"fig\" rid=\"F3\">Figures 3K–O</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 7</xref>).</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Alpha-cells contribute to CXCL10 expression in islets of new-onset T1D patients. <bold>(a)</bold> Triple immunofluorescence analysis of insulin (INS, green, panel A), glucagon (GCG, blue, panel B), and CXCL10 (red, panel C) of pancreatic islets in T1D DiViD cases. Panel E: digital zoom-in of overlapping (merge) channels, showing colocalization of CXCL10 and insulin (yellow pixels) indicated by yellow arrow and of CXCL10 and glucagon (magenta pixels) indicated by red arrow. Scale bar = 50 μm. Scale bar zoom-in = 20 μm. <bold>(b)</bold> Colocalization analysis of CXCL10 and insulin (green dots) and CXCL10 and glucagon (blue dots) in pancreatic islets of T1D DiViD cases. A total of <italic>n</italic> = 50 ICIs from 6 DiViD cases were analyzed for both CXCL10-insulin and CXCL10-glucagon colocalization rate. Values are reported as the percentage of overlapping CXCL10-insulin or CXCL10-glucagon pixels over total insulin or glucagon positive pixels, according to Mander's Coefficient calculation. Exact <italic>p</italic>-value was calculated using Wilcoxon matched-pairs signed rank test. <bold>(c)</bold> Colocalization plots of CXCL10-insulin (left) and CXCL10-glucagon (right) of a recent-onset diabetic DiViD individual ICI (Case-1). Positive pixels for CXCL10 (red), insulin (green), and glucagon (blue), alongside with colocalizing pixels (CXCL10-insulin: yellow; CXCL10-glucagon: magenta), are reported in the plots. Significant colocalizing pixels are within the area delimited by white lines, representing background and threshold levels relative to each channel. Each pixel is reported as a gray-scale RGB intensity value (0–255).</p></caption><graphic xlink:href=\"fendo-11-00630-g0005\"/></fig><p>In order to quantify the contribution of beta- and alpha-cells to the overall expression of CXCL10 in pancreatic islets of T1D subjects, we analyzed the colocalization rate of CXCL10-insulin and CXCL10-glucagon in ICIs detected in all DiViD cases. Such analysis demonstrated that CXCL10-glucagon colocalization rate was significantly higher compared to CXCL10-insulin [CXCL10-GCG 36.5 ± 17.1% vs. CXCL10-INS 23.6 ± 18.9% (mean ± SD) (<xref ref-type=\"fig\" rid=\"F5\">Figures 5b,c</xref>)], thus demonstrating that alpha-cells significantly contribute, together with beta-cells, to CXCL10 expression in pancreatic islets of T1D subjects.</p></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Several studies reported that CXCL10 expression is increased in <italic>in-vitro</italic> cultured pancreatic islets upon inflammatory stresses (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>, <xref rid=\"B35\" ref-type=\"bibr\">35</xref>), as well as in pancreatic islets of animal models of autoimmune diabetes (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>) and in donors with T1D (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>–<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). However, data are lacking regarding CXCL10 intra-islet expression pattern in T1D. Such context prompted us to further investigate CXCL10 expression in pancreas sections of NOD mice and of T1D subjects from DiViD study, in order to better define CXCL10 intra-islet distribution.</p><p>In the present study, we confirmed that CXCL10 was expressed in pancreatic islets but not in exocrine tissue in T1D, while its expression was not observed in pancreas of healthy donors. Our data are in line with previous reports showing increased expression of CXCL10 in pancreatic islets in T1D (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>–<xref rid=\"B25\" ref-type=\"bibr\">25</xref>).</p><p>Interestingly, our data suggest that both beta- and alpha-cells contribute to CXCL10 expression in T1D pancreatic islets, both in diabetic NOD mice and in DiViD T1D subjects.</p><p>In 12- to 21-week-old new-onset diabetic NOD mice, CXCL10 was expressed in pancreatic islets, but not in exocrine tissue, and significantly increased vs. age-matched normoglycaemic NOD mice.</p><p>Our results show a significant increase in the proportion of alpha-cells expressing CXCL10 in new-onset diabetic vs. normoglycaemic NOD mice, thus potentially suggesting that a higher rate of alpha-cells are subjected to inflammatory stresses and respond by activating CXCL10 transcription.</p><p>These findings are mirrored in pancreata of T1D DiViD subjects compared to healthy multiorgan donors collected within the EUnPOD network of INNODIA consortium. In line with previous studies (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>–<xref rid=\"B26\" ref-type=\"bibr\">26</xref>), we confirmed that CXCL10 was specifically expressed in pancreatic islets of T1D subjects and absent in non-diabetic controls. In ICIs, CXCL10 expression was observed both in beta- and in alpha-cells. As expected, in all DiViD cases analyzed, most of the ICIs (95%) showed positivity for CXCL10, in line with previous observations which attributed a more aggressive insulitis and inflammation to those islets containing residual beta-cells (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Of interest, in ICIs we observed a higher proportion of CXCL10 positive alpha-cells compared to beta-cells, suggesting a critical contribution of alpha-cells to the pancreatic islet expression of CXCL10. To this regard, it should be underlined that Mander's colocalization coefficient is independent of absolute signal as it measures the fraction of one protein that colocalizes with a second protein; therefore, it is unlikely that the differences observed in the colocalization rates are dependent on beta- or alpha-cell mass modifications.</p><p>Strikingly, the expression of CXCL10 was also clearly observed in alpha-cells of IDIs where beta-cells were absent and inflammation was lower or not present, as shown previously (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>–<xref rid=\"B40\" ref-type=\"bibr\">40</xref>) and in the present manuscript as well (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figures 4a,b</xref>). Based on manual counting of IDI-CXCL10<sup>pos</sup> in each DiViD case, we observed that Case-1, Case-2, and Case-5 revealed a higher fraction of IDI-CXCL10<sup>pos</sup> among all IDIs detected; this result is consistent between the two non-consecutive pancreatic sections analyzed. Conversely, a substantial heterogeneity between the two sections was observed in Case-3, Case-4, and Case-6, mainly due to the different rate of ICI-CXCL10<sup>pos</sup>, clearly evident in Case-6. Despite the high heterogeneity, overall, Case-3, Case-4, and Case-6 showed the lowest proportion of IDI-CXCL10<sup>pos</sup> (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary File 1</xref>). In an effort aimed at looking for specific characteristics correlated with CXCL10-based DiViD cases patterning, we found that Case-6, showing the lowest rate of CXCL10<sup>pos</sup> islets (considering both sections and independently of its cellular distribution) (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary File 1</xref>), also exhibited the lowest expression of <italic>HLA-ABC</italic> genes among DiViD cases, as previously reported by Richardson S and colleagues (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). Additionally, in Case-3, classified by having high residual beta-cell content, severe insulitis and high expression of HLA Class-I (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>), we observed the highest proportion of ICI-CXCL10<sup>pos</sup> among all DiViD cases.</p><p>Collectively, these results suggest that, although residual beta-cells drive severe pancreatic islet inflammation leading to a global CXCL10 increase, the expression of this chemokine in alpha cells could represent a phenomenon not strictly dependent on beta-cell content. Of note, a very high level of heterogeneity was observed among cases analyzed and among different paraffin blocks of the same case, in line with the heterogeneous nature of the disease, previously highlighted by several studies assaying the same cases (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>, <xref rid=\"B41\" ref-type=\"bibr\">41</xref>).</p><p>In support of our data, CXCL10 hyperexpression in DiViD cases was also previously observed at the mRNA level, being its expression significantly increased in laser-captured microdissected islets of T1D donors compared to non-diabetic controls (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>); of note, CXCL10 hyperexpression was reported to be significantly associated to peri-islet insulitis microdissected tissue rather than to pancreatic islets core. Such results are in line with our data; indeed, it is likely that CXCL10 hyperexpression observed in peri-islet/insulitic microdissected tissue from T1D donors was mostly derived from alpha-cells clusters which are more closely associated to the peri-islets basement membrane (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). In addition, our results exclude an overlapping between insulitic immune cells and CXCL10 expression as shown by CD45-CXCL10 immunofluorescence staining in T1D DiViD sections (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 4a</xref>).</p><p>In support to our findings, CXCL10 expression in alpha-cells was previously reported by Tanaka et al. in Japanese fulminant diabetes cases (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>) and, more recently, by Moin et al. (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>) in pancreatic islets of multiorgan donors with chronic pancreatitis, thus confirming and extending the observation of CXCL10 expression in alpha-cells in autoimmune diabetes.</p><p>Of interest, our data corroborate the increasing importance attributed to alpha-cells in the pathogenesis and progression of T1D. Alterations of several genes alongside with functional defects have been observed in alpha-cells obtained from T1D donors. These include alterations of alpha-cells phenotypic-maintenance genes and defects in glucagon secretion (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>). We can speculate that inflammation may contribute to the activation of several signaling pathways, which alter alpha-cells phenotype and activate innate inflammatory responses leading to CXCL10 expression. As a matter of fact, CXCL10 is not the only pro-inflammatory molecule expressed by alpha-cells; indeed, it has been reported that alpha-cells can express also IL-1β (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>) as well as IL-6 (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>), thus potentially contributing to the pro-inflammatory islet microenvironment causing preferential homing of T-lymphocytes in pancreas in T1D (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>). In turn, increased immune cell migration and then inflammation could enhance beta-cell antigenicity through higher HLA Class-I expression and novel peptides exposure to the immune system (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>), thus generating a critical positive feedback loop.</p><p>An additional layer of evidence, supporting the expression of CXCL10 by alpha-cells, is given by their molecular equipment needed to induce those signaling pathways which lead to CXCL10 transcriptional activation. Indeed, analysis of transcriptome datasets comparing beta- and alpha-cells gene expression, showed an almost equal expression levels of those receptors and intracellular molecules which initiate the signaling cascades leading to CXCL10 transcriptional activation, such as IFNAR1, IFNAR2, IFNGR, TYK2, TNFRSF1A, and IL-1R (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>–<xref rid=\"B53\" ref-type=\"bibr\">53</xref>), thus demonstrating the potential ability of alpha-cells to respond to the inflammatory milieu and potentially activate CXCL10 pathway.</p><p>However, several open questions remain. Firstly, the potential role of CXCL10 beside its effects on immune cells recruitment needs to be clarified; several reports attributed a role for CXCL10 in proliferation and angiogenesis (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>). Particularly, it has been reported that CXCL10 can modulate vascular angiogenesis (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>), also through the inhibition of VEGF-A (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>). Angiogenesis has been linked to beta-cell regeneration through the re-arrangement of islet microenvironment, thus hypothesizing a role for islet CXCL10 as a factor involved in the modulation of beta-cell regeneration (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>).</p><p>Secondly, the presence of CXCL10 in IDIs with no sign of inflammation may suggest that CXCL10 transcriptional activation is not only induced by cytokines and inflammatory mediators but may be caused by the exposure to additional factors. In this regard, alternative signaling pathways and receptors (e.g., TLR4) have been reported for the induction of CXCL10 (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>).</p><p>Thirdly, the co-existence of IDI-CXCL10<sup>pos</sup> and IDI-CXCL10<sup>neg</sup> indicates a high level of heterogeneity involving also pancreatic islets alpha-cells expressing CXCL10; the identification of those factors determining the expression of CXCL10 in alpha-cells and how these correlate with individual islet phenotype would be of major importance to understand the role of this chemokine in T1D.</p><p>In conclusion, we have shown that chemokine CXCL10 is expressed also by alpha-cells which represent important contributors to the expression of CXCL10 in pancreatic islets. These results further underline the role of alpha-cells in T1D pathogenesis and progression and suggest the need to advance our knowledge regarding function and dysfunction of these cells in pancreatic islet autoimmunity.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>All datasets generated for this study are included in the article/<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Material</xref>.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by Norwegian Governments Regional Ethics Committee. Written informed consent was obtained from all individuals with type 1 diabetes after they had received oral and written information from the diabetologist and the surgeon separately. EUnPOD multiorgan donors' pancreata not suitable for clinical purposes were obtained with informed written consent by organ donors' next-of-kin and processed with the approval of the local ethics committee of the Pisa University. Specific consent to the publication of the data was obtained from the participants or by organ donors' next-of-kin. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Ethics Committee of the KU Leuven. All animal procedures were performed in accordance with the NIH guidelines for the care and use of laboratory animals. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>LN, NB, and GS performed the experiments, analyzed the data, and wrote the manuscript. GG analyzed the data and contributed to the scientific discussion. GL performed the experiments during the revision stage and contributed to the scientific discussion. LN, GS, and FD reviewed the manuscript and designed experiments. LK and KD provided support for DiViD cohort and contributed to the scientific discussion. CG and LO reviewed the manuscript, provided support for animal models, and contributed to the scientific discussion. CM reviewed the manuscript and contributed to the scientific discussion. LM and PM reviewed the manuscript, provided support for EUnPOD donors, and contributed to the scientific discussion. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with the authors LN, LM, FD, GS, KD, PM, CG, LO, and CM.</p></sec></body><back><ack><p>The Secretarial help of Maddalena Prencipe was greatly appreciated.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This project has received funding from the Innovative Medicines Initiative 2 (IMI2) Joint Undertaking under Grant Agreement No. 115797-INNODIA and No. 945268 INNODIA HARVEST. This joint undertaking receives support from the Union's Horizon 2020 research and innovation programme and EFPIA, JDRF, and the Leona M. and Harry B. Helmsley Charitable Trust. FD was supported by the Italian Ministry of University and Research (No. 2017KAM2R5_003). GS was supported by the Italian Ministry of University and Research (No. 201793XZ5A_006).</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fendo.2020.00630/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fendo.2020.00630/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Table_1.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM2\"><media xlink:href=\"Data_Sheet_1.PDF\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coppieters</surname><given-names>KT</given-names></name><name><surname>Dotta</surname><given-names>F</given-names></name><name><surname>Amirian</surname><given-names>N</given-names></name><name><surname>Campbell</surname><given-names>PD</given-names></name><name><surname>Kay</surname><given-names>TWH</given-names></name><name><surname>Atkinson</surname><given-names>MA</given-names></name><etal/></person-group>. <article-title>Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients</article-title>. <source>J Exp Med</source>. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Oncol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. 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contrib-type=\"author\"><name><surname>Cortellini</surname><given-names>Alessio</given-names></name><xref ref-type=\"aff\" rid=\"aff17\"><sup>17</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/868770/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Quadrini</surname><given-names>Silvia</given-names></name><xref ref-type=\"aff\" rid=\"aff18\"><sup>18</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Palmieri</surname><given-names>Giuseppe</given-names></name><xref ref-type=\"aff\" rid=\"aff19\"><sup>19</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/226775/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Pigozzo</surname><given-names>Jacopo</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ascierto</surname><given-names>Paolo Antonio</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/153043/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Vitale</surname><given-names>Maria Grazia</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Strippoli</surname><given-names>Sabino</given-names></name><xref ref-type=\"aff\" rid=\"aff9\"><sup>9</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ferrucci</surname><given-names>Pier Francesco</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Berardi</surname><given-names>Rossana</given-names></name><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/65410/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Randon</surname><given-names>Giovanni</given-names></name><xref ref-type=\"aff\" rid=\"aff8\"><sup>8</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Cardone</surname><given-names>Pietro</given-names></name><xref ref-type=\"aff\" rid=\"aff10\"><sup>10</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Schinzari</surname><given-names>Giovanni</given-names></name><xref ref-type=\"aff\" rid=\"aff14\"><sup>14</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Silvestris</surname><given-names>Franco</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Tucci</surname><given-names>Marco</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff9\"><sup>9</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/739037/overview\"/></contrib><on-behalf-of>the Italian Melanoma Intergroup (IMI)</on-behalf-of></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro</institution>, <addr-line>Bari</addr-line>, <country>Italy</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Medical Oncology Unit, Department of Oncology and Hematology, Azienda Ospedaliera Papa Giovanni XXIII Hospital</institution>, <addr-line>Bergamo</addr-line>, <country>Italy</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Melanoma Oncology Unit</institution>, <addr-line>Veneto Institute of Oncology, Scientific Institute for Research, Hospitalization and Healthcare, Padua</addr-line>, <country>Italy</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Melanoma, Cancer Immunotherapy and Development Therapeutics, Istituto Nazionale Tumori IRCCS Fondazione Pascale</institution>, <addr-line>Naples</addr-line>, <country>Italy</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Department of Oncology, Azienda Sanitaria Universitaria del Friuli Centrale</institution>, <addr-line>Udine</addr-line>, <country>Italy</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Division of Melanoma, Sarcoma and Rare Tumors, European Institute of Oncology, Scientific Institute for Research, Hospitalization and Healthcare</institution>, <addr-line>Milan</addr-line>, <country>Italy</country></aff><aff id=\"aff7\"><sup>7</sup><institution>Oncology Clinic, Università Politecnica delle Marche</institution>, <addr-line>Ancona</addr-line>, <country>Italy</country></aff><aff id=\"aff8\"><sup>8</sup><institution>Melanoma Medical Oncology Unit, Department of Medical Oncology and Hematology, National Institute of Tumori</institution>, <addr-line>Milan</addr-line>, <country>Italy</country></aff><aff id=\"aff9\"><sup>9</sup><institution>IRCCS Giovanni Paolo II, Cancer Institute</institution>, <addr-line>Bari</addr-line>, <country>Italy</country></aff><aff id=\"aff10\"><sup>10</sup><institution>Department of Medical Sciences, Dermatologic Clinic, University of Turin</institution>, <addr-line>Turin</addr-line>, <country>Italy</country></aff><aff id=\"aff11\"><sup>11</sup><institution>First Division of Medical Oncology, IRCCS Regina Elena National Cancer Institute</institution>, <addr-line>Rome</addr-line>, <country>Italy</country></aff><aff id=\"aff12\"><sup>12</sup><institution>Medical Oncology Department, Santa Chiara Hospital, University of Pisa</institution>, <addr-line>Pisa</addr-line>, <country>Italy</country></aff><aff id=\"aff13\"><sup>13</sup><institution>Medical Oncology and Hematology Unit, Humanitas Cancer Center, Humanitas Clinical and Research Center, IRCCS</institution>, <addr-line>Rozzano</addr-line>, <country>Italy</country></aff><aff id=\"aff14\"><sup>14</sup><institution>Medical Oncology, Fondazione Policlinico Universitario ‘Agostino Gemelli’ IRCCS</institution>, <addr-line>Rome</addr-line>, <country>Italy</country></aff><aff id=\"aff15\"><sup>15</sup><institution>Medical Oncology, ASST-Sette Laghi, Ospedale di Circolo e Fondazione Macchi</institution>, <addr-line>Varese</addr-line>, <country>Italy</country></aff><aff id=\"aff16\"><sup>16</sup><institution>Medical Oncology Unit, Santa Croce and Carle Teaching Hospital</institution>, <addr-line>Cuneo</addr-line>, <country>Italy</country></aff><aff id=\"aff17\"><sup>17</sup><institution>Department of Biotechnological and Applied Clinical Sciences, San Salvatore Hospital, University of L’Aquila</institution>, <addr-line>L’Aquila</addr-line>, <country>Italy</country></aff><aff id=\"aff18\"><sup>18</sup><institution>Medical Oncology Unit, Azienda Sanitaria Locale Frosinone</institution>, <addr-line>Frosinone</addr-line>, <country>Italy</country></aff><aff id=\"aff19\"><sup>19</sup><institution>Unit of Cancer Genetics, Institute of Genetic and Biomedical Research, National Research Council</institution>, <addr-line>Sassari</addr-line>, <country>Italy</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Wen-Qing Li, Peking University Cancer Hospital, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Kathleen Marie Kokolus, University at Buffalo, United States; John August D’Orazio, University of Kentucky, United States</p></fn><corresp id=\"c001\">*Correspondence: Marco Tucci, <email>marco.tucci@uniba.it</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Skin Cancer, a section of the journal Frontiers in Oncology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>1652</elocation-id><history><date date-type=\"received\"><day>07</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Mannavola, Mandala, Todisco, Sileni, Palla, Minisini, Pala, Morgese, Di Guardo, Stucci, Guida, Indini, Quaglino, Ferraresi, Marconcini, Tronconi, Rossi, Nigro, Occelli, Cortellini, Quadrini, Palmieri, Pigozzo, Ascierto, Vitale, Strippoli, Ferrucci, Berardi, Randon, Cardone, Schinzari, Silvestris and Tucci.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Mannavola, Mandala, Todisco, Sileni, Palla, Minisini, Pala, Morgese, Di Guardo, Stucci, Guida, Indini, Quaglino, Ferraresi, Marconcini, Tronconi, Rossi, Nigro, Occelli, Cortellini, Quadrini, Palmieri, Pigozzo, Ascierto, Vitale, Strippoli, Ferrucci, Berardi, Randon, Cardone, Schinzari, Silvestris and Tucci</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><sec><title>Background</title><p>We performed a multicenter retrospective observational study to investigate the impact of clinical–pathological features and therapeutic strategies on both the complications and survival of patients with bone metastases (BMs) from malignant melanoma.</p></sec><sec><title>Patients and Methods</title><p>A total of 305 patients with melanoma and radiological evidence of BMs were retrospectively enrolled from 19 Italian centers. All patients received conventional treatments in accordance with each own treating physician’s practice. Both univariate and multivariate models were used to explore the impact of melanoma features, including skeletal-related events (SREs), and different treatments on both overall survival (OS) and time-to-SREs. The chi-squared test evaluated the suitability of several parameters to predict the occurrence of SREs.</p></sec><sec><title>Results</title><p>Eighty-three percent of patients had metachronous BMs. The prevalent (90%) bone metastatic site was the spine, while 45% had involvement of the appendicular skeleton. Forty-seven percent experienced at least one SRE, including palliative radiotherapy (RT) in 37% of cases. No melanoma-associated factor was predictive of the development of SREs, although patients receiving early treatment with bone-targeted agents showed 62% lower risk and delayed time of SRE occurrence. Median OS from the diagnosis of bone metastasis was 10.7 months. The multivariate analysis revealed as independent prognostic factors the number of BMs, number of metastatic organs, baseline lactate dehydrogenase levels, and treatment with targeted therapy or immunotherapy. Subgroup analyses showed the best OS (median = 16.5 months) in the subset of patients receiving both immunotherapy and palliative RT.</p></sec><sec><title>Conclusion</title><p>Based on our results, patients undergoing immunotherapy and palliative RT showed an OS benefit suggestive of a possible additive effect. The apparent protective role of bone targeting agent use on SREs observed in our analysis should deserve prospective evaluation.</p></sec></abstract><kwd-group><kwd>melanoma</kwd><kwd>bone metastases</kwd><kwd>SREs</kwd><kwd>immunotherapy</kwd><kwd>bisphosphonates</kwd><kwd>denosumab</kwd></kwd-group><counts><fig-count count=\"2\"/><table-count count=\"5\"/><equation-count count=\"0\"/><ref-count count=\"40\"/><page-count count=\"11\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Innovative therapies have improved the survival of patients with unresectable metastatic cutaneous melanoma (CM). However, the prognosis remains poor in those harboring negative prognostic factors, such as poor performance status (PS), high tumor burden, brain metastases, and high baseline levels of lactate dehydrogenase (LDH) (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>). Also, the genomic landscape of melanoma influences the clinical evolution as well as innate and acquired drug resistance, thus representing an up-growing field of interest (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>).</p><p>The impact of bone disease (BD) in melanoma has been scarcely investigated. Data from clinical trials indicate that the skeleton is the fourth site of metastasis after lung, liver, and brain, that occurs in about 11–18% of patients (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Bone metastases (BMs) generate typical skeletal-related events (SREs), such as severe bone pain, pathological fractures, spinal cord compression, hypercalcemia, and need for radiotherapy (nRT) or surgery to the bone, and are common in breast, prostate, and lung cancers while being so far a clinical challenge in other malignancies like melanoma (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Moreover, the potential beneficial effect of novel anti-melanoma agents on BMs and SREs is, at present, unclear (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>).</p><p>Data collected from the SEER (Surveillance, Epidemiology and End Results) database and large series from individual medical centers demonstrated that BMs from CM are frequently observed in young patients and are associated with elevated LDH, while the prognosis is apparently poor and almost similar to that of patients developing brain or liver metastases (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). In addition, BMs occur in patients with CM, while they are rarely detected in those with mucosal, uveal, and acral melanoma. Furthermore, BMs are frequently revealed at diagnosis of the metastatic disease as involving single or multiple sites and frequently affect the axial skeleton.</p><p>The pathogenesis of BMs is regulated by the interplay among malignant cells, osteoclasts, osteoblasts, and immune cells. Epithelial tumor cells, indeed, produce high RANK-L (the receptor activator of nuclear factor-kB ligand) levels that engulf the bone metastatic sites, thus interfering with osteoclasts through the RANK receptor that is also expressed by cancer cells like melanoma and regulated by interferon (IFN)-γ (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Systemic agents for the treatment of BMs include inhibitors of bone resorption and anabolic signals, namely, bone-targeting agents (BTAs), aimed at restoring the physiological bone turnover that results profoundly impaired in patients with skeletal colonization. Their use in breast, prostate, and lung cancers is currently well supported in various clinical settings (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>), while their effective contribution in patients with BMs from melanoma is still debated. Recent studies reported the synergistic effect of RANK-L blockade combined with immune checkpoint inhibition in patients with BMs from melanoma, suggesting a potential clinical benefit from this strategy (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Based on preliminary evidence (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>), clinical trials are currently ongoing and are aimed at exploring the overall therapeutic effect of denosumab with immunotherapy (NCT-03161756; EudraCT-2016-001925-15).</p><p>Growing interest in melanoma has also been oriented in combining an immune checkpoint inhibitor (ICI) with either systemic or local treatment to maximize the antitumor response. Ongoing clinical trials, for example, are exploring the association of targeted therapy and immunotherapy (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>–<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). They were supported by preclinical and translational studies showing that BRAF and MEK inhibition has immune-modulating effects that increase tumor T cell infiltration as well as tumor antigen exposition and PD-L1 expression (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Another attractive scenario is combining immunotherapy with radiotherapy (RT), the latter being often used in the case of BMs. The rationale is that tumor irradiation induces cell death, provoking local release of tumor-derived antigens that, in turn, promote T cell cross-priming by dendritic cells and long-term immunological memory (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). This priming of the immune system by RT can synergize with immunotherapy. Consequently, trials evaluating combinations of immunotherapy and RT have progressively increased in the last years, both in melanoma and other malignancies (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>).</p><p>Herein, we completed a retrospective multicentric survey in an Italian melanoma population bearing BMs to investigate the potential impact of clinical–pathological features and the therapeutic strategies on survival.</p></sec><sec id=\"S2\"><title>Patients and Methods</title><sec id=\"S2.SS1\"><title>Study Population</title><p>In this observational multicenter study, we retrospectively enrolled 305 patients with a diagnosis of melanoma and radiological evidence of BD. All patients received standard treatments in accordance with each own treating physician’s practice in 19 Italian Centers from November 1984 to March 2019. Clinical data were collected throughout the disease course and included features of melanoma, BM detection, access to systemic treatments (immunotherapy, target therapy, or chemotherapy), nRT, as well as date of first SRE and death. When available, other variables, including age, sex, melanoma primary site, histological parameters (e.g., histotype, Breslow, ulceration, number of mitoses, lymphocyte infiltrate, and nodal stage), BRAF/NRAS status, LDH levels, calcemia, time to appearance of BMs, presence of extraosseous metastases, and SRE type, were assessed. Specific information about the systemic treatments (e.g., drug sequences, objective responses, or time of disease progression) were not considered since these are out of the scope of this survey. All patients had written informed consent for clinical data collection and use for research purposes according to each own center’s rules. Ethical approval was not required for this study according to local legislation.</p></sec><sec id=\"S2.SS2\"><title>Statistical Analysis</title><p>Descriptive statistics were used for patient demographics and incidence of SREs. The chi-squared test analyzed the relationship of SRE occurrence with the clinical and pathological features of melanoma patients. Survival analyses were completed by the Kaplan–Meier method. Factors for analyses were identified <italic>a priori</italic> and included baseline clinical features and known prognostic factors for metastatic melanoma or BD. The univariate analysis (log-rank test) explored the clinical variables as prognostic factors for overall survival (OS) and time to SREs from BM diagnosis. Patients who did not experience a SRE were censored at the date of death or at the last follow-up visit for the analysis of time to develop SRE. All significant variables in the univariate model were used to build the multivariate analysis of survival using the Cox proportional hazards regression. Patients that were lost in follow-up were not considered for survival analyses. The statistics were completed with the Medcalc software (version 12.7.0.0). A <italic>p</italic> value < 0.05 was considered significant.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Baseline Demographic Features</title><p>In total, 305 patients with cutaneous (<italic>n</italic> = 290), mucosal (<italic>n</italic> = 6), and uveal (<italic>n</italic> = 9) melanoma were enrolled in the study. The basal clinical and pathological features of the population are described in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. Of note is that the median age at diagnosis was 56 years and 63.3% were male (<italic>n</italic> = 193). A BRAF mutation was documented in 59%, while NRAS mutation was present in 36% of patients. Almost 22% (<italic>n</italic> = 23/105) of patients had LDH levels >2 times the upper limit of the normal range (ULN) at the time of melanoma diagnosis.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Patient demographics and basal melanoma characteristics in the study population.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Frequency</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Percentage</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\">Age at melanoma diagnosis (<italic>n</italic> = 305)</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\">Median, 56 (range = 18–86) years</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Sex (<italic>n</italic> = 305)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">193</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">63.3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">112</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36.7</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Primary site (<italic>n</italic> = 305)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Limbs</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Head and neck</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Trunk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">134</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">43.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Occult</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11.5</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mucosa</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Uvea</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.0</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Histology (<italic>n</italic> = 239)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SSM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">88</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nodular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39.7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Acral</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lentigo Maligna</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mucosal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.5</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Uveal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Other</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13.0</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Breslow depth (<italic>n</italic> = 239)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤1 mm</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11.7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">>1–2 mm</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">>2–4 mm</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.5</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">>4 mm</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37.7</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Ulceration (<italic>n</italic> = 239)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Present</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">138</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">57.7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Absent</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">101</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">42.3</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Number of mitosis (<italic>n</italic> = 201)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">>1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">155</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">77.1</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Tumor-infiltrating lymphocytes (<italic>n</italic> = 169)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Brisk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Absent or not brisk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">132</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78.1</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Lymph node mts (<italic>n</italic> = 263)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">133</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50.6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N+</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">130</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49.4</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>BRAF status (<italic>n</italic> = 285)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mutated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">168</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">58.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wild type</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">117</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">41.1</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>NRAS status (<italic>n</italic> = 59)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mutated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wild type</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">64.4</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>LDH levels (<italic>n</italic> = 105)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤2 × ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">>2 × ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.9</td></tr></tbody></table><table-wrap-foot><attrib><italic>LDH, lactate dehydrogenase; ULN, upper limit of normal; SSM, superficial spreading melanoma; mts, metastases.</italic></attrib></table-wrap-foot></table-wrap><p>The majority of patients (97%) had extraosseous metastases, which included 247 patients (81%) with more than three metastatic sites and 73 patients (24%) with brain metastases. The onset of BMs and the diagnosis of primary melanoma were mostly metachronous (83%). The prevalent bone metastatic site was the spine (90%), while only 45% of patients had involvement of the appendicular skeleton, including 10% of them with both axial and appendicular colonization. The majority of patients (61%, <italic>n</italic> = 183/301) showed less than five BMs, while 39% (<italic>n</italic> = 118) harbored more lesions. Calcium levels at the time of BM diagnosis were usually normal, whereas 37% of patients (<italic>n</italic> = 95/254) expressed LDH levels > 2 times the ULN.</p><p>Almost half of the patients received BTAs, including bisphosphonates (40.8%, <italic>n</italic> = 119/292) or denosumab (6.8%, <italic>n</italic> = 20). With regard to systemic treatments, 33% of patients (<italic>n</italic> = 96/291) received only targeted therapy, 39% (<italic>n</italic> = 114) only ICIs, and 20.6% (<italic>n</italic> = 60) both ICIs and targeted agents, whereas a minority (7%, <italic>n</italic> = 21) underwent only chemotherapy (CHT). Data are summarized in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>.</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Characteristics of metastatic disease in the study population.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Frequency</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Percentage</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Number of metastatic organs (<italic>n</italic> = 305)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≥3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">247</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">81.0</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Presence of extraosseous mts (<italic>n</italic> = 305)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yes, without brain</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">223</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">73.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yes, with brain</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.9</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Time of BM diagnosis (<italic>n</italic> = 305)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synchronous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Metachronous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">255</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">83.6</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Sites of BM (<italic>n</italic> = 300)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Axial</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">165</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">55</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Appendicular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Both</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">106</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.3</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Number of BM (<italic>n</italic> = 301)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">183</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≥5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">118</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39.2</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Calcaemia at BM diagnosis (<italic>n</italic> = 235)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">218</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">>ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.2</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>LDH levels at BM diagnosis (<italic>n</italic> = 254)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤2 × ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">159</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62.6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">>2 × ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37.4</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Use of BTA (<italic>n</italic> = 292)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">153</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">52.4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bisphosphonates</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">119</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Denosumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.8</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Systemic treatment (<italic>n</italic> = 291)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Targeted therapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Immunotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">114</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Targeted and ICIs</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chemotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.2</td></tr></tbody></table><table-wrap-foot><attrib><italic>BTA, bone-targeting agents; BM, bone metastases; ICIs, immune checkpoint inhibitors; mts; metastases; LDH, lactate dehydrogenase; ULN, upper limit of normal.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS2\"><title>Bone Disease and Development of SREs</title><p>As shown in <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1A</xref>, 47% (<italic>n</italic> = 137/291) of patients developed at least one SRE, which included nRT in 37% (<italic>n</italic> = 109), fractures in 12% (<italic>n</italic> = 35), spinal cord injury in 7% (<italic>n</italic> = 21), and surgery in 3% (<italic>n</italic> = 8), while hypercalcemia occurred in a single case (< 1%). Thirty-four patients (12%) experienced two or more SREs.</p><p>The next set of analyses (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>) explored the features of melanoma patients who experienced SREs. The median age was 59 years and 65% were male. The majority of them showed metachronous BMs (87%) located in the axial skeleton (56%) as well as LDH levels ≤ 2 times the ULN (67%) and normal calcemia (89%) at the time of BM detection. Notably, 66% of patients had less than five BMs, while extraosseous metastases occurred in about 95% of patients. Among patients who experienced at least one SRE, almost 54% were previously treated with BTAs such as bisphosphonates.</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Patient demographics and BM characteristics in the population who experienced SREs.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Frequency</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Percentage</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Age at BM diagnosis (<italic>n</italic> = 137)</bold></td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Median, 59 years</bold></td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Sex (<italic>n</italic> = 137)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">65.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.0</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>BRAF status (<italic>n</italic> = 131)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mutated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">54.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wild type</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45.8</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>NRAS status (<italic>n</italic> = 27)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mutated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40.7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Wild type</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59.3</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>LDH levels at BM diagnosis (<italic>n</italic> = 109)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤2 × ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">>2 × ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.0</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Calcaemia at BM diagnosis (<italic>n</italic> = 104)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤ ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89.4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">> ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.6</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>BM and melanoma diagnosis (<italic>n</italic> = 137)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synchronous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Metachronous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">119</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">86.9</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>SRE and BM diagnosis (<italic>n</italic> = 129)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synchronous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.5</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Metachronous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70.5</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Localization of BM (<italic>n</italic> = 133)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Axial</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">74</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">55.6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Appendicular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Both</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.6</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Number of BM (<italic>n</italic> = 134)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">66.4</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≥5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.6</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Presence of extraosseous mts (<italic>n</italic> = 137)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">130</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">94.9</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Use of BTA (<italic>n</italic> = 137)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">46</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bisphosphonates</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">48.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Denosumab</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.1</td></tr></tbody></table><table-wrap-foot><attrib><italic>BM, bone metastases; BTA, bone-targeting agents; LDH, lactate dehydrogenase; mts, metastases; SRE, skeletal-related events; ULN, upper limit of normal.</italic></attrib></table-wrap-foot></table-wrap><p>Neither clinical parameters nor tumor-related variables correlated with SREs (data not shown). However, patients who were early treated with BTAs showed a minor SRE occurrence with respect to the untreated patients [20% vs. 39.6%; odds ratio (OR) = 0.38, 95% confidence interval (CI) = 0.2–0.72, <italic>p</italic> = 0.003] (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1B</xref>). Moreover, the univariate analysis (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>) investigated those factors putatively associated with longer time to SRE development and revealed a correlation with the axial localization of BMs [hazard ratio (HR) = 0.61, 95% CI = 0.33–1.13, <italic>p</italic> = 0.05] and with previous use of BTAs (HR = 0.41, 95% CI = 0.26–0.66, <italic>p</italic> = 0.001). However, only the use of BTAs before the development of SREs was confirmed as an independent prognostic factor (<italic>p</italic> = 0.003).</p><table-wrap id=\"T4\" position=\"float\"><label>TABLE 4</label><caption><p>Univariate and multivariate analyses of factors associated with time to SRE in patients with BM from melanoma.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Factors</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Effect tested</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Univariate analysis</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Multivariate analysis</td></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95% CI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95% CI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"8\" rowspan=\"1\"><bold>Demographics</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Age (years)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤55 vs. >55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.22</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.86–1.74</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.25</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Sex</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male vs. Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.80–1.61</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" colspan=\"8\" rowspan=\"1\"><bold>Baseline melanoma characteristics</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Histology</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SSM vs. Nodular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.96</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61–1.50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.85<sup>a</sup></td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mucosal vs. Nodular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.23–3.94</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Acral vs. Nodular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.47–4.34</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Uveal vs. Nodular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.17–1.43</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Others vs. Nodular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.81</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.34–1.93</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> BRAF genotype</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">V600 vs. Wild type</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.52–1.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> NRAS genotype</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mutated vs. Wild type</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.65–2.90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.33</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" colspan=\"8\" rowspan=\"1\"><bold>Characteristics present at BM diagnosis</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Time of diagnosis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synchronous vs. Metachronous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.47–1.20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Localization of BM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Axial vs. Extra-axial</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.33–1.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.05</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.36–1.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.39</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Number of BM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><5 vs. ≥5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.98</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.68–1.41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.91</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Calcaemia</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤ULN vs. >ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.28–1.72</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.35</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> LDH levels</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤2× ULN vs. > 2× ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.66–1.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.00</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" colspan=\"8\" rowspan=\"1\"><bold>Treatment and SRE</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Use of BTA before SRE</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yes vs. No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.26–0.66</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.001</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.43</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.24–0.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.003</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Systemic treatment</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Targeted/ICIs vs. CHT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.51–2.44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.78</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>BM, bone metastases; BTA, bone-targeting agents; CHT, chemotherapy; ICIs, immune checkpoint inhibitors; LDH, lactate dehydrogenase; SSM, superficial spreading melanoma; SRE, skeletal-related events; ULN, upper limit of normal. <sup><italic>a</italic></sup>Logrank test. Bold values are indicate to p < 0.05.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS3\"><title>Factors Associated With Overall Survival</title><p>At the time of data lock, 182 patients were deceased and 108 were still alive, while the other 15 resulted lost in follow-up. The median OS was 10.7 months (<xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2</xref>). <xref rid=\"T5\" ref-type=\"table\">Table 5</xref> describes data from both univariate and multivariate analyses of the factors potentially correlated with prognosis. The univariate analysis revealed a worse prognosis in males (<italic>p</italic> = 0.03) and in patients with melanoma of the trunk (<italic>p</italic> = 0.05) as well as a number of five or more BMs (<italic>p</italic> < 0.0001), high LDH levels at metastatic BD diagnosis (<italic>p</italic> < 0.0001), and evidence of three or more metastatic sites (<italic>p</italic> = 0.0027), or brain metastasis (<italic>p</italic> = 0.0002). In addition, patients who received new agents (targeted therapy and/or immunotherapy) showed OS improvement with respect to those treated with CHT (<italic>p</italic> < 0.0001). In addition, neither detrimental impact on OS was determined by the occurrence of SREs, nor in the case of multiple SREs (data not shown). The multivariate analysis confirmed as positive strong independent prognostic factors less than five skeletal metastasis (HR = 0.56, 95% CI = 0.39–0.79, <italic>p</italic> = 0.0013; <xref ref-type=\"supplementary-material\" rid=\"FS3\">Supplementary Figure S3</xref>), limited LDH values (HR = 0.49, 95% CI = 0.34–0.71, <italic>p</italic> = 0.0001), less than three metastatic sites (HR = 0.58, 95% CI = 0.34–0.99, <italic>p</italic> = 0.047), and treatments with targeted agents and/or ICIs (HR = 0.32, 95% CI = 0.17–0.58, <italic>p</italic> = 0.0002).</p><table-wrap id=\"T5\" position=\"float\"><label>TABLE 5</label><caption><p>Univariate and multivariate analyses of baseline factors associated with OS in patients with BM from melanoma.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Factors</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Effect tested</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Univariate analysis</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Multivariate analysis</td></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95% CI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95% CI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"8\" rowspan=\"1\"><bold>Demographics</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Age (years)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤55 vs. >55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56–1.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Sex</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male vs. Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.05–1.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.03</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.43</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.97–2.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.08</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"8\" rowspan=\"1\"><bold>Baseline melanoma characteristics</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Histology</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SSM vs. Nodular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.42–0.89</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mucosal vs. Nodular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.45–5.87</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Acral vs. Nodular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.23–1.34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07<sup><italic>a</italic></sup></td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Uveal vs. Nodular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.38–2.39</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Others vs. Nodular</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.34–1.85</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Melanoma site</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Limbs vs. Trunk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.47–0.97</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56–1.35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Others vs. Trunk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.70–1.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.05</bold><sup><italic>a</italic></sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.69–1.69</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.73</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Occult vs. Trunk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.65</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.41–1.03</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.41–1.38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.36</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> BRAF genotype</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">V600 vs. Wild type</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.89–1.64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.22</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> NRAS genotype</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mutated vs. Wild type</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48–1.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.65</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" colspan=\"8\" rowspan=\"1\"><bold>Characteristics present at BM diagnosis</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Time of diagnosis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synchronous vs. Metachronous</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.73–1.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Localization of BM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Axial vs. Extra-axial</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.76–1.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.46</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Number of BM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><5 vs. ≥5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.34–0.64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><0.0001</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.39–0.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.0013</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Calcaemia</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤ULN vs. >ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.23–1.26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> LDH levels</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">≤2 × ULN vs. >2 × ULN</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.30–0.60</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><0.0001</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.49</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.34–0.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.0001</bold></td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"8\" rowspan=\"1\"><bold>Characteristics of metastatic disease</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Number of metastatic organs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><3 vs. ≥3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.40–0.77</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.0027</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.34–0.99</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.047</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Presence of visceral mts</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yes with Brain vs. Yes w/o Brain</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.23–2.62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.0002<sup><italic>a</italic></sup></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.88–2.01</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.18</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No vs. Yes w/o Brain</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.21–0.87</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.13–2.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.49</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"8\" rowspan=\"1\"><bold>Treatment and SRE</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Use of BTA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yes vs. No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.60–1.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> SRE occurrence</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yes vs. No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.85</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64–1.14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.28</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"> Systemic treatment</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Targeted/ICIs vs. CHT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.10–0.57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><0.0001</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.17–0.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.0002</bold></td></tr></tbody></table><table-wrap-foot><attrib><italic>BM, bone metastases; BTA, bone targeting agents; CHT, chemotherapy; ICIs, immune checkpoint inhibitors; LDH, lactate dehydrogenase; mts, metastases; SSM, superficial spreading melanoma; SRE, skeletal-related events; ULN, upper limit of normal; w/o, whitout. <sup><italic>a</italic></sup>Logrank test. Bold values are indicate to p < 0.05.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS4\"><title>Impact of Melanoma-Dedicated Treatments on Overall Survival</title><p>The next set of analyses explored the role of systemic treatments and RT on OS. As shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>, patients receiving CHT alone underwent worsened survival as compared to those treated with new agents, including ICIs and/or targeted drugs (HR = 4.15, CI = 1.75–9.90, <italic>p</italic> < 0.0001). In detail, the median OS (mOS) were 16.5 (95% CI = 10.0–23.3), 13.0 (95% CI = 9.2–16.6), 9.0 (95% CI = 7.3–11.5) and 4.0 months (95% CI = 2.2-5.4) in patients comprehensively treated with (i) ICIs only, (ii) ICIs and targeted therapy, (iii) targeted therapy only, or (iv) CHT only, respectively.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Overall survival by systemic treatment in the study population <bold>(A)</bold> and BRAF-mutated population <bold>(B)</bold>. <italic>mOS</italic>, median overall survival; <italic>CHT</italic>, chemotherapy.</p></caption><graphic xlink:href=\"fonc-10-01652-g001\"/></fig><p>Further investigations were dedicated to the BRAF-mutated population (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). The median OS in patients who received at least one ICI (14.2 months, 95% CI = 9.7–18.0) resulted increased with respect to those treated with targeted therapy alone (8.8 months, 95% CI = 7.0–11.5), although the differences were not statistically significant (HR = 0.75, 95% CI = 0.51–1.10, <italic>p</italic> = 0.15).</p><p>Regarding bone-specific treatments (<xref ref-type=\"supplementary-material\" rid=\"FS4\">Supplementary Figure S4</xref>), patients who received BTAs showed only a modest benefit in terms of mOS as compared to the untreated ones (11 vs. 9 months), but not statistically significant differences were found (HR = 0.80, 95% CI = 0.60–1.06, <italic>p</italic> = 0.13). On the other hand, mOS was quite similar between patients who underwent RT (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>) vs. those who were never treated (10.4 vs. 9.2 months). We also verified (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>) whether or not different combinations of RT with new therapies interfered with survival. To this purpose, we divided the study population into four groups based on the treatment received: (A) ICIs and RT (<italic>n</italic> = 73); (B) ICIs without RT (<italic>n</italic> = 94); (C) targeted therapy only without ICIs (<italic>n</italic> = 64); and (D) targeted therapy plus RT and never ICIs (<italic>n</italic> = 30). We found that patients in group A achieved the best mOS (16 months, 95% CI = 10.4–20.7) with respect to either group B (13 months; HR = 0.78, 95% CI = 0.52–1.17, <italic>p</italic> = 0.23), group C (11 months; HR = 0.68, 95% CI = 7.0–14.5, <italic>p</italic> = 0.08), or group D (8.1 months; HR = 0.5, 95% CI = 0.29–0.86, <italic>p</italic> = 0.013).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Subset analyses of overall survival by radiotherapy need <bold>(A)</bold> and combinations of radiotherapy and new therapies <bold>(B)</bold>. <italic>mOS</italic>, median overall survival; <italic>RT</italic>, radiotherapy.</p></caption><graphic xlink:href=\"fonc-10-01652-g002\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>The development of BMs frequently occurs in cancer with a negative impact on the quality of life and survival. Appropriate algorithms for the management of BD mainly derive from extensive prospective trials with patients harboring tumors that frequently metastasize to the skeleton, such as breast, prostate, lung, and kidney cancers (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Otherwise, a definite evidence-based strategy for the treatment of BMs from other malignancies endowed with lower osteotropic propensity does not exist, while apparently innovative drugs such as cilengitide failed to provide satisfactory results to be applied for routine clinical practice (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>, <xref rid=\"B23\" ref-type=\"bibr\">23</xref>). Particularly, the impact of BD in melanoma has been poorly investigated, as well as the potential therapeutic strategies such as either RT or BTA use. Moreover, another unanswered question from recent registrative clinical trials in melanoma concerns the possible impact of ICIs or targeted agents in patients with BM.</p><p>The present study was aimed at exploring retrospectively the characteristics of melanoma patients bearing BD. As a result of the available literature to the topic, ours appears as a large retrospective study investigating the features of BMs in melanoma. The definition of the effective incidence of BMs in melanoma, however, is out of the scope of the study and, therefore, was not investigated.</p><p>The baseline demographic data in our melanoma population demonstrated that BMs occurred primarily in males with prevalent involvement of the axial skeleton. Almost all patients (97%) showed extraosseous metastases. Among them, 83% developed metachronous bone and visceral metastases, while in 13% of them BMs were discovered in consequence of a SRE occurrence. The frequency of patients harboring a BRAF mutation (59%) was almost in line with that observed in the general melanoma population. On the other hand, the relative higher frequency observed for mutated NRAS (35.6%) should not be considered as a major propensity of these patients in developing BMs since the NRAS mutational status was available only in less than 20% of cases.</p><p>Other studies describing a modest incidence of bone involvement at diagnosis of metastatic melanoma probably reflect the fact that the BD was not properly suspected and investigated at diagnosis. However, one-fourth (23.9%) of melanoma patients from our series showed a brain involvement which is instead reported in about 40–60% of the general melanoma population (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). This apparent difference from our data may probably imply specific molecular mechanisms which critically drive the osteotropism of melanoma cells like those affecting the SDF1/CXCR7/CXCR4 pathway, as recently proven (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>).</p><p>The median survival from the onset of BM was 10.7 months, but our analysis revealed that patients receiving innovative therapies including ICIs and/or targeted agents showed a better prognosis (9.0–16.5 months) than those undergoing CHT (4.0 months). The patients analyzed in this study (<italic>n</italic> = 290) apparently showed a lower OS with respect to those enrolled in recent phase 3 clinical trials with new agents resulting in 5-year survival rates higher than 50%. However, it is noteworthy that our survey refers to the time from BM diagnosis because the information relative to the time of a comprehensive diagnosis of metastatic disease was not available. The lack of a control group without BMs also restrains the possibility of exploring the real impact of the BD in the general melanoma prognosis. Moreover, our real-world experience includes patients with clinical features that are generally excluded from clinical trials, including a suboptimal PS or brain involvement, thus reducing the possibility of a direct comparison between various case studies.</p><p>The data from large clinical trials with recent 5-year survival updates and pooled analyses defined major negative prognostic factors in metastatic melanoma that included elevated LDH levels, poor PS, and three or more metastatic sites. Despite the lack of information regarding the PS of our population, our multivariate model was in line with these results, while revealing for the first time that an elevated number of BM (five or more skeletal lesions) significantly harms the survival. Additionally, the mutational status of melanoma patients bearing BM did not apparently influence OS, although a positive trend was seen in BRAF-mutated patients treated with immunotherapy (14.2 months) as compared to those treated with targeted therapy alone (8.8 months). These results, however, are conditioned by an <italic>a priori</italic> selection bias since the clinician therapeutic decisions were driven by individual prognostic factors; therefore, they should not lead to considering a superiority of immunotherapy in this setting.</p><p>SREs are major complications of BMs that restrain the quality of life in cancer patients. The 2-year cumulative incidence of SREs in breast, prostate, and lung tumors ranges from 41 to 54%, and more than one half of patients develop a SRE at cancer diagnosis or thereafter. In the majority of these tumors, SREs dramatically impact on cancer-specific survival (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Similarly to previous studies (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>–<xref rid=\"B31\" ref-type=\"bibr\">31</xref>), we observed that at least half of patients with BMs developed a SRE, whose nRT predominantly occurred in 37% of the studied population, while 12% of these patients experienced more than a single SRE. Moreover, in the majority of patients (85%), SREs followed the diagnosis of BMs, with about 50% of patients experiencing one SRE within 1 year from diagnosis.</p><p>No melanoma-associated factors were predictive for SRE development in our cohort, although we observed that those receiving primary treatment with BTAs showed a 62% reduced risk of experiencing SREs and delayed time to their occurrence. This apparent protective effect of BTAs in reducing the risk of SREs sounds very impressive if compared to other osteotropic tumors whose relative risk reduction ranges from 15 to 30% (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>). However, it is almost difficult to speculate on the possible more potent effect of BTAs in the treatment of melanoma BMs due to the retrospective nature of our study. Finally, a possible effect of BTAs on OS was not demonstrated in our melanoma population. Other studies investigated the efficacy of combining immunotherapy with anti-RANK-L monoclonal antibodies in this setting of patients, providing encouraging though weak results that undoubtedly require further investigation (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Based on our data, the use of BTAs should be thus suggested at the time of BM detection since it reduces the incidence and delays the development of SREs.</p><p>In a different fashion from other tumors, SREs had no impact on the survival of our melanoma patients bearing BMs, including the nRT that was the most recurrent complication. The modest effect of RT on prognosis was previously demonstrated since the majority of patients showed lower survival and required further treatments to stabilize the skeletal complications (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). However, relevant data from our study concerns the protective effect in terms of survival for patients receiving RT and immunotherapy, independently of targeted agents. Therefore, it is conceivable that the OS benefit observed in these patients could have balanced the worse prognosis of those combining RT and targeted therapy without ICIs.</p><p>Benefits derived from the combination of RT and immunotherapy have been widely described as abscopal effect in melanoma (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>) as well as in other malignancies (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>) and reflects the regression of non-irradiated metastatic lesions at a distance from the primary site of irradiation. The putative synergic effect of RT and immunotherapy gained by our population is also suggested by the similar effect of immunotherapy vs. targeted therapy in the absence of RT, although both a defect of enrollment and radiological imaging as well as the retrospective nature of the analysis are probably limiting factors in our study. However, this combined effect also results from restoration of the immune cell activity in their systemic anticancer effect. In addition, it has been demonstrated that T cells may protect against bone loss (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>), while IFN-γ counterbalances the osteoclastogenesis by interfering with RANK signaling (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). Finally, osteoblasts are also influenced by local T-helper 2 cells through parathormone (PTH) production (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>), and a role of plasmacytoid dendritic cells has also been described (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>).</p></sec><sec id=\"S5\"><title>Conclusion</title><p>In conclusion, we identified the number of BM as a novel prognostic factor in metastatic melanoma and observed that both reduced risk and delay in SRE development occur in patients early treated with BTAs. Despite the SREs not impacting on the survival of melanoma patients with BM, those receiving immunotherapy and requiring palliative RT obtained a major extent of benefit in terms of OS. Further prospective studies are thus needed to understand the effective role of immunotherapy in melanoma patients with BD. However, our preliminary observation suggests that palliative RT on symptomatic BMs may potentially reinforce the immune response and T cell activity in patients treated with ICIs, while the complementary activity of BTAs requires further investigation.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>MT, MM, VS, MP, AM, LP, FMo, LD, MG, AI, PQ, VF, RM, MCT, ER, ON, MO, AC, SQ, GP, JP, PA, MV, SS, PF, RB, GR, PC, and GS contributed to the provision of study materials or patients. AT did the data collection. FMa contributed to the statistical analysis. FMa and MT did the data interpretation and manuscript writing. All authors are accountable for all aspects of the work, gave final approval of the manuscript, and contributed to revising the manuscript critically.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was funded by the “Precision Medicine” project.</p></fn></fn-group><sec id=\"S10\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fonc.2020.01652/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fonc.2020.01652/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"FS1\"><label>FIGURE S1</label><caption><p><bold>(A)</bold> Percentage of patients experiencing different skeletal related events (SREs) in the study population. <bold>(B)</bold> Incidence of SREs according to the use of bone-targeted agents (BTAs). nRT, need for radiotherapy; <sup>∗∗</sup><italic>p</italic> < 0.01.</p></caption><media xlink:href=\"Image_1.JPEG\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS2\"><label>FIGURE S2</label><caption><p>Kaplan–Meier overall survival estimate in the study population (<italic>n</italic> = 290). mOS, median overall survival.</p></caption><media xlink:href=\"Image_2.JPEG\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS3\"><label>FIGURE S3</label><caption><p>Overall survival by number of bone metastases 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Pharmacol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Pharmacol.</journal-id><journal-title-group><journal-title>Frontiers in Pharmacology</journal-title></journal-title-group><issn pub-type=\"epub\">1663-9812</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33041826</article-id><article-id pub-id-type=\"pmc\">PMC7523510</article-id><article-id pub-id-type=\"doi\">10.3389/fphar.2020.581011</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Pharmacology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Hematopoietic Cell Kinase (HCK) Is Essential for NLRP3 Inflammasome Activation and Lipopolysaccharide-Induced Inflammatory Response <italic>In Vivo</italic></article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Kong</surname><given-names>Xiangxi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/992706\"/></contrib><contrib contrib-type=\"author\"><name><surname>Liao</surname><given-names>Yajin</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/1036531\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhou</surname><given-names>Lujun</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Ying</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Cheng</surname><given-names>Jinbo</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn001\">\n<sup>*</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/858545\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yuan</surname><given-names>Zengqiang</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn001\">\n<sup>*</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/829739\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Shukun</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn001\">\n<sup>*</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<sup>1</sup>\n<institution>The Brain Science Center, Beijing Institute of Basic Medical Sciences</institution>, <addr-line>Beijing</addr-line>, <country>China</country>\n</aff><aff id=\"aff2\">\n<sup>2</sup>\n<institution>Center on Translational Neuroscience, College of Life & Environmental Science, Minzu University of China</institution>, <addr-line>Beijing</addr-line>, <country>China</country>\n</aff><aff id=\"aff3\">\n<sup>3</sup>\n<institution>Laboratory of Oral Microbiota and Systemic Diseases, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine</institution>, <addr-line>Shanghai</addr-line>, <country>China</country>\n</aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Francisco Lopez-Munoz, Camilo José Cela University, Spain</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Tadayoshi Karasawa, Jichi Medical University, Japan; Dong-Yun Ouyang, Jinan University, China</p></fn><corresp id=\"fn001\">*Correspondence: Jinbo Cheng, <email xlink:href=\"mailto:cheng_jinbo@126.com\" xlink:type=\"simple\">cheng_jinbo@126.com</email>; Zengqiang Yuan, <email xlink:href=\"mailto:zyuan620@yahoo.com\" xlink:type=\"simple\">zyuan620@yahoo.com</email>; Shukun Wang, <email xlink:href=\"mailto:wangsk2020@126.com\" xlink:type=\"simple\">wangsk2020@126.com</email>\n</corresp><fn fn-type=\"other\" id=\"fn002\"><p>This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>581011</elocation-id><history><date date-type=\"received\"><day>07</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>20</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Kong, Liao, Zhou, Zhang, Cheng, Yuan and Wang</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Kong, Liao, Zhou, Zhang, Cheng, Yuan and Wang</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Activation of the NLRP3 inflammasome results in caspase 1 cleavage, which subsequently leads to IL-1<italic>β</italic> and IL-18 secretion, as well as pyroptosis, and aberrant activation of the inflammasome is involved in several diseases such as type 2 diabetes, atherosclerosis, multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease. NLRP3 activity is regulated by various kinases. Genetic and pharmacological inhibition of the hematopoietic cell kinase (HCK), a member of the Src family of non-receptor tyrosine kinases (NRTKs) primarily expressed in myeloid cells, has previously been shown to ameliorate inflammation, indicating that it may be involved in the regulation of microglia function. However, the underlying mechanism is not known. Hence, in this study, we aimed to investigate the role of HCK in NLRP3 inflammasome activation. We demonstrated that HCK silencing inhibited NLRP3 inflammasome activation. Furthermore, the HCK-specific inhibitor, A419259, attenuated the release of IL-1<italic>β</italic> and caspase 1(P20) from the macrophages and microglia and reduced the formation of the apoptosis-associated speck-like protein with a CARD domain (ASC) oligomer. We also observed that HCK binds to full length NLRP3 and its NBD(NACHT) and LRR domains, but not to the PYD domain. <italic>In vivo</italic>, the HCK inhibitor attenuated the LPS-induced inflammatory response in the liver of LPS-challenged mice. Collectively, these results suggested that HCK plays a critical role in NLRP3 inflammasome activation. Our results will enhance current understanding regarding the effectiveness of HCK inhibitors for treating acute inflammatory diseases.</p></abstract><kwd-group><kwd>nod-like receptor family protein 3</kwd><kwd>inflammasome</kwd><kwd>hematopoietic cell kinase</kwd><kwd>macrophage</kwd><kwd>microglia</kwd><kwd>A419259</kwd></kwd-group><counts><fig-count count=\"5\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"40\"/><page-count count=\"12\"/><word-count count=\"5548\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>The Nod-like receptor family protein 3 (NLRP3) inflammasome is a cytosolic protein complex composed of NLRP3, the adaptor protein apoptosis-associated speck-like protein with a CARD domain (ASC), and caspase 1, which are rapidly assembled in response to both infection with pathogens and endogenous “danger signals” such as monosodium urate, alum, silica, reactive oxygen species, amyloid <italic>β</italic>, and cholesterol (<xref rid=\"B7\" ref-type=\"bibr\">Duewell et al., 2010</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Spreafico et al., 2010</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Tschopp and Schroder, 2010</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Peeters et al., 2013</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Cho et al., 2014</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Shaw et al., 2014</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Kong et al., 2017</xref>). Activation of the NLRP3 inflammasome promotes the maturation and release of several proinflammatory cytokines, including interleukin-1<italic>β</italic> (IL-1<italic>β</italic>) and IL-18. Extracellular secretion of IL-1<italic>β</italic> and IL-18 requires two distinct signals: the signal driven by pattern recognition receptors, which induces the expression of pro-IL-1<italic>β</italic> and pro-IL-18 mRNAs (signal 1) and activation of the inflammasome (signal 2), which stimulates the cleavage of caspase 1 (<xref rid=\"B5\" ref-type=\"bibr\">Davis and Ting, 2010</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Schroder and Tschopp, 2010</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Lamkanfi and Dixit, 2012</xref>). Many studies have shown that the NLRP3 inflammasome is involved in diseases such as type 2 diabetes, atherosclerosis, colitis, Parkinson’s disease, and multiple sclerosis, as well as psychological disorders (<xref rid=\"B10\" ref-type=\"bibr\">Guarda et al., 2009</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Duewell et al., 2010</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Lamkanfi and Dixit, 2012</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Heneka et al., 2013</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Zhou et al., 2016</xref>).</p><p>Various protein kinases, including PAK1, PKA, PKC, PKD, PKR, BTK, PyK2, IRAK, Syk, and JNK1 have been reported to be required for inflammasome activation (<xref rid=\"B1\" ref-type=\"bibr\">Basak et al., 2005</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Lu et al., 2012</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Ito et al., 2015</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Chung et al., 2016</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Spalinger et al., 2016</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Swanson and Ting, 2016</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Stutz et al., 2017</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Magnotti et al., 2019</xref>). A protein tyrosine phosphatase, non-receptor type 22 (PTPN22), interacts with and dephosphorylates NLRP3 at tyrosine 861, thereby activating the inflammasome (<xref rid=\"B32\" ref-type=\"bibr\">Spalinger et al., 2016</xref>). Phosphatase PP2A dephosphorylates NLRP3 at serine 295 and negatively regulates its activity (<xref rid=\"B34\" ref-type=\"bibr\">Stutz et al., 2017</xref>). Interestingly, phosphorylation of NLRP3 at serine 295 by different upstream kinases differentially regulates NLRP3 inflammasome activation. PKA-induced NLRP3 phosphorylation at serine 295 significantly blocks nigericin-induced inflammasome activation, although PKD-induced serine 295 phosphorylation at the Golgi apparatus is required for inflammasome activation. In addition to NLRP3, phosphorylation of ASC and caspase 1 is also involved in NLRP3 activation. Studies have shown that Syk is involved in the tyrosine phosphorylation of murine ASC, which promotes ASC speck formation and NLRP3/AIM2 activation (<xref rid=\"B4\" ref-type=\"bibr\">Chung et al., 2016</xref>). In addition, Pyk2, BTK, IKK<italic>α</italic>, and IKKi also participate in inflammasome activation by phosphorylating ASC (<xref rid=\"B23\" ref-type=\"bibr\">Martin et al., 2014</xref>). Overall, phosphorylation of NLRP3 by specific kinases diversely regulates inflammasome activation in the presence of different stimuli, which controls the temporal and spatial activation of the NRLP3 inflammasome.</p><p>The hematopoietic cell kinase (HCK), a member of the Src family of non-receptor tyrosine kinases (NRTKs), is primarily expressed in myeloid cells. HCK is highly expressed in macrophages, which is further augmented during macrophage activation. HCK is involved in diverse inflammatory responses (<xref rid=\"B8\" ref-type=\"bibr\">English et al., 1993</xref>). Knocking down of endogenous <italic>Hck</italic> in the murine macrophage cell line BAC1.2F5 interferes with the lipopolysaccharide (LPS)-induced <italic>Tnfa</italic> expression, without altering TNF-α release (<xref rid=\"B29\" ref-type=\"bibr\">Scholz et al., 2000</xref>). Furthermore, A419259, a specific pharmacological inhibitor of HCK, ameliorated bone destruction associated with inflammation (<xref rid=\"B16\" ref-type=\"bibr\">Kim et al., 2020</xref>). Therefore, we speculated that HCK might also be involved in the regulation of microglia (resident macrophages of the central nervous system) function and progression of Alzheimer’s disease-like neuropathology (<xref rid=\"B19\" ref-type=\"bibr\">Lim et al., 2018</xref>). However, the underlying mechanism is not known.</p><p>Hence, in this study, we aimed to investigate the role of HCK in NLRP3 inflammasome activation. HCK physically interacted with NLRP3, leading to the induction of ASC oligomerization and caspase- 1 activation in a kinase activity-dependent manner <italic>in vitro</italic>. We also observed that endogenous inhibition of HCK activity alleviated LPS-induced inflammatory response in the liver. These results might enhance our understanding regarding the mechanism of NLRP3 inflammasome activation and the effectiveness of HCK inhibitors for treating acute inflammatory diseases.</p></sec><sec sec-type=\"materials|methods\" id=\"s2\"><title>Materials and Methods</title><sec id=\"s2_1\"><title>Mice</title><p>C57BL/6J mice were purchased from Charles River Co., Ltd. Male mice were used at about 8 weeks of age. All animal experiments were approved by and conformed to the guidelines of the institutional animal care and use committee of the Beijing Institute of Basic Medical Sciences.</p></sec><sec id=\"s2_2\"><title>Cell Preparation and Culture</title><p>HEK293T cells were maintained in basic high glucose (Gibco) Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (BI) and 1% penicillin/streptomycin (Gibco). Bone marrow-derived macrophages (BMDMs) were prepared as described previously (<xref rid=\"B25\" ref-type=\"bibr\">Oh et al., 2019</xref>) after sacrificing and immersing mice in 75% ethanol for 5 min. The mid-back skin was clipped and removed from the lower part of the body, and the tissue remaining on the pelvic and femoral bones was cleaned. The end of each bone was cut. The bone marrow was expelled from both ends of the bone using a 1 ml syringe filled with bone marrow growth medium. The cells were centrifuged at 350<italic>g</italic> for 5 min, and the red blood cells were lysed in ammonium chloride-potassium (ACK) buffer (0.154 M NH<sub>4</sub>Cl, 10 mM KHCO<sub>3</sub>, 0.1 mM EDTA) for 3 min. Subsequently, the cells were centrifuged at 350<italic>g</italic> for 5 min and resuspended in complete basic high glucose DMEM supplemented with macrophage colony stimulating factor (MCSF) (20 ng/ml) (Peprotech). The medium was changed with fresh MCSF-supplemented medium after every 3 days. The cells were ready for use in a week and were cultured for 3 weeks.</p><p>Murine peritoneal macrophages (PMs) were prepared as below. Briefly, mice were sacrificed and then immersed in 75% ethanol for 5 min. Five milliliters Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco) was injected into the abdominal cavities after clipping the abdominal skin and exposing the abdomens. Next, the mice were shaken mildly to extract the liquid from the abdominal cavities. The collected cells were centrifuged at 350 × <italic>g</italic> for 5 min, and the re-suspended cells were cultured and maintained in complete RPMI-1640 medium (supplemented with heat-inactivated 10% FBS and 1% penicillin/streptomycin).</p><p>Microglia were prepared from the cortex of mice at the P0 stage as described previously (<xref rid=\"B2\" ref-type=\"bibr\">Cheng et al., 2017</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Wu et al., 2019</xref>). The microglia were maintained in basic high glucose DMEM supplemented with heat-inactivated 20% FBS and 1% penicillin/streptomycin (Gibco).</p></sec><sec id=\"s2_3\"><title>Regents and Antibodies</title><p>LPS (L2630) and ATP (A7699) were purchased from Sigma-Aldrich. Nigericin (HY-127019), JANEX-1 (HY-15508), and A419259 (HY-15764) were from MedChemExpress (Monmouth, NJ, USA). Vigofect (T001) was from Vigorous (Beijing, China). The Lipofectamine™ RNAiMAX transfection reagent was from ThermoFisher (Waltham, MA, USA). The anti-rabbit ASC antibody (1:1000; #67824) was from Cell Signaling Technology (Cambridge, MA, USA). The anti-mouse NLRP3 (1:1,000; AG-20B-0014) and anti-mouse caspase 1 (p20) (1:1,000; AG-20B-0042) antibodies were from AdipoGen (San Diego, CA, USA). The anti-mouse IL-1<italic>β</italic> antibody (1:1,000; AF-401-NA) was from R&D Systems (Minneapolis, MN, USA). The anti-rabbit HCK antibody (1:500; A14537) was from Abclonal (Wuhan, China) and anti-rabbit HCK antibody (1:200; 11600-1-AP) was from ProteinTech (Wuhan, China). Anti-mouse <italic>β</italic>-tubulin (1:2,000; CW0098A), anti-mouse GAPDH (1:3000; CW0100M), goat-anti mouse horse radish peroxidase (HRP) IgG(H + L) (1:5,000; CW0110S), and goat anti-rabbit HRP IgG (H + L) (1:5,000; CW0103S) were from CWBiotech (Beijing, China). The anti-mouse FLAG antibody (1:1,000; F1804) was from Sigma-Aldrich and anti-mouse MYC antibody (M047-3) was from MBL (Woburn, MA, USA). Goat anti-mouse IgG HRP (L) and goat anti-mouse HRP(Fc) were from Invitrogen.</p></sec><sec id=\"s2_4\"><title>CellTiter-Glo<sup>®</sup> Luminescent Cell Viability Assay</title><p>PMs were seeded in a 96-well plate (1 × 10<sup>4</sup> cells/well), and A419259 (0.01, 0.1, 0.25, 0.5, and 1 μM) was added to the medium after 12 h. After 4 h, 100 μl of the CellTiter-Glo<sup>®</sup> luminescent (G7570, Promega, USA) solution was added to the 96-well plate and mixed gently on an orbital shaker at room temperature for 10 min. The resulting fluorescence was measured using a multiscan spectrophotometer (TECAN, SPARK 10M).</p></sec><sec id=\"s2_5\"><title>Inflammasome Activation Assays</title><p>BMDMs were seeded at the density of 1–2 × 10<sup>5</sup> cells/well in 96-well plates, iBMDMs at 1 × 10<sup>6</sup> cells/well in 6-well plates, PMs at 1 × 10<sup>6</sup> cells/well in 24-well plates or 5 × 10<sup>6</sup> cells/well 6-well plates, respectively, and THP1 cells at 1 × 10<sup>6</sup> cells/well in 12-well plates. The following day, the medium was replaced, and cells were stimulated with 500 ng/ml LPS for 3 h. The medium was removed and replaced with serum-free medium containing dimethyl sulfoxide (DMSO; 1:1,000) and 1 μM A419259 and incubated for 1 h. The cells were then stimulated with the following inflammasome activators: 2.5 mM ATP (30–45 min) and 5 μM nigericin (30–45 min).</p></sec><sec id=\"s2_6\"><title>Plasmids</title><p>3×FLAG-tagged ASC, 3×FLAG-tagged NLRP3, 3×FLAG-tagged PYD, 3×FLAG-tagged NBD, 3×FLAG-tagged LRRs, and MYC-tagged HCK were subcloned into the pCMV10 expression vector.</p></sec><sec id=\"s2_7\"><title>SiRNA-Mediated Gene Silencing</title><p>BMDMs were planted in 96-well plates (1 × 10<sup>5</sup> cells/ml) and THP1 cells were planted in 12-well plates (5 × 10<sup>5</sup> cells/ml). The overnight medium was replaced after 12 h, and 40 nM siRNA was added to each well along with the Lipofectamine™ RNAiMAX transfection reagent according to the manufacturer’s guidelines. All the siRNAs were from GenePharma (Shanghai, China). The sequences used for HCK knockdown are shown in <xref rid=\"T1\" ref-type=\"table\">\n<bold>Table 1</bold>\n</xref>.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>SiRNA target sequences.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Name</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Target Sequence (5′–3′)</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>siHck1</italic>\n<break/>\n<italic>siHck2</italic>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GGAGCCCAGCAUGUUGC<break/>CAAAGGUGGGCCGUUCC</td></tr></tbody></table></table-wrap></sec><sec id=\"s2_8\"><title>Transfection and Immunoprecipitation</title><p>The HEK293T cells were transfected with the plasmids expressing FLAG-NLRP3, FLAG-ASC, or MYC-HCK by using Vigofect. After 36 h, the cells were collected and resuspended in lysis buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet-P40, 5% glycerol, protease inhibitor cocktail, and phosphatase inhibitor) at 4°C for 30 min. Then, the cell lysates were centrifuged at 20,000g at 4°C for 5 min. The supernatants were incubated with Pierce™ protein A/G magnetic beads (ThemoFisher) (incubated with 1 µg anti-mouse FLAG antibody or anti-mouse MYC antibody for 1h at room temperature) overnight at 4°C. The supernatants were discarded and the magnetic beads were washed thrice with lysis buffer. Then, each sample was dissolved in 60 μl 1× sodium dodecyl sulfate (SDS) loading buffer and heated in a 100°C dry heater for 10 min.</p><p>LPS-primed iBMDMs (5 × 10<sup>6</sup> cells/well) (treated or not treated with 1 μM A419259 and stimulated with 5 μM nigericin for 1 h) were lysed with lysis buffer at 4°C for 30 min in 6 cm plate. Then, the cell lysates were centrifuged at 20,000g at 4°C for 5 min. The supernatants were incubated with Pierce™ protein A/G magnetic each (ThemoFisher) (incubated with 1 μg anti-HCK or 1 μg anti-ASC antibody for 1 h at room temperature) overnight at 4°C. The supernatants were discarded and the magnetic beads were washed thrice with lysis buffer. Then, the samples were dissolved in 60 μl 1× SDS loading buffer.</p></sec><sec id=\"s2_9\"><title>Cross-Linking of the ASC Oligomer</title><p>LPS-primed PMs (5 × 10<sup>6</sup> cells/well) were treated with 1 μM A419259 and stimulated with 5 μM nigericin for 30 min in 6-well plate. The cells were lysed with 0.5 ml lysis buffer (50 mM Tris-HCl pH 7.6, 0.5% Triton X-100) for 20 min and centrifuged at 6,000g for 15 min. The pellets were washed thrice with Tris-buffered saline (TBS) and resuspended in TBS containing 2 mM disuccinimidyl suberate (DDS) for cross-linking at 37°C for 45 min. Next, the pellets were centrifuged at 6,000 × <italic>g</italic> for 15 min and dissolved in 50 μl 1× SDS loading buffer.</p></sec><sec id=\"s2_10\"><title>Immunoblotting</title><p>Cells were lysed using the cell lysis buffer used for immunoprecipitation and then boiled in × SDS loading buffer. The cell culture supernatants were concentrated using methanol/chloroform and the pellets were lysed in the SDS sample buffer. Equal amounts of total protein were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) analysis, and immunoblotting with the appropriate antibodies was performed as described previously. For immunoblotting, the nitrocellulose blot with the transferred proteins was incubated with HRP-conjugated antibody in 5% skim milk-TBS-Tween 20 (0.1%) for 1 h with shaking. After three washes, the antigen–antibody complexes were visualized using enhanced chemiluminescence (ECL) reagents.</p></sec><sec id=\"s2_11\"><title>Enzyme-Linked Immunosorbent Assay (ELISA)</title><p>Supernatants from cell culture or serum were assayed for mouse TNF-α (AF-410-NA, R&D Systems) and IL-1<italic>β</italic> (MLB00C, R&D Systems) levels according to the manufacturer’s instructions.</p></sec><sec id=\"s2_12\"><title>Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)</title><p>Total RNA was extracted using the Trizol reagent (Invitrogen). cDNA was obtained using <italic>TransScript</italic>\n<sup>®</sup>II One-Step gDNA removal and cDNA synthesis super mix (TransGen Biotech, Beijing, China), following the manufacturer’s instructions. QRT-PCR analysis was performed in a LightCycler 480 (Roche) using specific primers, 40 ng cDNA, and the Ultra SYBR master mix (HighROX) (Cwbiotech). Relative mRNA expression was obtained using the ΔΔCt method. <italic>Actb</italic> or <italic>Gapdh</italic> was used as reference genes. The primers used for murine genes are shown in <xref rid=\"T2\" ref-type=\"table\">\n<bold>Table 2</bold>\n</xref>.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Primers for RT-qPCR.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Name</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Sequence (5′–3′)</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Nlrp3-</italic>forward<break/>\n<italic>Nlrp3-</italic>reverse<break/>\n<italic>Caspase1</italic>-forward<break/>\n<italic>Caspase1</italic>-reverse<break/>\n<italic>Il1b-</italic>forward<break/>\n<italic>Il1b-</italic>reverse<break/>\n<italic>Il-18</italic>-forward<break/>\n<italic>Il-18</italic>-reverse<break/>\n<italic>Asc</italic>-forward<break/>\n<italic>Asc</italic>-reverse<break/>\n<italic>Tnf-a-</italic>forward<break/>\n<italic>Tnf-a-</italic>reverse<break/>\n<italic>Il6-</italic>forward<break/>\n<italic>Il6-</italic>reverse<break/>\n<italic>Il10-</italic>forward<break/>\n<italic>Il10-</italic>reverse<break/>\n<italic>Il4-</italic>forward<break/>\n<italic>Il4-</italic>reverse<break/>\n<italic>Hck-</italic>forward<break/>\n<italic>Hck-reverse</italic>\n<break/>\n<italic>Actb-</italic>forward<break/>\n<italic>Actb-</italic>reverse<break/>\n<italic>Gapdh-</italic>forward<break/>\n<italic>Gapdh-</italic>reverse</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ATTACCCGCCCGAGAAAGG<break/>TCGCAGCAAAGATCCACACAG<break/>ACAAGGCACGGGACCTATG<break/>TCCCAGTCAGTCCTGGAAATG<break/>GCAACT GTTCCTGAACTCAACT<break/>ATCTTTTGGGGTCCGTCAACT<break/>GACTCTTGCGTCAACTTCAAGG<break/>CAGGCTGTCTTTTGTCAACGA<break/>CTTGTCAGGGGATGAACTCAAAA<break/>GCCATACGACTCCAGATAGTAGC<break/>CCCTCACACTCAGATCATCTTCT<break/>GCTACGACGTGGGCTACAG<break/>CCAAGAGGTGAGTGCCTTCCC<break/>CTGTTGTTCAGACTCTCTCCCT<break/>GCTCTTACTGACTGGCATGAG<break/>CGCAGCTCTAGGA GCATGTG<break/>GGTCTCAACCCCCAGCTAGT<break/>GCCGATGATCTCTCTCAAGTGAT<break/>TCCTCCGAGATGGAAGCAAG<break/>ACAGTGCGACCACAATGGTAT<break/>GGCTGTATTCCCCTCCATC<break/>CCAGTTGGTAACAATGCCATGT<break/>AGGTCGGTGTGAACGGATTTG<break/>TGTAGACCATGTAGTTGAGGTCA</td></tr></tbody></table></table-wrap></sec><sec id=\"s2_13\"><title>Immunofluorescence</title><p>Murine PMs (5 × 10<sup>5</sup> cells/well) were seeded on coverslips coated with 1× poly-L-lysine-ornithine in 24-well plate. LPS-primed murine PMs were treated with 1 μM A419259 for 1 h, stimulated with 1 mM nigericin for 30 min, and then fixed and permeabilized using 4% formaldehyde and 0.5% Triton X-100. The cells were incubated overnight with anti-rabbit ASC antibody (1:500; CST). Next, the cells were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG antibody (1:400). Nuclei were counterstained with DAPI. The cells were examined under a laser scanning confocal microscope (Nikon A1).</p></sec><sec id=\"s2_14\"><title>\n<italic>In Vivo</italic> LPS Challenge</title><p>C57BL/6 mice were injected intraperitoneally (i.p.) with 30 mg/kg A419259 or the vehicle control (DMSO/PBS) 24 h 12 h before i.p. injection of 5 mg/kg LPS or PBS. Mice were sacrificed after 3 h, and the tissues were stored at −80°C and prepared for immunoblotting.</p></sec><sec id=\"s2_15\"><title>Statistical Analysis</title><p>All values were expressed as the mean ± SEM. All statistical analyses were performed using GraphPad Prism version 6.0 software. The significance of differences was assessed by unpaired Student’s t test or one-way or two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test as indicated. P value < 0.05 was considered significant. Error bars represent SEM.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec id=\"s3_1\"><title>HCK Is an Upstream Regulator of the NLRP3 Inflammasome</title><p>Studies have shown that certain NRTKs are involved in the activation of the NLRP3 inflammasome. Hence, we constructed a NRTK siRNA library and designed a screening process (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1A</bold>\n</xref>). We transfected macrophages with siRNAs, and the supernatants were collected 72 h after LPS and nigericin (a NLRP3 activator) stimulation; the levels of secreted IL-1<italic>β</italic> and TNF-α were detected using ELISA. The results showed that the knockdown of <italic>Hck</italic> and Janus protein tyrosine kinase 1 (<italic>Jak1</italic>) and <italic>Jak3</italic> significantly reduced the levels of IL-1<italic>β</italic> in the supernatant (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figures 1B, C</bold>\n</xref>). However, production of TNF-α, an inflammasome-independent cytokine, was not affected, suggesting that knocking down of <italic>Hck</italic>, <italic>Jak1</italic>, and <italic>Jak3</italic> inhibited the release of IL-1<italic>β via</italic> inflammasome activation.</p><fig id=\"f1\" position=\"float\"><label>Figure 1</label><caption><p>Screening of HCK as a regulator for the NLRP3 inflammasome. <bold>(A)</bold> Flowchart showing the candidate siRNA library targeting 31 putative tyrosine kinases in BMDM. IL-1<italic>β</italic> and TNF-α levels were determined. <bold>(B, C)</bold> Enzyme-linked immunosorbent assay (ELISA) for IL-1<italic>β</italic>\n<bold>(B)</bold> or TNF-α <bold>(C)</bold> in supernatants from LPS-primed (500 ng, 3 h) PMs transfected with control siRNA (scrambled) or siRNAs specific for the individual tyrosine kinases post-nigericin (5 μM) stimulation for 30 min.</p></caption><graphic xlink:href=\"fphar-11-581011-g001\"/></fig></sec><sec id=\"s3_2\"><title>The HCK Inhibitor Attenuated NLRP3 Inflammasome-Induced Inflammatory Response in Macrophages and Microglia</title><p>Wang et al. has reported that <italic>Jak1</italic> knockdown blocked the phosphorylation of NF-<italic>κ</italic>B p65 in the nuclei and that of NF-<italic>κ</italic>B I<italic>κ</italic>B<italic>α</italic> in the cytoplasm and attenuated NLRP3 inflammasome activation (<xref rid=\"B37\" ref-type=\"bibr\">Wang et al., 2017</xref>). A previous study has shown that HCK, NF-<italic>κ</italic>B signaling, and JAK are triggered by the <italic>Myd88</italic> mutant (kinase activation mutant) in B cells (<xref rid=\"B24\" ref-type=\"bibr\">Ngo et al., 2011</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Yang et al., 2016</xref>), and MYD88 acts upstream of NLRP3. Hence, to further confirm these results, we used JAK3 inhibitor (JANEX-1) and HCK inhibitor (A419259, the Src family kinases specific inhibitor) to verify whether HCK and JAK3 were involved in the regulation of NLRP3 inflammasome activation. Interestingly, we observed that HCK inhibition, but not JAK3 inhibition, significantly reduced LPS/nigericin-mediated upregulation of IL-1<italic>β</italic> secretion in the cell culture supernatants, and A419259 did not affect the cell viability (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figures 2A–C</bold>\n</xref>). Intriguingly, A419259 did not inhibit the LPS-induced IL-1<italic>β</italic> and NLRP3 expression (<xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Supplementary Figure 1A</bold>\n</xref>). In addition to nigericin, the NLRP3 inflammasome can be also activated by ATP, silica, monosodium urate, and calcium (<xref rid=\"B6\" ref-type=\"bibr\">Dostert et al., 2008</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Franchi et al., 2009</xref>; <xref rid=\"B12\" ref-type=\"bibr\">He et al., 2012</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Rossol et al., 2012</xref>; <xref rid=\"B13\" ref-type=\"bibr\">He et al., 2016</xref>). Hence, we selected ATP as another secondary signaling stimulation to confirm the results obtained using nigericin. As expected, ATP treatment of LPS-primed macrophages increased intracellular cleaved caspase 1 expression and the level of secreted IL-1<italic>β</italic> in the culture medium, both of which were considerably attenuated by the HCK inhibitor, A419259 (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2D</bold>\n</xref>). The protein levels of NLRP3, pro-caspase 1, and pro-IL-1<italic>β</italic> were not significantly affected (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2D</bold>\n</xref>).</p><fig id=\"f2\" position=\"float\"><label>Figure 2</label><caption><p>HCK inhibition attenuated NLRP3 inflammasome-induced inflammatory response in macrophages and microglia. <bold>(A)</bold> PMs were seeded in a 96-well plate (1 × 10<sup>4</sup> cells/well) plate. After 12 h, the cells were treated with A419259 for 4 h, and cell viability was detected. <bold>(B)</bold> ELISA for cleaved IL-1<italic>β</italic> (P17) in the supernatants of LPS-primed (500 ng, 2 h) PMs treated with JAK3(JANEX-1) or HCK(A419259) inhibitor for 1 h and stimulated with nigericin for 30 min. <bold>(C)</bold> Immunoblotting analysis of cleaved IL-1<italic>β</italic> (P17) in the supernatants from LPS-primed (500 ng, 2 h) PMs treated with JAK3(JANEX-1) or HCK(A419259) inhibitor for 1 h and stimulated with nigericin for 30 min, followed by immunoblotting for pro-IL-1<italic>β</italic> or NLRP3 antibody. <bold>(D)</bold> Immunoblotting analysis of cleaved IL-1<italic>β</italic> (P17) and cleaved caspase 1 (P20) in the supernatants of LPS-primed (500 ng, 2 h) PMs treated for 1 h with the HCK inhibitor (A419259) and then stimulated with 1.5 mM ATP for 30 min, followed by immunoblotting analysis with pro-IL-1<italic>β</italic>, pro-caspase 1, NLRP3, or <italic>β</italic>-tubulin antibodies in whole cell lysates. <bold>(E, F)</bold> Immunoblotting analysis of cleaved IL-1<italic>β</italic> or cleaved caspase 1 in the supernatants of LPS-primed (500 ng, 2 h) microglia treated for 1 h with HCK inhibitors (A419259) and then stimulated with 5 μM nigericin <bold>(E)</bold> or 1.5 mM ATP <bold>(F)</bold> for 30 min. The cell lysates were immunoblotted with antibodies against pro-IL-1<italic>β</italic>, pro-caspase 1, NLRP3, or <italic>β</italic>- tubulin in cell lysates. Data from <bold>(A</bold>–<bold>F)</bold> are representative of at least three independent experiments. Data show means ± SEM. ***<italic>P</italic> < 0.001, ns, <italic>P</italic> > 0.05, Student’s <italic>t</italic>-test.</p></caption><graphic xlink:href=\"fphar-11-581011-g002\"/></fig><p>Furthermore, deficiency of HCK in PMs could suppress the IL-1<italic>β</italic> secretion and caspase1 activation without affecting the expression levels of NLRP3, pro-IL-1<italic>β</italic> and caspase1 (<xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Supplementary Figures 2A, B</bold>\n</xref>). We also transfected the human monocyte cell line, THP1, with <italic>Hck</italic>-specific siRNA or mock control and observed that <italic>Hck</italic> knockdown significantly decreased IL-1<italic>β</italic> secretion in the LPS-primed THP-1 cells treated with nigericin without altering the expression levels of NLRP3 and pro-IL-1<italic>β</italic> (<xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Supplementary Figure 3A</bold>\n</xref>). The HCK inhibitor also attenuated the secretion of IL-1<italic>β</italic> in the culture medium of nigericin-stimulated LPS-primed THP1 (<xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Supplementary Figure 3B</bold>\n</xref>).</p><p>The microglia perform functions similar to those of macrophages in the central nervous system. Hence, we speculated that A419259 might also inhibit NLRP3 inflammasome activation in the microglia. We used nigericin and ATP as secondary signaling stimuli to activate the NLRP3 inflammasome. The results showed that secretion of cleaved IL-1<italic>β</italic> and cleaved caspase 1 from the A419259-treated group was lower than that from the group treated with LPS and nigericin or ATP (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figures 2E, F</bold>\n</xref>\n<bold>)</bold>.</p><p>Taken together, these results demonstrated that HCK was involved in NLRP3 inflammasome activation and subsequent IL-1<italic>β</italic> production, indicating that NLRP3 inflammasome activation depended on HCK kinase activity.</p></sec><sec id=\"s3_3\"><title>HCK Inhibition Reduced Inflammasome Assembly</title><p>Oligomerization of ASC is an initiating event of NLRP3 inflammasome activation and is responsible for the recruitment and activation of caspase 1. We speculated that ASC oligomerization might be affected by HCK. Hence, we analyzed ASC oligomerization <italic>via</italic> ASC specks staining and DSS-induced ASC cross-linking assay. The results showed that compared to the siCtrl cells, the number of ASC specks was decreased after inflammasome activation in PMs transfected with siHCK (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figures 3A, B</bold>\n</xref>). Furthermore, DSS-induced ASC oligomerization also showed decrease in HCK deficiency PMs (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3C</bold>\n</xref>). In Addition, A419259 could suppress the ASC specks and DSS-induced ASC oligomerization formation as well (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figures 3D–F</bold>\n</xref>). These results indicated that deficiency of HCK and HCK kinase activity inhibition could reduce ASC oligomerization and inflammasome assembly. To further decipher the mechanism underlying HCK-mediated regulation of ASC oligomerization, we analyzed the interaction between HCK and ASC. We observed lack of direct interaction between ASC and HCK using reciprocal immunoprecipitation (<xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Supplementary Figures 4A–C</bold>\n</xref>). However, HCK inhibition markedly mitigated the interaction between ASC and NLRP3, indicating that HCK might directly affect NLRP3 function during inflammasome activation (<xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Supplementary Figures 4C, D</bold>\n</xref>).</p><fig id=\"f3\" position=\"float\"><label>Figure 3</label><caption><p>HCK promotes the oligomerization of ASC. <bold>(A, B)</bold> Immunostaining of ASC in the LPS-primed PMs transfected with control siRNA (Mock) or siHCK for 3 h, followed by stimulation with nigericin for 30 min. <bold>(C)</bold> Immunoblotting analysis of the purified cross-linked ASC oligomers from the whole cell lysates in the LPS-primed (500 ng, 3 h) PMs transfected with control siRNA (Mock) or siHCK for 72 h, followed by stimulation with 5 μM nigericin for another 30 min <bold>(D, E)</bold> Immunostaining of ASC in the LPS-primed PMs after 1 h-incubation with A419259, followed by stimulation with Nigericin for 30 min. Nuclei were counterstained with DAPI. Fluorescence was imaged using confocal microscopy. <bold>(F)</bold> Immunoblotting analysis of the purified cross-linked ASC oligomers from the whole cell lysates in the LPS-primed (500 ng, 2 h) PMs treated with HCK inhibitor (A419259) for 1 h, followed by stimulation with 5 μM nigericin for another 30 min. Scale bar is 50 μm in all figures. Arrowheads denote ASC specks, and the percentage of ASC speck is shown in <bold>(B</bold>, <bold>E)</bold>. Data from <bold>(A</bold>–<bold>F)</bold> are representative of at least three independent experiments. Data show means ± SEM, ***<italic>P</italic> < 0.001, Student’s <italic>t</italic>-test.</p></caption><graphic xlink:href=\"fphar-11-581011-g003\"/></fig></sec><sec id=\"s3_4\"><title>HCK Interacted With NLRP3 Independent of PYD</title><p>Next, we aimed to investigate the mechanism underlying HCK-mediated regulation of NLRP3 inflammasome activation. The MYC-HCK, FLAG-NLRP3, and FLAG-ASC plasmids were transfected into HEK293T cells. The results showed that full length HCK bound to full length NLRP3 (<xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4A</bold>\n</xref>). NLRP3 consists of three domains, namely, the PYD domain at the N-terminus, NBD (NACHT) domain in the middle, and LRRs at the C-terminus. To further characterize the domain of NLRP3 that interacts with HCK, we constructed plasmids harboring truncated NLRP3, including FLAG-PYD, FLAG-NBD, and FLAG-LRRs. Results of co-immunoprecipitation and immunoblotting experiments showed that HCK interacted with NBD and LRRs, but not PYD (<xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4B</bold>\n</xref>). Furthermore, endogenous HCK interacted with NLRP3 in HEK293T cells. Interestingly, A419259 reduced the interaction between HCK and NLRP3 in iBMDMs (<xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4C</bold>\n</xref>). We also observed that HCK phosphorylation increased when the NLRP3 inflammasome was activated by nigericin (<xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4D</bold>\n</xref>). Overall, these results suggested that HCK could bind to NLRP3 <italic>via</italic> NBD and LRR domains.</p><fig id=\"f4\" position=\"float\"><label>Figure 4</label><caption><p>HCK interacts with NLRP3 independent of its PYD domain. <bold>(A)</bold> HEK293T cells were transfected with MYC-HCK (WT) and FLAG-NLRP3, and the interaction between HCK and NLRP3 was analyzed using immunoprecipitation (IP) with anti-FLAG antibody and immunoblotting (IB) with the anti-FLAG and anti-MYC antibodies. <bold>(B)</bold> HEK293T cells were transfected with MYC-HCK (WT) and FLAG-NLRP3 or its truncated forms (FLAG-PYD, FLAG-NBD, or FLAG-LRRs, followed by immunoprecipitation (IP) with anti-FLAG and immunoblotting (IB) with anti-FLAG or anti-MYC antibodies. <bold>(C)</bold> LPS-primed PMs were treated with A419259 and then stimulated with 5 μM nigericin for 30 min, followed by immunoprecipitation (IP) with HCK antibody and immunoblotting (IB) with antibodies against HCK, phosphorylated tyrosine, NLRP3, ASC or <italic>β</italic>-tubulin. <bold>(D)</bold> The normalized levels of HCK phosphorylation were quantified. Data from <bold>(A</bold>–<bold>C)</bold> are representative of at least three independent experiments. Data show means ± SEM, *<italic>P</italic> > 0.05, **<italic>P</italic> < 0.01, Student’s <italic>t</italic>-test.</p></caption><graphic xlink:href=\"fphar-11-581011-g004\"/></fig></sec><sec id=\"s3_5\"><title>A419259 Reduced Inflammasome Activation <italic>In Vivo</italic> in Mice</title><p>HCK activation led to NLRP3 inflammasome complex assembly and activation. HCK was phosphorylated, which then bound to NLRP3, when the macrophages were stimulated with LPS (<xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Supplementary Figure 5</bold>\n</xref>). In the presence of the secondary signal, HCK promoted NLRP3 inflammasome complex assembly and activation, and consequently, pro-IL-1<italic>β</italic> was cleaved by caspase 1 (P20) and secreted. To assess whether the effects of HCK on inflammasome activation <italic>in vitro</italic> could be translated <italic>in vivo</italic>, LPS-induced mouse models of inflammasome activation were used (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5A</bold>\n</xref>). We evaluated serum IL-1<italic>β</italic> production in mice after i.p. injection of LPS and observed that LPS injection markedly increased serum IL-1<italic>β</italic> level, which was abolished by A419259 pre-treatment (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5B</bold>\n</xref>). Consistent with our previous results, mice treated with A419259 showed reduced response to LPS, with lower amounts of secreted IL-1<italic>β</italic> than in vehicle-treated mice. We analyzed the liver damage <italic>via</italic> HE staining. A419259 could markedly decrease the inflammatory cells’ infiltration in the liver (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5C</bold>\n</xref>). In addition, the protein levels of NLRP3 and caspase 1 (P20) in the liver decreased when the mice were treated with A419259 (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figures 5D, E</bold>\n</xref>). LPS treatment induced strong inflammatory response, as was evident from the increase in the levels of <italic>Nlrp3</italic>, <italic>Il-1β, Il-6</italic>, and <italic>Il-10</italic> mRNAs in the liver, which were mitigated by A419259 treatment (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5F</bold>\n</xref>). However, <italic>Caspas1, Asc, Tnf-a</italic>, and <italic>Il-4</italic> expression did not differ between vehicle-treated and LPS-treated mice (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5F</bold>\n</xref>). Collectively, these results confirmed the involvement of HCK during inflammasome activation <italic>in vivo</italic>, and showed that the HCK kinase inhibitor, A419259, attenuated LPS-induced inflammatory response in the liver.</p><fig id=\"f5\" position=\"float\"><label>Figure 5</label><caption><p>The HCK inhibitor A419259 attenuated LPS-induced inflammatory response <italic>in vivo.</italic>\n<bold>(A)</bold> Schematic graph illustrating the experimental timelines. <bold>(B)</bold> ELISA of the serum cleaved IL-1<italic>β</italic> (P17) from mice intraperitoneally injected with LPS (5 mg/kg of body weight) with or without A419259 (30 mg/kg of body weight) (n = 7 for each group). <bold>(C)</bold> Representative images of liver H&E staining (three mice, nine slices each group). <bold>(D)</bold> Immunoblotting analysis of cleaved caspase 1 (P20), NLRP3, or GAPDH in the livers of mice intraperitoneally injected with LPS with or without A419259 (n = 7 for each group). <bold>(E)</bold> Analysis of the changes in NLRP3 or cleaved caspase 1 (P20) protein level. <bold>(F)</bold> qRT-PCR analysis of <italic>nlrp3, Caspase1, Il-1β, Il-18, Asc, Tnf-α, Il-6, Il-10</italic>, and <italic>Il-4</italic> in the livers of mice intraperitoneally injected with LPS with or without A419259 (n = 7 for each group). Data show means ± SEM, *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01, ***<italic>P <</italic> 0.001. ns, <italic>P</italic> > 0.05, Student’s <italic>t</italic>-test.</p></caption><graphic xlink:href=\"fphar-11-581011-g005\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>NRTKs have been shown to play important roles in NLRP3 inflammasome activation. However, the functions and regulatory mechanisms of NRTKs during NLRP3 inflammasome activation are still not clear. Ito et al. showed that the tyrosine kinase BTK promoted ASC oligomerization by interacting with ASC and NLRP3 and activated the NLRP3 inflammasome (<xref rid=\"B15\" ref-type=\"bibr\">Ito et al., 2015</xref>). However, Mao et al. revealed that BTK deficiency augmented NLRP3 inflammasome activation by inhibiting the PP2A-mediated dephosphorylation of serine 5 in the pyrin domain of NLRP3 (<xref rid=\"B22\" ref-type=\"bibr\">Mao et al., 2020</xref>). Hence, regulation of NLRP3 phosphorylation requires further investigations. In this study, we identified a new tyrosine kinase, HCK, which activated the NLRP3 inflammasome <italic>via</italic> interaction with NLRP3 and promotion of ASC oligomerization.</p><p>Post-translational modifications of the NLRP3 complex include phosphorylation, acetylation, ubiquitination, oligomerization, and proteolytic cleavage, among which phosphorylation has been studied extensively. Phosphorylation mediated by diverse protein kinases has been reported to be involved in NLRP3 inflammasome activation. The protein tyrosine phosphatase, PTPN22, dephosphorylates NLRP3 <italic>via</italic> direct interaction with NLRP3 Tyr861 and induces NLRP3 inflammasome activation (<xref rid=\"B32\" ref-type=\"bibr\">Spalinger et al., 2016</xref>). The phosphatase PP2A is involved in the dephosphorylation of Ser5 and negatively regulates NLRP3 inflammasome activity (<xref rid=\"B23\" ref-type=\"bibr\">Martin et al., 2014</xref>). Serine 295 phosphorylation of NLRP3 performs dual functions with respect to NLRP3 inflammasome activation. PKA-induced Ser295 phosphorylation in human significantly blocks nigericin-induced inflammasome activation; however, PKD-induced Ser295 phosphorylation at the Golgi apparatus is required for inflammasome activation (<xref rid=\"B11\" ref-type=\"bibr\">Guo et al., 2016</xref>). Hara and Tsuchiya showed that signaling pathway-dependent kinases, Syk and JNK, activate the NLRP3 inflammasome by activating caspase 1 [13].</p><p>HCK of the NRTK family is localized in the cytoplasm and executes its functions as a kinase. It is generally activated <italic>via</italic> binding with target proteins, which it subsequently phosphorylates. We speculate that HCK might directly phosphorylate NLRP3. However, we failed to detect the HCK-induced NLRP3 phosphorylation change by Phos-Tag analysis and phosphorylation mass-spectrometry (data not shown). Hara et al. have recently reported that tyrosine phosphorylation of human ASC-Y144 (corresponding to Y146 in mouse ASC) is required for inflammasome activation, and that phosphorylated ASC can be detected in the Triton X-insoluble fraction (<xref rid=\"B4\" ref-type=\"bibr\">Chung et al., 2016</xref>). We also found that HCK could not lead to tyrosine phosphorylation of ASC by using Phos-Tag analysis (data not shown). In this study, we observed that HCK regulated the activation of the NLRP3 inflammasome by interacting with NLRP3. We could not detect any interaction between HCK and ASC; however, the oligomerization of ASC in PMs decreased when the kinase activity of HCK was inhibited. HCK did not interact with ASC when the inflammasome was not activated. Therefore, we believe that HCK might indirectly promote ASC oligomerization. How HCK regulates NLRP3 activation post-binding warrants and the oligomerization of ASC need to further investigate.</p><p>Recent studies have shown that the activation of the NLRP3 inflammasome is involved in the progression of various inflammatory diseases, such as type 2 diabetes, atherosclerosis, and Muckle-Wells syndrome (<xref rid=\"B7\" ref-type=\"bibr\">Duewell et al., 2010</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Lamkanfi and Dixit, 2012</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Kong et al., 2017</xref>). Furthermore, the inflammasome is also reported to be involved in neurodegenerative diseases such as multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease. Various studies have demonstrated that the anti-IL-1<italic>β</italic> antibody is effective in treating some of these disorders. HCK plays important roles in inflammatory response and is activated in tumor-associated immune cells. Genetic ablation of <italic>Hck</italic> in mice reduced cytokine expression in response to LPS stimulation and moderately increased the susceptibility of <italic>Hck</italic> knockout mice to infection (<xref rid=\"B27\" ref-type=\"bibr\">Poh et al., 2015</xref>). In this study, we observed that the HCK kinase activity inhibitor, A419259, restrained NLRP3 inflammasome activity in macrophages and microglia and attenuated the LPS-induced inflammatory response <italic>in vivo</italic>. Thus, A419259 might be a candidate drug for treating NLRP3 inflammasome activation-related diseases. Further investigations into the mechanism of HCK-mediated NLRP3 inflammasome activation are required to show HCK inhibition as a new strategy for the treatment of inflammatory disease.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by Beijing Institute of Basic Medical Science.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>XK, ZY, JC, and SW conceived and designed the project and drafted the manuscript. XK, YL, and LZ performed the experiments. All authors contributed to the article and approved the submitted version.</p></sec><sec sec-type=\"funding-information\" id=\"s8\"><title>Funding</title><p>This work was supported by grants from the National Nature Science Foundation of China (81671274 and 31600946 to SW; No. 81930029 to ZY; No.81400987 to JC; No. 81701187 to YL).</p></sec><sec id=\"s9\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><title>Acknowledgments</title><p>We thank Dr. Zhengfan Jiang (Peking university, Beijing, China) for gifting iBMDM.</p></ack><sec id=\"s10\" sec-type=\"supplementary-material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fphar.2020.581011/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fphar.2020.581011/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"DataSheet_1.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name><surname>Basak</surname><given-names>C.</given-names></name><name><surname>Pathak</surname><given-names>S. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33042005</article-id><article-id pub-id-type=\"pmc\">PMC7523511</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00609</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Severe Hypoglycemia: Is It Still a Threat for Children and Adolescents With Type 1 Diabetes?</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Urakami</surname><given-names>Tatsuhiko</given-names></name><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/675295/overview\"/></contrib></contrib-group><aff><institution>Department of Pediatrics, Nihon University School of Medicine</institution>, <addr-line>Tokyo</addr-line>, <country>Japan</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Valentino Cherubini, Azienda Ospedaliero Universitaria Ospedali Riuniti, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Stefano Zucchini, Sant'Orsola-Malpighi Polyclinic, Italy; Maurizio Delvecchio, Giovanni XXIII Children's Hospital, Italy</p></fn><corresp id=\"c001\">*Correspondence: Tatsuhiko Urakami <email>urakami.tatsuhiko@nihon-u.ac.jp</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Pediatric Endocrinology, a section of the journal Frontiers in Endocrinology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>609</elocation-id><history><date date-type=\"received\"><day>31</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Urakami.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Urakami</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Severe hypoglycemia is defined as a condition with serious cognitive dysfunction, such as a convulsion and coma, requiring external help from other persons. This condition is still lethal and is reported to be the cause of death in 4–10% in children and adolescents with type 1 diabetes. The incidence of severe hypoglycemia in the pediatric population was previously reported as high as more than 50–100 patient-years; however, there was a decline in the frequency of severe hypoglycemia during the past decades, and relationship with glycemic control became weaker than previously reported. A lot of studies have shown the neurological sequelae with severe hypoglycemia as cognitive dysfunction and abnormalities in brain structure. This serious condition also provides negative psychosocial outcomes and undesirable compensatory behaviors. Various possible factors, such as younger age, recurrent hypoglycemia, nocturnal hypoglycemia, and impaired awareness of hypoglycemia, are possible risk factors for developing severe hypoglycemia. A low HbA<sub>1c</sub> level is not a predictable value for severe hypoglycemia. Prevention of severe hypoglycemia remains one of the most critical issues in the management of pediatric patients with type 1 diabetes. Advanced technologies, such as continuous glucose monitoring (CGM), intermittently scanned CGM, and sensor-augmented pump therapy with low-glucose suspend system, potentially minimize the occurrence of severe hypoglycemia without worsening overall glycemic control. Hybrid closed-loop system must be the most promising tool for achieving optimal glycemic control with preventing the occurrence of severe hypoglycemia in pediatric patients with type 1 diabetes.</p></abstract><kwd-group><kwd>severe hypoglycemia</kwd><kwd>children and adolescents</kwd><kwd>type 1 diabetes</kwd><kwd>risk factor</kwd><kwd>advanced technology</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"129\"/><page-count count=\"11\"/><word-count count=\"8917\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Hypoglycemia is a commonly observed acute complication in the management of type 1 diabetes. It is a major barrier to achieve optimal glycemic control (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>) and may affect quality of life in the patients (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). Minimizing hypoglycemia is an important objective in the management of type 1 diabetes, and this can be attained by evaluating the risk factors and preventing them, although intensive glycemic management (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>).</p><p>Severe hypoglycemia is defined as a condition with serious cognitive dysfunction, such as a convulsion and coma, requiring external help from other persons to provide glucose and glucagon or take other correction assistance. Severe hypoglycemic coma is defined as the subgroup of severe hypoglycemia related to a convulsion or unconsciousness (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Severe hypoglycemia is still lethal and is reported to be the cause of death in 4% to 10% (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>–<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). It may be associated with permanent brain damage and is related to cognitive dysfunction and abnormalities in brain structure particularly in young children with type 1 diabetes (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>–<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). The International Society for Pediatric and Adolescent Diabetes Clinical Practice Consensus Guidelines 2018 has recommended that a target of HbA<sub>1c</sub> should be <7.0% (53 mmol/mol) in patients who can access contemporary technologies of insulin treatment and the potency to regular self-checking blood glucose and/or the use of continuous glucose monitoring (CGM) (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Whereas, careful attention must be poured to avoid severe hypoglycemia, glucose targets must be increased in patients with the risk factors for severe hypoglycemia (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B17\" ref-type=\"bibr\">17</xref>).</p><p>The main purpose of this review is to evaluate possible risk factors and the neurological sequelae in severe hypoglycemia and to introduce the advanced technologies to minimize the occurrence of severe hypoglycemia in children and adolescents with type 1 diabetes.</p></sec><sec id=\"s2\"><title>Research Methods</title><p>Briefly, we performed the literature research by MEDLINE and EMBASE covering the period between 1980 and 2019. The search terms were “type 1 diabetes,” “children,” “adolescents,” and “hypoglycemia” or “severe hypoglycemia.” Language restriction was applied in English.</p></sec><sec id=\"s3\"><title>Incidence</title><p>High incidence of severe hypoglycemia was shown by the Diabetes Control and Complications Trial (DCCT) in 1997 (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>); i.e., the incidence of hypoglycemia that needed treatment assistance was 61.2 per 100 patient-years in patients receiving intensive treatment and 18.7 per 100 patient-years in those receiving conventional treatment, respectively, with a relative risk of 3.28. The relative risk for coma and/or seizure was 3.02 for intensive treatment. High incidence was also demonstrated in large pediatric cohorts in Australia (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>) and Colorado (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>) in the early 2000s. However, the incidence has been decreasing over time. A population-based cohort of Western Australia demonstrated that the incidence of severe hypoglycemia was 17.3 per 100 patient-years in 2001 and 5.8 per 100 patient-years in 2006, and a 12% annual rate of decrease was observed during the study period (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). A similar decreased trend was also observed in children and adolescents in Germany and Australia (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>) and in Japan (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). A recent Italian study conducted in 29 diabetes centers during 2011–2012 reported less incidence of 7.7 per 100 patient-years (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>), whereas another Italian-center study showed higher incidence of 12.6 per 100 patient-years in 1990 and 16.5 per 100 patient-years in 2010, respectively (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Development of treatment regimens might contribute to decrease in the incidence of severe hypoglycemia; however, despite the advent of new insulin regimens, severe hypoglycemia still remained a relevant risk and a current threat for patients with type 1 diabetes and their family members (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Incidence of severe hypoglycemia over time.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Report</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Year</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Incidence<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>References</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DCCT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1984–1993</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B18\" ref-type=\"bibr\">18</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Conventional</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18.7</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Intensive</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61.2</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bulsara MK</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1992</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B19\" ref-type=\"bibr\">19</xref>)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2002</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.6</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rewers A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1996–2000</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B20\" ref-type=\"bibr\">20</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">O'Connell SM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B21\" ref-type=\"bibr\">21</xref>)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2006</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.8</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Karges B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1995</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B22\" ref-type=\"bibr\">22</xref>)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2012</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.6</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Urakami T</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2003–2013</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B23\" ref-type=\"bibr\">23</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cherubini V</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2011–2012</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B24\" ref-type=\"bibr\">24</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maltoni G</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1990</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B25\" ref-type=\"bibr\">25</xref>)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2010</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.5</td><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><fn id=\"TN1\"><label>*</label><p><italic>Per 100 patient-years</italic>.</p></fn></table-wrap-foot></table-wrap><p>On the other hand, previous studies showed that high incidence of severe hypoglycemia was related to a lower HbA<sub>1c</sub> level (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>); however, this association has recently weakened as reported in large longitudinal cohorts (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>). A cross-sectional analysis of 3 contemporary pediatric diabetes registry databases showed no inverse correlation between a mean HbA<sub>1c</sub> level and risk factors for severe hypoglycemia in children and adolescents with type 1 diabetes (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). It is possible that the advanced technologies over the past decades could be enabling better glycemic control without increase in the risk of severe hypoglycemia. Such advances could include the introduction and increased use of insulin analogs, insulin pump therapy, increased frequency of self-monitoring of blood glucose, and use of CGM.</p></sec><sec id=\"s4\"><title>Morbidity</title><sec><title>Neurological Outcomes</title><sec><title>Cognitive Function</title><p>Resent meta-analyses of the literature indicated that young patients with type 1 diabetes tended to show mildly lower overall intellectual function than healthy controls and that the domains of executive functions, learning, memory, and processing speed were also impaired (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>). On the other hand, larger difference in cognitive function was found in the subset of young patients with certain risk factors, including younger onset age and greater exposures to both severe hypoglycemia and hyperglycemia (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>).</p><p>Several studies in pediatric patients with type 1 diabetes demonstrated frequent episodes of severe hypoglycemia were related to worse performance than healthy controls on certain attention tasks, such as overall cognitive function, and verbal and visual memory. Particularly, children with certain risk factors, including younger onset age and frequent episodes of severe hypoglycemia, tended to develop cognitive dysfunction. Lin et al. (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>) found that severe hypoglycemia with early onset of type 1 diabetes below 6 years of age adversely affected verbal abilities, working memory, and processing speed later in life than healthy controls. Another study also showed that younger onset age (<5 years) was related to deficit of cognitive function (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). On the other hand, other studies demonstrated that frequent episodes of severe hypoglycemia particularly affected distinct memory function, when these episodes appeared before 5 years of age, and were related to full-scale IQ scores, processing speed, working memory, and perceptual reasoning (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Furthermore, severe hypoglycemia with a convulsion was associated with greater performance deficits, including attention tasks, overall cognitive function, and verbal and visual memory (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). Blasetti et al. (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>) indicated that prior episodes of severe and frequent hypoglycemia were mostly related to decreased learning and memory in young patients with type 1 diabetes using a meta-analysis.</p><p>On the other hand, some studies have demonstrated that severe hypoglycemia is unlikely to affect cognitive function. The Epidemiology of Diabetes Interventions and Complications follow-up study, conducted 18 years after the DCCT, reported that cognitive function did not decrease over the extended period in the youngest patients, although relatively high frequencies of severe hypoglycemia (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). A cross-sectional (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>) and longitudinal follow-up research in the same population-based cohort (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>) also did not elucidate a decrease in full-scale IQ scores, although executive function and fluid intelligence may be insufficient. On the other hand, other studies demonstrated that cognitive dysfunction also occurred with hyperglycemia other than hypoglycemia (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>–<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). A large study of younger children (4–10 years of age) with a short period of type 1 diabetes (mean 2.5 years) reported cognitive differences than age-matched healthy children (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). There were differences in full-scale IQ scores and executive functioning even after adjustment of parent IQ scores and internalizing mood symptom levels. The degree of exposure to hyperglycemia was relatively related to performance in these domains. The long-duration impact of hyperglycemia may play an additional role for cognitive outcomes in pediatric patients with type 1 diabetes.</p></sec><sec><title>Brain Structure</title><p>The association of structural abnormalities of brain accompanied by severe hypoglycemia has been shown, although there is increasing evidence that the brain changes were observed even without significant episodes of hypoglycemia among young patients with type 1 diabetes. Pell et al. (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>) reported an interaction between age and brain volume with youth with type 1 diabetes but with the occurrence of dysglycemia. Greater hippocampal volumes (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>) and decreased gray and white matter volumes were observed in children experienced hypoglycemic seizures (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). However, another study showed that brain changes were observed both with hypoglycemia and with hyperglycemia. Episodes of severe hypoglycemia were related to decreased gray matter volume in the left superior temporal region, whereas frequent episodes of hyperglycemia were related to decreased gray matter volume in the right cuneus and precuneus, decreased white matter volume in the right posterior parietal region, and increased gray matter volume in the right prefrontal region (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). These findings suggest that regional differences of brain volume might be related to both hypoglycemia and hyperglycemia.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Regional brain volume differences associated with hyperglycemia and severe hypoglycemia in youth with type 1 diabetes (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). <bold>(A)</bold> Regions with smaller gray matter volume in diabetic youth with severe hypoglycemia compared with those in diabetic youth without severe hypoglycemia. <bold>(B)</bold> Regions of less gray matter, <bold>(C)</bold> more gray matter, and <bold>(D)</bold> less white matter associated with greater hyperglycemia exposure.</p></caption><graphic xlink:href=\"fendo-11-00609-g0001\"/></fig><p>Recent reports also indicated the relation between frequent episodes of severe hypoglycemia and the increased risk of later-onset epilepsy (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>, <xref rid=\"B43\" ref-type=\"bibr\">43</xref>), and although the causative mechanisms were not elucidated, metabolic brain adaptations to frequent severe hypoglycemia might be the cause of later-onset epilepsy (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>).</p></sec></sec><sec><title>Psychological Outcomes</title><p>Severe hypoglycemia is likely to provide negative psychosocial outcomes and undesirable compensatory behaviors (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B45\" ref-type=\"bibr\">45</xref>). The fear toward severe hypoglycemia may induce anxiety, and in many children and family members, significant degrees of anxiety possibly lead to confusion in daily living activities and inadequate management of diabetes (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>). Children with type 1 diabetes and family members have risks of increased anxiety, insufficient sleep, and impaired quality of life (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B47\" ref-type=\"bibr\">47</xref>, <xref rid=\"B48\" ref-type=\"bibr\">48</xref>). Fear of severe hypoglycemia, particularly during night, must be the most serious problem in family members of younger children with type 1 diabetes (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). This fear may lead them to accept high blood glucose levels, and suboptimal glycemic control with behaviors avoiding hypoglycemia resulted in inadequate glycemic control (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B50\" ref-type=\"bibr\">50</xref>, <xref rid=\"B51\" ref-type=\"bibr\">51</xref>). The use of CGM and sensor-augmented pump can decrease the fear of hypoglycemia, although the studies conducted in children are limited (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>, <xref rid=\"B52\" ref-type=\"bibr\">52</xref>).</p></sec><sec><title>Risk Factors for Developing Severe Hypoglycemia</title><p>Various factors can affect severe hypoglycemia. Possible risk factors for developing severe hypoglycemia are shown in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><sec><title>Younger Age</title><p>Various studies have indicated that younger children tend to have more frequent and/or more serious episodes of hypoglycemia than later-onset patients with type 1 diabetes (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>–<xref rid=\"B57\" ref-type=\"bibr\">57</xref>). Younger children are likely to have more physical activities and less consumption of food, showing fluctuating blood glucose profiles, which increase risks for developing hypoglycemia (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). They lack counterregulatory hormone responses to subsequent hypoglycemia via autonomic function (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>). Deficits of counterregulatory hormone responses also cause autonomic failure. Consequently, younger children are at risk for frequent and severe hypoglycemia. Neurological damages accompanied by severe hypoglycemia are more common and more severe in younger children with type 1 diabetes (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). The onset age of diabetes might play a role for early exposure to severe hypoglycemia, as earlier exposure can occur in those with younger onset of type 1 diabetes (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>).</p></sec><sec><title>Nocturnal Hypoglycemia</title><p>The counterregulatory hormone responses to hypoglycemia attenuate during sleep (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>, <xref rid=\"B58\" ref-type=\"bibr\">58</xref>), and patients with type 1 diabetes tend to be less awakened by hypoglycemia compared with healthy subjects (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>). The fear of nocturnal hypoglycemia often provides anxiety and emotional stress, interfering sleep and lowering quality of life in family members, which is the most common cause of distress in family members (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>).</p><p>Earlier reports showed a high frequency of nocturnal hypoglycemia up to 40% during any nights in pediatric patients with type 1 diabetes (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>–<xref rid=\"B63\" ref-type=\"bibr\">63</xref>), whereas recent studies have demonstrated lowering frequencies of 15–25% during any nights (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>, <xref rid=\"B64\" ref-type=\"bibr\">64</xref>). Half of these hypoglycemic events were undetected by patients themselves, families, and caregivers (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>, <xref rid=\"B65\" ref-type=\"bibr\">65</xref>). The Juvenile Diabetes Research Foundation (JDRF) found recurrent and prolonged nocturnal hypoglycemia during 8.5% of nights in both children and adults, but more extended in children (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>). The use of insulin pump can decrease nocturnal hypoglycemia (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>), and this is further decreased using sensor-augmented pump with the control algorithms, which can suspend basal insulin delivery with sensor-detected (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>) or sensor-predicted hypoglycemia (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>).</p></sec><sec><title>Impaired Awareness of Hypoglycemia</title><p>Impaired awareness of hypoglycemia is an acquired complication of insulin treatment, whereby the potency to detect the onset of hypoglycemia is decreased or absent (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). Deficit of the counterregulatory hormone responses to hypoglycemia frequently coexist. Development of impaired awareness of hypoglycemia increases the risk for severe hypoglycemia more. This condition was reported to exist in approximately a quarter of adults with type 1 diabetes. Children and adolescents had similar prevalence of 19–37% (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B71\" ref-type=\"bibr\">71</xref>, <xref rid=\"B72\" ref-type=\"bibr\">72</xref>). However, a recent study has shown reduction in the prevalence over time, i.e., 33% in 2002 vs. 21% in 2015 in the same population-based cohort (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>). Although the prevalence of impaired awareness of hypoglycemia has decreased, it is still a major risk factor for developing severe hypoglycemia. Patients with impaired awareness of hypoglycemia have a 6-fold increase in the prevalence of severe hypoglycemia (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>).</p><p>It has been known that impaired awareness of hypoglycemia is related to decrease in glycemic thresholds for the release of counterregulatory hormones and induction of adrenergic warning signs. Korytkowski et al. (<xref rid=\"B75\" ref-type=\"bibr\">75</xref>) reported that a 2- to 3-fold decrease in the epinephrine responses was related to the loss of adrenergic warning symptoms against hypoglycemia. On the other hand, loss of autonomic symptoms precedes the neuroglycopenic symptoms, and patients are less likely to recognize hypoglycemia. Hypoglycemia tends to be prolonged when the awareness of low blood glucose is impaired. Patients can develop hypoglycemic seizures if unrecognized and prolonged conditions continue for more than 2–4 h (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>).</p><p>Most events of severe hypoglycemia occur during nighttime, because sleep more strongly impairs the counterregulatory hormone responses to hypoglycemia in patients with type 1 diabetes as well as normal subjects (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>). On the other hand, the glycemic threshold for neuroglycopenia does not change as much with the intensity of treatment, glycemic control, or with prior hypoglycemia (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>–<xref rid=\"B79\" ref-type=\"bibr\">79</xref>).</p><p>Avoidance of severe hypoglycemia for 2–3 weeks can reverse impaired awareness of hypoglycemia (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>), which is difficult to achieve in practice with current intensive insulin treatment in children with type 1 diabetes. Advanced technologies, such as the use of CGM (<xref rid=\"B80\" ref-type=\"bibr\">80</xref>) or sensor-augmented pump with control algorithms including suspend functions (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>, <xref rid=\"B81\" ref-type=\"bibr\">81</xref>), could reduce the rate of severe hypoglycemia in patients showing impaired awareness of hypoglycemia.</p></sec><sec><title>Frequent Episodes of Hypoglycemia</title><p>Most children with type 1 diabetes have isolated episodes of severe hypoglycemia; however, a few experience recurrent episodes of severe hypoglycemia. Frequent episodes of hypoglycemia are related to defective counterregulatory hormone responses to subsequent decrease in blood glucose concentrations. Therefore, prior episodes of frequent hypoglycemia are considered as an important risk factor for subsequent severe hypoglycemia (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Both defective counterregulatory hormone responses and impaired awareness of hypoglycemia cause hypoglycemia-associated autonomic failure related to recurrent hypoglycemia, resulting in subsequent severe hypoglycemia (<xref rid=\"B82\" ref-type=\"bibr\">82</xref>–<xref rid=\"B84\" ref-type=\"bibr\">84</xref>). In DCCT, analysis of 424 intensively treated patients found that longer duration of diabetes, glycemic control, and prior severe hypoglycemia were related to the occurrence of severe hypoglycemia (<xref rid=\"B85\" ref-type=\"bibr\">85</xref>). JDRF reported that the higher rate of severe hypoglycemia was related to severe hypoglycemia that occurred in the last 6 months (<xref rid=\"B86\" ref-type=\"bibr\">86</xref>). Therefore, prior episodes of recurrent hypoglycemia can be one of the important predictors of subsequent severe hypoglycemia.</p></sec><sec><title>Glycemic Control</title><p>In the 1990s, strict glycemic control was evaluated to affect the frequency of severe hypoglycemia (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>), particularly in younger children (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>). However, data from 2000 to 2009 in the Western Australian Children's Diabetes Database were analyzed, and there was a decline in the frequency of severe hypoglycemia, and relationship with glycemic control became weaker than previously reported (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>). The reduction in the severe hypoglycemia may have resulted from improvement in management of diabetes during the past decades. The correlation between glycemic control and the risk of severe hypoglycemia seems to be weaker, without increased risk of severe hypoglycemia associated with improvement of glycemic control (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>–<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). The association of a low HbA<sub>1c</sub> level is not a predictable value for severe hypoglycemia in pediatric patients with type 1 diabetes (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). Therefore, adequate glycemic control can be attained without increasing episodes of severe hypoglycemia.</p></sec><sec><title>Coexisting Morbidities</title><p>Coexisting morbidities, including hypothyroidism (<xref rid=\"B87\" ref-type=\"bibr\">87</xref>), celiac disease (<xref rid=\"B88\" ref-type=\"bibr\">88</xref>), and Addison disease (<xref rid=\"B89\" ref-type=\"bibr\">89</xref>, <xref rid=\"B90\" ref-type=\"bibr\">90</xref>), have been reported to be possible risk factors for severe hypoglycemia. The use of a gluten-free diet and adequate treatment of Addison disease and hypothyroidism can decrease the rate of severe hypoglycemia. Rarely, intentional self-administration of insulin to cause hypoglycemia, i.e., factitious hypoglycemia, can introduce recurrent and serious hypoglycemia and should be diagnosed as having psychological problems including eating disorders or psychiatric disease (<xref rid=\"B91\" ref-type=\"bibr\">91</xref>).</p></sec><sec><title>Urgent Treatment of Severe Hypoglycemia</title><p>Urgent treatment is required when severe hypoglycemia occurs and can be effectively reversed by injection of glucagon, which can be administered intravenously, intramuscularly, or subcutaneously (<xref rid=\"B92\" ref-type=\"bibr\">92</xref>, <xref rid=\"B93\" ref-type=\"bibr\">93</xref>). Family members and caregivers have difficulties in preparation and administration of glucagon, because glucagon reconstitution with sterile water is required in the current preparations. To resolve these problems, an intranasal glucagon preparation has been tried in children (<xref rid=\"B94\" ref-type=\"bibr\">94</xref>) and adults (<xref rid=\"B95\" ref-type=\"bibr\">95</xref>) with type 1 diabetes and was revealed to be a promising alternative to intramuscular glucagon. Glucagon cannot be available in areas with limited resources, and in the areas where glucagon may not be available, glucose gel or in powder form is used.</p><p>On the other hand, glucose must be administered intravenously more than a few minutes to reverse hypoglycemia. Rapid infusion or excessive concentration (i.e., 50%) can cause an excessive osmotic alteration, leading to hyperosmolar injury of brain (<xref rid=\"B96\" ref-type=\"bibr\">96</xref>).</p></sec></sec><sec><title>Advanced Technologies for Prevention of the Occurrence of Severe Hypoglycemia and Decrease in Risk Factors for Developing Severe Hypoglycemia</title><p>Prevention of severe hypoglycemia remains one of the most critical issues in the management of pediatric patients with type 1 diabetes. Closed-loop system is probably the best technology for prevention of hypoglycemia; however, in the initial step toward closed-loop system, CGM or integrated CGM and insulin pump have enabled patients with type 1 diabetes to further decrease hypoglycemia (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Possible technologies to prevent the occurrence of severe hypoglycemia and to decrease in the risk factors for developing severe hypoglycemia are shown in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><sec><title>CGM</title><p>Several studies have reported that CGM can reduce hypoglycemic events with a concomitant improvement in HbA<sub>1c</sub> in patients with type 1 diabetes, regardless of age (<xref rid=\"B97\" ref-type=\"bibr\">97</xref>–<xref rid=\"B99\" ref-type=\"bibr\">99</xref>). A randomized controlled multicenter study demonstrated reduction in time spent in hypoglycemia concomitant with a decrease in HbA<sub>1c</sub> in both children and adults with type 1 diabetes (<xref rid=\"B98\" ref-type=\"bibr\">98</xref>). A multicenter analysis of 3,553 subjects from the German-Austrian-Swiss-Luxembourgian Diabetes Prospective Follow-up registry demonstrated that initiation and regular use of CGM in children and adolescents with type 1 diabetes were associated with reduction in both diabetic ketoacidosis and severe hypoglycemia with modest improvement in glycemic control (<xref rid=\"B100\" ref-type=\"bibr\">100</xref>). Although the use of CGM can decrease the episodes of severe hypoglycemia in adult patients (<xref rid=\"B80\" ref-type=\"bibr\">80</xref>, <xref rid=\"B101\" ref-type=\"bibr\">101</xref>), this effect is not elucidated in pediatric patients (<xref rid=\"B102\" ref-type=\"bibr\">102</xref>). Moreover, JDRF (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>) reported frequent and often prolonged hypoglycemia, particularly during nighttime, in pediatric patients with type 1 diabetes, although using CGM; i.e., hypoglycemic events occurred in 8.5% during nights, and the duration of hypoglycemia over 2 h was 23% of the nights. Adolescents have a high acoustic arousal threshold from sleep (<xref rid=\"B103\" ref-type=\"bibr\">103</xref>) and therefore could have severe hypoglycemic events during nighttime (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>). Buckingham et al. (<xref rid=\"B104\" ref-type=\"bibr\">104</xref>) reported that 71% of youth wearing CGM did not respond to nighttime alarms. On the other hand, Ly et al. (<xref rid=\"B105\" ref-type=\"bibr\">105</xref>) reported that CGM with preset alarms improved epinephrine response in adolescents with type 1 diabetes, who had impaired awareness of hypoglycemia and a risk for nocturnal hypoglycemia. This study suggests that CGM might be a useful tool to relieve impaired awareness of hypoglycemia and potently avoid severe hypoglycemia in adolescents with type 1 diabetes.</p><p>Intermittently scanned CGM (isCGM; FreeStyle Libre; Abbott Diabetes Care, Alameda, CA, USA) has similar methodology to show continuous glucose measurements as ambulatory glucose profiles retrospectively at the time of checking. Glucose trend can be observed after intermittently scanning the sensor. IsCGM is approved in a number of countries for use, but there were a few clinical studies showing the effect on glycemic control in pediatric patients (<xref rid=\"B106\" ref-type=\"bibr\">106</xref>–<xref rid=\"B109\" ref-type=\"bibr\">109</xref>). These studies on isCGM have demonstrated a similar effect on maintaining adequate glycemic control as when using CGM, but decrease in the time spent in hypoglycemia seems difficult on multiple daily injections of insulin without using the advanced technologies, such as a sensor-augmented pump with low-glucose suspension or a hybrid closed-loop system (<xref rid=\"B109\" ref-type=\"bibr\">109</xref>).</p></sec><sec><title>Sensor-Augmented Pump Therapy With Low Glucose Suspension and That With Predictive Low Glucose Suspension</title><p>Sensor-augmented pump with low-glucose suspension further decreases the time spent in hypoglycemia and the occurrence of severe hypoglycemia. If the users ignore the alarm sounds, a low-glucose suspend system automatically suspends basal insulin delivery for up to 2 h in response to sensor-detected hypoglycemic events, after which basal insulin delivery is resumed at the programmed rate. A low-glucose suspend system can reduce the time of hypoglycemia, particularly during nighttime (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>, <xref rid=\"B110\" ref-type=\"bibr\">110</xref>, <xref rid=\"B111\" ref-type=\"bibr\">111</xref>). This function also decreases moderate to severe hypoglycemic events, particularly in patients with impaired awareness of hypoglycemia (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>). Furthermore, hyperglycemia, deterioration in overall glycemic control, and development to ketoacidosis are low frequencies (<xref rid=\"B97\" ref-type=\"bibr\">97</xref>, <xref rid=\"B111\" ref-type=\"bibr\">111</xref>).</p><p>Predictive low-glucose management system, the MiniMed 640G System (Medtronic, Northridge, CA, USA), suspends basal insulin delivery with the hypoglycemia prediction algorithm. Basal insulin delivery is usually suspended when sensor glucose level is within 3.9 mmol/L (70 mg/dL) above the patient-set low limit and is predicted to be 1.1 mmol/L (20 mg/dL) above this low limit for 30 min. When patients do not interfere, accompanied by the pump suspension, the insulin delivery resumes after the suspend period of 2 h or less at the programmed rate (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). The use of predictive low-glucose suspension more effectively decreases the rate of hypoglycemia and the risk of severe hypoglycemia in patients with type 1 diabetes (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>, <xref rid=\"B68\" ref-type=\"bibr\">68</xref>, <xref rid=\"B112\" ref-type=\"bibr\">112</xref>–<xref rid=\"B117\" ref-type=\"bibr\">117</xref>). Buckingham et al. (<xref rid=\"B117\" ref-type=\"bibr\">117</xref>) reported that predictive low-glucose suspension prevented hypoglycemia on 75% of nights and in 84% of predicted events in adolescents and young adults with type 1 diabetes. In the study, there was mild ketosis in a few cases when the insulin pump was suspended for 1.5–2 h, and serum ketone bodies returned to normal range with resumption of basal insulin delivery. On the other hand, Abraham et al. (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>) demonstrated that predictive low-glucose suspension was related to reduction in hypoglycemia than single use of sensor-augmented pump in a 6-month, multicenter, randomized controlled trial for pediatric patients with type 1 diabetes. This decline was observed both during daytime and nighttime. Episodes of hypoglycemia with a sensor-glucose value <3.5 mmol/L (63 mg/dL) for over 20 min also reduced with predictive low-glucose suspension than single use of sensor-augmented pump. Deterioration of glycemic control was not found in the use of the predictive low-glucose suspension. These findings suggest that a sensor-augmented pump therapy with low-glucose management, especially with predictive low-glucose suspension, is an important promising tool to decrease the frequency in the occurrence of hypoglycemia and the risks for severe hypoglycemia without worsening overall glycemic control in pediatric patients with type 1 diabetes.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Predictive low-glucose management system. The MiniMed 640G System (Medtronic, Northridge, CA, USA).</p></caption><graphic xlink:href=\"fendo-11-00609-g0002\"/></fig></sec><sec><title>Hybrid Closed-Loop System</title><p>In 2016, the Food and Drug Administration approved the first closed-loop system, the MiniMed 670G System (Medtronic), for patients 14 years or older in the United States. Hybrid closed-loop system, commonly referred to as an artificial pancreas, is an automated insulin delivery management, combined with CGM and insulin delivery without patient intervention. The system uses a proprietary proportional-integral-derivative controller with insulin feedback to calculate insulin dosages continually according to CGM levels (<xref rid=\"B118\" ref-type=\"bibr\">118</xref>–<xref rid=\"B120\" ref-type=\"bibr\">120</xref>). Several studies have been handled on the closed-loop systems and demonstrate improvement of glycemic control with decrease in the rate of hypoglycemia and the occurrence of severe hypoglycemia in both adults and children, especially at nighttime (<xref rid=\"B121\" ref-type=\"bibr\">121</xref>–<xref rid=\"B125\" ref-type=\"bibr\">125</xref>). In an open-label, randomized, crossover study design, the use of a closed-loop system significantly increased the time spent in the target range [3.9–10 mmol/L (70–180 mg/dL)], whereas the time spent in hypoglycemic range significantly decreased [<3.9 mmol/L (<70 mg/dL)] during both daytime and nighttime in adolescents with type 1 diabetes (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>) (<xref rid=\"B125\" ref-type=\"bibr\">125</xref>). The use of hybrid closed-loop system is in general effective and safe particularly at nighttime, and allows enough sleep and reduces the burden of diabetes management during overnight. The closed-loop system must be one of the most promising technologies to attain optimal glycemic control with minimizing the episodes of hypoglycemia, as well as occurrence of severe hypoglycemia. Although the majority of the systems include single-hormone insulin, dual-hormone systems, which infuse both insulin and glucagon, have also been in the research phase (<xref rid=\"B126\" ref-type=\"bibr\">126</xref>, <xref rid=\"B127\" ref-type=\"bibr\">127</xref>).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Closed-loop system. The FlorenceD2A closed-loop system (University of Cambridge, Cambridge, UK) (<xref rid=\"B125\" ref-type=\"bibr\">125</xref>). Median sensor glucose <bold>(A)</bold> and insulin delivery <bold>(B)</bold> during closed-loop insulin delivery period (solid red line and red-shaped area) and sensor-augmented pump period (dashed black line and gray-shaped area) from midnight to midnight. The glucose range 3.9–10.0 mmol/L (70–180 mg/dL) is denoted by horizontal dashed lines <bold>(A)</bold>.</p></caption><graphic xlink:href=\"fendo-11-00609-g0003\"/></fig><p>In summary, the incidence of severe hypoglycemia has been markedly declined in recent years, but still a lethal condition. Minimizing the risk factors for development of severe hypoglycemia is an important objective to prevent the occurrence of severe hypoglycemia in pediatric patients with type 1 diabetes. The new concept of time spent within target glucose range (time in range) will be in general used to evaluate the glucose trend and the quality of metabolic control (<xref rid=\"B128\" ref-type=\"bibr\">128</xref>, <xref rid=\"B129\" ref-type=\"bibr\">129</xref>). Achieving the target range [3.9–10 mmol/L (70–180 mg/dL)] more than 70% with minimizing severe hypoglycemia <1% is crucial in the management of not only adults but also children and adolescents with type 1 diabetes (<xref rid=\"B128\" ref-type=\"bibr\">128</xref>). This can be achieved through advanced diabetes technologies even in pediatric patients.</p></sec></sec></sec><sec id=\"s5\"><title>Author Contributions</title><p>The author confirms being the sole contributor of this work and has approved it for publication.</p></sec><sec id=\"s6\"><title>Conflict of Interest</title><p>The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The author declares that this study received funding from Novo Nordisk Pharma Ltd., Eli Lilly Japan K.K., Terumo Corp., and JCR Pharmaceuticals Co., Ltd. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"systematic-review\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Pharmacol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Pharmacol.</journal-id><journal-title-group><journal-title>Frontiers in Pharmacology</journal-title></journal-title-group><issn pub-type=\"epub\">1663-9812</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33041799</article-id><article-id pub-id-type=\"pmc\">PMC7523512</article-id><article-id pub-id-type=\"doi\">10.3389/fphar.2020.560920</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Pharmacology</subject><subj-group><subject>Systematic Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Effects of Jianpi Bushen Therapy for Treatment of CKD Anemia: A Meta-Analysis of Randomized Controlled Trials</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Liang</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/974928\"/></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Chengyin</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhou</surname><given-names>Yu</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Qi</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Zilin</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhu</surname><given-names>Xiaoyun</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ba</surname><given-names>Yuanming</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn001\">\n<sup>*</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<sup>1</sup>\n<institution>Department of Nephropathy, Hubei Provincial Hospital of TCM</institution>, <addr-line>Wuhan</addr-line>, <country>China</country>\n</aff><aff id=\"aff2\">\n<sup>2</sup>\n<institution>Clinical College of Chinese Medicine, Hubei University of Chinese Medicine</institution>, <addr-line>Wuhan</addr-line>, <country>China</country>\n</aff><aff id=\"aff3\">\n<sup>3</sup>\n<institution>Department of Oncology, Hubei Provincial Hospital of TCM</institution>, <addr-line>Wuhan</addr-line>, <country>China</country>\n</aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Jianping Chen, Guangzhou University of Chinese Medicine, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Mingze Qin, Shenyang Pharmaceutical University, China; Qian Zhou, Nanjing University of Chinese Medicine, China</p></fn><corresp id=\"fn001\">*Correspondence: Yuanming Ba, <email xlink:href=\"mailto:1723426138@qq.com\" xlink:type=\"simple\">1723426138@qq.com</email>\n</corresp><fn fn-type=\"other\" id=\"fn002\"><p>This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>560920</elocation-id><history><date date-type=\"received\"><day>11</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Li, Li, Zhou, Xu, Wang, Zhu and Ba</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Li, Li, Zhou, Xu, Wang, Zhu and Ba</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><sec><title>Objectives</title><p>To evaluate the efficacy of Traditional Chinese Medicine, specifically Jianpi Bushen (JPBS) therapy, for treatment of patients with chronic kidney disease (CKD) anemia.</p></sec><sec><title>Methods</title><p>Randomized controlled trials of JPBS therapy for CKD anemia were searched and selected from seven electronic databases. The Cochrane collaboration tool was used to conduct methodological quality assessment. RevMan v5.3 software was utilized to perform data analysis.</p></sec><sec><title>Results</title><p>In total, 12 randomized controlled trials with 799 patients met the meta-analysis criteria. The aggregated results indicated that JPBS therapy is beneficial for CKD anemia by improving the clinical efficacy rate [risk ratio (RR) = 1.23, 95% confidence interval (CI): (1.14, 1.33), <italic>P</italic> < 0.00001] and hemoglobin (Hb) [weighted mean difference (WMD) = 9.55, 95% CI: (7.97, 11.14), <italic>P</italic> < 0.00001], serum ferritin (SF) [WMD = 6.22, 95% CI: (2.65, 9.79), <italic>P</italic> = 0.0006], red blood cell (RBC) [WMD = 0.31, 95% CI: (0.24, 0.38), <italic>P</italic> < 0.00001], hematocrit (HCT) [WMD = 2.95, 95% CI: (2.36, 3.54), <italic>P</italic> < 0.00001], serum creatinine (SCr) [WMD = 64.57, 95% CI: (33.51, 95.64), <italic>P</italic> < 0.0001], and blood urea nitrogen (BUN) levels [WMD = 3.76, 95% CI: (2.21, 5.31), <italic>P <</italic>0.00001]. Furthermore, evidence suggests that JPBS therapy is safe and does not increase adverse reactions compared with western medicine (WM) alone.</p></sec><sec><title>Conclusion</title><p>This study found that JPBS therapy has a positive effect on the treatment of CKD anemia. However, more well-designed, double-blind, large-scale randomized controlled trials are needed to assess the efficacy of JPBS therapy in the treatment of CKD anemic patients.</p></sec></abstract><kwd-group><kwd>chronic kidney disease anemia</kwd><kwd>Jianpi Bushen therapy</kwd><kwd>meta-analysis</kwd><kwd>randomized controlled trials (RCT)</kwd><kwd>Traditional Chinese Medicine</kwd></kwd-group><counts><fig-count count=\"12\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"41\"/><page-count count=\"10\"/><word-count count=\"3809\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Anemia is one of the most common complications in patients with chronic kidney disease (CKD), and its prevalence progressively increases as the glomerular filtration rate declines (<xref rid=\"B13\" ref-type=\"bibr\">Hsu et al., 2002</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Li et al., 2016</xref>). Chronic kidney disease anemia is associated with increased risk of reduced quality of life, cardiovascular disease, cognitive impairment, and mortality (<xref rid=\"B12\" ref-type=\"bibr\">Hörl, 2013</xref>). One of the main causes of CKD anemia is a decrease in the renal production of erythropoietin (EPO). In addition, iron, which is an important raw material for hemoglobin production, is often deficient in CKD patients due to impaired dietary iron absorption and increased iron loss (<xref rid=\"B1\" ref-type=\"bibr\">Babitt and Lin, 2012</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Gafter-Gvili et al., 2019</xref>). Thus, erythropoiesis-stimulating agent (ESA) administration and iron supplementation are the primary Western medicine (WM) treatments for CKD patients with anemia. Although they are effective drugs that clearly avoid the need of blood transfusions, the controversy and safety issues have been raised for both drugs in recent years, especially when they are given at high doses. Evidence has shown that intravenous iron contributes to cardiovascular morbidity and mortality in patients with end-stage renal disease (ESRD) (<xref rid=\"B15\" ref-type=\"bibr\">Kuragano et al., 2014</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Bailie et al., 2015</xref>). Treatment with ESAs has also been linked to stroke, thrombotic events, and hypertension in CKD patients (<xref rid=\"B14\" ref-type=\"bibr\">Koulouridis et al., 2013</xref>). Moreover, 5–10% of patients exhibit an inadequate response to ESAs (<xref rid=\"B7\" ref-type=\"bibr\">Costa et al., 2008</xref>), resulting in an increased risk of faster progression to ESRD and all-cause mortality (<xref rid=\"B30\" ref-type=\"bibr\">Solomon et al., 2010</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Minutolo et al., 2012</xref>). Current WM treatment is also only designed to increase hemoglobin production, not necessarily to improve renal function. The production of EPO will decline with the decrease in glomerular filtration rate and progress of CKD, thereby worsening patient anemia (<xref rid=\"B27\" ref-type=\"bibr\">Prasad-Reddy et al., 2017</xref>). Hence, faced with the limitations of current treatment, complementary and/or alternative medicines that improve renal function and alleviate anemia are urgently needed.</p><p>Chinese herbal medicine (CHM) has been widely used to treat anemia (<xref rid=\"B4\" ref-type=\"bibr\">Bradley et al., 1999</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Chen et al., 2018</xref>). According to Traditional Chinese Medicine (TCM) theory, blood deficiency is the main feature of anemia and spleen and kidney deficiencies are the basic pathological features. Based on the relationship between the spleen and kidney and blood generation, classic TCM therapy for the treatment of anemia involves invigorating the spleen and reinforcing the kidney (known as Jianpi Bushen, JPBS). For instance, <xref rid=\"B34\" ref-type=\"bibr\">Wu et al. (2016)</xref> found that treatments based on kidney support had a positive effect on treating chronic aplastic anemia. <xref rid=\"B21\" ref-type=\"bibr\">Lin et al. (1990)</xref> also found the therapy of warming and tonifying the spleen and kidney improve hemopoietic function of chronic aplastic anemia patients. In recent decades, several clinical trials have evaluated the efficacy and renoprotection of JPBS therapy for the treatment of anemia in CKD (<xref rid=\"B33\" ref-type=\"bibr\">Wang, 2013</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Li, 2016</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Zhang, 2017</xref>). Therefore, in the current study, we performed a meta-analysis to assess JPBS therapy for patients with CKD anemia. The results reported here will help lay a foundation for the treatment of CKD anemia with JPBS therapy.</p></sec><sec sec-type=\"materials|methods\" id=\"s2\"><title>Materials and Methods</title><sec id=\"s2_1\"><title>Database Searching</title><p>Seven different databases (i.e., PubMed, Cochrane Library, EMBASE, Wanfang Database, China National Knowledge Infrastructure, Chinese Science and Technique Journals Database, and China Biological Medicine Database) were independently searched from inception to April 2019 (by authors YZ and QX). The search terms used were: (jianpi OR bushen OR invigorating spleen OR reinforcing kidney) AND (renal anemia OR anemia of chronic kidney disease OR anemia of renal disease). These terms were translated into Chinese when searching the Chinese databases. Additional studies missed by the electronic search were also searched manually.</p></sec><sec id=\"s2_2\"><title>Inclusion Criteria</title><p>The inclusion criteria were as follows: (1) randomized controlled trial (RCT); (2) patients diagnosed with CKD anemia according to the KDIGO Clinical Practice Guideline for Anemia in Chronic Kidney Disease (<xref rid=\"B24\" ref-type=\"bibr\">McDonald, 2012</xref>) and the Diagnosis and Treatment of Renal Anemia with Chinese Expert Consensus (Revision 2018) (<xref rid=\"B29\" ref-type=\"bibr\">Renal Physicians Association Branch of China Consensus Group of Experts on the Diagnosis and Treatment of Renal Anemia, 2018</xref>), with no limitations related to age, ethnicity, gender, or primary disease; (3) adoption of JPBS therapy combined with WM (iron supplements, ESAs) as the treatment group, with WM alone applied as the control group. Treatment needed to last at least eight weeks and the sample size needed to be ≥15; (4) primary outcomes to include clinical effective rate and hemoglobin (Hb) level, secondary outcomes to include serum ferritin (SF), red blood cell (RBC), hematocrit (HCT), serum creatinine (SCr), blood urea nitrogen (BUN) levels and number of adverse events; and (5) results available in either English or Chinese.</p></sec><sec id=\"s2_3\"><title>Exclusion Criteria</title><p>The exclusion criteria were as follows: (1) non-randomized controlled trial; (2) study subjects did not meet the diagnostic criteria of renal anemia; (3) both treatment and control groups were treated with CHM only; and (4) studies were duplicated publications or articles with unavailable data.</p></sec><sec id=\"s2_4\"><title>Quality Assessment and Data Extraction</title><p>The first author, year of publication, region, sample size, age, intervention, follow-up time, and main outcomes of each eligible study were collected by two authors (YZ and QX). Based on bias risk assessment of the Cochrane collaboration tool, the methodological quality of the selected studies was evaluated by YZ and QX. The assessed items included: (1) method of random allocation and allocation concealment; (2) blinding method of participants and personnel; (3) blinding method of outcome assessment; (4) integrity of data; (5) selectivity of result reporting; and (6) other biases. For any disagreement, the issue was resolved by discussion with a third author (CL).</p></sec><sec id=\"s2_5\"><title>Data Analysis</title><p>RevMan v5.3 from Cochrane was employed to analyze data. For dichotomous outcomes, risk ratio (RR) with a 95% confidence interval (CI) was used. For continuous outcomes, mean difference (MD) with a 95% confidence interval (CI) was adopted. Data heterogeneity was assessed by χ<sup>2</sup> and <italic>I</italic>\n<sup>2</sup> tests. If heterogeneity existed in pooled studies (<italic>I</italic>\n<sup>2</sup> ≥ 50%), a random model was applied; if not, a fixed model was applied. Statistically significant differences were considered at <italic>P</italic> < 0.05. Publication bias was evaluated by funnel plots and calculated using Begg’s/Egger’s tests through STATA v15.0. Sensitivity analysis was conducted by removing individual studies to assess the stability of the results.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec id=\"s3_1\"><title>Search Results and Study Characteristics</title><p>\n<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1</bold>\n</xref> shows a flow diagram of the study. Based on our database search, 356 articles reporting on CKD anemia treatment using JPBS therapy were identified. Of these articles, 167 duplicated publications were excluded. After going through the titles and abstracts, another 169 articles were excluded due to non-CKD anemia, non-human studies, and clinical experience reports and reviews. The 20 remaining full-text articles were further screened, with another eight eliminated because of non-RCT or incomplete data. Finally, a total of 12 articles with 799 patients were enrolled in this meta-analysis (<xref rid=\"B16\" ref-type=\"bibr\">Li et al., 2003</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Cheng et al., 2005</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bao et al., 2008</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Zhou et al., 2008</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Liang, 2009</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Qu, 2011</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Wang, 2012</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Liu, 2013</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Wang, 2013</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Li, 2016</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Du, 2017</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Zhang, 2017</xref>). Among them, 418 patients received JPBS therapy and 381 patients received WM treatment. The main characteristics of the enrolled articles are presented in <xref rid=\"T1\" ref-type=\"table\">\n<bold>Table 1</bold>\n</xref>.</p><fig id=\"f1\" position=\"float\"><label>Figure 1</label><caption><p>Flow chart of study selection process.</p></caption><graphic xlink:href=\"fphar-11-560920-g001\"/></fig><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Characteristics of RCTs included in the study.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Study ID</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Region</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Sample size (T/C)</th><th valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Age (y)</th><th valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Gender (M/F)</th><th valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Intervention</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Duration(weeks)</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Outcome</th></tr><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">C</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">C</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">T</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">C</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B32\" ref-type=\"bibr\">Wang, 2012</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40/40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">51.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17/23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19/21</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">a,b,e,f,g</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B8\" ref-type=\"bibr\">Du, 2017</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15/15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49.28 ± 8.24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50.15 ± 5.26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8/7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8/7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">b,d,e,f,g</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B16\" ref-type=\"bibr\">Li et al., 2003</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30/30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45.8 ± 19.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">47.5 ± 13.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21/9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20/10</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">a,b,e,f,g</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B28\" ref-type=\"bibr\">Qu, 2011</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27/25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">b,d,e,f,g</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B37\" ref-type=\"bibr\">Zhang, 2017</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30/30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17/13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12/18</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">a,b,c,e,f,g</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B22\" ref-type=\"bibr\">Liu, 2013</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27/27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">52.70 ± 15.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">42.66 ± 13.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17/10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15/12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">a,b,e,f</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B19\" ref-type=\"bibr\">Li, 2016</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60/60</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44.6 ± 12.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44.3 ± 12.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">38/22</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40/20</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">a,b,c,d,e,f,g</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B3\" ref-type=\"bibr\">Bao et al., 2008</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34/27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">47.5 ± 10.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">46.8 ± 11.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18/16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15/12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">a,b,e,f,g</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B33\" ref-type=\"bibr\">Wang, 2013</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31/31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14/17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16/15</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">a,b,d,e,f,g</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B6\" ref-type=\"bibr\">Cheng et al., 2005</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30/30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">47.8 ± 14.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">46.3 ± 12.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14/16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15/15</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">a,b,d,e,f,g</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B41\" ref-type=\"bibr\">Zhou et al., 2008</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">56/30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44.21 ± 12.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44.25 ± 11.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30/26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18/12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">a,b,e,f</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">\n<xref rid=\"B20\" ref-type=\"bibr\">Liang, 2009</xref>\n</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">China</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">38/36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">JPBS+WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">WM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">a,b,e,f,g</td></tr></tbody></table><table-wrap-foot><p>T, treatment group; C, control group; NA, not available; M/F, male/female; CHM, Chinese herbal medicine; JPBS, Jianpi Bushen; WM, western medicine; outcomes: a, clinical efficacy; b, Hb; c, SF; d, RBC; e, HCT; f, SCr; g, BUN.</p></table-wrap-foot></table-wrap></sec><sec id=\"s3_2\"><title>Risk of Bias</title><p>As shown in <xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figures 2</bold>\n</xref> and <xref ref-type=\"fig\" rid=\"f3\">\n<bold>3</bold>\n</xref>, the methodological quality of the included articles was relatively low. All enrolled articles claimed to be randomized, but only four described their random methods (<xref rid=\"B16\" ref-type=\"bibr\">Li et al., 2003</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Cheng et al., 2005</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Li, 2016</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Du, 2017</xref>). No article mentioned allocation concealment or blinding methods. All articles had complete data, and there was no selective reporting or other biases.</p><fig id=\"f2\" position=\"float\"><label>Figure 2</label><caption><p>Risk of bias graph.</p></caption><graphic xlink:href=\"fphar-11-560920-g002\"/></fig><fig id=\"f3\" position=\"float\"><label>Figure 3</label><caption><p>Risk of bias summary.</p></caption><graphic xlink:href=\"fphar-11-560920-g003\"/></fig></sec><sec id=\"s3_3\"><title>Effects of Intervention</title><sec id=\"s3_3_1\"><title>Clinical Efficacy Rate</title><p>Clinical efficacy rates were reported in 10 studies (<xref rid=\"B16\" ref-type=\"bibr\">Li et al., 2003</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Cheng et al., 2005</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bao et al., 2008</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Zhou et al., 2008</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Liang, 2009</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Wang, 2012</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Liu, 2013</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Wang, 2013</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Li, 2016</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Zhang, 2017</xref>), totaling 717 patients (376 cases in the JPBS therapy group and 341 cases in the WM treatment group). Results indicated that the clinical efficacy rates of the JPBS therapy group were higher than those of the WM treatment group [RR = 1.23, 95% CI: (1.14, 1.33), <italic>P</italic> < 0.00001, <xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4</bold>\n</xref>].</p><fig id=\"f4\" position=\"float\"><label>Figure 4</label><caption><p>Forest plot of clinical efficacy rate.</p></caption><graphic xlink:href=\"fphar-11-560920-g004\"/></fig></sec><sec id=\"s3_3_2\"><title>Hb Levels</title><p>Hb data were provided in all studies (<xref rid=\"B16\" ref-type=\"bibr\">Li et al., 2003</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Cheng et al., 2005</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bao et al., 2008</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Zhou et al., 2008</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Liang, 2009</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Qu, 2011</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Wang, 2012</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Liu, 2013</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Wang, 2013</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Li, 2016</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Du, 2017</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Zhang, 2017</xref>), totaling 799 patients (418 cases in the JPBS therapy group and 381 cases in the WM treatment group). As data heterogeneity was not found (<italic>I</italic>\n<sup>2</sup> = 39%, <italic>P</italic> = 0.08), the fixed effect model was applied (<xref ref-type=\"fig\" rid=\"f5\">\n<bold>Figure 5</bold>\n</xref>). Results indicated that the increase in serum Hb levels was greater in the JPBS therapy group than in the WM treatment group [WMD = 9.55, 95% CI: (7.97, 11.14), <italic>P</italic> < 0.00001].</p><fig id=\"f5\" position=\"float\"><label>Figure 5</label><caption><p>Forest plot of Hb levels.</p></caption><graphic xlink:href=\"fphar-11-560920-g005\"/></fig></sec><sec id=\"s3_3_3\"><title>SF Levels</title><p>SF data were provided in two studies (<xref rid=\"B19\" ref-type=\"bibr\">Li, 2016</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Zhang, 2017</xref>), totaling 180 patients (90 cases in the JPBS therapy group and 90 cases in the WM treatment group). As data heterogeneity was not found (<italic>I</italic>\n<sup>2</sup> = 0%, <italic>P</italic> = 0.70), the fixed effect model was applied (<xref ref-type=\"fig\" rid=\"f6\">\n<bold>Figure 6</bold>\n</xref>). Results indicated that the increase in serum SF levels was greater in the JPBS therapy group than in the WM treatment group [WMD = 6.22, 95% CI: (2.65, 9.79), <italic>P</italic> = 0.0006].</p><fig id=\"f6\" position=\"float\"><label>Figure 6</label><caption><p>Forest plot of SF levels.</p></caption><graphic xlink:href=\"fphar-11-560920-g006\"/></fig></sec><sec id=\"s3_3_4\"><title>RBC Levels</title><p>RBC data were provided in four studies, totaling 272 patients (136 cases in the JPBS therapy group and 136 cases in the WM treatment group) (<xref rid=\"B33\" ref-type=\"bibr\">Wang, 2013</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Li, 2016</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Du, 2017</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Zhang, 2017</xref>). No significant heterogeneity was found across the studies (<italic>I</italic>\n<sup>2</sup> = 9%, <italic>P</italic> = 0.35), and thus the fixed effect model was used (<xref ref-type=\"fig\" rid=\"f7\">\n<bold>Figure 7</bold>\n</xref>). Results indicated that the increase in serum RBC levels was greater in the JPBS therapy group than in the WM treatment group [WMD = 0.31, 95% CI: (0.24, 0.38), <italic>P</italic> < 0.00001].</p><fig id=\"f7\" position=\"float\"><label>Figure 7</label><caption><p>Forest plot of RBC levels.</p></caption><graphic xlink:href=\"fphar-11-560920-g007\"/></fig></sec><sec id=\"s3_3_5\"><title>HCT Levels</title><p>HCT data were provided in all included studies, totaling 799 patients (418 cases in the JPBS therapy group and 381 cases in the WM treatment group) (<xref rid=\"B16\" ref-type=\"bibr\">Li et al., 2003</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Cheng et al., 2005</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bao et al., 2008</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Zhou et al., 2008</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Liang, 2009</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Qu, 2011</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Wang, 2012</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Liu, 2013</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Wang, 2013</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Li, 2016</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Du, 2017</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Zhang, 2017</xref>). The fixed effect model was applied as no data heterogeneity was found among the studies (<italic>I</italic>\n<sup>2</sup> = 0%, <italic>P</italic>= 0.82) (<xref ref-type=\"fig\" rid=\"f8\">\n<bold>Figure 8</bold>\n</xref>). Compared with that in the WM treatment group, the level of serum HCT showed greater improvement in the JPBS therapy group (WMD = 2.95, 95% CI: (2.36, 3.54), <italic>P</italic> < 0.00001).</p><fig id=\"f8\" position=\"float\"><label>Figure 8</label><caption><p>Forest plot of HCT levels.</p></caption><graphic xlink:href=\"fphar-11-560920-g008\"/></fig></sec><sec id=\"s3_3_6\"><title>SCr Levels</title><p>SCr data were reported in all studies, totaling 799 patients (418 cases in the JPBS therapy group and 381 cases in the WM treatment group) (<xref rid=\"B16\" ref-type=\"bibr\">Li et al., 2003</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Cheng et al., 2005</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bao et al., 2008</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Zhou et al., 2008</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Liang, 2009</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Qu, 2011</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Wang, 2012</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Liu, 2013</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Wang, 2013</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Li, 2016</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Du, 2017</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Zhang, 2017</xref>). Data heterogeneity was found among the studies (<italic>I</italic>\n<sup>2</sup> = 64%, <italic>P</italic> = 0.002), and thus we applied the random effect model (<xref ref-type=\"fig\" rid=\"f9\">\n<bold>Figure 9</bold>\n</xref>). Compared with that in the WM treatment group, the level of serum SCr showed greater improvement in the JPBS therapy group (WMD = 64.57, 95% CI: (33.51, 95.64), <italic>P</italic> < 0.0001).</p><fig id=\"f9\" position=\"float\"><label>Figure 9</label><caption><p>Forest plot of SCr levels.</p></caption><graphic xlink:href=\"fphar-11-560920-g009\"/></fig></sec><sec id=\"s3_3_7\"><title>BUN Levels</title><p>BUN data were reported in 10 studies, totaling 659 patients (335 cases in the JPBS therapy group and 324 cases in the WM treatment group) (<xref rid=\"B16\" ref-type=\"bibr\">Li et al., 2003</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Cheng et al., 2005</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bao et al., 2008</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Liang, 2009</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Qu, 2011</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Wang, 2012</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Wang, 2013</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Li, 2016</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Du, 2017</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Zhang, 2017</xref>). Based on data heterogeneity analysis (<italic>I</italic>\n<sup>2</sup> = 63%, <italic>P</italic> = 0.004), the random effect model was adopted (<xref ref-type=\"fig\" rid=\"f10\">\n<bold>Figure 10</bold>\n</xref>). Results showed that the level of serum BUN decreased in the JPBS therapy group compared with that in the WM treatment group [WMD = 3.76, 95% CI: (2.21, 5.31), <italic>P</italic> < 0.00001].</p><fig id=\"f10\" position=\"float\"><label>Figure 10</label><caption><p>Forest plot of BUN levels.</p></caption><graphic xlink:href=\"fphar-11-560920-g010\"/></fig></sec></sec><sec id=\"s3_4\"><title>Adverse Event Reporting</title><p>Two studies reported adverse events. <xref rid=\"B20\" ref-type=\"bibr\">Liang (2009)</xref> reported six cases of high blood pressure in the control group. <xref rid=\"B19\" ref-type=\"bibr\">Li (2016)</xref> reported seven cases of high blood pressure in the JPBS therapy group (adverse event frequency of 7/60), as well as one case of allergic reaction and eight cases of high blood pressure in the control group (adverse event frequency of 9/60). The Renal Association Clinical Practice Guideline on Anemia of Chronic Kidney Disease suggests there may be an increase in the risk of allergic reaction during intravenous iron injection and of hypertension with ESA administration (<xref rid=\"B25\" ref-type=\"bibr\">Mikhail et al., 2017</xref>). Thus, the adverse events reported above may be caused by WM treatment. Based on the adverse events reported in the selected studies, JPBS therapy appears to be safe and does not show an increase in adverse reactions compared with WM alone.</p></sec><sec id=\"s3_5\"><title>Sensitivity Analysis</title><p>Due to the considerable heterogeneity in the SCr and BUN results found among the studies, sensitivity analysis was carried out to investigate the source of this data heterogeneity. Sensitivity analysis suggested that exclusion of any one study for each outcome did not alter the overall results, indicating that the conclusions were robust (<xref ref-type=\"fig\" rid=\"f11\">\n<bold>Figure 11</bold>\n</xref>).</p><fig id=\"f11\" position=\"float\"><label>Figure 11</label><caption><p>Sensitivity analysis plots of <bold>(A)</bold> SCr and <bold>(B)</bold> BUN.</p></caption><graphic xlink:href=\"fphar-11-560920-g011\"/></fig></sec><sec id=\"s3_6\"><title>Publication Bias</title><p>Publication bias was assessed based on the clinical efficacy and Hb, SCr, and BUN results. The clinical efficacy funnel plot revealed a slight asymmetry and Egger’s (<italic>t</italic> = 5.61, <italic>P</italic> = 0.001) and Begg’s tests (<italic>z</italic> = 2.15, <italic>P</italic> = 0.032) indicated possible publication bias (<xref ref-type=\"fig\" rid=\"f12\">\n<bold>Figures 12A–C</bold>\n</xref>). Visual assessment of funnel plots and Egger’s (Hb: <italic>t</italic> = 1.86, <italic>P</italic> = 0.093; SCr: <italic>t</italic> = 1.77, <italic>P</italic> = 0.107; BUN: <italic>t</italic> = 0.89, <italic>P</italic> = 0.398) and Begg’s test results (Hb: <italic>z</italic> = 1.85, <italic>P</italic> = 0.064; SCr: <italic>z</italic> = 1.85, <italic>P</italic> = 0.064; BUN: <italic>z</italic> = 0.89, <italic>P</italic> = 0.371) showed no publication bias for Hb, SCr, and BUN (<xref ref-type=\"fig\" rid=\"f12\">\n<bold>Figures 12D–L</bold>\n</xref>).</p><fig id=\"f12\" position=\"float\"><label>Figure 12</label><caption><p>Publication bias plots. <bold>(A)</bold> Funnel plot of clinical efficacy rate; <bold>(B)</bold> Egger’s plot of clinical efficacy rate; <bold>(C)</bold> Begg’s plot of clinical efficacy rate; <bold>(D)</bold> Funnel plot of Hb; <bold>(E)</bold> Egger’s plot of Hb; <bold>(F)</bold> Begg’s plot of Hb; <bold>(G)</bold> Funnel plot of SCr; <bold>(H)</bold> Egger’s plot of SCr<bold>; (I)</bold> Begg’s plot of SCr; <bold>(J)</bold> Funnel plot of BUN; <bold>(K)</bold> Egger’s plot of BUN; <bold>(L)</bold> Begg’s plot of BUN.</p></caption><graphic xlink:href=\"fphar-11-560920-g012\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Anemia is a common and serious complication for millions of patients with CKD (<xref rid=\"B23\" ref-type=\"bibr\">Locatelli et al., 2013</xref>). However, debate continues about the best strategies for the management of CKD anemia; for example, <xref rid=\"B30\" ref-type=\"bibr\">Solomon et al. (2010)</xref> found that treatment of CKD anemia with ESAs is inferior to that using placebo. In this context, current treatment of CKD anemic patients only reduces the risk of blood transfusion, rather than managing anemia and its associated risks. Nowadays, CHM is frequently applied to improve the symptoms and signs associated with CKD anemia. In accordance with TCM theory, the pathogenesis of CKD anemia is related to deficiencies of the spleen and kidney. Therefore, invigorating the spleen and reinforcing the kidney (Jianpi Bushen) are the main principles of TCM treatment for CKD anemia. In recent years, an increasing number of randomized controlled trials have been conducted using JPBS therapy for the treatment of CKD anemia, providing opportunities for objective and comprehensive evaluation of this method. Here, we performed a meta-analysis to clarify the efficacy of this treatment strategy.</p><p>The main findings of our meta-analysis were as follows: (1) compared with WM alone, JPBS therapy combined with WM improved the clinical efficacy rate; (2) compared with WM alone, JPBS therapy combined with WM significantly improved serum Hb, SF, RBC, and HCT levels in patients suffering CKD anemia; (3) adjunctive treatment with JPBS therapy decreased serum SCr and BUN levels in CKD anemic patients; and (4) JPBS therapy did not increase adverse reactions compared with WM alone. These results suggest that JPBS therapy may have beneficial effects in the management of CKD anemic patients.</p><p>The significant improvements in clinical efficacy rate and Hb, SF, RBC, HCT, SCr, BUN levels may be attributed to the promotion of hemoglobin formation and the renoprotective effects of CHM. In the included studies, the most frequently used Chinese medicines with the JPBS therapy were Astragali Radix, Angelicae Sinensis Radix, Codonopsis Radix, and Lycium Barbarum (<xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Supplementary Tables 1</bold>\n</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">\n<bold>2</bold>\n</xref>). The main components of Astragali Radix, i.e., flavonoids and polysaccharides, are reported to promote erythroid differentiation, increase fetal hemoglobin synthesis, and regulate EPO expression (<xref rid=\"B35\" ref-type=\"bibr\">Yang et al., 2010</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Zheng et al., 2011</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Zhang et al., 2017</xref>). Angelicae Sinensis Radix polysaccharides are thought to exhibit an anti-anemic effect, which can restore EPO production and improve iron availability <italic>via</italic> stabilizing HIF2α protein and suppressing inflammation (<xref rid=\"B31\" ref-type=\"bibr\">Wang et al., 2018</xref>). Codonopsis Radix polysaccharides significantly elevate peripheral blood Hb levels in mice (<xref rid=\"B11\" ref-type=\"bibr\">Gao et al., 2018</xref>). In respect of renoprotective effects, <xref rid=\"B38\" ref-type=\"bibr\">Zhao et al. (2016)</xref> reported that Lycium Barbarum polysaccharides improve renal function and alleviate kidney inflammation in diabetic rabbits. Therefore, a combination of these CHMs could theoretically alleviate anemia and improve renal function, with a clear therapeutic effect on CDK anemia.</p><p>The results of the present study are consistent with research from <xref rid=\"B17\" ref-type=\"bibr\">Li et al. (2004)</xref>. However, our study varies from previous published meta-analysis related to CKD anemia. For example, <xref rid=\"B39\" ref-type=\"bibr\">Zhao et al. (2017)</xref> suggested that Danggui Buxue Decoction combined with WM may be more effective in the treatment of CKD anemia than WM alone. However, they did not specifically reveal the TCM pathogenesis of CKD anemia and JPBS therapy. Based on another meta-analysis, <xref rid=\"B9\" ref-type=\"bibr\">Elliott et al. (2017)</xref> reported that ESAs may not have robust or reproducible benefits on renal function in humans. In our meta-analysis, JPBS therapy combined with WM showed positive effects on renal function. However, there are several limitations to this study. First, all included studies were conducted in China, which may limit the wide application of the results. Second, the methodological quality of the enrolled articles was generally low, with none providing a description of allocation concealment or blinding method, which may affect the credibility of the results. Third, there was a potential publication bias in regard to the clinical efficacy rate, which may be due to the flawed research design of small studies or the lack of publication of small studies with negative results. Lastly, data heterogeneity among the studies, including for partial results, was also significant, although sensitivity analysis indicated that our results were stable and reliable. The heterogeneity among the studies may be due to differences in treatment dose, intervention duration, and primary disease of CKD.</p><p>Our study demonstrated that JPBS therapy shows good potential for the treatment of CKD anemia, with the possibility of alleviating anemia while improving renal function. However, further research is needed to explore the possible mechanisms of JPBS therapy in the treatment of CKD anemia.</p></sec><sec id=\"s5\"><title>Conclusion</title><p>The results of this study indicate that JPBS therapy combined with WM is more effective than WM alone in terms of inducing remission in CKD anemia, especially in regard to clinical efficacy rates and improvement in Hb, SF, RBC, HCT, SCr, and BUN levels. However, our conclusions should be interpreted with some caution due to the relatively poor quality of the included studies. Therefore, to determine the clinical value of JPBS therapy in the treatment of CKD anemia, further standard, double-blind, randomized studies are needed.</p></sec><sec id=\"s6\"><title>Author Contributions</title><p>LL and YB designed the study. YZ, QX, and CL contributed to the data collection and data analysis. Results were interpreted by ZW and XZ. LL drafted the original manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec sec-type=\"funding-information\" id=\"s7\"><title>Funding</title><p>This study was supported by Academic Experience Inheritance of the Sixth National Group of Old Chinese Medicine Experts of the State Administration of Traditional Chinese Medicine [No. 2017 (29)].</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><title>Acknowledgments</title><p>We thank the two reviewers for their helpful comments, which have significantly improved the quality of this manuscript.</p></ack><sec id=\"s9\" sec-type=\"supplementary-material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fphar.2020.560920/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fphar.2020.560920/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Table_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM2\"><media xlink:href=\"Table_2.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name><surname>Babitt</surname><given-names>J. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Oncol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Oncol.</journal-id><journal-title-group><journal-title>Frontiers in Oncology</journal-title></journal-title-group><issn pub-type=\"epub\">2234-943X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33042796</article-id><article-id pub-id-type=\"pmc\">PMC7523513</article-id><article-id pub-id-type=\"doi\">10.3389/fonc.2020.01528</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Oncology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>A Myristoyl Amide Derivative of Doxycycline Potently Targets Cancer Stem Cells (CSCs) and Prevents Spontaneous Metastasis, Without Retaining Antibiotic Activity</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Ózsvári</surname><given-names>Béla</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/763336/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Magalhães</surname><given-names>Luma G.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/980199/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Latimer</surname><given-names>Joe</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/376256/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Kangasmetsa</surname><given-names>Jussi</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/983771/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Sotgia</surname><given-names>Federica</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/269370/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Lisanti</surname><given-names>Michael P.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/269289/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Translational Medicine, School of Science, Engineering and Environment (SEE), University of Salford</institution>, <addr-line>Manchester</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Salford Antibiotic Research Network, School of Science, Engineering and Environment (SEE), University of Salford</institution>, <addr-line>Manchester</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Eurofins Integrated Discovery UK Ltd.</institution>, <addr-line>Essex</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Lunella Biotech, Inc.</institution>, <addr-line>Ottawa, ON</addr-line>, <country>Canada</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Anna Sebestyén, Semmelweis University, Hungary</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Anna Maria Tokes, Semmelweis University, Hungary; Chris Albanese, Georgetown University, United States</p></fn><corresp id=\"c001\">*Correspondence: Federica Sotgia <email>fsotgia@gmail.com</email></corresp><corresp id=\"c002\">Michael P. Lisanti <email>michaelp.lisanti@gmail.com</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Cancer Metabolism, a section of the journal Frontiers in Oncology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>1528</elocation-id><history><date date-type=\"received\"><day>23</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>16</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Ózsvári, Magalhães, Latimer, Kangasmetsa, Sotgia and Lisanti.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Ózsvári, Magalhães, Latimer, Kangasmetsa, Sotgia and Lisanti</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Here, we describe the chemical synthesis and biological activity of a new Doxycycline derivative, designed specifically to more effectively target cancer stem cells (CSCs). In this analog, a myristic acid (14 carbon) moiety is covalently attached to the free amino group of 9-amino-Doxycycline. First, we determined the IC<sub>50</sub> of Doxy-Myr using the 3D-mammosphere assay, to assess its ability to inhibit the anchorage-independent growth of breast CSCs, using MCF7 cells as a model system. Our results indicate that Doxy-Myr is >5-fold more potent than Doxycycline, as it appears to be better retained in cells, within a peri-nuclear membranous compartment. Moreover, Doxy-Myr did not affect the viability of the total MCF7 cancer cell population or normal fibroblasts grown as 2D-monolayers, showing remarkable selectivity for CSCs. Using both gram-negative and gram-positive bacterial strains, we also demonstrated that Doxy-Myr did not show antibiotic activity, against <italic>Escherichia coli</italic> and <italic>Staphylococcus aureus</italic>. Interestingly, other complementary Doxycycline amide derivatives, with longer (16 carbon; palmitic acid) or shorter (12 carbon; lauric acid) fatty acid chain lengths, were both less potent than Doxy-Myr for the targeting of CSCs. Finally, using MDA-MB-231 cells, we also demonstrate that Doxy-Myr has no appreciable effect on tumor growth, but potently inhibits tumor cell metastasis <italic>in vivo</italic>, with little or no toxicity. In summary, by using 9-amino-Doxycycline as a scaffold, here we have designed new chemical entities for their further development as anti-cancer agents. These compounds selectively target CSCs, e.g., Doxy-Myr, while effectively minimizing the risk of driving antibiotic resistance. Taken together, our current studies provide proof-of-principle, that existing FDA-approved drugs can be further modified and optimized, to successfully target the anchorage-independent growth of CSCs and to prevent the process of spontaneous tumor cell metastasis.</p></abstract><kwd-group><kwd>cancer stem-like cells (CSCs)</kwd><kwd>Doxycycline</kwd><kwd>myristic acid</kwd><kwd>fatty acylation</kwd><kwd>cancer cell metastasis</kwd><kwd>prophylaxis of metastasis</kwd><kwd>9-amino-Doxycycline</kwd><kwd>antimitoscins</kwd></kwd-group><counts><fig-count count=\"14\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"33\"/><page-count count=\"14\"/><word-count count=\"7141\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Cancer stem cells (CSCs) are thought to be the root cause of recurrence, metastasis, and drug-resistance, in a host of cancer types (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). As a consequence, there is an unmet need to develop new therapeutics to target and selectively kill CSCs, while avoiding side effects, especially severe chemo-toxicity. Metastasis is believed to be driven by this unique sub-population of cancer-initiating cells (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B3\" ref-type=\"bibr\">3</xref>). CSCs have the ability to generate <italic>de novo</italic> tumors in immuno-deficient host organisms. Moreover, they have the capacity to engage in anchorage-independent growth, which facilitates their invasive spread throughout the various tissues and organ systems, resulting in local, and distant disseminated lesions (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Remarkably, these metastatic lesions are resistant to both chemo-therapy and radiation treatments. Unfortunately, the Achilles' heel of these pro-metastatic CSCs remains largely unknown. As a consequence, currently there are no anti-cancer drugs that are FDA-approved for the prevention of metastasis.</p><p>Over the last 5 years, we identified that mitochondria in CSCs may be a novel tractable therapeutic target, for inhibiting their anchorage-independent growth (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). More specifically, increased mitochondrial biogenesis may facilitate efficient high energy production, resulting in the rapid propagation of CSCs (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). In addition, within metastatic lymph nodes isolated from breast cancer patients, disseminated cancer cells show elevated levels of mitochondrial activity, especially Complex IV activation, as revealed by functional activity assays (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>).</p><p>Interestingly, mitochondrial biogenesis is critically linked to the activity of mitochondrial ribosomal proteins, that functionally translate key mitochondrial proteins, which are genetically encoded by mitochondrial DNA (mt-DNA); this includes 13 proteins that are necessary to functionally maintain OXPHOS and mitochondrial ATP synthesis (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B13\" ref-type=\"bibr\">13</xref>).</p><p>The reason that mitochondria have their own DNA and specific machinery for protein translation is that they originally evolved from engulfed aerobic bacteria, over the last 1.4 billion years, after invading eukaryotic cells. This evolutionary symbiotic relationship has certain functional consequences that could be safely exploited to achieve anti-mitochondrial therapy, specifically targeting CSCs. For example, because of the similarities between bacteria and mitochondria, a sub-set of bacteriostatic antibiotics block mitochondrial protein translation, as a manageable side effect (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). In this context, Doxycycline inhibits the activity of the small mito-ribosome, while Azithromycin blocks the large mito-ribosome, both as off-target side effects (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Similarly, Doxycycline and Azithromycin both inhibit the propagation of CSCs, in an anchorage-independent fashion, in numerous breast cancer cell lines, as well as in cell lines derived from many other solid tumor types (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). As such, we have suggested that these side-effects could be re-purposed to clinically target and therapeutically eradicate CSCs.</p><p>In direct support of this notion, a Phase II clinical trial has documented that brief treatment with Doxycycline, in early breast cancer patients, is indeed sufficient to significantly decrease the content of CSCs in the tumor mass, by employing CD44-staining as an established marker of CSCs in ER(+) patients (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Overall, the response rate approached 90%, resulting in reductions of up to nearly 67% in CSC tumor burden (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Quantitatively similar results were obtained in HER2(+) patients, using ALDH1 as a CSC marker. As such, blocking mitochondrial protein synthesis may be a viable approach for targeting and removing CSCs <italic>in vivo</italic>, prior to surgical excision, possibly preventing the development of metastases.</p><p>Consistent with the above observations, other groups have shown that Doxycycline treatment effectively reduces the expression of a panel of CSC markers, in breast cancer cell lines, such as CD44, ALDH, Oct4, Sox2, and Nanog (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>, <xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Moreover, Doxycycline treatment functionally inhibits multiple CSC signaling pathways, including Wnt, Notch, Hedgehog, and STAT1/3-signaling (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>).</p><p>Here, we describe a medicinal chemistry approach to design novel therapeutics to more selectively target CSCs. More specifically, we show that several new potent Doxycycline analogs can be generated by attaching a fatty acid onto 9-amino-Doxycycline, which is a relatively straightforward chemical modification. Importantly, these analogs, such as Doxy-Myr, lack antibiotic activity, but more potently target CSCs and effectively prevent metastasis, in an <italic>in vivo</italic> pre-clinical model. Therefore, Doxy-Myr could be used to eradicate CSCs, without exerting the selective pressures required for the development of antimicrobial resistance and without significant toxicity.</p><p>To better describe this general class of novel compounds which are lipid-modified FDA-approved antibiotics, we propose the term Antimitoscins, to specifically reflect their intrinsic anti-mitochondrial activity.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Materials</title><p>MCF7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (ATCC). hTERT-BJ1 fibroblasts were as we previously described (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Cells were cultured in DMEM, supplemented with 10% fetal calf serum (FCS), Glutamine and Pen/Strep.</p></sec><sec><title>Chemical Synthesis</title><p>Custom-chemical syntheses were performed by Eurofins Integrated Discovery UK Ltd., (Essex, UK). Conventional peptide synthesis methods were used to covalently attach each free fatty acid to 9-amino-Doxycycline. The desired reaction products were identified, chromatographically purified and the chemical structures were validated, by using a combination of NMR and mass spectrometry. The IUPAC names for the chemical compounds are as follows.</p><sec><title>Doxycycline</title><p>(4S,5S,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide.</p></sec><sec><title>Doxycycline-Myr</title><p>(4S,5S,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-9-(tetradecanoylamino)-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide.</p></sec><sec><title>Doxycycline-Laur</title><p>(4S,5S,6R,12aS)-4-(dimethylamino)-9-(dodecanoylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide.</p></sec><sec><title>Doxycycline-Pal</title><p>(4S,5S,6R,12aS)-4-(dimethylamino)-9-(hexadecanoylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide.</p></sec><sec><title>Doxycycline-TPP</title><p>[6-[[(5R,6S,7S,10aS)-9-carbamoyl-7-(dimethylamino)-1,6,8,10a,11-pentahydroxy-5-methyl-10,12-dioxo-5a,6,6a,7-tetrahydro-5H-tetracen-2-yl]amino]-6-oxo-hexyl]-triphenyl-phosphonium oxalate.</p></sec><sec><title>9-Amino-Doxycycline</title><p>(4S,5S,6R,12aS)-9-amino-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide.</p><p>9-Amino-Doxycycline was synthesized, essentially as previously described (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>).</p><p>See <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Materials and Methods</xref>, and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplemental Figure 1</xref>, for further details on the chemical synthesis of these Doxycycline derivatives.</p></sec></sec><sec><title>3D-Mammosphere Assay</title><p>The mammosphere assay is considered as the gold-standard for functionally measuring “stemness” and CSC propagation in breast cancer cells. Single cells are first plated at low density on low-attachment plates and >90% of “bulk” cancer cells die under these conditions, in a process of programmed cell death, termed anoikis. Only stem-like cells survive and propagate in suspension. Each 3D mammosphere is formed from a single CSC. Test compounds are added at the moment of single cell plating and then the number of 3D mammospheres are counted 5 days after plating. More specifically, a single cell suspension of MCF7 cells was prepared using enzymatic (1x Trypsin-EDTA, Sigma Aldrich) and manual disaggregation (25-gauge needle) (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Cells were then plated at a density of 500 cells/cm<sup>2</sup> in mammosphere medium (DMEM-F12/B27/ EGF (20-ng/ml)/PenStrep) in non-adherent conditions, in culture dishes coated with (2-hydroxyethylmethacrylate) (poly-HEMA, Sigma). Cells were then grown for 5 days and maintained in a humidified incubator at 37°C at an atmospheric pressure in 5% (v/v) carbon dioxide/air. After 5 days in culture, spheres >50 μm were counted using an eye-piece graticule, and the percentage of cells plated which formed spheres was calculated and is referred to as percent mammosphere formation, normalized to vehicle-alone treated controls. Mammosphere assays were performed in triplicate and repeated three times independently.</p></sec><sec><title>Fluorescence Imaging</title><p>Fluorescent images were taken after 72 h of incubation of MCF7 cells treated with either Doxycycline or Doxy-Myr (both at 10 μM), or vehicle control. Cell cultures were imaged with the EVOS Cell Imaging System (Thermo Fisher Scientific, Inc.), using the GFP channel. No fluorescent dye was used before imaging, therefore, any changes in signal were exclusively due to the auto-fluorescent nature of the Doxycycline compounds.</p></sec><sec><title>Cell Viability Assay</title><p>The Sulphorhodamine (SRB) assay is based on the measurement of cellular protein content (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). After treatment for 72 h in 96-well plates (8,000 cells/well), cells were fixed with 10% trichloroacetic acid (TCA) for 1 h in the cold room, and were dried overnight at room temperature. Then, cells were incubated with SRB for 15 min, washed twice with 1% acetic acid, and air dried for at least 1 h. Finally, the protein-bound dye was dissolved in a 10 mM Tris, pH 8.8, solution and read using the plate reader at 540-nm.</p></sec><sec><title>Cell Proliferation</title><p>Briefly, MCF7 cells were seeded in each well (10,000 cells/well) and employed to assess the efficacy of Doxycycline and Doxy-Myr, using RTCA (real-time cell analysis), via the measurement of cell-induced electrical impedance plate (Acea Biosciences Inc.) (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). This approach allows the quantification of the onset and kinetics of the cellular response. Experiments were repeated several times independently, using quadruplicate samples for each condition.</p></sec><sec><title>Cell Cycle Analysis</title><p>We performed cell-cycle analysis on MCF7 cells treated with Doxycycline, Doxy-Myr, or vehicle-alone. Briefly, after trypsinization, the re-suspended cells were incubated with 10 ng/ml of Hoechst solution (Thermo Fisher Scientific) for 40 min at 37°C under dark conditions. Following a 40 min period, the cells were washed and re-suspended in PBS Ca/Mg for acquisition on the Attune NxT flow cytometer (Thermo Scientific). We analyzed 10,000 events per condition. Gated cells were manually-categorized into cell-cycle stages (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>).</p></sec><sec><title>Bacterial Growth Assays</title><p>Briefly, antibiotic activity was assessed using standard assay systems (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>–<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). The antibiotic activity of Doxycycline analogs was determined experimentally, using Resazurin (R7017; Sigma-Aldrich, Inc.) as an indicator of bacterial metabolism/vitality, in a 96-well plate format, using <italic>Escherichia coli</italic> and <italic>Staphylococcus aureus</italic>. The minimum inhibitory concentration (MIC) for the studied compounds was determined using the broth microdilution method, the reference susceptibility test for rapidly growing aerobic or facultative microorganism(s). The assays were performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The test compounds and positive control (doxycycline, Sigma Aldrich #D1822) stock solutions were prepared at 25 mM in DMSO and serially diluted (2-fold dilution from 200 to 1.56 μM) in cation adjusted Mueller Hinton Broth (MHB, Sigma Aldrich #90922) in 96-well transparent plates (VWR #734-2781) into a final volume of 50 μL/well. <italic>Staphylococcus aureus</italic> (ATCC 29213) and <italic>E. coli</italic> (ATCC 25922) cultures were maintained on Mueller Hinton Agar (MHA, Sigma Aldrich #70191). A single colony of each strain was then grown overnight at 37 °C in MHB until OD<sub>600</sub> ~ 0.6–0.8 and diluted into MHB to a concentration of 106 colony forming units (CFU)/mL, which was equivalent to an OD<sub>600</sub> ~ 0.01. Diluted inocula (50 μL) were transferred to the wells of the previously prepared 96-well plates containing the test compounds, negative control (1% DMSO in MHB) and positive control (Doxycycline). Final wells volume was 100 μL, final concentrations for the testing compounds were between 100 and 0.78 μM and final microorganism concentration was 5 × 10<sup>5</sup> CFU/mL. Subsequently, 10 μL of one negative control well was plated in a petri dish containing MHA to check CFU and the purity of the cultures. The plates were incubated at 37°C for 24 h after which 20 μL of resazurin solution (0.2 mg/mL) was added to the wells followed by 1 h 30 min incubation at 37°C. The OD<sub>570</sub> and OD<sub>600</sub> were measured in a microplate reader (BMG FLUOstar Omega). The ratio between OD<sub>570</sub> and OD<sub>600</sub> was determined and the MIC represents the lowest concentration of compound that inhibited bacterial growth (OD<sub>570</sub>/OD<sub>600</sub> ratio inferior to the average ratio determined for negative control wells). MIC values were determined by three independent experiments.</p></sec><sec><title>Assays for Tumor Growth, Metastasis, and Embryo Toxicity</title><p>These xenograft assays were carried out, essentially as previously described, without any major modifications (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>–<xref rid=\"B27\" ref-type=\"bibr\">27</xref>).</p><sec><title>Preparation of Chicken Embryos</title><p>Fertilized White Leghorn eggs were incubated at 37.5°C with 50% relative humidity for 9 days. At that moment (E9), the chorioallantoic membrane (CAM) was dropped down by drilling a small hole through the eggshell into the air sac, and a 1 cm<sup>2</sup> window was cut in the eggshell above the CAM.</p></sec><sec><title>Amplification and Grafting of Tumor Cells</title><p>The MDA-MB-231 tumor cell line was cultivated in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. On day E9, cells were detached with trypsin, washed with complete medium and suspended in graft medium. An inoculum of 1 × 10<sup>6</sup> cells was added onto the CAM of each egg (E9) and then eggs were randomized into groups.</p></sec><sec><title>Tumor Growth Assays</title><p>At day 18 (E18), the upper portion of the CAM was removed from each egg, washed in PBS and then directly transferred to paraformaldehyde (fixation for 48 h) and weighed. For tumor growth assays, at least 10 tumor samples were collected and analyzed per group (<italic>n</italic> ≥ 10).</p></sec><sec><title>Metastasis Assays</title><p>On day E18, a 1 cm<sup>2</sup> portion of the lower CAM was collected to evaluate the number of metastatic cells in 8 samples per group (<italic>n</italic> = 8). Genomic DNA was extracted from the CAM (commercial kit) and analyzed by qPCR with specific primers for Human Alu sequences. Calculation of Cq for each sample, mean Cq, and relative amounts of metastases for each group are directly managed by the Bio-Rad® CFX Maestro software. A one-way ANOVA analysis with post-tests was performed on all the data.</p></sec><sec><title>Embryo Tolerability Assay</title><p>Before each administration, the treatment tolerability was evaluated by scoring the number of dead embryos.</p></sec></sec><sec><title>Statistical Analysis</title><p>Statistical significance was determined using the Student's <italic>t</italic>-test, values of <0.05 were considered significant. Data are shown as the mean ± SEM, unless stated otherwise. Also, ANOVA was conducted, where appropriate.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Generating New Analogs of Doxycycline for Targeting CSCs: Doxy-Myr and Doxy-TPP</title><p>Doxycycline is known to function as an inhibitor of the propagation of CSCs, through its ability to inhibit the small mitochondrial ribosome, which is an off-target side-effect (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Normally, Doxycycline is used as a broad-spectrum bacteriostatic antibiotic to treat a wide range of infections caused by gram-negative and gram-positive bacteria. Therefore, we sought to optimize the ability of Doxycycline for the targeting of CSCs, while minimizing its antibiotic activity, to derive a new chemical entity to selectively target CSCs.</p><p>As a first step, we synthesized 9-amino-Doxycycline, to which we covalently attached either a 14 carbon fatty acid moiety (myristic acid) or a six carbon spacer arm containing tri-phenyl-phosphonium (TPP). The chemical structures of these two Doxycycline analogs, as well as the parent compound, are all shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>. We speculated that the TPP-moiety would better target Doxy-TPP to mitochondria in CSCs. In contrast, the addition of myristic acid could act as a membrane targeting signal, possibly leading to the increased retention of Doxy-Myr within membranous compartments, such as the plasma membrane, the endoplasmic reticulum (ER), the Golgi apparatus, and/or mitochondria.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Chemical structures of two new Doxycycline derivatives: Doxy-Myr and Doxy-TPP. Note that Doxy-Myr contains a 14-carbon fatty acid (myristate) covalently attached to 9-amino-Doxycycline and Doxy-TPP contains a TPP-moiety attached via a 6-carbon spacer to 9-amino-Doxycycline.</p></caption><graphic xlink:href=\"fonc-10-01528-g0001\"/></fig><p>To determine the functional activity of these Doxycycline analogs, we used the 3D-mammosphere assay, to assess their ability to inhibit the 3D anchorage-independent propagation of MCF7 CSCs. Interestingly, Doxy-TPP was not more potent that Doxycycline itself, so further assays with Doxy-TPP were not carried out (data not shown).</p><p><xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref> shows a direct comparison of Doxycycline with Doxy-Myr. Remarkably, Doxy-Myr was >5-fold more potent than Doxycycline, with an IC<sub>50</sub> of 3.46 μM. In contrast, Doxycycline had an IC<sub>50</sub> of 18.1 μM. Therefore, Doxy-Myr is more potent for targeting the 3D anchorage-independent propagation of CSCs.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Doxy-Myr more potently inhibits the anchorage-independent propagation of CSCs. <bold>(A)</bold> MCF7 cells were plated under anchorage-independent growth conditions and the number of 3D-mammospheres were counted after 5 days. Note that Doxycycline and Doxy-Myr both inhibited 3D-mammosphere formation, all relative to vehicle-alone controls. However, Doxy-Myr was >5-fold more potent than Doxycycline (IC-50 of 3.46 vs. 18.1 μM). <bold>(B)</bold> Representative phase images of 3D-mammospheres are shown. Bar = 200 μm.</p></caption><graphic xlink:href=\"fonc-10-01528-g0002\"/></fig><p>To experimentally test the hypothesis that Doxy-Myr was better retained within cells, we took advantage of the observation that Doxycycline is fluorescent (Ex. 390–425 nm/Em. 520–560 nm). Doxy-Myr was more easily detected and retained in monolayer MCF7 cells, when compared to Doxycycline or cells treated with vehicle alone (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). Doxy-Myr fluorescence showed a peri-nuclear staining pattern, consistent with its partitioning and retention within intracellular membranous compartments. This observation could mechanistically explain its increased potency. No nuclear staining for Doxy-Myr was observed, indicating that it was predominantly excluded from the nucleus.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Doxy-Myr is better retained within cells and reveals a peri-nuclear staining pattern. MCF7 cells were cultured for 72 h as 2D-monolayers, in the presence of Doxycycline or Doxy-Myr, at a concentration of 10 μM. Vehicle-alone controls were processed in parallel. Then, MCF7 cells were washed twice with PBS and subjected to live cell imaging to capture the auto-fluorescent signal retained within cells. Note that Doxy-Myr showed the strongest intracellular retention, and was concentrated within peri-nuclear intracellular compartments. No nuclear staining was observed. Quantitation of mean pixel intensity, using Image J software, revealed that relative to Doxycycline, Doxy-Myr showed a near 3-fold increase in intracellular fluorescence.</p></caption><graphic xlink:href=\"fonc-10-01528-g0003\"/></fig></sec><sec><title>Doxy-Myr Is Non-toxic in 2D-Monolayers of MCF7 Cells and Normal Human Fibroblasts</title><p>To further assess the effects of Doxy-Myr on 2D-cell growth, we next used MCF7 cells and normal human fibroblasts (hTERT-BJ1) treated over a period of 3 days. <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref> shows that both Doxycycline and Doxy-Myr had no appreciable effects on cell viability, as determined over the concentration range of 5–20 μM.</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Doxy-Myr does not affect the viability of MCF7 cells or normal fibroblasts, when grown as 2D-monolayers. To determine the effects of Doxy-Myr on cell viability, we next treated MCF7 cells and normal human fibroblasts (hTERT-BJ1) over a period of 3 days. Note that Doxycycline and Doxy-Myr had no appreciable effects on cell viability, in the concentration range of 5–20 μM. <bold>(A)</bold> MCF7 cells, <bold>(B)</bold> hTERT-BJ1 cells.</p></caption><graphic xlink:href=\"fonc-10-01528-g0004\"/></fig><p>Potential 2D-effects on cell proliferation and the cell cycle were also determined using MCF7 cell monolayers. Clearly, Doxycycline and Doxy-Myr did not inhibit the proliferation of MCF7 cells, as assessed using the xCELLigence (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). Similarly, relative to the parent compound Doxycycline, Doxy-Myr did not have any significant effects on reducing cell cycle progression in 2D-monolayers of MCF7 cells (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>).</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Doxy-Myr does not inhibit the proliferation of MCF7 cell 2D-monolayers. Potential effects of Doxycycline and Doxy-Myr on cell proliferation were determined using MCF7 cell monolayers. Note that Doxycycline and Doxy-Myr did not inhibit the proliferation of MCF7 cells, as assessed using the xCELLigence, in the concentration range of 5–20 μM. *<italic>p</italic> < 0.05.</p></caption><graphic xlink:href=\"fonc-10-01528-g0005\"/></fig><fig id=\"F6\" position=\"float\"><label>Figure 6</label><caption><p>Doxy-Myr does not induce cell cycle arrest, in MCF7 2D-monolayers. MCF7 cells were cultured for 72 h as 2D-monolayers, in the presence of Doxycycline or Doxy-Myr, at a concentration of 10 μM. Vehicle-alone controls were processed in parallel. Note that relative to the parent compound Doxycycline, Doxy-Myr did not have any significant effects on reducing cell cycle progression in 2D-monolayers of MCF7 cells. Representative FACS cell cycle profiles are shown.</p></caption><graphic xlink:href=\"fonc-10-01528-g0006\"/></fig><p>Therefore, overall Doxy-Myr did not significantly reduce the viability, proliferation or cell cycle progression of 2D-monolayers of MCF7 cells, indicating that its effects were specific for cell propagation under 3D anchorage-independent growth conditions.</p></sec><sec><title>Doxy-Laur and Doxy-Pal Are Less Potent Than Doxy-Myr in Targeting CSCs</title><p>We also synthesized two other new Doxycycline analogs to study the influence of the fatty acid chain length on their functional activity. These two analogs included Doxy-Laur (harboring a 12 carbon fatty acid) and Doxy-Pal (harboring a 16 carbon fatty acid) (<xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref>). Therefore, we directly compared the functional inhibitory activity of Doxy-Myr, Doxy-Laur, and Doxy-Pal in the 3D-mammosphere assay, using MCF7 cells. Interestingly, <xref ref-type=\"fig\" rid=\"F8\">Figure 8</xref> demonstrates that the rank order potency is: Doxy-Myr > Doxy-Laur > Doxy-Pal > Doxycycline, with no direct correlation observed between chain length and activity. As such, the addition of myristic acid (a 14 carbon fatty acid) appears to be the optimal chain length modification.</p><fig id=\"F7\" position=\"float\"><label>Figure 7</label><caption><p>Chemical structures of two other Doxycycline derivatives: Doxy-Laur and Doxy-Pal. We created two other new Doxycycline analogs by varying the chain length of the fatty acid attached to 9-amino-Doxycycline. These two analogs included Doxy-Laur (harboring a 12 carbon fatty acid) and Doxy-Pal (harboring a 16 carbon fatty acid). Their chemical structures are as shown.</p></caption><graphic xlink:href=\"fonc-10-01528-g0007\"/></fig><fig id=\"F8\" position=\"float\"><label>Figure 8</label><caption><p>Rank order potency of the new Doxycycline derivatives. Note that Doxy-Myr is the most potent Doxycycline derivative for targeting CSC propagation, as assayed using the 3D mammosphere assay to quantitatively measure anchorage-independent growth. The rank order potency is Doxy-Myr > Doxy-Laur > Doxy-Pal > Doxycycline.</p></caption><graphic xlink:href=\"fonc-10-01528-g0008\"/></fig></sec><sec><title>Doxy-Myr, Doxy-Laur, and Doxy-Pal Lack Antibiotic Activity Against Common Gram-Negative and Gram-Positive Bacteria</title><p>Doxycycline is a well-established broad-spectrum antibiotic, that is routinely used for therapeutically targeting both gram-negative and gram-positive bacterial infections. As a consequence, we also assessed the antibiotic activity of the Doxycycline analogs, as compared to the parent compound Doxycycline.</p><p><xref ref-type=\"fig\" rid=\"F9\">Figure 9</xref> reveals that, as expected, Doxycycline potently and effectively inhibits the growth of both gram-negative (<italic>E. coli</italic>) and gram-positive (<italic>S. aureus</italic>) micro-organisms. Minimum inhibitory concentrations were 3.125 μM (1.3 mg/L) and 12.5 μM (5.5 mg/L), respectively. However, in striking contrast, Doxy-Myr, Doxy-Laur, and Doxy-Pal did not show any obvious antibiotic activity, in the same concentration range. Minimum inhibitory concentrations of the Doxycycline analogs were >100 μM (>65 mg/L). The clinical breakpoints for tetracyclines against <italic>E. coli</italic> and <italic>S. aureus</italic> are between 0.5–2.0 mg/L and 2.0–6.0 mg/L, respectively (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>).</p><fig id=\"F9\" position=\"float\"><label>Figure 9</label><caption><p>Doxy-Myr, Doxy-Laur and Doxy-Pal do not show any residual antibiotic activity. The novel Doxycycline analogs (Doxy-Myr, Doxy-Laur, and Doxy-Pal) were screened against a gram-positive (<italic>S. aureus</italic>; ATCC 29213) and a gram-negative (<italic>E. coli</italic>; ATCC 25922) strain of bacteria to verify the maintenance of the antibiotic activity, when compared to the parent compound. While Doxycycline presented MIC values of 3.125 μM against <italic>E. coli</italic> and 12.5 μM against <italic>S. aureus</italic>, none of the Doxycycline analogs inhibited bacterial growth up to the concentration of 100 μM.</p></caption><graphic xlink:href=\"fonc-10-01528-g0009\"/></fig><p>Therefore, these simple chemical modifications of Doxycycline have successfully removed its ability to act as a functional antibiotic, while simultaneously increasing its specificity for targeting CSCs.</p></sec><sec><title>Doxy-Myr Potently Inhibits Cancer Cell Metastasis <italic>in vivo</italic>, Without Significant Toxicity</title><p>To experimentally evaluate the functional effects of Doxycycline and Doxy-Myr <italic>in vivo</italic>, we next used MDA-MB-231 cells and the well-established chorio-allantoic membrane (CAM) assay in chicken eggs, to quantitatively measure tumor growth and metastasis (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). MDA-MB-231 breast cancer cells were used for our <italic>in vivo</italic> studies, as they are estrogen-independent, intrinsically more aggressive, form larger tumors and are significantly more migratory, invasive and metastatic. As such, they are a better <italic>in vivo</italic> model, for simultaneously evaluating both tumor growth and spontaneous metastasis. Moreover, we have previously demonstrated that Doxycycline also effectively inhibits the 3D anchorage-independent growth of MDA-MB-231 cells (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>).</p><p>Briefly, an inoculum of 1 × 10<sup>6</sup> MDA-MB-231 cells was added onto the Upper CAM of each egg (day E9) and then eggs were randomized into groups. On day E10, tumors were detectable and they were then treated daily for 8 days with vehicle alone (1% DMSO in PBS), Doxycycline or Doxy-Myr. After 8 days of drug administration, on day E18 all tumors were weighed, and the Lower CAM was collected to evaluate the number of metastatic cells, as analyzed by qPCR with specific primers for Human Alu sequences.</p><p>Morphologically, the CAM is constructed of two opposing sheets of epithelial cells, which are separated by a middle stromal layer, containing blood vessels and lymphatics. One epithelial layer is of ectodermal origin, while the other epithelial layer is of mesodermal/endodermal origin. Importantly, movement of metastatic MDA-MB-231 cells from the Upper CAM to the Lower CAM involves their migration away from the primary tumor, cellular invasion, intravasation, extravasation, and the formation of a new distant lesion, all of the normal steps that are key features of spontaneous tumor cell metastasis (see <xref ref-type=\"supplementary-material\" rid=\"SM3\">Supplemental Figure 2</xref>).</p><p><xref ref-type=\"fig\" rid=\"F10\">Figure 10</xref> shows the effects of Doxycycline and Doxy-Myr on MDA-MB-231 tumor growth. Note that that they both did not show any significant effects on tumor growth, as a result of the 8-day period of drug administration.</p><fig id=\"F10\" position=\"float\"><label>Figure 10</label><caption><p>Doxycycline and Doxy-Myr have no effect on tumor growth. MDA-MB-231 cells and the well-established chorio-allantoic membrane (CAM) assay in chicken eggs were used to quantitatively measure tumor growth. An inoculum of 1 × 10<sup>6</sup> MDA-MB-231 cells was added onto the Upper CAM of each egg (on Day E9) and then eggs were then randomized into groups. On day E10, tumors were detectable and they were then treated daily for 8 days with vehicle alone (1% DMSO in PBS), Doxycycline or Doxy-Myr. After 8 days of drug administration, on day E18 all tumors were weighed. Note that Doxycycline and Doxy-Myr did not have any significant effects on tumor growth. Averages are shown ± SEM. NS, not significant.</p></caption><graphic xlink:href=\"fonc-10-01528-g0010\"/></fig><p>However, both Doxycycline and Doxy-Myr showed significant effects on MDA-MB-231 cancer cell metastasis. <xref ref-type=\"fig\" rid=\"F11\">Figure 11</xref> illustrates that Doxycycline inhibited metastasis (by 44–57.5%). In contrast, Doxy-Myr inhibited metastasis (by 85–87%), at the same concentrations tested. Interestingly, the effects of Doxy-Myr on metastasis were significantly more pronounced.</p><fig id=\"F11\" position=\"float\"><label>Figure 11</label><caption><p>Doxycycline and Doxy-Myr selectively target and prevent cancer metastasis. MDA-MB-231 cells and the well-established chorio-allantoic membrane (CAM) assay in chicken eggs were used to quantitatively measure spontaneous tumor metastasis. An inoculum of 1 × 10<sup>6</sup> MDA-MB-231 cells was added onto the Upper CAM of each egg (on day E9) and then eggs were then randomized into groups. On day E10, tumors were detectable and they were then treated daily for 8 days with vehicle alone (1% DMSO in PBS), Doxycycline or Doxy-Myr. After 8 days of drug administration, the Lower CAM was collected to evaluate the number of metastatic cells, as analyzed by qPCR with specific primers for Human Alu sequences. Note that Doxycycline and Doxy-Myr both showed significant effects on MDA-MB-231 metastasis. However, Doxy-Myr was clearly more effective than Doxycycline in inhibiting metastasis. Averages are shown ± SEM. ****<italic>p</italic> < 0.0001.</p></caption><graphic xlink:href=\"fonc-10-01528-g0011\"/></fig><p>Surprisingly, little or no embryo toxicity was observed for Doxycycline and Doxy-Myr (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Therefore, we conclude that Doxy-Myr can be further developed as an anti-metastatic agent, selectively inhibiting tumor metastasis, without showing significant toxicity or antibiotic activity.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Chick embryo toxicity of Doxycycline and Doxy-Myr.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Group. #</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Group description</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Total</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Alive</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Dead</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>% Alive</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>% Dead</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Neg. Ctrl.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">83.33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.67</td></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Doxy, 0.125 mM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">84.62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.38</td></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Doxy, 0.250 mM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76.92</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.08</td></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Doxy-Myr, 0.125 mM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78.57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.43</td></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Doxy-Myr, 0.250 mM</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92.31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.69</td></tr></tbody></table></table-wrap></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Here, we report the chemical synthesis and biological activity of several new Doxycycline analogs, modified to increase their effectiveness in the targeting of CSCs. The most promising compound was Doxy-Myr, a Doxycycline analog in which a myristic acid (14 carbon) moiety is covalently attached to the free amino group of 9-amino-Doxycycline. We analyzed the potency of Doxy-Myr, using the 3D-mammosphere assay, to assess its potential inhibitory effects on the anchorage-independent propagation of breast CSCs. Overall, we observed that Doxy-Myr is >5-fold more potent than Doxycycline, the parent compound. Moreover, Doxy-Myr showed better intracellular retention, and was specifically localized within a peri-nuclear membranous compartment. In striking contrast, when MCF7 breast cancer cells or normal fibroblasts were grown as 2D-monolayers, Doxy-Myr did not reveal any effects on cell viability or proliferation, highlighting its unique selectivity for targeting the 3D-propagation of CSCs. In addition, we evaluated other Doxycycline analogs, with longer (16 carbon; palmitic acid) or shorter (12 carbon; lauric acid) chain lengths (<xref ref-type=\"fig\" rid=\"F12\">Figure 12</xref>). However, these two analogs were less effective than Doxy-Myr for targeting of CSCs. Finally, using MDA-MB-231 cells, we demonstrated that Doxy-Myr has no appreciable effects on tumor growth, but potently inhibits tumor cell metastasis <italic>in vivo</italic>, with little or no chick embryo toxicity.</p><fig id=\"F12\" position=\"float\"><label>Figure 12</label><caption><p>Schematic diagram highlighting our systematic approach to generating Doxycycline derivatives, to target CSCs and prevent metastasis. Lipid moieties were covalently conjugated to 9-amino-Doxycycline. Importantly, these three Doxycycline derivatives lack anti-microbial activity.</p></caption><graphic xlink:href=\"fonc-10-01528-g0012\"/></fig><p>Our results also showed that the lipophilic amide substituents in Doxycycline on the C9 of the Tetracycline (TC) skeleton led to the loss of its antibacterial activity. Previously published structure-activity relationship (SAR) studies have shown that chemical modification of the TC skeleton on C9 can be tolerated, leading to diverse antibacterial activity, as is exemplified by the antibiotic Tigecycline. The lipophilicity of the TCs seems to play a key role in the biological potency of this family of drugs. There is a trend of decreased antibiotic activity with the increase of lipophilicity, specifically against gram-negative species, with eventual loss of activity when high lipophilicity is achieved, which could partially explain the loss of activity, we observed after the addition of fatty acids to the Doxycycline scaffold.</p><p>Importantly, our improvement in the biological properties of these Doxycycline analogs for targeting CSCs and the associated loss of the anti-microbial activity, make these new analogs extremely promising, because tetracycline resistance among gram-negative and gram-positive pathogens requires exposure to inhibitory concentrations as a selective pressure (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>–<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). MICs exceeding 65 mg/L also suggest that these analogs might be non-inhibitory to members of the human microbiome, the complex community of microorganisms which exerts wide-ranging effects on human immunity and disease (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>, <xref rid=\"B30\" ref-type=\"bibr\">30</xref>).</p><p>Interestingly, a previous study successfully used the parent compound, Doxycycline, to prevent bone metastasis in a mouse model, by employing MDA-MD-231 cells (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). However, these authors did not examine the effects of Doxycycline on tumor growth, but only focused on bone metastasis. They attributed the efficacy of Doxycycline to its tropism for bone and to its ability to act as a protease inhibitor for lysosomal cysteine proteinases, the cathepsins, and MMPs, because Doxycycline behaves as a zinc chelator.</p><p>In contrast, herein, we have demonstrated that Doxycycline and Doxy-Myr both act as inhibitors of metastasis, by targeting the 3D anchorage-independent growth of CSCs, which is a completely different molecular mechanism (<xref ref-type=\"fig\" rid=\"F13\">Figures 13</xref>, <xref ref-type=\"fig\" rid=\"F14\">14</xref>). However, as predicted, Doxy-Myr was significantly more effective than Doxycycline, at the same concentrations examined. As such, based on these functional observations, we propose that this overall lipid modification strategy may be generally applicable, to facilitate the development and discovery of other drugs, for effectively preventing tumor progression, recurrence, and distant metastasis.</p><fig id=\"F13\" position=\"float\"><label>Figure 13</label><caption><p><italic>In vitro</italic> inhibition of 3D-growth predicts <italic>in vivo</italic> inhibition of metastasis. Here, we used two complementary breast cancer cell lines for our <italic>in vitro</italic> screening (MCF7) and <italic>in vivo</italic> (MDA-MB-231) validation assays. More specifically, Doxy-Myr had no effect on 2D-growth <italic>in vitro</italic> and no effect on tumor growth <italic>in vivo</italic>. Conversely, we showed that Doxy-Myr potently inhibited 3D-growth <italic>in vitro</italic>, which directly correlated with inhibition of metastasis <italic>in vivo</italic>. In support of this observation, 3D anchorage-independent growth is thought to be a required step for metastasis <italic>in vivo</italic>. N.E., no effect.</p></caption><graphic xlink:href=\"fonc-10-01528-g0013\"/></fig><fig id=\"F14\" position=\"float\"><label>Figure 14</label><caption><p>Summary: Properties of Doxy-Myr. Briefly, Doxy-Myr is a lipid modified Doxycycline derivative. Our results show that Doxy-Myr potently targets CSCs and selectively prevents metastasis, without affecting tumor growth. Moreover, Doxy-Myr was non-toxic in the chick embryo assay and did not affect the viability of normal cells, or MCF7 cells, grown as a 2D-monolayer. Importantly, Doxy-Myr lacked antibiotic activity, and did not affect the growth of gram-positive (<italic>E. coli</italic>) or gram-negative (<italic>S. aureus</italic>) organisms.</p></caption><graphic xlink:href=\"fonc-10-01528-g0014\"/></fig><p>Moreover, our current results are consistent with recent studies showing that prophylaxis with other classes of mitochondrial inhibitors is indeed sufficient to prevent metastasis, using the same pre-clinical xenograft model, with little or no effect on tumor growth and minimal toxicity (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>).</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>All datasets generated for this study are included in the article/<xref ref-type=\"sec\" rid=\"s8\">Supplementary Material</xref>.</p></sec><sec id=\"s6\"><title>Author Contributions</title><p>ML and FS conceived and initiated this project, they selected the clinically-approved drug Doxycycline for chemical modification and optimization by medicinal chemistry. JK performed the custom-chemical syntheses. The phenotypic drug screening and the majority of other wet-lab experiments described in this paper were performed by BÓ. LM determined the antibiotic activity of Doxycycline and its derivatives. BÓ, JK, and LM generated the final figures for the paper. ML and FS wrote the first draft of the manuscript, which was then further edited and approved by BÓ, LM, JL, JK, FS, and ML. ML generated the schematic summary diagrams. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s7\"><title>Conflict of Interest</title><p>JK is employed by the company Eurofins Integrated Discovery UK Ltd. FS holds a part-time affiliation with Lunella Biotech, Inc. ML holds a part-time affiliation with Lunella Biotech, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>We are grateful to Rumana Rafiq, for her kind and dedicated assistance, in keeping the Translational Medicine Laboratory at Salford running smoothly. We would like to thank the Foxpoint Foundation (Canada) and the Healthy Life Foundation (UK) for their philanthropic donations toward new equipment and infrastructure, in the Translational Medicine Laboratory at the University of Salford. We are thankful to Inovotion, Inc. (Grenoble, France), for independently performing the tumor growth and metastasis studies, using the CAM assay, as well as evaluating chicken embryo toxicity, through a research contract with Lunella Biotech, Inc. (Ottawa, Canada).</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by research grant funding, provided by Lunella Biotech, Inc. The funder, Lunella Biotech, Inc., provided the necessary monetary resources to carry out the current study.</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s8\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fonc.2020.01528/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fonc.2020.01528/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.PDF\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM2\"><label>Supplemental Figure 1</label><caption><p>Chemical synthesis of Doxycycline derivatives.</p></caption><media xlink:href=\"Image_1.tiff\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM3\"><label>Supplemental Figure 2</label><caption><p>CAM model for measuring tumor growth, metastasis, and embryo toxicity. On day E9, an inoculum of 1 million MDA-MB-231 breast tumor cells was layered on top of the Upper CAM and was allowed to form a primary tumor. Potential therapeutics were applied for a period of 8-days. Then, on day E18, the primary tumor was harvested from the upper CAM and the magnitude of distant metastases was quantitated in the Lower CAM, by performing qPCR with specific primers for recognizing Human Alu sequences. In order for the cells to metastasize, from the Upper CAM to the Lower CAM, it has been established that they need to undergo migration, invasion, intravasation, extravasation, and secondary lesion formation. Toxicity was measured by scoring embryo viability on day E18. See <italic>Materials and Methods</italic> for further details. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Rev Soc Bras Med Trop</journal-id><journal-id journal-id-type=\"iso-abbrev\">Rev. Soc. Bras. Med. Trop</journal-id><journal-id journal-id-type=\"publisher-id\">rsbmt</journal-id><journal-title-group><journal-title>Revista da Sociedade Brasileira de Medicina Tropical</journal-title></journal-title-group><issn pub-type=\"ppub\">0037-8682</issn><issn pub-type=\"epub\">1678-9849</issn><publisher><publisher-name>Sociedade Brasileira de Medicina Tropical - SBMT</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32997052</article-id><article-id pub-id-type=\"pmc\">PMC7523519</article-id><article-id pub-id-type=\"doi\">10.1590/0037-8682-0216-2020</article-id><article-id pub-id-type=\"other\">00353</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Major Article</subject></subj-group></article-categories><title-group><article-title>Health-risk assessment of Portuguese man-of-war (<italic>Physalia\nphysalis</italic>) envenomations on urban beaches in São Luís city, in the\nstate of Maranhão, Brazil</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Cavalcante</surname><given-names>Mayana Mendes e Silva</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rodrigues</surname><given-names>Zulimar Márita Ribeiro</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hauser-Davis</surname><given-names>Rachel Ann</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Siciliano</surname><given-names>Salvatore</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Haddad</surname><given-names>Vidal</given-names><suffix>Júnior</suffix></name><xref ref-type=\"aff\" rid=\"aff5\">\n<sup>5</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0001-6223-1785</contrib-id><name><surname>Nunes</surname><given-names>Jorge Luiz Silva</given-names></name><xref ref-type=\"aff\" rid=\"aff6\">\n<sup>6</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<label>1</label>Universidade Federal do Maranhão, Programa de Pós-Graduação em Saúde\ne Ambiente, São Luís, MA, Brasil.</aff><aff id=\"aff2\">\n<label>2</label>Universidade Federal do Maranhão, Núcleo de Estudos e Pesquisas\nAmbientais, São Luís, MA, Brasil.</aff><aff id=\"aff3\">\n<label>3</label>Instituto Oswaldo Cruz/Fiocruz, Laboratório de Avaliação e Promoção\nda Saúde Ambiental, Rio de Janeiro, RJ, Brasil.</aff><aff id=\"aff4\">\n<label>4</label>Instituto Oswaldo Cruz/Fiocruz, Laboratório de Biodiversidade, Rio\nde Janeiro, RJ, Brasil.</aff><aff id=\"aff5\">\n<label>5</label>Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento\nde Dermatologia e Radioterapia, Botucatu, SP, Brasil.</aff><aff id=\"aff6\">\n<label>6</label>Universidade Federal do Maranhão, Departamento de Oceanografia e\nLimnologia, São Luís, MA, Brasil.</aff><author-notes><corresp id=\"c1\"><bold>Corresponding author</bold>: Dr. Jorge Luiz Silva Nunes.\n<bold>e-mail:</bold><email>silvanunes@yahoo.com</email></corresp><fn fn-type=\"con\" id=\"fn1\"><p>\n<bold>Authors’ contributions:</bold>\n<bold>MMSC:</bold> conception and design of the study, acquisition of data,\ndrafting the article, analysis and interpretation of data;\n<bold>ZMRR:</bold> approval of the version to be submitted;\n<bold>RAHD:</bold> drafting the article, English translation;\n<bold>SS:</bold> drafting the article; <bold>VHJ:</bold> final approval\nof the version to be submitted; <bold>JLSN:</bold> conception and design of\nthe study, drafting the article; Final approval of the version to be\nsubmitted.</p></fn><fn fn-type=\"COI-statement\" id=\"fn3\"><p>\n<bold>Conflicts of interest:</bold> The authors declare no conflicts of\ninterest.</p></fn></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><year>2020</year></pub-date><volume>53</volume><elocation-id>e20200216</elocation-id><history><date date-type=\"received\"><day>05</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>13</day><month>7</month><year>2020</year></date></history><permissions><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the\nCreative Commons Attribution License</license-p></license></permissions><abstract><title>Abstract</title><sec><title>INTRODUCTION:</title><p> The Portuguese man-of-war (<italic>Physalia physalis</italic>) is a\ncosmopolitan species, with a widespread distribution and responsible for a\ngreat number of injuries caused by cnidarians worldwide, including Brazil.\nGeoprocessing technology, however, has never been used to assess the spatial\ndistribution of these animals on beaches. The aim of this study was to carry\nout a health risk assessment of Portuguese man-of-war (<italic>P.\nphysalis</italic>) envenomations on the São Marcos and Calhau beaches in\nSão Luís city, in the state of Maranhão, Brazil. </p></sec><sec><title>METHODS: </title><p>This is a descriptive and quantitative study concerning primary data on the\noccurrence of the Portuguese man-of-war (<italic>P. physalis</italic>) and\nhuman envenomations in the studied places, conducted over a two-year period\nin São Luís, Maranhão, northeastern Brazil. </p></sec><sec><title>RESULTS:</title><p> Envenomations mainly occurred on beaches presenting high density of\n<italic>P. physalis</italic> during the dry period. Vinegar has been\nincorporated as a first aid, according to recommendations set by the\nBrazilian Ministry of Health. </p></sec><sec><title>CONCLUSIONS:</title><p> In order to improve prevention and control actions of human envenomation,\nrisk areas for this type of envenomation should be clearly indicated as\nalert areas. Inclusion of the geographical location of the envenomation in\nthe Notification/Investigation SINAN Form was suggested for allowing the\ncontinuity of studies involving this public health issue.</p></sec></abstract><kwd-group><title>Keywords:</title><kwd>Physalia physalis</kwd><kwd>Geographic mapping</kwd><kwd>Venomous animals</kwd><kwd>Injury prevention</kwd><kwd>Public health</kwd></kwd-group><counts><fig-count count=\"4\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"32\"/></counts></article-meta></front><body><sec sec-type=\"intro\"><title>INTRODUCTION</title><p>The Portuguese man-of-war (<italic>Physalia physalis</italic>) is a conspicuous\ncolonial cnidarian with a bluish-pink coloration. It presents a gas vesicle called a\npneumatophore with a triangular shape and folds in the upper portion<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>. Its multiple tentacles are capable of firing thousands of intracellular\norganelles (cnida) filled with venom and used for predation or defense. This species\nis responsible for a high number of envenomations, which may lead to severe injuries\namong humans and even death<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>.</p><p>The pneumatophore enables these organisms to move either by drifting with currents or\nsailing on wind using the crest of its floater as a sail. The geographical\ndistribution is cosmopolitan and widespread, throughout the Indian Ocean and the\nSouth Atlantic<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>.</p><p>\n<italic>P. physalis</italic> causes several socioeconomic problems in countries such\nas New Zealand, Australia, Portugal, Mexico, France, Spain, Chile<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref>, and Brazil, due to human envenomations and consequent impacts on the local\ntourist economy<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>.</p><p>The Portuguese man-of-war is recognized as a cause of envenomation in bathers, mainly\nin northern and northeastern Brazil<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>. Colonies have been registered along the entire Brazilian coast<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B16\" ref-type=\"bibr\">\n<sup>16</sup>\n</xref>, and envenomation outbreak records have been described in the states of Rio\nGrande do Sul, Paraná, São Paulo, Rio de Janeiro, Pernambuco and Maranhão<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>.</p><p>The clinical manifestations of cnidarian injuries are typical, with the appearance of\nlinear plaques comprising edema and erythema and intense pain immediately after\ncontact with the animals on beaches. Systemic phenomena are rare, although dyspnea,\nmalaise, nausea, vomiting, headache, chills, drowsiness, arterial hypotension, and\ncardiac arrhythmias have been reported<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>. In one case, an anomalous reaction manifested by purpuric papules was also\nobserved after the initial phase of envenoming<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref>.</p><p> In Brazil, envenomations caused by venomous animals are included in the Compulsory\nNotification List (<italic>Lista de Notificação Compulsória</italic> - LNC) as set\nby Ordinance 204 from February 17<sup>th</sup>, 2016 by the Brazilian Health\nMinistry, and must be notified and recorded in the Injury Notification Information\nSystem (<italic>Sistema de Informação de Agravos de Notificação</italic> -\nSINAN)<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>. However, Portuguese man-of-war envenomation cases registered in the SINAN\nindicate important sub-notifications compared to regional and local surveys. Due to\na gap in the epidemiology of accidents caused by aquatic animals in Brazil, the\nlimited information available in the literature is the result of cross-sectional\nstudies, active case searches or research in medical files, presenting several\nrestrictions inherent to the collection of secondary data<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>. This, therefore, makes it difficult to accurately determine the extent of\nthe problem in the country and plan preventive actions.</p><p>Portuguese man-of-war envenomations are common on the beaches of São Luís, in the\nstate of Maranhão, Brazil, particularly during the second half of the year, when\nthese organisms are frequently found. However, few actions have been carried out to\nalert the population about preventive measures and prompt care in case of accidental\ncontact with these animals<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref>. Since injured people search for the lifeguard department as the first\nlocation for first-aid measures, the Marine Fire Brigade (<italic>Batalhão de\nBombeiros Marinhos</italic> - BBMar) health team and fire brigade need to be\nnotified of local envenomations. These data, however, are not present in the SINAN\ndatabase.</p><p>In this regard, geoprocessing technology has never been used to assess the\ndistribution of these animals and human envenomations in São Luís, and may prove a\nvaluable tool in this context. Thus, the aim of this study was to assess the\nemerging health risk due to Portuguese man-of-war (<italic>P. physalis</italic>)\nenvenomation on the São Marcos and Calhau beaches in São Luís, Maranhão, Brazil\nduring the years 2015 and 2016.</p></sec><sec sec-type=\"methods\"><title>METHODS</title><p>This was a descriptive and quantitative study concerning primary data on the\noccurrence of the Portuguese man-of-war (<italic>P. physalis</italic>) and cases of\nhuman envenomation. About 7.0 km were monitored, considering the extension of the\nSão Marcos (2°29’17.70” S, 44°17’04.53” W) and Calhau (2°28’58.65” S, 44°15’15.61”\nW) urban beaches, in São Luís, Maranhão, Brazil, during 2015 and 2016 (<xref ref-type=\"fig\" rid=\"f1\">Figure 1</xref>).</p><p>\n<fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1:</label><caption><title>Map of the study area indicating the São Marcos and Calhau beaches,\nSão Luís, Maranhão, Brazil.</title></caption><graphic xlink:href=\"1678-9849-rsbmt-53-e20200216-gf1\"/></fig>\n</p><p>The data were obtained by means of an intensive search procedure parallel to the tide\nline for counting <italic>P. physalis</italic> stranded in the sand and marking\ntheir geographical coordinates using a portable global positioning system device.\nSampling was conducted every fortnight, at weekends and at the flood tide, as these\nanimals are more frequently found in this type of tide. The data were geoprocessed\nusing the QGIS version 16.1 software and the kernel density estimator was used to\nidentify and analyze case concentrations and to statistically validate the risk of\nenvenomation events through geolocality proximity calculations.</p><p>Information concerning envenomations caused by Portuguese man-of-war was obtained\nthrough the application of a specific questionnaire. These data were collected in\nlifeguard department posts on the two evaluated beaches, from August 2015 to\nDecember 2016, on weekends and holidays, when the number of beachgoers was higher.\nData on envenomation (place of occurrence, month, year, and first-aid measures) were\nobtained.</p><sec><title>Ethical considerations</title><p>The study protocol was approved by the Ethics Committee of the Federal University\nof Maranhão (no. 1.625.949), and met the ethical principles for conducting\nresearch involving human beings, according to the Brazilian National Health\nCouncil (<italic>Conselho Nacional de Saúde</italic> - CNS) resolution no.\n466/2012.</p></sec></sec><sec sec-type=\"results\"><title>RESULTS</title><p>From January 2015 to December 2016, a total of 1,929 Portuguese man-of-war specimens\nwere found on the two evaluated beaches, 663 in 2015 and 1,273 in 2016. Portuguese\nman-of-war were most frequent in August (90), September (89), and October (140)\n2015, and in August (212), September (324), and November (272) 2016 (<xref ref-type=\"fig\" rid=\"f2\">Figure 2</xref>).</p><p> A total of 66 human envenomations were identified from August 2015 to December 2016:\n27 in Calhau and 39 in São Marcos. The months with the highest number of occurrences\nwere January (11 cases), September (9 cases), and November (19 cases) (<xref ref-type=\"fig\" rid=\"f2\">Figure 2</xref>).</p><p>\n<fig id=\"f2\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2:</label><caption><title>Temporal distribution of Portuguese man-of-war (<italic>Physalia\nphysalis</italic>) specimens and human envenomation cases on Calhau\nand São Marcos beaches, São Luís, Maranhão, in 2015 and 2016.</title></caption><graphic xlink:href=\"1678-9849-rsbmt-53-e20200216-gf2\"/></fig>\n</p><p>Calhau beach presented a high density of Portuguese man-of-war specimens (1,002\norganisms) located near the Calhau lifeguard station (between 2º28'53.980''S,\n44º15'6.005\"W and 2°28'55,200\"S, 44°14'57.152\"W), while São Marcos presented a\ndensity of 927 organisms, mostly concentrated on the stretch between 2º28'55,200''S,\n44º14'57,152''W and 2º 28 '55,200\"S, 44°14 '57.152 \"W (<xref ref-type=\"fig\" rid=\"f3\">Figure 3</xref>).</p><p>\n<fig id=\"f3\" orientation=\"portrait\" position=\"float\"><label>FIGURE 3:</label><caption><title>Distribution map of Portuguese man-of-war (<italic>Physalia\nphysalis</italic>) specimens on Calhau and São Marcos beaches, São\nLuís, Maranhão, in 2015 and 2016.</title></caption><graphic xlink:href=\"1678-9849-rsbmt-53-e20200216-gf3\"/></fig>\n</p><p>The highest occurrence of Portuguese man-of-war specimens coincided with the highest\noccurrence of human envenomations on Calhau beach, as both are close to the\nlifeguard station. On the other hand, the area presenting the highest occurrence of\nPortuguese man-of-war colonies at São Marcos did not coincide with the area with the\nhighest envenomation frequency, which was near the fire brigade. A total of 47\nenvenomations occurred in people living in São Luís and 19 in non-residents (<xref ref-type=\"fig\" rid=\"f4\">Figure 4</xref>).</p><p>\n<fig id=\"f4\" orientation=\"portrait\" position=\"float\"><label>FIGURE 4:</label><caption><title>Map indicating the distribution of human Portuguese man-of-war\n(<italic>Physalia physalis</italic>) envenomation cases on Calhau\nand São Marcos beaches, São Luís, Maranhão, from August 2015 to December\n2016.</title></caption><graphic xlink:href=\"1678-9849-rsbmt-53-e20200216-gf4\"/></fig>\n</p><p>The main response measures adopted after Portuguese man-of-war envenomations at the\nlifeguard stations were as follows: topical vinegar (acetic acid) application at the\nsite (50 cases); freshwater, silver sulphadiazine, and lidocaine (4 cases); vinegar\nand silver sulphadiazine (2 cases); freshwater and vinegar (2 cases); vinegar,\nsilver sulphadiazine, and lidocaine application (1 case); freshwater, vinegar and\nsea water (1 case) and vinegar and sea water (1 case). Moreover, 5 cases did not\nreceive first-aid measures. The bathers themselves carried out all the freshwater\napplications reported herein. </p></sec><sec sec-type=\"discussion\"><title>DISCUSSION</title><p>The high number of <italic>P. physalis</italic> organisms found during the two years\nof this assessment indicate a risk of human envenomations on the beaches in the\nstudy region. The largest Portuguese man-of-war agglomerations generally occur\nduring the dry season (August to December), as reported previously by Haddad\n<italic>et al.</italic>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>in Brazil, Loten <italic>et al.</italic>\n<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref> in Australia, Labadie <italic>et al</italic>.<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref> in France, Ferrer <italic>et al.</italic>\n<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref> in Spain, and Araya <italic>et al.</italic>\n<xref rid=\"B25\" ref-type=\"bibr\">\n<sup>25</sup>\n</xref> in Chile.</p><p>Some studies point out that the environmental conditions that surround the Portuguese\nman-of-war agglomerations are not yet well known<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>, and the most common explanations for coastal agglomerations comprise\nassociations between drought periods and transport phenomena (<italic>e.g.</italic>\nwinds, ocean currents)<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B26\" ref-type=\"bibr\">\n<sup>26</sup>\n</xref>.</p><p>Portuguese man-of-war agglomerations have been reported in São Luís during periods of\ndrought and strong winds (trade winds) present in the second half of the year, as\nreported by Ferreira-Bastos <italic>et al.</italic>\n<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref> and Nunes and Mendonça<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref>, which may explain most of the <italic>P. physalis</italic> agglomerations\nreported herein. In addition, Maranhão is located in the <italic>P.\nphysalis</italic> distribution area range of the tropical zone<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref>.</p><p>Furthermore, the high marine primary production in Maranhão promotes high marine\ntrophic web productivity<xref rid=\"B28\" ref-type=\"bibr\">\n<sup>28</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B29\" ref-type=\"bibr\">\n<sup>29</sup>\n</xref>, attracting several species to this region, may also be a cause for\nPortuguese man-of-war agglomerations, as <italic>P. physalis</italic> are predatory\norganisms and require surplus food resources to increase their frequency in certain\nlocations<xref rid=\"B30\" ref-type=\"bibr\">\n<sup>30</sup>\n</xref>.</p><p>The higher density sites of Portuguese man-of-war organisms coincided with the sites\nwhere the greatest number of human envenomations occurred, while the months\ncomprising the highest <italic>P. physalis</italic> sampling coincided with the dry\nseason, when more envenomations were noted. On the other hand, the greatest number\nof envenomations was also observed in January, the rainy season, which might be\nconnected with the school holidays, and the increased number of people on the\nbeaches, as reported previously by Ferreira-Bastos <italic>et al</italic>.<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>. </p><p>The highest frequency of injured people was noted at São Marcos beach, even with a\nlower Portuguese man-of-war density compared to the Calhau beach. Envenomation\nrecords indicate that most cases occurred near the lifeguard department, probably\ndue to search for treatment at the nearest location to the envenomation site. This\nis important so that treatment measures can be carried out successfully. In the\npresent study, the first-aid measures applied by the lifeguard department correspond\nto the Brazilian Ministry of Health recommendations. However, it was not always this\nway. In 2013, Ferreira-Bastos <italic>et al</italic>.<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>reported that the first-aid measures applied by the lifeguard department did\nnot correspond to the Brazilian Ministry of Health recommendations.</p><p>After envenomation prevention actions developed by the “<italic>Physalia</italic>\nProject” popularly known in the region as the “Portuguese man-of-war Project”, a\nchange in first-aid measures was applied at São Luís beaches. The main use of\nvinegar was incorporated into first-aid, as evidenced herein, in agreement with the\nBrazilian Ministry of Health recommendations. The use of silver sulfadiazine,\nlidocaine, and the application of freshwater, on the other hand, have no scientific\nbasis and are contraindicated in cnidarian envenomation cases<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>.</p><p>It is interesting to note that most injured people were São Luís residents displaying\nlittle information about <italic>P. physalis</italic> or envenomation\nmechanisms<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>. Analyses of bathing conditions carried out by the Environmental State\nDepartment of Maranhão directly affect envenomation Portuguese man-of-war cases in\nurban São Luís beaches, as adequate signs on proper or improper bathing conditions\nlead to changes in the number of bathers in the area<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>.</p><p> In addition, urban beaches in São Luís also suffer from intense anthropogenic\nimpacts, with several domestic sewage discharge points along the study area, 36 in\nSão Marcos and 15 in Calhau<xref rid=\"B31\" ref-type=\"bibr\">\n<sup>31</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B32\" ref-type=\"bibr\">\n<sup>32</sup>\n</xref>, making these beaches less attractive to tourists, explaining most of the\nenvenomations that occur among São Luís residents. This problem reflects not only\nhealth consequences but also tourism disturbances induced by the numerous beach\nclosings, with huge economic impacts. Therefore, it does not only affect local\nauthorities, but also local traders and beach goers.</p><p>According to Ferreira-Bastos <italic>et al.</italic>\n<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>, injuries afflicted by Portuguese man-of-war specimens along the coast of\nSão Luís have been reported as a real problem for bathers, although the authorities\ndo not consider this a significant issue, despite compulsory notification being\nrequired and the fact that every year envenomation cases are reported during\nspecific months. </p><p>Despite this, the number of recorded envenomations in this study was low, perhaps due\nto media disclosure coverage on these events being common in São Luís<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>, probably instructing the population on the fact that prompt care is\nrequired. Other factors leading to a low number of reports may include the fact that\nlocal beach bar employees and owners have also been instructed on how to act in\nthese situations, diluting lifeguard actions. Moreover, a lack of lifeguard\nawareness about the importance of notifications may lead to important\nsub-notification of human Portuguese man-of-war envenomations. </p><p>By the end of 2016, the beaches of Calhau and São Marcos were marked with signs\nindicating adequate bathing areas, which may increase the number of Portuguese\nman-of-war envenomations. Thus, it is clear that understanding the spatial\ndistribution of Portuguese man-of-war is paramount for the implementation of\nprevention measures and, in order to improve control actions, risk areas for this\ntype of accident, namely the sections between 2º28'53.980''S, 44º15'6.005''W and\n2º28'55.200''S, 44º14'57.152''W, on Calhau beach and 2º28'55.200''S, 44º14'57.152''W\nand 2º28'55.200''S, 44º14'57.152''W, on São Marcos beach, should be clearly\nindicated as alert areas, and the inclusion of the geographical location of each\nenvenomation should be reported in the envenomation notification form, allowing for\nthe continuity of studies involving this public health issue.</p><p>The study of Portuguese man-of-war envenomations has always been neglected by the\nagencies responsible for the knowledge, control and prevention of diseases involving\nthese animals. Thus, we suggest this piece of evidence to the Surveillance Service\nwith lifeguard teams for the purposes of updating and clinical training, as well as\nawareness about the importance of epidemiological data.</p></sec></body><back><ack><title>ACKNOWLEDGMENTS</title><p>The authors acknowledge the Federal University of Maranhão (UFMA) and the “Projeto\n<italic>Physalia</italic>” research project for providing technical support for\nthe development and implementation of this study.</p></ack><fn-group><fn fn-type=\"supported-by\" id=\"fn2\"><p>\n<bold>Financial support:</bold> The authors are very grateful to the Coordenação\nde Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing financial\nsupport.</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id=\"B1\"><label>1</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bardi</surname><given-names>J</given-names></name><name><surname>Marques</surname><given-names>AC</given-names></name></person-group><article-title>Taxonomic redescription of the Portuguese man-of-war, Physalia\nphysalis (Cnidaria, Hydrozoa, Siphonophorae, Cystonectae) from\nBrazil</article-title><source>Iheringia Ser. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Rev Soc Bras Med Trop</journal-id><journal-id journal-id-type=\"iso-abbrev\">Rev. Soc. Bras. Med. Trop</journal-id><journal-id journal-id-type=\"publisher-id\">rsbmt</journal-id><journal-title-group><journal-title>Revista da Sociedade Brasileira de Medicina Tropical</journal-title></journal-title-group><issn pub-type=\"ppub\">0037-8682</issn><issn pub-type=\"epub\">1678-9849</issn><publisher><publisher-name>Sociedade Brasileira de Medicina Tropical - SBMT</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32997047</article-id><article-id pub-id-type=\"pmc\">PMC7523520</article-id><article-id pub-id-type=\"doi\">10.1590/0037-8682-0027-2020</article-id><article-id pub-id-type=\"other\">00349</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Major Article</subject></subj-group></article-categories><title-group><article-title>Exploring local and global regression models to estimate the spatial variability of Zika and Chikungunya cases in Recife, Brazil</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0003-4540-9531</contrib-id><name><surname>dos Anjos</surname><given-names>Rafael Silva</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nóbrega</surname><given-names>Ranyére Silva</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ferreira</surname><given-names>Henrique dos Santos</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>de Lacerda</surname><given-names>António Pais</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>de Sousa-Neves</surname><given-names>Nuno</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<label>1</label>Universidade Federal de Pernambuco, Departamento de Ciências Geográficas, Recife, PE, Brasil.</aff><aff id=\"aff2\">\n<label>2</label>University of Lisbon, Department of Medicine, Lisbon, Portugal. </aff><aff id=\"aff3\">\n<label>3</label>University of Évora, Department of Landscape, Environment and Planning, Évora, Portugal.</aff><author-notes><corresp id=\"c1\"><bold>Corresponding author:</bold> Rafael Silva dos Anjos. <bold>e-mail:</bold><email>anjos.rsa@gmail.com</email></corresp><fn fn-type=\"con\" id=\"fn1\"><p>\n<bold>Authors’ contributions:</bold>\n<bold>RSA:</bold> Elaboration of the manuscript, production of the models and maps, analysis and interpretation of data; <bold>RSN:</bold> contribution to analyze the relationships between the factors and the arboviruses, constructions of the ideas, elaboration of the manuscript; <bold>HSF</bold>: Contribution to analyze the relationships between the factors and the arboviruses, constructions of the ideas; <bold>APL:</bold> Contribution to analyze the relationships between the factors and the arboviruses, approach about how the diseases is related to some factors, constructions of the ideas; <bold>NSN:</bold> Creation of the methodology, method, elaboration of the manuscript, constructions of the ideas. </p></fn><fn fn-type=\"COI-statement\" id=\"fn3\"><p>\n<bold>Conflict of Interest:</bold> The authors declare that there is no conflict of interest.</p></fn></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><year>2020</year></pub-date><volume>53</volume><elocation-id>e20200027</elocation-id><history><date date-type=\"received\"><day>23</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>11</day><month>8</month><year>2020</year></date></history><permissions><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License</license-p></license></permissions><abstract><title>Abstract</title><sec><title>INTRODUCTION:</title><p> In this study, we aim to compare spatial statistic models to estimate the spatial distribution of Zika and Chikungunya infections in the city of Recife, Brazil. We also aim to establish the relationship between the diseases and the analyzed geographical conditions. </p></sec><sec><title>METHODS:</title><p> The models were defined by combining three categories: type of spatial unit, calculation of the dependent variable format, and estimation methods (Geographical Weighted Regression [GWR] and Ordinary Least Square [OLS]). We identified the most accurate model to estimate the spatial distribution of the diseases. After selecting the model that provided best results, the relationship between the geographical conditions and the incidence of the diseases was analyzed. </p></sec><sec><title>RESULTS:</title><p> It was observed that the matrix of 100 meters (as the spatial unit) showed the highest efficiency to estimate the diseases. The best results were observed in the models that utilized the kernel density estimation (as the calculation of the dependent variable). In all models, the GWR method showed the best results. By considering the OLS coefficient values, it was observed that all geographical conditions are related to the incidence of Zika and Chikungunya, while the GWR coefficient values showed where this relationship was more noticeable. </p></sec><sec><title>CONCLUSIONS:</title><p> The model that utilized the combination of the matrix of 100 meters, kernel density estimation (as the calculation of the dependent variable) and GWR method showed the highest efficiency in estimating the spatial distribution of the diseases. The coefficient values showed that all analyzed geographical conditions are related to the illnesses’ incidence. </p></sec></abstract><kwd-group><title>Keywords:</title><kwd>Spatial Analysis</kwd><kwd>Public Health</kwd><kwd>Spatial Interaction</kwd></kwd-group><counts><fig-count count=\"2\"/><table-count count=\"2\"/><equation-count count=\"2\"/><ref-count count=\"21\"/></counts></article-meta></front><body><sec sec-type=\"intro\"><title>INTRODUCTION</title><p>Over the last decade, some viral diseases have gained notoriety due to their serious consequences for infected people in many countries, becoming a global health problem. Among the viruses causing some of these diseases are the Zika virus (ZIKV) and the chikungunya virus (CHIKV). Both viruses are transmitted primarily by <italic>Aedes</italic> mosquitoes<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>. </p><p>It is important to note that a variety of ecological and economic factors could contribute to arbovirus epidemics in Brazil. One example is poor sanitation infrastructure, which is common in the country and usually leads to the creation of several mosquito breeding sites<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>. Even in poor neighborhoods, there is a high spatial variability in the cases of arbovirus diseases. Conditions related to vector proliferation and infection by arboviruses are complex and involve both individual and environmental characteristics that vary from place to place<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>. </p><p>By identifying where the Zika and the chikungunya disease cases are located, public authorities can apply insecticides for mosquito control primarily in high‐risk areas, decreasing the transmission of these viruses. Furthermore, public educational campaigns encourage communities at risk to engage in preventive behaviors, as people who are aware of the risk in their neighborhood are more likely to eliminate potential breeding sites in their homes, apply insect repellent, dress appropriately to avoid bites, and avoid the outdoors during mosquito feeding hours<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>.</p><p>Zika fever is an exanthematous disease, related to Dengue, West Nile, and Yellow fever. This infection can last one week, with symptoms similar to Chikungunya and Dengue, including mild fever rash, arthralgia, arthritis, myalgia, headache, conjunctivitis, and edema. Severe cases involving hospitalization are uncommon, and deaths are rare<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref>.</p><p>Chikungunya is another arbovirus illness caused by the chikungunya virus that belongs to the genus <italic>Alphavirus</italic> of the Togaviridae family. It is transmitted predominantly by <italic>Aedes aegypti</italic> and <italic>Aedes albopictus mosquitoes</italic>. However, in some areas, transmission by <italic>Culex, Mansonia</italic>, and <italic>Anopheles</italic> species has also been observed<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>. Around 50-97% of individuals infected with chikungunya develop clinical disease with fever and arthralgia<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>.</p><p>Since Zika and chikungunya have the same conditions to their proliferation, such as transmission vector, this study grouped the notified cases of these illnesses. </p><p>Given the above, we aim to compare spatial statistic models to estimate the spatial distribution of Zika and chikungunya infections in the city of Recife (Pernambuco, Brazil). In addition, we also aim to establish the relationship between the illnesses’ incidence and the analyzed geographical conditions.</p></sec><sec sec-type=\"methods\"><title>METHODS</title><sec><title>Study Area</title><p>Recife, located in the northeast of Brazil, is the country’s fifth largest urban agglomeration and capital of the state of Pernambuco. Its urban layout is predominantly constituted by the coast and the urban rivers<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>\n<italic>.</italic> Recife has 1,637,834 million residents in an area of 218.4 km² <xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>. </p></sec><sec><title>Data </title><p>The Health Secretary of Recife provided all information on Zika and chikungunya cases, which were confirmed by diagnostic clinic and laboratory. From 2015 until 2017, 4,861 cases (124 cases of Zika and 4,737 cases of chikungunya) were geocoded. About 17% of all cases were not geocoded due to inconsistency of information related to the address. Cases notified in penitentiaries were eliminated from the geocoding.</p><p>The explanatory variables included in the analysis were based on three categories: </p><p>\n<list list-type=\"bullet\"><list-item><p>\n<italic>Social/Economic</italic>: persons per household, head of household income, population density, which were provided by the Brazilian Institute of Geography and Statistics (IBGE). </p></list-item><list-item><p>\n<italic>Environmental/Infrastructural</italic>: non-building structures, slope and green spaces. The two first factors were provided by the Light Detection and Ranging (LIDAR), with 1 m of spatial resolution. Green spaces were delimited by the Normalized Difference Vegetation Index (NDVI) from Sentinel satellite images with 10 m of spatial resolution. In all raster maps, the mean of the values was calculated for each spatial unit (census areas or matrix with cells of 100 m).</p></list-item><list-item><p>\n<italic>Climatic Factors:</italic> Annual rainfall data from 2015 until 2017 was provided by the Brazilian Center for Monitoring and Early Warnings of Natural Disasters (CEMADEN). </p></list-item></list>\n</p><p>The dependent and independent variables were grouped according to the spatial unit adopted in Recife. All these variables were transformed in an index, according to the following equation: </p><p>\n<disp-formula id=\"e1\"><mml:math id=\"M1\"><mml:mi>I</mml:mi><mml:mi>n</mml:mi><mml:mi>d</mml:mi><mml:mi>e</mml:mi><mml:mi>x</mml:mi><mml:mi> </mml:mi><mml:msub><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mi>I</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mfrac bevelled=\"true\"><mml:mrow><mml:mi> </mml:mi><mml:mfenced separators=\"|\"><mml:mrow><mml:msub><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mi> </mml:mi><mml:mo>-</mml:mo><mml:mi> </mml:mi><mml:mi> </mml:mi><mml:mi> </mml:mi></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mi>a</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfenced></mml:mrow><mml:mrow><mml:mi> </mml:mi><mml:msub><mml:mrow><mml:mi>V</mml:mi></mml:mrow><mml:mrow><mml:mi>s</mml:mi><mml:mi>t</mml:mi><mml:mi>d</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfrac><mml:mi> </mml:mi></mml:math><label>(1)</label></disp-formula>\n</p><p>Here, <italic>V</italic>\n<sub><italic>i</italic></sub> is the value of the independent variable in the spatial unit <italic>i, Va</italic> is the average of the variable <italic>V</italic> in all census areas, and <italic>Vstd</italic> is the standard deviation of the variable in all spatial units.</p></sec><sec><title>Spatial Units</title><p>Three main types of spatial units were utilized in this study: census areas, census areas without green spaces, and a cell matrix of 100 meters. The census area was provided by Brazilian Institute of Geography and Statistics (IBGE). For the creation of the census areas without green spaces, the green spaces in question were removed by utilizing the NDVI raster map described above. </p><p>The cell matrix of 100 meters was created to increase the details of the values derived from the raster maps by each spatial unit. Thus, a spatial unit smaller than census areas can show more precisely spatial variability between variables (such as slope, green spaces, rainfall) and the diseases. </p></sec><sec><title>Statistical Modelling</title><p>The global regression model (Ordinary Least Squares [OLS] and the local regression model (Geographically Weighted Regression [GWR]) were applied to explore risk conditions and their relationship with the spatial distribution of Zika and chikungunya cases in Recife. The OLS is a method of least square that comes from the fact that these estimates minimize the sum of squared residuals<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>. The technique of linear regressions takes no account of location in its analysis of relationships between variables<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>. </p><p>The GWR method is an alternative that utilizes regression, by adding the location to all variables<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref>. Thus, GWR coefficients values can show where the relationship between the independent and dependent variables is more significant. The GWR function is based on the following:</p><p>\n<disp-formula id=\"e2\"><mml:math id=\"M2\"><mml:msub><mml:mrow><mml:mi>Y</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mi> </mml:mi><mml:mrow><mml:mo stretchy=\"false\">∑</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>X</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mi>j</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mrow><mml:msub><mml:mrow><mml:mi>β</mml:mi></mml:mrow><mml:mrow><mml:mi>j</mml:mi><mml:mi> </mml:mi></mml:mrow></mml:msub><mml:mfenced separators=\"|\"><mml:mrow><mml:msub><mml:mrow><mml:mi>p</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfenced><mml:mo>+</mml:mo><mml:mi> </mml:mi><mml:msub><mml:mrow><mml:mi>ℇ</mml:mi><mml:mi> </mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi></mml:mrow></mml:msub></mml:math><label>(2)</label></disp-formula>\n</p><p>In the presented formula, <italic>i</italic> is the geographical location of the <italic>i</italic>\n<sup>th</sup> spatial unit; <italic>Y</italic> is the dependent variable; <italic>X</italic> is a matrix containing a set of independent or predictor variable; ℇ is a random vector whose distribution is N (0, σ<sup>2</sup> ); and β<sub>j</sub> is a vector of regression coefficient of the variable <italic>j</italic>\n<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref>. Here <italic>p</italic>\n<sub><italic>i</italic></sub> is the geographical location of the <italic>i</italic>th spatial unit. These <italic>β</italic>\n<sub><italic>j</italic></sub>\n<italic>(p</italic>\n<sub><italic>i</italic></sub>\n<italic>)</italic> would themselves contain coefficients to be estimated. </p><p>It is important to highlight that the estimates of the coefficients of each spatial unit are related to the bandwidth adopted (quantity of spatial unit adopted or distance of the location <italic>i</italic>). </p><p>The models analyzed were divided by combining three categories: spatial unit (as described above), calculation of the dependent variable, and method to estimate the diseases (GWR or OLS). </p><p>The calculation of the dependent variable was divided in four: number of cases by each spatial unit and the mean of the kernel density by spatial unit, with radius of 500, 700 and 900 meters. </p><p>Three parameters were used to identify the efficiency of the models: Sum of Residuals, AICC and the adjusted R<sup>2</sup>. </p><p>The sum of residuals (S. Res) is the absolute sum of the squared residuals, derived by the difference between estimates and observed values.</p><p>The Akaike Information Criterion Correction (AICC) is a measure of model performance for comparing different regression models. This parameter and the sum of residuals are appropriate to identify the best model method of the regression (global or local). </p><p>The last parameter, adjusted R<sup>2</sup> is a measure of goodness of fit. Its value varies from 0.0 to 1.0, with higher values being preferable. It was used because, when an extra explanatory is added to the model, the denominator is not altered, but the numerator is. Calculations for the adjusted R<sup>2</sup> value normalize the numerator and denominator by their degrees of freedom, solving the alteration problem. In this study, the adjusted R<sup>2</sup> was utilized to identify the best model, while the AICC and S. Res were adopted to identify the best method of each model (GWR or OLS).</p><p>To analyze the global and local relationship between the variables and the diseases, it was necessary to choose the best model according to the adjusted R<sup>2</sup>. Thus, it was possible to identify the intensity and spatial variability - utilizing the GWR method - of the coefficient values for each variable.</p><p>Three statistical variables were used to analyze the relationship between the variables and the diseases in the OLS method: robust probability,Variance Inflation Factor (VIF), and the Koenker Statistic. The robust probability indicates whether a coefficient is statistically significant (p < 0.01). The VIF values (>7.5) indicate redundancy among explanatory variables. If the Koenker Statistic indicates values smaller than 0.01, the relationships modeled are not consistent (either due to non-stationarity or heteroskedasticity)<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>.</p></sec></sec><sec sec-type=\"results\"><title>RESULTS</title><p>By analyzing the results in <xref rid=\"t1\" ref-type=\"table\">Table 1</xref>, it was observed that the cell matrix of 100 meters showed the best efficiency to estimate the diseases in both methods (OLS and GWR), excepting Model 3, which utilized the number of cases by cell matrix of 100 meters. By comparing the adjusted R<sup>2</sup> between Model 1 (census areas as spatial unit) and Model 6 (cell matrix of 100 meters), it is possible to notice an improvement of 0.09 to 0.35 (OLS method) and 0.47 to 0.92 (GWR method).</p><p>\n<table-wrap id=\"t1\" orientation=\"portrait\" position=\"float\"><label>TABLE 1:</label><caption><title>Models categorized by combination of spatial unit, calculation of dependent variable, and method to estimate the Zika and chikungunya cases. </title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Model </th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Method</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Bandwidth (m)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Spatial Unit</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Dependent Variable</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Adjusted R2</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">S. Res</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">AICC</th></tr></thead><tbody><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">OLS</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">census areas</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">number of cases</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.092</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 134</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 024</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GWR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">500</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">census areas</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">number of cases</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.47</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">593</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">OLS</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">census areas without green spaces</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">number of cases</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.095</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 133</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 022</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GWR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">500</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">census areas without green spaces</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">number of cases</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.47</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">595</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">47</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">OLS</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">cell matrix of 100 meters </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">number of cases</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 121</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">52 935</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GWR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">500</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">cell matrix of 100 meters </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">number of cases</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.19</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 280</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 110</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">OLS</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">cell matrix of 100 meters </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">kernel density estimation (500 m)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.36</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 461</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45 912</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GWR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">500</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">cell matrix of 100 meters </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">kernel density estimation (500 m)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.89</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 631</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 135</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">OLS</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">cell matrix of 100 meters </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">kernel density estimation (700 m)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.367</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 717</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45 767</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GWR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">700</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">cell matrix of 100 meters </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">kernel density estimation (700 m)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.92</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 743</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 067</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">OLS</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"> </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">cell matrix of 100 meters </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">kernel density estimation (900 m)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.359</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 949</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45 886</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">GWR</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">900</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">cell matrix of 100 meters </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">kernel density estimation (900 m)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.92</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 855</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8 036</td></tr></tbody></table></table-wrap>\n</p><p>In respect to the calculation of the dependent variable, the results which presented higher adjusted R<sup>2</sup> values numbers were observed in the models that utilized the kernel density estimation. This fact highlights the difference between models that utilize number of cases by spatial unit and the mean of kernel density estimation by spatial unit. However, considering the adjusted R<sup>2</sup> in OLS method, the radius of kernel density of 700 meters showed the best efficiency. Both Models 5 and 6 (with radii of 700 and 900 meters, respectively) had the same adjusted R<sup>2</sup> of 0.92. </p><p>In all models, the GWR method showed the best results, when the combination of all three parameters was analyzed: adjusted R<sup>2</sup>, sum of residuals, and the AICC. One particularity was that in Model 3, the OLS and GWR methods had similar adjusted R<sup>2</sup> values. </p><p>By analyzing the adjusted R<sup>2</sup> values of the models, in general, it was identified that the best results were shown in Model 5. This model had the spatial unit of cell matrix with 100 meters and kernel density with a radius of 700 meters. </p><p>The importance of the coefficients and parameters presented in the OLS method lies in the possibility of identifying how many variables were spatially significant and which variables were more significant to estimate the diseases. However, it is worth noting that these parameters show the global relationship between the geographical conditions and the diseases. </p><p>In this case, in the OLS method, it was observed that the VIF was low, indicating no problems with multicollinearity in all variables. Thus, this parameter shows that no variables were redundant. </p><p>Another point to consider is that the robust probability showed significant spatial autocorrelation for all variables. This significant spatial autocorrelation shows that the all variables improved the estimates of Zika and chikungunya cases. </p><p>The Koenker Statistic value for all variables was statistically significant (p <0.01), showing that relationships modeled are not consistent due to non-stationarity. This fact can indicate that the GWR model could show better estimations, leading to the thought that regression methods based on location (GWR method) are better than global regression methods to estimate the diseases, such as OLS.</p><p>In Model 5, three factors presented positive coefficients in the OLS method (<xref rid=\"t2\" ref-type=\"table\">Table 2</xref>): persons per households, population density, and slope. Other independent variables, such as green spaces, head of household income, nonbuilding structures and precipitation, had negative coefficients. </p><p>\n<table-wrap id=\"t2\" orientation=\"portrait\" position=\"float\"><label>TABLE 2:</label><caption><title>Independent variables utilized and their relationship with Zika and chikungunya cases according to coefficient, robust probability, and VIF in the OLS method (Model 5).</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th align=\"justify\" rowspan=\"1\" colspan=\"1\">Variable</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Coefficient</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Robust Probability</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">VIF</th></tr></thead><tbody><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Persons per household</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.320608</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.000000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.252564</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Head of household Income</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-0.179351</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.000000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.154345</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Rainfall</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-0.120486</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.000000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.141473</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Nonbuilding Structures</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-0.120486</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.000005</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.578071</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Slope</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.058705</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.000000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.240517</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Population Density</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.468012</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.000000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.567344</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Green Spaces</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-0.230611</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.000000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.547128</td></tr></tbody></table></table-wrap>\n</p><p>By considering the coefficients’ spatial distribution of GWR method, it is possible to identify which variables presented spatial heterogeneity. Thus, it enables the recognition of disparities between GWR and OLS coefficient values. By analyzing the GWR coefficients in <xref ref-type=\"fig\" rid=\"f1\">Figure 1</xref>, it is possible to identify that their values can be different according to the geographic location. Although the independent variables showed similarities between GWR and OLS coefficients, some regions presented an inverse relationship. </p><p>\n<fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1:</label><caption><title>Maps of GWR coefficient values divided by the geographical conditions analyzed.</title></caption><graphic xlink:href=\"1678-9849-rsbmt-53-e20200027-gf1\"/></fig>\n</p><p>Although the coefficient values of the OLS method presented an inverse relationship between non-building structures and the diseases, it is possible to see that some regions (east and southwest, for example) presented positive coefficient values in GWR method. However, negative coefficient values were found in most spatial units (62%). The coefficient values in GWR varied from -0.5 to 0.5, while the coefficient value in the OLS method was -0.12.</p><p>The negative coefficients for the variable head of household income in the OLS model was confirmed by the GWR coefficient values in most census areas. By comparison, the coefficient value in OLS method was -0.17, while the coefficient values in GWR method varied from -2.5 to 2.7. However, about 54% of the spatial units showed negative coefficient values in GWR method. </p><p>By analyzing population density, in most spatial units, the coefficient value in GWR model is positive, mainly where the disease rates were high, confirming the coefficient values in the OLS model (0.46). At the center of the municipality, one of the regions showed lower population density but higher Zika and chikungunya rates than its neighboring areas. This fact resulted in a negative coefficient value in the GWR map. The coefficient values in GWR method varied from -0.1 to 1.4. Despite that, about 96% of the spatial units showed positive coefficient values. </p><p>Regarding the relationship between arbovirus diseases and persons per household, in most spatial units, the coefficient was positive in the GWR model (57%), agreeing with the coefficient values in the OLS model. It is important to note that, although some spatial units presented high persons per household rates, the arbovirus rates were low, causing a negative coefficient in GWR model. </p><p>The relationship between slope and arboviruses presented positive and negative coefficients, without a spatial distribution pattern. However, about 60% of spatial units showed negative coefficients. This result differs from the OLS coefficient values. </p><p>The inversely proportional relationship between green spaces and arboviruses found in the OLS method is also present in all spatial units. The areas where the highest positive coefficient values occurred (mainly in the northeast of the city) showed low presence of green spaces and low Zika and chikungunya rates. About 81% of spatial units showed negative coefficient values in the GWR method. </p><p>Regarding the rainfall variable, the OLS coefficient values showed that regions with lower precipitation rates had higher Zika and chikungunya rates. However, the coefficient values in the GWR method presented negative values for 50% of the spatial units studied. </p><p>By analyzing the differences between the estimated and observed cases of Zika and chikungunya in <xref ref-type=\"fig\" rid=\"f2\">Figure 2</xref>, some particularities became evident. The OLS method presented the greatest difference when compared to the observed data, to which the GWR method showed similarities in the spatial distribution of the arboviruses. However, it was possible to identify the spatial variability of the diseases in both models, where the highest Zika and chikungunya rates were observed in the northeastern and some central areas of Recife.</p><p>\n<fig id=\"f2\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2:</label><caption><title>Density of estimated (GWR model and OLS model) and observed cases of Zika and chikungunya in Recife, Brazil.</title></caption><graphic xlink:href=\"1678-9849-rsbmt-53-e20200027-gf2\"/></fig>\n</p></sec><sec sec-type=\"discussion\"><title>DISCUSSION</title><p>By analyzing the results in <xref rid=\"t1\" ref-type=\"table\">Table 1</xref>, it was observed that the efficiency of the models can be assessed according to the spatial unit, the calculation of dependent variable, and the method of the model. </p><p>By analyzing the different parameters utilized in all models, it was possible to identify some particularities. The adoption of spatial units smaller than census areas can improve the estimates of the diseases studied. Some geographical conditions, which derive from the raster maps, have high spatial variability inside of the census areas. Thus, by adopting the cell matrix of 100 meters as the spatial unit instead of the census areas, the relationship between these conditions and the diseases can be more precise, improving the adjusted R<sup>2</sup> values. </p><p>Another point to consider is that census areas without green spaces as the spatial unit does not improve the calculation of the population density, resulting in best estimates. </p><p>The calculation of the dependent variable was another important parameter to estimate the Zika and chikungunya rates. The models that adopted the mean of kernel density to calculate the Zika and chikungunya rates presented better estimates than models that utilized the number of cases by spatial unit. This fact shows that the methods utilized (OLS and GWR) have a sensitivity related to the spatial variability of the diseases. By adopting the number of cases, their spatial variability was abrupt between the spatial units, decreasing the proximity with the spatial variability of the independent variables. This occurs because the variations of the geographical conditions are smooth, demanding this behavior of the dependent variable. It is important to note that the adoption of the mean of kernel density can be justified by two factors: (a) the best efficiency to estimate the Zika and chikungunya cases, utilizing the adjusted R<sup>2</sup> values as parameter; and (b) the regionalization of the rates.</p><p>Regarding the search radius adopted (500, 700 and 900 meters) in kernel density, the adjusted R<sup>2</sup> values were similar in all models. However, the model that utilized the search radius of 700 meters presented slightly better efficiency than others models. </p><p>The third parameter analyzed in the model were the OLS and GWR methods. In all models, the GWR method showed the best results, according to the analysis of the sum of residuals, AICC, and adjusted R<sup>2</sup> values. Although both methods estimate a dependent variable, their purposes are different. When the results of the OLS method estimates are good, the coefficient values are an important aspect to analyze the global relationship between the geographical conditions and the diseases. However, the Koenker Statistic value in Model 5 indicated that the coefficient values can be very different according to location, justifying the GWR method. On the other hand, the GWR method can indicate where the relationship was more notable or inverse of the OLS coefficient values. </p><p>Others studies showed that this efficiency in GWR method is better than the OLS method. For example, the correlation between some factors and notifications of Dengue in Pakistan was 0.37 in OLS model and 0.49 in GWR model<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref>. An aspect to consider is that these correlations can vary in space and time. For example, the correlation between population density, slope, altimetry, and malaria varied from 0.47 to 0.83, between 2001 and 2007<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>. </p><p>By considering all the parameters discussed and their efficiencies to estimate the Zika and chikungunya cases, in general, Model 5 presented the best results to estimate the diseases. This model had a cell matrix of 100 meters as spatial unit and mean of kernel density with 700 meters of search radius as calculation of the dependent variable. </p><p>By exploring the coefficients of both the OLS and GWR methods, some particularities were noteworthy. </p><p>Regarding to the GWR and OLS coefficient of nonbuilding structures, the negative coefficient values can be explained because in areas where the distance among buildings is low, the opportunities of the mosquito to infect the households are increased. Another point to consider is that slum areas have the lowest rates of nonbuilding structures in Recife, justifying the correlation between the highest rates of Zika and lowest rates of nonbuilding structures. It is important to note that the building density can be an indicator of the temperature due to the latent heat that is absorbed in areas with high building density. </p><p>A relationship between income and spatial distribution of Zika and chikungunya cases was observed in both coefficients methods (OLS and GWR). However, income is not a predominant factor in some census areas in the north of the city. This fact could be related to the decrease of arboviruses cases in areas where the low-income population is located. The heterogeneity of variables related to arboviral disease is observed in other studies, in which some socioeconomic indicators were not statistically significant in categorizing risk areas<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>. However, studies showed that the income rate is the factor that best characterized the risk areas for arbovirus diseases such as Dengue, for example<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>. </p><p>The relationship between population density and some diseases can be explained by other studies that revealed areas with higher human population densities had lower socioeconomic position, poorer water and sanitation conditions, and frequent tire capping facilities, but had reasonable infrastructure<xref rid=\"B16\" ref-type=\"bibr\">\n<sup>16</sup>\n</xref>. For example, in Porto Rico, it was identified that the poor population, population density, urbanized areas, and high temperature presented a correlation of 0.78 with incidence of Zika, in GWR model<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>. Another study in Pakistan verified that among variables such as altimetry, distance from rivers and population density, the last factor showed the highest correlation with notifications of Dengue<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref>.</p><p>In general, the correlation between persons per household and incidence of Zika and chikungunya diseases in both methods showed a directly proportional correlation. Results were detected in Ecuador, where the relation between Zika and persons per households was statistically significant<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>. In Venezuela, it was observed that the relation between Dengue and persons per household was directly proportional<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>. </p><p>Historically, the regions with high slope degree or flat areas (such as mangrove) are inhabited by a low-income population in Recife<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>. This fact resulted in a high spatial variation of the GWR coefficient values. In any case, the OLS method showed positive coefficients, highlighting that the regions with high slope degree are more susceptible to proliferation of Zika and chikungunya cases in Recife.</p><p>Another point to consider is that the mosquito <italic>Aedes aegypti</italic> prefers urbanized areas where the presence of vegetation cover is more limited. Thus, the green spaces can be a factor correlated with urbanization and, consequently, related to spatial distribution of the arboviruses. This result is similar to the study that identified the highest chikungunya incidence is in regions with highest percentages of urbanized land in Rio de Janeiro<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>. </p><p>A second factor to highlight is that the <italic>Aedes aegypti</italic> preferences for human blood decrease with the increase of vegetation cover. Moreover, it is important to highlight that most slum areas are highly compact, displaying the highest roof coverage and low vegetation cover rate<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>. </p><p>Some models identified that, while an increase in temperature should shift the <italic>Aedes aegypti</italic> boundaries towards higher latitudes, the increase of rainfall would be detrimental to its enlargement<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>. In most studies that analyzed the relationship between climatic parameters and arboviruses, it was identified that these studies are related to the arboviruses temporal distribution, disregarding the spatial distribution, due to the lack of meteorological well-distributed stations across the cities. However, the temporal analyses indicate that extremely intense rainstorms that produce a high volume of precipitation in a few hours may flush out larvae, leading to decreased vector abundance and arbovirus transmission<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>. These observations can suggest that the regions with low precipitation volume are better for the proliferation of mosquitoes. </p><p>Given the above considerations, the model that utilized the combination of the matrix of 100 meters (as the spatial unit), kernel density estimation (KDE) (as the calculation of the dependent variable) and Geographical Weighted Regression (as the estimation method) showed the highest efficiency to estimate the spatial distribution of Zika and chikungunya cases. </p><p>It is important to note that, although the GWR method presented best results, regression linear methods, such as OLS, can show the global relationship between geographical conditions and some diseases. On the other hand, the GWR coefficient values show where this relationship is more evident. </p><p>By comparing the GWR and OLS coefficient values in Recife, three geographical conditions showed a notable agreement in most spatial units in both methods: non-building structures, population density, and green spaces (more than 60% of spatial units). 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Rev Soc Bras Med Trop</journal-id><journal-id journal-id-type=\"iso-abbrev\">Rev. Soc. Bras. Med. Trop</journal-id><journal-id journal-id-type=\"publisher-id\">rsbmt</journal-id><journal-title-group><journal-title>Revista da Sociedade Brasileira de Medicina Tropical</journal-title></journal-title-group><issn pub-type=\"ppub\">0037-8682</issn><issn pub-type=\"epub\">1678-9849</issn><publisher><publisher-name>Sociedade Brasileira de Medicina Tropical - SBMT</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32997053</article-id><article-id pub-id-type=\"pmc\">PMC7523521</article-id><article-id pub-id-type=\"doi\">10.1590/0037-8682-0314-2020</article-id><article-id pub-id-type=\"other\">00354</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Major Article</subject></subj-group></article-categories><title-group><article-title>Cost analysis of smear microscopy and the Xpert assay for tuberculosis diagnosis: average turnaround time</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Figueredo</surname><given-names>Lida Jouca de Assis</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0001-7245-4472</contrib-id><name><surname>de Miranda</surname><given-names>Silvana Spíndola</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>dos Santos</surname><given-names>Lucas Benício</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Manso</surname><given-names>Caroline Gontijo Gonçalves</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Soares</surname><given-names>Valéria Martins</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Alves</surname><given-names>Suely</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Vater</surname><given-names>Maria Cláudia</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kritski</surname><given-names>Afrânio Lineu</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Carvalho</surname><given-names>Wânia da Silva</given-names></name><xref ref-type=\"aff\" rid=\"aff5\">\n<sup>5</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>de Pádua</surname><given-names>Cristiane Menezes</given-names></name><xref ref-type=\"aff\" rid=\"aff5\">\n<sup>5</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>de Almeida</surname><given-names>Isabela Neves</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<label>1</label>Universidade Federal de Minas Gerais, Laboratório de Pesquisa em Micobactérias, Belo Horizonte, MG, Brasil.</aff><aff id=\"aff2\">\n<label>2</label>Universidade Federal de Minas Gerais, Grupo de Pesquisa em Micobacterioses, Faculdade de Medicina, Belo Horizonte, MG, Brasil.</aff><aff id=\"aff3\">\n<label>3</label>Fundação Hospitalar do Estado de Minas Gerais, Hospital Júlia Kubistchek, Laboratório de Microbiologia, Belo Horizonte, MG, Brasil.</aff><aff id=\"aff4\">\n<label>4</label>Universidade Federal do Rio de Janeiro, Programa Acadêmico de Tuberculose, Rio de Janeiro, RJ, Brasil.</aff><aff id=\"aff5\">\n<label>5</label>Universidade Federal de Minas Gerais, Faculdade de Farmácia, Belo Horizonte, MG, Brasil.</aff><author-notes><corresp id=\"c1\"><bold>Corresponding author:</bold> Dra. Silvana Spíndola de Miranda. <bold>e-mail</bold>: <email>silvanaspindola@gmail.com</email></corresp><fn fn-type=\"con\" id=\"fn2\"><p>\n<bold>Authors’ contributions:</bold>\n<bold>LJAF:</bold> data collection, development of experiments, building the database, monitoring cost analysis, PhD student, and article writing. <bold>SSM</bold>: study conception and design, promotion of study financing, data analysis, and article writing. <bold>LBS:</bold> data collection, typing and conferencing of the database, and article writing. <bold>VMS:</bold> data collection, typing and conferencing of the database, and article review. <bold>SA:</bold> cost calculation, cost chain modeling, database conferencing, and article review. <bold>MCV:</bold> co-coordination of cost study, cost chain modeling, database conferencing, and article writing. <bold>ALK</bold>: study conception and design, and article review. <bold>WSC</bold>: study conception and design, promotion of study financing, data analysis, and article writing. <bold>CMP:</bold> statistical analysis, adjustment of the database, and article writing. <bold>INA</bold>: coordination of the data collected, adjustment of the database, modeling of the cost analyses, data analysis, and article writing. All the authors read and approved the final manuscript.</p></fn><fn fn-type=\"COI-statement\" id=\"fn3\"><p>\n<bold>Conflicts of interest:</bold> The authors declare no conflicts of interests.</p></fn></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><year>2020</year></pub-date><volume>53</volume><elocation-id>e20200314</elocation-id><history><date date-type=\"received\"><day>17</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License</license-p></license></permissions><abstract><title>Abstract</title><sec><title>INTRODUCTION:</title><p> Rapid and accurate tuberculosis detection is critical for improving patient diagnosis and decreasing tuberculosis transmission. Molecular assays can significantly increase laboratory costs; therefore, the average time and economic impact should be evaluated before implementing a new technology. The aim of this study was to evaluate the cost and average turnaround time of smear microscopy and Xpert assay at a university hospital. </p></sec><sec><title>METHODS:</title><p> The turnaround time and cost of the laboratory diagnosis of tuberculosis were calculated based on the mean cost and activity based costing (ABC). </p></sec><sec><title>RESULTS:</title><p> The average turnaround time for smear microscopy was 16.6 hours while that for Xpert was 24.1 hours. The Xpert had a mean cost of USD 17.37 with an ABC of USD 10.86, while smear microscopy had a mean cost of USD 13.31 with an ABC of USD 6.01. The sensitivity of smear microscopy was 42.9% and its specificity was 99.1%, while the Xpert assay had a sensitivity of 100% and a specificity of 96.7%.</p></sec><sec><title>CONCLUSIONS:</title><p> The Xpert assay has high accuracy; however, the turnaround time and cost of smear microscopy were lower than those of Xpert.</p></sec></abstract><kwd-group><title>Keywords:</title><kwd>Mycobacterium tuberculosis</kwd><kwd>Molecular diagnostic</kwd><kwd>Pulmonary tuberculosis</kwd><kwd>Cost analysis</kwd><kwd>Health system</kwd></kwd-group><counts><fig-count count=\"2\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"27\"/></counts></article-meta></front><body><sec sec-type=\"intro\"><title>INTRODUCTION</title><p>Tuberculosis (TB) has existed for millennia and remains a major global health problem<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>. The World Health Organization (WHO) estimated that in 2018, approximately 10 million people had developed TB, and that TB was responsible for an estimated 1.2 million deaths among human immunodeficiency virus (HIV) negative patients and an additional 251 thousand deaths among patients living with HIV<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>.</p><p>Brazil is one of 30 countries with a high TB burden, accounting for 84% of the global total number of cases<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>. In 2018, 78,652 new cases were registered, and only 34% of them were tested using the Xpert<sup>®</sup> MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) (Xpert). Moreover, 79% of the new patients knew their HIV status, and 87% of the identified cases were pulmonary TB<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>.</p><p>TB typically affects the lungs, but can also spread to other sites<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>. The diagnosis of extrapulmonary TB remains a challenge, since the total number of bacteria in extrapulmonary specimens is often lower than that present in pulmonary specimens<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>. Furthermore, the collection of extrapulmonary material requires invasive procedures, and it is usually difficult to obtain additional samples<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>.</p><p>Accurate, rapid detection of TB is critical for improving patient care and decreasing TB transmission<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>. The Xpert assay is an automated molecular test that can detect both TB and rifampicin resistance, generally within two hours after starting the test, with minimal hands-on technical time<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>.</p><p>The WHO issued initial recommendations concerning the Xpert test in early 2011<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>. In a systematic review, its sensitivity in detecting TB in pulmonary samples ranged from 58% to 100%, whereas the specificity ranged from 86% to 100%<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>.</p><p>The authors highlighted that the Xpert test is expensive, and that further research is needed to evaluate its use in TB programs and to assess if this investment could help start treatment promptly and improve health outcomes<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>. Despite the WHO recommendations for the use of the Xpert system, sputum smear microscopy remains widely used in many countries for the rapid diagnosis of TB under routine conditions. This method has a variable sensitivity ranging from 32% to 89% and a specificity between 85% and 100%<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>. </p><p>TB control programs must balance costs with performance characteristics and the need for rapid results<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>. Intensive implementation of molecular assays may lead to significant increases in laboratory cost. Selective implementation of molecular assays could be considered for some settings<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>, and accurate and rapid detection of TB is critical for improving patient outcomes (increased cure and decreased mortality rates, additional drug resistance, treatment failure, and relapse) and decreasing TB transmission<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>.</p><p>Thus, the aim of this study was to evaluate and compare the costs and average time to completion of smear microscopy and Xpert assay at a teaching hospital.</p></sec><sec sec-type=\"methods\"><title>METHODS</title><sec><title>Study design, variables and study site</title><p>The present study assessed respiratory and non-respiratory specimens (n=1009) that had been received at the Research Laboratory in Mycobacteria of the School of Medicine of the Federal University of Minas Gerais (UFMG), between November 2014 and November 2015. Of these, 141 patients accepted to participate, signed the term of free informed consent, and were included in the study of the accuracy and time of laboratory tests and in the analysis of clinical and sociodemographic characteristics.</p><p>Clinical and sociodemographic data were obtained using a standardized questionnaire and a review of the patients’ records on their behavior (alcohol and tobacco use), HIV infection, and any abnormalities on pulmonary imaging (chest x-ray or computed tomography). A pattern suggestive of TB was the presence of cavitation, infiltrate in upper lobe and apical segment of the lower lobe or mediastinal enlargement, or increased hilar lymph node or miliary pattern or pleural effusion or confluent parenchymal opacities, confluent parenchymal opacities, characterized as a budding tree<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref>. </p><p>These patients received medical care at the teaching hospital of the UFMG, a public and general university hospital that conducts educational, research, and medical care activities. This institution consists of one hospital unit and seven outpatient care centers<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>. Direct sputum smear examinations that were urgently requested were excluded from clinical specimens, since at the hospital, the Xpert system is not used immediately.</p></sec><sec><title>Processing of clinical samples</title><p>The specimens were processed following the standard <italic>N-acetyl-1-cysteine</italic> and sodium hydroxide (NALC/NaOH) method with a final NaOH concentration of 1%<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>. After this step, the sediment was resuspended in 1.0 to 1.5 mL of sterile water and used for smear microscopy, Xpert testing, and culture tests. </p><p>\n<italic>Smear microscopy:</italic> All smears prepared from decontaminated samples were stained with auramine O and analyzed by fluorescence microscopy<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>. </p><p>\n<italic>Xpert</italic>\n<sup><italic>®</italic></sup>\n<italic>MTB/RIF</italic>: This assay was performed according to the manufacturer’s instructions (Cepheid, Sunnyvale, CA, USA). Briefly, a sample reagent was added in a 3:1 ratio to ≥0.5 mL of decontaminated specimen. The closed tube was agitated manually twice during a 15-min incubation at room temperature. Then, 2 mL of the inactivated sample reagent-sample mixture was transferred to the Xpert test cartridge. The cartridges were inserted into the Xpert device, and the automatically generated results were read after 90 min<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>. </p></sec><sec><title>Average time: smear microscopy and Xpert</title><p>To calculate the average time to the release of test results, we took into consideration the hour when the samples were received in the collection sector and the hour when test results were released in the system. This period includes the times of receipt of samples, internal registration, the process of decontamination/centrifugation of the samples, separation of the aliquots for each diagnostic test, execution of the tests, release of results by the technician, and release of the results into the computerized system of the hospital.</p><p>The times during which tests were performed urgently via direct smear, at night, on weekends and holidays were not considered; only those performed routinely with NALC/NaOH were considered.</p></sec><sec><title>Cost analysis</title><p>The cost analysis of the TB laboratory diagnosis was based on the mean cost and activity based costing (ABC). The mean cost was calculated by dividing the total costs by the quantity produced over a determined period of time<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref>, which considered the total number of examinations performed in a month. The ABC principle is suitable for complex organizations, such as hospitals, where products use consumer resources in a highly heterogeneous manner<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref>, therefore covering as many direct and indirect costs as possible through cost drivers. The component cost evaluation and calculations were performed as described by Almeida et al. 2017 and the costs were expressed in USD, using the conversion rate of USD 1.00 = R$ 4.03, as established by the Central Bank of Brazil in 2019 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.bcb.gov.br/\">https://www.bcb.gov.br/</ext-link>)<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>. </p></sec><sec><title>Statistical analysis</title><p>Descriptive analysis was performed to characterize the study population concerning the selected variables. Measures of central tendency and dispersion were used for continuous variables, and absolute and relative frequencies for categorical variables. </p><p>The sensitivity and specificity of <italic>Xpert</italic>\n<sup><italic>®</italic></sup>\n<italic>MTB/RIF</italic> and smear microscopy with their 95% confidence intervals (CIs) were estimated, comparing the results to those of the phenotypic identification test as a standard method<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>. All analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).</p></sec><sec><title>Ethical considerations</title><p>The study protocol was approved by the Research Ethics Committee of the UFMG (protocol numbers CAAE-11821913.6.000.5257 and CAAE 0223.2412.7.1001.5149, and DEPE/HC protocol number 139/12).</p></sec></sec><sec sec-type=\"results\"><title>RESULTS</title><p>The study included 141 patients’ clinical samples: 100 pulmonary specimens and 41 extrapulmonary specimens (<xref ref-type=\"fig\" rid=\"f1\">Figure 1</xref>); 66% (93/141) of the patients were inpatients and 34% (48/141) were outpatients. </p><p>\n<fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1:</label><caption><title>The distribution of clinical samples.</title></caption><graphic xlink:href=\"1678-9849-rsbmt-53-e20200314-gf1\"/></fig>\n</p><p>The sociodemographic characteristics of these patients are presented in <xref rid=\"t1\" ref-type=\"table\">Table 1</xref>. Most patients were male, aged 46 to 60 years (median=46.0), single, had non-white skin, had nine or fewer years of formal education, and 25 had pulmonary TB. Between 20% and 40% of the patients were reported to be current users of alcohol and tobacco, respectively. Regarding radiological patterns, 27% presented presumed TB abnormalities. </p><p>\n<table-wrap id=\"t1\" orientation=\"portrait\" position=\"float\"><label>TABLE 1:</label><caption><title>Distribution of sociodemographic characteristics (n=141)*</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"2\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">Frequency </th></tr><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Variables</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">Total number of patients </th></tr><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">%</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Sociodemographic </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Age, years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">18-35</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28.7</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">36-45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.9</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">46-60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27.2</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">61-88</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Gender </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Male </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">83</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">58.9</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Skin color </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Non-white</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">84</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">80.0</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">White</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Education, years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Illiterate </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.2</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">≤ 9 </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">63.9</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">≥10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27.9</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Marital status</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Married </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">57</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">48.3</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Single </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">61</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">51.7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Behavioral </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Alcohol use (CAGE)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Positive</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">40.7</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Negative</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">59.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Tobacco use</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Never</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Current </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ever </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Clinical</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prescribed TB therapy </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">RHZE</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Special regimen</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.7</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">116</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">82.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">HIV status</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Positive</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13.5</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Negative</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Unknown result/not tested</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">78</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">55.3</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Radiological patterns</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Suggestive of TB</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">38</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Without abnormalities</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Other abnormalities </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26.2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Not performed </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">59</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">41.8</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN1\"><p>\n<bold>CAGE</bold> alcohol questionnaire; <bold>TB:</bold> tuberculosis; <bold>RHZE:</bold> rifampicin, isoniazid, pyrazinamide, ethambutol. *missing values were excluded.</p></fn></table-wrap-foot></table-wrap>\n</p><p>Accuracy assessment of the smear microscopy revealed a sensitivity of 42.9% (95% CI, 24.5-61.2) and a specificity of 99.1% (95% CI, 97.2-100.0). The accuracy assessment of the Xpert assay revealed a sensitivity of 100% (95% CI, 1.00-1.00) and a specificity of 96.7% (95% CI, 93.53-99.88).</p><p>Positive Xpert assays and negative culture test results were observed for the samples of two patients in retreatment, while 11 positive culture test results for nontuberculous mycobacteria (NTM) were not detected by the Xpert system. </p><p>The average time to smear microscopy results was 16.6 hours and the median time was 9.3 hours (standard deviation, 15.1 hours; range, 1.2-48 hours). The mean time to completion of the Xpert assay was 24.1 hours, with a median time of 24 hours (standard deviation, 18.2 hours; range, 2-72 hours). </p><sec><title>Cost analysis</title><p>The mean cost of the Xpert assay was USD 17.37 with an ABC value of USD 10.86, while the mean cost of the smear microscopy was USD 13.31 with an ABC value of USD 6.01. Proportionally, the impact of cost components of smear microscopy was lower than that of Xpert, except for the human resources component. The component cost that impacted the mean costs and ABC values of Xpert and smear microscopy are described in <xref ref-type=\"fig\" rid=\"f2\">Figure 2</xref>.</p><p>\n<fig id=\"f2\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2:</label><caption><title>The impact of ABC components.</title></caption><graphic xlink:href=\"1678-9849-rsbmt-53-e20200314-gf2\"/></fig>\n</p></sec></sec><sec sec-type=\"discussion\"><title>DISCUSSION</title><p>The study revealed that the average time to completion of the Xpert assay result was longer than that of sputum smear microscopy. This is because the slide is read immediately, but the sample undergoes processing in the Xpert system. In addition, the fact that in our laboratory we use the equipment with only four modules might also play a role. Nevertheless, although smear microscopy is cheaper than the Xpert assay, the latter is more accurate.</p><p>The advantage of the Xpert system is that it offers the possibility for a fully automated test after insertion of the clinical specimen into the cartridge. Assay steps (DNA extraction, amplification, and detection) are independent of manual technical interventions, thus minimizing analytical errors and improving quality control<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>. </p><p>The sensitivity and specificity values found in this study (100% and 96.7%, respectively) are similar to the results of a meta-analysis that included 27 studies with a total of 9500 specimens analyzed using the Xpert system, and which presented a range of sensitivity between 58% and 100% and specificity between 86% and 100%<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>. </p><p>The specificity was found to be lower than the sensitivity due to the presence of <italic>M. tuberculosis</italic> complex DNA in two cases of treatment control patients who presented negative culture tests. Therefore, the recommendation not to use the Xpert system for treatment control should be reinforced, except for probable TB in new cases, if resistance to rifampicin or multidrug resistance is suspected.</p><p>In this study, the Xpert system did not show false positive results among NTM isolates, mainly due to the increase in prevalence of disease-associated NTM, as has already been described by other authors<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>. Therefore, NTM should be suspected in conditions where smear microscopy tests yield positive results and are undetected by the Xpert assay<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>. Our data agree with the results of a meta-analysis that evaluated 14 studies including 180 NTM cases and showed that the false positivity of the Xpert assay for these pathogens was only 0.6%<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>, as well as another study that demonstrated a specificity of 97.8%<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>. </p><p>The average times extrapolated from the results (16.3 and 24 hours for smear microscopy and Xpert assay, respectively) are due to the fact that the laboratory does not work at night, on weekends, and holidays with processing of samples with NALC/NaOH; thus, the clinical samples are stored for the test to be carried out on the next business day. The recommendation of the Ministry of Health and reinforced by other authors is that routine results should be released within 24 hours<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>, which was seen from the mean of our results.</p><p>The ABC value of the Xpert assay was higher than that of smear microscopy and the input was the component with the greatest impact. However, it should be noted that the Xpert system is subsidized in Brazil<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref>. Efforts are being made to increase the coverage area of the Xpert system worldwide through subsidies to make it available in developing and underdeveloped countries, where health systems work under heavy economic constraints<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref>. </p><p>This is mainly due to the demand of exclusive inputs from the supplier that are imported and undergo exchange variation, which is a dynamic and sensitive variable to the economic scenario of each country<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref>.</p><p>When incorporating new technology and comparing it with already existing technology, it is important to evaluate all operational and technical aspects. Managers need to be aware of the relevant outcomes of TB treatment and bacillus transmission in the community when evaluating the advantages and disadvantages of incorporating new diagnostic tests<xref rid=\"B25\" ref-type=\"bibr\">\n<sup>25</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B26\" ref-type=\"bibr\">\n<sup>26</sup>\n</xref>. </p><p>The results described in this paper should alert Brazilian researchers to the need for the development and validation of a national molecular test. Although the Xpert system has high accuracy in the diagnosis of TB, national technological independence is necessary to ensure that cost is not a variable that hinders or even impedes the incorporation of a tool that is effective for the rapid and accurate diagnosis of TB<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref>. </p><p>The results should not be extrapolated to other scenarios owing to differences in the components analyzed. Thus, each site must perform its own analysis.</p><p>In conclusion, the Xpert assay has high accuracy; however, the time to sputum smear test results and the ABC value were lower than those observed for the Xpert system.</p></sec></body><back><ack><title>ACKNOWLEDGMENTS</title><p>We are grateful to the UFMG Postgraduate Program in Infectology and Tropical Medicine, to the Pro Rectory Research/UFMG, and to the Brazilian Tuberculosis Network (REDE TB). We are also grateful to the Minas Gerais Research Support Foundation (FAPEMIG) and the National Research Council.</p></ack><fn-group><fn fn-type=\"financial-disclosure\" id=\"fn1\"><p>\n<bold>Financial support:</bold> This research was funded by the Minas Gerais Research Support Foundation (FAPEMIG) process numbers: APQ-03266-13 and APQ-00094-12 and the National Research Council (CNPq) (process numbers: 446796/2014 and 310174/2014-7) and the Coordination of Improvement of Higher Level Personnel of the Ministry of Education of Brazil (CAPES). 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Rev Soc Bras Med Trop</journal-id><journal-id journal-id-type=\"iso-abbrev\">Rev. Soc. Bras. Med. Trop</journal-id><journal-id journal-id-type=\"publisher-id\">rsbmt</journal-id><journal-title-group><journal-title>Revista da Sociedade Brasileira de Medicina Tropical</journal-title></journal-title-group><issn pub-type=\"ppub\">0037-8682</issn><issn pub-type=\"epub\">1678-9849</issn><publisher><publisher-name>Sociedade Brasileira de Medicina Tropical - SBMT</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32997050</article-id><article-id pub-id-type=\"pmc\">PMC7523522</article-id><article-id pub-id-type=\"doi\">10.1590/0037-8682-0205-2020</article-id><article-id pub-id-type=\"other\">00351</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Major Article</subject></subj-group></article-categories><title-group><article-title>The clinical and molecular diagnosis of childhood and adolescent\npulmonary tuberculosis in referral centers</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0003-2727-520X</contrib-id><name><surname>Aurilio</surname><given-names>Rafaela Baroni</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Luiz</surname><given-names>Ronir Raggio</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Land</surname><given-names>Marcelo Gerardin Poirot</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Cardoso</surname><given-names>Claudete Aparecida Araújo</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kritski</surname><given-names>Afrânio Lineu</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sant’Anna</surname><given-names>Clemax Couto</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<label>1</label>Universidade Federal do Rio de Janeiro, Departamento de Pediatria da\nFaculdade de Medicina, Instituto de Puericultura e Pediatria Martagão Gesteira, Rio\nde Janeiro, RJ, Brasil.</aff><aff id=\"aff2\">\n<label>2</label>Universidade Federal do Rio de Janeiro, Instituto de Estudos de\nSaúde Coletiva, Rio de Janeiro, RJ, Brasil.</aff><aff id=\"aff3\">\n<label>3</label>Universidade Federal Fluminense, Faculdade de Medicina, Departamento\nde Saúde Materno Infantil, Niterói, RJ, Brasil.</aff><aff id=\"aff4\">\n<label>4</label>Universidade Federal do Rio de Janeiro, Programa Acadêmico de\nTuberculose da Faculdade de Medicina, Instituto de Doenças do Tórax/Hospital\nUniversitário Clementino Fraga Filho, Rio de Janeiro, RJ, Brasil.</aff><author-notes><corresp id=\"c1\"><bold>Corresponding author:</bold> Dra. Rafaela Baroni Aurilio.\n<bold>e-mail</bold>: <email>rafabaroni@yahoo.com.br</email></corresp><fn fn-type=\"con\" id=\"fn1\"><p>\n<bold>Author Contributions:</bold>\n<bold>RBA:</bold> conception and design of the study, acquisition of data,\nanalysis and interpretation of data, and drafting the article; RRL: analysis\nand interpretation of data and final approval of the manuscript version to\nbe submitted; MGL: analysis and interpretation of data and final approval of\nthe version to be submitted; CAAC: acquisition of data, analysis and\ninterpretation of data, drafting the article, and final approval of the\nversion to be submitted; ALK: analysis and interpretation of data, drafting\nthe article and final approval of the version to be submitted; CCS: analysis\nand interpretation of data, drafting the article; conception and design of\nthe study and final approval of the version to be submitted.</p></fn><fn fn-type=\"COI-statement\" id=\"fn2\"><p>\n<bold>Conflict of Interest:</bold> The authors declare that they have no\nconflict of interest.</p></fn></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><year>2020</year></pub-date><volume>53</volume><elocation-id>e20200205</elocation-id><history><date date-type=\"received\"><day>05</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>26</day><month>6</month><year>2020</year></date></history><permissions><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the\nCreative Commons Attribution License</license-p></license></permissions><abstract><title>Abstract</title><sec><title>INTRODUCTION:</title><p> The diagnostic accuracy of Xpert MTB/RIF (Xpert) in pulmonary tuberculosis\n(PTB) in children is lower than in adults. In Brazil, the diagnosis of PTB\nis based on a diagnostic score system (DSS). This study aims to study the\nrole of Xpert in children and adolescents with PTB symptoms.</p></sec><sec><title>METHODS: A </title><p>cross-sectional study was conducted in 3 referral centers to TB. Children and\nadolescents (0-19 years old) whose respiratory samples were submitted to\nXpert were included. Statistical analysis (bivariate and logistic\nregression) to assess the simultaneous influence of TB-related variables on\nthe occurrence of Xpert detectable in TB cases was done. To evaluate the\nagreement or disagreement between Xpert results with acid-fast bacillus\n(AFB) and cultures, κ method was used (significancy level of 5%).</p></sec><sec><title>RESULTS:</title><p> Eighty-eight patients were included in the study and PTB occurred in 43\npatients (49%) and Xpert was detectable in 21 patients (24%). Adolescents\nand positive culture results were independent predictive variables of Xpert\npositivity. DSS sensitivity compared with the final diagnosis of TB was 100%\n(95% CI, 88.1-100%), specificity was 97.2% (95% CI, 85.5-99.9%). The\naccuracy of the method was 98.5% (95% CI, 91.7-99.9%). </p></sec><sec><title>CONCLUSIONS:</title><p> Xpert contributed to diagnosis in 9% of patients with AFB and in culture\nnegative cases. DSS indicated relevance for this diagnostic approach of\nintrathoracic TB (ITB) in reference centers for presenting data both with\nhigh sensitivity and specificity.</p></sec></abstract><kwd-group><title>Keywords:</title><kwd>Tuberculosis</kwd><kwd>Diagnosis</kwd><kwd>Polymerase chain reaction</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"3\"/><equation-count count=\"0\"/><ref-count count=\"22\"/></counts></article-meta></front><body><sec sec-type=\"intro\"><title>INTRODUCTION</title><p>In 2018, 72,788 new cases of tuberculosis (TB) were registered in Brazil<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>. According to World Health Organization, out of the 22 countries that\nrepresent 80% of TB cases in the world, Brazil occupies 16<sup>th</sup>\nposition<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>. In 2018, in the state of Rio de Janeiro, the incidence of TB was 66.3\ncases/100,000 inhabitants. The percentage of patients that are younger than 14 years\nwas 8% of this total<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>.</p><p>Most children who develop primary TB, are paucibacillary and are usually unable to\nexpectorate, hence not allowing bacteriological identification. Since 2002, in\nBrazil, the diagnosis of intrathoracic TB (ITB) in children is recommended by the\nNational Tuberculosis Control Program (NTCP) based on a diagnostic scoring system\n(DSS) that includes clinical symptoms, epidemiological TB history, tuberculin skin\ntest (TST) result, radiological findings, nutritional state, and does not require\nbacteriological confirmation<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>. Based on the final punctuation (≥ 40 points (very likely pulmonary TB);\n30-35 points (possible pulmonary TB); ≤ 25 points (unlikely pulmonary TB)), it\nallows for the start of a treatment for ITB according to the total points. After the\nimplementation of this strategy, several studies carried out its validation and\nobtained sensitivity greater than 80% and specificity between 70-90%<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>. More details about DSS are shown in <inline-supplementary-material mime-subtype=\"jpeg\" mimetype=\"image\" xlink:href=\"1678-9849-rsbmt-53-e20200205-suppl1.jpg\" id=\"d38e267\" content-type=\"local-data\">Supplementary Figure\n1</inline-supplementary-material>.</p><p>Since 2014, the molecular diagnosis of TB in Brazil is conducted through Xpert\nMTB/RIF system (Xpert)<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>. In the meta-analysis study conducted by Detjen et al.<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref> comparing 15 ITB studies conducted in children and adolescents, the\nsensitivity and specificity of Xpert compared to the cultures, were 62% (95%\nconfidence interval [CI], 51-73%) and 98% (95% CI, 97-99%) in spontaneous or induced\nsputum (IS), respectively, and for gastric lavage (GL), 62% (95% CI, 51-73%) and 98%\n(95% CI, 96-99%), respectively. Compared with acid-fast bacilli (AFB) smear test,\nXpert sensitivity was 36% higher in sputum/induced sputum (IS) samples and 44%\nhigher in GL samples. The detection limits of Xpert in children is lower than in\nadults with ITB. In another systematic review<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>, the positivity of TB based on Xpert among children ranged from 2-17% in IS,\n5-51% in GL, and 3-8% in nasopharyngeal aspirate (NPA).</p><p>The aim of this study was to describe the diagnostic assessment of ITB and the role\nof Xpert in children and adolescents with ITB symptoms who visited referral centers\nin the state of Rio de Janeiro, Brazil.</p></sec><sec sec-type=\"methods\"><title>METHODS</title><p>The study was cross-sectional. The data were collected in three referral centers of\nTB situated in the state of Rio de Janeiro (Hospital Municipal Raphael de Paula e\nSouza, Hospital Universitário Antônio Pedro of Fluminense Federal University and\nInstituto de Puericultura e Pediatria Martagão Gesteira at Federal University of Rio\nde Janeiro) from October 2014 to March 2019. </p><p>Patients aged 0-19 years with clinical-radiological symptoms of ITB were included in\nthe study and one respiratory sample from these patients that could reflect ITB\ndisease was submitted to Xpert<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>. The specimen could be sputum, IS, GL, bronchial lavage, bronchoalveolar\nlavage (BAL), and pleural effusions (PE). Patients whose clinical specimens were\ninsufficient or contaminated for analysis were excluded.</p><p>Patients with ITB symptoms were evaluated by physicians from hospitals and had\nsub-acute or chronic respiratory infections (≥ 14 days) and abnormal chest\nradiographs. They may or may not have other elements suggestive of ITB, such as\ncontact with adults with ITB, positive TST, and undernutrition. Only 1 sample from\neach patient underwent Xpert test.</p><p>For each patient included in the study, data were collected and DSS<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>was performed retrospectively by the main researcher (RBA). The variables\nanalyzed were age, history of contact with ITB (in the last 2 years), and DSS result\n(very probable TB when the sum was ≥ 40; possible ITB, 30 or 35; ITB unlikely, ≤ 25\npoints)<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>, TST (negative and positive), Xpert result (detectable and undetectable),\nsmear AFB, and mycobacterial culture in respiratory specimens.</p><p>Through clinical, radiological, and laboratory criteria during the first care and\nfollow-up at referral centers, the patients were classified after 60 days of\nfollow-up according to the treatment responses as either final diagnosis of TB -\nfavorable evolution with anti-TB treatment, or non-TB - with other pulmonary\ndiseases that show satisfactory evolution without anti-TB treatment.</p><p>Although the Ministry of Health in Brazil<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>adopts an age of 10 years to categorize children and adolescents, in this\narticle, we consider the following: children are patients younger than 11 years and\nadolescents are those aged 11 to 19 years (cutoff point selected by ROC curve). </p><p>A database was elaborated using the Microsoft Office Excel program (2010 version) and\ndata analysis was performed using the SPSS statistical software package v 21. Data\nanalysis was performed using descriptive statistics, with frequencies for continuous\nvariables and percentages for categorical variables. For bivariate analysis,\nstatistical significance was calculated using the χ<sup>2</sup> test or Fisher’s\ntest whenever appropriate. The level of significance was set to 5%. Logistic\nregression analysis was performed to assess the simultaneous influence of TB-related\nvariables on the occurrence of detectable Xpert values in cases which indicate a\nfinal diagnosis of TB. The choice of variables for modeling was based on the\nsignificance level of bivariate analysis of up to 20%. The collinearity between the\ncandidate variables which enter the model was evaluated by a correlation matrix. The\ncutoff point, which was used to dichotomize the continuous variable age, was\ndetermined by the receiver operating characteristic (ROC) curve. Sensitivity and\nspecificity of DSS relative to final ITB diagnosis were performed using scores ≥ 30\nor < 30 points. To evaluate the agreement between Xpert results (detectable or\nundetectable) with AFB smear test and culture (results from both the methods were\nclassified as positive or negative), κ method was used.This project was approved by\nthe Research Ethics Committee of IPPMG/Universidad e Federal do Rio de Janeiro\n(961.452).</p></sec><sec sec-type=\"results\"><title>RESULTS</title><p>Eighty-eight patients with ITB symptoms were included in the study. The median age of\npatients was 105 months (interquartile range [IQR]: 34-168 months). Contact with TB\nwas reported in 41 (53%) of 77 patients and positive TST was observed in 25 (39%) of\n64 patients. There were no cases of ITB associated with extra-thoracic TB . Of the\n88 patients studied, 48 (54%) were children, and 40 (46%) were classified as\nadolescents.</p><p>Of the 88 patients included, 65 had DSS analyzed by the main researcher (RBA): 23\npatients (35%) achieved 40 points or more; 7 patients (11%) were between 30 and 35\npoints; and 35 patients (54%) had 25 points or less. Xpert was detectable among 21\n(24%) of 88 cases, positive AFB smears were identified in 10 cases (12%), and\nculture in 17 (20%) (<xref rid=\"t1\" ref-type=\"table\">Table 1</xref>). Resistance\nresult by Xpert was observed in 4 (10%) out of 40 adolescents, and 4 (19%) out of 21\ntotal patients with positivity by Xpert. The population distribution in relation to\nXpert positivity is shown in <xref ref-type=\"fig\" rid=\"f1\">Figure 1</xref>.</p><p>\n<table-wrap id=\"t1\" orientation=\"portrait\" position=\"float\"><label>TABLE 1:</label><caption><title>Bivariate analyses showing clinical and laboratory characteristics in\nchildren and adolescents with intrathoracic tuberculosis symptoms.\n</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"2\"/><col span=\"8\"/></colgroup><thead><tr><th align=\"left\" colspan=\"2\" rowspan=\"1\">\n</th><th align=\"center\" colspan=\"8\" rowspan=\"1\">Patients with ITB symptoms n=88 </th></tr><tr><th align=\"left\" colspan=\"2\" rowspan=\"1\">\n</th><th align=\"center\" colspan=\"4\" rowspan=\"1\">Children (n=48) </th><th align=\"center\" colspan=\"4\" rowspan=\"1\">Adolescents (n=40) </th></tr><tr><th align=\"left\" colspan=\"2\" rowspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">ITB</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Non ITB</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>p</italic> value</bold>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">ITB</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Non ITB</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>p</italic> value</bold>\n</th></tr><tr><th align=\"left\" colspan=\"2\" rowspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">(n=21)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">n=27)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">(n=22)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">(n=18)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th></tr></thead><tbody><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">TST (n=64)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7(46.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4(17.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.073*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (83.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4(28.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.005</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8(53.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19(82.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2(16.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10(71.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"center\" rowspan=\"2\" colspan=\"1\">Contact with PTB (n= 77)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14(66.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9(37.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.051</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11(68.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7(43.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.154</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7(33.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15(62.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5(31.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9(56.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Xpert (n=88)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Detectable</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7(33.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.002*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (63.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Undetectable</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14(66.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27(100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">41</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8 (36.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18(100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">AFB (n=82)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3(15.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1(4.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.309*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6(30.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.022*</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17(85.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24(96.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">41</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14(70.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17(100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Culture n=83)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8(42.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9(40.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.005*</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11(57.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26(100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13(59.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16(100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">DSS (n=65)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">≥30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18(100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11 (100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1(7.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"><30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23(100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12(92.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN1\"><p>\n<bold>ITB:</bold> intra-thoracic tuberculosis; <bold>Non ITB:</bold>\nnon- intrathoracic tuberculosis; <bold>TST:</bold> tuberculin skin\ntest; <bold>PTB:</bold> pulmonary tuberculosis; <bold>Xpert:</bold>\nXpert MTB-RIF system; <bold>AFB:</bold> acid-fast bacilli;\n<bold>DSS:</bold> diagnostic scoring system.</p></fn></table-wrap-foot></table-wrap>\n</p><p>\n<fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1:</label><caption><title>Population distribution in relation to Xpert test results in 88\npatients with intrathoracic tuberculosis symptoms.</title></caption><graphic xlink:href=\"1678-9849-rsbmt-53-e20200205-gf1\"/></fig>\n</p><p> Xpert was detectable in 7 (15%) out of 48 children, including 3/7 samples from BAL,\n3/7 specimens from GL, and 1/7 from PE samples, and 14 (35%) out of 40 adolescents,\nwith 11/14 from specimens taken from the sputum and IS and the other 3 samples were\nfrom PE and BAL. The final diagnosis of ITB was established in 43 (49%) of the 88\ncases. The characteristics of ITB and non-ITB patients are described in <xref rid=\"t1\" ref-type=\"table\">Table 1</xref>.</p><p>In 25 (58%) of the 43 patients with ITB, the time from specimen collection to the\ndelivery of the Xpert results to the health team ranged from 0 to 22 days, with a\nmedian of 1 day (IQR: 1-6 days). In 10 (40%) of these 25 cases studied, treatment\nwas started before receiving the laboratory Xpert results.</p><p>The comparison among Xpert, AFB smear and culture techniques are described in <xref rid=\"t2\" ref-type=\"table\">Table 2</xref>. In 81 patients who did all three\nmethods simultaneously, Xpert was detectable in 7 patients who were ABF and culture\nnegative. The <inline-supplementary-material mime-subtype=\"jpeg\" mimetype=\"image\" xlink:href=\"1678-9849-rsbmt-53-e20200205-suppl2.jpg\" id=\"d38e732\" content-type=\"local-data\">Supplementary Table\n1</inline-supplementary-material> shows the results of Xpert compared with\nTB-related characteristics in the group with final TB diagnosis (bivariate\nanalysis). Based on these results, logistic regression analysis was performed.</p><p>\n<table-wrap id=\"t2\" orientation=\"portrait\" position=\"float\"><label>TABLE 2:</label><caption><title>Comparison of Xpert results with acid-fast bacilli and culture\nmethods</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"2\"/><col span=\"4\"/></colgroup><thead><tr><th align=\"center\" colspan=\"2\" rowspan=\"1\">Bacteriological results </th><th align=\"center\" colspan=\"4\" rowspan=\"1\">Patients with ITB symptoms </th></tr><tr><th align=\"left\" colspan=\"2\" rowspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Xpert</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>Xpert</italic>\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>kappa</italic>\n</th></tr><tr><th align=\"left\" colspan=\"2\" rowspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Detectable (n= 21)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Undetectable (n= 67)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">(CI 95%)</th></tr></thead><tbody><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">AFB (n=82)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5(26.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5(8.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.220</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14(73.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">58(92.0%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">72</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(0.194 - 0.247)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Culture (n=83)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13(61.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4(6.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.592</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8(38.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">58(93.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">66</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(0.537 - 0.646)</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN2\"><p>\n<bold>ITB:</bold> intrathoracic tuberculosis; <bold>AFB:</bold>\nacid-fast bacilli; <bold>Xpert:</bold> Xpert MTB/RIF system.</p></fn></table-wrap-foot></table-wrap>\n</p><p>In the multivariate analysis, it was observed that patient age older than 11 years\nand positive culture result were independent characteristics of Xpert positivity\n(<xref rid=\"t3\" ref-type=\"table\">Table 3</xref>). In 35 patients with DSS score\nof less than 30, Xpert was undetectable in 100% of cases. In 30 patients with a\nscore of 30 or greater, Xpert was detectable in 15 cases (50%). DSS sensitivity\ncompared with the final diagnosis of TB was 100% (95% CI, 88.1-100%), specificity\nwas 97.2% (95% CI, 85.5-99.9%) and the accuracy of this method was 98.5% (95% CI,\n91.7-99.9%). </p><p>\n<table-wrap id=\"t3\" orientation=\"portrait\" position=\"float\"><label>TABLE 3:</label><caption><title>Multivariate analyses: the association of variables with a higher\nchance for Xpert positivity. </title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Predictor characteritic</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">OR</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">95% CI</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><italic>p</italic> value</bold>\n</th></tr></thead><tbody><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Positive culture</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.820</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.751 - 39.186</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.008</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Age ≥ 11 years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.177</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.926 - 18.846</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.063</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Constant</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.211</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.024</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN3\"><p>\n<bold>OR:</bold> Odds ratio; <bold>CI:</bold> confidence\ninterval.</p></fn></table-wrap-foot></table-wrap>\n</p></sec><sec sec-type=\"discussion\"><title>DISCUSSION</title><p>Our study was conducted in three referral centers for children and adolescents with\nITB in the state of Rio de Janeiro. The positive AFB smear result of 12% obtained in\nour study is higher than the values obtained by previous studies which ranged\n​​between 1.6-4.8%<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref>. Comparing the three methods used in our study, almost 25% of our patients\nwith detectable Xpert also had a positive AFB smear with little agreement by κ\nindex. This finding is similar to a previous study that showed higher performance of\na molecular test than AFB smear test for the diagnosis of ITB among children and\nadolescents<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref>. On the other hand, more than half of the patients with detectable Xpert\nalso had a positive culture result, which could be explainable since the detection\nlimit of the culture method could range from 10 to 100 colony-forming units\n(CFUs)/mL and approaches the values of the molecular test, which is 131 CFUs/mL<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>. Xpert contributed to diagnosis in 7 cases (9%) where both AFB and culture\nmethods were negative. The evaluation of Xpert results exclusively in pulmonary\nspecimens from patients who were younger than 15 years in South Africa<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>, showed similar positivity to our culture result (71.4% of the samples with\ndetectable Xpert in IS and 65.1% in NPA). The evaluation of symptomatic contacts\nwith active ITB disease cases in West Africa<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref> detected lower Xpert positivity result of 42% in samples with positive\nculture. </p><p>In the present study, Xpert positivity was 24%, which is much higher than the 2.4%\nvalue obtained by Togun et al. in West Africa<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref> analyzing IS samples from patients younger than 15 years who had previous\ncontact with active ITB patients or respiratory symptoms and/or positive TST. It is\npossible that the low positivity of this study compared with our study was due to\nthe fact that only IS was used which could generate samples with low bacillary load\nin children. Similarly, based on studies in India, a low positivity rate of Xpert\nwas observed. Raizada et al.<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref> obtained 10.4% Xpert positivity in referral centers among symptomatic\nrespiratory patients without compatible radiological imaging, and Das et al.<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref>, analyzing samples from GL, IS, cerebrospinal fluid and lymph node from\npatients who are younger than 15 years, found only 11% Xpert positivity. In these\nstudies, Xpert positivity lower than what was observed by our study could be due to\nthe fact that, in the first study, the authors included only patients under 15 years\nof age with TB symptoms without enhancing the radiological and, in the second study,\nthe authors indistinctly analyzed respiratory and extrapulmonary specimens. The\ninclusion criteria adopted by us were stricter than those adopted by other authors\nincluding cases with signs and symptoms suggestive of TB associated with other\nradiological findings suggestive of the disease, which increased Xpert positivity.\nOn the other hand, in the meta-analysis study performed by Detjen et al.<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref> in patients aged 0 to 15 years with presumed PTB, with or without suggestive\nradiological imaging, Xpert positivity was measured to be 11%.</p><p>Specifically analyzing the Xpert positivity of 15% observed among children, the\nhighest detection rates occurred in specimens collected from GL and BAL. Ioos et\nal.<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref> in a systematic review with children younger than 15 years, observed that\nXpert detection in respiratory specimens from patients with presumed ITB was higher\nin GL samples, however, BAL and PE were not evaluated. </p><p>Among the adolescents included in the present study, Xpert positivity was observed to\nbe 35%, which is higher than 15.7% found in the study conducted in Rio de Janeiro\nCity<xref rid=\"B16\" ref-type=\"bibr\">\n<sup>16</sup>\n</xref>, which included adolescents with ITB symptoms, in the year of the\nimplementation of the laboratory method as routine procedure in TB bacteriological\nidentification. Lower Xpert performance was expected since any person to be\nevaluated for ITB was included in the study, regardless of their epidemiological\nhistory, laboratory results, and chest radiograph imaging results.</p><p>In the current study, 10% of drug resistance was detected among adolescents with ITB\nsymptoms. Analyzing rifampicin resistance (RIF-resistance) detected by Xpert among\ncases in which this method was positive, our result was 19%, similar to the 16.6%\nreported by Raizada et al.<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref>, in India for patients under 14 years of age. In both these studies,\nrespiratory samples were exclusively analyzed and the studies were conducted in TB\nunits. </p><p>Analyzing the results based on age groups, the entire children group with ITB\npresented a DSS result of 30 or greater, allowing the start of anti-TB treatment. In\ncontrast, in those individuals with no ITB, all of them had a score of less than 30.\nIn the ITB group, bacteriological identification was observed among 33% of the\npatients, although this age group typically is paucibacillary. Also, in adolescents\nwith ITB, almost every population had DSS value of 30 or greater, and a little more\nthan half the population had detectable Xpert results. These findings support the\nvalidation of DSS obtained in other studies and, at the same time, guide the careful\nuse of Xpert, preceded by DSS, in children and adolescents treated at referral\ncenters<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>. DSS has been standardized for over a decade in Brazil. Our data confirmed\nthe high sensitivity and specificity of DSS, and it would be justifiable to\nroutinely adopt this system to make a decision to start ITB treatment. Diagnosis\nwithout bacteriological or molecular confirmation is still a reality in endemic\nareas of TB. A study conducted by Oliveira et al<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>in Rio de Janeiro analyzing adolescents showed that the clinical diagnosis of\nTB was decisive in patients with greater complexity (diagnosis at hospital level,\ncarriers of human immunodeficiency virus, or combined forms of TB) and they had a\nnegative Xpert value. In addition, the time from clinical sample collection to start\nof treatment by the health care team varied from 8 days (IQR, 6-14 days) in the\nXpert-positive/culture-positive group to 12 days (IQR, 5-36 days) in the\nXpert-negative/culture-negative group. In contrast, our shortest interval occurred\nsince this study was conducted at a referral center. In addition, we found that 40%\nof the anti-TB treatment group was instituted based on the clinical presentation and\nDSS, indicating its relevance on the diagnostic approach of ITB in this population\nin primary care units, such as referral centers, for presenting the results with\nboth high sensitivity and specificity<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>.</p><p>Although the positive culture and age above 11 being independent variables for Xpert\npositivity, the small sample size has OR with very wide 95% CI, meaning low\nprecision. </p><p>One of the limitations was the collection of a only a single sample from each patient\nfor Xpert analysis. Perhaps Xpert positivity percentages would be higher if more\nthan one sample was collected per patient<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>. Another limitation of the study is that it was conducted only in referral\ncenters, not including basic health units. It is probable that this situation\nprovided a classification bias since we used more criteria to select eligible\npatients, generating greater positivity of laboratory methods in the pediatric\ngroup. There is a perspective to increase the diagnostic capacity of new molecular\ntests via Xpert Ultra, which was built recently using the same methodology as Xpert,\nbut with higher sensitivity due to its lower detection limit for\n<italic>Mycobacterium tuberculosis</italic> (similar to the lower limit of\nculture)<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>. Its use in combined specimens might be able to increase the sensitivity in\ncases of bacteriologically confirmed ITB, but in cases with low bacillary load, the\ndetection of RMP resistance might be impaired<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref>.</p><p>Future studies with patients from primary care units in our country might provide a\ncomparison of Xpert results with our patients.</p></sec></body><back><ack><title>ACKNOWLEDGMENTS</title><p>CCS was supported by the Conselho Nacional Conselho Nacional de Pesquisa e\nDesenvolvimento (Grant # 305147/2016-1).</p></ack><fn-group><fn fn-type=\"financial-disclosure\" id=\"fn3\"><p>CCS was supported by the Conselho Nacional de Pesquisa e Desenvolvimento (Grant #\n305147/2016-1).</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id=\"B1\"><label>1</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>Ministério da Saúde (MS). 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Rev Soc Bras Med Trop</journal-id><journal-id journal-id-type=\"iso-abbrev\">Rev. Soc. Bras. Med. Trop</journal-id><journal-id journal-id-type=\"publisher-id\">rsbmt</journal-id><journal-title-group><journal-title>Revista da Sociedade Brasileira de Medicina Tropical</journal-title></journal-title-group><issn pub-type=\"ppub\">0037-8682</issn><issn pub-type=\"epub\">1678-9849</issn><publisher><publisher-name>Sociedade Brasileira de Medicina Tropical - SBMT</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32997046</article-id><article-id pub-id-type=\"pmc\">PMC7523523</article-id><article-id pub-id-type=\"doi\">10.1590/0037-8682-0562-2019</article-id><article-id pub-id-type=\"other\">00348</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Major Article</subject></subj-group></article-categories><title-group><article-title>The use of the circulating cathodic antigen (CCA) urine cassette\nassay for the diagnosis and assessment of cure of <italic>Schistosoma\nmansoni</italic> infections in an endemic area of the Amazon\nregion</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0003-2197-7323</contrib-id><name><surname>de Sousa</surname><given-names>Sergei Rodrigo Magalhães</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nogueira</surname><given-names>Joyce Favacho Cardoso</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dias</surname><given-names>Isabelle Helena Lima</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Fonseca</surname><given-names>Álvaro Luan Santana</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Favero</surname><given-names>Vivian</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Geiger</surname><given-names>Stefan Michael</given-names></name><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Enk</surname><given-names>Martin Johannes</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<label>1</label>Universidade do Estado do Pará, Programa de Pós-Graduação Strictu\nSensu em Biologia Parasitária na Amazônia, Belém, PA, Brasil. </aff><aff id=\"aff2\">\n<label>2</label>Instituto Evandro Chagas/SVS/MS, Laboratório de Parasitoses\nIntestinais, Esquistossomose e Malacologia, Secção de Parasitologia, Ananindeua, PA,\nBrasil. </aff><aff id=\"aff3\">\n<label>3</label>Pontifícia Universidade Católica do Rio Grande do Sul, Programa de\nPós-Graduação em Medicina e Ciências da Saúde, Laboratório de Parasitologia\nBiomédica, Porto Alegre, RS, Brasil. </aff><aff id=\"aff4\">\n<label>4</label>Universidade Federal de Minas Gerais, Departamento de Parasitologia,\nBelo Horizonte, MG, Brasil. </aff><author-notes><corresp id=\"c1\"><bold>Corresponding author:</bold> Msc. Sergei Rodrigo Magalhães de\nSousa<bold>.</bold><bold>e-mail:</bold><email>rodrigo.bio.uepa@gmail.com</email></corresp><fn fn-type=\"con\" id=\"fn3\"><p>\n<bold>Authors’ contributions:</bold>\n<bold>MJE and SMG</bold> participated in the design of the study.\n<bold>SRMS, IHLD, ÁLSF, VF, JFNC, and MJE</bold> participated in the\nacquisition, analysis, and interpretation of data and drafting of the\nmanuscript. All authors read and approved the final manuscript.</p></fn><fn fn-type=\"COI-statement\" id=\"fn4\"><p>\n<bold>Conflict of interest:</bold> The authors declare that they have no\ncompeting interests.</p></fn></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><year>2020</year></pub-date><volume>53</volume><elocation-id>e20190562</elocation-id><history><date date-type=\"received\"><day>07</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>20</day><month>7</month><year>2020</year></date></history><permissions><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the\nCreative Commons Attribution License</license-p></license></permissions><abstract><title>Abstract</title><sec><title>INTRODUCTION</title><p> Schistosomiasis is a poverty-related disease that affects people in 78\ncountries worldwide. This study aimed to evaluate the point-of-care\ncirculating cathodic antigen (POC-CCA) test performance using sensitive\nparasitological methods as a reference standard (RS) in individuals before\nand after treatment. </p></sec><sec><title>METHODS</title><p> The RS was established by combining the results of 16 Kato-Katz slides and\nthe Helmintex<sup>®</sup> method. Positivity rates of the POC-CCA test and\nKato-Katz and Helmintex<sup>®</sup> methods were calculated before treatment\nand 30 days afterward. Furthermore, the sensitivity, specificity, accuracy,\nand <italic>kappa</italic> coefficient before treatment were determined by\ncomparing the methods. The cure rate was defined 30 days after treatment.\n</p></sec><sec><title>RESULTS</title><p> Among the 217 participants, the RS detected a total of 63 (29.0%) positive\nindividuals. The POC-CCA test identified 79 (36.4%) infections. The\nevaluation of POC-CCA test performance in relation to the RS revealed a\nsensitivity of 61.9%, specificity of 74.0%, accuracy of 70.5%, and\n<italic>kappa</italic> coefficient of 0.33. Out of the 53 remaining\nparticipants after treatment, a total of 45 (81.1%) showed egg negative\nresults, and 8 (18.9%) were egg positive according to the RS. A total of 5\n(9.4%) egg-positive and 37 (69.8%) egg-negative individuals were positive by\nthe POC-CCA test. </p></sec><sec><title>CONCLUSIONS</title><p> Our data show that the POC-CCA test has potential as an auxiliary tool for\nthe diagnosis of <italic>Schistosoma mansoni</italic> infection, yielding\nbetter results than 16 Kato-Katz slides from three different stool samples.\nHowever, the immunochromatographic test lacks sufficient specificity and\nsensitivity for verifying the cure rate after treatment.</p></sec></abstract><kwd-group><title>Keywords:</title><kwd>Schistosoma mansoni</kwd><kwd>Kato-Katz</kwd><kwd>Helmintex<sup>®</sup></kwd><kwd>POC-CCA</kwd><kwd>Treatment</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"4\"/><equation-count count=\"0\"/><ref-count count=\"29\"/></counts></article-meta></front><body><sec sec-type=\"intro\"><title>INTRODUCTION</title><p>In 2012, the World Health Assembly adopted a resolution that predicts the\ninterruption of schistosomiasis transmission<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>. In 2015, a total of 118.5 million school-aged children and 100.2 million\nadults were indicated for preventive chemotherapy with praziquantel<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>. Schistosomiasis affects 78 countries worldwide, and according to the World\nHealth Organization (WHO), preventive chemotherapy is required in 52 endemic\ncountries with moderate to high disease transmission rates. A total of 90 million\nindividuals were treated in 2016 due to the expansion of control interventions<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>.</p><p>\n<italic>Schistosoma mansoni</italic> is the only species found in the Americas,\nwhere it is believed that more than 25 million individuals are at risk of\ninfection<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>. Brazil has the largest area and is responsible for 95% of cases<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>. From 2010 to 2016, regular schistosomiasis control in Brazil revealed a\npositivity rate of 4.4%<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref>. Studies conducted in 2015 and 2018 revealed an estimated 1.5 million\ninfected people<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>, indicating an overall schistosomiasis prevalence rate of approximately 1.0%\nin Brazil<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>.</p><p>A precise and efficient diagnosis is an important tool for the treatment and control\nof schistosomiasis<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>. The currently recommended method for quantitative diagnosis of <italic>S.\nmansoni</italic> is Kato-Katz (KK) fecal thick-smear slides, which are supposed\nto detect eggs in infected individuals feces<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>. However, this method has limitations and may underestimate the infection\nrate by up to 74.0%<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>.</p><p>In 2007, a new diagnostic method named Helmintex<sup>®</sup> (HTX), developed\nspecifically for use in areas where the <italic>S. mansoni</italic> egg burden was\nreduced, showed high sensitivity due to the use of a large amount of feces (30\ngrams) and several concentration steps that resulted in the isolation of <italic>S.\nmansoni</italic> eggs through interaction with paramagnetic particles in a\nmagnetic field<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>. Even with a 100% sensitivity for up to 1.3 eggs per gram (EPG) loads<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>, its application on a large scale presented some difficulties; thus, many\naspects of the HTX method were optimized, aiming to make it more efficient<xref rid=\"B16\" ref-type=\"bibr\">\n<sup>16</sup>\n</xref>. The HTX application confirmed this method as a high sensitivity diagnostic\ntool in endemic areas<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>.</p><p>Aiming to solve the dilemma for fast and accurate diagnosis of schistosomiasis, a\npoint-of-care (POC) urine test was developed for the detection of schistosome\ncirculating cathodic antigen (CCA). According to data presented in the\nmanufacturer’s manual of the POC-CCA test, the sensitivity rate may vary from 70% to\n100%, depending on the intensity of infection<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>. When the POC-CCA test was compared with the KK method, it was reported that\na single urine test showed a sensitivity equivalent to that of six<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref> or nine KK slides<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>. However, recent studies on the performance of POC-CCA also showed\ncontroversial results, with reduced accuracy and elevated false negative or false\npositive rates, particularly in low prevalence areas<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>.</p><p>The WHO and the Department of Control of Neglected Tropical Diseases in 2015\nsuggested the use of the POC-CCA test in endemic countries, along with the KK\nmethod, for monitoring and evaluation of control programs, whose goal is the\nelimination of schistosomiasis as a public health problem<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>. A published review<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref> on the use of KK as an RS to evaluate the performance of the POC-CCA test\nfor the diagnosis of <italic>S. mansoni</italic> infections showed that most of the\nstudies were conducted in Africa. To date, ten studies on POC-CCA performance for\nthe diagnosis of intestinal schistosomiasis have been conducted in Brazil. Only a\nfew of them used the HTX method as an RS<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>. Hence, there is an urgent need to better evaluate the performance of the\nPOC-CCA test, using more sensitive parasitological methods as RSs, such as the KK\ntechnique and modified HTX methods. Therefore, the present study aimed to evaluate\nthe POC-CCA test performance using more sensitive parasitological methods as\nreference standards (RSs) among individuals before and after treatment.</p></sec><sec sec-type=\"methods\"><title>METHODS</title><sec><title>Study area and population</title><p>The present study was conducted from March to October 2014 in the community of\nPaxiba, municipality of Turiaçú, Maranhão State in Brazil, located 152 km from\nthe capital São Luis. It is part of the Amazon region characterized by a\ntropical climate, with temperatures ranging from 16ºC to 36.4ºC and an annual\naverage rainfall between 191.9 mm and 218.2 mm<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref>.</p><p>All 235 community residents were invited to participate in this study. A sample\nsize calculation was not necessary because the entire community was enrolled.\nPrevious surveys conducted by the Brazilian Schistosomiasis Control Program\nreported a positivity rate of 5.0%.</p><p>To be enrolled in the present study, each participant had to deliver stool and\nurine samples. In addition, children younger than 2 years were excluded.</p></sec><sec><title>Biological sample collection procedures</title><p>Among the 217 participants, one morning urine sample and three stool samples were\ncollected on consecutive days at two time points: before treatment and 30 days\nafterward. It is important to note that only egg-positive individuals were\nre-examined 30 days after treatment. All biological samples were identified,\nstored in properly cooled cases, and transported to the Instituto Evandro Chagas\nSVS/MS. All laboratory procedures were carried out at the Laboratório de\nParasitoses Intestinais Esquistossomose e Malacologia, located at the Instituto\nEvandro Chagas SVS/MS. </p></sec><sec><title>Kato-Katz method (Katz et al., 1972)</title><p>A commercial KK kit (HelmTest; Biomanguinhos, Brazil) was used to prepare slides\nwith fecal smears, according to the manufacturer’s instructions.</p><p>A total of 16 KK fecal thick smears were prepared: twelve from the first fecal\nsample, two from the second, and two from the third. The 12 slides from the\nfirst sample summed up to 500 mg of examined fecal matter. Together with the\nslides from samples two and three, a total of approximately 667 mg of feces was\nanalyzed.</p><p>EPG values were calculated based on the number of eggs counted on 16 slides from\ndifferent samples.</p><p>The HTX test was also performed using the first sample. The remaining biological\nsamples were frozen and stored at the Biobank of the Parasitology Section of\nInstituto Evandro Chagas -Pará State.</p></sec><sec><title>Helmintex<sup>®</sup>\n</title><p>Described by Teixeira <italic>et al</italic>. (2007)<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref> and modified by Favero <italic>et al.</italic> (2017)<xref rid=\"B16\" ref-type=\"bibr\">\n<sup>16</sup>\n</xref>, the HTX method was specifically developed for <italic>S.\nmansoni</italic> egg detection. This method consists of concentration steps\nthat aim to select eggs among sediments by applying paramagnetic beads that bind\nto the schistosome eggshell. After this process, a magnetic field is applied,\nand the eggs can be separated from the remaining sediment. The final material\nwas added to a 3% ninhydrin solution and spread on filter paper to quantify the\neggs by reading under a microscope. In the present study, an average of five\nfilters was examined per sample. Eggs were identified correctly following the\nproposed criteria based on egg elements such as shape, presence of spike,\napproximated size, well-defined shell, space between the miracidium and shell,\nand purple color of the miracidium<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>. </p></sec><sec><title>POC-CCA</title><p>The urine-CCA cassette is recommended for the qualitative detection of an active\n<italic>Schistosoma</italic> infection, as it is more specific for\n<italic>S. mansoni</italic> infections. In the present study, the first\nversion of the test (lot number: 50182) was used, provided by the Brazilian\nMinistry of Health from Rapid Medical Diagnostics, Pretoria, South Africa.</p><p>Only one drop of first morning urine was required for the examination. According\nto the manufacturer’s recommendation, one drop of buffer was added<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>. The test result was reported 20 minutes after adding the buffer to the\nsamples. All the results of the immunochromatographic test were interpreted as\npositive, considering the development of a second pink line parallel to the\ncontrol line; otherwise, the test result was considered negative, according to\nthe manufacturer’s recommendations<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>. It is important to note that a weak pink line, classified as a ‘trace’\nresult, was included in the analysis, first as a positive result and second as a\nnegative result. Three experienced and properly trained laboratory staff\nanalyzed the POC-CCA test to ensure quality test results.</p></sec><sec><title>Reference standard</title><p>The RS was composed of a total of 16 KK slides and HTX method analysis to\nmaximize the detection of egg-positive individuals infected with <italic>S.\nmansoni</italic>. All positive individuals confirmed by the RS were\nclassified as true positives.</p></sec><sec><title>Statistical analysis</title><p>Statistical tests were performed using the program OpenEpi version 3.01 by\nEpidemiologic Statistics for Public Health (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.openepi.com/Menu/OE_Menu.htm\">https://www.openepi.com/Menu/OE_Menu.htm</ext-link> updated in 2013). The\nresults were paired in 2 × 2 tables with 95% confidence intervals (CIs). The\nrates of positivity, sensitivity, specificity, positive and negative predictive\nvalues, and accuracy were calculated to verify the performance of POC-CCA\ncompared to the RS. The kappa index was calculated to evaluate the concordance\nbetween the tests in analysis and RS, following the classification criteria\nrecommended by Landis and Koch (1977)<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref>, with concordance values considered as bad (<0.20), weak (0.21-0.40),\nmoderate (0.41-0.60), good (0.61-0.80), and excellent (>0.81).</p></sec><sec><title>Ethical considerations</title><p>The study was part of a multicenter project, with participants from the States of\nPará, Minas Gerais, and Rio Grande do Sul. The project was submitted to the\nethics committee and approved (CAAE: 21824513.9.3001.0019). All participants\nwere informed about the objectives and invited to participate voluntarily, and\nenrolled individuals signed the consent form. All individuals with positive test\nresults detected according to the RS were treated with praziquantel, following\nthe guidelines of the Brazilian Ministry of Health, with 60 mg/kg for children\nand 50 mg/kg for adults<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>.</p></sec></sec><sec sec-type=\"results\"><title>RESULTS</title><sec><title>Positivity rate by RS</title><p>After applying the exclusion criteria, of 235 individuals invited to participate\nin this study, only 217 remained. Among those, 111 (51.1%) were males and 106\n(48.9%) were females. In relation to age, 59 participants were aged 21-40 years,\nwhile 20 participants were older than 60 years (<inline-supplementary-material mime-subtype=\"jpeg\" mimetype=\"image\" xlink:href=\"1678-9849-rsbmt-53-e20190562-suppl1.jpg\" id=\"d38e687\" content-type=\"local-data\">Supplementary Table 1</inline-supplementary-material>). </p><p>The RS detected a total of 63 individuals with positive results, yielding a\npositivity rate of 29.0% among the 217 participants. Based on the number of\npositives and positivity rate in relation to sex, 38 (34.2%) males were infected\nand 25 (23.6%) females were egg-positive.</p><p>The positivity rate by age group determined by the RS was highest among\nindividuals aged 11-20 years (n=26). </p></sec><sec><title>Positivity rate by Kato-Katz and Helmintex<sup>®</sup> methods</title><p>The analysis via the KK technique using 16 slides of different samples among the\n217 participants showed that 31 (14.3%) individuals were <italic>S.\nmansoni</italic> egg positive. Using a single KK slide, a total of 12 (5.5%)\negg-positive individuals were detected, which increased further to 17 (7.8%)\nafter reading two slides. The parasitic load assessed via 16 slides from\ndifferent samples revealed that all the participants had a low parasite load,\nwith less than 100 EPG of feces.</p><p>In relation to the HTX method, a total of 53 (24.4%) egg-positive individuals\nwere identified.</p></sec><sec><title>Positivity rate by POC-CCA</title><p>Apart from the parasitological examination, a rapid urine test was applied for\nthe detection of CCA. The POC-CCA test identified 79 infected individuals,\nresulting in a positivity rate of 36.4%. Based on the number of positives and\npositivity rate in relation to sex, a total of 48 (60.8%) males and 31 (39.2%)\nfemales were egg positive. The distribution of positive individuals according to\nage group is shown in <inline-supplementary-material mime-subtype=\"jpeg\" mimetype=\"image\" xlink:href=\"1678-9849-rsbmt-53-e20190562-suppl1.jpg\" id=\"d38e712\" content-type=\"local-data\">Supplementary Table 1</inline-supplementary-material>.</p></sec><sec><title>Accuracy analysis and POC-CCA test “trace” results</title><p>Evaluation of the POC-CCA test performance in relation to the RS revealed a\nsensitivity of 61.9%. In comparison with each of the parasitological tests, 16\nKK slides and the HTX<sup>®</sup> method, the sensitivity of the POC-CCA test\ndecreased from 80.6% to 56.6%. Additional data regarding the accuracy analysis\nare described in <xref rid=\"t1\" ref-type=\"table\">Table 1</xref>.</p><p>\n<table-wrap id=\"t1\" orientation=\"portrait\" position=\"float\"><label>TABLE 1:</label><caption><title>Comparison of the number of individuals detected with intestinal\nschistosomiasis by the reference standard, KK technique, and HTX in\nrelation to positive individuals detected by the rapid urine test\n(POC-CCA).</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"2\"/><col span=\"1\"/><col span=\"2\"/><col span=\"1\"/><col span=\"2\"/><col span=\"1\"/></colgroup><thead><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">POC-CCA</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">Reference Standard </th><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">KK 16S 3SA </th><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">HTX<sup>®</sup>\n</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th></tr></thead><tbody><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Positive</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">79</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">54</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">79</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">46</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">79</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Negative</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">114</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">138</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">132</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">138</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">115</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">138</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">63</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">154</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">217</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">186</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">217</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">164</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">217</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Sensitivit</bold>y</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">61.9% (95% <italic>CI</italic>:\n49.5 - 72.9) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">80.6% (95% <italic>CI</italic>:\n63.7 - 90.8) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">56.6% (95% <italic>CI</italic>:\n43.3 - 69.0) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Specificity</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">74.0% (95% <italic>CI</italic>:\n66.6 - 80.3) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">71.0% (95% <italic>CI</italic>:\n64.1 - 77.0) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">70.1% (95<italic>% CI</italic>:\n62.7 - 76.6) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>PPV</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">49.4% (95% <italic>CI</italic>:\n38.6 - 60.1) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">31.6% (95% <italic>CI</italic>:\n22.4 - 42.5) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">38.0% (95% <italic>CI</italic>:\n28.1 - 49.0) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>NPV</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">82.6% (95% <italic>CI</italic>:\n75.4 - 88.0) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">95.6% (95% <italic>CI</italic>:\n90.8 - 98.0) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">83.3% (95% <italic>CI</italic>:\n76.2 - 88.6) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Kappa index</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">0.33 (95% <italic>CI</italic>:\n0.20 - 0.46) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">0.31 (95% <italic>CI</italic>:\n66.0 - 77.9) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">0.22 (95% <italic>CI</italic>:\n0.10 - 0.36) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Accuracy</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">70.5% (95% <italic>CI</italic>:\n64.1 - 76.2) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">72.3% (95% <italic>CI</italic>:\n0.20 - 0.42) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">66.8% (95% <italic>CI</italic>:\n60.3 - 72.7)</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN1\"><p>\n<bold>POC-CCA:</bold> Point-of-care circulating cathodic antigen\ntest; <bold>PPV:</bold> Positive predictive value;\n<bold>NPV:</bold> Negative predictive value;\n<bold>RS:</bold> composed of a total of 16 KK slides and 30\ngrams of fecal matter, examined by the HTX method; <bold>16S\n3SA:</bold> Sixteen slides, twelve slides from the first\nsample, two slides from the second sample, and two slides from\nthe third sample; <bold>HTX:</bold> Helmintex<sup>®</sup>\nmethod.</p></fn></table-wrap-foot></table-wrap>\n</p><p>In the comparisons between the POC-CCA and RS test results, a total of 39\nindividuals were found to be egg positive. This number decreased to 25 and 30\negg positive individuals when the test was compared to each of the\nparasitological methods (e.g., the KK and HTX methods, respectively). More\ndetails are shown in <xref rid=\"t1\" ref-type=\"table\">Table 1</xref>, <xref rid=\"t2\" ref-type=\"table\">Table 2</xref>, and <xref ref-type=\"fig\" rid=\"f1\">Figure 1</xref>.</p><p>\n<table-wrap id=\"t2\" orientation=\"portrait\" position=\"float\"><label>TABLE 2:</label><caption><title>Number of individuals with intestinal schistosomiasis, as\ndetected by one (1S 1st SA) or two (2S 1st SA) fecal thick smears\nand concordance and accuracy with the rapid urine test\n(POC-CCA).</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"3\"/><col span=\"3\"/></colgroup><thead><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">POC-CCA</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">1S 1<sup>st</sup> AS </th><th align=\"center\" colspan=\"3\" rowspan=\"1\">2S 1<sup>st</sup> AS </th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th></tr></thead><tbody><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Positive</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">69</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">79</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">64</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">79</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Negative</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">136</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">138</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">136</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">138</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">205</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">217</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">200</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">217</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Sensitivity</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">83.3% (95% <italic>CI</italic>:\n55.2 - 95.3) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">88.2% (95% <italic>CI</italic>:\n65.6 - 96.7) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Specificity</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">66.3% (95% <italic>CI</italic>:\n59.6 - 72.4) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">68.0% (95% <italic>CI</italic>:\n61.2 - 74.1) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>PPV</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">12.6% (95% <italic>CI</italic>:\n7.0 - 21.7) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">19.0% (95% <italic>CI</italic>:\n11.8 - 29.0) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>NPV</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">98.5% (95% <italic>CI</italic>:\n94.9 - 99.6) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">98.5% (95% <italic>CI</italic>:\n94.9 - 99.6) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Kappa index</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">0.13 (95% <italic>CI</italic>:\n0.06 - 0.21) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">0.21 (95% <italic>CI</italic>:\n0.12 - 0.30) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Accuracy</bold>\n</td><td align=\"center\" colspan=\"3\" rowspan=\"1\">67.3% (95% <italic>CI</italic>:\n60.8 - 73.1) </td><td align=\"center\" colspan=\"3\" rowspan=\"1\">69.5% (95% <italic>CI</italic>:\n63.2 - 75.3)</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN2\"><p>\n<bold>POC-CCA:</bold> Point-of-care circulating cathodic antigen\ntest; <bold>1S 1</bold>\n<sup>st</sup>\n<bold>SA:</bold> One slide of the first sample; <bold>2S\n1</bold>\n<sup>st</sup>\n<bold>SA:</bold> Two slides of the first sample;\n<bold>PPV:</bold> Positive predictive value;\n<bold>NPV:</bold> Negative predictive value</p></fn></table-wrap-foot></table-wrap>\n</p><p>\n<fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1:</label><caption><title>True positive results detected by the POC-CCA test confirmed by\ndifferent coproscopic methods.<bold>POC-CCA TPR:</bold> Point of\ncare circulating cathodic antigen true positive results; coproscopic\nmethods: (<bold>1S 1</bold>\n<sup>st</sup>\n<bold>SA:</bold> One slide of the first sample; <bold>2S 1</bold>\n<sup>st</sup>\n<bold>SA:</bold> Two slides of the first sample; <bold>16S\n3SA:</bold> Sixteen slides of three different samples, twelve\nslides of the first sample, two slides of the second sample and two\nslides of the third sample; <bold>HTX</bold>\n<sup>®</sup>\n<bold>:</bold> Helmintex<sup>®</sup> method); <bold>RS:</bold>\nReference standard</title></caption><graphic xlink:href=\"1678-9849-rsbmt-53-e20190562-gf1\"/></fig>\n</p><p>Analysis of the POC-CCA test showed a total of 42 weak positive results out of 79\npositive individuals detected, which were classified as “trace” results. Out of\nthese 42 trace results, a total of 15 were classified as egg positive and 27 as\negg negative when compared with the RS (<xref rid=\"t3\" ref-type=\"table\">Table\n3</xref>).</p><p>\n<table-wrap id=\"t3\" orientation=\"portrait\" position=\"float\"><label>TABLE 3:</label><caption><title>Positive and negative results determined by the reference\nstandard in relation to the POC-CCA test results, considering trace\nresults as negative.</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"3\"/></colgroup><thead><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">POC-CCA</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">Reference standard </th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th></tr></thead><tbody><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Positive</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Negative</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">140</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">180</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">63</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">154</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">217</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN3\"><p>\n<bold>POC-CCA:</bold> Point-of-care circulating cathodic antigen\ntest; Reference standard: composed of a total of 16 KK slides\nand 30 grams of fecal matter, examined by HTX.</p></fn></table-wrap-foot></table-wrap>\n</p></sec><sec><title>Parasitological and immunological results 30 days after treatment</title><p>Out of 63 positive individuals detected by the RS, only 53 continued in the\nevaluation 30 days after treatment. Out of the 53 participants, a total of 45\n(81.1%) individuals had egg negative results, and 8 (18.9%) were still egg\npositive according to the RS. Using 16 slides, KK tests of different samples\nrevealed no infected individuals. The HTX method detected a total of 8 (18.9%)\negg-positive individuals after 30 days of treatment. A total of 5 (9.4%)\negg-positive and 37 (69.8%) egg-negative individuals were detected when the\nPOC-CCA test was used. Furthermore, 11 (20.7%) trace results were detected.\n<xref rid=\"t4\" ref-type=\"table\">Table 4</xref> shows the relation between\nPOC-CCA and the RS results after treatment.</p><p>\n<table-wrap id=\"t4\" orientation=\"portrait\" position=\"float\"><label>TABLE 4:</label><caption><title>Concordance between POC-CCA results in relation to the reference\nstandard, 16 slides from different samples and HTX results 30 days\nafter treatment.</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"3\"/><col span=\"3\"/><col span=\"3\"/></colgroup><thead><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">POC-CCA</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">RS </th><th align=\"center\" colspan=\"3\" rowspan=\"1\">KK (16S 3SA) </th><th align=\"center\" colspan=\"3\" rowspan=\"1\">HTX </th></tr><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th></tr></thead><tbody><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (11)*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16 (11)*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (11)*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Negative</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN4\"><p>\n<bold>RS:</bold> Reference standard; <bold>16S 3SA:</bold>\nSixteen slides, twelve slides from the first sample, two slides\nfrom the second sample and two slides from the third sample;\n<bold>HTX:</bold> Helmintex<sup>®</sup> method.\n<bold>*</bold>Trace results classified as positive.</p></fn></table-wrap-foot></table-wrap>\n</p></sec></sec><sec sec-type=\"discussion\"><title>DISCUSSION</title><p>Over the last 30 years, efforts made by the Brazilian Schistosomiasis Control Program\nhave contributed to decreasing the positivity rate and individual parasite loads in\nendemic areas, consequently hampering <italic>S. mansoni</italic> egg detection by\nthe KK method<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>. The use of two slides under these circumstances does not provide\nsatisfactory diagnostic performance to proceed toward elimination of schistosomiasis\nas a public health problem<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>.</p><p>A study conducted by Lindholz <italic>et al</italic>. (2018)<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref> used three different diagnostic methods (the KK technique, HTX method, and\nPOC-CCA test) and compared the tests performances among individuals with low\nparasite loads, where the HTX<sup>®</sup> yielded more sensitive results than two KK\nslides.</p><p>Another Brazilian study used a RS combining 18 KK slides of three different stool\nsamples, the saline gradient technique, and the HTX method in an endemic area in the\nnorthern part of Minas Gerais State<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>. The HTX method yielded better results than any combination of KK slides and\nmuch better results than the saline gradient technique.</p><p>Our study also demonstrated that the HTX method detected 22 positive results on more\nthan 16 KK slides. On the other hand, the KK method confirmed a total of 9 positive\nindividuals who had not been identified by the HTX method. Therefore, the\ncombination of both methods was chosen as the RS because it maximizes the detection\nof egg positive samples.</p><p>Currently, the POC-CCA test demonstrated better results than two KK slides<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>. In a study conducted by Sousa <italic>et al</italic>. (2019)<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref> in a low prevalence area with low individual parasite loads, it was noticed\nthat the POC-CCA test had similar rates of detection of infected individuals when\ncompared with 9 slides from a single stool sample or 6 slides from 3 different\nsamples. However, the increase in sensitivity had shown an improvement of\nperformance of the POC-CCA test with an increase in the amount of feces analyzed.\nThus, it was concluded that it was necessary to improve the RS to better evaluate\nthe CCA test.</p><p>The present study indicated an increase in the sensitivity of the POC-CCA test when\nthe coproscopic techniques were combined. Therefore, it is notable that a total of\n39 true positives were detected by the POC-CCA test when compared with our RS. This\nvalue yielded a better detection rate than 16 KK slides from three different stool\nsamples, which identified 31 positives (<xref ref-type=\"fig\" rid=\"f1\">Figure\n1</xref>). This is a higher rate than that reported by Sousa <italic>et\nal</italic>. (2019)<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>.</p><p>Studies carried out in moderate and high prevalence areas showed that the POC-CCA\ntest is more sensitive than the KK method and can be used for screening and\ngeographical mapping of <italic>S</italic>. <italic>mansoni</italic> infections. A\nsensitivity rate of up to 99.5% was reached when a latent class analysis model was\nused<xref rid=\"B25\" ref-type=\"bibr\">\n<sup>25</sup>\n</xref>. However, the sensitivity of the POC-CCA test was compromised when it was\napplied in a low prevalence area with low parasite load. Oliveira <italic>et\nal.</italic> (2018)<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref> and Lindholz <italic>et al.</italic> (2018)<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref> described a decreased sensitivity of the POC-CCA test when the parasite load\nwas below 100 EPG. </p><p>Our study showed that the highest concordance was reached when compared with our\nestablished RS. In this context, if only the immunochromatographic test would be\nused for the diagnosis of an active <italic>S. mansoni</italic> infection, 40\npositive individuals out of a total of 79 would show false positive results in\ncomparison to the RS, and 24 egg positive individuals would not be detected by\nPOC-CCA. This means that approximately 50% of them would be treated unnecessarily.\nIn contrast, by using only two KK slides and comparing them to the RS, a total of 46\npositive results would be missed.</p><p>A total of 16 (25.4%) egg-positive individuals were not detected when the “trace”\nresult was classified as a negative result. This result revealed that the POC-CCA\ntest detects more infected individuals when “trace” results are classified as\npositive results, as also described by Prada <italic>et al</italic>. 2018<xref rid=\"B26\" ref-type=\"bibr\">\n<sup>26</sup>\n</xref>. In contrast, when “trace” results were considered negative, the POC-CCA\ntest showed better performance in the detection of true negative results. At this\npoint, the interpretation of “trace” results may be ambiguous, as described by\nClements <italic>et al</italic>. (2017)<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref>.</p><p>In our study, the area of prevalence may reach a different classification, depending\non the applied diagnostic method, and would result in a completely different\nrecommended strategy for the treatment of the population<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>. Our data reinforce the need to associate different diagnostic tools to\nimprove the detection of <italic>S. mansoni</italic> in individuals with low\nparasitic load, as recommended by Bezerra <italic>et al</italic>. (2018)<xref rid=\"B28\" ref-type=\"bibr\">\n<sup>28</sup>\n</xref>.</p><p>Thirty days after treatment, all the KK results were negative, and only HTX indicated\negg positivity. The scenarios observed in this study and other publications\nevaluating the presence of <italic>Schistosoma</italic> infection after treatment\ndemonstrated that infections with low parasite loads were more frequently detected\nwhen a larger amount of feces was analyzed<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B29\" ref-type=\"bibr\">\n<sup>29</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>.</p><p>The performance of the POC-CCA test 30 days after treatment shows poor detection of\npositive individuals. Out of 14 false positive results, 11 were “trace” results and\nclassified as positive. The high frequency of false positive results could be\nexplained by a small number of surviving worm couples, which stopped releasing eggs\nbecause of the damaging effects of praziquantel or by surviving juvenile forms of\nthe parasite that are not susceptible to praziquantel treatment. However, more\nstudies need to be conducted to elucidate the mechanism underlying this observation.\nIn relation to the loss of 6 (75.0%) egg-positive individuals, as detected by RS\nafter treatment, would be the possibility of prolonged release of eggs, even after\nkilling adult worms, which would result in a decrease in circulating antigens and in\nhuman blood within 2 to 3 weeks after treatment<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>. However, the present study did not seek to elucidate this mechanism.</p></sec><sec sec-type=\"conclusions\"><title>CONCLUSION</title><p>In summary, our data indicated that the POC-CCA test has potential as an auxiliary\ntool for the diagnosis of <italic>S. mansoni</italic> infections, revealing better\nresults than 16 KK slides from three different stool samples. However, the\nimmunochromatographic test was found not to be a specific and sufficiently sensitive\ntool to verify the cure rate after praziquantel treatment. This is in discordance\nwith the findings of Prada <italic>et al</italic>. (2018)<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref>, which suggested the CCA test as a better predictor of prevalence after\ntreatment. In relation to the performance of the KK technique, this study revealed\nthat this method lacks accuracy due to the decrease in the individual parasite load\nin a previously treated population. In contrast, the HTX method revealed solid\nresults as a potential alternative and additional tool for the evaluation of cure\nafter treatment of <italic>S. mansoni</italic> infections.</p></sec></body><back><ack><title>ACKNOWLEDGMENTS</title><p>The authors would like to thank the team of the Instituto Evandro Chagas/SVS/MS for\ntheir support and cooperation in this study.</p></ack><fn-group><fn fn-type=\"financial-disclosure\" id=\"fn2\"><p>\n<bold>Financial support:</bold> This work was supported by the Fundação Amazônia\nde Amparo a Estudos e Pesquisa (Fapespa), Conselho Nacional de Desenvolvimento\nCientífico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de\nNível Superior (CAPES), and Ministério da Ciência, Tecnologia e Inovação.\nMCTI/CNPq/MS-SCTIE-Decit n° 40/2012.</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id=\"B1\"><label>1</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>World Health Organization (WHO)</collab></person-group><source>Schistosomiasis: progress report 2001-2011 and strategic plan\n2012-2020</source><publisher-loc>Geneva</publisher-loc><publisher-name>WHO</publisher-name><year>2013</year><size units=\"pages\">74 p</size></element-citation></ref><ref id=\"B2\"><label>2</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>World Health Organization (WHO)</collab></person-group><source>Schistosomiasis and soil-transmitted helminths: number of people treated\nin 2015</source><publisher-loc>Geneva</publisher-loc><publisher-name>WHO</publisher-name><year>2016</year><size units=\"pages\">16 p</size></element-citation></ref><ref id=\"B3\"><label>3</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>World Health Organization (WHO)</collab></person-group><source>Schistosomiasis: Key facts</source><publisher-loc>Geneva</publisher-loc><publisher-name>WHO</publisher-name><year>2019</year><size units=\"pages\">1p</size></element-citation></ref><ref id=\"B4\"><label>4</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Noya</surname><given-names>O</given-names></name><name><surname>Katz</surname><given-names>N</given-names></name><name><surname>Pointier</surname><given-names>JP</given-names></name><name><surname>Theron</surname><given-names>A</given-names></name><name><surname>Noya</surname><given-names>BA</given-names></name></person-group><article-title>Schistosomiasis in America</article-title><source>PLoS Negl Trop Dis</source><year>2015</year><volume>2</volume><fpage>16</fpage><lpage>17</lpage></element-citation></ref><ref id=\"B5\"><label>5</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Katz</surname><given-names>N</given-names></name></person-group><source>Inquérito nacional de prevalência da esquistossomose mansoni e\ngeo-helmintoses (2010-2015)</source><publisher-loc>Belo Horizonte</publisher-loc><publisher-name>Inst René Rachou (Fiocruz)</publisher-name><year>2018</year><size units=\"pages\">76 p</size></element-citation></ref><ref id=\"B6\"><label>6</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>World Health Organization (WHO)</collab></person-group><source>Sixty-fifth world health assembly</source><publisher-loc>Geneva</publisher-loc><publisher-name>WHO</publisher-name><year>2012</year><size units=\"pages\">6p</size></element-citation></ref><ref id=\"B7\"><label>7</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>Brasil</collab></person-group><source>Programa de Vigilância e Controle da Esquistossomose</source><publisher-name>BR</publisher-name><publisher-loc>Brasília, DF</publisher-loc><year>2017</year></element-citation></ref><ref id=\"B8\"><label>8</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Vlas</surname><given-names>SJ</given-names></name><name><surname>Gryseels</surname><given-names>B</given-names></name></person-group><article-title>Underestimation of Schistosoma mansoni\nprevalences</article-title><source>Parasitol Today</source><year>1992</year><volume>8</volume><issue>4</issue><fpage>274</fpage><lpage>277</lpage><pub-id pub-id-type=\"pmid\">15463638</pub-id></element-citation></ref><ref id=\"B9\"><label>9</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>Ministério da Saúde (MS). 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Rev Soc Bras Med Trop</journal-id><journal-id journal-id-type=\"iso-abbrev\">Rev. Soc. Bras. Med. Trop</journal-id><journal-id journal-id-type=\"publisher-id\">rsbmt</journal-id><journal-title-group><journal-title>Revista da Sociedade Brasileira de Medicina Tropical</journal-title></journal-title-group><issn pub-type=\"ppub\">0037-8682</issn><issn pub-type=\"epub\">1678-9849</issn><publisher><publisher-name>Sociedade Brasileira de Medicina Tropical - SBMT</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32997051</article-id><article-id pub-id-type=\"pmc\">PMC7523524</article-id><article-id pub-id-type=\"doi\">10.1590/0037-8682-0214-2020</article-id><article-id pub-id-type=\"other\">00352</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Major Article</subject></subj-group></article-categories><title-group><article-title>Snakebites in Rio Branco and surrounding region, Acre, Western Brazilian Amazon</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>de Oliveira</surname><given-names>Laiane Parente</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Moreira</surname><given-names>José Genivaldo do Vale</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sachett</surname><given-names>Jacqueline de Almeida Gonçalves</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Monteiro</surname><given-names>Wuelton Marcelo</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff5\">\n<sup>5</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Meneguetti</surname><given-names>Dionatas Ulises de Oliveira</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff6\">\n<sup>6</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-2191-7817</contrib-id><name><surname>Bernarde</surname><given-names>Paulo Sérgio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff7\">\n<sup>7</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<label>1</label>Universidade Federal do Acre, Programa de Pós-Graduação Stricto Sensu em Ciências da Saúde na Amazônia Ocidental, Rio Branco, AC, Brasil.</aff><aff id=\"aff2\">\n<label>2</label>Universidade Federal do Acre, Centro Multidisciplinar, Cruzeiro do Sul, AC, Brasil.</aff><aff id=\"aff3\">\n<label>3</label>Universidade do Estado do Amazonas, Manaus, AM, Brasil.</aff><aff id=\"aff4\">\n<label>4</label>Fundação Alfredo da Matta, Diretoria de Ensino e Pesquisa, Manaus, AM, Brasil.</aff><aff id=\"aff5\">\n<label>5</label>Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, AM, Brasil.</aff><aff id=\"aff6\">\n<label>6</label>Universidade Federal do Acre, Colégio de Aplicação, Rio Branco, AC, Brasil.</aff><aff id=\"aff7\">\n<label>7</label>Universidade Federal do Acre, Campus Floresta, Centro Multidisciplinar, Laboratório de Herpetologia, Cruzeiro do Sul, AC, Brasil.</aff><author-notes><corresp id=\"c1\"><bold>Corresponding author:</bold> Paulo Sérgio Bernarde. <bold>e-mail:</bold><email>SnakeBernarde@hotmail.com</email></corresp><fn fn-type=\"con\" id=\"fn1\"><p>\n<bold>Authors’ contributions:</bold>\n<bold>LPO:</bold> conception and design of the study, acquisition of data, analysis and interpretation of data, drafting the article, final approval of the version to be submitted; <bold>JGVM:</bold> conception and design of the study, analysis and interpretation of data, final approval of the version to be submitted; <bold>JAGS:</bold> conception and design of the study, analysis and interpretation of data, final approval of the version to be submitted; <bold>WMM:</bold> conception and design of the study, analysis and interpretation of data, final approval of the version to be submitted; <bold>DUOM:</bold> conception and design of the study, analysis and interpretation of data, final approval of the version to be submitted; <bold>PSB:</bold> conception and design of the study, analysis and interpretation of data, drafting the article, final approval of the version to be submitted. </p></fn><fn fn-type=\"COI-statement\" id=\"fn2\"><p>\n<bold>Conflict of interest:</bold> The authors declare that there is no conflict of interest.</p></fn></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><year>2020</year></pub-date><volume>53</volume><elocation-id>e20200214</elocation-id><history><date date-type=\"received\"><day>01</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>17</day><month>7</month><year>2020</year></date></history><permissions><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License</license-p></license></permissions><abstract><title>Abstract</title><sec><title>INTRODUCTION</title><p> Snakebites are considered a neglected tropical disease in many countries in Latin America, including Brazil. As few studies have assessed snakebites in the Amazon region and especially in the state of Acre, epidemiological studies are of great importance. The present study aimed to describe the epidemiological characteristics of snakebites in the Rio Branco region, observing their characteristics in rural and urban areas and their correlation with rainfall and river outflow. </p></sec><sec><title>METHODS</title><p> This retrospective, descriptive study analyzed epidemiological information obtained from snakebite notifications registered on the Information System for Notifiable Diseases that occurred from March, 2018 to February, 2019. The cases of snakebite were correlated with rainfall and flow. </p></sec><sec><title>RESULTS</title><p> A total of 165 cases of snakebite were registered in the period. Most cases were caused by <italic>Bothrops</italic> and affected mainly individuals of the male sex who were between 21 and 30 years old. Most of the snakebites occurred in Rio Branco (71.52%; 29 cases per 100,000 inhabitants). Of these, 60.2% occurred in the urban area and 39.8% in the rural area and the majority occurred during the rainy season.</p></sec><sec><title>CONCLUSIONS</title><p> Although studies have shown that a majority of cases occur in rural areas, in this study, urbanization of snakebites was observed. The <italic>Bothrops</italic> genus was responsible for the highest number of snakebites and, during the rainy season, bites occurred more frequently. Educational prevention campaigns, population advice, and first aid in case of snakebites for the population are thus suggested.</p></sec></abstract><kwd-group><title>Keywords:</title><kwd>Ophidism</kwd><kwd>Snake bite</kwd><kwd>Epidemiology</kwd><kwd>Amazon</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"39\"/></counts></article-meta></front><body><sec sec-type=\"intro\"><title>INTRODUCTION</title><p>Snakebites are considered a neglected tropical disease in many countries in Africa, Asia, and Latin America<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>. Estimates show that more than 5 million snakebites occur every year in the world, with approximately 2 million envenomations which result in 94,000 deaths<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>. This information on the estimation of cases per snakebite may be underestimated when the existence of non-notified cases is considered, and thus may represent data exceeding those mentioned above<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>.</p><p>In Brazil, there are 405 known species of snakes<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>. Of these, sixty-six venomous snakes belong to the families Elapidae (genera: <italic>Micrurus</italic>, and <italic>Leptomicrurus</italic>), and Viperidae (genera: <italic>Bothrocophias</italic>, <italic>Bothrops</italic>, <italic>Crotalus</italic>, and <italic>Lachesis</italic>)<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>. Most of the snakebites recorded in the Amazon are caused by <italic>Bothrops</italic> (88.7%), with <italic>B. atrox</italic> being responsible for most cases of envenomation<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>.</p><p>In the state of Acre, the region known as Alto Juruá is characterized by a high incidence of snakebites<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>. Pierini et al.<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref> found a prevalence of 13% for cases of snakebites in traditional populations (extractivists, riverines, and indigenous). In this region, the prevalence of snakebites is associated mainly with the activities of agriculture and extractivism, which are practiced by people living in rural areas and forests and who are thus more vulnerable<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>.</p><p>In a retrospective study conducted at the Regional Hospital of Juruá in Cruzeiro do Sul in the state of Acre, Bernarde and Gomes<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref> recorded the occurrence of 195 cases of snakebites attended to at the hospital in the two-year period (August, 2007 to July, 2009). In the municipality of Tarauacá, also in the state of Acre, a retrospective study<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref> showed a high coefficient of morbidity due to snakebites in 2016 (72.5 cases per 100,000 inhabitants), which is higher than the coefficients recorded in municipalities such as Cruzeiro do Sul and Rio Branco. Although it is considered a health problem, there is still a knowledge gap regarding snakebites and their relationship with environmental conditions in the region of Rio Branco. The last study carried out in the region was conducted in 2002 and only contemplated epidemiological and clinical characteristics<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>. In this respect, the present study aimed to describe not only the epidemiological and clinical characteristics of snakebites in the region of Rio Branco, Acre, Western Amazonia, but also the characteristics of the accidents that occurred in rural and urban areas and their correlation with rainfall and low seasonal river levels.</p></sec><sec sec-type=\"methods\"><title>METHODS</title><p>The study was carried out using a retrospective approach and focused on the epidemiology of snakebites which occurred in the period from March, 2018 to February, 2019 and on the correlation of these cases with the respective rainfall and low seasonal river levels, the period from 2009 to 2018 was used.</p><p>The epidemiological data obtained were collected from the SINAN (Health Information Notification System) registry in the epidemiological surveillance sector for the municipality of Rio Branco, Acre, and included the cases dealt with in the accident and Emergency Hospital of Rio Branco. Accident and Emergency Hospital Rio Branco, which is the reference Center for the treatment of cases of snakebites occurring in the region of Acre Vale. This hospital treats victims of snakebites mainly from Rio Branco (<xref ref-type=\"fig\" rid=\"f1\">Figure 1</xref>), but may also receive patients from other nearby municipalities (Assis Brasil, Bujari, Capixaba, Plácido De Castro, Porto Acre, Senador Guiomard, Sena Madureira and Xapuri) or further away (e.g., Boca do Acre, Jordão, Tarauacá and even from Porto Velho, state of Rondônia). The municipality of Rio Branco, Acre, has an estimated population of 407,319<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref>, its territory is 8,834,942 km<sup>2</sup> and it is located in northwestern Brazil. According to the city plan, it can be observed that over the years, there has been an urban growth which has invaded the surrounding rural areas<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref>. However, despite this urbanization, some road systems located inside the urban area of Rio Branco still have characteristics of the old rural area (<xref ref-type=\"fig\" rid=\"f1\">Figure 1</xref>).</p><p>\n<fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1:</label><caption><title>Map of Brazil and State of Acre and location of the municipality of Rio Branco (above) and the delimitation of its urban area (below).</title></caption><graphic xlink:href=\"1678-9849-rsbmt-53-e20200214-gf1\"/></fig>\n</p><p>From the Health Information Registry, the following variables were obtained: year and month of occurrence, the identification of the snake type (type of snakebite), location of accident (urban or rural) in the municipality of occurrence, and data on socio-demographics such as age and gender, in addition to clinical data such as the anatomical region affected, the time that had elapsed between the accident and care being administered, the number of vials used, local and systemic manifestations, the type of serum used in the treatment of victims and the severity of the envenomation. The region’s rainfall data and seasonal river levels were obtained from the sites of the National Water Agency - ANA<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref> and the National Institute of Meteorology - INMET<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>. </p><p>The ratio of morbidity rates (per 100,000 inhabitants) was calculated by dividing the number of people who had suffered snakebites by the number of people living in the region during the period, multiplied by 100,000. Only the cases that occurred in the municipality of Rio Branco were considered, since some of the cases may have been treated in the municipalities of origin and not in the Accident and Emergency Hospital in Rio Branco. With respect to the correlation of rainfall, seasonal river levels, and snakebites, during the period from 2009 to 2018, the null hypothesis (H<sub>0</sub>), that there is no statistically significant correlation (p < 0.05) between the variables in focus, was tested using the Spearman non-parametric test, since a previous check linked the acceptance of the assumptions with the analysis of the bias of the parametric statistics<xref rid=\"B16\" ref-type=\"bibr\">\n<sup>16</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>.</p><p> This research is part of the project named \"Snakebites occurring in Rio Branco and region (Acre)\" approved by the Committee on Ethics and Research with Human Subjects at Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (approval no. 3.223.051).</p></sec><sec sec-type=\"results\"><title>RESULTS</title><p>In the period from March, 2018 to February, 2019, 165 cases of snakebites were recorded, the majority (76.36%) were classified as being caused by <italic>Bothrops</italic>, followed by non-venomous snakes (9.70%), <italic>Micrurus</italic> (1.82%), and <italic>Lachesis</italic> (1.21%). Many of the snakebites occurred in the municipality of Rio Branco (71.52%; 29 cases per 100,000 inhabitants). Snakebites were almost as common in the urban area (46.06%) as the rural area (53.94%) and a greater proportion occurred during the rainy season (64.24%) (<xref rid=\"t1\" ref-type=\"table\">Table 1</xref>).</p><p>\n<table-wrap id=\"t1\" orientation=\"portrait\" position=\"float\"><label>TABLE 1:</label><caption><title>Clinical and epidemiological characteristics of the cases of snakebites occurring in Rio Branco and the surrounding region (Acre - Brazil) during the period from March, 2018 to February, 2019.</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics (n = 165)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">%</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Snake genus</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Bothrops</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">126</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">76.36%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Lachesis</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.21%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Micrurus</italic>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.82%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Non-venomous / dry bite</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9.70%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Ignored</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.91%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Season</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Rainy (November to April)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">106</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">64.24%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Dry (May to October)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">59</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35.76%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Sex</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Male</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">107</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">64.85%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Female</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">58</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35.15%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Municipality of occurrence</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Rio Branco</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">118</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">71.52%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Bujari</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21.28%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Porto Acre</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14.89%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Senador Guiomard</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.64%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Sena Madureira</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10.64%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Capixaba</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.51%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Assis Brasil</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.26%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Jordão </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.26%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Tarauacá</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.26%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Xapuri</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2,13%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Plácido de Castro</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.13%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Boca do Acre</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.51%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Porto Velho</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.51%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Area of occurrence</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Rural</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">89</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53.94%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Urban</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">76</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">46.06%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age group, years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 0 to 10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.15%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 11 to 20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 21 to 30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.42%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 31 to 40</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.97%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 41 to 50</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12.73%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 51 to 60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.67%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> > 60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.06%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Occupation</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Farmer</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21.21%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Student</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">32.12%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Housewife</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.15%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Other</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31.52%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Time to hospital care </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 0 to 1 hour</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22.42%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 1 to 3 hours</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">48</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">29.09%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 3 to 6 hours</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.76%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 6 to 12 hours </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.03%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 12 to 24 hours</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.27%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> > 24 hours</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18.18%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Ignored</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.24%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Anatomical region of the bite</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Foot</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">91</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">55.15%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Leg</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">46</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27.88%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Hand</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.48%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Arm</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.03%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Head</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.42%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Thorax</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Ignored</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.42%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Classification of envenomation</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Light</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">120</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">72.73%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Moderate</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">36</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21.82%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Severe</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5.45%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Local manifestations and complications</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Pain</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">152</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">92.12%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Edema</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">120</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">72.73%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Bruising</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.82%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Cellulitis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Necrosis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Systemic manifestations and complications</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Change in clotting time (performed clotting exams, n = 38)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23.68%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Headache</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.03%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Bleeding</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.42%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Nausea</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.21%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Paresthesia </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.21%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Odynophagia</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Angioedema</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Hemorrhagic cerebrovascular stroke</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Dyspnea</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Eyelid ptosis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Decreased level of consciousness</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Somnolence</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Dizziness</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Blurred vision</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61%</td></tr></tbody></table></table-wrap>\n</p><p>Regarding the distribution of accidents, it was observed that the cases of snakebites occurred more often among males (64.85%) aged 21 to 30 years (22.42%). The main occupations of the victims were students (32.12% of the cases) and farmers (21.21%). </p><p>The largest proportion of the victims (29.09%) was treated in the hospital within the first three hours after the accident. However, 18.18% received late care, and were treated after 24 hours and most of the cases were classified as light (77.73%).</p><p>The lower limbs, feet (55.15%) and legs (27.88%) were the most affected. Local manifestations and complications consisted mainly of pain (92.12%) and edema (72.73%). Other manifestations also occurred, such as headache (3.03%), paresthesia (1.21%) and nausea (1.21%). Systemic manifestations and complications, such as changes in blood clotting time (23.68%), bleeding time (2.42%), dyspnoea (0.61%), palpebral ptosis (0.61%), haemorrhagic cerebrovascular stroke (0.61%) and decreased level of consciousness (0.61%) were also observed. During the study period no deaths were recorded.</p><p>Compared to the region as a whole, 71.52% of the cases of snakebites occurred in the municipality of Rio Branco. Of these cases, 60.17% occurred in the urban area and 39.83% in the rural area (<xref rid=\"t2\" ref-type=\"table\">Table 2</xref>). Snakebites caused by <italic>Bothrops</italic> were frequent both in the urban area (74.65%) and in the rural area (78.72%); there was no record of the occurrence of a snakebite caused by <italic>Lachesis</italic> in the urban area; and in the rural area there were no recorded bites caused by <italic>Micrurus</italic>.</p><p>\n<table-wrap id=\"t2\" orientation=\"portrait\" position=\"float\"><label>TABLE 2:</label><caption><title>Comparison of clinical and epidemiological manifestations of snakebites occurring in urban and rural areas in the municipality of Rio Branco (Acre, Brazil) during the period from March, 2018 to February, 2019.</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics (n = 118)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Urban</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Rural</th></tr><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">71 (60.17%)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">47 (39.83%)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Snake genus</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Bothrops</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">53 (74.65%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37 (78.72%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Lachesis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (4.26%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Micrurus</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1.41%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Non-venomous / dry bite</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8 (11.27%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (8.51%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Ignored</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 (12.68%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (8.51%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Season</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Rainy (November to April)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45 (63.38%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">36 (76.6%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Dry (May to October)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26 (36.62%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">11 (23.4%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Sex</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">41 (57.75%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33 (70.2%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Female</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30 (42.25%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (29.8%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age group, years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">0 to 10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (23.94%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (8.5%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">11 to 20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (18.31%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15 (31.91%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">21 to 30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (14.08%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 (19.15%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">31 to 40</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (19.72%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8 (17.02%0</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">41 to 50</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (14.08%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (12.77%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">51 to 60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 (7.04%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (2.13%%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">> 60</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (2.82%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (8.51%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Occupation</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Farmer</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (2.82%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (29.79%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Student</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27 (38.03%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18 (38.30%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Housewife</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Other</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">42 (59.15%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15 (31.91%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Time to hospital care </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">0 to 1 hour</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24 (33.80%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12 (25.53%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">1 to 3 hours</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">23 (32.39%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12 (25.53%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">3 to 6 hours</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 (12.68%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6 (12.77%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">6 to 12 hours </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (2.82%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (6.38%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">12 to 24 hours</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (9.86%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (2.13%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">> 24 hours</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (5.63%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9 (19.15%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Ignored</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (2.82%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (8.51%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Anatomical region of the bite</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Foot</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">41 (57.75%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25 (53.19%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Leg</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22 (30.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (27.66%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Hand</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (4.23%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 (10.64%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Arm</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (2.82%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (2.13%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Head</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (2.13%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Thorax</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1.41%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Ignored</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (2.82%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (4.26%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Classification of envenomation</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Light</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">63 (88.73%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33 (70.21%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Moderate</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (9.86%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12 (25.53%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Severe</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1.41%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (4.26%)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Local manifestations and complications</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Pain</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">65 (91.54%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43 (91.49%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Edema</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">49 (69.01%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34 (72.34%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Bruising</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (2.13%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Necrosis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1.41%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Compartment syndrome</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Manifestations and systemic complications </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Headache</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1.41%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (2.13%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Paresthesia</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (2.82%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Angioedema</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1.41%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Nausea</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1.41%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Dizziness</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (2.13%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Change in clotting time</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (9.09%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (22.22%)</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Bleeding</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (2.13%)</td></tr></tbody></table></table-wrap>\n</p><p>In relation to the historical series of snakebites (2009 to 2018), it has been found that the highest number of snakebites tends to concentrate in the months when the highest average precipitation and lowest river level occur. In the Spearman’s correlation analysis, there was a positive correlation between the variable snakebites with seasonal river level (<italic>r</italic> = 0.61, n = 120, p < 0.01) and with precipitation (<italic>r</italic> = 0.30, n = 120, p < 0.01). There was also a positive correlation between seasonal river level and precipitation (<italic>r</italic> = 0.49, n = 120, p < 0.01).</p><p>Snakebites occurred mainly in males, and a higher proportion of cases in females occurred in the urban area (42.5%) compared to rural areas (29.8%) (Pearson’s chi-squared = 5.8182, p = 0.0159). The 0-10 year age group (23.94%) in the urban area was the most affected; and in the rural area the 11-20 year age group was the most prevalent age group (31.91%). In the urban area, the majority of victims of snakebites had “other” listed as their occupation (59.15%), while in rural areas most of the victims were students (38.30%) and farmers (29.79%) (<xref rid=\"t2\" ref-type=\"table\">Table 2</xref>). </p><p>In the urban area, the largest proportion of the victims (66.19%) were treated in the hospital in the period up to 3 hours after the accident; however, in the rural area, the proportion of care given in the same period was 51.06%. The highest percentage of mild cases was registered in the urban area (88.73%) and moderate and severe in rural areas (25.53% and 4.26%, respectively) (Pearson chi-squared = 11.375, p = 0.0034).</p></sec><sec sec-type=\"discussion\"><title>DISCUSSION</title><p>During a period of 12 months, 165 cases were recorded in the Rio Branco region, more than that reported for the municipality of Tarauacá (29 cases) by Saboia and Bernarde<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref> and for the Cruzeiro do Sul region (133 cases) by Mota-da-Silva et al.<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>. However, a lower morbidity ratio was observed in this study for the city of Rio Branco (29 cases/100,000 inhabitants) when compared to Tarauacá (72.5 cases/100,000 inhabitants) and Cruzeiro do Sul (76.2/100,000 inhabitants). This is probably due to a larger population who live in rural areas and forests and agricultural and extractive activities in these last two regions<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>, in relation to the capital. In relation to the previous study conducted in Rio Branco by Moreno et al.<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>, which recorded 89 cases, a reduction in the number of cases per inhabitants was observed, from 35.1/100,000 inhabitants, to 29/100,000 inhabitants. This reduction may be associated with the urbanization process of accidents, which are now more frequent in this area.</p><p>Most of the registered accidents were attributed to <italic>Bothrops</italic> (76.36%), which corroborates information in the literature which states that this type of envenomation is the most frequent in the Amazon<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>. As expected, envenomation by <italic>Micrurus</italic> and <italic>Lachesis</italic> were uncommon (less than 2% each), and corroborates findings by other authors for the same region<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>. There was no record of an envenomation involving <italic>Crotalus</italic> in this study. This was expected, since the rattlesnake (<italic>Crotalus durissus</italic>), is not reported in Acre<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>, since it inhabits areas of cerrado, a type of vegetation that is absent in the state of Acre<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>.</p><p>The snakebites analyzed in the present study had a greater occurrence during the rainy season (64,24%) and the historical series (2009-2018) was statistically associated with the low seasonal river levels and rainfall, thus confirming the observations of other studies<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B24\" ref-type=\"bibr\">\n<sup>24</sup>\n</xref>. This predominance of snakebites in the wettest months is probably related to the increase in snake activity in this period, due to the greater availability of prey and reproductive activities<xref rid=\"B25\" ref-type=\"bibr\">\n<sup>25</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B26\" ref-type=\"bibr\">\n<sup>26</sup>\n</xref>. In addition, a greater chance of encounters between snakes and humans can be due to certain agricultural and extractive activities developed in this period, and the fact that flooding causes these animals to search for dry land areas<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref>. </p><p>The fact that the higher frequency of victims were male and bitten on the lower limbs corresponded to the epidemiological profile observed for the Brazilian Amazon<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>, as well as for the other regions of Brazil<xref rid=\"B28\" ref-type=\"bibr\">\n<sup>28</sup>\n</xref>.</p><p>In regard to the locations of the accidents, it is noted that, in general, the rural areas showed slightly more than half of the cases (53.94%), and when analyzing only the municipality of Rio Branco, there was a greater predominance in the urban area (60.17%), contrary to what was observed in other studies, in which there is a greater predominance of snakebites in rural areas (more than 85% of the cases)<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>. This urbanization of snakebites was also observed for Belém do Pará in the eastern Amazon<xref rid=\"B29\" ref-type=\"bibr\">\n<sup>29</sup>\n</xref>, where 66% of cases were recorded in the urban area. This situation may have occurred due to the demographic and spatial growth of the city of Rio Branco, the decentralization of the urban area<xref rid=\"B30\" ref-type=\"bibr\">\n<sup>30</sup>\n</xref>, disordered expansion, and exodus from rural to urban areas<xref rid=\"B31\" ref-type=\"bibr\">\n<sup>31</sup>\n</xref>, where more than 90% of the population is concentrated<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref>. There is also the existence of families living in high risk areas on the banks of the Acre River and associated streams, which are vulnerable to flooding. There is also occupation in the form of invasions of permanent preservation areas close to Environmental Protection Areas<xref rid=\"B31\" ref-type=\"bibr\">\n<sup>31</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B33\" ref-type=\"bibr\">\n<sup>33</sup>\n</xref>, which may also contribute to these accidents. Another factor that can contribute to snakebites in urban areas are the green areas (forest fragments) and floodplains present in cities, which favor the occurrence and encounter of some species of snakes<xref rid=\"B29\" ref-type=\"bibr\">\n<sup>29</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B34\" ref-type=\"bibr\">\n<sup>34</sup>\n</xref>. The city of Rio Branco has some patches of forest and rivers<xref rid=\"B35\" ref-type=\"bibr\">\n<sup>35</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B36\" ref-type=\"bibr\">\n<sup>36</sup>\n</xref>, which may explain some of the snakebites in the urban area.</p><p>Analyzing the time elapsed from the moment of the snakebite to the time of hospital care, most of the victims received care in the first six hours (67.27%), which indicates care occurring earlier when compared to the previous study by Moreno et al.<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>. Their study indicates that only 58.3% of the cases were treated in this period. This decrease in the “time to treatment” was also described in a study carried out by Mota-da-Silva et al.<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref> in the Alto Juruá region (60% treated within six hours), who attributed the cause to the increase in the number of ambulances and improvements in roads and telephone services as possible factors for this improvement in time.</p><p>A marked and expected difference was the greater speed of care of cases occurring in the urban area, which were closer to the hospital units than those in the rural area. In the urban área, 78.87% of the cases received care in the first six hours and in the rural area 63.83%. For the 24-hour period after the accident, the proportion was lower in the urban area (5.63%) and higher in the rural area (19.15%). This demonstrates that in the Amazon, some cases of snakebites occur in locations so far from urban centers, that they result in delays in hospital care or even lack of access to antivenom<xref rid=\"B7\" ref-type=\"bibr\">\n<sup>7</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref>. These are often due to displacement difficulties and, as such, may contribute to the severity of cases<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B37\" ref-type=\"bibr\">\n<sup>37</sup>\n</xref>. In the urban area, there was a predominance of moderate and severe cases in the rural area, which is probably associated with greater speed of care.</p><p>Regarding the severity of snakebites, the frequency of mild (72.73%), moderate (21.82%) and severe (5.45%) corroborates with the study by Mota-da-Silva et al.<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref> conducted in the Alto Juruá region (50%, 36.5%, and 13.5%, respectively) which shows that the highest occurrence of accidents was mild, followed by moderate and severe. However, these findings differ from what was observed in the previous study conducted in Rio Branco in 2002, in which moderate cases (48.6%) accounted for the majority of accidents, followed by mild (31.2%) and severe (20.2%) cases<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>. This may be due to an improvement in the structure of the health care network in the state of Acre with serum availability and training of professionals for more appropriate clinical management of snakebites. </p><p>The most frequent symptoms presented in this study were pain (92.12%) and edema (72.73%), which are the most commonly observed in cases of <italic>Bothrops</italic> envenomations<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B38\" ref-type=\"bibr\">\n<sup>38</sup>\n</xref>. Other symptoms and even complications observed less frequently in this study, such as cellulitis, ecchymosis, hemorrahage and necrosis, are also observed in <italic>Bothrops</italic> envenomations<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B38\" ref-type=\"bibr\">\n<sup>38</sup>\n</xref>. The blood clotting time observed (23.68%) was lower than that recorded in the previous study by Moreno et al.<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref> (43.1%) for Rio Branco and Tarauacá (55.5%)<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref> and the Alto Juruá region (82.5%)<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>. This low frequency may have been due to the low sample number of tests performed, only 38, compared to that of the other three studies (90 to 129 tests performed)<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B11\" ref-type=\"bibr\">\n<sup>11</sup>\n</xref>.</p><p>Some differences were observed between the snakebites that occurred in the urban and rural areas of Rio Branco. Among them, the two laquetic accidents which occurred in the rural area and though none occurred in the urban area, which was to be expected, since the snake <italic>Lachesis muta</italic> is a species associated with the dense forests<xref rid=\"B39\" ref-type=\"bibr\">\n<sup>39</sup>\n</xref>. More cases attributed to non-venomous snakes or “dry bites” were present in the urban area, where it is more common to record accidents with species that are not of medical interest<xref rid=\"B34\" ref-type=\"bibr\">\n<sup>34</sup>\n</xref>. A higher proportion of cases among females occurred in the urban area compared to rural areas, which may be related to the fact that a large portion of the accidents in cities occur in households<xref rid=\"B34\" ref-type=\"bibr\">\n<sup>34</sup>\n</xref> and in the rural area which is a more occupational setting, involving mostly farming activities and the extraction process which is carried out mainly by males<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B28\" ref-type=\"bibr\">\n<sup>28</sup>\n</xref>. This same hypothesis could explain the higher proportion of children (under 10 years old) bitten in the urban area (23.94%) than in the rural area (8.5%) in the present study.</p><p>Snakebites mainly affect adult males and tend to occur in the lower limbs. Although several studies have shown that most cases occur in rural areas, this study observed an urbanization of ophidism in the city of Rio Branco. The genus <italic>Bothrops</italic> was responsible for the highest number of snakebites and during the rainy season the accidents occur with a higher frequency, presenting a positive correlation with these events in the studied region. The performance of retrospective studies is important, among other reasons, because this information, when properly collected and recorded in the notification forms, becomes a valuable source of epidemiological data, and provides greater reliability and the possibility of better understanding about a given health problem. Educational snakebites prevention campaigns, population advice and first aid in case of this type of accidents for the populations of rural and urban areas are thus suggested.</p></sec></body><back><ack><title>ACKNOWLEDGEMENTS</title><p>We are grateful to the Epidemiological Surveillance Sector for the municipality of Rio Branco and Hospital of Rio Branco for allowing access to the information in the notification forms.</p></ack><ref-list><title>REFERENCES</title><ref id=\"B1\"><label>1</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chippaux</surname><given-names>JP</given-names></name></person-group><article-title>Incidence and mortality due to snakebite in the Americas</article-title><source>PLoS Negl Trop Dis</source><year>2017</year><volume>11</volume><issue>6</issue><elocation-id>e0005662</elocation-id><pub-id pub-id-type=\"pmid\">28636631</pub-id></element-citation></ref><ref 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Rev Soc Bras Med Trop</journal-id><journal-id journal-id-type=\"iso-abbrev\">Rev. Soc. Bras. Med. Trop</journal-id><journal-id journal-id-type=\"publisher-id\">rsbmt</journal-id><journal-title-group><journal-title>Revista da Sociedade Brasileira de Medicina Tropical</journal-title></journal-title-group><issn pub-type=\"ppub\">0037-8682</issn><issn pub-type=\"epub\">1678-9849</issn><publisher><publisher-name>Sociedade Brasileira de Medicina Tropical - SBMT</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32997049</article-id><article-id pub-id-type=\"pmc\">PMC7523525</article-id><article-id pub-id-type=\"doi\">10.1590/0037-8682-0051-2020</article-id><article-id pub-id-type=\"other\">00350</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Major Article</subject></subj-group></article-categories><title-group><article-title>Rapid detection of <italic>Mycobacterium tuberculosis</italic> in children using blood and urine specimens</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-9087-358X</contrib-id><name><surname>da Costa-Lima</surname><given-names>Juliana Figueirêdo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff5\">\n<sup>5</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Pimentel</surname><given-names>Lílian Maria Lapa Montenegro</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Santos</surname><given-names>Fabiana Cristina Fulco</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Salazar</surname><given-names>Marcela Pereira</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Duarte</surname><given-names>Rafael Silva</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mello</surname><given-names>Fernanda Carvalho de Queiroz</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff5\">\n<sup>5</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Schindler</surname><given-names>Haiana Charifker</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<label>1</label>Instituto Aggeu Magalhães/Fundação Oswaldo Cruz, Laboratório de Imunoepidemiologia, Departamento de Imunologia, Recife, PE, Brasil. </aff><aff id=\"aff2\">\n<label>2</label>Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo Góes, Laboratório de Micobactérias, Rio de Janeiro, RJ, Brasil. </aff><aff id=\"aff3\">\n<label>3</label>Universidade Federal do Rio de Janeiro, Instituto de Doenças do Tórax, Faculdade de Medicina, Rio de Janeiro, RJ, Brasil. </aff><aff id=\"aff4\">\n<label>4</label>Universidade Federal de Pernambuco, Hospital das Clínicas, Departamento Materno Infantil, Recife, PE, Brasil. </aff><aff id=\"aff5\">\n<label>5</label>Universidade Federal do Rio de Janeiro, Programa de Pós-Graduação Stricto Sensu em Clínica Médica, Rio de Janeiro, RJ, Brasil.</aff><author-notes><corresp id=\"c1\"><bold>Corresponding author:</bold> Dra. Juliana F. Costa-Lima. <bold>e-mail:</bold><email>jfcl@cpqam.fiocruz.br</email> / <email>jujufig@hotmail.com</email></corresp><fn fn-type=\"con\" id=\"fn2\"><p>\n<bold>Authors’ contributions:</bold>\n<bold>JFCL:</bold> the main author; performed all the technical steps of the work: collection and processing of clinical samples, DNA extraction, STNPCR technique, data analysis, writing and critical review of the intellectual content of the manuscript. <bold>LMLM:</bold> research supervisor, study design, data collection and analysis, writing and review of the manuscript. <bold>MPS</bold> and <bold>FCFS:</bold> technical activities, such as collection and processing of clinical samples, extraction of DNA, preparation of amplification reactions for the STNPCR technique, monitoring and reading culture tests. <bold>RSD</bold> and <bold>FCQM:</bold> clinical supervisors, study design, data analysis, on writing and review of the manuscript. <bold>HCS:</bold> coordinator of the study, design, data analysis, writing and critical review of relevant intellectual content of the manuscript; physician in the care and clinical classification of patients and in clinical supervision. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.</p></fn></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><year>2020</year></pub-date><volume>53</volume><elocation-id>e20200051</elocation-id><history><date date-type=\"received\"><day>15</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>17</day><month>7</month><year>2020</year></date></history><permissions><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License</license-p></license></permissions><abstract><title>Abstract</title><sec><title>INTRODUCTION: </title><p>Laboratory and clinical features of childhood tuberculosis (TB) are non-specific and establishing an accurate diagnosis remains a challenge. This study evaluated a Single tube nested-PCR (STNPCR) to detect genomic DNA of <italic>Mycobacterium tuberculosis</italic> complex in blood and urine. </p></sec><sec><title>METHODS: </title><p>Biological samples were obtained from children (<15 years old) with clinical suspicion of pulmonary and extrapulmonary TB at public hospitals in Recife-Pernambuco, Brazil. Cultures yielded negative results in a majority of childhood TB cases, which are generally paucibacillary. A set of clinical, epidemiological, radiological, and laboratory criteria with evident clinical improvement after anti-TB treatment were frequently used to define childhood TB cases. </p></sec><sec><title>RESULTS:</title><p> Ninety children with clinical suspicion were enrolled in this study (44 with TB and 46 without TB). The pulmonary TB group had 20 confirmed cases and 46 negative controls, while the extrapulmonary TB group had 24 confirmed cases. The STNPCR showed sensitivities to pulmonary and extrapulmonary TB of 47.4% and 52.2% (blood) and 38.8% and 20% (urine), respectively. Considering the low performance of STNPCR on separate samples, we decided to perform a combined analysis (parallel sensitivity analysis) of the results from blood and urine samples. The parallel sensitivity increased to 65% in blood and 62.5% in urine. The specificity in both samples ranged from 93.5-97.8%. </p></sec><sec><title>CONCLUSIONS: </title><p>Although STNPCR showed moderate sensitivity, the specificity is high; therefore, the test can be used as an auxiliary tool to diagnose TB in children. It is a rapid test that demonstrated better performance than other diagnostic tests in paucibacillary samples as it does in childhood tuberculosis.</p></sec></abstract><kwd-group><title>Keywords:</title><kwd>Childhood tuberculosis</kwd><kwd>Diagnosis</kwd><kwd>Blood</kwd><kwd>Urine</kwd><kwd>PCR</kwd><kwd>STNPCR</kwd></kwd-group><counts><fig-count count=\"0\"/><table-count count=\"4\"/><equation-count count=\"0\"/><ref-count count=\"49\"/></counts></article-meta></front><body><sec sec-type=\"intro\"><title>INTRODUCTION</title><p>According to the World Health Organization (WHO), 6.9% of all tuberculosis (TB) cases were notified among children (2016). The WHO estimated that more than 200,000 (16%) deaths from TB occurred among HIV-negative<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref> children, which makes TB one of the top 10 causes of death among children<xref rid=\"B2\" ref-type=\"bibr\">\n<sup>2</sup>\n</xref>. Brazil is ranked as 25<sup>th</sup> among 30 countries with the highest prevalences of TB, which are responsible for 82% of TB cases worldwide and 75% of childhood TB cases<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B3\" ref-type=\"bibr\">\n<sup>3</sup>\n</xref>. According to the Brazilian Notification of Injury Information System (<italic>Sistema de Informação de Agravos de Notificação - SINAN</italic>), in 2018, more than 94,000 cases of TB were registered in Brazil, where almost 7,900 confirmed cases occurred among patients younger than 19 years<xref rid=\"B4\" ref-type=\"bibr\">\n<sup>4</sup>\n</xref>.</p><p>Childhood TB does not have accurate epidemiological data, because of difficulty in establishing a diagnosis<xref rid=\"B5\" ref-type=\"bibr\">\n<sup>5</sup>\n</xref>. Most cases of TB in this age group are pulmonary and only 20% of total cases are extrapulmonary. However, the more severe forms are extrapulmonary and occur frequently among children as miliary TB and TB meningitis (mainly among children younger than 6 years). Peripheral lymph nodes and TB meningitis are the most frequent manifestations among children<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>.</p><p>The diagnosis of TB is based on the identification of <italic>Mycobacterium tuberculosis</italic> bacilli through smear microscopy, culture, or Xpert<sup>®</sup> MTB/RIF assay<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>. However, the diagnosis of TB in childhood is a big challenge, as it has no accurate gold standard diagnostic method<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B12\" ref-type=\"bibr\">\n<sup>12</sup>\n</xref>. Conventional techniques used in the diagnosis of adult patients present low sensitivity and specificity when applied to children. Bacteriological confirmation is not possible, because childhood TB is paucibacillary. Thus, very frequently, anti-TB treatment is initiated with no bacteriological confirmation<xref rid=\"B13\" ref-type=\"bibr\">\n<sup>13</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>.</p><p>Children younger than 10 years normally have no expectoration and have clinical signs and radiological examination findings that are complex to interpret. In this paucibacillary group, conventional diagnostic methods have poor sensitivity and specificity. The younger the child, the more unspecific the clinical symptoms are and the higher the risk of gravity of TB and death. The opposite also occurs: the older the child is, the more similar is the disease to the adult form<xref rid=\"B16\" ref-type=\"bibr\">\n<sup>16</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B17\" ref-type=\"bibr\">\n<sup>17</sup>\n</xref>. </p><p>There is almost no validated score for the diagnosis of childhood TB. In 2002, the Brazilian Ministry of Health (MH) recommended the use of a scoring system, based on clinical, radiological, epidemiological criteria and tuberculin skin test results to diagnose pulmonary TB in children and adolescents<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B18\" ref-type=\"bibr\">\n<sup>18</sup>\n</xref>. The score proposed by the MH of Brazil, which is known to confirm the diagnosis of TB in children and teenagers, is one of the most widely validated worldwide and which has consistent sensitivity and specificity<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>.</p><p>Since 2011, the WHO has recommended the use of Xpert<sup>®</sup> MTB/RIF (Cepheid, CA, USA) as a rapid test for the diagnosis of pulmonary TB and resistance to rifampicin<xref rid=\"B19\" ref-type=\"bibr\">\n<sup>19</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>. More recently, the Xpert MTB/RIF assay was recommended for the diagnosis of pulmonary TB in children. The Xpert<sup>®</sup> MTB/RIF assay has been evaluated in 13 studies that included a total of 3,347 specimens for the diagnosis of pediatric pulmonary TB, whose sensitivity varied from 55% to 90% for expectorated sputum, from 40% to 100% for induced sputum, and from 40% to 100% for gastric lavage or aspirate<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>. The MH of Brazil also recommends the use of molecular rapid tests for suspected cases of pulmonary TB when scores do not confirm the diagnosis<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>.</p><p>Molecular methods have been developed for use in the detection of bacilli and confirmation of disease. The use of polymerase chain reaction (PCR) assays has been proposed in a few studies to increase the sensitivity and specificity of the diagnosis of childhood tuberculosis<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B22\" ref-type=\"bibr\">\n<sup>22</sup>\n</xref>. Generally, they have high sensitivities and specificities using clinical samples and produce rapid results<xref rid=\"B9\" ref-type=\"bibr\">\n<sup>9</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B25\" ref-type=\"bibr\">\n<sup>25</sup>\n</xref>. These techniques detect a specific genome DNA region in the microorganism. </p><p>Thus, it is necessary to evaluate a new system for the detection of <italic>M. tuberculosis</italic> among children, particularly because of the limitations of conventional diagnostic methods. Based on the difficulty of diagnosis of childhood TB, among patients who do not have sputum expectoration, this study proposed the use of non-invasive samples, blood and urine, to detect the DNA of <italic>M. tuberculosis</italic> by single tube PCR (STNPCR)<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref> and evaluated it in comparison with other conventional tests in clinically suspected pulmonary and extrapulmonary TB. Blood and urine can be collected from outpatients regardless of the site of infection<xref rid=\"B26\" ref-type=\"bibr\">\n<sup>26</sup>\n</xref>.</p><p>The aim of the present study was to evaluate the new system of STNPCR for the detection of <italic>Mycobacterium tuberculosis</italic> DNA in alternative clinical specimens (blood and urine) via minimally invasive methods, other than sputum collection from children and adolescents with clinically suspected pulmonary or extrapulmonary TB.</p></sec><sec sec-type=\"methods\"><title>METHODS</title><sec><title>Study design, population, and setting</title><p>The present prospective study was conducted among 90 children with an initial clinical suspicion of pulmonary or extrapulmonary TB, up to 15 years of age, both sexes, and who spontaneously sought out hospitals or primary health care centers in Recife-Pernambuco, Brazil, considered a high-risk area for TB. It was developed at the laboratory of Immunoepidemiology of the Aggeu Magalhães Institute/Oswaldo Cruz Foundation (IAM/FIOCRUZ), between September 2009 and April 2014. </p></sec><sec><title>Classification of groups</title><p>All patients were prospectively followed and classified into two groups (active TB and not TB) using clinical, laboratorial, and therapeutic response criteria<xref rid=\"B27\" ref-type=\"bibr\">\n<sup>27</sup>\n</xref> according to the American Thoracic Society guidelines<xref rid=\"B28\" ref-type=\"bibr\">\n<sup>28</sup>\n</xref>, WHO guidelines<xref rid=\"B1\" ref-type=\"bibr\">\n<sup>1</sup>\n</xref>, and the Brazilian MH guidelines<xref rid=\"B6\" ref-type=\"bibr\">\n<sup>6</sup>\n</xref>. The attending physicians at hospitals made the final double-blind diagnoses. </p></sec><sec><title>Eligibility criteria</title><p>\n<bold>Active TB (pulmonary and extrapulmonary):</bold> Patients with clinical symptoms or radiological images compatible with TB, epidemiological history of contact with a person with bacilipherous TB, with or without isolation of <italic>M. tuberculosis</italic> on acid-fast bacilli (AFB) or on culture, or with clinical improvement consequent to specific anti-TB treatment (reference standard used).</p><p>\n<bold>Not TB:</bold> Patients with a diagnosis of another disease other than TB and without isolation of <italic>M. tuberculosis</italic>.</p></sec><sec><title>Biological samples</title><p>All children had one sample of total blood (3-5 mL collected in a vacuum tube with anti-coagulant EDTA) and/or three samples of urine (10 mL/day, on three consecutive days), which were merged at the laboratory to be analyzed as an unique sample on STNPCR. All samples were collected before the initiation of specific treatment.</p></sec><sec><title>Blood - Isolation of peripheral blood mononuclear cells and plasma</title><p>Peripheral blood mononuclear cells (PBMCs) and plasma were separated from whole blood at room temperature by the density gradient method using Ficoll-Paque<sup>TM</sup> Plus (GE Healthcare, Sweden). The erythrocytes were discarded and the PBMCs and plasma layers were separated to be used in DNA extraction and afterwards in STNPCR. </p></sec><sec><title>Urine decontamination</title><p>Urine samples were decontaminated using the Petroff’s method with 4% NaOH<xref rid=\"B29\" ref-type=\"bibr\">\n<sup>29</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B30\" ref-type=\"bibr\">\n<sup>30</sup>\n</xref>.</p></sec><sec><title>DNA extraction</title><p>DNA extraction was performed using the commercial QIAmp DNA mini kit (QIAGENGmbH, Hilden, Germany), as per the manufacturer’s recommendation. The DNA was extracted from PBMC, plasma, and urine samples. For all DNA extractions, a negative control tube (with TE buffer and no DNA template) were used to evaluate possible cross-contamination. </p></sec><sec><title>Molecular STNPCR system</title><p>The STNPCR system was based on the principles put forth by Abath et al.<xref rid=\"B31\" ref-type=\"bibr\">\n<sup>31</sup>\n</xref> and Costa-Lima et al.<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref> using an IS6110 insertion sequence (GenBank accession no. X52471) as a target to detect DNA from <italic>M. tuberculosis</italic> complex. The set of outer primers used were TJ5 (5′-CCGCAAAGTGTGGCTAAC-3′) and TJ3 (5′-ATCCCCTATCCGTATGGTG-3′) with an amplified fragment of 409 bp. The inner primers were OLI5 (5′-AACGGCTGATGACCAAAC-3′) and STAN3 (5′-GTCGAGTACGCCTTCTTGTT-3′), amplifying a 316 bp fragment<xref rid=\"B32\" ref-type=\"bibr\">\n<sup>32</sup>\n</xref>. These sets of primers were previews used on a conventional nested PCR system and on STNPCR of blood and urine<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B33\" ref-type=\"bibr\">\n<sup>33</sup>\n</xref>.</p></sec><sec><title>Ethical considerations</title><p>The study protocol was approved by the ethics committee (CAAE 08381812.4.00005190) of IAM/FIOCRUZ. Written informed consent was obtained from all guardians or legal representatives of each child who agreed to participate in the research and authorized the collection of clinical samples.</p></sec><sec><title>Statistical analysis</title><p>The database was prepared using SPSS Statistics version 20.0.0 (IBM Corp., Armonk, NY, USA), in which all crosstabs and frequencies and other analyses, excluding the sensitivities, specificities, and predictive values (screening tests) were assessed. For screening tests, the free software OpenEpi version 2.3.1 was used.</p><p>For statistical analyses, PBMC and plasma samples were considered as unique samples named “blood sample” and their sensitivity, specificity, and predictive values were analyzed in parallel<xref rid=\"B34\" ref-type=\"bibr\">\n<sup>34</sup>\n</xref>. In the same way, the “blood samples” plus urine samples were evaluated in parallel.</p></sec></sec><sec sec-type=\"results\"><title>RESULTS</title><sec><title>Clinical, epidemiological, and laboratorial results</title><p>Forty-four (47.3%) children were diagnosed with active TB from a total of 90. The mean age was 7.5 ± 4.9 years (range, 0-15). Only one child was known to have a coinfection of HIV and TB; for the remaining children, HIV tests were not performed. The inpatients (51.6%) were the majority in relation to outpatients. AFB tests were performed among 11 (12.8%) children, with only one positive result (a 14-year-old girl). Regarding culture, 86% of children underwent culture for at least one clinical sample (urine, sputum, pleural fluid, and/or other fluids). Culture test results were positive in only 10 (11%) of them. A majority of cultures were performed on urine (66.7%), with positive results in 6.9% (4/58). Other epidemiological and demographic data are detailed in <xref rid=\"t1\" ref-type=\"table\">Table 1</xref>. The chi-squared analysis of type of hospital admission, result of skin test, and treatment response, yielded <italic>p</italic> values of 0.005, 0.02 and <0.001, respectively. Each child provided a mean of 2.8 clinical samples for analysis. Most cases were of pulmonary TB, followed by peripheral lymph node TB. All clinical manifestations of TB are detailed in <xref rid=\"t2\" ref-type=\"table\">Table 2</xref>.</p><p>\n<table-wrap id=\"t1\" orientation=\"portrait\" position=\"float\"><label>TABLE 1:</label><caption><title>Frequency of clinical and epidemiological characteristics of children (<italic>n</italic> = 90) and their respective prevalence ratio and <italic>p-value</italic> (95% CI) on a Poisson binary regression related to confirmation of TB</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">TB</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Not TB</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Prevalence ratio - </th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n<italic>p-value</italic>\n</th></tr><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Poisson binary </th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th></tr><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">regression (CI)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Hospital admission</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Outpatients</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26 (59.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16 (34.8%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Inpatients</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18 (40.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30 (65.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.29 (1.06-1.57)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.01</bold>\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>44</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>46</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"justify\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Sex</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18 (40.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25 (54.3%)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Female</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26 (59.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">21 (45.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.77 (0.52-1.17)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.23 </td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>44</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>46</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Age (by years)</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>44</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>46</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.02 (1-1.05)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.053</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Age (by age groups)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">From 0 to 5 years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (22.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22 (47.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">From 6 to 10 years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (29.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8 (17.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">From 11 to 15 years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19 (43.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (30.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.15 (1.02-1.31)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.029</bold>\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">No age (by years) information</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (4.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (4.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Scar of BCG vaccine</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">31 (70.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26 (56.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (2.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (6.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.73 (0.47-1.14)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.171</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Not verified</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12 (27.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17 (37%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>44</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>46</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>90</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Type of contact with bacillipherous TB</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Contact with bacillipherous</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16 (55.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19 (61.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">No contact with bacillipherous</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (44.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (32.3%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.37 (0-0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold><0.001</bold>\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Unable to assess</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (-)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (5.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>29</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>31</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>60</bold>\n</td><td align=\"justify\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>AFB results (all samples)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Positive†</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Negative</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 (11.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.61 (0.45-0.83)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.002</bold>\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Not realized</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">33 (87.8%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">46 (100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>44</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>46</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>90</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Culture (all samples)</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Positive</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2 (4.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1 (2.2%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Negative</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15 (34.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18 (39.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.81 (0.46-1.42)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.46</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Not realized</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27 (61.4%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">27 (58.7%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>44</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>46</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>90</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Result of skin test</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">< 10 mm</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (29.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15 (32.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">> 10 mm</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">18 (41%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5 (10.9%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.73 (0.57-0.93)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>0.013</bold>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Not realized</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13 (29.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26 (56.5%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>44</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>46</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>90</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Clinical form of TB</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Pulmonary TB</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20 (42.6%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Extrapulmonary TB</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">24 (51.1%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">Not TB</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">46 (100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td align=\"justify\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>44</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>46</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>90</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN1\"><p>\n<bold>*</bold>AFB and culture performed at IAM/FIOCRUZ, independent of results from the reference laboratory; <bold>†</bold> Patient had result for AFB of one cross; <bold>AFB:</bold> acid fast bacilli; <bold>BCG:</bold> Bacillus Calmette-Guérin; <bold>TB:</bold> tuberculosis</p></fn></table-wrap-foot></table-wrap>\n</p><p>\n<table-wrap id=\"t2\" orientation=\"portrait\" position=\"float\"><label>TABLE 2:</label><caption><title>Clinical forms of tuberculosis among children.</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Clinical form </th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Frequency</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Percentage</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pulmonar</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">45.5%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pleural</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.8%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Peripheral lymph node</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.9%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Meningoencephalitis</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.5%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Bone </td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.5%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Articular</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.3%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cutaneous</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.3%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Abdominal</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.5%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pericardial</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.3%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Intestinal</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.3%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Miliary</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.3%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Other</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.8%</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Total</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>44</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>100%</bold>\n</td></tr></tbody></table></table-wrap>\n</p></sec><sec><title>Single tube nested PCR</title><p>This study evaluated the performance of STNPCR in each clinical sample collected: PBMC, plasma, and urine. Regarding loss criteria (insufficient material, hemolyzed blood, or other), four patients had no blood sample, in two other patients it was not possible to separate PBMCs, and eight children did not have urine samples. The sensitivities of tests varied from 26% to 50% and specificities from 94% to 100% (<xref rid=\"t3\" ref-type=\"table\">Table 3</xref>). Calculating in parallel, the sensitivity of “blood sample” plus urine was above 60%. The results for each sample analyzed alone and in parallel are detailed in <xref rid=\"t3\" ref-type=\"table\">Table 3</xref>. According to the clinical form of TB, accuracy was evaluated via STNPCR on “blood samples” alone and with urine (20 with pulmonary TB, 24 with extrapulmonary TB, and 46 with no TB), calculated in parallel (<xref rid=\"t3\" ref-type=\"table\">Table 3</xref>). Four patients had no blood (neither PBMC nor plasma) collected, two others had no PBMCs isolated from plasma, and eight children had no urine sample, all of whom were included in the study with only one sample (blood or urine). The positive and negative predictive values for this population (<italic>n=</italic>90) were 90.3% (28/31) and 72.9% (43/59), for all biological samples analyzed in parallel.</p><p>\n<table-wrap id=\"t3\" orientation=\"portrait\" position=\"float\"><label>TABLE 3:</label><caption><title>Performance of STNPCR by type of biological sample and among patients with and without TB (pulmonary and extrapulmonary).</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"1\"/><col span=\"2\"/></colgroup><thead><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">Clinical samples</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Nº of samples</th><th align=\"center\" colspan=\"2\" rowspan=\"1\">Patients with and without TB (all clinical forms) </th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Sensitivity</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Specificity</th></tr></thead><tbody><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">PBMC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">84</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">39%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">95.4%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25.7, 54.3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">84.5, 98.7</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(16/41)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(41/43)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Plasma</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">86</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26.2%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15.3, 41.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">92, 100</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(11/42)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(44/44)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Blood sample (PBMC plasma)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">86</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">50%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">95.5%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">35.5, 64.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">84.9, 98.7</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(21/42)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(42/44)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Urine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">82</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28.2%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">97.7%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16.5, 43.8</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">87.9, 99.6</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(11/39)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(42/43)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Blood sample + urine</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">90</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">63.6%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">93.5%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">48.9, 76.2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">82.5, 97.8</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(28/44)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(43/46)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Clinical samples</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Patients (n)</bold>\n</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Pulmonary TB (95% CI)</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Se</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Sp</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Blood samples*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">63</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">47.4%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">95.5%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(27.3, 68.3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(84.9, 98.7)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9/19</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">42/44</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Urine samples</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">62</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">38.8%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">97.7%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(19.2, 59)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(87.9, 99.6)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7/19</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">42/43</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Blood and urine* <italic>per</italic> patient</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">66</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">65%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">93.5%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(43.3, 81.9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(82.5, 97.8)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13/20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43/46</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Clinical samples</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Patients (n)</bold>\n</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Extrapulmonary TB (95% CI)</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Se</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Sp</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Blood samples*</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">67</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">52.2%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">95.5%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(33, 70.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(84.9, 98.7)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">12/23</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">42/44</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Urine samples</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">65</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">97.8%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(8.1, 41.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(88.4, 99.6)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4/20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">44/45</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Blood and urine* <italic>per</italic> patient</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">70</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">62.5%</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">93.5%</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(42.7, 78.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">(82.5, 97.8)</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">15/24</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43/46</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN2\"><p>*Patient was considered positive when at least one sample yielded positive results on STNPCR. Se: Sensitivity; Sp: Specificity; CI: confidence interval; STNPCR: single tube nested PCR; PBMC: peripheral blood mononuclear cell.</p></fn></table-wrap-foot></table-wrap>\n</p><p>Fifty-six patients had their diagnoses confirmed via cultures performed at the Central Laboratory of Pernambuco, which is a reference center for the diagnosis of TB. The other 34 patients (with no microbiological test confirmation), had TB diagnoses confirmed by therapeutic empirical tests. From this group (therapeutic empirical test) of 34 patients, thirty responded positively and 4 had no response. The four children who had no responses to specific treatment also presented negative PCR results. In addition, all 30 who had responded to specific treatment had positive PCR results (<xref rid=\"t4\" ref-type=\"table\">Table 4</xref>). </p><p>\n<table-wrap id=\"t4\" orientation=\"portrait\" position=\"float\"><label>TABLE 4:</label><caption><title>Accuracy of treatment response <italic>versus</italic> STNPCR result among patients (“blood sample” and urine samples).</title></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/><col span=\"3\"/></colgroup><thead><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">tSTNPCR</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">Treatment response </th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">results</th><th align=\"center\" colspan=\"3\" rowspan=\"1\">\n</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">\n</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">No</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Total</th></tr></thead><tbody><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Positive</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Negative</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">34</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Parameter</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Estimate</bold>\n</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>95% CI</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Sensitivity</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">66.7%</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">(48.8, 80.8) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Specificity</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">100%</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">(51, 100) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>Treatment response</bold>\n</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n<bold>TB</bold>\n</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">\n<bold>Not TB</bold>\n</td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">30 (68.2%)</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">0 (-) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0 (-)</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">4 (8.7%) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Diagnosed by microbiologic tests</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14 (31.8%)</td><td align=\"center\" colspan=\"2\" rowspan=\"1\">42 (91.3%) </td></tr><tr><td align=\"center\" rowspan=\"1\" colspan=\"1\">Total</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">44 (100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">46 (100%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">\n</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN3\"><p>\n<bold>CI:</bold> confidence interval; <bold>STNPCR:</bold> single tube nested PCR; <bold>TB:</bold> tuberculosis.</p></fn></table-wrap-foot></table-wrap>\n</p></sec></sec><sec sec-type=\"discussion\"><title>DISCUSSION</title><p>In pediatric samples, it is not easy to confirm the presence of bacilli, similar to patients with extrapulmonary TB and TB-HIV coinfection, because they are paucibacillary. These groups, including cases of drug resistant TB, are responsible for the increase in morbidity and mortality due to TB in developing countries<xref rid=\"B35\" ref-type=\"bibr\">\n<sup>35</sup>\n</xref>.</p><p>Regarding samples of reference in adults, sputum is not easy to collect in children because they frequently swallow rather than expectorate it<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>. Some studies advise the use of induced sputum instead for children with pulmonary TB<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref> and the gastric lavage for extrapulmonary TB<xref rid=\"B36\" ref-type=\"bibr\">\n<sup>36</sup>\n</xref>. In addition, both of these biological specimens are invasive samples and are not collected at a primary care health center<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B36\" ref-type=\"bibr\">\n<sup>36</sup>\n</xref>. On the other hand, one of the best ways to reduce the mortality due to childhood TB is to develop tests that could be run on accessible clinical specimens such as urine and blood<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B37\" ref-type=\"bibr\">\n<sup>37</sup>\n</xref>.</p><p>A control strategy for TB, particularly in regions with high endemicity, is to develop diagnostic tests which are rapid, sensitive, specific, and inexpensive for use in public health service. It would provide better detection of cases associated with effective treatment, leading to decreased transmission and cases of drug resistant TB. Nucleic acid amplification tests for the diagnosis of pediatric TB could reduce the mortality rate by 6.8%<xref rid=\"B37\" ref-type=\"bibr\">\n<sup>37</sup>\n</xref>. </p><p>The new technology, Xpert MTB/RIF assay, which should replace smear microscopy at Primary Health Care Centers<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>, is already being used in several countries with a high TB burden. The Xpert MTB/RIF assay yields similar results to the findings of this study on blood and urine collected from children with pulmonary TB<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>. The major disadvantage of Xpert MTB/RIF assay in children is the lowest sensitivity and specificity in respiratory or other samples<xref rid=\"B38\" ref-type=\"bibr\">\n<sup>38</sup>\n</xref>. In STNPCR, blood and urine are validation samples and can detect both pulmonary and extrapulmonary TB with similar accuracy. Other studies used these samples to detect TB by PCR and showed the importance of using them<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B26\" ref-type=\"bibr\">\n<sup>26</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B33\" ref-type=\"bibr\">\n<sup>33</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B39\" ref-type=\"bibr\">\n<sup>39</sup>\n</xref>\n<sup>-</sup>\n<xref rid=\"B41\" ref-type=\"bibr\">\n<sup>41</sup>\n</xref> and assisting the diagnosis of TB cases. </p><p>The culture only confirmed TB cases in 11% of pediatric patients. Besides that, culture takes too long to yield results, taking up to eight weeks<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>. Instead, STNPCR can yield test results in up to one day (from sample collection to result) or fewer, demonstrating that it can be used as an auxiliary tool for early diagnosis in children. Generally, for childhood TB, clinical samples are considered as reference standards if the culture yields negative results and the anti-TB therapy can be initiated based on clinical evidence<xref rid=\"B8\" ref-type=\"bibr\">\n<sup>8</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B21\" ref-type=\"bibr\">\n<sup>21</sup>\n</xref>.</p><p>For negative bacteriological cases associated with a strong clinical suspicion of the disease, the empirical treatment is initiated in approximately 30% of suspected TB cases<xref rid=\"B42\" ref-type=\"bibr\">\n<sup>42</sup>\n</xref>. As demonstrated by this study, most patients with active TB who responded to specific treatment had positive results on STNPCR. Considering empirical therapy as the gold standard, STNPCR presented a sensitivity of approximately 67% and a specificity of 100%. This may be evidence that the use of STNPCR in blood and urine samples may improve laboratory confirmation of cases and avoid the initiation of empirical treatment, which alone has a sensitivity ranging from 20 to 80% in adults<xref rid=\"B43\" ref-type=\"bibr\">\n<sup>43</sup>\n</xref>. In children, when the risk of death by TB is high, it is highly recommended that empirical treatment be initiated, regardless of confirmation by a diagnostic test<xref rid=\"B14\" ref-type=\"bibr\">\n<sup>14</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B15\" ref-type=\"bibr\">\n<sup>15</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B44\" ref-type=\"bibr\">\n<sup>44</sup>\n</xref>. It is mainly because there does not exist a single diagnostic test with good accuracy for childhood TB. In the present study, almost 12% of non-TB patients could have avoided unnecessary treatment if negative STNPCR results were considered as diagnostic. It can be concluded that the sensitivity and specificity of STNPCR compared with the clinical, laboratory, and epidemiological criteria or with treatment response were statistically the same, ranging from 63.6 to 66.7% and 93.5 to 100%, respectively.</p><p>The STNPCR is a molecular test which detects DNA circulating from <italic>M. tuberculosis</italic> complex in paucibacillary samples. Although, as a molecular test, it does not distinguish viable bacteria cells from non-viable cells, or even from free fragments of nucleic acids in samples. Therefore, STNPCR does not differentiate between active and latent TB. Therefore, the clinical features are paramount to confirm childhood active or latent TB<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B45\" ref-type=\"bibr\">\n<sup>45</sup>\n</xref>. </p><p>Based on the difficulty of diagnosis of childhood TB among patients who do not expectorate sputum, we tested non or minimal invasive samples to detect <italic>M. tuberculosis</italic>. Blood and urine can be collected from outpatients regardless of the site of infection.</p><p>Some evidence shows the presence of DNA fragments circulating in blood and urine<xref rid=\"B35\" ref-type=\"bibr\">\n<sup>35</sup>\n</xref>. These fragments are derived from cell-free nucleic acids of bacilli and result from breakdown of these microorganisms or dead human cells, which contained bacilli, and go on to circulate in blood. Some of these fragments of DNA pass through the kidney and are excreted in the urine as transrenal DNA<xref rid=\"B35\" ref-type=\"bibr\">\n<sup>35</sup>\n</xref>.</p><p>According to the results, plasma increases by 21% the sensitivity of PBMC when analyzed in parallel as “blood sample”. When the only clinical sample available for collection would be blood, it must be processed by molecular testing using PBMC and plasma separated and analyzed in parallel, together. Eight children had no urine samples because samples were self-collected at the patients’ homes and some of them did not return to the health care service with the biological sample.</p><p>Analyzing the three samples (PBMC, plasma, and urine) isolated yielded no statistical difference between their sensitivities. However, the parallel sensitivity of “blood sample” + urine together was higher than that of plasma or urine alone. Among children with difficult diagnostic interpretation, the collection of more than one clinical sample must be considered to increase the sensitivity of STNPCR. Studies have demonstrated that the combination of two or more different clinical samples from the same patient increases<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B26\" ref-type=\"bibr\">\n<sup>26</sup>\n</xref> the performance of STNPCR. </p><p>Related to the clinical type of TB (pulmonary and extrapulmonary), “blood sample” yielded the most positive results on STNPCR. The urine added around 30% of sensitivity to “blood samples” on parallel sensitivity of STNPCR results. Although this biological sample has a low isolated sensitivity, when associated with “blood sample” it increased the global sensitivity of patients test results<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref>.</p><p>It was observed in the study that sensitivity of urine in all patients was much lower than that of “blood sample”, but not statistically different. Probably, this difference in results depends on the differences in the physiopathology of clinical disease forms. The sensitivities of “blood sample” added to urine sample, analyzed in parallel, tend to be higher than that of only one isolated sample, both for the pulmonary and extrapulmonary groups. There were no renal TB cases; however, this does not mean that positivity on urine samples indicated false-positive results. The STNPCR does not distinguish between bacilli which are integral than fragments of it (free DNA)<xref rid=\"B35\" ref-type=\"bibr\">\n<sup>35</sup>\n</xref>. </p><p>In genitourinary TB, PCR is probably the most promising method of detecting <italic>M. tuberculosis</italic>\n<xref rid=\"B23\" ref-type=\"bibr\">\n<sup>23</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B46\" ref-type=\"bibr\">\n<sup>46</sup>\n</xref>. In this study, the sensitivity of urine culture corroborates another study that found that culture usually does not yield a sensitivity of more than 40%<xref rid=\"B46\" ref-type=\"bibr\">\n<sup>46</sup>\n</xref>. Only one positive urine culture for <italic>M. tuberculosis</italic> also yielded positive results on STNPCR. However, the three other urine cultures positive for nontuberculous mycobacteria strain were all-negative on STNPCR for <italic>M. tuberculosis</italic> complex<italic>.</italic>\n</p><p>For childhood TB, it is difficult to use just one reference test to confirm the disease. A set of criteria is used in almost all cases. Thus, the reference test is subjective and the accuracy of STNPCR could be underestimated. Therefore, to better evaluate the performance of STNPCR instead of the limitations of culture, the authors decided to also consider the treatment response as the gold standard to reflect in fact how childhood TB diagnosis is defined. In these groups of patients, the sensitivity found was similar, but the specificity was 100%. The false-negative samples on molecular test can be associated with the paucibacillary nature of samples or with a possible low efficacy on DNA extraction methods which could not minimize the inhibitory factors<xref rid=\"B47\" ref-type=\"bibr\">\n<sup>47</sup>\n</xref>.</p><p>In this analysis, the results demonstrated that STNPCR is exceptionally reliable in confirming TB because the sensitivity found was 60% and the positive predictive value was 100%. However, not only sensitivity and specificity should be considered to implement a new diagnostic tool; the cost and ease of implementation must be well evaluated<xref rid=\"B48\" ref-type=\"bibr\">\n<sup>48</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B49\" ref-type=\"bibr\">\n<sup>49</sup>\n</xref>. </p><p>The development of better diagnostic methods is a consensus and remains a significant priority for children<xref rid=\"B10\" ref-type=\"bibr\">\n<sup>10</sup>\n</xref>\n<sup>,</sup>\n<xref rid=\"B20\" ref-type=\"bibr\">\n<sup>20</sup>\n</xref>. The proposed method has the great advantage of using clinical samples that are available from most children and obtained via minimally invasive methods. Moreover, this system is fast, sensitive, and specific for use in the diagnosis of TB among children with any clinical form of disease. STNPCR is indicated as an auxiliary tool to help confirm TB in children. Therefore, more studies of the cost-effectiveness of using STNPCR are needed to evaluate the possibility of its implementation in public health services. </p></sec></body><back><ack><title>ACKNOWLEDGEMENTS</title><p>The authors would like to thank Dr. Marta Maciel Lyra for her commitment to sending patients with indications of TB to this study and for her continuing contribution to theclinical accompaniment of patients at a medical care center specialized in TB. We also thank the institutions that funded this study.</p></ack><fn-group><fn fn-type=\"financial-disclosure\" id=\"fn1\"><p>\n<bold>Financial support:</bold> All phases of this study were supported by Conselho Nacional de DesenvolvimentoCientífico e Tecnológico (grant number 456761/2014-4), ICOHRTA AIDS/TB-NIH(Grant #5U2RTW006883-02 AI066994 and #U2RTW006885), FACEPE/PPSUS,FIOCRUZ RID-08, and IAM/FIOCRUZ.</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id=\"B1\"><label>1</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>World Health Organization (WHO)</collab></person-group><source>Global Tuberculosis Report 2017</source><publisher-loc>Geneva</publisher-loc><publisher-name>WHO</publisher-name><year>2017</year><size units=\"pages\">147p</size><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.who.int/tb/publications/global_report/gtbr2017_main_text.pdf\">https://www.who.int/tb/publications/global_report/gtbr2017_main_text.pdf</ext-link></element-citation></ref><ref id=\"B2\"><label>2</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buonsenso</surname><given-names>D</given-names></name><name><surname>Lancella</surname><given-names>L</given-names></name><name><surname>Delogu</surname><given-names>G</given-names></name><name><surname>Krystofiak</surname><given-names>A</given-names></name><name><surname>Testa</surname><given-names>A</given-names></name><name><surname>Ranno</surname><given-names>O</given-names></name><etal/></person-group><article-title>A twenty-year retrospective study of pediatric tuberculosis in two tertiary hospitals in Rome</article-title><source>Pediatr Infect Dis J</source><year>2012</year><volume>31</volume><issue>10</issue><fpage>1022</fpage><lpage>1026</lpage><pub-id pub-id-type=\"pmid\">22668805</pub-id></element-citation></ref><ref id=\"B3\"><label>3</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>Ministério da Saúde (MS). 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content-type=\"http://credit.casrai.org/\">Funding Acquisition</role><role content-type=\"http://credit.casrai.org/\">Writing – Review & Editing</role><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-5770-8363</contrib-id><xref ref-type=\"aff\" rid=\"a1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lawlor</surname><given-names>Deborah A</given-names></name><role content-type=\"http://credit.casrai.org/\">Conceptualization</role><role content-type=\"http://credit.casrai.org/\">Funding Acquisition</role><role content-type=\"http://credit.casrai.org/\">Supervision</role><role content-type=\"http://credit.casrai.org/\">Writing – Review & Editing</role><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-6793-2262</contrib-id><xref ref-type=\"aff\" rid=\"a6\">6</xref><xref ref-type=\"aff\" rid=\"a7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sheldon</surname><given-names>Trevor A</given-names></name><role content-type=\"http://credit.casrai.org/\">Conceptualization</role><role content-type=\"http://credit.casrai.org/\">Funding Acquisition</role><role content-type=\"http://credit.casrai.org/\">Supervision</role><role content-type=\"http://credit.casrai.org/\">Writing – Review & Editing</role><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-7479-5913</contrib-id><xref ref-type=\"aff\" rid=\"a8\">8</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wright</surname><given-names>John</given-names></name><role content-type=\"http://credit.casrai.org/\">Conceptualization</role><role content-type=\"http://credit.casrai.org/\">Funding Acquisition</role><role content-type=\"http://credit.casrai.org/\">Methodology</role><role content-type=\"http://credit.casrai.org/\">Supervision</role><role content-type=\"http://credit.casrai.org/\">Writing – Review & Editing</role><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-9572-7293</contrib-id><xref ref-type=\"aff\" rid=\"a1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Pickett</surname><given-names>Kate E</given-names></name><role content-type=\"http://credit.casrai.org/\">Conceptualization</role><role content-type=\"http://credit.casrai.org/\">Funding Acquisition</role><role content-type=\"http://credit.casrai.org/\">Methodology</role><role content-type=\"http://credit.casrai.org/\">Supervision</role><role content-type=\"http://credit.casrai.org/\">Writing – Review & Editing</role><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-8066-8507</contrib-id><xref ref-type=\"aff\" rid=\"a4\">4</xref></contrib><contrib contrib-type=\"author\"><collab>on behalf of the Bradford Institute for Health Research COVID-19 Scientific Advisory Group</collab></contrib><aff id=\"a1\">\n<label>1</label>Bradford Institute for Health Research, Bradford Teaching Hospitals NHS Foundation Trust, Bradford Royal Infirmary, Duckworth Lane, Bradford, BD9 6RJ, UK</aff><aff id=\"a2\">\n<label>2</label>Faculties of Life Sciences and Health Studies, University of Bradford, Richmond Road, Bradford, BD7 1DP, UK</aff><aff id=\"a3\">\n<label>3</label>Leeds Clinical Trials Research Unit, University of Leeds, Leeds, LS2 9JT, UK</aff><aff id=\"a4\">\n<label>4</label>Department of Health Sciences, University of York, Seebohm Rowntree Building, University of York, Heslington, York, YO10 5DD, UK</aff><aff id=\"a5\">\n<label>5</label>Hull York Medical School, University of York, Heslington, YO10 5DD, UK</aff><aff id=\"a6\">\n<label>6</label>Medical Research Council Integrative Epidemiology Unit, University of Bristol, Oakfield House, Oakfield Grove, Bristol, BS8 2BN, UK</aff><aff id=\"a7\">\n<label>7</label>Population Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK</aff><aff id=\"a8\">\n<label>8</label>Institute of Population Health Sciences, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, Yvonne Carter Building, 58 Turner Street, London, E1 2AB, UK</aff></contrib-group><author-notes><corresp id=\"c1\"><label>a</label><email xlink:href=\"mailto:Rosie.McEachan@bthft.nhs.uk\">Rosie.McEachan@bthft.nhs.uk</email></corresp><corresp id=\"c2\"><label>b</label><email xlink:href=\"mailto:Josie.Dickerson@bthft.nhs.uk\">Josie.Dickerson@bthft.nhs.uk</email></corresp><fn fn-type=\"COI-statement\"><p>\n<bold>Competing interests: </bold>D.A. Lawlor has received research support from several national and international government and charity funders, as well as Roche Diagnostics and Medtronic Ltd for research unrelated to this protocol paper. No competing interests were disclosed by other authors.</p></fn></author-notes><pub-date pub-type=\"epub\"><day>13</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>13</day><month>8</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>5</volume><elocation-id>191</elocation-id><history><date date-type=\"accepted\"><day>27</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright: © 2020 McEachan RRC et al.</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"wellcomeopenres-5-17704.pdf\"/><abstract><p>The UK COVID-19 lockdown has included restricting social movement and interaction to slow the spread of disease and reduce demand on NHS acute services. It is likely that the impacts of restrictions will hit the least advantaged disproportionately and will worsen existing structural inequalities amongst deprived and ethnic minority groups.</p><p> The aim of this study is to deliver rapid intelligence to enable an effective COVID-19 response, including co-production of interventions, that address key issues in the City of Bradford, UK, and nationally. In the longer term we aim to understand the impacts of the response on health trajectories and inequalities in these.</p><p> In this paper we describe our approach and protocol. We plan an adaptive longitudinal mixed methods approach embedded with Born in Bradford (BiB) birth cohorts which have rich existing data (including questionnaire, routine health and biobank). All work packages (WP) interact and are ongoing. WP1 uses co-production and engagement methods with communities, decision-makers and researchers to continuously set (changing) research priorities and will, longer-term, co-produce interventions to aid the City’s recovery. In WP2 repeated quantitative surveys will be administered during lockdown (April-June 2020), with three repeat surveys until 12 months post-lockdown with an ethnically diverse pool of BiB participants (parents, children aged 9-13 years, pregnant women: total sample pool N=7,652, N=5,154, N=1,800). A range of health, social, economic and education outcomes will be assessed. In WP3 priority topics identified in WP1 and WP2 will be explored qualitatively. Initial priority topics include children’s mental wellbeing, health beliefs and the peri/post-natal period. Feedback loops will ensure findings are fed directly to decision-makers and communities (via WP1) to enable co-production of acceptable interventions and identify future priority topic areas. Findings will be used to aid development of local and national policy to support recovery from the pandemic and minimise health inequalities.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>COVID-19</kwd><kwd>coronavirus</kwd><kwd>children</kwd><kwd>family</kwd><kwd>mental health</kwd><kwd>health inequality</kwd><kwd>ethnicity</kwd><kwd>social determinants of health</kwd></kwd-group><funding-group><award-group id=\"fund-1\" xlink:href=\"http://dx.doi.org/10.13039/501100000265\"><funding-source>Medical Research Council</funding-source><award-id>MR/N024391/1</award-id></award-group><award-group id=\"fund-2\" xlink:href=\"http://dx.doi.org/10.13039/501100000274\"><funding-source>British Heart Foundation</funding-source><award-id>CS/16/4/32482</award-id></award-group><award-group id=\"fund-3\"><funding-source>UK Prevention Research Partnership</funding-source><award-id>MR/S037527/1</award-id></award-group><award-group id=\"fund-4\" xlink:href=\"http://dx.doi.org/10.13039/501100014338\"><funding-source>National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care Yorkshire and Humber</funding-source><award-id>NIHR200166</award-id></award-group><award-group id=\"fund-5\" xlink:href=\"http://dx.doi.org/10.13039/501100000269\"><funding-source>Economic and Social Research Council</funding-source><award-id>MR/N024391/1</award-id></award-group><award-group id=\"fund-6\" xlink:href=\"http://dx.doi.org/10.13039/501100000272\"><funding-source>National Institute for Health Research</funding-source></award-group><award-group id=\"fund-7\" xlink:href=\"http://dx.doi.org/10.13039/100004440\"><funding-source>Wellcome Trust</funding-source><award-id>101597</award-id></award-group><award-group id=\"fund-8\" xlink:href=\"http://dx.doi.org/10.13039/501100013529\"><funding-source>National Lottery Community Fund</funding-source></award-group><award-group id=\"fund-9\" xlink:href=\"http://dx.doi.org/10.13039/100014013\"><funding-source>UK Research and Innovation</funding-source></award-group><funding-statement>This work was supported by the Wellcome Trust [101597; Principal Investigator DAL; Co-Investigators JWr, RM]; a joint grant from the UK Medical Research Council (MRC) and UK Economic and Social Science Research Council (ESRC) [MR/N024391/1; joint Principal Investigators KP and DAL; Co-Investigators JWr, RM, JW]; a British Heart Foundation Clinical Study grant [CS/16/4/32482; PrincipaI Investigator: DAL; Co-Investigators JWr, JWes, RM]; the National Institute for Health Research under its Applied Research Collaboration Yorkshire and Humber [NIHR200166; PI: JWr; CIs KP, RM, JD, CC]; ActEarly UK Prevention Research Partnership Consortium [MR/S037527/1; Co-Principal Investigator JWr and Andrew Hayward; Co-Investigators JWes, RM, JD, KP, TS, LS]; the NIHR Clinical Research Network through research delivery support for this study; the National Lottery Community Fund, which provided funding for BiBBS through the Better Start Bradford programme; and UKRI Covid19 Research & Innovation Call, Medical Research Council (Principal Investigator JD; Co-investigators SB, LS, TS, MB, RM, JW, KP).</funding-statement><funding-statement><italic>The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</italic></funding-statement></funding-group></article-meta></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>The UK, alongside countries throughout the world, is facing an unprecedented national emergency due to the rapid spread of the COVID-19 virus. Ethnic minority groups, and those living in deprived areas are bearing the brunt of the virus with increased mortality rates as a result of the disease compared with more affluent and White British populations\n<sup><xref rid=\"ref-1\" ref-type=\"bibr\">1</xref></sup>. The increase in mortality in ethnic minority groups is likely to be due to a complex interplay of existing health co-morbidities and the pernicious social determinants of health including deprivation and poverty, which are more prevalent in these groups. The immediate response to the threat of the virus has been a stringent lockdown (implemented on 23\n<sup><sup>rd</sup></sup> March 2020), effectively limiting people to their homes, followed by ongoing restrictions on daily life. As a result of the lockdown measures, schools have closed and many businesses have been unable to trade, resulting in high numbers of employed staff being ‘furloughed’, with other small businesses or self-employed workers unable to generate an income for prolonged periods. In the second half of March 2020, the Department for Work and Pensions recorded 950,000 new Universal Credit claims, which is a significant increase, and suggests unemployment rose sharply even before more stringent lockdown restrictions were introduced\n<sup><xref rid=\"ref-2\" ref-type=\"bibr\">2</xref></sup>.</p><p>Whilst the lockdown measures have been successful in reducing the spread of the virus, there is a growing recognition of the wider impact of the COVID-19 response on vulnerable populations. Likely impacts from the restrictions imposed on these populations to limit the spread of COVID-19 may include worsening physical and mental health, a lack of access to health and other services, and economic insecurities including financial, food, housing and employment insecurities. The potential for increasing health inequalities is significant. Once the initial pandemic is under control, attention must turn to how to support vulnerable communities to emerge from this crisis and ameliorate the detrimental impacts on health, wellbeing and economic security.</p><p>The recovery from the COVID-19 pandemic will require intelligence on the health, social and economic impacts on vulnerable populations to be made available quickly to key policy and decision makers so that they can develop and implement policies and interventions to mitigate against potential longer term impacts of the COVID-19 pandemic. As budgets will be limited, it is likely that implementation of ‘recovery’ strategies will need to be prioritised to those in greatest need.</p><p>In order to make decisions about which policies to implement and when, it is vital that decision makers have access to information not only on the likelihood and severity of potential impacts, but also on the receptiveness and capacity of communities to engage with and benefit from policy interventions. Lived experiences and in-depth qualitative research will be key to knowing how best to help those who are most affected and most in need.</p><p>The\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.borninbradford.nhs.uk/\">Born in Bradford (BiB) research programme</ext-link> is in a unique position to be able to study the impact of the COVID-19 response on a key vulnerable population: pregnant women and families with pre-school, primary and/or secondary school aged children living in a highly deprived and ethnically diverse city. BiB has been following the health and wellbeing of over 36,000 Bradford residents since 2007. It hosts three birth cohort studies\n<sup><xref rid=\"ref-3\" ref-type=\"bibr\">3</xref>–\n<xref rid=\"ref-5\" ref-type=\"bibr\">5</xref></sup> (see\n<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>) as well as an internationally recognised programme of applied health research with a focus on health inequalities in deprived and ethnic minority populations.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"anchor\"><label>Table 1. </label><caption><title>Description of Born in Bradford research infrastructure.</title></caption><table frame=\"hsides\" rules=\"groups\" content-type=\"article-table\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cohort</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Description</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Number</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Questionnaire data</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Routine Data\n<break/>(health and\n<break/>education)</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Recent data collection?\n<break/>(prior to March 2020)</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Born in Bradford\n<break/>Family Cohort\n<break/> Study\n<sup><xref rid=\"ref-3\" ref-type=\"bibr\">3</xref>,\n<xref rid=\"ref-6\" ref-type=\"bibr\">6</xref></sup>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">A prospective birth cohort which is\n<break/> tracking the health and wellbeing of\n<break/>over 13,500 children, and their parents,\n<break/> born at Bradford Royal Infirmary\n<break/> between March 2007 and January\n<break/> 2011. The health of these children is\n<break/>being tracked from pregnancy through\n<break/> childhood and into adult life. </td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">13,776\n<break/>Children\n<break/>\n<break/>12,453\n<break/>Mothers\n<break/>\n<break/>3,353\n<break/> Fathers</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yes- baseline and\n<break/> multiple time points\n<break/> on sub-samples from\n<break/> 6 months to 11 years</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yes – ‘Growing Up’ study\n<break/> follow-up collected\n<break/> between 2017–2020:\n<break/>N~5000 parents; N~7500\n<break/>children aged between\n<break/> 6–11\n<sup><xref ref-type=\"other\" rid=\"tf1\">a</xref></sup>.</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Born in Bradford’s\n<break/> Better Start\n<sup><xref rid=\"ref-4\" ref-type=\"bibr\">4</xref></sup>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Experimental birth cohort study in three\n<break/> deprived, multi-ethnic wards within\n<break/>Bradford. Currently recruiting.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Target\n<break/>N=5000\n<break/>Current\n<break/>N~2900\n<sup><xref ref-type=\"other\" rid=\"tf1\">b</xref></sup>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yes – baseline and\n<break/> one follow-up (to date).\n<break/> Others are planned. </td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yes –recruitment of\n<break/>pregnant women at routine\n<break/> pregnancy clinic (~26–28\n<break/> weeks gestation)\n<break/>\n<break/>Also follow up ~N=600\n<break/> collected Summer 2019\n<break/> (infants aged between\n<break/> 1–3 years)</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">BiB 4 All\n<sup><xref rid=\"ref-5\" ref-type=\"bibr\">5</xref></sup>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Birth cohort focusing on routine\n<break/> data linkage for research purposes.\n<break/>Currently recruiting.</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Current\n<break/>N~2000\n<sup><xref ref-type=\"other\" rid=\"tf1\">b</xref></sup>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">No</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Yes</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Routine information only</td></tr></tbody></table><table-wrap-foot><fn><p id=\"tf1\">Notes:\n<sup>a</sup> This planned follow-up had to be stopped at the start of lockdown and has not been able to restart yet;\n<sup>b</sup> Recruitment ongoing daily, figures rounded to nearest 100 as of 31st May 2020.</p></fn></table-wrap-foot></table-wrap><p>Participants in all BiB cohorts have consented to the use of their routine health and education data and to be contacted for future research. Recent recruitment\n<sup><xref rid=\"ref-4\" ref-type=\"bibr\">4</xref>,\n<xref rid=\"ref-5\" ref-type=\"bibr\">5</xref></sup> and follow-ups of our cohort participants\n<sup><xref rid=\"ref-6\" ref-type=\"bibr\">6</xref>,\n<xref rid=\"ref-7\" ref-type=\"bibr\">7</xref></sup> means that we have a detailed understanding of the physical and mental health, social, and economic circumstances of our families since index pregnancies/births, including data collected in the recent ‘pre-pandemic’ and ‘pre-lockdown’ period (2016-March 2020). The wealth of existing data can provide details on how life-course environmental, social and biological factors influence resilience and adverse responses to COVID-19 and its management. The recent pre-pandemic data can act as an “immediately pre-COVID baseline” to explore how the pandemic response will influence a range of outcomes in the short, medium and longer term. We also have the opportunity to follow our participants prospectively throughout the COVID-19 crisis to understand the impact of the crisis on health and well-being trajectories through this unpredictable time.</p><sec><title>Aim and objectives</title><p>Our aim is to rapidly collect key information across a range of BiB research infrastructure platforms to provide information in the short term to support policy and decision makers to deliver an effective COVID-19 urgent response in the City of Bradford, and nationally, and in the longer term to better understand the wider societal impacts of the COVID-19 response on health trajectories and inequalities in these.</p><p>Our objectives are to:</p><p>1. Work with stakeholders, communities and researchers to identify key issues of concern, research priorities, key topics and knowledge gaps to ensure our research addresses key issues to help plan the City’s recovery to COVID-19.</p><p>2. Collect quantitative information with BiB cohort participants to identify the health, social, education and economic impacts of the COVID-19 response for vulnerable families.</p><p>3.Collect qualitative data over time from cohort participants and other Bradford communities to explore in more detail issues related to the impact of COVID-19 response.</p><p>4. Feedback emerging findings to inform the local and national response, and adapt research methods as required in response to changing contexts and priorities.</p><p>We plan to address a range of research questions over the short term (6 months), medium term (6-12 months) and longer term (12 months onwards). We have provided illustrations of the type of research questions we will be able to answer in\n<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>, but in line with our adaptive methods, these may be modified or expanded dependent on community and stakeholder priorities, changes in the virus infection rate and response to this (including subsequent local or national epidemics and further local/national lockdowns) and the changing context as our research progresses.</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"anchor\"><label>Table 2. </label><caption><title>Illustrative research questions.</title></caption><table frame=\"hsides\" rules=\"groups\" content-type=\"article-table\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Time frame</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Research questions</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Shorter term\n<break/>(6 months post\n<break/> lock-down)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">   •  What behaviour changes are people making to their daily lives during the COVID-19 response, and how are they\n<break/> coping with these changes?\n<break/>   •  What is the impact of the COVID-19 response on families’ physical and mental health?\n<break/>   •  What is the impact of the COVID-19 response on families’ economic (e.g. financial, food, housing and\n<break/> employment) security.\n<break/>   •  What is the impact of the COVID-19 response on families’ access to key services (e.g. health, social care,\n<break/> education)\n<break/>   •  Are some groups of families (e.g. those living in deprived area, ethnic minority groups, key workers) at greater risk\n<break/> of experiencing short-term negative effects from the COVID-19 response? How might these negative effects be\n<break/> mitigated?\n<break/>   •  Are there any benefits of the COVID-19 response for different groups of families?</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Medium term\n<break/> (12 months post\n<break/> lock-down)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">   •  What is the impact of the Government’s response to the COVID-19 pandemic on the physical and mental health,\n<break/>wellbeing, economic security (financial, food and employment) on, and access to key services by, families living in\n<break/> Bradford?\n<break/>   •  Are some families (e.g. ethnic minority groups, deprived, those with insecure/low income jobs) at greater risk of\n<break/> negative impacts from the pandemic?\n<break/>   •  Are there protective factors (e.g. social support, job security) that make some families more resilient to the\n<break/> impacts?\n<break/>   •  What should the priorities of policy and decision makers be to reduce the impacts of the COVID-19 response on\n<break/> vulnerable families now, and in future epidemics?</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Longer term\n<break/> (2–3 years post\n<break/>lock-down)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">   •  What are the longer term impacts of the COVID-19 response on health, social, education and economic\n<break/> outcomes?\n<break/>   •  Are there inequalities in the longer-term recovery of families?</td></tr></tbody></table></table-wrap></sec></sec><sec sec-type=\"methods\"><title>Methods</title><sec><title>Setting</title><p>With a population of over 530,000, Bradford is the fifth largest metropolitan district in England\n<sup><xref rid=\"ref-8\" ref-type=\"bibr\">8</xref></sup>. It is an ethnically diverse and young city situated in the North of England. Almost half of the births in the city are to women of South Asian (mostly of Pakistani heritage) and there are an increasing number of families in the city from Central and Eastern European backgrounds\n<sup><xref rid=\"ref-4\" ref-type=\"bibr\">4</xref></sup>. Almost one-third of the city’s population is aged under 20\n<sup><xref rid=\"ref-8\" ref-type=\"bibr\">8</xref></sup>.</p><p>Bradford faces some important challenges, making its population vulnerable to the wider impacts of the COVID-19 and its management response. It has some of the highest levels of poverty and ill-health in England. It has an accelerating prevalence of diabetes and cardiovascular disease\n<sup><xref rid=\"ref-9\" ref-type=\"bibr\">9</xref></sup>, due in part to its large South Asian population who are most at risk of these diseases. Almost a quarter of Bradford children live in poverty and 24% are obese at age 10/11\n<sup><xref rid=\"ref-10\" ref-type=\"bibr\">10</xref></sup>. There are specific structural characteristics in Bradford that make the community especially vulnerable to COVID-19, for example, a large proportion of households are classed as overcrowded\n<sup><xref rid=\"ref-11\" ref-type=\"bibr\">11</xref></sup>. </p><p>In England during emergencies, multi-agency groups, comprising of senior officers from organisations such as the emergency services, local authorities, NHS and community and voluntary sectors come together to co-ordinate the immediate response to and recovery from an emergency\n<sup><xref rid=\"ref-12\" ref-type=\"bibr\">12</xref></sup>. In Bradford District, a Bradford District Gold group was established in response to the COVID-19 emergency following Government guidance. This is a group of senior officers from organisations across the District including emergency services, Local Authority, NHS, and community and voluntary sector which is coordinating the District response and recovery to COVID-19.</p><p>To support Bradford District Gold a\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.bradfordresearch.nhs.uk/c-sag/\">COVID-19 Scientific Advisory Group (C-SAG)</ext-link> has been established to harness the research expertise and infrastructure of Bradford Institute for Health Research (BIHR) (including Born in Bradford), NHS and Local Authority partners to support Bradford District Gold. Bradford District C-SAG operates in two forms, bringing together health and business intelligence, commissioning, public health, policy and health research expertise in a multi-agency C-SAG and researcher expertise, including from Born in Bradford, in a BIHR C-SAG.</p><p>The C-SAG also benefits from the recently formed UK Prevention Research Partnership ActEarly consortium\n<sup><xref rid=\"ref-13\" ref-type=\"bibr\">13</xref></sup>. Working in close partnership with Born in Bradford, ActEarly focusses on early life changes to improve the health and opportunities for children living in two contrasting areas with high levels of child poverty; Bradford, West Yorkshire and Tower Hamlets, London. In each of these areas, ActEarly is working with local communities, local authorities and other national organisations to understand how we can help families’ live healthier lives, with a particular focus on delivering system level change. Crucially, the consortium formally brings together decision makers across health and education with researchers and communities. This existing forum provides a platform for early implementation of research findings and recommendations into practice.</p><p>Both the BiB research programme and ActEarly use their findings to develop new and practical ways to work with families and health professionals to improve the health and wellbeing of vulnerable populations. We work in close partnership with city, regional and national policy and decision makers in health, education, environment and social care. Together, we are a ‘people powered’ research programme using engagement, co-production and dissemination to ensure communities and stakeholders have a major voice in determining our research priorities.</p></sec><sec><title>Study design</title><p>In order to achieve our aims and objectives we plan, and have started, an adaptive longitudinal approach using a mixed methods convergent triangulation design. The study comprises four inter-linked work packages (WP) running in parallel, including: WP1) ongoing community consultations and co-production with key stakeholders (communities, community and voluntary sector organisations, decision makers, health and education professionals) and existing BiB research community groups; WP2) repeated longitudinal quantitative data collection with BiB families enrolled in our key birth cohorts (see\n<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>); WP3) detailed longitudinal qualitative data collection with population sub-groups; and WP4) a feedback loop to ‘flex’ future research priorities according to community and stakeholder priorities.</p><p>The Bradford District Gold group and national bodies (e.g. Department for Education, Public Health England, Royal College of Midwifery) will have a direct influence in setting our research focus, interpreting and disseminating findings (See\n<xref ref-type=\"fig\" rid=\"f1\">Figure 1</xref>).</p><fig fig-type=\"figure\" id=\"f1\" orientation=\"portrait\" position=\"anchor\"><label>Figure 1. </label><caption><title>Overview of planned adaptive research methodology.</title></caption><graphic xlink:href=\"wellcomeopenres-5-17704-g0000\"/></fig></sec><sec><title>Objective 1/work-package 1: co-production and engagement</title><p>Co-production of research priorities with communities and decision makers underpins the entirety of our adaptive research activities. Our approach to co-production is based on our Act Early ‘city collaboratory’ approach\n<sup><xref rid=\"ref-13\" ref-type=\"bibr\">13</xref></sup>, and will be used in the short term to identify key research priorities and knowledge gaps, and in the longer term to co-produce interventions and initiatives to mitigate poor outcomes and health inequalities. In order to develop acceptable and feasible initiatives that have the best chance of success, we need to ensure equality and engagement of communities, stakeholders/decision makers and researchers.</p><p>Genuine co-production is predicated on reciprocal and trusting relationships between communities and other key stakeholders, ongoing dialogue, joint ownership of decision making, sharing of power and continuous reflection. It is therefore not a short term process, but one that takes time to build. As such, it is impossible to outline exactly how the co-production process will work at the outset of a programme of work. We will convene a multi-disciplinary community led steering group (including citizens, community and voluntary sector organisations, and service providers) to help us set our initial research priorities and design later stages of our research programme.</p><p>Throughout the research programme we will also seek to use varied methods of engagement and consultation (also termed ‘soft intelligence gathering’) to collect views and lived experiences of key community groups and seldom-heard communities to ensure a broad range of community perspectives are taken into consideration in the identification of research priorities and co-production activities. Our community engagement research team (SI, AR) are experienced researchers and Bradford residents who have spent many years developing genuine and trusted relationships with local community and voluntary sector organisations. We will use these links to create a direct channel of communication to discuss emerging issues, concerns and community priorities using a range of communication platforms (e.g. email, phone, text, Twitter, Facebook, WhatsApp, local media). We illustrate one example of this approach in relation to identifying immediate community concerns related to the COVID-19 lockdown in\n<xref ref-type=\"other\" rid=\"B1\">Box 1.</xref>\n</p><p>\n<boxed-text content-type=\"website\" id=\"B1\" position=\"float\" orientation=\"portrait\"><caption><title>Box 1. Engagement in Action – Soft intelligence gathering to explore issues experienced as a result of the lock down in key vulnerable or seldom heard communities in Bradford</title></caption><p>\n<bold>Methods:</bold> Informal telephone interviews with 13 key community leaders (for example, religious leaders, voluntary sector organisations, local councillors) covering a range of deprived communities within Bradford. Interviews focused on what main issues arising from ‘lock-down’ restrictions will be in short, medium and longer term. Key community groups represented included White British, South Asian, Eastern European Roma community, and Refugee and Asylum seekers. We were interested in exploring differences amongst communities</p><p>\n<bold>Headline findings:</bold>\n</p><p>\n<underline>Lock-down rules and accessing information:</underline>\n</p><p>→ Families living in multi-generational households find it difficult to stick to social distancing rules.</p><p>→ Awareness of rules for social distancing amongst some Eastern European Roma groups is low, in part due to low literacy levels.</p><p>→ Hoaxes and fake news regarding COVID-19 are spreading via social media channels which are causing anxiety and worry, particularly amongst South Asian Families.</p><p>\n<underline>Exacerbation of existing financial insecurity and poverty:</underline>\n</p><p>→ There were concerns across all groups of the impact of reduction in income, particularly amongst self-employed and small businesses. People reported problems in accessing financial support packages from the government.</p><p>→ Many people in Eastern European and Roma communities have ‘cash-in-hand’ jobs or agency work, and are not eligible for benefits. They may fall through the cracks in terms of receiving support</p><p>→ Food poverty was an issue particularly for larger families. For other families who need to access foodbanks, ‘essential’ items (e.g. sanitary products, soap, toothpaste) were not always available in food parcels. Families were not always able to access free school meals for children.</p><p>\n<underline>Accessing services, including those tackling food insecurity:</underline>\n</p><p>→ Families not using services which may be available (e.g. food banks), due to stigma, and / or difficulties of referral system</p><p>→ There is reduced capacity of voluntary and community sector organisations to deliver services as many are reliant on volunteers who are now not able to help due to lock-down restrictions.</p><p>\n<underline>Mental health</underline>\n</p><p>→ The impact of the COVID-19 pandemic on mental health was felt to be an important issue both in the short-term and longer term.</p><p>→ This impact is caused directly by worry and anxiety about the virus, and also indirectly by impact on financial security.</p><p>→ Face to face access to organizations for support with welfare and housing has been curtailed and this is posing a particular problem for refugee and asylum seekers.</p><p>→ Loss and grief of loved ones and friends has had an impact too as lockdown has interrupted the usual grieving process of attending funeral/burials and the mourning period.</p><p>\n<underline>Home and learning environment</underline>\n</p><p>→ For families with children, parents are struggling to access learning materials, particularly on line (which is affecting children’s education) and struggling to keep children occupied.</p><p>→ Many families do not have reliable internet access or are not able to keep a phone in credit.</p><p>\n<underline>Addiction</underline>\n</p><p>→ Individuals who have problems with addiction, who may have previously resorted to criminal means to pay for their addiction via shop-lifting or other petty crimes and can no longer do so, may turn to more extreme methods if not given help.</p><p>\n<bold>How these findings have been used:</bold> Key findings have been fed back to District Gold and are being used to inform the next phase of living with COVID-19 and laying the foundations for scenario planning for a better future for the District\n<sup><xref rid=\"ref-14\" ref-type=\"bibr\">14</xref></sup>. They have been used to develop survey instruments and more detailed qualitative protocols to explore some of these issues in more detail (see below). We have also shared findings with local voluntary sector organisations who have reported quickly flexing provision of services in response to key issues, for example, provision of laptops for children of families in greatest need to assist education at home, and provision of ‘essential’ sanitary and hygiene products in food parcels. We have also shared findings back to participants. One participant shared the following comment:\n<italic>“Thank you for getting in touch we were feeling that our needs were getting ignored until you gave us a voice. We will be happy to help again”</italic>\n</p><p>See\n<xref rid=\"ref-15\" ref-type=\"bibr\">15</xref> for a full copy of the report.</p></boxed-text>\n</p><p>We will harness our established research advisory groups including BiB Parent Governors (BiB Parents with children aged 9–13), BiB Young Ambassadors (BiB Children aged 9–13) and our Community Research Advisory Group (BiBBS parents with children aged 0–5). We will also use emerging findings from our quantitative research arm. For example, free text questions embedded within our large-scale quantitative surveys will ask communities about their key worries or concerns, allowing us to collect a representative sense of feeling amongst Bradford families in a way not possible by closed response questions.</p><p>Stakeholder/decision maker views and priorities will be shared via a parallel multi-agency COVID-19 scientific advisory group (including Local Authority, NHS Commissioner and Provider representatives, chaired by CC) and the Bradford District Gold (of which JW is a member). When necessary, direct input by key members will be arranged. Finally, research priorities will be shared via the BIHR C-SAG group (described above).</p><p>Our community steering group will consider collective views and priorities from all groups and together with the research team will use these to shape the direction and content of future research plans, including both quantitative and qualitative research elements. In this way we will ensure that we reflect local community needs, and provide information to decision makers than can be acted upon. Our experience to date has shown that whilst many priorities may be similar across the diverse communities within Bradford, stakeholders, and researchers, there are certain unique issues which are particularly pertinent to seldom heard or under-served communities. Without systematic exploration in a targeted way to understand the nuances in views and differences in response to the COVID-19 lockdown presented by diverse groups, there is a risk that these points may not get the consideration they deserve at decision making forums, which can potentially further widen health inequalities.</p><p>We will work with our communities, stakeholders and decision makers to jointly interpret our emerging findings, and ensure that they are disseminated in meaningful and sensitive ways. Researchers will discuss findings with members of Bradford District Gold, the multi-agency C-SAG and citizens to provide additional perspectives to support interpretation and to collectively identify final recommendations for local action in conjunction with our community steering group. Our aspiration in the longer term is to support Bradford District Gold in the development and evaluation of initiatives and interventions to mitigate against worsening outcomes and inequalities with subsequent waves and repeat epidemics, and to aid the city recovery from COVID-19 by ensuring genuine co-production with communities and stakeholders.</p></sec><sec><title>Objective 2/work-package 2: quantitative surveys of the impact of covid-19 response</title><p>The main aim of this quantitative arm of our adaptive research protocol is to understand the wider impact of the COVID-19 Government response on vulnerable families using the Born in Bradford birth cohort research infrastructure (BiB, BiBBS, BiB4All, see\n<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><p>\n<bold><italic>Population.</italic></bold> Our sample will be drawn from the participants in our existing birth cohorts who have engaged in recent data collection to enable us to build on immediate pre-COVID-19 data (as well as other existing data from index pregnancies/birth): BiB Growing Up (BiBGU, follow-up data collection wave), data collected 2017–2020, Born in Bradford’s Better Start (BiBBS) 2016–2020 and the Born in Bradford routine data cohort (BiB4All) 2018–2020, only routine health data available for baseline). All participants in these three studies will be invited to take part in one of three key surveys:</p><list list-type=\"bullet\"><list-item><p>Sample 1: Parents – Parents of children aged 9–13 years in BiBGU and parents of children aged 0–5 years in BiBBS (total sample pool: N=7,652)</p></list-item><list-item><p>Sample 2: Children - Children of parents in the above BiBGU sample aged 9–13 years (N=5,154)</p></list-item><list-item><p>Sample 3: Perinatal -Women in the perinatal period (pregnancy and up to 12 months post-partum) in BiBBS and BiB4All (N=1,800)</p></list-item></list><p>For samples 1 and 2, surveys are planned at four time points over a one-year period with an immediate lockdown survey (April-June 2020) recently completed, and follow-up in September 2020, January 2021 and April 2021 planned.</p><p>For sample 3, pregnant women will be recruited throughout a 12 month period (June 2020-May 2021) with ongoing follow-up planned at 3, 6 and 9 months post-partum. In June-July 2020 a sub-sample of women who gave birth during the lockdown period (April-June 2020) will be recruited at the 3 months post-partum time point and followed up at 6 and 9 months post-partum.</p><p>The exact timing of the follow-up data collection periods will be flexed in response to the changing COVID-19 situation and research priorities emerging from WP1.</p><p>\n<bold><italic>Eligibility criteria</italic></bold>\n</p><p>Inclusion:</p><list list-type=\"bullet\"><list-item><p>Participant has been recruited to one of the above cohort studies and has consented to be followed-up for future research.</p></list-item><list-item><p> For data collection via phone calls: participant is able to speak English or language also spoken by some members of the research team (e.g. Urdu/Mirpuri; Punjabi; Hungarian; Romanian). </p></list-item></list><p>Exclusion:</p><p>Any participant who has:</p><list list-type=\"bullet\"><list-item><p>Withdrawn from the study;</p></list-item><list-item><p>Moved out of the Bradford District area;</p></list-item><list-item><p>Miscarried (BiBBS/BiB4All), had a still birth (BiBBS/BiB4All), or child death (BiBGU & BiBBS & BiB4All) recorded.</p></list-item></list><p>\n<bold><italic>Mode of survey delivery.</italic></bold> The survey for samples 1 and 3 will be completed primarily by phone with options for online and postal completion. Where participants are non-responsive by phone or email, postal questionnaires will be sent out with a stamped address envelope. Follow-ups will be conducted where postal questionnaires have not been returned within a reasonable timeframe (1-2 weeks). The survey for sample 2 will be completed by postal questionnaire addressed to the child’s parent.</p><p>\n<bold><italic>Questionnaire domains.</italic></bold> Key questionnaire domains for each of the surveys used in round 1 of data collection are summarised in\n<xref rid=\"T3\" ref-type=\"table\">Table 3</xref> below. The selected domains focus on capturing a wide range of potential impacts of the COVID-19 response across physical and mental health, living circumstances and economic, food and housing insecurity. In order to further contribute to the priority setting, open-ended questions in the survey asks about the participants’ main worries, challenges and any positive experiences as a result of the COVID-19 lockdown. Copies of the questionnaires are available on\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.bradfordresearch.nhs.uk/findings-and-resources/\">our website</ext-link> and as\n<italic>Extended data</italic>\n<sup><xref rid=\"ref-18\" ref-type=\"bibr\">18</xref></sup>. We envisage that core content of the questionnaires will be repeated in each follow-up wave of data collection, but part of the nature of our adaptive research protocol is that we will be ready to ‘flex’ future waves of data collection to support collection of data on key topics identified by the first round of the survey, qualitative work and our co-production and engagement work-stream (including consideration of Bradford District Gold) as ‘priority’ areas.</p><table-wrap id=\"T3\" orientation=\"portrait\" position=\"anchor\"><label>Table 3. </label><caption><title>Key questionnaire domains.</title></caption><table frame=\"hsides\" rules=\"groups\" content-type=\"article-table\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Domain</th><th align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Parent\n<break/>Sample</th><th align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Perinatal\n<break/>Sample</th><th align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Child\n<break/> Sample</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Key demographics (e.g. age, ethnicity, index of multiple deprivation,\n<break/> socio-economic status, employment)</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Household composition (e.g. household member clinically vulnerable\n<break/> to COVID-19; relationship status; housing tenure)</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Housing quality and access to outdoor space</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Insecurity of employment, finances, home & food</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Physical health (including general health, health anxieties, health\n<break/> behaviours and whether self-isolated due to COVID-19)</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Mental health (including depression [PHQ-8\n<sup><xref rid=\"ref-16\" ref-type=\"bibr\">16</xref></sup> and anxiety GAD-7]\n<sup><xref rid=\"ref-17\" ref-type=\"bibr\">17</xref></sup>)</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Family Relationships and Social Support </td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Peer support and bullying </td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Parenting competence</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Child behaviour</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Loneliness & social support</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Access and use of key services</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Physical activity</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Main worries (recorded as free text)</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Pregnancy related health and stress</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Pregnancy/baby related worries and concerns and changes to\n<break/> perinatal care</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Birth plans and breastfeeding intentions</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Experiences of perinatal services </td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Take up of baby immunisations</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">The mother-child relationship (attachment)</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x\n<xref ref-type=\"other\" rid=\"tf2\">*</xref>\n</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Breastfeeding </td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x\n<xref ref-type=\"other\" rid=\"tf2\">*</xref>\n</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Social support and contact with baby groups / other new mums.</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\">x\n<xref ref-type=\"other\" rid=\"tf2\">*</xref>\n</td><td align=\"right\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><fn><p id=\"tf2\">Notes: *postpartum survey only.</p></fn></table-wrap-foot></table-wrap><p>\n<bold><italic>Analysis.</italic></bold> Descriptive statistical analysis will be used to assess health, wellbeing, economic and social outcomes. Multivariable regression analyses will be used to model change from pre-COVID-19 baseline. Longitudinal trajectories of outcomes and their predictors across all survey waves will be estimated using appropriate marginal and mixed methods. All statistical analysis will be carried out using Stata 15\n<sup><xref rid=\"ref-19\" ref-type=\"bibr\">19</xref></sup>.</p><p>Free text questions on worries, challenges and positive experiences will be analysed using thematic analyses\n<sup><xref rid=\"ref-20\" ref-type=\"bibr\">20</xref></sup>, employing an inductive approach where coding and theme development will be driven by the content of the responses. Codebooks will be created by a single researcher (BL) and tested by a group of researchers to test the strength and validity. Adjustments will be made, through discussion of the researchers, throughout analysis to ensure that the codebooks are reflective of the all responses.</p></sec><sec><title>Objective 3/work-package 3: longitudinal qualitative methods</title><p>The main aim of the qualitative arm of this adaptive research proposal is to gain a deeper understanding of the impact of COVID-19 and the COVID-19 Government response on families in Bradford on key priority topic areas, using the Born in Bradford infrastructure as a starting point. In the medium term, the initial results and analysis of this research will support the District Gold in delivering an effective response to those families most in need. In the longer term, this information will allow a better understanding of the wider societal impacts of COVID-19 that will allow local services to prioritise their recovery of services, identify additional interventions, and inform local policy to improve resilience.</p><p>The content and focus of the initial qualitative priorities have been developed in partnership with communities and stakeholders using methods outlined in objective one. The soft intelligence gathered from communities (see\n<xref ref-type=\"other\" rid=\"B1\">Box 1</xref>) was supplemented with other sources of information:</p><list list-type=\"simple\"><list-item><label>a) </label><p>Analysis of the free text responses from the first 350 parents in sample 1 of the survey on their main worries, challenges and positive experiences. In this analysis we found there was a lot of health anxiety around catching COVID-19, concerns about finances and job uncertainty, increased mental load, concerns about children’s mental health and their education as well as practical concerns such as food shopping. The analysis also found that families were enjoying spending more time together and enjoying a slower pace of life.</p></list-item><list-item><label>b) </label><p>Soft intelligence gathering with members of District Gold. Brief 15–20 minute phone calls with nine members of Bradford’s District Gold to assess what their priorities were for qualitative research in Bradford in response to COVID-19. Short interviews were conducted by BL in April 2020. We first asked what they thought about the three priorities identified from our very early free text analysis of worries and concerns from our parent survey: family food security, children’s education (with a focus on children with special educational needs and disability[SEND]) and access to and experience of public/voluntary services. We then asked what would be their choice of three priorities and how they envisaged the qualitative work helping and informing their work at District Gold. These responses were recorded in note form and written up by BL. A rapid thematic analysis of the responses was conducted by BL and LS. Priorities identified were around health inequalities, poverty, domestic violence, child mental health, ethnic minority communities’ experiences and the people of Bradford’s relationship to health services as a result of COVID-19 (due to an apparent increase in mistrust and misinformation).</p></list-item><list-item><label>c) </label><p>Researchers within the BIHR C-SAG identified pregnancy and the post-partum period as a potentially challenging experience during the COVID-19 pandemic. Pregnant women were identified as a group vulnerable to COVID-19 which had increased health anxieties, alongside reduced access to face to face healthcare and reduced social support due to social distancing and restricted hospital visiting.</p></list-item></list><p>The information on priorities received from all sources was collated and reviewed by the C-SAG group to identify the following initial priority research areas: 1) adolescent mental health, 2) health beliefs, 3) pregnancy, birth and the postnatal period, 4) impact on those families already experiencing high levels of poverty and financial insecurity.</p><p>The C-SAG agreed that detailed, longitudinal qualitative research would be particularly valuable for priorities 1 to 3 at this time. Whilst the C-SAG acknowledged the clear importance of poverty and financial insecurity, the group was aware of a\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.nuffieldfoundation.org/project/poverty-covid-19-families-low-income\">recently funded mixed methods</ext-link> study exploring poverty in Bradford in the context of larger families which was being repurposed to address responses to the COVID-19 pandemic. For these reasons it was decided not to burden communities by instigating separate research on this priority area at this time.</p><p>Below we provide more detail on the three priority areas taken forward in the first rounds of qualitative data collection. Future topics for qualitative exploration will guided by our more formal co-production processes and our community led multi-disciplinary steering group. </p><p>\n<bold><italic>Priority 1: Children’s mental wellbeing.</italic></bold> The first of the selected priorities is children’s mental wellbeing under lockdown. Our consultations suggested that there was particular concern about secondary school age children, including the impact of social isolation, boredom, low mood and anxiety. Our definition of ‘mental wellbeing’ is broad and will be iterated as fieldwork progresses. It is important to state that we are not intending to focus the study on participants with a clinical assessment of depression or anxiety. That is, we are interested in understanding mental wellbeing concerns in the widest sense.</p><p>We are planning to conduct interviews with 20 families who have participated in both the adult and child COVID-19 surveys. The sample will be made up of two groups, the first will include children who reported moderate to low mental wellbeing in their survey and parents who raised concerns about their child(ren)’s mental wellbeing in the parent survey, and the second will include children who reported medium to high mental wellbeing in their survey and parents who did not raise concerns about their child(ren)’s mental wellbeing. We have chosen 20 in the first instance as we think this will ensure we can undertake data collection and analysis within a limited time frame whilst enabling us to obtain a diverse sample of BiB families (in terms of ethnicity, location, socio-economic background). As we are focusing on secondary school age children, these families will be from the BiBGU cohort, as the oldest children in this cohort are now aged 13 years old. BL (a post-doctoral Research Fellow with expertise in qualitative methods) will conduct two short interviews via phone/video with each family, one with a parent and the other with the child (accompanied by a parent, sibling or by themselves, whichever they prefer). The interview will focus on the child’s day-to-day life under lockdown and how they have been feeling. There will be the opportunity to do follow-up interviews post lockdown.</p><p>\n<bold><italic>Priority 2: Health beliefs.</italic></bold> A priority that came through strongly in our consultation with Bradford Gold and communities was around people’s relationship to health services during the pandemic. This is a broad topic which covers changes in access to health services (and the factors affecting this), misinformation about COVID-19 spreading (especially hoax health information via WhatsApp), patients being scared to attend hospital, mistrust of health services currently, the heavy impact of COVID-19 on ethnic minority communities and bereavement/grief.</p><p>For this work, we will be sampling a range of communities in Bradford but with a particular focus on the South Asian population who seem to be more adversely affected by the above. We will conduct interviews with community leaders/trusted individuals embedded within specific communities as a starting point, then using theoretically driven snowball sampling to focus on the most affected groups. We estimate that we will conduct around 15–25 interviews but this will be determined by our assessment of data saturation. The interviews will mainly be conducted by BL except for interviews in Punjabi/Urdu where they will be carried out by other experienced qualitative researchers with these language skills.</p><p>\n<bold><italic>Priority 3: Pregnancy, birth and the post-natal period.</italic></bold> This priority area will explore how the COVID-19 situation has impacted on women and partner’s experiences of pregnancy, birth and the postnatal period. A longitudinal method will be employed with interviews conducted by experienced qualitative research fellows during pregnancy, 3, 6 and 9 months post-partum. Interviews will be semi-structured with women and their partners being asked to talk about the issues that have been most important to them and/or that they are most concerned about. If they are not covered by the participant, questions and prompts that relate to the domains in the quantitative survey will be asked (see\n<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). These will be used flexibly to fit with the flow of the interview. A sub-sample of 20–30 women participating in the quantitative COVID-19 pregnancy survey will be recruited. Purposive sampling will ensure a balance of women from Pakistani and White British backgrounds and a balance from the BiBBS and BiB4All birth cohorts. In addition, 10–15 partners of the women participants will be recruited.</p><p>\n<bold><italic>Analysis.</italic></bold> For priorities 1 and 2, the process of analysis will be on-going throughout recruitment and interviews and will be recorded in a research diary. The interview transcriptions will be coded manually by the lead researchers (BL, LS) independently of each other at first to ensure validity. They will take a thematic, inductive approach which will involve multiple readings of the transcripts and exploration of participant’s meanings, keeping in mind the research objectives and the survey findings. The researchers will then come together to compare and discuss emergent findings and resolve any differences through consensus. The codes that are developed will be categorised under preliminary themes, which will be ordered into themes and sub-themes to produce findings.</p><p>For priority 3, given the longitudinal nature of the study, and the focus on changes in experiences over time, the ‘pen portrait’ approach\n<sup><xref rid=\"ref-21\" ref-type=\"bibr\">21</xref></sup> will be used to analyse data gathered from each woman and partner. Further inductive thematic analysis across the pen portraits will allow the researchers to compare and contrast findings from men and women and across other sampling criteria. Analysis will be led by a researcher, with regular discussion and interpretation of emerging themes within a multi-disciplinary team comprising experts in maternal and child health as well as qualitative methodology.</p></sec><sec><title>Objective 4/work-package 4: feedback and flex the research programme to key topic areas</title><p>Given the many uncertainties associated with the COVID-19 pandemic it is essential that our research remains responsive, evolving and flexible to continue to meet new priority research objectives and provide a meaningful contribution to the District and wider national response within the research framework. It is likely priority topic areas will continue to emerge, and change over the duration of the research. Our co-production and engagement work (work-package 1) will ensure that we continue to work with communities and stakeholders to identify topics that are most meaningful. Also, merging quantitative and qualitative findings, continued engagement and soft intelligence gathering will all shape future research activities, for example the content of follow-up quantitative surveys, and future qualitative work. Engagement with a broad range of stakeholders will ensure that a balance between immediate, medium and longer term concerns and research priorities is maintained.</p></sec><sec><title>Dissemination plans</title><p>We will use a range of tailored strategies to maximise dissemination and accessibility of our findings. A key priority is to provide rapid evidence to decision makers. Locally, we have well established channels for communication (see also\n<italic>Setting</italic>). We will produce rapid briefing notes and reports based on emerging findings, which will be regularly updated. These will be distributed via Bradford District Gold and multi-agency C-SAG groups. Where briefing notes do not contain sensitive information or breach confidentiality they will be published on our\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.bradfordresearch.nhs.uk/findings-and-resources/\">C-SAG resources page</ext-link>. Further afield, we are already engaging with key stakeholders nationally (including Public Health England, Department for Education and Schools, Association of Directors of Public Health) and regionally (West Yorkshire Health and Care Partnership, and Yorkshire and Humber Applied Research Collaboration). Internationally, we will engage with the International Network for Research on Inequalities and Child Health, the International Society for Social Pediatrics, and Unicef, to feed into international comparative research, dissemination and policy-making.</p><p>For communities we will build on our established communication platforms, including regular newsletters with BiB families, social media channels (Facebook, Twitter and YouTube). We will create ‘in a nutshell’ findings and post these on our\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://borninbradford.nhs.uk/our-findings/\">Born in Bradford website</ext-link>.</p><p>For academic audiences we will publish our findings in open access, peer reviewed journals.</p></sec><sec><title>Ethical statement</title><p>The work described in work-package two has already been approved by the Health Research Authority and Bradford/Leeds research ethics committee (BiB Growing Up study 16/YH/0320; BiBBS study 15/YH/0455; BiB4All study 17/YH/0202). Ethical approval for work carried out in work-package three has been approved as an amendment to the BiB Growing Up study for children’s mental wellbeing, and as an amendment to BiBBS and BiB4All for the pregnancy, birth and postnatal period. Ethical approval for the health beliefs qualitative study has been submitted to the University of York Health Sciences ethics committee.</p><p>All participants will be provided with information about the study and contact details for the research team. Verbal consent will be taken for questionnaires completed over the phone. For online or postal questionnaire participants will informed that by completing the questionnaire they are providing consent to participate (referred to as implicit consent). This approach has been approved by our local ethics committee. Child questionnaires are sent to the parent of the child, return of the questionnaire will be taken as implicit consent by the parents. Respondents will be reassured that they do not need to answer any questions that they do not wish to and will be free to stop the survey at any time. For qualitative interviews, information sheets will be given to the participants, and verbal consent will be audio- recorded at the start of the interview, no data will be collected unless consent is recorded. For interviews with children, verbal consent from both them and their parent will be recorded. Due to social distancing requirements, all interviews will be done by phone or video.</p><p>It is possible that questions in the survey and in the interviews may cover sensitive and/or upsetting issues. All participants will be provided with a ‘useful contacts’ sheet including signposting to services able to give support for mental health, domestic violence, child abuse and education needs. It is also possible that these studies may uncover safeguarding concerns of the participant or their family. It is made clear in the information provided that confidentiality may be broken if the researcher is concerned about the safety of the participant or their family. In such cases the researcher follows the safeguarding policy of Bradford Teaching Hospitals NHS Foundation Trust.</p></sec><sec><title>Study governance</title><p>A COVID-19 Scientific Advisory Group has been convened for the Bradford Institute for Health Research, which hosts Born in Bradford. This work will be overseen by this group, and by the BiB Executive Committee. In parallel we will regularly report progress to, and receive overview and scrutiny of our plans from, our existing established community research advisory groups.</p></sec><sec><title>Data security and sharing</title><p>Quantitative data: All collected data will be pseudonymised and will be linked to existing research data for each participant. Data will be stored on secure NHS computer drives and in compliance with all data laws. This data will be added to the BiB and BiBBS data resources and shared with researchers anonymously as per\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://borninbradford.nhs.uk/research/how-to-access-data/\">existing procedures</ext-link>.</p><p>Qualitative data: Interviews will be audio-recorded and transcribed by an organisation with a privacy agreement in place. Names of interviewees and any other names mentioned within the interviews will be pseudonymised and other identifying information will be removed. Pseudoymised interview data will be stored on a secure server at Bradford Teaching Hospitals for research purposes and may be accessed as per existing procedures to access BiB data.</p></sec><sec><title>Study status</title><p>The study is currently ongoing (commenced April 2020) and data collection is due to complete by May 2021.</p></sec></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>We outline an adaptive research protocol harnessing the power of the Born in Bradford research infrastructure to provide rapid intelligence on the impact of restrictions imposed to limit the spread of COVID-19 in a city with high numbers of vulnerable, deprived multi-ethnic families. By definition, whilst earlier stages of the research programme are well-specified, later stages will be developed in close partnership with communities and decision makers using emerging findings and responding to priority topic areas in real time. This type of research relies on building trusting, and genuine partnerships between researchers, communities and decision-makers. We have spent many years in Bradford developing these close relationships and can now use our ‘City of Research’ infrastructure to help inform local and national recovery from the COVID-19 pandemic.</p></sec><sec sec-type=\"data-availability\"><title>Data availability</title><sec><title>Underlying data</title><p>No underlying data are associated with this article.</p></sec><sec><title>Extended data</title><p>Harvard Dataverse: Extended data for BiB COVID19 Study Protocol:\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://doi.org/10.7910/DVN/UQ3KDF\">https://doi.org/10.7910/DVN/UQ3KDF</ext-link>\n<sup><xref rid=\"ref-18\" ref-type=\"bibr\">18</xref></sup>.</p><p>This project contains the following extended data:</p><list list-type=\"bullet\"><list-item><p>WP2 BiB covid 19_invitation letter_v1 31.03.2020.pdf (Covering letter for BiB COVID-19 questionnaires)</p></list-item><list-item><p>WP2 BiB-Child-Questionnaire-Round-1-May-2020.pdf (BiB COVID-19 Children's questionnaire administered during lock-down, May-June 2020)</p></list-item><list-item><p>WP2 BiB-Covid-19-pregnancy-questionnaire_postal_v1.pdf (BiB COVID-19 Pregnancy questionnaire administered June 2020 onwards)</p></list-item><list-item><p>WP2 BiB_BiBBS COVID-19 FamilyQuestionnaire_v1.pdf (BiB COVID-19 Family questionnaire administered during lock-down, April - June 2020)</p></list-item><list-item><p>WP2 Telephone script for questionniares.pdf (Introductory telephone script for questionnaires completed over the phone)</p></list-item><list-item><p>WP3 BiB Covid19_Parent Interview Guide_Child_wellbeing_V2.0.pdf (Parent interview topic guide for children's well-being qualitative interviews, work-package 3)</p></list-item><list-item><p>WP3 Child information sheet_Child_wellbeing.pdf (Child information sheet and consent script for child well-being qualitative interviews, work-package 3)</p></list-item><list-item><p>WP3 Parent information sheet_Child_wellbeing.pdf (Parent information sheet and consent script for child well-being qualitative interviews, work-package 3)</p></list-item><list-item><p>WP3 Child Interview Guide_Child_wellbeing_V2.0.pdf (Child interview topic guide for child well-being qualitative interviews, work-package 3)</p></list-item><list-item><p>WP3 COVID 19 Pregnancy Interview Info Sheet (Partners) v3 03.06.20.pdf (Pregnancy qualitative study: Information sheet for partners, work-package 3)</p></list-item><list-item><p>WP3 COVID 19 Pregnancy Interview Info Sheet Mothers v3 03.06.20.pdf (Pregnancy qualitative study: Information sheet for Mothers, work-package 3)</p></list-item><list-item><p>WP3 Pregnancy Partners_topic guide_covid study_V1.0.pdf (Pregnancy qualitative study: Interview topic guide for partners, work-package 3)</p></list-item><list-item><p>WP3 Pregnancy Women_topic guide_covid study_V1.0.pdf (Pregnancy qualitative study: Interview topic guide for mothers (work-package 3)</p></list-item><list-item><p>WP3 Health Beliefs Info Sheet and Consent Form.pdf (Information sheet and consent form for health belief qualitative interviews, work-package 3)</p></list-item><list-item><p>WP3 Health Beliefs Topic Guide.pdf (Interview topic guide for health belief qualitative interviews, work-package 3)</p></list-item></list><p>Data are available under the terms of the\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/publicdomain/zero/1.0/\">Creative Commons Zero \"No rights reserved\" data waiver</ext-link> (CC0 1.0 Public domain dedication).</p></sec></sec></body><back><ack><title>Acknowledgments</title><p>Born in Bradford is only possible because of the enthusiasm and commitment of the children and parents in BiB. We are grateful to all the participants, health professionals and researchers who have made Born in Bradford happen.</p><p>We acknowledge the input of the wider BIHR COVID-19 Scientific Advisory Group in the preparation of this protocol which includes (in addition to the named authors): Abigail Dutton, Bo Hou, Brian Kelly, Tom Lawton, Dan Mason, Michael McCooe, Mark Mon-Williams, Gill Santorelli, Najma Siddiqi, Kuldeep Sohal, Kathryn Willan.</p><p>This report is independent research partly funded by the National Institute for Health Research Yorkshire and Humber ARC. The views expressed in this publication are those of the authors and not necessarily those of the National Institute for Health Research or the Department of Health and Social Care. 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technique.</article-title>\n<source><italic toggle=\"yes\">BMC Med Res Methodol.</italic></source>\n<year>2019</year>;<volume>19</volume>(<issue>1</issue>):<fpage>169</fpage>.\n<pub-id pub-id-type=\"doi\">10.1186/s12874-019-0810-0</pub-id>\n<!--<pub-id pub-id-type=\"pmcid\">6679485</pub-id>-->\n<pub-id pub-id-type=\"pmid\">31375082</pub-id></mixed-citation></ref></ref-list></back><sub-article id=\"report39953\" article-type=\"peer-review\"><front-stub><article-id pub-id-type=\"doi\">10.21956/wellcomeopenres.17704.r39953</article-id><title-group><article-title>Reviewer response for version 1</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Wake</surname><given-names>Melissa</given-names></name><xref ref-type=\"aff\" rid=\"r39953a1\">1</xref><role>Referee</role><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-7501-9257</contrib-id></contrib><aff id=\"r39953a1\">\n<label>1</label>Murdoch Children's Research Institute, Melbourne, Vic, Australia</aff></contrib-group><author-notes><fn fn-type=\"COI-statement\"><p>\n<bold>Competing interests: </bold>No competing interests were disclosed.</p></fn></author-notes><pub-date pub-type=\"epub\"><day>30</day><month>10</month><year>2020</year></pub-date><permissions><copyright-statement>Copyright: © 2020 Wake M</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><related-article related-article-type=\"peer-reviewed-article\" id=\"d38e2269\" ext-link-type=\"doi\" xlink:href=\"10.12688/wellcomeopenres.16129.1\">Version 1</related-article><custom-meta-group><custom-meta><meta-name>recommendation</meta-name><meta-value>approve</meta-value></custom-meta></custom-meta-group></front-stub><body><p>Thank you for the opportunity to review the Born in Bradford COVID-19 Research Study. This will comprise a unique resource regarding the evolving impacts of the COVID-19 pandemic on children and families in a relatively disadvantaged, multi-ethnic British city during this extraordinary period. What is especially compelling is that, due to their rolling recruitment over many years, the three cohorts combined span all of childhood plus pregnancy. While I am aware of many other longitudinal child cohorts pressed into COVID service, most can report on only the narrow age band reflecting their birth window - when COVID-19 impacts are likely to differ profoundly by a child's and family's age and stage. </p><p> The methods are sound and appropriate, and the mixed methods and city-wide collaboration will provide a richness, breadth and depth not otherwise achievable. The speed of response in mounting this is admirable.</p><p> I would have liked to see a greater emphasis on teasing out any positives of the pandemic. While recognising that the methods appropriately reflect the initial consultations, potential benefits may not have been uppermost in mind so could be missed by this adaptive methodology. These may include reduced preterm birth, lower rates of other infections and asthma, less school stress, more family time (and more time in general), community connectedness and clearer air. </p><p> I also wondered why children (unlike parents) are not asked about their own physical health or loneliness?    </p><p> Out of interest, I wondered how the authors will balance the need to feed back findings in almost real time to the community with academic publication? </p><p> It will be interesting to see how the high frequency of questionnaires (multiple times in a single year) play out in short and long term response rates. Our own family focus groups here in Australia have indicated 'COVID fatigue' and a strong wish to think about something (anything!) else - especially about better times to come.  </p><p> A few minor typos (eg 'pseudomysed' at bottom of p12) and punctuation inconsistencies could be tidied up.</p><p> Congratulations to the BiB team on an impressive response and protocol.</p><p>Is the study design appropriate for the research question?</p><p>Yes</p><p>Is the rationale for, and objectives of, the study clearly described?</p><p>Yes</p><p>Are sufficient details of the methods provided to allow replication by others?</p><p>Yes</p><p>Are the datasets clearly presented in a useable and accessible format?</p><p>Not applicable</p><p>Reviewer Expertise:</p><p>Population child health</p><p>I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.</p></body></sub-article><sub-article id=\"report40154\" article-type=\"peer-review\"><front-stub><article-id pub-id-type=\"doi\">10.21956/wellcomeopenres.17704.r40154</article-id><title-group><article-title>Reviewer response for version 1</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Wolfe</surname><given-names>Ingrid</given-names></name><xref ref-type=\"aff\" rid=\"r40154a1\">1</xref><role>Referee</role><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-4717-7634</contrib-id></contrib><aff id=\"r40154a1\">\n<label>1</label>Department of Women's and Children's Health, School of Life Course Sciences, Faculty of Life Sciences and Medicine, King's College London, London, UK</aff></contrib-group><author-notes><fn fn-type=\"COI-statement\"><p>\n<bold>Competing interests: </bold>No competing interests were disclosed.</p></fn></author-notes><pub-date pub-type=\"epub\"><day>28</day><month>9</month><year>2020</year></pub-date><permissions><copyright-statement>Copyright: © 2020 Wolfe I</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><related-article related-article-type=\"peer-reviewed-article\" id=\"d38e2378\" ext-link-type=\"doi\" xlink:href=\"10.12688/wellcomeopenres.16129.1\">Version 1</related-article><custom-meta-group><custom-meta><meta-name>recommendation</meta-name><meta-value>approve</meta-value></custom-meta></custom-meta-group></front-stub><body><p>Thank you for asking me to review the Born in Bradford COVID-19 Research Study protocol for an adaptive mixed methods research study to gather actionable intelligence on the impact of COVID-19 on health inequalities among families living in Bradford. Reading this review was an enjoyable and educational experience, and as very occasionally happens, I am left inspired and more informed by conducting this review. </p><p> The Bradford Institute and Act Early initiative are well positioned to deliver the plans set out in this protocol. Actionable practical research, co-designed and co-produced, delivering rapid intelligence to decision-makers, and within an agile adaptive research design that allows iterative learning and revision is an example for us all to follow in such challenging times. </p><p> The rationale and methods are clearly set out, and could – indeed should – be reproduced elsewhere. The research design comprises co-production of research priorities, followed by mixed methods research with appropriate analytic methods, and finally a nicely-thought through feedback loop for co-design of interventions and/or further iterations of research. The four work packages set out in a logic model that allows continuous feedback and adaptation is well thought through and nicely designed both to speak to decision makers to support them in policy-making, and to inform research priorities for longer term work. </p><p> My comments and suggestions are minor. The introduction section describes the interaction between the COVID-19 pandemic and other factors – comorbidities, ethnicity, and social determinants. These interdependencies can be described as syndemic, which could provide a useful perspective in analysis (see for example Horton R. Offline: COVID-19 is not a pandemic DOI\n<sup><xref rid=\"rep-ref-40154-1\" ref-type=\"bibr\">1</xref></sup>). My only other comment is that the one of the three initial priority research areas is described as\n<italic>adolescent</italic> mental health in one area (page 10, penultimate paragraph), and\n<italic>children’s</italic> mental wellbeing in another (page 11 second para). The former seems right, given that the sampling is from secondary schools; it may seem quibbling to point this out, but to a purist child health researcher it might stick out. </p><p> I commend this protocol without hesitation, am confident that it will deliver actionable findings to local decision makers, and probably national ones too. I look forward to reading the results.</p><p>Is the study design appropriate for the research question?</p><p>Yes</p><p>Is the rationale for, and objectives of, the study clearly described?</p><p>Yes</p><p>Are sufficient details of the methods provided to allow replication by others?</p><p>Yes</p><p>Are the datasets clearly presented in a useable and accessible format?</p><p>Yes</p><p>Reviewer Expertise:</p><p>Child health systems and policy research; paediatrics.</p><p>I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.</p></body><back><ref-list><title>References</title><ref id=\"rep-ref-40154-1\"><label>1</label><mixed-citation publication-type=\"journal\">\n:\n<article-title>Offline: COVID-19 is not a pandemic</article-title>.\n<source><italic toggle=\"yes\">The Lancet</italic></source>.<year>2020</year>;<volume>396</volume>(<issue>10255</issue>) :\n<elocation-id>10.1016/S0140-6736(20)32000-6</elocation-id>\n<pub-id pub-id-type=\"doi\">10.1016/S0140-6736(20)32000-6</pub-id>\n</mixed-citation></ref></ref-list></back></sub-article><sub-article id=\"report40153\" article-type=\"peer-review\"><front-stub><article-id pub-id-type=\"doi\">10.21956/wellcomeopenres.17704.r40153</article-id><title-group><article-title>Reviewer response for version 1</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>McKinley</surname><given-names>Michelle</given-names></name><xref ref-type=\"aff\" rid=\"r40153a1\">1</xref><role>Referee</role><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-3386-1504</contrib-id></contrib><aff id=\"r40153a1\">\n<label>1</label>Centre for Public Health, Queen's University Belfast, Belfast, UK</aff></contrib-group><author-notes><fn fn-type=\"COI-statement\"><p>\n<bold>Competing interests: </bold>No competing interests were disclosed.</p></fn></author-notes><pub-date pub-type=\"epub\"><day>17</day><month>9</month><year>2020</year></pub-date><permissions><copyright-statement>Copyright: © 2020 McKinley M</copyright-statement><copyright-year>2020</copyright-year><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><related-article related-article-type=\"peer-reviewed-article\" id=\"d38e2510\" ext-link-type=\"doi\" xlink:href=\"10.12688/wellcomeopenres.16129.1\">Version 1</related-article><custom-meta-group><custom-meta><meta-name>recommendation</meta-name><meta-value>approve</meta-value></custom-meta></custom-meta-group></front-stub><body><p>The Born in Bradford (BiB) COVID-19 research study is a timely piece of research that aims to provide information in the short term to support policy and decision makers to deliver an effective COVID-19 urgent response in Bradford and nationally in the UK, and in the longer term will allow a better understanding of the wider societal impacts of the COVID-19 response on health trajectories and inequalities. The adaptive longitudinal mixed methods design will allow an agile response to COVID-19 developments and community and stakeholder priorities. The short, medium and longer term research questions will be addressed by four inter-linked work packages, focused around the BiB cohorts, that will run in parallel. These comprise: WP1) ongoing community consultations and co-production with key stakeholders (communities, community and voluntary sector organisations, decision makers, health and education professionals) and existing BiB research community groups; WP2) repeated longitudinal quantitative data collection with BiB families enrolled in BiB birth cohorts; WP3) detailed longitudinal qualitative data collection with population sub-groups; and WP4) a feedback loop to ‘flex’ future research priorities according to community and stakeholder priorities.</p><p> The rationale and objectives of the study are clearly described and the methods are clear and well justified. The existing BiB research infrastructure has been built up over many years and this COVID-19 research study sits within this infrastructure and is uniquely placed to provide ‘actionable intelligence’ on health inequalities in a timely manner. This is a robust, well written and presented protocol and I have no edits to suggest apart from one minor suggestion to include some information on whether participants are given any payments/vouchers/reimbursements for their participation in the research or describe other approaches that are used to encourage participation.</p><p>Is the study design appropriate for the research question?</p><p>Yes</p><p>Is the rationale for, and objectives of, the study clearly described?</p><p>Yes</p><p>Are sufficient details of the methods provided to allow replication by others?</p><p>Yes</p><p>Are the datasets clearly presented in a useable and accessible format?</p><p>Yes</p><p>Reviewer Expertise:</p><p>Nutrition, obesity, behaviour change</p><p>I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.</p></body></sub-article></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Psychiatry</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Psychiatry</journal-id><journal-title-group><journal-title>Frontiers in Psychiatry</journal-title></journal-title-group><issn pub-type=\"epub\">1664-0640</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33093833</article-id><article-id pub-id-type=\"pmc\">PMC7523537</article-id><article-id pub-id-type=\"doi\">10.3389/fpsyt.2020.537596</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Psychiatry</subject><subj-group><subject>Conceptual Analysis</subject></subj-group></subj-group></article-categories><title-group><article-title>Traumatic Bereavements: Rebalancing the Relationship to the Deceased and the Death Story Using the Two-Track Model of Bereavement</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Rubin</surname><given-names>Simon Shimshon</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn001\">\n<sup>*</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/909648\"/></contrib><contrib contrib-type=\"author\"><name><surname>Malkinson</surname><given-names>Ruth</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff5\">\n<sup>5</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Witztum</surname><given-names>Eliezer</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib></contrib-group><aff id=\"aff1\">\n<sup>1</sup>\n<institution>International Laboratory for the Study of Loss, Bereavement and Human Resilience, University of Haifa</institution>, <addr-line>Haifa</addr-line>, <country>Israel</country>\n</aff><aff id=\"aff2\">\n<sup>2</sup>\n<institution>School of Psychological Sciences, University of Haifa</institution>, <addr-line>Haifa</addr-line>, <country>Israel</country>\n</aff><aff id=\"aff3\">\n<sup>3</sup>\n<institution>Zramim, Postgraduate Psychotherapy Program, University of Haifa</institution>, <addr-line>Haifa</addr-line>, <country>Israel</country>\n</aff><aff id=\"aff4\">\n<sup>4</sup>\n<institution>Department of Psychology, Max Stern Emek Jezreel College</institution>, <addr-line>Emek</addr-line>, <country>Israel</country>\n</aff><aff id=\"aff5\">\n<sup>5</sup>\n<institution>Mitra–Israel Center for REBT</institution>, <addr-line>Rehovot</addr-line>, <country>Israel</country>\n</aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Clare Killikelly, University of Zurich, Switzerland</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Katie Moraes de Almondes, Federal University of Rio Grande do Norte, Brazil; Kossigan Cyrille Kokou-Kpolou, University of Picardie Jules Verne, France</p></fn><corresp id=\"fn001\">*Correspondence: Simon Shimshon Rubin, <email xlink:href=\"mailto:Rubin@psy.haifa.ac.il\" xlink:type=\"simple\">Rubin@psy.haifa.ac.il</email>\n</corresp><fn fn-type=\"other\" id=\"fn002\"><p>This article was submitted to Psychopathology, a section of the journal Frontiers in Psychiatry</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>537596</elocation-id><history><date date-type=\"received\"><day>02</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>17</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Rubin, Malkinson and Witztum</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Rubin, Malkinson and Witztum</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Bereavements that occur under external traumatic circumstances increase the risk for dysfunction, trauma symptomatology, as well as disordered and prolonged grief. While the majority of individuals who have experienced traumatic bereavements do not meet formal criteria for posttraumatic stress disorder (PTSD), persistent complex bereavement disorder (PCBD), or prolonged grief disorder (PGD), the degree of distress and dysfunction for these bereaved can be quite significant. The assessment and intervention paradigms in use with traumatic bereavements often prioritize the trauma and bypass the centrality of the interpersonal loss. By using a bifocal approach in conceptualizing bereavement, the Two-Track Model of Bereavement (TTMB) rebalances the approach to the class of traumatic bereavements. Track I examines biopsychosocial functioning and symptoms of trauma, and track II focuses on the nature of the ongoing relationship with the deceased and the death story that may also have elements of traumatic response. The model and its application serve to identify both adaptive and maladaptive responses to loss along both axes to optimally focus interventions where needed. The story of the death, the psychological relationship with the deceased, and the presence of biopsychosocial difficulties each have a part to play in assessment and intervention. A case study of assessment and intervention following traumatic bereavement due to suicide illustrates how attention to each of these factors in the TTMB can facilitate change. Ultimately, the relational bond with the deceased is a major vector in grief and mourning. Assessment and intervention with traumatic bereavements require attention to dysfunction and symptoms of trauma as well as to the death story and the state of the relationship to the deceased.</p></abstract><kwd-group><kwd>bereavement</kwd><kwd>prolonged grief disorder</kwd><kwd>traumatic bereavement</kwd><kwd>Two-Track Model of Bereavement</kwd><kwd>posttraumatic stress disorder</kwd><kwd>continuing bonds</kwd><kwd>suicide</kwd><kwd>psychotherapy</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"3\"/><equation-count count=\"0\"/><ref-count count=\"65\"/><page-count count=\"12\"/><word-count count=\"7606\"/></counts></article-meta></front><body><sec id=\"s1\"><title>Introduction</title><p>Bereavement following the death of a loved one is universal. How one grieves and mourns that loss, and how a broad array of variables influence grief and its outcome have received increasing attention and specification. Since the beginning of the 20<sup>th</sup> century, the field of thanatology has undergone tectonic changes in the way bereavement is understood. One revolution in bereavement is the break with Freud’s (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>) view of mourning as a process leading to the minimization of the emotional connection to the deceased. In place of a view that breaking the bonds (decathexis) with the deceased is adaptive, grieving is recognized as the reworking and maintenance of the attachment bond rather than its severance and has been designated the <italic>continuing bonds</italic> paradigm (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>–<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). The psychological processes in grief and mourning are related to the life-change reorganization that requires adaptation to the absence of the deceased’s physical presence (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>–<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). As a life stressor of major proportions, the death of a significant attachment figure puts the bereaved at risk of developing a broad variety of difficulties and dysfunctions including, but not limited to, depressive disorders. At the same time, the majority of bereaved respond to loss without sustained difficulties (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>).</p><p>The estimated prevalence of bereaved at risk to develop variations of prolonged grief disorder (PGD) and complications of grief generally range between 7-15% across all categories with traumatic circumstances of loss yielding significantly higher estimates (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>–<xref rid=\"B13\" ref-type=\"bibr\">13</xref>).</p></sec><sec id=\"s2\"><title>Traumatic Bereavements </title><p>The term <italic>traumatic bereavements</italic> originated in response to clinical reality where bereavements occurring under traumatic circumstances resulted in the familiar mixture of symptom pattern of PTSD together with acute grief (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Traumatic bereavements increase the risk for dysfunction and symptomatic difficulties and complicated grief (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B15\" ref-type=\"bibr\">15</xref>–<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Traumatic bereavement is a term that stresses the interface of traumatic circumstances with bereavement (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>). The potential significance of the interplay between the traumatic and bereavement elements in a variety of loss has received significant attention in the literature over the years, but consensus on diagnosis, research, and intervention remain elusive (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>–<xref rid=\"B22\" ref-type=\"bibr\">22</xref>).</p><p>In a meta-analysis studying the efficacy of grief interventions for a variety of bereavements, both traumatic loss and child loss demonstrated benefits from intervention while “uncomplicated losses” did not show benefit (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). This linkage would support the understanding that both trauma and the death of a child pose extraordinary challenges for post-death grieving and adaptation. Child loss has itself been described as a traumatic loss (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>). In our own studies of heightened grief scores with the two-track bereavement questionnaire for complicated grief years after the loss, we noted that the elevated scores that characterized only 5% of adults bereaved of their parents, applied to 10% of the spousal bereaved and to fully 25% of those who had lost children (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). These results support a position that the loss of a child is a loss of traumatic proportions in comparison with other family losses.</p><p>The interplay of trauma and bereavement is complex. In research examining adults who were not physically present but were bereaved in the 9-11 attacks, approximately 3 years later 43% received a classification of complicated grief with PTSD among the major comorbid conditions (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). A recent study looked at the presence of symptoms of prolonged grief disorder, posttraumatic stress disorder, and depression for 458 persons presenting for treatment at a Dutch clinic specializing in trauma (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). In the bereaved group, 45% reported a history of violent loss. For their bereaved sample, the authors report clinically relevant PGD scores for 28%, PTSD for 78% and depression for 28%. This overlap between symptoms of trauma and of grief in a clinical setting focusing on trauma is a repeated and robust finding. It underscores the overlap between the two classes of response.</p><p>In the framework of the revisions and discussions preceding the DSM -5 (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>–<xref rid=\"B31\" ref-type=\"bibr\">31</xref>), suggestions to include complicated grief and prolonged grief were rejected. Instead, a new term was added and specified in the category of conditions for further study–persistent complex bereavement disorder (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). This term was put forth to replace terms in use such as pathological grief and complicated grief and was structured with proposed criteria ordered in the current DSM format. It is particularly significant to note that following specified criteria, the diagnostician is requested to address whether the circumstances of death are traumatic:<disp-quote><p>\n<italic>Specify if:</italic>\n</p><p>\n<italic>“With traumatic bereavement: Bereavement due to homicide or suicide with persistent distressing preoccupations regarding the traumatic nature of the death (often in response to loss reminders), including the deceased’s last moments, degree of suffering and mutilating injury, or the malicious or intentional nature of the death”</italic>. [(<xref rid=\"B32\" ref-type=\"bibr\">32</xref>), pp.789–792]</p></disp-quote>\n</p><p>The parallel diagnostic formulation for complications of grief in the ICD 11 is classified for the first time under - 6B42 prolonged grief disorder (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). This emphasizes the criteria extended time and dysfunction such that “The disturbance causes significant impairment in personal, family, social, educational, occupational, or other important areas of functioning” (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>). In contrast to the DSM-5, the ICD-11 excludes the circumstances of traumatic bereavement from its definition thus ignoring significant component of traumatic bereavement (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). <xref rid=\"T1\" ref-type=\"table\">\n<bold>Table 1</bold>\n</xref> presents the diagnostic criteria for PCBD and PGD. It is important to note that proposed changes for a revised DSM-5 currently under consideration include creating a formal diagnosis of PGD in the DSM section II (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). We agree with those advocating for the inclusion of PGD in a DSM-5 revision in the chapter on trauma and stressor-related disorders rather than in the chapter on depressive disorders (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Irrespective of any decision on where to place the bereavement diagnosis, we believe it valuable for any modification of the DSM regarding disordered bereavement to retain the specification “with traumatic bereavement” as an important qualifier. The significance of the intersection of trauma and bereavement are consistent findings in the clinical and research literatures (e.g., <xref rid=\"B35\" ref-type=\"bibr\">35</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>A comparison of the PCBD diagnosis in DSM-5 (2013) and the PGD diagnosis of ICD-11 (2019).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Category</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">DSM-5 persistent complex bereavement-related disorder (PCBD– conditions for further study)</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ICD PGD (2019)</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Event</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Death of a close other</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Death of a close other</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Time frame and degree</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">12 months; “on most days to a clinically significant degree”</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6 months and clearly exceeds norms for culture/context</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Response focus</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1 of yearning/longing; sorrow/pain in response to death; preoccupation with deceased or circumstances of death.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Longing for the deceased or persistent preoccupation with the deceased</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Symptoms</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6 of the following: 1) marked difficulty accepting the death; 2) disbelief or emotional numbness over the loss; 3) difficulty with positive reminiscing about deceased; 4) bitterness or anger related to the loss; 5) maladaptive appraisals about oneself in relation to deceased or death; 6) excessive avoidance of reminders of the loss; 7) desire to die to be with deceased; 8) difficulty trusting other people since the death; 9) feeling alone or detached from other people since the death; 10) feeling that life is meaningless or empty without deceased or belief that one cannot function without deceased; 11) confusion about one’s role or diminished identity; 12) difficulty pursue interests or plan for future since loss</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Persistent and pervasive grief response characterized by longing for the deceased or persistent preoccupation with the deceased accompanied by intense emotional pain (e.g., sadness, guilt, anger, denial, blame, difficulty accepting the death, feeling one has lost a part of one’s self, an inability to experience positive mood, emotional numbness, difficulty in engaging with social, or other activities).</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Traumatic death circumstance</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Specify if: With traumatic bereavement with persistent distressing preoccupations regarding the traumatic nature of death</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No specifier</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Degree of impairment</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Clinically significant in social, occupational or other important areas of functioning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Significant impairment in personal, family, social, educational, occupational, or other important functioning</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Qualifier</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Beyond expected norms for relevant cultural, religious, or developmental stage</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exceeds social, cultural, or religious norms</td></tr></tbody></table></table-wrap><p>It is premature to determine the extent to which the changes in nomenclature of the DSM and the ICD will impact the ways in which practitioners and researchers approach trauma and bereavement (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>). In our experience in Israel and internationally, the emphasis on trauma and PTSD in the context of bereavement has the paradoxical effect of obscuring the centrality of the interpersonal relationship bond in these circumstances (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>, <xref rid=\"B40\" ref-type=\"bibr\">40</xref>). One might say that from the trauma perspective, bereavement under traumatic circumstances that results in the familiar symptom pattern of PTSD is PTSD. From the bereavement perspective, however, the symptom pattern of PTSD following the death of a significant relational figure is but one aspect of what may actually be predominantly dysfunctional grief and disordered mourning (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>). Furthermore, we believe that there are additional aspects of traumatizing elements that operate in traumatic bereavement. In some cases, the circumstances of the traumatic death events are highly intermeshed with the interpersonal relationship to the deceased that itself has strong conflictual and negative characteristics (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). Bereavements following troubled interpersonal relationships are one example of this. While the DSM-5 describes both homicide and suicide as qualifying as traumatic circumstances, in reality there are important distinctions to be made. One major difference relates to the fact that the person who dies from suicide is both the one who took life and the one whose life was taken. For many of the bereaved, this combination complicates the grief. Overall, assessment and intervention benefit from attention to greater specificity regarding the complicating and traumatizing features involved in bereavement (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>).</p><p>The literature on trauma and post-trauma has changed how both clinicians and the lay public think about life-threatening events and their impact (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B44\" ref-type=\"bibr\">44</xref>). With the passage of time and the expanding literature base, clinical practitioners and the general public are better informed as to the incidence, prevalence, and pernicious deleterious effects of exposure to traumatic events (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>, <xref rid=\"B46\" ref-type=\"bibr\">46</xref>). Similarly, awareness of the various intervention programs and their reported efficacy has increased as well (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B47\" ref-type=\"bibr\">47</xref>, <xref rid=\"B48\" ref-type=\"bibr\">48</xref>).</p><p>Alongside the generally positive effect of the expanding wellspring of knowledge and expertise that has accrued to date in the trauma field, there is a less welcome side effect to this phenomenon as it relates to bereavement. Specifically, since major life threatening events directed either at the self or at a loved one are considered events of significant magnitude as to satisfy the criterion for a traumatic stressor, this categorization is often seen to encompass the death of a loved one. Once the death of a loved one is categorized as a major stressor of potentially traumatic proportions, the interpersonal and attachment issues involved in bereavement may be downplayed or missed (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>, <xref rid=\"B49\" ref-type=\"bibr\">49</xref>). For example, combat soldiers whose comrades have fallen in battle report attention and follow-up related to the post-trauma and minimal consideration of the grief and mourning for fallen comrades (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>).</p><p>We shall return to amplify these points with clinical material after the presentation of the Two-Track Model of Bereavement (TTMB).</p></sec><sec id=\"s3\"><title>Two-Track Model of Bereavement (TTMB): A Model for Research and Practice</title><p>The TTMB was developed in order to make sense of research and clinical data that demonstrated successful adaptation to life post loss while maintaining a strong connection to the relationship with the deceased in the present (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>, <xref rid=\"B52\" ref-type=\"bibr\">52</xref>). The model itself addresses response to interpersonal loss from a bifocal perspective considering both the biopsychosocial functioning of the bereaved (track I), and the nature of the ongoing relational bond to the deceased (track II), across the life cycle (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>, <xref rid=\"B52\" ref-type=\"bibr\">52</xref>). Track I’s focus on biopsychosocial dysfunction reflects both the medico-psychiatric attention to human suffering and the psychological perspectives rooted in the broad domain of mental health, stress, and trauma literatures. Included here are the biological, behavioral, cognitive, emotional, intrapersonal, and interpersonal ways of one’s being in the world. These can be negatively affected following loss for extended periods (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). In cases where there is reason to suspect manifestations of post-trauma, inquiry into the triad of re-experiencing, avoiding, and hyper-alertness is warranted a as part of the assessment (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Viewed most broadly, the exploration of negative and positive changes in the general domain of function span a wide range of areas. The biospsychosocial approach shares much with the general clinical evaluation of persons facing stressful challenges to their previous mode of living in the world and the way in which they made sense of its meaning (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). These challenges are not fundamentally unique to the bereavement experience and the stresses it brings with it. Thus, exposure to natural disasters, combat, sexual victimization, terror, and a variety of life-threatening situations would warrant the explorations of this first domain in many of the same ways as with the assessment of responses post-bereavement.</p><p>The second track of the model, the relationship to the deceased, traces its origins and significance to a wholly different set of assumptions and postulates. In modern terminology, we would stress the relevance of the attachment and object relations literature and the nature of the attachment bond as central to what is unique to bereavement and loss (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>, <xref rid=\"B54\" ref-type=\"bibr\">54</xref>). The core insight related to track II of this model is that reworking the relationship to the deceased and the continuing bond with the deceased post-death is a critical feature for understanding response to bereavement. This is the central feature of what makes the loss process unique. The second domain of the two-track framework prioritizes the nature of the relationship to the deceased and the current status of the emotional bond to the deceased. How the psychological experience of the relationship and its accessibility have changed following death are central pieces of this approach (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). An additional focus of track II is rooted in the story of the death and how it has been integrated and assimilated by the bereaved. Particularly in cases of traumatic deaths, the events surrounding the death may remain unassimilated with potentially serious consequences (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B22\" ref-type=\"bibr\">22</xref>). In some cases, this unintegrated and traumatic segment interferes with the processing of the relationship and the access to the broad range of memories, associations, and emotions concerning the life course with the deceased. <xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1</bold>\n</xref> illustrates the domains of the TTMB as they unfold over time.</p><fig id=\"f1\" position=\"float\"><label>Figure 1</label><caption><p>Illustration of the two-track model of bereavement (TTMB)<xref ref-type=\"fn\" rid=\"fn1\">\n<sup>1</sup>\n</xref>.</p></caption><graphic xlink:href=\"fpsyt-11-537596-g001\"/></fig><p>The importance of combining the two perspectives of functioning and relationship in broad ways formed the basis for the TTMB (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B51\" ref-type=\"bibr\">51</xref>, <xref rid=\"B52\" ref-type=\"bibr\">52</xref>). In this model, the process of adaptation to interpersonal loss is linked to the disruption of homeostatic functioning that accompanies major life stressors on the one hand but also as a byproduct of relating and reconfiguring aspects of the relational bond and attachment system with the deceased on the other hand. The TTMB facilitates the assessment of both functioning and the nature of the continuing attachment to the deceased when significant others die—and this across the entire course of the bereaved person’s lifetime. <xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2</bold>\n</xref> conveys the mix of independence and interdependence of the two tracks.</p><fig id=\"f2\" position=\"float\"><label>Figure 2</label><caption><p>The TTMB in traumatic bereavement<xref ref-type=\"fn\" rid=\"fn2\">\n<sup>2</sup>\n</xref>.</p></caption><graphic xlink:href=\"fpsyt-11-537596-g002\"/></fig><p>The clinical implications of the model derive directly from its binocular focus. The extent to which potential psychological interventions should privilege one or both domains of the response to loss remains an important clinical question (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). The basic elements of the assessment schema of the TTMB and a rubric for clinician ratings are presented in <xref rid=\"T2\" ref-type=\"table\">\n<bold>Table 2</bold>\n</xref>. The 10 domains of track I’s biopsychosocial functioning and track II’s 11 domains beginning with the death story are relevant in assessing the response to loss at any point in time following the loss. <xref rid=\"T2\" ref-type=\"table\">\n<bold>Table 2</bold>\n</xref> summarizes the scoring scale for clinician assessment of bereaved individuals.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>The TTMB clinician rating scale.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Track I: Biopsychosocial difficulties in functioning and post-trauma</th><th valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Clinician evaluation</th><th valign=\"top\" colspan=\"3\" align=\"center\" rowspan=\"1\">Track II: Ongoing relationship to the deceased and death story</th><th valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Clinician evaluation</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Traumatic responses</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">1. Death story difficulties/unintegrated/interferes with connection to deceased</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2. Anxious affect and cognitions</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">2. Yearning and closeness</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3. Depressive affect and cognitions</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">3. Imagery/memory of deceased</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4. Somatic and Health</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">4. Positive affect</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5. Familial relationships</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">5. Negative/unmodulated affect</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6. General interpersonal</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">6. Preoccupation/avoidance (specify)</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7. Lowered self-esteem and distress</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">7. Idealization/devaluation (specify)</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8. Involvement in life</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">8. Conflict in the relationship</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9. Meaning in life</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">9. Loss trajectory (rate each: shock, searching, disorganization, and <break/>reorganization)</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">10. Strengths (growth/resilience)</td><td valign=\"top\" rowspan=\"2\" colspan=\"2\" align=\"left\"/><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">10. Self-system difficulties vis-à-vis deceased and/or death story (specify)</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" colspan=\"3\" align=\"left\" rowspan=\"1\">11. Memorialization</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\"/></tr><tr><td valign=\"top\" colspan=\"7\" align=\"left\" rowspan=\"1\">\n<bold>Clinician scoring key</bold>\n</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Absent/mild/limited</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Moderate</td><td valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Severe/strong</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Continue to monitor</td><td valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Insufficient information</td></tr></tbody></table></table-wrap><p>In assessing the impact of loss in cases of traumatic bereavements, the objective circumstances of the loss, with attendant symptomology characteristic of post-trauma are part of the biopsychosocial response. At the same time, however, inquiry and assessment of the death story are highly significant in assessing how the ongoing relationship to the deceased is affected by that (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>, <xref rid=\"B56\" ref-type=\"bibr\">56</xref>). Thus parallel focus is also relevant in relationships where the losses are not death but involve significant changes in requiring adjustment due to major changes in the attachment figure (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B57\" ref-type=\"bibr\">57</xref>). For example, in a recent publication, the rationale for a two-track model of dementia grief (TTM-DG) showed how the particular circumstances involved in caring/caretaking for a close family member suffering from dementia was best understood from the bifocal perspective of the TTMB (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>). In particular, the ongoing loss of personhood and identity for a person suffering from cognitive decline and dementia is a challenge and loss for those closest to him or her. For a fuller elaboration of the current applications of the model in clinical work and research, see <italic>Working With the Bereaved: Multiple Lenses on Loss and Mourning</italic> (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>).</p><p>Focusing on the extent of behavioral difficulties and symptoms of various types is valuable and is sensitive to the ways that bereavement challenges the bereaved to readjust to a new external reality. Whether or not the bereavement response meets criteria for a formal diagnosis, however, many persons seeking assistance after the death of a close person can benefit from the bifocal approach advocated. Many aspects of the relationship to the deceased both pre and post loss require us to learn more about whom was lost. This includes attention to what aspects of that relationship were lost for the bereaved and what portion of that relationship remains active and available to the bereaved after the death. In the case of circumstances of traumatic bereavement, the linkage of trauma with bereavement has often had the paradoxical effect of obscuring the decidedly interpersonal and intrapersonal impact of the loss and replacing it with a focus on the trauma elements (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>, <xref rid=\"B59\" ref-type=\"bibr\">59</xref>).</p></sec><sec id=\"s4\"><title>Working Clinically With the TTMB in a Case of Traumatic Bereavement</title><p>The following brief case study of assessment and intervention illustrate the use of the TTMB in a case of traumatic bereavement. In particular, the clinical example illustrates how bereavement can operate as a complex series of traumatizing experiences that have roots and links to both tracks of the TTMB.</p><p>Helen, a 36 year old woman, came for treatment to SSR 2 years after the suicide of her husband David. In the initial session, Helen said she was only now ready to get help. Early in the first session, she spontaneously described how she had discovered her husband’s suicide and body. Returning home from a brief business trip abroad, she had opened the door to the master bathroom where she was assaulted by “horrific sights and smells and waves of nausea.” Her husband’s decaying body was lying in the bathtub several days after he had killed himself. He had slit his wrists and the blood flow into the water had left everything a discolored pink after the water had drained away. Her affect as she described her experiences conveyed minimal emotion. While it was clear that the experiences were distressing and painful, her reporting was done in a factual way that was devoid of emotion.</p><disp-quote><p>Helen: “Years later, I close my eyes, [and sometimes still] see his body lying there and the red color of the bathtub surrounding him. I have dreams of being physically tortured by someone wearing his face. I have intrusive thoughts about him, this used to be maybe 100 times a day, but now it is much much less. I did most of my work from home and barely went out. I sat in the dark, self-medicated with alcohol and marijuana. I wanted to suffer so I did not see any mental health professionals”.</p></disp-quote><p>In the initial meetings, Helen shared her personal history and details about her husband and their time together. She was the eldest of two daughters who grew up in an intact family. She had always been “a bit of a tomboy, interested in sports, computers, and mathematics.” Previous losses included the death of her father when she was 19; the departure of her younger sister for a job overseas, and her mother’s subsequent remarriage and move to the sister’s city of residence. Helen had many strengths and was successful during her years of education, and eventually took her skills set to work in the computer world of high-tech which required a fair amount of international travel. Helen had been introduced to her future husband by a mutual friend and they immediately forged a connection. Soon they were living together, and they married a year later. All told, they had been together for 12 years before David’s suicide. David had been raised in a small town, had done well in school and college, eventually entering into government service. At age 30 he had begun to suffer from depressive episodes. He was seen by a mental health professional and prescribed medications. He stopped both soon thereafter. The couple had weathered several of David’s depressions including one for which he was hospitalized briefly. Several weeks before her business trip abroad, David had again gone into what seemed to be a depressive mood, but he assured her that his depression was under control and there was no need to see anyone. He said he would be fine so she need not be concerned for his wellbeing. During her stay overseas, they were in contact for the first 2 days before he told her that he was going to be incommunicado for several days as he was going to hike in an area where there was no cellphone coverage. Upon her return, she found his lifeless body at home.</p><p>At the time of her request for therapy, Helen exhibited some symptoms of stress and depressive affect, but these did not significantly interfere significantly with her day to day functioning. She had seen a physician for medication for sleep difficulties the first year, but was now sleeping relatively well although there were the occasional nightmares reported. As indicated above, she continued to have intrusive thoughts and images of the death scene on a daily basis, but these were no longer interfering with her functioning. She continued to avoid places that reminded her of David’s depressions and she had withdrawn from interactions with their mutual friends. She described heightened vigilance and anxiety in response to phone calls. Not infrequently, and particularly when the phone rang at odd hours, she felt a reflexive fear that the call was about bad news affecting her family overseas. These responses were much reduced from what she had experienced for many months following David’s suicide.</p><p>Queried about the nature of the memories, thoughts and feelings about the relationship with David, Helen said that she felt shut down and unable to connect with their good times together. She felt guilty for having left him to go on her business trip, denied feelings of anger vis-a-vis the suicide or his deception and the way he hid his state of mind from her. The memories of David and the recollections of the times when she had enjoyed his company eluded her along with the feelings of pleasure they once held.</p><sec id=\"s4_1\"><title>Assessment and Intervention Plan With the TTMB</title><p>The diagnostic categories applicable to Helen in the DSM-5 were from the trauma and stressor related disorders (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Helen’s current functioning involved significant suffering and many symptoms associated with post-trauma although not sufficiently meeting criteria for a DSM-5 diagnosis of PTSD. The experience of her husband’s suicide met the criterion for exposure to a traumatic event; there were intrusions of the death scene; avoidance of places that triggered memories; negative alterations in mood, and a degree of hyper alertness as manifest in her response to phone ringing. The circumstances and degree of clinically significant distress met conditions for “Other Specified Trauma- and Stressor-Related Disorder” with the further specification of persistent complex bereavement disorder. We would point out that this mix of trauma and bereavement in the more formal diagnosis reflects clinical, conceptual and diagnostic overlap, and interpenetration.</p><p>From the perspective of the TTMB, Helen had significant distress on many of the track I biopsychosocial categories. Feelings of anxiety, depression, a sense that her life was adrift and without meaning, reduced involvement in social interactions with family and friends, along with her lowered self-esteem reflected the extent to which her biopsychosocial functioning was impaired. At present, on the trauma dimension, there were elements of the triad of intrusions/re-experiencing, avoidance, and hyperarousal. These were significant and consistent with a residue of active post-trauma.</p><p>Examining track II’s death story and relational bond with her deceased husband conveyed significant difficulties. In the quote included earlier, Helen graphically described David’s suicide and the experience of finding his body. The story of the death emphasized the shocking elements, but nowhere was she able to integrate this experience into a broader narrative. There were no indications of some degree of acceptance and assimilation of this experience with the bulk of her relationship with David. Helen returned repeatedly to the question of why he had killed himself in the way that he did without caring about what it would be like for her to find him like that. The narrative of his death did not fit into her description of his depressions and struggles with suicidal thoughts. It was as if this experience began and stopped with the discovery of his body days after his death.</p><p>The story of the relationship with David and the limited degree to which memories of their lives together were accessible to her indicated that the relationship bond required therapeutic attention. Helen had difficulty accessing the wellspring of memories of their relationship. Memories of the positive elements of their relationship were few and vague. There was little in the way of perspective or the ability to reference the thick history of their years together. Negative and conflictual elements of the connection with David were present and associated with pain. She felt guilty over having left David to go overseas and also a sense of general unworthiness and failure as a spouse. Despite the objective dislocation that David’s death had caused her, she repeated stated that she was unable to be angry at him for what he had done. With access to positive memory and emotions of their relationship constricted and with a painful focus on David’s suicide in the fore, the relationship domain was problematic. In this condition, progression toward adaptive grief and mourning were stymied (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>).</p><p>Helen was having difficulties on track I and track II and the question of how to focus intervention with respect to these variables was highly relevant. On track I, the elements of trauma, depressive thoughts and emotions, lowered self-esteem, constricted connection with life tasks, connecting to meaning in life, were all rated by SSR as elevated and of moderate severity. The combination of these variables, together with limited family support and other interpersonal relationships, powerfully affected Helen’s ability to engage in her life. These track I biopsychosocial difficulties convey significant dysfunction and distress and intervention would need to take them into account.</p><p>On track II, Helen had not integrated the death story nor had she been able to retell the story with any degree of flexibility or perspective. The ongoing sense of shock in the way the death event was experienced interfered strongly with her ability to fully describe her husband and the many years of their relationship. This aspect of the death-related trauma is rated on track II because of its significance in impeding grief and mourning and for its interference with access to the interpersonal relationship and the continuing bond with the deceased. The relationship with David was scored as highly significant with the variables related to the death story, negative affect, preoccupation with the deceased, conflict in the relationship, and assault to the self-system as a result of the relationship being coded as of severe proportions. Yearning was pronounced, while positive affect, memories of the good times, and progress toward memorialization of the deceased were limited. <xref rid=\"T3\" ref-type=\"table\">\n<bold>Table 3</bold>\n</xref> gives the clinician scoring of Helen at the end of the intake evaluations on the TTMB variables discussed.</p><table-wrap id=\"T3\" position=\"float\"><label>Table 3</label><caption><p>Helen–Clinical Assessment with the TTMB rating scale at beginning of treatment.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Track I: Biopsychosocial functioning </th><th valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Clinician evaluation</th><th valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Track II: Relationship to the deceased and death story</th><th valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Clinician evaluation</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1. Traumatic responses</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Moderate</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">1. Death story</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Severe</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2. Anxious affect/cognitions</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Mild</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">2. Yearning and closeness</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Moderate</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3. Depressive affect/cognitions</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Moderate</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">3. Imagery/memory</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Limited</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4. Somatic and health</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Mild</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">4. Positive Affect</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Limited</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5. Familial relationships</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Mild/limited</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">5. Negative Affect</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Severe</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6. General interpersonal</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Mild/limited</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">6. Preoccupation/Avoidance</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Severe</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7. Lowered self-esteem</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Moderate</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">7. Idealization/devaluation</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Mild–deval</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8. Involvement in life</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Moderate</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">8. Conflict in the relationship</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Severe</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9. Meaning in life</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Moderate</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">9. Loss Trajectory (Shock, Searching, Disorganization, and <break/>Reorganization)</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Sh- Severe<break/>Se – Limited<break/>Dis- Moderate<break/>ReO - Limited</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">10. Strengths (growth/resilience)</td><td valign=\"top\" rowspan=\"2\" colspan=\"2\" align=\"left\">Limited</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">10. Self-system</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Severe</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">11. Memorialization</td><td valign=\"top\" colspan=\"2\" align=\"left\" rowspan=\"1\">Limited</td></tr><tr><td valign=\"top\" colspan=\"7\" align=\"left\" rowspan=\"1\">\n<bold>Clinician scoring key</bold>\n</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Absent/mild/limited</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Moderate</td><td valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Severe/strong</td><td valign=\"top\" colspan=\"2\" align=\"center\" rowspan=\"1\">Continue to monitor</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Insufficient information</td></tr></tbody></table></table-wrap></sec><sec id=\"s4_2\"><title>The Treatment Plan Based on the TTMB</title><p>In considering a treatment plan, once again, the TTMB framework was used in formulating the intervention plan. Helen’s response to the traumatic loss 2 years post-death was characterized by significant difficulties in functioning, features of traumatic stress, and relation based elements that served to reinforce each other. They limited her ability to function and her ability to grieve in ways that facilitated working through of aspects of the loss. With difficulties on both tracks, it was important to formulate an intervention plan that would take into account both domains of the TTMB.</p><p>The focus on track I’s biopsychosocial functioning and trauma suggested a range of intervention possibilities. The treatment components most relevant for the biopsychosocial difficulties combined elements of supportive psychotherapy with a variety of techniques and exercises that have been associated that included techniques drawn from evidence based treatment strategies (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>, <xref rid=\"B61\" ref-type=\"bibr\">61</xref>).</p><p>Concurrent with interventions related to track I, the focus on track II drew attention to the importance of transforming the death story. The frozen and shocking death scene effectively locked the death by suicide event into a kind of video clip that did not develop into a story. Finding ways to help Helen take the death story and continue on beyond the death scene, ultimately integrating it to the broader story of David’s life, death, and memory were an overarching goal of the track II intervention. The goals of treatment here emerged squarely from the track II conceptualization of the significance of the continuing bond to, and the dynamic relationship with, the complex of memories, thoughts and emotions linked to the deceased. This connection can serve as a positive and supportive presence for the bereaved across the life span (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). <xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3</bold>\n</xref> illustrates the double focus of the intervention plan and goals.</p><fig id=\"f3\" position=\"float\"><label>Figure 3</label><caption><p>Overview of the assessment and intervention plan with Helen.</p></caption><graphic xlink:href=\"fpsyt-11-537596-g003\"/></fig><p>Whatever the foci and plans for therapy, it was important to get Helen’s cooperation and to enlist her in the treatment plan. Following the initial sessions of history taking, the therapist shared the basic outline of the TTMB as a way of thinking about responses to loss with the goal of psychoeducation and helping Helen make sense of her response to David’s suicide. Following a discussion of the model, the predominant areas of difficulty and dysfunction that she experienced were shared back with her. Therapist and patient were in agreement on the need for intervention, and the areas in her life that were particularly troubling. The goals of treatment were to help her feel better, to assist her with the various domains of functioning that troubled here, to ease the pain and shock of her memory of David’s death, and to help her regain a more flexible and supportive connection with his memory.</p></sec><sec id=\"s4_3\"><title>Critical Junctures in Helen’s Treatment</title><p>The initial weeks of treatment were devoted to building the therapeutic alliance with Helen. In this initial stage of therapy she filled in details of her current life and the nature of the challenges she faced as well as her feelings of estrangement and longing for David. In order to assist her in reducing her anxiety, depression, and tension, one set of interventions were aimed at increasing her ability to modulate her affect. Self-observation, journaling, and release of tension <italic>via</italic> physical exercise were prescribed, and she was able to follow through on all these (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). The benefits of mindfulness were explained, and practice in the sessions was introduced (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>). Even though she did not continue with mindfulness, the focus on allowing emotions and thoughts to be seen as reflecting the ebb and flow of the mind rather than as thoughts to engage or avoid were helpful to her. Above all, encouraging her to share her thoughts and feelings about those encounters and areas of her life that troubled her without the requirement that they reflect the trauma or the loss were beneficial. This allowed her to explore her thoughts regarding meaning in life, her mixed feelings toward family and friends, and the nature of difficulties in her work and home that were of benefit to her. As she followed through with regular exercise, sporadic periods of mindfulness at home, sharing her thoughts in the sessions and journaling occasionally at home, her mood and sense of self began to improve. These in turn contributed to the strengthening of the therapeutic alliance.</p><p>The next stage of treatment began after the therapeutic alliance and initial benefits from treatment were in place. Building on the therapeutic alliance, the major intervention focus directed at Helen’s relationship to David and the manner of his death became the center of this next phase of treatment.</p><p>The techniques used in psychotherapy for bereavement are myriad (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>). In situations where the relationship is constrained or moribund finding ways to open possibilities for change and flow in the relationship can be transformative. Chair work where the bereaved and deceased “interact” and letter writing to the deceased are among the better known of these techniques (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>, <xref rid=\"B63\" ref-type=\"bibr\">63</xref>). From the TTMB perspective, in the case of Helen, because the circumstances of her husband’s death were so traumatic, they effectively stymied her ability to reorganize her relationship to him. The therapeutic technique was designed to permit Helen to soften the boundaries between the living and the dead, the real and the imaginary, and to introduce the possibility of change and the evolution of the relationship. One could think of this intervention technique as a way of seeking to open “transitional space and fluidity” into what had been a frozen and “dead” relationship (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>). At the same time, this intervention technique had potential to allow for the evolution in the story of David’s death and the discovery of his body that had heretofore remained traumatic, stuck, and repetitious.</p><p>Some 3 months into the therapy, having made gains in emotion regulation, having taken up walking for an hour each day, and having attempted mindfulness but deferred it for later, the more direct focus on Track II’s relationship began. The letter writing that involved having Helen write a letter to David with the goal of facilitating the reconnection and expansion of the current relationship. (See chapter 10, pp 151–171 in (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>) for a fuller description of this type of intervention.)</p><p>The first letter she composed was straightforward:<disp-quote><p>Dear David,</p><p>There are so many things I want to say to you…. things I’ve thought these past several years…. I want to be angry at you for what you did, what you promised me you would never do. I thought you were getting better, …</p><p>You promised you wouldn’t hurt yourself, because you said you didn’t want to hurt me. So why did you do it? …. And to do it like that, for me to find you? … how could you never consider what it would do to me, to find you like that?</p><p>I said I want to be angry at you, and as much as I want answers to these questions the truth is I can’t be angry with you I just miss you so much. All I think about is what I would do or give up to have another 5 minutes together, just to talk to you and hug you.</p></disp-quote>\n</p><p>In this first letter, the connection is made, and the longing and wish for reconnection are present. She introduces the possibility of complaints directed toward David for his act of suicide, but anger and disappointment are mentioned though not experienced. The longing for the reconnection are very much present.</p><p>The next letter was written two weeks later</p><disp-quote><p>Dear David,</p><p>Starting these letters is always so difficult. It feels strange, like I’m speaking to you again back when you were alive. … And yet this also feels more impersonal, as even though I feel I am talking to you, I feel less close to you then I did in the past. Maybe that is because I am angry over what you did. … The truth is that I don’t know what has caused it but my feelings for you have changed, and though I think of you constantly still it is not through the same rose colored glasses I once did.</p><p>I find myself feeling frustrated more and more by the fallout of your suicide. For so long I blamed you … How could you not consider what would happened to me, how utterly your actions would damage and destroy my life. (2nd letter).</p></disp-quote></sec><sec id=\"s4_4\"><title>Commentary</title><p>The degree of change between letter one and two is striking. Both letters speak to the ability of Helen to enter into the letter writing exercise and to find herself opening up with her emotions and entering into a dialogue with David. This entrance into a living dialogue brings with it potential for change. The emotion of anger that is denied in letter one emerges strongly in letter 2. More importantly, she indicates that an idealized picture of David referred to in the rose colored glasses of letter two have been replaced by a more sober assessment. The shift in how David is viewed and the change it brings with it in the relationship are pivotal in allowing grief to progress as Helen has more freedom to think about and experience a broader range of emotion than she had earlier. The second letter served as a transformative experience for Helen. Following the letter, Helen spoke about remembering more of her time together with David, and he felt both nearer to her and more alive than she had experienced him for a long time. The broadened access to memories of David, the anger focused on David instead of only on herself, and the story of the depressions that became part of the narrative around the suicide all reflected the movement toward grieving and mourning. Connecting more fully to David also allowed her to become more whole as well.</p><p>The sense of betrayal and assault that Helen experienced in response to the suicide were a traumatizing experience for her. Most importantly, the act of suicide served to shatter the person and internal working model of the David she had known. Not only were the memories and connections to him less available, they were also colored by the action that changed the way she viewed him and their relationship. So that in addition to the trauma of the discovery of the dead body and the assault on the senses involved with that, there was an additional trauma, and one that directly related to the experience of who her husband was for her. Helen in effect was dealing with a double trauma, one of external characteristics of the encounter with the death scene, and one that was rooted in the traumatic encounter with her experience of who her husband was for her, and his actions toward her. On the one hand, the Track I trauma has an objective component of exposure to David’s death and his dead body. On the other hand, the Track II trauma was also centered around the relationship with David and the betrayal that his suicide brought to Helen. The death story remained one that she had been unable to integrate and weave into the story of their relationship.</p></sec><sec id=\"s4_5\"><title>A Brief Note on the Treatment Conclusion</title><p>Helen’s initial letters to David were followed by more letters, by chair work where she took both roles in the dialogue with her deceased husband, and by her newly recovered ability to talk about their relationship. She continued her journaling, exercise, and self-care activities. The reconnection to the relationship with David allowed her to integrate the death story with the relationship in a way that prioritized David’s struggles with depression as most significant, and the mode of death and death scene as secondary. Positive memories, humorous incidents, and discussion of the painful periods of depression came up regularly. Treatment continued for another half-year. Notable changes followed and included a deeper engaged with her work, family, and friends; increasing positive emotions; and reduction in traumatic imagery and dreams. Yearning for David was less pronounced, the death story was better integrated with the life story, and Helen found increased support in the memory of the good times in her relationship with David. In the concluding sessions, her experience of the therapist and therapeutic relationship, and how these contributed to her improvement, were part of the closing of this treatment.</p></sec></sec><sec id=\"s5\"><title>Concluding Remarks</title><p>The interplay between trauma and bereavement in traumatic bereavements has received much attention but consensus remains elusive (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>). Attention to the extreme stresses involved in bereavements occurring under traumatic circumstances is critical to understanding both adaptive and maladaptive bereavement responses. At the same time, the decidedly interpersonal aspects of bereavement, grief and mourning should remain squarely in the center of the understanding of traumatic loss. It is a mark of the maturation of the bereavement field that many clinicians with a bereavement focus pay particular attention to circumstances of trauma. The traumas involved in the death circumstances can be joined by traumas related to the psychological representations of the attachment bond and interpersonal relationship. Any number of bereavement circumstances can result in the bereaved responding with a combination of trauma-related elements. These may be most appropriately understood from a combination of the biopsychosocial (including stress and PTSD), the degree of integration of the story of the death, and the degree and types of psychological engagement with the continuing bond with the deceased. We advocate for the use of the TTMB because its conceptual and applied perspective address the relevant biopsychosocial functioning, death narrative, and relationship with the deceased variables (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B56\" ref-type=\"bibr\">56</xref>).</p><p>Clinical work is often geared to assessment and interventions seeking to restore balanced biopsychosocial functioning. In interventions following disordered and maladaptive bereavement, the rebalancing and reworking of the relationship to the deceased may proceed without any direct assistance or awareness of the therapist. In other situations, therapy may focus on the ongoing relationship to the deceased without particular attention to the biopsychosocial status of the griever. It is most efficacious, however, for clinicians to use a balanced approach such as that of the TTMB to address both function and the relationship to the deceased. Such an approach facilitates the return to more adequate function, growth, and adaptation to loss over time.</p></sec><sec id=\"s6\"><title>Author Contributions</title><p>SSR, RM, and EW contributed to the design and organization of the article. SSR supplied the case material. RM and EW commented and expanded the discussion of the case. All three are responsible for <xref rid=\"T1\" ref-type=\"table\">\n<bold>Table 1</bold>\n</xref> and <xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figures 2</bold>\n</xref> and <xref ref-type=\"fig\" rid=\"f3\">\n<bold>3</bold>\n</xref>. SSR provided <xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1</bold>\n</xref> and <xref rid=\"T2\" ref-type=\"table\">\n<bold>Tables 2</bold>\n</xref> and <xref rid=\"T3\" ref-type=\"table\">\n<bold>3</bold>\n</xref>. EW spearheaded the consideration of the diagnostic issues in the DSM and the ICD, was in charge of references, and fact checked all material. SSR was the lead author who did the most of the writing and editing. RM and EW expanded and critiqued each draft leading to the final product. The authors view this as a team effort. The order of the names reflects not only the extent of contributions on the one hand but also the three names testify to the fact that “the whole is more than the sum of its parts.”</p></sec><sec id=\"s7\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><title>Acknowledgments</title><p>The authors thank Alexander Manevich for his valuable assistance at all stages of the manuscript preparation.</p></ack><fn-group><fn id=\"fn1\"><label>1</label><p>The assessment over time waves represent pictorially how a series of time points would look if graphed. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Psychiatry</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Psychiatry</journal-id><journal-title-group><journal-title>Frontiers in Psychiatry</journal-title></journal-title-group><issn pub-type=\"epub\">1664-0640</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33093834</article-id><article-id pub-id-type=\"pmc\">PMC7523538</article-id><article-id pub-id-type=\"doi\">10.3389/fpsyt.2020.540738</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Psychiatry</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Interaction of Background Noise and Auditory Hallucinations on Phonemic Mismatch Negativity (MMN) and P3a Processing in Schizophrenia</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Francis</surname><given-names>Ashley M.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/919701\"/></contrib><contrib contrib-type=\"author\"><name><surname>Knott</surname><given-names>Verner J.</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/54628\"/></contrib><contrib contrib-type=\"author\"><name><surname>Labelle</surname><given-names>Alain</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/131093\"/></contrib><contrib contrib-type=\"author\"><name><surname>Fisher</surname><given-names>Derek J.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"fn001\">\n<sup>*</sup>\n</xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/56136\"/></contrib></contrib-group><aff id=\"aff1\">\n<sup>1</sup>\n<institution>Department of Psychology, Saint Mary’s University</institution>, <addr-line>Halifax, NS</addr-line>, <country>Canada</country>\n</aff><aff id=\"aff2\">\n<sup>2</sup>\n<institution>Royal Ottawa Mental Health Centre</institution>, <addr-line>Ottawa, ON</addr-line>, <country>Canada</country>\n</aff><aff id=\"aff3\">\n<sup>3</sup>\n<institution>Department of Psychology, Carleton University</institution>, <addr-line>Ottawa, ON</addr-line>, <country>Canada</country>\n</aff><aff id=\"aff4\">\n<sup>4</sup>\n<institution>Department of Psychology, Mount Saint Vincent University</institution>, <addr-line>Halifax, NS</addr-line>, <country>Canada</country>\n</aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Kiyoto Kasai, University of Tokyo, Japan</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Yegang Hu, Shanghai Jiao Tong University, China; Tsuyoshi Araki, University of Tokyo, Japan</p></fn><corresp id=\"fn001\">*Correspondence: Derek J. Fisher, <email xlink:href=\"mailto:Derek.Fisher@MSVU.CA\" xlink:type=\"simple\">Derek.Fisher@MSVU.CA</email>\n</corresp><fn fn-type=\"other\" id=\"fn002\"><p>This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>540738</elocation-id><history><date date-type=\"received\"><day>06</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>17</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Francis, Knott, Labelle and Fisher</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Francis, Knott, Labelle and Fisher</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Auditory hallucinations (AHs) are among the cardinal symptoms of schizophrenia (SZ). During the presence of AHs aberrant activity of auditory cortices have been observed, including hyperactivation during AHs alone and hypoactivation when AHs are accompanied by a concurrent external auditory competitor. Mismatch negativity (MMN) and P3a are common ERPs of interest within the study of SZ as they are robustly reduced in the chronic phase of the illness. The present study aimed to explore whether background noise altered the auditory MMN and P3a in those with SZ and treatment-resistant AHs.</p><sec><title>Methods</title><p>MMN and P3a were assessed in 12 hallucinating patients (HPs), 11 non-hallucinating patients (NPs) and 9 healthy controls (HCs) within an auditory oddball paradigm. Standard (P = 0.85) and deviant (P = 0.15) stimuli were presented during three noise conditions: silence (SL), traffic noise (TN), and wide-band white noise (WN).</p></sec><sec><title>Results</title><p>HPs showed significantly greater deficits in MMN amplitude relative to NPs in all background noise conditions, though predominantly at central electrodes. Conversely, both NPs and HPs exhibited significant deficits in P3a amplitude relative to HCs under the SL condition only.</p></sec><sec><title>Significance</title><p>These findings suggest that the presence of AHs may specifically impair the MMN, while the P3a appears to be more generally impaired in SZ. That MMN amplitudes are specifically reduced for HPs during background noise conditions suggests HPs may have a harder time detecting changes in phonemic sounds during situations with external traffic or “real-world” noise compared to NPs.</p></sec></abstract><kwd-group><kwd>schizophrenia</kwd><kwd>hallucinations</kwd><kwd>mismatch negativity</kwd><kwd>electroencephalography</kwd><kwd>ERP</kwd><kwd>event-related potentials</kwd><kwd>P3a</kwd><kwd>P300</kwd></kwd-group><counts><fig-count count=\"4\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"105\"/><page-count count=\"12\"/><word-count count=\"6232\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><sec id=\"s1_1\"><title>Schizophrenia</title><p>Schizophrenia (SZ) is a debilitating neurological disease (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>) that affects approximately 1% of the world’s population (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>), with the global prevalence of SZ rising by over 5% percent in 26 years (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>). SZ is a multifaceted disease that affects one’s behavior, cognition, and intellectual functioning. Among the cardinal symptoms of SZ are auditory hallucinations (AH), being reported in 50%–80% of diagnosed cases (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>–<xref rid=\"B10\" ref-type=\"bibr\">10</xref>).</p><p>AHs are described as an aural sensory experience occurring during an awake state in the absence of external stimulation, over which the individual feels they have no control and which have a sense of realism (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B11\" ref-type=\"bibr\">11</xref>). AHs are believed to be a consequence of disruptions in the metacognitive processes that categorizes self-generated and external information sources, in addition to further deficits in information processing (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>–<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). The exact cause and mechanism of AHs, however, still remain unclear (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). It has been suggested that AHs may compete against external auditory stimuli for attentional resources (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>); this is supported by the findings that AHs and auditory cortex activity are diminished in cases where external speech competitors are presented (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>–<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). While AHs have been explored using many different methodologies, electroencephalography (EEG) has proven effective at capturing concurrent rapid changes in cortical activity (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>).</p></sec><sec id=\"s1_2\"><title>EEG</title><p>EEG is a non-invasive procedure that measures the summation of the neural activity of large groups of neurons firing; Event-related potentials (ERPs) are derived from EEG by assessing neuroelectric activity elicited simultaneously in response to stimuli (e.g., light or tones). ERPs are an exceptionally sensitive method to index cognition due to their temporal sensitivity and can be used to help supplement and comprehend behavioral observations.</p></sec><sec id=\"s1_3\"><title>MMN</title><p>Within SZ, one of the most commonly studied ERPs is the auditory mismatch negativity [MMN; (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>–<xref rid=\"B26\" ref-type=\"bibr\">26</xref>)]. MMN is well suited in that it does not require a behavioral response by the participant (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). MMN is elicited by any detectable change in an auditory stimulus, occurring regardless of conscious awareness to the change (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B29\" ref-type=\"bibr\">29</xref>). MMN is a fronto-central negatively peaking waveform with a typical latency of 90–250 ms (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>–<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). It was previously suggested that this waveform is produced when there is a discriminatory difference between the auditory stimuli presented and the memory trace of previous stimuli (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). The more recent prediction error models of the MMN suggest this waveform is elicited when the incoming stimulus violates a prediction based on a sensory model of the current auditory environment (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>).</p><p>MMN amplitude has been shown to be robustly reduced in individuals with SZ when compared to healthy controls (HCs) (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>, <xref rid=\"B35\" ref-type=\"bibr\">35</xref>). It appears MMN deficits are particularly robust in SZ among other psychotic disorders. Previous work has reported MMN amplitude reductions were most prominent in patients with SZ when compared to individuals with bipolar disorder (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>, <xref rid=\"B37\" ref-type=\"bibr\">37</xref>). Previous studies measuring MMN alterations in major psychiatric disorders (e.g., depression, obsessive-compulsive disorder) have found similar findings, suggesting the decreased amplitude in MMN may be specific to the SZ population (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>–<xref rid=\"B41\" ref-type=\"bibr\">41</xref>).</p><p>MMN has also been found to index brain deterioration in the SZ clinical populations (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>–<xref rid=\"B44\" ref-type=\"bibr\">44</xref>). MMN amplitude reduction has been conceptualized to be associated with gray matter loss in the supra-temporal cortex, as well as widespread loss across the cortex (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B45\" ref-type=\"bibr\">45</xref>, <xref rid=\"B46\" ref-type=\"bibr\">46</xref>). In addition, greater reductions of MMN amplitudes have been observed in later stages of illness (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>–<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). The reduction of MMN amplitudes can in part be attributed to the deficient functioning of <italic>N-</italic>methyl-D-aspartate (NMDA) glutamate receptor (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>–<xref rid=\"B52\" ref-type=\"bibr\">52</xref>).</p><p>Given the heterogeneity of SZ, there has been interest in how specific symptoms, such as AHs, contribute to brain-based changes in this illness (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). Previous research has found a link between severity of AH trait (i.e., predisposition to experience AHs) and MMN amplitude deficits (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B53\" ref-type=\"bibr\">53</xref>, <xref rid=\"B54\" ref-type=\"bibr\">54</xref>), suggesting that an increase in AH trait is associated overall decrease in MMN amplitude (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>–<xref rid=\"B55\" ref-type=\"bibr\">55</xref>). The link between AH trait, the MMN and neuroanatomy has been previously reported by Salisbury et al. (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B56\" ref-type=\"bibr\">56</xref>), who reported that gray matter loss near Heschl’s gyrus is associated with both an overall increase in AHs and a decrease in MMN amplitude. While increased hallucinatory trait has been linked to MMN deficits, the link between AH state and MMN (i.e., MMN amplitudes being reduced during a concurrent AH experience) is significantly more tenuous. This suggests that reductions in MMN amplitude are associated with brain-based changes associated with the production of hallucinations, rather than AHs usurping auditory processing resources within a limited capacity system.</p></sec><sec id=\"s1_4\"><title>P3a</title><p>The P300a, or P3a, is an ERP that often follows the MMN (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>) and is often observed between 200 and 650 ms. P3a is a positive waveform with a stereotypical fronto-central scalp distribution (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>–<xref rid=\"B60\" ref-type=\"bibr\">60</xref>). The auditory P3a is conceptualized as the directing of one’s attention to a deviant or novel sound (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>, <xref rid=\"B61\" ref-type=\"bibr\">61</xref>). Additionally, P3a can be elicited when there is a particular significance to the auditory stimulus either socially or emotionally, both of which are related to pre-existing information in long term memory (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>).</p><p>Commonly the P3a is elicited in experimental research through an active three-stimulus auditory oddball paradigm, in which the participant is presented with a string of standard stimulus with non-target distractors, and target tones for which they are told to identify (<xref rid=\"B61\" ref-type=\"bibr\">61</xref>–<xref rid=\"B63\" ref-type=\"bibr\">63</xref>). P3a can, however, be elicited through the means of a passive 2- or 3-stimulus-auditory oddball-paradigm (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>–<xref rid=\"B66\" ref-type=\"bibr\">66</xref>).</p><p>Reductions in P3a amplitude have been shown in individuals with chronic SZ (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>, <xref rid=\"B67\" ref-type=\"bibr\">67</xref>), after their first episode of psychosis (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>–<xref rid=\"B70\" ref-type=\"bibr\">70</xref>) as well as in individuals classified as being high risk for developing SZ (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>–<xref rid=\"B73\" ref-type=\"bibr\">73</xref>). This evidence suggests that P3a could serve as an important marker of SZ, including for those at risk of developing SZ. P3a is elicited in a similar manner to MMN whereby a combination of standard and deviant auditory stimuli is presented passively. P3a is elicited when attention is directed at a deviating tone. While MMN and P3a are associated, evidence shows that they are not dependant on each other and can occur in absence of one another (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>, <xref rid=\"B74\" ref-type=\"bibr\">74</xref>, <xref rid=\"B75\" ref-type=\"bibr\">75</xref>).</p><p>While pure tone stimuli are typically used to elicit the MMN and P3a, both can be elicited by any auditory deviant, including spectrally-complex phonetic stimuli (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>–<xref rid=\"B78\" ref-type=\"bibr\">78</xref>). Numerous studies have examined MMNs elicited by speech sounds to probe language-based processes in healthy and clinical populations (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>, <xref rid=\"B74\" ref-type=\"bibr\">74</xref>, <xref rid=\"B74\" ref-type=\"bibr\">74</xref>, <xref rid=\"B76\" ref-type=\"bibr\">76</xref>, <xref rid=\"B79\" ref-type=\"bibr\">79</xref>, <xref rid=\"B80\" ref-type=\"bibr\">80</xref>), with many employing phonemes (i.e., the simplest unit of speech with linguistic meaning) as standard and deviant stimuli. The earliest study to use a phonemic paradigm to investigate the MMN in SZ reported significant deficits in MMN amplitudes elicited by a cross-phoneme change in SZ patients [vs. HCs; (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>)]. In a follow-up study examining phonemic MMN in SZ patients with AHs, SZ patients with no hallucinations, and HCs (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>), SZ patients were found to have significant deficits in MMN amplitudes when compared to HCs; however, no significant differences were found between the two patients subgroups (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>).</p><p>It is speculated that P3a is altered by neurobiological instabilities such as those shown in SZ (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B66\" ref-type=\"bibr\">66</xref>) while other studies have shown a reduction in P3a amplitudes being linked to increased disease duration and an increase in AHs (<xref rid=\"B82\" ref-type=\"bibr\">82</xref>, <xref rid=\"B83\" ref-type=\"bibr\">83</xref>). Of the studies that have measured P3a with a SZ sample, reductions in P3a amplitude <italic>via</italic> the traditional oddball paradigm (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>, <xref rid=\"B84\" ref-type=\"bibr\">84</xref>–<xref rid=\"B86\" ref-type=\"bibr\">86</xref>) have been previously reported as well as P3a amplitude reductions when using a phonetic paradigm (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>). A study measuring P3a amplitude and latency with the traditional oddball phoneme paradigm in HCs showed that P3a amplitude and latency were correlated with recall of verbal information and working memory performance (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>), similar to what was found for MMN. To date, little research has been done to measure P3a amplitudes in SZ with and without AHs. The literature that does exist shows those with AH had decreased P3a amplitudes compared to those without AHs and HCs (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>).</p></sec><sec id=\"s1_5\"><title>Objectives and Hypotheses</title><p>The current study aimed to expand our understanding of the neurological workings of those with SZ, and elaborate on how AHs affect auditory processing by studying a group of individuals who report experiencing AHs and a group who do not. In order to better understand how auditory functions may change under different auditory conditions, MMN and P3a were examined under different background noise conditions [white noise (WN), “real-life” traffic noise (TN), and silence (SL)]. Compared to HCs it is expected that both patient groups will have significant reductions in both the MMN and P3a amplitudes; furthermore, it is expected that individuals currently experiencing AH will show the greatest deficits in MMN and P3a generation. Finally, we hypothesize that MMN and P3a will be reduced under conditions of auditory competition (TN and WN) compared to SL in all groups.</p></sec></sec><sec id=\"s2\"><title>Methods</title><sec id=\"s2_1\"><title>Study Participants</title><sec id=\"s2_1_1\"><title>Experimental Participants</title><p>Experimental participants were comprised of twenty-four individuals between the ages of 18–65 presenting with a primary diagnosis of SZ. Participants were recruited through the Outpatient Schizophrenia Clinic of the Royal Ottawa Mental Health Centre in Ottawa Ontario. Data from these participants in response to different auditory paradigms have previously been reported (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>, <xref rid=\"B77\" ref-type=\"bibr\">77</xref>, <xref rid=\"B87\" ref-type=\"bibr\">87</xref>); none of the neurophysiological data reported herein has previously been published.</p><p>Participants were assessed by their primary care physician with respect to inclusion/exclusion criteria for the study and had to be deemed as stable for four weeks prior to testing. Clinical history and ratings for the Positive and Negative Syndrome Scale (PANSS) were used to classify participants into the hallucinating (HP) or non-hallucinating (NP) condition. HPs (n = 12) were patients who reported a certain and consistent history of AHs over the course of their illness history. HPs exhibited a score of ≥3, which equates to a mild or greater hallucinatory experience on the hallucination item of the PANSS positive symptom scale. NPs (n = 12) were patients displaying a score of 1 on the same hallucination item of the PANSS and displaying no previous consistent AH history.</p><p>Confirmation of AHs was completed upon arrival to the laboratory through the Psychotic Symptom Rating Scale [PSYRATS; (<xref rid=\"B88\" ref-type=\"bibr\">88</xref>)]. The PSYRATS allowed us to quantify the frequency, duration, and loudness as well as other aspects of the AHs. HP and NPs were equivalent on age, gender, PANSS score [Positive Scale, Negative Scale and General Psychopathology Scale; (<xref rid=\"B89\" ref-type=\"bibr\">89</xref>)] and medication dosage (converted to chlorpromazine equivalents; CPZ). All participants had to present with normal hearing according to the audiometric assessment conducted on the study day requiring thresholds of 25 dB (SPL) or less to pure tones of 500, 1,000, and 2,000 Hz. A summary of participant demographic data can be found in <xref rid=\"T1\" ref-type=\"table\">\n<bold>Table 1</bold>\n</xref>.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Summary of participant demographic data (mean ± SD).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HP</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NP</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HC</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44.25 (10.90)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45.09 (10.70)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40.44 (12.96)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sex<break/>male (female)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8(4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9(2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4(5)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rx in CPZ equivalent (mg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">500.00 (231.60)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">327.27 (161.42)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PANSS positive*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.45 (3.40)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11.18 (4.31)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PANSS negative</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14.45 (4.21)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13.27 (5.24)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PANSS general*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.73 (6.38)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.18 (4.51)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PANSS hallucination item*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.64 (0.64)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.00 (0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PSYRATS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.67 (4.16)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr></tbody></table><table-wrap-foot><fn><p>*significant difference between groups (p <.05).</p></fn></table-wrap-foot></table-wrap><sec id=\"s2_1_1_1\"><title>Exclusion Criteria</title><p>Participants were excluded if they met any of the following: Co-morbid DSM- IV TR Axis I disorder; total PANSS score >65; present account of drug abuse/dependence; history of head injury; diagnosis of epilepsy or any other form of neurological disorder; treatment using electroconvulsive therapy (ECT) within the previous year; extrapyramidal symptoms (EPS); significant cardiac illness; or abnormal audiometric assessment.</p></sec></sec><sec id=\"s2_1_2\"><title>Control Participants</title><p>HCs were 12 volunteers who displayed normal hearing. As was the case with the clinical participants, data from these unaffected controls recorded in response to different auditory paradigms have previously been reported (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>, <xref rid=\"B77\" ref-type=\"bibr\">77</xref>, <xref rid=\"B87\" ref-type=\"bibr\">87</xref>). Participants were required to self-report psychiatric, medical, neurological, and alcohol/drug use/abuse histories as well as no current regular medication use (with the exception of oral contraceptives). Controls were matched to the experimental sample on measures of age, gender, and NART scores.</p></sec></sec><sec id=\"s2_2\"><title>ERP Recording</title><p>ERPs of interest were extracted from the EEG activity, which was recorded for each participant using an electrode cap with Ag<sup>+</sup>/Ag<sup>+</sup>-Cl<sup>−</sup> ring electrodes at 32 scalp sites according to the 10–20 system of electrode placement, including three midline sites [frontal (F<sub>z</sub>), central (C<sub>z</sub>), parietal (P<sub>z</sub>)], three left hemisphere [frontal (F<sub>3</sub>), central (C<sub>3</sub>), temporal (T<sub>7</sub>)] and three right hemisphere [frontal (F<sub>4</sub>), central (C<sub>4</sub>), temporal (T<sub>8</sub>)]. Electrodes were also placed on right and left mastoid, as well as nose and mid-forehead to serve as ground and reference channels respectively. Electro-oculogram activity was recorded from supra-/sub-orbital and external canthi sites <italic>via</italic> bipolar channels. All electrode impedances were below 5 kΩ; all electrical activity was recorded using BrainVision Recorder software with an amplifier bandpass setting of 0.1-30 Hz and digitized at 500 Hz. Data was then stored on a hard-drive for offline analysis using the BrainVision Analyzer software.</p></sec><sec id=\"s2_3\"><title>Neurophysiological Battery</title><p>MMN and P3a were assessed during three background conditions: SL, wide-band WN, and TN. The consonant-vowel (CV) syllables used to elicit MMN and P3a activity was identical to that of (<xref rid=\"B90\" ref-type=\"bibr\">90</xref>). The standard “ba’” (510 stimuli; P = 0.85) and deviant “da” (90 stimuli; P = 0.15) phonemes were presented in one block of 600 stimuli for each condition; presentation orders of background noise conditions (TN, SL, and wideband WN) were randomized and counter-balanced using a Latin square design. Stimuli were presented with an interstimulus interval (ISI) of 550–600 ms in a pseudo-randomized order ensuring that there was a minimum of three standard tones between each deviant.</p><p>The CV stimuli were created by having a male speak into a studio microphone and digitizing the audio stimuli using the Audacity program (22-kHz sampling rate, 11-kHz anti-aliasing filter, 12 dB/octave); edited to 150-ms duration. Spectral analysis of the CV stimuli showed them to display most of their power in the 100–2,000 Hz range, with very little power above 4,000 Hz. This was confirmed by Fast Fourier Transform (FFT) analysis, which showed most of the auditory energy to exist in the 100–2,000 Hz range in both CVs.</p><p>The WN and TN backgrounds were created within the hospital Audio-Visual department by digitizing from a pre-recorded sound effects audio tape and transferring ~12-min segments of each noise type to (CD) disc. Background noise was presented through external speakers and computer-presented CV stimuli (delivered using a digital-to-audio sampling rate of 16 kHz) were delivered binaurally through headphones, with the peak intensity of the CV signals being 10-dB sound pressure level (SPL), higher than the background noise intensity of 60 dB (SPL). Therefore, the signal-to-noise ratio (SNR) was +10 dB in the combined conditions. While the stimuli were presented, participants were asked to watch a silent, neutral emotive video and ignore the auditory phoneme stimuli being presented; each block lasted approximately 6 min.</p><p>Following each block, participants were asked to rate the hallucinations experienced during the recordings on the following three dimensions; 1) duration (0 = no AHs; 7 = continuous AHs); 2) loudness (0 = not audible; 7 = extremely loud); and 3) clarity (0 = unintelligible; 7 = very clear). See <xref rid=\"T2\" ref-type=\"table\">\n<bold>Table 2</bold>\n</xref> for mean hallucination scores for each noise condition.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Mean (± SE) state hallucination ratings separated by noise conditions.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">State Hallucination rating </th><th valign=\"top\" colspan=\"3\" align=\"center\" rowspan=\"1\">Noise condition</th></tr><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Silence</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Traffic Noise</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">White Noise</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Duration</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.08 (0.57)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.83 (0.61)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.50 (0.57)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Loudness</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.50 (0.29)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.75 (0.30)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.91 (0.45)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Clarity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.75 (0.54)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.83 (0.40)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.08 (0.54)</td></tr></tbody></table></table-wrap></sec><sec id=\"s2_4\"><title>EEG Data Processing</title><p>Data from all 32 scalp sites was re-referenced from a common reference to the nose channel before being segmented into standard and deviant tones for each of the three noise conditions (SL, TN, and WN). Digital filters were then applied using a band pass of 0.15–20 Hz (<xref rid=\"B91\" ref-type=\"bibr\">91</xref>). Electrical epochs (500-ms period, commencing 100-ms pre-stimulus) were corrected for eye movement (residual movement and blinks) using the Gratton & Coles algorithm (<xref rid=\"B92\" ref-type=\"bibr\">92</xref>), and baseline corrected using the pre-stimulus period (i.e., −100–0 ms); following these corrections, epochs exceeding ± 100 µV were excluded from further analysis. Following this, the data was put through an artifact rejection process whereby any epochs with data exceeding a 20µV/ms voltage step or exceeding ±75 µV within the epoch were excluded and the data was then averaged. Difference waves were generated by subtracting the waveforms of the standard stimulus away from waveforms generated by the deviant stimulus for each of the three conditions.</p><sec id=\"s2_4_1\"><title>MMN Processing</title><p>MMN waveforms were individually assessed in difference waves within a custom selection window from 100 to 270 ms; this window was chosen by observing the grand average of the data. MMN peaks were picked as the most negative point within the window and the output was the average within five voltage points (10 ms) to the left and right of the peaks amplitude. MMN amplitude and latency were measured at scalp site F<sub>z</sub> which is the site of maximum amplitude.</p></sec><sec id=\"s2_4_2\"><title>P3a Processing</title><p>P3a was analyzed through similar means and a peak detection window of 200-450 ms was identified for the difference waveforms this window was chosen by observing the grand average of the data. P3a was then picked as the largest positive peak within the given window of time. P3a amplitudes were quantified as the average within five voltage points (10 ms) to the right and left of the positive peak. P3a latency and amplitude were measured at C<sub>z</sub>, as this was the site of maximum amplitude.</p></sec></sec><sec id=\"s2_5\"><title>Study Procedure</title><p>Participants completed informed consent before attending the laboratory session. Following the completion of informed consent, participants were asked to complete demographic information. In addition, participants were measured on general medical/health, handedness [Edinburg Handedness Inventory; (<xref rid=\"B93\" ref-type=\"bibr\">93</xref>)], the National Adult Reading Test [NART; (<xref rid=\"B94\" ref-type=\"bibr\">94</xref>)] which provides an approximation of premorbid Full-Scale I.Q. Upon arrival to the laboratory, EEG electrodes were applied and participants completed the experiment.</p><p>Testing took place between 11 am and 2 pm and participants were asked to refrain from drug use (tobacco, alcohol, and cannabis), and medication (with the exception of anti-psychotics and adjunct drugs) beginning at midnight the night before their testing session.</p><p>Study procedures were conducted following clearance by both the Royal Ottawa Mental Health Center and Carleton University (CU) Research Ethics Boards.</p></sec><sec id=\"s2_6\"><title>Statistical Analysis</title><p>Some participants had to be excluded following data analysis due to uninterpretable data. More specifically, three HCs and one non-hallucinating participant were lost, leaving final groups of n = 12 for HPs, n = 11 for NPs, and n = 9 for HCs.</p><p>Analysis was carried out by using the Statistical Package for the Social Sciences (SPSS; SPSS Inc., Chicago, IL). MMN and P3a amplitudes were assessed with mixed analysis of variance (ANOVA), with one between group measure (3 levels: HC, HP, and NP) and three-within group factors 1. Noise (3 levels: SL, TN, WN), 2. Site (3 levels: left, midline, right), and 3. Region (2 levels: Frontal and Central). <italic>A priori</italic> planned comparisons were conducted to quantify MMN and P3a amplitude differences between groups.</p><p>Latency was analyzed with an additional mixed ANOVA with the same between groups measure as amplitude (3 levels: HC, HP, and NP) and one-within groups factor (noise—3 levels: SL, TN, and WN). <italic>A priori</italic> planned comparisons were conducted to determine group differences in latency during each task. For both amplitudes and latencies, effect sizes are stated using Hedges’<italic>g</italic> to account for uneven groups.</p><p>To assess the relationship between state hallucination ratings and noise condition, an ANOVA was performed with two within subject measures [1. noise condition (3 levels: SL, traffic, and WN) and 2. Hallucination state rating (3 levels: duration, loudness, and clarity)].</p><p>Correlational results were conducted to measure correlations between behavioral and demographic measures and MMN and P3a amplitudes. Bivariate Correlations (Spearmans’s rho) using a two-tailed significance level were run to analyse correlations between demographic and hallucination data with our ERP data. Additional correlations were performed between hallucination ratings and all scalp sites within each condition (SL, TN, and WN) to determine if there were any relationships between hallucinations experienced during the experiment in their duration, loudness and clarity to the P3a and MMN amplitude changes experienced.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec id=\"s3_1\"><title>MMN Amplitude</title><p>There was a main effect of region, F(1,29) = 38.68 <italic>p</italic> < 0.001, due to larger amplitudes being shown in the frontal (M = −1.24 µV, SD = 1.12) compared to the central (M = −0.67 µV, SD = 0.97) regions. The effect of region was further shown when analyzing group differences by pairwise comparisons; HPs (M = −0.39 µV, SD= 1.06) showed a significant deficit over HCs (M = −0.89 µV, SD = 0.71; <italic>p</italic> = 0.029, <italic>g</italic> = 0.54), however, only for the central region. Finally, the frontal region showed significant differences between amplitudes during the SL (M = −1.34 µV, SD = 1.34) and TN (M = −0.88 µV, SD = 0.92, <italic>p</italic> = 0.029, <italic>g</italic> = 0.40) conditions, as well as the TN (M = −0.88 µV, SD = 0.92) and WN conditions (M = −1.51 µV, SD = 1.10, <italic>p</italic> = 0.001, <italic>g</italic> = 0.62).</p><p>While no main effect of group was shown, planned pairwise comparisons revealed significant differences at site C<sub>4</sub> within the silent condition between HCs (M = −1.04, SD = 0.62) and HPs (M = −0.025, SD = 1.01, <italic>p =</italic> 0.013, <italic>g</italic> = 1.17) and HPs (M = −0.025 µV, SD =1.01) and NPs (M = −1.0 µV, SD = 0.86, <italic>p</italic> = 0.011, <italic>g =</italic> 1.04; <xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1</bold>\n</xref>), no significant differences were observed between NPs and HCs.</p><fig id=\"f1\" position=\"float\"><label>Figure 1</label><caption><p>Grand averaged subtracted waveforms showing MMN and P3a across six scalp sites (F<sub>3</sub>, F<sub>z</sub>, F<sub>4</sub>, C<sub>3</sub>, C<sub>z</sub>, C<sub>4</sub>) for all participant groups (HC, HP, NP) during the silence condition.</p></caption><graphic xlink:href=\"fpsyt-11-540738-g001\"/></fig><p>Additional significant differences resulting from pairwise comparisons were seen between HPs (M<sub>Traf</sub> = 0.088 µV, SD<sub>Traf</sub> = 0.80) and NPs (M<sub>Traf</sub> = −0.98 µV, SD<sub>Traf</sub> = 1.02; <italic>p</italic> = 0.010, <italic>g</italic> = 1.17) and HPs (M<sub>Traf</sub> = 0.088 µV, SD<sub>Traf</sub> = 0.80) and HCs (M<sub>Traf</sub> = −0.79 µV, SD<sub>Traf</sub> = 0.69; <italic>p</italic> = 0.039, <italic>g</italic> = 1.16) during the traffic condition however, only at site C<sub>3</sub> (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2</bold>\n</xref>). Additionally, at site C<sub>z</sub>, HPs (M<sub>Traf</sub> = −0.31 µV, SD<sub>Traf</sub> = 1.02) showed significant reductions in MMN amplitude compared to NPs (M<sub>Traf</sub> = −1.11, SD<sub>Traf</sub> = 0.74;<italic>p</italic> = 0.031; <italic>g</italic> = 0.89) in the traffic condition (<xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2</bold>\n</xref>). Finally, significant differences were also observed at site C<sub>4</sub> between HCs (M<sub>WN</sub> = −1.28, SD<sub>WN</sub> = 0.58) and the two patient groups (M<sub>NP</sub> = −0.35, SD<sub>NP</sub> = 0.66; <italic>p</italic> = 0.043, <italic>g</italic> = 1.49; M<sub>HP</sub> = −0.27, SD<sub>HP</sub> = 1.38; <italic>p</italic> = 0.027, <italic>g</italic> = 0.91) in the WN condition (<xref ref-type=\"fig\" rid=\"f3\">\n<bold>Figure 3</bold>\n</xref>). See <xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Table 3</bold>\n</xref> in supplemental materials for average MMN amplitudes within each group (HC, HP, and NP) under each noise condition (SL, traffic and WN).</p><fig id=\"f2\" position=\"float\"><label>Figure 2</label><caption><p>Grand averaged subtracted waveforms showing MMN and P3a across six scalp sites (F<sub>3,</sub> F<sub>z,</sub> F<sub>4,</sub> C<sub>3,</sub> C<sub>z,</sub> C<sub>4</sub>) for all participant groups (HC, HP, NP) during the traffic condition.</p></caption><graphic xlink:href=\"fpsyt-11-540738-g002\"/></fig><fig id=\"f3\" position=\"float\"><label>Figure 3</label><caption><p>Grand averaged subtracted waveforms showing MMN and P3a across six scalp sites (F<sub>3</sub>, F<sub>z</sub>, F<sub>4</sub>, C<sub>3</sub>, C<sub>z</sub>, C<sub>4</sub>) for all participant groups (HC, HP, NP) during the white noise condition.</p></caption><graphic xlink:href=\"fpsyt-11-540738-g003\"/></fig></sec><sec id=\"s3_2\"><title>MMN Latencies</title><p>A main effect of condition was found, F(2, 58) = 10.93, <italic>p</italic> < 0.001, due to longer latencies in the WN (M = 199.19 ms SD = 41.03) compared to the SL (M = 156.70 ms, SD = 44.25) and traffic conditions (M = 162.18 ms, SD = 45.17).</p></sec><sec id=\"s3_3\"><title>P3a Amplitudes</title><p>While there was no main effect of group, there were significant differences in the amplitudes present in HC between the SL condition (M = 1.39 µV, SD = 0.91) and the two noise conditions (M<sub>Traf</sub> = 0.18 µV, SD<sub>Traf</sub> = 0.82, <italic>p</italic> = 0.008, <italic>g</italic> = 1.40; M<sub>WN</sub> = 0.25 µV, SD<sub>WN</sub> = 0.93; <italic>p</italic> = 0.003, <italic>g</italic> = 1.24).</p><p>Planned comparisons showed larger amplitudes for HCs (M = 1.39 µV, SD = 0.91) compared to NPs (M = 0.34 µV, SD = 1.33; <italic>p</italic> = 0.022, <italic>g</italic> = 0.90), under the SL condition (<xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1</bold>\n</xref>) and HPs (M = 0.77 µV, SD = 1.25) compared to NPs (M = 0.039 µV, SD = 0.82) during the TN condition (<italic>p</italic> = 0.036, <italic>g</italic> = 0.69; <xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2</bold>\n</xref>). Followed up to individual sites, during the SL condition HCs exhibited larger amplitudes then the NPs at F<sub>z</sub> (M<sub>HC</sub> = 1.30 µV, SD<sub>HC</sub> = 0.61; M<sub>NP</sub> = −0.056 µV, SD<sub>NP</sub> = 1.54; <italic>p</italic> = 0.028, <italic>g</italic> = 1.11) and C<sub>z</sub> (M<sub>HC</sub> = 2.70 µV, SD<sub>HC</sub> = 1.63; M<sub>NP</sub> = 1.22, SD<sub>NP</sub> = 1.49; <italic>p</italic> = 0.032, <italic>g</italic> = 0.95; <xref ref-type=\"fig\" rid=\"f1\">\n<bold>Figure 1</bold>\n</xref>). Additionally, HPs (M = 1.14 µV, SD = 1.42) exhibited a larger P3a than NPs (M = −0.056 µV, SD = 1.54) at site F<sub>z</sub> during the SL condition, <italic>p</italic> = 0.035, <italic>g</italic> = 0.81. Finally, during the TN condition, HPs exhibited larger amplitudes then the NPs at F<sub>3</sub> (M<sub>HP</sub> = 0.97 µV, SD<sub>HP</sub> = 1.03; M<sub>NP</sub> = −0.24, SD<sub>NP</sub> = 0.78; <italic>p</italic> = 0.003, <italic>g</italic> = 1.32) and F<sub>z</sub> (M<sub>HP</sub> = 0.97 µV, SD<sub>HP</sub> = 0.91; M<sub>NP</sub> = 0.19, SD<sub>NP</sub> = 0.79; <italic>p</italic> = 0.019, <italic>g</italic> = 0.84; <xref ref-type=\"fig\" rid=\"f2\">\n<bold>Figure 2</bold>\n</xref>). See supplemental materials for <xref ref-type=\"supplementary-material\" rid=\"SM1\">\n<bold>Table 4</bold>\n</xref> reporting P3a amplitude grand averages for each participant group (HC, HP, and NP) under each noise condition (SL, TN, and WN).</p></sec><sec id=\"s3_4\"><title>P3a Latencies</title><p>There was a main effect of task found F(2,58) = 4.94, <italic>p</italic> = 0.010 due to longer latencies being present during the WN condition (M = 286.57 ms, SD = 51.83) compared to the other two conditions (M<sub>silence</sub> = 256.79 ms, SD<sub>silence</sub> = 47.39; M<sub>Traf</sub> = 258.37 ms, SD<sub>Traf</sub> = 54.29) conditions. <italic>Follow up</italic> comparisons revealed this was limited to the HC condition, <italic>p</italic>-values ranging from <italic>p</italic> = 0.021 to <italic>p</italic> = 0.007.</p></sec><sec id=\"s3_5\"><title>MMN Correlations</title><p>PANSS negative symptom scores were positively correlated (i.e., decreased MMN amplitude with increased PANSS score) at site F<sub>4</sub> during the traffic condition (r = .42, p = .043).</p><p>PSYRATS scores were positively correlated (i.e., decreased MMN amplitude with increased PSYRATS score) at sites C<sub>3</sub> (r = .46, p = .028) and C<sub>Z</sub> (r = .42, p = .048) in the traffic condition.</p><p>Age was also positively correlated with frontal site F<sub>4</sub> in the WN condition (<italic>r</italic> = 0.49, <italic>p</italic> = 0.004). Finally, the neuroleptic dosage (as measured in chlorpromazine equivalents) was significantly correlated with F<sub>4</sub> (<italic>r</italic> = 0.42, <italic>p</italic> = 0.048) in the SL condition. Additionally, a negative correlation (r = −.49, p = .018) was present between the latency during the WN condition and neuroleptic dosage.</p><p>Additional bivariate correlational analysis using spearman’s rho was used to examine the relationship between duration, loudness and clarity of any hallucinations present during the experiment and their correlation with MMN amplitude at all sites separated by condition. There were no significant findings between measures of hallucinatory state and MMN amplitude.</p></sec><sec id=\"s3_6\"><title>P3a Correlations</title><p>Age was significantly correlated with C<sub>3</sub> (<italic>r</italic> = −0.36, <italic>p</italic> = 0.043) in the SL condition. Neuroleptic dosage was found to significantly correlate with P3a amplitude at C<sub>3</sub> (<italic>r</italic> = 0.52, <italic>p</italic> = 0.010), however, only in the SL condition.</p><p>Additional bivariate correlational analysis using Spearman’s rho was used to examine the relationship between duration, loudness, and clarity of any hallucinations present during the experiment and their correlation with P3a amplitude at all sites separated by condition. There were significant correlations between the clarity (<italic>r</italic> = −0.69, <italic>p</italic> = 0.013; <xref ref-type=\"fig\" rid=\"f4\">\n<bold>Figure 4</bold>\n</xref>) of hallucinations and amplitude at site F<sub>3</sub> during the TN condition.</p><fig id=\"f4\" position=\"float\"><label>Figure 4</label><caption><p>Scatter plot showing the negative relationship between F<sub>3</sub> and state hallucination clarity in hallucinating participants.</p></caption><graphic xlink:href=\"fpsyt-11-540738-g004\"/></fig></sec><sec id=\"s3_7\"><title>Hallucination State Ratings</title><p>There was no main effect of noise condition on hallucination state ratings F(2,22) = 0.55, <italic>p</italic> = 0.58.</p></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>This study assessed the relationship between MMN and P3a amplitudes and latencies in a SZ sample with and without treatment-resistant AHs. Not surprising, MMN amplitude was decreased overall in both patient samples when compared to HCs which has been a consistent finding across the literature (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>, <xref rid=\"B36\" ref-type=\"bibr\">36</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>–<xref rid=\"B44\" ref-type=\"bibr\">44</xref>, <xref rid=\"B54\" ref-type=\"bibr\">54</xref>, <xref rid=\"B95\" ref-type=\"bibr\">95</xref>, <xref rid=\"B96\" ref-type=\"bibr\">96</xref>). Interestingly, this finding was only significant for the HP group in comparison to the NP group and, furthermore, degree of MMN reduction was associated with trait-level markers of AH severity (as indexed by the PSYRATS). These findings are consistent with work by Ford et al. (<xref rid=\"B84\" ref-type=\"bibr\">84</xref>), which showed that Brodmann area 41 (i.e., transverse temporal gyri of Heschl), the temporal site of MMN generation, was functioning at a higher level in non-hallucinators compared to the hallucinators.</p><p>Furthermore, decreases in amplitude tended to be condition specific; more specifically, while HPs had significantly reduced amplitudes in comparison to NPs and HCs, this finding was restricted to the traffic and WN conditions. This suggests that the brain of individuals with AHs may have an overall harder time processing changes in phonetic stimuli when background noise is present, perhaps due to structural or functional differences in cortical function that underlie AHs. Furthermore, when background noise becomes more complex or irregular (TN condition) individuals with AH may struggle with secondary cortical processing as opposed to during the SL conditions (<xref rid=\"B97\" ref-type=\"bibr\">97</xref>). It has been previously shown that AHs are reduced when external speech is present (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>–<xref rid=\"B22\" ref-type=\"bibr\">22</xref>), with a corresponding deactivation of auditory cortex suggesting generalized cortical hypofunction due to sensory overload when AHs compete with external auditory stimulation (<xref rid=\"B98\" ref-type=\"bibr\">98</xref>, <xref rid=\"B99\" ref-type=\"bibr\">99</xref>). Our findings suggest that auditory processing network is more taxed in the HP group during the traffic condition in comparison to all other conditions, and based on our correlations, it does not appear that there is a lack of resources present in this group but rather that this could outline a deficit in automatic primary or secondary cortical processing in a SZ sample. This is only the case, however, for those experiencing AH, which represent a specific neurological challenge faced by this particular sample. In addition to AHs, MMN amplitude was reduced in those clinical participants with greater PANSS negative symptom scores, consistent with previous work in both those with SZ (<xref rid=\"B100\" ref-type=\"bibr\">100</xref>) and a ketamine model of SZ in healthy participants (<xref rid=\"B101\" ref-type=\"bibr\">101</xref>). In future studies, it would be beneficial to include a time course component to measure the onset and offset of AHs for the duration of each task, as this would allow for further analysis on how state AHs impact MMN and P3a amplitude and latency.</p><p>Previous studies have analyzed background noise and its overall effects on cognitive processing in individuals with SZ; one study in particular used urban and social noise conditions to determine the cognitive burden on patients (<xref rid=\"B102\" ref-type=\"bibr\">102</xref>). Both the social and urban noise conditions were comparable to our TN condition. Wright et al. (<xref rid=\"B102\" ref-type=\"bibr\">102</xref>) found that patients had significantly reduced psychomotor speed, attention, working, and verbal memory in the presence of background noise from both of their conditions. The presence of background noise inhibiting the ability of verbal and working memory could play a part in the brain’s ability to detect differences, whether that be through comparison to a memory trace or a predictive model of the auditory environment. Furthermore, verbal memory has been shown to be impacted in MMN processing when phonemic stimuli are used (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B54\" ref-type=\"bibr\">54</xref>, <xref rid=\"B103\" ref-type=\"bibr\">103</xref>, <xref rid=\"B104\" ref-type=\"bibr\">104</xref>). Additionally, Dittmann-Balcar, Thienel, and Schall (<xref rid=\"B105\" ref-type=\"bibr\">105</xref>) found that MMN was reduced in participants if they were simultaneously performing an auditory discrimination task. While our participants were told to ignore the auditory tones being presented, this could support our finding of seeing significant reductions in the TN condition for our HPs—due to the nature of the TN and WN conditions being more auditorily demanding than the SL condition. It could also have to do with the fact that individuals with SZ are known to have complications with weighting and processing incoming auditory stimuli, likely due to a delay in the brain’s ability to transmit and process incoming stimuli and then act on said stimuli (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>).</p><p>P3a amplitudes were significantly larger in the SL background noise condition when compared to both active noise conditions (TN and WN); however, this was most prominent in the NPs. In contrast to HPs, who showed specific deficits in early auditory change processing during the presence of background noise, this suggests that NPs are impaired in later attention switching or stimuli categorization processes when background noise is applied. This finding appears to conflict with the findings of Fisher et al. (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>) which showed the largest P3a deficits in the HP condition; however, this study only examined P3a response to single-vowel phonemes under conditions of no background noise.</p><p>While there was no association between P3a amplitudes and hallucinatory trait (either <italic>via</italic> group status or correlations with the PSYRATS), clarity of state hallucinations was negatively correlated with P3a amplitude at frontal site F<sub>3</sub> during the traffic condition while MMN amplitude was not correlated with state hallucination ratings. This suggests that the P3a is more sensitive to hallucinatory state, while the MMN appears to be more strongly linked to hallucinatory trait within our sample, similar to previous reports by our group in an acutely ill sample (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>).</p></sec><sec id=\"s5\"><title>Limitations and Summary</title><p>This study does not go without its limitations, most notably the small sample size and the modest level of hallucinatory activity expressed by our SZ patients; it is unclear whether more severe hallucinatory activity would produce a stronger activation with background noise. While it is difficult to induce hallucinations, future studies may wish to recruit participants with more severe histories of AHs. In addition to this, the presence of active hallucinations varied in our HP participants during the three noise conditions; in future studies, it would be beneficial to measure those actively experiencing AHs vs. those who are not experiencing active hallucinations. This would allow for a more clear understanding on the impact of active AHs on MMN and P3a amplitude and latency. Furthermore, the medicated status of the patients could have limited the observations, although there was no significant difference in neuroleptic dosage between SZ groups. Future studies may also wish to incorporate structural neuroimaging techniques, such as magnetic resonance imaging and/or diffusion tensor imaging, in order to explore the potential underlying neuroanatomical correlates of observed deficits in acoustic change detection. A final limitation is the retrospective measure of hallucinatory state; a better method may be to ask participants to signal the onset and offset of hallucinations during stimulation.</p><p>Nevertheless, the study did find interesting and novel findings suggesting those with SZ and treatment-resistent AHs show earlier (i.e., MMN) deficits in the context of background noise, while these same conditions affect later processes (i.e., P3a) more strongly in those without AHs. The P3a findings contradicted our hypotheses, as it was anticipated that the HPs would show the greatest deficit on all measures. Overall, this suggests that the presence or absence of persistent AHs is associated with differing function of the auditory processing stream, with important implications for personalized treatments.</p></sec><sec sec-type=\"data-availability\" id=\"s6\"><title>Data Availability Statement</title><p>The datasets generated for this study will not be made publicly available because participants did not consent to the sharing or public availability of data.</p></sec><sec id=\"s7\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by Research Ethics Board of the Royal Ottawa Mental Health Centre and Department of Psychology Research Ethics Board, Carleton University. The patients/participants provided their written informed consent to participate in this study.</p></sec><sec id=\"s8\"><title>Author Contributions</title><p>AF wrote all drafts of the manuscript and analyzed the data set. DF contributed to study design, collected all data, and contributed to analysis and interpretation of finding, as well as editing all drafts. VK and AL contributed to design of study and editing of final draft of the manuscript.</p></sec><sec id=\"s9\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><sec id=\"s10\" sec-type=\"supplementary-material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fpsyt.2020.540738/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fpsyt.2020.540738/full#supplementary-material</ext-link>\n</p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"DataSheet_1.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005528</article-id><article-id pub-id-type=\"pmc\">PMC7523539</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10110</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Internal Medicine</subject></subj-group><subj-group><subject>Radiology</subject></subj-group><subj-group><subject>Oncology</subject></subj-group></article-categories><title-group><article-title>Acute Liver Failure With Severe Lactic Acidosis Secondary to Infiltrative Diffuse Large B-Cell Lymphoma: An Imaging-Negative Presentation</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Hafner</surname><given-names>Andre</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Eaton</surname><given-names>David B</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nInternal Medicine, University of South Dakota Sanford School of Medicine, Rapid City, USA </aff><aff id=\"aff-2\">\n<label>2</label>\nInternal Medicine, Monument Health, Rapid City, USA </aff><author-notes><corresp id=\"cor1\">\nAndre Hafner <email>andre.hafner@coyotes.usd.edu</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10110</elocation-id><history><date date-type=\"received\"><day>4</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Hafner et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Hafner et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/38880-acute-liver-failure-with-severe-lactic-acidosis-secondary-to-infiltrative-diffuse-large-b-cell-lymphoma-an-imaging-negative-presentation\">This article is available from https://www.cureus.com/articles/38880-acute-liver-failure-with-severe-lactic-acidosis-secondary-to-infiltrative-diffuse-large-b-cell-lymphoma-an-imaging-negative-presentation</self-uri><abstract><p>Liver involvement by non-Hodgkin’s lymphoma is common in late stage disease but rarely results in severe hepatic dysfunction. Here, we discuss a case of acute liver failure (ALF) with severe lactic acidosis in a 75-year-old female with diffuse large B-cell lymphoma (DLBCL). The patient was admitted with nausea, fevers, and mild acidosis. Although radiographic imaging did not demonstrate any liver abnormality, the patient soon developed signs of ALF along with severe lactic acidosis. Despite initiation of chemotherapy, the patient deteriorated quickly and was ultimately put on comfort measures. This case highlights an uncommon manifestation of DLBCL and suggests that an accelerated timeline for beginning chemotherapy may be warranted in patients with high clinical suspicion of secondary hepatic lymphoma.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>dlbcl</kwd><kwd>lactic acidosis</kwd><kwd>acute liver failure</kwd><kwd>secondary hepatic lymphoma</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL), which is the most common hematologic malignancy worldwide [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. Despite this relative frequency, DLBCL should be recognized as a heterogeneous grouping of various subtypes that share a common morphology but differ in terms of prognosis and treatment response [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. In most cases, initial symptoms are nonspecific and patients frequently present with late stage disease. Signs and symptoms can be focal or systemic and reflect both metabolic derangements and manifestations of underlying organ damage [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. Although liver involvement is estimated to occur in 16%-46% of patients with DLBCL, it very rarely leads to acute liver failure (ALF) [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. In the majority of cases, liver involvement represents extranodal extension of the disease rather than a primary hepatic lymphoma [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>].</p><p>Our case represents an example of malignancy-associated ALF with associated lactic acidosis and liver function derangements. Of note, the patient did not display hepatomegaly and did not show any signs of liver involvement on ultrasound or CT imaging prior to liver failure. Given the occult presentation of hepatic infiltration in this patient, we believe this case represents a valuable addition to the literature and emphasizes the importance of strong clinical suspicion in the absence of radiographic findings.</p></sec><sec sec-type=\"cases\"><title>Case presentation</title><p>A 75-year-old female with generalized abdominal pain and fatigue presented to the emergency room after lab studies at an urgent care showed acute kidney injury. Past medical history was significant for hypertension, hypercholesterolemia, restless leg syndrome, and peptic ulcer disease. Home medications included atorvastatin, losartan-hydrochlorothiazide, pramipexole, and bupropion. Review of systems was positive for 12 pounds of weight loss over the previous two weeks, subjective fever, and decreased appetite.</p><p>On presentation, vital signs showed heart rate of 96 beats/minute, blood pressure of 121/74 mmHg, a temperature of 37°C, and oxygen saturation of 95% on room air. Physical exam was remarkable for diffuse abdominal tenderness without guarding. EKG showed sinus rhythm and no ST elevation or arrhythmias. A CT scan of the abdomen and pelvis without IV contrast was significant for extensive lymphadenopathy in the retroperitoneum and the para-esophageal area near the splenic hilum highly suspicious for lymphoma. Aggressive IV fluids were initiated, and the patient’s condition improved to the point where she was discharged after two days with close follow-up and scheduled lymph node biopsy.</p><p>The patient’s condition soon worsened, however, and she returned to the ER two days later with recurrent epigastric and right upper quadrant (RUQ) abdominal pain, vomiting, diarrhea, and chest pain. Laboratory studies showed an elevated anion gap of 14 mmol/L and a mildly elevated aspartate aminotransferase (ALT) of 84 U/L. The patient was admitted and started on IV fluids. Blood cultures were drawn. A GI pathogen panel was collected and was found to be negative. Serology tests for hepatitis A, B, and C were negative. Over the following three days, the patient continued to experience abdominal pain, nausea, weakness, and fevers. The mild acidosis initially resolved, but the liver enzymes continued to rise. The patient developed hypoglycemia and worsening thrombocytopenia. CT-guided para-aortic lymph node biopsy was performed on day 4, and pathology confirmed stage IV NHL, diffuse large B cell type (Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>).</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Retroperitoneal lymph node biopsy showing diffuse proliferation of large lymphocytes (hematoxylin-eosin staining; H&E)</title></caption><graphic xlink:href=\"cureus-0012-00000010110-i01\"/></fig><p>On hospital day 5, the patient was transferred to the ICU due to the onset of progressive metabolic acidosis and dyspnea concerning for sepsis or ischemic bowel. In addition, the patient’s worsening confusion, hypoglycemia, and coagulopathy indicated possible ALF (Table <xref rid=\"TAB1\" ref-type=\"table\">1</xref>). </p><table-wrap id=\"TAB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Laboratory data showing deterioration of liver function </title><p>WBC, white cell count; Hb, hemoglobin; ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase; INR, international normalized ratio; aPTT, activated partial thromboplastin time</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Labs</td><td rowspan=\"1\" colspan=\"1\">Day 1 (admission)</td><td rowspan=\"1\" colspan=\"1\">Day 5</td><td rowspan=\"1\" colspan=\"1\">Day 7</td></tr><tr><td rowspan=\"1\" colspan=\"1\">WBC (10<sup>3</sup>/µL)</td><td rowspan=\"1\" colspan=\"1\">5.2</td><td rowspan=\"1\" colspan=\"1\">6.3</td><td rowspan=\"1\" colspan=\"1\">3.3</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Hb (g/dL)</td><td rowspan=\"1\" colspan=\"1\">10.1</td><td rowspan=\"1\" colspan=\"1\">10.1</td><td rowspan=\"1\" colspan=\"1\">7.2</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Platelets (10<sup>3</sup>/µL)</td><td rowspan=\"1\" colspan=\"1\">97</td><td rowspan=\"1\" colspan=\"1\">93</td><td rowspan=\"1\" colspan=\"1\">24</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">AST (U/L)</td><td rowspan=\"1\" colspan=\"1\">84</td><td rowspan=\"1\" colspan=\"1\">453</td><td rowspan=\"1\" colspan=\"1\">3,219</td></tr><tr><td rowspan=\"1\" colspan=\"1\">ALT (U/L)</td><td rowspan=\"1\" colspan=\"1\">47</td><td rowspan=\"1\" colspan=\"1\">109</td><td rowspan=\"1\" colspan=\"1\">618</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">ALP (U/L)</td><td rowspan=\"1\" colspan=\"1\">148</td><td rowspan=\"1\" colspan=\"1\">159</td><td rowspan=\"1\" colspan=\"1\">285</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Total bilirubin (mg/dL)</td><td rowspan=\"1\" colspan=\"1\">1.12</td><td rowspan=\"1\" colspan=\"1\">1.89</td><td rowspan=\"1\" colspan=\"1\">2.76</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Glucose (mg/dL)</td><td rowspan=\"1\" colspan=\"1\">87</td><td rowspan=\"1\" colspan=\"1\">70</td><td rowspan=\"1\" colspan=\"1\">165</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Prothrombin time (s)</td><td rowspan=\"1\" colspan=\"1\">--</td><td rowspan=\"1\" colspan=\"1\">22.6</td><td rowspan=\"1\" colspan=\"1\">26.5</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">INR</td><td rowspan=\"1\" colspan=\"1\">--</td><td rowspan=\"1\" colspan=\"1\">2.0</td><td rowspan=\"1\" colspan=\"1\">2.3</td></tr><tr><td rowspan=\"1\" colspan=\"1\">aPTT (s)</td><td rowspan=\"1\" colspan=\"1\">--</td><td rowspan=\"1\" colspan=\"1\">33.2</td><td rowspan=\"1\" colspan=\"1\">30</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Fibrinogen (mg/dL)</td><td rowspan=\"1\" colspan=\"1\">--</td><td rowspan=\"1\" colspan=\"1\">--</td><td rowspan=\"1\" colspan=\"1\">163</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Anion gap (mmol/L)</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">30</td><td rowspan=\"1\" colspan=\"1\">17</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Lactate (mmol/L)</td><td rowspan=\"1\" colspan=\"1\">--</td><td rowspan=\"1\" colspan=\"1\">17.5</td><td rowspan=\"1\" colspan=\"1\">8.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">pH</td><td rowspan=\"1\" colspan=\"1\">--</td><td rowspan=\"1\" colspan=\"1\">7.09</td><td rowspan=\"1\" colspan=\"1\">7.40</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">HCO<sub>3</sub>- (mmol/L)</td><td rowspan=\"1\" colspan=\"1\">20</td><td rowspan=\"1\" colspan=\"1\">13</td><td rowspan=\"1\" colspan=\"1\">26</td></tr></tbody></table></table-wrap><p>The patient was started on a bicarb drip and empiric antibiotics. Later that day, the patient underwent a diagnostic laparoscopy followed by laparoscopic cholecystectomy and multiple liver biopsies. During the surgery, the liver was noted to look grossly abnormal showing micro to moderate nodularity (Figure <xref ref-type=\"fig\" rid=\"FIG2\">2</xref>). There was no evidence of bowel ischemia, perforation, or inflammatory changes. Following surgery, the patient developed hemodynamic instability, most likely due to severe acidosis and liver failure. The patient was started on vasopressors and remained intubated with mechanical ventilation. </p><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>Intraoperative image of the liver showing micro to moderate nodularity</title></caption><graphic xlink:href=\"cureus-0012-00000010110-i02\"/></fig><p>It was assumed at this point that the liver dysfunction was related to malignant hepatic infiltration. Chemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) along with rasburicase was initiated on day 6 with appropriate dose reductions for renal and liver failure. During the following two days, the patient suffered seizures and worsening uremia. Platelet count worsened acutely, and this was attributed to underlying hepatic failure and possible disseminated intravascular coagulation rather than drug toxicity. The patient was started on continuous renal replacement therapy on day 7 due to oliguria and progressive renal failure.</p><p>Over the following four days, the patient continued to deteriorate clinically. Blood cultures did not show growth and the patient was transitioned off antibiotics. Multiple transfusions of fresh-frozen plasma (FFP) and platelets were required due to thrombocytopenia and coagulopathy. Although there was initial success in controlling the patient’s metabolic lactic acidosis and hemodynamic status, she remained unresponsive despite being off all sedation. Ultimately, the family decided to transition the patient to comfort care measures on day 11 and the patient expired two days later.</p></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>ALF is an uncommon diagnosis that is largely associated with viral hepatitis, toxic ingestion, or drug reactions [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. It is defined by findings of coagulopathy (international normalized ratio [INR] > 1.5) and encephalopathy in a patient without pre-existing liver disease and an illness lasting less than 26 weeks. Malignant infiltration of the liver is a rare cause of ALF but may be more common than previously reported. Although one frequently cited single center study completed in 1998 reported a incidence of 0.44% [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>], two more recent multicenter studies completed in 2012 and 2015 report an incidence of 1.4% [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>] and 1.8% [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>], respectively. ALF in this setting carries a particularly poor prognosis with median survival from time of admission being reported as low as six days.</p><p>Determining the etiology of ALF is critical for guiding treatment but can be especially challenging in cases of malignant infiltrative disease. Symptoms and laboratory findings of ALF are nonspecific; however, a complete history should give some indication of possible malignant etiology, such as a history of B-symptoms or unexplained weight loss [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>]. Physical exam may show lymphadenopathy or hepatosplenomegaly. Initial serology should exclude common viral etiologies. Most cases of hepatic lymphoma are secondary rather than primary to the liver; therefore, CT imaging should demonstrate enlarged lymph nodes and delineate their pathologic extent. However, in patients with NHL ultrasound and CT findings can be unreliable in demonstrating liver involvement [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>,<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. This is particularly challenging in patients with a diffuse pattern of liver involvement and nodules less than 1 cm in size who may only have findings of hepatomegaly or no liver findings at all. Further characterization of the malignancy with imaging should involve a combined positron emission tomography/CT (PET/CT), which has been shown to have a sensitivity approaching 100% for splenic involvement of lymphoma and is vital for determining the extranodal extent of disease [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]. Further imaging with MRI is not part of the standard protocol for suspected hepatic lymphomas, but may be considered to further characterize an incidental liver finding in the absence of clinical symptoms. A PubMed search involving the keywords secondary hepatic lymphoma (SHL) and NHL did not yield any studies that investigated the sensitivity or specificity of MRI in the setting of suspected hepatic lymphoma.</p><p>In our case, CT of the abdomen with contrast did not show any liver abnormalities (Figure <xref ref-type=\"fig\" rid=\"FIG3\">3</xref>). Although liver involvement was strongly suspected due to clinical and laboratory signs of ALF, it was only during exploratory surgery that the liver was noted to look grossly abnormal. PET/CT was delayed indefinitely due to the acuity of the patient’s condition and biopsy later confirmed diffuse malignant infiltration (Figure <xref ref-type=\"fig\" rid=\"FIG4\">4</xref>).</p><fig fig-type=\"figure\" id=\"FIG3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><title>CT with IV contrast reveals normal appearing liver and moderate splenomegaly</title></caption><graphic xlink:href=\"cureus-0012-00000010110-i03\"/></fig><fig fig-type=\"figure\" id=\"FIG4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><title>A needle core biopsy of the liver showing diffuse infiltration by tumor cells</title></caption><graphic xlink:href=\"cureus-0012-00000010110-i04\"/></fig><p>An important consideration for the differential diagnosis in this patient relates to the finding of severe lactic acidosis. This ominous sign has been shown to be associated with a poor prognosis in patients with lymphoma [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]. The underlying etiology of lactic acidosis can be multifactorial and is often elusive. Infectious, metabolic, and ischemic causes must all be considered. Sepsis is a major concern in patients with a hematologic malignancy and is associated with varying degrees of liver dysfunction. Metabolic effects of lymphoma can also elevate lactic acid due to either increased glycolysis in cancer cells via the Warburg effect or due to tumors that outgrow their blood supply [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>].</p><p>In our case, multiple blood cultures were negative both before and after the onset of lactic acidosis and our patient had no response to broad-spectrum antibiotics. No source of infection was identified. Therefore, it appears that the lactic acidosis was due to a combination of metabolic effects and localized ischemia. Indeed, one mechanism of liver damage in lymphoma is extensive infiltration of the sinusoids and hepatic vasculature leading to diffuse hepatic necrosis [<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>]. Finally, decreased lactate clearance due to impaired liver and renal function likely contributed to the acidosis [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>].</p><p>Due to the infrequency of ALF secondary to lymphoma, there are no definite guidelines for treatment. SHL is characteristic of late stage disease, and the International Prognostic Index (IPI) indicates an age-adjusted overall survival of 21% in a high risk patient above 80 years of age [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. Thus, earlier diagnosis and treatment should be the primary goal. A search of the literature found five cases of survival in patients with ALF associated with extranodal DLBCL [<xref rid=\"REF16\" ref-type=\"bibr\">16</xref>-<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>]. All five of these patients received a variation of standard therapy based off the R-CHOP regimen, but specific treatment differed significantly from one patient to the next. In our case, R-CHOP with dose reductions and rasburicase was initiated on hospital day 7 but was unable to reverse the course of the patient’s disease. </p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>SHL is a rare but important cause of ALF that carries a very poor prognosis. Confirmatory biopsy can be difficult to obtain in the setting of ALF, and the underlying etiology is not always forthcoming on imaging. Our case review suggests that even in the absence of hepatomegaly or radiographic abnormalities, a constellation of findings including lactic acidosis, coagulopathy, and hypoglycemia should raise strong suspicion for secondary hepatic involvement leading to ALF. Although there is no standard treatment for ALF in the setting of SHL, early initiation of chemotherapeutic agents has been demonstrated to result in long-term survival in a minority of patients.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"journal\"><article-title>New insights into the epidemiology of non-Hodgkin lymphoma and implications for therapy</article-title><source>Expert Rev Anticancer Ther</source><person-group><name><surname>Chihara</surname><given-names>D</given-names></name><name><surname>Nastoupil</surname><given-names>LJ</given-names></name><name><surname>Williams</surname><given-names>JN</given-names></name><name><surname>Lee</surname><given-names>P</given-names></name><name><surname>Koff</surname><given-names>JL</given-names></name><name><surname>Flowers</surname><given-names>CR</given-names></name></person-group><fpage>531</fpage><lpage>544</lpage><volume>15</volume><year>2015</year><pub-id pub-id-type=\"pmid\">25864967</pub-id></element-citation></ref><ref id=\"REF2\"><label>2</label><element-citation publication-type=\"journal\"><article-title>Diffuse large B-cell lymphoma—more than a diffuse collection of large B cells: an entity in search of a meaningful classification</article-title><source>Arch Pathol Lab Med</source><person-group><name><surname>Gurbaxani</surname><given-names>S</given-names></name><name><surname>Anastasi</surname><given-names>J</given-names></name><name><surname>Hyjek</surname><given-names>E</given-names></name></person-group><fpage>1121</fpage><lpage>1134</lpage><volume>133</volume><year>2009</year><uri xlink:href=\"https://pubmed.ncbi.nlm.nih.gov/19642739/\">https://pubmed.ncbi.nlm.nih.gov/19642739/</uri><pub-id pub-id-type=\"pmid\">19642739</pub-id></element-citation></ref><ref id=\"REF3\"><label>3</label><element-citation publication-type=\"journal\"><article-title>Clinical features of non-Hodgkins lymphoma presenting with acute liver failure: a report of five cases and review of published experience</article-title><source>Am J Gastroenterol</source><person-group><name><surname>Lettieri</surname><given-names>CJ</given-names></name><name><surname>Berg</surname><given-names>BW</given-names></name></person-group><fpage>1641</fpage><lpage>1646</lpage><volume>98</volume><year>2003</year><uri xlink:href=\"https://pubmed.ncbi.nlm.nih.gov/12873593/\">https://pubmed.ncbi.nlm.nih.gov/12873593/</uri><pub-id pub-id-type=\"pmid\">12873593</pub-id></element-citation></ref><ref id=\"REF4\"><label>4</label><element-citation publication-type=\"journal\"><article-title>Liver in systemic disease</article-title><source>World J Gastroenterol</source><person-group><name><surname>Shimizu</surname><given-names>Y</given-names></name></person-group><fpage>4111</fpage><lpage>4119</lpage><volume>14</volume><year>2008</year><uri xlink:href=\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725369/#:~:text=Moreover%2C%20the%20liver%20may%20be,caused%20by%20the%20systemic%20disease.\">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725369/#:~:text=Moreover%2C%20the%20liver%20may%20be,caused%20by%20the%20systemic%20disease.</uri><pub-id pub-id-type=\"pmid\">18636653</pub-id></element-citation></ref><ref id=\"REF5\"><label>5</label><element-citation publication-type=\"journal\"><article-title>Lymphoma and hematological conditions: I. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005530</article-id><article-id pub-id-type=\"pmc\">PMC7523540</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10112</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Genetics</subject></subj-group><subj-group><subject>Internal Medicine</subject></subj-group><subj-group><subject>Neurology</subject></subj-group></article-categories><title-group><article-title>Primary Periodic Paralyses: A Review of Etiologies and Their Pathogeneses</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Farooque</surname><given-names>Umar</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Cheema</surname><given-names>Asfand Yar</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kumar</surname><given-names>Ranjeet</given-names></name><xref ref-type=\"aff\" rid=\"aff-3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Saini</surname><given-names>Gagandeep</given-names></name><xref ref-type=\"aff\" rid=\"aff-4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kataria</surname><given-names>Saurabh</given-names></name><xref ref-type=\"aff\" rid=\"aff-5\">5</xref><xref ref-type=\"aff\" rid=\"aff-6\">6</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nNeurology, Dow University of Health Sciences, Karachi, PAK </aff><aff id=\"aff-2\">\n<label>2</label>\nMedicine, Lahore Medical and Dental College, Lahore, PAK </aff><aff id=\"aff-3\">\n<label>3</label>\nInternal Medicine, Liaquat University of Medical and Health Sciences, Jamshoro, PAK </aff><aff id=\"aff-4\">\n<label>4</label>\nInternal Medicine, Government Medical College, Patiala, IND </aff><aff id=\"aff-5\">\n<label>5</label>\nNeurology and Neurocritical Care, University of Missouri Health Care, Columbia, USA </aff><aff id=\"aff-6\">\n<label>6</label>\nNeurology, West Virginia University, Morgantown, USA </aff><author-notes><corresp id=\"cor1\">\nUmar Farooque <email>umarfarooque65@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10112</elocation-id><history><date date-type=\"received\"><day>23</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Farooque et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Farooque et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/39068-primary-periodic-paralyses-a-review-of-etiologies-and-their-pathogeneses\">This article is available from https://www.cureus.com/articles/39068-primary-periodic-paralyses-a-review-of-etiologies-and-their-pathogeneses</self-uri><abstract><p>Periodic paralyses are a group of disorders characterized by episodes of muscle paralyses. They are mainly divided as primary (hereditary) and secondary (acquired) periodic paralyses. Primary periodic paralyses occur as a result of mutations in genes encoding subunits of muscle membrane channel proteins such as sodium, calcium, and potassium channels, resulting in impairment of their properties. Primary periodic paralyses are further classified on the basis of affected ion channels and other associated complications. Some of these periodic paralyses are hyperkalemic periodic paralysis (Na-channel mutation), hypokalemic periodic paralysis (Na- or Ca-channel mutation), Andersen’s syndrome (K-channel mutation), etc.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>periodic paralysis</kwd><kwd>primary periodic paralysis</kwd><kwd>hyperkalemic periodic paralysis</kwd><kwd>hypokalemic periodic paralysis</kwd><kwd>andersen’s syndrome</kwd><kwd>congenital myasthenic syndrome</kwd><kwd>paramyotonia congenita</kwd><kwd>humans</kwd><kwd>etiology</kwd><kwd>pathogenesis</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction and background</title><p>Periodic paralyses are a group of heterogeneous disorders characterized by flaccid paralysis and episodic attacks of muscle weakness. Periodic paralyses are mainly classified as primary and secondary periodic paralyses. Primary periodic paralyses are hereditary in which genes encoding channel protein subunits of skeletal muscle membrane are mutated, such as the muscular sodium, potassium or calcium channels, or the SCL4A1 protein [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. Secondary periodic paralyses are, however, acquired that occur secondarily, as the name suggests, to either thyrotoxic periodic paralysis or some other renal, suprarenal, or non-renal causes, such as gastroenteritis [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. </p><p>The objective of this study is to review the etiologies and pathogeneses of primary periodic paralyses and discuss other related aspects.</p></sec><sec sec-type=\"review\"><title>Review</title><p>Etiologies and pathogeneses of primary periodic paralyses</p><p>Primary periodic paralyses are caused due to mutations in genes encoding subunits of channel proteins of skeletal muscle membrane or endoplasmic reticulum like sodium, potassium, and calcium channels [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. Following is an overview of the structure and properties of these ion channels.</p><p>Sodium Channels</p><p>The muscle membrane sodium channels, extremely crucial for muscle membrane potentials, are composed of an α‐subunit and a β‐subunit [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. α‐Subunit consists of four homologous domains (DI-DIV) surrounding a central ion pore. Each domain comprises of six transmembrane segments (S1-S6). Sodium channels are voltage-gated highly selectively permeable channels initiating depolarization by activation (open pore) through conformational changes followed by inactivation (closed pore) blocking sodium current and causing muscle membrane to repolarize. A slight delay in inactivation of these channels can cause myotonia and muscle weakness, which is further intensified by exercise and low temperature [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. Mutations in genes encoding sodium channels are responsible for hyperkalemic periodic paralysis, a part of hypokalemic periodic paralysis, paramyotonia congenita, and congenital myasthenic syndrome (CMS) [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>].</p><p>Calcium Channels</p><p>The human calcium channels (CACNL1A3) are composed of five subunits (α1, α2, β, γ, and δ) and play an important role in excitation-contraction coupling through its association with ryanodine receptors of the endoplasmic reticulum. They act as calcium release channels and regulate intracellular calcium levels in response to changes in membrane potential. A mutation in genes that encode muscle calcium channels causes hypokalemic periodic paralysis [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>].</p><p>Potassium Channels</p><p>The potassium channels are composed of four subunits arranged as mono‐ or heteromers forming a central ion-conducting pore. These channels are found in skeletal muscles, heart, and brain and are responsible for the maintenance of resting membrane potential playing an important role in repolarization and hyperpolarization required in myoblast fusion. A mutation in the potassium channel gene located on chromosome 17 results in Andersen-Tawil syndrome, causing hyperexcitability followed by inexcitability of skeletal muscle membrane [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>].</p><p>Primary periodic paralyses are of various types differentiated on the basis of different gene mutations and channel proteins affected by these mutations. Several different types of primary periodic paralysis include hypokalemic, hyperkalemic, Anderson’s Syndrome, paramyotonia congenita, CMS, etc. [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>].</p><p>Hyperkalemic periodic paralysis</p><p>Hyperkalemic periodic paralysis is an autosomal dominant disease in which individuals suffer from paralytic episodes in limbs and high serum potassium levels (>5 mmol/L) [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. These episodes range from 15 minutes to 4 hours and may also affect eyes, throat, and trunk but respiratory and cardiac muscles are not known to be affected [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>,<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. About half of the individuals suffering from hyperkalemic periodic paralysis report their first paralytic attack in the first decade of life usually at the age of 10 years, which becomes more frequent and severe with time up until the age of 50 years. A paralytic attack can be triggered by potassium-rich food or post-exercise rest and be intensified by low temperature and emotional stress. Most patients also have mild myotonia during each episode and some of these develop chronic progressive myopathy. Most of the older patients develop permanent muscle weakness [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>].</p><p>Pathogenesis</p><p>Several different mutations may be responsible for hyperkalemic periodic paralysis. The most common mutation occurs in skeletal muscle sodium channel gene SCN4A on chromosome 17. As a consequence, fast inactivation of sodium channels is either incomplete or slowed down abnormally enhancing sodium flow making muscle fibers more incline to depolarization. This prolonged depolarization is the reason why these individuals experience muscle weakness and myotonia. High extracellular potassium level causes mild depolarization of muscle fibers that keeps mutant sodium channels in the active state causing repetitive muscle action potentials which are perceived as myotonia. If the depolarization becomes more extensive, both normal and mutant sodium channels are held in a closed state stopping all the action potentials that leads to muscle paralysis and weakness [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>].</p><p>Hypokalemic periodic paralysis</p><p>Hypokalemic periodic paralysis, the most common periodic paralysis, is an autosomal dominant disorder characterized by episodes of flaccid muscle weakness lasting several hours to few days and low serum potassium level (<3.5 mmol/L) [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>,<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. The first attack occurs somewhere between 2 and 30 years and might occur once in a lifetime but are usually recurring daily, weekly, monthly, etc. Paralytic attacks can be worsened by low temperature, anxiety, excessive salt ingestion, lack of exercise, consumption of glucosteroids or alcohol, and anesthetic methods. Some patients may suffer from long-lasting interictal muscle weakness [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>].</p><p>Pathogenesis</p><p>Hypokalemic periodic paralysis occurs as a result of a mutation in the α‐subunit of the DHP‐receptor (CACNA1S) gene on chromosome 1 or the α‐subunit of the sodium channel (SCN4A) gene on chromosome 17 [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>].</p><p>Andersen-Tawil syndrome</p><p>Andersen-Tawil Syndrome (cardiodysrhythmic periodic paralysis or potassium‐sensitive periodic paralysis) is an uncommon hereditary autosomal dominant disorder characterized by episodes of flaccid paralysis, with space of an hour to few days between each episode, along with complications like cardiac arrhythmias (like bigeminy, long QT syndrome, prolonged QUc‐interval, ventricular arrhythmias, cardiac arrest, or sudden cardiac death) and several physical deformations (like short stature, a broad forehead, hypertelorism, low‐set ears, broad‐base nose, micrognathia, cleft palate, molar hypoplasia, enamel discoloration, tapered‐curved fingers, clinodactyly, syndactyly, or scoliosis) [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. Other uncommon associations include short palpebral fissures, persisting primary dentition, oligodontia, thin upper lip, unilateral hypoplasia or dysplasia of a kidney, dilated cardiomyopathy, heart block, semilunar valve abnormalities, bicuspid aortic valve, coarctation of the aorta, valvular pulmonary stenosis, small hands or feet, or joint laxity [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]. The signs of disease usually start to appear between 2 and 18 years of age [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>].</p><p>Pathogenesis</p><p>Andersen’s syndrome occurs as a result of a mutation in gene KCNJ2 located on chromosome 17 encoding potassium channel Kir2.1 [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>]. The mutation hinders flow or completely blocks the Kir2.1 channels leading to prolonged terminal phase muscle action potential. The mutation can also disturb the transport of Kir2.1 channel to muscle membrane. Prolonged depolarization, in the presence of hypokalemia, can cause cardiac arrhythmias [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. Other complications of Andersen’s syndrome occur as a result of the tetramerization of mutant Kir2.1 allele with wild‐type Kir2.1, Kir2.2, and Kir2.3 channels [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>].</p><p>Paramyotonia congenita</p><p>Paramyotonia congenita is an autosomal dominant disorder characterized by persistent non-progressive myotonia and muscle weakness mainly in eyelids, neck, and upper limb muscles, which is induced by cold temperature and further worsened by exercise [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. These episodes of weakness may last from minutes to several hours even after removing the inducing factors. The severity or frequency is not affected by potassium intake. Only triggering factors are cold temperature and voluntary activity [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>].</p><p>Pathogenesis</p><p>Paramyotonia congenita is caused by mutations in gene SCN4A located on chromosome 17 encoding skeletal muscle sodium channels. Voltage-sensitive part of domain IV is the most affected location; others including the cytoplasmic surface of transmembrane segments S2, S3, and S4. The most typical cause of paramytonia congenita is mutation segment S4 in which arginine is replaced by other amino acids nullifying its positive charge. These mutant channels are stabilized in an inactive state by low temperature, which explains the temperature sensitivity of the disease [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>].</p><p>Congenital myasthenic syndrome</p><p>CMS is a heterogeneous hereditary disorder characterized by episodes of respiratory or bulbar paralysis and generalized muscle weakness. Other complications include facial weakness, ptosis, feeding difficulties, poor suck, and even arthrogryposis. Cardiac and smooth muscles are not affected. Complications start appearing at birth or soon after with varying severities [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>].</p><p>Pathogenesis</p><p>CMS is caused by mutations at several genes encoding proteins of the myoneural junction. These include several subunits of the acetylcholine receptor (CHNRE, CHRNA1, CHRNB1, and CHRND), the collagenic tail subunit of the acetylcholinesterase (COLQ), choline acetyltransferase (CHAT), rapsyn (RAPSN), or sodium channel [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>].</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>This review summarizes the causes of various types of primary periodic paralyses and shines light over their complications, inducing factors, and pathogenesis at the gene level. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005527</article-id><article-id pub-id-type=\"pmc\">PMC7523541</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10109</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cardiology</subject></subj-group><subj-group><subject>Emergency Medicine</subject></subj-group><subj-group><subject>Internal Medicine</subject></subj-group></article-categories><title-group><article-title>Malignant Hypertension Without End-Organ Damage Secondary to Stressful Condition in a Female</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Hussain</surname><given-names>Hussain</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Fadel</surname><given-names>Aya</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nInternal Medicine, Cardiology Clinic, Pasadena, USA </aff><aff id=\"aff-2\">\n<label>2</label>\nInternal Medicine, Florida International University, Hialeah Hospital, Miami, USA </aff><author-notes><corresp id=\"cor1\">\nHussain Hussain <email>hussain1986hussain@yahoo.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10109</elocation-id><history><date date-type=\"received\"><day>18</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Hussain et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Hussain et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/39949-malignant-hypertension-without-end-organ-damage-secondary-to-stressful-condition-in-a-female\">This article is available from https://www.cureus.com/articles/39949-malignant-hypertension-without-end-organ-damage-secondary-to-stressful-condition-in-a-female</self-uri><abstract><p>Malignant hypertension (hypertensive emergency), is an extreme elevation of blood pressure under certain conditions that can lead to organ damage and other serious consequences. It is a common condition that affects about one in three Americans, according to the Centers for Disease Control and Prevention. An elevation of systolic blood pressure above 180 mmHg and diastolic blood pressure above 120 mmHg is considered a hypertensive emergency. This article addresses the case of a 61-year-old female patient who presented to the ER with a semicomatose and gasping condition and response to painful stimuli with an unclear voice. She also had unstable vital signs, with a blood pressure of 370/200 mmHg, a pulse rate of 115, a respiratory rate of 22, and a pulse oximetry of 96%, but no fever. Her son provided a brief history and reported that a stressful condition had occurred at home one hour before; she had begun to scream and been brought to the ER by ambulance in the condition described above. Cardiac monitoring and an electrocardiograph were performed and indicated a normal condition besides the unstable vital signs. Oxygen was administered via a nasal cannula with 20 mg of intravenous hydralazine, and the patient’s blood pressure improved progressively. Moreover, she regained consciousness with no end-organ damage. A hypertensive emergency is usually associated with end-organ damage, such as heart, kidney, eye, or brain damage. However, in this case, despite the extreme elevation of her blood pressure, the patient suffered no organ damage. It is essential to manage the extreme elevation of blood pressure as soon as possible and monitor the patient for consequences.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>malignant hypertension</kwd><kwd>hypertensive emergency</kwd><kwd>physiological changes in stress</kwd><kwd>hypertension</kwd><kwd>catecholamines</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Hypertension is the most common condition in the United States. It affects one in three American adults, according to the Centers for Disease Control and Prevention [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. Blood pressure is the pressure of the blood pushing against the arterial wall during its passage, and blood pressure varies depending on the activities performed. Hypertension increases the risk of heart disease, stroke, and other conditions. Systolic blood pressure measures arterial pressure during heartbeats, which normally should be less than 120 mmHg, whereas diastolic blood pressure measures arterial pressure between heartbeats or during heart rest, which should be less than 80 mmHg.</p><p>When blood pressure exceeds 120/80 mmHg, several readings reveal hypertension. Different levels of blood pressure occur: level one is considered normal, while level two indicates that a patient is at risk of prehypertension when his or her systolic blood pressure is between 120 mmHg and 129 mmHg and diastolic blood pressure is below 80 mmHg. Various evaluations consider blood pressure above 130/80 mmHg a hypertensive condition [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. Hypertensive urgency occurs when blood pressure is equal to or above 180/110 mmHg with no organ damage and can be lowered safely within a few hours using medications. A hypertensive emergency entails elevated blood pressure equal to or above 180/110 mmHg and end-organ damage; mean arterial blood pressure should thus be lowered in the ICU over thirty minutes-one hour by up to 25% to prevent imminent organ damage [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>].</p></sec><sec sec-type=\"cases\"><title>Case presentation</title><p>Clinical history</p><p>A 61-year-old female patient presented to the ER with a semicomatose and gasping condition. She was holding her right hand over the right side of her chest and responded only to painful stimuli with an unclear voice. Her son provided a brief history and reported that a stressful condition had occurred at home an hour before; she had been arguing with one of her family members for about 30 minutes, which had initiated her presentation. Her son also reported that she had been moving her right arm and chest erratically and screaming during the ambulance ride. Upon their arrival at the ER, the paramedics also offered a brief history. They could not report accurate vital signs from their ride to the hospital, stating that the patient’s blood pressure could not be read due to a device error; her other vital signs had included a respiratory rate of 24, heart rate of 126, and pulse oximetry of 94%. The paramedics had administered oxygen via a nasal cannula and set an intravenous line in the ambulance.</p><p>After her son and the paramedics supplied their information, the patient was attached to the cardiac monitor, which immediately rendered no signs of cardiac ischemia via electrocardiography (Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>). A physical examination indicated an obese, agitated, semicomatose, and gasping condition. She was moving her right arm and holding the right side of her chest intermittently. A cardiovascular examination revealed a regular rhythm and normal heart sounds with no murmur or rubs. Pulmonary, abdominal, and neurological examinations were also normal.</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Electrocardiography</title></caption><graphic xlink:href=\"cureus-0012-00000010109-i01\"/></fig><p>The vital signs recorded were surprising since, while the patient had no fever, her blood pressure was 370/191 mmHg, respiratory rate 22, heart rate 115, and pulse oximetry 95%. Immediate verification of her blood pressure revealed the same reading. A decision to administer 20 mg of intravenous hydralazine and monitor her vital signs under the highest care was made. The first ampule of hydralazine was administered, and after five minutes, the patient’s blood pressure dropped to 330/182 mmHg. The second ampule of hydralazine was then administered, and after another five minutes, her blood pressure dropped to 300/180 mmHg. Finally, the decision to administer 40 mg of hydralazine in two dosages ten minutes apart was made. The patient’s blood pressure dropped to 220/160 mmHg; she began to regain consciousness gradually, with no gasping, and her right arm movement subsided. Her vital signs indicated a heart rate of 118, a respiratory rate of 20, and unchanged pulse oximetry. Another electrocardiograph rendered no ischemia. However, five minutes after the last dose of hydralazine, another ampule was administered in response to unchanged blood pressure since the previous reading; the patient’s blood pressure then dropped to 171/130. She began to feel better and wanted to know what had happened to her. She was alert to time, place, and persons a few minutes later.</p><p>The patient’s current vital signs were as follows: blood pressure of 170/128 mmHg, respiratory rate of 17, heart rate of 119, and pulse oximetry of 94% on room air. She received 50 mg of oral metoprolol and provided a full history. She had been on 10 mg/day of lisinopril for the last five years for hypertension, and she denied having any chronic diseases such as diabetes mellitus, asthma, stroke, angina, and liver or kidney problems. Repeated physical, neurological, cardiac, pulmonary, and gastroenterological examinations were normal. Her blood pressure dropped to 150/100 mmHg 20 minutes after the latest reading. Her vital signs then revealed a heart rate of 105, respiratory rate of 17, and pulse oximetry of 95%. Computer tomography rendered normal findings and no signs of a stroke (Figure <xref ref-type=\"fig\" rid=\"FIG2\">2</xref>). Other measurements included 1 mg of creatinine and 25 mg of blood urea nitrogen (BUN). An echocardiograph and abdominal ultrasound were both within the normal parameters. Furthermore, an ophthalmological consultation rendered normal findings.</p><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>CT scan of the brain</title></caption><graphic xlink:href=\"cureus-0012-00000010109-i02\"/></fig><p>Before the patient was discharged, her vital signs demonstrated a blood pressure of 130/84 mmHg, a respiratory rate of 16, a heart rate of 79, a pulse oximetry of 97% on room air, and no fever. She was discharged the following day after achieving a stable condition. All laboratory results, such as her complete blood count and comprehensive metabolic panel, included normal limits. She was scheduled for an MRI five days after her discharge, and the results were within normal limits.</p></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>Pathophysiology</p><p>Hypertension is not only a common condition but also the leading cause of global death, accounting for 10.4 million deaths annually. This condition affects the high-income more than the low-income population and men more than women. The prevalence of blood pressure increases and adverse impacts on cardiovascular morbidity and mortality are increasing globally regardless of income (Figure <xref ref-type=\"fig\" rid=\"FIG3\">3</xref>) [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>].</p><fig fig-type=\"figure\" id=\"FIG3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><title>Stages of blood pressure</title><p>Credit: American Heart Association</p></caption><graphic xlink:href=\"cureus-0012-00000010109-i03\"/></fig><p>While hypertension is a chronic condition that affects the entire body, the most common organ affected is the heart. Blood pressure is the product of cardiac output and systemic vascular resistance; hypertension may involve an increase in cardiac output, an increase in systemic vascular resistance, or both. Aging individuals have stiffer blood vessels. The loss of elasticity form chronic exposure to pressure from blood passage plays a vital role in their pathophysiology (Figure <xref ref-type=\"fig\" rid=\"FIG4\">4</xref>).</p><fig fig-type=\"figure\" id=\"FIG4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><title>Blood vessel elasticity in response to systolic and diastolic pressure changes</title><p>Credit: Nature's Real Health</p></caption><graphic xlink:href=\"cureus-0012-00000010109-i04\"/></fig><p>Chronic pressure on the vascular wall leads to an increase in the alpha adrenoreceptor or an increase in the release of peptides such as angiotensin and endothelin, followed by an increase in systolic calcium in the vascular smooth muscle, which causes vasoconstriction. However, angiotensin and endothelin factors lead to an increase in vascular smooth muscle mass in terms of vascular remodeling (Figure <xref ref-type=\"fig\" rid=\"FIG5\">5</xref>). Vascular resistance and vascular stiffness produce an imposed load on the left ventricle; left ventricle hypertrophy, coronary arterial disease, and ventricle diastolic dysfunction occur as a result [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>].</p><fig fig-type=\"figure\" id=\"FIG5\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><title>Blood vessel wall in response to high pressure</title><p>Credit: USA vascular center.</p></caption><graphic xlink:href=\"cureus-0012-00000010109-i05\"/></fig><p>Hypertensive patients have an increased release of and sensitivity to norepinephrine, as well as an increased response to stressful stimuli and decreased baroreceptor sensitivity [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. Two types of hypertension occur: idiopathic hypertension, which includes 90% of hypertension cases, and secondary hypertension, which results from multiple diseases, including renovascular hypertension, acromegaly, obstructive sleep apnea, adrenal tumors, and congenital adrenal hyperplasia; using drugs such as cocaine, amphetamine, oral contraceptive pills, and decongestants; and herbal therapy (Figure <xref ref-type=\"fig\" rid=\"FIG6\">6</xref>) [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>].</p><fig fig-type=\"figure\" id=\"FIG6\" orientation=\"portrait\" position=\"float\"><label>Figure 6</label><caption><title>Mechanism of high blood pressure</title><p>Credit: https://prevent-<ext-link ext-link-type=\"uri\" xlink:href=\"hypertency.blogspot.com/2012/12/pathophysiology-of-essential.html\">hypertency.blogspot.com/2012/12/pathophysiology-of-essential.html</ext-link></p></caption><graphic xlink:href=\"cureus-0012-00000010109-i06\"/></fig><p>The regulation of blood pressure is a complex mechanism. Neurogenic control including the dorsal medulla (nucleus solitarius), pons, and midbrain, decreases the efferent sympathetic activity, which finally leads to bradycardia and vasodilation when the arterial baroreceptors respond to vessel wall distention by increasing the afferent impulse activity.</p><p>A hypertensive emergency is a true medical emergency that requires prompt treatment to reduce blood pressure and prevent organ damage. The pathophysiology is not well understood; nevertheless, the failure of normal autoregulation and an abrupt rise in systemic vascular resistance are often the first steps in the disease process. An increase in systemic vascular resistance occurs with the release of vasoconstrictors from the vessel wall. Incremental pressure changes in the vessel lead to endothelial damage. Local intravascular activation of coagulation cascade releases more vasoconstrictors, and fibrinoid necrosis of small blood vessels. If this process is not prevented as soon as possible, further vascular injury, tissue ischemia, and autoregulation dysfunction occur [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>].</p><p>The risk factors for hypertension include age (the most important one), race (African Americans are more vulnerable), family history, obesity, alcohol, diet, a sedentary lifestyle, and stress [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. While the causes of hypertensive emergency are extensive, the most important ones are medication noncompliance, stroke, eclampsia, adrenal tumor, angina, medication interaction, and stress [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>].</p><p>Stress is now a major predicament, whether it is related to environmental or psychological factors, as both can trigger a hormonal change in the body. The initial response to stress begins in the amygdala, which receives input from various brain centers, including visual and auditory systems. In other words, when an individual experiences a critical situation, data is transmitted to the amygdala, which interprets that information and sends it to the hypothalamus; the hypothalamus then communicates with the rest of the body via the autonomic nervous system to control vital signs such as breathing, blood pressure, heart rate, and the dilation or constriction of blood vessels. The autonomic nervous system is divided into sympathetic (fight-or-flight response) and parasympathetic (rest or digest) systems. The activation of the sympathetic nervous system during emotional stress ultimately sends an impulse to the adrenal medulla to release catecholamines, including epinephrine and norepinephrine, which both lead to an increase in vital signs such as elevated blood pressure, tachypnea, and tachycardia. If the stressors persist, the surge in catecholamines also persists, resulting in more damage to the vascular endothelium and worsening the condition [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]. These catecholamines usually affect alpha and beta receptors. Alpha receptor activation in the blood vessels leads to vasoconstriction and elevated blood pressure, whereas beta receptor activation in the heart or other organs leads to variable changes, such as the contraction or relaxation of smooth muscles and an increased heart rate (Figure <xref ref-type=\"fig\" rid=\"FIG7\">7</xref>) [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>].</p><fig fig-type=\"figure\" id=\"FIG7\" orientation=\"portrait\" position=\"float\"><label>Figure 7</label><caption><title>Physiological changes in response to stress</title><p>Credit: <ext-link ext-link-type=\"uri\" xlink:href=\"www.studyblue.com\">www.studyblue.com</ext-link></p></caption><graphic xlink:href=\"cureus-0012-00000010109-i07\"/></fig><p>The first organ that a hypertensive emergency affects is the kidney, which suffers from ischemia and activates the renin-angiotensin-aldosterone system (RAAS). The condition therefore worsens due to the further elevation of blood pressure. In addition to the activation of the RAAS, other factors such as endothelin, vasopressin, and catecholamines play a vital role in the pathogenesis of hypertensive emergency [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>].</p><p>The goal is to find any organ damage (including angina, myocardial infarction, stroke, acute kidney injury, hypertensive encephalopathy, eclampsia, preeclampsia, left ventricular failure, pulmonary edema, aortic dissection, and retinopathy) and attempt to prevent the progression as fast as possible. The patient could present with shortness of breath, chest pain, confusion, coma, vomiting, or vision changes. A physical examination should initially focus on any cardiac symptoms, such as gallops, murmurs, and S3 sounds; a lung examination on rales; and a neurological examination on seizures. Moreover, a mental status funduscopic examination should exclude papilledema and other conditions. Monitoring the patient’s vital signs is an important step. Indeed, the patient should be managed in the ICU under high care and supervision [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>,<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>].</p><p>First, lowering the patient’s blood pressure should be done within minutes by lowering the mean arterial pressure by up to 25% in the first thirty minutes and if the patient remains stable then further lowering can be done over the next hour. Attaching the patient to a cardiac monitor is a crucial step in blood pressure management [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]. Once the patient’s blood pressure has been lowered to a safe level, investigations into his or her full history and a physical examination should be conducted. The laboratory investigations include a complete blood count (CBC); a comprehensive metabolic panel (CMP); electrocardiography; echocardiography; brain, chest, and abdomen images, including CT scans or MRI; a drug panel; a thyroid-stimulating hormone (TSH) test; a catecholamine blood test; and a growth hormone test [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>]. Various antihypertensive medications are available for use in hypertensive crises (Figure <xref ref-type=\"fig\" rid=\"FIG8\">8</xref>) [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>].</p><fig fig-type=\"figure\" id=\"FIG8\" orientation=\"portrait\" position=\"float\"><label>Figure 8</label><caption><title>Antihypertensive medications</title><p>Credit: Merck Manuals [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]</p></caption><graphic xlink:href=\"cureus-0012-00000010109-i08\"/></fig></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>A hypertensive emergency usually presents with elevated blood pressure and end-organ damage, including heart, eye, kidney, and brain damage. Remarkably, the current case presented with the usual elevated blood pressure but without end-organ damage, which makes it a unique case to report. The patient was successfully treated with hydralazine and suffered no complications, including stroke and myocardial infarction.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"website\"><article-title>What is malignant hypertension (hypertensive emergency)?</article-title><date-in-citation content-type=\"access-date\"><month>7</month><year>2020</year></date-in-citation><year>2019</year><uri xlink:href=\"https://www.healthline.com/health/malignant-hypertension\">https://www.healthline.com/health/malignant-hypertension</uri></element-citation></ref><ref 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005505</article-id><article-id pub-id-type=\"pmc\">PMC7523542</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10079</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Internal Medicine</subject></subj-group><subj-group><subject>Neurosurgery</subject></subj-group><subj-group><subject>Oncology</subject></subj-group></article-categories><title-group><article-title>Malignant Epithelioid Neoplasm of the Brain</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Jog</surname><given-names>Abhishrut P</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Ronderos</surname><given-names>Diana M</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ali</surname><given-names>Asghar</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Niazi</surname><given-names>Masooma</given-names></name><xref ref-type=\"aff\" rid=\"aff-3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Diaz-Fuentes</surname><given-names>Gilda</given-names></name><xref ref-type=\"aff\" rid=\"aff-4\">4</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nInternal Medicine, BronxCare Health System, Bronx, USA </aff><aff id=\"aff-2\">\n<label>2</label>\nPulmonology and Critical Care, BronxCare Health System, Bronx, USA </aff><aff id=\"aff-3\">\n<label>3</label>\nPathology, BronxCare Health System, Bronx, USA </aff><aff id=\"aff-4\">\n<label>4</label>\nPulmonary and Critical Care, BronxCare Health System, Bronx, USA </aff><author-notes><corresp id=\"cor1\">\nDiana M. Ronderos <email>drondero@bronxleb.org</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>27</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10079</elocation-id><history><date date-type=\"received\"><day>18</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Jog et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Jog et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/34905-malignant-epithelioid-neoplasm-of-the-brain\">This article is available from https://www.cureus.com/articles/34905-malignant-epithelioid-neoplasm-of-the-brain</self-uri><abstract><p>Malignant epithelioid tumors have been described in various organ systems, but are rarely seen in the brain. They are aggressive tumors and have high mortality. In certain cases, the immunohistochemistry (IHC) findings may not be sufficient to clarify the diagnosis. In these cases, next-generation genetic sequencing may play a role in clarifying the diagnosis. In addition to lab testing, a thorough history and physical exam are necessary to rule out other sources of the tumor such as melanoma. Patients presenting with neurological symptoms are cared for by a wide variety of physicians, hence it is important to raise awareness of rare tumors in order to provide timely and appropriate management and referral for these patients. We present the case of a middle-aged woman who was diagnosed with a ‘malignant epithelioid neoplasm’ of the brain, a rare variety of tumors. We also give the clinical course of this illness.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>primary brain tumors</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>In the United States, the annual incidence of all tumors involving the brain is 46 per 100,000, and that of primary brain tumors is 15 per 100,000. Out of the total 600,000 cancer-related deaths that occur annually in the country, around 20,000 are attributable to primary brain tumors. The most common types of primary intracranial neoplasms are meningioma, pituitary tumors, and gliomas [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. Epithelioid neoplasms are not a very well described entity of brain tumors. Literature review reveals descriptions of epithelioid neoplasms in a variety of organ systems, but only a few in the brain, with further paucity of data on malignant forms.</p><p>We present the case of a middle-aged woman who was diagnosed with a ‘malignant epithelioid neoplasm’ and give the clinical course, diagnosis, and outcome of this illness.</p></sec><sec sec-type=\"cases\"><title>Case presentation</title><p>A 57-year-old female with a history of hypertension and HIV, compliant with highly active antiretroviral therapy (HAART) (CD4- 392 with undetectable viral load) presented with sudden onset left-sided weakness. Clinical exam revealed a power of 2/5 in the left upper and lower extremities, along with facial deviation to the right side. A thorough history and physical exam (head to toe, including back, palms, soles, and anal region) was found to be negative as well. The patient had no history of a similar episode or of any tumors in the past. There was no history of exposure to ionizing radiation or a known carcinogen in the past. Additionally, the patient did not have a history of any familial ailment predisposing to cancer.</p><p>Non-contrast CT head found a large hemorrhagic stroke measuring 5.7 x 3.8 x 3.8 cm in the right centrum semiovale resulting in inferior displacement and effacement of the right lateral ventricle and approximately 8 mm of right-to-left midline shift (Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>). A right parietooccipital craniotomy and removal of an intracerebral clot were performed.</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Non-contrast CT head</title><p>Large acute hemorrhage in the right centrum semiovale measuring approximately 5.7 x 3.8 x 3.8 cm resulting in inferior displacement and effacement of the right lateral ventricle and approximately 8 mm of right-to-left midline shift.</p></caption><graphic xlink:href=\"cureus-0012-00000010079-i01\"/></fig><p>Grossly the tissue was light tan in color and soft in consistency. Microscopic examination of hematoxylin and eosin-stained sections of the specimen showed an epithelioid and focally spindle-cell neoplasm that was mainly distributed in the perivascular Virchow-Robin spaces and in the leptomeningeal spaces (Figures <xref ref-type=\"fig\" rid=\"FIG2\">2</xref>, <xref ref-type=\"fig\" rid=\"FIG3\">3</xref>). The tumor was found to grow in sheets and fascicles. In the perivascular areas, the tumor showed a concentric growth pattern around blood vessels. Focally, there was a myxoid matrix deposition. The neoplastic cells were epithelioid and markedly pleomorphic, with a round to spindle-shaped, often irregular, hyperchromatic nuclei and occasionally prominent eosinophilic nucleoli. The neoplastic cells had moderate amounts of eosinophilic, sometimes granular, cytoplasm and showed distinct cell boundaries in some areas, but appeared syncytial in others. Multinucleated cells were also seen focally. Multiple mitotic figures were noted (including atypical mitoses). The surrounding brain parenchyma showed reactive astrocytosis, foamy macrophages, edema, and perivascular inflammation. CD45 highlighted many tumor-infiltrating immune cells and CD68 highlighted many tumor-infiltrating macrophages. The Ki 67 proliferation index was elevated, with up to 20% of cells labeling positive.</p><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>Light microscopy showing perivascular arrangement of tumor cells</title><p>In the perivascular areas, the tumor cells show a concentric growth pattern around blood vessels (H&E, magnification x 200). H&E, hemotoxylin and eosin.</p></caption><graphic xlink:href=\"cureus-0012-00000010079-i02\"/></fig><fig fig-type=\"figure\" id=\"FIG3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><title>Light microscopy showing epithelioid cells</title><p>The tumor is comprised of predominantly epithelioid cells and focally spindle cells growing in sheets and fascicles. The cells are pleomorphic with eosinophilic cytoplasm and multiple mitotic figures (arrow). H&E magnification x200. H&E, hemotoxylin and eosin.</p></caption><graphic xlink:href=\"cureus-0012-00000010079-i03\"/></fig><p>On IHC, the following markers were found to be positive in our patient: vimentin, caldesmon, Melan-A, Human Melanoma Black (HMB)-45, S 100, SOX 2, oligodendrocyte transcription factor 2 (Olig 2), platelet-derived growth factor receptor (PDGFR) alpha, cluster of differentiation (CD) 34, integrase interactor 1 (INI-1) (preserved) (Figure <xref ref-type=\"fig\" rid=\"FIG4\">4</xref>).</p><fig fig-type=\"figure\" id=\"FIG4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><title>Immunoreactivity to vimentin</title><p>The tumor cells are strongly immunoreactive to vimentin (immunohistochemistry, magnification x 400).</p></caption><graphic xlink:href=\"cureus-0012-00000010079-i04\"/></fig><p>The following markers were found to be negative in our patient: GFAP, CD 31, cytokeratin (CK), CK CAM 5.2, p40, p63, ETS-related gene (ERG), Desmin, spinal muscular atrophy (SMA), smooth muscle myosin (SMMS-1), calponin, myogenin, myoblast determination protein 1 (Myo D1), microphthalmia-associated transcription factor (MITF), tyrosinase, SOX 10, c-kit, signal transducer and activator of transcription (STAT 6), PR, ER, CD 10, Epstein-Barr virus (EBV), human herpesvirus (HHV)-8.</p><p>The findings did not conform to a specific diagnosis but were suggestive of an entity called a PEComa (Perivascular Epithelioid Cell neoplasm). A diagnosis of a ‘malignant epithelioid neoplasm’ was made.</p><p>A CT abdomen-pelvis and CT chest were done to look for a possible primary source but were found to be negative. MRI brain following the resection showed post-operative changes and mild fluid collection.</p><p>Despite the initial improvement, the patient complained of gradually worsening dizziness over the next four months. A repeat MRI showed a large hemorrhage in the cingulate gyrus bilaterally just above the posterior third of the body of the corpus callosum. Mass effect compression of the corpus callosum and a large amount of vasogenic edema extending into both parietal lobe white matter was present. Following the injection of gadolinium contrast, prominent enhancement within the region of the hemorrhage was seen. This was strongly suspicious of hemorrhagic neoplasm.</p><p>A stereotactic biopsy was performed. The tissue showed only reactive gliosis. The patient did not opt for further treatment. She was re-admitted three months later due to a fall. MRI brain on this admission showed an enhancing mass involving the posterior aspect of the corpus callosum extending to both parietal lobes measuring 5.1 x 3.9 x 4.3 cm, a progression from the prior MRI (Figure <xref ref-type=\"fig\" rid=\"FIG5\">5</xref>). Additionally, a bilateral pulmonary embolism was found. The hospital course was also complicated by status epilepticus. The patient’s condition deteriorated within the next month and she succumbed to the illness.</p><fig fig-type=\"figure\" id=\"FIG5\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><title>Magnetic resonance imaging of the brain</title><p>A: large hemorrhage in the cingulate gyrus (red arrow); B: mass effect compresses the corpus callosum (yellow arrow); C and D: a large amount of vasogenic edema extends into both parietal lobe white matter (white arrows).</p></caption><graphic xlink:href=\"cureus-0012-00000010079-i05\"/></fig></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>Immunohistochemical (IHC) staining was helpful in sifting through various tumor differentials that arose in this case. Glioblastoma, metastatic melanoma, angiosarcoma, epithelioid sarcoma, sarcomatoid carcinoma, meningeal hemangiopericytoma, and paraganglioma were all considerations. Glioblastomas typically show GFAP positivity [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. In this case, glial fibrillary acidic protein (GFAP) was negative. For melanocytic tumors, S 100 is the most sensitive marker. HMB-45, Melan-A, tyrosinase, and MITF demonstrate good specificity but not as good sensitivity as S-100 in melanomas [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. In our case, S 100, HMB-45, and Melan A were positive, whereas tyrosinase and MITF were negative. Additionally, more recently, Willis et al showed the usefulness of the marker SOX 10 in identifying metastatic melanomas [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. There was a 100% sensitivity and specificity in diagnosing melanomas. In our case, SOX 10 was negative. A melanoma had also been ruled out clinically. ERG and CD31 negativity went against angiosarcoma [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. The preserved INI-1 goes against epithelioid sarcoma [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. Sarcomatoid carcinomas typically show positivity for CK [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>], which was not the case in our patient. Absence of STAT- 6 goes against a diagnosis of meningeal hemangiopericytoma [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. Chromogranin and synaptophysin studies which are usually positive in paragangliomas were not available; however, imaging was not suggestive of that [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>].</p><p>The tumor that was characterized as a ‘malignant epithelioid neoplasm’ showed some features of the tumor entity called PEComa. The World Health Organization (WHO) defines PEComa as: ‘mesenchymal tumors composed of histologically and immunohistochemically distinctive perivascular epithelioid cells (PECs)' [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>]. Microscopically, the perivascular epithelioid cells are characterized by a radial arrangement around a blood vessel as seen in this case. They may show the presence of two components: an immediate perivascular component that appears epithelioid and a spindle shape appearing component, away from the vessel. They have a clear to granular, lightly eosinophilic cytoplasm. The nuclei are usually round to oval, centrally placed, and normochromic and may display mild to a significant amount of nuclear atypia and mitotic activity [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]. IHC in PEComa’s classically shows strong positivity for melanocytic markers, HMB-45 and Melan A, and myogenic markers such as SMA, desmin, Myo D1 [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. In this case, HMB-45 and Melan A were weakly positive. Caldesmon was the myogenic marker that was positive, whereas the other myoid markers were absent. Even though the tumor does not fit all the characteristics of a PEComa, an atypical presentation of this rare tumor is a possibility. No other source of malignancy was found in this case, despite an extensive search.</p><p>In our case, a few points are suggestive of the malignant potential: tumor size > 5 cm, the Ki 67 index is 20%, multiple mitoses including atypical mitotic bodies are present, recurrent growth of the tumor with hemorrhagic transformation and rapid and fatal course of the illness.</p><p>Treatment decisions for intracranial neoplasms are individualized and need a multidisciplinary approach consisting of medical oncology, radiation oncology, and neurosurgery. They are based on tumor type, location, malignancy potential, patient’s age, and physical condition. Treatment commonly includes surgery, radiotherapy, chemotherapy, or a combination [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]. Treatment for most PEComa’s has been through surgical excision. The recognition of PEComa has important management implications as some of them respond to mammalian target of rapamycin (mTOR) inhibitors such as sirolimus or everolimus [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>]. In our case, the patient had a surgical resection resulting in a temporary decrease of symptoms; however, the tumor grew back within the next four months.</p><p>The patient was HIV-positive. The most common primary tumor in HIV-positive individuals is a primary central nervous system (CNS) lymphoma. The occurrence of this malignant epithelioid neoplasm in the patient may be unrelated to the viral infection, and an association between the two has not been described in the literature. However, it is worth further investigation. The patient had multiple risk factors for the development of a pulmonary embolism including immobility due to the stroke, tumor, and surgery in the form of craniotomy. Greater clarity about the tumor can be obtained through next-generation sequencing, which was not done in this case.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>Malignant epithelioid tumors are uncommon tumors in the brain. They are aggressive tumors and have high mortality. In certain cases, the IHC findings may not be sufficient to clarify the diagnosis. In these cases, next-generation genetic sequencing may play a role in clarifying the diagnosis. In addition to lab testing, a thorough history and physical exam are necessary to rule out other sources of the tumor such as melanoma. Patients presenting with neurological symptoms are cared for by a wide variety of physicians, hence it is important to raise awareness of rare tumors in order to provide timely and appropriate management and referral for these patients.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"book\"><article-title>Adams and Victor's Principles of Neurology, 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005515</article-id><article-id pub-id-type=\"pmc\">PMC7523543</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10094</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Obstetrics/Gynecology</subject></subj-group><subj-group><subject>Oncology</subject></subj-group></article-categories><title-group><article-title>Transvaginal Saline Contrast Sonohystography to Investigate Postmenopausal Bleeding: A Systematic Review</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Tahmasebi</surname><given-names>Farshad</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Stewart</surname><given-names>Sarah</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mitra</surname><given-names>Anita</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Morje</surname><given-names>Mridula</given-names></name><xref ref-type=\"aff\" rid=\"aff-3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sayasneh</surname><given-names>Ahmad</given-names></name><xref ref-type=\"aff\" rid=\"aff-4\">4</xref><xref ref-type=\"aff\" rid=\"aff-5\">5</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nObstetrics and Gynaecology, Princess Royal University Hospital (PRUH), King’s College Hospital NHS Foundation Trust, London, GBR </aff><aff id=\"aff-2\">\n<label>2</label>\nObstetrics and Gynaecology, Institute of Reproductive and Developmental Biology, Department of Surgery & Cancer, Imperial College, London, GBR </aff><aff id=\"aff-3\">\n<label>3</label>\nObstetrics and Gynaecology, St Thomas’ Hospital, London, GBR </aff><aff id=\"aff-4\">\n<label>4</label>\nGynaecological Oncology, Guy's and St Thomas' NHS Foundation Trust, London, GBR </aff><aff id=\"aff-5\">\n<label>5</label>\nSchool of Life Course Sciences, Faculty of Life Sciences and Medicine, King's College London, London, GBR </aff><author-notes><corresp id=\"cor1\">\nFarshad Tahmasebi <email>tfarshad667@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>28</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10094</elocation-id><history><date date-type=\"received\"><day>15</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Tahmasebi et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Tahmasebi et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/37131-transvaginal-saline-contrast-sonohystography-to-investigate-postmenopausal-bleeding-a-systematic-review\">This article is available from https://www.cureus.com/articles/37131-transvaginal-saline-contrast-sonohystography-to-investigate-postmenopausal-bleeding-a-systematic-review</self-uri><abstract><p>Transvaginal ultrasound (TVUS) is the initial investigation of choice for postmenopausal bleeding (PMB), followed by diagnostic hysteroscopy and endometrial sampling if abnormalities are detected. Saline contrast sonohysterography (SCSH) - injection of saline through the cervix into the uterine cavity prior to TVUS - allowed increased diagnostic accuracy in women with PMB in several small, heterogeneous studies.</p><p>The objectives of the current study were to evaluate the diagnostic accuracy of SCSH in women with PMB, comparing findings with surgical and pathological reports, highlight the necessity of SCSH in guiding clinical decision-making, and establish if there is an increase/decrease in the number of hysteroscopies performed for PMB and, hence, the adherence of clinicians to imaging referral guidelines.</p><p>The search strategy included formulating search terms identifying all synonyms of SCSH and postmenopause. The databases searched were MEDLINE, Embase, and the Cochrane Library.</p><p>Only studies comparing SCSH to an alternative method were selected. The studies were screened and data analysis performed using content analysis. Data reduction was performed through systematic coding and the generation of themes</p><p>We identified 18 studies, comprising 974 women, using SCSH to evaluate the endometrial cavity in women with PMB; most support SCSH improving diagnostic accuracy through delineating intracavitary structures.</p><p>In effect, SCSH could be a first-line investigative modality to assess the uterine cavity once a larger, well-designed study has been conducted to clarify its specificity, sensitivity, and positive predictive value (PPV). Owing to its relatively non-invasive nature and potentially high diagnostic accuracy, SCSH could allow for more accurate decisions regarding the need for further investigation and subsequent management.</p><p>Tweetable abstract</p><p>\"Saline contrast sonohysterography improves the diagnostic accuracy of the endometrium in postmenopausal bleeding.\"</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>saline infusion sonography</kwd><kwd>post-menopausal bleed</kwd><kwd>endometrial cancer</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction and background</title><p>Transvaginal ultrasonography (TVUS) significantly improved the accurate diagnosis of intrauterine abnormalities. In women with postmenopausal bleeding (PMB), a measurement of endometrial thickness (ET) reliably distinguishes a women’s risk of endometrial cancer. An ET of ≤4 mm decreases the likelihood of endometrial cancer by 10-fold in users and non-users of hormone replacement therapy [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>-<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. In high‐risk women (ET > 5 mm), the evaluation of endometrial morphology and vascularisation using gray‐scale and Doppler ultrasound imaging refines endometrial cancer risk [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>-<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. Most studies reporting on ultrasonography of the uterine cavity are small, with sometimes conflicting results regarding sonohysterography, potentially due to heterogeneity in study design and population. Large multicentre studies are necessary to clarify the role of sonohysterography in the assessment of endometrial morphology and vascularisation to differentiate endometrial and intracavitary pathologies.</p><p>Abnormal uterine bleeding (AUB), defined as either metrorrhagia, vaginal bleeding separated from expected menses or menorrhagia, subjective complaints of either increased duration and/or volume [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. In peri-menopausal women, variations in bleeding may be secondary to physiological hormonal changes or neoplastic changes, either benign or malignant.</p><p>Postmenopausal bleeding (PMB), or bleeding occurring >12 months after menopause, occurs immediately after menopause in approximately 10% of cases [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>], signaling endometrial carcinoma in around 10% of cases [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>-<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>], or benign conditions, such as endometrial polyps, in 20%-40% [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]. Endometrial carcinoma is the most common gynecological malignancy, and 95% of women present with PMB [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>-<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]. Endometrial cancer often presents at an early stage, allowing curative surgical intervention; therefore, early, accurate diagnosis is paramount.</p><p>Historically, investigation of PMB was through dilatation and curettage (D&C) [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. Ultrasonography reduced investigatory invasiveness. Endometrial biopsy and hysteroscopy have almost completely replaced D&C. Outpatient endometrial biopsy reduces costs in diagnostic work-up, without affecting life expectancy [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]. Despite numerous studies investigating PMB, a consensus regarding the most accurate and efficient diagnostic pathway remains elusive [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>,<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>-<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>].</p><p>TVUS may visualize the endometrium in postmenopausal women poorly, and distortion of the cavity by pathological abnormalities can make assessment difficult. Such limitations have led to a growing interest in saline contrast sonohysterography (SCSH).</p><p>SCSH involves the instillation of sterile saline, a negative contrast agent, into the uterus through a hysterosalpingography catheter prior to TVUS. Uterine cavity distension is ‘optimal’ if fluid clearly distends the cavity, ‘suboptimal’ if the cavity is minimally distended, and ‘failed’ if no fluid is observed within the cavity.</p><p>Compared to TVUS, SCSH has been reported in premenopausal women to allow easier differentiation of polyps, submucous fibroids, and endometrial lesions that emerge clearly in anechoic saline [<xref rid=\"REF16\" ref-type=\"bibr\">16</xref>-<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>]. Fortunately, it requires widely available, inexpensive equipment, and has good reported diagnostic performance in the bleeding uterus [<xref rid=\"REF18\" ref-type=\"bibr\">18</xref>-<xref rid=\"REF19\" ref-type=\"bibr\">19</xref>].</p></sec><sec sec-type=\"review\"><title>Review</title><p>Materials and methods</p><p>The electronic bibliographic databases, MEDLINE, Embase, and the Cochrane Library, were searched (Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials (CENTRAL), Cochrane Methodology Register) on the accuracy of SCSH and other diagnostic tools in women with PMB using Medical Subject Headings (MeSH) and text words for SCSH (SCSH; Saline Infusion Sonography, Saline Infusion*; Saline Infusion Hysterosonography, Saline Contrast Sonohysterography)’, and ‘post-menopause’ (post-menopause*, menopause, post-menopause). We used the PICO model for the search strategy (Patient: post-menopausal, Intervention: saline infusion sonography, saline contrast sonohysterography, Comparison: other diagnostic modalities (outpatient/inpatient hysteroscopy or endometrial biopsy), Outcome: accuracy of saline contrast sonohysterography). The search strategy was adapted for use with other bibliographic databases in combination with database-specific filters for controlled trials, where available. Studies published from January 1980 to 2017 were considered.</p><p>Only texts in English were included. The reference lists of all known primary and review articles were examined for relevant citations not captured by electronic searches. Studies directly comparing the diagnostic accuracy of SCSH and other diagnostic modalities in women with PMB were included. Studies reporting a single technique were excluded.</p><p>Exclusion criteria included hormone therapy within the last six months, previous abnormal endometrial biopsy, cervical pathology on speculum examination, abnormal cervical smear, and history or evidence suggestive of active pelvic infection.</p><p>Content analysis was chosen as the data analysis method because of its strengths in methodically cataloging and summarizing substantial amounts of data and text. The overall approach to this method of analysis was the interpretation of patterns occurring within texts, particularly with the context of the sample data. Thus, the research coded the data in three phases: (1) absorption in the text and data, (2) reduction of the data through systematic coding and generation of themes, and (3) interpretation of findings. Using a deductive approach, data analysis was initiated with a preconceived coding template, organized according to an existing structure. Such a structure was premised on the purpose of this systematic review, to evaluate the diagnostic accuracy of SCSH in the evaluation of intracavitary abnormalities in postmenopausal women with AUB. Thus, the results of the content analysis were qualitative, summarizing key findings both by patient population and by recommendation of SCSH as a diagnostic tool.</p><p>Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref> shows the process of identifying studies and the methods used.</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Identification and method</title></caption><graphic xlink:href=\"cureus-0012-00000010094-i01\"/></fig><p>Results</p><p>We included 18 studies comprising 974 women in this systematic review. SCSH was considered a valuable diagnostic tool for PMB [<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>-<xref rid=\"REF22\" ref-type=\"bibr\">22</xref>] and advocated as an initial test rather than hysteroscopy to triage patients with PMB, SCSH could be the sole initial test, with endometrial biopsy used only for patients with a symmetrically thickened endometrium [<xref rid=\"REF23\" ref-type=\"bibr\">23</xref>]. However, one study argued that the feasibility of using SCSH as compared with diagnostic hysteroscopy is low in postmenopausal women [<xref rid=\"REF24\" ref-type=\"bibr\">24</xref>].</p><p>Others promoted SCSH in combination with TVUS as an initial diagnostic step for women with PMB [<xref rid=\"REF25\" ref-type=\"bibr\">25</xref>-<xref rid=\"REF26\" ref-type=\"bibr\">26</xref>] showing SCSH improves the diagnostic accuracy of TVUS through the better visualization of small intracavitary tumors; however, their populations were mostly perimenopausal women [<xref rid=\"REF27\" ref-type=\"bibr\">27</xref>-<xref rid=\"REF28\" ref-type=\"bibr\">28</xref>].</p><p>Miller et al. [<xref rid=\"REF29\" ref-type=\"bibr\">29</xref>] and Mihm et al. [<xref rid=\"REF30\" ref-type=\"bibr\">30</xref>] claimed that SCSH is valuable for patient selection and procedure planning for hysteroscopy, but did not define their population by menopausal status, therefore, these studies may not advocate SCSH in a specifically postmenopausal group. Takac et al. argued that SCSH is not a superior diagnostic tool to TVUS, a conclusion which contradicts other studies [<xref rid=\"REF31\" ref-type=\"bibr\">31</xref>].</p><p>Overall, the consensus is that SCSH is a useful initial diagnostic tool to investigate PMB, but a consensus is lacking regarding its sensitivity, specificity, and positive predictive value (PPV) for detecting uterine pathology.</p><p>Postmenopausal Population</p><p>Of the 18 studies analyzed, eight focused primarily on postmenopausal women.</p><p>Evaluation 1: SCHS as equal or superior to hysteroscopy, TVUS, and D&C: Epstein et al. evaluated 105 women with PMB and ET >5 mm, comparing hysteroscopy, TVUS, and SCSH [<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>]. The study suggested that SCSH could replace hysteroscopy for diagnosing focal intrauterine lesions in women with PMB and SCSH was superior to TVUS at distinguishing between benign and malignant endometria. They found a 96% correlation between SCSH and hysteroscopy at identifying focal lesions, with 80% sensitivity for polyps. A conventional ultrasound had 49% sensitivity, with a false-positive rate of 19%. When discriminating between benign and malignant lesions, hysteroscopy (84%) was more successful than SCSH (66%) and conventional ultrasound (40%). Neither hysteroscopy nor SCSH could dependably differentiate between benign and malignant focal lesions, with SCSH failing in almost 20% of cases; two-thirds of this failure was attributed to cervical stenosis and another one-quarter to problems with backflow or distension.</p><p>Goldstein et al. concurred SCSH is as accurate a diagnostic tool as hysteroscopy and frequently more effective than TVUS [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. This article was a summation of a panel discussion, not a quantitative study. Despite this limitation, the panel was monolithic in its assessment of SCSH as the appropriate tool to diagnose a focal abnormality, highlighting its safety, ease of the procedure, and equal detection rates for focal abnormalities with hysteroscopy. They noted that SCSH could be used to distinguish the nature of the focal abnormality and hysteroscopy would be used to remove it. They advocated SCSH for triaging patients, distinguishing diffuse and focal endometrial thickening, and concurring that SCSH was more sensitive than TVUS or endometrial biopsy (EMB) at detecting focal abnormalities in women with PMB.</p><p>Karsidag et al. argued that SCSH is a superior diagnostic tool to blind D&C and TVUS, with equal accuracy to hysteroscopy in diagnosing focal intrauterine lesions in 36 women with recurrent PMB following normal D&C [<xref rid=\"REF22\" ref-type=\"bibr\">22</xref>]. Results showed blind D&C had 47% sensitivity and 68% specificity for diagnosing focal intracavitary abnormalities, with 57% PPV and 59% negative predictive value (NPV) while missing 65% of polyps and submucous fibroids. TVUS, had a sensitivity of 63%, a specificity of 78%, a PPV of 89%, and an NPV of 41%. TVUS failed to identify 10 patients with polyps and misdiagnosed two normal patients with polyps. SCSH had a sensitivity of 93%, a specificity of 56%, a PPV of 86%, an NPV of 71%, and a diagnostic accuracy of 83%. When comparing SCSH with hysteroscopy, sensitivity was 88%, specificity was 75% while PPV was 97% and NPV was 43%. This concluded that SCSH and hysteroscopy are equally valuable diagnostic tools, but SCSH cannot reliably discriminate between benign and malignant focal lesions. Conversely, SCSH can distinguish between the endometrium and myometrium, making it more appropriate for classifying the degree of fibroid extension.</p><p>Takac et al. were outliers arguing that SCSH is not superior to TVUS [<xref rid=\"REF31\" ref-type=\"bibr\">31</xref>]. Fifty-three postmenopausal women with endometrial cancer were examined preoperatively with TVUS and SCSH to assess myometrial invasion. No statistically significant difference in the accuracy of SCSH (85%) as compared to TVUS (81%) was seen when correlated with definitive histopathologic findings. Regarding endometrioid adenocarcinomas, TVUS correctly predicted myometrial invasion in 85% as compared to SCSH 80%. SCSH had better specificity (72%) than TVUS (76%) and better sensitivity (96%) than TVUS (86%). Both modalities demonstrated 83.3% accuracy when predicting the depth of myometrial invasion in 15 cases of G1 tumors.</p><p>All four studies agreed that SCSH is a useful diagnostic tool that is easier, more cost-efficient, less invasive, and as effective as hysteroscopy. Three of four found SCSH superior to TVUS, D&C, and EMB, advocating it as an initial triage for patients with PMB.</p><p>Evaluation 2: SCSH in conjunction with TVUS: Two studies recommended SCSH in conjunction with TVUS; one assessed the combination as comparable to hysteroscopy, the other judged SCSH with TVUS as superior to EMB.</p><p>Inal et al. tested the diagnostic efficacy of TVUS and SCSH in the evaluation of uterine cavities in 60 tamoxifen-treated, asymptomatic, postmenopausal breast cancer patients [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. TVUS and SCSH failed in four (6.7%), detected a normal endometrium in 38 (63.3%) (hysteroscopy: 66.7%), and polypoid structures in 18 (30%) (hysteroscopy: 33.3%). When compared to hysteroscopy, TVUS with SCSH had 90% sensitivity, 100% specificity, 100% PPV, and 95% NPV.</p><p>Dubinsky et al. compared TVUS and SCSH with EMB, in 81 postmenopausal women with ET > 5 mm [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. TVUS and SCSH confirmed 45 lesions: 23 pedunculated endometrial masses (16 endometrial polyps, 4 fibroids, and 3 carcinomas) and 22 inhomogeneous sessile lesions. Of these 45 cases, 41 had false-negative aspiration biopsies. Researchers noted TVUS and SCSH failed in only eight of 89 cases, primarily due to cervical stenosis. Researchers, therefore, advocated TVUS and SCSH as an initial diagnostic tool for postmenopausal women. However, women with a positive diagnosis subsequently underwent hysteroscopy or hysterectomy, emphasizing the role and limitations of SCHS.</p><p>Evaluation 3: SCHS with endometrial biopsy: O’Connell et al. compared the combined diagnostic reliability of SCHS and EMB with fractional curettage and hysteroscopy for the initial evaluation of women with PMB [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. The combination of SCSH and EMB positively correlated with the surgical findings in > 95% (sensitivity 94%, specificity 96%, CI: 91% - 99%). Furthermore, no patients with endometrial hyperplasia or cancer were misdiagnosed. SCSH alone had a 92% positive correlation, however, sensitivity was conspicuously hindered by the omission of EMB. Therefore, the combination was recommended for initial evaluation, noting SCSH could be the sole initial triage, with EMB used for patients with symmetrically thickened endometrium.</p><p>Outlier: outcome-based study: Meng et al. looked at the clinical outcomes of 119 women with PMB who underwent SCSH [<xref rid=\"REF32\" ref-type=\"bibr\">32</xref>]. Findings indicated a statistically significant connection between a positive SCSH and more aggressive treatment; a negative SCSH resulted in more conservative treatment in 95%. Of the 80 women with a positive SCSH, abnormalities included polyps (42), submucosal fibroids (6), endometrial thickening (8), >2 of the above (7), or other; debris, adenomyosis, or indeterminate findings (17).</p><p>Perimenopausal Population</p><p>This systematic review focuses on postmenopausal women, however, one study of 159 perimenopausal patients with AUB was included [<xref rid=\"REF33\" ref-type=\"bibr\">33</xref>]. It concurs SCSH is reliable at investigating AUB and differentiating the need for medical therapy from surgery. SCSH was highly sensitive (98.9%) and specific (76.4%) at distinguishing between women with intrauterine lesions from those with normal or atrophic endometrium. SCSH was accurate in the diagnosis of submucosal fibroid (sensitivity 87.8%, specificity 95%) and polyps (sensitivity 89.6%, specificity 90.7%). Although SCSH recognized only 40% of endometrial cancers, all these patients had abnormalities during SCHS, indicating a zero false‐negative rate.</p><p>Combination of Pre-, Peri-, and Postmenopausal Populations</p><p>Four studies with mixed patient populations were included. While these studies evaluations are valuable, ascertaining the applicability of their findings exclusively to postmenopausal women is difficult.</p><p>Evaluation 1: SCHS as an accurate diagnostic tool (with caveats): Two studies argued that SCSH was a more effective diagnostic tool than TVUS. Yildizhan et al. compared the diagnostic efficacy of TVUS and SCSH [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. They showed that SCSH is more effective than TVUS. TVUS cannot distinguish between polyps, submucosal fibroids, and hyperplasia. They also failed to identify the exact location of growths and polyps. However, the authors were able to comment on the distinction between pre- and postmenopausal patients, arguing that while the feasibility of using SCSH in premenopausal women is comparable with diagnostic hysteroscopy, it is low in postmenopausal women. The authors recommend EMB as the primary method for diagnosing endometrial hyperplasia and carcinoma in postmenopausal women.</p><p>Dubinsky et al. included 88 pre-, peri-, and postmenopausal women [<xref rid=\"REF34\" ref-type=\"bibr\">34</xref>]. The authors concluded SCSH was an appropriate, effective diagnostic tool but not as valuable or suitable for detecting carcinoma. The PPV for carcinoma was 16%, suggesting that women with multifocal or sessile lesions should undergo guided biopsy, and benign-appearing polyps should be removed to control bleeding and eliminate the risk of carcinoma in situ. However, using SCSH, women with a benign endometrial disease could seek medical therapy, avoiding invasive diagnostic procedures. The inclusive patient population makes it unclear if results can be connected to women’s menopausal status.</p><p>Evaluation 2: SCHS in combination with TVUS: Schwarzler et al. argued that SCSH improves the diagnostic accuracy of TVUS through the better visualization of small intracavitary tumors [<xref rid=\"REF27\" ref-type=\"bibr\">27</xref>]. Studying 100 patients, researchers found that the sensitivity and NPV of TVUS (67% and 71%, respectively) improved considerably (87% and 86%, respectively) with SCSH. Moreover, the number of false-negative findings for this type of pathology reduced significantly. The addition of SCSH to TVUS did not significantly enhance the detection of malignant and premalignant changes in the endometrium. Moreover, a sizeable proportion of the study population were premenopausal women.</p><p>Pasrija et al. studied 58 women, finding that SCSH enhanced the diagnostic accuracy of TVUS [<xref rid=\"REF28\" ref-type=\"bibr\">28</xref>]. Results indicated that TVUS alone had 84.8% sensitivity, 79% specificity, 82.4% PPV, and 82% NPV. The addition of SCSH increased sensitivity to 94.1%, specificity to 88.5%, PPV to 91.4%, and NPV to 92%. When compared to hysteroscopy or hysterectomy, SCSH findings missed one endometrial polyp and one endocervical polyp, as well as diagnosed a false-positive growth. Importantly, 89.6% of women were perimenopausal.</p><p>Population Unknown</p><p>This is the final category of two studies that did not define their population’s menopausal status.</p><p>Evaluation 1: SCHS should be used in certain cases: Miller et al. argued SCSH should be the diagnostic tool when TVUS shows normal ET or if endometrial biopsy findings are benign [<xref rid=\"REF29\" ref-type=\"bibr\">29</xref>]. Ultimately, SCSH is a sensitive (95%) method for detecting focal intracavitary abnormalities and is valuable for patient selection and procedure planning for hysteroscopy.</p><p>Evaluation 2: SCSH with EMB:<italic> </italic>Mihm et al. studied 113 women, finding the combination of EMB and SCSH had 97% sensitivity and 70.2% specificity for the detection of abnormal pathologic features, with 82.1% PPV and 94.3% NPV as compared to hysteroscopy/curettage or hysterectomy [<xref rid=\"REF30\" ref-type=\"bibr\">30</xref>]. Researchers argued that the combination of EMB and SCSH allows patients to circumvent more aggressive procedures, offering a more conservative treatment option. Authors acknowledged not stratifying by menopausal status, could alter treatment decisions.</p><p>Discussion</p><p>Main Findings</p><p>PMB is estimated to account for 5%-10% of gynecologic visits, which is an increasing trend due to the increased prevalence of HRT. Thirty percent (30%) of PMB is secondary to exogenous estrogen, another 30% due to atrophic endometritis or vaginitis, while 40% is due to endometrial hyperplasia (10%), endometrial polyps (10%), submucous fibromyomas (10%), and uterine malignancies (10 %). Most cancers causing PMB are endometrial in origin, accounting for 5%-8% of all PMB cases [<xref rid=\"REF35\" ref-type=\"bibr\">35</xref>].</p><p>SCSH has been proposed as a means for the nonsurgical identification of intracavity abnormalities [<xref rid=\"REF25\" ref-type=\"bibr\">25</xref>]. Wolman et al. reported a prospective double-blind study of 47 postmenopausal patients evaluated first by SCSH and then hysteroscopy [<xref rid=\"REF36\" ref-type=\"bibr\">36</xref>]. SCSH proved accurate in the diagnosis of intraluminal masses with 86% sensitivity and 87% specificity.</p><p>The evaluation of PMB is evolving as newer imaging techniques like SCSH gain acceptance. Many algorithms for the evaluation of PMB have been introduced, most suggest starting with TVUS to assess ET, followed by SCSH to define abnormalities prior to surgical intervention or biopsy.</p><p>Numerous studies have shown ET ≤5 mm on TVUS has a high NPV for endometrial carcinoma, virtually excluding significant pathology. However, TVUS does not adequately define focal lesions.</p><p>SCSH is consistently reported as significantly better or equal to TVUS in defining intrauterine abnormalities. With several small studies reporting the diagnostic accuracy of SCSH in an exclusively postmenopausal population, ee sought to systematically analyze this data.</p><p>Strengths and limitations</p><p>In this review of 18 studies, including 974 women evaluating the use of SCSH to investigate postmenopausal bleeding, the sensitivity in individual studies ranged from 67.7%-97%, specificity from 46%-100%, with PPV ranging from 16-100 [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>-<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>].</p><p>It was also observed that in a sub-analysis of one study, including 159 peri-menopausal women with AUB, SCSH was highly sensitive (98.9%) and specific (76.4%) in the distinction between women with intrauterine lesions and those with normal or atrophic endometrium (98.9% and 76.4%, respectively). SCSH was also accurate in the diagnosis of submucosal fibroid (sensitivity 87.8%, specificity 95%) and polyps (sensitivity 89.6%, specificity 90.7%) [<xref rid=\"REF37\" ref-type=\"bibr\">37</xref>]. The review showed SCSH was in near-perfect agreement (96%) with hysteroscopy in the diagnosis of focally growing lesions. Similarly, Epstein et al. (2001), in diagnosing endometrial polyps, both SCHS and hysteroscopy had a sensitivity of approximately 80% while conventional ultrasound was 49% with a false-positive rate of 19% [<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>]. However, some authors found hysteroscopy (84%) was more successful than SCHS (44%) and conventional ultrasound (60%) in discriminating between benign and malignant lesions. Results were split between specificity (76% with TVUS and 72% with SCHS) and sensitivity (86% with TVUS and 96% with SCHS) [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>,<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>].</p><p>Likewise, findings were similar in predicting the depth of myometrial invasion in 15 cases (83.3%) of G1 tumors by both SCHS and TVUS. Takac et al. [<xref rid=\"REF31\" ref-type=\"bibr\">31</xref>] and Goldstein et al. [<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>] similarly concurred that SCHS is an equally effective diagnosis tool as hysteroscopy and frequently a more effective diagnostic tool than TVUS.\nTable <xref rid=\"TAB1\" ref-type=\"table\">1</xref> summarizes all the evidence found during our research while Table <xref rid=\"TAB2\" ref-type=\"table\">2</xref> lists all the studies included in the research.</p><table-wrap id=\"TAB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Summary of evidence </title><p>PPV: positive predictive value</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Paper</td><td rowspan=\"1\" colspan=\"1\">Final No. of patients</td><td rowspan=\"1\" colspan=\"1\">Sensitivity</td><td rowspan=\"1\" colspan=\"1\">Specificity</td><td rowspan=\"1\" colspan=\"1\">PPV</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Transvaginal hysterosonography: comparison with biopsy in the evaluation of postmenopausal bleeding</td><td rowspan=\"1\" colspan=\"1\">148</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">Unknown</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Saline contrast sonohysterography as the first-line investigation for women with uterine bleeding</td><td rowspan=\"1\" colspan=\"1\"> Unknown</td><td rowspan=\"1\" colspan=\"1\">76.525</td><td rowspan=\"1\" colspan=\"1\">95.325</td><td rowspan=\"1\" colspan=\"1\">Unknown</td></tr><tr><td rowspan=\"1\" colspan=\"1\">An evaluation of sonohysterography and diagnostic hysteroscopy for the assessment of intrauterine pathology</td><td rowspan=\"1\" colspan=\"1\">98</td><td rowspan=\"1\" colspan=\"1\">87</td><td rowspan=\"1\" colspan=\"1\">91</td><td rowspan=\"1\" colspan=\"1\">92</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Triage of abnormal postmenopausal bleeding: A comparison of endometrial biopsy and transvaginal sonohysterography versus fractional curettage with hysteroscopy</td><td rowspan=\"1\" colspan=\"1\">100</td><td rowspan=\"1\" colspan=\"1\">94</td><td rowspan=\"1\" colspan=\"1\">96</td><td rowspan=\"1\" colspan=\"1\">96</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Saline infusion sonography (SCSH) or office hysteroscopy: Which one is the best? A prospective randomized study</td><td rowspan=\"1\" colspan=\"1\">53</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">Unknown</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Prediction of benign and malignant endometrial disease: hysterosonographic pathologic correlation</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">89</td><td rowspan=\"1\" colspan=\"1\">46</td><td rowspan=\"1\" colspan=\"1\">16</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Transvaginal sonography, saline contrast sonohysterography and hysteroscopy for the investigation of women with postmenopausal bleeding and endometrium > 5 mm</td><td rowspan=\"1\" colspan=\"1\">105</td><td rowspan=\"1\" colspan=\"1\">67.7</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">71.3</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Evaluation of the woman with postmenopausal bleeding</td><td rowspan=\"1\" colspan=\"1\">N/A</td><td rowspan=\"1\" colspan=\"1\">N/A</td><td rowspan=\"1\" colspan=\"1\">N/A</td><td rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td rowspan=\"1\" colspan=\"1\">The accuracy of endometrial biopsy and saline sonohysterography in the determination of the cause of abnormal uterine bleeding</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">97</td><td rowspan=\"1\" colspan=\"1\">70.2</td><td rowspan=\"1\" colspan=\"1\">82.1</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Saline contrast hysterosonography in abnormal uterine bleeding: a systematic review and meta-analysis</td><td rowspan=\"1\" colspan=\"1\">2278</td><td rowspan=\"1\" colspan=\"1\">95</td><td rowspan=\"1\" colspan=\"1\">88</td><td rowspan=\"1\" colspan=\"1\">LR + = 8.23</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Prospective study of saline infusion sonohysterography in the evaluation of perimenopausal and postmenopausal women with abnormal uterine bleeding</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">94.1</td><td rowspan=\"1\" colspan=\"1\">88.5</td><td rowspan=\"1\" colspan=\"1\">91.4</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Diagnostic accuracy of sonohysterography in the evaluation of uterine cavities in tamoxifen-administered asymptomatic postmenopausal breast cancer patients with endometrial thickness ≥5 mm</td><td rowspan=\"1\" colspan=\"1\">60</td><td rowspan=\"1\" colspan=\"1\">90</td><td rowspan=\"1\" colspan=\"1\">100</td><td rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td rowspan=\"1\" colspan=\"1\">The short-term clinical outcomes after saline infusion sonohysterography in women with postmenopausal bleeding</td><td rowspan=\"1\" colspan=\"1\">119</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">Unknown</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Transvaginal ultrasonography with and without saline infusion in the assessment of myometrial invasion of endometrial cancer</td><td rowspan=\"1\" colspan=\"1\">53</td><td rowspan=\"1\" colspan=\"1\">72</td><td rowspan=\"1\" colspan=\"1\">96</td><td rowspan=\"1\" colspan=\"1\">95</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Transvaginal ultrasonography and saline infusion sonohysterography for the detection of intrauterine lesions in pre- and post-menopausal women with abnormal uterine bleeding</td><td rowspan=\"1\" colspan=\"1\">104</td><td rowspan=\"1\" colspan=\"1\">91.45</td><td rowspan=\"1\" colspan=\"1\">95.9</td><td rowspan=\"1\" colspan=\"1\">93.45</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Ultrasound and sonohysterography in the evaluation of abnormal vaginal bleeding</td><td rowspan=\"1\" colspan=\"1\">N/A</td><td rowspan=\"1\" colspan=\"1\">N/A</td><td rowspan=\"1\" colspan=\"1\">N/A</td><td rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Transvaginal sonography, sonohysterography, and hysteroscopy for investigation of focal intrauterine lesions in women with recurrent postmenopausal bleeding after dilatation & curettage</td><td rowspan=\"1\" colspan=\"1\">36</td><td rowspan=\"1\" colspan=\"1\">93</td><td rowspan=\"1\" colspan=\"1\">56</td><td rowspan=\"1\" colspan=\"1\">86</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">A prospective comparison of transvaginal ultrasound, saline infusion sonohysterography, and diagnostic hysteroscopy in the evaluation of endometrial pathology</td><td rowspan=\"1\" colspan=\"1\">98</td><td rowspan=\"1\" colspan=\"1\">91.8</td><td rowspan=\"1\" colspan=\"1\">60</td><td rowspan=\"1\" colspan=\"1\">LR + = 2.29</td></tr></tbody></table></table-wrap><table-wrap id=\"TAB2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><title>Summary of included studies</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Paper</td><td rowspan=\"1\" colspan=\"1\">Year</td><td colspan=\"2\" rowspan=\"1\">Authors</td><td colspan=\"2\" rowspan=\"1\">Final No. of patients</td><td colspan=\"2\" rowspan=\"1\">Pre-meno No. of patients</td><td colspan=\"2\" rowspan=\"1\">Post-meno No. of patients</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Transvaginal hysterosonography: comparison with biopsy in the evaluation of postmenopausal bleeding</td><td colspan=\"2\" rowspan=\"1\">1995</td><td colspan=\"2\" rowspan=\"1\">Dubinsky et al. [<xref rid=\"REF25\" ref-type=\"bibr\">25</xref>]</td><td colspan=\"2\" rowspan=\"1\">148</td><td colspan=\"2\" rowspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">148</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Saline contrast sonohysterography as the first-line investigation for women with uterine bleeding</td><td colspan=\"2\" rowspan=\"1\">1997</td><td colspan=\"2\" rowspan=\"1\">Bernard et al. [<xref rid=\"REF33\" ref-type=\"bibr\">33</xref>]</td><td colspan=\"2\" rowspan=\"1\">Unknown</td><td colspan=\"2\" rowspan=\"1\">109</td><td rowspan=\"1\" colspan=\"1\">53</td></tr><tr><td rowspan=\"1\" colspan=\"1\">An evaluation of sonohysterography and diagnostic hysteroscopy for the assessment of intrauterine pathology</td><td colspan=\"2\" rowspan=\"1\">1998</td><td colspan=\"2\" rowspan=\"1\">Schwarzler et al. [<xref rid=\"REF27\" ref-type=\"bibr\">27</xref>]</td><td colspan=\"2\" rowspan=\"1\">98</td><td colspan=\"2\" rowspan=\"1\">70</td><td rowspan=\"1\" colspan=\"1\">28</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Triage of abnormal postmenopausal bleeding: A comparison of endometrial biopsy and transvaginal sonohysterography versus fractional curettage with hysteroscopy</td><td colspan=\"2\" rowspan=\"1\">1998</td><td colspan=\"2\" rowspan=\"1\">O'Connell et al. [<xref rid=\"REF23\" ref-type=\"bibr\">23</xref>]</td><td colspan=\"2\" rowspan=\"1\">100</td><td colspan=\"2\" rowspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Saline infusion sonography (SCSH) or office hysteroscopy: which one is the best? A prospective randomized study</td><td colspan=\"2\" rowspan=\"1\">1998</td><td colspan=\"2\" rowspan=\"1\">Timmermans et al. [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]</td><td colspan=\"2\" rowspan=\"1\">53</td><td colspan=\"2\" rowspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">53</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Prediction of benign and malignant endometrial disease: hysterosonographic pathologic correlation</td><td colspan=\"2\" rowspan=\"1\">1995</td><td colspan=\"2\" rowspan=\"1\">Dubinsky et al. [<xref rid=\"REF34\" ref-type=\"bibr\">34</xref>]</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\">88</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Transvaginal sonography, saline contrast sonohysterography, and hysteroscopy for the investigation of women with postmenopausal bleeding and endometrium > 5 mm</td><td colspan=\"2\" rowspan=\"1\">2001</td><td colspan=\"2\" rowspan=\"1\">Epstein et al. [<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>]</td><td colspan=\"2\" rowspan=\"1\">105</td><td colspan=\"2\" rowspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">105</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Evaluation of the woman with postmenopausal bleeding</td><td colspan=\"2\" rowspan=\"1\">2001</td><td colspan=\"2\" rowspan=\"1\">Goldstein et al. [<xref rid=\"REF21\" ref-type=\"bibr\">21</xref>]</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">The accuracy of endometrial biopsy and saline sonohysterography in the determination of the cause of abnormal uterine bleeding</td><td colspan=\"2\" rowspan=\"1\">2002</td><td colspan=\"2\" rowspan=\"1\">Mihm et al. [<xref rid=\"REF30\" ref-type=\"bibr\">30</xref>]</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\">113</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Saline contrast hysterosonography in abnormal uterine bleeding: A systematic review and meta-analysis</td><td colspan=\"2\" rowspan=\"1\">2003</td><td colspan=\"2\" rowspan=\"1\">de Kroon et al. [<xref rid=\"REF38\" ref-type=\"bibr\">38</xref>]</td><td colspan=\"2\" rowspan=\"1\">2278</td><td colspan=\"2\" rowspan=\"1\">2278</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Prospective study of saline infusion sonohysterography in the evaluation of perimenopausal and postmenopausal women with abnormal uterine bleeding</td><td colspan=\"2\" rowspan=\"1\">2004</td><td colspan=\"2\" rowspan=\"1\">Pasrija et al. [<xref rid=\"REF28\" ref-type=\"bibr\">28</xref>]</td><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"2\" rowspan=\"1\">52</td><td rowspan=\"1\" colspan=\"1\">6</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Diagnostic accuracy of sonohysterography in the evaluation of uterine cavities in tamoxifen administered asymptomatic postmenopausal breast cancer patients with endometrial thickness ≥ 5 mm</td><td colspan=\"2\" rowspan=\"1\">2006</td><td colspan=\"2\" rowspan=\"1\">Inal et al. [<xref rid=\"REF26\" ref-type=\"bibr\">26</xref>]</td><td colspan=\"2\" rowspan=\"1\">60</td><td colspan=\"2\" rowspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">60</td></tr><tr><td rowspan=\"1\" colspan=\"1\">The short-term clinical outcomes after saline infusion sonohysterography in women with postmenopausal bleeding</td><td colspan=\"2\" rowspan=\"1\">2006</td><td colspan=\"2\" rowspan=\"1\">Meng et al. 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[<xref rid=\"REF22\" ref-type=\"bibr\">22</xref>]</td><td colspan=\"2\" rowspan=\"1\">36</td><td colspan=\"2\" rowspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">36</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">A prospective comparison of transvaginal ultrasound, saline infusion sonohysterography and diagnostic hysteroscopy in the evaluation of endometrial pathology</td><td colspan=\"2\" rowspan=\"1\">2010</td><td colspan=\"2\" rowspan=\"1\">Grimbizis et al. [<xref rid=\"REF39\" ref-type=\"bibr\">39</xref>]</td><td colspan=\"2\" rowspan=\"1\">98</td><td colspan=\"2\" rowspan=\"1\">77</td><td rowspan=\"1\" colspan=\"1\">21</td></tr></tbody></table></table-wrap></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>In conclusion, we suggest that SCSH could be used as the first-line investigative modality to evaluate the uterine cavity in PMB, owing to its relatively non-invasive nature and potentially high diagnostic accuracy to allow for more accurate decisions about the need for further investigation and subsequent preoperative planning. In cases where an endometrial lesion is detected, SCSH can act as a multipurpose investigation to both diagnose and triage the patient, assisting in decision-making regarding the need for further invasive surgical management. Hence, the role of SCHS should be to triage patients for more or less invasive therapy where an endometrial lesion is detected. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005526</article-id><article-id pub-id-type=\"pmc\">PMC7523545</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10108</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology/Diabetes/Metabolism</subject></subj-group><subj-group><subject>Internal Medicine</subject></subj-group></article-categories><title-group><article-title>A Literature Review on Thyrotoxic Periodic Paralysis</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Iqbal</surname><given-names>Qasim Z</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Niazi</surname><given-names>Muhammad</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zia</surname><given-names>Zeeshan</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sattar</surname><given-names>Saud Bin Abdul</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nInternal Medicine, Northwell Health, New York, USA </aff><author-notes><corresp id=\"cor1\">\nQasim Z. Iqbal <email>q.z.iqbal@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10108</elocation-id><history><date date-type=\"received\"><day>11</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Iqbal et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Iqbal et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/39453-a-literature-review-on-thyrotoxic-periodic-paralysis\">This article is available from https://www.cureus.com/articles/39453-a-literature-review-on-thyrotoxic-periodic-paralysis</self-uri><abstract><p>Thyrotoxic periodic paralysis (TPP) is a rare disease of the muscles that presents with painless weakness of the muscles. The patients usually have hypokalemia and hyperthyroidism with elevations in the level of triiodothyronine (T3) and thyroxine (T4). The muscle weakness is usually transient, and the patients in many cases suffer from recurrent episodes of muscle paralysis. This flaccid muscle paralysis predominantly affects the proximal and lower extremities group of muscles more than the distal and upper extremity muscles. This condition is one of the drastic complications of Graves's disease and, unfortunately, may require admission and treatment in the critical care units. It is often not recognized during the initial attack in the American population as the prevalence is very low among the Caucasian population and people from North America. However, while the prevalence is extremely low in the Caucasian population, it is known to be 10 times more common among the Asian population when compared with the Caucasian population. Furthermore, while the diseases of the thyroid gland are more common in females, this rare disease predominantly affects male sex. It is treated by reversing the hypokalemia, which can in itself prove to be fatal if not corrected quickly, and this is followed by treatment to restore the euthyroid state. A literature review on this reversible cause of muscle weakness is very important to better understand this disease.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>thyrotoxicity</kwd><kwd>endocrinology</kwd><kwd>hyperthyroidism</kwd><kwd>reversible cause of muscle weakness</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction and background</title><p>Thyrotoxic periodic paralysis (TPP) is a rare disease of the muscles. It presents with acute painless weakness of the muscles. The muscle weakness is recurrent and reversible, lasting from hours to sometimes days. This muscle weakness is known to be more severe in the proximal and lower extremity muscles. It is usually not recognized during the initial attack as the prevalence is very low among various populations especially the Caucasian population. Most cases of TPP are seen in patients with Grave's disease. Interestingly, despite the general higher incidence of hyperthyroidism found in females, this rare disease is more common in males. The patients would have a combination of hypokalemia and hyperthyroidism. The management focuses on the earlier and optimal replacement of potassium to avoid fatal complications of hypokalemia such as cardiac arrhythmias. This is then followed by restoration of the euthyroid state in the patient. Our review emphasizes the importance of both diagnosing and treating TPP early as this would decrease both mortality and morbidity in these patients.</p></sec><sec sec-type=\"review\"><title>Review</title><p>TPP is a rare disease of the muscles that presents with an acute painless weakness of the muscles. The patients affected usually have both hypokalemia and hyperthyroidism. This muscle weakness is found to be more severe in the proximal muscles and in the lower extremities. TPP is often not recognized at first attack due to a very low prevalence among the Caucasian population and since it is usually associated with mild symptoms of hyperthyroidism [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>].</p><p>This disease is found to be more common among the Asian population. It occurs in 0.1-0.2% of hyperthyroid patients in North America and is 10 times more common among the Asian population and males [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. The high incidence of TPP among the Asian population is associated with the human leukocyte antigen DRw8 isotype (HLA-DRw8). This shows that the main cause may be genetic, but the exact mechanism behind is still unknown [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>].</p><p>The control of potassium ion in the body is mainly by two ion channels, namely sodium-potassium (Na-K) pump and inward rectifying potassium channel (Kir). Both these channels work together in tight control to maintain serum potassium levels. Na-K pump pumps sodium into the cells (influx), whereas Kir channels control outward flow of potassium ions, i.e., efflux from the cells [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. The latter channels are inhibited by catecholamines and insulin, causing hypokalemia. Mutations of Kir can also contribute to hypokalemia. According to one recent study, loss of function mutation of the Kir channel (Kir 2.6) was found in approximately 33% of patients with TPP [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. The severity of the disease is not found to be correlated with the TPP, although muscle paralysis is known to be resolved once the euthyroid state is achieved. Hyperthyroid patients are known to have a high beta-adrenergic activity that pumps potassium inside the cells, causing hypokalemia that results in muscle weakness. Total potassium levels inside the body remain the same. There is no excessive gastrointestinal (GI) or urinary loss of potassium [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. The best diagnostic test to diagnose the cause behind hypokalemia is 24-hour urine potassium and potassium-to-creatinine ratio (K/Cr). Former is actually impractical in acute settings where potassium is actively being replete in the patients, whereas K/Cr is very useful, and a value of less than 13 in the setting of hypokalemia favors either GI loss or transcellular shift [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>,<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>].</p><p>Other precipitants of TPP include hyperinsulinemia state such as carbohydrate-rich meals, obesity, and catecholamine surge (recurrent episodes are more common during the early morning) [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>,<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. There is a reported case where a person had muscle paralysis every Monday possibly because of the intake of large amounts of meals on weekends [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. Hypokalemia can also be caused by androgens as they increase the activity of Na-K pump, whereas the estrogen and progesterone decrease it [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>,<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>]. This can be one of the reasons behind TPP being common among males. Hypomagnesemia and hypokalemia are also found to be associated with TPP. Similarly, there are case reports where patients developed TPP after high-dose steroids [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]. In another instance, patients developed TPP after starting antiretroviral therapy for AIDS [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>] or interferon-gamma therapy [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>].</p><p>The diagnosis is based on clinical presentation and by excluding other disorders associated with low potassium. High serum triiodothyronine (T3) and thyroxine (T4), low serum thyroid-stimulating hormone (TSH) levels, and thyroid uptake scan showing symmetric diffuse uptake are all part of the diagnostic evaluation. Furthermore, abnormal electrocardiogram (ECG) and electromyogram (EMG) findings accompanied by biochemical evidence of hypokalemia can further help in diagnosis. Electromyography study of the muscles usually shows a myopathic pattern of the muscle weakness that completely resolves during remission. There are reported cases where TPP results in rhabdomyolysis with normal creatinine phosphokinase levels. Muscle biopsy of skeletal muscles, if performed, has shown different structural changes, including, but are not restricted to, sarcolemmal nuclear proliferation, vacuolation, atrophy of muscle fibers, fatty infiltration, and mitochondrial changes on the histology [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>,<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>]. All these structural changes interfere with muscle contraction, leading to its paralysis.</p><p>One of the important and most feared complications associated with thyrotoxicosis that interestingly might be overlooked frequently is hypokalemia-induced cardiac arrhythmias. However, these are found to be more common among patients with pre-existing cardiac diseases. According to one study by Manthri et al. [<xref rid=\"REF16\" ref-type=\"bibr\">16</xref>], in which they studied the incidence and prevention of TPP in patients of hyperthyroidism, 44 of 100 patients presented with muscle weakness and one of them died due to ventricular arrhythmia with ECG findings consistent with hypokalemia.</p><p>The disease severity can vary from mild weakness to quadriplegia to total paralysis. There is in most cases no family history of muscle paralysis. Interestingly, the bulbar, respiratory, and ocular muscles are in most cases spared. There is, however, an interesting case report of a patient who exhibited upper motor neuron signs (hyperreflexia, ankle clonus, positive Babinski sign), which resolved after treatment of the underlying hyperthyroidism [<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>,<xref rid=\"REF18\" ref-type=\"bibr\">18</xref>]. The deep tendon reflexes in these patients can be diminished to absent. There are some case reports where patients would present acutely with monoplegia highly mimicking a stroke [<xref rid=\"REF19\" ref-type=\"bibr\">19</xref>]. Cognitive and sensory functions usually remain normal. The start of muscle paralysis frequently coincides with hyperthyroidism, but there are reported cases where TPP is seen in patients with subclinical hyperthyroidism, which was later diagnosed when further workup for hyperthyroidism is done.</p><p>The differential diagnosis of TPP would include familial hypokalemic periodic paralysis (FHPP). Since the patient needs to be hyperthyroid for TPP, it occurs at a later age when compared to FHPP. In a study, 605 of the patient developed paralysis before the age of 16 years in FHPP and 79% of patients developed paralysis in-between the ages of 29-39 years. Importantly, normal serum potassium levels in between episodes of muscle weakness differentiate TPP with FHPP. Moreover, FHPP is distinguished by the presence of ionic channel genes such as calcium voltage-gated channel subunit alpha 1 S (CACNA1S), sodium voltage-gated channel alpha subunit 4 (SCN4A), and potassium voltage-gated channel subfamily E regulatory subunit 3 (KCNE3) [<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>].</p><p>The management of TPP can be divided into acute and definite treatment. While managing the acute episodes, potassium can be repleted intravenously to relieve the symptoms of hypokalemia. However, unfortunately, this can result in rebound hyperkalemia when potassium shifts extracellularly [<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>,<xref rid=\"REF21\" ref-type=\"bibr\">21</xref>]. This is because patients with TPP do not actually have a total body deficiency of potassium; hence, close attention must be given to potassium replacement, as overly aggressive treatment can result in rebound hyperkalemia. Therefore, for this reason, no more than 60 milliequivalents of potassium should be given in the first 24 hours. Non-selective beta-blockers such as propranolol can also be used to block the effect of catecholamines on ion channels. The definitive therapy includes surgery (thyroidectomy), radioactive ablation, or anti-thyroid medications such as methimazole or propylthiouracil. Lastly, currently, there is no evidence of the benefit of using potassium supplements prophylactically to avoid TPP episodes.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>TPP is a rare disease of muscles that presents with acute painless weakness of muscles predominantly affecting the proximal and lower extremity muscles. It is usually found in patients with hypokalemia and hyperthyroidism. This condition is very rare in Caucasian populations and is very commonly misdiagnosed in western countries because of its similarities to familial periodic paralysis. It can be an initial presentation of thyrotoxicosis and the muscle weakness is reversible; hence, a better understanding of the disease would help us in both diagnosing and treating it early. In acute episodes, potassium can be given carefully through intravenous access, and non-selective beta-blockers can be used to block the effect of catecholamines on ion channels, resulting in the resolution of muscle weakness. The definitive therapy, however, could include surgery (thyroidectomy), radioactive ablation, or anti-thyroid medications such as methimazole or propylthiouracil.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"website\"><article-title>Thyrotoxic periodic paralysis</article-title><date-in-citation content-type=\"access-date\"><month>8</month><year>2020</year></date-in-citation><year>2020</year><uri xlink:href=\"https://www.uptodate.com/contents/thyrotoxic-periodic-paralysis.\">https://www.uptodate.com/contents/thyrotoxic-periodic-paralysis.</uri></element-citation></ref><ref id=\"REF2\"><label>2</label><element-citation publication-type=\"journal\"><article-title>Thyrotoxic periodic paralysis</article-title><source>South Med 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005525</article-id><article-id pub-id-type=\"pmc\">PMC7523546</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10107</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Trauma</subject></subj-group></article-categories><title-group><article-title>Hemorrhagic Anuria With Acute Kidney Injury After a Single Dose of Acetazolamide: A Case Study of a Rare Side Effect</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Lawson</surname><given-names>Christy</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Morris</surname><given-names>Leisa</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wilson</surname><given-names>Vera</given-names></name><xref ref-type=\"aff\" rid=\"aff-3\">3</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Burns</surname><given-names>Bracken</given-names><suffix>Jr</suffix></name><xref ref-type=\"aff\" rid=\"aff-4\">4</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nSurgery, Quillen College of Medicine, East Tennesse State University, Johnson City, USA </aff><aff id=\"aff-2\">\n<label>2</label>\nTrauma, Ballad Health Trauma Services, Johnson City, USA </aff><aff id=\"aff-3\">\n<label>3</label>\nPharmacy, Ballad Health Trauma Services, Johnson City, USA </aff><aff id=\"aff-4\">\n<label>4</label>\nSurgery, Quillen College of Medicine, East Tennessee State University, Johnson City, USA </aff><author-notes><corresp id=\"cor1\">\nBracken Burns Jr <email>burnsjb@etsu.edu</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10107</elocation-id><history><date date-type=\"received\"><day>14</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Lawson et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Lawson et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/33536-hemorrhagic-anuria-with-acute-kidney-injury-after-a-single-dose-of-acetazolamide-a-case-study-of-a-rare-side-effect\">This article is available from https://www.cureus.com/articles/33536-hemorrhagic-anuria-with-acute-kidney-injury-after-a-single-dose-of-acetazolamide-a-case-study-of-a-rare-side-effect</self-uri><abstract><p>Acetazolamide (ACZ) is a relatively commonly used medication in critical illness, glaucoma and altitude sickness. ACZ is sometimes used in the intensive care unit to assist with the treatment of metabolic alkalosis in ventilated patients. This is a case report of a patient who received two doses of ACZ, one week apart, for metabolic alkalosis and subsequently developed renal colic and dysuria that progressed to hemorrhagic anuria and acute kidney injury. This is an incredibly rare side effect of ACZ therapy, and has been reported in a few case reports in the literature, but usually is associated with a longer duration of therapy. This case resolved entirely within 24 hours with aggressive fluid therapy. Clinicians using ACZ therapy for any reason should be aware of this rare but significant side effect.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>acetazolamide</kwd><kwd>hemorrhagic anuria</kwd><kwd>acute kidney injury</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Acetazolamide (ACZ) is a carbonic anhydrase inhibitor. It works to cause an accumulation of carbonic acid in the proximal kidney, preventing its breakdown, and causes lowering of blood pH and resorption of sodium, bicarbonate, and chloride with their subsequent excretion into the urine [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. It is given either by oral or intravenous means, in either 125mg, 250mg or 500mg doses [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. It is used in the treatment of glaucoma, altitude sickness, periodic paralysis, epilepsy, congestive heart failure, and idiopathic intracranial hypertension [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. Additionally, it is often utilized in an off-label manner in patients on mechanical ventilation who have primary or mixed metabolic alkalosis as an aid to help correct pH, and allow the patient more efficient ventilator weaning [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>].</p><p>ACZ has a number of very common side effects, such as nausea, vomiting, fatigue, abdominal pain, and paresthesias. Rarer, more severe side effects have been described, such as Stevens-Johnson Syndrome [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. There have been a few case reports in the literature, dating back to the 1950s, that describe hemorrhagic anuria and acute kidney injury (AKI) as a rare side effect of ACZ, but this has typically been seen in patients who receive over 1200mg within 48 hours [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>].</p></sec><sec sec-type=\"cases\"><title>Case presentation</title><p>A 38-year-old male with no significant past medical history who took no medications and had allergies to penicillin, cefepime, and cantaloupe, fell backward from the bed of a moving truck onto his head. He sustained a significant blunt head trauma, with bilateral subdural hematomas, temporal bone fracture, nasal and orbital bone fractures, effacement of cisterns, cerebral compression, and possible diffuse axonal injury. He was intubated and placed on mechanical ventilation for airway protection due to his severe traumatic brain injury and coma, and placed in the surgical intensive care unit. His hospital course was relatively uncomplicated, though he did remain on the ventilator for 12 days and required tracheostomy placement due to prolonged decreased mental status. After tracheostomy placement on hospital day 8, he was being weaned fairly aggressively from the ventilator and was showing signs of improvement from a mental status perspective.</p><p>On hospital day 9, the patient had an arterial blood gas that revealed a pH of 7.53, a pCO2 of 38, a pO2 of 67, a bicarbonate level of 28 with a base excess of +4. In an effort to help his primarily metabolic alkalosis, a single dose of ACZ was given, 500mg intravenously. He had received the same 500mg intravenous dose of ACZ on hospital day 3 for a similar indication, a week prior to this dose. At this point, he had an indwelling foley catheter, a normal creatinine, and was producing over 2 L a day of clear urine.</p><p>On hospital day 11, the patient was successfully on tracheostomy collar and was more lucid and able to communicate relatively clearly. He began complaining of severe bilateral flank pain with dysuria and had a significantly decreased urine output of only 275mL of cloudy, sediment containing urine. A urinalysis revealed red cells, no white cells, no nitrates, and no bacteria. He had some mucoid-like discharge from the indwelling foley catheter with irrigation. A computed tomography of the abdomen and pelvis with renal stone protocol was obtained to evaluate for kidney stones. The images revealed no evidence of obstructive uropathy, a decompressed bladder, and several small, nonobstructing renal calculi in bilateral kidney lower poles. The urinary catheter was exchanged and the bladder was irrigated. Only the irrigation and some blood-stained mucous was evacuated. Urinary analgesia was given to the patient and aggressive fluid therapy was started. His creatinine, which had been 0.48 on the morning laboratory panel, increased to 2.0 on an evening laboratory panel with a blood urea nitrogen (BUN):creatinine ratio of 19.5:1, which can be interpreted as normal or can also indicate post-renal disease.</p><p>Due to the fact that the patient remained afebrile, had been on aztreonam for suspected pneumonia, and had no findings on urinalysis to indicate a urinary tract infection, a thorough medication review for nephrotoxic medications was conducted. The patient had received a 24-hour course of vancomycin on hospital day 4-5, as part of empiric therapy for a fever and leukocytosis workup. It had been stopped on hospital day 5, with appropriate monitoring and clearing of troughs. No other nephrotoxic medications had been administered during the patient’s hospital course.</p><p>A literature search revealed the case reports on hemorrhagic anuria and AKI with ACZ administration, as documented in the discussion, so fluid therapy was initiated. The following day, hospital day 10, the patient had near complete resolution of his renal colic and dysuria symptoms, had returned to a normal urine output of 1000mL in 12 hours and was producing clear urine again. His creatinine had decreased to 1.64. Over the next 48 hours, his creatinine returned to normal and his urine output also returned to normal. See Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref> for a representation of urine output and creatinine over the duration of his brief course of AKI.</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Correlation Between Creatinine and Urine Output from Hospital Day 11 to Hospital Day 15</title></caption><graphic xlink:href=\"cureus-0012-00000010107-i01\"/></fig></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>This rare side effect of ACZ has been discussed in several case reports or case series throughout the decades. The first mention of renal lesions from ACZ administration was documented by Glushien and Fisher in 1956. In their paper, a patient with Hodgkin’s disease received a four-day course of ACZ at 750mg per day, for a total of 3g of the medication. In this patient, the ACZ was utilized as a diuretic. Postmortem, the patient was found to have coagulated blood products and sulfonamide crystals similar in appearance to crystallized ACZ within the renal tubules [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>].</p><p>The next mention of a patient with renal impairment after ACZ administration also mentions this sulfonamide crystalluria. This was found in a 59-year-old patient who received a six-day course of ACZ (unknown dosing) for glaucoma treatment. The patient reported severe renal colic type pain, hematuria and had azotemia and anuria on clinical assessment, without evidence of kidney stones. He also had bilateral ureteral obstruction requiring catheterization. This patient responded to oral bicarbonate therapy, fluid resuscitation, and urinary catheterization, with return to baseline renal function by discharge [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>].</p><p>In a 1975 case study, Howlett described a 69-year-old patient who received a two-week course of ACZ, 1000mg per day, for glaucoma. He presented several days later with anuria, azotemia, nausea, and severe flank pain. Urinalysis revealed red blood cells, but this case differed in the fact that they did not discover sulfonamide crystals within the urine. Again, the patient responded to oral bicarbonate therapy and fluid resuscitation, with normalization of kidney function [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. Several other patients were described in a case series in 1978, with clinical presentations and laboratory assessments similar to the aforementioned patients who also received high dose ACZ for glaucoma management. They again had restoration of kidney function with oral bicarbonate and aggressive hydration. In this case series, the patients had antegrade urograms that revealed dilated renal pelvises with mucosal swelling and sludge-like material within the ureters that were causing blockages [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>].</p><p>In a 1989 paper on ACZ-induced AKI, a patient who presented after taking high-dose ACZ, again for glaucoma, presented with the previously described symptoms of severe flank pain, azotemia, nausea, and vomiting with AKI. This patient had no radiographic evidence of obstruction of the renal system, and underwent a kidney biopsy. This biopsy demonstrated tubular damage with cellular debris and intratubular crystal deposition. This patient also responded well to supportive measures with fluid rehydration, and this kidney biopsy that demonstrated crystal deposits similar to that found on the previous study patients’ assessments lends further proof to the idea that ACZ can cause renal impairment, theoretically due to this crystal deposition within the renal tubules [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>].</p><p>In all the case studies described, the patients were receiving high-dose ACZ therapy. The only case study that demonstrated similar findings with low-dose ACZ therapy is a 2014 case study that describes a patient who developed severe AKI and bilateral flank pain after ACZ administration for high altitude sickness. He received a total dose of 1200mg in 48 hours of ACZ, a far lower dose than previously described. This patient also had no radiographic evidence of ureteral obstruction. All other tests done on this patient to elucidate the cause of the renal failure were negative. The patient required two sessions of hemodialysis and aggressive fluid resuscitation, but subsequently had return to normal renal function [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>].</p><p>The patient described in this case study received two single 500mg doses of ACZ, a week apart, yet presented almost identically to these previously described cases. He had flank pain, azotemia, AKI, and red blood cells on urinalysis. He also had no evidence of ureteral obstruction on CT imaging. On urinary catheterization and irrigation of the bladder, he was noted to have mucoid discharge and significant sediment within the irrigation fluid, without urine output. This fluid was, unfortunately, not sent for analysis, so the nature of the sediment is not described. The only other nephrotoxic medication this patient had received was a 24-hour course of vancomycin, however this had been discontinued full six days prior to the presentation of AKI. Perhaps the most clear similarity in this patient’s presentation to those cases of AKI attributed to ACZ described in the literature was his complete and rapid resolution of symptoms and the return of his creatinine to baseline with IV fluid hydration.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>The “classic” presentation of ACZ-induced “hemorrhagic anuria” includes anuria, bilateral, severe flank pain, red cells on urinalysis, and no radiographic evidence of ureteral obstruction. This has been described rather robustly in the literature in the form of case series or studies, and is generally reported with high-dose ACZ administration. Our case illustrates that this presentation can occur with even a single, relatively low dose of ACZ, even in a patient with no pre-existing renal dysfunction. Clinicians should be aware of this rare side effect with the potential for significant morbidity, as well as the concept that supportive measures with aggressive fluid rehydration and possibly oral bicarbonate can rapidly reverse the AKI seen in this patient population.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study. East Tennessee State University issued approval NA. This study received exemption from East Tennessee State University Institutional Review Board.</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"book\"><article-title>Acetazolamide</article-title><source>Stat Pearls</source><date-in-citation content-type=\"access-date\"><month>5</month><year>2020</year></date-in-citation><person-group><name><surname>Farzam</surname><given-names>K</given-names></name><name><surname>Abdullah</surname><given-names>M</given-names></name></person-group><publisher-loc>Treasure Island</publisher-loc><publisher-name>Stat Pearls Publishing</publisher-name><year>2019</year><uri xlink:href=\"https://www.ncbi.nlm.nih.gov/books/NBK532282/\">https://www.ncbi.nlm.nih.gov/books/NBK532282/</uri></element-citation></ref><ref id=\"REF2\"><label>2</label><element-citation publication-type=\"journal\"><article-title>Adverse effects of intravenous acetazolamide administration for evaluation of 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005529</article-id><article-id pub-id-type=\"pmc\">PMC7523547</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10111</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Internal Medicine</subject></subj-group><subj-group><subject>Obstetrics/Gynecology</subject></subj-group><subj-group><subject>Hematology</subject></subj-group></article-categories><title-group><article-title>Idiopathic Bilateral Ovarian Vein Thrombosis in a Non-Pregnant Healthy Patient: A Case Report and Review of the Literature</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Basit</surname><given-names>Abdul</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kaur</surname><given-names>Pushpinder</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Villanueva</surname><given-names>Diana M</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Tahir</surname><given-names>Muhammad</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sonnenschine</surname><given-names>Mark</given-names></name><xref ref-type=\"aff\" rid=\"aff-3\">3</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nInternal Medicine, Coney Island Hospital, Brooklyn, USA </aff><aff id=\"aff-2\">\n<label>2</label>\nPathology, Case Western Reserve University School of Medicine, Cleveland, USA </aff><aff id=\"aff-3\">\n<label>3</label>\nHematology, Coney Island Hospital, Brooklyn, USA </aff><author-notes><corresp id=\"cor1\">\nMuhammad Tahir <email>tahir_786kb@yahoo.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10111</elocation-id><history><date date-type=\"received\"><day>18</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Basit et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Basit et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/39762-idiopathic-bilateral-ovarian-vein-thrombosis-in-a-non-pregnant-healthy-patient-a-case-report-and-review-of-the-literature\">This article is available from https://www.cureus.com/articles/39762-idiopathic-bilateral-ovarian-vein-thrombosis-in-a-non-pregnant-healthy-patient-a-case-report-and-review-of-the-literature</self-uri><abstract><p>Ovarian vein thrombosis (OVT) is a potentially life-threatening condition, and it is typically related to the peripartum period; however, it is also associated with pelvic inflammatory disease, recent pelvic or abdominal surgery, inflammatory bowel disease, thrombophilia, malignancy, and sepsis. Idiopathic isolated OVT is rare and is usually presented as case reports in the medical literature. In this report, we present a case of bilateral OVT in a postmenopausal female with no identifiable risk factors and normal coagulation profile workup to highlight the importance of considering it as a differential diagnosis in female patients presenting with abdominal pain. Early identification can prevent potentially life-threatening complications. Management is often conservative, and the choice of anticoagulation is based on the patient’s medical conditions. In this particular scenario, the patient was managed with low molecular weight heparin (LMWH) and transitioned to direct oral anticoagulant (DOAC) before discharge.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>ovarian vein thrombosis</kwd><kwd>bilateral thrombosis</kwd><kwd>idiopathic ovarian vein thrombosis</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>The first case of ovarian vein thrombosis (OVT) was reported in 1956 by Trang et al. [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. OVT is mainly associated with postpartum complications. Other associated etiologies include pelvic or abdominal surgeries, pelvic infection, sepsis, trauma, malignancy, and hypercoagulable states. The incidence rate increases with cesarean delivery, and untreated thrombosis can cause pulmonary embolism, increasing mortality up to 4% [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. The most common presenting complaint of OVT is abdominal pain associated with gastrointestinal symptoms. Early diagnosis is very important; abdomen and pelvic CT with intravenous contrast remains the main choice of imaging to successfully differentiate it from and rule out other pathological diseases related to female pelvic anatomy. The treatment of choice is anticoagulation, and the use of antibiotics is also indicated if signs and symptoms of pelvic infection are present [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>,<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>].</p></sec><sec sec-type=\"cases\"><title>Case presentation</title><p>Our patient was a 41-year-old African American woman, G6P6006 (all non-complicated vaginal deliveries and last delivery had been seven years ago). Her past medical history was unremarkable except for the chronic pelvic pain that she had been experiencing for the past five years. She presented to the emergency department complaining of sudden onset of worsening abdominal pain in the bilateral iliac fossa region radiating to the pubic symphysis. The pain was sharp and constant, associated with nausea and constipation. Her last menstrual period had been two years ago, and there had been no recent or remote history of contraceptive use, hematuria, trauma, prolonged traveling, surgeries, or infection. Upon questioning, there was no significant family history of any hematological disorders.</p><p>On physical examination, her oral temperature was 36.6 °C; she had a heart rate of 67 bpm, respiratory rate of 20 bpm, blood pressure of 132/77 mmHg, and oxygen saturation of 99% on room air. The patient was alert and oriented with remarkable findings of the left and right lower quadrant tenderness that was appreciated on palpation without the signs of guarding. The remaining systemic and neurological examinations were unremarkable. No abnormalities were reported on initial clinical laboratory investigations. Prothrombin time (PT), international normalized ratio (INR), and activated partial thromboplastin time (aPTT) were within normal limits. Since the patient initially presented with abdominal pain, hypercoagulable studies were not ordered. Later on, all hypercoagulability workups including but not limited to antithrombin III, protein C and S, and all the clotting factors were within normal limits. The patient was also evaluated for pelvic infections and complete blood count (CBC), erythrocytes sedimentation rate (ESR), and C-reactive protein (CPR) along with pro-calcitonin, which all came back normal. Gynecological consultation was ordered to find out the existence of any cervical or uterine pathologies and all the possible causes of pelvic infection were ruled out.</p><p>Abdominal and pelvic CT with oral and intravenous contrast revealed an apparent filling defect in the right and left gonadal veins, as shown in Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>. Another CT of the abdomen coronal revealed bilateral OVT as shown in Figure <xref ref-type=\"fig\" rid=\"FIG2\">2</xref>. Therefore, the diagnosis of bilateral OVT was made, and blood samples were obtained for coagulation profile and the patient was started on therapeutic low molecular weight heparin (LMWH) followed by direct oral anticoagulant (DOAC). The patient’s abdominal pain significantly improved by the third day of starting the anticoagulation, and she was discharged home with oral anticoagulation on apixaban. The patient is scheduled for follow-up at three- and six-month intervals, and the plan is to complete six months of anticoagulation therapy provided the thrombosis is resolved at the end of the treatment period.</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>CT image showing filling defect in the right and left gonadal vein (red arrows)</title><p>CT: computed tomography</p></caption><graphic xlink:href=\"cureus-0012-00000010111-i01\"/></fig><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>CT of the abdomen (coronal section) showing bilateral OVT as indicated by the red arrows</title><p>CT: computed tomography; OVT: ovarian vein thrombosis; AO: abdominal aorta; IVC: inferior vena cava; LRV: left renal vein</p></caption><graphic xlink:href=\"cureus-0012-00000010111-i02\"/></fig></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>The incidence of OVT ranges from 0.05% to 0.18% in normal vaginal deliveries but it goes up to 2% with cesarean deliveries and twin pregnancies [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>-<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>], with 90% of the cases involving right-sided ovarian vein [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. OVT is a rare yet potentially fatal condition with a 52% mortality rate if left untreated [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. It can extend to inferior vena cava or renal veins due to anatomical proximity. According to one report, the incidence of pulmonary embolism arising from OVT is 33%, with ~4% mortality rate. Other complications include sepsis and chronic pelvic pain [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>,<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>,<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. The classical Virchow’s triad can explain the pathophysiology and its increased incidence associated with pregnancy. During pregnancy, the blood flow in ovarian veins increases significantly, and the venous valves become incompetent. At the same time, the sudden change after childbirth causes venous collapse and stasis [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. The possible etiologies for underlying causes for increased right-sided OVT are related to its longer course, more incompetent valve, and direct pressure from the gravid uterus [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. Hypercoagulability also increases during pregnancy, sepsis, and malignancy or in vivo hypercoagulable state, leading to thrombosis. The surgical procedure causes intimal injury of endothelium and hence prone to thrombosis.</p><p>Idiopathic OVT is very rare, and no causative factor has been identified. The first case of idiopathic OVT was reported in 2004 by Yildirim et al. [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. Since then, 12 cases of unilateral idiopathic OVT have been reported. Upon careful review of these reported cases, we observed that few had risk factors that may lead to OVT. In one of the reported cases, the patient had polycystic ovarian syndrome (PCOS) and recent use of oral contraceptive (OCP), which had been discontinued eight weeks before the presentation [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>], while in another reported case, the patient had a recent history of pelvic inflammatory disease with evident bilateral hydrosalpinx and had undergone bilateral laparoscopic salpingectomy three weeks before the presentation, contributing to significant risk factors [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>]. One more patient had experienced recent significant trauma, and even at the time of presentation with OVT, had healing bruises at the abdominal wall [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>].</p><p>Among the reported cases, ~65% had right-sided isolated OVT, including one with a 3-cm mass [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]. None of them had a fever at presentation, while only a few had mild leukocytosis. Otherwise, the initial lab workup remained normal. Abdominal pain was consistently present in all the reported cases and almost always indicated the laterality of the OVT. Furthermore, most of the cases were acute to subacute. However, in the previously reported case of bilateral thrombosis [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>] and our presented case, patients had a long-standing history of abdominal pain, going back to several years, and presentation was characterized by acute over chronic abdominal pain.</p><p>To the best of our knowledge, only one case of idiopathic bilateral ovarian thrombosis has been reported previously [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. Moreover, in the previously reported case, the patient had a medical history of bilateral pulmonary embolism five years prior to presenting with bilateral OVT, which was one week after childbirth, and also had several inconclusive CT angiograms of the chest performed because of chest pain and elevated D dimers. Our case was unique as the patient had no previous medical or family history of any previous thrombosis, no recent history of sepsis, hospitalization, traveling, oral contraceptives, trauma, surgery or pelvis infection, or any remote history of unexplained abortion. The patient had chronic bilateral flank pain, similar to the previously reported case, for almost three years. All hypercoagulability workups remained negative, including beta 2 glycoprotein 1 immunoglobulin G (IgG), IgA, IgM antibodies, factor IX, V, VIII, XI, XII assay, factor V Leiden mutation, activated protein C (APC) resistance, AT III activity/antigen, D dimers, fibrinogen levels, hexagonal phase lupus assay, lupus anticoagulant panel including anticardiolipin, methylmalonic acid, homocysteine levels, plasmin F levels, protein C and S antigen, thrombin time, platelets number, bleeding time, and prothrombin time (INR). Furthermore, after an extensive examination of the history and workup, the patient was deemed to have idiopathic bilateral OVT.</p><p>Different imaging modalities can make the diagnosis of OVT. Doppler ultrasound (DUS) remains the first choice, as it is non-invasive, inexpensive, and readily available in the emergency department. However, it is operator-dependent, and body habitus, overlying structures, and bowel gas patterns can interfere with imaging [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>,<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. The sensitivity of the DUS is around ~50% only [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. Also, DUS cannot examine the whole length of the ovarian veins [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. Hence, further imaging modality is warranted. CT venogram or CT abdomen pelvis with contrast is also sensitive and specific for the diagnosis and is time- and cost-effective. Magnetic resonance angiography (MRA) remains the gold standard but is expensive and not widely available. In all the reported cases, almost all the patients were successfully diagnosed with abdomen and pelvis CT with intravenous contrast, making it the imaging of choice for the diagnosis of OVT.</p><p>Management of OVT is mainly conservative with anticoagulation and antibiotics indicated only if the pelvic infection is present. Surgical interventions, including the insertion of an inferior vena cava filter or ovarian veins ligation, might be warranted if there is evidence of persistent thrombosis with a high risk of complications and anticoagulation failure, or if a contraindication to anticoagulation is present. Among all the reported cases of OVT, the majority were treated initially with unfractionated heparin, with warfarin being added later on. No complications or failure of anticoagulation was reported. Furthermore, serial follow-ups showed the resolution of OVT. Only one case was managed by DOAC (rivaroxaban) [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>], with complete resolution of bilateral OVT at six weeks. In our case, the patient was initially started on therapeutic LMWH and later switched to DOAC (apixaban).</p><p>No guidelines are available regarding the management of OVT. However, multiple authors [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>,<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>] have argued that the lower-limb deep vein thrombosis (DVT) guidelines are applicable in this scenario. The duration of treatment and choice of anticoagulation remains an essential aspect of management. The duration of the anticoagulation is typically three to six months for transient or provoked conditions. In comparison, lifelong anticoagulation might be needed for persistent or hypercoagulable states. Since the introduction of DOAC, these agents have become a widely used method of anticoagulation. However, their safety during childbearing age is concerning. Animal studies have shown increased fetal toxicities, and no human studies are available. Nevertheless, regarding DOAC, the summary of product characteristics (SPCs) recommends against its usage during pregnancy and lactation. Warfarin is also contraindicated during pregnancy, and proper contraception use is mandatory for usage during childbearing age. LMWH is an alternative if pregnancy occurs, and DOAC or warfarin should be discontinued immediately [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. The studies included for literature review were collected from PubMed and are listed below in Table <xref rid=\"TAB1\" ref-type=\"table\">1</xref>.</p><table-wrap id=\"TAB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Summary of included studies of ovarian vein thrombosis with details of signs, symptoms, diagnostic and therapeutic interventions, and complications and follow-ups</title><p>DOAC: direct oral anticoagulant; PMHx: past medical history; CT: computed tomography; US: ultrasound; PCOS: polycystic ovarian syndrome; OCPs: oral contraceptive pills; IV: intravenous</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">No.</td><td rowspan=\"1\" colspan=\"1\">Author</td><td rowspan=\"1\" colspan=\"1\">Age at the time of presentation (years)</td><td rowspan=\"1\" colspan=\"1\">Duration of pelvic pain with constitutional signs and symptoms</td><td rowspan=\"1\" colspan=\"1\">Imaging modality used for diagnosis</td><td rowspan=\"1\" colspan=\"1\">Ovary with thrombus</td><td rowspan=\"1\" colspan=\"1\">Treatment</td><td rowspan=\"1\" colspan=\"1\">Complications/follow-up</td></tr><tr><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">Trang et al. [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]</td><td rowspan=\"1\" colspan=\"1\">47</td><td rowspan=\"1\" colspan=\"1\">1 day of acute back pain radiating to the abdomen with dyspnea</td><td rowspan=\"1\" colspan=\"1\">CT abdomen and pelvis with IV contrast</td><td rowspan=\"1\" colspan=\"1\">Left</td><td rowspan=\"1\" colspan=\"1\">Low molecular weight heparin, followed by DOAC (rivaroxaban) for 3 months</td><td rowspan=\"1\" colspan=\"1\">Follow-up at 2 months showed complete resolution of thrombus</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">Alalqam et al. [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]</td><td rowspan=\"1\" colspan=\"1\">42</td><td rowspan=\"1\" colspan=\"1\">1 day of acute left iliac fossa pain, nausea, constipation with abdominal tenderness and guarding</td><td rowspan=\"1\" colspan=\"1\">US abdomen, Doppler US, CT abdomen and pelvis with IV contrast</td><td rowspan=\"1\" colspan=\"1\">Left</td><td rowspan=\"1\" colspan=\"1\">Low molecular weight heparin, followed by warfarin for 6 months</td><td rowspan=\"1\" colspan=\"1\">Follow-up at 1 year showed complete resolution of thrombus</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> 3</td><td rowspan=\"1\" colspan=\"1\">Garcia et al. [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]</td><td rowspan=\"1\" colspan=\"1\">35</td><td rowspan=\"1\" colspan=\"1\">2 days of worsening left flank pain. History of low intensity left flank pain for 5 years. PMHx: bilateral pulmonary embolism 5 years ago, several non-diagnostic CT chests with elevated D dimer levels</td><td rowspan=\"1\" colspan=\"1\">CT abdomen and pelvis with IV contrast</td><td rowspan=\"1\" colspan=\"1\">Bilateral ovarian vein thrombosis</td><td rowspan=\"1\" colspan=\"1\">DOACs (rivaroxaban) for 3 months</td><td rowspan=\"1\" colspan=\"1\">Follow-up US at 6 and 12 weeks showed complete resolution of thrombus</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">Kodali et al. [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]</td><td rowspan=\"1\" colspan=\"1\">40</td><td rowspan=\"1\" colspan=\"1\">1 day of acute abdominal pain, with nausea. PMHx: significant abdominal wall trauma 2 months prior to presentation</td><td rowspan=\"1\" colspan=\"1\">CT abdomen and pelvis with IV contrast</td><td rowspan=\"1\" colspan=\"1\">Right</td><td rowspan=\"1\" colspan=\"1\">Low molecular weight heparin followed by warfarin for 6 months</td><td rowspan=\"1\" colspan=\"1\">No follow-up/complication details were provided</td></tr><tr><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">Yildirim et al. [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]</td><td rowspan=\"1\" colspan=\"1\">36</td><td rowspan=\"1\" colspan=\"1\">Peritoneal signs were present for 2 days</td><td rowspan=\"1\" colspan=\"1\">US, Doppler US, then CT abdomen pelvis with contrast</td><td rowspan=\"1\" colspan=\"1\">Right</td><td rowspan=\"1\" colspan=\"1\">Unfractionated heparin, followed by warfarin for 6 months</td><td rowspan=\"1\" colspan=\"1\">Calcification of thrombus was identified after 6 months of presentation</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">Stafford et al. [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]</td><td rowspan=\"1\" colspan=\"1\">42</td><td rowspan=\"1\" colspan=\"1\">1 day of acute abdominal pain with nausea, with local rebound tenderness</td><td rowspan=\"1\" colspan=\"1\">CT abdomen pelvis with contrast</td><td rowspan=\"1\" colspan=\"1\">Right</td><td rowspan=\"1\" colspan=\"1\">Unfractionated heparin, followed by warfarin</td><td rowspan=\"1\" colspan=\"1\">Follow-up US at 2 months showed complete resolution of thrombus</td></tr><tr><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">Doherty et al. [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]</td><td rowspan=\"1\" colspan=\"1\">29</td><td rowspan=\"1\" colspan=\"1\">8 months of acute over chronic abdominal pain</td><td rowspan=\"1\" colspan=\"1\">Doppler US, CT abdomen and pelvis with IV contrast</td><td rowspan=\"1\" colspan=\"1\">Left</td><td rowspan=\"1\" colspan=\"1\">Warfarin therapy for 6 months</td><td rowspan=\"1\" colspan=\"1\">No evidence of thrombus was found on follow-up at 2 months</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">8</td><td rowspan=\"1\" colspan=\"1\">Murphy et al. [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]</td><td rowspan=\"1\" colspan=\"1\">27</td><td rowspan=\"1\" colspan=\"1\">2 week’s subacute abdominal pain with low appetite and nausea, vomiting. PMHx: PCOS treated with OCPs, self-discontinuation 2 months prior to presentation. Pt had one copy of the C677T mutation of the MTHFR gene with no A1298C mutation</td><td rowspan=\"1\" colspan=\"1\">US abdomen was negative while CT with oral and IV contrast concluded the diagnosis</td><td rowspan=\"1\" colspan=\"1\">Right</td><td rowspan=\"1\" colspan=\"1\">No details provided</td><td rowspan=\"1\" colspan=\"1\">No follow-up/complication details were provided</td></tr><tr><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">Heavrin et al. [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>]</td><td rowspan=\"1\" colspan=\"1\">29</td><td rowspan=\"1\" colspan=\"1\">3 days of acute abdominal pain, and tenderness with voluntary guarding, nausea, and vomiting. PMHx: 3 weeks earlier diagnosed with PID, and bilateral hydrosalpinx. Underwent D&C, and bilateral laparoscopic salpingectomy</td><td rowspan=\"1\" colspan=\"1\">CT abdomen and pelvis with contrast</td><td rowspan=\"1\" colspan=\"1\">Left</td><td rowspan=\"1\" colspan=\"1\">Initially, the patient left against medical advice and presented 1 week later with chest pain; diagnosed with pulmonary embolism by CT chest with IV contrast. 6 months of anticoagulation with no details about the choice of anticoagulant</td><td rowspan=\"1\" colspan=\"1\">No complications at 18-month follow-up and no recurrence with complete resolution of the previous thrombosis</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">10 </td><td rowspan=\"1\" colspan=\"1\">Harris et al. [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]</td><td rowspan=\"1\" colspan=\"1\">53</td><td rowspan=\"1\" colspan=\"1\">1 week of subacute abdominal pain with a 3-cm palpable mass in the right lower quadrant</td><td rowspan=\"1\" colspan=\"1\">CT abdomen and pelvis with IV contrast</td><td rowspan=\"1\" colspan=\"1\">Right</td><td rowspan=\"1\" colspan=\"1\">Warfarin for 5 months</td><td rowspan=\"1\" colspan=\"1\">CT showed persistence of thrombosis with no further extension beyond ovarian veins</td></tr><tr><td rowspan=\"1\" colspan=\"1\">11</td><td rowspan=\"1\" colspan=\"1\">Chebl et al. [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]</td><td rowspan=\"1\" colspan=\"1\">79</td><td rowspan=\"1\" colspan=\"1\">7 days of subacute abdominal pain</td><td rowspan=\"1\" colspan=\"1\">CT abdomen and pelvis with IV contrast</td><td rowspan=\"1\" colspan=\"1\">Right</td><td rowspan=\"1\" colspan=\"1\">Warfarin for 3-6 months</td><td rowspan=\"1\" colspan=\"1\">No follow-up/complication details were provided</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\">Khishfe et al. [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>]</td><td rowspan=\"1\" colspan=\"1\">30</td><td rowspan=\"1\" colspan=\"1\">1 day of acute abdominal pain with nausea and local tenderness</td><td rowspan=\"1\" colspan=\"1\">CT abdomen and pelvis with IV contrast</td><td rowspan=\"1\" colspan=\"1\">Right</td><td rowspan=\"1\" colspan=\"1\">Warfarin and antibiotics therapy</td><td rowspan=\"1\" colspan=\"1\">No follow-up/complication details were provided</td></tr></tbody></table></table-wrap></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>OVT is a unique entity usually associated with the peripartum and postpartum period. Bilateral idiopathic OVT is a very rare condition that can lead to potentially fatal complications, and hence early diagnosis is very important. Hypercoagulability workup should be performed if no obvious risk factor has been identified, and anticoagulation remains the mainstay of the treatment along with antibiotics if needed. The choice of anticoagulation depends on individual risk factors, childbearing age status, and the underlying cause of OVT.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study</p></fn></fn-group><ack><p>We extend our special thanks to Dr. Jayshree for providing input for radiological images selection and also to Dr. Aaron Douen for reviewing the article and providing his scholarly contributions to make this possible.</p></ack><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"journal\"><article-title>Idiopathic left ovarian vein thrombosis</article-title><source>J Investig Med High Impact Case Rep</source><person-group><name><surname>Trang</surname><given-names>N</given-names></name><name><surname>Kalluri</surname><given-names>M</given-names></name><name><surname>Bajaj</surname><given-names>T</given-names></name><name><surname>Petersen</surname><given-names>G</given-names></name></person-group><fpage>2324709620947257</fpage><volume>8</volume><year>2020</year></element-citation></ref><ref id=\"REF2\"><label>2</label><element-citation publication-type=\"journal\"><article-title>The challenges of diagnosing idiopathic ovarian vein thrombosis: case report</article-title><source>Int J Surg Case Rep</source><person-group><name><surname>Alalqam</surname><given-names>MM</given-names></name><name><surname>Al Abbas</surname><given-names>R</given-names></name><name><surname>Abualsaud</surname><given-names>AS</given-names></name><name><surname>AlQattan</surname><given-names>AS</given-names></name><name><surname>Almabyouq</surname><given-names>F</given-names></name></person-group><fpage>63</fpage><lpage>65</lpage><volume>60</volume><year>2019</year><pub-id pub-id-type=\"pmid\">31203001</pub-id></element-citation></ref><ref id=\"REF3\"><label>3</label><element-citation publication-type=\"journal\"><article-title>Ovarian vein and caval thrombosis</article-title><source>Tex Heart Inst J</source><person-group><name><surname>Takach</surname><given-names>TJ</given-names></name><name><surname>Cervera</surname><given-names>RD</given-names></name><name><surname>Gregoric</surname><given-names>ID</given-names></name></person-group><fpage>579</fpage><lpage>582</lpage><volume>32</volume><year>2005</year><uri xlink:href=\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1351836/\">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1351836/</uri><pub-id pub-id-type=\"pmid\">16429909</pub-id></element-citation></ref><ref id=\"REF4\"><label>4</label><element-citation publication-type=\"journal\"><article-title>A rare case of idiopathic bilateral ovarian vein thrombosis</article-title><source>J Vasc Surg Venous Lymphat Disord</source><person-group><name><surname>Garcia</surname><given-names>R</given-names></name><name><surname>Gasparis</surname><given-names>AP</given-names></name><name><surname>Loh</surname><given-names>SA</given-names></name><name><surname>Labropoulos</surname><given-names>N</given-names></name></person-group><fpage>567</fpage><lpage>570</lpage><volume>5</volume><year>2017</year><pub-id pub-id-type=\"pmid\">28623997</pub-id></element-citation></ref><ref id=\"REF5\"><label>5</label><element-citation publication-type=\"journal\"><article-title>Diagnosis and management of ovarian vein thrombosis in a healthy individual: a case report and a literature review</article-title><source>J Thromb Haemost</source><person-group><name><surname>Kodali</surname><given-names>N</given-names></name><name><surname>Veytsman</surname><given-names>I</given-names></name><name><surname>Martyr</surname><given-names>S</given-names></name><name><surname>Lu</surname><given-names>K</given-names></name></person-group><fpage>242</fpage><lpage>245</lpage><volume>15</volume><year>2017</year><pub-id pub-id-type=\"pmid\">27930855</pub-id></element-citation></ref><ref id=\"REF6\"><label>6</label><element-citation publication-type=\"journal\"><article-title>Isolated idiopathic ovarian vein thrombosis: a rare case</article-title><source>Int Urogynecol J Pelvic Floor Dysfunct</source><person-group><name><surname>Yildirim</surname><given-names>E</given-names></name><name><surname>Kanbay</surname><given-names>M</given-names></name><name><surname>Ozbek</surname><given-names>O</given-names></name><name><surname>Coskun</surname><given-names>M</given-names></name><name><surname>Boyacioglu</surname><given-names>S</given-names></name></person-group><fpage>308</fpage><lpage>310</lpage><volume>16</volume><year>2005</year><pub-id pub-id-type=\"pmid\">16211369</pub-id></element-citation></ref><ref id=\"REF7\"><label>7</label><element-citation publication-type=\"journal\"><article-title>Idiopathic ovarian vein thrombosis: a rare cause of pelvic pain - 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Psychiatry</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Psychiatry</journal-id><journal-title-group><journal-title>Frontiers in Psychiatry</journal-title></journal-title-group><issn pub-type=\"epub\">1664-0640</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33093836</article-id><article-id pub-id-type=\"pmc\">PMC7523554</article-id><article-id pub-id-type=\"doi\">10.3389/fpsyt.2020.558655</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Psychiatry</subject><subj-group><subject>Hypothesis and Theory</subject></subj-group></subj-group></article-categories><title-group><article-title>Should Behavior Harmful to Others Be a Sufficient Criterion of Mental Disorders? Conceptual Problems of the Diagnoses of Antisocial Personality Disorder and Pedophilic Disorder</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Münch</surname><given-names>Ricarda</given-names></name><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/970373\"/></contrib><contrib contrib-type=\"author\"><name><surname>Walter</surname><given-names>Henrik</given-names></name><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/19822\"/></contrib><contrib contrib-type=\"author\"><name><surname>Müller</surname><given-names>Sabine</given-names></name><xref ref-type=\"author-notes\" rid=\"fn001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/26802\"/></contrib></contrib-group><aff id=\"aff1\"><institution>Research Division of Mind and Brain, Department of Psychiatry and Psychotherapy, CCM, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health</institution>, <addr-line>Berlin</addr-line>, <country>Germany</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Manuel Trachsel, University of Zurich, Switzerland</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Jennifer Helen Radden, University of Massachusetts Boston, United States; Stella Reiter-Theil, University of Basel, Switzerland</p></fn><corresp id=\"fn001\">*Correspondence: Sabine Müller, <email xlink:href=\"mailto:mueller.sabine@charite.de\" xlink:type=\"simple\">mueller.sabine@charite.de</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p>This article was submitted to Social Psychiatry and Psychiatric Rehabilitation, a section of the journal Frontiers in Psychiatry</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>558655</elocation-id><history><date date-type=\"received\"><day>03</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Münch, Walter and Müller</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Münch, Walter and Müller</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Generally, diseases are primarily harmful to the individual herself; harm to others may or may not be a secondary effect of diseases (<italic>e.g.</italic>, in case of infectious diseases). This is also true for mental disorders. However, both ICD-10 and DSM-5 contain two diagnoses which are primarily defined by behavior harmful to others, namely Pedophilic Disorder and Antisocial (or Dissocial) Personality Disorder (ASPD or DPD). Both diagnoses have severe conceptual problems in the light of general definitions of mental disorder, like the definition in DSM-5 or Wakefield’s “harmful dysfunction” model. We argue that in the diagnoses of Pedophilic Disorder and ASPD the criterion of harm to the individual is substituted by the criterion of harm to others. Furthermore, the application of the criterion of dysfunction to these two diagnoses is problematic because both heavily depend on cultural and social norms. Therefore, these two diagnoses fall outside the general disease concept and even outside the general concept of mental disorders. We discuss whether diagnoses which primarily or exclusively ground on morally wrong, socially inacceptable, or criminal behavior should be eliminated from ICD and DSM. On the one side, if harming others is a sufficient criterion of a mental disorder, the “evil” is pathologized. On the other side, there are practical reasons for keeping these diagnoses: first for having an official research frame, second for organizing and financing treatment and prevention. We argue that the criteria set of Pedophilic Disorder should be reformulated in order to make it consistent with the general definition of mental disorder in DSM-5. This diagnosis should only be applicable to individuals that are distressed or impaired by it, but not solely based on behavior harmful to others. For ASPD, we conclude that the arguments for eliminating it from the diagnostic manuals overweigh the arguments for keeping it.</p></abstract><kwd-group><kwd>antisocial personality disorder</kwd><kwd>psychopathy</kwd><kwd>dissocial personality disorder</kwd><kwd>pedophilic disorder</kwd><kwd>pedophilia</kwd><kwd>diagnostic criteria</kwd><kwd>definition of mental disorder</kwd><kwd>harmful behavior</kwd></kwd-group><counts><fig-count count=\"0\"/><table-count count=\"3\"/><equation-count count=\"0\"/><ref-count count=\"90\"/><page-count count=\"15\"/><word-count count=\"10861\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Generally, diseases are primarily harmful to the diseased individual herself either by being directly life-threatening or at least life-shortening, or by causing pain or suffering, or by impairing her ability to live in human symbiotic communities (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Harm to others, however, may or may not be a secondary effect of diseases. A typical example are infectious diseases which harm the infected individual and possibly others as well. A mere infection, however, is not called a disease as long as it is not and will not be harmful to the infected individual herself, even if it poses a risk to others as a secondary effect. This is evident from the example of asymptomatic carriers of pathogens. Although they may transmit the pathogen to others and harm particularly vulnerable, <italic>e.g.</italic> immunosuppressed people, medicine does not regard them as ill.<xref ref-type=\"fn\" rid=\"fn1\"><sup>1</sup></xref> Therefore, such persons should be described as being ‘disease-causing’ for others, rather than as being ‘diseased’ themselves.</p><p>If this is true for diseases in general, that they are primarily harmful to the individual herself, it should also be true for mental disorders as long as they are viewed as a subset of diseases. This is reflected in frequently cited attempts to formulate a general definition of mental disorder, like the definition in the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>) or the “harmful dysfunction” model by Wakefield (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Both definitions characterize a mental disorder by, broadly speaking, a dysfunction in mental processes that is associated with harm to the affected individual.</p><p>For some psychiatric diagnoses, however, it is questionable whether the presupposition of harm to the individual really applies. We will show that several diagnoses essentially rely on behavior that is harmful to others, but not necessarily to the individual herself. This is especially true for the diagnoses “Antisocial Personality Disorder” (ASPD) in DSM-5 (or “Dissocial Personality Disorder” in ICD-10) and “Pedophilic Disorder” in DSM-5 and ICD-11.<xref ref-type=\"fn\" rid=\"fn2\"><sup>2</sup></xref> Instead, as we will show, another disease criterion comes in here: the criterion of “harm to others”.</p><p>In the case of Pedophilic Disorder, harm to others is a sufficient criterion. In the case of ASPD, it is a necessary one and, as we will argue, practically also a sufficient one. In addition to the harm criterion, getting another meaning, we will argue that the criterion of a mental dysfunction is unclear in these diagnoses. Thus, the diagnoses of ASPD and Pedophilic Disorder fall out of the general concept of diseases and even out of the general concept of mental disorders. Are they accordingly rather “moral disorders” than clinical disorders?<xref ref-type=\"fn\" rid=\"fn3\"><sup>3</sup></xref> If this is true, psychiatry contributes to a “medicalization” of morally wrong behavior (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). The conceptual problems of ASPD and Pedophilic Disorder lead to the fundamental question which criteria define a mental disorder.</p><p>The aim of this paper is to discuss whether behavior harmful to others should be a sufficient criterion of mental disorder as it is the case in the diagnoses of ASPD and Pedophilic Disorder. If we come to the conclusion that this should not be the case, the question arises whether ASPD and Pedophilic Disorder should be eliminated from the diagnostic manuals.</p></sec><sec id=\"s2\"><title>Mental Disorders and Their Diagnostic Manuals</title><p>In probably no other specialty of medicine has the concept of “disease” been as contested as in psychiatry. Even though in psychiatry the term “disorder” is predominantly used, it can be regarded as synonymous to “disease”, especially regarding the practical consequences. Apart from the fundamental question whether there’s such a thing as “mental disorders” at all (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>), and hence, whether psychiatry is a part of medicine at all, the nature and definition of mental disorders in general have been discussed (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B8\" ref-type=\"bibr\">8</xref>, <xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Other controversies concern the disorder status of specific mental conditions, the most famous example probably being the removal of homosexuality from DSM in 1973 (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B11\" ref-type=\"bibr\">11</xref>). A still missing stringent scientific basis and the important role of values (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>) bring psychiatry into a position to constantly question its own presumptions about the concept of mental disorder.</p><p>Mental disorders are classified in two classification systems: First, the International Classification of Diseases and Related Health Problems, 10<sup>th</sup> revision (ICD-10), by the World Health Organization (WHO) (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Second, for mental disorders only, the Diagnostic and Statistical Manual of Mental Disorders, 5<sup>th</sup> edition (DSM-5), published by the American Psychiatric Association (APA) (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>). The latter is “viewed as representing the cutting-edge of the field” (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Both manuals define the current state of the art in psychiatric diagnostics and thus have a huge impact on clinical use but also on public discussions about mental health and finally, through their use in forensic settings, even on court rulings. The practical implications of the diagnostic manuals thus range from the funding of treatments by the public health system to the assessment of someone’s capacity to work, and indirectly to the evaluation of diminished criminal responsibility.<xref ref-type=\"fn\" rid=\"fn4\"><sup>4</sup></xref></p><p>The diagnoses in both diagnostic manuals rely on polythetic criteria sets, of which a specified number of criteria needs to apply for a specified period of time. Since the neurobiological underpinnings and the etiology of many mental disorders are still scarcely understood, the diagnostic criteria sets consist of observable and subjective symptoms. Contrary to most cases in “somatic medicine”, there are only few additional objective tests in psychiatry to support a suspected diagnosis (<italic>e.g.</italic> for dementias or autoimmune encephalitis).</p><p>Given their importance in the diagnostic process, the selection and exact formulation of the criteria of mental disorders are crucial. Changes in these criteria sets have a huge impact on the prevalence of certain mental disorders and on the lives of many individuals. It is thus not surprising that every revision of the diagnostic manuals is accompanied by extended controversies about the inclusion or elimination of diagnoses and the formulation of the diagnostic criteria sets (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Frances (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>), for example, sharply criticizes a “diagnostic inflation” in psychiatry which he thinks was intensified by DSM-5 by adding more diagnoses and expanding the existing ones.</p></sec><sec id=\"s3\"><title>Mental Disorders Harmful to Others</title><p>The most contested diagnoses in DSM and ICD are probably the paraphilias (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>) and Cluster B-personality disorders (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>).<xref ref-type=\"fn\" rid=\"fn5\"><sup>5</sup></xref> Especially Pedophilic Disorder and Antisocial Personality Disorder (ASPD) or Dissocial Personality Disorder (in ICD-10) are highly controversial diagnoses. Some authors question their status as clinical disorders [for ASPD, see Charland (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>)] or even their place in the manuals [for Pedophilic Disorder, see Green (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>)].</p><p>Pedophilic Disorder and ASPD are particularly contested because both diagnoses are highly linked to socially deviant or even criminal behavior. Persons with ASPD and pedophilic sexual offenders have a significantly increased risk of (re-)offending (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>–<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Sadler (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>) calls such diagnoses “vice-laden” disorders, vice being understood in a “technical sense—as simply criminal and/or immoral thought or conduct” (p. 452) by the legal and moral standards of the respective society. The notion of “vice-ladenness” indicates that those disorders imply thoughts and behaviors typically described and assessed in moral and/or legal rather than in medical terms.</p><p>Pedophilic Disorder and ASPD are not the only mental disorders associated with behaviors usually described in moral terms and potentially harmful to others, though. A person suffering from schizophrenia, for example, will presumably show in some way socially deviant behavior and may even cause harm to others when, for example, following the commands of imperative voices. The crucial point, however, is that in the case of schizophrenia the symptoms described in the diagnostic criteria set are “relatively immune to misconstrual as vice” (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>) (p. 9). Immoral or harmful behavior is not a defining criterion of the disorder, rather it may or may not be a secondary effect of it. In contrast, for ASPD and Pedophilic Disorder, behavior that is morally wrong and primarily harmful to others is a central part of the diagnosis: they are “vice-laden” at their core.</p><sec id=\"s3_1\"><title>Pedophilic Disorder</title><p>In DSM-IV, the diagnosis of pedophilia required that the fantasies, sexual urges, or behaviors involving children cause clinically significant distress or impairment in social, occupational, or other important areas of functioning (Criterion B). This criterion was changed in DSM-IV-TR so that it was then sufficient to have acted on the sexual urges. From DSM-IV-TR to DSM-5, all criteria remained unchanged after the proposed changes were declined (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>) (<xref rid=\"T1\" ref-type=\"table\"><bold>Table 1</bold></xref>).<xref ref-type=\"fn\" rid=\"fn6\"><sup>6</sup></xref></p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Comparison of the diagnostic criteria of pedophilic disorder and pedophilia in the DSM-IV, DSM-IV-TR, and DSM-5.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DSM-IV—Pedophilia (302.2)</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DSM-IV-TR—Pedophilia (302.2)</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DSM-5—Pedophilic Disorder (302.2)</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">A. Over a period of at least 6 months, recurrent, intense sexually arousing fantasies, sexual urges, or behaviors involving sexual activity with a prepubescent child or children (generally age 13 years or younger).<break/>B. The fantasies, sexual urges, or behaviors cause clinically significant distress or impairment in social, occupational, or other important areas of functioning.<break/>C. The person is at least age 16 years and at least 5 years older than the child or children in Criterion A.<break/>Note: Do not include an individual in late adolescence involved in an ongoing sexual relationship with a 12- or 13-year-old. (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">A. Over a period of at least 6 months, recurrent, intense sexually arousing fantasies, sexual urges, or behaviors involving sexual activity with a prepubescent child or children (generally age 13 years or younger).<break/>B. <italic>The person has acted on these sexual urges, or the sexual urges or fantasies cause marked distress or interpersonal difficulty</italic>.<break/>C. The person is at least age 16 years and at least 5 years older than the child or children in Criterion A.<break/>Note: Do not include an individual in late adolescence involved in an ongoing sexual relationship with a 12- or 13-year-old. (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">A. Over a period of at least 6 months, recurrent, intense sexually arousing fantasies, sexual urges, or behaviors involving sexual activity with a prepubescent child or children (generally age 13 years or younger).<break/>B. The individual has acted on these sexual urges, or the sexual urges or fantasies cause marked distress or interpersonal difficulty.<break/>C. The person is at least age 16 years and at least 5 years older than the child or children in Criterion A.<break/>Note: Do not include an individual in late adolescence involved in an ongoing sexual relationship with a 12- or 13-year-old. (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>)</td></tr></tbody></table><table-wrap-foot><p>Text that has been changed from the previous version is shown in italics.</p></table-wrap-foot></table-wrap><p>DSM-5, however, introduced a distinction between Pedophilia and Pedophilic Disorder. Pedophilia denotes the mere sexual preference for prepubescent children (Criterion A) and is not considered a mental disorder anymore. Pedophilic Disorder is Pedophilia with either personal distress or interpersonal difficulty, or sexual acts involving prepubescent children (Criterion B).</p><p>ICD-11, which has been presented by the WHO in 2019 and will foreseeably come into effect on 1 January 2022, adjusted the criteria of “Pedophilic Disorder” to the DSM-5 criteria (<xref rid=\"T2\" ref-type=\"table\"><bold>Table 2</bold></xref>). Except for the time criterion (the sexual attraction to children must be present for at least 6 months), which is only required in DSM-5, the criteria in ICD-11 and DSM-5 are basically the same (<xref rid=\"T1\" ref-type=\"table\"><bold>Tables 1</bold></xref> and <xref rid=\"T2\" ref-type=\"table\"><bold>2</bold></xref>).</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Comparison of the diagnostic criteria of pedophilic disorder and pedophilia in ICD-10 and ICD-11.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ICD-10— Pedophilia (F 65.4)</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ICD-11 – Pedophilic Disorder (6D32)</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">A sexual preference for children, usually of prepubertal or early pubertal age. Some pedophiles are attracted only to girls, others only to boys, and others again are interested in both sexes. (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Pedophilic disorder is characterized by a sustained, focused, and intense pattern of sexual arousal—as manifested by persistent sexual thoughts, fantasies, urges, or behaviors—involving pre-pubertal children</italic>.<break/><italic>In addition, in order for Pedophilic Disorder to be diagnosed, the individual must have acted on these thoughts, fantasies or urges or be markedly distressed by them</italic>.<break/><italic>This diagnosis does not apply to sexual behaviors among pre- or post-pubertal children with peers who are close in age</italic>. (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>)</td></tr></tbody></table><table-wrap-foot><p>Text that has been changed from the previous version is shown in italics.</p></table-wrap-foot></table-wrap><p>The age limit mentioned by DSM-5 (13 years) is clearly below the age of sexual consent, which ranges between 14 and 18 years in most countries (in the US states, for example, it ranges between 16 and 18 years). This means that the criterion of “has acted on these sexual urges” is equivalent to committing a criminal act.</p><p>This, however, does not apply to all countries in the world. According to the UNICEF child marriage report from 2014, about 250 million women alive today were married before age 15 (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). In some countries, this is even covered by law as it is allowed to marry before age 18 (in some cases there is no minimum age at all) under certain circumstances (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). This shows that not in every country sexual intercourse with children age 13 or younger is considered a criminal offense. Therefore, the legal and social reactions which individuals, who sexually abuse children, will have to face differ. Of course, even though tolerated by law in some countries, sexual acts involving children are harmful and should be legally forbidden all over the world.</p><p>Most researchers emphasize the difference between pedophilic interests and sexual offending against children. Not all individuals with pedophilic interests sexually approach children, and not all child molesters have “recurrent and intense” pedophilic interests; about half of the cases of sexual abuse of children are committed by presumably non-pedophilic offenders.<xref ref-type=\"fn\" rid=\"fn7\"><sup>7</sup></xref></p><p>However, both criteria A and B of Pedophilic Disorder contain a behavioral aspect that is sufficient for the respective criterion to be fulfilled. The use of the conjunction “or” before “behaviors” in criterion A makes it possible to meet this criterion solely by repeated acts of sexual behavior involving children (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). Regarding criterion B, sexual acts involving children are also sufficient to fulfill this criterion. This means that repeated sexual behavior involving children is sufficient to fulfill both criteria.</p><p>According to the criteria in DSM-5 and ICD-11, a diagnosis of Pedophilic Disorder requires neither suffering from the sexual fantasies, urges, or behaviors towards children nor experiencing any impairment in social, occupational or other important activities. The diagnosis can be made solely on grounds of behavior harmful to others. This has been criticized as a confusion of “mental disorder” and “crime” (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>) or “immoral behavior” (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>).</p></sec><sec id=\"s3_2\"><title>Antisocial Personality Disorder (ASPD)</title><p>In an attempt to define reliably measurable personality traits, the DSM focused on behavior in the definition of ASPD, which was intended to be an equivalent of psychopathy (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>). Psychopathy, conceptualized by the Hare Psychopathy Checklist Revised (PCL-R) (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>), contains much more interpersonal and affective symptoms than ASPD (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B43\" ref-type=\"bibr\">43</xref>) but is not a diagnosis in ICD-10 or DSM-5 (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>).<xref ref-type=\"fn\" rid=\"fn8\"><sup>8</sup></xref> Almost all criteria of ASPD in DSM-5 refer to behavior primarily harmful to others (<xref rid=\"T3\" ref-type=\"table\"><bold>Table 3</bold></xref>). In accordance with the diagnostic criteria required for all personality disorders, the antisocial personality traits must be “inflexible, maladaptive, and persistent and cause significant functional impairment or subjective distress” (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>).</p><table-wrap id=\"T3\" position=\"float\"><label>Table 3</label><caption><p>Comparison of the diagnostic criteria of Antisocial Personality Disorder (DSM-5) and Dissocial Personality Disorder (ICD-10).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DSM-5—Antisocial Personality Disorder (301.7)</th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ICD-10—Dissocial Personality Disorder (F60.2)</th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">A. A pervasive pattern of disregard for and violation of the rights of others, occurring since age 15 years, as indicated by three (or more) of the following:<break/>1. Failure to conform to social norms with respect to lawful behaviors, as indicated by repeatedly performing acts that are ground for arrest.<break/>2. Deceitfulness, as indicated by repeated lying, use of aliases, or conning others for personal profit or pleasure.<break/>3. Impulsivity or failure to plan ahead.<break/>4. Irritability and aggressiveness, as indicated by repeated physical fights or assaults.<break/>5. Reckless disregard for safety of self or others.<break/>6. Consistent irresponsibility, as indicated by repeated failure to sustain consistent work behavior or honor financial obligations.<break/>7. Lack of remorse, as indicated by being indifferent to or rationalizing having hurt, mistreated or stolen from another.<break/>B. The individual is at least age 18 years.<break/>C. There is evidence of conduct disorder with onset before age 15 years.<break/>D. The occurrence of antisocial behavior is not exclusively during the course of schizophrenia or bipolar disorder.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Personality disorder, usually coming to attention because of a gross disparity between behavior and the prevailing social norms, and characterized by (at least three of the following criteria)<break/>1. Callous unconcern for the feelings of others.<break/>2. Gross and persistent attitude of irresponsibility and disregard for social norms, rules, and obligations.<break/>3. Incapacity to maintain enduring relationships, though having no difficulty in establishing them.<break/>4. Very low tolerance to frustration and a low threshold for discharge of aggression, including violence.<break/>5. Incapacity to experience guilt, or to profit from adverse experience, particularly punishment.<break/>6. Marked proneness to blame others, or to offer plausible rationalizations for the behavior bringing the subject into conflict with society.<break/>There may be persistent irritability as an associated feature. Conduct disorder during childhood and adolescence, though not invariably present, may further support the diagnosis.</td></tr></tbody></table></table-wrap><p>The equivalent of ASPD in ICD-10, Dissocial Personality Disorder (DPD), refers less to behavioral and more to affective symptoms than ASPD in its criteria set (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>) (<xref rid=\"T3\" ref-type=\"table\"><bold>Table 3</bold></xref>). However, as Kröber and Lau (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>) note, most of the criteria can still be “easily derived from the criminal behavior itself” (p. 681).</p><p>The general criteria of personality disorders in ICD-10 require that “the disorder leads to considerable personal distress but this may only become apparent late in its course” and “the disorder is usually, but not invariably, associated with significant problems in occupational and social performance” (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>) (p. 202).<xref ref-type=\"fn\" rid=\"fn9\"><sup>9</sup></xref></p><p>However, Habermeyer states that persons with antisocial or dissocial personality traits subjectively do not suffer from their abnormalities and show little willingness to get treated (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). This is accentuated for inmates with high values on the Psychopathy Checklist (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Many, if not the overwhelming majority of subjects with psychopathy are perfectly content with and identify with their traits; there is no subjective suffering involved in psychopathy (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). Because there is nothing painful or ego-dystonic in psychopathic symptoms, it is unlikely that a psychopath would seek or endure treatment (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). Also persons with ASPD rarely seek treatment (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>, <xref rid=\"B46\" ref-type=\"bibr\">46</xref>), indicating that they usually do not feel significantly distressed or impaired by their condition. This becomes evident from the description of the self-image of people with dissocial or antisocial personality traits by Müller-Isberner et al.: “These people generally see themselves as autonomous, strong loners. Some see themselves as exploited and mistreated by society and justify harming others by saying that they themselves are being harassed. Others see themselves as robbers in a world where the motto is ‘eat and be eaten’ or ‘the winner takes it all’ and where it is normal or even desirable and necessary to violate social rules.”<xref ref-type=\"fn\" rid=\"fn10\"><sup>10</sup></xref> (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>) (p. 373).</p><p>This raises the question whether the diagnosis of ASPD could be made for anyone at all if the criteria of subjective distress and/or functional impairment were strictly applied. In clinical practice, distress can be presumed if someone seeks help voluntarily. The question is why this person seeks help and what distresses her. According to the literature on antisocial personality cited above, it is probably not her antisocial personality. However, subjective distress “in general” is not sufficient to make this specific diagnosis, even if all the other criteria of ASPD apply. According to DSM-5, the subjective distress must be caused by the antisocial personality traits.</p><p>It could be objected that a lack of personal distress in ASPD is precisely part of its psychopathology, in the sense that not recognizing one’s own problems is even more pathological than recognizing them. However, the general problem with this argument is that it allows the attribution of mental disorders to persons without personal distress from the outside. Even though there are cases in which this can be justified (<italic>e.g.</italic> in the case of severe psychosis/delusions where the individual doesn’t recognize her psychosis/delusions), there is a high risk of misusing psychiatric diagnoses for pathologizing socially deviant or nonconformist behavior.</p><p>The questionable personal distress in ASPD is especially relevant in the forensic context where the prevalence of ASPD is much higher than in the general population. The base rate in the population is estimated at 2%, whereas the prevalence among male prisoners is estimated at between 47 and 80% (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B48\" ref-type=\"bibr\">48</xref>). Prisoners are certainly distressed. However, distress because of the legally justified consequences of antisocial behavior, like a loss of freedom, must not be confused with distress because of the antisocial personality traits themselves (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). Distress because of society’s negative reaction to deviant behavior is not a sign of a mental disorder. Rather, it is normal. We suspect that the criterion of subjective distress and/or impairment often is not considered correctly when the diagnosis of ASPD is made, especially not in forensic contexts. The great difference between the prevalence of ASPD in the general population and among male prisoners indicates a strong correlation between ASPD and imprisonments. This means that either most criminals have a mental disorder or that ASPD is a construct mainly depicting criminal behavior.</p><p>We conclude that, strictly speaking, many persons diagnosed with ASPD in fact only have antisocial personality traits, which are not a mental disorder according to DSM-5. This conclusion is supported by the observation of Herpertz that a lack of considering the general definition of personality disorder and instead a focus on the easily applicable specific criteria lists led to an “inflationary diagnosis frequency” of personality disorders (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). We suspect that, especially in the case of ASPD, many persons are mistakenly classified as “mentally ill” because of a wrongful interpretation or even neglect of the distress/impairment criterion.</p><p>ICD-10 and DSM-5 present a categorial classification of personality disorders with ASPD/Dissocial Personality Disorder being a distinct disorder-entity. This categorial approach to personality disorders, however, is broadly contested (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). DSM-5 already introduced an alternative “hybrid” model for personality disorders, mixing categorial and dimensional approaches.<xref ref-type=\"fn\" rid=\"fn11\"><sup>11</sup></xref></p><p>According to the alternative model, the typical features of ASPD are “a failure to conform to lawful and ethical behavior, and an egocentric, callous lack of concern for others, accompanied by deceitfulness, irresponsibility, manipulative-ness, and/or risk taking” (p. 763). Psychopathy is described as a distinct variant that is “marked by a lack of anxiety or fear and by a bold interpersonal style that may mask maladaptive behaviors (<italic>e.g.</italic>, fraudulence).” (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>) (p. 765).</p><p>ICD-11 goes even further in replacing the categorial model by a dimensional one (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). According to this model, the diagnosis of a personality disorder comprises three steps. First, the general criteria of a personality disorder must be met (“problems in functioning of aspects of the self […], and/or interpersonal dysfunction […] that have persisted over an extended period of time (<italic>e.g.</italic>, 2 years or more)”, “the disturbance is manifest in patterns of cognition, emotional experience, emotional expression, and behaviour that are maladaptive”, “the disturbance is associated with substantial distress or significant impairment in personal, family, social, educational, occupational or other important areas of functioning” (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>)). Then, the severity of this general personality disorder must be determined (mild, moderate, severe). Eventually, the specific underlying personality structure is assessed according to five personality domains (negative affectivity, detachment, dissociality, disinhibition, anakastia). Thus, in ICD-11, there will be no category “Dissocial Personality Disorder” anymore. Instead, dissocial and disinhibited traits and behaviors may be a specifier among others in a diagnosis of a (general) personality disorder.</p></sec><sec id=\"s3_3\"><title>Interim Conclusion</title><p>In both the definitions of ASPD and Pedophilic Disorder behavior harmful to others or even criminal behavior is a criterion for the diagnosis of a mental disorder. For Pedophilic Disorder, even though harming others (for a period of at least 6 months) is not a necessary criterion, it can be a sufficient one. For ASPD, repeated harming of others is a necessary criterion, and—not formally, but practically—also a sufficient one.</p><p>The key question is: Should criminal behavior/harm to others be a sufficient criterion of a mental disorder? Or does this lead to a “medicalization” of vice conditions, meaning that “all problematic deviance reflects human illness or injury, including criminality and ‘immoral’ conduct” (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>) (p. 12)? The crucial point is: can behavior harmful to others alone indicate the presence of a mental disorder? Or is this rather an attempt to “pathologize the morally wrong”? We will come back to this question later.</p><p>The conceptual problems of Pedophilic Disorder and ASPD lead directly to a more fundamental question: which criteria define a mental disorder?</p></sec></sec><sec id=\"s4\"><title>The Definition of Mental Disorder</title><sec id=\"s4_1\"><title>The General Concept of Disease</title><p>If psychiatry claims to be a part of medicine, a general definition of disease should be the basis of a definition of mental disorders. Hucklenbroich developed a profound reconstruction of the general concept of disease (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>). He distinguishes four levels of the concept of disease. The first level is the life-world and personal concept of disease (person X is ill). On the second level, a distinction can be made between healthy and pathological life processes (X is pathological). At the third level, reference is made to a standard model of the human organism (X is pathologically altered). At the fourth level, disease entities and categories are postulated (X is a disease). The basis of the determination of disease entities is an etiopathogenetic model that comprises an identification of primary causes and the typical clinical course.</p><p>According to this reconstruction, life processes that meet four criteria can be described as pathological: 1. They are states, processes, or procedures in individuals, 2. which are attributable to the organism, not the environment, 3. which take place independently of the will and knowledge of the affected individuals, and 4. for which there is at least one non-pathological alternative course.</p><p>To determine which processes are diseases, Hucklenbroich distinguishes positive and negative disease criteria. Positive criteria of a disease are: 1. lethality; 2. pain, discomfort, suffering; 3. disposition for 1 or 2; 4. inability to reproduce; 5. inability to live together. The two negative criteria of disease, which determine a condition as non-pathological, are 1. universal occurrence and inevitability, <italic>e.g.</italic> gender, intrauterine and ontogenetic phases, pregnancy, menopause, old age, natural death; 2. knowingly and intentionally self-induced behavior (as long as self-determination is not diminished), <italic>e.g.</italic> suicide, value judgements, risky behavior, abstinence, intentional lying.</p><p>Hucklenbroich argues that this general concept of disease also applies to mental disorders, even though an etiopathogenetic disease model like in “somatic” medicine is still missing in psychiatry (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). According to his model, especially the positive criteria 2 and 5 are relevant for mental disorders. Mental disorders are often associated with significant pain, discomfort or suffering. Additionally, they may impair the ability to live together with others in a community. However, Hucklenbroich notes that due to the lack of knowledge about the etiopathogenesis of mental disorders there are still diverging concepts of mental disorder (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>).</p></sec><sec id=\"s4_2\"><title>The DSM-5 Definition of Mental Disorder</title><p>One of the mostly cited definitions of mental disorder is given in DSM-5. While conceding that “no definition can capture all aspects of all disorders in the range contained in DSM-5” (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>) (p. 20), it is stated that the definition is rather meant to formulate elements required for considering something a mental disorder:<disp-quote><p>“A mental disorder is a syndrome characterized by <bold>clinically significant disturbance</bold> in an individual’s cognition, emotion regulation, or behavior that reflects a <bold>dysfunction in the psychological, biological, or developmental processes underlying mental functioning</bold>. Mental disorders are usually associated with significant <bold>distress or disability in social, occupational, or other important activities</bold>. An expectable or culturally approved response to a common stressor or loss, such as the death of a loved one, is not a mental disorder. <bold>Socially deviant behavior</bold> (<italic>e.g.</italic> political, religious, or sexual) and <bold>conflicts that are primarily between the individual and society are not mental disorders</bold> unless the deviance or conflict results from a dysfunction in the individual, as described above.” (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>) (p. 20, emphasis added)</p></disp-quote></p><p>The definition starts with 1. an observable symptom level (“clinically significant disturbance”) that is 2. caused by an underlying dysfunction in the “mental domain” of an individual, and that has 3. some expected consequences, namely distress or disability in important activities of daily life. The rest of the definition specifies circumstances under which certain conditions are not deemed mental disorders: Socially deviant behavior and conflicts between the individual and society, which are not the result of a dysfunction, are not considered mental disorders.</p><p>The last point seems to be crucial. Pedophilic Disorder and ASPD are, prima facie, conditions that are mainly based on a conflict between the individual and other individuals and/or society.<xref ref-type=\"fn\" rid=\"fn12\"><sup>12</sup></xref> A person with Pedophilic Disorder could argue that his sexual orientation simply does not fit in his society’s current concepts of approved sexual relationships while denying that sexual contacts with children are actually harmful to them.<xref ref-type=\"fn\" rid=\"fn13\"><sup>13</sup></xref> Or a person diagnosed with ASPD could argue that he does not feel bothered by his antisocial behavior because he has many advantages by it, although he might come into conflict with the law unless he is careful.</p><p>According to DSM-5, socially deviant behavior can be a sign of a mental disorder only if it results from a dysfunction in the individual’s “psychological, biological, or developmental processes underlying mental functioning”. However, the behavioral symptoms described in the diagnoses of ASPD and Pedophilic Disorder can have very different causes. Indeed, the lack of differentiation between the different causes of mental disorders is a fundamental problem of the nominalistic approach of DSM and ICD.</p><p>If hypersexual and even pedophilic behavior occurred in previously normal people after a brain tumor, a brain trauma, or epilepsy surgery, the brain pathology probably causally contributed to the abnormal behavior (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>, <xref rid=\"B55\" ref-type=\"bibr\">55</xref>). This is reflected in the differentiation between “developmental” and “acquired” pedophilia in the literature where acquired pedophilia is etiologically associated with a structural brain abnormality and developmental pedophilia is not (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>, <xref rid=\"B56\" ref-type=\"bibr\">56</xref>). However, the diagnostic manuals do not differentiate between these two types of pedophilia, as the diagnoses are symptom-based and do not consider etiology.</p><p>Also for antisocial behavior, there are associations between damage of the prefrontal cortex, be it due to a head injury or due to neurodegeneration like in Frontotemporal Dementia, and the occurrence of antisocial behavior in previously normal people (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>). Cases of severe ventromedial prefrontal lobe epilepsy have been described that were associated with persistent antisocial behavior that was reversible after epilepsy surgery (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>). In these cases, abnormal behavior is associated with a brain pathology which suggests a causal link between this pathology and the deviant behavior.</p><p>On the other hand, someone can behave in the same way for completely different reasons. For example, someone could live in a subculture where it is normal to behave in an antisocial or even criminal way to be “successful”. If it is normal in the social environment to make a living from, for example, drug dealing or criminal financial transactions, it could be reasonable to follow this tradition. Another example is someone who shows hypersexual behavior because he simply has no reason to confine himself due to money and power. In these cases, there is no reason to assume an underlying pathology. It is rather a morally questionable behavior.</p><p>The point here is: the fact that there are cases of brain pathologies leading to disinhibited or antisocial behavior doesn’t imply that all people behaving in the same way have a brain pathology.</p></sec><sec id=\"s4_3\"><title>Wakefield’s “Harmful Dysfunction” Model</title><p>The question of the underlying dysfunction in ASPD and Pedophilic Disorder seems to be crucial for defending their status as mental disorders. A frequently cited concept related to the DSM definition of mental disorder is Wakefield’s “harmful dysfunction” model (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). This model assumes that a mental condition can be classified as a mental disorder when two criteria apply: Firstly, it is the result of a dysfunction, understood in an evolutionary sense as the failure of a process to perform the function it was biologically designed for; secondly, it is harmful to the individual according to sociocultural standards (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). By this definition, Wakefield tries to escape definitional problems by combining, as he calls it, a “value term” (harm) and a “scientific and factual” term (dysfunction) (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). The idea is to evade two problems: On the one hand, a mere “scientific” concept of mental disorder leads to the problem that every deviation from a scientifically defined standard could be viewed as a mental disorder even though the affected individual is neither suffering nor impaired. On the other hand, a mere value-based concept of mental disorders entails the risk of pathologizing socially disvalued behavior. Thus, according to Wakefield, only a harmful dysfunction represents a mental disorder, not a dysfunction without any harm to the individual nor something evaluated as harmful (according to sociocultural standards) but without representing a dysfunction.</p><p>We will come back to the notion of dysfunction in Pedophilic Disorder and ASPD later. Regarding the harm criterion, ASPD and Pedophilic Disorder are special since most mental disorders are primarily harmful to the affected individual. For “vice-laden” disorders like ASPD and Pedophilic Disorder, however, the “harm-criterion” primarily concerns others. Of course, some persons with Pedophilic Disorder might experience personal distress, probably after having internalized the society’s negative attitude towards pedophilia. Some persons with ASPD, however, may even enjoy real benefits through their special personality traits, both in terms of income and reproductive success. Malon (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>) introduces the concept of “dangerous dysfunction” instead of “harmful dysfunction” in the case of Pedophilic Disorder, arguing that it is actually the concept of “dangerous dysfunction” that explains the presence of Pedophilic Disorder in DSM.</p></sec><sec id=\"s4_4\"><title>Alternative Definitions of Mental Disorder</title><p>In the diagnoses of ASPD and Pedophilic Disorder, harm to the individual in the sense of personal distress or impairment is not necessarily implied. However, harm to the individual might be present even without the person concerned being aware of it. The philosopher Graham (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>) states that having a mental disorder does not necessarily comprise the recognition of its harmfulness by the affected individual herself. According to Graham, a mental disorder is a disability, dysfunction or impairment in one or more basic mental or psychological faculties or capacities of a person that has harmful or potentially harmful consequences for the person concerned (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>) (p. 28). It is a disorder because it is harmful in the sense that the person is worse off with the disorder than without the disorder, that she cannot control it, and that it cannot be removed by using additional psychological resources, <italic>e.g.</italic> by simply “pulling oneself together”.</p><p>Insofar, a person with Pedophilic Disorder could be regarded as worse off with the disorder than without it because having it means that either he has to abstain from fulfilling sexual relationships his whole life or he will commit a criminal act and possibly be punished for it. However, this argument is valid only for pedophilic persons living in societies which condemn and regularly punish child sexual abuse. In the case of ASPD, one could argue that the person is worse off with the disorder than without it because he is, for example, not able to have good relationships with other people. This, however, presupposes a certain model of good relationships and a “good life”, and therefore is value-laden and moralistic.</p><p>Heinz et al. (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>, <xref rid=\"B61\" ref-type=\"bibr\">61</xref>) argue for a differentiation between mental diseases in a narrow sense and states of suffering or disorders in a broader sense that do not meet the criteria of a disease. This differentiation, however, is not made by DSM and ICD where the notion of mental disorder is used for all diagnoses. Heinz et al. demand that the notion of mental disease should only be applied when life-relevant functional abilities are impaired and the affected person suffers from it or is impaired in her ability to cope with everyday life. Applying such a standard, many currently classifiable disorders are not diseases in this sense (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>, <xref rid=\"B61\" ref-type=\"bibr\">61</xref>). However, they are more or less easily classifiable states of suffering for which psychotherapeutic help and possibly drugs can be offered (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>, <xref rid=\"B61\" ref-type=\"bibr\">61</xref>). In this sense, Pedophilic Disorder and ASPD are not mental diseases.</p></sec><sec id=\"s4_5\"><title>What is a Mental Dysfunction?</title><p>The concept of mental dysfunction is central in most definitions of mental disorder. However, there is no consistent definition of this concept. For example, DSM-5 uses the notion of dysfunction without elucidating it.</p><p>Schramme (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>) distinguishes four models of mental functions. The first model, for which Wakefield’s concept of dysfunction is the most prominent example, is based on evolutionary psychology. According to Wakefield, mental functions result from selection processes and thus enable individuals to solve problems of adaptation (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Schramme rightly criticizes the historical orientation of this theory: Some processes may have been adaptive to past environments but not to our present environment. The second model of mental functions comes from cognitive psychology. Functions in this sense are best understood in formal terms as “input–output-relations”, not in any teleological sense. Schramme notes that this theory hardly applies to the concept of mental disorder, because it does not imply “normativity”, that means, it has no concept of how a mental function should work, and thus no concept of dysfunction. The third model supports a goal theory of function and is close to Boorse’s disease theory that identifies survival and reproduction as the highest goals of organisms (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). Mental functions are thus understood through their relation to these goals. In contrast to evolutionary psychology, this model does not refer to the evolutionary selection of these functions but evaluates them with regard to the present environment. Schramme, however, criticizes that this model lacks a plausible model of the “psychological species design” with regard to survival and reproduction. The fourth model is the ‘value-theory’, for which there is no established psychological account. This model determines functions according to their contribution to human welfare and the good human life. A mental function thus allows for the individual to live a good life. However, such a theory is always at risk of confounding a certain way of life with mental health.</p></sec></sec><sec id=\"s5\"><title>Discussing the Disorder Status of Pedophilic Disorder and ASPD</title><p>As we have argued, in both the definitions of ASPD and Pedophilic Disorder behavior harmful to others or even criminal behavior is a criterion for the diagnosis of a mental disorder.</p><p>If we thus conclude that ASPD and Pedophilic Disorder are just a “medicalization” of vice conditions, we have to ask whether and, if so, how these diagnoses can still be justified within a medical model.</p><sec id=\"s5_1\"><title>Neurobiological Findings in Pedophilic Disorder and ASPD</title><p>The most influential argument to justify the diagnoses of ASPD and Pedophilic Disorder within a medical model seems to be a “conservative” one. These diagnoses are well established, they have a long clinical tradition and some prognostic utility (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). This supports the argument that they should only be changed if there is strong empirical evidence that another nosological construct is more valid than the established ones.</p><p>The idea of a validation of the existing nosological constructs is pursued by researchers investigating underlying neurobiological and neuropsychological alterations in persons with ASPD or Pedophilic Disorder. There is a growing body of research indicating that there might be deviations in the brains of persons with ASPD and Pedophilic Disorder. However, the interpretation of these findings needs to be handled with care: Are the neurobiological deviations a sign of a pathology, or a sign of a vulnerability, or a consequence of a disease, or only a normal variant? And further, can these neurobiological differences causally explain the behavior (at least partly)?</p><p>For ASPD, studies show structural and functional deviations mainly in the areas of the amygdala, the striatum and the prefrontal cortex (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>, <xref rid=\"B57\" ref-type=\"bibr\">57</xref>, <xref rid=\"B63\" ref-type=\"bibr\">63</xref>). Genetic etiological studies suggest an association of a gene x environment interaction of MAOA enzyme deficiency and childhood maltreatment with antisocial behavior (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>, <xref rid=\"B63\" ref-type=\"bibr\">63</xref>). Evidence for developmental factors in the etiology of ASPD comes from studies that suggest a link between prenatal factors, such as birth complications, maternal smoking and alcohol consumption during pregnancy, or prenatal nutritional deficiency, and the occurrence of antisocial and violent behavior (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>, <xref rid=\"B64\" ref-type=\"bibr\">64</xref>). Also, an association between maltreatment during childhood and maternal withdrawal in infancy and ASPD has been found (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>). These findings suggest, that biological and social factors play a role in the development of ASPD, while “the presence of both factors exponentially increases the rates of antisocial and violent behavior” (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>) (p. 4).</p><p>For Pedophilic Disorder, reduced amygdala volumes were found in several studies (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>, <xref rid=\"B66\" ref-type=\"bibr\">66</xref>). The association between pedophilia and increased rates of left-handedness, more head injuries before age thirteen, and lower intelligence suggest that neurodevelopmental factors play a role in the development of pedophilia (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>). These findings support, though do not prove, the idea of underlying neurobiological alterations in Pedophilic Disorder.</p><p>However, most of the studies have severe methodological flaws.</p><p>For Pedophilic Disorder, most of the studies show a sampling bias in investigating only incarcerated pedophilic child sexual offenders with very scant evidence on non-offending pedophiles (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>, <xref rid=\"B66\" ref-type=\"bibr\">66</xref>). It is thus not clear whether alterations found in the brains of pedophilic child sexual offenders are causally contributing to their pedophilic preference itself or whether they are rather associated with offending in general by, for example, contributing to diminished behavioral control or lower intelligence. The latter assumption is supported by a MRT study by Schiffer et al. (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>), which provided first evidence that child sexual offending in pedophilia rather than pedophilia alone is associated with structural brain differences. Their study was published in the context of the German multi-sided research network NeMUP that investigated differences between pedophilic and non-pedophilic men, between child sexual offenders and non-offenders, and between convicted and non-convicted (pedophilic) child offenders.<xref ref-type=\"fn\" rid=\"fn14\"><sup>14</sup></xref></p><p>In the case of ASPD, the main methodological problem seems to be confounding variables, since most of the persons with ASPD show psychiatric comorbidities like substance use disorder or mood disorders (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). Another problem is the questionable homogeneity of persons that fulfill the criteria of ASPD. A study by Gregory et al. (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>), for example, found significant differences in gray matter volume in the prefrontal cortex between offenders with ASPD and additional psychopathic traits and offenders with ASPD without psychopathic traits, but not between offenders with ASPD without psychopathic traits and non-offenders.</p><p>These findings show the need for better study designs to get more reliable results. However, even if we get better results, we still face the general problem of interpreting neurobiological differences as indicated above. The finding of a neurobiological difference is not equivalent to a dysfunction, understood in psychological terms. The question of dysfunction is superior to it. An atypical structure or function of the amygdala, for example, is not per se dysfunctional or pathological. The assessment of its dysfunctionality depends on its assumed effects on the psychological and behavioral level, and how these effects are evaluated. An atypical function of the amygdala could even be evaluated as advantageous because it is associated with less anxiety.</p></sec><sec id=\"s5_2\"><title>Dysfunction in Pedophilic Disorder and ASPD</title><p>A crucial point in any discussion about the disorder status of a mental condition is the question if there is a convincing model of dysfunction, understood in psychological terms.</p><sec id=\"s5_2_1\"><title>Pedophilic Disorder</title><p>With regard to pedophilia, one could argue under an evolutionary account of dysfunction, that it is a form of a sexual dysfunction, assuming that the biologically defined function of sexual arousal (<italic>i.e.</italic> the reason the mechanism of sexual arousal was selected for) lies in its contribution to (potential) reproduction (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>) (p. 499), which is clearly not the case in pedophilic sexual behavior. This, however, is an insufficient model of the function of human sexuality. Human sexuality has important functions beyond reproduction, particularly promoting pair bonding and fulfilling emotional needs. Many forms of sexuality that do not pursue reproduction are broadly accepted, <italic>e.g.</italic> sexual intercourse of infertile people, under birth control, or homosexuality. Furthermore, there is no reason not to use a certain function for other, possibly purely hedonistic purposes that have nothing to do with its evolutionary function. The fact that a function is used for other than the alleged evolutionary purposes does not mean that this is dysfunctional.</p><p>Some pedophilic men actually state that they are not only interested in sexual contact with children but also look for romantic relationships with them (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>). The dysfunction in Pedophilic Disorder thus cannot simply stem from the fact that the sexual arousal is not associated with (potential) reproduction. The concept of a dysfunction in an evolutionary sense falls too short here.</p><p>According to DSM-5 and ICD-11, a pedophilic sexual interest is only deemed a mental disorder when it leads to subjective distress or impairment, or has been acted upon.</p><p>To assume that having certain sexual fantasies or urges is not pathological but acting according to them is, seems inconsistent. It might be explained by the implicit assumption that there is another dysfunction involved, namely an impaired ability to control one’s behavior. To illustrate this point: if a heterosexual teleiophilic man (<italic>i.e.</italic> a man sexually attracted to physically mature individuals) sexually assaults a woman, it is not generally supposed that he must be mentally disordered because he couldn’t control his sexual urges. For it is just as possible that he thought the assault was justified, <italic>e.g.</italic> because the woman dressed “lewdly”. There is no reason to regard the case of the heterosexual teleiophilic sexual offender differently from the case of a pedophilic sexual offender who is convinced that his behavior is morally justified, or who just does not respect the rights of children.</p><p>Moser (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>) rightly argues that a diagnosis of a paraphilia does not imply a lack of the ability to control one’s behavior: “Those individuals who cannot control their sexual impulses may qualify for another diagnosis based upon their inability to control their impulses, but not based upon the specific sexual behavior.” (p. 323).</p><p>This analysis shows that a model of dysfunction measured by moral standards is employed for Pedophilic Disorder. This argument is supported by the fact that the appraisal of sexual activities with children depends on historical and cultural contexts and has been accepted at varying times and cultures (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). This, of course, does not morally justify sexual acts with children. Only cultural relativists would conclude that sexual acts involving children are morally permissible because they are accepted in some cultures. We, however, regard child sexual abuse as a violation of universal human rights, including children’s rights. Thus, the fact that child sexual abuse is not sanctioned in some countries is no valid argument against its moral wrongness and its legal prohibition.</p></sec><sec id=\"s5_2_2\"><title>ASPD</title><p>In the case of ASPD, one could argue that antisocial behavior represents a dysfunction in social functioning. This argument implicitly presupposes that prosocial behavior is normal human behavior. However, under an evolutionary account, in many or even most societies during human history antisocial behavior was probably “adaptive” because it was the “normal and efficient” way to success, both in terms of reproduction and material wealth. Only in civilized societies governed by the rule of law, antisocial behavior becomes less adaptive than prosocial behavior and is considered abnormal and dysfunctional.</p><p>Some authors suggested that psychopathy could also be understood in evolutionary terms due to frequency-based selection as “adaptive” behavior (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>, <xref rid=\"B75\" ref-type=\"bibr\">75</xref>). According to this idea, a society with a prosocial majority can tolerate a small number of psychopaths that pursue their goals without being restrained by “other-regarding norms”. Reimer (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>) argues that the typical personality traits of psychopaths, like experiencing less anxiety and being able to resist attempts of “moral” social reinforcing, can also be understood as advantageous under a pro-individualist account of human existence. Maibom argues that psychopathy is not a disorder at all, but “from a certain perspective, what we call deficits are actually advantages” (<xref rid=\"B75\" ref-type=\"bibr\">75</xref>) (p. 34).</p></sec></sec></sec><sec id=\"s6\"><title>Practical Arguments for Considering Pedophilic Disorder and ASPD as Mental Disorders</title><p>Classifying something as a mental disorder is not only a theoretical question, but also has practical implications that need to be considered.</p><p>Most persons with Pedophilic Disorder and ASPD don’t seek help (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B43\" ref-type=\"bibr\">43</xref>). For ASPD, individuals presumably often don’t feel pain and thus have no motivation to change their condition (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>). For Pedophilic Disorder, the possible reasons for not seeking help range from not feeling distressed by it, or not recognizing its potential harmfulness towards others to a lack of knowledge about possibilities to get help and shame and fear of stigmatization (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>).</p><p>However, as the study of Levenson et al. (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>) also shows, some persons with Pedophilic Disorder are willing to get help. As an example, the Dunkelfeld (“dark field”) project in Berlin, Germany, a voluntary prevention project for pedophilic men at risk of offending, shows that a significant number of pedophiles seeks help (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>).</p><p>In many countries, the diagnosis of a mental disorder justifies treatment within the publicly funded health system. For that reason, the diagnoses of ASPD and Pedophilic Disorder can serve a useful purpose for individuals who feel distressed by their condition. If the health system with its long clinical experience can offer help, then it should do so (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>).</p><p>However, the question is whether we need the diagnoses of Pedophilic Disorder and ASPD so that these persons can get help. For social problems social institutions outside the health system could be conceivable that offer help. Even if these diagnoses were removed from the diagnostic manuals, people could get help within the health system for comorbid conditions like depression or anxiety disorder if these mainly cause their personal distress. In the case of paraphilias, Moser et al. argue that “other psychological characteristics describe these individuals and their concerns more accurately than their sexual interests do” (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Indeed, 93% of a sample of pedophilic sex offenders showed psychiatric comorbidities, mostly mood and anxiety disorders and substance use disorders (<xref rid=\"B78\" ref-type=\"bibr\">78</xref>). ASPD is also associated with anxiety disorders and substance use disorders. For the latter a prevalence of 80–85% among persons with ASPD was reported (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>).</p><p>One could object that these comorbidities possibly are a consequence of the Pedophilic Disorder or ASPD and therefore the focus of treatment should be the Pedophilic Disorder or ASPD as the primary condition. However, the fact that there are almost no effective treatments for Pedophilic Disorder or ASPD yet indicates that what actually can be treated within the health system might rather be associated disorders like depression, anxiety, or substance use disorder and not ASPD or Pedophilic Disorder itself.</p><p>Both, ASPD and Pedophilic Disorder, are supposed to be associated, besides others, with neurodevelopmental factors (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>, <xref rid=\"B66\" ref-type=\"bibr\">66</xref>), which makes it difficult to therapeutically intervene as late as in adulthood. The goal of therapies is thus rather the prevention of future deviant behavior in order to avoid harm to others. As Seto (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>) puts it regarding Pedophilic Disorder: “Instead of a ‘cure’, the focus of treatments for nonoffending individuals with pedophilia or hebephilia is the development of more effective self-management, to prevent sexual offending.” (p. 209).</p><p>The idea of drug treatment with antiandrogens or GnRH analogs (androgen deprivation therapy, ADT) in Pedophilic Disorder is not to change sexual preference but to reduce sex drive and thereby reduce the risk of (re-)offending. There is, until now, very limited evidence of the efficacy of ADT, and the level of willingness to undergo this kind of treatment is quite low (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>, <xref rid=\"B80\" ref-type=\"bibr\">80</xref>). Furthermore, according to a review of studies on behavioral and cognitive–behavioral treatments of pedophilia, there is no reliable evidence of their long-term efficacy (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). There are, however, few hints that it might be possible to actually modify sexual interest in children by, for example, strengthening self-esteem, coping skills, emotional self-regulation, and relationship skills in order to enable men with a sexual interest in children to fulfill their emotional and sexual needs with adult partners (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>). Studies on specific techniques, like masturbatory reconditioning in order to suppress deviant sexual interests and/or enhance normative sexual interests, show scant evidence of their efficacy to date (<xref rid=\"B82\" ref-type=\"bibr\">82</xref>).</p><p>For ASPD, a meta-analysis by Wilson (<xref rid=\"B83\" ref-type=\"bibr\">83</xref>) shows no significant effects of treatments. A lack of high-quality studies and small sample sizes might contribute to these findings. Better designed studies with larger sample sizes are required for future research.</p><p>It seems necessary to classify ASPD and Pedophilic Disorder as mental disorders in order to facilitate further research on them, gain better insights into their etiology, and develop new therapies. The example of the “psychopathy”-concept, however, shows that there can be a lot of research on a concept without being an official diagnosis in DSM and ICD (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>). The psychopathy-checklist (PCL-R) is widely used in forensic contexts to reliably assess the risk potential of criminals with psychopathic traits (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Since psychopathy does not need to be a diagnosis in DSM and ICD to be a broadly applied concept, it seems that ASPD and Pedophilic Disorder do not need it either.</p><p>Similar to psychopathy, ASPD and Pedophilic Disorder are most relevant in forensic contexts (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>). Apart from clinical utility, the forensic implications of these diagnoses need to be considered. According to Sexually Violent Predator laws in many U.S. states, sex offenders with a “mental abnormality” and a high risk of re-offending can be indefinitely committed after the prison sentence to protect society from them (<xref rid=\"B84\" ref-type=\"bibr\">84</xref>). Even though “mental abnormality” is a legal term referring to an impairment in emotional and volitional capacity that predisposes to the commission of criminal sexual acts and not synonymous with “mental disorder” (<xref rid=\"B85\" ref-type=\"bibr\">85</xref>), the diagnosis of a paraphilic disorder, as specified in DSM, is practically mostly accepted as sufficient to ascertain “mental abnormality” (<xref rid=\"B86\" ref-type=\"bibr\">86</xref>). Regarding these severe consequences, the definition of the paraphilic disorders in DSM seems especially critical.</p></sec><sec sec-type=\"conclusions\" id=\"s7\"><title>Conclusions</title><sec id=\"s7_1\"><title>“Vice-Laden Disorders” in Psychiatry</title><p>Diagnoses that primarily rely on behavior harmful to others, like Pedophilic Disorder and ASPD, fall out of the general disease concept. They even do not meet the general criteria of mental disorders as defined by DSM-5 or the “harmful dysfunction” model by Wakefield. Neither the criterion of harm to the individual himself, nor the criterion of a dysfunction are met in these two diagnoses.<xref ref-type=\"fn\" rid=\"fn15\"><sup>15</sup></xref> Instead, they rely on another disease criterion: the criterion of harm to others. Psychiatry brings itself into great conceptual difficulties by making behavior harmful to others/criminal behavior a central part of the definition of some mental disorders, while at the same time lacking a clear concept of dysfunction in these cases. When diagnoses are formulated in a way that makes it possible to apply them to mere antisocial and criminal behavior, psychiatry is at risk of confounding the medical and the moral.</p><p>Furthermore, the purely behavioral diagnoses do not reveal whether the behavior is based on a mental dysfunction or whether it was chosen voluntarily or for specific reasons.</p><p>Therefore, the formulation of the criteria sets of “vice-laden” disorders needs to be done very cautiously in order to avoid a confusion between criminal/immoral behavior and mental disorder. It should not be possible that harming others/criminal behavior defines a mental disorder. A psychiatric diagnosis should not only rely on observable behavior, but consider psychological, cognitive, or affective factors as well.</p><p>After considering the arguments for and against the disorder-status of Pedophilic Disorder and ASPD, we come to different conclusions regarding both diagnoses.</p></sec><sec id=\"s7_2\"><title>The Disorder-Status of Pedophilic Disorder</title><p>In the case of Pedophilic Disorder, we think that the diagnosis should be kept but reformulated in accordance with the general definition of mental disorder in DSM-5 in order to make it consistent with a medical model of mental disorder. This means it should only be applicable to individuals that are distressed or impaired by it so that they can get treatment within the health system. It should not be possible to make the diagnosis solely based on behavior harmful to others. Therefore, we suggest reformulating Criterion B of Pedophilic Disorder as follows: “The sexual urges or fantasies cause marked distress or interpersonal difficulty (<italic>e.g.</italic> in the context of occupation, family life, friendships, intimate life).” That means, the criterion “The individual has acted on these sexual urges” is cancelled.</p><p>Our suggested reformulation of Criterion B is indeed consistent with the form it already had in DSM-IV. As De Block et al. (<xref rid=\"B87\" ref-type=\"bibr\">87</xref>) note, the DSM-IV diagnostic criteria were “by far the most consistent vis-à-vis the DSM’s own definition of mental disorder” (p. 291). It was, however, criticized that this criteria set leads to the situation that someone acting on his pedophilic interests without feeling distressed would not be considered mentally ill (<xref rid=\"B88\" ref-type=\"bibr\">88</xref>). O’Donohoe et al. (<xref rid=\"B89\" ref-type=\"bibr\">89</xref>) argue that rather the lack of experiencing subjective distress when being sexually attracted to children than the experience of distress is a sign of psychological problems. They do not accept that, according to DSM-IV, a “contended pedophile” does not meet the criteria of a mental disorder. They argue that a person sexually interested in children must be considered in some way socially impaired “because societal norms dictate that it is abnormal for a person to be sexually interested in children” (p. 102). They clearly want to classify pedophilia as a mental disorder for social and forensic rather than for medical reasons. Their postulation that “a single instance of sexual behavior with a child should be sufficient to label someone as having a disorder” (<xref rid=\"B89\" ref-type=\"bibr\">89</xref>) (p. 103) confounds criminal behavior with mental disorder.</p><p>If pedophilia by itself is not a mental disorder according to DSM-5, then acting according to it cannot be a mental disorder unless there is clear evidence of a dysfunction of volitional control. Impairment of volitional control, however, is not implied in the diagnosis of a paraphilic disorder (<xref rid=\"B85\" ref-type=\"bibr\">85</xref>). If we assume that sometimes such impairment is given, then it probably stems from another disease (like <italic>e.g.</italic> dementia, a brain tumor or mental retardation). If there is no such impairment, we have to assume that this person acted deliberately, and it is not clear why this should be a sign of a mental disorder rather than simply a criminal act.</p><p>The DSM-5 warns of the dangers of using a diagnostic manual developed for clinical purposes in the forensic context. For assigning mental disorder in the legal sense “additional information is usually required beyond that contained in the DSM-5 diagnosis, which might include information about the individual’s functional impairments and how these impairments affect the particular abilities in question” (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>) (p. 25).</p><p>It is important to note that there is a difference between a mental disorder and the US-American legal concept of “mental abnormality”.</p><p>We suggest that it should be possible to diagnose a “mental abnormality” in the forensic sense for a person with pedophilia who is neither distressed nor impaired by his pedophilic condition (<italic>i.e.</italic>, who fulfills criterion A but not B according to our suggestion). Even though this person does not meet the criteria of a mental disorder as suggested by us, he might still meet the concept of “mental abnormality” if there is evidence of a high risk of reoffending. We thus suggest that this difference in clinical and forensic use is clearly annotated in the diagnostic criteria of Pedophilic Disorder in DSM. This suggestion is important with regard to other countries than the USA. The DSM is used worldwide for research, and therefore its diagnostic criteria should not be distorted in order to adapt them to the US legal system. In Germany, for example, no diagnosis of a mental disorder is required to order preventive detention after imprisonment; rather the assessment of danger and the prognosis of the probability of recidivism is decisive.</p><p>Our intention is not to protect the “contented pedophile”, as long as he is dangerous, from preventive detention or to downplay the harm that child molesters do to their victims in any sense. On the other hand, our suggestion is not meant to preclude the detained child molester from getting treatment if at some point he starts to show insight into his problems and wants to get treated. Rather, we want to separate the medical aspects of Pedophilic Disorder from the societal and forensic implications.</p><p>To summarize, our suggestion is as follows. We agree with the differentiation between Pedophilia and Pedophilic Disorder in DSM-5 and suggest adding a category “Pedophilia with mental abnormality” for forensic purposes. Thus, we suggest defining Pedophilia as pedophilic preference without distress/impairment; Pedophilic Disorder as pedophilic preference with distress/impairment; and Pedophilia with mental abnormality as pedophilic preference with sexual offending and high risk of re-offending with or without distress/impairment.</p></sec><sec id=\"s7_3\"><title>The Disorder-Status of ASPD</title><p>In the case of ASPD, however, we think that the arguments to remove it as a distinct diagnosis from the diagnostic manuals are stronger than the ones to keep it. Especially the presumed lack of personal distress of individuals with ASPD and the strong correlation with criminal behavior and incarceration indicate that this diagnosis is more of a social than a mere health-related problem.</p><p>We agree with Kröber and Lau (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>) who said: “If those with antisocial personalities, like anyone else, are subject to social influences and learning processes, they act as rational and competent citizens; their decision against behaving in compliance with standards should not be considered as pathologic.” (p. 687).</p><p>Herpertz and Sass (<xref rid=\"B90\" ref-type=\"bibr\">90</xref>) warn of the consequences of confounding antisocial behavior with “real” disorders in forensic psychiatry: “If the forensic psychiatrist fails to distinguish clearly between simple antisocial behaviour and a profound disturbance in personality, psychiatry runs the risk of being charged with handling all kinds of recurrent social deviance and delinquency. This would greatly hamper our capacity to treat those offenders who show real and treatable mental disorders.” (<xref rid=\"B90\" ref-type=\"bibr\">90</xref>).</p><p>As Gert & Culver (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>) put it: “If psychiatry is to take its place as a branch of medicine, mental disorders, like physical disorders, should be limited to conditions that cause harm to the person with the disorder.” (p. 489).</p><p>We think that the implementation of a dimensional model of personality disorders, as introduced by ICD-11, will mitigate the problem of attributing a diagnosis of mental disorder to mere criminal behavior. The ICD-11 does not contain the diagnosis “Dissocial Personality Disorder” anymore. Antisocial or dissocial personality traits will then be a specifier among others in the diagnosis of a general personality disorder. Thus, with this new model, the focus will hopefully be more on the cognitive, affective and interpersonal dimensions of personality disorders while avoiding an overly focus on deviant behavior.</p><p>To summarize: We suggest removing ASPD from the DSM, and support the planned removal of the diagnosis DPD from the ICD-11.</p></sec><sec id=\"s7_4\"><title>Practical Implications</title><p>Our suggestion to remove or reformulate the “vice-laden” diagnoses does not imply the demand for stopping research on them—quite the contrary. Especially in the forensic context, it is important to find opportunities to effectively prevent their harmful consequences and develop treatment methods insofar this is possible. The concept of psychopathy shows that an official diagnosis is not necessary for research to be done on forensically relevant conditions.</p><p>Regarding Pedophilic Disorder, our suggestions strongly support therapeutic offers (like the Dunkelfeld project) for people who feel distressed or impaired by their condition and seek help.</p><p>Regarding antisocial behavior, we think that it is much more of a social problem that has to be addressed more by other societal systems than the health system.</p><p>Finally, our suggestions have legal implications in some legal systems. Particularly for the USA, we suggest adding the category of “pedophilia with mental abnormality” in DSM for forensic use in order to separate the clinical and forensic aspects of pedophilia. However, the requirements of the legal systems in some countries are no valid argument against clear conceptual differentiations in the psychiatric diagnostic systems.</p></sec></sec><sec id=\"s8\"><title>Author Contributions</title><p>Development of the concept (SM, RM). Writing of the paper (RM). Literature research (RM, SM). Discussion of the concept (RM, SM, HW). Editing the text (SM, HW). All authors contributed to the article and approved the submitted version.</p></sec><sec sec-type=\"funding-information\" id=\"s9\"><title>Funding</title><p>ERA-NET NEURON and the Federal Ministry of Education and Research (BMBF) of Germany funded the work of SM (grant number: 01GP1621A). The research of RM and HW did not receive any specific grant from funding agencies in the public, commercial, or not-for profit sectors. We acknowledge support from the German Research Foundation (DFG) and Open Access Publication Fund of Charité—Universitätsmedizin Berlin.</p></sec><sec id=\"s10\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn id=\"fn1\"><label>1</label><p>Contrary to medical mainstream opinion, Hucklenbroich regards asymptomatic carriers of infectious diseases as ill. According to his theory (see <italic>The general concept of disease</italic>), asymptomatic carriers fall within the scope of disease criterion 5 (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>).</p></fn><fn id=\"fn2\"><label>2</label><p>The rationale of our argumentation applies to other diagnoses as well, like for example “Coercive sexual sadism disorder” in ICD-11. We have chosen Pedophilic Disorder and ASPD because they are the most questioned and relevant diagnoses.</p></fn><fn id=\"fn3\"><label>3</label><p>Charland (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>) argues that only the personality disorders in Clusters A and C are genuine clinical disorders. In contrast, he considers the Cluster B disorders (which include antisocial, borderline, histrionic, and narcissistic personality disorder) as moral disorders since their definitions are “morally loaded” and they require “moral treatment”.</p></fn><fn id=\"fn4\"><label>4</label><p>A psychiatric diagnosis <italic>per se</italic> is not a reason for assuming a lack of criminal responsibility or diminished responsibility but it is part of the forensic examination. According to German criminal law, “[a] person acts without guilt who, at the time the criminal act is committed, is incapable of understanding the wrongfulness of his or her action or is incapable of acting in accordance with this understanding due to mental illness, due to a profound disturbance of consciousness, or due to mental retardation or another serious mental abnormality” [Section 20, German Criminal Code, English translation cited from (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>)]. Diminished responsibility is present in the case of a diminished capability of the offender to understand the wrongfulness of an action or to act in accordance with this understanding due to one of the reasons indicated in Section 20 and may lead to mitigated penalty (Section 21, German Criminal Code). Section 20 lists four mental conditions that are necessary prerequisites for assuming a lack of criminal responsibility. However, these mental conditions are not equivalent to specific psychiatric diagnoses. They are legal terms that refer to psychiatric diagnoses (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>).</p></fn><fn id=\"fn5\"><label>5</label><p>The general concept of the personality disorders has been criticized fundamentally. Lieb criticizes the concept of personality disorder as contradictory in itself and as harmful to the patient and to the therapeutic relationship (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>).</p></fn><fn id=\"fn6\"><label>6</label><p>It was proposed to include the attraction to pubescent children and rename the diagnosis “pedohebephilic disorder”, to include a victim count and the use of child pornography in criterion B, and to include the specifiers “in remission” and “in controlled environment” (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). After the refusal of these changes, it was criticized that Pedophilic Disorder is the only Paraphilic Disorder in DSM-5 that lacks the specifiers “in full remission” and “in controlled environment” (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>). Further criticism was directed against the refusal to include the attraction to pubescent children (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). These discussions, however, are not in the focus of this paper.</p></fn><fn id=\"fn7\"><label>7</label><p>Data on the proportion of pedophilic and non-pedophilic child sexual offenders are quite scarce and come from small studies. According to Seto et al. (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>), in a sample of 100 child pornography offenders (where the authors assumed a high probability of pedophilic interest due to phallometric responses), 57% were not known to have had sexual contact with children. Conversely, the prevalence of pedophilic preference among identified child sexual offenders is estimated at about 40–50% (based on their sexual arousal to stimuli depicting children or their sexual offense history) (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). First (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>) notes that “compared with other paraphilic disorders, child molestation is even more likely to occur for nonparaphilic reasons”. Nonparaphilic reasons may be “a lack of more preferred sexual opportunities, hypersexuality, indiscriminate sexual interests, or disinhibition as a result of substance use or other factors” (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>) (p. 393). Knack et al. (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>) name “a general anti-social orientation”, “a sexual interest in coercion”, “attitudes accepting of sex between adults and children”, and “indiscriminate or opportunistic sexual behaviours” as reasons for non-pedophilic child sexual abuse (p. 183). Strassberg et al. found that non-pedophilic child molesters are more likely to show psychopathic traits than pedophilic child molesters (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>).</p></fn><fn id=\"fn8\"><label>8</label><p>ASPD and psychopathy are largely overlapping concepts. According to Ogloff (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>), 81% of persons diagnosed with psychopathy also meet the criteria of ASPD, whereas only 38% of the persons with ASPD receive a diagnosis of psychopathy. This indicates that the population of persons diagnosed with psychopathy can more or less be considered a subset of the population of persons diagnosed with ASPD. Exceptions are typically fraudulent personalities (or so-called “white collar offenders”) who are psychopaths but do not meet the criteria of dissocial or antisocial personality disorder (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>).</p></fn><fn id=\"fn9\"><label>9</label><p>For the sake of clarity, we will mainly refer to Antisocial Personality Disorder in this paper, even though many of the points made equally apply to Dissocial Personality Disorder. However, because of the stronger focus on behavior in ASPD compared with Dissocial Personality Disorder, we consider the diagnosis of ASPD as more problematic.</p></fn><fn id=\"fn10\"><label>10</label><p>Translated by Sabine Müller.</p></fn><fn id=\"fn11\"><label>11</label><p>The alternative model for personality disorders in DSM-5 has been developed for further research (Section III). In the alternative model, personality disorders are generally characterized by impairments in personality functioning (Criterion A) and pathological personality traits (Criterion B). Personality functioning (Criterion A) involves self-functioning (identity and self-direction) and interpersonal functioning (empathy and intimacy). For each of these four elements, five levels of impairment (ranging from no impairment to extreme impairment) can be differentiated. Pathological personality traits (Criterion B) are organized in five broad domains, namely negative affectivity, detachment, antagonism, disinhibition, and psychotism. The impairments in personality functioning and personality trait expression are relatively inflexible and pervasive across a broad range of personal and social situations (Criterion C). They are relatively stable with onset in at least adolescence or early adulthood (Criterion D), cannot be better explained by another mental disorder (Criterion E), are not attributable to the physiological effects of a substance or another medical condition (Criterion F), and not better understood as normal for an individual’s developmental stage or sociocultural environment (Criterion G) (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>) (pp. 761–3).</p></fn><fn id=\"fn12\"><label>12</label><p>As soon as a crime is committed against an individual person, the perpetrator comes into conflict not only with the victim but also with the society whose moral or legal norms have been violated.</p></fn><fn id=\"fn13\"><label>13</label><p>As an example, in Germany there were fierce debates about the harmfulness of sexual interactions between adults and children in the 1960s to the 1980s. Some sexologists, psychologists, and psychiatrists denied the harmfulness of sexual interactions with children (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>). Pedophilic activists demanded the abolition of the legal age limit of sexual interactions, a position that was supported even by several leaders of the Green party (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>).</p></fn><fn id=\"fn14\"><label>14</label><p>The NeMUP researchers found that executive dysfunctions are related to offense status rather than pedophilic preference (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>). Furthermore, they revealed that offenders and non-offenders differed in age, intelligence, educational level and experience of childhood sexual abuse, whereas pedophiles and non-pedophiles mainly differed in sexual characteristics (<italic>e.g.</italic>, additional paraphilias) (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>). When they compared convicted and non-convicted pedophilic child sexual offenders, they found only two significant differences between the two groups. The convicted offenders had a higher interest in prepubescent children and had committed significantly more sexual offenses against children compared to non-convicted subjects (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). However, significant differences regarding clinical characteristics, inhibition performances, neuronal activation, empathy and impulsiveness between the two groups were not found (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>).</p></fn><fn id=\"fn15\"><label>15</label><p>However, this conclusion is not equally applicable to definitions of mental disorder that do not require that the individual recognizes the harmful consequences of his condition, like the definition of Graham (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1</label><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name><surname>Hucklenbroich</surname><given-names>P</given-names></name></person-group> “<article-title>Disease entity” as the key theoretical concept of medicine</article-title>. <source>J Med Philos</source> (<year>2014</year>) <volume>39</volume>(<issue>6</issue>):<page-range>609–33</page-range>.  <pub-id pub-id-type=\"doi\">10.1093/jmp/jhu040</pub-id>\n</mixed-citation></ref><ref id=\"B2\"><label>2</label><mixed-citation publication-type=\"book\">\n<person-group person-group-type=\"author\"><name><surname>Hucklenbroich</surname><given-names>P</given-names></name></person-group> “<article-title>Die wissenschaftstheoretische Struktur der medizinischen Krankheitslehre</article-title>”. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"letter\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Evol Lett</journal-id><journal-id journal-id-type=\"doi\">10.1002/(ISSN)2056-3744</journal-id><journal-id journal-id-type=\"publisher-id\">EVL3</journal-id><journal-title-group><journal-title>Evolution Letters</journal-title></journal-title-group><issn pub-type=\"epub\">2056-3744</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33014421</article-id><article-id pub-id-type=\"pmc\">PMC7523560</article-id><article-id pub-id-type=\"doi\">10.1002/evl3.188</article-id><article-id pub-id-type=\"publisher-id\">EVL3188</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Letter</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Letters</subject></subj-group></article-categories><title-group><article-title>A novel seed dispersal mode of <italic>Apostasia nipponica</italic> could provide some clues to the early evolution of the seed dispersal system in Orchidaceae</article-title><alt-title alt-title-type=\"left-running-head\">K. SUETSUGU</alt-title><alt-title alt-title-type=\"right-running-head\">SEED DISPERSAL BY CRICKETS IN <italic>APOSTASIA</italic></alt-title></title-group><contrib-group><contrib id=\"evl3188-cr-0001\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Suetsugu</surname><given-names>Kenji</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-7943-4164</contrib-id><xref ref-type=\"aff\" rid=\"evl3188-aff-0001\">\n<sup>1</sup>\n</xref><address><email>kenji.suetsugu@gmail.com</email></address></contrib></contrib-group><aff id=\"evl3188-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">Department of Biology</named-content>\n<named-content content-type=\"organisation-division\">Graduate School of Science</named-content>\n<institution>Kobe University</institution>\n<city>Kobe</city>\n<named-content content-type=\"country-part\">Hyogo</named-content>\n<postal-code>657–8501</postal-code>\n<country country=\"JP\">Japan</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label>E‐mail: <email>kenji.suetsugu@gmail.com</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>02</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><month>10</month><year>2020</year></pub-date><volume>4</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1002/evl3.v4.5</issue-id><fpage>457</fpage><lpage>464</lpage><history><date date-type=\"received\"><day>13</day><month>4</month><year>2020</year></date><date date-type=\"rev-recd\"><day>08</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>08</day><month>7</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 The Authors. Evolution Letters published by Wiley Periodicals LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB). <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Evolution Letters</italic> published by Wiley Periodicals, LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB).</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:EVL3-4-457.pdf\"/><abstract><title>Abstract</title><p>Despite being one of the most diverse families, scant attention has been paid to the seed dispersal system in Orchidaceae, owing to the widely accepted notion that wind dispersal is the dominant strategy. However, the indehiscent fruits, with seeds immersed in fleshy tissue, evoke the possibility of endozoochory in Apostasioideae, the earliest diverging lineage of orchids. In the present study, I investigated the seed dispersal system of <italic>Apostasia nipponica</italic> by direct observation, time‐lapse photography, and investigation of the viability of seeds passing through the digestive tract of orthopterans. This study revealed a previously undocumented seed dispersal system in <italic>A. nipponica</italic>, in which the cricket, <italic>Eulandrevus ivani</italic>, and the camel cricket, <italic>Diestrammena yakumontana</italic>, consume the fruit and defecate viable seeds. Orthopterans are rarely considered seed dispersers, but the gross fruit morphology and pigmentation patterns of some <italic>Apostasia</italic> species parallel those seen in <italic>A. nipponica</italic>, suggesting that similar seed dispersal systems could be widespread among <italic>Apostasia</italic> species. Whether seed dispersal by orthopteran frugivores is common in Apostasioideae warrants further investigation.</p></abstract><kwd-group kwd-group-type=\"author-generated\"><kwd id=\"evl3188-kwd-0001\">Apostasioideae</kwd><kwd id=\"evl3188-kwd-0002\">camel cricket</kwd><kwd id=\"evl3188-kwd-0003\">cricket</kwd><kwd id=\"evl3188-kwd-0004\">endozoochory</kwd><kwd id=\"evl3188-kwd-0005\">Orchidaceae</kwd><kwd id=\"evl3188-kwd-0006\">seed disperser</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source><institution-wrap><institution>Japan Society for the Promotion of Science </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/501100001691</institution-id></institution-wrap></funding-source><award-id>17H05016</award-id></award-group></funding-group><counts><fig-count count=\"2\"/><table-count count=\"1\"/><page-count count=\"8\"/><word-count count=\"4747\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>October 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.9.1 mode:remove_FC converted:29.09.2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><boxed-text position=\"float\" content-type=\"box\" orientation=\"portrait\"><caption><title>Impact summary</title></caption><p>Seed dispersal is a key evolutionary process and central theme in terrestrial plant ecology. Animal‐mediated seed dispersal, most frequently by birds and mammals, benefits seed plants by ensuring efficient and directional transfer of seeds without relying on random abiotic factors such as wind and water. In return for these seed dispersal services, many plants provide nutritional rewards in the form of fleshy fruits. Orchids are unique among terrestrial plants in that their seedlings are completely dependent on fungi for their nutritive needs until they are mature enough to photosynthesize. Therefore, orchids usually produce remarkably small seeds that lack endosperm and are dispersed in air like dust particles. Therefore, the prevailing assumption is that orchid seeds are dispersed by wind. However, because indehiscent fruits with hard seed coats are common within the subfamily Apostasioideae, the earliest diverging clade in Orchidaceae, animal‐mediated seed dispersal can be an ancestral trait in the clade. Here, I present evidence for seed dispersal by crickets and camel crickets in <italic>Apostasia nipponica</italic> (Apostasioideae). To my knowledge, this is the first detailed report of a seed dispersal system in Apostasioideae. Owing to many plesiomorphic characters and the earliest diverging phylogenetic position, members of Apostasioideae have been extensively studied to understand their floral structure, taxonomy, biogeography, and genome. The interaction described here, importantly, provides some clues to the animals that may have participated in the seed dispersal of the ancestors of orchids. Given that the origin of crickets and camel crickets precedes the evolution of orchids, they are among the candidates for seed dispersers of the ancestors of extant orchids. Ancestral character‐state reconstruction analysis, with more data on the seed dispersal systems of other apostasioids, can provide deeper insights into the early evolution of the seed dispersal system in Orchidaceae.</p></boxed-text><p>Orchidaceae is one of the largest and most diverse families of flowering plants, with more than 28,000 known species spanning 763 genera (Christenhusz and Byng <xref rid=\"evl3188-bib-0007\" ref-type=\"ref\">2016</xref>). This high species diversity is likely linked to its specialized pollination syndromes, symbiotic associations with mycorrhizal fungi, colonization of epiphytic habitats, tropical and cordillera distribution, and its use of crassulacean acid metabolism (Givnish et al. <xref rid=\"evl3188-bib-0017\" ref-type=\"ref\">2015</xref>; Zhang et al. <xref rid=\"evl3188-bib-0042\" ref-type=\"ref\">2018</xref>). Consequently, researchers have paid considerably more attention to orchid pollination and orchid‐mycorrhizal symbiosis than to orchid seed dispersal (Zhang et al. <xref rid=\"evl3188-bib-0042\" ref-type=\"ref\">2018</xref>), even though orchids also exhibit diverse fruit morphology (Dirks‐Mulder et al. <xref rid=\"evl3188-bib-0011\" ref-type=\"ref\">2019</xref>).</p><p>Limited attention has been paid to the mode of orchid seed dispersal, probably owing to the dogma that wind seed dispersal is the dominant strategy (Arditti and Ghani <xref rid=\"evl3188-bib-0001\" ref-type=\"ref\">2000</xref>). Orchid seeds are very small and extremely light, and are produced in large numbers (Jersáková and Malinová <xref rid=\"evl3188-bib-0023\" ref-type=\"ref\">2007</xref>). These seeds do not possess an endosperm but instead usually have large internal air spaces that allow them to float in the air column (Arditti and Ghani <xref rid=\"evl3188-bib-0001\" ref-type=\"ref\">2000</xref>). In addition, orchid seeds are usually winged or filiform, evolved to be potentially carried with air currents (Kiyohara et al. <xref rid=\"evl3188-bib-0025\" ref-type=\"ref\">2012</xref>; Fan et al. <xref rid=\"evl3188-bib-0014\" ref-type=\"ref\">2020</xref>). Furthermore, most orchid seeds have thin papery coats formed by a single layer of nonlignified dead cells (Molvray and Chase <xref rid=\"evl3188-bib-0030\" ref-type=\"ref\">1999</xref>). It has been considered that the fragile thin seed coats cannot withstand the digestive fluids of animals (Garay <xref rid=\"evl3188-bib-0015\" ref-type=\"ref\">1964</xref>; McCormick et al. <xref rid=\"evl3188-bib-0028\" ref-type=\"ref\">2013</xref>), in contrast to the thick seed coats in indehiscent fruits, which are considered an adaptation for endozoochory (Jordano <xref rid=\"evl3188-bib-0024\" ref-type=\"ref\">1995</xref>).</p><p>Although indehiscent fruits and seeds with hard seed coats are rare in Orchidaceae, they can be found in some species of several subfamilies. Using molecular studies, orchids have been divided into five subfamilies: Apostasioideae, Vanilloideae, Cypripedioideae, Orchidoideae, and Epidendroideae (Givnish et al. <xref rid=\"evl3188-bib-0017\" ref-type=\"ref\">2015</xref>). Indehiscent fruits with seeds covered with a hard seed coat (Arditti and Ghani <xref rid=\"evl3188-bib-0001\" ref-type=\"ref\">2000</xref>; Molvray and Chase <xref rid=\"evl3188-bib-0030\" ref-type=\"ref\">1999</xref>) include those of <italic>Selenipedium</italic> (Cypripedioideae), <italic>Vanilla</italic> and <italic>Cyrtosia</italic> (Vanilloideae), and <italic>Palmorchis</italic> and <italic>Yoania</italic> (Epidendroideae). Among these orchids, <italic>C. septentrionalis</italic> and two <italic>Yoania</italic> species are dispersed by frugivorous birds and camel crickets, respectively (Suetsugu et al. <xref rid=\"evl3188-bib-0037\" ref-type=\"ref\">2015</xref>; Suetsugu <xref rid=\"evl3188-bib-0035\" ref-type=\"ref\">2018a</xref>,<xref rid=\"evl3188-bib-0036\" ref-type=\"ref\">b</xref>).</p><p>Notably, the subfamily Apostasioideae commonly has indehiscent fruits with hard, crustose black seed coats (Molvray and Chase <xref rid=\"evl3188-bib-0030\" ref-type=\"ref\">1999</xref>). Apostasioids are the earliest diverging subfamily of orchids and consist of only two genera (<italic>Apostasia</italic> and <italic>Neuwiedia</italic>), with only about 20 species distributed in southeastern Asia, Japan, and northern Australia (Chen et al. <xref rid=\"evl3188-bib-0006\" ref-type=\"ref\">2017</xref>). All <italic>Apostasia</italic> and most <italic>Neuwiedia</italic> species investigated to date are known to possess berries with hard seed coats (Kocyan and Endress <xref rid=\"evl3188-bib-0026\" ref-type=\"ref\">2001</xref>; Molvray and Chase <xref rid=\"evl3188-bib-0030\" ref-type=\"ref\">1999</xref>). Consequently, it has been suspected that the fruits are consumed by animals (Clements <xref rid=\"evl3188-bib-0009\" ref-type=\"ref\">1999</xref>). In addition, some other traits, such as inconspicuous fruits at the ground level, might be associated with endozoochory by terrestrial invertebrates, given that camel crickets disperse the seeds of several mycoheterotrophic plants with similar fruit presentation (Suetsugu <xref rid=\"evl3188-bib-0035\" ref-type=\"ref\">2018a</xref>,<xref rid=\"evl3188-bib-0036\" ref-type=\"ref\">b</xref>). In fact, orthopteran visitors (i.e., crickets and camel crickets) were observed feeding on the ripe fruits of <italic>Apostasia nipponica</italic> in my preliminary field investigation.</p><p>Apostasioids are well known for several unique traits, such as a nonresupinate flower with an actinomorphic perianth and pollen grains that do not form pollinia (Kocyan and Endress <xref rid=\"evl3188-bib-0026\" ref-type=\"ref\">2001</xref>; Zhang et al. <xref rid=\"evl3188-bib-0043\" ref-type=\"ref\">2017</xref>), although they also share some synapomorphies with other orchids, such as small seeds with a reduced embryo and mycoheterotrophic protocorms (Kristiansen et al. <xref rid=\"evl3188-bib-0027\" ref-type=\"ref\">2001</xref>). These characters have been considered ancestral in orchids, given that they are similar to those found in the members of Hypoxidaceae (which is closely related to Orchidaceae; Zhang et al. <xref rid=\"evl3188-bib-0043\" ref-type=\"ref\">2017</xref>). Similarly, the presence of an indehiscent fruit with a thick seed coat, found in most <italic>Apostasia</italic> and <italic>Neuwiedia</italic> species, may be plesiomorphic in Orchidaceae (Molvray and Chase <xref rid=\"evl3188-bib-0030\" ref-type=\"ref\">1999</xref>). Intriguingly, Orthoptera is one of the oldest insect orders, and its fossil records are available from the Late Carboniferous era (Gorochov et al. <xref rid=\"evl3188-bib-0020\" ref-type=\"ref\">2006</xref>); the origin of crickets and camel crickets predates the evolution of orchids (Givnish et al., <xref rid=\"evl3188-bib-0017\" ref-type=\"ref\">2015</xref>, <xref rid=\"evl3188-bib-0018\" ref-type=\"ref\">2016</xref>; Song et al. <xref rid=\"evl3188-bib-0034\" ref-type=\"ref\">2015</xref>). Therefore, crickets and camel crickets are arguably among the candidate seed dispersers for the ancestors of extant orchids, if they defecate viable seeds.</p><p>In the present study, I investigated the seed dispersal system of <italic>Apostasia nipponica</italic> to demonstrate a potential mutualism between crickets/camel crickets and <italic>A. nipponica</italic>. Specifically, I investigated whether (i) <italic>A. nipponica</italic> fruits are mainly consumed by crickets and camel crickets through direct observation and time‐lapse photography, (ii) the seeds defecated by crickets and camel crickets remain viable by checking their viability by 2,3,5‐triphenyl tetrazolium chloride (TTC) staining, and (iii) the fruits and seeds of <italic>A. nipponica</italic> are adapted for dispersal by animals through anatomical investigations using microtome sectioning to demonstrate endozoochory between crickets/camel crickets and <italic>A. nipponica</italic>.</p><sec id=\"evl3188-sec-0020\"><title>Material and Methods</title><sec id=\"evl3188-sec-0030\"><title>FIELD STUDY</title><p>\n<italic>Apostasia nipponica</italic> develops fleshy, succulent, indehiscent fruits that mature approximately 12 months after flowering (Fig. <xref rid=\"evl3188-fig-0001\" ref-type=\"fig\">1</xref>).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3188-fig-0001\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>(A) An <italic>Apostasia nipponica</italic> plant. (B) Cross section of an <italic>A. nipponica</italic> fruit (Scale bar = 500 μm). (C) Cross section of an <italic>A. nipponica</italic> seed (Scale bar = 50 μm). The thickened lignified tissues stained by Safranin O were indicated by arrows. (D) Cricket <italic>Eulandrevus ivani</italic> feces containing <italic>A. nipponica</italic> seeds (indicated by arrows) (Scale bar = 1 mm).</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"EVL3-4-457-g001\"/></fig><p>Field studies were carried out on Yakushima Island, Kagoshima Prefecture, Japan. The study site contained approximately 10 fruiting individuals of <italic>A. nipponica</italic>, with each plant having one to four mature fruits. The population size in the present study was slightly limited because <italic>A. nipponica</italic> is very rare throughout its distribution area and its population size is typically very small. Direct observations were made by walking around the study site and by sitting next to fruiting patches to observe the behavior of potential fruit visitors, in July 2015. The total period of direct observation was approximately 30 h, covering the time between sunset and sunrise, because a preliminary investigation indicated that fruit consumption occurred primarily at night. In addition, consumers of <italic>A. nipponica</italic> fruit were also investigated using the interval‐programming function of a waterproof digital camera (Optio WG40; Pentax, Japan) from July to August 2019. In front of each fruiting individual, a single time‐lapse camera was set to acquire photographs at 50 s intervals, because a direct observation revealed that both crickets and camel crickets typically spent several minutes feeding within a single patch. The cameras captured pictures for approximately 12 h and covered the time between sunset (19:00) and sunrise (5:30), but the observation period varied slightly depending on camera conditions. In total, 41,429 photographs were captured over 575.40 h of monitoring. Only the species that were observed consuming fruits were designated as fruit consumers in the 2015 and 2019 studies.</p></sec><sec id=\"evl3188-sec-0040\"><title>SEED VIABILITY</title><p>Three individuals of the cricket <italic>Eulandrevus ivani</italic> and three individuals of the camel cricket <italic>Diestrammena yakumontana</italic> were captured after they had consumed the fruits of <italic>A. nipponica</italic> in the field site. After capture, they were kept in separate enclosures and excrements were collected after 48 h, they were examined under a dissection microscope, and the number of intact seeds were counted. The intact seeds were subsequently tested by TTC viability staining, as previously described for dust‐like seeds (de Vega et al. <xref rid=\"evl3188-bib-0010\" ref-type=\"ref\">2011</xref>). The viability of defecated seeds was compared with that of the same number of seeds collected directly from the fruit. The differences in viability between seeds from fruits and excrements were assessed using a generalized linear model with a binomial error structure and a logit link. The statistical analysis was performed using R software version 3.6.0 (R Development Core Team <xref rid=\"evl3188-bib-0032\" ref-type=\"ref\">2019</xref>).</p></sec><sec id=\"evl3188-sec-0050\"><title>FRUIT AND SEED ANATOMY</title><p>Fruit samples were fixed in formalin‐acetic acid‐alcohol, dehydrated using an ethanol series, and then embedded in Technovit 7100 resin (Kulzer, Wertheim, Germany) for microtome sectioning. Serial resin sections were cut at a thickness of 4‐5 μm, stained with Safranin O embedded in an Entellan mounting medium (Merck, Darmstadt, Germany), and examined under an Olympus BX‐51 microscope (Olympus, Tokyo, Japan). Using this staining technique, lignified tissues and secondary cell walls were stained red (Zhong and Ye <xref rid=\"evl3188-bib-0044\" ref-type=\"ref\">2007</xref>).</p></sec></sec><sec id=\"evl3188-sec-0060\"><title>Results</title><p>Both direct observation and time‐lapse photography revealed that the fruits of <italic>Apostasia nipponica</italic> were consumed by the cricket, <italic>Eulandrevus ivani</italic>, and the camel cricket, <italic>Diestrammena yakumontana</italic> (Figs. <xref rid=\"evl3188-fig-0002\" ref-type=\"fig\">2</xref> and S1; Table <xref rid=\"evl3188-tbl-0001\" ref-type=\"table\">1</xref>). In addition, direct observations showed that the field mouse, <italic>Apodemus argenteus</italic>, completely ignored the <italic>A. nipponica</italic> fruits while in their vicinity. In some cases, the entire fruits were consumed during a single visit by a cricket or camel cricket (Fig. <xref rid=\"evl3188-fig-0002\" ref-type=\"fig\">2</xref>). Although it might be possible to detect other foragers by setting the cameras to a shorter frame interval, I consider this to be unlikely, because no additional feeding marks were left on the fruits between the frames with orthopteran visitors. The possibility of water dispersal was also excluded, because no decaying fruits were observed during the study period; rather, the crickets or camel crickets consumed almost all the mature fruits. In particular, time‐lapse photography revealed that, among the nine fruits monitored, six were entirely consumed by crickets or camel crickets. In addition, among the other three fruits, two disappeared despite not showing any signs of decay during the period when the digital camera was not set. Therefore, they were also likely consumed by animals, whereas Apostasioideae seeds have sometimes been considered to be dispersed by water when their indehiscent fruits decay (Wood <xref rid=\"evl3188-bib-0040\" ref-type=\"ref\">1999</xref>). The remaining one fruit was left intact. Therefore, seed dispersal by cricket and camel cricket is the dominant seed dispersal system of <italic>A. nipponica</italic> in the investigated population.</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3188-fig-0002\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Sequential photographs of the cricket <italic>Eulandrevus ivani</italic> consuming an <italic>Apostasia nipponica</italic> fruit (indicated by arrows). Photos were obtained using time‐lapse photography.</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"EVL3-4-457-g002\"/></fig><table-wrap id=\"evl3188-tbl-0001\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>The orthopteran species involved in seed dispersal, and total number of fruit visitations, of <italic>Apostasia nipponica</italic>\n</p></caption><table frame=\"hsides\" rules=\"groups\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><thead><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"1\" colspan=\"1\">Orthopteran species</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Times visited (2015)</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Times visited (2019)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Eulandrevus ivani</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3 (8)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Diestrammena yakumontana</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">9 (24)</td></tr></tbody></table><table-wrap-foot><fn id=\"evl3188-tbl1-note-0001\"><p>In time‐lapse photography (2019), numbers and numbers in parentheses are individuals that fed on the fruits and total numbers of frames captured orthopteran visitors, respectively. The orthopteran visitors were regarded as the same individual when captured in consecutive frames.</p></fn></table-wrap-foot><permissions><copyright-holder>John Wiley & Sons, Ltd.</copyright-holder></permissions></table-wrap><p>All crickets and camel crickets collected during fruit consumption defecated <italic>A. nipponica</italic> seeds in their excrements; each excrement of crickets and camel crickets contained 14.5 ± 9.5 and 10.1 ± 6.5 (<italic>n</italic> = 20 and <italic>n</italic> = 10, mean ± SD) seeds, respectively. Each individual defecated 96.3 ± 80.6 and 33.7 ± 13.3 seeds within 48 h. Microscopic observation revealed that the seeds recovered from the feces of camel cricket remained intact, with normal (i.e., not deformed) embryos, similar to the samples collected from intact fruits. In addition, TTC staining confirmed these observations, and no significant difference was detected in the viability of seeds defecated by crickets (21.4% ± 6.5%), those defecated by camel crickets (25.7% ± 4.1%), and those collected directly from intact fruits (22.6% ± 6.0%). Microtome sectioning confirmed that <italic>A. nipponica</italic> seeds were embedded in the fleshy pulp, which likely facilitates the ingestion of seeds by crickets or camel crickets when they feed on the fleshy pulp. In addition, Safranin O staining showed that the seeds of <italic>A. nipponica</italic> possess a thickened lignified testa that is absent in the dust‐like seeds of most orchids (Fig. <xref rid=\"evl3188-fig-0001\" ref-type=\"fig\">1</xref>). The thickened lignified tissue that coats its seeds is probably an adaptation to protect the seeds from digestion by the fruit consumers.</p></sec><sec id=\"evl3188-sec-0070\"><title>Discussion</title><p>Seed dispersal by animals is a complex mutualistic interaction involving a great diversity of plant and animal species (Howe and Smallwood <xref rid=\"evl3188-bib-0022\" ref-type=\"ref\">1982</xref>; Chen et al. <xref rid=\"evl3188-bib-0004\" ref-type=\"ref\">2018</xref>). However, the importance of seed dispersal by invertebrates, with the exception of ants, has received comparatively little attention (Bronstein et al. <xref rid=\"evl3188-bib-0002\" ref-type=\"ref\">2006</xref>; de Vega et al. <xref rid=\"evl3188-bib-0010\" ref-type=\"ref\">2011</xref>). Therefore, discoveries of uncommon mechanisms of seed dispersal by invertebrates such as wetas, beetles, cockroaches, camel crickets, and slugs usually evoke public curiosity toward animal‐plant mutualisms (Duthie et al. <xref rid=\"evl3188-bib-0012\" ref-type=\"ref\">2006</xref>; Midgley et al. <xref rid=\"evl3188-bib-0029\" ref-type=\"ref\">2015</xref>; Uehara and Sugiura <xref rid=\"evl3188-bib-0039\" ref-type=\"ref\">2017</xref>; Chen et al. <xref rid=\"evl3188-bib-0004\" ref-type=\"ref\">2018</xref>; Suetsugu <xref rid=\"evl3188-bib-0035\" ref-type=\"ref\">2018a</xref>,<xref rid=\"evl3188-bib-0036\" ref-type=\"ref\">b</xref>).</p><p>This study revealed a previously undocumented seed dispersal system in <italic>A. nipponica</italic>, in which its orthopteran visitors consume fruits and excrete viable seeds. The interaction is probably stable at least in the investigated site, because similar results were obtained in different years. The results suggest a stable interaction that constitutes a mutualism, wherein both partners benefit from the association—orthopteran visitors obtain nutrients from the pulp and <italic>A. nipponica</italic> achieves dispersal of seeds from the parent plant (de Vega et al. <xref rid=\"evl3188-bib-0010\" ref-type=\"ref\">2011</xref>). The seeds of <italic>A. nipponica</italic> are coated with lignified tissue that in all likelihood protects the seeds as they pass through the digestive tract of crickets and camel crickets. Although neither the cricket nor camel cricket can fly, they potentially transport the seeds long distances owing to their remarkable jumping abilities (Heads and Martins‐Neto <xref rid=\"evl3188-bib-0021\" ref-type=\"ref\">2007</xref>). Despite the traditional view that the minute, dust‐like, and wind‐dispersed orchid seeds can travel long distances, both genetic and experimental researches have indicated that orchids have limited dispersal ability; orchid seeds often fall close to the maternal plant (within a few meters), particularly in understory species (Chung et al. <xref rid=\"evl3188-bib-0008\" ref-type=\"ref\">2004</xref>; Trapnell et al. <xref rid=\"evl3188-bib-0038\" ref-type=\"ref\">2004</xref>; Brzosko et al. <xref rid=\"evl3188-bib-0003\" ref-type=\"ref\">2017</xref>). Given that <italic>A. nipponica</italic> fruits are produced close to the ground in dark understory environments, seed dispersal by crickets is probably a successful strategy in <italic>A. nipponica</italic>, occurring under closed canopies where the wind speed is low (Givnish et al. <xref rid=\"evl3188-bib-0016\" ref-type=\"ref\">2005</xref>).</p><p>With more than 25,700 extant species, the Orthoptera is the most diverse order among the polyneopteran insect lineages (Song et al. <xref rid=\"evl3188-bib-0034\" ref-type=\"ref\">2015</xref>). Orthopteran insects occupy every conceivable terrestrial habitat outside the Polar Regions and play integral roles in ecosystems. Because orthopterans are often considered to be detrimental to plants, their participation in mutualistic interactions, such as seed dispersal, has rarely been explored. However, crickets and camel crickets, which are abundant in subtropical and tropical forests, are commonly attracted to fruits on the forest floor (Santana et al. <xref rid=\"evl3188-bib-0033\" ref-type=\"ref\">2016</xref>; Suetsugu <xref rid=\"evl3188-bib-0035\" ref-type=\"ref\">2018a</xref>,<xref rid=\"evl3188-bib-0036\" ref-type=\"ref\">b</xref>). It has been shown that crickets consume the arils of arillate seeds and abandon the seeds in other locations, thereby acting as secondary epizoochorous seed dispersers (Santana et al. <xref rid=\"evl3188-bib-0033\" ref-type=\"ref\">2016</xref>). These recent discoveries suggest that mutualistic systems involving unexpected taxa might be more common than previously thought.</p><p>Curiously, camel crickets are also seed dispersers of several other orchids such as <italic>Yoania amagiensis</italic> and <italic>Yoania japonica</italic> (Suetsugu <xref rid=\"evl3188-bib-0035\" ref-type=\"ref\">2018a</xref>,<xref rid=\"evl3188-bib-0036\" ref-type=\"ref\">b</xref>). This is noteworthy because all orchid seeds lack endosperms; their embryos contain only marginal carbon reserves, owing to their initial mycoheterotrophy (Zhang et al. <xref rid=\"evl3188-bib-0043\" ref-type=\"ref\">2017</xref>). Orchids increase the chances of encounters with host fungi by minimizing maternal investment in individual seeds while maximizing the number of seeds (Eriksson and Kainulainen <xref rid=\"evl3188-bib-0013\" ref-type=\"ref\">2011</xref>). Endozoochory can occur whenever seeds are swallowed whole and are resilient enough to remain intact after passage through the gut of animals (de Vega et al. <xref rid=\"evl3188-bib-0010\" ref-type=\"ref\">2011</xref>; McCormick et al. <xref rid=\"evl3188-bib-0028\" ref-type=\"ref\">2013</xref>). Therefore, the small size of orchid seeds enabled by mycoheterotrophic germination may be a predisposition to the evolution of endozoochory by small animals. Whether seed dispersal by orthopterans is common in the members of Apostasioideae remains to be investigated. Nonetheless, the gross fruit morphology and pigmentation pattern of some <italic>Apostasia</italic> species, such as <italic>Apostasia shenzhenica</italic> and <italic>Apostasia fogangica</italic>, parallel those seen in <italic>A. nipponica</italic> (Chen and Liu <xref rid=\"evl3188-bib-0005\" ref-type=\"ref\">2011</xref>; Yin et al. <xref rid=\"evl3188-bib-0041\" ref-type=\"ref\">2016</xref>), suggesting that similar seed dispersal systems could be widespread among these species.</p><p>Despite being the most diverse plant family, many aspects of the evolutionary history of Orchidaceae remain obscure. In particular, because of their extremely minute size, the orchid seeds lack a definitive fossil record (Gołaszewska et al. <xref rid=\"evl3188-bib-0019\" ref-type=\"ref\">2019</xref>). Therefore, the interaction described here provides some clues regarding the animals that may have participated in the seed dispersal of ancestral clades of orchids. The family Gryllidae (crickets) represents early diverging clades within Orthoptera and is considered to have separated from other groups in the Triassic, approximately 240 million years ago, whereas the family Rhaphidophoridae (camel crickets) is thought to have originated approximately 140 million years ago, based on molecular studies (Song et al. <xref rid=\"evl3188-bib-0034\" ref-type=\"ref\">2015</xref>). In contrast, orchids appear to have diverged from the common ancestor of all other members of Asparagales approximately 112 million years ago (Givnish et al., <xref rid=\"evl3188-bib-0017\" ref-type=\"ref\">2015</xref>, <xref rid=\"evl3188-bib-0018\" ref-type=\"ref\">2016</xref>). Therefore, both crickets and camel crickets were arguably available when orchids originated, and they are among the candidates for seed dispersers of the ancestor of extant orchids.</p><p>Owing to many plesiomorphic characters and the earliest diverging phylogenetic position, members of Apostasioideae have been extensively studied to understand their floral structure, taxonomy, biogeography, and genome (Kocyan and Endress <xref rid=\"evl3188-bib-0026\" ref-type=\"ref\">2001</xref>; Chen and Liu <xref rid=\"evl3188-bib-0005\" ref-type=\"ref\">2011</xref>; Niu et al. <xref rid=\"evl3188-bib-0031\" ref-type=\"ref\">2017</xref>; Zhang et al. <xref rid=\"evl3188-bib-0043\" ref-type=\"ref\">2017</xref>). Hoverer, there is still a lack of information regarding seed dispersal in the subfamily. Here, I describe seed dispersal of Apostasioideae members by animals for the first time. Whether seed dispersal by animals (and particularly by orthopteran fruit feeders) is common in these orchids warrants further investigation. Although zoochory has also evolved secondarily from wind dispersal, at least twice within other orchid subfamilies (Suetsugu et al. <xref rid=\"evl3188-bib-0037\" ref-type=\"ref\">2015</xref>; Suetsugu <xref rid=\"evl3188-bib-0035\" ref-type=\"ref\">2018a</xref>,<xref rid=\"evl3188-bib-0036\" ref-type=\"ref\">b</xref>), it is even possible that animal‐mediated seed dispersal is an ancestral trait in Apostasioideae, given that indehiscent fruits with hard seed coat are common within the clade (Molvray and Chase <xref rid=\"evl3188-bib-0030\" ref-type=\"ref\">1999</xref>). Ancestral character‐state reconstruction analysis, with more data on the seed dispersal systems of other apostasioids, will provide deeper insights into the early evolution of the seed dispersal system in Orchidaceae.</p></sec><sec id=\"evl3188-sec-0080\"><title>AUTHOR CONTRIBUTIONS</title><p>KS conceived and designed the study, conducted field study and laboratory experiments, and wrote the manuscript.</p></sec><sec sec-type=\"data-availability\" id=\"evl3188-sec-0100\"><title>DATA ARCHIVING</title><p>The data that support the findings of this study are available from the corresponding author on request.</p></sec><sec sec-type=\"COI-statement\" id=\"evl3188-sec-0110\"><title>CONFLICT OF INTEREST</title><p>The author declares no conflict of interest.</p></sec><sec id=\"evl3188-sec-0120\"><p>\n<bold>Associate Editor: C. Moreau</bold>\n</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting information</title><supplementary-material content-type=\"local-data\"><caption><p>\n<bold>Figure S1</bold>. Sequential photographs of the camel cricket <italic>Diestrammena yakumontana</italic> consuming an <italic>Apostasia nipponica</italic> fruit (indicated by arrows).</p></caption><media xlink:href=\"EVL3-4-457-s001.jpg\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\"><caption><p>\n<bold>Table S1</bold>. Raw data of the number of intact seeds and viable seeds defecated by the cricket and camel cricket collected in its natural habitat during fruit consumption.</p><p>\n<bold>Table S2</bold>. The number of intact seeds in each excrement defecated by the cricket and camel cricket collected in its natural habitat during fruit consumption.</p></caption><media xlink:href=\"EVL3-4-457-s002.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"evl3188-sec-0090\"><title>ACKNOWLEDGMENTS</title><p>The author thanks Drs. T. Givnish, G. Lim, and Y. Imada for their constructive comments. I thank K. Tetsuka and H. Okada for help with field study and insect identification, respectively. I also thank Dr. T. Yamamoto for technical support in determining fruit and seed anatomy. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"letter\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Evol Lett</journal-id><journal-id journal-id-type=\"doi\">10.1002/(ISSN)2056-3744</journal-id><journal-id journal-id-type=\"publisher-id\">EVL3</journal-id><journal-title-group><journal-title>Evolution Letters</journal-title></journal-title-group><issn pub-type=\"epub\">2056-3744</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33014418</article-id><article-id pub-id-type=\"pmc\">PMC7523561</article-id><article-id pub-id-type=\"doi\">10.1002/evl3.193</article-id><article-id pub-id-type=\"publisher-id\">EVL3193</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Letter</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Letters</subject></subj-group></article-categories><title-group><article-title>How female × male and male × male interactions influence competitive fertilization in <italic>Drosophila melanogaster</italic>\n</article-title><alt-title alt-title-type=\"left-running-head\">S. LÜPOLD ET AL.</alt-title></title-group><contrib-group><contrib id=\"evl3193-cr-0001\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Lüpold</surname><given-names>Stefan</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-5069-1992</contrib-id><xref ref-type=\"aff\" rid=\"evl3193-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"evl3193-aff-0002\">\n<sup>2</sup>\n</xref><address><email>stefan.luepold@ieu.uzh.ch</email></address></contrib><contrib id=\"evl3193-cr-0002\" contrib-type=\"author\"><name><surname>Reil</surname><given-names>Jonathan Bradley</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-9511-960X</contrib-id><xref ref-type=\"aff\" rid=\"evl3193-aff-0003\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"evl3193-aff-0004\">\n<sup>4</sup>\n</xref></contrib><contrib id=\"evl3193-cr-0003\" contrib-type=\"author\"><name><surname>Manier</surname><given-names>Mollie K.</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-9114-8649</contrib-id><xref ref-type=\"aff\" rid=\"evl3193-aff-0002\">\n<sup>2</sup>\n</xref><xref ref-type=\"aff\" rid=\"evl3193-aff-0005\">\n<sup>5</sup>\n</xref></contrib><contrib id=\"evl3193-cr-0004\" contrib-type=\"author\"><name><surname>Zeender</surname><given-names>Valérian</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-0468-9242</contrib-id><xref ref-type=\"aff\" rid=\"evl3193-aff-0001\">\n<sup>1</sup>\n</xref></contrib><contrib id=\"evl3193-cr-0005\" contrib-type=\"author\"><name><surname>Belote</surname><given-names>John M.</given-names></name><xref ref-type=\"aff\" rid=\"evl3193-aff-0002\">\n<sup>2</sup>\n</xref></contrib><contrib id=\"evl3193-cr-0006\" contrib-type=\"author\"><name><surname>Pitnick</surname><given-names>Scott</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-8973-6166</contrib-id><xref ref-type=\"aff\" rid=\"evl3193-aff-0002\">\n<sup>2</sup>\n</xref></contrib></contrib-group><aff id=\"evl3193-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">Department of Evolutionary Biology and Environmental Studies</named-content>\n<institution>University of Zurich</institution>\n<city>Zurich</city>\n<postal-code>CH‐8057</postal-code>\n<country country=\"CH\">Switzerland</country>\n</aff><aff id=\"evl3193-aff-0002\">\n<label><sup>2</sup></label>\n<named-content content-type=\"organisation-division\">Department of Biology</named-content>\n<institution>Syracuse University</institution>\n<city>Syracuse</city>\n<named-content content-type=\"country-part\">New York</named-content>\n<postal-code>13244</postal-code>\n</aff><aff id=\"evl3193-aff-0003\">\n<label><sup>3</sup></label>\n<named-content content-type=\"organisation-division\">Department of Entomology</named-content>\n<institution>Cornell University</institution>\n<city>Ithaca</city>\n<named-content content-type=\"country-part\">New York</named-content>\n<postal-code>14853</postal-code>\n</aff><aff id=\"evl3193-aff-0004\">\n<label><sup>4</sup></label>\n<named-content content-type=\"organisation-division\">Department of Plant and Environmental Protection Sciences</named-content>\n<institution>University of Hawaii at Mānoa</institution>\n<city>Honolulu</city>\n<named-content content-type=\"country-part\">Hawaii</named-content>\n<postal-code>96822</postal-code>\n</aff><aff id=\"evl3193-aff-0005\">\n<label><sup>5</sup></label>\n<named-content content-type=\"organisation-division\">Department of Biological Sciences</named-content>\n<institution>George Washington University</institution>\n<city>Washington</city>\n<named-content content-type=\"country-part\">DC</named-content>\n<postal-code>20052</postal-code>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label>E‐mail: <email>stefan.luepold@ieu.uzh.ch</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>04</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><month>10</month><year>2020</year></pub-date><volume>4</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1002/evl3.v4.5</issue-id><fpage>416</fpage><lpage>429</lpage><history><date date-type=\"received\"><day>29</day><month>7</month><year>2019</year></date><date date-type=\"rev-recd\"><day>02</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>13</day><month>8</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 The Authors. Evolution Letters published by Wiley Periodicals LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB). <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Evolution Letters</italic> published by Wiley Periodicals, LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB).</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:EVL3-4-416.pdf\"/><abstract><title>Abstract</title><p>How males and females contribute to joint reproductive success has been a long‐standing question in sexual selection. Under postcopulatory sexual selection, paternity success is predicted to derive from complex interactions among females engaging in cryptic female choice and males engaging in sperm competition. Such interactions have been identified as potential sources of genetic variation in sexually selected traits but are also expected to inhibit trait diversification. To date, studies of interactions between females and competing males have focused almost exclusively on genotypes and not phenotypic variation in sexually selected traits. Here, we characterize within‐ and between‐sex interactions in <italic>Drosophila melanogaster</italic> using isogenic lines with heritable variation in both male and female traits known to influence competitive fertilization. We confirmed, and expanded on, previously reported genotypic interactions within and between the sexes, and showed that several reproductive events, including sperm transfer, female sperm ejection, and sperm storage, were explained by two‐ and three‐way interactions among sex‐specific phenotypes. We also documented complex interactions between the lengths of competing males’ sperm and the female seminal receptacle, which are known to have experienced rapid female‐male co‐diversification. Our results highlight the nonindependence of sperm competition and cryptic female choice and demonstrate that complex interactions between the sexes do not limit the ability of multivariate systems to respond to directional sexual selection.</p></abstract><kwd-group kwd-group-type=\"author-generated\"><kwd id=\"evl3193-kwd-0001\">Cryptic female choice</kwd><kwd id=\"evl3193-kwd-0002\">ejaculate‐female interactions</kwd><kwd id=\"evl3193-kwd-0003\">female reproductive tract</kwd><kwd id=\"evl3193-kwd-0004\">genetic compatibility</kwd><kwd id=\"evl3193-kwd-0005\">postcopulatory sexual selection</kwd><kwd id=\"evl3193-kwd-0006\">sperm competition</kwd><kwd id=\"evl3193-kwd-0007\">trait diversification</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source>U.S. National Science Foundation</funding-source><award-id>DEB‐1145965</award-id><award-id>DEB‐1655840</award-id></award-group><award-group id=\"funding-0002\"><funding-source><institution-wrap><institution>Swiss National Science Foundation </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/501100001711</institution-id></institution-wrap></funding-source><award-id>PA00P3_134191</award-id><award-id>PP00P3_170669</award-id></award-group><award-group id=\"funding-0003\"><funding-source><institution-wrap><institution>Directorate for Biological Sciences </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/100000076</institution-id></institution-wrap></funding-source><award-id>DEB‐1145965</award-id><award-id>DEB‐1655840</award-id></award-group></funding-group><counts><fig-count count=\"5\"/><table-count count=\"1\"/><page-count count=\"14\"/><word-count count=\"11046\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>October 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.9.1 mode:remove_FC converted:29.09.2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><boxed-text position=\"float\" content-type=\"box\" orientation=\"portrait\"><caption><title>Impact Summary</title></caption><p>For species with internal fertilization and female promiscuity, postcopulatory sexual selection (PSS) is believed to depend, in part, on complex interactions between rival males and between the sexes. Although little investigated, clarifying such interactions is critical as they may limit the efficacy of PSS in the diversification of reproductive traits (e.g., ejaculate biochemistry and sperm, genitalia and female reproductive tract morphology). Here, we resolve how sex‐specific traits and their interactions contribute to key reproductive events and outcomes related to competitive fertilization success, including traits known to have experienced rapid diversification. Our results provide novel insights into the operation and complexity of PSS and demonstrate that the processes of sperm competition and cryptic female choice are not independent selective forces. Simultaneously, the complex interactions between the sexes do not necessarily limit rapid trait diversification in multivariate systems.</p></boxed-text><sec id=\"evl3193-sec-0020\"><p>Because females of most species mate with multiple males within reproductive cycles (Arnqvist and Rowe <xref rid=\"evl3193-bib-0006\" ref-type=\"ref\">2005</xref>; Taylor et al. <xref rid=\"evl3193-bib-0102\" ref-type=\"ref\">2014</xref>), sexual selection, encompassing both male‐male competition and female choice, can continue after mating in the form of sperm competition and cryptic female choice, respectively (Parker <xref rid=\"evl3193-bib-0075\" ref-type=\"ref\">1970a</xref>; Eberhard <xref rid=\"evl3193-bib-0030\" ref-type=\"ref\">1996</xref>). As for premating sexual selection, a longstanding goal of studies of postcopulatory sexual selection (PSS) has been to characterize genetic variation in male traits and female preferences that are believed to be the targets of sexual selection, and to identify how such variation relates to differential reproductive success. Explicit demonstration of the means by which phenotypes affect fitness is a prerequisite for resolving the selective processes (Endler <xref rid=\"evl3193-bib-0031\" ref-type=\"ref\">1986</xref>; Sober <xref rid=\"evl3193-bib-0100\" ref-type=\"ref\">1993</xref>). However, compared to premating sexual selection (Andersson <xref rid=\"evl3193-bib-0004\" ref-type=\"ref\">1994</xref>; Jennions et al. <xref rid=\"evl3193-bib-0045\" ref-type=\"ref\">2001</xref>; Kokko and Jennions <xref rid=\"evl3193-bib-0048\" ref-type=\"ref\">2003</xref>), our causal, mechanistic understanding of how variation in postcopulatory sexual traits translates into fitness, and so how PSS contributes to trait diversification, is relatively scant (Howard et al. <xref rid=\"evl3193-bib-0044\" ref-type=\"ref\">2009</xref>; Lüpold and Pitnick <xref rid=\"evl3193-bib-0054\" ref-type=\"ref\">2018</xref>).</p><p>Our understanding of PSS is limited partly due to challenges of observing its processes within the female reproductive tract (FRT) and of discriminating between competing sperm (Manier et al. <xref rid=\"evl3193-bib-0063\" ref-type=\"ref\">2010</xref>, <xref rid=\"evl3193-bib-0064\" ref-type=\"ref\">2013a</xref>,<xref rid=\"evl3193-bib-0065\" ref-type=\"ref\">b</xref>). However, two further, interrelated aspects of PSS cloud our understanding of its role in both maintaining genetic variation and driving evolutionary diversification. First, sex‐specific mediators of competitive fertilization success tend to be multivariate, potentially including a multitude of genitalic, seminal fluid, sperm, and FRT morphological, physiological, neurological, and/or biochemical traits, any of which may influence sperm transfer, storage, maturation, motility, longevity, and contribution to fertilization (Snook <xref rid=\"evl3193-bib-0098\" ref-type=\"ref\">2005</xref>; Poiani <xref rid=\"evl3193-bib-0087\" ref-type=\"ref\">2006</xref>; Pitnick et al. <xref rid=\"evl3193-bib-0083\" ref-type=\"ref\">2009a</xref>,<xref rid=\"evl3193-bib-0084\" ref-type=\"ref\">b</xref>; Carmel et al. <xref rid=\"evl3193-bib-0015\" ref-type=\"ref\">2016</xref>). Second, because sperm competition takes place within the FRT, the competitiveness of ejaculates is likely to depend in large measure on their interactions with the female (Ravi Ram and Wolfner <xref rid=\"evl3193-bib-0088\" ref-type=\"ref\">2007</xref>; Pitnick et al. <xref rid=\"evl3193-bib-0084\" ref-type=\"ref\">2009b</xref>, <xref rid=\"evl3193-bib-0085\" ref-type=\"ref\">2020</xref>; Sirot and Wolfner <xref rid=\"evl3193-bib-0097\" ref-type=\"ref\">2015</xref>). Any variation in the FRT environment may change the conditions under which sperm compete, and therefore, shift the relative competitive advantage between males (Eberhard <xref rid=\"evl3193-bib-0030\" ref-type=\"ref\">1996</xref>; Firman et al. <xref rid=\"evl3193-bib-0034\" ref-type=\"ref\">2017</xref>; Lüpold and Pitnick <xref rid=\"evl3193-bib-0054\" ref-type=\"ref\">2018</xref>). That such female × male interactions can influence patterns of sperm precedence has been demonstrated in diverse species with both internal (Lewis and Austad <xref rid=\"evl3193-bib-0051\" ref-type=\"ref\">1990</xref>; Wilson et al. <xref rid=\"evl3193-bib-0108\" ref-type=\"ref\">1997</xref>; Clark et al. <xref rid=\"evl3193-bib-0024\" ref-type=\"ref\">1999</xref>; Miller and Pitnick <xref rid=\"evl3193-bib-0070\" ref-type=\"ref\">2002</xref>; Nilsson et al. <xref rid=\"evl3193-bib-0073\" ref-type=\"ref\">2003</xref>; Birkhead et al. <xref rid=\"evl3193-bib-0010\" ref-type=\"ref\">2004</xref>; Chow et al. <xref rid=\"evl3193-bib-0018\" ref-type=\"ref\">2010</xref>; Delbare et al. <xref rid=\"evl3193-bib-0028\" ref-type=\"ref\">2017</xref>) and external fertilization (Turner and Montgomerie <xref rid=\"evl3193-bib-0104\" ref-type=\"ref\">2002</xref>; Evans and Marshall <xref rid=\"evl3193-bib-0032\" ref-type=\"ref\">2005</xref>; Rosengrave et al. <xref rid=\"evl3193-bib-0091\" ref-type=\"ref\">2008</xref>; Simmons et al. <xref rid=\"evl3193-bib-0096\" ref-type=\"ref\">2009</xref>; Alonzo et al. <xref rid=\"evl3193-bib-0003\" ref-type=\"ref\">2016</xref>). Further evidence for such interactions comes from studies of conspecific sperm precedence (Howard et al. <xref rid=\"evl3193-bib-0044\" ref-type=\"ref\">2009</xref>; Manier et al. <xref rid=\"evl3193-bib-0064\" ref-type=\"ref\">2013a</xref>,<xref rid=\"evl3193-bib-0065\" ref-type=\"ref\">b</xref>,<xref rid=\"evl3193-bib-0066\" ref-type=\"ref\">c</xref>). In fact, mounting evidence suggests that competitive fertilization events may rarely be independent of female effects (Eberhard <xref rid=\"evl3193-bib-0030\" ref-type=\"ref\">1996</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0057\" ref-type=\"ref\">2016</xref>).</p><p>Considering these interactions among multivariate traits and between sexes, PSS is expected to favor the maintenance of genetic variation in reproductive characters and to inhibit strong directional selection on specific traits for three reasons. First, having numerous traits contributing to a fitness outcome can dilute the strength of selection on any single trait. Second, directional sexual selection on males should be limited if their mating or competitive fertilization success is influenced by their compatibility with females rather than their intrinsic quality (Birkhead <xref rid=\"evl3193-bib-0009\" ref-type=\"ref\">1998</xref>; Pitnick and Brown <xref rid=\"evl3193-bib-0081\" ref-type=\"ref\">2000</xref>; Tregenza and Wedell <xref rid=\"evl3193-bib-0103\" ref-type=\"ref\">2000</xref>; Neff and Pitcher <xref rid=\"evl3193-bib-0072\" ref-type=\"ref\">2005</xref>; Oh and Badyaev <xref rid=\"evl3193-bib-0074\" ref-type=\"ref\">2006</xref>). Third, many interacting traits provide the requisite conditions for nontransitive competitive outcomes in the manner of a rock‐paper‐scissors game (Maynard Smith <xref rid=\"evl3193-bib-0067\" ref-type=\"ref\">1982</xref>), which further limits the strength of directional selection (Clark <xref rid=\"evl3193-bib-0021\" ref-type=\"ref\">2002</xref>). Nontransitivity in competitive fertilization success has been experimentally demonstrated for <italic>Drosophila melanogaster</italic> by using fixed‐chromosome lines (Clark et al. <xref rid=\"evl3193-bib-0024\" ref-type=\"ref\">1999</xref>, <xref rid=\"evl3193-bib-0025\" ref-type=\"ref\">2000</xref>; Zhang et al. <xref rid=\"evl3193-bib-0112\" ref-type=\"ref\">2013</xref>; Reinhart et al. <xref rid=\"evl3193-bib-0089\" ref-type=\"ref\">2015</xref>), and for domestic fowl (<italic>Gallus gallus domesticus</italic>) by using artificial insemination (Birkhead et al. <xref rid=\"evl3193-bib-0010\" ref-type=\"ref\">2004</xref>).</p><p>Genotypic interactions between the sexes could be pervasive or even ubiquitous. If true, then we predict that even where competitive fertilization success is determined purely by raffle‐based sperm competition, it would function as a loaded raffle (Parker <xref rid=\"evl3193-bib-0077\" ref-type=\"ref\">1990</xref>) due to differential compatibility between each male's sperm and the FRT (e.g., Pitnick et al. <xref rid=\"evl3193-bib-0085\" ref-type=\"ref\">2020</xref>). This assumption is the basis for the contention that, relative to premating sexual selection, PSS intrasexual competition and intersexual choice are a false dichotomy and fall more on a continuum (e.g., Eberhard <xref rid=\"evl3193-bib-0030\" ref-type=\"ref\">1996</xref>; Arnqvist <xref rid=\"evl3193-bib-0005\" ref-type=\"ref\">2014</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0057\" ref-type=\"ref\">2016</xref>; Lüpold and Pitnick <xref rid=\"evl3193-bib-0054\" ref-type=\"ref\">2018</xref>). The position of the two selective processes along this continuum is likely to be determined by the relative contributions of sex‐specific effects to three‐way interactions between physically or biochemically interacting traits of females and competing males. However, without a detailed understanding of the underlying processes it is near impossible to determine whether variation in reproductive outcomes between females and competing males is primarily attributable to, for example, genotype‐ or condition‐dependent postcopulatory female biases toward certain sperm phenotypes or differential performance of competing sperm within these different selective environments. Hence, unlike male‐male contest competition, for example, where males may compete over access to mating opportunities even before females arrive, male‐male competition at the postcopulatory stage would rarely escape direct female involvement, particularly in internal fertilizers. Due to the predicted interactions between females and males, we also see great potential for the same, or tightly linked, ejaculate traits to be favored by both inter‐ and intrasexual processes of selection as sperm would be most competitive in a selective environment that is more favorable to them.</p><p>In an alternative scenario, ejaculate‐female interactions could be more limited in scope, for example, by being restricted to processes of cryptic female choice. If so, we would predict such limitation to contribute to the discreteness of processes underlying sperm competition and cryptic female choice, respectively, and that traits of sperm competition should have a greater potential to respond to directional sexual selection than those of cryptic female choice. If these selective processes are largely independent rather than intertwined as described above, there should, at least in principle, also be greater scope for intra‐ and intersexual selection to target separate ejaculate traits, even though ejaculate traits may rarely evolve independently (Gómez Montoto et al. <xref rid=\"evl3193-bib-0040\" ref-type=\"ref\">2011</xref>; Fitzpatrick et al. <xref rid=\"evl3193-bib-0035\" ref-type=\"ref\">2012</xref>; Lüpold <xref rid=\"evl3193-bib-0053\" ref-type=\"ref\">2013</xref>; Lymbery et al. <xref rid=\"evl3193-bib-0059\" ref-type=\"ref\">2018</xref>; Liao et al. <xref rid=\"evl3193-bib-0052\" ref-type=\"ref\">2019</xref>).</p><p>To date, it has been impossible to empirically disentangle predictions about the relative importance of sperm competition and cryptic female choice, or about the extent to which they are discrete selective processes. The main impediments in such exploration are that all studies demonstrating female × male interactions in the pattern of sperm precedence or competitive fertilization success have done so without investigating any specific traits (i.e., by using genetically discrete lines or geographic populations), or else have examined only a single pair of interacting, sex‐specific traits, such as sperm length and female seminal receptacle (SR) length (Miller and Pitnick <xref rid=\"evl3193-bib-0070\" ref-type=\"ref\">2002</xref>) or genetic variation in male sex peptide and its female receptor (Zhang et al. <xref rid=\"evl3193-bib-0112\" ref-type=\"ref\">2013</xref>). Knowledge of the sex‐specific traits contributing to PSS processes and of the degree to which interactions between competing males and the females, respectively, mediate those processes is too limited for any system to make clear predictions about their influence on diversification and the maintenance of variation.</p><p>To advance our understanding of PSS, we set the goals of (1) identifying many of the putative sex‐specific targets of PSS (e.g., copulation duration; the number, length, and in vivo swimming velocity of sperm; female remating interval; fecundity; sperm‐storage organ morphometry; and sperm storage, ejection, and use), (2) quantifying their genetic variation, and (3) determining their contribution to variation in competitive fertilization success within a multivariate framework. To accomplish these goals, we embarked on a three‐stage research program using isogenic populations of <italic>D. melanogaster</italic> with sperm that express either green (GFP) or red (RFP) fluorescent protein in their sperm heads (Manier et al. <xref rid=\"evl3193-bib-0063\" ref-type=\"ref\">2010</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>, <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>; Ala‐Honkola et al. <xref rid=\"evl3193-bib-0002\" ref-type=\"ref\">2013</xref>). The fluorescent tags allow direct visualization of living sperm within the FRT while discriminating between sperm from competing males within twice‐mated females, as well as tracking the spatiotemporal fate of both males’ sperm throughout remating and progeny production by females. In the first stage of this program, we held the female genetic background (i.e., isoline) constant and competed males from different isolines to resolve male‐mediated contributions to competitive fertilization success (Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>). We demonstrated that longer and slower sperm are better at displacing sperm of a previous male from the female SR (i.e., primary sperm‐storage organ), or resisting such displacement by incoming sperm of a subsequent mate (Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>). In the second stage, we resolved female‐mediated contributions by holding the genetic background of all males constant while competing their ejaculates within females from different isolines (Lüpold et al. <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>). This study revealed how females can strongly bias sperm storage between males by varying the time between remating and ejecting a mass containing excess second‐male and displaced first‐male sperm from their bursa copulatrix before initiating oviposition (Lüpold et al. <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>). Because in <italic>D. melanogaster</italic> paternity is shared among males in proportion to their sperm representation within the SR (Civetta <xref rid=\"evl3193-bib-0019\" ref-type=\"ref\">1999</xref>; Manier et al. <xref rid=\"evl3193-bib-0063\" ref-type=\"ref\">2010</xref>, <xref rid=\"evl3193-bib-0066\" ref-type=\"ref\">2013c</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>, <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>), female sperm ejection is a key element of cryptic female choice in this species (Snook and Hosken <xref rid=\"evl3193-bib-0099\" ref-type=\"ref\">2004</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>). Here, in the third and final installment, we report on experiments in which the genetic backgrounds of both competing males and females were systematically varied to identify genotypic effects and interactions while resolving the contribution of multivariate traits to female × male, male × male, and female × male × male interactions to variation in competitive fertilization success. Specifically, after competing different male genotypes in different female genetic backgrounds, we examined how the interactions between male (e.g., sperm length and number) and female attributes (e.g., remating interval or SR length) influence reproductive events known to affect competitive fertilization (e.g., timing of female postmating sperm ejection or sperm storage).</p></sec><sec id=\"evl3193-sec-0030\"><title>Materials and Methods</title><sec id=\"evl3193-sec-0040\"><title>EXPERIMENTAL MATERIAL</title><p>We performed all experiments with LH<sub>m</sub> populations of <italic>D. melanogaster</italic> that express a protamine labeled with either GFP or RFP in sperm heads (Manier et al. <xref rid=\"evl3193-bib-0063\" ref-type=\"ref\">2010</xref>), which permit discriminating sperm from different males and quantifying sperm within the FRT. Using random individuals from large population cages (all backcrossed to the LH<sub>m</sub> wild type for six generations; Manier et al. <xref rid=\"evl3193-bib-0063\" ref-type=\"ref\">2010</xref>), we generated isogenic lines (“isolines”; Parsons and Hosgood <xref rid=\"evl3193-bib-0078\" ref-type=\"ref\">1968</xref>; David et al. <xref rid=\"evl3193-bib-0026\" ref-type=\"ref\">2005</xref>) by 15 generations of full‐sibling inbreeding, thus yielding theoretical homozygosity levels of 96% (Falconer <xref rid=\"evl3193-bib-0033\" ref-type=\"ref\">1989</xref>). To avoid inbreeding effects, we crossed independent pairs of isogenic lines (i.e., males of one isoline with virgin females of another) to create repeatable heterozygous F<sub>1</sub> genotypes for the experiments described here. Male and female reproductive traits were previously characterized for these crosses and shown to be heritable, and replicated mating trials within given genotype combinations generate repeatable results (Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>, <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>, <xref rid=\"evl3193-bib-0049\" ref-type=\"ref\">2016</xref>). Based on these assays, we selected parental isolines that captured most of the variance in both male and female reproductive traits among genotypes when creating heterozygotes. In total, our experimental population consisted of six female RFP genotypes, an independent set of six first‐male RFP genotypes, and three second‐male GFP genotypes. We reared all flies at low densities in replicate vials with standard cornmeal‐molasses‐agar medium supplemented with yeast, collected them as virgins upon eclosion, and aged them for 3 (males) or 4 days (females) before their first mating. All males were mated once to a nonexperimental female on the day before their first experimental mating to avoid sexual naiveté (Bjork et al. <xref rid=\"evl3193-bib-0011\" ref-type=\"ref\">2007</xref>).</p></sec><sec id=\"evl3193-sec-0050\"><title>SPERM COMPETITION EXPERIMENT</title><p>We have repeatedly shown that paternity (i.e., P<sub>2</sub>) in <italic>D. melanogaster</italic> (including in these isogenic lines) is directly proportional to the respective numbers of sperm from two competing males remaining in storage (S<sub>2</sub>), particularly within the SR, after females have ejected any excess second‐male and displaced first‐male sperm, thus following a fair raffle among stored sperm (Manier et al. <xref rid=\"evl3193-bib-0063\" ref-type=\"ref\">2010</xref>, <xref rid=\"evl3193-bib-0066\" ref-type=\"ref\">2013c</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>, <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>). Therefore, we focused our efforts on investigating how female × male, male × male, and female × male × male interactions influence the process of sperm displacement until it is interrupted by female sperm ejection (Manier et al. <xref rid=\"evl3193-bib-0063\" ref-type=\"ref\">2010</xref>, <xref rid=\"evl3193-bib-0065\" ref-type=\"ref\">2013b</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>) and used S<sub>2</sub> as a proxy of P<sub>2</sub>. Throughout the text, we refer to S<sub>2</sub> within the SR as the “fertilization set” (Parker <xref rid=\"evl3193-bib-0077\" ref-type=\"ref\">1990</xref>).</p><p>Within each of eight replicates, examined in four temporal blocks of two full replicate sets (staggered by 2 days), we mated virgin females each to an RFP male and, 2 days later, to a GFP male in all 108 possible combinations between genotypes (total <italic>N</italic> = 864 trios tested). Females not remating within 4 hours were given additional 4‐hour remating opportunities on days 3–5 after the first mating. Immediately after the end of the second mating, we removed the male from the mating vial, isolated the female in a glass three‐well spot plate beneath a glass coverslip, and checked for sperm ejection every 10 min for up to 5 hours using a stereomicroscope. We recorded the time to sperm ejection, immediately removed the female from the well and froze it for later quantification of stored sperm, and transferred the ejected mass to phosphate‐buffered saline (PBS) on a microscope slide and sealed the coverslip with rubber cement.</p><p>For all dissected females, we counted the sperm of both competitors across the different organs of the FRT (bursa copulatrix, SR, and paired spermathecae with ducts) and determined the total number of sperm for each male in all female sperm‐storage organs combined, and the proportion of total sperm derived from the second male (S<sub>2</sub>) across all storage organs and within the SR, respectively. Combining these counts with those of the ejected masses further permitted calculating the number of first‐male resident sperm at the time of remating, the number of second‐male sperm transferred, and both the absolute and relative number of each male's sperm stored and ejected, respectively.</p><p>Finally, for each of the nine male genotypes, we dissected six males after measuring their thorax length, retrieved sperm from their seminal vesicles using a fine probe, fixed the sample on a microscope slide with a mixture of methanol and acetic acid (3:1 [v/v]), rinsed it with PBS, and mounted it under a coverslip in glycerol and PBS (80:20 [v/v]). We measured the length of five sperm per male using the segmented line tool in ImageJ version 1.47 at 200× magnification under the dark‐field optics of an Olympus BX‐60 microscope. For each of the six female genotypes, we measured the thorax length of each of eight females, and dissected their reproductive tract into PBS on a microscope slide and covered it with a glass coverslip with clay at the corners, allowing the SR to be flattened to two dimensions without stretching. We then measured SR length using ImageJ at 200× magnification under an Olympus BX‐60 microscope with Nomarski DIC optics. Both sperm and SR length are significantly heritable (Miller and Pitnick <xref rid=\"evl3193-bib-0070\" ref-type=\"ref\">2002</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>, <xref rid=\"evl3193-bib-0053\" ref-type=\"ref\">2013</xref>, <xref rid=\"evl3193-bib-0057\" ref-type=\"ref\">2016</xref>).</p></sec><sec id=\"evl3193-sec-0060\"><title>STATISTICAL ANALYSES</title><p>We performed all analyses using the statistical software package R version 3.4.3 (R Core Team <xref rid=\"evl3193-bib-0113\" ref-type=\"ref\">2017</xref>). We conducted analyses both at the genotypic and trait levels. We used the genotypic analyses primarily for comparison with previous studies on female × male interactions explaining sperm precedence patterns, which had all been done at this level (see Introduction). Based on previous studies of <italic>D. melanogaster</italic> that have documented genotypic nontransitivity in sperm precedence (Clark et al. <xref rid=\"evl3193-bib-0024\" ref-type=\"ref\">1999</xref>, <xref rid=\"evl3193-bib-0025\" ref-type=\"ref\">2000</xref>; Zhang et al. <xref rid=\"evl3193-bib-0112\" ref-type=\"ref\">2013</xref>), we explicitly predicted a female × male × male interaction in the fertilization set, as well as in reproductive patterns and events leading to this final outcome. The only exceptions were the number of first‐male sperm still residing in the FRT at remating and the progeny produced up to that point, for which we had no a priori expectations of second‐male effects beyond the timing of remating and therefore omitted all second‐male effects in the model. For each focal variable, we conducted either a linear mixed‐effects model (LMM) with the temporal blocking as a four‐level random effect or, for the proportional data of S<sub>2</sub>, a generalized LMM (GLMM) with a binomial error distribution, a logit link, and an additional observation‐level random effect to account for overdispersion. Each model included the female and first‐male and second‐male genotypes with all two‐ and three‐way interactions as fixed effects, except where only females and first males and their interaction were predicted a priori and so any second‐male contributions were excluded. We used conditional <italic>F</italic> tests (LMM) or Wald <italic>χ</italic>\n<sup>2</sup> tests (GLMM) to test for significant main and interactive genotypic effects.</p><p>In a second set of analyses (henceforth “traits analyses”), we examined the interrelationships between the male and female reproductive traits themselves. Due to their complexity and a lack of specific information on how precisely traits should interact to explain focal traits, these analyses were necessarily somewhat exploratory. Thus, instead of null‐hypothesis significance testing based on a priori predictions, we used an information‐theoretic approach to account for model uncertainty and identify the most plausible model(s) (Burnham and Anderson <xref rid=\"evl3193-bib-0014\" ref-type=\"ref\">2002</xref>; Grueber et al. <xref rid=\"evl3193-bib-0041\" ref-type=\"ref\">2011</xref>; Richards et al. <xref rid=\"evl3193-bib-0090\" ref-type=\"ref\">2011</xref>; Symonds and Moussalli <xref rid=\"evl3193-bib-0101\" ref-type=\"ref\">2011</xref>). These analyses were again based on (G)LMMs, accounting for genetic nonindependence by including each represented genotype and the female × male × male genotypic combination as random effects (and an observation‐level random effect in GLMMs). For each analysis, we generated a model set with all combinations of predictors and interactions (up to a maximum of three‐way interactions for interpretability) from a global model using the <italic>dredge</italic> function implemented in the <italic>MuMIn</italic> package (Bartón <xref rid=\"evl3193-bib-0008\" ref-type=\"ref\">2017</xref>). We ranked these models by their Akaike information criterion with sample size adjustment (AIC<sub>c</sub>; Burnham and Anderson <xref rid=\"evl3193-bib-0014\" ref-type=\"ref\">2002</xref>; Grueber et al. <xref rid=\"evl3193-bib-0041\" ref-type=\"ref\">2011</xref>; Symonds and Moussalli <xref rid=\"evl3193-bib-0101\" ref-type=\"ref\">2011</xref>) and limited our confidence model set to candidates within ΔAIC<sub>c</sub> ≤ 6 of the best model (Bolker et al. <xref rid=\"evl3193-bib-0012\" ref-type=\"ref\">2009</xref>; Richards et al. <xref rid=\"evl3193-bib-0090\" ref-type=\"ref\">2011</xref>; Symonds and Moussalli <xref rid=\"evl3193-bib-0101\" ref-type=\"ref\">2011</xref>), which largely corresponded to cumulative Akaike weights ≥ 0.95. To reduce the retention of overly complex models, we excluded, using the <italic>nested</italic> function in the <italic>MuMIn</italic> package, those models that simply represented more complex versions (e.g., one additional parameter) of any model with a lower AIC<sub>c</sub> value (Burnham and Anderson <xref rid=\"evl3193-bib-0014\" ref-type=\"ref\">2002</xref>; Richards et al. <xref rid=\"evl3193-bib-0090\" ref-type=\"ref\">2011</xref>).</p><p>Although the primary goal was to determine which explanatory variables and interactions were represented in the confidence model set and thus likely to contribute to the variation in the focal trait, we additionally calculated the natural (conditional) averages and 95% confidence intervals of their coefficients as well as their relative variable importance across the confidence model set (Burnham and Anderson <xref rid=\"evl3193-bib-0014\" ref-type=\"ref\">2002</xref>; Grueber et al. <xref rid=\"evl3193-bib-0041\" ref-type=\"ref\">2011</xref>). We standardized all predictors (mean = 0; SD = 0.5) to infer standardized effect sizes (Gelman <xref rid=\"evl3193-bib-0039\" ref-type=\"ref\">2008</xref>; Grueber et al. <xref rid=\"evl3193-bib-0041\" ref-type=\"ref\">2011</xref>).</p><p>Finally, we used piecewise structural equation modeling (or confirmatory path analyses; Shipley <xref rid=\"evl3193-bib-0093\" ref-type=\"ref\">2009</xref>) in the R package <italic>piecewiseSEM</italic> (Lefcheck <xref rid=\"evl3193-bib-0049\" ref-type=\"ref\">2016</xref>) to visualize how the numerous male and female traits directly or indirectly influence the fertilization set (S<sub>2</sub> in SR) as a proxy of fitness outcomes (Manier et al. <xref rid=\"evl3193-bib-0063\" ref-type=\"ref\">2010</xref>, <xref rid=\"evl3193-bib-0066\" ref-type=\"ref\">2013c</xref>). This approach decomposes a network of relationships into the simple or multiple linear regressions for each response and allows for combinations of different model structures such as LMM and GLMM. Each regression is assessed separately before being combined to evaluate the entire structural equation model (Lefcheck <xref rid=\"evl3193-bib-0049\" ref-type=\"ref\">2016</xref>).</p></sec></sec><sec id=\"evl3193-sec-0070\"><title>Results</title><sec id=\"evl3193-sec-0080\"><title>GENOTYPIC ANALYSES</title><p>We examined how the different genotypes (six females, six first males, and three second‐males), and particularly any two‐ or three‐way interactions between them, contributed to variation in reproductive parameters (e.g., sperm transferred or stored, timing of female sperm ejection), using (G)LMMs with the four temporally separated blocks as a random factor. We omitted in all analyses females that did not complete the experiment (death or loss), had no recorded remating by day 5, or post hoc showed no evidence of successful sperm transfer during the first mating (e.g., no progeny produced between matings and no first‐male sperm found at remating). These exclusions reduced our sample size from 864 to 744 successful mating trios, but additional missing data (e.g., no ejected mass found) resulted in varying sample sizes between analyses.</p><p>We first tested for the contribution of three‐way genotypic interactions to total S<sub>2</sub> (i.e., the proportional representation of the second‐male's sperm among all sperm retained by the female in both spermathecae and the SR after sperm ejection) and to the fertilization set (i.e., proportional representation in the SR only) (Manier et al. <xref rid=\"evl3193-bib-0063\" ref-type=\"ref\">2010</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>, <xref rid=\"evl3193-bib-0053\" ref-type=\"ref\">2013</xref>). In a GLMM with a binomial error distribution and an additional observation‐level random factor to remove overdispersion, variation in total S<sub>2</sub> was explained by a female × first‐male interaction (<italic>N</italic> = 577; <italic>χ</italic>\n<sup>2</sup>\n<sub>25</sub> = 39.72, <italic>P</italic> = 0.031) and a weak trend for a three‐way interaction (<italic>χ</italic>\n<sup>2</sup>\n<sub>25</sub> = 63.50, <italic>P</italic> = 0.095; Table S1). The fertilization set was influenced by a three‐way interaction (<italic>N</italic> = 589; <italic>χ</italic>\n<sup>2</sup>\n<sub>50</sub> = 69.63, <italic>P</italic> = 0.035), a male × male interaction (<italic>χ</italic>\n<sup>2</sup>\n<sub>10</sub> = 21.65, <italic>P</italic> = 0.017), and a first‐male main effect (<italic>χ</italic>\n<sup>2</sup>\n<sub>5</sub> = 16.17, <italic>P</italic> = 0.006; Table S2; Fig. <xref rid=\"evl3193-fig-0001\" ref-type=\"fig\">1</xref>).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3193-fig-0001\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Interactions between female, first‐male and second‐male genotypes explaining variation in the fertilization set. Each dot represents the cross‐specific mean.</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"EVL3-4-416-g001\"/></fig><p>Next, in LMMs, we focused on two‐ and three‐way interactions between members of the mating trio on specific reproductive traits predicted to contribute to S<sub>2</sub>. We found no interactive effects on traits leading up to the females’ remating (all <italic>P</italic> > 0.16; Tables S3–S5). Rather, the female remating interval, ranging between 2 and 5 days, was explained solely by the female genotype (<italic>F</italic>\n<sub>5,633.35</sub> = 6.20, <italic>P</italic> < 0.0001; Table S3). Further, controlling for the remating interval and omitting the second‐male genotypes due to no a priori expectation of their contribution, the number of progeny produced between the first and second mating was influenced primarily by the female genotype (<italic>F</italic>\n<sub>5,687.32</sub> = 20.54, <italic>P</italic> < 0.0001), with a weak contribution of the first‐male genotype (<italic>F</italic>\n<sub>5,687.20</sub> = 2.11, <italic>P</italic> = 0.063; Table S4). Similarly, accounting for the number of progeny produced as a proxy of sperm use, the number of first‐male sperm still residing in the FRT at remating was also explained by both the female (<italic>F</italic>\n<sub>5,509.31</sub> = 10.60, <italic>P</italic> < 0.0001) and first‐male main effects (<italic>F</italic>\n<sub>5,509.32</sub> = 2.31, <italic>P</italic> = 0.043; Table S5).</p><p>All later reproductive stages exhibited at least some interactive effects, albeit no statistically significant three‐way interaction. For example, the duration of the second copulation was explained primarily by the second‐male's genotype (<italic>F</italic>\n<sub>5,630.50</sub> = 55.05, <italic>P</italic> < 0.0001), but also by a female × first‐male interaction (<italic>F</italic>\n<sub>25,630.61</sub> = 1.70, <italic>P</italic> = 0.019; Table S6). The number of sperm transferred during these copulations was explained by a trend for a three‐way interaction between all individuals of a mating trio (<italic>F</italic>\n<sub>50,446.07</sub> = 1.35, <italic>P</italic> = 0.062) in addition to a strong second‐male main effect (<italic>F</italic>\n<sub>5,446.04</sub> = 8.27, <italic>P</italic> = 0.0003; Table S7). Finally, the time to female sperm ejection after remating was determined by the female (<italic>F</italic>\n<sub>5,537.40</sub> = 20.99, <italic>P</italic> < 0.0001) and second‐male genotypes (<italic>F</italic>\n<sub>2,537.41</sub> = 3.20, <italic>P</italic> = 0.041) and their interaction (<italic>F</italic>\n<sub>5,537.49</sub> = 2.20, <italic>P</italic> = 0.017; Table S8).</p></sec><sec id=\"evl3193-sec-0090\"><title>TRAITS ANALYSES</title><p>In the traits analyses, focusing on the interrelationships between the male and female reproductive traits themselves, we used multimodel inference based on LMMs or GLMMs that included each represented genotype, the female × male × male genotypic combination, and the temporal blocks as random effects. After selecting the confidence model set, we averaged the coefficients using natural model averaging (Burnham and Anderson <xref rid=\"evl3193-bib-0014\" ref-type=\"ref\">2002</xref>; Grueber et al. <xref rid=\"evl3193-bib-0041\" ref-type=\"ref\">2011</xref>; for details, see Material and Methods).</p><p>Our first traits analysis focused on the number of sperm transferred by the second males, which we predicted to depend on female size, copulation duration, the number of first‐male sperm residing within the FRT, and their interactions (Lüpold et al. <xref rid=\"evl3193-bib-0055\" ref-type=\"ref\">2011</xref>, <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>). The parsimonious confidence set consisted of two models on second‐male sperm transfer (<italic>N</italic> = 557, ΔAIC<sub>c</sub> ≤ 0.64), represented by strong positive effects of copulation duration (<italic>β</italic> = 0.26; 95% confidence interval [0.10–0.42]) and the number of resident sperm (<italic>β</italic> = 0.48 [0.32–0.63]), and a weak trend for an interaction between them (<italic>β</italic> = 0.25 [–0.05 to 0.55]; Table S9). These results suggest that males transfer more sperm when there are numerous resident sperm in the FRT, by prolonging copulation. Next, we tested the prediction that the time to female sperm ejection should be influenced by the joint effects of SR length and the differences (second – first male) in sperm length and number between males (<italic>N</italic> = 529). Here, only the difference in sperm length had an effect (<italic>β</italic> = 0.20 [0.05–0.36]; Table S10). Further examination using only the absolute sperm lengths of both males rather than the difference between them revealed that this effect was driven primarily by the second male's sperm length (LMM, <italic>N</italic> = 529; first male: <italic>β</italic> = –0.01 [–0.09 to 0.10]; second‐male: <italic>β</italic> = 0.10 [0.03–0.17]). This result suggests that longer second‐male sperm might prolong the time to female sperm ejection and thus the sperm displacement phase, which was previously shown to increasingly bias sperm storage toward the second male (Lüpold et al. <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>).</p><p>We further predicted that the relative numbers of first‐ and second‐male sperm stored by females after sperm ejection (i.e., total S<sub>2</sub>) should be explained by the relative sperm lengths and the numbers of first‐male resident and second‐male transferred sperm at the end of copulation (Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>), coupled with the timing of female sperm ejection (Lüpold et al. <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>). Using candidate models derived from a GLMM (<italic>N</italic> = 505 observations across all 108 genotypic combinations) including all interactions between ejection time and the between‐male differences in sperm length and numbers, respectively, and with a binomial error distribution and logit link as well as an observation‐level random effect to account for overdispersion, total S<sub>2</sub> increased with both ejection time (<italic>β</italic> = 0.43 [0.27–0.60]) and the difference in sperm numbers (<italic>β</italic> = 0.69 [0.51–0.86]), and was further influenced by an interaction between these two predictors (<italic>β</italic> = 0.48 [0.16–0.79]; ΔAIC<sub>c</sub> = 6.99; Table S11 and Fig. <xref rid=\"evl3193-fig-0002\" ref-type=\"fig\">2</xref>). This interaction suggests that by delaying or precipitating ejection, females can amplify or dampen, respectively, the competitive advantage of the second male's larger ejaculate.</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3193-fig-0002\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Conditional effects plot of the two‐way interaction between the time from remating to female sperm ejection and the difference (second male – first male) in sperm number explaining variance in total S<sub>2</sub>. The plot indicates that the effects of relative sperm numbers and ejection time reinforce one another, with the greatest change in S<sub>2</sub> when the second male transfers a disproportionate amount of sperm and the female waits relatively long to eject excess or displaced sperm. The shaded areas around lines depict the 95% confidence intervals.</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"EVL3-4-416-g002\"/></fig><p>As mentioned previously, it is important to discriminate between total S<sub>2</sub> throughout the FRT and the fertilization set in the SR. Here, we repeated the above analysis for S<sub>2</sub> in the SR, but additionally included SR length and female thorax length as predictors, to examine more complex links between sex‐specific traits explaining relative sperm numbers in the fertilization set. To limit model complexity, we restricted interactions to two‐ and three‐way interactions and further limited all models to a maximum of 10 parameters (including interactions). Although the full model set contained 1294 different models, this was reduced to only 22 models (Table S12) by removing models that were simply more complex versions of any model with a lower AIC<sub>c</sub> value (Burnham and Anderson <xref rid=\"evl3193-bib-0014\" ref-type=\"ref\">2002</xref>; Richards et al. <xref rid=\"evl3193-bib-0090\" ref-type=\"ref\">2011</xref>). The resulting confidence model set (ΔAIC<sub>c</sub> ≤ 6; Bolker et al. <xref rid=\"evl3193-bib-0012\" ref-type=\"ref\">2009</xref>; Symonds and Moussalli <xref rid=\"evl3193-bib-0101\" ref-type=\"ref\">2011</xref>) consisted of seven models, with female thorax length (<italic>β</italic> = 0.35 [0.11–0.59]), SR length (<italic>β</italic> = –0.56 [–0.79 to –0.32]), the time to sperm ejection (<italic>β</italic> = 0.87 [0.63–1.11]), and difference in the number of sperm between males (<italic>β</italic> = 0.76 [0.51–1.00]) being the most important predictors. The difference in sperm length appeared unimportant as a main effect but contributed to all three interactions whose 95% confidence interval excluded zero after model averaging (Tables <xref rid=\"evl3193-tbl-0001\" ref-type=\"table\">1</xref> and S12). For example, together with female thorax length and the difference in sperm number, it formed a three‐way interaction, meaning that in a small female, any increasing bias in sperm numbers toward the second male will have a strong effect on S<sub>2</sub> if second male has relatively long sperm, but a weaker effect if he has short sperm (Fig. <xref rid=\"evl3193-fig-0003\" ref-type=\"fig\">3</xref>). In large females, however, the effect of relative sperm length on S<sub>2</sub> tends to reverse. Further, the interaction between the difference in sperm length and SR length (Tables <xref rid=\"evl3193-tbl-0001\" ref-type=\"table\">1</xref> and S12) means that if second males have shorter sperm than their rivals, any increase in SR length reduces S<sub>2</sub>, whereas SR length has a much weaker effect on the fertilization set if second males have relatively longer sperm (Fig. <xref rid=\"evl3193-fig-0004\" ref-type=\"fig\">4</xref>). This result corroborates Miller and Pitnick's (<xref rid=\"evl3193-bib-0070\" ref-type=\"ref\">2002</xref>) findings using populations of <italic>D. melanogaster</italic> with experimentally evolved, exaggerated sperm and SR lengths as well as subsequent studies that also predicted significant effects of interactions between sperm lengths of both males and female SR length on the fertilization set (Pattarini et al. <xref rid=\"evl3193-bib-0079\" ref-type=\"ref\">2006</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>, <xref rid=\"evl3193-bib-0057\" ref-type=\"ref\">2016</xref>).</p><table-wrap id=\"evl3193-tbl-0001\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Model‐averaged coefficients of the analysis on the fertilization set (i.e., S<sub>2</sub> within the female SR) following sperm ejection, including the standardized effects of the difference (second – first male) in sperm length (ΔSL) and in sperm number (ΔSN), the time to female sperm ejection (EJT), female SR length (SRL), and female thorax length (FTL). <italic>N</italic> = 508, including all 108 genotypic combinations. See Table S12 for full details</p></caption><table frame=\"hsides\" rules=\"groups\"><col align=\"left\" span=\"1\"/><col align=\"char\" char=\".\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><thead><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"1\" colspan=\"1\">Parameter</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Estimate</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">SE</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">95% CI</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Intercept</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">2.35</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(2.02, 2.68)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ΔSN</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">0.76</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(0.51, 1.00)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">EJT</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">0.87</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(0.63, 1.11)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SRL</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">−0.56</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(−0.79, −0.32)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">FTL</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">0.35</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(0.11, 0.59)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ΔSL × FTL</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">−0.55</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(−1.02, −0.09)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ΔSL</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">–0.10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.17</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(–0.43, 0.22)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ΔSN × EJT</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">0.40</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(–0.09, 0.88)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ΔSL × SRL</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">0.48</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(0.02, 0.94)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ΔSL × ΔSN × FTL</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">−1.12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.47</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(−2.03, −0.21)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ΔSN × FTL</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">0.21</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.24</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(–0.26, 0.69)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ΔSL × ΔSN</td><td align=\"char\" rowspan=\"1\" colspan=\"1\">0.18</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0.25</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">(–0.31, 0.66)</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd.</copyright-holder></permissions></table-wrap><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3193-fig-0003\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Conditional effects plot of the three‐way interaction between the female thorax length and the relative differences in sperm length and sperm number, respectively, explaining variance in the fertilization set (S<sub>2</sub> within the female SR). Differences are shown from the second male's perspective (i.e., second male – first male). The plot depicts how any increase in the number of sperm transferred by the second male relative to the first‐male sperm residing in storage is met with a greater change in S<sub>2</sub> for relatively long sperm in small females (A) but for short sperm in large females (C), with a transitional stage for intermediate female size (B).</p></caption><graphic id=\"nlm-graphic-5\" xlink:href=\"EVL3-4-416-g003\"/></fig><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3193-fig-0004\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Conditional effects plot of the two‐way interaction between SR length and the difference (second male – first male) in sperm length explaining variance in the fertilization set (S<sub>2</sub> within the female SR). The plot indicates that the second male's sperm representation in the SR declines with any increase in SR length if his sperm are shorter than those of his rival, but less so when he has relatively longer sperm. The shaded areas around lines depict the 95% confidence intervals.</p></caption><graphic id=\"nlm-graphic-7\" xlink:href=\"EVL3-4-416-g004\"/></fig></sec><sec id=\"evl3193-sec-0100\"><title>STRUCTURAL EQUATION MODEL</title><p>Finally, we used a piecewise structural equation modeling approach to better visualize both direct and indirect effects of these numerous male and female traits on one another, and particularly on the fertilization set. This analysis revealed a complex network of interrelationships (Fig. <xref rid=\"evl3193-fig-0005\" ref-type=\"fig\">5</xref>; see Fig. S1 for visualization of the individual models). Not surprisingly, relative sperm numbers in the FRT immediately after copulation were a good predictor of relative sperm numbers in the SR after ejection, confirming previous results (Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>, <xref rid=\"evl3193-bib-0053\" ref-type=\"ref\">2013</xref>). Interestingly, longer SRs indirectly decreased S<sub>2</sub> by storing more resident sperm at the time of remating, contrasting with previous studies suggesting that longer SRs are associated with increased S<sub>2</sub>, particularly when the second male has longer sperm (Miller and Pitnick <xref rid=\"evl3193-bib-0070\" ref-type=\"ref\">2002</xref>). However, as shown in the simpler models above, it seems likely that SR length interacts with other reproductive traits, and as a result, can affect S<sub>2</sub> both positively and negatively depending on the context (note that for simplicity, interactions between traits are omitted in our structural equation models).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3193-fig-0005\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><p>Visual representation of the confirmatory path analysis examining direct and indirect effects on the fertilization set (i.e., S<sub>2</sub> within the female seminal receptacle). The width of each arrow is proportional to its corresponding coefficient (in bold, ± standard error). Black, solid arrows depict positive effects, whereas red, dashed arrows represent negative effects. All effects shown were statistically significant (<italic>P</italic> ≤ 0.04); all others (<italic>P</italic> ≥ 0.09) are omitted for better visibility (see Fig. S1 for all relationships examined). This also includes any paths involving the lengths of first‐ and second‐male sperm and the female thorax, which had significant effects within some of the individual models but not after combining models.</p></caption><graphic id=\"nlm-graphic-9\" xlink:href=\"EVL3-4-416-g005\"/></fig></sec></sec><sec id=\"evl3193-sec-0110\"><title>Discussion</title><p>There is a growing paradigm in sexual selection theory that emphasizes interactions between the sexes (e.g., Clark <xref rid=\"evl3193-bib-0021\" ref-type=\"ref\">2002</xref>; Arnqvist and Rowe <xref rid=\"evl3193-bib-0006\" ref-type=\"ref\">2005</xref>; Ravi Ram and Wolfner <xref rid=\"evl3193-bib-0088\" ref-type=\"ref\">2007</xref>; Howard et al. <xref rid=\"evl3193-bib-0044\" ref-type=\"ref\">2009</xref>; Arnqvist <xref rid=\"evl3193-bib-0005\" ref-type=\"ref\">2014</xref>). Advancing such theory is currently limited by our understanding of genotypic interactions between the sexes and of concomitant functional interactions among those sex‐specific traits that impact competitive reproductive outcomes. Here, we identified PSS mechanisms that involve female × male interactions and resolved sex‐specific traits that underlie those mechanisms. We note that these findings are conservative, as with only six respective first‐male and female genotypes and three second‐male genotypes, we worked with limited genetic variation. For logistical reasons, our study also excluded a number of sex‐specific traits that are expected to contribute to PSS, including genitalic traits (e.g., House and Simmons <xref rid=\"evl3193-bib-0043\" ref-type=\"ref\">2003</xref>; Kamimura <xref rid=\"evl3193-bib-0046\" ref-type=\"ref\">2005</xref>; Wojcieszek and Simmons <xref rid=\"evl3193-bib-0109\" ref-type=\"ref\">2011</xref>; Simmons and Fitzpatrick <xref rid=\"evl3193-bib-0095\" ref-type=\"ref\">2019</xref>), FRT secretions (Gasparini and Pilastro <xref rid=\"evl3193-bib-0038\" ref-type=\"ref\">2011</xref>; Alonzo et al. <xref rid=\"evl3193-bib-0003\" ref-type=\"ref\">2016</xref>; Rosengrave et al. <xref rid=\"evl3193-bib-0092\" ref-type=\"ref\">2016</xref>; Lehnert et al. <xref rid=\"evl3193-bib-0050\" ref-type=\"ref\">2017</xref>), as well as male seminal‐fluid proteins (Clark et al. <xref rid=\"evl3193-bib-0023\" ref-type=\"ref\">1995</xref>; Chapman et al. <xref rid=\"evl3193-bib-0016\" ref-type=\"ref\">2000</xref>; Nakadera et al. <xref rid=\"evl3193-bib-0071\" ref-type=\"ref\">2014</xref>) and their female receptors (Sirot and Wolfner <xref rid=\"evl3193-bib-0097\" ref-type=\"ref\">2015</xref>; McDonough et al. <xref rid=\"evl3193-bib-0068\" ref-type=\"ref\">2016</xref>). Hence, although this study represents important progress toward understanding the targets and selective dynamics of PSS, there is much more work to be done. It is also important to note that experimental investigation of interactions such as these is analytically complex, every currently available approach having its own limitations and challenges. Although our different statistical approaches (i.e., null‐hypothesis significance testing, information theoretic using AIC‐based multi‐model inference, and path analyses) resulted in similar patterns, there were quantitative differences in the degree to which female × male interactions explained variation in reproductive outcomes. Because there was some phenotypic variation within the heterozygous F<sub>1</sub> genotypes for all traits examined, and we had only few genotypes, we expect the “traits analyses” to be more sensitive to detecting interactions than the genotypic analyses.</p><p>Our investigation corroborated previous work in <italic>D. melanogaster</italic> showing that reproductive outcomes are mediated by male‐specific (Clark et al. <xref rid=\"evl3193-bib-0023\" ref-type=\"ref\">1995</xref>; Fiumera et al. <xref rid=\"evl3193-bib-0036\" ref-type=\"ref\">2005</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>; Civetta and Ranz <xref rid=\"evl3193-bib-0020\" ref-type=\"ref\">2019</xref>), female‐specific (Clark and Begun <xref rid=\"evl3193-bib-0022\" ref-type=\"ref\">1998</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>; Ala‐Honkola and Manier <xref rid=\"evl3193-bib-0001\" ref-type=\"ref\">2016</xref>; Chen et al. <xref rid=\"evl3193-bib-0017\" ref-type=\"ref\">2019</xref>), and interactive effects between the sexes and competing males (Clark et al. <xref rid=\"evl3193-bib-0024\" ref-type=\"ref\">1999</xref>; Mack et al. <xref rid=\"evl3193-bib-0062\" ref-type=\"ref\">2002</xref>; Miller and Pitnick <xref rid=\"evl3193-bib-0070\" ref-type=\"ref\">2002</xref>; Bjork et al. <xref rid=\"evl3193-bib-0011\" ref-type=\"ref\">2007</xref>; Reinhart et al. <xref rid=\"evl3193-bib-0089\" ref-type=\"ref\">2015</xref>), patterns that have also been studied extensively in a diversity of taxa (reviewed in Arnqvist and Rowe <xref rid=\"evl3193-bib-0006\" ref-type=\"ref\">2005</xref>; Snook <xref rid=\"evl3193-bib-0098\" ref-type=\"ref\">2005</xref>; Oh and Badyaev <xref rid=\"evl3193-bib-0074\" ref-type=\"ref\">2006</xref>; Howard et al. <xref rid=\"evl3193-bib-0044\" ref-type=\"ref\">2009</xref>; Pitnick et al. <xref rid=\"evl3193-bib-0084\" ref-type=\"ref\">2009b</xref>; Pizzari and Parker <xref rid=\"evl3193-bib-0086\" ref-type=\"ref\">2009</xref>; Arnqvist <xref rid=\"evl3193-bib-0005\" ref-type=\"ref\">2014</xref>; Firman et al. <xref rid=\"evl3193-bib-0034\" ref-type=\"ref\">2017</xref>). In support of these interactive effects on relative paternity shares (P<sub>2</sub>) among genotypes, we found that a three‐way interaction between the different genotypes of a mating trio significantly contributed to variation in the fertilization set, which is a consequence of all other traits examined (e.g., the number of sperm transferred by first males, retained and used by females, the number of sperm transferred by second males, and the outcome of sperm displacement activity following remating) and arguably the strongest correlate of variation in male and female fitness (Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>, <xref rid=\"evl3193-bib-0053\" ref-type=\"ref\">2013</xref>; Manier et al. <xref rid=\"evl3193-bib-0064\" ref-type=\"ref\">2013a</xref>,<xref rid=\"evl3193-bib-0066\" ref-type=\"ref\">c</xref>). This interaction likely explains the previously reported nontransitivity in P<sub>2</sub> (Clark et al. <xref rid=\"evl3193-bib-0024\" ref-type=\"ref\">1999</xref>, <xref rid=\"evl3193-bib-0025\" ref-type=\"ref\">2000</xref>; Zhang et al. <xref rid=\"evl3193-bib-0112\" ref-type=\"ref\">2013</xref>; Reinhart et al. <xref rid=\"evl3193-bib-0089\" ref-type=\"ref\">2015</xref>). Importantly, our analyses allowed us to decompose the underlying processes leading to this outcome, which we here discuss in turn.</p><p>The remating interval of females, which is arguably one of the strongest determinants of the intensity of PSS (Boorman and Parker <xref rid=\"evl3193-bib-0013\" ref-type=\"ref\">1976</xref>; Simmons <xref rid=\"evl3193-bib-0094\" ref-type=\"ref\">2001</xref>; Taylor et al. <xref rid=\"evl3193-bib-0102\" ref-type=\"ref\">2014</xref>), was mediated by females alone in our analysis, which might be due to unusually little variation, with 55% of all remating events (410 of 744) occurring 2 days after the first mating, regardless of genotypic combination, and another 26% (197 of 744) on the third day. However, both the number of progeny produced by the female before remating and the number of resident, first‐male sperm remaining in storage at the time of remating were influenced by the female genotype and at least a weak first‐male effect. This female effect is unsurprising and could be the result of variation in female fecundity, sperm retention or sensitivity to seminal‐fluid proteins after the initial mating, or fertilization efficiency (Pitnick et al. <xref rid=\"evl3193-bib-0082\" ref-type=\"ref\">2001</xref>; Pischedda et al. <xref rid=\"evl3193-bib-0080\" ref-type=\"ref\">2012</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>; Delbare et al. <xref rid=\"evl3193-bib-0028\" ref-type=\"ref\">2017</xref>). The male effect might be attributable to variation in first‐male ejaculate size or seminal fluid composition (Avila et al. <xref rid=\"evl3193-bib-0007\" ref-type=\"ref\">2011</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0056\" ref-type=\"ref\">2012</xref>). The lack of an interactive effect on female fecundity is consistent with Pischedda et al.’s (<xref rid=\"evl3193-bib-0080\" ref-type=\"ref\">2012</xref>) study, but contrasts with Delbare et al.’s (<xref rid=\"evl3193-bib-0028\" ref-type=\"ref\">2017</xref>) study, both of which crossed inbred lines of the same five geographically (and genetically) distant populations of <italic>D. melanogaster</italic> in a 5 × 5 factorial design. Hence, insufficient genetic variation is unlikely to be the only explanation for the absence of contributing interactions in our study.</p><p>The duration of the second copulation was determined predominantly by the genotype of the copulating male, with a significant female × first‐male interaction. Male control of copulation duration has been reported in multiple arthropod species (e.g., Yasui <xref rid=\"evl3193-bib-0111\" ref-type=\"ref\">1994</xref>; Wilder and Rypstra <xref rid=\"evl3193-bib-0107\" ref-type=\"ref\">2007</xref>; Holwell <xref rid=\"evl3193-bib-0042\" ref-type=\"ref\">2008</xref>), including <italic>Drosophila</italic> (e.g., MacBean and Parsons <xref rid=\"evl3193-bib-0060\" ref-type=\"ref\">1966</xref>, <xref rid=\"evl3193-bib-0061\" ref-type=\"ref\">1967</xref>), and is often related to ejaculate transfer and ultimately competitive fertilization success (Parker <xref rid=\"evl3193-bib-0076\" ref-type=\"ref\">1970b</xref>; Dickinson <xref rid=\"evl3193-bib-0029\" ref-type=\"ref\">1986</xref>; Wolf et al. <xref rid=\"evl3193-bib-0110\" ref-type=\"ref\">1989</xref>; García‐González and Gomendio <xref rid=\"evl3193-bib-0037\" ref-type=\"ref\">2004</xref>; Wang et al. <xref rid=\"evl3193-bib-0105\" ref-type=\"ref\">2008</xref>; reviewed in Weggelaar et al. <xref rid=\"evl3193-bib-0106\" ref-type=\"ref\">2019</xref>). The female × first‐male interaction might be related to the number of first‐male sperm residing within the FRT, in that females with a long SR tended to store more first‐male sperm at the time of remating. These patterns combined might then explain the trend for a three‐way interaction in second‐male sperm transfer, with the number of sperm transferred by second males increasing with both the duration of their copulation and the number of first‐male sperm still residing within the female, including a weak interaction between them (Table S9). That male <italic>D. melanogaster</italic> adjust their ejaculate size to the presence or absence of a competitor's sperm has previously been documented (Lüpold et al. <xref rid=\"evl3193-bib-0055\" ref-type=\"ref\">2011</xref>) and is a taxonomically widespread phenomenon (meta‐analyzed in Kelly and Jennions <xref rid=\"evl3193-bib-0047\" ref-type=\"ref\">2011</xref>). Our results suggest that sperm allocation might be even more sophisticated than previously realized by responding to the quantity of rival sperm in the FRT, even though the mechanism(s) underlying such nuanced adjustments remain(s) elusive.</p><p>After copulation, the timing of female ejection was explained by both the female and second‐male genotype, including their interaction. Females that eject sperm later allow more time for second‐male sperm to enter storage and further displace first‐male sperm. Because longer sperm are better at displacing, and resisting displacement by, shorter sperm (Miller and Pitnick <xref rid=\"evl3193-bib-0070\" ref-type=\"ref\">2002</xref>; Pattarini et al. <xref rid=\"evl3193-bib-0079\" ref-type=\"ref\">2006</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>; Manier et al. <xref rid=\"evl3193-bib-0065\" ref-type=\"ref\">2013b</xref>), longer ejection times benefit males with longer sperm and perhaps also the female through indirect fitness benefits (Lüpold et al. <xref rid=\"evl3193-bib-0057\" ref-type=\"ref\">2016</xref>). Indeed, our traits analysis revealed that the difference in sperm length between competing males influenced the timing of female sperm ejection (Table S10), while further engaging in interactions with female size, SR length, and the difference in sperm number to explain a significant proportion of the variance in the fertilization set (Tables S11 and S12). Although the mechanism(s) underlying the delay in female sperm ejection after mating with a long‐sperm male remain(s) unknown, this pattern, combined with a genetic correlation between female sperm ejection and SR length (Lüpold et al. <xref rid=\"evl3193-bib-0057\" ref-type=\"ref\">2016</xref>), provides a possible functional explanation for the heightened precedence of relatively long sperm in a long compared to short SR reported previously (Miller and Pitnick <xref rid=\"evl3193-bib-0070\" ref-type=\"ref\">2002</xref>).</p><p>Because sperm ejection is a principal means by which females influence paternity in <italic>Drosophila</italic> (Snook and Hosken <xref rid=\"evl3193-bib-0099\" ref-type=\"ref\">2004</xref>; Manier et al. <xref rid=\"evl3193-bib-0063\" ref-type=\"ref\">2010</xref>, <xref rid=\"evl3193-bib-0064\" ref-type=\"ref\">2013a</xref>,<xref rid=\"evl3193-bib-0065\" ref-type=\"ref\">b</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0058\" ref-type=\"ref\">2013</xref>) and many other taxa (reviewed in Dean et al. <xref rid=\"evl3193-bib-0027\" ref-type=\"ref\">2011</xref>), these results provide strong support for the expectation that cryptic female choice processes will evolve mechanisms entailing interactions between mating partners (Arnqvist <xref rid=\"evl3193-bib-0005\" ref-type=\"ref\">2014</xref>; Firman et al. <xref rid=\"evl3193-bib-0034\" ref-type=\"ref\">2017</xref>).</p><p>The present study examined only a small proportion of the sex‐specific phenotypes suspected of influencing competitive fertilization success. Nevertheless, all measured male and female reproductive traits contributed to the competitive fertilization set or at least to some reproductive event known to determine it. Further, interactions identified here between competing males and between sexes were shown to explain a significant amount of variation in several key reproductive events, including those generally considered functional components of both sperm competition (e.g., the number of sperm inseminated during remating by a female) and cryptic female choice (e.g., sperm ejection time). Moreover, sperm length, which is recognized as a PSS ornament that interacts functionally and evolutionarily with SR length (Miller and Pitnick <xref rid=\"evl3193-bib-0070\" ref-type=\"ref\">2002</xref>; Pattarini et al. <xref rid=\"evl3193-bib-0079\" ref-type=\"ref\">2006</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0057\" ref-type=\"ref\">2016</xref>), did so in a consistent manner in the present study and was further shown to interact with sperm number and SR length in determining competitive fertilization success (i.e., the fertilization set). Our results thus provide further evidence that sperm competition, oftentimes considered to operate between males alone, may in fact rarely be independent of female effects (Eberhard <xref rid=\"evl3193-bib-0030\" ref-type=\"ref\">1996</xref>; Arnqvist <xref rid=\"evl3193-bib-0005\" ref-type=\"ref\">2014</xref>; Lüpold et al. <xref rid=\"evl3193-bib-0057\" ref-type=\"ref\">2016</xref>; Firman et al. <xref rid=\"evl3193-bib-0034\" ref-type=\"ref\">2017</xref>), thereby supporting the idea that sperm competition and cryptic female choice are likely to represent a false dichotomy. Because the identified interactions included sperm and SR length, which have been shown to represent one of the most extreme co‐diversifying systems of male ornamentation and female preference (Lüpold et al. <xref rid=\"evl3193-bib-0057\" ref-type=\"ref\">2016</xref>), multivariate systems with complex interactions between the sexes might not be limited in their ability to respond to directional sexual selection. This scenario would be the case particularly if these interactions are also context dependent (e.g., due to condition‐dependent ejaculate composition or female sperm‐use dynamics), such that even antagonistic effects on S<sub>2</sub> between traits (e.g., see Fig. <xref rid=\"evl3193-fig-0005\" ref-type=\"fig\">5</xref>) do not necessarily restrict the opportunity for selection on, and thus the evolution of, sex‐specific traits. This raises the question of whether the evolution of extreme phenotypes under directional selection, possibly reflected by the widespread sex‐specific main effects in our analyses, has been facilitated by the combination of limited nontransitivity between genotypes and a complex interplay between sex‐specific, heritable, and likely condition‐dependent traits, which might have helped maintain considerable genetic variation within populations. Finally, our study illustrates both the benefits and empirical challenges of quantifying the contribution of interactions to the operation of sexual selection.</p></sec><sec id=\"evl3193-sec-0130\"><title>AUTHOR CONTRIBUTIONS</title><p>SL, MKM, JMB, and SP conceived the research. SL, JBR, MKM, VZ, and SP performed the research. SL analyzed the data. SL and SP drafted the manuscript, which all authors edited and approved.</p></sec><sec sec-type=\"COI-statement\" id=\"evl3193-sec-0140\"><title>CONFLICT OF INTEREST</title><p>The authors declare no conflict of interest.</p></sec><sec sec-type=\"data-availability\" id=\"evl3193-sec-0150\"><title>DATA ARCHIVING</title><p>All data are deposited in the Dryad Repository (<ext-link ext-link-type=\"uri\" xlink:href=\"https://doi.org/10.5061/dryad.x3ffbg7gq\">https://doi.org/10.5061/dryad.x3ffbg7gq</ext-link>).</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting information</title><supplementary-material content-type=\"local-data\"><caption><p>\n<bold>Supplementary Figure S1</bold>: Visual representation of the GLMM models used in the confirmatory path analysis, with arrows connecting all predictors with the response variable examined.</p><p>\n<bold>Supplementary Table S1</bold>: Results of a generalized linear mixed model with binomial error distribution representing the genotypic effects on total S<sub>2</sub> after female sperm ejection (<italic>N</italic> = 577 across all 108 genotypic mating combinations).</p><p>\n<bold>Supplementary Table S2</bold>: Results of a generalized linear mixed model with binomial error distribution representing the genotypic effects on S<sub>2</sub> within the seminal receptacle (i.e., the “fertilization set”) after female sperm ejection (<italic>N</italic> = 589 across all 108 genotypic mating combinations).</p><p>\n<bold>Supplementary Table S3</bold>: Results of a linear mixed‐effects model (with the temporal block as a four‐level random factor) analyzing the genotypic effects and interactions on the female remating interval.</p><p>\n<bold>Supplementary Table S4</bold>: Results of a linear mixed‐effects model (with the temporal block as a four‐level random factor) analyzing the genotypic effects and interactions on the number of offspring produced between copulations, controlling for the female remating interval.</p><p>\n<bold>Supplementary Table S5</bold>: Results of a linear mixed‐effects model (with the temporal block as a four‐level random factor) analyzing the genotypic effects and interactions on the number of 1st‐male sperm residing in the female reproductive tract at remating, controlling for the number of progeny produced prior to remating.</p><p>\n<bold>Supplementary Table S6</bold>: Results of a linear mixed‐effects model (with the temporal block as a four‐level random factor) analyzing the genotypic effects and interactions on the duration of the second copulation.</p><p>\n<bold>Supplementary Table S7</bold>: Results of a linear mixed‐effects model (with the temporal block as a four‐level random factor) analyzing the genotypic effects and interactions on the number of sperm transferred by the second male.</p><p>\n<bold>Supplementary Table S8</bold>: Results of a linear mixed‐effects model (with the temporal block as a four‐level random factor) analyzing the genotypic effects and interactions on the time to female sperm ejection.</p><p>\n<bold>Supplementary Table S9</bold>: Results of the information‐theoretic analyses examining the effects of copulation duration (C), the number of 1st‐male sperm residing in the FRT at remating (R) and female thorax length (T) on the number of sperm transferred by the second male (N = 557).</p><p>\n<bold>Supplementary Table S10</bold>: Results of the information‐theoretic analyses examining the effects of the difference in sperm length (L), the difference in sperm number (N) and seminal receptacle length (S) on the time to female sperm ejection (N = 527).</p><p>\n<bold>Supplementary Table S11</bold>: Results of the information‐theoretic analyses examining the effects of the difference in sperm length (L), the difference in sperm number (N) and the time to female sperm ejection (E) on the relative numbers of sperm stored between males (i.e., total S<sub>2</sub>, <italic>N</italic> = 505).</p><p>\n<bold>Supplementary Table S12</bold>: Results of the information‐theoretic analyses examining the effects of the difference in sperm length (L), the difference in sperm number (N), the time to female sperm ejection (E), female SR length (S) and female thorax length (T) on S<sub>2</sub> within the SR (<italic>N</italic> = 508). Each model was a GLMM with the temporal block as a four‐level random factor. An observation‐level random effect was included to account for overdispersion.</p></caption><media xlink:href=\"EVL3-4-416-s001.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"evl3193-sec-0120\"><title>ACKNOWLEDGMENTS</title><p>We thank W. T. Starmer and S. Nakagawa for insightful discussions, and the reviewers and associate editor for their constructive comments. This research was supported by a generous gift by M. Weeden and J. Weeden to Syracuse University and grants from the U.S. National Science Foundation (DEB‐1145965 to SP, SL, JMB, and MKM and DEB‐1655840 to SP) and the Swiss National Science Foundation (PA00P3_134191 and PP00P3_170669 to SL).</p></ack><ref-list id=\"evl3193-bibl-0001\"><title>LITERATURE CITED</title><ref id=\"evl3193-bib-0001\"><mixed-citation publication-type=\"journal\" id=\"evl3193-cit-0001\">\n<string-name>\n<surname>Ala‐Honkola</surname>, <given-names>O.</given-names>\n</string-name> and <string-name>\n<given-names>M. K.</given-names>\n<surname>Manier</surname>\n</string-name>. <year>2016</year>\n<article-title>Multiple mechanisms of cryptic female choice act on intraspecific male variation in <italic>Drosophila simulans</italic>\n</article-title>. <source xml:lang=\"en\">Behav. Ecol. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"letter\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Evol Lett</journal-id><journal-id journal-id-type=\"doi\">10.1002/(ISSN)2056-3744</journal-id><journal-id journal-id-type=\"publisher-id\">EVL3</journal-id><journal-title-group><journal-title>Evolution Letters</journal-title></journal-title-group><issn pub-type=\"epub\">2056-3744</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33014419</article-id><article-id pub-id-type=\"pmc\">PMC7523562</article-id><article-id pub-id-type=\"doi\">10.1002/evl3.189</article-id><article-id pub-id-type=\"publisher-id\">EVL3189</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Letter</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Letters</subject></subj-group></article-categories><title-group><article-title>Footprints of local adaptation span hundreds of linked genes in the Atlantic silverside genome</article-title><alt-title alt-title-type=\"left-running-head\">A. P. WILDER ET AL.</alt-title></title-group><contrib-group><contrib id=\"evl3189-cr-0001\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Wilder</surname><given-names>Aryn P.</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-4970-6891</contrib-id><xref ref-type=\"aff\" rid=\"evl3189-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"evl3189-aff-0002\">\n<sup>2</sup>\n</xref><address><email>awilder@sandiegozoo.org</email></address></contrib><contrib id=\"evl3189-cr-0002\" contrib-type=\"author\"><name><surname>Palumbi</surname><given-names>Stephen R.</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-5195-1947</contrib-id><xref ref-type=\"aff\" rid=\"evl3189-aff-0003\">\n<sup>3</sup>\n</xref></contrib><contrib id=\"evl3189-cr-0003\" contrib-type=\"author\"><name><surname>Conover</surname><given-names>David O.</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-8083-7155</contrib-id><xref ref-type=\"aff\" rid=\"evl3189-aff-0004\">\n<sup>4</sup>\n</xref></contrib><contrib id=\"evl3189-cr-0004\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Therkildsen</surname><given-names>Nina Overgaard</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-6591-591X</contrib-id><xref ref-type=\"aff\" rid=\"evl3189-aff-0001\">\n<sup>1</sup>\n</xref><address><email>nt246@cornell.edu</email></address></contrib></contrib-group><aff id=\"evl3189-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">Department of Natural Resources</named-content>\n<institution>Cornell University</institution>\n<city>Ithaca</city>\n<named-content content-type=\"country-part\">New York</named-content>\n<postal-code>14853</postal-code>\n</aff><aff id=\"evl3189-aff-0002\">\n<label><sup>2</sup></label>\n<institution>Current address: San Diego Zoo Institute for Conservation Research</institution>\n<city>Escondido</city>\n<named-content content-type=\"country-part\">California</named-content>\n<postal-code>92027</postal-code>\n</aff><aff id=\"evl3189-aff-0003\">\n<label><sup>3</sup></label>\n<named-content content-type=\"organisation-division\">Department of Biology, Hopkins Marine Station</named-content>\n<institution>Stanford University</institution>\n<city>Pacific Grove</city>\n<named-content content-type=\"country-part\">California</named-content>\n<postal-code>93950</postal-code>\n</aff><aff id=\"evl3189-aff-0004\">\n<label><sup>4</sup></label>\n<named-content content-type=\"organisation-division\">Department of Biology</named-content>\n<institution>University of Oregon</institution>\n<city>Eugene</city>\n<named-content content-type=\"country-part\">Oregon</named-content>\n<postal-code>97403</postal-code>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label>E‐mail: <email>nt246@cornell.edu</email>; <email>awilder@sandiegozoo.org</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>19</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><month>10</month><year>2020</year></pub-date><volume>4</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1002/evl3.v4.5</issue-id><fpage>430</fpage><lpage>443</lpage><history><date date-type=\"received\"><day>20</day><month>11</month><year>2019</year></date><date date-type=\"rev-recd\"><day>09</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>13</day><month>7</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 The Authors. Evolution Letters published by Wiley Periodicals LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB). <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Evolution Letters</italic> published by Wiley Periodicals, LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB).</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:EVL3-4-430.pdf\"/><abstract><title>Abstract</title><p>The study of local adaptation in the presence of ongoing gene flow is the study of natural selection in action, revealing the functional genetic diversity most relevant to contemporary pressures. In addition to individual genes, genome‐wide architecture can itself evolve to enable adaptation. Distributed across a steep thermal gradient along the east coast of North America, Atlantic silversides (<italic>Menidia menidia</italic>) exhibit an extraordinary degree of local adaptation in a suite of traits, and the capacity for rapid adaptation from standing genetic variation, but we know little about the patterns of genomic variation across the species range that enable this remarkable adaptability. Here, we use low‐coverage, whole‐transcriptome sequencing of Atlantic silversides sampled along an environmental cline to show marked signatures of divergent selection across a gradient of neutral differentiation. Atlantic silversides sampled across 1371 km of the southern section of its distribution have very low genome‐wide differentiation (median <italic>F</italic>\n<sub>ST</sub> = 0.006 across 1.9 million variants), consistent with historical connectivity and observations of recent migrants. Yet almost 14,000 single nucleotide polymorphisms (SNPs) are nearly fixed (<italic>F</italic>\n<sub>ST</sub> > 0.95) for alternate alleles. Highly differentiated SNPs cluster into four tight linkage disequilibrium (LD) blocks that span hundreds of genes and several megabases. Variants in these LD blocks are disproportionately nonsynonymous and concentrated in genes enriched for multiple functions related to known adaptations in silversides, including variation in lipid storage, metabolic rate, and spawning behavior. Elevated levels of absolute divergence and demographic modeling suggest selection maintaining divergence across these blocks under gene flow. These findings represent an extreme case of heterogeneity in levels of differentiation across the genome, and highlight how gene flow shapes genomic architecture in continuous populations. Locally adapted alleles may be common features of populations distributed along environmental gradients, and will likely be key to conserving variation to enable future responses to environmental change.</p></abstract><kwd-group kwd-group-type=\"author-generated\"><kwd id=\"evl3189-kwd-0001\">Cline</kwd><kwd id=\"evl3189-kwd-0002\">divergent selection</kwd><kwd id=\"evl3189-kwd-0003\">gene flow</kwd><kwd id=\"evl3189-kwd-0004\">linkage disequilibrium</kwd><kwd id=\"evl3189-kwd-0005\">supergenes</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source><institution-wrap><institution>Villum Fonden </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/100008398</institution-id></institution-wrap></funding-source><award-id>Postdoctoral fellowship</award-id></award-group><award-group id=\"funding-0002\"><funding-source><institution-wrap><institution>National Science Foundation </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/100000001</institution-id></institution-wrap></funding-source><award-id>OCE‐1434325</award-id><award-id>OCE‐1756316</award-id></award-group></funding-group><counts><fig-count count=\"5\"/><table-count count=\"1\"/><page-count count=\"14\"/><word-count count=\"10553\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>October 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.9.1 mode:remove_FC converted:29.09.2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><boxed-text position=\"float\" content-type=\"box\" orientation=\"portrait\"><caption><title>Impact Statement</title></caption><p>The Atlantic silverside shows a remarkable degree of local adaptation in phenotypic traits, but the genomic basis is unknown. Whole transcriptome sequence data from silversides sampled across their distribution reveal thousands of single nucleotide polymorphisms nearly fixed for opposite alleles across locations, comprising over 1% of variants in the transcriptome, concentrated into large blocks of tightly linked genes with functions related to well‐described adaptations. Absolute divergence was elevated across these genes, indicating the effects of diversifying selection acting on these loci under gene flow, and demographic models that include both migration and selection best fit the transcriptome‐wide data. Because most of the genome shows minimal geographic differentiation due to ongoing gene flow, these massive blocks of fixed differences comprise one of the most striking examples reported to date of how gene flow and selection cluster adaptive variation across the genome. The geographic distribution of variation suggests that protection of populations across environmental gradients may help preserve a reservoir of adaptive potential in the face of human impact.</p></boxed-text><p>Local adaptation is a critical generator of ecologically relevant genetic and phenotypic variation. Gene flow can both facilitate and counteract adaptation (Tigano and Friesen <xref rid=\"evl3189-bib-0074\" ref-type=\"ref\">2016</xref>). Studying the interplay between the homogenizing effect of gene flow and diversifying effect of selection allows an examination of adaptation to contemporary pressures (Whitlock <xref rid=\"evl3189-bib-0079\" ref-type=\"ref\">2015</xref>), and the mechanisms key to shaping the potential response to future environmental shifts (Hoffmann and Sgro <xref rid=\"evl3189-bib-0035\" ref-type=\"ref\">2011</xref>). Theoretical models predict that the ability to adapt to local conditions despite ongoing gene flow depends on the genetic basis of adaptive traits and overall levels of genetic variability (Haldane <xref rid=\"evl3189-bib-0031\" ref-type=\"ref\">1930</xref>; Yeaman <xref rid=\"evl3189-bib-0082\" ref-type=\"ref\">2015</xref>), but we still have a limited understanding of the genomic patterns underlying adaptive divergence in highly connected natural populations, particularly for complex traits with a multigenic basis.</p><p>The Atlantic silverside (<italic>Menidia menidia</italic>), a marine fish distributed across a steep thermal gradient along the east coast of North America (Baumann and Doherty <xref rid=\"evl3189-bib-0007\" ref-type=\"ref\">2013</xref>), presents a valuable natural system for studying rapid evolution and local adaptation in the face of gene flow. A substantial body of evidence from lab and field studies has demonstrated that this very abundant species (with effective population sizes in the millions; Lou et al. <xref rid=\"evl3189-bib-0048\" ref-type=\"ref\">2018</xref>) exhibits a remarkable degree of local adaptation in numerous traits across latitudes (Conover et al. <xref rid=\"evl3189-bib-0019\" ref-type=\"ref\">2009</xref>). Among the most widely recognized is north‐south variation in growth rate: short growing seasons in the north are associated with genetically determined faster growth rates, compared to longer growing seasons and slower intrinsic growth rates in the south. These genetic differences counteract environmental influences on growth rates, resulting in similar body sizes despite vast differences in local environmental conditions across the species range (Conover and Present <xref rid=\"evl3189-bib-0017\" ref-type=\"ref\">1990</xref>; Arnott et al. <xref rid=\"evl3189-bib-0003\" ref-type=\"ref\">2006</xref>). Generally termed counter‐gradient variation, this pattern is also seen in many other fish, frogs, and marine invertebrates (Conover and Schultz <xref rid=\"evl3189-bib-0014\" ref-type=\"ref\">1995</xref>; Conover et al. <xref rid=\"evl3189-bib-0019\" ref-type=\"ref\">2009</xref>). Counter‐gradient and co‐gradient variation (in which the environment enhances the genetic effect on the phenotype) in myriad other traits have been described in Atlantic silversides, including vertebral number, temperature‐dependent sex determination, swimming performance, lipid storage, and spawning behavior (Conover and Baumann <xref rid=\"evl3189-bib-0015\" ref-type=\"ref\">2009</xref>). Common garden experiments have established that these traits are genetically based and can vary over less than 100 km (Conover and Present <xref rid=\"evl3189-bib-0017\" ref-type=\"ref\">1990</xref>; Hice et al. <xref rid=\"evl3189-bib-0034\" ref-type=\"ref\">2012</xref>).</p><p>Importantly, gene flow appears to be high across much of the geographic region where trait divergence is observed. Mitochondrial sequence data define three regional subdivisions of the Atlantic silverside distribution: south of Cape Cod, the Gulf of Maine, and the Gulf of St. Lawrence, with limited substructure within each (Mach et al. <xref rid=\"evl3189-bib-0049\" ref-type=\"ref\">2011</xref>; Lou et al. <xref rid=\"evl3189-bib-0048\" ref-type=\"ref\">2018</xref>; Fig. <xref rid=\"evl3189-fig-0001\" ref-type=\"fig\">1A</xref>). Demographic modeling has suggested that the Gulf of Maine and Gulf of St. Lawrence populations were established from the south after the Last Glacial Maximum, whereas localities extending from Cape Cod to Florida were occupied and connected by ongoing gene flow through the Last Glacial Maximum (Lou et al. <xref rid=\"evl3189-bib-0048\" ref-type=\"ref\">2018</xref>).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3189-fig-0001\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Genetic variation of Atlantic silversides across the east coast of North America. (A) Collection sites in Georgia (GA), North Carolina (NC), New York (NY), Gulf of Maine (GoM), and Gulf of St. Lawrence (GSL). Broken lines indicate boundaries for previously identified phylogeographic regions. (B) Principal components 1 and 2 of transcriptome‐wide variation among individuals, colored by sampling locations in panel A. (C) Transcriptome‐wide distribution of global <italic>F</italic>\n<sub>ST</sub> across all populations (yellow bars) fit to a <italic>χ</italic>\n<sup>2</sup> distribution (blue line) expected under neutrality. The inset zooms in on the tail of the distribution. (D) Boxplots of the frequency of the southern alleles at SNPs in the top 10% of the global <italic>F</italic>\n<sub>ST</sub> distribution (left panel) and outlier SNPs (right panel).</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"EVL3-4-430-g001\"/></fig><p>Under gene flow, strong linkage among co‐adapted loci can preserve adaptive trait complexes that otherwise might be broken apart by recombination (Dobzhansky and Dobzhansky <xref rid=\"evl3189-bib-0022\" ref-type=\"ref\">1970</xref>; Kirkpatrick and Barton <xref rid=\"evl3189-bib-0043\" ref-type=\"ref\">2006</xref>; Yeaman <xref rid=\"evl3189-bib-0081\" ref-type=\"ref\">2013</xref>), a mechanism that has been proposed to underlie adaptation in diverse taxa (Samuk et al. <xref rid=\"evl3189-bib-0067\" ref-type=\"ref\">2017</xref>; Wellenreuther and Bernatchez <xref rid=\"evl3189-bib-0078\" ref-type=\"ref\">2018</xref>). Studies in the Atlantic silverside have provided evidence of correlations among locally adaptive traits, raising the possibility that suites of co‐adapted alleles associated with a latitudinal gradient may be maintained by genetic linkage (Walsh et al. <xref rid=\"evl3189-bib-0076\" ref-type=\"ref\">2006</xref>; Hice et al. <xref rid=\"evl3189-bib-0034\" ref-type=\"ref\">2012</xref>; Salinas et al. <xref rid=\"evl3189-bib-0066\" ref-type=\"ref\">2012</xref>). In a laboratory experiment, for example, size‐selective harvest of Atlantic silversides led to striking evolution over just four generations, not only in growth rates but also in several correlated traits (Conover and Munch <xref rid=\"evl3189-bib-0016\" ref-type=\"ref\">2002</xref>; Walsh et al. <xref rid=\"evl3189-bib-0076\" ref-type=\"ref\">2006</xref>). We recently found that this selective response was associated with a dramatic shift in alleles across one cluster of hundreds of genes held together in tight linkage disequilibrium (LD) (Therkildsen et al. <xref rid=\"evl3189-bib-0073\" ref-type=\"ref\">2019</xref>). With reference data from wild populations, we also showed that this same genomic block is nearly fixed for opposite alleles across a natural cline in growth rates between the northern and southern extents of the silverside range, suggesting that the rapid multi‐trait response to selection in the experiment was fueled by pre‐existing variation and genomic architecture shaped by diversifying selection across the latitudinal cline (Therkildsen et al. <xref rid=\"evl3189-bib-0073\" ref-type=\"ref\">2019</xref>). Yet, this targeted screen revealed only how genomic regions that responded to experimental selection vary among wild silverside populations. It did not explore the broader signatures of natural selection underlying the pronounced local adaptation of multiple traits described in the wild, nor evaluate to what extent such signatures are linked, a feature we may predict to be abundant based on high gene flow in this system.</p><p>Here, we investigate for the first time the genome‐wide patterns of variation and genomic architecture that underlie the remarkable adaptive divergence maintained in Atlantic silversides despite high connectivity across latitudes. Using low‐coverage, whole genome sequence data mapped to a reference transcriptome, we characterize genomic differentiation and signatures of selection across the four locations referenced in Therkildsen et al. (<xref rid=\"evl3189-bib-0073\" ref-type=\"ref\">2019</xref>), plus one intermediate sampling locality near a common biogeographic breakpoint for marine species. We expect that divergent selection under gene flow should enhance both differentiation (<italic>F</italic>\n<sub>ST</sub>) and absolute divergence (<italic>d<sub>XY</sub></italic>) of locally adaptive alleles (Cruickshank and Hahn <xref rid=\"evl3189-bib-0020\" ref-type=\"ref\">2014</xref>; Delmore et al. <xref rid=\"evl3189-bib-0021\" ref-type=\"ref\">2018</xref>), and we predict tighter linkage among adaptive alleles where gene flow is higher (Yeaman and Whitlock <xref rid=\"evl3189-bib-0083\" ref-type=\"ref\">2011</xref>; Samuk et al. <xref rid=\"evl3189-bib-0067\" ref-type=\"ref\">2017</xref>). Shaped by a latitudinal gradient and steep thermal cline, genetic variation in this species has allowed for pronounced spatial and temporal adaptive responses, and may serve as the foundation for future adaptive shifts in the face of climate change.</p><sec id=\"evl3189-sec-0020\"><title>Methods</title><sec id=\"evl3189-sec-0030\"><title>LIBRARY PREPARATION AND BIOINFORMATICS</title><p>Adult silversides were collected during the spring spawning period between 2005 and 2007 from five locations: Jekyll Island, Georgia (GA; <italic>n</italic> = 48), Oregon Inlet, North Carolina (NC; <italic>n</italic> = 47), Patchogue, New York (NY; <italic>n</italic> = 49), Minas Basin, Nova Scotia in the Gulf of Maine (GoM; <italic>n =</italic> 50), and Magdalen Island, Quebec in the Gulf of St. Lawrence (GSL; <italic>n</italic> = 42; Fig. <xref rid=\"evl3189-fig-0001\" ref-type=\"fig\">1A</xref>). Our first round of sequencing included all individuals from four of these locations: GA, NY, GoM, and GSL. A separate barcoded genomic library was prepared for each individual as described in Therkildsen and Palumbi (<xref rid=\"evl3189-bib-0072\" ref-type=\"ref\">2017</xref>). The libraries were pooled in equimolar amounts and sequenced using paired‐end 125 bp reads on an Illumina HiSeq instrument, randomly distributing sample libraries across lanes. Whole‐genome sequence reads were mapped to an Atlantic silverside reference transcriptome (Therkildsen and Baumann <xref rid=\"evl3189-bib-0071\" ref-type=\"ref\">2020</xref>) to a final average depth of 1.5×. Because several clusters of single nucleotide polymorphisms (SNPs) showed fixation for either the reference or alternate allele in each of our four original populations (details below), precluding robust analysis of LD, we added a second round of sequencing of samples from an intermediate location, NC, located near Cape Hatteras, a region of steep thermal change and a phylogeographic break for a number of marine species (Briggs and Bowen <xref rid=\"evl3189-bib-0008\" ref-type=\"ref\">2012</xref>; Pappalardo et al. <xref rid=\"evl3189-bib-0058\" ref-type=\"ref\">2015</xref>). For these samples, individually barcoded genomic libraries were prepared with a similar protocol and sequenced using paired‐end 75 bp reads (see Methods in the Supporting Information for details), and mapped using the same parameters as for the other samples.</p><p>We provide a concise overview of each step in our analysis here; additional details are provided in the Methods section of the Supporting Information. To avoid calling spurious variants due to differences in read length (Leigh et al. <xref rid=\"evl3189-bib-0047\" ref-type=\"ref\">2018</xref>), biallelic SNPs were called across the four original populations (GA, NY, GoM, and GSL; <italic>n</italic> = 189) in the program ANGSD version 0.912 (Korneliussen et al. <xref rid=\"evl3189-bib-0045\" ref-type=\"ref\">2014</xref>), and downstream analyses were performed using this set of SNPs for all five populations. The filtered dataset comprised 1,904,119 biallelic SNPs across the ∼52 MB transcriptome. Contigs had a mean of 92.57 SNPs and an average of ∼1 SNP per 24 bp (the average contig length was 2537bp), and high overall nucleotide diversity within all populations (Table S1). Except where noted, all downstream analyses were based on genotype likelihoods to incorporate uncertainty in genotyping.</p></sec><sec id=\"evl3189-sec-0040\"><title>POPULATION GENOMIC ANALYSIS</title><p>Major and minor alleles, minor allele frequencies (MAF), and <italic>F</italic>\n<sub>ST</sub> were estimated in ANGSD from genotype likelihoods for each SNP with data for at least 10 individuals per population (Korneliussen et al. <xref rid=\"evl3189-bib-0045\" ref-type=\"ref\">2014</xref>). To identify <italic>F</italic>\n<sub>ST</sub> outlier SNPs in the transcriptome, we compared the global <italic>F</italic>\n<sub>ST</sub> (estimated from MAF output by ANGSD) to an expected <italic>χ</italic>\n<sup>2</sup> distribution fit to ∼33,000 SNPs (randomly sampled and pruned for LD), trimming 5% from each side of the <italic>F</italic>\n<sub>ST</sub> distribution in OutFLANK (Whitlock and Lotterhos <xref rid=\"evl3189-bib-0080\" ref-type=\"ref\">2015</xref>). We then applied the <italic>χ</italic>\n<sup>2</sup> fit to all SNPs across the transcriptome to identify <italic>F</italic>\n<sub>ST</sub> outliers using a false discovery rate of <italic>q</italic> < 0.05. Absolute divergence (<italic>d<sub>XY</sub></italic>) for each contig was estimated at all SNPs from MAF output by ANGSD using the CalcDxy.R script in ngsTools (Fumagalli et al. <xref rid=\"evl3189-bib-0028\" ref-type=\"ref\">2014</xref>), accounting for the length of each contig. Principal components analysis (PCA) was performed by computing eigenvectors in R from the covariance matrix between individuals estimated in PCAngsd (Meisner and Albrechtsen <xref rid=\"evl3189-bib-0051\" ref-type=\"ref\">2018</xref>). We also performed admixture analysis using PCAngsd, which estimates the optimal number of clusters (<italic>K</italic>) and the posterior assignment probabilities of individuals to each cluster. We estimated interpopulation ancestry between NY and GSL, and between GA and NY from a subset of 100,000 SNPs under <italic>K</italic> = 4 in the program <italic>entropy</italic> (Gompert et al. <xref rid=\"evl3189-bib-0030\" ref-type=\"ref\">2014</xref>). To evaluate whether low genome‐wide differentiation reflects connectivity through gene flow, we used the Moments pipeline (Jouganous et al. 2017; Leaché et al. 2019) to fit and compare simple demographic models to the two‐dimensional site frequency spectrum (2dSFS) between GA and NY. One SNP per contig was randomly selected to generate the empirical 2dSFS, and competing models were ranked by Akaike information criterion (AIC). We compared eight evolutionary models for the two populations with the following migration scenarios: (1) no migration, (2) symmetrical migration, and (3) secondary contact. We extended each of these three scenarios in three models allowing for reduced Ne across a proportion of the genome due to background/positive selection, and extended the two gene flow scenarios in two models allowing for decreased migration across a proportion of the genome due to, for example, barrier loci (Rougeux et al. <xref rid=\"evl3189-bib-0065\" ref-type=\"ref\">2017</xref>).</p><p>Within each intermediate latitude population (NC, NY, and GoM), we estimated LD between all pairs of <italic>F</italic>\n<sub>ST</sub> outlier SNPs (identified in the OutFLANK analysis above) using default settings in the program ngsLD (Fox et al. <xref rid=\"evl3189-bib-0027\" ref-type=\"ref\">2019</xref>). LD analysis within GSL and GA was not meaningful because of the near fixation for one allele at almost all outlier SNPs. We used the <italic>D</italic>′ statistic output by ngsLD and filtered out SNPs with MAF <0.1. To define blocks of LD, we first estimated the mean <italic>D</italic>′ value between pairs of contigs with more than two outlier SNPs and then performed hierarchical clustering of pairwise mean LD between contigs in each population in the R package <italic>hclust</italic>. We anchored contigs from the silverside transcriptome onto the genome of a related species, the medaka (<italic>Oryzias latipes</italic>), to approximate the relative positions of silverside contigs. Comparisons of fish genomes tend to show few interchromosomal rearrangements (Amores et al. <xref rid=\"evl3189-bib-0001\" ref-type=\"ref\">2014</xref>; Rondeau et al. <xref rid=\"evl3189-bib-0063\" ref-type=\"ref\">2014</xref>; Pettersson et al. <xref rid=\"evl3189-bib-0059\" ref-type=\"ref\">2019</xref>), and thus we expect to sort contigs correctly to chromosomes, but we do not expect to infer the location of a contig within a chromosome. Although we leveraged the medaka genome to infer the likelihood that LD clusters are physically linked on the same chromosome, all analyses of LD blocks were based on hierarchical clustering of silverside contigs without using any genomic information from medaka. To visualize haplotype patterns within LD blocks, we created heatmaps showing the most likely genotypes (inferred from genotype likelihoods for each individual at outlier SNPs), defining the “southern” allele as the major allele in GA.</p><p>We used snpEff to predict the functional effects of SNPs (Cingolani et al. <xref rid=\"evl3189-bib-0010\" ref-type=\"ref\">2012</xref>), and tested for enrichment of variant types in the top 1% tail of the <italic>F</italic>\n<sub>ST</sub> distribution, and among SNPs in LD blocks, relative to their proportions across the rest of the transcriptome using a <italic>χ</italic>\n<sup>2</sup> goodness‐of‐fit test. Contigs were annotated with gene functions based on sequence similarity to reference fish species. We tested for enrichment of Biological Processes GO terms in each LD block by scoring each gene with the sum of outlier SNPs, and tested significance using the gene score resampling (GSR) method in the program <italic>ErmineJ</italic> (Gillis et al. <xref rid=\"evl3189-bib-0029\" ref-type=\"ref\">2010</xref>).</p></sec></sec><sec id=\"evl3189-sec-0050\"><title>Results and Discussion</title><sec id=\"evl3189-sec-0060\"><title>NEAR FIXATION OF OPPOSITE ALLELES ACROSS HUNDREDS OF GENES DESPITE LOW GENOME‐WIDE DIFFERENTIATION</title><p>All five populations formed distinct clusters along principal components of genome‐wide variation (Fig. <xref rid=\"evl3189-fig-0001\" ref-type=\"fig\">1B</xref>), but the overall level of differentiation was relatively low (median <italic>F</italic>\n<sub>ST</sub> range 0.006‐0.036 between neighboring populations). Set within this background of low genome‐wide differentiation, 32,384 of the ∼1.9 M total SNPs (1.70%) showed greater differentiation than expected from a neutral <italic>χ</italic>\n<sup>2</sup> distribution (false discovery rate <italic>q</italic> < 0.05 in OutFLANK; Fig. <xref rid=\"evl3189-fig-0001\" ref-type=\"fig\">1C</xref>), pointing to nonneutral evolutionary forces acting on the genome. Although most differentiated SNPs follow a cline in allele frequencies across latitudes, the extreme <italic>F</italic>\n<sub>ST</sub> outlier SNPs (<italic>q</italic> < 0.05) mostly differentiate GA from all the populations to the north (Fig. <xref rid=\"evl3189-fig-0001\" ref-type=\"fig\">1D</xref>).</p><p>The majority of the 32,384 outlier SNPs showed strong LD patterns in populations at the center of the geographic range where allele frequencies are intermediate. Hierarchical clustering of LD across contigs revealed that 1171 out of 1710 contigs with extreme outlier SNPs cluster into one of four distinct blocks of high LD (Fig. S1). Anchoring contigs along the medaka (<italic>Oryzias latipes</italic>) genome indicate that the contigs in the four LD blocks predominantly map to four different medaka chromosomes (Fig. <xref rid=\"evl3189-fig-0002\" ref-type=\"fig\">2</xref>). Three large blocks (comprising 263, 255, and 391 contigs, respectively) that are polymorphic in the NC population correspond to medaka chromosome (Chr) 8, 18, and 24 (89.5%, 82.3%, and 91.2% of mappable contigs map to these chromosomes, respectively; Fig. <xref rid=\"evl3189-fig-0002\" ref-type=\"fig\">2A</xref>). In GoM, another distinct LD block of 263 contigs maps mostly to medaka Chr 11 (72.9% of mappable contigs; Fig. <xref rid=\"evl3189-fig-0002\" ref-type=\"fig\">2C</xref>).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3189-fig-0002\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Linkage disequilibrium (LD) between pairs of contigs with outlier SNPs in North Carolina (NC), New York (NY), and Gulf of Maine (GoM). Pixels are colored by mean <italic>D</italic>′ across outlier SNPs in each contig pair. (White pixels show contigs with fewer than two outlier SNPs with MAF > 0.1). Hierarchical clustering of LD between contig pairs clusters all these contigs into four LD blocks (see Fig. S1), indicated by row panels in the symmetrical matrix. Column panels indicate the chromosome each contig maps to in the medaka genome, showing large proportions of contigs in LD blocks mapping to a single medaka chromosome. GA and GSL are not shown because the near fixation of alleles in these populations limits the power of LD analysis.</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"EVL3-4-430-g002\"/></fig><p>The membership of genes in LD blocks (hereafter referred to as LD blocks 8, 11, 18, and 24) was defined without any a priori information about chromosomal locations in the medaka genome, and thus the high degree of co‐localization on single medaka chromosomes suggests that the contigs within each LD block are likely to be physically linked in the Atlantic silverside genome. Silversides and medaka diverged ∼75 million years ago (Near et al. <xref rid=\"evl3189-bib-0055\" ref-type=\"ref\">2012</xref>), but the species share the same chromosome number, 2<italic>n</italic> = 48 (Warkentine et al. <xref rid=\"evl3189-bib-0077\" ref-type=\"ref\">1987</xref>), and comparisons of genomes both within the Atherinomorpha clade (to which both species belong; Amores et al. <xref rid=\"evl3189-bib-0001\" ref-type=\"ref\">2014</xref>; Miller et al. <xref rid=\"evl3189-bib-0052\" ref-type=\"ref\">2019</xref>) and fishes more broadly (Rondeau et al. <xref rid=\"evl3189-bib-0063\" ref-type=\"ref\">2014</xref>; Pettersson et al. <xref rid=\"evl3189-bib-0059\" ref-type=\"ref\">2019</xref>), tend to show few interchromosomal rearrangements. Thus, although potential intrachromosomal rearrangements limit our ability to infer the order of genes within chromosomes, the nonrandom clustering of outlier SNPs on medaka chromosomes (Fig. <xref rid=\"evl3189-fig-0003\" ref-type=\"fig\">3A</xref>), and strong LD among silverside contigs mapping to the same medaka chromosomes (but lower LD among those mapping to different chromosomes; Fig. <xref rid=\"evl3189-fig-0002\" ref-type=\"fig\">2</xref>), suggests that the medaka genome provides a reasonable approximation of the genomic location of our contigs.</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3189-fig-0003\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Variation among pairs of Atlantic silverside populations across contigs anchored to the medaka genome, showing overrepresentation of differentiated SNPs on the four medaka chromosomes corresponding to LD blocks in Figure <xref rid=\"evl3189-fig-0002\" ref-type=\"fig\">2</xref>. Few interchromosomal rearrangements between fish genomes suggest that contigs sort correctly to chromosomes, but we do not expect to robustly infer the location of a contig within a chromosome. (A) Observed versus expected number of global <italic>F</italic>\n<sub>ST</sub> outlier SNPs on each medaka chromosome. (B‐F) <italic>F</italic>\n<sub>ST</sub> at individual SNPs for (B) GoM versus GSL, (C) NY versus GoM, (D) NC versus NY, (E) GA versus NC, and (F) GA versus NY, with colored lines showing sliding mean <italic>F</italic>\n<sub>ST</sub>. (G‐I) Comparison of the distribution of <italic>d<sub>XY</sub></italic> between population pairs for contigs in LD blocks 8, 11, 18, and 24 and contigs outside blocks (OB). Asterisks above violin plots indicate significantly elevated <italic>d<sub>xy</sub></italic> in blocks relative to OB; dashes indicate significantly reduced <italic>d<sub>xy</sub></italic> relative to OB. <sup>***/‐‐‐</sup>\n<italic>P</italic> < 1× 10<sup>–4</sup>; <sup>**</sup>\n<italic><sup>/‐‐</sup> P</italic> < 1× 10<sup>–3</sup>; <sup>*/‐</sup>\n<italic>P</italic> < 0.05.</p></caption><graphic id=\"nlm-graphic-5\" xlink:href=\"EVL3-4-430-g003\"/></fig><p>Pairwise comparisons show that the most striking heterogeneity in genomic differentiation is found within the southernmost phylogeographic region (Lou et al. <xref rid=\"evl3189-bib-0048\" ref-type=\"ref\">2018</xref>), between GA and NY (Fig. <xref rid=\"evl3189-fig-0003\" ref-type=\"fig\">3F</xref>). Separated by ∼1500 km of coastline, the three populations sampled in this region had the lowest baseline levels of differentiation (genome‐wide median <italic>F</italic>\n<sub>ST</sub> = 0.009, 0.007, and 0.006 between GA and NC, NC and NY, and GA and NY, respectively), yet 13,734 SNPs were fixed or nearly fixed (<italic>F</italic>\n<sub>ST</sub> > 0.95) for opposite alleles between GA and NY. SNPs in the top 1% of the <italic>F<sub>ST</sub></italic> distribution were disproportionately nonsynonymous (NS) variants (29.1% more NS variants than the rest of the transcriptome; <italic>χ</italic>\n<sup>2</sup> goodness of fit test; <italic>P</italic> < 2.2 × 10<sup>–16</sup>; Table S2), and the vast majority (99.1%) of them clustered into LD blocks 8, 18, and 24 (with 82.0% mapping to medaka chromosomes 8, 18, and 24, Fig. <xref rid=\"evl3189-fig-0003\" ref-type=\"fig\">3A</xref>). There was a clear north‐south split of alleles at these blocks across the range; the three northern populations share the NY allele at almost all outlier SNPs, whereas the opposite alleles are found almost exclusively in GA. In NC, near Cape Hatteras, a region of steep thermal transition, northern and southern alleles on blocks 8, 18, and 24 were at intermediate frequencies (Figs. <xref rid=\"evl3189-fig-0003\" ref-type=\"fig\">3D</xref> and <xref rid=\"evl3189-fig-0003\" ref-type=\"fig\">3E</xref>).</p><p>Genes in the three LD blocks show elevated levels of absolute divergence (<italic>d<sub>XY</sub></italic>) between GA and NY compared to the rest of the genome (Fig. <xref rid=\"evl3189-fig-0003\" ref-type=\"fig\">3I</xref>). This pattern is consistent with a model of differential gene flow where selection promotes divergence at locally adaptive loci, whereas gene flow homogenizes the rest of the genome (Nachman and Payseur <xref rid=\"evl3189-bib-0054\" ref-type=\"ref\">2012</xref>; Cruickshank and Hahn <xref rid=\"evl3189-bib-0020\" ref-type=\"ref\">2014</xref>). By contrast, among populations where alleles at these LD blocks are shared (i.e., NY, GoM, and GSL), <italic>d<sub>XY</sub></italic> across these loci is reduced relative to genome‐wide levels (Fig. <xref rid=\"evl3189-fig-0003\" ref-type=\"fig\">3G‐H</xref>), indicating linked selection on a shared allele in both populations or in an ancestral population (Irwin et al. <xref rid=\"evl3189-bib-0036\" ref-type=\"ref\">2016</xref>; Delmore et al. <xref rid=\"evl3189-bib-0021\" ref-type=\"ref\">2018</xref>).</p><p>The LD blocks are also enriched for functional variants relative to the rest of the transcriptome, consistent with selection on functional genetic variation (Table S3). LD block 18 had 35.1% more NS variants and LD block 24 had 16.4% more NS variants than expected from transcriptome‐wide proportions (<italic>χ</italic>\n<sup>2</sup> goodness‐of‐fit, both <italic>P</italic> < 2.2 × 10<sup>–16</sup>; Table S3). Although LD block 8 did not show significant enrichment of NS variants, it had 9.0% more UTR variants (<italic>χ</italic>\n<sup>2</sup> goodness‐of‐fit, <italic>P</italic> < 2.2 × 10<sup>–16</sup>; Table S3). The extreme degree of differentiation, enrichment of functional variation, and elevated levels of absolute divergence across the large LD blocks exceed expectations under neutral evolution (Figs. <xref rid=\"evl3189-fig-0001\" ref-type=\"fig\">1C</xref> and <xref rid=\"evl3189-fig-0003\" ref-type=\"fig\">3G‐I</xref>), and support a role for selection in shaping them.</p></sec><sec id=\"evl3189-sec-0070\"><title>EVIDENCE OF SUPPRESSED RECOMBINATION WITHIN LARGE LD BLOCKS</title><p>In the three blocks that are polymorphic along the southern coastline, we quantified genotype likelihoods at outlier SNPs to evaluate the distribution of alleles among individuals. In LD blocks 18 and 24, homozygous individuals had either the northern or southern allele (but not both) at virtually all SNPs, but because the sequencing at ∼1× coverage captures only one allele on average, heterozygous genotypes were a mosaic of the two alleles. In GA and NY, virtually all copies of locally rare alleles at these SNPs were present in the same few individuals (Figs. <xref rid=\"evl3189-fig-0004\" ref-type=\"fig\">4A</xref> and <xref rid=\"evl3189-fig-0004\" ref-type=\"fig\">B</xref>). There was a cline in frequencies of northern and southern haplotypes of both blocks across the three southern populations, with few observations of heterozygotes outside of NC. In LD block 24, four individuals in three populations outside of NC carried both haplotypes (Fig. <xref rid=\"evl3189-fig-0004\" ref-type=\"fig\">4A</xref>), and in LD block 18, there was one heterozygote in GA (of <italic>n</italic> = 48 individuals), and no southern haplotypes north of NC (Fig. <xref rid=\"evl3189-fig-0004\" ref-type=\"fig\">4B</xref>). Block 8 showed looser associations of alleles across SNPs, but the distinct north‐south haplotype structure was largely maintained, and we observed no southern haplotypes in the three northern populations (Fig. <xref rid=\"evl3189-fig-0004\" ref-type=\"fig\">4C</xref>). The fact that heterozygous individuals appear to be heterozygous across the entire blocks, and homozygous individuals are homozygous across them, suggests very strong linkage of northern and southern alleles (respectively) across extended haplotypes.</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3189-fig-0004\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Distribution of genotypes across LD blocks. Genotype likelihoods for silverside individuals (rows) at each outlier SNP (columns) in (A) LD block 24, (B) LD block 18, (C) LD block 8, and (D) LD block 11. Barplots (left) show the average genotype (number of southern alleles) across SNPs for each individual, colored by sampling origin (red: GA, <italic>n</italic> = 48; orange: NC, <italic>n</italic> = 47; purple: NY, <italic>n</italic> = 49; green: GoM, <italic>n</italic> = 50; blue: GSL, <italic>n</italic> = 42). Because of low read depth, heterozygotes appear as mosaics of each of the three possible genotypes (NN, NS, and SS) and have an average number of southern alleles of ∼1 across SNPs.</p></caption><graphic id=\"nlm-graphic-7\" xlink:href=\"EVL3-4-430-g004\"/></fig><p>Long haplotypes in almost perfect LD within populations imply that divergent alleles have limited recombination. Such large swaths (spanning hundreds of genes) of maintained LD point to inversions or other mechanisms of recombination suppression, which may maintain linked genes as stable polymorphisms. Inversions that capture multiple locally adapted alleles can have a selective advantage, and are particularly important when gene flow is high (Noor et al. <xref rid=\"evl3189-bib-0056\" ref-type=\"ref\">2001</xref>; Kirkpatrick and Barton <xref rid=\"evl3189-bib-0043\" ref-type=\"ref\">2006</xref>). An alternative explanation for high LD is recent admixture of differentiated alleles, but LD decays very rapidly when recombination is unrestricted (Hartl et al. <xref rid=\"evl3189-bib-0032\" ref-type=\"ref\">1997</xref>). Yet in NC where frequencies of northern and southern alleles were intermediate and heterozygosity was high (providing plenty of opportunity for recombination), the distinct haplotypes are maintained, suggesting suppressed recombination is maintaining LD across these haplotypes. This conclusion is further corroborated by our previous report of a strong response to size selection on LD block 24 in a laboratory experiment (Therkildsen et al. <xref rid=\"evl3189-bib-0073\" ref-type=\"ref\">2019</xref>; the other LD blocks were not identified in the experiment). Established from silversides collected near NY, the initially rare southern haplotype showed a dramatic increase in frequency under strong selection, yet did not recombine with the northern haplotype over 10 generations despite high heterozygosity in the experimental population.</p><p>Recombination suppression (e.g., within inversions) may lead to the accumulation of deleterious mutations, providing an alternate explanation for NS variant enrichment seen across these blocks. But because most individuals were homozygous for northern or southern alleles (Fig. <xref rid=\"evl3189-fig-0004\" ref-type=\"fig\">4</xref>), recombination within inversion types can facilitate purging of these mutations (Otto and Lenormand <xref rid=\"evl3189-bib-0057\" ref-type=\"ref\">2002</xref>; Kirkpatrick <xref rid=\"evl3189-bib-0042\" ref-type=\"ref\">2010</xref>), making this explanation less plausible than one of divergent selection on adaptive variation. Although having a contiguous genome will elucidate the genomic mechanisms underlying the recombination suppression revealed in these analyses, the transcriptome data make a clear case for strong selection on suites of genes in LD.</p></sec><sec id=\"evl3189-sec-0080\"><title>ATLANTIC SILVERSIDES AS AN EXTREME EXAMPLE OF THE GENOMIC PATTERNS UNDERLYING LOCAL ADAPTATION</title><p>The exact width of the highly linked blocks of divergence in the Atlantic silverside genome remains to be determined, but alignment to the medaka genome suggests that they could span much of the four chromosomes on which they are found (Fig. <xref rid=\"evl3189-fig-0003\" ref-type=\"fig\">3</xref>). The total length of the 908 contigs that make up the three LD blocks (8, 18, and 24), which are highly differentiated across the southern region between GA and NY, amount to ∼2.88 MB in length. However, the reference contigs exclude introns and intergenic regions that intersperse the transcriptome sequence, indicating that these footprints must span multiple megabases of the Atlantic silverside genome.</p><p>Localized genomic regions of elevated intraspecific differentiation have now been identified in numerous species, but often the stretches of elevated differentiation span smaller genomic regions, as is the case for the stickleback (<italic>Gasterosteus aculeatus</italic>; Jones et al. <xref rid=\"evl3189-bib-0038\" ref-type=\"ref\">2012</xref>) and African honeybee (Wallberg et al. <xref rid=\"evl3189-bib-0075\" ref-type=\"ref\">2017</xref>). In other species, such as the Atlantic cod (<italic>Gadus morhua</italic>), Atlantic herring (<italic>Clupea harengus</italic>), and maize (<italic>Zea mays</italic>), Mb‐scale inversions are under strong divergent selection, but rarely approach fixation of opposite haplotypes between populations (Fang et al. <xref rid=\"evl3189-bib-0025\" ref-type=\"ref\">2012</xref>; Pettersson et al. <xref rid=\"evl3189-bib-0059\" ref-type=\"ref\">2019</xref>; Kess et al. <xref rid=\"evl3189-bib-0041\" ref-type=\"ref\">2020</xref>). The genomic breadth and magnitude of allele frequency differentiation (i.e., near fixation of opposite alleles across Mb‐scale regions against an almost complete absence of genome‐wide differentiation, as seen in the southern part of the range) represents, to our knowledge, one of the most extreme examples reported to date of the degree to which differentiation of locally adapted populations can vary across the genome (compare to other notable examples in Table <xref rid=\"evl3189-tbl-0001\" ref-type=\"table\">1</xref>). The increasing availability of genome‐wide SNP data for many species will make the identification of clusters of differentiated alleles easier, and determine whether adaptation to large‐scale environmental differentiation is regularly associated with extreme differentiation across gene clusters.</p><table-wrap id=\"evl3189-tbl-0001\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Examples of intraspecific differentiation generated by local adaptation in the face of gene flow across populations</p></caption><table frame=\"hsides\" rules=\"groups\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><thead><tr style=\"border-bottom:solid 1px #000000\" valign=\"bottom\"><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Species</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Differentiation driven by selection</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Size of differentiated region</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Adaptation</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">Genome‐wide differentiation</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">References</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Zea mays</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">AF range ∼0.2‐0.9 across altitudinal cline</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">∼50 MB inversion (700 genes)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Associated with environmental variables and phenotypic traits</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Isolation by distance</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fang et al. <xref rid=\"evl3189-bib-0025\" ref-type=\"ref\">2012</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Clupea harengus</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Max <italic>F</italic>\n<sub>ST</sub> ∼0.9</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">7.2 MB</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Salinities and spawning times</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Median <italic>F</italic>\n<sub>ST</sub> = 0.032</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Barrio et al. <xref rid=\"evl3189-bib-0006\" ref-type=\"ref\">2016</xref>; Pettersson et al. <xref rid=\"evl3189-bib-0059\" ref-type=\"ref\">2019</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Gadus morhua</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Max <italic>F</italic>\n<sub>ST</sub> of SNPs within inversions ∼0.7‐0.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Several MB, ∼4% of genome</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Salinity, temperature, and migration ecotypes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Neutral <italic>F</italic>\n<sub>ST</sub> = 0.0012</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hemmer‐Hansen et al. <xref rid=\"evl3189-bib-0033\" ref-type=\"ref\">2013</xref>; Kess et al. <xref rid=\"evl3189-bib-0041\" ref-type=\"ref\">2020</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Drosophila melanogaster</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">AF range ∼0.2‐1.0 across latitudinal cline</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">∼5‐15 MB inversion</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Size, development, fecundity, other traits</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>F</italic>\n<sub>ST</sub> ∼0.027‐0.044</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fabian et al. <xref rid=\"evl3189-bib-0024\" ref-type=\"ref\">2012</xref>; Kapun et al. <xref rid=\"evl3189-bib-0040\" ref-type=\"ref\">2016</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Apis mellifera</italic>\n</p>\n<p>subspecies</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Two putative inversions, <italic>F</italic>\n<sub>ST</sub> = 0.7 and 0.8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">100s of Kb</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Highland habitat</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>F</italic>\n<sub>ST</sub> = 0.05‐0.07</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Wallberg et al. <xref rid=\"evl3189-bib-0075\" ref-type=\"ref\">2017</xref>\n</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Menidia menidia</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>F</italic>\n<sub>ST</sub> > 0.95 for 13,734 SNPs</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Unknown, likely megabases</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">GO terms related to known adaptations (e.g. lipid storage, metabolism, spawning)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<p>Median <italic>F</italic>\n<sub>ST</sub> = 0.006</p>\n<p>(GA vs. NY)</p>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">This study</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd.</copyright-holder></permissions></table-wrap></sec><sec id=\"evl3189-sec-0090\"><title>MULTIPLE ADAPTIVE CLINES ACROSS A GRADIENT OF GENOME‐WIDE POPULATION DIFFERENTIATION</title><p>In the middle of the silverside range between NY and GoM, genome‐wide differentiation was intermediate (median <italic>F</italic>\n<sub>ST</sub> = 0.024), but there were no SNPs fixed for opposite alleles (max <italic>F</italic>\n<sub>ST</sub> = 0.93). The most highly differentiated SNPs fell into contigs that formed a tight LD block corresponding to medaka Chr 11 (Fig. <xref rid=\"evl3189-fig-0002\" ref-type=\"fig\">2C</xref>). Genes within LD block 11 (spanning 263 contigs that cumulatively encompass 0.70 MB of the transcriptome) had significantly elevated levels of <italic>d<sub>XY</sub></italic> between NY and GoM (Fig. <xref rid=\"evl3189-fig-0003\" ref-type=\"fig\">3H</xref>), and 5.8% more NS variants than the rest of the transcriptome (<italic>χ</italic>\n<sup>2</sup> goodness‐of‐fit, <italic>P</italic> < 1 × 10<sup>–3</sup>; Table S3), but unlike the three LD blocks that distinguish the southern population (GA) from the northern, alleles of LD block 11 transition at a higher latitude, with the southern haplotype predominant in the three southern populations, and the northern haplotype predominant in the North. The different geographic breakpoints among the LD blocks on different chromosomes suggest that they are driven by separate selection pressures across latitudes, consistent with multiple, distinct latitudinal clines for different phenotypic traits in Atlantic silversides (Conover et al. <xref rid=\"evl3189-bib-0019\" ref-type=\"ref\">2009</xref>; Hice et al. <xref rid=\"evl3189-bib-0034\" ref-type=\"ref\">2012</xref>).</p><p>In contrast to the highly connected southern coastline, the two most northerly samples, GoM and GSL, had the highest background level of differentiation among neighboring locations (median <italic>F</italic>\n<sub>ST</sub> = 0.036), reflecting the history of independent colonization of GoM and GSL from the southern coastline (Lou et al. <xref rid=\"evl3189-bib-0048\" ref-type=\"ref\">2018</xref>). Despite the higher level of background differentiation, no SNP was fixed for opposite alleles (max <italic>F</italic>\n<sub>ST</sub> = 0.91), and there was little obvious clustering of highly differentiated SNPs on medaka chromosomes (Fig. <xref rid=\"evl3189-fig-0003\" ref-type=\"fig\">3B</xref>).</p></sec><sec id=\"evl3189-sec-0100\"><title>LONG‐TERM AND CONTEMPORARY MIGRATION AND GENE FLOW</title><p>Admixture analysis of all SNPs indicate the genomic data best fit a model of <italic>K</italic> = 3 populations, with a gradient in posterior assignment to populations 1 and 2 of individuals in GA, NC, and NY (Fig. <xref rid=\"evl3189-fig-0005\" ref-type=\"fig\">5</xref>). When SNPs on the four LD blocks are excluded from the analysis, individuals from GA, NC, and NY all show high assignment (approximately >75%) to population 1. Similarly, PCA of SNPs excluding those in LD blocks shows much less separation and more overlap of the three populations south of Cape Cod (Fig. S3). Furthermore, genome‐wide levels of differentiation south of Cape Cod appear to increase steadily with geographic distance, but the slope is far shallower when differentiation within LD blocks is excluded (Fig. S4). Cumulatively, these results indicate high connectivity across this region, with differentiation of the LD blocks accounting for most of the structure.</p><p>The 2dSFS, which summarizes the number of SNPs with a given frequency in two populations, can be shaped by both neutral and nonneutral forces. We compared the fit of 2dSFS generated from demographic models with and without gene flow, and including the heterogeneous effects of selection and differential introgression on the genome (Rougeaux et al. <xref rid=\"evl3189-bib-0065\" ref-type=\"ref\">2017</xref>), to the observed 2dSFS between GA and NY. All models that allowed for reduced effective population size (<italic>N</italic>\n<italic><sub>e</sub></italic>) across a proportion of the genome due to selection fit consistently better than models that did not, and models that included migration fit better than those that did not (Table S4; Fig. S5). The best ranking model was one of symmetrical migration with ∼12% of the genome having <italic>N</italic>\n<italic><sub>e</sub></italic> reduced to 0.013 that of the rest of the genome. Overall, the SFS of models that included the effects of selection were able to capture the highly differentiated alleles along the edges of the SFS (Figs. <xref rid=\"evl3189-fig-0005\" ref-type=\"fig\">5C</xref> and <xref rid=\"evl3189-fig-0005\" ref-type=\"fig\">5D</xref>), suggesting that these sites were under recurrent selection (Charlesworth <xref rid=\"evl3189-bib-0009\" ref-type=\"ref\">2009</xref>), whereas models that did not include heterogeneity in the genome underpredicted the frequency of these sites in the SFS (Fig. <xref rid=\"evl3189-fig-0005\" ref-type=\"fig\">5E</xref>).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3189-fig-0005\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><p>Admixture analysis under an optimally fitted model of <italic>K</italic> = 3 populations shows a cline in posterior assignment of individuals from GA, NC, and NY using all genome‐wide SNPs (A), but shows high assignment of individuals from these locations to one cluster using only SNPs outside LD blocks (B). Observed 2dSFS for GA and NY (C) showed the best fit to a model of symmetrical migration with reduced <italic>N</italic>\n<italic><sub>e</sub></italic> at a subset of loci due to selection (D), compared to other candidate models such as symmetrical migration without selection (E), and those of divergence in isolation without migration. Models that included the effects of selection were able to capture the highly differentiated alleles along the edges of the 2dSFS (D), whereas models that did not include heterogeneity in the genome underpredicted the frequency of these sites in the 2dSFS (E).</p></caption><graphic id=\"nlm-graphic-9\" xlink:href=\"EVL3-4-430-g005\"/></fig><p>The finding of ongoing migration between these two populations corroborates the more detailed demographic history reconstructed with Approximate Bayesian computation based on full mitochondrial genome sequences of these same individuals (Lou et al. <xref rid=\"evl3189-bib-0048\" ref-type=\"ref\">2018</xref>). Continuous migration models also outperformed secondary contact models, and the optimal estimate for the duration of time in isolation in the best models of secondary contact was small (Table S4). This structure differs from the classic clines in metabolic genes of <italic>Fundulus heteroclitus</italic> along the same coastline (Powers and Place <xref rid=\"evl3189-bib-0061\" ref-type=\"ref\">1978</xref>; Ropson et al. <xref rid=\"evl3189-bib-0064\" ref-type=\"ref\">1990</xref>), which occur across a geographic region shaped by secondary contact, making it difficult to fully disentangle effects of selection from demographic history in <italic>F. heteroclitus</italic> (Strand et al. <xref rid=\"evl3189-bib-0070\" ref-type=\"ref\">2012</xref>). In contrast, the fixation of opposite alleles among Atlantic silverside populations spans a coastline (GA to NY) with neither a historical mitochondrial lineage split (Lou et al. <xref rid=\"evl3189-bib-0048\" ref-type=\"ref\">2018</xref>) nor evidence of a period of divergence in isolation in the nuclear genome, and therefore is unlikely to be driven by secondary contact.</p><p>In addition to long‐term migration along the southern coastline, we observed four individuals with very rare haplotypes on outlier chromosomes relative to their capture location; at least two likely stem from recent migration events. One silverside from GSL (of <italic>n</italic> = 42) appears to have originated near NY based on transcriptome‐wide variation (Figs. <xref rid=\"evl3189-fig-0001\" ref-type=\"fig\">1A</xref> and S3B). It was homozygous for southern haplotypes on LD block 11 and heterozygous on LD block 24 (a combination of haplotypes observed in some NY individuals), and thus was likely a first‐generation migrant. In GA (<italic>n</italic> = 48), one individual was heterozygous on LD blocks 18 and 24, and comprised the only observation of northern haplotypes on these blocks in GA. Heterozygosity on two LD blocks suggests that this individual may be a recent cross between GA and NY (or an intermediate location). Two NY individuals (of <italic>n</italic> = 49) were heterozygous for only LD block 24 and no other blocks, indicating that they were either F2 backcrosses from a GA migrant or there is stable polymorphism of both LD block 24 haplotypes in NY. Estimates of intersource population ancestry of individuals from GA and NY support the inference that these heterozygous individuals are interpopulation hybrids resulting from recent admixture (i.e., have at least one nonadmixed parent; Fig. S2A), whereas the individual sampled in GSL shows high posterior assignment to NY with no evidence of interpopulation ancestry (Fig. S2B).</p><p>Observations of at least two recent long‐distance migration events out of fewer than 200 fish further support ongoing gene flow as an explanation for the limited neutral structure, and indicate not only local stepping‐stone migration, but also frequent long‐distance dispersal events that may stem from the winter migration offshore to the continental shelf at northern latitudes (Clarke et al. <xref rid=\"evl3189-bib-0011\" ref-type=\"ref\">2010</xref>). Evidence of historical connectivity through demographic modeling (Lou et al. <xref rid=\"evl3189-bib-0048\" ref-type=\"ref\">2018</xref>) as well as contemporary gene flow is bolstered by direct dispersal estimates based on otolith chemistry (Clarke et al. <xref rid=\"evl3189-bib-0011\" ref-type=\"ref\">2010</xref>). Elevated absolute divergence (<italic>d<sub>XY</sub></italic>) within LD blocks suggests selection against maladaptive gene flow at these loci (Cruickshank and Hahn <xref rid=\"evl3189-bib-0020\" ref-type=\"ref\">2014</xref>). The nearly complete absence of alternate haplotypes across LD blocks, except in recent migrants or their offspring, between otherwise highly connected populations (e.g., GA and NY) indicates very rapid selection against migrant haplotypes at these loci.</p></sec><sec id=\"evl3189-sec-0110\"><title>GREATER CLUSTERING OF OUTLIER LOCI ACROSS REGIONS WITH HIGH GENE FLOW</title><p>The geographic region most strongly connected by gene flow was where we found the most and the largest outlier blocks. Further north, gene flow appears to be more restricted, yet much smaller chromosomal regions show elevated differentiation. Prior phenotypic work demonstrates adaptive clines through both the northern and southern part of the Atlantic silverside range (Hice et al. <xref rid=\"evl3189-bib-0034\" ref-type=\"ref\">2012</xref>). Although the full genomic architecture underlying these adaptations remains to be uncovered, our findings here of more clustered genomic signatures of selection in the South are consistent with theoretical predictions that higher gene flow results in more linked architecture, usually by inversions (Kirkpatrick and Barton <xref rid=\"evl3189-bib-0043\" ref-type=\"ref\">2006</xref>). Support for this prediction has been shown at the interspecific level in <italic>Drosophila</italic>, passerines, rodents, and <italic>Helianthus</italic> sunflowers, where sympatric species with greater potential for introgression have more inversion polymorphisms that differentiate them than allopatric species (reviewed in Wellenreuther and Bernatchez <xref rid=\"evl3189-bib-0078\" ref-type=\"ref\">2018</xref>). The strongest evidence for intraspecific adaptive inversions comes from environmental clines, for example, inversions associated with climatic gradients in <italic>Drosophila melanogaster</italic>, <italic>Anopheles</italic> mosquitoes, and <italic>Mimulus guttatus</italic>, and with biotic and abiotic clines in <italic>Littorina saxatilis</italic> (Faria et al. <xref rid=\"evl3189-bib-0026\" ref-type=\"ref\">2018</xref>; Wellenreuther and Bernatchez <xref rid=\"evl3189-bib-0078\" ref-type=\"ref\">2018</xref>), reflecting inversions as a common mechanism for adaptive divergence along continuous environmental gradients.</p></sec><sec id=\"evl3189-sec-0120\"><title>GENES IN LD BLOCKS HAVE FUNCTIONS RELATED TO MULTIPLE ADAPTATIONS</title><p>Near perfect LD across blocks of highly differentiated SNPs indicates that alleles at these loci are nearly always associated (even if not physically linked). To examine potential functions associated with the LD blocks, we tested for GO enrichment of genes within each of them relative to the rest of the genome. Highly differentiated genes (ranked by the number of OutFLANK outlier SNPs) in LD block 8 were enriched for genes involved in humoral immune response (26 significant terms in total; Table S5). Genes involved in renal filtration were also enriched in this block, possibly reflecting adaptation to local salinity conditions, as the kidney is a major osmoregulatory organ in teleosts (Marshall and Grosell <xref rid=\"evl3189-bib-0050\" ref-type=\"ref\">2006</xref>). LD block 18 was enriched for terms related to metabolic processes, mirroring variation in metabolic rates across latitudes in Atlantic silversides (Conover and Present <xref rid=\"evl3189-bib-0017\" ref-type=\"ref\">1990</xref>; Arnott et al. <xref rid=\"evl3189-bib-0003\" ref-type=\"ref\">2006</xref>), and a number of terms related to synapse maturation and neurotransmitter function, possibly reflecting temperature adaptations in nervous system function (Baldwin <xref rid=\"evl3189-bib-0005\" ref-type=\"ref\">1971</xref>; Montgomery and Macdonald <xref rid=\"evl3189-bib-0053\" ref-type=\"ref\">1990</xref>; Pörtner et al. <xref rid=\"evl3189-bib-0060\" ref-type=\"ref\">2007</xref>), as well as cartilage morphogenesis (29 significant terms; Table S6). On Chr. 24, enriched terms (of 47 significant terms) were related to regulation of lipid storage, reflecting lipid content variation across latitudes demonstrated by common garden experiments (Schultz and Conover <xref rid=\"evl3189-bib-0068\" ref-type=\"ref\">1997</xref>). Other notable enriched terms were related to development and differentiation of Sertoli cells, which play a central role in spermatogenesis in other fish (Rolland et al. <xref rid=\"evl3189-bib-0062\" ref-type=\"ref\">2009</xref>; Bahamonde et al. <xref rid=\"evl3189-bib-0004\" ref-type=\"ref\">2016</xref>), providing a potential link to variation in spawning periods in Atlantic silversides (Table S7; Conover and Kynard <xref rid=\"evl3189-bib-0012\" ref-type=\"ref\">1984</xref>). LD block 24 was also associated with body size in experimental silverside populations (Therkildsen et al. <xref rid=\"evl3189-bib-0073\" ref-type=\"ref\">2019</xref>), indicating a link to variation in growth rate. LD block 11 was enriched for genes that regulate T cell‐mediated immunity and other immune functions (22 significant terms; Table S8). Selective sweeps on immune genes have been shown in a number of other teleost species (e.g., Jensen et al. <xref rid=\"evl3189-bib-0037\" ref-type=\"ref\">2008</xref>; Kjærner‐Semb et al. <xref rid=\"evl3189-bib-0044\" ref-type=\"ref\">2016</xref>). Other notable terms enriched in LD block 11 overlapped with functions in other blocks, for example, glomerulus morphogenesis and lipid organization, and a number of significant GO terms in LD block 11 were related to cardiac function, known to be a limiting factor in temperature tolerance in other fish species (Steinhausen et al. <xref rid=\"evl3189-bib-0069\" ref-type=\"ref\">2008</xref>; Anttila et al. <xref rid=\"evl3189-bib-0002\" ref-type=\"ref\">2014</xref>), and may play a role in adaptation to the steep thermal cline along the Atlantic coast.</p><p>These functional enrichment analyses suggest that the highly differentiated LD blocks may underlie some of the trait divergence already described for the Atlantic silverside. Prior work on both wild and experimental silverside populations have suggested strong correlations among locally adapted traits (Walsh et al. <xref rid=\"evl3189-bib-0076\" ref-type=\"ref\">2006</xref>; Hice et al. <xref rid=\"evl3189-bib-0034\" ref-type=\"ref\">2012</xref>; Salinas et al. <xref rid=\"evl3189-bib-0066\" ref-type=\"ref\">2012</xref>). Artificial selection on growth rate resulted in concomitant shifts in other traits related to growth efficiency, morphology, and fecundity (Walsh et al. <xref rid=\"evl3189-bib-0076\" ref-type=\"ref\">2006</xref>; Salinas et al. <xref rid=\"evl3189-bib-0066\" ref-type=\"ref\">2012</xref>). Our results here provide an indication that genes underlying multiple complex traits may co‐locate to the same LD blocks, potentially representing co‐adapted gene complexes (Kirkpatrick and Barton <xref rid=\"evl3189-bib-0043\" ref-type=\"ref\">2006</xref>).</p><p>It is important to point out that if these LD blocks are associated with inversions, genes within the inversions are not independent. It is possible that clusters of genes with similar functions (from tandem duplications, e.g.) captured within an inversion may lead to apparent enrichment of those functions simply because the genes are physically linked. Additional research is underway to determine whether the LD blocks are associated with chromosomal inversions, as well as genotype‐phenotype‐environment analyses with increased sampling along the latitudinal cline to tease apart the targets of selection. Nonetheless, multiple lines of evidence point to an adaptive role for the LD blocks, and the functional enrichment results give an indication of the major functions of genes within them, even if their adaptive roles remain unclear.</p></sec><sec id=\"evl3189-sec-0130\"><title>IMPLICATIONS</title><p>Virtually all the traits known to vary genetically in Atlantic silversides are now known to do so in numerous other species (Conover et al. <xref rid=\"evl3189-bib-0018\" ref-type=\"ref\">2005</xref>). The genomic patterns generated by local adaptation in silversides are an extreme case of what may be a common feature of wide‐ranging populations distributed across highly connected environmental gradients; thus, Atlantic silversides are a useful model not only for studying adaptive variation vital to preserving population health and adaptive potential, but also for identifying and conserving functional variation in managed and harvested marine species. Although the Atlantic silverside is only caught in commercial fisheries to a limited extent, this species demonstrates how even the most striking adaptations may be cryptic: field observations of similar sizes at maturity across the range mask huge adaptive variation in this key life history trait (Conover and Schultz <xref rid=\"evl3189-bib-0014\" ref-type=\"ref\">1995</xref>), and even the megabase‐scale haplotypes underlying local adaptation were not detected when marker density was low (Mach et al. <xref rid=\"evl3189-bib-0049\" ref-type=\"ref\">2011</xref>). Without information from laboratory experiments and genomic analysis, low genome‐wide differentiation may prompt a decision to manage highly divergent alleles and phenotypes as if they were a nearly panmictic unit, when managing them as interdependent populations with unique adaptive variation would be warranted. This example underscores the importance of incorporating large‐scale genomic data (and, when possible, common garden experiments) to identify and manage adaptive variation, particularly in widely distributed species.</p><p>Our findings also highlight the critical interplay between gene flow and selection in shaping the genomic architecture that lays the foundation for the response to future environmental shifts. Artificial selection has demonstrated the high adaptive potential of Atlantic silversides (Conover and Van Voorhees <xref rid=\"evl3189-bib-0013\" ref-type=\"ref\">1990</xref>; Conover and Munch <xref rid=\"evl3189-bib-0016\" ref-type=\"ref\">2002</xref>), and shown that the genomic variation leveraged for rapid adaptation depends on standing variation available in the population (Therkildsen et al. <xref rid=\"evl3189-bib-0073\" ref-type=\"ref\">2019</xref>). Gene flow may accelerate an adaptive response to climate change by providing relevant genetic variation, especially along a thermal gradient. However, tight linkage of co‐adapted alleles, an architecture associated with selection under gene flow (Kirkpatrick <xref rid=\"evl3189-bib-0042\" ref-type=\"ref\">2010</xref>; Samuk et al. <xref rid=\"evl3189-bib-0067\" ref-type=\"ref\">2017</xref>), may also constrain an adaptive response if linked traits are antagonistic to the direction of selection (Etterson and Shaw <xref rid=\"evl3189-bib-0023\" ref-type=\"ref\">2001</xref>). Beneficial alleles linked to maladaptive traits will not rapidly dissociate if recombination is suppressed between alternate alleles. This interplay is becoming increasingly relevant as humans alter both the environment and natural levels of gene flow, and organisms must rapidly adapt in response.</p></sec></sec><sec id=\"evl3189-sec-0150\"><title>AUTHOR CONTRIBUTIONS</title><p>NOT and SRP designed the study. NOT and APW generated the data. APW and NOT analyzed the data with input from SRP. DOC collected the samples. All authors wrote the manuscript.</p></sec><sec sec-type=\"data-availability\" id=\"evl3189-sec-0160\"><title>DATA ARCHIVING</title><p>The genomic sequence data described in this article have been archived and are publicly available in the NCBI Short Read Archive under Bioproject ID PRJNA376564. Sample locality data and transcriptome‐wide SNP data can be accessed through the Dryad repository <ext-link ext-link-type=\"uri\" xlink:href=\"https://doi.org/10.5061/dryad.jm63xsj7w\">https://doi.org/10.5061/dryad.jm63xsj7w</ext-link>.</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting information</title><supplementary-material content-type=\"local-data\"><caption><p>\n<bold>Table S1</bold>. Nucleotide diversity statistics within populations of Atlantic silversides based on pairwise differences (π) and number of polymorphic sites (Watterson's θ) across the transcriptome.</p><p>\n<bold>Table S2</bold>. Pearson's χ2 contingency tables and goodness of fit significance tests for enrichment of variant types in the top 1% of the FST distribution between pairs of neighboring populations relative to the genome‐wide distribution of variant types.</p><p>\n<bold>Table S3</bold>. Pearson's χ2 contingency tables and goodness of fit significance tests for enrichment of untranslated region (UTR), non‐synonymous (NS), or synonymous (S) variant types in LD blocks 8, 18, 24 and 11 (D‐G), relative to the genome‐wide distribution of variant types.</p><p>\n<bold>Table S4</bold>. Fits and parameter estimates for demographic models of the 2dSFS of GA and NY, showing the best of five independent runs ranked by AIC.</p><p>\n<bold>Table S5</bold>. Enrichment of Biological Processes Gene Ontology (GO) terms among genes scored by summing the number of OutFLANK outlier SNPs in LD block 8.</p><p>\n<bold>Table S6</bold>. Enrichment of Biological Processes Gene Ontology (GO) terms among genes scored by summing the number of OutFLANK outlier SNPs in LD block 18.</p><p>Table S7. Enrichment of Biological Processes Gene Ontology (GO) terms among genes scored by summing the number of OutFLANK outlier SNPs in LD block 24.</p><p>\n<bold>Table S8</bold>. Enrichment of Biological Processes Gene Ontology (GO) terms among genes scored by summing the number of OutFLANK outlier SNPs in LD block 11.</p><p>\n<bold>Figure S1</bold>. Pairwise linkage disequilibrium (LD) between outlier SNPs on silverside contigs (genes) in A) North Carolina (NC), B) New York (NY), and C) Gulf of Maine (GoM).</p><p>\n<bold>Figure S2</bold>. A) Interpopulation ancestry analysis of individuals from NY and GA showing high interpopulation ancestry of one individual from GA (the individual that was heterozygous for southern and northern alleles on Chr 8, 18, and 24), and two NY individuals (heterozygous for southern and northern alleles on Chr 24).</p><p>\n<bold>Figure S3</bold>. Principal components (PC) 1 and 2 using A) all SNPs, B) all SNPs outside of LD blocks, and C‐F) SNPs in LD blocks 8, 18, 24, and 11. Note that for Blocks 18, 24, and 11, individuals cluster into three distinct groups on PC1, likely corresponding to the two homozygous and the heterozygous state for each LD block.</p><p>\n<bold>Figure S4</bold>. Isolation by distance between all pairs of silverside populations, showing differentiation across genome‐wide SNPs (circles) and differentiation of SNPs outside LD blocks (triangles).</p><p>\n<bold>Figure S5</bold>. Fit of demographic models to the joint SFS of silversides from GA and NY, colored by the number of SNPs in each combination of minor alleles in GA and NY.</p><p>\n<bold>Figure S6</bold>. Fit of the top‐ranked demographic model (symmetrical migration with heterogeneous Ne) to the empirical 2dSFS for GA and NY (blue vertical line), relative to 100 Poisson‐sampled 2dSFS simulated under the model (gray bars), showing a good fit of the model to the data.</p></caption><media xlink:href=\"EVL3-4-430-s001.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"evl3189-sec-0140\"><title>ACKNOWLEDGMENTS</title><p>We would like to thank H. Baumann for assistance with transporting samples; B. Sheets and H. Borchardt‐Wier for help with DNA extractions and sample preparation; and A. Jacobs and A. Tigano for helpful suggestions on analyses and earlier versions of the manuscript. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"letter\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Evol Lett</journal-id><journal-id journal-id-type=\"doi\">10.1002/(ISSN)2056-3744</journal-id><journal-id journal-id-type=\"publisher-id\">EVL3</journal-id><journal-title-group><journal-title>Evolution Letters</journal-title></journal-title-group><issn pub-type=\"epub\">2056-3744</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33014420</article-id><article-id pub-id-type=\"pmc\">PMC7523563</article-id><article-id pub-id-type=\"doi\">10.1002/evl3.191</article-id><article-id pub-id-type=\"publisher-id\">EVL3191</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Letter</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Letters</subject></subj-group></article-categories><title-group><article-title>Hybridization and introgression between toads with different sex chromosome systems</article-title><alt-title alt-title-type=\"left-running-head\">C. DUFRESNES ET AL.</alt-title></title-group><contrib-group><contrib id=\"evl3191-cr-0001\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Dufresnes</surname><given-names>Christophe</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-8497-8908</contrib-id><xref ref-type=\"aff\" rid=\"evl3191-aff-0001\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"evl3191-aff-0002\">\n<sup>2</sup>\n</xref><address><email>Christophe.Dufresnes@hotmail.fr</email></address></contrib><contrib id=\"evl3191-cr-0002\" contrib-type=\"author\"><name><surname>Litvinchuk</surname><given-names>Spartak N</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-7447-6691</contrib-id><xref ref-type=\"aff\" rid=\"evl3191-aff-0003\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"evl3191-aff-0004\">\n<sup>4</sup>\n</xref></contrib><contrib id=\"evl3191-cr-0003\" contrib-type=\"author\"><name><surname>Rozenblut‐Kościsty</surname><given-names>Beata</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-9950-1571</contrib-id><xref ref-type=\"aff\" rid=\"evl3191-aff-0005\">\n<sup>5</sup>\n</xref></contrib><contrib id=\"evl3191-cr-0004\" contrib-type=\"author\"><name><surname>Rodrigues</surname><given-names>Nicolas</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-1588-4465</contrib-id><xref ref-type=\"aff\" rid=\"evl3191-aff-0006\">\n<sup>6</sup>\n</xref></contrib><contrib id=\"evl3191-cr-0005\" contrib-type=\"author\"><name><surname>Perrin</surname><given-names>Nicolas</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-7756-6323</contrib-id><xref ref-type=\"aff\" rid=\"evl3191-aff-0006\">\n<sup>6</sup>\n</xref></contrib><contrib id=\"evl3191-cr-0006\" contrib-type=\"author\"><name><surname>Crochet</surname><given-names>Pierre‐André</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-0422-3960</contrib-id><xref ref-type=\"aff\" rid=\"evl3191-aff-0007\">\n<sup>7</sup>\n</xref></contrib><contrib id=\"evl3191-cr-0007\" contrib-type=\"author\"><name><surname>Jeffries</surname><given-names>Daniel L</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-1701-3978</contrib-id><xref ref-type=\"aff\" rid=\"evl3191-aff-0006\">\n<sup>6</sup>\n</xref></contrib></contrib-group><aff id=\"evl3191-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">LASER</named-content>\n<named-content content-type=\"organisation-division\">College of Biology and the Environment</named-content>\n<institution>Nanjing Forestry University</institution>\n<city>Nanjing</city>\n<country country=\"CN\">People's Republic of China</country>\n</aff><aff id=\"evl3191-aff-0002\">\n<label><sup>2</sup></label>\n<named-content content-type=\"organisation-division\">Department of Animal and Plant Sciences</named-content>\n<institution>University of Sheffield</institution>\n<city>Sheffield</city>\n<country country=\"GB\">United Kingdom</country>\n</aff><aff id=\"evl3191-aff-0003\">\n<label><sup>3</sup></label>\n<institution>Institute of Cytology</institution>\n<institution>Russian Academy of Sciences</institution>\n<city>Saint Petersburg</city>\n<country country=\"RU\">Russia</country>\n</aff><aff id=\"evl3191-aff-0004\">\n<label><sup>4</sup></label>\n<institution>Dagestan State University</institution>\n<city>Makhachkala</city>\n<country country=\"RU\">Russia</country>\n</aff><aff id=\"evl3191-aff-0005\">\n<label><sup>5</sup></label>\n<named-content content-type=\"organisation-division\">Department of Evolutionary Biology and Conservation of Vertebrates</named-content>\n<named-content content-type=\"organisation-division\">Faculty of Biological Sciences</named-content>\n<institution>University of Wrocław</institution>\n<city>Wrocław</city>\n<country country=\"PL\">Poland</country>\n</aff><aff id=\"evl3191-aff-0006\">\n<label><sup>6</sup></label>\n<named-content content-type=\"organisation-division\">Department of Ecology & Evolution</named-content>\n<institution>University of Lausanne</institution>\n<city>Lausanne</city>\n<country country=\"CH\">Switzerland</country>\n</aff><aff id=\"evl3191-aff-0007\">\n<label><sup>7</sup></label>\n<named-content content-type=\"organisation-division\">CEFE</named-content>\n<institution>Univ. Montpellier, CNRS, EPHE, IRD</institution>\n<named-content content-type=\"street\">Univ Paul Valéry Montpellier 3</named-content>\n<city>Montpellier</city>\n<country country=\"FR\">France</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label><email>Christophe.Dufresnes@hotmail.fr</email><break/></corresp></author-notes><pub-date pub-type=\"epub\"><day>19</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><month>10</month><year>2020</year></pub-date><volume>4</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1002/evl3.v4.5</issue-id><fpage>444</fpage><lpage>456</lpage><history><date date-type=\"received\"><day>16</day><month>4</month><year>2020</year></date><date date-type=\"rev-recd\"><day>28</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>7</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 The Authors. Evolution Letters published by Wiley Periodicals LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB). <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Evolution Letters</italic> published by Wiley Periodicals, LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB).</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:EVL3-4-444.pdf\"/><abstract><title>Abstract</title><p>The growing interest in the lability of sex determination in non‐model vertebrates such as amphibians and fishes has revealed high rates of sex chromosome turnovers among closely related species of the same clade. Can such lineages hybridize and admix with different sex‐determining systems, or could the changes have precipitated their speciation? We addressed these questions in incipient species of toads (Bufonidae), where we identified a heterogametic transition and characterized their hybrid zone with genome‐wide markers (RADseq). Adult and sibship data confirmed that the common toad <italic>B. bufo</italic> is female heterogametic (ZW), while its sister species the spined toad <italic>B. spinosus</italic> is male heterogametic (XY). Analysis of a fine scale transect across their parapatric ranges in southeastern France unveiled a narrow tension zone (∼10 km), with asymmetric mitochondrial and nuclear admixture over hundreds of kilometers southward and northward, respectively. The geographic extent of introgression is consistent with an expansion of <italic>B. spinosus</italic> across <italic>B. bufo</italic>’s former ranges in Mediterranean France, as also suggested by species distribution models. However, widespread cyto‐nuclear discordances (<italic>B. spinosus</italic> backrosses carrying <italic>B. bufo</italic> mtDNA) run against predictions from the dominance effects of Haldane's rule, perhaps because Y and W heterogametologs are not degenerated. Common and spined toads can thus successfully cross‐breed despite fundamental differences in their sex determination mechanisms, but remain partially separated by reproductive barriers. Whether and how the interactions of their XY and ZW genes contribute to these barriers shall provide novel insights on the debated role of labile sex chromosomes in speciation.</p></abstract><kwd-group kwd-group-type=\"author-generated\"><kwd id=\"evl3191-kwd-0001\"><italic>Bufo bufo</italic></kwd><kwd id=\"evl3191-kwd-0002\"><italic>Bufo spinosus</italic></kwd><kwd id=\"evl3191-kwd-0003\">hybrid zone</kwd><kwd id=\"evl3191-kwd-0004\">RADseq</kwd><kwd id=\"evl3191-kwd-0005\">reproductive isolation</kwd><kwd id=\"evl3191-kwd-0006\">sex chromosome turnover</kwd><kwd id=\"evl3191-kwd-0007\">speciation</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source><institution-wrap><institution>Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/501100001711</institution-id></institution-wrap></funding-source><award-id>31003A_166323</award-id><award-id>P2LAP3_171818</award-id></award-group></funding-group><counts><fig-count count=\"4\"/><table-count count=\"2\"/><page-count count=\"13\"/><word-count count=\"9183\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>October 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.9.1 mode:remove_FC converted:29.09.2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><boxed-text position=\"float\" content-type=\"box\" orientation=\"portrait\"><caption><title>Impact Summary</title></caption><p>In mammals, sex is determined by a pair of XX/XY sex chromosomes: the Y chromosome triggers male development, hence males are XY and females are XX. This is the reverse in birds, where females carry the heterogametolog (noted W); females are ZW and males are ZZ. Because the Y and W are transmitted clonally and degenerate, sex chromosomes usually evolve faster than the rest of the genome, and eventually contribute genetic incompatibilities between diverging lineages. However, in other vertebrates such as amphibians and fishes, sex chromosomes are reshuffled frequently and closely related lineages may differ in their mechanisms of sex determination. Can they still hybridize and mix their genomes with different sex chromosomes? We addressed this question in two toad taxa, <italic>Bufo bufo</italic>, and <italic>Bufo spinosus</italic>. By screening thousands of genetic markers across males and females, we first showed that <italic>B. bufo</italic> is ZW while <italic>B. spinosus</italic> is XY, evidence that one species experienced a shift of sex chromosomes since their initial divergence several million years ago. We then accurately characterized their hybrid zone in southeastern France with hundreds of species‐diagnostic markers along a densely sampled transect. Despite their different sex‐determining systems, the two species can successfully hybridize, but only across a narrow transition (10km wide) where reproductive isolation (genetic incompatibilities) likely prevent their gene pools from merging. Parts of the genome diffused hundreds of kilometers inside species ranges, which is consistent with a northern expansion of the southern species (<italic>B. spinosus</italic>) into the range of its counterpart (<italic>B. bufo</italic>). Importantly, we report asymmetric patterns of admixture between the nuclear compared to the maternally inherited mitochondrial DNA, which could result from uneven sex ratio in hybrids of varying gametolog composition (XW, YW, XZ, YZ). Our study suggests that the evolution of different sex chromosomes did not seal reproductive barriers between these toads and offer a promising framework to understand whether and how the interactions of their XY and ZW genes contribute to their speciation.</p></boxed-text><p>Almost a century after the conception of Haldane's rule (Haldane <xref rid=\"evl3191-bib-0027\" ref-type=\"ref\">1922</xref>), the role played by sex chromosomes in speciation remains an exciting topic for evolutionary biologists (Payseur et al. <xref rid=\"evl3191-bib-0048\" ref-type=\"ref\">2018</xref>). Because they usually evolve faster (due to less efficient purifying selection and/or enhanced adaptation), bear more genes, and are hemizygous in the heterogametic sex (XY males and ZW females), X and Z chromosomes are expected to disproportionally contribute to genetic incompatibilities underlying post‐zygotic isolation (hybrid unviability and sterility), in comparison to the rest of the genome (Qvarnström and Bailey <xref rid=\"evl3191-bib-0054\" ref-type=\"ref\">2009</xref>; Schilthuizen et al. <xref rid=\"evl3191-bib-0059\" ref-type=\"ref\">2011</xref>; Beukeboom and Perrin <xref rid=\"evl3191-bib-0009\" ref-type=\"ref\">2014</xref>). Empirical evidence for this effect is readily observed in mammals (Payseur et al. <xref rid=\"evl3191-bib-0047\" ref-type=\"ref\">2004</xref>), birds (Storchová et al. <xref rid=\"evl3191-bib-0065\" ref-type=\"ref\">2010</xref>), and <italic>Drosophila</italic> (Presgraves <xref rid=\"evl3191-bib-0049\" ref-type=\"ref\">2008</xref>), where sex determination is stable and sex chromosomes have been decaying for tens/hundreds of million years (Beukeboom and Perrin <xref rid=\"evl3191-bib-0009\" ref-type=\"ref\">2014</xref>). In contrast, the effect is more elusive in taxa with young, less stable sex chromosomes (Lima <xref rid=\"evl3191-bib-0035\" ref-type=\"ref\">2014</xref>). Hybrid incompatibilities may quickly accumulate on recently evolved Y chromosomes in the early stages of differentiation (Dufresnes et al. <xref rid=\"evl3191-bib-0021\" ref-type=\"ref\">2016</xref>; Hu and Filatov <xref rid=\"evl3191-bib-0028\" ref-type=\"ref\">2016</xref>; Filatov <xref rid=\"evl3191-bib-0022\" ref-type=\"ref\">2018</xref>), but remain essentially autosomal when recombination prevents X–Y divergence (Macaya‐Sanz et al. <xref rid=\"evl3191-bib-0038\" ref-type=\"ref\">2011</xref>; Gerchen et al. <xref rid=\"evl3191-bib-0024\" ref-type=\"ref\">2018</xref>). Taxonomic groups that exhibit a high diversity of sex chromosomes, with both male (XY) and female (ZW) heterogamety, thus hold the key to understanding how sex‐linked genes affect reproductive isolation and speciation (Filatov <xref rid=\"evl3191-bib-0022\" ref-type=\"ref\">2018</xref>; Ogata et al. <xref rid=\"evl3191-bib-0044\" ref-type=\"ref\">2018</xref>).</p><p>Due to the lability of sex determination in fishes (Kitano and Peichel <xref rid=\"evl3191-bib-0034\" ref-type=\"ref\">2012</xref>) and amphibians (Miura <xref rid=\"evl3191-bib-0042\" ref-type=\"ref\">2017</xref>), closely related lineages sometimes possess different sex chromosome systems. What happens when these systems meet in secondary contact? Given the importance of sex‐linked genes in hybrid incompatibilities (Qvarnström and Bailey <xref rid=\"evl3191-bib-0054\" ref-type=\"ref\">2009</xref>), carrying non‐homologous sex chromosomes should generally increase reproductive isolation and thus the probability to speciate. For instance, genic conflicts could drastically alter sex determination and gametogenesis in hybrids, causing intersex or sterile individuals, respectively, in turn building strong post‐zygotic barriers. Combining independently evolved sex chromosomes could also promote new combinations of sexual characters. In sticklebacks, the evolution of neo‐sex chromosomes has driven inter‐species phenotypic divergence, which triggered speciation events (Kitano et al. <xref rid=\"evl3191-bib-0033\" ref-type=\"ref\">2009</xref>; Kitano and Peichel <xref rid=\"evl3191-bib-0034\" ref-type=\"ref\">2012</xref>). Alternatively, the systems may remain fully compatible (e.g., if one supersedes the other), resulting in porous species boundaries and admixed populations, as documented in the Japanese wrinkled frog <italic>Glandirana rugosa</italic> (Miura et al. <xref rid=\"evl3191-bib-0040\" ref-type=\"ref\">1998</xref>; Ogata et al., <xref rid=\"evl3191-bib-0043\" ref-type=\"ref\">2003</xref>; Miura <xref rid=\"evl3191-bib-0041\" ref-type=\"ref\">2007</xref>,<xref rid=\"evl3191-bib-0044\" ref-type=\"ref\">2018</xref>). These opposite outcomes illustrate the difficulty to predict the consequences of colliding sex‐determining systems on the speciation process, which so far have been studied only in a handful of cases.</p><p>We focused on these fascinating aspects in two incipient species of toads, <italic>Bufo bufo</italic> and <italic>B. spinosus</italic>. These widespread amphibians form a 900‐km‐long transition across France, which has been extensively characterized using morphology, mitochondrial DNA (mtDNA) and various sets of nuclear markers (Arntzen et al., <xref rid=\"evl3191-bib-0004\" ref-type=\"ref\">2016</xref>, <xref rid=\"evl3191-bib-0005\" ref-type=\"ref\">2017</xref>, <xref rid=\"evl3191-bib-0006\" ref-type=\"ref\">2018</xref>; van Riemsdijk et al. <xref rid=\"evl3191-bib-0068\" ref-type=\"ref\">2019</xref>). Previous studies uncovered puzzling patterns of genetic diversity and introgression. On the one hand, analyses of local transects quantified abrupt shifts in allele frequencies that may be mediated by reproductive isolation and/or demographic processes such as hybrid zone movement (Arntzen et al. <xref rid=\"evl3191-bib-0004\" ref-type=\"ref\">2016</xref>; van Riemsdijk et al. <xref rid=\"evl3191-bib-0068\" ref-type=\"ref\">2019</xref>). On the other hand, the widespread presence of “<italic>bufo</italic>” mtDNA and allozyme alleles among southern populations (assigned to <italic>B. spinosus</italic>), hundreds of kilometers away from the identified contact zones, has been interpreted as a sign of widespread admixture, initially blurring their validity as distinct species (García‐Porta et al. <xref rid=\"evl3191-bib-0023\" ref-type=\"ref\">2012</xref>, but see Recuero et al. <xref rid=\"evl3191-bib-0055\" ref-type=\"ref\">2012</xref> and Arntzen et al. <xref rid=\"evl3191-bib-0003\" ref-type=\"ref\">2013</xref>). Arntzen et al. (<xref rid=\"evl3191-bib-0005\" ref-type=\"ref\">2017</xref>) tentatively reconciled these conflicting observations under a biogeographic scenario involving multiple spatial shifts of the hybrid zone, following the successive expansions of each species from their respective Iberian (<italic>B. spinosus</italic>), and Alpine/Balkan glacial refugia (<italic>B. bufo</italic>) since the last ice age. But because previous phylogeographic studies were limited to mtDNA and a few introns (Arntzen et al. <xref rid=\"evl3191-bib-0005\" ref-type=\"ref\">2017</xref>) or allozymes (García‐Porta et al. <xref rid=\"evl3191-bib-0023\" ref-type=\"ref\">2012</xref>), it remains unclear whether “foreign” alleles genotyped as far as the Mediterranean coast and the Jura Mountains reflect past introgression or shared ancestral polymorphism. The high genetic resolution now offered by high‐throughput genomic approaches has the potential to distinguish among these hypotheses and better understand the factors that mediate admixture at this iconic hybrid zone.</p><p>Among potential incompatibilities, <italic>B. bufo</italic> and <italic>B. spinosus</italic> may exhibit fundamental differences in their sex‐determining systems. Common toads from the <italic>B. bufo</italic> complex were proposed to be female‐heterogametic (ZW) early on (Ponse <xref rid=\"evl3191-bib-0051\" ref-type=\"ref\">1942</xref>), which was for long considered the rule among bufonids (e.g., Malone and Fontenot <xref rid=\"evl3191-bib-0039\" ref-type=\"ref\">2008</xref>). Nevertheless, sex chromosome turnovers and male‐heterogamety do occur in this family (Stöck et al. <xref rid=\"evl3191-bib-0063\" ref-type=\"ref\">2011</xref>). In <italic>Bufo</italic>, the experiments of Ponse (<xref rid=\"evl3191-bib-0052\" ref-type=\"ref\">1950</xref>) and Rostand (<xref rid=\"evl3191-bib-0056\" ref-type=\"ref\">1952</xref>,<xref rid=\"evl3191-bib-0057\" ref-type=\"ref\">1953</xref>) suggested contradictory patterns of heterogamety depending on the geographic origins of specimens. Recently, Skorinov et al. (<xref rid=\"evl3191-bib-0061\" ref-type=\"ref\">2018</xref>) characterized a karyotypic dimorphism specific to males in <italic>B. spinosus</italic>, based on a few individuals. Hence, <italic>B. bufo</italic> and <italic>B. spinosus</italic> could differ in their heterogametic sex chromosomes (ZW versus XY), which in turn would allow testing whether such a recent turnover have affected their propensity to admix at range margins, and precipitated their incipient speciation.</p><p>In this study, we revisit the <italic>B. bufo</italic>/<italic>spinosus</italic> hybrid zone with genome‐wide data (RADseq), specifically to (i) confirm a putative heterogametic transition of sex‐determining systems between the two species and (ii) illuminate their patterns of admixture along an extensive transect in southeastern France. We predict substantial introgression mediated by geographic and demographic processes if the different sex‐determining systems between the two species remained compatible and did not seal their reproductive barriers.</p><sec id=\"evl3191-sec-0020\"><title>Methods</title><sec id=\"evl3191-sec-0030\"><title>FIELD SAMPLING</title><p>A total of 576 individuals of common and spined toads were captured in February–April 2016 from 67 localities between French Catalonia and Western Switzerland, broadly covering the <italic>B. bufo</italic>/<italic>spinosus</italic> transition (Table S1). Most individuals were live adults (<italic>n</italic> = 525), subsequently released after DNA collection with non‐invasive buccal swabs. Additional tissue samples were also obtained from road kills (<italic>n</italic> = 21) and small larvae (<italic>n</italic> = 30), fixed in 70–96% ethanol. To find sex‐linked markers, our sampling scheme involved an extensive set of males and females (<italic>n</italic> ≥ 20 of each sex) from two localities far from the contact zone, and confirmed as pure <italic>B. spinosus</italic> (loc. 3; Aumelas, France: 43.5743°N, 3.6442°E) and pure <italic>B. bufo</italic> (loc. 21; Aubonne, Switzerland: 46.5083°N, 6.3691E). Beyond general characteristics (size, color, body shape), sex was diagnosed by the presence (males) or absence (females) of nuptial pads on the first fingers, a dimorphism particularly marked during the breeding season (Dufresnes <xref rid=\"evl3191-bib-0019\" ref-type=\"ref\">2019</xref>).</p><p>In order to get additional evidence for the sex‐determining system of <italic>B. bufo</italic>, we also analyzed phenotypically sexed offspring obtained from a controlled cross—sex‐specific polymorphism is expected to be more readily detectable among sibs than randomly sampled adults of various ancestry. One mating pair (amplexus) was captured at loc. 21 and kept in a large container (525 L) until spawning (a few days), after which both parents were released in their place of capture. Tadpoles were raised until metamorphosis and a subset of 60 froglets (Gosner stage > 42–45) was euthanized by overdose of MS222 (0.15 g/L, buffered with sodium bicarbonate 0.3g/L) and fixed in 70% ethanol. Tissue samples (hindlegs) were collected for downstream genetic analyses. Froglets were then sexed by dissection and histological analysis of their gonads (Fig. S1). Gonads were first post‐fixed for 2 h in Bouin's solution (Sigma) and rinsed in 70% ethanol. The gonads were photographed using a cooled Carl Zeiss Axio‐Cam HRc CCD camera mounted on a Stemi SV11 (Zeiss) microscope. Afterward, gonads were dehydrated, embedded in paraplast, sectioned into 7‐μm slices, stained with Mallory's trichrome, and staged according to Ogielska and Kotusz (<xref rid=\"evl3191-bib-0045\" ref-type=\"ref\">2004</xref>) and Haczkiewicz and Ogielska (<xref rid=\"evl3191-bib-0026\" ref-type=\"ref\">2013</xref>).</p></sec><sec id=\"evl3191-sec-0040\"><title>LABWORK</title><p>The DNA of all samples was extracted using the Qiagen Biosprint robotic workstation. Four separate genomic libraries were prepared following the double‐digest RADseq protocol fully described in Brelsford et al. (<xref rid=\"evl3191-bib-0012\" ref-type=\"ref\">2016a</xref>). Briefly, this consists of enzyme digestion (here using <italic>SbfI</italic> and <italic>MseI</italic>), ligation of individual barcodes (on the <italic>SbfI</italic> end), amplification of the ligated fragments, and size selection between 400 and 500 bp. Library 1 included 41 adult male and female reference samples of <italic>B. bufo</italic>. Library 2 included 40 adult male and female reference samples of <italic>B. spinosus</italic>. Library 3 included 35 <italic>B. bufo</italic> metamorphs that could be confidently sexed. Bufonid samples from other projects completed these libraries (up to 96 samples) and each library was sequenced on two Illumina HiSeq 2500 lanes (single read 125). Library 4 included 200 toad samples collected from 21 localities across the hybrid zone (Table S1) and was sequenced on three Illumina lanes (single read 125). Raw reads were quality‐checked and filtered with FastQC version 0.10.1 (Andrews <xref rid=\"evl3191-bib-0002\" ref-type=\"ref\">2010</xref>), and demultiplexed with Stacks version 1.48 (Catchen et al. <xref rid=\"evl3191-bib-0015\" ref-type=\"ref\">2013</xref>).</p><p>The majority of wild‐caught toads (<italic>n</italic> = 514) were mitotyped by sequencing a short fragment (∼500 bp) of the gene <italic>cytochrome‐b</italic> (<italic>cyt‐b</italic>), amplified with the following custom primers: CytB.Bufo.Mid.F (5’–ATTATTGCAGGCGCCTCAATA–3’) and CytB.Bufo.R (5’–AGTTTRTTTTCTGTGAGTCC–3’). PCRs were carried out in 25 μL reactions containing 3 μL of template DNA, 7.5 μL of multiplex master mix (Qiagen, containing buffer, dNTPs and hot‐start polymerase), 1 μL of each primer (10 μM), and were run as follows: 95°C for 15’; 35 cycles of 94°C for 30”, 53°C for 45” and 72°C for 1’; 72°C for 5’. Sequences were manually aligned in Seaview (Gouy et al. <xref rid=\"evl3191-bib-0025\" ref-type=\"ref\">2010</xref>) and matched against reference haplotypes from the two species (GenBank sequences from Recuero et al. <xref rid=\"evl3191-bib-0055\" ref-type=\"ref\">2012</xref>).</p><p>Finally, we further incorporated the average mitochondrial and nuclear allele frequencies of <italic>B. bufo</italic>/<italic>spinosus</italic> obtained for ∼200 populations of southeastern France and nearby Italy by Arntzen et al. (<xref rid=\"evl3191-bib-0005\" ref-type=\"ref\">2017</xref>, taken from their tables S1 and S4, respectively), for complementation and comparison.</p></sec><sec id=\"evl3191-sec-0050\"><title>SCREENING FOR SEX‐LINKED LOCI</title><p>Sexed adults and siblings of <italic>B. bufo</italic> (libraries 1 and 3), as well as adults of <italic>B. spinosus</italic> (library 2), were screened for putative sex‐linked markers, applying two independent approaches. The first approach, SLM finder, is an ad hoc pipeline developed to identify SNPs that show sex biases in allele frequencies (method I), heterozygosity (method II), or RADseq tags that are specific to only one sex (method III) (Breslford et al. <xref rid=\"evl3191-bib-0013\" ref-type=\"ref\">2016b</xref>; Jeffries et al. <xref rid=\"evl3191-bib-0031\" ref-type=\"ref\">2018</xref>). For this, loci were constructed and SNPs were called using Stacks version 1.48. Default parameters were used for the <italic>Ustacks</italic> and <italic>Cstacks</italic> modules, as these represented the best balance between stringency and data inclusion. For the <italic>Populations</italic> module, SNP genotypes were retained if the locus coverage was at least 8 reads (<italic>–m</italic> 8), if called for ≥80% of individuals of each sex (<italic>–r</italic> 0.8, <italic>–p</italic> 2) and if heterozygosity across the whole dataset was not greater than 75% (<italic>–max_obs_het</italic> 0.75), to remove over merged paralogous loci.</p><p>In order to setup the optimal parameters with which to identify sex‐linked markers, we ran SLM finder for 24 different combinations of the parameters underlying methods I and II (heterogametic and homogametic thresholds), and for six parameter values of method III (sex specificity threshold; Table S2). For each set, we performed a permutation test whereby male and female sex assignments were randomly shuffled across the entire sample set 100 times, SLM finder was run, and the number of putative sex‐linked markers obtained was recorded. This null distribution was then compared to the number of sex‐linked markers flagged on the observed data (i.e., real sex assignments). If this number fell above the 95th percentile of the null distribution, we considered sex linkage to be significant (<italic>P</italic> < 0.05). For a given method, we could further select the optimal parameter set as the one minimizing false positives, that is, showing the smallest proportion of sex‐linked markers in the null distribution relative to the number of sex‐linked markers in the observed data.</p><p>The second approach, RadSex (<ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/RomainFeron/RadSex\">https://github.com/RomainFeron/RadSex</ext-link>), identifies sex specific RADseq tags or alleles directly from the raw read data (i.e., without locus assembly or SNP calling), and compare their presence among samples of each sex. This approach has the advantage that it does not require the assembly of loci, which has two important consequences: (i) it is sensitive to only one parameter, namely the coverage of unique RAD tags (<italic>–min_cov</italic>), hence reducing the potential for parameter biases; (ii) complex loci that are usually discarded by assembling pipelines such as Stacks (e.g., indel or polyallelic markers) are considered in the analysis. The coverage table was created (<italic>radsex process</italic>) using a minimum coverage of 1 read (<italic>–min_cov</italic> 1). The distribution table, which summarizes the number of males and females in which each unique sequence is present (<italic>radsex distrib</italic>) was created using a minimum coverage of 5 reads (<italic>–min‐cov</italic> 5). These values allow to retain low coverage loci while removing reads that contain potential sequencing errors (present in single copies only) or PCR errors (present in several copies only). Markers showing statistically significant association with sex were identified via Chi‐squared test using Yate's correction for continuity and Bonferroni correction for multiple testing.</p></sec><sec id=\"evl3191-sec-0060\"><title>HYBRID ZONE ANALYSES</title><p>The 200 hybrid zone samples (library 4) were processed with the <italic>denovo.pl</italic> pipeline of Stacks. Default stacking parameters were again used for <italic>Ustacks</italic> and <italic>Cstacks</italic> modules. In a first step, we filtered for SNPs genotyped in at least 90% of the samples (–<italic>r</italic> 0.9) in all localities (–<italic>p</italic> 21), which yielded 8,560 SNPs (99.3% of matrix completeness). The genetic structure of this initial dataset was explored by a Principal Component Analysis (PCA) on individual genotypes with the R package <italic>adegenet</italic> (Jombart <xref rid=\"evl3191-bib-0032\" ref-type=\"ref\">2008</xref>). For specific hybrid zone analyses, we then called species‐diagnostic SNPs, as those fixed between populations at the edges of our study area, specifically, between loc. 1–3 (<italic>B. spinosus</italic>) and loc. 20–21 (<italic>B. bufo</italic>), retaining only one SNP per RADseq tag, which yielded 950 SNPs. We performed a PCA as above, and estimated the ancestry of individuals to either species with the Bayesian clustering algorithm of STRUCTURE (Pritchard et al. <xref rid=\"evl3191-bib-0053\" ref-type=\"ref\">2000</xref>), focusing on runs with <italic>K</italic> = 2. We applied the admixture model without pre‐assignment of samples, and performed 10 replicate chains of 100,000 iterations, after a burn‐in of 10,000. Replicates were combined with CLUMPP (Jakobsson and Rosenberg <xref rid=\"evl3191-bib-0030\" ref-type=\"ref\">2007</xref>). For each population, we computed observed heterozygosity (<italic>H<sub>o</sub></italic>) with the R package <italic>hierfstat</italic> (Goudet <xref rid=\"evl3191-bib-0029\" ref-type=\"ref\">2005</xref>), linkage disequilibrium (correlation coefficient <italic>R<sup>2</sup></italic>) averaged over all pairs of loci with the R package <italic>genetics</italic> (Warnes et al. <xref rid=\"evl3191-bib-0070\" ref-type=\"ref\">2013</xref>), and the admixture linkage disequilibrium (<italic>D’</italic>) as the variance in individual hybrid index (Barton and Gale <xref rid=\"evl3191-bib-0008\" ref-type=\"ref\">1993</xref>), here using the genome‐wide individual ancestry obtained with STRUCTURE (individual <italic>Q</italic> scores) as a proxy. <italic>H<sub>o</sub></italic> was also computed for each individual.</p><p>The hybrid zone was further characterized by fitting sigmoid clines to allele frequency changes along the northeast‐southwest geographic transect covered by our sampling, using the <italic>R</italic> package <italic>hzar</italic> (Derryberry et al. <xref rid=\"evl3191-bib-0018\" ref-type=\"ref\">2014</xref>). We first computed clines for mtDNA and the average nuclear ancestry obtained with STRUCTURE (population <italic>Q</italic> scores), and performed model selection (AIC) on clines with two (center <italic>c</italic> and width <italic>w</italic>) to six parameters (length <italic>δ</italic> and slope <italic>τ</italic> of the exponential tails). Maximum (<italic>P<sub>max</sub></italic>) and minimum (<italic>P<sub>min</sub></italic>) allele frequencies were set to 0 and 1, respectively, since the considered markers/indices were fixed between our edge populations. Second, to explore the heterogeneity of introgression throughout the genome, we fitted clines to each of the 950 SNPs individually, using models with six parameters for comparability. We specifically compared the tail parameters δ and τ to quantify whether introgression was asymmetric, which should be reflected by longer and steeper tails on one side of the cline compared to the other one (Barton and Gale <xref rid=\"evl3191-bib-0008\" ref-type=\"ref\">1993</xref>).</p></sec><sec id=\"evl3191-sec-0070\"><title>SPECIES DISTRIBUTION MODELING</title><p>To grasp whether environmental factors (adaptation to different ecological niches) contributed to mediate the transition between <italic>B. bufo</italic> and <italic>B. spinosus</italic>, we built species distribution models. We applied recent methodological recommendations to compute robust ecological niche models with MaxEnt 3.4.1 (Phillips et al. <xref rid=\"evl3191-bib-0050\" ref-type=\"ref\">2006</xref>), such as occurrence filtering, using multiple combinations of model parameters (features and regularization multipliers), and multiple statistical criteria for model selection (partial ROC, omission rates, and AICc).</p><p>A total of 14,504 localities known of <italic>B. bufo</italic> and <italic>B. spinosus</italic> were initially considered, combining our own records, museum collections, and previously published data. We first filtered this dataset to avoid spatial autocorrelation and duplication using NicheToolBox (Osorio‐Olvera et al. <xref rid=\"evl3191-bib-0046\" ref-type=\"ref\">2018</xref>). We then retained the localities at least 10 km (0.093°) apart (see Brown <xref rid=\"evl3191-bib-0014\" ref-type=\"ref\">2014</xref>), and the final dataset comprised 3212 records for <italic>B. bufo</italic> and 886 records for <italic>B. spinosus</italic> (see Fig. S2).</p><p>To compute the models, altitude and 19 bioclimatic layers representative of the climatic data over ∼1950–2000 were extracted from the WorldClim 1.4 database (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.worldclim.org\">http://www.worldclim.org</ext-link>). Ten additional layers were considered: the aridity index (Global Aridity and Potential Evapo‐Transpiration; <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.cgiar-csi.org/data/global-aridity-and-pet-database\">http://www.cgiar-csi.org/data/global-aridity-and-pet-database</ext-link>), the global percent of tree coverage (<ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/globalmaps/gm_ve_v1\">https://github.com/globalmaps/gm_ve_v1</ext-link>), and eight land cover variables (spatial homogeneity of global habitat, broadleaf forests, needleleaf forests, mixed forests, shrubs, barren, herbaceous, and cultivated vegetation; <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.earthenv.org/\">https://www.earthenv.org/</ext-link>). To also consider topography in the model, four landscape layers were calculated with QGIS: aspect, exposition, slope, and terrain roughness index. These layers had a 30 arc seconds spatial resolution. All analyses were conducted under the WGS 84 projection with species‐specific masks covering the areas of occurrence of <italic>B. bufo</italic> and <italic>B. spinosus</italic>.</p><p>To eliminate predictor collinearity before generating these models, we calculated Pearsons's correlation coefficients for all pairs of bioclimatic variables using ENMTools (Warren et al. <xref rid=\"evl3191-bib-0071\" ref-type=\"ref\">2010</xref>). For correlated pairs (│r│> 0.75), the variable that appeared the most biologically important for the species was retained. The resulting dataset contained ten bioclimatic variables for <italic>B. bufo</italic>: Bio 1 (annual mean temperature; °C × 10), Bio 2 (mean diurnal range; °C × 10), Bio 3 (isothermality; Bio2/Bio7 × 100), Bio7 (temperature annual range; °C × 10), Bio 8 (mean temperature of wettest quarter; °C × 10), Bio 14 (precipitation of driest month; mm), Bio 15 (precipitation seasonality; CV), Bio 16 (precipitation of wettest quarter; mm), Bio 18 (precipitation of warmest quarter; mm), and Bio 19 (precipitation of coldest quarter; mm); and five bioclimatic variables for <italic>B. spinosus</italic>: Bio 3, Bio 6 (minimum temperature of coldest month; °C × 10), Bio 8, Bio 14, and Bio 16. We then applied a jackknife analysis to estimate the relative contributions of variables to the MaxEnt model.</p><p>MaxEnt models were ran with 10 replicates. Model calibration consisted in the evaluation of models created with distinct regularization multipliers (0.5 to 6, at intervals of 0.5) and feature classes (resulted from all combinations of linear, quadratic, product, threshold, and hinge response types). The sets of variables consisted of 25 layers for <italic>B. bufo</italic> and 20 for <italic>B. spinosus</italic>. The best parameter settings were selected considering statistical significance (partial ROC), predictive power (omission rates E = 5%), and complexity level (AICc), obtained using the <italic>R</italic> package kuenm (Cobos et al. <xref rid=\"evl3191-bib-0016\" ref-type=\"ref\">2019</xref>).</p><p>To assess whether the parapatric ranges correspond to an environmental transition for the species, we examined how their occurrence probability varied among the sampled populations of our transect. In addition, we performed multivariate analyses (PCA, R package <italic>ade4</italic>) on the geoclimatic variables at occurrence records, contrasting those from France (where the transition is located) and the rest of the species ranges.</p></sec></sec><sec id=\"evl3191-sec-0080\"><title>Results</title><sec id=\"evl3191-sec-0090\"><title>SEX‐LINKED MARKERS</title><p>Testing different parameter combination using a permutation approach allowed to set up the optimal parameters set of SLM finder, which were used to identify putative sex‐linked markers from the adult and sibling datasets of <italic>B. bufo</italic>, as well as the adult dataset of <italic>B. spinosus</italic> (Fig. S3 and Table S2).</p><p>For <italic>B. bufo</italic>, the adult dataset comprised 146,292,576, reads after demultiplexing, which contained 35,084 SNPs after the Stacks processing (average coverage = 32.3 reads/locus). No support was found for either XY or ZW patterns of sex‐linkage in this dataset, regardless of the parameter combination (Table <xref rid=\"evl3191-tbl-0001\" ref-type=\"table\">1</xref>). In contrast, the RadSex approach highlighted one significantly sex‐linked marker consistent with a ZW system, that is, a sequence found among most of the females (19/20), but none of the males (0/21; Fig. <xref rid=\"evl3191-fig-0001\" ref-type=\"fig\">1</xref>, left panel). This sequence contains an indel polymorphism and was therefore discarded by the Stacks pipeline, which is why it was not present (and flagged) among the loci screened by SLM finder.</p><table-wrap id=\"evl3191-tbl-0001\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Number of SNPs consistent with a XY and a ZW system in <italic>B. bufo</italic> (adults: 21 males and 20 females; sibs: 18 males and 17 females) and <italic>B. spinosus</italic> (adults: 20 of each sex) identified by SLM Finder under the allele frequency (I), heterozygosity (II) and tag dropout (III) approaches. Higher numbers than expected by chance (<italic>P</italic> ≤ 0.05) are highlighted by asterisks, according to permutation tests (Fig. S3)</p></caption><table frame=\"hsides\" rules=\"groups\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\"/><th style=\"border-bottom:solid 1px #000000\" colspan=\"4\" align=\"left\" rowspan=\"1\">\n<italic>B. bufo</italic>\n</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" rowspan=\"1\" colspan=\"1\">I</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">II</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">III</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Total</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adults (23,007 loci)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XY</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ZW</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Sibs (11,465 loci)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XY</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ZW</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16*</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">15*</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">16</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\"/><th style=\"border-bottom:solid 1px #000000\" colspan=\"4\" align=\"left\" rowspan=\"1\">\n<italic>B. spinosus</italic>\n</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" rowspan=\"1\" colspan=\"1\">I</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">II</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">III</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Total</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Adults (28,089 loci)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">XY</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">90*</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">89*</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">104</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ZW</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd.</copyright-holder></permissions></table-wrap><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3191-fig-0001\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Sex‐specific presence of RAD variants in <italic>B. bufo</italic> (from the adult and sib datasets) and <italic>B. spinosus</italic> (adults only) as inferred by the RADsex approach. Differences reaching statistical significance are squared in red. In <italic>B. bufo</italic>, the analysis counted more tags specific to females (vertical axis) than males (horizontal axis), but a single one in significant proportions. In <italic>B. spinosus</italic>, tens of tags are significantly specific to males.</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"EVL3-4-444-g001\"/></fig><p>The <italic>B. bufo</italic> sibling dataset comprised 122,605,407 reads after demultiplexing, which contained 12,840 SNPs after the Stacks processing (average coverage = 39.1 reads/locus). SLM finder tests also found evidence for a ZW system from this data. With the optimal thresholds, the frequency and heterozygosity methods of SLM finder reported 16 and 15 putative ZW loci, respectively (15 supported by both methods; Table <xref rid=\"evl3191-tbl-0001\" ref-type=\"table\">1</xref>). Only one putative XY marker was identified (by the heterozygosity method), which fell within the expected rate of false positive (Fig. S3). Note that for the heterozygosity method, more relaxed parameters on the homogametic threshold yielded a significant male‐heterogametic signal (Fig. S3). This could reflect Z‐specific polymorphisms between the father's Zs (ZZ) and the mother's Z (ZW), hence mimicking an XY‐like allele segregation in the full‐sib clutch, as demonstrated for similar datasets (e.g., <italic>Rana montezumae</italic>, Jefrries et al. <xref rid=\"evl3191-bib-0031\" ref-type=\"ref\">2018</xref>). RadSex also suggested a ZW system among the sibling dataset: many tags were sequenced exclusively among the females (11–13/17), although the bias was not significant (Fig. <xref rid=\"evl3191-fig-0001\" ref-type=\"fig\">1</xref>, central panel).</p><p>The <italic>B. spinosus</italic> adult dataset comprised 452,840,434 reads after demultiplexing, which contained 28,090 SNPs (average coverage = 37.2 reads/locus). SLM finder found strong evidence for an XY system (Table <xref rid=\"evl3191-tbl-0001\" ref-type=\"table\">1</xref>). Altogether, a total of 104 XY loci were flagged, 89 of them being detected by both the allele frequency and the heterozygosity methods, and in highly significant numbers (Fig. S3b). In contrast, only four tags fit a ZW pattern, which was within random expectations. The RadSex results also recovered an XY system, with 151 significantly male‐specific RAD tags, but no female‐specific ones (Fig. <xref rid=\"evl3191-fig-0001\" ref-type=\"fig\">1</xref>, right panel). About a third (48 markers) corresponds to those recovered by SLM finder, while the rest were discarded by the Stacks pipeline (see Methods).</p></sec><sec id=\"evl3191-sec-0100\"><title>HYBRID ZONE ANALYSES</title><p>The distributions of the <italic>B. bufo</italic> and <italic>B. spinosus</italic> genomes in southeastern France are displayed in Fig. <xref rid=\"evl3191-fig-0002\" ref-type=\"fig\">2</xref>, according to RADseq, introns and mitochondrial markers. Our initial RADseq dataset (8,560 SNPs) recovered the two gene pools as the main source of genetic structure, which built the first PCA component (PC1 in Fig. S4). It also highlighted some intraspecific structure: the southernmost <italic>B. spinosus</italic> samples (loc. 1–2), and the northernmost <italic>B. bufo</italic> samples (loc. 20–21) form the second and third PCA components, respectively (Fig. S4).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3191-fig-0002\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>The hybrid zone between <italic>B. bufo</italic> (blue) and <italic>B. spinosus</italic> (red) in southern France. The left panel presents our nuclear clustering analyses based on RADseq data (950 species‐diagnostic loci, i. e. fixed between edge populations); the left inset map zooms in the area of contact. The middle panel reports population ancestry obtained from the analysis of intron markers BDNF, POMC, RAG1 and RPL3 (Arntzen et al. <xref rid=\"evl3191-bib-0005\" ref-type=\"ref\">2017</xref>); many individuals with <italic>B. spinosus</italic> or intermediate ancestries at these loci were sampled in ranges that clearly belong to <italic>B. bufo</italic> (according to the RADseq data). The right panel combines mitochondrial barcoding from ours and Arntzen et al. (<xref rid=\"evl3191-bib-0005\" ref-type=\"ref\">2017</xref>). The top right inset map shows the distribution of the two species in Western Europe (adapted from Dufresnes <xref rid=\"evl3191-bib-0019\" ref-type=\"ref\">2019</xref>).</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"EVL3-4-444-g002\"/></fig><p>As expected, the 950 SNPs fixed between the groups of populations at the edges of our transect (loc. 1–3 and loc. 20–21) mostly captured the genetic differentiation between the two species (63.8% of variance explained by PC1 in Fig. S4). The STRUCTURE ancestry coefficients obtained from this dataset closely matched the PC1 scores, indicative of robust inferences (Fig. S5). The clustering analysis supported a sharp nuclear transition between our loc. 14 and 15, located just 9 km apart in the hills north of Romans‐sur‐Isère (Fig. <xref rid=\"evl3191-fig-0002\" ref-type=\"fig\">2</xref>), associated with an increase of heterozygosity (<italic>H<sub>o</sub></italic>) and linkage disequilibrium (Fig. S6). Fainted traces of admixture were detected up to a hundred kilometers in either direction (loc. 7–14 and loc. 15–20, Fig. <xref rid=\"evl3191-fig-0002\" ref-type=\"fig\">2</xref>), but all ancestry coefficients reached above 0.9, even close to the putative center (Fig. <xref rid=\"evl3191-fig-0002\" ref-type=\"fig\">2</xref>, Fig. S7). Accordingly, the cline fitted to the average population ancestry (STRUCTURE's <italic>Q</italic>) along our transect was only 8.6 km wide, with the best model involving mirrored introgression tails on both sides (Fig. <xref rid=\"evl3191-fig-0003\" ref-type=\"fig\">3</xref> and Table <xref rid=\"evl3191-tbl-0002\" ref-type=\"table\">2</xref>). The geographic transition inferred from nuclear introns (Arntzen et al. <xref rid=\"evl3191-bib-0005\" ref-type=\"ref\">2017</xref>) is comparatively more variable, with genotypes assigned to <italic>B. spinosus</italic> found among populations of <italic>B. bufo</italic> (Fig. <xref rid=\"evl3191-fig-0002\" ref-type=\"fig\">2</xref>).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3191-fig-0003\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Geographic clines fitted to the proportion of mitochondrial DNA, nuclear genome average (STRUCTURE <italic>Q</italic>), and allele frequency at 950 species‐diagnostic SNPs (i.e., fixed between edge populations) along our <italic>B. bufo</italic>/<italic>spinosus</italic> transect running from Switzerland (NE) to French Catalonia (SW). Distances are given as the deviation from the median center.</p></caption><graphic id=\"nlm-graphic-5\" xlink:href=\"EVL3-4-444-g003\"/></fig><table-wrap id=\"evl3191-tbl-0002\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Summary of the cline analyses. For the mtDNA and nuclear <italic>Q</italic> scores, parameter estimates for <italic>w</italic> and <italic>c</italic> are provided with their 95% confidence interval. For the 950 species‐diagnostic SNPs, the median and the 95% distribution of parameters are shown</p></caption><table frame=\"hsides\" rules=\"groups\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><thead><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" rowspan=\"1\" colspan=\"1\">mtDNA</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Nuclear (<italic>Q</italic>)</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Nuclear (SNPs)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Best model</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">one tail (<italic>B. spinosus</italic>)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">mirrored tails</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Model likelihood</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−24.6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">−1.3</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Width <italic>w</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.4 (3.4‐35.1)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">8.6 (8.6‐14.8)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">11.7 (2.3–64.6)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Center <italic>c</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">203.6 (194.6–209.6)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">185.9 (183.5–186.3)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">184.1 (179.5–191.2)</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd.</copyright-holder></permissions></table-wrap><p>The mitochondrial transition was less clear cut: <italic>B. bufo</italic> mitotypes are widespread south of the nuclear transition, and even far within the range of <italic>B. spinosus</italic>, while no <italic>B. spinosus</italic> mtDNA was detected among <italic>B. bufo</italic> populations (Fig. <xref rid=\"evl3191-fig-0002\" ref-type=\"fig\">2</xref>). As a result, the best model for the mitochondrial cline involved an introgression tail on the <italic>B. spinosus</italic> side, a narrow width (11.4 km), and a center shifted about 20 km south compared to the nuclear cline (around Valence, near loc. 13; Figs. <xref rid=\"evl3191-fig-0002\" ref-type=\"fig\">2</xref>–<xref rid=\"evl3191-fig-0003\" ref-type=\"fig\">3</xref> and Table <xref rid=\"evl3191-tbl-0002\" ref-type=\"table\">2</xref>).</p><p>The clines fitted independently to each of the 950 species‐diagnostic loci are shown in Fig. <xref rid=\"evl3191-fig-0003\" ref-type=\"fig\">3</xref>. The width and center estimates followed unimodal distributions with medians of 11.7 and 184.1 km (equidistant to loc. 14 and 15), respectively (Table <xref rid=\"evl3191-tbl-0002\" ref-type=\"table\">2</xref>; Fig. S8). Introgression tails were significantly longer (higher <italic>δ</italic>) and steeper (higher <italic>τ</italic>) on the <italic>B. bufo</italic> than on the <italic>B. spinosus</italic> side (<italic>P</italic> ⋘ 0.05 for <italic>δ</italic> and <italic>P</italic> = 0.04 for <italic>τ</italic>; paired Wilcoxon signed ranked tests; Fig. S9).</p></sec><sec id=\"evl3191-sec-0110\"><title>SPECIES DISTRIBUTION MODELING</title><p>A total of 348 replicate models for each species were assessed for calibration, all of which were statistically significant when compared with a null model of random prediction. Of these significant models for <italic>B. bufo</italic>, a single model significantly met both the omission criterion of 5% and the AICc criteria (Table S3). Mean diurnal range (16%), broadleaf forest (16%), cultivated vegetation (15%), tree coverage (15%), and shrubs percent (13%) altogether contributed 75% of the model (Table S3). For <italic>B. spinosus</italic> no model met the omission criterion and a single model significantly met the AICc criteria (Table S3). Of the parameters included in the model, cultivated vegetation (38%), tree coverage (17%), mixed forest percent (13%), and mean temperature of wettest quarter (12%) altogether contributed 80% (Table S3).</p><p>The projected distributions of <italic>B. bufo</italic> and <italic>B. spinosus</italic> under the selected models are displayed in Fig. <xref rid=\"evl3191-fig-0004\" ref-type=\"fig\">4</xref>. For both species, large parts of France received intermediate occurrence probabilities, but most records fall within the edges of the geoclimatic parameter space of both species, supporting that these areas correspond to some environmental‐ecological boundaries (Fig. S10). This ecological transition was steeper for <italic>B. bufo</italic> than for <italic>B. spinosus</italic>: along our transect, the suitability for <italic>B. bufo</italic> significantly decreased with geographic distance, while the relationship was not significant for <italic>B. spinosus</italic> (Fig. S11).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3191-fig-0004\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Projected distributions of <italic>B. bufo</italic> and <italic>B. spinosus</italic> according to their bioclimatic models. Thick lines show range limits. The area of contact studied here (frame) is suitable for both species, particularly along the Rhône valley for <italic>B. spinosus</italic>.</p></caption><graphic id=\"nlm-graphic-7\" xlink:href=\"EVL3-4-444-g004\"/></fig></sec></sec><sec id=\"evl3191-sec-0120\"><title>Discussion</title><p>Can the evolution of distinct sex‐determining systems precipitate speciation events between diverging lineages? Our study shows that the heterogametic transition in the toads <italic>B. bufo</italic> (ZW) and <italic>B. spinosus</italic> (XY) was insufficient to prevent admixture, although their narrow hybrid zone implies some post‐zygotic isolation.</p><p>This recent turnover—the two species are 5–10 million years old (García‐Porta et al. <xref rid=\"evl3191-bib-0023\" ref-type=\"ref\">2012</xref>, Recuero et al. <xref rid=\"evl3191-bib-0055\" ref-type=\"ref\">2012</xref>)—bolsters the emerging view that sex chromosomes evolve rapidly within amphibian radiations, as seen from large‐scale studies in Ranid (Jeffries et al. <xref rid=\"evl3191-bib-0031\" ref-type=\"ref\">2018</xref>) and Hylid frogs (Dufresnes et al. <xref rid=\"evl3191-bib-0020\" ref-type=\"ref\">2015</xref>). In other bufonids, female heterogamety has been reported in the genus <italic>Rhinella</italic> (Abramyan et al. <xref rid=\"evl3191-bib-0001\" ref-type=\"ref\">2009</xref>), while <italic>Bufotes</italic> (Stöck et al. <xref rid=\"evl3191-bib-0063\" ref-type=\"ref\">2011</xref>), <italic>Strauchbufo</italic> (Deng and Shang <xref rid=\"evl3191-bib-0017\" ref-type=\"ref\">1984</xref>), and <italic>Duttaphrynus</italic> (Siripyasing et al. <xref rid=\"evl3191-bib-0060\" ref-type=\"ref\">2008</xref>) are male‐heterogametic. Female heterogamety is probably the ancestral state in <italic>Bufo</italic>, since ZW chromosomes were also characterized in <italic>B. gargarizans</italic> (Wen et al. <xref rid=\"evl3191-bib-0072\" ref-type=\"ref\">1983</xref>), a close Asian relative of <italic>B. bufo</italic> and <italic>B. spinosus</italic>. These recurrent heterogametic switches are rather uncommon: transitions are expected to preserve the heterogametic sex, due to mutation load selection and drift (Blaser et al. <xref rid=\"evl3191-bib-0010\" ref-type=\"ref\">2014</xref>; Saunders et al. <xref rid=\"evl3191-bib-0058\" ref-type=\"ref\">2018</xref>). In amphibians, phenotypic males recombine far less than females (Bufonids included, Stöck et al. <xref rid=\"evl3191-bib-0064\" ref-type=\"ref\">2013</xref>), so a Y should differentiate faster from the X than the W from the Z. Accordingly, we found much larger numbers of XY markers in <italic>B. spinosus</italic> than of ZW markers in <italic>B. bufo</italic>, despite stronger resources for the latter species. This also makes homomorphic ZW chromosomes more difficult to detect, which could contribute to why male heterogamety appear more prevalent in some amphibian groups (Miura <xref rid=\"evl3191-bib-0042\" ref-type=\"ref\">2017</xref>; but see Jeffries et al. <xref rid=\"evl3191-bib-0031\" ref-type=\"ref\">2018</xref>).</p><p>Whether this heterogametic transition involves the same chromosome pair remains to be determined. No genome assembly is yet available for bufonids, and insufficient numbers of putatively sex‐linked markers could be aligned to published references. The male‐specific karyotype dimorphism found in <italic>B. spinosus</italic> by Skorinov et al. (<xref rid=\"evl3191-bib-0061\" ref-type=\"ref\">2018</xref>) was on chromosome 6 (like in <italic>Strauchbufo radei</italic>, Deng and Shang <xref rid=\"evl3191-bib-0017\" ref-type=\"ref\">1984</xref>), but this needs clarification with additional samples. The amount of XY markers flagged here (Table <xref rid=\"evl3191-tbl-0001\" ref-type=\"table\">1</xref>) is comparable to that of homomorphic sex chromosomes identified in other amphibians with similar datasets and methodologies (Brelsford et al. <xref rid=\"evl3191-bib-0013\" ref-type=\"ref\">2016b</xref>; Jeffries et al. <xref rid=\"evl3191-bib-0031\" ref-type=\"ref\">2018</xref>). We also found only two Y‐specific tags (Table <xref rid=\"evl3191-tbl-0001\" ref-type=\"table\">1</xref>), which suggests little molecular differentiation between the Y and the X. Additional analyses will be needed to ascertain the identity and degree of differentiation/degeneracy of the sex chromosomes found in <italic>Bufo</italic>.</p><p>Despite their different sex‐determining systems, the reproductive barriers of <italic>B. spinosus</italic> (XY) and <italic>B. bufo</italic> (ZW) are not complete. They can still produce fertile offspring and experience gene flow at range margins. In Normandy, Trujillo et al. (<xref rid=\"evl3191-bib-0067\" ref-type=\"ref\">2017</xref>) documented an all‐hybrid population composed of reproducing males and females, some directly observed in amplexus. We detected slight peaks of linkage disequilibrium and heterozygosity in the closest contacting populations (Fig. S6), consistent with a recent origin of the introgression, as also expected given the currently overlapping distributions of the species (Arntzen et al. <xref rid=\"evl3191-bib-0007\" ref-type=\"ref\">2020</xref>). Yet, the narrow geographic transitions documented in southeastern France (∼10 km) and Normandy (∼20 km, Arntzen et al. <xref rid=\"evl3191-bib-0004\" ref-type=\"ref\">2016</xref>) suggest a dynamic of tension zones, where post‐zygotic selection against hybrids efficiently prevents the parental genomes to merge. Common toads are very mobile (Smith and Green <xref rid=\"evl3191-bib-0062\" ref-type=\"ref\">2005</xref>) so dispersal should be unaffected by landscape barriers: the alleles that successfully crossed the contact zone diffused far within species ranges (Fig. <xref rid=\"evl3191-fig-0002\" ref-type=\"fig\">2</xref>). Moreover, the transition does not follow the Rhône and the Isère rivers (as found by Arntzen et al., <xref rid=\"evl3191-bib-0005\" ref-type=\"ref\">2017</xref>, <xref rid=\"evl3191-bib-0007\" ref-type=\"ref\">2020</xref>), but was located in the woody hills north of the Isère and east of the Rhône (Fig. <xref rid=\"evl3191-fig-0002\" ref-type=\"fig\">2</xref>), and which offer many suitable habitats for common toads (C.D. and N.R. pers. obs.). Its present position potentially results from complex demographic processes during the Late Quaternary. The deep and asymmetric introgression at mitochondrial (on the <italic>B. spinosus</italic> side) and nuclear (on the <italic>B. bufo</italic> side) markers could indicate hybrid zone movement. van Riemsdijk et al. (<xref rid=\"evl3191-bib-0068\" ref-type=\"ref\">2019</xref>) dissected such movements in Normandy, while Arntzen et al. (<xref rid=\"evl3191-bib-0005\" ref-type=\"ref\">2017</xref>) interpreted the cytonuclear discordances in southeastern France as the consequences of successive range shifts since the last glaciation. This signal could also arise due to adaptive introgression (of <italic>B. bufo</italic> mtDNA) and/or mtDNA‐nuclear incompatibilities (with <italic>B. spinosus</italic> mtDNA) (Toews and Brelsford <xref rid=\"evl3191-bib-0066\" ref-type=\"ref\">2012</xref>), but here the geographic extent of the cytonuclear discordances remain compatible with neutral expectations (Bonnet et al. <xref rid=\"evl3191-bib-0011\" ref-type=\"ref\">2017</xref>). Our bioclimatic models bring indirect support to the movement hypothesis: <italic>B. bufo</italic> could have formerly occupied Mediterranean France (Fig. <xref rid=\"evl3191-fig-0004\" ref-type=\"fig\">4</xref>) but was replaced by a northeastward expansion of <italic>B. spinosus</italic> (which seemingly performs better in these ranges, Fig. S11), to the point that only its mtDNA remains in the south. <italic>Bufo spinosus</italic> may continue to locally expand further north if conditions become more suitable due to global warming. This is however specific to our particular study area: the 800‐km‐long <italic>Bufo</italic> transition spans heterogeneous environments from Provencal France to the British Channel, and distinct bioclimatic factors were shown to mediate the boundaries in other parts of the parapatric ranges (Arntzen et al. <xref rid=\"evl3191-bib-0007\" ref-type=\"ref\">2020</xref>). Beside ecology, whether phenotypic traits (acoustic or olfactive) contribute to some pre‐mating barriers also remains to be tested.</p><p>Although insufficient to fully prevent interspecies gene flow, can the different sex chromosomes of <italic>B. bufo</italic> and <italic>B. spinosus</italic> contribute to their mechanisms of post‐zygotic isolation? The dominance theory of Haldane's rule predicts lower fitness for offspring exposed to recessive sex‐linked incompatibilities in the heterogametic sex, in turn causing the large X‐effect on hybrid sterility (Schilthuizen et al. <xref rid=\"evl3191-bib-0059\" ref-type=\"ref\">2011</xref>). In our system, 75% of the progeny of females <italic>B. bufo</italic> (ZW) and males <italic>B. spinosus</italic> (XY) should carry a Y, a W, or both, and may thus be more sensitive than the reverse cross (XX <italic>B. spinosus</italic> female × ZZ <italic>B. bufo</italic> male) where only homogametologs (Z and X) segregate. Under these assumptions, hybrids from the <italic>B. spinosus</italic> matriline should have a greater fitness and thus be more abundant at the species transition. However, here we rather observed the opposite, that is, many <italic>B. spinosus</italic> backcrosses carrying <italic>B. bufo</italic> mtDNA. The dominance predictions of Haldane's rule may thus not apply to this system, perhaps because the heterogametologs are not sufficiently degenerated to bear enough recessive deleterious mutations. This was advocated to explain the absence of large X‐effects in green toads (Gerchen et al. <xref rid=\"evl3191-bib-0024\" ref-type=\"ref\">2018</xref>). Nevertheless, the cyto‐nuclear discordance we observed in the hybrid zone may still originate from conflicts between the genes involved in the sex determination cascade, and which could lead to differential sex‐ratio and fitness among the possible F1 hybrid genotypes (XZ, XW, YZ, YW) and those obtainable from backcrossing (YY, WW). For instance, the lack of introgression by <italic>B. spinosus</italic> mtDNA would fit the hypothesis that the XZ hybrids obtained from <italic>B. spinosus</italic> mothers (XX) and <italic>B. bufo</italic> fathers (ZZ) only develop as males. Finally, a role of the distinct sex chromosomes of <italic>B. bufo</italic> and <italic>B. spinosus</italic> in driving their divergence at secondary sexual traits (as in sticklebacks, Kitano et al. <xref rid=\"evl3191-bib-0033\" ref-type=\"ref\">2009</xref>) would be worth investigating, even though sexual dimorphism in amphibians seems controlled by the differential expression of autosomal genes rather than by sex‐linked polymorphisms (e.g., Ma et al. <xref rid=\"evl3191-bib-0036\" ref-type=\"ref\">2018a</xref>, <xref rid=\"evl3191-bib-0037\" ref-type=\"ref\">2018b</xref>; Veltsos et al. <xref rid=\"evl3191-bib-0069\" ref-type=\"ref\">2020</xref>).</p><p>Overall, the <italic>B. bufo</italic>/<italic>spinosus</italic> study system is somewhat reminiscent of the Japanese wrinkled frog (<italic>Glandirana rugosa</italic>), where closely related geographic forms bear homologous XY and ZW chromosomes and meet in secondary contacts, although without obvious incompatibilities (Miura <xref rid=\"evl3191-bib-0041\" ref-type=\"ref\">2007</xref>). In an extensive survey of this fascinating system, Ogata et al. (<xref rid=\"evl3191-bib-0044\" ref-type=\"ref\">2018</xref>) experimentally produced reciprocal F1s and backcrosses, showing that YZ and XW embryos developed in males and females, respectively, while XZ and YW embryos developed into either sex. Subsequent crosses between these unusual genotypes produced WW and YY embryos. All WW individuals died after hatching, and YY individuals did develop, but fewer reached sexual maturity than expected. It will be fascinating to obtain similar information from <italic>B. bufo</italic> × <italic>B. spinosus</italic> crosses and assess whether the same effects on sex‐determination and heterogametolog lethality apply, and how here it relates to the partial reproductive isolation between these species. While it is not presently possible to test for differential sex‐linked versus autosomal introgression with our anonymous species‐diagnostic RAD tags, an anchored reference genome would allow to locate these tags and perform such test to assess whether sex‐linked genes play a disproportionate role in driving incompatibilities (large X‐ and Z‐effects). By characterizing a heterogametic transition between these hybridizing common toads, our study thus provides the first step to understand how labile sex determination mechanisms can interact with speciation processes. We call to integrate this model system in a comparative framework and address the question more generally, notably by targeting other pairs of parapatric lineages that carry different sex chromosomes, as increasingly reported in anuran amphibians (e.g., <italic>Hyla orientalis</italic>/<italic>savignyi</italic> in Turkey, Dufresnes et al. <xref rid=\"evl3191-bib-0020\" ref-type=\"ref\">2015</xref>, 2020; <italic>Rana japonica</italic> in Japan, <italic>R. pipiens</italic> in the United States, Jeffries et al. <xref rid=\"evl3191-bib-0031\" ref-type=\"ref\">2018</xref>).</p></sec><sec id=\"evl3191-sec-0140\"><title>AUTHOR CONTRIBUTIONS</title><p>C.D., N.P., and P.A.C. designed the study. C.D., N.R., and P.A.C. conducted the field work. C.D., D.L.J., S.N.L., and B.R.K. performed the analyses. C.D. and D.L.J. wrote the article.</p></sec><sec sec-type=\"data-availability\" id=\"evl3191-sec-0150\"><title>DATA ARCHIVING</title><p>The mitochondrial barcoding data are available in Table S1. The RAD‐seq (raw sequence reads) were uploaded on the NCBI sequence read archive (SRA) under BioProject PRJNA542138.</p></sec><sec id=\"evl3191-sec-0160\"><p>Associate Editor: J. Mank</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting information</title><supplementary-material content-type=\"local-data\"><caption><p>\n<bold>Fig. S1</bold>: Anatomical examination of <italic>B. bufo</italic> gonads.</p><p>\n<bold>Fig. S2</bold>: Occurrence records used to build the species distribution models.</p><p>\n<bold>Fig. S3</bold>: Parameter testing for identification of sex‐linked markers with SLM finder.</p><p>\n<bold>Fig. S4</bold>: PCA of <italic>Bufo</italic> allele frequency in southeastern France.</p><p>\n<bold>Fig. S5</bold>: Correlation between STRUCTURE and PCA scores.</p><p>\n<bold>Fig. S6</bold>: Population averages of ancestry, heterozygosity and linkage disequilibrium indices along the hybrid zone transect.</p><p>\n<bold>Fig. S7</bold>: Triangle plot of individual heterozygosity <italic>vs</italic> nuclear ancestry.</p><p>\n<bold>Fig. S8</bold>: Distribution of the cline parameters.</p><p>\n<bold>Fig. S9</bold>: Comparison of the tail parameters of the clines between the <italic>B. bufo</italic> and the <italic>B. spinosus</italic> side.</p><p>\n<bold>Fig. S10</bold>: Multivariate analyses of environmental conditions at <italic>B. bufo</italic> and <italic>B. spinosus</italic> occurrence records.</p><p>\n<bold>Fig. S11</bold>: Relationship between species occurrence probability and distance along our transect in southeastern France.</p></caption><media xlink:href=\"EVL3-4-444-s001.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\"><caption><p>\n<bold>Table S1</bold>: Details on the samples included this study.</p></caption><media xlink:href=\"EVL3-4-444-s002.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\"><caption><p>\n<bold>Table S2</bold>: Parameter sets tested in SLM Finder.</p></caption><media xlink:href=\"EVL3-4-444-s003.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\"><caption><p>\n<bold>Table S3</bold>: Performance metrics and variable contributions of the species distribution models.</p></caption><media xlink:href=\"EVL3-4-444-s004.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"evl3191-sec-0130\"><title>ACKNOWLEDGMENTS</title><p>This study was only possible thanks to many fieldwork contributions by B. Adam, L. Beligné, D. Combrisson, L. Courmont, D. Dufresnes, M. Dufresnes, R. Duguet, S. Gardien, J.M. Ferro, C. Jacquier, P. Joly, M Lathuillière, Q. Martinez, A. Movia, G. Maillet, L. Parrain, N. Parrain, M. Pezin, M. Pajković, P. Priol, A. Roux, C. Roy, A. Sprumont, V. Thary. We also thank J.W. Arntzen for useful discussions and B. Wielstra for comments on a previous version of the manuscript. This study was funded by the Swiss National Science Foundation (grant no. 31003A_166323 to N.P. and fellowship no. P2LAP3_171818 to C.D.). S.N.L. was supported by the RFBR research project 20‐04‐00918.</p></ack><ref-list id=\"evl3191-bibl-0001\"><title>LITERATURE CITED</title><ref id=\"evl3191-bib-0001\"><mixed-citation publication-type=\"journal\" id=\"evl3191-cit-0001\">\n<string-name>\n<surname>Abramyan</surname>, <given-names>J.</given-names>\n</string-name>, <string-name>\n<given-names>T.</given-names>\n<surname>Ezaz</surname>\n</string-name>, <string-name>\n<given-names>J. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Evol Lett</journal-id><journal-id journal-id-type=\"doi\">10.1002/(ISSN)2056-3744</journal-id><journal-id journal-id-type=\"publisher-id\">EVL3</journal-id><journal-title-group><journal-title>Evolution Letters</journal-title></journal-title-group><issn pub-type=\"epub\">2056-3744</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33014417</article-id><article-id pub-id-type=\"pmc\">PMC7523564</article-id><article-id pub-id-type=\"doi\">10.1002/evl3.192</article-id><article-id pub-id-type=\"publisher-id\">EVL3192</article-id><article-categories><subj-group subj-group-type=\"overline\"><subject>Comment and Opinion</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Comment and Opinion</subject></subj-group></article-categories><title-group><article-title>The search for sexually antagonistic genes: Practical insights from studies of local adaptation and statistical genomics</article-title><alt-title alt-title-type=\"left-running-head\">F. RUZICKA ET AL.</alt-title></title-group><contrib-group><contrib id=\"evl3192-cr-0001\" contrib-type=\"author\" corresp=\"yes\"><name><surname>Ruzicka</surname><given-names>Filip</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-9089-624X</contrib-id><xref ref-type=\"aff\" rid=\"evl3192-aff-0001\">\n<sup>1</sup>\n</xref><address><email>Filip.Ruzicka@monash.edu</email></address></contrib><contrib id=\"evl3192-cr-0002\" contrib-type=\"author\"><name><surname>Dutoit</surname><given-names>Ludovic</given-names></name><xref ref-type=\"aff\" rid=\"evl3192-aff-0002\">\n<sup>2</sup>\n</xref><xref ref-type=\"author-notes\" rid=\"evl3192-note-0001\">\n<sup>†</sup>\n</xref></contrib><contrib id=\"evl3192-cr-0003\" contrib-type=\"author\"><name><surname>Czuppon</surname><given-names>Peter</given-names></name><xref ref-type=\"aff\" rid=\"evl3192-aff-0003\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"evl3192-aff-0004\">\n<sup>4</sup>\n</xref></contrib><contrib id=\"evl3192-cr-0004\" contrib-type=\"author\"><name><surname>Jordan</surname><given-names>Crispin Y.</given-names></name><xref ref-type=\"aff\" rid=\"evl3192-aff-0005\">\n<sup>5</sup>\n</xref></contrib><contrib id=\"evl3192-cr-0005\" contrib-type=\"author\"><name><surname>Li</surname><given-names>Xiang‐Yi</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-8662-0865</contrib-id><xref ref-type=\"aff\" rid=\"evl3192-aff-0006\">\n<sup>6</sup>\n</xref></contrib><contrib id=\"evl3192-cr-0006\" contrib-type=\"author\"><name><surname>Olito</surname><given-names>Colin</given-names></name><xref ref-type=\"aff\" rid=\"evl3192-aff-0007\">\n<sup>7</sup>\n</xref></contrib><contrib id=\"evl3192-cr-0008\" contrib-type=\"author\"><name><surname>Runemark</surname><given-names>Anna</given-names></name><xref ref-type=\"aff\" rid=\"evl3192-aff-0007\">\n<sup>7</sup>\n</xref></contrib><contrib id=\"evl3192-cr-0009\" contrib-type=\"author\"><name><surname>Svensson</surname><given-names>Erik I.</given-names></name><xref ref-type=\"aff\" rid=\"evl3192-aff-0007\">\n<sup>7</sup>\n</xref></contrib><contrib id=\"evl3192-cr-0007\" contrib-type=\"author\"><name><surname>Yazdi</surname><given-names>Homa Papoli</given-names></name><xref ref-type=\"aff\" rid=\"evl3192-aff-0007\">\n<sup>7</sup>\n</xref></contrib><contrib id=\"evl3192-cr-0010\" contrib-type=\"author\"><name><surname>Connallon</surname><given-names>Tim</given-names></name><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-1962-4951</contrib-id><address><email>tim.connallon@monash.edu</email></address><xref ref-type=\"aff\" rid=\"evl3192-aff-0001\">\n<sup>1</sup>\n</xref></contrib></contrib-group><aff id=\"evl3192-aff-0001\">\n<label><sup>1</sup></label>\n<named-content content-type=\"organisation-division\">School of Biological Sciences</named-content>\n<institution>Monash University</institution>\n<city>Clayton</city>\n<postal-code>VIC 3800</postal-code>\n<country country=\"AU\">Australia</country>\n</aff><aff id=\"evl3192-aff-0002\">\n<label><sup>2</sup></label>\n<named-content content-type=\"organisation-division\">Department of Zoology</named-content>\n<institution>University of Otago</institution>\n<city>Dunedin</city>\n<postal-code>9054</postal-code>\n<country country=\"NZ\">New Zealand</country>\n</aff><aff id=\"evl3192-aff-0003\">\n<label><sup>3</sup></label>\n<named-content content-type=\"organisation-division\">Institute of Ecology and Environmental Sciences, UPEC, CNRS, IRD, INRA</named-content>\n<institution>Sorbonne Université</institution>\n<city>Paris</city>\n<postal-code>75252</postal-code>\n<country country=\"FR\">France</country>\n</aff><aff id=\"evl3192-aff-0004\">\n<label><sup>4</sup></label>\n<named-content content-type=\"organisation-division\">Center for Interdisciplinary Research in Biology, CNRS, Collège de France</named-content>\n<institution>PSL Research University</institution>\n<city>Paris</city>\n<postal-code>75231</postal-code>\n<country country=\"FR\">France</country>\n</aff><aff id=\"evl3192-aff-0005\">\n<label><sup>5</sup></label>\n<named-content content-type=\"organisation-division\">School of Biomedical Sciences</named-content>\n<institution>University of Edinburgh</institution>\n<city>Edinburgh</city>\n<postal-code>EH8 9XD</postal-code>\n<country country=\"GB\">United Kingdom</country>\n</aff><aff id=\"evl3192-aff-0006\">\n<label><sup>6</sup></label>\n<named-content content-type=\"organisation-division\">Institute of Biology</named-content>\n<institution>University of Neuchâtel</institution>\n<city>Neuchatel</city>\n<postal-code>CH‐2000</postal-code>\n<country country=\"CH\">Switzerland</country>\n</aff><aff id=\"evl3192-aff-0007\">\n<label><sup>7</sup></label>\n<named-content content-type=\"organisation-division\">Department of Biology</named-content>\n<institution>Lund University</institution>\n<city>Lund</city>\n<postal-code>SE‐22362</postal-code>\n<country country=\"SE\">Sweden</country>\n</aff><author-notes><corresp id=\"correspondenceTo\"><label>*</label>E‐mail: <email>Filip.Ruzicka@monash.edu</email><email>tim.connallon@monash.edu</email><break/></corresp><fn id=\"evl3192-note-0001\"><label>†</label><p>Ludovic Dutoit is a co‐first author, along with Filip Ruzicka</p></fn></author-notes><pub-date pub-type=\"epub\"><day>31</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><month>10</month><year>2020</year></pub-date><volume>4</volume><issue>5</issue><issue-id pub-id-type=\"doi\">10.1002/evl3.v4.5</issue-id><fpage>398</fpage><lpage>415</lpage><history><date date-type=\"received\"><day>28</day><month>4</month><year>2020</year></date><date date-type=\"rev-recd\"><day>13</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>7</month><year>2020</year></date></history><permissions><!--<copyright-statement content-type=\"issue-copyright\"> © 2020 The Authors. Evolution Letters published by Wiley Periodicals LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB). <copyright-statement>--><copyright-statement content-type=\"article-copyright\">© 2020 The Authors. <italic>Evolution Letters</italic> published by Wiley Periodicals, Inc. on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB).</copyright-statement><license license-type=\"creativeCommonsBy\"><license-p>This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"file:EVL3-4-398.pdf\"/><abstract><title>Abstract</title><p>Sexually antagonistic (SA) genetic variation—in which alleles favored in one sex are disfavored in the other—is predicted to be common and has been documented in several animal and plant populations, yet we currently know little about its pervasiveness among species or its population genetic basis. Recent applications of genomics in studies of SA genetic variation have highlighted considerable methodological challenges to the identification and characterization of SA genes, raising questions about the feasibility of genomic approaches for inferring SA selection. The related fields of local adaptation and statistical genomics have previously dealt with similar challenges, and lessons from these disciplines can therefore help overcome current difficulties in applying genomics to study SA genetic variation. Here, we integrate theoretical and analytical concepts from local adaptation and statistical genomics research—including <italic>F</italic>\n<sub>ST</sub> and <italic>F</italic>\n<sub>IS</sub> statistics, genome‐wide association studies, pedigree analyses, reciprocal transplant studies, and evolve‐and‐resequence experiments—to evaluate methods for identifying SA genes and genome‐wide signals of SA genetic variation. We begin by developing theoretical models for between‐sex <italic>F</italic>\n<sub>ST</sub> and <italic>F</italic>\n<sub>IS</sub>, including explicit null distributions for each statistic, and using them to critically evaluate putative multilocus signals of sex‐specific selection in previously published datasets. We then highlight new statistics that address some of the limitations of <italic>F</italic>\n<sub>ST</sub> and <italic>F</italic>\n<sub>IS</sub>, along with applications of more direct approaches for characterizing SA genetic variation, which incorporate explicit fitness measurements. We finish by presenting practical guidelines for the validation and evolutionary analysis of candidate SA genes and discussing promising empirical systems for future work.</p></abstract><kwd-group kwd-group-type=\"author-generated\"><kwd id=\"evl3192-kwd-0001\">Evolutionary genomics</kwd><kwd id=\"evl3192-kwd-0002\">fitness variation</kwd><kwd id=\"evl3192-kwd-0003\"><italic>F</italic><sub>ST</sub></kwd><kwd id=\"evl3192-kwd-0004\"><italic>F</italic><sub>IS</sub></kwd><kwd id=\"evl3192-kwd-0005\">genomic inference of natural selection</kwd><kwd id=\"evl3192-kwd-0006\">intralocus sexual conflict</kwd><kwd id=\"evl3192-kwd-0007\">local adaptation</kwd></kwd-group><funding-group><award-group id=\"funding-0001\"><funding-source><institution-wrap><institution>Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/501100001711</institution-id></institution-wrap></funding-source><award-id>180145</award-id></award-group><award-group id=\"funding-0002\"><funding-source><institution-wrap><institution>Australian Research Council </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/501100000923</institution-id></institution-wrap></funding-source></award-group><award-group id=\"funding-0003\"><funding-source><institution-wrap><institution>Vetenskapsrådet </institution><institution-id institution-id-type=\"open-funder-registry\">10.13039/501100004359</institution-id></institution-wrap></funding-source><award-id>2016–03356</award-id></award-group></funding-group><counts><fig-count count=\"3\"/><table-count count=\"1\"/><page-count count=\"18\"/><word-count count=\"14703\"/></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>October 2020</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.9.1 mode:remove_FC converted:29.09.2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><boxed-text position=\"float\" content-type=\"box\" orientation=\"portrait\"><caption><title>Impact Summary</title></caption><p>Genome sequences carry a record of the evolutionary and demographic histories of natural populations. Research over the last two decades has dramatically improved our ability to detect genomic signals of adaptation by natural selection, including several widely‐used methods for identifying genes underlying local adaptation and quantitative trait variation. Yet the application of these methods to identify sexually antagonistic (SA) genes—wherein variants that are adaptive for one sex are maladaptive for the other—remains underexplored, despite the potential importance of SA selection as a mechanism for maintaining genetic variation. Indeed, several lines of evidence suggest that SA genetic variation is common within animal and plant populations, underscoring the need for analytical methods that can reliably identify SA genes and genomic signals of SA genetic variation. Here, we integrate statistics and experimental designs that were originally developed within the fields of local adaptation and statistical genomics and apply them to contexts of sex‐specific adaptation and SA genetic variation. First, we evaluate and extend statistical methods for identifying signals of SA variation from genome sequence data alone. We then apply these methods to reanalyze previously published data on allele frequency differences between sexes—a putative signal of SA selection. Second, we highlight more direct approaches for identifying SA genetic variation, which use experimental evolution and statistical associations between individual genetic variants and fitness. Third, we provide guidelines for the biological validation, evolutionary analysis, and interpretation of candidate SA polymorphisms. By building upon the strong methodological foundations of local adaptation and statistical genomics research, we provide a roadmap for rigorous analyses of genetic data in the context of sex‐specific adaptation, thereby facilitating insights into the role and pervasiveness of SA variation in adaptive evolution.</p></boxed-text><p>A population's evolutionary capacity for adaptation hinges upon the nature and extent of the genetic variation it harbors (Fisher <xref rid=\"evl3192-bib-0063\" ref-type=\"ref\">1930</xref>). In simple environments where selection is uniform over time, across space, and among different classes of individuals within the population, adaptation may proceed by fixing unconditionally beneficial mutations and eliminating deleterious ones. Yet species exist in complex environments, where opportunities for adaptation can be limited by genetic trade‐offs among traits and fitness components (Otto <xref rid=\"evl3192-bib-0121\" ref-type=\"ref\">2004</xref>; Gomulkiewicz and Houle <xref rid=\"evl3192-bib-0068\" ref-type=\"ref\">2009</xref>; Chevin <xref rid=\"evl3192-bib-0036\" ref-type=\"ref\">2013</xref>; Connallon and Hall <xref rid=\"evl3192-bib-0045\" ref-type=\"ref\">2018</xref>) or by gene flow and conflicting directional selection among habitats within a species’ range (Kirkpatrick and Barton <xref rid=\"evl3192-bib-0088\" ref-type=\"ref\">1997</xref>; Lenormand <xref rid=\"evl3192-bib-0095\" ref-type=\"ref\">2002</xref>; Duputié et al. <xref rid=\"evl3192-bib-0057\" ref-type=\"ref\">2012</xref>). Such contexts allow maladaptation to persist in spite of abundant genetic variation within the population (Walsh and Blows <xref rid=\"evl3192-bib-0171\" ref-type=\"ref\">2009</xref>).</p><p>“Sexually antagonistic” (SA) genetic variation—wherein alleles that are beneficial when expressed in one sex are harmful when expressed in the other—represents a particularly common form of genetic trade‐off (Rice and Chippindale <xref rid=\"evl3192-bib-0137\" ref-type=\"ref\">2001</xref>; Bonduriansky and Chenoweth <xref rid=\"evl3192-bib-0026\" ref-type=\"ref\">2009</xref>; Van Doorn <xref rid=\"evl3192-bib-0164\" ref-type=\"ref\">2009</xref>). SA genetic variation arises from sex differences in selection (a.k.a. sex‐specific selection) on traits that are genetically correlated between the sexes (Connallon and Clark <xref rid=\"evl3192-bib-0042\" ref-type=\"ref\">2014b</xref>), and may contribute substantially to fitness variation (Kidwell et al. <xref rid=\"evl3192-bib-0087\" ref-type=\"ref\">1977</xref>; Abbott <xref rid=\"evl3192-bib-0001\" ref-type=\"ref\">2011</xref>; Connallon and Clark <xref rid=\"evl3192-bib-0041\" ref-type=\"ref\">2014a</xref>; Olito et al. <xref rid=\"evl3192-bib-0120\" ref-type=\"ref\">2018</xref>) and maladaptation (Lande <xref rid=\"evl3192-bib-0092\" ref-type=\"ref\">1980</xref>; Matthews et al. <xref rid=\"evl3192-bib-0108\" ref-type=\"ref\">2019</xref>). Estimates of phenotypic selection suggest that sex differences in directional selection are common (Cox and Calsbeek <xref rid=\"evl3192-bib-0050\" ref-type=\"ref\">2009</xref>; Lewis et al. <xref rid=\"evl3192-bib-0096\" ref-type=\"ref\">2011</xref>; Gosden et al. <xref rid=\"evl3192-bib-0069\" ref-type=\"ref\">2012</xref>; Stearns et al. <xref rid=\"evl3192-bib-0155\" ref-type=\"ref\">2012</xref>; Morrissey <xref rid=\"evl3192-bib-0111\" ref-type=\"ref\">2016</xref>; De Lisle et al. <xref rid=\"evl3192-bib-0053\" ref-type=\"ref\">2018</xref>; Singh and Punzalan <xref rid=\"evl3192-bib-0150\" ref-type=\"ref\">2018</xref>), implying that many genetic variants affecting quantitative traits have SA effects on fitness. Likewise, estimates of genetic variation for fitness suggest that the genetic basis of female and male fitness components is partially discordant, with some multilocus genotypes conferring high fitness in one sex and low fitness in the other (Chippindale et al. <xref rid=\"evl3192-bib-0037\" ref-type=\"ref\">2001</xref>; reviewed in Connallon and Matthews <xref rid=\"evl3192-bib-0044\" ref-type=\"ref\">2019</xref>).</p><p>Although studies of sex‐specific selection indicate that SA alleles contribute to fitness variation in several animal and plant populations (e.g., Chippindale et al. <xref rid=\"evl3192-bib-0037\" ref-type=\"ref\">2001</xref>; Fedorka and Mousseau <xref rid=\"evl3192-bib-0061\" ref-type=\"ref\">2004</xref>; Svensson et al. <xref rid=\"evl3192-bib-0157\" ref-type=\"ref\">2009</xref>; Delph et al. <xref rid=\"evl3192-bib-0055\" ref-type=\"ref\">2011</xref>; Mokkonen et al. <xref rid=\"evl3192-bib-0110\" ref-type=\"ref\">2011</xref>; Berger et al. <xref rid=\"evl3192-bib-0019\" ref-type=\"ref\">2014</xref>), the population genetic basis of this fitness variation is largely unknown, leaving several important questions unanswered. For example, what fraction of genetic variance for fitness is attributable to SA alleles versus other classes of genetic variation (e.g., deleterious mutations)? Is SA genetic variation attributable to many small‐effect loci or to a few large‐effect loci? Are SA polymorphisms maintained under balancing selection, or are they transient and primarily evolving via mutation, directional selection, and drift? Are SA alleles randomly distributed across the genome or are they enriched on certain chromosome types (e.g., sex chromosomes)? These questions are part of the broader debate about the genetic basis of fitness variation and the evolutionary forces that maintain it (Lewontin <xref rid=\"evl3192-bib-0097\" ref-type=\"ref\">1975</xref>; Charlesworth and Hughes <xref rid=\"evl3192-bib-0031\" ref-type=\"ref\">1999</xref>), which is one of the oldest in this field and perhaps the most difficult to resolve (e.g., Lewontin <xref rid=\"evl3192-bib-0075\" ref-type=\"ref\">1975</xref>, p. 23).</p><p>As in most areas of evolutionary biology, research on SA selection is increasingly drawing upon genomics. A few studies have identified candidate SA polymorphisms with large effects on traits related to fitness (Roberts et al. <xref rid=\"evl3192-bib-0140\" ref-type=\"ref\">2009</xref>; Barson et al. <xref rid=\"evl3192-bib-0011\" ref-type=\"ref\">2015</xref>; Rostant et al. <xref rid=\"evl3192-bib-0143\" ref-type=\"ref\">2015</xref>; VanKuren and Long <xref rid=\"evl3192-bib-0165\" ref-type=\"ref\">2018</xref>; Pearse et al. <xref rid=\"evl3192-bib-0124\" ref-type=\"ref\">2019</xref>), and this handful of SA loci almost certainly represents the tip of the iceberg (Ruzicka et al. <xref rid=\"evl3192-bib-0146\" ref-type=\"ref\">2019</xref>). Many other studies have highlighted genomic patterns of expression or sequence diversity that could be indicative of sex‐specific selection (e.g., Innocenti and Morrow <xref rid=\"evl3192-bib-0079\" ref-type=\"ref\">2010</xref>; reviewed in Kasimatis et al. <xref rid=\"evl3192-bib-0081\" ref-type=\"ref\">2017</xref>; Mank <xref rid=\"evl3192-bib-0107\" ref-type=\"ref\">2017</xref>; Rowe et al. <xref rid=\"evl3192-bib-0144\" ref-type=\"ref\">2018</xref>).</p><p>Although there is optimism that genomics will facilitate the study of sex‐specific selection, we still face several challenges in applying genomic data to identify and characterize SA genetic variation. For example, some putative genomic signals of sex‐specific selection, such as sex‐biased gene expression, are ambiguous: at best, they may serve as indirect proxies of sex‐specific selection, or at worst provide no information about contemporary selection on each sex (Kasimatis et al. <xref rid=\"evl3192-bib-0081\" ref-type=\"ref\">2017</xref>; Rowe et al. <xref rid=\"evl3192-bib-0144\" ref-type=\"ref\">2018</xref>). Allele frequency differences between sexes (e.g., between‐sex <italic>F</italic>\n<sub>ST</sub>) may represent more straightforward genomic signatures of sex‐specific selection (e.g., Cheng and Kirkpatrick <xref rid=\"evl3192-bib-0034\" ref-type=\"ref\">2016</xref>; Lucotte et al. <xref rid=\"evl3192-bib-0101\" ref-type=\"ref\">2016</xref>; but see Bissegger et al. <xref rid=\"evl3192-bib-0024\" ref-type=\"ref\">2019</xref>; Kasimatis et al. <xref rid=\"evl3192-bib-0083\" ref-type=\"ref\">2020</xref>). Yet ambiguous null hypotheses for empirical estimates of between‐sex <italic>F</italic>\n<sub>ST</sub>, along with high statistical noise relative to biological signal in these estimates, raise questions about statistical power and the prevalence of false positives within such data (Kasimatis et al. <xref rid=\"evl3192-bib-0082\" ref-type=\"ref\">2019</xref>). In addition, we need to better understand the extent to which common pitfalls of genome sequence datasets—for example, mismapped reads (Tsai et al. <xref rid=\"evl3192-bib-0161\" ref-type=\"ref\">2019</xref>), sampling biases, hidden population structure, and effects of linkage and hitchhiking—yield artifactual signals of sex‐specific processes, and thus questionable candidate SA genes.</p><p>The challenges in applying genomics to sex‐specific selection have strong parallels in the fields of local adaptation and statistical genomics. Although the study of SA loci is still in its infancy, the fields of local adaptation and statistical genomics have already grappled intensively with many of the conceptual and methodological challenges that research on sex‐specific selection now faces (Hoban et al. <xref rid=\"evl3192-bib-0075\" ref-type=\"ref\">2016</xref>; Visscher et al. <xref rid=\"evl3192-bib-0168\" ref-type=\"ref\">2017</xref>). For example, local adaptation research has long emphasized the importance of clear null models for distinguishing genes involved in local adaptation from false positives that simply reside in the tails of neutral null distributions (Lewontin and Krakauer <xref rid=\"evl3192-bib-0098\" ref-type=\"ref\">1973</xref>; Günther and Coop <xref rid=\"evl3192-bib-0070\" ref-type=\"ref\">2013</xref>; Whitlock and Lotterhos <xref rid=\"evl3192-bib-0174\" ref-type=\"ref\">2015</xref>; Lohse <xref rid=\"evl3192-bib-0099\" ref-type=\"ref\">2017</xref>). Similarly, statistical genomics researchers have repeatedly warned that hidden population structure in genome‐wide association studies (GWAS) can lead to spurious conclusions about the genetic basis of quantitative traits, complex diseases, and the role of adaptation in population differentiation (Lander and Schork <xref rid=\"evl3192-bib-0093\" ref-type=\"ref\">1994</xref>; Price et al. <xref rid=\"evl3192-bib-0128\" ref-type=\"ref\">2010</xref>; Barton et al. <xref rid=\"evl3192-bib-0012\" ref-type=\"ref\">2019</xref>; Berg et al. <xref rid=\"evl3192-bib-0018\" ref-type=\"ref\">2019</xref>; Sohail et al. <xref rid=\"evl3192-bib-0153\" ref-type=\"ref\">2019</xref>). Lessons from local adaptation and statistical genomics research can therefore sharpen hypothesis framing, guide statistical methodology, and inform best practices for disentangling signal, noise, and artifacts in studies of sex‐specific selection.</p><p>Here, by drawing insights from local adaptation and statistical genomics research, we present practical guidelines for population genomic analyses of sex‐specific fitness variation. We first outline two statistics that can, in principle, provide <italic>indirect</italic> evidence of sex‐specific fitness effects of genetic variation: between‐sex <italic>F</italic>\n<sub>ST</sub>, which is sensitive to sex differences in viability selection and some components of reproductive success, and <italic>F</italic>\n<sub>IS</sub>, a measure of Hardy‐Weinberg deviations in diploids, which is sensitive to sex differences in overall selection (i.e., cumulative effects of viability, fertility, fecundity, and mating competition). We develop theoretical null models for each metric, provide an overview of their sampling distributions and statistical power, and present a reanalysis of published <italic>F</italic>\n<sub>ST</sub> data in light of our models. We also highlight complementary methods adapted from case‐control GWAS to overcome some of the limitations of these metrics. Second, we evaluate several <italic>direct</italic> approaches for characterizing sex‐specific genetic variation for fitness, which combine elements from quantitative genetics, reciprocal transplant studies, and experimental evolution. These direct approaches have been extensively employed to study the genetic basis of locally adapted phenotypes and quantitative traits, but rarely to identify SA loci. Third, we outline approaches for validating candidate genes and discuss best practices for the analysis and interpretation of their evolutionary histories.</p><sec id=\"evl3192-sec-0020\"><title>Indirect Approaches for Identifying SA Genes</title><p>Estimating fitness under natural conditions is difficult, rendering approaches for identifying SA genes that rely on fitness measurements (i.e., direct approaches; see section “Direct Approaches for Identifying SA Genes”) unfeasible for many populations. Any widely applicable approach must therefore make use of <italic>indirect</italic> empirical signals of SA selection in genome sequence data, which can now be collected for virtually any species.</p><p>Two specific patterns of genome sequence variation could be indicative of contemporary SA selection, as emphasized by several recent studies (e.g., Cheng and Kirkpatrick <xref rid=\"evl3192-bib-0034\" ref-type=\"ref\">2016</xref>; Lucotte et al. <xref rid=\"evl3192-bib-0101\" ref-type=\"ref\">2016</xref>; Eyer et al. <xref rid=\"evl3192-bib-0060\" ref-type=\"ref\">2019</xref>; Kasimatis et al. <xref rid=\"evl3192-bib-0082\" ref-type=\"ref\">2019</xref>). First, because sex differences in selection during the life cycle are expected to generate allele frequency differences between breeding females and males (i.e., the members of each sex that contribute to offspring of the next generation; see Box 1), allele frequency differences between <italic>samples</italic> of females and males of a population could be indicative of sex differences in selection—including SA selection, sex‐limited selection, or ongoing sexually concordant selection that differs in magnitude between the sexes. Ideally, inferences of sex‐specific selection from allele frequency estimates should be based on samples of breeding adults that have passed the filter of viability selection, sexual selection, and fertility/fecundity selection, although in practice genome sequences of random samples of adults are more readily obtainable and will reflect viability selection (Cheng and Kirkpatrick <xref rid=\"evl3192-bib-0034\" ref-type=\"ref\">2016</xref>; Kasimatis et al. <xref rid=\"evl3192-bib-0082\" ref-type=\"ref\">2019</xref>). Second, allele frequency differences between breeding females and males of a given generation elevate heterozygosity in offspring of the next generation relative to Hardy‐Weinberg expectations (Kasimatis et al. <xref rid=\"evl3192-bib-0082\" ref-type=\"ref\">2019</xref>). Inflated heterozygosity in a large random sample of offspring could therefore reflect sex differences in viability selection, sexual selection, and/or fertility and fecundity selection during the previous generation.</p><sec id=\"evl3192-sec-0030\"><title>SINGLE‐LOCUS SIGNALS OF SEX‐SPECIFIC SELECTION IN FIXATION INDICES</title><p>Fixation indices, which are widely applied in studies of population structure (Wright <xref rid=\"evl3192-bib-0179\" ref-type=\"ref\">1951</xref>), can be used to quantify allele frequency differences between sexes (<italic>F</italic>\n<sub>ST</sub>) and elevations in heterozygosity in the offspring of a given generation (<italic>F</italic>\n<sub>IS</sub>), each of which are predicted consequences of sex‐specific selection (Boxes 1‐2). Several studies have estimated <italic>F</italic>\n<sub>ST</sub> between sexes using gene sequences sampled from adults (Cheng and Kirkpatrick <xref rid=\"evl3192-bib-0034\" ref-type=\"ref\">2016</xref>; Lucotte et al. <xref rid=\"evl3192-bib-0101\" ref-type=\"ref\">2016</xref>; Flanagan and Jones <xref rid=\"evl3192-bib-0064\" ref-type=\"ref\">2017</xref>; Wright et al., <xref rid=\"evl3192-bib-0177\" ref-type=\"ref\">2018</xref>, <xref rid=\"evl3192-bib-0178\" ref-type=\"ref\">2019</xref>; Bissegger et al. <xref rid=\"evl3192-bib-0024\" ref-type=\"ref\">2019</xref>; Vaux et al. <xref rid=\"evl3192-bib-0166\" ref-type=\"ref\">2019</xref>) or from breeding individuals (Dutoit et al. <xref rid=\"evl3192-bib-0058\" ref-type=\"ref\">2018</xref>), yet it remains unclear how much information about SA selection is contained within these estimates. A major problem, as recently emphasized by Kasimatis et al. (<xref rid=\"evl3192-bib-0082\" ref-type=\"ref\">2019</xref>), is that the contribution of sex‐specific selection to allele frequency differentiation between the sexes may often be weak compared to effects of sampling error in allele frequency estimates. Indeed, simulations presented in several studies (e.g., Cheng and Kirkpatrick <xref rid=\"evl3192-bib-0034\" ref-type=\"ref\">2016</xref>; Lucotte et al. <xref rid=\"evl3192-bib-0101\" ref-type=\"ref\">2016</xref>; Connallon and Hall <xref rid=\"evl3192-bib-0045\" ref-type=\"ref\">2018</xref>; Kasimatis et al. <xref rid=\"evl3192-bib-0082\" ref-type=\"ref\">2019</xref>) confirm that signals of SA selection in between‐sex <italic>F</italic>\n<sub>ST</sub> are swamped by sampling error in small population genomic datasets. Nevertheless, without clearly defined probability distributions for between‐sex <italic>F</italic>\n<sub>ST</sub> and related metrics of allele frequency differentiation, it remains difficult to evaluate whether sampling error, by itself, is sufficient to account for the empirical distributions of population genomic metrics that are putatively associated with sex‐specific selection.</p><p>As we show in Appendix A (Supporting Information) (see Box 2), a null distribution for between‐sex <italic>F</italic>\n<sub>ST</sub> estimates at loci with no sex differences in selection conforms to a special case of Lewontin and Krakauer's (<xref rid=\"evl3192-bib-0098\" ref-type=\"ref\">1973</xref>) classic null model for <italic>F</italic>\n<sub>ST</sub> estimated between populations. An appealing feature of our two‐sex null model is its insensitivity to some of the simplifying assumptions inherent in Lewontin and Krakauer's original model (i.e., that subpopulations are independent; see Nei and Maruyama <xref rid=\"evl3192-bib-0118\" ref-type=\"ref\">1975</xref>; Robertson <xref rid=\"evl3192-bib-0142\" ref-type=\"ref\">1975</xref>; Charlesworth <xref rid=\"evl3192-bib-0029\" ref-type=\"ref\">1998</xref>; Beaumont <xref rid=\"evl3192-bib-0013\" ref-type=\"ref\">2005</xref>; Whitlock and Lotterhos <xref rid=\"evl3192-bib-0174\" ref-type=\"ref\">2015</xref>), or issues arising from genetic linkage (Charlesworth <xref rid=\"evl3192-bib-0029\" ref-type=\"ref\">1998</xref>), which do not affect the two‐sex null distribution when <italic>F</italic>\n<sub>ST</sub> is independently estimated per single nucleotide polymorphism (SNP), but can strongly impact the null for concatenated sequences (e.g., gene‐wide <italic>F</italic>\n<sub>ST</sub> estimates; see Booker et al. <xref rid=\"evl3192-bib-0028\" ref-type=\"ref\">2020</xref>; Appendix A [Supporting Information]). Another appealing feature of the null model for <italic>F</italic>\n<sub>ST</sub> is its insensitivity to the distribution of allele frequencies in the population, provided SNPs with very low minor allele frequencies (MAFs) are excluded from the analysis (e.g., MAFs < 0.05 for small datasets; MAFs < 0.01 for large datasets; see Appendices A and D [Supporting Information]). This insensitivity to MAF under the null is unique to <italic>F</italic>\n<sub>ST</sub>; other metrics of allele frequency differentiation strongly covary with MAF, so that simulations based on the MAF distribution of the study population are required to generate genome‐wide null predictions against which data can be compared (Appendix D [Supporting Information]; Figs. S6 and S7; see Berner <xref rid=\"evl3192-bib-0021\" ref-type=\"ref\">2019</xref> and Bissegger et al. <xref rid=\"evl3192-bib-0024\" ref-type=\"ref\">2019</xref> for examples).</p><p>By comparing the distribution of between‐sex <italic>F</italic>\n<sub>ST</sub> under the null (i.e., no sex differences in selection) with the corresponding distribution under SA selection (Box 2), we can formally evaluate the minimum strength of selection (<italic>s</italic>\n<sub>min</sub>, defined as the minimum cost, per sex, of inheriting the “wrong” SA allele) required for SA loci to reliably reside within the upper tail of the null distribution. For example, the 99th percentile for the null distribution is <mml:math id=\"nlm-math-7\"><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mrow><mml:mi mathvariant=\"normal\">ST</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mn>99</mml:mn><mml:mo>%</mml:mo></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msub><mml:mo>≈</mml:mo><mml:mn>3.32</mml:mn><mml:mo>/</mml:mo><mml:msub><mml:mi>n</mml:mi><mml:mi>H</mml:mi></mml:msub></mml:mrow></mml:math>, where <italic>n<sub>H</sub></italic> is the harmonic mean sample size of female‐ and male‐derived gene sequences, and <mml:math id=\"nlm-math-8\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> refers to an estimate of <italic>F</italic>\n<sub>ST</sub> (see Box 2; results are based on Nei's <xref rid=\"evl3192-bib-0117\" ref-type=\"ref\">1973</xref> estimator for <italic>F</italic>\n<sub>ST</sub>, which closely aligns with Wright's <xref rid=\"evl3192-bib-0179\" ref-type=\"ref\">1951</xref> definition for <italic>F</italic>\n<sub>ST</sub> between a pair of populations; see Appendix A [Supporting Information] for discussion of alternative <italic>F</italic>\n<sub>ST</sub> estimators). Roughly 1% of <italic>F</italic>\n<sub>ST</sub> estimates should fall above this threshold when there are no sex differences in selection. The probability that a SA locus resides within the tail of the null distribution depends on the allele frequencies at the locus, the strength of selection, and the sample size of individuals that are sequenced (Appendix A [Supporting Information]). In studies with large sample sizes (i.e., <italic>n<sub>H</sub></italic> = 10<sup>5</sup> or greater, as in some human genomic datasets: see Fig. <xref rid=\"evl3192-fig-0001\" ref-type=\"fig\">1</xref>), <mml:math id=\"nlm-math-9\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> for a SA locus with intermediate allele frequencies and a fitness effect of a few percent will reliably fall within the upper tail of the null distribution (Fig. <xref rid=\"evl3192-fig-0001\" ref-type=\"fig\">1</xref>; Appendix D [Supporting Information]). In contrast, studies where <italic>n<sub>H</sub></italic> < 10<sup>4</sup> require very strong selection (<italic>s</italic>\n<sub>min</sub> > 0.05) for SA loci to reliably reside within the upper tail of the null <italic>F</italic>\n<sub>ST</sub> distribution (Fig. <xref rid=\"evl3192-fig-0001\" ref-type=\"fig\">1</xref>), and are unlikely to identify individual SA loci (i.e., significant <italic>F</italic>\n<sub>ST</sub> outliers), even in cases where SA genetic polymorphism <italic>is</italic> common throughout the genome. Under scenarios of sex‐limited selection, or sexually concordant selection that differs in magnitude between the sexes, sample sizes required to reliably identify true <italic>F</italic>\n<sub>ST</sub> outliers must be even larger, as the degree of allele frequency differentiation under sex‐limited selection is roughly half the differentiation expected under SA selection, and differentiation is further muted under sexually concordant selection.</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3192-fig-0001\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Signal of SA selection relative to sampling error in <italic>F</italic>\n<sub>ST</sub> and <italic>F</italic>\n<sub>IS</sub> estimates. <bold>(A)</bold> Lines show the minimum strength of selection (<italic>s</italic>\n<sub>min</sub>; see Appendices A and B [Supporting Information]) that causes the expected values of <mml:math id=\"nlm-math-10\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> and <mml:math id=\"nlm-math-11\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub></mml:math> at an SA locus to reach the 5% tail (broken line) or the 1% tail (solid line) of the null distributions for <mml:math id=\"nlm-math-12\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> (black lines) and <mml:math id=\"nlm-math-13\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub></mml:math> (gray lines). Circles show the theoretical <italic>s</italic>\n<sub>min</sub> values (for the 5% tail) for published between‐sex <italic>F</italic>\n<sub>ST</sub> studies that include eight human datasets (Cheng and Kirkpatrick <xref rid=\"evl3192-bib-0034\" ref-type=\"ref\">2016</xref>; Lucotte et al. <xref rid=\"evl3192-bib-0101\" ref-type=\"ref\">2016</xref>; Kasimatis et al. <xref rid=\"evl3192-bib-0083\" ref-type=\"ref\">2020</xref>; Pirastu et al. <xref rid=\"evl3192-bib-0126\" ref-type=\"ref\">2020</xref>), four fish datasets (see Flanagan and Jones <xref rid=\"evl3192-bib-0064\" ref-type=\"ref\">2017</xref>; Wright et al., <xref rid=\"evl3192-bib-0177\" ref-type=\"ref\">2018</xref>, <xref rid=\"evl3192-bib-0178\" ref-type=\"ref\">2019</xref>; Bissegger et al. <xref rid=\"evl3192-bib-0024\" ref-type=\"ref\">2019</xref>; Vaux et al. <xref rid=\"evl3192-bib-0166\" ref-type=\"ref\">2019</xref>), and seven bird datasets (see Dutoit et al. <xref rid=\"evl3192-bib-0058\" ref-type=\"ref\">2018</xref>; Wright et al. <xref rid=\"evl3192-bib-0178\" ref-type=\"ref\">2019</xref>). The harmonic mean sample size, <italic>n<sub>H</sub></italic>, refers to the number of genes sequenced from diploid female and male samples in studies of between‐sex <italic>F</italic>\n<sub>ST</sub> (<italic>n<sub>H</sub></italic> = 2(1/<italic>n<sub>f</sub></italic> + 1/<italic>n<sub>m</sub></italic>)<sup>−1</sup>, where <italic>n<sub>j</sub></italic> = twice the number of individuals sampled from the <italic>j</italic>th sex); <italic>n</italic> refers to the sample size of offspring that are genotyped in studies of <italic>F</italic>\n<sub>IS</sub> (see Box 2). <bold>(B)</bold> Probability that <mml:math id=\"nlm-math-14\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> for an additive SA locus (i.e., <italic>h<sub>f</sub></italic> = <italic>h<sub>m</sub></italic> = ½) with intermediate equilibrium allele frequencies (<italic>p</italic>, <italic>q</italic> = 1/2) is in the top 5% tail of the null distribution for <mml:math id=\"nlm-math-15\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math>. |<italic>s<sub>m</sub></italic>| is the fitness cost of being homozygous for the “wrong” SA allele (see Appendices A and B [Supporting Information]). See Appendices C and D (Supporting Information) for further analyses of statistical power.</p></caption><graphic id=\"nlm-graphic-1\" xlink:href=\"EVL3-4-398-g001\"/></fig><p>A second putative signal of sex‐specific selection is an enrichment of heterozygotes among offspring cohorts, as inferred from high values of <italic>F</italic>\n<sub>IS</sub> (defined in Box 2; Appendix B [Supporting Information]). Although only a single study has used estimates of <italic>F</italic>\n<sub>IS</sub> (<mml:math id=\"nlm-math-16\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub></mml:math>) to test for sex‐specific selection (Eyer et al. <xref rid=\"evl3192-bib-0060\" ref-type=\"ref\">2019</xref>; see Boxes 1‐2), the potential for future applications warrants evaluation of signals of sex‐specific selection using this metric. Hardy‐Weinberg deviations in a sample, as captured by <mml:math id=\"nlm-math-17\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub></mml:math>, may arise from selection, nonrandom mating (e.g., inbreeding or population structure), or random sampling of genotypes from the population (Crow and Kimura <xref rid=\"evl3192-bib-0051\" ref-type=\"ref\">1970</xref>; Weir <xref rid=\"evl3192-bib-0173\" ref-type=\"ref\">1997</xref>; Lachance <xref rid=\"evl3192-bib-0091\" ref-type=\"ref\">2009</xref>). Statistical properties of Hardy‐Weinberg deviations in genotype samples are well established (Weir <xref rid=\"evl3192-bib-0173\" ref-type=\"ref\">1997</xref>), and easily adaptable for our point of interest: the distribution of <mml:math id=\"nlm-math-18\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub></mml:math> in a randomly mating population in which allele frequencies may differ between the female and male parents of a given generation (Box 2). As illustrated in Box 2, the sampling variance for <mml:math id=\"nlm-math-19\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub></mml:math> exceeds that of <mml:math id=\"nlm-math-20\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> by a substantial margin. Consequently, <mml:math id=\"nlm-math-21\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub></mml:math> has far less power than <mml:math id=\"nlm-math-22\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> to distinguish signal of SA selection from noise (Fig. <xref rid=\"evl3192-fig-0001\" ref-type=\"fig\">1A</xref>), let alone distinguishing sex‐limited selection or sexually concordant selection that differs in magnitude between the sexes. An additional issue is that signals of elevated heterozygosity are expected to be strongest among cohorts sampled at <italic>birth</italic>, yet selection occurring during the life cycle can potentially decrease heterozygosity, further dampening signals of sex‐specific selection in <mml:math id=\"nlm-math-23\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub></mml:math> estimates from adult samples.</p></sec><sec id=\"evl3192-sec-0040\"><title>\n<italic>F</italic>\n<sub>ST</sub> DISTRIBUTIONS AND MULTI‐LOCUS SIGNALS OF SEX‐SPECIFIC SELECTION</title><p>Genome scans for individually significant SA loci (<mml:math id=\"nlm-math-24\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> or <mml:math id=\"nlm-math-25\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub></mml:math> outliers) are severely underpowered unless SA loci segregate for intermediate frequency alleles with large fitness effects and sample sizes are very large (see above). Indeed, no empirical <italic>F</italic>\n<sub>ST</sub> study to date has yielded individually significant autosomal candidate SA SNPs that have survived corrections for multiple‐testing and rigorous controls for genotyping error and read‐mapping artifacts (see below).</p><p>Although <italic>F</italic>\n<sub>ST</sub> scans for individually significant SA loci are highly conservative, the full empirical distribution of <italic>F</italic>\n<sub>ST</sub> for autosomal SNPs may nonetheless carry a cumulative signature of sex‐specific selection at many loci—even in the absence of individually significant SA genes. For example, SNPs responding to sex‐specific selection (i.e., SNPs with sex‐specific fitness effects or SNPs in linkage disequilibrium with them) should inflate average <mml:math id=\"nlm-math-26\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> and the proportion of observations in upper quantiles predicted by null models (Fig. <xref rid=\"evl3192-fig-0002\" ref-type=\"fig\">2</xref>). An excess of observations in the upper quantiles of the theoretical null may imply an enrichment of SNPs responding to sex‐specific selection in the tail of the empirical <mml:math id=\"nlm-math-27\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> distribution, with SNPs in the tail representing interesting candidates for follow‐up analyses (see section “Validation and Follow‐up Analyses of Candidate SA Genes”). Moreover, the fraction of true versus false positives among candidates can be quantified. For example, if 2% of observed SNPs fall within the top 1% quantile of the theoretical null, this implies a 1:1 ratio of true to false positives within the top 2% of observations (i.e., a false discovery rate of 50%).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3192-fig-0002\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Reanalyses of genome‐wide distributions of between‐sex <italic>F</italic>\n<sub>ST</sub> for three vertebrate species. Each panel shows the ratio of observed versus permuted <mml:math id=\"nlm-math-28\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> for SNPs within 100 quantiles of the theoretical null model of <mml:math id=\"nlm-math-29\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math>. A ratio above one indicates an excess of observed <mml:math id=\"nlm-math-30\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> values in a given quantile. <bold>Theoretical data</bold>: <mml:math id=\"nlm-math-31\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mrow><mml:mi>S</mml:mi><mml:mi>T</mml:mi></mml:mrow></mml:msub></mml:math> values were simulated for 10<sup>5</sup> neutrally evolving loci with MAF above 5% in the dataset, and for 10<sup>3</sup> loci responding to SA selection prior to sampling of gene sequences (each SA‐responding locus had a SA fitness effect or was in perfect linkage disequilibrium with an SA locus). SA‐responding loci had intermediate allele frequencies in the population (<italic>p</italic> = ½). SA selection coefficients were drawn from an exponential distribution with mean of <italic>s</italic>\n<sub>avg</sub> = 0.03, with additive fitness effects (<italic>h</italic> = ½). The top 1% theoretical quantile is enriched by ∼50%, implying that ∼1/3 of SNPs above the 99% threshold of the null model are true positives. <bold>Empirical data</bold>: population samples from flycatchers, pipefish, and humans (1000 Genomes data) (see Figs. S1‐S4), excluding variants with MAF < 0.05; Fig. S5 illustrates the effect of including rare variants in the human reanalysis. For flycatcher data, the absence of values in some low‐<mml:math id=\"nlm-math-32\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mrow><mml:mi>S</mml:mi><mml:mi>T</mml:mi></mml:mrow></mml:msub></mml:math> quantiles is due to the small number of sampled sequences, which generates some discrepancy between the <italic>discrete</italic> distribution of <mml:math id=\"nlm-math-33\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> estimates (for both observed and permuted data) and the <italic>continuous</italic> theoretical null outlined in Box 2. The top 1% theoretical quantiles for flycatchers and pipefish are enriched by ∼50% and ∼100%, respectively, implying that ∼1/3 and ∼1/2 of SNPs above the 99% threshold of the null are “true” positives (which still require biological validation and filtering for putative artefacts, as described in the main text). Code and data are available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/ldutoit/male_female_fst\">https://github.com/ldutoit/male_female_fst</ext-link>.</p></caption><graphic id=\"nlm-graphic-3\" xlink:href=\"EVL3-4-398-g002\"/></fig><p>To test for elevation of empirical <mml:math id=\"nlm-math-34\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> estimates relative to our theoretical null model, we reanalyzed three representative datasets from previously published studies (collared flycatcher <italic>Ficedula albicollis</italic>: Dutoit et al. <xref rid=\"evl3192-bib-0058\" ref-type=\"ref\">2018</xref>; gulf pipefish <italic>Syngnathus scovelli</italic>: Flanagan and Jones <xref rid=\"evl3192-bib-0064\" ref-type=\"ref\">2017</xref>; human: The 1000 Genomes Project Consortium 2015 data used by Cheng and Kirkpatrick <xref rid=\"evl3192-bib-0034\" ref-type=\"ref\">2016</xref>; Code available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/ldutoit/male_female_fst\">https://github.com/ldutoit/male_female_fst</ext-link>). For human and flycatcher whole‐genome resequencing datasets, we used autosomal coding variants, excluding any SNP with missing data. For the pipefish RAD‐seq dataset, coding and noncoding variants with less than 50% missing data were included; sex‐linked regions are unknown in this species and could not be excluded. In all datasets, polymorphic sites with MAFs below 5% were also excluded, as sites with low MAF exhibit inflated sampling variances (see Whitlock and Lotterhos <xref rid=\"evl3192-bib-0174\" ref-type=\"ref\">2015</xref>; Appendix A [Supporting Information] and Figs. S5‐S7). Analyses were carried out in bedtools (Quinlan and Hall <xref rid=\"evl3192-bib-0131\" ref-type=\"ref\">2010</xref>), vcftools (Danecek et al. <xref rid=\"evl3192-bib-0052\" ref-type=\"ref\">2011</xref>), and R (R Core Team <xref rid=\"evl3192-bib-0132\" ref-type=\"ref\">2020</xref>).</p><p>For all three datasets, permuted <mml:math id=\"nlm-math-35\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> distributions (i.e., <mml:math id=\"nlm-math-36\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> calculated after randomly permuting sex labels across individuals) conform well to the theoretical null model for <mml:math id=\"nlm-math-37\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> (Figs. S1‐S4). For the 1000 Genomes human dataset, <mml:math id=\"nlm-math-38\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> observations (i.e., nonpermuted estimates) are indistinguishable from both the theoretical null and permuted distributions, with no enrichment of observations within the top quantiles of the null (Fig. <xref rid=\"evl3192-fig-0002\" ref-type=\"fig\">2</xref>; <mml:math id=\"nlm-math-39\"><mml:msup><mml:mi>χ</mml:mi><mml:mn>2</mml:mn></mml:msup></mml:math> tests; 5% tail: <italic>P</italic> = 0.18; 1% tail: <italic>P</italic> = 0.33; comparison between the nonpermuted mean <mml:math id=\"nlm-math-40\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> and the <mml:math id=\"nlm-math-41\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> means for 1000 permutations of the data: <italic>P</italic> > 0.05). In contrast, flycatcher and pipefish datasets show elevated <mml:math id=\"nlm-math-42\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> values relative to the 5% and 1% tails of their null distributions (Fig. <xref rid=\"evl3192-fig-0002\" ref-type=\"fig\">2</xref>; <mml:math id=\"nlm-math-43\"><mml:msup><mml:mi>χ</mml:mi><mml:mn>2</mml:mn></mml:msup></mml:math> tests; <italic>P</italic> = 4.33 × 10<sup>−9</sup> and <italic>P</italic> < 2.2 × 10<sup>−16</sup> for flycatcher; <italic>P</italic> < 2.2 × 10<sup>−16</sup> and <italic>P</italic> < 2.2 × 10<sup>−16</sup> for pipefish), and exhibit inflated means for observed <mml:math id=\"nlm-math-44\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> relative to the <mml:math id=\"nlm-math-45\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> means of 1000 permutations of the data (<italic>P</italic> < 0.05 for both datasets). Such enrichment shows that sampling error is not sufficient to explain empirical distributions of <mml:math id=\"nlm-math-46\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math>, and instead implies that many loci are responding to sex differences in selection (either directly or indirectly through hitchhiking with selected loci), or that a false signal of elevated <mml:math id=\"nlm-math-47\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> has been generated by population structure and/or data quality issues, as discussed below.</p></sec><sec id=\"evl3192-sec-0050\"><title>ACCOUNTING FOR SPURIOUS SIGNALS OF SEX‐SPECIFIC SELECTION</title><p>The analyses presented above suggest that <mml:math id=\"nlm-math-48\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math>, although severely underpowered for detecting individually significant outlier SNPs, may capture polygenic signals of sex‐specific selection. However, sex differences in selection should not be invoked as the cause of such elevations in <mml:math id=\"nlm-math-49\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math> without excluding artifacts that may generate similar patterns.</p><p>First, incorrect mapping of sex‐linked markers to autosomes can potentially lead to artificial inflation of <italic>F</italic>\n<sub>ST</sub> estimates. For example, Y‐ or W‐linked sequences may be erroneously mapped to sequence paralogs on autosomes (Tsai et al. <xref rid=\"evl3192-bib-0161\" ref-type=\"ref\">2019</xref>), resulting in artificially high <italic>F</italic>\n<sub>ST</sub> inferences at autosomal sites (Bissegger et al. <xref rid=\"evl3192-bib-0024\" ref-type=\"ref\">2019</xref>; Kasimatis et al. <xref rid=\"evl3192-bib-0083\" ref-type=\"ref\">2020</xref>). This problem can be mitigated in species with high‐quality reference genomes, where mismapped reads can be eliminated through quality‐filtering steps (i.e., removal of SNPs associated with low MAFs or extreme deviations from Hardy‐Weinberg expectations), removal of candidate regions showing high sequence similarity to sex chromosome sequences (Kasimatis et al. <xref rid=\"evl3192-bib-0083\" ref-type=\"ref\">2020</xref>), and excluding regions with sex‐biases in read coverage. However, mismapping is difficult to control for in species lacking high‐quality reference genomes, including those where the sex determination system is unknown (e.g., the pipefish dataset in Fig. <xref rid=\"evl3192-fig-0002\" ref-type=\"fig\">2</xref>), or where sex chromosomes are young or rapidly evolving. Moreover, the effects of demographic processes, including recent admixture events or sex‐biased migration, play out differently between sex chromosomes and autosomes (Hedrick <xref rid=\"evl3192-bib-0074\" ref-type=\"ref\">2007</xref>), so that caution is required in interpreting elevated between‐sex <italic>F</italic>\n<sub>ST</sub> on the X or Z, as has been reported in humans (e.g., Lucotte et al. <xref rid=\"evl3192-bib-0101\" ref-type=\"ref\">2016</xref>).</p><p>Second, sex differences in population structure—arising from the broad geographic sampling of individuals or recent migration into a single sampled population—can also generate signals of genetic differentiation between females and males in the absence of sex differences in selection (Box 1). Taxa with broad contemporary distributions (e.g., humans and <italic>Drosophila</italic>) often show significant genetic differentiation among populations. Uneven or unrepresentative sampling of individuals of each sex from a set of different locations can, by chance, inflate allele frequency differences between the sexes beyond expectations for a single, panmictic population. If loci showing high between‐sex <italic>F</italic>\n<sub>ST</sub> also exhibit high between‐population <italic>F</italic>\n<sub>ST</sub>, this could be indicative of population structure contributing to allele frequency divergence between the sexes in the empirical sample. Studies that sample individuals from a single population may also show artificially elevated between‐sex <italic>F</italic>\n<sub>ST</sub> if migration is sex biased (Box 1), which is common among animals (Trochet et al. <xref rid=\"evl3192-bib-0160\" ref-type=\"ref\">2016</xref>).</p><p>Sex‐specific population structure can be accounted for by leveraging the statistical framework of case‐control GWAS, in which associations between polymorphic variants and binary phenotypic states are quantified (e.g., presence or absence of a disease). The case‐control GWAS approach treats <italic>sex</italic> (female or male) as the binary phenotypic state and scans for loci with the strongest associations, which should exhibit elevated absolute odds ratios (see Appendix C [Supporting Information]; Kasimatis et al. <xref rid=\"evl3192-bib-0083\" ref-type=\"ref\">2020</xref>; Pirastu et al. <xref rid=\"evl3192-bib-0126\" ref-type=\"ref\">2020</xref>). Although the underlying logic is identical to between‐sex <italic>F</italic>\n<sub>ST</sub>, existing analytical methods for case‐control GWAS can take population structure and relatedness in the empirical sample into account by including kinships (or the top principal components derived from kinships) as covariates (Astle and Balding <xref rid=\"evl3192-bib-0007\" ref-type=\"ref\">2009</xref>; Price et al. <xref rid=\"evl3192-bib-0128\" ref-type=\"ref\">2010</xref>). The case‐control GWAS framework also permits estimation of SNP‐based heritability of the phenotype (i.e., sex; Yang et al. <xref rid=\"evl3192-bib-0180\" ref-type=\"ref\">2011</xref>; Speed et al. <xref rid=\"evl3192-bib-0154\" ref-type=\"ref\">2017</xref>), which can be used to quantify a genome‐wide signal of sex‐specific selection.</p><p>Despite the advantages of leveraging an existing statistical framework, using case‐control GWAS to test for associations between alternative alleles and sex does not sidestep all of the challenges faced by <italic>F</italic>\n<sub>ST</sub> and <italic>F</italic>\n<sub>IS</sub> statistics (see Appendices C and D [Supporting Information]). As with <italic>F</italic>\n<sub>ST</sub> and <italic>F</italic>\n<sub>IS</sub>, large sample sizes remain essential for discriminating between sampling variance and true signal of sex differences in selection (especially when selection is weak), and the methods perform poorly when MAFs are low. Additionally, association tests using odds ratios depend heavily on a normal approximation, and there is a deep and still evolving literature regarding hypothesis testing using these methods that users should be aware of (e.g., Haldane <xref rid=\"evl3192-bib-0071\" ref-type=\"ref\">1956</xref>; Wang and Shan <xref rid=\"evl3192-bib-0172\" ref-type=\"ref\">2015</xref>).</p></sec></sec><sec id=\"evl3192-sec-0060\"><title>Direct Approaches for Identifying SA Genes</title><p>In exceptional study systems, candidate SA polymorphisms can be identified through explicit statistical associations between genotypes and fitness. Such <italic>direct</italic> inference approaches present two major advantages over indirect methods. First, the inclusion of fitness measurements can potentially increase power to detect individual SA loci, relative to indirect methods (e.g., Fig. <xref rid=\"evl3192-fig-0001\" ref-type=\"fig\">1</xref>). Second, association tests can be conducted across many components of fitness (e.g., viability, fecundity, and mating success), facilitating identification of the life history stages and selective contexts affected by SA loci. We outline two general approaches for direct inference of sex‐specific selection—GWAS and evolve‐and‐resequence (E&R) studies—which have been extensively employed to identify genes associated with human trait variation and/or local adaptation (Long et al. <xref rid=\"evl3192-bib-0100\" ref-type=\"ref\">2015</xref>; Visscher et al. <xref rid=\"evl3192-bib-0168\" ref-type=\"ref\">2017</xref>), yet rarely to identify SA loci.</p><sec id=\"evl3192-sec-0070\"><title>GWAS OF SEX‐SPECIFIC FITNESS</title><p>GWAS quantify statistical associations between phenotypic variation and polymorphic SNPs throughout the genome. Using GWAS to identify SA loci further requires that data on fitness components and genotypes are collected from individuals of each sex. A major advantage of GWAS is the availability of statistically rigorous methods to identify candidate loci, including methods to control for covariates in analyses (Price et al. <xref rid=\"evl3192-bib-0128\" ref-type=\"ref\">2010</xref>), and approaches that correct for multiple testing (e.g., family‐wise or false discovery rate correction; Benjamini and Hochberg <xref rid=\"evl3192-bib-0015\" ref-type=\"ref\">1995</xref>) or that reduce the number of tests through gene‐based association analysis (Nagamine et al. <xref rid=\"evl3192-bib-0116\" ref-type=\"ref\">2012</xref>; Riggio et al. <xref rid=\"evl3192-bib-0139\" ref-type=\"ref\">2013</xref>; Bérénos et al. <xref rid=\"evl3192-bib-0016\" ref-type=\"ref\">2015</xref>). We discuss the application of GWAS to three dataset types: (i) datasets in which genotypes and phenotypes are each measured independently in each sex (e.g., humans); (ii) systems amenable to experimental manipulation, in which each genotype can be replicated among female and male carriers (e.g., isogenic or hemiclone fruit fly lines); and (iii) pedigreed populations, in which the genealogical relationships between all individuals are known (e.g., some sedentary vertebrate populations).</p><p>Where genotypic and phenotypic measurements are performed among independently sampled individuals of each sex, as in humans, SA loci can be identified by first performing a separate GWAS in each sex (“sex‐stratified” GWAS) (Fig. <xref rid=\"evl3192-fig-0003\" ref-type=\"fig\">3A</xref>), and then quantifying the difference between male‐ and female‐specific effect sizes (see also Gilks et al. <xref rid=\"evl3192-bib-0067\" ref-type=\"ref\">2014</xref>). Illustrating this approach, Winkler et al. (<xref rid=\"evl3192-bib-0176\" ref-type=\"ref\">2015</xref>) performed a sex‐stratified GWAS on several human anthropometric traits, then defined a <italic>t</italic>‐statistic as <mml:math id=\"nlm-math-50\"><mml:mrow><mml:mi>t</mml:mi><mml:mo>=</mml:mo><mml:mspace width=\"0.33em\"/><mml:mfrac><mml:mrow><mml:msub><mml:mi>β</mml:mi><mml:mi>M</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mi>F</mml:mi></mml:msub></mml:mrow><mml:mrow><mml:mi>√</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi>S</mml:mi><mml:msubsup><mml:mi>E</mml:mi><mml:mi>M</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>+</mml:mo><mml:mi>S</mml:mi><mml:msubsup><mml:mi>E</mml:mi><mml:mi>F</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>−</mml:mo><mml:mn>2</mml:mn><mml:mi>ρ</mml:mi><mml:mi>S</mml:mi><mml:msub><mml:mi>E</mml:mi><mml:mi>M</mml:mi></mml:msub><mml:mi>S</mml:mi><mml:msub><mml:mi>E</mml:mi><mml:mi>F</mml:mi></mml:msub></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mfrac></mml:mrow></mml:math>, where <mml:math id=\"nlm-math-51\"><mml:msub><mml:mi>β</mml:mi><mml:mi>M</mml:mi></mml:msub></mml:math> and <mml:math id=\"nlm-math-52\"><mml:msub><mml:mi>β</mml:mi><mml:mi>F</mml:mi></mml:msub></mml:math> are the sex‐specific effect sizes, <mml:math id=\"nlm-math-53\"><mml:mrow><mml:mi>S</mml:mi><mml:msub><mml:mi>E</mml:mi><mml:mi>M</mml:mi></mml:msub></mml:mrow></mml:math> and <mml:math id=\"nlm-math-54\"><mml:mrow><mml:mi>S</mml:mi><mml:msub><mml:mi>E</mml:mi><mml:mi>F</mml:mi></mml:msub></mml:mrow></mml:math> are the sex‐specific standard errors, and <mml:math id=\"nlm-math-55\"><mml:mi>ρ</mml:mi></mml:math> is the between‐sex rank correlation among genome‐wide loci. For each polymorphic site, <italic>P</italic>‐values were generated by comparing the observed <italic>t</italic> statistics to a null <italic>t</italic>‐distribution with no sex‐specific effects (where <mml:math id=\"nlm-math-56\"><mml:mrow><mml:mi mathvariant=\"normal\">E</mml:mi><mml:mrow><mml:mo stretchy=\"false\">[</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">]</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mi mathvariant=\"normal\">E</mml:mi><mml:mrow><mml:mo stretchy=\"false\">[</mml:mo><mml:mrow><mml:msub><mml:mi>β</mml:mi><mml:mi>M</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mi>F</mml:mi></mml:msub></mml:mrow><mml:mo stretchy=\"false\">]</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mn>0</mml:mn></mml:mrow></mml:math> under the null). This approach has been applied to nonfitness traits in humans (Randall et al. <xref rid=\"evl3192-bib-0133\" ref-type=\"ref\">2013</xref>; Myers et al. <xref rid=\"evl3192-bib-0115\" ref-type=\"ref\">2014</xref>; Winkler et al. <xref rid=\"evl3192-bib-0176\" ref-type=\"ref\">2015</xref>; Mitra et al. <xref rid=\"evl3192-bib-0109\" ref-type=\"ref\">2016</xref>; reviewed in Khramtsova et al. <xref rid=\"evl3192-bib-0086\" ref-type=\"ref\">2019</xref>), but has yet to be applied to fitness components (e.g., “number of children” phenotype in the UK Biobank; Sudlow et al. <xref rid=\"evl3192-bib-0156\" ref-type=\"ref\">2015</xref>).</p><fig fig-type=\"Figure\" xml:lang=\"en\" id=\"evl3192-fig-0003\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>GWAS as a direct method for identifying SA loci. <bold>(A)</bold> Manhattan plots of –log<sub>10</sub>\n<italic>P</italic>‐values from a GWAS of female and male fitness (data from Ruzicka et al. <xref rid=\"evl3192-bib-0146\" ref-type=\"ref\">2019</xref>), illustrating distributions of <italic>P</italic>‐values at or near candidate loci (circled) with male‐limited (left), female‐limited (middle), and SA (right) fitness effects. <bold>(B)</bold> Simulated phenotypic values of female and male fitness, before (left) and after (right) 45° rotation of the bivariate coordinate matrix to obtain sexually antagonistic (SA) and sexually concordant (SC) axes of phenotypic variation. Where fitness variation is predominantly SA (the top left of panel B), most variation is along the SA phenotypic axis (the top right of panel B). Where fitness variation is predominantly SC (the bottom left of panel B), most variation is along the SC phenotypic axis (the bottom right of panel B). In all panels, colors denote SA phenotypic values.</p></caption><graphic id=\"nlm-graphic-5\" xlink:href=\"EVL3-4-398-g003\"/></fig><p>In some experimental systems (e.g., fruit flies; flowering plants), the creation of isogenic or hemiclone lines (Abbott and Morrow <xref rid=\"evl3192-bib-0002\" ref-type=\"ref\">2011</xref>; Mackay et al. <xref rid=\"evl3192-bib-0103\" ref-type=\"ref\">2012</xref>; Berger et al. <xref rid=\"evl3192-bib-0019\" ref-type=\"ref\">2014</xref>) allows the same genotypes to be replicated and phenotypically assayed in carriers of each sex. Here, genotypes are effectively transplanted into male and female bodies or “environments,” analogous to the reciprocal transplantation of individuals sampled from different environments in local adaptation studies (Price et al. <xref rid=\"evl3192-bib-0129\" ref-type=\"ref\">2018</xref>). Identifying SA loci can then be achieved by transforming the bivariate coordinate system of male and female fitness values of a set of genotypes through matrix rotation (see Berger et al. <xref rid=\"evl3192-bib-0019\" ref-type=\"ref\">2014</xref>), which generates a univariate SA phenotype amenable to GWAS analysis (Fig. <xref rid=\"evl3192-fig-0003\" ref-type=\"fig\">3B</xref>). The approach is exemplified by a recent study in <italic>D. melanogaster</italic> (Ruzicka et al. <xref rid=\"evl3192-bib-0146\" ref-type=\"ref\">2019</xref>), which identified ∼230 candidate SA polymorphic sites.</p><p>In pedigreed vertebrate populations, such as Soay sheep (<italic>Ovis aries</italic>) or Florida scrub jays (<italic>Aphelocoma coerulescens</italic>), the genetic relationships between all individuals are known, and transmission of individual alleles across successive generations can be estimated (MacCluer et al. <xref rid=\"evl3192-bib-0102\" ref-type=\"ref\">1986</xref>). Because an individual's genetic contribution to future generations is a genuine representation of its Darwinian fitness, alleles transmitted more frequently by one sex relative to the other represent candidate SA variants. Analyses of pedigreed populations of Florida scrub jays have been used to identify alleles with above‐average transmission rates to descendants irrespective of sex (i.e., unconditionally beneficial alleles; Chen et al. <xref rid=\"evl3192-bib-0033\" ref-type=\"ref\">2019</xref>), yet this type of GWAS remains to be used to identify SA loci. It should be noted, however, that many pedigreed populations are necessarily small (given the logistics of monitoring them), which may hinder detection of loci affecting fitness variation.</p><p>Although GWAS‐based identification of candidate SA loci shows promise, two major drawbacks must be kept in mind. First, measurements that capture total lifetime reproductive success are difficult to obtain, and caution is required in interpreting results based on single fitness component (e.g., reproductive but not viability selection), which may correlate imperfectly with total fitness. Second, effect sizes are typically small for polygenic traits (Visscher et al. <xref rid=\"evl3192-bib-0168\" ref-type=\"ref\">2017</xref>), including fitness. Powerful GWAS of sex‐specific fitness may therefore be logistically prohibitive, and candidate SA loci will necessarily represent the subset of loci with particularly large fitness effects.</p></sec><sec id=\"evl3192-sec-0080\"><title>E&R WITH SEX‐LIMITED SELECTION</title><p>Experimental elimination of selection in one sex but not the other (i.e., sex‐limited selection) is a powerful way to identify SA selection in action. Various sex‐limited selection designs have been implemented, including (i) restricting transmission of the genome to the male line, and thereby removing selection through females (Rice, <xref rid=\"evl3192-bib-0136\" ref-type=\"ref\">1996</xref>, <xref rid=\"evl3192-bib-0134\" ref-type=\"ref\">1998</xref>; Prasad et al. <xref rid=\"evl3192-bib-0127\" ref-type=\"ref\">2007</xref>; Bedhomme et al. <xref rid=\"evl3192-bib-0014\" ref-type=\"ref\">2008</xref>; Abbott et al. <xref rid=\"evl3192-bib-0003\" ref-type=\"ref\">2010</xref>), or vice versa (Rice <xref rid=\"evl3192-bib-0135\" ref-type=\"ref\">1992</xref>), using <italic>Drosophila</italic> hemiclones; (ii) eliminating fitness variance in one sex (e.g., by enforcing random contributions to offspring number, or removing opportunity for mate choice) but not the other (Rundle et al. <xref rid=\"evl3192-bib-0145\" ref-type=\"ref\">2006</xref>; Morrow et al. <xref rid=\"evl3192-bib-0112\" ref-type=\"ref\">2008</xref>; Maklakov et al. <xref rid=\"evl3192-bib-0104\" ref-type=\"ref\">2009</xref>; Hollis et al. <xref rid=\"evl3192-bib-0076\" ref-type=\"ref\">2014</xref>; Immonen et al. <xref rid=\"evl3192-bib-0078\" ref-type=\"ref\">2014</xref>; Chenoweth et al. <xref rid=\"evl3192-bib-0035\" ref-type=\"ref\">2015</xref>; Veltsos et al. <xref rid=\"evl3192-bib-0167\" ref-type=\"ref\">2017</xref>); and (iii) applying sex‐limited artificial selection on a specific fitness component, such as mating success (Dugand et al. <xref rid=\"evl3192-bib-0056\" ref-type=\"ref\">2019</xref>), lifespan (Berg and Maklakov <xref rid=\"evl3192-bib-0017\" ref-type=\"ref\">2012</xref>; Chen and Maklakov <xref rid=\"evl3192-bib-0032\" ref-type=\"ref\">2014</xref>; Berger et al. <xref rid=\"evl3192-bib-0020\" ref-type=\"ref\">2016</xref>), reproductive tactic (Bielak et al. <xref rid=\"evl3192-bib-0023\" ref-type=\"ref\">2014</xref>), or mating investment (Pick et al. <xref rid=\"evl3192-bib-0125\" ref-type=\"ref\">2017</xref>).</p><p>To identify SA loci, sex‐limited selection can be combined with genotyping at multiple time points during experimental evolution within each selection regime (E&R), thereby connecting population genetic changes to the phenotypic responses accrued during experimental evolution. By tracking allele frequencies in male‐limited and female‐limited selection lines, alleles that show a significant time‐by‐treatment interaction point to candidate SA loci, and their frequency dynamics can be characterized using current analytical tools for E&R experiments (Wiberg et al. <xref rid=\"evl3192-bib-0175\" ref-type=\"ref\">2017</xref>; Vlachos et al. <xref rid=\"evl3192-bib-0170\" ref-type=\"ref\">2019</xref>). E&R is a powerful and proven approach for identifying the genomic basis of phenotypic variation and local adaptation (Turner et al. <xref rid=\"evl3192-bib-0162\" ref-type=\"ref\">2011</xref>; Long et al. <xref rid=\"evl3192-bib-0100\" ref-type=\"ref\">2015</xref>; Barghi et al. <xref rid=\"evl3192-bib-0009\" ref-type=\"ref\">2019</xref>). Moreover, because it is experimental, the issues of sex‐specific population structure that arise in between‐sex <italic>F</italic>\n<sub>ST</sub> studies (see section “Indirect Approaches for Identifying SA Genes”) can be minimized. Yet despite these advantages, we are not aware of any published study that has used E&R to identify SA loci (see Chenoweth et al. <xref rid=\"evl3192-bib-0035\" ref-type=\"ref\">2015</xref> for the closest effort to date).</p><p>Resequencing can be performed using population samples taken at multiple time points within a single generation (Svensson et al. <xref rid=\"evl3192-bib-0158\" ref-type=\"ref\">2018</xref>) or across multiple generations, with the latter approach benefitting from the fact that allele frequency responses to selection are cumulative over multiple generations. E&R, like GWAS, remains best suited for detecting loci with relatively large fitness effects. Selection on complex polygenic traits typically leads to small changes in allele frequencies at large numbers of loci, resulting in genomic signals of selection that are difficult to distinguish from genetic drift (Schlötterer et al. <xref rid=\"evl3192-bib-0148\" ref-type=\"ref\">2015</xref>). Consequently, the study organism, the number of replicates, the effective population sizes of selection and control lines, and the duration of experiments must be carefully considered in the design of E&R experiments (Baldwin‐Brown et al. <xref rid=\"evl3192-bib-0008\" ref-type=\"ref\">2014</xref>; Kofler and Schlötterer <xref rid=\"evl3192-bib-0090\" ref-type=\"ref\">2014</xref>; Kessner and Novembre <xref rid=\"evl3192-bib-0084\" ref-type=\"ref\">2015</xref>).</p></sec></sec><sec id=\"evl3192-sec-0090\"><title>Validation and Follow‐Up Analyses of Candidate SA Genes</title><p>Candidate SA genes and SNP sets enriched for SA alleles (i.e., identified using methods outlined above) provide context for addressing long‐standing questions about SA variation, including the genomic distribution, biological functions, and population genetic processes shaping SA polymorphisms. We focus on two specific issues in follow‐up analyses of putative SA variants. First, we outline approaches for biologically validating SA candidates—a crucial task given that candidate gene sets may include appreciable proportions of false positives and artifactual signals of sex‐specific selection (see section “Indirect Approaches for Identifying SA Genes”). Second, we discuss population genetic analyses and issues of interpretation with bearing on the evolutionary histories of SA genes.</p><sec id=\"evl3192-sec-0100\"><title>BIOLOGICAL VALIDATION OF SA GENES</title><p>Candidate SA loci can be directly validated in laboratory‐amenable taxa by experimentally manipulating each allele and measuring its sex‐specific fitness effect. A good example of experimental validation of naturally occurring SA genes is a study by VanKuren and Long (<xref rid=\"evl3192-bib-0165\" ref-type=\"ref\">2018</xref>), in which RNA interference and CRISPR‐Cas9 were used to demonstrate SA effects of tandem duplicate genes <italic>Apollo</italic> and <italic>Artemis</italic> on offspring production in <italic>D. melanogaster</italic>. Similarly, Akagi and Charlesworth (<xref rid=\"evl3192-bib-0005\" ref-type=\"ref\">2019</xref>) used manipulative molecular experiments to study candidate SA genes in several plant species. As a third example, several studies investigated a P450 transposable element insertion that upregulates the <italic>Cyp6g1</italic> gene and increases DDT resistance in <italic>D. melanogaster</italic> (Smith et al. <xref rid=\"evl3192-bib-0151\" ref-type=\"ref\">2011</xref>; Rostant et al. <xref rid=\"evl3192-bib-0143\" ref-type=\"ref\">2015</xref>; Hawkes et al. <xref rid=\"evl3192-bib-0073\" ref-type=\"ref\">2016</xref>). Although evidence for SA effects at this particular locus is mixed, the experimental approaches used—including measurements of sex‐specific fitness among isogenic lines and tracking the frequencies of each alternative allele in experimental cages—represent validation steps with potential for broad usage.</p><p>Direct experimental manipulation of candidate SA genes is not always feasible, and in instances where it is not, their biological validity can be assessed in other ways. In organisms harboring nonfunctional genomic material, it is possible to test whether candidate loci are enriched in genomic regions that are putatively functional (e.g., coding or regulatory) rather than inert (e.g., intergenic). Such “genic enrichment,” which is expected for SA polymorphisms with genuine phenotypic effects, has previously been used to strengthen validity of candidate alleles for local adaptation (Barreiro et al. <xref rid=\"evl3192-bib-0010\" ref-type=\"ref\">2008</xref>; Coop et al. <xref rid=\"evl3192-bib-0049\" ref-type=\"ref\">2009</xref>; Key et al. <xref rid=\"evl3192-bib-0085\" ref-type=\"ref\">2016</xref>). Another way to increase confidence in candidate loci is to look for multiple signals of SA selection. For example, candidates identified through elevated <italic>F</italic>\n<sub>ST</sub> that are also associated with SA fitness effects in a GWAS represent the best candidates for follow‐up evolutionary analyses (see below). Finally, if independent data exist on the sex‐specific phenotypic effects of individual mutations (e.g., in RNAi databases), these data can be mined to support the validity of candidate SA genes.</p></sec><sec id=\"evl3192-sec-0110\"><title>EVOLUTIONARY DYNAMICS OF SA GENES</title><p>We do not outline the range of population genetic analyses that could be used to describe the evolutionary dynamics of candidate SA loci, as these have been comprehensively reviewed elsewhere (e.g., Vitti et al. <xref rid=\"evl3192-bib-0169\" ref-type=\"ref\">2013</xref>; Fijarczyk and Babik <xref rid=\"evl3192-bib-0062\" ref-type=\"ref\">2015</xref>). Instead we provide guidance on some common issues that are likely to arise when analyzing patterns of genetic variation at SA loci and interpreting their mode of evolution.</p><p>First, we emphasize that the evolutionary dynamics of a contemporary SA gene may have, in the past, been governed by any combination of genetic drift, net directional selection (selection favoring fixation of one SA allele), or balancing selection (selection maintaining SA polymorphism). Theory often focuses on the conditions generating balancing selection at SA loci (e.g., Kidwell et al. <xref rid=\"evl3192-bib-0087\" ref-type=\"ref\">1977</xref>; Patten and Haig <xref rid=\"evl3192-bib-0123\" ref-type=\"ref\">2009</xref>; Fry <xref rid=\"evl3192-bib-0065\" ref-type=\"ref\">2010</xref>), leading some empirical studies to use signals of balancing selection (e.g., elevated Tajima's <italic>D</italic>) as indirect proxies for SA selection (e.g., Dutoit et al. <xref rid=\"evl3192-bib-0058\" ref-type=\"ref\">2018</xref>; Wright et al., <xref rid=\"evl3192-bib-0177\" ref-type=\"ref\">2018</xref>, <xref rid=\"evl3192-bib-0178\" ref-type=\"ref\">2019</xref>; Sayadi et al. <xref rid=\"evl3192-bib-0147\" ref-type=\"ref\">2019</xref>). However, whether contemporary candidate SA alleles evolved under balancing selection hinges upon both the historical pattern of sex‐specific selection and dominance at such loci (Kidwell et al. <xref rid=\"evl3192-bib-0087\" ref-type=\"ref\">1977</xref>; Connallon and Chenoweth <xref rid=\"evl3192-bib-0046\" ref-type=\"ref\">2019</xref>), which can be influenced by spatial and temporally varying selection (Connallon et al. <xref rid=\"evl3192-bib-0048\" ref-type=\"ref\">2019</xref>), and effective population size (Connallon and Clark, <xref rid=\"evl3192-bib-0039\" ref-type=\"ref\">2012</xref>, <xref rid=\"evl3192-bib-0041\" ref-type=\"ref\">2014a</xref>; Mullon et al. <xref rid=\"evl3192-bib-0113\" ref-type=\"ref\">2012</xref>). SA loci with large and symmetric selection coefficients or beneficial reversals of dominance (e.g., <italic>h<sub>f</sub></italic> = 1 and <italic>h<sub>m</sub></italic> = 0 in Box 1) are most conducive to balancing selection, whereas sufficient asymmetry between the sexes in the strength of selection (e.g., Mallet and Chippindale <xref rid=\"evl3192-bib-0105\" ref-type=\"ref\">2011</xref>; Mallet et al. <xref rid=\"evl3192-bib-0106\" ref-type=\"ref\">2011</xref>; Sharp and Agrawal <xref rid=\"evl3192-bib-0149\" ref-type=\"ref\">2013</xref>) should result in net directional selection that removes SA polymorphism rather than maintaining it (Kidwell et al. <xref rid=\"evl3192-bib-0087\" ref-type=\"ref\">1977</xref>; Kasimatis et al. <xref rid=\"evl3192-bib-0082\" ref-type=\"ref\">2019</xref>). Even when conditions for long‐term balancing selection are met, the <italic>efficacy</italic> of balancing selection relative to drift may often be weak at SA loci, leading to genetic diversity patterns that are indistinguishable from neutrally evolving loci (Connallon and Clark, <xref rid=\"evl3192-bib-0039\" ref-type=\"ref\">2012</xref>, <xref rid=\"evl3192-bib-0040\" ref-type=\"ref\">2013</xref>; Mullon et al. <xref rid=\"evl3192-bib-0113\" ref-type=\"ref\">2012</xref>). In short, loci under contemporary SA selection can have a broad range of possible evolutionary histories. As such, the typical mode of evolution operating at candidate SA loci <italic>cannot be assumed a priori</italic> and should instead be viewed as a question that must be resolved empirically.</p><p>Second, the detection of elevated polymorphism at SA loci does not necessarily imply balancing selection. For example, SA candidate loci may exhibit significantly elevated MAFs relative to non‐SA loci (Ruzicka et al. <xref rid=\"evl3192-bib-0146\" ref-type=\"ref\">2019</xref>), yet relaxed directional selection can account for this pattern if non‐SA loci encompass a mix of neutral sites and sites evolving under sexually concordant directional selection. To establish that SA loci are evolving under balancing selection, it is necessary to show that SA genetic variation is significantly elevated compared to confirmed neutral sites (e.g., short introns, Parsch et al. <xref rid=\"evl3192-bib-0122\" ref-type=\"ref\">2010</xref>) and cannot be accounted for by demographic or mutational processes (Andrés et al. <xref rid=\"evl3192-bib-0006\" ref-type=\"ref\">2009</xref>; DeGiorgio et al. <xref rid=\"evl3192-bib-0054\" ref-type=\"ref\">2014</xref>; Bitarello et al. <xref rid=\"evl3192-bib-0025\" ref-type=\"ref\">2018</xref>). On the other hand, significant <italic>reductions</italic> in polymorphism at SA loci, relative to neutral sites, do not necessarily rule out balancing selection either. Counterintuitively, when the equilibrium frequency of the minor allele is low (i.e., equilibrium MAF < 0.2, approximately), balanced polymorphisms can be lost more rapidly than neutral polymorphisms, leading to reduced genetic variation relative to neutral expectations (Robertson <xref rid=\"evl3192-bib-0141\" ref-type=\"ref\">1962</xref>; Mullon et al. <xref rid=\"evl3192-bib-0113\" ref-type=\"ref\">2012</xref>).</p><p>A third and final point is that nonrandom patterns of genetic variation at SA loci can be generated by ascertainment bias alone. For example, data filtering steps that remove low‐MAF SNPs (see “Indirect Approaches for Identifying SA Genes”) necessarily exclude rare SA variants from all downstream analyses. Elevated power to detect fitness effects among intermediate‐frequency sites in a GWAS can also generate a spurious positive relationship between candidate SA sites and MAF that might be mistaken for nonneutral evolution. Similarly, SA loci could be nonrandomly distributed across the genome (e.g., enriched in regions with low or high recombination), thereby generating spurious patterns in population genomic data that appear to indicate nonneutral evolution. It is therefore important to correct for such biases where possible by, for example, incorporating external data on recombination rate variation (Comeron et al. <xref rid=\"evl3192-bib-0038\" ref-type=\"ref\">2012</xref>; Elyashiv et al. <xref rid=\"evl3192-bib-0059\" ref-type=\"ref\">2016</xref>) or assessing evidence of trans‐species polymorphisms among SA loci before and after removal of CpG sites (Leffler et al. <xref rid=\"evl3192-bib-0094\" ref-type=\"ref\">2013</xref>).</p></sec></sec><sec id=\"evl3192-sec-0120\"><title>Moving Forward</title><p>We have critically assessed a broad range of methods for detecting genomic signatures of SA selection, including <italic>indirect methods</italic> based on genome sequence analysis (section “Indirect Approaches for Identifying SA Genes”) and <italic>direct methods</italic> based on associations between genome sequences and fitness measurements (section “Direct Approaches for Identifying SA Genes”). An inescapable conclusion from our indirect inference models is that very strong sex differences in selection or very large sample sizes are required to detect individual SA candidate polymorphisms with high confidence (Fig. <xref rid=\"evl3192-fig-0001\" ref-type=\"fig\">1</xref>; Appendices A‐D [Supporting Information]), in agreement with previous simulation studies of between‐sex <italic>F</italic>\n<sub>ST</sub> (Lucotte et al. <xref rid=\"evl3192-bib-0101\" ref-type=\"ref\">2016</xref>; Connallon and Hall <xref rid=\"evl3192-bib-0045\" ref-type=\"ref\">2018</xref>; Kasimatis et al. <xref rid=\"evl3192-bib-0082\" ref-type=\"ref\">2019</xref>). Nevertheless, estimates of the full distribution of <italic>F</italic>\n<sub>ST</sub> from previously published flycatcher and pipefish datasets reveal an intriguing elevation of genome‐wide <italic>F</italic>\n<sub>ST</sub> relative to our null models, which justifies future empirical studies of allele frequency differences between sexes (see below). Although an elevated signal of between‐sex <italic>F</italic>\n<sub>ST</sub> is not present in our reanalysis of human data, the absence of a signal is perhaps unsurprising given the small number of loci analyzed, following the removal of noncoding sequences and loci with rare polymorphisms. Genome‐wide analyses of sex‐specific selection in humans using larger datasets (Kasimatis et al. <xref rid=\"evl3192-bib-0083\" ref-type=\"ref\">2020</xref>), and examinations of sex‐biased expression among sites with elevated <italic>F</italic>\n<sub>ST</sub> (Cheng and Kirkpatrick <xref rid=\"evl3192-bib-0034\" ref-type=\"ref\">2016</xref>)—which were the focus of previous work, but not of our reanalysis—are therefore encouraged. With regard to direct methods for identifying SA genes, the substantial logistical challenge of accurately measuring fitness must be circumvented, yet the approach is powerful when feasible (see Ruzicka et al. <xref rid=\"evl3192-bib-0146\" ref-type=\"ref\">2019</xref>) and certain to be a key component of future work on the genetics of sex‐specific fitness variation.</p><p>Although there is little doubt that identifying and characterizing SA genes is challenging, there are several reasons for optimism. First, the low power of indirect metrics to detect selection at an individual locus level does not rule out the detection of a cumulative signal of polygenic sex differences in selection (e.g., Fig. <xref rid=\"evl3192-fig-0002\" ref-type=\"fig\">2</xref>). Although such an approach implies that candidate SA genes (e.g., those in the highest <italic>F</italic>\n<sub>ST</sub> quantiles) will include many false positives, elevated false discovery rates are not necessarily problematic if we are interested in the general properties of SA candidates relative to samples of putatively neutral (or non‐SA) loci. Nevertheless, in studies with low‐to‐moderate sample sizes, where many candidate genes will be false positives, researchers should minimally demonstrate that (i) the empirical distribution of the metric of interest differs significantly from its appropriate null (see Kasimatis et al. <xref rid=\"evl3192-bib-0082\" ref-type=\"ref\">2019</xref>; our reanalyses in section “Indirect Approaches for Identifying SA Genes”), (ii) putative signals of selection are not driven by sex‐specific population structure or other artefacts (see section “ Indirect Approaches for Identifying SA Genes”), and (iii) candidate loci are situated in putatively functional genome regions (see section “Validation and Follow‐up Analyses of Candidate SA Genes”).</p><p>Second, the power to detect SA genes using indirect metrics can often be increased in relatively simple ways. For example, pooled sequencing is a cost‐effective method for estimating allele frequencies from samples of many individuals (Schlötterer et al. <xref rid=\"evl3192-bib-0148\" ref-type=\"ref\">2015</xref>), and well‐suited for genome‐wide <italic>F</italic>\n<sub>ST</sub> scans (although not for <italic>F</italic>\n<sub>IS</sub> scans, as estimating <italic>F</italic>\n<sub>IS</sub> requires individual‐level genotype data). Researchers could, alternatively, focus attention toward large publicly available genomic datasets that are adequately powered for detecting loci under moderately strong SA selection (see Fig. <xref rid=\"evl3192-fig-0001\" ref-type=\"fig\">1</xref>), or toward genomic regions predicted to have relatively high statistical power. For example, studies of pseudoautosomal regions of recombining sex chromosomes have substantially higher power to detect <italic>F</italic>\n<sub>ST</sub> outliers driven by sex differences in selection (Qiu et al. <xref rid=\"evl3192-bib-0130\" ref-type=\"ref\">2013</xref>; Kirkpatrick and Guerrero <xref rid=\"evl3192-bib-0089\" ref-type=\"ref\">2014</xref>). Targeted sampling strategies may also amplify power to identify SA genes. For example, estimating allele frequencies among <italic>breeding</italic> adults—which have passed filters of viability selection and components of adult reproductive success—increases the number of episodes of selection that can contribute to allele frequency differentiation between sexes, improving the potential for detecting elevated between‐sex <italic>F</italic>\n<sub>ST</sub>.</p><p>Third, well‐chosen study systems can improve prospects for accurately measuring lifetime reproductive success and identifying SA loci through direct methods (GWAS or E&R). For example, difficulties in accurately measuring fitness under field conditions can be mitigated in pedigreed populations, where the genetic contribution of each individual to successive generations is known (provided the population is well monitored), and each genotype can therefore be associated with an accurate estimate of total lifetime reproductive success in each sex. Emerging approaches to infer pedigrees from genomic data alone (Snyder‐Mackler et al. <xref rid=\"evl3192-bib-0152\" ref-type=\"ref\">2016</xref>) may further facilitate identification of SA loci in the absence of long‐term monitoring efforts. In some experimental systems, such as laboratory‐adapted hemiclones of <italic>D. melanogaster</italic> (Rice et al. <xref rid=\"evl3192-bib-0138\" ref-type=\"ref\">2005</xref>; Abbott and Morrow <xref rid=\"evl3192-bib-0002\" ref-type=\"ref\">2011</xref>), relatively accurate measurements of outbred lifetime reproductive success are also possible. E&R is feasible for experimental organisms with short generation times and where large laboratory populations can be maintained (e.g., <italic>Drosophila</italic>, seed beetle <italic>Callosobruchus maculatus</italic>). Here, there is a relatively untapped opportunity to identify SA loci by combining sex‐limited selection (e.g., Rice <xref rid=\"evl3192-bib-0135\" ref-type=\"ref\">1992</xref>; Prasad et al. <xref rid=\"evl3192-bib-0127\" ref-type=\"ref\">2007</xref>; Morrow et al. <xref rid=\"evl3192-bib-0112\" ref-type=\"ref\">2008</xref>; Abbott et al. <xref rid=\"evl3192-bib-0003\" ref-type=\"ref\">2010</xref>; Bonel et al. <xref rid=\"evl3192-bib-0027\" ref-type=\"ref\">2018</xref>) with genotyping across multiple generations (e.g., Turner et al. <xref rid=\"evl3192-bib-0162\" ref-type=\"ref\">2011</xref>; Long et al. <xref rid=\"evl3192-bib-0100\" ref-type=\"ref\">2015</xref>; Barghi et al. <xref rid=\"evl3192-bib-0009\" ref-type=\"ref\">2019</xref>; Abbott et al. <xref rid=\"evl3192-bib-0004\" ref-type=\"ref\">2020</xref>).</p><p>Finally, despite notable exceptions (e.g., the dioecious plant <italic>Silene latifolia</italic>; Delph et al. <xref rid=\"evl3192-bib-0055\" ref-type=\"ref\">2011</xref>; Muyle et al. <xref rid=\"evl3192-bib-0114\" ref-type=\"ref\">2012</xref>), plant systems remain underused in research on SA selection. One advantage of plants is their greater amenability to field measurements of fitness components, as widely used in studies of local adaptation and species’ range limits (Hargreaves et al. <xref rid=\"evl3192-bib-0072\" ref-type=\"ref\">2014</xref>). Another advantage is the great diversity of reproductive systems in flowering plant species, the vast majority of which are hermaphroditic and susceptible to SA selection (Jordan and Connallon <xref rid=\"evl3192-bib-0080\" ref-type=\"ref\">2014</xref>; Tazzyman and Abbott <xref rid=\"evl3192-bib-0159\" ref-type=\"ref\">2015</xref>; Olito <xref rid=\"evl3192-bib-0119\" ref-type=\"ref\">2017</xref>; Olito et al. <xref rid=\"evl3192-bib-0120\" ref-type=\"ref\">2018</xref>), potentially leading to allele frequency differences between the pollen and ovules contributing to fertilization under haploid selection, and to elevated <italic>F</italic>\n<sub>IS</sub> among offspring. A third advantage of plants is their greater tendency to express genetic variation during the haploid stage of their life cycle (e.g., Immler and Otto <xref rid=\"evl3192-bib-0077\" ref-type=\"ref\">2018</xref>). Haploid (relative to diploid) expression is expected to inflate the contribution of genetic polymorphism to fitness variance and magnify evolutionary responses to selection, including within‐generation allele frequency divergence between sexes (Connallon and Jordan <xref rid=\"evl3192-bib-0043\" ref-type=\"ref\">2016</xref>). Exploiting plant systems may thereby increase statistical power to identify candidate SA genes or genomic signals of SA variation using direct (GWAS and E&R) or indirect inference approaches.</p><boxed-text position=\"float\" content-type=\"box\" orientation=\"portrait\"><caption><title>Box 1. Processes generating sexually divergent allele frequencies</title></caption><sec id=\"evl3192-sec-0130\"><label>1</label><p>Several evolutionary scenarios can lead to sex differences in the frequencies with which individual alleles are transmitted to offspring (Hedrick <xref rid=\"evl3192-bib-0074\" ref-type=\"ref\">2007</xref>; Úbeda et al. <xref rid=\"evl3192-bib-0163\" ref-type=\"ref\">2011</xref>; Connallon et al. <xref rid=\"evl3192-bib-0047\" ref-type=\"ref\">2018</xref>). We focus on two processes—sex differences in selection and sex‐biased migration—that may each commonly arise and affect estimates of allele frequency differences between sexes.</p><p>\n<bold><italic>Sex differences in selection</italic></bold> . Consider a single biallelic locus in which the focal allele (allele <italic>A</italic>) has a frequency of <italic>p</italic> at birth within a given generation; the alternative <italic>a</italic> allele has a frequency of 1 – <italic>p</italic>. Selection during the life cycle alters the allele frequencies in the set of adults that contribute offspring to the next generation. The frequency of the <italic>A</italic> allele in breeding females and males (respectively) is follows:\n<disp-formula id=\"evl3192-disp-0001\"><mml:math id=\"nlm-math-57\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mi>p</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mi>s</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mi>p</mml:mi><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mi>p</mml:mi></mml:mrow></mml:mfenced><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:mi>p</mml:mi><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mn>2</mml:mn><mml:msub><mml:mi>h</mml:mi><mml:mi>f</mml:mi></mml:msub></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced><mml:mo>+</mml:mo><mml:mi>O</mml:mi><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:msubsup><mml:mi>s</mml:mi><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>,</mml:mo><mml:msubsup><mml:mi>s</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>,</mml:mo><mml:msub><mml:mi>s</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:msub><mml:mi>s</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow></mml:mfenced></mml:mrow></mml:math></disp-formula>and\n<disp-formula id=\"evl3192-disp-0002\"><mml:math id=\"nlm-math-58\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mi>p</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mi>s</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mi>p</mml:mi><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mi>p</mml:mi></mml:mrow></mml:mfenced><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:mi>p</mml:mi><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mn>2</mml:mn><mml:msub><mml:mi>h</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced><mml:mo>+</mml:mo><mml:mi>O</mml:mi><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:msubsup><mml:mi>s</mml:mi><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>,</mml:mo><mml:msubsup><mml:mi>s</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>,</mml:mo><mml:msub><mml:mi>s</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:msub><mml:mi>s</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow></mml:mfenced><mml:mo>,</mml:mo></mml:mrow></mml:math></disp-formula>where <italic>s<sub>f</sub></italic> and <italic>s<sub>m</sub></italic> are female and male selection coefficients for the <italic>A</italic> allele, <italic>h<sub>f</sub></italic> and <italic>h<sub>m</sub></italic> are the dominance coefficients (Table <xref rid=\"evl3192-tbl-0001\" ref-type=\"table\">1</xref>), and <mml:math id=\"nlm-math-59\"><mml:mrow><mml:mi>O</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:msubsup><mml:mi>s</mml:mi><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>,</mml:mo><mml:msubsup><mml:mi>s</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>,</mml:mo><mml:msub><mml:mi>s</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:msub><mml:mi>s</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math> refers to second‐order terms in the selection coefficients, which are negligible (and can be ignored) when <italic>s<sub>f</sub></italic> and <italic>s<sub>m</sub></italic> are small, as expected for most loci (Charlesworth and Charlesworth <xref rid=\"evl3192-bib-0030\" ref-type=\"ref\">2010</xref>, p. 97).</p><table-wrap id=\"evl3192-tbl-0001\" xml:lang=\"en\" content-type=\"Table\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Sex‐specific relative fitness of genotypes of a biallelic locus</p></caption><table frame=\"hsides\" rules=\"groups\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\"/><th style=\"border-bottom:solid 1px #000000\" colspan=\"3\" align=\"left\" rowspan=\"1\">Genotype</th></tr><tr style=\"border-bottom:solid 1px #000000\"><th align=\"left\" rowspan=\"1\" colspan=\"1\"/><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>AA</italic>\n</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Aa</italic>\n</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>aa</italic>\n</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Female relative fitness</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 + <italic>s<sub>f</sub></italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 + <italic>s<sub>f</sub>h<sub>f</sub></italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male relative fitness</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 + <italic>s<sub>m</sub></italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1 + <italic>s<sub>m</sub>h<sub>m</sub></italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd.</copyright-holder></permissions></table-wrap><p>We expect allele frequency differences between breeding adults of each sex (<italic>p<sub>f</sub></italic> ≠ <italic>p<sub>m</sub></italic>) when the fitness effects of each allele differ between sexes, that is, (1) the same allele is favored in each sex but the strength of selection differs between sexes (e.g., <italic>p<sub>f</sub></italic> > <italic>p<sub>m</sub></italic> when <italic>s<sub>f</sub></italic> > <italic>s<sub>m</sub></italic> > 0); (2) alleles have sex‐limited fitness effects (e.g., <italic>p<sub>f</sub></italic> > <italic>p<sub>m</sub></italic> when <italic>s<sub>f</sub></italic> > 0 = <italic>s<sub>m</sub></italic>), or (3) alleles are sexually antagonistic (e.g., <italic>p<sub>f</sub></italic> > <italic>p<sub>m</sub></italic> when <italic>s<sub>f</sub></italic> > 0 > <italic>s<sub>m</sub></italic>). Allele frequencies are expected to remain equal between sexes (<italic>p<sub>f</sub></italic> = <italic>p<sub>m</sub></italic>) when genetic variation is neutral (<italic>s<sub>f</sub></italic> = <italic>s<sub>m</sub></italic> = 0), or selection and dominance coefficients are identical between sexes (<italic>s<sub>f</sub></italic> = <italic>s<sub>m</sub></italic> and <italic>h<sub>f</sub></italic> = <italic>h<sub>m</sub></italic>).</p><p>\n<bold><italic>Sex‐biased migration</italic></bold> . Consider an island population receiving new migrants each generation, with migration occurring before reproduction during the life cycle. At birth, the frequencies of <italic>A</italic> and <italic>a</italic> alleles in the island population are <italic>p</italic> and 1 – <italic>p</italic>, respectively. Let <italic>m<sub>f</sub></italic> and <italic>m<sub>m</sub></italic> represent the proportions of breeding females and breeding males on the island that are migrants. The expected frequency of the <italic>A</italic> allele in breeding females and males (respectively) will be\n<disp-formula id=\"evl3192-disp-0003\"><mml:math id=\"nlm-math-60\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:msub><mml:mi>m</mml:mi><mml:mi>f</mml:mi></mml:msub></mml:mrow></mml:mfenced><mml:mi>p</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mi>m</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>∼</mml:mo></mml:mover></mml:mrow></mml:math></disp-formula>and\n<disp-formula id=\"evl3192-disp-0004\"><mml:math id=\"nlm-math-61\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:msub><mml:mi>m</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow></mml:mfenced><mml:mi>p</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mi>m</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>∼</mml:mo></mml:mover><mml:mo>,</mml:mo></mml:mrow></mml:math></disp-formula>where <mml:math id=\"nlm-math-62\"><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>∼</mml:mo></mml:mover></mml:math> is the frequency of the <italic>A</italic> allele in migrant individuals. The identity, <mml:math id=\"nlm-math-63\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:msub><mml:mi>m</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>m</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>∼</mml:mo></mml:mover><mml:mo>−</mml:mo><mml:mi>p</mml:mi></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math>, implies that allele frequency differences between breeding females and males (<italic>p<sub>f</sub></italic> ≠ <italic>p<sub>m</sub></italic>) would require sex‐biased migration (<italic>m<sub>f</sub></italic> ≠ <italic>m<sub>m</sub></italic>) and allele frequency differences between migrant and resident (nonmigrant) individuals (<mml:math id=\"nlm-math-64\"><mml:mrow><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>∼</mml:mo></mml:mover><mml:mo>≠</mml:mo><mml:mi>p</mml:mi></mml:mrow></mml:math>).</p></sec></boxed-text><boxed-text position=\"float\" content-type=\"box\" orientation=\"portrait\"><caption><title>Box 2. Fixation indices (<italic>F</italic>\n<sub>ST</sub> and <italic>F</italic>\n<sub>IS</sub>) applied to sex differences</title></caption><sec id=\"evl3192-sec-0140\"><label>1</label><p>Allele frequency differences between breeding adults of each sex can be quantified by way of fixation indices, originally devised by Wright (<xref rid=\"evl3192-bib-0179\" ref-type=\"ref\">1951</xref>) for characterizing genetic differentiation among populations. We again consider a biallelic autosomal locus with the focal allele (<italic>A</italic>) at a frequency of <italic>p<sub>f</sub></italic> in breeding females and <italic>p<sub>m</sub></italic> in breeding males.</p><p>\n<bold><italic>Between‐sex F</italic><sub>ST</sub></bold> . <italic>F</italic>\n<sub>ST</sub> is a standardized measure of the allele frequency difference between the sexes:\n<disp-formula id=\"evl3192-disp-0005\"><mml:math id=\"nlm-math-65\"><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:msup><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow></mml:mfenced><mml:mn>2</mml:mn></mml:msup><mml:mrow><mml:mn>4</mml:mn><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:mrow></mml:mfenced></mml:mrow></mml:mfrac><mml:mo>,</mml:mo></mml:mrow></mml:math></disp-formula>where <mml:math id=\"nlm-math-66\"><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:math> = (<italic>p<sub>f</sub></italic> + <italic>p<sub>m</sub></italic>)/2 (Cheng and Kirkpatrick <xref rid=\"evl3192-bib-0034\" ref-type=\"ref\">2016</xref>). This definition applies to the entire population, and therefore differs from empirical estimates of <italic>F</italic>\n<sub>ST</sub> that are based upon samples of gene sequences from the population. Although there are several estimators of <italic>F</italic>\n<sub>ST</sub> (see Appendix A [Supporting Information]; Bhatia et al. <xref rid=\"evl3192-bib-0022\" ref-type=\"ref\">2013</xref>; Gammerdinger et al. <xref rid=\"evl3192-bib-0066\" ref-type=\"ref\">2020</xref>), we focus on the simplest:\n<disp-formula id=\"evl3192-disp-0006\"><mml:math id=\"nlm-math-67\"><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:msup><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi>f</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi>m</mml:mi></mml:msub></mml:mrow></mml:mfenced><mml:mn>2</mml:mn></mml:msup><mml:mrow><mml:mn>4</mml:mn><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>^</mml:mo></mml:mover></mml:mrow></mml:mfenced></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula>(Nei <xref rid=\"evl3192-bib-0117\" ref-type=\"ref\">1973</xref>), where <mml:math id=\"nlm-math-68\"><mml:msub><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi>f</mml:mi></mml:msub></mml:math> and <mml:math id=\"nlm-math-69\"><mml:msub><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi>m</mml:mi></mml:msub></mml:math> are the allele frequency estimates from samples of females and males, and <mml:math id=\"nlm-math-70\"><mml:mrow><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mo>=</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:msub><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi>f</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mo>/</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:math>. As we show in Appendix A (Supporting Information), the ratio\n<disp-formula id=\"evl3192-disp-0007\"><mml:math id=\"nlm-math-71\"><mml:mfrac><mml:mrow><mml:mn>4</mml:mn><mml:msub><mml:mi>n</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:msub><mml:mi>n</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:mrow></mml:mfenced><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mi>n</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:msub><mml:mi>p</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mi>f</mml:mi></mml:msub></mml:mrow></mml:mfenced><mml:mo>+</mml:mo><mml:msub><mml:mi>n</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:msub><mml:mi>p</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow></mml:mfenced></mml:mrow></mml:mfrac></mml:math></disp-formula>has an approximately noncentral chi‐squared distribution with one degree of freedom and noncentrality parameter of <mml:math id=\"nlm-math-72\"><mml:mrow><mml:mi>λ</mml:mi><mml:mo>=</mml:mo><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mi>f</mml:mi></mml:msub></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow><mml:msub><mml:mi>n</mml:mi><mml:mi>f</mml:mi></mml:msub></mml:mfrac><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow><mml:msub><mml:mi>n</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mfrac></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:math>, where <italic>n<sub>f</sub></italic> and <italic>n<sub>m</sub></italic> are the numbers of sequences derived from females and males, respectively. The approximation can break down when <italic>n<sub>f</sub></italic> and <italic>n<sub>m</sub></italic> are small or the minor (rarer) allele at the locus has a frequency close to zero. Under the statistical null distribution, the true allele frequencies do not differ between the sexes (<italic>p<sub>f</sub></italic> = <italic>p<sub>m</sub></italic>), and therefore <mml:math id=\"nlm-math-73\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">ST</mml:mi></mml:msub></mml:math>\n<mml:math id=\"nlm-math-74\"><mml:mrow><mml:mo>≈</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mn>0</mml:mn></mml:msub><mml:mo>/</mml:mo><mml:mn>2</mml:mn><mml:msub><mml:mi>n</mml:mi><mml:mi>H</mml:mi></mml:msub></mml:mrow></mml:math>, where <mml:math id=\"nlm-math-75\"><mml:msub><mml:mi>X</mml:mi><mml:mn>0</mml:mn></mml:msub></mml:math> is a chi‐squared random variable with one degree of freedom, and <italic>n<sub>H</sub></italic> = 2(1/<italic>n<sub>f</sub></italic> + 1/<italic>n<sub>m</sub></italic>)<sup>−1</sup> is the harmonic mean sample size.</p><p>\n<bold><italic>F</italic><sub>IS</sub><italic>in offspring</italic></bold>. <italic>F</italic>\n<sub>IS</sub> can be used to quantify deviations between the observed heterozygosity in a cohort of individuals before selection (e.g., individuals sampled and sequenced at birth) and the expected heterozygosity under Hardy‐Weinberg equilibrium. With random mating among breeding adults with female and male allele frequencies <italic>p<sub>f</sub></italic> and <italic>p<sub>m</sub></italic>, and ignoring effects of genetic drift or segregation distortion, the frequency of the <italic>A</italic> allele in offspring of the next generation will be <mml:math id=\"nlm-math-76\"><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:math> = (<italic>p<sub>f</sub></italic> + <italic>p<sub>m</sub></italic>)/2, and the proportion that is heterozygous will be <italic>P<sub>Aa</sub></italic> = <italic>p<sub>f</sub></italic>(1 – <italic>p<sub>m</sub></italic>) + <italic>p<sub>m</sub></italic>(1 – <italic>p<sub>f</sub></italic>). Under these conditions, <italic>F</italic>\n<sub>IS</sub> will be\n<disp-formula id=\"evl3192-disp-0008\"><mml:math id=\"nlm-math-77\"><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:msub><mml:mi>P</mml:mi><mml:mrow><mml:mi>A</mml:mi><mml:mi>a</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mn>2</mml:mn><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:mrow></mml:mfenced></mml:mrow></mml:mfrac><mml:mo>−</mml:mo><mml:mn>1</mml:mn><mml:mo>=</mml:mo><mml:mfrac><mml:msup><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow></mml:mfenced><mml:mn>2</mml:mn></mml:msup><mml:mrow><mml:mn>4</mml:mn><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:mrow></mml:mfenced></mml:mrow></mml:mfrac><mml:mo>,</mml:mo></mml:mrow></mml:math></disp-formula>where the final expression is equivalent to <italic>F</italic>\n<sub>ST</sub> between breeding females and males of the prior generation (Kasimatis et al. <xref rid=\"evl3192-bib-0082\" ref-type=\"ref\">2019</xref>). The above expression for <italic>F</italic>\n<sub>IS</sub> applies to the entire set of offspring in a population, whereas empirical estimates of <italic>F</italic>\n<sub>IS</sub> will be based on the genotypes of offspring sampled from the population. As shown in Appendix B (Supporting Information), estimates of <italic>F</italic>\n<sub>IS</sub> will be approximately normally distributed with a mean and variance of\n<disp-formula id=\"evl3192-disp-0009\"><mml:math id=\"nlm-math-78\"><mml:mrow><mml:mi mathvariant=\"normal\">E</mml:mi><mml:mfenced separators=\"\" open=\"[\" close=\"]\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub></mml:mfenced><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mn>2</mml:mn><mml:mi>n</mml:mi></mml:mrow></mml:mfrac><mml:mo>+</mml:mo><mml:mfrac><mml:msup><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow></mml:mfenced><mml:mn>2</mml:mn></mml:msup><mml:mrow><mml:mn>4</mml:mn><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover><mml:mfenced separators=\"\" open=\"(\" close=\")\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mover accent=\"true\"><mml:mi>p</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:mrow></mml:mfenced></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula>and\n<disp-formula id=\"evl3192-disp-0010\"><mml:math id=\"nlm-math-79\"><mml:mrow><mml:mi mathvariant=\"normal\">var</mml:mi><mml:mfenced separators=\"\" open=\"[\" close=\"]\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mrow><mml:mi>I</mml:mi><mml:mi>S</mml:mi></mml:mrow></mml:msub></mml:mfenced><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mi>n</mml:mi></mml:mfrac><mml:mo>,</mml:mo></mml:mrow></mml:math></disp-formula>where <italic>n</italic> is the number of offspring genotyped for the locus. The approximation applies when the sample size is large. Under a null model, in which offspring are outbred and mating is random, estimates <mml:math id=\"nlm-math-80\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mi mathvariant=\"normal\">IS</mml:mi></mml:msub></mml:math> will be normal with mean of 1/2<italic>n</italic> and variance of 1/<italic>n</italic>.</p></sec></boxed-text></sec><sec id=\"evl3192-sec-0160\"><title>AUTHOR CONTRIBUTIONS</title><p>All authors contributed to the conceptualization of the manuscript. TC, CO, and PC developed mathematical models. LD conducted data reanalyses, with input from TC and FR. TC and FR coordinated manuscript writing, with initial drafts of \"Direct approaches for identifying SA genes\" by AR, XYL, ES, and FR, initial drafts of \"Validation and follow‐up analyses of candidate SA genes\" by HPY and CYJ, and remaining sections drafted by TC and FR. All authors contributed to manuscript editing.</p></sec><sec sec-type=\"data-availability\" id=\"evl3192-sec-0170\"><title>DATA ARCHIVING</title><p>All code for reproducing reanalyses presented in the manuscript is available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/ldutoit/male_female_fst\">https://github.com/ldutoit/male_female_fst</ext-link>. Scripts and other data can be found on dryad: <ext-link ext-link-type=\"uri\" xlink:href=\"https://doi.org/10.5061/dryad.b2rbnzscc\">https://doi.org/10.5061/dryad.b2rbnzscc</ext-link>\n</p></sec><sec id=\"evl3192-sec-0180\"><label>1</label><p>Associate Editor: J. Mank</p></sec><sec sec-type=\"supplementary-material\"><title>Supporting information</title><supplementary-material content-type=\"local-data\"><caption><p>\n<bold>Figure S1</bold>. Permuted versus observed <mml:math id=\"nlm-math-1\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mrow><mml:mi>S</mml:mi><mml:mi>T</mml:mi></mml:mrow></mml:msub></mml:math> for simulated data.</p><p>\n<bold>Figure S2</bold>. Permuted versus observed <mml:math id=\"nlm-math-2\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mrow><mml:mi>S</mml:mi><mml:mi>T</mml:mi></mml:mrow></mml:msub></mml:math> for the flycatcher dataset.</p><p>\n<bold>Figure S3</bold>. Permuted versus observed <mml:math id=\"nlm-math-3\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mrow><mml:mi>S</mml:mi><mml:mi>T</mml:mi></mml:mrow></mml:msub></mml:math> for the pipefish dataset.</p><p>\n<bold>Figure S4</bold>. Permuted vs. observed <mml:math id=\"nlm-math-4\"><mml:msub><mml:mover accent=\"true\"><mml:mi>F</mml:mi><mml:mo>^</mml:mo></mml:mover><mml:mrow><mml:mi>S</mml:mi><mml:mi>T</mml:mi></mml:mrow></mml:msub></mml:math> for the human 1000 Genomes data.</p><p>\n<bold>Figure S5</bold>. Human 1000 Genomes data with SNPs with low minor allele frequencies (MAFs) included in the analysis.</p><p>\n<bold>Appendix A</bold>. Distribution of <italic>F<sub>ST</sub></italic> estimates.</p><p>\n<bold>Table S1</bold>. Sex‐specific relative fitness of genotypes of a biallelic locus with additive fitness effects in each sex.</p><p>\n<bold>Appendix B</bold>. Sex‐specific allele frequencies and <italic>F<sub>IS</sub></italic> estimates.</p><p>\n<bold>Appendix C</bold>. Case‐control GWAS and the Log‐Odds Ratio.</p><p>\n<bold>Figure S6</bold>. Probability that <mml:math id=\"nlm-math-5\"><mml:mi mathvariant=\"script\">L</mml:mi></mml:math>. ⁄<italic>SE</italic> for an additive SA locus.</p><p>\n<bold>Figure S7</bold>. Theoretical signal of multilocus SA polymorphism: the ratio of observed versus permuted absolute log odds ratio estimates within 100 quantiles of the theoretical null of <mml:math id=\"nlm-math-6\"><mml:mi mathvariant=\"script\">L</mml:mi></mml:math>. ⁄<italic>SE</italic>.</p><p>\n<bold>Appendix D</bold>. Comparison of null model distributions for different metrics allele frequency differentiation between sexes.</p></caption><media xlink:href=\"EVL3-4-398-s001.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"evl3192-sec-0150\"><title>ACKNOWLEDGMENTS</title><p>We thank A. Wright, H. Ellegren, and S. Flanagan for sharing data; S. Flanagan for support through pipefish data analysis; and I. Booksmythe, M. Kirkpatrick, and three anonymous reviewers for helpful comments on an earlier draft of this manuscript. The study was supported by funds from the Australian Research Council to TC and FR, the Swedish Research Council (VR: grant number 2016–03356) to EIS, and the Swiss National Science Foundation (Ambizione number 180145) to XYL. The New Zealand eScience Infrastructure (NeSI) provided support through their high‐performance computing facilities; NeSI and collaborator institutions are funded through the Ministry of Business, Innovation and Employment's Research Infrastructure program (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.nesi.org.nz\">https://www.nesi.org.nz</ext-link>). 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Epidemiology</journal-id><journal-id journal-id-type=\"iso-abbrev\">Epidemiology</journal-id><journal-id journal-id-type=\"publisher-id\">EDE</journal-id><journal-title-group><journal-title>Epidemiology (Cambridge, Mass.)</journal-title></journal-title-group><issn pub-type=\"ppub\">1044-3983</issn><issn pub-type=\"epub\">1531-5487</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32841985</article-id><article-id pub-id-type=\"pmc\">PMC7523568</article-id><article-id pub-id-type=\"art-access-id\">00016</article-id><article-id pub-id-type=\"doi\">10.1097/EDE.0000000000001248</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cardiopulmonary Epidemiology</subject></subj-group></article-categories><title-group><article-title>Explaining the Sex Effect on Survival in Cystic Fibrosis: a Joint Modeling Study of UK Registry Data</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Taylor-Robinson</surname><given-names>David</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">a</xref></contrib><contrib contrib-type=\"author\"><name><surname>Schlüter</surname><given-names>Daniela K.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">a</xref></contrib><contrib contrib-type=\"author\"><name><surname>Diggle</surname><given-names>Peter J.</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">b</xref></contrib><contrib contrib-type=\"author\"><name><surname>Barrett</surname><given-names>Jessica K.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">c</xref><xref ref-type=\"aff\" rid=\"aff4\">d</xref></contrib><aff id=\"aff1\">From the <label>a</label>Department of Public Health and Policy, University of Liverpool, Liverpool, United Kingdom</aff><aff id=\"aff2\"><label>b</label>CHICAS, Lancaster Medical School, Lancaster University, Lancaster, United Kingdom</aff><aff id=\"aff3\"><label>c</label>Department of Public Health and Primary Care, University of Cambridge, United Kingdom</aff><aff id=\"aff4\"><label>d</label>MRC Biostatistics Unit, University of Cambridge, United Kingdom.</aff></contrib-group><author-notes><corresp id=\"c1\">Correspondence: David Taylor-Robinson, Department of Public Health and Policy, Farr Institute@HeRC, University of Liverpool, Liverpool, United Kingdom. E-mail: <email xlink:href=\"david.taylor-robinson@liverpool.ac.uk\">david.taylor-robinson@liverpool.ac.uk</email>.</corresp></author-notes><pub-date pub-type=\"epub\"><day>06</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>11</month><year>2020</year></pub-date><volume>31</volume><issue>6</issue><fpage>872</fpage><lpage>879</lpage><history><date date-type=\"received\"><day>10</day><month>7</month><year>2019</year></date><date date-type=\"accepted\"><day>27</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">Creative Commons Attribution License 4.0 (CCBY)</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"ede-31-872.pdf\"/><abstract abstract-type=\"toc\"><p>Supplemental Digital Content is available in the text.</p></abstract><abstract><title>Background:</title><p>Male sex is associated with better lung function and survival in people with cystic fibrosis but it is unclear whether the survival benefit is solely due to the sex-effect on lung function.</p><sec><title>Methods:</title><p>This study analyzes data between 1996 and 2015 from the longitudinal registry study of the UK Cystic Fibrosis Registry. We jointly analyze repeated measurements and time-to-event outcomes to assess how much of the sex effect on lung function also explains survival. These novel methods allow examination of association between percent of forced expiratory volume in 1 second (%FEV1) and covariates such as sex and genotype, and survival, in the same modeling framework. We estimate the probability of surviving one more year with a probit model.</p></sec><sec><title>Results:</title><p>The dataset includes 81,129 lung function measurements of %FEV1 on 9,741 patients seen between 1996 and 2015 and captures 1,543 deaths. Males compared with females experienced a more gradual decline in %FEV1 (difference 0.11 per year 95% confidence interval [CI] = 0.08, 0.14). After adjusting for confounders, both overall level of %FEV1 and %FEV1 rate of change are associated with the concurrent hazard for death. There was evidence of a male survival advantage (probit coefficient 0.15; 95% CI = 0.10, 0.19) which changed little after adjustment for %FEV1 using conventional approaches but was attenuated by 37% on adjustment for %FEV1 level and slope in the joint model (0.09; 95% CI = 0.06, 0.12).</p></sec><sec><title>Conclusions:</title><p>We estimate that about 37% of the association of sex on survival in cystic fibrosis is mediated through lung function.</p></sec></abstract><kwd-group><kwd>Cystic fibrosis</kwd><kwd>Joint-modeling</kwd><kwd>Registry</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>SDC</meta-name><meta-value>T</meta-value></custom-meta><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><p>Male sex has been identified as a positive prognostic factor in cystic fibrosis.<sup><xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref></sup> An apparent effect of sex on morbidity and mortality in cystic fibrosis, with males having better outcomes, has been a common finding in large epidemiologic studies, first suggested in US centers,<sup><xref rid=\"R4\" ref-type=\"bibr\">4</xref></sup> and then confirmed in US population-level registry studies.<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref>,<xref rid=\"R6\" ref-type=\"bibr\">6</xref></sup> There have been similar findings in the UK,<sup><xref rid=\"R7\" ref-type=\"bibr\">7</xref></sup> with Barr et al in the UK<sup><xref rid=\"R8\" ref-type=\"bibr\">8</xref></sup> suggesting that despite overall improved survival in the 21st century, females continue to be more likely to die below the median age of death compared with males, a pattern that has persisted since the 1960s. There has been recent debate about the sex gap, suggesting that this may be narrowing over time as a result of improving treatment.<sup><xref rid=\"R9\" ref-type=\"bibr\">9</xref>,<xref rid=\"R10\" ref-type=\"bibr\">10</xref></sup> In terms of use of health services in cystic fibrosis, a large study in Canada further demonstrated decreased risk of hospitalization in males.<sup><xref rid=\"R11\" ref-type=\"bibr\">11</xref></sup> Our previous study of the UK Cystic Fibrosis Registry shows that the sex difference in cystic fibrosis outcomes is clearly apparent in the UK cystic fibrosis population<sup><xref rid=\"R12\" ref-type=\"bibr\">12</xref></sup> and this is also shown in a recent survival analysis using UK data.<sup><xref rid=\"R13\" ref-type=\"bibr\">13</xref>,<xref rid=\"R14\" ref-type=\"bibr\">14</xref></sup> The cause of the sex gap in survival remains unclear. Lung function, as measured by the percent predicted forced expiratory volume in 1 second (%FEV1) is commonly used as a measure of disease severity in cystic fibrosis, and has been shown to be related to survival. One explanation for the sex gap in cystic fibrosis survival is that this is explained by worse lung function in females. For instance, some studies suggest that females may be more likely to become colonized with <italic>Pseudomonas aeruginosa</italic> leading to lung damage at an earlier age,<sup><xref rid=\"R15\" ref-type=\"bibr\">15</xref></sup> and this may be related to the effect of estrogen.<sup><xref rid=\"R16\" ref-type=\"bibr\">16</xref></sup> A recent review of the sex gap in cystic fibrosis has suggested that the finding of lower female survival is evident in most studies, and that evidence to suggest closure of the gap in recent cohorts is less convincing than the data supporting its continued existence. The review does suggest that in cohorts of adults with late diagnosis, and conditional on survival to age 40 years, the sex gap appears to narrow or even be reversed.<sup><xref rid=\"R3\" ref-type=\"bibr\">3</xref></sup></p><p>In this study, we aim to quantify how aspects of an individual cystic fibrosis patient’s longitudinal profile of lung function are related to their survival prognosis; and to decompose the impact of sex on these joint outcomes. Joint modeling approaches are potentially of great utility in the context of studying outcomes in cystic fibrosis patients.<sup><xref rid=\"R17\" ref-type=\"bibr\">17</xref></sup> Survival is of central interest and analyses often seek to adjust for lung function as a time-varying covariate, which we know is measured imprecisely with clinically significant measurement error. Furthermore, the dynamics of lung function decline are also of interest, but there is potentially informative drop-out due to the direct link between lung function and survival prognosis. Together, these properties of cystic fibrosis data (measurement error and dropout) mean that separate analysis of repeated measurement and survival outcomes is potentially inefficient, because it does not exploit the dependence between the repeated measurement process and the hazard for survival, and leads to biased estimation of the association between the two because it ignores measurement error.<sup><xref rid=\"R18\" ref-type=\"bibr\">18</xref></sup> Joint modeling of lung function and survival offers an approach to address all of these issues.</p><p>Joint modeling has been applied to cystic fibrosis data in a few previous studies. The first of these was a center-level study by Schluchter and colleagues that modeled longitudinal FEV1 and survival simultaneously for a cohort of delF508 homozygous patients, but this study did not explore the sex effect.<sup><xref rid=\"R17\" ref-type=\"bibr\">17</xref></sup> Subsequently, cystic fibrosis data have been used to develop methods for joint modeling, including an approach that we previously developed.<sup><xref rid=\"R19\" ref-type=\"bibr\">19</xref></sup> In this study, we apply this novel approach for the joint modeling of lung function and survival and contrast this to commonly used approaches to adjusting for time-varying covariates in survival analyses. We use the joint model to test the hypothesis that the survival advantage for males is explained by the effect of sex on lung function.</p><sec sec-type=\"methods\"><title>METHODS</title><p>We undertook a longitudinal retrospective cohort study of individuals in the UK Cystic Fibrosis Registry, which records longitudinal health data on all people with cystic fibrosis in England, Wales, Scotland, and Northern Ireland. All UK cystic fibrosis centers and clinics routinely collect data in a standardized fashion. When patients with cystic fibrosis attend a new center in the UK, they or their parents consent to collection and storage of information on the patient’s health and treatment in the Cystic Fibrosis Registry.<sup><xref rid=\"R20\" ref-type=\"bibr\">20</xref></sup> In the UK, cystic fibrosis patients are seen in the outpatient clinic for a comprehensive annual review, including evaluation of clinical status and pulmonary function. The UK Cystic Fibrosis Registry is supported and coordinated by the UK Cystic fibrosis Trust. In the UK, the Registry is estimated to capture almost all of the cystic fibrosis population; any consenting patients attending National Health Service clinics will have annual data routinely collected into the database, 89% of whom have a “complete” dataset capturing key clinical parameters over time. The registry is carefully managed and curated to a high level of data quality, and is therefore ideally suited to the study of cystic fibrosis outcomes.<sup><xref rid=\"R20\" ref-type=\"bibr\">20</xref></sup></p><sec><title>Primary Outcome and Covariates</title><p>Our directed acyclic graph which informed the analysis, identifying variables in the minimally sufficient set of adjustments, is shown in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>. The primary outcomes were %FEV1, as per other studies that have explored the cystic fibrosis sex gap in outcomes,<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref></sup> and survival. Pulmonary function tests were measured annually at the review visit, and performed according to international recommendations, measuring forced expiratory volume in 1 second, expressed as a percentage of predicted values for sex, age, height, and ethnicity using the global lung function initiative reference equations.<sup><xref rid=\"R21\" ref-type=\"bibr\">21</xref></sup> We restricted the analysis to white patients under the age of 40 at last follow-up, with at least one lung function measurement between the start of 1996 and the end of 2015. We chose to apply an upper age limit to the analysis since the female sex gap has been shown to be present up to this point; and we have previously shown that random intercept and slope models make unrealistic assumptions when applied over long periods.<sup><xref rid=\"R22\" ref-type=\"bibr\">22</xref></sup> About 97% of people with cystic fibrosis in the UK are white. Non-white patients tend to have worse outcomes, but the numbers are small in the UK, restricting power to demonstrate subgroup effects.<sup><xref rid=\"R12\" ref-type=\"bibr\">12</xref></sup></p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1.</label><caption><p>Directed acyclic graph for the effect of sex on key outcomes in cystic fibrosis. We aim to use a joint model to test the hypothesis that there is a direct effect of sex on survival.</p></caption><graphic xlink:href=\"ede-31-872-g001\"/></fig><p>The primary exposure of interest was male sex. The time metameter was patient age at clinic visit at which the %FEV1 measure was taken. Other covariates in the analysis were genotype coded as the number of delta F508 alleles (0, 1, or 2) and dichotomized into 2 F508 alleles versus 0 or 1 alleles or not typed, and birth year which was treated as a continuous covariate and centered at the mean value (approximately 1986) to capture cohort effects.</p></sec><sec><title>Statistical Analysis</title><p>Full details of the joint modeling approach are provided in the supplementary eAppendix 1; <ext-link ext-link-type=\"uri\" xlink:href=\"http://links.lww.com/EDE/B714\">http://links.lww.com/EDE/B714</ext-link>. We applied the method developed by Barrett et al which allows exact likelihood inference for a wide range of random-effect specifications, and the code for fitting this model is available via the link (<ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/Jessbarrett/CysticFibrosisJM\">https://github.com/Jessbarrett/CysticFibrosisJM</ext-link>). Repeated %FEV1 measures on individuals are correlated, and this must be accommodated to obtain valid inferences. Furthermore, lung function is related to survival. Thus, repeated measurements of %FEV1 and survival were modeled jointly using shared random effects to account for the interdependence of the two processes.<sup><xref rid=\"R19\" ref-type=\"bibr\">19</xref></sup> The submodel for %FEV1 adjusted for the patient’s age at measurement, birth year, sex, and number of F508 alleles. Exploratory plots of the data are shown in eAppendix2; <ext-link ext-link-type=\"uri\" xlink:href=\"http://links.lww.com/EDE/B714\">http://links.lww.com/EDE/B714</ext-link>. Informed by these, we approximated time-trends with a quadratic time function, to accommodate nonlinear change over time.<sup><xref rid=\"R22\" ref-type=\"bibr\">22</xref></sup> Interactions were included between the linear time variables in age and birth year and all other covariates. The birth year was included to account for cohort effects and a survivor effect arising from left truncation of the data at the start of follow-up. The intercept (level of %FEV1 at age five) and age effect (annual linear change in %FEV1) were treated as normally distributed, correlated random effects to allow for individual intercepts and slopes. The random effects in the longitudinal model capture other sources of unmeasured heterogeneity not captured by the fixed effects.</p><p>Subjects entered the cohort at different ages so that patients contributed person-years to the analysis only at ages corresponding to their actual ages during the study period.</p><p>The probability of surviving one year was modeled using a probit link function. The survival submodel was adjusted for age halfway through the year, birth year, sex, and F508 alleles (dichotomized as previously). In addition, the survival submodel depended on a patient’s %FEV1 value halfway through the year and their %FEV1 rate of change, as estimated by the longitudinal %FEV1 model including the random effects. The time-to-event submodel is specified on a discrete-time scale modeling the conditional probability of surviving one year given that you have survived to the start of the year, whereby a positive coefficient means that an increase in the predictor leads to an increase in the predicted probability of survival. The effect of %FEV1 level and %FEV1 rate of change on survival were also expressed as hazard ratios (see the statistical eAppendix1; <ext-link ext-link-type=\"uri\" xlink:href=\"http://links.lww.com/EDE/B714\">http://links.lww.com/EDE/B714</ext-link> for details of estimating hazard ratios from our survival model). Note that for the hazard ratios, the direction of effect is reversed, i.e. a hazard ratio >1 means that an increase in the predictor gives a decreased probability of survival.</p><p>We assess whether there is a direct association between sex and survival according to the directed acyclic graph as shown in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>, after accounting for lung function. Our coefficient of interest in the joint model is the sex effect in the survival submodel, after adjustment for %FEV1 rate of change and overall level of %FEV1 (for full algebraic details see eAppendix1; <ext-link ext-link-type=\"uri\" xlink:href=\"http://links.lww.com/EDE/B714\">http://links.lww.com/EDE/B714</ext-link>). To demonstrate the utility of using a joint modeling approach to test our hypothesis, we assessed how the association between sex and survival changed depending on the modeling approach. Once we fitted the joint model to the data, we compared the association between sex and survival with that in a standard probit survival model, first without any adjustment for %FEV1, and then with %FEV1 added as a baseline time-invariant covariate (i.e. %FEV1 at first visit), and finally as a time-varying covariate. We estimated all model parameters by maximum likelihood and used generalized likelihood ratio statistics to compare nested models, and Wald statistics to test hypotheses about model parameters. We plotted residual diagnostics for the longitudinal and survival submodels, and an empirical variogram to check our model fit.</p></sec><sec><title>Ethics</title><p>National Health Service research ethics approval (Huntingdon Research Ethics Committee 07/Q0104/2) has been granted for the collection of data into the UK database. The cystic fibrosis Trust database committee approved the use of anonymized data in this study.</p></sec></sec><sec sec-type=\"results\"><title>RESULTS</title><sec><title>Population Characteristics</title><p>The dataset contained 81,129 lung function measures on 9,741 patients between 1996 and 2015 in the UK and captured 1,543 deaths. The median number of %FEV1 measures per person was 8 (range 1–25). About 87% of individuals had three or more follow-up measures with a total of 96,598 person-years of follow-up. The baseline characteristics of the population, stratified by sex, are shown in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1.</label><caption><p>Characteristics of Study Population by Sex: UK Cystic Fibrosis Registry</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" rowspan=\"1\" colspan=\"1\">Female</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Male</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">All</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">N (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4605 (47)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5136 (53)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">9741</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Observation, n (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37849 (47)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43280 (53)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">81129</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Observations per patient, mean (SD)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.2 (4.7)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.4 (4.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">8.3 (4.8)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Deaths, n (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">813 (53)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">730 (47)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1543</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Genotype</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> No. delta 508: 2, n (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2361 (51)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2752 (54)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5113 (53)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> No. delta 508: 1, n (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1718 (37)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1782 (354)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3500 (36)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> No. delta 508: 0, n (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">327 (7.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">354 (6.9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">681 (7.0)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> Missing</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">199 (4.3)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">248 (4.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">447 (4.6)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Birth cohort, n (%)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> <1960</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3 (0.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4 (0.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7 (0.1)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 1960–1964</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">68 (1.5)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">115 (2.2)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">183 (1.9)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 1965–1969</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">141 (3.1)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">199 (3.9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">340 (3.5)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 1970–1974</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">257 (5.6)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">335 (6.5)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">592 (6.1)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 1975–1979</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">365 (7.9)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">473 (9.2)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">838 (8.6)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 1980-1984</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">587 (13)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">686 (13)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1273 (13)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 1985–1989</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">668 (15)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">772 (15)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1440 (15)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"> 1990–1994</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">722 (16)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">749 (15)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1471 (15)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">  >1995</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1794 (39)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1803 (35)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3597 (37)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age at entry, yrs, mean (SD)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.2 (9.0)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20.0 (9.4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">19.6 (9.2)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age at diagnosis, yrs, mean (SD) (missing, n = 118)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0 (6.5)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0 (6.8)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3.0 (6.7)</td></tr></tbody></table></table-wrap></sec><sec><title>Associations of Covariates with Lung Function Trajectory</title><p>We explored the effect of covariates on %FEV1. Results from the %FEV1 submodel are shown in Table <xref rid=\"T2\" ref-type=\"table\">2</xref>. Random effect parameter estimates are reported in eAppendix2; <ext-link ext-link-type=\"uri\" xlink:href=\"http://links.lww.com/EDE/B714\">http://links.lww.com/EDE/B714</ext-link>. There was a borderline difference between males and females in the level of %FEV1 at age 5 with the difference in intercept of 0.90 (95% CI = 0.01, 1.80). %FEV<sub>1</sub> initially declined at a rate of −1.52 (95% CI = −1.58, −1.45) percentage points per year for females. Males experienced a more gradual decline in %FEV1 (difference 0.11 per year 95% CI= 0.08, 0.14). Lung function declined at a faster rate for patients with 2 F508 alleles compared with those with 0 or 1 F508 alleles (−0.35 percentage points per year 95% CI = −0.45, −0.25).</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2.</label><caption><p>Joint Model Results for the %FEV1 Submodel. Fixed Effects Estimates of Association of Covariates on Forced Expiratory Volume in 1 second as a Percentage of Predicted (%FEV1)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" rowspan=\"1\" colspan=\"1\">Estimate (95% CI)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Intercept at age 5 years</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">88 (87, 89)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age<sup><xref ref-type=\"table-fn\" rid=\"tab2fn1\">a</xref></sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−1.5 (−1.6, −1.4)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age squared</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.013 (0.011, 0.015)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Birth year</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.22 (0.15, 0.29)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.90 (0.01, 1.8)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">F508 alleles: 2 vs 0, 1 or not typed</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.080 (−1.17, 1.01)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age × male</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11 (0.08, 0.14)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age × F508 alleles: 2 vs 0, 1 or not typed</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.35 (−0.45, −−0.25)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Birth year × male</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.059 (−0.027, 0.15)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Birth year × F508 alleles: 2 vs 0, 1 or not typed</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.0040 (−0.067, 0.059)</td></tr></tbody></table><table-wrap-foot><fn fn-type=\"other\" id=\"tab2fn1\"><label>a</label><p>Age term corresponds to %FEV1 slope. The age × male term represents the age by sex interaction, i.e. the difference in slope for males compared with females</p></fn><fn fn-type=\"other\"><p>%FEV1 indicates percent forced expiratory volume.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Associations of Covariates with Survival</title><p>Table <xref rid=\"T3\" ref-type=\"table\">3</xref> shows the results from the survival models. The joint model (column 1) shows that higher overall levels of %FEV1 and a more gradual %FEV1 decline were associated with improved survival. Figure <xref ref-type=\"fig\" rid=\"F2\">2</xref> provides a visual illustration of the relationship between lung function and survival for individuals selected from the population with different %FEV1 trajectories (different random effects). For example, for 20-year old males born in 1980 with two F508 alleles and population-average %FEV1 level and %FEV1 decline, a 10-unit lower level of %FEV1 is associated with an increase in the concurrent hazard of death (HR 2.26 CI = 2.13, 2.41). Furthermore, those with a one-unit-per-year decline compared with no decline in %FEV1 have over a three-fold higher hazard of death for every year that passes (HR 3.67 CI = 3.31, 4.07).</p><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3.</label><caption><p>Parameter Estimates (95% CI) From the Probit Survival Models. A Positive Coefficient Means that an Increase in the Predictor Leads to an Increase in the Predicted Probability of Survival</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" rowspan=\"1\" colspan=\"1\">Joint model</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">No %FEV1</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Baseline %FEV1</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Time-varying %FEV1</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Intercept</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.6 (1.5, 1.7)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2.4 (2.3, 2.4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.3 (1.1, 1.4)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.37 (0.23, 0.50)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">%FEV1 (per 10 units)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.28 (0.26, 0.29)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18 (0.17, 0.19)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.32 (0.31, 0.33)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">%FEV1 slope</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45 (0.43, 0.48)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Age-5 (per 10 yrs)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.042 (−−0.059, −0.026)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.046 (−0.089, −0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.12 (−0.17, −0.08)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.059 (0.007, 0.111)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Birth year (per 10 yrs)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.26 (0.22, 0.30)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16 (0.12, 0.21)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.073 (−0.120, −0.025)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.039 (−0.011,0.090)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.092 (0.062, 0.122)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15 (0.10, 0.19)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.13 (0.09, 0.18)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14 (0.09, 0.19)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">F508 alleles: 2 vs 0, 1 or not typed</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14 (0.03, 0.25)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.13 (−0.17, −0.09)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.10 (−0.14, −0.06)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.042 (−0.089, 0.006)</td></tr></tbody></table><table-wrap-foot><fn fn-type=\"other\"><p>%FEV1 indicates percent forced expiratory volume.</p></fn></table-wrap-foot></table-wrap><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2.</label><caption><p>Estimated longitudinal trajectories and survival curves for 20-year-old males with varying intercepts and slopes, born in 1980, with 2 F508 alleles.</p></caption><graphic xlink:href=\"ede-31-872-g002\"/></fig><p>Figure <xref ref-type=\"fig\" rid=\"F3\">3</xref> visualizes the association of sex with lung function and survival in the joint model. Males have a more favorable %FEV1 and survival trajectory. Note that the estimated sex effects in Figure <xref ref-type=\"fig\" rid=\"F3\">3</xref> are population-averaged effects; that is, they describe average values of %FEV1 for subpopulations of individuals sharing the same explanatory characteristics, rather than for any one individual.</p><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>FIGURE 3.</label><caption><p>Estimated longitudinal trajectories and survival curves for males and females age 20, born in 1980 with 2 F508 alleles. Effect of sex on forced expiratory volume in 1 second as a percentage of predicted (%FEV1) and survival in the final joint model.</p></caption><graphic xlink:href=\"ede-31-872-g003\"/></fig></sec><sec><title>Association Between Female Sex and Survival Adjusted for Lung Function</title><p>In Table <xref rid=\"T3\" ref-type=\"table\">3</xref>, we compare the results of four possible survival models that adjust for %FEV1 in various ways. Contrasting the association of sex with survival across these models, in all three of the standard survival models there is a strong association between sex and survival in models without adjustment for %FEV1, and with adjustment for % FEV1 either as a baseline or as a time-varying covariate (effect sizes 0.15, 0.13, 0.14, i.e. males have better survival than females). By contrast, we observe less association between sex and survival after adjusting for %FEV1 (level and rate of decline) in the joint model, with an effect size attenuated by 37% to 0.09 (95% CI = 0.06, 0.12). Table <xref rid=\"T3\" ref-type=\"table\">3</xref> also shows that the genotype effect is reversed once we adjust for longitudinal lung function in the joint model. Residual diagnostics did not raise any concerns about model fit (eAppendix2; <ext-link ext-link-type=\"uri\" xlink:href=\"http://links.lww.com/EDE/B714\">http://links.lww.com/EDE/B714</ext-link>).</p></sec></sec><sec sec-type=\"discussion\"><title>DISCUSSION</title><p>We apply a novel joint modeling approach, for the first time, to show that about 40% of the association of female sex on survival in cystic fibrosis is explained by the effect of sex on lung function. Both increased rates of decline in lung function, and a decreased overall level of lung function, are associated with an increased risk of death. The strength of this analysis is the use of a joint modeling approach that leads to more robust estimation of both survival estimates and the rate of lung function decline. Our joint modeling approach allows exploration of how aspects of an individual cystic fibrosis patient’s longitudinal profile of %FEV1 are related to their survival prognosis. A key advantage of the joint model is that it estimates the relationship between characteristics of the “true” error-free underlying %FEV1 trace and survival. Our analysis here suggests that measurement error – in conventional approaches to adjusting for %FEV1 in survival analysis – has led to previous estimates of a substantial association between sex and survival. This overestimate was attenuated once error-free %FEV1 was taken into account in the joint model.</p><p>Many studies have explored survival in cystic fibrosis,<sup><xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R13\" ref-type=\"bibr\">13</xref></sup> and notable among these are the large studies from North America that have used Cox regression to estimate the effect of various covariates on survival chances in registry populations.<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref>,<xref rid=\"R6\" ref-type=\"bibr\">6</xref>,<xref rid=\"R23\" ref-type=\"bibr\">23</xref></sup> A large number of factors have been identified that may influence survival, with female sex identified most commonly as a risk factor. Other influences include poor respiratory function and risk factors for poor lung function such as <italic>P. aeruginosa</italic> infection status, homozygous delta F508 status, or heterozygous nondelta F508 status, non-white ethnicity, and low income. Many studies have investigated the impact of baseline time-invariant factors on survival, including a recent study using UK data by Keogh and colleagues which showed a clear sex gap, with worse survival for female patients.<sup><xref rid=\"R13\" ref-type=\"bibr\">13</xref></sup> Where studies have investigated time varying predictors such as lung function, these have been included in survival models as current values. To our knowledge, our study is one of the first to quantify the association of rate of lung function decline with survival using a joint modeling approach. Another recent study by Keogh et al used landmarking to predict survival in the UK population, and in a two-stage approach used fitted values from a longitudinal mixed-effects model for %FEV1 to estimate current values of %FEV1 for inclusion in the second-stage landmarking analysis.<sup><xref rid=\"R14\" ref-type=\"bibr\">14</xref></sup> The study found that current %FEV1 was the strongest predictor of survival. As in our joint model, this two-stage landmarking approach could also be used to estimate the association of the recent rate of decline of %FEV1 with survival.</p><p>There is a large literature on lung function decline in people with cystic fibrosis. Konstan et al have undertaken the largest studies to date of %FEV1 in both pediatric and adult cohorts,<sup><xref rid=\"R24\" ref-type=\"bibr\">24</xref>–<xref rid=\"R26\" ref-type=\"bibr\">26</xref></sup> using a mixed-effects regression approaches, to show that higher baseline %FEV1, <italic>P. aeruginosa</italic> colonization, female sex, and poor nutritional status were amongst the factors associated with a greater decline in lung function in people with cystic fibrosis.<sup><xref rid=\"R24\" ref-type=\"bibr\">24</xref></sup> Our analysis contributes to this literature by explicitly quantifying how longitudinal changes in lung function are related to survival. Both an increased rate of decline in lung function, and a decreased overall level of lung function are associated with an increased risk of death in people with cystic fibrosis.</p><p>Large national cystic fibrosis patient registry data in different countries have shown that survival for cystic fibrosis females was less than that for matched cystic fibrosis males, in an apparent sex gap. The etiology of this gap is poorly understood, with increased mortality in females seen even after correcting for lung function, suggesting the explanation is likely to be multifactorial. Our analysis adds to this literature, estimating that about 40% of the effect of sex on survival is explained by the impact of sex on lung function decline, which in turn influences survival. There are known effects of sex on lung function and acquisition of <italic>P. aeruginosa</italic> that we have previously identified in this dataset.<sup><xref rid=\"R12\" ref-type=\"bibr\">12</xref></sup> Others have suggested alternative biologic reasons, including the impact of estrogen and increased occurrence of cystic fibrosis-related diabetes (CFRD).<sup><xref rid=\"R3\" ref-type=\"bibr\">3</xref>,<xref rid=\"R8\" ref-type=\"bibr\">8</xref></sup> Social explanations proposed may relate to gender roles, such as a possible propensity to less exercise in childhood in girls, and an increased tolerance of poor nutritional status in adolescent girls with cystic fibrosis, fuelled by the societal pressure to appear thin.<sup><xref rid=\"R27\" ref-type=\"bibr\">27</xref></sup></p><p>Our analysis suggests that the sex gap may be partly explained by worse lung function in females. We suggest that standard approaches for adjusting for lung function in survival analysis may lead to insufficient adjustment, which may explain the difference between our results, and other large epidemiologic studies of cystic fibrosis. Because lung function is measured with significant error, the associations with other covariates in the analysis may be confounded by residual effects of lung function. In our joint model, which more precisely adjusts for underlying lung function, differences in lung function explain about 40% of the sex difference in mortality. For instance, one of the largest studies to explore the gender gap in cystic fibrosis was Rosenfeld and colleagues’ analysis of US registry data,<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref></sup> which showed a significant association of sex on survival and lung function. The authors show that pulmonary function was the only risk factor that explained a portion of the observed gender-related difference in survival. Among the subjects 1-20 years of age, females had a hazard ratio of 1.7 in the unadjusted Cox regression analysis, but this was attenuated to 1.5 while adjusting for %FEV1 as a time-varying covariate. The authors suggest that differences in pulmonary function did appear to explain a small portion of the excess female mortality, but no other factor further accounted for the gender gap. A more recent large study of US registry data identified a survival advantage for males compared with females, with a 19% (CI = 13%, 24%) lower adjusted risk for death in males as compared with females,<sup><xref rid=\"R28\" ref-type=\"bibr\">28</xref></sup> and the authors further highlight that the reasons for this are not well-understood. However, this analysis did not adjust for lung function at a time-varying covariate but focused on adjustment for baseline factors at the time of diagnosis, which can be used by clinicians at diagnosis to inform discussions about patient prognosis.</p><p>To our knowledge, this is the first study to explore the sex effect in cystic fibrosis using joint modeling approaches, though previous studies have used cystic fibrosis data to develop joint modeling methods.<sup><xref rid=\"R19\" ref-type=\"bibr\">19</xref></sup> Key strengths of this study include the population-wide coverage of the UK Cystic Fibrosis Registry and the high quality of the data.<sup><xref rid=\"R20\" ref-type=\"bibr\">20</xref></sup> Although we have not presented our analysis as formal mediation analysis, the steps we have undertaken map onto the Baron and Kenny<sup><xref rid=\"R29\" ref-type=\"bibr\">29</xref></sup> steps for mediation analysis, subject to a number of assumptions (see eAppendix 2; <ext-link ext-link-type=\"uri\" xlink:href=\"http://links.lww.com/EDE/B714\">http://links.lww.com/EDE/B714</ext-link>). Newer methods for mediation analysis based on the counterfactual framework, and software to implement them, are developing rapidly, and further work is warranted to understand how joint models can be used within the potential outcomes framework. Such approaches could be usefully applied to range of mediation questions in cystic fibrosis epidemiology. For example, while not the main focus of our analysis here, the reversal of the genotype effect in our joint model suggests that heterozygotes for del508, compared with those with one or no del508 alleles, have better survival after adjustment for lung function. Further analyses could explore decomposing the sex effect on survival through a potential effect of sex on <italic>P. aeruginosa</italic> acquisition which is known to impact lung function decline.</p><p>There are a number of limitations to our analysis: First, it relies on retrospective, routinely collected data. Second, our joint modeling approach assumes that %FEV1 measurements are independent of survival given an individual’s random-effect values, which may not be appropriate. Third, we did not explore the full range of mediating pathways through which sex may impact survival. Our main hypothesis related to the sex term in the survival submodel, and we did not seek to adjust for other potential downstream mediators of the association between sex and survival in the survival submodel, such as CFRD and infection status indicators, instead adjusting for slope and overall level of lung function as captured in our longitudinal submodel. Whilst the random effects included in the longitudinal component of our joint model capture residual underlying baseline heterogeneity they are unlikely to adequately capture time-varying effects such as infections. The impact of infection on survival, however, is likely to be largely mediated through impacts on lung function. A recent study has illustrated how left truncation can lead to biased estimates in the context of joint models.<sup><xref rid=\"R30\" ref-type=\"bibr\">30</xref></sup> We aimed to limit the impact of left truncation by including the birth year as a covariate in the longitudinal and survival submodels. The effect of birth year here is driven by both cohort effects and left truncation. Another limitation of our study is that it is debatable how transplanted individuals should be handled in a joint model.<sup><xref rid=\"R14\" ref-type=\"bibr\">14</xref></sup> For the purposes of this analysis, we included post-transplant FEV1 measures and deaths, but further work is needed to understand how best to take account of transplantation and post-transplant survival in the context of a joint modeling analysis.</p><p>In summary, we have applied the Barrett et al<sup><xref rid=\"R19\" ref-type=\"bibr\">19</xref></sup> approach to joint modeling in cystic fibrosis to address the question of how the sex effect on survival is explained by lung function. Our analysis suggests that if the lung function gap between males and females can be narrowed, this should also narrow the survival gap. Our analysis approach can be applied to similar etiologic questions in longitudinal Cystic Fibrosis Registry data and can be used to more accurately adjust for time-varying covariates measured with error, such as lung function, in survival analyses in cystic fibrosis and other conditions.</p></sec><sec><title>ACKNOWLEDGMENTS</title><p>We thank the UK Cystic Fibrosis Trust for access to the UK Cystic Fibrosis Registry and all the Centre Directors for the input of data to the registry. Elaine Gunn assisted with access to the data. JKB was supported by the MRC (grant number G0902100 and unit programme number MC_UU_00002/5). DTR is funded by the MRC on a Clinician Scientist Fellowship (MR/P008577/1). The work was supported by the Strategic Research Centre “EpiNet: Harnessing data to improve lives” funded by the Cystic Fibrosis Trust.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><supplementary-material content-type=\"local-data\" id=\"SD1\"><media id=\"s1\" content-type=\"document\" xlink:href=\"ede-31-872-s001.docx\" mimetype=\"application\" mime-subtype=\"docx\" orientation=\"portrait\" position=\"anchor\"/></supplementary-material></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p>D.T.R., D.K.S., and P.J.D. are supported by a UK cystic fibrosis Trust Strategic Research Centre grant. J.K.B. is supported by an MRC Unit programme. David Taylor-Robinson is funded by the MRC on a Clinician Scientist Fellowship (MR/P008577/1).</p></fn><fn fn-type=\"COI-statement\"><p>D.T.R., D.K.S., and P.J.D. are supported by a UK cystic fibrosis Trust Strategic Research Centre grant. The other author has no conflicts to report.</p></fn><fn fn-type=\"supplementary-material\"><p>Supplemental digital content is available through direct URL citations in the HTML and PDF versions of this article (<ext-link ext-link-type=\"uri\" xlink:href=\"www.epidem.com\">www.epidem.com</ext-link>).</p></fn><fn fn-type=\"equal\"><p>D.T.R., P.J.D., and J.K.B. are guarantors. D.T.R., P.J.D., and J.K.B. conceived and designed. D.T.R., D.K.S., P.J.D., and J.K.B. involved in data collection. D.T.R., D.K.S., P.J.D., and J.K.B. responsible for analysis and interpretation. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Bioeng Biotechnol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Bioeng. Biotechnol.</journal-id><journal-title-group><journal-title>Frontiers in Bioengineering and Biotechnology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-4185</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33042960</article-id><article-id pub-id-type=\"pmc\">PMC7523570</article-id><article-id pub-id-type=\"doi\">10.3389/fbioe.2020.01027</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Bioengineering and Biotechnology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Drug-Loaded Lipid-Coated Hybrid Organic-Inorganic “Stealth” Nanoparticles for Cancer Therapy</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Xue</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/988163/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Salzano</surname><given-names>Giuseppina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Qiu</surname><given-names>Jingwen</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Menard</surname><given-names>Mathilde</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Berg</surname><given-names>Kristian</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/300046/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Theodossiou</surname><given-names>Theodossis</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ladavière</surname><given-names>Catherine</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Gref</surname><given-names>Ruxandra</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Université Paris-Saclay, CNRS UMR 8214, Institut des Sciences Moléculaires d’Orsay</institution>, <addr-line>Orsay</addr-line>, <country>France</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital</institution>, <addr-line>Oslo</addr-line>, <country>Norway</country></aff><aff id=\"aff3\"><sup>3</sup><institution>University of Lyon, CNRS, UMR 5223, IMP</institution>, <addr-line>Villeurbanne</addr-line>, <country>France</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Gang Liu, Xiamen University, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Paolo Bigini, Mario Negri Institute for Pharmacological Research (IRCCS), Italy; Clara Mattu, Politecnico di Torino, Italy</p></fn><corresp id=\"c001\">*Correspondence: Ruxandra Gref, <email>ruxandra.gref@universite-paris-saclay.fr</email>; <email>ruxandra.gref@u-psud.fr</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Nanobiotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>1027</elocation-id><history><date date-type=\"received\"><day>28</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>06</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Li, Salzano, Qiu, Menard, Berg, Theodossiou, Ladavière and Gref.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Li, Salzano, Qiu, Menard, Berg, Theodossiou, Ladavière and Gref</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Hybrid porous nanoscale metal organic frameworks (nanoMOFs) made of iron trimesate are attracting increasing interest as drug carriers, due to their high drug loading capacity, biodegradability, and biocompatibility. NanoMOF surface modification to prevent clearance by the innate immune system remains still challenging in reason of their high porosity and biodegradable character. Herein, FDA-approved lipids and poly(ethylene glycol) (PEG)-lipid conjugates were used to engineer the surface of nanoMOFs by a rapid and convenient solvent-exchange deposition method. The resulting lipid-coated nanoMOFs were extensively characterized. For the first time, we show that nanoMOF surface modification with lipids affords a better control over drug release and their degradation in biological media. Moreover, when loaded with the anticancer drug Gem-MP (Gemcitabine-monophosphate), iron trimesate nanoMOFs acted as “Trojan horses” carrying the drug inside cancer cells to eradicate them. Most interestingly, the PEG-coated nanoMOFs escaped the capture by macrophages. In a nutshell, versatile PEG-based lipid shells control cell interactions and open perspectives for drug targeting.</p></abstract><kwd-group><kwd>metal organic frameworks</kwd><kwd>nanoparticles</kwd><kwd>lipids</kwd><kwd>poly(ethylene glycol)</kwd><kwd>stealth</kwd><kwd>sustained drug release</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Agence Nationale de la Recherche<named-content content-type=\"fundref-id\">10.13039/501100001665</named-content></funding-source><award-id rid=\"cn001\">ANR-14-CE08-0017</award-id><award-id rid=\"cn001\">ANR-16-CE18-0018</award-id><award-id rid=\"cn001\">ANR-10-LABX-0035</award-id></award-group></funding-group><counts><fig-count count=\"8\"/><table-count count=\"0\"/><equation-count count=\"2\"/><ref-count count=\"33\"/><page-count count=\"12\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Despite progresses in drug development and cancer biology, cancer mortality rate remains over 30%, and the morbidity much higher. Nanomedicine has shown great promise through drug delivery by achieving drug transcytosis, drug targeting and theranostics (<xref rid=\"B26\" ref-type=\"bibr\">Rosenblum et al., 2018</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Senapati et al., 2018</xref>). Nanoscale metal organic frameworks (nanoMOFs) recently emerged as an attracting class of hybrid nanomaterials for biomedical applications due to their biodegradability, biocompatibility, elevated drug loading capacity and high versatility in terms of architecture and physico-chemical properties (<xref rid=\"B18\" ref-type=\"bibr\">Horcajada et al., 2010</xref>, <xref rid=\"B19\" ref-type=\"bibr\">2012</xref>; <xref rid=\"B16\" ref-type=\"bibr\">He et al., 2015</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Rojas et al., 2019</xref>). NanoMOFs are formed by the self-assembly of metal centers and organic ligands, leading to the formation of open crystalline structures with regular and high porosities.</p><p>Iron (III) trimesate nanoMOFs (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> upper panel) are among the most widely studied MOFs for drug delivery (<xref rid=\"B18\" ref-type=\"bibr\">Horcajada et al., 2010</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Agostoni et al., 2013</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Baati et al., 2013</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Simon-Yarza et al., 2016</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Li et al., 2019a</xref>). Recently, they were shown to display several intrinsic properties of main interest in the nanomedicine field: radio-enhancement properties when submitted to γ-irradiation (<xref rid=\"B21\" ref-type=\"bibr\">Li et al., 2019a</xref>); they behaved as T<sub>2</sub>-weighted MRI imaging contrast agents (<xref rid=\"B18\" ref-type=\"bibr\">Horcajada et al., 2010</xref>) and they had intrinsic antibacterial effects killing intracellular bacteria (<xref rid=\"B22\" ref-type=\"bibr\">Li et al., 2019b</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p><bold>Upper panel</bold>: Schematic representation of the MIL-100(Fe) nanoMOF assembly from iron trimers and trimesic acid. Gem-MP was loaded by impregnation from aqueous solutions, penetrating inside the nanoMOFs through their largest windows (approx. 9 Å in size). Lipid molecules (DOPC and DSPE-PEG 2000) with larger molecular dimensions than the large windows were used to coat the nanoMOFs. <bold>Lower panel</bold>: Preparation steps of lipid-coated nanoMOFs. First, Gem-MP was loaded, into the nanoMOFs followed by their coating with lipid shells and PEG-lipid conjugates.</p></caption><graphic xlink:href=\"fbioe-08-01027-g001\"/></fig><p>In addition, iron trimesate nanoMOFs MIL-100 (Fe) (MIL stands for Material from Institut Lavoisier) exhibited high drug loading capacity soaking a variety of drugs from their aqueous solutions with yields close to 100%. In the case of Gemcitabine-monophosphate (Gem-MP), the drug payload reached ∼30 wt% with >98% drug loading efficiency (<xref rid=\"B24\" ref-type=\"bibr\">Rodriguez-Ruiz et al., 2015</xref>). Gem-MP, the active intermediate of Gem, is widely used in various carcinomas, including pancreatic cancer, bladder cancer, and non-small cell lung cancer. The administration of Gem-MP is of high interest for resistant cancer treatment since the phosphorylation of Gem can be a rate-limiting step especially difficult for resistant cancer cells. However, Gem-MP administration is hampered by its poor stability in biological media and low cellular uptake (<xref rid=\"B7\" ref-type=\"bibr\">Bouffard et al., 1993</xref>). In this challenging context, some of us showed that Gem-MP could be protected against degradation with increased cellular uptake by encapsulation in nanoMOFs (<xref rid=\"B24\" ref-type=\"bibr\">Rodriguez-Ruiz et al., 2015</xref>).</p><p>Surface modifications are essential to control drug release and modulate the <italic>in vivo</italic> fate of nanoMOFs in the living body. Silica coatings were employed in an attempt to control the release of loaded molecules from nanoMOFs MIL-101 (<xref rid=\"B29\" ref-type=\"bibr\">Taylor-Pashow et al., 2009</xref>). NanoMOFs were coated with lipid bilayers to improve their uptake by cancer cells (<xref rid=\"B33\" ref-type=\"bibr\">Wuttke et al., 2015</xref>) or with chitosan to increase their intestinal permeability (<xref rid=\"B17\" ref-type=\"bibr\">Hidalgo et al., 2017</xref>). Heparin coatings endowed the nanoMOFs with longer-blood circulation time (<xref rid=\"B6\" ref-type=\"bibr\">Bellido et al., 2015</xref>).</p><p>Poly(ethylene glycol) (PEG) based materials remain the most employed ones to engineer coatings able to prevent nanoparticles (NPs) clearance by the innate immune system, which is a prerequisite for biomedical applications (<xref rid=\"B15\" ref-type=\"bibr\">Gref et al., 1994</xref>, <xref rid=\"B13\" ref-type=\"bibr\">1995</xref>). However, as compared to dense polymeric NPs, the porous nanoMOFs are more challenging to be coated with PEG, because these linear chains readily penetrate within their porosity, inducing an uncontrolled “burst” drug release (<xref rid=\"B2\" ref-type=\"bibr\">Agostoni et al., 2015</xref>). There are still scarce examples of successful PEGylated nanoMOF formulations. PEG was crosslinked onto the nanoMOF’s surface to avoid its penetration inside the porous cores (<xref rid=\"B12\" ref-type=\"bibr\">Giménez-marqués et al., 2018</xref>) but resulted in a non-biodegradable coating. Alternatively, nanoMOFs were coated with inclusion complexes consisting of functionalized cyclodextrins (CDs) and PEG chains coupled to adamantine (<xref rid=\"B2\" ref-type=\"bibr\">Agostoni et al., 2015</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Aykac et al., 2017</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Cutrone et al., 2019a</xref>). Finally, comb-like copolymers consisting of polysaccharides grafted with moieties able to coordinate to the nanoMOFs and PEG chains were synthesized and anchored onto the nanoMOFs (<xref rid=\"B11\" ref-type=\"bibr\">Cutrone et al., 2019b</xref>). However, all these coatings imply sophisticated chemistry strategies and/or several preparation steps, which might restrict their further applications. Moreover, all the PEGylated (macro) molecules used in the previous studies are not approved by Food and Drug Administration (FDA).</p><p>In this context, we propose to engineer for the first time PEGylated coatings on nanoMOFs by using only FDA-approved materials using a convenient one-step method. To date, Doxil R and Onivyde R represent the only FDA-approved PEGylated NPs (<xref rid=\"B5\" ref-type=\"bibr\">Barenholz, 2012</xref>), where DSPE-PEG 2000 (1,2-distearoyl-<italic>sn</italic>-glycero-3-phosphoethanolamine-N-[amino (poly-ethylene glycol)-2000] sodium salt) were used in both cases. Herein, DSPE-PEG 2000 was used in combination with DOPC (1,2-dioleoyl-<italic>sn</italic>-glycero-3-phosphocholine) to functionalize the surface of iron trimesate MOFs (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> lower panel). Moreover, we show that the PEG-based coating have an impact on both drug release and nanoMOFs degradation, which was not the case with the coatings used so far. Finally, the coatings were able not only to reduce macrophage uptake <italic>in vitro</italic> but also to kill cancer cells.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Materials</title><p>Iron (III) chloride hexahydrate (98%) was purchased from Alfa Aesar (Schiltigheim, France). 1,3,5-benzenetricarboxylic acid (BTC, 95%) and absolute ethanol (99%) were from Sigma-Aldrich (Saint-Quentin-Fallavier, France). These materials were used for the synthesis of nanoMOFs. Amoxicillin (Amox) from Sigma-Aldrich (Saint-Quentin-Fallavier, France) and 2′,2′-difluorodeoxycytidine monophosphate (Gem-MP) from Toronto Research Chemicals (North York, Canada) were the drugs used in this study. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and (1,2-distearoyl-<italic>sn</italic>-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] sodium salt (DSPE-PEG 2000) were ordered from Avanti Polar Lipids (Alabama, United States) as coating materials. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich, Oslo, Norway) was used for toxicity evaluation of nanoMOFs. All the chemicals were used without further purification.</p></sec><sec id=\"S2.SS2\"><title>Cell Culture</title><p>Murine macrophage cell line J774A.1, <italic>CelluloNet biobank BB-0033-00072</italic>, were grown in RPMI-1640 medium (Thermo Fisher Scientific, Villebon-sur-Yvette, France) supplemented with 10% v/v decomplemented fetal bovine serum (FBS, Thermo Fisher Scientific, Villebon-sur-Yvette, France), 1% L-Gluthamine (Sigma-Aldrich, Oslo, Norway), and 1% (P/S, Sigma-Aldrich, Oslo, Norway) at 37°C in humidified air containing 5% CO<sub>2</sub>. SKOV3 ovarian cancer cell were cultivated in a RPMI-1640 media without phenol red supplemented with 10% FBS, 5% L-Gluthamine and 5% penicillin/streptomycin (P/S) at 37°C in a 5% CO<sub>2</sub> humidified atmosphere.</p></sec><sec id=\"S2.SS3\"><title>Synthesis and Characterization of MIL-100(Fe) NanoMOFs</title><p>Iron trimesate nanoMOFs was synthesized by microwave assisted hydrothermal reaction as previously described [6]. Briefly, 20 mL of aqueous mixture containing 6.0 mM of iron chloride hexahydrate and 4.02 mM of trimesic acid (TA, 1,3,5-benzenetricarboxylic acid) was heated at 130°C for 6 min under stirring. The reaction was carried out with the power of 1600 W (Mars-5, CEM, United States). The as-synthesized nanoMOFs were harvested by centrifugation (10,000 <italic>g</italic>, 15 min) and washed with absolute ethanol to remove the excessive TA until the supernatant became colorless. NanoMOFs were stored in ethanol at room temperature for further usage at the concentration of 18.2 mg/mL.</p><p>SEM images were acquired on a Zeiss SUPRA 55 VP field emission gun scanning electron microscope fitted with an EDAX EDS analytical system. It was set to a low voltage (1 kV) and low current (a few pA) in order not to damage the samples and to avoid any conductive coating that could bother direct observation of the samples. Secondary electron type detector was used to record the images.</p><p>Dynamic light scattering (DLS) measurements were performed at 25°C on a Malvern Zetasizer Nano-ZS instrument at 90° angle. The mean hydrodynamic diameter of the particles was determined in a diluted aqueous suspension at 50 μg/mL.</p><p>Nanoparticle tracking analysis (NTA) was performed on Malvern NanoSight (LM10 Instrument, Malvern Instruments Ltd., Orsay, France), which combines a conventional optical microscope with a laser to illuminate the NPs in Brownian motion. It is used to individually follow nanoMOFs to gain insight into their size distribution and concentration.</p><p>Zeta potential (ZP) of nanoMOFs were measured at 25°C using a Zetasizer Nano-ZS instrument at different pH ranging from 3 to 10. NanoMOFs was diluted to 100 μg/mL with 1 mM KCl. Measured electrophoretic mobilities were converted to zeta potential values according to the Smoluchowski equation. Nitrogen sorption measurements were performed on a Micromeritics Instruments ASAP 2020 at 77 K. Samples were degassed at 100°C for 15 h. BET surface area was calculated in the partial pressure range of 0.05 – 0.20 P/P<sub>0</sub>.</p></sec><sec id=\"S2.SS4\"><title>Drug Encapsulation in NanoMOFs</title><p>Drugs (Gem-MP and Amox) were loaded within nanoMOFs simply by impregnation of drug(s) aqueous solutions and nanoMOFs. Practically, nanoMOFs suspension (1.0 mg) were centrifuged for 10 min at 10,000 <italic>g</italic> and re-suspended in 1 mL of aqueous drug solutions (0.125 ∼ 1 mg/mL for Amox and 0.08 ∼ 0.2 mg/mL for Gem-MP) or water as a control. Different drug concentrations were used to optimize the drug encapsulation. After incubation at room temperature under gentle stirring for several hours (12 h for Amox and 4 h for Gem-MP), the nanoMOFs were recovered by centrifugation at 10,000 <italic>g</italic> for 10 min. The non-encapsulated drug in the supernatant was quantified by adapting previously described High Performance Liquid Chromatography (HPLC) methods (<xref rid=\"B21\" ref-type=\"bibr\">Li et al., 2019a</xref>, <xref rid=\"B22\" ref-type=\"bibr\">b</xref>). Specifically, HPLC analysis was performed on an Agilent system using a tunable UV absorbance detector. The injection volume of AMOX was 10 μL followed by eluant flow at a rate of 0.5 mL/min through a C18 Silica column (4.6 × 250 mm, 5 μm; Phenomenex) maintained at 30°C. The mobile phase consisted of 30% (v/v) methanol containing 5.2 mg/mL of sodium dihydrogene phosphate monohydrate. The pH was adjusted to 5 using phosphoric acid solution. AMOX were detected at 247 nm and retention times were 4.6 min. Similarly, Gem-MP was detected using the same Agilent system and column. The mobile phase was composed of 84% buffer [0.2 M (TEAA)]: 16% methanol. It was detected at 254 nm with an injection volume of 10 μl. The drug payload was calculated as Equation (1):</p><p>\n<disp-formula id=\"S2.E1\"><label>(1)</label><mml:math id=\"M1\"><mml:mrow><mml:mi>P</mml:mi><mml:mi>a</mml:mi><mml:mi>y</mml:mi><mml:mi>l</mml:mi><mml:mi>o</mml:mi><mml:mi>a</mml:mi><mml:mpadded width=\"+5.6pt\"><mml:mi>d</mml:mi></mml:mpadded><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mo>%</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>E</mml:mi><mml:mo>⁢</mml:mo><mml:mi>n</mml:mi><mml:mo>⁢</mml:mo><mml:mi>c</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>p</mml:mi><mml:mo>⁢</mml:mo><mml:mi>s</mml:mi><mml:mo>⁢</mml:mo><mml:mi>u</mml:mi><mml:mo>⁢</mml:mo><mml:mi>l</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+5.6pt\"><mml:mi>d</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>D</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi><mml:mo>⁢</mml:mo><mml:mi>u</mml:mi><mml:mo>⁢</mml:mo><mml:mi>g</mml:mi><mml:mo>⁢</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>g</mml:mi></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mo>⁢</mml:mo></mml:mrow><mml:mrow><mml:mi>n</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>n</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>M</mml:mi><mml:mo>⁢</mml:mo><mml:mi>O</mml:mi><mml:mo>⁢</mml:mo><mml:mi>F</mml:mi><mml:mo>⁢</mml:mo><mml:mi>s</mml:mi><mml:mo>⁢</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>g</mml:mi></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:mn>100</mml:mn></mml:mrow></mml:math></disp-formula></p></sec><sec id=\"S2.SS5\"><title>Surface Modification of NanoMOFs With DOPC Lipids and PEG-Lipid Conjugates</title><p>Surface modification was performed using a “green” method. To prepare DOPC coated nanoMOFs, 60 μl of nanoMOFs were mixed with 40 μl of DOPC alcoholic solution containing 100 μg of DOPC. Subsequently, 900 μl of water were rapidly added using an electronic pipette. The weight ratio between DOPC and nanoMOF was in the range of 1:20 ∼ 1:1. In the case of PEG-lipid conjugates coated nanoMOFs, 20 wt% of DOPC was replaced by DSPE-PEG 2000.</p></sec><sec id=\"S2.SS6\"><title>Characterization of Lipid Coated NanoMOFs</title><sec id=\"S2.SS6.SSS1\"><title>Lipid Quantification</title><p>DOPC quantification was performed by a colorimetric, enzymatic method (BIOLABO, Maizy, France) which is commonly used to determine the phospholipid amount in serum. This titration is based on the assay of the choline moiety of phospholipids. To do this, 10 μL of specimens or a standard solution were mixed with the reagents in the BIOLABO titration kit. The mixtures were stirred 10 min. at 37°C. Then, the absorbance at 500 nm of all samples was measured. The DOPC concentration was finally calculated as Eq. (2):</p><p>\n<disp-formula id=\"S2.E2\"><label>(2)</label><mml:math id=\"M2\"><mml:mrow><mml:mrow><mml:mi>D</mml:mi><mml:mo>⁢</mml:mo><mml:mi>O</mml:mi><mml:mo>⁢</mml:mo><mml:mi>P</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+1.7pt\"><mml:mi>C</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>c</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>n</mml:mi><mml:mo>⁢</mml:mo><mml:mi>c</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mi>n</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>i</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>n</mml:mi></mml:mrow><mml:mo>=</mml:mo><mml:mi/></mml:mrow><mml:mrow><mml:mrow><mml:mi>S</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>n</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi><mml:mo>⁢</mml:mo><mml:mpadded width=\"+1.7pt\"><mml:mi>d</mml:mi></mml:mpadded><mml:mo>⁢</mml:mo><mml:mi>c</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>n</mml:mi><mml:mo>⁢</mml:mo><mml:mi>c</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mi>n</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>i</mml:mi><mml:mo>⁢</mml:mo><mml:mi>o</mml:mi><mml:mo>⁢</mml:mo><mml:mi>n</mml:mi></mml:mrow><mml:mo>×</mml:mo><mml:mfrac><mml:mrow><mml:mi>A</mml:mi><mml:mo>⁢</mml:mo><mml:mi>b</mml:mi><mml:mo>⁢</mml:mo><mml:mi>s</mml:mi><mml:mo>⁢</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi>s</mml:mi><mml:mo>⁢</mml:mo><mml:mi>p</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mi>c</mml:mi><mml:mo>⁢</mml:mo><mml:mi>i</mml:mi><mml:mo>⁢</mml:mo><mml:mi>m</mml:mi><mml:mo>⁢</mml:mo><mml:mi>e</mml:mi><mml:mo>⁢</mml:mo><mml:mi>n</mml:mi></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mi>A</mml:mi><mml:mo>⁢</mml:mo><mml:mi>b</mml:mi><mml:mo>⁢</mml:mo><mml:mi>s</mml:mi><mml:mo>⁢</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:mi>s</mml:mi><mml:mo>⁢</mml:mo><mml:mi>t</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>n</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi><mml:mo>⁢</mml:mo><mml:mi>a</mml:mi><mml:mo>⁢</mml:mo><mml:mi>r</mml:mi><mml:mo>⁢</mml:mo><mml:mi>d</mml:mi></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:mn>100</mml:mn></mml:mrow></mml:math></disp-formula>\n</p></sec><sec id=\"S2.SS6.SSS2\"><title>NPs Concentration Measurements by NP Tracking Analysis (NTA)</title><p>The concentration of nanoMOFs modified with DOPC or PEG-lipid conjugates at different weight ratios was investigated by Nanosight (LM10 Instrument, Malvern Instruments Ltd., Orsay, France), which combines a conventional optical microscope with a laser to illuminate the NPs in Brownian motion. Of main interest here, the size distribution and concentration could be determined simultaneously. Results are expressed as means of five independent measurements.</p></sec><sec id=\"S2.SS6.SSS3\"><title>Colloid Stability Characterization by DLS</title><p>The colloid stability of the nanoMOFs before and after lipid surface modification was monitored in water every day during 3 weeks’ storage at 4°C. The stability in biological medium, including cell culture medium and phosphate buffer saline (PBS) used in this study, was also measured at 0, 0.5, 1, 2, 4, 6, and 8 h after incubation at 37°C.</p></sec><sec id=\"S2.SS6.SSS4\"><title>Drug Release and Degradation of nanoMOFs</title><p>Drug release was performed in PBS of different concentrations at 37°C. Briefly, drug loaded nanoMOFs were centrifuged at 10,000 <italic>g</italic> for 10 min and the pellet was re-dispersed in 1 mL water by vortex. Aliquots of 100 μL were taken and mixed with 900 μl of the media used for release. The final concentration of PBS was 1, 3, and 6 mM and nanoMOFs of 2.0 mg/mL. After different incubation times (30 min, 1 h, 2 h, 4 h, 6 h and 24 h), the suspensions were centrifuged and the supernatants were assessed by HPLC as previously described to determine the amount of released drug. Moreover, the trimesate release was also evaluated by HPLC. Briefly, trimesate was analyzed with a mobile phase consisting of 90% buffer (5.75 g/L of NH<sub>4</sub>H<sub>2</sub>PO<sub>4</sub>): 10% Acetonitrile containing 5 mM TBAP. The injection volume was 5 μl and the detection wavelength was set at 220 nm.</p></sec><sec id=\"S2.SS6.SSS5\"><title>Human Plasma Protein Adhesion Tests</title><p>Human serum albumin (HSA) was used in this study. NanoMOFs modified or not (300 μg/mL) were incubated with HSA at 100 μg/mL in 10 mM phosphate buffer at 37°C. The samples were centrifuged at 10,000 <italic>g</italic> for 5 min to remove the nanoMOFs after 1, 2, 4, 6, 8 and 12 h incubation. The excessive amounts of HSA in the supernatant were quantified using a bicinchoninic acid (BCA) assay.</p></sec><sec id=\"S2.SS6.SSS6\"><title>NanoMOF Internalization in Macrophage</title><p>NanoMOF internalization was quantified by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Macrophage cells (J774A.1) were seeded at a density of 2.0 × 10<sup>5</sup> cells per well in 24-well plates. Cells were cultured at 37°C in 5% CO<sub>2</sub> overnight for attachment. Cells were then incubated with 1 mL cell culture media containing nanoMOFs coated or not with lipids (nanoMOF concentration = 50 μg/mL). At the end of the 4 h incubation, the cells were washed with PBS for three times to eliminate the excess of MOFs. Cells were finally dried and digested using aqua regia (15 min under ultrasonic bath), Fe quantification was performed using an ICP-MS equipped with a triple quadrupole (Agilent 8800, Agilent Technologies, Japan). Fe and Co were added as internal standard on samples and calibration standards solution at a concentration of 10 μg/L. Isotopes were detected using “on-mass mode” (<sup>54</sup>Fe<sup>+</sup>, <sup>56</sup>Fe<sup>+</sup>, <sup>59</sup>Co<sup>+</sup>). Helium was introduced into the collision/reaction cell at a flow rate of 3 mL/min. Dwell time for each of the targeted isotopes was 1 s. Fe was quantified using external calibration prepared using certified 1000 mg/L Fe standard solution (Merck, Germany). Operation conditions were daily optimized using a tuning solution.</p></sec><sec id=\"S2.SS6.SSS7\"><title>Cytotoxicity Assessment</title><p>MTT assays were carried out on SKOV3 ovarian cancer cell line to investigate the cytotoxicity of NPs. The cells were plated in 96 well plates at a concentration of 10,000 cells per well. The media was removed after 24 h incubation and replaced by fresh media containing the MOFs nanoparticles at different concentrations. The cytotoxicity was assessed by MTT assay at 24, 48 and 72 h following the incubation of the cells with the MOFs. In brief, 100 μL of complete media containing 0.5 mg/mL of MTT were added to cells and incubated for 2 h at 37°C in a 5% CO<sub>2</sub> humidified atmosphere. Subsequently, the MTT media were removed and replaced by 100 μL of DMSO per well to dissolve the MTT-formazan crystals. The plates were shaken for 10 min at 350 rpm in a Heidolph Titramax 101 orbital shaker, and the absorbance at 595 nm was measured with the Tecan spark M10 plate reader. Each MTT experiment was reproduced three times.</p></sec></sec></sec><sec id=\"S3\"><title>Results and Discussion</title><sec id=\"S3.SS1\"><title>MIL-100 (Fe) nanoMOF Surface Modification and Characterization of Functionalized NanoMOFs</title><p>Iron trimesate nanoMOFs with mean diameters of 232 ± 14 nm and Brunauer–Emmett–Teller (BET) surface areas of 1519 ± 50 m<sup>2</sup>.g<sup>–1</sup> were successfully synthesized by a “green” organic solvent-free hydrothermal method exempt of toxic additives such as hydrofluoric acid (<xref rid=\"B1\" ref-type=\"bibr\">Agostoni et al., 2013</xref>). They were crystalline and exhibited a facetted morphology (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>) in agreement with previously reported data (<xref rid=\"B10\" ref-type=\"bibr\">Cutrone et al., 2019a</xref>, <xref rid=\"B11\" ref-type=\"bibr\">b</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Representative scanning electron microscope (SEM) images of nanoMOFs before and after lipid modification. <bold>(A)</bold> nanoMOFs; <bold>(B)</bold> nanoMOFs after modification with DOPC; <bold>(C)</bold> nanoMOFs after modification with DOPC and DSPE-PEG 2000. Scale bar: 100 μm.</p></caption><graphic xlink:href=\"fbioe-08-01027-g002\"/></fig><p>In an attempt to achieve “stealth” NPs, the as-synthesized nanoMOF were surface functionalized with PEG-lipid conjugates in a one-step procedure using a mixture of DSPE-PEG 2000 and DOPC. Lipids were associated within less than 2 min at room temperature by dispersing the nanoMOFs in an ethanolic aqueous solution containing both DSPE-PEG 2000 and DOPC, followed by a quick addition of water to favor lipid deposition onto the nanoMOF surface. Indeed, lipids were freely soluble in ethanol/water mixtures, but they readily precipitated upon progressive addition of water which drastically reduced their solubility, leading to precipitation onto the nanoMOF surfaces (<xref rid=\"B33\" ref-type=\"bibr\">Wuttke et al., 2015</xref>). DOPC-coated nanoMOFs were prepared as controls using the same method. The bare and coated nanoMOFs were characterized by a set of complementary methods.</p><p>Firstly, SEM images show that the lipid-coated nanoMOFs displayed similar shapes but with more rounded edges (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>) as compared to the uncoated ones (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>), possibly because surface modification. No significant differences were observed for the coated nanoMOFs with or without PEG-lipid conjugates (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). Secondly, EDX experiments were performed to detect the presence of elements specific to the MOF cores (C, O, Fe) and to the shells (C, O, N) in the top layers of the NPs (around 10 nm depth). The presence of the DOPC coating was evidenced by the detection of an N peak characteristic of DOPC which was not found with bare nanoMOFs (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S1</xref>). Interestingly, in the PEG shells obtained with the lipid mixtures, the relative O content was increased by a factor of 4 as compared to DOPC coatings (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S1</xref>) possibly due to the presence of PEG chains in the nanoMOFs’ top layers, as PEG has the highest O content from all the nanoMOF components. These data offer a straightforward proof for both the presence of DOPC and PEG-lipid conjugates in the nanoMOF top layers.</p><p>The amount of DOPC in the nanoMOFs was quantified by using a colorimetric enzymatic method. For this, the DOPC:nanoMOF weight ratio in the preparation procedure was varied from 1:20 to 1:1. As shown in <xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>, the amount of lipids associated to the nanoMOFs increased with the amount of lipids used in the coating procedure. A plateau was reached at a DOPC: nanoMOF weight ratio of 1:3, corresponding to 25 ± 4 wt% lipids associated to the nanoMOFs. These quantities of coating material are among the highest reported so far (<xref rid=\"B18\" ref-type=\"bibr\">Horcajada et al., 2010</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Agostoni et al., 2015</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Bellido et al., 2015</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Hidalgo et al., 2017</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Giménez-marqués et al., 2018</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Cutrone et al., 2019a</xref>, <xref rid=\"B11\" ref-type=\"bibr\">b</xref>). As comparison, phosphorylated cyclodextrin (CD-P) coatings on same iron trimesate nanoMOFs reached ∼ 17 wt% (<xref rid=\"B2\" ref-type=\"bibr\">Agostoni et al., 2015</xref>). The important lipid association could be possibly due to: i) the fast precipitation of lipids at the hydrophobic surface of nanoMOFs, and ii) the strong affinity of the phosphate groups in the lipids for the iron sites at the nanoMOFs’ surface.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Characterization of lipid-coated nanoMOFs. <bold>(A)</bold> Quantification of the amount of DOPC in the nanoMOFs; <bold>(B)</bold> Mean hydrodynamic diameter (red) and concentration (blue) of DOPC-coated nanoMOFs determined by NTA. <bold>(C)</bold> Zeta potential of nanoMOFs as a function of pH before (blue) and after lipid coating with (red) or without (green) the addition of DSPE-PEG 2000.</p></caption><graphic xlink:href=\"fbioe-08-01027-g003\"/></fig><p>The nanoMOFs, coated or not, were characterized by a set of complementary methods. First, X-ray powder diffraction (XRPD) showed that the crystalline structure of the nanoMOFs was preserved after surface modification (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S2</xref>). Dynamic light scattering (DLS) proved that there were no significant differences between the mean hydrodynamic diameters of nanoMOFs before and after surface functionalization (232 ± 14 nm, 241 ± 17 nm and 238 ± 11 nm for uncoated nanoMOFs, lipid coated nanoMOFs at a DOPC: nanoMOF weight ratio of 1:3, with and without DSPE-PEG 2000, respectively). Moreover, the BET surface areas were not affected by surface modification with lipids (1519 ± 50 m<sup>2</sup>.g<sup>–1</sup>, 1486 ± 70 m<sup>2</sup>.g<sup>–1</sup>, and 1547 ± 80 m<sup>2</sup>.g<sup>–1</sup> for uncoated nanoMOFs, lipid coated nanoMOFs with and without DSPE-PEG 2000, respectively), suggesting that the bulky lipids were located onto the nanoMOF’s external surfaces rather than into their porosity.</p><p>Before surface modification, the nanoMOF concentration was around (4 ± 0.8) × 10<sup>12</sup> particles/mL, as determined by Nanoparticle Tracking Analysis (NTA). Interestingly, the nanoMOF particle concentration did not change upon modification with lipids (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>, blue histograms), suggesting that the lipids adhered at their surface and did not remain into the suspension medium. Indeed, at DOPC:nanoMOFs weight ratios from 0:1 up to 1:3, both particle concentrations and mean hydrodynamic diameters were unaffected [(5.6 ± 0.8) × 10<sup>12</sup> particles/mL, and 210 ± 23 nm, respectively]. To support this hypothesis, DLS analysis of supernatants (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table S1</xref>) after particle centrifugation revealed that they were devoid of any lipid vesicles (<1% particles free).</p><p>However, addition of excess lipids (weight DOPC:nanoMOF ratio of 1:1) resulted in a dramatic increase of total particle concentration [from (4 ± 0.8) × 10<sup>12</sup> to (1.1 ± 0.4) × 10<sup>13</sup> particles/mL], presumably because the nanoMOF surfaces were saturated with lipids. Of note, the mean hydrodynamic diameter of the nanoMOFs was unaffected, only the polydispersity index (PdI) increased from 0.15 to 0.25, possibly because of the presence of lipid vesicles in excess. Note that the association of DSPE-PEG 2000 didn’t significantly influence the mean hydrodynamic diameter, nor the nanoMOF’s concentration (less than 10% variations) suggesting that the PEGylated lipids also attached onto the nanoMOFs. In conclusion, lipids were associated up to 25 ± 4 wt% without inducing any changes in nanoMOF porosities, size distribution, and crystallinity.</p><p>Interestingly, the presence of the coatings affected the nanoMOFs electrophoretic mobility, as shown by Zeta potential (ZP) investigations in <xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>. Indeed, the ZP of the uncoated nanoMOFs was strongly dependent upon the pH of the suspension medium, shifting from positive values (+ 23 ± 3 mV) at pH lower than 5 to negative values (−15 ± 3 mV) at basic pH. This could be probably due to the presence of both uncoordinated iron sites and terminal carboxyl groups of the trimesate ligands at the external nanoMOFs surface (<xref rid=\"B11\" ref-type=\"bibr\">Cutrone et al., 2019b</xref>). The ZP values were dramatically altered after surface modification (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>). DOPC-coated nanoMOFs displayed negative ZP values (−6 to −20 mV) whatever the pH in the range of 3 to 10, in line with data reported for DOPC liposomes (<xref rid=\"B9\" ref-type=\"bibr\">Chibowski and Szcześ, 2016</xref>). These results support the presence of DOPC lipid layers onto the nanoMOFs which shield their charged surface moieties. Interestingly, when the nanoMOFs were surface-functionalized with PEG chains, their ZP values were shifted to neutral (–1.6 ± 3.4 mV). This is in good agreement with other studies on PEG-coated NPs (<xref rid=\"B13\" ref-type=\"bibr\">Gref et al., 1995</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Thevenot et al., 2007</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Troutier and Ladavière, 2007</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Troutier-Thuilliez et al., 2009</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Bugnicourt et al., 2019</xref>).</p></sec><sec id=\"S3.SS2\"><title>Effect of the Coatings on the Colloidal Stability of NanoMOFs in Biological Media</title><p>As the majority of uncoated NPs, nanoMOFs suffer from poor stability in biological media, which hampers their biomedical applications. <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref> clearly shows that uncoated nanoMOFs undergo a fast aggregation in both phosphate buffer saline (PBS, pH = 7.4, 10 mM) and cell culture medium DMEM (Dulbecco’s Modified Eagle Medium) without fetal bovine serum (FBS), with the mean hydrodynamic diameters rapidly increasing to more than 1 μm within only 6 h at 37°C (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). No significant variation was observed for the mean hydrodynamic diameter of uncoated nanoMOFs in water in the first 1 h, however, they tended to aggregate upon storage (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S3</xref>). They were stable only in DMEM supplemented with 10% (v/v) FBS, possibly due to the formation of a protein corona at their surface preventing their aggregation (see section “Effect of surface functionalization of nanoMOFs on protein adsorption and macrophage uptake”).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Colloidal stability of nanoMOFs in different media, before <bold>(A)</bold> and after surface functionalization with DOPC <bold>(B)</bold> or PEG-lipid conjugates <bold>(C)</bold>. Mean hydrodynamic diameters of nanoMOF suspensions at 100 μg/mL were determined by DLS after 6 h incubation at 37°C. (Black: water; red: PBS; blue: DMEM supplemented with 10v/v% FBS; pink: DMEM without FBS).</p></caption><graphic xlink:href=\"fbioe-08-01027-g004\"/></fig><p>In contrast, DOPC coated nanoMOFs were stable both in water and DMEM. No aggregation was observed even after 3 weeks storage. However, they still underwent aggregation in PBS (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Remarkably, PEGylation allowed circumventing stability issues, whatever the suspension media (less than 10% diameter variation in PBS).</p><p>As all the coated and uncoated nanoMOFs were stable in DMEM supplemented with 10% (v/v) FBS, it was possible to explore further their cytotoxicity and interactions with cancer cell lines and macrophages. The PEGylated nanoMOFs exhibited excellent colloidal stability in all the tested biological media and thus appeared as optimal candidates for biological applications.</p></sec><sec id=\"S3.SS3\"><title>Control of Degradation and Drug Release by Lipid Coating</title><p>There is a general agreement on the fact that once the nanomaterials release their drug cargo, they should degrade to avoid accumulation inside the body (<xref rid=\"B19\" ref-type=\"bibr\">Horcajada et al., 2012</xref>). However, Fe-based nanoMOFs are reported to degrade rapidly in the biological media, because of coordination of various ions (phosphates, sulfates, etc.) to their iron sites, sometimes leading to uncontrolled “burst” drug release (<xref rid=\"B1\" ref-type=\"bibr\">Agostoni et al., 2013</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Li et al., 2017</xref>, <xref rid=\"B22\" ref-type=\"bibr\">2019b</xref>). It was therefore interesting to investigate if the hydrophobic lipid coatings could interfere with the rapid penetration of the aqueous degrading media inside the pores, thus allowing gaining better control upon the release and degradation mechanisms.</p><p>Degradation of nanoMOFs is generally monitored by the release of the constituting ligand trimesate (<xref rid=\"B1\" ref-type=\"bibr\">Agostoni et al., 2013</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Rodriguez-Ruiz et al., 2015</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Li et al., 2017</xref>). The degradation of the lipid-coated or bare nanoMOFs was studied by assaying ligand trimesate by HPLC in PBS (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>). In PBS 1 mM, uncoated nanoMOFs (blue curve, <xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>) underwent a fast degradation in the first 1 h at 37°C with around 15.5 ± 1.1% trimesate released, in agreement with previous reports (<xref rid=\"B20\" ref-type=\"bibr\">Li et al., 2017</xref>). It was discovered that in the same conditions, the lipid-coated nanoMOFs, with (red curve, <xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>) or without PEG-lipids conjugates (green curve, <xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>), exhibited much slower degradation profiles than the uncoated nanoMOFs, with only 10 ± 0.2% trimesate release in the first 1 h. This suggests a more progressive diffusion of the phosphate ions into the coated nanoMOFs, slowing down their degradation. However, the same plateau was reached after 24 h incubation, corresponding to a total complexation of the phosphates in the medium (<xref rid=\"B20\" ref-type=\"bibr\">Li et al., 2017</xref>). In conclusion, the shell efficiently delayed the degradation process.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Effect of surface modification on Gem-MP and trimesate release analyzed by HPLC. <bold>(A)</bold> Trimesate release in 1 mM PBS from nanoMOFs before (blue) and after lipid coating with (red) or without (green) coating with PEG-lipid conjugates after incubation at 37°C. <bold>(B)</bold> Gem-MP release from uncoated nanoMOFs in PBS with different molarities (orange: 6 mM, blue: 3 mM; dark blue: 1 mM). Gem-MP release in 1 mM <bold>(C)</bold> or 3 mM <bold>(D)</bold> PBS from nanoMOFs before (blue/dark blue) and after lipid coating with (red) or without (green) coating with PEG-lipid conjugates. In all cases <bold>(B–D)</bold>, phosphate concentration was adjusted to 10 mM after 72 h incubation at 37°C (red arrows), followed by further incubation for 4 h at 37°C.</p></caption><graphic xlink:href=\"fbioe-08-01027-g005\"/></fig><p>Then, the effect of lipid coatings on drug release was studied. Selected drug of interest was Gem-MP, a hydrophilic drug with low cell permeability. NanoMOFs acted as efficient “nanosponges”, soaking Gem-MP from their aqueous solution with almost perfect efficiency (>98%). Maximal loadings reached 25 wt% reflecting the strong interaction between the drug and the iron trimesate matrices. Advantageously, the lipid coating process didn’t induce any significant drug release (less than 3% variations before and after coating).</p><p>Gem-MP release is governed by a competition of coordination between the phosphate moieties in Gem-MP and free phosphates in PBS for the iron(III) Lewis acids of nanoMOFs (<xref rid=\"B1\" ref-type=\"bibr\">Agostoni et al., 2013</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2015</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Rodriguez-Ruiz et al., 2015</xref>). As expected, it was found that the higher the amount of phosphates, the higher the amount of drug released (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>). At low phosphate concentrations (PBS 1 mM or 3 mM), a plateau (around 40% or 80% Gem-MP release) was reached in 24 h, when all the phosphate molecules present in the release medium were complexed to the iron sites, as previously reported (<xref rid=\"B1\" ref-type=\"bibr\">Agostoni et al., 2013</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2015</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Rodriguez-Ruiz et al., 2015</xref>). When additional phosphates were added in the release medium, all the drug still remaining in the nanoMOFs was immediately released (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>, arrow). Gem-MP release was well correlated with particle degradation, resulting in trimesate release (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S4</xref>).</p><p>The presence of the lipid coating reduced the drug release from the nanoMOFs (<xref ref-type=\"fig\" rid=\"F5\">Figure 5C</xref>). For instance, after 6 h incubation in PBS 1 mM, around 30% Gm-MP was released from the coated nanoMOFs, in comparison to 40% with the uncoated ones. This is possibly due to the restricted diffusion of phosphates into the nanoMOFs because of the lipid coating. Similarly, after 6 h incubation in PBS 3 mM, around 50% Gem-MP was released from the coated nanoMOFs, in comparison to 78% with the uncoated ones (<xref ref-type=\"fig\" rid=\"F5\">Figure 5D</xref>). The Gem-MP release from coated nanoMOFs gradually increased in a sustained manner in the following days (<xref ref-type=\"fig\" rid=\"F5\">Figures 5C,D</xref>). All the remained drugs could be released out after 4 h incubation in concentrated phosphate buffer (10 mM PBS).</p><p>Similar results were found with another drug, amoxicillin (Amox). The amount of Amox released in the first hour of incubation release was reduced by a factor of two in the case of lipid-coated nanoMOFs as compared to the naked ones (<xref ref-type=\"fig\" rid=\"F6\">Figure 6A</xref>), confirmed by different dilution factors at 10, 20, and 40 (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>). However, in the presence of strongly complexing phosphates, the degradation was only delayed, but not avoided (<xref ref-type=\"fig\" rid=\"F6\">Figure 6C</xref>).</p><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>Effect of coating on Amox release in water <bold>(A,B)</bold> and in PBS <bold>(C)</bold>. <bold>(A)</bold> Release kinetics of Amox in water from nanoMOFs (1 mg/mL) before or after coating, with a dilution factor of 20; <bold>(B)</bold> Effect of dilution factor on Amox release after 4 h incubation at 37°C in water (Blue: uncoated nanoMOFs; red: DOPC coated nanoMOFs; Green: DOPC and PEG-lipid conjugate coated nanoMOFs).</p></caption><graphic xlink:href=\"fbioe-08-01027-g006\"/></fig></sec><sec id=\"S3.SS4\"><title>Cytotoxicity Assays of nanoMOFs on Ovarian Cancer Cells</title><p>All the studied nanoMOFs were non-toxic for the SKOV3 ovarian cancer cells up to 100 μg/mL (<xref ref-type=\"fig\" rid=\"F7\">Figure 7A</xref>, blue histograms), with more than 98% cell viability in 24 h, which is in agreement with the previously reported lack of toxicity of these materials (<xref rid=\"B18\" ref-type=\"bibr\">Horcajada et al., 2010</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Baati et al., 2013</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Bellido et al., 2015</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Giménez-marqués et al., 2018</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Li et al., 2019b</xref>). In contrast, as expected, the anticancer drug Gem-MP (20 μg/mL, <xref ref-type=\"fig\" rid=\"F7\">Figure 7A</xref>, brown histograms) exerted a cytotoxic effect with 45% cell viability after 48 h incubation, which further diminished to 29% in 72 h. Remarkably, Gem-MP loaded nanoMOFs showed a strong <italic>in vitro</italic> activity on SKOV3 ovarian cancer cells, higher than the free drug (<xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref>). At equivalent Gem-MP concentrations, whatever the drug loading (8 or 20 wt%) and the amount of nanoMOF in contact with the cells (10 to 100 μg/mL), the drug-loaded nanoMOFs outperformed the free drug in terms of toxicity on cancer cells (<xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure S5</xref>).</p><fig id=\"F7\" position=\"float\"><label>FIGURE 7</label><caption><p>Cytotoxcity measured by MTT assays of nanoMOFs (blue), anticancer efficacy of Gem-MP (brown), Gem-MP loaded nanoMOFs before (gray) and after coating with DOPC (red) or PEG-lipid conjugates (green). The experiments were performed on SKOV3 ovarian cancer cells, at different incubation times, with different concentrations nanoMOFs. <bold>(A)</bold> 100 μg/mL; <bold>(B)</bold> 30 μg/mL. Gem-MP loading was 20 wt%.</p></caption><graphic xlink:href=\"fbioe-08-01027-g007\"/></fig><p>This is in line with previous studies showing the efficient internalization of nanoMOFs bearing or not a lipid coating in pancreatic, breast, or bladder cancer cell lines (<xref rid=\"B24\" ref-type=\"bibr\">Rodriguez-Ruiz et al., 2015</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Wuttke et al., 2015</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Li et al., 2019a</xref>). It was recently shown that nanoMOFs acted as “Trojan horses” internalized by cancer cells, carrying their Gem-MP cargo to interfere with DNA (<xref rid=\"B21\" ref-type=\"bibr\">Li et al., 2019a</xref>). In this study it was shown that interestingly, the presence of a lipid coating (PEGylated or not) did not reduce the nanoMOF anticancer efficacy on SKOV3 ovarian cancer cells.</p></sec><sec id=\"S3.SS5\"><title>Effect of Surface Functionalization of NanoMOFs on Protein Adsorption and Macrophage Uptake</title><p>It is well known that intravenously administered NPs are readily covered by plasma proteins, creating the so-called “protein corona”, which plays a crucial role on the NPs’ biodistribution and <italic>in vivo</italic> fate (<xref rid=\"B14\" ref-type=\"bibr\">Gref et al., 2000</xref>). To gain insight on the influence of lipid coating of nanoMOFs on protein adsorption, HSA (human serum albumin), the most abundant protein in human blood plasma, was selected for this study.</p><p>NanoMOFs coated or not with DOPC lipids and PEG-lipid conjugates were incubated for 4 h at 37°C with HSA. After separation of the supernatants by centrifugation, the amount of non-adsorbed HSA was quantified using a BCA titration in order to determine the adsorbed HSA amounts onto nanoMOFs, lipid-modified or not. These amounts, expressed as μg/mg of nanoMOFs, are reported in <xref ref-type=\"fig\" rid=\"F8\">Figure 8A</xref>. In the case of uncoated nanoMOFs (<xref ref-type=\"fig\" rid=\"F8\">Figure 8A</xref>, blue curve), the amount of adsorbed HSA reached a plateau within 6 h, with around 50 μg HSA/mg nanoMOFs. Interestingly, lipid coating dramatically reduced HSA adsorption, to only ∼5 μg HSA/mg nanoMOFs (<xref ref-type=\"fig\" rid=\"F8\">Figure 8A</xref>, green curve), regardless of the addition of DSPE-PEG 2000 (<xref ref-type=\"fig\" rid=\"F8\">Figure 8A</xref>, red curve). To the best of our knowledge, these adsorbed HSA amounts are among the lowest reported with MIL-100 (Fe) nanoMOFs (<xref rid=\"B14\" ref-type=\"bibr\">Gref et al., 2000</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Cutrone et al., 2019b</xref>), suggesting that lipid-based coating on nanoMOFs is efficient to avoid albumin adsorption.</p><fig id=\"F8\" position=\"float\"><label>FIGURE 8</label><caption><p>HSA adsorption assayed by BCA assay <bold>(A)</bold> and J774 murine macrophage uptake <bold>(B)</bold> of nanoMOFs before and after lipid surface functionalization. <bold>(A)</bold> HSA adsorption by nanoMOFs before (blue) and after lipid coating with (red) or without (green) the addition of DSPE-PEG 2000. <bold>(B)</bold> NanoMOFs (before and after lipid coating, with or without addition of DSPE-PEG 2000) internalization inside murine macrophage J774 cells. 50 μg/mL nanoMOFs were incubated with 3 × 10<sup>5</sup> J774 cells for 4 h, and the amount of internalized nanoMOFs was determined by ICP-MS and expressed as a% of the initial nanoMOF amount in contact with the cells. Significant difference was observed for nanoMOFs before and after surface modification (<italic>p</italic> < 0.01).</p></caption><graphic xlink:href=\"fbioe-08-01027-g008\"/></fig><p>The potential “stealth” effect of the lipid-coated nanoMOFs, PEGylated or not, was evaluated on the murine macrophage cell line J774. Quantitative data on the amounts of nanoMOFs internalized by cells were obtained by ICP-MS, after extensive washing to remove the non-associated particles. An incubation time of 4 h was chosen as it corresponds to the typical blood circulation time of PEG-coated NPs (<xref rid=\"B11\" ref-type=\"bibr\">Cutrone et al., 2019b</xref>). Interestingly, the DOPC coating of nanoMOFs reduced their macrophage uptake by a factor of 2.4, from 75 ± 6% to 31 ± 3% (<xref ref-type=\"fig\" rid=\"F8\">Figure 8B</xref>). The nanoMOF functionalization with PEG chains was even more effective, reducing their interactions with macrophage to 21 ± 2%. Despite these great <italic>in-vitro</italic> results, it is widely known that numerous complex interactions can occur after NP administration in multicellular organisms. Therefore, <italic>in vivo</italic> studies need to be carried on to demonstrate the efficacy of the PEG coating to reduce reticuloendothelial system (RES) uptake.</p><p>Nevertheless, it has to be noted that, in similar experimental conditions, other coating materials showed higher interactions with macrophages, for instance, 41 ± 3% for CD-P coating, and 39 ∼ 24% for comb-like copolymers (<xref rid=\"B10\" ref-type=\"bibr\">Cutrone et al., 2019a</xref>, <xref rid=\"B11\" ref-type=\"bibr\">b</xref>). The advantage of lipid coating, demonstrated in this work, is a straightforward method, leading to efficient and stable coatings based on already FDA-approved materials. Lipid coatings on NPs are considered to be a promising strategy for the treatment of severe pathologies such as cancer (<xref rid=\"B23\" ref-type=\"bibr\">Luchini and Vitiello, 2019</xref>). In this study, the lipid coating not only afforded a control upon cell interaction but also provided a biocompatible protective barrier, modulating drug release and nanoMOF degradation. Of note, the nanoMOFs used in this study were shown to be biocompatible after intravenous administration in rats (<xref rid=\"B4\" ref-type=\"bibr\">Baati et al., 2013</xref>). However, the biocompatibility of the supermolecules assembled resulting from nanoMOFs coating with lipids has to be demonstrated <italic>in vivo</italic>.</p></sec></sec><sec id=\"S4\"><title>Conclusion</title><p>The surface of iron trimesate nanoMOFs was successfully modified with FDA approved DSPE-PEG 2000 in combination with DOPC by a fast solvent-exchange deposition method. We described herein the preparation and comprehensive characterization of the lipid modified NPs. We showed, for the first time, that the lipid surface modification of porous nanoMOFs reduced their tendency to degrade rapidly in PBS. Moreover, the coating of nanoMOFs with PEG-lipid conjugates successfully decreased their uptake by macrophages <italic>in vitro</italic> by a factor of 3.6. Finally, nanoMOFs acted as “Trojan horses” internalizing inside the cancer cells, and carrying their Gem-MP cargo to interfere with DNA.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>RG conceived the study. RG, XL, and GS designed the experiments. XL, GS, and JQ preformed the experiments. CL contributed to the lipid investigations. XL and RG wrote the manuscript. MM, KB, and TT contributed to the biological evaluations. All authors approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> Financial support for this work was provided the French National Research Agency (ANR-14-CE08-0017 and ANR-16-CE18-0018) and Euronanomed III (project PCInano). This work was also supported by a public grant overseen by the French National Research Agency as part of the “Investissements d’Avenir” program (Labex NanoSaclay, ANR-10-LABX-0035).</p></fn></fn-group><ack><p>We acknowledge Dr. B. Moreira-Alvarez and Dr. J. R. Encinar for their kind help with ICP-MS analysis. We also thank Dr. D. Constantin for help with XRPD experiments.</p></ack><sec id=\"S9\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fbioe.2020.01027/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fbioe.2020.01027/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Agostoni</surname><given-names>V.</given-names></name><name><surname>Chalati</surname><given-names>T.</given-names></name><name><surname>Horcajada</surname><given-names>P.</given-names></name><name><surname>Willaime</surname><given-names>H.</given-names></name><name><surname>Anand</surname><given-names>R.</given-names></name><name><surname>Semiramoth</surname><given-names>N.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Towards an improved anti-HIV activity of NRTI via Metal-Organic Frameworks nanoparticles.</article-title>\n<source><italic>Adv. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">J Clin Rheumatol</journal-id><journal-id journal-id-type=\"iso-abbrev\">J Clin Rheumatol</journal-id><journal-id journal-id-type=\"publisher-id\">RHU</journal-id><journal-title-group><journal-title>Journal of Clinical Rheumatology</journal-title></journal-title-group><issn pub-type=\"ppub\">1076-1608</issn><issn pub-type=\"epub\">1536-7355</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">31192860</article-id><article-id pub-id-type=\"pmc\">PMC7523572</article-id><article-id pub-id-type=\"publisher-id\">RHU50868</article-id><article-id pub-id-type=\"doi\">10.1097/RHU.0000000000001121</article-id><article-id pub-id-type=\"art-access-id\">00035</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Images</subject></subj-group></article-categories><title-group><article-title>Multiarticular Deforming and Erosive Tophaceous Gout With Severe Comorbidities</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Anghelescu</surname><given-names>Aurelian</given-names></name><degrees>MD, PhD</degrees></contrib></contrib-group><aff>From the Neurorehabilitation Clinic, Teaching Emergency Hospital “Bagdasar-Arseni,” Bucharest, Romania.</aff><author-notes><corresp id=\"corr1\">Correspondence: Aurelian Anghelescu, MD, PhD, “Carol Davila” University of Medicine and Pharmacy, Eroilor Sanitari Boulevard, No. 8, 5th Sector, Bucharest 050471, Romania. E-mail: <email>aurelian_anghelescu@yahoo.co.uk</email>.</corresp></author-notes><pub-date pub-type=\"ppub\"><month>10</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>13</day><month>6</month><year>2019</year></pub-date><volume>26</volume><issue>7</issue><fpage>e269</fpage><lpage>e271</lpage><permissions><copyright-statement>Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2019</copyright-year><copyright-holder>Wolters Kluwer Health, Inc. All rights reserved.</copyright-holder><license license-type=\"open-access\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">Creative Commons Attribution License 4.0 (CCBY)</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"rhu-26-e269.pdf\"/><custom-meta-group><custom-meta><meta-name>STATUS</meta-name><meta-value>ONLINE-ONLY</meta-value></custom-meta><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><p>Gout is an increasingly common chronic metabolic disorder, resulting from the inflammatory responses to monosodium urate crystals deposited in tissues.</p><p>Tophaceous gout can occur years after recurrent attacks of acute inflammatory arthropathies. Microscopically, the tophus (the cardinal feature of advanced gout) is a foreign body, granuloma-like structure that contains lumps of monosodium urate crystals and is surrounded by inflammatory cells and connective tissue.<sup><xref rid=\"bib1\" ref-type=\"bibr\">1</xref></sup></p><p>Multiarticular chronic tophaceous gout causes progressive joint damage and the development of multiple, severe destructive ulcerations and reduces the quality of life.</p><p>Although a few cases of erosive polyarticular tophaceous gout with severe osteolysis that require digital amputation have been identified in the literature, none of these have demonstrated extensive bone destruction.</p><p>A 57-year-old white man with chronic tophaceous multiarticular gout was referred to our neurorehabilitation clinic.</p><p>He presented with tophi on the right pinna and left knee, “white bumps” on the left elbow, multiple ulcerated tophi on his feet and hands, and necrotic lesions (distal right thumb and the distal phalanx of the little finger of the right hand). He was recently treated with vancomycin for a methicillin-resistant <italic>Staphylococcus aureus</italic> infection.</p><p>He had a medical history of heavy smoking, longstanding and suboptimally treated gout (since 2004), previous partial amputation of the fourth right finger (2015), arterial hypertension (since 2012), renal lithiasis (since 2014), moderate kidney disease, myocardial infarction and coronary stent (2012), left sylvian ischemic stroke, and right hemiplegia and aphasia (in December 2018).</p><p>Local physical and radiologic examination shows multiarticular deformities and deviations of the fingers and toes, soft-tissue involvement (multiple tophi, erosive and necrotic lesions; Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>, Fig. <xref ref-type=\"fig\" rid=\"F2\">2</xref>).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1</label><caption><p>A and B, Left hand. C and D, Right hand. E, Left foot. F and G, Right foot. H, Left elbow. I, Right pinna.</p></caption><graphic xlink:href=\"rhu-26-e269-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2</label><caption><p>A, Anteroposterior radiograph of the hands: calcified tophus (arrow), suggesting renal impairment. Multiple bilateral interphalangeal articular erosions, adjacent to tophus (asterisk). B, Anteroposterior radiograph of the feet: multiple bilateral interphalangeal (asterisk) and metatarsophalangeal joints erosions (left-right arrow).</p></caption><graphic xlink:href=\"rhu-26-e269-g002\"/></fig><p>Laboratory testing revealed uric acid (UA) 7.5 mg/dL (446 μmol/L), erythrocyte sedimentation rate 130 mm/h, white blood cells 6300/μL, 65.6% neutrophils, plasma fibrinogen 854 mg/dL, C-reactive protein 20.2 mg/L, and serum creatinine 1.70 mg/dL (glomerular filtration rate 44 mL/min per 1.73m<sup>2</sup>).</p><p>Patient's participation in the rehabilitation program was limited by his dysfunctional medical comorbidities and neurologic status.</p><p>Lifetime diet, long-term colchicine 0.5 mg twice daily and allopurinol 300 mg twice daily, neurotrophic factors, antihypertensive, antiplatelet therapy, and follow-up were indicated at discharge.</p><p>Gout represents a risk factor for multiple complications, including chronic kidney disease, diabetes mellitus, hypertension, dyslipidemia, and increased mortality.<sup><xref rid=\"bib1\" ref-type=\"bibr\">1</xref></sup></p><p>Gout is a risk factor for cardiocerebrovascular disease, compared with diabetes for incident ischemic stroke.<sup><xref rid=\"bib2\" ref-type=\"bibr\">2</xref></sup></p><p>Although UA is the primary endogenous antioxidant and free radical scavenger in the blood and is potentially neuroprotective during acute ischemic stroke, patients with cerebral ischemia and high UA levels (>380 μmol/L) or low UA levels (≤250 μmol/L) are 2 to 3 times more likely to have a poor outcomes than those with normal UA levels (250–380 μmol/L).<sup><xref rid=\"bib3\" ref-type=\"bibr\">3</xref></sup></p><p>The current standard of care for gout patients with accumulated comorbidities and chronic health problems requires a multidisciplinary/interdisciplinary team management approach.</p><p>Due to the favorable pharmaceutical control of hyperuricemia, the surgical treatment of tophaceous ulcerated gout is seldom necessary and is indicated only in severe cases that present with joint destruction, deformities, and infection.<sup><xref rid=\"bib4\" ref-type=\"bibr\">4</xref></sup></p><p>Gout management requires long-term, urate-lowering therapy to achieve a serum urate concentration of less than 5 mg/dL (300 μmol/L)<sup><xref rid=\"bib1\" ref-type=\"bibr\">1</xref></sup></p><p>Allopurinol, in combination with aspirin and lipid-lowering therapy, may improve endothelial function and inflammation and may reduce the occurrence of disabilities in patients with stroke.<sup><xref rid=\"bib5\" ref-type=\"bibr\">5</xref></sup></p></body><back><fn-group><fn fn-type=\"COI-statement\"><p>The author declares no conflict of interest.</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id=\"bib1\"><label>1</label><mixed-citation publication-type=\"journal\"><person-group><name><surname>Chhana</surname><given-names>A</given-names></name><name><surname>Dalbeth</surname><given-names>N</given-names></name></person-group>\n<article-title>The gouty tophus: a review</article-title>. <source><italic toggle=\"yes\">Curr Rheumatol Rep</italic></source>. <year>2015</year>;<volume>17</volume>:<fpage>19</fpage>.<pub-id pub-id-type=\"pmid\">25761926</pub-id></mixed-citation></ref><ref id=\"bib2\"><label>2</label><mixed-citation publication-type=\"journal\"><person-group><name><surname>Singh</surname><given-names>JA</given-names></name><name><surname>Ramachandaran</surname><given-names>R</given-names></name><name><surname>Yu</surname><given-names>S</given-names></name>, <etal/></person-group>\n<article-title>Is gout a risk equivalent to diabetes for stroke and myocardial infarction? A retrospective claims database study</article-title>. <source><italic toggle=\"yes\">Arthritis Res Ther</italic></source>. <year>2017</year>;<volume>19</volume>:<fpage>228</fpage>.<pub-id pub-id-type=\"pmid\">29041963</pub-id></mixed-citation></ref><ref id=\"bib3\"><label>3</label><mixed-citation publication-type=\"journal\"><person-group><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Huang</surname><given-names>ZC</given-names></name><name><surname>Lu</surname><given-names>TS</given-names></name>, <etal/></person-group>\n<article-title>Prognostic significance of uric acid levels in ischemic stroke patients</article-title>. <source><italic toggle=\"yes\">Neurotox Res</italic></source>. <year>2016</year>;<volume>29</volume>:<fpage>10</fpage>–<lpage>20</lpage>.<pub-id pub-id-type=\"pmid\">26376636</pub-id></mixed-citation></ref><ref id=\"bib4\"><label>4</label><mixed-citation publication-type=\"journal\"><person-group><name><surname>Falidas</surname><given-names>E</given-names></name><name><surname>Rallis</surname><given-names>E</given-names></name><name><surname>Bournia</surname><given-names>VK</given-names></name>, <etal/></person-group>\n<article-title>Multiarticular chronic tophaceous gout with severe and multiple ulcerations: a case report</article-title>. <source><italic toggle=\"yes\">J Med Case Rep</italic></source>. <year>2011</year>;<volume>5</volume>:<fpage>397</fpage>.<pub-id pub-id-type=\"pmid\">21854566</pub-id></mixed-citation></ref><ref id=\"bib5\"><label>5</label><mixed-citation publication-type=\"journal\"><person-group><name><surname>Britnell</surname><given-names>SR</given-names></name><name><surname>Chillari</surname><given-names>KA</given-names></name><name><surname>Brown</surname><given-names>JN</given-names></name></person-group>\n<article-title>The role of xanthine oxidase inhibitors in patients with history of stroke: a systematic review</article-title>. <source><italic toggle=\"yes\">Curr Vasc Pharmacol</italic></source>. <year>2018</year>;<volume>16</volume>:<fpage>583</fpage>–<lpage>588</lpage>.<pub-id pub-id-type=\"pmid\">28933307</pub-id></mixed-citation></ref></ref-list></back></article>\n"
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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Stroke</journal-id><journal-id journal-id-type=\"iso-abbrev\">Stroke</journal-id><journal-id journal-id-type=\"publisher-id\">STR</journal-id><journal-title-group><journal-title>Stroke</journal-title></journal-title-group><issn pub-type=\"ppub\">0039-2499</issn><issn pub-type=\"epub\">1524-4628</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32933420</article-id><article-id pub-id-type=\"pmc\">PMC7523579</article-id><article-id pub-id-type=\"art-access-id\">00011</article-id><article-id pub-id-type=\"doi\">10.1161/STROKEAHA.120.030208</article-id><article-categories><subj-group subj-group-type=\"hwp-journal-coll\"><subject>10152</subject><subject>10155</subject><subject>10162</subject><subject>10178</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Original Contributions</subject><subj-group><subject>Clinical and Population Sciences</subject></subj-group></subj-group></article-categories><title-group><article-title>Effect of Pre- and In-Hospital Delay on Reperfusion in Acute Ischemic Stroke Mechanical Thrombectomy</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Kaesmacher</surname><given-names>Johannes</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref ref-type=\"aff\" rid=\"aff2\">2</xref><xref ref-type=\"fn\" rid=\"fn01\">*</xref></contrib><contrib contrib-type=\"author\"><name><surname>Maamari</surname><given-names>Basel</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff3\">3</xref><xref ref-type=\"fn\" rid=\"fn01\">*</xref></contrib><contrib contrib-type=\"author\"><name><surname>Meinel</surname><given-names>Thomas R.</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Piechowiak</surname><given-names>Eike I.</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mosimann</surname><given-names>Pascal J.</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mordasini</surname><given-names>Pasquale</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Goeldlin</surname><given-names>Martina</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Arnold</surname><given-names>Marcel</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dobrocky</surname><given-names>Tomas</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Boeckh-Behrens</surname><given-names>Tobias</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Berndt</surname><given-names>Maria</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Michel</surname><given-names>Patrik</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Requena</surname><given-names>Manuel</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Benali</surname><given-names>Amel</given-names></name><degrees>MSc</degrees><xref ref-type=\"aff\" rid=\"aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Pierot</surname><given-names>Laurent</given-names></name><degrees>MD, PhD</degrees><xref ref-type=\"aff\" rid=\"aff8\">8</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mendes Pereira</surname><given-names>Vitor</given-names></name><degrees>MD, PhD</degrees><xref ref-type=\"aff\" rid=\"aff9\">9</xref></contrib><contrib contrib-type=\"author\"><name><surname>Boulouis</surname><given-names>Grégoire</given-names></name><degrees>MD, MSc</degrees><xref ref-type=\"aff\" rid=\"aff10\">10</xref></contrib><contrib contrib-type=\"author\"><name><surname>Brehm</surname><given-names>Alex</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff11\">11</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sporns</surname><given-names>Peter B.</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff11\">11</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ospel</surname><given-names>Johanna M.</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff12\">12</xref><xref ref-type=\"aff\" rid=\"aff13\">13</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gralla</surname><given-names>Jan</given-names></name><degrees>MD, MSc</degrees><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref ref-type=\"fn\" rid=\"fn02\">†</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Fischer</surname><given-names>Urs</given-names></name><degrees>MD, MSc</degrees><xref ref-type=\"aff\" rid=\"aff3\">3</xref><xref ref-type=\"fn\" rid=\"fn02\">†</xref></contrib><on-behalf-of>on behalf of the BEYOND-SWIFT Investigators</on-behalf-of><aff id=\"aff1\"><label>1</label>University Institute of Diagnostic and Interventional Neuroradiology (J.K., E.I.P., P.J.M., P. Mordasini, T.D., J.G.), University Hospital Bern, Inselspital, University of Bern, Switzerland.</aff><aff id=\"aff2\"><label>2</label>University Institute of Diagnostic and Interventional and Pediatric Radiology (J.K.), University Hospital Bern, Inselspital, University of Bern, Switzerland.</aff><aff id=\"aff3\"><label>3</label>Department of Neurology (B.M., T.R.M., M.G., M.A., U.F.), University Hospital Bern, Inselspital, University of Bern, Switzerland.</aff><aff id=\"aff4\"><label>4</label>Department of Diagnostic and Interventional Neuroradiology, Klinikum rechts der Isar, Technical University Munich, Germany (T.B.-B., M.B.).</aff><aff id=\"aff5\"><label>5</label>Department of Neurology, CHUV Lausanne, Switzerland (P. Michel).</aff><aff id=\"aff6\"><label>6</label>Department of Neurology, Vall d’Hebron University Hospital, Barcelona, Spain (M.R.).</aff><aff id=\"aff7\"><label>7</label>Department of Neuroradiology, CHU Montpellier, France (A. Benali).</aff><aff id=\"aff8\"><label>8</label>Department of Neuroradiology, CHU Reims, France (L.P.).</aff><aff id=\"aff9\"><label>9</label>Joint Department of Medical Imaging and Division of Neurosurgery, Toronto Western Hospital, University of Toronto, ON, Canada (V.M.P.).</aff><aff id=\"aff10\"><label>10</label>Department of Neuroradiology, Université Paris Descartes, Sainte Anne Hospital, France (G.B.).</aff><aff id=\"aff11\"><label>11</label>Department of Neuroradiology (A. Brehm, P.B.S.), University Hospital Basel, Switzerland.</aff><aff id=\"aff12\"><label>12</label>Department of Radiology (J.M.O.), University Hospital Basel, Switzerland.</aff><aff id=\"aff13\"><label>13</label>Department of Clinical Neuroscience, University of Calgary, Canada (J.M.O.).</aff></contrib-group><author-notes><corresp id=\"c1\">Correspondence to: Urs Fischer, MD, MSc, Department of Neurology, University of Bern, Freiburgstrasse 8, CH-3010, Switzerland, Email <email xlink:href=\"urs.fischer@insel.ch\">urs.fischer@insel.ch</email></corresp><corresp id=\"c2\">Johannes Kaesmacher, MD, University Institute of Diagnostic and Interventional Neuroradiology, University of Bern, Freiburgstrasse 8, CH-3010, Switzerland, Email <email xlink:href=\"johannes.kaesmacher@insel.ch\">johannes.kaesmacher@insel.ch</email></corresp></author-notes><pub-date pub-type=\"epub\"><day>16</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>10</month><year>2020</year></pub-date><volume>51</volume><issue>10</issue><fpage>2934</fpage><lpage>2942</lpage><history><date date-type=\"received\"><day>14</day><month>4</month><year>2020</year></date><date date-type=\"rev-recd\"><day>31</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>12</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 The Authors.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" specific-use=\"CC-BY\"><license-p><italic>Stroke</italic> is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the <ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\">Creative Commons Attribution</ext-link> License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"str-51-2934.pdf\"/><abstract abstract-type=\"toc\"><p>Supplemental Digital Content is available in the text.</p></abstract><abstract><sec><title>Background and Purpose:</title><p>Post hoc analyses of randomized controlled clinical trials evaluating mechanical thrombectomy have suggested that admission-to-groin-puncture (ATG) delays are associated with reduced reperfusion rates. Purpose of this analysis was to validate this association in a real-world cohort and to find associated factors and confounders for prolonged ATG intervals.</p></sec><sec><title>Methods:</title><p>Patients included into the BEYOND-SWIFT cohort (Bernese-European Registry for Ischemic Stroke Patients Treated Outside Current Guidelines With Neurothrombectomy Devices Using the Solitaire FR With the Intention for Thrombectomy; <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.clinicaltrials.gov\">https://www.clinicaltrials.gov</ext-link>; Unique identifier: NCT03496064) were analyzed (n=2386). Association between baseline characteristics and ATG was evaluated using mixed linear regression analysis. The effect of increasing symptom-onset-to-admission and ATG intervals on successful reperfusion (defined as Thrombolysis in Cerebral Infarction [TICI] 2b-3) was evaluated using logistic regression analysis adjusting for potential confounders.</p></sec><sec><title>Results:</title><p>Median ATG was 73 minutes. Prolonged ATG intervals were associated with the use of magnetic resonance imaging (+19.1 [95% CI, +9.1 to +29.1] minutes), general anesthesia (+12.1 [95% CI, +3.7 to +20.4] minutes), and borderline indication criteria, such as lower National Institutes of Health Stroke Scale, late presentations, or not meeting top-tier early time window eligibility criteria (+13.8 [95% CI, +6.1 to +21.6] minutes). There was a 13% relative odds reduction for TICI 2b-3 (adjusted odds ratio [aOR], 0.87 [95% CI, 0.79–0.96]) and TICI 2c/3 (aOR, 0.87 [95% CI, 0.79–0.95]) per hour ATG delay, while the reduction of TICI 2b-3 per hour increase symptom-onset-to-admission was minor (aOR, 0.97 [95% CI, 0.94–0.99]) and inconsistent regarding TICI 2c/3 (aOR, 0.99 [95% CI, 0.97–1.02]). After adjusting for identified factors associated with prolonged ATG intervals, the association of ATG delay and lower rates of TICI 2b-3 remained tangible (aOR, 0.87 [95% CI, 0.76–0.99]).</p></sec><sec><title>Conclusions:</title><p>There is a great potential to reduce ATG, and potential targets for improvement can be deduced from observational data. The association between in-hospital delay and reduced reperfusion rates is evident in real-world clinical data, underscoring the need to optimize in-hospital workflows. Given the only minor association between symptom-onset-to-admission intervals and reperfusion rates, the causal relationship of this association warrants further research.</p></sec><sec><title>Registration:</title><p>URL: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.clinicaltrials.gov\">https://www.clinicaltrials.gov</ext-link>. Unique identifier: NCT03496064.</p></sec></abstract><kwd-group><kwd>odds ratio</kwd><kwd>reperfusion</kwd><kwd>thrombectomy</kwd><kwd>workflow</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>SDC</meta-name><meta-value>T</meta-value></custom-meta><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><p>Achieving successful and ideally complete reperfusion is the most important modifiable predictor of good outcome in patients with acute ischemic stroke undergoing mechanical thrombectomy.<sup><xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R4\" ref-type=\"bibr\">4</xref></sup> A recent analysis of the HERMES (Highly Effective Reperfusion Evaluated in Multiple Endovascular Stroke Trials) collaboration suggested that the chances of achieving reperfusion decrease with prolonged admission-to-groin (ATG) intervals, with an estimated relative decrease in the odds of successful reperfusion of 22% per hour of in-hospital delay.<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref></sup></p><p>No significant effect on successful reperfusion was found for symptom-onset-to-groin-puncture (STG) intervals, however.<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref></sup> This discrepancy was attributed mostly to the strict inclusion criteria of the randomized controlled trials and uncertainties regarding the precision of the symptom onset, hence diluting a possibly significant effect of time elapsed from symptom onset.<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref></sup></p><p>In the current study, we aimed to quantify the effect of symptom-to-admission (STA), ATG, and STG intervals on rates of successful reperfusion in a real-world registry with less restrictive inclusion criteria and performed explorative analysis regarding potential confounders.</p><sec sec-type=\"methods\"><title>Methods</title><p>The data that support the findings of this study are available from the corresponding author upon reasonable request and after clearance by the local ethics committee.</p><sec><title>BEYOND-SWIFT Registry</title><p>The BEYOND-SWIFT (Bernese-European Registry for Ischemic Stroke Patients Treated Outside Current Guidelines With Neurothrombectomy Devices Using the Solitaire FR With the Intention for Thrombectomy) is an investigator-initiated, international, multicenter observational registry evaluating the outcome of mechanical thrombectomy patients (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.clinicaltrials.gov\">https://www.clinicaltrials.gov</ext-link>; Unique identifier: NCT03496064). Details of the registry have been published previously.<sup><xref rid=\"R6\" ref-type=\"bibr\">6</xref></sup> In short, 7 comprehensive stroke centers included patients treated with a Medtronic market-released neurothrombectomy device (applied as first-line device) for patients presenting with large vessel occlusion acute ischemic stroke. Table I in the <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.120.030208\">Data Supplement</ext-link> provides an overview of included patients and rates of available follow-up data for each center. Written informed consent was obtained from patients, or the institutional review board waived the need for patient consent, according to regulations at each center/country (Table I in the <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.120.030208\">Data Supplement</ext-link>). In Bern, ethical approval was given for pooling and analysis of the registry data (Kantonale Ethikkommission Bern, Bern, Switzerland; Local Ethics Committee Study Identifier: 2018-00766). For the present analysis, we also included 437 patients from another German center.<sup><xref rid=\"R7\" ref-type=\"bibr\">7</xref></sup></p><p>Of 2397 patients included into the registry, 2127 were treated for anterior circulation large vessel occlusion strokes. Of these, there were 1953 patients with full documentation of STA, ATG, and STG, and 1949 had available angiography runs to evaluate reperfusion success. Using the same selection criteria for the additional German center, of 547 patients, 437 were included.</p></sec><sec><title>Variables and Outcomes</title><p>Site of occlusion was classified into intracranial internal carotid artery, carotid T/L, and first/second segment of the middle cerebral artery. Reperfusion success was evaluated applying the extended Thrombolysis in Cerebral Infarction (TICI) scale with TICI 2b defined as ≥50% reperfusion of the initially hypoperfused target territory and TICI 2c defined as “near-complete perfusion except for slow flow in a few distal cortical vessels or presence of small distal cortical emboli.”<sup><xref rid=\"R8\" ref-type=\"bibr\">8</xref>,<xref rid=\"R9\" ref-type=\"bibr\">9</xref></sup> Reperfusion success was rated by an independent research fellow or was operator adjudicated depending on the centers’ standards. Alberta Stroke Program Early CT Scores (ASPECTS) were evaluated on noncontrast admission computed tomography images or diffusion-weighted imaging–based ASPECTS when patients underwent magnetic resonance imaging (MRI). Slow and fast progressors were dichotomized according to a median split of ASPECTS decay (estimated as ASPECTS regions infarcted/time from STA). Interventional characteristics were recorded including interventional technique, maneuver count, interventional complications, and time from groin puncture to reperfusion. Functional outcome was assessed at 3 months after the index event using the modified Rankin Scale (mRS), with mRS ≤2 defined as good functional outcome. Symptomatic intracranial hemorrhage was defined according to ECASS-II (European Co-Operative Acute Stroke Study-II) definition.<sup><xref rid=\"R10\" ref-type=\"bibr\">10</xref></sup> Primary outcome of this analysis was the rate of TICI 2b-3 with strata of ATG intervals. Secondary outcomes were the evaluation of a potential association between STA and STG and rates of TICI 2b-3. Explorative analyses were performed regarding rates of TICI 2c/3, rates of first-pass TICI 2c/3, periprocedural complications, utilizing >3 maneuvers, and occurrence of symptomatic intracranial hemorrhage with strata of ATG intervals.</p></sec><sec><title>Descriptive Statistics</title><p>Continuous variables are presented as mean±SD or median and interquartile range (IQR). Frequency counts are shown as percentage and n/N.</p></sec><sec><title>Logistic Regression Analysis</title><p>The effect of STA, ATG, and STG on TICI 2b-3 was evaluated using multivariate binary logistic regression analysis with STA/ATG included as continuous predictor variable and TICI 2b-3 as outcome variable. Mixed logistic regression analyses with random effects were omitted because preanalysis did not provide evidence for model superiority against simple logistic regression analysis (Table II in the <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.120.030208\">Data Supplement</ext-link>). In the first logistic regression model (model A), analyses were adjusted for age, sex, occlusion location (categorical variable: intracranial internal carotid artery/carotid T/L as reference versus M1 versus M2), and treatment with intravenous tPA (tissue-type plasminogen activator), as described previously.<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref></sup> Subgroup analyses were conducted for slow versus fast progressor in STA analyses and for computed tomography versus MRI in ATG analyses, utilizing interaction terms. Modeled predicted probabilities of TICI 2b-3 with increasing STA or ATG were displayed treating all other variables in the model at mean (continuous variables) or at balance (categorical variables). In model B, we incorporated all variables associated with ATG from the analysis outlined below together with stroke etiologic cause (according to the Trial of ORG 10172 in Acute Stroke Treatment criteria), interventional technique,<sup><xref rid=\"R11\" ref-type=\"bibr\">11</xref></sup> and year of patient treatment (see Table III in the <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.120.030208\">Data Supplement</ext-link> for a model overview). We also ran an additional sensitivity analysis including periprocedural complications and number of maneuvers into the model (model B*). All logistic regression models are adjusted for center. Outputs of logistic regression analyses are generally displayed as adjusted odds ratio (aOR) per 60-minute increase and corresponding 95% CIs.</p></sec><sec><title>Mixed Linear Regression Analysis</title><p>To find associations between baseline characteristics and prolonged ATG intervals, a mixed linear regression analysis was performed because a log-likelihood test revealed improved model fit as opposed to a simple linear regression model. Age, sex, direct admission versus transfer, functional dependence (mRS score >2) before the index event, admission National Institutes of Heath Stroke Scale (NIHSS) scores, STA, imaging modality, ASPECTS, intracranial occlusion, tandem occlusion, general anesthesia, intravenous tPA treatment, and year of treatment were included in the model. Alternatively, we implemented treatment eligibility according to early time window American Heart Association (AHA)/American Stroke Association (ASA) criteria, omitting variables included into eligibility criteria (age, admission NIHSS, etc). Early time window eligibility according to AHA/ASA criteria was defined as a compound criterion of meeting prestroke mRS score <2, internal carotid artery or M1 occlusion, age >18 years, NIHSS ≥6, ASPECTS ≥6, and time to groin ≤6 hours.<sup><xref rid=\"R12\" ref-type=\"bibr\">12</xref></sup> Center site was implemented as a random-effects variable.</p><p>Significance level was set to α=0.05. All tests are 2 sided. All analyses were conducted using STATA (v 15.1; Stata Corp, TX).</p></sec></sec><sec sec-type=\"results\"><title>Results</title><sec><title>Cohort</title><p>Two thousand three hundred eighty-six patients were included (median age, 74.7 years; IQR, 62.2–82.0; 51.2% women; Table <xref rid=\"T1\" ref-type=\"table\">1</xref>), of which 2008 (84.2%) were successfully reperfused (TICI 2b-3, including 54.4% with TICI 2c/3 reperfusions). Rates of successful reperfusion and TICI 2c/3 differed across centers (Figure I in the <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.120.030208\">Data Supplement</ext-link>) and by year of patient treatment (odds ratio per year increase after 2015, 1.16 [95% CI, 1.05–1.28]; Figure II in the <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.120.030208\">Data Supplement</ext-link>). Additionally, we observed an association between interventional technique applied and reperfusion success, with the highest rates of successful reperfusion or TICI 2c/3 observed in patients treated with stent retriever and a balloon guide catheter (Figure III in the <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.120.030208\">Data Supplement</ext-link>). Median STA delay was 150 minutes (IQR, 72–265 minutes), whereas median ATG interval was 73 minutes (IQR, 47–102 minutes). The relative percentage of ATG from STG was 33.0% (IQR, 16.4%–52.6%). Occlusion site was mostly M1 (57.2%, 1365 of 2386), followed by intracranial internal carotid artery/carotid T/L (26.2%, 625 of 2386) and M2 occlusions (16.6%, 396 of 2386).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1.</label><caption><p>Study Cohort</p></caption><graphic xlink:href=\"str-51-2934-g001\"/></table-wrap></sec><sec><title>Effect of STA and ATG on Successful Reperfusion</title><p>Overall, there was a small reduction in the likelihood of achieving TICI 2b-3 with increasing STA (aOR, 0.97 [95% CI, 0.94–0.99] per hour; Table <xref rid=\"T2\" ref-type=\"table\">2</xref>; Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>A). This association appeared stronger in fast progressors (aOR, 0.86 [95% CI, 0.79–0.95] per hour), without reaching statistical significance on interaction analysis (<italic>P</italic> for interaction, 0.081). With increasing ATG, there was a strong reduction in the rates of TICI 2b-3 (aOR, 0.87 [95% CI, 0.79–0.96] per hour; Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>C), corresponding to a 13% reduction in the odds of TICI 2b-3 per in-hospital hour delay. This association appeared more pronounced in patients undergoing MRI (aOR, 0.77 [95% CI, 0.65–0.91]), without reaching significance on interaction analysis (<italic>P</italic> for interaction, 0.102).</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2.</label><caption><p>Logistic Regression Analysis With STA/ATG as Predictor Variable and TICI 2b-3 as Outcome Variable</p></caption><graphic xlink:href=\"str-51-2934-g002\"/></table-wrap><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1.</label><caption><p><bold>Association of symptom-onset-to-admission (STA) and admission-to-groin-puncture (ATG) intervals with the probability of achieving Thrombolysis in Cerebral Infarction (TICI) 2b-3 or 2c/3.</bold> Adjusted predicted probabilities of TICI 2b-3 or 2c/3 according to STA, symptom-onset-to-groin puncture (STG), and ATG intervals in minutes (see Methods). <bold>A</bold>, A small association of increasing STA with decreasing odds of achieving TICI 2b-3 was found (adjusted odds ratio [aOR], 0.96 [95%, 0.94–0.99] per hour) while no statistically significant association between STA and the odds of achieving TICI 2c/3 was observed (aOR, 0.99 [95% CI, 0.97–1.02] per hour). <bold>B</bold>, A small association of increasing STG with decreasing odds of achieving TICI 2b-3 was found (aOR, 0.96 [95%, 0.94–0.99] per hour). <bold>C</bold>, With increasing ATG, there was a strong reduction in the rates of TICI 2b-3 (aOR, 0.87 [95% CI, 0.79–0.96] per hour), corresponding to a 13% reduction in the odds of TICI 2b-3 per in-hospital hour delay. This association was also stable when considering TICI 2c/3 as relevant end point (aOR, 0.87 [95% CI, 0.79–0.95] per hour).</p></caption><graphic xlink:href=\"str-51-2934-g003\"/></fig><p>For ATG, these association remained unchanged when considering TICI 2c/3 as relevant interventional end point (aOR, 0.87 [95% CI, 0.79–0.95]; Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>C), while no significant association of STA with rates of TICI 2c/3 was found (aOR, 0.99 [95% CI, 0.97–1.02]; Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>A).</p><p>For STG intervals, a significant association with TICI 2b-3 was found (Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>B), but this was partially attributed to the association of ATG intervals with TICI 2b-3 (see STG* in Table <xref rid=\"T2\" ref-type=\"table\">2</xref>).</p></sec><sec><title>Association of ATG With Secondary Interventional Outcomes</title><p>Every hour decrease in ATG was associated with reduced rates of first-pass TICI 2c/3 (aOR, 0.87 [95% CI, 0.77–0.98]). There was no statistically significant association of ATG with other interventional characteristics, including complications, rates of symptomatic intracranial hemorrhage, and utilization of >3 maneuvers (Figure <xref ref-type=\"fig\" rid=\"F2\">2</xref>).</p><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2.</label><caption><p><bold>Association of admission-to-groin-puncture (ATG) intervals with secondary procedural outcomes.</bold> A significant effect of ATG was found regarding rates of first-pass Thrombolysis in Cerebral Infarction (FP TICI) 2c/3 (adjusted odds ratio, 0.87 [95% CI, 0.77–0.98]), while no significant associations were observed for all other secondary outcomes. sICH indicates symptomatic intracranial hemorrhage.</p></caption><graphic xlink:href=\"str-51-2934-g004\"/></fig></sec><sec><title>Factors Associated With ATG</title><p>Of 2386 patients, 2240 were included in the random-effects linear regression analysis for identifying factors associated with ATG (Figure <xref ref-type=\"fig\" rid=\"F3\">3</xref>A and <xref ref-type=\"fig\" rid=\"F3\">3</xref>B). Transfer admissions (−28.1 [95% CI, −37.2 to −19.1] minutes), computed tomography versus MRI (−10.3 [95% CI, −21.4 to +0.8] minutes in the model without the compound AHA/ASA eligibility variable and −19.1 [95% CI, −29.1 to −9.1] minutes in the model with the compound AHA/ASA eligibility variable), higher admission NIHSS (−1.5 minutes per point increase [95% CI, −2.3 to −0.8 minutes]), and patient treatment in recent years (−15.3 minutes per year increase since 2015 [95% CI, −19.3 to −11.4 minutes]) were associated with shorter ATG intervals. In contrary, late presentation (+2.6 minutes per minute presentation delay [95% CI, +1.5 to +3.6 minutes]) and use of general anesthesia (+18.7 [95% CI, 8.4–29.0] minutes) were associated with longer ATGs. Implementing the AHA/ASA eligibility criteria to the model, conformance with the AHA/ASA early time window eligibility criteria was associated with shorter ATG intervals (−13.8 [95% CI, −21.6 to −6.1] minutes; Figure <xref ref-type=\"fig\" rid=\"F3\">3</xref>C).</p><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3.</label><caption><p><bold>Adjusted differences in admission-to-groin-puncture (ATG) intervals according to baseline and procedural variables.</bold>\n<bold>A</bold>, Categorical variables (effect scale, −60 to 60 min), patients receiving magnetic resonance imaging (MRI) or general anesthesia had increased ATG. <bold>B</bold>, Continuous variables (effect scale, −10 to 10 min), patients presenting late, patients with lower National Institutes of Health Stroke Scale (NIHSS), and patients treated in earlier years had increased ATG. <bold>C</bold>, Same model but functional dependence, age, Alberta Stroke Program Early CT Score (ASPECTS), admission NIHSS, and symptom-to-admission (STA) replaced by a compound variable of meeting American Heart Association (AHA)/American Stroke Association (ASA) guideline indication criteria. Patients meeting AHA/ASA guideline indication criteria on average had 14 min shorter ATG. CT indicates computed tomography; IV tPA, intravenous tissue-type plasminogen activator; M1, first segment of the middle cerebral artery; and M2, second segment of the middle cerebral artery. *Year of treatment implemented as continuous variable as per year increase since 2015.</p></caption><graphic xlink:href=\"str-51-2934-g005\"/></fig></sec><sec><title>Sensitivity Analyses Utilizing Refined Models for the Effect of ATG</title><p>We included 1822 of 2386 patients in the refined model B. When incorporating factors associated with prolonged ATG intervals, together with adjustment for stroke etiology, year of patient inclusion, and interventional technique, the effect of ATG on rates of TICI 2b-3 could still be detected, yielding a 13% relative decrease in the odds of TICI 2b-3 per hour of ATG delay (aOR, 0.87 [95% CI, 0.76–0.99]; Figure <xref ref-type=\"fig\" rid=\"F4\">4</xref>). Additionally implementing the number of maneuvers and periprocedural complications as covariates did not significantly change this association (model B*; aOR, 0.74 [95% CI, 0.59–0.92]).</p><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Figure 4.</label><caption><p><bold>Association of admission-to-groin-puncture (ATG) intervals with probability of Thrombolysis in Cerebral Infarction (TICI) 2b-3 in various models.</bold>\n<bold>A</bold>, Adjusted predicted probabilities of TICI 2b-3 with respect to increasing ATG intervals using model A (corresponding to the model used by Bourcier et al). <bold>B</bold>, Adjusted predicted probabilities of TICI 2b-3 applying a refined logistic regression model B, additionally adjusting for factors associated with increased ATG, stroke etiologic cause, interventional technique, and year of treatment (model B).</p></caption><graphic xlink:href=\"str-51-2934-g006\"/></fig></sec><sec><title>Clinical Outcomes With Respect to Admission to Reperfusion Intervals</title><p>There was a 19% relative odds reduction in achieving mRS score of 0 to 2 for every hour in-hospital delay (aOR, 0.81 [95% CI, 0.73–0.90]). There was no interaction regarding this effect and occlusion site (<italic>P</italic>=0.64) or AHA/ASA guideline eligibility (<italic>P</italic>=0.51). This effect was less pronounced when considering the time elapsed from symptom onset (aOR per hour delay, 0.96 [95% CI, 0.93–0.99]).</p></sec></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>This registry analysis has the following main findings: (1) the association of a reduced rate of TICI 2b-3 with increasing ATG is evident in real-world clinical data with a comparable effect size to what has been reported from randomized controlled trial data; (2) in contrast, the association between STA and TICI 2b-3 appears considerably weaker and is not consistent for other interventional end points (eg, TICI 2c/3); (3) patients with borderline indications not meeting early time window AHA/ASA guideline criteria were more likely to have prolonged ATG intervals, probably reflecting difficulties in treatment decision-making, which may also relate to pursuing the goal of TICI 2b-3 less rigorously; (4) even after correcting for such factors and other confounders (interventional technique, year of patient treatment) associated with prolonged ATG intervals, the effect of ATG on TICI 2b-3 remained statistically robust; (5) in high-volume centers ATG intervals are quiet long, and associated factors like the use of MRI or general anesthesia, as well as patient characteristics associated with delays, have been identified as potential targets for improvement programs.</p><p>Achieving successful reperfusion remains the most important modifiable predictor of outcome in patients presenting with acute ischemic stroke due to large vessel occlusion. Accordingly, identifying factors associated with decreased rates of successful reperfusion is important. In our study, each hour of ATG delay was associated with a relative decrease of 13% in the odds of TICI 2b-3, comparing slightly lower than the 22% odds reduction published by the HERMES collaboration.<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref></sup> Our findings support that this association is not only true for selected randomized controlled trial patients but also tangible in a real-world cohort of patients with less restrictive inclusion criteria. We found a weak association between STA and rates of TICI 2b-3 and observed a significant impact of STG interval on TICI 2b-3, although this effect seems mainly mediated by the association of ATG with TICI 2b-3.</p><p>One of the most important findings of the multicenter registry is that ATG intervals are quite long, with a median delay of 73 minutes. Allowing for varying effect sizes across centers, we were able to identify relevant factors associated with prolonged ATG intervals. These included the use of MRI and general anesthesia, as well as patient admission characteristics, such as low admission NIHSS, late presentation, and not meeting early time window AHA/ASA guideline criteria. Contrary to previous findings,<sup><xref rid=\"R13\" ref-type=\"bibr\">13</xref></sup> administration of intravenous tPA did not prolong the ATG interval. Even though treatment decisions in patients not meeting guideline criteria should be individualized, there seems to be a significant delay in ATG intervals for those patients. To reduce such delays is not only important for improving patients outcome but should be kept in mind when evaluating the outcome of patients presenting with borderline criteria subjected to mechanical thrombectomy. This real-world data indicate that in these patients, potential beneficial effects may be masked by associated delays in ATG intervals and respective lower rates of achieving TICI 2b-3. Corroborating previous studies, the use of admission MRI was associated with in-hospital delays,<sup><xref rid=\"R14\" ref-type=\"bibr\">14</xref>,<xref rid=\"R15\" ref-type=\"bibr\">15</xref></sup> further advocating the need for quality improvement program regarding sequence efficiency, as described recently.<sup><xref rid=\"R16\" ref-type=\"bibr\">16</xref></sup></p><p>Several possible causal relations between prolonged ATG intervals and reduced rates of TICI 2b-3 have previously been discussed,<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref></sup> such as thrombus modification over time (volume/extension,<sup><xref rid=\"R17\" ref-type=\"bibr\">17</xref></sup> histological features<sup><xref rid=\"R18\" ref-type=\"bibr\">18</xref></sup>), which might influence mechanical reperfusion efficacy.<sup><xref rid=\"R19\" ref-type=\"bibr\">19</xref>,<xref rid=\"R20\" ref-type=\"bibr\">20</xref></sup> At first sight, however, this may be contradictive to the only small association between STA and TICI 2b-3 because STA does usually constitute by far more of the time interval elapsed between symptom onset and the start of the intervention (≈2/3 in our cohort). Bourcier et al argued that the effect of STA may be diluted by uncertainties regarding the exact time point of symptom onset. Additionally, the authors argued that the effect of STA may be confounded by strict inclusion criteria of the randomized controlled trials (ie, only including patients with good collaterals, small infarct cores, incomplete occlusion pattern).<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref></sup> The latter phenomenon concerning the effect of time on functional outcome, often referred to as the time-reset effect, is indeed well known: admission/imaging-to-groin intervals have a stronger association with outcome, as opposed to STA intervals, as also observed in this registry.<sup><xref rid=\"R21\" ref-type=\"bibr\">21</xref></sup> Given the less restrictive selection criteria of our patient cohort, however, one would expect this bias to be weaker, thereby potentially unmasking an underlying association.</p><p>Our results provide evidence that prolonged ATG intervals are associated with borderline selection criteria. In these cases, it is reasonable to assume that decision-making is generally prolonged. The grit to achieve successful reperfusion in borderline indications may perhaps also diminish, which might explain decreased TICI 2b-3 with increasing ATG. It is noteworthy that the association of ATG with TICI 2b-3 persisted after adjusting for confounding factors associated with prolonged ATG intervals. However, this does not necessarily imply the absence of more ill-defined residual confounding factors related to more complex decision-making, such as difficult vascular anatomy, questionable life expectancy/comorbidities (cancer and dementia), or the search for a second/third opinion that may impede the dedication of subsequently pursuing TICI 2b-3 as rigorously as in more clear-cut cases.</p><p>Last, there is a paralleling development regarding improvements in rates of TICI 2b-3 and shorter ATG intervals in recent years following publication of the large randomized controlled trials in 2015. Hence, an association of ATG with TICI 2b-3 may simply reflect this technical development or alternatively is a proxy reflecting that more experienced centers with presumingly shorter ATG also have higher rates of TICI 2b-3. However, correcting for center and years of patient inclusion yielded a stable point estimate and 95% CI, suggesting that the association of ATG with rates of TICI 2b-3 is not merely explained by such technical developments or center-specific considerations.</p><p>Until further evidence becomes available, the causal relationship between prolonged ATG intervals and reduced rates of TICI 2b-3 with only a minor impact of STA remains elusive. More focus is needed on decision-making and workflow factors related to patients’ characteristics and prolonged ATG intervals. Before such evidence becomes available, we do not know to what extent dawdling diminishes reperfusion or if prolonged ATG intervals relate to patient characteristics, where reperfusion is less often achieved or less rigorously pursued to be achieved.</p><sec><title>Strengths and Limitations</title><p>Strengths of this analysis are the large cohort of patients and increased sample sizes in subgroups thereby increasing statistical power, as well as adequate modeling, enabling to explore factors associated with prolonged ATG intervals while correcting for confounders. Limitations of this study include a high percentage of incomplete complete workflow metrics and the retrospective nature of the registry. Most importantly, reperfusion success was not core laboratory adjudicated, impeding generalizability of the findings and comparability across centers. While this may be especially relevant for differentiating TICI 2b and TICI 3,<sup><xref rid=\"R22\" ref-type=\"bibr\">22</xref></sup> the agreement between operator and core laboratory for dichotomized TICI scores (ie, TICI 0/1/2a versus TICI 2b-3) is usually substantial<sup><xref rid=\"R22\" ref-type=\"bibr\">22</xref></sup>—an argument that supports the validity of our findings. Moreover, the association between prolonged ATG and reduced rates of, for example, TICI 2c/3 or first-pass TICI 2c/3 are interesting but should be handled with caution, since these variables were not defined as the primary outcome and, as secondary explorative analyses, are susceptible to α-error inflation.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>There is a great potential to reduce ATG, and potential targets for improvement can be deduced from observational data. The association between in-hospital delay and reduced reperfusion rates is evident in real-world clinical data, underscoring the need to optimize in-hospital workflows. Given the only minor association between STA and reperfusion rates and controversial pathophysiological considerations, the causal relationship of this association warrants further research.</p></sec></sec><sec><title>Sources of Funding</title><p>This study was supported by Medtronic (Dublin, Ireland). Medtronic did not take part in the conception, design, or article draft of this study. The work of Dr Kaesmacher was supported by the Swiss Academy of Medical Sciences/Bangerter Foundation and the Swiss Stroke Society.</p></sec><sec><title>Disclosures</title><p>Dr Fischer reports grants from Medtronic during the conduct of the study, grants from Medtronic, and other from Medtronic, Stryker, and CSL Behring outside the submitted work. Dr Gralla is a global principal investigator of STAR (Solitaire FR Thrombectomy for Acute Revascularisation), Clinical Event Committee member of the PROMISE study (Prospective, Multicenter, Observational, Single-Arm European Registry on the ACE Reperfusion Catheters and the Penumbra System in the Treatment of Acute Ischemic Stroke; Penumbra), and a principal investigator and consultant for the SWIFT DIRECT study (Solitaire With the Intention for Thrombectomy Plus Intravenous tPA Versus DIRECT Solitaire Stent-Retriever Thrombectomy in Acute Anterior Circulation Stroke; Medtronic) and receives Swiss National Science Foundation grants for magnetic resonance imaging in stroke. Dr Pierot reports personal fees from Balt, Phenox, and Microvention outside the submitted work. Dr Kaesmacher reports grants from Swiss Academy of Medical Sciences/Bangerter Foundation, Swiss Stroke Society, and Clinical Trial Unit Bern during the conduct of the study. Dr Michel reports grants from the Swiss National Science Foundation and the Swiss Heart Foundation during the conduct of the study; grants from European Thrombosis Investigator-Initiated Research program (Bristol-Myers Squibb/Pfizer), and personal fees from Medtronic used for research, outside the submitted work. Dr Arnold reports personal fees from Bayer, Bristol-Myers Squibb, Medtronic, Amgen, Daiichi Sankyo, Nestlé Health Sciences, Boehringer Ingelheim, and Covidien during the conduct of the study. Dr Mendes Pereira reports personal fees from Medtronic and Stryker during the conduct of the study. The other authors report no conflicts.</p></sec><sec><title>Supplemental Materials</title><p>Tables I–III</p><p>Figures I–III</p><p>Reference <xref rid=\"R6\" ref-type=\"bibr\">6</xref></p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><supplementary-material content-type=\"local-data\" id=\"SD1\"><media id=\"s1\" content-type=\"document\" xlink:href=\"str-51-2934-s001.pdf\" mimetype=\"application\" mime-subtype=\"pdf\" orientation=\"portrait\" position=\"anchor\"/></supplementary-material></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>\n<def-list><title>Nonstandard Abbreviations and Acronyms</title><def-item><term>AHA</term><def><p>American Heart Association</p></def></def-item><def-item><term>aOR</term><def><p>adjusted odds ratio</p></def></def-item><def-item><term>ASA</term><def><p>American Stroke Association</p></def></def-item><def-item><term>ASPECTS</term><def><p>Alberta Stroke Program Early CT Score</p></def></def-item><def-item><term>ATG</term><def><p>admission-to-groin-puncture</p></def></def-item><def-item><term>BEYOND-SWIFT</term><def><p>Bernese-European Registry for Ischemic Stroke Patients Treated Outside Current Guidelines With Neurothrombectomy Devices Using the Solitaire FR With the Intention for Thrombectomy</p></def></def-item><def-item><term>HERMES</term><def><p>Highly Effective Reperfusion Evaluated in Multiple Endovascular Stroke Trials</p></def></def-item><def-item><term>IQR</term><def><p>interquartile range</p></def></def-item><def-item><term>mRS</term><def><p>modified Rankin Scale</p></def></def-item><def-item><term>NIHSS</term><def><p>National Institutes of Health Stroke Scale</p></def></def-item><def-item><term>STA</term><def><p>symptom-onset-to-admission</p></def></def-item><def-item><term>STG</term><def><p>symptom-onset-to-groin</p></def></def-item><def-item><term>TICI</term><def><p>Thrombolysis in Cerebral Infarction</p></def></def-item><def-item><term>tPA</term><def><p>tissue-type plasminogen activator</p></def></def-item></def-list>\n</p></fn><fn fn-type=\"equal\" id=\"fn01\"><label>*</label><p>Drs Kaesmacher and Maamari share first authorship.</p></fn><fn fn-type=\"equal\" id=\"fn02\"><label>†</label><p>Drs Gralla and Fischer share last authorship.</p></fn><fn fn-type=\"financial-disclosure\" id=\"fn03\"><p>For Sources of Funding and Disclosures, see page 2941.</p></fn><fn fn-type=\"presented-at\" id=\"fn04\"><p>Presented in part at the German Society of Neuroradiology Annual Meeting, Frankfurt am Maim, Germany, October 9–12, 2019.</p></fn><fn fn-type=\"supplementary-material\" id=\"fn05\"><p>The Data Supplement is available with this article at <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.120.030208\">https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.120.030208</ext-link>.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liebeskind</surname><given-names>DS</given-names></name><name><surname>Bracard</surname><given-names>S</given-names></name><name><surname>Guillemin</surname><given-names>F</given-names></name><name><surname>Jahan</surname><given-names>R</given-names></name><name><surname>Jovin</surname><given-names>TG</given-names></name><name><surname>Majoie</surname><given-names>CB</given-names></name><name><surname>Mitchell</surname><given-names>PJ</given-names></name><name><surname>van der Lugt</surname><given-names>A</given-names></name><name><surname>Menon</surname><given-names>BK</given-names></name><name><surname>San Román</surname><given-names>L</given-names></name><etal/></person-group>; 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Epidemiology</journal-id><journal-id journal-id-type=\"iso-abbrev\">Epidemiology</journal-id><journal-id journal-id-type=\"publisher-id\">EDE</journal-id><journal-title-group><journal-title>Epidemiology (Cambridge, Mass.)</journal-title></journal-title-group><issn pub-type=\"ppub\">1044-3983</issn><issn pub-type=\"epub\">1531-5487</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32826524</article-id><article-id pub-id-type=\"pmc\">PMC7523582</article-id><article-id pub-id-type=\"art-access-id\">00007</article-id><article-id pub-id-type=\"doi\">10.1097/EDE.0000000000001239</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Methods</subject></subj-group></article-categories><title-group><article-title>Quantitative Bias Analysis for a Misclassified Confounder</article-title><subtitle>A Comparison Between Marginal Structural Models and Conditional Models for Point Treatments</subtitle></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Nab</surname><given-names>Linda</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">a</xref></contrib><contrib contrib-type=\"author\"><name><surname>Groenwold</surname><given-names>Rolf H. H.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">a</xref><xref ref-type=\"aff\" rid=\"aff2\">b</xref></contrib><contrib contrib-type=\"author\"><name><surname>van Smeden</surname><given-names>Maarten</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">a</xref></contrib><contrib contrib-type=\"author\"><name><surname>Keogh</surname><given-names>Ruth H.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">c</xref></contrib><aff id=\"aff1\">From the <label>a</label>Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands</aff><aff id=\"aff2\"><label>b</label>Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands</aff><aff id=\"aff3\"><label>c</label>Department of Medical Statistics, London School of Hygiene and Tropical Medicine, London, United Kingdom.</aff></contrib-group><author-notes><corresp id=\"c1\">Correspondence: Linda Nab, Department of Clinical Epidemiology, Leiden University Medical Center, Postzone C7-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail: <email xlink:href=\"l.nab@lumc.nl\">l.nab@lumc.nl</email>.</corresp></author-notes><pub-date pub-type=\"epub\"><day>05</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>11</month><year>2020</year></pub-date><volume>31</volume><issue>6</issue><fpage>796</fpage><lpage>805</lpage><history><date date-type=\"received\"><day>11</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>17</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">Creative Commons Attribution License 4.0 (CCBY)</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"ede-31-796.pdf\"/><abstract abstract-type=\"toc\"><p>Supplemental Digital Content is available in the text.</p></abstract><abstract><p>Observational data are increasingly used with the aim of estimating causal effects of treatments, through careful control for confounding. Marginal structural models estimated using inverse probability weighting (MSMs-IPW), like other methods to control for confounding, assume that confounding variables are measured without error. The average treatment effect in an MSM-IPW may however be biased when a confounding variable is error prone. Using the potential outcome framework, we derive expressions for the bias due to confounder misclassification in analyses that aim to estimate the average treatment effect using an marginal structural model estimated using inverse probability weighting (MSM-IPW). We compare this bias with the bias due to confounder misclassification in analyses based on a conditional regression model. Focus is on a point-treatment study with a continuous outcome. Compared with bias in the average treatment effect in a conditional model, the bias in an MSM-IPW can be different in magnitude but is equal in sign. Also, we use a simulation study to investigate the finite sample performance of MSM-IPW and conditional models when a confounding variable is misclassified. Simulation results indicate that confidence intervals of the treatment effect obtained from MSM-IPW are generally wider, and coverage of the true treatment effect is higher compared with a conditional model, ranging from overcoverage if there is no confounder misclassification to undercoverage when there is confounder misclassification. Further, we illustrate in a study of blood pressure-lowering therapy, how the bias expressions can be used to inform a quantitative bias analysis to study the impact of confounder misclassification, supported by an online tool.</p></abstract><kwd-group><kwd>Inverse probability weighting</kwd><kwd>Marginal structural models</kwd><kwd>Misclassification</kwd><kwd>Point-treatment study</kwd><kwd>Quantitative bias analysis</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>SDC</meta-name><meta-value>T</meta-value></custom-meta><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><p>The aim of many observational epidemiologic studies is to estimate a causal relation between an exposure and an outcome, through careful control for confounding. In the case of a point-treatment, that is, estimating the effect of a treatment at a single time point on a subsequent outcome, many methods exist that aim to estimate average treatment effects. These include traditional conditional regression analysis and marginal structural models estimated using inverse probability weighting (MSMs-IPW).<sup><xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref></sup> Unlike conditional regression, MSMs extend to estimation of joint treatment effects over multiple time points in longitudinal settings with time-dependent confounding.<sup><xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R3\" ref-type=\"bibr\">3</xref></sup></p><p>To obtain valid inference, MSMs-IPW, like other methods to control for confounding, assume that confounding variables are measured without error, an assumption hardly ever warranted in observational epidemiologic research.<sup><xref rid=\"R4\" ref-type=\"bibr\">4</xref>–<xref rid=\"R7\" ref-type=\"bibr\">7</xref></sup> A type of measurement error is classification error, which occurs when categorical variables are misclassified. For instance, smoking status (smoker versus nonsmoker) is prone to classification error but has been used as a confounding variable in studies investigating dialysis on mortality<sup><xref rid=\"R8\" ref-type=\"bibr\">8</xref></sup> and iron supplement use during pregnancy on anemia at delivery.<sup><xref rid=\"R9\" ref-type=\"bibr\">9</xref></sup> Another example of the use of a potentially misclassified confounding variable is alcohol use during pregnancy (yes versus no) in studies investigating associations between exposure to triptans during fetal life and risk of externalizing and internalizing behaviors in children.<sup><xref rid=\"R10\" ref-type=\"bibr\">10</xref></sup> In all aforementioned examples, MSMs were used to estimate the exposure–outcome relation, but the assumption of error-free confounding variables is possibly violated and may lead to bias in the treatment effect estimator.</p><p>There is a substantial literature on bias due to measurement error in confounding variables in conditional regression analyses,<sup><xref rid=\"R11\" ref-type=\"bibr\">11</xref>–<xref rid=\"R15\" ref-type=\"bibr\">15</xref></sup> but the impact of measurement error in confounding variables in causal inference methods, such as MSMs-IPW, has not received much attention. One exception is a study by Regier et al<sup><xref rid=\"R16\" ref-type=\"bibr\">16</xref></sup> that showed by means of a simulation study that measurement error in continuous confounding variables can introduce bias in the average treatment effect estimator in a point-treatment study. McCaffrey et al<sup><xref rid=\"R17\" ref-type=\"bibr\">17</xref></sup> proposed a weighting method to restore the treatment effect estimator when covariates are measured with error.</p><p>We provide a discussion of measurement error in a confounding variable. In addition, we derive expressions that quantify the bias in the average treatment effect estimator if a dichotomous confounding variable is misclassified, focusing on a point-treatment study with a continuous outcome. These expressions allow us (1) to quantify the bias due to classification error in a confounding variable in MSMs-IPW and to compare this with the bias from a conditional regression analysis and (2) to inform quantitative bias analyses.<sup><xref rid=\"R18\" ref-type=\"bibr\">18</xref>–<xref rid=\"R20\" ref-type=\"bibr\">20</xref></sup> We use simulation results to study the finite sample performance of an marginal structural model estimated using inverse probability weighting (MSM-IPW) compared with that of conditional regression models if classification error in a confounding variable is present. We illustrate our quantitative bias analysis in a study of the effect of blood pressure-lowering drugs on blood pressure.</p><sec><title>SETTINGS AND IMPACT OF MEASUREMENT ERROR, NOTATION, AND ASSUMPTIONS</title><p>Let <inline-graphic xlink:href=\"ede-31-796-i001.jpg\"/> denote the treatment indicator and <inline-graphic xlink:href=\"ede-31-796-i002.jpg\"/> the outcome. Let there be a variable <inline-graphic xlink:href=\"ede-31-796-i003.jpg\"/> that confounds the association between treatment and outcome and suppose that, instead of confounding variable <inline-graphic xlink:href=\"ede-31-796-i004.jpg\"/>, the error-prone confounding variable <inline-graphic xlink:href=\"ede-31-796-i005.jpg\"/> is observed. We consider two settings in which measurement error in confounding variables may occur and discuss the impact of measurement error in both settings.</p><sec><title>Settings and Impact of Measurement Error</title><p>The directed acyclic graph (DAG) in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref> illustrates setting 1. In this setting, treatment initiation is based on the error-prone confounding variable. Consider, for example, a study investigating the relation between the use of antidepressant drugs (<inline-graphic xlink:href=\"ede-31-796-i006.jpg\"/>) and the risk of a hip fracture (<inline-graphic xlink:href=\"ede-31-796-i007.jpg\"/>).<sup><xref rid=\"R21\" ref-type=\"bibr\">21</xref></sup> Benzodiazepine use may be a confounding variable but is prone to classification error because only prescription information may be available and over-the-counter use is often unknown. The clinician initiating the antidepressant drugs might not know their patient’s over-the-counter use and initiates treatment based on the observed error-prone benzodiazepine use (<inline-graphic xlink:href=\"ede-31-796-i008.jpg\"/>) instead of actual use (<inline-graphic xlink:href=\"ede-31-796-i009.jpg\"/>), as depicted in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>A. Here, conditioning on the error-prone <italic>L</italic>* will block the backdoor path from treatment <inline-graphic xlink:href=\"ede-31-796-i010.jpg\"/> to outcome <inline-graphic xlink:href=\"ede-31-796-i011.jpg\"/>. Thus, it is sufficient to control for the error-prone confounding variable to estimate the causal effect of treatment on outcome. This means that measurement error in a confounding variable will not always lead to bias.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>FIGURE 1.</label><caption><p>Measurement error <inline-graphic xlink:href=\"ede-31-796-i012.jpg\"/> in variable <inline-graphic xlink:href=\"ede-31-796-i013.jpg\"/> that confounds the association between treatment <inline-graphic xlink:href=\"ede-31-796-i014.jpg\"/> and outcome <inline-graphic xlink:href=\"ede-31-796-i015.jpg\"/> in two settings illustrated in directed acyclic graphs. A, Setting 1: Treatment <inline-graphic xlink:href=\"ede-31-796-i016.jpg\"/> is initiated based on the error-prone confounding variable <inline-graphic xlink:href=\"ede-31-796-i017.jpg\"/>. B, Setting 2: Treatment <inline-graphic xlink:href=\"ede-31-796-i018.jpg\"/> is initiated based on confounding variable <inline-graphic xlink:href=\"ede-31-796-i019.jpg\"/>.</p></caption><graphic xlink:href=\"ede-31-796-g001\"/></fig><p>The DAG in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>B illustrates setting 2, in which treatment initiation is based on <inline-graphic xlink:href=\"ede-31-796-i020.jpg\"/>, but only a proxy of <inline-graphic xlink:href=\"ede-31-796-i021.jpg\"/> is observed (<italic>L</italic>*). An example here might be a study investigating the effect of influenza vaccination (<inline-graphic xlink:href=\"ede-31-796-i022.jpg\"/>) on mortality (<inline-graphic xlink:href=\"ede-31-796-i023.jpg\"/>) in the elderly population.<sup><xref rid=\"R22\" ref-type=\"bibr\">22</xref></sup> Frailty (<inline-graphic xlink:href=\"ede-31-796-i024.jpg\"/>) possibly confounds the association between influenza vaccination and mortality. Frailty is observed by a clinician, but only a proxy of frailty (<italic>L</italic>*) may be available in electronic health records, as depicted in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>B. Here, conditioning on <italic>L</italic>* will not fully adjust for confounding by <inline-graphic xlink:href=\"ede-31-796-i025.jpg\"/>, because conditioning on <italic>L</italic>* does not block the backdoor path from <inline-graphic xlink:href=\"ede-31-796-i026.jpg\"/> to <inline-graphic xlink:href=\"ede-31-796-i027.jpg\"/> via <inline-graphic xlink:href=\"ede-31-796-i028.jpg\"/></p></sec><sec><title>Notation and Assumptions</title><p>We will now continue investigating the impact of classification error in setting 2, by focusing on the setting where <inline-graphic xlink:href=\"ede-31-796-i029.jpg\"/> is a dichotomous confounding variable and <inline-graphic xlink:href=\"ede-31-796-i030.jpg\"/> a continuous outcome. We use the potential outcomes framework.<sup><xref rid=\"R23\" ref-type=\"bibr\">23</xref>,<xref rid=\"R24\" ref-type=\"bibr\">24</xref></sup> Let <inline-graphic xlink:href=\"ede-31-796-i031.jpg\"/> denote the outcome that an individual would have had if treatment <inline-graphic xlink:href=\"ede-31-796-i032.jpg\"/> was set to <inline-graphic xlink:href=\"ede-31-796-i033.jpg\"/>, and let <inline-graphic xlink:href=\"ede-31-796-i034.jpg\"/> denote the outcome if treatment <inline-graphic xlink:href=\"ede-31-796-i035.jpg\"/> was set to <inline-graphic xlink:href=\"ede-31-796-i036.jpg\"/>. We assume that <italic>L</italic>* is nondifferentially misclassified with respect to the outcome (<inline-graphic xlink:href=\"ede-31-796-i037.jpg\"/>) and to the treatment (<inline-graphic xlink:href=\"ede-31-796-i038.jpg\"/>). Let <inline-graphic xlink:href=\"ede-31-796-i039.jpg\"/> denote the sensitivity of <italic>L</italic>* and <inline-graphic xlink:href=\"ede-31-796-i040.jpg\"/>, the specificity of <italic>L</italic>* (i.e., <inline-graphic xlink:href=\"ede-31-796-i041.jpg\"/>). We also denote the probability of treatment given the level of <inline-graphic xlink:href=\"ede-31-796-i042.jpg\"/> by <inline-graphic xlink:href=\"ede-31-796-i043.jpg\"/> and the prevalence of <inline-graphic xlink:href=\"ede-31-796-i044.jpg\"/> by <inline-graphic xlink:href=\"ede-31-796-i045.jpg\"/>. Here, we assume that <inline-graphic xlink:href=\"ede-31-796-i046.jpg\"/> because we are not interested in populations where <inline-graphic xlink:href=\"ede-31-796-i047.jpg\"/> is present or absent in everyone. Finally, we assume no measurement error in exposure and outcome.</p><p>We also assume that the following causal assumptions are satisfied to recover the causal effect of treatment on the outcome. Under the consistency assumption, we require that we observe <inline-graphic xlink:href=\"ede-31-796-i048.jpg\"/> if the individual is not exposed or <inline-graphic xlink:href=\"ede-31-796-i049.jpg\"/> if the individual is exposed.<sup><xref rid=\"R25\" ref-type=\"bibr\">25</xref></sup> Further, we assume that the potential outcome <inline-graphic xlink:href=\"ede-31-796-i050.jpg\"/> for an individual does not depend on treatments received by other individuals and that there are not multiple versions of treatment, also referred to as Stable Unit Treatment Value Assumption.<sup><xref rid=\"R26\" ref-type=\"bibr\">26</xref></sup> Additionally, we assume conditional exchangeability, i.e., given any level of <inline-graphic xlink:href=\"ede-31-796-i051.jpg\"/>, if the untreated group had in fact received treatment, then their expected outcome would have been the same as that in the treated, and vice versa.<sup><xref rid=\"R25\" ref-type=\"bibr\">25</xref></sup> In notation, <inline-graphic xlink:href=\"ede-31-796-i052.jpg\"/>, for <inline-graphic xlink:href=\"ede-31-796-i053.jpg\"/>. Finally, we assume <inline-graphic xlink:href=\"ede-31-796-i054.jpg\"/> for <inline-graphic xlink:href=\"ede-31-796-i055.jpg\"/> (positivity).<sup><xref rid=\"R27\" ref-type=\"bibr\">27</xref></sup></p><p>For causal contrasts, we compare expected potential outcomes (i.e., counterfactual outcomes) under the two different treatments. The average causal effect of the treatment on the outcome is <inline-graphic xlink:href=\"ede-31-796-i056.jpg\"/>. Under the above defined assumptions, the conditional effect of treatment <inline-graphic xlink:href=\"ede-31-796-i057.jpg\"/> on outcome <inline-graphic xlink:href=\"ede-31-796-i058.jpg\"/> can be defined through the following linear model:</p><disp-formula id=\"M1\"><graphic xlink:href=\"ede-31-796-g002.jpg\" position=\"float\" orientation=\"portrait\"/><label>(1)</label></disp-formula><p>Estimates for <inline-graphic xlink:href=\"ede-31-796-i059.jpg\"/> in the above model can be obtained by fitting a conditional regression model. Alternatively, the effect of treatment <inline-graphic xlink:href=\"ede-31-796-i060.jpg\"/> on outcome <inline-graphic xlink:href=\"ede-31-796-i061.jpg\"/> may be estimated by fitting an MSM:</p><disp-formula id=\"M2\"><graphic xlink:href=\"ede-31-796-g003.jpg\" position=\"float\" orientation=\"portrait\"/><label>(2)</label></disp-formula><p>Estimates for <inline-graphic xlink:href=\"ede-31-796-i062.jpg\"/> in the above model can be obtained by IPW estimation: by fitting a linear regression model for <inline-graphic xlink:href=\"ede-31-796-i063.jpg\"/> on <inline-graphic xlink:href=\"ede-31-796-i064.jpg\"/> where the contribution of each individual is weighted by 1 over the probability of that individual’s observed treatment given <inline-graphic xlink:href=\"ede-31-796-i065.jpg\"/>, estimating the marginal treatment effect.<sup><xref rid=\"R2\" ref-type=\"bibr\">2</xref></sup> Because our focus is on linear models and we make the simplifying assumption that the effect of <inline-graphic xlink:href=\"ede-31-796-i066.jpg\"/> on <inline-graphic xlink:href=\"ede-31-796-i067.jpg\"/> does not vary between strata of <inline-graphic xlink:href=\"ede-31-796-i068.jpg\"/>, the conditional and marginal treatment effects, denoted by <inline-graphic xlink:href=\"ede-31-796-i069.jpg\"/> in model equations 1 and 2, respectively, are equal. This is not generally true for nonlinear models due to noncollapsibility.<sup><xref rid=\"R2\" ref-type=\"bibr\">2</xref></sup> We assume that the effect of <inline-graphic xlink:href=\"ede-31-796-i070.jpg\"/> on <inline-graphic xlink:href=\"ede-31-796-i071.jpg\"/> does not vary between strata of <inline-graphic xlink:href=\"ede-31-796-i072.jpg\"/>, to derive bias expressions that are easier to use in practice and require fewer parameters.<sup><xref rid=\"R28\" ref-type=\"bibr\">28</xref></sup></p></sec></sec><sec><title>QUANTIFICATION OF BIAS DUE TO CLASSIFICATION ERROR IN A CONFOUNDING VARIABLE</title><p>Our aim is to study the effect of using the misclassified confounding variable <italic>L</italic>* in place of the confounding variable <inline-graphic xlink:href=\"ede-31-796-i073.jpg\"/> in the conditional regression model or in the model for the weights used to fit the MSM on the average treatment effect estimator in the setting where <inline-graphic xlink:href=\"ede-31-796-i074.jpg\"/>, not <italic>L</italic>*, influences treatment initiation (setting 2 above).</p><sec><title>Conditional Model</title><p>By the law of total expectation, the expected value of the outcome <inline-graphic xlink:href=\"ede-31-796-i075.jpg\"/> given treatment <inline-graphic xlink:href=\"ede-31-796-i076.jpg\"/> and <italic>L</italic>* is (see eAppendix 1; <ext-link ext-link-type=\"uri\" xlink:href=\"http://links.lww.com/EDE/B698\">http://links.lww.com/EDE/B698</ext-link> section 1 for further detail),</p><disp-formula id=\"M3\"><graphic xlink:href=\"ede-31-796-g004.jpg\" position=\"float\" orientation=\"portrait\"/></disp-formula><disp-formula id=\"M4\"><graphic xlink:href=\"ede-31-796-g005.jpg\" position=\"float\" orientation=\"portrait\"/></disp-formula><disp-formula id=\"M5\"><graphic xlink:href=\"ede-31-796-g006.jpg\" position=\"float\" orientation=\"portrait\"/></disp-formula><p>where <inline-graphic xlink:href=\"ede-31-796-i077.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i078.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i079.jpg\"/> represent the coefficients of the linear model <inline-graphic xlink:href=\"ede-31-796-i080.jpg\"/>, modeling the mean of <inline-graphic xlink:href=\"ede-31-796-i081.jpg\"/> times <italic>L</italic>* (i.e., <inline-graphic xlink:href=\"ede-31-796-i082.jpg\"/>) given <inline-graphic xlink:href=\"ede-31-796-i083.jpg\"/> and <italic>L</italic>* (see next paragraph for an explanation of why these appear). The coefficient for treatment <inline-graphic xlink:href=\"ede-31-796-i084.jpg\"/> in the above model is <inline-graphic xlink:href=\"ede-31-796-i085.jpg\"/>, and is therefore biased for the parameter of interest (i.e., <inline-graphic xlink:href=\"ede-31-796-i086.jpg\"/>). By rewriting <inline-graphic xlink:href=\"ede-31-796-i087.jpg\"/> in terms of <inline-graphic xlink:href=\"ede-31-796-i088.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i089.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i090.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i091.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i092.jpg\"/> (see eAppendix 1 section 1), we find that the bias due to classification error in <italic>L</italic>* in the average treatment effect in a conditional regression model is as follows:</p><disp-formula id=\"M6\"><graphic xlink:href=\"ede-31-796-g007.jpg\" position=\"float\" orientation=\"portrait\"/></disp-formula><disp-formula id=\"M7\"><graphic xlink:href=\"ede-31-796-g008.jpg\" position=\"float\" orientation=\"portrait\"/><label>(3)</label></disp-formula><p>where <inline-graphic xlink:href=\"ede-31-796-i093.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i094.jpg\"/> (see eAppendix 1 section 1 for a derivation).</p><p>We focused on a model for <inline-graphic xlink:href=\"ede-31-796-i095.jpg\"/> conditional on <inline-graphic xlink:href=\"ede-31-796-i096.jpg\"/> and <italic>L</italic>* which includes only main effects of <inline-graphic xlink:href=\"ede-31-796-i097.jpg\"/> and <italic>L</italic>*, as this is typically done in practice when replacing <inline-graphic xlink:href=\"ede-31-796-i098.jpg\"/> with <italic>L</italic>*. In fact, it can be shown that when the model for <inline-graphic xlink:href=\"ede-31-796-i099.jpg\"/> given <inline-graphic xlink:href=\"ede-31-796-i100.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i101.jpg\"/> includes only main effects of <inline-graphic xlink:href=\"ede-31-796-i102.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i103.jpg\"/>, the implied correctly specified model for <inline-graphic xlink:href=\"ede-31-796-i104.jpg\"/> given <inline-graphic xlink:href=\"ede-31-796-i105.jpg\"/> and <italic>L</italic>* also includes an interaction between <inline-graphic xlink:href=\"ede-31-796-i106.jpg\"/> and <italic>L</italic>*, explaining the appearance of <inline-graphic xlink:href=\"ede-31-796-i107.jpg\"/>, and <inline-graphic xlink:href=\"ede-31-796-i108.jpg\"/> in the above because the interaction is not modeled. See eAppendix 1 section 1 for the bias in case an interaction is modeled</p></sec><sec><title>Marginal Structural Model Estimated Using Inverse Probability Weighting</title><p>An MSM-IPW proceeds by fitting a linear regression for outcome <inline-graphic xlink:href=\"ede-31-796-i109.jpg\"/> on treatment <inline-graphic xlink:href=\"ede-31-796-i110.jpg\"/>, where the contribution of each individual is weighted by 1 over the probability of that individual’s observed treatment given misclassified <italic>L</italic>*.<sup><xref rid=\"R2\" ref-type=\"bibr\">2</xref></sup> An estimator for the average treatment effect <inline-graphic xlink:href=\"ede-31-796-i111.jpg\"/> is as follows:</p><disp-formula id=\"M8\"><graphic xlink:href=\"ede-31-796-g009.jpg\" position=\"float\" orientation=\"portrait\"/></disp-formula><disp-formula id=\"M9\"><graphic xlink:href=\"ede-31-796-g010.jpg\" position=\"float\" orientation=\"portrait\"/></disp-formula><p>It can be shown that <inline-graphic xlink:href=\"ede-31-796-i112.jpg\"/> Consequently, the bias in the average treatment effect in an MSM-IPW is as follows:</p><disp-formula id=\"M10\"><graphic xlink:href=\"ede-31-796-g011.jpg\" position=\"float\" orientation=\"portrait\"/><label>(4)</label></disp-formula><p>We refer to eAppendix 1 section 2 for a derivation of the above formula.</p></sec><sec><title>Exploration of Bias</title><p>To study the bias due to misclassification from the conditional model and MSM-IPW, we explore bias expressions (equations 3 and 4).</p><sec><title>Null Bias</title><p>To confirm the derived bias expressions, we consider three trivial conditions where bias in the average treatment effect estimator is expected to be null, in line with general understanding of causal inference.<sup><xref rid=\"R29\" ref-type=\"bibr\">29</xref></sup> (1) If there is no classification error in <italic>L</italic>*, i.e., specificity is 1 (<inline-graphic xlink:href=\"ede-31-796-i113.jpg\"/>) and sensitivity is 1 (<inline-graphic xlink:href=\"ede-31-796-i114.jpg\"/>), it follows that <inline-graphic xlink:href=\"ede-31-796-i115.jpg\"/> corresponds to <italic>L</italic>*, irrespective of treatment level (i.e., <inline-graphic xlink:href=\"ede-31-796-i116.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i117.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i118.jpg\"/>, and <inline-graphic xlink:href=\"ede-31-796-i119.jpg\"/>). (2) If the true relation between <inline-graphic xlink:href=\"ede-31-796-i120.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i121.jpg\"/> is null (i.e., <inline-graphic xlink:href=\"ede-31-796-i122.jpg\"/> is zero, thus there is no arrow from <inline-graphic xlink:href=\"ede-31-796-i123.jpg\"/> to <inline-graphic xlink:href=\"ede-31-796-i124.jpg\"/> in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>B). (3) If <inline-graphic xlink:href=\"ede-31-796-i125.jpg\"/> does not affect the probability of receiving treatment (i.e., <inline-graphic xlink:href=\"ede-31-796-i126.jpg\"/>, thus there is no arrow from <inline-graphic xlink:href=\"ede-31-796-i127.jpg\"/> to <inline-graphic xlink:href=\"ede-31-796-i128.jpg\"/> in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>B), the probability that <inline-graphic xlink:href=\"ede-31-796-i129.jpg\"/> is 1 depends on the value of <italic>L</italic>* but no longer on <inline-graphic xlink:href=\"ede-31-796-i130.jpg\"/> (i.e., <inline-graphic xlink:href=\"ede-31-796-i131.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i132.jpg\"/>). Bias is null under these conditions for both models (MSM-IPW and conditional model). Because the bias expressions are strictly monotonic, the bias in an MSM-IPW cannot be negative if the bias in the conditional model is positive and vice versa (i.e., the bias will be in the same direction for both models).</p><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2.</label><caption><p>Visualization of the direction and magnitude of the bias in the average treatment effect estimator in relation to the prevalence of treatment among individuals with the confounding variable present. In this visualization, the confounding variable <inline-graphic xlink:href=\"ede-31-796-i133.jpg\"/> is misclassified with a sensitivity of 0.9 and specificity of 0.95. Consequently, the average treatment effect estimated in an MSM-IPW or conditional regression model is biased, independent of true average treatment effect. The prevalence of <inline-graphic xlink:href=\"ede-31-796-i134.jpg\"/> is 50% (i.e., <inline-graphic xlink:href=\"ede-31-796-i135.jpg\"/>). The direction and magnitude of the bias depend on (1) the strength and direction of the association between <inline-graphic xlink:href=\"ede-31-796-i136.jpg\"/> and treatment (denoted by <inline-graphic xlink:href=\"ede-31-796-i137.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i138.jpg\"/>, here set at <inline-graphic xlink:href=\"ede-31-796-i139.jpg\"/> in the left-hand-side plot and <inline-graphic xlink:href=\"ede-31-796-i140.jpg\"/> in the right-hand-side plot); and (2) the strength and direction of the association between <inline-graphic xlink:href=\"ede-31-796-i141.jpg\"/> and the outcome (denoted by <inline-graphic xlink:href=\"ede-31-796-i142.jpg\"/> in the text and here set at <inline-graphic xlink:href=\"ede-31-796-i143.jpg\"/>). Larger values of <inline-graphic xlink:href=\"ede-31-796-i144.jpg\"/> will result in steeper curves; <inline-graphic xlink:href=\"ede-31-796-i145.jpg\"/> will mirror the graph in <inline-graphic xlink:href=\"ede-31-796-i146.jpg\"/>.</p></caption><graphic xlink:href=\"ede-31-796-g012\"/></fig></sec><sec><title>Equal Biases</title><p>The bias in the average treatment effect in a conditional regression analysis is equal to that in an MSM-IPW if bias in both models is null (see above). We also see that bias expressions (equations 3 and 4) show that bias for the two methods is equal if the term between curly brackets in equation 3 is equal to 1, which is the case if (1) <inline-graphic xlink:href=\"ede-31-796-i147.jpg\"/>; (2) <inline-graphic xlink:href=\"ede-31-796-i148.jpg\"/>; and (3) <inline-graphic xlink:href=\"ede-31-796-i149.jpg\"/>. If conditions (1) and/or (2) are met, there is no bias in an MSM-IPW nor in a conditional model. Under condition (3), bias is generally non-null (except if, for example, <inline-graphic xlink:href=\"ede-31-796-i150.jpg\"/>, see Null Bias).</p></sec><sec><title>Sign and Magnitude of Bias</title><p>Figures <xref ref-type=\"fig\" rid=\"F2\">2</xref>–<xref ref-type=\"fig\" rid=\"F4\">4</xref> illustrate the contributions to bias in the average treatment effect due to misclassification components (sensitivity and specificity) and due to confounding components (prevalence of confounding variable, strength of association between confounding variable and treatment and outcome) in a conditional model and an MSM-IPW, obtained by using the bias expressions.</p><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>FIGURE 3.</label><caption><p>Visualization of the magnitude of the bias in the average treatment effect estimator in relation to the prevalence of a confounding variable. In this visualization, the confounding variable <inline-graphic xlink:href=\"ede-31-796-i151.jpg\"/> is misclassified with a sensitivity of 0.9 and specificity of 0.95. Consequently, the average treatment effect estimated in an MSM-IPW or conditional regression model is biased, independent of true average treatment effect. The confounding variable is positively associated with treatment (i.e., here <inline-graphic xlink:href=\"ede-31-796-i152.jpg\"/>, where <inline-graphic xlink:href=\"ede-31-796-i153.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i154.jpg\"/>), and outcome (denoted by <inline-graphic xlink:href=\"ede-31-796-i155.jpg\"/> in the text and here set at <inline-graphic xlink:href=\"ede-31-796-i156.jpg\"/>). The magnitude of the bias depends on the prevalence of the confounding variable (i.e., <inline-graphic xlink:href=\"ede-31-796-i157.jpg\"/>). Larger values of <inline-graphic xlink:href=\"ede-31-796-i158.jpg\"/> will result in steeper curves.</p></caption><graphic xlink:href=\"ede-31-796-g013\"/></fig><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>FIGURE 4.</label><caption><p>Visualization of the magnitude of the bias in the average treatment effect estimator in relation to specificity and sensitivity of a misclassified confounding variable. In this visualization, the prevalence of the confounding variable <inline-graphic xlink:href=\"ede-31-796-i159.jpg\"/> is 50% (i.e., <inline-graphic xlink:href=\"ede-31-796-i160.jpg\"/>), the association between <inline-graphic xlink:href=\"ede-31-796-i161.jpg\"/> and treatment (denoted by <inline-graphic xlink:href=\"ede-31-796-i162.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i163.jpg\"/>) and outcome is positive (denoted by <inline-graphic xlink:href=\"ede-31-796-i164.jpg\"/> in the text and here set at <inline-graphic xlink:href=\"ede-31-796-i165.jpg\"/>). Given these values, if <inline-graphic xlink:href=\"ede-31-796-i166.jpg\"/> is misclassified, the average treatment effect estimated in an MSM-IPW or conditional regression model is biased, independent of true average treatment effect. The magnitude of the bias depends on the specificity and sensitivity of <inline-graphic xlink:href=\"ede-31-796-i167.jpg\"/> and is maximal if sensitivity equals 1 − specificity. The strength of the association between <inline-graphic xlink:href=\"ede-31-796-i168.jpg\"/> and treatment is greater in the right-hand-side plot (<inline-graphic xlink:href=\"ede-31-796-i169.jpg\"/>) compared with the left-hand-side plot (<inline-graphic xlink:href=\"ede-31-796-i170.jpg\"/>), and consequently, bias is greater. Larger values of <inline-graphic xlink:href=\"ede-31-796-i171.jpg\"/> will result in steeper curves.</p></caption><graphic xlink:href=\"ede-31-796-g014\"/></fig><p>Figure <xref ref-type=\"fig\" rid=\"F2\">2</xref> shows that (1) the bias is positive if both the association between <inline-graphic xlink:href=\"ede-31-796-i172.jpg\"/> and treatment and <inline-graphic xlink:href=\"ede-31-796-i173.jpg\"/> and outcome are positive (i.e., <inline-graphic xlink:href=\"ede-31-796-i174.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i175.jpg\"/>, respectively) and (2) the bias is greater if the difference between <inline-graphic xlink:href=\"ede-31-796-i176.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i177.jpg\"/> is greater (i.e., if the strength of the association between <inline-graphic xlink:href=\"ede-31-796-i178.jpg\"/> and treatment is greater). In contrast, the bias is negative if <inline-graphic xlink:href=\"ede-31-796-i179.jpg\"/>, whereas <inline-graphic xlink:href=\"ede-31-796-i180.jpg\"/> is positive. In case <inline-graphic xlink:href=\"ede-31-796-i181.jpg\"/>, Figure <xref ref-type=\"fig\" rid=\"F2\">2</xref> is mirrored in <inline-graphic xlink:href=\"ede-31-796-i182.jpg\"/>, and consequently, bias is negative if <inline-graphic xlink:href=\"ede-31-796-i183.jpg\"/> and positive if <inline-graphic xlink:href=\"ede-31-796-i184.jpg\"/>. An increment in <inline-graphic xlink:href=\"ede-31-796-i185.jpg\"/> will result in greater bias and steeper curves in Figure <xref ref-type=\"fig\" rid=\"F2\">2</xref>. Figure <xref ref-type=\"fig\" rid=\"F3\">3</xref> shows that the magnitude of the bias depends on the prevalence of <inline-graphic xlink:href=\"ede-31-796-i186.jpg\"/>. Further, it shows that bias is greater if the strength of association between <inline-graphic xlink:href=\"ede-31-796-i187.jpg\"/> and treatment is greater. Figure <xref ref-type=\"fig\" rid=\"F4\">4</xref> shows that, generally, the bias is greater if <italic>L</italic>* has lower specificity and sensitivity. Moreover, for a fixed sensitivity, bias is minimal if specificity equals 1 and is maximal if 1 − specificity equals sensitivity; by fixing specificity, bias is minimal if sensitivity equals 1 and is maximal if sensitivity equals 1 − specificity. Figure <xref ref-type=\"fig\" rid=\"F4\">4</xref> shows that the bias is greater if the strength of the association between <inline-graphic xlink:href=\"ede-31-796-i188.jpg\"/> and treatment is greater. An increment in <inline-graphic xlink:href=\"ede-31-796-i189.jpg\"/> will result in greater bias and steeper curves in Figure <xref ref-type=\"fig\" rid=\"F4\">4</xref>. An online application can be used to obtain bias plots for other combinations of the parameters available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://lindanab.shinyapps.io/SensitivityAnalysis\">https://lindanab.shinyapps.io/SensitivityAnalysis</ext-link>.</p></sec></sec><sec><title>Simulation Study</title><p>We conducted a simulation study to study the finite sample properties of MSMs estimated using IPW and conditional models if there is classification error in the confounding variable. Five thousand data sets were generated with sample sizes of 1,000 and 100, using the following data-generating mechanisms:</p><disp-formula id=\"M11\"><graphic xlink:href=\"ede-31-796-g015.jpg\" position=\"float\" orientation=\"portrait\"/></disp-formula><disp-formula id=\"M12\"><graphic xlink:href=\"ede-31-796-g016.jpg\" position=\"float\" orientation=\"portrait\"/></disp-formula><p>We studied five different scenarios, of which the parameters values can be found in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>. In all scenarios, the average treatment effect <inline-graphic xlink:href=\"ede-31-796-i190.jpg\"/> (estimand) is 1 and the association between the confounding variable <inline-graphic xlink:href=\"ede-31-796-i191.jpg\"/> and outcome <inline-graphic xlink:href=\"ede-31-796-i192.jpg\"/> is 2 (i.e., <inline-graphic xlink:href=\"ede-31-796-i193.jpg\"/>). In scenario 0, we assume no classification error. In scenarios 1–4, we assume that <italic>L</italic>* has a specificity of 0.95 (i.e., <inline-graphic xlink:href=\"ede-31-796-i194.jpg\"/>) and a sensitivity of 0.90 (i.e., <inline-graphic xlink:href=\"ede-31-796-i195.jpg\"/>). In scenario 1, bias in the average treatment effect estimator is expected to be negative because the probability of receiving treatment given that <inline-graphic xlink:href=\"ede-31-796-i196.jpg\"/> is not present is greater than receiving treatment given that <inline-graphic xlink:href=\"ede-31-796-i197.jpg\"/> is present, and the association between <inline-graphic xlink:href=\"ede-31-796-i198.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i199.jpg\"/> is positive (i.e., <inline-graphic xlink:href=\"ede-31-796-i200.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i201.jpg\"/>). In contrast, in scenarios 2 and 3, bias in the average treatment effect estimator is expected to be positive, because <inline-graphic xlink:href=\"ede-31-796-i202.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i203.jpg\"/>. Further, after investigation of Figure <xref ref-type=\"fig\" rid=\"F3\">3</xref>, we expect that bias in the average treatment effect estimated in a conditional model is greater than that in an MSM-IPW in scenarios 2 and 3. Finally, in scenario 4, we expect that bias in the average treatment effect from the conditional model is equal to that in an MSM-IPW.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>TABLE 1.</label><caption><p>Values of the Parameters in the Five Different Simulation Scenarios</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Scenario</th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"ede-31-796-i204.jpg\"/></th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"ede-31-796-i205.jpg\"/></th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"ede-31-796-i206.jpg\"/></th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"ede-31-796-i207.jpg\"/></th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"ede-31-796-i208.jpg\"/></th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"ede-31-796-i209.jpg\"/></th><th align=\"center\" rowspan=\"1\" colspan=\"1\"><inline-graphic xlink:href=\"ede-31-796-i210.jpg\"/></th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.50</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.50</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.90</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.50</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.90</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.90</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.80</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.90</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.80</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.50</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.90</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.45</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.50</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td></tr></tbody></table></table-wrap><sec><title>Model Estimation and Performance Measures</title><p>We obtained the average treatment effect <inline-graphic xlink:href=\"ede-31-796-i211.jpg\"/> (estimand) by fitting a conditional model using conditional regression and by fitting an MSM-IPW, both using the misclassified <italic>L</italic>* instead of <inline-graphic xlink:href=\"ede-31-796-i212.jpg\"/> from the data-generating mechanism. For the MSM-IPW analysis, we used the R package ipw.<sup><xref rid=\"R30\" ref-type=\"bibr\">30</xref>,<xref rid=\"R31\" ref-type=\"bibr\">31</xref></sup> Performance of both models was evaluated in terms of the bias, the mean squared error of the estimated treatment effect (MSE), the percentages of 95% confidence intervals that contain the true value of the estimand (coverage), the empirical standard deviation of the estimated treatment effects (empSE), and mean model-based standard error of the estimated treatment effect. We estimated robust model-based standard errors of the average treatment effect in an MSM-IPW using the R package survey.<sup><xref rid=\"R32\" ref-type=\"bibr\">32</xref></sup> We calculated Monte Carlo standard errors for all performance measures,<sup><xref rid=\"R33\" ref-type=\"bibr\">33</xref></sup> using the R package rsimsum.<sup><xref rid=\"R34\" ref-type=\"bibr\">34</xref></sup> Additionally, we calculated the theoretical bias of the average treatment effect in both methods based on the bias expressions (equations 3 and 4).</p></sec></sec></sec><sec sec-type=\"results\"><title>RESULTS</title><p>Table <xref rid=\"T2\" ref-type=\"table\">2</xref> shows the results of the simulation study. Bias found in the simulation study corresponds to the theoretical bias derived from the bias expressions. The empirical standard deviation of the average treatment effect estimates (empSE) from the MSM-IPW is equal to or greater than that from the conditional model. Yet, in the scenarios where bias in the average treatment effect in the MSM-IPW was smaller than bias in the conditional model (scenarios 2 and 3), empSE of both methods was equal, and hence, MSE is smaller for one method if also bias is smaller. Furthermore, the (robust) model-based standard errors of the average treatment effect in an MSM-IPW are conservative and greater than the empirical standard errors, because the uncertainty in estimating the treatment weights is not taken into account. Allowing for the estimation of the weights will shrink the standard errors.<sup><xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R35\" ref-type=\"bibr\">35</xref></sup> We chose not to use a less conservative standard error estimation for MSM-IPW, such as bootstrapping, because our goal was to frame this simulation as investigating the properties of the commonly used MSM-IPW estimation procedure. Consequently, confidence intervals of the treatment effect obtained in an MSM-IPW are generally wider and coverage of the true treatment effect is higher compared with a conditional model, ranging from overcoverage if there is no classification error to smaller undercoverage when there is classification error.</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>TABLE 2.</label><caption><p>Results of Simulation Study Studying the Finite Sample Properties of a marginal structural model estimated using inverse probability weighting (MSM-IPW) and a CM If There Is Classification Error in the Confounding Variable</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Method</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Sample Size</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Scenario<sup>a</sup></th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Bias (Formula)<sup>b</sup></th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Bias</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">MSE<sup>c</sup></th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Coverage</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">empSE<sup>d</sup></th><th align=\"center\" rowspan=\"1\" colspan=\"1\">modelSE<sup>e</sup></th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">MSM-IPW</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1,000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00 (0.000)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.99 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.42</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.42 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.18 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 (0.000)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.67 (0.007)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.004)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 (0.000)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.68 (0.007)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.10 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.99 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.22 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.31 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.42</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.42 (0.005)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29 (0.005)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.78 (0.006)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.34 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.37 (0.001)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.14 (0.004)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.94 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29 (0.004)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.84 (0.005)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.26 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.28 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15 (0.004)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.95 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.31 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CM</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1,000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00 (0.000)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.95 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.34 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.12 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 (0.000)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.46 (0.007)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.32</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.32 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.11 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.02 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.03 (0.000)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.49 (0.007)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.07 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">100</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.00 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.05 (0.001)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.95 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.22 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.22 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.34</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−0.33 (0.004)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.19 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.73 (0.006)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.29 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.27 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.16 (0.004)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.09 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.90 (0.004)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.32</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.32 (0.004)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.17 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.74 (0.006)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.26 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.25 (0.000)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.15 (0.003)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.08 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.90 (0.004)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24 (0.002)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.24 (0.000)</td></tr></tbody></table><table-wrap-foot><fn fn-type=\"other\" id=\"tab2fn1\"><label>a</label><p>In all scenarios, the average treatment effect (estimand) is 1 (<inline-graphic xlink:href=\"ede-31-796-i213.jpg\"/>) and the effect of the confounding variable on the outcome is 2 (<inline-graphic xlink:href=\"ede-31-796-i214.jpg\"/>). Five thousand data sets were generated. Monte Carlo standard errors are shown between brackets. In scenario 0, there is no classification error (specificity and sensitivity of the misclassified confounding variable are 1, i.e., <inline-graphic xlink:href=\"ede-31-796-i215.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i216.jpg\"/>). In scenarios 1–4, the specificity of the misclassified confounding variable is 0.95 (i.e., <inline-graphic xlink:href=\"ede-31-796-i217.jpg\"/>) and the sensitivity is 0.9 (i.e., <inline-graphic xlink:href=\"ede-31-796-i218.jpg\"/>). The prevalence of the confounding variable (<inline-graphic xlink:href=\"ede-31-796-i219.jpg\"/>) and the probability of receiving treatment if the confounding is not present or present (<inline-graphic xlink:href=\"ede-31-796-i220.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i221.jpg\"/>, respectively) are set as follows in the scenarios: scenario 0: <inline-graphic xlink:href=\"ede-31-796-i222.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i223.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i224.jpg\"/>; scenario 1: <inline-graphic xlink:href=\"ede-31-796-i225.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i226.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i227.jpg\"/>; scenario 2: <inline-graphic xlink:href=\"ede-31-796-i228.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i229.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i230.jpg\"/>; scenario 3: <inline-graphic xlink:href=\"ede-31-796-i231.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i232.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i233.jpg\"/>; and scenario 4: <inline-graphic xlink:href=\"ede-31-796-i234.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i235.jpg\"/>, <inline-graphic xlink:href=\"ede-31-796-i236.jpg\"/>.</p></fn><fn fn-type=\"other\" id=\"tab2fn2\"><label>b</label><p>Bias based on bias expressions (equations 3 and 4) in the text.</p></fn><fn fn-type=\"other\" id=\"tab2fn3\"><label>c</label><p>Mean squared error.</p></fn><fn fn-type=\"other\" id=\"tab2fn4\"><label>d</label><p>Empirical standard error.</p></fn><fn fn-type=\"other\" id=\"tab2fn5\"><label>e</label><p>Model-based standard error.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>ILLUSTRATION: QUANTITATIVE BIAS ANALYSIS OF CLASSIFICATION ERROR IN A CONFOUNDING VARIABLE</title><p>Quantitative bias analysis provides a tool to incorporate uncertainty in study results due to systematic errors.<sup><xref rid=\"R18\" ref-type=\"bibr\">18</xref>,<xref rid=\"R20\" ref-type=\"bibr\">20</xref></sup> Using an example study of blood pressure-lowering therapy, we illustrate how the bias expressions (equations 3 and 4) can be used to perform a quantitative bias analysis for misclassification of a confounding variable.</p><sec><title>Application</title><p>For our illustration we use data of the National Health And Nutritional Examination Survey (NHANES),<sup><xref rid=\"R36\" ref-type=\"bibr\">36</xref>,<xref rid=\"R37\" ref-type=\"bibr\">37</xref></sup> more details can be found in the Supplement 2; <ext-link ext-link-type=\"uri\" xlink:href=\"http://links.lww.com/EDE/B698\">http://links.lww.com/EDE/B698</ext-link>. Specifically, we study the effect of diuretic use (<inline-graphic xlink:href=\"ede-31-796-i237.jpg\"/>) in comparison to beta blocker use <inline-graphic xlink:href=\"ede-31-796-i238.jpg\"/> on systolic blood pressure (<inline-graphic xlink:href=\"ede-31-796-i239.jpg\"/>) using two approaches: by inverse weighting with the propensity for diuretic or beta blocker use given self-reported categorical body mass index (BMI) (<italic>L</italic>*) and using a conditional linear regression with adjustment for self-reported categorical BMI. For this illustration, we categorize self-reported BMI into two distinct categories: underweight/normal weight (BMI <inline-graphic xlink:href=\"ede-31-796-i240.jpg\"/> (<inline-graphic xlink:href=\"ede-31-796-i241.jpg\"/>)) and overweight/obese (BMI ≥25 (<inline-graphic xlink:href=\"ede-31-796-i242.jpg\"/>)). However, we stress that one should preferably not categorize BMI in most practical applications.<sup><xref rid=\"R38\" ref-type=\"bibr\">38</xref></sup> Moreover, we assume that dichotomizing self-reported BMI does not introduce differential misclassification.<sup><xref rid=\"R7\" ref-type=\"bibr\">7</xref></sup></p><p>We assume that blood pressure-lowering therapy is initiated based on the true BMI (<inline-graphic xlink:href=\"ede-31-796-i243.jpg\"/>) instead of the observed self-reported BMI (setting 2, Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>B). Further, we consider BMI the only confounding variable, and treatment and outcome to be measured without error, which is a simplification of reality. Additionally, we assume that the classification error in self-reported BMI category is nondifferential for the subject’s treatment or blood pressure (given true BMI category). Expert knowledge is needed to inform this assumption. To quantify how large the bias in the average treatment effect estimator is expected to be due to classification error in self-reported BMI category, we perform a quantitative bias analysis using the bias expressions (equations 3 and 4).</p></sec><sec><title>Average Treatment Effect</title><p>Table <xref rid=\"T3\" ref-type=\"table\">3</xref> shows the average treatment effect of diuretics use in comparison to beta blocker use on mean systolic blood pressure. In an MSM-IPW, we estimated an average treatment effect (95% confidence interval [CI]) of −3.52 (−1.21, −5.74). In a conditional regression model, we estimated an average treatment effect (95% CI) of −3.48 (−1.27, −5.76).</p><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>TABLE 3.</label><caption><p>Average Treatment Effect of Diuretics Use Compared with Beta Blocker Use on Mean Systolic Blood Pressure in NHANES<sup><xref rid=\"R36\" ref-type=\"bibr\">36</xref>,<xref rid=\"R37\" ref-type=\"bibr\">37</xref></sup></p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Model</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">Effect Size (CI)</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Unadjusted</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−4.03 (−6.30, −1.76)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Marginal structural model<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−3.52 (−1.21, −5.74)</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Conditional model<sup>b</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">−3.48 (−1.27, −5.76)</td></tr></tbody></table><table-wrap-foot><fn fn-type=\"other\" id=\"tab3fn1\"><label>a</label><p>Estimated in a marginal structural model, by inverse weighting with the propensity for diuretic or beta blocker use given self-reported categorized body mass index (BMI).</p></fn><fn fn-type=\"other\" id=\"tab3fn2\"><label>b</label><p>Estimated in a conditional regression model with adjustment for self-reported categorical BMI.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Quantitative Bias Analysis</title><p>To inform the quantitative bias analysis, we need to make assumptions on the sensitivity and specificity of the self-reported BMI and that classification errors are nondifferential with respect to blood pressure and treatment. For the purpose of this illustration, we speculate ranges for the sensitivity and specificity of self-reported BMI category of 0.90 to 0.98. In practice, these parameters should be informed by reports in the literature and/or a researcher’s expert experience. Researchers may also decide to investigate how extreme the misclassification (measured using sensitivity and specificity) would need to be to change the conclusions of their study. We refer to the Shiny application (introduced in the subsequent section) for other choices for the sensitivity and specificity of self-reported BMI category.</p><p>By uniformly sampling from the range of plausible values of <inline-graphic xlink:href=\"ede-31-796-i244.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i245.jpg\"/> and using the bias expressions (equations 3 and 4), a distribution of possible biases is obtained (eAppendix 2; <ext-link ext-link-type=\"uri\" xlink:href=\"http://links.lww.com/EDE/B698\">http://links.lww.com/EDE/B698</ext-link> for further details). The solid line in Figure <xref ref-type=\"fig\" rid=\"F5\">5</xref> shows the distribution of bias in an MSM-IPW. Mean bias is −0.31, and median bias is −0.30 (interquartile range, −0.40 to −0.20). We also considered sampling <inline-graphic xlink:href=\"ede-31-796-i246.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i247.jpg\"/> from a trapezoidal (with modes at one third and two thirds between the minimum and maximum) or a symmetrical triangular distribution. Sampling from these distributions results in mean bias approximately equal to when uniform sampling is applied, but with less spread (dashed and dotted line in Figure <xref ref-type=\"fig\" rid=\"F5\">5</xref>). This result suggests that the results in Table <xref rid=\"T3\" ref-type=\"table\">3</xref> are not affected much by the classification error in self-reported BMI category. In the NHANES, anthropometric measures were also taken by trained technicians. The average treatment effect when BMI measures taken by trained technicians were used instead of self-reported BMI measures is given in eAppendix 2; <ext-link ext-link-type=\"uri\" xlink:href=\"http://links.lww.com/EDE/B698\">http://links.lww.com/EDE/B698</ext-link>.</p><fig id=\"F5\" orientation=\"portrait\" position=\"float\"><label>FIGURE 5.</label><caption><p>Density of predicted bias due to classification error in self-reported BMI category in NHANES.<sup><xref rid=\"R36\" ref-type=\"bibr\">36</xref>,<xref rid=\"R37\" ref-type=\"bibr\">37</xref></sup> Bias in the average treatment effect of diuretics use compared with beta blocker use on mean systolic blood pressure by inverse weighting with the propensity for diuretic or beta blocker use given self-reported categorical BMI (MSM-IPW), and using a conditional linear regression with adjustment for self-reported categorical BMI. The specificity and sensitivity of self-reported BMI category range from 0.90 to 0.98 and are sampled from a uniform distribution, trapezoidal (with modes on one third and two third), and symmetrical triangular distribution.</p></caption><graphic xlink:href=\"ede-31-796-g017\"/></fig></sec></sec><sec><title>SHINY APPLICATION: AN ONLINE TOOL FOR STUDYING THE IMPACT OF A MISCLASSIFIED VARIABLE</title><p>We developed an online tool for creating bias plots (Figures <xref ref-type=\"fig\" rid=\"F2\">2</xref>–<xref ref-type=\"fig\" rid=\"F4\">4</xref>) and performing quantitative bias analyses (illustrated in the previous section), available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://lindanab.shinyapps.io/SensitivityAnalysis\">https://lindanab.shinyapps.io/SensitivityAnalysis</ext-link>. The bias plots can be used to predict the implications of classification error in a confounding variable in specific study settings by varying the strength of association between the confounding variable and treatment and between the confounding variable and outcome; prevalence of the confounding variable; and specificity and sensitivity of the misclassified confounding variable. The quantitative bias analysis can be used for studying the impact of classification error in a confounding variable at the analysis stage of a study and to investigate how sensitive conclusions are to the assumption of no classification error. These bias plots can also be used to inform decisions about measurement methods or choice of variables to be extracted in the planning stage of studies.</p></sec><sec sec-type=\"discussion\"><title>DISCUSSION</title><p>Inverse probability weighting and conditional models are both important and frequently used tools to adjust for confounding variables in observational studies. In this article, we derived expressions for the bias in the average treatment effect in an MSM-IPW and a conditional model. These expressions can inform quantitative bias analyses for bias due to a misclassified confounding variable.</p><p>Quantitative bias analysis of misclassified confounding variables is one example of quantitative bias analyses for observational epidemiologic studies. Several approaches exist to assess sensitivity of causal conclusions to unmeasured confounding.<sup><xref rid=\"R28\" ref-type=\"bibr\">28</xref>,<xref rid=\"R39\" ref-type=\"bibr\">39</xref>,<xref rid=\"R40\" ref-type=\"bibr\">40</xref></sup> These aim to quantify the impact of violations of the assumption of no unmeasured confounding, although our approach aims to quantify the impact of violations of the assumption that all confounding variables are measured without error.</p><p>Several methods have been proposed to adjust for measurement error in covariates in MSMs-IPW. Pearl<sup><xref rid=\"R41\" ref-type=\"bibr\">41</xref></sup> developed a general framework for causal inference in the presence of error-prone covariates, which yields weighted estimators in the case of a dichotomous confounding variable measured with error. The framework relies on a joint distribution of the outcome and the confounding variable. Conversely, the weighting method proposed by McCaffrey et al<sup><xref rid=\"R17\" ref-type=\"bibr\">17</xref></sup> does not require a model for the outcome. Additionally, regression calibration,<sup><xref rid=\"R42\" ref-type=\"bibr\">42</xref></sup> simulation-extrapolation,<sup><xref rid=\"R43\" ref-type=\"bibr\">43</xref>,<xref rid=\"R44\" ref-type=\"bibr\">44</xref></sup> and multiple imputation<sup><xref rid=\"R45\" ref-type=\"bibr\">45</xref></sup> have been proposed for correcting for measurement error in covariates of MSMs. These methods assume that the measurement error model is known, which may often be unrealistic. In this context, it is also important to mention previous studies of the impact of measurement error in the exposure or the end point in MSMs, which has been studied by Babanezhad et al<sup><xref rid=\"R46\" ref-type=\"bibr\">46</xref></sup> and Shu and Yi,<sup><xref rid=\"R47\" ref-type=\"bibr\">47</xref></sup> respectively.</p><p>If treatment is allocated based on an error-prone confounding variable, the treatment effect will not be biased (see DAG in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>A). However, investigators should be careful in concluding that covariate measurement error will not affect their analysis. Suppose that there is an unmeasured variable <inline-graphic xlink:href=\"ede-31-796-i248.jpg\"/> that acts as a confounding variable between the error-prone covariate <italic>L</italic>* and treatment <inline-graphic xlink:href=\"ede-31-796-i249.jpg\"/>. Conditioning on <italic>L</italic>* will then open a path between <inline-graphic xlink:href=\"ede-31-796-i250.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i251.jpg\"/> via. <inline-graphic xlink:href=\"ede-31-796-i252.jpg\"/> and thus confound the relation between <inline-graphic xlink:href=\"ede-31-796-i253.jpg\"/> and <inline-graphic xlink:href=\"ede-31-796-i254.jpg\"/>.</p><p>This article considered classification error in a dichotomous confounding variable in a point-treatment study with a continuous outcome. The same principles apply to measurement error in a categorical or continuous confounding variable or when multiple confounding variables are considered, although more elaborate assumptions should then be made.<sup><xref rid=\"R48\" ref-type=\"bibr\">48</xref></sup> Moreover, we assumed that the relation between exposure and outcome does not vary between strata of the confounding variable, i.e., that there is no treatment effect modification. Future research could extend our bias expressions by relaxing this simplifying assumption, therefore extending our results to more general settings.</p><p>MSMs-IPW are increasingly applied to longitudinal data to estimate the joint effects of treatment at multiple time points on a subsequent outcome, including time-dependent outcomes, addressing the problem of time-dependent confounding.<sup><xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R3\" ref-type=\"bibr\">3</xref></sup> There has been little work to understand or correct for the impact of misclassified or mismeasured confounding variables in this more complex setting. Our results extend directly to the time-dependent setting when the aim is to estimate the effect of a current treatment on a time-dependent outcome measured at the next time point.<sup><xref rid=\"R49\" ref-type=\"bibr\">49</xref></sup> An area for future work is to extend our results to the setting in which the aim is to estimate the joint effects of treatment at multiple time points and to the time-dependent setting with time-varying treatments and confounding variables. An additional factor to consider in the time-varying setting is the impact of stabilized versus unstabilized weights on the bias if both numerator and denominator of the stabilized weights involve conditioning on an error-prone covariate.</p><p>The bias expressions derived in this article can be used to assess bias due to classification error in a dichotomous confounding variable. If classification error in confounding variables is suspected, a quantitative bias analysis provides an opportunity to quantitatively inform readers on the possible impact of such errors on causal conclusions.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><supplementary-material content-type=\"local-data\" id=\"SD1\"><media id=\"s1\" content-type=\"document\" xlink:href=\"ede-31-796-s001.pdf\" mimetype=\"application\" mime-subtype=\"pdf\" orientation=\"portrait\" position=\"anchor\"/></supplementary-material></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p>L.N., R.H.H.G., and M.v.S. were supported by grants from the Netherlands Organization for Scientific Research (ZonMW-Vidi project 917.16.430) and Leiden University Medical Center. R.H.K. was supported by a Medical Research Council Methodology Fellowship (MR/M014827/1) and a UK Research and Innovation Future Leaders Fellowship (MR/S017968/1). This work received support from Stichting Jo Kolk Studiefonds and Leids Universiteits Fonds in the form of a travel grant</p></fn><fn fn-type=\"COI-statement\"><p>The authors report no conflicts of interest.</p></fn><fn fn-type=\"supplementary-material\"><p>Supplemental digital content is available through direct URL citations in the HTML and PDF versions of this article (<ext-link ext-link-type=\"uri\" xlink:href=\"www.epidem.com\">www.epidem.com</ext-link>).</p></fn><fn fn-type=\"other\"><p>Data and code availability: The data and code used for the simulation study and quantitative bias analysis have been made publicly and can be accessed via. <ext-link ext-link-type=\"uri\" xlink:href=\"www.github.com/LindaNab/memsm\">www.github.com/LindaNab/memsm</ext-link>.</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id=\"R1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Ann Bot</journal-id><journal-id journal-id-type=\"iso-abbrev\">Ann. Bot</journal-id><journal-id journal-id-type=\"publisher-id\">annbot</journal-id><journal-title-group><journal-title>Annals of Botany</journal-title></journal-title-group><issn pub-type=\"ppub\">0305-7364</issn><issn pub-type=\"epub\">1095-8290</issn><publisher><publisher-name>Oxford University Press</publisher-name><publisher-loc>US</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32185391</article-id><article-id pub-id-type=\"pmc\">PMC7523593</article-id><article-id pub-id-type=\"doi\">10.1093/aob/mcaa033</article-id><article-id pub-id-type=\"publisher-id\">mcaa033</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Original Articles</subject></subj-group><subj-group subj-group-type=\"category-taxonomy-collection\"><subject>AcademicSubjects/SCI01210</subject></subj-group></article-categories><title-group><article-title>Mechanisms behind species-specific water economy responses to water level drawdown in peat mosses</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Bengtsson</surname><given-names>Fia</given-names></name><xref ref-type=\"aff\" rid=\"AF0001\">1</xref><xref ref-type=\"corresp\" rid=\"c1\"/><!--<email>fia.bengtsson@ebc.uu.se</email>--></contrib><contrib contrib-type=\"author\"><name><surname>Granath</surname><given-names>Gustaf</given-names></name><xref ref-type=\"aff\" rid=\"AF0001\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Cronberg</surname><given-names>Nils</given-names></name><xref ref-type=\"aff\" rid=\"AF0002\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rydin</surname><given-names>Håkan</given-names></name><xref ref-type=\"aff\" rid=\"AF0001\">1</xref></contrib></contrib-group><aff id=\"AF0001\"><label>1</label>\n<institution>Department of Plant Ecology and Evolution, Evolutionary Biology Centre, Uppsala University</institution>, Norbyvägen 18D, SE-752 36 Uppsala, <country country=\"SE\">Sweden</country></aff><aff id=\"AF0002\"><label>2</label>\n<institution>Department of Biology, Lund University</institution>, Ecology Building, SE-22362 Lund, <country country=\"SE\">Sweden</country></aff><author-notes><corresp id=\"c1\">For correspondence. E-mail <email>fia.bengtsson@ebc.uu.se</email></corresp></author-notes><pub-date pub-type=\"ppub\"><month>7</month><year>2020</year></pub-date><pub-date pub-type=\"epub\" iso-8601-date=\"2020-03-18\"><day>18</day><month>3</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>18</day><month>3</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>126</volume><issue>2</issue><fpage>219</fpage><lpage>230</lpage><history><date date-type=\"received\"><day>19</day><month>3</month><year>2019</year></date><date date-type=\"rev-request\"><day>09</day><month>8</month><year>2019</year></date><date date-type=\"editorial-decision\"><day>25</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>2</month><year>2020</year></date><date date-type=\"corrected-typeset\"><day>01</day><month>4</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020. Published by Oxford University Press on behalf of the Annals of Botany Company.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"cc-by\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"mcaa033.pdf\"/><abstract><title>Abstract</title><sec id=\"s0100\"><title>Background and Aims</title><p>The ecosystem engineers <italic>Sphagnum</italic> (peat mosses) are responsible for sequestering a large proportion of carbon in northern peatlands. Species may respond differently to hydrological changes, and water level changes may lead to vegetation shifts in peatlands, causing them to revert from sinks to sources of carbon. We aimed to compare species-specific responses to water level drawdown within <italic>Sphagnum</italic>, and investigate which traits affect water economy in this genus.</p></sec><sec id=\"s0101\"><title>Methods</title><p>In a mesocosm experiment, we investigated how water level drawdown affected water content (WC) in the photosynthetically active apex of the moss and maximum quantum yield of photosystem II (i.e. <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub>) of 13 <italic>Sphagnum</italic> species. Structural traits were measured, and eight anatomical traits were quantified from scanning electron microscopy micrographs.</p></sec><sec id=\"s0102\"><title>Key Results</title><p>Mixed-effects models indicated that at high water level, large leaves were the most influential predictor of high WC, and at low water level WC was higher in species growing drier in the field, with larger hyaline cell pore sizes and total pore areas associated with higher WC. Higher stem and peat bulk density increased WC, while capitulum mass per area and numerical shoot density did not. We observed a clear positive relationship between <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> and WC in wet-growing species.</p></sec><sec id=\"s0103\"><title>Conclusions</title><p>While we found that most hummock species had a relatively high water loss resistance, we propose that some species are able to maintain a high WC at drawdown by storing large amounts of water at a high water level. Our result showing that leaf traits are important warrants further research using advanced morphometric methods. As climate change may lead to more frequent droughts and thereby water level drawdowns in peatlands, a mechanistic understanding of species-specific traits and responses is crucial for predicting future changes in these systems.</p></sec></abstract><kwd-group><kwd><italic>Sphagnum</italic></kwd><kwd>bulk density</kwd><kwd>moss water content</kwd><kwd>ecohydrology</kwd><kwd>hyaline cell</kwd><kwd>leaf anatomy</kwd><kwd>pore size</kwd><kwd>water retention</kwd></kwd-group><funding-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>Swedish Research Council</institution><institution-id institution-id-type=\"DOI\">10.13039/501100004359</institution-id></institution-wrap></funding-source><award-id>2015-05174</award-id></award-group></funding-group><counts><page-count count=\"12\"/></counts></article-meta></front><body><sec id=\"s1\"><title>INTRODUCTION</title><p>Climate change is projected to lead to regional changes in precipitation (<xref rid=\"CIT0029\" ref-type=\"bibr\">Meehl <italic>et al.</italic>, 2007</xref>). More frequent droughts and thereby water level drawdowns in peatlands (<xref rid=\"CIT0011\" ref-type=\"bibr\">Granath <italic>et al.</italic>, 2016</xref>) will affect carbon storage and sequestration (<xref rid=\"CIT0006\" ref-type=\"bibr\">Charman <italic>et al.</italic>, 2013</xref>) and possibly lead to vegetation shifts. In most Northern peatlands, the ground vegetation is dominated by peat mosses (<italic>Sphagnum</italic>) that engineer these ecosystems. To predict shifts in <italic>Sphagnum</italic> assemblages, we need to understand the relationships between species-specific plant traits and water economy in peatland ecosystems.</p><p>As ecosystem engineers, <italic>Sphagnum</italic> mosses acidify, sequester carbon and nutrients from and waterlog their surroundings, thereby forming their own habitats (<xref rid=\"CIT0040\" ref-type=\"bibr\">Rydin and Jeglum, 2013</xref>). A thick peat layer will store water and prevent the surface moss layer from drying out, and thereby maintain moss photosynthesis and sustain growth (<xref rid=\"CIT0048\" ref-type=\"bibr\">Waddington <italic>et al.</italic>, 2015</xref>). Each <italic>Sphagnum</italic> shoot bears a capitulum at the top, which is where the apical meristem is located and where photosynthesis occurs in the most recently formed and tightly packed branches. The optimum capitulum water content for <italic>Sphagnum</italic> photosynthesis, at least in measurements of detached capitula, lies between 650 and 1211 % of plant dry mass (<xref rid=\"CIT0003\" ref-type=\"bibr\">Bengtsson <italic>et al.</italic>, 2016</xref>). This is high in comparison with other bryophytes (<xref rid=\"CIT0050\" ref-type=\"bibr\">Wang and Bader, 2018</xref>). The rate of CO<sub>2</sub> uptake is reduced to 50 % as water content is reduced by about 50 % from this optimum (<xref rid=\"CIT0039\" ref-type=\"bibr\">Rydin, 1993</xref>; <xref rid=\"CIT0044\" ref-type=\"bibr\">Schipperges and Rydin, 1998</xref>).</p><p>\n<italic>Sphagnum</italic> species have different niches related to the height above the water table (HWT) at which they grow. These niches are phylogenetically constrained, with most species of subgenus (section) <italic>Acutifolia</italic> growing at a higher HWT (hummock species) and most subgenus <italic>Cuspidata</italic> species growing at a lower HWT (hollow species) (<xref rid=\"CIT0017\" ref-type=\"bibr\">Johnson <italic>et al.</italic>, 2015</xref>). The species that grow higher above the water table have adaptations to avoid and/or tolerate desiccation, while hollow species are dependent on wet periods to sustain growth (<xref rid=\"CIT0044\" ref-type=\"bibr\">Schipperges and Rydin, 1998</xref>; <xref rid=\"CIT0027\" ref-type=\"bibr\">Mazziotta, <italic>et al.</italic>, 2019</xref>). Hollow species cannot survive at higher positions, but occur scattered in hummocks together with hummock species, because they receive water from their neighbours (<xref rid=\"CIT0038\" ref-type=\"bibr\">Rydin, 1985</xref>; <xref rid=\"CIT0037\" ref-type=\"bibr\">Robroek <italic>et al.</italic>, 2007</xref>).</p><p>Although there is evidence of <italic>Sphagnum</italic> species being able to gradually increase tolerance to desiccation (<xref rid=\"CIT0014\" ref-type=\"bibr\">Hájek and Vicherová, 2013</xref>), their primary strategy is to avoid desiccation so that they can continue photosynthesizing in sunny conditions without damage to the photosynthetic apparatus (<xref rid=\"CIT0026\" ref-type=\"bibr\">Marschall and Proctor, 2004</xref>; <xref rid=\"CIT0012\" ref-type=\"bibr\">Hájek, 2014</xref>). This is achieved with a combination of large water storage capacity, high water retention, and capillary forces that replenish the evaporating surface water from below. The large amount of small extracellular spaces in hummock species profiles is thought to be the cause of efficient capillarity and water retention in these species (<xref rid=\"CIT0016\" ref-type=\"bibr\">Hayward and Clymo, 1982</xref>). In combination with long pendant branches that act as wicks, small pore spaces make it possible for hummock species to grow high above the water table even though they have no vascular tissue. Smaller pore sizes cause a higher bulk density (BD), i.e. more dry mass per volume (<xref rid=\"CIT0016\" ref-type=\"bibr\">Hayward and Clymo, 1982</xref>; <xref rid=\"CIT0009\" ref-type=\"bibr\">Golubev and Whittington, 2018</xref>). The BD of the <italic>Sphagnum</italic> surface layer (near-surface BD), <italic>Sphagnum</italic> litter (recently dead moss) and peat (more or less humified material) are considered key traits in studies about <italic>Sphagnum</italic> hydrology (<xref rid=\"CIT0016\" ref-type=\"bibr\">Hayward and Clymo, 1982</xref>; <xref rid=\"CIT0047\" ref-type=\"bibr\">Thompson and Waddington, 2008</xref>). Also, the numerical density is often described as a factor controlling the rate of water loss in <italic>Sphagnum</italic> since small, densely growing shoots decrease evaporation by forming a smooth surface (<xref rid=\"CIT0007\" ref-type=\"bibr\">Elumeeva <italic>et al.</italic>, 2011</xref>; <xref rid=\"CIT0021\" ref-type=\"bibr\">Laing <italic>et al.</italic>, 2014</xref>).</p><p>A key to the <italic>Sphagnum</italic> species’ ability to form and grow in peatlands is their hyaline cells, which are large, dead, tubular cells with strong walls with a capacity to hold water. It should be noted that approx. 90 % of the water content in <italic>Sphagnum</italic> carpets is held in extracellular pore spaces, and only around 10 % of the water is held in hyaline cells (<xref rid=\"CIT0016\" ref-type=\"bibr\">Hayward and Clymo, 1982</xref>). However, the water inside the hyaline cells is tightly held and is the last water to be lost by evaporation. In combination, the hyaline cells and the extracellular pore spaces create a large water-holding capacity, where plant mass only constitutes 2 % of the near-surface volume (<xref rid=\"CIT0016\" ref-type=\"bibr\">Hayward and Clymo, 1982</xref>).</p><p>In <italic>Sphagnum</italic> hyaline cells, water moves passively in and out through pores (<xref rid=\"CIT0025\" ref-type=\"bibr\">Malcolm, 1996</xref>). The pores may, however, affect water retention as well as capillarity, since smaller pores reduce water loss from the cell, while they also impede rehydration of the cells (<xref rid=\"CIT0023\" ref-type=\"bibr\">Lewis, 1988</xref>). The distribution of the pores on dorsal or ventral leaf sides renders them more or less exposed, and pores on either side could have different effects on water loss and replenishment. Similarly, the drought-sensitive chlorophyll cells can be exposed on one side more than the other. Species growing at a low HWT generally have chlorophyll cells more exposed on the dorsal side of the leaves. In the wet habitat this gives easier access of CO<sub>2</sub>, as diffusion is low in water. In contrast, species growing at a high HWT instead expose the chlorophyll cells on the ventral side of the leaf, which gives them more protection against desiccation (<xref rid=\"CIT0035\" ref-type=\"bibr\">Rice and Schuepp, 1995</xref>). However, this pattern is phylogenetically confounded, and empirical evidence for this mechanism is still lacking. Furthermore, protecting the chlorophyll cells by exposing them on the inside may decrease the risk of photoinhibition, which can be tracked by measuring <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub>, a variable known to be correlated with <italic>Sphagnum</italic> capitulum water content (<xref rid=\"CIT0012\" ref-type=\"bibr\">Hájek, 2014</xref>). <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> [maximum potential quantum yield of photosystem II (PSII)] is a proxy of stress on the photosynthetic apparatus, where a value below a species maximum (often between 0.7 and 0.8 in <italic>Sphagnum</italic> and varying among species, <xref rid=\"CIT0019\" ref-type=\"bibr\">Korrensalo <italic>et al.</italic>, 2016</xref>) indicates a damaged or downregulated PSII (<xref rid=\"CIT0030\" ref-type=\"bibr\">Murchie and Lawson, 2013</xref>).</p><p>In this study, we investigate the relationships between processes related to water economy of <italic>Sphagnum</italic>, and the traits potentially influencing these processes. We compare a large set of species from different habitats along the microtopographic (HWT) gradient. In a mesocosm experiment, we test how species differ in their ability to maintain capitulum water content as the water table is lowered, and how water content is related to <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub>. First, we hypothesize that the higher above the water table a species is growing in the field, the higher is its ability to retain capitulum water when the water table is lowered. Secondly, we ask whether differences in water-holding capacity and water retention among species are best explained by anatomical traits at the micro-scale, such as leaf size and pore area of leaves, or structural features at the macro-scale, such as surface-peat BD, <italic>Sphagnum</italic> shoot numerical density and the area-based mass of the capitulum section (CMA). Thirdly, we ask at what capitulum water content <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> starts to drop, and whether this differs between species depending on their HWT niche.</p></sec><sec sec-type=\"materials\" id=\"s2\"><title>MATERIALS AND METHODS</title><sec id=\"s3\"><title>Species and study sites</title><p>The field sampling was performed during the second half of August 2015, mainly at the mire complex Kulflyten in central-southern Sweden (59°54′N, 15°50′E), where the many <italic>Sphagnum</italic> niches make sampling of many species possible within the same ecosystem. Two species were sampled at a small rich fen, Glon, situated at the coastal land uplift area on the Swedish east coast (60°31′N, 17°55′E). The mean July and December temperatures, respectively, are 16.6 °C and –2.6 °C at Kulflyten, and 16.8 °C and –1.0 °C at Glon. Annual precipitation (1982–2013) averages 733 mm at Kulflyten and 649 mm at Glon (<xref rid=\"CIT0045\" ref-type=\"bibr\">SMHI, 2014</xref>).</p><p>We chose 13 <italic>Sphagnum</italic> species representing different niches along the wetness gradient of mires and three of the larger <italic>Sphagnum</italic> clades (subgenera: <italic>Acutifolia</italic>, <italic>Cuspidata</italic> and <italic>Sphagnum</italic>). At Kulflyten we included species growing on the open bog (<italic>S. fuscum</italic>, <italic>S. rubellum</italic>, <italic>S. balticum</italic>, <italic>S. tenellum</italic>, <italic>S. cuspidatum</italic>, <italic>S. majus</italic> and <italic>S. magellanicum</italic>), on the pine bog (<italic>S. magellanicum</italic>), in the slightly minerotrophic fen soak on the bog expanse (<italic>S. lindbergii</italic> and <italic>S. papillosum</italic>), in the lagg fen on the mire margin (<italic>S. fallax</italic> anf <italic>S. angustifolium</italic>) and in the spruce swamp forest surrounding the mire (<italic>S. girgensohnii</italic>). In the rich fen we sampled <italic>S. warnstorfii</italic>, which only occurs in rich fens, and <italic>S. fuscum</italic> from high, probably ombrotrophic hummocks<italic>. Sphagnum fuscum</italic> was sampled both from the bog and from the rich fen, and we refer to them as ‘<italic>S. fuscum</italic> bog’ and ‘<italic>S. fuscum</italic> fen’. <italic>Sphagnum magellanicum</italic> was sampled on the open bog and pine bog, and we refer to them as ‘<italic>S. magellanicum</italic> open’ and ‘<italic>S. magellanicum</italic> pine’. Recently <italic>S. magellanicum</italic> was sub-divided into several species, two of which are found in the northern hemisphere: <italic>S. medium</italic> and <italic>S. divinum</italic> (<xref rid=\"CIT0015\" ref-type=\"bibr\">Hassel <italic>et al.</italic>, 2018</xref>). Our <italic>S. magellanicum</italic> samples contain both species in both habitats (Kjell Ivar Flatberg, pers. comm.), so we treat them as <italic>S. magellanicum sensu lato</italic> (<italic>s.l.</italic>).</p></sec><sec id=\"s4\"><title>Sampling and experimental set-up</title><p>Each species was sampled in 4–5 patches, which generally overlap with patches used in <xref rid=\"CIT0003\" ref-type=\"bibr\">Bengtsson <italic>et al.</italic> (2016</xref>, <xref rid=\"CIT0004\" ref-type=\"bibr\">2018</xref>). At each patch, we carefully cut out a core from the mire surface and placed it in a PVC pipe (height = 21 cm, diameter = 16 cm).</p><p>After sampling, the cores were kept in plastic boxes in the shade of the lagg fen and supplied with water from the lagg. A week after the start of sampling, the cores were brought from the field to a climate room, where deionized water was added so that the moss capitula were 5 cm above the water table and vascular plants were cut from the surface. Temperature in the growth room was set to 15 °C and the photosynthetic photon flux density (PPFD) was 100–130 μmol m<sup>–2</sup> s<sup>–1</sup>. The cores were sprayed with deionized water every couple of days until the start of the experiment so that the capitula would be rinsed free from residues that would otherwise accumulate in the absence of rain and cause their branch tips to blacken. The cores were randomized in the lab with three or four cores per box, but in a way that the same species would not occur twice in a box. Prior to the experiment, a few samples needed extra peat added to the base to raise them to the top of the pipe, and some were cut (from the bottom) to lower the capitula to the height of the pipe.</p><p>During sampling at Kulflyten we measured HWT at each sampling patch, while for the fen we only had HWT data from 2012. As we also had HWT data from Kulflyten 2012, we estimated the fen HWT for 2015 by assuming the same between-year difference of HWT for the fen as for Kulflyten.</p></sec><sec id=\"s5\"><title>Water level drawdown experiment</title><p>The experiment was designed to test (1) the water-holding capacity of the <italic>Sphagnum</italic> mosses and their ability to retain water in their capitula; and (2) if water content in the capitulum is related to the stress response of PSII. The experiment started 1 week after the cores were brought to the lab. The water table was raised to 20 mm below the tips of the capitula and kept at that level for 4 d, to allow the shoots to equilibrate before measurements. The water level was then successively lowered to 50, 100, 150 and 200 mm below the capitula, and after each treatment level the mesocosms were allowed to equilibrate for 3 d before subsequent measurements. The water level was then lowered to the next level on the fourth day.</p><p>After equilibration with the water level, the lights were turned off, and after 30 min of dark acclimatisation we measured fluorescence yield (maximum quantum yield of PSII, <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub>; see <xref rid=\"CIT0010\" ref-type=\"bibr\">Granath <italic>et al.</italic>, 2009</xref> for details) with a pulse-modulated fluorometer under very weak light conditions holding the fibre optic cable steady 11 mm from the top of a capitulum (Mini-PAM photosynthesis yield analyser; Walz, Germany). Five capitula per sample had been individually marked with plastic toothpicks, and these were used for fluorescence yield measurements throughout the experiment. The measurements were then averaged per core for each water level.</p><p>The capitulum water content measurements were destructive: three samples per core were collected, and their positions were marked to avoid later samples being a neighbouring shoot. Each sample was immediately put in a small plastic water-tight vial of known weight. The vial was weighed with the sample and the sample was then dried (65 °C) and weighed, and water content (g g<sup>–1</sup> dry moss) was calculated.</p></sec><sec id=\"s6\"><title>Structural measurements</title><p>Once the experiments were finished, the peat cores were drained and dried until no further weight loss was detected (65 °C). We measured numerical density (ND; number of capitula per cm<sup>2</sup>) using a circle with a diameter of 4 cm, and calculated the mean from four different measurements on each core. The capitulum layer (top 1.5–2 cm) was cut off and weighed to give the area-based mass of the capitulum layer (g cm<sup>–2</sup>; referred to as capitulum mass area, CMA), which is the same variable as ‘capitulum biomass’ in <xref rid=\"CIT0021\" ref-type=\"bibr\">Laing <italic>et al.</italic> (2014)</xref> and similar to ‘canopy mass area’ used for non-<italic>Sphagnum</italic> mosses by <xref rid=\"CIT0049\" ref-type=\"bibr\">Waite and Sack (2010)</xref>. Additionally, bulk density (g cm<sup>–3</sup>) was measured in three cut out and dried sections: 5 cm immediately below the capitula (BD1), the next 5 cm below this (BD2) and the remaining core below (BD3).</p></sec><sec id=\"s7\"><title>Leaf trait measurements</title><p>We measured branch leaf traits from scanning electron microscopy (SEM) micrographs using the software ImageJ (Fiji; <xref rid=\"CIT0043\" ref-type=\"bibr\">Schindelin <italic>et al.</italic>, 2012</xref>). To obtain the images, we used moss material from one sample per species, collected in 2013 at the same patches as the experimental material. We used branches just beneath the capitulum of a shoot, and picked out leaves from the middle of a normal looking spreading branch. The moss leaves were mounted on aluminium stubs for SEM using glue – no other preparation of the material was performed – and images were acquired under 5000 V in the scanning electron microscope (Hitachi SU3500).</p><p>For each sample, we measured morphological traits on two leaves of which one showed the dorsal side and one showed the ventral side. Leaf length and width were estimated as the average from the two leaves (µm). On each leaf surface, 20 pores were randomly chosen and the area (µm<sup>2</sup>) and diameter (µm) in the longest direction of each of these pores were measured (<xref ref-type=\"fig\" rid=\"F1\">Fig. 1</xref>). The total number of pores on the leaf was counted, and the pore area was calculated as a percentage of the leaf area measured using the software ImageJ. Chlorophyll cell exposure (proportion of leaf area) was measured along the widest section of the leaves, and calculated as the summed widths of the exposed chlorophyll cells divided by the total leaf width. If parts of the section were not clearly hyaline or chlorophyll cells, they were excluded (<xref ref-type=\"fig\" rid=\"F1\">Fig. 1</xref>).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1.</label><caption><p>(A) A <italic>Sphagnum</italic> shoot with a capitulum at the top; side view. (B) Capitulum with apical meristem and tightly packed branches; view from above. (C) Branch with overlapping leaves (A–C, <italic>S. fallax</italic>). (D) Leaf, one cell layer thick, constituting hyaline and chlorophyll cells (<italic>S. fuscum</italic>; ventral side). (E) Leaf measurements in ImageJ illustrated by a ventral leaf side of <italic>S. fuscum</italic>. Leaf length and width were measured (mm). Along the widest section of the leaf (purple vertical line). the widths of exposed chlorophyll cells were measured. A grid was set up (teal) and grid squares were randomized to choose 20 pores (yellow numbers). Areas (µm<sup>2</sup>; purple) and diameters of pores along the leaf length were measured. Illustrations: Fia Bengtsson.</p></caption><graphic xlink:href=\"mcaa033f0001\"/></fig></sec><sec id=\"s8\"><title>Statistical analysis</title><p>We used a mixed-effects model to estimate species-specific responses in capitulum water content (WC) to water level drawdown, with an interaction specified for the fixed factors water level (WL) and species. Sample (i.e. core) ID was included as a random factor to account for repeated sampling and specified as varying intercept and slope. We extracted three WC responses to WL below the moss surface for each sample: WC at WL 20 mm (WC<sub>20</sub>), WC at WL 200 mm (WC<sub>200</sub>) and the slope of the regression lines between WC and WL during the lowering of the water level from 20 to 200 mm (WC<sub>slope</sub>, extracted from the mixed-effect model for each sample).</p><p>To quantify the influence of morphological and structural variables on WC<sub>slope</sub>, WC<sub>20</sub> and WC<sub>200</sub>, we fitted linear mixed models (LMMs) with species as a random factor. Bulk density and structural traits (e.g. ND) were recorded for all samples, while data on leaf anatomical variables were scored only at the species level (or species–environment combinations, e.g. <italic>S. magellanicum</italic>). Hence, we had predictors on both the sample and group level in our models, and many predictors were strongly intercorrelated. To test these predictors and explore possible alternative models, while considering the sample sizes and intercorrelations, we used a three-stage modelling approach where predictors were included based on the research questions and what we know about <italic>Sphagnum</italic> biology. First we defined the overall model structure specification. We avoided strong collinearity by only allowing either ND or CMA (sample-level variables), either leaf length or width, and only one anatomical character out of six in a model. For the response WC<sub>20</sub>, only the top bulk density section (BD1) was included since the lower sections were completely water saturated during this part of the experiment. Secondly, we identified four models per response variable to test the predictors we expected to be important based on (eco-)hydrological models (BD, ND or CMA) or earlier literature (leaf width, and leaf dorsal pore size or area as they are more exposed to evaporation compared with the ventral side). Leaf width, rather than leaf length, was chosen because a wider leaf makes it possible to hold more water by enabling a more curved leaf. Dorsal chlorophyll exposure was strongly correlated with pore area/size (<italic>r</italic> > 0.7), which are variables directly related to water storage (<xref rid=\"CIT0013\" ref-type=\"bibr\">Hájek and Beckett, 2008</xref>; <xref rid=\"CIT0028\" ref-type=\"bibr\">McCarter and Price, 2014</xref>), while chlorophyll exposure is not expected to affect water content (<xref rid=\"CIT0013\" ref-type=\"bibr\">Hájek and Beckett, 2008</xref>). We therefore decided to exclude these variables from the models, but they are included in the publicly available data set for future analyses. Thirdly, we ran all possible model combinations, given the defined model structure above, and ranked the models based on the AICc (Akaike information crtiterion corrected for sample size). The two models with the lowest AICc were included as best predicting models among our set of models (<xref rid=\"CIT0005\" ref-type=\"bibr\">Burnham and Anderson, 2004</xref>). All models within four AICc units from the lowest AICc model can be found in <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary data Table S1</xref>.</p><p>To investigate the relationship between <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> and water content for each species, we largely followed the approach described by <xref rid=\"CIT0013\" ref-type=\"bibr\">Hájek and Beckett (2008)</xref> and fitted an asymptotic function {<italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> = <italic>a </italic>− (<italic>a </italic>− <italic>b</italic>) × exp[−log<sub>e</sub>(<italic>c</italic>)] × WC} to the data. Here, WC is the measured water content, <italic>a</italic> is the asymptotic maximum <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> value, <italic>b</italic> is the intercept and <italic>c</italic> describes the relative increase in <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> while WC is increasing. This function is a slightly different asymptotic function from that which <xref rid=\"CIT0013\" ref-type=\"bibr\">Hájek and Beckett (2008)</xref> used but it fitted our data better and converges more easily. The function was fitted as a non-linear model using generalized least squares where a compound symmetry correlation structure was applied to account for within-sample dependence. The point when a lower water content in the capitula results in a decline of <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> was defined as 0.95<italic>a</italic> (<xref rid=\"CIT0013\" ref-type=\"bibr\">Hájek and Beckett, 2008</xref>). We used this point estimate of water content as a way to rank the species along the tissue water content variable to indicate in which order they are affected by losing water, rather than claiming that this is a stress threshold for each species.</p><p>All analyses were performed in the statistical software R v.3.4.0 (<xref rid=\"CIT0033\" ref-type=\"bibr\">R Core Team, 2017</xref>), and LMMs were fitted using the lme4 package (version 1.1-17; <xref rid=\"CIT0002\" ref-type=\"bibr\">Bates <italic>et al.</italic>, 2015</xref>). Classic residual checks were performed, and the response WC<sub>slope</sub> was multiplied by –1 and log<sub>e</sub> transformed to produce a homogenous error distribution. For the LMMs, <italic>R</italic><sup>2</sup> values (conditional and marginal) and AICc values were extracted using the package MuMIn (version 1.42.1, <xref rid=\"CIT0001\" ref-type=\"bibr\">Bartoń, 2018</xref>). For AICc values, we fitted the models with maximum likelihood, while restricted maximum likelihood (REML) was used to extract coefficient estimates and <italic>R</italic><sup>2</sup> values. The non-linear models were fitted using the gnls function in the nlme package (version 3.1-142, <xref rid=\"CIT0031\" ref-type=\"bibr\">Pinheiro <italic>et al.</italic>, 2019</xref>), and a pseudo-<italic>R</italic><sup>2</sup> was calculated based on the residuals (<xref rid=\"CIT0022\" ref-type=\"bibr\">Lefcheck, 2016</xref>): <italic>σ</italic><sub>fitted</sub>/(<italic>σ</italic><sub>fitted</sub> + <italic>σ</italic><sub>residuals</sub>). We used the function Anova in the package car to produce <italic>P</italic>-values for fixed factors (version 3.0-0, <xref rid=\"CIT0008\" ref-type=\"bibr\">Fox and Weisberg, 2011</xref>). Principal component analysis (PCA) of the leaf traits of the species was performed using the function prcomp in base R. Data is available from the Figshare repository, doi: 10.6084/m9.figshare.12034353.</p></sec></sec><sec id=\"s9\"><title>RESULTS</title><sec id=\"s10\"><title>Water content at water level drawdown</title><p>The water content measurements at 20 and 200 mm were uncorrelated (<italic>r </italic>= 0.11 for WC<sub>20</sub> vs. WC<sub>200</sub>; d.f. = 71, <italic>P</italic> = 0.4). We used the slope of the regression for each species between water content (WC) and water level (WL) (WC<sub>slope</sub>; <xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>) as an integrated response to changes in water level. By statistical necessity WC<sub>slope</sub> is correlated with the other measures; more strongly so for WC<sub>20</sub> (<italic>r</italic> = –0.84, <italic>P</italic> < 0.0001) than for WC<sub>200</sub> (<italic>r</italic> = 0.43, <italic>P</italic> < 0.001).</p><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2.</label><caption><p>Capitulum water content measured in samples of 13 <italic>Sphagnum</italic> species as the water table was lowered from 20 to 200 mm below the surface. Lines illustrate the linear relationship between capitulum water content and water level; the slope of the line defines the parameter WC<sub>slope</sub> (g g<sup>–1</sup> mm<sup>–1</sup>) at the species level; <italic>n</italic> = 4–5 per species. Note that in subsequent statistical analyses, individual values of WC<sub>slope</sub> were used for each sample. See <xref ref-type=\"fig\" rid=\"F3\">Fig. 3</xref> for the values of WC<sub>slope</sub>.</p></caption><graphic xlink:href=\"mcaa033f0002\"/></fig><p>Our fitted LMM identified differences between species in WC<sub>slope</sub> (species × WL interaction: <italic>F</italic><sub>15,277</sub> = 171.4, <italic>P</italic> < 0.0001). This model explained 91 % (<italic>R</italic><sup>2</sup><sub>conditional</sub>) of the total variation in capitulum WC, while species and WL together explained 84 % of the total variation (<italic>R</italic><sup>2</sup><sub>marginal</sub>), and WL alone explained 54 %. Ranking species’ WC response to WL (i.e. WC<sub>slope</sub>) according to their HWT positions in the field, from driest (hummock) to wettest (hollow), revealed that the lawn-hollow species had steeper slopes than the hummock species (<xref ref-type=\"fig\" rid=\"F3\">Fig. 3</xref>). There were a few exceptions though: the hollow-dwelling <italic>S. fallax</italic> and <italic>S. lindbergii</italic> had slopes more similar to the hummock species, and one of the driest growing species, <italic>S. magellanicum</italic> pine had the steepest negative slope of all species.</p><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Fig. 3.</label><caption><p>The slopes (g g<sup>–1</sup> mm<sup>–1</sup>) of the regression lines between capitulum water content and water level (WC<sub>slope</sub>; <xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>) in the water level drawdown experiment plotted with mean and 95 % confidence interval for each species. The species are ranked according to their mean height above the water table (HWT; shown on the right side of the figure). WC<sub>slope</sub> is based on five repeated measurements of 4–5 cores per species (<italic>n</italic> = 365).</p></caption><graphic xlink:href=\"mcaa033f0003\"/></fig></sec><sec id=\"s11\"><title>The effects of leaf traits and structural traits on water holding and water retention</title><p>We summarize the ranges and means of the measured structural and anatomical traits in Supplementary data <xref ref-type=\"supplementary-material\" rid=\"sup1\">Table S2</xref>. From our pre-defined models for each response, we found that, overall, the results were fairly robust to model specification (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Our pre-defined models (i.e. hypothesized models) predicted the responses WC<sub>20</sub> and WC<sub>slope</sub> better than they did WC<sub>200</sub>, and these models were similar to the models ranked highest according to AICc values (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). For WC<sub>20</sub>, the predictors in our four pre-defined models explained 50–55 % of total variation, for WC<sub>slope</sub> 51–66 % and for WC<sub>200</sub> 11–16 %.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1.</label><caption><p>Mixed-effects models of the responses WC<sub>20</sub>, WC<sub>200</sub> and WC<sub>slope</sub>, including as predictors: (1) one of the anatomical leaf traits (<italic>n</italic> = 15); (2) either leaf length or width (<italic>n</italic> = 15); (3) either capitulum mass per area (CMA) or shoot numerical density (ND) (<italic>n</italic> = 73); and (4) one or more of the bulk density (BD) sections (<italic>n</italic> = 73)</p></caption><table frame=\"vsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\">Models</th><th rowspan=\"1\" colspan=\"1\">Intercept</th><th rowspan=\"1\" colspan=\"1\">Dorsal pore area (%)</th><th rowspan=\"1\" colspan=\"1\">Dorsal pore size (µm)</th><th rowspan=\"1\" colspan=\"1\">Dorsal pore number</th><th rowspan=\"1\" colspan=\"1\">Ventral pore area (%)</th><th rowspan=\"1\" colspan=\"1\">Ventral pore size (µm)</th><th rowspan=\"1\" colspan=\"1\">Ventral pore number</th><th rowspan=\"1\" colspan=\"1\">Leaf length</th><th rowspan=\"1\" colspan=\"1\">Leaf width (mm)</th><th rowspan=\"1\" colspan=\"1\">CMA (g cm<sup>–2</sup>)</th><th rowspan=\"1\" colspan=\"1\">ND (cm<sup>–2</sup>)</th><th rowspan=\"1\" colspan=\"1\">BD1 (g cm<sup>–3</sup>)</th><th rowspan=\"1\" colspan=\"1\">BD2 (g cm<sup>–3</sup>)</th><th rowspan=\"1\" colspan=\"1\">BD3 (g cm<sup>–3</sup>)</th><th rowspan=\"1\" colspan=\"1\">\n<italic>R</italic>\n<sup>2</sup>m/ <italic>R</italic><sup>2</sup>c</th><th rowspan=\"1\" colspan=\"1\">d.f.</th><th rowspan=\"1\" colspan=\"1\">AICc</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Response: WC<sub>20</sub></td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Rank 1 AICc</td><td rowspan=\"1\" colspan=\"1\">12.25 ± 1.68</td><td rowspan=\"1\" colspan=\"1\">–0.12 ± 0.06</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">11.79 ± 2.13</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">107.5 ± 37.5</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">55/74</td><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">363.2</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Rank 2 AICc</td><td rowspan=\"1\" colspan=\"1\">14.90 ± 2.22</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.38 ± 0.17</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">8.69 ± 2.38</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.37 ± 0.21</td><td rowspan=\"1\" colspan=\"1\">118.7 ± 40.2</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">57/75</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">363.3</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 1</td><td rowspan=\"1\" colspan=\"1\">12.03 ± 1.84</td><td rowspan=\"1\" colspan=\"1\">–0.11 ± 0.06</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">11.56 ± 2.26</td><td rowspan=\"1\" colspan=\"1\">0.12 ± 0.35</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">108.1 ± 38.0</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">55/74</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">365.6</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 2</td><td rowspan=\"1\" colspan=\"1\">12.73 ± 1.77</td><td rowspan=\"1\" colspan=\"1\">–0.11 ± 0.06</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">11.22 ± 2.24</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.19 ± 0.21</td><td rowspan=\"1\" colspan=\"1\">120.8 ± 40.5</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">55/74</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">364.6</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 3</td><td rowspan=\"1\" colspan=\"1\">12.57 ± 2.27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.19 ± 0.15</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">12.51 ± 2.62</td><td rowspan=\"1\" colspan=\"1\">0.05 ± 0.37</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">112.1 ± 38.6</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">51/74</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">367.9</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 4</td><td rowspan=\"1\" colspan=\"1\">12.90 ± 2.07</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.16 ± 0.15</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">11.97 ± 2.67</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.15 ± 0.23</td><td rowspan=\"1\" colspan=\"1\">121.2 ± 41.5</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">50/74</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">367.4</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Response: log<sub>e</sub>WC<sub>slope</sub></td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Rank 1 AICc</td><td rowspan=\"1\" colspan=\"1\">–3.26 ± 0.19</td><td rowspan=\"1\" colspan=\"1\">–0.02 ± 0.01</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">1.25 ± 0.27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">10.2 ± 2.5</td><td rowspan=\"1\" colspan=\"1\">–8.6 ± 2.2</td><td rowspan=\"1\" colspan=\"1\">64/87</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">25.2</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Rank 2 AICc</td><td rowspan=\"1\" colspan=\"1\">–2.85 ± 0.27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.07 ± 0.02</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">1.38 ± 0.29</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">10.3 ± 2.5</td><td rowspan=\"1\" colspan=\"1\">–8.8 ± 2.2</td><td rowspan=\"1\" colspan=\"1\">62/87</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">26.3</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 1</td><td rowspan=\"1\" colspan=\"1\">–3.34 ± 0.22</td><td rowspan=\"1\" colspan=\"1\">–0.02 ± 0.01</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">1.29 ± 0.28</td><td rowspan=\"1\" colspan=\"1\">–0.01 ± 0.04</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">3.6 ± 4.6</td><td rowspan=\"1\" colspan=\"1\">9.2 ± 3.0</td><td rowspan=\"1\" colspan=\"1\">–8.2 ± 2.3</td><td rowspan=\"1\" colspan=\"1\">66/87</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">29.3</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 2</td><td rowspan=\"1\" colspan=\"1\">–3.33 ± 0.23</td><td rowspan=\"1\" colspan=\"1\">–0.02 ± 0.01</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">1.27 ± 0.28</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.003 ± 0.024</td><td rowspan=\"1\" colspan=\"1\">3.8 ± 4.8</td><td rowspan=\"1\" colspan=\"1\">9.0 ± 2.9</td><td rowspan=\"1\" colspan=\"1\">–8.1 ± 2.3</td><td rowspan=\"1\" colspan=\"1\">65/87</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">29.4</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 3</td><td rowspan=\"1\" colspan=\"1\">–3.20 ± 0.29</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.04 ± 0.02</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">1.46 ± 0.36</td><td rowspan=\"1\" colspan=\"1\">–0.01 ± 0.04</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">3.2 ± 4.7</td><td rowspan=\"1\" colspan=\"1\">9.5 ± 3.0</td><td rowspan=\"1\" colspan=\"1\">–8.5 ± 2.3</td><td rowspan=\"1\" colspan=\"1\">52/86</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">35.7</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 4</td><td rowspan=\"1\" colspan=\"1\">–3.22 ± 0.29</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.04 ± 0.02</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">1.44 ± 0.37</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">0.0004 ± 0.03</td><td rowspan=\"1\" colspan=\"1\">3.2 ± 4.9</td><td rowspan=\"1\" colspan=\"1\">9.3 ± 3.0</td><td rowspan=\"1\" colspan=\"1\">–8.5 ± 2.3</td><td rowspan=\"1\" colspan=\"1\">51/86</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">35.8</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Response: WC<sub>200</sub></td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">Rank 1 AICc</td><td rowspan=\"1\" colspan=\"1\">3.69 ± 1.92</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">0.32 ± 0.15</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">33.9 ± 13.9</td><td rowspan=\"1\" colspan=\"1\">19/64</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">336.1</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Rank 2 AICc</td><td rowspan=\"1\" colspan=\"1\">6.16 ± 1.17</td><td rowspan=\"1\" colspan=\"1\">0.10 ± 0.06</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">33.6 ± 13.9</td><td rowspan=\"1\" colspan=\"1\">17/64</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">337.2</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 1</td><td rowspan=\"1\" colspan=\"1\">5.47 ± 1.91</td><td rowspan=\"1\" colspan=\"1\">0.11 ± 0.06</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">0.92 ± 2.40</td><td rowspan=\"1\" colspan=\"1\">–0.01 ± 0.32</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">12.5 ± 40.5</td><td rowspan=\"1\" colspan=\"1\">1.4 ± 26.5</td><td rowspan=\"1\" colspan=\"1\">32.8 ± 20.2</td><td rowspan=\"1\" colspan=\"1\">16/66</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">346.9</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 2</td><td rowspan=\"1\" colspan=\"1\">5.27 ± 2.00</td><td rowspan=\"1\" colspan=\"1\">0.11 ± 0.06</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">1.05 ± 2.46</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">0.05 ± 0.21</td><td rowspan=\"1\" colspan=\"1\">10.7 ± 42.0</td><td rowspan=\"1\" colspan=\"1\">1.7 ± 26.0</td><td rowspan=\"1\" colspan=\"1\">32.8 ± 20.1</td><td rowspan=\"1\" colspan=\"1\">16/68</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">346.9</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 3</td><td rowspan=\"1\" colspan=\"1\">4.61 ± 2.27</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">0.21 ± 0.15</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.15 ± 2.65</td><td rowspan=\"1\" colspan=\"1\">0.05 ± 0.33</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">11.4 ± 40.8</td><td rowspan=\"1\" colspan=\"1\">–2.3 ± 26.3</td><td rowspan=\"1\" colspan=\"1\">36.0 ± 20.1</td><td rowspan=\"1\" colspan=\"1\">11/66</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">348.0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Model 4</td><td rowspan=\"1\" colspan=\"1\">4.70 ± 2.23</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">0.21 ± 0.16</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.07 ± 2.74</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">–0.01 ± 0.22</td><td rowspan=\"1\" colspan=\"1\">11.7 ± 42.3</td><td rowspan=\"1\" colspan=\"1\">–1.6 ± 25.9</td><td rowspan=\"1\" colspan=\"1\">36.1 ± 20.0</td><td rowspan=\"1\" colspan=\"1\">11/66</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">348.0</td></tr></tbody></table><table-wrap-foot><fn id=\"fn-0100\"><p>Each model estimate is shown as ± s.e. Species is included as a random factor (<italic>n</italic> = 15). The models are arranged under each response starting with the models resulting with the lowest AIC, and then our original/hypothesized models (see the Materials and Methods). Marginal and conditional <italic>R</italic><sup>2</sup>s (<italic>R</italic><sup>2</sup>m, <italic>R</italic><sup>2</sup>c) show the amount of the total variance that is explained by predictors, and predictors and species, respectively. AICc is a measure of model fit where the lowest value indicates the best relative fit</p></fn></table-wrap-foot></table-wrap><p>The canopy traits (ND and CMA) were not important predictors in our models, and the coefficients showed large standard errors (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). In contrast, leaf width was consistently important, except for WC<sub>200</sub>. Pore area (dorsal or ventral) was also a meaningful variable in our models but could be interchanged by ventral pore size for WC<sub>slope</sub> and WC<sub>200</sub>.</p><p>The bulk density variables, which were measured at different depths of the cores, showed strong effects on all WC responses (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). For WC<sub>slope</sub>, the two lower BD sections had larger effects in the pre-defined models than the top section, and the top section was not included in the highest ranked AICc models. For WC<sub>200</sub>, only BD3 was included in the top AICc models.</p></sec><sec id=\"s12\"><title>The relationship between <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> and capitulum WC</title><p>Species-specific asymptotic regression models showed an overall good fit of the relationship between <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> and capitulum WC (<xref ref-type=\"fig\" rid=\"F4\">Fig. 4</xref>). The species that had a poor fit, i.e. low pseudo-<italic>R</italic><sup>2</sup>, were species that lost little water during the experiment (mainly the hummock species). Their narrow range in WC made it difficult to fit a model that explains much of the variation. In contrast, some hollow species with a larger WC range showed good model fit, e.g. <italic>S. tenellum</italic> (pseudo-<italic>R</italic><sup>2</sup> = 92 %), <italic>S. majus</italic> (pseudo-<italic>R</italic><sup>2</sup> = 85 %) and <italic>S. cuspidatum</italic> (pseudo-<italic>R</italic><sup>2</sup> = 74 %) (<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary data Table S3</xref>). From these models, we calculated at what WC a decline of <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> occurred for each species. We found no clear relationship with HWT among species, but the variation among wet-dwelling species was higher than among the dry-dwelling species (<xref ref-type=\"fig\" rid=\"F5\">Fig. 5</xref>).</p><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Fig. 4.</label><caption><p>Capitulum water content as a predictor of chlorophyll fluorescence (<italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub>) under a water table drawdown. The lines show the fitted asymptotic regression line for each species based on five repeated measurements of 4–5 samples (<italic>n</italic> = 20–25 per panel).</p></caption><graphic xlink:href=\"mcaa033f0004\"/></fig><fig id=\"F5\" orientation=\"portrait\" position=\"float\"><label>Fig. 5.</label><caption><p>The capitulum water content when <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> starts to decline during desiccation, plotted against mean sampled HWT in the field for each species.</p></caption><graphic xlink:href=\"mcaa033f0005\"/></fig></sec></sec><sec id=\"s13\"><title>DISCUSSION</title><sec id=\"s14\"><title>WC at water level drawdown and adaptations to different HWT niches</title><p>We interpret our three water content responses to represent maximum water-holding capacity (WC<sub>20</sub>), water retention capacity (WC<sub>slope</sub>) and desiccation avoidance (WC<sub>200</sub>).</p><p>We expected hummock species to show desiccation avoidance during the water table drawdown as this prolongs the period in which they can continue to grow during dry spells. In line with our prediction, most species in our experiment that were sampled high above the water table retained water during drawdown, leading to high WC<sub>200</sub>, which fits the theory of hummock species avoiding desiccation (<xref rid=\"CIT0026\" ref-type=\"bibr\">Marschall and Proctor, 2004</xref>; <xref rid=\"CIT0012\" ref-type=\"bibr\">Hájek, 2014</xref>). Also, species with a higher maximum water-holding capacity lost more water during the WT drawdown, and consequently exhibited a similarly low WC<sub>200</sub> to the other species. However, there was one exception to the hollow–hummock pattern with slower water loss in hummock species; the hummock-growing <italic>S. magellanicum</italic> pine had a steep WC<sub>slope</sub>. We interpret this as an alternative strategy for a hummock species: to avoid desiccation by having a higher maximum water-holding capacity, rather than losing water at a slow pace. To our knowledge, there are no prior observations of this alternative strategy of hummock species for survival high above the water table.</p><p>\n<xref rid=\"CIT0007\" ref-type=\"bibr\">Elumeeva <italic>et al.</italic> (2011)</xref> found that although colonies of the hummock species <italic>S. fuscum</italic> lost water more slowly than hollow species, the evaporation rate was on a par with that of the hollow species <italic>S. lindbergii</italic>. We also found that <italic>S. fuscum</italic> behaves as expected for a hummock species, and that <italic>S. lindbergii</italic> behaved more like a hummock species than a hollow species in terms of water loss. Similarly, the hollow-dwelling species <italic>S. fallax</italic> behaved differently from other hollow species as it lost water to a similar degree as hummock species. If these water economy traits are important in the field, our finding should not be surprising as <italic>S. fallax</italic> and <italic>S. angustifolium</italic>, which is a hummock-growing species in the same part of the mire as <italic>S. fallax</italic>, are closely related and supports the hypothesis of phylogenetic constraint on niche evolution in <italic>Sphagnum</italic> (<xref rid=\"CIT0017\" ref-type=\"bibr\">Johnson <italic>et al.</italic>, 2015</xref>).</p></sec><sec id=\"s15\"><title>Factors affecting water holding and retention</title><p>We found that leaf width was among the most important traits increasing maximum water-holding capacity and water retention, as most models supported this trait as a predictor. For WC<sub>20</sub> and WC<sub>slope</sub>, leaf width was included in the models with the lowest AIC, while it was excluded from the top WC<sub>200</sub> models. Larger leaves can be associated with larger holding capacity, suggesting greater pore space between the leaves. As a comparison, <xref rid=\"CIT0041\" ref-type=\"bibr\">Såstad and Flatberg (1993)</xref> found evidence of shorter leaves in drier conditions within <italic>S. strictum</italic>, and that more curved leaves retained water more efficiently (i.e. the convexity of leaves, or how cucullate they are), and they argued that curvature is more likely to be the trait with most influence on water economy than leaf area.</p><p>Leaf curvature directly influences water-holding capacity by creating larger pore spaces that do not drain easily (<xref rid=\"CIT0025\" ref-type=\"bibr\">Malcolm, 1996</xref>). The subgenus <italic>Sphagnum</italic> has ‘hooded’ and strongly curved leaves that provide higher water holding-capacity. In the species we sampled, curvature and leaf size are strongly related. <italic>Sphagnum magellanicum</italic> pine had the greatest water-holding capacity and the fastest water loss, indicating a large pore space but mainly consisting of larger spaces that drain rapidly during water level drawdown (<xref rid=\"CIT0028\" ref-type=\"bibr\">McCarter and Price, 2014</xref>). This species had the second driest realized niche at our field sites (high hummocks in the pine bog), while <italic>S. magellanicum</italic> open grew closer to the water table on the open bog. This intraspecific variation does not seem to exactly fit the recent division of <italic>S. magellanicum</italic> into two species in Europe (<xref rid=\"CIT0015\" ref-type=\"bibr\">Hassel <italic>et al.</italic>, 2018</xref>), since both <italic>S. divinum</italic> and <italic>S. medium</italic> appeared in both habitats.</p><p>We speculate that hummock formation for this type of species in the pine bog without the high bulk density usually associated with hummock species is possible through leaf trait-driven high maximum water-holding capacity. In the pine bog, evaporation is lower as the tree canopy reduces incoming radiation and wind (<xref rid=\"CIT0048\" ref-type=\"bibr\">Waddington <italic>et al.</italic>, 2015</xref>). This makes it possible for <italic>S. magellanicum</italic> pine to take advantage of rain events through a large storage capacity, and, because competition for light is more important under a canopy, looser colonies and larger shoots are beneficial (<xref rid=\"CIT0027\" ref-type=\"bibr\">Mazziotta <italic>et al.</italic>, 2019</xref>). In open habitats, higher BD and a smoother moss surface are necessary, because higher BD results in stronger capillary forces and a smoother canopy hampers evaporation (<xref rid=\"CIT0007\" ref-type=\"bibr\">Elumeeva <italic>et al.</italic>, 2011</xref>; <xref rid=\"CIT0021\" ref-type=\"bibr\">Laing <italic>et al.</italic>, 2014</xref>; <xref rid=\"CIT0048\" ref-type=\"bibr\">Waddington <italic>et al.</italic>, 2015</xref>). The anatomical traits related to hyaline pore size and area were important for predicting water content and retention in both pre-defined models and those selected by AICc.</p><p>At lower water tables (lower water potential), the extracellular water pool will drain fast and the proportion of smaller pore spaces and their connectivity becomes important for the maintenance of water in the capitula (<xref rid=\"CIT0028\" ref-type=\"bibr\">McCarter and Price, 2014</xref>). This occurs around a WC of roughly 1000 % (<xref rid=\"CIT0016\" ref-type=\"bibr\">Hayward and Clymo, 1982</xref>; <xref rid=\"CIT0034\" ref-type=\"bibr\">Rezanezhad <italic>et al.</italic>, 2016</xref>), which suggests that our drawdown had less impact on intracellular water storage (here we include hyaline cells) as many species’ water content did not decrease far below 1000 %. Hyaline cell pore size and area, and chlorophyll cell placement have previously been discussed to affect the water loss of the hyaline cells (<xref rid=\"CIT0023\" ref-type=\"bibr\">Lewis, 1988</xref>), but otherwise the literature on the adaptive value of pore area is scarce. <xref rid=\"CIT0024\" ref-type=\"bibr\">Li <italic>et al.</italic> (1992)</xref> found a greater number of pores in dry conditions (within species). However, we did not detect any influence of pore numbers on water storage/retention, indicating that pore size/area should be further studied. In our models we did not include chlorophyll cell placement as it was strongly correlated with pore-related variables, but this is also a variable to explore in future studies. In addition, our PCA (<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary data Fig. S1</xref>) indicates that these traits aggregate at subgenus level, where small pore size and pore area and exposure of chlorophyll cells to the dorsal side are associated with species belonging to subgenus <italic>Cuspidata</italic>. Thus, it remains to be determined to what extent these leaf traits are mechanistically important for the water economy of <italic>Sphagnum</italic>, the plasticity in these trait values and the potential covariation with phylogeny.</p><p>Higher near-surface BD should increase capillarity and water retention, and thereby maintain a high capitulum WC during a water table drawdown. The top BD section (2–7 cm) strongly influenced water content at 2 cm water level, but was not important for predicting the water retention and content under water level drawdown. This is somewhat expected as the lower sections will determine the conductivity of the peat water column (smaller pore sizes means greater capillarity), thereby upholding capitulum WC in the event of drought (<xref rid=\"CIT0016\" ref-type=\"bibr\">Hayward and Clymo, 1982</xref>). More surprising was the weak influence of ND on WC. However, this may be a consequence of minimal air movement in the lab environment, while in the field ND increases water-holding capacity by decreasing the evaporation through increased surface evenness (<xref rid=\"CIT0007\" ref-type=\"bibr\">Elumeeva <italic>et al.</italic>, 2011</xref>; <xref rid=\"CIT0048\" ref-type=\"bibr\">Waddington <italic>et al.</italic>, 2015</xref>).</p><p>Geometric morphometrics are still not widely used for research on bryophyte traits although there are many potential applications (<xref rid=\"CIT0046\" ref-type=\"bibr\">Stanton and Reeb, 2016</xref>). For example, measuring traits on the convex <italic>Sphagnum</italic> leaves could be done more accurately using 3-D image processing. More advanced morphometric analyses could, in addition to more accurate measurements of anatomical traits, be used to estimate the intra- and extracellular holding capacity of a <italic>Sphagnum</italic> leaf (<xref rid=\"CIT0013\" ref-type=\"bibr\">Hájek and Beckett, 2008</xref>). In addition to the anatomical traits, traits at the shoot level, such as branch lengths and fascicle density along the stem, contribute to water economy traits (<xref rid=\"CIT0016\" ref-type=\"bibr\">Hayward and Clymo, 1982</xref>; <xref rid=\"CIT0042\" ref-type=\"bibr\">Såstad <italic>et al.</italic>, 1999</xref>). Morphometric analyses could also be used to estimate traits at the canopy scale, as has been done using laser scanning microscopy (<xref rid=\"CIT0036\" ref-type=\"bibr\">Rice <italic>et al.</italic>, 2005</xref>). Traits at all these levels have bearing on the adaptations to different heights above the water table.</p></sec><sec id=\"s16\"><title>Changes in chlorophyll fluorescence related to water content</title><p>Being able to sustain photosynthesis at lower water content and the ability to restore photosynthesis after severe droughts are manifestations of desiccation tolerance. Here, we used <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> as an indicator of water stress in <italic>Sphagnum</italic> as chlorophyll fluorescence has been used as a functional trait in <italic>Sphagnum</italic> before, for example in <xref rid=\"CIT0020\" ref-type=\"bibr\">Laine <italic>et al.</italic> (2011)</xref>, <xref rid=\"CIT0018\" ref-type=\"bibr\">Kangas <italic>et al.</italic> (2014)</xref> and <xref rid=\"CIT0019\" ref-type=\"bibr\">Korrensalo <italic>et al.</italic> (2016)</xref>.</p><p>\n<italic>F</italic>\n<sub>v</sub>/<italic>F</italic><sub>m</sub> could be modelled from water content quite well in hollow species, while in hummock species the water table drawdown did not affect water content enough to cause substantial effects on <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> (<xref ref-type=\"fig\" rid=\"F4\">Fig. 4</xref>). These results support the idea of hummock species not only resisting desiccation, but also tolerating it. These stabilizing processes result in high variation in the <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> response to water table drawdown among species growing high above the water table, and high among species growing closer to the water table (<xref ref-type=\"fig\" rid=\"F5\">Fig. 5</xref>). The environment on hummocks is very constrained, which limits how species can respond to change, whereas in hollows species multiple strategies are possible. However, it should be noted that we performed a short-term experiment and the capitulum water content may need to be low for a longer time to damage PSII. Some mosses, particularly the hummock species, may also have ‘hardened’ and developed desiccation tolerance (<xref rid=\"CIT0014\" ref-type=\"bibr\">Hájek and Vicherová, 2013</xref>) during the slow desiccation in these species. This may have contributed to the relatively small decreases in chlorophyll fluorescence that we found.</p></sec><sec id=\"s17\"><title>Conclusions</title><p>We found overall support for the hypothesis that there is a negative correlation between HWT position in the field and capitulum water retention under water table drawdown. Furthermore, we suggest that higher water content under drought can be achieved either by slow water loss or by a high maximum water-holding capacity. We found that chlorophyll fluorescence is linked to water content, but in our models this relationship was only strong for hollow species.</p><p>We found some evidence that anatomical traits explain the water economy responses. In particular, leaf width was important for maximum water-holding capacity and water retention, which we attributed to leaf convexity and large extracellular spaces. Cell-level traits linked to hyaline pore size and pore area also appeared to influence water-holding capacity and retention. However, leaf and cell trait variation was largely correlated with species identity, which limits mechanistic inference, and further studies are needed to test our results. Bulk density largely controlled the water retention curve and capitulum water content, but bulk density at different depths affected different responses. We conclude that quantification of anatomical traits with advanced morphometric methods is worth pursuing to improve our mechanistic understanding of the ecohydrology of <italic>Sphagnum</italic>-dominated peatlands.</p></sec></sec><sec sec-type=\"supplementary-material\" id=\"s18\"><title>SUPPLEMENTARY DATA</title><p>Supplementary data are available online at <ext-link ext-link-type=\"uri\" xlink:href=\"https://academic.oup.com/aob\">https://academic.oup.com/aob</ext-link> and consist of the following: Table S1: mixed-effects models of the responses WC<sub>20</sub>, WC<sub>200</sub> and WC<sub>slope</sub>. Table S2: the ranges and means of the traits measured for 13 <italic>Sphagnum</italic> species. Table S3: species-specific asymptotic regression models were fitted for the relationship between <italic>F</italic><sub>v</sub>/<italic>F</italic><sub>m</sub> and water content. Figure S1: PCA of the leaf traits of the <italic>Sphagnum</italic> species.</p><supplementary-material content-type=\"local-data\" id=\"sup1\"><label>mcaa033_suppl_Supplementary_Material</label><media xlink:href=\"mcaa033_suppl_supplementary_material.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><sec id=\"s0104\"><title>FUNDING</title><p>This study was supported by the Swedish Research Council (contract 2015-05174 to H.R.) and by a scholarship from The Royal Swedish Academy of Sciences to F.B.</p></sec></body><back><ack id=\"a1\"><title>ACKNOWLEDGEMENTS</title><p>Thanks to Joseph Anderson, Paul Eisenblätter, Fei Mengjie, Aaron and Idunn Wan, who helped in the field and lab, and Magni Olsen Kyrkjeeide and Kjell Ivar Flatberg who checked whether our <italic>S. magellanicum s.l.</italic> samples morphologically fit <italic>S. divinum</italic> or <italic>S. medium</italic>. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Access Microbiol</journal-id><journal-id journal-id-type=\"hwp\">acmi</journal-id><journal-id journal-id-type=\"publisher-id\">acmi</journal-id><journal-title-group><journal-title>Access Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">2516-8290</issn><publisher><publisher-name>Microbiology Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005869</article-id><article-id pub-id-type=\"pmc\">PMC7523622</article-id><article-id pub-id-type=\"publisher-id\">000105</article-id><article-id pub-id-type=\"doi\">10.1099/acmi.0.000105</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Chalcones, stilbenes and ketones have anti-infective properties via inhibition of bacterial drug-efflux and consequential synergism with antimicrobial agents</article-title><alt-title alt-title-type=\"recto-page-foot\"><ext-link ext-link-type=\"uri\" xlink:href=\"http://acmi.microbiologyresearch.org\">http://acmi.microbiologyresearch.org</ext-link></alt-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-1438-1241</contrib-id><name><surname>Hellewell</surname><given-names>Lauren</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-1165-9295</contrib-id><name><surname>Bhakta</surname><given-names>Sanjib</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><aff id=\"aff1\">\n<label><sup>1</sup>​</label>\n<institution content-type=\"department\">Division of Biosciences</institution>, <institution>Institue of Structual and Molecular Biology, University College London</institution>, <addr-line content-type=\"city\">London, WC1E 6PT</addr-line>, <country>UK</country>\n</aff><aff id=\"aff2\">\n<label><sup>2</sup>​</label>\n<institution content-type=\"department\">Department of Biological Sciences</institution>, <institution>Institute of Structural and Molecular Biology, Birkbeck, University of London</institution>, <addr-line content-type=\"city\">London, WC1E 7HX</addr-line>, <country>UK</country>\n</aff></contrib-group><author-notes><corresp id=\"cor1\"><bold>*Correspondence:</bold> Sanjib Bhakta, <email xlink:href=\"mailto:s.bhakta@bbk.ac.uk\">s.bhakta@bbk.ac.uk</email></corresp></author-notes><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>18</day><month>2</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>18</day><month>2</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>2</volume><issue>4</issue><elocation-id>acmi000105</elocation-id><history><date date-type=\"received\"><day>03</day><month>10</month><year>2019</year></date><date date-type=\"accepted\"><day>21</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 The Authors</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"acmi-2-105.pdf\"/><abstract><p>With antimicrobial resistance creating a major public health crisis, the designing of novel antimicrobial compounds that effectively combat bacterial infection is becoming increasingly critical. Interdisciplinary approaches integrate the best features of whole-cell phenotypic evaluation to validate novel therapeutic targets and discover new leads to combat antimicrobial resistance. In this project, whole-cell phenotypic evaluation such as testing inhibitors on bacterial growth, viability, efflux pump, biofilm formation and their interaction with other drugs were performed on a panel of Gram-positive, Gram-negative and acid-fast group of bacterial species. This enabled additional antimicrobial activities of compounds belonging to the flavonoid family including ketones, chalcones and stilbenes, to be identified. Flavonoids have received renewed attention in literature over the past decade, and a variety of beneficial effects of these compounds have been illuminated, including anti-cancer, anti-inflammatory, anti-tumour as well as anti-fungal and anti-bacterial. However, their mechanisms of action are yet to be identified. In this paper, we found that the compounds belonging to the flavonoid family exerted a range of anti-infective properties being identified as novel efflux pump inhibitors, whilst offering the opportunity to be used in combination therapy. The compound 2-phenylacetophenone displayed broad-spectrum efflux pump inhibition activity, whilst trans-chalcone, displayed potent activity against Gram-negative and mycobacterial efflux pumps causing inhibition higher than known potent efflux pump inhibitors, verapamil and chlorpromazine. Drug-drug interaction studies also highlighted that 2-phenylacetophenone not only has the potential to work additively with known antibacterial agents that affect the cell-wall and DNA replication but also trans-chalcone has the potential to work synergistically with anti-tubercular agents. Overall, this paper shows how whole-cell phenotypic analysis allows for the discovery of new antimicrobial agents and their consequent mode of action whilst offering the opportunity for compounds to be repurposed, in order to contribute in the fight against antimicrobial resistance.</p></abstract><kwd-group><kwd>antimicrobial resistance</kwd><kwd>efflux pumps</kwd><kwd>synergism</kwd><kwd>chalcone</kwd><kwd>stilbene</kwd><kwd>ketone</kwd><kwd>whole-cell phenotypic evaluation</kwd><kwd>new drug discovery</kwd></kwd-group><funding-group><award-group id=\"ID0EJEAG285\"><funding-source>GCRF</funding-source><award-id>105123-21</award-id><principal-award-recipient>Sanjib Bhakta</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OpenAccessEmbargo</meta-name><meta-value>0</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"s1\"><title>Impact statement</title><p>This whole-cell phenotypic evaluation-based research project aims to exploit a group of compounds for the early-stage drug discovery against a selected panel of WHO priority bacterial pathogens. Our goal was to identify ‘hits’ which would be worthy of further optimisation as adjuvants for existing antibiotic therapy. This project has provided the first step in addressing an important issue, i.e. the urgent need for alternative approaches to combat antimicrobial drug resistance (AMR).</p></sec><sec sec-type=\"intro\" id=\"s2\"><title> Introduction</title><p>Bacterial resistance is a serious problem that is a well-established public health concern [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]. Most of the antibiotics that are available in the market today were discovered in the golden antibiotic era; in the period from 1940’s to 1960’s [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]. It was a very successful period for discovering different classes of antibiotics; mostly antibiotics from natural sources, and also some synthetic analogues [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]. Most importantly, this period marked progress in medical microbiology that heightened our understanding of the bacteria itself and the molecular mechanisms of antibiotics. However, since then, only four new classes of antibiotics have been approved for sale [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. Pathogens have since developed a number of mechanisms which render said antibiotics unusable against certain bacterial infections [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]. Consequently, the majority of the new drugs currently in clinical settings are derivatives of the chemical classes of known antibiotics and are already met with underlying antibiotic resistance mechanisms [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]. Although there are strategies in place for controlling the expansion of antibiotic resistance, including the prevention of the misuse of antibiotics [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>], other novel tactics need to be brought forward, such as the development of new infectives from brand new chemical classes. Therefore, how do we accelerate the discovery pipeline of new drugs with novel mechanisms of antibiotic action? [<xref rid=\"R8\" ref-type=\"bibr\">8, 9</xref>] Drug discovery via whole-cell phenotypic evaluation has been the most successful method for discovering antibiotics with a novel chemical class such as cephalosporin, rifampicin and vancomycin. The Waksman platform is a well-known example of whole cell screening platform that was used to screen soil-derived <italic>Streptomycetes</italic> for antibacterial activity which has successfully discovered antibiotics such as streptomycin and neomycin [<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]. In addition, emerging methods using transcript quantification, public databases, combined with machine-learning tools, are providing ground-breaking new and alternative screening strategies by augmenting phenotypic screening results [<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]. Whole-cell evaluation offers a number of advantages over other drug discovery methods which includes the evaluation of a compounds’ efficiency in penetrating bacterial cell-walls and for discovering novel efflux inhibitors [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]. In addition, whole-cell evaluation is a sensible platform combined with target drug design for the discovery of compounds that are prodrug which require intracellular metabolism to become active [<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]. Alternative platforms primarily focusing on specific biomolecule targets, disregarding whole-cell screening, have therefore faced several challenges in new antibiotic development. Lastly, whole-cell phenotypic screening also allows for identification of various other mechanisms of action known antimicrobial agents could have. Many biologically active compounds have been shown to have more than one endogenous mechanism of action. This is termed by ‘pleiotropy’ which looks into the ability of the biologically active compounds to implement more than one mechanism of action [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]. With this knowledge, known and putative antimicrobial compounds belonging to the flavonoid family [<xref rid=\"R15\" ref-type=\"bibr\">15–19</xref>], were investigated in this study to evaluate their whole-cell phenotypic effect, with the aim to fill the existing research gap of identifying any additional mechanisms of action the compounds may possess.</p></sec><sec id=\"s3\"><title>Materials</title><sec id=\"s3-1\"><title>Bacterial strains</title><p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">Escherichia coli</ext-link>\n</named-content></italic> K12 (<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic>) strain, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">Bacillus subtilis</ext-link>\n</named-content></italic> strain 168 (<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">B. subtilis</ext-link>\n</named-content></italic>), <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">Mycobacterium smegmatis</ext-link>\n</named-content></italic> mc<sup>2</sup>155 ATCC700084 (<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic>), <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6319\">Mycobacterium aurum</ext-link>\n</named-content></italic> ATC:NC 10437 (<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6319\">M. aurum</ext-link>\n</named-content></italic>) and <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10880\">Mycobacterium bovis</ext-link>\n</named-content></italic> BCG Pasteur ATCC 35734 (<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10880\">M. bovis</ext-link>\n</named-content></italic> BCG), purchased from UK National Collection of Type Cultures (NCTC) were used as a representative for Gram-negative, Gram-positive and mycobacterium species.</p></sec><sec id=\"s3-2\"><title>Compound library</title><p>The compound library used in this study included; trimethoprim, methotrexate, pyrimethamine, 2-chloro-4,6-diamino-1,3,5-triazine, 2-phenylacetophenone, trans-chalcone, trans-stilbene, trans-stilbene oxide, luteolin, 2,4-diaminoquinazine and triamterene, <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1.</label><caption><p>The compound library used in this study. MW; molecular weight, HBD; hydrogen bond donors, HBA; hydrogen bond acceptors, LogP; partition coefficient. Compounds in this library fall into categories; diaminopyrimidines, diaminopteridines, diaminotriazines, diaminoquinazolines, flavonoids, stilbenes and deoxybenzoins. All compounds in this library adhere to Lipinski's rule of five</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Drug</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Structure</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Formula</p>\n</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Properties</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trimethoprim</bold>\n</p>\n<p>(<bold>Compound 1/C1</bold>)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<inline-graphic xlink:href=\"acmi-2-105-ilg001.jpg\"/>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>C<sub>14</sub>H<sub>18</sub>N<sub>4</sub>O<sub>3</sub>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Form: Powder</p>\n<p>MW: 290.32 g/mol</p>\n<p>HBD: 2</p>\n<p>HBA: 7</p>\n<p>LogP: 0.9</p>\n<p>Solvent: DMSO</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Methotrexate</bold>\n</p>\n<p>(<bold>Compound 2/C2</bold>)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<inline-graphic xlink:href=\"acmi-2-105-ilg002.jpg\"/>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>C<sub>20</sub>H<sub>22</sub>N<sub>8</sub>O<sub>5</sub>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Form: Yellow powder</p>\n<p>MW: 454.44 g/mol</p>\n<p>HBD: 5</p>\n<p>HBA: 12</p>\n<p>LogP: −1.9</p>\n<p>Solvent: DMSO</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Pyrimethamine</bold>\n</p>\n<p>(<bold>Compound 3/C3</bold>)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<inline-graphic xlink:href=\"acmi-2-105-ilg003.jpg\"/>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>C<sub>12</sub>H<sub>13</sub>CIN<sub>4</sub>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Form: Solid</p>\n<p>MW: 248.72 g/mol</p>\n<p>HBD: 2</p>\n<p>HBA: 4</p>\n<p>LogP: 2.7</p>\n<p>Solvent: DMSO</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2-chloro- 4,6 -diamino- 1,3,5 triazine</bold>\n</p>\n<p>(<bold>Compound 4/C4</bold>)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<inline-graphic xlink:href=\"acmi-2-105-ilg004.jpg\"/>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>C<sub>3</sub>H<sub>4</sub>CIN<sub>5</sub>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Form: Powder</p>\n<p>MW: 145.55 g/mol</p>\n<p>HBD: 2</p>\n<p>HBA: 3</p>\n<p>LogP: 0.25</p>\n<p>Solvent: DMSO</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2-phenylacetophenone</bold>\n</p>\n<p>(<bold>Compound 5/C5</bold>)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<inline-graphic xlink:href=\"acmi-2-105-ilg005.jpg\"/>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>C<sub>14</sub>H<sub>12</sub>O</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Form: light yellow crystalline powder</p>\n<p>MW: 196.249 g/mol</p>\n<p>HBD: 0</p>\n<p>HBA: 1</p>\n<p>LogP: 3.2</p>\n<p>Solvent: DMSO</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-chalcone</bold>\n</p>\n<p>(<bold>Compound 6/C6</bold>)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<inline-graphic xlink:href=\"acmi-2-105-ilg006.jpg\"/>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>C<sub>15</sub>H<sub>12</sub>O</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Form: Powder</p>\n<p>MW: 208.26 g/mol</p>\n<p>Solvent: DMSO</p>\n<p>HBD: 0</p>\n<p>HBA: 1</p>\n<p>LogP: 4.0</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-stilbene</bold>\n</p>\n<p>(<bold>Compound 7/C7</bold>)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<inline-graphic xlink:href=\"acmi-2-105-ilg007.jpg\"/>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>C<sub>14</sub>H<sub>12</sub>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Form: White crystalline powder</p>\n<p>MW: 180.25 g/mol</p>\n<p>Solvent: DMSO</p>\n<p>HBD: 0</p>\n<p>HBA: 0</p>\n<p>LogP: 4.8</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-stilbene oxide</bold>\n</p>\n<p>(<bold>Compound 8/C8</bold>)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<inline-graphic xlink:href=\"acmi-2-105-ilg008.jpg\"/>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>C<sub>14</sub>H<sub>12</sub>O</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Form: White crystalline powder</p>\n<p>MW: 196.24 g/mol</p>\n<p>Solvent: DMSO</p>\n<p>HBD: 0</p>\n<p>HBA: 1</p>\n<p>LogP: 3.5</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Luteolin</bold>\n</p>\n<p>(<bold>Compound 9/C9</bold>)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<inline-graphic xlink:href=\"acmi-2-105-ilg009.jpg\"/>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>C<sub>15</sub>H<sub>10</sub>O<sub>6</sub>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Form: Yellow powder</p>\n<p>MW: 286.24 g/mol</p>\n<p>Solvent: DMSO</p>\n<p>HBD: 4</p>\n<p>HBA: 6</p>\n<p>LogP: 2.5</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2,4- diaminoquinazine</bold>\n</p>\n<p>(<bold>Compound 10/C10</bold>)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<inline-graphic xlink:href=\"acmi-2-105-ilg010.jpg\"/>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>C<sub>8</sub>H<sub>8</sub>N<sub>4</sub>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Form: White powder</p>\n<p>MW: 160.18 g/mol</p>\n<p>Solvent: DMSO</p>\n<p>HBD: 2</p>\n<p>HBA: 4</p>\n<p>LogP: 1</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Triamterene</bold>\n</p>\n<p>(<bold>Compound 11/C11</bold>)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<inline-graphic xlink:href=\"acmi-2-105-ilg011.jpg\"/>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>C<sub>12</sub>H<sub>11</sub>N<sub>7</sub>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Form: Yellow powder</p>\n<p>MW: 253.269 g/mol</p>\n<p>Solvent: Formic acid</p>\n<p>HBD: 3</p>\n<p>HBA: 7</p>\n<p>LogP: 0.98</p>\n</td></tr></tbody></table></table-wrap><p>The controls (existing antibiotics) used in this study included; ampicillin, kanamycin, isoniazid, ethambutol and rifampicin.</p></sec></sec><sec sec-type=\"methods\" id=\"s4\"><title>Methods</title><sec id=\"s4-1\"><title>Resazurin microtiter plate assay (REMA)</title><p>The inhibitor compounds were further tested using a whole-cell phenotypic screen, REMA following modified [<xref rid=\"R20\" ref-type=\"bibr\">20, 21</xref>]. In brief an additional fluorescence reading step was included to optimise the method. This enabled elimination of any compound and dye interference. The reading of the florescence was done using a plate reader (FLUOstar OPTIMA, BMG Labtech, UK) with the settings as follows; 544 nm and 590 nm for excitation and detection of fluorescence (emission), gain 2200 and a temperature of 37 °C. The MIC was determined as the lowest concentration of the drug at which no growth of bacteria was observed. The experiment was performed in triplicate.</p></sec><sec id=\"s4-2\"><title>Cytotoxicity and selectivity index</title><p>For the evaluation of the cytotoxicity profiles an established laboratory protocol was followed. In brief, the cytotoxicity assay was performed in a 96-well plate. Two-fold serial dilutions were made by transferring 100 µl from the first row to the next row containing 100 µl of complete RPMI media. This step was repeated until the second last row as the last row was kept as a control in which no compound was added (media only). Each compound was tested in biological triplicates, using three separate cell culture lines. In each well, 100 µl of cell line (RAW264.7 or THP1), which had been passaged and normalised to a concentration of 5×10<sup>5</sup> cells ml<sup>−1</sup>, was added. The plate was then incubated in a CO<sub>2</sub> incubator. Following a 48 h incubation, the plate was washed with 1XPBS by pipetting and fresh complete RPMI was added to each well. Then 30 µl of freshly prepared resazurin solution at 0.01 % was added to the media and this plate was then incubated overnight in the CO<sub>2</sub> incubator. The following day, the colour change was observed, and fluorescence intensity was measured using FLUOstar OPTIMA microplate reader at λexc=540 nm, λemi=590 nm. The growth inhibitory concentration was determined based on a resazurin fluorescence assay. The 50 % growth inhibitory concentration (GIC<sub>50</sub>) is the concentration which gave fluorescence at the midpoint of the highest fluorescence detected and the lowest fluorescence.</p><p>The selectivity index/antibacterial selectivity was determined as the ratio between the GIC<sub>50</sub> on macrophage (RAW 264.7 and THP1) and the MIC observed in the REMA assays by using the formula: SI=GIC µg ml<sup>−1</sup>/MIC µg ml<sup>–1</sup>.</p></sec><sec id=\"s4-3\"><title>Efflux pump analysis</title><p>The EtBr efflux assay to test the inhibitor compounds (at ¼ × MIC) was performed in a 96-well flat bottom plate (Greiner Bio-One). Meanwhile for controls using known efflux inhibitor chlorpromazine and verapamil had a final concentration of ½ × MIC for each organism used.</p><p>The method was performed using the standard laboratory protocol. Bacterium cultures; <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">B. subtilis</ext-link>\n</named-content></italic>, <italic>M. smegmatis, M. aurum</italic> and <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10880\">M. bovis</ext-link>\n</named-content></italic> BCG were passaged at least twice, and grown over night until the OD<sub>600</sub> reached 0.8. The OD was adjusted to 0.4 by taking equal volumes of the culture and M7H9 (5 ml of each), making sure the final volume of the suspension remained at 10 ml. The cells were collected by centrifugation at a speed of 3000 <bold><italic>g</italic></bold> for 10 min after which the supernatant was removed by decanting. The pellet was resuspended with in 10 ml of sterile 1 × PSB, using a vortex to distribute the cells uniformly.</p><p>The test samples contained 500 µl of culture resuspended in 1 × PBS at OD 0.4, 2.5 µl of 80 % glucose (to keep the cells viable and metabolically active), 1.25 µg ml<sup>−1</sup> EtBr (as the substrate for the efflux pumps) and the compounds being tested were at ¼ × MIC concentrations. Blank samples contained all the components mentioned above except the bacterial suspension, which was replaced with 1 × PBS, required to correct the intrinsic florescence of the inhibitors. All the test samples and blanks were prepared in triplicate. The continuous reading of the florescence was done using a plate reader (FLUOstar OPTIMA, BMG Labtech, UK) with the settings as follows; 540 nm and 590 nm for excitation and detection of fluorescence (emission), gain 2200, a temperature of 37 °C and a cycle of measurement every 60 s for a total period of 60 min.</p></sec><sec id=\"s4-4\"><title>Biofilm inhibition analysis</title><p>Biofilm growth was started by thawing cryopreserved bacterial cell culture, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic>, followed by passaging three times in M7H9 supplemented with ADC and grown until OD reached the upper mid log phase (OD 2.0). This is when molecules within the bacterial population switch on a communication system known as quorum sensing. This culture was therefore used and diluted 1 : 100 times in Sauton’s media. This included triplicates of each concentration for each compound along-side triplicates of a positive control (diluted cell culture in Sauton’s media), a 1 % DMSO control (M7H9 supplemented with ADC, diluted cell culture and 20 ul of DMSO) and a negative control (M7H9 supplemented with ADC alone). Then 2 ml of the diluted cell culture was added to each Eppendorf tube along with specific volumes of each compound to make up certain concentration ranges. Three compounds were tested at varying concentrations; trans-chalcone 500–31.25 µg ml<sup>−1</sup>, 2-phenylacetophenone 1000–125 µg ml<sup>−1</sup> and trans-stilbene oxide 1000–125 µg ml<sup>−1</sup>. The plastic test tubes were then incubated for 5 days at 35 ˚C, then biofilm formation was then examined for direct, visible phenotypic changes to the biofilm.</p></sec><sec id=\"s4-5\"><title>Drug-drug interaction</title><p>Compounds showing potent efflux pump inhibition on each Gram-positive, Gram-negative and mycobacterium models used in this study, were used in combination with clinically relevant compounds. Synergy testing was performed using the checkerboard assay [<xref rid=\"R22\" ref-type=\"bibr\">22–25</xref>]. This was used to determine the interaction and potency of two test articles when used concurrently. This method determined the effect on potency of the combination of antibiotics in comparison to their individual activities. This comparison was then represented as the Fractional Inhibitory Concentration (FIC) index value.</p><p>To quantify the interactions between the antimicrobial and efflux inhibitors being tested, the FIC index (the combination of antibiotics that produced the greatest change from the individual antimicrobials MIC) value is calculated for each strain and antibiotic combination: A/MIC<sub>A</sub>+B/MIC<sub>B</sub>=FIC<sub>A</sub>+ FI<sub>CB</sub>=FIC index, where A and B are the MIC of each antimicrobial in combination (in a single well), and MIC<sub>A</sub> and MIC<sub>B</sub> are the MIC of each drug individually. This FIC index value was then used to categorise the interaction of the two antibiotics tested [<xref rid=\"R26\" ref-type=\"bibr\">26</xref>].</p></sec></sec><sec sec-type=\"results\" id=\"s5\"><title>Results</title><sec id=\"s5-1\"><title>Minimum inhibitory concentration values</title><p>The minimum inhibitory concentration values displayed for the known compounds; trimethoprim, methotrexate and pyrimethamine when tested against the panel of bacterium varied from <0.98 to >500 µg ml<sup>−1</sup>. This was in line with what had previously been reported using various independent screening techniques. The other selected chemical entities (chalcones, stilbenes and ketones) within this study also displayed poor growth inhibitory activities when tested on each bacterial model, except trans-chalcone which was found to be selectively potent against slow-growing mycobacteria. Triamterene was abandoned from the study at this point as its dissolvent, formic acid, was causing bacterial growth inhibition instead of the compound itself, <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>.</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2.</label><caption><p>MIC values of compounds of interest investigated using REMA analysis; against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> K12, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">B. subtilis</ext-link>\n</named-content></italic> strain 168, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic> mc<sup>2</sup> 155<italic>, M. aurum</italic> ATC: NC 10437 and <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10880\">M. bovis</ext-link>\n</named-content></italic> BCG. ‘–‘ represents ‘not determined’; ‘*’ represents ‘abandoned due to insolubility’</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Compound</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>MIC (µg/mL)</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic>\n</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">B. subtilis</ext-link>\n</named-content></italic>\n</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic>\n</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6319\">M. aurum</ext-link>\n</named-content></italic>\n</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10880\">M. bovis</ext-link>\n</named-content></italic> BCG</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trimethoprim</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p><0.98</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.91</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>7.81</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>62.5</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>500</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Methotrexate</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>250</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>125</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Pyrimethamine</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>25</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>12.5</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.13</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.13</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>50</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2-chloro-4,6-diamino-1,3,5-triazine</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>250</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2-phenylacetophenone</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>250</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-chalcone</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>125</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>62.5</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>15.60</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-stilbene</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-stilbene oxide</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>250</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Luteolin</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2,4-diaminoquinazoline</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>62.50</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>250</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>250</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>250</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>250</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Triamterene</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>*</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>*</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>*</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>*</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>*</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Isoniazid</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>15.60</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p><0.98</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p><0.98</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Ampicillin</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1.95</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p><0.98</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Kanamycin</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>15.63</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>7.81</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr></tbody></table></table-wrap></sec><sec id=\"s5-2\"><title>Cytotoxicity and therapeutic potential evaluated</title><p>All test compounds investigated in this study were found poor in terms of their bacterial growth inhibitory properties and revealed a low selectivity index (SI) when tested against both THP-1 and RAW 264.7 cell lines. From these results the GIC<sub>50</sub> and selectivity index for each compound was established, <xref rid=\"T3 T4\" ref-type=\"table\">Tables 3 and 4</xref>. As expected, trimethoprim, methotrexate and pyrimethamine had high SI scores for all bacterium used in this study. This correlates with previously published results and were already on clinical trial.</p><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3.</label><caption><p>GIC<sub>50</sub> scores</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Compound</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RAW 264.7 Marine Macrophage Cell Line</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>THP-1 Mammalian Macrophage Cell Line</p>\n</th></tr><tr><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>GIC<sub>50</sub> (µg/ml)</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>GIC<sub>50</sub> (µg/ml)</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trimethoprim</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Methotrexate</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Pyrimethamine</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>100</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>100</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2-chloro-4,6-diamino-1,3,5-triazine</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>125</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2-phenylacetophenone</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>125</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>250</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-chalcone</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>7.81</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>15.63</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-stilbene</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-stilbene oxide</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>125</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>500</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Luteolin</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>31.25</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>500</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2,4-diaminoquinazoline</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>125</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>125</p>\n</td></tr></tbody></table></table-wrap><table-wrap id=\"T4\" orientation=\"portrait\" position=\"float\"><label>Table 4.</label><caption><p>Selectivity index (SI) of each compound. RAW 264.7 and THP-1 cell line selectivity index values are displayed below for each bacterium for the two cell lines used against a representative of Gram-ve, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> (<italic>Ec</italic>); Gram +ve, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">B. subtilis</ext-link>\n</named-content></italic> (<italic>Bs</italic>) and Acid-fast bacilli, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10880\">M. bovis</ext-link>\n</named-content></italic> BCG (<italic>Mb</italic>)</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Compounds</p>\n</th><th align=\"center\" valign=\"top\" colspan=\"3\" rowspan=\"1\">\n<p>SI (RAW 264.7 Cell Line)</p>\n</th><th align=\"center\" valign=\"top\" colspan=\"3\" rowspan=\"1\">\n<p>SI (THP-1 Cell Line)</p>\n</th></tr><tr><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Ec</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Bs</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Mb</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Ec</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Bs</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Mb</italic>\n</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trimethoprim</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>510</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>510</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>510</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>510</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Methotrexate</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>4</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>4</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>4</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>4</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Pyrimethamine</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>16</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>16</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2-chloro-4,6-diamino-1,3,5-triazine</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.25</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.25</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.5</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2-phenylacetophenone</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.25</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.5</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.5</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-chalcone</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.02</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.02</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.5</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.03</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.03</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-stilbene</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Trans-stilbene oxide</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.25</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.25</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Luteolin</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.06</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.06</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.06</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2,4-diaminoquinazoline</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.5</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.5</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.5</p>\n</td></tr></tbody></table></table-wrap></sec><sec id=\"s5-3\"><title>Identification of novel efflux pump inhibitors</title><p>Compounds 2-phenylacetophenone, trans-chalcone, trans-stilbene and trans-stilbene oxide were found to inhibit the accumulation assay at ¼ MIC for a variety of Gram-positive, Gram-negative and mycobacterial species, having a better effect than two known efflux pump inhibitors, verapamil and chlorpromazine. From these results a lead compound was established which displayed the highest inhibitory activity for each bacterium. The 2-phenylacetophenone displayed broad-spectrum activity causing high inhibitory readings for the Gram-negative representative, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic>, as well as the mycobacterium species; <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6319\">M. aurum</ext-link>\n</named-content></italic> and <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10880\">M. bovis</ext-link>\n</named-content></italic> BCG. Whilst 2-phenylacetophenone matched the efflux pump inhibition activity of known control chlorpromazine when tested against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> in the first 30 min, the compound displayed significantly higher efflux inhibitory activity compared to controls chlorpromazine and verapamil when used against the mycobacterium species (<xref ref-type=\"fig\" rid=\"F1 F2 F3 F4\">Figs 1–4</xref>). Trans-chalcone displayed high efflux pump inhibition within the first 30 min of analysis against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">B. subtilis</ext-link>\n</named-content></italic> which matched known control chlorpromazine, <xref ref-type=\"fig\" rid=\"F5\">Fig. 5</xref>. All other compounds displayed negative results, no efflux pump inhibition matching the bacterial control or causing little efflux pump inhibition.</p><fig fig-type=\"figure\" id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1.</label><caption><p>Accumulation assay using EtBr with <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> K12. The bacterial control consisted of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> K12 cells with growth supplements and ethidium bromide (EtBr).</p></caption><graphic xlink:href=\"acmi-2-105-g001\"/></fig><fig fig-type=\"figure\" id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Fig. 4.</label><caption><p>Accumulation assay using EtBr with <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10880\">M. bovis</ext-link>\n</named-content></italic> BCG. The bacterial control consisted of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10880\">M. bovis</ext-link>\n</named-content></italic> BCG Pasteur cells with growth supplements and ethidium bromide (EtBr).</p></caption><graphic xlink:href=\"acmi-2-105-g002\"/></fig><fig fig-type=\"figure\" id=\"F5\" orientation=\"portrait\" position=\"float\"><label>Fig. 5.</label><caption><p>Accumulation assay using EtBr with <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">B. subtilis</ext-link>\n</named-content></italic> Strain 168. The bacterial control consisted of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">B. subtilis</ext-link>\n</named-content></italic> Strain 168 cells with growth supplements and ethidium bromide (EtBr).</p></caption><graphic xlink:href=\"acmi-2-105-g003\"/></fig><fig fig-type=\"figure\" id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2.</label><caption><p>Accumulation assay using EtBr with <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic> mc<sup>2</sup> 155. The bacterial control consisted of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic> mc<sup>2</sup> 155 cells with growth supplements and ethidium bromide (EtBr).</p></caption><graphic xlink:href=\"acmi-2-105-g004\"/></fig><fig fig-type=\"figure\" id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Fig. 3.</label><caption><p>Accumulation assay using EtBr with <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6319\">M. aurum</ext-link>\n</named-content></italic> ATC: NC 10437. The bacterial control consisted of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6319\">M. aurum</ext-link>\n</named-content></italic> ATC: NC 10437 cells with growth supplements and ethidium bromide (EtBr).</p></caption><graphic xlink:href=\"acmi-2-105-g005\"/></fig></sec><sec id=\"s5-4\"><title>Selected inhibitors ablated mycobacterial biofilm formation</title><p>Significant inhibition of biofilm formation for <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic> was seen at 2× MIC of log phase cells for 2-phenylacetophenone and trans-chalcone. Lower concentrations of each compound did not change cell viability, but physiological changes were observed, indicating that some endogenous mechanisms were influenced, <xref ref-type=\"fig\" rid=\"F6\">Fig. 6</xref>.</p><fig fig-type=\"figure\" id=\"F6\" orientation=\"portrait\" position=\"float\"><label>Fig. 6.</label><caption><p>Biofilm inhibition study using <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic> mc<sup>2</sup> 155 cells against trans-chalcone, 2-phenylacephenone and trans-stilbene oxide. Positive control (+ve); <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic> cells and growth media alone, negative control (-ve); growth media alone. The red arrow indicates biofilm formation.</p></caption><graphic xlink:href=\"acmi-2-105-g006\"/></fig></sec><sec id=\"s5-5\"><title>Potent novel efflux pump and biofilm formation inhibitors displayed synergistic properties</title><p>Compounds showing significant efflux pump activity were analysed for their synergistic activities. The compound 2-phenylacetophenone displayed potent efflux pump activity for <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> and therefore was tested for interactions with kanamycin and ampicillin. Potent activity for this compound was also seen when used against mycobacterial models in this study and therefore was used in combination with ethambutol, isoniazid and rifampicin against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic>. Trans-chalcone displayed potent efflux activity which was also seen across all bacterial models used in this study and was used in combination with kanamycin and ampicillin against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> and <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">B. subtilis</ext-link>\n</named-content></italic> as well as with ethambutol, isoniazid and rifampicin against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic>. Trans-stilbene showed potent activity against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">B. subtilis</ext-link>\n</named-content></italic> and so was used in combination with kanamycin and ampicillin to measure their interaction. Lastly, trans-stilbene oxide displayed potent efflux pump activity against mycobacterium models used in this study and so was used in combination with ethambutol, isoniazid and rifampicin against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic>. The FIC index value was used to interpret what effect each compound combination was having on each bacterium; a value ≤0.5 displayed synergistic properties, >displayed additive properties whilst values of 1–4 and >4 were shown to have indifferent and antagonistic properties, <xref rid=\"T5\" ref-type=\"table\">Table 5</xref>. The 2-phenylacetophenone had additive effects when used in conjunction with kanamycin as well as ampicillin when used against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic>. Trans-chalcone and trans-stilbene, however, had an antagonistic effect when used in conjunction with kanamycin and ampicillin against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">B. subtilis</ext-link>\n</named-content></italic>. Also 2-phenylacetophenone had synergistic effects when used with ethambutol and isoniazid against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic>, whilst having additive properties when used in conjunction with rifampicin. Trans-stilbene oxide, however, displayed either indifferent or antagonistic results when used in conjunction with anti-mycobacterial agents against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic>. To our interest, trans-chalcone displayed synergistic properties when used with all clinically relevant anti-mycobacterial agents used in this study, ethambutol, isoniazid and rifampicin when used against <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6403\">M. smegmatis</ext-link>\n</named-content></italic>.</p><table-wrap id=\"T5\" orientation=\"portrait\" position=\"float\"><label>Table 5.</label><caption><p>Synergy analysis with compounds showing potent efflux pump inhibition activity and known antibiotics. The FIC index (the combination of antibiotics that produced the greatest change from the individual antimicrobials MIC) value is calculated for each strain and antibiotic combination: A/MIC<sub>A</sub>+B/MIC<sub>B</sub>=FIC<sub>A</sub>+ FI<sub>CB</sub>=FIC Index. This FIC Index value was then used to categorise the interaction of the two antibiotics tested. <italic>Ec; E. coli, Bs; B. subtilis, Ms; M. smegmatis</italic>\n</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Clinically relevant compounds</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Putative inhibitors</p>\n</th><th align=\"center\" valign=\"top\" colspan=\"3\" rowspan=\"1\">\n<p>FIC Index Value</p>\n</th></tr><tr><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Ec</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Bs</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Ms</italic>\n</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"3\" colspan=\"1\">\n<p>\n<bold>Kanamycin</bold>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2-phenylacetophenone</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1.00</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>Trans-chalcone</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>4</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Trans-stilbene</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>4</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"3\" style=\"background:#CCCCCC\" colspan=\"1\">\n<p>\n<bold>Ampicillin</bold>\n</p>\n</td><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>2-phenylacetophenone</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.99</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Trans-chalcone</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>4</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>Trans-stilbene</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>4</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"3\" colspan=\"1\">\n<p>\n<bold>Ethambutol</bold>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2-phenylacetophenone</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.38</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>Trans-chalcone</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.38</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Trans-stilbene oxide</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>4</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"3\" style=\"background:#CCCCCC\" colspan=\"1\">\n<p>\n<bold>Isoniazid</bold>\n</p>\n</td><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>2-phenylacetophenone</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.26</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Trans-chalcone</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.5</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>Trans-stilbene oxide</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>>4</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"3\" colspan=\"1\">\n<p>\n<bold>Rifampicin</bold>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2-phenylacetophenone</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.56</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>Trans-chalcone</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" style=\"background:#CCCCCC\" rowspan=\"1\" colspan=\"1\">\n<p>0.38</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Trans-stilbene oxide</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4.00</p>\n</td></tr></tbody></table></table-wrap></sec></sec><sec sec-type=\"discussion\" id=\"s6\"><title>Discussion</title><p>The overall aim of this study was to evaluate compounds using whole-cell phenotypic assays in order to establish any other possible mechanisms of action known and putative inhibitory compounds exhibited on a panel of Gram-positive, Gram-negative and mycobacterial species. Whilst discoveries on the molecular basis of disease provides numerous pathways in identifying new antibiotics, developing completely new antimicrobial compounds takes a significant amount of effort, money and time. Therefore, it is critical to improve these strategies to reduce both the time frame and costs of drug discovery, with the answer being drug repurposing. The use of unsuitable models and methodologies often leads to the disregard of molecules from drug development pipelines in spite of these molecules harbouring important/relevant properties that may not necessarily be anti-bacterial but may have the capacity to potentiate current treatment/reverse resistance. Therefore, it is essential that in parallel to extracting and synthesising novel chemical entities, methodologies for testing these molecules are optimised to glean maximum information on their effects against microbes, making sure that all available avenues are explored to pump-prime new antibiotic discovery, development and drug-repositioning [<xref rid=\"R27\" ref-type=\"bibr\">27</xref>].</p><p>Efflux pumps play an important role in antibiotic resistance as well as in biofilm formation [<xref rid=\"R28\" ref-type=\"bibr\">28, 29</xref>]. Consequently, results in this paper are significant in the fight against antibiotic resistance, displaying various chemical classes such as; chalcones, ketones and stilbenes, to have potential activity against such mechanisms. These are classes which have not been previously identified as efflux pump inhibitors. From efflux pump inhibition combined with synergism analysis it can be deduced that 2-phenylacetophenone has the potential to work synergistically/additively with known antibacterial agents that affect the cell wall and DNA replication across Gram-negative and mycobacterial species. Whilst trans-chalcone has the potential to work synergistically with mycobacterial agents. These results ultimately display how deoxybenzoins derivatives and chalcone chemical classes have the potential to work in combination with various known antimicrobial agents to enhance treatment and help eradicate antibacterial resistance which has not previously been looked into. However, further validation would be needed across a wider range of bacterial species, or in combination with other antibacterial agents. Another significant result includes the identification of new compounds that inhibit biofilm development could enable decreasing tuberculosis treatment that further will have a global impact in reduction of antibiotic resistance due to compliance with TB long term treatment [<xref rid=\"R30\" ref-type=\"bibr\">30, 31</xref>]. To enhance the validity of the biofilm method, it would be more appropriate to compare the MIC of stationary phase cells to biofilm MIC values. This is because as stationary phase cells are more drug tolerant for many reasons, such as having a slow metabolism, the MIC of stationary phase cells would differ from that of logarithmic phase cells. When intertwined with literature evidence that biofilms originate from a stationary population, it would therefore be more appropriate to compare MIC values between stationary phase cells and biofilms than logarithmic phase cells [<xref rid=\"R32\" ref-type=\"bibr\">32–34</xref>]. To further validation of the biofilm assay, future work would include biochemical analysis of the biofilm formation. Looking into the chemical nature of the biofilm would indicate the changing properties of the biofilm and possibly reveal which endogenous mechanism is being influenced by the specific compounds mentioned in this paper.</p><p>Unfortunately, all putative compounds looked into in this study displayed higher toxicity results. However, this was an expected result for many chemical classes involved in this study as many are labelled as anti-cancer drugs, such as chalcones and stilbenes [<xref rid=\"R35\" ref-type=\"bibr\">35, 36</xref>]. This therefore leaves the question; with other mechanisms of action being highlighted within this piece of research, could these compounds classes be chemically re-engineered to have lower toxicity rates and be used in a variety of ways? Compounds that target bacterial growth, efflux pump activity and biofilm formation are of great importance for combating and eradicating bacterial infections from a broad range of species. Specific compounds present in this study exhibit all of these mechanisms.</p><p>This study has highlighted the importance of whole-cell phenotypic evaluation in identifying other possible mechanisms of actions for inhibitor molecules whilst indicating how the chemical classes of ketones, chalcones and stilbenes have the potential to help in the fight against antimicrobial resistance being novel efflux pump and biofilm inhibitors.</p></sec></body><back><ack id=\"ack1\"><title>Funding information</title><p>This project was partly supported by a Global Challenges Research Fund to SB at Birkbeck.</p></ack><ack id=\"ack2\"><title>Acknowledgements</title><p>Authors would like to thank Parmiss Akra Ahmed for critically reading the manuscript, Dr Arundhati Maitra, Liam Tom Martin for helpful discussions and Marie Maugueret-Minerve, the Microbiology Laboratory Manager for her technical assistance. SB is a Cipla Distinguished Fellow in Pharmaceutical Sciences.</p></ack><ack id=\"ack3\"><title>Author contributions</title><p>SB and LH conceived and designed the project. LH performed all the experiments and recorded observation in SB’s mycobacteria research laboratory at Birkbeck, University of London; LH and SB interpreted results and wrote the manuscript jointly.</p></ack><ack id=\"ack4\"><title>Conflicts of interest</title><p content-type=\"COI-statement\">The authors declare that there are no conflicts of interest.</p></ack><fn-group><fn id=\"FN1\"><p>Abbreviations: ADC, albumin dextrose catalase; AMR, antimicrobial resistance; BCG, bacillus Calmette-Guérin; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; EtBr, ethidium bromide; FIC, fractional inhibitory concentration; GIC, growth inhibitory concentration; M7H9, Middlebrook; MIC, minimum inhibitory concentration; NCTC, National Collection of Type Cultures; OD, optical density; PBS, phosphate-buffered saline; REMA, resazurin microtiter plate assay; RPMI, Roswell Park Memorial Insitute; SI, selectivity index; WHO, World Health 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Access Microbiol</journal-id><journal-id journal-id-type=\"hwp\">acmi</journal-id><journal-id journal-id-type=\"publisher-id\">acmi</journal-id><journal-title-group><journal-title>Access Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">2516-8290</issn><publisher><publisher-name>Microbiology Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005867</article-id><article-id pub-id-type=\"pmc\">PMC7523623</article-id><article-id pub-id-type=\"publisher-id\">000103</article-id><article-id pub-id-type=\"doi\">10.1099/acmi.0.000103</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>First hybrid complete genome of <italic>Aeromonas veronii</italic> reveals chromosome-mediated novel structural variant <italic>mcr-3.30</italic> from a human clinical sample</article-title><alt-title alt-title-type=\"recto-page-foot\"><ext-link ext-link-type=\"uri\" xlink:href=\"http://acmi.microbiologyresearch.org\">http://acmi.microbiologyresearch.org</ext-link></alt-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-8667-7132</contrib-id><name><surname>Ragupathi</surname><given-names>Naveen Kumar Devanga</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"equal\" rid=\"equal-contrib1\">†</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sethuvel</surname><given-names>Dhiviya Prabaa Muthuirulandi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"equal\" rid=\"equal-contrib1\">†</xref></contrib><contrib contrib-type=\"author\"><name><surname>Anandan</surname><given-names>Shalini</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Murugan</surname><given-names>Dhivya</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Asokan</surname><given-names>Kalaiarasi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Neethi Mohan</surname><given-names>Ramya Gajaraj</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Vasudevan</surname><given-names>Karthick</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-5545-606X</contrib-id><name><surname>D</surname><given-names>Thirumal Kumar</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-5971-8290</contrib-id><name><surname>C</surname><given-names>George Priya Doss</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Veeraraghavan</surname><given-names>Balaji</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><aff id=\"aff1\">\n<label><sup>1</sup>​</label>\n<institution content-type=\"department\">Department of Clinical Microbiology</institution>, <institution>Christian Medical College</institution>, <addr-line content-type=\"city\">Vellore – 632004</addr-line>, <country>India</country>\n</aff><aff id=\"aff2\">\n<label><sup>2</sup>​</label>\n<institution content-type=\"department\">School of Bio Sciences and Technology</institution>, <institution>Vellore Institute of Technology</institution>, <addr-line content-type=\"city\">Vellore – 632014</addr-line>, <country>India</country>\n</aff></contrib-group><author-notes><fn fn-type=\"equal\" id=\"equal-contrib1\"><label>†</label><p>These authors contributed equally to this work</p></fn><corresp id=\"cor1\"><bold>*Correspondence:</bold> Balaji Veeraraghavan, <email xlink:href=\"mailto:esbl@cmcvellore.ac.in\">esbl@cmcvellore.ac.in</email></corresp><fn fn-type=\"accession-no\"><p>This complete genome project has been deposited at GenBank under the accession number CP032839 (Plasmid accession number CP032840).</p></fn></author-notes><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>17</day><month>2</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>2</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>2</volume><issue>4</issue><elocation-id>acmi000103</elocation-id><history><date date-type=\"received\"><day>19</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>13</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 The Authors</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"acmi-2-103.pdf\"/><abstract><p>Recent findings demonstrate the origin of the plasmid-mediated colistin resistance gene <italic>mcr-3</italic> from aeromonads. The present study aimed to screen for plasmid-mediated colistin resistance among 30 clinical multidrug-resistant (MDR) <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> spp. PCR was used to screen for the presence of <italic>mcr-1</italic>, <italic>mcr-2</italic>, <italic>mcr-3</italic> and <italic>mcr-4</italic>, which revealed <italic>mcr-3</italic> in a colistin-susceptible isolate (FC951). All other isolates were negative for <italic>mcr</italic>. Sequencing of FC951 revealed that the <italic>mcr-3</italic> (<italic>mcr-3.30</italic>) identified was different from previously reported variants and had 95.62 and 95.28 % nucleotide similarity with <italic>mcr-3.3</italic> and <italic>mcr-3.10</italic>. Hybrid assembly using IonTorrent and MinION reads revealed structural genetic information for <italic>mcr-3.30</italic> with an insertion of IS<italic>As18</italic> within the gene. Due to this, <italic>mcr-3.30</italic> was non-expressive, which makes FC951 susceptible to colistin. Further, <italic>in silico</italic> sequence and protein structural analysis confirmed the new variant. To the best of our knowledge, this is the first report on a novel <italic>mcr-3</italic> variant from India. The significant role of <italic>mcr</italic>-like genes in different <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> species remains unknown and requires additional investigation to obtains insights into the mechanism of colistin resistance.</p></abstract><kwd-group><kwd><italic>mcr-3</italic></kwd><kwd>colistin</kwd><kwd><italic>Aeromonas</italic></kwd><kwd>IS<italic>As18</italic></kwd><kwd><italic>bla</italic>OXA-12</kwd><kwd><italic>bla</italic>CEPH-A3</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>OpenAccessEmbargo</meta-name><meta-value>0</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>\n<italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> spp. are ubiquitous and are known to cause gastroenteritis, wound infections and septicaemia; they are commonly known as ‘jacks of all trades’. Aeromonads are universally resistant to the penicillin group of antibiotics (penicillin, ampicillin, carbenicillin and ticarcillin) and are generally susceptible to tetracyclines and quinolones [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]. Recently, increasing resistance to third-generation cephalosporins and carbapenems has been reported [<xref rid=\"R2\" ref-type=\"bibr\">2, 3</xref>].</p><p>The recent discovery of plasmid-mediated colistin resistance genes (<italic>mcr</italic>) has attracted global attention. A study reported that the <italic>mcr-3</italic> identified in <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">Escherichia coli</ext-link>\n</named-content></italic> is similar to the gene present in <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> species and suggested that it might have originated from aeromonads [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. It should be noted that most <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> species are susceptible to colistin, whereas <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3058\">Aeromonas jandaei</ext-link>\n</named-content></italic> and <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">Aeromonas hydrophila</ext-link>\n</named-content></italic> have been reported to be intrinsically resistant to polymyxins [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. However, the role of <italic>mcr</italic>-like genes in different <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> species is not clearly understood and requires further investigation.</p><p>This study examined the presence of plasmid-mediated colistin resistance among <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> spp. using PCR and structural analysis with next-generation sequencing. The nucleotide sequences obtained using experimental methods were translated into a protein sequence, and the 3D structure was modelled using <italic>in silico</italic> approaches to understand the structural changes in the different variants of <italic>mcr</italic>.</p></sec><sec sec-type=\"methods\" id=\"s2\"><title>Methods</title><sec id=\"s2-1\"><title>Isolates and identification</title><p>A total of 30 <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> spp. isolated from stool specimens collected from January to December 2017 from symptomatic patients attending the Christian Medical College, Vellore were included in the study. Isolation and identification of the genus and species were carried out using a standard culture and biochemical tests [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>].</p></sec><sec id=\"s2-2\"><title>Antimicrobial susceptibility testing (AST)</title><sec id=\"s2-2-1\"><title>Disc diffusion</title><p>AST testing was carried out using the Kirby–Bauer disk diffusion method. The antimicrobial agents tested were trimethoprim/sulfamethoxazole (1.25/23.75 µg), tetracycline (30 µg), ciprofloxacin (5 µg), cefotaxime (30 µg), imipenem (10 µg) and meropenem (10 µg) (Oxoid, UK). Quality control (QC) strains (<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10789\">Klebsiella pneumoniae</ext-link>\n</named-content></italic> ATCC 700603, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.2553\">Pseudomonas aeruginosa</ext-link>\n</named-content></italic> ATCC 27853 and <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> ATCC 25922) were included in all batches, as recommended by the Clinical and Laboratory Standards Institute (CLSI-M45) [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>].</p></sec></sec><sec id=\"s2-3\"><title>Minimum inhibitory concentration (MIC)</title><p>The colistin MIC was determined for the studied isolates by broth microdilution and interpreted using the CLSI 2017 breakpoint recommendation [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]. <italic>mcr-1</italic>-positive <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> with the expected range 4–8 µg ml<sup>−1</sup>, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> ATCC 25922 (0.25–2 µg ml<sup>−1</sup>) and <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.2553\">P. aeruginosa</ext-link>\n</named-content></italic> ATCC 27853 (0.5–4 µg ml<sup>−1</sup>) were used as QC) strains for colistin MIC determination.</p></sec><sec id=\"s2-4\"><title>Screening of <italic>mcr</italic> genes by PCR</title><p>The presence of <italic>mcr-1</italic>, <italic>mcr-2</italic>, <italic>mcr-3</italic> and <italic>mcr-4</italic> encoding for plasmid-mediated colistin resistance was screened by PCR as described previously [<xref rid=\"R4\" ref-type=\"bibr\">4, 6–8</xref>].</p></sec><sec id=\"s2-5\"><title>Next-generation sequencing</title><p>The isolate that was positive for <italic>mcr</italic> was selected for next-generation sequencing to analyse colistin resistance determinants and other genetic factors. A QIAamp DNA Mini kit (Qiagen, Hilden, Germany) was used for genomic DNA extraction. Whole-genome sequencing (WGS) of the isolate was performed with 400 bp read chemistry using an IonTorrent Personal Genome Machine (PGM) (Life Technologies, Carlsbad, CA, USA) as per the manufacturer’s instructions. Data were assembled <italic>de novo</italic> using Assembler SPAdes v.5.0.0.0 embedded in Torrent Suite Server v.5.0.3. Sequence annotation was performed using PATRIC, the bacterial bioinformatics database and analysis resource (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.patricbrc.org\">http://www.patricbrc.org</ext-link>), and the National Center for Biotechnology Information’s (NCBI’s) Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP, <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html\">http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html</ext-link>).</p><p>The CGE server (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.cbs.dtu.dk/services\">http://www.cbs.dtu.dk/services</ext-link>) and PATRIC [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>] were employed for downstream analysis. ResFinder 2.1 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://cge.cbs.dtu.dk//services/ResFinder/\">https://cge.cbs.dtu.dk//services/ResFinder/</ext-link>) was used to analyse the resistance gene profile [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]. Antimicrobial resistance genes were also screened using the Antibiotic Resistance Genes Database (ARDB) and the Comprehensive Antibiotic Resistance Database (CARD) through PATRIC. PlasmidFinder 1.3 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://cge.cbs.dtu.dk//services/PlasmidFinder/\">https://cge.cbs.dtu.dk//services/PlasmidFinder/</ext-link>) was used to screen for the presence of plasmids [<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]. The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas genes was identified from the genome using the CRISPRFinder tool [<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]. The MultiLocus Sequence Typing (MLST) 1.8 tool was employed for sequence type analysis (<ext-link ext-link-type=\"uri\" xlink:href=\"https://cge.cbs.dtu.dk//services/MLST/\">https://cge.cbs.dtu.dk//services/MLST/</ext-link>) [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]. The genome was screened for insertion sequence elements using ISFinder (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www-is.biotoul.fr/blast.php\">https://www-is.biotoul.fr/blast.php</ext-link>) [<xref rid=\"R13\" ref-type=\"bibr\">13</xref>].</p></sec><sec id=\"s2-6\"><title>MinION Oxford Nanopore sequencing</title><p>DNA library preparation and sequencing was prepared using SQK-LSK108 Kit R9 version (Oxford Nanopore Technologies, Oxford, UK) using a 1D sequencing method according to the manufacturer’s protocol. Sequencing was performed using the FLO-MIN106 R9 flow cell in the MinION Mk 1B sequencer. MinKNOW software v 1.15.1 (Oxford Nanopore Technologies, Oxford, UK) was employed in a Windows platform to perform sequencing and raw data (fast5 files) were obtained.</p></sec><sec id=\"s2-7\"><title>MinION sequence analysis</title><p>The fast5 files were generated from MinION sequencing and the reads were base-called with Albacore 2.0.1 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://nanoporetech.com/about-us/news/new-basecaller-now-performs-raw-basecalling-improved-sequencing-accuracy\">https://nanoporetech.com/about-us/news/new-basecaller-now-performs-raw-basecalling-improved-sequencing-accuracy</ext-link>). Furthermore, the adapters were trimmed using Porechop (<ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/rrwick/Porechop\">https://github.com/rrwick/Porechop</ext-link>). Canu 1.7 [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>] was used for MinION error correction and assembly with a genome size of 5.0 m as input. After <italic>de novo</italic> assembly, the contigs were polished with Nanopolish 0.10.1 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/jts/nanopolish\">https://github.com/jts/nanopolish</ext-link>).</p></sec><sec id=\"s2-8\"><title>Hybrid assembly using IonTorrent and MinION reads</title><p>To increase the accuracy and completeness of he genome, a hybrid assembly using both IonTorrent and MinION reads with Unicycler (v0.4.6) was performed [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]. By default, Unicycler utilizes SPAdes [<xref rid=\"R16\" ref-type=\"bibr\">16</xref>] to assemble the short reads with different k-mers and filter out the low-depth regions. Subsequently, it trims and generates the short-read assembly graph. In addition, it uses Miniasm [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>] and Racon [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>] to assemble the MinION long reads and further the reads were bridged to determine all the genome repeats and produce complete genome assembly. The short reads were also polished with multiple rounds of Pilon [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>] to reduce the base-level errors. After assembly, the assembly statistics and average nucleotide identity of different assemblies were evaluated using the Quast [<xref rid=\"R20\" ref-type=\"bibr\">20</xref>] and OrthoANI 0.93.1 tools [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>], respectively.</p></sec><sec id=\"s2-9\"><title>\n<italic>In silico</italic> sequence analysis</title><p>The <italic>mcr</italic> nucleotide sequences obtained from the experimental techniques were translated to a protein sequence using the online Expasy Translate tool (<ext-link ext-link-type=\"uri\" xlink:href=\"https://web.expasy.org/translate/\">https://web.expasy.org/translate/</ext-link>). The percentage identity of known MCR variants in comparison with novel protein was identified using <sc>blast</sc> search. The conserved amino acid region from the closely related variants was determined using Clustal Omega.</p></sec><sec id=\"s2-10\"><title>\n<italic>In silico</italic> structure analysis</title><p>The sequences of the MCR variant was used to model the 3D structure of the proteins. The 3D structures of the variants were modelled using Swiss-Model [<xref rid=\"R22\" ref-type=\"bibr\">22</xref>]. The translated variant sequences were given as the input for the 3D variant modelling. The rampage server was used to evaluate the quality of the modelled variants [<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]. Finally, the MetaPocket server was used to predict the active pocket of the novel variant [<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]. The structure visualization was performed using PyMOL.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec id=\"s3-1\"><title>Antimicrobial susceptibility</title><p>The resistance profiles for the studied <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> isolates are presented in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. The colistin MIC was determined for all the isolates and this showed 30 % of the isolates were resistant to colistin. The MIC was identified to be 0.5 µg ml<sup>−1</sup> (susceptible) for the isolate that was positive for <italic>mcr-3</italic>.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1.</label><caption><p>Antimicrobial susceptibility of the selected <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> spp.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Sample no.</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Sample ID</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Age/sex</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Organism</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Resistance pattern (disc diffusion)</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Colistin MIC (µg ml<sup>−1</sup>)</p>\n</th></tr></thead><tbody><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC3340</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>29M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> spp.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-SXT-TAX</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4R</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC193</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.26210\">A. dhakensis</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-IMI-MEM</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>≥64R</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC199</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>76M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC284</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>75M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> spp.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-IMI-MEM</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>16R</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC728</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>71M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3052\">A. caviae</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-TET-TAX-IMI-MEM-CIP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.5S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC729</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>66M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3052\">A. caviae</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-TET-SXT-TAX-IMI-MEM-CIP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.5S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>7</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC715</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>34F</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3052\">A. caviae</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>32R</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>8</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC850</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>27M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3072\">A. veronii</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-TET-SXT-IMI-MEM-CIP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>9</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>*FC951</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>55M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3072\">A. veronii</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-TET-IMI-MEM</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.5S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>10</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC1239</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>21F</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.26210\">A. dhakensis</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-TAX-IMI-MEM</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>≥64R</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>11</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC1520</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>45F</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>32R</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>12</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC1169</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>13</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC599</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>57F</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-SXT</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p> 2S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>14</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC814</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>55F</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-IMI</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>32R</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>15</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC2457</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-TET-SXT</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>16</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC538</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>58M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-IMP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>17</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC771</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3052\">A. caviae</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-TAX-CIP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4R</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>18</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC578</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>26F</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3052\">A. caviae</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>19</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC377</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>22F</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-SXT-IMI-MEM</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>20</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC1245</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>42F</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-IMI</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>21</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC788</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-TAX-IMI</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>8R</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>22</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC157</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>58M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3072\">A. veronii</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.5S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>23</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC390</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>9F</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3052\">A. caviae</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>24</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC1411</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-IMI</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>25</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC523</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-TET</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>26</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC1999</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>54F</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-IMI-MEM</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.5S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>27</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC2051</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>28M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3052\">A. caviae</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>28</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC2019</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>60M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3052\">A. caviae</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>29</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC906</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>55M</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP-TET</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.5S</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>30</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>FC1435</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>73F</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10566\">A. hydrophila</ext-link>\n</named-content></italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AMP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>>32R</p>\n</td></tr></tbody></table><table-wrap-foot><fn id=\"tbl1fn1\"><p>*Isolate sequenced: R, resistant; S, susceptible; AMP, ampicillin; TET, tetracycline; SXT, trimethoprim/sulfamethoxazole; CIP, ciprofloxacin; TAX, cefotaxime; IMI, imipenem; MEM, meropenem.</p></fn></table-wrap-foot></table-wrap></sec><sec id=\"s3-2\"><title>Screening of <italic>mcr</italic> genes</title><p>Of the 30 isolates screened for <italic>mcr-1</italic>, <italic>mcr-2</italic>, <italic>mcr-3</italic> and <italic>mcr-4</italic>, only one isolate (FC951) was positive for <italic>mcr-3</italic>.</p></sec></sec><sec id=\"s4\"><title>Next-generation sequencing</title><p>The <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3072\">A. veronii</ext-link>\n</named-content></italic> (FC951) hat was positive for <italic>mcr-3</italic> by PCR was sequenced using IonTorrent PGM. Analysis of <italic>mcr-3</italic> revealed only 95.6 % identity against the reference sequences in the database (henceforth termed <italic>mcr-3.30</italic>). Further, ISFinder revealed that IS<italic>As18</italic> belongs to the IS<italic>4</italic> family next to <italic>eptA,</italic> aka <italic>mcr-3</italic>.</p><p>However, the <italic>mcr-3.30</italic> was split in to two contigs (IonTorrent). Generally, the IonTorrent assembly is highly accurate, but the assembly had too many fragments. The long-read sequencing of FC951 using MinION resulted in a complete genome (a chromosome and a plasmid), but with errors. Hybrid assembly using IonTorrent and MinION reads in Unicycler resulted a complete chromosomal contig for FC951 with increased accuracy and few errors. Interestingly, analysis of the complete genome revealed <italic>mcr-3.30</italic> integrated in the chromosome along with the insertion of IS<italic>As18</italic> within <italic>mcr-3.30</italic>. The structure of the genetic environment of <italic>mcr-3.30</italic> is shown in <xref ref-type=\"fig\" rid=\"F1\">Fig. 1a</xref>.</p><fig fig-type=\"figure\" id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1.</label><caption><p>(a) Genetic environment of <italic>mcr-3.30</italic> with an insertion of IS<italic>As18</italic> transposase (1141 bp) leading to disruption of <italic>mcr-3.30</italic> function. (b) CRISPR Cas system identified in FC951 and arrangement of Cas genes.</p></caption><graphic xlink:href=\"acmi-2-103-g001\"/></fig><p>The Quast analysis showed the N50 and N75 value of the hybrid assembly to be 4 660 178, which is approximately 96 % of the total assembly length. In addition, ANI is calculated using different assembly methods. It is evident that the closeness between IonTorrent and hybrid assembly is about 99.92 %, which represents the high accuracy of the hybrid assembly. The hybrid assembly generated a 4.66 Mbp chromosome length single contig. In contrast, the IonTorrent-only assembly produced an assembly with more than 300 contigs and only 139 contigs >1000 bp. It was clear that hybrid assembly has its own advantages, with improved accuracy and a reduced error rate with genome completeness compared to MinION-only or IonTorrent-only assembly.</p><p>Moreover, annotation of the extra-chromosomal sequences from MinION could not be designated as a complete plasmid, although it showed 21 % similarity with a previously reported <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.2216\">Xanthomonas citri</ext-link>\n</named-content></italic> plasmid (CP020883.1).</p><p>Further analysis of resistance genes using ResFinder revealed the presence of <italic>bla</italic>\n<sub>OXA12</sub> and <italic>bla</italic>\n<sub>CEPH-A3</sub> genes. A CRISPR Cas system was identified in FC951 and the arrangements of the genes were as shown in <xref ref-type=\"fig\" rid=\"F1\">Fig. 1b</xref>. Notably, the sequence type of FC951 was identified to be novel, ST-515.</p><p>This complete genome project has been deposited at GenBank under the accession number CP032839 (plasmid accession number CP032840).</p><sec id=\"s4-1\"><title>\n<italic>In silico</italic> sequence analysis</title><p>The <italic>mcr-3</italic> nucleotide and protein sequence identified in this study was compared with the previously reported variants <italic>mcr-3.1</italic>–<italic>mcr-3.10</italic> using <sc>blast</sc> search (KY924928.1, NMWW01000143.1, MF495680, NQCO01000074.1, MF489760, MF598076.1, MF598077.1, MF598078.1, MF598080.1 and MG214531) and identified to be a novel variant (<italic>mcr-3.30</italic>). The nucleotide percentage identity matrix for the <italic>mcr</italic> gene variants was as given in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>. From the analysis, <italic>mcr-3.3</italic> and <italic>mcr-3.10</italic> were found to be highly identical (95.62 and 95.28 %, respectively. The most conserved amino acid region among the three variants (<italic>mcr-3.3</italic>, <italic>mcr-3.10</italic> and <italic>mcr-3.30</italic>) was identified using Clustal Omega. The region of amino acids from LEU-359 to ILE-427 was found to be the largest conserved sequence among them.</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2.</label><caption><p>Percentage identity matrix of <italic>mcr-3</italic> variants in comparison with FC951 <italic>mcr-3.30</italic>\n</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.30</italic> (FC951)</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.6</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.3</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.8</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.7</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.5</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.4</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.1</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.2</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.9</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.10</italic>\n</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.30</italic> (FC951)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>100</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.6</italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>94.07</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>100</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.3</italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>95.62</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>98.15</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>100</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.8</italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>95.01</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>98.64</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>98.95</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>100</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.7</italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>92.6</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.33</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.09</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>93.97</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>100</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.5</italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>94.07</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>93.72</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.32</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.22</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.69</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>100</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.4</italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>94.13</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>93.72</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.32</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.22</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.82</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>99.75</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>100</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.1</italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>94.2</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>93.78</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.38</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.28</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.88</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>99.82</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>99.94</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>100</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.2</italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>94.2</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>93.84</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.44</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.34</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.82</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>99.88</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>99.88</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>99.94</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>100</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.9</italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>94.54</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.39</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.75</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.9</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>96.62</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>97.72</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>97.85</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>97.91</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>97.85</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>100</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcr-3.10</italic>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>95.28</bold>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.7</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>96.55</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.33</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>96.56</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>98.65</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>98.77</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>98.83</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>98.77</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>99.08</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>100</p>\n</td></tr></tbody></table></table-wrap></sec><sec id=\"s4-2\"><title>\n<italic>In silico</italic> structure analysis</title><p>The 3D structures were modelled using the sequences for the variants <italic>mcr-3.1</italic>–<italic>mcr-3.10</italic> using the Swiss-Model server. The template used and its respective PDB ID, coverage range and coverage identity are tabulated in <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>. Further, the quality of the modelled variants was evaluated using the rampage server. The percentage of the amino acids in the favoured region, the percentage of amino acids in the allowed region and the percentage of amino acids in the outlier region are tabulated in <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>. Superimposed structural evaluation of MCR variant 3.30 against the closely related MCR variant 3.3 and MCR variant 3.10 was visualized using PyMOL (<xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>). In addition, LEU53, ILE164, ALA57, TYR175, GLN186, ILE189, VAL176, GLY179, ALA192, PHE32, LEU50, VAL61, ARG180, VAL178, ASN182, LEU185, PHE46, LEU36, SER183, VAL29, GLY28, PRO51, LEU54, ASN25, ALA21, TRP26, GLU188, LEU24, LEU58, PRO191, ASN193, VAL195, PHE65 and ASN196 were identified as the active site of the MCR-3.30 variant using the Meta Pocket server (Fig. S1, available in the online version of this article).</p><fig fig-type=\"figure\" id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2.</label><caption><p>(a) Superimposed structural evaluation of MCR variant 3.30 against the closely related MCR variant 3.3 and MCR variant 3.10, (b) 3D structure of MCR variant 3.3, (c) 3D structure of MCR variant 3.10 and (d) 3D structure of MCR variant 3.30.</p></caption><graphic xlink:href=\"acmi-2-103-g002\"/></fig><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3.</label><caption><p>List of parameters considered for modelling the variants using Swiss-Model and Ramachandran plot evaluation of the structures using <sc>rampage</sc>\n</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Variants of <italic>mcr</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Template PDB</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Coverage range</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Coverage</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Identity</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>% of amino acids in the favoured region</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>% of amino acids in the allowed region</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>% of amino acids in the outlier region</p>\n</th></tr></thead><tbody><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5FGN</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6–544</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.98</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>37.55</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.2 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4.9 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.9 %</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.2</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5FGN</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6–545</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.98</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>37.55</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.2 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4.9 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.9 %</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.3</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5FGN</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1–546</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.99</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>35.82</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.0 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5.1 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.9 %</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.4</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5FGN</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6–544</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.98</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>37.55</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.4 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4.7 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.9 %</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.5</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5FGN</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6–546</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.97</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>40.27</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.9 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.6 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.6 %</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.6</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5FGN</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6–546</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.97</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>39.69</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.7 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.6 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.8 %</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.7</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5FGN</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6–546</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.97</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>40.08</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.1 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4.1 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.8 %</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.8</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5FGN</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6–546</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.97</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>39.69</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.7 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.6 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>80.0 %</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.9</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5FGN</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6–546</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.97</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>40.08</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>95.3 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4.0 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>80.0 %</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.10</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5FGN</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6–544</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.97</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>37.74</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>94.6 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4.5 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>90.0 %</p>\n</td></tr><tr><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.30</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5FGN</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6–469</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.98</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>36.24</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>93.7 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4.6 %</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1.7 %</p>\n</td></tr></tbody></table></table-wrap></sec></sec><sec sec-type=\"discussion\" id=\"s5\"><title>Discussion</title><p>Resistance to colistin is mainly mediated via alteration of the lipopolysaccharides (LPS) of the bacterial outer membrane. The alterations include mutations in lipid A-modifying genes. The most commonly reported mutations are in the <italic>mgrB</italic> gene and are therefore not transferable through horizontal gene transfer [<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]. However, in 2015, the first plasmid-mediated colistin resistance gene (<italic>mcr-1</italic>) was reported [<xref rid=\"R26\" ref-type=\"bibr\">26</xref>], which belongs to the phosphoethanolamine transferase enzyme family (<italic>Ept</italic>A). <italic>mcr-1</italic> was identified in <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> from human patients and animals in China. In 2016, another study reported the mobilizable colistin resistance gene, <italic>mcr-2,</italic> from porcine and bovine <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> isolates in Belgium [<xref rid=\"R27\" ref-type=\"bibr\">27</xref>]. Further, <italic>mcr-3</italic> and <italic>mcr-4</italic> were identified in <italic>E. coli, Klebsiella</italic> spp. and <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3291\">Salmonella</ext-link>\n</named-content></italic> spp. [<xref rid=\"R4\" ref-type=\"bibr\">4, 28</xref>]. Recently, <italic>mcr-5</italic> was identified in <italic><named-content content-type=\"subspecies\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3292\">Salmonella enterica</ext-link>\n</named-content></italic> subsp. enterica serovar Paratyphi B isolated from poultry in Denmark [<xref rid=\"R29\" ref-type=\"bibr\">29</xref>].</p><p>Several mobile colistin resistance genes have been identified, but only <italic>mcr-1</italic> and <italic>mcr-3</italic> have been reported with a high number of variants in GenBank database. A recent study highlighted the importance of a third mobile colistin resistance gene, <italic>mcr-3</italic> in <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10568\">Aeromonas salmonicida</ext-link>\n</named-content></italic>, due to its resemblance to various other phosphoethanolamine transferases in <italic><named-content content-type=\"family\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3091\">Enterobacteriaceae</ext-link>\n</named-content></italic> and also suggested that this resistance gene might have already been widely disseminated [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. Here we discuss a novel variant of <italic>mcr-3</italic> identified in <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3072\">A. veronii</ext-link>\n</named-content></italic> isolated from a clinical specimen.</p><p>The novel <italic>mcr-3</italic> variant identified in this study exhibited ≤95 % nucleotide sequence similarity to all other previously reported <italic>mcr</italic>-3 variants, and is henceforth named <italic>mcr-3.30. In silico</italic> protein sequence comparison revealed the novelty of MCR-3.30. The superimposed protein structure comparison with MCR-3.3 and MCR-3.10 further confirmed the MCR-3.30 variant. Major structural changes were observed in domain 2. Similar comparisons of MCR-3 and MCR-1 protein structures were previously reported by [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. Knowledge of the 3D structure of proteins is now in great demand in the field of computer-aided drug discovery (CADD). It helps researchers in identifying new drugs. In this study, we have used homology modelling technique to model the 3D structures of MCR variants.</p><p>\n<italic>mcr-3</italic> was first identified in a 261 kb IncHI2-type plasmid pWJ1 from <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. However, initially, known plasmid replicons were not identified in FC951 harbouring <italic>mcr-3.30</italic> via PlasmidFinder. Later, sequence assembly and alignment with pWJ1 revealed an Inc-W like replicase gene in the extrachromosomal region, along with TraI and TrbN genes responsible for multi-functional conjugation and conjugal transfer proteins.</p><p>There had been a previous report on the chromosome integration of the <italic>mcr-3</italic> variant in <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3072\">A. veronii</ext-link>\n</named-content></italic> [<xref rid=\"R30\" ref-type=\"bibr\">30</xref>]. In this study, a major insertion of IS<italic>As18</italic> (1141 bp) belonging to the IS<italic>4</italic> family of insertional elements was found within <italic>mcr-3.30</italic>. IS<italic>As18</italic> had previously only been reported in <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10568\">A. salmonicida</ext-link>\n</named-content></italic> as a transposase [<xref rid=\"R31\" ref-type=\"bibr\">31</xref>]. The entire <italic>mcr-3.30</italic> region, including the insertion, was flanked by <italic>eamA</italic> and <italic>dgkA</italic>. These genes encode a metabolite transporter and a diacylglycerol kinase, respectively [<xref rid=\"R32\" ref-type=\"bibr\">32</xref>]. The <italic>mcr-3.30</italic> genetic environment also had other insertional elements, such as IS<italic>As19</italic>, IS<italic>As20</italic>, IS<italic>630</italic>, IS<italic>Kpn15</italic>, IS<italic>5</italic> and IS<italic>3</italic> transposase.</p><p>However, <italic>mcr-3.30</italic> disruption caused by the insertion of IS<italic>As18</italic> has rendered it non-expressive. Accordingly, the isolate FC951, in spite of harbouring <italic>mcr-3.30,</italic> was phenotypically susceptible to colistin. Similarly, a previous study by Pham Thanh <italic>et al</italic>. identified a deactivated <italic>mcr-1</italic> due to the disruption of the gene by a 22 bp duplication in colistin-susceptible <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3333\">Shigella sonnei</ext-link>\n</named-content></italic> [<xref rid=\"R33\" ref-type=\"bibr\">33</xref>]. The gene was found to be reactivated by conjugation experiments. resulting in a colistin-resistant phenotype. Another study by Liassine <italic>et al</italic>. reported <italic>mcr-1</italic> in susceptible <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> with an unknown cause of susceptibility [<xref rid=\"R34\" ref-type=\"bibr\">34</xref>]. A recent study showed the presence of <italic>mcr-1</italic> in susceptible <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> due to the insertion of 1329 bp transposon IS10R and found that this could not be reactivated. As shown from these studies, the inactivated gene can be restored upon colistin exposure, particularly in settings where colistin use is high. This phenomenon emphasizes the importance of phenotypic confirmation despite detection of the gene in molecular screening [<xref rid=\"R33\" ref-type=\"bibr\">33, 35</xref>]. In contrast, the isolates of the present study that were resistant to colistin by MIC were negative for screened <italic>mcr</italic>; this could be due to other chromosomal mechanisms that need to be explored.</p><p>There are no reports of <italic>mcr</italic> variants other than <italic>mcr-1</italic> from India. Various Indian centres have reported colistin-resistant strains in hospitalized patients. So far, there are only published reports of colistin resistance in <italic><named-content content-type=\"family\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3091\">Enterobacteriaceae</ext-link>\n</named-content></italic> and non-fermenters such as <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10789\">K. pneumoniae</ext-link>\n</named-content></italic> and <italic>A. baumannii</italic> from India.</p><p>Mutation in the <italic>mgr</italic>B gene is the most common resistance mechanism in <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10789\">K. pneumoniae</ext-link>\n</named-content></italic> and mutation in the <italic>lpx</italic>A/D, <italic>lps</italic>B and <italic>pmr</italic>B genes is responsible for resistance in <italic>A. baumannii</italic>. On the other hand, the presence of plasmid-mediated <italic>mcr</italic> confers resistance in <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">E. coli</ext-link>\n</named-content></italic> [<xref rid=\"R25\" ref-type=\"bibr\">25, 36, 37</xref>]</p><p>Apart from the colistin resistance gene, the genome analysis revealed the presence of other resistance genes, such as <italic>bla</italic>\n<sub>OXA-12</sub>, belonging to class D beta-lactamase in FC951, which confers resistance to ampicillin and is known to be naturally produced by <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3058\">Aeromonas jandaei</ext-link>\n</named-content></italic> and has strong activity against oxacillin [<xref rid=\"R38\" ref-type=\"bibr\">38</xref>]. The isolate also harboured <italic>bla</italic>\n<sub>CEPH-A3</sub>, which is the most common metallo-beta-lactamase (MBL) produced by <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3044\">Aeromonas</ext-link>\n</named-content></italic> species responsible for carbapenem resistance.</p><sec id=\"s5-1\"><title>Conclusion</title><p>To the best of our knowledge, this is the first report on a novel <italic>mcr-3</italic> variant (<italic>mcr-3.30</italic>) at the structural level, in comparison with the known variants (MCR-3.3–MCR-3.10). The <italic>mcr-3.30</italic> identified in this study is non-functional for colistin resistance due to the insertion of IS<italic>As18</italic> within the gene. Further, this is the first complete genome sequence of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3072\">A. veronii</ext-link>\n</named-content></italic> from India, and the first hybrid genome of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3072\">A. veronii</ext-link>\n</named-content></italic> globally. These findings support extended screening of known and further exploration of unknown colistin resistance mechanisms in this pathogen as well as in other Gram-negative pathogens.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Data</title><supplementary-material content-type=\"local-data\" id=\"supp1\"><caption><title>Supplementary material 1</title></caption><media xlink:href=\"acmi-2-103-s001.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"ack1\"><title>Funding information</title><p>The authors received no specific grant from any funding agency.</p></ack><ack id=\"ack2\"><title>Acknowledgements</title><p>The authors gratefully acknowledge Christian Medical College, Vellore for providing laboratory space and facilities.</p></ack><ack id=\"ack3\"><title>Conflicts of interest</title><p content-type=\"COI-statement\">The authors declare that there are no conflicts of interest.</p></ack><ack id=\"ack4\"><title>Ethical statement</title><p>The study was approved by the Institutional Review Board of the Christian Medical College, Vellore (83-i/11/13)</p></ack><fn-group><fn id=\"FN1\"><p>Abbreviations: ARDB, antibiotic resistance genes database; 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Access Microbiol</journal-id><journal-id journal-id-type=\"hwp\">acmi</journal-id><journal-id journal-id-type=\"publisher-id\">acmi</journal-id><journal-title-group><journal-title>Access Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">2516-8290</issn><publisher><publisher-name>Microbiology Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005865</article-id><article-id pub-id-type=\"pmc\">PMC7523624</article-id><article-id pub-id-type=\"publisher-id\">000099</article-id><article-id pub-id-type=\"doi\">10.1099/acmi.0.000099</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Molecular characterization of the first reported <italic>Aichivirus</italic> A in Australia</article-title><alt-title alt-title-type=\"recto-page-foot\"><ext-link ext-link-type=\"uri\" xlink:href=\"http://acmi.microbiologyresearch.org\">http://acmi.microbiologyresearch.org</ext-link></alt-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-7549-3343</contrib-id><name><surname>Northill</surname><given-names>Judith A.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-8918-8905</contrib-id><name><surname>Simmons</surname><given-names>Russell J.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Genge</surname><given-names>Doris</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-4506-8610</contrib-id><name><surname>Moore</surname><given-names>Frederick A.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><aff id=\"aff1\">\n<label><sup>1</sup>​</label>\n<institution>Public Health Virology, Forensic and Scientific Services</institution>, <addr-line content-type=\"city\">Coopers Plains, QLD</addr-line>, <country>Australia</country>\n</aff></contrib-group><author-notes><corresp id=\"cor1\"><bold>*Correspondence:</bold> Judith A. Northill, <email xlink:href=\"mailto:judy.northill@health.qld.gov.au\">judy.northill@health.qld.gov.au</email></corresp></author-notes><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>11</day><month>2</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>11</day><month>2</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>2</volume><issue>4</issue><elocation-id>acmi000099</elocation-id><history><date date-type=\"received\"><day>10</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>20</day><month>12</month><year>2019</year></date></history><permissions><copyright-statement>© 2020 The Authors</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"acmi-2-099.pdf\"/><abstract><p>A novel real-time reverse transcription polymerase chain reaction (RT-rPCR) assay was developed to detect <italic>Aichivirus A</italic> (AiV-A) based on four complete genomes. The assay successfully detected AiV-A in a sample from a patient with acute gastroenteritis in January 2008. Screening of 756 samples submitted for norovirus testing during May 2008 detected a further 23 AiV-A-positive samples from 18 individual patients. Genotyping using novel primers targeting the 3C–3D junction region identified AiV-A genotype B. Further sequencing of the VP1 region supported the 3C–3D result. All three assays proved useful to support foodborne outbreak investigations. This is the first report of AiV-A detection in Australia.</p></abstract><kwd-group><kwd><italic>Aichivirus</italic></kwd><kwd>real-time RT-PCR</kwd><kwd>gastroenteritis</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>OpenAccessEmbargo</meta-name><meta-value>0</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>\n<italic>Aichivirus A</italic> (AiV-A) viruses were first detected in samples from an oyster-related gastroenteritis outbreak in Aichi, Japan in 1989 [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]. AiV-A viruses are species of the family <italic>Picornaviridae</italic> sharing the genus <italic>Kobuvirus</italic> with five other species identified as <italic>Aichivirus B</italic>–<italic>F</italic> [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]. AiV-A are a group of single-stranded, positive-sense RNA viruses that cluster into two confirmed genotypes, A and B [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>], with a third genotype proposed [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>].</p><p>The first complete AiV-A genome was determined in 1991 [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]. AiV-A has since been identified in many countries including, Bangladesh, Thailand, Vietnam [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>], Germany, Brazil [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>], France [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>], Tunisia [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>], Hungary [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>], PR China [<xref rid=\"R10\" ref-type=\"bibr\">10</xref>], Finland [<xref rid=\"R11\" ref-type=\"bibr\">11</xref>], Spain [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>], India [<xref rid=\"R13\" ref-type=\"bibr\">13</xref>] and Sweden [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]. These global viruses have also been reported in polluted water in Venezuela [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>] and sewage in Tunisia [<xref rid=\"R16\" ref-type=\"bibr\">16</xref>].</p><p>Here we report the first detection of AiV-A in Australia. We detail the development of a novel screening RT-rPCR assay and two new conventional agarose gel-based RT-PCRs for genotyping in the 3C–3D and VP1 regions. We discuss the amino acid differences between genotypes within the capsid sequences that support previous reports [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>] and report additional sequences of AiV-A that further aid assay development or improvements for the identification of foodborne gastroenteritis clusters.</p></sec><sec sec-type=\"methods\" id=\"s2\"><title>Methods</title><sec id=\"s2-1\"><title>Specimens</title><p>The initial AiV-A detected sample type was a faecal sample from a female patient. The further study was conducted on 756 samples. The majority were faecal samples (739), while the remaining 17 were vomitus.</p></sec><sec id=\"s2-2\"><title>Nucleic acid extraction</title><p>Viral RNA was extracted from a 10 % faecal suspension using the Qiagen BioRobot Universal System and the QIAamp Virus BioRobot MDx kit (Qiagen, Australia).</p></sec><sec id=\"s2-3\"><title>Real-time RT-PCR for detection of AiV-A RNA (AiV-A RT-rPCR)</title><p>A novel RT-rPCR was designed for AiV-A (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>) using Primer Express software (Life Technologies, USA). The 106 nt target region was located in the AiV-A 3′ UTR (GenBank accession number DQ028632) and the chosen probe and primers were aligned and compared to other sequence information available in 2007. Assay-specific synthetic templates (Geneworks Pty Ltd, Australia) were devised as a long-term consistent control. Details of the method to produce these binary controls are outlined in a previous publication [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]. Both controls were prepared and titrated for use (data not shown). A cycle threshold (<italic>C</italic>\n<sub>T</sub>) of <40 indicated a positive result with a <italic>C</italic>\n<sub>T</sub>>40 indicating that the template was not detected. The reaction mix was formulated using the Invitrogen SuperScript III Platinum One-Step Quantitative RT-PCR System (Life Technologies, Australia), 300 nM of both forward and reverse primer and 150 nM of probe, and 5 µl of RNA extract or diluted synthetic control was added for a final volume of 20 µl [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]. RT-rPCR was performed using a Corbett Rotor-Gene 6000 (Qiagen, Australia) with cycling conditions described elsewhere [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>].</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1.</label><caption><p>Diagnostic and genotyping oligonucleotides</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Assay name</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Oligonucleotide name</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Sequence (5′−3′)</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><sup>a</sup></italic>Location</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"3\" colspan=\"1\">\n<p>AiV-A RT-rPCR<sup><italic>b</italic>,<italic>c</italic></sup>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AiV-F-8046</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>TGCTTCGGCACGCTTAGTT</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>8046–8064 (3′ UTR)</p>\n</td></tr><tr><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AiV-R-8151</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>TGCARTACAACCAYGGCTTAGG</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>8120–8151 (3′ UTR)</p>\n</td></tr><tr><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AiV-8082-FAM</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6FAM-CACTCCTCCATGGTGATATAAAGACCAC-TAMRA</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>8082–8109 (3′ UTR)</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>3C–3D RT-PCR<sup><italic>d</italic></sup>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AiV-6213F</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>ACTGGGCCACCCTCCAGACG</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6228–6247 (3C)</p>\n</td></tr><tr><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AiV-7044R</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>GGTTGATTTCAGCTTGGAGTTC</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>7058–7037 (3D)</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"3\" colspan=\"1\">\n<p>VP1 RT-PCR<sup><italic>d</italic></sup>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AiV-2789F</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>TTCACCATCCCCTTCATCTC</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2804–2823 (VP3)</p>\n</td></tr><tr><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AiV-4302R</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>GCAAGGGAGACAGAATTTGC</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4317–4298 (2B)</p>\n</td></tr><tr><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AiV-3867R</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>GGGGAGACCTTGCGGATRGC</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3882–3863 (2A)</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Yamashita 3C-3D RT-PCR<sup><italic>e</italic></sup>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AICHI-6261</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>ACACTCCCACCTCCCGCCAGTA</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6276–6297 (3C)</p>\n</td></tr><tr><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>AICHI-6779</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>GGAAGAGCTGGGTGTCAAGA</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6794–6775 (3D)</p>\n</td></tr></tbody></table><table-wrap-foot><fn id=\"tbl1fn1\"><p>\n<italic>a,</italic> Location is based on the GenBank AiV-A strain, accession number DQ028632.</p></fn><fn id=\"tbl1fn2\"><p>\n<italic>b,</italic> Designed in 2007 using an alignment of available sequences, AB010145, AB040749, AY747174 and DQ028632.</p></fn><fn id=\"tbl1fn3\"><p>\n<italic>c,</italic> Designed using Primer Express software (Life Technologies, USA).</p></fn><fn id=\"tbl1fn4\"><p>\n<italic>d,</italic> Designed using Primer3 (<ext-link ext-link-type=\"uri\" xlink:href=\"http://primer3.ut.ee/\">http://primer3.ut.ee/</ext-link>).</p></fn><fn id=\"tbl1fn5\"><p>\n<italic>e,</italic> Published by Yamashita <italic>et al</italic>. [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>].</p></fn></table-wrap-foot></table-wrap></sec><sec id=\"s2-4\"><title>Conventional RT-PCRs for initial subgenomic sequencing and full-genome Sanger sequencing</title><p>A conventional RT-PCR from a published set of primers (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>) was used to generate a 519 bp product [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]. This RT-PCR is referred to as the ‘Yamashita 3C–3D conventional RT-PCR’. The reaction mix was formulated using the Invitrogen SuperScript III One-Step RT-PCR System with Platinum <italic>Taq</italic> High Fidelity (Life Technologies, Brisbane Australia). To this mix, 5 µl of extracted RNA was added for a total reaction volume of 20 µl. For mix details and cycling times see the detailed protocol published elsewhere [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]. This RT-PCR was used to confirm the first detected sample by AiV-A RT-rPCR, but it could not detect all the subsequent samples, so a new set of primers was designed for this region.</p><p>Further specific sets of oligonucleotides (available upon request) were designed using the complete genome of four AiV-A strains (AB010145, AB040749, AY747174 and DQ028632) as a guide. Multiple RT-PCRs were performed using 500 nM of primers and the Invitrogen Superscript III One-Step RT-PCR System with Platinum <italic>Taq</italic> kit. The cycling times were 55 °C for 15 min, 94 °C for 2 min, followed by 40 cycles of 94 °C for 15 s, 52 °C for 30 s and 68 °C for 20 s. Amplified products from these RT-PCRs were purified using the Qiagen QIAQuick PCR Purification kit (Qiagen, Australia) and sequencing was performed using the Big Dye Terminator Cycle Sequencing Ready Reaction kit version 3.1 (Applied Biosystems, Australia) and an ABI3130xl Genetic Analyser (Applied Biosystems, Australia). The genome ends were determined using the 5′/3′ RACE kit, second Generation (Roche, Germany).</p></sec><sec id=\"s2-5\"><title>Conventional RT-PCRs for additional subgenomic sequencing</title><p>Two new conventional RT-PCRs were designed for genotyping. Firstly, a newly designed RT-PCR targeting the AiV-A 3C–3D junction region (3C–3D RT-PCR) proved highly successful. The primers, AiV-6213F and AiV-7044R (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), produced an 832 bp sequence that extends beyond most of the partial 3C–3D sequences publicly available in GenBank (<xref ref-type=\"fig\" rid=\"F1\">Fig. 1</xref>). The reaction mix was formulated using the Invitrogen SuperScript III One-Step RT-PCR System with Platinum <italic>Taq</italic> High Fidelity (Life Technologies, Australia). To this mix, 5 µl of extracted RNA was added for a total reaction volume of 25 µl. For mix details and cycling times see the detailed protocol published elsewhere [<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]. The 3C–3D RT-PCR was used to confirm all the samples detected by the RT-rPCR.</p><fig fig-type=\"figure\" id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1.</label><caption><p>Schematic of AiV-A based on the sequence from GenBank AB040749. The target regions of the 3C–3D RT-PCR, VP1 RT-hnPCR and AiV RT-rPCR are underlined. UTR, untranslated region; L, non-structural leader protein; VP0, VP3 and VP1, structural viral proteins, P1; 2A, 2B and 2C,non-structural proteins, P2; 3A, 3B, 3C and 3D, non-structural proteins, P3.</p></caption><graphic xlink:href=\"acmi-2-099-g001\"/></fig><p>The second assay was a conventional hemi-nested RT-PCR targeting the VP1 region of AiV-A (VP1 RT-hnPCR). The VP1 is the most variable of the structural proteins [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>] and ideally suited for genotyping purposes. This novel RT-PCR was designed using available sequences with the second round providing coverage of the full-length VP1 coding region. The RT-PCR reagents were prepared using the Invitrogen SuperScript III One-Step RT-PCR System with Platinum <italic>Taq</italic> High Fidelity kit (Life Technologies, Australia) and primers AiV-2789F and AiV-4302R (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), with 5 µl of RNA added for a final reaction volume of 20 µl. A second-round PCR was amplified using the forward primer above, AiV-2789F, and AiV-3867R (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The reaction mix was formulated using the MyFi mix [Bioline (Aust) Pty Ltd, Australia] and 5 µl of the first-round PCR product diluted 1/100 for a total reaction volume of 20 µl. For mix details, primer concentration and cycling times see the detailed protocol published elsewhere [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]. The first-round PCR product was 1514 bp and the second round was 1079 bp in size.</p></sec><sec id=\"s2-6\"><title>Nucleotide sequence analysis and phylogenetics</title><p>Overlapping sequences were assembled using Sequencher 5.0 (Gene Codes Corporation, USA) to construct a near full-length genome. Sequence alignment and phylogenetic analysis for both the 3C–3D and VP1 regions were performed using <sc>mega</sc>7 (22), removing the primer sequences. The 3C–3D sequences from the published RT-PCR (519 bp) and the new 3C-3C RT-PCR (832 bp) were analysed with GenBank sequences and trimmed to 477 nucleotides to accommodate the shorter genotype C sequences, DQ145759 and LN612592 (<xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>). The VP1 RT-hnPCR sequences (1079 bp) were aligned with shorter GenBank sequences and trimmed to 641 nucleotides for analysis (<xref ref-type=\"fig\" rid=\"F3\">Fig. 3</xref>). Both phylogenetic trees were calculated using the maximum-likelihood method with 500 bootstrap replications using <sc>mega</sc>7 [<xref rid=\"R22\" ref-type=\"bibr\">22</xref>].</p><fig fig-type=\"figure\" id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2.</label><caption><p>Phylogenetic analysis of 477 bp sequences in the 3C–3D junction region of AiV-A. Strains marked with a blue diamond relate to this study. MG200054 is the first detected Australian AiV-A (red triangle). JQ792240–JQ792249, MG200055–MG200058 and MK830033–MK830037 were the sequences detected in the May 2008 study. Note: a 477 bp fragment was analysed to accommodate the shorter genotype C GenBank sequences.</p></caption><graphic xlink:href=\"acmi-2-099-g002\"/></fig><fig fig-type=\"figure\" id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Fig. 3.</label><caption><p>Phylogenetic analysis of 641 bp sequences in the VP1 region of AiV-A. Strains marked with a diamond relate to this study. MG200054 is the first detected Australian AiV-A (red triangle). Sequences MG237919, MG253822–5 and MK799849–MK799850 were the sequences detected in May 2008.</p></caption><graphic xlink:href=\"acmi-2-099-g003\"/></fig></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><p>The new RT-rPCR assay successfully detected AiV-A reference material supplied by T. Yamashita, Aichi Prefectural Institute of Public Health, Japan. Synthetically produced controls, included in each run alongside no-template controls, were used to monitor ongoing assay performance during routine use of the assay. The RT-rPCR was added to a suite of assays employed to investigate suspected foodborne outbreaks of unknown origin.</p><sec id=\"s3-1\"><title>First detection of an AiV-A virus</title><p>In January 2008, a patient in Queensland presented with gastroenteritis of unknown aetiology. Testing of the RNA extract from a stool sample (FSS693) identified AiV-A at 29 cycles. The Yamashita 3C–3D RT-PCR was used to confirm the AiV-A RT-rPCR result. Sanger sequencing confirmed AiV-A and genotyping using the 519 bp sequence placed it in AiV-A genotype B (<xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>). This region of the genome is widely used [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>] and the majority of partial GenBank sequences include this area. The nucleotide sequence was deposited in the GenBank database under accession number EU715251 and later updated with the longer sequence MG200054.</p><p>Further genomic sequencing of sample FSS693 was then conducted using sets of specific primers designed based on the complete genome of available AiV-A strains. Only the 5′ end of sample FSS693 could not be determined due to its difficult stem–loop structure [<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]. Phylogenetic analysis of both the 3C–3D and capsid region showed that it belonged to genotype B and had an amino acid insertion of a proline in VP0 at position 223. The amino acid sites 219–223 varied throughout the available sequences, and FSS693 had an STNSP motif, the same amino acid sequence identified in three Bangladesh sequences, EU143274–EU143276. The full-length capsid region, VP0, VP3 and VP1, was analysed with additional capsid sequences from GenBank. The two groups differed at the nucleotide level by 11–14 %; FSS693 differed by 5–6 % compared to other genotype B sequences.</p><p>Isolation of FSS693 was also attempted by three blind passages in Vero cells, however it proved unsuccessful, with no CPE observed and an increase in <italic>C</italic>\n<sub>T</sub>s when each passage was tested using the AiV-A RT-rPCR, until it was not detected on the final passage.</p></sec><sec id=\"s3-2\"><title>Further screening for <italic>Aichivirus</italic> A viruses</title><p>The detection of AiV-A in January 2008 prompted the investigation of additional faecal samples to gauge whether the virus was in circulation among in samples tested in the Queensland community. During May 2008, 756 faecal and vomit samples from 650 patients (55 % female) were submitted for norovirus testing and each of these samples was also screened using the AiV-A RT-rPCR as they arrived. The age distribution of patients is skewed to each end (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>).</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2.</label><caption><p>Age distribution of patients</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Age group (years)</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>No.</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Percentage of population</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p><1–4</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>154</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>23.7</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5–19</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>44</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6.8</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>20–39</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>47</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>7.2</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>40–59</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>75</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>11.5</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>60–99</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>330</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>50.8</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Totals</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>650</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>100</p>\n</td></tr></tbody></table></table-wrap><p>There were 23 positive samples detected from a total of 18 individual patients (3 % of norovirus requests). The 18 patients ranged in age from 2 to 89 years, with 50 % of patients (<italic>n</italic>=9) aged 61–89 years old. The remaining patients were either children aged 2–18 years (<italic>n</italic>=6) or adults aged 19–60 years (<italic>n</italic>=3). Sample types included 1 vomitus and the remaining 22 were faecal samples. From the 18 patients with AiV-A, 7 were also co-infected with norovirus genotype 2. This is comparable to previous studies reporting viral co-infection, such as AiV-A with astrovirus, rotavirus or norovirus [<xref rid=\"R4\" ref-type=\"bibr\">4, 7, 24</xref>]. In one study two patients were co-infected with three viruses, AiV-A, rotavirus and astrovirus [<xref rid=\"R24\" ref-type=\"bibr\">24</xref>].</p><p>These samples were also genotyped using the 832 bp amplified product of the 3C–3D RT-PCR. All 18 patients’ viruses grouped within AiV-A genotype B; however, sequence variation was seen within the group of samples (<xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>). There were 2 sequences, MK830034 and MG237919, with a pairwise distance of 6 % compared to the remaining 16 sequences from this study. Sequences generated were deposited with GenBank.</p></sec><sec id=\"s3-3\"><title>Analysis of Aichivirus A VP1</title><p>Eight of the Queensland May 2008 samples were tested using the VP1 RT-hnPCR. Seven were detected and able to be further sequenced. The sequences obtained were analysed alongside sequences from GenBank and a phylogenetic tree was created using <sc>mega</sc>7 (<xref ref-type=\"fig\" rid=\"F3\">Fig. 3</xref>). A 641 bp length sequence region was aligned to accommodate the shorter GenBank sequences. All seven sequences were genotype B, which confirmed the 3C–3D result. The sequences fell into two distinct clades within genotype B, with two sequences having a pairwise nucleotide distance of 5–7 % compared to the remaining five sequences from this study. The sequences generated were deposited with GenBank.</p></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Often there are undiagnosed cases of gastroenteritis and to address gaps in testing a screening assay for AiV-A was proposed. We designed an RT-rPCR for AiV-A in 2007 based on sequences available at the time. The AiV-A RT-rPCR allowed us to detect AiV-A in a faecal sample followed by identification of a further 18 cases from samples submitted for norovirus testing. This observational investigation of patients with AiV-A infection involved those aged from 2 to 89 years. While small, the findings revealed that AiV-A infects children and adults of all ages. Seroprevalence studies in Germany and Japan have shown that up to 80 % of adults have antibodies for AiV-A [<xref rid=\"R7\" ref-type=\"bibr\">7, 25</xref>]. Our study also revealed seven dual infections with noroviruses, with many previous studies reporting co-infection as commonplace, raising the question of whether AiV-A viruses are passengers or pathogens [<xref rid=\"R4\" ref-type=\"bibr\">4, 7, 24</xref>]. Recent studies have been unable to identify a causative role for AiV-A in gastroenteritis [<xref rid=\"R26\" ref-type=\"bibr\">26, 27</xref>], but longitudinal cohort studies are needed to better assess the role of AiV-As in acute gastroenteritis. Our first AiV-A-positive sample tested negative for <italic>Norovirus</italic>, <italic>Mamastrovirus 1</italic> and <italic>Rotavirus A</italic>, and conventional enteric bacteria. Eleven patients from the subsequent investigation had gastroenteritis of unknown aetiology, however; exhaustive testing for other viruses associated with gastroenteritis was not carried out, and further studies are warranted in Australia to determine the sustained ongoing burden of AiV-A in our population.</p><p>AiV-A viruses can be assigned to three genotypes, designated A, B and C [<xref rid=\"R3\" ref-type=\"bibr\">3, 4</xref>]. All 19 positive Queensland-detected viruses contained genotype B viruses according to sequences generated by our 3C–3D RT-PCR. Interestingly, the sequences demonstrated variability, falling into distinct clades within genotype B (<xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>). The 3C–3D RT-PCR may, therefore, be useful for contact tracing purposes in addition to its role in confirming the screening assay results. Genotype B viruses have been reported worldwide [<xref rid=\"R7\" ref-type=\"bibr\">7, 10, 13, 28</xref>]. From our 3C–3D phylogenetic tree (<xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>), Queensland viruses were seen to group together with sequences from predominantly East Asian and Southeast Asian countries.</p><p>The VP1 sequence is the most variable of the AiV-A regions [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>], making it difficult to sequence but a valuable source of data for genotyping when sequencing is successful. Most recent sequences in GenBank are partial VP1 sequences, ending before a region of approximately 40 nucleotides with a high number of cytosines. Our study generated seven partial VP1 sequences, which were all genotype B, with five grouping together with sequences from China and Thailand and two most like sequences from Brazil and Bangladesh (<xref ref-type=\"fig\" rid=\"F3\">Fig. 3</xref>). This supports the hypothesis that AiV-A is circulating throughout the community, possibly through multiple introductions over time, and not as a single strain or outbreak of the virus.</p><p>Further scrutiny of the VP0, VP3 and VP1 capsid regions of FSS693 revealed the formation of clades within both the genotype A and B lineages; however, the number of publicly available full-length capsid sequences is limited. The first four genotype A clades were reported in 2008 [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>] when the full capsid gene was analysed, and additional full-length capsid sequences from Thailand, Taiwan, Viet Nam, Germany and China are now available in GenBank. There is still no full-length genotype C sequence, only short VP1 and 3C–3D sequences from Burkina Faso and France (Mali travel history [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]). The mean percentage nucleotide distance between the two genotypes for the capsid sequences is 12–14 %, with FSS693 having a mean percentage difference of 5–6 % to other genotype B sequences. The reported amino acid insertion [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>] in the VP0 of genotype B sequences was also observed in the FSS693 sequence. Amino acid sites 219–223 vary among the sequences analysed, with FSS693 having the same STNSP motif as that seen in three Bangladeshi sequences.</p><p>To the best of our knowledge, these are the first AiV-As detected from acutely ill patients to be characterized in Australia. These novel assays support the detection of <italic>Aichivirus A</italic> when used to identify a pathogen in cases of gastroenteritis of unknown aetiology and their use highlights the need for more research on these rarely sought viruses. Our novel RT-rPCR diagnostic and conventional sequencing assays provided valuable information for understanding the identification, global spread and genetic variability of AiV-As circulating in Australia.</p></sec></body><back><ack id=\"ack1\"><title>Funding information</title><p>This work was supported by Forensic and Scientific Services, Health Support Queensland.</p></ack><ack id=\"ack2\"><title>Acknowledgements</title><p>We thank T. Yamashita, Aichi Prefectural Institute of Public Health, Japan, for providing reference material and Ian M. Mackay for critical review of the manuscript.</p></ack><ack id=\"ack3\"><title>Author contributions</title><p>J.N. planned the study, conceived and designed experiments, analysed data and wrote the manuscript. All authors conducted experiments and reviewed and approved the final version of the manuscript.</p></ack><ack id=\"ack4\"><title>Conflicts of interest</title><p content-type=\"COI-statement\">The authors declare that there are no conflicts of interest.</p></ack><ack id=\"ack5\"><title>Ethical statement</title><p>This study was conducted on samples previously collected and submitted for related gastrointestinal disease testing. Ethical clearance was granted by the Forensic and Scientific Services Human Ethics Committee, 2008, and it was decided that ethical permission was not required from individuals as the project was deemed to be method development and process improvement activity.</p></ack><fn-group><fn id=\"FN1\"><p>Abbreviations: AiV-A, Aichivirus A; PCR, Polymerase chain reaction; RT-PCR, Reverse transcription polymerase chain reaction; RT-rPCR, Reverse transcription real-time polymerase chain reaction; UTR, Untranslated region.</p></fn><fn id=\"FN2\" fn-type=\"accession-no\"><p>Sequences have been deposited with GenBank and assigned the following accession numbers: JQ792241–249; MG200054–058; MG237919; MG253822–825; MK799849–852; and MK830033–037.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>1</label><element-citation publication-type=\"journal\"><person-group 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Access Microbiol</journal-id><journal-id journal-id-type=\"hwp\">acmi</journal-id><journal-id journal-id-type=\"publisher-id\">acmi</journal-id><journal-title-group><journal-title>Access Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">2516-8290</issn><publisher><publisher-name>Microbiology Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005864</article-id><article-id pub-id-type=\"pmc\">PMC7523625</article-id><article-id pub-id-type=\"publisher-id\">000097</article-id><article-id pub-id-type=\"doi\">10.1099/acmi.0.000097</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Case Report</subject></subj-group></article-categories><title-group><article-title>When good bacteria behave badly: a case report of <italic>Bacillus clausii</italic> sepsis in an immunocompetant adult</article-title><alt-title alt-title-type=\"recto-page-foot\"><ext-link ext-link-type=\"uri\" xlink:href=\"http://acmi.microbiologyresearch.org\">http://acmi.microbiologyresearch.org</ext-link></alt-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-2329-5692</contrib-id><name><surname>Princess</surname><given-names>Isabella</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-7819-2944</contrib-id><name><surname>Natarajan</surname><given-names>T.</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ghosh</surname><given-names>Siddhartha</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><aff id=\"aff1\">\n<label><sup>1</sup>​</label>\n<institution content-type=\"department\">Department of Microbiology</institution>, <institution>Apollo Speciality Hospitals</institution>, <addr-line content-type=\"city\">Vanagaram, Chennai – 600095</addr-line>, <country>India</country>\n</aff><aff id=\"aff2\">\n<label><sup>2</sup>​</label>\n<institution content-type=\"department\">Department of Neurosciences</institution>, <institution>Apollo Speciality Hospitals</institution>, <addr-line content-type=\"city\">Vanagaram, Chennai – 600095</addr-line>, <country>India</country>\n</aff></contrib-group><author-notes><corresp id=\"cor1\"><bold>*Correspondence:</bold> Isabella Princess, <email xlink:href=\"mailto:isadear@gmail.com\">isadear@gmail.com</email></corresp></author-notes><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>3</day><month>2</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>3</day><month>2</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>2</volume><issue>4</issue><elocation-id>acmi000097</elocation-id><history><date date-type=\"received\"><day>19</day><month>10</month><year>2019</year></date><date date-type=\"accepted\"><day>03</day><month>12</month><year>2019</year></date></history><permissions><copyright-statement>© 2020 The Authors</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"acmi-2-097.pdf\"/><abstract><p>Reports of unusual microorganisms causing human infections are on the rise due to transitions in epidemiological trends. Commensal/normal flora which are otherwise termed as ‘good bacteria’ are now causing infections in different group of patients, mostly immunocompromised individuals. Various host and environmental factors play a pivotal role in microbial transmigration from their normal habitat into the blood and other body sites. We report one such ‘good bacterium’ associated with sepsis in a patient who was given the same bacterium in the form of probiotics.</p></abstract><kwd-group><kwd><italic>Bacillus clausii</italic></kwd><kwd>probiotics</kwd><kwd>blood culture</kwd><kwd>sepsis</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>OpenAccessEmbargo</meta-name><meta-value>0</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Probiotics are live microorganisms which have the ability to colonize the human gut and provide health benefits [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]. Organisms commonly used as probiotics include <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.5320\">Lactobacillus</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.7677\">Bifidobacterium</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4857\">Bacillus</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.5525\">Enterococcus</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3092\">Escherichia</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.5605\">Streptococcus</ext-link>\n</named-content></italic> or fungi such as <italic>Saccharomyces boulardi</italic> [<xref rid=\"R2\" ref-type=\"bibr\">2, 3</xref>]. The beneficial effects of probiotics documented in the literature include: synthesis of vitamins, prevention of colonization of pathogenic bacteria, antagonization of other bacteria by secreting bacteriocins and other antibacterial substances, and stimulation of secretory IgA antibodies which antagonize other pathogens [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]. These benefits make them a potential option in treating antibiotic-associated colitis, critically ill and surgical patients, and diarrhoea in children and adults.</p><p>Although the beneficial effects of probiotics are well publicized, the risk and drawbacks due to the same is almost always neglected. There have been reports of various probiotic bacteria such as <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10618\">Bacillus subtilis</ext-link>\n</named-content></italic> causing sepsis and a few recent reports from India and the Philippines have surfaced on <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">Bacillus clausii</ext-link>\n</named-content></italic> sepsis in immunocompromised individuals and in neonates [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. We encountered a diabetic but otherwise normal adult developing sepsis with <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic>, the source of which was traced to be the same organism given to her in the form of probiotics. To the best of our knowledge, this is the first report of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> causing sepsis in an immunocompetent adult.</p></sec><sec sec-type=\"cases\" id=\"s2\"><title>Case report</title><p>Our patient is a middle-aged type II diabetic, not a known hypertensive, with no history of any other chronic illness and no other significant past or family history. The patient presented to the emergency department with acute-onset frontal headache of 1 day duration. A diagnosis of cerebral vein thrombosis with right parietal intraparenchymal bleeding with oedema and midline shift was made. An emergency decompressive craniotomy was done and the patient was gradually weaned off the ventilator, the tracheostomy tube was decannulated, she was mobilized and her Glasgow Coma Scale (GCS) improved to 15/15.</p><p>After 26 days, the patient underwent autologous and mesh cranioplasty. She then developed subgaleal haematoma 7 days after the cranioplasty for which re-exploration and evacuation of the haematoma was performed. Recurrent subgaleal and extradural haematoma with midline shift developed with a drop in GCS to 5/15. The patient underwent emergency re-exploration and evacuation of the haematoma with bone flap removal with significant blood loss which was corrected with transfusion of 6 units of blood and blood products. The patient was being continuously monitored in the Neurosurgical intensive care unit and treated with appropriate medication after four surgical procedures.</p><p>On the third post-operative day of the last surgery, a tracheal secretion grew <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10789\">Klebsiella pneumoniae</ext-link>\n</named-content></italic> which was multidrug-resistant. Drain fluid and pus taken from the surgical site also grew multidrug-resistant <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10789\">K. pneumoniae</ext-link>\n</named-content></italic> which was successfully eliminated with antimicrobial therapy. The patient was treated with appropriate antibiotics and with the probiotic Enterogermina (<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> spore suspension of 2 billion/5 ml, Strains: O/C, N/R, SIN and T) [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>] due to loose stools on broad-spectrum antibiotics. After about 10 days, the patient developed fever after which two sets of blood cultures were drawn from two different sites (peripheral venipuncture). Procalcitonin as determined by semi-quantitative immunochromatography was >0.5 to ≤2 ng ml<sup>−1</sup>. Two aerobic blood culture bottles grew Gram-positive bacilli within 48 h which were isolated in pure culture and identified as <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> by a matrix assisted laser desorption ionization time of flight (MALDI-TOF; bioMeriéux) assay (<xref ref-type=\"fig\" rid=\"F1\">Fig. 1</xref>). Growth was observed 11 days after initiation of the probiotic containing spores of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic>. Phenotypic drug susceptibility testing for determination of MIC using an E test was performed using Clinical Laboratory Standards Institute (CLSI) M45 guidelines [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]. The isolate was susceptible to ciprofloxacin (MIC: 0.38 µg ml<sup>−1</sup>) and vancomycin (MIC: 0.5 µg ml<sup>−1</sup>) but resistant to penicillin (MIC: 32 µg ml<sup>−1</sup>). All other recommended antibiotics were tested using Kirby Bauer disc diffusion but are not reported because there are no interpretative CLSI guidelines. In order to determine the origin of the blood isolate, the probiotic was subjected to identification following aerobic culture. We were able to isolate and identify the organism as <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> using MALDI-TOF with the susceptibility pattern of the probiotic strain perfectly matching the blood isolate (<xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>). These phenotypically correlative findings between the clinical isolate and the probiotic stain established that the probable source of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> in blood to be from the probiotic. The patient was treated with teicoplanin, which showed an MIC of 0.094 µg ml<sup>−1</sup>, although it could not be reported as susceptible due to the unavailability of interpretative guidelines. The patient responded well to antibiotic therapy and became afebrile with very good resolution of sepsis within 48 h of teicoplanin initiation. Repeat blood cultures drawn 2 weeks after initial blood cultures showed no growth, thereby signifying adequate response to antibiotic therapy.</p><fig fig-type=\"figure\" id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1.</label><caption><p>Culture characteristics of the <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">Bacillus clausii</ext-link>\n</named-content></italic> isolate from blood on (a) blood agar (colony variants) and (b) chocolate agar.</p></caption><graphic xlink:href=\"acmi-2-097-g001\"/></fig><fig fig-type=\"figure\" id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2.</label><caption><p>Antibiotic susceptibility testing of the clinical isolate (right) and probiotic strain (left)</p></caption><graphic xlink:href=\"acmi-2-097-g002\"/></fig></sec><sec sec-type=\"discussion\" id=\"s3\"><title>Discussion</title><p>The usefulness and disadvantages of using <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> spores as probiotics in critically ill patients is discussed here. It is noteworthy that <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> in our patient is definitely a rare occurrence compared to previous reports of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> sepsis. Our patient did not have any underlying immunocompromising condition such as malignancy, corticosteroid therapy, transplantation or immunodeficiency syndromes. This observation is contrary to previously published reports of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> associated with sepsis in individuals with one of the above listed immunocompromising conditions [<xref rid=\"R7\" ref-type=\"bibr\">7, 8</xref>]. Therefore it is noteworthy that <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> used as probiotics can transmigrate from the gut and enter the bloodstream of an individual irrespective of his/her underlying immunocompromised state.</p><p>Another major lesson from our patient is the need to send at least two sets of blood cultures in clinically suspected sepsis patients [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]. Additional blood culture bottles increase the sensitivity and yield of microorganisms causing sepsis [<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]. It is common practice to ignore Gram-positive bacilli as a contaminant from one bottle of blood because <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4857\">Bacillus</ext-link>\n</named-content></italic> is one of the most common contaminants [<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]. In our patient, the organism was isolated from two (aerobic) bottles out of four (two aerobic and two anaerobic) bottles of blood sent for culture. In order to establish the pathogen it is convenient to collect an adequate quantity of blood, and convincing evidence would be isolation of the organism of questionable significance from more than one blood culture bottle.</p><p>In previously published reports, vancomycin, a glycopeptide group antibiotic, has been used in treating patients with <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> sepsis. Our patient was treated with another glycopeptide, teicoplanin, and successful elimination of the bacterium from blood was achieved. Since interpretative guidelines for teicoplanin are not given by the CLSI, the same could not be interpreted from the patient's isolate. However, following successful treatment in our patient, it can be postulated that teicoplanin is an effective alternative in treating sepsis due to <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic>. A major challenge in treating <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> sepsis is the limited therapeutic options. This is due to the fact that <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> is known to carry multiple drug-resistant genes [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>], and could explain treatment failure in patients from previously published reports. Initiation of an appropriate antibiotic as well as adequate dosage was another positive aspect of our case which resulted in better patient outcome. Therefore, thorough knowledge of resistance patterns is mandatory when dealing with infections caused by exotic/unusual organisms.</p><p>Good bacteria can turn bad, good bacteria can carry drug-resistant genes [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>], and good bacteria can be recalcitrant to therapy and require high-end antibiotic therapy. Bacterial strains used as probiotics can become virulent and establish themselves as pathogens. The mechanism of virulence remains questionable especially in normal individuals, warranting further research on this issue.</p><sec id=\"s3-1\"><title>Conclusion</title><p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> may be safe as a probiotic but its use in immunocompromised and chronically ill patients is questionable. This therapeutic dilemma is due to its probable ability to transmigrate from the gut into the bloodstream and cause sepsis, as experienced by our patient. There are no previous reports of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> sepsis among adults with no known underlying chronic disease or immunocompromising condition. This case report can thus be taken as a wake-up call for making judicious use of this probiotic even in normal individuals. We conclude by stating that use of probiotics containing <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.4891\">B. clausii</ext-link>\n</named-content></italic> spores in critically ill patients is not always beneficial; rather its use should be used with caution.</p></sec></sec></body><back><ack id=\"ack1\"><title>Funding information</title><p>The authors received no specific grant from any funding agency.</p></ack><ack id=\"ack2\"><title>Conflicts of interest</title><p content-type=\"COI-statement\">The authors declare that there are no conflicts of interest.</p></ack><fn-group><fn id=\"FN1\"><p>Abbreviations: CLSI, Clinical Laboratory Standards Institute; GCS, Glasgow Coma Scale; MALDI-TOF, matrix assisted laser desorption ionization time of flight; MIC, Minimum Inhibitory Concentration.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>1</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sanders</surname><given-names>ME</given-names></name></person-group><article-title>Probiotics: definition, sources, selection, and uses</article-title><source>Clin Infect Dis</source><year>2008</year><volume>46</volume><fpage>S58</fpage><lpage>S61</lpage><pub-id pub-id-type=\"doi\">10.1086/523341</pub-id><pub-id pub-id-type=\"pmid\">18181724</pub-id></element-citation></ref><ref id=\"R2\"><label>2</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elshaghabee</surname><given-names>FMF</given-names></name><name><surname>Rokana</surname><given-names>N</given-names></name><name><surname>Gulhane</surname><given-names>RD</given-names></name><name><surname>Sharma</surname><given-names>C</given-names></name><name><surname>Panwar</surname><given-names>H</given-names></name><etal>et al</etal></person-group><article-title>\n<italic>Bacillus</italic> as potential probiotics: status, concerns, and future perspectives</article-title><source>Front Microbiol</source><year>2017</year><volume>8</volume><elocation-id>1490</elocation-id><pub-id pub-id-type=\"doi\">10.3389/fmicb.2017.01490</pub-id><pub-id pub-id-type=\"pmid\">28848511</pub-id></element-citation></ref><ref id=\"R3\"><label>3</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gupta</surname><given-names>V</given-names></name><name><surname>Garg</surname><given-names>R</given-names></name></person-group><article-title>Probiotics</article-title><source>Indian J Med Microbiol</source><year>2009</year><volume>27</volume><fpage>202</fpage><lpage>209</lpage><pub-id pub-id-type=\"doi\">10.4103/0255-0857.53201</pub-id><pub-id pub-id-type=\"pmid\">19584499</pub-id></element-citation></ref><ref id=\"R4\"><label>4</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oggioni</surname><given-names>MR</given-names></name><name><surname>Pozzi</surname><given-names>G</given-names></name><name><surname>Valensin</surname><given-names>PE</given-names></name><name><surname>Galieni</surname><given-names>P</given-names></name><name><surname>Bigazzi</surname><given-names>C</given-names></name></person-group><article-title>Recurrent septicemia in an immunocompromised patient due to probiotic strains of <italic>Bacillus subtilis</italic>\n</article-title><source>J Clin Microbiol</source><year>1998</year><volume>36</volume><fpage>325</fpage><lpage>326</lpage><pub-id pub-id-type=\"doi\">10.1128/JCM.36.1.325-326.1998</pub-id><pub-id pub-id-type=\"pmid\">9431982</pub-id></element-citation></ref><ref id=\"R5\"><label>5</label><element-citation publication-type=\"web\"><person-group><collab>Enterogermina, Bacillus clausii – Sanofi</collab></person-group><article-title>Patient information leaflet</article-title><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.enterogermina.in//media/EMS/Conditions/Consumer%20Healthcare/Brands/Enterogermina-India/Prodotti/pdf/product.pdf?la=en\">https://www.enterogermina.in//media/EMS/Conditions/Consumer%20Healthcare/Brands/Enterogermina-India/Prodotti/pdf/product.pdf?la=en</ext-link><comment>on: 16/08/2019</comment></element-citation></ref><ref id=\"R6\"><label>6</label><element-citation publication-type=\"book\"><person-group><collab>CLSI</collab></person-group><source>Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Access Microbiol</journal-id><journal-id journal-id-type=\"hwp\">acmi</journal-id><journal-id journal-id-type=\"publisher-id\">acmi</journal-id><journal-title-group><journal-title>Access Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">2516-8290</issn><publisher><publisher-name>Microbiology Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005870</article-id><article-id pub-id-type=\"pmc\">PMC7523626</article-id><article-id pub-id-type=\"publisher-id\">000106</article-id><article-id pub-id-type=\"doi\">10.1099/acmi.0.000106</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Case Report</subject></subj-group></article-categories><title-group><article-title>The case report of <italic>Mycobacterium arupense</italic> wound infection in diabetes mellitus patients; the first report and literature review</article-title><alt-title alt-title-type=\"recto-page-foot\"><ext-link ext-link-type=\"uri\" xlink:href=\"http://acmi.microbiologyresearch.org\">http://acmi.microbiologyresearch.org</ext-link></alt-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Navid</surname><given-names>Sepehr</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sadegh-Ehdaei</surname><given-names>Bahar</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Shabani</surname><given-names>Mehdi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hasani</surname><given-names>Melika</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mirzaei</surname><given-names>Arezoo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ghazvini</surname><given-names>Kiarash</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Youssefi</surname><given-names>Masoud</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-1208-8479</contrib-id><name><surname>Keikha</surname><given-names>Masoud</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>4</sup>\n</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><aff id=\"aff1\">\n<label><sup>1</sup>​</label>\n<institution content-type=\"department\">Department of Microbiology</institution>, <institution>School of Medicine, Isfahan Medical University</institution>, <addr-line content-type=\"city\">Isfahan</addr-line>, <country>Iran</country>\n</aff><aff id=\"aff2\">\n<label><sup>2</sup>​</label>\n<institution content-type=\"department\">M.Sc. of Molecular Genetics</institution>, <institution>Sana Institue of Higher Education</institution>, <addr-line content-type=\"city\">Sari</addr-line>, <country>Iran</country>\n</aff><aff id=\"aff3\">\n<label><sup>3</sup>​</label>\n<institution content-type=\"department\">Antimicrobial Resistance Research Center</institution>, <institution>Mashhad University of Medical Sciences</institution>, <addr-line content-type=\"city\">Mashhad</addr-line>, <country>Iran</country>\n</aff><aff id=\"aff4\">\n<label><sup>4</sup>​</label>\n<institution content-type=\"department\">Department of Microbiology and Virology</institution>, <institution>Faculty of Medicine, Mashhad University of Medical Sciences</institution>, <addr-line content-type=\"city\">Mashhad</addr-line>, <country>Iran</country>\n</aff></contrib-group><author-notes><corresp id=\"cor1\"><bold>*Correspondence:</bold> Masoud Keikha, <email xlink:href=\"mailto:masoud.keykha90@gmail.com\">masoud.keykha90@gmail.com</email></corresp></author-notes><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>17</day><month>2</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>2</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>2</volume><issue>4</issue><elocation-id>acmi000106</elocation-id><history><date date-type=\"received\"><day>05</day><month>11</month><year>2019</year></date><date date-type=\"accepted\"><day>09</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 The Authors</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"acmi-2-106.pdf\"/><abstract><p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">Mycobacterium arupense</ext-link>\n</named-content></italic> is among the opportunist pathogens of atypical mycobacteria emergence (atypical mycobacteria) that is one of the isolated and reported environmental and clinical specimens. Numerous cases of osteo-articular infections of this bacterium are reported nowadays, while the pulmonary infection is rare. We identified <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">Mycobacterium arupense</ext-link>\n</named-content></italic> in non-healing wound infection of an elderly woman with history of diabetes mellitus. She has negative tests for HIV, HBV and HCV, but was positive for HTLV-1. The patient was referred according to mild-fever, non-healing, destructive, and swelled lesion on her left foot. The mycobacterial wounds infection was suspected due to her non-conclusive previous treatment. The pathology, acid-fast staining, conventional and 16S rRNA sequencing confirmed the micro-organism to be <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic>. Finally, the patient recovered following two-week consumption of clarithromycin, ethambutol and rifabutin. The results of this study provide evidence on the potential pathogenicity, clinical outcomes and treatment of infections caused by this bacterium.</p></abstract><kwd-group><kwd><italic>Mycobacterium arupense</italic></kwd><kwd>diabetes mellitus</kwd><kwd>wound infection</kwd><kwd>16S rRNA</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>OpenAccessEmbargo</meta-name><meta-value>0</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Non-tuberculosis mycobacteria (NTM) are a group of ‘Mycobacteria’ that live in environmental resources such as saprophytes and that enter their body through inhalation and traumatic inclusion, causing the mycobacterosis infection [<xref rid=\"R1\" ref-type=\"bibr\">1, 2</xref>]. The incidence rate of NTM infections is increasing nowadays [<xref rid=\"R3\" ref-type=\"bibr\">3, 4</xref>]. The improved diagnostic methods, especially the molecular diagnostic methods, and the increased number of immune-disorders have increased the rate of NTM infections [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>].</p><p>\n<italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">Mycobacterium arupense</ext-link>\n</named-content></italic> was first isolated from a tendon sample in 2006 and identified by Cloud <italic>et al</italic>. [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]. <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> is part of the <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6406\">M. terrae</ext-link>\n</named-content></italic> complex and is very similar to <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6384\">M. nonchromogenicum</ext-link>\n</named-content></italic> [<xref rid=\"R6\" ref-type=\"bibr\">6, 7</xref>]. Identification of this type of clinical sample is quite difficult due to the similarity of phenotypic tests with the members of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.6406\">M. terrae</ext-link>\n</named-content></italic> complex. However, the 16S rRNA gene in <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> is as a signature sequence and the sequencing of housekeeping genes, especially 16S rRNA, is able to correctly identify this species [<xref rid=\"R6\" ref-type=\"bibr\">6, 8</xref>]. According to the American Thoracic Society (ATS) guidelines, it is recommended that the NTM isolates isolated from clinical specimens should be identified to the species level for the final diagnosis, accurate identification, patient management, appropriate treatment and epidemiological goals [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>].</p><p>There are numerous reports about the <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> isolation from respiratory, tenosynovitis, osteoarticular, osteomyelitis and disseminated infections [<xref rid=\"R7\" ref-type=\"bibr\">7, 8, 10–12</xref>]. The present study was the first case report of cutaneous infection by <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> in a HTLV-1-infected diabetic patient (HTLV-1 infected).</p></sec><sec id=\"s2\"><title>Case presentation</title><p>A 51-year-old woman referred to Al-Zahra Hospital in Isfahan (Isfahan, Iran) in June 2018 due to non-healing foot ulcers in her left foot. She was a housewife living in a rural area near Faridan, Iran, working on farms and having a previous experience of foot ulcers. However, she stated that her recent foot ulcer had not healed in the last 1.5 months. The patient had a history of diabetes mellitus (since 2011). On initial examination, the patient had a mild fever (37.8 °C), and a swollen, necrotic ulcer was evident on the toes, and according to the patient, the lesions were not very painful. Sampling was done from the ulcer. Based on the microbiology lab reports, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.11043\">Staphylococcus aureus</ext-link>\n</named-content></italic> and <italic>Klebsiella pneumonia</italic> were isolated and penicillin, doxycycline, imipenem and bandage with Betadine were prescribed for the patient.</p><p>The patient returned again about a month later due to failure to respond to treatment, although she reported painful scarring and pale discharge; lesion depth was 1.5 cm and also extended to her sole (<xref ref-type=\"fig\" rid=\"F1\">Fig. 1</xref>). The patient had a temperature of 38.2 °C and according to MRI abdominal cavity and chest X-ray, she had no signs of inflammation in her lungs and internal organs.</p><fig fig-type=\"figure\" id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1.</label><caption><p>Wound lesion on the right foot of the patient. (A) lesions on the toes, (B) extended lesion on her sole.</p></caption><graphic xlink:href=\"acmi-2-106-g001\"/></fig><p>The Fasting Blood Sugar (FBS) level was 126 mg dl<sup>–1</sup>; also Count Blood Cell (CBC) included: WBC: 11 500 μl<sup>–1</sup>, RBC: 4500 μl<sup>–1</sup>, Hb: 15.3 g dl<sup>–1</sup>, HCT: 44 % and transferases hepatic abnormalities were slightly elevated; patient CRP and ESR were also evaluated at 61 mg l<sup>–1</sup> and 56 mm h<sup>–1</sup>, respectively. The patient had negative signs of HIV, HCV, and HBV, but the signs of HTLV-1 was positive (the titer of HTLV-1 virus in the patient blood was 12.8 copies per 100 cells).</p><p>The pathology evaluation revealed the presence of granuloma. Blood culture of the patient was negative, and wound exudate samples were examined using Gram-staining and Ziehl-Neelsen staining. Acid-fast bacilli were confirmed in the wound exudate, and subsequently, wound exudate samples were cultured on blood agar and Lowenstein Jensen slant. Two weeks later small colonies appeared on LJ enriched in Sauton's broth (<xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>).</p><fig fig-type=\"figure\" id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2.</label><caption><p>Colony morphology (a) and acid-fast staining (b) of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">Mycobacterium arupense</ext-link>\n</named-content></italic>.</p></caption><graphic xlink:href=\"acmi-2-106-g002\"/></fig><p>The considered isolate was identified as rapidly growing mycobacteria (RGM) due to the growth rate (<7 days), lack of pigment production, negative results for niacin and nitrate reductase as well as urease and heat stable catalase production (68 °C). Molecular analysis was performed to identify to the species level. Simple boiling method was used to extract the DNA, the amplification of nearly full-length of 16S rRNA was performed by primers pA: 5′ AG-AGA GTTTGATCCTGGCTCAG-3′ and pI: 5′-TGCACACAGGCCACAAGGGA-3′ according to Rogall <italic>et al</italic>., and the sequence of PCR product was analysed [<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]. NTM spp. can be differentiated by high-precision via nucleotide sequence of the hypervariable regions A (125-270) and B (408-503) of the 16S rRNA. In addition, the nucleotide sequence of rRNA gene of a short helix region is between the 451–482 positions that is a signature for RGM [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]. Based on the results by Blast, it was found that the partial sequence 16S rRNA of the considered isolation was 100 % similar to <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">Mycobacterium arupense</ext-link>\n</named-content></italic> (DQ157760). A phylognic-relationship analysis based on closely related mycobacterial species also identified the isolate as <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> as accession number: MN865166 (<xref ref-type=\"fig\" rid=\"F3\">Fig. 3</xref>).</p><fig fig-type=\"figure\" id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Fig. 3.</label><caption><p>16S rRNA sequence-based phylogenetic tree of our isolate and closely related mycobacterial species which are conducted using <sc>mega</sc> with the neighbour-joining method and K2P distance.</p></caption><graphic xlink:href=\"acmi-2-106-g003\"/></fig><p>Drug susceptibility test (DST) was performed according to CLSI M24-A2 recommendations by the broth micro-dilution method. Based on the DST results, the considered isolate was sensitive to clarithromycin, ethambutol, and rifabutin antibiotics and resistant to isoniazid, rifampicin, amikacin, moxifloxacin, ciprofloxacin and linezolid. Finally, the treatment was done with purulent drainage, initiation with clarithromycin, ethambutol and rifabutin together with once applying of interferon alpha to reduce the proviral load HTLV-1. After two weeks of antibiotictherapy the foot wound infection of the patient recovered and the patient was discharged with personal consent.</p></sec><sec sec-type=\"discussion\" id=\"s3\"><title>Discussion</title><p>There are various evidences of isolation of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> from environmental samples nowadays, such as surface water, soil, fish tanks, animal urine, and duck houses [<xref rid=\"R15\" ref-type=\"bibr\">15, 16</xref>]. Despite the widespread presence of this bacterium, there have been limited reports of human infections with <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> [<xref rid=\"R16\" ref-type=\"bibr\">16, 17</xref>]. The present study was the first case report of a diabetic person foot ulcer infection by <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic>. Due to the limitations of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic>'s clinical reports, there is no standard guideline for the treatment of infections of this bacterium [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]. According to the review of the literature, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> infections are more common in people with immune-disorders (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1.</label><caption><p>The summaries of clinical case reports of infection with <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic>\n</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>The authors</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Cases</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Risk factors</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Diagnostic method</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Treatment</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Duration</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Clinical outcome</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Location</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Year</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Ref</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Lopez <italic>et al</italic>.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Tenosynovitis</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Multiple immunomodulatory drugs</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Culture</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Clarithromycin (500 mg 2×/d)</p>\n<p>Ethambutol (1,200 mg/d)</p>\n<p>Rifabutin (300 mg/d)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>12</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>improved</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>USA</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2016</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Tsai <italic>et al</italic>.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Tenosynovitis</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Diabetes mellitus</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Sequencing of 16S rRNA</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Clarithromycin (500 mg every 12 h), moxifloxacin (400 mg daily), rifabutin (300 mg daily), ciprofloxacin</p>\n<p>(400 mg every 12 h), and ethambutol</p>\n<p>(1000 mg daily)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>improved</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Taiwan</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2008</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Slany <italic>et al</italic>.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Pulmonary (3 cases)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Diabetes mellitus (case 1) Chronic gastritis (case 2)</p>\n<p>–</p>\n<p>(case 3)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Culture and 16S rRNA sequencing</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Tuberculosis therapy</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1–3 months</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>improved</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Czech Republic</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2010</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Lee <italic>et al</italic>.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Tenosynovitis</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Puncture injury</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>16S rRNA sequencing</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Clarithromycin, ethambutol, and rifampin</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<sc>nr</sc>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<sc>nr</sc>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>South Korea</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2014</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Heidarieh <italic>et al</italic>.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Pulmonary</p>\n<p>(case 1)</p>\n<p>Disseminated (case 2)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>HIV-infected</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Culture and 16S rRNA sequencing</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Clarithromycin, ethambutol, and rifampin</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<sc>nr</sc>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<sc>nr</sc>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Iran</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2013</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Seidl <italic>et al</italic>.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Osteoarticular</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Traumatic knee arthrotomy</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Culture</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Azithromycin, rifampin, and ethambutol</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>24</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>improved</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Colorado</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2014</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Beam <italic>et al</italic>.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Flexor Tenosynovitis</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Hypertension and hyperlipidemia</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>16S rRNA sequencing</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Rifabutin, ethambutol, and clarithromycin and surgical drainage</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>improved</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>USA</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2014</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R27\" ref-type=\"bibr\">27</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Neonakis <italic>et al</italic>.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Pulmonary</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Large deficiency of the mitral valve and hypertension</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Hsp65-RFLP</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Rifabutin, ethambutol, and clarithromycin</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<sc>nr</sc>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<sc>nr</sc>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Greece</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2009</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Legouta <italic>et al</italic>.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Osteomyelitis</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Immunocompetent</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Culture and hsp65 and 16S rRNA sequencing</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Ciprofloxacin, ethambutol, amikacin</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>12 month</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>improved</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>France</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2012</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R28\" ref-type=\"bibr\">28</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Senda et al.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Tenosynovitis</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Arterial hypertension</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>DNA–DNA hybridization</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Surgery and Rifampin, ethambutol</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>14</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>improved</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Japan</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2015</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R29\" ref-type=\"bibr\">29</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Zhou <italic>et al</italic>.</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Pleural effusion</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Immunocompetent</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>16S rRNA sequencing</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Capreomycin and moxifloxacin</p>\n<p>(No NTM treatment)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<sc>nr</sc>\n</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>improved</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>China</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2018</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]</p>\n</td></tr></tbody></table></table-wrap><p>Currently, human infections caused by <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> are divided into two categories: pulmonary and extra-pulmonary infections. Based on the existing reports, most of these people have trauma, HIV, or corticosteroids use [<xref rid=\"R17\" ref-type=\"bibr\">17, 18</xref>]. Regarding the limited available information, it is not possible to fully understand the clinical significance, clinical outcome and duration of treatment of this bacterium [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>].</p><p>However, surgical and antimicrobial therapy methods are commonly used for tenosynovitis and osteo-articular infections, whereas disseminated infections initiated with rifabutin, clatrithomycin and ethambutol have had satisfactory results. Furthermore, treatment regarding the pulmonary infection is based on ethambutol, clatrithomycin, rifabutin and drug susceptibility test; TMP-SXT results were also varied (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><p>According to the review of the literatures, the duration of treatment for <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> infections varies between 6 and 24 months, depending on the type of infection and the involved tissue, and includes a combination of surgery and antibiotic therapy. No signs of relapse or re-infection were reported after the treatment (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Also, most reports have shown that <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> clinical isolates are sensitive to clarithromycin, rifabutin, ethambutol and rarely to quinolones (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><p>In a study on <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> infections in cancer patients, Hamal <italic>et al</italic>. observed that the clinical outcome showed no significant difference between the treated <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> infected cancer patients treated and the untreated group; there were no reports of relapse or death from <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]. Vasireddy <italic>et al</italic>. reported in their studies 10 strains of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> tissue specimens that most of these patients had experience of trauma or using corticosteroids [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]. Currently, <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> is considered an emergent pathogen for osteoarticular infection. However, the role of this bacterium as a respiratory system pathogen is still unknown [<xref rid=\"R20\" ref-type=\"bibr\">20–22</xref>]. Pulmonary infections caused by <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> have been so far observed only in immune-deficiency patients (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><p>In this study, we present the first report of an unusual cutaneous infection caused by <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> in Iran. Patient's immune system of the present study was weakened by infection with HTLV-1 and diabetes mellitus, and according to the evidence, this bacterium is more likely to cause opportunistic infections in the individuals with immune system deficiency.</p><p>Identification of <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> is very important in TB-indemic regions, especially in Iran. Due to the slow growth of mycobacterium tuberculosis in the developing countries such as Iran, the considered patient affected by TB is reported only by observing acid-fast bacilli in smears of clinical specimens and considering a TB-endemic area [<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]. This report demonstrates the importance of culture and identification to the species level of mycobacteria for appropriate diagnosis and treatment [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]. Based on the available evidence, two reports of infection with <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.10098\">M. arupense</ext-link>\n</named-content></italic> in Iran have been reported indicating circulation of this bacterium in this geographical area [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]. The study was also the first report of cutaneous infection by this bacterium, indicating the potential pathogenicity of this microorganism.</p><p>Finally, the importance of molecular methods in identifying NTM spp. should be mentioned. Conventional and culture methods are expensive due to the slow growing nature of mycobacteria, and are not quite appropriate due to their inconclusive state, whereas molecular methods, especially 16S rRNA sequencing, are able to identify NTM species in high accuracy, in addition to being non-expensive and fast [<xref rid=\"R24\" ref-type=\"bibr\">24</xref>].</p></sec></body><back><ack id=\"ack1\"><title>Funding information</title><p>The authors received no specific grant from any funding agency.</p></ack><ack id=\"ack2\"><title>Acknowledgements</title><p>Written informed consent for publication of their clinical details was obtained from the patient.</p></ack><ack id=\"ack3\"><title>Author contributions</title><p>Author Contributions: Study concept and design: M. K; drafting of the manuscript: K. G., M. Y., B. S.; analysis and interpretation of data: S. N; revised the manuscript: M. Sh., M. H.</p></ack><ack id=\"ack4\"><title>Conflicts of interest</title><p content-type=\"COI-statement\">The authors declare that there are no conflicts of interest.</p></ack><ack id=\"ack5\"><title>Ethical statement</title><p>The study was approved by the Ethics Committee of Mashhad University of Medical Sciences (IR.MUMS.REC.1398.2165442).</p></ack><fn-group><fn id=\"FN1\"><p>Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency viruses; HTLV-1, human T-cell lymphotropic virus type 1; NTM, Nontuberculous mycobacteria.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>1</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Keikha</surname><given-names>M</given-names></name><name><surname>Ghazvini</surname><given-names>K</given-names></name></person-group><article-title>Comment on “nontuberculous mycobacterial infection after lung transplantation: a report of four 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"brief-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Access Microbiol</journal-id><journal-id journal-id-type=\"hwp\">acmi</journal-id><journal-id journal-id-type=\"publisher-id\">acmi</journal-id><journal-title-group><journal-title>Access Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">2516-8290</issn><publisher><publisher-name>Microbiology Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005868</article-id><article-id pub-id-type=\"pmc\">PMC7523627</article-id><article-id pub-id-type=\"publisher-id\">000104</article-id><article-id pub-id-type=\"doi\">10.1099/acmi.0.000104</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Short Communication</subject></subj-group></article-categories><title-group><article-title>Sensitivity of shotgun metagenomics to host DNA: abundance estimates depend on bioinformatic tools and contamination is the main issue</article-title><alt-title alt-title-type=\"recto-page-foot\"><ext-link ext-link-type=\"uri\" xlink:href=\"http://acmi.microbiologyresearch.org\">http://acmi.microbiologyresearch.org</ext-link></alt-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-7448-0869</contrib-id><name><surname>McArdle</surname><given-names>Andrew J.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-9878-4007</contrib-id><name><surname>Kaforou</surname><given-names>Myrsini</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><aff id=\"aff1\">\n<label><sup>1</sup>​</label>\n<institution content-type=\"department\">Section of Paediatric Infectious Disease, Department of Infectious Disease</institution>, <institution>Imperial College London</institution>, <addr-line content-type=\"city\">London W2 1PG</addr-line>, <country>UK</country>\n</aff></contrib-group><author-notes><corresp id=\"cor1\"><bold>*Correspondence:</bold> Andrew J. McArdle, <email xlink:href=\"mailto:a.mcardle@imperial.ac.uk\">a.mcardle@imperial.ac.uk</email></corresp></author-notes><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>17</day><month>2</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>2</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>2</volume><issue>4</issue><elocation-id>acmi000104</elocation-id><history><date date-type=\"received\"><day>04</day><month>11</month><year>2019</year></date><date date-type=\"accepted\"><day>14</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 The Authors</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"acmi-2-104.pdf\"/><abstract><p>A recent study reported that increasing host DNA abundance and reducing read depth impairs the sensitivity of detection of low-abundance micro-organisms by shotgun metagenomics. The authors used DNA from a synthetic bacterial community with abundances varying across several orders of magnitude and added varying proportions of host DNA. However, the use of a marker-gene-based abundance estimation tool (MetaPhlAn2) requires considerable depth to detect marker genes from low-abundance organisms. Here, we reanalyse the deposited data, and place the study in the broader context of low microbial biomass metagenomics. We opted for a fast and sensitive read binning tool (Kraken 2) with abundance estimates from Bracken. With this approach all organisms are detected even when the sample comprises 99 % host DNA and similarly accurate abundance estimates are provided (mean squared error 0.45 vs. 0.3 in the original study). We show that off-target genera, whether contaminants or misidentified reads, come to represent over 10 % of reads when the sample is 99 % host DNA and exceed counts of many target genera. Therefore, we applied Decontam, a contaminant detection tool, which was able to remove 61 % of off-target species and 79 % of off-target reads. We conclude that read binning tools can remain sensitive to low-abundance organisms even with high host DNA content, but even low levels of contamination pose a significant problem due to low microbial biomass. Analytical mitigations are available, such as Decontam, although steps to reduce contamination are critical.</p></abstract><kwd-group><kwd>metagenomics</kwd><kwd>deep sequencing</kwd><kwd>taxonomy</kwd></kwd-group><funding-group><award-group id=\"ID0EKWAE280\"><funding-source>Lee Foundation</funding-source><principal-award-recipient>Andrew James McArdle</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OpenAccessEmbargo</meta-name><meta-value>0</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"s1\"><title>Data Summary</title><p>NCBI sequence read archive accession PRJNA521492</p></sec><sec sec-type=\"intro\" id=\"s2\"><title>Introduction</title><p>The study of metagenomics and microbiomes has yielded impressive insights into the microbiology of the environment and of multicellular organisms in health and disease [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>].</p><p>Although more expensive than amplicon-based microbiome approaches (e.g. 16S rRNA gene sequencing), shotgun metagenomics is increasingly gaining prominence. Benefits include no PCR-related bias, greater specificity of identifications and representation of diversity, and ability to detect organisms from all kingdoms [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]. Additionally, metagenomic sequences can be analysed functionally, and whole or partial metagenomes can be reconstructed with greater depth of sequencing.</p><p>However, high-depth sequencing does not guarantee abundant microbial reads. Challenges most frequently arise when microbial biomass is low [<xref rid=\"R3\" ref-type=\"bibr\">3–5</xref>]. In this case, total DNA will be limited, and few reads may be obtained. Furthermore, the quantity of contaminant organisms is likely to remain constant (as the processes that cause contamination should not be not associated with the determinants of host DNA proportion), and thus their relative contribution will increase. The same problem can arise when samples are dominated by DNA from a host organism – in these cases, host sequencing reads may vastly outnumber those from microbes.</p><p>Although techniques exist to mitigate this by selectively depleting host DNA, usually by removing free DNA before lysis [<xref rid=\"R6\" ref-type=\"bibr\">6–9</xref>], they are in their infancy and could also deplete DNA from dead or damaged organisms, which would include those under immune attack [<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]. Depleting host DNA would not reduce the impact of contamination occurring prior to depletion.</p><p>In this context, we commend Pereira-Marques <italic>et al</italic>. on their insightful study into the effects of host DNA and read depth on microbial abundance estimates from shotgun metagenomics [<xref rid=\"R11\" ref-type=\"bibr\">11</xref>].</p><p>The authors evaluated the impact of a range of amounts of host DNA and sequencing depths on microbiome taxonomic profiling using shotgun metagenomic sequencing, from synthetic samples where bacterial DNA from 20 species of varying abundances was spiked with varying amounts of murine DNA. Sequencing was performed to achieve 5.5 Gb per sample.</p><p>The authors showed that increasing proportions of host DNA (10, 90 and 99 %) led to decreased sensitivity in detecting very low- and low-abundance species, increasing the number of undetected species.</p><p>Although not stated, we anticipate the authors may have selected MetaPhlAn2 for their analysis because by detecting clade-specific marker genes of known number per organism, relative abundances within a sample can be directly estimated [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]. Despite this advantage, we are concerned that relying upon a small number of marker genes will render the approach less resilient to the pitfalls of reduced depth than read binning approaches.</p><p>Consequently, we applied Kraken, a fast and sensitive read binning tool [<xref rid=\"R13\" ref-type=\"bibr\">13</xref>], which performed well in recent benchmarks [<xref rid=\"R14\" ref-type=\"bibr\">14, 15</xref>]. Advantageously, a partner tool (Bracken) also exists for relative abundance estimation [<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]. We obtained the variable-length trimmed reads from the study (NCBI sequence read archive accession PRJNA521492) and built a Kraken database comprising NCBI RefSeq bacterial, fungal, viral, archaeal and mouse genome sequences with core vector elements (downloaded on 5 July 2019 using included scripts). This resulted in 18 834 operational taxonomic units (OTUs). Kraken (version 2.0.8-beta) was then run with default settings, followed by Bracken.</p><p>For each sample we categorized reads assigned to any microbial OTU as microbial. We follow the sample naming conventions of the original analysis: MS=microbial sample; SS10=10 % host DNA; SS90=90 % host DNA; SS99=99 % host DNA.</p></sec><sec id=\"s3\"><title>Sensitivity</title><p>All expected organisms (<italic>n</italic>=20) were detected in all samples. This contrasts with the results presented by Pereira-Marquez <italic>et al</italic>. where nine of the 20 species became undetectable in SS99.</p><p>Over 75 % of microbial reads were allocated to the known species (on target), except in sample SS99 where this fell to 67 %. Other species of the expected genera represented much fewer than 1 % of microbial reads in all samples. Fewer than 2 % of microbial reads were assigned to OTUs outside of the lineage of the expected genera (off target), except for SS99 where this was 12 % (Table S1, available in the online version of this article).</p></sec><sec id=\"s4\"><title>Relative abundance</title><p>Crude assigned read counts are not a guide to relative abundance because of varying genome size, and because reads from different organisms may be assigned at the species level at differing rates due to homology. Bracken was developed to overcome the second limitation by reallocating reads assigned to higher levels. We apply Bracken here at the species level to estimate abundance and then correct for genome size. The Bracken database was built for a read length of 150 (the median length of the trimmed reads).</p><p>Bracken estimated that over 98 % of microbial reads were on-target (species) in MS and SS10. In SS90 this fell to 96.8 % and in SS99 to 83.3 %.</p><p>We normalized abundances by genome size (obtained from NCBI genomes at <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/genome\">https://www.ncbi.nlm.nih.gov/genome</ext-link>) for the target species, discounting the small proportion of off-target reads. In MS, the ratios of observed/expected relative abundance was between 0.5 and 2 for 16 of the 20 species, compared to 17 in the published study (<xref ref-type=\"fig\" rid=\"F1\">Fig. 1</xref> and Table S2). The mean squared relative error for MetaPhlAn was 0.3 and for Bracken was 0.45.</p><fig fig-type=\"figure\" id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1.</label><caption><p>Taxonomic profile of the synthetic metagenome samples determined with Kraken 2, and expressed as the relative abundance of species in a heat map. Actual abundances are presented as per the original publication based on the theoretical number of genome copies present. Species are listed from highest to lowest expected relative abundances. MS=microbial sample; SS10=10 % host DNA; SS90=90 % host DNA; SS99=99 % host DNA.</p></caption><graphic xlink:href=\"acmi-2-104-g001\"/></fig><p>Changes in relative abundance due to host DNA abundance were modest, even in SS99 where 12 of 20 organisms were within 10 % of the estimate from MS (mean squared relative error 0.02; Table S2).</p><p>We found the association of variation in observed/expected ratio with genome GC content to be similar to the original report (<italic>r</italic>=−0.74 vs. −0.85; data not shown).</p></sec><sec id=\"s5\"><title>Other species</title><p>Using Bracken recalculated reads, off-target genera (<italic>n</italic>=1 336) could be classified into synthetic-associated (MS:SS99 >10 : 1), host-associated (SS99:MS >10 : 1) or non-specific. Over 92 % of reads were from host- or synthetic-associated genera. Synthetic-associated genera contributed 0.8 % of microbial reads in MS, and host-associated genera less than 0.01 %. Host-associated genera contributed 11.5 % of microbial reads in SS99 (despite being only 0.2 % of murine reads), and synthetic-associated genera 1 %.</p><p>The top four synthetic-associated genera were <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3329\">Shigella</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3291\">Salmonella</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3130\">Citrobacter</ext-link>\n</named-content></italic> and <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3202\">Klebsiella</ext-link>\n</named-content></italic>. These are all likely to represent misclassified <italic><named-content content-type=\"species\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3093\">Escherichia coli</ext-link>\n</named-content></italic> reads. The top four host-associated genera were <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3371\">Pasteurella</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.2493\">Halomonas</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.2481\">Alcanivorax</ext-link>\n</named-content></italic> and <italic>Mycobacteria. Alcanivorax</italic> and <italic><named-content content-type=\"family\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3370\">Pasteurellaceae</ext-link>\n</named-content></italic> have previously been reported to contaminate DNA extraction kits [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]. We note that host DNA was extracted in the laboratory whereas the microbial DNA was obtained commercially, and thus different contaminants are unsurprising.</p><p>The target genera with lowest read counts in SS99 were <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.32706\">Schaalia</ext-link>\n</named-content></italic> and <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.507\">Deinococcus</ext-link>\n</named-content></italic> (36 and 37 reads respectively). Fifty-four off-target genera had 36 or more reads. The most abundant off-target genus (<italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.3371\">Pasteurella</ext-link>\n</named-content></italic>) contributed 11 530 reads, greater than 13 of 17 target genera.</p></sec><sec id=\"s6\"><title>Low microbial biomass</title><p>The greater sensitivity of this read binning approach reveals the underlying problem of high relative contamination in the samples with high host DNA content. The problem can now be reframed as one of low (proportionate) microbial biomass and potential mitigations can be considered.</p><p>The challenge of low microbial biomass samples, introduced earlier, has been more extensively studied in rRNA amplification-based approaches than shotgun metagenomics. Nonetheless, many of the problems are shared, and we direct readers to a recent review by Eisenhofer <italic>et al</italic>. [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]. Pre-analytical mitigations include appropriate controls, as described therein.</p><p>Analytical mitigations for 16S rRNA gene studies were explored in a recent publication [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]. The authors investigated filtering based on relative abundance thresholds in negative controls: Decontam [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>], an approach based on the inverse relationship between the relative abundance of contaminants and total microbial DNA; and SourceTracker [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>], which takes a Bayesian approach using external or internal community references.</p><p>In summary, it was found that simple censoring of thresholded negative control OTUs discriminated contaminant and target sequence variants poorly. The Decontam approach discriminated better, correctly classifying all target sequence clusters, and up to 90.4 % of contaminant sequence clusters. SourceTracker performed poorly without external references (a typical scenario), identifying less than 1 % of contaminant sequence clusters.</p><p>Although limited by few samples and no duplicates, we applied Decontam to the Bracken-normalized species counts, using the frequency-based approach. Input DNA concentration was replaced by the total microbial read counts (because all samples had been normalized to 0.2 ng ml<sup>−1</sup>). None of the 20 target species were classified as contaminants. In total, 2636 of 4319 (61 %) off-target species were classified as contaminants, and these accounted for 92 % of off-target reads in SS99 and 68 % in SS90. Only 11 % of off-target reads in SS10 and 5 % in MS were classified as contaminants, unsurprisingly, as these reads are dominated by synthetic-associated genera.</p><p>In SS99, the least abundant genera, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.32706\">Schaalia</ext-link>\n</named-content></italic> and <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.507\">Deinococcus</ext-link>\n</named-content></italic>, retained 35 and 34 reads, respectively. Only seven off-target genera had 34 or more reads, comprising the four synthetic-associated genera above, with <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.13074\">Cronobacter</ext-link>\n</named-content></italic>, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.22608\">Nitrosopumilus</ext-link>\n</named-content></italic> and <italic>Enterobacter. Shigella</italic> had the most reads at 1303, exceeding 10 of 17 target genera.</p></sec><sec id=\"s7\"><title>Interpretation</title><p>The marker gene approach employed by MetaPhlAn is very sensitive to read depth, and hence to host DNA abundance. In contrast, the read binning approach employed by Kraken 2 detects organisms across the >2 000-fold range of relative abundances even with 99 % host DNA content.</p><p>Genome-size normalization of Bracken-estimated read counts provides similarly accurate estimates of relative abundance to MetaPhlAn. The untrimmed reads (not available) may give better results as they would all be of the same length, which is expected by Bracken.</p><p>We demonstrate that the large relative contribution of contaminants when microbial reads are in a minority is a greater concern, representing around 10 % of microbial reads in SS99 with contaminant genera exceeding the counts of some target genera.</p><p>However, the frequency-based Decontam approach allows nearly four-fifths of these off-target reads to be excluded. Furthermore, many of those that remain may represent misclassified target reads.</p><p>It is important to note that the literature does not demonstrate supremacy of read binning approaches in all regards. Walsh <italic>et al</italic>. [<xref rid=\"R20\" ref-type=\"bibr\">20</xref>] showed in a low-complexity food microbiome that Metaphlan 2 was sensitive and also more specific than Kraken.</p></sec><sec id=\"s8\"><title>Concluding remarks</title><p>The appropriate selection of analytical tools is vital for accurate and sensitive metagenome analysis. For samples with low microbial biomass, reducing contamination is a priority, although mitigation is possible. Techniques to selectively remove host DNA are required, but thorough benchmarking is awaited.</p></sec><sec id=\"s10\"><title>Data bibliography</title><p>Code and output from Kraken and Bracken are available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/andrewjmc/pereira_marques_reanalysis\">https://github.com/andrewjmc/pereira_marques_reanalysis</ext-link>\n</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Data</title><supplementary-material content-type=\"local-data\" id=\"supp\"><caption><title>Supplementary material 1</title></caption><media xlink:href=\"acmi-2-104-s001.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"ack1\"><title>Funding information</title><p>A.J.M. is funded by the Wellcome Trust/Imperial College London 4i PhD Programme. M.K. is funded by the Wellcome Trust (Sir Henry Wellcome Fellowship grant 206508/Z/17/Z) and supported by the Imperial College BRC.</p></ack><ack id=\"ack2\"><title>Author contributions</title><p>A.J.M. conceptualized the study, performed analyses and prepared the original draft. M.K. provided supervision. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Access Microbiol</journal-id><journal-id journal-id-type=\"hwp\">acmi</journal-id><journal-id journal-id-type=\"publisher-id\">acmi</journal-id><journal-title-group><journal-title>Access Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">2516-8290</issn><publisher><publisher-name>Microbiology Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005872</article-id><article-id pub-id-type=\"pmc\">PMC7523628</article-id><article-id pub-id-type=\"publisher-id\">000109</article-id><article-id pub-id-type=\"doi\">10.1099/acmi.0.000109</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Case Report</subject></subj-group></article-categories><title-group><article-title>A case of canine cutaneous pythiosis in Thailand</article-title><alt-title alt-title-type=\"recto-page-foot\"><ext-link ext-link-type=\"uri\" xlink:href=\"http://acmi.microbiologyresearch.org\">http://acmi.microbiologyresearch.org</ext-link></alt-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Chindamporn</surname><given-names>Ariya</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kammarnjessadakul</surname><given-names>Patcharee</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kesdangsakonwut</surname><given-names>Sawang</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Banlunara</surname><given-names>Wijit</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>3</sup>\n</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><aff id=\"aff1\">\n<label><sup>1</sup>​</label>\n<institution content-type=\"department\">Department of Microbiology</institution>, <institution>Faculty of Medicine, Chulalongkorn University</institution>, <addr-line content-type=\"city\">Bangkok 10330</addr-line>, <country>Thailand</country>\n</aff><aff id=\"aff2\">\n<label><sup>2</sup>​</label>\n<institution content-type=\"department\">Department of Microbiology, Faculty of Medical Technology</institution>, <institution>Huachiew Chalermprakiet University</institution>, <addr-line content-type=\"city\">Samut Prakan, 10540</addr-line>, <country>Thailand</country>\n</aff><aff id=\"aff3\">\n<label><sup>3</sup>​</label>\n<institution content-type=\"department\">Department of Pathology</institution>, <institution>Faculty of Veterinary Science, Chulalongkorn University</institution>, <addr-line content-type=\"city\">Bangkok 10330</addr-line>, <country>Thailand</country>\n</aff></contrib-group><author-notes><corresp id=\"cor1\"><bold>*Correspondence:</bold> Wijit Banlunara, <email xlink:href=\"mailto:wijit.k@chula.ac.th\">wijit.k@chula.ac.th</email></corresp></author-notes><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>14</day><month>2</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>14</day><month>2</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>2</volume><issue>4</issue><elocation-id>acmi000109</elocation-id><history><date date-type=\"received\"><day>23</day><month>10</month><year>2019</year></date><date date-type=\"accepted\"><day>24</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 The Authors</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"acmi-2-109.pdf\"/><abstract><sec><title>Introduction</title><p>\n<italic>Pythium insidiosum</italic> causes pythiosis in humans and animals in tropical and subtropical climates. The clinical manifestations in humans are mostly systemic, vascular or ocular forms, in contrast to animals, which are cutaneous, subcutaneous and gastrointestinal forms. The highest incidence of human cases is reported in Thailand, however, no canine pythiosis has been documented yet.</p></sec><sec><title>Case presentation</title><p> A female, mixed-breed, stray dog showed severe extensive ulcerative haemorrhagic dermatitis at the perineum involving the anus and tail. On cytology, there were sparse branching septate fungal hyphae. The tissue samples were subjected to polymerase chain reaction and gene sequencing for fungal identification.</p></sec><sec><title>Conclusion</title><p> The results of the internal transcribed spacer 1 and 2 (ITS1 and ITS2) gene had 99 % homology to <italic>Pythium insidiosum</italic> (accession no. FJ17396) and the <italic>COX2</italic> gene (accession no. GQ451572). The phylogenetic tree of both genes was classified in clade A<sub>TH.</sub> This is the first fully documented diagnosis of canine cutaneous pythiosis in Thailand.</p></sec></abstract><kwd-group><kwd>dog</kwd><kwd>skin</kwd><kwd>phylogenetic analysis</kwd><kwd><italic>Pythium insidiosum</italic></kwd><kwd>Thailand</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>OpenAccessEmbargo</meta-name><meta-value>0</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>\n<italic>Pythium insidiosum</italic>, an oomycetes organism, is the only species in the genus <italic>Pythium</italic> causing destructive malady in humans and animals in tropical and subtropical areas. The clinical manifestations of humans are systemic, vascular and ocular forms. In contrast, the clinical presentations of animal pythiosis are cutaneous, subcutaneous and gastrointestinal forms. Based on accumulated information to date, there have been reports in some livestocks, companion and wild animals. Those infected animal species were bovine, canine, feline, equine and avian (Californian nestling white-faced ibis; <italic>Plegadis chihi</italic>) [<xref rid=\"R1\" ref-type=\"bibr\">1–7</xref>]. Also, spectacle bears from zoos were infected [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]. These cases were documented from all continents, mainly, in tropical and subtropical regions such as America: Texas, Costa Rica, Brazil, Argentina; Asia: India, Indonesia, Japan; Australia and the last continent in 2005, was in Africa [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]. Canine cutaneous and subcutaneous forms had been widely reported in the USA and Brazil [<xref rid=\"R2\" ref-type=\"bibr\">2–6, 9–11</xref>]. However, an exceptional pulmonary case in a Staffordshire terrier was documented [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]. Even though the first human case in Thailand was reported in 1985 and the highest incidence of human cases is in Thailand, no canine case has been documented yet [<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]. Most of these human cases were diagnosed by direct examination or histopathology, showing non-septate hyphae, and the diagnosis was confirmed by rapid growth on the medium and/or molecular techniques. All the <italic>P. insidiosum</italic> isolates documented in Thailand demonstrated that their phylogenetic trees were classified in clade B<sub>TH</sub> (clade II) and clade C<sub>TH </sub>(clade III) based on the universal primers, named internal transcribed spacer regions (ITS) 2 - ITS 4, and the primers in mitochondrial inner membrane, named the cytochrome oxidase 2 (<italic>COX 2</italic>) gene, which generates the sister clade, providing a better classification over the ITS primers [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]. This is the first canine cutaneous pythiosis pathology report with supporting molecular findings.</p></sec><sec sec-type=\"cases\" id=\"s2\"><title>Case report</title><p>A female, mixed-breed, stray dog, approximately 2–5 years old, was brought to a private small animal hospital in the western vicinity of Bangkok with serious skin lesions, presenting at the perineum, involving the anus and tail. The gross skin lesions showed severe extensive ulcerative haemorrhagic dermatitis (<xref ref-type=\"fig\" rid=\"F1\">Fig. 1a–b</xref>). The cytology samples were prepared from the lesions during the first sampling under the permission of the owner and following the guidelines for the use of animal tissue for the scientific purpose of Chulalongkorn University animal care and use committee. The method used was direct examination using potassium hydroxide (KOH). The rest of the samples were further submitted for fungal culture, and fungal identification using polymerase chain reaction and phylogenetic analysis according to the previously mentioned method [<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]. Unfortunately, the animal died after the first visit and necropsy was not allowed. The cytology samples were stained with Giemsa stain. There were eosinophilic contorted septate hyphae, measuring 3–5 μm in diameter (<xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>). Similar fungal hyphae were also observed in the submerged creamy rapid growth, 24 h, colony on Sabouraud dextrose agar. In this step, zoospore production was induced and two-flagellate zoospores were demonstrated (<xref ref-type=\"fig\" rid=\"F3\">Fig. 3</xref>). However, the production of zoospores is not the definite identification for <italic>P. insidiosum,</italic> so both the isolate and tissue samples were submitted to amplify the regions of ITS1 and ITS 2 by using polymerase chain reaction. The DNA-sequences of amplicons from both samples showed identical 891 bp. After alignment, these sequences had 99 % homology to <italic>P. insidiosum</italic> (accession no. GQ475490, MTPI04) and its accession number is FJ17396, strain PAC2. The sequences of the isolate from the 550 bp. <italic>COX2</italic> amplicon was identified as <italic>P. insidiosum</italic> (accession no. GQ451572, strain PAC2). Each amplicon was analysed and used to construct the phylogenetic tree (<xref ref-type=\"fig\" rid=\"F4\">Fig. 4a, b</xref>). Both sequences belonged to clade A<sub>TH</sub>.</p><fig fig-type=\"figure\" id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1.</label><caption><p>(a) Skin lesions present at the perineum and tail of a dog (arrows). (b) Gross diagnosis is severe extensive ulcerative haemorrhagic dermatitis (left recumbency, posterior view of lesions).</p></caption><graphic xlink:href=\"acmi-2-109-g001\"/></fig><fig fig-type=\"figure\" id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2.</label><caption><p>Cytologic finding of the lesion shows contorted branching septate fungal hypha. (Giemsa stain).</p></caption><graphic xlink:href=\"acmi-2-109-g002\"/></fig><fig fig-type=\"figure\" id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Fig. 3.</label><caption><p>Zoospores of <italic>P. insidiosum</italic> and zoospore germination shows short hypha (arrow) (bar=20 µm).</p></caption><graphic xlink:href=\"acmi-2-109-g003\"/></fig><fig fig-type=\"figure\" id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Fig. 4.</label><caption><p>The phylogenetic tree was generated using neighbour-joining (NJ) analysis. Based on 10 000 replicates of bootstrap. (a) COX2 and (b) ITS region (*).</p></caption><graphic xlink:href=\"acmi-2-109-g004\"/></fig></sec><sec sec-type=\"discussion\" id=\"s3\"><title>Discussion</title><p>It is interesting that both sequences belonged to clade A<sub>TH</sub>. This study is the first documented canine cutaneous pythiosis case, which was infected by clade A<sub>TH</sub>. From our knowledge up to the present, the members in clade A<sub>TH</sub> are isolates from the USA, but not from Asia [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]. It is in contrast to the clades from the environment and all clinical isolates in Thailand, which belong to clade B<sub>TH </sub>and C<sub>TH</sub> [<xref rid=\"R13\" ref-type=\"bibr\">13, 14</xref>]. This report will draw the attention that not only human cases, but also animal cases, which pythiosis should be included as endemic in Thailand. This is a new knowledge of clade A<sub>TH </sub>\n<italic>P. insidiosum</italic> occurrence in Thailand. However, the limitation of this case was the exact history.</p></sec></body><back><ack id=\"ack1\"><title>Funding information</title><p>This work received no specific grant from any funding agency.</p></ack><ack id=\"ack2\"><title>Acknowledgements</title><p>The authors are thankful to Dr Navaporn Worasilchai for taking the zoospores’ picture.</p></ack><ack id=\"ack3\"><title>Conflicts of interest</title><p content-type=\"COI-statement\">The authors declare that there are no conflicts of interest.</p></ack><ack id=\"ack4\"><title>Ethical statement</title><p>The cytologic sampling and photographs were performed under the permission of the dog’s owner and the study has already been approved by the guidelines for the use of animal tissue for the scientific purpose of Chulalongkorn University animal care and use committee.</p></ack><ref-list><title>References</title><ref id=\"R1\"><label>1</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaffin</surname><given-names>MK</given-names></name><name><surname>James</surname><given-names>S</given-names></name><name><surname>McMullan</surname><given-names>WC</given-names></name></person-group><article-title>Cutaneous pythiosis in the horse</article-title><source>Vet Clin North Am <italic toggle=\"yes\">Equine Pract</italic></source><year>1995</year><volume>11</volume><fpage>91</fpage><lpage>103</lpage><pub-id pub-id-type=\"doi\">10.1016/S0749-0739(17)30334-6</pub-id><pub-id pub-id-type=\"pmid\">7634168</pub-id></element-citation></ref><ref id=\"R2\"><label>2</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gaastra</surname><given-names>W</given-names></name><name><surname>Lipman</surname><given-names>LJA</given-names></name><name><surname>De Cock</surname><given-names>AWAM</given-names></name><name><surname>Exel</surname><given-names>TK</given-names></name><name><surname>Pegge</surname><given-names>RBG</given-names></name><etal>et al</etal></person-group><article-title>\n<italic>Pythium insidiosum</italic>: an overview</article-title><source>Vet Microbiol</source><year>2010</year><volume>146</volume><fpage>1</fpage><lpage>16</lpage><pub-id pub-id-type=\"doi\">10.1016/j.vetmic.2010.07.019</pub-id><pub-id pub-id-type=\"pmid\">20800978</pub-id></element-citation></ref><ref id=\"R3\"><label>3</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maia</surname><given-names>LA</given-names></name><name><surname>Olinda</surname><given-names>RG</given-names></name><name><surname>Araújo</surname><given-names>TF</given-names></name><name><surname>Firmino</surname><given-names>PR</given-names></name><name><surname>Nakazato</surname><given-names>L</given-names></name><etal>et al</etal></person-group><article-title>Cutaneous pythiosis in a donkey (<italic>Equus asinus</italic>) in Brazil</article-title><source>J VET Diagn Invest</source><year>2016</year><volume>28</volume><fpage>436</fpage><lpage>439</lpage><pub-id pub-id-type=\"doi\">10.1177/1040638716651467</pub-id><pub-id pub-id-type=\"pmid\">27271986</pub-id></element-citation></ref><ref id=\"R4\"><label>4</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mercer</surname><given-names>J</given-names></name><name><surname>White</surname><given-names>A</given-names></name><name><surname>Kennis</surname><given-names>B</given-names></name></person-group><article-title>Successful management of cutaneous pythiosis in a dog with subsequent cutaneous vasculitis</article-title><source>Vet Rec Case Rep</source><year>2014</year><volume>2</volume><elocation-id>e000143</elocation-id><pub-id pub-id-type=\"doi\">10.1136/vetreccr-2014-000143</pub-id></element-citation></ref><ref id=\"R5\"><label>5</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Neto</surname><given-names>RT</given-names></name><name><surname>de M.G. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Access Microbiol</journal-id><journal-id journal-id-type=\"hwp\">acmi</journal-id><journal-id journal-id-type=\"publisher-id\">acmi</journal-id><journal-title-group><journal-title>Access Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">2516-8290</issn><publisher><publisher-name>Microbiology Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005871</article-id><article-id pub-id-type=\"pmc\">PMC7523629</article-id><article-id pub-id-type=\"publisher-id\">000107</article-id><article-id pub-id-type=\"doi\">10.1099/acmi.0.000107</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Toward a genetic system in the marine cyanobacterium <italic>Prochlorococcus</italic>\n</article-title><alt-title alt-title-type=\"recto-page-foot\"><ext-link ext-link-type=\"uri\" xlink:href=\"http://acmi.microbiologyresearch.org\">http://acmi.microbiologyresearch.org</ext-link></alt-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-1326-9928</contrib-id><name><surname>Laurenceau</surname><given-names>Raphaël</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-7768-1313</contrib-id><name><surname>Bliem</surname><given-names>Christina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-1072-6828</contrib-id><name><surname>Osburne</surname><given-names>Marcia S.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff3\">\n<sup>†</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0001-5308-1818</contrib-id><name><surname>Becker</surname><given-names>Jamie W.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff4\">\n<sup>‡</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-2638-823X</contrib-id><name><surname>Biller</surname><given-names>Steven J.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff5\">\n<sup>§</sup>\n</xref></contrib><contrib contrib-type=\"author\"><name><surname>Cubillos-Ruiz</surname><given-names>Andres</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff6\">\n<sup>#</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff7\">\n<sup>¶</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff8\">\n<sup>**</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-1480-2445</contrib-id><name><surname>Chisholm</surname><given-names>Sallie W.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref><xref ref-type=\"corresp\" rid=\"cor2\">*</xref></contrib><aff id=\"aff1\">\n<label><sup>1</sup>​</label>\n<institution content-type=\"department\">Department of Civil and Environmental Engineering</institution>, <institution>Massachusetts Institute of Technology</institution>, <addr-line content-type=\"city\">Cambridge, MA</addr-line>, <country>USA</country>\n</aff><aff id=\"aff2\">\n<label><sup>2</sup>​</label>\n<institution content-type=\"department\">Department of Biology</institution>, <institution>Massachusetts Institute of Technology</institution>, <addr-line content-type=\"city\">Cambridge, MA</addr-line>, <country>USA</country>\n</aff><aff id=\"aff3\">\n<label><sup>†</sup>​</label>Present address: Department of Molecular Biology and Microbiology Tufts University School of Medicine, Boston, MA, USA</aff><aff id=\"aff4\">\n<label><sup>‡</sup>​</label>Present address: Department of Biology, Haverford College, Haverford, PA, USA</aff><aff id=\"aff5\">\n<label><sup>§</sup>​</label>Present address: Department of Biological Sciences, Wellesley College, Wellesley, MA, USA</aff><aff id=\"aff6\">\n<label><sup>#</sup>​</label>Present address: Institute for Medical Engineering and Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA</aff><aff id=\"aff7\">\n<label><sup>¶</sup>​</label>Present address: Broad Institute of MIT and Harvard, Cambridge, MA, USA</aff><aff id=\"aff8\">\n<label><sup>**</sup>​</label>Present address: Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA</aff></contrib-group><author-notes><corresp id=\"cor1\"><bold>*Correspondence:</bold> Raphaël Laurenceau, <email xlink:href=\"mailto:raphaellaurenceau@gmail.com\">raphaellaurenceau@gmail.com</email></corresp><corresp id=\"cor2\"><bold>*Correspondence:</bold> Sallie W. Chisholm, <email xlink:href=\"mailto:chisholm@mit.edu\">chisholm@mit.edu</email></corresp></author-notes><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>19</day><month>2</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>19</day><month>2</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>2</volume><issue>4</issue><elocation-id>acmi000107</elocation-id><history><date date-type=\"received\"><day>11</day><month>11</month><year>2019</year></date><date date-type=\"accepted\"><day>30</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 The Authors</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"acmi-2-107.pdf\"/><abstract><p>As the smallest and most abundant primary producer in the oceans, the cyanobacterium <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically. Multiple attempts have been made to develop an efficient genetic transformation method for <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> over the years; all have been unsuccessful to date, despite some success with their close relative, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic>. To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.</p></abstract><kwd-group><kwd>genetic system</kwd><kwd>intractable bacteria</kwd><kwd><italic>Prochlorococcus</italic></kwd></kwd-group><funding-group><award-group id=\"ID0EYWAE284\"><funding-source>Simons Foundation</funding-source><award-id>337262, 509034SCFY17, 329108</award-id><principal-award-recipient>Sallie W. Chisholm</principal-award-recipient></award-group><award-group id=\"ID0EFVAE283\"><funding-source>National Science Foundation</funding-source><award-id>1645061</award-id><principal-award-recipient>Sallie W. Chisholm</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OpenAccessEmbargo</meta-name><meta-value>0</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>While environmental microbiology has been revolutionized by the rapid pace of improved sequencing technologies [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>], the number of genetically tractable model organisms has lagged behind [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]. The dearth of such organisms has limited our progress since most ‘omics’ analyses rely on comparisons with model organisms for their interpretation [<xref rid=\"R1\" ref-type=\"bibr\">1, 3–5</xref>]. Isolation of pure (axenic) cultures from the wild has proved to be a significant challenge and developing genetic tools for these isolates has been even more difficult.</p><p>Countless attempts have demonstrated that the creation of successful genetic transformation protocols requires tedious trial and error, and methods developed for one strain are often unsuccessful in close relatives (for a few examples see: [<xref rid=\"R6\" ref-type=\"bibr\">6–13</xref>]). There is no single solution for all bacteria, and no way to predict which techniques will succeed [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]. Thus, there is a pressing need for broad-spectrum, automatized, and standardized genetic transformation procedures [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>] that could enable downstream genetic screening methods such as transposon insertion sequencing [<xref rid=\"R5\" ref-type=\"bibr\">5, 16–18</xref>]. Such projects are not suitable for graduate students or post-docs because of the high risk involved, nor are they easily funded.</p><p>\n<italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> is the most abundant cyanobacterium worldwide, dominating vast regions of the global oceans [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]. It may account for up to 50 % of the chlorophyll in oligotrophic ocean regions [<xref rid=\"R20\" ref-type=\"bibr\">20, 21</xref>] and is responsible for around 8.5 % of global ocean primary productivity [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]. Our understanding of <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> biology and ecology has been greatly facilitated by the rise of metagenomic, metatranscriptomic, and metaproteomic studies of ocean samples over the past decade [<xref rid=\"R22\" ref-type=\"bibr\">22–26</xref>]. Because of its high relative abundance, it often dominates databases derived from surface microbial communities in the oceans [<xref rid=\"R27\" ref-type=\"bibr\">27</xref>], and has emerged as one of the best-described marine microorganisms with more than one thousand complete or nearly complete genomes available [<xref rid=\"R28\" ref-type=\"bibr\">28–30</xref>]. <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> is a perfect example of the imbalance between the availability of genomic data and the dearth of genetic tools.</p><p>\n<italic>Prochlorococcus’</italic> numerical dominance in oligotrophic oceans is attributed to its small size (hence high surface/volume ratio), which enhances its ability to compete for limiting nutrients [<xref rid=\"R31\" ref-type=\"bibr\">31</xref>], and its vast genomic diversity [<xref rid=\"R32\" ref-type=\"bibr\">32–37</xref>], which expands the niche dimensions of the <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> meta-population [<xref rid=\"R28\" ref-type=\"bibr\">28, 38, 39</xref>]. Ecologically meaningful units within the meta-population can be found at all levels of phylogeny, both deeply rooted – such as their adaptation to different light levels – and in the ‘leaves of the tree’ where differences in the presence/absence of specific nutrient acquisition genes can be found [<xref rid=\"R28\" ref-type=\"bibr\">28, 37, 38, 40</xref>]. Although we have been able to unravel the selective pressures that shape the distributions of genes of obvious ecological relevance, many intriguing stories are undoubtedly obscured by our inability to assign functions to the numerous unannotated genes in these cells; for each new <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strain isolated, or wild single-cell sequenced, roughly 100 new genes are added to the <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> pangenome – the vast majority of which are of unknown function [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>].</p><p>In addition to its ecological and biogeochemical relevance, the biology of <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> is of interest because of its potential as a chassis for synthetic biology applications [<xref rid=\"R41\" ref-type=\"bibr\">41</xref>]. This cell is the simplest photosynthetic ‘machine’ designed by nature – an attractive foundation for manipulation [<xref rid=\"R41\" ref-type=\"bibr\">41–44</xref>]. It has the smallest genome of any oxygenic phototroph as well as an efficient carbon concentrating mechanism [<xref rid=\"R45\" ref-type=\"bibr\">45</xref>]. Indeed, a recent study screening the metabolic potential of cyanobacteria for biofuel production ranked various <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strains as the top candidates [<xref rid=\"R46\" ref-type=\"bibr\">46</xref>]. Furthermore, their diversity makes them ideal subjects for studying the properties of small-genome organism consortia for bioproduction [<xref rid=\"R43\" ref-type=\"bibr\">43</xref>].</p><p>Progress in studying <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> from these different perspectives has been hampered by our inability to develop a robust genetic system. Although we and others have worked on this over many years, very limited progress has been made. Possible reasons behind these failures include the cell’s slow growth rate (roughly one doubling per day under optimal laboratory conditions), their reluctance to grow axenically on solid media, specific media requirements [<xref rid=\"R47\" ref-type=\"bibr\">47, 48</xref>], sensitivity to trace metal contamination [<xref rid=\"R49\" ref-type=\"bibr\">49, 50</xref>] and reactive oxygen species [<xref rid=\"R51\" ref-type=\"bibr\">51</xref>], unusual membrane composition [<xref rid=\"R52\" ref-type=\"bibr\">52</xref>], and their apparent reduced homologous recombination potential [<xref rid=\"R53\" ref-type=\"bibr\">53</xref>].</p><p>From our perspective, a significant impediment to rapid progress is that investigators generally do not publish negative results or partial progress; as a result, considerable time is wasted by others in rediscovering what does not work. Thus, before describing our progress, we first report experiments that failed. We initially tried to build upon the only reported successful (albeit inefficient) <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> genetic transformation method, based on conjugation from an <italic>E. coli</italic> donor strain [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>]. This method proved unsuccessful in our hands, probably because the six month multi-step procedure is vulnerable to user variability. From there we moved on to explore the use of electroporation and were able to find appropriate conditions for delivering DNA into living <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cells – developing a method that should be applicable to a wide range of microorganisms. The next step will be to develop genetic tools compatible with the host that will also bypass the cell’s defences against exogenous DNA, which are abundant in cyanobacteria [<xref rid=\"R55\" ref-type=\"bibr\">55</xref>]. We report the results of some initial attempts, in which transposomes were delivered into <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cells.</p></sec><sec sec-type=\"results|discussion\" id=\"s2\"><title>Results and discussion</title><sec id=\"s2-1\"><title>Selecting an antibiotic for <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic>\n</title><p>Finding a suitable selectable marker is a prerequisite for the selection of transformants. We subjected different <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strains to various common antibiotics in order to find their minimum inhibitory concentration (MIC), revealing that some antibiotics were more effective than others (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). While <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strain MED4 was resistant to nalidixic acid, it was highly sensitive to ciprofloxacin and chloramphenicol (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Kanamycin, which has been used as a selective marker for various marine <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> strains [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>] and a LLIV clade <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strain [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>] had a MIC of 50 µg ml<sup>−1</sup>, and failed to suppress the emergence of spontaneous kanamycin-resistant colonies at a high frequency in our hands, regardless of the <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strain. We ultimately focused on streptomycin, which can be used in combination with spectinomycin to reduce the appearance of spontaneous resistance mutants [<xref rid=\"R56\" ref-type=\"bibr\">56, 57</xref>].</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1.</label><caption><p>Minimum inhibitory concentration (MIC) of various antibiotics on different <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strains</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" style=\"background:#F2F2F2\" rowspan=\"1\" colspan=\"1\">\n<p>Antibiotic</p>\n</th><th align=\"left\" valign=\"top\" style=\"background:#F2F2F2\" rowspan=\"1\" colspan=\"1\">\n<p>Cellular target</p>\n</th><th align=\"left\" valign=\"top\" style=\"background:#F2F2F2\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strain</p>\n</th><th align=\"left\" valign=\"top\" style=\"background:#F2F2F2\" rowspan=\"1\" colspan=\"1\">\n<p>Minimum inhibitory concentration (MIC), µg ml<sup>−1</sup>\n</p>\n</th><th align=\"left\" valign=\"top\" style=\"background:#F2F2F2\" rowspan=\"1\" colspan=\"1\">\n<p>Appearance of resistant mutants above MIC</p>\n</th><th align=\"left\" valign=\"top\" style=\"background:#F2F2F2\" rowspan=\"1\" colspan=\"1\">\n<p>Reference</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Nalidixic Acid</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>DNA gyrase,</p>\n<p>A subunit</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>MED4</p>\n<p>MIT9313</p>\n<p>MIT9215</p>\n<p>NATL2A</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Resistant (MIC >100)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Yes</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This study</p>\n<p>[<xref rid=\"R95\" ref-type=\"bibr\">95</xref>]</p>\n<p>[<xref rid=\"R95\" ref-type=\"bibr\">95</xref>]</p>\n<p>[<xref rid=\"R95\" ref-type=\"bibr\">95</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Ciprofloxacin</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>DNA gyrase,</p>\n<p>A subunit and Topoisomerase IV</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>MED4</p>\n<p>MIT0604</p>\n<p>MIT9312</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.5</p>\n<p>0.5</p>\n<p>0.5</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Yes</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This study</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Chloramphenicol</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Protein synthesis (50S ribosome)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>MED4</p>\n<p>MIT0604</p>\n<p>MIT9312</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0.5</p>\n<p>0.5</p>\n<p>0.1–0.5</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>No</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This study</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Kanamycin</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Protein synthesis (30S ribosome)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>MED4</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>25–50</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Yes</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This study</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Streptomycin</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Protein synthesis (30S ribosome)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>MED4</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5–10</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Yes</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This study</p>\n</td></tr></tbody></table></table-wrap></sec><sec id=\"s2-2\"><title>Attempts at <italic>E. coli</italic> mediated conjugation</title><p>\n<italic>E. coli</italic> mediated conjugation has been successfully used to transform several marine <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> strains (which are closely related to <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic>) [<xref rid=\"R6\" ref-type=\"bibr\">6, 58</xref>] and has been reported to work for one LLIV clade <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strain [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>]. We first attempted <italic>E. coli</italic> conjugation with <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> via a filter mating procedure which did not yield any conjugants in our hands, even after incubating for two months. We next used a liquid mating procedure, testing different <italic>E. coli</italic> donor strains carrying plasmids with distinct antibiotic resistance genes on various <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> and <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> recipient strains (see Methods for details, and strains and plasmids in Table 2), but again obtained no conjugants.</p><p>One of the limitations of our mating experiments was the low survival frequency of <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> during filter mating. Indeed, while a <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> strain could readily recover after the mating procedure, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strains were often lost at this step. The switch to a liquid mating procedure (see Methods) improved survivability but still did not yield transconjugants. In an effort to find an alternative route to conjugation with <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic>, we developed a mixed approach where mating was performed inside an agar stab (see Methods for details). Briefly, concentrated cultures of the donor and receiver strains were mixed and injected together inside 1 ml of polymerized agar - the ‘agar stab’ (Fig. S1, available in the online version of this article). We reasoned that this agar stab environment would provide a solid medium favoring conjugation while mitigating the drawback of desiccation on top of a filter. After 24 h incubation, the cells were removed from the agar with a pipet tip, resuspended in liquid medium, and plated on antibiotic selection plates.</p><p>We attempted this procedure with a conjugative plasmid - pBAMD1-4 [<xref rid=\"R59\" ref-type=\"bibr\">59</xref>] - containing a resistance gene to spectinomycin/streptomycin. Plasmid pBAMD1-4 is a suicide vector containing the mini-Tn5 transposition system. This transposition system, encoded on vector pRL27, was previously used successfully in <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> strain WH8102 [<xref rid=\"R60\" ref-type=\"bibr\">60</xref>] and by Tolonen <italic>et al</italic>. in <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strain MIT9313 [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>]. Though this protocol could not be reproduced in our hands, it shows that Tn5 transposition can function in <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic>. In addition, we cloned the <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> promoter for Rubisco protein, P <italic>ccmk</italic>, in front of a fluorescent reporter <italic>gfp</italic> or <italic>yfp</italic> gene to facilitate the characterization of conjugants (see Methods for details). Using this ‘agar stab’ procedure, a <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> strain and two <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strains survived and were able to grow on plates in the absence of antibiotics. After 30 days, we obtained transconjugant colonies of <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> WH8102, but did not obtain transconjugants for either <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strain (SB and NATL2A). We thus abandoned conjugation as a feasible mechanism of genetic transfer and moved on to electroporation. This required the development of a method to assess DNA delivery into live cells.</p></sec><sec id=\"s2-3\"><title>Development of a dead cell stain for <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic>\n</title><p>In the absence of an established method to transform <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic>, we decided to focus on optimizing the initial step required for any genetic system: DNA delivery. A screening procedure for DNA entry must be able to differentiate between live and dead cells since the delivery of DNA inside dead cells would yield false positives. We needed a dead cell stain that emitted in the blue/violet wavelength range (between 400–500 nm wavelength) so that it could be differentiated from chlorophyll autofluorescence (650–700 nm), and fluorescein-labelled oligonucleotide fluorescence (495–555 nm). While Sytox green (ThermoFisher Scientific) has been used successfully with <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> [<xref rid=\"R51\" ref-type=\"bibr\">51</xref>], we found that under certain conditions, such as cells recovering from stress events, it does not effectively distinguish live from dead cells [<xref rid=\"R61\" ref-type=\"bibr\">61</xref>]. We next tried Sytox blue (ThermoFisher Scientific), which emits around 480 nm, but the signal was below the detection limit of the Guava easyCyte HT flow cytometer. We then turned to an amine reactive stain – the Live/Dead fixable Violet stain (ThermoFisher Scientific), which was developed primarily for eukaryotic cells and emits fluorescence at 452 nm. This stain reacts with amine residues from proteins; it labels the exposed amine groups at the surface of both live and dead cells, but only dead cells that allow the dye to penetrate their membrane reveal labelling in the cytoplasm, greatly increasing the overall signal. This stain identified dead <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cells in a robust and highly reproducible way (<xref ref-type=\"fig\" rid=\"F1\">Fig. 1</xref>), representing a new addition to the <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> tool kit.</p><fig fig-type=\"figure\" id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1.</label><caption><p>Efficacy of Live/Dead stain in differentiating live and dead <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cells. (a) Growth of the culture as measured by bulk relative red fluorescence as a function of time. Blue arrows indicate when samples were taken for treatment and flow cytometric analysis. The third growth curve indicates when, in the growth curve, a sub-sample was taken for the heat-shock measurement. RBCF: Relative Bulk Chlorophyll Fluorescence. (b) The gates in the upper flow cytometry panels delineate the <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cell population, while the rectangular gates in the lower plots indicate the increase in the number of dead cells as the culture progresses from exponential to stationary phase culture, and also after heat shock (bottom right). Percentages of dead cells measured in each population are indicated.</p></caption><graphic xlink:href=\"acmi-2-107-g001\"/></fig></sec><sec id=\"s2-4\"><title>Development of an efficient electroporation procedure</title><p>While electroporation is one of the most powerful techniques for delivering DNA to the inside of living cells [<xref rid=\"R14\" ref-type=\"bibr\">14, 62</xref>], and protocols have been developed for marine bacteria [<xref rid=\"R63\" ref-type=\"bibr\">63–66</xref>], it is a harsh treatment, typically resulting in significant cell loss [<xref rid=\"R67\" ref-type=\"bibr\">67</xref>]. Further, the required removal of salts results in osmotic stress - particularly challenging for a notoriously temperamental marine bacterium like <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> [<xref rid=\"R47\" ref-type=\"bibr\">47</xref>]. This stress must be mitigated using an osmoprotectant, and we explored the efficacy of different osmoprotectants as we pursued this approach.</p><sec id=\"s2-4-1\"><title>Selecting an osmoprotectant</title><p>We tested the ability of <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> to survive exposure to most commonly used electroporation osmoprotectants (glycerol, sorbitol, and PEG8000, see Methods for details). Cells washed in glycerol or PEG8000 did not survive any better than cells washed with MilliQ water, while sorbitol allowed cells to recover as fast as the seawater media control (<xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>). Of note, the initial cell density had a major impact on recovery: a starting density of 3.3×10<sup>7</sup> cells µl<sup>−1</sup> yielded only 0–5 % recovery, whereas one of 3.7×10<sup>8</sup> cells µl<sup>−1</sup> yielded 53±4% recovery, possibly due to more efficient pelleting or mitigation of oxidative stress at higher densities. Washes with sorbitol at the higher densities, however, yielded sufficient cell numbers for electroporation.</p><fig fig-type=\"figure\" id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2.</label><caption><p>Effects of different osmoprotectants on the survival of <italic>Prochlorococcus. Prochlorococcus</italic> strain MED4 cells were washed three times in glycerol 10 % (v/v), sorbitol 18.2 % (w/v) (corresponds to 1 M), and PEG 8000 5 % (w/v), placed back in their growth medium (Pro99), and culture growth was monitored using bulk fluorescence. Washes were also performed without any protectant in MilliQ water as a negative control, and in the growth medium Pro99 as a positive control. Error bars show the standard deviation of triplicate samples.</p></caption><graphic xlink:href=\"acmi-2-107-g002\"/></fig></sec><sec id=\"s2-4-2\"><title>Electroporation optimization</title><p>Using sorbitol as the osmoprotectant buffer, we next set up a method to screen for the most efficient electroporation conditions. Briefly, exponentially growing cultures were harvested and suspended in sorbitol. Fluorescein-labelled DNA was added to the washed cells, which were electroporated at variable electric fields and time constants while keeping the capacitance and resistance fixed. Cells were then transferred to fresh media for recovery and were used for dead cell staining and flow cytometry analysis (see <xref ref-type=\"fig\" rid=\"F3\">Fig. 3a</xref> and Methods section for details). We initially attempted to deliver a fluorescein-labelled plasmid in these optimization experiments; however, <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> exhibits strong autofluorescence in the 650–700 nm range due to chlorophyll [<xref rid=\"R68\" ref-type=\"bibr\">68, 69</xref>], but also a lower background autofluorescence in the green range used to detect fluorescein. This residual autofluorescence was sufficient to mask the weak signal obtained from fluorescein-labelled plasmids and prevented their reproducible detection. We thus used fluorescein-labelled oligonucleotides as our probe for DNA delivery (see Methods for detail). Oligonucleotides can be delivered in much higher amounts than plasmids, allowing us to increase the signal to noise ratio significantly. Although oligonucleotide delivery cannot predict plasmid delivery quantitatively - especially considering that plasmid size is inversely correlated with delivery efficiency [<xref rid=\"R70\" ref-type=\"bibr\">70</xref>] - their identical molecular composition allows them to be a convenient proxy to assess the best conditions for delivery of DNA molecules by electroporation.</p><fig fig-type=\"figure\" id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Fig. 3.</label><caption><p>Screening workflow for examining the efficiency of DNA delivery into cells via electroporation. (a) DNA delivery screening workflow. Light green oval shapes represent <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cells; oval shapes filled with bright green and short wavy lines correspond to cells that have incorporated the fluorescently labelled oligonucleotides; red dotted lines represent non-viable cells compromised by the electroporation treatment; solid purple line corresponds to non-viable cells stained with the dead-cell stain. (b) Percentage of live cells (assessed by the Live/Dead violet stain) with detectable levels of fluorescein-labelled oligonucleotides as well as the % of cells surviving the electric shock, as a function of electric field intensity. (c) Same as b but varying the time constant of the electroporation shock.</p></caption><graphic xlink:href=\"acmi-2-107-g003\"/></fig><p>We next tested a wide range of electroporation conditions. Using a fixed time constant of 12.5 ms, we found electric fields of 7 kV cm<sup>–1</sup> to be the most efficient condition for balancing the delivery of DNA inside cells while minimizing cell death (<xref ref-type=\"fig\" rid=\"F3\">Fig. 3b</xref>). We then varied the time constant, and found a peak delivery rate at about 15 ms; cell mortality appeared to increase linearly with the time constant (<xref ref-type=\"fig\" rid=\"F3\">Fig. 3c</xref>). This broad exploration of electric fields and time constants allowed us to find the most promising conditions for DNA delivery using a minimum number of samples. Considering the two-week time frame needed to grow enough cell material, and the limited number of samples that can be tested in parallel, this approach proved particularly advantageous.</p><p>We next focused on further optimizing electroporation conditions over the narrow range between 7–8 kV cm<sup>–1</sup> (<xref ref-type=\"fig\" rid=\"F4\">Fig. 4</xref>). Increasing the time constant from 12.5 ms to 25 ms at 7 kV cm<sup>–1</sup> marginally increased the delivery rate, but decreased cell survival more than two-fold. On the other hand, using 8 kV cm<sup>–1</sup> with a shorter time constant of 5 ms increased the number of survivors but reduced the DNA delivery rate to that of the non-electroporated control (<xref ref-type=\"fig\" rid=\"F4\">Fig. 4a</xref>). Thus, taking into account both the DNA delivery efficiency and cell survival, we determined that 7 kV cm<sup>–1</sup>; 12.5 ms was the most efficient condition for electroporation (<xref ref-type=\"fig\" rid=\"F4\">Fig. 4b</xref>). That such small variations in either the electric field or the time constant had dramatic effects was unexpected, and unusual for optimization of electroporation conditions [<xref rid=\"R64\" ref-type=\"bibr\">64, 71–73</xref>].</p><fig fig-type=\"figure\" id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Fig. 4.</label><caption><p>Fine-scale optimization of electroporation conditions. (a) Cell survival following electric pulse (assessed by dead-cell stain, left axis), and percentage of survivors with detectable levels of fluorescein-labelled oligonucleotides (right axis). (b) Efficiency of DNA delivery into cells for these electroporation conditions. t-test p value is indicated for the best electroporation conditions compared to the control. Note that a single electroporation reaction is performed on approximately ~10<sup>9</sup> cells; thus, 2 % represents a large number of potential transformants.</p></caption><graphic xlink:href=\"acmi-2-107-g004\"/></fig></sec></sec><sec id=\"s2-5\"><title>Development of a pour-plating procedure for axenic <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cultures</title><p>Assuming that mutants will one day be generated, we will need a procedure for selecting them on solid media. To date, the only way to grow <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> on solid media has been to use a ‘helper strain’ of heterotrophic bacteria, typically <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.2806\">Alteromonas</ext-link>\n</named-content></italic> [<xref rid=\"R48\" ref-type=\"bibr\">48, 51, 74</xref>]. <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> does not encode catalase, thus the ‘helper’ strain provides this function – removing reactive oxygen species from the media, reducing oxidative stress, and thereby facilitating growth [<xref rid=\"R75\" ref-type=\"bibr\">75</xref>]. Since this approach then requires re-purification of axenic <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> after obtaining a mutant, we sought to develop a plating protocol that would eliminate the helper cells and the need for this additional purification step. We approached this in two ways. First, because autoclaving agar produces reactive oxygen species [<xref rid=\"R76\" ref-type=\"bibr\">76</xref>], we sterilized the low melting point agar solution in ultrapure water using three rounds of boiling in a microwave before adding it to Pro99 medium resulting in a 0.3 % agar solution. Second, we included pyruvate in the medium, which serves to quench reactive oxygen species and is known to enhance the survival of liquid axenic <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cultures at low cell densities [<xref rid=\"R77\" ref-type=\"bibr\">77, 78</xref>]. With these modifications, we were able to obtain pour plates containing axenic <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> colonies (see Methods for details). The colonies typically took 2–3 months to become visible to the naked eye (Fig. S2), and could then be picked and transferred into small (~5 ml) volumes of liquid Pro99 medium; they grew as expected and were verified as axenic. This combination of microwave sterilization of the agar and addition of pyruvate to the plating medium allowed us to reproducibly obtain axenic colonies for all the <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strains tested so far (MED4, MIT9313, MIT0604, MIT1314, MIT9312, and SB).</p></sec><sec id=\"s2-6\"><title>Transformation attempts by transposome electroporation</title><p>The vast majority of genetic systems developed for bacteria rely on the use of replicative plasmids. Replicative plasmids offer a versatile platform to introduce and express exogenous genes in the target bacterial strain, allowing the development of further technologies for gene knockouts, such as Lambda Red recombineering or CRISPR-Cas9 [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]. However, in order to replicate, plasmids require an origin of replication compatible with the host. A common strategy for engineering a replicative plasmid is to use an origin of replication sequence derived from a wild plasmid found in the target bacterial host or to use a ‘broad host range’ origin of replication derived from conjugative plasmids [<xref rid=\"R79\" ref-type=\"bibr\">79</xref>]. As of yet, no wild plasmids have been found associated with <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cells, which are under intense selective pressure for genome minimization [<xref rid=\"R28\" ref-type=\"bibr\">28, 31</xref>]. Further, broad host range plasmids have yielded mediocre maintenance in closely related marine <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> strains [<xref rid=\"R56\" ref-type=\"bibr\">56</xref>] and have failed to replicate at all in <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>].</p><p>An alternative strategy is to use integrative plasmids to serve as delivery vectors, relying on the target cell’s homologous recombination machinery to recombine the exogenous DNA into the host genome [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]. This process has been successful in generating gene knockouts in marine <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> strains [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>] but required a lengthy procedure for <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>], which was not reproducible in our hands (see section above on conjugation attempts). We thus opted for a strategy that relies minimally on the host’s genetic abilities - the use of transposase enzymes to integrate foreign DNA into the target host chromosome. The transposase gene and transposon DNA can either be introduced on a plasmid vector so that transposase expression and activity occurs within the target cells (for example, plasmid pBAMD14 [<xref rid=\"R59\" ref-type=\"bibr\">59</xref>] used in conjugation attempts), or the transposase/transposon protein-DNA complex (the transposome) can be prepared <italic>in vitro</italic> and delivered inside the host by electroporation [<xref rid=\"R80\" ref-type=\"bibr\">80</xref>].</p><p>We used the EZ-Tn5 transposition system (Lucigen), which has worked successfully for several challenging bacteria [<xref rid=\"R81\" ref-type=\"bibr\">81–84</xref>]. Transposon DNA inserts were designed using genetic segments optimized for cyanobacteria [<xref rid=\"R56\" ref-type=\"bibr\">56</xref>], the mini-Tn5-vector pBAMD [<xref rid=\"R59\" ref-type=\"bibr\">59</xref>], and sequences designed specifically for <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic>, such as the strong Rubisco promoter P <italic>ccmk</italic> [<xref rid=\"R85\" ref-type=\"bibr\">85</xref>] or the codon-optimized resistance genes for spectinomycin/streptomycin (<italic>aadA</italic>) and chloramphenicol resistance (<italic>cat</italic>) (see Methods for details). Each of these constructs was tested in two <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strains (MED4 and MIT9313), as well as in several other strains selected for a lower likelihood of degrading incoming DNA by means of a restriction-modification system (MIT1314, MIT0604, MIT9215). However, no transformants were obtained in any of these attempts, despite growth of electroporated strains on the no-antiobiotic control plates.</p></sec><sec id=\"s2-7\"><title>Looking toward the future</title><p>While suitable DNA delivery conditions is a significant step forward, we have not yet achieved a successful genetic transformation of <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic>. Among the remaining hurdles are overcoming host defences against foreign genetic material such as restriction-modification systems, and optimizing expression of the antibiotic selection marker and the stable integration of foreign DNA inside the chromosome. We are confident, however, that effective solutions can be found to overcome these hurdles. Solutions might come from new transposome systems [<xref rid=\"R86\" ref-type=\"bibr\">86</xref>], which could reveal more efficient means than the Tn5 transposition; from the delivery of Cpf1-RNA complexes inside cells [<xref rid=\"R87\" ref-type=\"bibr\">87, 88</xref>]; from using more systematic approaches to evade restriction-modification defences [<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]; through the discovery of yet unknown defence systems [<xref rid=\"R89\" ref-type=\"bibr\">89</xref>] that are hampering transformation; or finally by harnessing endogenous mobile genetic elements, which have been tailored by evolution to work efficiently in these minimal cells. The latter is the direction we are currently exploring.</p></sec></sec><sec sec-type=\"methods\" id=\"s3\"><title>Methods</title><sec id=\"s3-1\"><title>Culture conditions</title><p>Axenic <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> MED4 cells were grown under constant light flux (30–40 μmol photons m<sup>−2</sup> s<sup>−1</sup>) at 24 °C in natural seawater-based Pro99 medium containing 0.2-µm-filtered Sargasso Sea water, amended with Pro99 nutrients (N, P, and trace metals) prepared as previously described [<xref rid=\"R47\" ref-type=\"bibr\">47</xref>]. Growth was monitored using bulk culture fluorescence measured with a 10AU fluorometer (Turner Designs).</p></sec><sec id=\"s3-2\"><title>Testing different antibiotics</title><p>MED4 cells were grown in Pro99 medium with a Sargasso seawater base, at a light level of 25 μmol photons m<sup>−2</sup> s<sup>−1</sup>. Growth was monitored by measuring bulk chlorophyll fluorescence and was compared to a no-drug control culture.</p></sec><sec id=\"s3-3\"><title>\n<italic>E. coli</italic> mediated conjugation</title><p>For conjugation via filter mating, we followed the procedure described by Tolonen <italic>et al</italic>. [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>]. Conjugation was performed using <italic>E. coli</italic> 1100–2 carrying plasmid pRK2 (as the conjugation vector) and pRL153 (kanamycin-resistant), as well as <italic>E. coli</italic> BW19851 carrying BAC conjugation plasmid pMBD14 [<xref rid=\"R90\" ref-type=\"bibr\">90</xref>] (chloramphenicol-resistant) - on receiver strain <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> 8102, and <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strains MIT9313 and MED4. As described above, in our hands, the method did not yield any conjugants after incubation of exconjugants for two months. For conjugation via liquid mating, we used the same donor and receiver strains, and the following procedure: 1 ml of log-phase donor <italic>E. coli</italic> was added to 25 ml of log-phase <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> or <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> receiver strain and the co-culture was left to grow overnight. Antibiotic was then directly added to the co-culture at the same concentration used on solid media (kanamycin 50 µg ml<sup>−1</sup>, chloramphenicol 10 µg ml<sup>−1</sup>). The addition of nalidixic acid at 50 µg ml<sup>−1</sup> was also attempted to remove the <italic>E. coli</italic> donor strain. Cultures were then grown for 45–60 days but did not yield conjugants.</p><p>For conjugation via ‘agar stab mating’, we used <italic>E. coli</italic> donor strain BW20767 carrying plasmids pBAMD1-4-GFP or pBAMD1-4-YFP for mating with <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> WH8102 and <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> strains NATL2A and SB. Then 25 ml of log-phase receiver strain was pelleted by centrifugation at 5000 <bold><italic>g</italic></bold> for 15 min at 20 °C and resuspended in 200 µl Pro99 medium. Log phase <italic>E. coli</italic> donor strain was mixed at a 1 : 1 cell ratio with the receiver strain, and 20 µl of the mixture was injected inside a 1 ml volume of polymerized Pro99 with 1 % agar - the ‘agar stab’ (Fig. S1). After 24 h incubation, cells were sucked out of the agar using a pipet tip, resuspended in liquid Pro99 medium, and plated for selection with 5 µg ml<sup>−1</sup> of spectinomycin and streptomycin (combined 1 : 1; plating procedure below).</p></sec><sec id=\"s3-4\"><title>Plasmids and mini-Tn5 transposon construction</title><p>We used plasmid mini-Tn5 pBAMD1-4 [<xref rid=\"R59\" ref-type=\"bibr\">59</xref>] to construct transposon tools for insertion into <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> genomes. Genes encoding the GFPmutII and YFP reporters, shown to fluoresce efficiently in a wide range of cyanobacteria [<xref rid=\"R56\" ref-type=\"bibr\">56</xref>] were cloned by Gibson assembly behind the strong <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> P <italic>ccmk</italic> promoter for Rubisco protein [<xref rid=\"R91\" ref-type=\"bibr\">91</xref>], well conserved in various <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> and <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.655\">Synechococcus</ext-link>\n</named-content></italic> strains [<xref rid=\"R85\" ref-type=\"bibr\">85</xref>]. The pBAMD1-4 plasmid was linearized by digestion with HindIII-HF and EcoRI-HF (New England Biolabs #R3104S and #R3101S) at 37 °C for 2 h in Cutsmart buffer. The GFPmutII-encoding fragment was amplified from plasmid pCV0001 DNA using primers RL017 and RL018; the YFP-encoding fragment was amplified from plasmid pCV0021 using primers RL020 and RL021. The P <italic>ccmk</italic> promoter was amplified from <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> MED4 genomic DNA using primers RL015 and RL016 for the P <italic>ccmk-gfpmutII</italic> construct, and RL015 and RL019 for the P <italic>ccmk-yfp</italic> construct. The three-part assembly reaction (plasmid-promoter-insert) was performed using the Gibson assembly master mix 2x (New England Biolabs #E2611S), following the manufacturer’s instructions. The mixture was transformed in One Shot PIR1 Chemically Competent <italic>E. coli</italic> (Thermofisher #C101010). For the construction of pBAMD1-4-Pccmk (comprising the P <italic>ccmk</italic> promoter inserted within the transposon in front of the antibiotic resistance <italic>aadA</italic> gene, but no fluorescent marker gene) P <italic>ccmk</italic> was amplified from <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> MED4 genomic DNA using primers RL121 and RL122, which contain EcoRI and SwaI restriction sites, respectively. Both pBAMD1-4 and the insert were digested with EcoRI-HF and SwaI-HF (New England Biolabs #R0604S and # R3101S), ligated, and transformed in PIR1 <italic>E. coli</italic> as described above.</p></sec><sec id=\"s3-5\"><title>Live/Dead violet staining protocol</title><p>For each sample labelled, 100 µl of culture were transferred to an Eppendorf tube containing 900 µl of filtered Pro99 medium. Then 1 µl of reconstituted LIVE/DEAD Fixable Violet Dead Cell Stain (Thermo Fisher Scientific) was added to the 1 ml cell suspension and incubated for 30 min at room temperature in the dark. Cells were pelleted by centrifugation at 10 000 <bold><italic>g</italic></bold> for 5 min at 20 °C and resuspended in 0.5 ml of fresh Pro99. Samples were then serially diluted and immediately run on the flow cytometer. A positive control (untreated exponentially growing cell culture) and negative control (1 ml of cells from the same culture heated to 80 °C for 5 min) were included with each run to gate the live and dead cell populations.</p></sec><sec id=\"s3-6\"><title>Flow cytometry</title><p>Cell abundances, viability (Live/Dead violet staining and chlorophyll fluorescence), and fluorescein-labelled oligonucleotide delivery were measured on a Guava easyCyte 12HT flow cytometer (EMD Millipore, Billerica, MA). Cells were excited with a blue 488 nm laser for measuring chlorophyll fluorescence (692/40 nm), size (forward scatter) and fluorescein fluorescence (525/30 nm); and with a violet 405 nm laser to measure the violet dead stain fluorescence (448/50 nm). All flow cytometry files were analysed using FlowJo software version 7.6.5 (FlowJo, LLC, Ashland, OR, USA) and the following workflow: (1) <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cells were counted by a defined gate on the red fluorescence (chlorophyll) versus forward scatter (size) plot. (2) From these selected events, viable cells were counted by a defined threshold on the blue fluorescence (violet dead cell stain). The threshold was adjusted for each run using the positive (untreated cells) and negative (heat-shocked cells) control. (3) From the ‘viable cell’ subset of events, the cells into which DNA was delivered were counted by a defined threshold on the green fluorescence (fluorescein-labelled oligonucleotides). The threshold was adjusted so that <1 % of events from the negative control (cells that did not enter into contact with the oligonucleotides) were included. The dead cell control comprised cells killed by heat treatment at 80 °C for 5 min. The flow cytometry plots were generated using FlowJo.</p></sec><sec id=\"s3-7\"><title>Testing different osmoprotectants</title><p>For each osmoprotectant tested, 2 ml of triplicate exponentially growing MED4 culture were harvested by centrifugation at 13 000 <bold><italic>g</italic></bold> for 10 min. Under sterile conditions, pellets were resuspended by gently pipetting up and down in a volume of 100 µl of either solutions 10 % (v/v) glycerol in water; 18.2 % (w/v) sorbitol in water (1M); 5 % (w/v) PEG8000 in water; Pro99 medium (positive control); or MilliQ water (negative control). The washing was repeated once before cells were centrifuged again, resuspended into 1 ml of Pro99 medium, and inoculated in 5 ml of Pro99. The tubes were placed back in the incubator, and recovery was monitored by measuring bulk culture fluorescence. Of note, all osmoprotectant solutions were filter sterilized through 0.2 µm supor membrane filters. Cultures that recovered were checked for the presence of heterotrophic contamination using purity broths, as previously described [<xref rid=\"R47\" ref-type=\"bibr\">47</xref>] to ensure that recovery was not facilitated by a contaminating heterotrophic partner [<xref rid=\"R74\" ref-type=\"bibr\">74</xref>].</p></sec><sec id=\"s3-8\"><title>Electroporation procedure</title><p>Exponentially growing cultures were harvested by centrifugation at 7000 <bold><italic>g</italic></bold> for 15 min at 20 °C. Then 50 ml culture pellets were washed three times in 5 ml (1 M sorbitol solution) by centrifugation at 5000 <bold><italic>g</italic></bold> for 8 min at 20 °C. The final resuspension volume was calculated so that cells were concentrated 1000 times compared to the initial culture volume. Then 500 ng of 90 bp fluorescein-labelled oligonucleotides (sequence: cctcataacaagcagcgctcatagtattaggaatatcgtgaaattcaagatctaagaatatttttttatttaaatttttcaaaattttta) were added to 50 µl of the washed cells and electroporated in 2 mm gap cuvettes at variable electric fields and time constants while keeping the capacitance and resistance fixed at 25 µF and 200 ohms. One ml of fresh Pro99 medium at room temperature was immediately added to the cuvette, and cells were transferred to a culture tube containing 4 ml Pro99 supplemented with 5 mM glucose and 5 mM pyruvate for recovery. Samples were then directly stained with Live/Dead violet and analysed by flow cytometry. We initially tried to deliver a fluorescently labelled pUC19 plasmid (using the fluorescein LabelIT Tracker kit, MirusBio), but the signal to noise ratio was not sufficient to detect the plasmid.</p><p>For transposome delivery, 2 µl of EZ-Tn5 (Lucigen) <italic>in vitro</italic>-assembled transposomes were added to the 50 µl cell suspension in sorbitol before electroporation. Addition of DOTAP liposomal transfection reagent (Millipore Sigma) and Type One Inhibitor (Lucigen), following the manufacturer’s instructions, were also attempted, as they were shown to favour successful transposome transformation in the cyanobacterium <italic>Arthrospira platensis C1</italic> [<xref rid=\"R92\" ref-type=\"bibr\">92, 93</xref>]. After transposome delivery, cells were left to recover for 24 h before plating.</p></sec><sec id=\"s3-9\"><title>Pour plating of axenic <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> cultures</title><p>To obtain single colonies from an axenic culture, serial dilutions of an exponentially growing cyanobacterial culture grown in Pro99 medium (as described above) were pour-plated in the plating medium (Pro99 medium supplemented with 0.05 % (wt/vol) Pyruvate and 3.75 mM TAPS (pH 8) and containing 0.3 % low melting point (LMP) agarose. To prepare the plating medium, we first sterilized a 3 % LMP agar solution in ultrapure water independently by three rounds of boiling in a microwave (~20 s each, to avoid boiling-over and loss of volume). We then added the melted LMP agar 1 : 10 in the Pro99 medium base supplemented with pyruvate and TAPS. The mixture was maintained at ~28 °C in a water bath to cool before plating. Of importance, the LMP agarose was microwave sterilized and not autoclaved to avoid creating reactive oxygen species likely to inhibit <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> growth [<xref rid=\"R76\" ref-type=\"bibr\">76</xref>]. Microwave-based boiling was sufficient to prevent the appearance of contaminants in the nutrient-poor Pro99 medium. For antibiotic selection after transposome delivery, 1 ml of ‘recovering’ cells (post electroporation, see above) were centrifuged and resuspended in 100 µl of fresh Pro99, and pour plated in 10 ml plating medium supplemented with the antibiotic. Kanamycin was used at 50 µg ml<sup>−1</sup>, and aminoglycoside resistance (encoded by <italic>aadA</italic>) selected with a combination of streptomycin and spectinomycin at 5 µg ml<sup>−1</sup> total concentration (2.5 µg ml<sup>−1</sup> each). Individual colonies typically became visible after 40–60 days. They were picked using a sterile pipet tip and inoculated into 5 ml of fresh Pro99 medium. The efficiency of colony formation from a diluted culture is variable and generally low (~1 %), an effect that seems alleviated by plating a higher number of cells, as an axenic lawn of cells would grow significantly faster, typically visible after 15–30 days.</p></sec><sec id=\"s3-10\"><title>Transposome preparation</title><p>The chloramphenicol resistance gene <italic>cat</italic> and the spectinomycin/streptomycin resistance gene <italic>aadA</italic> were codon-optimized for <italic><named-content content-type=\"genus\">\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://doi.org/10.1601/nm.650\">Prochlorococcus</ext-link>\n</named-content></italic> using the codon usage table from the Kazusa database, and the OPTIMIZER online tool [<xref rid=\"R94\" ref-type=\"bibr\">94</xref>]. The codon-optimized sequence, which is preceded by the P <italic>ccmk</italic> promoter and flanked by the Tn5 mosaic end, and were synthesized through the Genscript gene synthesis service (see sequence in supplementary data).</p><p>Transposome DNA containing the antibiotic resistance cassette and the mosaic ends were amplified using the template plasmids and primers (all 5′ phosphorylated) listed in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>:</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2.</label><caption><p>Strains, plasmids and primers. Antibiotic markers: Ap<sup>R</sup>, ampicillin; Cm<sup>R</sup>, chloramphenicol; Gm<sup>R</sup>, gentamicin; Km<sup>R</sup>, kanamycin; Sm<sup>R</sup>, streptomycin; Sp<sup>R</sup>, spectinomycin; and Tet<sup>R</sup>, tetracycline</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" style=\"background:#D9D9D9\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>E. coli</italic> strains</p>\n</th><th align=\"left\" valign=\"top\" style=\"background:#D9D9D9\" rowspan=\"1\" colspan=\"1\">\n<p>Description/relevant characteristics</p>\n</th><th align=\"left\" valign=\"top\" style=\"background:#D9D9D9\" rowspan=\"1\" colspan=\"1\">\n<p>Reference</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1100–2</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>mcrA- endA-; host for pRK24 and pRL153</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R54\" ref-type=\"bibr\">54</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>BW19851</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Tra- Tet<sup>R</sup> Sm<sup>R</sup> Pir+; host for pRL27</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R96\" ref-type=\"bibr\">96</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>BW20767</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>RP4–2-Tc::Mu-1 kan::Tn7 integrant leu-63::IS10 recA1 zbf-5 creB510 hsdR17 endA1 thi uidA (∆MluI)::pir+; host for pRL27 and pBAMD derivatives</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R96\" ref-type=\"bibr\">96</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>PIR1</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>∆lac169 rpoS(Am) robA1 creC510 hsdR514 endA recA1 uidA(∆MluI)::pir-116; host for pBAMD derivatives</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Thermofisher #C101010</italic>\n</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Plasmids</bold>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p> </p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p> </p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>pRK24</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Tet<sup>R</sup> Amp<sup>R</sup> RP4 conjugal plasmid</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R54\" ref-type=\"bibr\">54</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>pRL153</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Kan<sup>R</sup> RSF1010 derivative</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R54\" ref-type=\"bibr\">54</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>pCV0001</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>RSF1010 derivative, Sm<sup>R</sup>/Sp<sup>R</sup>, GFPmut2 under a PconII promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R56\" ref-type=\"bibr\">56</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>pCV0021</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>RSF1010 derivative, Sm<sup>R</sup>/Sp<sup>R</sup>, YFP under a PconII promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R56\" ref-type=\"bibr\">56</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>pMBD1-4</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>BAC conjugation plasmid, Cm<sup>R</sup></italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R90\" ref-type=\"bibr\">90</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>pBAMD1-4-GFP</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Derived from pBAMD1-4</italic> [<xref rid=\"R59\" ref-type=\"bibr\">59</xref>]. <italic>Mini-Tn5 delivery plasmid; ori(R6K); Ap<sup>R</sup> Sm<sup>R</sup>/Sp<sup>R</sup> ; GFPmut2</italic> [<xref rid=\"R56\" ref-type=\"bibr\">56</xref>] <italic>placed under P ccmk promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>pBAMD1-4-YFP</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Derived from pBAMD1-4</italic> [<xref rid=\"R59\" ref-type=\"bibr\">59</xref>]. <italic>Mini-Tn5 delivery plasmid; ori(R6K); Ap<sup>R</sup> Sm<sup>R</sup>/Sp<sup>R</sup> ; YFP</italic> [<xref rid=\"R56\" ref-type=\"bibr\">56</xref>] <italic>placed under P ccmk promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>pBAMD1-4-Pccmk</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Sm<sup>R</sup>/Sp<sup>R</sup> directly under P ccmk promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>pRL27</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Kan<sup>R</sup>; mini-Tn5 oriR6K</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R96\" ref-type=\"bibr\">96</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Transposomes</bold>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p> </p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p> </p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>T1</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Sm<sup>R</sup>/Sp<sup>R</sup> ; GFPmut2 under a P conII promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>T2</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Sm<sup>R</sup>/Sp<sup>R</sup> ; YFP under a P conII promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>T3</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Sm<sup>R</sup>/Sp<sup>R</sup> ; GFPmut2 under P ccmk promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>T4</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Sm<sup>R</sup>/Sp<sup>R</sup> ; YFP under P ccmk promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>T5</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Codon-optimized Cm<sup>R</sup> under P ccmk promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>T6</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic><R6Kγori/KAN-2>from Lucigen (#TSM08KR); Kan<sup>R</sup></italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>[<xref rid=\"R80\" ref-type=\"bibr\">80</xref>]</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>T7</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Sm<sup>R</sup>/Sp<sup>R</sup> under P ccmk promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>T8</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>Codon-optimized Sm<sup>R</sup>/Sp<sup>R</sup> under P ccmk promoter</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>Primers</bold> (5’>3’)</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p> </p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p> </p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL005</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>CTGTCTCTTATACACATCTCAACCCTGATGCGTGGAGACCGAAACCTT</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL006</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>CTGTCTCTTATACACATCTCAACCATCAAAGCTTCAAAAAGGCCATCC</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL007</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>CTGTCTCTTATACACATCTCAACCCTGAGCTAGCGGCGCGCCAAAAAA</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL008</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>CTGTCTCTTATACACATCTCAACCATCATCTAGAAAGCTTCAAAAAGG</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL015</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>TAGGCCGCGGCCGCGCGTTGATGCACTAACTAATTTCGATAAGTATTGACATATCAATAG</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL016</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>CCTTTACTCATTGTTTCTGTAGCCATTGCCTACTAATTACTAAATG</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL017</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>CTACAGAAACAATGAGTAAAGGAGAAGAACTTTTCACTGGAG</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL018</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>GCTTTGGCGGCCGCATTATTATTTGTATAGTTCATCCATGCCATGTGTAATCC</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL019</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>GCTCACCATTGTTTCTGTAGCCATTGCCTACTAATTACTAAATG</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL020</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>ACAGAAACAATGGTGAGCAAGGGCGAG</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL021</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>GCTTTGGCGGCCGCATTACTTGTACAGCTCGTCCATGCC</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL079</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>CTGTCTCTTATACACATCTTTGTGTCTCAGG</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL080</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>CTGTCTCTTATACACATCTTTGTGTCTCAGG</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL081</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>CTGTCTCTTATACACATCTCAACCATCATTG</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL121</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>ATCGGAATTCTTGATGCACTAACTAATTTCGA</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL122</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>CGATATTTAAATTGTTTCTGTAGCCATTGCCTAC</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>RL140</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<italic>CTGTCTCTTATACACATCTTTGATGCAC</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>This work</p>\n</td></tr></tbody></table></table-wrap><list list-type=\"bullet\"><list-item><p>Transposome T1 amplified using RL005 and RL006 primers from pCV0001 template DNA</p></list-item><list-item><p>Transposome T2 amplified using RL007 and RL008 primers from pCV0021 template DNA</p></list-item><list-item><p>Transposome T3 amplified using RL079 and RL080 primers from pBAMD1-4-GFP template DNA</p></list-item><list-item><p>Transposome T4 amplified using RL079 and RL080 primers from pBAMD1-4-YFP template DNA</p></list-item><list-item><p>Transposome T5 amplified using RL081 and RL080 primers from the synthesized codon-optimized chloramphenicol resistance cassette template DNA</p></list-item><list-item><p>Transposome T7 amplified using RL079 and RL080 primers from the pBAMD1-4-Pccmk template DNA</p></list-item><list-item><p>Transposome T8 amplified using RL140 and RL080 primers from the synthesized codon-optimized Spectinomycin/streptomycin resistance cassette template DNA.</p></list-item></list><p>PCR mixtures were digested for 1 h at 37 °C with DpnI (New England Biolabs) to remove the template plasmids. PCR products were then purified using the QIAquick PCR Purification Kit (Qiagen), eluted in TE buffer at pH 7.5, and DNA purity was assessed using a NanoDrop ND8000 spectrophotometer. Using the same kit, samples were often purified a second time to remove detectable impurities. Transposomes were formed using the EZ-Tn5 Transposase (Lucigen) following the manufacturer’s instruction. Then 4 µl of EZ-Tn5 transposase preparation was mixed with 2 µl of transposon DNA at 100 µg µl<sup>−1</sup> plus 2 µl of 100 % glycerol and the mixture was incubated for 30 min at room temperature. The transposomes were either electroporated immediately or frozen at −20 °C for later use. The commercial transposome R6Kγori/KAN-2 from Lucigen kit #TSM08KR was used according to the manufacturer’s instructions.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Data</title><supplementary-material content-type=\"local-data\" id=\"supp1\"><caption><title>Supplementary material 1</title></caption><media xlink:href=\"acmi-2-107-s001.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"ack1\"><title>Funding information</title><p>This study was supported in part by the National Science Foundation (NSF-EDGE – 1645061 to SWC) and the Simons Foundation (Life Sciences Project Award IDs 337262 and 509034SCFY17 and SCOPE Award ID 329108, to SWC).</p></ack><ack id=\"ack2\"><title>Acknowledgements</title><p>We gratefully acknowledge Benjamin M. Woolston for sharing his method for DNA delivery screening in live cells, which was critical to the development of the procedure in <italic>Prochlorococcus</italic>. We thank Paulo A. Garcia and Cullen R. Buie for the helpful discussions on determining the optimal electroporation parameters. We thank Bianca Brahamsha for her help, and specifically for her guidance regarding antibiotic selection. We thank Sebastien Rodrigue for his participation and his many ideas on how to address this challenge.</p></ack><ack id=\"ack3\"><title>Conflicts of interest</title><p content-type=\"COI-statement\">The authors declare that there are no conflicts of interest.</p></ack><fn-group><fn id=\"FN1\"><p>Abbreviations: GFP, Green Fluorescent Protein; PEG, Polyethylene glycol; YFP, Yellow Fluorescent Protein.</p></fn><fn id=\"FN2\"><p>Supplementary material is available with the online version of this article.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>1</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dick</surname><given-names>GJ</given-names></name><name><surname>Lam</surname><given-names>P</given-names></name></person-group><article-title>Omic approaches to microbial geochemistry</article-title><source>Elements</source><year>2015</year><volume>11</volume><fpage>403</fpage><lpage>408</lpage><pub-id 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"brief-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Access Microbiol</journal-id><journal-id journal-id-type=\"hwp\">acmi</journal-id><journal-id journal-id-type=\"publisher-id\">acmi</journal-id><journal-title-group><journal-title>Access Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">2516-8290</issn><publisher><publisher-name>Microbiology Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005866</article-id><article-id pub-id-type=\"pmc\">PMC7523630</article-id><article-id pub-id-type=\"publisher-id\">000101</article-id><article-id pub-id-type=\"doi\">10.1099/acmi.0.000101</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Short Communication</subject></subj-group></article-categories><title-group><article-title>Incrimination of <italic>Aedes aegypti</italic> and <italic>Aedes albopictus</italic> as vectors of dengue virus serotypes 1, 2 and 3 from four states of Northeast India</article-title><alt-title alt-title-type=\"recto-page-foot\"><ext-link ext-link-type=\"uri\" xlink:href=\"http://acmi.microbiologyresearch.org\">http://acmi.microbiologyresearch.org</ext-link></alt-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Chetry</surname><given-names>Sumi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-0651-2739</contrib-id><name><surname>Patgiri</surname><given-names>Saurav Jyoti</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bhattacharyya</surname><given-names>Dibya Ranjan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0002-2815-3303</contrib-id><name><surname>Dutta</surname><given-names>Prafulla</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">\n<sup>1</sup>\n</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">https://orcid.org/0000-0003-4504-4704</contrib-id><name><surname>Kumar</surname><given-names>N. Pradeep</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">\n<sup>2</sup>\n</xref></contrib><aff id=\"aff1\">\n<label><sup>1</sup>​</label>\n<institution content-type=\"department\">ICMR-Regional Medical Research Centre</institution>, <institution>North East Region</institution>, <addr-line content-type=\"city\">Dibrugarh</addr-line>, <country>Assam, India</country>\n</aff><aff id=\"aff2\">\n<label><sup>2</sup>​</label>\n<institution>ICMR-Vector Control Research Centre</institution>, <addr-line content-type=\"city\">Puducherry</addr-line>, <country>India</country>\n</aff></contrib-group><author-notes><corresp id=\"cor1\"><bold>*Correspondence:</bold> Saurav Jyoti Patgiri, <email xlink:href=\"mailto:saurav.patgiri@gmail.com\">saurav.patgiri@gmail.com</email></corresp></author-notes><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>26</day><month>3</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>26</day><month>3</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>2</volume><issue>4</issue><elocation-id>acmi000101</elocation-id><history><date date-type=\"received\"><day>20</day><month>11</month><year>2019</year></date><date date-type=\"accepted\"><day>03</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 The Authors</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License.</license-p></license></permissions><self-uri content-type=\"pdf\" xlink:href=\"acmi-2-101.pdf\"/><abstract><p>Dengue is an important vector borne disease with a great public health concern worldwide. Northeast India has experienced dengue almost every year for a decade. As studies on dengue vectors from this region are limited, we undertook an investigation to detect natural infection of the dengue virus (DENV) in potential dengue vectors of this region. Adult <italic>Aedes</italic> mosquitoes which were collected were subjected to RT-PCR for detection of infecting dengue serotype. Minimum infection rate was also determined for each positive pool. Out of the total 6229 adult <italic>Aedes</italic> mosquitoes collected, <italic>Aedes aegypti</italic> (63.3 %) was abundant in comparison to <italic>Aedes albopictus</italic> (36.7 %). These specimens (515 mosquito pools) were subjected to RT-PCR for detection of DENV-1, 2, 3 and 4. RT-PCR revealed the existence of DENV in both male as well as female mosquito pools suggesting natural transovarial transmission of DENV in this region. A total of 54 pools tested were positive for DENV-1, 2, 3 serotypes. This study revealed the occurence of DENV in both the potential dengue vectors from this region along with evidence of transovarial transmission which helps in persistence of the virus in nature.</p></abstract><kwd-group><kwd><italic>Aedes albopictus</italic></kwd><kwd>Aedes aegypti</kwd><kwd>Dengue virus</kwd><kwd>Transovarial transmission</kwd><kwd>Northeast India</kwd></kwd-group><funding-group><award-group id=\"ID0E2XAE282\"><funding-source>Indian Council of Medical Research</funding-source><award-id>Ref. No. VIR/9/2017/ECD-1 dated 21.08.17</award-id><principal-award-recipient>N. Pradeep Kumar</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OpenAccessEmbargo</meta-name><meta-value>0</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Dengue is an important vector borne disease causing significant morbidity as well as mortality worldwide. A rough estimate of dengue cases occurring every year stands at approximately 390 million and the majority of these are asymptomatic [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]. Dengue is caused by dengue virus (DENV), five serotypes of which are known to cause human infection universally, each antigenically distinct from each other.</p><p>Northeast India comprises eight states and except Sikkim, all the other states have reports of DENV activity in the past with the four common serotypes of DENV (DENV-1 to 4) being prevalent in the region [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>].</p><p>The virus is transmitted by <italic>Aedes</italic> (<italic>Ae</italic>.) mosquitoes, particularly <italic>Ae. aegypti</italic> and <italic>Ae. albopictus</italic> in a human-mosquito-human cycle or by transovarial transmission [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]. Both the vectors are day time feeders. While <italic>Ae. aegypti</italic> breeds mainly in man-made breeding habitats (solid waste), <italic>Ae. albopictus</italic> usually prefers a natural habitat and also breeds with <italic>Ae. aegypti</italic>. Mostly indoor habitat is preferred which is resistant to climatic variations and hence increases the longevity of the mosquitoes [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>].</p><p>Dengue is widespread in India, especially in the urban environment, with outbreaks being reported previously from many areas [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]. The occurrence of dengue case reports from the northeastern states of India has increased gradually over the years. Probable factors contributing to this could be migration of population from areas where dengue is endemic and increased unplanned urbanization and industrialization which are responsible for creating favourable breeding habitats for the vector mosquitoes. Transovarial transmission (TOT) in dengue is already well established and there are reports from many areas throughout the world [<xref rid=\"R6\" ref-type=\"bibr\">6–8</xref>]. Knowledge of transovarial transmission contributing a dengue outbreak is useful in dengue surveillance to explore the possibility of development of models for early warning [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>].</p><p>Though <italic>Aedes</italic> is prevalent in northeast India, their vectorial status is less documented. Earlier, <italic>Ae. aegypti</italic> was incriminated from Guwahati, Assam for DENV-1 and 2 [<xref rid=\"R9\" ref-type=\"bibr\">9–11</xref>]. The role of <italic>Ae. albopictus</italic> in dengue transmission is not known in this region till date. This study was therefore conducted to evaluate the status of the two major dengue vectors in dengue endemic areas of four northeastern states viz. Assam, Arunachal Pradesh, Meghalaya and Nagaland.</p></sec><sec sec-type=\"methods\" id=\"s2\"><title>Methods</title><sec id=\"s2-1\"><title>Mosquito sample collection and identification</title><p>Adult <italic>Aedes</italic> mosquitoes were collected during the period of February, 2018 to February, 2019 in four study sites namely Guwahati (Assam), Pasighat (Arunachal Pradesh), Tura (Meghalaya) and Dimapur (Nagaland). Collection of mosquitoes was conducted in different indoor and outdoor resting habitats using a suction tube. After collection, the mosquitoes were transported to the laboratory, identified to species level using standard identification keys and pooled separately on the basis of study site, day of collection, species, gender and abdominal status (fed and unfed in case of female mosquitoes only) [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>].</p></sec><sec id=\"s2-2\"><title>RNA extraction</title><p>Maximum 20 mosquitoes of same day, location, species, gender and abdominal condition were pooled together. If the number of mosquitoes for one pool exceeded 20, subsequent pools were made with the extra specimens accordingly. Each pool was stored in 50 µl Tri reagent at 4 °C until use. Prior to RNA isolation, the mosquitoes in the pool were thoroughly homogenized till no visible parts remained. After homogenization, RNA isolation was done using Tri reagent as per manufacturer’s protocol [<xref rid=\"R13\" ref-type=\"bibr\">13, 14</xref>].</p></sec><sec id=\"s2-3\"><title>Reverse transcription polymerase chain reaction (RT-PCR)</title><p>The total RNA, extracted from the mosquito pools were initially converted to cDNA followed by PCR for DENV capsid pre-membrane region [<xref rid=\"R15\" ref-type=\"bibr\">15, 16</xref>]. The amplified product was again subjected to a hemi-nested PCR, to detect the serotype involved. The presence of individual serotypes in the pools was established by the detection of amplification products having molecular weights 482, 119, 290 and 392 bp for each of the four dengue serotypes (DENV 1–4) respectively using gel electrophoresis.</p></sec><sec id=\"s2-4\"><title>Minimum Infection Rate (MIR) calculation</title><p>Minimum Infection Rate (MIR) was calculated by assuming that a positive mosquito pool contains a single infected mosquito. MIR was calculated as the ‘ratio of the total number of positive pools to the number of tested mosquitoes, multiplied by 1000’ [<xref rid=\"R17\" ref-type=\"bibr\">17, 18</xref>].</p></sec><sec id=\"s2-5\"><title>Statistical analysis</title><p>Independent samples <italic>t</italic>-test was used for analysing any difference in the means of gender-wise distribution and MIR values of <italic>Aedes</italic> mosquitoes across the four study sites. IBM SPSS Statistics 20.0 was used for all statistical analysis. A <italic>P</italic> value <0.05 was considered as statistically significant.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec id=\"s3-1\"><title>Entomological investigation</title><p>A total of 6229 adult <italic>Aedes</italic> mosquitoes were collected from the four study sites. Maximum mosquitoes could be collected from Pasighat, Arunachal Pradesh. The number of mosquitoes collected from Pasighat, Arunachal Pradesh was 2253 (<italic>Ae. aegypti</italic>- 134, <italic>Ae. albopictus</italic>- 2119), Dimapur, Nagaland was 1902 (<italic>Ae. aegypti</italic>- 1858, <italic>Ae. albopictus</italic>- 44), Guwahati, Assam was 1597 (<italic>Ae. aegypti</italic>- 1484, <italic>Ae. albopictus</italic>- 113) and from Tura, Meghalaya, 477 mosquitoes could be collected (<italic>Ae. aegypti</italic>- 470, <italic>Ae. albopictus</italic>- 7). Gender wise distribution of total mosquitoes collected from different locations is given in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. For <italic>Ae. Aegypti,</italic> it was seen that 52.5 % (<italic>n</italic>=2073) of the total mosquitoes collected belonged to the female gender with no significant gender difference in the distribution amongst the four states (<italic>P</italic>=0.869). Majority of the female mosquitoes (90.4 %, <italic>n</italic>=1873) were in the unfed state. For <italic>Ae. Albopictus,</italic> female mosquitoes comprised 79.2 % (<italic>n</italic>=1809) and no significant difference was observed in the gender-wise distribution across the study areas (<italic>P</italic>=0.467). Unfed mosquitoes comprised 95.2 % of the total female mosquito population.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1.</label><caption><p>Species and gender wise distribution of <italic>Aedes</italic> mosquitoes collected from four study sites</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"3\" colspan=\"1\">\n<p>Location</p>\n</th><th align=\"center\" valign=\"top\" colspan=\"5\" rowspan=\"1\">\n<p>\n<italic>Ae. aegypti</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"3\" colspan=\"1\">\n<p>Total (%)</p>\n</th><th align=\"center\" valign=\"top\" colspan=\"5\" rowspan=\"1\">\n<p>\n<italic>Ae. albopictus</italic>\n</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"3\" colspan=\"1\">\n<p>Total (%)</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"3\" colspan=\"1\">\n<p>Grand Total</p>\n</th></tr><tr><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Male (%)</p>\n</th><th align=\"center\" valign=\"top\" colspan=\"3\" rowspan=\"1\">\n<p>Female</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Two-tailed <italic>p</italic> value</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Male (%)</p>\n</th><th align=\"center\" valign=\"top\" colspan=\"3\" rowspan=\"1\">\n<p>Female</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Two-tailed <italic>p</italic> value</p>\n</th></tr><tr><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Total (%)</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Fed</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Unfed</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Total (%)</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Fed</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Unfed</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Guwahati</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>662 (44.6)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>822 (55.4)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>85</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>737</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"4\" colspan=\"1\">\n<p>0.869</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>1484</bold> (<bold>92.9</bold>)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>52 (46)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>61 (54)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>58</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"4\" colspan=\"1\">\n<p>0.467</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>113</bold> (<bold>7.1</bold>)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>1597</bold>\n</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Pasighat</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>64 (47.8)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>70 (52.2)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>65</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>134</bold> (<bold>5.9</bold>)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>411 (19.4)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1708 (80.6)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>83</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1625</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2119</bold> (<bold>94.1</bold>)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2253</bold>\n</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Dimapur</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>932 (50.2)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>926 (49.8)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>81</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>845</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>1858</bold> (<bold>97.7</bold>)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>7 (15.9)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>37 (84.1)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>36</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>44</bold> (<bold>2.3</bold>)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>1902</bold>\n</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Tura</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>215 (45.7)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>255 (54.3)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>29</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>226</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>470</bold> (<bold>98.5</bold>)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4 (57.1)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3 (42.9)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>7</bold> (<bold>1.5</bold>)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>477</bold>\n</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Grand Total</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1873</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2073</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>200</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1873</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>3946</bold> (<bold>63.3</bold>)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>474</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1809</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>87</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1722</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>2283</bold> (<bold>36.7</bold>)</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>\n<bold>6229</bold>\n</p>\n</td></tr></tbody></table></table-wrap><p>It was observed that distribution of the collected mosquitoes varied in the four study sites of northeast India. <italic>Ae. aegypti</italic> (63.3 %) was observed to be more abundant in comparison to <italic>Ae. albopictus</italic> (36.7 %). In Pasighat, Arunachal Pradesh, <italic>Ae. albopictus</italic> (94.1 %) was predominant as this small township contains and is surrounded by a greater density of natural vegetation as compared to the other three study sites. Again, in Dimapur, Nagaland, <italic>Ae. aegypti</italic> was predominating (97.7 %). Distribution of <italic>Ae. aegypti</italic> and <italic>Ae. albopictus</italic> in the four study areas is shown in <xref ref-type=\"fig\" rid=\"F1\">Fig. 1</xref>. Month-wise collection pattern of <italic>Aedes</italic> mosquito showed that maximum mosquitoes could be collected during pre-monsoon season i.e. April-June, 2018 (<xref ref-type=\"fig\" rid=\"F2\">Fig. 2</xref>).</p><fig fig-type=\"figure\" id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1.</label><caption><p>Graphical representation of distribution of <italic>Ae. aegypti</italic> and <italic>Ae. albopictus</italic> in the four study sites.</p></caption><graphic xlink:href=\"acmi-2-101-g001\"/></fig><fig fig-type=\"figure\" id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2.</label><caption><p>Graphical representation of month-wise <italic>Aedes</italic> collection.</p></caption><graphic xlink:href=\"acmi-2-101-g002\"/></fig></sec><sec id=\"s3-2\"><title>Molecular analysis</title><p>A total of 515 pools made from the collected mosquitoes were included in the final analysis. During this study DENV-1, 2 and 3 were detected from northeast India (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Mono-infection with DENV-1, 2 and 3 serotypes was detected in three pools, 46 pools and one pool respectively. DENV-1 and 2 co-infection was observed in three pools, while DENV-2 and 3 co-infection was observed in a single pool (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Unlike other states, only DENV-2 serotype was detected from Meghalaya.</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2.</label><caption><p>State wise distribution of DENV serotypes detected</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>Location where DENV positivity found</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>No. of mosquito pools screened for DENV</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>No. of mosquito pools positive for DENV</p>\n</th><th align=\"left\" valign=\"bottom\" rowspan=\"1\" colspan=\"1\">\n<p>Serotypes detected</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Dimapur, Nagaland</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>151</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>5</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>DENV-1 (1 pool), DENV-2 (4 pools)</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Guwahati, Assam</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>134</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>19</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>DENV-1 (1 pool), DENV-2 (16 pools), DENV-3 (1 pool), DENV-2 and 3 co-infection (1 pool)</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Pasighat, Arunachal Pradesh</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>168</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>29</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>DENV-1 (1 pool), DENV-2 (25 pools), DENV-1 and 2 co-infection (3 pools)</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Tura, Meghalaya</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>62</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>DENV-2</p>\n</td></tr></tbody></table></table-wrap></sec><sec id=\"s3-3\"><title>Estimation of Minimum Infection Rate (MIR)</title><p>DENV infection was detected in male as well as female mosquito pools. The calculated species and gender wise MIR of each serotype is tabulated below (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). In case of DENV-1, MIR values recorded ranged from 1.2 to 142.9 across the four sites with no significant difference (<italic>P</italic>=0.292) in the values between male and female <italic>Aedes</italic> mosquitoes. In case of DENV-2, MIR values ranging from 1.1 to 49.2 were observed; however, male and female <italic>Aedes</italic> populations in the study sites did not exhibit any significant difference in the MIR values (<italic>P</italic>=0.721). For DENV-3, the MIR of the single positive pool of <italic>Ae. aegypti</italic> (female) collected from Assam was 2.4. Positivity observed in male mosquitoes during the study is a direct indication of natural transovarial transmission (TOT) of the virus in this region.</p><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3.</label><caption><p>State wise minimum infection rate (MIR) of serotypes detected</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>State</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Mosquito species</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Gender</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Total no. of mosquitoes</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>No. of positive pools for DENV-1</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>MIR for DENV-1</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Two-tailed <italic>p</italic> value</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>No. of positive pools for DENV-2</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>MIR for DENV-2</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Two-tailed <italic>p</italic> value</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>No. of positive pools for DENV-3</p>\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>MIR for DENV-2</p>\n</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Nagaland</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"8\" colspan=\"1\">\n<p>\n<italic>Ae. aegypti</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Male</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>932</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"16\" colspan=\"1\">\n<p>0.292</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.2</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"16\" colspan=\"1\">\n<p>0.721</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Female</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>926</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1.1</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Assam</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Male</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>662</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>7</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>10.6</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Female</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>822</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1.2</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>6</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>7.3</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2.4</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Arunachal Pradesh</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Male</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>64</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>46.9</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Female</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>70</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>14.3</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>28.6</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Meghalaya</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Male</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>215</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Female</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>255</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3.9</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Nagaland</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"8\" colspan=\"1\">\n<p>\n<italic>Ae. albopictus</italic>\n</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Male</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>7</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>142.9</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Female</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>37</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Assam</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Male</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>52</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>19.2</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Female</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>61</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>49.2</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Arunachal Pradesh</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Male</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>411</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2.4</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>9</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>21.9</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Female</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1708</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>2</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>1.2</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>14</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>8.2</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"2\" colspan=\"1\">\n<p>Meghalaya</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Male</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>4</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>Female</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>3</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>0</p>\n</td><td align=\"char\" char=\".\" valign=\"top\" rowspan=\"1\" colspan=\"1\">\n<p>–</p>\n</td></tr></tbody></table><table-wrap-foot><fn id=\"tbl3fn1\"><p>MIR: Minimum infection rate.</p></fn></table-wrap-foot></table-wrap></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Dengue has been prevalent in northeast India for at least a decade. Increasing case load with newer geographic invasions every year along with simultaneous circulation of all the four serotypes increases the chances of occurrence of a severe outbreak in near future. <italic>Aedes</italic> vectors are highly prevalent in northeast India. <italic>Ae. aegypti</italic> has already been incriminated as a vector of DENV-1 and 2 from Assam [<xref rid=\"R10\" ref-type=\"bibr\">10, 11</xref>]. However, their prevalence and distribution from other northeastern states remains unknown. This is a maiden report which establishes <italic>Ae. albopictus</italic> and <italic>Ae. aegypti</italic> as DENV vectors in these four northeastern states.</p><p>DENV-1, 2 and 3 were detected from <italic>Aedes</italic> mosquito populations during the study. No mosquito pool was positive for DENV-4 in the current study. Surveillance in a greater number of mosquito pools over a wider geographical area is required to yield confirmation about the actual status of DENV-4 circulating in the area. Previous studies have also shown that DENV-2 was the predominant serotype in this region and DENV-4, the rarest [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]. In India as a whole, DENV-4 serotype is not very common and from northeast India, previous reports are confined to Arunachal Pradesh and Manipur [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]. Moreover, the serotype replacement phenomenon observed in relation to dengue virus might also be responsible for the absence of DENV-4 serotype in the current study [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]. In Meghalaya, only the DENV-2 serotype was observed, which is in concurrence with the DENV serotype recorded in human cases from the state previously [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>].</p><p>For DENV-1 and DENV-2, positivity in field collected male mosquitoes suggests natural TOT of <italic>Aedes</italic> mosquitoes of this region. TOT is common in case of <italic>Aedes</italic> and this observation is comparable to earlier studies from this region [<xref rid=\"R3\" ref-type=\"bibr\">3, 10</xref>]. Although no significant difference in the MIR values was observed between male and female <italic>Aedes</italic> mosquitoes, the mere detection of DENV in the male population could have an important epidemiological role, as the males have the potential to transmit the DENV infection to females through venereal transmission [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]. During unfavourable seasons, even low MIR might be adequate to maintain the virus in circulation [<xref rid=\"R20\" ref-type=\"bibr\">20</xref>].</p><p>The presence of multiple serotypes in mosquito pools collected from three of the four states studied implies that the possibility of secondary dengue infections occurring in human subjects residing in the area cannot be ignored. Previous Indian studies from different states have observed secondary infection rates varying from <10 % to as high as >75 % with an overall proportion of 42.9 % (95 % CI, 33.7–52.6) [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]. Considering that secondary infections by a different serotype of DENV can result in increased chances of development of severe forms of dengue such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), vector control measures become increasingly important to prevent human fatalities [<xref rid=\"R22\" ref-type=\"bibr\">22, 23</xref>].</p><p>DENV detection in <italic>Aedes</italic> mosquitoes is a routine surveillance practice in different countries including India [<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]. For effective epidemiological and clinical management of DENV cases, it is crucial to detect the virus in both field collected <italic>Aedes</italic> mosquitoes and in human samples in a particular area. As treatment for dengue is mainly symptomatic and no effective vaccine is available till date, the only way left to tackle dengue is by vector control and personal protection from mosquito bites. Health officials from a dengue endemic area should educate the community about different measures for mosquito control. Also, routine virological studies should be conducted to have an understanding of the circulating serotypes of DENV.</p></sec></body><back><ack id=\"ack1\"><title>Funding information</title><p>This study was funded by Indian Council of Medical Research (Ref. No. VIR/9/2017/ECD-1 dated 21.08.17).</p></ack><ack id=\"ack2\"><title>Acknowledgement</title><p>The authors acknowledge the help and cooperation of the respective district administrations where the study was carried out and the dedicated project staff involved in the study. The authors are also grateful to Mr Karuna Gogoi for his help in performing statistical analysis.</p></ack><ack id=\"ack3\"><title>Author contributions</title><p>N.P.K., S.J.P., P.D. and D.R.B. contributed in conceptualization of the study. S.C., S.J.P., P.D., D.R.B. and N.P.K. contributed equally in conducting the study, analysis and validation of the results, drafting and critically reviewing the manuscript.</p></ack><ack id=\"ack4\"><title>Conflicts of interest</title><p content-type=\"COI-statement\">The author declare no competing financial or non-financial interests.</p></ack><fn-group><fn id=\"FN1\"><p>Abbreviations: DENV, Dengue Virus; DHF, Dengue Hemorrhagic Fever; DSS, Dengue Shock Syndrome; MIR, Minimum Infection Rate; TOT, Trans-ovarial Transmission.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.3 20210610//EN\" \"JATS-archivearticle1-3-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" dtd-version=\"1.3\" article-type=\"research-article\"><?properties open_access?><?properties manuscript?><processing-meta base-tagset=\"archiving\" mathml-version=\"3.0\" table-model=\"xhtml\" tagset-family=\"jats\"><restricted-by>pmc</restricted-by></processing-meta><front><journal-meta><journal-id journal-id-type=\"nlm-journal-id\">8213295</journal-id><journal-id journal-id-type=\"pubmed-jr-id\">1156</journal-id><journal-id journal-id-type=\"nlm-ta\">Biomed Pharmacother</journal-id><journal-id journal-id-type=\"iso-abbrev\">Biomed Pharmacother</journal-id><journal-title-group><journal-title>Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie</journal-title></journal-title-group><issn pub-type=\"ppub\">0753-3322</issn><issn pub-type=\"epub\">1950-6007</issn></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32559853</article-id><article-id pub-id-type=\"pmc\">PMC7523634</article-id><article-id pub-id-type=\"doi\">10.1016/j.biopha.2020.110229</article-id><article-id pub-id-type=\"manuscript\">NIHMS1628938</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>DKK2 blockage-mediated immunotherapy enhances anti-angiogenic therapy of <italic>Kras</italic> mutated colorectal cancer</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Hu</surname><given-names>Jiajia</given-names></name><xref ref-type=\"aff\" rid=\"A1\">a</xref><xref ref-type=\"aff\" rid=\"A2\">b</xref><xref rid=\"FN1\" ref-type=\"author-notes\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Zhengting</given-names></name><xref ref-type=\"aff\" rid=\"A3\">c</xref><xref rid=\"FN1\" ref-type=\"author-notes\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Zhengxi</given-names></name><xref ref-type=\"aff\" rid=\"A2\">b</xref><xref ref-type=\"aff\" rid=\"A4\">d</xref><xref rid=\"FN1\" ref-type=\"author-notes\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Ao</given-names></name><xref ref-type=\"aff\" rid=\"A2\">b</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sun</surname><given-names>Jing</given-names></name><xref ref-type=\"aff\" rid=\"A5\">e</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zheng</surname><given-names>Minhua</given-names></name><xref ref-type=\"aff\" rid=\"A5\">e</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wu</surname><given-names>Jibo</given-names></name><xref ref-type=\"aff\" rid=\"A2\">b</xref><xref ref-type=\"aff\" rid=\"A6\">f</xref></contrib><contrib contrib-type=\"author\"><name><surname>Shen</surname><given-names>Tianli</given-names></name><xref ref-type=\"aff\" rid=\"A2\">b</xref><xref ref-type=\"aff\" rid=\"A7\">g</xref></contrib><contrib contrib-type=\"author\"><name><surname>Qiao</surname><given-names>Ju</given-names></name><xref ref-type=\"aff\" rid=\"A8\">h</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lin</surname><given-names>Li</given-names></name><xref ref-type=\"aff\" rid=\"A6\">f</xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Biao</given-names></name><xref ref-type=\"aff\" rid=\"A1\">a</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wu</surname><given-names>Dianqing</given-names></name><xref ref-type=\"aff\" rid=\"A2\">b</xref><xref rid=\"CR1\" ref-type=\"corresp\">*</xref></contrib><contrib contrib-type=\"author\"><name><surname>Xiao</surname><given-names>Qian</given-names></name><xref ref-type=\"aff\" rid=\"A2\">b</xref><xref rid=\"CR1\" ref-type=\"corresp\">*</xref></contrib></contrib-group><aff id=\"A1\"><label>a</label>Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China</aff><aff id=\"A2\"><label>b</label>Department of Pharmacology and Vascular Biology and Therapeutic Program, Yale School of Medicine, New Haven, CT, United States</aff><aff id=\"A3\"><label>c</label>Department of Gastroenterology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China</aff><aff id=\"A4\"><label>d</label>Department of Orthodontics, Shanghai Ninth People’s Hospital, School of Stomatology, Shanghai Key Laboratory of Stomatology, Shanghai Jiao Tong University, Shanghai, China</aff><aff id=\"A5\"><label>e</label>Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China</aff><aff id=\"A6\"><label>f</label>Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China</aff><aff id=\"A7\"><label>g</label>Department of General Surgery, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, China</aff><aff id=\"A8\"><label>h</label>Department of Mechanical and Industrial Engineering, Northeastern University, Boston, MA, United States</aff><author-notes><fn fn-type=\"equal\" id=\"FN1\"><label>1</label><p id=\"P1\">These authors contributed equally to this article.</p></fn><fn fn-type=\"con\" id=\"FN2\"><p id=\"P2\">Authors’contributions</p><p id=\"P3\">Q.X. was involved in the design of the study, conduction of experiments, acquisition of data, analysis of data, interpretation of data and writing of the manuscript. J.H. was involved in the conduction of experiments, acquisition of data, analysis of data, interpretation of data and writing of the manuscript. W.Z. and Z.C. were involved in providing patient samples, and acquisition of data. A.L., J.W., T.S., and J.Q were involved in the acquisition of data. J.S. and M.Z. were involved in providing patient samples. L.L was involved in setting up of study logistics. B.L. was involved in the recruitment of patients. D.W. was involved in the financing of the project and writing of the manuscript. All authors read and approved the final manuscript.</p></fn><corresp id=\"CR1\"><label>*</label>Corresponding authors. <email>dianqing.wu@yale.edu</email> (D. Wu), <email>qian.xiao@yale.edu</email> (Q. Xiao).</corresp></author-notes><pub-date pub-type=\"nihms-submitted\"><day>17</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>20</day><month>5</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>7</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>01</day><month>7</month><year>2021</year></pub-date><volume>127</volume><fpage>110229</fpage><lpage>110229</lpage><!--elocation-id from pubmed: 10.1016/j.biopha.2020.110229--><permissions><license><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\" specific-use=\"textmining\" content-type=\"ccbylicense\">https://creativecommons.org/licenses/by/4.0/</ali:license_ref><license-p>This is an open access article under the CC BY license (<ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\">https://creativecommons.org/licenses/BY/4.0/</ext-link>).</license-p></license></permissions><abstract id=\"ABS1\"><p id=\"P4\">There are limited options for targeted therapies for colorectal cancer (CRC). Anti-EGFR therapy is limited to CRC without KRAS mutations. Even worse, most of CRC are refractory to currently immune checkpoint blockade. DKK2, which is upregulated in CRC, was recently found to suppress host immune responses, and its blockage effectively impeded tumor progression in benign genetic CRC models in our previous study. Here, our recent study demonstrated that in human CRC tumor samples expressing high levels of DKK2, DKK2 blockade caused stronger activation of tumor infiltrating CD8<sup>+</sup> T cells in <italic>ex vivo</italic> culture. Intriguingly, we observed a correlation of high DKK2 expression with increased lymph node metastasis prevalence in these CRC patients as well. Furthermore, in a mouse genetic CRC model with mutations in APC and KRAS, which more closely mimics advanced human CRC, we confirmed the tumor inhibitory effect of DKK2 blockade, which significantly retarded tumor progression and extended survival, with increased immune effector cell activation and reduced angiogenesis. Based on this, we performed a combined administration of DKK2 blockade with sub-optimal anti-VEGFR treatment and observed a synergetic effect on suppressing tumor angiogenesis and progression, as well as extending survival, better than those of every single therapy. Thus, this study provides further evidence for the potential therapeutic application of DKK2 blockade in the clinical treatment of human CRC.</p></abstract><kwd-group><kwd>DKK2</kwd><kwd>KRAS</kwd><kwd>APC</kwd><kwd>Anti-VEGFR</kwd><kwd>Immune activation</kwd><kwd>Tumor microenvironment</kwd><kwd>Therapeutic approaches</kwd></kwd-group></article-meta></front><body><sec id=\"S1\"><label>1.</label><title>Introduction</title><p id=\"P5\">Colorectal cancer (CRC) is the third most common cancer in women and men, and is the third leading cause of cancer death in the United States, accounting for approximately 9% of all cancer deaths [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]. Treatment for colon cancer is based largely on the stages of cancer, and the major therapies include surgery resection, radiation, chemotherapies, and targeted therapies. The targeted therapies for CRC include Bevacizumab (Avastin), which targets VEGF and blood vessel formation, and Cetuximab (Erbitux), which targets epidermal growth factor receptor) [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]. However, these anti-EGFR drugs don’t work in CRC that has mutations (defects) in the KRAS, NRAS or BRAF gene [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>].</p><p id=\"P6\">Tumor growth is often restricted due to their metabolic demands for oxygen and nutrients, which have limited diffusion. For further growth, tumors require increased blood vessel formation, <italic>i.e</italic>., angiogenesis. Angiogenesis is not only necessary for tumors to grow, but also contributes to the spread of bloodborne metastases [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>–<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. Vascular endothelial growth factor (VEGF) is a family of soluble protein growth factors, which consist of key mediators of angiogenesis and lymphangiogenesis in the context of tumor biology [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>–<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]. VEGF not only drives angiogenesis but also serves as a survival factor for endothelial cells and promotes the abnormal phenotype of blood vessels in tumors [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]. Anti-VEGF agents have been used widely in the treatment of many solid cancers [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>,<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]. However, major adverse events have been reported with the systemic administration of anti-VEGF monoclonal antibodies, including hypertension, impaired wound healing, hemorrhage and thrombosis, cardiac impairment, stroke, gastrointestinal perforations, and kidney disease [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>,<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]. These side effects reduce patient compliance and limit the application of anti-VEGF, particularly at the most effective dosage, on tumor therapy [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]. In colorectal cancer patients, even with bevacizumab combination with chemotherapy regimens, the median progression-free survival of metastatic colorectal cancer patients remains less than one year [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]. Therefore, improvement of the response rate and survival with new targeted agents remains an area of active investigation.</p><p id=\"P7\">Recently, immunotherapy, particularly the immune checkpoint inhibitors, has provided a significant advance in cancer treatment. However, the currently approved checkpoint inhibitors including anti-PD1/PD-L1 and anti-CTLA4 are only efficacious for CRC with a high level of microsatellite instability (MSI-H), which are only accounted for less than 10 % CRC cases. Most CRC, which belongs to the microsatellite stable (MSI-S) group, are refractory to the treatment. We recently showed that DKK2, a canonical Wnt signaling pathway antagonist [<xref rid=\"R10\" ref-type=\"bibr\">10</xref>,<xref rid=\"R11\" ref-type=\"bibr\">11</xref>], which is upregulated in CRC, promoted tumor progression by suppressing the activation of immune effector cells [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]. We demonstrated that the DKK2 blockade with the anti-DKK2 antibody 5F8 was effective in suppressing tumor progression using a syngeneic CRC model grafted with mouse colon cancer cell MC38 and a genetic benign CRC model using the Apc<sup>Min/+</sup> mice [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>,<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]. The gradual accumulation of genetic alterations in driver genes, such as APC, KRAS, SMAD4, TP53, and <italic>PIK3A</italic>, underlie the development and malignant progression of human colorectal cancers [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>–<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]. In this study, we revealed that enhanced cytotoxic T cell response of anti-DKK2 antibody 5F8 treatment is dependent on DKK2 expression in human colorectal cancer samples. Intriguingly, we observed a correlation between high DKK2 expression and increased lymph node metastasis prevalence in these CRC patients as well. Furthermore, we tested the effect of the DKK2 blockade on a mouse genetic CRC model that more closely mimics advanced human CRC. We found that the anti-DKK2 antibody 5F8 inhibited tumor progression in an advanced colon cancer model by modifying tumor immune microenvironment. Besides the regulation of DKK2 on cytotoxic immune cells, we also observed that 5F8 antibody treatment impaired tumor vasculature at the late stages of tumor progression.</p><p id=\"P8\">A growing number of pre-clinical studies on combination therapies targeting both immune escape and tumor vascularization were proved to have promising effects [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>,<xref rid=\"R17\" ref-type=\"bibr\">17</xref>–<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]. Those preclinical studies could help to identify the most promising candidates for clinical trials and offer new insights into the biological mechanisms that underlie drug synergies. Thus, we also investigated the combination effect of DKK2 blockage on VEGF-A/VEGFR blockade-mediated suppression of tumor progression. Interestingly, we found a synergetic effect on suppressing tumor progression, as well as extending survival, by a combination administration of DKK2 blockade with a suboptimal dose of anti-VEGFR <italic>via</italic> activation of CD8<sup>+</sup> T/NK cells and impaired tumor angiogenesis.</p></sec><sec id=\"S2\"><label>2.</label><title>Materials and methods</title><sec id=\"S3\"><label>2.1.</label><title>Antibodies</title><p id=\"P9\">mouse CD4–PE (eBioscience, 12-0042-82), mouse NK1.1–allophycocyanin (BioLegend, 108,710), mouse CD8a–PE–cyanine 7 (eBioscience, 25-0081-82), mouse CD69–PE (Biolegend, 104508), human/mouse granzyme B–FITC (BioLegend, 515403), mouse CD314 (NKG2D)-PE-cyanine 7 (eBioscience, 25-5882-81), mouse IFN-γ–PE (eBioscience, 12-7311-81), mouse CD45-eFluor 450 (eBioscience, 48-0459-41), mouse CD107a-V450 (BD, 560648), mouse CD8a-allophycocyanin (eBioscience, 17-0081-81), mouse CD279 (PD-1)-PE (BioLegend, 135205), mouse CD19–PE–cyanine 7 (eBioscience, 25-0193-81), Ki67 (Abcam, ab15580), cleaved caspase-3 (Asp175) (CST, 9661S), CD31 (Abcam ab, 28364), FITC-labeled AffiniPure F(ab)2 fragment donkey anti-mouse IgG (H + L) (Jackson Lab, 715-096-151), and Alexa Fluor 647-labeled AffiniPure F(ab)2 fragment goat anti-rabbit IgG (H + L) (Jackson Lab, 111-606-045). Mouse monoclonal antibody (mAb) to DKK2 (5F8) was generated by standard hybridoma technology through immunization of mice with a synthetic peptide (KLNSIKSSLGGETPGC) of human DKK2 at AbMax (Beijing, China). Therapeutic antibodies to VEGFR were Clone DC101 (BioXcell, BE0060) with rat IgG1 isotype control (BioXcell, BE0091) as the control IgG.</p></sec><sec id=\"S4\"><label>2.2.</label><title>Human colon tumor sample study</title><p id=\"P10\">Sixteen patients with CRC, including 6 women and 10 men, were enrolled and pathologically diagnosed with colorectal adenocarcinoma at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from March 2018 to February 2019. Written informed consent was provided by all patients. This study protocol was following the approved guidelines and was approved by the Human Ethics Committee and the Research Ethics Committee of Ruijin Hospital. Fresh tumor and adjacent normal tissue samples (at least 2 cm from matched tumor tissues) were surgically resected from the above-described patients. Their ages ranged from 37 to 87 with a median of 63. None of them was treated with chemotherapy or radiation before tumor resection. The stages of these patients were classified according to the guidance of the AJCC version. Among these patients, one was diagnosed at stage I, seven at stage II, and eight at stage III. Among these patients, 8 had positive lymph nodes. None of those patients had distal metastasis, as evidenced by the enhanced computerized tomography (CT) results for abdomen, chest and pelvic areas before surgery. The available clinical characteristics are summarized in <xref rid=\"SD2\" ref-type=\"supplementary-material\">Supplementary Table 1</xref>.</p></sec><sec id=\"S5\"><label>2.3.</label><title>Ex vivo cell culture</title><p id=\"P11\">Fresh tumors and adjacent normal tissues were collected from surgical specimens after microscopical examination of the tissue by a pathologist. All tissues were washed by washing buffer (DMEM containing 10 % FBS and 65 mM DTT) in a shaker with the speed of 200 r/m at 37 °C for 15 min, and then were washed by cold 1*PBS thrice to remove DTT. Tissues were cut into small pieces (approximately 1 mm<sup>3</sup>) in the RPMI-1640 medium with 10 % fetal bovine serum, then were divided into 2 parts. One was incubated with anti-DKK2 antibody (40 μg/mL) and the other with control IgG, in the incubator at 37 °C for 18 h. After that, the tissue suspensions were enzymatically digested by Collagenase VIII at the concentration of 1 μg/mL in the incubator at 37 °C for one hour. The dissociated cells were subsequently passed through a 70-μm cell-strainer and centrifuged at 500<italic>g</italic> for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer and incubated on ice for 2 min to lyse red blood cells. After washing twice with cold 1*PBS, the cell pellets were re-suspended in sorting buffer (1*PBS supplemented with 2% FBS) and passed through a 40-μm cell-strainer. Lymphocytes were isolated by Ficoll density gradient technique according to standard protocol. Cells were re-suspended in sorting buffer for later FACS analysis.</p></sec><sec id=\"S6\"><label>2.4.</label><title>In situ hybridization</title><p id=\"P12\"><italic>In situ</italic> hybridization detection of DKK2 mRNA was carried out using the following reagents acquired from Advanced Cell Diagnostics, INC based on provided protocol: RNAscope® Target Retrieval Reagents (Cat#322000), RNAscope® Pretreat Reagents- H<sub>2</sub>0<sub>2</sub> and ProteasePlus (Cat# 322330), RNAscope® 2.5 HD Detection Reagent- RED (Cat#322360), RNAscope® Wash Buffer Reagents (Cat#310091), BioCare EcoMount (Cat#320409), ImmECatdge™Hydrophobic Barrier Pen (Cat#310018) and the mouse Dkk2probe (#404841).</p></sec><sec id=\"S7\"><label>2.5.</label><title>Mice</title><p id=\"P13\">Kras<sup>LSL-G12D</sup> (B6.129S4-Krastm4Tyj/J), and Apc<sup>fl/fl</sup> (C57BL/6-Apctm1Tyj/J) mice [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>,<xref rid=\"R22\" ref-type=\"bibr\">22</xref>] were acquired from the Jackson Laboratory. Wild-type C57BL/6 mice were purchased from Envigo (Harlan). Mice were housed in specific-pathogen-free conditions and cared for following US National Institutes of Health guidelines, and all procedures were approved by the Yale University Animal Care and Use Committee.</p></sec><sec id=\"S8\"><label>2.6.</label><title>Mouse model of colon cancer</title><p id=\"P14\">Kras<sup>LSL-G12D</sup> (B6.129S4-Krastm4Tyj/J), and Apc<sup>fl/fl</sup> (C57BL/6-Apctm1Tyj/J) mice were backcrossed to <italic>C57BL/6</italic> background 3 generations, then Kras<sup>LSL-G12D</sup> mice were crossed with Apc<sup>fl/fl</sup> mice. For adenoviral infection of the colonic epithelium [<xref rid=\"R23\" ref-type=\"bibr\">23</xref>], mice were fasted overnight and anesthetized using 2% isoflurane. The distal colon was washed with PBS <italic>via</italic> the I.V. catheter through the anus. Then, 100 μL trypsin was injected into the colon for 10–15 min. The lining of the distal colon was then mechanically abraded using a small caliber brush. After washing with PBS, 10<sup>9</sup> pfu of adenovirus in 100 μL PBS was injected into the colon for 30 min through the anus. Four weeks later, Kras<sup>LSL-G12D</sup>; Apc<sup>fl/fl</sup> mice were randomly divided into two groups (17 each) for intraperitoneal injection with either control IgG antibody (10 mg/kg) or anti-DKK2 antibody (10 mg/kg) every week for another 6 weeks. 6 pairs of mice were used for flow cytometry analysis, 6 pairs of mice were used for gross inspection and histopathological examination, and rest 5 pairs of mice were used for survival study. Mice were housed in specific-pathogen-free conditions and cared for following US National Institutes of Health guidelines, and all procedures were approved by the Yale University Animal Care and Use Committee.</p></sec><sec id=\"S9\"><label>2.7.</label><title>Tumor syngeneic graft</title><p id=\"P15\">MC38 cell line was purchased from Kerafast (Cat#ENH204-FP). MC38 colon carcinoma cells (0.5 × 10<sup>6</sup>) were mixed with BD Matrigel (Matrix Growth Factor Reduced) (BD, 354230) in 100 μL and injected subcutaneously (s.c.) into the right flanks of the backs of female C57BL/6 mice (8–10 weeks old). Tumor growth was measured with calipers, and size was expressed as one-half of the product of perpendicular length and square width in cubic millimeters. For antibody treatment, control IgG, anti-DKK2 (5F8) and anti-VEGFR antibodies were diluted in PBS, and 100 μL was injected intraperitoneally (i.p.) at the intervals indicated in the figures. For survival tests, mice were euthanized when the tumor size exceeded 1500 mm<sup>3</sup>.</p></sec><sec id=\"S10\"><label>2.8.</label><title>Histology</title><p id=\"P16\">Murine organs were fixed in phosphate-buffered 4% formaldehyde and embedded in paraffin. 5-μm thick sections were stained with hematoxylin and eosin (H&E). Immunohistochemistry on 5-μm paraffin sections using antibodies against Ki67 (Abcam, ab15580), cleaved caspase-3 (Asp175) (CST, 9661S), CD31 (Abcam ab, 28364), was performed as described [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>,<xref rid=\"R24\" ref-type=\"bibr\">24</xref>,<xref rid=\"R25\" ref-type=\"bibr\">25</xref>].</p></sec><sec id=\"S11\"><label>2.9.</label><title>Flow cytometry</title><p id=\"P17\">Cells in single-cell suspension were fixed with 2% PFA (Santa-Cruz sc-281692). After washing with a Flow Cytometry Staining Buffer (eBioscience 00-4222-26), cells were stained with antibodies for cell surface marker for 1 h on ice in the dark. For staining of intracellular proteins, the cells were washed and resuspended in the Permeabilization Buffer (BD 554723) and stained by antibodies in the Permeabilization Buffer for 1 h on ice in the dark. The cells were then pelleted and resuspended in the Flow Cytometry Staining Buffer for flow cytometry analysis [<xref rid=\"R26\" ref-type=\"bibr\">26</xref>,<xref rid=\"R27\" ref-type=\"bibr\">27</xref>].</p></sec><sec id=\"S12\"><label>2.10.</label><title>Statistical analysis and study design</title><p id=\"P18\">Minimal group sizes for tumor progression studies were determined <italic>via</italic> power calculations with the DSS Researcher’s Toolkit with an α of 0.05 and power of 0.8. Animals were grouped unblinded, but randomized, and investigators were blinded for the qualification experiments. No samples or animals were excluded from the analysis. Assumptions concerning the data including normal distribution and similar variation between experimental groups were examined for appropriateness before statistical tests were conducted. Comparisons between two groups were performed by unpaired, two-tailed t-tests. Comparisons between more than two groups were performed by oneway ANOVA, whereas comparisons with two or more independent variable factors were performed by two-way ANOVA followed by Bonferroni’s post hoc correction using Prism 6.0 software (GraphPad). Statistical tests were done with biological replicates. P < 0.05 was considered statistically significant.</p></sec></sec><sec id=\"S13\"><label>3.</label><title>Results</title><sec id=\"S14\"><label>3.1.</label><title>DKK2 blockade-induced enhancement of cytotoxic T cell responses are correlated with DKK2 expression in human colorectal cancer</title><p id=\"P19\">Our previous experiments demonstrated that DKK2 blockade with the anti-DKK2 antibody 5F8 was effective in suppressing tumor progression by enhancing immune effector CD8 + T cell activation, in a syngeneic CRC model grafted with mouse colon cancer cell MC38 and a genetic benign CRC model using the Apc<sup>Min/+</sup> mice [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]. To verify whether DKK2 blockade activates infiltrating CD8<sup>+</sup> T cells in human colorectal cancers, we collected 16 human colorectal tumor samples (<xref rid=\"SD2\" ref-type=\"supplementary-material\">Supplementary Table 1</xref>). One half of the surgically resected tumors were minced and cultured <italic>ex vivo</italic> in the presence of control IgG or 5F8 for 16 h, followed by an analysis of tumor infiltrating CD8<sup>+</sup> T by flow cytometry. The other half of the tumors were sectioned for histological examination (<xref rid=\"SD2\" ref-type=\"supplementary-material\">Supplementary Table 1</xref>) and DKK2 expression analysis <italic>via in situ</italic> hybridization (<xref rid=\"F1\" ref-type=\"fig\">Fig. 1A</xref>). Based on DKK2 mRNA levels in tumor sections, the samples were divided into the DKK2<sup>high</sup> and DKK2<sup>low</sup> groups (<xref rid=\"F1\" ref-type=\"fig\">Fig. 1A</xref>). Anti-DKK2 treatment induced significant increases in GZMB and CD69 over control IgG treatment in the DKK2<sup>high</sup>, but not DKK2<sup>low</sup> sample group (<xref rid=\"F1\" ref-type=\"fig\">Fig. 1D</xref>–<xref rid=\"F1\" ref-type=\"fig\">G</xref>), suggesting that DKK2 blockade activates infiltrating CD8<sup>+</sup> T cells in human colorectal cancers that express high levels of DKK2. Interestingly, the patients with metastases in juxtaintestinal lymph nodes have higher expression levels of DKK2 in the original tumors than those without lymph node metastases (<xref rid=\"F1\" ref-type=\"fig\">Fig. 1B</xref>), although there was no significant difference in the size of the original tumors between the DKK2<sup>high</sup> and DKK2<sup>low</sup> groups (<xref rid=\"F1\" ref-type=\"fig\">Fig. 1C</xref>). This result suggests that DKK2 may play a role in colorectal cancer metastasis.</p></sec><sec id=\"S15\"><label>3.2.</label><title>DKK2 blockade inhibits tumor progression in Kras<sup>G12D/+</sup>; Apc<sup>fl/fl</sup></title><p id=\"P20\"><italic>mouse</italic> To further evaluate the effect of the DKK2 blockade on a mouse model that more closely mimics advanced human CRC, we tested the anti-DKK2 antibody 5F8 in mice carrying the floxed Kras<sup>G12D/+</sup> and Apc<sup>fl/fl</sup> alleles. Upon administration of the Adeno-Cre virus to colons of these mice, tumors were formed at colons due to the lack of APC and expression of KRAS<sup>G12D</sup>, an activated KRAS mutant. Treatment of these mice with 5F8 significantly reduced tumor number and tumor size (<xref rid=\"F2\" ref-type=\"fig\">Fig. 2A</xref>–<xref rid=\"F2\" ref-type=\"fig\">C</xref>) and extended survival (<xref rid=\"F2\" ref-type=\"fig\">Fig. 2D</xref>) compared with mice treated with an isotype control antibody. Flow cytometry analysis of tumor infiltrated leukocytes revealed that the DKK2 blockade did not significantly affect the infiltration of CD45<sup>+</sup> (<xref rid=\"F3\" ref-type=\"fig\">Fig. 3A</xref>, <xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 1A</xref>), CD4<sup>+</sup> T cells, CD8<sup>+</sup> T or natural killing (NK) cells (<xref rid=\"F3\" ref-type=\"fig\">Fig. 3B</xref>) in the tumors. As same as in human colon cancer tissue, 5F8 treatment significantly increased the expression of Granzyme B (GZMB) in CD8<sup>+</sup> T cells, as well as in NK cells (<xref rid=\"F3\" ref-type=\"fig\">Fig. 3C</xref>). We also observed significant increases in levels of other activation markers of CD8<sup>+</sup> T cells and NK cells, including CD69, CD107a, CD314, and IFN-γ in CD8<sup>+</sup> T cells (<xref rid=\"F3\" ref-type=\"fig\">Fig. 3E</xref>) and CD107a, CD69, and CD314 in NK cells (<xref rid=\"F3\" ref-type=\"fig\">Fig. 3D</xref>). We did not observe any significant changes in levels of CTLA-4 or PD-1 in 5F8-treated CD8<sup>+</sup> T cells compared with those from isotype IgG treated mice (<xref rid=\"F3\" ref-type=\"fig\">Fig. 3E</xref>). These results suggest that the DKK2 blockade inhibits tumor progression in this advanced mouse CRC model accompanied by increased CD8<sup>+</sup> T cell and NK cell activation.</p></sec><sec id=\"S16\"><label>3.3.</label><title>DKK2 blockade inhibits tumor angiogenesis but increased immune effector cell activation</title><p id=\"P21\">The effects of DKK2 blockade on tumor proliferation, apoptosis, and neovascularization were assessed. Consistent with our previous study [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>], DKK2 blockade increased the number of apoptotic cells (<xref rid=\"F4\" ref-type=\"fig\">Fig. 4C</xref>) without significant effects on tumor cell proliferation (<xref rid=\"F4\" ref-type=\"fig\">Fig. 4D</xref>). Unexpectedly, we found that there is a significant reduction of tumor neovascularization in the anti-DKK2 antibody treatment group (<xref rid=\"F4\" ref-type=\"fig\">Fig. 4A</xref>–<xref rid=\"F4\" ref-type=\"fig\">B</xref>). DKK2 blockade significantly increased IFNγ expression in CD8<sup>+</sup> T (<xref rid=\"F3\" ref-type=\"fig\">Fig. 3E</xref>) and NK cells [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]. Studies have demonstrated that IFNγ not only impedes tumor growth by acting directly on cancer cells [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>,<xref rid=\"R29\" ref-type=\"bibr\">29</xref>], it also acts on the tumor stroma and tumor angiogenesis for effective rejection of large, established tumors [<xref rid=\"R30\" ref-type=\"bibr\">30</xref>–<xref rid=\"R33\" ref-type=\"bibr\">33</xref>]. The previous study also indicated that DKK2 directly promotes angiogenesis in rodent and human endothelial cells [<xref rid=\"R34\" ref-type=\"bibr\">34</xref>–<xref rid=\"R37\" ref-type=\"bibr\">37</xref>]. We were also able to reproduce the effects of DKK2 on angiogenesis using the tube formation assay <italic>in vitro</italic> and the Matrigel plug assay <italic>in vivo</italic>. This <italic>in vitro</italic> assay revealed that treatment of the cells with DKK2 protein profoundly enhanced tube formation in endothelial cells (<xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 2A</xref>–<xref rid=\"SD1\" ref-type=\"supplementary-material\">G</xref>). DKK2 also significantly increases angiogenesis compared with controls in the <italic>in vivo</italic> assay (<xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 3A</xref>–<xref rid=\"SD1\" ref-type=\"supplementary-material\">B</xref>). Taking these results together, DKK2 can promote angiogenesis, and anti-DKK2 antibody treatment may inhibit tumor angiogenesis directly and indirectly at the late stage of tumor progression.</p></sec><sec id=\"S17\"><label>3.4.</label><title>Synergetic effect of combination treatment of anti-DKK2 and anti-VEGFR on the syngeneic graft tumor model</title><p id=\"P22\">Given that anti-VEGF signaling is an approved therapy for human CRC, we tested if the combination therapy including VEGFR blockade and DKK2 blockade in the MC38 tumor syngeneic graft model could have a synergetic effect. Firstly, we applied previously reported an effective dosage of anti-VEGFR (10 mg/kg) [<xref rid=\"R38\" ref-type=\"bibr\">38</xref>–<xref rid=\"R40\" ref-type=\"bibr\">40</xref>] to the MC38 tumor syngeneic model in the presence or absence of 5F8 on Day 6 after tumor incubation. Both DKK2 blockade and VEGFR blockade showed significant tumor-suppressive effects, and the VEGFR blockade demonstrated a superior anti-tumor effect compared to the DKK2 blockade (<xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 4A</xref>–<xref rid=\"SD1\" ref-type=\"supplementary-material\">B</xref>). The combination treatment showed numeric, but not statistically significant, further retardation of tumor suppression compared to anti-VEGFR treatment alone (<xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 4A</xref>–<xref rid=\"SD1\" ref-type=\"supplementary-material\">B</xref>). Analysis of tumor sections revealed that the VEGFR blockade showed a strong inhibitory effect on neovascularization compared to the control IgG group or DKK2 blockade group. The combination treatment showed no further enhancement in the anti-angiogenesis effect (<xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 4C</xref>–<xref rid=\"SD1\" ref-type=\"supplementary-material\">D</xref>). From a clinical perspective, the purpose of combination therapy is to reduce the dose of a single therapeutic drug and thereby reduce the toxic side effects. We then tested a sub-optimal dose (2.5 mg/kg) of anti-VEGFR [<xref rid=\"R38\" ref-type=\"bibr\">38</xref>–<xref rid=\"R40\" ref-type=\"bibr\">40</xref>]. This dose of anti-VEGFR showed a smaller inhibitory effect on tumor progression (<xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 5A</xref>) than that of the full therapeutic dose but still had a tumor inhibitory effect that the tumor weight was lighter than that of the control IgG group at 19 days when tumors were collected for flow cytometry analyses (<xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 5B</xref>). However, this sub-optimal dose of anti-VEGFR did not provide significant long-term survival benefit over the control antibody (<xref rid=\"F5\" ref-type=\"fig\">Fig. 5A</xref> & <xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 5C</xref>–<xref rid=\"SD1\" ref-type=\"supplementary-material\">D</xref>). DKK2 blockage showed a stronger effect on tumor suppression than this sub-optimal dose of anti-VEFGR (<xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 5A</xref>–<xref rid=\"SD1\" ref-type=\"supplementary-material\">B</xref>) and extended survival (<xref rid=\"F5\" ref-type=\"fig\">Fig. 5A</xref> & <xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 5C</xref>–<xref rid=\"SD1\" ref-type=\"supplementary-material\">D</xref>). The combination yielded significant additional long-term survival benefit compared with individual blockade (<xref rid=\"F5\" ref-type=\"fig\">Fig. 5A</xref>, <xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 5C</xref>–<xref rid=\"SD1\" ref-type=\"supplementary-material\">D</xref>), even though the combination did not exert a significantly further effect on tumor weight compared with DKK2 blockade alone at Day 19 (<xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 5A</xref>–<xref rid=\"SD1\" ref-type=\"supplementary-material\">B</xref>). Analysis of tumor-infiltrating lymphocytes showed that DKK2 blockades led to increased levels of GZMB in tumor-infiltrating CD8<sup>+</sup> T cells and NK cells (<xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 5E</xref>–<xref rid=\"SD1\" ref-type=\"supplementary-material\">H</xref>). Importantly, the combination blockade resulted in the further increased level in IFNγ expression in these cells while DKK2 blockade alone increased the level of IFNγ in both CD8<sup>+</sup> T cells and NK cells (<xref rid=\"F5\" ref-type=\"fig\">Fig. 5B</xref>–<xref rid=\"F5\" ref-type=\"fig\">D</xref>, <xref rid=\"SD1\" ref-type=\"supplementary-material\">Supplementary Fig. 1B</xref>). This effect of the combination of IFNγ expression suggests that VEGF-A/VEGFR plays an important role in the development of an immunosuppressive microenvironment involved in CD8<sup>+</sup> T/NK cell dys-function. Although the sub-optimal dose of VEGFR blockade only showed a trend inhibitory effect on neovascularization and tumor proliferation, compared with the control IgG group (<xref rid=\"F5\" ref-type=\"fig\">Fig. 5E</xref>–<xref rid=\"F5\" ref-type=\"fig\">F</xref>), the combination treatment had a strong anti-angiogenesis effect (<xref rid=\"F5\" ref-type=\"fig\">Fig. 5E</xref>). Consistently, the number of Ki67 positive cells was also decreased in the combo treatment group, compared to the control IgG group or single antibody treatment group (<xref rid=\"F5\" ref-type=\"fig\">Fig. 5F</xref>). Further examination of cleaved caspase3 showed that the combo treatment group has a dramatic increased cleaved caspase3+ cells (<xref rid=\"F5\" ref-type=\"fig\">Fig. 5G</xref>). Given these results, the DKK2 blockade can be used with anti-VEGFR therapy and even suboptimal doses of anti-VEGFR would provide additional benefits to the immunomodulatory effects of DKK2 blockade in treating colon tumors.</p></sec></sec><sec id=\"S18\"><label>4.</label><title>Discussion</title><p id=\"P23\">In this study, we examined the effects of the DKK2 antibody on tumor progression and microenvironments in human CRC samples and mouse CRC models. We found that DKK2 blockade retarded tumor progression by increasing cytotoxic immune cell activation and suppressing tumor angiogenesis at the late tumor progression stage. We also found that the combination of anti-DKK2 can be used with anti-VEGFR in a mouse CRC model and notably DKK2-blockade can augment anti-tumor efficacy of sub-optimal dosage of anti-VEGFR by further reducing tumor angiogenesis and enhancing anti-tumor immunity.</p><p id=\"P24\">In our previous study, we uncovered the function of DKK2 in promoting tumor progression by suppressing NK cell and CD8<sup>+</sup> T cell activation in Apc<sup>Min/+</sup> mouse intestinal tumor model [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]. It was not determined if anti-DKK2 blockage could also gain the similar tumor inhibitory effect in advanced colon tumors. With this study, we found that there is an enhanced cytotoxic T cell response after anti-DKK2 antibody treatment, which is dependent on DKK2 expression <italic>via in vitro</italic> human colorectal cancer tissue culture system (<xref rid=\"F1\" ref-type=\"fig\">Fig. 1</xref>). Meanwhile, we showed that the DKK2 blockade has the same anti-tumor effects in the Kras<sup>G12D/+</sup>; Apc<sup>fl/fl</sup> mice (<xref rid=\"F2\" ref-type=\"fig\">Fig. 2</xref>). Interestingly, we observed that there was a correlation between lymph node metastasis and DKK2 expression in the original tumors (<xref rid=\"F1\" ref-type=\"fig\">Fig. 1B</xref>). These findings are consistent with previous studies which demonstrated that DKK2 positively regulates tumor cell metastasis [<xref rid=\"R34\" ref-type=\"bibr\">34</xref>–<xref rid=\"R36\" ref-type=\"bibr\">36</xref>,<xref rid=\"R41\" ref-type=\"bibr\">41</xref>].</p><p id=\"P25\">Tumors can grow only to a small size before their metabolic demands are restricted due to the diffusion limit of oxygen and nutrients [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]. To grow beyond this size, the tumor switches to an angiogenic phenotype and attracts blood vessels from the surrounding stroma [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R3\" ref-type=\"bibr\">3</xref>,<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]. This process is a prerequisite for the further outgrowth of the tumor. The previous study demonstrated that DKK2 has distinct functions of DKK1 [<xref rid=\"R34\" ref-type=\"bibr\">34</xref>]. DKK2 induction promoted angiogenesis in cultured human endothelial cells and in <italic>in vivo</italic> assays using mice, while DKK1 suppressed angiogenesis and was repressed upon induction of morphogenesis [<xref rid=\"R34\" ref-type=\"bibr\">34</xref>,<xref rid=\"R36\" ref-type=\"bibr\">36</xref>]. Those previous studies indicated that DKK2 directly promotes angiogenesis in rodent and human endothelial cells [<xref rid=\"R34\" ref-type=\"bibr\">34</xref>–<xref rid=\"R37\" ref-type=\"bibr\">37</xref>]. In this study, we also reproduced the pro-angiogenic effects of recombinant DKK2 proteins. On the other hand, studies also demonstrated that IFNγ not only impedes tumor growth by acting directly on cancer cells [<xref rid=\"R28\" ref-type=\"bibr\">28</xref>,<xref rid=\"R29\" ref-type=\"bibr\">29</xref>], it also acts on the tumor stroma and tumor angiogenesis for effective rejection of large, established tumors [<xref rid=\"R30\" ref-type=\"bibr\">30</xref>–<xref rid=\"R33\" ref-type=\"bibr\">33</xref>]. We have shown that DKK2 blockade significantly increased IFNγ expression in both CD8<sup>+</sup> T and NK cells [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]. This suggests that the DKK2 blockade can also inhibit angiogenesis indirectly through its regulation on IFNγ. Thus, the marked anti-angiogenic effects observed in 5F8-treated mouse CRC models may be due to both direct and indirect effects DKK2 blockade on angiogenesis. Given that the DKK2 blockade had insignificant effects on tumor angiogenesis at the early stages of tumor progression [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>], most of the anti-tumor effects of DKK2 blockade are likely the results of its effects on modulation of tumor immune microenvironments.</p><p id=\"P26\">VEGF signaling plays an important role in tumor angiogenesis and cancer growth, researchers have made numerous attempts to decrease its expression and prevent tumor growth [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>–<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]. Recent research has shown that blood endothelial cells forming the tumor vessels can actively suppress the recruitment, adhesion, and activity of T cells [<xref rid=\"R42\" ref-type=\"bibr\">42</xref>–<xref rid=\"R44\" ref-type=\"bibr\">44</xref>]. Likewise, during tumorigenesis, the lymphatic vasculature undergoes dramatic remodeling that facilitates the metastatic spreading of cancer cells and immunosuppression [<xref rid=\"R42\" ref-type=\"bibr\">42</xref>,<xref rid=\"R44\" ref-type=\"bibr\">44</xref>,<xref rid=\"R45\" ref-type=\"bibr\">45</xref>]. More and more emerging evidence is highlighting the major role played by tumor-associated blood or lymphatic vasculature in thwarting immunosurveillance mechanisms and antitumor immunity [<xref rid=\"R42\" ref-type=\"bibr\">42</xref>–<xref rid=\"R44\" ref-type=\"bibr\">44</xref>]. This indicates that targeting of tumor vasculature might improve the efficacy of cancer immunotherapy. Considering that anti-DKK2 enhanced activation of cytotoxic immune cells, and VEGF-A/VEGFR was involved in the inhibition of dendritic cell maturation, accumulation of MDSC, and induction of T<sub>reg</sub> cells, and CD8<sup>+</sup> T cell activation/exhaustion [<xref rid=\"R38\" ref-type=\"bibr\">38</xref>,<xref rid=\"R46\" ref-type=\"bibr\">46</xref>–<xref rid=\"R50\" ref-type=\"bibr\">50</xref>], whether combination treatment with immune modulators may provide a better solution for Anti-VEGF therapy? Our study demonstrated that VEGFR blockage further increased the expression level of IFNγ in both CD8<sup>+</sup> T cells and NK cells when it combined with immunomodulator anti-DKK2 antibody 5F8, compared to single antibody treatment. It is of note that the combination of anti-DKK2 antibody and VEGFR blockade induced a strong antitumor effect.</p><p id=\"P27\">The dose of anti-VEGF agents matters [<xref rid=\"R51\" ref-type=\"bibr\">51</xref>–<xref rid=\"R53\" ref-type=\"bibr\">53</xref>]. Preclinical data indicate that high doses of anti-VEGF agents lead to increased deposition of the extracellular matrix that, together with hypoxia, can promote the infiltration of immunosuppressive and/or pro-tumor immune cells, such as monocytic and granulocytic MDSCs [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>–<xref rid=\"R56\" ref-type=\"bibr\">56</xref>]. These results suggest that high-dose anti-VEGF therapy limits the benefits of chemotherapy in mouse models and patients with metastatic colorectal cancer (CRC) or advanced-stage NSCLC [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>,<xref rid=\"R57\" ref-type=\"bibr\">57</xref>,<xref rid=\"R58\" ref-type=\"bibr\">58</xref>]. By contrast, the use of lower doses of anti-angiogenic agents, for instance, as low as one-quarter of the doses that induce anti-vascular effects has the potential to induce prolonged vessel normalization [<xref rid=\"R59\" ref-type=\"bibr\">59</xref>,<xref rid=\"R60\" ref-type=\"bibr\">60</xref>]. The results of two retrospective studies have shown that low-dose bevacizumab (< 3.6 mg/kg per week) in combination with chemoradiotherapy confers a greater survival benefit than high-dose bevacizumab in patients with glioblastoma [<xref rid=\"R61\" ref-type=\"bibr\">61</xref>–<xref rid=\"R64\" ref-type=\"bibr\">64</xref>]. Our study also has proved that the use of lower sub-optimal doses of the anti-VEGFR antibody, combined with the anti-DKK2 antibody, could significantly modify the immune response environment.</p><p id=\"P28\">Our study provided a novel therapeutic approach combining VEGFR blockade and DKK2 blockade, which target the tumor vasculature and boosting immune cells’ killing capacity to help to overcome immunotherapy resistance. More work is needed to further elucidate the mechanism by which DKK2 regulates endothelial cells and its crosstalk with VEGF-A/VEGFR pathway during tumor progression. The potential combinatorial strategies using antiangiogenic and immunotherapy approaches also need to be further evaluated.</p></sec><sec id=\"S19\"><label>5.</label><title>Conclusions</title><p id=\"P29\">There are limited options for targeted therapies for colorectal cancer (CRC). Anti-EGFR therapy is limited to CRC without KRAS mutations. Even worse, most of CRC are refractory to currently immune checkpoint blockade. Our recent study demonstrated that in human CRC tumor samples expressing high levels of DKK2, DKK2 blockade caused stronger activation of tumor infiltrated CD8<sup>+</sup> T cells in <italic>ex vivo</italic> culture. In a mouse genetic CRC model with mutations in APC and KRAS, which more closely mimics advanced human CRC, we confirmed the tumor inhibitory effect of DKK2 blockade, which significantly retarded tumor progression and extended survival, with increased immune effector cell activation and reduced angiogenesis. Based on this, our study provided a novel therapeutic approach combining VEGFR blockade and DKK2 blockade, which target the tumor vasculature and boosting immune cells’ killing capacity to help to overcome immunotherapy resistance. Thus, this study provides further evidence for the potential therapeutic application of DKK2 blockade in the clinical treatment of human CRC.</p></sec><sec sec-type=\"supplementary-material\" id=\"SM1\"><title>Supplementary Material</title><supplementary-material content-type=\"local-data\" id=\"SD1\"><label>Supp1</label><media xlink:href=\"NIHMS1628938-supplement-Supp1.docx\" orientation=\"portrait\" id=\"d40e960\" position=\"anchor\"/></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SD2\"><label>supp2</label><media xlink:href=\"NIHMS1628938-supplement-supp2.xlsx\" orientation=\"portrait\" id=\"d40e963\" position=\"anchor\"/></supplementary-material></sec></body><back><ack id=\"S20\"><title>Acknowledgment</title><p id=\"P30\">We thank Michelle Orsulak for technical assistance.</p><p id=\"P31\">Funding</p><p id=\"P32\">This work was supported by the foundation from the CSC Scholarships (No. 201806235042) the foundation of talent plan A for Guangci Excellent Youth of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (No. GCQN-2017-A12); National Cancer Institute (CA214703); and Transcenta Inc.</p></ack><fn-group><fn id=\"FN3\"><p id=\"P33\">Ethics approval and consent to participate</p><p id=\"P34\">This study protocol was following the approved guidelines and was approved by the Human Ethics Committee and the Research Ethics Committee of Ruijin Hospital. Written informed consent was provided by all patients in this study. All procedures involving animals were approved by the Yale University Animal Care and Use Committee.</p></fn><fn fn-type=\"COI-statement\" id=\"FN5\"><p id=\"P35\">Declaration of Competing Interest</p><p id=\"P36\">Transcenta Inc, who receives a license for the anti-DKK2 antibody supports this work.</p></fn><fn id=\"FN7\"><p id=\"P37\">Appendix A. Supplementary data</p><p id=\"P38\"><xref rid=\"SD2\" ref-type=\"supplementary-material\">Supplementary material</xref> related to this article can be found, in the online version, at doi:<ext-link ext-link-type=\"uri\" xlink:href=\"https://doi.org/10.1016/j.biopha.2020.110229\">https://doi.org/10.1016/j.biopha.2020.110229</ext-link>.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>[1]</label><mixed-citation publication-type=\"journal\"><name><surname>Siegel</surname><given-names>RL</given-names></name>, <name><surname>Miller</surname><given-names>KD</given-names></name>, <name><surname>Jemal</surname><given-names>A</given-names></name>, <article-title>Cancer statistics, 2019</article-title>, <source>CA Cancer J. 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Oncol</source>\n<volume>33</volume> (<issue>10</issue>) (<year>2015</year>) <fpage>1197</fpage>–<lpage>1213</lpage>.<pub-id pub-id-type=\"pmid\">25713439</pub-id></mixed-citation></ref></ref-list></back><floats-group><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1.</label><caption><p id=\"P39\">Enhanced cytotoxic response induced by anti-DKK2 treatment is dependent on Dkk2 expression in human colorectal cancer. (A) DKK2 mRNA in human intestinal tumor sections was detected by <italic>in situ</italic> hybridization. Based on DKK2 mRNA expression, the patients were divided into 2 groups: DKK2<sup>high</sup> and DKK2<sup>low</sup>. Scale bars are 100 μm. (B) DKK2 expression in patients with or without juxtaintestinal lymph nodes metastasis. (C) Comparison of tumor size in DKK2<sup>high</sup> and DKK2<sup>low</sup> group. (D) Representative flow cytometry of granzyme B in Control IgG or anti-DKK2 (5F8) treatment of DKK2<sup>high</sup> and DKK2<sup>low</sup> group. (E) Fold change of granzyme B (anti-DKK2/Control IgG) in DKK2<sup>high</sup> and DKK2<sup>low</sup> group. (F) Representative flow cytometry of CD69 in Control IgG or anti-DKK2 (5F8) treatment of DKK2<sup>high</sup> and DKK2<sup>low</sup> group. (G) Fold change of CD69 (anti-DKK2/Control IgG) in DKK2<sup>high</sup> and DKK2<sup>low</sup> group (Two-sided Student’s t-test). (*P < 0.05; **P < 0.01; ***P < 0.001).</p></caption><graphic xlink:href=\"nihms-1628938-f0001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2.</label><caption><p id=\"P40\">Anti-DKK2 antibody inhibited tumor progression in Kras<sup>G12D/+</sup>; Apc<sup>fl/fl</sup> intestine tumor mouse model. (A–D) Kras<sup>G12D/+</sup>; Apc<sup>fl/fl</sup> mice (n = 20) were administrated with the adeno-cre virus through the anus at age 10 weeks. When mice were 12-week-old, mice were divided into 2 groups randomly. Then mice were treated with anti-DKK2 (5F8) or an isotype antibody (IgG) (10 mg/kg i.p. once per week). (A–C) 5 pairs of mice were sacrificed at 16-week-old, tumors in the colon were counted and measured under a stereomicroscope after staining with methylene blue (<italic>P</italic> = 0.01; two-tailed Student’s <italic>t</italic>-test; <italic>n</italic> = 5). (D) 5 pairs of mice were monitored, and their survival was recorded (<italic>P</italic> = 0.02; two-sided Mantel-Cox log-rank test; <italic>n</italic> = 5). Data are presented as mean ± s.e.m.</p></caption><graphic xlink:href=\"nihms-1628938-f0002\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Fig. 3.</label><caption><p id=\"P41\">Administration of anti-DKK2 antibody enhanced cytotoxicity of NK and CD8<sup>+</sup> T cells. (A–F) Flow cytometry analysis of tumor-in-filtrating lymphocytes in Kras<sup>G12D/+</sup>; Apc<sup>fl/fl</sup> mice. Leukocytes from tumors from Kras<sup>G12D/+</sup>; Apc<sup>fl/fl</sup> mice injected with of 5F8 or IgG (10 mg/Kg, i.p.) were prepared and analyzed by flow cytometry. (A) percentage of CD45 was shown. (B) CD4, CD8, NK were pre-gated from CD45<sup>+</sup> population. (C) Granzyme B expression was measured in both CD8 and NK cells. (D) additional markers of tumor infiltrated NK1.1<sup>+</sup> cells were described. (E) Flow cytometry analysis of additional markers of tumor infiltrated CD8<sup>+</sup> T cells. Data are presented as means ± sem (Two-sided Student’s t-test; n = 5) (*P < 0.05; **P < 0.01; ***P < 0.001).</p></caption><graphic xlink:href=\"nihms-1628938-f0003\"/></fig><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Fig. 4.</label><caption><p id=\"P42\">Anti-DKK2 treatment impaired tumor vasculature in Kras<sup>G12D/+</sup>; Apc<sup>fl/fl</sup> mice. (A) Histological sections of colon tumors collected from Kras<sup>G12D/+</sup>; Apc<sup>fl/fl</sup> mice injected with of 5F8 or IgG (10 mg/Kg, i.p. once per week) were stained with anti-CD31 antibody together with DAPI. Scale bars are 100 μm. Five independent sections per mouse were quantified from five mice per group. (B) Quantification of CD31 area (%) for each group. Data are presented as a means ± sem (Two-sided Student t-test). (*P < 0.05; **P < 0.01; ***P < 0.001). (C–D) Histological sections of colon tumors collected from Kras<sup>G12D/+</sup>; Apc<sup>fl/fl</sup> mice injected with of 5F8 or IgG (10 mg/Kg, i.p. once per week) were stained with anti-Cleaved caspase-3 or Ki67 antibody together with DAPI. Scale bars are 100 μm. Five independent sections per mouse were quantified from five mice per group. Quantification of the number of Cleaved caspase-3 positive or Ki67 positive for each group. Data are presented as a means ± sem (Two-sided Student t-test). (*P < 0.05; **P < 0.01; ***P < 0.001).</p></caption><graphic xlink:href=\"nihms-1628938-f0004\"/></fig><fig id=\"F5\" orientation=\"portrait\" position=\"float\"><label>Fig. 5.</label><caption><p id=\"P43\">Combination treatment of anti-VEGF and anti-DKK2 further enhanced the activation of cytotoxic immune cells. (A) Augmented anti-tumor effects of DKK2 and VEGFR blockade combination in the MC38 tumor model. C57BL/6 mice were inoculated s.c. with MC38 cells. Treatment of IgG (12.5 mg/kg), anti-DKK2 (10 mg/kg)+IgG (2.5 mg/kg), anti-VEGFR (2.5 mg/kg)+IgG (10 mg/kg), and anti-DKK2 (10 mg/kg)+anti-VEGFR (2.5 mg/kg) in 100 μL was done at every 4 days starting Day 12. Survival was evaluated by the two-sided Log-rank (Mantel-Cox) multiple comparison test with Bonferroni correction (P = 0.005, IgG <italic>vs</italic> combo; P = 0.011, IgG <italic>vs</italic> anti-DKK2, P = 0.10 IgG <italic>vs</italic> anti-VEGFR; P = 0.046, combo <italic>vs</italic> anti-DKK2; P = 0.023, combo <italic>vs</italic> anti-VEGFR). (B–D) Effects of the antibody treatments on cytotoxic immune cells. C57BL/6 mice were inoculated s.c. with the MC38 cells. Treatments of anti-DKK2 (10 mg/kg, i.p) and/or anti-VEGFR (2.5 mg/kg, i.p) were done at Days 12, 15 and 18. Tumors were collected for flow cytometry analysis on Day 19. Flow data are presented as means ± sem (two-way Anova). (*P < 0.05; **P < 0.01; ***P < 0.001). (E) Histological sections of tumors from Figure 6A were stained with anti-CD31 antibody together with DAPI. Scale bars are 100 μm. Five independent sections per mouse were quantified from five mice per group. Quantification of CD31 area (%) for each group. Data are presented as means ± sem (Sidak’s multiple comparisons test). (*P < 0.05; **P < 0.01; ***P < 0.001). (F–G) Histological sections of tumors from Figure 6A were stained with anti-Ki67 or anti-cleaved caspase3 antibody together with DAPI. Scale bars are 100 μm. Five independent sections per mouse were quantified from five mice per group. Quantification of Ki67 number for each group. Data are presented as means ± sem (Bonferroni’s multiple comparisons test). (*P < 0.05; **P < 0.01; ***P < 0.001).</p></caption><graphic xlink:href=\"nihms-1628938-f0005\"/></fig><boxed-text id=\"BX1\" position=\"float\" orientation=\"portrait\"><caption><title>Consent for publication</title></caption><p id=\"P44\">The consent forms from the individuals who participated in this study will be provided upon request.</p></boxed-text></floats-group></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">STAR Protoc</journal-id><journal-id journal-id-type=\"iso-abbrev\">STAR Protoc</journal-id><journal-title-group><journal-title>STAR Protocols</journal-title></journal-title-group><issn pub-type=\"epub\">2666-1667</issn><publisher><publisher-name>Elsevier</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33000001</article-id><article-id pub-id-type=\"pmc\">PMC7523635</article-id><article-id pub-id-type=\"pii\">S2666-1667(20)30079-4</article-id><article-id pub-id-type=\"doi\">10.1016/j.xpro.2020.100092</article-id><article-id pub-id-type=\"publisher-id\">100092</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Protocol</subject></subj-group></article-categories><title-group><article-title>AbSeq Protocol Using the Nano-Well Cartridge-Based Rhapsody Platform to Generate Protein and Transcript Expression Data on the Single-Cell Level</article-title></title-group><contrib-group><contrib contrib-type=\"author\" id=\"au1\"><name><surname>Erickson</surname><given-names>Jami R.</given-names></name><email>jrericks@fredhutch.org</email><xref rid=\"aff1\" ref-type=\"aff\">1</xref><xref rid=\"cor1\" ref-type=\"corresp\">∗</xref></contrib><contrib contrib-type=\"author\" id=\"au2\"><name><surname>Mair</surname><given-names>Florian</given-names></name><email>fmair@fredhutch.org</email><xref rid=\"aff1\" ref-type=\"aff\">1</xref><xref rid=\"cor2\" ref-type=\"corresp\">∗∗</xref></contrib><contrib contrib-type=\"author\" id=\"au3\"><name><surname>Bugos</surname><given-names>Grace</given-names></name><xref rid=\"aff1\" ref-type=\"aff\">1</xref><xref rid=\"aff3\" ref-type=\"aff\">3</xref></contrib><contrib contrib-type=\"author\" id=\"au4\"><name><surname>Martin</surname><given-names>Jody</given-names></name><email>jody_martin@bd.com</email><xref rid=\"aff2\" ref-type=\"aff\">2</xref><xref rid=\"fn1\" ref-type=\"fn\">4</xref><xref rid=\"cor3\" ref-type=\"corresp\">∗∗∗</xref></contrib><contrib contrib-type=\"author\" id=\"au5\"><name><surname>Tyznik</surname><given-names>Aaron J.</given-names></name><xref rid=\"aff2\" ref-type=\"aff\">2</xref></contrib><contrib contrib-type=\"author\" id=\"au6\"><name><surname>Nakamoto</surname><given-names>Margaret</given-names></name><xref rid=\"aff2\" ref-type=\"aff\">2</xref></contrib><contrib contrib-type=\"author\" id=\"au7\"><name><surname>Mortimer</surname><given-names>Stefanie</given-names></name><xref rid=\"aff2\" ref-type=\"aff\">2</xref></contrib><contrib contrib-type=\"author\" id=\"au8\"><name><surname>Prlic</surname><given-names>Martin</given-names></name><email>mprlic@fredhutch.org</email><xref rid=\"aff1\" ref-type=\"aff\">1</xref><xref rid=\"aff3\" ref-type=\"aff\">3</xref><xref rid=\"fn2\" ref-type=\"fn\">5</xref><xref rid=\"cor4\" ref-type=\"corresp\">∗∗∗∗</xref></contrib><aff id=\"aff1\"><label>1</label>Fred Hutchinson Cancer Research Center, Vaccine and Infectious Disease Division, Seattle, WA 98109, USA</aff><aff id=\"aff2\"><label>2</label>BD Biosciences, La Jolla, CA 92037, USA</aff><aff id=\"aff3\"><label>3</label>Department of Immunology, University of Washington, Seattle, WA 98195, USA</aff></contrib-group><author-notes><corresp id=\"cor1\"><label>∗</label>Corresponding author <email>jrericks@fredhutch.org</email></corresp><corresp id=\"cor2\"><label>∗∗</label>Corresponding author <email>fmair@fredhutch.org</email></corresp><corresp id=\"cor3\"><label>∗∗∗</label>Corresponding author <email>jody_martin@bd.com</email></corresp><corresp id=\"cor4\"><label>∗∗∗∗</label>Corresponding author <email>mprlic@fredhutch.org</email></corresp><fn id=\"fn1\"><label>4</label><p id=\"ntpara0010\">Technical Contact</p></fn><fn id=\"fn2\"><label>5</label><p id=\"ntpara0015\">Lead Contact</p></fn></author-notes><pub-date pub-type=\"pmc-release\"><day>25</day><month>8</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on <pub-date\n\t\t\t\t\t\tpub-type=\"epub\">.--><pub-date pub-type=\"collection\"><day>18</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>8</month><year>2020</year></pub-date><volume>1</volume><issue>2</issue><elocation-id>100092</elocation-id><permissions><copyright-statement>© 2020 The Author(s)</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"CC BY\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</license-p></license></permissions><abstract id=\"abs0010\"><title>Summary</title><p>By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 10<sup>4</sup> cells using the nano-well based Rhapsody platform. We also include a workflow for sample multiplexing, which uniquely identifies the initial source of cells (such as tissue type or donor) in the downstream analysis after upstream pooling.</p><p>For complete information on the use and execution of this protocol, please refer to <xref rid=\"bib7\" ref-type=\"bibr\">Mair et al. (2020)</xref>.</p></abstract><abstract abstract-type=\"graphical\" id=\"abs0015\"><title>Graphical Abstract</title><fig id=\"undfig1\" position=\"anchor\"><graphic xlink:href=\"fx1\"/></fig></abstract><abstract abstract-type=\"author-highlights\" id=\"abs0020\"><title>Highlights</title><p><list list-type=\"simple\" id=\"ulist0010\"><list-item id=\"u0010\"><label>•</label><p id=\"p0010\">Step-by-step protocol for obtaining combined transcript and protein single-cell data</p></list-item><list-item id=\"u0015\"><label>•</label><p id=\"p0015\">Protocol includes considerations for data analysis</p></list-item><list-item id=\"u0020\"><label>•</label><p id=\"p0020\">Sample multiplexing strategy to reduce batch effects is described</p></list-item><list-item id=\"u0025\"><label>•</label><p id=\"p0025\">Discussion of enrichment strategies for rare cell types or human tissues</p></list-item></list></p></abstract><abstract abstract-type=\"teaser\" id=\"abs0025\"><p>By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 10<sup>4</sup> cells using the nano-well based Rhapsody platform. We also include a workflow for sample multiplexing, which uniquely identifies the initial source of cells (such as tissue type or donor) in the downstream analysis after upstream pooling.</p></abstract></article-meta></front><body><sec id=\"sec1\"><title>Before You Begin</title><p id=\"p0030\">This protocol should be read in full prior to starting an experiment. While this protocol may take over 12 h to complete, there are several stopping points that allow experimentation to be segmented over several days if needed. New versions of products discussed within this protocol are continuing to emerge. Ensure you are taking current recommendations on best practices from the product manufacturer.</p><p id=\"p0035\">In addition to standard lab equipment (including a PCR machine, consumables, etc.), access to a Rhapsody Express instrument is needed, as well as Rhapsody-specific reagents including the nano-well cartridges, oligonucleotide-labeled antibodies, library preparation reagents, and a panel of primers targeting the genes of interest. If samples will be multiplexed (optional and described in the protocol), then Sample Tag antibodies are needed as well. A full list of reagents is provided in the <xref rid=\"sec8\" ref-type=\"sec\">Key Resources Table</xref>. Furthermore, for quantification and quality control of intermediate and final PCR products a Qubit and TapeStation instrument is required.</p><p id=\"p0040\">As outlined in the protocol, it is critical at several steps to use laboratory practices, reagents, and workspaces that are suitable for working with RNA to avoid contamination with RNAse and subsequent mRNA degradation. Finally, it is essential to carefully consider the number of cells that will be analyzed and, if required, include a cell subset enrichment approach (such as FACS-based purification of cells) prior to starting the workflow. These enrichment techniques can also provide cost savings as AbSeq and mRNA sequencing reads can be limited to the cells of interest. Of note, there are multiple approaches to ensure antibodies used for enrichment do not interfere with oligo-conjugated AbSeq reagents, including choices of non-competing clones. These considerations and others are discussed in more detail in the section “<xref rid=\"sec5\" ref-type=\"sec\">Limitations</xref>”. For additional background on the technology underlying surface protein detection using oligo-nucleotide-labeled antibodies, we refer the reader to the following publications (<xref rid=\"bib8\" ref-type=\"bibr\">Peterson et al., 2017</xref>; <xref rid=\"bib10\" ref-type=\"bibr\">Stoeckius et al., 2017</xref>).</p><sec id=\"sec1.1\"><title>Defrosting Cryopreserved Peripheral Blood Mononuclear Cells (PBMCs)</title><p id=\"p0045\"><disp-quote><p><inline-graphic xlink:href=\"fx2.gif\"/><bold>Timing: 30 min</bold></p></disp-quote><list list-type=\"simple\" id=\"olist0010\"><list-item id=\"o0010\"><label>1.</label><p id=\"p0050\">Prepare media for defrosting cells</p></list-item><list-item id=\"o0015\"><label>a.</label><p id=\"p0055\">Warm the following reagents in a 37°C water bath:</p></list-item><list-item id=\"o0020\"><label>i.</label><p id=\"p0060\">RPMI 1640 (Thermo Cat # 11875119)</p></list-item><list-item id=\"o0025\"><label>ii.</label><p id=\"p0065\">Fetal Bovine Serum (FBS)</p></list-item><list-item id=\"o0030\"><label>iii.</label><p id=\"p0070\">L-Glutamine</p></list-item><list-item id=\"o0035\"><label>iv.</label><p id=\"p0075\">Penicillin-Streptomycin</p></list-item><list-item id=\"o0040\"><label>b.</label><p id=\"p0080\">Make complete media by adding 10% FBS, 1% L-Glutamine, and 1% penicillin-streptomycin to RMPI 1640.</p></list-item><list-item id=\"o0045\"><label>2.</label><p id=\"p0085\">Obtain PBMC vials for the desired number of samples from liquid nitrogen.</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> The sample multiplexing capability is currently limited to a maximum of 12 different samples.</p></disp-quote><list list-type=\"simple\" id=\"olist0015\"><list-item id=\"o0050\"><label>3.</label><p id=\"p0090\">Immediately place the PBMC vials into a 37°C water bath.</p></list-item><list-item id=\"o0055\"><label>4.</label><p id=\"p0095\">For each vial, remove the PBMCs from the water bath when a small ice pellet remains.</p></list-item><list-item id=\"o0060\"><label>5.</label><p id=\"p0100\">Slowly add 1 mL of warm complete media to the PBMCs in the cryovial in a dropwise manner.</p></list-item><list-item id=\"o0065\"><label>6.</label><p id=\"p0105\">Transfer the contents of the vial dropwise to a 15 mL conical tube with 10 mL of pre-warmed complete media.</p></list-item><list-item id=\"o0070\"><label>7.</label><p id=\"p0110\">Using 1 mL of warmed complete media, rinse the vial to ensure collection of all cells and add to the 15 mL conical tube.</p></list-item><list-item id=\"o0075\"><label>8.</label><p id=\"p0115\">Centrifuge cells at 250 × <italic>g</italic> for 5 min.</p></list-item><list-item id=\"o0080\"><label>9.</label><p id=\"p0120\">Decant the supernatant.</p></list-item><list-item id=\"o0085\"><label>10.</label><p id=\"p0125\">Resuspend pellet for each sample in 5 mL of warm complete media.</p></list-item><list-item id=\"o0090\"><label>11.</label><p id=\"p0130\">Count cells using Trypan blue to ascertain cell viability.</p></list-item><list-item id=\"o0095\"><label>12.</label><p id=\"p0135\">Take an aliquot containing the desired number of cells for analysis from each sample (it is recommended to start with at least three times the cell number that should be later loaded on the cartridge) and transfer them to new 1.5 mL LoBind tubes.</p></list-item><list-item id=\"o0100\"><label>13.</label><p id=\"p0140\">Centrifuge the tubes at 400 × <italic>g</italic> for 5 min and remove the supernatant.</p></list-item><list-item id=\"o0105\"><label>14.</label><p id=\"p0145\">Resuspend the cell pellets in 180 μL of Sample Buffer.</p></list-item></list><disp-quote><p><bold><italic>Optional:</italic></bold> If enrichment of certain cell populations is desired, use a cell sorter following standard practices (<xref rid=\"bib5\" ref-type=\"bibr\">Cossarizza et al., 2019</xref>) and for each target population collect at least three times the cell number that should be later loaded on the cartridge. Keep sorted cells on ice, wash them after sorting and then resuspend them in 180 μL of cold Sample Buffer.</p></disp-quote><disp-quote><p><bold><italic>Note:</italic></bold> all subsequent protocol steps can also be used with immune cells isolated from solid tissue samples after sort enrichment, or from fresh peripheral blood samples.</p></disp-quote></p></sec></sec><sec id=\"sec8\"><title>Key Resources Table</title><p id=\"p1135\"><table-wrap position=\"float\" id=\"undtbl1\"><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan=\"3\"><bold>Antibodies</bold></td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td>BD AbSeq antibody-oligos</td><td>BD Biosciences</td><td>various</td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td colspan=\"3\"><bold>Biological Samples</bold></td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td>Cryopreserved peripheral blood mononuclear cells</td><td>HIV Vaccine Trials Network, Fred Hutchinson Cancer Research Center</td><td>N/A</td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td colspan=\"3\"><bold>Chemicals, Peptides, and Recombinant Proteins</bold></td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td>Ethyl alcohol, Pure (200 proof, molecular biology grade)</td><td>Sigma-Aldrich</td><td>E7023-500ML</td></tr><tr><td>RPMI 1640</td><td>Major Supplier</td><td>n/a</td></tr><tr><td>Fetal Bovine Serum</td><td>Major Supplier</td><td>n/a</td></tr><tr><td>L-Glutamine</td><td>Major Supplier</td><td>n/a</td></tr><tr><td>Penicillin-Streptomycin</td><td>Major Supplier</td><td>n/a</td></tr><tr><td>Cell Staining Buffer</td><td>Major Supplier</td><td>n/a</td></tr><tr><td>Trypan blue Solution, 0.4%</td><td>ThermoFisher Scientific</td><td>15250061</td></tr><tr><td>70% ethyl alcohol or 70% isopropyl alcohol</td><td>Major Supplier</td><td>n/a</td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td colspan=\"3\"><bold>Critical Commercial Assays</bold></td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td>BD Rhapsody Cartridge Reagent Kit</td><td>BD Biosciences</td><td>633731</td></tr><tr><td>BD Rhapsody Cartridge Kit</td><td>BD Biosciences</td><td>633733</td></tr><tr><td>BD Single-Cell Multiplexing Kit – Human Sample Tag 12</td><td>BD Biosciences</td><td>633781</td></tr><tr><td>BD Rhapsody cDNA Kit</td><td>BD Biosciences</td><td>633732</td></tr><tr><td>BD Rhapsody Targeted Amplification Kit</td><td>BD Biosciences</td><td>633734</td></tr><tr><td>BD Rhapsody Targeted Primer Panel</td><td>BD Biosciences</td><td>various</td></tr><tr><td>BD Rhapsody Supplemental Panel</td><td>BD Biosciences</td><td>633742</td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher Scientific</td><td>Q32851</td></tr><tr><td>SPRIselect Reagent</td><td>Beckman Coulter</td><td>B23318</td></tr><tr><td>High Sensitivity D5000 ScreenTape</td><td>Agilent</td><td>5067-5592</td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td colspan=\"3\"><bold>Software and Algorithms</bold></td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td>BD Rhapsody Targeted Analysis Pipeline</td><td>SevenBridges</td><td>jiewho/bd-public-project/bd-rhapsody-analysis-pipeline</td></tr><tr><td>R</td><td>The R Project for Statistical Computing</td><td>v3.6.3</td></tr><tr><td>RStudio</td><td>RStudio</td><td>v1.2.5042</td></tr><tr><td>Seurat</td><td>Satija Lab, NYU Genome Center</td><td><ext-link ext-link-type=\"uri\" xlink:href=\"https://satijalab.org/seurat/\" id=\"intref0040\">https://satijalab.org/seurat/</ext-link></td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td colspan=\"3\"><bold>Other</bold></td></tr><tr><td colspan=\"3\"><hr/></td></tr><tr><td>BD Rhapsody Express Single-Cell System</td><td>BD Biosciences</td><td>633707</td></tr><tr><td>6-Tube Magnetic Separation Rack for 1.5 mL tubes</td><td>New England Biolabs</td><td>S1506S</td></tr><tr><td>Large magnetic separation stand</td><td>V&P Scientific, Inc.</td><td>VP 772FB-1</td></tr><tr><td>Clear acrylic cylinder adapter for 15 mL tube magnet</td><td>V&P Scientific, Inc.</td><td>VP 772FB-1A</td></tr><tr><td>Low-profile magnetic separation stand for 0.2 mL, 8-strip tubes</td><td>Permagen Labware</td><td>MSB08</td></tr><tr><td>Themomixer (16°C–37°C, 1,200 rpm)</td><td>Eppendorf</td><td>5382000023 & 5360000038</td></tr><tr><td>Qubit Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q32866</td></tr><tr><td>Heat block capable of 80°C</td><td>VWR</td><td>10153-348</td></tr><tr><td>4200 TapeStation</td><td>Agilient Technologies</td><td>G2991AA</td></tr><tr><td>Thermal cycler with heated lid</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Water bath</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Laminar flow hood</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Digital timer</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Pipettes (P2, P10, P20, P200, P1000)</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Multi-channel pipette, 2–20 μL</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Microcentrifuge for 1.5–2.0 mL tubes</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Microcentrifuge for 0.2 mL tubes</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Centrifuge and rotor for 15 mL tubes</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Vortexer</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Pipet-Aid</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Falcon Tube with Cell Strainer Cap</td><td>Thermo Fisher Scientific</td><td>352235</td></tr><tr><td>Improved Neubauer Hemocytometer</td><td>INCYTO</td><td>DHC-N01-5</td></tr><tr><td>DNA LoBind Tubes, 1.5 mL</td><td>Eppendorf</td><td>0030.08051</td></tr><tr><td>DNA LoBind Tubes, 5 mL</td><td>Eppendorf</td><td>0030108310</td></tr><tr><td>Low retention filtered pipette tips, 10 μL</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Low retention filtered pipette tips, 200 μL</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Low retention filtered pipette tips, 1,000 μL</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Gilson PIPETMAN Tipack Filtered Tips, 100–1,200 μL</td><td>Thermo Fisher Scientific</td><td>F171803G</td></tr><tr><td>Gilson PIPETMAN Tipack Filtered Tips, 500–5,000 μL</td><td>Thermo Fisher Scientific</td><td>F161370G</td></tr><tr><td>Qubit Asssay Tubes</td><td>Thermo Fisher Scientific</td><td>Q32856</td></tr><tr><td>0.2 mL PCR 12-strip tubes</td><td>Major supplier</td><td>n/a</td></tr><tr><td>10 mL sterile serological pipettes</td><td>Major supplier</td><td>n/a</td></tr><tr><td>Premoistened cleaning wipes with 70% ethyl alcohol or 70% isopropyl alcohol</td><td>Major supplier</td><td>n/a</td></tr></tbody></table></table-wrap><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> BD Stain Buffer contains Sodium Azide. Contact with acidic solutions and metal compounds over time may form potentially explosive metal azides. Should any of this material be introduced into a sanitary sewer system, flush with copious amounts of water.</p></disp-quote></p></sec><sec id=\"sec2\"><title>Materials and Equipment</title><p id=\"p0150\"><disp-quote><p><bold><italic>Alternatives:</italic></bold> Agencourt AMPure XP magnetic beads (Beckman Coulter, Cat. No. A63880) can be used instead of SPRISelect Reagent. Instead of the BD Rhapsody Express, the Rhapsody Single-Cell Analysis System (BD Cat. No. 633701) can be used, which provides additional QC steps by imaging cells in the wells of the cartridge. Furthermore, the 2100 Bioanalyzer (Agilient Technologies Cat. No. G2940CA) can be used instead of the 4200 TapeStation. If a Bioanalyzer will be used, High Sensitivity Kit for Bioanalyzer (Aligent 5067-4626) can be used in place of High Sensitivity D5000 ScreenTape. Instead of using the package Seurat (<xref rid=\"bib4\" ref-type=\"bibr\">Butler et al., 2018</xref>) in R and RStudio, SeqGeq (BD Biosciences) can be used to analyze data. A comprehensive review of analysis packages for single-cell analysis pipelines is provided on <ext-link ext-link-type=\"uri\" xlink:href=\"https://osca.bioconductor.org/\" id=\"intref0010\">https://osca.bioconductor.org/</ext-link> (<xref rid=\"bib1\" ref-type=\"bibr\">Amezquita et al., 2020</xref>).</p></disp-quote></p></sec><sec id=\"sec3\"><title>Step-By-Step Method Details</title><sec id=\"sec3.1\"><title>Preparing the Cartridge</title><p id=\"p0155\"><disp-quote><p><inline-graphic xlink:href=\"fx2.gif\"/><bold>Timing: 30 min</bold></p></disp-quote></p><p id=\"p0160\">The cartridge is essential for partitioning of single cells. There are two key steps to preparing the cartridge before addition of cells: priming the cartridge and treating the surface. It is possible to run two cartridges in parallel using a single Rhapsody Express instrument.<list list-type=\"simple\" id=\"olist0020\"><list-item id=\"o0110\"><label>1.</label><p id=\"p0165\">From the Rhapsody cDNA kit (Cat. No. 633773), thaw the following reagents at room temperature (15°C–25°C), then place on ice:</p></list-item><list-item id=\"o0115\"><label>a.</label><p id=\"p0170\">Nuclease-free water</p></list-item><list-item id=\"o0120\"><label>b.</label><p id=\"p0175\">RT Buffer</p></list-item><list-item id=\"o0125\"><label>c.</label><p id=\"p0180\">RT 0.1 M DTT</p></list-item><list-item id=\"o0130\"><label>d.</label><p id=\"p0185\">dNTP</p></list-item><list-item id=\"o0135\"><label>e.</label><p id=\"p0190\">RNase Inhibitor</p></list-item><list-item id=\"o0140\"><label>f.</label><p id=\"p0195\">Bead RT/PCR Enhancer</p></list-item><list-item id=\"o0145\"><label>g.</label><p id=\"p0200\">10× Exonuclease I buffer</p></list-item><list-item id=\"o0150\"><label>h.</label><p id=\"p0205\">Bead resuspension buffer</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> Only remove enzymes from −20°C when in use.</p></disp-quote></p><p id=\"p0210\">Place on ice:<list list-type=\"simple\" id=\"olist0025\"><list-item id=\"o0155\"><label>i.</label><p id=\"p0215\">Sample Buffer</p></list-item><list-item id=\"o0160\"><label>j.</label><p id=\"p0220\">1 M DTT</p></list-item><list-item id=\"o0165\"><label>k.</label><p id=\"p0225\">Lysis Buffer</p></list-item><list-item id=\"o0170\"><label>l.</label><p id=\"p0230\">Cell Capture Beads</p></list-item><list-item id=\"o0175\"><label>m.</label><p id=\"p0235\">Bead Wash Buffer</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> Ensure that the Eppendorf SmartBlock Plate is installed on the thermomixer and is set to 21°C.</p></disp-quote><list list-type=\"simple\" id=\"olist0030\"><list-item id=\"o0180\"><label>2.</label><p id=\"p0240\">Set a heatblock or additional thermomixer with the Eppendorf SmartBlock 1.5 mL to 80°C.</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> All steps performed on the cartridge will only use electronic Gilson pipettes. Regular pipettes cannot be used for these steps, as the rate of flow into the cartridge is carefully set and controlled by the Gilson pipettes to maximize cell and bead retention during loading and wash steps.</p></disp-quote><list list-type=\"simple\" id=\"olist0035\"><list-item id=\"o0185\"><label>3.</label><p id=\"p0245\">Priming the Rhapsody Cartridge</p></list-item><list-item id=\"o0190\"><label>a.</label><p id=\"p0250\">Push the cartridge into the far end of the Express instrument tray to match the cartridge and tray notches. Lay the cartridge flat and release it. Ensure that the cartridge is flat in the tray and the barcode faces out.</p></list-item><list-item id=\"o0195\"><label>b.</label><p id=\"p0255\">Move the left slider to the middle (0) position on the Express instrument. The Retrieval (top) magnet and Lysis (bottom) magnets are away from the cartridge tray.</p></list-item><list-item id=\"o0200\"><label>c.</label><p id=\"p0260\">Move the front slider to <bold>OPEN</bold></p></list-item><list-item id=\"o0205\"><label>d.</label><p id=\"p0265\">Remove the cap of a waste collection container (PN 650000090) and insert both the container and a new 5 mL LoBind Tube (Eppendorf cat. no. 0030108310) for bead retrieval into the appropriate slots in the drawer. Secure the cap of the 5 mL LoBind Tube to the holder (<xref rid=\"fig1\" ref-type=\"fig\">Figure 1</xref>).</p></list-item></list></p><p id=\"p0270\"><list list-type=\"simple\" id=\"olist0040\"><list-item id=\"o0210\"><label>e.</label><p id=\"p0275\">Move the front slider to <bold>WASTE</bold></p></list-item><list-item id=\"o0215\"><label>f.</label><p id=\"p0280\">Insert the tip of the pipette perpendicular to the port, seal the pipette tip against the gasket, and then load the cartridge with 700 μL of 100% (absolute) ethyl alcohol using the Gilson P1200M pipette in <bold>Prime/Treat</bold> mode (<xref rid=\"fig2\" ref-type=\"fig\">Figure 2</xref>).</p></list-item></list><fig id=\"fig1\"><label>Figure 1</label><caption><p>Setting Up the BD Rhapsody Express</p><p>BD Rhapsody Express showing proper cartridge position with 5 mL LoBind Tube for collection of Cell Capture Beads and waste container installed. During the workflow, the slider needs to be moved either to “Waste” or to “Beads” as indicated in the protocol.</p></caption><graphic xlink:href=\"gr1\"/></fig></p><p id=\"p0285\"><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> ethyl alcohol should be steadily flowing through the cartridge. If ethyl alcohol is not going into the cartridge, the amount of pressure on the gasket from the pipette needs to be modified to allow reagent flow.</p></disp-quote><list list-type=\"simple\" id=\"olist0045\"><list-item id=\"o0220\"><label>g.</label><p id=\"p0290\">Load the cartridge with 700 μL of air using the Gilson P1200M pipette in <bold>Prime/Treat</bold> mode.</p></list-item><list-item id=\"o0225\"><label>h.</label><p id=\"p0295\">Load the cartridge with 700 μL of Cartridge Wash Buffer 1 (PN 650000060) with the Gilson P1200M pipette in <bold>Prime/Treat</bold> mode (<xref rid=\"fig2\" ref-type=\"fig\">Figure 2</xref>).</p></list-item><list-item id=\"o0230\"><label>i.</label><p id=\"p0300\">Leave the cartridge on the tray at 15°C–25°C for 1 min.</p></list-item><list-item id=\"o0235\"><label>4.</label><p id=\"p0305\">Treating the surface of the cartridge</p></list-item><list-item id=\"o0240\"><label>a.</label><p id=\"p0310\">Load the cartridge with 700 μL of air using the Gilson P1200M pipette in <bold>Prime/Treat</bold> mode.</p></list-item><list-item id=\"o0245\"><label>b.</label><p id=\"p0315\">Load the cartridge with 700 μL of Cartridge Wash Buffer 1(PN 650000060) using the Gilson P1200M pipette in <bold>Prime/Treat</bold> mode (<xref rid=\"fig2\" ref-type=\"fig\">Figure 2</xref>).</p></list-item><list-item id=\"o0250\"><label>c.</label><p id=\"p0320\">Leave the cartridge on the tray at 15°C–25°C for 10 min.</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> If using two cartridges with a single instrument for one experiment, start the priming/ treating of the second cartridge during this 10-min incubation and repeat the steps above to prepare the second cartridge.</p></disp-quote><list list-type=\"simple\" id=\"olist0050\"><list-item id=\"o0255\"><label>d.</label><p id=\"p0325\">Load the cartridge with 700 μL of air using the Gilson P1200M pipette in <bold>Prime/Treat</bold> mode.</p></list-item><list-item id=\"o0260\"><label>e.</label><p id=\"p0330\">Load the cartridge with 700 μL of Cartridge Wash Buffer 2 (PN 650000061) using the Gilson P1200M pipette in <bold>Prime/Treat</bold> mode (<xref rid=\"fig2\" ref-type=\"fig\">Figure 2</xref>).</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> The cartridge can be stored at 15°C–25°C for ≤4 h. You can leave the cartridge on the tray. The performance of the cartridge has not been validated at 15°C–25°C storage for >4 h.</p></disp-quote><fig id=\"fig2\"><label>Figure 2</label><caption><p>Loading the Cartridge</p><p>Insert the pipette perpendicular to the cartridge with enough pressure to properly seal the orange gasket with the pipette tip, then dispense the pipette contents. The pipette needs to be set to “Prime/Treat”.</p></caption><graphic xlink:href=\"gr2\"/></fig></p></sec><sec id=\"sec3.2\"><title>Perform Sample Multiplexing (Optional) and Ab-Oligo Staining</title><p id=\"p0335\"><disp-quote><p><inline-graphic xlink:href=\"fx2.gif\"/><bold>Timing: 1.5 h</bold></p></disp-quote></p><p id=\"p0340\">This protocol describes use of antibodies conjugated to oligonucleotides (Ab-Oligo) for sample multiplexing and surface protein profiling. Each Ab-Oligo contains an antibody-specific barcode, a poly(A) tail for bead capture and additional sequences for PCR amplification and library generation. There are two different methods to utilize multiplexing and surface protein profiling: co-labeling or sequential labeling. Co-labeling will save time, however, sequential labeling is more economical and can additionally reduce batch effects resulting from inconsistent staining. The following steps describe sequential Ab-Oligo labeling of 20,000–1 million cells. Up to 100 antibodies can be pooled together per staining reaction. If no sample multiplexing is required, proceed directly to step 8 (Fc receptor blocking).<list list-type=\"simple\" id=\"olist0055\"><list-item id=\"o0265\"><label>5.</label><p id=\"p0345\">Use the cell suspensions derived from different samples (or from sorted populations) from the “Defrosting cryopreserved Peripheral Blood Mononuclear Cells (PBMCs)” section, and make sure that each sample is resuspended in 180 μL of Cell staining Buffer.</p></list-item><list-item id=\"o0270\"><label>6.</label><p id=\"p0350\">Labeling samples with sample multiplexing antibodies</p></list-item><list-item id=\"o0275\"><label>a.</label><p id=\"p0355\">Prepare a spreadsheet listing which sample is going to be labeled with which Sample Tag (1–12).</p></list-item><list-item id=\"o0280\"><label>b.</label><p id=\"p0360\">Quick-spin Sample Tag tubes to collect the contents at the bottom.</p></list-item><list-item id=\"o0285\"><label>c.</label><p id=\"p0365\">For each sample, transfer 180 μL cell suspension to the corresponding Sample Tag tube. Pipette-mix.</p></list-item><list-item id=\"o0290\"><label>d.</label><p id=\"p0370\">Incubate at room temperature (15°C–25°C) for 20 min.</p></list-item><list-item id=\"o0295\"><label>e.</label><p id=\"p0375\">Transfer each labeled cell suspension to a 1.5 mL LoBind tube.</p></list-item><list-item id=\"o0300\"><label>f.</label><p id=\"p0380\">Add 1 mL Cell Staining Buffer to labeled cells and pipette-mix.</p></list-item><list-item id=\"o0305\"><label>g.</label><p id=\"p0385\">Centrifuge each tube at 400 × <italic>g</italic> for 5 min.</p></list-item><list-item id=\"o0310\"><label>h.</label><p id=\"p0390\">Pipette off the supernatant taking care to not disturb the cell pellet.</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> Pipetting off supernatant as opposed to decanting will reduce cell loss during washes. However, it is critical to wash the cells well (i.e., leave as little supernatant as possible).</p></disp-quote><list list-type=\"simple\" id=\"olist0060\"><list-item id=\"o0315\"><label>i.</label><p id=\"p0395\">Repeat steps 6f–6h for a total of three washes</p></list-item><list-item id=\"o0320\"><label>j.</label><p id=\"p0400\">Resuspend the cell pellet cells in 100 μL cold Cell staining buffer</p></list-item><list-item id=\"o0325\"><label>7.</label><p id=\"p0405\">Pooling the samples after labeling with multiplexing antibodies</p></list-item></list><disp-quote><p><bold><italic>Optional:</italic></bold> if the individual cell samples were obtained from different sources and the cell concentration is unknown, count the cells from step 6j using Trypan blue to determine the mixing ratio as required.</p></disp-quote><list list-type=\"simple\" id=\"olist0065\"><list-item id=\"o0330\"><label>a.</label><p id=\"p0410\">Pool all the individual samples from 6j into a new 1.5 mL LoBind tube.</p></list-item><list-item id=\"o0335\"><label>b.</label><p id=\"p0415\">Add additional Sample Buffer to bring the total volume to 1.5 mL.</p></list-item><list-item id=\"o0340\"><label>c.</label><p id=\"p0420\">Centrifuge the tube at 400 × <italic>g</italic> for 5 min.</p></list-item><list-item id=\"o0345\"><label>d.</label><p id=\"p0425\">Pipette off the supernatant taking care to not disturb the cell pellet.</p></list-item><list-item id=\"o0350\"><label>e.</label><p id=\"p0430\">Resuspend the cells in 100 μL cold staining buffer.</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> Samples containing myeloid and B cells should be treated with Fc-blocking reagent prior to Ab-Oligo staining (<xref rid=\"bib2\" ref-type=\"bibr\">Andersen et al., 2016</xref>).</p></disp-quote><list list-type=\"simple\" id=\"olist0070\"><list-item id=\"o0355\"><label>8.</label><p id=\"p0435\">Blocking non-specific Fc Receptor Ab-Oligo Binding.</p></list-item><list-item id=\"o0360\"><label>a.</label><p id=\"p0440\">Pipette reagents into a new 1.5 mL LoBind tube on ice (<xref rid=\"tbl1\" ref-type=\"table\">Table 1</xref>):</p></list-item></list></p><p id=\"tspara0025\">Reagents should be added sequencially, as listed in the table, into a 1.5 mL LoBind Tube on ice. Mix can be scaled according to number of samples.<list list-type=\"simple\" id=\"olist0185\"><list-item id=\"o0810\"><label>b.</label><p id=\"p1170\">Pipette-mix Fc Block master mix, and briefly centrifuge.</p></list-item><list-item id=\"o0815\"><label>c.</label><p id=\"p1175\">Add 100 μL Fc block master mix to the cells from step 7e.</p></list-item><list-item id=\"o0820\"><label>d.</label><p id=\"p1180\">Incubate cells on ice for 10 min</p></list-item><list-item id=\"o0825\"><label>e.</label><p id=\"p1185\">Add 1 mL of cold staining buffer to wash</p></list-item><list-item id=\"o0830\"><label>f.</label><p id=\"p1190\">Centrifuge the tube at 400 × <italic>g</italic> for 5 min</p></list-item><list-item id=\"o0835\"><label>g.</label><p id=\"p1195\">Pipette off the supernatant taking care to not disturb the cell pellet</p></list-item><list-item id=\"o0840\"><label>h.</label><p id=\"p1200\">Resuspend the cells in 100 μL cold staining buffer.</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> When handling low cell numbers, the washing steps 8e–h can be omitted.</p></disp-quote></p><p id=\"p1945\"><list list-type=\"simple\" id=\"olist0175\"><list-item id=\"o0795\"><label>9.</label><p id=\"p1155\">Preparing 2× BD AbSeq antibody-oligo labeling master mix on ice</p></list-item><list-item id=\"o0800\"><label>a.</label><p id=\"p1160\">Centrifuge BD AbSeq Ab-Oligos in a tabletop centrifuge at 400 × g for 30 s and place on ice.</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> Alternatively<bold>,</bold> Ab-Oligos can be placed into a Latch Rack for 500 μL Tubes (ThermoFisher Scientific, Cat. No. 4890) on ice and be centrifuged in the Latch Rack with a plate adapter. Further, tubes can be uncapped and re-capped with an 8-Channel Screw Cap Tube Capper (Thermo Fisher Scientific Cat. No. 4105MAT) and aliquoted with a multi-channel pipette.</p></disp-quote><list list-type=\"simple\" id=\"olist0180\"><list-item id=\"o0805\"><label>b.</label><p id=\"p1165\">In pre-amplification workspace, pipette reagents into a new 1.5 mL LoBind Tube on ice (<xref rid=\"tbl2\" ref-type=\"table\">Table 2</xref>):</p></list-item></list></p><p id=\"tspara0050\">Master mix can be scaled based on number of samples.<list list-type=\"simple\" id=\"olist0195\"><list-item id=\"o0890\"><label>c.</label><p id=\"p1250\">Pipette-mix the 2× AbSeq labeling master mix, and place back on ice.</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> The final working concentrations for the Ab-Oligos are between 0.1 μg and 1 μg per stain depending on the antibody clone and have been optimized by the manufacturer. For an example of final working concentrations on a select set of Ab-Oligos, see Supplemental Table 4 in (<xref rid=\"bib7\" ref-type=\"bibr\">Mair et al., 2020</xref>).</p></disp-quote><list list-type=\"simple\" id=\"olist0190\"><list-item id=\"o0845\"><label>10.</label><p id=\"p1205\">Labeling samples with AbSeq Ab-oligos</p></list-item><list-item id=\"o0850\"><label>a.</label><p id=\"p1210\">In a new 1.5 mL LoBind tube, combine 100 μL pooled cell suspension labeled with Sample Tags (obtained from step 7e or 8h) and 100 μL 2× AbSeq labeling master mix. Pipette-mix.</p></list-item><list-item id=\"o0855\"><label>b.</label><p id=\"p1215\">Incubate on Ice for 30 min.</p></list-item><list-item id=\"o0860\"><label>11.</label><p id=\"p1220\">Washing Labeled Cells</p></list-item><list-item id=\"o0865\"><label>a.</label><p id=\"p1225\">Add 1 mL Cell staining Buffer to labeled cells and pipette-mix.</p></list-item><list-item id=\"o0870\"><label>b.</label><p id=\"p1230\">Centrifuge the tube at 400 × g for 5 min.</p></list-item><list-item id=\"o0875\"><label>c.</label><p id=\"p1235\">Pipette off the supernatant taking care to not disturb the cell pellet.</p></list-item><list-item id=\"o0880\"><label>d.</label><p id=\"p1240\">Repeat steps 11a–c for a total of 3 washes.</p></list-item><list-item id=\"o0885\"><label>e.</label><p id=\"p1245\">Resuspend pellet in 100 μL cold Sample Buffer and use 10 μL to perform a Trypan blue viability staining. Ideally, if planning to load a full cartridge, the cell count should be >300,000 cells/mL with a viability >90%.</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> Cells must be resuspended in Sample Buffer (and not Staining Buffer)</p></disp-quote><disp-quote><p><bold><italic>Note:</italic></bold> Sufficient post-labeling washes are important for reducing noise that comes from residual unbound antibodies being captured onto 3’ capture beads during single-cell capture. However, some cell loss occurs with each additional wash. Users can choose to perform more or fewer washes depending on the abundance of their sample. If the user has an excess of cells for the experiment, it is recommended that the stain and wash steps occur in a 5 mL polystyrene tube. This will allow an increase in wash volume to 3 mL instead of 1 mL.</p></disp-quote></p><p id=\"p0445\"><table-wrap position=\"anchor\" id=\"tbl1\"><label>Table 1</label><caption><p>2× Fc Block Master Mix</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Component</th><th>1 Sample (μL)</th><th>1 Sample + 20% Overage (μL)</th></tr></thead><tbody><tr><td>Cell staining buffer</td><td>90.0</td><td>108.0</td></tr><tr><td>Human Fc Block (Cat. No. 5642220)</td><td>10.0</td><td>12.0</td></tr><tr><td>Total</td><td>100</td><td>120.0</td></tr></tbody></table></table-wrap></p><p id=\"p0450\"><table-wrap position=\"anchor\" id=\"tbl2\"><label>Table 2</label><caption><p>2× AbSeq labeling Master Mix</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Component</th><th>1 Sample (μL)</th><th>1 Sample + 10% Overage (μL)</th><th>2 Samples + 10% Overage (μL)</th></tr></thead><tbody><tr><td>Per ab-oligo</td><td>2.0</td><td>2.2</td><td>4.4</td></tr><tr><td>Stain Buffer (N= no. ab-oligos)</td><td>100.0 - (2.0 ∗ N)</td><td>110 - (2.2 ∗ N)</td><td>220 – (4.4 ∗ N)</td></tr><tr><td>Total</td><td>100</td><td>110</td><td>220</td></tr></tbody></table></table-wrap></p></sec><sec id=\"sec3.3\"><title>Loading of Cells onto the Cartridge and Retrieval of Cell Capture Beads Containing mRNA and Feature Barcodes</title><p id=\"p0455\"><disp-quote><p><inline-graphic xlink:href=\"fx2.gif\"/><bold>Timing: 1 h</bold></p></disp-quote></p><p id=\"p0460\">This protocol describes the steps to isolate and capture mRNA and feature barcodes from partitioned cells on the Rhapsody cartridge. Single-cell partitioning is achieved by loading cells at densities that are low enough to achieve a single cell per well distribution in the cartridge. Each cartridge contains >200,000 nanowells, thus the likelihood of having more than one cell in a single well depends on the cell density (see table below). There are different approaches to identify multiplets based on factors such as number of genes or unique molecular identifiers present compared to the rest of the sample, as well as computational approaches (<xref rid=\"bib1\" ref-type=\"bibr\">Amezquita et al., 2020</xref>). Identifying multiplets can be significantly improved if several samples are multiplexed prior to loading on the cartridge because any sample with two or more Sample Tags is easily identified as a multiplet (<xref rid=\"bib11\" ref-type=\"bibr\">Stoeckius et al., 2018</xref>).</p><p id=\"p0465\">Refer to <xref rid=\"tbl3\" ref-type=\"table\">Table 3</xref> to determine an acceptable multiplet rate for the number of captured cells on retrieved Cell Capture Beads:<table-wrap position=\"float\" id=\"tbl3\"><label>Table 3</label><caption><p>Expected Multiplet Rate for a Given Cell Load</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Number of Captured Cells on Retrieved Cell Capture Beads (Target)</th><th>Estimated Multiplet Rate (%)</th></tr></thead><tbody><tr><td>100</td><td>0.0</td></tr><tr><td>500</td><td>0.1</td></tr><tr><td>1,000</td><td>0.2</td></tr><tr><td>5,000</td><td>1.2</td></tr><tr><td>10,000</td><td>2.4</td></tr><tr><td>20,000</td><td>4.7</td></tr></tbody></table></table-wrap></p><p id=\"tspara0075\">An approximation of the percentage of multiplets that will occur within a cartridge based on the number of cells loaded. Because there are a fixed number of nanowells in a cartridge, the likelihood of multiplets increases with the number of loaded cells. As discussed in the main text, multiplets can only efficiently be removed when using sample multiplexing.<list list-type=\"simple\" id=\"olist0215\"><list-item id=\"o0980\"><label>12.</label><p id=\"p1340\">According to the number of cells counted in step 11e, take the appropriate volume containing the targeted cell number + 30% (i.e., if planning to capture 20,000 cells per cartridge, take 26,000 cells. If planning to load two cartridges in parallel take 52,000 cells).</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> 30% is an approximate number that should allow a more accurate prediction of the number of cells collected after. This number takes into account cell loss from both washes and cartridge loading (only 575 μL of the 650 μL single-cell suspension will be loaded onto the Rhapsody Cartridge).</p></disp-quote><list list-type=\"simple\" id=\"olist0200\"><list-item id=\"o0895\"><label>13.</label><p id=\"p1255\">Add Sample Buffer to bring the volume of the cell suspension to 650 μL (or 1,300 μL if planning to load two cartridges in parallel)</p></list-item><list-item id=\"o0900\"><label>14.</label><p id=\"p1260\">Load the cells onto the cartridge.</p></list-item><list-item id=\"o0905\"><label>a.</label><p id=\"p1265\">Load the cartridge on the tray with 700 μL of air using the Gilson P1200M pipette in <bold>Prime/Treat</bold> mode.</p></list-item><list-item id=\"o0910\"><label>b.</label><p id=\"p1270\">Change the mode of the Gilson P1200M pipette to <bold>Cell Load.</bold></p></list-item><list-item id=\"o0915\"><label>c.</label><p id=\"p1275\">With a manual pipette, gently pipet the cell suspension up and down to mix.</p></list-item><list-item id=\"o0920\"><label>d.</label><p id=\"p1280\">On the Gilson P1200M, press the pipette button once to aspirate 40 μL of air, immerse the pipette tip in cell suspension, and then press the button again to aspirate <bold>575</bold> μL of cold cell suspension (<xref rid=\"fig2\" ref-type=\"fig\">Figure 2</xref>).</p></list-item><list-item id=\"o0925\"><label>e.</label><p id=\"p1285\">Insert the tip of the pipette perpendicular to the port, seal the pipette tip against the gasket, and then press the button a third time to dispense 615 μL of air and cells.</p></list-item><list-item id=\"o0930\"><label>f.</label><p id=\"p1290\">Incubate at room temperature (15°C–25°C) for 15 min. During the 15-min incubation, prepare Cell Capture Beads.</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> If planning to load two cartridges at the same time using only one Rhapsody Express instrument, load cartridge #2 at the 14-min mark of step 14f by repeating steps 14a–f. This is essential to ensure you have correct timing for subsequent steps.</p></disp-quote><list list-type=\"simple\" id=\"olist0205\"><list-item id=\"o0935\"><label>15.</label><p id=\"p1295\">Preparing Cell Capture Beads</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> Keep the Cell Capture Beads on ice before use. For maximum recovery, do not vortex samples containing Cell Capture Beads. Gently mix suspensions with Cell Capture Beads by pipette only. Use low retention pipette tips and LoBind tubes only.</p></disp-quote><list list-type=\"simple\" id=\"olist0210\"><list-item id=\"o0940\"><label>a.</label><p id=\"p1300\">Place Cell Capture Bead tube on magnet for 1 min, then remove storage buffer.</p></list-item><list-item id=\"o0945\"><label>b.</label><p id=\"p1305\">Remove tube from magnet, and pipette 750 μL cold Sample Buffer into the tube.</p></list-item><list-item id=\"o0950\"><label>c.</label><p id=\"p1310\">Pipette-mix and place on ice.</p></list-item><list-item id=\"o0955\"><label>16.</label><p id=\"p1315\">Loading the Cell Capture Beads</p></list-item><list-item id=\"o0960\"><label>a.</label><p id=\"p1320\">Change the mode of the Gilson P1200M pipette to <bold>Prime/Treat.</bold></p></list-item><list-item id=\"o0965\"><label>b.</label><p id=\"p1325\">At the end of the 15 cell-load incubation, load the cartridge with 700 μL of air using the P1200M pipette in <bold>Prime/Treat</bold> mode.</p></list-item><list-item id=\"o0970\"><label>c.</label><p id=\"p1330\">Change the mode of the Gilson P1200M pipette to <bold>Bead Load.</bold></p></list-item><list-item id=\"o0975\"><label>d.</label><p id=\"p1335\">Use a P1000 standard pipette to gently pipet the Cell Capture Beads in cold Sample Buffer (PN 650000062) up and down to mix, and, using the Rhapsody P1200M pipette in <bold>Bead Load</bold> mode, immediately load the cartridge with 630 μL of beads (<xref rid=\"fig3\" ref-type=\"fig\">Figure 3</xref>).</p></list-item><list-item id=\"o0365\"><label>e.</label><p id=\"p0475\">Let the beads settle in the cartridge on the tray at room temperature (15- 25°C) for 3 min.</p></list-item><list-item id=\"o0370\"><label>f.</label><p id=\"p0480\">Place cartridge on the Eppendorf SmartBlock Plates.</p></list-item><list-item id=\"o0375\"><label>g.</label><p id=\"p0485\">Shake the cartridge at room temperature (21°C) for 15 s at 1,000 rpm.</p></list-item></list></p><p id=\"p0470\"><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> Do not change timing and use only SmartBlock Plate for shaking.</p></disp-quote><list list-type=\"simple\" id=\"olist0080\"><list-item id=\"o0380\"><label>h.</label><p id=\"p0490\">Blot outlet drip with lint-free wipe.</p></list-item><list-item id=\"o0385\"><label>i.</label><p id=\"p0495\">Return cartridge to Express instrument and wait 30 s.</p></list-item><list-item id=\"o0390\"><label>j.</label><p id=\"p0500\">Change the mode of the Gilson P1200M pipette to <bold>Wash.</bold></p></list-item><list-item id=\"o0395\"><label>k.</label><p id=\"p0505\">Load the cartridge with 700 μL of air using the Gilson P1200M pipette in <bold>Wash</bold> mode.</p></list-item><list-item id=\"o0400\"><label>l.</label><p id=\"p0510\">Load the cartridge with 700 μL of cold Sample Buffer using the Gilson P1200M pipette in <bold>Wash</bold> mode (<xref rid=\"fig2\" ref-type=\"fig\">Figure 2</xref>).</p></list-item><list-item id=\"o0405\"><label>m.</label><p id=\"p0515\">Repeat steps 16k and 16l once for a total of two washes.</p></list-item><list-item id=\"o0410\"><label>n.</label><p id=\"p0520\">Replace Eppendorf SmartBlock Plates adapter with Eppendorf SmartBlock 1.5 mL adaptor and heat thermoblock to 37°C.</p></list-item><list-item id=\"o0415\"><label>17.</label><p id=\"p0525\">Lysing the cells</p></list-item><list-item id=\"o0420\"><label>a.</label><p id=\"p0530\">Add 75.0 μL of 1 M DTT (PN 650000063) to one bottle of 15 mL Lysis Buffer (PN 650000064), and then check the <bold>Add DTT</bold> box on the Lysis Buffer label. <bold>The Lysis Buffer with DTT must be used within 24 h.</bold></p></list-item><list-item id=\"o0425\"><label>b.</label><p id=\"p0535\">Briefly vortex the lysis mix and place it on ice.</p></list-item><list-item id=\"o0430\"><label>c.</label><p id=\"p0540\">Move the left slider to <bold>LYSIS.</bold> The (bottom) magnet is now in the up position and is in contact with the cartridge.</p></list-item><list-item id=\"o0435\"><label>d.</label><p id=\"p0545\">Change the mode on the Rhapsody P1200M pipette to <bold>Lysis.</bold></p></list-item><list-item id=\"o0440\"><label>e.</label><p id=\"p0550\">Load the cartridge with 550 μL of Lysis Buffer with DTT using the Rhapsody P1200M pipette in <bold>Lysis</bold> mode (<xref rid=\"fig2\" ref-type=\"fig\">Figure 2</xref>).</p></list-item><list-item id=\"o0445\"><label>f.</label><p id=\"p0555\">Leave the cartridge on the tray at 15°C–25°C for <bold>2 min</bold>.</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> If cell lysis step exceeds 2 min, noise from diffusion of transcripts into neighboring wells can increase.</p></disp-quote><list list-type=\"simple\" id=\"olist0085\"><list-item id=\"o0450\"><label>18.</label><p id=\"p0560\">During 2-min lysis step:</p></list-item><list-item id=\"o0455\"><label>a.</label><p id=\"p0565\">Ensure that a 5 mL LoBind Tube (Eppendorf cat. no. 0030108310) was inserted into the drawer for bead retrieval.</p></list-item><list-item id=\"o0460\"><label>b.</label><p id=\"p0570\">Set the mode on the Gilson P5000M pipette to <bold>Retrieval.</bold></p></list-item><list-item id=\"o0465\"><label>c.</label><p id=\"p0575\">Move the front slider to <bold>BEADS.</bold></p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> If the slider is not set to BEADS, then sample will go into the waste and the experiment is lost.</p></disp-quote><list list-type=\"simple\" id=\"olist0090\"><list-item id=\"o0470\"><label>19.</label><p id=\"p0580\">Retrieving the Cell Capture Beads from the cartridge</p></list-item><list-item id=\"o0475\"><label>a.</label><p id=\"p0585\">Move the left slider to <bold>RETRIEVAL.</bold> The (top) magnet is now in the down position and is in contact with the cartridge (<xref rid=\"fig4\" ref-type=\"fig\">Figure 4</xref>).<fig id=\"fig4\"><label>Figure 4</label><caption><p>BD Rhapsody Set to Retrieval</p><p>When the left slider is set to RETRIEVAL, the top magnet will sit atop the cartridge.</p></caption><graphic xlink:href=\"gr4\"/></fig></p></list-item><list-item id=\"o0480\"><label>b.</label><p id=\"p0595\">Leave the Retrieval magnet in the down position for 30 s.</p></list-item><list-item id=\"o0485\"><label>c.</label><p id=\"p0600\">During these 30 s, use the Gilson P5000M pipette to aspirate 5,000 μL of Lysis Buffer with DTT.</p></list-item><list-item id=\"o0490\"><label>d.</label><p id=\"p0605\">At the end of the 30 s, press down on the Gilson P5000M pipette to seal the pipette tip against the gasket of the cartridge to avoid leaks (<xref rid=\"fig5\" ref-type=\"fig\">Figure 5</xref>).<fig id=\"fig5\"><label>Figure 5</label><caption><p>Bead Collection</p><p>After cell lysis, collect the mRNA and oligo-bound beads using the P5000M pipette. Make sure that the magnet is in the correct position and the tube slider is set to “Beads”.</p></caption><graphic xlink:href=\"gr5\"/></fig></p></list-item><list-item id=\"o0495\"><label>e.</label><p id=\"p0615\">Move the left slider to the middle (<bold>0</bold>) position, and <italic>immediately</italic> load the cartridge with 4,950 μL of Lysis Buffer with DTT using the Gilson P5000M pipette. The Retrieval (top) magnet is in its full up position and is away from the cartridge. The Cell Capture Beads bound to the captured mRNA and feature barcodes are flushed through the cartridge and collected in the 5 mL LoBind tube.</p></list-item><list-item id=\"o0500\"><label>f.</label><p id=\"p0620\">Remove the pipette tip from the inlet gasket of the cartridge before pressing the dial button once to purge the tip. Discard the pipette tip.</p></list-item><list-item id=\"o0505\"><label>g.</label><p id=\"p0625\">Move the front slider to <bold>OPEN</bold>, and then remove and cap the 5 mL LoBind Tube.</p></list-item><list-item id=\"o0510\"><label>h.</label><p id=\"p0630\">Uncap the tube and place it on the large magnetic separation stand fitted with the 15 mL tube adapter for 1 min. Proceed immediately to Washing Cell Capture Beads.</p></list-item><list-item id=\"o0515\"><label>i.</label><p id=\"p0635\">Appropriately dispose of cartridge, waste collection container, and Lysis Buffer with DTT.</p></list-item><list-item id=\"o0520\"><label>20.</label><p id=\"p0640\">Washing Cell Capture Beads</p></list-item><list-item id=\"o0525\"><label>a.</label><p id=\"p0645\">After 1-min incubation leaving the 5 mL tube containing retrieved Cell Capture Beads on large magnet, remove all but 1 mL of supernatant without disturbing beads (<xref rid=\"fig6\" ref-type=\"fig\">Figure 6</xref>).<fig id=\"fig6\"><label>Figure 6</label><caption><p>Washing Cell Capture Beads</p><p>Allow beads to settle next to the magnet, and remove lysis buffer without disturbing the beads.</p></caption><graphic xlink:href=\"gr6\"/></fig></p></list-item><list-item id=\"o0530\"><label>b.</label><p id=\"p0655\">Remove tube from magnet. Gently pipette-mix beads, and transfer to a new 1.5 mL LoBind tube.</p></list-item><list-item id=\"o0535\"><label>c.</label><p id=\"p0660\">If there are still beads left in the 5 mL tube, add 0.5 mL Lysis Buffer with DTT, rinse 5 mL tube, and transfer to 1.5 mL LoBind tube from step 20b.</p></list-item><list-item id=\"o0540\"><label>d.</label><p id=\"p0665\">Place tube on magnet for ≤ 2 min and remove supernatant.</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> Avoid leaving Lysis Buffer or bubbles in tube. Lysis Buffer might cause the reverse transcription reaction to fail.</p></disp-quote><list list-type=\"simple\" id=\"olist0110\"><list-item id=\"o0545\"><label>e.</label><p id=\"p0670\">Remove tube from magnet and pipette 1 mL of cold Bead Wash Buffer into tube. Pipet mix.</p></list-item><list-item id=\"o0550\"><label>f.</label><p id=\"p0675\">Place tube on 1.5 mL tube magnet for ≤2 min and remove supernatant.</p></list-item><list-item id=\"o0555\"><label>g.</label><p id=\"p0680\">Remove tube from magnet, and pipette 1 mL cold Bead wash Buffer into tube. Pipette-mix, and place on ice.</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> Start reverse transcription ≤30 min after washing retrieve Cell Capture Beads with Bead Wash Buffer. If a second cartridge is used, then repeat steps 17a–20g with the second cartridge within this time.</p></disp-quote><fig id=\"fig3\"><label>Figure 3</label><caption><p>Loading the Cell Capture Beads</p><p>Hold pipette vertically and with enough pressure to seal the gasket while quickly dispensing beads to avoid settling. The pipette needs to be set to “Bead Load”.</p></caption><graphic xlink:href=\"gr3\"/></fig></p></sec><sec id=\"sec3.4\"><title>Performing Reverse Transcription and Exonuclease I Treatment on the Cell Capture Beads</title><p id=\"p0685\"><disp-quote><p><inline-graphic xlink:href=\"fx2.gif\"/><bold>Timing: 90 min</bold></p></disp-quote></p><p id=\"p0690\">This protocol describes the steps to link cell barcodes to mRNA and feature barcodes by reverse transcription, which yields a stable sample and preserving single-cell information for downstream library generation steps. If two cartridges were used in parallel, the two sets of Cell Capture Beads should be kept separate and be treated as two independent libraries.<list list-type=\"simple\" id=\"olist0115\"><list-item id=\"o0560\"><label>21.</label><p id=\"p0695\">Performing reverse transcription</p></list-item><list-item id=\"o0565\"><label>a.</label><p id=\"p0700\">In the pre-amplification workspace, in a new 1.5 mL LoBind Tube that is on ice, pipette the components in the following order to prepare the cDNA mix (<xref rid=\"tbl4\" ref-type=\"table\">Table 4</xref>):</p></list-item></list></p><p id=\"p0691\">Add reagents sequencially, as listed in table, into a 1.5 mL LoBind tube on ice. Volumes can be further scaled up if more than 2 cDNA libraries need to be produced.<list list-type=\"simple\" id=\"olist0220\"><list-item id=\"o0985\"><label>b.</label><p id=\"p1345\">Gently vortex and centrifuge the mix, and then put it back on ice.</p></list-item><list-item id=\"o0990\"><label>c.</label><p id=\"p1350\">Place the tube of washed beads on the 1.5 mL tube magnet for ≤2 min, and then carefully remove and appropriately discard the supernatant without disturbing the beads while leaving the tube on the magnet.</p></list-item><list-item id=\"o0995\"><label>d.</label><p id=\"p1355\">Use a low retention tip to pipette 200 μL of the cDNA mix to resuspend the beads. Gently mix the suspension by pipette only. Do not vortex.</p></list-item><list-item id=\"o1000\"><label>e.</label><p id=\"p1360\">Transfer the bead suspension to a new 1.5 mL LoBind Tube.</p></list-item><list-item id=\"o1005\"><label>f.</label><p id=\"p1365\">Ensure that the SmartBlock Thermoblock 1.5 mL or equivalent is installed on the thermomixer.</p></list-item><list-item id=\"o1010\"><label>g.</label><p id=\"p1370\">Incubate the suspension on the thermomixer at 1,200 rpm and 37°C for 20 min.</p></list-item><list-item id=\"o1015\"><label>h.</label><p id=\"p1375\">After incubation, put the tube on ice.</p></list-item><list-item id=\"o1020\"><label>22.</label><p id=\"p1380\">Treating the Cell Capture Beads with Exonuclease I</p></list-item><list-item id=\"o1025\"><label>a.</label><p id=\"p1385\">In the pre-amplification workspace, prepare the Exonuclease I mix in a new 1.5 mL LoBind Tube that is on ice by adding the components in the following order (<xref rid=\"tbl5\" ref-type=\"table\">Table 5</xref>):</p></list-item></list><table-wrap position=\"float\" id=\"tbl4\"><label>Table 4</label><caption><p>cDNA Master Mix</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Component</th><th>1 Library (μL)</th><th>1 Library + 20% Overage (μL)</th><th>2 Libraries + 10% Overage (μL)</th></tr></thead><tbody><tr><td>Nuclease-Free Water</td><td>106.7</td><td>128.0</td><td>235.0</td></tr><tr><td>RT Buffer</td><td>40.0</td><td>48.0</td><td>88.0</td></tr><tr><td>dNTP</td><td>20.0</td><td>24.0</td><td>44.0</td></tr><tr><td>RT 0.1 M DTT</td><td>10.0</td><td>12.0</td><td>22.0</td></tr><tr><td>RT/PCR Enhancer</td><td>3.3</td><td>4.0</td><td>7.3</td></tr><tr><td>RNase Inhibitor</td><td>10.0</td><td>12.0</td><td>22.0</td></tr><tr><td>Reverse Transcriptase</td><td>10.0</td><td>12.0</td><td>22.0</td></tr><tr><td>Total</td><td>200.0</td><td>240.0</td><td>440.0</td></tr></tbody></table></table-wrap></p><p id=\"tspara0105\">Into a 1.5 mL LoBind tube on ice, add reagents sequencially, as listed in table. Exonuclease I mix can be scaled up to accommodate more than two samples.<list list-type=\"simple\" id=\"olist0230\"><list-item id=\"o1050\"><label>b.</label><p id=\"p1410\">Gently vortex and centrifuge the mix, and then put it back on ice.</p></list-item><list-item id=\"o1055\"><label>c.</label><p id=\"p1415\">Place the tube of beads with cDNA mix on the 1.5 mL tube magnet for ≤2 min, and then carefully remove and appropriately discard the supernatant without disturbing the beads and while leaving the tube on the magnet.</p></list-item><list-item id=\"o1060\"><label>d.</label><p id=\"p1420\">Remove the tube from the magnet, and then use a low retention tip to pipette 200 μL of Exonuclease I mix to the tube, gently resuspend the beads by pipette only. Do not vortex.</p></list-item><list-item id=\"o1065\"><label>e.</label><p id=\"p1425\">Incubate the suspension on the thermomixer at 1,200 rpm and on the thermomixer at 1,200 rpm and 37°C for 30 min.</p></list-item><list-item id=\"o1070\"><label>23.</label><p id=\"p1430\">Inactivating Exonuclease I</p></list-item><list-item id=\"o1075\"><label>a.</label><p id=\"p1435\">Transfer the bead suspension with Exonuclease I to the thermomixer or heat block in the pre-amplification workspace at 80°C (no shaking) for 20 min.</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> If the thermomixer is the same as that used for the 37˚C step put the samples on ice until that temperature is reached rather than leaving the tubes in the thermomixer as the temperature ramps up.</p></disp-quote><list list-type=\"simple\" id=\"olist0225\"><list-item id=\"o1030\"><label>b.</label><p id=\"p1390\">Put the bead suspension on ice for ∼1 min.</p></list-item><list-item id=\"o1035\"><label>c.</label><p id=\"p1395\">Place the tube on the 1.5 mL tube magnet until the solution is clear (≤1 min).</p></list-item><list-item id=\"o1040\"><label>d.</label><p id=\"p1400\">Carefully remove and appropriately discard the supernatant without disturbing the beads while leaving the tube on the magnet.</p></list-item><list-item id=\"o1045\"><label>e.</label><p id=\"p1405\">Remove the tube from the magnet, and with a low retention tip, pipette 200 μL of cold Bead Resuspension Buffer to gently resuspend the beads. Do not vortex.</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx4.gif\"/><bold>Pause Point:</bold> The Exonuclease I-treated beads can be stored at 2°C–8°C for ≤3 months.</p></disp-quote></p><p id=\"p0710\"><table-wrap position=\"anchor\" id=\"tbl5\"><label>Table 5</label><caption><p>Exonuclease I Mix</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Component</th><th>1 Library (μL)</th><th>1 Library + 20% Overage (μL)</th><th>2 Libraries + 10% Overage (μL)</th></tr></thead><tbody><tr><td>Nuclease-Free Water</td><td>170.0</td><td>204.0</td><td>374.0</td></tr><tr><td>10 Exonuclease I Buffer</td><td>20.0</td><td>24.0</td><td>44.0</td></tr><tr><td>Exonuclease I</td><td>10.0</td><td>12.0</td><td>22.0</td></tr><tr><td>Total</td><td>200.0</td><td>240.0</td><td>440.0</td></tr></tbody></table></table-wrap></p></sec><sec id=\"sec3.5\"><title>Library Preparation – Multiplexed Targeted mRNA, AbSeq, and Sample Tags</title><p id=\"p0715\"><disp-quote><p><inline-graphic xlink:href=\"fx2.gif\"/><bold>Timing: 7 h 30 min</bold></p></disp-quote></p><p id=\"p0720\">This section describes the steps for PCR amplification of the gene targets of interest and the captured features barcodes, as well as the fragment size selection to separate the mRNA library from the AbSeq library and Sample Tag libraries (<xref rid=\"fig7\" ref-type=\"fig\">Figure 7</xref>). Different gene panels, including custom gene sets can be used depending on the experimental question.<list list-type=\"simple\" id=\"olist0120\"><list-item id=\"o0570\"><label>24.</label><p id=\"p0725\">Thaw the following reagents at room temperature (15°C–25°C) and then place on ice:</p></list-item><list-item id=\"o0575\"><label>a.</label><p id=\"p0730\">Nuclease-Free water</p></list-item><list-item id=\"o0580\"><label>b.</label><p id=\"p0735\">Bead RT/PCR enhancer</p></list-item><list-item id=\"o0585\"><label>c.</label><p id=\"p0740\">Elution Buffer</p></list-item><list-item id=\"o0590\"><label>d.</label><p id=\"p0745\">Universal Oligo</p></list-item><list-item id=\"o0595\"><label>e.</label><p id=\"p0750\">Library Forward Primer</p></list-item><list-item id=\"o0600\"><label>f.</label><p id=\"p0755\">Library Reverse Primer 1–4 (depending on what reverse primer desired)</p></list-item><list-item id=\"o0605\"><label>g.</label><p id=\"p0760\">PCR1 Panel Supplement (optional)</p></list-item><list-item id=\"o0610\"><label>h.</label><p id=\"p0765\">PCR2 Panel Supplement (optional)</p></list-item><list-item id=\"o0615\"><label>i.</label><p id=\"p0770\">Bead Resuspension Buffer</p></list-item><list-item id=\"o0620\"><label>j.</label><p id=\"p0775\">Sample Tag PCR1 Primer</p></list-item><list-item id=\"o0625\"><label>k.</label><p id=\"p0780\">Sample Tag PCR2 Primer</p></list-item><list-item id=\"o0630\"><label>l.</label><p id=\"p0785\">BD AbSeq Primer</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> Only remove enzymes from −20°C when in use.</p></disp-quote><list list-type=\"simple\" id=\"olist0125\"><list-item id=\"o0635\"><label>25.</label><p id=\"p0790\">Perform PCR1</p></list-item><list-item id=\"o0640\"><label>a.</label><p id=\"p0795\">In pre-amplification workspace, pipette reagents into a new 1.5 mL LoBind Tube on ice (<xref rid=\"tbl6\" ref-type=\"table\">Table 6</xref>):</p></list-item></list><fig id=\"fig7\"><label>Figure 7</label><caption><p>Library Preparation Workflow</p><p>Overview of each PCR required for library preparation. After PCR1, DNA double-sided size selection will split the transcripts based on amplicon size into mRNA or Sample Tag/AbSeq libraries. PCR2 is then preformed to further enrich both mRNA and Sample Tag libraries. Finally, each library is indexed for sequencing.</p></caption><graphic xlink:href=\"gr7\"/></fig></p><p id=\"tspara0115\">Combine all components into a 1.5 mL LoBind tube on ice. If more than 2 PCR reactions are needed, volumes can be scaled accordingly.<list list-type=\"simple\" id=\"olist0235\"><list-item id=\"o1080\"><label>b.</label><p id=\"p1440\">Gently vortex mix, briefly centrifuge, and place back on ice.</p></list-item><list-item id=\"o1085\"><label>c.</label><p id=\"p1445\">Place tube of Exonuclease I-treated beads in Bead Resuspension Buffer (Cat. No. 650000066) on 1.5 mL magnet for <2 min. Remove supernatant.</p></list-item><list-item id=\"o1090\"><label>d.</label><p id=\"p1450\">Remove tube from magnet and resuspend beads in 200 μL PCR1 reaction mix. Do not vortex.</p></list-item><list-item id=\"o1095\"><label>e.</label><p id=\"p1455\">Ensuring that the beads are fully resuspended, pipette 50 μL PCR1 reaction mix with beads into each of four 0.2 mL PCR tubes.</p></list-item><list-item id=\"o1100\"><label>f.</label><p id=\"p1460\">Bring reaction mix to the post-amplification workspace.</p></list-item><list-item id=\"o1105\"><label>g.</label><p id=\"p1465\">Program the thermal cycler (<xref rid=\"tbl7\" ref-type=\"table\">Table 7</xref>):</p></list-item></list></p><p id=\"tspara0165\">Depending on the number of cells targeted, a different number of cycles should be used to ensure sufficient quantity of library and limit PCR bias. This table is based on resting PBMCs, and should be adjusted accordingly for different cell types. Note that the targeted cell number is lower than the actual number of cells loaded on the cartridge (as calculated in step 12).<list list-type=\"simple\" id=\"olist0265\"><list-item id=\"o1290\"><label>h.</label><p id=\"p1650\">Ramp heated lid and heat block of post-amplification thermal cycler to ≥95°C by starting the thermal cycler program and then pausing it.</p></list-item><list-item id=\"o1295\"><label>i.</label><p id=\"p1655\">For each 0.2 mL PCR tube, gently pipette-mix, immediately place tube in thermal cycler, and unpause the thermal cycler program.</p></list-item></list></p><p id=\"tspara0125\">Program a thermocycler to run the above steps. See <xref rid=\"tbl8\" ref-type=\"table\">Table 8</xref> for suggested number of cycles. Do not use fast cycling mode.<disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> The thermocycler should be at 95°C when the tubes are added to ensure amplification of on-bead molecules.</p></disp-quote><disp-quote><p><inline-graphic xlink:href=\"fx4.gif\"/><bold>Pause Point:</bold> Once the PCR1 cycle is complete, the reaction can be stored at 4°C. Purification of PCR1 must occur ≤24 h after PCR1.</p></disp-quote><list list-type=\"simple\" id=\"olist0240\"><list-item id=\"o1110\"><label>j.</label><p id=\"p1470\">After PCR, briefly centrifuge tubes.</p></list-item><list-item id=\"o1115\"><label>k.</label><p id=\"p1475\">Pipette-mix and combine the four reactions into a new 1.5 mL LoBind Tube.</p></list-item><list-item id=\"o1120\"><label>l.</label><p id=\"p1480\">Place the 1.5 mL tube on magnet for 2 min and carefully pipette the <bold>supernatant</bold> (PCR1 products) into a new 1.5 mL LoBind Tube without disturbing the beads.</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> (Optional) Remove tube with the Cell Capture Beads from magnet, and pipette 200 μL cold Bead Resuspension Buffer into tube. Pipette-mix. Do not vortex. Store beads at 2°C–8°C in post-amplification workspace. Beads can be sub-sampled or undergo multiple rounds of amplification with different primer panels.</p></disp-quote><list list-type=\"simple\" id=\"olist0245\"><list-item id=\"o1125\"><label>26.</label><p id=\"p1485\">Perform double-sided SPRISelect bead purification to separate the shorter AbSeq and Sample Tag PCR1 products (∼170 bp) from the longer mRNA targeted PCR1 products (350–800 bp).</p></list-item><list-item id=\"o1130\"><label>a.</label><p id=\"p1490\">Prepare 5 mL fresh 80% (v/v) ethyl alcohol by combining 4.0 mL absolute ethyl alcohol, molecular biology grade (major supplier) with 1.0 mL nuclease-free water (major supplier). Vortex tube for 10 s.</p></list-item><list-item id=\"o1135\"><label>b.</label><p id=\"p1495\">Vortex SPRISelect Reagent at high speed 1 min until beads are fully resuspended.</p></list-item><list-item id=\"o1140\"><label>c.</label><p id=\"p1500\">Pipette 140 μL SPRISelect beads into the tube with 200 μL PCR1 products (step 25l). Pipette-mix 10 times.</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> SPRISelect beads should be pipetted very carefully. To ensure appropriate size selection, it is essential that only the exact amount of SPRISelect beads is added to PCR1 products. Do not immerse pipette tips with SPRISelect bead droplets on the outside into PCR1 products. Instead, place tip on side of tube and slowly expel all liquid, replace pipette tip, and pipette-mix.</p></disp-quote><list list-type=\"simple\" id=\"olist0250\"><list-item id=\"o1145\"><label>d.</label><p id=\"p1505\">Incubate at room temperature (15°C–25°C) for 5 min.</p></list-item><list-item id=\"o1150\"><label>e.</label><p id=\"p1510\">Place 1.5 mL LoBind Tube on magnet for 5 min.</p></list-item><list-item id=\"o1155\"><label>f.</label><p id=\"p1515\">Keeping tube on magnet, transfer the 400 μL supernatant (AbSeq PCR1 and Sample Tag products) to a new 1.5 mL tube without disturbing beads (mRNA targeted PCR1 products).</p></list-item><list-item id=\"o1160\"><label>g.</label><p id=\"p1520\">Store the supernatant at room temperature (15°C–25°C) while purifying and eluting the mRNA targeted PCR1 products (27a–27h below), then purify the AbSeq and Sample Tag PCR1 products.</p></list-item><list-item id=\"o1165\"><label>27.</label><p id=\"p1525\">Purifying mRNA targeted PCR1 products</p></list-item><list-item id=\"o1170\"><label>a.</label><p id=\"p1530\">Keeping tube on magnet, gently add 500 μL fresh 80% ethyl alcohol to the tube of SPRISelect beads bound with mRNA targeted PCR1 products and incubate 30 s. Remove supernatant.</p></list-item><list-item id=\"o1175\"><label>b.</label><p id=\"p1535\">Repeat step 27a once for two washes.</p></list-item><list-item id=\"o1180\"><label>c.</label><p id=\"p1540\">Keeping tube on magnet, use a small-volume pipette to remove residual supernatant from tube, and discard.</p></list-item><list-item id=\"o1185\"><label>d.</label><p id=\"p1545\">Air-dry beads at room temperature (15°C–25°C) for 5 min.</p></list-item><list-item id=\"o1190\"><label>e.</label><p id=\"p1550\">Remove tube from magnet and resuspend bead pellet in 30 μL of Elution Buffer (Cat. No. 91-1084). Vigorously pipette-mix until beads are uniformly dispersed. Small clumps do not affect performance.</p></list-item><list-item id=\"o1195\"><label>f.</label><p id=\"p1555\">Incubate at room temperature (15°C–25°C) for 2 min, and briefly centrifuge.</p></list-item><list-item id=\"o1200\"><label>g.</label><p id=\"p1560\">Place tube on magnet until solution is clear, usually ≤30 s.</p></list-item><list-item id=\"o1205\"><label>h.</label><p id=\"p1565\">Pipette the eluate (∼30 μL) into a new 1.5 mL LoBind Tube (purified mRNA targeted PCR1 products).</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx4.gif\"/><bold>Pause Point:</bold> Store at 2°C–8°C before proceeding in ≤24 h or at –25°C to –15°C for ≤6 months.</p></disp-quote><list list-type=\"simple\" id=\"olist0255\"><list-item id=\"o1210\"><label>28.</label><p id=\"p1570\">Purifying combined AbSeq/Sample Tag PCR1 products</p></list-item><list-item id=\"o1215\"><label>a.</label><p id=\"p1575\">Pipette 100 μL SPRISelect beads into the tube with 400 μL AbSeq/Sample Tag PCR1 products from step 26g above. Pipette-mix 10 times.</p></list-item><list-item id=\"o1220\"><label>b.</label><p id=\"p1580\">Incubate at room temperature (15°C–25°C) for 5 min.</p></list-item><list-item id=\"o1225\"><label>c.</label><p id=\"p1585\">Place on magnet for 5 min.</p></list-item><list-item id=\"o1230\"><label>d.</label><p id=\"p1590\">Keeping tube on magnet, remove supernatant.</p></list-item><list-item id=\"o1235\"><label>e.</label><p id=\"p1595\">Keeping tube on magnet, gently add 500 μL fresh 80% ethyl alcohol, and incubate 30 s. Remove supernatant.</p></list-item><list-item id=\"o1240\"><label>f.</label><p id=\"p1600\">Repeat step 28e once for two washes.</p></list-item><list-item id=\"o1245\"><label>g.</label><p id=\"p1605\">Keeping tube on magnet, use a small-volume pipette to remove residual supernatant from tube, and discard.</p></list-item><list-item id=\"o1250\"><label>h.</label><p id=\"p1610\">Air-dry beads at room temperature (15°C–25°C) for 5 min.</p></list-item><list-item id=\"o1255\"><label>i.</label><p id=\"p1615\">Remove tube from magnet and resuspend bead pellet in 30 μL Elution Buffer (Cat. No. 91-1084). Vigorously pipette-mix until beads are uniformly dispersed. Small clumps do not affect performance.</p></list-item><list-item id=\"o1260\"><label>j.</label><p id=\"p1620\">Incubate at room temperature (15°C–25°C) for 2 min, and briefly centrifuge.</p></list-item><list-item id=\"o1265\"><label>k.</label><p id=\"p1625\">Place tube on magnet until solution is clear, usually ≤30 s.</p></list-item><list-item id=\"o1270\"><label>l.</label><p id=\"p1630\">Pipette the eluate (∼30 μL) into a new 1.5 mL LoBind Tube (purified AbSeq/Sample Tag PCR1 products).</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx4.gif\"/><bold>Pause Point:</bold> Store at 2°C–8°C before proceeding in ≤24 h or at –25°C to –15°C for ≤6 months.</p></disp-quote><list list-type=\"simple\" id=\"olist0260\"><list-item id=\"o1275\"><label>29.</label><p id=\"p1635\">Quantify AbSeq/Sample Tag PCR1 products</p></list-item><list-item id=\"o1280\"><label>a.</label><p id=\"p1640\">Dilute an aliquot (∼2 μL) 1:1 with nuclease-free water, and run on the Agilent TapeStation with the High Sensitivity D5000 ScreenTape.</p></list-item><list-item id=\"o1285\"><label>b.</label><p id=\"p1645\">Measure the yield of the largest peak of the AbSeq/Sample Tag PCR1 products (∼170 bp, <xref rid=\"fig8\" ref-type=\"fig\">Figure 8</xref>).</p></list-item></list></p><p id=\"p0800\"><table-wrap position=\"anchor\" id=\"tbl6\"><label>Table 6</label><caption><p>PCR1 Reaction Mix for Targeted mRNA, AbSeq, and Sample Tags</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Component</th><th>1 Library (μL)</th><th>1 Library + 20% Overage (μL)</th><th>2 Libraries + 10% Overage (μL)</th></tr></thead><tbody><tr><td>PCR MasterMix</td><td>100.0</td><td>120.0</td><td>220</td></tr><tr><td>Universal Oligo</td><td>20.0</td><td>24.0</td><td>44</td></tr><tr><td>Bead RT/PCR Enhancer</td><td>12.0</td><td>14.4</td><td>26.4</td></tr><tr><td>PCR1 primer panel</td><td>40.0</td><td>48.0</td><td>88</td></tr><tr><td>(Optional) PCR1 Panel Supplement</td><td>10</td><td>12</td><td>22</td></tr><tr><td>Sample Tag PCR1 Primer</td><td>1.2</td><td>1.4</td><td>2.64</td></tr><tr><td>BD AbSeq Primer</td><td>12.0</td><td>14.4</td><td>26.4</td></tr><tr><td>Nuclease-Free Water</td><td>Up to 14.8</td><td>Up to 17.8</td><td>Up to 32.6</td></tr><tr><td>Total</td><td>200.0</td><td>240.0</td><td>440</td></tr></tbody></table></table-wrap></p><p id=\"p0805\"><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> Do not use fast cycling mode</p></disp-quote><table-wrap position=\"float\" id=\"tbl7\"><label>Table 7</label><caption><p>Thermocycler Program for PCR1</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Step</th><th>Cycles</th><th>Temperature</th><th>Time</th></tr></thead><tbody><tr><td>Hot Start</td><td>1</td><td>95°C</td><td>3 min</td></tr><tr><td>Denaturation</td><td rowspan=\"3\">10–15</td><td>95°C</td><td>30 s</td></tr><tr><td>Annealing</td><td>60°C</td><td>3 min</td></tr><tr><td>Extension</td><td>72°C</td><td>1 min</td></tr><tr><td>Final extension</td><td>1</td><td>72°C</td><td>5 min</td></tr><tr><td>Hold</td><td>1</td><td>4°C</td><td>∞</td></tr></tbody></table></table-wrap><table-wrap position=\"float\" id=\"tbl8\"><label>Table 8</label><caption><p>Suggested PCR1 Cycles for Given Cell Inputs</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>No. Cells Targeted on Cartridge</th><th>Suggested PCR Cycles for PCR1</th></tr></thead><tbody><tr><td>500</td><td>15</td></tr><tr><td>1,000</td><td>14</td></tr><tr><td>2,500</td><td>13</td></tr><tr><td>5,000</td><td>12</td></tr><tr><td>10,000</td><td>11</td></tr><tr><td>20,000</td><td>10</td></tr></tbody></table></table-wrap></p><p id=\"p0810\"><list list-type=\"simple\" id=\"olist0130\"><list-item id=\"o0645\"><label>c.</label><p id=\"p0815\">Dilute an aliquot of AbSeq/Sample Tag PCR1 products to 0.1–1.1 ng/μL with Elution Buffer (Cat. No. 91-1084) before index PCR of AbSeq PCR1 products. Use undiluted AbSeq/Sample Tag PCR1 products for Sample Tag PCR2 amplification.</p></list-item><list-item id=\"o0650\"><label>30.</label><p id=\"p0820\">Perform PCR2 of targeted mRNA (<xref rid=\"tbl9\" ref-type=\"table\">Table 9</xref>) and Sample Tag products (<xref rid=\"tbl10\" ref-type=\"table\">Table 10</xref>). The AbSeq PCR1 products do not require additional amplification beyond index PCR.</p></list-item></list><fig id=\"fig8\"><label>Figure 8</label><caption><p>Example of AbSeq/Sample Tag PCR1 Products</p><p>The largest peak should be approximately 170 bp. Other base pair lengths present are indicative of incomplete double-sided selection.</p></caption><graphic xlink:href=\"gr8\"/></fig></p><p id=\"p0825\"><list list-type=\"simple\" id=\"olist0135\"><list-item id=\"o0655\"><label>a.</label><p id=\"p0830\">In pre-amplification workspace, pipette reagents into a new 1.5 mL LoBind Tube on ice:</p></list-item></list><table-wrap position=\"float\" id=\"tbl9\"><label>Table 9</label><caption><p>Targeted mRNA PCR2 Reaction Mix</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Component</th><th>1 Library (μL)</th><th>1 Library + 20% Overage (μL)</th><th>2 Libraries + 10% Overage (μL)</th></tr></thead><tbody><tr><td>PCR MasterMix</td><td>25.0</td><td>30.0</td><td>55</td></tr><tr><td>Universal Oligo</td><td>2.0</td><td>2.4</td><td>4.4</td></tr><tr><td>PCR2 primer panel</td><td>10.0</td><td>12.0</td><td>22</td></tr><tr><td>(Optional) PCR2 Panel Supplement</td><td>2.5</td><td>3</td><td>5.5</td></tr><tr><td>Nuclease-Free Water</td><td>Up to 8.0</td><td>Up to 9.6</td><td/></tr><tr><td>Total</td><td>45.0</td><td>54.0</td><td>99</td></tr></tbody></table></table-wrap><table-wrap position=\"float\" id=\"tbl10\"><label>Table 10</label><caption><p>Sample Tag PCR2 Reaction Mix</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Component</th><th>1 Library (μL)</th><th>1 Library + 20% Overage (μL)</th><th>2 Libraries + 10% Overage (μL)</th></tr></thead><tbody><tr><td>PCR MasterMix</td><td>25.0</td><td>30.0</td><td>55</td></tr><tr><td>Universal Oligo</td><td>2.0</td><td>2.4</td><td>4.4</td></tr><tr><td>Sample Tag PCR2 Primer</td><td>3.0</td><td>3.6</td><td>6.6</td></tr><tr><td>Nuclease-Free Water</td><td>18.0</td><td>9.6</td><td>39.6</td></tr><tr><td>Total</td><td>45.0</td><td>54.0</td><td>99</td></tr></tbody></table></table-wrap></p><p id=\"tspara0175\">This reaction will further amplify the mRNA products. Add all components to a 1.5 mL LoBind tube on ice.</p><p id=\"tspara0185\">This reaction will amplify the Sample Tag products from PCR1. Combine all components from this table into a 1.5 mL LoBind tube on ice.<list list-type=\"simple\" id=\"olist0270\"><list-item id=\"o1300\"><label>b.</label><p id=\"p1660\">Gently vortex mix, briefly centrifuge, and place back on ice.</p></list-item><list-item id=\"o1305\"><label>c.</label><p id=\"p1665\">Bring PCR2 mixes into post-amplification workspace.</p></list-item><list-item id=\"o1310\"><label>d.</label><p id=\"p1670\">In two separate, new 0.2 mL PCR tubes:</p></list-item><list-item id=\"o1315\"><label>i.</label><p id=\"p1675\">mRNA targeted PCR1 products: Pipette 5.0 μL products into 45.0 μL mRNA targeted PCR2 reaction mix.</p></list-item><list-item id=\"o1320\"><label>ii.</label><p id=\"p1680\">Sample Tag PCR1 products: Pipette 5.0 μL products into 45.0 μL Sample Tag PCR2 reaction mix.</p></list-item><list-item id=\"o1325\"><label>e.</label><p id=\"p1685\">Gently vortex, and briefly centrifuge.</p></list-item><list-item id=\"o1330\"><label>f.</label><p id=\"p1690\">Program the thermal cycler (<xref rid=\"tbl11\" ref-type=\"table\">Table 11</xref>).</p></list-item></list></p><p id=\"tspara0205\">Program a thermocycler to this program without using fast cycling mode.<disp-quote><p><inline-graphic xlink:href=\"fx4.gif\"/><bold>Pause Point:</bold> PCR2 products can be stored at 2°C–8°C for ≤24 h or stored at −25°C to −15°C for ≤6 months.</p></disp-quote><list list-type=\"simple\" id=\"olist0275\"><list-item id=\"o1335\"><label>31.</label><p id=\"p1695\">Purify targeted mRNA and Sample Tag PCR2 products</p></list-item><list-item id=\"o1340\"><label>a.</label><p id=\"p1700\">Vortex SPRISelect beads at high speed 1 min until beads are fully resuspended.</p></list-item><list-item id=\"o1345\"><label>b.</label><p id=\"p1705\">Briefly centrifuge PCR2 products.</p></list-item><list-item id=\"o1350\"><label>c.</label><p id=\"p1710\">To 50.0 μL PCR2 products pipette:</p></list-item><list-item id=\"o1355\"><label>i.</label><p id=\"p1715\">mRNA targeted PCR2 products: <bold>40 μL</bold> SPRISelect beads.</p></list-item><list-item id=\"o1360\"><label>ii.</label><p id=\"p1720\">Sample Tag PCR2 products: <bold>60 μL</bold> SPRISelect beads.</p></list-item><list-item id=\"o1365\"><label>d.</label><p id=\"p1725\">Pipette-mix 10 times, and incubate at room temperature (15°C–25°C) for 5 min.</p></list-item><list-item id=\"o1370\"><label>e.</label><p id=\"p1730\">Place tube on strip tube magnet for 3 min. Remove supernatant.</p></list-item><list-item id=\"o1375\"><label>f.</label><p id=\"p1735\">Keeping tube on magnet, gently add 200 μL fresh 80% ethyl alcohol into tube, and incubate 30 s. Remove supernatant.</p></list-item><list-item id=\"o1380\"><label>g.</label><p id=\"p1740\">Repeat step 31f once for two washes.</p></list-item><list-item id=\"o1385\"><label>h.</label><p id=\"p1745\">Keeping tube on magnet, use a small-volume pipette to remove residual supernatant from tube and discard.</p></list-item><list-item id=\"o1390\"><label>i.</label><p id=\"p1750\">Air-dry beads at room temperature (15°C–25°C) for 3 min.</p></list-item><list-item id=\"o1395\"><label>j.</label><p id=\"p1755\">Remove tubes from magnet, and resuspend bead pellet in 30 μL Elution Buffer (Cat. No. 91-1084). Pipette-mix until beads are fully resuspended.</p></list-item><list-item id=\"o1400\"><label>k.</label><p id=\"p1760\">Incubate at room temperature (15°C–25°C) for 2 min, and briefly centrifuge.</p></list-item><list-item id=\"o1405\"><label>l.</label><p id=\"p1765\">Place tubes on magnet until solution is clear, usually ≤30 s.</p></list-item><list-item id=\"o1410\"><label>m.</label><p id=\"p1770\">Pipette entire eluate (∼30 μL) of each sample into two separate new 1.5 mL LoBind Tubes (purified mRNA targeted PCR2 and Sample Tag PCR2 products).</p></list-item></list><disp-quote><p><inline-graphic xlink:href=\"fx4.gif\"/><bold>Pause Point:</bold> Store at 2°C–8°C before proceeding on the same day or at –25°C to –15°C for ≤6 months.</p></disp-quote><list list-type=\"simple\" id=\"olist0280\"><list-item id=\"o1415\"><label>n.</label><p id=\"p1775\">Estimate the concentration of each sample by quantifying 2 μL of the PCR2 products with a Qubit™ Fluorometer using the Qubit dsDNA HS Assay Kit.</p></list-item><list-item id=\"o1420\"><label>o.</label><p id=\"p1780\">Dilute an aliquot of the products with Elution Buffer (Cat. No. 91-1084):</p></list-item><list-item id=\"o1425\"><label>i.</label><p id=\"p1785\">mRNA targeted PCR2 products: 0.2–2.7 ng/μL.</p></list-item><list-item id=\"o1430\"><label>ii.</label><p id=\"p1790\">Sample Tag PCR2 products: 0.1–1.1 ng/μL.</p></list-item><list-item id=\"o1435\"><label>32.</label><p id=\"p1795\">Perform index PCR to prepare final libraries</p></list-item><list-item id=\"o1440\"><label>a.</label><p id=\"p1800\">In pre-amplification workspace, prepare the three libraries + 20% overages of the final amplification mix for each of the three products. Pipette reagents into a new 1.5 mL LoBind Tube on ice (<xref rid=\"tbl12\" ref-type=\"table\">Table 12</xref>):</p></list-item></list></p><p id=\"tspara0215\">Combine all components in a 1.5 mL LoBind Tube on ice. Utilize a different reverse primer for each sample that will be run on the same sequencing flow cell. The mRNA, AbSeq, and Sample Tag libraries from the same cartridge should use the same reverse primer.<list list-type=\"simple\" id=\"olist0285\"><list-item id=\"o1445\"><label>b.</label><p id=\"p1805\">Gently vortex mix, briefly centrifuge, and place back on ice.</p></list-item><list-item id=\"o1450\"><label>c.</label><p id=\"p1810\">Bring index PCR mixes to post-amplification workspace.</p></list-item><list-item id=\"o1455\"><label>d.</label><p id=\"p1815\">In three separate, new 0.2 mL PCR tubes:</p></list-item><list-item id=\"o1460\"><label>i.</label><p id=\"p1820\">mRNA targeted PCR2 products: Pipette 3.0 μL of 0.2–2.7 ng/μL products into 47.0 μL index PCR mix.</p></list-item><list-item id=\"o1465\"><label>ii.</label><p id=\"p1825\">Sample Tag PCR2 products: Pipette 3.0 μL of 0.1–1.1 ng/μL products into 47.0 μL index PCR mix.</p></list-item><list-item id=\"o1470\"><label>iii.</label><p id=\"p1830\">AbSeq PCR1 products: Pipette 3.0 μL of 0.1–1.1 ng/μL products into 47.0 μL index PCR mix.</p></list-item><list-item id=\"o1475\"><label>e.</label><p id=\"p1835\">Gently vortex and briefly centrifuge.</p></list-item><list-item id=\"o1480\"><label>f.</label><p id=\"p1840\">Program the thermal cycler. (<xref rid=\"tbl13\" ref-type=\"table\">Tables 13</xref> and <xref rid=\"tbl14\" ref-type=\"table\">14</xref>)</p></list-item></list></p><p id=\"tspara0225\">Program a thermocycler to run this program. Refer to <xref rid=\"tbl14\" ref-type=\"table\">Table 14</xref> for cycle numbers.</p><p id=\"tspara0240\">This table is a suggestion for the number of index cycles to use depending on the concentration of each library. It is possible you may need to adjust the number of cycles per experiment.<disp-quote><p><inline-graphic xlink:href=\"fx4.gif\"/><bold>Pause Point:</bold> The Index PCR product can be stored at 2°C–8°C for ≤24 h, or at −25°C to −15°C for ≤6 months.</p></disp-quote><list list-type=\"simple\" id=\"olist0290\"><list-item id=\"o1485\"><label>33.</label><p id=\"p1845\">Purify index PCR products</p></list-item><list-item id=\"o1490\"><label>a.</label><p id=\"p1850\">Vortex SPRISelect beads at high speed 1 min until beads are fully resuspended.</p></list-item><list-item id=\"o1495\"><label>b.</label><p id=\"p1855\">Briefly centrifuge index PCR products.</p></list-item><list-item id=\"o1500\"><label>c.</label><p id=\"p1860\">To 50.0 μL of each of the individual index PCR products pipette:</p></list-item><list-item id=\"o1505\"><label>i.</label><p id=\"p1865\">mRNA targeted library: <bold>35 μL</bold> SPRISelect beads.</p></list-item><list-item id=\"o1510\"><label>ii.</label><p id=\"p1870\">AbSeq and Sample Tag libraries: <bold>40 μL</bold> SPRISelect beads.</p></list-item><list-item id=\"o1515\"><label>d.</label><p id=\"p1875\">Pipette-mix 10 times. Incubate at room temperature (15°C–25°C) for 5 min.</p></list-item><list-item id=\"o1520\"><label>e.</label><p id=\"p1880\">Place each tube on strip tube magnet for 3 min. Remove supernatant.</p></list-item><list-item id=\"o1525\"><label>f.</label><p id=\"p1885\">Keeping tube on magnet, for each tube, gently add 200 μL fresh 80% ethyl alcohol into tube and incubate 30 s. Remove supernatant.</p></list-item><list-item id=\"o1530\"><label>g.</label><p id=\"p1890\">Repeat step 33f for a second wash</p></list-item><list-item id=\"o1535\"><label>h.</label><p id=\"p1895\">Keeping tubes on magnet, use a small-volume pipette to remove residual supernatant from tube, and discard.</p></list-item><list-item id=\"o1540\"><label>i.</label><p id=\"p1900\">Air-dry beads at room temperature (15°C–25°C) for 3 min.</p></list-item><list-item id=\"o1545\"><label>j.</label><p id=\"p1905\">Remove tubes from magnet and resuspend each bead pellet in 30 μL Elution Buffer (Cat. No. 91-1084). Pipette-mix until beads are fully resuspended.</p></list-item><list-item id=\"o1550\"><label>k.</label><p id=\"p1910\">Incubate at room temperature (15°C–25°C) for 2 min, and briefly centrifuge.</p></list-item><list-item id=\"o1555\"><label>l.</label><p id=\"p1915\">Place tubes on magnet until solution is clear, usually ≤30 s.</p></list-item><list-item id=\"o1560\"><label>m.</label><p id=\"p1920\">For each tube, pipette entire eluates (∼30 μL) into three separate new 1.5 mL LoBind Tubes (final sequencing libraries).</p></list-item><list-item id=\"o1565\"><label>34.</label><p id=\"p1925\">Perform quality control on the final sequencing libraries</p></list-item><list-item id=\"o1570\"><label>a.</label><p id=\"p1930\">Estimate the concentration of each sample by quantifying 2 μL of the final sequencing library with a Qubit Fluorometer using the Qubit dsDNA HS Kit, following manufacture’s protocol, to obtain an approximate concentration of PCR products to dilute for quantification on an Agilent 4200 TapeStation. Follow the manufacturer’s instructions. The expected concentration of the libraries is >1.5 ng/μL.</p></list-item><list-item id=\"o1575\"><label>b.</label><p id=\"p1935\">Measure the average fragment size of the mRNA targeted library by using the Agilent TapeStation with the High Sensitivity D5000 tape following the manufacturer’s instructions.</p></list-item><list-item id=\"o1580\"><label>c.</label><p id=\"p1940\">The final mRNA targeted library should show a fragment distribution that depends on the panel used (<xref rid=\"fig9\" ref-type=\"fig\">Figure 9</xref>). The expected size of Sample Tag index PCR product is 290 bp (<xref rid=\"fig10\" ref-type=\"fig\">Figure 10</xref>). You might observe a smaller peak of ∼270 bp, which corresponds to AbSeq product. The expected size of AbSeq index PCR products is ∼270 bp (<xref rid=\"fig11\" ref-type=\"fig\">Figure 11</xref>).</p></list-item></list></p><p id=\"p0835\"><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> Do not use fast cycling mode:</p></disp-quote><table-wrap position=\"float\" id=\"tbl11\"><label>Table 11</label><caption><p>Thermocycler Program for PCR2</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Step</th><th>Cycles</th><th>Temperature</th><th>Time</th></tr></thead><tbody><tr><td>Hot Start</td><td>1</td><td>95°C</td><td>3 min</td></tr><tr><td>Denaturation</td><td rowspan=\"3\">10</td><td>95°C</td><td>30 s</td></tr><tr><td>Annealing</td><td>60°C</td><td>3 min</td></tr><tr><td>Extension</td><td>72°C</td><td>1 min</td></tr><tr><td>Final extension</td><td>1</td><td>72°C</td><td>5 min</td></tr><tr><td>Hold</td><td>1</td><td>4°C</td><td>∞</td></tr></tbody></table></table-wrap></p><p id=\"p0840\"><disp-quote><p><bold><italic>Note:</italic></bold> when preparing the libraries from two independent cartridges, prepare two separate master mixes with unique reverse primers. For information on ordering additional indices beyond the four indices included with the reagent kit contact <ext-link ext-link-type=\"uri\" xlink:href=\"mailto:scomix@bdscomix.bd.com\" id=\"intref0015\">scomix@bdscomix.bd.com</ext-link>.</p></disp-quote><table-wrap position=\"float\" id=\"tbl12\"><label>Table 12</label><caption><p>Indexing PCR Reaction Mix</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Component</th><th>1 Library (μL)</th><th>For Three Libraries (mRNA, AbSeq, Sample Tag) + 10% Overage (μL)</th></tr></thead><tbody><tr><td>PCR MasterMix</td><td>25.0</td><td>82.5</td></tr><tr><td>Library Forward Primer</td><td>2.0</td><td>6.6</td></tr><tr><td>Library Reverse Primer</td><td>2.0</td><td>6.6</td></tr><tr><td>Nuclease-Free Water</td><td>18.0</td><td>59.4</td></tr><tr><td>Total</td><td>47.0</td><td>155.1</td></tr></tbody></table></table-wrap></p><p id=\"p0845\"><disp-quote><p><inline-graphic xlink:href=\"fx3.gif\"/><bold>CRITICAL:</bold> Do not use fast cycling mode:</p></disp-quote><table-wrap position=\"float\" id=\"tbl13\"><label>Table 13</label><caption><p>Thermocycler Program for Indexing PCR</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Step</th><th>Cycles</th><th>Temperature</th><th>Time</th></tr></thead><tbody><tr><td>Hot Start</td><td>1</td><td>95°C</td><td>5 min</td></tr><tr><td>Denaturation</td><td rowspan=\"3\">6–8</td><td>95°C</td><td>30 s</td></tr><tr><td>Annealing</td><td>60°C</td><td>30 s</td></tr><tr><td>Extension</td><td>72°C</td><td>30 s</td></tr><tr><td>Final extension</td><td>1</td><td>72°C</td><td>1 min</td></tr><tr><td>Hold</td><td>1</td><td>4°C</td><td>∞</td></tr></tbody></table></table-wrap><table-wrap position=\"float\" id=\"tbl14\"><label>Table 14</label><caption><p>Suggested PCR Cycle Numbers for Indexing PCR</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Conc. Index PCR Input for Targeted mRNA Libraries (ng/μL)</th><th>Conc. Index PCR Input for Sample Tag and AbSeq Libraries 9 (ng/μL)</th><th>Suggested PCR Cycles</th></tr></thead><tbody><tr><td>1.2–2.7</td><td>0.5–1.1</td><td>6</td></tr><tr><td>0.6–1.2</td><td>0.25–0.5</td><td>7</td></tr><tr><td>0.2–0.6</td><td>0.1–0.25</td><td>8</td></tr></tbody></table></table-wrap></p><p id=\"p0850\"><list list-type=\"simple\" id=\"olist0140\"><list-item id=\"o0660\"><label>d.</label><p id=\"p0855\">To calculate the concentration in nM, use the following equation:</p></list-item></list><disp-formula id=\"ufd1\">C = X × 1×10<sup>6</sup> × (1/MW) × (1/S)</disp-formula>Where<fig id=\"fig9\"><label>Figure 9</label><caption><p>Example of Final mRNA Library TapeStation Trace</p><p>The major peak length will depend on the PCR panels used. If you detect some transcripts with longer base pair lengths, this is typically not an issue as shorter transcripts amplify more efficiently than longer products.</p></caption><graphic xlink:href=\"gr9\"/></fig><fig id=\"fig10\"><label>Figure 10</label><caption><p>Example of Final Sample Tag Library TapeStation Trace</p><p>The expected size of the final Sample Tag library is approximately 290 bp. If you observe a smaller peak (around 270 bp), it is likely those transcripts are AbSeq products.</p></caption><graphic xlink:href=\"gr10\"/></fig><fig id=\"fig11\"><label>Figure 11</label><caption><p>Example of Final AbSeq Library TapeStation Trace</p><p>The expected size of the AbSeq final library is around 270bp.</p></caption><graphic xlink:href=\"gr11\"/></fig></p><p id=\"p0860\">X = the concentration of the library calculated by Qubit in ng/μL</p><p id=\"p0865\">1 × 10<sup>6</sup> is used to convert μL to L,</p><p id=\"p0870\">MW = the molecular weight of dsDNA</p><p id=\"p0875\">S = the average size of your library determined by the Tape Station or Bioanalyzer.</p><p id=\"p0880\">For example, if the Qubit reading for an mRNA library is 45 ng/μL with an average library size of 585 bp, the equation would be:<disp-formula id=\"ufd2\">C = 45 ng/μL × 1×10<sup>6</sup> μL/L × (1/660 mol/g) × (1/585 bp)</disp-formula><disp-formula id=\"ufd3\">C = 116.6 nM</disp-formula><list list-type=\"simple\" id=\"olist0145\"><list-item id=\"o0665\"><label>35.</label><p id=\"p0885\">Combining libraries for sequencing (Multiplexing)</p></list-item><list-item id=\"o0670\"><label>a.</label><p id=\"p0890\">To determine how much of each sample to pool for sequencing, first dilute each sample to the recommended concentration for library pooling according to the Illumina guide (usually 1–4 nM, <xref rid=\"tbl15\" ref-type=\"table\">Table 15</xref>).</p></list-item></list></p><p id=\"tspara0250\">Suggested flowcell loading concentrations for libraries and PhiX for different Illumina systems.<table-wrap position=\"float\" id=\"tbl15\"><label>Table 15</label><caption><p>Sequencing Flowcell Loading and PhiX Concentration</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Illumina System</th><th>Sequencing Flowcell Loading Concentration</th><th>Phix Concentration</th></tr></thead><tbody><tr><td>MiSeq V2</td><td>6–10 pM</td><td>10%</td></tr><tr><td>MiSeq V3</td><td>6–10 pM</td><td>10%</td></tr><tr><td>MiniSeq High or Mid Output</td><td>1–1.5 pM</td><td>20%</td></tr><tr><td>NextSeq High or Mid Output</td><td>1–1.5 pM</td><td>20%</td></tr><tr><td>HiSeq 2500</td><td>7–15 pM</td><td>10%</td></tr><tr><td>HiSeq 3000/4000</td><td>3 nM</td><td>15%</td></tr><tr><td>NovaSeq 6000</td><td>2 nM</td><td>20%</td></tr></tbody></table></table-wrap></p><p id=\"p0895\"><list list-type=\"simple\" id=\"olist0150\"><list-item id=\"o0675\"><label>b.</label><p id=\"p0900\">Next determine how many reads each sample requires. To do this, multiply the target cell number by the desired number of reads per cell. For example, if you wanted to sequence sample one mRNA library at 5,000 reads per cell, and had a target number of 10,000 cells, the number of reads for the sample would be 5,000 × 10,000 = 5,000,000 reads.</p></list-item><list-item id=\"o0680\"><label>c.</label><p id=\"p0905\">Determine the fraction of the reads will go toward each sample. This can be done by dividing by each sample by the total reads needed across all samples. If you wanted to multiplex the Sample Tag, mRNA, and AbSeq libraries from a sample and each has 5,000,000 reads, 50,000,000 reads, and 150,000,000 reads respectively, the total reads is 205,000,000. The fraction of reads that will go to the Sample Tag is 0.02.</p></list-item><list-item id=\"o0685\"><label>d.</label><p id=\"p0910\">To determine the volume (μL) of each sample diluted to the flowcell loading concentration needed to pool for the final multiplexed library, multiply the fraction of reads by the total volume of the library needed. For example, to calculate the volume of Sample Tag (from step 35c) you need in your final library of 150 μL, multiply 0.02 by 150 to determine you will need 3.66 μL.</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> If the volume of library requires is prohibitively small a higher volume can be used for the pooling. For example, if only 5 μL is needed, a 100 μL pool can be made, and 5 μL of that pool can be taken into sequencing.</p></disp-quote><list list-type=\"simple\" id=\"olist0155\"><list-item id=\"o0690\"><label>e.</label><p id=\"p0915\">Pipette each required amount of sample into a LoBind 1.5 mL tube.</p></list-item><list-item id=\"o0695\"><label>36.</label><p id=\"p0920\">Sequence the libraries</p></list-item><list-item id=\"o0700\"><label>a.</label><p id=\"p0925\">Recommended sequencing depths are as follows:</p></list-item><list-item id=\"o0705\"><label>i.</label><p id=\"p0930\">mRNA: 5,000–10,000 reads per cell</p></list-item><list-item id=\"o0710\"><label>ii.</label><p id=\"p0935\">Sample Tag: 600–1,000 reads per cell</p></list-item><list-item id=\"o0715\"><label>iii.</label><p id=\"p0940\">AbSeq: 400–1,000 reads per cell per antibody</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> The recommended sequencing depths are provided as a starting point, since the number of reads that should be targeted is not a fixed value, but rather depends on several factors. First, the number of reads needed per cell for each mRNA library will depend on how many primers are included in your panel and the abundance of the transcripts of interest. If the goal is to capture rare transcripts as much as possible, a higher read per cell sequencing target may be beneficial. Similarly, the targeted reads per cell for each AbSeq library will depend on the makeup of the antibody panel. Proteins that are expressed at high levels will use proportionally more reads, which can leave other antibodies in the panel poorly resolved, particularly if they target proteins that are expressed at low levels.</p></disp-quote></p></sec><sec id=\"sec3.6\"><title>Primary Analysis Pipeline</title><p id=\"p0945\"><disp-quote><p><inline-graphic xlink:href=\"fx2.gif\"/><bold>Timing: 1 h to upload data; run time varies</bold></p></disp-quote><list list-type=\"simple\" id=\"olist0160\"><list-item id=\"o0720\"><label>37.</label><p id=\"p0950\">Download fastq files from BaseSpace from the “File” menu using the BaseSpace Sequence Hub Downloader or obtain Fastq files from your sequencing core.</p></list-item><list-item id=\"o0725\"><label>38.</label><p id=\"p0955\">Create Seven Bridges login by going to <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.sevenbridges.com/bdgenomics/\" id=\"intref0020\">http://www.sevenbridges.com/bdgenomics/</ext-link></p></list-item><list-item id=\"o0730\"><label>39.</label><p id=\"p0960\">Create a new Project in Seven Bridges by clicking on the “Projects” tab and selecting “Create a project”.</p></list-item><list-item id=\"o0735\"><label>40.</label><p id=\"p0965\">Upload the fastq files using the Seven Bridges Uploader by clicking “Upload Files” and selecting the appropriate Fastq files (R1 and R2 are required.)</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> the uploader can be downloaded from the “Data Tools” section of the tab “Data”.</p></disp-quote><list list-type=\"simple\" id=\"olist0165\"><list-item id=\"o0740\"><label>41.</label><p id=\"p0970\">In the “Files” tab of the project, add the fasta reference files for the mRNA and AbSeq panels to the project</p></list-item><list-item id=\"o0745\"><label>a.</label><p id=\"p0975\">Generate AbSeq reference file by going to <ext-link ext-link-type=\"uri\" xlink:href=\"http://abseq-ref-gen.genomics.bd.com/\" id=\"intref0025\">http://abseq-ref-gen.genomics.bd.com/</ext-link> and selecting the antibodies that were used in your experiment. Use the resulting fasta-file and upload using the Seven Bridges Uploader.</p></list-item><list-item id=\"o0750\"><label>b.</label><p id=\"p0980\">Copy the appropriate mRNA fasta reference file (based on the gene panel that you used) to the project from the “Demo Project” in the “Projects” section of file addition</p></list-item><list-item id=\"o0755\"><label>42.</label><p id=\"p0985\">In the “Apps” section of the project, click on “add app” and select the “BD Rhapsody™ Targeted Analysis Pipeline” by clicking “run.</p></list-item><list-item id=\"o0760\"><label>43.</label><p id=\"p0990\">In the new screen, navigate to the “Inputs” section on the left hand side, select the AbSeq .fasta file as the “AbSeq Reference”, the .fastq files for the “Reads”, and the mRNA .fasta file as the “Reference”.</p></list-item><list-item id=\"o0765\"><label>44.</label><p id=\"p0995\">In the “App Settings” section select “Single-Cell Multiplexing Kit- Human” from the dropdown menu in “Multiplexing_Settings” to enable Sample Tag calling. Annotate the Sample Tags as appropriate for your experiment.</p></list-item><list-item id=\"o0770\"><label>45.</label><p id=\"p1000\">Select “Run” to trigger the analysis pipeline. Depending on the size of the experiment, the run can take several hours up to a day.</p></list-item><list-item id=\"o0775\"><label>46.</label><p id=\"p1005\">Once analysis is complete, output files will populate in the “Outputs” section of the analysis.</p></list-item><list-item id=\"o0780\"><label>a.</label><p id=\"p1010\"><italic>“RSEC_MolsPerCell.csv”</italic> and <italic>“DBEC_MolsPerCell.csv”</italic> files can be found in the “Data Tables” section.</p></list-item></list><disp-quote><p><bold><italic>Note:</italic></bold> RSEC_MolsPerCell file can be used regardless of sequencing depth. DBEC_MolsPerCell file should be used if the mean RSEC sequencing depth is at least 6. When comparing files from two different pipeline outputs, RSEC file should be used unless all genes being compared underwent the same correction (either RSEC or DBEC). This information can be found in the #Targets# section of the Metrics Summary<italic>.</italic></p></disp-quote><list list-type=\"simple\" id=\"olist0170\"><list-item id=\"o0785\"><label>b.</label><p id=\"p1015\"><italic>“Sample_Tag_Calls.csv”</italic> can be found in the “Multiplex” section.</p></list-item><list-item id=\"o0790\"><label>c.</label><p id=\"p1020\">Zip files containing the RSEC_MolsPerCell files for only the cells associated with each Sample Tag can be downloaded from the “Multiplex” section.</p></list-item></list></p></sec></sec><sec id=\"sec4\"><title>Expected Outcomes</title><p id=\"p1025\">Type of data that are generated: The protocol presented here details sample and library preparation for the detection of targeted transcript and surface protein expression at the single-cell level. After sequencing and pre-processing of the Fastq files, the final output will essentially yield a large and sparse data matrix containing the molecule counts for mRNA and the proteins for each cell (MolsPerCell.csv).</p><p id=\"p1030\">For the analysis of these complex data sets, there are different options available. Users that prefer a graphical user interface can use commercial software packages such as SeqGeq or BioTuring (among others). If more versatile options are required it is recommended to use the R environment and dedicated packages for single-cell analysis which are commonly available on Bioconductor. A full description of this process and all available analysis approaches is beyond the scope of this protocol and we refer the reader to recent reviews which also include excellent online tutorials and links to additional resources (<xref rid=\"bib1\" ref-type=\"bibr\">Amezquita et al., 2020</xref>; <xref rid=\"bib6\" ref-type=\"bibr\">Luecken and Theis, 2019</xref>).</p><p id=\"p1035\">The exact number of cells that are called in the sequencing data will be less than the number of cells loaded on the cartridge. The number depends on the sample type, accuracy of cell counts and viability of the cells loaded. For a typical experiment with human leukocytes, after quality control filtering and removal of multiplets, users can expect to recover data for about 60% of the total cells loaded.</p><p id=\"p1040\">The final number of gene targets varies depending on the design of the target transcriptomic assay but can be up to 499 target genes. We reported that with an assay targeting 492 genes, 2,000–4,000 reads per cell from the transcriptomic portion of the library delivered sufficient resolution<sup>1</sup>. Like the transcriptomic analysis, the number of proteins targeted can be customized for specific experimental aims. We previously reported that with an assay targeting 41 proteins, read depths of 200–400 reads/antibody/cell from the protein portion of the library results in sufficient resolution<sup>1</sup>.</p></sec><sec id=\"sec5\"><title>Limitations</title><sec id=\"sec5.1\"><title>Limitations to Assessing Protein Expression</title><p id=\"p1045\">Antibody-based probes are powerful tools for detection of specific protein targets. However, meaningful interpretation of sequencing-based protein measurements requires rigorous validation. In some instances we observed that the same antibody clone can yield different expression patterns in sequencing-based analysis relative to conventional cytometry (<xref rid=\"bib7\" ref-type=\"bibr\">Mair et al., 2020</xref>). Furthermore, the sequencing read depth in context of the chosen antibody panel is of particular importance here. Highly expressed proteins (e.g., lineage markers such as CD4, CD8, HLA-DR) will use a significant portion of the sequencing reads, which may make it difficult to resolve less abundant proteins (such as IL-7Rα) if the chosen read depth is too low. While there is no general consensus yet for a minimum read depth in all experimental settings, it is recommended to calculate with at least 200–400 reads per antibody per cell as a rule of thumb (<xref rid=\"bib7\" ref-type=\"bibr\">Mair et al., 2020</xref>).</p></sec><sec id=\"sec5.2\"><title>Limitations to Assessing Transcript Expression</title><p id=\"p1050\">Our previous comparison between whole transcriptome and targeted transcript analysis demonstrated that targeted analysis can be highly sensitive in detection of low-abundance transcripts, but some genes may be under-represented compared to whole transcriptome analysis (and vice versa). This may result from different amplification efficiencies between multiplexed targeted primers used in the Rhapsody platform and the template-switch process in other WTA platforms. A lack of a signal for a specific transcript (by either WTA or targeted transcriptomics) cannot necessarily be interpreted as absence of transcript expression. Further, transcripts that contain internal poly A stretches can also artificially inflate the number of captured transcript molecules with WTA that would otherwise correctly not be captured in a 3’ end targeted approach. Differences in poly A site expression (location and total number) due to tissue types and cell states (i.e., activated versus resting) can cause inherent heterogeneity in abundance and identity of observed transcripts. Because of these issues one possible strategy is to first use WTA on the sample of interest to inform locations of poly A sites to include in the primer design of targeted mRNA panels to better correlate these two types of data. Finally, it is important to remember in this context that the dynamic range of expression for proteins spans about 6–7 orders of magnitude, whereas transcript copy numbers span about two orders of magnitude (<xref rid=\"bib3\" ref-type=\"bibr\">Azimifar et al., 2014</xref>; <xref rid=\"bib9\" ref-type=\"bibr\">Schwanhausser et al., 2011</xref>), with a mean copy number of ∼4 copies per cell, which are inherently difficult to detect with any single-cell analysis approach.</p></sec><sec id=\"sec5.3\"><title>Limitations to the Number of Cells that Can Be Analyzed and Enrichment Strategies</title><p id=\"p1055\">If the experimental goal is an exploratory snapshot of all immune cells, one may start with a bulk immune cell population (e.g., human peripheral blood lymphocytes). However, if a specific and possibly rare cell subset is of interest, then enriching for these cells may be necessary to obtain meaningful data. For example, human dendritic cell subsets might represent only 10–100 cells in a typical PBMC sample of 20,000 cells, thus requiring enrichment for meaningful analysis.</p><p id=\"p1060\">If enrichment by cell sorting (FACS) is performed, it is important to ensure that the used fluorochrome-conjugated antibodies do not overlap or interfere with the antibody clones used in the downstream oligo-antibody assay. This can be tested e.g., by prior co-staining of different antibody clones by flow cytometry and selecting only compatible clones. As an alternative, if the same clones have to be used, it is possible to evaluate the cell staining ratios of fluorochrome-conjugated antibodies with AbSeq antibodies coupled with complementary sequence-conjugated fluorochrome (Flow Proxy) to determine an optimal ratio for cell sorting while maintaining AbSeq staining. In these scenarios, fluorochrome-conjugated Abs can be stained simultaneously with oligo-conjugated AbSeq reagents saving researchers time and minimize handling and potential perturbations to cells.</p><p id=\"p1065\">Importantly, if magnetic enrichment protocols are used, positive selection magnetic bead enrichment should be avoided while negative selection/depletion is preferred. Ferrous particles that remain on cells from enrichment may interfere with downstream processing in the nano-well-cartridge.</p></sec></sec><sec id=\"sec6\"><title>Troubleshooting</title><sec id=\"sec6.1\"><title>Problem 1</title><p id=\"p1070\">Final library concentration lower than what is required for sequencing.</p></sec><sec id=\"sec6.2\"><title>Potential Solution</title><p id=\"p1075\">If the final library has too low of a concentration, the indexing PCR can be re-done with the PCR2 products using more than the recommended 6–8 cycles. The additional number of cycles will depend on how much more the final library concentration needs to increase. It is recommended that you start with just one or two extra cycles more to avoid as much PCR amplification bias as possible. Another option is to return to PCR1 products, or completely remake the library from the saved exonuclease treated beads.</p></sec><sec id=\"sec6.3\"><title>Problem 2</title><p id=\"p1080\">Tape Station traces are showing peaks that are not very clean (see <xref rid=\"fig12\" ref-type=\"fig\">Figure 12</xref>).<fig id=\"fig12\"><label>Figure 12</label><caption><p>Example of a Poor-Quality Trace</p><p>This is an example of a library that had an incomplete double-sided selection. The library should undergo another round of cleanup.</p></caption><graphic xlink:href=\"gr12\"/></fig></p></sec><sec id=\"sec6.4\"><title>Potential Solution</title><p id=\"p1085\">This is generally due to poor SPRISelect bead clean up. Ensure you are using exactly the indicated volume of beads and there are no SPRISelect bead droplets on your pipette tips. The SPRISelect bead clean up can be repeated on a sample that has already been cleand up, using the same ratio of beads to sample as before, although some sample loss should be expected.</p></sec><sec id=\"sec6.5\"><title>Problem 3</title><p id=\"p1090\">Visualization of AbSeq data in FlowJo does not show the expected expression patterns.</p></sec><sec id=\"sec6.6\"><title>Potential Solution</title><p id=\"p1095\">One of the possible analysis options is to convert the molecule per cell CSV-file into the flow cytometry standard (FCS) file format by using R packages such as flowCore or premessa. This allows the visualization of bivariate plots in the popular software package FlowJo. However, the data ranges for RNA and protein expression are widely different, and thus it is important to adjust the transformation of these two molecule classes appropriately. One option is to choose the arcsinh transform in FlowJo and adjusting the co-factor as required to achieve bimodal expression for well-defined protein markers (e.g., CD3).</p></sec><sec id=\"sec6.7\"><title>Problem 4</title><p id=\"p1100\">No signal obtained for oligo-antibody conjugate after prior enrichment with cell sorting.</p></sec><sec id=\"sec6.8\"><title>Potential Solution</title><p id=\"p1105\">The reason for this can be that a competing fluorescent-labeled antibody clone was used for cell enrichment, preventing the subsequent binding of the oligo-antibody conjugate to its target. As discussed in more detail in the section “<xref rid=\"sec5\" ref-type=\"sec\">Limitations</xref>”, if the same antibody targets are needed for enrichment as well as for readout during the multi-omic workflow, it is recommended to use different antibody clones. Experimenters need to perform prior co-staining experiments using these clones in a conventional flow cytometry experiment to test whether the clones are compatible (e.g., for targeting human CD3, the two clones OKT3 and SK7 can be used).</p></sec><sec id=\"sec6.9\"><title>Problem 5</title><p id=\"p1110\">Poor resolution obtained for oligo-antibody signal.</p></sec><sec id=\"sec6.10\"><title>Potential Solution</title><p id=\"p1115\">For some antibodies and experimental setups, the final concentration as pre-determined by the manufacturer might be suboptimal. In this case, it is recommended to titer the oligo-antibody conjugate on a conventional flow cytometer using an oligo-nucleotide coupled to fluorochromes (oligo-dT-fluorochrome).</p></sec></sec><sec id=\"sec7\"><title>Resource Availability</title><sec id=\"sec7.1\"><title>Lead Contact</title><p id=\"p1120\">Further information and requests for resources and reagents that are not commercially available should be directed to and will be fulfilled by the Lead Contact, Martin Prlic (<ext-link ext-link-type=\"uri\" xlink:href=\"mailto:mprlic@fredhutch.org\" id=\"intref0030\">mprlic@fredhutch.org</ext-link>).</p></sec><sec id=\"sec7.2\"><title>Materials Availability</title><p id=\"p1125\">This study did not generate new unique reagents.</p></sec><sec sec-type=\"data-availability\" id=\"sec7.3\"><title>Data and Code Availability</title><p id=\"p1130\">The published article includes all code generated during this study. 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The graphical abstract was created using <ext-link ext-link-type=\"uri\" xlink:href=\"http://BioRender.com\" id=\"intref0045\">BioRender.com</ext-link>. J.R.E. was supported by <funding-source id=\"gs1\"><institution-wrap><institution-id institution-id-type=\"doi\">10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source> (NIH), United States, T32 AI007509-20. F.M. was supported through The American Association of Immunologists (AAI), United States, Intersect Fellowship Program for Computational Scientists and Immunologists.</p><sec id=\"sec9\"><title>Author Contributions</title><p id=\"p1145\">J.R.E., F.M., G.B., J.M., A.J.T., S.M., M.N., and M.P. wrote the original draft.</p></sec><sec sec-type=\"COI-statement\" id=\"sec10\"><title>Declaration of Interests</title><p id=\"p1150\">J.R.E., F.M. G.B., and M.P. declare no competing interests. J.M., A.J.T., S.M., and M.N. are employees of BD Biosciences.</p></sec></ack></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Genet</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Genet.</journal-id><journal-title-group><journal-title>Frontiers in Genetics</journal-title></journal-title-group><issn pub-type=\"epub\">1664-8021</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33093841</article-id><article-id pub-id-type=\"pmc\">PMC7523636</article-id><article-id pub-id-type=\"doi\">10.3389/fgene.2020.00984</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Genetics</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Genotypic and Phenotypic Analysis in Chinese Cohort With Autosomal Recessive Osteogenesis Imperfecta</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Shan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/863574/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Cao</surname><given-names>Yixuan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/826854/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Han</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1060281/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Lulu</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/985601/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ren</surname><given-names>Xiuzhi</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Mi</surname><given-names>Huan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Yanzhou</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Guan</surname><given-names>Yun</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1061652/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhao</surname><given-names>Feiyue</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Mao</surname><given-names>Bin</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/824875/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Tao</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>You</surname><given-names>Yi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Guan</surname><given-names>Xin</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Yujiao</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Xue</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/829779/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhao</surname><given-names>Xiuli</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/751075/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College</institution>, <addr-line>Beijing</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>The People’s Hospital of Wuqing District</institution>, <addr-line>Tianjin</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Shandong Provincial Hospital Affiliated to Shandong University</institution>, <addr-line>Jinan</addr-line>, <country>China</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine</institution>, <addr-line>Baltimore, MD</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Oliver Semler, Universitätsklinikum Köln, Germany</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Annalisa Vetro, Meyer University Hospital, University of Florence, Italy; Hao Zhang, Shanghai Sixth People’s Hospital, China</p></fn><corresp id=\"c001\">*Correspondence: Xiuli Zhao, <email>xiulizhao@ibms.pumc.edu.cn</email></corresp><corresp id=\"c002\">Xue Zhang, <email>xuezhang@pumc.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Genetics of Common and Rare Diseases, a section of the journal Frontiers in Genetics</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>984</elocation-id><history><date date-type=\"received\"><day>31</day><month>10</month><year>2019</year></date><date date-type=\"accepted\"><day>04</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Li, Cao, Wang, Li, Ren, Mi, Wang, Guan, Zhao, Mao, Yang, You, Guan, Yang, Zhang and Zhao.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Li, Cao, Wang, Li, Ren, Mi, Wang, Guan, Zhao, Mao, Yang, You, Guan, Yang, Zhang and Zhao</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Osteogenesis imperfecta (OI) is a rare heritable skeletal disorder which is mainly caused by defected type I collagen. Autosomal recessive OI (AR-OI) is caused by mutations of genes that are responsible for type I collagen modification and folding, and is often associated with more severe phenotypes. Due to the limited number of recessive OI patients, it has been difficult to study the mutation spectrum as well as the correlation of genotype and phenotype. This study recruited a Chinese cohort of 74 AR-OI families, aiming to establish the mutation spectrum and to examine the genotypic and phenotypic correlation. We identified 82 variants including 25 novel variants and 57 HGMD reported variants in these AR-OI patients, using whole exome sequencing/panel sequencing combined with Sanger sequencing. Pathogenic mutations were found at <italic>WNT1</italic> (<italic>n</italic> = 30, 40.54%), <italic>SERPINF1</italic> (<italic>n</italic> = 22, 29.73%), <italic>FKBP10</italic> (<italic>n</italic> = 10, 13.51%), <italic>CRTAP</italic> (<italic>n</italic> = 3, 4.05%), <italic>P3H1</italic> (<italic>n</italic> = 3, 4.05%), <italic>SERPINH1</italic> (<italic>n</italic> = 2, 2.70%), <italic>SEC24D</italic> (<italic>n</italic> = 3, 4.05%), and <italic>PLOD2</italic> (<italic>n</italic> = 1, 1.35%) respectively. Thus, <italic>WNT1</italic> represents the most frequent pathogenic gene of AR-OI in Chinese population. The most common clinical manifestations of AR-OI patients include walking problem (72.86%), scoliosis (65.28%) and frequent fractures (fractures ≥2/year) (54.05%). Interestingly, ptosis represents a unique phenotype of patients carrying <italic>WNT1</italic> variants, and it was rare in patients harboring other pathogenic genes. Our study expanded the mutation spectrum of AR-OI and enriched the knowledge of genotypic and phenotypic correlation in Chinese cohort with AR-OI.</p></abstract><kwd-group><kwd>autosomal recessive osteogenesis imperfecta</kwd><kwd>mutation spectrum</kwd><kwd>phenotype</kwd><kwd><italic>WNT1</italic></kwd><kwd>Chinese cohort</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source><award-id rid=\"cn001\">81472053</award-id></award-group></funding-group><counts><fig-count count=\"4\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"29\"/><page-count count=\"13\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Osteogenesis imperfecta (OI), also known as brittle bone disease, is a genetically and clinically heterogeneous skeletal disorder. Typical clinical manifestations include low bone mass, frequent fractures, short stature, blue sclerea, and bone deformity. More than 90% of OI cases are caused by a defect of type I collagen, and therefore OI is known as an autosomal dominant inherited disease due to a mutation in <italic>COL1A1</italic> or <italic>COL1A2</italic>, which encodes alpha 1 or alpha 2 chains of type I collagen (<xref rid=\"B20\" ref-type=\"bibr\">Marini et al., 2007</xref>). Multiple genes have been reported to contribute to the development of autosomal recessive OI (AR-OI), including <italic>SERPINF1</italic>, <italic>LEPRE1</italic>, <italic>CRTAP</italic>, <italic>PPIB</italic>, <italic>FKBP10</italic>, <italic>BMP1</italic>, <italic>SP7</italic>, <italic>PLOD2</italic>, <italic>TMEM38B</italic>, <italic>P4HB</italic>, <italic>SERPINH1</italic>, <italic>SEC24D</italic>, <italic>WNT1</italic>, <italic>CREB3L1</italic>, and <italic>SPARC</italic> (<xref rid=\"B9\" ref-type=\"bibr\">Kang et al., 2017</xref>). Although the incidence rate of AR-OI is < 10% of the whole OI population, the clinical manifestations of AR-OI are much more severe than the dominant OI patients (<xref rid=\"B16\" ref-type=\"bibr\">Liu et al., 2017</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Li et al., 2019</xref>).</p><p>Synthesis of type I collagen is a sophisticated process. With the development of next generation sequencing, remarkable progress has been made in identifying new genes associated with the modification and folding of type I collagen. These genes have formed a unique biological network, and deficiency in any individual gene may lead to recessive OI. Propeptides encoded by <italic>COL1A1</italic> and <italic>COL1A2</italic> undergo post-translational modifications in the endoplasmic reticulum, followed by transportation to Golgi and cleavage of N-/C- terminal propeptides. Consequently, collagen fibers are formed to further build up the collagen matrix. Defect in any step during the type I collagen synthesis can lead to the development of OI (<xref rid=\"B9\" ref-type=\"bibr\">Kang et al., 2017</xref>). For example, deficiency of <italic>CRTAP</italic>, <italic>P3H1</italic> or <italic>PPIB</italic>, the components of collagen prolyl 3-hydroxylation complex, will lead to defects in post translational modification of unfolded collagen alpha-chains (<xref rid=\"B21\" ref-type=\"bibr\">Morello et al., 2006</xref>). Mutations in <italic>FKBP10</italic>, <italic>SERPINH1</italic> or <italic>BMP1</italic> cause defects in collagen folding and crosslinking (<xref rid=\"B12\" ref-type=\"bibr\">Koide et al., 2006</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Schwarze et al., 2013</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Kang et al., 2017</xref>). Alterations in <italic>SP7</italic> (<xref rid=\"B3\" ref-type=\"bibr\">Azetsu et al., 2017</xref>), <italic>WNT1</italic> (<xref rid=\"B11\" ref-type=\"bibr\">Keupp et al., 2013</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Laine et al., 2013</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Pyott et al., 2013</xref>) or <italic>CREB3L1</italic> (<xref rid=\"B23\" ref-type=\"bibr\">Murakami et al., 2009</xref>) affect osteoblast differentiation (<xref rid=\"B7\" ref-type=\"bibr\">Forlino and Marini, 2016</xref>). Dysfunction of PEDF, which is encoded by <italic>SERPINF1</italic>, will damage the bone homeostasis (<xref rid=\"B1\" ref-type=\"bibr\">Akiyama et al., 2010</xref>).</p><p>Although mutation spectrums on autosomal dominant OI have been established in large cohorts of Chinese (<xref rid=\"B14\" ref-type=\"bibr\">Li et al., 2019</xref>), Swedish (<xref rid=\"B15\" ref-type=\"bibr\">Lindahl et al., 2015</xref>) and Canadian/American populations (<xref rid=\"B4\" ref-type=\"bibr\">Bardai et al., 2016</xref>), mutation spectrum on AR-OI remains unclear due to its rare incidence. There was only one report about the molecular spectrum based on findings from a small cohort of 19 recessive OI patients from Mediterranean (<xref rid=\"B26\" ref-type=\"bibr\">Rauch et al., 2010</xref>). Here, we examined the mutation spectrum in 74 AR-OI families, the largest cohort worldwide to our knowledge. Current findings expand our knowledge about the gene spectrum and phenotypic spectrum of AR-OI, and provide important information for genetic diagnosis of AR-OI patients.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Subjects</title><p>We recruited 74 AR-OI probands from a cohort of 1095 OI patients (646 families) in mainland China. These patients displayed typical clinical manifestations of OI: recurrent fractures, short stature, bone malformation, with or without extra-skeletal manifestations such as blue sclera, hearing loss and dentinogenesis imperfecta. After obtaining approval from Institutional Review Board (IRB) of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China (015-2015) and informed consent from all participants/legal guardians of children under 18, peripheral blood samples were collected from all available family members of AR-OI patients. Skin samples were collected from some of the patients based on the availability.</p><p>Clinical parameters of probands were recorded at their first visit, including age, gender, height, times of fractures, presence of ptosis, scoliosis, blue sclerae, dentinogenesis imperfecta, and ability of walking. Height was also converted to Z-score calculated based on the age and medium height of Chinese population (<xref rid=\"B14\" ref-type=\"bibr\">Li et al., 2019</xref>).</p></sec><sec id=\"S2.SS2\"><title>Variant Nomenclature</title><p>Variants were named according to the nomenclature provided by Human Genome Variation Society<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup>. Genomic DNA and cDNA sequences of <italic>SERPINF1</italic> (NC_000017.10), <italic>CRTAP</italic> (NC_000003.11), <italic>SERPINH1</italic> (NC_000011.9), <italic>FKBP10</italic> (NC_000017.10), <italic>PLOD2</italic> (NC_000003.11), <italic>WNT1</italic> (NC_000012.11), <italic>SEC24D</italic> (NC_000004.11), <italic>P3H1</italic> (NC_000001.11) were obtained from National Center for Biotechnology Information (NCBI) reference sequence and University of California, Santa Cruz (UCSC) Genome browser database<sup><xref ref-type=\"fn\" rid=\"footnote2\">2</xref></sup>.</p></sec><sec id=\"S2.SS3\"><title>Whole Exome Sequencing (WES)</title><p>Samples from 59 probands underwent WES process. Genomic DNA was extracted from leukocytes using a standard sodium dodecyl sulfate-proteinase <sc>K</sc>-phenol/chloroform extraction method. 1–3 μg genomic DNA was used for WES as described previously (<xref rid=\"B14\" ref-type=\"bibr\">Li et al., 2019</xref>). DNA was fragmented as 150 bp, and the primers and adapters were then ligated to the DNA fragments to construct libraries. The analysis was performed on singletons, looking on previously known genes as filtering criteria. Sequencing was carried out on HiSeq 4000 System (Illumina). SAMtools mpileup and bcftools were used for variant calling and SNP/Indels identification. Control-FREEC was utilized for CNV detection.</p></sec><sec id=\"S2.SS4\"><title>Genomic Panel Sequencing</title><p>Fifteen probands were examined by genomic panel sequencing. Customized panel sequencing including 184 genes related to the monogenic disorders focused in our lab was conducted as described previously (<xref rid=\"B14\" ref-type=\"bibr\">Li et al., 2019</xref>). 21 OI-related genes were included with 18 recessive OI genes. Sequencing was performed on the HiSeq 2500 System (Illumina, San Diego, CA, United States). All reference sequences were based on the GRCh37/hg19 assembly of the human genome.</p></sec><sec id=\"S2.SS5\"><title>Confirmation of Pathogenic Mutation by Sanger Sequencing</title><p>To verify the candidate mutations detected by WES or Panel Sequencing, genomic DNA was amplified following the PCR program: 95°C for 3 min; 94°C for 30 s, 58°C for 30 s, 72°C for 50 s (35 cycles); 72°C for 8 min. PCR amplified fragments was subjected to Sanger Sequencing based on Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, United States). Sequence was analyzed using CodonCode Aligner (version 6.0.2.6; CodonCode, Centerville, MA, United States).</p></sec><sec id=\"S2.SS6\"><title>Isolation and Culturing of Dermal Fibroblasts</title><p>To test the splicing effect caused by two variants in <italic>FKBP10</italic> in proband PUMC-121 on endogenous level, skin samples of the proband and an ethnically matched healthy individual were collected from fresh skin biopsies. Human fibroblasts were maintained at 37°C and 5% CO<sub>2</sub> and supplied with F-12 supplemented with 1% <sc>L</sc>-glutamine, 20% fetal bovine serum, 1% sodium pyruvate, and 1% penicillin-streptomycin.</p></sec><sec id=\"S2.SS7\"><title>RNA Level Analysis</title><p>When a mutation was located in an intron, RNA level analysis would be conducted. Total RNA was isolated from peripheral blood or skin fibroblasts using Trizol reagent (Invitrogen, Cat. No. 15596018) followed by cDNA preparation using GoScript<sup>TM</sup> Reverse Transcription System (Promega, Cat. No. A5001), according to the manufacturer’s instructions. cDNA was used for sequencing or for quantitative PCR analysis.</p></sec><sec id=\"S2.SS8\"><title>Quantitative PCR (qPCR)</title><p>qPCR was carried out to detect large fragment deletion and mRNA expression level of <italic>FKBP10</italic> gene. PCR amplification was carried out with SYBR Premix ExTaq (Takara, Bio., Dalian, China) and primer pairs (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Table S1</xref>) according to the manufacturer’s protocol. The reactions were monitored continuously in a Rotor-Gene 6000 instrument (Qiagen, Hilden, Germany) according to the following program: 95°C for 3 min followed by 40 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 20 s. The relative copy number (RCN) of the targeted sequence was normalized to the expression levels of GAPDH by calculating the ΔCt (Ct<sub>gene of interest</sub> - Ct<sub>GAPDH</sub>), and RCN = 2<sup>–ΔΔCt</sup>.</p></sec><sec id=\"S2.SS9\"><title>T-Clone Sequencing</title><p>T-Clone sequencing was used when Sanger sequencing results showed interlaced alleles. Briefly, The PCR product of target samples with disrupted signals was linked to the pMD19-T vector. The ligation reaction contained 4 μl Solution I (Takara, Shiga, Japan), 1 μl pMD19-T vector and 5 μl purified PCR product. DNA Sanger sequencing was performed followed by vector ligation, <italic>E. coli</italic> transformation and bacterial culturing.</p></sec><sec id=\"S2.SS10\"><title>Statistical Analysis</title><p>Statistical analysis was conducted for age of first onset, times of fractures, frequency of fractures, height, height Z-score and ptosis for the patients with pathogenic genes <italic>WNT1</italic>, <italic>SERPINF1</italic>, and <italic>FKBP10</italic>. GraphPad Prism (version 6.00; GraphPad Software, La Jolla, CA, United States) was used for statistical analysis and was performed for <italic>n</italic> ≥ 9. Differences between three or more groups were analyzed by one-way ANOVA, and comparisons between two groups were analyzed by student <italic>t</italic>-test. Graphs were presented as average ± standard deviation, and <italic>p</italic> < 0.05 was considered as significant difference.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Phenotypic Characterization of AR-OI</title><p>We recruited 74 probands (39 males, 34 females, and one gender unknown fetus) with AR-OI from 60 non-consanguineous families and 14 consanguineous families. Except for the fetus, their ages ranged from 6 months to 35 years old and their parents were confirmed to be unaffected. The clinical manifestations of the 74 probands, including frequent fractures, scoliosis, short stature, blue sclerae, ptosis, disability to walk and dentinogenesis imperfecta (DI) were recorded and analyzed (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Phenotypes of all probands were summarized in <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Table S2</xref>. These patients presented severe phenotypes: most of them showed disability to walk (72.86%, <xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>) and scoliosis (65.28%, <xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>). Nearly half of them experienced more than 2 fracture times per year (54.05%, <xref ref-type=\"fig\" rid=\"F1\">Figure 1F</xref>), 38.03% of them had dentinogenesis imperfecta (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>), and 38.03% of patients presented extremely low short statures with a Z score less than −4 SD (corresponding to type III OI). Compared to dominant inherited OI, blue sclerae was less frequent (32.43%) in AR-OI individuals. A unique phenotype, ptosis (23.88%), was observed in recessive inherited OI patients (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>), but was absent in dominant OI patients. None of AR-OI individuals displayed hearing loss or intellectual disability.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Phenotypic characteristics of AR-OI patients <bold>(A–F)</bold>. AR-OI patients had severe clinical manifestations including ptosis <bold>(A)</bold>, dentinogenesis imperfecta <bold>(B)</bold>, severe limb deformity and disability to walk independently <bold>(C)</bold>, scoliosis <bold>(D)</bold>, skeleton deformity <bold>(E)</bold>, and frequent fractures <bold>(F)</bold>. <bold>(G)</bold> Percentage of the presence of each clinical manifestation in AR-OI probands.</p></caption><graphic xlink:href=\"fgene-11-00984-g001\"/></fig></sec><sec id=\"S3.SS2\"><title>Genotypic Characterization of Recessive OI</title><sec id=\"S3.SS2.SSS1\"><title>Variants Identification of Recessive OI</title><p>Variants of the 74 probands were identified in <italic>WNT1</italic> (<italic>n</italic> = 30), <italic>SERPINF1</italic> (<italic>n</italic> = 22), <italic>FKBP10</italic> (<italic>n</italic> = 10), <italic>SERPINH1</italic> (<italic>n</italic> = 2), <italic>CRTAP</italic> (<italic>n</italic> = 3), <italic>PLOD2</italic> (<italic>n</italic> = 1), <italic>P3H1</italic> (<italic>n</italic> = 3), and <italic>SEC24D</italic> (<italic>n</italic> = 3) (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and <xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). Mutations in <italic>WNT1</italic> showed the highest percentage (40.54%) in this cohort, followed by mutations in <italic>SERPINF1</italic> (29.73%) and <italic>FKBP10</italic> (13.51%, <xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Identification of mutations of recessive osteogenesis imperfecta in Chinese population.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Proband number</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Family history</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Consan- guineous</bold></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Allele origin</bold></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Variant type</bold></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Variant position</bold></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Nucleotide change</bold></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Amino acid change</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Novelty</bold></td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"9\" rowspan=\"1\"><bold><italic>WNT1</italic></bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-3 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.397G > A; c.677C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ala133Thr; p.Ser226Leu</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.506dupG; c.506dupG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Cys170Leufs*6; p.Cys170Leufs*6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.506dupG; c.506dupG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Cys170Leufs*6; p.Cys170Leufs*6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.301C > T; c.382T > G</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg101Cys; p.Phe128Val</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-128 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>De novo</italic>; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nonsense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.189delG; c.620G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Leu64*; p.Arg207His</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-145</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 4; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.677C > T; c.677C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ser226Leu; p.Ser226Leu</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-154</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.466delC; c.466delC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg156Glyfs*43; p.Arg156Glyfs*43</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-158</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.216dupA; c.506G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg73Thrfs*82; p.Gly169Asp</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-217</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.620G > A; c.620G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg207His; p.Arg207His</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-221 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.506dupG; c.506dupG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Cys170Leufs*6; p.Cys170Leufs*6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-226</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.620G > A; c.620G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg207His; p.Arg207His</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-230</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; nonsense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.301C > T; c.681C > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg101Cys; p.Cys227*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-245</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Splicing; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Intron 1; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.105-2A > G; c.674G > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.?; p.Gly225Val</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-247</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 4; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.677C > T; c.677C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ser226Leu; p.Ser226Leu</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-258</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.10delT; c.677C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Trp4Glyfs*35; p.Ser226Leu</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-274</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.301C > T; c.937C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg101Cys; p.Arg313Cys</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-277</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; exon 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.2T > C; c.216dupA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.?; p.Arg73Thrfs*82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-281 (<italic>n</italic> = 3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; nonsense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 4; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.770T > C; c.774C > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Leu257Pro; p.Try258*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-327</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Splicing; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Intron 1; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.104 + 1G > A; c.501G > C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.?; p.Trp167Cys</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-329</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.371C > T; c.620G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Thr124Met; p.Arg207His</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-471 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 4; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.677C > T; c.677C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ser226Leu; p.Ser226Leu</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-490</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.620G > A; c.620G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg207His; p.Arg207His</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-491</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 4; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c. 557A > T; c. 557A > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Asp186Val; p.Asp186Val</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-554</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 4; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.677C > T; c.677C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ser226Leu; p.Ser226Leu</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-555</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.397G > A; c.986G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ala133Thr; p.Cys329Thr</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-572 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2;exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.301C > T; c.710C > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg101Cys; p.Pro237His</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-586 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.493T > C; c.677C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Trp165Arg; p.Ser226Leu</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-612</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 4; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.677C > T; c.677C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ser226Leu; p.Ser226Leu</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-618</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.514G > T; c.590T > C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Asp172Tyr; p.Leu197Pro</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-630</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.397G > A; c.505_506del</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ala133Thr; (p.Gly169Leufs*6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; N</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"9\" rowspan=\"1\"><bold><italic>SERPINF1</italic></bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.261_265 dupGGCCC; c.879delC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Leu89Argfs*26; p.Thr294Profs*8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-33 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inframe; inframe</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.271_279dup; c.271_279dup</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ala91_Ser93dup; p.Ala91_Ser93dup</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-74</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; exon 1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.1A > G; c.1A > G</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.?; p.?</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-150 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; nonsense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.248_249delTC; c.397C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Leu83Glnfs*28; p.Gln133*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-255 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nonsense; nonsense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 6; exon 6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.907C > T; c.907C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg303*; p.Arg303*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-275</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inframe; inframe</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 6; exon 6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.829_831 delTTC; c.829_831 delTTC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Phe277del; p.Phe277del</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-306</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.261_265 dupGGCCC; c.261_265 dupGGCCC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Leu89Argfs*26; p.Leu89Argfs*26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-331</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; exon 1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.77dupC; c.77dupC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Glu27Glyfs*38; p.Glu27Glyfs*38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-381</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Splicing; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Intron 2; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.283 + 1G > T; c.498-499delCA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.?; p.Arg167Serfs*35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-348</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 7; exon 7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.1193dupT; c.1193dupT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg399Glufs*31; p.Arg399Glufs*31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-422</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 6; exon 6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.839dupT; c.839dupT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Lys281Glufs*20; p.Lys281Glufs*20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-496</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; exon 1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.77dupC; c.77dupC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Glu27Glyfs*38; p.Glu27Glyfs*38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-482</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.271_279del; c.748_763del</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ala91_Ser93del; p.Val250Trpfs*14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-495</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; exon 1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.77dupC; c.77dupC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Glu27Glyfs*38; p.Glu27Glyfs*38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-413</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nonsense; nonsense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 6; exon 6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.808G > T; c.808G > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Gly270*; p.Gly270*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-527 (<italic>n</italic> = 3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nonsense; nonsense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 6; exon 6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.907C > T; c.907C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg303*; p.Arg303*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-546</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 6; exon 6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chr17:1,679,402-1,680,079; Chr17:1,679,402-1,680,079 (hg19)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.?; p.?</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-585 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nonsense; nonsense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 4; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.553C > T; c.553C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Gln185*; p.Gln185*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-595</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 6; exon 6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.863_866 dupTGAT; c.879delC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Glu290Aspfs*12; p.Thr294Profs*8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-607</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.167C > G; c.271_279dup</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ala56Gly; p.Ala91_Ser93dup</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-611</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nonsense; nonsense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; exon 1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.79G > T; c.79G > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Glu27*; p.Glu27*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-624</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nonsense; nonsense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 3; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.397C > T; c.397C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Gln133*; p.Gln133*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"9\" rowspan=\"1\"><bold><italic>FKBP10</italic></bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-68 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 5; exon 5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.809_843del; c.813_814delGA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Leu270Glnfs*91; p.Glu271Aspfs*101</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-121</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Splicing; splicing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Intron 5; exon 6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.918-6T > G; c.1016G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ser306Argfs*18; p.Ser306Argfs*18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-157</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.344G > A; c.831delC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg115Gln; p.Gly278Alafs*20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-207 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; gross deletion</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.831delC; chr17: g.39974881 _39980318del (hg19)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Gly278Alafs*20; p.?</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-405</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.320_353del; c.344G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.107_118del; p.Arg115Gln</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-431</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 2; exon 5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c. 344G > A; c.831dupC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg115Gln; p.Gly278Argfs*95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-525</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 5; exon 8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.831dupC; c.1390delG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Gly278Argfs*95; p.Glu464Argfs*67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-536</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; frameshift</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 5; exon 5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.831dupC; c.831dupC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Gly278Argfs*95; p.Gly278Argfs*95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-605</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Splicing; splicing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Intron 5; intron 5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.918-3C > G; c.918-3C > G</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.?; p.?</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-606</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; splicing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; intron 5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.1A > G; c.918-6T > G</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Met1Val; p.Ser306Argfs*18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; R</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"9\" rowspan=\"1\"><bold><italic>SERPINH1</italic></bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-285</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.149T > G; c.1214G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Leu50Arg; p.Arg405His</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-324 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; exon 3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.589G > C; c.800T > C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Gly197Arg; p.Leu267Pro</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"9\" rowspan=\"1\"><bold><italic>CRTAP</italic></bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-118 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nonsense; splicing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; intron 1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.202G > T; c.471 + 4delA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Glu68*; p.?</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-456 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Splicing; splicing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Intron 6; intron 6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.1153-3C > G; c.1153-3C > G</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Gly385Argfs*46; p.Gly385Argfs*46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-582</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; splicing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; intron 6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.18_25 dupGG GGGCCG; c.1153-3C > G</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Ala9Glyfs*7; p.Gly385Argfs*46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"9\" rowspan=\"1\"><bold><italic>PLOD2</italic></bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-320</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 18; exon 18</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.1856G > A; c.1856G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Arg619His; p.Arg619His</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"9\" rowspan=\"1\"><bold><italic>P3H1</italic></bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-566</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nonsense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon3; exon14</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.652G > T; c.1948G > C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Glu218*; p.Gly650Arg</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-590</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nonsense; nonsense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon3; exon15</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.652G > T; c.2164C > T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Glu218*; p.Gln722*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-597</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; splicing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 9; intron 14</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.1466T > C; c.1915-1G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Leu489Pro; p?</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"9\" rowspan=\"1\"><bold><italic>SEC24D</italic></bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-266 (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Y</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 16; exon 21</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.2185G > A; c.2869A > G</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Val729Met; p.Thr957Ala</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N; N</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-204</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Maternal; paternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Missense; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 6; exon7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.875C > T; c.938G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Pro292Leu; p.Arg313His</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; R</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PUMC-514</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Paternal; maternal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frameshift; missense</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Exon 1; exon17</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">c.113dupC; c.2296G > A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">p.Thr39Asnfs*16; p.Gly766Ser</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">R; N</td></tr></tbody></table><table-wrap-foot><attrib><italic>* For each proband, <italic>n</italic> = 1 unless stated on the table; <italic>n</italic> = number of patients; In lane Family history/Consanguineous, Y = Yes, N = No; In lane Novelty, R = Reported, N = Novel. * The novelty was recorded according to HGMD database (professional 2020.01).</italic></attrib></table-wrap-foot></table-wrap><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Genotypic characterization of AR-OI patients. <bold>(A)</bold> Mutation distribution in Chinese AR-OI patients. <bold>(B)</bold> The number of pedigrees in each pathogenic gene. <bold>(C)</bold> Distribution of mutation types in Chinese AR-OI patients.</p></caption><graphic xlink:href=\"fgene-11-00984-g002\"/></fig><p>Four major types of variants were found in this cohort, including missense, nonsense, frameshift and splicing mutations. We analyzed the number of each type of variant in each pathogenic gene and found that missense mutation was the main variant type in <italic>WNT1</italic>, <italic>SERPINH1</italic> and <italic>SEC24D</italic>, and frameshift was frequently observed in <italic>SERPINF1</italic> and <italic>FKBP10</italic> (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). Most variants were located at exons, but a homozygous intronic variant c.1153-3C > G was found in <italic>CRTAP</italic> (PUMC-456) (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1</xref>). Sequencing analysis of RT-PCR product of RNA isolated from dermal fibroblasts of this proband and his father confirmed that variant c.1153-3C > G led to an insertion of AG in the mutant transcript (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1C</xref>).</p></sec><sec id=\"S3.SS2.SSS2\"><title>Establishment of Mutation Spectrums of <italic>WNT1</italic>, <italic>SERPINF1</italic>, and <italic>FKBP10</italic></title><p>We identified 82 variants including 25 novel variants and 57 HGMD (professional 2020.01) reported variants (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Note that there were 36 variants reported in our previous study (<xref rid=\"B14\" ref-type=\"bibr\">Li et al., 2019</xref>). Because most variants are located in <italic>WNT1</italic>, <italic>SERPINF1</italic> and <italic>FKBP10</italic>, the mutation spectrums of these genes were established (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Mutation spectrum of <italic>WNT1</italic>\n<bold>(A)</bold>, <italic>SERPINF1</italic>\n<bold>(B)</bold>, and <italic>FKBP10</italic>\n<bold>(C)</bold> of Chinese AR-OI patients. Black boxes represent the exons in each gene and lines represent the introns. Variants found in this study are listed above the boxes with novel variants in red and reported variants in black. Variants that have been reported previously are also listed under the boxes. Notation *digit indicates the number of probands carrying the same variant.</p></caption><graphic xlink:href=\"fgene-11-00984-g003\"/></fig><sec id=\"S3.SS2.SSS2.Px1\"><title><italic>WNT</italic>1</title><p>We found 28 variants in <italic>WNT1</italic> from 30 probands, including 21 reported variants and 7 novel variants (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and <xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). There were some hotspot variants in <italic>WNT1</italic>: homozygous mutation c.506dupG(p.Cys170Leufs<sup>∗</sup>6) was found in three unrelated non-consanguineous families (PUMC-10, 21, and 221), leading to the production of truncated protein; Mutation c.677C > T(p.Ala133Thr) was observed in PUMC-3, 145, 247, 258, 471, and 554; Mutation c.620G > A (p.Arg207His) occurred in two probands (PUMC-128 and 329) and three non-consanguineous families (PUMC-217, 226, and 490), representing a hotspot in this Chinese cohort.</p></sec><sec id=\"S3.SS2.SSS2.Px2\"><title><italic>SERPINF</italic>1</title><p>Eight consanguineous and 14 non-consanguineous families were identified with mutations in <italic>SERPINF1</italic>, including 21 variants (14 reported variants and 7 novel variants) (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and <xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). A known homozygous mutation, c.77dupC (p.Glu27Glyfs<sup>∗</sup>38), was found in three non-consanguineous families, which resulted in frameshift and truncated protein. Interestingly, there were three siblings from a consanguineous family (PUMC-527) identified with same homozygotes of a novel variant, c.907C > T in <italic>SERPINF1</italic>, but they exhibited extremely different phenotypes (<xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2</xref>). The proband had severe scoliosis and unable to walk independently with more than 45 times of fracture. However, his elder sister, harboring the same genotype, did not present any OI phenotype at all with only once fracture lifetime.</p></sec><sec id=\"S3.SS2.SSS2.Px3\"><title><italic>FKBP1</italic>0</title><p>Thirteen variants in <italic>FKBP10</italic>, including 10 known variants and 3 novel variants, were identified in 10 non-consanguineous families (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and <xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>). Hotspot c.831indelC was identified in PUMC-157, 207, 431, 525 and 536, and these variants led to a premature termination codon. Hotspot c.344G > A was identified in PUMC-157, 405, and 431. Among the patients with variants in <italic>FKBP10</italic>, three special cases needed to be highlighted. Case I, Patient PUMC-405 was a compound heterozygote with mutations in <italic>FKBP10</italic>. Sanger sequencing analysis by software <italic>Chromas</italic> showed disrupted signals in exon 2 in <italic>FKBP10</italic> (<xref ref-type=\"supplementary-material\" rid=\"FS4\">Supplementary Figure S4A</xref>), and two alleles were separated by T clone sequencing of the mutant region: c.320_353del(p.107_118del) with a 34 bp deletion (<xref ref-type=\"supplementary-material\" rid=\"FS4\">Supplementary Figure S4B</xref>), and a missense mutation c.344G > A(p.Arg115Gln) within the deleted region (<xref ref-type=\"supplementary-material\" rid=\"FS4\">Supplementary Figure S4C</xref>); Case II, Patient PUMC-207 harbored a gross deletion and an indel variant in <italic>FKBP10</italic>. The compound mutations found in proband PUMC-207 were inherited from the parents: the indel of c.831delC was inherited from the mother and the gross deletion of chr17: g.39974881_39980318del (hg19) was inherited from the father, which was confirmed in breakpoint analysis using Gap-PCR and Sanger sequencing (<xref ref-type=\"supplementary-material\" rid=\"FS5\">Supplementary Figure S5</xref>); Case III, Patient PUMC-121 was previously reported that an intronic variant c.918-6T > G and a variant in adjacent exon c.1016G > A in <italic>FKBP10</italic> similarly drove to skipping of exon 6, indicated by a minigene assay (<xref ref-type=\"supplementary-material\" rid=\"FS6\">Supplementary Figures S6A,B</xref>) (<xref rid=\"B14\" ref-type=\"bibr\">Li et al., 2019</xref>). Based on the sequencing results from cDNA (<xref ref-type=\"supplementary-material\" rid=\"FS6\">Supplementary Figure S6C</xref>) and RNA isolated from the patient’s dermal fibroblasts in the follow up study, we found that variant c.918-6T > G caused skipping of exon 6 and variant c.1016G > A caused partial skipping of exon 6. This was confirmed by qPCR analysis (<xref ref-type=\"supplementary-material\" rid=\"FS6\">Supplementary Figure S6D</xref>).</p></sec></sec></sec><sec id=\"S3.SS3\"><title>Correlation Between Genotypic and Phenotypic Changes</title><p>In order to answer whether there is a correlation between genotypes and observed phenotypes in AR-OI patients, we first analyzed the sites of fractures in patients harboring different pathogenic genes. We did not find significant difference in fracture locations among patients with different causative genes (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). We then analyzed the phenotypes including fracture times, scoliosis, height, blue sclerae, DI, walking ability, and ptosis in these patients. Statistical analysis was conducted for <italic>WNT1</italic>, <italic>SERPINF1</italic> and <italic>FKBP10</italic>, with > 3 probands (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Table S3</xref>), and only ptosis was significantly different between groups.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Correlation analysis of genotype and phenotype of Chinese AR-OI patients. <bold>(A)</bold> Analysis of fracture sites in patients with variants in <italic>WNT1</italic>, <italic>SERPINF1</italic>, <italic>FKBP10</italic>, <italic>SERPINH1</italic>, <italic>CRTAP</italic>, <italic>PLOD2</italic>, <italic>P3H1</italic>, and <italic>SEC24D</italic>. <bold>(B)</bold>. Fracture frequencies in patients with and without ptosis phenotype. Comparison of age of first onset <bold>(C)</bold> and height <bold>(D)</bold> of patients with variants in <italic>WNT1</italic>, <italic>SERPINF1</italic>, and <italic>FKBP10</italic> (*<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01).</p></caption><graphic xlink:href=\"fgene-11-00984-g004\"/></fig><sec id=\"S3.SS3.SSS1\"><title>Patients With <italic>WNT1</italic> Mutation Had Ptosis Phenotype</title><p>We noticed that AR-OI patients carrying <italic>WNT1</italic> mutations were prone to developing ptosis, with 46.67% penetrance (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Table S3</xref>). This manifestation was common in patients with a homozygous function loss of <italic>WNT1</italic>, but was rare in patients with a loss of <italic>SERPINF1</italic>. Ptosis distribution displayed significant difference in different genes (<italic>p</italic> = 0.0042). In patients with <italic>WNT1</italic> mutations, those with ptosis also showed more severe skeletal dysfunctions including higher fracture frequency (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>) and unable to walk independently (80%), as compared to those without ptosis.</p></sec><sec id=\"S3.SS3.SSS2\"><title>Patients With Variants in <italic>SERPINF1</italic> Presented a Distinct Phenotype</title><p>Compared to patients with mutations in <italic>WNT1</italic> and <italic>FKBP10</italic>, patients with variants in <italic>SERPINF1</italic> showed significantly delayed first fracture age (<italic>p</italic> = 0.0072, <xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>) and taller height (<italic>p</italic> = 0.0460, <xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref>).</p></sec></sec></sec><sec id=\"S4\"><title>Discussion</title><p>With the development of next generation sequencing, genes associated with OI have been well-studied and identified. We examined mutation spectrum of AR-OI in a large cohort of 74 Chinese OI families. Current study unraveled 25 novel variants from 82 identified variants, and demonstrated <italic>WNT1</italic> mutation as the most frequent mutation in all AR-OI genes. Furthermore, these AR-OI patients showed a unique phenotype, ptosis.</p><p>Type I collagen is a heterotrimer structure with two alpha 1 chains and one alpha 2 chain. Dominant OI is caused by mutations in <italic>COL1A1</italic> or <italic>COL1A2</italic>, and phenotypes can vary from mild to moderate/severe depending on the whether the collagen defect was caused by haplo-insufficiency or helical mutations that led to dominant negative effect (<xref rid=\"B26\" ref-type=\"bibr\">Rauch et al., 2010</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Lindahl et al., 2015</xref>). In AR-OI, alteration occurs during post translational modification progress when a triple helix pro-collagen is already formed, and hence most of these mutations affect both the synthesis of the whole type I collagen protein and bone homeostasis. Accordingly, AR-OI patients often exhibit more severe phenotypes than dominant OI, including significantly earlier first fracture age, higher fracture frequency, higher percentage of scoliosis and higher proportion of disability to walk (<xref rid=\"B14\" ref-type=\"bibr\">Li et al., 2019</xref>). Consistent with previous findings, more severe clinical manifestations were also observed in a cohort of Chinese patients with recessive OI in current study, including a high rate of scoliosis, disability of independent walking, extreme short stature, and high facture frequency (<xref ref-type=\"fig\" rid=\"F1\">Figure 1G</xref>).</p><p>Current study in Chinese AR-OI patients suggests that <italic>WNT1</italic> is the most frequently involved gene, followed by <italic>SERPINF1</italic> and <italic>FKBP10</italic>. These findings differ from that in Caucasian population, which showed that <italic>SERPINF1</italic> was the highest, followed by <italic>CRTAP</italic> (<xref rid=\"B4\" ref-type=\"bibr\">Bardai et al., 2016</xref>). The most distinct phenotypic characteristic in Chinese AR-OI patients was a unique ptosis phenotype, which was presented almost exclusively in patients associated with <italic>WNT1</italic> variants (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Table S3</xref>). This result is consistent with recent finding that ptosis was a hallmark for OI patients with <italic>WNT1</italic> mutations from Indian and Turkish families (<xref rid=\"B24\" ref-type=\"bibr\">Nampoothiri et al., 2019</xref>), and that from seven Chinese families (<xref rid=\"B18\" ref-type=\"bibr\">Lu et al., 2018</xref>). <italic>WNT1</italic> signaling was critical for the cross talk between osteoblastic lineage and osteoclastic-lineage cells (<xref rid=\"B13\" ref-type=\"bibr\">Laine et al., 2013</xref>). In addition to its vital importance in bone homeostasis (<xref rid=\"B19\" ref-type=\"bibr\">Luther et al., 2018</xref>), <italic>WNT1</italic> also plays an essential role in neurological development (<xref rid=\"B6\" ref-type=\"bibr\">Faqeih et al., 2013</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Aldinger et al., 2016</xref>). Because patients displayed ptosis all suffered neurological dysfunctions (<xref rid=\"B24\" ref-type=\"bibr\">Nampoothiri et al., 2019</xref>), it was postulated that ptosis phenotype may be associated with impaired brain or nerve development. Although we did not observe any intellectual problem in AR-OI patients with ptosis phenotype, these patients developed more severe skeletal phenotypes than those without ptosis (<xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref>). No correlation was found between ptosis and mutation type or variant location. The mechanism of the development of ptosis in patients with <italic>WNT1</italic> variant-induced AR-OI remains to be elucidated in future studies.</p><p>The severity of skeletal phenotype was compared in patients with <italic>WNT1</italic>, <italic>SERPINF1</italic>, and <italic>FKBP10</italic> variants. Patients with variants in <italic>SERPINF1</italic> had the highest number of average fracture times (30.77 ± 6.626) and highest frequency of fractures (4.193 ± 0.7986 n/year). However, their stature is the tallest (108.7 ± 3.154 cm, <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Table S3</xref>). <xref rid=\"B22\" ref-type=\"bibr\">Mrosk et al. (2018)</xref> reported that the phenotypic severity of genes associated with OI is: <italic>WNT1</italic> > <italic>SERPINF1</italic> > <italic>FKBP10</italic>, based on findings from 50 OI families, including 24 recessive OI families. In the current study, patients with <italic>WNT1</italic> variants presented the shortest height (97.40 ± 3.889) and highest percentage of scoliosis 80.00%, but did not show any significant difference in other parameters such as height Z-score, blue sclerae, dentinogenesis imperfecta and disability of independent walking from patients with variants in <italic>SERPINF1</italic> and <italic>FKBP10</italic> (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Table S3</xref>). It needs to be noted that patients with <italic>FKBP10</italic> variants all presented a short neck phenotype and severe spinal malformation (<xref ref-type=\"supplementary-material\" rid=\"FS3\">Supplementary Figure S3</xref>). FKBP10 is known to function as a molecular chaperone that interacts with collagen (<xref rid=\"B8\" ref-type=\"bibr\">Ishikawa et al., 2008</xref>), but how it affects the cervical vertebra development remains unclear.</p><p>Recurrent variants were found in <italic>WNT1</italic> (c.506dupG, c.677C > T, and c.620G > A), <italic>SERPINF1</italic> (c.77dupC, c.907C > T) and <italic>FKBP10</italic> (c.831indelC and c.344G > A). All the homozygous of these recurrent variants corresponded to a severe phenotype (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Table S2</xref>). In particularly, OI patients with the novel variant c.907C > T in <italic>SERPINF1</italic> presented the most severe phenotypes: with 45–100 times of fractures, severe scoliosis and extremely short stature (Z-score < −8) (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Table S2</xref>). Previous studies also identified some recurrent variants, such as c.506dupG in <italic>WNT1</italic>, but did not find a clear correlation with the severity of phenotype (<xref rid=\"B17\" ref-type=\"bibr\">Liu et al., 2016</xref>). Variant c.506dupG in <italic>WNT1</italic> was found to be a hotspot region in 5 families among 10 total families examined (<xref rid=\"B24\" ref-type=\"bibr\">Nampoothiri et al., 2019</xref>). <xref rid=\"B10\" ref-type=\"bibr\">Kelley et al. (2011)</xref> reported the recurrent variant, c.344G > A in <italic>FKBP10</italic> from a Caucasian child, who presented multiple fractures and a short stature. Further studies are needed to establish a causal-relationship between hotspot variant sites and phenotypes in these patients.</p><p>Frameshift occupied a high proportion in <italic>SERPINF1</italic> and <italic>FKBP10</italic> mutations (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>), suggesting that the two genes may have a distinct mechanism for OI development. In particular, most of variants located in <italic>SERPINF1</italic> may be involved in occurrence of nonsense/frameshift mediated pre-termination codon (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), and these patients had very severe skeletal phenotypes, which is in line with previous findings (<xref rid=\"B5\" ref-type=\"bibr\">Becker et al., 2011</xref>). <italic>SERPINF1</italic> encodes pigment epithelium-derived factor (PEDF), which can inhibit osteoclastogensis by regulating osteoprotegerin expression (<xref rid=\"B1\" ref-type=\"bibr\">Akiyama et al., 2010</xref>) and enhancing pre-osteoblast differentiation (<xref rid=\"B29\" ref-type=\"bibr\">Toru Akiyama et al., 2003</xref>). The truncated PEDF protein which mainly results in the absence of C terminal of PEDF, led to the malfunction of protein (<xref rid=\"B28\" ref-type=\"bibr\">Shao et al., 2003</xref>). Therefore, nonsense/frameshift mutations in <italic>SERPINF1</italic> may lead to truncated PEDF and severe OI phenotype.</p><p>In summary, we examined the largest cohort (<italic>n</italic> = 74) of Chinese AR-OI probands and established the mutation spectrum of the three most frequent pathogenic genes (<italic>WNT1</italic>, <italic>SERPINF1</italic>, <italic>FKBP10</italic>) in this population. We also identified 25 novel variants and 7 hotspot variants. Genotypic and phenotypic analysis unraveled ptosis as a unique phenotype in patients with <italic>WNT1</italic> variants. Furthermore, patients with ptosis or hotspot variants showed more severe skeletal phenotypes than others. Current findings may enrich our understanding of the genetic basis of AR-OI, and our study provides new knowledge for a precise diagnosis of this disease in Chinese population.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The datasets for this article are not publicly available due to concerns regarding participant/patient anonymity. Requests to access the datasets should be directed to the corresponding author.</p></sec><sec id=\"S6\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by Institutional Review Board (IRB) of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China (015-2015). Written informed consent to participate in this study was provided by all participants/legal guardians of children under 18.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>SL, YC, and HW performed the experiment and wrote the manuscript. XR and YW collected the samples of patients. HM, YG, FZ, LL, BM, TY, YYo, XG, and YYa conducted data analysis. XZho and XZhn designed and supervised this research. All authors performed critical reading and approved the final version of manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This study was supported by grants from National Key Research and Development Program of China (2016YFE0128400 and 2016YFC0905100), CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-3-003), and National Natural Science Foundation of China (81472053).</p></fn></fn-group><ack><p>We thank all the OI patients and their families for their participation.</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.hgvs.org/munomen\">http://www.hgvs.org/munomen</ext-link></p></fn><fn id=\"footnote2\"><label>2</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://genome.ucsc.edu/\">http://genome.ucsc.edu/</ext-link></p></fn></fn-group><sec id=\"S10\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fgene.2020.00984/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fgene.2020.00984/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"FS1\"><label>FIGURE S1</label><caption><p>Dermal fibroblast analysis confirmed the sequencing result of intronic variant c.1153-3C > G in <italic>CRTAP</italic>. <bold>(A)</bold> Pedigree information was shown in the family tree. <bold>(B)</bold> Results from genomic DNA sequencing showed an intronic mutation of c.1153-3C > G in <italic>CRTAP</italic> in the proband II1. Parents of the proband were heterozygous carrier of the same variant. <bold>(C)</bold> Sequencing of dermal fibroblasts of I1 and II1 confirmed an insertion of AG in the mutant transcript.</p></caption><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS2\"><label>FIGURE S2</label><caption><p>A consanguineous family that had the same variant but presented variable expressivity. <bold>(A)</bold> The pedigree of consanguineous family PUMC-527. <bold>(B)</bold> A photo of patients II1 (mild) and II2 (severe) with a wide difference in their phenotypes. <bold>(C)</bold> Genotype of all the familial members revealed by DNA sequencing.</p></caption><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS3\"><label>FIGURE S3</label><caption><p>Recessive OI patients with variants in <italic>FKBP10</italic> exhibited a unique short neck phenotype <bold>(A–C)</bold>. Photo records and X-ray examinations of three individuals with <italic>FKBP10</italic> variants. <bold>(A)</bold> Short neck (PUMC-68), <bold>(B)</bold> short neck and severe deformity of spinal (PUMC-68), <bold>(C)</bold> compressible vertebrae (PUMC-121).</p></caption><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS4\"><label>FIGURE S4</label><caption><p>A case of micro-deletion and missense mutation in the same region in <italic>FKBP10</italic>. <bold>(A)</bold> Sanger DNA sequencing of proband PUMC-405 showed disrupted signal in exon 2 in <italic>FKBP10</italic>. Two mutations was separated after T-clone sequencing: c.320_353del(p.107_118del) <bold>(B)</bold> and c.344G > A(p.Arg115Gln) which was located within the deletion region <bold>(C)</bold>.</p></caption><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS5\"><label>FIGURE S5</label><caption><p>A case of gross deletion in <italic>FKBP10</italic>. <bold>(A)</bold> Sanger sequencing results of the proband and his parents. <bold>(B)</bold> An intragenic deletion was found in the proband and his father indicated by quantitative real-time PCR. The values presented as triplicate determinations ± SD. RCN, relative copy number. <bold>(C)</bold> Breakpoint analysis showed that breakpoint was found in proband and his father (640 bp), while absence in the mother of proband because of the existence of original fragment. M: Marker, 640 bp: fragment length of the deletion junction product, 304 bp: control. <bold>(D)</bold> Sequence chromatograms of the g.41818632_41824069del breakpoint.</p></caption><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS6\"><label>FIGURE S6</label><caption><p>Identification of the splicing effect of compound mutations in <italic>FKBP10</italic>. <bold>(A)</bold> Sanger sequencing indicated a missense variant c.1016G > A(p.Arg339Gln) in exon 6 and an intronic variants c.918-6T > G in adjacent intron 5 in <italic>FKBP10</italic> in patient PUMC-121. <bold>(B)</bold> Minigene assay showed that both c.918-6T > G and c.1016G > A led in skipping of exon 6. <bold>(C)</bold> Sequencing result of RT-PCR product from RNA extracted from the patient’s dermal fibroblasts. <bold>(D)</bold> Relative normalized expression of exon 6, exons 4–5, and exons 7–8 comparing the patient and control. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Bioeng Biotechnol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Bioeng. Biotechnol.</journal-id><journal-title-group><journal-title>Frontiers in Bioengineering and Biotechnology</journal-title></journal-title-group><issn pub-type=\"epub\">2296-4185</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33042969</article-id><article-id pub-id-type=\"pmc\">PMC7523645</article-id><article-id pub-id-type=\"doi\">10.3389/fbioe.2020.568186</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Bioengineering and Biotechnology</subject><subj-group><subject>Mini Review</subject></subj-group></subj-group></article-categories><title-group><article-title>3D Printing of Metal/Metal Oxide Incorporated Thermoplastic Nanocomposites With Antimicrobial Properties</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Abudula</surname><given-names>Tuerdimaimaiti</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1019838/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Qurban</surname><given-names>Rayyan O.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1024027/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bolarinwa</surname><given-names>Sherifdeen O.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1066665/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Mirza</surname><given-names>Ahmed A.</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1066600/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Pasovic</surname><given-names>Mirza</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1029300/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Memic</surname><given-names>Adnan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/992373/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Center of Nanotechnology, King Abdulaziz University</institution>, <addr-line>Jeddah</addr-line>, <country>Saudi Arabia</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Biochemistry, Faculty of Science, King Abdulaziz University</institution>, <addr-line>Jeddah</addr-line>, <country>Saudi Arabia</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Physics, Faculty of Science, King Abdulaziz University</institution>, <addr-line>Jeddah</addr-line>, <country>Saudi Arabia</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University</institution>, <addr-line>Jeddah</addr-line>, <country>Saudi Arabia</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Department of Electrical and Computer Engineering, Faculty of Engineering, King Abdulaziz University</institution>, <addr-line>Jeddah</addr-line>, <country>Saudi Arabia</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Eun-Bum Cho, Seoul National University of Science and Technology, South Korea</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Fatemeh Kabirian, Materials and Energy Research Center, Iran; Shun Duan, Beijing University of Chemical Technology, China</p></fn><corresp id=\"c001\">*Correspondence: Adnan Memic, <email>amemic@kau.edu.sa</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Biomaterials, a section of the journal Frontiers in Bioengineering and Biotechnology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>568186</elocation-id><history><date date-type=\"received\"><day>31</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>13</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Abudula, Qurban, Bolarinwa, Mirza, Pasovic and Memic.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Abudula, Qurban, Bolarinwa, Mirza, Pasovic and Memic</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Three-dimensional (3D) printing has experienced a steady increase in popularity for direct manufacturing, where complex geometric items can be produced without the aid of templating tools, and manufacturing waste can be remarkably reduced. While customized medical devices and daily life items can be made by 3D printing of thermoplastics, microbial contamination has been a serious obstacle during their usage. A very clever approaches to overcome this challenge is to incorporate antimicrobial metal or metal oxide (M/MO) nanoparticles within the thermoplastics during or prior to 3D printing. Many M/MO nanoparticles can prevent contamination from a wide range of microorganism, including antibiotic-resistant bacteria via various antimicrobial mechanisms. Additionally, they can be easily printed with thermoplastic without losing their integrity and functionality. In this mini review, we summarize recent advancements and discuss future trends related to the development of 3D printed antimicrobial thermoplastic nanocomposites by addition of M/MO nanoparticles.</p></abstract><kwd-group><kwd>3D printing</kwd><kwd>antimicrobial</kwd><kwd>metals</kwd><kwd>metal oxides</kwd><kwd>thermoplastics</kwd><kwd>nanocomposites</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">King Abdulaziz University<named-content content-type=\"fundref-id\">10.13039/501100004054</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"1\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"75\"/><page-count count=\"8\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Three-dimensional (3D) printing has garnered great interest among not only manufacturers for quick prototyping but also amongst the general public (<xref rid=\"B32\" ref-type=\"bibr\">MacDonald and Wicker, 2016</xref>). According to Wohlers Report 2020, the size of the global 3D printing market is $13.7 billion, exhibiting 25% annual growth since 2014 (<xref rid=\"B68\" ref-type=\"bibr\">Wohlers Talk, 2020</xref>). The technology allows for end-use products with complex shapes to be precisely fabricated by a layered additive approach without templating tools such as molds (<xref rid=\"B32\" ref-type=\"bibr\">MacDonald and Wicker, 2016</xref>; <xref rid=\"B61\" ref-type=\"bibr\">Valino et al., 2019</xref>). Accordingly, customized production using this technique can remarkably reduce the cost for low-volume manufacturing and even provide opportunities for customers to make their own “mini factory” (<xref rid=\"B13\" ref-type=\"bibr\">Gibson et al., 2014</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Memic et al., 2017</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Poelma and Rolland, 2017</xref>). Moreover, traditional subtractive manufacturing causes massive materials waste, which can be reduced by as much as 90% when 3D printing is utilized (<xref rid=\"B61\" ref-type=\"bibr\">Valino et al., 2019</xref>). Due to these advantages, 3D printing has become a promising alternative for producing aerospace, automotive, healthcare and consumer devices (<xref rid=\"B32\" ref-type=\"bibr\">MacDonald and Wicker, 2016</xref>; <xref rid=\"B33\" ref-type=\"bibr\">Martin et al., 2017</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Gerdes et al., 2020</xref>). However, current 3D printing potential is hampered by limited material choices that meet required performance specifications for a particular application (<xref rid=\"B32\" ref-type=\"bibr\">MacDonald and Wicker, 2016</xref>). Therefore, developing multifunctional advanced materials that can easily satisfy end-use needs has become one of the central foci for researchers in this field.</p><p>Thermoplastics can be inexpensive, easy to process, chemically stable, lightweight, and flexible materials making them very attractive for 3D printing applications (<xref rid=\"B45\" ref-type=\"bibr\">Poelma and Rolland, 2017</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Blok et al., 2018</xref>). Many personal and medical devices are mainly made of thermoplastics (<xref rid=\"B5\" ref-type=\"bibr\">Blok et al., 2018</xref>; <xref rid=\"B61\" ref-type=\"bibr\">Valino et al., 2019</xref>), making them the main material source for customer-level desktop printing systems. Furthermore, they are easily used in 3D printing applications, and are printable via different approaches such as selective laser sintering (SLS), stereolithography (SLA) and fused deposition modeling (FDM). Among them, FDM is one of the most popular and cost-effective printing techniques and is almost unique for printing of thermoplastics (<xref rid=\"B61\" ref-type=\"bibr\">Valino et al., 2019</xref>). However, microbial contamination still represent a challenge for biomedical applications of 3D printing, especially for handheld and medical devices (<xref rid=\"B16\" ref-type=\"bibr\">González-Henríquez et al., 2019a</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Memic et al., 2019</xref>). In general, contamination associated with medical devices has been a serious issue in clinical treatment. Bacterial contamination can pose a critical threat to patients and considerably increase the healthcare cost due to need for reoperation and/or replacement of infected devices (<xref rid=\"B62\" ref-type=\"bibr\">Vasilev et al., 2009</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Tamayo et al., 2016</xref>). For example, urinary catheters, indwelling vascular catheters and mechanical ventilation are responsible for 95% of urinary tract infections, 87% of bloodstream infections and 86% of pneumonia cases, respectively (<xref rid=\"B48\" ref-type=\"bibr\">Richards et al., 1999</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Weinstein and Darouiche, 2001</xref>). Accumulation of bacteria on polymeric substrates could cause the formation of biofilms in which bacteria are 1,000 to 10,000 times more resistant to antibiotics than those not in a biofilm (<xref rid=\"B62\" ref-type=\"bibr\">Vasilev et al., 2009</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Gnanadhas et al., 2015</xref>). Thus, it is vital to develop 3D printable antimicrobial thermoplastic materials to minimize risk of infection during their usage.</p><p>The current standard to fight infections, namely the usage of antibiotics, is facing a challenge attributed to ongoing mutation of bacterial microorganisms. As mutations are constantly occurring, so is the development of resistance to antibiotics (<xref rid=\"B15\" ref-type=\"bibr\">Gold et al., 2018</xref>). Alternatively, integration of metals and metal oxides (M/MO) within thermoplastics is one of the most promising approaches to design flexible plastic devices with antimicrobial properties (<xref rid=\"B11\" ref-type=\"bibr\">Emamifar, 2011</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Tamayo et al., 2016</xref>). Most of the essential metals such as copper, zinc, magnesium and their oxides have strong biocidal effects, while other non-essential M/MO including silver, gold and cerium oxide are popular antibacterial agents (<xref rid=\"B3\" ref-type=\"bibr\">Al-Shawafi et al., 2017</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Gold et al., 2018</xref>). Recent advancements in nanotechnology have enabled the synthesis of M/MO in the nano-size range, which greatly enhance their antimicrobial performance (<xref rid=\"B34\" ref-type=\"bibr\">Martinez-Gutierrez et al., 2010</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Gold et al., 2018</xref>). Low concentrations, ranging from 0.1 to 4% of M/MO nanoparticles added in the polymer matrix can be functionally sufficient for prevention of most infections; a concentration often compatible with mammalian cells with negligible toxicity (<xref rid=\"B34\" ref-type=\"bibr\">Martinez-Gutierrez et al., 2010</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Diez-Pascual and Diez-Vicente, 2017</xref>). Additionally M/MO nanoparticles inhibit or stunt bacterial growth via various mechanisms dissimilar to antibiotics, which would require a specific type of bacteria to undergo multiple gene mutations to generate any kind of resistance, if any at all (<xref rid=\"B15\" ref-type=\"bibr\">Gold et al., 2018</xref>). Furthermore, M/MO do not chemically deteriorate or thermally degrade in the temperature range of 3D printing of thermoplastics (<xref rid=\"B47\" ref-type=\"bibr\">Rhim et al., 2013</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Palza, 2015</xref>). To best of our knowledge, this is one of the first reviews to summarize the progress made in this sub-field.</p><p>In this mini review, we will summarize the latest progress, advances and trends related to M/MO induced thermoplastic composites fabricated using 3D printing. First, we will discuss general aspects of the antimicrobial mechanism of thermoplastic nanocomposites. Next, we will describe the incorporation of nanoparticles in 3D printing and discuss key factors affecting antimicrobial activity of thermoplastic composites. Finally, we will highlight the impact of 3D printed antimicrobial thermoplastics in different applications and give insights for future developments. The overall purpose of this mini review is to highlight the impact of antimicrobial thermoplastic composites on progressive application of 3D printing.</p></sec><sec id=\"S2\"><title>Antimicrobial Mechanism of the Nanocomposites</title><p>Antimicrobial activity of metals such as silver and copper has been observed since ancient times, as they were used for water sterilization and wound healing (<xref rid=\"B43\" ref-type=\"bibr\">Palza, 2015</xref>; <xref rid=\"B56\" ref-type=\"bibr\">Tamayo et al., 2016</xref>). However, the broad potential of M/MO antimicrobial agents has more recently been recognized due to the advancements in nanotechnology (<xref rid=\"B20\" ref-type=\"bibr\">Hasan et al., 2018</xref>). Metals and their oxides can form nanoparticles through numerous top-down and bottom-top synthesis approaches including sol-gel, co-precipitation, electrochemical, green synthesis, microwave, and sonochemical amongst other approaches (<xref rid=\"B51\" ref-type=\"bibr\">Salah et al., 2019</xref>). M/MO nanoparticles exhibit significantly superior antibacterial performance compared to the micro/macro particles or the bulk surface, often correlated with their smaller size and their high surface area that aid in their bioactivity and mechanism of action discussed below (<xref rid=\"B15\" ref-type=\"bibr\">Gold et al., 2018</xref>). Additionally, M/MO nanoparticles have advantages including the prevention of biofilm formation by inhibiting planktonic growth, altering lipids and proteins, damaging DNA, and interfering with enzyme activities of bacteria (<xref rid=\"B15\" ref-type=\"bibr\">Gold et al., 2018</xref>). Hence, antibiotic-resistant bacteria could be effectively inhibited by M/MO nanoparticles, as they exert antimicrobial activities via other mechanisms different from ones exerted by antibiotics (<xref rid=\"B46\" ref-type=\"bibr\">Raghunath and Perumal, 2017</xref>). However, over-dosage and/or an immediate release of the nanoparticles could cause injuries to certain eukaryotic cells (<xref rid=\"B15\" ref-type=\"bibr\">Gold et al., 2018</xref>).</p><p>Incorporation of M/MO nanoparticles into polymers could improve overall antibacterial efficacy of the nanocomposite, due to the synergetic polymer and nanoparticle effects (<xref rid=\"B56\" ref-type=\"bibr\">Tamayo et al., 2016</xref>). In most M/MO thermoplastic nanocomposites, the antimicrobial mechanism is mainly associated with toxicity of M/MO nanoparticles (<xref rid=\"B52\" ref-type=\"bibr\">Sánchez-López et al., 2020</xref>). On the other hand, polymer matrix as a support regulates the release behavior of the nanoparticles and ions as a response to bacterial adhesion on the composite surface (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1</xref>). Namely, water molecules could diffuse from the bacterial medium into the polymer matrix, and cause release of metal ions. Although antimicrobial mechanisms of M/MO are still a subject of meticulous research, owing to the continuous mutation of bacteria, there are three major mechanisms (<xref rid=\"B15\" ref-type=\"bibr\">Gold et al., 2018</xref>).</p><sec id=\"S2.SS1\"><title>Metal Ion Limitation</title><p>Metal and metal oxide nanoparticles can inhibit bacterial growth by attacking the electronegative phospholipid bilayers of the bacterial cell wall with electropositive metal ions (<xref rid=\"B6\" ref-type=\"bibr\">Chandrangsu et al., 2017</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Raghunath and Perumal, 2017</xref>). They release cations to the anionic sites of the cell membrane and neutralize charges within the cell via electrostatic interaction. This charge difference favors accumulation of metallic ions, which in turn permeates the cell membrane and recruit intrinsic metals to be adsorbed into it (<xref rid=\"B46\" ref-type=\"bibr\">Raghunath and Perumal, 2017</xref>). In response, the homeostatic system in the cell releases more ions from its storage to make up for the loss. This cycle continues until the cell is starved of its essential metals and metal-dependent metabolic processes come to a halt (<xref rid=\"B15\" ref-type=\"bibr\">Gold et al., 2018</xref>). This antimicrobial mechanism has been demonstrated in iron oxide and zinc-based nanoparticles (<xref rid=\"B46\" ref-type=\"bibr\">Raghunath and Perumal, 2017</xref>).</p></sec><sec id=\"S2.SS2\"><title>Biomolecular Interaction</title><p>Another bactericidal mechanism of M/MO nanoparticles is through interaction with DNA and cytosolic enzymes of various microorganism (<xref rid=\"B46\" ref-type=\"bibr\">Raghunath and Perumal, 2017</xref>). Metals and their ions could crosslink DNA and disrupt its helical structure halting bacterial proliferation. Furthermore, metals and their ions could directly bind to enzymes and disrupt their tertiary structures, which in turn disrupt digestion and respiration processes (<xref rid=\"B25\" ref-type=\"bibr\">Imlay, 2014</xref>). As a result, bacterial cells start to lose metabolic activity and die. Metal ions like copper (I), zinc (II), manganese (II), and iron (II) have been reported to exhibit this antibacterial mechanism (<xref rid=\"B55\" ref-type=\"bibr\">Skaar and Raffatellu, 2015</xref>).</p></sec><sec id=\"S2.SS3\"><title>Reactive Oxygen Species (ROS) Formation</title><p>Many transitional metals and their oxides have strong redox properties allowing them to gain electrons from reactive donor sites (<xref rid=\"B46\" ref-type=\"bibr\">Raghunath and Perumal, 2017</xref>). Hence the intermolecular interaction between M/MO nanoparticles and the bacterial cells initiates the formation of many ROS such as hydroxyl radicals (OH<sup>•</sup>), superoxide anion (O<sup>–</sup><sub>2</sub>) and hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). Further propogation of ROS via different reactions increase their concentrations, causing oxidative stress (<xref rid=\"B40\" ref-type=\"bibr\">Nel et al., 2006</xref>). This stress mediates the damage of cell macromolecules, distortion of proteins and lipids, alteration of DNA/RNA, inhibition of enzymes activities, and eventually death of the microbe (<xref rid=\"B15\" ref-type=\"bibr\">Gold et al., 2018</xref>). For example, silver, gold (<xref rid=\"B72\" ref-type=\"bibr\">Zheng et al., 2017</xref>), zinc oxide (<xref rid=\"B21\" ref-type=\"bibr\">He et al., 2011</xref>) and magnesium oxide have been reported to prompt ROS formation (<xref rid=\"B15\" ref-type=\"bibr\">Gold et al., 2018</xref>).</p></sec></sec><sec id=\"S3\"><title>3D Printing of Thermoplastic Nanocomposites</title><p>In general, 3D printing is a process to build a structure by adding materials layer-by-layer according to a computer aided design (CAD) model (<xref rid=\"B28\" ref-type=\"bibr\">Jiménez et al., 2019</xref>). Initially a CAD model can be created by various 3D design software and stored in a format adapted for 3D printing (<xref rid=\"B61\" ref-type=\"bibr\">Valino et al., 2019</xref>). Afterwards, the designed model is transferred to slicer programs to set the printing options such as the thickness of the model, the height of the layers, filling ratio and printing speed, which can be saved as a G-code file (<xref rid=\"B28\" ref-type=\"bibr\">Jiménez et al., 2019</xref>). Finally, various types of 3D printers use the G-code file to build the models to be ready for post-manufacturing removal of supports, sanding and filing processes (<xref rid=\"B18\" ref-type=\"bibr\">Guo et al., 2019</xref>).</p><p>Fused deposition modeling (FDM), one of the most common 3D printing techniques, applies thermoplastic based polymers and nanocomposites (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). In this technique, filaments are heat-melted and extruded from a nozzle and allowed to solidify in a printing bed. The hot nozzle commonly moves in X and Y direction to build the first layer, while succeeding layers are continuously being built by moving the nozzle up in the Z direction (<xref rid=\"B41\" ref-type=\"bibr\">Ngo et al., 2018</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Jiménez et al., 2019</xref>). Addition of M/MO nanoparticles is usually achieved during formation of filaments prior to 3D printing. Melt-blending, solvent-casting and surface coating (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S2</xref>) are few of the approaches to mix the nanoparticles with thermoplastics (<xref rid=\"B17\" ref-type=\"bibr\">González-Henríquez et al., 2019b</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Schematic illustration of 3D printing of M/MO incorporated thermoplastic nanocomposites using FDM process. Composite filaments can be made by extruding mixture of the M/MO nanoparticles with the thermoplastics. Then filaments are heat-melted and extruded from a nozzle and allowed to solidify in a printing bed. Pre-programmed computer aided design (CAD) model is used to control the printing process.</p></caption><graphic xlink:href=\"fbioe-08-568186-g001\"/></fig><p>Another approach to print thermoplastic nanocomposites is SLS, in which a laser selectively sinters the particles of a polymer powder and fuses them together, building the intended part layer-by-layer (<xref rid=\"B36\" ref-type=\"bibr\">Mazzoli, 2013</xref>). The process starts with the powder and the build area being heated to just near the melting temperature of the polymer. Then, a recoating blade spreads a thin layer of powder over the build platform (<xref rid=\"B44\" ref-type=\"bibr\">Paul and Anand, 2012</xref>). Next, a laser fuses powder polymers together. After scanning each cross-section of the component, the power bed is lowered down to the next layer, and another layer is built on top. This process is repeated until the intended model is completed (<xref rid=\"B44\" ref-type=\"bibr\">Paul and Anand, 2012</xref>; <xref rid=\"B61\" ref-type=\"bibr\">Valino et al., 2019</xref>). For this approach, addition of powder M/MO nanoparticles with the polymer powder is done by mechanical mixers such as rotary tumblers (<xref rid=\"B60\" ref-type=\"bibr\">Turner et al., 2020</xref>). The mixing process is easier and can result in a more uniform distribution of nanoparticles when compared to FDM. Yet another method utilized to create antibacterial thermoplastic nanocomposite 3D printed models is SLA. In this method, M/MO nanoparticles are first dispersed into the monomer solution prior to its polymerization (<xref rid=\"B59\" ref-type=\"bibr\">Totu et al., 2017</xref>). The resin is then deposited layer-by-layer based on the designed model. In final step the polymerization is effectuated by UV or other light sources (<xref rid=\"B67\" ref-type=\"bibr\">Weng et al., 2016</xref>). In addition, liquid deposition molding (LDM), solvent-cast 3D printing (SCP) and melt electrowriting (MEW) can also be used to print the thermoplastic nanocomposites (<xref rid=\"B22\" ref-type=\"bibr\">Hewitt et al., 2019</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Nonato et al., 2019</xref>). However, regardless of the printing approach and method of nanocomposite preparation much research remains to be done.</p></sec><sec id=\"S4\"><title>Factors Influencing Antimicrobial Properties of Nanocomposites</title><p>As discussed above, M/MO nanoparticles can play a key role in the antibacterial properties of the thermoplastic nanocomposites, in which, their size, ionic status and concentration, strongly influence the antimicrobial performance of the nanocomposite (<xref rid=\"B50\" ref-type=\"bibr\">Salah et al., 2020</xref>). Moreover, ionic release of M/MO nanoparticles is critical to the balance between immediate antimicrobial activity and long-term efficacy of the nanocomposites. Accordingly, polymer properties affecting the ionic release of M/MO nanoparticles such as hydrophilicity, density and crystallinity should be carefully considered in the design of antimicrobial nanocomposites for different applications (<xref rid=\"B56\" ref-type=\"bibr\">Tamayo et al., 2016</xref>).</p><p>Antimicrobial performance of nanocomposites can significantly differ depending on the M/MO incorporated into the same polymer matrix. For example, <xref rid=\"B39\" ref-type=\"bibr\">Muwaffak et al. (2017)</xref> suggests that silver in polycaprolactone (PCL) exhibits a superior antibacterial effect against <italic>Staphylococcus aureus</italic> when compared to copper or zinc. Studies also showed that antibacterial activity of the nanocomposite can be greatly enhanced by size reduction of these nanoparticles. For example, polylactic acid (PLA) filled with 10% silver wires can inhibit only 50% of <italic>Escherichia coli</italic> growth when the wire diameter is 330 nm (<xref rid=\"B63\" ref-type=\"bibr\">Walker et al., 2020</xref>), while 100% inhibition of the same bacterial strain can be achieved by adding only 1% silver nanowires with the average diameter of 60 nm (<xref rid=\"B4\" ref-type=\"bibr\">Bayraktar et al., 2019</xref>). Furthermore, oxidized or ionized metal particles are recommended for higher antibacterial efficacy over pure metal particles. For instance, <xref rid=\"B9\" ref-type=\"bibr\">Delgado et al. (2011)</xref> investigated the effect of adding copper or copper oxide on antimicrobial activity of polypropylene nanocomposite. Their study revealed that copper oxide fillers are much more efficient at eliminating bacteria when compared to copper fillers, when tested against <italic>Escherichia coli</italic> (<xref rid=\"B9\" ref-type=\"bibr\">Delgado et al., 2011</xref>). Finally, increasing concentration of the antibacterial nanoparticles is a common strategy to enhance overall antibacterial efficiency of the nanocomposites (<xref rid=\"B29\" ref-type=\"bibr\">Kalakonda et al., 2017</xref>). Nonetheless, it is important to consider other effects of the printed nanocomposites, such as biocompatibility and environmental safety according to their potential applications.</p></sec><sec id=\"S5\"><title>Applications</title><p>3D printed antimicrobial thermoplastic nanocomposites have ample application potential in the medical and healthcare fields, as they can provide an on-demand support for the customization and personalization of medical devices (<xref rid=\"B75\" ref-type=\"bibr\">Zuniga and Cortes, 2020</xref>). Specifically, they are very attractive for surgical prosthesis since some of the thermoplastics (e.g., PLA, PCL) have been extensively studied for tissue engineering and regenerative medicine application (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). With the integration of M/MO nanoparticles, bacterial infection can be avoided during surgical implantation, which otherwise could affect tissue growth, postpone surgical recovery, upsurge risks of complications, and even cause death (<xref rid=\"B1\" ref-type=\"bibr\">Abudula et al., 2020</xref>). For example, <xref rid=\"B73\" ref-type=\"bibr\">Zou et al. (2020)</xref> prepared nanocomposite scaffolds containing poly(lactic-co-glycolic acid) (PLGA), copper/zinc based zeolitic-imidazolate-frameworks (Cu@ZIP-8) by FDM-based 3D printing for infected bone repair application. They suggested that a sustainable release of Cu@ZIP-8 in aqueous media ensures long-term antibacterial efficacy of the developed scaffolds (<xref rid=\"B73\" ref-type=\"bibr\">Zou et al., 2020</xref>). <xref rid=\"B2\" ref-type=\"bibr\">Afghah et al. (2020)</xref> developed a PCL-poly propylene succinate (PCL-PPSu) composite with silver-doped biocidal properties, which can be printable by FDM. They suggested that the printed scaffolds can be potentially applied for skin tissue engineering due to their inhibition effects against various microorganism, a good cyto-compatibility and biodegradability (<xref rid=\"B2\" ref-type=\"bibr\">Afghah et al., 2020</xref>). Similarly, <xref rid=\"B59\" ref-type=\"bibr\">Totu et al. (2017)</xref> developed an antibacterial poly methylmethacrylate/TiO<sub>2</sub> nanocomposite that was employed for the fabrication of complete denture sets prototyped by SLA 3D printing. Although the composite only contained 0.4% of TiO<sub>2</sub> nanoparticles, it exhibited sufficient antimicrobial activity against <italic>Candida</italic> species. They also tested these nanocomposites with real patients and demonstrated that this manufacturing approach is a promising treatment option for patients diagnosed with complete edentulism (<xref rid=\"B59\" ref-type=\"bibr\">Totu et al., 2017</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Cristache et al., 2020</xref>). <xref rid=\"B60\" ref-type=\"bibr\">Turner et al. (2020)</xref> introduced a SLS printing of antibacterial nanocomposite by mixing polyamide 12 with 1% of commercially available B65003 silver additives to make delicate, complex, and personalized devices for medical and healthcare applications (<xref rid=\"B60\" ref-type=\"bibr\">Turner et al., 2020</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Examples of 3D printing of metal/metal oxide incorporated thermoplastic nanocomposites with antibacterial properties for various applications.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\">Materials<hr/></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Printing method</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Application</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">References</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Thermoplastic</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">M/MO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Other(s)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLGA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Copper</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ZIF-8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HT-LDM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Infected bone repair</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B73\" ref-type=\"bibr\">Zou et al. (2020)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PMMA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Titanium oxide</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SLA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Digital dentistry</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B8\" ref-type=\"bibr\">Cristache et al. (2020)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLLA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Silver</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HNTs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Infected bone repair</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B19\" ref-type=\"bibr\">Guo et al. (2020)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PCL-PPSu</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Silver</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FDM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Skin tissue engineering</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B2\" ref-type=\"bibr\">Afghah et al. (2020)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ABS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zinc</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FDM + casting</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Infected bone repair</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B7\" ref-type=\"bibr\">Cockerill et al. (2020)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyamide 12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Silver</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Personalized devices</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B60\" ref-type=\"bibr\">Turner et al. (2020)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Copper-zinc alloy</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PWF</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FDM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Handled devices</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B69\" ref-type=\"bibr\">Yang et al. (2020)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLA/PGA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Silver</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MBG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Infected bone repair</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B54\" ref-type=\"bibr\">Shuai et al. (2019)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zinc oxide</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SCP</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Food packaging</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B42\" ref-type=\"bibr\">Nonato et al. (2019)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PCL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Copper</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bio glass</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LDM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Infected bone repair</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B65\" ref-type=\"bibr\">Wang et al. (2019)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Copper</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FDM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Finger prosthesis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B71\" ref-type=\"bibr\">Young et al. (2019)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Silver</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FDM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Public health</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B4\" ref-type=\"bibr\">Bayraktar et al. (2019)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLA/PGA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Silver</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Grapheme</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SLS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bone tissue engineering</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B53\" ref-type=\"bibr\">Shuai et al. (2018)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PLA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Copper</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FDM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Finger prosthesis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B74\" ref-type=\"bibr\">Zuniga (2018)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PMMA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Titanium oxide</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SLA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Digital dentistry</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B59\" ref-type=\"bibr\">Totu et al. (2017)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PBAT/PLA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Silver</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Egg-shell</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FDM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Food packaging</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B58\" ref-type=\"bibr\">Tiimob et al. (2017)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ABS</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Zinc oxide</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FDM</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Toys</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B30\" ref-type=\"bibr\">León-Cabezas et al. (2017)</xref></td></tr></tbody></table><table-wrap-foot><attrib><italic>PLGA, Poly(lactic-co-glycolic acid); PMMA, Poly(methyl methacrylate); PLLA, Poly-L-lactide; PCL, Poly (caprolactone); PPSu, Poly propylene succinate; ABS, Acrylonitrile butadiene styrene; PLA, Poly (lactic acid); PGA, Polyglycolide; PBAT, Poly(butylene adipate-co-terephthalate); M/MO, Metal/metal oxide; ZIF-8, Zinc based zeolitic-imidazolate-frameworks; HNTs, Halloysite nanotubes; PWF, Particleboard wood flour; MBG, Mesoporous bioactive glass; HT, High temperature; LDM, Liquid deposition modeling; SLA, Stereolithography; SLS, Selective laser sintering; FDM, Fused deposition modeling; SCP, Solvent-cast 3D printing.</italic></attrib></table-wrap-foot></table-wrap><p>Likewise, tuning the mechanical properties by incorporation of M/MO nanoparticles could be of great benefit especially to the food-packaging industry. More specifically, nanocomposites can be a versatile alternative to traditionally manufactured plastics for reducing food contamination and provide safer degradable options for packaging. For example, <xref rid=\"B58\" ref-type=\"bibr\">Tiimob et al. (2017)</xref> incorporated silver nanoparticles into a polymer blend composed of 70% poly butylene-co-adipate-terephthalate and 30% PLA to design 3D printed nanocomposites by FDM. Their research suggested that the developed composites can be used for food packaging due to their robust mechanical properties and ability to control growth of microorganisms in food (<xref rid=\"B58\" ref-type=\"bibr\">Tiimob et al., 2017</xref>).</p><p>Finally, 3D printed antibacterial nanocomposites can also be widely integrated in daily-life products (<xref rid=\"B23\" ref-type=\"bibr\">Huang et al., 2016</xref>). Many of the devices and tools that are used routinely in day-to-day tasks can be made with antibacterial materials to reduce the potential negative effects of pathogenic bacteria and fungi. Notably, objects associated with public use can be made safer by 3D printing with antimicrobial nanocomposites. <xref rid=\"B69\" ref-type=\"bibr\">Yang et al. (2020)</xref> fabricated a particleboard wood flour/PLA composite filament reinforced by copper-zinc alloy nanoparticles for FDM-based 3D printing. They suggested that the developed filament can be used to print personalized furniture, public benches and toys, owing to its good antimicrobial properties and environmental safety (<xref rid=\"B69\" ref-type=\"bibr\">Yang et al., 2020</xref>). In short, these examples provide a clear vision of how 3D printing of antimicrobial nanocomposites can be applied to make imaginative and customized products.</p></sec><sec id=\"S6\"><title>Challenges and Future Scope</title><p>In conclusion, M/MO nanoparticles as antimicrobial fillers can greatly increase the value of 3D printed thermoplastic products. Nonetheless, challenges remain in commercializing these thermoplastic nanocomposites for a wider range of applications. For example, one of the critical issues related to their clinical application is that many antibacterial tests have been performed <italic>in vitro</italic>, in which some factors that influence antibacterial performance <italic>in vivo</italic>, such as inflammation, are neglected (<xref rid=\"B64\" ref-type=\"bibr\">Wang et al., 2016</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Jiao et al., 2019</xref>). A similar challenge is having comprehensive compatibility testing of these nanocomposites that are essential for their use with the human body. Long-term exposure and biosafety remain a major challenge when consider their application in the medical field, particularly toxic effect of M/MO on a wide range of cells and tissue need to be assessed. For example, <xref rid=\"B24\" ref-type=\"bibr\">Ickrath et al. (2017)</xref> suggested cytotoxic and genotoxic effects of zinc oxide nanoparticles after long-term and repetitive exposure to human mesenchymal stem cells. Therefore, currently reported short-term, single cell-line reference data is often not enough to provide a generalized biocompatibility profile of the developed materials in many if not most cases (<xref rid=\"B56\" ref-type=\"bibr\">Tamayo et al., 2016</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Rtimi et al., 2019</xref>). In addition, burst-release of antimicrobial agents (i.e., metal ions or particles) can represent a significant limitation of the 3D printed composites. One way to overcome this challenge would be by entrapping antibacterial particles with an additional polymer layer using coaxial nozzle configurations (<xref rid=\"B70\" ref-type=\"bibr\">Yao et al., 2019</xref>). Meanwhile, release properties of antibacterial components could be also controlled by stimuli-responsive materials, especially if the nanocomposites can be release in response to bacterial adhesion to the material (<xref rid=\"B17\" ref-type=\"bibr\">González-Henríquez et al., 2019b</xref>). In addition, controlled release of nanoparticles as ions or metallic particles from the polymer matrix is often key to maintaining the balance between antimicrobial performance and mammalian cytocompatibility (<xref rid=\"B57\" ref-type=\"bibr\">Tamayol et al., 2017</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Mas-Moruno et al., 2019</xref>).</p><p>Other challenges include addition of M/MO nanoparticles into 3D printed thermoplastics often through a multistage process. For example, the nanoparticles first need to be synthesized, then homogeneously dispersed into the polymer matrix and finally 3D printed. This not only increases the total manufacturing cost, but can also deteriorate 3D print quality by inducing filler defects, poor adhesion and particle agglomeration or uneven distribution. Alternatively, <italic>in situ</italic> synthesis of nanoparticles could possibly be a way to overcome such challenges (<xref rid=\"B43\" ref-type=\"bibr\">Palza, 2015</xref>). The concept of <italic>in situ</italic> printing has recently emerged, in which personalized healthcare devices and prosthesis can be directly printed on the defected tissue or organ without 3D scanning and computational design. Particularly, robotic assisted <italic>in situ</italic> printing could be one of the most exciting techniques introduced in the future, which would elevate novelty of 3D printing to a new level (<xref rid=\"B31\" ref-type=\"bibr\">Ma et al., 2020</xref>). Finally, there are many possible application areas yet to be studied using 3D printed nanocomposites, particularly considering their capacity to tailor the product design on a personal level (<xref rid=\"B26\" ref-type=\"bibr\">Ishack and Lipner, 2020</xref>). Specifically, there is a lot of potential for developing personal protective equipment, including masks, ventilators tubes, gloves, which could be especially important during the current global pandemic (<xref rid=\"B75\" ref-type=\"bibr\">Zuniga and Cortes, 2020</xref>).</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>The manuscript was written and edited with contribution from all authors. All authors have given approval to the final version of the manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This project was funded by the Science and Technology Unit – King Abdulaziz University – Kingdom of Saudi Arabia award number UE-41-106.</p></fn></fn-group><ack><p>This project was funded by the Science and Technology Unit – King Abdulaziz University – Kingdom of Saudi Arabia award number UE-41-106.</p></ack><sec id=\"S10\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fbioe.2020.568186/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fbioe.2020.568186/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"FS1\"><media xlink:href=\"Image_1.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abudula</surname><given-names>T.</given-names></name><name><surname>Gauthaman</surname><given-names>K.</given-names></name><name><surname>Hammad</surname><given-names>A. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Biol Reprod</journal-id><journal-id journal-id-type=\"iso-abbrev\">Biol. Reprod</journal-id><journal-id journal-id-type=\"publisher-id\">biolreprod</journal-id><journal-title-group><journal-title>Biology of Reproduction</journal-title></journal-title-group><issn pub-type=\"ppub\">0006-3363</issn><issn pub-type=\"epub\">1529-7268</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32529252</article-id><article-id pub-id-type=\"pmc\">PMC7523691</article-id><article-id pub-id-type=\"doi\">10.1093/biolre/ioaa075</article-id><article-id pub-id-type=\"publisher-id\">ioaa075</article-id><article-categories><subj-group subj-group-type=\"category-taxonomy-collection\"><subject>AcademicSubjects/MED00773</subject><subject>AcademicSubjects/SCI01070</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Contraceptive Special Issue</subject></subj-group></article-categories><title-group><article-title>Mechanism of semen liquefaction and its potential for a novel non-hormonal contraception<xref ref-type=\"author-notes\" rid=\"afn1\"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Anamthathmakula</surname><given-names>Prashanth</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Winuthayanon</surname><given-names>Wipawee</given-names></name><xref rid=\"cor1\" ref-type=\"corresp\"/><xref ref-type=\"aff\" rid=\"aff1\"/><!--<email>w.winuthayanon@wsu.edu</email>--></contrib></contrib-group><aff id=\"aff1\">\n<institution>School of Molecular Biosciences</institution>, Center for Reproductive Biology, College of Veterinary Medicine, <institution>Washington State University</institution>, Pullman, WA 99164, <country country=\"US\">USA</country></aff><author-notes><corresp id=\"cor1\"><bold>Correspondence</bold>: School of Molecular Biosciences, Center for Reproductive Biology, College of Veterinary Medicine, Washington State University, Biotechnology/Life Science Building, 1770 Stadium Way, Pullman, WA 99164, USA. Tel: +15093358296; E-mail: <email>w.winuthayanon@wsu.edu</email></corresp><fn id=\"afn1\"><p>\n<bold>Grant Support</bold>: This study is partly supported by the Eunice Kennedy Shriver NIH/NICHD R01HD097087 to WW.</p></fn></author-notes><pub-date pub-type=\"ppub\"><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"epub\" iso-8601-date=\"2020-05-14\"><day>14</day><month>5</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>14</day><month>5</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>103</volume><issue>2</issue><fpage>411</fpage><lpage>426</lpage><history><date date-type=\"received\"><day>14</day><month>2</month><year>2020</year></date><date date-type=\"rev-recd\"><day>8</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>12</day><month>5</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"cc-by\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"ioaa075.pdf\"/><abstract><title>Abstract</title><p>Semen liquefaction is a proteolytic process where a gel-like ejaculated semen becomes watery due to the enzymatic activity of prostate-derived serine proteases in the female reproductive tract. The liquefaction process is crucial for the sperm to gain their motility and successful transport to the fertilization site in Fallopian tubes (or oviducts in animals). Hyperviscous semen or failure in liquefaction is one of the causes of male infertility. Therefore, the biochemical inhibition of serine proteases in the female reproductive tract after ejaculation is a prime target for novel contraceptive development. Herein, we will discuss protein components in the ejaculates responsible for semen liquefaction and any developments of contraceptive methods in the past that involve the liquefaction process.</p></abstract><abstract abstract-type=\"teaser\"><p>We propose inhibition of semen liquefaction has the potential to be developed as a non-hormonal contraceptive method.</p></abstract><kwd-group><kwd>semen liquefaction</kwd><kwd>semenogelins</kwd><kwd>kallikrein-related peptidase</kwd><kwd>prostate-specific antigen</kwd><kwd>sperm motility</kwd><kwd>contraceptive</kwd><kwd>fertility</kwd></kwd-group><funding-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>National Institutes of Health</institution><institution-id institution-id-type=\"DOI\">10.13039/100000002</institution-id></institution-wrap></funding-source><award-id>R01HD097087</award-id></award-group></funding-group><counts><page-count count=\"16\"/></counts></article-meta></front><body><sec id=\"sec1\"><title>Introduction</title><p>The fate of ejaculated spermatozoa in humans is very different from that in rodents. Male mice and rats ejaculate sperm and accessory gland secretions (e.g., seminal vesicle, prostate) directly into the uterus and produce a copulatory plug, which is not liquefied in vivo. In humans, however, the ejaculate is deposited in the anterior wall of the vagina, which later liquefies, and the sperm gain their motility to transport to the upper female reproductive tract for fertilization (reviewed in [<xref rid=\"ref1\" ref-type=\"bibr\">1</xref>]).</p><p>In humans, the semen is a fluid conglomerate consisting of two major components: the cellular fraction (consisting of spermatozoa, migrating leucocytes, immature germ cells, and epithelial cells) and acellular fraction consisting of seminal plasma and extracellular vesicles (epididymosomes and prostasomes) (<xref rid=\"f1\" ref-type=\"fig\">Figure 1</xref>) (reviewed in [<xref rid=\"ref2\" ref-type=\"bibr\">2</xref>]). Human semen consists of approximately 2–5% spermatozoa and 98–95% seminal plasma, have a minimum volume of 2 mL, a pH of 7.2–8.0 and contain 200–500 million spermatozoa. The liquefaction process requires proteins present in the acellular fraction (seminal plasma) of the semen. Therefore, before describing the process, we will discuss necessary protein components present in the seminal plasma that are involved in the liquefaction process.</p></sec><sec id=\"sec1a\"><title>Seminal plasma</title><p>The seminal plasma is rich in sugars, glycans, lipids, inorganic ions, metabolites, cell-free DNA, microRNAs, peptides, and proteins, which are secreted from seminal vesicles, prostate, epididymis, and bulbourethral glands (reviewed in [<xref rid=\"ref3\" ref-type=\"bibr\">3</xref>]). Seminal vesicles contribute to ~65% of the semen volume and are rich in semenogelins (SEMGs), fibronectin, prostaglandins, cytokines, and fructose, while the prostatic secretions are rich in proteolytic enzymes, citrate, and lipids and contribute to ~25% of the total volume of seminal fluid (<xref rid=\"f1\" ref-type=\"fig\">Figure 1</xref>). The semen has an alkaline pH (7.2–8.0) from seminal vesicles and prostate secretions containing basic polyamines such as spermine, spermidine, and putrescine, which counteract the vaginal acidity and are important for sperm survival. Secretions from bulbourethral glands (contain mucins, galactose, sialic acid) contribute to ~1% of semen volume and act as lubricants enabling efficient sperm transfer (reviewed in [<xref rid=\"ref2\" ref-type=\"bibr\">2</xref>]). The seminal plasma proteins play an important role in semen coagulation, sperm motility, capacitation, acrosome reaction, and immune activity suppression in the female reproductive tract (reviewed in [<xref rid=\"ref3\" ref-type=\"bibr\">3</xref>]). Here, we will discuss the function of key factors present in the seminal plasma that are involved in the semen liquefaction process.</p><fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Fluid components in human ejaculate. The majority of semen is made up of seminal vesicle fluid (~65%; containing semenolgelins or SEMGs and fibronectin) and prostatic fluid (~25%; containing pro-kallikrein (Pro-KLK) enzymes and Zn<sup>2+</sup>). Epididymal fluid and testis make up to ~10% of the semen, while bulbourethral gland (mostly secretes mucinous proteins) is only 1%.</p></caption><graphic xlink:href=\"ioaa075f1\"/></fig><sec id=\"sec2\"><title>Seminal vesicle secretions: SEMGs</title><p>Semenogelin proteins (encoded by <italic>SEMG1</italic> and <italic>SEMG2</italic> genes) are secreted from seminal vesicles [<xref rid=\"ref4\" ref-type=\"bibr\">4</xref>]. SEMG1 and SEMG2 are the two major proteins of the seminal coagulum and represent 20–40% of the seminal plasma proteins [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>, <xref rid=\"ref6\" ref-type=\"bibr\">6</xref>]. SEMG1, a predominant 52 kDa protein, contains a single cysteine residue at position 239 (Cys<sup>239</sup>) and forms intermolecular disulfide bridges with the less abundant SEMG2 (exist as non-glycosylated 71 kDa and glycosylated 76 kDa) at Cys<sup>159</sup> and Cys<sup>360</sup> residues, resulting in high molecular weight complex SEMGs (reviewed in [<xref rid=\"ref7\" ref-type=\"bibr\">7</xref>]). Upon ejaculation, semen immediately turns into a gelatinous meshwork of crosslinking SEMGs. As a result, sperm are entrapped within the seminal coagulum. The N-terminal fragment of SEMG1 was originally identified as the region of seminal plasma motility inhibitor (SPMI) [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>, <xref rid=\"ref9\" ref-type=\"bibr\">9</xref>]. In addition, the C-terminal fragment of SEMG1 containing Cys<sup>239</sup> (164–283 amino acids) was found to have significant inhibitory effects on both motility and progressive motility of intact live human spermatozoa [<xref rid=\"ref10\" ref-type=\"bibr\">10</xref>]. Accordingly, O’Rand et al. [<xref rid=\"ref11\" ref-type=\"bibr\">11</xref>] reported that recombinant human SEMG inhibits sperm progressive motility. In this context, a study by Yamakasi et al. [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>] also indicated that patients with higher number of SEMG-unbound spermatozoa can achieve successful pregnancy, making total SEMG-unbound sperm count a relevant parameter for in vivo fertilization. Additionally, SEMG peptides are also involved in other biological functions such as increasing sperm hyaluronidase activity [<xref rid=\"ref13\" ref-type=\"bibr\">13</xref>], antibacterial activity [<xref rid=\"ref14\" ref-type=\"bibr\">14</xref>], hyperpolarization, and permeability of sperm plasma membrane [<xref rid=\"ref15\" ref-type=\"bibr\">15</xref>].</p></sec><sec id=\"sec3\"><title>Prostatic fluid: kallikreins</title><p>Prostate-specific antigen (also known as kallikrein-related peptidase 3 or KLK3), prostatic acid phosphatase, and prostate secretory protein of 94 amino acids (PSP94) are the three predominant proteins in the prostate fluid secreted by the prostate gland [<xref rid=\"ref16\" ref-type=\"bibr\">16</xref>]. Tissue kallikrein-related peptidases (KLKs) are trypsin- and/or chymotrypsin-like serine proteases secreted by the prostatic epithelial cells. The KLK locus, the largest contiguous cluster of serine proteases, is localized on human chromosome 19 and encodes <italic>KLK1-15</italic>. Despite 36–77% homology among the 15 KLKs at the protein level, amino acid sequences surrounding the catalytic triad (His<sup>57</sup>, Asp<sup>102</sup>, and Ser<sup>195</sup>) are highly conserved among mammalian species (reviewed in [<xref rid=\"ref17\" ref-type=\"bibr\">17</xref>]). Tissue KLKs are different from plasma kallikrein, which is a liver-derived protease, encoded by <italic>KLKB1</italic> gene on human chromosome 4 [<xref rid=\"ref18\" ref-type=\"bibr\">18</xref>]. Shaw and Diamandis [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>] performed a comprehensive expression profiling of KLKs in human tissues and biological fluids and observed that the majority of KLKs (KLK1–5, 9, 11, 13–15) are expressed by the prostate and secreted into seminal plasma.</p><p>KLKs are initially synthesized as pre-pro-KLK proteins, and then the pre-peptides are removed during secretion. Pro-KLKs are inactive, and extracellular cleavage of their amino-terminal pro-peptide by proteolytic activation cascades governs the semen liquefaction mechanism. After secretion, the zymogen activation cascade is initiated by pro-KLK5, which undergoes autoactivation [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>] and subsequently activates downstream pro-KLK2, 3, 7, 8, and 14 [<xref rid=\"ref17\" ref-type=\"bibr\">17</xref>, <xref rid=\"ref21\" ref-type=\"bibr\">21</xref>] (<xref rid=\"f2\" ref-type=\"fig\">Figure 2</xref>). Activated KLK14, in turn, also activates pro-KLK5 in a positive feedback loop [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. Active KLK2 is a known activator of pro-KLK3 [<xref rid=\"ref22\" ref-type=\"bibr\">22–25</xref>] (<xref rid=\"f2\" ref-type=\"fig\">Figure 2</xref>). Additional prostatic KLKs such as KLK4 [<xref rid=\"ref26\" ref-type=\"bibr\">26</xref>] and KLK14 [<xref rid=\"ref27\" ref-type=\"bibr\">27</xref>, <xref rid=\"ref28\" ref-type=\"bibr\">28</xref>] have also been reported to activate pro-KLK3 and presumably aid in semen liquefaction. KLK14 is also involved in the activation of pro-KLK1 and KLK11 [<xref rid=\"ref27\" ref-type=\"bibr\">27</xref>]. While their role in semen liquefaction is not completely known, KLK11 is expressed in intermediate amounts in seminal plasma at concentrations ranging from 2 to 37 μg/mL [<xref rid=\"ref29\" ref-type=\"bibr\">29</xref>], compared to other KLKs (detailed below). Interestingly, KLK2 and KLK3 are only present in the primates [<xref rid=\"ref30\" ref-type=\"bibr\">30</xref>, <xref rid=\"ref31\" ref-type=\"bibr\">31</xref>] and have no known orthologs among the rodent KLKs.</p><fig id=\"f2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Signaling cascade of kallikrein 3 (KLK3) activation during liquefaction process. Pro-KLKs are secreted into the prostatic fluid. High concentration of Zn<sup>2+</sup> in prostatic fluid inactivates KLK3 activity. After ejaculation, prostatic and seminal vesicle fluids are combined. SEMGs are available to sequester Zn<sup>2+</sup> as SEMGs have higher affinity to Zn<sup>2+</sup> compared to KLKs. Pro-KLK5 undergoes autocleavage to rid of pro-peptide sequences and autoactivates. Subsequently, KLK5 activates pro-KLK2 and 3. KLK2 also potentially activates pro-KLK3. Activated KLK3 then hydrolyzes SEMGs into smaller fractions. After hydrolysis, semen becomes liquefied and sperm gain their motility to transport to the upper female reproductive tract for fertilization.</p></caption><graphic xlink:href=\"ioaa075f2\"/></fig><p>In human body, prostate glands accumulate the highest levels of Zn<sup>2+</sup> in prostatic epithelial cells in an androgen-dependent Zn<sup>2+</sup> cellular uptake, which is mediated by specific zinc transporters [<xref rid=\"ref32\" ref-type=\"bibr\">32</xref>]. The role of Zn<sup>2+</sup> in liquefaction process will be discussed in the following section. In addition to KLKs, prostatic secretions are also rich in other proteins, such as prostatic acid phosphatase and zinc-α2-glycoprotein, which are involved in proteolytic cleavage of SEMGs [<xref rid=\"ref33\" ref-type=\"bibr\">33</xref>], degradation of phospholipids [<xref rid=\"ref34\" ref-type=\"bibr\">34</xref>], and lipid mobilization [<xref rid=\"ref35\" ref-type=\"bibr\">35</xref>].</p></sec></sec><sec id=\"sec4\"><title>Semen liquefaction process</title><p>Semen liquefaction at the molecular level is characterized by progressive and site-specific cleavage of SEMGs into soluble low molecular weight proteins in the female reproductive tract [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>, <xref rid=\"ref36\" ref-type=\"bibr\">36</xref>]. Human semen usually liquefies within ~15 to 20 min post-ejaculation [<xref rid=\"ref37\" ref-type=\"bibr\">37</xref>] (<xref rid=\"f3\" ref-type=\"fig\">Figure 3</xref>) and is a necessary step for further sperm processes related to fertilization, such as capacitation [<xref rid=\"ref38\" ref-type=\"bibr\">38</xref>]. KLK3 is the major enzyme (staggering concentrations of 1290 μg/mL in seminal plasma [<xref rid=\"ref39\" ref-type=\"bibr\">39</xref>]) that hydrolyzes SEMGs and fibronectin and liquefies semen coagulum facilitating sperm motility [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>, <xref rid=\"ref10\" ref-type=\"bibr\">10</xref>, <xref rid=\"ref36\" ref-type=\"bibr\">36</xref>, <xref rid=\"ref39\" ref-type=\"bibr\">39–41</xref>]. KLK3 hydrolysis of SEMGs occurs preferentially at tyrosine, glutamine, and leucine and less commonly at other residues (histidine, aspartic acid, serine, and asparagine) [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>, <xref rid=\"ref41\" ref-type=\"bibr\">41</xref>]. Other members of the KLK family participating in the process of semen liquefaction include KLK2 (concentration of 10–100 μg/mL), KLK5, and KLK14 (both enzymes ranging from 1 to 10 ng/mL) [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>, <xref rid=\"ref21\" ref-type=\"bibr\">21</xref>, <xref rid=\"ref23\" ref-type=\"bibr\">23</xref>, <xref rid=\"ref27\" ref-type=\"bibr\">27</xref>, <xref rid=\"ref42\" ref-type=\"bibr\">42</xref>]. Active KLK2, 5, and 14 have been reported to cleave fibronectin and SEMGs in ex vivo and in vitro studies [<xref rid=\"ref21\" ref-type=\"bibr\">21</xref>, <xref rid=\"ref27\" ref-type=\"bibr\">27</xref>, <xref rid=\"ref28\" ref-type=\"bibr\">28</xref>, <xref rid=\"ref42\" ref-type=\"bibr\">42–44</xref>]. Additionally, KLK6, 7, and 13 also exhibit catalytic activity toward fibronectin [<xref rid=\"ref45\" ref-type=\"bibr\">45–47</xref>].</p><fig id=\"f3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Scanning images of human semen before and after liquefaction. The samples were fixed at (A–D) 3 min, (E–F) 6 min, and (G–H) 15 min after ejaculation. Images in (G–H) were taken from samples immediately after liquefaction. (A) 30× magnification, (B) 600×, (C) 3000×, (D) 2875×, (E) 1200×, (F) 3100×, and (H) 1200×. S, spermatozoon. (A–C) and (E–H) normally liquefying; (D) slowly liquefying. Reprint of original images with permission from [<xref rid=\"ref37\" ref-type=\"bibr\">37</xref>].</p></caption><graphic xlink:href=\"ioaa075f3\"/></fig><p>Semen liquefaction is also regulated by endogenous inhibitors such as Zn<sup>2+</sup> (<xref rid=\"f2\" ref-type=\"fig\">Figure 2</xref>) as well as protein C inhibitor (PCI). Prostatic KLKs are inactivated by allosteric reversible binding of Zn<sup>2+</sup> in the seminal plasma (reviewed in [<xref rid=\"ref2\" ref-type=\"bibr\">2</xref>]). Numerous studies have reported the ability of Zn<sup>2+</sup> to inhibit KLK2 [<xref rid=\"ref43\" ref-type=\"bibr\">43</xref>], KLK3 [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>, <xref rid=\"ref41\" ref-type=\"bibr\">41</xref>, <xref rid=\"ref48\" ref-type=\"bibr\">48</xref>], KLK5 [<xref rid=\"ref21\" ref-type=\"bibr\">21</xref>], and KLK14 [<xref rid=\"ref28\" ref-type=\"bibr\">28</xref>] activities. Moreover, the inhibitory effect of Zn<sup>2+</sup> on KLK3, 5, and 14 activities is reversible by SEMGs [<xref rid=\"ref21\" ref-type=\"bibr\">21</xref>, <xref rid=\"ref28\" ref-type=\"bibr\">28</xref>, <xref rid=\"ref48\" ref-type=\"bibr\">48</xref>].</p><p>Once the ejaculation cue is triggered, SEMG-containing seminal vesicle secretions and prostatic fluid enriched with Zn<sup>2+</sup> and KLKs are mixed with the sperm-enriched epididymal fluid to form a coagulum that entraps spermatozoa. Upon ejaculation, SEMGs sequester Zn<sup>2+</sup> ions from KLKs as SEMGs possess a higher affinity to Zn<sup>2+</sup>, leading to KLK disinhibition and activation of the proteolytic cascade resulting in semen liquefaction (reviewed in [<xref rid=\"ref2\" ref-type=\"bibr\">2</xref>]). Therefore, KLKs in concert with SEMGs regulate semen coagulation and liquefaction in a Zn<sup>2+</sup>-dependent manner. In addition to Zn<sup>2+</sup>, PCI has been shown to form a complex with SEMGs and KLKs to inhibit activities of KLKs in the seminal plasma [<xref rid=\"ref43\" ref-type=\"bibr\">43</xref>, <xref rid=\"ref49\" ref-type=\"bibr\">49</xref>]. However, biological contribution of PCI in human semen liquefaction is widely unknown and requires further investigations.</p></sec><sec id=\"sec5\"><title>Factors affecting semen liquefaction</title><p>Genetic variations of genes involved in the liquefaction process as well as biochemical disruption could lead to liquefaction defect. This includes factors affecting the production and activity of KLKs, SEMGs, Zn<sup>2+</sup>, endogenous protease inhibitors, and other pathological conditions in male accessory organs (<xref rid=\"TB1\" ref-type=\"table\">Table 1</xref>). As liquefaction process takes place in the female reproductive tract, local production of KLKs, endogenous protease inhibitors, and pathological conditions in the female tract could also be contributing factors for liquefaction defect. In clinical settings, the liquefaction time is of diagnostic importance if more than 1 h elapses without any change in the semen consistency [<xref rid=\"ref50\" ref-type=\"bibr\">50</xref>]. Any defects in the liquefaction process can lead to impaired semen liquefaction and ~12% of infertility patients have the symptom of non-liquefied semen [<xref rid=\"ref51\" ref-type=\"bibr\">51</xref>]. Here, we describe possible factors contributing to, or conditions resulting in defective semen liquefaction.</p><table-wrap id=\"TB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Physiological functions of proteins in male accessory gland secretions potentially involved in regulating fertility in humans and rodents.</p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Protein</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Gene (human)</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Gene (mouse)</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Function</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Phenotypes when mutated, overexpressed, or genetically ablated</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Seminal vesicles</bold>\n</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SEMG1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SEMG1</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Svs2</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SEMG1 forms intermolecular disulfide bridges with SEMG2 resulting in high molecular weight coagulum upon ejaculation [<xref rid=\"ref7\" ref-type=\"bibr\">7</xref>]. Inhibits sperm motility [<xref rid=\"ref10\" ref-type=\"bibr\">10</xref>, <xref rid=\"ref11\" ref-type=\"bibr\">11</xref>]. SVS2 is a known decapacitation factor and maintains sperm motility and sperm cholesterol levels, prevents spontaneous sperm capacitation, and is essential for sperm survival in the mouse uterus [<xref rid=\"ref66\" ref-type=\"bibr\">66</xref>, <xref rid=\"ref68\" ref-type=\"bibr\">68</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SEMG1</italic> variants (rs147894843, rs2301366) associated with infertility [<xref rid=\"ref57\" ref-type=\"bibr\">57</xref>, <xref rid=\"ref63\" ref-type=\"bibr\">63</xref>]. Elevated SEMG1 precursor reported in oligozoospermic men [<xref rid=\"ref64\" ref-type=\"bibr\">64</xref>]. <italic>Svs2</italic><sup>−/−</sup> mice are subfertile with defects in copulatory plug formation [<xref rid=\"ref68\" ref-type=\"bibr\">68</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SVS7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SVS7</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Pate4</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SVS7 in mouse is essential for copulatory plug formation in vivo [<xref rid=\"ref69\" ref-type=\"bibr\">69</xref>]. SVS7 enhances mouse sperm motility in vitro [<xref rid=\"ref70\" ref-type=\"bibr\">70</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No known mutation/phenotype reported in humans. <italic>Pate4</italic><sup>−/−</sup> mice are subfertile with defects in copulatory plug formation [<xref rid=\"ref69\" ref-type=\"bibr\">69</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SERPINE2/PN1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SERPINE2/PN1</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Serpine2/Pn1</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease inhibitor acts as a decapacitation factor [<xref rid=\"ref82\" ref-type=\"bibr\">82</xref>]. Inhibits protein tyrosine phosphorylation and sperm capacitation [<xref rid=\"ref82\" ref-type=\"bibr\">82</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Elevated PN1 levels in semen of men displaying seminal dysfunction [<xref rid=\"ref85\" ref-type=\"bibr\">85</xref>]. <italic>Pn1<sup>−/−</sup></italic> mice are infertile due to altered seminal protein composition and defects in copulatory plug formation [<xref rid=\"ref85\" ref-type=\"bibr\">85</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SPINK3/SPINK1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SPINK3/SPINK1</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Spink3/Spink1</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease inhibitor prevents premature acrosomal reaction and protects sperm in the uterine environment in mice [<xref rid=\"ref89\" ref-type=\"bibr\">89</xref>, <xref rid=\"ref90\" ref-type=\"bibr\">90</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No known mutation/phenotype involving fertility reported in humans or mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SPINKL</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No known ortholog</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Spinkl</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease inhibitor acts as decapacitation factor and enhances sperm motility in mice [<xref rid=\"ref97\" ref-type=\"bibr\">97</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No known mutation/phenotype involving fertility reported in humans or mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Testis and epididymis</bold>\n</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">EPPIN</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SPINLW1</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No known ortholog</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Localized on the sperm surface. Modulates KLK3 activity and acts as decapacitating factor [<xref rid=\"ref10\" ref-type=\"bibr\">10</xref>, <xref rid=\"ref78\" ref-type=\"bibr\">78</xref>, <xref rid=\"ref79\" ref-type=\"bibr\">79</xref>]. EPPIN-bound SEMG1 crucial for SEMGs degradation and initiation of progressive sperm motility [<xref rid=\"ref11\" ref-type=\"bibr\">11</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SPINLW1</italic> upregulated in caput epididymis of non-obstructive azoospermic patients [<xref rid=\"ref80\" ref-type=\"bibr\">80</xref>]. rs11594 variant associated with increased risk of idiopathic male infertility in Chinese–Han population [<xref rid=\"ref81\" ref-type=\"bibr\">81</xref>].</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Epididymis</bold>\n</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SPINK2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SPINK2</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Spink2</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease inhibitor protects sperm against protease activity during spermatogenesis</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Homozygous <italic>SPINK2</italic> mutation leads to azoospermia in men [<xref rid=\"ref86\" ref-type=\"bibr\">86</xref>]. Decreased <italic>SPINK2</italic> expression in azoospermic infertile men [<xref rid=\"ref87\" ref-type=\"bibr\">87</xref>]. <italic>Spink2</italic> mutant mice have elevated serine protease activity and exhibit impaired fertility [<xref rid=\"ref88\" ref-type=\"bibr\">88</xref>]. <italic>Spink2<sup>−/−</sup></italic> mice are azoospermic and infertile [<xref rid=\"ref86\" ref-type=\"bibr\">86</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SPINK5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SPINK5</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Spink5</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease inhibitor inhibits KLK5, 7, and 14 activities in corneocytes and regulates desquamation process [<xref rid=\"ref91\" ref-type=\"bibr\">91</xref>, <xref rid=\"ref92\" ref-type=\"bibr\">92</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No known mutation/phenotype reported involving fertility in humans or mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SPINK13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>SPINK13</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Spink13</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease inhibitor. Essential for acrosomal integrity, sperm maturation, and fertility in rats [<xref rid=\"ref96\" ref-type=\"bibr\">96</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No known mutation/phenotype reported in humans. <italic>Spink13</italic> knockdown rats demonstrate premature acrosomal reaction and reduced fertility [<xref rid=\"ref96\" ref-type=\"bibr\">96</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Prostate gland</bold>\n</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK1</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK1</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klk1/mGK6</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Serine protease</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low level observed in SHV samples [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. No known mutation/phenotype reported involving fertility in mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK2/hK2</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK2</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No known ortholog</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease cleaves fibronectin and SEMGs [<xref rid=\"ref42\" ref-type=\"bibr\">42</xref>, <xref rid=\"ref43\" ref-type=\"bibr\">43</xref>]. Activator of pro-KLK3 [<xref rid=\"ref22\" ref-type=\"bibr\">22–25</xref>]. Inhibited by Zn<sup>2+</sup> [<xref rid=\"ref43\" ref-type=\"bibr\">43</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low KLK2 seminal levels observed in men with abnormal liquefaction and SHV [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. SNP (rs2664155) associated with male infertility [<xref rid=\"ref61\" ref-type=\"bibr\">61</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK3/PSA</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK3</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No known ortholog</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease. Major enzyme hydrolyzes SEMGs and fibronectin and liquefies semen coagulum facilitating sperm motility [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>, <xref rid=\"ref10\" ref-type=\"bibr\">10</xref>, <xref rid=\"ref36\" ref-type=\"bibr\">36</xref>, <xref rid=\"ref39\" ref-type=\"bibr\">39–41</xref>]. Inhibited by Zn<sup>2+</sup> [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>, <xref rid=\"ref41\" ref-type=\"bibr\">41</xref>, <xref rid=\"ref48\" ref-type=\"bibr\">48</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low KLK3 level observed in men with SHV [<xref rid=\"ref55\" ref-type=\"bibr\">55</xref>, <xref rid=\"ref57\" ref-type=\"bibr\">57</xref>] and abnormal liquefaction [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. Reduced sperm motility observed in men with low seminal KLK3 levels [<xref rid=\"ref59\" ref-type=\"bibr\">59</xref>]. SNPs (rs266881, rs174776, rs1810020, rs266875, rs35192866) associated with male infertility [<xref rid=\"ref60\" ref-type=\"bibr\">60</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK4</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klk4</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease activates pro-KLK3 [<xref rid=\"ref26\" ref-type=\"bibr\">26</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">No known mutation/phenotype reported involving fertility in humans or mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK5</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK5</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klk5</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease. Initiates liquefaction cascade by activating downstream pro-KLK2, 3, 7, 8 and 14 [17, 21]. Cleaves fibronectin and SEMGs [<xref rid=\"ref21\" ref-type=\"bibr\">21</xref>, <xref rid=\"ref44\" ref-type=\"bibr\">44</xref>]. Inhibited by Zn<sup>2+</sup> [<xref rid=\"ref21\" ref-type=\"bibr\">21</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low level observed in SHV samples [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. No known mutation/phenotype reported involving fertility in mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK6</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klk6</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease exhibits catalytic activity towards fibronectin [<xref rid=\"ref46\" ref-type=\"bibr\">46</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low level observed in SHV samples in humans [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. No known mutation/phenotype reported involving fertility in mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK7</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klk7</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease exhibits catalytic activity towards fibronectin [<xref rid=\"ref47\" ref-type=\"bibr\">47</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK7</italic> (rs1654526) SNP associated with SHV in humans [<xref rid=\"ref57\" ref-type=\"bibr\">57</xref>]. Low level observed in SHV samples in humans [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. No known mutation/phenotype reported involving fertility in mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK8</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK8</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klk8</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low level observed in SHV samples [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. No known mutation/phenotype reported involving fertility in mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK10</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK10</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klk10</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<bold>Serine protease</bold>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low level observed in SHV samples [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. No known mutation/phenotype reported involving fertility in mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK12</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK12</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klk12</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK12</italic> (rs61742847) SNP associated with SHV [<xref rid=\"ref57\" ref-type=\"bibr\">57</xref>]. No known mutation/phenotype reported involving fertility in mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK13</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK13</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klk13</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease exhibits catalytic activity towards fibronectin [<xref rid=\"ref44\" ref-type=\"bibr\">44</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low seminal levels observed in men with abnormal liquefaction and SHV [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. No known mutation/phenotype reported involving fertility in mice</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK14</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK14</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Klk14</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease. Activates pro-KLK1, 3, 5 and 11 [20, 27, 28]. Cleaves fibronectin and SEMGs [<xref rid=\"ref27\" ref-type=\"bibr\">27</xref>, <xref rid=\"ref28\" ref-type=\"bibr\">28</xref>]. Inhibited by Zn<sup>2+</sup> [<xref rid=\"ref28\" ref-type=\"bibr\">28</xref>]</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Low seminal levels observed in men with clinically delayed liquefaction, SHV, and asthenospermia [<xref rid=\"ref28\" ref-type=\"bibr\">28</xref>, <xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. KLK14 inhibition by ACT<sub>G9</sub> delays semen liquefaction [<xref rid=\"ref27\" ref-type=\"bibr\">27</xref>]. No known mutation/phenotype reported in mice involving fertility</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">TGM4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">TGM4</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>Tgm4</italic>\n</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">A prostate-specific autoantigen plays a critical role in male reproduction and catalyzes the formation of N-ε-(γ-glutamyl)lysine cross-bridges between SEMGs in humans [<xref rid=\"ref72\" ref-type=\"bibr\">72</xref>] and SVS proteins in mice [<xref rid=\"ref75\" ref-type=\"bibr\">75</xref>], respectively</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">TGM4 autoantibodies are detected in subfertile adult male patients with autoimmune polyendocrine syndrome type 1, caused by mutations in autoimmune regulator (<italic>AIRE</italic>) gene [<xref rid=\"ref74\" ref-type=\"bibr\">74</xref>]. <italic>Tgm4</italic><sup>−/−</sup> mice are subfertile with defects in copulatory plug formation and seminal fluid viscosity [<xref rid=\"ref75\" ref-type=\"bibr\">75</xref>]. <italic>Aire<sup>−/−</sup></italic> mice develop TGM4 autoantibodies, compromised TGM4 secretion, prostatitis, and exhibit subfertility [<xref rid=\"ref74\" ref-type=\"bibr\">74</xref>].</td></tr></tbody></table></table-wrap><sec id=\"sec6\"><title>Semen hyperviscosity</title><p>According to WHO criteria, viscosity can be assessed in semen by observing the length of the thread formed by gently aspirating semen and allowing it to drop by gravity after 1-h incubation at room temperature [<xref rid=\"ref50\" ref-type=\"bibr\">50</xref>]. A normal sample leaves the pipette in small discrete drops, while in cases of semen hyperviscosity (SHV), the drop will form a thread greater than 2 cm long [<xref rid=\"ref50\" ref-type=\"bibr\">50</xref>]. Based on the thread length, SHV can be further classified into mild (2–4 cm), moderate (4–6 cm), and severe SHV (≥6 cm) [<xref rid=\"ref52\" ref-type=\"bibr\">52</xref>].</p><p>SHV has a prevalence of 12–32% in men with fertility problems [<xref rid=\"ref52\" ref-type=\"bibr\">52–54</xref>]. SHV negatively impacts semen quality and sperm motility because of the sperm-trapping effect of hyperviscous semen [<xref rid=\"ref53\" ref-type=\"bibr\">53</xref>, <xref rid=\"ref55\" ref-type=\"bibr\">55</xref>]. Biochemical analysis of rheological properties of semen indicated the presence of highly organized peptide cores complexed with oligosaccharide chains and disulfide bonds in hyperviscous semen compared to normal samples [<xref rid=\"ref56\" ref-type=\"bibr\">56</xref>]. Gopalkrishnan et al<italic>.</italic> [<xref rid=\"ref54\" ref-type=\"bibr\">54</xref>] found that in semen samples with abnormal viscosity, the sperm count, motility, and chromatin integrity were significantly decreased when compared to controls with normal semen viscosity [<xref rid=\"ref54\" ref-type=\"bibr\">54</xref>]. The etiology of SHV has often been attributed to male accessory gland infection, increased levels of leukocytes, and inflammation. Therefore, the composition of human seminal plasma is important in understanding the physiology of reproduction, and any alterations in seminal plasma may explain molecular mechanisms in some cases of infertility. The following section will discuss genetic variations of KLK enzymes that may contribute to SHV conditions in men and result in defective semen liquefaction.</p></sec><sec id=\"sec7\"><title>\n<italic>KLK</italic> mutations</title><p>Genetic factors may also influence the viscosity of seminal fluid. KLK3 level was significantly lower in SHV samples when compared to samples with normal viscosity [<xref rid=\"ref55\" ref-type=\"bibr\">55</xref>, <xref rid=\"ref57\" ref-type=\"bibr\">57</xref>] suggesting the association between prostatic enzymes and semen viscosity. In a recent study, genetic variation within <italic>KLK</italic> locus was found to be associated with SHV [<xref rid=\"ref57\" ref-type=\"bibr\">57</xref>]. <italic>KLK7</italic> (rs1654526) and <italic>KLK12</italic> (rs61742847) polymorphisms are significantly associated with SHV, while genetic variation in <italic>KLK3</italic> and <italic>KLK15</italic> was found to be three times higher in SHV samples than in controls [<xref rid=\"ref57\" ref-type=\"bibr\">57</xref>]. Emami et al. [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>] reported a possible role of KLKs in the pathogenesis of delayed semen liquefaction and SHV. Lower concentrations of KLK2, 3, 13, and 14 in men with abnormal liquefaction and KLK1, 2, 5–8, 10, 13, and 14 in individuals with SHV semen were observed [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. In agreement with these findings, men with low concentration of seminal KLK3 have reduced sperm motility [<xref rid=\"ref59\" ref-type=\"bibr\">59</xref>]. Accordingly, KLK14 levels are significantly lower in individuals with clinically delayed liquefaction and in asthenospermic infertile men [<xref rid=\"ref28\" ref-type=\"bibr\">28</xref>]. In addition, targeted inhibition of KLK14 activity by the pharmacological inhibitor ACT<sub>G9</sub> (based on serum KLK3 inhibitor α1-anti-chymotrypsin) in seminal plasma considerably delays semen liquefaction [<xref rid=\"ref28\" ref-type=\"bibr\">28</xref>].</p><p>Gupta et al. [<xref rid=\"ref60\" ref-type=\"bibr\">60</xref>] sequenced <italic>KLK3</italic> gene in 875 infertile and 290 fertile men and identified a total of 28 substitutions in <italic>KLK3</italic> coding region. Of 28 <italic>KLK3</italic> substitutions, 5 SNPs (rs266881, rs174776, rs1810020, rs266875, and rs35192866) appear to be strong risk factors for male infertility, while 1 SNP (c.206 + 235 T > C) is protective [<xref rid=\"ref60\" ref-type=\"bibr\">60</xref>]. Variations in other KLKs have also been correlated with male infertility. Lee and Lee [<xref rid=\"ref61\" ref-type=\"bibr\">61</xref>] performed genotypic association analysis in 218 non-obstructive azoospermic and 220 fertile controls and showed that a SNP in the <italic>KLK2</italic> intron 1 (+255 G > A, rs2664155) was associated with male infertility. Savblom et al. [<xref rid=\"ref62\" ref-type=\"bibr\">62</xref>] also reported the association of SNPs in <italic>KLK3</italic> and <italic>KLK2</italic> with concentrations of KLK3 and KLK2, respectively, in seminal plasma and serum. These studies indicate that genetic variations in <italic>KLK2</italic> and <italic>3</italic> could also directly affect their enzymatic activity and hence semen liquefaction, ultimately affecting fertility.</p></sec><sec id=\"sec8\"><title>\n<italic>SEMG</italic> mutations</title><p>Genetic alterations of <italic>SEMG1</italic> variant rs147894843 is involved in altered proteolytic activity, which may affect semen quality and liquefaction leading to infertility in men [<xref rid=\"ref57\" ref-type=\"bibr\">57</xref>]. In a recent study, the association between <italic>SEMG</italic> variants and male infertility was examined [<xref rid=\"ref63\" ref-type=\"bibr\">63</xref>]. In Chinese–Han male population, the <italic>SEMG1</italic> variant rs2301366 was associated with abnormal semen parameters such as semen volume, sperm concentration, sperm number per ejaculate, and sperm motility and more susceptible to infertility [<xref rid=\"ref63\" ref-type=\"bibr\">63</xref>]. In another study, a negative correlation between sperm motility and the proportion of SPMI (of SEMG sequence)-bound spermatozoa was also found in male subjects including infertile normozoospermics, asthenozoospermics, and oligozoospermics [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>]. These findings suggest that SEMGs remained on the sperm surface post-liquefaction might account for impaired sperm motility in infertile men. In a functional proteomic analysis of seminal plasma proteins, SEMG1 isoform b pre-pro-protein levels were elevated in oligozoospermic men with abnormal sperm morphology when compared to donors with normal sperm count and morphology [<xref rid=\"ref64\" ref-type=\"bibr\">64</xref>]. Interestingly, Thacker et al. [<xref rid=\"ref65\" ref-type=\"bibr\">65</xref>] analyzed the major proteins in the semen from fertile and infertile men and found that infertile men lacked SEMG2 precursor showing unique differences in the semen profile. Therefore, SEMGs are crucial for liquefaction process, and mutations in <italic>SEMGs</italic> may have a profound impact on sperm function that goes beyond liquefaction process.</p><p>In mice, seminal vesicle secretion 2 (SVS2), an ortholog of human SEMG1, is a major seminal vesicle secreted protein that acts as a decapacitation factor and controls sperm motility [<xref rid=\"ref66\" ref-type=\"bibr\">66</xref>]. SVS2 maintains sperm cholesterol levels and prevents spontaneous sperm capacitation in the uterus [<xref rid=\"ref67\" ref-type=\"bibr\">67</xref>]. In the oviduct, the removal of SVS2 from the sperm’s surface induces a decrease in cholesterol from the sperm membrane, thereby resulting in the ability of sperm to fertilize the eggs [<xref rid=\"ref67\" ref-type=\"bibr\">67</xref>]. <italic>Svs2<sup>−/−</sup></italic> mice are subfertile due to copulatory plug formation defect [<xref rid=\"ref68\" ref-type=\"bibr\">68</xref>]. Additionally, SVS2 has been demonstrated to protect sperm against the spermicidal uterine environment as the sperm from <italic>Svs2<sup>−/−</sup></italic> mice were killed in the uterine cavity and failed to reach the eggs in the oviduct [<xref rid=\"ref68\" ref-type=\"bibr\">68</xref>]. SVS7, also known as PATE4 (prostate and testis expression 4), is another major seminal vesicle secreted protein essential for copulatory plug formation [<xref rid=\"ref69\" ref-type=\"bibr\">69</xref>]. SVS7 has been shown to enhance mouse sperm motility in vitro [<xref rid=\"ref70\" ref-type=\"bibr\">70</xref>], and <italic>Svs7<sup>−/−</sup></italic> mice exhibit subfertility due to defects in copulatory plug formation ([<xref rid=\"ref69\" ref-type=\"bibr\">69</xref>], reviewed in [<xref rid=\"ref71\" ref-type=\"bibr\">71</xref>]).</p><p>In addition to SEMGs and SVSs, transglutaminases (TGMs) could also potentially involve in the enzymatic complex during liquefaction. In humans, SEMGs are important substrates for TGM [<xref rid=\"ref72\" ref-type=\"bibr\">72</xref>]. TGM catalyze protein crosslinking by formation of N-ε-(γ-glutamyl)lysine cross-bridges between lysine and glutamine residues of donor and acceptor proteins respectively (reviewed in [<xref rid=\"ref73\" ref-type=\"bibr\">73</xref>]). TGM4 is a prostate-specific autoantigen and plays a critical role in male reproduction. TGM4 autoantibodies are detected in subfertile adult men, and these patients elicit an autosomal recessive disorder caused by mutations in the autoimmune regulator (<italic>AIRE</italic>) gene [<xref rid=\"ref74\" ref-type=\"bibr\">74</xref>]. Accordingly, <italic>Aire<sup>−/−</sup></italic> mice develop TGM4 autoantibodies, have compromised TGM4 secretion, prostatitis, and are subfertile [<xref rid=\"ref74\" ref-type=\"bibr\">74</xref>]. In mice, the copulatory plug formation is mediated by TGM4, which catalyzes the formation of N-ε-(γ-glutamyl)lysine cross-bridges between SVS proteins ([<xref rid=\"ref75\" ref-type=\"bibr\">75</xref>], reviewed in [<xref rid=\"ref71\" ref-type=\"bibr\">71</xref>]). <italic>Tgm4<sup>−/−</sup></italic> male mice are subfertile due to faulty copulatory plug formation and seminal fluid viscosity [<xref rid=\"ref75\" ref-type=\"bibr\">75</xref>]. Several glutamine and lysine residues in SVS proteins 1–4 serve as substrates and are target sites for TGM4 cross-linking and may aid in copulatory plug formation, reviewed in [<xref rid=\"ref71\" ref-type=\"bibr\">71</xref>]. These findings indicate that functional SEMGs, SVSs, and TGM4 are required for normal male fertility in humans and mice.</p></sec><sec id=\"sec9\"><title>Prostatectomy</title><p>If KLK3 is the key executor of semen liquefaction process, a loss of KLK3 production due to surgical removal of prostate glands (prostatectomy) would result in a liquefaction defect and ultimately male infertility. In patients with localized prostate cancer, radical prostatectomy is performed, which involves the removal of the entire prostate gland, the seminal vesicles, and the vas deferens. As the continuity of the genital tract is disrupted, seminal emission and ejaculation is lost (anejaculation), leading to obstructive azoospermia, but spermatogenesis normally persists in these patients. Nerve sparing radical prostatectomy may also result in erectile dysfunction [<xref rid=\"ref76\" ref-type=\"bibr\">76</xref>]. Therefore, it is difficult to determine the absolute requirement of prostate-derived KLK3 in human reproduction. Or it is also possible that KLKs from other tissues (i.e., the female reproductive tract) can also contribute to the liquefaction process. This possibility will be discussed in a later section.</p></sec><sec id=\"sec10\"><title>Enzymatic activity of endogenous protease inhibitors from male accessory glands</title><p>Although endogenous protease inhibitors are not directly shown to be involved in semen liquefaction, studies suggest that their activity may affect functions of SEMGs and KLKs. Therefore, the importance of these endogenous protease inhibitors will be discussed briefly below.</p><sec id=\"sec11\"><title>Human epididymal protease inhibitor</title><p>Epididymal protease inhibitor (EPPIN) plays a critical role in sperm function and male fertility. EPPIN is a serine protease inhibitor containing both Kunitz-type and whey acidic protein (WAP)-type four disulfide core protease inhibitor consensus sequences and is expressed in testis and epididymis [<xref rid=\"ref77\" ref-type=\"bibr\">77</xref>]. In the ejaculate coagulum, EPPIN is localized on the sperm surface and bound to SEMG1 (at Cys<sup>239</sup>) where it acts as a decapacitating factor by modulating the activity of KLK3 [<xref rid=\"ref10\" ref-type=\"bibr\">10</xref>, <xref rid=\"ref78\" ref-type=\"bibr\">78</xref>, <xref rid=\"ref79\" ref-type=\"bibr\">79</xref>]. EPPIN-bound SEMG1 is critical for the degradation of SEMGs during semen liquefaction and for the initiation of progressive sperm motility in vivo [<xref rid=\"ref11\" ref-type=\"bibr\">11</xref>]. <italic>EPPIN</italic> gene (<italic>SPINLW1</italic>; serine protease inhibitor-like with Kunitz and WAP domains 1) is significantly upregulated in the caput epididymis of infertile men with non-obstructive azoospermia compared to fertile patients [<xref rid=\"ref80\" ref-type=\"bibr\">80</xref>]. Genetic variants of the <italic>EPPIN</italic> are also reported to be associated with idiopathic male infertility. In the Chinese–Han population, Ding et al. [<xref rid=\"ref81\" ref-type=\"bibr\">81</xref>] reported the association of <italic>EPPIN</italic> variant rs2231829 with decreased risk of idiopathic infertility, while variant rs11594 increased the risk of idiopathic male infertility with abnormal semen parameters such as semen volume, sperm concentration, and sperm motility. However, there are no differences in risk for these genotypes among men with normal semen parameters, suggesting that men with different <italic>EPPIN</italic> variants have either an elevated or a reduced frequency of abnormal sperm parameters [<xref rid=\"ref81\" ref-type=\"bibr\">81</xref>].</p></sec><sec id=\"sec12\"><title>Serpin peptidase inhibitor, clade E, member 2</title><p>Serpin peptidase inhibitor, clade E, member 2 (SERPINE2) is also known as Kunitz-type or protease nexin-1 (PN1) and is highly expressed in seminal vesicles [<xref rid=\"ref82\" ref-type=\"bibr\">82</xref>]. SERPINE2 has broad protease inhibitor activity against serine proteases (such as thrombin, plasminogen activators, trypsin, and plasmin) and has been demonstrated to block protein tyrosine phosphorylation and inhibit sperm capacitation [<xref rid=\"ref82\" ref-type=\"bibr\">82</xref>]. Protein tyrosine phosphorylation is essential for sperm functions such as motility, capacitation, hyperactivation, acrosome reaction, and fertilization [<xref rid=\"ref83\" ref-type=\"bibr\">83</xref>, <xref rid=\"ref84\" ref-type=\"bibr\">84</xref>]. <italic>Pn1<sup>−/−</sup></italic> mice are infertile due to altered seminal protein composition, which leads to inadequate semen coagulation and deficient vaginal plug formation upon copulation [<xref rid=\"ref85\" ref-type=\"bibr\">85</xref>]. On the other hand, abnormally high PN1 levels were reported in semen of men with seminal vesicle dysfunction when compared to seminal plasma from fertile men who had low PN1 levels, indicating that controlled extracellular proteolytic activity is important for fertility in humans [<xref rid=\"ref85\" ref-type=\"bibr\">85</xref>]. However, it remains unclear if SERPINs are involved in the liquefaction process in mammals.</p></sec><sec id=\"sec13\"><title>Serine protease inhibitor Kazal-type</title><p>Serine protease inhibitor Kazal-type 2 (SPINK2) is an acrosomal protein localized in the human mature spermatozoa [<xref rid=\"ref86\" ref-type=\"bibr\">86</xref>]. Comparative gene expression profiling of infertile men diagnosed with azoospermia showed that <italic>SPINK2</italic> expression was decreased fourfold compared with fertile men [<xref rid=\"ref87\" ref-type=\"bibr\">87</xref>]. Genetic variation of <italic>SPINK2</italic> is also reported to be associated with male infertility, where homozygous <italic>SPINK2</italic> mutation leads to azoospermia while haploinsufficiency can result in oligozoospermia [<xref rid=\"ref86\" ref-type=\"bibr\">86</xref>]. In mice, SPINK2 is expressed in the germ cells and epididymis where it protects the developing sperm against protease activity during spermatogenesis [<xref rid=\"ref88\" ref-type=\"bibr\">88</xref>]. <italic>Spink2</italic> mutant mice exhibit significantly impaired fertility accompanied by elevated serine protease activity [<xref rid=\"ref88\" ref-type=\"bibr\">88</xref>], while <italic>Spink2<sup>−/−</sup></italic> mice are azoospermic and infertile [<xref rid=\"ref86\" ref-type=\"bibr\">86</xref>].</p><p>SPINK3 is a seminal vesicle-secreted protease inhibitor, which binds to the plasma membrane of the mouse sperm and appears to have protective function against protease activity in the uterus [<xref rid=\"ref89\" ref-type=\"bibr\">89</xref>]. In addition, SPINK3 prevents the premature acrosomal reaction of the sperm until fertilization through reduction in endogenous nitric oxide [<xref rid=\"ref90\" ref-type=\"bibr\">90</xref>].</p><p>Although the role of human SPINKs in semen liquefaction is unknown, SPINK5 is known to specifically inhibit KLK5, 7, and 14 activities in corneocytes and regulate desquamation process [<xref rid=\"ref91\" ref-type=\"bibr\">91</xref>, <xref rid=\"ref92\" ref-type=\"bibr\">92</xref>]. The absence of SPINK5-mediated inhibition of KLK5, 7, and 14 is implicated in Netherton syndrome, a severe skin disorder with impaired keratinization and hair malformation [<xref rid=\"ref91\" ref-type=\"bibr\">91–93</xref>]. However, it is unclear if <italic>Spink</italic>5<sup>−/−</sup> mice are fertile [<xref rid=\"ref93\" ref-type=\"bibr\">93</xref>]. Similarly, SPINK6 is a potent inhibitor of KLK2, 4, 5, 6, 7, 12, 13, and 14 and plays an important role in skin barrier homeostasis [<xref rid=\"ref94\" ref-type=\"bibr\">94</xref>, <xref rid=\"ref95\" ref-type=\"bibr\">95</xref>]. In the context of semen liquefaction, preventing the activation of KLKs by SPINK5 and 6 could potentially lead to liquefaction defects.</p><p>Epididymal specific protein, SPINK13 is associated with the sperm membrane and essential for acrosomal integrity, sperm maturation, and fertility in rats [<xref rid=\"ref96\" ref-type=\"bibr\">96</xref>]. A related protease inhibitor, SPINKL (SPINK-like) is another seminal vesicle-secreted protease inhibitor reported to prevent premature sperm capacitation in mice [<xref rid=\"ref97\" ref-type=\"bibr\">97</xref>]. Nevertheless, the results obtained from these rodent and human studies highlight the importance of a balance between proteases and their regulation by inhibitors, which may disrupt liquefaction by suppressing protease activities of KLK in the semen.</p></sec></sec><sec id=\"sec14\"><title>Endogenous proteases and protease inhibitors in the female reproductive tract</title><p>After ejaculation, semen is exposed to numerous secretory proteins (including proteinases and proteinase inhibitors) from the female reproductive tract. The distribution of KLKs in female reproductive tract varies widely (<xref rid=\"TB2\" ref-type=\"table\">Table 2</xref>) [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>, <xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]. Immunohistochemical (IHC) studies revealed the expression of KLK5, 6, 11, 12, and 13 in the vaginal stratified squamous epithelium, cervical mucus-secreting epithelium, glandular epithelium of Fallopian tubes, and endometrium [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]. Additionally, in an earlier study using enzyme-linked immunosorbent assay (ELISA) performed in adult tissue, Shaw and Diamandis [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>] detected the presence of multiple KLKs in the vagina (KLK1, 5–14), cervix (KLK1, 4–6, 8, 11–14), uterus (KLK1, 4, 6, 9, 11–14), Fallopian tube (KLK1, 6, 7, 9–14), and ovary (KLK1, 6–8, 10, 11, 14) at varying concentrations. Cervical–vaginal fluid (CVF) hydrates the mucosa of the vagina and ectocervix. CVF contains large amounts of both endogenous proteases and protease inhibitors [<xref rid=\"ref99\" ref-type=\"bibr\">99</xref>, <xref rid=\"ref100\" ref-type=\"bibr\">100</xref>]. In the CVF, multiple KLKs (excluding KLK2, 4, and 9) are also detected using ELISA and proteomic analyses [<xref rid=\"ref99\" ref-type=\"bibr\">99</xref>, <xref rid=\"ref100\" ref-type=\"bibr\">100</xref>]. The presence of KLKs in the CVF is thought to be a combined secretory action by the tissues and glands in the female reproductive tract. Secretory protein levels of KLK11–13 in the CVF are remarkably high and only exceeded by KLK2 and KLK3 levels in seminal plasma. In addition, endogenous inhibitors such as α2-macroglobulin, SERPINs, and SPINKs that regulate KLK activity have also been detected in the CVF [<xref rid=\"ref99\" ref-type=\"bibr\">99</xref>, <xref rid=\"ref100\" ref-type=\"bibr\">100</xref>].</p><table-wrap id=\"TB2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Protease and protease inhibitors in the female reproductive tract.</p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Region</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Description</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Vagina</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK1, 5–14 detected by ELISA [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK5, 6, 11, 12, 13 expression detected using IHC in the vaginal stratified squamous epithelium [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Estradiol decreased KLK6, 10, and 11 levels in vaginal epithelial cells [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cervix</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK1, 4–6, 8, 11–14 detected by ELISA [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK5, 6, 11, 12, 13 expression detected using IHC in the mucus-secreting epithelium [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK5 and 12 cleave MUC4 and 5B in the endocervical epithelium leading to collagen remodeling [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Estradiol upregulated <italic>KLK4</italic>, <italic>5</italic>, and <italic>8</italic> expression in ectocervical cells [<xref rid=\"ref101\" ref-type=\"bibr\">101</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Uterus</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK1, 4, 6, 9, 11–14 detected by ELISA [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK5, 6, 11, 12, 13 expression detected using IHC in the glandular epithelial cells of the endometrium [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">\n<italic>KLK1</italic> expression is upregulated in endometrium during mid-menstrual cycle [<xref rid=\"ref102\" ref-type=\"bibr\">102</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fallopian tube</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK1, 6, 7, 9–14 detected by ELISA [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK5, 6, 11, 12, 13 expression detected using IHC in the glandular epithelium [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ovary</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK1, 6–8, 10, 11, 14 detected by ELISA [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CVF</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">KLK1, 3, 5–8, 10–15 detected using ELISA and proteomic analyses [<xref rid=\"ref99\" ref-type=\"bibr\">99</xref>, <xref rid=\"ref100\" ref-type=\"bibr\">100</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Progesterone increases KLK5–7, 11, and 12 levels in the CVF [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Serine protease inhibitor</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CVF</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">SPINK5 is specific inhibitor of KLK5, 7, and 14 activities [<xref rid=\"ref100\" ref-type=\"bibr\">100</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">SPINK6 is a potent inhibitor of KLK2, 4, 5, 6, 7, 12, 13, and 14 activities</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Estradiol upregulates <italic>SPINK5</italic> and <italic>SPINK6</italic> expression in ectocervical cells [<xref rid=\"ref101\" ref-type=\"bibr\">101</xref>]</td></tr></tbody></table></table-wrap><p>In the female reproductive tract, <italic>KLK</italic> expression is regulated by female steroid hormones [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>, <xref rid=\"ref101\" ref-type=\"bibr\">101</xref>]. Progesterone appears to stimulate KLK expression as levels of KLK5–7, 11, and 12 in CVF peaked after ovulation and positively correlated with the levels of progesterone [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]. In contrast, estradiol (E<sub>2</sub>) treatment decreased the concentrations of KLK6, 10, and 11 in a vaginal epithelium cell line [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>], but increased <italic>KLK4</italic>, <italic>5</italic>, and <italic>8</italic> in ectocervical cells [<xref rid=\"ref101\" ref-type=\"bibr\">101</xref>]. Moreover, <italic>KLK1</italic> is expressed at high concentrations in human endometrium during mid-menstrual cycle when circulating E<sub>2</sub> is elevated [<xref rid=\"ref102\" ref-type=\"bibr\">102</xref>]. Similarly, <italic>Klk1</italic> expression in mouse and rat uteri is stimulated by E<sub>2</sub> [<xref rid=\"ref103\" ref-type=\"bibr\">103</xref>]. In a recent study, Li et al<italic>.</italic> [<xref rid=\"ref101\" ref-type=\"bibr\">101</xref>] reported that the expression of <italic>KLK</italic>s (<italic>KLK4,5</italic>, and <italic>8</italic>) and proteinase inhibitors <italic>SPINK5</italic> and <italic>SPINK6</italic> in human ectocervical cells is regulated by E<sub>2</sub> in an estrogen receptor (ESR1)-dependent manner. Additionally, cell type-specific deletion of <italic>Esr1</italic> in the epithelial cells of the female reproductive tract (<italic>Wnt7a<sup>Cre/+</sup>Esr1</italic><sup>f/f</sup> mice) severely reduces the expression of uterine <italic>Klk</italic> genes (<italic>Klk1</italic> and <italic>Klk1b</italic>) [<xref rid=\"ref101\" ref-type=\"bibr\">101</xref>]. Although the anatomy between human and mice reproductive tract is different, the contribution of the female factor found in the <italic>Wnt7a<sup>Cre/+</sup>Esr1</italic><sup>f/f</sup> mice provides fundamental evidence that the exposure of post-ejaculated semen to the suboptimal microenvironment in the female reproductive tract leads to faulty liquefaction and subsequently causes a fertility defect. Therefore, it is possible that an imbalance between proteases and protease inhibitors due to abnormal estrogen signaling within the female reproductive environment may disrupt liquefaction, which could be one of the reasons for unexplained infertility observed in humans.</p><p>Mucins (MUC) are the primary glycoproteins comprising cervical mucus and are thought to influence sperm transport through the cervix and uterus as they allow sperm penetrance. Apart from contributing to activation of semen liquefaction cascade, KLK5 is also responsible for digestion of collagen and modification of mucins [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]. MUC4 and 5B are the major mucins in the endocervical epithelium and are cleaved by both KLK5 and 12 in vitro [<xref rid=\"ref98\" ref-type=\"bibr\">98</xref>]. Therefore, collective proteolytic action of KLKs from the seminal plasma and secretions from the female reproductive tract are crucial for normal semen liquefaction, sperm release, and transport to the site of fertilization in Fallopian tubes.</p></sec></sec><sec id=\"sec15\"><title>Development of inhibitors for semen liquefaction</title><p>Current contraceptive technologies fail to meet the needs for all women. Hormonal methods of contraception, including oral contraceptive pills (OCPs), dermal patches, injections, and implants, are highly effective and reversible. However, a critical drawback of hormonal contraceptives arises from concerns over the long-term effects of hormones on patient health [<xref rid=\"ref104\" ref-type=\"bibr\">104</xref>]. For instance, estrogen-containing OCPs have been linked to an increased risk of venous thrombosis [<xref rid=\"ref105\" ref-type=\"bibr\">105</xref>], breast cancer [<xref rid=\"ref106\" ref-type=\"bibr\">106</xref>], among other pathologies [<xref rid=\"ref107\" ref-type=\"bibr\">107</xref>, <xref rid=\"ref108\" ref-type=\"bibr\">108</xref>]. Uterine bleeding is also a common reason given for women to discontinue progestin-only regimens [<xref rid=\"ref109\" ref-type=\"bibr\">109</xref>]. The current over-the-counter contraceptives (condoms and spermicides) are associated with high failure rates [<xref rid=\"ref110\" ref-type=\"bibr\">110</xref>]. In addition, usage of spermicides can damage vaginal and cervical mucosa increasing the risk of viral infection in women [<xref rid=\"ref111\" ref-type=\"bibr\">111–113</xref>]. Therefore, there is a need for new non-hormonal vaginal contraceptives for women that can be used on demand. As mentioned above, pathophysiology, genetic inhibition, or biochemical inhibition of liquefaction process negatively impacts fertility in humans. Therefore, blocking KLK3 activity remains the prime candidate for the development of new contraceptives as it would prevent semen liquefaction and sperm transport in the female reproductive tract, potentially leading to clinical use.</p><p>Inhibition of key components regulating the coagulation and liquefaction has been previously assessed in vitro using pan-serine protease inhibitors. Early studies by Matsuda et al<italic>.</italic> [<xref rid=\"ref114\" ref-type=\"bibr\">114</xref>] demonstrated that treatment of human ejaculates with proteinase inhibitor, Fusan (6-amidino-2-naphtyl-6-guanidinobenzoate dihydrochloride), for 30 min inhibited liquefaction (<xref rid=\"f4\" ref-type=\"fig\">Figure 4A–D</xref>), caused solidification of semen, and completely inhibited sperm motility. Other studies focused on the use of commercially available synthetic serine protease inhibitors [such as 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) and phenylmethylsulfonyl fluoride (PMSF)], heavy metal cations (Zn<sup>2+</sup> and Hg<sup>2+</sup>), and heavy metal chelator 1,10-phenanthroline to partially or completely inhibit KLK3 activity in vitro [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>, <xref rid=\"ref115\" ref-type=\"bibr\">115</xref>]. However, these inhibitors have never been tested in in vivo models until recently.</p><fig id=\"f4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Treatment of pan-serine protease inhibitors in (A–D) human ejaculate and (E–F) female mice. (A) Human spermatozoa after liquefaction (336× magnification). Inhibition of liquefaction after Fusan treatment: (B) 1 mM Fusan (400×), (C) 10 mM Fusan (200×), (D) 10 mM Fusan (400×). (E, F) Female mice were transcervically treated with AEBSF before mating, and semen was collected ~8 h after mating. (E) Seminal volume was severely reduced in female mice treated with AEBSF compared to saline (Vehicle). (F) Total sperm number in the oviduct is significantly reduced in AEBSF-treated mice. (G) A lack of SEMG2 cleavage in the uteri from female mice treated with AEBSF. <sup>*</sup><italic>P</italic> < 0.05, unpaired <italic>t-</italic>test. Reprint of original (A)–(D) images with permission from [<xref rid=\"ref114\" ref-type=\"bibr\">114</xref>]. Images (E, F) were modified with permission from [<xref rid=\"ref101\" ref-type=\"bibr\">101</xref>] under the Creative Commons Attribution License.</p></caption><graphic xlink:href=\"ioaa075f4\"/></fig><p>Li et al. [<xref rid=\"ref101\" ref-type=\"bibr\">101</xref>] showed that AEBSF effectively inhibited semen liquefaction in vivo using a mouse model as there was a lack of SEMG2 cleavage in semen collected from uterus. This semen liquefaction blockade by AEBSF treatment caused severe reduction of sperm transport to the oviduct compared to vehicle treatment in vivo (<xref rid=\"f4\" ref-type=\"fig\">Figure 4E–G</xref>). As a proof-of-concept to determine whether protease inhibitors could potentially be used as a contraceptive, we performed a study using a pan-serine protease inhibitor (AEBSF). We showed that AEBSF (1) effectively and reversibly reduced fecundity in female mice; (2) acted as spermicide and inhibited sperm motility, resulting in a decreased fertilization in vivo and in vitro; and (3) was significantly less damaging to the vaginal epithelium (compared to N9) when treated for 10 min or three consecutive days in vivo in mice [<xref rid=\"ref116\" ref-type=\"bibr\">116</xref>]. This review is preceding the report on our AEBSF study in this Special Issue. Despite inhibitory capacity of AEBSF, their application as therapeutic agent is hampered due to a lack of selectivity. Therefore, increased interest for development of a highly potent and selective inhibitor toward KLK3 activity that would cause blockade of semen liquefaction and sperm transport within the female reproductive tract will have potential pharmaceutical utility as a novel contraceptive.</p><fig id=\"f5\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><p>Using of specific KLK3 inhibitors as a novel contraceptive method to block semen liquefaction process. KLK3 inhibitor will potentially attenuate semen liquefaction process as its activity will be specific to KLK3 and does not affect other KLKs or other serine proteases in semen as well as in the female reproductive tract.</p></caption><graphic xlink:href=\"ioaa075f5\"/></fig><p>Before moving on to the selective inhibitor for KLK3, it is important to note that anti-EPPIN was also developed as a male contraceptive; however, the goal was to decrease sperm motility and not inhibition of the semen liquefaction. Briefly, a study in <italic>Macaca radiata</italic> monkeys immunized with recombinant human EPPIN showed an effective and reversible male infertility without hormone disruption [<xref rid=\"ref117\" ref-type=\"bibr\">117</xref>]. Treatment of human spermatozoa with anti-EPPIN antibodies inhibited EPPIN–SEMG1 interaction and significantly decreased sperm motility [<xref rid=\"ref11\" ref-type=\"bibr\">11</xref>]. Furthermore, anti-EPPIN antibodies have been demonstrated to inhibit human sperm acrosomal reaction, reduce intracellular Ca<sup>2+</sup> concentration, and does not alter tyrosine phosphorylation of sperm proteins [<xref rid=\"ref118\" ref-type=\"bibr\">118</xref>]. The use of sperm surface EPPIN as a non-hormonal contraceptive target has led to the development of small organic compounds that could substitute for SEMG1 or anti-EPPIN antibodies and provide a reversible, short-lived pharmacological alternative. In this regard, EP055 is a 1,3,5-triazine compound that targets EPPIN on the surface of sperm and inhibits motility [<xref rid=\"ref119\" ref-type=\"bibr\">119</xref>]. Intravenous infusion of EP055 in male macaques demonstrated plasma half-life of 11 min and the drug being retained in semen for up to 78 h followed by recovery of sperm motility [<xref rid=\"ref119\" ref-type=\"bibr\">119</xref>]. Although EPPIN modulates the hydrolysis of SEMGs by KLK3, it is not an effective inhibitor of KLK3 activity [<xref rid=\"ref120\" ref-type=\"bibr\">120</xref>, <xref rid=\"ref121\" ref-type=\"bibr\">121</xref>]. Therefore, mode of action of anti-EPPIN will be different than that of semen liquefaction inhibition.</p></sec><sec id=\"sec16\"><title>Development of small molecule(s) inhibitors specifically for KLK3</title><p>KLK3 is an ideal target for the development of small-molecule inhibitors targeting its enzymatic activity that would allow development of non-invasive contraception technologies (<xref rid=\"f5\" ref-type=\"fig\">Figure 5</xref>). Most of the studies involved in generation of small molecule inhibitors of KLK3 were focused on their usage in targeted treatment of prostate cancer (<xref rid=\"TB3\" ref-type=\"table\">Table 3</xref>). The first KLK3 inhibitors reported in the literature used a homology model derived from porcine KLK to design and synthesize β-lactam analogs, which showed promising inhibitory activity with an IC<sub>50</sub> (inhibitor maximal inhibitory concentration) as low as 226 nM [<xref rid=\"ref122\" ref-type=\"bibr\">122</xref>]. To obtain mechanistic insights into the inhibition of KLK3, Singh et al. [<xref rid=\"ref123\" ref-type=\"bibr\">123</xref>, <xref rid=\"ref124\" ref-type=\"bibr\">124</xref>] showed that β-lactam based inhibitors compete with KLK3 substrates and form a stable covalent complex at the catalytic Ser<sup>189</sup> residue in a time-dependent manner. Other strategies include the use of azapeptides, which target both cysteine and serine proteases and effectively inhibit KLK3 activity with the K<sub>i</sub> (inhibition constant) as low as 500 nM [<xref rid=\"ref125\" ref-type=\"bibr\">125</xref>].</p><p>Using high-throughput screening of chemical libraries, Koistinen et al. [<xref rid=\"ref126\" ref-type=\"bibr\">126</xref>] screened 49 920 compounds to identify small drug-like molecules and pinpointed two compounds inhibiting KLK3-activity in a dose-dependent manner in human umbilical vein endothelial cells [<xref rid=\"ref126\" ref-type=\"bibr\">126</xref>]. These two active compounds contain either benzoxazinone or triazole derivatives and exhibit potent KLK3 inhibition with IC<sub>50</sub> of 300–500 nM but lack selectivity toward KLK3 activity [<xref rid=\"ref126\" ref-type=\"bibr\">126</xref>]. In addition, triazole derivatives have also been identified to inhibit other KLKs such as KLK5, 7, and 14, as well as matriptase (a transmembrane serine protease) [<xref rid=\"ref127\" ref-type=\"bibr\">127</xref>]. Therefore, the non-selective mechanism of serine protease inhibition by β-lactam analogs and azapeptides severely limited their development as therapeutic drugs as they possess off-target inhibitory activity.</p><p>LeBeau et al. generated a series of small-molecule active-site inhibitors of KLK3 using peptide aldehydes and boronic acid-based inhibitors containing Ser-Ser-Lys-Leu-Gln peptide present in SEMG2 (a natural specific substrate for KLK3) as a template. Peptide aldehyde inhibitors showed specific proteolytic activity toward KLK3 with more than 20-fold specificity than that of chymotrypsin but had a K<sub>i</sub> of 6.5 μM [<xref rid=\"ref128\" ref-type=\"bibr\">128</xref>]. Boronic acid modification led to the development of more potent inhibitors with high specificity and a K<sub>i</sub> of 65 nM, 100-fold lower than the aldehyde-modified peptide inhibitors [<xref rid=\"ref128\" ref-type=\"bibr\">128</xref>]. In vivo evaluation of this inhibitor through intravenous injection at a dose of 33 mg/kg for two cycles of three consecutive days in human prostate cancer xeno-grafted mice led to significant reduction in KLK3 serum levels (free KLK3 levels by 35% and total KLK3 levels by 30%), however, had minimal effect on tumor growth [<xref rid=\"ref128\" ref-type=\"bibr\">128</xref>]. Subsequent modification of this inhibitor by introducing the non-natural amino acid nor-leucine (Nle) exhibited potent KLK3 inhibition with a K<sub>i</sub> of 48 nM [<xref rid=\"ref129\" ref-type=\"bibr\">129</xref>].</p><table-wrap id=\"TB3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>Summary of key KLK3 inhibitors reported in the literature.</p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Type</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Relevance</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Agent</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Description/pharmacological data/therapeutic impact</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">β-lactam analogs</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Unclear</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">2-azetidinone</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">IC<sub>50</sub> = 226 nM [<xref rid=\"ref122\" ref-type=\"bibr\">122</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Benzoxazinone derivatives</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">K<sub>i</sub> = 300 nM. 30 times more selective compared to chymotrypsin (K<sub>i</sub> = 8.5 μM) [<xref rid=\"ref126\" ref-type=\"bibr\">126</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Triazole derivatives</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">K<sub>i</sub> = 500 nM. 10 times more selective compared to chymotrypsin (K<sub>i</sub> = 5.4 μM) [<xref rid=\"ref126\" ref-type=\"bibr\">126</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cysteine and serine protease inhibitors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Azapeptides</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">K<sub>i</sub> = 500 nM [<xref rid=\"ref125\" ref-type=\"bibr\">125</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Heavy metal cations</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Semen liquefaction</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Zn<sup>2+</sup></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits KLK3 activity at 10 mM [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">IC<sub>50</sub> = 20 μM [<xref rid=\"ref41\" ref-type=\"bibr\">41</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hg<sup>2+</sup></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits KLK3 activity at 10 mM [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">IC<sub>50</sub> = 150 μM [<xref rid=\"ref41\" ref-type=\"bibr\">41</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cu<sup>2+</sup></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">IC<sub>50</sub> = 150 μM</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cd<sup>2+</sup></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">IC<sub>50</sub> = 200 μM</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Co<sup>2+</sup></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">IC<sub>50</sub> = 500 μM</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Heavy metal chelator</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Semen liquefaction</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">1,10-phenanthroline</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits KLK3 activity at 50 mM [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Pan-serine protease inhibitors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Semen liquefaction</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">PMSF</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits KLK3 activity at 5 mM [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">AEBSF</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits KLK3 activity at 5 mM [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">PMSF</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits KLK3 activity at 20 mM [<xref rid=\"ref115\" ref-type=\"bibr\">115</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">AEBSF</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits KLK3 activity at 10 mM [<xref rid=\"ref115\" ref-type=\"bibr\">115</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Peptide aldehyde inhibitor</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Z-SSKLL-H</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">K<sub>i</sub> = 6.5 μM [<xref rid=\"ref128\" ref-type=\"bibr\">128</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Peptidyl boronic acid inhibitor</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Z-SSKL(boro)L</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">K<sub>i</sub> = 65 nM. 60 times more selective compared to chymotrypsin (K<sub>i</sub> = 3.9 μM). Reduction in free and total KLK3 serum levels in human prostate cancer xenografts produced in nude mice upon intravenous administration of 33 mg/kg dose for two cycles of three consecutive days/5 days [<xref rid=\"ref128\" ref-type=\"bibr\">128</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Z-SSKn(boro)L</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">K<sub>i</sub> = 48.4 nM. Norleucine substitution of Z-SSKL(boro)L [<xref rid=\"ref129\" ref-type=\"bibr\">129</xref>]</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"left\" rowspan=\"1\" colspan=\"1\">Ahx-FSQn(boro)Bpg</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">K<sub>i</sub> = 72 nM. Eight times more selective compared to chymotrypsin (K<sub>i</sub> = 580 nM). Reduction in free and total KLK3 serum levels in human prostate cancer xenografts produced in nude mice upon intravenous administration of 10 mg/kg dose for three cycles of five consecutive days/week [<xref rid=\"ref130\" ref-type=\"bibr\">130</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">RNA aptamer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Not applicable</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Synthetic RNA molecules (92 mer) selected from pools of random-sequence oligonucleotides to specifically bind active KLK3 [<xref rid=\"ref131\" ref-type=\"bibr\">131</xref>]</td></tr></tbody></table></table-wrap><p>To enhance selectivity of KLK3, Kostova et al. generated a peptidyl boronic acid-based selective KLK3 inhibitor containing a bromopropylglycine group, which had a K<sub>i</sub> of 72 nM and eightfold selectivity over chymotrypsin. Systemic administration of this compound at a dose of 10 mg/kg for three cycles of five consecutive days in nude mice with human prostate cancer xenografts showed minimal effect on tumor growth but led to significant reduction in serum KLK3 levels [<xref rid=\"ref130\" ref-type=\"bibr\">130</xref>]. Other novel KLK3-targeting therapeutic strategies involve the use of RNA aptamers, synthetic nucleic acid molecules, selected from pools of random oligonucleotides via the systematic evolution of ligands by exponential enrichment (SELEX) process to specifically target active KLK3 [<xref rid=\"ref131\" ref-type=\"bibr\">131</xref>]. However, the selectivity of RNA aptamers on KLK3 activity has not yet been tested in vivo<italic>.</italic></p><p>To date, numerous endogenous inhibitors of KLK3 activity with physiological significance ranging from metal ions (Zn<sup>2+</sup>) to proteinase inhibitors (SERPINs) have been reported, but none have been employed for the development of contraceptives to inhibit semen liquefaction in vivo. Additionally, the development of selective KLK3 inhibitors was focused for targeted treatment of prostate cancer. Although numerous small molecule and peptides to inhibit KLK3 activity have been developed, they may not be suitable for contraceptive purposes because the activity is not entirely specific to KLK3 but also bind to a great variety of proteases. An unusual feature of SERPINs is their ability to often inhibit non-target cysteine proteases, i.e., cross-class inhibition [<xref rid=\"ref132\" ref-type=\"bibr\">132</xref>]. In addition, peptide inhibitors are often pH-dependent, thus may not withstand the relatively low pH in vaginal microenvironment [<xref rid=\"ref133\" ref-type=\"bibr\">133</xref>].</p></sec><sec id=\"sec17\"><title>Conclusion</title><p>Human semen liquefaction is a post-ejaculation proteolytic process that changes semen from a gel-like coagulum to a watery consistency (liquefied) and is mainly governed by SEMGs and prostate-derived KLK enzymatic activities. The blockade of semen liquefaction prevents sperm migration in the female reproductive tract and is an unexplored target for both male and female contraception. Inhibition of semen liquefaction can be achieved by using molecules that can stabilize SEMGs (preventing hydrolysis), local delivery of exogenous metal ions (Zn<sup>2+</sup>), overexpression of endogenous protease inhibitors (SERPINs/SPINKs), or administration of synthetic serine protease inhibitors. Of the numerous key molecules involved in the liquefaction cascade, targeting KLK activities (i.e., KLK2, 3, 5, and 14) is a viable option due to the fact that these KLKs are produced specifically in the prostate gland, hence, providing a localized target for the development of a non-hormonal contraceptive.</p><p>One of the immediate possibilities is the use of specific KLK3 inhibitors that were previously developed for prostate cancer patients. KLK3 in the seminal plasma is secreted at extremely high concentration, relative to other KLKs, and is the key executor enzyme involved in semen liquefaction. Rather than using a pan inhibitor of KLK gene family, which could potentially lead to non-intended effects, studies focusing on the development of small drug-like molecules specifically inhibiting seminal KLK3 activity would prove useful in the development of novel non-steroidal, over-the-counter contraceptive with improved efficiency.</p></sec><sec id=\"sec18\"><title>Conflict of interest</title><p>The authors have declared that no conflict of interest exists.</p></sec></body><back><ref-list id=\"bib1\"><title>References</title><ref id=\"ref1\"><label>1.</label><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name name-style=\"western\"><surname>Suarez</surname><given-names>SS</given-names></name>, <name name-style=\"western\"><surname>Pacey</surname><given-names>AA</given-names></name></person-group>\n<article-title>Sperm transport in the female reproductive tract</article-title>. <source>Hum Reprod Update</source><year>2006</year>; <volume>12</volume>:<fpage>23</fpage>–<lpage>37</lpage>.<pub-id pub-id-type=\"pmid\">16272225</pub-id></mixed-citation></ref><ref id=\"ref2\"><label>2.</label><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name name-style=\"western\"><surname>Verze</surname><given-names>P</given-names></name>, <name name-style=\"western\"><surname>Cai</surname><given-names>T</given-names></name>, <name name-style=\"western\"><surname>Lorenzetti</surname><given-names>S</given-names></name></person-group>\n<article-title>The role of the prostate in male fertility, health and disease</article-title>. <source>Nat Rev Urol</source><year>2016</year>; <volume>13</volume>:<fpage>379</fpage>–<lpage>386</lpage>.<pub-id pub-id-type=\"pmid\">27245504</pub-id></mixed-citation></ref><ref id=\"ref3\"><label>3.</label><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name name-style=\"western\"><surname>Drabovich</surname><given-names>AP</given-names></name>, <name name-style=\"western\"><surname>Saraon</surname><given-names>P</given-names></name>, <name name-style=\"western\"><surname>Jarvi</surname><given-names>K</given-names></name>, <name name-style=\"western\"><surname>Diamandis</surname><given-names>EP</given-names></name></person-group>\n<article-title>Seminal plasma as a diagnostic fluid for male reproductive system disorders</article-title>. <source>Nat Rev Urol</source><year>2014</year>; <volume>11</volume>:<fpage>278</fpage>–<lpage>288</lpage>.<pub-id pub-id-type=\"pmid\">24709963</pub-id></mixed-citation></ref><ref id=\"ref4\"><label>4.</label><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name name-style=\"western\"><surname>Lilja</surname><given-names>H</given-names></name>, <name name-style=\"western\"><surname>Lundwall</surname><given-names>A</given-names></name></person-group>\n<article-title>Molecular cloning of epididymal and seminal vesicular transcripts encoding a semenogelin-related protein</article-title>. <source>Proc Natl Acad Sci U S A</source><year>1992</year>; <volume>89</volume>:<fpage>4559</fpage>–<lpage>4563</lpage>.<pub-id pub-id-type=\"pmid\">1584792</pub-id></mixed-citation></ref><ref id=\"ref5\"><label>5.</label><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name name-style=\"western\"><surname>Lilja</surname><given-names>H</given-names></name>, <name name-style=\"western\"><surname>Abrahamsson</surname><given-names>PA</given-names></name>, <name name-style=\"western\"><surname>Lundwall</surname><given-names>A</given-names></name></person-group>\n<article-title>Semenogelin, the predominant protein in human semen. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Biol Reprod</journal-id><journal-id journal-id-type=\"iso-abbrev\">Biol. Reprod</journal-id><journal-id journal-id-type=\"publisher-id\">biolreprod</journal-id><journal-title-group><journal-title>Biology of Reproduction</journal-title></journal-title-group><issn pub-type=\"ppub\">0006-3363</issn><issn pub-type=\"epub\">1529-7268</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32463099</article-id><article-id pub-id-type=\"pmc\">PMC7523692</article-id><article-id pub-id-type=\"doi\">10.1093/biolre/ioaa062</article-id><article-id pub-id-type=\"publisher-id\">ioaa062</article-id><article-categories><subj-group subj-group-type=\"category-taxonomy-collection\"><subject>AcademicSubjects/SCI01070</subject><subject>AcademicSubjects/MED00773</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Contraceptive Special Issue</subject></subj-group></article-categories><title-group><article-title>A cell-based high-content screen identifies isocotoin as a small molecule inhibitor of the meiosis-specific MEIOB–SPATA22 complex<xref ref-type=\"author-notes\" rid=\"afn1\"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Yang</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref ref-type=\"author-notes\" rid=\"afn2\"/></contrib><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Rong</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">2</xref><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref ref-type=\"author-notes\" rid=\"afn2\"/></contrib><contrib contrib-type=\"author\"><name><surname>Leu</surname><given-names>N Adrian</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Lei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ibragmova</surname><given-names>Ilsiya</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Schultz</surname><given-names>David C</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>P Jeremy</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref rid=\"cor1\" ref-type=\"corresp\"/><!--<email>pwang@vet.upenn.edu</email>--></contrib></contrib-group><aff id=\"aff1\"><label>1</label>\n<institution>Department of Biomedical Sciences</institution>, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania, USA</aff><aff id=\"aff2\"><label>2</label>\n<institution>School of Basic Medical Sciences</institution>, Wuhan University, Wuhan, Hubei Province, <country country=\"CN\">China</country></aff><aff id=\"aff3\"><label>3</label>\n<institution>High-Throughput Screening Core</institution>, University of Pennsylvania, Philadelphia, Pennsylvania, USA</aff><author-notes><corresp id=\"cor1\"><bold>Correspondence:</bold> Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA. Tel: (01) 215-746-0160; E-mail: <email>pwang@vet.upenn.edu</email></corresp><fn id=\"afn1\"><p>\n<bold>Grant Support:</bold> This work was supported by Lalor foundation postdoctoral fellowship to YX, China Scholarship Council fellowship 201906275011 to RL, National Institutes of Health grants R01HD084007 and R35GM118052 to PJW.</p></fn><fn id=\"afn2\"><p>Yang Xu, Rong Liu, contributed equally.</p></fn></author-notes><pub-date pub-type=\"ppub\"><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"epub\" iso-8601-date=\"2020-04-25\"><day>25</day><month>4</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>25</day><month>4</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>103</volume><issue>2</issue><fpage>333</fpage><lpage>342</lpage><history><date date-type=\"received\"><day>29</day><month>2</month><year>2020</year></date><date date-type=\"rev-recd\"><day>6</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>4</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"cc-by\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"ioaa062.pdf\"/><abstract><title>Abstract</title><p>MEIOB and SPATA22 are meiosis-specific proteins, interact with each other, and are essential for meiotic recombination and fertility. Aspartic acid 383 (D383) in MEIOB is critical for its interaction with SPATA22 in biochemical studies. Here we report that genetic studies validate the requirement of D383 for the function of MEIOB in mice. The <italic>Meiob</italic><sup>D383A/D383A</sup> mice display meiotic arrest due to depletion of both MEIOB and SPATA22 proteins in the testes. We developed a cell-based bimolecular fluorescence complementation (BiFC) assay, in which MEIOB and SPATA22 are fused to split YFP moieties and their co-expression in cultured cells leads to the MEIOB–SPATA22 dimerization and reconstitution of the fluorophore. As expected, the interaction-disrupting D383A substitution results in the absence of YFP fluorescence in the BiFC assay. A high-throughput screen of small molecule libraries identified candidate hit compounds at a rate of 0.7%. Isocotoin, a hit compound from the natural product library, inhibits the MEIOB–SPATA22 interaction and promotes their degradation in HEK293 cells in a dose-dependent manner. Therefore, the BiFC assay can be employed to screen for small molecule inhibitors that disrupt protein–protein interactions or promote degradation of meiosis-specific proteins.</p></abstract><abstract abstract-type=\"teaser\"><p>A cell-based bimolecular fluorescence complementation screening assay identifies isocotoin that promotes degradation of MEIOB and SPATA22.</p></abstract><kwd-group><kwd>meiosis</kwd><kwd>MEIOB</kwd><kwd>SPATA22</kwd><kwd>isocotoin</kwd><kwd>male contraception</kwd></kwd-group><funding-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>China Scholarship Council</institution><institution-id institution-id-type=\"DOI\">10.13039/501100004543</institution-id></institution-wrap></funding-source><award-id>201906275011</award-id></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>National Institutes of Health</institution><institution-id institution-id-type=\"DOI\">10.13039/100000002</institution-id></institution-wrap></funding-source><award-id>R01HD084007</award-id><award-id>R35GM118052</award-id></award-group></funding-group><counts><page-count count=\"10\"/></counts></article-meta></front><body><sec id=\"sec1\"><title>Introduction</title><p>Male reproduction is a multistep process involving hormonal regulation, spermatogenesis, and sperm maturation [<xref rid=\"ref1\" ref-type=\"bibr\">1</xref>]. In theory, each step is a potential site for disruption of male fertility, but some steps may be more amenable to intervention than others. To date, the development of hormonal male contraception has focused on inhibition of spermatogenesis by suppression of the hypothalamic–pituitary secretion of gonadotropins following testosterone replacement therapy [<xref rid=\"ref2\" ref-type=\"bibr\">2</xref>, <xref rid=\"ref3\" ref-type=\"bibr\">3</xref>]. In contrast, nonhormonal male contraceptive lead compounds target Sertoli cells and germ cells [<xref rid=\"ref4\" ref-type=\"bibr\">4–6</xref>]. Ideally, acceptable nonhormonal male contraceptives should be effective and reversible with minimal side effects. To date, no such male contraceptives are available. This is somewhat surprising, because spermatogenesis appears highly susceptible to perturbations and at least 4% of all genes are specifically involved in male reproduction [<xref rid=\"ref7\" ref-type=\"bibr\">7</xref>]. In particular, genetic studies in mice have identified more than 600 genes that are specifically or preferentially involved in the regulation of fertility, providing a plethora of potential molecular targets for contraception in humans [<xref rid=\"ref8\" ref-type=\"bibr\">8–10</xref>].</p><p>Most mouse genetic mutations affecting fertility have been characterized following targeted disruption in embryonic stem cells, forward genetic mutagenesis screens, and CRISPR/Cas9-mediated genome editing. Studies of these mouse mutants have defined a number of fundamental biological processes in germ cell development, gamete maturation, and fertilization [<xref rid=\"ref10\" ref-type=\"bibr\">10</xref>, <xref rid=\"ref11\" ref-type=\"bibr\">11</xref>]. While many of these mutants exhibit pleiotropic defects, a fraction of them give rise to “pure sterile” phenotypes with no observed somatic defects. Such “pure sterile” mutants potentially identify gene targets for developing specific male contraceptives with minimal side effects. Meiotic recombination, an essential process for spermatogenesis, could be targeted for male contraception, since most protein components of meiotic recombination are specifically expressed in germ cells.</p><p>MEIOB (meiosis-specific with oligonucleotide-binding motif) is a meiosis-specific single-stranded DNA-binding protein. We identified MEIOB in a genome-wide proteomics screen for meiotic chromatin-associated proteins in mice [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>]. SPATA22 (spermatogenesis associated 22) is also a meiosis-specific protein essential for meiotic recombination [<xref rid=\"ref13\" ref-type=\"bibr\">13–15</xref>]. Both <italic>Meiob</italic> and <italic>Spata22</italic> are specifically expressed in meiotic germ cells but not in somatic tissues. MEIOB and SPATA22 co-localize as foci on meiotic chromosomes [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>]. Both <italic>Meiob-</italic> and <italic>Spata22</italic>-deficient mice are viable and healthy. However, inactivation of either <italic>Meiob</italic> or <italic>Spata22</italic> causes meiotic failure in both males and females, thus resulting in “pure sterility” in mice [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>, <xref rid=\"ref16\" ref-type=\"bibr\">16</xref>]. Mutations in the human <italic>MEIOB</italic> gene cause infertility in men and primary ovarian insufficiency in women [<xref rid=\"ref17\" ref-type=\"bibr\">17–19</xref>]. In mouse testes, MEIOB interacts with SPATA22. In addition, chromatin localization of MEIOB and SPATA22 is mutually dependent [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>]. Based on these findings, blockade of MEIOB–SPATA22 interaction by chemical inhibitors could disrupt spermatogenesis, thus providing a novel male contraceptive target.</p><p>MEIOB exhibits homology (23% aa identity) with replication protein A1 (RPA1) in the OB (oligonucleotide-binding) domain and thus is a meiosis-specific paralogue of RPA1 [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>]. MEIOB forms a complex with SPATA22 and RPA [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. RPA, a heterotrimer of RPA1, RPA2, and RPA3, is a ubiquitously expressed ssDNA-binding complex, which is required for DNA replication, DNA repair, and meiotic recombination [<xref rid=\"ref21\" ref-type=\"bibr\">21–23</xref>]. Biochemical studies have shown that MEIOB and SPATA22 form an obligate complex through defined interaction domains [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. The SPATA22-binding domain (aa 294–450) in MEIOB is distinct from its ssDNA/RPA1-binding domain. The aspartic residue D383 in MEIOB is required for its interaction with SPATA22, as mutation of D383 to an alanine completely abolishes the MEIOB–SPATA22 interaction in cultured cells [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. The MEIOB-SPATA22 complex interacts with the RPA heterotrimeric complex, but their localizations to DNA double-strand breaks are independent [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>, <xref rid=\"ref23\" ref-type=\"bibr\">23</xref>].</p><p>Enzymes such as kinases have a defined docking pocket for their substrates and thus are traditionally favorite targets for drug development. In contrast, protein–protein interactions have not been traditionally targeted for therapeutic purposes, partly because of the low success in finding small molecules that can inhibit or disrupt the relatively large interfaces often involved in protein–protein interactions. However, despite these relatively large binding interfaces, the majority of binding energy usually involves only a few key residues within an interaction “hot spot” [<xref rid=\"ref24\" ref-type=\"bibr\">24</xref>]. Protein–protein interactions have been targeted for drug development [<xref rid=\"ref25\" ref-type=\"bibr\">25</xref>]. Notably, inhibitors that block protein interactions have been identified for chromatin-binding proteins and apoptosis proteins such as p53 and Bcl-2 [<xref rid=\"ref26\" ref-type=\"bibr\">26</xref>]. In addition, a small molecule, JQ1, blocks the binding of BRDT to acetylated histone, resulting in reversible contraceptive effects in mice [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. Given the largely untapped vast pharmacological space of the protein interactome, protein–protein interactions have become increasingly viable targets for drug development. Here we demonstrate that the D383 residue in MEIOB is essential for its interaction with SPATA22 in the testes. To screen for potential small molecule inhibitors of the MEIOB–SPATA22 interaction, we developed a bimolecular fluorescence complementation (BiFC) assay [<xref rid=\"ref27\" ref-type=\"bibr\">27–29</xref>]. High-content screening of compound libraries identified isocotoin as a hit compound for degradation of MEIOB and SPATA22.</p></sec><sec id=\"sec2\"><title>Materials and methods</title><sec id=\"sec3\"><title>Ethics statement</title><p>Mice were monitored daily in a barrier vivarium and under veterinarian care from University Laboratory Animal Resources at the University of Pennsylvania. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania.</p></sec><sec id=\"sec4\"><title>Generation of <italic>Meiob</italic><sup>D383A</sup> mice</title><p>The guide RNA (GTTGATCTTACTGACCACAC) was cloned into the px330 vector (Addgene) and transcribed in vitro (<xref rid=\"sup1\" ref-type=\"supplementary-material\">Supplementary Figure S1A</xref>) [<xref rid=\"ref30\" ref-type=\"bibr\">30</xref>]. The ssDNA repair template contains the D383A mutation (GAC to GCG): 5’-GAAGCCTCCAATACTTGTACAATTTGCAACCAAGATTCTTCCAGATTGAAATCTTTTTTTCTCAGTTTTGATGTGCTAGTTGATCTTACT<underline>GCG</underline>CACACTGGAACCCTCCATTCCTGCAGTCTCTCAGGAAGTATTGCTGAGGAAACTTTGGGCTGCACGGTAAATAGCAGAAAGGAAAAAATAG-3′. A 50 μl mixture of Cas9 mRNA (catalogue number L-7206, TriLink, 100 ng/μl), sgRNA (50 ng/μl), and ssDNA template (100 ng/μl) was used for microinjection of ~ 60 zygotes (1–2 picoliter per zygote). All zygotes were transferred into three recipient females. Twenty-two founder mice were obtained. The <italic>Meiob</italic><sup>D383A</sup> allele was genotyped by PCR (205 bp) with primers 5’-GATGTGCTAGTTGATCTTACTGCG-3′ and 5’-GATGGCTTTTCCTAAGATCGCTTC-3′. The wild-type allele was genotyped by PCR (205 bp) with primers 5’-GATGTGCTAGTTGATCTTACTGAC-3′ and 5’-GATGGCTTTTCCTAAGATCGCTTC-3′. The genomic DNA around the D383A mutation was amplified by PCR (655 bp) with primers 5’-GTGCGTCTATGGTCTAGATTTGTG-3′ and 5’-GGCAGCTCATAACCATCTTTAACT-3′, subcloned and sequenced. Sanger sequencing revealed frame-shift (FS) mutations in the apparently “wild-type” alleles (<xref rid=\"sup1\" ref-type=\"supplementary-material\">Supplementary Figure S1B and C</xref>). We obtained the following founders: <italic>Meiob</italic><sup>D383A/FS</sup>, <italic>Meiob</italic><sup>D383A/D383A</sup>, and <italic>Meiob</italic><sup>FS/FS</sup>. Four <italic>Meiob</italic><sup>D383A/FS</sup> males were bred with wild-type females, but none of them produced progenies.</p></sec><sec id=\"sec5\"><title>Generation of MEIOB–SPATA22 BiFC constructs</title><p>To generate the NYFP-MEIOB WT construct, NYFP (consisting of amino acids 1–173 of mVenus) (Addgene) was cloned into the HindIII/KpnI sites of the pcDNA3.1/myc-His B plasmid vector (Invitrogen), and mouse <italic>Meiob</italic> cDNA was cloned into the BamHI/EcoRI sites. The NYFP-MEIOB D383A construct was identical to the NYFP-MEIOB WT construct except for the point mutation D383A in MEIOB. To generate the CYFP-SPATA22 construct, CYFP (consisting of amino acids 155–239 of mVenus) was cloned into the HindIII/KpnI sites of the pcDNA3.1/myc-His B plasmid vector, and mouse <italic>Spata22</italic> cDNA was cloned into the BamHI/XhoI sites.</p><p>The BiFC WT construct consisted of NYFP-MEIOB WT, CYFP-SPATA22, and mCherry and was generated based on the NYFP-MEIOB WT construct. CYFP-SPATA22 was subcloned into the EcoRI/XbaI sites of the NYFP-MEIOB WT construct. mCherry was subcloned into the XbaI/ApaI sites. NYFP-MEIOB and CYFP-SPATA22 were separated by E2A peptide (GSGQCTNYALLKLAGDVESNPGP) inserted at the EcoRI site. CYFP-SPATA22 and mCherry were separated by F2A peptide (GSGVKQTLNFDLLKLAGDVESNPGP) inserted at the XbaI site. The BiFC D383A construct was identical to the BiFC WT construct except for the point mutation D383A in MEIOB. All the constructs were verified by Sanger sequencing on an ABI 3730 DNA analyzer.</p></sec><sec id=\"sec6\"><title>Establishment of Tet-inducible BiFC stable cell lines</title><p>The Flp-In T-REx-293 cell line was purchased from Thermo Fisher (catalogue number R78007) and maintained in DMEM/high glucose (MediaTech) supplemented with 10% FBS (Sigma), 1× penicillin–streptomycin (Invitrogen), 100 μg/ml zeocin, and 15 μg/ml blasticidin. The pcDNA5-BiFC construct was generated by subcloning the BiFC cassette containing NYFP-MEIOB-E2A-CYFP-SPATA22-F2A-mCherry into the NotI/XhoI sites of pcDNA5/FRT/TO-neo (catalogue number 41000, Addgene). To generate the BiFC MEIOB WT stable cell line, parental Flp-In T-Rex-293 cells were transfected with pPGKFLPobpA (catalogue number 13793, Addgene) and pcDNA5-BiFC at a 9:1 ratio. Twenty-four hours later, stable cell lines were selected under 400 μg/ml G418 and 15 μg/ml blasticidin for 10 days. A total of 15 individual clones were recovered in a 96-well plate through colony picking, and BiFC WT Clone No. 20 was saved for all experiments. This cell line (No. 20) was maintained in DMEM/high glucose supplemented with 10% FBS, 1× penicillin–streptomycin, 200 μg/ml G418, and 15 μg/ml blasticidin.</p><p>Likewise, we established Nef-NYFP-E2A-Nef-CYFP-F2A-mRFP BiFC tetracycline-inducible stable cell line (Clone No. 2) using the previously reported construct as a template [<xref rid=\"ref29\" ref-type=\"bibr\">29</xref>]. Nef forms a homodimer [<xref rid=\"ref29\" ref-type=\"bibr\">29</xref>].</p></sec><sec id=\"sec7\"><title>Compound libraries</title><p>We screened a total of 47 800 compounds, including 800 compounds from MicroSource natural product library, 3000 bioactive compounds from SelleckChem, 11 000 compounds from ChemDiv’s SMART library, and 33 000 compounds from ChemBridge’s Core set. Compounds were suspended in DMSO, arrayed in columns 3–22 of 384-well microplates, and stored at −20 °C. Library plates were thawed a maximum of 10× to maintain compound integrity.</p></sec><sec id=\"sec8\"><title>High-throughput screening</title><p>We seeded 4000 HEK293:FLP-InT-REx-BiFC#20 cells in a volume of 20 μl per well of 384-well clear-bottom, black-walled microplate (catalogue number 3764, Corning) precoated with 25 μg/ml collagen I using a Multidrop™ Combi Reagent Dispenser (Thermo Scientific). Cells were allowed to attach overnight at 37 °C, 5% CO<sub>2</sub> in a humidified chamber. Compounds (50 nl) were transferred to assay plates using a 384, 50 nl slotted pin tool (V&P Scientific) and a JANUS Automated Workstation (Perkin Elmer). Compounds/drugs were added to a final concentration of 20 μM in 0.2% DMSO. We dispensed 5 μl of 10 μg/ml tetracycline diluted in growth medium to columns 1–23 using a Multidrop™ Combi Reagent Dispenser (Thermo Scientific) for a final concentration of 2 μg/ml. Column 24 received 5 μl growth medium. Cells were incubated at 37 °C, 5% CO<sub>2</sub> for 24 h, and then fixed with 4% formaldehyde. Nuclei were stained with 4 μg/ml Hoechst 33342 dye (Sigma). Cells were imaged at 10× on an automated ImageXpress Micro (Molecular Devices, Sunnyvale CA).</p></sec><sec id=\"sec9\"><title>Data analysis</title><p>The total number cells, YFP+, RFP+, double positive, YFP integrated intensity, and RFP (mCherry) integrated intensity were quantified using a multiwavelength cell scoring algorithm in MetaXpress 5.3.0.5 (Molecular Devices, Sunnyvale, CA). For the pilot screening of the MicroSource and SelleckChem libraries, raw values from tetracycline-induced and non-induced wells were aggregated and used to calculate z’-factors for each assay plate, as a measure of assay performance and data quality. The total number of cells, YFP+, RFP+, double positive cells (YFP + RFP+), YFP integrated Intensity, RFP Integrated Intensity, and YFP/RFP intensity ratio were normalized to aggregated negative plate control wells (i.e., DMSO) and expressed as percentage of control [POC = ((Test well)/(DMSOavg)) × 100] and z-score [Z = (DMSOavg-Test well)/(DMSOsd)], a measure of standard deviations away from the mean of the aggregated negative controls, in Spotfire (PerkinElmer). Candidate hits were defined as compounds that inhibited YFP intensity by three standard deviations from the mean without affecting total number of cells and RFP intensity.</p><p>For the data analysis of screening of ChemDiv’s SMART library and ChemBridge’s core set, raw values from tetracycline-induced wells with DMSO (negative control) and isocotoin (positive control) were aggregated and used to calculate z’-factors for each assay plate, as a measure of assay performance and data quality. Candidate hits were defined by the following criteria: (1) YFP/RFP z-score ≥ −3; total cell POC > 80% (filter for cell toxicity); RFP integrated intensity −3 < z-score < 3 (secondary filter for toxicity and autofluorescence).</p></sec><sec id=\"sec10\"><title>Compound synthesis, induction of protein expression in stable cell lines, co-immunoprecipitation, and Western blot analysis</title><p>200 mg of isocotoin (C<sub>14</sub>H<sub>12</sub>O<sub>4</sub>; CAS# 81525-12-4; MW, 244) was synthesized at the Chemical and Nanoparticle Synthesis Core at the University of Pennsylvania. Isocotoin was dissolved in DMSO, aliquoted, and stored in lightproof tubes at −20 °C. VER-49009 was purchased from SelleckChem. The BiFC stable cell line #20 was used in all experiments. Expression of NYFP-MEIOB and CYFP-SPATA22 was induced by adding tetracycline (1 μg/ml) in the culture media. Cells were harvested for fluorescence imaging. YFP was imaged under the FITC channel, and mCherry was imaged under the Texas Red channel. Cells were lysed using the lysis buffer [62.5 mM Tris-HCl (pH 6.8), 3% SDS, 10% glycerol, and 5% 2-mercaptoethanol] for Western blotting analysis. Epitope-tagged MEIOB and SPATA22 expression constructs, transfection, and co-immunoprecipitation (<xref rid=\"f3\" ref-type=\"fig\">Figure 3D</xref>) were previously described [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. The following primary antibodies were used: rabbit anti-MEIOB antibody (1:250) [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>], rabbit anti-SPATA22 (1:200, catalogue number 16989-1-AP, ProteinTech Group), anti-RPA1 (1:50, catalogue number Ab87272, Abcam), anti-RPA2 (1:50, UP2436, custom-made) [<xref rid=\"ref23\" ref-type=\"bibr\">23</xref>], anti-V5 monoclonal (1:4000, catalogue number R960-25, Invitrogen), anti-FLAG monoclonal (1:10000, catalogue number F1804, Sigma), rabbit anti-GFP (1:1000, catalogue number ab290, Abcam), and mouse anti-ACTB monoclonal antibody (1:10000, catalogue number A5441, clone AC-15, Sigma). YFP fluorescence, mCherry fluorescence, and Western blot band quantifications were performed using ImageJ (<ext-link ext-link-type=\"uri\" xlink:href=\"https://imagej.nih.gov/ij/\">https://imagej.nih.gov/ij/</ext-link>). IC50 was calculated using GraphPad Prism 8.3.0 (GraphPad software, LLC). All quantification experiments were performed at least three times.</p></sec></sec><sec id=\"sec11\"><title>Results</title><sec id=\"sec12\"><title>The aspartic acid D383 in MEIOB is required in vivo</title><p>Our previous biochemical studies identified D383 as a critical residue for MEIOB–SPATA22 interaction [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. The D383 residue is evolutionarily conserved in diverse species from <italic>Drosophila</italic> to human (<xref rid=\"f1\" ref-type=\"fig\">Figure 1A</xref>). The D383A substitution in MEIOB abolished its interaction with SPATA22 when expressed in HEK293 cells [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. In the current study, we sought to test the functional consequence of this substitution <italic>in vivo</italic>. To generate the <italic>Meiob</italic><sup>D383A</sup> allele, a guide RNA flanking <italic>Meiob</italic> codon 383 and an ssDNA template containing D383A mutation (GAC—GCG) were designed for CRISPR/Cas9-mediated genome editing in mouse zygotes (<xref rid=\"sup1\" ref-type=\"supplementary-material\">Supplementary Figure S1A</xref>). Out of 22 founder mice, a total of 15 <italic>Meiob</italic><sup>D383A/FS</sup> (9 males and 6 females; FS, frame shift) and 4 <italic>Meiob</italic><sup>D383A/D383A</sup> (1 male and 3 females) mice were obtained. No <italic>Meiob</italic><sup>D383A/+</sup> mice were produced due to frame-shift nonsense mutations in the apparent “+” allele. No germline transmission of the <italic>Meiob</italic><sup>D383A</sup> point mutation was obtained due to the infertility of both <italic>Meiob</italic><sup>D383A/FS</sup> and <italic>Meiob</italic><sup>D383A/D383A</sup> mice in both sexes. However, we could not exclude the remote possibility that off-target mutations might contribute to the infertility phenotypes.</p><fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>The MEIOB D383A mutation causes meiotic arrest and infertility in mouse. (A) Conservation of the D383 residue in MEIOB across species. The conserved residues are highlighted in red. (B) Histological analysis of testes from 8-week-old wild-type and <italic>Meiob</italic><sup>D383A/D383A</sup> mice. Pac-like, pachytene-like. Scale bar, 50 μm. (C) Western blot analysis of wild-type and <italic>Meiob</italic><sup>D383A/D383A</sup> testes. ACTB serves as a loading control. (D–G) Immunofluorescence analysis of surface nuclear spreads of spermatocytes from adult wild-type and <italic>Meiob</italic><sup>D383A/FS</sup> (founder tag# 74). Spread nuclei of spermatocytes were immunostained with anti-MEIOB (D), anti-SPATA22 (E), anti-RPA1 (F), and anti-RPA2 (G). DNA was stained with DAPI. Scale bars, 10 μm.</p></caption><graphic xlink:href=\"ioaa062f1\"/></fig><p>All the <italic>Meiob</italic> mutant males exhibited sharply reduced testis size and meiotic arrest (<xref rid=\"f1\" ref-type=\"fig\">Figure 1</xref> and <xref rid=\"sup1\" ref-type=\"supplementary-material\">Supplementary Figure S1</xref>). In the <italic>Meiob</italic><sup>D383A/D383A</sup> testis, the most advanced spermatocytes were pachytene-like (<xref rid=\"f1\" ref-type=\"fig\">Figure 1B</xref>). Western blot analysis revealed that MEIOB and SPATA22 were absent in the <italic>Meiob</italic><sup>D383A/D383A</sup> testis. The <italic>Meiob</italic><sup>D383A/FS</sup> testes were also significantly smaller than the wild-type testes (<xref rid=\"sup1\" ref-type=\"supplementary-material\">Supplementary Figure S1B and C</xref>) and displayed meiotic arrest (<xref rid=\"sup1\" ref-type=\"supplementary-material\">Supplementary Figure S1D</xref>). The frame-shift mutations led to the C-terminal truncation of the SPATA22-binding domain (aa 294–450) of MEIOB and thus were expected to disrupt the interaction (<xref rid=\"sup1\" ref-type=\"supplementary-material\">Supplementary Figure S1B and C</xref>) [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. MEIOB and SPATA22 formed foci on meiotic chromosomes in wild-type spermatocytes but not in <italic>Meiob</italic><sup>D383A/FS</sup> spermatocytes (<xref rid=\"f1\" ref-type=\"fig\">Figure 1D and E</xref>). RPA interacts and colocalizes with MEIOB/SPATA22 in spermatocytes [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>]. In contrast, RPA2, a subunit of RPA, was abundantly expressed in the <italic>Meiob</italic><sup>D383A/D383A</sup> testis (<xref rid=\"f1\" ref-type=\"fig\">Figure 1C</xref>). Like in wild-type spermatocytes, RPA1 and RPA2 foci were abundant in <italic>Meiob</italic><sup>D383A/FS</sup> spermatocytes, showing that RPA localization is independent of MEIOB (<xref rid=\"f1\" ref-type=\"fig\">Figure 1F and G</xref>). Therefore, <italic>Meiob</italic><sup>D383A/D383A</sup> and <italic>Meiob</italic><sup>D383A/FS</sup> males displayed the same meiotic arrest phenotype as <italic>Meiob</italic><sup>−/−</sup> males [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>]. Taken together, our study of the point mutant mice demonstrates that the interaction-disrupting mutation (D383A) leads to degradation of MEIOB (D383A) and SPATA22 in testes.</p></sec><sec id=\"sec13\"><title>Development of a BiFC assay for MEIOB–SPATA22 interaction</title><p>The bimolecular fluorescence complementation (BiFC) assay is a cell-based fluorescence technology for characterizing protein–protein interactions [<xref rid=\"ref28\" ref-type=\"bibr\">28</xref>]. Yellow fluorescent protein (YFP) is partitioned into two parts: NYFP and CYFP. These two fragments are not fluorescent when separate but reconstitute the fluorophore and become fluorescent when brought into close proximity (<xref rid=\"f2\" ref-type=\"fig\">Figure 2A</xref>). To develop a cell-based BiFC assay for protein–protein interactions, we constructed a tricistronic multi-protein expression vector (<xref rid=\"f2\" ref-type=\"fig\">Figure 2B</xref>). In this construct, three proteins were expressed: NYFP-MEIOB (wild type), CYFP-SPATA22, and red fluorescent protein monomeric Cherry (mCherry). A “self-cleaving” 2A peptide was inserted between the fusion proteins [<xref rid=\"ref31\" ref-type=\"bibr\">31</xref>]. The 2A peptides are from picornavirus and are extremely rare in the mammalian genome. The “cleavage” caused by the 2A peptide is not proteolytic; instead it promotes ribosomal skipping by preventing the normal peptide bond formation between glycine and proline (<xref rid=\"f2\" ref-type=\"fig\">Figure 2A</xref>) [<xref rid=\"ref32\" ref-type=\"bibr\">32</xref>]. The 2A peptide-mediated cleavage does not affect the translation efficiency of the downstream coding sequences. mCherry serves as a marker of expression and thus as an internal reference protein. Two distinct 2A peptide linkers were used. The E2A linker (from equine rhinitis virus) was inserted between NYFP-MEIOB and CYFP-SPATA22, while the F2A linker (from foot-and-mouth disease virus) was engineered between CYFP-SPATA22 and mCherry (<xref rid=\"f2\" ref-type=\"fig\">Figure 2B</xref>). HEK293 cells were transfected with this multi-protein expression construct. Both green fluorescence (restored by MEIOB–SPATA22 interaction) and red fluorescence (mCherry) were observed in transfected cells (<xref rid=\"f2\" ref-type=\"fig\">Figure 2C</xref>). As a negative control, the interaction-disrupting point mutation (D383A) in MEIOB was introduced in the multi-protein expression vector (<xref rid=\"f2\" ref-type=\"fig\">Figure 2B</xref>). As expected, cells transfected with this mutant vector were positive for mCherry but negative for green fluorescence (YFP) (<xref rid=\"f2\" ref-type=\"fig\">Figure 2C</xref>). These results showed that the BiFC assay for the MEIOB–SPATA22 interaction was robust.</p><fig id=\"f2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>A cell-based high-content screening assay for MEIOB–SPATA22 interaction. (A) Bimolecular fluorescence complementation assay for protein–protein interactions. Binding of proteins A (MEIOB) and B (SPATA22) enables reconstitution of the fluorophore core of YFP. (B) Schematic design of a multi-protein tricistronic expression construct for the BiFC assay. Two self-cleaving 2A peptide sequences are from picornavirus. E2A: equine rhinitis A virus. F2A: foot-and-mouth disease virus. (C) Fluorescence readout of the BiFC assay. HEK293T cells were transfected with the wild-type construct or the mutant construct in the 384-well format. Cells were imaged 48 h post transfection. Only one well is shown per construct. Scale bar, 50 μm.</p></caption><graphic xlink:href=\"ioaa062f2\"/></fig><p>We next cloned the NYFP-MEIOB CYFP-SPATA22 mCherry insert from the BiFC vector (<xref rid=\"f2\" ref-type=\"fig\">Figure 2A</xref>) into the pcDNA5/FRT/TO vector (tetracycline-inducible). A stable cell line (No. 20) was generated by transfection of the Flp-In T-REx 293 cells (Thermo Fisher) with the pcDNA5-BiFC (wild type) vector and the Flp recombinase-expressing plasmid [<xref rid=\"ref33\" ref-type=\"bibr\">33</xref>]. The advantage of a stable Tet-inducible BiFC cell line is threefold: (1) inducible expression, (2) no need for transfection, and (3) increased reproducibility.</p></sec><sec id=\"sec14\"><title>High-throughput screening of compound libraries</title><p>We screened a total of 47 800 compounds from four libraries (MicroSource purified natural product library, SelleckChem bioactive library, ChemDiv’s SMART library, and ChemBridge’s Core set) using the BiFC stable cell line in the 384-well format. As reconstitution of the YFP fluorophore in the BiFC system is irreversible [<xref rid=\"ref28\" ref-type=\"bibr\">28</xref>], inhibitors must be added prior to the appearance of fluorescence. YFP fluorescence usually appears 8–12 h after transfection [<xref rid=\"ref29\" ref-type=\"bibr\">29</xref>], due to the time required for expression, protein–protein binding, YFP complementation, and fluorophore maturation [<xref rid=\"ref28\" ref-type=\"bibr\">28</xref>]. In our screen, compounds and tetracycline were added simultaneously to the cells. After 24 h, cells were fixed for imaging. In the pilot screen, we screened 800 purified natural products (MicroSource) and obtained 6 compound hits defined by 3 standard deviations from the aggregate mean of the YFP/mCherry ratio in all wells and more than 50% of cell viability (<xref rid=\"sup2\" ref-type=\"supplementary-material\">Supplementary Table S1</xref>). Isocotoin was the most potent compound identified with 60% inhibition of the YFP fluorescence. In the second pilot screen, we screened a library of 3000 small molecules (SelleckChem) with annotated biological activities and obtained three hits (<xref rid=\"sup2\" ref-type=\"supplementary-material\">Supplementary Table S2</xref>).</p><p>We next screened 44 000 compounds from ChemDiv’s SMART library and ChemBridge’s core set. Because there were no known inhibitors of the MEIOB–SPATA22 interaction, isocotoin identified in the pilot screen was used as a positive control in these large-scale screens. Compounds were added to a final concentration of 20 μM. We determined candidate hits by the following criteria: (a) the YFP/RFP ratio z-score was less than −3 (three standard deviations); (b) cell viability (total cell POC) was > 80%; (c) RFP integrated intensity z-score was between −3 and 3 (excluding toxicity and autofluorescence). By these criteria, a total of 323 compounds were identified at a hit rate of 0.7%: 143 from ChemDiv’s SMART library (<xref rid=\"sup2\" ref-type=\"supplementary-material\">Supplementary Table S3</xref>) and 180 from ChemBridge’s core set (<xref rid=\"sup2\" ref-type=\"supplementary-material\">Supplementary Table S4</xref>).</p></sec><sec id=\"sec15\"><title>Isocotoin inhibits the MEIOB–SPATA22 interaction</title><p>Isocotoin from the natural product library (C<sub>14</sub>H<sub>12</sub>O<sub>4</sub>; CAS# 81525-12-4; MW, 244; <xref rid=\"f3\" ref-type=\"fig\">Figure 3A</xref>) reduced the YFP fluorescence in tetracycline-treated BiFC cells (<xref rid=\"sup1\" ref-type=\"supplementary-material\">Supplementary Table S1</xref>). We retested the effect of isocotoin with resynthesized compound. In the BiFC cells, isocotoin (80 μM) sharply reduced the YFP fluorescence but had no effect on the mCherry red fluorescence (<xref rid=\"f3\" ref-type=\"fig\">Figure 3B</xref>). Isocotoin (80 uM) reduced the YFP/mCherry ratio by 37% (<xref rid=\"f3\" ref-type=\"fig\">Figure 3C</xref>). To test whether isocotoin inhibits the MEIOB–SPATA22 interaction, we performed co-immunoprecipitation with and without isocotoin (<xref rid=\"f3\" ref-type=\"fig\">Figure 3D</xref>). HEK293 cells were transfected separately with MEIOB and SPATA22. Protein lysates were incubated with DMSO, VER-49009, or isocotoin, prior to mixing and co-immunoprecipitation. VER-49009 was a hit compound from the SelleckChem library screening (<xref rid=\"sup2\" ref-type=\"supplementary-material\">Supplementary Table S2</xref>). VER-49009 is a potent inhibitor of HSP90, a molecular chaperone involved in stress responses, folding, and stability of client proteins [<xref rid=\"ref34\" ref-type=\"bibr\">34</xref>, <xref rid=\"ref35\" ref-type=\"bibr\">35</xref>]. Inhibition of HSP90 causes degradation of substrate proteins through the ubiquitin–proteasome pathway [<xref rid=\"ref36\" ref-type=\"bibr\">36</xref>]. Much less MEIOB was co-immunoprecipitated with SPATA22 in the presence of isocotoin than the DMSO control and VER-49009, showing that isocotoin inhibits the MEIOB–SPATA22 complex formation but VER-49009 does not (<xref rid=\"f3\" ref-type=\"fig\">Figure 3D</xref>).</p><fig id=\"f3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Isocotoin inhibits the MEIOB–SPATA22 interaction. (A) Chemical structure of isocotoin. (B) Isocotoin reduces the YFP fluorescence in the BiFC stable cell lines. The MEIOB–SPATA22 BiFC stable cells (No. 20) were treated for 24 h with tetracycline (1 μg/ml) alone or both isocotoin (80 μM) and tetracycline. Scale bar, 50 μm. (C) Quantification of YFP/mCherry fluorescence (ratio on the y-axis). Four images of each treatment (as in panel B) were quantified with ImageJ. ~ 1000 cells in each treatment were quantified. (D) Co-immunoprecipitation analysis with and without compounds. The experiment flowchart is shown on the left. HEK293 cells were transfected separately with MEIOB- and SPATA22-expressing plasmids. Epitope-tagged constructs were previously described [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. The final concentration of compounds in the lysate was 20 μM. VER-49009 is an inhibitor of HSP90 [<xref rid=\"ref35\" ref-type=\"bibr\">35</xref>].</p></caption><graphic xlink:href=\"ioaa062f3\"/></fig></sec><sec id=\"sec16\"><title>Isocotoin promotes the degradation of MEIOB and SPATA22</title><p>We examined the protein abundance in the BiFC cells treated with tetracycline and isocotoin simultaneously (<xref rid=\"f4\" ref-type=\"fig\">Figure 4A</xref>). Without tetracycline, MEIOB and SPATA22 were not expressed. Isocotoin reduced the abundance of both MEIOB and SPATA22 in a dose-dependent manner. The isocotoin IC50 was 75 ± 10 μM for MEIOB and 51 ± 8 μM for SPATA22. In this experiment, transcription was continuously induced by tetracycline (<xref rid=\"f4\" ref-type=\"fig\">Figure 4A</xref>). We next performed a pulse–chase experiment (<xref rid=\"f4\" ref-type=\"fig\">Figure 4B</xref>). The BiFC cells were treated with tetracycline for 8 h, washed, and subsequently treated with isocotoin for 16 h. Consistently, isocotoin decreased the abundance of MEIOB and SPATA22 in a dose-dependent manner, suggesting that isocotoin affects the protein stability after complex formation. As expected, the isocotoin IC50 was lower: 62 ± 6 μM for MEIOB and 37 ± 11 μM for SPATA22 (<xref rid=\"f4\" ref-type=\"fig\">Figure 4B</xref>). Furthermore, isocotoin promoted degradation of MEIOB and SPATA22 in short-term treatments (4–8 h) (<xref rid=\"f4\" ref-type=\"fig\">Figure 4C</xref>). HIV Nef protein forms homodimers [<xref rid=\"ref29\" ref-type=\"bibr\">29</xref>]. Isocotoin did not promote degradation of the Nef-YFP protein in the Nef-YFP BiFC cells, suggesting that isocotoin does not inhibit YFP (<xref rid=\"f4\" ref-type=\"fig\">Figure 4D</xref>). These results demonstrated that isocotoin causes degradation of MEIOB and SPATA22.</p><fig id=\"f4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Isocotoin promotes degradation of MEIOB and SPATA22. (A) Dose–response curve of isocotoin on the degradation of MEIOB and SPATA22. The BiFC stable cells were treated simultaneously with tetracycline (1 μg/ml) and various concentrations of isocotoin (0–80 μM) for 24 h. Note that transcription of <italic>Meiob</italic> and <italic>Spata22</italic> was continuous, due to the presence of tetracycline in the medium in the 24-h duration of experiments. The experiments were performed three times. Band intensity was quantified using ImageJ and plotted on the right. (B) Dose–response curve of isocotoin on the degradation of existing MEIOB and SPATA22. The BiFC stable cells were treated with tetracycline (1 μg/ml) for 8 h, washed, and cultured in fresh medium with isocotoin only (0–80 μM) for 16 h. The experiments were performed three times and plotted on the right. (C) Isocotoin promotes degradation of MEIOB/SPATA22 in short-term treatments. The BiFC stable cells were treated with tetracycline (1 μg/ml) alone or simultaneously with tetracycline (1 μg/ml) and isocotoin (80 μM) for 4, 6, and 8 h. Tet: tetracycline. (D) Lack of effect of isocotoin on degradation of Nef-YFP. The Nef-NYFP-Nef-CYFP BiFC stable cells were treated for 8 h and then collected for Western blot analysis. Note that VER-49009 lacks effect on the stability of Nef-YFP.</p></caption><graphic xlink:href=\"ioaa062f4\"/></fig></sec></sec><sec id=\"sec17\"><title>Discussion</title><p>We have demonstrated that the conserved D383 residue in MEIOB is essential for its function in meiosis in vivo. Importantly, both MEIOB and SPATA22 proteins were absent in the <italic>Meiob</italic><sup>D383A/D383A</sup> testes (<xref rid=\"f1\" ref-type=\"fig\">Figure 1C</xref>), showing that disruption of their interaction destabilizes both proteins in vivo. Therefore, small molecule inhibitors of their interaction would lead to their degradation and thus infertility. Because MEIOB and SPATA22 are only expressed in meiotic germ cells, specific inhibition of their interaction will probably not affect somatic cells [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>, <xref rid=\"ref13\" ref-type=\"bibr\">13</xref>]. In addition, since MEIOB and SPATA22 are not expressed in spermatogonial stem cells, the contraceptive effect of these inhibitors would be reversible in one cycle of spermatogenesis. As genetic and molecular studies indicate that these two proteins can be manipulated to result in a “pure sterile” phenotype, these two proteins constitute novel validated male contraceptive targets.</p><p>We have developed a cell-based BiFC assay for screening inhibitors of the MEIOB–SPATA22 interaction. The BiFC assay has been developed for investigating protein–protein interactions or protein dimerization and for chemical library screening [<xref rid=\"ref28\" ref-type=\"bibr\">28</xref>, <xref rid=\"ref29\" ref-type=\"bibr\">29</xref>, <xref rid=\"ref37\" ref-type=\"bibr\">37</xref>]. In our BiFC assay, a single tricistronic vector has the advantage of expressing all three proteins in the same cell (<xref rid=\"f2\" ref-type=\"fig\">Figure 2B</xref>). However, a single vector still requires transfection. Even though transfection efficiency in HEK293 cells is usually very high, protein expression still varies from cell to cell. We have generated a MEIOB–SPATA22 BiFC HEK293 stable cell line, in which transcription is tetracycline-inducible. The use of an inducible stable cell line significantly simplified the screening process and increased uniformity of protein expression levels. This BiFC stable cell line can be readily used for screening of more compounds in the future. Because this assay is cell-based, cytotoxic compounds can be excluded. In addition, autofluorescent compounds can also be excluded. Another advantage of this assay is that compounds identified are expected to be cell-permeable.</p><p>High-content screening of chemical libraries using our BiFC assay has identified compound hits for further study (<xref rid=\"sup2\" ref-type=\"supplementary-material\">Supplementary Tables S1</xref>–<xref rid=\"sup2\" ref-type=\"supplementary-material\">S4</xref>). Isocotoin was one of the most potent compounds identified in this screen. Isocotoin causes degradation of MEIOB and SPATA22 in the BiFC cell line (<xref rid=\"f4\" ref-type=\"fig\">Figure 4</xref>). Isocotoin inhibits the complex formation of MEIOB and SPAT22 (<xref rid=\"f3\" ref-type=\"fig\">Figure 3D</xref>). Intriguingly, isocotoin also promotes degradation of MEIOB and SPATA22 even after they form complexes (<xref rid=\"f4\" ref-type=\"fig\">Figure 4B</xref>). One possible explanation is that isocotoin not only inhibits the MEIOB–SPATA22 interaction but also promotes their degradation through an unknown mechanism even after they form a complex. The biochemical basis of action by isocotoin in the degradation of MEIOB and SPATA22 warrants further investigation. This study shows that the BiFC assay can be used for screening not only inhibitors of protein–protein interaction but also compounds that promote protein degradation.</p></sec><sec id=\"sec19\"><title>Authors’ contributions</title><p>YX, RL, and LZ carried out the experiments. NAL performed microinjection of zygotes. II and DCS performed the compound screen. YX, RL, II, and DCS analyzed data. PJW, YX, II, and DCS wrote the manuscript. All authors commented on the manuscript.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><supplementary-material content-type=\"local-data\" id=\"sup1\"><label>Figure_S1_ioaa062</label><media xlink:href=\"figure_s1_ioaa062.png\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"sup2\"><label>Table_S1_4_ioaa062</label><media xlink:href=\"table_s1_4_ioaa062.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"ioaa062-ack\"><title>Acknowledgment</title><p>We thank Kido Nwe and the Chemical and Nanoparticle Synthesis Core at the University of Pennsylvania for synthesis of isocotoin, Thomas Smithgall for the Nef BiFC plasmid, and Rui Guo for imaging histology slides.</p></ack><sec id=\"sec19a\"><title>Conflict of interest</title><p>The authors declare that they have no conflict of interest.</p></sec><ref-list id=\"bib1\"><title>References</title><ref id=\"ref1\"><label>1.</label><mixed-citation publication-type=\"book\">\n<person-group person-group-type=\"author\"><name name-style=\"western\"><surname>Russell</surname><given-names>LD</given-names></name>, <name name-style=\"western\"><surname>Ettlin</surname><given-names>RA</given-names></name>, <name name-style=\"western\"><surname>Sinha Hikim</surname><given-names>AP</given-names></name>, <name name-style=\"western\"><surname>Clegg</surname><given-names>ED</given-names></name></person-group>\n<source>Histological and Histopathological Evaluation of the Testis</source>. <publisher-loc>Clearwater, FL</publisher-loc>:\n<publisher-name>Cache River Press</publisher-name>; 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.3 20210610//EN\" \"JATS-archivearticle1-3-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" dtd-version=\"1.3\" xml:lang=\"en\" article-type=\"research-article\"><?properties open_access?><?properties manuscript?><processing-meta base-tagset=\"archiving\" mathml-version=\"3.0\" table-model=\"xhtml\" tagset-family=\"jats\"><restricted-by>pmc</restricted-by></processing-meta><front><journal-meta><journal-id journal-id-type=\"nlm-journal-id\">101672100</journal-id><journal-id journal-id-type=\"pubmed-jr-id\">44627</journal-id><journal-id journal-id-type=\"nlm-ta\">Clin Case Rep Rev</journal-id><journal-id journal-id-type=\"iso-abbrev\">Clin Case Rep Rev</journal-id><journal-title-group><journal-title>Clinical case reports and reviews</journal-title></journal-title-group><issn pub-type=\"epub\">2059-0393</issn></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32999736</article-id><article-id pub-id-type=\"pmc\">PMC7523693</article-id><article-id pub-id-type=\"manuscript\">NIHMS1607896</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>MEK inhibition with trametinib is a successful therapy in ganglioglioma</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Daniel</surname><given-names>Eliza Baird</given-names></name><degrees>BA</degrees><xref rid=\"A1\" ref-type=\"aff\">1</xref><xref rid=\"A2\" ref-type=\"aff\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ney</surname><given-names>Douglas E</given-names></name><xref rid=\"A3\" ref-type=\"aff\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Levy</surname><given-names>Jean M Mulcahy</given-names></name><xref rid=\"A1\" ref-type=\"aff\">1</xref><xref rid=\"A2\" ref-type=\"aff\">2</xref><xref rid=\"CR1\" ref-type=\"corresp\">*</xref></contrib></contrib-group><aff id=\"A1\"><label>1</label>Department of Pediatrics, University of Colorado Denver, Aurora, USA</aff><aff id=\"A2\"><label>2</label>The Morgan Adams Foundation Pediatric Brain Tumor Research Program, Children’s Hospital Colorado, Aurora, USA</aff><aff id=\"A3\"><label>3</label>Departments of Neurology and Neurosurgery, University of Colorado School of Medicine, Aurora, USA</aff><author-notes><corresp id=\"CR1\"><label>*</label><bold><italic toggle=\"yes\">Correspondence to:</italic></bold> Jean M Mulcahy Levy, UC Denver at Anschutz Medical Campus, Pediatrics Department, Mail Stop 8302, 12800 E. 19th Ave. Aurora, CO 80045, USA, Tel: (303)-724-3372; Fax: (303) 724-3363; Jean. <email>MulcahyLevy@ucdenver.edu</email></corresp></author-notes><pub-date pub-type=\"nihms-submitted\"><day>18</day><month>7</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>6</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>08</day><month>5</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>29</day><month>9</month><year>2020</year></pub-date><volume>6</volume><issue>2</issue><elocation-id>479</elocation-id><permissions><license><ali:license_ref xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\" specific-use=\"textmining\" content-type=\"ccbylicense\">https://creativecommons.org/licenses/by/4.0/</ali:license_ref><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract id=\"ABS1\"><p id=\"P1\">Gangliogliomas are predominantly low-grade primary brain tumors comprised of neuronal and glial components that are found in both pediatric and young adult populations. In the majority of cases, surgical resection of these tumors is curative. However, tumor location in eloquent centers of the brain can make surgical intervention inappropriate. Additionally, a subset of tumors progress to anaplastic ganglioglioma which carries a poor prognosis, despite resection. Activating mutations in the MAPK pathway, such as BRAF V600E, have been identified in many of these tumors. Tumors carrying such mutations have demonstrated susceptibility to MEK inhibition therapy. However, there remains a subset of ganglioglioma that do not contain a known mutation in the MAPK pathway and thus have not been targeted with MEK inhibition therapy. Here, we present a young adult ganglioglioma patient without identified MAPK pathway activation mutations who demonstrated a significant and sustained response to MEK inhibition with trametinib.</p></abstract><kwd-group><kwd>ganglioglioma</kwd><kwd>MEK inhibitor</kwd><kwd>Trametinib</kwd><kwd>BRAF</kwd><kwd>low-grade-glioma</kwd></kwd-group></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p id=\"P2\">Gangliogliomas are primary brain tumors most commonly arising in children and young adults less than 30 years old but are seen in the older adult population as well. Like pediatric patients, adult ganglioglioma are predominantly low-grade (91.4%) [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]. While they are generally associated with a favorable prognosis following gross total resection (GTR), a subset may progress to anaplastic ganglioglioma while other tumors are in eloquent centers of the brain and unable to be resected [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]. In adults it has been reported that only 59% of patients are able to achieve a GTR which can have an adverse effect on outcomes [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]. Ganglioglioma patients have been reported to have progression free survival (PFS) of 58% at 5 years and only 37% at 10 years with extent of resection most strongly associated with PFS. Reported medium time to progression was only 1.8 years with a subtotal resection (STR) while this extended to 16.7 years with a GTR [<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]. The role of chemotherapy and radiation for patients with STR is still under debate and maximal safe resection remains the gold-standard therapy [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>].</p><p id=\"P3\">In recent years there has been extensive work describing the genetic landscape and potential therapeutic targets in these tumors. The BRAF V600E mutation makes up a significant amount of the genetic alterations found in ganglioglioma, but other MAPK and non-MAPK pathway mutations have been described [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. Pekmezci et al. note that in the majority of cases, ganglioglioma showed BRAF alterations however, in tumors lacking identified BRAF mutations, 69% demonstrated mutations that are predicted to activate the MAPK pathway such as alterations in KRAS, FGFR2, NF1, and RAF1 [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>].</p><p id=\"P4\">Given the prevalence of MAPK activation described in gangliogliomas, MEK inhibitors such as trametinib may provide a significant therapeutic option. MEK inhibition in conjunction with BRAF inhibition has shown promising results in BRAF V600E mutation in adult anaplastic ganglioglioma [<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]. Additionally, trametinib therapy in other low-grade gliomas with known activation mutations in the MAPK/ERK pathway shows promising response rates and regimen tolerance in pediatric patients [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]. Ultimately, a subset (estimated to make up 10–60% of ganglioglioma) containing BRAF V600E/ MAPK mutations respond to MEK inhibition however effects of this treatment on other subsets harboring genetic alterations both within and outside of the MAPK pathway remains largely unknown [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. We present an adult GG patient without identified MAPK pathway activation mutations who demonstrated a significant and sustained response to MEK inhibition with trametinib</p></sec><sec id=\"S2\"><title>Case Report</title><p id=\"P5\">A 32-year-old woman presented with two to three years of progressive right-sided arm greater than leg weakness. Her history consisted of a bifrontal brain mass at the age of 11 years. At that time, she underwent biopsy, which was said to be consistent with germinoma, however, the pathology was not able to be re-reviewed. Adjuvant treatment consisted of craniospinal radiotherapy with resolution of disease without recurrence. At the time of her current presentation, MRI imaging showed an enhancing left thalamic mass (<xref rid=\"F1\" ref-type=\"fig\">Figure 1a</xref>). Biopsy was undertaken which was consistent with a WHO grade 1 ganglioglioma, negative for BRAF or other targetable mutations. Due to the location of the lesion she was not a candidate for attempt at GTR. She initiated treatment with trametinib 2 mg daily which was complicated by diffuse rash and photosensitivity which resolved after drug interruption to allow rash recovery and dose reduction to 1.5 mg daily. Follow-up at 6 months showed significant radiographic improvement (<xref rid=\"F1\" ref-type=\"fig\">Figure 1b</xref>) as well as improvement in strength. Clinical and radiographic response persists at 18 months post-initiation of trametinib and she continues on 1.5 mg daily.</p></sec><sec id=\"S3\"><title>Discussion</title><p id=\"P6\">Due to the great majority of characterized mutations in ganglioglioma identified as players in the MAPK pathway [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>], we hypothesized for this patient that ganglioglioma is largely a single pathway (MAPK) driven neoplasm. Thus, we predicted that tumors without an identified mutation within the MAPK pathway, such as this patient’s tumor, would also be responsive to MEK inhibition. The success of trametinib in this patient highlights the potential benefit of this class of drugs for patients with unresectable ganglioglioma.</p><p id=\"P7\">There are multiple ongoing studies of MEK inhibitors for central nervous system tumors in pediatric patients including the Pediatric MATCH (<ext-link xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT03213691\" ext-link-type=\"uri\">NCT03213691</ext-link>) and other trials (<ext-link xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT02285439\" ext-link-type=\"uri\">NCT02285439</ext-link>, <ext-link xlink:href=\"https://clinicaltrials.gov/ct2/show/NCT01089101\" ext-link-type=\"uri\">NCT01089101</ext-link>). And while there are several studies including targeting the MAPK pathway in brain metastatic melanoma and other tumors, there are currently no primary adult CNS tumor studies evaluating the potential of MEK inhibitors for patients with verified or presumed MAPK pathway activation. This case and the success of MEK inhibition in primary pediatric low-grade gliomas would suggest a potential avenue of new research for adult low-grade and ganglioglioma patients [<xref rid=\"R5\" ref-type=\"bibr\">5</xref>].</p></sec></body><back><ack id=\"S4\"><p id=\"P8\">Funding</p><p id=\"P9\">Supported by NIH/NCI (K08CA193982) and The Morgan Adams Foundation (EBD, JML).</p></ack><ref-list><title>References</title><ref id=\"R1\"><label>1.</label><mixed-citation publication-type=\"journal\"><name><surname>Varshneya</surname><given-names>K</given-names></name>, <name><surname>Sarmiento</surname><given-names>JM</given-names></name>, <name><surname>Nuno</surname><given-names>M</given-names></name>, <name><surname>Lagman</surname><given-names>C</given-names></name>, <name><surname>Mukherjee</surname><given-names>D</given-names></name>, <etal/> (<year>2016</year>) <article-title>A national perspective of adult gangliogliomas</article-title>. <source>J Clin Neurosci</source>\n<volume>30</volume>: <fpage>65</fpage>–<lpage>70</lpage>.<pub-id pub-id-type=\"pmid\">27083133</pub-id></mixed-citation></ref><ref id=\"R2\"><label>2.</label><mixed-citation publication-type=\"journal\"><name><surname>Beland</surname><given-names>B</given-names></name>, <name><surname>Tsang</surname><given-names>RY</given-names></name>, <name><surname>Sutherland</surname><given-names>G</given-names></name> (<year>2018</year>) <article-title>Unprecedented response to combination BRAF and MEK inhibitors in adult anaplastic ganglioglioma</article-title>. <source>J Neurooncol</source>\n<volume>137</volume>: <fpage>667</fpage>–<lpage>669</lpage>.<pub-id pub-id-type=\"pmid\">29335912</pub-id></mixed-citation></ref><ref id=\"R3\"><label>3.</label><mixed-citation publication-type=\"journal\"><name><surname>Compton</surname><given-names>JJ</given-names></name>, <name><surname>Laack</surname><given-names>NN</given-names></name>, <name><surname>Eckel</surname><given-names>LJ</given-names></name>, <name><surname>Schomas</surname><given-names>DA</given-names></name>, <name><surname>Giannini</surname><given-names>C</given-names></name>, <etal/> (<year>2012</year>) <article-title>Long-term outcomes for low-grade intracranial ganglioglioma: 30-year experience from the Mayo Clinic</article-title>. <source>J Neurosurg</source>\n<volume>117</volume>: <fpage>825</fpage>–<lpage>830</lpage>.<pub-id pub-id-type=\"pmid\">22957524</pub-id></mixed-citation></ref><ref id=\"R4\"><label>4.</label><mixed-citation publication-type=\"journal\"><name><surname>Pekmezci</surname><given-names>M</given-names></name>, <name><surname>Villanueva-Meyer</surname><given-names>JE</given-names></name>, <name><surname>Goode</surname><given-names>B</given-names></name>, <name><surname>Van Ziffle</surname><given-names>J</given-names></name> (<year>2018</year>) <article-title>The genetic landscape of ganglioglioma</article-title>. <source>Acta Neuropathol Commun</source>\n<volume>6</volume>: <fpage>47</fpage>.<pub-id pub-id-type=\"pmid\">29880043</pub-id></mixed-citation></ref><ref id=\"R5\"><label>5.</label><mixed-citation publication-type=\"journal\"><name><surname>Kondyli</surname><given-names>M</given-names></name>, <name><surname>Larouche</surname><given-names>V</given-names></name>, <name><surname>Saint-Martin</surname><given-names>C</given-names></name>, <name><surname>Ellezam</surname><given-names>B</given-names></name> (<year>2018</year>) <article-title>Trametinib for progressive pediatric low-grade gliomas</article-title>. <source>J Neurooncol</source>\n<volume>140</volume>: <fpage>435</fpage>–<lpage>444</lpage><pub-id pub-id-type=\"pmid\">30097824</pub-id></mixed-citation></ref></ref-list></back><floats-group><fig position=\"float\" id=\"F1\"><label>Figure 1.</label><caption><p id=\"P10\">Ganglioglioma without identified MAPK pathway activation mutations demonstrates a significant and sustained response to MEK inhibition with trametinib</p><p id=\"P11\"><bold>T1-weighted post-contrast axial images:</bold> (A) showing contrast enhancing lesion centered in the left thalamic region. (B) Follow-up imaging 6 months post-initiation of trametinib demonstrated a significant partial radiographic response</p></caption><graphic xlink:href=\"nihms-1607896-f0001\" position=\"float\"/></fig></floats-group></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Biol Reprod</journal-id><journal-id journal-id-type=\"iso-abbrev\">Biol. Reprod</journal-id><journal-id journal-id-type=\"publisher-id\">biolreprod</journal-id><journal-title-group><journal-title>Biology of Reproduction</journal-title></journal-title-group><issn pub-type=\"ppub\">0006-3363</issn><issn pub-type=\"epub\">1529-7268</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32543655</article-id><article-id pub-id-type=\"pmc\">PMC7523694</article-id><article-id pub-id-type=\"doi\">10.1093/biolre/ioaa107</article-id><article-id pub-id-type=\"publisher-id\">ioaa107</article-id><article-categories><subj-group subj-group-type=\"category-taxonomy-collection\"><subject>AcademicSubjects/MED00773</subject><subject>AcademicSubjects/SCI01070</subject></subj-group><subj-group subj-group-type=\"heading\"><subject>Contraceptive Special Issue</subject></subj-group></article-categories><title-group><article-title>Review of rationale and progress toward targeting cyclin-dependent kinase 2 (CDK2) for male contraception<xref ref-type=\"author-notes\" rid=\"afn1\"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Faber</surname><given-names>Erik B</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref ref-type=\"aff\" rid=\"aff2\">2</xref><xref ref-type=\"author-notes\" rid=\"afn2\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Nan</given-names></name><xref ref-type=\"author-notes\" rid=\"afn2\"/><xref ref-type=\"aff\" rid=\"aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Georg</surname><given-names>Gunda I</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">1</xref><xref rid=\"cor1\" ref-type=\"corresp\"/><!--<email>georg@umn.edu</email>--></contrib></contrib-group><aff id=\"aff1\"><label>1</label>\n<institution>Department of Medicinal Chemistry</institution>, College of Pharmacy, University of Minnesota–Twin Cities, Minneapolis, MN, USA</aff><aff id=\"aff2\"><label>2</label>\n<institution>Medical-Scientist Training Program</institution>, University of Minnesota Medical School, University of Minnesota–Twin Cities, Minneapolis, MN, USA</aff><author-notes><corresp id=\"cor1\"><bold>Correspondence:</bold> Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota–Twin Cities, 717 Delaware St. SE, Room 451, Minneapolis, MN 55414, USA. Tel: <phone>+1-612-626-6320</phone>; E-mail: <email>georg@umn.edu</email></corresp><fn id=\"afn1\"><p>\n<bold>Grant Support:</bold> This work was supported by NIH/NICHD 5 U01 HD080431, NIH/NICHD 1 R61 HD099743, and NIH/NIGMS R01 GM121515. E. Faber was supported by NIH/NIGMS training grants T32 GM008244 and T32 GM008700 as well as by NIH/NCI fellowship F30 CA232303.</p></fn><fn id=\"afn2\"><p>Erik B. Faber and Nan Wang contributed equally to this work.</p></fn></author-notes><pub-date pub-type=\"ppub\"><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"epub\" iso-8601-date=\"2020-06-16\"><day>16</day><month>6</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>16</day><month>6</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>103</volume><issue>2</issue><fpage>357</fpage><lpage>367</lpage><history><date date-type=\"received\"><day>19</day><month>4</month><year>2020</year></date><date date-type=\"rev-recd\"><day>3</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>19</day><month>4</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"cc-by\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"ioaa107.pdf\"/><abstract><title>Abstract</title><p>Cyclin-dependent kinase 2 (CDK2) is a member of the larger cell cycle regulating CDK family of kinases, activated by binding partner cyclins as its name suggests. Despite its canonical role in mitosis, <italic>CDK2</italic> knockout mice are viable but sterile, suggesting compensatory mechanisms for loss of CDK2 in mitosis but not meiosis. Here, we review the literature surrounding the role of CDK2 in meiosis, particularly a cyclin-independent role in complex with another activator, Speedy 1 (SPY1). From this evidence, we suggest that CDK2 could be a viable nonhormonal male contraceptive target. Finally, we review the literature of pertinent CDK2 inhibitors from the preclinical to clinical stages, mostly developed to treat various cancers. To date, there is no potent yet selective CDK2 inhibitor that could be repurposed as a contraceptive without appreciable off-target toxicity. To achieve selectivity for CDK2 over closely related kinases, developing compounds that bind outside the conserved adenosine triphosphate-binding site may be necessary.</p></abstract><abstract abstract-type=\"teaser\"><p>Cyclin-dependent kinase 2 (CDK2) is a validated and underexploited target for male contraception.</p></abstract><kwd-group><kwd>contraception</kwd><kwd>spermatogenesis</kwd><kwd>kinases</kwd><kwd>male reproductive tract</kwd><kwd>meiosis</kwd><kwd>meiotic arrest</kwd></kwd-group><funding-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>National Institutes of Health</institution><institution-id institution-id-type=\"DOI\">10.13039/100000002</institution-id></institution-wrap></funding-source><award-id>5 U01 HD080431</award-id><award-id>1 R61 HD099743</award-id><award-id>R01 GM121515</award-id><award-id>T32 GM008244</award-id><award-id>T32 GM008700</award-id><award-id>F30 CA232303</award-id></award-group></funding-group><counts><page-count count=\"11\"/></counts></article-meta></front><body><sec id=\"sec1\"><title>Introduction</title><p>The exploration of nonhormonal targets is considered a new and promising approach to discover and develop highly effective, well-tolerated, and reversible male and female contraceptive agents. Despite this possibility, a nonhormonal male contraceptive agent has not entered clinical trial or progressed toward investigational new drug-enabling preclinical development. The purpose of this review is to explore the evidence and remaining questions surrounding cyclin-dependent kinase 2 (CDK2) as a male nonhormonal contraceptive target. After reviewing the pertinent knockout models and biological evidence, we will briefly review the current literature regarding inhibitor development against CDK2, particularly focusing on selectivity and future approaches.</p></sec><sec id=\"sec2\"><title>Knockout models of <italic>CDK2</italic> are viable but sterile</title><p>CDK2 is a member of the CDK family involved in regulating the cell cycle. In mitotic cells, CDK2 is activated by phosphorylation and binds to E-type cyclins to progress from G1 into S phase, then subsequently binds A-type cyclins in S phase to advance the cell cycle [<xref rid=\"ref1\" ref-type=\"bibr\">1</xref>]. In particular, it was thought that CDK2/cyclin E complexes were necessary for the phosphorylation of retinoblastoma (RB) protein and the release of E2F transcription factors to initiate S phase and DNA synthesis [<xref rid=\"ref2\" ref-type=\"bibr\">2</xref>, <xref rid=\"ref3\" ref-type=\"bibr\">3</xref>]. Despite this role of CDK2 in cell division, unexpectedly two different <italic>CDK2</italic> knockout mouse models have a fully penetrant phenotype of sterility with otherwise normal development and lifespan, but a slightly smaller size after weaning [<xref rid=\"ref4\" ref-type=\"bibr\">4</xref>, <xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. Anatomical and histological samples, as well as mating behavior, are all normal compared to wild-type mice with the exception of the gonads [<xref rid=\"ref4\" ref-type=\"bibr\">4</xref>]. Primary mouse embryonic fibroblasts (MEFs) derived from this model are able to proliferate but have a delay into S phase [<xref rid=\"ref4\" ref-type=\"bibr\">4</xref>, <xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. Further analysis reveals that the CDK2 substrate RB remains phosphorylated, even at sites previously thought to be specific to CDK2, suggesting a compensatory mechanism by another kinase [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. Phosphorylation of the CDK2 substrate histone H1 in cyclin A immunoprecipitates is additionally observed, suggesting CDK1 compensates for <italic>CDK2</italic> loss in this latter case [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. From this evidence, CDK2 is necessary for meiosis but not mitosis. It also suggests that selective inhibition of CDK2 could be relatively nontoxic to somatic cells.</p><p>The compensatory mechanisms for <italic>CDK2</italic> loss were further explored. Other kinases can compensate for normal CDK2 activity in its absence, particularly CDK1 [<xref rid=\"ref6\" ref-type=\"bibr\">6–8</xref>]. CDK1 is indispensable and sufficient to drive the cell cycle [<xref rid=\"ref6\" ref-type=\"bibr\">6</xref>, <xref rid=\"ref7\" ref-type=\"bibr\">7</xref>]. In the absence of CDK2, CDK1 binds cyclin E, for example, and recapitulates the natural CDK2 kinase function [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>]. Further silencing of <italic>CDK1</italic> in the context of <italic>CDK2</italic> knockout MEFs results in decreased proliferation, suggesting that CDK1 is responsible for compensating <italic>CDK2</italic> loss [<xref rid=\"ref8\" ref-type=\"bibr\">8</xref>]. Knockout of <italic>CDK2</italic> also results in an earlier transcriptional activation of <italic>CDK1</italic> [<xref rid=\"ref9\" ref-type=\"bibr\">9</xref>]. Furthermore, full genetic substitution with <italic>CDK2</italic> at the <italic>CDK1</italic> locus results in a nonviable phenotype, emphasizing the unique importance of CDK1 to somatic cell proliferation [<xref rid=\"ref9\" ref-type=\"bibr\">9</xref>]. However, the meiotic role of CDK2 is dependent on its genetic locus and timing of gene expression, as <italic>CDK2<sup>−/−</sup></italic> mouse models with the <italic>CDK1<sup>+</sup></italic>/<italic>CDK2<sup>KI</sup></italic> haplotype at the <italic>CDK1</italic> locus recapitulates the viable, but sterile phenotype of previous <italic>CDK2<sup>−/−</sup></italic> mouse models [<xref rid=\"ref4\" ref-type=\"bibr\">4</xref>, <xref rid=\"ref5\" ref-type=\"bibr\">5</xref>, <xref rid=\"ref9\" ref-type=\"bibr\">9</xref>]. Overall, a contraceptive agent targeting CDK2 must be selective over CDK1 to have an appropriate toxicological profile.</p><p>Male <italic>CDK2<sup>−/−</sup></italic> mice lack late spermatocytes, spermatids, and sperm [<xref rid=\"ref4\" ref-type=\"bibr\">4</xref>, <xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. The seminiferous tubules decrease in size but spermatogonia are present, and both Sertoli and Leydig cells appear unaffected [<xref rid=\"ref4\" ref-type=\"bibr\">4</xref>, <xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. Histological analysis of wild-type and <italic>CDK2<sup>−/−</sup></italic> testes do not differ until P20, when germ cells complete meiosis I [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. Notably, germ cells do not differentiate in <italic>CDK2<sup>−/−</sup></italic> mice [<xref rid=\"ref4\" ref-type=\"bibr\">4</xref>]. Adult <italic>CDK2<sup>−/−</sup></italic> mice have atrophic testes 20% the weight and size of their wild-type littermates [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. In <italic>CDK2<sup>−/−</sup></italic> cellular models of spermatogenesis, all cells arrest in the mid-pachytene stage of prophase I (<xref rid=\"f1\" ref-type=\"fig\">Figure 1a</xref>) [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. In particular, unsynapsed chromosomes are observed in this stage, suggesting that <italic>CDK2<sup>−/−</sup></italic> spermatocytes have a defect in forming the axial element and many chromosomes are unpaired during this stage [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. While CDK2 is not required for the assembly of the synaptonemal complex, CDK2 regulates homologous pairing and synapsis, as well as formation of the sex body, double-strand break processing, and attachment of telomeres to the nuclear membrane [<xref rid=\"ref10\" ref-type=\"bibr\">10</xref>, <xref rid=\"ref11\" ref-type=\"bibr\">11</xref>].</p><fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>(a) Known CDK2 binding partner cyclin A2 is absent during prophase I. The knockout phenotype of <italic>CDK2</italic> and the knockout phenotype of <italic>SPDYA</italic> both arrest in mid-pachytene. The knockout phenotype of <italic>CCNA1</italic> arrests in diplotene. Image adapted with permission [<xref rid=\"ref72\" ref-type=\"bibr\">72</xref>]. (b and c) The cellular localization of CDK2 and E-type cyclins do not overlap during pachytene, the subphase of prophase I when <italic>CDK2</italic><sup>−/−</sup> cells arrest, where CDK2 is only found on telomeric ends. However, the localization of CDK2 and SPY1 do overlap in pachytene [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>, <xref rid=\"ref20\" ref-type=\"bibr\">20</xref>].</p></caption><graphic xlink:href=\"ioaa107f1\"/></fig><p>In female <italic>CDK2<sup>−/−</sup></italic> mice, severe atrophy of the ovaries is observed even before sexual maturity and increased in severity into adulthood, as <italic>CDK2<sup>−/−</sup></italic> ovaries are 15–20% the weight and size compared with their wild-type counterparts [<xref rid=\"ref4\" ref-type=\"bibr\">4</xref>, <xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. The oviduct and uterus are unaffected [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. No primordial follicles and corpora lutea are observed and oocytes are not developed, suggesting that embryonic development was affected in this tissue, as oocytes normally arrest in prophase I perinatally and continue meiosis later in adulthood [<xref rid=\"ref4\" ref-type=\"bibr\">4</xref>, <xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. A similar phenotype in wild-type and <italic>CDK2<sup>−/−</sup></italic> oocytes was observed until the pachytene stage and then diverged greatly by the diplotene stage of prophase I, a substage later than what is observed in spermatogenesis models [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>]. In particular, centromeres are randomly distributed around the nuclei in diplotene oocytes whereas they are normally located at discrete locations at this stage [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>].</p><p>In adult men, spermatogenesis and meiosis are ongoing processes, unlike oogenesis, which begins in utero and arrests in meiosis I. Because CDK2 acts in an early stage of meiosis I, it is a validated contraceptive target for men but remains unclear in women, as a reversible therapeutic targeting CDK2 might be inappropriately timed to affect oogenesis in adult women. To answer this question, two oocyte-specific conditional knockout mouse models were made: one for <italic>CDK2</italic> (Oo<italic>CDK2<sup>−/−</sup></italic>) and the other for <italic>CDK1</italic> (Oo<italic>CDK1<sup>−/−</sup></italic>) [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>]. In particular, primordial and further developed follicles are devoid of their respective proteins in these two models, and ovaries without <italic>CDK1</italic> or <italic>CDK2</italic> are morphologically indistinguishable from their wild-type counterparts. Ovulation and formation of corpora lutea are also unaffected. However, only Oo<italic>CDK1<sup>−/−</sup></italic> mice are infertile whereas Oo<italic>CDK2<sup>−/−</sup></italic> mice normally develop oocytes and are as fertile as their wild-type counterparts with normal litter sizes [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>]. CDK1 is necessary for germinal vesicle breakdown, as triggered by luteinizing hormone to resume meiosis, whereas CDK2 is not. Loss of <italic>CDK2</italic> does also not affect the sequential arrest in meiosis II before fertilization [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>]. Rescue of the Oo<italic>CDK1<sup>−/−</sup></italic> genotype with <italic>CDK1</italic> mRNA injection resumes meiosis appropriately, further signifying that CDK1 is responsible for resumption of meiosis postnatally [<xref rid=\"ref12\" ref-type=\"bibr\">12</xref>]. This would suggest that a contraceptive targeting CDK2 could be effective in adult men, but unlikely for adult women.</p></sec><sec id=\"sec3\"><title>CDK2 is necessary in spermatogenesis via a cyclin-independent mechanism</title><p>While the precise role of CDK2 in meiosis is unknown, it appears that its activation is cyclin-independent, unlike its role in interphase. Because CDK2 is known to bind to cyclins A and E in particular, and a role for cyclins in meiosis is established, it was assumed the CDK2 bound to cyclin was necessary to advance meiosis.</p><p>In both mice and humans, CDK2 binds two isoforms of cyclin A—cyclin A1 (CCNA1) and cyclin A2 (CCNA2). The cyclin A1 isoform is primarily expressed in the germ line and restricted to meiotic cells. It is distributed in the nucleus after mid-pachytene and then localized on the telomeres in late diplotene, persisting through metaphase of the first meiotic division but not though the second. Cyclin A1 demonstrates haploinsufficiency for mouse fertility and has been hypothesized to be responsible for instances of human oligospermia [<xref rid=\"ref13\" ref-type=\"bibr\">13</xref>]. Antibodies against cyclin A1 precipitate both CDK1 and CDK2 in testicular lysates [<xref rid=\"ref14\" ref-type=\"bibr\">14</xref>]. However, knockout models of <italic>CCNA1</italic> arrest in the later diplotene stage with the disappearance of the synaptonemal complex, differing in phenotype from that of <italic>CDK2</italic> knockouts, which arrest in the pachytene stage where the synaptonemal complex is still intact (<xref rid=\"f1\" ref-type=\"fig\">Figure 1a</xref>) [<xref rid=\"ref15\" ref-type=\"bibr\">15</xref>]. Within the testis of <italic>CCNA1<sup>−/−</sup></italic> models, CDK1 activity decreases by 80% while CDK2 activity only decreases modestly, suggesting that cyclin A1 acts primarily through CDK1 in the testis [<xref rid=\"ref15\" ref-type=\"bibr\">15</xref>]. Immuno-depletion of cyclin A1 in testis extracts also does not significantly alter the overall activity of CDK1 or CDK2, suggesting neither kinase is strongly dependent on cyclin A1 [<xref rid=\"ref15\" ref-type=\"bibr\">15</xref>]. Finally, cyclin A1 and CDK2 do not colocalize in prophase I of spermatogenesis, indicating that cyclin A1 is not the pertinent binding partner of CDK2 [<xref rid=\"ref16\" ref-type=\"bibr\">16</xref>].</p><p>In contrast, cyclin A2 is ubiquitously expressed in dividing somatic cells and restricted to the premeiotic S-phase in germ cells. Knockout models of <italic>CCNA2</italic> are embryonically lethal, making its precise role in spermatogenesis difficult to study [<xref rid=\"ref17\" ref-type=\"bibr\">17</xref>]. However, it is present only in spermatogonia and preleptotene spermatocytes (<xref rid=\"f1\" ref-type=\"fig\">Figure 1a</xref>) [<xref rid=\"ref13\" ref-type=\"bibr\">13</xref>]. In wild-type testicular lysates, anticyclin A2 antibodies precipitates CDK2 but not CDK1, and anti-CDK2 antibodies precipitate cyclin A2 as well as other polypeptides [<xref rid=\"ref13\" ref-type=\"bibr\">13</xref>]. The presence of CDK1 and its ability to bind cyclin A2 may compensate for the loss of CDK2 in the pre-pachytene stages of <italic>CDK2<sup>−/−</sup></italic> models. Since cyclin A2 is absent for all of prophase I and therefore is absent during the time <italic>CDK2<sup>−/−</sup></italic> spermatocytes arrest in mid-pachytene, it is unlikely CDK2-cyclin A2 is the pertinent complex responsible for <italic>CDK2<sup>−/−</sup></italic> spermatocyte arrest.</p><p>CDK2 also binds E-type cyclins, of which there are two in mammals, cyclins E1 and E2. Cyclins E1 and E2 are thought to have redundant functions in meiosis, as knockout mouse models of either cyclin E gene individually result in viable offspring. However, male <italic>CCNE2<sup>−/−</sup></italic> mice have reduced fertility, with a decreased testis size and a sperm count 50% to their wild-type counterparts [<xref rid=\"ref18\" ref-type=\"bibr\">18</xref>]. In contrast, double knockout models of <italic>CCNE1</italic> and <italic>CCNE2</italic> are embryonically lethal and conditional double knockout models yield a sterile and azoospermic phenotype in male mice [<xref rid=\"ref18\" ref-type=\"bibr\">18</xref>, <xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]. From this, the role of E-type cyclins in spermatogenesis was explored. E-type cyclins help spermatocytes progress through prophase I [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]. Cyclin E1 appears at pachytene of prophase I until diplotene while cyclin E2 is present from preleptotene and increases throughout most of prophase I [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]. Cyclin E1 is located on the sex chromosomes while cyclin E2 is not [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]. Although loss of <italic>CCNE1</italic> does not lead to infertility, it does disrupt the formation and progression of synapsis [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]. In contrast, loss of <italic>CCNE2</italic> does not affect fertility until the diplotene stage, but does result in heterologous chromosomal associations during pachytene involving a “one-to-one” chromosome connection on the telomeric ends [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]. E-type cyclins are also necessary for double-strand break repair as well as telomere structural integrity and stability [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>].</p><p>While E-type cyclins and CDK2 appear to be acting early in prophase I, it is unlikely their function is co-dependent. For example, CDK2 is distinctly localized on the telomeres and recombination nodules throughout prophase I [<xref rid=\"ref10\" ref-type=\"bibr\">10</xref>]. This is demonstrated in <xref rid=\"f1\" ref-type=\"fig\">Figure 1b</xref>, where the chromosomes are highlighted by red SYCP3 and CDK2 is highlighted in green at the ends of these chromosomes during the stages of prophase I [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. In contrast, <xref rid=\"f1\" ref-type=\"fig\">Figure 1c</xref> shows green E-type cyclins diffusely spread throughout the nucleus, suggesting that E-type cyclins and CDK2 are not colocalized during prophase I [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]. However, co-immunoprecipitation studies of cyclins E1 and E2 reveal an interaction with CDK2 [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]. While a catalytic function was not measured from these precipitates, it is suggested that E-type cyclins help CDK2 localize to the telomeres. <italic>CCNE1<sup>−/−</sup></italic> spermatocytes had similar CDK2 telomeric localization as wild-type spermatocytes, <italic>CCNE2<sup>−/−</sup></italic> spermatocytes had a 50% reduction in CDK2 telomeric localization, and double knockout <italic>CCNE1<sup>−/−</sup> CCNE2<sup>−/−</sup></italic> spermatocytes had a 93% reduction in CDK2 telomeric localization. While this would suggest that E-type cyclins are important for the appropriate localization of CDK2, it is unclear whether E-type cyclins localize CDK2 in particular or serve to stabilize the telomere in general. Whether E-type cyclins perform catalysis in association with CDK2 or serve a scaffolding function for CDK2 in spermatogenesis also remains to be determined. Finally, the phenotypes of E-type cyclin loss and <italic>CDK2<sup>−/−</sup></italic> differ. Complete ablation of E-type cyclin function in spermatocytes arrests cells in the early pachytene, near but distinct from the mid-pachytene arresting phenotype of <italic>CDK2<sup>−/−</sup></italic> spermatocytes [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>, <xref rid=\"ref19\" ref-type=\"bibr\">19</xref>]. Only 1.5% of E-type cyclin depleted spermatocytes progress into mid-pachytene [<xref rid=\"ref19\" ref-type=\"bibr\">19</xref>].</p></sec><sec id=\"sec4\"><title>CDK2 is necessary in meiosis via a complex with SPY1</title><p>Recently, an additional role of CDK2 in meiosis has been established. The protein Speedy 1 (SPY1), also known as Ringo A, can activate CDK2 by binding to the same site as cyclins (<xref rid=\"f2\" ref-type=\"fig\">Figure 2a</xref>) [<xref rid=\"ref21\" ref-type=\"bibr\">21</xref>]. Unlike cyclin activation, SPY1 activation of CDK2 does not require phosphorylation to initiate its catalytic function [<xref rid=\"ref22\" ref-type=\"bibr\">22</xref>]. SPY1 is expressed in all tissues, but its expression is only substantially increased in the testis (20 ± fold higher) [<xref rid=\"ref23\" ref-type=\"bibr\">23</xref>]. <italic>SPDYA<sup>−/−</sup></italic> mice have the same viable but sterile phenotype as of <italic>CDK2<sup>−/−</sup></italic> mice, with no additional observed abnormalities [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. The hypoplastic testes from <italic>SPDYA<sup>−/−</sup></italic> mice are four times smaller than their wild-type counterparts and while spermatogonia exist in the mutant mice, neither spermatozoa nor spermatids are observed [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>].</p><fig id=\"f2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>(a) SPY1 interacts with CDK2 along the same interface as cyclin (overlay of PDB IDs 1FIN and 5UQ1). (b) The chromosomal phenotypes of <italic>CDK2</italic><sup>−/−</sup> and <italic>SPDYA</italic><sup>−/−</sup> show nearly identical arresting phenotypes in mid-pachytene [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>].</p></caption><graphic xlink:href=\"ioaa107f2\"/></fig><p>In particular, <italic>SPDYA<sup>−/−</sup></italic> spermatocytes and oocytes arrest in the pachytene stage of prophase I, as is observed with <italic>CDK2<sup>−/−</sup></italic> spermatocytes (<xref rid=\"f2\" ref-type=\"fig\">Figure 2b</xref>) [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. <italic>SPDYA<sup>−/−</sup></italic> spermatocytes have more double-strand breaks and no crossing-over events, suggesting that SPY1 affects late recombination and homologous chromosome pairing [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. Additionally, <italic>SPDYA<sup>−/−</sup></italic> spermatocytes show nonhomologous chromosome pairing, failure of the telomeres to attach to the nuclear membrane, and telomere fusion, just like <italic>CDK2<sup>−/−</sup></italic> spermatocytes [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>, <xref rid=\"ref10\" ref-type=\"bibr\">10</xref>, <xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. SPY1 and CDK2 colocalize at the telomeres in spermatocytes from leptotene throughout pachytene, even on the asynapsed sex chromosomes [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. Interestingly, CDK2 does not localize on the telomeres in <italic>SPDYA<sup>−/−</sup></italic> spermatocytes, suggesting that SPY1 may help to localize CDK2 to the telomere. In wild-type spermatocytes, antibodies against SPY1 co-immunoprecipitated CDK2 and vice versa. In <italic>SPDYA<sup>−/−</sup></italic> spermatocytes, the activity of immunoprecipitated CDK2 decreased by 70%, highlighting the importance of the CDK2-SPY1 catalytic complex [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. Loss of localization and decreased CDK2 activity are thought to be the causes of infertility in <italic>SPDYA<sup>−/−</sup></italic> mice.</p><p>While the substrate profile of CDK2-SPY1 is incomplete, CDK2-SPY1 can phosphorylate the protein SUN1 in vitro [<xref rid=\"ref11\" ref-type=\"bibr\">11</xref>, <xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. Furthermore, <italic>SUN1</italic> knockout mouse models are very similar to the <italic>SPDYA</italic> knockout and <italic>CDK2</italic> knockout models, demonstrating nonhomologous pairing, increased double-strand breaks, and loss of telomeric attachment to the nuclear membrane [<xref rid=\"ref5\" ref-type=\"bibr\">5</xref>, <xref rid=\"ref10\" ref-type=\"bibr\">10</xref>, <xref rid=\"ref20\" ref-type=\"bibr\">20</xref>, <xref rid=\"ref24\" ref-type=\"bibr\">24</xref>]. SUN1 is important for localizing telomeres to the nuclear membrane in prophase I [<xref rid=\"ref24\" ref-type=\"bibr\">24–26</xref>]. In <italic>SPDYA<sup>−/−</sup></italic> spermatocytes, SUN1 does not localize to telomeres as it normally does [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. In wild-type spermatocytes, SUN1 associates with TERB1, another protein important for telomeric localization to the nuclear membrane [<xref rid=\"ref27\" ref-type=\"bibr\">27</xref>]. TERB1 localization is unperturbed on telomeres in <italic>SPDYA<sup>−/−</sup></italic> spermatocytes [<xref rid=\"ref20\" ref-type=\"bibr\">20</xref>]. It is hypothesized that phosphorylation of SUN1 by CDK2-SPY1 is necessary for the association of SUN1 with TERB1. This could be one mechanism by which the CDK2-SPY1 complex is necessary in spermatogenesis. The high concentration of SPY1 in the testis, coupled with a relatively healthy <italic>SPDYA</italic> knockout model and validated mechanism in spermatogenesis via CDK2 binding, suggests that targeting a SPY1-CDK2 complex could be a viable strategy to develop a safe, nonhormonal male contraceptive.</p></sec><sec id=\"sec5\"><title>CDK2 inhibitors</title><p>During the last two decades, CDK2 inhibitors have not been developed for contraception but instead predominantly to treat cancer. While 18 CDK2 inhibitors have entered clinical trials, none have been approved by the Food and Drug Administration. In the final sections of this review, we will summarize the information about CDK2 inhibitors that have entered clinical trials, selective CDK2 inhibitors in preclinical development, allosteric CDK2 inhibitors, and selected other CDK2 inhibitors with a view toward their potential use as contraceptive agents.</p></sec><sec id=\"sec6\"><title>CDK2 inhibitors in the clinic</title><p>Eighteen nonselective CDK inhibitors (<xref rid=\"TB1\" ref-type=\"table\">Table 1</xref>; Supplementary data, <xref rid=\"sup1\" ref-type=\"supplementary-material\">Figure S1</xref>) have entered clinical trials for the treatment of different types of cancer; 10 are currently in clinical trials and the others were terminated after phase I or II trials due to undesirable off-target toxicity and poor pharmacokinetic properties. First-generation CDK inhibitors include alvocidib, a flavonoid alkaloid, BMS-387032, a compound with a 2-aminothiazole-5-thiol core, PHA-793887, featuring a 1<italic>H</italic>-pyrazol-3-amine scaffold, and (<italic>R</italic>)-roscovitine, a purine. These inhibitors failed to show any significant clinical advantages, which has been attributed to off-target effects and inappropriate tumor type selection for testing [<xref rid=\"ref28\" ref-type=\"bibr\">28</xref>]. While (<italic>R</italic>)-roscovitine also inhibits CDK1/5/7/9, specific inhibition of CDK2 by this compound was directly linked to cancer cell apoptosis [<xref rid=\"ref29\" ref-type=\"bibr\">29</xref>]. A phase II study of (<italic>R</italic>)-roscovitine for Cushing disease was initiated in 2018 and is currently (April 2020) recruiting patients. The five other recorded phase I/II studies of (<italic>R</italic>)-roscovitine were either withdrawn or terminated at an early stage for unknown reasons [<xref rid=\"ref30\" ref-type=\"bibr\">30</xref>]. Similarly, the CDK1/2/4/5/7/9-targeting compound alvocidib was investigated in phase I/II studies, but did not show significant clinical advantages [<xref rid=\"ref31\" ref-type=\"bibr\">31</xref>]. However, phase I/II studies of combination therapies of alvocidib and venetoclax were initiated for the treatment of acute myeloid leukemia [<xref rid=\"ref32\" ref-type=\"bibr\">32</xref>]. BMS-387032 is a CDK2/7/9 inhibitor that underwent two phase I trials for the treatment of advanced solid tumors and B-cell malignancies [<xref rid=\"ref33\" ref-type=\"bibr\">33</xref>]. PHA-793887 was investigated in a phase I clinical trial in 2008 to study dose-escalation in patients with acute myeloid leukemia and myelodysplastic syndromes [<xref rid=\"ref34\" ref-type=\"bibr\">34</xref>]. However, this study was terminated early for unknown reasons. In order to achieve a higher selectivity for CDK1 and CDK2 and to further increase potency, a second-generation of CDK inhibitors were developed. In particular, dinaciclib targets CDK1/2/5/9 and has been most studied in the clinic among all second-generation CDK inhibitors, tested as a single agent or in combination with other therapies [<xref rid=\"ref35\" ref-type=\"bibr\">35</xref>]. Dinaciclib exhibited activity against a broad range of tumor cell lines including various breast, prostate, colon, and lung cancer cell lines [<xref rid=\"ref36\" ref-type=\"bibr\">36</xref>]. Dinaciclib is currently in phase I trials for the treatment of multiple myeloma, diffuse large B-cell lymphoma, and triple negative breast cancer. Various phase II and III trials of dinaciclib have been completed for the treatment of nonsmall cell lung cancer, advanced breast cancer, acute myeloid leukemia, acute lymphocytic leukemia, stage IV melanoma, and mantle cell lymphoma [<xref rid=\"ref37\" ref-type=\"bibr\">37–39</xref>]. The only completed phase III study of dinaciclib is a combination therapy of dinaciclib and ofatumumab for the treatment of refractory chronic lymphocytic leukemia in 2014, which concluded that dinaciclib had an acceptable safety and tolerability profile in these patients [<xref rid=\"ref40\" ref-type=\"bibr\">40</xref>]. The most common adverse effects of dinaciclib were neutropenia (35%), thrombocytopenia (20%), decreased neutrophil count (20%), febrile neutropenia (10%), pneumonia (5%), and sepsis (5%) [<xref rid=\"ref40\" ref-type=\"bibr\">40</xref>]. While this side effect profile may be acceptable for patients with cancer, it is unlikely to be adequate for an approved contraceptive.</p><table-wrap id=\"TB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>CDK2 inhibitors that have entered clinical trials.</p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">CDK2 inhibitors in the clinic</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Highest Phase</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Conditions</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">Targets</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">References</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Alvocidib hydrochloride (flavopiridol hydrochloride, DSP-2033, HL-275, HMR-1275, L86-8275, MDL-107826A, or NSC-649890)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase II<xref rid=\"tblfn1\" ref-type=\"table-fn\"><sup>a</sup></xref></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Leukemia, acute myeloid and myelodysplasia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/4/5/7/9, BCL-2, MCL-1 and XIAP</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref73\" ref-type=\"bibr\">73</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">AT7519</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase II<xref rid=\"tblfn1\" ref-type=\"table-fn\"><sup>a</sup></xref></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Refractory solid tumors and lymphoma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/5/9 and GSK3<inline-formula><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym} \n\\usepackage{amsfonts} \n\\usepackage{amssymb} \n\\usepackage{amsbsy}\n\\usepackage{upgreek}\n\\usepackage{mathrsfs}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}\n}{}$\\beta$\\end{document}</tex-math></inline-formula></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref74\" ref-type=\"bibr\">74</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">BMS-387032 (SNS-032)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase II</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Multiple myeloma, hematological cancer, and solid tumors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK2/7/9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref75\" ref-type=\"bibr\">75</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Couroupitine B (NSC-105327, indigo red, indigopurpurin, or indirubin)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase I</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Myeloid leukemia</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/4/5 and GSK3<inline-formula><tex-math id=\"M2\">\\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym} \n\\usepackage{amsfonts} \n\\usepackage{amssymb} \n\\usepackage{amsbsy}\n\\usepackage{upgreek}\n\\usepackage{mathrsfs}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}\n}{}$\\beta$\\end{document}</tex-math></inline-formula></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref76\" ref-type=\"bibr\">76</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Dinaciclib (MK-7965, NSC-727135, or SCH-727965)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase III<xref rid=\"tblfn1\" ref-type=\"table-fn\"><sup>a</sup></xref></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Advanced breast cancer, nonsmall cell lung cancer, acute lymphocytic leukemia, acute myeloid leukemia, mantle cell lymphoma, and stage IV melanoma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/5/9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref36\" ref-type=\"bibr\">36</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Fadraciclib (CYC-065)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase I<xref rid=\"tblfn1\" ref-type=\"table-fn\"><sup>a</sup></xref></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Advanced solid tumors, chronic lymphocytic leukemia, acute myeloid leukemia, and myelodysplastic syndromes</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK2/5/9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref77\" ref-type=\"bibr\">77</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">FN-1501</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase I<xref rid=\"tblfn1\" ref-type=\"table-fn\"><sup>a</sup></xref></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Acute myeloid leukemia and solid tumor</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK2/4/6 and FLT3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref78\" ref-type=\"bibr\">78</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">7-Hydroxystaurosporine (KRX-0601, KW-2401, NSC-638850 or UCN-01)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase II</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Small cell lung cancer, leukemia, lymphoma, leukemia, melanoma, ovarian cancers, and non-Hodgkin’s lymphoma</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/4/6, CHK1/2, PI3K, NHE, PDK1, and PKC</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref79\" ref-type=\"bibr\">79</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Milciclib (PHA-848125 or TZLS-201)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase II<xref rid=\"tblfn1\" ref-type=\"table-fn\"><sup>a</sup></xref></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Hepatocellular carcinoma and thymus</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/4/5/7, WEE1/2, and TRKA</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref48\" ref-type=\"bibr\">48</xref>, <xref rid=\"ref80\" ref-type=\"bibr\">80</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PF-06873600</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase II<xref rid=\"tblfn1\" ref-type=\"table-fn\"><sup>a</sup></xref></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Breast cancer and ovarian cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK2/4/6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref81\" ref-type=\"bibr\">81</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PHA-690509</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase I</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cancer</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref82\" ref-type=\"bibr\">82</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">PHA-793887</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase I</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Leukemia and solid tumors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/4/5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref83\" ref-type=\"bibr\">83</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Roniciclib (Bay-1000394)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase II</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Small cell lung cancer, ovarian cancer, and solid tumors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/3/4/9, FLT4, AURK1, JAK2/3 and MAP3K9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref47\" ref-type=\"bibr\">47</xref>, <xref rid=\"ref84\" ref-type=\"bibr\">84</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">(<italic>R</italic>)-Roscovitine (Seliciclib, CYC-202 or NSC-701554)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase II<xref rid=\"tblfn1\" ref-type=\"table-fn\"><sup>a</sup></xref></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Cushing syndrome, fibrosis, rheumatoid arthritis, and solid tumors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/5/7/9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref29\" ref-type=\"bibr\">29</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">R-547</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase I</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Solid tumors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref85\" ref-type=\"bibr\">85</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">TP-1287</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase I<xref rid=\"tblfn1\" ref-type=\"table-fn\"><sup>a</sup></xref></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Myeloid leukemia and solid tumors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/4/6/7/9, BIRC5, Mcl-1, Bcl-2, and XIAP</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref86\" ref-type=\"bibr\">86</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Voruciclib hydrochloride(P-1446)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase I<xref rid=\"tblfn1\" ref-type=\"table-fn\"><sup>a</sup></xref></td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Diverse advanced solid tumors, hematologic malignancies and relapsed/refractory B-cell malignancies</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/4/9</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref87\" ref-type=\"bibr\">87</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ZK-304709 (ZK-CDK)</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Phase I</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Solid tumors</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">CDK1/2/4/7/9 and FLK1/4</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref88\" ref-type=\"bibr\">88</xref>]</td></tr></tbody></table><table-wrap-foot><fn id=\"tblfn1\"><p>\n<sup>a</sup>Under active development.</p></fn></table-wrap-foot></table-wrap><p>7-Hydroxystaurosporine, an analog of the natural product staurosporine, is a CDK1/2/4/6 inhibitor that completed phase I studies for the treatment of relapsed or refractory acute myeloid leukemia and chronic myelogenous leukemia, which demonstrated that it can be safely administered but lacks clinical efficacy [<xref rid=\"ref41\" ref-type=\"bibr\">41</xref>, <xref rid=\"ref42\" ref-type=\"bibr\">42</xref>]. Moreover, a phase II study of 7-hydroxystaurosporine for the treatment of relapsed T-cell lymphomas was terminated [<xref rid=\"ref43\" ref-type=\"bibr\">43</xref>]. AT-7519 is a potent CDK1/2/5/9 inhibitor that was discontinued in phase II trials for the treatment of advanced solid tumors and multiple myeloma due to the low objective response rates for reduction in tumor burden [<xref rid=\"ref44\" ref-type=\"bibr\">44–46</xref>]. Roniciclib is a pan-CDK inhibitor that underwent phase II clinical trials for the treatment of small cell lung cancer [<xref rid=\"ref47\" ref-type=\"bibr\">47</xref>]. However, roniciclib demonstrated an unfavorable risk–benefit profile in patients with small cell lung cancer, leading to premature termination of the study [<xref rid=\"ref47\" ref-type=\"bibr\">47</xref>]. Another CDK inhibitor, milciclib, is currently undergoing phase II clinical trials for the treatment of thymic and hepatocellular carcinomas [<xref rid=\"ref48\" ref-type=\"bibr\">48</xref>, <xref rid=\"ref49\" ref-type=\"bibr\">49</xref>]. Finally, the dual CDK2/4/6 and FT3 inhibitor FN-1501 is in a phase I trial for the treatment of acute myeloid leukemia and solid tumors [<xref rid=\"ref50\" ref-type=\"bibr\">50</xref>].</p><p>Information about other drugs, which have not completed any clinical trials, are summarized in <xref rid=\"TB1\" ref-type=\"table\">Table 1</xref>. Of all the CDK2 inhibitors that have entered clinical trials, none are selective or safe enough to be repurposed for contraceptive indications.</p></sec><sec id=\"sec7\"><title>Selective type I inhibitors</title><p>The discovery of CDK2 inhibitors has primarily focused on targeting the adenosine triphosphate (ATP)-binding site (type I inhibitors). In <xref rid=\"TB2\" ref-type=\"table\">Table 2</xref>, inhibitors are summarized that are in preclinical development and have appreciable selectivity for CDK2 over other CDKs. Their chemical structures and code names are shown in Supplementary data, <xref rid=\"sup2\" ref-type=\"supplementary-material\">Figure S2</xref>. The inhibitors compound 73, CCT068127, NU2058, NU6102, and purvalanol B are purine analogs mimicking the adenosine in ATP. Several are strong inhibitors, such as NU6102 with an IC<sub>50</sub> of 6.0 nM [<xref rid=\"ref51\" ref-type=\"bibr\">51</xref>]. The introduction of a 4-sulfamoylalanilino group to the C2 position of NU2058 provided analog NU6102 that has enhanced selectivity for CDK2 to 50-fold greater over CDK1/4/5, which all share close structural homology with CDK2. Because CDK1 is necessary for somatic cells, a contraceptive CDK2 inhibitor should not inhibit CDK1. Furthermore, some kinases such as CDK7/CDK9 phosphorylate RNA polymerase II and therefore the inhibition of these off-target kinases may conceal the true pharmacological effects of purported CDK2 inhibitors including dinaciclib, (<italic>R</italic>)-roscovitine, and BMS-387032 [<xref rid=\"ref52\" ref-type=\"bibr\">52</xref>]. Purine-derived compound 73, carrying a biphenyl moiety at the 6-position, has superior selectivity for CDK2 over CDK1 (2000-fold) compared with (<italic>R</italic>)-roscovitine and dinaciclib, but still inhibits CDK9 [<xref rid=\"ref53\" ref-type=\"bibr\">53</xref>]. Purvalanol A and purvalanol B are purines with better selectivity toward CDK2 than (<italic>R</italic>)-roscovitine and alvocidib, which was achieved by modification of the 2-, 6-, and 9-positions of the purine core [<xref rid=\"ref54\" ref-type=\"bibr\">54</xref>].</p><table-wrap id=\"TB2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Inhibition by selective ATP-site CDK2 inhibitors against CDKs.</p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/></colgroup><thead><tr><th rowspan=\"2\" align=\"left\" colspan=\"1\">Compounds</th><th colspan=\"8\" align=\"center\" rowspan=\"1\">IC<sub>50</sub> (nM)</th><th rowspan=\"2\" align=\"center\" colspan=\"1\">References</th></tr><tr><th align=\"center\" rowspan=\"1\" colspan=\"1\">CDK2/cyc A</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">CDK1/cyc A</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">CDK3/cyc E</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">CDK4/cyc D1</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">CDK5/p25</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">CDK6/cyc D1</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">CDK7/cyc H</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">CDK9/cyc K</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">AZD5438</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">20</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">CCT068127</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">110(10<xref rid=\"tblfn2\" ref-type=\"table-fn\"><sup>a</sup></xref>)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1100<xref rid=\"tblfn3\" ref-type=\"table-fn\"><sup>b</sup></xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4800</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">70<xref rid=\"tblfn5\" ref-type=\"table-fn\"><sup>d</sup></xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6200<xref rid=\"tblfn6\" ref-type=\"table-fn\"><sup>e</sup></xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">520</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">90</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref55\" ref-type=\"bibr\">55</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Compound 51</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">7.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">38</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4.0</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6.6</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">>1000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref56\" ref-type=\"bibr\">56</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Compound 73</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">44</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">86 000<xref rid=\"tblfn3\" ref-type=\"table-fn\"><sup>b</sup></xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26<xref rid=\"tblfn4\" ref-type=\"table-fn\"><sup>c</sup></xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">28<xref rid=\"tblfn4\" ref-type=\"table-fn\"><sup>c</sup></xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">25</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref53\" ref-type=\"bibr\">53</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NU2058</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref89\" ref-type=\"bibr\">89</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">NU6102</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6<xref rid=\"tblfn2\" ref-type=\"table-fn\"><sup>a</sup></xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">146<xref rid=\"tblfn3\" ref-type=\"table-fn\"><sup>b</sup></xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">184</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">122</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">2530</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4120</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref51\" ref-type=\"bibr\">51</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Purvalanol B (NG-60)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6(9<xref rid=\"tblfn2\" ref-type=\"table-fn\"><sup>a</sup></xref>)</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6<xref rid=\"tblfn3\" ref-type=\"table-fn\"><sup>b</sup></xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10 000</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">6<xref rid=\"tblfn5\" ref-type=\"table-fn\"><sup>d</sup></xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref54\" ref-type=\"bibr\">54</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SCH-546909</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">1420</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref59\" ref-type=\"bibr\">59</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">SU9516</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">22</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">200</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref57\" ref-type=\"bibr\">57</xref>, <xref rid=\"ref90\" ref-type=\"bibr\">90</xref>]</td></tr></tbody></table><table-wrap-foot><p>- Not available.</p><fn id=\"tblfn2\"><p>\n<sup>a</sup>IC<sub>50</sub> against CDK2/cyclin E.</p></fn><fn id=\"tblfn3\"><p>\n<sup>b</sup>IC<sub>50</sub> against CDK1/cyclin B.</p></fn><fn id=\"tblfn4\"><p>\n<sup>c</sup>% inhibition at 100 <inline-formula><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym} \n\\usepackage{amsfonts} \n\\usepackage{amssymb} \n\\usepackage{amsbsy}\n\\usepackage{upgreek}\n\\usepackage{mathrsfs}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}\n}{}$\\mu$\\end{document}</tex-math></inline-formula>M.</p></fn><fn id=\"tblfn5\"><p>\n<sup>d</sup>IC<sub>50</sub> against CDK5/p35.</p></fn><fn id=\"tblfn6\"><p>\n<sup>e</sup>IC<sub>50</sub> against CDK6/D3.</p></fn></table-wrap-foot></table-wrap><p>More selective CDK2 inhibitors with other scaffolds have also been developed. CCT068127 has improved selectivity toward CDK2 compared with the parent (<italic>R</italic>)-roscovitine, but it inhibits CDK5/9 [<xref rid=\"ref55\" ref-type=\"bibr\">55</xref>]. CCT068127 forms additional hydrogen bonding interactions with the DFG-motif, explaining its improved potency compared with (<italic>R</italic>)-roscovitine. Thiazole core compound 51 displays high selectivity for CDK2 and CDK5 over other CDKs [<xref rid=\"ref56\" ref-type=\"bibr\">56</xref>]. SU9516 has a tri-substituted indolinone core that is 2-fold selective for CDK2 over CDK1 and more than 20-fold selective for CDK2 over CDK4 [<xref rid=\"ref57\" ref-type=\"bibr\">57</xref>]. AZD5438 contains an imidazole core and is selective for CDK1/2/9 [<xref rid=\"ref58\" ref-type=\"bibr\">58</xref>]. SCH-546909 is derived from a natural product and exhibits 10-fold selectivity for CDK2 over CDK4, but less than 2-fold selectivity for CDK2 over CDK1 [<xref rid=\"ref59\" ref-type=\"bibr\">59</xref>]. In summary, because no CDK2 inhibitor with substantial selectivity over other CDKs exists to date, if these compounds were to be used chronically, for example, as a contraceptive agent, then the off-target effects present a significant problem.</p></sec><sec id=\"sec8\"><title>Type II CDK2 inhibitors</title><p>Inhibition of CDK2 can also be achieved through stabilization of an inactive conformation with the DFG motif facing out (DFG-out) toward the solvent (type II). The energy differences between the conformations of the DFG-out activation loops of kinases [<xref rid=\"ref60\" ref-type=\"bibr\">60</xref>, <xref rid=\"ref61\" ref-type=\"bibr\">61</xref>] offer an opportunity to obtain type II inhibitors with improved selectivity compared with type I inhibitors. K03861, an aminopyrimidine-phenyl urea inhibitor (Supplementary data, <xref rid=\"sup3\" ref-type=\"supplementary-material\">Figure S3</xref>), was identified as a CDK2 type II inhibitor with a K<sub>d</sub> of 53 nM [<xref rid=\"ref62\" ref-type=\"bibr\">62</xref>] and is the first type II inhibitor of CDK2. K03861 competes with cyclin binding and stabilizes the inactive state of CDK2 by binding to the ATP-binding site and reaching toward a hydrophobic pocket located between the C-lobe and the alpha-C-helix (Supplementary data, <xref rid=\"sup1\" ref-type=\"supplementary-material\">Figure S4</xref>, PDB ID: 5A14). However, this compound was promiscuous and therefore not selective for CDK2.</p></sec><sec id=\"sec9\"><title>Type III and IV allosteric inhibitors</title><p>Although a majority of the previous CDK2-targeting efforts focused on the ATP-binding site, achieving high selectivity for CDK2 against other kinases in this manner is difficult due to the highly conserved nature of the kinase ATP-binding site, especially within the CDK family. The discovery of allosteric pockets provides an opportunity to attain a higher degree of selectivity toward CDK2, as these pockets are often less conserved.</p><p>Four allosteric pockets in CDK2 have been identified crystallographically (<xref rid=\"TB3\" ref-type=\"table\">Table 3</xref>) with ligands bound in each of them [<xref rid=\"ref63\" ref-type=\"bibr\">63–65</xref>]. Additionally, two allosteric pockets were putatively discovered by computational methods [<xref rid=\"ref66\" ref-type=\"bibr\">66</xref>, <xref rid=\"ref67\" ref-type=\"bibr\">67</xref>]. The best characterized allosteric pocket binds 8-anilino-1-naphthalene sulfonate (ANS), as confirmed by X-ray crystallography [<xref rid=\"ref63\" ref-type=\"bibr\">63</xref>]. In the allosteric pocket, an ANS molecule binds adjacent to the ATP-binding site and the C-helix (<xref rid=\"f3\" ref-type=\"fig\">Figure 3</xref>, PDB ID: 3PXQ). The sulfonic acid moiety of ANS forms hydrogen bonds and salt bridges through interactions with K33, D145, and F146, whereas the naphthalene and aniline rings form hydrophobic interactions with Y15, I35, L55, V64, and F80 (<xref rid=\"f3\" ref-type=\"fig\">Figure 3</xref>). A second ANS molecule binds adjacent to the first in the allosteric pocket and also interacts with C-helix residues (<xref rid=\"f3\" ref-type=\"fig\">Figure 3</xref>). The major residues interacting with the second ANS molecule are I52, L76, K56, and H71 (<xref rid=\"f3\" ref-type=\"fig\">Figure 3</xref>). At high concentrations of ANS, a third ANS molecule binds in the ATP-binding site (<xref rid=\"f3\" ref-type=\"fig\">Figure 3</xref>).</p><table-wrap id=\"TB3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>Allosteric inhibitors of CDK2.</p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/><col align=\"left\" span=\"1\"/></colgroup><thead><tr><th align=\"left\" rowspan=\"1\" colspan=\"1\">Compounds</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">PDB ID</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">The binding pocket of CDK2</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">K<sub>d</sub> (<inline-formula><tex-math id=\"M4\">\\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym} \n\\usepackage{amsfonts} \n\\usepackage{amssymb} \n\\usepackage{amsbsy}\n\\usepackage{upgreek}\n\\usepackage{mathrsfs}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}\n}{}$\\mu$\\end{document}</tex-math></inline-formula>M)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">CDK2 IC<sub>50</sub> (<inline-formula><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym} \n\\usepackage{amsfonts} \n\\usepackage{amssymb} \n\\usepackage{amsbsy}\n\\usepackage{upgreek}\n\\usepackage{mathrsfs}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}\n}{}$\\mu$\\end{document}</tex-math></inline-formula>M)</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">IC<sub>50</sub> (<inline-formula><tex-math id=\"M6\">\\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym} \n\\usepackage{amsfonts} \n\\usepackage{amssymb} \n\\usepackage{amsbsy}\n\\usepackage{upgreek}\n\\usepackage{mathrsfs}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}\n}{}$\\mu$\\end{document}</tex-math></inline-formula>M) of cell viability assays</th><th align=\"center\" rowspan=\"1\" colspan=\"1\">References</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">ANS</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">3PXZ, 3PY1, 3PXF, 3PXQ, 4EZ7</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ANS pocket</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">91</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref63\" ref-type=\"bibr\">63</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">B2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Noncatalytic pocket near the interface of the CDK2/cyclin A3</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">52</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">85<sup>a</sup></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref66\" ref-type=\"bibr\">66</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Compound 1</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5OSJ</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">An allosteric pocket adjacent to the cyclin binding interface</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref65\" ref-type=\"bibr\">65</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Compound 2</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">5FP6</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">The second ANS pocket</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref64\" ref-type=\"bibr\">64</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">Compound 3f</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ANS pocket</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">43</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref68\" ref-type=\"bibr\">68</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">DAALT</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">Interface of CDK2/cyclin E</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref67\" ref-type=\"bibr\">67</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">DPIT</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ANS pocket</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref70\" ref-type=\"bibr\">70</xref>]</td></tr><tr><td align=\"left\" rowspan=\"1\" colspan=\"1\">FLI-06</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"left\" rowspan=\"1\" colspan=\"1\">ANS pocket</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">-</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">71</td><td align=\"center\" rowspan=\"1\" colspan=\"1\">4<xref rid=\"tblfn8\" ref-type=\"table-fn\"><sup>b</sup></xref></td><td align=\"center\" rowspan=\"1\" colspan=\"1\">[<xref rid=\"ref69\" ref-type=\"bibr\">69</xref>]</td></tr></tbody></table><table-wrap-foot><p>- Not available.</p><fn id=\"tblfn7\"><p>\n<sup>a</sup>IC<sub>50</sub> with A549.</p></fn><fn id=\"tblfn8\"><p>\n<sup>b</sup>IC<sub>50</sub> with MDA-MB231.</p></fn></table-wrap-foot></table-wrap><fig id=\"f3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>The co-crystal structure of three molecules of ANS bind to CDK2 (PDB ID: 3PXQ) where one of them binds to the ATP-binding site (green), and the other two bind deeper within the allosteric pocket (yellow and cyan). The red cross is a water molecule. Black dotted lines indicate hydrogen bonds and salt bridges. Green dotted lines indicate hydrophobic interactions. Magentas residues interact with the ANS molecule deep within the allosteric pocket (yellow).</p></caption><graphic xlink:href=\"ioaa107f3\"/></fig><p>The experimental K<sub>d</sub> of ANS for CDK2 is 37 μM and its inhibitory potential against the active CDK2-cyclin A complex is weak with an IC<sub>50</sub> = 91 μM. Notably, the ANS pocket is inaccessible when CDK2 binds to cyclin A/E. Therefore, inhibitors targeting this binding site require high affinity to outcompete cyclins and drive the equilibrium back to inactive free CDK2. Since discovery of this previously unrecognized allosteric pocket, several inhibitors with different scaffolds targeting this pocket have been reported, including FLI-06, compound 3f, and DPIT (Supplementary data, <xref rid=\"sup1\" ref-type=\"supplementary-material\">Figure S5</xref>), although none of which have been confirmed by structural methods to bind in this pocket. Compound 3f has an IC<sub>50</sub> = 10 μM, representing the best inhibition against CDK2 of this series to date [<xref rid=\"ref68\" ref-type=\"bibr\">68</xref>]. In addition, compound 3f inhibits wild-type epidermal growth factor receptor (EGFR) as well as carcinogenic mutant L858R/T790M EGFR, with IC<sub>50</sub> values of 50 and 10 μM, respectively. Similarly, increasing concentrations of FLI-06 displaces ANS, suggesting that it binds in the same pocket [<xref rid=\"ref69\" ref-type=\"bibr\">69</xref>]. In cancer cell lines, FLI-06 inhibits cell viability with an IC<sub>50</sub> = 4 μM against MDA-MB231 cells and IC<sub>50</sub> = 4.5 μM against ZR-75-1 cells. The group that discovered FLI-06 also developed compound 3f. DPIT, a previously reported anti-HSV-1 agent, was later suggested to bind the ANS pocket via docking studies [<xref rid=\"ref70\" ref-type=\"bibr\">70</xref>]. None of these published allosteric inhibitors, however, has been crystallographically confirmed to bind to the ANS allosteric pocket. The only crystallographically confirmed fragment that binds to the ANS pocket is compound 2 [<xref rid=\"ref64\" ref-type=\"bibr\">64</xref>]. The alignment in Supplementary data, <xref rid=\"sup1\" ref-type=\"supplementary-material\">Figure S6</xref> shows that compound 2 binds in a similar location as the ANS molecule in the second allosteric ANS site within the allosteric pocket.</p><p>The other allosteric pockets were discovered computationally at the interface of CDK2 and cyclin E, next to the T-loop [<xref rid=\"ref67\" ref-type=\"bibr\">67</xref>, <xref rid=\"ref71\" ref-type=\"bibr\">71</xref>]. Short 5-mer peptide DAALT bind to the interface allosteric pocket with the strongest affinity of K<sub>d</sub> = 0.5 μM to prevent the protein–protein interaction of CDK2 and cyclins [<xref rid=\"ref67\" ref-type=\"bibr\">67</xref>]. Docking studies indicated that C177, K178, and Y180 are three key residues with the highest contribution toward binding these small peptides. These peptide inhibitors were suggested to induce the similar conformational change as cyclins, although this was never confirmed structurally [<xref rid=\"ref67\" ref-type=\"bibr\">67</xref>]. Another allosteric pocket near the CDK2/cyclin A3 interface has been suggested [<xref rid=\"ref66\" ref-type=\"bibr\">66</xref>]. Compound B2 (Supplementary data, <xref rid=\"sup1\" ref-type=\"supplementary-material\">Figure S5</xref>), obtained from a virtual high throughput screen, inhibits CDK2/cyclin A3 interaction with an IC<sub>50</sub> = 52 μM and exhibits weak antiproliferative activities against A549, HepG2, and MDA-MB-231cell lines [<xref rid=\"ref66\" ref-type=\"bibr\">66</xref>]. However, compound B2 contains a PAINS scaffold and an imine moiety that may be unstable in aqueous solution. Considering these factors and the lack of structural data for the presence of the pocket where compound B2 binds, more data are needed to confirm these results.</p><p>A crystallographically verified (PDB ID: 5OSJ) type IV acryl amide covalent inhibitor, (compound 1; Supplementary data, <xref rid=\"sup1\" ref-type=\"supplementary-material\">Figure S5</xref>) binds to an allosteric pocket adjacent to the cyclin binding interface, forming a covalent bond with Cys177, a conserved residue in CDK2 not present in the other CDK family members [<xref rid=\"ref65\" ref-type=\"bibr\">65</xref>]. However, the lead covalent inhibitor exhibits weak inhibition against CDK2-cyclin A2 with 83% inhibition at 0.5 mM. Covalent inhibitors may be acceptable as contraceptive agents if they are selective, but further studies are needed to validate this assertion.</p></sec><sec id=\"sec10\"><title>Remaining questions about CDK2 as a contraceptive target</title><p>While the importance of CDK2 in spermatogenesis is clear, its appeal as a contraceptive target remains more uncertain. The <italic>CDK2<sup>−/−</sup></italic> mouse model suggests that a CDK2-selective inhibitor would have minimal off-target effects due to compensation in somatic cells by CDK1. However, it is unclear what effect prolonged CDK2 inhibition would have on a healthy adult, as CDK2 aids in repairing DNA double-strand breaks, known causes of numerous cancers. Because spermatocytes that arrest in mid-pachytene undergo apoptosis, the reversibility of a selective CDK2 inhibitor as a contraceptive agent needs to be confirmed. However, because spermatogonia are unaffected, a selective CDK2 inhibitor should be reversible. Additionally, a CDK2 inhibitor that interacts with protein binding partners (e.g., cyclins) could serve to not only inhibit CDK2 but also sequester cyclins, which could be toxic, as other CDK family members like CDK1 might not be activated. An ideal CDK2 inhibitor for contraception could therefore be a PROTAC molecule, which degrades CDK2 and recapitulates the <italic>CDK2<sup>−/−</sup></italic> mouse model, or an inhibitor that disrupts the ability of CDK2 to bind to partner proteins like the cyclins or SPY1. Such an inhibitor could bind the ATP site or an allosteric site, as long as it is negatively cooperative with partner protein binding. Another option is to develop an inhibitor that targets the CDK2-SPY1 complex while not affecting the complexes of phosphorylated CDK proteins with cyclins.</p><p>Finally, the drug administration schedule and acceptability need to be considered. Because the role of CDK2 in spermatogenesis is at the beginning of meiosis I and the entire spermatogenesis process takes over a month in adult males, a selective CDK2-targeting contraceptive would not have an immediate contraceptive effect. The pharmacokinetic properties of a selective CDK2 inhibitor must be considered when dosing to ensure a high level of efficacy as a contraceptive. Since this would be a medication that will to be taken by otherwise healthy individuals chronically, the tolerance for undesirable side effects will be very limited.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><supplementary-material content-type=\"local-data\" id=\"sup1\"><label>fig_S1_ioaa107</label><media xlink:href=\"fig_s1_ioaa107.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"sup2\"><label>fig_S2_ioaa107</label><media xlink:href=\"fig_s2_ioaa107.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"sup3\"><label>fig_S3_ioaa107</label><media xlink:href=\"fig_s3_ioaa107.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"sup4\"><label>fig_S4_ioaa107</label><media xlink:href=\"fig_s4_ioaa107.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"sup5\"><label>fig_S5_ioaa107</label><media xlink:href=\"fig_s5_ioaa107.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"sup6\"><label>fig_S6_ioaa107</label><media xlink:href=\"fig_s6_ioaa107.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ref-list id=\"bib1\"><title>References</title><ref id=\"ref1\"><label>1.</label><mixed-citation publication-type=\"journal\">\n<person-group person-group-type=\"author\"><name name-style=\"western\"><surname>Morgan</surname><given-names>DO</given-names></name></person-group>\n<article-title>Principles of CDK regulation</article-title>. <source>Nature</source><year>1995</year>; 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"brief-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Brain</journal-id><journal-id journal-id-type=\"iso-abbrev\">Brain</journal-id><journal-id journal-id-type=\"publisher-id\">brainj</journal-id><journal-title-group><journal-title>Brain</journal-title></journal-title-group><issn pub-type=\"ppub\">0006-8950</issn><issn pub-type=\"epub\">1460-2156</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32875326</article-id><article-id pub-id-type=\"pmc\">PMC7523698</article-id><article-id pub-id-type=\"doi\">10.1093/brain/awaa221</article-id><article-id pub-id-type=\"publisher-id\">awaa221</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Reports</subject></subj-group><subj-group subj-group-type=\"category-taxonomy-collection\"><subject>AcademicSubjects/MED00310</subject><subject>AcademicSubjects/SCI01870</subject></subj-group></article-categories><title-group><article-title>Impairments of auditory scene analysis in posterior cortical atrophy</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Hardy</surname><given-names>Chris J D</given-names></name><xref ref-type=\"author-notes\" rid=\"awaa221-FM1\"/><xref ref-type=\"aff\" rid=\"awaa221-aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yong</surname><given-names>Keir X X</given-names></name><xref ref-type=\"author-notes\" rid=\"awaa221-FM1\"/><xref ref-type=\"aff\" rid=\"awaa221-aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Goll</surname><given-names>Johanna C</given-names></name><xref ref-type=\"aff\" rid=\"awaa221-aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Crutch</surname><given-names>Sebastian J</given-names></name><xref ref-type=\"aff\" rid=\"awaa221-aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Warren</surname><given-names>Jason D</given-names></name><xref ref-type=\"aff\" rid=\"awaa221-aff1\"/></contrib></contrib-group><aff id=\"awaa221-aff1\">\n<institution>Dementia Research Centre, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology</institution>, London, <country country=\"GB\">UK</country></aff><author-notes><fn id=\"awaa221-FM1\"><p>Chris J. D. Hardy and Keir X. X. Yong contributed equally to this work.</p></fn><corresp id=\"awaa221-cor1\">Correspondence to: Prof Jason D. Warren Dementia Research Centre, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK E-mail: <email>jason.warren@ucl.ac.uk</email></corresp></author-notes><pub-date pub-type=\"collection\"><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\" iso-8601-date=\"2020-09-01\"><day>01</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>01</day><month>9</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><!-- oupReleaseDelayRemoved from OA Article (10.1093/brain/awaa221awaa221ReportsAcademicSubjects/MED00310AcademicSubjects/SCI01870Impairments of auditory scene analysis in posterior cortical atrophyHardyChris J DYongKeir X XGollJohanna CCrutchSebastian JWarrenJason D\n Dementia Research Centre, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UKChris J. D. Hardy and Keir X. X. Yong contributed equally to this work.Correspondence to: Prof Jason D. Warren Dementia Research Centre, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK E-mail: jason.warren@ucl.ac.uk0920200109202001092020143926892695awaa221_Supplementary_DataSee Särkäö and Sihvonen (doi:10.1093/brain/awaa218) for a scientific commentary on this article.Posterior cortical atrophy (PCA) is thought of as a visual-led dementia. However, Hardy, Yong et al. show that people with PCA also have difficulties with auditory scene analysis – the central hearing processes that enable us to focus on a single person speaking in a crowded room – suggesting that PCA affects more than vision.110320202705202027052020© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.2020This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractAlthough posterior cortical atrophy is often regarded as the canonical ‘visual dementia’, auditory symptoms may also be salient in this disorder. Patients often report particular difficulty hearing in busy environments; however, the core cognitive process—parsing of the auditory environment (‘auditory scene analysis’)—has been poorly characterized. In this cross-sectional study, we used customized perceptual tasks to assess two generic cognitive operations underpinning auditory scene analysis—sound source segregation and sound event grouping—in a cohort of 21 patients with posterior cortical atrophy, referenced to 15 healthy age-matched individuals and 21 patients with typical Alzheimer’s disease. After adjusting for peripheral hearing function and performance on control tasks assessing perceptual and executive response demands, patients with posterior cortical atrophy performed significantly worse on both auditory scene analysis tasks relative to healthy controls and patients with typical Alzheimer’s disease (all P < 0.05). Our findings provide further evidence of central auditory dysfunction in posterior cortical atrophy, with implications for our pathophysiological understanding of Alzheimer syndromes as well as clinical diagnosis and management.See Särkäö and Sihvonen (doi:10.1093/brain/awaa218) for a scientific commentary on this article.Posterior cortical atrophy (PCA) is thought of as a visual-led dementia. However, Hardy, Yong et al. show that people with PCA also have difficulties with auditory scene analysis – the central hearing processes that enable us to focus on a single person speaking in a crowded room – suggesting that PCA affects more than vision.posterior cortical atrophyauditory scene analysishearingdementiaAlzheimer's diseaseAlzheimer's Research UKBrain Research Trust10.13039/501100000368The Wolfson FoundationAlzheimer’s SocietyAS-PG-16-007Economic and Social Research Council10.13039/501100000269ES/L001810/1National Institute for Health Research University College London Hospitals Biomedical Research CentreUCL Leonard Wolfson Experimental Neurology CentrePR/ylr/18575Action on Hearing Loss-Dunhill Medical Trust Pauline Ashley FellowshipPA23_HardyAlzheimer's Society10.13039/501100000320453 (AS-JF-18-003ESRC10.13039/501100000269NIHR10.13039/100006662ES/L001810/1Alzheimer’s Research UK Senior ResearchWellcome Trust10.13039/100004440091673/Z/10/Z) --><volume>143</volume><issue>9</issue><fpage>2689</fpage><lpage>2695</lpage><history><date date-type=\"received\"><day>11</day><month>3</month><year>2020</year></date><date date-type=\"rev-recd\"><day>27</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>5</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"cc-by\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"awaa221.pdf\"/><related-article related-article-type=\"companion\" id=\"d38e165\" ext-link-type=\"doi\" xlink:href=\"10.1093/brain/awaa218\"/><abstract><title>Abstract</title><p>Although posterior cortical atrophy is often regarded as the canonical ‘visual dementia’, auditory symptoms may also be salient in this disorder. Patients often report particular difficulty hearing in busy environments; however, the core cognitive process—parsing of the auditory environment (‘auditory scene analysis’)—has been poorly characterized. In this cross-sectional study, we used customized perceptual tasks to assess two generic cognitive operations underpinning auditory scene analysis—sound source segregation and sound event grouping—in a cohort of 21 patients with posterior cortical atrophy, referenced to 15 healthy age-matched individuals and 21 patients with typical Alzheimer’s disease. After adjusting for peripheral hearing function and performance on control tasks assessing perceptual and executive response demands, patients with posterior cortical atrophy performed significantly worse on both auditory scene analysis tasks relative to healthy controls and patients with typical Alzheimer’s disease (all <italic>P </italic><<italic> </italic>0.05). Our findings provide further evidence of central auditory dysfunction in posterior cortical atrophy, with implications for our pathophysiological understanding of Alzheimer syndromes as well as clinical diagnosis and management.</p></abstract><abstract abstract-type=\"teaser\"><p>See Särkäö and Sihvonen (doi:<related-article related-article-type=\"companion\" id=\"d38e180\" ext-link-type=\"doi\" xlink:href=\"10.1093/brain/awaa218\">10.1093/brain/awaa218</related-article>) for a scientific commentary on this article.</p><p>Posterior cortical atrophy (PCA) is thought of as a visual-led dementia. However, Hardy, Yong <italic>et al.</italic> show that people with PCA also have difficulties with auditory scene analysis – the central hearing processes that enable us to focus on a single person speaking in a crowded room – suggesting that PCA affects more than vision.</p></abstract><kwd-group><kwd>posterior cortical atrophy</kwd><kwd>auditory scene analysis</kwd><kwd>hearing</kwd><kwd>dementia</kwd><kwd>Alzheimer's disease</kwd></kwd-group><funding-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>Alzheimer's Research UK</institution></institution-wrap></funding-source></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>Brain Research Trust</institution><institution-id institution-id-type=\"DOI\">10.13039/501100000368</institution-id></institution-wrap></funding-source></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>The Wolfson Foundation</institution></institution-wrap></funding-source></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>Alzheimer’s Society</institution></institution-wrap></funding-source><award-id>AS-PG-16-007</award-id></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>Economic and Social Research Council</institution><institution-id institution-id-type=\"DOI\">10.13039/501100000269</institution-id></institution-wrap></funding-source><award-id>ES/L001810/1</award-id></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>National Institute for Health Research University College London Hospitals Biomedical Research Centre</institution></institution-wrap></funding-source></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>UCL Leonard Wolfson Experimental Neurology Centre</institution></institution-wrap></funding-source><award-id>PR/ylr/18575</award-id></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>Action on Hearing Loss-Dunhill Medical Trust Pauline Ashley Fellowship</institution></institution-wrap></funding-source><award-id>PA23_Hardy</award-id></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>Alzheimer's Society</institution><institution-id institution-id-type=\"DOI\">10.13039/501100000320</institution-id></institution-wrap></funding-source><award-id>453 (AS-JF-18-003</award-id></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>ESRC</institution><institution-id institution-id-type=\"DOI\">10.13039/501100000269</institution-id></institution-wrap></funding-source></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>NIHR</institution><institution-id institution-id-type=\"DOI\">10.13039/100006662</institution-id></institution-wrap></funding-source><award-id>ES/L001810/1</award-id></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>Alzheimer’s Research UK Senior Research</institution></institution-wrap></funding-source></award-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>Wellcome Trust</institution><institution-id institution-id-type=\"DOI\">10.13039/100004440</institution-id></institution-wrap></funding-source><award-id>091673/Z/10/Z</award-id></award-group></funding-group><counts><page-count count=\"7\"/></counts></article-meta></front><body><p>\n<bold>See Särkäö and Sihvonen (doi:<related-article related-article-type=\"companion\" id=\"d38e313\" ext-link-type=\"doi\" xlink:href=\"10.1093/brain/awaa218\">10.1093/brain/awaa218</related-article>) for a scientific commentary on this article.</bold>\n</p><sec sec-type=\"intro\"><title>Introduction</title><p>Posterior cortical atrophy (PCA) is conventionally defined as a syndrome characterized by progressive impairment of higher visual function, in particular visuoperceptual and visuospatial skills, often designated the ‘visual variant’ of Alzheimer’s disease (<xref rid=\"awaa221-B2\" ref-type=\"bibr\">Benson <italic>et al.</italic>, 1988</xref>; <xref rid=\"awaa221-B7\" ref-type=\"bibr\">Crutch <italic>et al.</italic>, 2017</xref>). Patients with PCA have particular difficulty interpreting and navigating ‘busy’, dynamic visual environments requiring parsing of multiple objects distributed in space (<xref rid=\"awaa221-B26\" ref-type=\"bibr\">Shakespeare <italic>et al.</italic>, 2013</xref>; <xref rid=\"awaa221-B31\" ref-type=\"bibr\">Yong <italic>et al.</italic>, 2018</xref>). Posterior cortical functions such as calculation, spelling and praxis are also affected in PCA (<xref rid=\"awaa221-B2\" ref-type=\"bibr\">Benson <italic>et al.</italic>, 1988</xref>; <xref rid=\"awaa221-B7\" ref-type=\"bibr\">Crutch <italic>et al.</italic>, 2017</xref>), along with other cognitive domains including language (<xref rid=\"awaa221-B6\" ref-type=\"bibr\">Crutch <italic>et al.</italic>, 2013</xref>), episodic memory (<xref rid=\"awaa221-B1\" ref-type=\"bibr\">Ahmed <italic>et al.</italic>, 2018</xref>), working memory (<xref rid=\"awaa221-B30\" ref-type=\"bibr\">Trotta <italic>et al.</italic>, 2019</xref>), executive functioning (<xref rid=\"awaa221-B25\" ref-type=\"bibr\">Putcha <italic>et al.</italic>, 2018</xref>), and visuo-vestibular integration (<xref rid=\"awaa221-B8\" ref-type=\"bibr\">Crutch <italic>et al.</italic>, 2018</xref>).</p><p>In the realm of sound, both PCA and typical amnestic Alzheimer’s disease adversely affect the processing of auditory spatial information, leading to impaired detection of sound source motion and localization of stationary sounds in space (<xref rid=\"awaa221-B13\" ref-type=\"bibr\">Golden <italic>et al.</italic>, 2015</xref>). Such deficits are likely to contribute to impaired navigation of complex, everyday environments in both PCA and typical amnestic Alzheimer’s disease, and may form part of a wider spectrum of central auditory dysfunction in these syndromes (<xref rid=\"awaa221-B6\" ref-type=\"bibr\">Crutch <italic>et al.</italic>, 2013</xref>; <xref rid=\"awaa221-B27\" ref-type=\"bibr\">Slattery <italic>et al.</italic>, 2019</xref>). Taken together, this evidence suggests that central auditory impairment may be more significant in PCA than generally recognized.</p><p>The process of auditory scene analysis (ASA) depends fundamentally on accurate and efficient parsing of the auditory environment into its constituent sound sources and patterns (<xref rid=\"awaa221-B4\" ref-type=\"bibr\">Bregman, 1994</xref>), such that these ‘auditory objects’ (<xref rid=\"awaa221-B17\" ref-type=\"bibr\">Griffiths and Warren, 2004</xref>) can be tracked, analysed and ultimately associated with meaning. One striking example of ASA in action is the well-known ‘cocktail party effect’ (exemplified by hearing one’s own name across a crowded room); however, a broadly similar task confronts the brain whenever we process a target sound under the non-ideal listening conditions of daily life. Because everyday auditory environments usually contain multiple, acoustically diverse sound sources that are superimposed and evolving over time, ASA demands substantial neural computational resources: it is therefore potentially vulnerable to the early effects of neurodegenerative pathologies. Patients with typical amnestic Alzheimer’s disease have a generic deficit of ASA, linked to degeneration of posterior temporo-parietal networks (<xref rid=\"awaa221-B14\" ref-type=\"bibr\">Goll <italic>et al.</italic>, 2012</xref>). On neuroanatomical and neuropathological grounds, impaired ASA is therefore anticipated to be a feature of PCA and indeed, might manifest earlier and more saliently in PCA than typical amnestic Alzheimer’s disease (<xref rid=\"awaa221-B2\" ref-type=\"bibr\">Benson <italic>et al.</italic>, 1988</xref>; <xref rid=\"awaa221-B7\" ref-type=\"bibr\">Crutch <italic>et al.</italic>, 2017</xref>; <xref rid=\"awaa221-B11\" ref-type=\"bibr\">Firth <italic>et al.</italic>, 2019</xref>). However, it is challenging to assess perception of auditory scenes in the clinic or laboratory and ASA has not been investigated before in PCA.</p><p>Here we used a previously devised neuropsychological paradigm (<xref rid=\"awaa221-B14\" ref-type=\"bibr\">Goll <italic>et al.</italic>, 2012</xref>) to assess ASA in patients with PCA, relative to patients with typical amnestic Alzheimer’s disease and healthy older individuals. This paradigm uses synthetic sound sources presented as dual auditory sequences to simulate the kinds of interactions between auditory objects that might occur in any natural auditory scene. Experimental tasks assessed two core, complementary cognitive operations underpinning ASA: ‘segregation’ (the parsing of superimposed, coincident sounds into separate sound objects, as in the cocktail party effect), and ‘grouping’ (the perceptual assembly of temporally-distributed sound events into a single auditory object, as when hearing out a melody for a particular voice or instrument in polyphonic music). We hypothesized that patients with PCA as a group would show significant deficits of ASA-segregation and ASA-grouping, relative both to healthy controls and to patients with typical amnestic Alzheimer’s disease.</p></sec><sec><title>Materials and methods</title><p>Twenty-one patients with PCA, 21 patients with typical amnestic Alzheimer’s disease (published previously: <xref rid=\"awaa221-B14\" ref-type=\"bibr\">Goll <italic>et al.</italic>, 2012</xref>) and 15 healthy age-matched individuals were included. All patients with PCA presented with visual difficulties and relative sparing of memory, language and insight, while patients with typical amnestic Alzheimer’s disease presented with episodic memory decline. Patients fulfilled criteria for the relevant diagnosis (<xref rid=\"awaa221-B10\" ref-type=\"bibr\">Dubois <italic>et al.</italic>, 2007</xref>; <xref rid=\"awaa221-B7\" ref-type=\"bibr\">Crutch <italic>et al.</italic>, 2017</xref>) (<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Table 1</xref>), corroborated by general neuropsychological assessment, CSF examination and brain MRI. Participant group characteristics are summarized in <xref rid=\"awaa221-T1\" ref-type=\"table\">Table 1</xref>; additional neuropsychological data for the PCA group are presented in <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Table 2</xref>. Disease-related atrophy profiles were assessed using voxel-based morphometry for each patient group (details in the <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary material</xref>). Study approval was granted by the institutional ethics committee; all participants gave informed consent following Declaration of Helsinki guidelines.\n</p><table-wrap id=\"awaa221-T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Summary of demographic, clinical, audiometric and auditory scene analysis task data for all participant groups</p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col valign=\"top\" align=\"left\" span=\"1\"/><col valign=\"top\" align=\"left\" span=\"1\"/><col valign=\"top\" align=\"left\" span=\"1\"/><col valign=\"top\" align=\"left\" span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th align=\"left\" rowspan=\"1\" colspan=\"1\">Controls</th><th align=\"left\" rowspan=\"1\" colspan=\"1\">PCA<xref ref-type=\"table-fn\" rid=\"tblfn2\"><sup>a</sup></xref></th><th align=\"left\" rowspan=\"1\" colspan=\"1\">tAD</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>General</bold>\n</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<italic>n</italic>, male: female</td><td rowspan=\"1\" colspan=\"1\">7:8</td><td rowspan=\"1\" colspan=\"1\">7:14</td><td rowspan=\"1\" colspan=\"1\">9:12</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Age, years</td><td rowspan=\"1\" colspan=\"1\">63.36 (6.4)</td><td rowspan=\"1\" colspan=\"1\">63.02 (9.5)</td><td rowspan=\"1\" colspan=\"1\">65.03 (7.9)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hearing loss, dB</td><td rowspan=\"1\" colspan=\"1\">7.9 (7.7)</td><td rowspan=\"1\" colspan=\"1\">\n<bold>17.0 (10.5)</bold>\n<xref ref-type=\"table-fn\" rid=\"tblfn6\">*</xref>\n</td><td rowspan=\"1\" colspan=\"1\">13.6 (6.9)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Symptom duration, years</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">\n<italic>3.65</italic> (<italic>2.3</italic>)</td><td rowspan=\"1\" colspan=\"1\">5.93 (2.5)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>Background neuropsychology</bold>\n</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">MMSE (/30)</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">\n<italic>18.48</italic> (<italic>4.6</italic>)</td><td rowspan=\"1\" colspan=\"1\">22.10 (4.2)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">RMT Words (<italic>z</italic>-score)<xref ref-type=\"table-fn\" rid=\"tblfn3\"><sup>b</sup></xref></td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">\n<bold>−2.15 (2.2)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">\n<bold>−2.65 (1.9)</bold>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">RMT Faces (<italic>z</italic>-score)<xref ref-type=\"table-fn\" rid=\"tblfn4\"><sup>c</sup></xref></td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">−1.95 (2.3)</td><td rowspan=\"1\" colspan=\"1\">\n<bold>−2.28 (2.0)</bold>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Digit span forward (/12)</td><td rowspan=\"1\" colspan=\"1\">8.40 (1.6)</td><td rowspan=\"1\" colspan=\"1\">\n<bold>6.81 (1.9)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">7.48 (2.3)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Digit span reverse (/12)</td><td rowspan=\"1\" colspan=\"1\">7.27 (1.3)</td><td rowspan=\"1\" colspan=\"1\">\n<bold><italic>3.14</italic> (<italic>1.3</italic>)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">\n<bold>5.24 (2.8)</bold>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">WASI Vocabulary (/72)<xref ref-type=\"table-fn\" rid=\"tblfn5\"><sup>d</sup></xref></td><td rowspan=\"1\" colspan=\"1\">71.36 (4.4)</td><td rowspan=\"1\" colspan=\"1\">\n<bold>54.47 (8.8)</bold>**</td><td rowspan=\"1\" colspan=\"1\">\n<bold>57.00 (14.8)</bold>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Graded naming test (/30)<xref ref-type=\"table-fn\" rid=\"tblfn5\"><sup>d</sup></xref></td><td rowspan=\"1\" colspan=\"1\">27.00 (3.3)</td><td rowspan=\"1\" colspan=\"1\">\n<bold>13.90 (4.6)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">\n<bold>13.95 (9.0)</bold>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Graded difficulty arithmetic (/24)<xref ref-type=\"table-fn\" rid=\"tblfn5\"><sup>d</sup></xref></td><td rowspan=\"1\" colspan=\"1\">15.55 (3.7)</td><td rowspan=\"1\" colspan=\"1\">\n<bold><italic>1.76</italic> (<italic>3.2</italic>)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">\n<bold>6.33 (4.9)</bold>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Single word comprehension (<italic>z</italic>-score)</td><td rowspan=\"1\" colspan=\"1\">–</td><td rowspan=\"1\" colspan=\"1\">0.23 (0.7)</td><td rowspan=\"1\" colspan=\"1\">\n<bold>−6.41 (7.7)</bold>***</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<bold>ASA tests</bold>\n</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\">ASA segregation<xref ref-type=\"table-fn\" rid=\"tblfn3\"><sup>b</sup></xref></td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"> ASA segregation test (/20)</td><td rowspan=\"1\" colspan=\"1\">19.07 (1.5)</td><td rowspan=\"1\" colspan=\"1\">\n<bold>12.14 (3.0)</bold>****</td><td rowspan=\"1\" colspan=\"1\">\n<bold>15.45 (4.2)</bold>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Task requirement control test (/10)</td><td rowspan=\"1\" colspan=\"1\">10.00 (0.01)</td><td rowspan=\"1\" colspan=\"1\">\n<bold><italic>8.95</italic> (<italic>1.2</italic>)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">10.00 (0.0)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Perceptual cue control test (/10)</td><td rowspan=\"1\" colspan=\"1\">9.67 (0.6)</td><td rowspan=\"1\" colspan=\"1\">8.71 (1.4)</td><td rowspan=\"1\" colspan=\"1\">9.35 (1.0)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">ASA grouping</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"> ASA grouping test (/20)<xref ref-type=\"table-fn\" rid=\"tblfn4\"><sup>c</sup></xref></td><td rowspan=\"1\" colspan=\"1\">18.67 (1.2)</td><td rowspan=\"1\" colspan=\"1\">\n<bold><italic>11.05</italic> (<italic>3.8</italic>)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">\n<bold>15.62 (3.8)</bold>\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Task requirement control test (/10)</td><td rowspan=\"1\" colspan=\"1\">9.93 (0.3)</td><td rowspan=\"1\" colspan=\"1\">\n<bold><italic>9.00</italic> (<italic>0.8</italic>)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">10.0 (0.0)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Perceptual cue control test (/10)</td><td rowspan=\"1\" colspan=\"1\">9.87 (0.4)</td><td rowspan=\"1\" colspan=\"1\">\n<bold><italic>7.90</italic> (<italic>1.9</italic>)</bold>\n</td><td rowspan=\"1\" colspan=\"1\">9.86 (0.5)</td></tr></tbody></table><table-wrap-foot><fn id=\"tblfn1\"><p>Mean (SD) data are presented unless otherwise indicated; maximum scores for neuropsychological tests are indicated in parentheses. Bold indicates significantly lower than healthy controls, <italic>P </italic><<italic> </italic>0.001 unless otherwise specified (for <italic>z</italic>-scores, bold indicates average performance outside 95% of the area under normal distribution, i.e. ±1.96); italics indicate significantly lower than the typical Alzheimer’s disease (tAD) group, <italic>P </italic>≤<italic> </italic>0.01 unless otherwise specified (statistical data including 95% CIs are presented in full in <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Table 3</xref>). Certain cognitive functions were assessed using different tests in the PCA and typical Alzheimer’s disease cohorts: the typical Alzheimer’s disease cohort was given the long-form Recognition Memory Test (RMT) for words and faces and the British Picture Vocabulary Scale (BPVS) for single word comprehension; the PCA group were given the short-form RMT for words and faces, and the Concrete Synonyms test for single word comprehension, and to give a comparable indication of how each syndromic group performed in these domains we derived <italic>z</italic>-scores using age-appropriate normative data (<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Table 3</xref>). Administration of the graded naming test differed across groups: control and typical Alzheimer’s disease participants were presented with items visually; participants with PCA were asked to name from verbal description. MMSE = Mini-Mental State Examination score; WASI = Wechsler Abbreviated Scale of Intelligence.</p></fn><fn id=\"tblfn2\"><label><sup>a</sup></label><p>CSF profiles of tau and amyloid-β were available for 13 patients with PCA and were consistent with Alzheimer’s pathology in 12 cases, based on local reference ranges (total tau/amyloid-β<sub>1-42</sub> ratio > 1). One participant with PCA showed clear response bias on the ASA-grouping task (<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary material</xref>); this participant was removed from analysis of the ASA-grouping test.</p></fn><fn id=\"tblfn3\"><label><sup>b</sup></label><p>Data were not available for one participant with typical Alzheimer’s disease.</p></fn><fn id=\"tblfn4\"><label><sup>c</sup></label><p>Data were not available for one participant with PCA.</p></fn><fn id=\"tblfn5\"><label><sup>d</sup></label><p>Data were not available for four healthy control participants.</p></fn><fn id=\"tblfn6\"><label>*</label><p>\n<italic>P </italic>=<italic> </italic>0.003 versus controls.</p></fn><fn id=\"tblfn7\"><label>**</label><p>\n<italic>P </italic>=<italic> </italic>0.002 versus controls.</p></fn><fn id=\"tblfn8\"><label>***</label><p>\n<italic>P </italic><<italic> </italic>0.001 versus PCA.</p></fn><fn id=\"tblfn9\"><label>****</label><p>\n<italic>P </italic>=<italic> </italic>0.012 versus typical Alzheimer’s disease.</p></fn></table-wrap-foot></table-wrap><p>To assess any effects of hearing loss on performance in the experimental tasks, all subjects underwent pure tone audiometry, following a previously described procedure (<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary material</xref>). For each participant, the mean audiometric threshold over five frequencies (0.5, 1, 2, 3, 4 kHz) was taken as a composite score for that participant’s peripheral hearing function, used as a covariate in subsequent analyses.</p><p>Auditory scene analysis was assessed using two main experimental tasks, as described previously (<xref rid=\"awaa221-B14\" ref-type=\"bibr\">Goll <italic>et al.</italic>, 2012</xref>) (details of tasks and stimuli are provided in the <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary material</xref> and <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Fig. 1</xref>): an ASA-segregation task, requiring separation of temporally concurrent sound objects based on spectral shape (an important acoustic cue to the perception of timbre, or sound identity; <xref ref-type=\"fig\" rid=\"awaa221-F1\">Fig. 1A</xref>); and an ASA-grouping task, requiring grouping of temporally spaced sound objects into a single stream on the basis of fundamental frequency (an important acoustic determinant of the perception of pitch; <xref ref-type=\"fig\" rid=\"awaa221-F1\">Fig. 1B</xref>). Prior to each of these ASA tests, we administered bespoke control tests using tasks designed to familiarize participants with the paradigm and to assess more general cognitive processes that might affect ASA: a ‘perceptual-cue’ control, to establish how well participants could discriminate changes in perceptual cues (timbre or pitch) driving the relevant ASA task; and a ‘task-response’ control, to establish how well participants could comply with the general response and working memory requirements of the relevant ASA test. All participants completed initial practice trials and only commenced the experimental tests once the experimenter was confident that participants understood the relevant tasks (<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary material</xref>).\n</p><fig id=\"awaa221-F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>\n<bold>Summary of the ASA paradigm and participant performance.</bold> <italic>Top</italic> panels depict the ASA paradigms (<bold>A</bold> and <bold>B</bold>); <italic>bottom</italic> panels show plots of individual participant performance on the respective ASA tests (<bold>C</bold> and <bold>D</bold>); see also <xref rid=\"awaa221-T1\" ref-type=\"table\">Table 1</xref> and further stimulus details in the <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary material</xref>. Conditions in all subtests of the ASA-segregation assessment (<bold>A</bold>) and ASA-grouping assessment (<bold>B</bold>) are shown. Each <italic>top</italic> panel signifies one trial on which a sequence of sounds (harmonic complexes) was presented; rectangles represent individual sound elements (width indicates relative duration and height indicates relative intensity (<bold>A</bold>) or fundamental frequency (f0, corresponding to pitch; <bold>B</bold>). <bold>A</bold> and <bold>B</bold> are intended as illustrative diagrams, and not to scale; the same range of intensity and pitch values was used in each test. In the main ASA-segregation test (<bold>A</bold>), sound sequences (each of total duration 10 s) containing two different timbres (coded as black and light grey) were presented, and 20 trials comprising two experimental conditions were created by varying the temporal pattern such that one timbre (designated the ‘target’) was either continuous (10 trials) or intermittent (10 trials); the task on each trial was to decide whether the target sounds were ‘long’ (i.e. continuous) or ‘on-off’ (i.e. intermittent). A perceptual-cue control test was created to establish that participants were reliably able to detect timbre changes: 10 sound sequences were presented, five with continuous fixed timbre and five with two alternating timbres, and the task on each trial was to decide if the sound was ‘same’ or ‘changing’. A task-requirement control test was used to establish that participants could comply with the requirement to report continuous and intermittent temporal patterns: 10 sequences of sounds were presented, five continuous and five intermittent, and the task on each trial was to decide whether the sound was ‘long’ (i.e. continuous) or ‘on-off’ (i.e. intermittent). In the main ASA-grouping test (<bold>B</bold>), 20 trials (each of total duration 12 s) were presented, each comprising two interleaved sound sequences with two different pitch values (coded as black and light grey; one designated the ‘target’ pitch) such that the target pitch sequence was either isochronous (fixed inter-sound interval, 10 trials) or anisochronous (randomly varying inter-sound interval, 10 trials); the task on each trial was to decide whether sounds with the target pitch were ‘even’ or ‘uneven’. A perceptual-cue control test was created to establish that participants were reliably able to detect pitch differences: 10 isochronous sequences were presented, five with fixed pitch and five with changing pitch, and the task on each trial was to decide if the pitch was ‘same’ or ‘changing’. A task-requirement control test was used to establish that participants could comply with the requirement to report even and uneven temporal patterns: 10 sequences of sounds with fixed pitch were presented, five isochronous and five anisochronous, and the task on each trial was to decide whether the sequence was ‘even’ or ‘uneven’. Participants were familiarized with the task and the targets for each test beforehand, to ensure they fully understood the experimental instructions (<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary material</xref>). Data plots show individual scores (circles) on the ASA-segregation main task (<bold>C</bold>) and ASA-grouping main task (<bold>D</bold>). For each group, horizontal lines indicate median score, oblongs code interquartile range and whiskers 95% confidence intervals; a score of 10 would correspond to chance performance. Control = healthy control group; PCA = patient group with posterior cortical atrophy; tAD = patient group with typical Alzheimer’s disease.</p></caption><graphic xlink:href=\"awaa221f1\"/></fig><p>The task on each trial was a two-alternative forced-choice decision. For the segregation task, participants were asked to report verbally whether target stimuli were ‘long’ (i.e. continuous) or ‘on-and-off’ (intermittent); for the grouping task, participants reported whether target stimuli were even or uneven (<xref ref-type=\"fig\" rid=\"awaa221-F1\">Fig. 1A and B</xref>). Details of the experimental protocol and examples of the stimuli are provided in the <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary material</xref>; all sounds were presented from a notebook computer binaurally via headphones at a comfortable listening level. No feedback about performance was given during the assessment and no time limit was imposed on subject responses.</p><p>Data were analysed in Stata version 14 (StataCorp). Demographic and clinical characteristics were compared between groups using ANOVA and Fisher’s exact tests; where the omnibus test was significant, <italic>post hoc</italic> comparisons between groups were investigated. Scores on each of the ASA and control tests were entered into separate analysis of covariance (ANCOVA) regression models, adjusting for peripheral hearing score and (for the ASA tests) also for corresponding control task scores; importantly, these control tasks were similar to the ASA tasks on more general perceptual, attentional, working memory, recall and response decision demands, allowing us to take account of these potentially confounding factors in the ASA group comparison. Pairwise group differences were assessed using planned comparisons that also adjusted for these covariates. Residuals from the ANCOVA models were not normally distributed, so we adopted a non-parametric approach that allowed for relaxation of the normality and heteroscedasticity assumptions made by ANCOVA; full details of this approach are given in the <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary material</xref>. We used Spearman’s ρ to assess associations between performance on each of the main ASA tasks and Mini-Mental State Examination (MMSE) in the PCA group. An alpha level of 0.05 was accepted as the statistical significance threshold for all tests.</p><sec sec-type=\"data-availability\"><title>Data availability</title><p>The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available as they include information that could compromise the privacy of the research participants.</p></sec></sec><sec sec-type=\"results\"><title>Results</title><p>Results for group comparisons of demographic and clinical characteristics and experimental test performance are summarized in <xref rid=\"awaa221-T1\" ref-type=\"table\">Table 1</xref> and additional data are presented in <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Tables 2 and 3</xref>; individual participant performance plots on the ASA tests are shown for each participant group in <xref ref-type=\"fig\" rid=\"awaa221-F1\">Fig. 1C and D</xref>. Syndromic groups showed the anticipated profiles of disease-related brain atrophy (<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Fig. 1</xref>).</p><p>Participant groups did not differ significantly in age [<italic>F</italic>(2,54) = 0.35, <italic>P = </italic>0.71] or gender (<italic>P = </italic>0.79). There was evidence of an overall difference in peripheral hearing function across groups [<italic>F</italic>(2,54) = 4.92, <italic>P = </italic>0.01], with the PCA group performing more poorly than healthy controls (<italic>t</italic> = −3.13, <italic>P = </italic>0.003). Relative to the typical amnestic Alzheimer’s disease group, the PCA group had a significantly shorter symptom duration [<italic>t</italic>(40) = −3.09, <italic>P = </italic>0.004] despite a lower MMSE score [<italic>t</italic>(40) = −2.66, <italic>P = </italic>0.01]. The PCA group performed significantly more poorly than both the healthy control group and the typical amnestic Alzheimer’s disease group on the bespoke tests controlling for task response and working memory demands and on one test controlling for perceptual discrimination ability (details in <xref rid=\"awaa221-T1\" ref-type=\"table\">Table 1</xref> and <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Table 1</xref>).</p><p>After adjusting for group differences in peripheral hearing function and control task performance, there was a significant group effect on performance in the main ASA-segregation test [<italic>F</italic>(2,50) = 12.12, <italic>P < </italic>0.001], the PCA group performing worse than both the healthy control group {adjusted difference, −6.40 [95% confidence intervals (CI): −8.54 to −4.41], <italic>t</italic> = −4.90, <italic>P < </italic>0.001} and the typical amnestic Alzheimer’s disease group [adjusted difference, −3.10 (95% CI: −5.49 to −0.74), <italic>t</italic> = −2.63; <italic>P </italic>=<italic> </italic>0.01].</p><p>There was also an overall group effect on performance in the main ASA-grouping test [<italic>F</italic>(2,50) = 8.91, <italic>P </italic>=<italic> </italic>0.001], the PCA group again performing worse than both the healthy control group [adjusted difference, −6.91 (95% CI: −11.27 to −3.48), <italic>t</italic> = −4.17, <italic>P < </italic>0.001] and the typical amnestic Alzheimer’s disease group [adjusted difference, −3.97 (95% CI: −8.12 to −0.14], <italic>t</italic> = −2.59; <italic>P </italic>=<italic> </italic>0.01]. Within the PCA group, performance on the main ASA tasks was not significantly associated with MMSE score (ASA-segregation, ρ  =  0.31, <italic>P </italic>=<italic> </italic>0.17; ASA-grouping, ρ  =  0.25, <italic>P </italic>=<italic> </italic>0.28). An analysis of hit rates for different sequence types in the main ASA tests within each patient group did not find evidence of response bias (<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Table 4</xref>).</p></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>We have presented evidence that patients with PCA perform worse on tasks of ASA-segregation and ASA-grouping than healthy older individuals or patients with typical amnestic Alzheimer’s disease. Our data suggest that this impairment is not attributable to peripheral hearing function, executive task demands or working memory capacity, nor to general disease severity. Rather, the findings support the concept of a multi-sensory breakdown in parsing the world at large in PCA.</p><p>Our findings corroborate earlier work suggesting that there may be a prominent auditory contribution to the PCA phenotype (<xref rid=\"awaa221-B6\" ref-type=\"bibr\">Crutch <italic>et al.</italic>, 2013</xref>; <xref rid=\"awaa221-B13\" ref-type=\"bibr\">Golden <italic>et al.</italic>, 2015</xref>; <xref rid=\"awaa221-B27\" ref-type=\"bibr\">Slattery <italic>et al.</italic>, 2019</xref>). The ASA-segregation and ASA-grouping tasks used here were developed to probe fundamental processes in the disambiguation and understanding of auditory scenes. We have previously shown that patients with typical amnestic Alzheimer’s disease are impaired on these tasks relative to healthy individuals of similar age (<xref rid=\"awaa221-B14\" ref-type=\"bibr\">Goll <italic>et al.</italic>, 2012</xref>). Current findings emphasize that these generic ASA component processes are substantially more severely impaired in PCA, underlining the profound involvement of posterior cortical functions in this syndrome. ASA impairment may have a common neuroanatomical and pathophysiological basis in PCA and typical amnestic Alzheimer’s disease, linked to involvement of the core temporo-parietal ‘default mode’ network (<xref rid=\"awaa221-B16\" ref-type=\"bibr\">Greicius <italic>et al.</italic>, 2004</xref>; <xref rid=\"awaa221-B14\" ref-type=\"bibr\">Goll <italic>et al.</italic>, 2012</xref>; <xref rid=\"awaa221-B7\" ref-type=\"bibr\">Crutch <italic>et al.</italic>, 2017</xref>); ASA may engage a generic interface between external sensory events and the internal monitoring and modelling of those events over time, instantiated by this network (<xref rid=\"awaa221-B14\" ref-type=\"bibr\">Goll <italic>et al.</italic>, 2012</xref>).</p><p>In addition to more severe ASA impairment, the PCA cohort here exhibited deficits of more elementary auditory processing not clearly apparent in patients with typical amnestic Alzheimer’s disease: namely, pure tone detection and pitch change perception. To our knowledge, audiometric deficits have not previously been identified as a feature of PCA but this corroborates emerging evidence that pure tone detection can be impaired even in canonical dementia syndromes (most notably, non-fluent/agrammatic variant primary progressive aphasia) that typically spare the peripheral auditory pathways (<xref rid=\"awaa221-B21\" ref-type=\"bibr\">Hardy <italic>et al.</italic>, 2016</xref>, <xref rid=\"awaa221-B18\" ref-type=\"bibr\">2019</xref>). The processing of pitch change (particularly in the context of a task requiring working memory) is likely to depend on temporo-parietal junctional cortices that are targeted in PCA (<xref rid=\"awaa221-B5\" ref-type=\"bibr\">Crutch <italic>et al.</italic>, 2012</xref>; <xref rid=\"awaa221-B24\" ref-type=\"bibr\">Plack <italic>et al.</italic>, 2014</xref>; <xref rid=\"awaa221-B12\" ref-type=\"bibr\">Golden <italic>et al.</italic>, 2016</xref>; <xref rid=\"awaa221-B11\" ref-type=\"bibr\">Firth <italic>et al.</italic>, 2019</xref>; <xref rid=\"awaa221-B23\" ref-type=\"bibr\">Phillips <italic>et al.</italic>, 2019</xref>): impaired pitch processing here may also indicate a more general impairment of magnitude estimation, as patients with PCA also have difficulties with other aspects of numerical and spatial magnitude encoding (<xref rid=\"awaa221-B9\" ref-type=\"bibr\">Delazer <italic>et al.</italic>, 2006</xref>; <xref rid=\"awaa221-B29\" ref-type=\"bibr\">Spotorno <italic>et al.</italic>, 2014</xref>; <xref rid=\"awaa221-B15\" ref-type=\"bibr\">González <italic>et al.</italic>, 2019</xref>) and these processes are likely to depend on neural mechanisms that are at least partly shared across modalities (<xref rid=\"awaa221-B22\" ref-type=\"bibr\">Kadosh <italic>et al.</italic>, 2008</xref>; <xref rid=\"awaa221-B3\" ref-type=\"bibr\">Bonn and Cantlon, 2012</xref>).</p><p>PCA participants were also impaired relative to healthy controls and typical amnestic Alzheimer’s disease participants on both control tasks controlling for specific task demands. Performance on these tasks was incorporated in the main analyses of ASA-segregation and ASA-grouping, but this deficit remains noteworthy. One parsimonious explanation may be that these tasks tax auditory working memory processes more affected in the PCA group (as indexed by reverse digit span, <xref rid=\"awaa221-T1\" ref-type=\"table\">Table 1</xref>).</p><p>From a clinical standpoint, these findings support the bedside impression that people with PCA have difficulty following speech and other sounds of interest particularly in busy auditory environments, and suggest that defective ASA is an even more salient limitation in PCA than in typical amnestic Alzheimer’s disease. However, further work is required to characterize the auditory phenotype of PCA more fully, and to evaluate the extent to which ASA impairments constitute a hallmark of Alzheimer’s disease pathology; CSF data were only available for 13 patients (62%), meaning that we lacked confirmation of pathophysiology in the remaining eight cases. Emerging evidence suggests that the logopenic variant of primary progressive aphasia, which is typically associated with Alzheimer’s disease pathology (<xref rid=\"awaa221-B28\" ref-type=\"bibr\">Spinelli <italic>et al.</italic>, 2017</xref>), may have a specific auditory phenotype (<xref rid=\"awaa221-B20\" ref-type=\"bibr\">Hardy <italic>et al.</italic>, 2018</xref>; <xref rid=\"awaa221-B8906446\" ref-type=\"bibr\">Johnson <italic>et al</italic>., 2020</xref>); it remains to be seen whether ASA might also be affected in this patient population. Deficits detected on these psychoacoustic tests across the Alzheimer’s disease spectrum should be correlated with structural and functional neuroimaging data to define their neural mechanisms.</p><p>In larger patient cohorts and future longitudinal studies, it will be of interest to determine how early auditory scene analysis deficits emerge, with a view to advancing diagnosis; and whether different subsyndromes of PCA [including those attributable to non-Alzheimer pathologies (<xref rid=\"awaa221-B7\" ref-type=\"bibr\">Crutch <italic>et al.</italic>, 2017</xref>)] have distinct auditory phenotypes, with neuropathological correlation. Finally, adopting a therapeutic perspective, there is the promise [based on work in typical amnestic Alzheimer’s disease (<xref rid=\"awaa221-B19\" ref-type=\"bibr\">Hardy <italic>et al.</italic>, 2017</xref>, <xref rid=\"awaa221-B20\" ref-type=\"bibr\">2018</xref>)] that residual auditory plasticity might be harnessed in PCA to benefit hearing under challenging listening conditions, via cognitive training and pharmacological modulation with acetylcholinesterase inhibitors. The deficits observed here should be compared with daily life symptoms of communication and disease burden to assess their functional relevance more directly. Strategies to support everyday activities (such as reading) in PCA often rely on auditory presentation. Understanding how to optimally combine auditory, visual, and other sensory cues may offer key insights in enabling navigation of the world at large by people with PCA and other forms of dementia.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><supplementary-material content-type=\"local-data\" id=\"sup1\"><label>awaa221_Supplementary_Data</label><media xlink:href=\"awaa221_supplementary_data.zip\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"ack1\"><title>Acknowledgements</title><p>The authors are grateful to all participants for their involvement.</p><sec><title>Funding</title><p>The Dementia Research Centre is supported by Alzheimer's Research UK, Brain Research Trust, and The Wolfson Foundation. This work was supported by the Alzheimer’s Society (AS-PG-16-007), Economic and Social Research Council (ES/L001810/1), the National Institute for Health Research University College London Hospitals Biomedical Research Centre, and the UCL Leonard Wolfson Experimental Neurology Centre (PR/ylr/18575). C.J.D.H. is supported by an Action on Hearing Loss-Dunhill Medical Trust Pauline Ashley Fellowship (PA23_Hardy). K.Y. is funded by the Alzheimer's Society, grant number 453 (AS-JF-18-003). S.J.C. was supported by a grant from ESRC/NIHR (ES/L001810/1) and an Alzheimer’s Research UK Senior Research Fellowship. 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><?properties manuscript?><front><journal-meta><journal-id journal-id-type=\"nlm-journal-id\">101767490</journal-id><journal-id journal-id-type=\"pubmed-jr-id\">49593</journal-id><journal-id journal-id-type=\"nlm-ta\">Transl Biophotonics</journal-id><journal-title-group><journal-title>Translational biophotonics</journal-title></journal-title-group><issn pub-type=\"epub\">2627-1850</issn></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005888</article-id><article-id pub-id-type=\"pmc\">PMC7523705</article-id><article-id pub-id-type=\"doi\">10.1002/tbio.201900005</article-id><article-id pub-id-type=\"manuscript\">NIHMS1593817</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Advances in Doppler optical coherence tomography and angiography</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Yan</given-names></name><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-3468-9235</contrib-id><xref ref-type=\"aff\" rid=\"A1\">1</xref><xref ref-type=\"aff\" rid=\"A2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Jason</given-names></name><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-7100-3220</contrib-id><xref ref-type=\"aff\" rid=\"A1\">1</xref><xref ref-type=\"aff\" rid=\"A2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Zhongping</given-names></name><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-4584-4560</contrib-id><xref ref-type=\"aff\" rid=\"A1\">1</xref><xref ref-type=\"aff\" rid=\"A2\">2</xref></contrib></contrib-group><aff id=\"A1\"><label>1</label>Beckman Laser Institute, University of California, Irvine, California</aff><aff id=\"A2\"><label>2</label>Department of Biomedical Engineering, University of California, Irvine, California</aff><author-notes><corresp id=\"CR1\"><bold>Correspondence:</bold> Zhongping Chen, University of California, Irvine, 1002 Health Sciences Road, Irvine, CA 92617. <email>z2chen@uci.edu</email></corresp></author-notes><pub-date pub-type=\"nihms-submitted\"><day>21</day><month>5</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>20</day><month>11</month><year>2019</year></pub-date><pub-date pub-type=\"ppub\"><month>12</month><year>2019</year></pub-date><pub-date pub-type=\"pmc-release\"><day>01</day><month>12</month><year>2020</year></pub-date><volume>1</volume><issue>1-2</issue><elocation-id>e201900005</elocation-id><permissions><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"https://onlinelibrary.wiley.com/doi/full/10.1002/tbio.201900005\"/><abstract id=\"ABS1\"><p id=\"P1\">Since the first demonstration of Doppler optical coherence tomography (OCT) in 1997, several functional extensions of Doppler OCT have been developed, including velocimetry, angiogram, and optical coherence elastography. These functional techniques have been widely used in research and clinical applications, particularly in ophthalmology. Here, we review the principles, representative methods, and applications of different Doppler OCT techniques, followed by discussion on the innovations, limitations, and future directions of each of these techniques.</p></abstract><abstract id=\"ABS2\" abstract-type=\"graphical\"><title>Graphical Abstract</title><p id=\"P2\"><graphic xlink:href=\"nihms-1593817-f0001.jpg\" position=\"anchor\" orientation=\"portrait\"/></p></abstract><kwd-group><kwd>angiography</kwd><kwd>Doppler</kwd><kwd>ophthalmology</kwd><kwd>optical coherence elastography</kwd><kwd>optical coherence tomography</kwd><kwd>phase-resolved</kwd></kwd-group></article-meta></front><body><sec id=\"S1\"><label>1 |</label><title>INTRODUCTION</title><p id=\"P3\">Optical coherence tomography (OCT) is an imaging technique that utilizes low-coherence light to capture structural images of biological tissue with high resolution in the micrometer scale [<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]. OCT has brought great impact to diagnosis and management of diseases in many fields of medicine by enabling the visualization, hence the quantification, of morphological changes caused by the disease. However, the structural changes of the tissue are often minuscule in the earlier stages of diseases, and it is challenging to diagnose these diseases at an early stage from a morphological change with OCT imaging. Physiological changes, such as microvascular distribution and blood flow velocity, could provide complementary information in addition to OCT that may improve the diagnostic yield in early detection.</p><p id=\"P4\">Based on the Doppler principle, Doppler OCT is a functional imaging technique that allows for quantifying the speed of moving particles with high spatial resolution and sensitivity in addition to structural imaging [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>–<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]. Doppler OCT was first demonstrated in 1997 [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>, <xref rid=\"R5\" ref-type=\"bibr\">5</xref>, <xref rid=\"R11\" ref-type=\"bibr\">11</xref>] in which a spectrogram method was applied to obtain Doppler frequency shift based on time-domain OCT. However, the long acquisition time as well as the conflict between spatial resolution and velocity sensitivity limited its application [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>, <xref rid=\"R13\" ref-type=\"bibr\">13</xref>]. The development of Fourier-domain OCT significantly increased the imaging speed of OCT [<xref rid=\"R14\" ref-type=\"bibr\">14</xref>, <xref rid=\"R15\" ref-type=\"bibr\">15</xref>]. In 2000, a phase-resolved method was proposed and demonstrated to image and quantify blood flow in which the Doppler shift could be calculated by observing the phase change between sequential A-lines in a B-scan or C-scan [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]. With the phase-resolved method, a high imaging speed, high spatial resolution and high-velocity sensitivity can be achieved. Chen et al. demonstrated the first in vivo imaging of vasculature and blood flow in patients using phase-resolved Doppler OCT [<xref rid=\"R16\" ref-type=\"bibr\">16</xref>, <xref rid=\"R17\" ref-type=\"bibr\">17</xref>]. However, this method is sensitive to the orientation and pulsatile nature of blood flow, and the results are the most optimal when the flow direction is aligned with the probe beam. The phase-resolved Doppler variance method was developed in 2000 to address this issue, which allows quantification of transverse flow [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>, <xref rid=\"R19\" ref-type=\"bibr\">19</xref>]. In addition, Doppler variance methods also enable visualizing small vessels down to the capillary level, making it ideal for OCT angiography (OCTA) applications [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>, <xref rid=\"R20\" ref-type=\"bibr\">20</xref>]. Based on the Doppler variance method, a number of extensions have been developed, including intensity-based Doppler variance, amplitude decorrelation, speckle variance, standard deviation (SD), intensity-based differentiation, phase variance and intensity and phase-based differentiation [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>–<xref rid=\"R8\" ref-type=\"bibr\">8</xref>, <xref rid=\"R10\" ref-type=\"bibr\">10</xref>, <xref rid=\"R20\" ref-type=\"bibr\">20</xref>–<xref rid=\"R34\" ref-type=\"bibr\">34</xref>].</p><p id=\"P5\">OCTA has the capability to visualize the microvasculature with high resolution (1–15 μm) and a moderate penetration depth (1–2 mm). It has become an attractive tool for angiography in ophthalmology, cancer and cerebral research due to advantages over conventional imaging methods: fast acquisition time, high spatial resolution, depth-resolved information, absolute flow measurement and non-invasiveness. The qualitative comparison with the current angiography modalities is summarized in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. In addition to angiography and flowmetry, Doppler OCT has also been extended to elastography application: namely, optical coherence elastography (OCE). Benefiting from the optical resolution enabled by phase-resolved OCT, OCE provides high spatial resolution at the micrometer-level and an axial displacement sensitivity on the order of subnanometer [<xref rid=\"R35\" ref-type=\"bibr\">35</xref>–<xref rid=\"R40\" ref-type=\"bibr\">40</xref>]. As such, it has been widely applied in biomedical research to provide quantitative assessment of tissue biomechanical properties [<xref rid=\"R41\" ref-type=\"bibr\">41</xref>–<xref rid=\"R51\" ref-type=\"bibr\">51</xref>].</p><p id=\"P6\">Doppler OCT and OCTA provide a noninvasive means for studying flowmetry, angiography and elastography with high spatial resolution and sensitivity and have been utilized in the fields of neurology, ophthalmology, cardiology and dermatology [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>, <xref rid=\"R16\" ref-type=\"bibr\">16</xref>, <xref rid=\"R18\" ref-type=\"bibr\">18</xref>, <xref rid=\"R40\" ref-type=\"bibr\">40</xref>–<xref rid=\"R42\" ref-type=\"bibr\">42</xref>, <xref rid=\"R44\" ref-type=\"bibr\">44</xref>, <xref rid=\"R46\" ref-type=\"bibr\">46</xref>–<xref rid=\"R50\" ref-type=\"bibr\">50</xref>, <xref rid=\"R59\" ref-type=\"bibr\">59</xref>–<xref rid=\"R61\" ref-type=\"bibr\">61</xref>]. In this review, we describe the methods, key advances, limitations, clinical applications and future directions of Doppler OCT.</p></sec><sec id=\"S2\"><label>2 |</label><title>PRINCIPLE OF DOPPLER OCT AND ANGIOGRAPHY</title><sec id=\"S3\"><label>2.1 |</label><title>Spectrogram</title><p id=\"P7\">Doppler OCT was first proposed for blood flow quantification in 1997 [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>, <xref rid=\"R5\" ref-type=\"bibr\">5</xref>]. Based on the Doppler principle, the frequency shift of the backscattered light from a moving particle can be generated, as shown in <xref rid=\"F1\" ref-type=\"fig\">Figure 1</xref>, and calculated using <xref rid=\"FD1\" ref-type=\"disp-formula\">Equation (1)</xref> [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>, <xref rid=\"R6\" ref-type=\"bibr\">6</xref>]:\n<disp-formula id=\"FD1\"><label>(1)</label><mml:math display=\"block\" id=\"M10\"><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>f</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mn>2</mml:mn><mml:mi>π</mml:mi></mml:mrow></mml:mfrac><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mstyle mathvariant=\"bold-italic\"><mml:mi>k</mml:mi></mml:mstyle><mml:mtext mathvariant=\"bold-italic\">s</mml:mtext></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mstyle mathvariant=\"bold-italic\"><mml:mi>k</mml:mi></mml:mstyle><mml:mstyle mathvariant=\"bold-italic\"><mml:mi>i</mml:mi></mml:mstyle></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>⋅</mml:mo><mml:mstyle mathvariant=\"bold-italic\"><mml:mi>V</mml:mi></mml:mstyle></mml:mrow></mml:math></disp-formula>\nwhere <bold><italic>k</italic></bold><sub><bold><italic>i</italic></bold></sub> and <bold><italic>k</italic></bold><sub><bold><italic>s</italic></bold></sub> are wave vectors of incoming and scattered light, respectively, and <bold><italic>V</italic></bold> is the velocity vector of the moving particles. Given the Doppler angle, <italic>θ</italic> (between the incident light beam and the flow direction), <xref rid=\"FD1\" ref-type=\"disp-formula\">Equation (1)</xref> is simplified to:\n<disp-formula id=\"FD2\"><label>(2)</label><mml:math display=\"block\" id=\"M11\"><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>f</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mn>2</mml:mn><mml:mo>×</mml:mo><mml:mi>n</mml:mi><mml:mo>×</mml:mo><mml:mi>V</mml:mi><mml:mo>×</mml:mo><mml:mtext>cos</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>θ</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mi>λ</mml:mi></mml:mfrac></mml:mrow></mml:math></disp-formula>\nwhere <italic>n</italic> is the tissue refractive index, and <italic>λ</italic> is the central wavelength of the light. In order to extract Doppler frequency shift, <italic>Δf</italic>, which is the difference between the carrier frequency from optical phase modulation, <italic>f</italic><sub>0</sub>, and the centroid, <italic>f</italic><sub><italic>c</italic></sub>, of the measured power spectrum, a short-time fast Fourier transformation (STFFT) or wavelet transformation can be applied to calculate the power spectrum of OCT signals [<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]. A detailed signal processing is shown in <xref rid=\"F2\" ref-type=\"fig\">Figure 2</xref>. With the spectrogram method, structural and velocity images can be obtained simultaneously, but the velocity sensitivity will be compromised by the increased spatial resolution or imaging speed.</p></sec><sec id=\"S4\"><label>2.2 |</label><title>Phase-resolved Doppler OCT</title><p id=\"P8\">To overcome the limitation of the spectrogram method, the phase-resolved Doppler OCT method was proposed in 2000 [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]. The Doppler frequency shift can be extracted by calculating the phase change in sequential A-lines using inter-A-lines or inter-frames. Deriving <italic>Δf</italic> through phase change can be achieved through <xref rid=\"FD3\" ref-type=\"disp-formula\">Equation (3)</xref>:\n<disp-formula id=\"FD3\"><label>(3)</label><mml:math display=\"block\" id=\"M12\"><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>f</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>φ</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn><mml:mo>×</mml:mo><mml:mi>π</mml:mi><mml:mo>×</mml:mo><mml:mi>Δ</mml:mi><mml:mi>T</mml:mi></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula>\nwhere <italic>ΔT</italic> is the time interval between sequential A-lines, and Δφ is the phase change. Δφ can be calculated using OCT complex data (<italic>F</italic><sub><italic>m</italic></sub> and <italic>F</italic><sub><italic>m</italic> + 1</sub>), as shown in <xref rid=\"FD4\" ref-type=\"disp-formula\">Equation (4)</xref>.\n<disp-formula id=\"FD4\"><label>(4)</label><mml:math display=\"block\" id=\"M13\"><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>φ</mml:mi><mml:mo>=</mml:mo><mml:msup><mml:mrow><mml:mtext>tan</mml:mtext></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mi>I</mml:mi><mml:mi>m</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>×</mml:mo><mml:msubsup><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mo>*</mml:mo></mml:msubsup></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mtext mathvariant=\"italic\">Re</mml:mtext><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>×</mml:mo><mml:msubsup><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mo>*</mml:mo></mml:msubsup></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mfrac></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow></mml:math></disp-formula>\nwhere <italic>F</italic><sub><italic>m</italic></sub> and <italic>F</italic><sub><italic>m</italic> + 1</sub> are the OCT complex data from same location but at a different time. Therefore, the longitudinal flow velocity can be determined by measuring the phase of the OCT signals as a function of time, as demonstrated by combining <xref rid=\"FD2\" ref-type=\"disp-formula\">Equations (2)</xref>, (<xref rid=\"FD3\" ref-type=\"disp-formula\">3</xref>) and (<xref rid=\"FD4\" ref-type=\"disp-formula\">4</xref>):\n<disp-formula id=\"FD5\"><label>(5)</label><mml:math display=\"block\" id=\"M14\"><mml:mrow><mml:mrow><mml:mi>V</mml:mi><mml:mo>×</mml:mo><mml:mtext>cos</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>θ</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>λ</mml:mi><mml:mo>×</mml:mo><mml:mi>Δ</mml:mi><mml:mi>φ</mml:mi></mml:mrow><mml:mrow><mml:mn>4</mml:mn><mml:mo>×</mml:mo><mml:mi>π</mml:mi><mml:mo>×</mml:mo><mml:mi>n</mml:mi><mml:mo>×</mml:mo><mml:mi>Δ</mml:mi><mml:mi>T</mml:mi></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:mfrac><mml:mi>λ</mml:mi><mml:mrow><mml:mn>4</mml:mn><mml:mo>×</mml:mo><mml:mi>π</mml:mi><mml:mo>×</mml:mo><mml:mi>n</mml:mi><mml:mo>×</mml:mo><mml:mi>Δ</mml:mi><mml:mi>T</mml:mi></mml:mrow></mml:mfrac></mml:mrow><mml:mspace linebreak=\"newline\"/><mml:mrow><mml:mo>×</mml:mo><mml:msup><mml:mrow><mml:mtext>tan</mml:mtext></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mi>I</mml:mi><mml:mi>m</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>×</mml:mo><mml:msubsup><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mo>*</mml:mo></mml:msubsup></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mtext mathvariant=\"italic\">Re</mml:mtext><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>×</mml:mo><mml:msubsup><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mo>*</mml:mo></mml:msubsup></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mfrac></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow></mml:mrow></mml:math></disp-formula></p><p id=\"P101\">The signal processing of phase-resolved Doppler is shown in <xref rid=\"F3\" ref-type=\"fig\">Figure 3</xref> [<xref rid=\"R62\" ref-type=\"bibr\">62</xref>, <xref rid=\"R63\" ref-type=\"bibr\">63</xref>]. With the phase-resolved Doppler OCT method, high-velocity sensitivity, high spatial resolution and high imaging speed can be achieved simultaneously, enabling real-time visualization and quantification of blood flow. Since the phase-resolved Doppler OCT method is sensitive to the orientation and pulsatile nature of blood flow, determining the Doppler angle plays an important role in accurate quantification of blood flow. Furthermore, this method cannot be applied when the Doppler angle is near 90°, which limits its application, such as for ocular blood flow imaging.</p></sec><sec id=\"S5\"><label>2.3 |</label><title>Doppler variance OCT</title><p id=\"P9\">To address the limitations of phase-resolved Doppler OCT to image transverse flow, Doppler variance method based on the bandwidth of Doppler frequency shift was proposed [<xref rid=\"R18\" ref-type=\"bibr\">18</xref>, <xref rid=\"R19\" ref-type=\"bibr\">19</xref>]. OCT incident probe-beam geometry causes a broadening of the Doppler frequency shift spectrum which can be used to quantify blood flow when the flow direction is near perpendicular to the probe beam. The principle is shown in <xref rid=\"F4\" ref-type=\"fig\">Figure 4</xref>, where Doppler bandwidth, B, is approximated by the differences between the Doppler shift generated by the red and blue beam.</p><p id=\"P10\">Therefore, the transverse flow velocity, <italic>V</italic><sub><italic>T</italic></sub> = <italic>V</italic>sin<italic>θ</italic>, can be quantified by <xref rid=\"FD6\" ref-type=\"disp-formula\">Equation (6)</xref> [<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]:\n<disp-formula id=\"FD6\"><label>(6)</label><mml:math display=\"block\" id=\"M15\"><mml:mrow><mml:mi>V</mml:mi><mml:mo>×</mml:mo><mml:mtext>sin</mml:mtext><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>θ</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mn>8</mml:mn><mml:mo>×</mml:mo><mml:mi>λ</mml:mi><mml:mo>×</mml:mo><mml:mi>σ</mml:mi></mml:mrow><mml:mrow><mml:mi>π</mml:mi><mml:mo>×</mml:mo><mml:mi>n</mml:mi><mml:mo>×</mml:mo><mml:msub><mml:mrow><mml:mtext>NA</mml:mtext></mml:mrow><mml:mrow><mml:mi>e</mml:mi><mml:mi>f</mml:mi><mml:mi>f</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula>\nwhere NA<sub><italic>eff</italic></sub> is the effective numerical aperture of the scan lens. The SD, <italic>σ</italic>, of the Doppler bandwidth can be determined by:\n<disp-formula id=\"FD7\"><label>(7)</label><mml:math display=\"block\" id=\"M16\"><mml:mrow><mml:msup><mml:mi>σ</mml:mi><mml:mn>2</mml:mn></mml:msup><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mo>∫</mml:mo><mml:mi>g</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>f</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>f</mml:mi><mml:mo>−</mml:mo><mml:mover accent=\"true\"><mml:mi>f</mml:mi><mml:mo>¯</mml:mo></mml:mover><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mi>d</mml:mi><mml:mi>f</mml:mi></mml:mrow><mml:mrow><mml:mo>∫</mml:mo><mml:mi>g</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>f</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:msup><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msup><mml:mi>d</mml:mi><mml:mi>f</mml:mi></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>2</mml:mn><mml:mi>π</mml:mi><mml:mi>Δ</mml:mi><mml:mi>T</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mfrac><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mfrac><mml:mrow><mml:mn>2</mml:mn><mml:mo>×</mml:mo><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>×</mml:mo><mml:msubsup><mml:mi>F</mml:mi><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mo>*</mml:mo></mml:msubsup></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:msup><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mo>+</mml:mo><mml:msup><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mfrac></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:math></disp-formula>\nwhere <italic>f</italic> is the Doppler shift, <inline-formula><mml:math display=\"inline\" id=\"M17\"><mml:mover accent=\"true\"><mml:mi>f</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:math></inline-formula> is averaged Doppler shift, and <italic>g</italic>(<italic>f</italic>) is the Doppler power spectrum. <xref rid=\"F5\" ref-type=\"fig\">Figure 5</xref> shows a representative angiogram from a rat cerebral cortex [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>].</p><p id=\"P11\">While <xref rid=\"FD5\" ref-type=\"disp-formula\">Equations (5)</xref>, (<xref rid=\"FD6\" ref-type=\"disp-formula\">6</xref>), and (<xref rid=\"FD7\" ref-type=\"disp-formula\">7</xref>) provide the back-bone for high-sensitivity flow measurement, the velocity range is limited due to phase wrapping and phase washout, which are the main challenges of Doppler OCT in flow velocity quantification. To address this, several phase calculation algorithms, such as the fast phase unwrapping method proposed by Schofield et al. have been developed [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>, <xref rid=\"R65\" ref-type=\"bibr\">65</xref>, <xref rid=\"R66\" ref-type=\"bibr\">66</xref>].</p></sec><sec id=\"S6\"><label>2.4 |</label><title>Angiogram</title><p id=\"P12\">OCTA is an extension of Doppler OCT that reconstructs the microvasculature by detecting the micro motions in biological tissue. These motions induced by the moving blood cells and plasma can generate fluctuations in the amplitude and phase of the interference signal that correspond to the flow velocity. The first OCTA based on Doppler variance OCT was demonstrated in 2001 [<xref rid=\"R17\" ref-type=\"bibr\">17</xref>, <xref rid=\"R18\" ref-type=\"bibr\">18</xref>], and since then various OCTA algorithms based on the detection of fluctuations in the amplitude and/or phase have been developed for the visualization of blood vessels. OCTA can be categorized into: (a) amplitude, including intensity-based Doppler variance, amplitude decorrelation, speckle variance, SD and intensity-based differentiation; (b) phase, including phase variance; and (c) both amplitude and phase, including phase-resolved Doppler variance, and intensity and phase-based differentiation. These algorithms are summarized in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>.</p><p id=\"P13\">In most cases, these algorithms have similar performances, although each is designed to utilize a particular scanning protocol for a specific application in a subfield of medicine, whose requirements vastly differ. In ophthalmology, for instance, the phase variance method is a more favorable approach for achieving a higher contrast-to-noise ratio than the amplitude decorrelation and speckle variance approaches [<xref rid=\"R25\" ref-type=\"bibr\">25</xref>], whereas in mouse brain imaging, intensity-based Doppler variance is a more suitable technique for mapping vasculature than phase-resolved Doppler variance [<xref rid=\"R67\" ref-type=\"bibr\">67</xref>]. <xref rid=\"F6\" ref-type=\"fig\">Figure 6</xref> shows a representative retinal angiogram (scan area: ~7 × 7 mm<sup>2</sup>) using intensity-based Doppler variance [<xref rid=\"R68\" ref-type=\"bibr\">68</xref>]. Microvascular network from millimeter-vessel down to single capillary can be clearly visualized.</p></sec></sec><sec id=\"S7\"><label>3 |</label><title>IMAGING PROTOCOL</title><p id=\"P14\">OCTA acquires multiple images in sequence to reveal the portion with fluctuations. Since this principle involves temporal imaging, the imaging protocol which determines the time interval between successive fluctuations plays a key factor in the signal-to-noise ratio (SNR) and dynamic range of OCTA. The two conventional imaging protocols are inter-frames and inter-A-lines, as depicted in <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>. In the inter-frame imaging protocol, neighboring B-scans are compared to extract vascular information. This protocol has a longer time interval Δ<italic>T</italic> as it utilizes the slow scan of the scanning apparatus. While this provides high sensitivity for the blood vessel with slow flow, prolonged time intervals may cause more motion-induced artifacts and phase wrapping, as well as signal saturation for the blood vessel with fast flow. On the contrary, neighboring A-lines are correlated in the inter-A-line method by using the fast scan of the scan setup to achieve a shorter time interval, and this allows for accurate quantification of fast flow while sacrificing the sensitivity for capillaries. For both imaging protocols, the scanning step needs to be much smaller with respect to lateral resolution (ie, the beam size) in order to achieve accurate angiography.</p><p id=\"P15\">Several averaging methods have also been incorporated in imaging protocols to enhance the sensitivity of OCTA, with split spectrum and volume averaging most predominantly used, as shown in <xref rid=\"T4\" ref-type=\"table\">Table 4</xref>. The split spectrum method divides the interference spectrum into several narrow spectra using a Gaussian window to generate several OCT images by performing Fourier transform for each sub-spectrum [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]. These OCT images are post-processed using an angiography algorithm and then averaged to improve the SNR. This method is computationally inexpensive but sacrifices spatial resolution. The split spectrum method improves the image contrast and continuity of vessels [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]. Conversely, volumetric averaging maintains the image spatial resolution and, therefore, can greatly improve the image quality, but it reduces the imaging speed. Nonetheless, volumetric averaging is particularly advantageous in visualizing the outer capillary plexus [<xref rid=\"R25\" ref-type=\"bibr\">25</xref>].</p></sec><sec id=\"S8\"><label>4 |</label><title>APPLICATIONS</title><sec id=\"S9\"><label>4.1 |</label><title>Ophthalmology</title><p id=\"P16\">OCT in ophthalmology is currently most well-adapted for clinical application. To date, many OCT devices with angiography are commercially available, including ZEISS Angioplex, Optovue AngioVue, Topcon, etc. These devices aid in visualizing the vascular anatomy to allow for better understanding of the pathophysiology of eye disease. The density, morphology and flow velocity of the vasculature in the retina are highly associated with disease pathology and being able to provide quantitative measurements of these parameters can therefore provide information for early detection, disease progression monitoring and treatment management. As such, OCTA is widely used in clinical research for characterizing various eye diseases, including: (a) dry age-related macular degeneration (AMD) where choriocapillaris flow and density are associated with the disease progression [<xref rid=\"R69\" ref-type=\"bibr\">69</xref>]; (b) wet AMD which is characterized by the presence of choroidal neovascularization [<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]; (c) diabetic retinopathy which exhibits abnormalities in choriocapillaris and/or retinal microvascular network [<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]; (d) retinal artery/vein occlusion in which non-perfusion in the capillary can be visualized; (e) glaucoma, which can be identified by an attenuated dense peripapillary microvascular network in both the superficial disc vasculature and the deeper lamina cribosa; (f) anterior segment ischemia (ASI) where iris vessel filling function and qualitative vessel density values can be evaluated to determine whether a patient is at risk to develop ASI during strabismus surgery [<xref rid=\"R70\" ref-type=\"bibr\">70</xref>, <xref rid=\"R71\" ref-type=\"bibr\">71</xref>]; and (g) ocular surface disorders where conjunctival and intrascleral vasculatures can be imaged for quantitative analysis of vessel density, vessel length density, vessel diameter index and fractal dimension of superficial- and deep-layer flows [<xref rid=\"R72\" ref-type=\"bibr\">72</xref>]. Currently, techniques, such as Hessian filtering, adaptive thresholding, variable interscan time analysis, machine learning and other numerical methods, have been utilized to quantify density, morphology, and flow velocity of the vasculature of the eye globe as well as suspicious lesion segmentation [<xref rid=\"R73\" ref-type=\"bibr\">73</xref>–<xref rid=\"R77\" ref-type=\"bibr\">77</xref>].</p><p id=\"P17\"><xref rid=\"F7\" ref-type=\"fig\">Figure 7</xref> shows the representative OCTA images of the aforementioned diseases, where degradation of microvasculature can be clearly visualized. Furthermore, it has also been demonstrated that the retinal vascular density is significantly lower in Alzheimer’s patients than healthy subjects, verifying the potential of OCTA in studying Alzheimer’s disease (AD) progression through quantification of retinal vasculature change correlated to neurodegeneration [<xref rid=\"R78\" ref-type=\"bibr\">78</xref>].</p></sec><sec id=\"S10\"><label>4.2 |</label><title>Neurology</title><p id=\"P18\">The nervous system is a complex network which is supplied with oxygen and nutrients through the blood vessel system to maintain physiological functions. Visualizing microvasculature and quantifying blood flow velocity using OCTA play an important role in studying physiological functions of the neuron system, including occurrences and progression of brain diseases, drug administration and responses of brain to external stimuli. Due to the limited penetration depth of OCT, most current research focuses on small animal models to study the mechanism of brain injury, disease progression, and evaluation of treatment strategies. Chen et al. demonstrated the first Doppler OCT image of brain microvasculature in 1999 [<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]. Liu et al. demonstrated the microvasculature from a healthy rat cortex with thinned skull, as shown in <xref rid=\"F8\" ref-type=\"fig\">Figure 8A</xref> [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]. Jia et al. studied cerebrovascular blood perfusion in a cerebral stroke rodent model using OCTA to better understand stroke as well as to optimize current therapies via treatment monitoring [<xref rid=\"R60\" ref-type=\"bibr\">60</xref>], as shown in <xref rid=\"F8\" ref-type=\"fig\">Figure 8B</xref>. To study traumatic brain injury (TBI), Jia et al reconstructed three-dimensional images of cerebral vasculatures in a TBI mouse model, demonstrating the microvasculature change in pre- and post-TBI mice that allows for exploring the mechanism of TBI rehabilitation [<xref rid=\"R61\" ref-type=\"bibr\">61</xref>]. Lin et al imaged the mouse brain from a 20-month 3xTg-AD model mouse to investigate the relationship between amyloid-β and vascular pathophysiology in which 3xTg-AD mice exhibited a vessel volume fraction decrease of 29% compared to the control mouse [<xref rid=\"R80\" ref-type=\"bibr\">80</xref>], as shown in <xref rid=\"F8\" ref-type=\"fig\">Figures 8C</xref> and <xref rid=\"F8\" ref-type=\"fig\">D</xref>.</p></sec><sec id=\"S11\"><label>4.3 |</label><title>Cancer angiogenesis</title><p id=\"P19\">Tumor growth and metastasis rely on angiogenesis to provide a sufficient supply of oxygen and nutrients as well as to remove the waste [<xref rid=\"R81\" ref-type=\"bibr\">81</xref>]. Microvasculature visualization and blood flow quantification allow for early detection, characterization and treatment optimization. Vakoc et al utilized OCTA to demonstrate microvasculature of different kinds of tumors, including in the breast, brain, colorectum, and skin, as shown in <xref rid=\"F9\" ref-type=\"fig\">Figures 9A</xref>–<xref rid=\"F9\" ref-type=\"fig\">C</xref> [<xref rid=\"R82\" ref-type=\"bibr\">82</xref>]. The tumors can be identified by the increase in vascular density. In addition, Vakoc et al. investigated the vascular dynamics based on dorsal tumor of a rat during anti-angiogenic therapy to further verify the capability of OCTA in which a decreased mean vessel diameter as well as a continuous expanding volume of tumor were demonstrated. Alison et al. reported a study in the investigation of tumor vasculature change in malignant iris melanomas and benign iris lesions [<xref rid=\"R83\" ref-type=\"bibr\">83</xref>]. Osman et al. developed an ultrahigh-speed endoscopic OCTA system for delineation of dysplastic margins at the gastroesophageal junction and demonstrated a sensitivity of 94% and a specificity of 69% for identifying dysplasia based on 32 patients [<xref rid=\"R84\" ref-type=\"bibr\">84</xref>]. Lee et al. reported the use of endoscopic OCTA to differentiate low-grade and high-grade dysplasia in Barrett’s esophagus from 32 patients [<xref rid=\"R85\" ref-type=\"bibr\">85</xref>].</p></sec><sec id=\"S12\"><label>4.4 |</label><title>Cilia motion</title><p id=\"P20\">Ciliary activity, characterized by the synchronized beating of ciliary cells, generates the primary driving force for mucosa transportation. The dysfunction of ciliary motion could lead to a number of severe diseases, including respiratory disorders and infertility. Doppler OCT has the capability of providing a noninvasive and high-sensitivity imaging tool for evaluation of cilia motion. Jing et al. developed a high-speed Doppler OCT system to visualize temporal cilia beating for studying the influence of external factors on cilia beating frequency, including temperature and albuterol, as shown in <xref rid=\"F10\" ref-type=\"fig\">Figure 10A</xref> [<xref rid=\"R86\" ref-type=\"bibr\">86</xref>]. Recently, He et al reported phase-resolved Doppler spectrally encoded interferometric microscopy for real-time visualization of surface dynamics of the oviduct to characterize the ciliary beating frequency in the oviduct, as shown in <xref rid=\"F10\" ref-type=\"fig\">Figure 10B</xref> [<xref rid=\"R87\" ref-type=\"bibr\">87</xref>].</p></sec><sec id=\"S13\"><label>4.5 |</label><title>Optical coherence elastography</title><p id=\"P21\">In addition to angiography and flowmetry, Doppler OCT has also been extended to the application of elastography. OCE has the same resolution as OCT, and its superior displacement sensitivity and high imaging speed make phase-resolved OCE a prominent technique for elasticity measurements. In OCE, an external or internal force is applied to induce a localized displacement, which is then detected by OCT. With its high sensitivity, phase-resolved Doppler OCT measures the phase change which is converted to relative displacement using <xref rid=\"FD8\" ref-type=\"disp-formula\">Equation (8)</xref>:\n<disp-formula id=\"FD8\"><label>(8)</label><mml:math display=\"block\" id=\"M18\"><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>d</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mi>λ</mml:mi><mml:mrow><mml:mn>4</mml:mn><mml:mi>π</mml:mi><mml:mi>n</mml:mi></mml:mrow></mml:mfrac><mml:mi>Δ</mml:mi><mml:mi>φ</mml:mi></mml:mrow></mml:math></disp-formula>\nwhere <italic>n</italic> is the tissue refractive index, and <italic>λ</italic> is the central wavelength of the light. The absolute displacement can be obtained by integrating the relative displacement, as shown in <xref rid=\"FD9\" ref-type=\"disp-formula\">Equation (9)</xref>:\n<disp-formula id=\"FD9\"><label>(9)</label><mml:math display=\"block\" id=\"M19\"><mml:mrow><mml:mi>d</mml:mi><mml:mo>=</mml:mo><mml:mo>∫</mml:mo><mml:mi>Δ</mml:mi><mml:mi>d</mml:mi><mml:mi>d</mml:mi><mml:mi>t</mml:mi><mml:mo>=</mml:mo><mml:mo>∫</mml:mo><mml:mfrac><mml:mrow><mml:mi>λ</mml:mi><mml:mi>Δ</mml:mi><mml:mi>φ</mml:mi></mml:mrow><mml:mrow><mml:mn>4</mml:mn><mml:mi>π</mml:mi><mml:mi>n</mml:mi></mml:mrow></mml:mfrac><mml:mi>d</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:math></disp-formula></p><p id=\"P102\">In addition, the intensity and phase variance methods described in <xref rid=\"S2\" ref-type=\"sec\">Section 2</xref> can also be used to visualize the displacement change, but calibration is required, and the sensitivity is relatively low. Besides displacement measurement, resonance frequency and elastic wave propagation have been proposed to calculate the Young’s modulus [<xref rid=\"R39\" ref-type=\"bibr\">39</xref>, <xref rid=\"R42\" ref-type=\"bibr\">42</xref>, <xref rid=\"R88\" ref-type=\"bibr\">88</xref>, <xref rid=\"R89\" ref-type=\"bibr\">89</xref>]. OCE has been widely applied in research to provide quantitative assessment of tissue biomechanical properties [<xref rid=\"R37\" ref-type=\"bibr\">37</xref>, <xref rid=\"R39\" ref-type=\"bibr\">39</xref>, <xref rid=\"R43\" ref-type=\"bibr\">43</xref>–<xref rid=\"R47\" ref-type=\"bibr\">47</xref>, <xref rid=\"R49\" ref-type=\"bibr\">49</xref>, <xref rid=\"R88\" ref-type=\"bibr\">88</xref>–<xref rid=\"R94\" ref-type=\"bibr\">94</xref>]. One of the main applications is in ophthalmology. Stefano et al reported the first in vivo human corneal elasticity with 10 subjects, demonstrating a difference in the induced anterior and posterior stromal displacements. Wu et al reported the ex vivo elasticity measurement of lens and investigated the correlation between lens elasticity and intraocular pressure using OCE. Recently, Li et al developed a swept source-based OCE system that is able to simultaneously assess the elasticities of the crystalline lens and the cornea in vivo (as shown in <xref rid=\"F11\" ref-type=\"fig\">Figures 11A</xref>–<xref rid=\"F11\" ref-type=\"fig\">C</xref>) [<xref rid=\"R95\" ref-type=\"bibr\">95</xref>]. For elasticity measurement of posterior eye, Qu et al reported the first in vivo quantitative elasticity map of the retina by displacement measurement (ie, the compression approach) in which a difference was demonstrated between healthy and damaged rabbit retina in 2018 [<xref rid=\"R45\" ref-type=\"bibr\">45</xref>]. In addition, He et al presented a quantitative method of mapping the mechanical elasticity of the posterior eye based on shear wave method in 2019 in which the elasticity of different layers of retina were quantified, as shown in <xref rid=\"F11\" ref-type=\"fig\">Figures 11D</xref>, <xref rid=\"F11\" ref-type=\"fig\">E</xref>. In addition to ophthalmology, OCT has been applied in cardiology. In 2012, Qi et al demonstrated the first OCE for quantification of plaque by a microscopic system, as shown in <xref rid=\"F11\" ref-type=\"fig\">Figures 11F</xref>–<xref rid=\"F11\" ref-type=\"fig\">I</xref> [<xref rid=\"R40\" ref-type=\"bibr\">40</xref>]. Qu et al reported an intravascular endoscopic OCE system in which a miniature focused ring transducer was assembled into an imaging probe to provide ultrasound excitation to detect atherosclerosis plaques [<xref rid=\"R48\" ref-type=\"bibr\">48</xref>]. The performance of the system and probe were validated using cadaver tissue. Furthermore, OCE has also been applied in blood coagulation, breast cancer, skin pathology and airway compliance for elasticity quantification as well as mechanobiology research to study the mechanical responses of microparticles [<xref rid=\"R96\" ref-type=\"bibr\">96</xref>–<xref rid=\"R101\" ref-type=\"bibr\">101</xref>]. <xref rid=\"F11\" ref-type=\"fig\">Figure 11</xref> shows representative OCE images from different applications.</p></sec></sec><sec id=\"S14\"><label>5 |</label><title>LIMITATIONS AND FUTURE DIRECTIONS OF DOPPLER OCT AND OCTA</title><p id=\"P22\">Phase-resolved Doppler OCT requires measurement of angles between the OCT detection beam and the blood vessels to quantify the flow velocity. Although quantification of the angle over large numbers of vessels is computer-intensive, Qi et al have demonstrated a volumetric vessel reconstruction approach which enable calculation of Doppler angles to determine the absolute blood flow velocity over a large field-of-view [<xref rid=\"R102\" ref-type=\"bibr\">102</xref>]. Alternatively, these problems can be solved by employing angle-independent imaging methods, such as multiple OCT detection beams [<xref rid=\"R103\" ref-type=\"bibr\">103</xref>, <xref rid=\"R104\" ref-type=\"bibr\">104</xref>] or synthetic subaperture [<xref rid=\"R105\" ref-type=\"bibr\">105</xref>] in which several Doppler angles are utilized to extract velocity components to calculate the absolute velocity. In angiography applications, such as the algorithms summarized in <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, the absolute velocity can be determined through pre-calibration (ie, experimentally defining the correlation between SD vs flow velocity); nonetheless, this can only be used for flow velocity within the dynamic range as faster flow can cause signal saturation [<xref rid=\"R30\" ref-type=\"bibr\">30</xref>].</p><p id=\"P23\">The dynamic range of Doppler OCT is confined by phase wrapping, as the phase shift is mathematically restricted to [−π, +π], limiting the ability to detect higher flow speed that is outside of the dynamic range. This issue can be addressed by increasing the Doppler angle or scanning speed or varying the time interval. However, these may degrade the image quality as well as increase the acquisition time. Recently, an automated phase unwrapping algorithm [<xref rid=\"R106\" ref-type=\"bibr\">106</xref>] has been proposed in which the magnitude of the phase shift gradient is calculated to correct the wrapping. In addition, phase wrapping correction and discontinuity improvement have also been demonstrated using a two-dimensional unwrapping method [<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]. In 2018, Wei et al reported a novel scanning pattern for achieving high dynamic range in which the improved flow dynamic range can be achieved by generating three B-scans of different time intervals [<xref rid=\"R107\" ref-type=\"bibr\">107</xref>].</p><p id=\"P24\">As Doppler OCT ultimately relies on determining the temporal phase shift of the interference signal, the phase stability of the imaging system is a critical key factor in obtaining accurate measurements. Spectral domain OCT (SD-OCT) is commonly considered as the optimal method to achieve high phase stability because of the static operation principle utilized by its spectrometer. While SD-OCT can provide high-precision phase measurements, it has the inherent disadvantage of phase washouts [<xref rid=\"R108\" ref-type=\"bibr\">108</xref>]. On the contrary, swept source OCT (SS-OCT) for phase measurements are more widely used as it can provide a higher imaging speed than SD-OCT. Although the operation of SS-OCT has less phase stability, several techniques have been reported to resolve this issue, including optimizing synchronization through the use of a lambda (wavelength) trigger and/or signal timing delay [<xref rid=\"R109\" ref-type=\"bibr\">109</xref>], and utilizing a common path setup [<xref rid=\"R110\" ref-type=\"bibr\">110</xref>].</p><p id=\"P25\">In order to perform the Doppler algorithm, multiple temporal data of either an A-line or a B-frame of the same location is required. In OCT, this is typically achieved via a scan apparatus, whose scan speed depends on the light source of SS-OCT and camera speed of SD-OCT. Because the blood flow is relatively slower than the physiological bulk motion, acquiring the temporal data often also captures motion-based artifacts. These artifacts can be corrected by using histogram-based methods to extract and remove the phase change induced by bulk motions [<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]. A scanning protocol has been proposed to remove the bulk motion from periodic physiological bulk motion in which a stitch scan protocol in the slow scan direction is applied to stagger bulk motion [<xref rid=\"R111\" ref-type=\"bibr\">111</xref>]. Volumetric averaging can also be applied to remove the bulk-motion artifacts, but it greatly increases the imaging time as several volumetric datasets are required. Furthermore, a motion-tracking sensor also can be a possible solution to remove the bulk motion [<xref rid=\"R112\" ref-type=\"bibr\">112</xref>].</p><p id=\"P26\">In Doppler-based OCTA, vascular permeability or leakage cannot be easily visualized due to the lack of image contrast as the amplitude/phase fluctuations of the interference signal are minimal in blood vessels with quasi-static blood flow. Recently, Winkelmann et al have reported a spectral contrast technique for OCTA in which spectral signatures of blood in the visible range are applied to achieve angiograms without the need of blood movement or temporal information [<xref rid=\"R113\" ref-type=\"bibr\">113</xref>]. In addition, the motion artifacts can be eliminated as only a single scan is required, but this method cannot quantify flow rates and also has limited penetration depth.</p><p id=\"P27\">Additionally, reconstruction of smaller vessels and capillaries is often challenging in OCTA as the induced signal fluctuations are more marginal. Several processing algorithms have been used to increase the SNR for imaging capillaries, chiefly by improving the imaging contrast or solving for blood vessel discontinuity [<xref rid=\"R27\" ref-type=\"bibr\">27</xref>, <xref rid=\"R114\" ref-type=\"bibr\">114</xref>]. Hessian-Frangi filter is one of the most commonly used techniques in OCTA for improving the visualization of discontinuous vasculature [<xref rid=\"R115\" ref-type=\"bibr\">115</xref>]. Tan et al demonstrated a modified Bayesian residual transform-based processing algorithm to reduce speckle noise and motion-related artifacts [<xref rid=\"R114\" ref-type=\"bibr\">114</xref>]. Recently, Lee et al incorporated artificial intelligence into OCTA and demonstrated increased detail of the superficial retinal vasculature [<xref rid=\"R77\" ref-type=\"bibr\">77</xref>].</p><p id=\"P28\">Since OCT is an optical imaging technique that mostly relies on light in the near-infrared spectrum, it has a shallow penetration depth (1–2 mm), which limits the utility of OCTA to only the superficial vasculature. Recently, Li et al constructed an SS-OCT system for intravascular imaging using a broadband laser with a center wavelength of 1.7 μm, demonstrating an extended penetration depth compared to conventional OCT systems utilizing shorter wavelength light sources [<xref rid=\"R116\" ref-type=\"bibr\">116</xref>]. Dual-axis OCT has also been proposed to improve penetration depth by Zhao et al [<xref rid=\"R117\" ref-type=\"bibr\">117</xref>]. In addition to hardware improvements, algorithms have been incorporated to extend the imaging depth. For instance, the scattering reflection matrix approach has been proposed to address the issue caused by multiple scattering although real-time imaging remains a challenge due to the required long acquisition time [<xref rid=\"R118\" ref-type=\"bibr\">118</xref>]. Lastly, multimodal imaging systems that incorporate OCT with ultrasound and/or photoacoustic to provide complementary information have also been reported [<xref rid=\"R119\" ref-type=\"bibr\">119</xref>, <xref rid=\"R120\" ref-type=\"bibr\">120</xref>].</p><p id=\"P29\">Several of the aforementioned limitations, including dynamic range, signal saturation and motion artifacts, can be improved by increasing imaging speed of the OCT system; overcoming these limitations will further facilitate the clinical translation of Doppler OCT techniques. Currently, a Fourier-domain mode-locking (FDML) laser with an A-line scan rate in the MHz range has been commercially available, enabling high-speed volumetric scanning for OCTA. An imaging speed of up to 4.7 volumes/s has been demonstrated using FDML, along with improved image contrast [<xref rid=\"R121\" ref-type=\"bibr\">121</xref>, <xref rid=\"R122\" ref-type=\"bibr\">122</xref>]. Lee et al. have also developed full-field OCT that applies parallel illumination to achieve high-speed <italic>en face</italic> imaging [<xref rid=\"R123\" ref-type=\"bibr\">123</xref>]. Data acquisition and processing speed can be further improved through both hardware and software optimizations, including using high-bandwidth digitizers and utilizing a parallel processing scheme.</p><p id=\"P30\">Currently, quantitative data analysis in both OCT and OCTA are computationally intensive and less efficient. The accuracy may suffer because of the large volume of data generated by high speed imaging systems. Machine learning has been used in segmentation of OCT structure as well as OCTA. The applications of artificial intelligence in OCT and OCTA, although still at an early stage, have great potential to increase the accuracy and efficiency of quantitative analysis [<xref rid=\"R73\" ref-type=\"bibr\">73</xref>, <xref rid=\"R77\" ref-type=\"bibr\">77</xref>, <xref rid=\"R124\" ref-type=\"bibr\">124</xref>].</p></sec><sec id=\"S15\"><label>6 |</label><title>SUMMARY</title><p id=\"P31\">Doppler OCT as a foundational basis of functional imaging provides noninvasive techniques for quantitative and dynamic evaluation of numerous tissue physiology and pathophysiology in vivo. In angiography and blood flowmetry, different combinations of the processing algorithm, averaging method, and scanning protocol are designed for specific applications, enabling detection and characterization of a broad spectrum of diseases. The best experimental results are often obtained by identifying the optimal balance between the acquisition time, imaging depth and field of view and system cost. In summary, functional extensions of OCT based on the Doppler principle reveal additional tissue characteristics that are not available through conventional OCT, and the reported literature as well as the current state of research have demonstrated Doppler OCT and OCTA as a promising clinical tool in vasculature visualization, flow velocity quantification, and elasticity measurement. Although Doppler OCT and OCTA have been widely applied in ophthalmology, a large number of clinical applications of this technology remains to be explored.</p></sec></body><back><ack id=\"S16\"><title>ACKNOWLEDGMENTS</title><p id=\"P32\">This work is supported by the National Institutes of Health (R01HL-125084, R01HL-127271, R01EY-026091, R01EY-028662), American Heart Association (18PRE34050021), and the National Science Foundation (DGE-1839285).</p><p id=\"P33\">FUNDING information</p><p id=\"P34\">American Heart Association, Grant/Award Number: 18PRE34050021; National Institutes of Health, Grant/Award Numbers: R01HL-125084, R01HL-127271, R01EY-026091, R01EY-028662; National Science Foundation, Grant/Award Number: DGE-1839285</p></ack><fn-group><fn fn-type=\"COI-statement\" id=\"FN1\"><p id=\"P35\">CONFLICT OF INTEREST</p><p id=\"P36\">Dr. Chen has a financial interest in OCT Medical Imaging, Inc., which, however, did not support this 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Since OCT detects only back-scattered light, <italic>θ</italic> is identical for both the incident and back-scattered light</p></caption><graphic xlink:href=\"nihms-1593817-f0006\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>FIGURE 2</label><caption><p id=\"P38\">Signal processing for structural and velocity images</p></caption><graphic xlink:href=\"nihms-1593817-f0007\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>FIGURE 3</label><caption><p id=\"P39\">Phase-resolved Doppler optical coherence tomography method</p></caption><graphic xlink:href=\"nihms-1593817-f0008\"/></fig><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>FIGURE 4</label><caption><p id=\"P40\">Phase-resolved variance Doppler optical coherence tomography</p></caption><graphic xlink:href=\"nihms-1593817-f0009\"/></fig><fig id=\"F5\" orientation=\"portrait\" position=\"float\"><label>FIGURE 5</label><caption><p id=\"P41\">Depth-encoded image of microvasculature of rat cerebral cortex. Adapted from Reference [<xref rid=\"R64\" ref-type=\"bibr\">64</xref>]</p></caption><graphic xlink:href=\"nihms-1593817-f0010\"/></fig><fig id=\"F6\" orientation=\"portrait\" position=\"float\"><label>FIGURE 6</label><caption><p id=\"P42\">Retinal angiogram using intensity-based Doppler variance. Adapted from Reference [<xref rid=\"R68\" ref-type=\"bibr\">68</xref>]</p></caption><graphic xlink:href=\"nihms-1593817-f0011\"/></fig><fig id=\"F7\" orientation=\"portrait\" position=\"float\"><label>FIGURE 7</label><caption><p id=\"P43\">(A) Dry AMD: decreased signal in the choriocapillaris corresponding to flow impairment, indicated by the white arrows. (B) Wet AMD: presence of abnormal blood vessels, indicated by the white dashed circle. (C) Diabetic retinopathy: enlarged foveal avascular zone and aneurysms, in which microaneurysms with non-proliferative diabetic retinopathy are labeled by red dashed circles. (D) Branch retinal artery occlusion: decreased capillary perfusion, indicated by the yellow arrow. (E) Chronic branch retinal vein occlusion: capillary non-perfusion, indicated by the white arrows. (F) Glaucoma: vascular impairments, indicated by the red arrows. (G) Iris vascular network. (H) Conjunctival and intrascleral vasculatures. (I) Retinal vascular from a patient with Alzheimer’s Disease, which has a lesser vascular density. Scale bars: 1 mm. Adapted from References [<xref rid=\"R24\" ref-type=\"bibr\">24</xref>, <xref rid=\"R26\" ref-type=\"bibr\">26</xref>, <xref rid=\"R72\" ref-type=\"bibr\">72</xref>, <xref rid=\"R78\" ref-type=\"bibr\">78</xref>, <xref rid=\"R79\" ref-type=\"bibr\">79</xref>]</p></caption><graphic xlink:href=\"nihms-1593817-f0012\"/></fig><fig id=\"F8\" orientation=\"portrait\" position=\"float\"><label>FIGURE 8</label><caption><p id=\"P44\">(A) The projection of vasculature of a rat cerebral cortex with thinned skull. The colormap is depth encoded. (B) The projection of vasculature from mouse brain in ischemic stroke. Left: baseline; middle: stroke; right: after 30 minutes. Blood perfusion restored partially, and occlusion still exists. The colormap is flow velocity encoded. (C) The vasculature projection of mouse brains from an AD model and healthy mouse. The colormap is depth encoded. (D)Images of vessel density differences between a control and an AD mouse, in which the AD mouse exhibited a vessel volume fraction decrease of 29% compared to the control mouse. Adapted from References [<xref rid=\"R8\" ref-type=\"bibr\">8</xref>, <xref rid=\"R60\" ref-type=\"bibr\">60</xref>, <xref rid=\"R80\" ref-type=\"bibr\">80</xref>]</p></caption><graphic xlink:href=\"nihms-1593817-f0013\"/></fig><fig id=\"F9\" orientation=\"portrait\" position=\"float\"><label>FIGURE 9</label><caption><p id=\"P45\">Vasculature of murine mammary carcinoma in breast (left), brain (middle), and dorsal skin (right) in which tissue microenvironments exhibit strikingly different vascular networks. Scale bar: 500 μm. The colormap is depth encoded. Adapted from Reference [<xref rid=\"R82\" ref-type=\"bibr\">82</xref>]</p></caption><graphic xlink:href=\"nihms-1593817-f0014\"/></fig><fig id=\"F10\" orientation=\"portrait\" position=\"float\"><label>FIGURE 10</label><caption><p id=\"P46\">(A) Doppler images of ciliary motion at temperatures of 25°C, 27°C, 29°C, 31°C, and 34°C, in which cilia beating frequency under different temperatures were observed. (B) Spatial distribution of the cilia beating frequency at 23°C, 26°C, 29°C, and 33°C in which temperature has a positive impact on ciliary activity. Adapted from References [<xref rid=\"R86\" ref-type=\"bibr\">86</xref>, <xref rid=\"R87\" ref-type=\"bibr\">87</xref>]</p></caption><graphic xlink:href=\"nihms-1593817-f0015\"/></fig><fig id=\"F11\" orientation=\"portrait\" position=\"float\"><label>FIGURE 11</label><caption><p id=\"P47\">(A) Cross-sectional raw data showing elastic wave propagation of retinal layers at different time points for an ex-vivo pig retina. (B) Elasticity map in rabbit retina in vivo. A different stiffness was demonstrated in different layers of the retina. (C) Doppler OCT image of rabbit cornea and crystalline lens. (D) and (E) Spatiotemporal Doppler OCT images of cornea and lens, respectively. (F) OCT structural and (G) Doppler OCT images of a human cadaver coronary artery. (H) Histological image and (d) close-up view of an atherosclerotic lesion. The red-colored region denoted by the blue arrow in (I) exhibits smaller phase and displacement and, therefore, indicates less elastic, stiffer tissue such as plaques. Scale bars: 1 mm. Adapted from References [<xref rid=\"R40\" ref-type=\"bibr\">40</xref>, <xref rid=\"R42\" ref-type=\"bibr\">42</xref>, <xref rid=\"R95\" ref-type=\"bibr\">95</xref>]</p></caption><graphic xlink:href=\"nihms-1593817-f0016\"/></fig><table-wrap id=\"T1\" position=\"float\" orientation=\"landscape\"><label>TABLE 1</label><caption><p id=\"P48\">Summary of current angiography methods</p></caption><table frame=\"below\" rules=\"none\"><colgroup span=\"1\"><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/></colgroup><thead><tr><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Performance method</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Lateral resolution</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Axial resolution</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Flow velocity sensitivity</th><th align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Invasiveness</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">ICG angiogram [<xref rid=\"R52\" ref-type=\"bibr\">52</xref>, <xref rid=\"R53\" ref-type=\"bibr\">53</xref>]</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">●●</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Laser Doppler flowmetry [<xref rid=\"R54\" ref-type=\"bibr\">54</xref>, <xref rid=\"R55\" ref-type=\"bibr\">55</xref>]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">●●</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">●</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">None</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Doppler ultrasound [<xref rid=\"R56\" ref-type=\"bibr\">56</xref>]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">●</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">●</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">●●</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">None</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Laser speckle [<xref rid=\"R57\" ref-type=\"bibr\">57</xref>, <xref rid=\"R58\" ref-type=\"bibr\">58</xref>]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">●●</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">None</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">●</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">None</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Doppler OCT [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>–<xref rid=\"R8\" ref-type=\"bibr\">8</xref>, <xref rid=\"R10\" ref-type=\"bibr\">10</xref>, <xref rid=\"R20\" ref-type=\"bibr\">20</xref>–<xref rid=\"R33\" ref-type=\"bibr\">33</xref>].</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">●●</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">●●</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">●●</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">None</td></tr></tbody></table><table-wrap-foot><fn id=\"TFN1\"><p id=\"P49\"><italic>Note</italic>: ●●●, excellent; ●●, good; ●, moderate.</p></fn></table-wrap-foot></table-wrap><table-wrap id=\"T2\" position=\"float\" orientation=\"landscape\"><label>TABLE 2</label><caption><p id=\"P50\">Summary of current algorithms of optical coherence tomography angiography</p></caption><table frame=\"below\" rules=\"none\"><colgroup span=\"1\"><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/></colgroup><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Method</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Algorithm</th><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Doppler variance</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Intensity-based [<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-formula><mml:math display=\"inline\" id=\"M1\"><mml:mrow><mml:msup><mml:mi>σ</mml:mi><mml:mn>2</mml:mn></mml:msup><mml:mo>=</mml:mo><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mfrac><mml:mrow><mml:mn>2</mml:mn><mml:mo>×</mml:mo><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow><mml:mo>×</mml:mo><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:msup><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mo>+</mml:mo><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:msup><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mfrac></mml:mrow></mml:math></inline-formula></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Phase-resolved [<xref rid=\"R6\" ref-type=\"bibr\">6</xref>, <xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-formula><mml:math display=\"inline\" id=\"M2\"><mml:mrow><mml:msup><mml:mi>σ</mml:mi><mml:mn>2</mml:mn></mml:msup><mml:mo>=</mml:mo><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mfrac><mml:mrow><mml:mn>2</mml:mn><mml:mo>×</mml:mo><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>×</mml:mo><mml:msubsup><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mo>*</mml:mo></mml:msubsup></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:msup><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mo>+</mml:mo><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:msup><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mfrac></mml:mrow></mml:math></inline-formula></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Amplitude decorrelation [<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-formula><mml:math display=\"inline\" id=\"M3\"><mml:mrow><mml:msup><mml:mi>σ</mml:mi><mml:mn>2</mml:mn></mml:msup><mml:mo>=</mml:mo><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:mfrac><mml:mrow><mml:mn>2</mml:mn><mml:mo>×</mml:mo><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow><mml:mo>×</mml:mo><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:msup><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mo>+</mml:mo><mml:msup><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mfrac></mml:mrow></mml:math></inline-formula></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Speckle variance [<xref rid=\"R29\" ref-type=\"bibr\">29</xref>]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-formula><mml:math display=\"inline\" id=\"M4\"><mml:mrow><mml:msup><mml:mi>σ</mml:mi><mml:mn>2</mml:mn></mml:msup><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mi>M</mml:mi></mml:mfrac><mml:mo>×</mml:mo><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:msup><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow><mml:mo>−</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mi>M</mml:mi></mml:mfrac><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>M</mml:mi></mml:msubsup><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:math></inline-formula></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">SD [<xref rid=\"R30\" ref-type=\"bibr\">30</xref>]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-formula><mml:math display=\"inline\" id=\"M5\"><mml:mrow><mml:mi>R</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msqrt><mml:mrow><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:msup><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow><mml:mo>−</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mi>M</mml:mi></mml:mfrac><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>M</mml:mi></mml:msubsup><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:msqrt></mml:mrow><mml:mrow><mml:mfrac><mml:mn>1</mml:mn><mml:mi>M</mml:mi></mml:mfrac><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow></mml:mfrac></mml:mrow></mml:math></inline-formula></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Differentiation</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Intensity-based [<xref rid=\"R22\" ref-type=\"bibr\">22</xref>]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-formula><mml:math display=\"inline\" id=\"M6\"><mml:mrow><mml:mi>I</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:mo>‖</mml:mo><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mrow><mml:mo>|</mml:mo><mml:mo>−</mml:mo><mml:mo>|</mml:mo></mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>‖</mml:mo></mml:mrow></mml:math></inline-formula></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Intensity and phase-based [<xref rid=\"R31\" ref-type=\"bibr\">31</xref>]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-formula><mml:math display=\"inline\" id=\"M7\"><mml:mrow><mml:mi>I</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:mrow><mml:mo>|</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>F</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>|</mml:mo></mml:mrow></mml:mrow></mml:math></inline-formula></td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Phase variance [<xref rid=\"R32\" ref-type=\"bibr\">32</xref>, <xref rid=\"R33\" ref-type=\"bibr\">33</xref>]</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-formula><mml:math display=\"inline\" id=\"M8\"><mml:mrow><mml:mi>P</mml:mi><mml:msub><mml:mi>V</mml:mi><mml:mi>v</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac><mml:msubsup><mml:mstyle><mml:mo>∑</mml:mo></mml:mstyle><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:msup><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>Δ</mml:mi><mml:msub><mml:mi>φ</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mi>M</mml:mi><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac><mml:mstyle stretchy=\"false\"><mml:munderover><mml:mo>∑</mml:mo><mml:mrow><mml:mi>m</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>M</mml:mi></mml:munderover></mml:mstyle><mml:mi>Δ</mml:mi><mml:msub><mml:mi>φ</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:math></inline-formula></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><inline-formula><mml:math display=\"inline\" id=\"M9\"><mml:mrow><mml:mi>Δ</mml:mi><mml:msub><mml:mi>φ</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>φ</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>φ</mml:mi><mml:mi>m</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula></td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><fn id=\"TFN2\"><p id=\"P51\"><italic>F</italic><sub><italic>m</italic></sub> and <italic>F</italic><sub><italic>m</italic> + 1</sub>: OCT complex data from the same location but at different time points. <italic>M</italic>: time repeated at one location. Δφ: phase change. <italic>σ</italic><sup>2</sup>: variance. <italic>R</italic>: SD. I: intensity of OCT signal.</p></fn></table-wrap-foot></table-wrap><table-wrap id=\"T3\" position=\"float\" orientation=\"landscape\"><label>TABLE 3</label><caption><p id=\"P52\">Summary of scanning protocols</p></caption><table frame=\"below\" rules=\"none\"><colgroup span=\"1\"><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/></colgroup><thead><tr><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Inter-A-lines</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Inter-frames</th></tr></thead><tbody><tr><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Principle</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><graphic xlink:href=\"nihms-1593817-t0002\"/></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><graphic xlink:href=\"nihms-1593817-t0003\"/></td></tr><tr><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Advantage</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Less motion artifact</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">High sensitivity for slow velocity</td></tr><tr><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Disadvantage</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Low sensitivity for slow velocity</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">More motion artifact</td></tr></tbody></table></table-wrap><table-wrap id=\"T4\" position=\"float\" orientation=\"landscape\"><label>TABLE 4</label><caption><p id=\"P53\">Summary of current average methods</p></caption><table frame=\"below\" rules=\"none\"><colgroup span=\"1\"><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/><col align=\"left\" valign=\"middle\" span=\"1\"/></colgroup><thead><tr><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\"/><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Principle</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Advantage</th><th align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">Disadvantage</th></tr></thead><tbody><tr><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Averaging method</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Split spectrum</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><graphic xlink:href=\"nihms-1593817-t0004\"/></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">High speed</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Low axial resolution</td></tr><tr><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Volumetric</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\"><graphic xlink:href=\"nihms-1593817-t0005\"/></td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">High sensitivity</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Long acquisition time</td></tr></tbody></table></table-wrap></floats-group></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">APL Photonics</journal-id><journal-id journal-id-type=\"coden\">APPHD2</journal-id><journal-title-group><journal-title>Apl Photonics</journal-title></journal-title-group><issn pub-type=\"epub\">2378-0967</issn><publisher><publisher-name>AIP Publishing LLC</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33015361</article-id><article-id pub-id-type=\"pmc\">PMC7523711</article-id><article-id pub-id-type=\"publisher-id\">5.0021270</article-id><article-id pub-id-type=\"doi\">10.1063/5.0021270</article-id><article-id pub-id-type=\"publisher-manuscript\">APP20-PS-CAP2020-00390</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Perspectives</subject></subj-group></article-categories><title-group><article-title>Engineering photonics solutions for COVID-19</article-title><alt-title alt-title-type=\"short-title\">Soler <italic>et al.</italic></alt-title><fn-group><fn fn-type=\"title-note\"><p>Note: This paper is part of the APL Photonics Special Topic on Coronavirus and\nPhotonics.</p></fn></fn-group></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Soler</surname><given-names>Maria</given-names></name><xref ref-type=\"aff\" rid=\"a1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Scholtz</surname><given-names>Alexis</given-names></name><xref ref-type=\"aff\" rid=\"a2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zeto</surname><given-names>Rene</given-names></name><xref ref-type=\"aff\" rid=\"a3\">3</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0001-9890-5104</contrib-id><name><surname>Armani</surname><given-names>Andrea M.</given-names></name><xref ref-type=\"aff\" rid=\"a2 a3\">2,3</xref><xref ref-type=\"corresp\" rid=\"cor1\">a)</xref><email/></contrib><aff id=\"a1\"><label>1</label><institution>Nanobiosensors and Bioanalytical Applications\nGroup (NanoB2A), Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, BIST\nand CIBER-BBN</institution>, Barcelona, <country>Spain</country></aff><aff id=\"a2\"><label>2</label><institution>Department of Biomedical Engineering, University\nof Southern California</institution>, Los Angeles, California 90089,\n<country>USA</country></aff><aff id=\"a3\"><label>3</label><institution>Mork Family Department of Chemical Engineering and\nMaterials Science, University of Southern California</institution>, Los Angeles,\nCalifornia 90089, <country>USA</country></aff></contrib-group><author-notes><corresp id=\"cor1\"><label>a)</label>Author to whom correspondence should be addressed:\n<email>armani@usc.edu</email></corresp></author-notes><pub-date pub-type=\"ppub\"><day>01</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>01</day><month>9</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"ppub\"/>. --><volume>5</volume><issue>9</issue><elocation-id seq=\"1\">090901</elocation-id><history><date date-type=\"received\"><day>08</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>17</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 Author(s).</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Author(s)</copyright-holder><license license-type=\"ccc\"><license-p>2378-0967/2020/5(9)/090901/14/<price>$0.00</price></license-p></license><license license-type=\"open\"><license-p>All article content, except where otherwise noted, is licensed under a Creative\nCommons Attribution (CC BY) license (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>).</license-p></license></permissions><abstract><p>As the impact of COVID-19 on society became apparent, the engineering and scientific\ncommunity recognized the need for innovative solutions. Two potential roadmaps emerged:\ndeveloping short-term solutions to address the immediate needs of the healthcare\ncommunities and developing mid/long-term solutions to eliminate the over-arching threat.\nHowever, in a truly global effort, researchers from all backgrounds came together in\ntackling this challenge. Short-term efforts have focused on re-purposing existing\ntechnologies and leveraging additive manufacturing techniques to address shortages in\npersonal protective equipment and disinfection. More basic research efforts with mid-term\nand long-term impact have emphasized developing novel diagnostics and accelerating\nvaccines. As a foundational technology, photonics has contributed directly and indirectly\nto all efforts. This perspective will provide an overview of the critical role that the\nphotonics field has played in efforts to combat the immediate COVID-19 pandemic as well as\nhow the photonics community could anticipate contributing to future pandemics of this\nnature.</p></abstract><funding-group><award-group><funding-source><named-content content-type=\"funder_name\">National Science Foundation</named-content><named-content content-type=\"funder_identifier\">http://dx.doi.org/10.13039/100000001</named-content></funding-source><award-id award-type=\"contract\">2028446</award-id></award-group><award-group><funding-source><named-content content-type=\"funder_name\">Agencia Estatal de\nInvestigación</named-content><named-content content-type=\"funder_identifier\">http://dx.doi.org/10.13039/501100011033</named-content></funding-source><award-id award-type=\"contract\">SEV-2017-0706</award-id></award-group><award-group><funding-source><named-content content-type=\"funder_name\">Generalitat de Catalunya</named-content><named-content content-type=\"funder_identifier\">http://dx.doi.org/10.13039/501100002809</named-content></funding-source></award-group><award-group><funding-source><named-content content-type=\"funder_name\">European Commission</named-content><named-content content-type=\"funder_identifier\">http://dx.doi.org/10.13039/501100000780</named-content></funding-source><award-id award-type=\"contract\">101003544</award-id></award-group></funding-group><counts><page-count count=\"14\"/></counts><custom-meta-group><custom-meta><meta-name>crossmark</meta-name><meta-value/></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"s1\"><label>I.</label><title>INTRODUCTION</title><p>From microscopy<xref rid=\"c1\" ref-type=\"bibr\"><sup>1–3</sup></xref> to optical\ncommunications,<xref rid=\"c4\" ref-type=\"bibr\"><sup>4–6</sup></xref> optical\ntechnologies permeate nearly every aspect of society. Therefore, when confronted with the\nchallenges of a global pandemic,<xref rid=\"c7\" ref-type=\"bibr\"><sup>7–12</sup></xref> it is not surprising that many solutions have been found in\nphotonic devices and developed by optical engineers. Although research and development\nefforts have been hindered by work from home conditions and manufacturing has been delayed\nby shortages in the supply chain,<xref rid=\"c13\" ref-type=\"bibr\"><sup>13</sup></xref>\nphotonics researchers and companies have made significant contributions to diagnostics and\npersonal protective equipment (PPE) by both adapting existing systems and inventing new\ntechnologies (<xref ref-type=\"fig\" rid=\"f1\">Fig. 1</xref>).<xref rid=\"c8\" ref-type=\"bibr\"><sup>8,10,14</sup></xref></p><fig fig-type=\"figure\" id=\"f1\" orientation=\"portrait\" position=\"float\"><label>FIG. 1.</label><caption><p>Overview of the different technological solutions being pursued to address\nCOVID-19.</p></caption><graphic xlink:href=\"APPHD2-000005-090901_1-g001\"/></fig><p>The short-term solutions have focused on inventing easily manufacturable biomedical devices\nand on addressing the global shortages in personal protective equipment (PPE) to reduce\nspread, particularly in healthcare settings. This work has included the development of\nfabric face masks, 3D printable face shields, and respirators for healthcare workers. While\noptical technologies played only a supporting role in the PPE fabrication efforts, they\ndirectly contributed to PPE re-use. Specifically, numerous approaches that leverage the\nability of ultraviolet-C (UV-C) or UV germicidal irradiation (UVGI) to serve as a\ndisinfection method were developed.<xref rid=\"c15\" ref-type=\"bibr\"><sup>15–17</sup></xref> In the face of the pandemic, many countries accelerated approvals\nof various technologies or granted emergency use authorization. As a result, these safety\nmeasures were available to the healthcare community within weeks. In addition, many existing\ntechnologies were either re-configured or adapted to more directly address COVID-19\nneeds.</p><p>The mid-term and long-term solutions emphasized developing methods for tracking and\naccelerating pharmacological solutions. Accurate tracking of infected individuals to reduce\nand to contain COVID-19 spread requires a combination of software and hardware\n(diagnostics). While software solutions were quickly launched, accurate diagnostics have\nbeen more challenging to deploy, in part, because of the numerous fundamental questions\nabout the pathophysiology of COVID-19 that remain unanswered. As a result, many diagnostics,\nincluding optical diagnostics, are finding an immediate use in understanding the nature of\nthe disease.<xref rid=\"c18\" ref-type=\"bibr\"><sup>18,19</sup></xref></p><p>In parallel, researchers are also pursuing the development of therapeutics and vaccines.\nWhile outside of the scope of this perspective, numerous potential strategies are being\ninvestigated with the hope that one will be proven effective quickly.<xref rid=\"c20\" ref-type=\"bibr\"><sup>20–33</sup></xref>\nAdditionally, it is notable that the fundamental approach to vaccine and therapeutic trials\nhas also been re-envisioned, greatly accelerating the potential timeline of\navailability.</p><p>This perspective will focus on the role of optics in healthcare, discussing technologies\nfor disinfection and diagnostics. For both topics, a brief background is followed by a more\nin-depth discussion on recent innovations and their impact to society. We end by discussing\nthe future prospects and open questions in the field.</p></sec><sec id=\"s2\"><label>II.</label><title>EXISTING HEALTHCARE MONITORING TECHNOLOGIES</title><p>Since the time of Hippocrates, diagnosis of disease has played a key role in medicine and\nhealthcare. Initial approaches relied on palpation and analyzing physical symptoms, such as\ntemperature. As technology progressed, physicians and scientists developed more advanced\ndiagnostic methods, for example, analyzing the cell shape and color of red blood cells. In\nthe modern era, medical diagnostics has transitioned from the cellular to the molecular\nlevel, and it typically relies on an integrated transducer platform to facilitate device\nfabrication. Additionally, with the development of instruments such as MRI, NMR, and CT,\nimaging is no longer limited to <italic>ex vivo</italic> and <italic>in vitro</italic>\nmethods.</p><p>Integrated diagnostic platforms for detecting either molecular or protein indicators of\ndisease based on electrical, mechanical, and optical transduction mechanisms have been\ndemonstrated. All three types of sensors have been used extensively in research\napplications. For example, mechanical sensor arrays based on cantilevers have been used to\nweigh individual cancer cells, investigating the efficacy of different therapeutics.\nElectrical sensors based on nanopore arrays have been used to analyze and sequence DNA, and\noptical sensors based on plasmonics have been used to understand antibody–antigen binding\nreactions. However, the majority of commercialized diagnostic systems that are utilized in\nmedical settings for disease diagnosis are based on optical technologies due to the\nsimplicity of the optical signal readout and compatibility with a wide range of sample\ntypes. For example, optical biosensing methods have increased the precision and accuracy of\ndisease diagnosis, optical detectors have enabled disease progression monitoring, and\nlaser-based treatments and therapeutics have improved the therapeutic efficacy and shortened\nrecovery times.<xref rid=\"c34\" ref-type=\"bibr\"><sup>34–38</sup></xref> In\nthe context of COVID-19, many existing technologies have been rapidly re-configured and\napplied for both diagnostics in a healthcare setting and at-home monitoring of disease\nprogression.</p><p>Most molecular diagnostic platforms for healthcare settings rely on detecting either the\nRNA or the DNA of the pathogen<xref rid=\"c39\" ref-type=\"bibr\"><sup>39–43</sup></xref> or the immune system response to the pathogen (antibody).<xref rid=\"c38\" ref-type=\"bibr\"><sup>38,44–47</sup></xref> Therefore, before a\ndiagnostic can be developed, it is necessary to either obtain and sequence the RNA or DNA or\nidentify the antibody generating the response. From a diagnostic perspective, it is more\nstraightforward to perform the former because it directly detects the virus. Additionally,\nRNA or DNA methods detect the active circulating pathogen, potentially in pre-symptomatic\npatients (<xref ref-type=\"fig\" rid=\"f2\">Fig. 2</xref>).<xref rid=\"c18\" ref-type=\"bibr\"><sup>18,27,39,48</sup></xref> In the case of COVID-19 and other\nhighly contagious pathogens, this ability is critical as it allows preventative measures to\nreduce transmission to be taken, potentially preventing or containing outbreaks.<xref rid=\"c12\" ref-type=\"bibr\"><sup>12,18</sup></xref></p><fig fig-type=\"figure\" id=\"f2\" orientation=\"portrait\" position=\"float\"><label>FIG. 2.</label><caption><p>The concentration of RNA and antibodies indicative of a SARS-CoV-2 infection varies\nwith time since infection. Adapted with permission from Lee <italic>et al.</italic>,\nFront. Immunol. <bold>11</bold>, 879 (2020). Copyright 2020 Author(s), licensed under a\nCreative Commons Attribution 4.0 License.</p></caption><graphic xlink:href=\"APPHD2-000005-090901_1-g002\"/></fig><p>In contrast, an antibody-based diagnostic infers the presence of the virus through the\nperson’s immune system response.<xref rid=\"c49\" ref-type=\"bibr\"><sup>49</sup></xref>\nTherefore, it is an indirect indicator of infection, and it is much more susceptible to\nincorrect findings. Additionally, it requires knowledge of the antibody that is specific to\nthe pathogen. Importantly, because antibody-based techniques will only show positive\ndiagnosis after the immune system has responded, these methods are unable to detect\npre-symptomatic infections (<xref ref-type=\"fig\" rid=\"f2\">Fig. 2</xref>). Therefore, in the\ncase of COVID-19, it is not surprising that the development and widespread adoption of an\nRNA-based test occurred well in advance of an antibody-based assay.<xref rid=\"c14\" ref-type=\"bibr\"><sup>14,19,50–53</sup></xref></p><p>Once the RNA sequence was established, conventional DNA methods, namely, reverse\ntranscription-polymerase chain reaction (RT-PCR), in combination with existing sample\nhandling and processing protocols, were leveraged to convert the viral RNA located in the\nnasal swab sample to DNA and to amplify the DNA concentration to detectable levels using\nRT-PCR (<xref ref-type=\"fig\" rid=\"f3\">Fig. 3</xref>).<xref rid=\"c32\" ref-type=\"bibr\"><sup>32,48,54,55</sup></xref> While RT-PCR increases the sample\nconcentration, detection is typically performed using quantitative-PCR (qPCR), which is a\nfluorescence-based sensing method.<xref rid=\"c42\" ref-type=\"bibr\"><sup>42,43</sup></xref> It is important to note that RT-PCR and qPCR are distinctly\ndifferent steps in this process; however, qPCR and RT-PCR can be performed simultaneously,\nallowing the concentration to be measured in real time. This approach is often called\nreal-time RT-PCR (or RT-qPCR) in shorthand.</p><fig fig-type=\"figure\" id=\"f3\" orientation=\"portrait\" position=\"float\"><label>FIG. 3.</label><caption><p>Comparison of PCR methods for diagnosis of COVID-19. (a) Two-step PCR. First, RNA is\nconverted to DNA through RT-PCR. Then, DNA is quantified using qPCR. This approach\nallows for optimization of both reactions, but it is more time-consuming than a one-step\nprocess. (b) One-step PCR. RT-PCR and qPCR are performed in the same vial in parallel.\nThis approach is faster, but the two reactions are not optimized. (c) Cartoon of the\ntype of data that are generated and analyzed. Images created with <ext-link ext-link-type=\"uri\" xlink:href=\"http://Biorender.com\">Biorender.com</ext-link>.</p></caption><graphic xlink:href=\"APPHD2-000005-090901_1-g003\"/></fig><p>As a fluorescent-based detection technology that relies primarily on the visible spectrum,\nqPCR (and RT-qPCR) has benefited greatly from numerous advances in optical technology,\nranging from optical sources to detectors as well as novel fluorophores. The reduction in\nfootprint and increased lifetime of LEDs relative to conventional bulbs has allowed for a\nreduction in system size and lowered maintenance and operating costs. For high performing\nsystems, the availability of narrow linewidth sources and high sensitivity detectors\ncovering a wide spectral range has enabled signal multiplexing. When combined with robotic\nsamplers for automated handling, a single instrument can analyze hundreds of samples per\nhour with low false positive/false negative rates. Although the technology required for PCR\ntechnologies has improved significantly, because PCR detects RNA and DNA directly from the\nvirus, PCR technologies can only detect active infections, and not past infections once the\npatient recovers.</p><p>An alternative method that can detect both active and recovered patients is based on\nantibody detection. These technologies detect the immune system’s response to the\ninfection.<xref rid=\"c56\" ref-type=\"bibr\"><sup>56</sup></xref> Therefore, the first step\nin developing the assay is antibody discovery<xref rid=\"c21\" ref-type=\"bibr\"><sup>21,29,44,57,58</sup></xref> and then establishing a reliable and high affinity\nantibody production line. In addition, while the terms “antibody” and “immunoglobulin” (Ig)\nare commonly used interchangeably, the immune system produces several types of antibodies\ndepending on the type of immune response and pathway.<xref rid=\"c56\" ref-type=\"bibr\"><sup>56</sup></xref> If sufficient information is known about the temporal nature of\nthe immune response, a diagnostic that indicates both the presence and the progression of a\ndisease can be developed, improving the diagnostic. An example of this approach is detecting\nboth the IgM and IgG antibodies that are produced at different timepoints during infection\n(<xref ref-type=\"fig\" rid=\"f2\">Fig. 2</xref>).<xref rid=\"c56\" ref-type=\"bibr\"><sup>56</sup></xref> However, these antibodies have very different structures and\naffinities, which contribute to their precision and accuracy when used in a sensor.\nTherefore, it is important to understand their biochemistry before architecting a sensor\nthat relies on this pair.</p><p>IgM is the largest antibody (<xref ref-type=\"fig\" rid=\"f4\">Fig. 4</xref>), comprised of\nfive monomers arranged in a ring, resulting in ten binding sites. It is poly-reactive and\nhas low-avidity, allowing it to respond quickly to unknown insults. The low avidity is\nfundamental to IgM’s operational principle and allows it to be the fastest responding\nantibody of the immune system. However, in the context of diagnostics, this property can\nresult in high false positives. At the same time, the rapid production of these IgM’s allows\nthem to be an indicator of active or recent infection, given their short lifetime. It takes\nthe immune system ∼1 week to produce IgM’s, and they only remain in the circulatory system\nfor approximately another week.</p><fig fig-type=\"figure\" id=\"f4\" orientation=\"portrait\" position=\"float\"><label>FIG. 4.</label><caption><p>Immunoglobulin structure. (a) IgM structure is comprised of five monomers with ten\nbinding sites. (b) IgG structure is comprised of one monomer with two binding sites. The\nbinding sites are located at the end of the F<sub>AB</sub> region of the monomer, in the\nvariable region. Images created with <ext-link ext-link-type=\"uri\" xlink:href=\"http://Biorender.com\">Biorender.com</ext-link>.</p></caption><graphic xlink:href=\"APPHD2-000005-090901_1-g004\"/></fig><p>In contrast, IgG is comprised of a single monomer with two adaptive binding sites (<xref ref-type=\"fig\" rid=\"f4\">Fig. 4</xref>). Because it is generated in response to a specific\ninsult, it has moderate to high avidity for that insult. While it takes a few weeks for IgG\nantibody concentrations to rise and stabilize, IgG’s can circulate at a constant\nconcentration for several months to a lifetime. Therefore, they provide a good indicator of\npast exposure. However, it is important to note that the concentration and avidity of IgG’s\ngenerated can be related to the magnitude of the initial insult and the host immune system\nresponse. Therefore, in the case of COVID-19, the concentration detected is not a reliable\nindicator of time since exposure because neither of these variables are known. On a side\nnote, IgG’s are also responsible for the long-term immunity provided by engineered vaccines,\nwhich induce a strong immune system response. However, it is not clear at this time if the\npresence of COVID-19 IgG’s indicates long-term immunity.<xref rid=\"c59\" ref-type=\"bibr\"><sup>59</sup></xref></p><p>The majority of commercialized optical sensors for COVID-19 antibody detection have focused\non leveraging existing instrumentation for signal readout, such as microplate readers or\nfluorescent imaging systems, or on developing point-of-care systems, including simple\ncolorimetric indicators. These strategies vary in their approach for IgG and IgM\nidentification, the complexity of the sample handling and chemistry protocols, the\ntime-to-result, and the information that can be provided (<xref rid=\"t1\" ref-type=\"table\">Table I</xref>). All of these factors contribute to the false positive and false negative\nrates, which determine the accuracy and precision (or reliability) of the finding.\nTherefore, it is important to recognize that the term “antibody test” is a very broad\nclassification given a large variety of diagnostic tests that rely on the detection of\nantibodies.</p><table-wrap id=\"t1\" orientation=\"portrait\" position=\"float\"><label>TABLE I.</label><caption><p>Overview of common diagnostic tests for COVID-19.</p></caption><table frame=\"hsides\" rules=\"groups\" border=\"1\"><colgroup span=\"1\"><col align=\"left\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/><col align=\"center\" span=\"1\"/></colgroup><thead><tr><th colspan=\"1\" rowspan=\"1\"/><th colspan=\"1\" rowspan=\"1\"/><th align=\"center\" colspan=\"1\" rowspan=\"1\">Time for</th><th align=\"center\" colspan=\"1\" rowspan=\"1\">Antibody</th><th align=\"center\" colspan=\"1\" rowspan=\"1\">Concentration</th><th align=\"center\" colspan=\"1\" rowspan=\"1\">Effectiveness</th></tr><tr><th align=\"center\" colspan=\"1\" rowspan=\"1\">Test</th><th align=\"center\" colspan=\"1\" rowspan=\"1\">Mechanism</th><th align=\"center\" colspan=\"1\" rowspan=\"1\">the test</th><th align=\"center\" colspan=\"1\" rowspan=\"1\">presence</th><th align=\"center\" colspan=\"1\" rowspan=\"1\">of antibodies</th><th align=\"center\" colspan=\"1\" rowspan=\"1\">of antibodies</th></tr></thead><tbody><tr><td colspan=\"1\" rowspan=\"1\">Rapid diagnostic</td><td colspan=\"1\" rowspan=\"1\">Substrate changes color to indicate</td><td colspan=\"1\" rowspan=\"2\">10 min–30 min</td><td colspan=\"1\" rowspan=\"2\">Yes</td><td colspan=\"1\" rowspan=\"2\">No</td><td colspan=\"1\" rowspan=\"2\">No</td></tr><tr><td colspan=\"1\" rowspan=\"1\">test<xref rid=\"c44\" ref-type=\"bibr\"><sup>44</sup></xref></td><td colspan=\"1\" rowspan=\"1\">the presence of antibody</td></tr><tr><td colspan=\"1\" rowspan=\"1\">Neutralization</td><td colspan=\"1\" rowspan=\"1\">Patient sample and virus are mixed</td><td colspan=\"1\" rowspan=\"3\">3 days–5 days</td><td colspan=\"1\" rowspan=\"3\">Yes</td><td colspan=\"1\" rowspan=\"3\">No</td><td colspan=\"1\" rowspan=\"3\">Yes</td></tr><tr><td colspan=\"1\" rowspan=\"1\">assay</td><td colspan=\"1\" rowspan=\"1\">with cells to determine the presence and</td></tr><tr><td colspan=\"1\" rowspan=\"1\">test<xref rid=\"c60\" ref-type=\"bibr\"><sup>60</sup></xref></td><td colspan=\"1\" rowspan=\"1\">efficacy of protecting cells</td></tr><tr><td colspan=\"1\" rowspan=\"1\">ELISA,<xref rid=\"c53\" ref-type=\"bibr\"><sup>53,61</sup></xref></td><td colspan=\"1\" rowspan=\"1\">Substrate changes or emits color a series</td><td colspan=\"1\" rowspan=\"3\">2 h–5 h</td><td colspan=\"1\" rowspan=\"3\">Yes</td><td colspan=\"1\" rowspan=\"3\">Yes</td><td colspan=\"1\" rowspan=\"3\">No</td></tr><tr><td colspan=\"1\" rowspan=\"1\">chemiluminescent</td><td colspan=\"1\" rowspan=\"1\">to indicate the presence of antibody;</td></tr><tr><td colspan=\"1\" rowspan=\"1\">immunoassay</td><td colspan=\"1\" rowspan=\"1\">of dilutions are run to obtain concentration</td></tr></tbody></table></table-wrap><p>Among the different antibody diagnostic methods, the rapid diagnostic test (RDT) is the\nmost commonly recognized by the general public.<xref rid=\"c19\" ref-type=\"bibr\"><sup>19,39,51,53</sup></xref> An example configuration of an RDT is shown in <xref ref-type=\"fig\" rid=\"f5\">Fig. 5</xref>. By simply monitoring the color of the two detection\nstrips (as well as the control strip), the user can make a diagnosis in 10 min–30 min with a\nsmall sample of blood. Not surprisingly, due to their quick response time, low cost, and\nease of use, these tests have quickly gained popularity. However, due to limitations with\nspecificity of the reactants, the false positive and false negative rates are significantly\nhigher than those in RT-qPCR.<xref rid=\"c14\" ref-type=\"bibr\"><sup>14</sup></xref> Therefore,\nwhile RDTs can be used as one piece of information, decisions regarding healthcare should\nnot rely solely on antibody test results until higher affinity antibodies have been\ndeveloped and can be reliably manufactured.<xref rid=\"c62\" ref-type=\"bibr\"><sup>62</sup></xref></p><fig fig-type=\"figure\" id=\"f5\" orientation=\"portrait\" position=\"float\"><label>FIG. 5.</label><caption><p>Schematic of the RDT. (a) The RDT has two diagnostic lanes and a control lane. The\nconjugation pad contains a COVID-19 antigen and a control antibody, both labeled with a\nmetal nanoparticle. The sample is wicked across the conjugation pad and then across all\nthree lanes. (b) If a strip changes color, it indicates that the antibody is present.\nImages created with <ext-link ext-link-type=\"uri\" xlink:href=\"http://Biorender.com\">Biorender.com</ext-link>.</p></caption><graphic xlink:href=\"APPHD2-000005-090901_1-g005\"/></fig><p>In diagnosing and monitoring COVID-19 progression, a base set of physical symptoms that are\nsimple to analyze has been identified, including temperature and blood oxygen saturation\nlevels among others.<xref rid=\"c62\" ref-type=\"bibr\"><sup>62</sup></xref> While both\nmeasurements can indicate multiple other illnesses, they can still provide information\nquickly and inexpensively, without requiring blood samples, nasal swabs, or expensive\nequipment. Additionally, once diagnosed, the measurements can allow disease progression to\nbe tracked from home, reducing the burden on the healthcare system.</p><p>Stand-off cameras based on infrared (8 <italic>µ</italic>m–14 <italic>µ</italic>m)\ndetectors have been used extensively in the scientific community for some time to perform\nthermal imaging, particularly in the atmospheric and aerospace communities but also in\nenvironmental sciences for tracking global climate change. However, these detectors were\nextremely large and expensive. Several years ago, in response to SARS, stand-off imaging\nsystems leveraging these thermal cameras were developed for airports and other high-traffic\nareas to easily and quickly monitor the temperatures of large populations of people.\nHowever, the shift to handheld units required a significant reduction in size and in cost as\nwell as the integration of self-referencing capability. This combination was only recently\naccomplished. Currently, stand-off measurements for monitoring temperature are a fundamental\ncomponent of many corporate and government COVID-19 monitoring strategies. However, body\ntemperature is not a perfect indicator as asymptomatic carriers can transmit the virus yet\nhave no discernable temperature increase.</p><p>Blood oxygen saturation monitoring, also known as pulse oximetry (or pulse-ox), is a way to\ndetermine the percent of hemoglobin that carries oxygen. This measure is an indicator of\nmany physical parameters, including lung function. While the most accurate method is to\ndirectly perform a gas analysis of arterial blood, this approach is also incredibly invasive\nand rarely performed. The standard of care is to measure the peripheral oxygen saturation\nlevel. While systems based on monitoring changes in reflectance and in transmission have\nbeen developed, measuring transmission is more commonly used due to its higher accuracy.\nSpecifically, the optical absorption of oxygenated hemoglobin is approximately an order of\nmagnitude higher than that of de-oxygenated hemoglobin in the red blood cells (620 nm–700\nnm), but in the near-IR, the values are nearly equal (800 nm–940 nm). Therefore, by\ncomparing the two signals, the percent of oxygenated hemoglobin can be calculated.</p><p>The initial optical pulse-ox systems date back to the early 1930s and 1940s, but the\nsystems were not in routine use in a medical setting until the 1970s and 1980s. Moreover,\nthese systems relied on precision optical sources and were extremely sensitive to patient\nmotion, limiting their use to hospital settings. In the 1990s, signal analysis technology\nwas developed to stabilize the signal against patient motion. However, these systems still\nrelied on precision light sources and complex control systems. With the advent of low-cost\nmicro-LEDs with low power requirements that could be directly integrated on-chip,\nfinger-clip pulse-ox devices were designed and developed. Inexpensive and suitable for\nat-home use, these systems have transformed cardiovascular care in both the hospital and the\nhome. Given the impact of COVID-19 on lung function, these easy to use pulse-ox devices are\nnow being used to monitor the progression of COVID-19 patients from home,<xref rid=\"c63\" ref-type=\"bibr\"><sup>63–65</sup></xref> and research is investigating\ntheir use as an “early warning system” for COVID-19.<xref rid=\"c66\" ref-type=\"bibr\"><sup>66</sup></xref></p></sec><sec id=\"s3\"><label>III.</label><title>EMERGING DIAGNOSTIC TECHNOLOGIES</title><p>A prominent area in integrated photonics focuses on the development of biological and\nchemical sensors for diagnostics for a wide range of diseases.<xref rid=\"c34\" ref-type=\"bibr\"><sup>34,37,47,67–71</sup></xref> Unlike the methods\nalready discussed, the emerging technologies directly leverage light–matter interactions in\nthe detection mechanism and have the possibility of being integrated with microfluidics for\nhigh-throughput sample delivery and analysis. High-throughput or multiplexing capability is\nof particular interest given diagnostic test shortages faced early on in the COVID-19\npandemic.<xref rid=\"c8\" ref-type=\"bibr\"><sup>8</sup></xref> Two commonly used methods to\ndetect and identify specific substances in clinical samples are the detection of refraction\nindex and optical transmission changes and the detection of optical scattering.</p><p>Beyond the well-known Surface Plasmon Resonance (SPR) biosensor originally commercialized\nby Biacore, new sensing systems based on plasmonic nanotechnology<xref rid=\"c72\" ref-type=\"bibr\"><sup>72,73</sup></xref> and silicon-based photonic rings and waveguides<xref rid=\"c74\" ref-type=\"bibr\"><sup>74,75</sup></xref> are continuously emerging to provide\nthe most appealing analytical features for rapid screening and diagnosis. As nanofabrication\nmethods and optical component integration have advanced, the portability of these platforms\nhas improved. Both sensors rely on the fundamentally simple concept of evanescent field\ndetection of refractive index change.</p><p>Briefly, an evanescent wave generated at the interface of a waveguide or a metallic\nnanostructure with the outer medium is able to probe minute variations of the dielectric\nrefractive index and transduce them into variations of certain light properties, such as\nresonance, intensity, or phase. By tethering specific bioreceptors (e.g., antibodies or DNA\nprobes) onto the sensor, the target analyte is captured from the sample. In this manner, the\nsame surface chemistry methods developed for the commercialized RDTs can be leveraged to\naccelerate the design and translation of these optical systems. Once the target analyte\nattaches to the sensor surface, the refractive index of the local environment changes. Over\na given concentration range, this change scales linearly with the analyte concentration.\nBecause of the role that available surface binding sites play in generating the detection\nsignal, one approach for controlling a sensor’s operating range is to alter the density and\ncomposition of binding sites. The idea of optimizing the surface chemistry to tailor the\nworking range is an emerging area of research.</p><p>The evanescent wave optical detection schemes with the highest sensitivity are those that\ntrack the frequency shift of resonance or interferometry signals. Both signals are\nfundamentally monitoring the local change in the refractive index caused by an analyte.\nResonance-shift methods monitor the change in a system’s resonance frequency, such as that\nof a plasmonic nanoparticle or waveguide, due to a local change in the refractive index\ncaused by an analyte. Interferometric methods monitor the phase change of an optical probe\nsignal due to local changes in the refractive index caused by an analyte.<xref rid=\"c35\" ref-type=\"bibr\"><sup>35,37,49</sup></xref> This approach delivers\nquantitative data in real time without the need of any fluorescent or colorimetric labeling\n(i.e., label-free assay). The increased sensitivity allows for a reduction in the requisite\nsample volume and reagents required, making them ideal tools for decentralized and\nhigh-throughput testing.<xref rid=\"c74\" ref-type=\"bibr\"><sup>74,76</sup></xref></p><p>In the last decade, label-free integrated photonic biosensors have demonstrated their\ncapabilities in analyzing clinically relevant materials and reporting specific detection of\nproteins, nucleic acids, or pathogens in human body fluids (serum, urine, saliva, etc.) with\noutstanding assay sensitivity ranging from attomolar to femtomolar (aM–fM) levels of RNA\nmolecules to less than ten bacterial cells, for example.<xref rid=\"c76\" ref-type=\"bibr\"><sup>76–79</sup></xref> They have also formed the foundation of\nportable diagnostic systems for viral pathogen detection for Ebola and malaria. This proven\nfeasibility together with their unique versatility positions photonic biosensor technologies\nas an attractive solution for novel COVID-19 diagnostics.</p><p>Notably, optical technologies are being developed for two types of detection of a\nSARS-CoV-2 viral infection: (1) direct and (2) indirect. With direct detection, either the\ncirculating viral RNA or the virus itself is detected. The first approach is similar to\nRT-qPCR in that the viral RNA is being detected. For these RNA sensors, specific RNA target\nsequences are identified by hybridization to a complementary nucleic acid sequence (i.e.,\nDNA) immobilized on the surface. If the biosensor is sensitive enough, it does not need\npre-amplification cycles based on PCR. This strategy represents a significant improvement\nover RT-qPCR and decreases the time-to-result from over an hour for RT-qPCR to 10 min–15 min\n(after RNA extraction). However, similar to qPCR, the sensor is detecting the copies of\nviral RNA per ml of blood. The second approach directly detects the circulating active viral\nparticles, circumventing the basic limitation of PCR that has resulted in false positives.\nNamely, PCR amplifies both circulating RNA and RNA in active viral particles. Therefore,\nafter a patient recovers, there can still be circulating RNA that can be amplified and\ndetected. This circulating RNA can result in false positive signals. By detecting only\nintact viral particles, this limitation is overcome.</p><p>Previous research on photonic biosensors has demonstrated the genomic analysis of other\nrespiratory virus infections: influenza, respiratory syncytial virus (RSV), and other\ncoronaviruses such as the original SARS-CoV or the human coronavirus OC43/229E, responsible\nfor common cold.<xref rid=\"c41\" ref-type=\"bibr\"><sup>41,80</sup></xref> However, in\nthose cases, it was necessary to perform prior or post-amplification procedures to enhance\nthe assay sensitivity. In general, genomic-based optical biosensing assays could provide an\nalternative method to RT-qPCR, meeting the sensitivity and specificity requirements for\nclinical diagnosis.</p><p>Soon after the COVID-19 outbreak, scientists started developing photonic biosensors for the\ndetection of the SARS-CoV-2 genomic material.<xref rid=\"c81\" ref-type=\"bibr\"><sup>81</sup></xref> In the previous work, the general strategy of direct viral RNA\ndetection using optical technologies has been demonstrated using plasmonic and silicon\nphotonic biosensors. Notably, this approach allows for quantification of the viral load, and\noverall sensitivities in the range of 10<sup>2</sup> copies/ml have been demonstrated.<xref rid=\"c82\" ref-type=\"bibr\"><sup>82–84</sup></xref> This prior work formed a\nfoundation for developing detection technologies for SARS-CoV-2, and one of the first works\npublished during the pandemic reported a nanoplasmonic biosensor for direct analysis of\nspecific RNA fragments (RdRp gene) of the novel coronavirus.<xref rid=\"c81\" ref-type=\"bibr\"><sup>81</sup></xref> The sensor, based on gold nanoislands, combines the localized SPR\nsensing with the plasmonic photothermal effect that enhances the selective hybridization of\ncomplementary sequences while reducing non-specific binding of similar targets [<xref ref-type=\"fig\" rid=\"f6\">Fig. 6(a)</xref>]. Qiu <italic>et al.</italic> showed a\ndetectability in the picomolar (pM) range, estimating a limit of detection for entire viral\nRNA strands around 10<sup>4</sup> copies/ml. The biosensor’s sensitivity could theoretically\nbe appropriate for direct testing of clinical specimens without PCR given that the viral\nload in throat/nasal swabs of COVID-19 positive patients is usually between 10<sup>5</sup>\nand 10<sup>6</sup> copies/ml.</p><fig fig-type=\"figure\" id=\"f6\" orientation=\"portrait\" position=\"float\"><label>FIG. 6.</label><caption><p>(a) Illustration of a dual-functional nanoplasmonic biosensor for COVID-19 RNA\nanalysis. Reproduced with permission from Qiu <italic>et al.</italic>, ACS Nano\n<bold>14</bold>, 5268 (2020). Copyright 2020 American Chemical Society. (b)\nIllustration of a nanophotonic bimodal waveguide interferometer for direct sensing of\nintact SARS-CoV-2 viruses. CoNVaT project.</p></caption><graphic xlink:href=\"APPHD2-000005-090901_1-g006\"/></fig><p>However, direct detection of the live virus particles is the ultimate goal. By\nfunctionalizing the sensor surface with specific receptors (e.g., antibodies) toward\nexternal proteins of the virus membrane, it is possible to capture intact virus entities\nthat circulate in the body fluids, providing straightforward information of the live viral\nload in the patient without the need for RNA extraction and/or fragmentation procedures. To\nour knowledge, nothing has been reported yet for the direct detection of SARS-CoV-2 viruses\nwith photonic biosensors, which is not surprising since the assay development requires\nhigh-quality, specific antibodies that are more difficult to produce than nucleic acid\nprobes. However, several substantial research efforts are underway.</p><p>CoNVat, one of the first large research projects in Europe specifically dedicated to\ndevelop advanced nanophotonic biosensors for diagnosis of coronavirus infection, is focused\non waveguide sensors. In particular, the goal of the research is to implement pioneering\nsilicon photonics interferometric technology, the Bimodal Waveguide (BiMW) biosensor,<xref rid=\"c38\" ref-type=\"bibr\"><sup>38</sup></xref> for SARS-CoV-2 detection [<xref ref-type=\"fig\" rid=\"f6\">Fig. 6(b)</xref>]. This technology will offer an integrated\napproach for accurate and quantitative diagnosis in less than 30 min.</p><p>One optical detection system has already proven effective in detecting viruses, including\nEbola and Marburg. It is based on a simple interferometric reflectance imaging sensor (IRIS)\nplatform.<xref rid=\"c13\" ref-type=\"bibr\"><sup>13,85–91</sup></xref> The IRIS can not only detect but also count individual viral\npathogens in a complex medium without time-consuming sample purification or preparation. The\nIRIS has a unique detection mode from the previously discussed refractometric and\nfluorescence-based systems. Notably, when the virus binds to the surface, its weight\ngenerates a detectable optical signal. This is a label-free approach, and the detection\nsignature is specific to the physical size and properties of the virus, providing a\nsecondary signature for identification. In the previous work, the researchers demonstrated a\nreal-time detection limit of 100 PFU/ml for vesicular stomatitis virus (VSV), and in\nresponse to the previous Ebola epidemic, they developed a 20 min assay for the Ebola virions\nat 1.5 × 10<sup>4</sup> PFU/ml sensitivity corresponding to an average cycle threshold (CT)\nof 23.1 on RT-PCR.<xref rid=\"c90\" ref-type=\"bibr\"><sup>90</sup></xref> Therefore, once\noptimized for SARS-CoV-2 detection, this optical system is ideally suited to accelerate\ndiagnosis.</p><p>In addition to direct detection of the viral load, optical sensors can also be used for\nindirect detection diagnostics or the detection of the body’s antibody response.<xref rid=\"c35\" ref-type=\"bibr\"><sup>35,37,67,69</sup></xref> As previously\ndiscussed, this type of assay is well-developed, and several relatively simple constructs\nthat can provide a simple yes/no determination have been deployed. In contrast, photonic\nbiosensors can quantify serum antibodies. In previous epidemics and pandemics, optical\nbiosensors were developed for serological analysis of viral infections<xref rid=\"c45\" ref-type=\"bibr\"><sup>45,92–94</sup></xref> or have been used during vaccine\ndevelopment.<xref rid=\"c95\" ref-type=\"bibr\"><sup>95–97</sup></xref> In the current\nCOVID-19 pandemic, the development of biosensing protocols for simple, rapid, and efficient\nserology testing could enable biomedical and epidemiology research efforts, accelerating the\ndiscovery of an optimum vaccine and improving our understanding of SARS-CoV-2 in humans.</p><p>However, like any emerging research technologies, there are numerous hurdles that must be\novercome before these potentially transformative devices can be translated. Notably, the\nintegration and full automation of operational sensing devices will require the\nestablishment of universal and reproducible protocols for bioassay performance and the\ndemonstration of reliability in large clinical studies. In some cases, multiple optical\ncomponents, including the sensing elements as well as on-chip optical sources and detectors\nor imagers, may be needed. By creating a cohesive package, the noise level can be reduced,\nimproving the overall system performance. This level of system complexity will leverage\nrecent advances in heterogeneous fabrication protocols being developed. Additionally, many\nof these systems can also be integrated with complementary electrical components, such as\nelectrophoresis, as well as microfluidics to move some aspects of sample preparation\non-chip.</p></sec><sec id=\"s4\"><label>IV.</label><title>DISINFECTION TECHNOLOGIES</title><p>While this immediate crisis is the catalyst, there is a global need for the development of\nimproved disinfection methods. Historically, the majority of antibiotic-resistant bacterial\ninfections originated in medical settings. However, in 2019, there was a dramatic shift. Due\nto the heroic efforts of the medical community to improve disinfection and sterilization,\nthe number of infections originating in medical settings decreased.<xref rid=\"c98\" ref-type=\"bibr\"><sup>98,99</sup></xref> However, the overall numbers continued their upward\ntrajectory, due to community-based transmission. Moreover, many disinfection protocols\nassume ready access to chemicals and generate significant environmental waste. Therefore, to\nincrease access to disinfection protocols in low-resource environments, reduce the\nenvironmental impact, and increase the usability, alternative methods must be developed and\nevaluated.<xref rid=\"c100\" ref-type=\"bibr\"><sup>100–102</sup></xref></p><p>When designing a disinfectant method, the first step is to evaluate the key components of\nthe biological contaminant. In the case of coronavirus, the critical elements that ensure\nthe stability, replication, and cell targeting ability are the envelope protein, RNA, and\nspike glycoprotein, respectively.<xref rid=\"c30\" ref-type=\"bibr\"><sup>30,103–105</sup></xref> If any of these components are destroyed, the virus will\nbe inactivated. Therefore, most disinfection methods are designed to target one or more of\nthese elements. There are three general categories: thermal, chemical, and radiation (<xref ref-type=\"fig\" rid=\"f7\">Fig. 7</xref>). While the focus of this review is on\nradiation-based methods, for comparison purposes, all three methods will be briefly\ndiscussed.<xref rid=\"c102\" ref-type=\"bibr\"><sup>102</sup></xref></p><fig fig-type=\"figure\" id=\"f7\" orientation=\"portrait\" position=\"float\"><label>FIG. 7.</label><caption><p>Overview of disinfection strategies. (a) Basic structure of coronavirus (and\nSARS-CoV-2), highlighting the key components needed for functioning. (b) Thermal and (c)\nchemical disinfection methods degrade the spike protein and the envelope protein. (d)\nIrradiation including UV-C degrades the RNA. Adapted with permission from Stadler\n<italic>et al.</italic>, Nat. Rev. Microbiol. <bold>1</bold>, 209 (2003). Copyright\n2003 Springer Nature. SARS-beginning to understand a new virus.</p></caption><graphic xlink:href=\"APPHD2-000005-090901_1-g007\"/></fig><p>Thermal methods primarily target the spike glycoprotein with secondary impact on the\nenvelope protein, depending on the thermal dose (heat and duration). Thermal methods were\nprobably the first disinfection method developed and can be as simple as boiling water or an\nopen flame. However, more rigorously controlled systems rely on ovens or other large\nchambers and can be either dry or moist heat. Given that they uniformly treat a sample, they\nare particularly suitable for linens, though they can be used for a myriad of supplies, such\nas surgical tools.</p><p>Chemical methods, including hydrogen peroxide and chlorine, target the spike glycoprotein\nand the envelope protein.<xref rid=\"c106\" ref-type=\"bibr\"><sup>106,107</sup></xref>\nBoth vapor-based and handwipe chemical methods have been developed. Due to their efficacy in\nremoving both monolayers and larger quantities of contaminants quickly, wipe-based chemical\nmethods are the preferred disinfection methods for surfaces or solid materials. However, one\ndrawback of chemical methods is that they only destabilize viruses that they directly\ninteract with. To improve efficacy in materials with complex topologies, such as linens, a\nhybrid system is frequently used, combining both thermal and chemical methods.</p><p>While technically being a chemical method, due to its distinct mechanism, ozone\n(O<sub>3</sub>)-based disinfection is its own category. As a powerful oxidant, ozone is\nable to destroy the glycoproteins and the envelope proteins as well as all nucleotide bases\ncomprising the RNA. Because ozone is a gas, it is effective on porous media as well as solid\nsurfaces. Additionally, the process does not require high temperatures, making it\nparticularly attractive. However, ozone is extremely reactive and readily returns to oxygen\ngas (O<sub>2</sub>). Therefore, it is very challenging to use.</p><p>The most recent development in disinfection systems is radiation-based systems. This\ncategory includes microwave, infrared (IR), and UV-C systems. Microwave-based and IR-based\nsystems typically operate in an indirect manner. Namely, the microwaves (or radio-frequency\nwaves) excite water, which thermally heats and destroys the spike glycoprotein. Thus,\nmicrowave radiation can be considered a moist thermal disinfection. Similarly, since IR\nsources are thermal sources, IR-systems can act as either dry or moist thermal systems. In\ncontrast, systems based on UV-C radiation operate in a completely different manner.</p><p>RNA is comprised of four nucleotides: Adenine (A), Guanine (G), Uracil (U), and Cytosine\n(C). They are grouped into two categories: purines (A, G) and pyrimidines (U, C). Typically,\npurines bind to pyrimidines (A–U, G–C). However, when RNA is exposed to 260 nm light, it\ninitiates binding between pyrimidines.<xref rid=\"c108\" ref-type=\"bibr\"><sup>108,109</sup></xref> By changing the fundamental structure of the RNA helix, RNA\nreplication is inhibited, preventing viruses from reproducing. Therefore, similar to\nchemical methods, the UV-C light must directly interact with a virus particle to be\neffective. However, unlike chemical methods, UV-C does not expose the material to any kind\nof toxic chemical. Additionally, it can be performed at low temperatures. For these reasons,\nthe initial application of UV-C was in the environmental field, namely, for air purification\nand water disinfection.<xref rid=\"c109\" ref-type=\"bibr\"><sup>109–114</sup></xref> However, the airborne contagiousness of SARS-CoV-2 increased\nthe demand for new universal disinfection techniques to be developed.</p><p>In the context of COVID-19, UV-C has proven particularly effective in disinfecting PPE that\nprotects healthcare workers against airborne pathogens, primarily masks, face shields, and\neyewear. This ability has helped address the unprecedented spike in demand for those types\nof PPE that has threatened the safety of healthcare workers. While governments worked to\nrelax import restrictions to ease the shock to PPE supply chains, during the worst part of\nthe initial COVID-19 crisis, healthcare workers in many countries were forced to reuse their\nPPE at a dramatic scale. Although many countries are past the initial COVID-19 peak,\noutbreaks are still expected to occur throughout the world until a vaccine is widely\navailable. This means that spurious PPE shortages will be a continuing reality for the time\nbeing, and it is worthwhile to investigate strategies that can enable safe PPE reuse in\ntimes of crisis. UV-C provides one path to disinfect PPE.</p><p>However, it is important to note that due to its universal damage mechanism that destroys\nboth DNA and RNA, UV-C can have significant negative health effects on people as well as\nviruses, including permanent blindness.<xref rid=\"c108\" ref-type=\"bibr\"><sup>108</sup></xref> Therefore, there are numerous safety standards governing the use of\nUV-C light sources in the workplace. For example, automatic shut-offs and the use of\neye-protection when using UV-C light sources are standard. However, many of these safety\nstandards are missing in consumer products that do not undergo the same scrutiny. Given the\nrapid deployment of UV-C based technologies into the consumer market, the lack of safety\nstandards is a growing concern.</p><p>One significant advantage of both thermal and radiation methods over chemical methods is\nthat both thermal and radiation have minimal consumables. This difference is notable for\nseveral reasons. First, the amount of waste generated and the resulting environmental impact\nare decreased as compared to those in chemical techniques. Additionally, the reliance on\nsupply chains and on manufacturing is reduced.</p><p>Unlike thermal treatments, UV disinfection protocols do not require large ovens, and the\nfundamental mechanism is amenable to high-throughput rates. They also may not necessarily\nrequire a large financial investment or lead time prior to their use because UV disinfectant\ntools come in a variety of forms, ranging from small handheld wands to semi-autonomous\nrobotic systems. A field hospital established in 48 hours to deal with a COVID-19 outbreak\nmay not be able to support the industrial-scale ovens with high power requirements for\nthermal disinfection or the chemical vapor stations with gas purification needs for chemical\ndisinfection, but it could purchase portable UV disinfection wands and enclosures. Depending\non the emitted UV power, the light simply needs to be held above the contaminated surface\nfor a fixed amount of time to ensure that the surface has been sterilized. This can be as\nlittle as 10 s–30 s depending on the type of UV source used.<xref rid=\"c17\" ref-type=\"bibr\"><sup>17,100,115–117</sup></xref></p><p>UV-C sources fall into two common categories: bulbs and LEDs. Xenon or mercury bulbs are\nthe most commonly used, having originally formed the foundation of the industry. While they\nare relatively inexpensive to make, they have high power requirements, which translate to\nhigh operating (or electrical) costs. Additionally, in comparison with that of alternative\nsources, their lifetime is short, and power cycling can further impact the lifetime. An\nemerging alternative is UV-C LEDs. High power LEDs have significantly reduced power demands\nand increased lifetime. They also have smaller footprints, allowing more compact (or\nhandheld) systems to be designed. However, they are much more expensive, increasing the\n“upfront” costs as compared to bulbs. Last, despite being called “high power,” the LED\noutput power is low to moderate when compared to that of bulbs. To account for this\ndifference, the exposure time when using LEDs is increased in order to achieve the same\ndose.<xref rid=\"c117\" ref-type=\"bibr\"><sup>117,118</sup></xref></p><p>Dosage is the product of intensity delivered to a surface and time. Physical factors such\nas the distance between the source and the surface and the source intensity profile play a\nrole in the calculation of how long a UV disinfectant tool needs to be used in order to be\neffective against different pathogens. Further complicating issues, different surface types\n(e.g., porous vs non-porous) require different dosages. Given the mechanism of UV-C,\nbiological factors also contribute. Namely, because UV-C is initiating dimerization of the\npyrimidines, if the concentration of pyrimidines is higher, a lower dose can be used. Thus,\npathogens containing DNA, which is a double helix structure, intrinsically require a lower\ndose than RNA-based pathogens, including viruses. For this reason, viral pathogens require\nthe highest dosages.</p><p>Last, there are two competing considerations: the desire to disinfect (damage) all viral\nand bacterial contaminants and the need to not damage the surface, particularly for repeated\nexposures. Therefore, determining the optimum dosage is a complex optimization problem that\nmust consider not only the specific pathogen but also the UV-C source, the surface\nproperties, and the specific application.</p><p>In the current climate, the two most common PPE disinfected using UV-C is N95 masks and\nface shields. These represent two very different surfaces, and their mechanism of acting as\nPPE is very different. Face shields are either glass or chemically resistant plastic,\nwhereas N95 masks are fibrous (natural or manmade). Therefore, while the face shield surface\nis considered non-porous and robust against relatively high doses of UV-C (when compared to\nporous materials), N95 masks and surgical masks are more delicate. Additionally, while\nchemical treatments, including chemical immersion and wipes, can be used on face shields,\nchemical compatibility can impact the porous structure of N95 masks, degrading the\nfiltration ability, particularly with repeated chemical disinfection. As a result, systems\nare typically configured for either masks or shields, but not both. Similarly, disinfection\nprotocols are typically optimized for a single material type.<xref rid=\"c119\" ref-type=\"bibr\"><sup>119</sup></xref></p><p>Thus far, three general approaches for PPE disinfection have been studied: (1)\nreconfiguring standard biosafety cabinets,<xref rid=\"c16\" ref-type=\"bibr\"><sup>16</sup></xref> (2) creating enclosed portable boxes with a single UV-C source or\narrays of UV-C sources,<xref rid=\"c17\" ref-type=\"bibr\"><sup>17</sup></xref> and (3) building\nlarge rooms with intense UV-C sources located in the center. The first two approaches are\ncapable of disinfecting a few to a few dozen N95 masks or face shields at a time, while the\nthird technique can disinfect hundreds of N95 masks at once. However, the infrastructure\nrequired for the third method is significantly more expensive.</p><p>Given the important role that the material plays in the N95 mask filtration efficacy, the\nUV-C dosage thresholds for material degradation have been extensively researched. In one\nstudy using biosafety cabinets as the UV-C source, the balance between the required dose to\ndisinfect and the mask integrity was investigated. It was shown the sterilizer cabinet had a\nslight impact on the filtration efficiency of N95 masks (from the standard 95% to 93%) after\n20 cycles. For an N95 mask, ∼4.5 J/cm<sup>2</sup> is the lowest dosage at which no physical\ndamage to N95 masks was observed, but this varies substantially and values up to 120\nJ/cm<sup>2</sup> may be acceptable in certain situations.<xref rid=\"c15\" ref-type=\"bibr\"><sup>15</sup></xref> This value establishes a “damage threshold” rule of thumb for\ncertain types of PPE.</p><p>Additionally, the environment around the mask during disinfection can play a role in\ndamage. It is common to perform thermal treatments in a moist or humid environment as the\nhumidity accelerates the disinfection process. However, the humidity also damages the masks,\nreducing the filtration to 80% after ten cycles.<xref rid=\"c15\" ref-type=\"bibr\"><sup>15</sup></xref> When this value is compared to 95% filtration for a dry oven or\nthe UV sterilization cabinet, it is evident that the environment is a key factor that should\nbe considered and controlled.</p><p>While the material damage threshold provides an upper limit to the total dose, the lower\nlimit is set by the minimum dosage required to disinfect a surface. In one study, it was\nestablished that a 17 mW/cm<sup>2</sup> treatment for 30 min (30.6 J/cm<sup>2</sup>) was\nenough to safely disinfect N95 masks against viral particles, allowing for ∼20 cycles of\nthis treatment before physical damage occurred (establishing a damage threshold around ∼600\nJ/cm<sup>2</sup>).<xref rid=\"c15\" ref-type=\"bibr\"><sup>15</sup></xref> This work provides\nthe current basis for all UV-based disinfection techniques when using N95 masks.</p><p>Given their reliability and environmental control, biosafety cabinets and fixed enclosures\nare the standard approach for performing UV-C disinfection in healthcare settings. However,\nUV-C is also particularly amenable to creating handheld platforms for consumer use. UV wands\nand small boxes for cellphones and other personal items are battery-powered and are ideal\nfor resource constrained healthcare settings as well as personal use.</p><p>To disinfect larger surfaces around a room and objects that cannot be moved, a mobile\nsystem is needed. Therefore, it is desirable to create an autonomous or a semi-autonomous\nplatform that can disinfect these rooms or other large spaces routinely. In general, current\nUV-C systems suitable for this task largely fall into one of the two categories: passive\nrobots that operate simply within their environments and active robots that interact with\ncomponents of their environments. Each type of robot has its own advantages and\ndisadvantages associated with its development, implementation, and utilization.</p><p>The passive robots are much simpler systems, whose designs typically involve a mobile base\nwith fixed vertical UV-C sources and are usually marketed as accomplishing “whole-room\ndisinfection.” A few of these systems existed commercially prior to the COVID-19 pandemic,\nmotivated by the need to reduce hospital acquired antibiotic-resistant bacterial\ninfections.<xref rid=\"c117\" ref-type=\"bibr\"><sup>117,120–122</sup></xref>\nThe fairly simplistic design of the most basic models allowed the manufacturing of these\nrobots to be rapidly increased, and systems were beginning to be broadly distributed within\na few weeks of the pandemic being formally acknowledged worldwide.</p><p>The design is primarily determined by the degree of autonomy, and the majority of systems\nrely on either pulsed xenon lamps<xref rid=\"c100\" ref-type=\"bibr\"><sup>100,123–125</sup></xref> or mercury gas bulbs.<xref rid=\"c125\" ref-type=\"bibr\"><sup>125</sup></xref> One study has suggested that the continuous dose of UV-C light\nprovided by the mercury bulbs is more effective at reducing pathogen levels than that\nprovided by pulsed xenon lamps.<xref rid=\"c125\" ref-type=\"bibr\"><sup>125</sup></xref> These\nlights are mounted upright on a mobile base, which ranges from a simple wheeled base that\nhas to be manually moved around to a robotic base that allows for more autonomous use. Many\nof these systems have built-in safety measures, including motion-detecting sensors or lidar\nto detect people and automatically shut off the bulbs to prevent harmful exposure. Some also\nintegrate sensory data and use software-based methods to enhance and optimize the delivery\nof UV-C light throughout the room, such as measuring reflected UV-C light to ensure that the\nlowest dose delivered throughout the room meets the minimum dosage required for\ndisinfection.<xref rid=\"c101\" ref-type=\"bibr\"><sup>101</sup></xref></p><p>Studies of these passive robots have confirmed some of the theoretical advantages of UV-C\ndisinfection. Through their “no touch” disinfection process, they remove the human error\nfrom the process of disinfection and allow for more frequent cleanings since a person is not\nrequired to be present during operation of the robot. However, these robots still suffer\nfrom limitations due to safety concerns; because they uniformly emit UV-C light throughout a\nroom, the room must be completely empty of people to avoid potential harm. In addition,\nbecause UV-C light diminishes in intensity proportional to the distance squared, some\nhospitals have found the added inconvenience of positioning high-touch surfaces (such as\nmobile computer stands, hospitals beds, and tables) around the UV-C robot<xref rid=\"c125\" ref-type=\"bibr\"><sup>125</sup></xref> or moving the UV-C robot to several\ndifferent positions within a room<xref rid=\"c123\" ref-type=\"bibr\"><sup>123</sup></xref> to\nbe necessary.</p><p>In addition, the inability of these passive robots to directly interact with their\nenvironments due to limited sensing and a complete lack of effectors significantly limits\ntheir disinfection capabilities to only exposed surfaces. As a result, key surfaces within\nhospital rooms that may be hidden from these passive robots remain dirty. These could\ninclude occluded, high-frequency contact surfaces, such as the surface of a counter hidden\nunder a box of gloves, the inside of an ADA door handle, or the back of a door. One study\nhas suggested that even some exposed surfaces are still not disinfected sufficiently,\nimplying that the robots may need to be “smarter” and interact more with the environment to\nprovide more thorough disinfection.<xref rid=\"c123\" ref-type=\"bibr\"><sup>123</sup></xref>\nWhile these robots still suffer from some limitations, the importance of the more simplistic\ndesign should not be understated, as it had allowed these robots to be successfully\ncommercialized and deployed in hospitals, rendering them immediately available during the\nCOVID-19 pandemic.</p><p>More recently, there has been a push to develop robots with end effectors to actively\nmanipulate their environments to allow for more comprehensive UV-C disinfection,\nparticularly for high-trafficked surfaces. These interactions can allow access to surfaces\nthat were occluded to the light provided by the passive robots. For instance, a robot with\narms that allow it to grab objects could move a box of gloves to disinfect the surface of a\ncounter underneath it or to pick up a keyboard and expose it to UV-C light from different\nangles. A robot with UV-C lights mounted to an arm, unlike the fixed UV-C lights of the\npassive robots, could actively position its arm to disinfect surfaces that are difficult to\naccess on immobilized objects or between objects.</p><p>Because these robots require more complex control algorithms to include the manipulation of\ntheir arms, development of these active robots has taken longer than the design of the\npassive robots, as evidenced by the availability of only passive robots in the commercial\nmarket. Furthermore, these robots are most advantageous if they are semi-autonomous or fully\nautonomous, freeing up a crucial staff member who would otherwise be occupied controlling or\ndirecting the robot. This requires the development of intricate planning algorithms, the\nintegration of sensory input for the movement of these robots’ manipulators, and the design\nand construction of the system.</p><p>Since the COVID-19 pandemic began, one Swiss startup has been circumnavigating the long\ndevelopment period by adapting a previous design. Rovenso has existing designs for mobile\nrobots for autonomous security and monitoring purposes, including a custom mobile base\ncapable of navigating varying terrain and even climbing stairs. Using a self-described\n“hack,” the Rovenso team has attached a UV-C light source to their ROVéo robot, which was\noriginally designed to autonomously monitor the security of industrial sites.<xref rid=\"c126\" ref-type=\"bibr\"><sup>126</sup></xref> Their design uses lidar to map the\nsurrounding environments and target highly used surfaces for UV-C disinfection. However,\nRovenso has designed their UV-C disinfection robot primarily for use in non-medical spaces\nwhere less stringent cleaning requirements are needed, such as offices and indoor\nworkspaces.</p><p>Although UV-C cannot and should not replace thermal and chemical disinfection in every\nscenario, UV-C can be used effectively in many situations where thermal and chemical methods\nare not possible or practical. Additionally, given the low consumable requirements, UV-C is\ncompatible with a wide range of operating environments and can provide an alternative\nmethod, particularly when supply chains are strained.</p></sec><sec id=\"s5\"><label>V.</label><title>FUTURE OUTLOOK AND CONCLUSION</title><p>Optical technologies have directly contributed to both preventing the spread of and\ndiagnosing COVID-19. This is truly indicative of the ubiquitous nature of photonics\ntechnologies. While many of the technologies discussed here are already in the community and\nin healthcare settings, many are still in nascent stages of development with their impact\nyet to be realized. Two areas that will experience significant growth are the coupling\nbetween photonics and robotics for automated disinfection and photonics, robotics, and\nartificial intelligence (AI) for high-throughput diagnostics. Both robotics and AI are\nexperiencing intellectual revolutions with synergistic advances, such as the development of\nautonomous robots. By combining these intelligent automated systems with photonic-based\ndisinfection or diagnostic technologies, our approach to disinfection and diagnostics could\nbe revolutionized.</p><p>As mentioned previously, one of the initial uses of UV-C disinfection was in environmental\napplications, including air and water purification, and this market remains the highest\nsector. Historically, the majority of these units were used in conjunction with filtration\nsystems to break down and remove contaminants related to the spread of airborne mold or\nother asthma-related particulates. However, given that COVID-19 spreads via airborne\ntransmission, the integration of UV-C into ventilation systems is one potential approach\nunder consideration for reducing transmission in indoor settings. UV-C would be particularly\nattractive for this application given its ability to disinfect without direct manual\nintervention. However, several challenges for long-term, effective operation must be\nconsidered, including the total dose required, the cost of operation, and the ability to\nreplace the UV-C source. In this type of system, high power UV LEDs would be the ideal\nsource, given their low cost of operation, their stable emission intensity over the\nlifetime, and their long operating lifetime. However, the initial cost of high power UV LEDs\nis significantly more than that of mercury bulbs. Thus, advances in photonics manufacturing\nwill greatly drive decision-making in this application. Last, it is important to note that\nthis type of system would not prevent direct person-to-person transmission. In this context,\nthe threat of infection from transmission through ventilation systems has not been\nrigorously established.</p><p>In a related application, performing disinfection of surfaces that we directly contact is\nmanually intensive. As a result, it is challenging to maintain a clean environment in\nhigh-traffic areas, such as public transit, public bathrooms, and sports arenas. Therefore,\nthese high contact points carry a high risk of transmission. An autonomous robot capable of\noperating independently would be able to reduce transmission risk by disinfecting more\nfrequently. This ability would not only reduce infections during the present COVID-19\npandemic but also improve overall healthcare in the future, as many seasonal flu viruses are\ntransmitted on surfaces. However, while this technology could have a clear positive societal\nimpact, it is also important to recognize and address the safety and ethical concerns.<xref rid=\"c127\" ref-type=\"bibr\"><sup>127,128</sup></xref> For example, an autonomous\nrobotic system that could potentially interact with people and pets would need to have\nintegrated safety measures to ensure that they were not exposed to the UV-C source. In\naddition, discussions on the complex ethical landscape in AI and robotics are currently\nongoing.</p><p>Throughout the COVID-19 pandemic, it has become evident that diagnosis is very complex,\nrelying on advances in biotechnology as well as sensing methods. Additionally, there are\nmany types of samples that can be analyzed. For example, while nasal swabs were the primary\ninitial samples, more recent antibody tests rely on blood. This diversity motivated the\ndevelopment of a range of robotic sample handling systems that required smaller sample\nvolumes and used less reagents per test. Additionally, the robotic systems accelerated the\nsample processing, enabling a significant increase in throughput. However, many of these\nrobotic systems relied on proprietary plasticware and biological reagents. As a result,\nshortages in either supply impacted diagnostic labs and have motivated many facilities to\ndevelop their own supply chains. This dependence on outside vendors, particularly for key\nreagents, is always an important consideration when developing a new diagnostic.</p><p>As discussed, in the antibody tests, both false positives and false negatives can occur.\nTherefore, to improve accuracy, it is necessary to evaluate not just a single test result,\nbut the result in the context of the patient’s other symptoms. Moreover, given the high\ntransmission risk, the time-to-result, or the speed with which a diagnostic test produces a\nresult, is paramount. While optical sensors produce results quickly, contextualization of\nthe results relies on additional information. By combining these results with a machine\nlearning-based AI optimized to contextualize the results, physicians will be armed with the\ntools needed to make higher accuracy treatment decisions on a faster timescale. Such a\nsystem not only would advance the present COVID-19 treatment strategy but also could change\nour approach to patient care.<xref rid=\"c62\" ref-type=\"bibr\"><sup>62,129</sup></xref>\nHowever, to be effective, machine learning systems require extensive learning libraries as\nwell as a better understanding of the disease pathophysiology.<xref rid=\"c19\" ref-type=\"bibr\"><sup>19,27,33</sup></xref> Because COVID-19 is so new, obtaining this\nkey information is extremely challenging.</p></sec><sec id=\"s6\"><title>AUTHORS’ CONTRIBUTIONS</title><p>All authors contributed equally to this work.</p></sec><sec sec-type=\"data-availability\" id=\"s7\"><title>DATA AVAILABILITY</title><p>Data sharing is not applicable to this article as no new data were created or analyzed in\nthis study.</p></sec></body><back><ack><title>ACKNOWLEDGMENTS</title><p>The authors thank Fakhar Singhera for assistance in preparing the figures in Biorender. The\nauthors would like to thank the National Science Foundation (Grant No. 2028446) and the\nCoNVaT project from the H2020 research and innovation programme of the European Commission\n(Project Grant No. 101003544). The ICN2 was funded by the CERCA programme/Generalitat de\nCatalunya. The ICN2 was supported by the Severo Ochoa Centres of Excellence programme,\nfunded by AEI (Grant No. SEV-2017-0706).</p></ack><ref-list><title>REFERENCES</title><ref id=\"c1\"><label>1.</label><mixed-citation publication-type=\"journal\"><string-name><given-names>J.</given-names>\n<surname>de Anda</surname></string-name>, <string-name><given-names>E. Y.</given-names>\n<surname>Lee</surname></string-name>, <string-name><given-names>C. K.</given-names>\n<surname>Lee</surname></string-name>, <string-name><given-names>R. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Genome Biol Evol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Genome Biol Evol</journal-id><journal-id journal-id-type=\"publisher-id\">gbe</journal-id><journal-title-group><journal-title>Genome Biology and Evolution</journal-title></journal-title-group><issn pub-type=\"epub\">1759-6653</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32853353</article-id><article-id pub-id-type=\"pmc\">PMC7523724</article-id><article-id pub-id-type=\"doi\">10.1093/gbe/evaa178</article-id><article-id pub-id-type=\"publisher-id\">evaa178</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Research Article</subject></subj-group><subj-group subj-group-type=\"category-taxonomy-collection\"><subject>AcademicSubjects/SCI01130</subject><subject>AcademicSubjects/SCI01140</subject></subj-group></article-categories><title-group><article-title>Divergent Evolution of Mutation Rates and Biases in the Long-Term Evolution Experiment with <italic>Escherichia coli</italic></article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0003-3370-092X</contrib-id><name><surname>Maddamsetti</surname><given-names>Rohan</given-names></name><xref ref-type=\"aff\" rid=\"evaa178-aff1\">e1</xref><xref ref-type=\"corresp\" rid=\"evaa178-cor1\"/><!--<email>rohan.maddamsetti@gmail.com</email>--></contrib><contrib contrib-type=\"author\"><name><surname>Grant</surname><given-names>Nkrumah A</given-names></name><xref ref-type=\"aff\" rid=\"evaa178-aff2\">e2</xref><xref ref-type=\"aff\" rid=\"evaa178-aff3\">e3</xref><xref ref-type=\"aff\" rid=\"evaa178-aff4\">e4</xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Zhang</surname><given-names>George</given-names></name><role>Associate Editor</role></contrib></contrib-group><aff id=\"evaa178-aff1\"><label>e1</label>\n<institution>Department of Biomedical Engineering, Duke University</institution>\n</aff><aff id=\"evaa178-aff2\"><label>e2</label>\n<institution>BEACON Center for the Study of Evolution in Action, Michigan State University</institution>\n</aff><aff id=\"evaa178-aff3\"><label>e3</label>\n<institution>Department of Microbiology and Molecular Genetics, Michigan State University</institution>\n</aff><aff id=\"evaa178-aff4\"><label>e4</label>\n<institution>Program in Ecology, Evolutionary Biology and Behavior, Michigan State University</institution>\n</aff><author-notes><corresp id=\"evaa178-cor1\">Corresponding author: E-mail: <email>rohan.maddamsetti@gmail.com</email>.</corresp></author-notes><pub-date pub-type=\"collection\"><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\" iso-8601-date=\"2020-08-27\"><day>27</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>27</day><month>8</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>12</volume><issue>9</issue><fpage>1591</fpage><lpage>1603</lpage><history><date date-type=\"accepted\"><day>21</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"cc-by\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"evaa178.pdf\"/><abstract><title>Abstract</title><p>All organisms encode enzymes that replicate, maintain, pack, recombine, and repair their genetic material. For this reason, mutation rates and biases also evolve by mutation, variation, and natural selection. By examining metagenomic time series of the Lenski long-term evolution experiment (LTEE) with <italic>Escherichia coli</italic> (Good BH, McDonald MJ, Barrick JE, Lenski RE, Desai MM. 2017. The dynamics of molecular evolution over 60,000 generations. Nature 551(7678):45–50.), we find that local mutation rate variation has evolved during the LTEE. Each LTEE population has evolved idiosyncratic differences in their rates of point mutations, indels, and mobile element insertions, due to the fixation of various hypermutator and antimutator alleles. One LTEE population, called Ara+3, shows a strong, symmetric wave pattern in its density of point mutations, radiating from the origin of replication. This pattern is largely missing from the other LTEE populations, most of which evolved missense, indel, or structural mutations in <italic>topA</italic>, <italic>fis</italic>, and <italic>dusB</italic>—loci that all affect DNA topology. The distribution of mutations in those genes over time suggests epistasis and historical contingency in the evolution of DNA topology, which may have in turn affected local mutation rates. Overall, the replicate populations of the LTEE have largely diverged in their mutation rates and biases, even though they have adapted to identical abiotic conditions.</p></abstract><kwd-group><kwd>experimental evolution</kwd><kwd>metagenomics</kwd><kwd>mutation</kwd></kwd-group><funding-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>National Science Foundation</institution><institution-id institution-id-type=\"DOI\">10.13039/100000001</institution-id></institution-wrap></funding-source><award-id>DEB-1951307</award-id></award-group></funding-group><counts><page-count count=\"13\"/></counts></article-meta></front><body><boxed-text id=\"evaa178-BOX1\" position=\"float\" orientation=\"portrait\"><sec><title>Significance</title><p>Bacteria often evolve elevated mutation rates during adaptation to challenging environments. Less is known about how mutation rates vary over the chromosome, and how those local biases evolve during adaptive evolution. To answer this question, we analyzed metagenomic data from an ongoing experiment with <italic>Escherichia coli</italic> in which 12 replicate populations of bacteria, started from a single clonal strain in 1988, were allowed to evolve for more than 30 years. We find that each replicate population has a different genomic distribution of observed mutations, indicating that local mutation rates have evolved idiosyncratically, even though each population has adapted to the same laboratory conditions. Intriguingly, our results indicate that adaptive mutations that change DNA topology may also affect local mutation rates.</p></sec></boxed-text><sec sec-type=\"intro\"><title>Introduction</title><p>Loci that modify DNA repair and recombination modify the evolutionary process. Therefore, one might ask whether natural selection adaptively tunes mutation and recombination rates. This idea—that second-order selection <italic>adaptively</italic> modifies the evolutionary process itself—is debated (<xref rid=\"evaa178-B51\" ref-type=\"bibr\">Tenaillon et al. 2001</xref>; <xref rid=\"evaa178-B29\" ref-type=\"bibr\">Lynch et al. 2016</xref>). Nonetheless, populations of <italic>Escherichia coli</italic>, engineered to have constitutive sexual recombination and elevated mutation rates, adapt faster than control populations in the laboratory (<xref rid=\"evaa178-B6\" ref-type=\"bibr\">Cooper 2007</xref>; <xref rid=\"evaa178-B41\" ref-type=\"bibr\">Peabody et al. 2016</xref>, <xref rid=\"evaa178-B42\" ref-type=\"bibr\">2017</xref>).</p><p>To study second-order selection on mutation rates, one can use experimental evolution. By running experiments in which replicate populations evolve under controlled conditions, with different starting mutation rates, one can ask whether particular mutation rates are favored over others (<xref rid=\"evaa178-B4\" ref-type=\"bibr\">Chao et al. 1983</xref>; <xref rid=\"evaa178-B28\" ref-type=\"bibr\">Loh et al. 2010</xref>; <xref rid=\"evaa178-B47\" ref-type=\"bibr\">Sprouffske et al. 2018</xref>). Here, we use metagenomic time series data from the Lenski long-term evolution experiment (LTEE) with <italic>E. coli</italic> to study how mutation rates evolve in real time.</p><p>In the LTEE, 12 populations of <italic>E. coli</italic>, descended from a common ancestral strain, have adapted for more than 73,000 generations to carbon-limited minimal media. Six of the populations are labeled Ara+, whereas the other six are labeled Ara−, based on the presence or absence of an evolutionarily neutral arabinose marker (<xref rid=\"evaa178-B25\" ref-type=\"bibr\">Lenski et al. 1991</xref>). The LTEE populations are strictly asexual. Some populations have evolved defects in DNA repair which vastly increase their point mutation rates. The causative hypermutator alleles likely went to fixation by linkage with highly beneficial mutations, rather than being beneficial per se (<xref rid=\"evaa178-B46\" ref-type=\"bibr\">Sniegowski et al. 1997</xref>; <xref rid=\"evaa178-B50\" ref-type=\"bibr\">Tenaillon et al. 2016</xref>). We refer to the LTEE populations that have evolved large increases in point mutation rates as “hypermutator populations,” and refer to the others as “nonmutator populations.”</p><p>Molecular evolution in the hypermutator populations of the LTEE is dominated by “genetic draft,” in which large numbers of nearly neutral passenger mutations hitchhike with a small number of beneficial driver mutations (<xref rid=\"evaa178-B37\" ref-type=\"bibr\">Neher 2013</xref>). This phenomenon has obscured the genomic signatures of adaptation in those populations (<xref rid=\"evaa178-B50\" ref-type=\"bibr\">Tenaillon et al. 2016</xref>; <xref rid=\"evaa178-B7\" ref-type=\"bibr\">Couce et al. 2017</xref>; <xref rid=\"evaa178-B18\" ref-type=\"bibr\">Good et al. 2017</xref>; <xref rid=\"evaa178-B31\" ref-type=\"bibr\">Maddamsetti et al. 2017</xref>). In this regime, also called “emergent neutrality” (<xref rid=\"evaa178-B44\" ref-type=\"bibr\">Schiffels et al. 2011</xref>), the evolutionary dynamics inferred from whole-population samples of the hypermutator populations (<xref rid=\"evaa178-B18\" ref-type=\"bibr\">Good et al. 2017</xref>) provides good data on mutation rates and biases, even though natural selection drives the dynamics. Here, we examined LTEE metagenomics data (<xref rid=\"evaa178-B18\" ref-type=\"bibr\">Good et al. 2017</xref>) for mutation rate variation and biases over the chromosome (<xref rid=\"evaa178-B14\" ref-type=\"bibr\">Foster et al. 2013</xref>; <xref rid=\"evaa178-B40\" ref-type=\"bibr\">Paul et al. 2013</xref>; <xref rid=\"evaa178-B20\" ref-type=\"bibr\">Jee et al. 2016</xref>; <xref rid=\"evaa178-B38\" ref-type=\"bibr\">Niccum et al. 2019</xref>).</p></sec><sec sec-type=\"results\"><title>Results</title><sec><title>Cumulative Number of Observed Mutations in Each Population Reveals Dynamics Caused by Both Hypermutator and Antimutator Alleles</title><p>We examined the number of observed mutations over time in each LTEE population (<xref ref-type=\"fig\" rid=\"evaa178-F1\">figs. 1 and 2</xref>, supplementary figs. S1–S3, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). These results show that mutation rates have evolved idiosyncratically over the LTEE. <xref ref-type=\"fig\" rid=\"evaa178-F1\">Figure 1<italic>A</italic></xref> shows the number of point mutations over time in each population. The rate of observed point mutations decreased in three of the six hypermutator populations (Ara−2, Ara+3, and Ara+6). The decrease in the rate of molecular evolution in these populations was previously ascribed to the evolution of antimutator alleles (<xref rid=\"evaa178-B50\" ref-type=\"bibr\">Tenaillon et al. 2016</xref>; <xref rid=\"evaa178-B18\" ref-type=\"bibr\">Good et al. 2017</xref>). Although antimutator alleles of <italic>mutY</italic> compensating for defects in <italic>mutT</italic> have been reported in Ara−1 (<xref rid=\"evaa178-B52\" ref-type=\"bibr\">Wielgoss et al. 2013</xref>), the change in slope observed at 40,000 generations in Ara−1 is subtle compared with the slope changes in Ara−2, Ara+3, and Ara+6.</p><fig id=\"evaa178-F1\" orientation=\"portrait\" position=\"float\"><label><sc>Fig</sc>. 1.</label><caption><p>Divergent evolution of mutation rates in the LTEE. Each panel shows the cumulative number of observed mutations, subdivided by mutation class, over time in each LTEE population. The top six panels show the nonmutator LTEE populations, and the bottom six panels show the hypermutator LTEE populations. (<italic>A</italic>) Point mutations are shown in red. (<italic>B</italic>) Indel mutations are shown in purple. (<italic>C</italic>) sv associated with transposons are shown in green, whereas those that are not associated with transposons are shown in gray.</p></caption><graphic xlink:href=\"evaa178f1\"/></fig><fig id=\"evaa178-F2\" orientation=\"portrait\" position=\"float\"><label><sc>Fig</sc>. 2.</label><caption><p>The dynamics of hypermutator and antimutator alleles affect the spectrum of observed point mutations over time. (<italic>A</italic>) Spectrum of point mutations over time in the hypermutator LTEE populations. (<italic>B</italic>) Inset figure showing nondominant point mutation spectra over time in the hypermutator LTEE populations.</p></caption><graphic xlink:href=\"evaa178f2\"/></fig><p>\n<xref ref-type=\"fig\" rid=\"evaa178-F1\">Figure 1<italic>B</italic></xref> shows the number of observed indel mutations over time in each population. Five of the six point-mutation hypermutator populations also show an indel hypermutator phenotype. These five populations all evolved defects in mismatch repair (MMR) (<xref rid=\"evaa178-T1\" ref-type=\"table\">table 1</xref> and <xref ref-type=\"fig\" rid=\"evaa178-F4\">fig. 4</xref>). The exception is Ara−1, which evolved a frameshift <italic>mutT</italic> allele (<xref rid=\"evaa178-T1\" ref-type=\"table\">table 1</xref> and <xref ref-type=\"fig\" rid=\"evaa178-F3\">fig. 3</xref>) that induces a high point mutation rate, absent a corresponding indel hypermutator phenotype.</p><fig id=\"evaa178-F3\" orientation=\"portrait\" position=\"float\"><label><sc>Fig</sc>. 3.</label><caption><p>Oxidative damage repair alleles in hypermutator LTEE populations. This visualization uses computer code from <xref rid=\"evaa178-B18\" ref-type=\"bibr\">Good et al. (2017)</xref>. Stars indicate the time (and allele frequency) at which mutations are reliably estimated to appear in the time series. The allele frequency trajectories for all observed mutations in the hypermutator populations are shown in gray. The allele frequency trajectories of de novo mutations (excepting synonymous mutations) in oxidative damage repair genes (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary file 1</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online) are colored and labeled in each population.</p></caption><graphic xlink:href=\"evaa178f3\"/></fig><table-wrap id=\"evaa178-T1\" orientation=\"portrait\" position=\"float\"><label>Table 1.</label><caption><p>Putative Hypermutator and Antimutator Alleles Described in the Text</p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col valign=\"top\" align=\"left\" span=\"1\"/><col valign=\"top\" align=\"left\" span=\"1\"/><col valign=\"top\" align=\"left\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/><col valign=\"top\" align=\"char\" char=\".\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/></colgroup><thead><tr><th rowspan=\"1\" colspan=\"1\">Population</th><th rowspan=\"1\" colspan=\"1\">Gene</th><th rowspan=\"1\" colspan=\"1\">DNA Repair Pathway</th><th rowspan=\"1\" colspan=\"1\">Appearance Time (Generations)</th><th rowspan=\"1\" colspan=\"1\">Position (bp)</th><th rowspan=\"1\" colspan=\"1\">Mutation</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Ara−1</td><td rowspan=\"1\" colspan=\"1\">\n<italic>uvrC</italic>\n</td><td rowspan=\"1\" colspan=\"1\">Oxidative damage repair</td><td rowspan=\"1\" colspan=\"1\">26,250</td><td rowspan=\"1\" colspan=\"1\">1,972,086</td><td rowspan=\"1\" colspan=\"1\">Q183P</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara−1</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutT</italic>\n</td><td rowspan=\"1\" colspan=\"1\">Oxidative damage repair</td><td rowspan=\"1\" colspan=\"1\">26,250</td><td rowspan=\"1\" colspan=\"1\">114,034</td><td rowspan=\"1\" colspan=\"1\">(C)<sub>6→7</sub></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara−1</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutY</italic>\n</td><td rowspan=\"1\" colspan=\"1\">Oxidative damage repair</td><td rowspan=\"1\" colspan=\"1\">28,750</td><td rowspan=\"1\" colspan=\"1\">2,988,792</td><td rowspan=\"1\" colspan=\"1\">L40W</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara−1</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutY</italic>\n</td><td rowspan=\"1\" colspan=\"1\">Oxidative damage repair</td><td rowspan=\"1\" colspan=\"1\">32,250</td><td rowspan=\"1\" colspan=\"1\">2,989,164</td><td rowspan=\"1\" colspan=\"1\">L164*</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara−2</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutL</italic>\n</td><td rowspan=\"1\" colspan=\"1\">MMR</td><td rowspan=\"1\" colspan=\"1\">2,250</td><td rowspan=\"1\" colspan=\"1\">4,375,786</td><td rowspan=\"1\" colspan=\"1\">(TGGCGC)<sub>3→4</sub></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara−2</td><td rowspan=\"1\" colspan=\"1\">\n<italic>uvrA</italic>\n</td><td rowspan=\"1\" colspan=\"1\">Oxidative damage repair</td><td rowspan=\"1\" colspan=\"1\">12,250</td><td rowspan=\"1\" colspan=\"1\">4,251,585</td><td rowspan=\"1\" colspan=\"1\">A407T</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara−2</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutT</italic>\n</td><td rowspan=\"1\" colspan=\"1\">Oxidative damage repair</td><td rowspan=\"1\" colspan=\"1\">13,750</td><td rowspan=\"1\" colspan=\"1\">114,113</td><td rowspan=\"1\" colspan=\"1\">R89H </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara−2</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutL</italic>\n</td><td rowspan=\"1\" colspan=\"1\">MMR</td><td rowspan=\"1\" colspan=\"1\">*This in-frame reversion fixes at 42,250 generations</td><td rowspan=\"1\" colspan=\"1\">4,375,781</td><td rowspan=\"1\" colspan=\"1\">(TGGCGC)<sub>3→2</sub></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara−3</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutS</italic>\n</td><td rowspan=\"1\" colspan=\"1\">MMR</td><td rowspan=\"1\" colspan=\"1\">34,750</td><td rowspan=\"1\" colspan=\"1\">2,753,768</td><td rowspan=\"1\" colspan=\"1\">Q606*</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara−3</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutY</italic>\n</td><td rowspan=\"1\" colspan=\"1\">Oxidative damage repair</td><td rowspan=\"1\" colspan=\"1\">48,250</td><td rowspan=\"1\" colspan=\"1\">2,989,624</td><td rowspan=\"1\" colspan=\"1\">Δ1 bp</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara−4</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutL</italic>\n</td><td rowspan=\"1\" colspan=\"1\">MMR</td><td rowspan=\"1\" colspan=\"1\">7,250</td><td rowspan=\"1\" colspan=\"1\">4,375,781</td><td rowspan=\"1\" colspan=\"1\">(TGGCGC)<sub>3→2</sub></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara+3</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutS</italic>\n</td><td rowspan=\"1\" colspan=\"1\">MMR</td><td rowspan=\"1\" colspan=\"1\">2,750</td><td rowspan=\"1\" colspan=\"1\">2,752,473</td><td rowspan=\"1\" colspan=\"1\">+G</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara+6</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutS</italic>\n</td><td rowspan=\"1\" colspan=\"1\">MMR</td><td rowspan=\"1\" colspan=\"1\">1,250</td><td rowspan=\"1\" colspan=\"1\">2,752,473</td><td rowspan=\"1\" colspan=\"1\">+G</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara+6</td><td rowspan=\"1\" colspan=\"1\">\n<italic>uvrA</italic>\n</td><td rowspan=\"1\" colspan=\"1\">Oxidative damage repair</td><td rowspan=\"1\" colspan=\"1\">4,750</td><td rowspan=\"1\" colspan=\"1\">4,250,341</td><td rowspan=\"1\" colspan=\"1\">I821M</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara+6</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutT</italic>\n</td><td rowspan=\"1\" colspan=\"1\">Oxidative damage repair</td><td rowspan=\"1\" colspan=\"1\">4,750</td><td rowspan=\"1\" colspan=\"1\">114,034</td><td rowspan=\"1\" colspan=\"1\">(C)<sub>6→5</sub></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara+6</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutY</italic>\n</td><td rowspan=\"1\" colspan=\"1\">Oxidative damage repair</td><td rowspan=\"1\" colspan=\"1\">31,750</td><td rowspan=\"1\" colspan=\"1\">2,988,917</td><td rowspan=\"1\" colspan=\"1\">Y82D</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Ara+6</td><td rowspan=\"1\" colspan=\"1\">\n<italic>mutY</italic>\n</td><td rowspan=\"1\" colspan=\"1\">Oxidative damage repair</td><td rowspan=\"1\" colspan=\"1\">49,750</td><td rowspan=\"1\" colspan=\"1\">2,989,297</td><td rowspan=\"1\" colspan=\"1\">C208W</td></tr></tbody></table></table-wrap><p>The hypermutator dynamics in Ara−2 are particularly striking. An antimutator allele eventually fixes, and reverts both the point and indel hypermutator phenotype back to ancestral or near ancestral levels (<xref ref-type=\"fig\" rid=\"evaa178-F1\">fig. 1<italic>A</italic> and <italic>B</italic></xref>). The hypermutator phenotype is caused by phase variation of a (TGGCGC)<sub>3</sub> repeat in <italic>mutL</italic> (<xref rid=\"evaa178-T1\" ref-type=\"table\">table 1</xref>). Reversions to the triplet state reverse the hypermutator phenotype. The number of new point and indel mutations in Ara−2 (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary figs. S1 and S2</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online) fluctuates with the allele frequency dynamics of this <italic>mutL</italic> repeat (<xref ref-type=\"fig\" rid=\"evaa178-F4\">fig. 4</xref>). Although fixations are usually irreversible in large asexual populations, phase variation is an exception: polymerases often slip on repetitive sequences, causing those repeats to expand or contract at relatively high rates (<xref rid=\"evaa178-B36\" ref-type=\"bibr\">Moxon et al. 2006</xref>).</p><fig id=\"evaa178-F4\" orientation=\"portrait\" position=\"float\"><label><sc>Fig</sc>. 4.</label><caption><p>MMR alleles in the hypermutator LTEE populations. This visualization uses computer code from <xref rid=\"evaa178-B18\" ref-type=\"bibr\">Good et al. (2017)</xref>. Stars indicate the time (and allele frequency) at which mutations are reliably estimated to appear in the time series. The allele frequency trajectories for all observed mutations in the hypermutator populations are shown in gray. The allele frequency trajectories of de novo mutations (except synonymous mutations) in MMR genes (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary file 1</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online) are colored and labeled in each population.</p></caption><graphic xlink:href=\"evaa178f4\"/></fig><p>At first glance, <xref ref-type=\"fig\" rid=\"evaa178-F1\">figure 1<italic>B</italic></xref> seems to show that Ara+6 fixed a mutation reverting the indel hypermutator phenotype. However, a close examination of the indel mutation rate and allele frequency dynamics in Ara+6 reveals that a super-hypermutator clade evolved within the first 1,000 generations (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S2</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Additional evidence for the super-hypermutator clade comes from the evolution and extinction of an A:T→G:C and G:C→A:T hypermutator phenotype (<xref ref-type=\"fig\" rid=\"evaa178-F2\">fig. 2</xref>) that parallels the evolution of the indel hypermutator phenotype. This super-hypermutator clade carries a frameshift allele of the MMR gene <italic>mutS</italic> (<xref rid=\"evaa178-T1\" ref-type=\"table\">table 1</xref> and <xref ref-type=\"fig\" rid=\"evaa178-F4\">fig. 4</xref>), is distinguished by marker alleles of the nucleotide excision repair genes <italic>uvrA</italic> and <italic>uvrB</italic> (<xref ref-type=\"fig\" rid=\"evaa178-F3\">fig. 3</xref>), and persists at low frequency until going extinct by 20,000 generations (<xref ref-type=\"fig\" rid=\"evaa178-F3\">figs. 3 and 4</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S2</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). The majority clade in Ara+6 evolved a mutation in <italic>mutT</italic> at 4,750 generations (<xref rid=\"evaa178-T1\" ref-type=\"table\">table 1</xref> and <xref ref-type=\"fig\" rid=\"evaa178-F3\">fig. 3</xref>) that causes a point mutation hypermutator phenotype without causing an indel hypermutator phenotype. The coexistence of clades with different hypermutator phenotypes, and the eventual extinction of the super-hypermutator clade, most reasonably explains the loss of the indel hypermutator phenotype from Ara+6.</p><p>\n<xref ref-type=\"fig\" rid=\"evaa178-F1\">Figure 1<italic>C</italic></xref> shows the number of observed structural mutations over time. As described in the original report for this data set (<xref rid=\"evaa178-B18\" ref-type=\"bibr\">Good et al. 2017</xref>), structural mutations (or structural variants, sv) are defined by junctions between two distinct locations in the reference genome. The vast majority of these structural mutations are caused by insertion sequence (IS) transpositions. Three of the canonical nonmutator populations (Ara−5, Ara−6, and Ara+1) show an IS hypermutator phenotype. The IS hypermutator phenotype in Ara+1 was reported previously (<xref rid=\"evaa178-B39\" ref-type=\"bibr\">Papadopoulos et al. 1999</xref>; <xref rid=\"evaa178-B50\" ref-type=\"bibr\">Tenaillon et al. 2016</xref>). In contrast, only one of the canonical hypermutator populations, Ara−3, shows an IS hypermutator phenotype. The rate of observed structural mutations in Ara−3 shows three different slopes. Ara−3 evolved an IS hypermutator phenotype very early in the LTEE. Around 30,000 generations, the IS rate intensifies, either due to genetic evolution, or as a consequence of stress induced by the citrate metabolic innovation that evolved around that time (<xref rid=\"evaa178-B3\" ref-type=\"bibr\">Blount et al. 2012</xref>, <xref rid=\"evaa178-B2\" ref-type=\"bibr\">2020</xref>). Finally, the IS rate decreases around 45,000 generations. More than 100 mutations go to fixation in the selective sweep at 45,000 generations in Ara−3, including mutations in the DNA repair genes <italic>recR</italic>, <italic>recE</italic>, <italic>ligA</italic>, <italic>uvrA</italic>, and <italic>ybaZ</italic>. The distinct IS rates observed in Ara−3 may, in part, reflect clonal interference between deeply diverged, competing lineages in that population (<xref rid=\"evaa178-B3\" ref-type=\"bibr\">Blount et al. 2012</xref>; <xref rid=\"evaa178-B26\" ref-type=\"bibr\">Leon et al. 2018</xref>), especially if those lineages have different IS transposition rates.</p><p>We also examined the spectrum of point mutations in each hypermutator population over time (<xref ref-type=\"fig\" rid=\"evaa178-F2\">fig. 2</xref>). Ara–1 and Ara+6 show a high frequency of A:T→C:G transversion mutations, characteristic of defects in <italic>mutT</italic> (<xref rid=\"evaa178-B49\" ref-type=\"bibr\">Tajiri et al. 1995</xref>; <xref rid=\"evaa178-B16\" ref-type=\"bibr\">Fowler et al. 2003</xref>; <xref rid=\"evaa178-B52\" ref-type=\"bibr\">Wielgoss et al. 2013</xref>). Ara–2, Ara–3, Ara–4, and Ara+3, which all have defects in MMR (<xref rid=\"evaa178-T1\" ref-type=\"table\">table 1</xref> and <xref ref-type=\"fig\" rid=\"evaa178-F4\">fig. 4</xref>), show a high frequency of A:T→G:C and G:C→A:T mutations. These findings are consistent with genomic analyses of LTEE hypermutators (<xref rid=\"evaa178-B7\" ref-type=\"bibr\">Couce et al. 2017</xref>). Furthermore, Ara−1, Ara−3, and Ara+6 all show late increases in the frequency of G:C→T:A transversion mutations, characteristic of defects in <italic>mutY</italic> (<xref rid=\"evaa178-B49\" ref-type=\"bibr\">Tajiri et al. 1995</xref>; <xref rid=\"evaa178-B16\" ref-type=\"bibr\">Fowler et al. 2003</xref>; <xref rid=\"evaa178-B52\" ref-type=\"bibr\">Wielgoss et al. 2013</xref>).</p><p>In examining <italic>mutT</italic>, we noticed that two of the three cases of <italic>mutT</italic> alleles arising to high frequency in the LTEE occur on an <italic>uvrA</italic> background (Ara−2 and Ara+6), whereas the third, in Ara−1, occurs on an <italic>uvrC</italic> background (<xref ref-type=\"fig\" rid=\"evaa178-F3\">fig. 3</xref>). The <italic>mutT</italic> allele in Ara−2 does not cause the characteristic <italic>mutT</italic> A:T→C:G hypermutator phenotype found in Ara−1 and Ara+6 (<xref ref-type=\"fig\" rid=\"evaa178-F2\">fig. 2</xref>), so its association with <italic>uvrA</italic> may be coincidental. However, the same <italic>uvrA</italic> substitution that goes to fixation with <italic>mutT</italic> in Ara+6 also occurs in a 40,000 generation isolate from the Ara−1 population called REL10939 (<xref rid=\"evaa178-B50\" ref-type=\"bibr\">Tenaillon et al. 2016</xref>), which suggests that this particular <italic>uvrA</italic> allele may be beneficial in those contexts. Furthermore, it has been reported that <italic>uvrA/mutT</italic> and <italic>uvrB</italic>/<italic>mutT</italic> double knockouts have a substantially lower mutation rate than <italic>mutT</italic> knockouts, in the presence of hydrogen peroxide (<xref rid=\"evaa178-B19\" ref-type=\"bibr\">Hori et al. 2007</xref>). Based on these observations, we hypothesize that the <italic>mutT</italic> alleles that successfully went to fixation in the LTEE may have evolved on an <italic>uvrABC</italic> genetic background that reduced the intensity of the <italic>mutT</italic> hypermutator phenotype.</p></sec><sec><title>Gene-Orientation Mutation Bias Evolves in the LTEE</title><p>Several reports indicate that mutation rates differ between the leading and lagging strands of the DNA replication bubble (<xref rid=\"evaa178-B24\" ref-type=\"bibr\">Lee et al. 2012</xref>; <xref rid=\"evaa178-B40\" ref-type=\"bibr\">Paul et al. 2013</xref>). Potential causes include asymmetry in nucleotide composition around the replication origin (GC skew) (<xref rid=\"evaa178-B34\" ref-type=\"bibr\">Marín and Xia 2008</xref>), context-dependent mutation rates that are asymmetric around the replication origin (<xref rid=\"evaa178-B48\" ref-type=\"bibr\">Sung et al. 2015</xref>), and head-on collisions between the replication and transcription molecular machinery (<xref rid=\"evaa178-B40\" ref-type=\"bibr\">Paul et al. 2013</xref>). Such reports motivated us to ask whether the LTEE metagenomics data showed evidence of gene-orientation mutation biases, such that genes oriented with (or against) the leading or lagging strand of DNA synthesis have different mutation rates.</p><p>Our null expectation is that the distribution of synonymous mutations on each strand of the chromosome should be related to the amount of coding sequence on each strand (i.e., the density of genes multiplied by their length). Furthermore, the spectrum of nucleotide substitutions on each strand should reflect local G:C content in the ancestral LTEE clone REL606: for example, G:C→A:T substitutions should be more common in G:C-rich regions. <xref ref-type=\"fig\" rid=\"evaa178-F5\">Figure 5<italic>A</italic></xref> shows this null expectation. Both the amount of coding sequence and G:C content per strand are asymmetric about the replication origin of REL606. At the replication origin, one DNA strand switches from leading to lagging, while its complement switches from lagging to leading. This switch occurs because DNA replication is bidirectional, such that two replisomes move in opposite directions from the replication origin. Even in the absence of gene-orientation mutation bias, <xref ref-type=\"fig\" rid=\"evaa178-F5\">figure 5<italic>A</italic></xref> shows that some asymmetry in the distribution of synonymous mutations over the replication origin is expected.</p><fig id=\"evaa178-F5\" orientation=\"portrait\" position=\"float\"><label><sc>Fig</sc>. 5.</label><caption><p>Gene-orientation mutation bias evolves in the LTEE. The <italic>x</italic> axis is the reference genome, centered on the replication origin, partitioned into 46 equally sized bins of ∼100 kb. In each labeled subfigure, top and bottom panels show genes occurring on each of the two strands of the chromosome, with the arbitrary labels 1 and −1. (<italic>A</italic>) The nucleotide composition of genes on the two strands of the chromosome of the LTEE ancestral clone REL606. (<italic>B</italic>) The genomic distribution of mutations within genes, summed over MMR-deficient LTEE populations (left panel) and MutT-deficient LTEE populations (right panel).</p></caption><graphic xlink:href=\"evaa178f5\"/></fig><p>The observed distributions of synonymous mutations on each strand of the chromosome are shown in <xref ref-type=\"fig\" rid=\"evaa178-F5\">figure 5<italic>B</italic></xref>. We separately analyzed MMR- and MutT-deficient hypermutator populations. In both cases, the number of observed mutations significantly differs between genes oriented with or against the movement of the replisome, based on comparing the expected ratio of mutations to the observed ratio of mutations. The MMR-deficient hypermutator populations show significantly more gene-orientation mutation bias than expected (two-tailed binomial test: observed ratio of 2,066:2,664 mutations vs. expected ratio of 1,730,238:2,066,587 nucleotides; <italic>P </italic>=<italic> </italic>0.0090), whereas the MutT-deficient hypermutator populations show significantly less gene-orientation bias than expected (two-tailed binomial test: observed ratio of 947:1,033 mutations vs. expected ratio of 1,730,238:2,066,587 nucleotides; <italic>P </italic>=<italic> </italic>0.0446). Note that these calculations do not account for the characteristic mutation spectra of MMR- and MutT-deficient hypermutators (<xref ref-type=\"fig\" rid=\"evaa178-F5\">fig. 5<italic>B</italic></xref>). For example, the extreme rate of A:T→C:G mutations seen in MutT-deficient hypermutators (<xref rid=\"evaa178-B15\" ref-type=\"bibr\">Foster et al. 2015</xref>) should cause A:T rich genes to mutate faster than A:T poor genes.</p></sec><sec><title>The Genomic Distribution of Observed Mutations in Ara+3 Shows a Strong, Symmetric Wave Pattern over the Origin of Replication</title><p>Multiple studies (<xref rid=\"evaa178-B45\" ref-type=\"bibr\">Sharp et al. 1989</xref>; <xref rid=\"evaa178-B22\" ref-type=\"bibr\">Lang and Murray 2011</xref>; <xref rid=\"evaa178-B14\" ref-type=\"bibr\">Foster et al. 2013</xref>; <xref rid=\"evaa178-B12\" ref-type=\"bibr\">Dillon et al. 2018</xref>; <xref rid=\"evaa178-B38\" ref-type=\"bibr\">Niccum et al. 2019</xref>) have reported correlations between local mutation rates and distance from the origin of replication. One hypermutator LTEE population, called Ara+3, shows a symmetric wave pattern reflected over <italic>oriC</italic> (<xref ref-type=\"fig\" rid=\"evaa178-F6\">fig. 6</xref>). Indeed, the genomic distribution of observed mutations in Ara+3 is significantly different from the genomic distribution of observed mutations summed over all hypermutator populations (two-sample Kolmogorov–Smirnov test: <italic>D</italic> = 0.0567, <italic>P </italic><<italic> </italic>10<sup>−14</sup>). The wave in Ara+3 has a trough-to-peak ratio of ∼25:75 (<xref ref-type=\"fig\" rid=\"evaa178-F6\">fig. 6</xref>). Excluding Ara+3, the genomic distribution of observed mutations summed over the remaining MMR-deficient LTEE populations shows a weak wave pattern, whereas the populations with defects in <italic>mutT</italic> shows no evidence of the wave pattern (<xref ref-type=\"fig\" rid=\"evaa178-F7\">fig. 7</xref>). The genomic distribution of observed mutations in the MMR-deficient populations (excluding Ara+3) is significantly different from the genomic distribution of observed mutations in the MutT-deficient populations (two-sample Kolmogorov–Smirnov test: <italic>D</italic> = 0.040916, <italic>P </italic><<italic> </italic>10<sup>−9</sup>).</p><fig id=\"evaa178-F6\" orientation=\"portrait\" position=\"float\"><label><sc>Fig</sc>. 6.</label><caption><p>One hypermutator LTEE population, Ara+3, shows a strong wave pattern of mutation rate variation centered on the replication origin. Each panel shows the genomic distribution of mutations observed in each hypermutator LTEE population in the metagenomics data. The <italic>x</italic> axis is the reference genome, centered on the replication origin, partitioned into 46 equally sized bins of ∼100 kb. Indels are in purple, missense mutations are in dark blue, noncoding mutations are blue green, nonsense mutations are sea green, sv are green, and synonymous mutations are yellow.</p></caption><graphic xlink:href=\"evaa178f6\"/></fig><fig id=\"evaa178-F7\" orientation=\"portrait\" position=\"float\"><label><sc>Fig</sc>. 7.</label><caption><p>MMR-deficient LTEE populations (excluding Ara+3) show a weak wave pattern, whereas MutT-deficient LTEE populations show no wave pattern. The left panel shows the genomic distribution of mutations observed in Ara−2, Ara−3, and Ara−4. The right panel shows the genomic distribution of mutations observed in Ara−1 and Ara+6. The <italic>x</italic> axis is the reference genome, centered on the replication origin, partitioned into 46 equally sized bins of ∼100 kb. Indels are in purple, missense mutations are in dark blue, noncoding mutations are blue green, nonsense mutations are sea green, sv are green, and synonymous mutations are yellow.</p></caption><graphic xlink:href=\"evaa178f7\"/></fig></sec><sec><title>Evidence for Epistasis and Historical Contingency in the Evolution of DNA Topology</title><p>Why does a strong wave pattern only appear in Ara+3? Others have hypothesized that local chromatin structure affects local mutation rates (<xref rid=\"evaa178-B14\" ref-type=\"bibr\">Foster et al. 2013</xref>; <xref rid=\"evaa178-B38\" ref-type=\"bibr\">Niccum et al. 2019</xref>). Furthermore, DNA topology has evolved in parallel in the LTEE, and artificially increasing DNA supercoiling is beneficial under LTEE conditions (<xref rid=\"evaa178-B9\" ref-type=\"bibr\">Crozat et al. 2005</xref>, <xref rid=\"evaa178-B8\" ref-type=\"bibr\">2010</xref>). Therefore, we hypothesized that mutations in genes that affect DNA topology might affect the wave pattern. To test this hypothesis, we examined the timing and distribution of mutations in <italic>topA</italic>, <italic>fis</italic>, and <italic>dusB</italic> (<italic>yhdG</italic>). We focused on these genes for several reasons. First, these loci show strong parallel evolution in the LTEE (<xref rid=\"evaa178-B8\" ref-type=\"bibr\">Crozat et al. 2010</xref>). Second, introducing evolved alleles of <italic>topA</italic> and <italic>fis</italic> into the ancestral genome are sufficient to confer a fitness benefit as well as additive changes to DNA topology (<xref rid=\"evaa178-B9\" ref-type=\"bibr\">Crozat et al. 2005</xref>). Finally, statistical analysis of the pattern of evolution for <italic>dusB</italic> and <italic>fis</italic> in the LTEE led to the discovery that <italic>dusB</italic> regulates <italic>fis</italic> expression (<xref rid=\"evaa178-B9\" ref-type=\"bibr\">Crozat et al. 2005</xref>, <xref rid=\"evaa178-B8\" ref-type=\"bibr\">2010</xref>). We excluded synonymous mutations from this analysis. We counted both fixations and mutations destined for extinction, because many beneficial mutations go extinct in large asexual populations due to clonal interference (<xref rid=\"evaa178-B17\" ref-type=\"bibr\">Gerrish and Lenski 1998</xref>; <xref rid=\"evaa178-B21\" ref-type=\"bibr\">Lang et al. 2013</xref>; <xref rid=\"evaa178-B27\" ref-type=\"bibr\">Levy et al. 2015</xref>; <xref rid=\"evaa178-B30\" ref-type=\"bibr\">Maddamsetti, Lenski, et al. 2015</xref>; <xref rid=\"evaa178-B1\" ref-type=\"bibr\">Ba et al. 2019</xref>).</p><p>All LTEE populations evolved missense, indel, or structural mutations in <italic>topA</italic>, <italic>fis</italic>, and <italic>dusB</italic> within the first 10,000 generations, except two: Ara+2 and Ara+3 (<xref ref-type=\"fig\" rid=\"evaa178-F8\">fig. 8</xref>). The timing and distribution of mutations in these genes across populations suggests epistasis and historical contingency (<xref rid=\"evaa178-B18\" ref-type=\"bibr\">Good et al. 2017</xref>). The early arrival times for mutations in these genes suggests that there is an early, limited window of opportunity for those mutations to go to fixation. Quantitative evidence comes from Ara+3, which has no missense, nonsense, indel, or structural mutations in <italic>topA</italic>, <italic>fis</italic>, and <italic>dusB</italic> whatsoever, despite its strong hypermutator phenotype. The probability of this event is <italic>P</italic> = (1−(<italic>t</italic>/<italic>g</italic>))<sup><italic>n</italic></sup>, where <italic>t</italic> is the effective mutational target size, <italic>g</italic> is the length of the chromosome (<italic>g = </italic>4,629,812), and <italic>n</italic> is the number of observed missense, indel, and structural mutations in Ara+3 (<italic>n </italic>=<italic> </italic>4,368). Given the wave pattern in Ara+3, the effective mutational target size of <italic>topA</italic>, <italic>fis</italic>, and <italic>dusB</italic> could be smaller than their combined physical target size (3,861 bp), say if they occurred in the trough of the wave. To take this into account, we partitioned the chromosome into bins, counted mutations per bin, and calculated the effective mutational target size by multiplying the physical target size (length) of <italic>topA</italic>, <italic>fis</italic>, and <italic>dusB</italic> by the number of mutations per base pair in their respective bins. These genes are significantly depleted of mutations in Ara+3, for bin sizes ranging from 100 kb to the entire chromosome (one-tailed randomization tests with 10,000 bootstraps: <italic>P </italic><<italic> </italic>0.05 in all cases).</p><fig id=\"evaa178-F8\" orientation=\"portrait\" position=\"float\"><label><sc>Fig</sc>. 8.</label><caption><p>The strong wave pattern in Ara+3 anticorrelates with mutations (excluding synonymous changes) in the DNA topology genes <italic>topA</italic>, <italic>fis</italic>, and <italic>dusB</italic>. This visualization uses computer code written by <xref rid=\"evaa178-B18\" ref-type=\"bibr\">Good et al. (2017)</xref>. The allele frequency trajectories for all observed mutations in the 12 LTEE populations are shown in gray. The allele frequency trajectories of de novo mutations in <italic>topA</italic>, <italic>fis</italic>, and <italic>dusB</italic> (excepting synonymous mutations) are colored and labeled in each population.</p></caption><graphic xlink:href=\"evaa178f8\"/></fig><p>The distribution of synonymous mutations in <italic>topA</italic>, <italic>fis</italic>, and <italic>dusB</italic> across the LTEE populations is interesting (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S4</xref> and <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). A single, synonymous A312A substitution in <italic>dusB</italic> went to fixation at ∼4,000 generations in Ara+3, simultaneously with alleles in the MMR genes <italic>mutS</italic> and <italic>mutH</italic> that apparently caused the early hypermutator phenotype in this population. No other synonymous mutations in <italic>dusB</italic> are observed in Ara+3. Furthermore, there is evidence of parallel evolution at this particular position in <italic>dusB</italic>. The same synonymous mutation occurs in Ara+6, and another synonymous mutation, one base pair downstream in the next codon, is the only synonymous mutation in <italic>topA</italic>, <italic>fis</italic>, or <italic>dusB</italic> observed in Ara−2 (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S4</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). This parallelism suggests that positive selection may be acting on these synonymous variants. Overall, it is striking how few synonymous mutations in <italic>topA</italic>, <italic>fis</italic>, and <italic>dusB</italic> occur in the hypermutator LTEE populations, which implies that synonymous variants in these genes may not be evolving neutrally. Indeed, STIMS (<xref rid=\"evaa178-B32\" ref-type=\"bibr\">Maddamsetti and Grant 2020</xref>) finds a significant signal of purifying selection on synonymous mutations in <italic>topA</italic>, <italic>fis</italic>, and <italic>dusB</italic> in Ara−1 and Ara−3 (one-tailed randomization test with 10,000 bootstraps: <italic>P </italic><<italic> </italic>0.0001).</p><p>We also examined the genes that encode the nucleoid-binding protein HU and the terminus-organizing protein MatP, as deletions of these loci were shown to affect the wave pattern (<xref rid=\"evaa178-B38\" ref-type=\"bibr\">Niccum et al. 2019</xref>). Notwithstanding the relevance of HU and MatP in <xref rid=\"evaa178-B38\" ref-type=\"bibr\">Niccum et al. (2019)</xref>, these genes show limited evidence of parallel evolution in the LTEE (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S5</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online).</p></sec><sec><title>Synonymous Nucleotide Diversity in Natural <italic>E. coli</italic> Populations Does Not Predict Mutation Rate Variation in the LTEE</title><p>Finally, we used the LTEE metagenomic data to revisit previous work, which found that the distribution of synonymous mutations in the LTEE does not reflect patterns of synonymous variation among natural <italic>E. coli</italic> isolates (<xref rid=\"evaa178-B33\" ref-type=\"bibr\">Maddamsetti et al. 2015</xref>). During our reanalysis, we found a potential coding error affecting the results of the Kolmogorov–Smirnov test reported in that paper. Therefore, we used Poisson regression to ask whether the estimates of synonymous nucleotide diversity <italic>θ</italic><sub>s</sub> published in <xref rid=\"evaa178-B35\" ref-type=\"bibr\">Martincorena et al. (2012)</xref>, when treated as gene-specific estimates of the point-mutation rate per base pair, predict the distribution of synonymous mutations observed in the LTEE. A null model in which mutations occur uniformly over the chromosome (Akaike’s Information Criterion, AIC = 8,529.6) fits the data far better than the <italic>θ</italic><sub>s</sub> model (AIC = 9,171.3). When we fit both models to Ara+3, we again find that the null model is better than the <italic>θ</italic><sub>s</sub> model at predicting the observed distribution of synonymous mutations (AIC = 2,168.2 for null model vs. AIC = 2,190.8 for <italic>θ</italic><sub>s</sub> model). This finding validates the conclusions reported in <xref rid=\"evaa178-B33\" ref-type=\"bibr\">Maddamsetti et al. (2015)</xref>, despite the potential problems in that analysis.</p></sec></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>By examining the distribution of observed mutations over more than 60,000 generations of the LTEE (<xref rid=\"evaa178-B18\" ref-type=\"bibr\">Good et al. 2017</xref>), we find that mutation rates and biases have diverged idiosyncratically, despite identical abiotic conditions. One LTEE population, Ara+3, shows strong evidence of the wave pattern in mutation rate variation. Similar patterns have been seen in mutation accumulation experiments with MMR-deficient strains of <italic>E. coli</italic> as well as in <italic>Vibrio</italic> bacteria (<xref rid=\"evaa178-B12\" ref-type=\"bibr\">Dillon et al. 2018</xref>; <xref rid=\"evaa178-B38\" ref-type=\"bibr\">Niccum et al. 2019</xref>). Our result shows that genomic biases in mutation rates evolve dynamically on laboratory timescales. It is likely that the identity and effects of many hypermutator and antimutator alleles in the LTEE remains unknown. For instance, we do not know what alleles, if any, cause the apparent late decrease in mutation rate seen in Ara+3. Experiments are needed, both to discover those unknown alleles, and to test for genetic interactions that modulate mutation rates in the LTEE, as we have hypothesized for alleles of <italic>uvrABC</italic> and <italic>mutT</italic>.</p><p>The divergence in the rates, biases, and spectra of mutations across replicate populations in this simple long-term evolution experiment makes one wonder about the scope of natural variation in mutation rates, biases, and spectra. An evolution experiment with replicate mouse microbiomes has indicated that microbial evolution in the gut is probably characterized by long-term maintenance of intraspecies genetic diversity, including mutation rate polymorphism (<xref rid=\"evaa178-B43\" ref-type=\"bibr\">Ramiro et al. 2020</xref>). Phylogenomic studies have also found extensive evidence for horizontal gene transfer in DNA repair genes (<xref rid=\"evaa178-B11\" ref-type=\"bibr\">Denamur et al. 2000</xref>), which suggests that polymorphism in DNA repair genes may cause extensive natural variation in mutation and recombination rates within and across bacterial (meta-) populations and communities.</p><p>We find statistical evidence for historical contingency and epistasis in the evolution of DNA topology in the LTEE, and for Ara+3 in particular. These findings suggest a relationship between local DNA topology and local mutation rate variation, consistent with the experiments reported by <xref rid=\"evaa178-B38\" ref-type=\"bibr\">Niccum et al. (2019)</xref>. These findings immediately suggest the need for experiments to test whether local DNA topology causes local mutation rate variation, and to test whether local DNA topology affects strand-specific and gene-orientation mutation biases.</p><p>A comparison of synonymous genetic variation estimated from natural <italic>E. coli</italic> isolates to the distribution of observed synonymous mutations in the LTEE confirms the conclusion of earlier work (<xref rid=\"evaa178-B33\" ref-type=\"bibr\">Maddamsetti et al. 2015</xref>) using richer data, and is consistent with other reports as well (<xref rid=\"evaa178-B24\" ref-type=\"bibr\">Lee et al. 2012</xref>; <xref rid=\"evaa178-B5\" ref-type=\"bibr\">Chen and Zhang 2013</xref>; <xref rid=\"evaa178-B29\" ref-type=\"bibr\">Lynch et al. 2016</xref>). In sum, gene-specific variation in synonymous nucleotide diversity <italic>θ</italic><sub>s</sub>, estimated from natural isolates of <italic>E. coli</italic>, does not predict the genomic distribution of synonymous mutations observed in the LTEE. In any case, the other results that we have presented, in addition to prior reports (<xref rid=\"evaa178-B14\" ref-type=\"bibr\">Foster et al. 2013</xref>; <xref rid=\"evaa178-B40\" ref-type=\"bibr\">Paul et al. 2013</xref>; <xref rid=\"evaa178-B48\" ref-type=\"bibr\">Sung et al. 2015</xref>; <xref rid=\"evaa178-B20\" ref-type=\"bibr\">Jee et al. 2016</xref>; <xref rid=\"evaa178-B38\" ref-type=\"bibr\">Niccum et al. 2019</xref>), strongly indicate that mutation rates vary over the <italic>E. coli</italic> chromosome.</p><p>These results add to the robust debate on the causes and consequences of mutation rate evolution. It is clear that a deeper understanding of the relationships among chromatin structure, genomic variation in mutation and recombination rates, and natural selection, and their consequences for short- and long-term genome evolution, will be a fruitful goal for further research.</p></sec><sec><title>Materials and Methods</title><p>Preprocessed LTEE metagenomic data, and associated analysis and visualization code was downloaded from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/benjaminhgood/LTEE-metagenomic\">https://github.com/benjaminhgood/LTEE-metagenomic</ext-link>. Analysis codes are available from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/rohanmaddamsetti/LTEE-purifying-selection/blob/master/mutation-rate-analysis.R\">https://github.com/rohanmaddamsetti/LTEE-purifying-selection/blob/master/mutation-rate-analysis.R</ext-link> and <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/rohanmaddamsetti/LTEE-purifying-selection/blob/master/metagenomics-library.R\">https://github.com/rohanmaddamsetti/LTEE-purifying-selection/blob/master/metagenomics-library.R</ext-link>. We systematically examined DNA repair genes in <italic>E. coli</italic> (<xref rid=\"evaa178-B13\" ref-type=\"bibr\">Eisen and Hanawalt 1999</xref>; <xref rid=\"evaa178-B23\" ref-type=\"bibr\">Lee et al. 2016</xref>; <xref rid=\"evaa178-B10\" ref-type=\"bibr\">Deatherage et al. 2018</xref>), as well as annotated DNA polymerases, and other proteins of the replisome. A table of these genes and their annotations is in <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary data</xref> file 1, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online. We cross-checked the evolutionary dynamics of alleles of these genes in the LTEE metagenomic data against the observed changes in mutation rates and spectra in each LTEE population. We also examined the LTEE genomic data (<xref rid=\"evaa178-B50\" ref-type=\"bibr\">Tenaillon et al. 2016</xref>) for mutations in these genes, using the R Shiny web app interface at <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.barricklab.org/shiny/LTEE-Ecoli\">www.barricklab.org/shiny/LTEE-Ecoli</ext-link>. In this manner, we curated a list of putative hypermutator and antimutator alleles in the LTEE (<xref rid=\"evaa178-T1\" ref-type=\"table\">table 1</xref>). Those alleles, and alleles of other genes in their respective DNA repair pathways, are shown in <xref ref-type=\"fig\" rid=\"evaa178-F3\">figures 3</xref> and <xref ref-type=\"fig\" rid=\"evaa178-F4\">4</xref>. <xref ref-type=\"fig\" rid=\"evaa178-F3\">Figure 3</xref> shows the evolutionary dynamics of alleles in genes encoding base excision repair, nucleotide excision repair, and degradation of oxidized nucleotide triphosphates. <xref ref-type=\"fig\" rid=\"evaa178-F4\">Figure 4</xref> shows the evolutionary dynamics of alleles in genes encoding DNA MMR. Data sets and analysis codes to replicate the findings and figures in this paper are available on the Dryad Digital Repository (DOI: https://doi.org/10.5061/dryad.kprr4xh2z.).</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><supplementary-material content-type=\"local-data\" id=\"sup1\"><label>evaa178_Supplementary_Data</label><media xlink:href=\"evaa178_supplementary_data.zip\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"ack1\"><title>Acknowledgments</title><p>We thank Richard Lenski and Helen Murphy for valuable discussions and advice. We thank Jeffrey Barrick for valuable discussions and comments on an earlier version of our manuscript. We also thank Benjamin Good for making preprocessed LTEE metagenomic data and analysis scripts accessible for the research community. The LTEE that generated the bacteria we used in this study is supported by a grant from the National Science Foundation (currently DEB-1951307) to Richard Lenski and Jeffrey Barrick. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Genome Biol Evol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Genome Biol Evol</journal-id><journal-id journal-id-type=\"publisher-id\">gbe</journal-id><journal-title-group><journal-title>Genome Biology and Evolution</journal-title></journal-title-group><issn pub-type=\"epub\">1759-6653</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32770231</article-id><article-id pub-id-type=\"pmc\">PMC7523725</article-id><article-id pub-id-type=\"doi\">10.1093/gbe/evaa165</article-id><article-id pub-id-type=\"publisher-id\">evaa165</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Genome Report</subject></subj-group><subj-group subj-group-type=\"category-taxonomy-collection\"><subject>AcademicSubjects/SCI01130</subject><subject>AcademicSubjects/SCI01140</subject></subj-group></article-categories><title-group><article-title>Complete Genome Sequence of the Polysaccharide-Degrading Rumen Bacterium <italic>Pseudobutyrivibrio xylanivorans</italic> MA3014 Reveals an Incomplete Glycolytic Pathway</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-2101-2452</contrib-id><name><surname>Palevich</surname><given-names>Nikola</given-names></name><xref ref-type=\"aff\" rid=\"evaa165-aff1\">e1</xref><xref ref-type=\"aff\" rid=\"evaa165-aff2\">e2</xref><xref ref-type=\"corresp\" rid=\"evaa165-cor1\"/><!--<email>nik.palevich@agresearch.co.nz</email>--></contrib><contrib contrib-type=\"author\"><name><surname>Maclean</surname><given-names>Paul H</given-names></name><xref ref-type=\"aff\" rid=\"evaa165-aff1\">e1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kelly</surname><given-names>William J</given-names></name><xref ref-type=\"aff\" rid=\"evaa165-aff3\">e3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Leahy</surname><given-names>Sinead C</given-names></name><xref ref-type=\"aff\" rid=\"evaa165-aff1\">e1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rakonjac</surname><given-names>Jasna</given-names></name><xref ref-type=\"aff\" rid=\"evaa165-aff2\">e2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Attwood</surname><given-names>Graeme T</given-names></name><xref ref-type=\"aff\" rid=\"evaa165-aff1\">e1</xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Ochman</surname><given-names>Howard</given-names></name><role>Associate Editor</role></contrib></contrib-group><aff id=\"evaa165-aff1\"><label>e1</label>\n<institution>AgResearch Limited, Grasslands Research Centre</institution>, Palmerston North, <country country=\"NZ\">New Zealand</country></aff><aff id=\"evaa165-aff2\"><label>e2</label>\n<institution>Institute of Fundamental Sciences, Massey University</institution>, Palmerston North, <country country=\"NZ\">New Zealand</country></aff><aff id=\"evaa165-aff3\"><label>e3</label>\n<institution>Donvis Limited</institution>, Palmerston North, <country country=\"NZ\">New Zealand</country></aff><author-notes><corresp id=\"evaa165-cor1\">Corresponding author: E-mail: <email>nik.palevich@agresearch.co.nz</email>.</corresp></author-notes><pub-date pub-type=\"collection\"><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\" iso-8601-date=\"2020-08-08\"><day>08</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>08</day><month>8</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>12</volume><issue>9</issue><fpage>1566</fpage><lpage>1572</lpage><history><date date-type=\"accepted\"><day>05</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"cc-by\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"evaa165.pdf\"/><abstract><title>Abstract</title><p>Bacterial species belonging to the genus <italic>Pseudobutyrivibrio</italic> are important members of the rumen microbiome contributing to the degradation of complex plant polysaccharides. <italic>Pseudobutyrivibrio xylanivorans</italic> MA3014 was selected for genome sequencing to examine its ability to breakdown and utilize plant polysaccharides. The complete genome sequence of MA3014 is 3.58 Mb, consists of three replicons (a chromosome, chromid, and plasmid), has an overall G + C content of 39.6%, and encodes 3,265 putative protein-coding genes (CDS). Comparative pan-genomic analysis of all cultivated and currently available <italic>P. xylanivorans</italic> genomes has revealed a strong correlation of orthologous genes within this rumen bacterial species. MA3014 is metabolically versatile and capable of growing on a range of simple mono- or oligosaccharides derived from complex plant polysaccharides such as pectins, mannans, starch, and hemicelluloses, with lactate, butyrate, and formate as the principal fermentation end products. The genes encoding these metabolic pathways have been identified and MA3014 is predicted to encode an extensive range of Carbohydrate-Active enZYmes with 78 glycoside hydrolases, 13 carbohydrate esterases, and 54 glycosyl transferases, suggesting an important role in solubilization of plant matter in the rumen.</p></abstract><kwd-group><kwd><italic>Pseudobutyrivibrio xylanivorans</italic></kwd><kwd>genome</kwd><kwd>rumen</kwd><kwd>bacteria</kwd><kwd>polysaccharide</kwd><kwd>enolase</kwd></kwd-group><funding-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>New Zealand Ministry of Business, Innovation and Employment New Economy Research</institution></institution-wrap></funding-source><award-id>C10X0803</award-id></award-group></funding-group><counts><page-count count=\"7\"/></counts></article-meta></front><body><boxed-text id=\"evaa165-BOX1\" position=\"float\" orientation=\"portrait\"><sec><title>Significance</title><p>In this article, we report the first high-quality and closed multireplicon genome of the rumen bacterium <italic>Pseudobutyrivibrio xylanivorans</italic> MA3014. Furthermore, we provide phenotypic characterization and comparative genotypic analyses with other rumen <italic>Pseudobutyrivibrio xylanivorans</italic>, a group of organisms with important roles in ruminal xylan and pectin breakdown. These analyses show that rumen <italic>Pseudobutyrivibrio xylanivorans</italic> encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) and binding proteins involved in the depolymerization and utilization of insoluble plant polysaccharides. The reconstruction of their metabolic pathways combined with metabolite analysis provides a deeper insight into their metabolism and adaptation to life in the rumen.</p></sec></boxed-text><sec sec-type=\"intro\"><title>Introduction</title><p>\n<italic>Pseudobutyrivibrio</italic> [family Lachnospiraceae, order Clostridiales] are anaerobic, monotrichous, butyrate-producing, curved rods, and have been isolated from or detected in the gastrointestinal tracts of various ruminants, monogastric animals, and humans (<xref rid=\"evaa165-B20\" ref-type=\"bibr\">Kopečný et al. 2003</xref>; <xref rid=\"evaa165-B41\" ref-type=\"bibr\">Willems and Collins 2009</xref>; <xref rid=\"evaa165-B12\" ref-type=\"bibr\">Henderson et al. 2015</xref>). <italic>Pseudobutyrivibrio</italic> are among a small number of rumen genera capable of utilizing the complex plant structural polysaccharide xylan (<xref rid=\"evaa165-B1\" ref-type=\"bibr\">Bryant and Small 1956</xref>; <xref rid=\"evaa165-B16\" ref-type=\"bibr\">Hungate 1966</xref>). Two species of <italic>Pseudobutyrivibrio</italic> are currently recognized; <italic>Pseudobutyrivibrio ruminis</italic> and <italic>Pseudobutyrivibrio xylanivorans</italic> (<xref rid=\"evaa165-B37\" ref-type=\"bibr\">Van Gylswyk et al. 1996</xref>; <xref rid=\"evaa165-B20\" ref-type=\"bibr\">Kopečný et al. 2003</xref>). <italic>Pseudobutyrivibrio xylanivorans</italic> is commonly found in domestic and wild ruminants and the type strain Mz 5<sup>T</sup> (DSM 14809) (<xref rid=\"evaa165-B20\" ref-type=\"bibr\">Kopečný et al. 2003</xref>; <xref rid=\"evaa165-B12\" ref-type=\"bibr\">Henderson et al. 2015</xref>) is able to utilize xylan, hemicellulose, and various oligo- and monosaccharides as substrates for growth (<xref rid=\"evaa165-B43\" ref-type=\"bibr\">Zorec et al. 2000</xref>). Gaining an insight into the role of these microbial primary plant polysaccharide fermenters is important for understanding rumen function. Here, we present the complete genome sequence of <italic>P. xylanivorans</italic> MA3014, a strain isolated from a New Zealand pasture-grazed dairy cow (<xref rid=\"evaa165-B25\" ref-type=\"bibr\">Noel 2013</xref>; <xref rid=\"evaa165-B32\" ref-type=\"bibr\">Seshadri et al. 2018</xref>), and describe its comparison with other representative <italic>P. xylanivorans</italic> genomes.</p></sec><sec><title>Materials and Methods</title><sec><title>Growth Conditions and Fermentation End Product Analysis</title><p>\n<italic>Pseudobutyrivibrio xylanivorans</italic> MA3014 was isolated from the rumen contents of fistulated Friesian dairy cattle and sequenced (<xref rid=\"evaa165-B25\" ref-type=\"bibr\">Noel 2013</xref>; <xref rid=\"evaa165-B32\" ref-type=\"bibr\">Seshadri et al. 2018</xref>). MA3014 was grown in RM02 medium (<xref rid=\"evaa165-B19\" ref-type=\"bibr\">Kenters et al. 2011</xref>) with 10 mM glucose and 0.1% yeast extract but without rumen fluid and culture purity was confirmed by Gram stain. The morphological features of MA3014 cells were determined by both scanning and transmission electron microscopy of cells grown on RM02 medium alone or with the addition of neutral detergent fraction of plant material as previously described (<xref rid=\"evaa165-B28\" ref-type=\"bibr\">Palevich et al. 2017</xref>, <xref rid=\"evaa165-B30\" ref-type=\"bibr\"> Palevich, Kelly, Leahy, et al. 2019</xref>).</p><p>Growth on soluble substrates was assessed as an increase in culture density OD<sub>600nm</sub> compared with cultures without carbon source added (all tested at 0.5% w/v final concentration), whereas total VFA production was used as an indicator of substrate utilization and growth for insoluble polymers (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S2</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). VFA production was determined from triplicate broth cultures grown overnight in RM02 medium with cellobiose as substrate and analyzed for formate, acetate, propionate, n-butyrate, iso-valerate, and lactate on a HP 6890 series GC (Hewlett Packard) with 2-ethylbutyric acid (Sigma-Aldrich, St. Louis, MO) as the internal standard. To derivatize formic, lactic, and succinic acids, samples were mixed with HCl ACS reagent (Sigma-Aldrich, St. Louis, MO) and diethyl ether, with the addition of <italic>N</italic>-methyl-<italic>N</italic>-<italic>t</italic>-butyldimethylsilyltri-fluoroacetamide (MTBSTFA) (Sigma-Aldrich, St. Louis, MO) (<xref rid=\"evaa165-B31\" ref-type=\"bibr\">Richardson et al. 1989</xref>).</p></sec><sec><title>Preparation of Genomic DNA for Whole-Genome Sequencing</title><p>Genomic DNA was extracted from freshly grown cells by a modification of the standard cell lysis method previously described (<xref rid=\"evaa165-B30\" ref-type=\"bibr\">Palevich, Kelly, Leahy, et al. 2019</xref>; <xref rid=\"evaa165-B32\" ref-type=\"bibr\">Seshadri et al. 2018</xref>), followed by phenol–chloroform extraction, and purification using the Qiagen Genomic-Tip 500 Maxi Kit (Qiagen, Hilden, Germany). Specificity of genomic DNA was verified by automated Sanger sequencing of the 16<italic>S</italic> rRNA gene following PCR amplification from genomic DNA. Total DNA amounts were determined using a NanoDrop ND-1000 (Thermo Scientific Inc.) and a Qubit Fluorometer dsDNA BR Kit (Invitrogen), in accordance with the manufacturer’s instructions. Genomic DNA integrity was verified by agarose gel electrophoresis and using a 2000 BioAnalyzer (Agilent).</p></sec><sec><title>Genome Sequencing, Assembly, and Comparison</title><p>\n<italic>Pseudobutyrivibrio xylanivorans</italic> MA3014 was selected for genome sequencing as a NZ strain and only representative member of <italic>P. xylanivorans</italic> from the Hungate1000 collection (<xref rid=\"evaa165-B32\" ref-type=\"bibr\">Seshadri et al. 2018</xref>; <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S1</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). The complete genome sequence of MA3014 was determined by pyrosequencing 3-kb mate paired-end (PE) sequence libraries using the 454 GS FLX platform with Titanium chemistry (Macrogen, Korea). Pyrosequencing reads provided 55× coverage of the genome and were assembled using the Newbler assembler (version 2.7, Roche 454 Life Sciences) which resulted in 116 contigs across 13 scaffolds. Gap closure was managed using the Staden package (<xref rid=\"evaa165-B35\" ref-type=\"bibr\">Staden et al. 1999</xref>) and gaps were closed using additional Sanger sequencing by standard and inverse PCR techniques. In addition, MA3014 genomic DNA was sequenced using shotgun sequencing of 2-kb PE sequence libraries using the Illumina MiSeq platform (Macrogen, Korea) which provided 677-fold sequencing coverage. A de novo assembly was performed using the assemblers Velvet version 3.0 (<xref rid=\"evaa165-B42\" ref-type=\"bibr\">Zerbino and Birney 2008</xref>), and EDENA version 3.120926 (<xref rid=\"evaa165-B13\" ref-type=\"bibr\">Hernandez et al. 2008</xref>). The resulting sequences were combined with the Newbler assembly using the Staden package and Geneious, version 8.1 (<xref rid=\"evaa165-B17\" ref-type=\"bibr\">Kearse et al. 2012</xref>). Genome assembly was confirmed by pulsed-field gel electrophoresis (<xref rid=\"evaa165-B26\" ref-type=\"bibr\">Palevich 2011</xref>; <xref rid=\"evaa165-B30\" ref-type=\"bibr\">Palevich, Kelly, Leahy, et al. 2019</xref>) and genome annotation was performed as described previously (<xref rid=\"evaa165-B18\" ref-type=\"bibr\">Kelly et al. 2010</xref>). Genome comparisons of orthologous gene clusters within <italic>Pseudobutyrivibrio</italic> genomes were performed using OrthoVenn version 2 (<xref rid=\"evaa165-B38\" ref-type=\"bibr\">Wang et al. 2015</xref>).</p></sec></sec><sec><title>Results and Discussion</title><sec><title>Genome Assembly, Properties, and Annotation</title><p>The genome of <italic>P. xylanivorans</italic> MA3014 was sequenced using short-read 454 GS FLX Titanium and Illumina technologies which generated 9.9 million PE reads (<xref rid=\"evaa165-T1\" ref-type=\"table\">table 1</xref>). The MA3014 assembly with high coverage of 677× was achieved using insert sizes that ranged between 238 bp (Illumina MiSeq) and 2.5 kb (454 GS-FLX Titanium). In total, 2.6 Gb of trimmed and filtered sequence data were retained for the reported assembly. The assembled, closed genome is 3,584,491 bp with an overall %G + C content of 39.6% and consists of three replicons (<xref rid=\"evaa165-B26\" ref-type=\"bibr\">Palevich 2011</xref>, <xref rid=\"evaa165-B27\" ref-type=\"bibr\">2016</xref>); a single chromosome (3,412,851 bp, %G + C 39.7), a chromid (PxyII, 88,942 bp, %G + C 36.9), and a plasmid (pNP95, 82,698 bp, %G + C 37.4). The overall genome statistics of MA3014 are similar to those from <italic>P. xylanivorans</italic> Mz 5<sup>T</sup> (DSM 14809) and NCFB 2399 (DSM 10317) (<xref rid=\"evaa165-B20\" ref-type=\"bibr\">Kopečný et al. 2003</xref>), are detailed in <xref rid=\"evaa165-T1\" ref-type=\"table\">table 1</xref>. Gene prediction from the MA3014 genome sequence resulted in a total of 3,365 genes annotated of which 3,265 (97.03%) were CDS, and 81 were various RNA genes such as 16<italic>S</italic>/23<italic>S</italic>/tRNAs and so on (<xref rid=\"evaa165-T1\" ref-type=\"table\">table 1</xref>). Putative functions were assigned to 2,364 (70.25%), whereas 901 CDS were annotated as hypothetical proteins or proteins of unknown function. In total, 840 (24.96%) genes have homology to proteins in the KEGG database, whereas 2,506 (74.47%) and 2,593 (77.06%) of annotated genes have well-defined PFAM and InterPro protein domains, respectively. In contrast, 153 (4.55%) of the annotated genes have identified type I signal peptide protein domain hits and are predicted to have extracellular functions. The MA3014 chromosome encodes 3,098 CDS, whereas the PxyII and pNP95 encode 96 and 71 genes, respectively. Overall, the coding region comprises 89.77% of the genome, which is typical of rumen <italic>Butyrivibrio</italic> and <italic>Pseudobutyrivibrio</italic> genomes sequenced within the Hungate1000 project (<xref rid=\"evaa165-B32\" ref-type=\"bibr\">Seshadri et al. 2018</xref>; <xref rid=\"evaa165-B29\" ref-type=\"bibr\">Palevich, Kelly, Leahy, et al. 2019</xref>).</p><table-wrap id=\"evaa165-T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Comparison of Assembly and Annotation Statistics for the <italic>Pseudobutyrivibrio xylanivorans</italic> MA3014, Mz 5<sup>T</sup>, and NCFB 2399 Genomes</p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col valign=\"top\" align=\"left\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/><col valign=\"top\" align=\"center\" span=\"1\"/></colgroup><thead><tr><th rowspan=\"2\" colspan=\"1\"/><th colspan=\"2\" rowspan=\"1\">MA3014<hr/></th><th colspan=\"2\" rowspan=\"1\">Mz 5<sup>T</sup> (DSM 14809)<hr/></th><th colspan=\"2\" rowspan=\"1\">NCFB 2399 (DSM 10317)<hr/></th></tr><tr><th rowspan=\"1\" colspan=\"1\">Value</th><th rowspan=\"1\" colspan=\"1\">% of Total<xref ref-type=\"table-fn\" rid=\"tblfn1\"><sup>a</sup></xref></th><th rowspan=\"1\" colspan=\"1\">Value</th><th rowspan=\"1\" colspan=\"1\">% of Total<xref ref-type=\"table-fn\" rid=\"tblfn1\"><sup>a</sup></xref></th><th rowspan=\"1\" colspan=\"1\">Value</th><th rowspan=\"1\" colspan=\"1\">% of Total<xref ref-type=\"table-fn\" rid=\"tblfn1\"><sup>a</sup></xref></th></tr></thead><tbody><tr><td colspan=\"7\" rowspan=\"1\">Genome project information</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Status</td><td colspan=\"2\" rowspan=\"1\">Complete</td><td colspan=\"2\" rowspan=\"1\">Draft</td><td colspan=\"2\" rowspan=\"1\">Draft</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Isolation source</td><td colspan=\"2\" rowspan=\"1\">Bovine rumen</td><td colspan=\"2\" rowspan=\"1\">Bovine rumen</td><td colspan=\"2\" rowspan=\"1\">Ovine rumen</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> BioSample ID</td><td colspan=\"2\" rowspan=\"1\">SAMN12605118</td><td colspan=\"2\" rowspan=\"1\">SAMN02745725</td><td colspan=\"2\" rowspan=\"1\">SAMN02910350</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> BioProject ID</td><td colspan=\"2\" rowspan=\"1\">PRJNA560993</td><td colspan=\"2\" rowspan=\"1\">PRJNA245713</td><td colspan=\"2\" rowspan=\"1\">PRJNA254867</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Assembly method</td><td colspan=\"2\" rowspan=\"1\">Newbler v. 2.3, Velvet v. 3.0, EDENA v. 3.1</td><td colspan=\"2\" rowspan=\"1\">vpAllpaths v. r46652</td><td colspan=\"2\" rowspan=\"1\">vpAllpaths v. r46652</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genome coverage</td><td colspan=\"2\" rowspan=\"1\">677×</td><td colspan=\"2\" rowspan=\"1\">472×</td><td colspan=\"2\" rowspan=\"1\">402×</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Sequencing technology</td><td colspan=\"2\" rowspan=\"1\">454 GS-FLX Titanium, Illumina MiSeq</td><td colspan=\"2\" rowspan=\"1\">Illumina HiSeq 2000, HiSeq 2500</td><td colspan=\"2\" rowspan=\"1\">Illumina HiSeq 2000, HiSeq 2500</td></tr><tr><td colspan=\"7\" rowspan=\"1\">Genome statistics</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genome size (bp)</td><td rowspan=\"1\" colspan=\"1\">3,584,491</td><td rowspan=\"1\" colspan=\"1\">100</td><td rowspan=\"1\" colspan=\"1\">3,420,924</td><td rowspan=\"1\" colspan=\"1\">100</td><td rowspan=\"1\" colspan=\"1\">3,213,944</td><td rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> DNA coding (bp)</td><td rowspan=\"1\" colspan=\"1\">3,217,653</td><td rowspan=\"1\" colspan=\"1\">89.77</td><td rowspan=\"1\" colspan=\"1\">3,122,173</td><td rowspan=\"1\" colspan=\"1\">91.27</td><td rowspan=\"1\" colspan=\"1\">2,947,505</td><td rowspan=\"1\" colspan=\"1\">91.71</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> DNA G+C (bp)</td><td rowspan=\"1\" colspan=\"1\">1,417,916</td><td rowspan=\"1\" colspan=\"1\">39.56</td><td rowspan=\"1\" colspan=\"1\">1,324,549</td><td rowspan=\"1\" colspan=\"1\">38.72</td><td rowspan=\"1\" colspan=\"1\">1,272,039</td><td rowspan=\"1\" colspan=\"1\">39.58</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> DNA replicons/scaffolds</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">100</td><td rowspan=\"1\" colspan=\"1\">57</td><td rowspan=\"1\" colspan=\"1\">100</td><td rowspan=\"1\" colspan=\"1\">34</td><td rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td colspan=\"7\" rowspan=\"1\">Genome annotations</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Total genes</td><td rowspan=\"1\" colspan=\"1\">3,365</td><td rowspan=\"1\" colspan=\"1\">100</td><td rowspan=\"1\" colspan=\"1\">3,150</td><td rowspan=\"1\" colspan=\"1\">100</td><td rowspan=\"1\" colspan=\"1\">2,965</td><td rowspan=\"1\" colspan=\"1\">100</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Protein-coding genes</td><td rowspan=\"1\" colspan=\"1\">3,265</td><td rowspan=\"1\" colspan=\"1\">97.03</td><td rowspan=\"1\" colspan=\"1\">3,078</td><td rowspan=\"1\" colspan=\"1\">97.71</td><td rowspan=\"1\" colspan=\"1\">2,890</td><td rowspan=\"1\" colspan=\"1\">97.47</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> RNA genes</td><td rowspan=\"1\" colspan=\"1\">81</td><td rowspan=\"1\" colspan=\"1\">2.41</td><td rowspan=\"1\" colspan=\"1\">72</td><td rowspan=\"1\" colspan=\"1\">2.29</td><td rowspan=\"1\" colspan=\"1\">75</td><td rowspan=\"1\" colspan=\"1\">2.53</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> rRNA operons</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> tRNA genes</td><td rowspan=\"1\" colspan=\"1\">53</td><td rowspan=\"1\" colspan=\"1\">1.58</td><td rowspan=\"1\" colspan=\"1\">55</td><td rowspan=\"1\" colspan=\"1\">1.75</td><td rowspan=\"1\" colspan=\"1\">49</td><td rowspan=\"1\" colspan=\"1\">1.65</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genes in internal clusters</td><td rowspan=\"1\" colspan=\"1\">689</td><td rowspan=\"1\" colspan=\"1\">20.48</td><td rowspan=\"1\" colspan=\"1\">329</td><td rowspan=\"1\" colspan=\"1\">10.44</td><td rowspan=\"1\" colspan=\"1\">248</td><td rowspan=\"1\" colspan=\"1\">8.36</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genes with function prediction</td><td rowspan=\"1\" colspan=\"1\">2,364</td><td rowspan=\"1\" colspan=\"1\">70.25</td><td rowspan=\"1\" colspan=\"1\">2,397</td><td rowspan=\"1\" colspan=\"1\">76.1</td><td rowspan=\"1\" colspan=\"1\">2,275</td><td rowspan=\"1\" colspan=\"1\">76.73</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genes with enzymes</td><td rowspan=\"1\" colspan=\"1\">733</td><td rowspan=\"1\" colspan=\"1\">21.78</td><td rowspan=\"1\" colspan=\"1\">761</td><td rowspan=\"1\" colspan=\"1\">24.16</td><td rowspan=\"1\" colspan=\"1\">739</td><td rowspan=\"1\" colspan=\"1\">24.92</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genes connected to KEGG pathways</td><td rowspan=\"1\" colspan=\"1\">840</td><td rowspan=\"1\" colspan=\"1\">24.96</td><td rowspan=\"1\" colspan=\"1\">879</td><td rowspan=\"1\" colspan=\"1\">27.9</td><td rowspan=\"1\" colspan=\"1\">858</td><td rowspan=\"1\" colspan=\"1\">28.94</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genes connected to KEGG Orthology (KO)</td><td rowspan=\"1\" colspan=\"1\">1,433</td><td rowspan=\"1\" colspan=\"1\">42.59</td><td rowspan=\"1\" colspan=\"1\">1,486</td><td rowspan=\"1\" colspan=\"1\">47.17</td><td rowspan=\"1\" colspan=\"1\">1,460</td><td rowspan=\"1\" colspan=\"1\">49.24</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genes assigned to COGs</td><td rowspan=\"1\" colspan=\"1\">2,404</td><td rowspan=\"1\" colspan=\"1\">71.44</td><td rowspan=\"1\" colspan=\"1\">1,933</td><td rowspan=\"1\" colspan=\"1\">61.37</td><td rowspan=\"1\" colspan=\"1\">1,883</td><td rowspan=\"1\" colspan=\"1\">63.51</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genes with Pfam domains</td><td rowspan=\"1\" colspan=\"1\">2,506</td><td rowspan=\"1\" colspan=\"1\">74.47</td><td rowspan=\"1\" colspan=\"1\">2,502</td><td rowspan=\"1\" colspan=\"1\">79.43</td><td rowspan=\"1\" colspan=\"1\">2,367</td><td rowspan=\"1\" colspan=\"1\">79.83</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genes with TIGRfam domains</td><td rowspan=\"1\" colspan=\"1\">850</td><td rowspan=\"1\" colspan=\"1\">25.26</td><td rowspan=\"1\" colspan=\"1\">894</td><td rowspan=\"1\" colspan=\"1\">28.38</td><td rowspan=\"1\" colspan=\"1\">883</td><td rowspan=\"1\" colspan=\"1\">29.78</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genes with InterPro domains</td><td rowspan=\"1\" colspan=\"1\">2,593</td><td rowspan=\"1\" colspan=\"1\">77.06</td><td rowspan=\"1\" colspan=\"1\">1,702</td><td rowspan=\"1\" colspan=\"1\">54.03</td><td rowspan=\"1\" colspan=\"1\">1,608</td><td rowspan=\"1\" colspan=\"1\">54.23</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genes with signal peptides</td><td rowspan=\"1\" colspan=\"1\">153</td><td rowspan=\"1\" colspan=\"1\">4.55</td><td rowspan=\"1\" colspan=\"1\">180</td><td rowspan=\"1\" colspan=\"1\">5.71</td><td rowspan=\"1\" colspan=\"1\">167</td><td rowspan=\"1\" colspan=\"1\">5.63</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Genes with transmembrane helices</td><td rowspan=\"1\" colspan=\"1\">818</td><td rowspan=\"1\" colspan=\"1\">24.31</td><td rowspan=\"1\" colspan=\"1\">837</td><td rowspan=\"1\" colspan=\"1\">26.57</td><td rowspan=\"1\" colspan=\"1\">816</td><td rowspan=\"1\" colspan=\"1\">27.52</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> CRISPR repeats</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">—</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Reference</td><td colspan=\"2\" rowspan=\"1\">This report</td><td colspan=\"4\" rowspan=\"1\">\n<xref rid=\"evaa165-B20\" ref-type=\"bibr\">Kopečný et al. (2003)</xref>\n</td></tr></tbody></table><table-wrap-foot><fn id=\"tblfn1\"><label>a</label><p>The total is based on either the size of the genome in base pairs or the total number of genes or CDS in the annotated genome.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Genome Comparison</title><p>A comparison of the <italic>P. xylanivorans</italic> MA3014 genome with the draft genomes of <italic>P. xylanivorans</italic> Mz 5<sup>T</sup> (DSM 14809) and NCFB 2399 (DSM 10317) (<xref rid=\"evaa165-B20\" ref-type=\"bibr\">Kopečný et al. 2003</xref>) is shown in <xref rid=\"evaa165-T1\" ref-type=\"table\">table 1</xref>. The MA3014 genome is larger than both Mz 5<sup>T</sup> and NCFB 2399, containing 187 and 375 more CDS, respectively. A feature of MA3014 is the presence of a chromid or secondary chromosome which is also found in other well-characterized <italic>Butyrivibrio</italic> genomes (<xref rid=\"evaa165-B18\" ref-type=\"bibr\">Kelly et al. 2010</xref>; <xref rid=\"evaa165-B29\" ref-type=\"bibr\">Palevich, Kelly, Ganesh, et al. 2019</xref>). Chromids are replicons with %G + C content similar to that of their main chromosome, but have plasmid-type maintenance and replication systems, are usually smaller than the chromosome (but larger than plasmids) and contain genes essential for growth along with several core genus-specific genes (<xref rid=\"evaa165-B10\" ref-type=\"bibr\">Harrison et al. 2010</xref>). The PxyII replicon has been designated as a chromid of MA3014 as it possesses all of these characteristics and contains genes encoding enzymes that have a role in carbohydrate metabolism and transport. Since the PxyII chromid is 2,834 bp smaller than the Bhu II chromid of <italic>Butyrivibrio hungatei</italic> MB2003, it is now the smallest chromid reported for bacteria. Several plasmid replication genes have been identified in the Mz 5<sup>T</sup> draft genome but not in NCFB 2399 therefore the presence of extrachromosomal elements requires experimental validation in these <italic>P. xylanivorans</italic> strains.</p><p>Comparison of MA3014, Mz 5<sup>T</sup>, and NCFB 2399 genomes based on COG category (<xref rid=\"evaa165-T1\" ref-type=\"table\">table 1</xref>) and synteny analysis (<xref ref-type=\"fig\" rid=\"evaa165-F1\">fig. 1<italic>A</italic> and <italic>B</italic></xref>), show that these <italic>Pseudobutyrivibrio</italic> strains are genetically similar. Overall, the average nucleotide identity based on the synteny analysis for MA3014 compared with Mz 5<sup>T</sup> was 81.2%, with 80.7% for MA3014 and NCFB 2399 (<xref ref-type=\"fig\" rid=\"evaa165-F1\">fig. 1<italic>A</italic> and <italic>B</italic></xref>). Despite the differences in genome sizes of MA3014 and Mz 5<sup>T</sup>, the predicted metabolism and actual carbohydrate utilization phenotypes of these two rumen bacteria are comparable. A BlastP (e-value cut-off 10<sup>−5</sup>) comparison of MA3014, Mz 5<sup>T</sup>, and NCFB 2399 scaffolds with at least a single one-to-one ortholog shared among the genomes revealed a strong correlation of orthologous genes among these species. Most of the predicted MA3014 genes were found to have homologs in the other two strains (2,356; 73%), with the <italic>P. xylanivorans</italic> species represented by 768 orthologous clusters and 1,996 single-copy genes. In total, 2,036 core genes were found to be orthologous among the three <italic>P. xylanivorans</italic> genomes compared, with only 58 genes found to be unique to MA3014. In comparison, only 27 and 19 genes were found to be unique to Mz 5<sup>T</sup> and NCFB 2399, respectively. Genomic comparisons with other species within the genera <italic>Butyrivibrio</italic> and <italic>Pseudobutyrivibrio</italic> have revealed strong syntenies between their genomes (<xref rid=\"evaa165-B30\" ref-type=\"bibr\">Palevich, Kelly, Leahy, et al. 2019</xref>), indicating a shared origin and subsequent divergent evolution among these rumen bacteria.</p><fig id=\"evaa165-F1\" orientation=\"portrait\" position=\"float\"><label><sc>Fig</sc>. 1.</label><caption><p>(<italic>A</italic> and <italic>B</italic>) Genome synteny analysis. Alignment of the <italic>Pseudobutyrivibrio xylanivorans</italic> MA3014 genome against the draft genomes of <italic>P. xylanivorans</italic> Mz 5<sup>T</sup> (<italic>A</italic>) and <italic>P. xylanivorans</italic> NCFB 2399 (<italic>B</italic>). The general statistics and levels of completeness for each genome assembly are detailed in <xref rid=\"evaa165-T1\" ref-type=\"table\">table 1</xref>. Whenever the two sequences agree, a colored line or dot is plotted. Units displayed in base pairs. Color codes: Blue, forward sequence; red, reverse sequence. (<italic>C</italic>) Fermentation pathways in rumen <italic>Pseudobutyrivibrio</italic>. Bcd-Etf, butyryl-CoA dehydrogenase/electron-transferring flavoprotein; Ech, <italic>Escherichia coli</italic> hydrogenase-3-type hydrogenase; Fd, ferredoxin; Fd<sub>ox</sub>, oxidized Fd; Fd<sub>red</sub>, reduced Fd; Glo, glyoxalase; MsgA, methylglyoxal synthase; NAD, nicotinamide adenine dinucleotide; NAD<sub>ox</sub>, oxidized NAD; NAD<sub>red</sub>, reduced NAD; NifJ, nitrogen fixation J; Rnf, <italic>Rhodobacter</italic> nitrogen fixation; ATPase, F<sub>0</sub>F<sub>1</sub>-ATPsynthase.</p></caption><graphic xlink:href=\"evaa165f1\"/></fig></sec><sec><title>Polysaccharide Degradation</title><p>The Carbohydrate-Active enZYmes (CAZymes) database was used to identify glycoside hydrolases (GHs), glycosyl transferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and carbohydrate-binding protein module (CBM) families within the MA3014 genome. Additional manual curation and analysis of the functional domains of enzymes involved in the breakdown or synthesis of complex carbohydrates, has revealed the polysaccharide-degrading potential of this rumen bacterium (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S3</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Overall, the CAZyme profile of MA3014 is similar to other <italic>Pseudobutyrivibrio</italic> but is not as extensive as those of <italic>Butyrivibrio</italic> (<xref rid=\"evaa165-B27\" ref-type=\"bibr\">Palevich 2016</xref>; <xref rid=\"evaa165-B30\" ref-type=\"bibr\">Palevich, Kelly, Leahy, et al. 2019</xref>). Approximately 4.5% of the MA3014 genome (146 CDSs) is predicted to encode 26 secreted (25 GHs and one CE) and 120 intracellular (63 GHs, 12 CEs, and 54 GTs) proteins dedicated to polysaccharide degradation. The enzymatic profiles of MA3014 and Mz 5<sup>T</sup> are almost identical, as both possess the same genes encoding predicted secreted and intracellular CAZymes in their genomes. The majority (48) of MA3014 genes encoding intracellular proteins involved in polysaccharide breakdown (excluding GTs), had corresponding homologs in Mz 5<sup>T</sup>. The most abundant Pfam domains included GH families (GH3, GH13, and GH43) and CE1, most of which did not contain signal sequences and were therefore predicted to be located intracellularly. Similarly, CAZymes with predicted roles in xylan (GH8, GH51, GH115), dextrin, and starch (GH13 and GH77) degradation families were also predicted to be located mostly intracellularly.</p><p>Growth experiments showed MB2003 to be metabolically versatile and able to grow on a wide variety of monosaccharides and disaccharides (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S2</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). However, unlike Mz 5<sup>T</sup> (<xref rid=\"evaa165-B20\" ref-type=\"bibr\">Kopečný et al. 2003</xref>), MA3014 was unable to utilize the insoluble substrate pectin for growth. This difference is due to Mz 5<sup>T</sup> possession of four pectate lyases (one PL1 and three PL3) predicted to be involved in pectin degradation and utilization. MA3014 is predicted to breakdown starch and xylan based on four large (>1,000 aa) cell-associated enzymes (<xref rid=\"evaa165-B18\" ref-type=\"bibr\">Kelly et al. 2010</xref>) shown to be significantly up-regulated in related <italic>B. hungatei</italic> MB2003 and <italic>Butyrivibrio proteoclasticus</italic> B316<sup>T</sup> cells grown on xylan (<xref rid=\"evaa165-B29\" ref-type=\"bibr\">Palevich, Kelly, Ganesh, et al. 2019</xref>). These are: α-amylase <italic>amy13E</italic> (FXF36_11320), arabinogalactan endo-1,4-β-galactosidase <italic>agn53A</italic> (FXF36_02635), xylosidase/arabinofuranosidase <italic>xsa43D</italic> (FXF36_08285), endo-1,4-β-xylanase xyn10A (FXF36_14365). These enzymes contain multiple cell wall-binding repeat domains (CW-binding domain, Pfam01473) at their C-termini that are predicted to anchor the protein to the peptidoglycan cell membrane (<xref rid=\"evaa165-B4\" ref-type=\"bibr\">Dunne et al. 2012</xref>). Interestingly, among the MA3014 homologues all but <italic>xyn10A</italic> are smaller than 1,000 aa and none contain CW-binding domains. However, <italic>xyn10A</italic> contains a CBM9 (Pfam06452), with <italic>xyn10B</italic> containing a CBM13 (Pfam00652) and CBM2 (Pfam00553) domains respectively with predicted xylan-binding functions.In addition, the secreted α-amylase <italic>amy13E</italic> (FXF36_11320) contains a CBM26 (Pfam16738) domain with predicted starch-binding functions (<xref rid=\"evaa165-B24\" ref-type=\"bibr\">McCartney et al. 2004</xref>; <xref rid=\"evaa165-B7\" ref-type=\"bibr\">Gilbert et al. 2013</xref>).</p><p>\n<italic>Pseudobutyrivibrio xylanivorans</italic> MA3014 cells grown in liquid media supplemented with plant material revealed the copious production of exopolysaccharides (EPS). EPS production has been reported in <italic>Butyrivibrio</italic> strains and the EPS is composed of the neutral sugars rhamnose, fucose, mannose, galactose, and glucose (<xref rid=\"evaa165-B34\" ref-type=\"bibr\">Stack 1988</xref>), made from recycled breakdown products of plant polysaccharides. Our findings also show the presence of cytoplasmic inclusions, similar to those seen in B316<sup>T</sup> and other <italic>Butyrivibrio</italic> strains containing glycogen-like material (<xref rid=\"evaa165-B15\" ref-type=\"bibr\">Hespell et al. 1993</xref>). The MA3014 genome encodes a complete repertoire of genes for glycogen synthesis and degradation, suggesting that a variety of complex oligosaccharides resulting from extracellular hydrolysis are metabolized within the cell and that glycogen has a role in the storage of excess carbohydrate.</p></sec><sec><title>Enolase Loss and Metabolic Flexibility</title><p>An extremely unusual feature of MA3014 is that it lacks an enolase gene. The pathway for butyrate production requires a complete Embden–Meyerhof–Parnas (EMP) glycolytic pathway, including an enolase (<italic>eno</italic>, EC4.2.1.11), which converts 2-phospho-<sc>d</sc>-glycerate to phosphoenolpyruvate in the second to last step. Of all 21 <italic>Pseudobutyrivibrio</italic> genomes sequenced in the Hungate1000 project, only <italic>P. xylanivorans</italic> MA3014 and <italic>P. ruminis</italic> AD2017 lack a detectable enolase gene, which was confirmed using PCR screens with <italic>eno</italic>-specific primers (<xref rid=\"evaa165-B18\" ref-type=\"bibr\">Kelly et al. 2010</xref>; <xref rid=\"evaa165-B30\" ref-type=\"bibr\">Palevich, Kelly, Ganesh, et al. 2019</xref>). The methylglyoxal shunt and uronic acid metabolic pathways (<xref ref-type=\"fig\" rid=\"evaa165-F1\">fig. 1<italic>C</italic></xref>), have been suggested as alternatives to the EMP pathway (<xref rid=\"evaa165-B3\" ref-type=\"bibr\">Cooper 1984</xref>; <xref rid=\"evaa165-B18\" ref-type=\"bibr\">Kelly et al. 2010</xref>; <xref rid=\"evaa165-B29\" ref-type=\"bibr\">Palevich, Kelly, Ganesh, et al. 2019</xref>). In this pathway, the dihydroxyacetone phosphate is transformed to pyruvate via methylglyoxal and <sc>d</sc>-lactate dehydrogenase encoded by <italic>ldhA</italic>. The MA3014 genome contains methylglyoxal synthase, <italic>mgsA</italic> (FXF36_12340), glyoxalases <italic>gloA/B</italic> (FXF36_00730, FXF36_01130, and FXF36_09530), and both <sc>d</sc>- and <sc>l</sc>-lactate dehydrogenases <italic>ldh</italic> (FXF36_04170 and FXF36_11135) genes. In addition, MA3014 has the same set of genes as the previously reported and well-characterized <italic>B. hungatei</italic> MB2003 and <italic>B. proteoclasticus</italic> B316<sup>T</sup> for the production of butyrate, formate, acetate, and lactate (<xref rid=\"evaa165-B18\" ref-type=\"bibr\">Kelly et al. 2010</xref>; <xref rid=\"evaa165-B28\" ref-type=\"bibr\">Palevich et al. 2017</xref>; <xref rid=\"evaa165-B29\" ref-type=\"bibr\">Palevich, Kelly, Ganesh, et al. 2019</xref>).</p><p>In some butyrate-forming anaerobes, crotonyl-CoA reduction is linked to electron transport phosphorylation via flavin-based electron-bifurcating <italic>ech</italic> and <italic>rnf</italic> complexes which act as transmembrane ion pumps (<xref rid=\"evaa165-B14\" ref-type=\"bibr\">Herrmann et al. 2008</xref>; <xref rid=\"evaa165-B21\" ref-type=\"bibr\">Li et al. 2008</xref>; <xref rid=\"evaa165-B39\" ref-type=\"bibr\">Welte et al. 2010</xref>; <xref rid=\"evaa165-B2\" ref-type=\"bibr\">Buckel and Thauer 2013</xref>). A recent analysis of the Hungate1000 data set (<xref rid=\"evaa165-B9\" ref-type=\"bibr\">Hackmann and Firkins 2015</xref>; <xref rid=\"evaa165-B32\" ref-type=\"bibr\">Seshadri et al. 2018</xref>; <xref rid=\"evaa165-B30\" ref-type=\"bibr\">Palevich, Kelly, Leahy, et al. 2019</xref>), found that <italic>Pseudobutyrivibrio</italic> and <italic>Butyrivibrio</italic> genomes encode both Ech and Rnf homologs proposed to act in concert with NifJ and Bcd-Etf to form an electrochemical potential and drive ATP synthesis (<xref rid=\"evaa165-B36\" ref-type=\"bibr\">Tremblay et al. 2012</xref>; <xref rid=\"evaa165-B8\" ref-type=\"bibr\">Gutekunst et al. 2014</xref>). This allows these rumen bacteria to generate ∼4.5 ATP/glucose in total, one of the highest yields for anaerobic fermentation of glucose (<xref rid=\"evaa165-B2\" ref-type=\"bibr\">Buckel and Thauer 2013</xref>). Given the importance of <italic>eno</italic>, <italic>Pseudobutyrivibrio</italic> and <italic>Butyrivibrio</italic> may be displaying an example of environment-specific evolution by gene loss that warrants further investigation into the alternative pathways that permit ATP generation. The genome sequence of <italic>P. xylanivorans</italic> MA3014 presented here is consistent with the genome architecture of other sequenced <italic>Pseudobutyrivibrio</italic> strains and is a valuable resource for future studies regarding bacterial-driven plant-fiber degradation in ruminants.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><p>\n<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary data</xref> are available at <italic>Genome Biology and Evolution</italic> online.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><supplementary-material content-type=\"local-data\" id=\"sup1\"><label>evaa165_Supplementary_Data</label><media xlink:href=\"evaa165_supplementary_data.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"ack1\"><title>Acknowledgments</title><p>The <italic>Pseudobutyrivibrio xylanivorans</italic> MA3014 genome sequencing project was funded by the New Zealand Ministry of Business, Innovation and Employment New Economy Research Fund program: accessing the uncultured rumen microbiome (Contract No. C10X0803). Electron microscopy was conducted with the assistance of the Manawatū Microscopy and Imaging Centre at Massey University, Palmerston North, New Zealand. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"correction\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Physiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Physiol.</journal-id><journal-title-group><journal-title>Frontiers in Physiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-042X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33041837</article-id><article-id pub-id-type=\"pmc\">PMC7523729</article-id><article-id pub-id-type=\"doi\">10.3389/fphys.2020.00943</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Physiology</subject><subj-group><subject>Correction</subject></subj-group></subj-group></article-categories><title-group><article-title>Corrigendum: Alveolar Dynamics and Beyond – the Importance of Surfactant Protein C and Cholesterol in Lung Homeostasis and Fibrosis</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Sehlmeyer</surname><given-names>Kirsten</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/955222/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ruwisch</surname><given-names>Jannik</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/899781/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Roldan</surname><given-names>Nuria</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/923744/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Lopez-Rodriguez</surname><given-names>Elena</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/862378/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Institute of Functional and Applied Anatomy, Hannover Medical School</institution>, <addr-line>Hanover</addr-line>, <country>Germany</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Biomedical Research in Endstage and Obstructive Lung Disease Hannover, Member of the German Centre for Lung Research</institution>, <addr-line>Hanover</addr-line>, <country>Germany</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Alveolix AG and ARTORG Center, University of Bern</institution>, <addr-line>Bern</addr-line>, <country>Switzerland</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Institute of Functional Anatomy, Charité – Universitätsmedizin Berlin</institution>, <addr-line>Berlin</addr-line>, <country>Germany</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Yu Ru Kou, National Yang-Ming University, Taiwan</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Argen Mamazhakypov, Max Planck Institute for Heart and Lung Research, Germany; Bela Suki, Boston University, United States</p></fn><corresp id=\"c001\">*Correspondence: Elena Lopez-Rodriguez <email>elena.lopez-rodriguez@charite.de</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Respiratory Physiology, a section of the journal Frontiers in Physiology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work</p></fn></author-notes><pub-date pub-type=\"epub\"><day>15</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>15</day><month>9</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>11</volume><elocation-id>943</elocation-id><history><date date-type=\"received\"><day>28</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>14</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Sehlmeyer, Ruwisch, Roldan and Lopez-Rodriguez.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Sehlmeyer, Ruwisch, Roldan and Lopez-Rodriguez</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><related-article related-article-type=\"corrected-article\" id=\"d38e191\" ext-link-type=\"doi\" xlink:href=\"10.3389/fphys.2020.00386\" journal-id=\"Front Physiol\" journal-id-type=\"nlm-ta\" vol=\"11\" page=\"386\">A Corrigendum on <article-title>Alveolar Dynamics and Beyond – the Importance of Surfactant Protein C and Cholesterol in Lung Homeostasis and Fibrosis</article-title> by Sehlmeyer, K., Ruwisch, J., Roldan, N., and Lopez-Rodriguez, E. (2020). Front. Physiol. 11:386. doi: <object-id>10.3389/fphys.2020.00386</object-id></related-article><kwd-group><kwd>surfactant protein C</kwd><kwd>pulmonary fibrosis</kwd><kwd>alveolar dynamics</kwd><kwd>lipid metabolism</kwd><kwd>alveolar macrophages</kwd><kwd>cholesterol</kwd><kwd>metaflammation</kwd></kwd-group><counts><fig-count count=\"0\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"0\"/><page-count count=\"6\"/><word-count count=\"3782\"/></counts></article-meta></front><body><p>In the original article, there was a mistake in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> as published. <bold>References on tables are misplaced</bold>.</p><p>In <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, the cited reference Glasser et al., 2008 should be Glasser et al., 2001. The cited reference Glasser and Senft, 2009 should be Glasser et al., 2003. The cited reference Glasser et al., 2013b should be Lawson et al., 2005. The cited reference Glasser et al., 2013a should be Madala et al., 2011. The cited references Bridges et al., 2003 should be Glasser et al., 2008. The cited reference Lawson et al., 2011 should be Glasser et al., 2009. The cited reference Nureki et al., 2018 should be Glasser et al., 2013b. The cited reference Venosa et al., 2019 should be Glasser et al., 2013a. The cited reference Katzen et al., 2019 should be Bridges et al., 2003. The cited reference Jin et al., 2018 should be Lawson et al., 2011. The cited reference Conkright et al., 2002 in the third row (section Models with incomplete proSP-C processing) should be Nureki et al., 2018. The cited reference Conkright et al., 2002 in the fourth row should be Venosa et al., 2019. The cited reference Conkright et al., 2002 in the fifth row should be Katzen et al., 2019. The cited reference Conkright et al., 2002 in the sixth row should be Jin et al., 2018. And the cited reference Thouvenin et al., 2010 should be Conkright et al., 2002.</p><p>In <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>, the cited reference Cottin et al., 2011 should be Thomas et al., 2002. The cited reference Ono et al., 2011 should be Tredano et al., 2004. The cited reference Kuse et al., should be Abou Taam et al., 2009. The cited reference Avital et al., 2014 should be Thouvenin et al., 2010. The cited reference van Hoorn et al., 2014 should be Cottin et al., 2011. The cited reference Hevroni et al., 2015 should be Ono et al., 2011. The cited reference Salerno et al., 2016 should be Kuse et al., 2013. The cited reference Chibbar et al., 2004 should be Avital et al., 2014. The cited reference Stevens et al., 2005 should be van Hoorn et al., 2014. The cited reference Glasser et al., 2001 should be Hevroni et al., 2015. The cited reference Glasser et al., 2003 should be Salerno et al., 2016. The cited reference Lawson et al., 2005 should be Chibbar et al., 2004. And the last cited reference Lawson et al., 2005 should be Stevens et al., 2005. The corrected <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> appears below.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>SP-C related mouse models.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" colspan=\"2\" rowspan=\"1\"><bold>Mouse model</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>General results</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Lung morphology</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>BALF</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Lung mechanics</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>SP-C null mutants</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glasser et al., 2001</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Generation of SP-C null mutant mice, Swiss black background</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Viable, normal growth and reproducibility.Reduced stability of small bubbles but normal activity at standard bubble size</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Indistinguishable from controls</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reduced hysteresitivity at each end-expiratory pressure</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glasser et al., 2003</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SP-C null mutant mice, 129/Sv background</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reduced health and fecundity</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">From 2 month: enlargement of alveoli, irregular alveolar septation, multifocal cellular infiltrates. From 6 month: type 2 cell hyperplasia, interstitial thickening, peribronchiolar and perivascular monocytic infiltration. Intracellular lipid inclusions in macrophages and AE2C, cytoplasmic crystals in macrophages</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased macrophage number</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased lung volumes at higher pressures, increased hysteresivity, increased airway resistance and tissue damping</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>2nd hit models</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lawson et al., 2005</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Intratracheal bleomycin application, Swiss black background</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Higher mortality and weight loss, more pronounced fibrosis and delayed resolution</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased number of inflammatory cells, fibrotic foci (collagen, fibroblasts, destroyed septa), enhanced collagen deposition; delayed resolution of fibrosis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased neutrophil counts</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Madala et al., 2011</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bleomycin and rapamycin, S129S6 background</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Preventive and therapeutic treatment with rapamycin failed to reduce bleomycin induced tissue inflammation and collagen deposition</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glasser et al., 2008</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Instillation of <italic>Pseudomonas aeruginosa</italic>, 129S6 and FVB/N strain</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reduced survival of 2-week-old mice, increased bacterial colony counts in 2-week-old 129S6 but not in FVB/N mice</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased inflammation, tissue and airway infiltrates (neutrophils and enlarged macrophages with cytoplasmic inclusions)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased total cell counts: neutrophils; large foamy macrophages</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glasser et al., 2009</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Respiratory syncytial virus infection, 129S6 and FVB/N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Higher susceptibility to RSV and delayed resolution of induced changes in lung morphology in both strains</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">More extensive interstitial thickening, air space consolidation, goblet cell hyperplasia</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased total cell counts: polymorphonuclear leucocytes, lymphocytes, enlarged foamy mononuclear cells</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glasser et al., 2013b</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RSV infection, expression of SP-C inducible by doxycycline (on 129S6; <italic>55.3/Sftpc-/-</italic>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SP-C expression reduced RSV-induced tissue inflammation and inflammatory cell counts and increased viral clearance</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Diffuse alveolar and interstitial infiltrates in doxycycline untreated mice, reduced inflammation in doxycycline treated mice</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reduced total cell counts and percentage of neutrophil counts in doxycycline -treated mice</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glasser et al., 2013a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LPS challenge, 129S6 background</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">More intense airway and airspace inflammation, delayed resolution of tissue inflammation</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">More intense cellular infiltration, perivascular edema, fragmentation of alveolar septae; residual inflammation 30 days post LPS exposure</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased total cell counts without LPS challenge (reduced by application of Survanta)</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Models with incomplete proSP-C processing</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conkright et al., 2002</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Expression of SP-C<sub>24−57</sub> HA, FVB/N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Delayed/arrested lung development and lethal neonatal respiratory distress syndrome</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bridges et al., 2003</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Deletion of exon 4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Not viable</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fetal lung tissue: disrupted lung organogenesis, branching morphogenesis, dose-dependent cell cytotoxicity</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lawson et al., 2011</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conditional expression of L188Q upon doxycycline; intratracheal bleomycin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No spontaneous pulmonary fibrosis; more extensive fibrosis in response to bleomycin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased apoptosis, total lung collagen, higher number of myofibroblasts after bleomycin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cell numbers unaltered in bleomycin treated WT and mutant mice</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">More reduced static lung compliance in bleomycin treated L188Q mice than challenged controls</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nureki et al., 2018</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conditional mouse mutant, constitutive and inducible I73T expression (by Tamoxifen), C57BL/6J</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased early mortality, spontaneous acute alveolitis, parenchymal injury, fibrotic remodeling</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Constitutive I73T expression: diffuse parenchymal lung remodeling; disrupted embryonic lung architecture Induced expression: acute, diffuse lung injury after tamoxifen, partial recovery but development of fibrotic phenotype</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Constitutive expression: age-dependent increases in BALF cellularity<break/> Induced expression: increased total cell counts, early macrophage accumulation, followed by polymorphonuclear cells and eosinophilia, milder increase in total lymphocytes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Induced expression: restrictive pattern (PV loops), decreased static compliance</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Venosa et al., 2019</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conditional mouse mutant, I73T expression induced by Tamoxifen; Local and i.v application of clodronate</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Multiphasic and multicellular alveolitis; local clodronate application reduced survival, i.v. clodronate improved survival and reduced eosinophilia</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Early reduction of macrophages, followed by accumulation of immature macrophages, neutrophils and eosinophils</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Katzen et al., 2019</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Constitutive and conditional C121G mutant inducible by tamoxifen, C57BL/6J</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Constitutive expression: lethal postnatal respiratory failure<break/> Conditional expression in adult mice: dose-dependent morbidity and mortality, multiphasic polycellular alveolitis with increased BALF cell counts</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Constitutive: distorted architecture, enlarged airspaces, interstitial widening, inflammatory infiltrates, proteinaceous fluid Conditional expression: acute diffuse lung injury, partial recovery but spontaneous fibrotic lung remodeling</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conditional expression: polycellular alveolitis, increased total cell counts, early macrophage increase, followed by neutrophils and eosinophils, milder increase in lymphocytes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Restrictive pattern: decline in static lung compliance</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Jin et al., 2018</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sterile injury model (surfactant protein C-thymidine kinase) induced by ganciclovir in presence (SPC-TK) and absence (SPC-TK/SPC-KO) of SP-C expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased injury and higher mortality in absence than in presence of SP-C expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Diffuse alveolar damage qualitatively similar but more pronounced in SPC-TK/SPC-KO</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total cell counts unaltered in SPC-TK/SPC-KO and SPC-TK, higher neutrophils and lymphocyte cell counts in SPC-TK/SPC-KO</td><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Lung mechanics and BALF cells data from patients.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Variant</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>BALF cells</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Lung mechanics</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Reference</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">L188Q</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TLC 52%, DLCo 51% (male patient, onset 20 years); FVC 21% (female patient, onset 17 years)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Thomas et al., 2002</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">85% M, 12% L, 3% N</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tredano et al., 2004</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R167Q</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">84% M, 11% L, 5% N</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">92% M, 7% N, 1% L, 0% E</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FRC: 69% (8 months), 77% (13 years)DLCo: 25% (8 months), 51% (13 years</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Abou Taam et al., 2009</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">30% M, 60% N, 10% L, 0% E (Moraxella catarrhalis)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FRC: 138% (36 months), DLCo: 111% (33 months), 128% (36 months), 156% (42 months)</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">82% M, 13% N, 3% L, 2% E</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FRC: 120% (26 months), 128% (35 months), 73% (39 months) DLCo: 98% (26 months), 89% (35 months), 164% (39 months)</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">84% M, 5% N, 11% L, 0% E</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">93% M, 1% N, 6% L, 0% E</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FRC 112% (26 months), DLCo: 87% (26 months)</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">15x I73T, 1x V39A, c.325- 1G>A, c.424delC, c.435G>C (Q145H), L188P, C189Y, L194P</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">70 ± 5% M, 8 ± 2% L, 18 ± 4% N, total: 379 ± 56 x 10<sup>3</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">82% patients with SpO<sub>2</sub> testing <95%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Thouvenin et al., 2010</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">40% M, 57% N, 3% L (mother 32 years)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FVC 62%, TLC 77%, FEV1 83%, RV 108%, DLCo 33%, PaO<sub>2</sub> room air 11.3 kPa, PaO<sub>2</sub> after 10 min exercise (35W): 7.3 kPa</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cottin et al., 2011</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">74% M, 20% N, 4% L, 2% E (child, 3 months)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">G100S</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BAL cell count (100.000 cells/ml): 2.4, 90% M, 7.5% L, 2.5% N, 0% E; CD4/CD8 ratio: 1,7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VC 72.2%, FEV1 84.1% DLCo: 69.3%</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ono et al., 2011</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BAL cell count (100.000 cells/ml): 2, 86% M, 12% L, 1% N, 1% E, CD4/CD8 ratio: 1.6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VC 85%, FEV1 90.3% DLCo: not available</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BAL cell count (100.000 cells/ml):1.4, 91% M, 5.8% L, 2.4%N, 0.8%E, CD4/CD8 ratio: 1.5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VC 96.6%, FEV1 85% DLCo: 65.2%</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BAL cell count (100.000 cells/ml): 1.21, 54.2% M, 10.1%L, 34.5% N, 1.2% E, CD4/CD8 ratio: 0.25</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VC 42.5%, FEV1 92.9% DLCo: 38.5%</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BAL cell count (100.000 cells/ml): 3.85, 80% M, 17.3% L, 1.1% N, 1.6% E, CD4/CD8 ratio: 0.6 (time diagnosis)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">VC 65.3%, FEV1 83.3% DLCo: not available (at time diagnosis)</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Y104H</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">91% M, 8% L, 1% N</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FVC 85%, DLCo 89%, oxygen saturation 97% to 95% (with exercise)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Kuse et al., 2013</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">16 years: 90% FVC, 86% TLC, 96% DLCo, 96% VO<sub>2</sub> max; 37 years: FVC 65%, TLC 91%, DLCo 42%, VO<sub>2</sub>max: 5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Avital et al., 2014</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I38F</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 years: FVC 77%, TLC 90%, DLCo 108%, VO<sub>2</sub>max 78%; 32 years: FVC 94%, TLC 96%, DLCo 82%, VO<sub>2</sub>max 69%, high breathing reserve: 115 l/min, saturation 100% at peak exercise</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7 years: FVC 59%, TLC 95% DLCo not available, VO<sub>2</sub>max 80%, 28 years: FVC 46%, TLC 48%, DLCo 58%, VO<sub>2</sub>max 79%</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8 years: FVC 69%, TLC 100%, DLCo 107%, VO<sub>2</sub>max 83%, 29 years: FVC 102%, TLC 106%, DLCo 95%, VO<sub>2</sub>max 83%</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">V39L</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">16 years: FVC 88%, TLC 95%, DLCo 109%, VO<sub>2</sub>max 93%; 37 years: 94% FVC, 96% TLC, 82% DLCo, 91% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">C121F</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Infiltration of granulocytes and alveolar macrophages</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">van Hoorn et al., 2014</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4 months:88% oxygen saturation, respiratory rate 85, V<sub>T</sub> 6.0 ml/kg, V<sub>E</sub> 507 ml/min/kg, Crs 2.96 ml/cmH<sub>2</sub>O, Crs/kg 0.76/kg, VC 92 ml(52%), TLC 196 ml(74%), FRC 128 ml(110%), RV 104 ml(99%), V<sub>max</sub>FRC 416 ml/s (263%), FEF<sub>75</sub> 410 ml/s (207%), FEF<sub>85</sub> 295 ml/s(258%)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hevroni et al., 2015</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I38F</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.3 months: 91% oxygen saturation, respiratory rate 77, V<sub>T</sub> 6.3 ml/kg, V<sub>E</sub> 484 ml/min/kg, Crs 2.26 ml/cmH<sub>2</sub>O, Crs/kg 0.59/kg, VC 28 ml(69%), TLC 211 ml(94%), FRC 138 ml(125%), RV 108 ml(109%), V<sub>max</sub>FRC 343 ml/s (245%), FEF<sub>75</sub> 579 ml/s (334%), FEF<sub>85</sub> 477 ml/s (476%)</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I73T</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Normal cytology and lipid index lipid-laden alveolar macrophages</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Salerno et al., 2016</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">L188E</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Normal lung volumes, diffusion capacity 18% of predicted</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chibbar et al., 2004</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">E66K</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Increased cellularity with foamy mononuclear cell</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stevens et al., 2005</td></tr></tbody></table><table-wrap-foot><p><italic>Summary of data from patients suffering fibrosis related to a SP-C mutation. Changes in BALF cells and lung mechanics are summarized for the available data. Crs, respiratory system compliance; PaO2, partial pressure of oxygen; RV, residual volume; VC, vital capacity; VE, minute ventilation; VT, tidal volume; VO2 max, maximal oxygen uptake; FEF75, Forced expiratory flow at 75% of forced vital capacity; FEF85, Forced expiratory flow at 85% of forced vital capacity. M, macrophage; N, neutrophil; L, lymphocyte; E, eosinophil</italic>.</p></table-wrap-foot></table-wrap><p>In the original article some references are misplaced.</p><p>In section “<bold>Inflammatory Response Under Impaired Lung Mechanics – Where Sterols Come into Play</bold>,” <bold>paragraph</bold> 5, the cited reference Ertunc and Hotamisligil, 2016 was incorrectly placed. It should be Jin et al., 2018. In paragraph 6, the cited reference Veldhuizen et al., 1996, 1997; Vazquez De Anda et al., 2000; Maitra et al., 2002 should be Ertunc and Hotamisligil, 2016. The cited reference Fessler and Summer, 2016 should be Veldhuizen et al., 1996, 1997; Vazquez De Anda et al., 2000; Maitra et al., 2002. The cited reference Glasser et al., 2003; Hamvas et al., 2004; Lawson et al., 2004; Stevens et al., 2005; Henderson et al., 2013; Liptzin et al., 2015; Salerno et al., 2016 should be Fessler and Summer, 2016. The cited reference Cassel et al., 2008 should be Glasser et al., 2003; Hamvas et al., 2004; Lawson et al., 2004; Stevens et al., 2005; Henderson et al., 2013; Liptzin et al., 2015; Salerno et al., 2016. In <bold>paragraph 7</bold>, the cited reference Vilaysane et al., 2010; Wree et al., 2014; Lv et al., 2018 should be Cassel et al., 2008. The cited reference Zhou et al., 2013 should be Gasse et al., 2007. The cited reference So et al., 2007; Ertunc and Hotamisligil, 2016 should be Vilaysane et al., 2010; Wree et al., 2014; Lv et al., 2018. The cited reference; Gasse et al., 2007; Robert et al., 2016 should be So et al., 2007; Ertunc and Hotamisligil, 2016.</p><p>In section “<bold>SP-C Modifications in Animal Models</bold>,” <bold>paragraph 1</bold>, the cited reference Lawson et al., 2005; Glasser et al., 2008, 2013a,b; Glasser and Senft, 2009; Ruwisch et al., 2020 should be Glasser et al., 2003; Ruwisch et al., 2020. The cited reference Jolley et al., 1999 should be Glasser et al., 2001; Glasser et al., 2003. The cited reference Lawson et al., 2005; Glasser et al., 2008, 2013a,b; Glasser and Senft, 2009 should be Jolley et al., 1999. In <bold>paragraph 2</bold>, the cited reference Lawson et al., 2005 should be Lawson et al., 2005; Glasser et al., 2008; Glasser et al., 2009; Glasser et al., 2013a,b. The cited reference Madala et al., 2011 should be Lawson et al., 2005. The cited reference Glasser et al., 2008 should be Madala et al., 2011. The cited reference Glasser et al., 2009 should be Glasser et al., 2008. The cited reference Glasser and Senft, 2009 should be Glasser et al., 2009. The cited reference Glasser et al., 2013b should be Glasser et al., 2008; Glasser et al., 2009. The cited reference Glasser et al., 2008 should be Glasser et al., 2013b. In <bold>paragraph 3</bold>, the cited reference Glasser et al. (2001), Bridges et al. (2003), should be Glasser et al., 2001, 2008. The cited reference Lawson et al., 2011, should be Bridges et al., 2003; in <bold>line 17</bold>, first Nureki et al., 2018 should be Lawson et al., 2011; in <bold>line 35</bold>, Venosa et al., 2019 should be Nureki et al., 2018. The cited reference Katzen et al., 2019 should be Venosa et al., 2019. The cited reference Nogee et al., 2001 should be Katzen et al., 2019.</p><p>In section “<bold>SP-C Mutations in Human Patients</bold>,” <bold>paragraph 1</bold>, the cited reference Ono et al. (2011), Litao et al. (2017) should be Nogee et al., 2001; in <bold>line 3</bold> Thomas et al., 2002; Ono et al., 2011; Kuse et al., 2013; Avital et al., 2014; Hevroni et al., 2015 should be Nogee et al., 2001; in <bold>line 9</bold> Thomas et al., 2002; Ono et al., 2011; Kuse et al., 2013; Avital et al., 2014; Hevroni et al., 2015 should be Ono et al., 2011; Litao et al., 2017. The cited reference Cottin et al., 2011; Ono et al., 2011 should be Ono et al., 2011; Kuse et al., 2013; Avital et al., 2014; Hevroni et al., 2015. The cited reference Cottin et al., 2011, Hevroni et al., 2015 should be Cottin et al., 2011; Ono et al., 2011. The cited reference Thomas et al., 2002; Abou Taam et al., 2009; Cottin et al., 2011; Ono et al., 2011; Avital et al., 2014 should be Cottin et al., 2011; Hevroni et al., 2015. The cited reference Thouvenin et al., 2010; Hevroni et al., 2015 should be Thomas et al., 2002; Abou Taam et al., 2009; Cottin et al., 2011; Ono et al., 2011; Avital et al., 2014, and Abou Taam et al., 2009 should be Thouvenin et al., 2010; Hevroni et al., 2015. The cited reference Avital et al., 2014 should be Abou Taam et al., 2009. The cited reference Lawson et al. (2004) should be Avital et al., 2014. The cited reference Cottin et al., 2011 should be Lawson et al., 2004 and Nogee et al. (2001), Chibbar et al. (2004), Hamvas et al. (2004), Lawson et al. (2004), Cameron et al. (2005), Stevens et al. (2005), Soraisham et al. (2006), Mechri et al. (2010), Thouvenin et al. (2010), Citti et al. (2013), Park et al. (2018) should be Cottin et al., 2011.</p><p>In <bold>paragraph 2</bold>, the cited reference Korfei et al., 2008 should be Nogee et al., 2001; Chibbar et al., 2004; Hamvas et al., 2004; Cameron et al., 2005; Stevens et al., 2005; Soraisham et al., 2006; Mechri et al., 2010; Thouvenin et al., 2010; Citti et al., 2013; Litao et al., 2017; Park et al., 2018. The cited reference Wambach et al., 2010 should be Korfei et al., 2008. The cited reference Amin et al., 2001 should be Wambach et al., 2010. The cited reference Chibbar et al., 2004; Hamvas et al., 2004; Tredano et al., 2004; Rosen and Waltz, 2005; Stevens et al., 2005; Bullard and Nogee, 2007; Guillot et al., 2009; Mechri et al., 2010; Cottin et al., 2011; Citti et al., 2013; Henderson et al., 2013; Hepping et al., 2013; Turcu et al., 2013; Akimoto et al., 2014; Avital et al., 2014; van Hoorn et al., 2014; Kroner et al., 2015; Liptzin et al., 2015; Peca et al., 2015; Griese et al., 2016; Liu et al., 2016; Hayasaka et al., 2018 should be Amin et al., 2001. The cited reference Lawson et al., 2004; Setoguchi et al., 2006; Markart et al., 2007; van Moorsel et al., 2010; Cottin et al., 2011; Ono et al., 2011; Kuse et al., 2013 should be Chibbar et al., 2004; Hamvas et al., 2004; Tredano et al., 2004; Rosen and Waltz, 2005; Stevens et al., 2005; Bullard and Nogee, 2007; Guillot et al., 2009; Mechri et al., 2010; Cottin et al., 2011; Citti et al., 2013; Henderson et al., 2013; Hepping et al., 2013; Turcu et al.,2013; Akimoto et al., 2014; Avital et al., 2014; van Hoorn et al., 2014; Kroner et al., 2015; Liptzin et al., 2015; Peca et al., 2015; Griese et al., 2016; Liu et al., 2016; Hayasaka et al., 2018 and Hamvas et al., 2004; Abou Taam et al., 2009; Mechri et al., 2010; Cottin et al., 2011; Litao et al., 2017 should be Lawson et al., 2004; Setoguchiet al., 2006; Markart et al., 2007; van Moorsel et al., 2010; Cottin et al., 2011; Ono et al., 2011; Kuse et al., 2013. In <bold>paragraph 3</bold>, the cited reference Amin et al., 2001 should be Hamvas et al., 2004; Abou Taam et al., 2009; Mechri et al., 2010; Cottin et al., 2011; Litao et al., 2017. The cited reference Lawson et al., 2004; Stevens et al., 2005; Hamvas, 2010; Henderson et al., 2013 should be Amin et al., 2001. The cited reference Liptzin et al., 2015; Salerno et al., 2016 should be Lawson et al., 2004; Stevens et al., 2005; Henderson et al., 2013. The cited reference Tredano et al., 2004; Abou Taam et al., 2009; Thouvenin et al., 2010; Ono et al., 2011 should be Liptzin et al., 2015; Salerno et al., 2016. The cited reference Thouvenin et al., 2010; Cottin et al., 2011 should be Abou Taam et al., 2009; Thouvenin et al., 2010; Ono et al., 2011. The cited reference Tredano et al., 2004; Ono et al., 2011 should be Thouvenin et al., 2010; Cottin et al., 2011 and Ruwisch et al., 2020 should be Ono et al., 2011.</p><p>In section “<bold>Lung Fibrosis and Cholesterol</bold>” the cited reference Xu et al., 2012 should be Kreuter et al., 2018. The cited reference Baritussio et al., 1980 should be Kreuter et al., 2018. The cited reference Turley et al., 1981 should be Baritussio et al., 1980. The cited reference Milos et al., 2016 should be Turley et al., 1981. The cited reference Liao and Laufs, 2004; Jain and Ridker, 2005 should be Kreuter et al., 2018 and Thomas et al., 2002 should be Liao and Laufs, 2004; Jain and Ridker, 2005.</p><p>The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.</p></body><back><ref-list><title>References</title><p>All references are correctly listed in the published reference list of the publication.</p></ref-list></back></article>\n"
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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Genome Biol Evol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Genome Biol Evol</journal-id><journal-id journal-id-type=\"publisher-id\">gbe</journal-id><journal-title-group><journal-title>Genome Biology and Evolution</journal-title></journal-title-group><issn pub-type=\"epub\">1759-6653</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32785673</article-id><article-id pub-id-type=\"pmc\">PMC7523730</article-id><article-id pub-id-type=\"doi\">10.1093/gbe/evaa164</article-id><article-id pub-id-type=\"publisher-id\">evaa164</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Research Article</subject></subj-group><subj-group subj-group-type=\"category-taxonomy-collection\"><subject>AcademicSubjects/SCI01130</subject><subject>AcademicSubjects/SCI01140</subject></subj-group></article-categories><title-group><article-title>Alignment-Integrated Reconstruction of Ancestral Sequences Improves Accuracy</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Aadland</surname><given-names>Kelsey</given-names></name><xref ref-type=\"aff\" rid=\"evaa164-aff1\"/></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-9490-6019</contrib-id><name><surname>Kolaczkowski</surname><given-names>Bryan</given-names></name><xref ref-type=\"corresp\" rid=\"evaa164-cor1\"/><!--<email>bryank@ufl.edu</email>--><xref ref-type=\"aff\" rid=\"evaa164-aff1\"/></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>dos Reis</surname><given-names>Mario</given-names></name><role>Associate Editor</role></contrib></contrib-group><aff id=\"evaa164-aff1\">\n<institution>Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida</institution>\n</aff><author-notes><corresp id=\"evaa164-cor1\">Corresponding author: E-mail: <email>bryank@ufl.edu</email>.</corresp></author-notes><pub-date pub-type=\"collection\"><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\" iso-8601-date=\"2020-08-12\"><day>12</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>12</day><month>8</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>12</volume><issue>9</issue><fpage>1549</fpage><lpage>1565</lpage><history><date date-type=\"accepted\"><day>03</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"cc-by\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"evaa164.pdf\"/><abstract><title>Abstract</title><p>Ancestral sequence reconstruction (ASR) uses an alignment of extant protein sequences, a phylogeny describing the history of the protein family and a model of the molecular-evolutionary process to infer the sequences of ancient proteins, allowing researchers to directly investigate the impact of sequence evolution on protein structure and function. Like all statistical inferences, ASR can be sensitive to violations of its underlying assumptions. Previous studies have shown that, whereas phylogenetic uncertainty has only a very weak impact on ASR accuracy, uncertainty in the protein sequence alignment can more strongly affect inferred ancestral sequences. Here, we show that errors in sequence alignment can produce errors in ASR across a range of realistic and simplified evolutionary scenarios. Importantly, sequence reconstruction errors can lead to errors in estimates of structural and functional properties of ancestral proteins, potentially undermining the reliability of analyses relying on ASR. We introduce an alignment-integrated ASR approach that combines information from many different sequence alignments. We show that integrating alignment uncertainty improves ASR accuracy and the accuracy of downstream structural and functional inferences, often performing as well as highly accurate structure-guided alignment. Given the growing evidence that sequence alignment errors can impact the reliability of ASR studies, we recommend that future studies incorporate approaches to mitigate the impact of alignment uncertainty. Probabilistic modeling of insertion and deletion events has the potential to radically improve ASR accuracy when the model reflects the true underlying evolutionary history, but further studies are required to thoroughly evaluate the reliability of these approaches under realistic conditions.</p></abstract><kwd-group><kwd>ancestral sequence reconstruction</kwd><kwd>alignment uncertainty</kwd><kwd>alignment integration</kwd></kwd-group><funding-group><award-group award-type=\"grant\"><funding-source><institution-wrap><institution>National Science Foundation</institution><institution-id institution-id-type=\"DOI\">10.13039/100000001</institution-id></institution-wrap></funding-source><award-id>1817942</award-id></award-group></funding-group><counts><page-count count=\"17\"/></counts></article-meta></front><body><boxed-text id=\"evaa164-BOX1\" position=\"float\" orientation=\"portrait\"><sec><title>Significance</title><p>Ancestral sequence reconstruction is a powerful technique for directly investigating the sequence, structure and function of ancient molecules to better understand molecular-evolutionary processes. However, ancestral reconstruction may not always be accurate under challenging conditions that make it difficult to correctly align protein sequences. We found that integrating information from many different alignment methods can produce reliable ancestral sequence reconstructions, even when the individual protein alignments have many errors. Our study suggests alignment-integrated methods may be an important approach for improving ancestral sequence reconstruction accuracy under challenging conditions.</p></sec></boxed-text><sec><title>Introduction</title><p>Aside from happening upon a piece of preserved ancient DNA (<xref rid=\"evaa164-B36\" ref-type=\"bibr\">Meyer et al. 2016</xref>) or reversing the arrow of time (<xref rid=\"evaa164-B37\" ref-type=\"bibr\">Micadei et al. 2019</xref>), ancestral sequence reconstruction (ASR) is the only available technique for directly investigating the sequence, structure and function of ancient molecules. Because ASR studies rely on statistical inferences of ancestral sequences that cannot be validated directly, the accuracy with which ancestral protein sequences can be inferred has been a major concern of the ASR research community (<xref rid=\"evaa164-B18\" ref-type=\"bibr\">Hall 2006</xref>; <xref rid=\"evaa164-B45\" ref-type=\"bibr\">Randall et al. 2016</xref>; <xref rid=\"evaa164-B15\" ref-type=\"bibr\">Eick et al. 2017</xref>). Previous studies have suggested that ASR is expected to be highly accurate in many cases (<xref rid=\"evaa164-B45\" ref-type=\"bibr\">Randall et al. 2016</xref>; <xref rid=\"evaa164-B58\" ref-type=\"bibr\">Vialle et al. 2018</xref>). Interestingly, studies have shown that the accuracy of the phylogenetic tree describing the evolutionary history of the protein family has only a very weak impact on ASR accuracy and generally only affects the most statistically ambiguous sequence positions (<xref rid=\"evaa164-B20\" ref-type=\"bibr\">Hanson-Smith et al. 2010</xref>). This largely counterintuitive result is due to the fact that the same evolutionary scenarios that make the phylogenetic tree uncertain also make ancestral sequences more similar across different phylogenies.</p><p>Some studies have suggested that there may be a trade-off between sequence reconstruction accuracy and the accuracy with which some structural and functional properties of the sequence can be inferred (<xref rid=\"evaa164-B60\" ref-type=\"bibr\">Williams et al. 2006</xref>; <xref rid=\"evaa164-B35\" ref-type=\"bibr\">Matsumoto et al. 2015</xref>; <xref rid=\"evaa164-B1\" ref-type=\"bibr\">Arenas et al. 2017</xref>). Specifically, maximum-a-posteriori (MAP) ASR [also referred to as maximum-likelihood {ML} ASR], which reconstructs the most accurate protein sequences, can produce biased inferences of structural stability. This stability bias can be alleviated using a sampling approach that randomly generates ancestral sequences from the posterior probability (PP) distributions at each site. However, this sampling approach produces sequences that are less accurate than MAP reconstruction, which can impact inferences of other structural or functional properties (<xref rid=\"evaa164-B15\" ref-type=\"bibr\">Eick et al. 2017</xref>).</p><p>One recent study found that the alignment of extant protein sequences forming the basis for phylogenetic inference and ASR can have a potentially strong affect on ASR accuracy (<xref rid=\"evaa164-B58\" ref-type=\"bibr\">Vialle et al. 2018</xref>). That ASR accuracy depends on alignment accuracy is concerning, as the “correct” alignment of extant protein sequences can hardly ever be known with certainty, and there are few reliable methods for diagnosing alignment error or ambiguity (<xref rid=\"evaa164-B12\" ref-type=\"bibr\">Dickson et al. 2010</xref>; <xref rid=\"evaa164-B42\" ref-type=\"bibr\">Penn et al. 2010</xref>). It is currently unknown whether the same alignment errors that cause ASR errors also impact the inferred structural or functional properties of reconstructed ancestral sequences, and no general methodologies exist to alleviate the impact of alignment error on ASR.</p><p>Here, we develop and evaluate a novel ASR approach that combines information from many different sequence alignments to infer “alignment-integrated” ancestral sequences. Although this approach does not completely eliminate the impact of alignment errors on ASR accuracy, we found that integrating sequence alignments reduces both ASR errors and errors in the structural and functional properties of inferred ancestral sequences, often performing as well as structure-guided sequence alignment. Our study suggests that, particularly for cases in which diverse structures of different protein family members are not available to guide the alignment process, integrating different alignments can be a reliable approach for mitigating the impact of alignment errors on ASR accuracy.</p></sec><sec><title>Results and Discussion</title><sec><title>Alignment Errors Vary with Alignment Method and Protein Domain Family</title><p>To assess the impact of alignment errors on ASR accuracy, we used structural alignments of individual protein domains to simulate sequence data along empirical domain-family phylogenies (see <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S1 and fig. S1</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online), with sequence composition and insertion–deletion (indel) patterns inferred from the structural alignment (see Materials and Methods section). Simulated data were then aligned using a variety of sequence-based methods as well as a “structure-guided” approach that used the original structural alignment to “seed” the alignment of additional sequences (see Materials and Methods section). Comparing sequence-alignments and structure-guided alignments to the correct simulated alignment allowed us to evaluate the extent to which the simulation conditions generated alignment errors that could potentially impact ASR accuracy.</p><p>In general, both sequence-alignments and structure-guided alignments underestimated the correct alignment length by placing fewer gaps in the alignment, resulting in overestimation of the proportion of variable and parsimony-informative positions (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S2 and fig. S2</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Across the five different protein-domain families used in this study, inferred alignments underestimated alignment length by 1.3-fold, on average (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>6.41e<sup>−4</sup>), and the number of gaps by 1.2-fold (<italic>P </italic>=<italic> </italic>1.69e<sup>−3</sup>). The proportion of variable sites was overestimated by 1.8-fold (<italic>P </italic>=<italic> </italic>4.97e<sup>−19</sup>), and the proportion of parsimony-informative sites was overestimated by 1.9-fold (<italic>P </italic>=<italic> </italic>2.28e<sup>−13</sup>). Structure-guided alignments were no different from sequence-alignment methods in any of the calculated alignment attributes (<italic>t</italic>-test <italic>P </italic>><italic> </italic>0.10), suggesting structure-guided and sequence-alignment methods tend to make similar errors in alignment length and the numbers of variable and parsimony-informative sites.</p><p>Although the general trend of alignment length underestimation is strongly supported by our data and is consistent with results from a previous study (<xref rid=\"evaa164-B16\" ref-type=\"bibr\">Fletcher and Yang 2010</xref>), we observed significant variation in alignment errors, both across protein domain families and across alignment methods (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S2 and fig. S2</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). For example, ClustalW tended to underestimate alignment length to a greater degree than other sequence-alignment methods (by 2.3-fold on average, vs. 1.1-fold for other methods; <italic>t</italic>-test <italic>P </italic><<italic> </italic>0.034). Across all alignment methods, the caspase activation and recruitment domain (CARD) protein domain family’s correct alignment length was underestimated to a greater degree (1.9-fold) than the other protein domain families (1.2-fold; <italic>t</italic>-test <italic>P </italic><<italic> </italic>0.06). In contrast to this general trend of alignment length underestimation, mafft, probalign and tcoffee tended to overestimate the lengths of the correct DSRM1 and DSRM2 protein domain family alignments (by 1.3-fold; <italic>P </italic><<italic> </italic>4.07e<sup>−4</sup>). These results suggest different alignment methods produce different types of alignment errors for different protein domain families. However, variation in alignment accuracy across replicate data sets of the same protein domain family was low, with the standard deviation (SD) never exceeding 11% of the inferred mean value of each alignment attribute (see <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S2</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). This suggests that most of the variation in alignment accuracy is expected to be due to the particular interaction between a chosen alignment algorithm and the way a protein domain family has evolved, rather than stochastic variation in the simulated evolutionary process.</p><p>We quantified the distance of each inferred alignment from the correct simulated alignment using a position-wise distance metric, which estimates the probability that a randomly selected residue from a randomly selected sequence was aligned to an incorrect residue from another randomly selected sequence (<xref rid=\"evaa164-B5\" ref-type=\"bibr\">Blackburne and Whelan 2012</xref>). In general, the results of this distance-based alignment assessment (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S3 and fig. S3</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online) were consistent with those of more traditional alignment metrics (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S2 and fig. S2</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Across all protein domain families and alignment methods, the probability of randomly selecting an incorrectly aligned residue was 0.31. However, there was strong variation in alignment distances across both domain families and alignment methods (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S3 and fig. S3</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). The CARD family produced larger average alignment distances than the other domain families (0.72 across alignment methods, vs. 0.21 for the other domain families; <italic>t</italic>-test <italic>P </italic><<italic> </italic>9.67e<sup>−6</sup>). Across protein domains, structure-guided alignments were >1.25-fold closer to the correct alignment than any of the sequence-alignment methods (<italic>t</italic>-test <italic>P </italic><<italic> </italic>0.033). There were no detectable systematic differences in alignment distances among sequence-alignment methods, which produced average distances between 0.24 (msaprobs and probcons) and 0.43 (probalign; one-factor ANOVA <italic>P </italic>=<italic> </italic>0.92).</p><p>Overall, these results suggest the test cases used in this study cover a range of alignment difficulties that do not strongly favor particular sequence-alignment algorithms over others and represent a reasonable test suite for assessing the impact of alignment errors on ASR under realistic conditions. Structure-guided alignment methods have been shown to out-perform sequence-alignment in previous studies (<xref rid=\"evaa164-B26\" ref-type=\"bibr\">Kim and Lee 2007</xref>), which has typically been attributed to the generally stronger conservation of protein structure versus sequence (<xref rid=\"evaa164-B23\" ref-type=\"bibr\">Ingles-Prieto et al. 2013</xref>). Our alignment-distance results are consistent with these findings, but more traditional alignment metrics did not strongly differentiate structure-guided from sequence-alignment methods, suggesting the structure-guided approach might produce only marginally better alignments under the challenging conditions used in this study.</p></sec><sec><title>Alignment Errors Reduce ASR Accuracy</title><p>Each set of empirical simulation conditions (see Materials and Methods section) was used to generate replicate correctly aligned extant protein sequences at the tips of the phylogeny and correct ancestral sequences at every internal node. We assessed ASR error rates at each node on the phylogeny by comparing the ancestral sequence inferred using only an alignment of the extant protein sequences to the correct simulated ancestral sequence at that node. Three types of ASR errors were considered: 1) residue errors, in which both the inferred and correct ancestral sequences have an amino-acid residue at the given alignment position, but the residues differ; 2) insertion errors, in which the inferred ancestral sequence has a residue at the given alignment position, but the correct ancestral sequence has a gap character (“−”), and 3) deletion errors, in which the inferred ancestral sequence has a gap character, but the correct ancestral sequence has an amino-acid residue. The ASR error rate for an inferred ancestral sequence was calculated as the number of ASR errors, divided by the length of the pairwise alignment of the correct to the inferred ancestral sequence (i.e. errors/site). The expected ASR error rate for a given ancestral node was calculated as the mean error rate over replicates.</p><p>Given a sequence alignment, phylogenetic tree and statistical model of the molecular-evolutionary process, reconstruction of the most likely residue at each position in the alignment and node on the phylogeny has been well-described, as has the assessment of statistical confidence in the residue reconstruction (<xref rid=\"evaa164-B62\" ref-type=\"bibr\">Yang et al. 1995</xref>; <xref rid=\"evaa164-B28\" ref-type=\"bibr\">Koshi and Goldstein 1996</xref>). However, due to the way gap characters are encoded in most phylogenetic models, standard ASR does not reconstruct historical insertions or deletions (indels), resulting in ancestral sequences with no gap characters (<xref rid=\"evaa164-B18\" ref-type=\"bibr\">Hall 2006</xref>). Unfortunately, the methodological details of ancestral indel reconstruction are poorly described in many published ASR studies (<xref rid=\"evaa164-B7\" ref-type=\"bibr\">Chang et al. 2002</xref>; <xref rid=\"evaa164-B17\" ref-type=\"bibr\">Gaucher et al. 2003</xref>; <xref rid=\"evaa164-B6\" ref-type=\"bibr\">Bridgham et al. 2006</xref>; <xref rid=\"evaa164-B59\" ref-type=\"bibr\">Voordeckers et al. 2012</xref>; <xref rid=\"evaa164-B57\" ref-type=\"bibr\">Tan et al. 2016</xref>), making it difficult to assess how ancestral gaps were inferred. Although some methods have been developed that infer ancestral gaps as part of a more complex likelihood model (<xref rid=\"evaa164-B46\" ref-type=\"bibr\">Redelings and Suchard 2005</xref>; <xref rid=\"evaa164-B21\" ref-type=\"bibr\">Herman et al. 2014</xref>; <xref rid=\"evaa164-B22\" ref-type=\"bibr\">Holmes 2017</xref>; <xref rid=\"evaa164-B52\" ref-type=\"bibr\">Shim and Larget 2018</xref>), these approaches are largely untested and have not been adopted in many ASR studies. Many historical ASR studies probably used maximum-parsimony reconstruction of presence–absence ancestral states or a parsimony-like subjective criterion to place ancestral gap characters (<xref rid=\"evaa164-B18\" ref-type=\"bibr\">Hall 2006</xref>; <xref rid=\"evaa164-B19\" ref-type=\"bibr\">Hanson-Smith and Johnson 2016</xref>). Other studies have suggested using ML (MAP) reconstruction of presence–absence states to infer ancestral gaps (<xref rid=\"evaa164-B2\" ref-type=\"bibr\">Ashkenazy et al. 2012</xref>), which is the approach we take here (see Materials and Methods section).</p><p>Assuming the correct simulated alignment, we found that site-wise MAP reconstruction of ancestral gap states generated error rates comparable with residue reconstruction (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary tables S4–S8 and figs. S4–S7</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Averaged across all protein domain families and ancestral nodes, ASR error rates were low when the correct alignment was known in advance (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S4</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online), and the rate of residue-reconstruction errors (6.18e<sup>−3</sup> errors/site) was slightly worse than the rate of erroneous insertions (8.49e<sup>−4</sup> errors/site) or deletions (8.17e<sup>−4</sup>; <italic>t</italic>-test <italic>P </italic><<italic> </italic>0.013). This pattern was generally observed across all the protein domain families (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary tables S4–S8 and figs. S4–S7</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Only in the case of the CARD family was the residue reconstruction error rate (3.37e<sup>−3</sup>) slightly lower than the rate of erroneously inferred insertions (3.55e<sup>−3</sup>) or deletions (3.44e<sup>−3</sup>), and these differences were not statistically significant (<italic>t</italic>-test <italic>P </italic>><italic> </italic>0.31). For all other domain families, residue reconstruction error rates were slightly higher than indel reconstruction error rates (<italic>P </italic><<italic> </italic>0.039). These results suggest site-wise MAP reconstruction of ancestral gap states—although failing to accurately model statistical dependencies among contiguous gaps (<xref rid=\"evaa164-B2\" ref-type=\"bibr\">Ashkenazy et al. 2012</xref>)—provides a robust methodology for systematically inferring ancestral insertions and deletions under realistic conditions, provided the alignment is accurate.</p><p>When the alignment is not known in advance and must be inferred from the sequence data, we found errors in ASR were higher overall and increased with increasing distance from the correct alignment (<xref ref-type=\"fig\" rid=\"evaa164-F1\">fig. 1</xref>; <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary tables S4–S8 and figs. S4–S7</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Total ASR error rates were >4.8-fold higher when ancestral sequences were inferred using sequence-alignment methods (<italic>t</italic>-test <italic>P </italic><<italic> </italic>5.78e<sup>−3</sup>). Even when structure-guided alignments were used to infer ancestral sequences, total ASR error rates were 4.4-fold higher than when the correct alignment was known in advance (<italic>t</italic>-test <italic>P </italic><<italic> </italic>6.84e<sup>−3</sup>). These results were generally consistent across ASR error types and protein domain families (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary tables S4–S8 and figs. S4–S7</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Residue errors increased by at least 2.6-fold when sequence-alignment was used, compared with the correct alignment (<italic>t</italic>-test <italic>P </italic><<italic> </italic>0.013), and indel errors increased by 12.3- and 12.7-fold, respectively (<italic>t</italic>-test <italic>P </italic><<italic> </italic>4.37e<sup>−3</sup>). In all cases, structure-guided alignments produced ancestral sequences that had >1.5-fold fewer errors than sequence-alignment methods (<italic>t</italic>-test <italic>P </italic><<italic> </italic>8.43e<sup>−3</sup>).</p><fig id=\"evaa164-F1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1</label><caption><p>Errors in ASR correlate with alignment errors. We simulated protein evolution using empirically derived conditions from five protein domain families and aligned the resulting sequences using structure-guided and seven different sequence-alignment methods. We measured the position-wise distance of each alignment from the correct simulated alignment (<italic>x</italic> axis), which estimates the probability of selecting two incorrectly aligned residues at random. We used each alignment to infer the most likely ancestral sequence at each node on the phylogeny and compared the inferred ancestral sequence to the correct simulated sequence to estimate ASR error rates. ASR errors were divided into four categories: 1) residue errors, in which both correct and inferred ancestral sequences have a residue at a given position in the alignment, but the residues differ; 2) insertion errors, in which the inferred sequence has a residue at a given alignment position, but the correct sequence has a gap; 3) deletion errors, in which the inferred sequence has a gap, but the correct sequence has a residue, and 4) total errors. Sequence-wide error rates (errors/site; <italic>y</italic> axis) were computed by dividing the number of errors by the length of the pairwise alignment of the inferred and correct ancestral sequences. Dotted lines indicate least-squares linear regressions.</p></caption><graphic xlink:href=\"evaa164f1\"/></fig><p>In general, there was a strong correlation between an inferred alignment’s distance from the correct alignment and total ASR error rate (<xref ref-type=\"fig\" rid=\"evaa164-F1\">fig. 1</xref>; <italic>r</italic><sup>2</sup> > 0.78, mean <italic>r</italic><sup>2</sup> = 0.88). Similarly high correlations were observed for rates of insertion and deletion errors (<italic>r</italic><sup>2</sup> > 0.76, mean <italic>r</italic><sup>2</sup> > 0.85). However, residue errors were less strongly correlated with the distance from the correct alignment (mean <italic>r</italic><sup>2</sup> = 0.55), largely because muscle and probcons residue-errors were only very weakly correlated with alignment distance (<italic>r</italic><sup>2</sup> < 0.23). Interestingly, whereas error rate increased with alignment distance at roughly the same rate for alignment algorithms other than ClustalW (slope of the best-fit regression line was 0.25–0.45 for total ASR error rate), ClustalW’s total ASR error rate increased much more rapidly as the alignment diverged from the correct alignment (slope = 1.08; ANCOVA <italic>P </italic><<italic> </italic>1.63e<sup>−3</sup>). ClustalW’s ASR error rates were generally higher than the other alignment methods, even at comparable alignment distances (see <xref ref-type=\"fig\" rid=\"evaa164-F1\">fig. 1</xref>), suggesting that, in addition to an alignment’s distance from the correct alignment, the specific types of alignment errors made can also affect how alignment errors impact ASR accuracy.</p><p>Our results confirm that ASR accuracy can be negatively impacted by alignment errors (<xref rid=\"evaa164-B58\" ref-type=\"bibr\">Vialle et al. 2018</xref>) and suggest structure-guided alignment—although not a panacea—generally produces more accurate ancestral sequences than sequence-alignment methods.</p></sec><sec><title>Alignment-Integrated ASR Improves Accuracy</title><p>Unlike the phylogeny (<xref rid=\"evaa164-B20\" ref-type=\"bibr\">Hanson-Smith et al. 2010</xref>), our results and those of a previous study (<xref rid=\"evaa164-B58\" ref-type=\"bibr\">Vialle et al. 2018</xref>) strongly suggest the sequence alignment—which can never be known with certainty in practice—can have a strong impact on ASR. We hypothesized that integrating over alignment uncertainty could potentially alleviate this negative impact. To test this hypothesis, we developed a heuristic approach that reconstructs ancestral residues and gap states by integrating information from the seven sequence-alignment methods examined in this study, placing equal prior weight on each alignment (see Materials and Methods section).</p><p>We found that integrating information from many sequence-alignment algorithms reduced ASR error rates, compared with relying on any single sequence-alignment method (<xref ref-type=\"fig\" rid=\"evaa164-F2\">fig. 2</xref>; <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary tables S4–S8 and figs. S4–S7</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). On average, integrating over alignment uncertainty improved total ASR error rates by >1.3-fold, compared with choosing any single sequence-alignment method (<italic>t</italic>-test <italic>P </italic><<italic> </italic>0.022). Although alignment-integrated ASR always generated fewer errors in residue and deletion reconstructions, improvements in these types of ASR errors were generally small and not always statistically significant. The fold-improvement in residue reconstruction error rates ranged from 1.1 (compared with tcoffee; <italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.17) to 1.8 (ClustalW; <italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.012), whereas the improvement in deletion error rates ranged from 1.1-fold (probcons; <italic>P </italic>=<italic> </italic>0.18) to 2.1-fold (probalign; <italic>P </italic>=<italic> </italic>8.43e<sup>−3</sup>). The most dramatic reduction in ASR error rate was observed for insertion errors, for which alignment–integration improved error rates by >2.9-fold, compared with single sequence-alignment methods (<italic>t</italic>-test <italic>P </italic><<italic> </italic>7.04e<sup>−3</sup>).</p><fig id=\"evaa164-F2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2</label><caption><p>Alignment-integrated and structure-guided approaches produce fewer ASR errors than single sequence-alignment methods. We simulated extant and ancestral sequences for five protein domain families, using empirically derived conditions, and aligned the resulting extant sequence data using the correct simulated alignment, structure-guided, and seven different sequence-alignment methods. We used each alignment to infer the most likely ancestral sequence at each node on the phylogeny. In addition, alignment-integrated ancestral sequences were generated by combining inferences from the seven sequence-alignment methods. In each case, we compared the inferred ancestral sequence to the correct simulated ancestral sequence to estimate ASR error rates (expected errors/site). Error rate distributions were calculated over 10 replicate simulations and all nodes on each of the five protein domain family phylogenies. We plot the distributions of total- (top), residue-, insertion-, and deletion-errors (bottom) for each alignment method.</p></caption><graphic xlink:href=\"evaa164f2\"/></fig><p>The same general pattern was consistently observed across all of the protein domain families examined in this study (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary tables S5–S8 and figs. S4–S7</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online): compared with choosing a single sequence-alignment method, alignment–integration improved overall ASR error rates in all cases (by >1.1-fold), primarily by reducing the rate of insertion errors (by >1.8-fold; <italic>t</italic>-test <italic>P </italic><<italic> </italic>0.022). In the case of the DSRM3 and RNA recognition domain (RD) families, the improvement in total ASR error rate was not always statistically significant, compared with some of the sequence-alignment methods. In both cases, mafft, msaprobs, muscle, and probcons were statistically equivalent to alignment–integration (<italic>t</italic>-test <italic>P </italic>><italic> </italic>0.057), and probalign was equivalent to alignment–integration for the RD family (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.063).</p><p>These results suggest integrating over different sequence-alignment methods generally improves the accuracy of ASR, compared with choosing a single sequence-alignment method, primarily by reducing the rate of erroneously inferred insertions. The improvement in total ASR accuracy may be small for highly conserved protein families or other scenarios in which sequence-based alignments are generally accurate.</p><p>Interestingly, integrating over many sequence-alignments slightly improved ASR error rates, even compared with the highly accurate structure-guided alignment approach. Overall, total ASR error rates were reduced by 1.2-fold using alignment–integration, compared with structure-guided alignment (<xref ref-type=\"fig\" rid=\"evaa164-F2\">fig. 2</xref>; <italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.035), even though each of the individual sequence-alignments was farther away from the correct alignment than was the structure-guided alignment (see <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S3 and fig. S3</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Compared with structure-guided alignment, alignment–integration produced 1.2-fold more residue reconstruction errors (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.059), the same number of deletion errors (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.088), and 3.0-fold fewer insertion errors (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>7.68e<sup>−3</sup>). This improvement in insertion error rate was observed for all domain families except the RD (<italic>P </italic><<italic> </italic>0.033; see <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S7 and fig. S6</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online), and total ASR error rates were never significantly better using the structure-guided alignment (<italic>t</italic>-test <italic>P </italic>><italic> </italic>0.11). These results suggest alignment–integration is a promising technique for reducing ASR error rates, even for protein families for which diverse structural data are not available to generate structure-guided alignments.</p><p>Protein sequence alignments are inferred using diverse methodologies, and new alignment methods are developed regularly. Most widely used methods rely on heuristic strategies to place gap characters, with a rough “guide tree” being used to order pairwise alignments (<xref rid=\"evaa164-B9\" ref-type=\"bibr\">Chatzou et al. 2016</xref>). However, some methods have extended phylogenetic models to explicitly incorporate indel events within a probabilistic framework (<xref rid=\"evaa164-B46\" ref-type=\"bibr\">Redelings and Suchard 2005</xref>; <xref rid=\"evaa164-B32\" ref-type=\"bibr\">Loytynoja 2014</xref>). We evaluated the accuracy of ancestral sequences reconstructed from two different “phylogenetically aware” probabilistic alignment methods: PRANK, which uses an indel model assuming a rough guide-tree approximation of the phylogeny (<xref rid=\"evaa164-B33\" ref-type=\"bibr\">Loytynoja and Goldman 2008</xref>; <xref rid=\"evaa164-B32\" ref-type=\"bibr\">Loytynoja 2014</xref>), and BAli-Phy, which uses Bayesian co-estimation of phylogeny and sequence alignment (<xref rid=\"evaa164-B46\" ref-type=\"bibr\">Redelings and Suchard 2005</xref>). Because BAli-Phy is extremely computationally costly (<xref rid=\"evaa164-B41\" ref-type=\"bibr\">Nute et al. 2019</xref>), analyses were conducted assuming the correct phylogenetic tree. To facilitate comparisons, PRANK and BAli-Phy were used only to generate sequence alignments, with ancestral sequences reconstructed using the same approach as for other alignment programs (see Materials and Methods section).</p><p>Interestingly, we found that using the MAP alignment generated by BAli-Phy to reconstruct ancestral sequences was statistically indistinguishable from assuming the correct simulated alignment (see <xref ref-type=\"fig\" rid=\"evaa164-F2\">fig. 2</xref>; <italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.16), whereas PRANK alignments generated using a similar indel model (but without conditioning on the correct phylogenetic tree topology) resulted in among the least accurate ancestral sequences (<xref ref-type=\"fig\" rid=\"evaa164-F2\">fig. 2</xref>). These results are consistent with those of a recent study examining protein sequence alignment accuracy, in which BAli-Phy generated highly accurate sequence alignments from simulated data, whereas PRANK did not (<xref rid=\"evaa164-B41\" ref-type=\"bibr\">Nute et al. 2019</xref>). Although these results suggest probabilistic modeling of indel events could be a productive strategy for improving the accuracy of protein sequence alignment and ancestral reconstruction, future studies will be required to determine why similar approaches can produce very different results.</p></sec><sec><title>Alignment–Integration Increases ASR Ambiguity</title><p>It is common practice in many ASR studies to reconstruct “plausible alternative” states at positions with ambiguous reconstructions, to evaluate the impact of ASR uncertainty on downstream analyses (<xref rid=\"evaa164-B15\" ref-type=\"bibr\">Eick et al. 2017</xref>). ASR errors that are only weakly supported are likely to be identified by this approach, whereas errors with very high PP will likely be accepted as “correct,” potentially undermining the validity of downstream structural or functional analyses.</p><p>We found that integrating many sequence-alignment methods, in addition to reducing ASR error rates, also reduced the PPs of erroneous ancestral states, when they were inferred (<xref ref-type=\"fig\" rid=\"evaa164-F3\">fig. 3<italic>A</italic>;</xref><xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S9</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). On average, the PP of erroneously inferred ancestral states was >0.9 when any single sequence-alignment or the structure-guided alignment was used to infer ancestral sequences. In contrast, the alignment-integrated approach had an average PP of 0.67 for erroneous ancestral states, which was much more similar to that of the correct alignment (0.65; <italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.066). Interestingly, this strong similarity between the alignment-integrated approach and the correct alignment was primarily confined to residue errors, for which alignment–integration produced an average PP of 0.59, and the correct alignment’s mean PP was 0.57 (<italic>P </italic>=<italic> </italic>0.093). In the case of insertion or deletion errors, the correct alignment produced low average PPs for erroneously inferred ancestral states (<0.43), whereas alignment–integration’s PPs were much higher (>0.71, on average; <italic>t</italic>-test <italic>P </italic><<italic> </italic>6.85e<sup>−3</sup>). Even in these cases, however, alignment–integration produced much more weakly supported errors than any single sequence-alignment or structure-guided alignment, whose mean PPs for erroneously inferred ancestral states were always >0.9 (<italic>t</italic>-test <italic>P </italic><<italic> </italic>8.15e<sup>−3</sup>).</p><fig id=\"evaa164-F3\" orientation=\"portrait\" position=\"float\"><label>Fig. 3</label><caption><p>Alignment-integrated ASR generates lower statistical confidence in erroneous ancestral states and stronger support for the correct state when errors are made. We used the correct simulated alignment, seven different sequence-alignment methods, structure-guided alignment, and alignment–integration to infer ancestral protein sequences from five empirically derived simulation conditions. Total- (top), residue-, insertion-, and deletion- (bottom) error rates (expected errors/site; <italic>x</italic> axis) were calculated by comparing the inferred ancestral sequence to the correct simulated sequence at each node on the phylogeny. We used kernel density estimation to calculate the frequency distributions (<italic>y</italic> axis) of PPs for erroneous MAP ancestral states (left) and the correct ancestral state (right) when the correct state was not the inferred MAP state.</p></caption><graphic xlink:href=\"evaa164f3\"/></fig><p>When ASR errors were made, alignment–integration also generated much stronger support for the correct ancestral state than any of the other alignment strategies, including the correct alignment (<xref ref-type=\"fig\" rid=\"evaa164-F3\">fig. 3<italic>B</italic>;</xref><xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S10</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). On average, the PP of the correct ancestral state using sequence-alignment or structure-guided alignment was <0.046 when the correct state was not the MAP reconstruction. Assuming the correct alignment improved the mean PP of the correct state by ∼3-fold (to 0.14; <italic>t</italic>-test <italic>P </italic><<italic> </italic>8.42e<sup>−3</sup>). However, alignment–integration further increased the mean PP of the correct ancestral state by 2.4-fold, compared with the correct alignment (<italic>P </italic>=<italic> </italic>5.78e<sup>−3</sup>). Importantly, alignment–integration increased the PP of the correct state to 0.33, on average, which is higher than the cutoff of 0.2–0.3 commonly used to identify plausible alternative ancestral reconstructions (<xref rid=\"evaa164-B15\" ref-type=\"bibr\">Eick et al. 2017</xref>). This large increase in statistical support for the correct ancestral state when errors were made by alignment–integration was most pronounced for deletion errors (mean PP 0.46) and less pronounced for insertion (mean PP = 0.30) or residue errors (mean PP = 0.21). For all types of errors, however, alignment integration produced >1.3-fold higher PPs for the correct ancestral state, compared with the correct alignment (<italic>t</italic>-test <italic>P </italic><<italic> </italic>0.021) and >3.1-fold higher support than any other alignment method (<italic>P </italic><<italic> </italic>4.78e<sup>−3</sup>).</p><p>Although alignment–integration is obviously not a panacea, these results suggest, in addition to improving ASR accuracy, alignment–integration might be an important approach for “exposing” some potential errors to downstream robustness analysis by reducing the statistical support for erroneously inferred ancestral states and increasing the PP of the correct ancestral state when it is not inferred as the MAP state.</p><p>The generally favorable increase in statistical ambiguity when ASR errors are made by alignment–integration does come at the cost of increased ambiguity for correctly inferred ancestral states (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S11 and fig. S8</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). On average, correct ancestral state inferences were made with high statistical confidence using any of the methods examined in this study (>0.97 mean PP). However, alignment–integration generated lower statistical confidence in correct ancestral state inferences than any of the other methods, all of which had >0.99 mean PP for correctly inferred states (<italic>t</italic>-test <italic>P </italic><<italic> </italic>4.07e<sup>−3</sup>). All of the ASR methods exhibited stronger statistical support for correctly inferred ancestral gap states versus correctly inferred amino-acid residues (<italic>t</italic>-test <italic>P </italic><<italic> </italic>0.011). However, this difference was more pronounced for alignment–integration, compared with the other ASR methods. When using alignment–integration, the mean PP of correctly inferred residues was 0.84 (>0.94 for the other methods), whereas the mean PP was 0.98 for gap states correctly inferred by alignment-integrated ASR. These results suggest the reduced susceptibility to ASR error enjoyed by alignment–integration is also associated with increased ambiguity in reconstructed amino-acid residues, even when they are correctly inferred. This increased ASR ambiguity could potentially increase the operational costs of evaluating robustness to uncertainty as part of a typical ASR study.</p></sec><sec><title>Alignment–Integration Improves Structural and Functional Inferences</title><p>In many ASR studies, the actual ancestral sequences are only of secondary interest, being commonly used to better understand how the protein’s structural and functional properties evolved (<xref rid=\"evaa164-B15\" ref-type=\"bibr\">Eick et al. 2017</xref>). In some cases, researchers may decide to tolerate additional errors in sequence reconstruction, provided they result in more accurate inferences of specific structural or functional properties (<xref rid=\"evaa164-B60\" ref-type=\"bibr\">Williams et al. 2006</xref>; <xref rid=\"evaa164-B35\" ref-type=\"bibr\">Matsumoto et al. 2015</xref>; <xref rid=\"evaa164-B1\" ref-type=\"bibr\">Arenas et al. 2017</xref>). To investigate the potential impact of alignment errors on the accuracy of downstream structural and functional investigations, we generated structural models of ancestral DSRM1 protein sequences inferred using each alignment method and estimated each protein’s structural stability and double-stranded RNA (dsRNA)-binding affinity using computational approaches (see Materials and Methods section).</p><p>We found alignment-integrated ASR generally improved computational estimates of structural stability and RNA-binding affinity, compared with relying on a single sequence-alignment method (<xref ref-type=\"fig\" rid=\"evaa164-F4\">fig. 4;</xref><xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S12 and fig. S9</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Aside from probcons, alignment–integration had significantly smaller errors in inferred structural stability than sequence-alignment methods (>1.14-fold; <italic>t</italic>-test <italic>P </italic><<italic> </italic>0.048), performing similarly to structure-guided alignment (<xref ref-type=\"fig\" rid=\"evaa164-F4\">fig. 4</xref>; <italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.19). In this case, alignment–integration and probcons produced equivalent errors in structural stability estimates (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.12). We observed similar results for estimated dsRNA-binding affinity (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S9</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Alignment–integration produced >1.14-fold smaller errors in affinity estimates, compared with all sequence-alignment methods other than msaprobs (<italic>t</italic>-test <italic>P </italic><<italic> </italic>0.041). Binding affinity errors were equivalent, on average, among alignment-integrated, structure-guided, and msaprobs alignments (<italic>t</italic>-test <italic>P </italic>><italic> </italic>0.055).</p><fig id=\"evaa164-F4\" orientation=\"portrait\" position=\"float\"><label>Fig. 4</label><caption><p>Alignment-integrated and structure-guided approaches produce less error in inferred structural properties of ancestral proteins than single sequence-alignment methods. We simulated replicate extant and ancestral sequences by evolving an RNA-binding protein domain along its empirically determined phylogeny, using a structure-guided alignment to determine the amino-acid composition and pattern of insertions/deletions. Ancestral sequences were inferred using the correct simulated alignment, structure-guided alignment, seven different sequence-alignment methods, and alignment–integration. We modeled the structure of each ancestral sequence and estimated its structural stability (Δ<italic>G</italic>) using a computational approach. Errors in structural stability were calculated by comparing values estimated from the correct ancestral sequences to those estimated using each alignment method. Error rate distributions were calculated over 10 replicate simulations and all nodes on the phylogeny.</p></caption><graphic xlink:href=\"evaa164f4\"/></fig><p>As expected, having the correct sequence alignment improved inferences of ancestral structural and functional properties in all cases (<italic>t</italic>-test <italic>P </italic><<italic> </italic>0.017) but did not completely alleviate errors in structural stability or binding affinity estimates. On average, stability and affinity estimates deviated by >25% from the values inferred using the correct ancestral sequences (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S12 and fig. S10</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). The mean structural stability (Δ<italic>G</italic>) of correct ancestral DSRM1 domains was 0.087 cal/(mol × K) per residue (i.e. the change in per-residue free energy of the native state, compared with misfolded or unfolded states, calculated using a contact-based energy model; see Materials and Methods section), and structural stability estimates were typically 29.7% away from the correct values. Similarly, dsRNA-affinity estimates were, on average, 26.7% away from the values inferred using the correct ancestral sequences.</p><p>Together, these results suggest ambiguity or bias in the ASR process can itself contribute to errors in downstream structural and functional inferences under challenging conditions (<xref rid=\"evaa164-B60\" ref-type=\"bibr\">Williams et al. 2006</xref>; <xref rid=\"evaa164-B1\" ref-type=\"bibr\">Arenas et al. 2017</xref>). Alignment errors appear to exacerbate errors in estimated structural stability and binding affinity of ancestral proteins, but structure-guided alignment or alignment–integration significantly reduced these errors.</p><p>There was a weak but significant positive correlation between ASR errors and errors in structural and functional estimates for all alignment methods (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S13 and fig. S11</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Errors in the inference of the ancestral sequence explained 40% of the variation in structural stability error (<italic>r</italic><sup>2</sup> < 0.78) and 29% of the variation in binding affinity error (<italic>r</italic><sup>2</sup> < 0.64). The mean slope of the best-fit regression line across all alignment methods was 0.44 for structural stability and 4.25 for binding affinity, and all slopes were significantly greater than zero (<italic>t</italic>-test <italic>P </italic><<italic> </italic>2.50e<sup>−3</sup>). There were some differences in both correlation and slope across alignment methods. For example, ClustalW, mafft, probalign and tcoffee showed weaker correlations between ASR error rates and structural stability errors (<italic>r</italic><sup>2</sup> < 0.29), while the remaining alignments had generally higher correlations (<italic>r</italic><sup>2</sup> > 0.41). Similarly, the slope of the best-fit regression line varied from a minimum of 0.17 (probalign) to a maximum of 0.74 (for the correct alignment). Similar results were observed for the correlation between ASR error rate and binding affinity errors: correlation varied between <italic>r</italic><sup>2</sup> = 0.021 (ClustalW) and <italic>r</italic><sup>2</sup> = 0.64 (correct alignment), and slope varied from 1.07 (ClustalW) to 7.00 (probalign; <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S12</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Qualitatively similar results were observed when considering different types of ASR sequence errors (see <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S13 and fig. S11</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online).</p><p>These results suggest the overall rate of sequence reconstruction errors is positively correlated with errors in estimates of structural and functional properties of ancestral sequences. The generally lower magnitudes of structural and functional errors observed for the structure-guided and alignment-integrated methods can be at least partially explained by their generally lower sequence-error rates. However, precisely how sequence-reconstruction errors translate into errors in structural or functional estimates is expected to be complex in realistic cases, and the specific types of sequence-reconstruction errors made by different alignment algorithms likely also plays a role in determining errors in structural or functional estimates.</p></sec><sec><title>Integrating PP Distributions Improves ASR Accuracy</title><p>To begin systematically investigating the factors impacting ASR accuracy, we varied the branch lengths and indel rates along a minimal three-taxon phylogeny with equal branch lengths and the same indel rate on all branches (<xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S12</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Sequences were simulated using the JTT+G evolutionary model, and indels were placed randomly along the sequence. The ancestral sequence at the only node on the phylogeny was reconstructed using the correct simulated alignment, seven sequence-alignment methods and the alignment-integrated approach (see Materials and Methods section).</p><p>Results from this three-taxon simulation were largely consistent with those obtained using larger, more realistic phylogenies (<xref ref-type=\"fig\" rid=\"evaa164-F5\">fig. 5</xref>; supplementary figs. S13–S16, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Across all simulation conditions, alignment–integration slightly improved ASR accuracy by 1.06-fold, compared with choosing a single sequence-alignment strategy at random (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>4.01e<sup>−9</sup>). Even when the optimal sequence-alignment strategy was chosen for each set of simulation conditions, alignment–integration improved ASR accuracy by 1.03-fold (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>3.33e<sup>−4</sup>). Although the improvement in alignment-integrated ASR accuracy was typically small in this case (∼1%, on average), it was consistent across the vast majority of simulation conditions. Only under 5/64 conditions was the best sequence-alignment method as accurate or more accurate than alignment–integration, and most of these conditions had short branch lengths and low indel rates, leading to very low ASR errors across all methods.</p><fig id=\"evaa164-F5\" orientation=\"portrait\" position=\"float\"><label>Fig. 5</label><caption><p>Alignment-integrated ASR produces low rates of reconstruction errors in simplified three-taxon simulations. We simulated protein sequences along a three-taxon phylogeny with equal branch lengths (<italic>x</italic> axis) and the same indel rate (<italic>y</italic> axis) across the phylogeny (see <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S12</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). The most likely ancestral sequence at the single node on the phylogeny was inferred using the correct simulated alignment, seven different sequence-alignment methods, and alignment–integration. Total- (top), residue-, insertion-, and deletion- (bottom) error rates were calculated by comparing inferred ancestral sequences to the correct simulated ancestral sequence. For each set of simulation conditions, we calculated the difference in error rates between the correct alignment versus the least-erroneous sequence-alignment method (left column) or versus alignment–integration (right column). Positive values (blue) indicate that the correct alignment produced more errors than the given inference method, and negative values (red) indicate that the correct alignment produced fewer errors.</p></caption><graphic xlink:href=\"evaa164f5\"/></fig><p>As expected, ASR error rates increased with increasing branch lengths for all methods (linear regression slope >0.38, <italic>r</italic><sup>2</sup> > 0.85, <italic>t</italic>-test <italic>P </italic><<italic> </italic>1.09e<sup>−3</sup>; <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S14 and figs. S13–S16</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). For short branch lengths, ASR error rates were weakly correlated with increasing indel rates: when branches were <0.6 substitutions/site, linear regression slope was >0.19 (<italic>r</italic><sup>2</sup> > 0.68, <italic>t</italic>-test <italic>P </italic><<italic> </italic>0.012; <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary table S14 and figs. S13–S16</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). However, the correlation between ASR error and indel rates was not observed for branch lengths >0.6 (<italic>t</italic>-test <italic>P </italic>><italic> </italic>0.044).</p><p>Interestingly, alignment–integration appeared slightly more accurate than using the correct sequence alignment under some conditions, improving ASR error rates by ∼0.6%, on average, compared with the correct alignment (<xref ref-type=\"fig\" rid=\"evaa164-F5\">fig. 5</xref>); however, this difference was not statistically significant (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.42). We did observe slightly lower insertion error rates using alignment–integration, compared with the correct alignment (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>9.56e<sup>−3</sup>). Residue errors were more frequent for alignment–integration (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>4.85e<sup>−8</sup>), and there was no overall difference in deletion error rates between the two methods (<italic>t</italic>-test <italic>P </italic>=<italic> </italic>0.25).</p><p>These results suggest alignment–integration can consistently improve ASR error rates, compared with single sequence-alignment methods, even under an extremely simplified three-taxon model system with random indels. Under some of these simplified conditions, alignment–integration can produce error rates comparable with those obtained when the correct alignment is known in advance.</p><p>Results from three-taxon simulations suggest that simple “majority-rule” is sufficient to explain most of the cases in which alignment–integration improves ASR accuracy (<xref ref-type=\"fig\" rid=\"evaa164-F6\">fig. 6</xref>). On average, when one of the alignment methods produced an error that was not present in the alignment-integrated ancestral sequence, 66.3% of the other sequence-alignment methods reconstructed the correct ancestral state. This result was consistent across all simulation conditions (standard error 0.002; see <xref ref-type=\"fig\" rid=\"evaa164-F6\">fig. 6</xref>). Interestingly, residue-reconstruction errors tended to have a weaker majority in favor of the correct ancestral residue; on average only 60.0% of other alignments recovered the correct ancestral residue when one alignment made a residue-reconstruction error (<italic>z</italic>-test <italic>P </italic>=<italic> </italic>9.17e<sup>−32</sup>). Insertion errors had the strongest majority in favor of the correct ancestral state, with, 82.5% of alternative alignments recovering the correct gap state when one alignment erroneously inferred an insertion at that position (<italic>z</italic>-test <italic>P </italic><<italic> </italic>1.75e<sup>−48</sup>).</p><fig id=\"evaa164-F6\" orientation=\"portrait\" position=\"float\"><label>Fig. 6</label><caption><p>“Majority-rule” explains the majority of cases in which alignment–integration improves ASR error rates. We simulated protein sequences along a simplified three-taxon phylogeny, varying the branch length and indel rate (see <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S12</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Ancestral sequences were inferred using seven different sequence-alignment methods and alignment–integration. Total- (top), residue-, insertion-, and deletion- (bottom) errors were calculated by comparing inferred ancestral sequences to the correct simulated ancestral sequence. Here, we consider only those cases in which a single sequence-alignment method produces an ASR error that is “repaired” by alignment–integration. <italic>Left panel</italic>: For each branch length (<italic>x</italic> axis) and indel rate (<italic>y</italic> axis), we plot the proportion of alternative sequence-alignment methods that inferred the correct ancestral state when one sequence-alignment method generated an ASR error that was not found in the alignment-integrated ancestral sequence. <italic>Right panel</italic>: Across all simulation conditions, we consider all cases in which a sequence-alignment method makes an error, and that error is repaired by alignment–integration. We report the proportion of such cases in which 1) the majority of alternative sequence-alignment methods infer the correct ancestral state (“correct”; blue), 2) the majority of alternative sequence-alignment methods infer an incorrect ancestral state, but that state is different from the original error (“different”; orange), 3) the majority of alternative sequence-alignment methods infer the same incorrect ancestral state (“same”; green), and 4) other scenarios (“other”; red).</p></caption><graphic xlink:href=\"evaa164f6\"/></fig><p>When alignment–integration was able to “repair” an ASR error made by a single sequence-alignment method, 78.4% of these repairs were explainable by majority-rule (<xref ref-type=\"fig\" rid=\"evaa164-F6\">fig. 6</xref>). However, in 8.1% of cases, the majority of alternative alignments also produced ASR errors, but the errors differed across alignments. Interestingly, in 11.5% of cases, the majority of sequence-alignments actually produced the same ASR error, even though alignment–integration reconstructed the correct ancestral state. Although the specific proportions differed somewhat across different types of ASR errors (see <xref ref-type=\"fig\" rid=\"evaa164-F6\">fig. 6</xref>), the pattern of ASR error repairs due to alignment–integration was consistent: most repairable errors (70.0–97.0%, depending on error type) could be attributed to majority-rule, with smaller proportions of errors being repaired by alignment–integration when most sequence-alignments generate different ASR errors (0.2–11.4%) or when most alignments make the same ASR error (2.6–16.3%).</p><p>For cases in which majority-rule could not explain alignment–integration repair of the ASR error, we observed an upward shift in the PP of the correct ancestral state, compared with similar scenarios that were not repaired by alignment–integration (<xref ref-type=\"fig\" rid=\"evaa164-F7\">fig. 7</xref>). The proportion of cases in which the correct ancestral state had PP <0.1 fell from 0.65 when alignment–integration did not repair the ASR error to 0.51 when alignment–integration repaired the ASR error, even though the majority of sequence-alignments produced an erroneous ancestral state (<italic>z</italic>-test <italic>P </italic><<italic> </italic>1.0e<sup>−20</sup>). Similarly, the proportion of correct ancestral states with PP <0.05 fell from 0.48 when alignment–integration did not repair the error to 0.21 when it did (<italic>z</italic>-test <italic>P </italic><<italic> </italic>1.0e<sup>−20</sup>).</p><fig id=\"evaa164-F7\" orientation=\"portrait\" position=\"float\"><label>Fig. 7</label><caption><p>Alignment–integration can “repair” ASR errors when the correct ancestral state is not strongly disfavored across sequence-alignment methods. We simulated protein sequences along a three-taxon phylogeny, varying the branch length and indel rate (see <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S12</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Ancestral sequences were inferred using seven sequence-alignment methods and alignment–integration. Total- (top), residue-, insertion-, and deletion- (bottom) errors were calculated by comparing inferred ancestral sequences to the correct ancestral sequence. Here, we consider only those cases in which a single sequence-alignment method produces an ASR error, and the majority of alternative sequence-alignment methods do not infer the correct ancestral state. We further divide such cases into 1) errors that are “repaired” by alignment–integration (blue) and 2) errors that are not repaired by alignment–integration (orange). Under each scenario, we estimate the frequency distribution of the PP of the correct ancestral state by kernel density estimation.</p></caption><graphic xlink:href=\"evaa164f7\"/></fig><p>When alignment–integration was able to repair an ASR error by mechanisms other than majority-rule, we observed a large peak in frequency at the PP making the ancestral reconstruction maximally ambiguous (<xref ref-type=\"fig\" rid=\"evaa164-F7\">fig. 7</xref>), which occurs at 0.05 for residue- and deletion-errors (for which the correct ancestral state is one of the 20 amino-acid residues) and at 0.5 for insertion-errors (for which the correct ancestral state is the gap state). This suggests that a relatively large proportion of ASR errors which majority-rule fails to repair—but which alignment–integration still repairs by other mechanisms—occur at highly ambiguous positions with relatively flat PP distributions across many possible ancestral states. Interestingly, the peak at 0.5 PP for insertion-errors was particularly pronounced when alignment–integration repaired the ASR error (<xref ref-type=\"fig\" rid=\"evaa164-F7\">fig. 7</xref>), suggesting “balance-of-probability” repairs may be particularly efficacious in cases of insertion errors, which may contribute to alignment–integration’s very low insertion error rates (see <xref ref-type=\"fig\" rid=\"evaa164-F2\">fig. 2</xref>).</p><p>Overall, these results suggest majority-rule accounts for ∼80% of cases in which alignment–integration is able to “repair” an ancestral reconstruction error generated by a single sequence-alignment method, but more subtle effects of integrating PP distributions also contribute to improved ASR accuracy by alignment–integration. When the correct ancestral state does not have very low PP across all sequence alignments, alignment–integration can sometimes repair ASR errors, even when the majority of sequence alignments reconstruct the wrong ancestral state.</p></sec></sec><sec><title>Conclusions</title><p>For future ASR studies, our results add to the emerging evidence that alignment errors cannot always be ignored when evaluating the accuracy of ASR (<xref rid=\"evaa164-B58\" ref-type=\"bibr\">Vialle et al. 2018</xref>), and in practice, sequence-alignment methods cannot always be relied upon to generate alignments accurate enough to ensure reliable ASR. When multiple structures from across the protein family are available, our results suggest that structure-guided alignment is an efficient approach for improving ASR accuracy, but many protein families lack the rich empirical structural data necessary for structure-guided alignment. In these cases, we recommend that future studies make some effort to evaluate the impact of alignment ambiguity on ASR results. The alignment–integration approach we present here is one mechanism for incorporating alignment ambiguity into ASR studies, which so-far appears to perform well across a variety of realistic and simplified model problems.</p><p>The empirically derived simulation conditions used in this study represent realistic but highly challenging ASR problems, with generally long branches and large phylogenies (see <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S1</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online), which are expected to contribute to elevated alignment and ASR error rates (<xref rid=\"evaa164-B58\" ref-type=\"bibr\">Vialle et al. 2018</xref>). Under less challenging conditions, standard ASR methodology has been found to be highly reliable (<xref rid=\"evaa164-B20\" ref-type=\"bibr\">Hanson-Smith et al. 2010</xref>; <xref rid=\"evaa164-B45\" ref-type=\"bibr\">Randall et al. 2016</xref>; <xref rid=\"evaa164-B58\" ref-type=\"bibr\">Vialle et al. 2018</xref>), and alignment–integration is unlikely to provide any benefits under conditions in which most sequence-alignment methods are extremely accurate.</p><p>Alignment–integration is computationally costly, as many different alignments need to be inferred, and phylogenetic model parameters and ancestral sequences need to be computed using each alignment before being combined. Alignments that are very similar to one another may be at best redundant and at worst could bias the ASR toward a “false consensus.” Similarly, wildly inaccurate alignments could introduce statistical noise or generate biased results when included in the integration process. The identification of a set of alignment algorithms that tend to produce highly accurate but different alignments is expected to be important for reducing the computational demands of alignment-integrated ASR while maintaining its useful statistical properties and low error rates.</p><p>In many of our analyses, specific sequence-alignment algorithms are able to generate ancestral sequences that are nearly as accurate as those generated by alignment integration or structure-guided alignment, suggesting that using a single sequence-alignment method may be adequate, even in some challenging cases. However, the specific alignment algorithm that will perform well for a specific ASR study may be difficult to determine in practice and would likely require comparisons with other alignment algorithms, which would be nearly as computationally costly as alignment-integrated ASR. Because there is no known alignment method that performs optimally in all cases, we recommend that, at minimum, future ASR studies that rely on a single sequence alignment strongly justify the specific approach chosen as the most appropriate for the study.</p><p>Our cursory analysis of probabilistic alignment methods suggests Bayesian co-estimation of the alignment and phylogenetic tree has the potential to provide exceptionally accurate ASRs, at least under some conditions. However, we remain cautious about recommending this approach for a number of reasons. First, our analyses were conditioned on the correct phylogenetic tree, which is hardly ever known with certainty, and the accuracy of ASR under the more realistic case of joint alignment–phylogeny co-estimation has not been investigated. Second, the existing implementation of this approach is extraordinarily computationally intensive, which might necessitate trade-offs in practice that could partially undermine the method’s high accuracy. Finally, a recent study of alignment accuracy found that BAli-Phy’s co-estimation approach was accurate only when sequence data was simulated and not when biological benchmark data sets were used to evaluate alignment accuracy (<xref rid=\"evaa164-B41\" ref-type=\"bibr\">Nute et al. 2019</xref>), suggesting the exceptional accuracy of this approach could be partially explained by strong similarity between the simulation model and that used to analyze the data, which might not translate into high accuracy on biological data. By using Bayesian Markov chain Monte Carlo to sample alignments, phylogenies, and ancestral sequences from the PP distribution, BAli-Phy implements an elegant approach at alignment-integrated ASR with a stronger formal justification than the heuristic method we present here. However, it is unknown whether integrating over the uncertainty associated with a single alignment model will accrue the same benefits as integrating many different alignment algorithms. Future studies will be needed to address these questions before the BAli-Phy approach or similar methods can be recommended in general for ASR.</p></sec><sec><title>Materials and Methods</title><sec sec-type=\"data-availability\"><title>Software and Data Availability</title><p>All analyses presented in this study were performed using objective, transparent, reproducible algorithms documented in readable source code. All input data and analysis/visualization scripts are freely available under the General Public License as open-access documentation associated with this publication at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/bryankolaczkowski/airas\">https://github.com/bryankolaczkowski/airas</ext-link></p></sec><sec><title>Empirical Sequence Simulations</title><p>Empirical structures of diverse CARD, double-stranded RNA-binding motif (DSRM), and RDs were obtained from the protein data bank (<xref rid=\"evaa164-B4\" ref-type=\"bibr\">Berman et al. 2000</xref>) and edited to remove any ligands or structural data from outside the annotated domain of interest. Structures from each domain family were aligned using the iterative_structural_align function in MODELLER v9.19 (<xref rid=\"evaa164-B50\" ref-type=\"bibr\">Sali and Blundell 1993</xref>; <xref rid=\"evaa164-B34\" ref-type=\"bibr\">Madhusudhan et al. 2009</xref>) to generate a multiple sequence alignment based primarily on structural superposition. This alignment was further edited manually to ensure that all aligned residues overlapped in the aligned structures.</p><p>Sequence data sets and consensus phylogenies for each domain family were curated from previous studies of DSRM (<xref rid=\"evaa164-B11\" ref-type=\"bibr\">Dias et al. 2017</xref>), RIG-like receptor (<xref rid=\"evaa164-B39\" ref-type=\"bibr\">Mukherjee et al. 2014</xref>; <xref rid=\"evaa164-B44\" ref-type=\"bibr\">Pugh et al. 2016</xref>), and CARD (<xref rid=\"evaa164-B27\" ref-type=\"bibr\">Korithoski et al. 2015</xref>) families. Sequences were aligned to the structure-based alignment using the −seed option in mafft ginsi v7.402 (<xref rid=\"evaa164-B25\" ref-type=\"bibr\">Katoh et al. 2002</xref>), and sequence regions not globally alignable to the structure-based alignment were trimmed. To simulate sequences with more realistic distributions of insertions and deletions (indels) across the sequence, we used the distribution of indels in the structure-guided alignment to determine the placement of indels in simulated sequences. Positions in the structure-guided alignment having at least three contiguous nongap residues were considered impermissible to indels for the purposes of sequence simulation, whereas indels were allowed at all other positions in the alignment.</p><p>Simulation of 10 replicate data sets for each protein domain family—including correct ancestral sequences at each node on the phylogeny—was performed using indel‐Seq‐Gen v2.1.03 (<xref rid=\"evaa164-B56\" ref-type=\"bibr\">Strope et al. 2006</xref>), assuming the consensus phylogeny, the JTT evolutionary model (<xref rid=\"evaa164-B24\" ref-type=\"bibr\">Jones et al. 1992</xref>) and a four-category discrete gamma model of among-site rate variation with shape parameter <italic>α</italic> = 1.75 (<xref rid=\"evaa164-B61\" ref-type=\"bibr\">Yang 1994</xref>). For each replicate, the root sequence was generated randomly from the structure-guided multiple sequence alignment by sampling amino-acid residues at each position based on the frequency of the amino-acid at that position. Columns in the multiple sequence alignment with >50% gap characters were not sampled when generating root sequences. Insertions and deletions were generated at permissible positions using the distributions from (<xref rid=\"evaa164-B8\" ref-type=\"bibr\">Chang and Benner 2004</xref>), with a maximum indel size of 2 for CARD and DSRM3 domains, 4 for DSRM1 and DSRM2 domains, and 5 for the RD.</p></sec><sec><title>Sequence Alignment</title><p>Simulated sequences were aligned using ClustalW v2.1 (<xref rid=\"evaa164-B53\" ref-type=\"bibr\">Sievers et al. 2011</xref>), mafft ginsi v7.402 (<xref rid=\"evaa164-B25\" ref-type=\"bibr\">Katoh et al. 2002</xref>), msaprobs v0.9.7 (<xref rid=\"evaa164-B31\" ref-type=\"bibr\">Liu et al. 2010</xref>), muscle v3.8.31 (<xref rid=\"evaa164-B14\" ref-type=\"bibr\">Edgar 2004</xref>), probalign v1.4 (<xref rid=\"evaa164-B48\" ref-type=\"bibr\">Roshan and Livesay 2006</xref>), probcons v1.12 (<xref rid=\"evaa164-B13\" ref-type=\"bibr\">Do et al. 2005</xref>), and tcoffee v10.00.r1613 (<xref rid=\"evaa164-B40\" ref-type=\"bibr\">Notredame et al. 2000</xref>), all with default parameters. In addition to sequence-based alignments, structure-guided alignments were generated by aligning each set of simulated sequences to the structure-based alignment (see above) using the −seed option in mafft ginsi (<xref rid=\"evaa164-B25\" ref-type=\"bibr\">Katoh et al. 2002</xref>). Alignment errors were quantified by measuring the distance of each sequence alignment from the correct simulated alignment, using the d_pos option in MetAl v1.1, which estimates the probability that a randomly selected residue aligns to an incorrect position in a randomly selected sequence (<xref rid=\"evaa164-B5\" ref-type=\"bibr\">Blackburne and Whelan 2012</xref>).</p></sec><sec><title>Ancestral Sequence Reconstruction</title><p>Ancestral sequences were reconstructed from each alignment using marginal reconstruction (<xref rid=\"evaa164-B62\" ref-type=\"bibr\">Yang et al. 1995</xref>) implemented in RAxML v8.2.10 (<xref rid=\"evaa164-B55\" ref-type=\"bibr\">Stamatakis 2014</xref>), assuming the correct phylogeny and evolutionary model but estimating branch lengths and model parameters from each input data set. Each sequence alignment was converted to a binary presence–absence alignment, and ancestral gap states were inferred by marginal reconstruction using the BINCAT model in RAxML (<xref rid=\"evaa164-B30\" ref-type=\"bibr\">Lewis 2001</xref>; <xref rid=\"evaa164-B54\" ref-type=\"bibr\">Stamatakis 2006</xref>), assuming the correct tree topology with branch lengths and model parameters estimated from each data set. If the PP of the gap state was >0.5 in the presence–absence reconstruction, that position was reconstructed as a gap character; otherwise, the position was reconstructed as whichever amino-acid residue had the largest PP in the sequence reconstruction.</p><p>Alignment-integrated ASRs were produced by respectively combining sequence-reconstruction PPs and presence–absence PPs across all sequence-alignment methods (excluding data from structure-guided and correct alignments), assuming equal prior weights over sequence alignments. Let <italic>P<sub>i</sub></italic> be the prior weight of alignment method <italic>i</italic>, and <italic>P</italic>(<italic>j</italic>, <italic>k</italic>, <italic>m</italic> | <italic>i</italic>) be the probability of ancestral state <italic>j</italic> at sequence position <italic>k</italic> and node <italic>m</italic> on the phylogeny, assuming alignment method <italic>i</italic>. Then, the heuristic “alignment-integrated posterior probability” of ancestral state <italic>j</italic> at position <italic>k</italic> and node <italic>m</italic> is given by:\n<disp-formula id=\"E1\"><mml:math id=\"M1\"><mml:mi>P</mml:mi><mml:mfenced separators=\"|\"><mml:mrow><mml:mi>j</mml:mi><mml:mo>,</mml:mo><mml:mi>k</mml:mi><mml:mo>,</mml:mo><mml:mi>m</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mrow><mml:munder><mml:mo stretchy=\"false\">∑</mml:mo><mml:mrow><mml:mi>i</mml:mi></mml:mrow></mml:munder><mml:mrow/></mml:mrow><mml:mi>P</mml:mi><mml:mfenced separators=\"∣\"><mml:mrow><mml:mi>j</mml:mi><mml:mo>,</mml:mo><mml:mi>k</mml:mi><mml:mo>,</mml:mo><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi></mml:mrow></mml:mfenced><mml:msub><mml:mrow><mml:mi>P</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi></mml:mrow></mml:msub><mml:mo>.</mml:mo></mml:math></disp-formula></p><p>Here, we set the prior weight of each alignment method <italic>P<sub>i</sub></italic> = 1/<italic>n</italic>, where <italic>n</italic> is the number of alignment methods.</p><p>Alignment–integration requires mapping all sequence alignments to one another, so that homologous columns from different alignments can be integrated. This was done using the −merge option in mafft ginsi.</p><p>After respective integration of sequence- and presence–absence reconstructions, the MAP ancestral sequence was generated as described above for single sequence-alignments.</p><p>ASR errors were calculated by comparing the MAP reconstructed ancestral state to the correct simulated ancestral state. For each inferred ancestral sequence, we calculated the number of errors divided by the length of the alignment generated by mapping the inferred ancestral sequence to the correct ancestral sequence. In addition to total ASR error rates, we also separately calculated the three possible types of ASR errors: 1) residue errors, in which both correct and inferred sequences have amino-acid residues at the same alignment position, but the inferred residue is different from the correct residue; 2) insertion errors, in which the correct ancestral sequence has a gap character, but the inferred sequence has a residue at that position, and 3) deletion errors, in which the correct sequence has a residue, but the inferred sequence has a gap. For each ancestral node on the phylogeny, we calculated the expected ASR error rate as the mean over 10 replicate data sets. Differences in error rates among methods across all replicates and nodes on the phylogeny were assessed using the two-tailed independent two-sample <italic>t</italic>-test, assuming unequal variances. Gaussian kernel density estimates were generated using least squares cross validation to estimate the smoothing parameter (<xref rid=\"evaa164-B49\" ref-type=\"bibr\">Rudemo 1982</xref>).</p></sec><sec><title>Probabilistic Sequence Alignment</title><p>Probabilistic sequence alignments were inferred using PRANK v170427 (<xref rid=\"evaa164-B32\" ref-type=\"bibr\">Loytynoja 2014</xref>), with default parameters, and BAli-Phy v3.5 (<xref rid=\"evaa164-B46\" ref-type=\"bibr\">Redelings and Suchard 2005</xref>). BAli-Phy analyses were conducted assuming the correct phylogeny, the JTT+G evolutionary model and the rs07 indel model (<xref rid=\"evaa164-B47\" ref-type=\"bibr\">Redelings and Suchard 2007</xref>). Following the approach described in <xref rid=\"evaa164-B41\" ref-type=\"bibr\">Nute et al. (2019)</xref>, we concatenated the Markov chain Monte Carlo samples from 32 independent BAli-Phy runs, each executed for a minimum of 1,000 generations, after discarding the first 25% of samples from each run. The MAP alignment calculated over all BAli-Phy runs was used to reconstruct ancestral sequences, using the approach outlined above.</p></sec><sec><title>Structural Modeling and RNA-Affinity Estimation</title><p>Structural homology models of DSRM1 domains were generated using MODELLER v9.19 (<xref rid=\"evaa164-B50\" ref-type=\"bibr\">Sali and Blundell 1993</xref>). We used multi-template modeling (<xref rid=\"evaa164-B29\" ref-type=\"bibr\">Larsson et al. 2008</xref>), assuming the structures and structure-based alignment generated for the DSRM1 domain simulations (see above). For each ancestral sequence, 50 models were generated and ranked using the MODELLER objective function, DOPE and DOPEHR assessment scores (<xref rid=\"evaa164-B51\" ref-type=\"bibr\">Shen and Sali 2006</xref>). Each score was normalized by dividing it by its SD across the 50 models, and we chose the best structural model as that with the optimal mean of normalized scores.</p><p>The structural stability of each protein structural model was inferred using DeltaGREM 2009, which estimates the change in free-energy/sequence-length of a given protein structure, compared with a statistical model of misfolded or unfolded protein ensembles, using a contact-based energy function (<xref rid=\"evaa164-B38\" ref-type=\"bibr\">Minning et al. 2013</xref>; <xref rid=\"evaa164-B3\" ref-type=\"bibr\">Bastolla 2014</xref>). We calculated structural stability errors as the absolute value of the difference in estimated stabilities between the correct ancestral sequence’s structural model and that of the inferred ancestral sequence. The expected stability error for each node on the phylogeny was calculated as the mean over 10 replicates.</p><p>DSRM1–dsRNA-binding affinities were inferred using a previously developed statistical machine learning approach (<xref rid=\"evaa164-B10\" ref-type=\"bibr\">Dias and Kolazckowski 2015</xref>). For each ancestral sequence, a structural homology model was generated as described above, but including the dsRNA ligand from PDBID 5N8L. The p<italic>K</italic>d = −log<sub>10</sub>(<italic>K</italic>d) was estimated using a support-vector machine trained on a large ensemble of protein–RNA and protein–DNA complexes with associated empirically determined binding affinities. Errors in affinity predictions were calculated as the absolute value of the difference in estimated affinities between the correct ancestral sequence’s protein–RNA structural model and that of the inferred ancestral sequence, with expected errors calculated as the mean over 10 replicates.</p><sec><title>Three-Taxon Simulations</title><p>The JTT+G model (four-category discrete gamma approximation with shape parameter <italic>α</italic> = 1.75) was used to simulate 100 replicate data sets along three-taxon phylogenies with branch lengths ranging from 0.1 to 0.8 substitutions/site (see <xref ref-type=\"supplementary-material\" rid=\"sup1\">supplementary fig. S9</xref>, <xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary Material</xref> online). Starting sequences of 200 residues were generated at random from the JTT amino-acid frequency distribution and “evolved” along the phylogeny using indel-seq-gen v2.1.03 (<xref rid=\"evaa164-B56\" ref-type=\"bibr\">Strope et al. 2006</xref>). Insertions and deletions were generated at random, with the indel rate varying from 0.001 to 0.05 times the branch length (<xref rid=\"evaa164-B43\" ref-type=\"bibr\">Pervez et al. 2014</xref>). Indel length was capped at 20 residues, with the length distribution of insertions and deletions taken from <xref rid=\"evaa164-B8\" ref-type=\"bibr\">Chang and Benner (2004)</xref>.</p></sec></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><p>\n<xref ref-type=\"supplementary-material\" rid=\"sup1\">Supplementary data</xref> are available at <italic>Genome Biology and Evolution</italic> online.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><supplementary-material content-type=\"local-data\" id=\"sup1\"><label>evaa164_Supplementary_Data</label><media xlink:href=\"evaa164_supplementary_data.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack id=\"ack1\"><title>Acknowledgments</title><p>This material is based upon work supported by the National Science Foundation under Grant No. (1817942). 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Neurophotonics</journal-id><journal-id journal-id-type=\"iso-abbrev\">Neurophotonics</journal-id><journal-id journal-id-type=\"coden\">NEUROW</journal-id><journal-id journal-id-type=\"publisher-id\">NPh</journal-id><journal-title-group><journal-title>Neurophotonics</journal-title></journal-title-group><issn pub-type=\"ppub\">2329-423X</issn><issn pub-type=\"epub\">2329-4248</issn><publisher><publisher-name>Society of Photo-Optical Instrumentation Engineers</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33029548</article-id><article-id pub-id-type=\"pmc\">PMC7523733</article-id><article-id pub-id-type=\"doi\">10.1117/1.NPh.7.3.035011</article-id><article-id pub-id-type=\"publisher-manuscript\">NPh-20029R</article-id><article-id pub-id-type=\"publisher-id\">20029R</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Research Papers</subject></subj-group><subj-group subj-group-type=\"SPIE-art-type\"><subject>Paper</subject></subj-group></article-categories><title-group><article-title>Short-channel regression in functional near-infrared spectroscopy is more effective when considering heterogeneous scalp hemodynamics</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Wyser</surname><given-names>Dominik</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">a</xref><xref ref-type=\"aff\" rid=\"aff2\">b</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref><xref ref-type=\"other\" rid=\"b1\"/><email>wyserd@ethz.ch</email></contrib><contrib contrib-type=\"author\"><name><surname>Mattille</surname><given-names>Michelle</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">a</xref><xref ref-type=\"other\" rid=\"b2\"/><email>mattillm@student.ethz.ch</email></contrib><contrib contrib-type=\"author\"><name><surname>Wolf</surname><given-names>Martin</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">b</xref><xref ref-type=\"other\" rid=\"b3\"/><email>martin.wolf@alumni.ethz.ch</email></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-0760-7054</contrib-id><name><surname>Lambercy</surname><given-names>Olivier</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">a</xref><xref ref-type=\"other\" rid=\"b4\"/><email>olivier.lambercy@hest.ethz.ch</email></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-1748-4852</contrib-id><name><surname>Scholkmann</surname><given-names>Felix</given-names></name><xref ref-type=\"aff\" rid=\"aff2\">b</xref><xref ref-type=\"aff\" rid=\"aff3\">c</xref><xref ref-type=\"other\" rid=\"b5\"/><email>felix.scholkmann@usz.ch</email></contrib><contrib contrib-type=\"author\"><name><surname>Gassert</surname><given-names>Roger</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">a</xref><xref ref-type=\"other\" rid=\"b6\"/><email>roger.gassert@hest.ethz.ch</email></contrib><aff id=\"aff1\"><label>a</label><institution>ETH Zurich</institution>, Department of Health Sciences and Technology, Rehabilitation Engineering Laboratory, Zurich, <country>Switzerland</country></aff><aff id=\"aff2\"><label>b</label><institution>University Hospital Zurich, University of Zurich</institution>, Department of Neonatology, Biomedical Optics Research Laboratory, Zurich, <country>Switzerland</country></aff><aff id=\"aff3\"><label>c</label><institution>University of Bern</institution>, Institute of Complementary and Integrative Medicine, Bern, <country>Switzerland</country></aff></contrib-group><author-notes><corresp id=\"cor1\"><label>*</label>Address all correspondence to Dominik Wyser, E-mail: <email>relab.publications@hest.ethz.ch</email></corresp></author-notes><pub-date pub-type=\"epub\"><day>29</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>7</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>29</day><month>9</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>7</volume><issue>3</issue><elocation-id>035011</elocation-id><history><date date-type=\"received\"><day>27</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>4</day><month>9</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 The Authors</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>The Authors</copyright-holder><license license-type=\"open-access\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\"><license-p>Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.</license-p></license></permissions><self-uri xlink:title=\"pdf\" xlink:href=\"NPH_7_3_035011.pdf\"/><abstract><title>Abstract.</title><p><bold>Significance:</bold> The reliability of functional near-infrared spectroscopy (fNIRS) measurements is reduced by systemic physiology. Short-channel regression algorithms aim at removing systemic “noise” by subtracting the signal measured at a short source–detector separation (mainly scalp hemodynamics) from the one of a long separation (brain and scalp hemodynamics). In literature, incongruent approaches on the selection of the optimal regressor signal are reported based on different assumptions on scalp hemodynamics properties.</p><p><bold>Aim:</bold> We investigated the spatial and temporal distribution of scalp hemodynamics over the sensorimotor cortex and evaluated its influence on the effectiveness of short-channel regressions.</p><p><bold>Approach:</bold> We performed hand-grasping and resting-state experiments with five subjects, measuring with 16 optodes over sensorimotor areas, including eight 8-mm channels. We performed detailed correlation analyses of scalp hemodynamics and evaluated 180 hand-grasping and 270 simulated (overlaid on resting-state measurements) trials. Five short-channel regressor combinations were implemented with general linear models. Three were chosen according to literature, and two were proposed based on additional physiological assumptions [considering multiple short channels and their Mayer wave (MW) oscillations].</p><p><bold>Results:</bold> We found heterogeneous hemodynamics in the scalp, coming on top of a global close-to-homogeneous behavior (correlation 0.69 to 0.92). The results further demonstrate that short-channel regression always improves brain activity estimates but that better results are obtained when heterogeneity is assumed. In particular, we highlight that short-channel regression is more effective when combining multiple scalp regressors and when MWs are additionally included.</p><p><bold>Conclusion:</bold> We shed light on the selection of optimal regressor signals for improving the removal of systemic physiological artifacts in fNIRS. We conclude that short-channel regression is most effective when assuming heterogeneous hemodynamics, in particular when combining spatial- and frequency-specific information. A better understanding of scalp hemodynamics and more effective short-channel regression will promote more accurate assessments of functional brain activity in clinical and research settings.</p></abstract><kwd-group><title>Keywords:</title><kwd>functional near-infrared spectroscopy</kwd><kwd>short-channel regression</kwd><kwd>scalp hemodynamics</kwd><kwd>brain–computer interface</kwd><kwd>physiological systemic artifacts</kwd><kwd>general linear model</kwd></kwd-group><counts><fig-count count=\"10\"/><table-count count=\"1\"/><ref-count count=\"79\"/><page-count count=\"23\"/></counts><custom-meta-group><custom-meta><meta-name>running-head</meta-name><meta-value>Wyser et al.: Short-channel regression in functional near-infrared spectroscopy…</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"sec1\"><label>1</label><title>Introduction</title><p>Functional near-infrared spectroscopy (fNIRS) enables the noninvasive measurement of human brain activity by monitoring concentration changes of oxygenated hemoglobin (<inline-formula><mml:math id=\"math1\"><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">O</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula>) and deoxygenated hemoglobin (HHb) in the blood.<xref rid=\"r1\" ref-type=\"bibr\"><sup>1</sup></xref><named-content content-type=\"online\"><xref rid=\"r2\" ref-type=\"bibr\"/><xref rid=\"r3\" ref-type=\"bibr\"/></named-content><named-content content-type=\"print\"><sup>–</sup></named-content><xref rid=\"r4\" ref-type=\"bibr\"><sup>4</sup></xref> fNIRS has evolved from a tool for basic research to a widely used technique to investigate brain activity in nonconstrained environments.<xref rid=\"r5\" ref-type=\"bibr\"><sup>5</sup></xref><sup>,</sup><xref rid=\"r6\" ref-type=\"bibr\"><sup>6</sup></xref> Despite its versatile use, there remain several challenges, in particular, the sensitivity of continuous-wave fNIRS to hemodynamic changes of non-neuronal origin.<xref rid=\"r2\" ref-type=\"bibr\"><sup>2</sup></xref><sup>,</sup><xref rid=\"r7\" ref-type=\"bibr\"><sup>7</sup></xref><named-content content-type=\"online\"><xref rid=\"r8\" ref-type=\"bibr\"/><xref rid=\"r9\" ref-type=\"bibr\"/></named-content><named-content content-type=\"print\"><sup>–</sup></named-content><xref rid=\"r10\" ref-type=\"bibr\"><sup>10</sup></xref> These are often referred to as physiological “noise” or “interference” and include systemic activities, such as cardiac pulsation (1 to 2 Hz), respiration (0.2 to 0.4 Hz), low-frequency oscillations (<inline-formula><mml:math id=\"math2\"><mml:mrow><mml:mo form=\"prefix\">∼</mml:mo><mml:mn>0.1</mml:mn><mml:mtext>  </mml:mtext><mml:mi>Hz</mml:mi></mml:mrow></mml:math></inline-formula>) and very low-frequency oscillations (0.01 to 0.05 Hz),<xref rid=\"r11\" ref-type=\"bibr\"><sup>11</sup></xref> and an increase in blood flow through sympathetic nervous activity.<xref rid=\"r12\" ref-type=\"bibr\"><sup>12</sup></xref> These artifacts generate signal changes that may mimic or mask true task-evoked hemodynamic responses (HRs) and may lead to false positives or false negatives.<xref rid=\"r8\" ref-type=\"bibr\"><sup>8</sup></xref><sup>,</sup><xref rid=\"r10\" ref-type=\"bibr\"><sup>10</sup></xref><sup>,</sup><xref rid=\"r13\" ref-type=\"bibr\"><sup>13</sup></xref> This challenge has been acknowledged and its significance recognized in the recent years by the fNIRS community.<xref rid=\"r8\" ref-type=\"bibr\"><sup>8</sup></xref> Although the susceptibility to non-neuronal signals is specific to the measurement principle of fNIRS, all technologies that infer brain activity via hemodynamic changes, i.e., fNIRS, functional magnetic resonance imaging, and positron emission tomography, are affected.</p><p>As a main contributor to low-frequency oscillations, Mayer waves (MW) are rhythmic hemodynamic oscillations in arterial blood pressure,<xref rid=\"r14\" ref-type=\"bibr\"><sup>14</sup></xref> and are presumably the main reason why it is not possible to recover a functional HR in some subjects.<xref rid=\"r15\" ref-type=\"bibr\"><sup>15</sup></xref> Cardiac and respiratory signals can be removed with low-pass filters, when adequately selected for the specific measurement protocols and task/stimulus durations.<xref rid=\"r16\" ref-type=\"bibr\"><sup>16</sup></xref><sup>,</sup><xref rid=\"r17\" ref-type=\"bibr\"><sup>17</sup></xref> The removal of the other systemic signals is more difficult and requires the application of more elaborate signal processing since their frequency contents overlap with the functional HR.<xref rid=\"r18\" ref-type=\"bibr\"><sup>18</sup></xref><named-content content-type=\"online\"><xref rid=\"r19\" ref-type=\"bibr\"/></named-content><named-content content-type=\"print\"><sup>–</sup></named-content><xref rid=\"r20\" ref-type=\"bibr\"><sup>20</sup></xref> Short-channel regression methods have been proposed as a means to separate cerebral from systemic activity.<xref rid=\"r21\" ref-type=\"bibr\"><sup>21</sup></xref><sup>,</sup><xref rid=\"r22\" ref-type=\"bibr\"><sup>22</sup></xref> Through the separate measurement of the scalp hemodynamics by means of a short-separation (SS) channel (typically <inline-formula><mml:math id=\"math3\"><mml:mrow><mml:mo form=\"prefix\"><</mml:mo><mml:mn>15</mml:mn><mml:mtext>  </mml:mtext><mml:mi>mm</mml:mi></mml:mrow></mml:math></inline-formula> and ideally 8.4-mm length<xref rid=\"r23\" ref-type=\"bibr\"><sup>23</sup></xref><sup>,</sup><xref rid=\"r24\" ref-type=\"bibr\"><sup>24</sup></xref>), a signal that predominantly contains systemic and minimal brain activity is obtained. To extract the contribution of the brain from a long-separation (LS) fNIRS measurement (typically 30 mm), the SS is subtracted from the LS signal. Short-channel regression has been shown to significantly improve the quality of the recovered functional brain activity.<xref rid=\"r18\" ref-type=\"bibr\"><sup>18</sup></xref><sup>,</sup><xref rid=\"r21\" ref-type=\"bibr\"><sup>21</sup></xref><sup>,</sup><xref rid=\"r22\" ref-type=\"bibr\"><sup>22</sup></xref><sup>,</sup><xref rid=\"r25\" ref-type=\"bibr\"><sup>25</sup></xref></p><p>However, conflicting approaches on how to apply these scalp regressors can be found in the literature, especially, on the assumed spatial distribution of the scalp hemodynamics, and there is currently no consensus on which approach is better. Systemic artifacts are typically not constrained locally, but they affect the whole brain and extracerebral tissues, and are thus considered “global.” As a consequence, it is often assumed that global noise is distributed homogeneously over the entire scalp layer and thus that a single global signal can be used for the short-channel regression.<xref rid=\"r26\" ref-type=\"bibr\"><sup>26</sup></xref><named-content content-type=\"online\"><xref rid=\"r27\" ref-type=\"bibr\"/><xref rid=\"r28\" ref-type=\"bibr\"/><xref rid=\"r29\" ref-type=\"bibr\"/></named-content><named-content content-type=\"print\"><sup>–</sup></named-content><xref rid=\"r30\" ref-type=\"bibr\"><sup>30</sup></xref> In contrast, it was repeatedly shown that scalp hemodynamics follow spatially heterogeneous patterns,<xref rid=\"r9\" ref-type=\"bibr\"><sup>9</sup></xref><sup>,</sup><xref rid=\"r17\" ref-type=\"bibr\"><sup>17</sup></xref><sup>,</sup><xref rid=\"r31\" ref-type=\"bibr\"><sup>31</sup></xref><named-content content-type=\"online\"><xref rid=\"r32\" ref-type=\"bibr\"/><xref rid=\"r33\" ref-type=\"bibr\"/><xref rid=\"r34\" ref-type=\"bibr\"/><xref rid=\"r35\" ref-type=\"bibr\"/><xref rid=\"r36\" ref-type=\"bibr\"/><xref rid=\"r37\" ref-type=\"bibr\"/></named-content><named-content content-type=\"print\"><sup>–</sup></named-content><xref rid=\"r38\" ref-type=\"bibr\"><sup>38</sup></xref> i.e., different short-channel signals are measured at different locations on the scalp. An additional complexity is observed when considering that the physiological artifacts also show frequency-dependent spatial variations,<xref rid=\"r36\" ref-type=\"bibr\"><sup>36</sup></xref> with a noteworthy effect observed for the MW-frequency band (i.e., average time lags up to 2 s between ipsilateral head regions<xref rid=\"r36\" ref-type=\"bibr\"><sup>36</sup></xref>). It is not entirely understood yet how the scalp hemodynamics behave spatially and how strongly their frequency-specific behavior affects the effectiveness of short-channel regression, raising the question of how to optimally apply short-channel regressors.</p><p>In this work, we investigate the scalp hemodynamics above sensorimotor areas with respect to the two differing assumptions of their spatial distribution (heterogeneous versus homogeneous) and their effectiveness for short-channel regression. Through the combination of local and global regressors, we strive to regress systemic signals of cerebral and extracerebral tissue origin more completely. We further hypothesize that the separate consideration of temporal heterogeneities in form of MW oscillations enables a more optimal regression of systemic signals by reducing the physiological noise. To do so, we propose an algorithm based on a general linear model (GLM) that makes use of nonlinear least squares. This work is important as it paves the way for the implementation of more accurate short-channel regression that could increase the overall quality of fNIRS measurements, allowing more wide-spread application of this technology. This can particularly help to promote clinical applications by making measurements more reliable at the individual level.</p></sec><sec id=\"sec2\"><label>2</label><title>Methods</title><p>To investigate scalp hemodynamics over sensorimotor areas and its influence on the estimation of the functional HR, we conducted an fNIRS experiment to obtain resting-state and motor-execution measurements. The study was conducted in accordance with the Declaration of Helsinki Ethical Principles and Good Clinical Practices, and all subjects provided informed consent.</p><sec id=\"sec2.1\"><label>2.1</label><title>Subjects and Data Acquisition</title><p>Five male, right-handed volunteers (aged <inline-formula><mml:math id=\"math4\"><mml:mrow><mml:mn>27</mml:mn><mml:mo>±</mml:mo><mml:mn>4.7</mml:mn></mml:mrow></mml:math></inline-formula> years, range: 24 to 35 years) participated in the experiment. Subjects were seated in front of a computer screen in a dark room, with their hands lying comfortably on their lap [<xref ref-type=\"fig\" rid=\"f1\">Fig. 1(a)</xref>]. The experiment was split into one 15-min resting-state run and two 15-min runs of motor-execution tasks. The resting-state run was motivated by the need for subject-specific rest data to which an artificial HR could later be added for further validation of the regressors. One motor-execution run consisted of 20 block-designed trials each involving 16 s of self-paced opening and closing of the right hand at <inline-formula><mml:math id=\"math5\"><mml:mrow><mml:mo form=\"prefix\">∼</mml:mo><mml:mn>1</mml:mn><mml:mtext>  </mml:mtext><mml:mi>Hz</mml:mi></mml:mrow></mml:math></inline-formula> (onscreen instructions: green indicator and command “move hand”), followed by an intertrial time that was randomized to 14, 16, or 18 s (“rest”). The subjects were instructed to perform a smooth grasping movement. Two seconds before trial start, the subjects were informed about the upcoming task (“get ready”) to ensure they were paying attention. During data acquisition, subjects were instructed to sit still and to move only their right hand when the instruction “move hand” was displayed. Subject 5 performed only one motor-execution run due to discomfort.</p><fig id=\"f1\" orientation=\"portrait\" position=\"float\"><label>Fig. 1</label><caption><p>Experimental setup. (a) Subjects were seated in front of a screen with the NIRSport system mounted. (b) Optode arrangement on the head. Gray areas indicate the locations of the SS channels (<inline-formula><mml:math id=\"math6\"><mml:mrow><mml:msub><mml:mrow><mml:mi>SS</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula> to <inline-formula><mml:math id=\"math7\"><mml:mrow><mml:msub><mml:mrow><mml:mi>SS</mml:mi></mml:mrow><mml:mrow><mml:mn>8</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula>), dashed green lines the LS channels that were measured, and the two orange lines the selected channels with highest <inline-formula><mml:math id=\"math8\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values. The selected channel for subject 1, 2, 3, and 5 was anterior to the C3 location (thick orange line), and superior for subject 4 (thin orange line). (c) Sensitivity map of the measurement arrangement.</p></caption><graphic xlink:href=\"NPh-007-035011-g001\"/></fig><p>The measurements were performed with the NIRSport system (NIRx Medizintechnik GmbH, Berlin, Germany). The system measured with eight sources and eight detectors at two wavelengths (760 and 850 nm), with a sampling frequency of 7.8125 Hz. For the conduction of this study, an fNIRS cap enabling the measurement with SS and LS channels was deployed. The cap was provided by the manufacturer and enabled the concomitant measurement with 20 LS channels with a source–detector separation of 30 mm and 8 SS channels of 8-mm distance that were located around each light source [<inline-formula><mml:math id=\"math9\"><mml:mrow><mml:msub><mml:mrow><mml:mi>SS</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula> to <inline-formula><mml:math id=\"math10\"><mml:mrow><mml:msub><mml:mrow><mml:mi>SS</mml:mi></mml:mrow><mml:mrow><mml:mn>8</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula> in <xref ref-type=\"fig\" rid=\"f1\">Fig. 1(b)</xref>]. The optodes were placed over the left and right parietal lobes covering the sensorimotor cortex. Two sources (<inline-formula><mml:math id=\"math11\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>3</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math12\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>7</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>) were placed over the left and right primary motor cortices on C3 and C4 according to the international 10–20 system of electrode placement [<xref ref-type=\"fig\" rid=\"f1\">Fig. 1(b)</xref>]. The other optodes were located over the left and right premotor cortices (<inline-formula><mml:math id=\"math13\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>4</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math14\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>8</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>), supplementary motor area (<inline-formula><mml:math id=\"math15\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math16\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>5</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>), and primary somatosensory cortices (<inline-formula><mml:math id=\"math17\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>2</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math18\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>6</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>). Center-to-center distances<xref rid=\"r33\" ref-type=\"bibr\"><sup>33</sup></xref> between LS and SS channels were 15, 34, 54, 62, and 75 mm per hemisphere.</p></sec><sec id=\"sec2.2\"><label>2.2</label><title>Data Processing</title><p>Data were processed using custom-built MATLAB scripts (Version 2018b, MathWorks, Massachussets, US). Channels with low signal quality were excluded from data analysis. They were identified based on the evaluation of the cardiac signal by the method of Perdue et al.<xref rid=\"r39\" ref-type=\"bibr\"><sup>39</sup></xref> More specifically, a Gaussian curve was fitted into the frequency spectrum of <inline-formula><mml:math id=\"math19\"><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">O</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula> between 0.6 and 1.8 Hz, and a peak signal of <inline-formula><mml:math id=\"math20\"><mml:mrow><mml:mo form=\"prefix\">≥</mml:mo><mml:mn>12</mml:mn><mml:mtext>  </mml:mtext><mml:mi>dB</mml:mi></mml:mrow></mml:math></inline-formula> was marked as good signal quality. This approach enables the detection of channels that have a strong optical signal but lack of physiological signal content, which may occur in SS channels. For subjects 2 and 3, the channels <inline-formula><mml:math id=\"math21\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>6</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math22\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>8</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> were removed due to low signal quality, respectively. <inline-formula><mml:math id=\"math23\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>8</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> was excluded for subject 4 due to repeated data dropout of the corresponding sensor.</p><p>Motion artifacts were minimized with a movement artifact reduction algorithm involving detection by moving standard deviation and removal by spline fitting.<xref rid=\"r40\" ref-type=\"bibr\"><sup>40</sup></xref> After applying the modified Beer–Lambert law,<xref rid=\"r41\" ref-type=\"bibr\"><sup>41</sup></xref> with differential pathlength factors of 6.1 at 760 nm and 5.6 at 850 nm,<xref rid=\"r42\" ref-type=\"bibr\"><sup>42</sup></xref><sup>,</sup><xref rid=\"r43\" ref-type=\"bibr\"><sup>43</sup></xref> the concentration changes of <inline-formula><mml:math id=\"math24\"><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">O</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula> and HHb were filtered bidirectionally (MATLAB: filtfilt; finite impulse response, order 1000<xref rid=\"r16\" ref-type=\"bibr\"><sup>16</sup></xref>) at two passbands with regard to the signal of interest:<xref rid=\"r15\" ref-type=\"bibr\"><sup>15</sup></xref><sup>,</sup><xref rid=\"r36\" ref-type=\"bibr\"><sup>36</sup></xref> HR band (0.01 to 0.3 Hz) and MW band (0.07 to 0.14 Hz). It is important to note that a narrow definition of the HR band was chosen in this work and little power (<inline-formula><mml:math id=\"math25\"><mml:mrow><mml:mo form=\"prefix\"><</mml:mo><mml:mi>‱</mml:mi></mml:mrow></mml:math></inline-formula>) of the HR may exceed this range, as well as that the frequency spectrum will change for study protocols with different block durations. In this work, only <inline-formula><mml:math id=\"math26\"><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">O</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula> was investigated, because it is influenced to a higher degree by confounding factors than HHb.<xref rid=\"r15\" ref-type=\"bibr\"><sup>15</sup></xref><sup>,</sup><xref rid=\"r18\" ref-type=\"bibr\"><sup>18</sup></xref><sup>,</sup><xref rid=\"r32\" ref-type=\"bibr\"><sup>32</sup></xref><sup>,</sup><xref rid=\"r44\" ref-type=\"bibr\"><sup>44</sup></xref></p><p>The LS channel with the strongest cerebral activation during the hand-grasping experiment according to GLM <inline-formula><mml:math id=\"math27\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-statistics (see Sec. <xref ref-type=\"sec\" rid=\"sec2.5\">2.5</xref>) was selected for the subsequent analysis. For all subjects, the selected channel originated from the source placed over C3 (<inline-formula><mml:math id=\"math28\"><mml:mrow><mml:msub><mml:mrow><mml:mi>SS</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula>). The corresponding detector optode was located anterior for subjects 1, 2, 3, and 5 [thick orange line in <xref ref-type=\"fig\" rid=\"f1\">Fig. 1(b)</xref>], and superior for subject 4 (thin orange line). For all subjects, the channel flagged for the strongest activation was located over the expected primary motor cortex area of the brain.</p></sec><sec id=\"sec2.3\"><label>2.3</label><title>Short-Channel Regression Using GLM</title><p>The GLM method is a commonly known technique that allows the regression of short channels and that is relatively easy to implement.<xref rid=\"r12\" ref-type=\"bibr\"><sup>12</sup></xref><sup>,</sup><xref rid=\"r38\" ref-type=\"bibr\"><sup>38</sup></xref><sup>,</sup><xref rid=\"r45\" ref-type=\"bibr\"><sup>45</sup></xref><named-content content-type=\"online\"><xref rid=\"r46\" ref-type=\"bibr\"/></named-content><named-content content-type=\"print\"><sup>–</sup></named-content><xref rid=\"r47\" ref-type=\"bibr\"><sup>47</sup></xref> It is known that the regression of different regressor signals may alter the shape of the recovered signal.<xref rid=\"r36\" ref-type=\"bibr\"><sup>36</sup></xref> Therefore, we applied five regressor sets incorporating different assumptions on the spatial distribution of systemic interference. Three approaches were implemented according to literature and relied on the standard GLM method [i.e., (i) one local regressor, (ii) one global regressor, and (iii) one local and one global regressor], whereas two approaches were based on physiological assumptions and made use of an adapted GLM method using non-negative least squares [i.e., (iv) one local, one global and one MW-bandpass filtered global regressor, and (v) all available SS channels and their MW-bandpass filtered signal].</p><sec id=\"sec2.3.1\"><label>2.3.1</label><title>Classical GLM</title><p>In GLM, the measurements (<inline-formula><mml:math id=\"math29\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">Y</mml:mi></mml:mrow></mml:math></inline-formula>), the explanatory variables (<inline-formula><mml:math id=\"math30\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow></mml:math></inline-formula>), and the error term (<inline-formula><mml:math id=\"math31\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">ε</mml:mi></mml:mrow></mml:math></inline-formula>) are linked to each other according to <disp-formula id=\"e001\"><mml:math id=\"math32\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">Y</mml:mi><mml:mo>=</mml:mo><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi mathvariant=\"bold-italic\">β</mml:mi><mml:mo>+</mml:mo><mml:mi mathvariant=\"bold-italic\">ε</mml:mi><mml:mo>,</mml:mo></mml:mrow></mml:math><label>(1)</label></disp-formula>where <inline-formula><mml:math id=\"math33\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">β</mml:mi></mml:mrow></mml:math></inline-formula> is the model parameters/weights. The design matrix <inline-formula><mml:math id=\"math34\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow></mml:math></inline-formula> consists of a constant-offset array (<inline-formula><mml:math id=\"math35\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>C</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula>), a modeled HR (<inline-formula><mml:math id=\"math36\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>HR</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula>), and a nuisance regressor from the superficial scalp layer (<inline-formula><mml:math id=\"math37\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>SCALP</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula>) <disp-formula id=\"e002\"><mml:math id=\"math38\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mo>=</mml:mo><mml:mo stretchy=\"false\">[</mml:mo><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi mathvariant=\"normal\">C</mml:mi></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>HR</mml:mi></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>SCALP</mml:mi></mml:msub><mml:mo stretchy=\"false\">]</mml:mo><mml:mo>.</mml:mo></mml:mrow></mml:math><label>(2)</label></disp-formula></p><p>To solve Eq. (1) and obtain an estimated parameter <inline-formula><mml:math id=\"math39\"><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">β</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">^</mml:mo></mml:mrow></mml:mover></mml:mrow></mml:math></inline-formula>, ordinary least squares using the Moore–Penrose pseudoinverse is applied <disp-formula id=\"e003\"><mml:math id=\"math40\"><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">β</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">^</mml:mo></mml:mrow></mml:mover><mml:mo>=</mml:mo><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msup><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mo>⊤</mml:mo></mml:mrow></mml:msup><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:msup><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mo>⊤</mml:mo></mml:mrow></mml:msup><mml:mi mathvariant=\"bold-italic\">Y</mml:mi><mml:mo>.</mml:mo></mml:mrow></mml:math><label>(3)</label></disp-formula></p><p>An estimated, cerebral activity <inline-formula><mml:math id=\"math41\"><mml:mrow><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">y</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">^</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>C</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula> is obtained after subtraction of <inline-formula><mml:math id=\"math42\"><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>SCALP</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">β</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">^</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>SCALP</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula> from an LS measurement <inline-formula><mml:math id=\"math43\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">y</mml:mi><mml:mi>LS</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula>.</p><p>Three approaches based on literature used the standard GLM method: (i) selecting a local regressor (<inline-formula><mml:math id=\"math44\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>SCALP</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>SS</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula>) from the most proximal SS channel <inline-formula><mml:math id=\"math45\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>3</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> (method: <inline-formula><mml:math id=\"math46\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>),<xref rid=\"r18\" ref-type=\"bibr\"><sup>18</sup></xref> (ii) using a global regressor (<inline-formula><mml:math id=\"math47\"><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>SCALP</mml:mi></mml:mrow></mml:msub><mml:mo>≔</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>PCA</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula>) derived from the first principal component after principal component analysis (PCA) of all SS channels (method: <inline-formula><mml:math id=\"math48\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>PCA</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>),<xref rid=\"r29\" ref-type=\"bibr\"><sup>29</sup></xref> and (iii) the combination of the local and the global PCA regressor (<inline-formula><mml:math id=\"math49\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>SCALP</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mo stretchy=\"false\">[</mml:mo><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>SS</mml:mi></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>PCA</mml:mi></mml:msub><mml:mo stretchy=\"false\">]</mml:mo></mml:mrow></mml:math></inline-formula>) to assume separate superficial and global brain noise (method: <inline-formula><mml:math id=\"math50\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula>).<xref rid=\"r17\" ref-type=\"bibr\"><sup>17</sup></xref></p></sec><sec id=\"sec2.3.2\"><label>2.3.2</label><title>Non-negative GLM</title><p>The two other GLM methods were based on the assumption of phase-shifted oscillations in the MW band.<xref rid=\"r12\" ref-type=\"bibr\"><sup>12</sup></xref> This facilitates the reduction of residuals, as shown in <xref ref-type=\"fig\" rid=\"f2\">Fig. 2</xref>.Two sinusoidal oscillations at 0.1 Hz with time lags of 0.5 s [Pearson’s <inline-formula><mml:math id=\"math51\"><mml:mrow><mml:mi>r</mml:mi><mml:mo>=</mml:mo><mml:mn>0.95</mml:mn></mml:mrow></mml:math></inline-formula>, <xref ref-type=\"fig\" rid=\"f2\">Fig. 2(a)</xref>] and 1 s [<inline-formula><mml:math id=\"math52\"><mml:mrow><mml:mi>r</mml:mi><mml:mo>=</mml:mo><mml:mn>0.80</mml:mn></mml:mrow></mml:math></inline-formula>, <xref ref-type=\"fig\" rid=\"f2\">Fig. 2(b)</xref>] are represented, respectively, to simulate two phase-shifted MW signals. After regression,<xref rid=\"r21\" ref-type=\"bibr\"><sup>21</sup></xref> the introduced time lags lead to new oscillations with residual magnitudes of 30% and 60% of the original signal. It is worthy to note that the residuals would have had zero magnitude without the presence of phase shift. In fNIRS signals, similar delays for the MW oscillations are observed<xref rid=\"r36\" ref-type=\"bibr\"><sup>36</sup></xref> [<xref ref-type=\"fig\" rid=\"f2\">Figs. 2(c)</xref> and <xref ref-type=\"fig\" rid=\"f2\">2(d)</xref>].</p><fig id=\"f2\" orientation=\"portrait\" position=\"float\"><label>Fig. 2</label><caption><p>Simulations and sample signals of MWs. Subtraction of two signals in the MW band using least-squares minimization. The black line shows the reference signal, the blue line shows the regressor signal, and the red line shows the residual after subtraction of the other two signals. (a) and (b) Simulated signals at 0.1 Hz with time lags of 0.5 and 1 s between reference and regressor signal, respectively. The regressed signal (red line) has residual magnitudes of 30% and 60% of the original signals. (c) and (d) Example of a MW-bandpass filtered <inline-formula><mml:math id=\"math53\"><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">O</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula> measurement, showing one LS channel over the primary motor cortex (<inline-formula><mml:math id=\"math54\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>3</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, black line) in comparison to a SS channel located over the primary motor cortex (<inline-formula><mml:math id=\"math55\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>4</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, blue line) or supplementary motor area. The regressed signal (red line) shows the influence of location-dependent phase shift.</p></caption><graphic xlink:href=\"NPh-007-035011-g002\"/></fig><p>Non-negative least squares<xref rid=\"r48\" ref-type=\"bibr\"><sup>48</sup></xref><named-content content-type=\"online\"><xref rid=\"r49\" ref-type=\"bibr\"/></named-content><named-content content-type=\"print\"><sup>–</sup></named-content><xref rid=\"r50\" ref-type=\"bibr\"><sup>50</sup></xref> was applied for the estimation of <inline-formula><mml:math id=\"math56\"><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">β</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">^</mml:mo></mml:mrow></mml:mover></mml:mrow></mml:math></inline-formula> to reduce the risk of overfitting when including additional regressors. Non-negative least squares (MATLAB: lsqnonneg) iteratively minimizes the least-squared error between observation and the expected values by setting negative values to 0.<xref rid=\"r51\" ref-type=\"bibr\"><sup>51</sup></xref> It prevents the arbitrary combination of multiple regressors by forcing only positive linear combinations of physiological regressor signals. This approach is based on the physical foundation that concentration changes measured by fNIRS must be a summation of physiological signals originating from cortical and noncortical regions.<xref rid=\"r21\" ref-type=\"bibr\"><sup>21</sup></xref> Conceptually, this reflects the fact that a regressor (e.g., scalp signal) is not able to “generate” photons but only to absorb them. Applying non-negative least squares is not a necessity when considering multiple (phase shifted) regressors and similar results may be obtained with classical GLM, but the constraints preclude unrealistic outcomes of GLM regression.<xref rid=\"r52\" ref-type=\"bibr\"><sup>52</sup></xref> Equation (1) is then redefined as <disp-formula id=\"e004\"><mml:math id=\"math57\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">Y</mml:mi><mml:mo>=</mml:mo><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi mathvariant=\"bold-italic\">β</mml:mi><mml:mo>+</mml:mo><mml:mi mathvariant=\"bold-italic\">ε</mml:mi><mml:mspace depth=\"0.0ex\" height=\"0.0ex\" width=\"1em\"/><mml:mtext>subject to</mml:mtext><mml:mtext>  </mml:mtext><mml:mi mathvariant=\"bold-italic\">β</mml:mi><mml:mo>≥</mml:mo><mml:mn>0</mml:mn><mml:mo>.</mml:mo></mml:mrow></mml:math><label>(4)</label></disp-formula></p><p>To prevent a biased estimate by restricting <inline-formula><mml:math id=\"math58\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>C</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math59\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>HR</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula> to be multiplied with positive values only, <inline-formula><mml:math id=\"math60\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow></mml:math></inline-formula> was extended with their negative duplication <disp-formula id=\"e005\"><mml:math id=\"math61\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mo>=</mml:mo><mml:mo stretchy=\"false\">[</mml:mo><mml:mo>±</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">C</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mo>±</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>HR</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>SCALP</mml:mi></mml:mrow></mml:msub><mml:mo stretchy=\"false\">]</mml:mo><mml:mo>.</mml:mo></mml:mrow></mml:math><label>(5)</label></disp-formula></p><p>The fourth (iv) GLM approach (method: <inline-formula><mml:math id=\"math62\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mrow><mml:mi>GLM</mml:mi></mml:mrow><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula>, exemplarily shown in <xref ref-type=\"fig\" rid=\"f3\">Fig. 3</xref>) is an extension of <inline-formula><mml:math id=\"math63\"><mml:mrow><mml:msup><mml:mrow><mml:mi>GLM</mml:mi></mml:mrow><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula> with a third scalp signal being the phase-shifted PCA-MW signal <inline-formula><mml:math id=\"math64\"><mml:mrow><mml:msubsup><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>PCA</mml:mi><mml:mrow><mml:mi>MW</mml:mi><mml:mo>,</mml:mo><mml:mi>PS</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula> (<inline-formula><mml:math id=\"math65\"><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>SCALP</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mo stretchy=\"false\">[</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>SS</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>PCA</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msubsup><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>PCA</mml:mi></mml:mrow><mml:mrow><mml:mi>MW</mml:mi><mml:mo>,</mml:mo><mml:mi>PS</mml:mi></mml:mrow></mml:msubsup><mml:mo stretchy=\"false\">]</mml:mo></mml:mrow></mml:math></inline-formula>). This approach considers delays in the MW band between the global component and the LS signal. The <inline-formula><mml:math id=\"math66\"><mml:mrow><mml:msubsup><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>PCA</mml:mi><mml:mrow><mml:mi>MW</mml:mi><mml:mo>,</mml:mo><mml:mi>PS</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula> was obtained as follows: first, <inline-formula><mml:math id=\"math67\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>PCA</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math68\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">y</mml:mi><mml:mi>LS</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula> were bandpass filtered in the MW band to obtain <inline-formula><mml:math id=\"math69\"><mml:mrow><mml:msubsup><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>PCA</mml:mi><mml:mi>MW</mml:mi></mml:msubsup></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math70\"><mml:mrow><mml:msubsup><mml:mi mathvariant=\"bold-italic\">y</mml:mi><mml:mi>LS</mml:mi><mml:mi>MW</mml:mi></mml:msubsup></mml:mrow></mml:math></inline-formula>. Second, <inline-formula><mml:math id=\"math71\"><mml:mrow><mml:msubsup><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>PCA</mml:mi><mml:mi>MW</mml:mi></mml:msubsup></mml:mrow></mml:math></inline-formula> was phase shifted in the range of <inline-formula><mml:math id=\"math72\"><mml:mrow><mml:mo form=\"prefix\">±</mml:mo><mml:mn>3</mml:mn><mml:mtext>  </mml:mtext><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow></mml:math></inline-formula>. Third, ordinary least squares was iteratively applied for the three bandpass-filtered signals, <inline-formula><mml:math id=\"math73\"><mml:mrow><mml:msubsup><mml:mi mathvariant=\"bold-italic\">y</mml:mi><mml:mi>LS</mml:mi><mml:mi>MW</mml:mi></mml:msubsup></mml:mrow></mml:math></inline-formula>, <inline-formula><mml:math id=\"math74\"><mml:mrow><mml:msubsup><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>PCA</mml:mi><mml:mi>MW</mml:mi></mml:msubsup></mml:mrow></mml:math></inline-formula>, and phase-shifted <inline-formula><mml:math id=\"math75\"><mml:mrow><mml:msubsup><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>PCA</mml:mi><mml:mi>MW</mml:mi></mml:msubsup></mml:mrow></mml:math></inline-formula> to find the optimally delayed signal <inline-formula><mml:math id=\"math76\"><mml:mrow><mml:msubsup><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>PCA</mml:mi><mml:mrow><mml:mi>MW</mml:mi><mml:mo>,</mml:mo><mml:mi>PS</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula> that minimizes the residual errors.</p><fig id=\"f3\" orientation=\"portrait\" position=\"float\"><label>Fig. 3</label><caption><p>Example of GLM regression. The time course of an LS signal <inline-formula><mml:math id=\"math77\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">y</mml:mi><mml:mi>LS</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula> (only <inline-formula><mml:math id=\"math78\"><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">O</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula> was investigated in this work) is regressed with the GLM approach (the <inline-formula><mml:math id=\"math79\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mi>GLM</mml:mi><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula> method is shown as an example). The design matrix <inline-formula><mml:math id=\"math80\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow></mml:math></inline-formula> consists of a constant offset <inline-formula><mml:math id=\"math81\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>C</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula>, a model of the HR function (<inline-formula><mml:math id=\"math82\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>HR</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula>, the time and dispersion derivatives are not shown), and the scalp regressors <inline-formula><mml:math id=\"math83\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>SCALP</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula>. The estimated cerebral activity <inline-formula><mml:math id=\"math84\"><mml:mrow><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">y</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">^</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>C</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula> is obtained by performing least-squares minimization and subtracting <inline-formula><mml:math id=\"math85\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>SCALP</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula> from <inline-formula><mml:math id=\"math86\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">y</mml:mi><mml:mi>LS</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula>. The shaded areas represent the hand-grasping trials, whereas the white areas represent rest phases.</p></caption><graphic xlink:href=\"NPh-007-035011-g003\"/></fig><p>The fifth (v) approach (method: <inline-formula><mml:math id=\"math87\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mrow><mml:mi>GLM</mml:mi></mml:mrow><mml:mrow><mml:mi>multiSS</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula>) was an attempt to minimize systemic interference by including all spatial and temporal information available from the scalp measurements. It was not based on an underlying physiological model but was an approach to extract maximal information available from the superficial layer. The scalp regressor matrix was constructed from all SS channels <inline-formula><mml:math id=\"math88\"><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>multiSS</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula> and their phase-shifted MW signal <inline-formula><mml:math id=\"math89\"><mml:mrow><mml:msubsup><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>multiSS</mml:mi></mml:mrow><mml:mrow><mml:mi>MW</mml:mi><mml:mo>,</mml:mo><mml:mi>PS</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:math></inline-formula>, obtained the same way as described in the previous paragraph for (iv) (<inline-formula><mml:math id=\"math90\"><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>SCALP</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mo stretchy=\"false\">[</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>multiSS</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msubsup><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>multiSS</mml:mi></mml:mrow><mml:mrow><mml:mi>MW</mml:mi><mml:mo>,</mml:mo><mml:mi>PS</mml:mi></mml:mrow></mml:msubsup><mml:mo stretchy=\"false\">]</mml:mo></mml:mrow></mml:math></inline-formula>).</p></sec></sec><sec id=\"sec2.4\"><label>2.4</label><title>Simulation of Hemodynamic Response Time Series</title><p>A major challenge when analyzing fNIRS measurements is that no ground truth for neuronal activity is available. Therefore, we performed simulations where a blocked design of two conditions (task and rest) convolved with the HR function was superimposed on the measurements from the resting-state run.<xref rid=\"r22\" ref-type=\"bibr\"><sup>22</sup></xref> The double gamma kernel had an amplitude of <inline-formula><mml:math id=\"math91\"><mml:mrow><mml:mn>0.3</mml:mn><mml:mtext>  </mml:mtext><mml:mi>μ</mml:mi><mml:mi mathvariant=\"normal\">M</mml:mi></mml:mrow></mml:math></inline-formula> for <inline-formula><mml:math id=\"math92\"><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">O</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula>,<xref rid=\"r18\" ref-type=\"bibr\"><sup>18</sup></xref> an onset delay of 0.1 s, and a time-to-peak of 6.7 s.<xref rid=\"r53\" ref-type=\"bibr\"><sup>53</sup></xref> For each of the 5 subjects, 18 trials of 16 s were simulated and superimposed on the LS channel located over C3, and intertrial times were randomized between 14 and 18 s identical to the motor-execution run. The simulations were repeated three times with randomized intertrial durations. In total, 270 trials were simulated.</p><p>We deliberately used real resting-state measurements instead of purely simulated systemic signals, because we expected that simulated noise could only insufficiently reflect heterogeneous behavior in the scalp and brain layers. A possible limitation of this approach is that resting-state measurements may contain spontaneous neural activity with amplitudes comparable to functional brain activity,<xref rid=\"r7\" ref-type=\"bibr\"><sup>7</sup></xref><sup>,</sup><xref rid=\"r54\" ref-type=\"bibr\"><sup>54</sup></xref><sup>,</sup><xref rid=\"r55\" ref-type=\"bibr\"><sup>55</sup></xref> hampering the efficacy of the regression. Therefore, it is important to investigate the performance of the different GLM approaches through simulations and actual motor-execution runs.</p></sec><sec id=\"sec2.5\"><label>2.5</label><title>Data Analysis</title><p>The normalized MW amplitude <inline-formula><mml:math id=\"math93\"><mml:mrow><mml:msubsup><mml:mi>A</mml:mi><mml:mi>MW</mml:mi><mml:mi mathvariant=\"normal\">N</mml:mi></mml:msubsup></mml:mrow></mml:math></inline-formula> was calculated as the ratio of the amplitudes of the MW oscillations divided by the amplitude of the cardiac signal. These signal amplitudes were obtained with the square root of the signal power (MATLAB: bandpower) for the frequency ranges of 0.07 to 0.14 Hz and 0.6 to 2 Hz, respectively. For every subject, the median value of all LS measurements with high signal quality (a Gaussian peak fit with strength above 10 dB<xref rid=\"r39\" ref-type=\"bibr\"><sup>39</sup></xref>) served as representative value.</p><p>Pearson’s correlation coefficient (<inline-formula><mml:math id=\"math94\"><mml:mrow><mml:mi>r</mml:mi></mml:mrow></mml:math></inline-formula>) was calculated for all combinations of the 8 SS channels and the selected LS channel. For the subjects with two runs (subjects 1 to 4), the mean of both runs was calculated after applying Fisher <inline-formula><mml:math id=\"math95\"><mml:mrow><mml:mi>z</mml:mi></mml:mrow></mml:math></inline-formula>-transformation to compensate for skewness effects, followed by backward transformation.<xref rid=\"r18\" ref-type=\"bibr\"><sup>18</sup></xref> Interchannel time lags were obtained from cross-correlation analysis with maximal time lags of <inline-formula><mml:math id=\"math96\"><mml:mrow><mml:mo form=\"prefix\">±</mml:mo><mml:mn>5</mml:mn><mml:mtext>  </mml:mtext><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow></mml:math></inline-formula>.</p><p>For evaluation of the regression results, GLM was applied on all regressed signals. The GLM approach as shown in Eqs. (1) and (3) was used, with the design matrix <inline-formula><mml:math id=\"math97\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow></mml:math></inline-formula> consisting only of <inline-formula><mml:math id=\"math98\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi mathvariant=\"normal\">C</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math99\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mi>HR</mml:mi></mml:msub></mml:mrow></mml:math></inline-formula> (i.e., <inline-formula><mml:math id=\"math100\"><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mi>SCALP</mml:mi></mml:mrow></mml:msub><mml:mo>≔</mml:mo><mml:mn>0</mml:mn></mml:mrow></mml:math></inline-formula>). In this work, we tested the null hypothesis that during a trial the recovered LS signal did not change.<xref rid=\"r56\" ref-type=\"bibr\"><sup>56</sup></xref> From the solution of the GLM model, we calculated for every run (i) the <inline-formula><mml:math id=\"math101\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-value as <inline-formula><mml:math id=\"math102\"><mml:mrow><mml:mi>t</mml:mi><mml:mo>=</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:msup><mml:mrow><mml:mi mathvariant=\"bold-italic\">c</mml:mi></mml:mrow><mml:mrow><mml:mo>⊤</mml:mo></mml:mrow></mml:msup><mml:mover accent=\"true\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">β</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">^</mml:mo></mml:mrow></mml:mover><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">/</mml:mo><mml:mo stretchy=\"false\">[</mml:mo><mml:mi>var</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi mathvariant=\"bold-italic\">ε</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:msup><mml:mrow><mml:mi mathvariant=\"bold-italic\">c</mml:mi></mml:mrow><mml:mrow><mml:mo>⊤</mml:mo></mml:mrow></mml:msup><mml:msup><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msup><mml:mrow><mml:mi mathvariant=\"bold-italic\">X</mml:mi></mml:mrow><mml:mrow><mml:mo>⊤</mml:mo></mml:mrow></mml:msup><mml:mi mathvariant=\"bold-italic\">X</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:mi mathvariant=\"bold-italic\">c</mml:mi><mml:mo stretchy=\"false\">]</mml:mo></mml:mrow></mml:math></inline-formula>,<xref rid=\"r57\" ref-type=\"bibr\"><sup>57</sup></xref> with <inline-formula><mml:math id=\"math103\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">c</mml:mi></mml:mrow></mml:math></inline-formula> being the contrast vector, and <inline-formula><mml:math id=\"math104\"><mml:mrow><mml:mi mathvariant=\"bold-italic\">ε</mml:mi></mml:mrow></mml:math></inline-formula> the estimated residuals, (ii) the Pearson’s correlation coefficient (<inline-formula><mml:math id=\"math105\"><mml:mrow><mml:mi>r</mml:mi></mml:mrow></mml:math></inline-formula>) between recovered and fitted time courses, and (iii) the root-mean-square error (RMSE) of the residuals. Statistical differences between regression methods were determined using two-tailed, paired <inline-formula><mml:math id=\"math106\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-tests. Fisher <inline-formula><mml:math id=\"math107\"><mml:mrow><mml:mi>z</mml:mi></mml:mrow></mml:math></inline-formula> transformation was applied on correlation data prior to the <inline-formula><mml:math id=\"math108\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-test.</p><p>To fathom the potential of the regression approaches for protocols that are based on single-trial evaluation, such as may be the case for brain–computer interfaces (BCI), we additionally calculated a trial-based contrast-to-noise ratio (CNR) metric. The results of the CNR evaluation are presented in the <xref ref-type=\"sec\" rid=\"sec6\">Appendix</xref>.</p></sec></sec><sec id=\"sec3\"><label>3</label><title>Results</title><p>The results section is split into three parts. First, the spatiotemporal distribution of scalp hemodynamics over sensorimotor areas is presented with respect to the incongruent assumptions of heterogeneity and homogeneity (Sec. <xref ref-type=\"sec\" rid=\"sec3.1\">3.1</xref>). Second, the performance of the five GLM regression approaches is reported for simulated HRs, which were overlaid on actual resting-state measurements (Sec. <xref ref-type=\"sec\" rid=\"sec3.2\">3.2</xref>). Third, the performance of the same regression methods is presented for a hand-grasping experiment (Sec. <xref ref-type=\"sec\" rid=\"sec3.3\">3.3</xref>).</p><sec id=\"sec3.1\"><label>3.1</label><title>Behavior of Scalp Hemodynamics over Sensorimotor Areas</title><p>The normalized MW amplitudes (<inline-formula><mml:math id=\"math109\"><mml:mrow><mml:msubsup><mml:mi>A</mml:mi><mml:mi>MW</mml:mi><mml:mi mathvariant=\"normal\">N</mml:mi></mml:msubsup></mml:mrow></mml:math></inline-formula>) for <inline-formula><mml:math id=\"math110\"><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">O</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula> for the resting-state and motor-execution measurements are shown in <xref rid=\"t001\" ref-type=\"table\">Table 1</xref>, showing that the MW amplitudes varied substantially between subjects. Large MW amplitudes were present for subjects 3 and 5, indicating that it may be more difficult to recover an HR within the strong physiological noise, as compared to the other subjects with weaker MW oscillations.</p><table-wrap id=\"t001\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Normalized amplitude of MWs for <inline-formula><mml:math id=\"math111\"><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">O</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula>. Median normalized amplitude in the MW band (<inline-formula><mml:math id=\"math112\"><mml:mrow><mml:msubsup><mml:mi>A</mml:mi><mml:mi>MW</mml:mi><mml:mi mathvariant=\"normal\">N</mml:mi></mml:msubsup></mml:mrow></mml:math></inline-formula>) for all LS channels with good signal quality for <inline-formula><mml:math id=\"math113\"><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">O</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula>. The MW amplitude was normalized using the amplitude of the cardiac signal. In parenthesis, the interquartile ranges are shown.</p></caption><!--OASIS TABLE HERE--><table frame=\"hsides\" rules=\"groups\"><colgroup><col/><col/><col/><col/><col/><col/></colgroup><thead><tr><th valign=\"top\"> </th><th valign=\"top\">Subject 1</th><th valign=\"top\">Subject 2</th><th valign=\"top\">Subject 3</th><th valign=\"top\">Subject 4</th><th valign=\"top\">Subject 5</th></tr></thead><tbody><tr><td>Resting state</td><td>0.58 (0.52 to 0.61)</td><td>0.52 (0.39 to 0.57)</td><td>1.18 (0.89 to 1.44)</td><td>0.81 (0.71 to 1.03)</td><td>1.61 (1.46 to 2.47)</td></tr><tr><td>Motor execution</td><td>0.54 (0.51 to 0.59)</td><td>0.47 (0.39 to 0.64)</td><td>1.28 (0.97 to 1.40)</td><td>0.79 (0.62 to 1.02)</td><td>1.06 (0.92 to 1.48)</td></tr></tbody></table></table-wrap><p>Correlation and phase-shift analysis of the eight SS channels in comparison with a reference LS channel placed over primary motor cortex are shown in <xref ref-type=\"fig\" rid=\"f4\">Fig. 4(a)</xref>. For the HR and the MW bands during resting state, globally high Pearson’s <inline-formula><mml:math id=\"math114\"><mml:mrow><mml:mi>r</mml:mi></mml:mrow></mml:math></inline-formula> (<inline-formula><mml:math id=\"math115\"><mml:mrow><mml:mo form=\"prefix\">></mml:mo><mml:mn>0.8</mml:mn></mml:mrow></mml:math></inline-formula>) was observed with a weak interhemispheric symmetry, where the SS optodes placed over C3 (i.e., <inline-formula><mml:math id=\"math116\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math117\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>3</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>) and C4 (i.e., <inline-formula><mml:math id=\"math118\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>5</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math119\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>7</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>) were most similar to the LS channel. The symmetric pattern was more pronounced for the motor-execution measurements, with the same short channels (i.e., <inline-formula><mml:math id=\"math120\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, <inline-formula><mml:math id=\"math121\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>3</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, <inline-formula><mml:math id=\"math122\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>5</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, and <inline-formula><mml:math id=\"math123\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>7</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>), being the closest and the symmetrically opposite SS channels to the reference LS channel, exhibiting the highest correlations, and the frontal (i.e., <inline-formula><mml:math id=\"math124\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>5</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math125\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>8</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>) and occipital (i.e., <inline-formula><mml:math id=\"math126\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>2</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math127\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>6</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>) channels having lower correlations.</p><fig id=\"f4\" orientation=\"portrait\" position=\"float\"><label>Fig. 4</label><caption><p>Group-level correlation analysis of SS channels. Group-averaged correlation values and time lags for the resting-state and right-hand grasping experiments. All correlations of the SS channels were calculated with respect to the LS channel placed over the left primary motor cortex (M1, orange lines). (a) The correlations and time lags are projected onto a standard head. The colored squares indicate the location of the SS channels. (b) Correlations for different experimental states (resting state versus motor execution), frequency bands (HR versus MW band) and spatial conditions (ipsilateral, contralateral, and global). For every state, the channel with the highest correlation was selected. The gray dots and red lines indicate individual subjects (subjects 1 to 5 in horizontal order) and the group median values, respectively.</p></caption><graphic xlink:href=\"NPh-007-035011-g004\"/></fig><p>Short channels with lower correlations tended to have larger time lags with respect to the reference LS channel. <inline-formula><mml:math id=\"math128\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>3</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> had an average time lag of <inline-formula><mml:math id=\"math129\"><mml:mrow><mml:mn>0.2</mml:mn><mml:mo>±</mml:mo><mml:mn>0.37</mml:mn><mml:mtext>  </mml:mtext><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow></mml:math></inline-formula> (resting state) and <inline-formula><mml:math id=\"math130\"><mml:mrow><mml:mn>0.58</mml:mn><mml:mo>±</mml:mo><mml:mn>0.44</mml:mn><mml:mtext>  </mml:mtext><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow></mml:math></inline-formula> (motor execution) in the HR band, and <inline-formula><mml:math id=\"math131\"><mml:mrow><mml:mn>0.25</mml:mn><mml:mo>±</mml:mo><mml:mn>0.27</mml:mn><mml:mtext>  </mml:mtext><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow></mml:math></inline-formula> (resting state) and <inline-formula><mml:math id=\"math132\"><mml:mrow><mml:mn>0.51</mml:mn><mml:mo>±</mml:mo><mml:mn>0.36</mml:mn><mml:mtext>  </mml:mtext><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow></mml:math></inline-formula> (motor execution) in the MW band. A delay between the LS and the SS channels was observed for all frequency bands and conditions at a group level. <xref ref-type=\"fig\" rid=\"f4\">Figure 4(a)</xref> graphically shows the last columns of the unshifted correlation matrices for the motor-execution runs shown in <xref ref-type=\"fig\" rid=\"f5\">Fig. 5</xref> (upper panels).</p><fig id=\"f5\" orientation=\"portrait\" position=\"float\"><label>Fig. 5</label><caption><p>Group-level correlation matrix during hand-grasping experiment. The upper two panels show the unshifted correlation values for all channel-pairs, and the lower two panels were obtained for optimally phase-shifted signals. The diameter of the colored circles corresponds to Pearson’s correlation <inline-formula><mml:math id=\"math133\"><mml:mrow><mml:mi>r</mml:mi></mml:mrow></mml:math></inline-formula>: a bigger circle implies a higher correlation (first numbers in the upper-right triangles). The color relates to the time shift in seconds (the lower number in parenthesis): red indicates a positive, blue a negative time lag. A blue circle indicates that the horizontal signal labeled on the left side (row) lags behind the vertical signal labeled on the top (column). <inline-formula><mml:math id=\"math134\"><mml:mrow><mml:msub><mml:mrow><mml:mi>SS</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula> to <inline-formula><mml:math id=\"math135\"><mml:mrow><mml:msub><mml:mrow><mml:mi>SS</mml:mi></mml:mrow><mml:mrow><mml:mn>8</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula> denote the short-separation channels, LS is the long-separation channel placed over C3.</p></caption><graphic xlink:href=\"NPh-007-035011-g005\"/></fig><p>Correlation values for three different spatial conditions (contralateral, ipsilateral, and global) were investigated for the same frequency bands (i.e., MW and HR bands) and experiments (i.e., resting state and motor execution) as before, and are shown in <xref ref-type=\"fig\" rid=\"f4\">Fig. 4(b)</xref>. For the contralateral (i.e., left hemisphere for the right-hand grasping task) and ipsilateral (i.e., right hemisphere for the right-hand grasping task) cases, only the SS channels with the highest correlations to the reference LS channel were used. For the global condition, the correlation between the reference LS channel and the PCA model was calculated. The correlation values were high (<inline-formula><mml:math id=\"math136\"><mml:mrow><mml:mi>r</mml:mi><mml:mo>></mml:mo><mml:mn>0.9</mml:mn></mml:mrow></mml:math></inline-formula>) during resting state and both frequency bands for all three conditions, with slightly higher values for the contralateral than the ipsilateral or global conditions. The motor-execution experiment exhibited decreased correlation values for all conditions and frequency bands, with a much stronger effect for the HR band than the MW band. Among all conditions, the correlations remained relatively high, with a minimum correlation of 0.72 for subject 2 (motor execution and ipsilateral side). Typically, the closest SS channels were most similar to the reference LS channel, and the MW band was less influenced by the motor-execution tasks than the HR band. The global PCA model showed consistently lower correlations than the contralateral condition, but still achieved values in the same order of magnitude (i.e., <inline-formula><mml:math id=\"math137\"><mml:mrow><mml:mi>r</mml:mi><mml:mo>≥</mml:mo><mml:mn>0.74</mml:mn></mml:mrow></mml:math></inline-formula>).</p><p><xref ref-type=\"fig\" rid=\"f5\">Figure 5</xref> shows that the channels most similar to the reference LS channel are <inline-formula><mml:math id=\"math138\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, <inline-formula><mml:math id=\"math139\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>3</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, <inline-formula><mml:math id=\"math140\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>5</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, and <inline-formula><mml:math id=\"math141\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>7</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, which are located closest and symmetrically opposite to the LS channel. Correlations between SS channels follow a symmetrical pattern with opposite SS channels showing the highest correlation, i.e., the SS channel pairs <inline-formula><mml:math id=\"math142\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> versus <inline-formula><mml:math id=\"math143\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>5</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, <inline-formula><mml:math id=\"math144\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>2</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> versus <inline-formula><mml:math id=\"math145\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>6</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, <inline-formula><mml:math id=\"math146\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>3</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> versus <inline-formula><mml:math id=\"math147\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>7</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>, and <inline-formula><mml:math id=\"math148\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>4</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> versus <inline-formula><mml:math id=\"math149\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>8</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>. While time lags among the SS channels show positive and negative values, the LS channel consistently lagged behind the SS channels in the range of <inline-formula><mml:math id=\"math150\"><mml:mrow><mml:mo form=\"prefix\">−</mml:mo><mml:mn>0.19</mml:mn></mml:mrow></mml:math></inline-formula> to <inline-formula><mml:math id=\"math151\"><mml:mrow><mml:mo form=\"prefix\">−</mml:mo><mml:mn>0.79</mml:mn><mml:mtext>  </mml:mtext><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow></mml:math></inline-formula> for the MW band. The correlation coefficients for the MW band were larger than for the HR band, especially when the signals are phase shifted (lower tables in <xref ref-type=\"fig\" rid=\"f5\">Fig. 5</xref>).</p></sec><sec id=\"sec3.2\"><label>3.2</label><title>Simulation of Hemodynamic Response Time Series</title><p>In this section, we report on the effectiveness of five GLM regression approaches to remove physiological artifacts from simulated measurements. For each subject, a total of 54 HRs were simulated, which were overlaid on resting-state measurements of a reference LS channel over the left primary motor cortex. All regression approaches were applied on the same time courses, but used different sets of SS signals (all obtained from resting-state SS channels). For each subject, a <inline-formula><mml:math id=\"math152\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-value using a separate GLM model was calculated, as well as correlation (<inline-formula><mml:math id=\"math153\"><mml:mrow><mml:mi>r</mml:mi></mml:mrow></mml:math></inline-formula>) and RMSE between the recovered and the ideal HR. Normalized <inline-formula><mml:math id=\"math154\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values were obtained from the ratio of every regression method and the reference method <inline-formula><mml:math id=\"math155\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>. A larger value indicates either an HR with a stronger amplitude and/or a less noisy signal in reference to <inline-formula><mml:math id=\"math156\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>.</p><p>All GLM regression approaches enabled the more effective estimation of brain estimates in comparison to the original LS signal (i.e., no regression applied), expressed as significant differences for all metrics in <xref ref-type=\"fig\" rid=\"f6\">Fig. 6</xref>. No significant difference between <inline-formula><mml:math id=\"math157\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>PCA</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math158\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> was observed. There was no difference in normalized <inline-formula><mml:math id=\"math159\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values despite some intersubject variability: subjects 2 and 3 benefited especially from the global regressor and subjects 4 and 5 from the local regressor. The improvement in normalized <inline-formula><mml:math id=\"math160\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values for <inline-formula><mml:math id=\"math161\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula> was small, but overall its performance was equal (subjects 1, 4, and 5) or better (subjects 2 and 3) than the benchmark <inline-formula><mml:math id=\"math162\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> method. <inline-formula><mml:math id=\"math163\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula> benefited from local and global regressors by combining the regressor signals of the two simpler methods <inline-formula><mml:math id=\"math164\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> than <inline-formula><mml:math id=\"math165\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>PCA</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>.</p><fig id=\"f6\" orientation=\"portrait\" position=\"float\"><label>Fig. 6</label><caption><p>Effectiveness of brain activity estimates for resting-state simulations. Box plots show the (a) normalized and (b) absolute values. Normalized <inline-formula><mml:math id=\"math166\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values were obtained as the ratio of <inline-formula><mml:math id=\"math167\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values between each GLM regression and the <inline-formula><mml:math id=\"math168\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> approach (being the most common of the used methods). <inline-formula><mml:math id=\"math169\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values were obtained after run-wise analysis of the entire time course by means of a GLM. The gray dots and red lines indicate individual subjects (subjects 1 to 5 in horizontal order) and the group median values, respectively. Results are shown for the simulated time courses, where artificial HRs were superimposed on resting-state measurements. A total of 270 trials were evaluated.</p></caption><graphic xlink:href=\"NPh-007-035011-g006\"/></fig><p>The additional inclusion of MW oscillations in <inline-formula><mml:math id=\"math170\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mrow><mml:mi>GLM</mml:mi></mml:mrow><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math171\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mi>GLM</mml:mi><mml:mi>multiSS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> further improved the estimates of brain activity. In particular, both methods lead to significant improvements in normalized <inline-formula><mml:math id=\"math172\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values with a median change of +14% and +31%, respectively, in comparison to <inline-formula><mml:math id=\"math173\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>. Although there was no significant difference of absolute values in <xref ref-type=\"fig\" rid=\"f6\">Fig. 6(b)</xref>, a trend toward the higher effectiveness of the same approaches is visible.</p></sec><sec id=\"sec3.3\"><label>3.3</label><title>Motor-Execution Task</title><p>The block averages of relative concentration changes for the five subjects in <xref ref-type=\"fig\" rid=\"f7\">Fig. 7</xref> illustrate the influence of the short-channel regression on the brain estimates. The signals were averaged over all hand-grasping trials obtained during the two runs (when applicable). All subjects showed an HR in the reference LS channel (placed over the left primary motor cortex) during the right-hand grasping task, and a shape more similar to the ideal HR was obtained after short-channel regression. The SS channels (<inline-formula><mml:math id=\"math174\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>3</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula> was used by way of illustration) exhibited a relatively flat curve for subjects 2 to 4 and showed a larger activation for subjects 1 and 5 as an indicator of strong systemic activity.</p><fig id=\"f7\" orientation=\"portrait\" position=\"float\"><label>Fig. 7</label><caption><p>Block average time traces for the motor-execution experiment. Block-averaged <inline-formula><mml:math id=\"math175\"><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">O</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula> and HHb responses from all hand-grasping trials are shown for a time segment of 34 s for each of the five subjects. Gray bars indicate the 16-s hand-grasping task, white bars indicate rest. (a) Averages (<inline-formula><mml:math id=\"math176\"><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">O</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula>) for the original LS channel (located over primary motor cortex), the five investigated GLM approaches, and the closest SS channel (<inline-formula><mml:math id=\"math177\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>3</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>) are shown. (b) Close-up plots of relative concentration changes of <inline-formula><mml:math id=\"math178\"><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">O</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mi>Hb</mml:mi></mml:mrow></mml:math></inline-formula> and HHb (red and blue lines with median value and median absolute deviation) for the LS channel, the SS channel (<inline-formula><mml:math id=\"math179\"><mml:mrow><mml:msub><mml:mi>SS</mml:mi><mml:mn>3</mml:mn></mml:msub></mml:mrow></mml:math></inline-formula>) and the regressed signal using <inline-formula><mml:math id=\"math180\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mi>GLM</mml:mi><mml:mi>multiSS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>.</p></caption><graphic xlink:href=\"NPh-007-035011-g007\"/></fig><p>All GLM methods achieved better estimates of brain activity than the unregressed original LS signal in <xref ref-type=\"fig\" rid=\"f8\">Fig. 8</xref>. However, for the motor-execution experiment, the improvements in <inline-formula><mml:math id=\"math181\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-value and correlation were less pronounced than for the simulations (Sec. <xref ref-type=\"sec\" rid=\"sec3.2\">3.2</xref>), and mainly the RMSE metric showed a statistically significant improvement. For subject 4, applying short-channel regression led to no or small changes in <inline-formula><mml:math id=\"math182\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-value while the RMSE strongly decreased. This presumably is a consequence of the strong systemic activation and an accompanying decrease of signal amplitude after regression. No superiority of <inline-formula><mml:math id=\"math183\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>PCA</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> over <inline-formula><mml:math id=\"math184\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> was observed, with a slight, nonsignificant trend toward <inline-formula><mml:math id=\"math185\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>PCA</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>. Although the median normalized <inline-formula><mml:math id=\"math186\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values for <inline-formula><mml:math id=\"math187\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>PCA</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math188\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> were nearly identical on a group level, there were distinct intersubject differences, e.g., with subject 2 profiting strongly from the global regressor (<inline-formula><mml:math id=\"math189\"><mml:mrow><mml:mo form=\"prefix\">+</mml:mo><mml:mn>79</mml:mn><mml:mo>%</mml:mo></mml:mrow></mml:math></inline-formula> for <inline-formula><mml:math id=\"math190\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>PCA</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>). <inline-formula><mml:math id=\"math191\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>GLM</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula> performed similarly to <inline-formula><mml:math id=\"math192\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>PCA</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>. No significant difference in <inline-formula><mml:math id=\"math193\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values was observed but significantly lower RMSE.</p><fig id=\"f8\" orientation=\"portrait\" position=\"float\"><label>Fig. 8</label><caption><p>Effectiveness of brain activity estimates for the hand-grasping experiment. Box plots show (a) normalized and (b) absolute values. Normalized <inline-formula><mml:math id=\"math194\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values were obtained as the ratio of <inline-formula><mml:math id=\"math195\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values between each GLM regression and the <inline-formula><mml:math id=\"math196\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> approach (being the most common of the used methods). <inline-formula><mml:math id=\"math197\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values were obtained after run-wise analysis of the entire time course by means of a GLM. The gray dots and red lines indicate individual subjects (subjects 1 to 5 in horizontal order) and the group median values, respectively. Results are shown for the simulated time courses, where artificial HRs were superimposed on resting-state measurements. A total of 180 trials were evaluated.</p></caption><graphic xlink:href=\"NPh-007-035011-g008\"/></fig><p>The additional inclusion of MW oscillations in <inline-formula><mml:math id=\"math198\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mrow><mml:mi>GLM</mml:mi></mml:mrow><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula> led to better brain activity estimates in comparison to the <inline-formula><mml:math id=\"math199\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> benchmark, expressed as a 15% median improvement in normalized <inline-formula><mml:math id=\"math200\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-value and a significant decrease in RMSE. <inline-formula><mml:math id=\"math201\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mi>GLM</mml:mi><mml:mi>multiSS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> returned the best estimated brain activity, with an overall improvement in normalized <inline-formula><mml:math id=\"math202\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-value of 29% and significant changes in correlation and RMSE compared to <inline-formula><mml:math id=\"math203\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>. Both methods were more effective on single-subject level, showing an improvement in all metrics observable for every subject.</p></sec></sec><sec id=\"sec4\"><label>4</label><title>Discussion</title><p>A better understanding of systemic hemodynamics is crucial for the correct interpretation of fNIRS measurements and, thus, the future usability of fNIRS for research and clinical applications. While short-channel regression has been identified as an important step in making fNIRS technology more applicable, the important question of which systemic signals to regress remains insufficiently addressed. In this work, we investigated in detail the fNIRS signals from 8 SS channels (8-mm source–detector separation) over sensorimotor brain areas by comparing them with a reference LS signal (separation: 30 mm, located over the left primary motor cortex) during resting-state and a hand-grasping experiment. We evaluated five GLM regression approaches that made use of different regressor signals, which were selected from literature and physiological assumptions. We proposed two regression approaches based on non-negative least squares to include additional spatial and temporal information of the scalp (i.e., multiple scalp regressors and their phase-shifted MW signals) compared to state-of-the-art approaches. We show the improved effectiveness to remove physiological noise, thereby shedding light on the optimal selection of scalp regressors to obtain better estimates of functional brain activation.</p><sec id=\"sec4.1\"><label>4.1</label><title>Heterogeneous versus Homogeneous Scalp Hemodynamics</title><p>With respect to the incongruent assumptions on the spatial distribution of scalp hemodynamics (heterogeneous<xref rid=\"r9\" ref-type=\"bibr\"><sup>9</sup></xref><sup>,</sup><xref rid=\"r17\" ref-type=\"bibr\"><sup>17</sup></xref><sup>,</sup><xref rid=\"r28\" ref-type=\"bibr\"><sup>28</sup></xref><sup>,</sup><xref rid=\"r31\" ref-type=\"bibr\"><sup>31</sup></xref><named-content content-type=\"online\"><xref rid=\"r32\" ref-type=\"bibr\"/><xref rid=\"r33\" ref-type=\"bibr\"/><xref rid=\"r34\" ref-type=\"bibr\"/><xref rid=\"r35\" ref-type=\"bibr\"/><xref rid=\"r36\" ref-type=\"bibr\"/><xref rid=\"r37\" ref-type=\"bibr\"/></named-content><named-content content-type=\"print\"><sup>–</sup></named-content><xref rid=\"r38\" ref-type=\"bibr\"><sup>38</sup></xref> versus homogeneous<xref rid=\"r22\" ref-type=\"bibr\"><sup>22</sup></xref><sup>,</sup><xref rid=\"r26\" ref-type=\"bibr\"><sup>26</sup></xref><sup>,</sup><xref rid=\"r27\" ref-type=\"bibr\"><sup>27</sup></xref><sup>,</sup><xref rid=\"r29\" ref-type=\"bibr\"><sup>29</sup></xref><sup>,</sup><xref rid=\"r30\" ref-type=\"bibr\"><sup>30</sup></xref>) reported in literature for fNIRS experiments, we found evidence for a location-specific behavior of the scalp hemodynamics. This supports the hypothesis of heterogeneity. However, we also observed a global spatial distribution with close-to-homogeneous characteristics, mainly when analyzing the resting-state measurements. In particular, we showed that the hemodynamics are more similar for close and symmetrical (interhemispheric) scalp regions and that the propagation of the hemodynamics is delayed between regions. MW oscillations were delayed between different SS channels and in reference to the reference LS channel, and correlations improved after applying phase shifting. From these findings, we conclude that scalp hemodynamics at different locations can be interpreted as a superposition of the same physiologically originating signals (e.g., MWs, task-evoked systemic activation), however, with slight variations of the same signals at every location due to time lags, nonstationarities and nonlinearities.</p><p>Our results are in agreement with recent publications investigating scalp hemodynamics. We confirm the findings of Gagnon et al.<xref rid=\"r33\" ref-type=\"bibr\"><sup>33</sup></xref> that an LS channel over the contralateral motor region is better correlated to close than more distant SS channels, however, the phenomenon was less prominent in our case. Zhang et al.<xref rid=\"r36\" ref-type=\"bibr\"><sup>36</sup></xref> also found, for a resting-state measurement with short channels distributed over the whole head, higher correlations for close and symmetrical short channels among themselves. In particular, they showed a more homogeneous behavior for the MW band than for the whole-frequency band, similar to what we observed for the MW and HR bands. They further observed time lags of 0.35 and 0.83 s for close and symmetrical short channels in the MW band, which is in the same range as our findings (0.19 to 0.79 s). Furthermore, for the local and the symmetrical configurations in the MW band, they measured relatively large correlation values (<inline-formula><mml:math id=\"math204\"><mml:mrow><mml:mi>r</mml:mi><mml:mo>≥</mml:mo><mml:mo>∼</mml:mo><mml:mn>0.78</mml:mn></mml:mrow></mml:math></inline-formula>), being in the same range as our correlations (<inline-formula><mml:math id=\"math205\"><mml:mrow><mml:mi>r</mml:mi><mml:mo>≥</mml:mo><mml:mn>0.75</mml:mn></mml:mrow></mml:math></inline-formula>).</p><p>Based on the evidence from this work and in line with literature, it is hard to deny the observed heterogeneity in scalp hemodynamics over sensorimotor brain regions. However, this clearly comes on top of an underlying homogeneous behavior, as suggested by Sato et al.,<xref rid=\"r29\" ref-type=\"bibr\"><sup>29</sup></xref> and depending on the intended application, this simplified assumption of homogeneity might be a reasonable compromise. Since there is no strict threshold for when homogeneity ends and heterogeneity begins, similar observations may lead to different conclusions on the spatial distribution of scalp hemodynamics. We believe that the incongruent assumptions of homogeneity and heterogeneity can be explained by different experimental conditions, research hypotheses, and evaluation approaches. Multiple factors may further influence the observed spatial patterns and should be investigated in more detail, e.g., the used fNIRS instrumentation, the optode locations (frontal, temporal, or occipital regions), the experimental protocol (resting state versus functional task), or the selection of evaluation metrics.</p><p>In the first part of this work, we conclusively demonstrated that scalp hemodynamics follow a heterogeneous distribution. While this was proposed before for baseline/resting-state measurements for the entire head on a larger scale<xref rid=\"r36\" ref-type=\"bibr\"><sup>36</sup></xref> or the contralateral sensorimotor regions only,<xref rid=\"r33\" ref-type=\"bibr\"><sup>33</sup></xref> we extended the notion of heterogeneity to left and right sensorimotor areas, and different experimental protocols (resting state and motor execution) and frequency bands (HR and MW bands). We showed that heterogeneity is also observable in the MW oscillations and that phase shifting increased correlations but also that the heterogeneous pattern was manifested less prominently in the MW than the HR band. These findings underline the importance of taking into account heterogeneous scalp hemodynamics during short-channel regression in order to remove physiological noise as thoroughly as possible.</p></sec><sec id=\"sec4.2\"><label>4.2</label><title>Physiological Explanations for Scalp Heterogeneities</title><p>The observed interhemispheric symmetry between SS channels is consistent with the literature.<xref rid=\"r36\" ref-type=\"bibr\"><sup>36</sup></xref><sup>,</sup><xref rid=\"r58\" ref-type=\"bibr\"><sup>58</sup></xref> We assume this observation to be a consequence of location-specific characteristics of the underlying vessels. In particular, the scalp is predominantly supplied from the left and right external carotid arteries (except parts of the frontal regions, which are supplied from the internal carotid arteries), which branch into multiple larger and smaller arteries to supply different scalp areas. The scalp regions above the sensorimotor cortices are supplied from a tree-like structure consisting of the superficial temporal arteries and its smaller branches. It is possible that symmetrical interhemispheric arteries and arterioles have more similar path lengths and sympathetic mediation<xref rid=\"r12\" ref-type=\"bibr\"><sup>12</sup></xref> than closer vessels on the same hemisphere, explaining the location-specific perfusion and the reported symmetry. A similar structure is observed for the venous vessels as well. Furthermore, Yücel et al.<xref rid=\"r15\" ref-type=\"bibr\"><sup>15</sup></xref> showed that the amplitudes of SS signals (in the MW band) can differ across the head and explained this by the anatomy of the underlying vessels and the relative positioning of the optodes: If a larger artery is located below an optode, stronger MW oscillations are expected. We preclude other factors that may lead to the reported symmetry: (1) It is unlikely that significant cerebral activation was co-registered with a 7-mm SS channel (the expected sensitivity to the brain is <inline-formula><mml:math id=\"math206\"><mml:mrow><mml:mo form=\"prefix\"><</mml:mo><mml:mn>1</mml:mn><mml:mo>%</mml:mo></mml:mrow></mml:math></inline-formula><xref rid=\"r24\" ref-type=\"bibr\"><sup>24</sup></xref>). (2) The influence of emissary veins, which connect scalp and cerebral venous tissue for pressure equalization, is not entirely understood, but it was hypothesized that their diameter is too small to generate strong hemodynamic changes.<xref rid=\"r12\" ref-type=\"bibr\"><sup>12</sup></xref> (3) Instrumentation noise is not expected to generate symmetrical patterns.</p><p>The exact mechanism and function of MW are still subject to discussion, but it is known that they are spontaneous oscillations in arterial pressure linked to the baroreceptor loop.<xref rid=\"r14\" ref-type=\"bibr\"><sup>14</sup></xref><sup>,</sup><xref rid=\"r59\" ref-type=\"bibr\"><sup>59</sup></xref><sup>,</sup><xref rid=\"r60\" ref-type=\"bibr\"><sup>60</sup></xref> The baroreceptor loop is a homeostatic mechanism responsible for the regulation of the blood pressure. Thus, MW is a global phenomenon that is observed all over the body. It is important to note that “global” does not necessarily imply homogeneity in the entire body or particularly the brain and scalp. Therefore, our observation of heterogeneity in scalp MW oscillations is not in disagreement with the existing knowledge about MW, most of all since the heterogeneity was only weakly manifested and comes on top of a global close-to-homogeneous distribution. This observation may again be a consequence of slight variations in the anatomy and innervation of the measured vessels. Other physiological contributors than MW exist that may generate signal heterogeneities in the MW band, but whose contribution cannot be separated from MW in this work. (1) Vasomotion designates spontaneous changes in the vasomotor tone and generates similarly strong oscillations than MW.<xref rid=\"r59\" ref-type=\"bibr\"><sup>59</sup></xref><sup>,</sup><xref rid=\"r60\" ref-type=\"bibr\"><sup>60</sup></xref> The exact interplay between MW and vasomotion remains unclear,<xref rid=\"r12\" ref-type=\"bibr\"><sup>12</sup></xref><sup>,</sup><xref rid=\"r15\" ref-type=\"bibr\"><sup>15</sup></xref> and it is a challenging task to separate the two phenomena. (2) Evoked systemic activation may lead to spurious activation in the MW-frequency band.<xref rid=\"r19\" ref-type=\"bibr\"><sup>19</sup></xref></p><p>In our measurements, we observed that the reference LS signal was lagging behind the SS signals, similarly to what was reported in Kirilina et al.<xref rid=\"r12\" ref-type=\"bibr\"><sup>12</sup></xref> This is an interesting observation since the LS signal is a superposition of signals from superficial and deep-layer origin. A constant (negative) time lag between the LS and SS channels implies that either the systemic activity is more delayed in the cerebral compartment compared to the scalp, or spontaneous signals (spontaneous neural activity or spontaneous vasomotion) are co-registered.<xref rid=\"r28\" ref-type=\"bibr\"><sup>28</sup></xref><sup>,</sup><xref rid=\"r61\" ref-type=\"bibr\"><sup>61</sup></xref><sup>,</sup><xref rid=\"r62\" ref-type=\"bibr\"><sup>62</sup></xref> Kirilina et al.<xref rid=\"r12\" ref-type=\"bibr\"><sup>12</sup></xref> hypothesized that the time lag between cerebral and extracerebral tissue is a consequence of different vascular path lengths and possible delayed sympathetic mediation between the deep and superficial layers. We support this hypothesis and want to emphasize that the brain and scalp are supplied by different arteries (i.e., from the internal and external carotid, respectively), which already branch relatively low at the level of the neck (i.e., carotid sinus). Since these time lags between tissue layers reduce the effectiveness of the short-channel regression, it will be important to further elucidate the interconnection between cerebral and extracerebral systemic signals and investigate further ways to assess them as well as to include them as regressors.</p></sec><sec id=\"sec4.3\"><label>4.3</label><title>Effect of Heterogeneous Scalp Hemodynamics on Short-Channel Regression</title><p>The effect of five different scalp regressors on the outcome of short-channel regression was investigated. All five GLM approaches significantly improved the signal quality of the recovered time series by reducing the physiological noise. This was expressed as increase in <inline-formula><mml:math id=\"math207\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values and Pearson’s correlation (<inline-formula><mml:math id=\"math208\"><mml:mrow><mml:mi>r</mml:mi></mml:mrow></mml:math></inline-formula>) and a decrease in RMSE, when comparing the recovered with a modeled HR. The ability to improve signal quality of fNIRS measurements based on short-channel regression is undisputed in the community<xref rid=\"r2\" ref-type=\"bibr\"><sup>2</sup></xref> and was confirmed with this work.</p><p>No advantage or disadvantage for the use of a global regressor (<inline-formula><mml:math id=\"math209\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>PCA</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>) over the conventional local regressor (<inline-formula><mml:math id=\"math210\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>) was observed for our experimental conditions. Similarly, Tian et al.<xref rid=\"r28\" ref-type=\"bibr\"><sup>28</sup></xref> were not able to show the superiority of a local over a global regressor. However, these results are different from Erdoğan et al.<xref rid=\"r17\" ref-type=\"bibr\"><sup>17</sup></xref> and Goodwin et al.,<xref rid=\"r37\" ref-type=\"bibr\"><sup>37</sup></xref> where the global regressors performed worse. These presumably contradicting findings again may be explained by different experimental conditions, and we believe that particularly the way that the global regressor is calculated has a strong influence on the results. The small difference between <inline-formula><mml:math id=\"math211\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>PCA</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math212\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> is consistent with our observations that the scalp hemodynamics shows a global distribution close to homogeneity over sensorimotor areas.</p><p>We confirmed that the combined use of a local and global regressor (<inline-formula><mml:math id=\"math213\"><mml:mrow><mml:msup><mml:mrow><mml:mi>GLM</mml:mi></mml:mrow><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula>) led to improved effectiveness of removing systemic artifacts in comparison to <inline-formula><mml:math id=\"math214\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math215\"><mml:mrow><mml:msup><mml:mrow><mml:mi>GLM</mml:mi></mml:mrow><mml:mrow><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula>.<xref rid=\"r17\" ref-type=\"bibr\"><sup>17</sup></xref> The improved performance can be explained by the physiological assumption that the LS fNIRS signal is a combination of extracerebral and cerebral components, and while the local regressor better covers the superficial component, the global regressor may introduce additional information more similar to the cerebral signal. Therefore, it is suggested whenever possible to consider the multilayered behavior of fNIRS signals and include both local and global regressors.</p><p>Building on the finding of delayed MW oscillations between different scalp regions and between cerebral and superficial compartments, two of the five regression approaches attempted to specifically consider temporal heterogeneity of the fNIRS signals. By including the phase-shifted MW signal from the scalp as a separate regressor, we proposed a way to independently consider one of the strongest contributors to the degradation of fNIRS measurements and to take into account their delayed oscillations.<xref rid=\"r19\" ref-type=\"bibr\"><sup>19</sup></xref><sup>,</sup><xref rid=\"r28\" ref-type=\"bibr\"><sup>28</sup></xref> Non-negative least squares enabled the inclusion of additional information by finding a sparse solution to the linear equation model by setting the weights of “unuseful” regressors to zero, thereby, inherently applying a channel selection procedure. Both introduced approaches (<inline-formula><mml:math id=\"math216\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mrow><mml:mi>GLM</mml:mi></mml:mrow><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math217\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mi>GLM</mml:mi><mml:mi>multiSS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>) achieved higher <inline-formula><mml:math id=\"math218\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values compared to the other three, state-of-the-art approaches. It was observed that the effectiveness of the different regressors are subject-dependent, e.g., subject 3 improved strongly when the MW were time shifted and subject 2 benefited from the PCA regressor in the motor-execution experiments. Interestingly, the results even improved for the subjects who showed strong activations already before short-channel regression (i.e., subjects 1 and 2). The benefit of using phase-shifted regressor signals confirms the results of Tian et al.<xref rid=\"r28\" ref-type=\"bibr\"><sup>28</sup></xref> and von Lühmann et al.,<xref rid=\"r63\" ref-type=\"bibr\"><sup>63</sup></xref> who found improved regression outcomes when a separate adaptive filter for MW or a phase-shifting approach based on canonical correlation analysis (CCA) was applied.</p><p>Our findings highlight that short-channel regression can be further improved when making more sophisticated assumptions based on human physiology. This may be important for brain research when a more exact estimation of the location and magnitude of the HR is necessary, but also for single-trial applications (e.g., BCI) requiring “clean” signals to minimize false positives and false negatives.</p></sec><sec id=\"sec4.4\"><label>4.4</label><title>Limitations and Outlook</title><p>This study is limited due to the relatively low number of participants. Nevertheless, a comprehensive view of the generalizability of our results is obtained. The subjects had very different signal contents: subjects 1 and 2 had strong HRs, subjects 3 and 5 had large MW amplitudes, and subject 5 had very strong task-evoked systemic activation. Nevertheless, the two <inline-formula><mml:math id=\"math219\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:mmultiscripts><mml:mi>GLM</mml:mi></mml:mmultiscripts></mml:mrow></mml:math></inline-formula> approaches improved the results for all subjects and conditions, implying that no negative effect from their application should be expected.</p><p>The MW-bandpass filtered signal may not solely include MW oscillations, but also contributions from task-evoked systemic activation and the HR (contained in the LS channel). Therefore, there is a risk that removing specific frequencies may alter the magnitude or slope of the HRs after regression.<xref rid=\"r32\" ref-type=\"bibr\"><sup>32</sup></xref> This risk is small for the investigated protocol with block durations of 30 to 34 s but will increase when shorter block durations would be used due to more overlapping frequency contents between functional activation and MW.<xref rid=\"r64\" ref-type=\"bibr\"><sup>64</sup></xref> Furthermore, the computational power required for the <inline-formula><mml:math id=\"math220\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:mmultiscripts><mml:mi>GLM</mml:mi></mml:mmultiscripts></mml:mrow></mml:math></inline-formula> approaches is higher, mainly due to the bandpass filtering and phase shifting of the regressor channels. We proposed the application of non-negative least squares also for the regression of other biosignals (e.g., blood pressure<xref rid=\"r10\" ref-type=\"bibr\"><sup>10</sup></xref><sup>,</sup><xref rid=\"r65\" ref-type=\"bibr\"><sup>65</sup></xref> and peripheral fNIRS measurements<xref rid=\"r38\" ref-type=\"bibr\"><sup>38</sup></xref>).</p><p>In the simulation part of this work, we did not assume any systemic task-evoked activation, which minimizes the possibility to overcompensate systemic signals. It is known that during a functional task, the autonomic nervous system activity is increased,<xref rid=\"r66\" ref-type=\"bibr\"><sup>66</sup></xref><sup>,</sup><xref rid=\"r67\" ref-type=\"bibr\"><sup>67</sup></xref> which leads to a task-evoked reaction in the SS channels.<xref rid=\"r13\" ref-type=\"bibr\"><sup>13</sup></xref><sup>,</sup><xref rid=\"r34\" ref-type=\"bibr\"><sup>34</sup></xref> The strength of systemic activation is influenced by task-evoked changes in the mean arterial blood pressure,<xref rid=\"r8\" ref-type=\"bibr\"><sup>8</sup></xref><sup>,</sup><xref rid=\"r68\" ref-type=\"bibr\"><sup>68</sup></xref> and thus different protocols may lead to different scalp artifact patterns. Also, different sets of global/local regressor signals and ways of calculating them could be considered and could alter the brain activity estimates. For example, a global regressor could be combined with local scalp signals, which are obtained from the residuals after regression of the global signal from each SS signal.</p><p>In this study, we deliberately used a simple regression method and performed the regression offline on the entire dataset. Future studies should compare our findings with other approaches proposed in the literature, such as autoregressive models,<xref rid=\"r47\" ref-type=\"bibr\"><sup>47</sup></xref> Bayesian filtering,<xref rid=\"r69\" ref-type=\"bibr\"><sup>69</sup></xref> or the recently proposed temporally embedded CCA (tCCA).<xref rid=\"r63\" ref-type=\"bibr\"><sup>63</sup></xref> The tCCA approach also considers latencies in the regressor signals and may offer an alternative to the proposed selection of the optimal delay based on least-squares minimization between LS and SS signals. For real-time applications, we suggest to elaborate on the compatibility of the proposed approach (non-negative weight estimation) with algorithms that inherently consider phase shifts by design,<xref rid=\"r18\" ref-type=\"bibr\"><sup>18</sup></xref><sup>,</sup><xref rid=\"r27\" ref-type=\"bibr\"><sup>27</sup></xref><sup>,</sup><xref rid=\"r28\" ref-type=\"bibr\"><sup>28</sup></xref> e.g., adaptive filters.<xref rid=\"r70\" ref-type=\"bibr\"><sup>70</sup></xref> Alternatively, the phase shifts of the MW signals could be calculated for a baseline measurement<xref rid=\"r71\" ref-type=\"bibr\"><sup>71</sup></xref> when applied with a sliding window approach.<xref rid=\"r30\" ref-type=\"bibr\"><sup>30</sup></xref> Real-time algorithms may further improve regression performance because of the nonstationary and nonlinear nature of SS signals.<xref rid=\"r18\" ref-type=\"bibr\"><sup>18</sup></xref><sup>,</sup><xref rid=\"r72\" ref-type=\"bibr\"><sup>72</sup></xref></p><p>The hardware employed provided SS channels next to the sources but not next to the detectors. Results may have looked different with additional SS channels located at the detectors.<xref rid=\"r34\" ref-type=\"bibr\"><sup>34</sup></xref><sup>,</sup><xref rid=\"r73\" ref-type=\"bibr\"><sup>73</sup></xref> Therefore, it will be important for future applications to apply hardware that captures scalp hemodynamics at both source and detector sites.<xref rid=\"r74\" ref-type=\"bibr\"><sup>74</sup></xref><sup>,</sup><xref rid=\"r75\" ref-type=\"bibr\"><sup>75</sup></xref></p></sec></sec><sec id=\"sec5\"><label>5</label><title>Conclusion</title><p>With this work, we aimed to shed light on the behavior of scalp hemodynamics over the sensorimotor cortex and the influence of scalp regressors on short-channel regression. The better understanding of the systemic physiology enhances the estimates of functional cerebral activation, and is an important step in promoting routine application of fNIRS, especially at the individual level as required in clinical applications. We conclude that</p><list list-type=\"simple\"><list-item><label>1.</label><p>The scalp hemodynamics follows a heterogeneous and frequency-specific behavior. These heterogeneities are superimposed on a global, close-to-homogeneous distribution.</p></list-item><list-item><label>2.</label><p>The introduction of an adapted GLM approach using non-negative least squares enabled the inclusion of multiple regressors with a reduced risk of overfitting compared to state-of-the-art methods. We tested five regressor combinations and found that better performance was achieved when assuming heterogeneous scalp hemodynamics. In particular, we showed the benefit of considering delayed MWs in the regression to compensate for their phase-shifted oscillations between different scalp regions and compartments (cerebral versus superficial).</p></list-item></list><p>With this work, we highlighted the importance of applying short-channel regression and present a way to unite the multilayered behavior of systemic signals in a regression algorithm. We proposed an easy-to-implement short-channel regression method based on GLM and showed the benefit of including multichannel and multifrequency information. We encourage all future fNIRS studies to concomitantly capture SS channels to reduce the influence of systemic physiology.</p></sec><sec id=\"sec6\"><label>6</label><title>Appendix: Single-Trial Evaluation</title><p>A CNR metric was calculated to investigate the effectiveness in improving single-trial estimation of brain activity. A high CNR value gives an indication on the ability of single-trial classification, for example in the frame of a BCI.</p><p>The trial-based CNR metric was established in reference to Saager et al.,<xref rid=\"r73\" ref-type=\"bibr\"><sup>73</sup></xref> where for every trial, a specific curve (e.g., a skewed Gaussian curve<xref rid=\"r37\" ref-type=\"bibr\"><sup>37</sup></xref><sup>,</sup><xref rid=\"r73\" ref-type=\"bibr\"><sup>73</sup></xref>) was fitted. We adapted the method by fitting an artificial HR into the segmented trials instead of a Gaussian curve. Fitting an artificial HR has the advantage that the ideal shape has a stronger influence on the results than more simplistic functions or frequency-based CNR approaches.<xref rid=\"r27\" ref-type=\"bibr\"><sup>27</sup></xref><sup>,</sup><xref rid=\"r44\" ref-type=\"bibr\"><sup>44</sup></xref><sup>,</sup><xref rid=\"r76\" ref-type=\"bibr\"><sup>76</sup></xref> For every trial, we iteratively minimized (MATLAB: fminsearchbnd) the RMSE between the artificial response and a selected segment (i.e., from trial onset until the next intertrial end).<xref rid=\"r77\" ref-type=\"bibr\"><sup>77</sup></xref> The simulated HR was created by convolution of the boxcar and the double gamma function.<xref rid=\"r78\" ref-type=\"bibr\"><sup>78</sup></xref> During the optimization procedure, amplitude of response, delay of response, delay of undershoot, and a constant offset of the HR function were varied, whereas all other variables of the double gamma kernel were kept constant. To preserve realistic shapes, we restricted the boundaries for the delay of response and the delay of undershoot to: 4 to 10 s and 10 to 20 s.<xref rid=\"r77\" ref-type=\"bibr\"><sup>77</sup></xref> For every trial, three trial-wise metrics were obtained:<xref rid=\"r79\" ref-type=\"bibr\"><sup>79</sup></xref> (i) the CNR by calculating the ratio of the fitted artificial response’s amplitude (<inline-formula><mml:math id=\"math221\"><mml:mrow><mml:msup><mml:mi>S</mml:mi><mml:mi>i</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>) divided by the <inline-formula><mml:math id=\"math222\"><mml:mrow><mml:msup><mml:mi>RMSE</mml:mi><mml:mi mathvariant=\"normal\">i</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> (<inline-formula><mml:math id=\"math223\"><mml:mrow><mml:msup><mml:mi>CNR</mml:mi><mml:mi>i</mml:mi></mml:msup><mml:mo>=</mml:mo><mml:msup><mml:mi mathvariant=\"normal\">S</mml:mi><mml:mi>i</mml:mi></mml:msup><mml:mo stretchy=\"false\">/</mml:mo><mml:msup><mml:mi>RMSE</mml:mi><mml:mi>i</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>), (ii) Pearson’s correlation coefficient (<inline-formula><mml:math id=\"math224\"><mml:mrow><mml:msup><mml:mi>r</mml:mi><mml:mi>i</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>) between artificial and recovered responses, and (iii) <inline-formula><mml:math id=\"math225\"><mml:mrow><mml:msup><mml:mi>RMSE</mml:mi><mml:mi>i</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> of the segment’s residuals. A higher single-trial CNR increases the chance to flag a trial as active,<xref rid=\"r37\" ref-type=\"bibr\"><sup>37</sup></xref> and, prospectively, implies a reduction of needed repetitions to detect significant differences between tasks, or a more reliable classification during BCI applications. A CNR value of <inline-formula><mml:math id=\"math226\"><mml:mrow><mml:mo form=\"prefix\">≥</mml:mo><mml:mn>2.5</mml:mn></mml:mrow></mml:math></inline-formula> was empirically found to have a probability higher than 95% that the recovered signal contains an activation, as calculated with a one-sided <inline-formula><mml:math id=\"math227\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-test on randomized tasks during rest condition.</p><p>Results in <xref ref-type=\"fig\" rid=\"f9\">Figs. 9(a)</xref> and <xref ref-type=\"fig\" rid=\"f10\">10(a)</xref> indicate a similar performance of the CNR metric as for the run-wise <inline-formula><mml:math id=\"math228\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:math></inline-formula>-values from the main body of this work. All short-channel regression methods achieved higher CNR values compared to the original case. <inline-formula><mml:math id=\"math229\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math230\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>PCA</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> did not show a significant difference. The methods <inline-formula><mml:math id=\"math231\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mi>GLM</mml:mi><mml:mrow><mml:mi>SS</mml:mi><mml:mo>+</mml:mo><mml:mi>PCA</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:math></inline-formula> and <inline-formula><mml:math id=\"math232\"><mml:mrow><mml:mi>n</mml:mi><mml:mi>n</mml:mi><mml:msup><mml:mi>GLM</mml:mi><mml:mi>multiSS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> using non-negative least squares and including separately MW signals performed best.</p><fig id=\"f9\" orientation=\"portrait\" position=\"float\"><label>Fig. 9</label><caption><p>CNR for resting-state simulations. (a) Box plots show absolute values of CNR, correlation, and RMSE. The gray dots and red lines indicate individual subjects (subjects 1 to 5 in horizontal order) and the group median values, respectively. (b) CNR, RMSE, and correlation (<inline-formula><mml:math id=\"math233\"><mml:mrow><mml:mi>r</mml:mi></mml:mrow></mml:math></inline-formula>) values were obtained for each trial between an iteratively fitted artificial HR and the actual signal, and the results for each GLM regression approach (<inline-formula><mml:math id=\"math234\"><mml:mrow><mml:mi>y</mml:mi></mml:mrow></mml:math></inline-formula> axis) are depicted with respect to the reference <inline-formula><mml:math id=\"math235\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> method (<inline-formula><mml:math id=\"math236\"><mml:mrow><mml:mi>x</mml:mi></mml:mrow></mml:math></inline-formula> axis). Results are obtained for a total of <inline-formula><mml:math id=\"math237\"><mml:mrow><mml:mi>n</mml:mi><mml:mo>=</mml:mo><mml:mn>270</mml:mn></mml:mrow></mml:math></inline-formula> simulated HRs.</p></caption><graphic xlink:href=\"NPh-007-035011-g009\"/></fig><fig id=\"f10\" orientation=\"portrait\" position=\"float\"><label>Fig. 10</label><caption><p>CNR for the hand-grasping experiment. (a) Box plots show absolute values of CNR, correlation, and RMSE. The gray dots and red lines indicate individual subjects (subjects 1 to 5 in horizontal order) and the group median values, respectively. (b) CNR, RMSE, and correlation (<inline-formula><mml:math id=\"math238\"><mml:mrow><mml:mi>r</mml:mi></mml:mrow></mml:math></inline-formula>) values were obtained for each trial between an iteratively fitted artificial HR and the actual signal, and the results for each GLM regression approach (<inline-formula><mml:math id=\"math239\"><mml:mrow><mml:mi>y</mml:mi></mml:mrow></mml:math></inline-formula> axis) are depicted with respect to the reference <inline-formula><mml:math id=\"math240\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> method (<inline-formula><mml:math id=\"math241\"><mml:mrow><mml:mi>x</mml:mi></mml:mrow></mml:math></inline-formula> axis). Results are obtained for a total of <inline-formula><mml:math id=\"math242\"><mml:mrow><mml:mi>n</mml:mi><mml:mo>=</mml:mo><mml:mn>180</mml:mn></mml:mrow></mml:math></inline-formula> hand-grasping trials.</p></caption><graphic xlink:href=\"NPh-007-035011-g010\"/></fig><p>In <xref ref-type=\"fig\" rid=\"f9\">Figs. 9(b)</xref> and <xref ref-type=\"fig\" rid=\"f10\">10(b)</xref>, scatter plots indicate the ability to improve single-trial estimation of brain activity in comparison to the <inline-formula><mml:math id=\"math243\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula> method. The percentages indicate the number of trials that achieved a higher (upper triangle) or smaller (lower triangle) value compared to <inline-formula><mml:math id=\"math244\"><mml:mrow><mml:msup><mml:mi>GLM</mml:mi><mml:mi>SS</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>. Here, the previous findings are confirmed, with all regression methods outperforming the original signal, and the non-negative GLM methods achieving best results.</p></sec></body><back><ack><title>Acknowledgments</title><p>We thank Tanya Bafna for her pioneering work on this publication. 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J.</given-names></name><etal>et al.</etal></person-group>, “<article-title>Event-related fMRI: characterizing differential responses</article-title>,” <source>NeuroImage</source>\n<volume>7</volume>(<issue>1</issue>), <fpage>30</fpage>–<lpage>40</lpage> (<year>1998</year>).<pub-id pub-id-type=\"coden\">NEIMEF</pub-id><issn>1053-8119</issn><pub-id pub-id-type=\"doi\">10.1006/nimg.1997.0306</pub-id><pub-id pub-id-type=\"pmid\">9500830</pub-id></mixed-citation></ref><ref id=\"r79\"><label>79.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gagnon</surname><given-names>L.</given-names></name><etal>et al.</etal></person-group>, “<article-title>Quantification of the cortical contribution to the NIRS signal over the motor cortex using concurrent NIRS-fMRI measurements</article-title>,” <source>NeuroImage</source>\n<volume>59</volume>(<issue>4</issue>), <fpage>3933</fpage>–<lpage>3940</lpage> (<year>2012</year>).<pub-id pub-id-type=\"coden\">NEIMEF</pub-id><issn>1053-8119</issn><pub-id pub-id-type=\"doi\">10.1016/j.neuroimage.2011.10.054</pub-id><pub-id pub-id-type=\"pmid\">22036999</pub-id></mixed-citation></ref></ref-list><bio id=\"b1\"><p><bold>Dominik Wyser</bold> received his BSc and MSc degrees in mechanical engineering from ETH Zurich, Switzerland, in 2012 and 2014, respectively. In 2014, he joined the Rehabilitation Engineering Lab at ETH Zurich, where he is currently pursuing his PhD on the topic of BCI, in collaboration with the Biomedical Optics Research Lab at the University Hospital Zurich. He is working on the development of an fNIRS instrument for the real-time detection of motion intention.</p></bio><bio id=\"b2\"><p><bold>Michelle Mattille</bold> received her BSc degree in mechanical engineering from ETH Zurich, Switzerland, in 2019. She joined the Rehabilitation Engineering Lab at ETH Zurich from 2017 to 2019 for several projects, where she worked on signal processing and classification of fNIRS data. Currently, she is pursuing her MSc degree in mechanical engineering at ETH Zurich.</p></bio><bio id=\"b3\"><p><bold>Martin Wolf</bold> is a professor of biomedical optics at the University of Zurich. He received his PhD from ETH Zurich. He heads the Biomedical Optics Research Laboratory, which specializes in developing techniques to measure and quantitatively image oxygenation of brain, muscle, tumors, and other tissues. His aim is to translate these techniques to clinical application for the benefit of adult patients and preterm infants.</p></bio><bio id=\"b4\"><p><bold>Olivier Lambercy</bold> received his MSc degree in microengineering from the Ecole Polytechnique Fédérale de Lausanne, Switzerland, in 2005 and his PhD in mechanical engineering from the National University of Singapore in 2009. In 2009, he joined the Rehabilitation Engineering Laboratory at ETH Zurich as a senior research associate. He is associate editor of the <italic>Journal of NeuroEngineering and Rehabilitation</italic>. His research interests are in medical and rehabilitation robotics, human motor control, and human–machine interaction.</p></bio><bio id=\"b5\"><p><bold>Felix Scholkmann</bold> received his PhD from the University of Zurich, Switzerland, in 2014. He is a research associate at the University Hospital Zurich (Biomedical Optics Research Laboratory, Department of Neonatology) and University of Bern. His research focuses on biomedical signal processing, biophotonics (development and application of NIRS), neuroscience, and integrative physiology and biophysics.</p></bio><bio id=\"b6\"><p><bold>Roger Gassert</bold> is a professor of rehabilitation engineering in the Department of Health Sciences and Technology at ETH Zurich. He received his MSc degree in microengineering and his PhD in neuroscience robotics from the Ecole Polytechnique Fédérale de Lausanne in 2002 and 2006, respectively. His research interests are in physical human–robot interaction, rehabilitation and neuroscience robotics, noninvasive brain–robot interfaces and assistive technology.</p></bio></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005541</article-id><article-id pub-id-type=\"pmc\">PMC7523737</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10127</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Infectious Disease</subject></subj-group><subj-group><subject>Pulmonology</subject></subj-group><subj-group><subject>Public Health</subject></subj-group></article-categories><title-group><article-title>Death due to Cardiac Arrest in a Young Female With Highly Suspected COVID-19: A Case Report</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Mohamed</surname><given-names>Sherif</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Abo El-Hassan</surname><given-names>Osama</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rizk</surname><given-names>Magda</given-names></name><xref ref-type=\"aff\" rid=\"aff-3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ismail</surname><given-names>Jumana H</given-names></name><xref ref-type=\"aff\" rid=\"aff-4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Baioumy</surname><given-names>Aml</given-names></name><xref ref-type=\"aff\" rid=\"aff-4\">4</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nChest Diseases and Tuberculosis, Faculty of Medicine, Assiut University, Assiut, EGY </aff><aff id=\"aff-2\">\n<label>2</label>\nPulmonary Medicine, Faculty of Medicine, Cairo University, Cairo, EGY </aff><aff id=\"aff-3\">\n<label>3</label>\nAnaesthesiology, Faculty of Medicine, Cairo University, Cairo, EGY </aff><aff id=\"aff-4\">\n<label>4</label>\nChest Diseases, Faculty of Medicine, Cairo University, Cairo, EGY </aff><author-notes><corresp id=\"cor1\">\nSherif Mohamed <email>saawm220@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>30</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10127</elocation-id><history><date date-type=\"received\"><day>17</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Mohamed et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Mohamed et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/39748-death-due-to-cardiac-arrest-in-a-young-female-with-highly-suspected-covid-19-a-case-report\">This article is available from https://www.cureus.com/articles/39748-death-due-to-cardiac-arrest-in-a-young-female-with-highly-suspected-covid-19-a-case-report</self-uri><abstract><p>Despite the common clinical presentations of the pandemic coronavirus disease of 2019 (COVID-19) being well-known, there remain issues on its atypical or rare presentations. Moreover, despite the known risk factors for severe COVID-19 are cardiovascular disease, diabetes mellitus, hypertension, chronic lung disease, and advanced age, still younger patients suffer from this disease. Herein, we present a case report of a 28-year-old female patient who was presented to the ED with cardiac arrest, then died within 12 hours. First swab testing by reverse transcription-polymerase chain reaction (RT-PCR) came negative. However, she has typical CT features of COVID-19 pneumonia, along with an echocardiographic picture of acute cor pulmonale. Though it is rare, cardiac arrest can happen in young apparently healthy patients with COVID-19. As COVID-19 patients are commonly having clotting disorders, endothelial and organ dysfunction, coagulopathy, and liable for pulmonary thromboembolism (PTE), it is important to select those COVID-19 patients who are at higher risk of PTE, and practice CT pulmonary angiography (CTPA) for the diagnosis of PTE, especially in case of significant increase of D-dimer values.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>covid-19</kwd><kwd>cardiac arrest</kwd><kwd>mortality</kwd><kwd>young</kwd><kwd>female</kwd><kwd>false-negative</kwd><kwd>pulmonary embolism</kwd><kwd>coagulopathy</kwd><kwd>case report</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Despite the fact that common classical clinical presentations of the pandemic coronavirus disease of 2019 (COVID-19) are well-known, there remain issues on its atypical or rare presentations. In a report to the United States Centers for Disease Control and Prevention (CDC) of over 370,000 confirmed COVID-19 cases [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>], the reported symptoms in descending order were as follows; cough (50%), fever (subjective or >100.4°F/38°C; 43%), myalgia (36%), headache (34%), and dyspnea (29%). Less common symptoms included sore throat (20%), diarrhea (19%), nausea and/or vomiting (12%), and loss of smell or taste, abdominal pain, and rhinorrhea (<10%) [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>].</p><p>Severe COVID-19 can occur in otherwise healthy individuals of any age, but it predominantly occurs in adults with advanced age or underlying medical comorbidities [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. Recently, the CDC has created a list of certain comorbidities that have been associated with severe disease (defined as infection resulting in hospitalization, admission to the ICU, intubation or mechanical ventilation, or death) [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. Established risk factors include: cancer, chronic kidney disease, chronic obstructive pulmonary disease (COPD), immunocompromised state from a solid organ transplant, obesity, serious cardiovascular disease, heart failure, coronary artery disease, cardiomyopathies, sickle cell disease, and type 2 diabetes mellitus. </p><p>Herein, we present a case report of a 28-year-old female patient who presented to the ED with cardiac arrest and then died within 12 hours. Swab testing by reverse transcription-polymerase chain reaction (RT-PCR) came negative on the next day. However, she had typical CT features of COVID-19 pneumonia. This atypical and severe presentation of COVID-19 in a young patient could have significant impacts on diagnostic and therapeutic strategies of such patients. </p></sec><sec sec-type=\"cases\"><title>Case presentation</title><p>A 28-year-old Egyptian female patient was brought by her husband to the ED in a state of cardiac arrest. Cardio-pulmonary resuscitation (CPR) was immediately started and she revived after seven minutes. She was intubated and mechanically ventilated. History was taken from the husband that the patient is a nonsmoker, with two days history of cough and mild shortness of breath, with no fever. Two hours before arrest she felt marked shortness of breath, chest tightness, then developed fainting attack with marked pallor and cold extremities. No history of close contact to COVID-19 suspected or confirmed case in the last two weeks. No history of convulsions, vomiting, headache, or gastrointestinal symptoms. No past history of any chronic medical or pulmonary illnesses. After resuscitation, baseline clinical examination of the chest, heart abdomen was unremarkable, except for tachycardia. No clinical signs were suggestive of deep venous thrombosis (DVT) of the lower limbs. As the patient's symptoms were mainly respiratory -- at the time of COVID-19 pandemic -- the resuscitating team decided to do urgent plain CT of the brain and chest before transferring the patient to the ICU. CT brain was unremarkable, whereas surprisingly CT chest revealed extensive bilateral wide-spread, more peripherally situated parenchymal ground glass opacities (GGOs) and consolidations in almost all lobes of both lungs (Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>).</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>CT chest images of cardiac arrest in a young female patient.</title><p>A&B: Axial views showing bilateral mosaic changes and peripheral ground glass opacities (GGOs) in both upper lobes (yellow arrows) with consolidation of the left upper lobe (red arrow). C&D: Coronal views confirming the same findings.</p></caption><graphic xlink:href=\"cureus-0012-00000010127-i01\"/></fig><p>At the ICU, the patient was on mechanical ventilation, FIO2 of 100%, with vital signs of blood pressure 70/40 mmHg, temperature 35.5°C, respiratory rate of 35 cycles/minute, and O2 saturation of 88%. The treating and ICU teams decided ventilator strategy for acute respiratory distress syndrome (ARDS), prone positioning, IV vasopressors, septic workup, coagulation profile, and to do tracheal secretions swabbing for RT-PCR. Her laboratory workup revealed normal total white blood count (WBC) with relative neutropenia and lymphocytosis, hemoglobin (Hb) level of 11.2 g/dL, and normal platelet count. She had normal renal functions and electrolytes with double fold rising of liver enzymes. D-Dimer was 6.1 ug/mL. Serum testing for pregnancy (B-HCG) came negative. Electrocardiography (ECG) revealed right ventricular strain and right bundle branch block (RBBB). Bedside 2D echocardiography was carried out and revealed a picture of acute cor pulmonale (Figure <xref ref-type=\"fig\" rid=\"FIG2\">2</xref>).</p><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>Echocardiography shows acute cor pulmonale.</title><p>Hugely dilated right side of the heart. Dilated right ventricular dimension, with reverted Brenheimic effect and paradoxical septal motion. Normal left ventricular chamber internal dimensions with good systolic function. No regional resting wall motion abnormalities.</p><p>RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium</p></caption><graphic xlink:href=\"cureus-0012-00000010127-i02\"/></fig><p>Ejection fraction (EF) was 64%. Despite all measures, the patient remained hypoxic and hypotensive for 12 hours after intubation. Then, she had a second cardiac arrest, and CPR was initiated; however, she did not revive and was declared dead after 12 hours from her intubation. Next day, the result of RT-PCR came negative for SARS-CoV-2. A consent was obtained from the patient husband for publication of this case report.</p></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>Herein, we present a case report of a 28-year-old female who presented with cardiac arrest, with typical CT findings of COVID-19 pneumonia, then died within 12 hours. This atypical and severe presentation for COVID-19 is strange and uncommon for many reasons: (1) young age; (2) female; (3) apparently healthy with apparently good underlying cardiopulmonary reserve and no underlying risk factors of COVID-19 or its severe form; (4) rapid deterioration in few hours, up to death; and (5) first sample was negative for SARS-CoV-2 by RT-PCR. Unfortunately, there was no time for a second confirmatory sample.</p><p>Our current knowledge for clinical presentation of COVID-19 is that 81% of infected individuals have mild symptoms, 14% have severe symptoms requiring hospitalization, while 5% become critically ill requiring mechanical ventilation. These differences in response are likely the result of degree of viral load, host immune response, age of the patient, and presence of co-morbidities [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. However, because the obligate receptor for the virus spike protein, human angiotensin converting enzyme (ACE-2) is expressed in epithelial cells throughout the body, including the lungs, heart, kidney, and even the endothelium [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]; it is not surprising that 8%-28% of patients with COVID-19 infections have evidence of cardiac injury with elevated troponin [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. Some patients with COVID-19 infections die from cardiac arrest, likely as a result of a combination of primary cardiac involvement, or manifestation of systemic involvement such as severe hypoxia, multi-organ dysfunction syndrome, or systemic inflammatory response syndrome [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. Acute pulmonary embolism (PE), reported in COVID-19 patients, has been shown to be a cause of clinical deterioration in viral types of pneumonia, as well [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>].</p><p>Patients with COVID-19 often show clotting disorders, with organ dysfunction and coagulopathy, resulting in higher mortality [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. Critical data came from the analysis of coagulation tests in samples collected on admission and during the hospital stay of COVID-19 patients. Nonsurvivors had significantly higher D-dimer and fibrinogen-degradation product (FDP) levels, and longer prothrombin time (PT) vs. survivors on admission [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]. Moreover, the clinical diagnosis of disseminated intravascular coagulation (DIC) was observed in nonsurvivors during late stages of hospitalization [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]. Moreover, endothelial dysfunction is a key determinant in hypertension, thrombosis, and DIC, which is common in patients with severe COVID-19 [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>].</p><p>Back to the clinical presentation of our case report, we thought that the patient had the diagnosis of severe COVID-19 disease, despite that swab came negative. International guidelines have reported that a considerable number of patients could have false negative testing for SARS-CoV-2, depending on the type of collected specimen [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]. Supporting this observation, recent studies had shown the diagnostic significance of CT chest in diagnosing COVID-19 patients who had initial negative testing result(s) by RT-PCR [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. Despite that the cause of death in this patient is not clear, we think that it is attributed to massive PE. This possibility is supported by the bedside echocardiographic findings and high D-Dimer levels. Unfortunately, time was not enough to carry out CT pulmonary angiography (CTPA) to confirm this diagnosis. Recently, autopsy studies performed for COVID-19 patients were shown. In patients who died from COVID-19-associated respiratory failure, the histologic pattern in the peripheral lung was diffuse alveolar damage with perivascular T-cell infiltration. The lungs also showed distinctive vascular features, consisting of severe endothelial injury associated with the presence of intracellular virus and disrupted cell membranes. Histologic analysis of pulmonary vessels showed widespread thrombosis with microangiopathy [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>].</p><p>Unfortunately, the patient’s husband did not agree for post-mortem examination of our patient. Lessons that could be learned from this case is that, it is important to select COVID-19 patients at higher risk of PE, and practice CTPA for the diagnosis of pulmonary thromboembolism (PTE) especially in case of significant increase of D-dimer values. Anticoagulation could be a necessary therapy to control and reduce pro-thrombotic events, as well as to prevent PE, in patients with COVID-19 [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>].</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>Despite it is rare, cardiac arrest could happen in young apparently healthy patients with COVID-19. The clinicians dealing with suspected or confirmed COVID-19 cases should always be alert. As COVID-19 patients are commonly having clotting disorders, endothelial and organ dysfunction, coagulopathy, and liable for PTE. We recommend that it is of crucial importance to select those COVID-19 patients at higher risk of PTE and practice CT pulmonary angiography for the diagnosis of PTE, especially in case of significant increase of D-dimer values. If no clear contraindication(s), anticoagulation should be started early to control and reduce pro-thrombotic events in patients with COVID-19.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"journal\"><article-title>Coronavirus disease 2019 case surveillance - United States, January 22-May 30</article-title><source>MMWR Morb Mortal Wkly Rep</source><person-group><name><surname>Stokes</surname><given-names>EK</given-names></name><name><surname>Zambrano</surname><given-names>LD</given-names></name><name><surname>Anderson</surname><given-names>KN</given-names></name><etal/></person-group><fpage>759</fpage><lpage>765</lpage><volume>69</volume><year>2020</year><pub-id pub-id-type=\"pmid\">32555134</pub-id></element-citation></ref><ref id=\"REF2\"><label>2</label><element-citation publication-type=\"website\"><article-title>Coronavirus Disease 2019 (COVID- 19): Who Is at Increased Risk for Severe Illness? - People of Any Age with Underlying Medical Conditions. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005540</article-id><article-id pub-id-type=\"pmc\">PMC7523738</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10126</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology/Diabetes/Metabolism</subject></subj-group><subj-group><subject>Internal Medicine</subject></subj-group><subj-group><subject>Public Health</subject></subj-group></article-categories><title-group><article-title>Behavior and Practices of Type 2 Diabetic Patients Regarding Obesity: A Cross-Sectional Study</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Akbar</surname><given-names>Quratulain</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Ahmed Khan</surname><given-names>Bilal</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rajput</surname><given-names>Bakhtawar Saleem</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Jatoi</surname><given-names>Nadia Nazir</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Elahi</surname><given-names>Sadia</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gain</surname><given-names>Abbas Mustafa</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Amjad</surname><given-names>Arooba</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Akbar</surname><given-names>Dureshahwar</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nazir</surname><given-names>Maaz Bin</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gianchand</surname><given-names>Naveed</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nInternal Medicine, Dow Medical College, Dow University of Health Sciences, Karachi, PAK </aff><author-notes><corresp id=\"cor1\">\nBilal Ahmed Khan <email>ronaldorooneyberbatov@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>30</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10126</elocation-id><history><date date-type=\"received\"><day>21</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>30</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Akbar et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Akbar et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/38334-behavior-and-practices-of-type-2-diabetic-patients-regarding-obesity-a-cross-sectional-study\">This article is available from https://www.cureus.com/articles/38334-behavior-and-practices-of-type-2-diabetic-patients-regarding-obesity-a-cross-sectional-study</self-uri><abstract><p>Background</p><p>Obesity is a major public health concern and is associated with incident cardiovascular diseases. A very few studies around the globe have assessed how type 2 diabetic (T2D) patients comprehend obesity. Our study aims to evaluate the concerns and behaviors of T2D patients regarding obesity in a developing country like Pakistan.</p><p>Methods</p><p>A cross-sectional study was conducted in Karachi during the period of December to February 2020 in which T2D patients were assessed for their comprehension of how obesity affects their disease and concerns, as well as their practices such as weight loss activities and dietary habits. Data analysis was performed using Statistical Package for the Social Sciences Version 24 (IBM Corp., Armonk, NY, USA).</p><p>Results</p><p>Of 417 T2D patients inducted in our study, 265 (63.5%) knew their ideal body weight, whereas only 221 (52.9%) knew how to measure it. Among those who were willing to lose weight, this was mostly due to a wish to avoid further complications of obesity (N=155 [73.1%]) and also peer/family pressures (N=124 [58.5%]) among other reasons. More obese (N=68 [43.6%]) than non-obese participants (N=87 [33.3%]) were willing to consult a doctor to help them reduce weight. Participants had adopted various strategies to reduce weight, of which increasing exercise (N=242 [85.8%]) and healthy eating (N=162 [57.4%]) were most popular.</p><p>Conclusions</p><p>There is a need to address barriers to weight loss among T2D patients in Pakistan and to provide patients with pragmatic guidelines on how to make sustainable lifestyle changes to help reduce and maintain their body weight.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>type 2 diabetes</kwd><kwd>obesity</kwd><kwd>diabetes mellitus</kwd><kwd>diabetes complications</kwd><kwd>health behavior</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>The World Health Organization (WHO) has labeled obesity a global epidemic. Obesity is classified as a body mass index (BMI) of ≥30 kg/m2 and is a major risk factor for insulin resistance leading to type 2 diabetes mellitus (T2DM) [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. Many epidemiological studies support an association between T2DM and obesity [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>,<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>].</p><p>According to the WHO, the prevalence of T2DM in Pakistan for the year 2000 was 5.2 million, and for 2030, it is estimated to be 13.8 million. One-fourth of the population of Pakistan can be categorized as overweight or obese with the use of Indo-Asian-specific BMI cutoff values [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. Developing or transitional economies are experiencing globalization of food markets, growing fast-food chains, and a rising trend of street vendors providing food items at affordable prices [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. This is also the case for Pakistan, especially in urban areas, making this population susceptible to obesity and its associated complications.</p><p>Obesity and T2DM together are associated with significant morbidity and mortality due to cardiovascular disease [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. Numerous measurements of obesity, with BMI, waist circumference, waist-to-hip ratio, and waist-to-stature ratio, are associated with the risk of T2DM [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. In obese patients with T2DM, weight loss has a positive impact on the control of hyperglycemia [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. Patient enthusiasm and knowledge are important factors in the management of both obesity and T2DM, and it is therefore important to evaluate the behavior and practices of T2DM patients with regard to obesity and related disorders.</p><p>There is a paucity of literature on this topic from developing countries such as Pakistan. The aim of this study is to evaluate the behavior and practices of type 2 diabetic (T2D) patients regarding obesity in Karachi, Pakistan.</p></sec><sec sec-type=\"materials|methods\"><title>Materials and methods</title><p>A cross-sectional, population-based study was conducted in two tertiary care hospitals of Karachi (Civil Hospital and Pakistan Steel Hospital) during the period of December to February 2020 after getting approval from the Institutional Review Board of Dow University of Health Sciences. The sample population consisted of T2DM patients selected through convenience sampling. A sample size of 450 was taken through <ext-link ext-link-type=\"uri\" xlink:href=\"openepi.com\">openepi.com</ext-link> with a 97% confidence level. Out of this, 417 completed the questionnaire fully, yielding a co-operation rate of 92.9%. Only residents of Karachi who were suffering from T2DM were included in this study. Patients with type 1 DM and gestational DM were excluded.</p><p>A structured questionnaire was used to carry out data collection. The questionnaire was available in both English and Urdu (the national language of Pakistan) to reduce linguistic barriers. Before data collection was started, the questionnaire was reviewed for relevance and completeness by two physicians independently, and a pilot study was also conducted and relevant changes were made. The questionnaire was divided into four sections containing 29 questions. The first section of the questionnaire consisted of sociodemographic information such as age, gender, weight, height, and level of education, as well as personal and family history of present medical illness. The second section dealt with comprehension regarding obesity, ideal body weight, BMI thresholds, and risk factors/causes of obesity. The third section assessed the behavior of the participants concerning obesity with coexisting T2DM and their willingness to lose weight, make dietary changes, and adopt exercise. The final section inquired about their weight loss regime (if any), dietary habits, and practices such as measurement of weight, blood pressure, and blood glucose level. Only recent and relevant information was asked in the questionnaire in order to minimize recall bias.</p><p>Verbal or written consent was taken from all participants after assuring them of complete confidentiality. The study investigators verbally interviewed those participants who were illiterate and completed the questionnaire on their behalf based on their oral responses. Interviewer bias was reduced here by training all interviewers to approach and interact with participants in a neutral and non-judgmental manner prior to carrying out data collection. No imputation method was used to account for missing data; incomplete questionnaires were excluded from the analysis.</p><p>T2DM was defined by the WHO definition, i.e., maintained fasting blood glucose of ≥7.0 mmol/L [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]. Respondents were categorized as obese (BMI ≥ 30) or non-obese (BMI < 30) per the WHO criteria. Data were analyzed using Statistical Package for the Social Sciences (SPSS) Version 24.0 (IBM Corp., Armonk, NY, USA).</p></sec><sec sec-type=\"results\"><title>Results</title><p>Of the 417 T2D patients who participated in this study, 156 (37.4%) were obese. A majority of participants had a family history of either hypertension (n=262 [62.8%]) or T2DM (n=246 [60.0%]). The baseline characteristics of the study population are summarized in Table <xref rid=\"TAB1\" ref-type=\"table\">1</xref>.</p><table-wrap id=\"TAB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Baseline characteristics of the study population</title></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nMale\n</td><td rowspan=\"1\" colspan=\"1\">\nFemale\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nGender\n</td><td rowspan=\"1\" colspan=\"1\">\n200 (49.5%)\n</td><td rowspan=\"1\" colspan=\"1\">\n217 (50.5%)\n</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"3\" rowspan=\"1\">\nAge\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<30 years\n</td><td rowspan=\"1\" colspan=\"1\">\n10\n</td><td rowspan=\"1\" colspan=\"1\">\n14\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n31-50 years\n</td><td rowspan=\"1\" colspan=\"1\">\n72\n</td><td rowspan=\"1\" colspan=\"1\">\n94\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n51-60 years\n</td><td rowspan=\"1\" colspan=\"1\">\n70\n</td><td rowspan=\"1\" colspan=\"1\">\n50\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n>60 years\n</td><td rowspan=\"1\" colspan=\"1\">\n48\n</td><td rowspan=\"1\" colspan=\"1\">\n55\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nLevel of education\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nNo formal education\n</td><td rowspan=\"1\" colspan=\"1\">\n102\n</td><td rowspan=\"1\" colspan=\"1\">\n92\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\n<12th grade (secondary school)\n</td><td rowspan=\"1\" colspan=\"1\">\n33\n</td><td rowspan=\"1\" colspan=\"1\">\n44\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nGraduate\n</td><td rowspan=\"1\" colspan=\"1\">\n59\n</td><td rowspan=\"1\" colspan=\"1\">\n49\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nPost-graduate\n</td><td rowspan=\"1\" colspan=\"1\">\n9\n</td><td rowspan=\"1\" colspan=\"1\">\n22\n</td></tr></tbody></table></table-wrap><p>More than half the participants in our sample did not know the difference between obese and overweight (n=220 [52.8%]). Of the 265 participants who knew what their ideal body weight should be 221 (83.4%) knew how to measure it. High-calorie intake and lack of exercise were considered to be causes of obesity by a majority (n=264 [63.3%] and n=260 [62.4%], respectively) (Table <xref rid=\"TAB2\" ref-type=\"table\">2</xref>).</p><table-wrap id=\"TAB2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><title>T2DM patients regarding obesity</title><p>T2DM, type 2 diabetes mellitus</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nNon-obese (n=261)\n</td><td rowspan=\"1\" colspan=\"1\">\nObese (n=156)\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nDo you know the difference between obesity and overweight?\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nYes\n</td><td rowspan=\"1\" colspan=\"1\">\n140 (53.6%)\n</td><td rowspan=\"1\" colspan=\"1\">\n80 (51.3%)\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nDo you know the normal blood glucose level?\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nYes\n</td><td rowspan=\"1\" colspan=\"1\">\n174 (66.7%)\n</td><td rowspan=\"1\" colspan=\"1\">\n91 (58.3%)\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nDo you know the ideal body weight? And how to measure it?\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nYes/Yes\n</td><td rowspan=\"1\" colspan=\"1\">\n102 (39.1%)\n</td><td rowspan=\"1\" colspan=\"1\">\n66 (42.3%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nYes/No\n</td><td rowspan=\"1\" colspan=\"1\">\n59 (22.6%)\n</td><td rowspan=\"1\" colspan=\"1\">\n38 (24.4%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nNo/Yes\n</td><td rowspan=\"1\" colspan=\"1\">\n29 (11.1%)\n</td><td rowspan=\"1\" colspan=\"1\">\n24 (15.5%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nNo/No\n</td><td rowspan=\"1\" colspan=\"1\">\n71 (27.2%)\n</td><td rowspan=\"1\" colspan=\"1\">\n28 (17.9%)\n</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"3\" rowspan=\"1\">\nWhat do you think are the causes of obesity?\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nSlow metabolism\n</td><td rowspan=\"1\" colspan=\"1\">\n90 (34.5%)\n</td><td rowspan=\"1\" colspan=\"1\">\n73 (46.8%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nHigh-calorie intake\n</td><td rowspan=\"1\" colspan=\"1\">\n167 (64.0%)\n</td><td rowspan=\"1\" colspan=\"1\">\n97 (62.2%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nLack of exercise\n</td><td rowspan=\"1\" colspan=\"1\">\n163 (62.5%)\n</td><td rowspan=\"1\" colspan=\"1\">\n97 (62.2%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nFamily history\n</td><td rowspan=\"1\" colspan=\"1\">\n81 (31.0%)\n</td><td rowspan=\"1\" colspan=\"1\">\n47 (3.1%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nNo specific reasons\n</td><td rowspan=\"1\" colspan=\"1\">\n22 (8.4%)\n</td><td rowspan=\"1\" colspan=\"1\">\n11 (7.1%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nNone of the above\n</td><td rowspan=\"1\" colspan=\"1\">\n9 (3.4%)\n</td><td rowspan=\"1\" colspan=\"1\">\n3 (1.9%)\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nWhat do you think are the risk factors for development of obesity?\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nDiabetes mellitus\n</td><td rowspan=\"1\" colspan=\"1\">\n119 (45.6%)\n</td><td rowspan=\"1\" colspan=\"1\">\n97 (62.2%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nCardiovascular diseases\n</td><td rowspan=\"1\" colspan=\"1\">\n88 (33.7%)\n</td><td rowspan=\"1\" colspan=\"1\">\n99 (63.5%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nHypertension\n</td><td rowspan=\"1\" colspan=\"1\">\n98 (37.5%)\n</td><td rowspan=\"1\" colspan=\"1\">\n55 (35.3%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nHigh cholesterol\n</td><td rowspan=\"1\" colspan=\"1\">\n150 (57.5%)\n</td><td rowspan=\"1\" colspan=\"1\">\n93 (59.6%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nJoint pains/arthritis\n</td><td rowspan=\"1\" colspan=\"1\">\n95 (36.4%)\n</td><td rowspan=\"1\" colspan=\"1\">\n65 (41.7%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nNone of the above\n</td><td rowspan=\"1\" colspan=\"1\">\n20 (7.7%)\n</td><td rowspan=\"1\" colspan=\"1\">\n10 (6.4%)\n</td></tr></tbody></table></table-wrap><p>It was found that more obese participants were willing to reduce their weight (n=86 [55.1%]) than non-obese participants (n=126 [48.3%]) (Table <xref rid=\"TAB3\" ref-type=\"table\">3</xref>). Among those who were willing to lose weight, this was mostly due to a wish to avoid further complications of obesity (n=155 [73.1%]) and also due to peer/family pressures (n=124 [58.5%]). Moreover, more obese (n=68 [43.6%]) than non-obese participants (n=87 [33.3%]) were willing to consult a doctor to help them reduce weight.</p><table-wrap id=\"TAB3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><title>Behavior of obese and non-obese T2DM patients regarding obesity</title><p>T2DM, type 2 diabetes mellitus</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nNon-obese (n=261)\n</td><td rowspan=\"1\" colspan=\"1\">\nObese (n=156)\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nAre you willing to reduce your weight?\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nYes\n</td><td rowspan=\"1\" colspan=\"1\">\n126 (48.3%)\n</td><td rowspan=\"1\" colspan=\"1\">\n86 (55.1%)\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nIf yes, then kindly select one or more of the following:\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nDue to peer/family pressure\n</td><td rowspan=\"1\" colspan=\"1\">\n55 (21.1%)\n</td><td rowspan=\"1\" colspan=\"1\">\n69 (44.2%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nAvoid further complications\n</td><td rowspan=\"1\" colspan=\"1\">\n80 (30.7%)\n</td><td rowspan=\"1\" colspan=\"1\">\n75 (48.1%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nTo do daily chores more efficiently\n</td><td rowspan=\"1\" colspan=\"1\">\n60 (23.0%)\n</td><td rowspan=\"1\" colspan=\"1\">\n48 (30.8%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nTo fit in better in the society\n</td><td rowspan=\"1\" colspan=\"1\">\n18 (6.9%)\n</td><td rowspan=\"1\" colspan=\"1\">\n29 (18.6%)\n</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"3\" rowspan=\"1\">\nAre you willing to consult a doctor to reduce your weight?\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nYes\n</td><td rowspan=\"1\" colspan=\"1\">\n87 (33.3%)\n</td><td rowspan=\"1\" colspan=\"1\">\n68 (43.6%)\n</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"3\" rowspan=\"1\">\nDo you think overweight people should try to lose their weight?\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nYes\n</td><td rowspan=\"1\" colspan=\"1\">\n213 (81.6%)\n</td><td rowspan=\"1\" colspan=\"1\">\n121 (77.6%)\n</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"3\" rowspan=\"1\">\nHow important do you think it is for an obese person with diabetes to lose weight?\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nVery Important\n</td><td rowspan=\"1\" colspan=\"1\">\n202 (77.4%)\n</td><td rowspan=\"1\" colspan=\"1\">\n134 (85.9%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nNo need to lose weight\n</td><td rowspan=\"1\" colspan=\"1\">\n30 (11.5%)\n</td><td rowspan=\"1\" colspan=\"1\">\n14 (9.0%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nDiabetes and obesity are not related\n</td><td rowspan=\"1\" colspan=\"1\">\n26 (10.0%)\n</td><td rowspan=\"1\" colspan=\"1\">\n8 (5.1%)\n</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"3\" rowspan=\"1\">\nDo you think having proper knowledge of your condition and treatment can help you reach a controllable state?\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nYes\n</td><td rowspan=\"1\" colspan=\"1\">\n192 (73.6%)\n</td><td rowspan=\"1\" colspan=\"1\">\n99 (63.5%)\n</td></tr></tbody></table></table-wrap><p>A greater percentage of obese (n=72 [46.2%]) than non-obese patients (n=57 [21.8%]) reported multiple previous attempts at losing weight. Participants had adopted various strategies to reduce weight, of which increasing exercise (n=242 [85.8%]) and healthy eating (n=162 [57.4%]) were most popular. Eating habits of patients, i.e., number of meals per day and diet composition, are shown in Figures <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>, <xref ref-type=\"fig\" rid=\"FIG2\">2</xref>, respectively.</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Number of meals per day taken by study participants</title></caption><graphic xlink:href=\"cureus-0012-00000010126-i01\"/></fig><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>Diet composition of study participants</title></caption><graphic xlink:href=\"cureus-0012-00000010126-i02\"/></fig><p>A greater proportion of obese participants exercised daily (n=71 [45.5%]) or had followed a special diet (n=65 [41.7%]) as compared to non-obese ones. Tables <xref rid=\"TAB4\" ref-type=\"table\">4</xref>, <xref rid=\"TAB5\" ref-type=\"table\">5</xref> show the self-care practices of T2DM patients regarding obesity.</p><table-wrap id=\"TAB4\" orientation=\"portrait\" position=\"float\"><label>Table 4</label><caption><title>Practices of obese and non-obese T2DM patients regarding obesity</title><p>T2DM, type 2 diabetes mellitus</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nNon-obese (n=261)\n</td><td rowspan=\"1\" colspan=\"1\">\nObese (n=156)\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nHave you ever tried to lose weight in the past?\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nYes, many times\n</td><td rowspan=\"1\" colspan=\"1\">\n57 (21.8%)\n</td><td rowspan=\"1\" colspan=\"1\">\n72 (46.2%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nYes, occasionally\n</td><td rowspan=\"1\" colspan=\"1\">\n98 (37.5%)\n</td><td rowspan=\"1\" colspan=\"1\">\n55 (35.3%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nNever tried\n</td><td rowspan=\"1\" colspan=\"1\">\n106 (40.6%)\n</td><td rowspan=\"1\" colspan=\"1\">\n29 (17.6%)\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nIf yes, which of the following options have you followed to reduce your weight?\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nExercise\n</td><td rowspan=\"1\" colspan=\"1\">\n137 (52.5%)\n</td><td rowspan=\"1\" colspan=\"1\">\n105 (67.3%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nWeight-reducing medication\n</td><td rowspan=\"1\" colspan=\"1\">\n31 (11.9%)\n</td><td rowspan=\"1\" colspan=\"1\">\n32 (20.5%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nAvoiding meals\n</td><td rowspan=\"1\" colspan=\"1\">\n69 (26.4%)\n</td><td rowspan=\"1\" colspan=\"1\">\n49 (31.4%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nEating healthy/reducing calorie intake\n</td><td rowspan=\"1\" colspan=\"1\">\n100 (38.3%)\n</td><td rowspan=\"1\" colspan=\"1\">\n62 (39.7%)\n</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"3\" rowspan=\"1\">\nIf you said yes to exercise, how often do you exercise?\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nDaily\n</td><td rowspan=\"1\" colspan=\"1\">\n85 (32.6%)\n</td><td rowspan=\"1\" colspan=\"1\">\n71 (45.5%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nFew times a week\n</td><td rowspan=\"1\" colspan=\"1\">\n39 (15.0%)\n</td><td rowspan=\"1\" colspan=\"1\">\n27 (17.3%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nFew times a month\n</td><td rowspan=\"1\" colspan=\"1\">\n13 (4.9%)\n</td><td rowspan=\"1\" colspan=\"1\">\n7 (4.5%)\n</td></tr><tr style=\"background-color:#ccc\"><td colspan=\"3\" rowspan=\"1\">\nHave you ever been on a diet in an attempt to lose weight?\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nYes\n</td><td rowspan=\"1\" colspan=\"1\">\n91 (34.9%)\n</td><td rowspan=\"1\" colspan=\"1\">\n65 (41.7%)\n</td></tr></tbody></table></table-wrap><table-wrap id=\"TAB5\" orientation=\"portrait\" position=\"float\"><label>Table 5</label><caption><title>Self-monitoring practices of obese and non-obese T2DM patients </title><p>T2DM, type 2 diabetes mellitus</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\n \n</td><td rowspan=\"1\" colspan=\"1\">\nNon-obese (n=261)\n</td><td rowspan=\"1\" colspan=\"1\">\nObese (n=156)\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nHow often do you check your blood pressure?\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nDaily\n</td><td rowspan=\"1\" colspan=\"1\">\n28 (10.7%)\n</td><td rowspan=\"1\" colspan=\"1\">\n24 (15.4%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nAlternate days\n</td><td rowspan=\"1\" colspan=\"1\">\n40 (15.3%)\n</td><td rowspan=\"1\" colspan=\"1\">\n21 (13.5%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nTwice a week\n</td><td rowspan=\"1\" colspan=\"1\">\n49 (18.8%)\n</td><td rowspan=\"1\" colspan=\"1\">\n41 (26.3%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nOnce a week\n</td><td rowspan=\"1\" colspan=\"1\">\n71 (27.2%)\n</td><td rowspan=\"1\" colspan=\"1\">\n29 (18.6%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nUndocumented\n</td><td rowspan=\"1\" colspan=\"1\">\n63 (24.1)\n</td><td rowspan=\"1\" colspan=\"1\">\n41 (26.3%)\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nHow often do you check your blood glucose level?\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nDaily\n</td><td rowspan=\"1\" colspan=\"1\">\n39 (14.9%)\n</td><td rowspan=\"1\" colspan=\"1\">\n25 (16.0%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nAlternate days\n</td><td rowspan=\"1\" colspan=\"1\">\n41 (15.7%)\n</td><td rowspan=\"1\" colspan=\"1\">\n31 (19.9%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nTwice a week\n</td><td rowspan=\"1\" colspan=\"1\">\n49 (18.8%)\n</td><td rowspan=\"1\" colspan=\"1\">\n41 (26.3%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nOnce a week\n</td><td rowspan=\"1\" colspan=\"1\">\n71 (27.2%)\n</td><td rowspan=\"1\" colspan=\"1\">\n29 (18.6%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nUndocumented\n</td><td rowspan=\"1\" colspan=\"1\">\n61 (24.4%)\n</td><td rowspan=\"1\" colspan=\"1\">\n30 (19.2%)\n</td></tr><tr><td colspan=\"3\" rowspan=\"1\">\nHow often do you check your weight?\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nDaily\n</td><td rowspan=\"1\" colspan=\"1\">\n23 (8.8%)\n</td><td rowspan=\"1\" colspan=\"1\">\n34 (21.8%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nAlternate days\n</td><td rowspan=\"1\" colspan=\"1\">\n20 (7.7%)\n</td><td rowspan=\"1\" colspan=\"1\">\n4 (2.6%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nTwice a week\n</td><td rowspan=\"1\" colspan=\"1\">\n24 (9.2%)\n</td><td rowspan=\"1\" colspan=\"1\">\n25 (16.0%)\n</td></tr><tr><td rowspan=\"1\" colspan=\"1\">\nOnce a week\n</td><td rowspan=\"1\" colspan=\"1\">\n55 (21.1%)\n</td><td rowspan=\"1\" colspan=\"1\">\n32 (20.5%)\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">\nUndocumented\n</td><td rowspan=\"1\" colspan=\"1\">\n139 (55.6%)\n</td><td rowspan=\"1\" colspan=\"1\">\n61 (39.1%)\n</td></tr></tbody></table></table-wrap></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>Obesity among T2DM patients is an alarming issue rising steadily worldwide and needs to be promptly addressed with efficient measures to reduce its economic burden [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>]. A study carried out recently showed that being overweight also increases the risk of having cardiovascular and coronary diseases in a diabetic patient [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]. To deal with this issue, there is a need to educate people with T2DM about obesity and body weight reducing measures as patient education influences their health practices [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. In our study, the majority of diabetics had a family history of obesity (129 [30.9%]) and hyper-cholesterolemia (134 [32.2%]) as proven by many types of researches that the fat-mass gene has a strong association with diabetes [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>].</p><p>In this study, a considerable proportion of participants (n=220 [52.8%]) were unaware of the difference between overweight and obese or about how body weight is measured. This could be since in Pakistan, not every household has a weighing instrument as it is considered an unimportant resource to have, and gyms and places where they are available are unfortunately less popular. This is also reflected in the lack of consistency in weight self-monitoring practices among our participants. Many patients in our study were not regularly monitoring their blood pressure, weight, or blood sugar levels, underscoring the importance of the routine availability of appropriate instruments. Having a weighing machine is as important as having a glucometer in every household with T2DM patients since obesity can increase the risk of developing T2DM complications. Furthermore, clinicians should perceive the need for guidance, medication referral, and advice for weight loss regimens to be able to treat diabetes in their obese patients [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>].</p><p>The lack of knowledge regarding ideal body weight in this study was surprisingly more profound as compared with a study carried out on newly diagnosed patients in Ghana. However, a satisfactory understanding of weight measurement techniques was found in patients from Ghana [<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>]. The majority of our patients knew that increased calorie intake (264 [63.3%]) and lack of exercise (260 [62.4%]) were risk factors for obesity, as also shown by studies carried out in Bangladesh [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>], Ghana [<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>], and South Africa [<xref rid=\"REF16\" ref-type=\"bibr\">16</xref>].</p><p>Obese T2DM patients were more willing to reduce weight as opposed to non-obese patients and were motivated by both health and social reasons. This could be due to health-related complications already faced by these patients, and a wish to fit in a society where body-shaming is highly prevalent [<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>]. Unfortunately, only 43.6% (n=68) of obese T2DM patients in our study shared a positive attitude toward consulting their physician for weight-loss counseling, making it imperative for physicians to initiate the discussion about the impact of obesity on the general health of T2DM patients [<xref rid=\"REF18\" ref-type=\"bibr\">18</xref>].</p><p>More obese patients were seen to monitor their blood glucose (157 [37.6%]) and blood pressure (156 [37.4%]) levels daily as compared to non-obese ones, possibly due to an understanding that they were at higher risk of heart disease and hypertension than non-obese T2DM patients. In Pakistan, three meals are typically consumed in a day, and fried oily food and dairy products are part of the normal diet regimen. This dietary pattern is consistent with our study findings and a previous study conducted in Pakistan in Aga Khan Hospital Karachi [<xref rid=\"REF19\" ref-type=\"bibr\">19</xref>]. The popular trend of only “eating out” in Pakistan further makes it challenging for T2DM patients to avoid high-calorie foods [<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>].</p><p>According to a study carried out in Malaysia [<xref rid=\"REF21\" ref-type=\"bibr\">21</xref>], most diabetic patients showed a positive attitude toward making lifestyle changes; however, they failed to implement them. Another study conducted in Pakistan similarly found that although T2DM patients had profound knowledge about their disease, they failed to apply their knowledge to change their behavior [<xref rid=\"REF22\" ref-type=\"bibr\">22</xref>]. This highlights the need for public health initiatives that go beyond increasing health awareness and motivate and guide patients on how to make sustainable changes to their diet and physical activity routine.</p><p>There have been many successfully executed lifestyle modification programs, such as the U.S. Diabetes Prevention Program (US DPP) and Finnish Diabetes Prevention Program, which provide their participants with weekly guidelines on ways to reduce weight and eat healthily [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>,<xref rid=\"REF23\" ref-type=\"bibr\">23</xref>-<xref rid=\"REF25\" ref-type=\"bibr\">25</xref>]. Based on these models, similar campaigns can and should be developed in Pakistan too given its current burden of these two chronic illnesses.</p><p>Besides large-scale efforts to increase public awareness, physicians also have a critical role in addressing barriers to weight loss among T2DM patients and the need to provide patients with pragmatic guidelines and support on this issue. Physicians should further make appropriate referrals to nutritionists, physical therapists, and bariatric surgery, as needed.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>This study might aid in the application of diabetic education programs, giving more stress on elder and younger groups of patients and fortify the patients to have more connection with concerning physicians so that complications at the early stage of the disease could be prevented.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study</p></fn></fn-group><fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"book\"><article-title>The Asia-Pacific Perspective: Redefining Obesity and Its Treatment</article-title><person-group><name><surname>World Health</surname><given-names>Organization</given-names></name></person-group><publisher-loc>Sydney</publisher-loc><publisher-name>Health Communications Australia</publisher-name><year>2000</year><uri xlink:href=\"https://apps.who.int/iris/handle/10665/206936\">https://apps.who.int/iris/handle/10665/206936</uri></element-citation></ref><ref id=\"REF2\"><label>2</label><element-citation publication-type=\"journal\"><article-title>Body mass index and diabetes in Asia: a cross-sectional pooled analysis of 900,000 individuals in the Asia cohort consortium</article-title><source>PLoS 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005535</article-id><article-id pub-id-type=\"pmc\">PMC7523739</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10119</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Internal Medicine</subject></subj-group><subj-group><subject>Rheumatology</subject></subj-group></article-categories><title-group><article-title>Coevality of Systemic Lupus Erythematosus With Sickle Cell Trait: A Not So Uncommon Entity</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Das</surname><given-names>Dhriti Sundar</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sahoo</surname><given-names>Debananda</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nGeneral Medicine, All India Institute of Medical Sciences, Bhubaneswar, IND </aff><aff id=\"aff-2\">\n<label>2</label>\nInternal Medicine, All India Institute of Medical Sciences, Bhubaneswar, IND </aff><author-notes><corresp id=\"cor1\">\nDhriti Sundar Das <email>recdhriti@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10119</elocation-id><history><date date-type=\"received\"><day>18</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Das et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Das et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/29319-coevality-of-systemic-lupus-erythematosus-with-sickle-cell-trait-a-not-so-uncommon-entity\">This article is available from https://www.cureus.com/articles/29319-coevality-of-systemic-lupus-erythematosus-with-sickle-cell-trait-a-not-so-uncommon-entity</self-uri><abstract><p>The coexistence of systemic lupus erythematosus (SLE) with sickle cell trait is quite sparingly reported in literature. Here, we narrate the case of a 17-year-old girl from Eastern India with sickle cell trait who presented with acute lupus pneumonitis. The challenges to the final diagnosis of SLE with sickle cell trait were because of the often lesser degree of clinical suspicion at the outset. In this report, we discuss this not so uncommon combination of conditions and review related literature. This girl, who was a known case of sickle cell trait, presented with fever, cough, shortness of breath with subsequent rashes, oral ulceration, high erythrocyte sedimentation rate (ESR) and proteinuria. After ruling out infective causes, she was found to be antinuclear antibody (ANA) positive and with stage 4 lupus nephritis. Emphasis should be given to the presence of autoimmune conditions in patients with sickle hemoglobinopathies, including sickle cell trait wherein atypical or systemic involvement may occur. Such association holds more importance as sickle hemoglobinopathies is one of the major hemoglobinopathies reported in this part of the country.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>sickle cell trait</kwd><kwd>systemic lupus erythematosus</kwd><kwd>lupus pneumonitis</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Sickle hemoglobinopathies, a group of commonly encountered genetic disorders that include sickle cell trait, sickle cell disease, and sickle Beta−Thalassemia, are prevalent in this eastern part of India [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>,<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. Due to the multisystem involvement and manifestations, the diagnosis of lupus in patients with hemoglobinopathies may often get delayed [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>,<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. We report a case of known sickle cell trait whose clinical features and subsequent presentations lead to the diagnosis of lupus.</p></sec><sec sec-type=\"cases\"><title>Case presentation</title><p>A 17-year-old girl presented with low-grade fever, not associated with chills and rigor, cough with expectoration for two weeks, non-radiating chest pain, and breathlessness of three days duration. She was a known case of sickle cell trait. The cough was associated with hemoptysis. She had no history of joint pain or photosensitivity rashes. Past history revealed cervical lymphadenopathy a few months back with biopsy showing reactive lymphadenitis; apart from this, no other past similar illness was reported. Family history was not suggestive. She had no history of tuberculosis or any contact history of tuberculosis. On examination, pulse was 112/min, blood pressure 136/86 mmHg, oxygen saturation being 90% on room air and respiratory rate being 30 per minute. On auscultation, chest revealed bilateral crepitations and wheezes. Cardiac and neurological examinations were unremarkable. Subsequently, her saturation dropped to 84% on room air. She was started on IV antibiotics, nebulization with salbutamol, isotonic normal saline. Further evaluation revealed high erythrocyte sedimentation rate (ESR; 151 mm at the end of the first hour) with anemia, rashes over both feet, bilateral cervical lymphadenopathy, painless oral ulcer and amenorrhea. Her initial total leucocyte counts and renal function test were normal. There was mild thrombocytopenia (1.26 lakh per cumm) and hypoproteinemia with hypoalbuminemia. Routine urinalysis showed 3+ proteinuria. Chest x-ray showed bilateral patchy infiltrates (Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>). Peripheral blood smear showed features of hemolytic anemia with target cells. Direct Coombs test was positive. Serum ferritin was high. The test for antinuclear antibodies (ANA) was positive (3+; mixed pattern), and dsDNA was also positive, fulfilling the American College of Rheumatology criteria for the diagnosis of systemic lupus erythematosus (SLE). Ultrasound of the abdomen showed only splenomegaly. Computed tomography (CT) scan of thorax revealed bilateral ground-glass opacities with right pleural effusion with bilateral mediastinal, axillary lymphadenopathy (Figure <xref ref-type=\"fig\" rid=\"FIG2\">2</xref>). Sputum culture showed no growth. Sputum was negative for acid-fast bacilli (AFB) and cartridge-based nucleic acid amplification test (CBNAAT). Twenty-four-hour urine protein was 3521 mg. Echocardiography was normal. Perinuclear antineutrophil cytoplasmic antibodies (P-ANCA) and cytoplasmic antineutrophil cytoplasmic antibodies (C-ANCA) were negative. The patient responded with pulse methylprednisolone and then converted to oral steroids and started on hydroxychloroquine. The patient was discharged to follow up with renal biopsy report, which revealed proliferative and sclerosing lesions involving more than 50 percent of glomeruli suggestive of stage 4 lupus nephritis. However, the red blood cells (RBCs) within the vessels did not show evidence of sickling. </p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Chest x-ray posteroanterior (PA) view showing bilateral patchy infiltrates</title></caption><graphic xlink:href=\"cureus-0012-00000010119-i01\"/></fig><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>CT thorax showing bilateral ground-glass opacities</title></caption><graphic xlink:href=\"cureus-0012-00000010119-i02\"/></fig></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>The present case of sickle cell trait (SCT) and SLE highlights the importance of diagnosing such association with utmost clinical suspicion. The diagnosis of SCT is usually made long before SLE is evident [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. The coexistence of these diseases is of great interest, but the paucity of cases that are being reported from this part of the country implies it is uncommon. Moreover, since sickle hemoglobinopathies are the major hemoglobinopathies in this eastern state of India (Odisha), therefore such association holds more importance and relevance from clinical and management point of view of such cases in this part of the country [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. With the overlapping clinical features and reporting of ANA positivity in patients with sickle cell disease (SCD), diagnosing lupus is indeed very challenging to the clinicians at large [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>,<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. However, very few cases of lupus are reported with the SCT variant of hemoglobinopathies [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. Patients with SCD have an abnormal alternate pathway of the complement system and this may make them prone to the development of autoimmune diseases [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>,<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. The array of symptoms of both the conditions may, at times, be confusing to substantially delineate out the primary disease and therefore, one should have a high index of clinical suspicion on encountering such symptomatology to make an early diagnosis. The high titers of ANA positivity in sickle hemoglobinopathies especially taxes physicians in current clinical practice.</p><p>Large clinico-epidemiological studies are indeed required to delve more into such association of immune disorders in patients with sickle hemoglobinopathies.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>This report highlights the importance of having a high degree of clinical suspicion in diagnosing such cases at the earliest possible time and starting treatment without any further delay to prevent complications. Such association holds more relevance in our setting as sickle hemoglobinopathies are the commonest hemoglobinopathies in this eastern state of India, i.e., Odisha.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study</p></fn></fn-group><ack><p>I would like to acknowledge all the faculties, residents, and staff of the department of General Medicine, All India Institute of Medical Sciences (AIIMS), Bhubaneswar, for support related to patient care and follow up.</p></ack><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"journal\"><article-title>Spectrum of hemoglobinopathies in the state of Orissa, India: a ten years cohort study</article-title><source>J Assoc 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005531</article-id><article-id pub-id-type=\"pmc\">PMC7523740</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10114</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Infectious Disease</subject></subj-group></article-categories><title-group><article-title>The Role of Sex in the Risk of Mortality From COVID-19 Amongst Adult Patients: A Systematic Review</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Kelada</surname><given-names>Monica</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Anto</surname><given-names>Ailin</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dave</surname><given-names>Karishma</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Saleh</surname><given-names>Sohag N</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nInfectious Diseases, Imperial College London, London, GBR </aff><aff id=\"aff-2\">\n<label>2</label>\nPharmacology, Imperial College London, London, GBR </aff><author-notes><corresp id=\"cor1\">\nAilin Anto <email>ailin.anto18@imperial.ac.uk</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10114</elocation-id><history><date date-type=\"received\"><day>17</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Kelada et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Kelada et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/36138-the-role-of-sex-in-the-risk-of-mortality-from-covid-19-amongst-adult-patients-a-systematic-review\">This article is available from https://www.cureus.com/articles/36138-the-role-of-sex-in-the-risk-of-mortality-from-covid-19-amongst-adult-patients-a-systematic-review</self-uri><abstract><p>A worldwide outbreak of coronavirus disease 2019 (COVID-19), identified as being caused by the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), was classified as a Public Health Emergency of International Concern by the World Health Organisation (WHO) on January 30, 2020. Initial sex-disaggregated mortality data emerging from the Wuhan province of China identified male sex as a risk factor for increased COVID-19 mortality.  </p><p>In this systematic review, we aimed to assess the role of sex in the risk of mortality from COVID-19 in adult patients through comparison of clinical markers and inflammatory indexes.  </p><p>A systematic search was conducted on the following databases: PubMed, WHO COVID-19 database, Ovid MEDLINE, and Web of Science between the dates of June 15, 2020, and June 30, 2020. Key search terms used included: “sex”, “gender”, “SARS-COV-2”, “COVID” and “mortality”. We accepted the following types of studies concerning adult COVID-19 patients: retrospective cohort, observational cohort, case series, and applied research. Further studies were extracted from reference searching. The risk of bias was determined using the National Institutes of Health Quality Assessment Tool for Observational Cohort, Cross-Sectional Studies, and Case Series. </p><p>We identified a total of 16 studies published between January 2020 and June 2020 for analysis in this systematic review. Our study population consisted of 11 cohort studies, four case series, and one genetic study, including a total of 76,555 participants. Ten of the studies included in this review observed a higher risk of mortality among males compared to females, and eight of these studies found this risk to be statistically significant.   </p><p>Sex-disaggregated COVID-19 mortality data identifies male patients with comorbidities as being at an increased risk of mortality worldwide. Further investigation revealed differences in immune response regulated by sex hormones, angiotensin-converting enzyme 2 (ACE2) expression, and health behaviours as contributing factors to increased risk of mortality from COVID-19 among males.   </p><p>Nine out of the 16 studies included were conducted in China. In order to comprehensively assess sex-differences in the risk of mortality from COVID-19, more studies will need to be conducted worldwide. Sex-disaggregated COVID-19 data published in the medical literature is limited, however it has become evident that male sex is an important risk factor for mortality. Further exploration into the impact of sex on this pandemic is required in order to develop targeted therapies, as well as public health policies, and to prevent sex bias in treatment.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>covid-19</kwd><kwd>sex</kwd><kwd>gender</kwd><kwd>mortality</kwd><kwd>review</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction and background</title><p>In December 2019, a rise in pneumonia cases of unknown aetiology was seen in the Wuhan province of China. Approximately one month later, the coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the pathogenic source of the disease [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. The virus is transmitted by talking, coughing, sneezing, aerosols and is now thought to be airborne [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. The rapidly spreading and contagious nature of the virus led to a Public Health Emergency of International Concern being declared by the World Health Organisation (WHO) as of January 30, 2020 [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>].   </p><p>Mild illness may present with symptoms such as fever, malaise, headache, muscle pain, dry cough, and sore throat. However, more severe disease may progress to Acute Respiratory Distress Syndrome (ARDS) and death [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>].  </p><p>As of June 30, 2020, England had 39,177 COVID-19 fatalities of which 56.9% were males and 43.1% were females [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. Further exploration into the impact of sex on this pandemic is required to develop targeted therapies and public health policies. Sex-disaggregated analysis of the COVID-19 outbreak is important, as mortality data from 49 countries indicate that males have a higher overall mortality rate than females [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. Although male and female susceptibility is the same, it has become evident that the male sex is an important risk factor for mortality [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>].</p><p>In this systematic review we aimed to assess the role of sex in the risk of mortality from COVID-19 in adult patients through comparison of clinical markers and inflammatory indexes.</p></sec><sec sec-type=\"review\"><title>Review</title><p>Methodology</p><p>Search Strategy   </p><p>A systematic search was conducted on the following electronic databases: PubMed, WHO COVID-19 database, Ovid MEDLINE and Web of Science between the dates of June 15, 2020, and June 30, 2020. This systematic review was conducted in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines  and has been registered on PROSPERO (identifier number: CRD42020196076) [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>].  </p><p>A search strategy containing keywords was implemented across all search domains. Our search strategy consists of the following keywords: “male”, “female”, “men”, “women”, “sex”, “gender”, “corona”, “COVID-19”, “Cov2”, “SARS”, “SARS-COV-2”, “SARS-2”, “SARS-corona”, “severe acute respiratory syndrome”, “hormone”, “androgen”, “testosterone”, “oestrogen”, “estrogen”, “oestradiol”, “estradiol”, “ACE2”, “ACE-2”, “TMPRSS2”, “mortality”, “morbidity”, “death”.   One example of a search string used is: “((COVID-19) OR (Coronavirus)) AND ((Gender) OR (sex) OR (male) OR (female)) AND ((mortality) OR (death rate))”.  </p><p>Selection Criteria  </p><p>The preliminary search yielded 1092 papers. After removal of duplicates, 655 abstracts and titles were screened for relevancy, through which 598 papers were excluded. The remaining 57 papers underwent full text screening. An additional 25 papers were retrieved through screening of relevant references. A total of 82 papers underwent full text screening. After applying inclusion and exclusion criteria, 16 papers were selected for analysis in this systematic review.   </p><p>We accepted the following types of studies: retrospective cohort, observational cohort, case series, and applied research. Papers from any country concerning confirmed COVID-19 adult patients were accepted if they were written in English.  Papers concerning paediatric (being under 18 years of age) and pregnant patients were excluded. Papers addressing other viral respiratory diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), were also excluded.  </p><p>Our outcome of interest was mortality rate of males and females diagnosed with COVID-19. Indicators that we regarded as potential reasons for the difference in mortality included: immunoglobulin G (IgG) antibody levels, immune cell levels, inflammatory indexes, receptor expression, hormone levels, behavioural factors , and disease severity.  </p><p>Two reviewers screened the abstracts and titles retrieved from the searches. The third reviewer conducted a final check on the relevance of the papers against the inclusion and exclusion criteria. Data retrieved from the studies were collated into tabular form outlining the study design and main findings.  Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref> shows the PRISMA flow diagram detailing the study identification, screening, and selection process. </p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>PRISMA flow diagram </title><p>PRISMA flow diagram to show study identification, screening, inclusion and exclusion against specific pre-defined criteria. </p><p>COVID-19: coronavirus disease 2019, WHO: World Health Organization, SARS: severe acute respiratory syndrome, MERS: Middle East respiratory syndrome</p></caption><graphic xlink:href=\"cureus-0012-00000010114-i01\"/></fig><p>Risk of Bias Assessment  </p><p>Risk of bias was assessed using the National Institutes of Health (NIH) Quality Assessment Tool for Cohort, Cross-Sectional Studies and Case series [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. Two review authors assessed the risk of bias and this was reviewed by the third author.</p><p>Results</p><p>We identified a total of 16 studies published between January 2020 and June 2020 for analysis in this systematic review. Our study population consisted of 11 cohort studies, four case series and one genetic study, including a total of 76,555 participants. The characteristics and main outcomes of each study are summarised in Table <xref rid=\"TAB1\" ref-type=\"table\">1</xref>.  </p><table-wrap id=\"TAB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Sixteen inclusion studies detailing study country, type, population, purpose and main outcomes.</title><p>SARS-CoV-2: severe acute respiratory syndrome coronavirus 2, COVID-19: coronavirus disease 2019, ACE2: angiotensin-converting enzyme 2, IgG: immunoglobulin G, OR: odds ratio</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Reference </td><td rowspan=\"1\" colspan=\"1\">Country </td><td rowspan=\"1\" colspan=\"1\">Study Design </td><td rowspan=\"1\" colspan=\"1\">Sample Population </td><td rowspan=\"1\" colspan=\"1\">%  Males </td><td rowspan=\"1\" colspan=\"1\">%  Females </td><td rowspan=\"1\" colspan=\"1\">Study Purpose </td><td rowspan=\"1\" colspan=\"1\">Outcome/Conclusions </td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Qin L. et al (2020) [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]      </td><td rowspan=\"1\" colspan=\"1\">China  </td><td rowspan=\"1\" colspan=\"1\">Retrospective Cohort Study     </td><td rowspan=\"1\" colspan=\"1\">548 COVID-19 inpatients  </td><td rowspan=\"1\" colspan=\"1\">50.9  </td><td rowspan=\"1\" colspan=\"1\">49.1  </td><td rowspan=\"1\" colspan=\"1\">To investigate how the effect of sex on inflammation affects COVID-19 outcomes </td><td rowspan=\"1\" colspan=\"1\">Mortality was higher in males (22.2% vs 10.4%). Relative Risk for mortality was 2.2 for males compared to females. Males had higher rates of lymphopenia, thrombocytopenia. Multiple linear regression method revealed greater levels of inflammation indexes in males.  </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Asfahan S. et al (2020) [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]       </td><td rowspan=\"1\" colspan=\"1\">China  </td><td rowspan=\"1\" colspan=\"1\">Retrospective Cohort study  </td><td rowspan=\"1\" colspan=\"1\">44,672 COVID-19 inpatients  </td><td rowspan=\"1\" colspan=\"1\">51.4  </td><td rowspan=\"1\" colspan=\"1\">48.6  </td><td rowspan=\"1\" colspan=\"1\">To describe mortality characteristics for COVID-19 from publicly available data from China   </td><td rowspan=\"1\" colspan=\"1\">81% of deaths were in the ages above 60 years, 63.8% of deaths were males. Logistic regression risk factors showed that only age and comorbidities significantly affected mortality. Sex and occupation when adjusted for other factors were not significant predictors of mortality - although case fatality rate was higher in males (2.8%) compared to females (1.7%).  </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Borobia AM. et al (2020) [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>]   </td><td rowspan=\"1\" colspan=\"1\">Spain  </td><td rowspan=\"1\" colspan=\"1\">Retrospective Cohort Study  </td><td rowspan=\"1\" colspan=\"1\">2226 COVID-19 in-patients  </td><td rowspan=\"1\" colspan=\"1\">48.2  </td><td rowspan=\"1\" colspan=\"1\">51.8  </td><td rowspan=\"1\" colspan=\"1\">To describe the clinical characteristics of hospitalized patients with COVID-19  </td><td rowspan=\"1\" colspan=\"1\">Mortality was higher in males than females (26.6% vs. 15.1%). Compared with the entire cohort, the patients admitted to the ICU had a higher male/female ratio (3.2 vs. 0.93). In multivariate logistic regression model, male sex was associated with higher probability of death from COVID-19. </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Yang X. et al (2020) [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]      </td><td rowspan=\"1\" colspan=\"1\">China  </td><td rowspan=\"1\" colspan=\"1\">Retrospective Cohort Study  </td><td rowspan=\"1\" colspan=\"1\"> 52 critically ill COVID-19 adults  </td><td rowspan=\"1\" colspan=\"1\">67  </td><td rowspan=\"1\" colspan=\"1\">33  </td><td rowspan=\"1\" colspan=\"1\">To describe the clinical course and outcome of critically ill patients with COVID-19  </td><td rowspan=\"1\" colspan=\"1\">67% of critically ill patients were males. (Critically ill patients were defined as those admitted to the Intensive Care Unit (ICU) who required mechanical ventilation or had a fraction of inspired oxygen of at least 60% or more)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Li X. et al (2020) [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]  </td><td rowspan=\"1\" colspan=\"1\">China  </td><td rowspan=\"1\" colspan=\"1\">Retrospective Cohort Study  </td><td rowspan=\"1\" colspan=\"1\">269 severe COVID-19 inpatients  </td><td rowspan=\"1\" colspan=\"1\">56.9  </td><td rowspan=\"1\" colspan=\"1\">43.1  </td><td rowspan=\"1\" colspan=\"1\">To evaluate the severity on admission, complications and outcomes of COVID-19 patients  </td><td rowspan=\"1\" colspan=\"1\">Multivariable Cox proportional hazards regression analysis showed that male sex is a risk factor (adjusted HR, 1.7; 95% CI, 1.0-2.8). Other risk factors identified: Older age, leukocytosis, hyperglycaemia, high lactate dehydrogenase, high corticosteroid dose, cardiac injury.  </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Shi Y.et al (2020) [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]      </td><td rowspan=\"1\" colspan=\"1\">China  </td><td rowspan=\"1\" colspan=\"1\">Retrospective Cohort study   </td><td rowspan=\"1\" colspan=\"1\">49 severe COVID-19 inpatients  </td><td rowspan=\"1\" colspan=\"1\">73.5  </td><td rowspan=\"1\" colspan=\"1\">26.5  </td><td rowspan=\"1\" colspan=\"1\">To explore host risk factors. To establish a score system to identify high risk patients  </td><td rowspan=\"1\" colspan=\"1\">Out of the 49 severe patients, 36 were male and 13 were female. There were significantly more males among the severe cases (P=0.003). On multivariate analysis, male sex is associated with severe disease at admission (OR 3.68 [95% CI 1.75–7.75], P = 0.001).</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Liu Y. (2020) [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>]      </td><td rowspan=\"1\" colspan=\"1\">China  </td><td rowspan=\"1\" colspan=\"1\">Retrospective Cohort study  </td><td rowspan=\"1\" colspan=\"1\">245 COVID-19 patients  </td><td rowspan=\"1\" colspan=\"1\">46.5  </td><td rowspan=\"1\" colspan=\"1\">53.5  </td><td rowspan=\"1\" colspan=\"1\">To investigate whether neutrophil-to-lymphocyte ratio (NLR) can serve as a predictor of hospital mortality  </td><td rowspan=\"1\" colspan=\"1\">The odd ratio for mortality was 1.10 in males (CI 1.02-1.10; P=0.016). Sensitivity analysis was used to convert NLR into a categorical tertile variable. Males made up 61% of tertile 3 (most severe tertile). NLR is an independent risk factor for mortality especially in males. </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\">Jin J. et al (2020) [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]     </td><td rowspan=\"2\" colspan=\"1\">China  </td><td rowspan=\"2\" colspan=\"1\">Retrospective Cohort study   </td><td rowspan=\"1\" colspan=\"1\">43 case series hospitalised patients     </td><td rowspan=\"1\" colspan=\"1\">51.2  </td><td rowspan=\"1\" colspan=\"1\">48.8  </td><td rowspan=\"2\" colspan=\"1\">To compare the severity and mortality between male and female patients with COVID-19   </td><td rowspan=\"1\" colspan=\"1\">Males and females had the same susceptibility. Male cases were generally more serious than female (P = 0.035). Males were more prone to dying (χ2 test, P = 0.016). 37 patients died, 70.3% were male, 29.7% were female.  </td></tr><tr><td rowspan=\"1\" colspan=\"1\">37 cases from public data set of the first patients who died of COVID-19 and 1019 who survived     </td><td rowspan=\"1\" colspan=\"1\">50.8  </td><td rowspan=\"1\" colspan=\"1\">49.2  </td><td rowspan=\"1\" colspan=\"1\">The number of males who died from COVID-19 is 2.4 times that of females (70.3 vs. 29.7%, P = 0.016).     </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Zeng F. et al (2020) [<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>]     </td><td rowspan=\"1\" colspan=\"1\">China  </td><td rowspan=\"1\" colspan=\"1\">Retrospective Cohort study   </td><td rowspan=\"1\" colspan=\"1\">331 COVID-19 inpatients  </td><td rowspan=\"1\" colspan=\"1\">38.4  </td><td rowspan=\"1\" colspan=\"1\">61.6  </td><td rowspan=\"1\" colspan=\"1\">To investigate if there is a difference in serum IgG antibody between males and females  </td><td rowspan=\"1\" colspan=\"1\">IgG antibody tended to be stronger in female patients in the early phase of infection. In female patients, the concentration of COVID-19 IgG antibody continuously increased from mild to severe status and then decreased in recovering patients. In male patients, the IgG antibody rose from mild to general status and then decreased from general status patients to recovering patients. Females patients generated a high level of COVID-19 IgG antibody relative to male patients in severe status. Female level was higher in week 2-4 but after week 4 there was no longer a difference between the sexes.</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Li M. et al (2020) [<xref rid=\"REF16\" ref-type=\"bibr\">16</xref>]      </td><td rowspan=\"1\" colspan=\"1\">China  </td><td rowspan=\"1\" colspan=\"1\">Retrospective Cohort study  </td><td rowspan=\"1\" colspan=\"1\">31 GTEx normal tissues     </td><td rowspan=\"1\" colspan=\"1\"> N/A </td><td rowspan=\"1\" colspan=\"1\"> N/A </td><td rowspan=\"1\" colspan=\"1\">To investigate the difference in ACE2 expression in different human tissue to understand COVID-19 mechanism of infection  </td><td rowspan=\"1\" colspan=\"1\">No significant difference found in ACE2 expression between males and females. Negative correlation found between ACE2 and CD8<sup>+</sup> cells, interferon response and B cells in female lungs. Positive correlation found between ACE2 and CD8<sup>+</sup>, interferon response and B cells in male lungs.  </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Docherty A. et al (2020) [<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>]    </td><td rowspan=\"1\" colspan=\"1\">UK  </td><td rowspan=\"1\" colspan=\"1\">Observational Cohort Study  </td><td rowspan=\"1\" colspan=\"1\">20,133 COVID in-patients     </td><td rowspan=\"1\" colspan=\"1\">60  </td><td rowspan=\"1\" colspan=\"1\">40  </td><td rowspan=\"1\" colspan=\"1\">To characterise the clinical features of patients admitted to hospital with COVID-19 and explore risk factors associated with mortality.  Independent risk factors for mortality were increasing age, male sex, and chronic comorbidity, including obesity.    </td><td rowspan=\"1\" colspan=\"1\">Multivariable Cox proportional hazards ratio model: Female sex was associated with lower mortality (0.81 hazard ratio, CI (95%) 0.75 to 0.86, P<0.001).     </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Fagone P. et al (2020) [<xref rid=\"REF18\" ref-type=\"bibr\">18</xref>]    </td><td rowspan=\"1\" colspan=\"1\">Italy</td><td rowspan=\"1\" colspan=\"1\">Genetic Study</td><td rowspan=\"1\" colspan=\"1\">134 healthy lung tissue samples  </td><td rowspan=\"1\" colspan=\"1\">70</td><td rowspan=\"1\" colspan=\"1\">30</td><td rowspan=\"1\" colspan=\"1\">Aimed to identify a specific gene signature characterising SARS-COV-2 infection, employing gene term enrichment analysis on the differentially expressed genes.   </td><td rowspan=\"1\" colspan=\"1\">Gene term enrichment analysis for the 94 upregulated genes identified three significantly altered pathways upon SARS-CoV-2 infection: “cytokine-mediated signalling pathway”, “IL-17 signalling pathway”, and “defence response to other organism”. Comparison of transcriptomic profile of lung tissue from healthy women and men revealed female lung tissues has a more similar phenotype to that induced upon SARS-CoV-2 infection than identical tissues from men.  </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Rastrelli G. et al (2020) [<xref rid=\"REF19\" ref-type=\"bibr\">19</xref>]     </td><td rowspan=\"1\" colspan=\"1\">Italy  </td><td rowspan=\"1\" colspan=\"1\">Case Series  </td><td rowspan=\"1\" colspan=\"1\">31 male COVID-19 inpatients recovered in Respiratory Intensive care [RICU]).  </td><td rowspan=\"1\" colspan=\"1\">100  </td><td rowspan=\"1\" colspan=\"1\">0  </td><td rowspan=\"1\" colspan=\"1\">To estimate the association between Testosterone level and COVID-19 clinical outcomes (outlined as conditions requiring transfer to higher or lower intensity of care).   </td><td rowspan=\"1\" colspan=\"1\">Total Testosterone (TT) and calculated Free Testosterone (cFT) showed a significant decline according to worsening outcomes. Linear regressions showed that for each nmol/L decrease in TT and 10 pmol/L decrease in cFT, the probability of worse clinical outcomes increased [T (p= 0.017), cFT (p=0.007)]. Lower baseline levels of TT and cFT levels predict poor mortality in COVID-19 infected males admitted to Respiratory Intensive Care Unit (RICU).</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Richardson S. et al (2020) [<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>]    </td><td rowspan=\"1\" colspan=\"1\">USA  </td><td rowspan=\"1\" colspan=\"1\">Case Series  </td><td rowspan=\"1\" colspan=\"1\">5700 COVID-19 in-patients  </td><td rowspan=\"1\" colspan=\"1\">60.3  </td><td rowspan=\"1\" colspan=\"1\">39.7  </td><td rowspan=\"1\" colspan=\"1\">To describe the clinical characteristics and outcomes of patients with COVID-19.  </td><td rowspan=\"1\" colspan=\"1\">Mortality rates were higher for male compared with female patients at every 10-year age interval older than 20 years. Mortality was 0% (0/20) for male and female patients younger than 20 years. </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Suleyman G. et al (2020) [<xref rid=\"REF21\" ref-type=\"bibr\">21</xref>]      </td><td rowspan=\"1\" colspan=\"1\">USA  </td><td rowspan=\"1\" colspan=\"1\">Case Series  </td><td rowspan=\"1\" colspan=\"1\">463 COVID-19 in-patients  </td><td rowspan=\"1\" colspan=\"1\">44.1  </td><td rowspan=\"1\" colspan=\"1\">55.9  </td><td rowspan=\"1\" colspan=\"1\">To describe clinical characteristics and outcomes of COVID-19 patients. To perform a comparative analysis of hospitalised and ambulatory patient populations  </td><td rowspan=\"1\" colspan=\"1\">Male sex was independently associated with ICU admission (OR= 2.0, P=0.006) and significantly associated with mortality (OR=1.8, P= 0,03). Multivariable analysis of patient characteristics and need for mechanical ventilation in ICU showed high association with male sex (OR=2.9, P<0.001).  </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Grasselli G. et al (2020) [<xref rid=\"REF22\" ref-type=\"bibr\">22</xref>]  </td><td rowspan=\"1\" colspan=\"1\">Italy</td><td rowspan=\"1\" colspan=\"1\">Case Series</td><td rowspan=\"1\" colspan=\"1\">1591 COVID-19 in-patients admitted to ICU  </td><td rowspan=\"1\" colspan=\"1\">82</td><td rowspan=\"1\" colspan=\"1\">18</td><td rowspan=\"1\" colspan=\"1\">To characterise patients requiring ICU admission from COVID-19  </td><td rowspan=\"1\" colspan=\"1\">The majority of critically ill patients admitted to ICU were older men. 82% of COVID-19 inpatients admitted to ICU from February 20<sup>th </sup>to March 8<sup>th</sup> 2020 were male. The median age of patients admitted to ICU was 63 years old.   </td></tr></tbody></table></table-wrap><p>Sex and Mortality From COVID-19  </p><p>Ten studies from the database search observed higher risk of mortality amongst males compared to females. Eight studies found male sex to be significantly associated with increased risk of mortality from COVID-19. One study found no significant association between male sex and mortality after adjusting for confounders. Figure <xref ref-type=\"fig\" rid=\"FIG2\">2</xref> indicates which studies have observed an increased risk of mortality in males and those in which this association is significant.  </p><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>Male sex and mortality from COVID-19</title><p>This figure shows which of the included studies observed an increased risk of mortality due to male sex, and which studies observed a significant association (p<0.05).  </p></caption><graphic xlink:href=\"cureus-0012-00000010114-i02\"/></fig><p>Risk of Bias Assessment Results</p><p>Table <xref rid=\"TAB2\" ref-type=\"table\">2</xref> summarises the risk of bias results assessed using the NIH quality assessment tool [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>].</p><table-wrap id=\"TAB2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><title>Results of the National Institutes of Health Quality Assessment Tool for Cohort, Cross-Sectional Studies and Case series [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>] for the 16 inclusion studies.</title><p>Cohort and Cross-Sectional Studies: 1. Was the research question or objective in this paper clearly stated? 2. Was the study population clearly specified and defined? 3. Was the participation rate of eligible persons at least 50%? 4. Were all the subjects selected or recruited from the same or similar populations? Were inclusion and exclusion criteria for being in the study prespecified and applied uniformly? 5. Was a sample size justification, power description, or variance and effect estimates provided? 6. Were the exposure(s) of interest measured prior to the outcome(s) being measured? 7. Was the timeframe sufficient so that one could reasonably expect to see an association between exposure and outcome if it existed? 8. For exposures that can vary in amount or level, did the study examine different levels of the exposure as related to the outcome? 9. Were the exposure measures (independent variables) clearly defined, valid, reliable, and implemented consistently? 10. Was the exposure(s) assessed more than once over time? 11. Were the outcome measures (dependent variables) clearly defined, valid, reliable, and implemented consistently across all study participants? 12. Were the outcome assessors blinded to the exposure status of participants? 13. Was loss to follow-up after baseline 20% or less? 14. Were key potential confounding variables measured and adjusted statistically for their impact on the relationship between exposure(s) and outcome(s)? </p><p>Case Series: 1. Was the study question or objective clearly stated? 2. Was the study population clearly and fully described, including a case definition? 3. Were the cases consecutive? 4. Were the subjects comparable? 5. Was the intervention clearly described? 6. Were the outcome measures clearly defined, valid, reliable, and implemented consistently across all study participants? 7. Was the length of follow-up adequate? 8. Were the statistical methods well-described? 9. Were the results well-described? </p></caption><table frame=\"hsides\" rules=\"groups\"><colgroup span=\"1\"><col span=\"1\"/></colgroup><tbody><tr style=\"background-color:#ccc\"><td colspan=\"15\" rowspan=\"1\">NIH Risk of Bias Assessment</td></tr><tr><td colspan=\"15\" rowspan=\"1\">Cohort and Cross-Sectional Studies</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Reference</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">8</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">11</td><td rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\">13</td><td rowspan=\"1\" colspan=\"1\">14</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Qin L. et al </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NR</td><td rowspan=\"1\" colspan=\"1\">Y</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Asfahan S. et al  </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NR</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Borobia AM. et al </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NR</td><td rowspan=\"1\" colspan=\"1\">Y</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Yang X. et al  </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NR</td><td rowspan=\"1\" colspan=\"1\">Y</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Li X. et al  </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NR</td><td rowspan=\"1\" colspan=\"1\">Y</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Shi Y. et al  </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NR</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">N</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Liu Y. et al  </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Jin J. et al </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Zeng F. et al  </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">NR</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NR</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Li M. et al </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NR</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">NR</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Docherty A. et al   </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NR</td><td rowspan=\"1\" colspan=\"1\">Y</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Fagone P. et al </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">NR</td></tr><tr><td colspan=\"15\" rowspan=\"1\">Case Series</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Reference</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">8</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Rastrelli G. et al</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Richardson S. et al  </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Suleyman G. et al </td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">NA</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Grasselli G. et al</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\">Y</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td colspan=\"15\" rowspan=\"1\">Key:  Y = Yes, N = No,  NA = Not applicable,  NR = Not reported</td></tr></tbody></table></table-wrap><p>Discussion</p><p>We undertook a comprehensive search of the literature concerning sex-disaggregated mortality from COVID-19 in order to summarise the potential underlying reasons for the disparity in mortality rate. We found that published evidence suggests the female sex has a protective role against COVID-19 mortality. Main reasons for this finding include the higher levels of the circulating form of ACE2 in females, the immunostimulatory effect of female sex hormones, more rapid clearance of pathogens by the female immune system and females tending to display disease-preventing behaviours.  </p><p>Sex Differences in ACE2 Expression</p><p>Differences in angiotensin-Converting Enzyme 2 (ACE2) expression between sexes is thought to contribute to the higher male mortality rate. ACE2 degrades Angiotensin II into Angiotensin 1-7, counteracting the Renin-Angiotensin-System (RAS) axis. This reduces the effects of the RAS axis which usually increases blood pressure, sympathetic tone, vasoconstriction, inflammation, and fibrosis. ACE2 also serves as the primary receptor for SARS-CoV-2 cellular invasion [<xref rid=\"REF23\" ref-type=\"bibr\">23</xref>]. The viral spike protein contains the S1 domain, which serves as a receptor-binding portion, and the S2 domain which facilitates cellular-viral fusion [<xref rid=\"REF24\" ref-type=\"bibr\">24</xref>,<xref rid=\"REF25\" ref-type=\"bibr\">25</xref>]. The high affinity of SARS-CoV-2 for the ACE2 receptor facilitates viral spread between person to person [<xref rid=\"REF26\" ref-type=\"bibr\">26</xref>]. It is also thought that SARS-CoV-2 infection downregulates ACE2 expression, reducing its protective role and explaining the progression of patients into ARDS [<xref rid=\"REF23\" ref-type=\"bibr\">23</xref>].  </p><p>ACE2 is expressed on the PAR region of the X chromosome, which has a greater chance of escaping X chromosomal inactivation [<xref rid=\"REF27\" ref-type=\"bibr\">27</xref>]. ACE2 is also upregulated by oestrogen leading to disparity in ACE2 expression in some organs between the sexes [<xref rid=\"REF27\" ref-type=\"bibr\">27</xref>,<xref rid=\"REF28\" ref-type=\"bibr\">28</xref>]. The paradox of ACE2 upregulation yet lower female mortality can be explained by a few theories. There are two types of ACE2: the membrane-bound, which provides the viral entry point, and the circulating which has a cardiovascular protective function. It is thought that females express more of the circulating ACE2 providing protection against disease progression into ARDS [<xref rid=\"REF29\" ref-type=\"bibr\">29</xref>].   </p><p>Vikse et al. suggest that the testes may serve as a reservoir for SARS-CoV-2, delaying viral clearance and increasing the likelihood of systemic tissue damage. The high levels of ACE2 expression and the immune-privileged nature of this organ concur with this theory [<xref rid=\"REF30\" ref-type=\"bibr\">30</xref>]. It is also thought that amino acid substitutions can influence viral S1-ACE2 interaction and viral infectivity. Due to hemizygosity in males, carrying a viral-boosting allelic variant of ACE2 may lead to increased susceptibility to severe disease [<xref rid=\"REF31\" ref-type=\"bibr\">31</xref>,<xref rid=\"REF32\" ref-type=\"bibr\">32</xref>]. Li et al. propose that increased male mortality can be attributed to the increased likelihood of a cytokine storm in the lungs, accelerating progression into ARDS. They found a positive correlation between ACE2 expression and immune cell levels (Natural Killer (NK) cells, CD8+ cells) in male lung tissue whereas the opposite was found in females [<xref rid=\"REF16\" ref-type=\"bibr\">16</xref>].  </p><p>Role of Sex Hormones</p><p>Previous literature shows that differences in sex hormones impact the immune system and therefore may play a role in SARS-CoV-2 clearance. It is thought that testosterone (T) has both protective and adverse effects on mortality risk.   </p><p>Low levels of T appear to be linked with increased susceptibility of respiratory diseases [<xref rid=\"REF33\" ref-type=\"bibr\">33</xref>]. Rastrelli et al. demonstrate that low levels of T and circulating free testosterone (cFT) are predictors for adverse outcomes and mortality from COVID-19 [<xref rid=\"REF19\" ref-type=\"bibr\">19</xref>]. This concurs with existing literature in which an association between hypogonadism and proinflammatory cytokine levels is observed [<xref rid=\"REF33\" ref-type=\"bibr\">33</xref>,<xref rid=\"REF34\" ref-type=\"bibr\">34</xref>]. Severe infections are also associated with a reduction in numbers of CD4<sup>+</sup> T cells, CD8<sup>+</sup> T cells, B cells, and NK cells. The presence of androgen receptors (AR) on these cells suggests that T is important in their function [<xref rid=\"REF34\" ref-type=\"bibr\">34</xref>].  </p><p>In addition to this, T plays a complex role in coagulation which could affect male mortality rate. Intravascular thrombosis and endothelial dysfunction complicate COVID-19 prognosis. Published evidence indicates this occurs more frequently in males than females [<xref rid=\"REF35\" ref-type=\"bibr\">35</xref>].  T augments activation and aggregation of platelets by increasing platelet expression of thromboxane A2 receptors [<xref rid=\"REF36\" ref-type=\"bibr\">36</xref>].  In contrast, a negative correlation between serum T levels and platelet reactivity has been discovered by an ex vivo study [<xref rid=\"REF37\" ref-type=\"bibr\">37</xref>]. T enhances the production of endothelial nitric oxide, a potent vasodilator and inhibitor of platelet recruitment. Mean platelet volume, a biological indicator of platelet activation, is seen to be increased in hypogonadal males [<xref rid=\"REF38\" ref-type=\"bibr\">38</xref>]. Therefore, it could be hypothesised that T protects males against new thrombotic events in COVID-19, an effect that is lost through hypogonadism [<xref rid=\"REF33\" ref-type=\"bibr\">33</xref>].   </p><p>T has a cardioprotective role and promotes myocardial health. Thus, males with hypogonadism are predisposed to increased cardiovascular risk from COVID-19. T is vital in regulating glucose and maintaining favourable lipid metabolism [<xref rid=\"REF35\" ref-type=\"bibr\">35</xref>]. Furthermore, being a rapid onset vasodilator, T reduces blood pressure by blocking calcium channel opening. Males with cardiovascular diseases (CVD) tend to have low serum T levels [<xref rid=\"REF39\" ref-type=\"bibr\">39</xref>]. This further illustrates the importance of T in protecting against chronic CVD, as well as acute cardiac injury, which is typically associated with severe COVID-19 disease [<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>,<xref rid=\"REF33\" ref-type=\"bibr\">33</xref>,<xref rid=\"REF40\" ref-type=\"bibr\">40</xref>].</p><p>Although hypogonadism appears to be a risk factor for mortality, a contradictory ‘Testosterone driven COVID-19’ theory exists [<xref rid=\"REF28\" ref-type=\"bibr\">28</xref>]. Transmembrane Protease Serine 2 (TMPRSS2) cleaves the viral S protein at two sites allowing penetration changes on which viral entry into cells depends. It is thought that increased male mortality could be attributed to the androgen regulation of TMPRSS2. There is discourse in the literature as some papers find that there is no significant difference in TMPRSS2 expression in the lungs between the sexes [<xref rid=\"REF41\" ref-type=\"bibr\">41</xref>]. However, other papers find that males have significantly higher (P=0.029) expression of TMPRSS2 at the pulmonary level which may lead to viral progression and poorer outcomes [<xref rid=\"REF32\" ref-type=\"bibr\">32</xref>].  </p><p>Oestrogen (E) is thought to have a protective role against COVID-19 mortality in females. Lower female mortality could be attributed to E stimulating immune cell development, namely B cells, leading to humoral anti-viral responses. E receptors, present on various leukocytes induce pro-inflammatory cytokine production such as interleukin (IL)-12, tumor necrosis factor-alpha (TNF-alpha) and chemokine (C-C motif) ligand 2 (CCL2) [<xref rid=\"REF42\" ref-type=\"bibr\">42</xref>]. The activated lymphocytes and alveolar macrophages increase type 1 and 2 interferon (IFN) production, reducing viral load.   </p><p>Scotland et al. suggest that E may also affect leukocyte function. They found that female mice have an increased number of resident T lymphocytes and that their tissue macrophages have a higher density of toll-like receptor (TLR), specifically TLR2 and TLR4; this allows rapid detection and elimination of pathogens [<xref rid=\"REF43\" ref-type=\"bibr\">43</xref>]. Channappanavar et al. also demonstrate E’s protective role as they found oophorectomy or treating female mice with an ER antagonist resulted in increased mortality from SARS-CoV-1, whereas gonadectomy did not affect mortality [<xref rid=\"REF44\" ref-type=\"bibr\">44</xref>]. This finding may also be applicable to SARS-Cov-2, providing a potential explanation for higher male COVID-19 mortality. </p><p>Sex Differences in Immune Regulation </p><p>A study conducted by Zeng et al. highlights that females produce more serum (SARS-CoV-2) IgG in comparison to males in severe disease status [<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>]. TLR7, a pattern recognition receptor, is expressed on the X chromosome and can bypass X chromosomal inactivation [<xref rid=\"REF42\" ref-type=\"bibr\">42</xref>]. Female X chromosomal homozygosity results in a greater gene dosage and expression of TLR7, allowing for stronger antigen detection [<xref rid=\"REF45\" ref-type=\"bibr\">45</xref>]. TLR7 presenting plasmacytoid dendritic cells in females produce more type 1 IFN following ligand stimulation when compared to males [<xref rid=\"REF45\" ref-type=\"bibr\">45</xref>]. In the presence of TLR7, type 1 IFN enhances B cell-mediated immunoglobulin secretion as well as proliferation [<xref rid=\"REF46\" ref-type=\"bibr\">46</xref>]. These biological processes provide an explanation for higher serum IgG in females.  </p><p>Gene term enrichment analysis of the genes upregulated in a SARS-CoV-2 infection in human lung epithelium identify the ‘cytokine-mediated signalling pathway’ as the most significantly altered pathway [<xref rid=\"REF18\" ref-type=\"bibr\">18</xref>]. Qin et al. observe that COVID-19 disease severity is positively correlated with inflammation [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. Following viral invasion of the lungs, aberrant release of inflammatory cytokines (soluble IL-2, IL-6, IL-8, IL-10) and proteins (LDH, ferritin, high-sensitivity CRP [hs-CRP]) damage the alveolar epithelial cell barrier causing oedema and hypoxia leading to ARDS [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>,<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>]. Inflammatory indexes within this cohort are significantly higher in males. This is worth noting as mortality within this cohort was twice as likely in males and this could be as a result of the immunopathogenic damage caused by excess cytokine storms promoting acute lung injury [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>].  </p><p>Gene term enrichment analysis may provide further explanation for these sex-based differences in cytokine expression. Several Differentially Expressed Genes (DEGs) identified upon SARS-CoV-2 infection of human lung epithelium are found to be modulated by sex hormones. Neutrophil chemotactic factor CXCL1 and dendritic cell chemotactic factor CCL20 are significant DEGs upregulated in SARS-CoV-2 infection. Both factors are regulated by AR, providing further evidence for the role of excess cytokine storms observed in males increasing mortality [<xref rid=\"REF18\" ref-type=\"bibr\">18</xref>].   </p><p>SARS-CoV-2 infection is known to result in significant lymphocytopenia, the extent of which differs between the sexes [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>,<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>,<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>,<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>]. Yang et al. observe lymphocytopenia in 80% of their most critically ill patients [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]. Qin et al. similarly note that male COVID-19 patients have a lower overall lymphocyte count compared to females when adjusting for age and comorbidity [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. Additionally, previous research finds that females have higher CD4<sup>+</sup> T cell counts than age-matched males, and after in vitro stimulation females produce higher numbers of activated CD4<sup>+</sup> T cells [<xref rid=\"REF44\" ref-type=\"bibr\">44</xref>]. The greater reserve of CD4<sup>+</sup> lymphocytes, combined with lower risk of lymphocytopenia in SARS-CoV-2 infection, may potentially decrease the risk of mortality from COVID-19 in females. </p><p>Sex Differences in Behaviour</p><p>Behavioural differences are also thought to contribute to the difference in COVID-19 mortality between sexes. Males tend to partake in higher-risk behaviours such as smoking and drinking alcohol; the WHO reports that 40% of males worldwide smoke compared to 9% of females [<xref rid=\"REF47\" ref-type=\"bibr\">47</xref>]. Additionally, it is thought that females are more likely to follow hygiene and preventative routines [<xref rid=\"REF48\" ref-type=\"bibr\">48</xref>].  </p><p>A study by Guan et al. finds an association between disease severity and smoking. Smokers make up a greater proportion of severe COVID-19 patients compared to non-severe patients (16.9% and 11.8%, respectively) [<xref rid=\"REF49\" ref-type=\"bibr\">49</xref>]. However, it is worth noting that in this study sex is not taken into consideration. Bagone et al. observe an association between cigarette smoke and increased heme oxygenase-1 induction (HO-1) of lung fibroblasts in mice. HO-1 is thought to have anti-viral and cytoprotective properties [<xref rid=\"REF50\" ref-type=\"bibr\">50</xref>]. This confounds the previous understanding of the relationship between smoking and SARS-CoV-2 infection. Therefore, further research and clarification are needed to determine the precise mechanisms of this relationship.   </p><p>Strengths and limitations</p><p>Our review has several strengths. The search strategy we implemented contained a comprehensive list of search words resulting in an in-depth analysis of available evidence. We adopted a pragmatic search strategy approach, appropriate for an ongoing pandemic setting, which aligned with PRISMA guidelines and WHO recommendations for rapidly reviewing evidence in the context of emergencies. Papers also underwent an extensive appraisal before being included in our review as two reviewers assessed potential studies while a third reviewer verified this. Although the risk of bias assessment showed most of the studies used had little or no bias, we found that disease severity was measured differently between studies. As a result, comparison of patients between studies must be done carefully. </p><p>There are several limitations to our review. We limited our electronic database search due to the dynamic and rapidly evolving nature of the COVID-19 pandemic, therefore we included publications only in English between January 2020 and June 2020. Our search strategy was also focused solely on sex and COVID-19 mortality; no comparisons were made regarding the role of sex in the SARS and MERS outbreaks. Additionally, nine out of the 16 studies used were conducted in China. In order to comprehensively assess sex differences in the risk of mortality from COVID-19, more studies will need to be conducted worldwide.  </p><p>Sex-disaggregated COVID-19 data published in medical literature is limited however it has become evident that the male sex is an important risk factor for mortality. Further exploration into the impact of sex on this pandemic is required in order to develop targeted therapies, as well as public health policies, and to prevent sex bias in treatment.    </p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>In conclusion, data emerging worldwide suggests that the male sex has a significant role in increasing risk of COVID-19 mortality amongst adult patients. This association may be explained by the findings that males tend to have lower serum IgG antibody generation, decreased CD4<sup>+ </sup>T cell reserves, and lower circulating ACE2 expression when compared to females.  Male sex is also found to be associated with increased disease severity upon hospital admission, higher rates of ICU admission, and increased clinical markers such as lymphopenia and inflammatory indexes. Conversely, female sex is found to play a significant role in lowering risk of mortality from COVID-19.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><ack><p>Author Contributions:\nMK, AA, and KD contributed equally to this systematic review. MK, AA, KD collected and analysed data. The risk of bias assessment of included studies was conducted by AA and KD; MK verified this. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005532</article-id><article-id pub-id-type=\"pmc\">PMC7523742</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10115</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Internal Medicine</subject></subj-group><subj-group><subject>Gastroenterology</subject></subj-group></article-categories><title-group><article-title>Pharmacological Prevention of Post-Endoscopic Retrograde Cholangiopancreatography Pancreatitis: Where Do We Stand Now?</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Ahmad</surname><given-names>Wiqas</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Okam</surname><given-names>Nkechi A</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Torrilus</surname><given-names>Chenet</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rana</surname><given-names>Dibyata</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Khatun</surname><given-names>Mst. Khaleda</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Jahan</surname><given-names>Nusrat</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nInternal Medicine, California Institute of Behavioral Neurosciences & Psychology, Fairfield, USA </aff><author-notes><corresp id=\"cor1\">\nWiqas Ahmad <email>wiqasahmad642@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10115</elocation-id><history><date date-type=\"received\"><day>9</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Ahmad et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Ahmad et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/39291-pharmacological-prevention-of-post-endoscopic-retrograde-cholangiopancreatography-pancreatitis-where-do-we-stand-now\">This article is available from https://www.cureus.com/articles/39291-pharmacological-prevention-of-post-endoscopic-retrograde-cholangiopancreatography-pancreatitis-where-do-we-stand-now</self-uri><abstract><p>Post-endoscopic retrograde cholangiopancreatography pancreatitis (PEP) is the most frequently occurring complication of endoscopic retrograde cholangiopancreatography (ERCP). PEP is associated with significant morbidity and mortality; that is why the prevention of PEP is essential. Pharmacoprevention holds a central position in PEP prophylaxis. The current literature explores the efficacy of various pharmacological agents in preventing PEP, their routes of administration, and the correct administration timing. Data was collected on PubMed using regular keywords, the latter yielded 2077 papers. After applying inclusion and exclusion criteria, 218 papers were selected and screened and 28 studies were finally chosen after the removal of duplicate and irrelevant studies. The selected 28 articles comprised 25 randomized clinical trials and three systematic reviews.</p><p>The study concludes that rectal non-steroidal anti-inflammatory drugs (NSAIDs) administered before ERCP are effective in preventing PEP in high-risk patients. The efficacy of rectal NSAIDs in low to medium risk group is not well established. A combination of rectal NSAIDs and intravenous hydration provides improved prophylaxis against PEP in high-risk patients than NSAIDs alone. Nafamostat, sublingual nitrates, and intravenous hydration are potential alternatives in patients with contraindications to NSAIDs.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>: post ercp pancreatitis</kwd><kwd>post ercp pancreatitis and nsaids</kwd><kwd>pharmacologic prophylaxis in post ercp pancreatitis</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction and background</title><p>Endoscopic retrograde cholangiopancreatography (ERCP) is a procedure that is used in the diagnosis and management of hepatobiliary and pancreatic disorders [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. The most frequently occurring complication of ERCP is pancreatitis, and its incidence is approximately 2 to 7% [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. However, post-endoscopic retrograde cholangiopancreatography pancreatitis (PEP) is severe in 0.1 to 0.5% cases only [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. PEP has significant financial and social repercussions. The annual expenditure of PEP is estimated to be around 0.20 billion USD and the annual fatality is about 0.7% in the United States of America [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. PEP has been described as the development of new pancreatic-type epigastric pain associated with at least three times rise in serum amylase/lipase levels occurring 24 hours post ERCP, with pain sufficiently severe to require hospital admission or prolong the stay of an admitted patient [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. According to the European Society of Gastrointestinal Endoscopy (ESGE), risk factors associated with increased risk of PEP are female gender, suspected sphincter of Oddi dysfunction, prior episode of pancreatitis, prolonged cannulation time, passing guide wire into pancreatic duct more than once and injection of contrast into the pancreatic duct [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>].</p><p>Different postulated factors may act independently or aggregately to trigger PEP, but the end point of all these mechanisms is to activate inflammatory pathways. Targeting these pathways by preventive treatment options like drugs or addressing various technical issues in ERCP can be used to prevent PEP [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. Even after improvements in techniques and expertise of endoscopists, the incidence of PEP is still high. Although pancreatic stent placement is an effective way of decreasing the risk of PEP in high-risk patients, it is difficult to perform, and usually, it is done at the end of ERCP. There is no standard preventive strategy to avoid this complication. That is why more than 35 different drugs have been studied in clinical trials until now [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. Non-steroidal anti-inflammatory drugs (NSAIDs) are the only class of drugs that have been effective in preventing PEP. Though indomethacin administration through the rectal route has been commonly practiced in the past few years, suitable candidates for this drug and appropriate administration timing have not been appropriately established [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>].</p><p>In this review, we aim to explore pharmacological agents that are effective in the prevention of PEP. Although NSAIDs have shown promising results in various studies, it is still unknown whether all NSAIDs are equally effective and whether or not all patients should receive prophylaxis. Additionally, we endeavor to explore other pharmacological agents besides NSAIDs that are useful in PEP prophylaxis. And finally, we will evaluate whether a combination of NSAIDs with potential pharmacological options offers any additional protection against PEP.</p></sec><sec sec-type=\"review\"><title>Review</title><p>Literature was searched in PubMed using regular keywords for data collection. Table <xref rid=\"TAB1\" ref-type=\"table\">1</xref> shows regular keywords and their records.</p><table-wrap id=\"TAB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Showing search results of regular keywords.</title><p>ERCP: endoscopic retrograde cholangiopancreatography, NSAIDs: non-steroidal anti-inflammatory drugs</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Regular keyword</td><td rowspan=\"1\" colspan=\"1\">Total  Records Available</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Post ERCP Pancreatitis</td><td rowspan=\"1\" colspan=\"1\">       1810</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Post ERCP Pancreatitis and NSAIDs</td><td rowspan=\"1\" colspan=\"1\">        190</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Pharmacologic Prophylaxis in Post ERCP Pancreatitis</td><td rowspan=\"1\" colspan=\"1\">          77</td></tr></tbody></table></table-wrap><p>The following inclusion criteria were used in study selection for this review article: human subjects only, studies related to PEP only, papers published in English language and within the last 10 years, only randomized clinical trials (RCTs) and systematic reviews (SR), all studies available in full-text form.</p><p>The following exclusion criteria were used: animal studies, studies published in languages other than English, observational studies, case reports, case series, meta-analysis, non-RCT trials.</p><p>Table <xref rid=\"TAB2\" ref-type=\"table\">2</xref> summarizes results after applying inclusion and exclusion criteria for the regular keyword “Post ERCP Pancreatitis.”</p><table-wrap id=\"TAB2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><title>Applying inclusion /exclusion criteria for selection of studies. </title><p>RCT: randomized clinical trial, ERCP: endoscopic retrograde cholangiopancreatography</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Regular keyword- Post ERCP Pancreatitis             </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Total Records                                               1810</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Inclusion /Exclusion                                </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Humans                                                        1467</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">English Language                                        1352                 </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Published within the last ten years             792  </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">RCTs and Systematic Reviews                    161</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Full Text                                                        158</td></tr></tbody></table></table-wrap><p>Table <xref rid=\"TAB3\" ref-type=\"table\">3</xref> shows results after applying the inclusion/exclusion criteria for the regular keyword “Post ERCP Pancreatitis and NSAIDs.”</p><table-wrap id=\"TAB3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><title>Applying inclusion/exclusion criteria for selection of studies.</title><p>RCT: randomized clinical trial, ERCP: endoscopic retrograde cholangiopancreatography, NSAIDs: non-steroidal anti-inflammatory drugs</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Regular keyword- Post ERCP Pancreatitis and NSAIDs</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Total Records                                              190</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Inclusion /Exclusion</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Humans                                                        160</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">English Language                                        154  </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Published within the last ten years           135  </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">RCTs and Systematic Reviews                   48  </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Full text                                                        48</td></tr></tbody></table></table-wrap><p>Table <xref rid=\"TAB4\" ref-type=\"table\">4</xref> shows results after applying the inclusion/exclusion criteria for the regular keyword “Pharmacologic prophylaxis in Post ERCP pancreatitis.”</p><table-wrap id=\"TAB4\" orientation=\"portrait\" position=\"float\"><label>Table 4</label><caption><title>Applying inclusion/exclusion criteria for selection of studies.</title><p>RCT: randomized clinical trial, ERCP: endoscopic retrograde cholangiopancreatography</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Regular keyword- Pharmacologic prophylaxis in Post ERCP Pancreatitis</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Total Records                                                77</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Inclusion /Exclusion</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Humans                                                         64</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">English Language                                         63  </td></tr><tr><td rowspan=\"1\" colspan=\"1\">RCTs and Systematic Reviews                    12  </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Published within the last 10 years               34</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Full Text                                                         12</td></tr></tbody></table></table-wrap><p>A total of 147 articles were excluded from the study either because they were duplicate studies or did not address the outcome of interest (pharmacological prevention in PEP). After a refined search, 71 articles were considered eligible for inclusion. We reviewed all 71 studies, and excluded 43 studies either because data extraction was not possible, studies were still ongoing and results were not available, or they included both systematic review and meta-analysis in one study. Finally, 28 studies were included, which comprised 25 RCTs and three systematic reviews. Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref> shows the selection process of studies.</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Showing selection process of studies for this review.</title><p>RCT: randomized clinical trial, SR: systematic review</p></caption><graphic xlink:href=\"cureus-0012-00000010115-i01\"/></fig><p>Discussion</p><p>Pharmacological prophylaxis is a crucial component of post-ERCP pancreatitis (PEP) prevention, and a significant number of pharmacological agents have been investigated in PEP prophylaxis. The literature review would explore the efficacy of different pharmacological agents in the prevention of PEP, their route of administration, and the correct timing of administration.</p><p>Non-Steroidal Anti-Inflammatory Drugs (NSAIDs)</p><p>Non-steroidal anti-inflammatory drugs (NSAIDs) are the only class of drugs that has shown significant benefit in PEP prophylaxis. In the current literature review, eight studies explored NSAIDs' role in PEP prevention. Table <xref rid=\"TAB5\" ref-type=\"table\">5</xref> summarizes the main results of these studies.</p><table-wrap id=\"TAB5\" orientation=\"portrait\" position=\"float\"><label>Table 5</label><caption><title>Summarizing results of studies.</title><p>ERCP: endoscopic retrograde cholangiopancreatography,  PEP: post endoscopic retrograde cholangiopancreatography pancreatitis,  NSAIDs: non-steroidal anti-inflammatory drugs.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Author/ year</td><td rowspan=\"1\" colspan=\"1\">Sample Size</td><td rowspan=\"1\" colspan=\"1\">Drug Used</td><td rowspan=\"1\" colspan=\"1\">Route and Timing  Of Drug Administration</td><td rowspan=\"1\" colspan=\"1\">Main Points</td><td rowspan=\"1\" colspan=\"1\">P -value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Andrade-Davila et al.,2015 [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>].</td><td rowspan=\"1\" colspan=\"1\">166</td><td rowspan=\"1\" colspan=\"1\">indomethacin</td><td rowspan=\"1\" colspan=\"1\">Rectal (administered after  ERCP)</td><td rowspan=\"1\" colspan=\"1\">Study was conducted in patients at high risk of PEP.PEP developed in 4.87 % (indomethacin group) vs. 20.23 % (Placebo group) . Rectal indomethacin effectively decreased risk of PEP.</td><td rowspan=\"1\" colspan=\"1\">P=0.01</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Elmunzer et al. ,2012 [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>].</td><td rowspan=\"1\" colspan=\"1\">602</td><td rowspan=\"1\" colspan=\"1\">indomethacin</td><td rowspan=\"1\" colspan=\"1\">Rectal  (administered after ERCP)</td><td rowspan=\"1\" colspan=\"1\">PEP developed in 9.2% (indomethacin group) vs. 16.9 % (placebo group).Rectal indomethacin Significantly reduced PEP development  in patients at high risk of developing PEP.</td><td rowspan=\"1\" colspan=\"1\">P=0.005</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Mansour Ghanaei et al. ,2016 [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>].</td><td rowspan=\"1\" colspan=\"1\">     324</td><td rowspan=\"1\" colspan=\"1\">  naproxen          </td><td rowspan=\"1\" colspan=\"1\">   Rectal (Before ERCP)</td><td rowspan=\"1\" colspan=\"1\">PEP occurred more commonly in the Placebo group than the naproxen group.</td><td rowspan=\"1\" colspan=\"1\"> P<0.01</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Otsuka T et al. ,2012 [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>].</td><td rowspan=\"1\" colspan=\"1\">104</td><td rowspan=\"1\" colspan=\"1\">diclofenac</td><td rowspan=\"1\" colspan=\"1\"> Rectal (Before ERCP)</td><td rowspan=\"1\" colspan=\"1\">PEP developed in 3.9 %( diclofenac group) vs. 18.9 %( control group). The study showed that a low dose of the diclofenac effectively reduced the risk of PEP.</td><td rowspan=\"1\" colspan=\"1\">P=0.017</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kubilian NM et al. ,2015 [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>].</td><td rowspan=\"1\" colspan=\"1\">N/A</td><td rowspan=\"1\" colspan=\"1\">    NSAIDs</td><td rowspan=\"1\" colspan=\"1\">   Rectal (Before/After ERCP)</td><td rowspan=\"1\" colspan=\"1\"> This systematic review concluded that rectal NSAIDs reduce the risk of PEP in the high risk group.</td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Levenick et al. ,2016 [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>].</td><td rowspan=\"1\" colspan=\"1\">449</td><td rowspan=\"1\" colspan=\"1\">indomethacin</td><td rowspan=\"1\" colspan=\"1\">Rectal (during procedure drug was given)</td><td rowspan=\"1\" colspan=\"1\">The study assessed the prophylactic benefit of indomethacin in all patients irrespective of their risk of developing PEP (around 2/3 of the patents were at average risk of PEP.)Incidence of PEP in indomethacin group (7.2 %) and placebo group (4.9 %) indicated that indomethacin in this study did not prevent risk of developing PEP.</td><td rowspan=\"1\" colspan=\"1\">P=0.33</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dobronte Z et al. , 2014 [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>].</td><td rowspan=\"1\" colspan=\"1\">686</td><td rowspan=\"1\" colspan=\"1\">indomethacin</td><td rowspan=\"1\" colspan=\"1\"> Rectal (Before ERCP)</td><td rowspan=\"1\" colspan=\"1\">Incidence of PEP in the control group (6.9%) was not significantly different from the indomethacin group (5.8%). In this study indomethacin did not prove effective in PEP prevention.</td><td rowspan=\"1\" colspan=\"1\">P=0.54</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Mohammad Alizadeh et al. ,2017 [<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>]  </td><td rowspan=\"1\" colspan=\"1\">372</td><td rowspan=\"1\" colspan=\"1\">diclofenac/ indomethacin/naproxen</td><td rowspan=\"1\" colspan=\"1\">Rectal (Before ERCP)</td><td rowspan=\"1\" colspan=\"1\">Incidence rate of PEP was 2.4 % in  the diclofenac group,3.4 % in the indomethacin group and 10.3 % in naproxen group. This study showed that diclofenac and indomethacin are more effective in reducing PEP</td><td rowspan=\"1\" colspan=\"1\">P =0.001</td></tr></tbody></table></table-wrap><p>Many of the RCTs and the systematic analysis we reviewed in this study have shown that NSAIDs are effective in reducing the risk of PEP [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>,<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>-<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>], although a few RCTs have shown that NSAIDs are not useful in PEP prevention [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>,<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>,<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>]. NSAIDs provide improved prophylaxis in patients at high risk of developing PEP, as demonstrated by multiple studies [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>,<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]. The study conducted by Levenick et al. in patients at average risk of ERCP showed that rectal NSAIDs may not have prophylactic benefits in average-risk patients [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]. Among NSAIDs, diclofenac and indomethacin are more effective than naproxen in PEP prophylaxis [<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>]. The administration route is also significant as NSAIDs administered via the rectal route effectively prevents PEP [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>,<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>,<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>,<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. In contrast, the administration of NSAIDs through other routes has not been found useful [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>,<xref rid=\"REF16\" ref-type=\"bibr\">16</xref>,<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>]. There is still some controversy regarding the appropriate timing of NSAIDs administration (before/during/after ERCP). However, the analysis of most of the studies in our review showed that NSAIDs administration before ERCP is the most suitable option. The recommendation from the European Society for Gastrointestinal Endoscopy (ESGE) is that all patients undergoing ERCP should receive prophylactic rectal indomethacin or diclofenac provided they have no contraindications to NSAIDs [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>].</p><p>NSAIDs are cheap, readily available, and highly effective in PEP prevention; therefore, they should be used in all patients at high risk of developing PEP, provided they do not have any conditions that may preclude its use. Rectal NSAIDs, in conjunction with intravenous hydration, are another viable alternative that can be used in high-risk patients. There is little evidence to support the use of prophylactic rectal NSAIDs in low to medium risk patients. Further studies in this regard may be a possible area of research for the future.</p><p>Intravenous Hydration</p><p>Intravenous hydration can prevent PEP by maintaining sufficient perfusion to the pancreas and thereby suppressing the inflammatory cascade within the pancreas [<xref rid=\"REF18\" ref-type=\"bibr\">18</xref>]. Recent evidence indicates that hydration with Ringer's lactate (RL) solution is better than normal saline because RL solution decreases the risk of developing systemic inflammatory response more than normal saline [<xref rid=\"REF19\" ref-type=\"bibr\">19</xref>].</p><p>A systematic review performed by Smeets et al. evaluated the role of hydration in PEP prophylaxis and revealed that intravenous hydration offers some protection against PEP. However, the evidence was not entirely conclusive [<xref rid=\"REF18\" ref-type=\"bibr\">18</xref>]. In 2018, Park et al. conducted an RCT, the results of the study showed that aggressive hydration with Ringer's lactate prevents PEP occurrence in patients with average to high risk [<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>].</p><p>Intravenous hydration is a potential option for patients at high risk of developing PEP, particularly if they also have contraindications to NSAIDs.</p><p>Combination of Rectal NSAIDs and Intravenous Hydration</p><p>Though rectal NSAIDs alone have shown encouraging results in PEP prophylaxis but still PEP occurs despite NSAIDs usage in high-risk patients. A study conducted by Hosseini et al. has demonstrated that a combination of rectal indomethacin with intravenous normal saline significantly reduced the incidence of PEP [<xref rid=\"REF21\" ref-type=\"bibr\">21</xref>]. Another study conducted by Mok et al. revealed that the combination of Ringer’s lactate and rectal indomethacin was also quite effective in preventing PEP in patients at high risk of PEP [<xref rid=\"REF22\" ref-type=\"bibr\">22</xref>].</p><p>Therefore, it seems reasonable that in patients at high risk of PEP, both rectal NSAIDs and intravenous hydration are used.</p><p>Somatostatin</p><p>Somatostatin acts by inhibiting secretions from the pancreas and its role in preventing PEP has been explored in various studies. A study conducted by Bai et al. suggested that somatostatin effectively reduces amylase levels and prevents PEP [<xref rid=\"REF23\" ref-type=\"bibr\">23</xref>], while a systematic review revealed that somatostatin has moderate benefit in PEP prophylaxis [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. Two other studies showed that somatostatin is not useful in preventing PEP [<xref rid=\"REF24\" ref-type=\"bibr\">24</xref>,<xref rid=\"REF25\" ref-type=\"bibr\">25</xref>].</p><p>Currently, somatostatin is not recommended for PEP prevention because there is little evidence that it is useful in reducing the risk of PEP.</p><p>Antioxidants</p><p>A systematic review was conducted to determine the role of antioxidants such as allopurinol, beta carotene, selenite, pentoxifylline, and N-acetyl cysteine in the prevention PEP and concluded that antioxidants are not useful in PEP prophylaxis [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>].</p><p>Protease Inhibitors</p><p>Protease inhibitors have been considered a possible treatment option in acute pancreatitis due to these drugs' ability to block different enzymes and inflammatory cascade in the pancreas [<xref rid=\"REF26\" ref-type=\"bibr\">26</xref>]. Various protease inhibitors like nafamostat and ulinastatin have also been investigated in the prevention of post-ERCP pancreatitis.</p><p>Nafamostat has been found effective in reducing the incidence of PEP [<xref rid=\"REF26\" ref-type=\"bibr\">26</xref>]. Another study done by Park and his team showed that both nafamostat and ulinastatin are equally useful in PEP prophylaxis [<xref rid=\"REF27\" ref-type=\"bibr\">27</xref>]. Kubilian and his team also found in their systematic review that nafamostat effectively prevents PEP [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>].</p><p>Though nafamostat appears promising in PEP prophylaxis, it has not been widely used because it is quite expensive and needs to be administered through the intravenous route.</p><p>Nitrates</p><p>Nitroglycerin is a drug that reduces the sphincter of Oddi pressure by causing smooth muscle relaxation, and it also enhances blood flow to the pancreas [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>].</p><p>Katsinelos et al. found that administering a combination of nitroglycerin and glucagon makes it more likely to selectively cannulate common bile duct and reduce PEP incidence [<xref rid=\"REF28\" ref-type=\"bibr\">28</xref>].</p><p>A systemic review performed by Kubilian and his team showed mixed results; they analyzed seven studies of which three RCTs showed a benefit of nitroglycerin in decreasing PEP while four RCTs did not demonstrate any benefit. The two studies that used sublingual nitrates revealed that it is effective in reducing PEP [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>].</p><p>The evidence for nitrate efficacy is modest and is not recommended for PEP prophylaxis. However, sublingual nitrates offer a potential prophylactic option in those with contraindications to NSAIDs. Further studies in the future will help define the role of sublingual nitrates in PEP prophylaxis clearly.</p><p>Limitations</p><p>The current literature review has few limitations. Only randomized clinical trials and systematic reviews conducted after 2010 were included. Many studies assessed the function of pharmacoprevention in high-risk patients developing PEP so that potential research may be done to investigate the role of pharmacoprevention in low to medium risk groups.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>Pharmacoprevention is an effective method for the prevention of PEP. The review concludes that rectal NSAIDs administered before ERCP are successful in preventing PEP in high-risk populations. The use of NSAIDs by other routes is not effective in PEP prophylaxis. The effectiveness of NSAIDs in patients with low to medium risk is not well established, and this could be a possible area for further investigations. Intravenous hydration with lactated ringer solution provides some prophylaxis, but a combination of rectal NSAIDs and intravenous hydration is a more suitable option for high-risk patients. Possible options are available in patients with contraindications to NSAIDs such as intravenous hydration, nafamostat and sublingual nitrates, but further studies in the future are needed to elucidate the role of these drugs in PEP prophylaxis.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"journal\"><article-title>No benefit of oral diclofenac on post-endoscopic retrograde cholangiopancreatography pancreatitis</article-title><source>Dig Dis 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005537</article-id><article-id pub-id-type=\"pmc\">PMC7523743</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10122</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cardiac/Thoracic/Vascular Surgery</subject></subj-group><subj-group><subject>Internal Medicine</subject></subj-group><subj-group><subject>Pulmonology</subject></subj-group></article-categories><title-group><article-title>Intrapleural Fibrinolytic Therapy Improves Results With Talc Slurry Pleurodesis</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Bellini</surname><given-names>Alyssa</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tarrazzi</surname><given-names>Francisco</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tami</surname><given-names>Catherine</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Patino</surname><given-names>Sanja H</given-names></name><xref ref-type=\"aff\" rid=\"aff-3\">3</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Block</surname><given-names>Mark</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nDepartment of Surgery, University of California Davis, Sacramento, USA </aff><aff id=\"aff-2\">\n<label>2</label>\nDivision of Thoracic Surgery, Memorial Healthcare, Hollywood, USA </aff><aff id=\"aff-3\">\n<label>3</label>\nInternal Medicine, Florida Atlantic University Charles E. Schmidt College of Medicine, Boca Raton, USA </aff><author-notes><corresp id=\"cor1\">\nMark Block <email>mblock@mhs.net</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10122</elocation-id><history><date date-type=\"received\"><day>18</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Bellini et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Bellini et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/38088-intrapleural-fibrinolytic-therapy-improves-results-with-talc-slurry-pleurodesis\">This article is available from https://www.cureus.com/articles/38088-intrapleural-fibrinolytic-therapy-improves-results-with-talc-slurry-pleurodesis</self-uri><abstract><p>Objective</p><p>Talc slurry pleurodesis (TSP) can lead to permanent small loculations. Intrapleural tissue plasminogen activator (tPA) breaks down loculations, and therefore may improve results but may also inhibit pleurodesis. tPA was given with and after talc slurry to promote more uniform talc distribution and eliminate loculations.</p><p>Methods</p><p>Charts were reviewed for patients treated with TSP with or without tPA. Chest x-rays after TSP were compared to chest x-rays before and graded as “worse”, “same”, or “better”. Incidence of need for repeat TSP was recorded.</p><p>Results</p><p>There were 52 patients, eight with bilateral effusions, for a study cohort of 60 effusions. One-third of the effusions were malignant. No patients experienced significant bleeding. Results were better than baseline for 14 (26%) patients given tPA, but not for patients that never received tPA. The addition of tPA 4-6 mg with talc slurry resulted in no patients requiring repeat TSP. When tPA was given after talc slurry, a delay of three days was associated with the lowest incidence of repeat TSP (3/14, 21%).</p><p>Conclusions</p><p>There were no significant complications from tPA use to supplement TSP, and tPA may improve results without interfering with pleurodesis. A prospective trial is warranted.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>talc slurry pleurodesis</kwd><kwd>talc pleurodesis</kwd><kwd>tpa</kwd><kwd>tissue plasminogen activator</kwd><kwd>pleural effusion</kwd><kwd>loculated pleural effusion</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Recurrent pleural effusion is a common clinical problem, with an estimated incidence of 1.5 million per year in the United States [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. If optimal management of the underlying medical condition does not resolve the effusion, then options for palliation are intermittent thoracentesis, indwelling pleural catheter, or pleurodesis. Chemical pleurodesis causes an inflammatory response that produces fibrin adhesion and fibrosis, obliterating the pleural space [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. Of the many agents available for chemical pleurodesis, a Cochrane Review concluded that talc is the agent of choice [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. Bedside pleurodesis (talc slurry) is easier to perform and less costly than operative pleurodesis (talc poudrage), and may yield similar outcomes [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>-<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. A critical concern is that uneven distribution of talc may lead to uneven pleurodesis and a multiloculated effusion [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>-<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>].</p><p>Tissue plasminogen activator (tPA) is a fibrinolytic that has been shown to be effective for drainage of loculated pleural effusions [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>-<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. Based on our favorable experience with intrapleural tPA for management of complex pleural effusions [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>], we started using it to eliminate loculated collections that developed after pleurodesis. To the best of our knowledge, this had not been attempted before. Our initial experience was promising and safe, and therefore, our service started the routine use of tPA to supplement talc slurry pleurodesis (TSP), giving it either with and/or after talc slurry. We hypothesized that administration with talc slurry would improve distribution of talc throughout the pleural space, and that it would break down loculated collections if given after talc slurry. The potential downside of giving it after talc slurry was that it might interfere with effective pleurodesis, and thus, dosing and timing were variable. This report is a retrospective analysis to determine if objective findings support the continued use of tPA to supplement TSP, and if a more rigorous prospective investigation is warranted.</p></sec><sec sec-type=\"materials|methods\"><title>Materials and methods</title><p>Institutional Review Board approval was obtained for this retrospective review. Charts were reviewed for patients treated with TSP for recurring pleural effusions between March 2014 and December 2016. Demographics and clinical data were collected, and imaging results recorded.</p><p>Technique of talc slurry pleurodesis</p><p>All patients had pigtail chest tubes (sizes 8-14 Fr) inserted under image guidance for recurring pleural effusion. TSP was performed by injection of talc slurry (5 mg sterile asbestos-free talc mixed with 20 mg lidocaine 1% in 60 ml normal saline) through the chest tube and then clamping the tube for 6 hours. Beginning in April 2014, tPA (2-6 mg) was also given through the chest tube either with the talc slurry and/or at an interval after. Patients were followed daily for chest tube output and with imaging (chest x-ray and/or chest CT). Management decisions regarding when to remove the chest tube, whether to administer additional tPA, or whether to repeat TSP were made by the clinician based on daily evaluations and did not follow a clearly defined protocol.</p><p>Assessment of results</p><p>Results of TSP were determined based on imaging and the need for repeat pleurodesis. Qualitative outcome assessments (“worse”, “same”, “better”) were made by comparing the end-result imaging (chest x-rays after TSP at the time of chest tube removal) to chest x-rays showing the best lung expansion before starting pleurodesis (after chest tube placement but before TSP). Duration of chest tube drainage was also recorded.</p><p>Statistics</p><p>Statistical analyses were not performed because this is a retrospective review with a high degree of variability in treatment decisions for each patient, and therefore sample sizes for groups with similar treatment are relatively small. Furthermore, the intent of the review is to determine if there is sufficient evidence to warrant a statistically valid prospective evaluation, rather than to definitively evaluate efficacy.</p></sec><sec sec-type=\"results\"><title>Results</title><p>Fifty-seven patients had TSP for recurring pleural effusion, of which five either expired or were discharged to hospice within four days, and thus, were excluded from analysis. Eight of the remaining 52 patients had bilateral TSP for bilateral pleural effusions, yielding a study cohort of 60 interventions. Of these, seven effusions were treated with talc slurry without tPA (“TSP without tPA”), and 53 effusions were treated with talc slurry and tPA (“TSP with tPA”; Table <xref rid=\"TAB1\" ref-type=\"table\">1</xref>). Heart failure and cancer each accounted for slightly more than a third of the effusions. Gender distribution was approximately equal and most effusions (65%) were right-sided. No patients experienced bleeding in response to tPA sufficient to cause hemodynamic instability.</p><table-wrap id=\"TAB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Patient Characteristics</title><p>TSP: talc slurry pleurodesis, tPA: tissue plasminogen activator, CHF: congestive heart failure, ESRD: end-stage renal disease</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Characteristic</td><td rowspan=\"1\" colspan=\"1\">All</td><td rowspan=\"1\" colspan=\"1\">TSP without tPA</td><td rowspan=\"1\" colspan=\"1\">TSP with tPA</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Total cases, n</td><td rowspan=\"1\" colspan=\"1\">60</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">53</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Mean age, years; (range)</td><td rowspan=\"1\" colspan=\"1\">73</td><td rowspan=\"1\" colspan=\"1\">77 (58-90)</td><td rowspan=\"1\" colspan=\"1\">72 (37-99)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Sex, n</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Male</td><td rowspan=\"1\" colspan=\"1\">27 (45%)</td><td rowspan=\"1\" colspan=\"1\">5 (71%)</td><td rowspan=\"1\" colspan=\"1\">22 (42%)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Female</td><td rowspan=\"1\" colspan=\"1\">33 (55%)</td><td rowspan=\"1\" colspan=\"1\">2 (29%)</td><td rowspan=\"1\" colspan=\"1\">31 (58%)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Etiology, n</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">CHF</td><td rowspan=\"1\" colspan=\"1\">22</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">18</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Malignant</td><td rowspan=\"1\" colspan=\"1\">21</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">19</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Cirrhosis</td><td rowspan=\"1\" colspan=\"1\">8</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">7</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">ESRD</td><td rowspan=\"1\" colspan=\"1\">8</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">8</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Sickle cell</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">1</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Laterality, n</td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td><td rowspan=\"1\" colspan=\"1\"> </td></tr><tr><td rowspan=\"1\" colspan=\"1\">Right</td><td rowspan=\"1\" colspan=\"1\">38</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">33</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Left</td><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">6</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Bilateral</td><td rowspan=\"1\" colspan=\"1\">8</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">7</td></tr></tbody></table></table-wrap><p>Results of TSP with and without tPA were assessed by evaluating radiographic results, incidence of pleurodesis failure (as measured by need for repeat talc slurry), and duration of chest tube drainage. The proportion of patients with a satisfactory (“same or better”) radiographic result was similar whether or not tPA was used (72% “Any tPA” vs. 71% “Without tPA”), but tPA use was associated with improvement (“better”) in 26% of cases (Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>), compared to no improvement in cases in which tPA was not used (Table <xref rid=\"TAB2\" ref-type=\"table\">2</xref>). At the same time, tPA use was not associated with a higher incidence of pleurodesis failure, as measured by the need for repeat TSP (57% of cases not receiving tPA compared to 43% of cases receiving tPA). Among the effusions treated with TSP and tPA, the lowest rate of failure was seen in the group that received tPA only after talc slurry (33%), and the highest rate was in the group that received tPA both with and after talc slurry (48%). Although tPA use was associated with a one day longer median duration of chest tube drainage compared to no tPA (nine days vs. eight days), when tPA was given only with talc slurry median duration was shorter (five days). Maximum chest tube duration was much longer when tPA was given (35 days vs. 10 days), reflecting ongoing management with tPA dosing and repeat talc slurry administration in that one patient. The frequency of use of various doses of tPA, from 2-6 mg, is shown in Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>.</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Frequency of use of different doses of tissue plasminogen activator (tPA).</title><p>The dose of tPA given both with the slurry and after the slurry varied from 2-6 mg. The number of times each dose was used is shown, with empty bars indicating doses given with talc slurry, and filled bars doses given after talc slurry.</p></caption><graphic xlink:href=\"cureus-0012-00000010122-i01\"/></fig><table-wrap id=\"TAB2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><title>Results of TSP with or without tPA</title><p>TSP: talc slurry pleurodesis, tPA: tissue plasminogen activator</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td colspan=\"2\" rowspan=\"1\"> </td><td colspan=\"4\" rowspan=\"1\">                        Chest x-ray Result</td><td rowspan=\"2\" colspan=\"1\">  Number needing repeat TSP,   N (%)</td><td rowspan=\"2\" colspan=\"1\">  Chest tube duration (days), Median (range)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Group</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Worse,    (N)</td><td rowspan=\"1\" colspan=\"1\">Same , (N)</td><td rowspan=\"1\" colspan=\"1\">Better,  N(%)</td><td rowspan=\"1\" colspan=\"1\">Same or better, N (%)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Without tPA</td><td rowspan=\"1\" colspan=\"1\">7</td><td rowspan=\"1\" colspan=\"1\">     2</td><td rowspan=\"1\" colspan=\"1\">     5</td><td rowspan=\"1\" colspan=\"1\">0 (0)</td><td rowspan=\"1\" colspan=\"1\">5 (71)</td><td rowspan=\"1\" colspan=\"1\">4 (57)</td><td rowspan=\"1\" colspan=\"1\">8 (3-10)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Any tPA</td><td rowspan=\"1\" colspan=\"1\">53</td><td rowspan=\"1\" colspan=\"1\">     15</td><td rowspan=\"1\" colspan=\"1\">     24</td><td rowspan=\"1\" colspan=\"1\">14 (26)</td><td rowspan=\"1\" colspan=\"1\">38 (72)</td><td rowspan=\"1\" colspan=\"1\">23 (43)</td><td rowspan=\"1\" colspan=\"1\">9 (2-35)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">   Only with Slurry</td><td rowspan=\"1\" colspan=\"1\">15</td><td rowspan=\"1\" colspan=\"1\">     4</td><td rowspan=\"1\" colspan=\"1\">     6</td><td rowspan=\"1\" colspan=\"1\">5 (33)</td><td rowspan=\"1\" colspan=\"1\">11 (73)</td><td rowspan=\"1\" colspan=\"1\">6 (40)</td><td rowspan=\"1\" colspan=\"1\">5 (2-18)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">   Only after Slurry</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">     2</td><td rowspan=\"1\" colspan=\"1\">     5</td><td rowspan=\"1\" colspan=\"1\">2 (22)</td><td rowspan=\"1\" colspan=\"1\">7 (78)</td><td rowspan=\"1\" colspan=\"1\">3 (33)</td><td rowspan=\"1\" colspan=\"1\">9 (5-31)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">   With & After Slurry</td><td rowspan=\"1\" colspan=\"1\">29</td><td rowspan=\"1\" colspan=\"1\">     9</td><td rowspan=\"1\" colspan=\"1\">     13</td><td rowspan=\"1\" colspan=\"1\">7 (24)</td><td rowspan=\"1\" colspan=\"1\">20 (69)</td><td rowspan=\"1\" colspan=\"1\">4 (48)</td><td rowspan=\"1\" colspan=\"1\">12 (5-35)</td></tr></tbody></table></table-wrap><p>To evaluate the hypothesis that giving tPA with talc slurry might improve talc distribution and mesothelial contact, we looked at results in the 15 cases where tPA was given only with talc slurry (Table <xref rid=\"TAB3\" ref-type=\"table\">3</xref>). The lowest dose (2 mg) was associated with a high proportion of satisfactory radiographic results (78%), but also a high incidence of pleurodesis failure (67%). In contrast, the higher doses of 4-6 mg were associated with a high proportion of satisfactory results (67%) and no pleurodesis failures.</p><table-wrap id=\"TAB3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><title>Effect of tPA dose when given with talc slurry</title><p>TSP: talc slurry pleurodesis, tPA: tissue plasminogen activator</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"2\" colspan=\"1\"> tPA Dose (mg)</td><td rowspan=\"2\" colspan=\"1\"> N</td><td colspan=\"4\" rowspan=\"1\">                     Chest x-ray Result</td><td rowspan=\"2\" colspan=\"1\">  Number needing repeat TSP, N (%)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"> Worse, N</td><td rowspan=\"1\" colspan=\"1\"> Same, N</td><td rowspan=\"1\" colspan=\"1\"> Better, N</td><td rowspan=\"1\" colspan=\"1\">Same or better, N (%)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">9</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">7 (78)</td><td rowspan=\"1\" colspan=\"1\">6 (67)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">2 (57)</td><td rowspan=\"1\" colspan=\"1\">0</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">0</td></tr><tr><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">2 (100)</td><td rowspan=\"1\" colspan=\"1\">0</td></tr></tbody></table></table-wrap><p>The concern with giving tPA after talc slurry is that the fibrinolytic effect might interfere with effective pleurodesis, and therefore, we evaluated the effect of timing between talc slurry and tPA on the incidence of TSP failure (Table <xref rid=\"TAB4\" ref-type=\"table\">4</xref>). Without tPA, the failure rate was 57% (Table <xref rid=\"TAB2\" ref-type=\"table\">2</xref>), and when tPA was given only with talc slurry it was 40%. When given after talc slurry, the lowest rate of pleurodesis failure was seen with an interval of three days (21%) (Figure <xref ref-type=\"fig\" rid=\"FIG2\">2</xref> and Figure <xref ref-type=\"fig\" rid=\"FIG3\">3</xref>), while intervals of five days or more were associated with a very high 82% failure rate.</p><table-wrap id=\"TAB4\" orientation=\"portrait\" position=\"float\"><label>Table 4</label><caption><title>Effect of interval between talc slurry and tPA on incidence of repeat TSP</title><p>TSP: talc slurry pleurodesis, tPA: tissue plasminogen activator</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Interval to First Dose of tPA (days)</td><td rowspan=\"1\" colspan=\"1\">N</td><td rowspan=\"1\" colspan=\"1\">Incidence of Repeat TSP N (%)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">0</td><td rowspan=\"1\" colspan=\"1\">15</td><td rowspan=\"1\" colspan=\"1\">6 (40)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\">8</td><td rowspan=\"1\" colspan=\"1\">3 (37)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">1 (33)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">3 (21)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">2</td><td rowspan=\"1\" colspan=\"1\">1 (50)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">≥5</td><td rowspan=\"1\" colspan=\"1\">11</td><td rowspan=\"1\" colspan=\"1\">9 (82)</td></tr></tbody></table></table-wrap><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>TSP + tPA for recurrent malignant pleural effusion.</title><p>A 60-year-old woman 17 years post right upper lobectomy and 12 years post radio-frequency ablation for separate primary lung cancers, presented with a recurrent left malignant pleural effusion (day -2). A pigtail chest tube was inserted on day -1 and TSP performed with tPA, 4 mg, on day 0. The chest tube was removed on day +3. Follow-up imaging showed no recurrence (days +29 and +93).</p><p>TSP: talc slurry pleurodesis, tPA: tissue plasminogen activator</p></caption><graphic xlink:href=\"cureus-0012-00000010122-i02\"/></fig><fig fig-type=\"figure\" id=\"FIG3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><title>tPA after TSP for drainage of developing loculation. </title><p>A 67-year-old man presented with hepatic hydrothorax (day -8). A pigtail chest tube was placed with output >2,000 cc/day. TSP with tPA, 6 mg, was given on day 0. Lateral chest x-ray on day +2 demonstrated accumulating loculation so tPA, 6 mg, was given on day +3. Lateral chest x-ray on day +4 demonstrated resolution of the effusion. Chest tube removed on day +8 with follow-up chest x-ray on day +16 showing resolution.</p><p>TSP: talc slurry pleurodesis, tPA: tissue plasminogen activator</p></caption><graphic xlink:href=\"cureus-0012-00000010122-i03\"/></fig></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>Despite the growing popularity of indwelling pleural catheters for management of recurrent pleural effusion, pleurodesis remains an important option. Patients may not be able or willing to care for an indwelling catheter, or the effusion may be the result of a chronic condition, such as heart, liver or renal failure, in which case spontaneous pleurodesis with an indwelling catheter is unlikely. In these circumstances, chemical pleurodesis may be the best option. Talc has been the preferred agent, and although intraoperative administration as talc poudrage is the traditional technique, good clinical evidence indicates that bedside talc slurry is equally effective [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>-<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. Unfortunately, as of this writing, sterile talc for pleurodesis is no longer available. Nevertheless, the underlying physiology of pleurodesis should be the same regardless of agent, and therefore, strategies to optimize results with TSP are relevant for bedside chemical pleurodesis with other agents.</p><p>An important deterrent to the use of pleurodesis is that results are sometimes suboptimal. Patients often develop residual loculated effusions that can become permanent if not aggressively drained, and occasionally, pleurodesis is incomplete, requiring re-administration of the pleurodesis agent. Several strategies have been used to address these problems. For example, the argument in favor of talc poudrage is that intraoperative administration optimizes delivery of talc throughout the pleural space, minimizing the likelihood of developing loculations [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>-<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]. However, clinical studies do not support that poudrage is any better than slurry [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>-<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. Additionally, from a physiologic standpoint, it seems very likely that as soon as the operation is over, the chest tube is clamped, and the patient is awakened, any fluid remaining in the pleural space and any effusion that accumulates over the ensuing hours will rapidly redistribute the talc, as if it had been injected in a slurry. Another strategy is to frequently change the patient’s position while the chest tube is clamped, hoping that gravity will assist with dissemination of the talc. Although an attractive concept, studies indicate this does not improve results and that pleural circulation distributes an agent independent of positioning [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>-<xref rid=\"REF16\" ref-type=\"bibr\">16</xref>]. Better strategies are needed, and effectively addressing these problems should make pleurodesis a more attractive option.</p><p>Over the past decade, fibrinolytic therapy has proven an effective technique for eliminating pleural fluid loculations [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>-<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. Fibrinolytics have also been given prior to pleurodesis to improve lung expansion and achieve a better result [<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>]. In a randomized controlled study of streptokinase compared to saline before TSP, Saydam and colleagues found that thrombolytic therapy was associated with improved lung expansion by CT imaging, decreased dyspneic symptoms and lower recurrence [<xref rid=\"REF18\" ref-type=\"bibr\">18</xref>]. To the best of our knowledge, there is no report of using thrombolytics with and/or after the chemical pleurodesis agent.</p><p>The good experience we have had with tPA for management of complex pleural effusions led us to begin using tPA to promote drainage of loculated collections that developed after administration of talc slurry. Our early experience was promising and safe. Therefore, we considered the strategy of using tPA with TSP more deliberately and developed two hypotheses. First, best results with chemical pleurodesis are achieved when the sclerosing agent comes in contact with the greatest surface area of the mesothelial lining [<xref rid=\"REF19\" ref-type=\"bibr\">19</xref>,<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>]. Fibrinolytics may enhance this process by breaking down small loculations to improve access of the agent to a greater surface area, and by lysing any fibrin coating that would inhibit direct contact of the agent with mesothelial cells. This hypothesis suggests that administration of tPA with talc slurry, not just before, would improve results. And second, because fibrinolytics effectively break down pleural fluid loculations, administration of tPA as these loculations develop during pleurodesis may contribute to a more uniform result. However, this effect may also inhibit pleurodesis. Pleural mesothelial cells normally produce urokinase and tPA, making the pleural space fibrinolytic. The inflammatory cascade induced by pleurodesis changes the pleural coagulation-fibrinolysis balance and causes the formation of fibrin links between the pleural surfaces. Fibroblasts proliferate in the fibrin mesh and produce collagen, creating a well-vascularized connective tissue bond between the visceral and parietal pleura [<xref rid=\"REF19\" ref-type=\"bibr\">19</xref>-<xref rid=\"REF21\" ref-type=\"bibr\">21</xref>]. This bond eliminates the pleural space, and thus prevents reaccumulation of pleural fluid. Administration of tPA during this process may eliminate developing loculations, but probably also degrades the fibrin mesh and prevents these last steps of pleurodesis from occurring. However, this effect is probably time-sensitive. The inflammation necessary for pleurodesis is likely an ongoing process over many days, so that a brief, properly timed period of fibrinolysis may eliminate developing loculations at a time when inflammation is continuing, allowing subsequent fibrin formation to complete pleurodesis.</p><p>To evaluate the first hypothesis, we looked specifically at cases in which tPA was given only with talc slurry. Results were promising, with some patients realizing a radiographic outcome that was better than before pleurodesis. Furthermore, with higher doses of tPA, no patient required repeat pleurodesis. Although maximum chest tube duration was very long, the median was less than when no tPA was used. The improvement in radiographic result and decreased need for repeat pleurodesis suggest that tPA may improve distribution of the pleurodesis agent and facilitate a more homogeneous pleurodesis.</p><p>To evaluate the second hypothesis, we looked at cases in which tPA was given after talc slurry. These results showed a high proportion of satisfactory radiographic results, with some patients better than before pleurodesis. The incidence of repeat pleurodesis, an indication that the tPA probably interfered with pleurodesis, was low when the tPA was given early after talc slurry (two to three days), but high when given at an interval of five days or more. These findings suggest that if fibrinolytic is given during an early window, loculated collections can be effectively drained while ongoing inflammation will continue the process of pleurodesis.</p><p>There are several other important findings in this review. First, tPA did not precipitate significant bleeding in any patient, either in the pleural space or as a result of systemic absorption, even in patients with malignant effusions. Our experience with these 60 cases is that this treatment is safe. Second, pigtail catheters rather than larger chest tubes were used in all patients, demonstrating that chest tube size is unimportant for effective drainage of the pleural space. And third, duration of chest tube drainage was exceptionally long in a few cases. This is a highly undesirable outcome and likely reflects the ad hoc approach towards chest tube management in these patients.</p><p>The important limitation to this study is that it is a retrospective review of a series of patients treated without a clear, prospective protocol. As a result, there is considerable variability in the dosing and timing of tPA from one patient to the next, in the criteria used to determine if repeat TSP was indicated, and when the chest tube could be removed.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>We believe definitive conclusions about efficacy are not reasonable and that statistical analyses are of questionable value given the variability in the dosing and timing of tPA from one patient to the next, in the criteria used to determine if repeat TSP was indicated, and when the chest tube could be removed. However, we also believe that these findings provide sufficient evidence to conclude that tPA with pleurodesis is safe and may improve results of TSP. As a result, we are undertaking a formal, prospective evaluation using an investigational protocol based on this experience. Unfortunately, talc is no longer available, but the underlying physiology should be the same regardless of agent. In the interval since talc became unavailable, we have been using doxycycline for pleurodesis and are undertaking a prospective evaluation with this agent.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study. Memorial Healthcare System Institutional Review Board issued approval 005764. This minimal risk study meets the criteria for expedited review as outlined in 45 CFR 46.110(b)(1) or 21 CFR 56.110(b)(1). 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005536</article-id><article-id pub-id-type=\"pmc\">PMC7523744</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10121</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Gastroenterology</subject></subj-group><subj-group><subject>General Surgery</subject></subj-group></article-categories><title-group><article-title>Metal Stent Insertion for Malignant Obstruction of a Colostomy</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Wehbeh</surname><given-names>Antonios</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rahal</surname><given-names>Mahmoud</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Fatima</surname><given-names>Hala</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nGastroenterology and Hepatology, Indiana University School of Medicine, Indianapolis, USA </aff><aff id=\"aff-2\">\n<label>2</label>\nInternal Medicine, Indiana University School of Medicine, Indianapolis, USA </aff><author-notes><corresp id=\"cor1\">\nAntonios Wehbeh <email>wehbehantonios@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10121</elocation-id><history><date date-type=\"received\"><day>26</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Wehbeh et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Wehbeh et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/36104-metal-stent-insertion-for-malignant-obstruction-of-a-colostomy\">This article is available from https://www.cureus.com/articles/36104-metal-stent-insertion-for-malignant-obstruction-of-a-colostomy</self-uri><abstract><p>A 47-year-old female with metastatic cervical cancer and diverting colostomy presented with abdominal distention and minimal stool output from her colostomy. A computed tomography (CT) scan revealed a metastatic mass causing partial obstruction at the colostomy level and significant proximal colonic dilation. Her obstruction was relieved by the endoscopic placement of a metal stent through the stoma, with the stent’s distal edge visible externally but not protruding beyond skin level. Two months later, the stent remained patent and did not migrate. This case highlights a viable palliative treatment option for patients who are not operative candidates.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>colostomy</kwd><kwd>colonic obstruction</kwd><kwd>metal stent</kwd><kwd>colonoscopy</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Malignant obstruction of colostomy is a rare occurrence that is traditionally treated with surgical intervention. However, when surgery is not a feasible option, the recommendations for its management are unclear [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. Self-expandable metallic stents (SEMS) have been used for the relief of malignant colonic obstruction [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>], yet only a few reports describing the use of stents for colostomy obstruction are available [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>, <xref rid=\"REF3\" ref-type=\"bibr\">3</xref>, <xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. In this article, we report a case where a colonic stent was successfully placed to relieve malignant stomal obstruction due to metastatic cervical cancer. </p><p>This case report was presented as a poster at the 2019 American College of Gastroenterology (ACG) Annual Meeting, San Antonio, Texas, and it was published as an accepted meeting presentation in The American Journal of Gastroenterology, October 2019, supplement issue.</p></sec><sec sec-type=\"cases\"><title>Case presentation</title><p>A 47-year-old female with a past history of hypertension and metastatic cervical cancer presented with abdominal distention and minimal stool output from her ostomy for two weeks duration. She described having small amounts of liquid stools which gradually decreased to the point of only mucous discharge from her ostomy. She had anorexia, abdominal cramping, and bloating with food intake, but no nausea or vomiting. Three years prior, she was diagnosed with stage IIB cervical cancer after presenting with abnormal vaginal bleeding. Her disease progressed rapidly despite chemotherapy and radiation. It was complicated by rectovaginal fistula for which she underwent laparoscopic diverting colostomy years prior to presentation. The patient had recently noted an enlarging mass near the ostomy site which was biopsied and showed metastatic adenocarcinoma of endocervical origin.</p><p>On presentation, examination revealed a soft but distended abdomen with palpable peristomal subcutaneous masses. A CT scan of the abdomen and pelvis showed a large heterogeneous mass adjacent to the colostomy with both intra-abdominal and extra-abdominal components. The mass was causing compression and partial obstruction at the level of the colostomy with significant proximal colonic dilation measuring up to 8 cm in the cecum and ascending colon (Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>).</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Axial cuts of an abdominal CT scan showing obstruction at the level of the colostomy by a mass (orange arrow), resulting in significant proximal colonic dilation (white arrow) </title></caption><graphic xlink:href=\"cureus-0012-00000010121-i01\"/></fig><p>As a temporizing measure, a red rubber catheter was passed through the stoma to irrigate and decompress the colon. Given the extensive metastatic disease, she was not a candidate for any operative intervention, and subsequently, endoscopic stent placement was pursued.</p><p>Colonoscopy was performed under moderate sedation. Examination showed a severe stenosis 4 cm in length at the surgical stoma which was traversed with the adult colonoscope. A 25 mm x 6 cm covered self-expandable metal stent (SEMS) was successfully placed with the distal edge visible externally but not protruding beyond skin level (Figures <xref ref-type=\"fig\" rid=\"FIG2\">2a</xref>, <xref ref-type=\"fig\" rid=\"FIG2\">2b</xref>).</p><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>Colonoscopy showing severe stenosis at the stoma (a), treated with placement of a covered metal stent (b)</title></caption><graphic xlink:href=\"cureus-0012-00000010121-i02\"/></fig><p>No complications occurred during or after the procedure. After the placement of the stent, her abdominal discomfort resolved. She started to have good stool output and tolerated a regular diet.</p><p>She presented two months later with draining fistula tracts around the ostomy, and a CT scan showed a patent stent still in place without recurrent colonic obstruction (Figures <xref ref-type=\"fig\" rid=\"FIG3\">3a</xref>, <xref ref-type=\"fig\" rid=\"FIG3\">3b</xref>).</p><fig fig-type=\"figure\" id=\"FIG3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><title>Abdominal CT scan showing the metal stent in place, as seen on axial (a) and sagittal cuts (b)</title></caption><graphic xlink:href=\"cureus-0012-00000010121-i03\"/></fig></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>Stoma construction is a common procedure performed in the setting of colon cancer, trauma, diverticulitis, ischemic colitis, inflammatory bowel disease, and fistulas [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. Stenosis and malignant colostomy obstruction are among the complications of stoma construction [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>, <xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. Surgery is usually the traditional treatment of these complications, however, there is limited data available regarding the management of patients who are poor surgical candidates.</p><p>SEMS can be used both as a bridge to surgery or as a palliative measure in patients with a malignant colonic obstruction due to metastatic disease and unresectable colon cancer tumor. There is no data on whether SEMS has lower morbidity or mortality when compared to palliative surgery in this group of patients. There are a few case reports describing the use of either covered or uncovered SEMS for colostomy obstruction, all of which remained patent until the patients’ death 1.5 - 6 months later [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>, <xref rid=\"REF3\" ref-type=\"bibr\">3</xref>, <xref rid=\"REF4\" ref-type=\"bibr\">4</xref>].</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>In conclusion, metal stent insertion for malignant obstruction of colostomy could be a viable palliative treatment option in patients who are not operative candidates. Further studies are needed to assess the effectiveness and safety of metal stents in improving obstructive symptoms compared with palliative surgery.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"journal\"><article-title>Insertion of self expandable metal stent for malignant stomal obstruction in a patient with advanced colon cancer</article-title><source>Clin Endosc</source><person-group><name><surname>Wi</surname><given-names>JO</given-names></name><name><surname>Shin</surname><given-names>SJ</given-names></name><name><surname>Yoo</surname><given-names>JH</given-names></name><etal/></person-group><fpage>448</fpage><lpage>450</lpage><volume>45</volume><year>2012</year><pub-id pub-id-type=\"pmid\">23251899</pub-id></element-citation></ref><ref id=\"REF2\"><label>2</label><element-citation publication-type=\"journal\"><article-title>Stent as bridge to surgery for left-sided malignant colonic obstruction reduces adverse events and stoma rate compared with emergency surgery: results of a systematic review and meta-analysis of randomized controlled trials</article-title><source>Gastrointest Endosc</source><person-group><name><surname>Arezzo</surname><given-names>A</given-names></name><name><surname>Passera</surname><given-names>R</given-names></name><name><surname>Secco</surname><given-names>GL</given-names></name><etal/></person-group><fpage>416</fpage><lpage>426</lpage><volume>86</volume><year>2017</year><pub-id pub-id-type=\"pmid\">28392363</pub-id></element-citation></ref><ref id=\"REF3\"><label>3</label><element-citation publication-type=\"journal\"><article-title>Insertion of a self-expanding metal stent for a stomal stenosis</article-title><source>Endoscopy</source><person-group><name><surname>Kim</surname><given-names>WH</given-names></name><name><surname>Kwon</surname><given-names>CI</given-names></name><name><surname>Kim</surname><given-names>JW</given-names></name><name><surname>Lee</surname><given-names>C</given-names></name></person-group><fpage>143</fpage><lpage>144</lpage><volume>2</volume><year>2012</year></element-citation></ref><ref id=\"REF4\"><label>4</label><element-citation publication-type=\"journal\"><article-title>A metal stent in a colostomy obstruction</article-title><source>GE Port J Gastroenterol</source><person-group><name><surname>Gils</surname><given-names>NAV</given-names></name><name><surname>Cornelissen</surname><given-names>JGHM</given-names></name><name><surname>Tan</surname><given-names>A</given-names></name></person-group><fpage>214</fpage><lpage>215</lpage><volume>25</volume><year>2018</year><pub-id pub-id-type=\"pmid\">29998173</pub-id></element-citation></ref><ref id=\"REF5\"><label>5</label><element-citation publication-type=\"journal\"><article-title>Stoma complications: A multivariate analysis</article-title><source>Am Surg</source><person-group><name><surname>Duchesne</surname><given-names>JC</given-names></name><name><surname>Wang</surname><given-names>YZ</given-names></name><name><surname>Weintraub</surname><given-names>SL</given-names></name><name><surname>Boyle</surname><given-names>M</given-names></name><name><surname>Hunt</surname><given-names>JP</given-names></name></person-group><fpage>961</fpage><lpage>966</lpage><volume>11</volume><year>2002</year><uri xlink:href=\"https://www.ncbi.nlm.nih.gov/pubmed/12455788\">https://www.ncbi.nlm.nih.gov/pubmed/12455788</uri></element-citation></ref><ref id=\"REF6\"><label>6</label><element-citation publication-type=\"journal\"><article-title>Complications of abdominal stoma surgery</article-title><source>Dis Colon Rectum</source><person-group><name><surname>Shellito</surname><given-names>Shellito</given-names></name><name><surname>P.C</surname><given-names>P.C</given-names></name></person-group><fpage>1562</fpage><lpage>1572</lpage><volume>41</volume><year>1998</year><pub-id pub-id-type=\"pmid\">9860339</pub-id></element-citation></ref><ref id=\"REF7\"><label>7</label><element-citation publication-type=\"journal\"><article-title>Stoma complications: a literature overview</article-title><source>Colorectal Dis</source><person-group><name><surname>Shabbir</surname><given-names>J</given-names></name><name><surname>Britton</surname><given-names>DC</given-names></name></person-group><fpage>958</fpage><lpage>964</lpage><volume>12</volume><year>2010</year><pub-id pub-id-type=\"pmid\">19604288</pub-id></element-citation></ref></ref-list></back></article>\n"
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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005534</article-id><article-id pub-id-type=\"pmc\">PMC7523745</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10118</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cardiac/Thoracic/Vascular Surgery</subject></subj-group><subj-group><subject>Cardiology</subject></subj-group><subj-group><subject>Internal Medicine</subject></subj-group></article-categories><title-group><article-title>Effect of Gender on the Outcomes of ST-Elevation Myocardial Infarction at a Tertiary Care Hospital in Riyadh, Saudi Arabia</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Alharbi</surname><given-names>Mohammed S</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Alanazi</surname><given-names>Bander K</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Alquhays</surname><given-names>Ibrahim A</given-names></name><xref ref-type=\"aff\" rid=\"aff-3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Alhamied</surname><given-names>Nawaf A</given-names></name><xref ref-type=\"aff\" rid=\"aff-4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Al Shimemeri</surname><given-names>Abdullah</given-names></name><xref ref-type=\"aff\" rid=\"aff-5\">5</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nMedicine, Qassim University, Buraiydah, SAU </aff><aff id=\"aff-2\">\n<label>2</label>\nMedicine, King Faisal University, Al Ahsa, SAU </aff><aff id=\"aff-3\">\n<label>3</label>\nInternal Medicine, King Faisal University, Al Ahsa, SAU </aff><aff id=\"aff-4\">\n<label>4</label>\nMedicine, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, SAU </aff><aff id=\"aff-5\">\n<label>5</label>\nInternal Medicine: Critical Care, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, SAU </aff><author-notes><corresp id=\"cor1\">\nMohammed S. Alharbi <email>mohammedsaadov@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10118</elocation-id><history><date date-type=\"received\"><day>23</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Alharbi et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Alharbi et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/37878-effect-of-gender-on-the-outcomes-of-st-elevation-myocardial-infarction-at-a-tertiary-care-hospital-in-riyadh-saudi-arabia\">This article is available from https://www.cureus.com/articles/37878-effect-of-gender-on-the-outcomes-of-st-elevation-myocardial-infarction-at-a-tertiary-care-hospital-in-riyadh-saudi-arabia</self-uri><abstract><p>Objective</p><p>This study aimed to evaluate the impact of gender on the outcomes among ST elevation myocardial infarction patients at King Abdulaziz Medical City in Riyadh, Saudi Arabia.</p><p>Methods</p><p>This retrospective study analyzed the data of 900 patients (770 males and 130 females) admitted between January 2016 and December 2018 diagnosed with ST-elevation myocardial infarction (STEMI). We recorded the baseline characteristics, comorbidities, treatment, complications, and mortality for all patients, and compared these data between female and male patients.</p><p>Results</p><p>The baseline characteristics: BMI and age were higher in females and were statistically significant (p = 0.0001). We found a higher incidence of heart failure in females than in males which was statistically significant (p = 0.0010). In addition, the mortality rate was higher in female than in male patients, although this difference was not statistically significant (p = 0.3850).</p><p>Conclusion</p><p>In conclusion, despite the advances in the technology and the use of novel reperfusion therapies females were associated with poorer outcomes after adjustment of the baseline characteristics and risk factors. In other words, heart failure, mitral regurgitation, and arrhythmias were higher in females with significant p values.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>st elevation myocardial infarction</kwd><kwd>myocardial reperfusion</kwd><kwd>complications</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Globally, around 3 million people sustain ST-elevation myocardial infarction (STEMI) annually, while around 4 million people suffer from non-ST elevation myocardial infarction (NSTEMI) [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. The rate of STEMI in men is twice as high as that in women [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. Continuous advancements in reperfusion therapy have resulted in a significant decline in the mortality and related complications in patients with STEMI [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>,<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. Multiple studies have investigated the risk factors of mortality, complications, and prognostic factors after STEMI and the results in some studies showed that female gender may adversely affect the clinical outcome in STEMI and it is associated with higher mortality and more complications [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>,<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. While the others attributed the difference in the baseline characteristics to be the main reason beyond the poorer outcome rather than the gender itself [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>,<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>].</p><p>The structure of the left ventricle differs between the genders, and females are more prone to heart failure due to concentric remodeling despite preserved LV ejection fractions [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>,<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>]. Women show a higher incidence of mechanical failures, such as mitral regurgitation and ventricular septal rupture [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]. In most studies, female patients with STEMI also had a higher age and concurrent disease burden, such as diabetes, hypertension, etc., than males [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>,<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>,<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>,<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>].</p><p>It is not clearly understood whether left ventricular systolic dysfunction after myocardial infarction is responsible for the poorer clinical outcomes and higher rates of complications in women [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>].</p><p>However, other studies did not find a correlation between gender and clinical outcome, they attributed the higher mortality in female patients to a higher age and Killip class at the time of admission, other concurrent diseases, and confounding factors [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>,<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>,<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>]. The health status of female patients at the time of myocardial infarction has also been found to affect the outcomes [<xref rid=\"REF16\" ref-type=\"bibr\">16</xref>].</p><p>Few recent studies have studied gender differences that may affect the prognosis after STEMI in female patients in details. Given the ambiguity in the literature, we need to understand gender-based variations so that tailored therapeutic approaches can be designed. Further, in view of the financial and socio-economic burden of myocardial infarction and coronary disease, it is important to study the mechanisms and the underlying causes that affect clinical outcomes in women. This study aimed to evaluate the impact of gender on the outcomes among ST elevation myocardial infarction patients at King Abdulaziz Medical City in Riyadh, Saudi Arabia.</p></sec><sec sec-type=\"materials|methods\"><title>Materials and methods</title><p>This study was approved by the ethics committee of King Abdullah International Medical Research Center (No. RSS19/038/R) and conformed to the ethical principles outlined in the Declaration of Helsinki. We performed a retrospective chart review of all adult patients (n = 900) with a final diagnosis of STEMI admitted to King Abdulaziz Medical City, Riyadh, Saudi Arabia, between January 2016 and December 2018.</p><p>We extracted the following data from the hospital’s BestCare database using a customized data collection sheet: demographic data such as age, gender, nationality, height, weight, body mass index (BMI). Moreover, data regarding comorbidities such as hypertension, hyperlipidemia, and diabetes were collected as well as risk factors such as the history of smoking, renal failure, chronic obstructive pulmonary disease, and stroke. We also recorded the cardiac troponins, creatine kinase (CK), CK-MB isoenzyme, electrocardiography (ECG) findings, and echocardiographic data which included: left ventricular ejection fraction (LVEF), significant mitral regurgitation, and any other post-myocardial infarction abnormalities. We further noted the details of treatment, such as coronary artery bypass graft, percutaneous coronary intervention, intravenous fibrinolysis, and angioplasty.</p><p>Statistical analysis</p><p>We summarized categorical variables as number (percentage) and numerical variables (continuous variables) as mean and standard deviation (SD). The normality assumptions were assessed for all numerical variables using statistical test. We compared categorical variables using the chi square or Fisher exact test, normally distributed numerical variables with the t test, and other quantitative variables with the Mann-Whitney U test. The differences in baseline characteristics between the genders are expected in the observational studies.</p><p>For the adjustment of these differences, a propensity score was generated for the age, BMI, conservative management, diabetes mellitus (DM), hypertension (HTN), dyslipidemia, renal failure and stroke.</p><p>Multivariate logistic regression was used to find out the relationship between gender and the different complications considered in this study, adjusting for the generated propensity score. The odds ratios (OR) and estimates with the 95% confidence intervals (CI) were reported for the associations. We assessed model fit using the Hosmer-Lemeshow goodness-of-fit test. We considered a P-value of < 0.05 statistically significant and used SAS® software, version 9.4 (SAS Institute Inc., Cary, NC, USA) for all statistical analyses.</p></sec><sec sec-type=\"results\"><title>Results</title><p>The data of 900 patients with STEMI - 770 males and 130 females - were analyzed. Females had a higher mean age; 65.7 ± 13.5 for females vs. 58.9 ± 12.8 years for males (p = 0.0001) and BMI was higher in females compared to males and the results were statistically significant (p = 0.0001) as shown in Table <xref rid=\"TAB1\" ref-type=\"table\">1</xref>. The baseline treatment provided to male and female patients with STEMI is shown in Table <xref rid=\"TAB2\" ref-type=\"table\">2</xref>. No statistically significant differences were observed in the treatment provided except for the conservative line of treatment (p = 0.0308). Table <xref rid=\"TAB3\" ref-type=\"table\">3</xref> presents the results of the statistical analysis for the risk factors for STEMI among male and female patients.</p><table-wrap id=\"TAB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Age and body mass index of patients with STEMI.</title><p>STEMI: ST-elevation myocardial infarction</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Variables</td><td rowspan=\"1\" colspan=\"1\">Male (n = 770) 85.55%</td><td rowspan=\"1\" colspan=\"1\">Female (n = 130) 14.44%</td><td rowspan=\"1\" colspan=\"1\">p-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Age mean ± S.D.</td><td rowspan=\"1\" colspan=\"1\">58.9 ± 12.8</td><td rowspan=\"1\" colspan=\"1\">65.7 ± 13.5</td><td rowspan=\"1\" colspan=\"1\">0.0001</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Body mass index (BMI)</td><td rowspan=\"1\" colspan=\"1\">28.3 ± 5.8</td><td rowspan=\"1\" colspan=\"1\">30.5 ± 6.4</td><td rowspan=\"1\" colspan=\"1\">0.0001</td></tr><tr><td colspan=\"4\" rowspan=\"1\">Mann-Whitney U test is used to calculate the p-value.</td></tr></tbody></table></table-wrap><table-wrap id=\"TAB2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><title>Baseline treatment provided to the patients.</title><p>^ Chi-Square Test; ^^ Fisher's Exact Test.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Baseline treatment</td><td rowspan=\"1\" colspan=\"1\">Male</td><td rowspan=\"1\" colspan=\"1\">Female</td><td rowspan=\"1\" colspan=\"1\">p-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Percutaneous coronary intervention (PCI)</td><td rowspan=\"1\" colspan=\"1\">660 (85.7%)</td><td rowspan=\"1\" colspan=\"1\">107 (82.3%)</td><td rowspan=\"1\" colspan=\"1\">0.1911^^</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Thrombolysis</td><td rowspan=\"1\" colspan=\"1\">78 (10.1%)</td><td rowspan=\"1\" colspan=\"1\">11 (8.5%)</td><td rowspan=\"1\" colspan=\"1\">0.5326^^</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Coronary artery bypass surgery (CABG)</td><td rowspan=\"1\" colspan=\"1\">72 (9.4%)</td><td rowspan=\"1\" colspan=\"1\">8 (6.2%)</td><td rowspan=\"1\" colspan=\"1\">0.2257^^</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Conservative management</td><td rowspan=\"1\" colspan=\"1\">48 (6.2%)</td><td rowspan=\"1\" colspan=\"1\">15 (11.5%)</td><td rowspan=\"1\" colspan=\"1\">0.0308^^</td></tr></tbody></table></table-wrap><table-wrap id=\"TAB3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><title>Risk factors for complications in STEMI in male and female gender.</title><p>^ Chi-Square Test; ^^ Fisher's Exact Test.</p><p>STEMI: ST-elevation myocardial infarction</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Risk factors</td><td rowspan=\"1\" colspan=\"1\">Male (N = 770)</td><td rowspan=\"1\" colspan=\"1\">Female (N = 130)</td><td rowspan=\"1\" colspan=\"1\">p-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Diabetes mellitus</td><td rowspan=\"1\" colspan=\"1\">449 (58.3%)</td><td rowspan=\"1\" colspan=\"1\">104 (79.8%)</td><td rowspan=\"1\" colspan=\"1\">0.0001 ^</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Hypertension</td><td rowspan=\"1\" colspan=\"1\">417 (54.1%)</td><td rowspan=\"1\" colspan=\"1\">104 (79.8%)</td><td rowspan=\"1\" colspan=\"1\">0.0001 ^</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dyslipidemia</td><td rowspan=\"1\" colspan=\"1\">472 (61.2%)</td><td rowspan=\"1\" colspan=\"1\">92 (70.5%)</td><td rowspan=\"1\" colspan=\"1\">0.0103 ^</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Previous ischemic heart disease</td><td rowspan=\"1\" colspan=\"1\">159 (20.6%)</td><td rowspan=\"1\" colspan=\"1\">23 (17.6%)</td><td rowspan=\"1\" colspan=\"1\">0.0726 ^</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Family history</td><td rowspan=\"1\" colspan=\"1\">33 (4.2%)</td><td rowspan=\"1\" colspan=\"1\">8 (5.8%)</td><td rowspan=\"1\" colspan=\"1\">0.1293 ^</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Smoking</td><td rowspan=\"1\" colspan=\"1\">373 (48.4%)</td><td rowspan=\"1\" colspan=\"1\">12 (8.0%)</td><td rowspan=\"1\" colspan=\"1\">0.0001 ^</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Renal failure</td><td rowspan=\"1\" colspan=\"1\">73 (9.5%)</td><td rowspan=\"1\" colspan=\"1\">19 (14.6%)</td><td rowspan=\"1\" colspan=\"1\">0.0267 ^</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Chronic obstructive pulmonary disease</td><td rowspan=\"1\" colspan=\"1\">14 (1.8%)</td><td rowspan=\"1\" colspan=\"1\">3 (2.3%)</td><td rowspan=\"1\" colspan=\"1\">0.2344 ^^</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Stroke</td><td rowspan=\"1\" colspan=\"1\">46 (6.0%)</td><td rowspan=\"1\" colspan=\"1\">12 (9.2%)</td><td rowspan=\"1\" colspan=\"1\">0.0575 ^</td></tr></tbody></table></table-wrap><p>The complications observed in the two groups are shown in Table <xref rid=\"TAB4\" ref-type=\"table\">4</xref>. Heart failure was the most common complication in both genders, but its frequency was higher in females than in males with 54 (41.5%) compared to 280 (27%) with significant p-value (p = 0.0010). Mitral regurgitation was the second most frequent complication in both genders, occurring in 41 (31.5%) of females and 169 (21.9%) of males (p = 0.0201). Arrhythmias were diagnosed in 36 (27.7%) of females and 158 (20.5%) in males (p = 0.768). Mortality was higher in females than males 13 (10%) vs. 59 (7.7%), but this difference was not statistically significant (p = 0.3850).</p><table-wrap id=\"TAB4\" orientation=\"portrait\" position=\"float\"><label>Table 4</label><caption><title>Complications</title><p>-Denominator of the percentage is the total number of patients. ^^Chi-square test/**Fisher Exact test is used to calculate the P-value. $ Propensity score adjusted Logistic regression is used to calculate Odds ratio and p-value. aOR*: Adjusted Odds ratio. Male subjects are considered as reference category.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Complications</td><td rowspan=\"1\" colspan=\"1\">Male (N = 770)</td><td rowspan=\"1\" colspan=\"1\">Female (N = 130)</td><td rowspan=\"1\" colspan=\"1\">p-value</td><td rowspan=\"1\" colspan=\"1\">aOR</td><td rowspan=\"1\" colspan=\"1\">9% CI</td><td rowspan=\"1\" colspan=\"1\">p-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Heart failure</td><td rowspan=\"1\" colspan=\"1\">208 (27.0%)</td><td rowspan=\"1\" colspan=\"1\">54 (41.5%)</td><td rowspan=\"1\" colspan=\"1\">0.0010^^</td><td rowspan=\"1\" colspan=\"1\">1.73</td><td rowspan=\"1\" colspan=\"1\">(1.149, 2.590)</td><td rowspan=\"1\" colspan=\"1\">0.0085<sup>$</sup>\n</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Mitral regurgitation</td><td rowspan=\"1\" colspan=\"1\">169 (21.9%)</td><td rowspan=\"1\" colspan=\"1\">41 (31.5%)</td><td rowspan=\"1\" colspan=\"1\">0.0201^^</td><td rowspan=\"1\" colspan=\"1\">1.34</td><td rowspan=\"1\" colspan=\"1\">(0.874, 2.081)</td><td rowspan=\"1\" colspan=\"1\">0.175</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Tricuspid regurgitation</td><td rowspan=\"1\" colspan=\"1\">232 (30.1%)</td><td rowspan=\"1\" colspan=\"1\">41 (31.5%)</td><td rowspan=\"1\" colspan=\"1\">0.8609^</td><td rowspan=\"1\" colspan=\"1\">1.03</td><td rowspan=\"1\" colspan=\"1\">(0.678, 1.577)</td><td rowspan=\"1\" colspan=\"1\">0.875</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Arrhythmia</td><td rowspan=\"1\" colspan=\"1\">158 (20.5%)</td><td rowspan=\"1\" colspan=\"1\">36 (27.7%)</td><td rowspan=\"1\" colspan=\"1\">0.0768^^</td><td rowspan=\"1\" colspan=\"1\">1.48</td><td rowspan=\"1\" colspan=\"1\">(0.951, 2.328)</td><td rowspan=\"1\" colspan=\"1\">0.081</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Cardiac arrest</td><td rowspan=\"1\" colspan=\"1\">104 (13.5%)</td><td rowspan=\"1\" colspan=\"1\">20 (15.4%)</td><td rowspan=\"1\" colspan=\"1\">0.6009^^</td><td rowspan=\"1\" colspan=\"1\">1.25</td><td rowspan=\"1\" colspan=\"1\">(0.727, 2.175)</td><td rowspan=\"1\" colspan=\"1\">0.411</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Cardiogenic shock</td><td rowspan=\"1\" colspan=\"1\">78 (10.1%)</td><td rowspan=\"1\" colspan=\"1\">14 (10.8%)</td><td rowspan=\"1\" colspan=\"1\">0.8610^^</td><td rowspan=\"1\" colspan=\"1\">1.23</td><td rowspan=\"1\" colspan=\"1\">(0.660, 2.321)</td><td rowspan=\"1\" colspan=\"1\">0.506</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Died</td><td rowspan=\"1\" colspan=\"1\">59 (7.7%)</td><td rowspan=\"1\" colspan=\"1\">13 (10%)</td><td rowspan=\"1\" colspan=\"1\">0.3850^^</td><td rowspan=\"1\" colspan=\"1\">1.16</td><td rowspan=\"1\" colspan=\"1\">(0.596, 2.290)</td><td rowspan=\"1\" colspan=\"1\">0.651</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Heart block</td><td rowspan=\"1\" colspan=\"1\">57 (7.4%)</td><td rowspan=\"1\" colspan=\"1\">8 (6.2%)</td><td rowspan=\"1\" colspan=\"1\">0.5907^^</td><td rowspan=\"1\" colspan=\"1\">0.69</td><td rowspan=\"1\" colspan=\"1\">(0.316, 1.522)</td><td rowspan=\"1\" colspan=\"1\">0.362</td></tr></tbody></table></table-wrap></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>Only a few studies have tried to explain the higher mortality in female patients with STEMI as compared to male patients [<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>-<xref rid=\"REF19\" ref-type=\"bibr\">19</xref>]. The potential reasons suggested by these authors included a delayed response, differences in the medical attention given to female patients as compared to male patients, or in distinct pathophysiological processes related to the female gender [<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>]. In our study, we did not find a statistically significant difference in the mortality between the genders, although females had a higher mortality risk than male patients. As described in previous studies, the women in our study had a higher mean age at the time of presentation than men [<xref rid=\"REF18\" ref-type=\"bibr\">18</xref>,<xref rid=\"REF19\" ref-type=\"bibr\">19</xref>]. Heart failure was more frequent in female than in male patients. Male patients showed higher levels of myocardial injury marker proteins in the blood, such as CK, CK-MB, and troponin C type I. The frequency of previous coronary interventions and findings on ECG did not significantly differ between the genders.</p><p>Similar to other studies, risk factors such as hypertension, diabetes mellitus, and dyslipidemia were more prevalent in females [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>,<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>,<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>,<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]. This higher risk profile might be the cause of the higher incidence rate of complications after STEMI such as heart failure, mitral regurgitation, arrhythmias, and death in females. We believe this is more likely than gender itself being an independent prognostic factor.</p><p>The findings in our study are consistent with those of Ng and Lansky [<xref rid=\"REF19\" ref-type=\"bibr\">19</xref>], Pedersen et al. [<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>], and, more recently Kanic et al. [<xref rid=\"REF21\" ref-type=\"bibr\">21</xref>] who also found a correlation between higher age and more frequent comorbidities in women. Females in our study were older than males at the time of diagnosis, which carries a higher risk of comorbidities. The cardio-protective effect of estrogen before menopause is thought to be the cause of this observation [<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>]. Even though higher rates of smoking have been reported in males, the mortality rate is higher in females [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>,<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>].</p><p>In our study, the treatment modalities were similar in both groups except that conservative management was more frequent in females. The high incidence of mitral regurgitation that we found in female patients was also documented by other authors [<xref rid=\"REF16\" ref-type=\"bibr\">16</xref>]. The significant difference in the incidence of heart failure in our study with 41.5% in females as compared to 27% in males is often attributed to structural differences between the genders [<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>-<xref rid=\"REF20\" ref-type=\"bibr\">20</xref>].</p><p>Study limitations</p><p>As the study design was retrospective in character, the time elapsed between the start of symptoms and beginning of treatment in our patients could not be determined. Moreover, the results of the study is based on one center thus we could not collect a larger sample.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>In conclusion, despite the advances in the technology and the use of novel reperfusion therapies, females were associated with poorer outcomes after adjustment of the baseline characteristics and risk factors. In other words, heart failure, mitral regurgitation, and arrhythmias were higher in females with significant p values. Further studies are needed to support the results of this research as it was a single center study.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study. King Abdullah International Medical Research Center issued approval (RSS19/038/R). After reviewing your submitted research proposal and related documents, the IRB has approved the submission.</p></fn></fn-group><fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group><ack><p>Ramesh Kumar, PhD. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005556</article-id><article-id pub-id-type=\"pmc\">PMC7523746</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10686</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Internal Medicine</subject></subj-group><subj-group><subject>Infectious Disease</subject></subj-group><subj-group><subject>Rheumatology</subject></subj-group></article-categories><title-group><article-title>Rhabdomyolysis in COVID-19: Report of Four Cases</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Singh</surname><given-names>Balraj</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kaur</surname><given-names>Parminder</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mechineni</surname><given-names>Ashesha</given-names></name><xref ref-type=\"aff\" rid=\"aff-3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Maroules</surname><given-names>Michael</given-names></name><xref ref-type=\"aff\" rid=\"aff-4\">4</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nHematology/Oncology, Saint Joseph's University Medical Center, Paterson, USA </aff><aff id=\"aff-2\">\n<label>2</label>\nCardiology, Saint Joseph's University Medical Center, Paterson, USA </aff><aff id=\"aff-3\">\n<label>3</label>\nInternal Medicine, Saint Joseph's University Medical Center, Paterson, USA </aff><aff id=\"aff-4\">\n<label>4</label>\nHematology/Oncology, Saint Joseph's University Medical Center, Paterson, USA </aff><author-notes><corresp id=\"cor1\">\nBalraj Singh <email>bsriar9@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>27</day><month>9</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>9</month><year>2020</year></pub-date><volume>12</volume><issue>9</issue><elocation-id>e10686</elocation-id><history><date date-type=\"accepted\"><day>27</day><month>9</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Singh et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Singh et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/41837-rhabdomyolysis-in-covid-19-report-of-four-cases\">This article is available from https://www.cureus.com/articles/41837-rhabdomyolysis-in-covid-19-report-of-four-cases</self-uri><abstract><p>Coronavirus disease 2019 (COVID-19) is a global public health emergency. COVID-19 is most well known for affecting the respiratory system, although it can also result in several extrapulmonary manifestations. Limited literature is available regarding rhabdomyolysis in COVID-19. We report four cases of rhabdomyolysis in COVID-19 patients. High index of suspicion is required for the appropriate clinical scenario to recognize this life-threatening situation so that complications can be avoided</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>covid 19</kwd><kwd>sars-cov-2 (severe acute respiratory syndrome coronavirus -2)</kwd><kwd>rhabdomyolysis</kwd><kwd>viral myositis</kwd><kwd>coronavirus</kwd><kwd>coronavirus disease</kwd><kwd>atypical presentation</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the coronavirus disease 2019 (COVID-19), has infected millions of people and has caused more than hundreds of thousands of deaths globally. COVID-19 can present with a spectrum of clinical manifestations, including fever, myalgia, cough, dyspnea, and less frequently headache, diarrhea, nausea, and vomiting [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. Although respiratory symptoms predominate, widespread organ-specific manifestations of COVID-19 are increasingly being reported. We report four cases of rhabdomyolysis in COVID-19.</p></sec><sec sec-type=\"cases\"><title>Case presentation</title><p>COVID-19 was diagnosed by nasopharyngeal swab real-time reverse transcription polymerase chain reaction (RT-PCR). Relevant clinical characteristics and laboratory values of the four cases are summarized in Table <xref rid=\"TAB1\" ref-type=\"table\">1</xref>. All patients had elevated creatine kinase at presentation</p><table-wrap id=\"TAB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Clinical characteristics and laboratory values of the four cases</title><p>Reference ranges are as follows: CK 30-223 unit/L, white cell count 4.5-11 K/mm<sup>3</sup>, hemoglobin 12-16 g/dL, platelets 140-440 K/mm<sup>3</sup>, troponin less than 0.03 ng/ml, sodium 135-145 mEq/ L, potassium 3.5-5 mEq/L, phosphorous 2.5-5 mg/dL, BUN 7-23 mg/dL,  creatinine 0.6-1.30 mg/dL, aspartate transaminase 13-39 U/L, alanine transaminase 7-52 U/L, ESR 0-32 mm/hr, CRP less than 10 mg/L, ferritin 12-300 ng/mL, LDH 140-271 U/L, d-dimer less than 0.5, urinalysis blood negative, urinalysis red blood cells 0-3/HPF.</p><p>CK, creatine kinase; BUN, blood urea nitrogen; AST, aspartate transaminase; ALT, alanine transaminase; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; LDH, lactate dehydrogenase; ND, not done; HPF, high power field</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Variable</td><td rowspan=\"1\" colspan=\"1\">Case 1</td><td rowspan=\"1\" colspan=\"1\">Case 2</td><td rowspan=\"1\" colspan=\"1\">Case 3</td><td rowspan=\"1\" colspan=\"1\">Case 4</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Age</td><td rowspan=\"1\" colspan=\"1\">67</td><td rowspan=\"1\" colspan=\"1\">39</td><td rowspan=\"1\" colspan=\"1\">43</td><td rowspan=\"1\" colspan=\"1\">70</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Sex</td><td rowspan=\"1\" colspan=\"1\">Male</td><td rowspan=\"1\" colspan=\"1\">Male</td><td rowspan=\"1\" colspan=\"1\">Male</td><td rowspan=\"1\" colspan=\"1\">Male</td></tr><tr><td rowspan=\"1\" colspan=\"1\">History of alcohol abuse/substance abuse/trauma/exertion</td><td rowspan=\"1\" colspan=\"1\">None</td><td rowspan=\"1\" colspan=\"1\">None</td><td rowspan=\"1\" colspan=\"1\">None</td><td rowspan=\"1\" colspan=\"1\">None</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Influenza/parainfluenza/enterovirus/adenovirus</td><td rowspan=\"1\" colspan=\"1\">Negative</td><td rowspan=\"1\" colspan=\"1\">Negative</td><td rowspan=\"1\" colspan=\"1\">Not done</td><td rowspan=\"1\" colspan=\"1\">Not done</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Home medications</td><td rowspan=\"1\" colspan=\"1\">None</td><td rowspan=\"1\" colspan=\"1\">Amlodipine, clonidine, hydrochlorothiazide, furosemide</td><td rowspan=\"1\" colspan=\"1\">Hydralazine  </td><td rowspan=\"1\" colspan=\"1\">None</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Intubated</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">No</td><td rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CK at presentation (U/L)</td><td rowspan=\"1\" colspan=\"1\">589</td><td rowspan=\"1\" colspan=\"1\">4,330</td><td rowspan=\"1\" colspan=\"1\">8,636</td><td rowspan=\"1\" colspan=\"1\">5,008</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Peak CK</td><td rowspan=\"1\" colspan=\"1\">19,773</td><td rowspan=\"1\" colspan=\"1\">4,330</td><td rowspan=\"1\" colspan=\"1\">9,793</td><td rowspan=\"1\" colspan=\"1\">5,008</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Day of hospitalization corresponding to peak CK</td><td rowspan=\"1\" colspan=\"1\">Day 4</td><td rowspan=\"1\" colspan=\"1\">Day 1</td><td rowspan=\"1\" colspan=\"1\">Day 2</td><td rowspan=\"1\" colspan=\"1\">Day 1</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">White cell count (K/mm<sup>3</sup>)</td><td rowspan=\"1\" colspan=\"1\">8.7</td><td rowspan=\"1\" colspan=\"1\">8.2</td><td rowspan=\"1\" colspan=\"1\">8.2</td><td rowspan=\"1\" colspan=\"1\">11.8</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Hemoglobin (g/dL)</td><td rowspan=\"1\" colspan=\"1\">13.4</td><td rowspan=\"1\" colspan=\"1\">14.9</td><td rowspan=\"1\" colspan=\"1\">11.8</td><td rowspan=\"1\" colspan=\"1\">15.7</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Platelets (K/mm<sup>3</sup>)</td><td rowspan=\"1\" colspan=\"1\">161</td><td rowspan=\"1\" colspan=\"1\">136</td><td rowspan=\"1\" colspan=\"1\">115</td><td rowspan=\"1\" colspan=\"1\">147</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Troponin (ng/mL)</td><td rowspan=\"1\" colspan=\"1\">0.02</td><td rowspan=\"1\" colspan=\"1\">0.4</td><td rowspan=\"1\" colspan=\"1\">0.4</td><td rowspan=\"1\" colspan=\"1\">0.3</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Sodium (mEq/L)</td><td rowspan=\"1\" colspan=\"1\">139</td><td rowspan=\"1\" colspan=\"1\">134</td><td rowspan=\"1\" colspan=\"1\">134</td><td rowspan=\"1\" colspan=\"1\">144</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Potassium (mEq/L)</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">4</td><td rowspan=\"1\" colspan=\"1\">5.1</td><td rowspan=\"1\" colspan=\"1\">3.5</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Phosphorous (mg/dL)</td><td rowspan=\"1\" colspan=\"1\">2.8</td><td rowspan=\"1\" colspan=\"1\">ND</td><td rowspan=\"1\" colspan=\"1\">8.8</td><td rowspan=\"1\" colspan=\"1\">3.5</td></tr><tr><td rowspan=\"1\" colspan=\"1\">BUN (mg/dL)</td><td rowspan=\"1\" colspan=\"1\">18</td><td rowspan=\"1\" colspan=\"1\">57</td><td rowspan=\"1\" colspan=\"1\">74</td><td rowspan=\"1\" colspan=\"1\">32</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Creatinine (mg/dL)</td><td rowspan=\"1\" colspan=\"1\">1.16</td><td rowspan=\"1\" colspan=\"1\">3.8</td><td rowspan=\"1\" colspan=\"1\">20</td><td rowspan=\"1\" colspan=\"1\">1.68</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Acute renal replacement therapy</td><td rowspan=\"1\" colspan=\"1\">Yes</td><td rowspan=\"1\" colspan=\"1\">No</td><td rowspan=\"1\" colspan=\"1\">No</td><td rowspan=\"1\" colspan=\"1\">No</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">AST (U/L)</td><td rowspan=\"1\" colspan=\"1\">46</td><td rowspan=\"1\" colspan=\"1\">131</td><td rowspan=\"1\" colspan=\"1\">142</td><td rowspan=\"1\" colspan=\"1\">179</td></tr><tr><td rowspan=\"1\" colspan=\"1\">ALT (U/L)</td><td rowspan=\"1\" colspan=\"1\">30</td><td rowspan=\"1\" colspan=\"1\">65</td><td rowspan=\"1\" colspan=\"1\">43</td><td rowspan=\"1\" colspan=\"1\">53</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">ESR (mm/hr)</td><td rowspan=\"1\" colspan=\"1\">53</td><td rowspan=\"1\" colspan=\"1\">43</td><td rowspan=\"1\" colspan=\"1\">84</td><td rowspan=\"1\" colspan=\"1\">63</td></tr><tr><td rowspan=\"1\" colspan=\"1\">CRP (mg/L)</td><td rowspan=\"1\" colspan=\"1\">157.9</td><td rowspan=\"1\" colspan=\"1\">85.5</td><td rowspan=\"1\" colspan=\"1\">249</td><td rowspan=\"1\" colspan=\"1\">363.3</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Ferritin (ng/mL)</td><td rowspan=\"1\" colspan=\"1\">574</td><td rowspan=\"1\" colspan=\"1\">1,170</td><td rowspan=\"1\" colspan=\"1\">5,217</td><td rowspan=\"1\" colspan=\"1\">1,780</td></tr><tr><td rowspan=\"1\" colspan=\"1\">LDH (U/L)</td><td rowspan=\"1\" colspan=\"1\">459</td><td rowspan=\"1\" colspan=\"1\">907</td><td rowspan=\"1\" colspan=\"1\">478</td><td rowspan=\"1\" colspan=\"1\">1475</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">D-dimer</td><td rowspan=\"1\" colspan=\"1\">0.62</td><td rowspan=\"1\" colspan=\"1\">1.92</td><td rowspan=\"1\" colspan=\"1\">1.06</td><td rowspan=\"1\" colspan=\"1\">3.11</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Urinalysis blood  </td><td rowspan=\"1\" colspan=\"1\">ND</td><td rowspan=\"1\" colspan=\"1\">Large blood</td><td rowspan=\"1\" colspan=\"1\">ND</td><td rowspan=\"1\" colspan=\"1\">Large blood</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Urinalysis red blood cells (per HPF)  </td><td rowspan=\"1\" colspan=\"1\">ND</td><td rowspan=\"1\" colspan=\"1\">4-5</td><td rowspan=\"1\" colspan=\"1\">ND</td><td rowspan=\"1\" colspan=\"1\">No red blood cells</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Outcome</td><td rowspan=\"1\" colspan=\"1\">Died</td><td rowspan=\"1\" colspan=\"1\">Died</td><td rowspan=\"1\" colspan=\"1\">Died</td><td rowspan=\"1\" colspan=\"1\">Died</td></tr></tbody></table></table-wrap><p>Case 1 </p><p>A 67-year-old male patient with a past medical history of hypertension presented to the emergency department complaining of fever and shortness of breath. On initial examination, the patient was in severe respiratory distress, tachypneic, using accessory muscles, saturating at approximately 80% on room air, and was intubated. Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref> shows the creatine kinase levels corresponding to the day of hospitalization.</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Creatine kinase levels corresponding to the day of hospitalization</title></caption><graphic xlink:href=\"cureus-0012-00000010686-i01\"/></fig><p>Chest X-ray performed at baseline showed bilateral hazy infiltrates. Chest CT showed extensive ground-glass opacity and consolidation in the lungs bilaterally. The patient was started on azithromycin, ceftriaxone, hydroxychloroquine, and fluids. On day 3, the patient developed acute renal failure and was started on hemodialysis. The patient had a complicated hospital course, requiring ventilatory support throughout the hospitalization and unfortunately passed away on day 21. </p><p>Case 2 </p><p>A 39-year-old male patient with a history of hypertension presented to the emergency department with fever, muscle aches, shortness of breath, and altered mental status. On initial examination, the patient was in severe respiratory distress, tachypneic, using accessory muscles, hypoxic, and was intubated. Urinalysis was positive for large blood with four to five red blood cells (reference: 0-3) seen microscopically. Chest X-ray showed extensive confluent bilateral pulmonary infiltrates. The patient passed away due to cardiac arrest on day 1. </p><p>Case 3 </p><p>A 43-year-old male patient with a past medical history of end-stage renal disease on hemodialysis comes to the emergency room with complaints of cough, shortness of breath muscle aches, and fever of two days of duration. Vital signs on presentation were heart rate of 107 beats per minute, blood pressure of 94/61 mm Hg, oxygen saturation 99% on room air, and temperature of 37.1 degree Celsius. On physical examination, diffuse bilateral rhonchi were present. Figure <xref ref-type=\"fig\" rid=\"FIG2\">2</xref> shows the creatine kinase levels corresponding to the day of hospitalization.</p><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>Creatine kinase levels corresponding to the day of hospitalization</title></caption><graphic xlink:href=\"cureus-0012-00000010686-i02\"/></fig><p>Chest x ray showed multifocal ill-defined parenchymal opacities bilaterally, most notably over mid and lower lung zones with increased interstitial thickening. The patient was started ceftriaxone, hydroxychloroquine, and azithromycin. The patient passed away due to cardiac arrest on day 2. </p><p>Case 4 </p><p>A 70-year-old male patient with no significant past medical history presented to the emergency department with chief complaints of shortness of breath and cough of eight days of duration. Vital signs on presentation were heart rate of 101 beats per minute, blood pressure of 94/61 mm Hg, respiratory rate 20 breaths per minute, oxygen saturation 87% on room air, and temperature of 37.2 degree Celsius. The patient was intubated due to worsening respiratory status. Figure <xref ref-type=\"fig\" rid=\"FIG3\">3</xref> shows the creatine kinase levels corresponding to the day of hospitalization.</p><fig fig-type=\"figure\" id=\"FIG3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><title>Creatine kinase levels corresponding to the day of hospitalization</title></caption><graphic xlink:href=\"cureus-0012-00000010686-i03\"/></fig><p>Urinalysis was positive for large blood with no red blood cells seen microscopically. Chest x-ay showed bilateral hazy infiltrates throughout both lung fields. The patient was treated with fluids, intravenous methylprednisolone 40 mg q12 hours, and ceftriaxone. Hydroxychloroquine and azithromycin were not started due to prolonged corrected Q-T interval of 503 ms. The patient was extubated on day 8; however, he required reintubation on day 11. The patient had a complicated hospital course - septic shock, gastrointestinal bleed, and after discussion with the patient’s family, he was made comfort care and died on day 17.</p></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>Rhabdomyolysis is described as the rapid breakdown of skeletal muscles resulting in release of cell degradation products and intracellular elements within the bloodstream and extracellular space. Rhabdomyolysis can be caused by multiple etiologies, such as trauma, marked exertion in untrained individuals, hyperthermia, metabolic myopathies, drugs or toxins, infections, or electrolyte disorders [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. The usual symptoms include muscle pain, weakness, and dark urine. The laboratory diagnosis is based on the measurement of creatine kinase in serum or plasma. A prompt diagnosis is a prerequisite for successful treatment and avoiding complications. Identification of the triggering event is of paramount importance. Early and aggressive fluid resuscitation is the cornerstone of management. Potential complications of rhabdomyolysis include fluid and electrolyte abnormalities, kidney failure, liver injury, cardiac arrhythmias, compartment syndrome, and disseminated intravascular coagulation [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>].</p><p>Viruses that have been associated with rhabdomyolysis include influenza, parainfluenza, coxsackievirus, Epstein-Barr, herpes simplex, adenovirus, echovirus, human immunodeficiency virus, and cytomegalovirus [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>,<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. Case reports of SARS-CoV-2 causing rhabdomyolysis have been described. Rhabdomyolysis has been described both as a presenting feature and a late complication of COVID-19. Chedid et al. described a case of a 51-year-old male patient with severe rhabdomyolysis leading to acute kidney injury (AKI) as the primary presenting feature of COVID-19 [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. Jin and Tong reported a 60-year-old man with COVID-19, who was found to have rhabdomyolysis on day 9 of hospitalization when he developed pain and weakness of his lower extremities [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. Possible mechanisms of action of viral-induced myositis include direct viral invasion or myotoxic cytokines or immune mediated [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>,<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]. Further studies are needed to understand the pathogenesis.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>We report four cases of rhabdomyolysis in COVID-19 patients. Our cases add to the limited literature regarding rhabdomyolysis in COVID-19. Clinicians should be aware of the life-threatening manifestation of COVID-19 so that timely diagnoses can be made, and complications avoided.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"journal\"><article-title>Clinical characteristics of coronavirus disease 2019 in China</article-title><source>N Engl J 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005533</article-id><article-id pub-id-type=\"pmc\">PMC7523747</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10116</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Plastic Surgery</subject></subj-group><subj-group><subject>Orthopedics</subject></subj-group><subj-group><subject>Trauma</subject></subj-group></article-categories><title-group><article-title>Negative Pressure Wound Therapy With Flap Reconstruction for Extensive Soft Tissue Loss in the Foot: A Case Report</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Vellingiri</surname><given-names>Kishore</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>S</surname><given-names>Nagakumar J</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hongaiah</surname><given-names>Deepak</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nOrthopaedics, Sri Devaraj Urs Academy of Higher Education and Research, Kolar, IND </aff><aff id=\"aff-2\">\n<label>2</label>\nPlastic Surgery, Sri Devaraj Urs Academy of Higher Education and Research, Kolar, IND </aff><author-notes><corresp id=\"cor1\">\nNagakumar J. S <email>jamathinagi@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10116</elocation-id><history><date date-type=\"received\"><day>19</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Vellingiri et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Vellingiri et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/38480-negative-pressure-wound-therapy-with-flap-reconstruction-for-extensive-soft-tissue-loss-in-the-foot-a-case-report\">This article is available from https://www.cureus.com/articles/38480-negative-pressure-wound-therapy-with-flap-reconstruction-for-extensive-soft-tissue-loss-in-the-foot-a-case-report</self-uri><abstract><p>Negative pressure wound therapy (NPWT) can create the healing granulation tissue that will form a wound bed for the skin graft, thereby reducing the volume of the soft tissue defect. The application of uniform negative pressure, which is delivered by vacuum-assisted closure (VAC) therapy, induces a physical response (macrostrain) and a biological response (microstrain). The patient in the current case report presented with an alleged history of a road traffic accident, sustaining a crush injury to his right heel pad, resulting in an open comminuted fracture of the right calcaneum with bone loss. A total of seven days of NPWT was allowed. Negative pressure sponge dressing was then applied in this region and adhesive drapes were sealed. Once sealed, suction was set at the continuous pressure of -125 mm Hg. The authors noted that the benefits significantly outweigh the costs of the VAC system, making it an essential treatment option for patients similar to the one presented in this case report.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>negative pressure wound therapy</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Negative pressure wound therapy (NPWT), also known as topical negative pressure therapy, has revolutionized the world of wound care. NPWT aids in forming granulation tissue, which serves as a wound bed for the skin graft and reduces the volume of soft tissue defects [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. We report a case of a crush injury to the right heel pad, which caused an open comminuted fracture of the right calcaneum with bone loss. Initial debridement with regular dressing was done, which was associated with poor outcome. So a single set of seven days of vacuum-assisted closure (VAC) was tried for this patient after secondary debridement, which, in turn, turned out to be a game-changer for the patient with extensive soft tissue loss in the foot. VAC can thus potentially replace microsurgical soft tissue transfer, reduce the risk of infection, and aid in salvaging the limb.</p><p>The abstract of the case was accepted for paper presentation at the 53rd Annual Conference of Tamilnadu Orthopaedic Association (TNOACON) held from February 7 to 9, 2020, at Kingston Engineering College, Vellore, India. The abstract is scheduled to be published in the online supplement of the Tamilnadu Orthopaedic Association Journal.</p></sec><sec sec-type=\"cases\"><title>Case presentation</title><p>A 22-year-old male patient was brought to R L Jalappa Hospital & Research Centre, affiliated with Sri Devaraj Urs Medical College, Kolar, India. The patient presented with an alleged history of a road traffic accident. He sustained a crush injury to his right heel pad, resulting in an open comminuted fracture of the right calcaneum with bone loss (Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>). The range of motion at the right ankle and the subtalar joint was painful and restricted. Dorsalis pedis artery pulsation was palpable, and distal sensation was intact. Active toe movements were present. The capillary refill time was normal. All other long bones and joints were clinically normal. There were no other injuries anywhere in the body.</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Crush injury of the right heel pad exposing the underlying soft tissue and bone.</title></caption><graphic xlink:href=\"cureus-0012-00000010116-i01\"/></fig><p>At the emergency department, triple antibiotic prophylaxis was administered. It consisted of intravenous (IV) administration of augmentin 1.2 g, amikacin 500 mg, and metronidazole 100 ml. Wound wash was given with six liters of sterile saline at the bedside and the wound site dressed with moist gauze. The patient was stabilized with a below-knee splint and sent for radiographs. Preoperative radiographs are provided in Figure <xref ref-type=\"fig\" rid=\"FIG2\">2</xref>. After imaging was completed and informed consent obtained, the patient underwent thorough wound debridement and NPWT was given. The initial operative encounter consisted of the patient being operated under spinal anesthesia. Gross wound contaminants were removed, including all devitalized bone. Three liters of sterile saline were then used to irrigate the wound. Once complete, all wounds were left open and a wound VAC was applied. Figure <xref ref-type=\"fig\" rid=\"FIG3\">3</xref> shows the clinical picture of the right heel pad after three days following initial debridement.</p><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>Plain radiographs of the right foot lateral view showing comminuted right calcaneum fracture with bone loss.</title></caption><graphic xlink:href=\"cureus-0012-00000010116-i02\"/></fig><fig fig-type=\"figure\" id=\"FIG3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><title>Clinical picture of the right heel pad after three days following initial debridement.</title></caption><graphic xlink:href=\"cureus-0012-00000010116-i03\"/></fig><p>Vacuum-assisted closure (VAC) technique</p><p>The wounds were then lightly packed with sponge and sealed with adhesive drapes. The sterile sponge was applied over the exposed part of the bone and hardware. When sealing the sponge, care was taken to lay drapes over the skin with minimal skin tension. Suction was set at -125 mm of Hg. A total of seven days of NPWT was allowed [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. The vacuum-assisted closure device is shown in Figure <xref ref-type=\"fig\" rid=\"FIG4\">4</xref>.</p><fig fig-type=\"figure\" id=\"FIG4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><title>Vacuum-assisted closure (VAC) device</title></caption><graphic xlink:href=\"cureus-0012-00000010116-i04\"/></fig><p>Hospital course</p><p>Postoperatively, the patient received intravenous augmentin 1.2 g twice daily for seven days, amikacin 500 mg twice daily for seven days, and metronidazole 100 ml thrice daily for three days. It was followed by oral Amoxiclav 625 mg twice daily for seven days. The patient was taken to the operating room one more time for wound debridement (Figure <xref ref-type=\"fig\" rid=\"FIG5\">5</xref>), followed by reverse sural artery flap placement (Figure <xref ref-type=\"fig\" rid=\"FIG6\">6</xref>). Assessment of the wound during the time of secondary debridement demonstrated a progressive wound bed with granulation tissue and a decrease in overall wound size. Postoperatively, the patient stayed in the hospital for seven days for flap monitoring. The surgical site healed well (Figures <xref ref-type=\"fig\" rid=\"FIG7\">7</xref>-<xref ref-type=\"fig\" rid=\"FIG10\">10</xref>). Active dorsiflexion of the right ankle was present. The patient is being followed at regular intervals. Outpatient follow-up ensued at two weeks, one month, and three months.</p><fig fig-type=\"figure\" id=\"FIG5\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><title>Clinical picture of the right heel pad after seven days of secondary wound debridement and with negative pressure wound therapy</title></caption><graphic xlink:href=\"cureus-0012-00000010116-i05\"/></fig><fig fig-type=\"figure\" id=\"FIG6\" orientation=\"portrait\" position=\"float\"><label>Figure 6</label><caption><title>Clinical picture of the right heel pad reconstruction with reverse sural artery flap</title></caption><graphic xlink:href=\"cureus-0012-00000010116-i06\"/></fig><fig fig-type=\"figure\" id=\"FIG7\" orientation=\"portrait\" position=\"float\"><label>Figure 7</label><caption><title>Clinical picture of the right heel pad lateral aspect at three months follow-up</title></caption><graphic xlink:href=\"cureus-0012-00000010116-i07\"/></fig><fig fig-type=\"figure\" id=\"FIG8\" orientation=\"portrait\" position=\"float\"><label>Figure 8</label><caption><title>Clinical picture of the right heel pad medial aspect at three months follow-up</title></caption><graphic xlink:href=\"cureus-0012-00000010116-i08\"/></fig><fig fig-type=\"figure\" id=\"FIG9\" orientation=\"portrait\" position=\"float\"><label>Figure 9</label><caption><title>Clinical picture of the right heel pad reconstructed with reverse sural artery flap</title></caption><graphic xlink:href=\"cureus-0012-00000010116-i09\"/></fig><fig fig-type=\"figure\" id=\"FIG10\" orientation=\"portrait\" position=\"float\"><label>Figure 10</label><caption><title>Clinical picture of the right heel pad plantar aspect at three months follow-up</title></caption><graphic xlink:href=\"cureus-0012-00000010116-i10\"/></fig></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>The VAC drape helps in providing a wound healing environment. VAC GranuFoam dressings contract under negative pressure. It provides direct and complete contact with the wound bed. The VAC canister collects the wound exudate. Four-hundred to 600 micron-reticulated pores help distribute pressure through the wound bed [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>-<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. VAC GranuFoam dressing - Black is hydrophobic, and its open-pore nature (400-600 microns) provides a uniform application of negative pressure at the wound site. It helps in wound contraction. VAC - white foam dressing is hydrophilic, pre-moistened with sterile water, and is a denser foam with a greater pore size distribution. It requires a higher pressure of 125-175 mm of Hg to provide adequate negative pressure therapy distribution. The application of uniform negative pressure delivered by VAC therapy induces a physical response (macrostrain) and a biological response (microstrain). Macrostrain draws wound edges together and removes exudate and infectious material. It reduces edema, and promotes perfusion. Microstrain creates tissue micro deformation, causing cells to stretch [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. Cell stretch leads to the migration of cells and proliferation that result in granulation tissue formation.</p><p>VAC therapy is indicated in acute wounds - traumatic, partial-thickness burns, flaps, and grafts, in subacute dehisced wounds, and in chronic conditions - diabetic ulcers, pressure injuries, and venous insufficiency ulcers. VAC therapy contraindications include foam dressing directly in contact with exposed blood vessels, anastomotic sites, organs or nerves, malignancy in the wound, non-enteric and unexplored fistulas, necrotic tissue with eschar present, sensitivity to silver (VAC GranuFoam silver dressing only), and untreated osteomyelitis.</p><p>Nicks B A et al. in a study proved the management of acute wounds varies based on location and characteristics [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>]. A systematic approach gives the best framework for managing acute wounds. NPWT helps in providing temporary wound coverage during the period of debridement and closure of the wound.</p><p>Careful selection of the patient and the procedure, along with the appropriate technique, is imperative for NPWT success. The wound healing process involves coagulation, inflammation followed by a migratory and proliferative stage, and, finally, remodeling. NPWT is shown to increase cell proliferation, increase local perfusion, increase granulation, and clear wound infections earlier. It will decrease hospital length of stay, and expedite the final closure of the wound. Khurram M F et al. demonstrated that VAC therapy accelerates the healing process by granulation tissue formation and reduces morbidity and pain in pediatric patients [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. The sizes of soft tissue defects, reduced from 69.18 cm<sup>2</sup> to 50.73 cm<sup>2</sup> after VAC in their study. Yadav S et al. proposed that this novel VAC technique helps in the management of difficult wounds [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. It has always been a cause of concern for the treating clinicians and VAC delivers at par or better results as compared to the conventional techniques. Webster J et al. in their study urged the need for larger, well-designed, and well-conducted trials to evaluate the effects of NPWT for use on clean, closed surgical wounds [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]. The trials should initially focus on wounds that are difficult to heal like sternal wounds or incisions on obese patients. Huang C et al. in their study suggested that for better care of patients, it is essential to stay updated on advancements in mechanobiology, cell therapy, and biofilms [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>]. The optimal parameters for specific wounds, including interface material, the waveform of suction application, and the amount of suction, are to be analyzed. The disadvantages of VAC are increased cost, additional technical requirements, and required inpatient monitoring of the system.</p><p>Treating patients with extensive soft tissue damage and high-grade compound fractures is challenging, as it requires a complex approach with plastic, orthopedic and vascular-reconstructive procedures. Management involves combinations of closure after wound debridement by secondary intention, VAC device usage, and various reconstructive plastic surgery methods. We authors noted that the benefits significantly outweigh the costs of the VAC system, making it an essential treatment option for patients similar to the one presented in this case report.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>The patient in our case report had better clinical and functional outcomes following NPWT for extensive soft tissue loss in the foot. NPWT is relatively a safer technique with less chance of infection than traditional dressing methods. Our hypothesis is that wound debridement followed by a single set of VAC placement for seven days should be tried for wound needing soft tissue coverage. Hence, it is a feasible and valuable method in the treatment of compound fractures with massive soft tissue defects.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study. Sri Devaraj Urs Medical College, Institutional Ethics Committee issued approval DMC/KLR/IEC/138/2019-20</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"journal\"><article-title>The ongoing and worldwide challenge of pediatric trauma</article-title><source>Int J Critical Illn Inj Sci</source><person-group><name><surname>Sharar</surname><given-names>SR</given-names></name></person-group><fpage>111</fpage><lpage>113</lpage><volume>2</volume><year>2012</year><pub-id pub-id-type=\"pmid\">23181203</pub-id></element-citation></ref><ref id=\"REF2\"><label>2</label><element-citation publication-type=\"journal\"><article-title>Circumferential negative pressure wound therapy with instillation and dwell prior to delayed flap coverage for a type IIIB open tibia 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] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005538</article-id><article-id pub-id-type=\"pmc\">PMC7523748</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10124</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Urology</subject></subj-group></article-categories><title-group><article-title>Does Diversion in Poorly Functioning Obstructed Kidneys in Adults Favors Reconstructive Surgeries Over Ablative Procedures? A Prospective Study</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Kalra</surname><given-names>Sidhartha</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mehra</surname><given-names>Ketan</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Muruganandham</surname><given-names>Kaliyaperumal</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dorairajan</surname><given-names>Lalgudi N</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Manikandan</surname><given-names>Ramanitharan</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dhanapathi</surname><given-names>Halanaik</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sreenivasan Kodakkattil</surname><given-names>Sreerag</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nUrology and Renal Transplantation, Jawaharlal Institute of Post Graduate Medical Education and Research, Pondicherry, IND </aff><aff id=\"aff-2\">\n<label>2</label>\nNuclear Medicine, Jawaharlal Institute of Post Graduate Medical Education and Research, Pondicherry, IND </aff><author-notes><corresp id=\"cor1\">\nSidhartha Kalra <email>sid6121984@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10124</elocation-id><history><date date-type=\"received\"><day>12</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Kalra et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Kalra et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/39493-does-diversion-in-poorly-functioning-obstructed-kidneys-in-adults-favors-reconstructive-surgeries-over-ablative-procedures-a-prospective-study\">This article is available from https://www.cureus.com/articles/39493-does-diversion-in-poorly-functioning-obstructed-kidneys-in-adults-favors-reconstructive-surgeries-over-ablative-procedures-a-prospective-study</self-uri><abstract><p>Objective</p><p>In obstructed poorly functioning kidneys, management depends on the recovery potential of the kidney. Some kidneys have good recovery capability and diversion may unfold the real condition of the kidney. This study evaluated whether pre-operative drainage for six weeks results in improvement of renal function in unilateral obstructed poorly functioning kidney with split renal function (SRF) less than 20%.</p><p>Methods</p><p>This was a prospective interventional study conducted between March 2013 and December 2015. All patients between 15 and 65 years, with unilaterally obstructed kidney with SRF ≤20% underwent percutaneous nephrostomy (PCN) drainage for six weeks. Patients having post-drainage SRF of ≥15% and per day urine output from PCN > 400 ml were considered for the reconstructive procedure. Nephrectomy was performed in cases with SRF <15% after considering patient preferences.</p><p>Results</p><p>Twelve of 17 patients had improvement in SRF; four had no change while one had a decrease in SRF after drainage. The mean improvement in glomerular filtration rate (GFR) and SRF was 1.4 ml/min and 3%, respectively (P = 0.08). Three out of seven patients with SRF of ≥15% showed an improvement of 5% or more while none of the patients with SRF <15% had such an improvement. Eight patients had final SRF <15% and underwent nephrectomy. Factors such as pre-existing SRF, duration of symptoms, kidney size, transverse pelvic diameter, 24-hour urinary output, and etiology for obstruction were not significant in predicting functional improvement.</p><p>Conclusion</p><p>Diversion and decompression of poorly functioning kidneys do not result in a significant functional improvement in obstructed kidneys with SRF <15%.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>poorly functioning kidneys</kwd><kwd>nephrectomy</kwd><kwd>percutaneous nephrostomy</kwd><kwd>pelviureteral junction obstruction</kwd><kwd>pyeloplasty</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Poorly functioning obstructed kidney in adults is a common problem encountered by the urologist. Surgical management of these kidneys is usually based on the differential renal function calculated on the diuretic renogram [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. The decision to remove or preserve a poorly functioning obstructed kidney depends on its function. It is recommended to reassess the function of these kidneys at four to six weeks after the relief of obstruction. However, this recommendation is based on an animal study conducted by Kerr in 1954 [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>]. There have been conflicting views addressing this issue in humans. Few reports consider that there is a functional recovery after drainage while others are of the view that there is no chance of improvement of function in poorly functioning obstructed kidneys.</p><p>There is still a considerable debate as at what level of split renal function (SRF) should a kidney preserved. Few authors consider SRF of less than 10% as the threshold for reconstruction [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. In contrast, few authors opine that drainage in patients with SRF <30% may not have significant improvement [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. At our institution, we routinely consider a cutoff of SRF <15% for the ablative procedure while the decision for SRF between 15% and 20% is left to individual surgeon’s discretion and patient preferences. Limited literature is available on pre-operative factors that could predict functional improvement in patients with the affected renal unit after drainage [<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. Additionally, there has been inconsistency in the accurate measurement of SRF in poorly functioning hydronephrotic kidneys and extrapolating these results to a functional outcome [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>].</p><p>The present study was designed to evaluate whether drainage for six weeks results in improvement of renal function in unilateral obstructed poorly functioning kidney with SRF <20%. Factors associated with functional recovery and correlation of glomerular function (measured by diethylene triamine penta-acetic acid [DTPA] scan [Gamma camera method]) and creatinine clearance (CRCL) were also assessed.</p></sec><sec sec-type=\"materials|methods\"><title>Materials and methods</title><p>This was a prospective interventional study conducted at the Department of Urology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry between March 2013 and December 2015. All patients presenting with unilateral obstructed poorly functioning kidney, SRF ≤20% measured by technetium-99m DTPA (99mTc DTPA) and age ranged from 15 to 65 years were included in this study. Patients with bilateral obstruction, obstruction in a solitary kidney, malignant obstruction, pyonephrotic kidneys, and deranged renal functions were excluded from the study.</p><p>Baseline demographic characteristics including age, sex, etiological factor, and duration of symptoms were recorded. Eligible patients underwent a renal function test and abdominal ultrasonography to check for kidney size and dilatation of the pelvicalyceal system at presentation. The renal function tests were performed by a 99mTc DTPA scan using the gamma camera method.</p><p>Ultrasound-guided percutaneous nephrostomy (PCN) was performed under local anesthesia on obstructed moiety as a daycare procedure. Any associated complications such as infection, bleeding requiring admission, PCN blockade, or slipping were recorded and treated. Patients were on regular follow-up and at six weeks, a repeat DTPA scan was performed to note any change in renal function after drainage, and subsequently, patients were admitted for definitive management. CRCL of the drained moiety was estimated using the formula CrCl = (UCr x V)/SCr (serum creatinine-adjusted for body surface area), where UCr represents 24-hour urinary creatinine of the drained moiety and V represents 24-hour urine volume from PCN.</p><p>Patients with post-drainage SRF ≥15% and per day urine output post-PCN of more than 400 ml were considered for the reconstructive procedure. Nephrectomy was performed if SRF was less than 15%. Various factors predicting functional recoveries such as age, pre-existing SRF, duration of symptoms, renal morphometric parameters, daily PCN urinary output, and etiology were recorded and analyzed. The correlation between glomerular filtration rate (GFR) measured by DTPA scan and urinary CRCL was also assessed.</p><p>The sample size of 22 was considered sufficient to detect a difference in SRF of 10% and an SD of 12.6 from baseline. Calculations were based on α of 0.01 and power of 80%. The statistical analysis was performed using professional statistics package EPI Info 7.0 version (CDC, Atlanta, USA) for windows. Descriptive data were represented as mean (SD) for numeric variables, percentages, and proportions for categorical variables. Wilcoxon’s rank test was used to compare SRF/GFR of the pre- and post-drainage poorly functioning kidney. Spearman’s correlation coefficient was used to assess the association between improvement in SRF with age, daily PCN urinary output, kidney size and transverse parenchymal diameter, and also the correlation between GFR calculated on DTPA and by urinary CRCL method. Simple linear regression was applied when the correlation was found to be statistically significant. Independent T-test was used to assess the significance of various factors in predicting functional recovery. Values of p < 0.05 was considered statistically significant.</p></sec><sec sec-type=\"results\"><title>Results</title><p>A total of 21 patients who fulfilled the inclusion and exclusion criteria were enrolled in the study. Two patients withdrew their consent and two patients got operated at a different institution during the follow-up period of six weeks for personal reasons. Hence, 17 patients (80%) who completed the intervention were analyzed.</p><p>Of the total 17 patients, 10 (58.52%) were males and seven (41.18%) were females (Table <xref rid=\"TAB1\" ref-type=\"table\">1</xref>). The overall mean (SD) age was 36.12 (12.16) years. The mean duration symptoms were 9.81 months, the kidney size was 11.97 cm, and the mean transverse pelvic diameter was 5.14 cm. The mean serum creatinine was 1.08 mg/dL. A total of 10 (58.82%) patients had primary pelviureteric junction obstruction (PUJO), four had calculus disease, and three had upper ureteric stricture as the cause of obstruction (Table <xref rid=\"TAB2\" ref-type=\"table\">2</xref>).</p><table-wrap id=\"TAB1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><title>Demographics and clinical characteristics at admission</title><p>SD: standard deviation.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Characteristics</td><td rowspan=\"1\" colspan=\"1\">N = 17</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Age in years (SD)</td><td rowspan=\"1\" colspan=\"1\">36.12 (12.16)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Male (%)</td><td rowspan=\"1\" colspan=\"1\">10 (58.82)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Female (%)</td><td rowspan=\"1\" colspan=\"1\">7 (41.18)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Duration of symptoms in months (SD)</td><td rowspan=\"1\" colspan=\"1\">9.81 (5.54)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Kidney size in cm (SD)</td><td rowspan=\"1\" colspan=\"1\">11.97 (2.4)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Transverse Pelvic diameter in cm (SD)</td><td rowspan=\"1\" colspan=\"1\">5.14 (3.521)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Mean serum creatinine in mg/dL (SD)</td><td rowspan=\"1\" colspan=\"1\">1.08 (0.17)</td></tr></tbody></table></table-wrap><table-wrap id=\"TAB2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><title>Etiology of obstruction</title><p>PUJO: pelviureteric junction obstruction.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Diagnosis</td><td rowspan=\"1\" colspan=\"1\">Number (%)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">PUJO (%)</td><td rowspan=\"1\" colspan=\"1\">10 (58.82)</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Renal and upper ureteric calculi (%)</td><td rowspan=\"1\" colspan=\"1\">4 (23.53)</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Upper ureteric stricture (%)</td><td rowspan=\"1\" colspan=\"1\">3 (17.65)</td></tr></tbody></table></table-wrap><p>The PCN was successfully performed in all 17 patients and none of the patients had urinary tract infection or bleeding after PCN. The PCN drainage was kept for six weeks and then definitive surgery was performed.</p><p>The mean (SD) GFR pre-drainage calculated by DTPA scan was 8.21 (4.5) ml/min and the SRF was 13.0% (4.35). The mean (SD) GFR post-drainage was 9.60 (5.97) ml/min and the SRF was 16.12% (7.35). Twelve (70.59%) of 17 patients had improvement in SRF; four (23.53%) had no change while one (5.88%) had a decrease in SRF after drainage. The mean improvement in GFR and SRF was 1.4 ml/min and 3%, respectively. This improvement in GFR and SRF was statistically significant (p = 0.008).</p><p>The mean (SD) age in patients showing improvement in SRF was 34.58 (7.17) compared to 39.80 (20.63) years in patients showing no improvement. There was a negative correlation between the two, however, statistically insignificant (p = 0.438; Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>). The mean (SD) urinary drainage from PCN was 773.53 (537.10) ml. Twelve patients with improvement in SRF had the mean output of 883.33 ml, while five patients with no improvement in SRF had the mean drainage of 510. However, this difference in urine output production in patients showing improvement versus no improvement was not statistically significant (p = 0.20; Table <xref rid=\"TAB3\" ref-type=\"table\">3</xref>).</p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Graph showing age and improvement in SRF</title><p>SRF: split renal function.</p></caption><graphic xlink:href=\"cureus-0012-00000010124-i01\"/></fig><table-wrap id=\"TAB3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><title>Comparison of patients with improvement versus no improvement after PCN</title><p>PCN: percutaneous nephrostomy.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Parameters</td><td rowspan=\"1\" colspan=\"1\">Improvement</td><td rowspan=\"1\" colspan=\"1\">No improvement</td><td rowspan=\"1\" colspan=\"1\">P-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Patients, number</td><td rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\">5</td><td rowspan=\"1\" colspan=\"1\">0.008</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Age (years)</td><td rowspan=\"1\" colspan=\"1\">34.58 (7.17)</td><td rowspan=\"1\" colspan=\"1\">39.80 (20.63)</td><td rowspan=\"1\" colspan=\"1\">0.438</td></tr><tr><td rowspan=\"1\" colspan=\"1\">Duration of symptoms (months)</td><td rowspan=\"1\" colspan=\"1\">9.92 (5.91)</td><td rowspan=\"1\" colspan=\"1\">9.5 (5.00)</td><td rowspan=\"1\" colspan=\"1\">0.902</td></tr><tr style=\"background-color:#ccc\"><td rowspan=\"1\" colspan=\"1\">Kidney size (cm)</td><td rowspan=\"1\" colspan=\"1\">12.23 (2.42)</td><td rowspan=\"1\" colspan=\"1\">11.34 (2.70)</td><td rowspan=\"1\" colspan=\"1\">0.512</td></tr><tr><td rowspan=\"1\" colspan=\"1\">PCN urinary output (mL)</td><td rowspan=\"1\" colspan=\"1\">883.33 (562.60)</td><td rowspan=\"1\" colspan=\"1\">510 (400.63)</td><td rowspan=\"1\" colspan=\"1\">0.201</td></tr></tbody></table></table-wrap><p>Ten patients with initial SRF of less than 15% showed a mean (SD) percent improvement in SRF of 14.1% (24.33) contrary to 36.6% (33.31) mean percent change in SRF seen in seven patients with initial SRF more than 15%. This difference was not statistically significant (p = 0.127)</p><p>The mean (SD) GFR calculated by urinary CRCL method was 7.92 (5.22) compared to 9.60 (5.97) calculated on DTPA. An almost perfect positive correlation was found between GFR calculated by urinary CRCL and DTPA method with a correlation coefficient of 0.93 approaching close to the value of 1 (Figure <xref ref-type=\"fig\" rid=\"FIG2\">2</xref>).</p><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>Graph showing correlation between GFR by CRCL and DTPA scan</title><p>GFR: glomerular filtration rate, CRCL: creatinine clearance, DTPA: diethylene triamine penta-acetic acid.</p></caption><graphic xlink:href=\"cureus-0012-00000010124-i02\"/></fig><p>No adverse events were recorded in the form of bleeding, infection PCN blockage, and slippage in any participants of the study. At a one-year follow-up interval, none of the patients who underwent the reconstructive procedure had any further deterioration in renal function and required any additional procedure.</p></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>This prospective study evaluated whether drainage for six weeks results in improvement of renal function in unilateral obstructed poorly functioning kidney with ≤20%. The results showed that PCN drainage did not result in a significant functional improvement and clinical outcome in obstructed kidneys with SRF ≤15%.</p><p>Surgical management of poorly functioning obstructed kidneys in adults is usually based on the differential renal function calculated on the diuretic renogram. Although drainage decreases hydronephrosis and improves the excretory pattern, a significant increase in differential renal function occurs only in some patients. Various factors that could predict the functional recovery of these kidneys after drainage are not conclusively defined in the literature. With the advancement in the field of diagnostic imaging, most of these cases are detected early in life. Thus, most of the studies concerning this issue are focused on pediatric patients rather than adults.</p><p>Various studies have shown different independent predictors for the improvement and resolution of renal function. In a previous study, Gupta et al. analyzed the usefulness of PCN drainage in patients with ureteropelvic junction obstruction having SRF <10% (n = 20) [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. Twelve of 17 kidneys showed improvement in function after drainage while the rest of the five units did not show any functional recovery. Gupta et al. concluded that drainage in such kidneys may help in differentiating kidney which is likely to improve and not all such kidneys should be removed [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. In another study, Ransley et al. included 112 patients (142 kidneys) with prenatally diagnosed hydronephrosis [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. Out of these nine kidneys had a poor function on renogram at three months and underwent pigtail drainage. Three of these nine kidneys recovered with sufficient function and subsequently underwent pyeloplasty. Ransley et al. recommended attempting drainage in cases where SRF is <20% but >10% [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. However, kidneys having SRF <10% should be directly considered for an ablative procedure without trying drainage.</p><p>Overall, most of these studies were conducted in the pediatric age group where the kidneys have the potential for development. Mayor et al. in their study found an association of early drainage and improvement in function with age [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>]. In another study Chandrasekharam et al. also concluded that functional recovery was significantly higher in patients less than one year of age undergoing pyeloplasty [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>]. In contrast, MacNeily et al. did not find the patient’s age as a predicting factor in functional recovery [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>].</p><p>Ortapamuk et al. studied the improvement in differential function in adult obstructed kidneys with PUJO [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>]. Patients were distributed into two groups according to baseline differential function. A total of 22 patients had an initial SRF of >30% (group 1) while 10 patients had SRF <30% (group 2). Improvement in SRF was found to be significant when there was a 5% increment in differential function over the baseline. They found a statistically significant improvement in differential function in group 1 with differential function more than 30 compared to the other group. However, no significant correlation between functional recovery with ultrasonography findings, clinical presentation, and age among the two groups was found. They concluded that improvement in SRF in adult obstructed kidney depends on the initial level of SRF and patients with differential function <30% may not benefit much from drainage procedure.</p><p>Recently, Zhang et al. retrospectively analyzed 53 patients with unilateral PUJO with SRF <10% who underwent PCN as the drainage procedure and patients were considered as improved if there was a functional recovery of >10% and daily PCN drainage was >400 ml [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. At six weeks of drainage, 30 of 53 (56.6%) patients showed an improvement in the mean GFR and SRF. The before and after PCN, the mean (SD) GFR was 3.57 (2.79) and 14.05 (5.42) ml/min/1.73 m<sup>2</sup>, and the mean (SD) SRF was 4.53% (3.21) and 16.07% (5.49). Authors concluded that such poorly functioning obstructed kidneys should be considered for drainage especially in young adults (age <35 years). Moreover, diuretic renography with 99mTc-DTPA was not a reliable assessment of renal function in these obstructed kidneys.</p><p>In the present study, we evaluate the functional recovery after drainage in the unilaterally obstructed kidney with SRF ≤20%. In this study, 12 of 17 patients (70%) showed a statistically significant improvement in SRF after drainage. The mean (SD) pre- and post-drainage SRF was 13.0% (4.35) and 16.12% (7.35), respectively. However, only nine of these 12 patients had the final SRF ≥15% and underwent the reconstructive procedure. The rest of the three patients despite showing percent change in SRF (post-pre-drainage/pre-drainage SRF) had final SRF of <15% and who had nephrectomy. In total, 10 out of 17 patients (53%) who had an initial SRF <15% showed a mean (SD) percent improvement in SRF of 14.1% (24.32) while seven patients with initial SRF >15% had 36.6% (33.3) improvement in SRF. This difference was statistically insignificant (p=0.13).</p><p>However, taking into account the criteria of 5% improvement in SRF as significant, three out of seven patients (42%) with initial SRF of >15% showed such improvement while none of the patients with SRF <15% showed improvement of 5% or more. A negative correlation was observed between age and functional recovery although statistically insignificant. The mean (SD) age in patients with improvement in SRF was 34.58 (7.17) years in comparison to 39.80 (20.63) years in patients with no functional recovery. Zhang et al. in their study found a statistically significant association of improvement in SRF with age [<xref rid=\"REF3\" ref-type=\"bibr\">3</xref>]. In this particular study, 24 out of 29 adults with a mean (SD) age of 28.28 (4.88) years (82.8%) showed improvement in SRF; however, only six of 24 (25%) patients with a mean (SD) age 51.21 (10.69) years showed improvement.</p><p>A positive correlation was observed between the percent change in SRF and 24-hour urinary output from PCN. However, the difference in urinary output from PCN among patients having functional improvement versus those with no change in function was statistically insignificant. Thus, PCN urinary output as an independent factor may not be adequate for predicting the salvage ability of these kidneys and assessment of a repeat differential function could be necessary before considering definitive management. Other factors such as duration of symptoms, kidney size, transverse pelvic diameter, and etiology were not found to be significant in predicting the functional improvement after drainage in the present study. This observation was similar to a previous report by Ortapamuk et al. [<xref rid=\"REF4\" ref-type=\"bibr\">4</xref>].</p><p>The mean (SD) GFR estimation by CRCL at six weeks post-drainage was 7.92 (5.22) ml/min which was slightly less compared to 9.60 (5.97) ml/min calculated on DTPA scan. However, an almost perfect positive correlation was seen between the two on statistical analysis with a correlation coefficient of 0.93 reaching the value of 1. A similar association was reported by Ulibarri et al. who calculated GFR in post-renal transplant patients by DTPA, CRCL method, and GFR-estimating equations [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. They observed a significant correlation between GFR estimated by the CRCL method and DTPA scan. DTPA scan although not a definitive assessment of SRF and GFR in such poorly functioning kidney can still be a fair guide of renal function.</p><p>Pre-existing SRF was found to be statistically insignificant in predicting the functional recovery in our study. However, it was seen that most of the patients with initial SRF <10% did not show a significant improvement after drainage with only two out of 10 patients (20%) reaching the salvageable limit of 15%. Both of them had an initial SRF of 14%. Although a statistically significant mean improvement of 3% in SRF and 1.4 ml/min in GFR was seen in this study, it was not sufficient to extrapolate to a clinical improvement.</p><p>Nayyar et al. in 2016 studied 32 patients with SRF <20% who underwent pyeloplasty for pelviureteral junction obstruction [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]. In their study, the mean follow-up was 26.8 months and none of the patients required any re-intervention for obstructive drainage, intractable pain, or deteriorating function. There was a success rate of 93.7% and 40.6% showed significant improvement in SRF which was >5% over perioperative period. In their study, only one patient (3.1%) had the deterioration of function. They concluded that pyeloplasty provided a high rate of success both functional and morphologically in poorly functioning kidney with pelviureteral junction as the etiology. This study was different from our study as it considered only pelviureteral junction obstruction as the etiology and also they proceeded with the definitive surgery instead of pre-operative drainage.</p><p>Thus, the crux of the problem not only lies in accurately measuring the differential function of these poorly functioning obstructed kidney but also in deciding the definitive treatment. Since, subjecting these kidneys to a reconstructive procedure has the future risk of infection, the persistence of symptoms and a further decrease in function with time.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>PCN drainage did not result in a significant functional improvement and clinical outcome in obstructed kidneys with SRF <15%. Diuretic renography with a DTPA scan provided a fair estimate of function in these obstructed units. An almost perfect positive correlation of GFR estimation by DTPA scan and CRCL method was observed. A negative correlation was seen between age and functional recovery, however, statistically insignificant. Factors such as duration of symptoms, kidney size, transverse pelvic diameter, and etiology are not predictive of functional recovery after drainage. Further prospective studies with a larger sample size are warranted to confirm these results.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study. Institution Ethics Committee of JIPMER, Puducherry issued approval JIP/IEC/SC/2/301/2013. The study was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki. Written informed consent was obtained from all patients before patient enrollment.</p></fn></fn-group><fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group><ref-list><title>References</title><ref id=\"REF1\"><label>1</label><element-citation publication-type=\"journal\"><article-title>The use of renal scintigraphy in assessing the potential for recovery in the obstructed renal tract in children</article-title><source>BJU Int</source><person-group><name><surname>Thompson</surname><given-names>A</given-names></name><name><surname>Gough</surname><given-names>DC</given-names></name></person-group><fpage>853</fpage><lpage>856</lpage><volume>87</volume><year>2001</year><pub-id pub-id-type=\"pmid\">11412226</pub-id></element-citation></ref><ref id=\"REF2\"><label>2</label><element-citation 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cureus</journal-id><journal-id journal-id-type=\"issn\">2168-8184</journal-id><journal-title-group><journal-title>Cureus</journal-title></journal-title-group><issn pub-type=\"epub\">2168-8184</issn><publisher><publisher-name>Cureus</publisher-name><publisher-loc>Palo Alto (CA)</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33005539</article-id><article-id pub-id-type=\"pmc\">PMC7523749</article-id><article-id pub-id-type=\"doi\">10.7759/cureus.10125</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Internal Medicine</subject></subj-group><subj-group><subject>Infectious Disease</subject></subj-group><subj-group><subject>Epidemiology/Public Health</subject></subj-group></article-categories><title-group><article-title>Profile of Patients Suspected to be COVID-19: A Retrospective Analysis of Early Pandemic Data</article-title></title-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Muacevic</surname><given-names>Alexander</given-names></name></contrib><contrib contrib-type=\"editor\"><name><surname>Adler</surname><given-names>John R</given-names></name></contrib></contrib-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Goel</surname><given-names>Ashish</given-names></name><xref ref-type=\"aff\" rid=\"aff-1\">1</xref><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Raizada</surname><given-names>Alpana</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bansal</surname><given-names>Kamakshi</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gaur</surname><given-names>Nikhil</given-names></name><xref ref-type=\"aff\" rid=\"aff-2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Abraham</surname><given-names>Jyotika</given-names></name><xref ref-type=\"aff\" rid=\"aff-3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yadav</surname><given-names>Anil</given-names></name><xref ref-type=\"aff\" rid=\"aff-4\">4</xref></contrib></contrib-group><aff id=\"aff-1\">\n<label>1</label>\nInternal Medicine, University Respati Yogyakarta, Yogyakarta, IDN </aff><aff id=\"aff-2\">\n<label>2</label>\nInternal Medicine, University College of Medical Sciences, Delhi, IND </aff><aff id=\"aff-3\">\n<label>3</label>\nNursing, Guru Teg Bahadur Hospital, Delhi, IND </aff><aff id=\"aff-4\">\n<label>4</label>\nInternal Medicine, Guru Teg Bahadur Hospital, Delhi, IND </aff><author-notes><corresp id=\"cor1\">\nAlpana Raizada <email>alpanaraizadakharya@gmail.com</email></corresp></author-notes><pub-date date-type=\"pub\" publication-format=\"electronic\"><day>29</day><month>8</month><year>2020</year></pub-date><pub-date date-type=\"collection\" publication-format=\"electronic\"><month>8</month><year>2020</year></pub-date><volume>12</volume><issue>8</issue><elocation-id>e10125</elocation-id><history><date date-type=\"received\"><day>4</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020, Goel et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Goel et al.</copyright-holder><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/3.0/\"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri xlink:href=\"https://www.cureus.com/articles/38803-profile-of-patients-suspected-to-be-covid-19-a-retrospective-analysis-of-early-pandemic-data\">This article is available from https://www.cureus.com/articles/38803-profile-of-patients-suspected-to-be-covid-19-a-retrospective-analysis-of-early-pandemic-data</self-uri><abstract><p>Background and Objectives</p><p>Coronavirus disease 2019 (COVID-19), a global public health emergency of profound magnitude, has brought life to an unprecedented near-standstill. The clinical profile of the disease is still emerging and is marked by considerable geographical variability in terms of transmissibility, clinical profile, virulence, and mortality of the disease. As clinical data is being reported from around the globe, it becomes important to focus on local subjects in a global milieu, lest one misses the trees for the forest.</p><p>Our study is a short retrospective analysis of the demographic and clinical profiles of subjects presenting with a mild flu-like illness to our hospital who were tested for COVID-19. It compares the differences in age and sex of those who tested positive with those negative. In addition, it reviews the length of time it might take for a case testing positive on reverse transcriptase-polymerase chain reaction (RT-PCR) test to become negative.</p><p>Methodology</p><p>A retrospective analysis of data from adults who presented to our hospital with a mild flu-like illness between the months of March and May 2020 was conducted to understand the disease profile. The nasal/oropharyngeal swabs were collected from each patient and were transported to state-approved laboratories chain for RT-PCR analysis. Information was collected from reports received, clinical information forms, and sample collection forms that were being maintained as a part of the clinical management protocol. Data were analysed using Stata software, version 13 (StataCorp LLC, College Station, TX, USA).</p><p>Observations and Results</p><p>Three thousand twenty-six subjects presented to our hospital with either mild flu-like symptoms or with suspected exposure to a confirmed case of COVID-19. The subjects had a mean age of 37.3 (± 15.1) years and 1,805 (60.3%) were males. A regression analysis revealed an adjusted odds of 1.6 (95% confidence interval (CI): 1.2, 2.1) for testing positive for males as compared to females. For every one year increase in age, the odds for testing positive increased by 1.02 (95% CI: 1.01, 1.03).</p><p>Of the 2,592 individuals for whom data was available, 201 (7.6%) were found positive on RT-PCR analysis. Those testing positive were significantly older (41.0 years vs 36.8 years; p = 0.001) and more likely to be male (number: 138; 9.0% vs 6.7%; p = 0.05). Cough, followed by fever, was a common presenting feature.</p><p>A survival time analysis using data from 54 participants documented 455 days of the total observation period. A median time of eight days was required for the test to convert from positive to negative if the patient remained mildly symptomatic and did not develop a severe complicated illness. The time to conversion did not differ with age or sex.</p><p>Conclusions</p><p>Our analysis shows that patients with COVID-19 have presented with milder symptoms and have recovered well. The low test positivity rate is indicative of the early phase of the pandemic in the country and is a reflection of active infection control measures.</p></abstract><kwd-group kwd-group-type=\"author\"><kwd>coronavirus disease 2019 (covid-19)</kwd><kwd>reverse transcription polymerase chain reaction (rt-pcr)</kwd></kwd-group></article-meta><notes><p content-type=\"disclaimer\">The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.</p></notes></front><body><sec sec-type=\"intro\"><title>Introduction</title><p>Coronaviruses, a family of viruses that range from the common cold to the coronavirus disease 2019 (COVID-19) and include the Middle East respiratory syndrome (MERS) coronavirus and the severe acute respiratory syndrome (SARS) coronavirus, has rapidly become a common household name in the space of a few months [<xref rid=\"REF1\" ref-type=\"bibr\">1</xref>]. COVID-19 was declared a global public health emergency and has brought life to an unprecedented near-standstill owing to an unexpected, practically global lockdown. While comparisons have been drawn to the Spanish flu pandemic of 1918, the public health interventions in the present pandemic have been both brisk and far more severe.</p><p>Being an entirely new virus, a clinical profile of the disease is now emerging and has been characterized largely from Chinese studies [<xref rid=\"REF2\" ref-type=\"bibr\">2</xref>-<xref rid=\"REF5\" ref-type=\"bibr\">5</xref>]. As COVID-19 paves its way into India (there are over 178,014 active cases at the time of writing this paper), the disease dynamics in a naïve Indian population are only now being elaborated [<xref rid=\"REF6\" ref-type=\"bibr\">6</xref>-<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. Considerable geographical variability has been noticed in the transmissibility, clinical profile, virulence, and mortality of COVID-19. Consequently, the COVID-19 dashboard, maintained by the Johns Hopkins Bloomberg School, indicates that while the case fatality rate in the United Kingdom is 13.9%, Australia has managed to escape with 1.4% [<xref rid=\"REF8\" ref-type=\"bibr\">8</xref>]. India has had just over 14,000 deaths with the case fatality rate consistently under 3.5%. When the differences are so wide and cannot be explained by the preparedness of healthcare systems alone, it becomes important to explore host-characteristics. As clinical data emerge from areas outside of China, it becomes important to focus on local subjects in a global milieu, lest one misses the trees for the forest [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>-<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]. A recent case series of 21 patients from Delhi answers some questions but also raises several more unanswered questions [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>].</p><p>We present a short retrospective analysis of the demographic and clinical profiles of subjects presenting with a mild flu-like illness to our hospital who were tested for COVID-19. It compares the differences in age and sex of those who tested positive with those negative. In addition, it reviews the length of time it might take for a case testing positive on reverse transcriptase-polymerase chain reaction (RT-PCR) test to become negative.</p></sec><sec sec-type=\"materials|methods\"><title>Materials and methods</title><p>The proposal was reviewed by the Institutional Ethics Committee-Human Research (IEC-HR), University College of Medical Sciences, University of Delhi, Delhi (#IEC-HR/2020/44/2R), and a waiver of consent was approved for the present work.</p><p>A retrospective analysis of data from subjects who presented to our hospital with mild flu-like illness between the months of March and May 2020 was conducted to understand the disease profile. Data from adults who presented to our hospital (a non-COVID-designated facility during March through May 2020) in Delhi were included. Nasal/oropharyngeal swabs were collected from each patient and were transported to state-approved laboratories, maintaining the appropriate cold chain for RT-PCR analysis. </p><p>Information was collected from the reports received, clinical information forms, and sample collection forms that were being maintained as a part of the clinical management protocol. Data were analyzed using Stata software, version 13 (StataCorp LLC, College Station, Texas, USA). Differences in proportions by groups (sex, clinical symptoms) were tested by using chi-square tests and the differences in means for age were examined using the t-test. The data was set-up as a survival time analysis to determine the median duration required for conversion from a positive to a negative test.</p></sec><sec sec-type=\"results\"><title>Results</title><p>Data were available for 3,026 subjects who presented to our hospital with either mild flu-like symptoms or with suspected exposure to a confirmed case of COVID-19 during the early phases of the pandemic. The subjects had a mean age of 37.3 (± 15.1) years and 1,805 (60.3%) were males. The mean age was similar in males and females. A regression analysis revealed an adjusted odds of 1.6 (95% confidence interval (CI): 1.2, 2.1) for testing positive for males as compared to females. Every one year increase in age increased the odds for testing positive by 1.02 (95% CI: 1.01, 1.03)</p><p>Of the 2,592 individuals on whom data was available, 201 (7.6%) were found to be positive on RT-PCR analysis and reports were inconclusive for 12 (0.5%). Those testing positive were significantly older (41.0 years vs 36.8 years; p = 0.001) and more likely to be male (n: 138, 9.0% vs 6.7%; p = 0.05). The age distribution of patients based on the test report is presented in Figure <xref ref-type=\"fig\" rid=\"FIG1\">1</xref>. </p><fig fig-type=\"figure\" id=\"FIG1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><title>Boxplot showing the distribution of age over the test report</title></caption><graphic xlink:href=\"cureus-0012-00000010125-i01\"/></fig><p>Limited data were available on the clinical profile of 31 patients who had tested positive. The mean age of this group was 39.2 years (± 16.7) and there were 25 (80.1%) males. Only one patient had symptoms for more than a week. Most individuals who were found to be positive presented in the first week of illness. Among these 31 patients who tested positive, the commonest presenting features were cough (seen in 16 patients, 51%), fever (seen in eight patients, 26%), myalgia (seen in three patients, 10%), and breathlessness (seen in three patients, 10%) with some overlap between symptoms. Ten patients who were found to be positive did not have a clear history of exposure at presentation. </p><p>A survival time analysis was conducted, including available data from 54 patients who contributed 455 days of the total observation period. Once a patient tested positive for COVID-19, it took a median time of eight days (interquartile range (IQR): 5, 11 days) for the test to become negative if the patient remained mildly symptomatic and did not develop a severe complicated illness. The maximum time to test negative was 23 days over a total observation time of 455 days contributed by a total of 54 patients. The time to conversion did not differ with age or sex as shown in Figure <xref ref-type=\"fig\" rid=\"FIG2\">2</xref>.</p><fig fig-type=\"figure\" id=\"FIG2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><title>Kaplan-Meier estimate showing time to recovery after testing positive</title></caption><graphic xlink:href=\"cureus-0012-00000010125-i02\"/></fig></sec><sec sec-type=\"discussion\"><title>Discussion</title><p>In this retrospective analysis, we report that among subjects presenting to the hospital with a mild flu-like illness, those who tested positive for COVID-19 were significantly older and more likely to be men. Patients who tested positive took a median of eight days to become negative for COVID-19 by RT-PCR.</p><p>In a retrospective study by Xiao et al. of 301 confirmed COVID-19 patients hospitalized at Tongji Hospital in Wuhan, China, the median age was 58 years and 51.2% were male [<xref rid=\"REF12\" ref-type=\"bibr\">12</xref>]. The median period between onset of symptoms and positive severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RT-PCR results was 16 days (IQR: 10 - 23, n = 301). The median period of RT-PCR results to turn negative was 20 days (IQR: 17 - 24; n = 216). Infected patients ≥ 65 years old stayed contagious longer (22 days vs 19 days, p = 0.015).</p><p>In another study conducted by Liu et al. at Wuhan in the early days of the pandemic, 4,880 cases were tested for SARS-CoV-2 [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]. The median age was 50 years (IQR: 27) with 46.13% of the subjects being male. Out of the 4,880 patients, 1,875 (38.42%) were positive by RT-PCR. Males had a significantly higher positivity rate than females (40.43% vs 36.71%, respectively; p < 0.01) [<xref rid=\"REF13\" ref-type=\"bibr\">13</xref>]. </p><p>Escalera-Antezana et al. evaluated 152 subjects in a retrospective cross-sectional analysis and found that 12 (7.9%) of them returned positive results by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>]. The median age of the infected subjects was 39 years (IQR: 25 - 43) and half of them were male. Most subjects remained mildly symptomatic and all recovered. </p><p>The earliest data to emerge from across the world have consistently described patients who have presented with a milder illness and have rapidly recovered [<xref rid=\"REF9\" ref-type=\"bibr\">9</xref>, <xref rid=\"REF15\" ref-type=\"bibr\">15</xref>-<xref rid=\"REF16\" ref-type=\"bibr\">16</xref>]. Kim et al. observed 213 COVID-19 patients in South Korea and found that 19% remained asymptomatic [<xref rid=\"REF14\" ref-type=\"bibr\">14</xref>]. Others had a cough (40%) and hypoxia (40%). Joshi et al. described nine relatively younger men in a small case series from Nepal who had a mean age of 20 - 40 years [<xref rid=\"REF15\" ref-type=\"bibr\">15</xref>]. </p><p>In a retrospective analysis of 95 patients hospitalized during three weeks in March in the United Kingdom, Tomlins et al. reported that less than half of them could be discharged and one in five patients died [<xref rid=\"REF17\" ref-type=\"bibr\">17</xref>]. They reported that diabetes, cerebrovascular, and cardiovascular illness were associated with a poorer outcome. Most of their patients presented with fever and cough, while anosmia was under-represented. Those presenting with breathlessness had a greater chance of dying. While mortality appears to be considerably higher in their study as compared to other international reports, it is noteworthy that their patients were older (mean: 75 years).</p><p>Goyal et al. retrospectively analysed data from 393 patients, with a mean age of 62 years, hospitalized in New York with common symptoms of cough, fever, and breathlessness [<xref rid=\"REF11\" ref-type=\"bibr\">11</xref>]. Forty patients (10.2%) died, and obese men had more severe disease.</p><p>Colaneri et al. from Italy retrospectively studied 44 COVID-19-positive patients and nearly 39% of the patients in their study developed a serious illness, while two (4.5%) died [<xref rid=\"REF10\" ref-type=\"bibr\">10</xref>].</p><p>Closer home, in a series of 21 case studies, Gupta et al. documented a mean age of 40.3 years with male preponderance (66.7%) [<xref rid=\"REF7\" ref-type=\"bibr\">7</xref>]. The most common clinical presentation was cough and fever. However, 43.9% of individuals were asymptomatic. Hypertension, followed by diabetes, was the most common comorbidity seen.</p><p>The COVID-19 patients in India are younger as compared to their Western counterparts. Male preponderance has been seen worldwide. The proportion of asymptomatic individuals seems to be substantial and may be attributable to active contact tracing and surveillance in the country. Time taken to document COVID-19 negativity by RT-PCR in our study is lesser and may be explained by the relatively younger age composition in our study. However, we did not find any difference in time to negativity based on age.</p><p>The test positivity rate of 7.6% represents the early phase of the pandemic in India and is also a marker of the prompt availability of free testing facilities and active surveillance by the government. </p><p>The present study is the first analysis of structured patient data inclusive of those who tested negative for COVID-19 from India. The study can be used as a baseline to assess changes in test positivity rates overtime that may show the transition of the pandemic from one phase to another. The study provides important inputs for informed decision-making in patient management and helps in understanding the local trend of the disease.</p><p>There is limited data available since, during the early part of the pandemic, data collection and electronic registries were not as robust, leading to gaps in information.</p></sec><sec sec-type=\"conclusions\"><title>Conclusions</title><p>Our analysis shows that patients with COVID-19 have presented with milder symptoms and have recovered well. Of the individuals who were tested for COVID-19, 7.6% were found to be COVID-19 positive and most patients recovered after a mild illness. If global trends are to be believed, the pandemic could still be in its early stages in Delhi and the numbers have remained low due to the early, brisk, and effective public health measures taken by the State. However, there is little room for complacence and the disease may take a more severe form in the not so distant future, as has been sadly realized by other countries.</p></sec></body><back><fn-group content-type=\"competing-interests\"><fn fn-type=\"COI-statement\"><p>The authors have declared that no competing interests exist.</p></fn></fn-group><fn-group content-type=\"other\"><title>Human Ethics</title><fn fn-type=\"other\"><p>Consent was obtained by all participants in this study. Institutional Ethics Committee-Human Research (IEC-HR), University College of Medical Sciences, University of Delhi, Delhi issued approval IEC-HR/2020/44/2R. Decision: Accept in present form</p></fn></fn-group><fn-group content-type=\"other\"><title>Animal Ethics</title><fn fn-type=\"other\"><p><bold>Animal subjects:</bold> All authors have confirmed that this study did not involve animal subjects or tissue.</p></fn></fn-group><ack><p>The authors wish to thank Professor Karen Bandeen-Roche, Chair, Department of Biostatistics at Johns Hopkins Bloomberg School of Public Health, Baltimore, MD for her guidance in statistical analysis. We also wish to thank Mr. Sandeep, Nursing Officer at the Guru Teg Bahadur Hospital for his help to assemble the data. The samples of the patients were tested at Lady Hardinge Medical College, Delhi; Max Pathology Labs, Delhi, and at the Institute of Liver and Biliary Sciences, Delhi during the early part of the pandemic before the facility was initiated at our hospital. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991483</article-id><article-id pub-id-type=\"pmc\">PMC7523752</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-08574</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022456</article-id><article-id pub-id-type=\"art-access-id\">22456</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3800</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Systematic Review</subject></subj-group></article-categories><title-group><article-title>Efficacy and safety of Chinese herbal compound in the treatment of functional constipation</article-title><subtitle>A protocol for systematic review and meta-analysis</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0003-4416-8334</contrib-id><name><surname>Ji</surname><given-names>Lijiang</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Fan</surname><given-names>Yihua</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Linhui</given-names></name><degrees>MB</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Bai</surname><given-names>Huiwen</given-names></name><degrees>MB</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Weng</surname><given-names>Liping</given-names></name><degrees>MB</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhao</surname><given-names>Ping</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Department of Anorectal Surgery, Changshu Hospital Affiliated to Nanjing University of Chinese Medicine, Changshu, Jiangsu province</aff><aff id=\"aff2\"><label>b</label>First Teaching Hospital of Tianjin University of Traditional Chinese Medicine</aff><aff id=\"aff3\"><label>c</label>Tianjin University of Traditional Chinese Medicine, Tianjin, China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Ping Zhao, Changshu Hospital Affiliated to Nanjing University of Chinese Medicine, No.6 Huang-He Road, Changshu, Suzhou 215500, Jiangsu province, China (e-mail: <email>zhaoping201106@163.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22456</elocation-id><history><date date-type=\"received\"><day>29</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>31</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22456.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>Functional constipation refers to constipation without organic lesions caused by dietary factors, mood depression, changes in life rules, and poor bowel habits. Clinically, constipation is mainly manifested by changes of stool texture, difficulty or lack of bowel movement, and dry stool. Sometimes, it can be accompanied by abdominal distension and abdominal discomfort. Chinese herbal compound is a prescription which is composed of 2 or more medicinal flavors and is designed for relatively certain diseases and syndromes. Clinical studies have shown that TCM compounds have a good therapeutic effect on functional constipation, but there is no evidence of evidence-based medicine. The purpose of this study is to systematically evaluate the efficacy and safety of TCM compounds in the treatment of functional constipation, and to provide evidence-based basis for the clinical application of TCM compounds in the treatment of constipation.</p></sec><sec sec-type=\"methods\"><title>Methods:</title><p>A systematic search was performed on English database (PubMed, Embase, Web of Science, the Cochrane Library) and Chinese database (CNKI, Wanfang, Weipu (VIP), CBM), in addition to the manual retrieval of Baidu and Google academic for randomized controlled trials (RCTs) on the treatment of functional constipation with Chinese herbal compound. The retrieval time limit was from the establishment of the database to August 2020. Two researchers independently screened the literature, extracted the data and evaluated the quality of the included studies. A meta-analysis was performed using RevMan5.3 software.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>In this study, the efficacy and safety of TCM herbal compounds in the treatment of functional constipation were evaluated by the overall effective rate, recovery rate, colonic transmission function, and other indicators.</p></sec><sec sec-type=\"conclusion\"><title>Conclusions:</title><p>This study will provide reliable evidence-based evidence for the clinical application of Chinese herbal compound in the treatment of functional constipation.</p></sec><sec><title>OSF Registration number:</title><p>DOI 10.17605/OSF.IO/D5ECF.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>bowel function</kwd><kwd>Chinese herbal compound</kwd><kwd>functional constipation</kwd><kwd>protocol</kwd><kwd>systematic review</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>the Suzhou Municipal Science and Technology Bureau Supporting Project</funding-source><award-id>No.SYS2019004</award-id><principal-award-recipient>Lijiang Ji</principal-award-recipient></award-group><award-group id=\"award2\" award-type=\"Fundref\"><funding-source>the Suzhou Municipal Science and Technology Bureau Supporting Project</funding-source><award-id>SYSD2019195</award-id><principal-award-recipient>Lijiang Ji</principal-award-recipient></award-group><award-group id=\"award3\" award-type=\"Fundref\"><funding-source>the Suzhou Municipal Science and Technology Bureau Supporting Project</funding-source><award-id>SYSD2018199</award-id><principal-award-recipient>Lijiang Ji</principal-award-recipient></award-group><award-group id=\"award4\" award-type=\"Fundref\"><funding-source>Changshu Municipal Science and Technology Bureau Supporting Project</funding-source><award-id>No. CS201808</award-id><principal-award-recipient>Lijiang Ji</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Functional constipation (FC) is a common functional bowel disorder in clinical practice, manifesting as straining during defecation, lumpy or hard stools and infrequent bowel movements, in the absence of evident organic or structural diseases.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> Functional constipation can not only cause hemorrhoids, anal fissure, colon cancer, and other disease syndromes, but also induce and aggravate cardiovascular and cerebrovascular diseases, leading to acute myocardial infarction, cerebrovascular accident, and other diseases, endangering life.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> Existing studies have shown that the prevalence of functional constipation seems to increase gradually in the age of 50, and reaches the maximum after the age of 70, and the prevalence of functional constipation is generally higher in females than in males.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> The etiology of functional constipation is very complex, including: abnormal functions of colon, rectum, anal canal and pelvic floor muscles,<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> intestinal flora imbalance,<sup>[<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> mental depression and psychological anxiety,<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> smoking and alcohol abuse,<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup> poor bowel habits, poor dietary habits<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> and dietary structure,<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> gender and age, and genetic factors. In clinical practice, western medicine mainly treats functional constipation by adjusting diet structure, biofeedback therapy,<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> drugs, surgery, etc.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup></p><p>Traditional Chinese medicine compound is composed of 2 or more herbs, which is one of the main intervention methods in TCM clinic. Based on the theory of traditional Chinese medicine, the compound medicine addresses both symptoms and root causes by adjusting the zang-fu organs, Yin and Yang, Qi and blood, and using the medicine according to syndrome differentiation. Rich experience has been accumulated in the treatment of constipation with traditional Chinese medicine, which can provide individualized treatment programs according to different conditions of patients to restore intestinal function, with safety and effectiveness, few adverse reactions and no drug dependence.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup></p><p>At present, although many clinical studies have shown that Traditional Chinese medicine has a significant effect on functional constipation, with high cure rate, low recurrence rate, and few adverse reactions, however, the number of clinical trials is small, and there are differences in research design and efficacy, which to some extent affect the promotion of this therapy.</p><p>Therefore, in this study, we conducted a meta-analysis to investigate the effects of TCM compounds on bowel function and quality of life in patients with functional constipation, so as to provide a reliable evidence-based basis for the treatment of functional constipation by TCM compounds.</p></sec><sec><label>2</label><title>Methods</title><sec><label>2.1</label><title>Protocol register</title><p>This protocol of systematic review and meta-analysis has been drafted under the guidance of the preferred reporting items for systematic reviews and meta-analyses protocols (PRISMA-P). Moreover, it has been registered on open science framework (OSF) on August 28, 2020. (Registration number: DOI 10.17605/OSF.IO/D5ECF).</p></sec><sec><label>2.2</label><title>Ethics</title><p>Since this is a protocol with no patient recruitment and personal information collection, the approval of the ethics committee is not required.</p></sec><sec><label>2.3</label><title>Eligibility criteria</title><sec><label>2.3.1</label><title>Types of studies</title><p>We will collected all available randomized controlled trails(RCTs) on Chinese herbal compound treatment for functional constipation, regardless of blinding, publication status, region, but Language will be restricted to Chinese and English.</p></sec><sec><label>2.3.2</label><title>Types of participants</title><list list-type=\"simple\"><list-item><label>1.</label><p>In accordance with the diagnostic criteria of Rome(II, III, IV) for functional constipation;</p></list-item><list-item><label>2.</label><p>No restriction on age, race or gender;</p></list-item><list-item><label>3.</label><p>No other complications.</p></list-item></list></sec><sec><label>2.3.3</label><title>Types of interventions</title><p>The treatment group: use traditional Chinese medicine compound intervention (decoction, Chinese patent medicine) only or combined with western medicine;</p><p>The control group: western medicine intervention or placebo.</p></sec><sec><label>2.3.4</label><title>Types of outcome measures</title><list list-type=\"simple\"><list-item><label>1.</label><p>Main outcome: Overall effective rate, recovery rate and colonic transmission function;</p></list-item><list-item><label>2.</label><p>Secondary outcome: Symptom improvement, recurrence rate, quality of life score, and intestinal flora level.</p></list-item></list></sec></sec><sec><label>2.4</label><title>Exclusion criteria</title><list list-type=\"simple\"><list-item><label>1.</label><p>Studies published repeatedly;</p></list-item><list-item><label>2.</label><p>Studies which were abstracts and conference papers, and in which the original data cannot be obtained;</p></list-item><list-item><label>3.</label><p>Study whose data was incomplete or where there were obvious errors that cannot be handled after contacting the author;</p></list-item><list-item><label>4.</label><p>Study with wrong random method;</p></list-item><list-item><label>5.</label><p>Organic constipation or constipation due to other secondary causes;</p></list-item><list-item><label>6.</label><p>Intervention measures other than traditional Chinese medicine compound or combined with other therapies (such as acupuncture, massage, qigong exercise, psychological therapy).</p></list-item></list></sec><sec><label>2.5</label><title>Search strategy</title><p>“Traditional Chinese medicine”(zhong yao), “functional constipation”(gong neng xing bian mi) and other Chinese search terms were used for retrieval in Chinese databases, including CNKI, Wanfang Data Knowledge Service Platform, VIP Information Chinese Journal Service Platform, and China Biomedical Database. English retrieval words such as “traditional Chinese medicine”, “Chinese patent medicine”, “Chinese medicine prescription”, “functional constipation” were used for retrieval in English databases, including PubMed, EMBASE, Web of Science and the Cochrane Library. In addition, manual retrieval was performed in Baidu and Google academic. The retrieval time was from the establishment of the database to August 2020, and all domestic and foreign literatures on the treatment of functional constipation with Traditional Chinese medicine were collected. Take PubMed as an example, and the retrieval strategy is shown in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Search strategy in PubMed database.</p></caption><graphic xlink:href=\"medi-99-e22456-g001\"/></table-wrap></sec><sec><label>2.6</label><title>Data screening and extraction</title><p>Cochrane Collaboration System Reviewer Manual Version 5.0 was used as a reference for the method of selection in the study. According to the PRISMA flow chart, EndNote X7 document management software was utilized by 2 researchers to independently screen the documents based on the above inclusion and exclusion criteria before mutual check. Those difficult to determine whether included in the study, would be discussed and judged with a third researcher. At the same time, Excel 2013 was used to extract relevant information, including: ① Clinical features (title, first author, publication year and month, sample size, sex ratio, average age, average course of disease); ② Intervention measures: The name, composition, dosage form, frequency and course of treatment of the Chinese herbal compound used in the treatment group, the name, frequency, and course of treatment of western medicine;</p><p>The name, administration method, frequency, and course of treatment of western medicine used in the control group; ③ Evaluation factors of risk bias in randomized controlled studies; ④ Observation indicators. The literature screening process is shown in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Flow diagram.</p></caption><graphic xlink:href=\"medi-99-e22456-g002\"/></fig></sec><sec><label>2.7</label><title>Literature quality evaluation</title><p>Built-in Risk bias evaluation tool of Review Manager 5.3 Software (the Cochrane collaborations tool for assessing risk of bias) was used to assess the risk bias in the included studies. Two researchers determined the literatures from 3 levels, including low-risk, unclear, and high-risk based on the performance of the included literature in the above evaluation items. After completion, they would recheck. In case of a disagreement, they would discuss. If no agreement could be reached, a decision would be made in consultation with researchers from the third party.</p></sec><sec><label>2.8</label><title>Statistical analysis</title><sec><label>2.8.1</label><title>Data analysis and processing</title><p>The RevMan 5.3 software provided by the Cochrane Collaboration was used for statistical analysis. ① For dichotomous variables, relative risk (RR) was used for statistics. For continuous variables, weighted mean difference (WMD) was selected when the tools and units of measurement indicators are the same, standardized mean difference (SMD) was selected with different tools or units of measurement, and all the above were represented by effect value and 95% Confidence interval (CI). ② Heterogeneity test: Q test was used to qualitatively determine inter-study heterogeneity. If <italic>P</italic> ≥ .1, there was no inter-study heterogeneity, If <italic>P</italic> < .1, it indicated inter-study heterogeneity. At the same time, <italic>I</italic><sup>2</sup> value was used to quantitatively evaluate the inter-study heterogeneity. If <italic>I</italic><sup><italic>2</italic></sup> ≤ 50%, the heterogeneity was considered to be good, and the fixed-effect model was adopted. If <italic>I</italic><sup><italic>2</italic></sup> > 50%, it was considered to have significant heterogeneity, the source of heterogeneity would be explored through subgroup analysis or sensitivity analysis. If there was no obvious clinical or methodological heterogeneity, it would be considered as statistical heterogeneity, and the random-effect model would be used for analysis. Descriptive analysis was used if there was significant clinical heterogeneity between the 2 groups and subgroup analysis was not available.</p></sec><sec><label>2.8.2</label><title>Dealing with missing data</title><p>If data is missing or incomplete, we will contact the corresponding author to obtain the missing data. If not, this study will be removed.</p></sec><sec><label>2.8.3</label><title>Subgroup analysis</title><p>Subgroup analysis was carried out according to the treatment group of Chinese herbal compound assisted other therapies and Chinese herbal compound treatment only; subgroup analysis was carried out according to the age of the patients, which can be divided into 4 subgroups: minors, young people, middle-aged people, and elderly people. Subgroup analysis was carried out according to the clinical classification of functional constipation, which can be divided into 3 subgroups: normal type, slow type, and defecation disorder type. Subgroup analysis was carried out according to the TCM syndromes, which can be divided into 5 subgroups: xu mi(deficient constipation), re mi(constipation due to heat), leng mi(constipation due to interior cold), qi mi(constipation due to qi stagnation), xue mi(constipation with blood deficiency syndrome). Subgroup analysis was carried out according to the types of Chinese herbal compound used.</p></sec><sec><label>2.8.4</label><title>Sensitivity analysis</title><p>In order to test the stability of meta-analysis results of outcomes, a one-by-one elimination method will be adopted for sensitivity analysis.</p></sec><sec><label>2.8.5</label><title>Assessment of reporting biases</title><p>For the major outcome indicators, if the included study was ≥10, funnel plot was used to qualitatively detect publication bias. Eggers and Beggs test are used to quantitatively assess potential publication bias.</p></sec><sec><label>2.8.6</label><title>Evidence quality evaluation</title><p>The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) will be used to assess the quality of evidence. It contains 5 domains (bias risk, consistency, directness, precision, and publication bias). And the quality of evidence will be rated as high, moderate, low, and very low.</p></sec></sec></sec><sec><label>3</label><title>Discussion</title><p>Functional constipation belongs to the categories of “constipation” (Bian mi) and “difficulty in defecation” (Da bian nan) in Traditional Chinese medicine. In the early stage of TCM, a lot of experience has been accumulated in the clinical treatment of functional constipation, and many effective therapies have been designed.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> According to traditional medicine, the etiology and pathogenesis of constipation can be divided into 5 aspects:</p><list list-type=\"simple\"><list-item><label>1.</label><p>“Re mi”(constipation due to heat) caused by gastrointestinal heat and body fluid consumption.</p></list-item><list-item><label>2.</label><p>“Qi mi”(constipation due to qi stagnation) caused by stagnation of the qi and abnormal ventilation and depression.</p></list-item><list-item><label>3.</label><p>“Leng mi”(constipation due to interior cold) caused by excrescent Yin and cold stagnating in the gastrointestinal tract.</p></list-item><list-item><label>4.</label><p>“Yang xu mi”(constipation with yang deficiency syndrome) caused by Qi and Yang deficiency, Yang qi cannot pushing and defecation time prolonging.</p></list-item><list-item><label>5.</label><p>“Xue xu mi”(constipation with blood deficiency syndrome) caused by Yin and blood deficiency, which leads to defecation difficulties.<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup></p></list-item></list><p>Through treatment based on syndrome differentiation, TCM often uses tonifying, Catharsis, and warming methods in clinical treatment of functional constipation with different syndromes. Studies have shown that Buzhong Yiqi Decoction can regulate the secretion of gastrointestinal hormones, elevate the level of Human serum Substance P (SP), reduce the level of Plasma Motilin (MTL), enhance gastrointestinal motility, and improve gastrointestinal motility disorders.<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]</sup> Both Xuanfei Tongbian Decoction and Maziren Pill could regulate the serum enteric neurotransmitter level by up-regulating SP and down-regulating nitric oxide (NO),<sup>[<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</sup> improve gastrointestinal motility, and play a role in the treatment of functional constipation.<sup>[<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]</sup> Yiqi Kaimi Prescription and Qishen Gutuo Mixture can increase the anal resting pressure, restore the anorectal sensory function and the coordination of pelvic floor muscle groups,<sup>[<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</sup> and thus restore the normal process of voluntary defecation.<sup>[<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]</sup> Wenyang Xuanfei Prescription can significantly improve the expression level of Cajal mesenchymal cell-specific protein C-Kit and Stem Cell Factor (SCF) in rat colon by tonifying kidney and warming Yang, shorten defecation time, and promote intestinal peristalsis.<sup>[<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</sup> Xiaochaihu Decoction can relieve anxiety and depression symptoms of some patients with functional constipation, improve their defecation ability, and improve constipation.<sup>[<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]</sup> Yiqi Xuanfei Tongbian Prescription can regulate gastrointestinal flora level, inhibit the release of inflammatory factors, promote the release of gastrointestinal secretions, and improve gastrointestinal function.<sup>[<xref rid=\"R22\" ref-type=\"bibr\">22</xref>]</sup> Sini Powder and Yunchang Capsule can shorten the colon transmission time and improve constipation symptoms.<sup>[<xref rid=\"R23\" ref-type=\"bibr\">23</xref>,<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]</sup> A variety of traditional Chinese medicine compound components can also regulate the expression of aquaporins (AQPs) in the intestine and maintain the balance of water and liquid metabolism in the body.<sup>[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]</sup></p><p>At present, there are many widely reported trials on different TCM compounds for the treatment of functional constipation, and TCM compounds have achieved good clinical efficacy in the treatment of functional constipation,<sup>[<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]</sup> but there is a lack of systematic and correct evaluation. Therefore, it is necessary to objectively evaluate the effect of TCM compounds on patients with functional constipation through evidence-based medicine, promote the treatment of TCM, and provide scientific and evidence-based TCM prescriptions for clinical practice. However, this study also has some limitations. The composition, dosage and course of treatment of the Chinese herbal compound included in the study were inconsistent, there may be some clinical heterogeneity, and the follow-up time is short, so it is difficult to evaluate the long-term efficacy of the Chinese herbal compound. In addition, many studies did not involve allocation-hiding and blind methods, and the methodological quality was poor, with some bias. At the same time, due to the limitation of language ability, only English and Chinese literature were searched, and studies in other languages may be ignored, which may lead to certain publication bias.</p></sec><sec><title>Author contributions</title><p><bold>Data curation:</bold> Lijiang Ji, Yihua Fan.</p><p><bold>Funding acquisition:</bold> Ping Zhao.</p><p><bold>Investigation:</bold> Ping Zhao.</p><p><bold>Resources:</bold> Linhui Li, Huiwen Bai.</p><p><bold>Software:</bold> Linhui Li, Huiwen Bai, Liping Weng.</p><p><bold>Supervision:</bold> Ping Zhao.</p><p><bold>Writing – original draft:</bold> Lijiang Ji, Yihua Fan.</p><p><bold>Writing – review & editing:</bold> Lijiang Ji.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: AQPs = aquaporins, CI = confidence interval, CNKI = China Knowledge Network, FC = Functional constipation, GRADE = The Grading of Recommendations Assessment, Development, and Evaluation, MTL = level of Plasma Motilin, NO = nitric oxide, OSF = open science framework, PRISMA-P = preferred reporting items for systematic reviews and meta-analyses protocols, RCTs = randomized controlled trails, RR = relative risk, SCF = Recombinant Stem Cell Factor, SMD = standardized mean difference, SP = substance P, VIP = VIP Information Chinese Journal Service Platform, WMD = weighted mean difference.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Ji L, Fan Y, Li L, Bai H, Weng L, Zhao P. Efficacy and safety of Chinese herbal compound in the treatment of functional constipation: a protocol for systematic review and meta-analysis. <italic>Medicine</italic>. 2020;99:39(e22456).</p></fn><fn fn-type=\"supported-by\"><p>This work is supported by the Suzhou Municipal Science and Technology Bureau Supporting Project (No.SYS2019004, SYSD2019195, SYSD2018199), and Changshu Municipal Science and Technology Bureau Supporting Project (No. CS201808).</p></fn><fn fn-type=\"other\"><p>The private information from individuals will not be published. This systematic review also will not involve endangering participant rights. Ethical approval is not required. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991443</article-id><article-id pub-id-type=\"pmc\">PMC7523753</article-id><article-id pub-id-type=\"publisher-id\">MD-D-19-05851</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022326</article-id><article-id pub-id-type=\"art-access-id\">22326</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3400</subject></subj-group><subj-group><subject>Research Article</subject><subject>Clinical Case Report</subject></subj-group></article-categories><title-group><article-title>Fabry disease with early-onset ventricular dilation</article-title><subtitle>A case report</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Tang</surname><given-names>Jia</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wu</surname><given-names>Chao</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Cao</surname><given-names>Jian</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Liang</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Saranathan.</surname><given-names>Maya</given-names></name></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Peking Union Medical College Hospital (PUMCH)</aff><aff id=\"aff2\"><label>b</label>Department of Cardiology, Fuwai Hospital, National Center for Cardiovascular Diseases</aff><aff id=\"aff3\"><label>c</label>Department of Radiology</aff><aff id=\"aff4\"><label>d</label>Department of Cardiology, Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Science (CAMS) and Peking Union Medical College (PUMC), Beijing, China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Liang Wang, Department of Cardiology of Peking Union Medical College Hospital, No.1 Shuaifuyuan, Dongcheng District, Beijing 100730, China (e-mail: <email>wzmwll@sina.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22326</elocation-id><history><date date-type=\"received\"><day>30</day><month>7</month><year>2019</year></date><date date-type=\"rev-recd\"><day>26</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22326.pdf\"/><abstract><title>Abstract</title><sec><title>Rationale:</title><p>The most common cardiac involvement of Fabry disease (FD) is left ventricular hypertrophy (LVH), which usually occurs in male patients over the age of 30. In rare cases, it can progress to ventricular dilation in the late stage of the disease.</p></sec><sec><title>Patient concerns:</title><p>A 16-year-old boy presenting with recurrent extremity pain and chest distress was admitted to our hospital. Imaging examinations revealed ventricular dilation.</p></sec><sec><title>Diagnosis:</title><p>α-Galactosidase A enzyme assay and <italic>GLA</italic> gene sequencing confirmed the diagnosis of FD and revealed a novel mutation c.76_77insT.</p></sec><sec><title>Interventions:</title><p>The patient was treated using metoprolol (23.75 mg qd) and angiotensin-converting enzyme inhibitor (fosinopril sodium 5 mg qd). He refused enzyme replacement therapy for financial reasons.</p></sec><sec><title>Outcomes:</title><p>The echocardiography, electrocardiography, renal function, and routine blood and urine tests performed 20 months after the patients discharge from hospital showed no significant changes. The patient reported a slow and gradual decrease in the frequency and degree of pain and chest distress, starting approximately 24 months after discharge.</p></sec><sec><title>Lessons:</title><p>Cardiac involvement of FD can progress rapidly in some cases. Screening for FD should be considered in patients with unexplained ventricular dilation, especially in those with a history of typical FD manifestations.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>Fabry disease</kwd><kwd>ventricular dilation</kwd><kwd>T1 mapping</kwd><kwd>myocardial fibrosis</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Fabry disease (FD) is an X-linked recessive lysosomal storage disease caused by a deficiency of the α-galactosidase A enzyme (encoded by the <italic>GLA</italic> gene). The disease can affect multiple organs, including the heart, brain, and kidneys.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> The most common cardiac involvement is concentric left ventricular hypertrophy (LVH), which usually occurs in male patients over the age of 30.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> In rare cases, hypertrophic cardiomyopathy can progress to a dilated phase in the late stage of the disease.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>,<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> In the present report, we describe the case of a 16-year-old boy diagnosed with FD who developed ventricular dilation. To our knowledge, this is the first reported case of early-onset ventricular dilation in FD, suggesting that cardiac involvement can progress relatively rapidly in some cases.</p></sec><sec><label>2</label><title>Case presentation</title><p>A 16-year-old male Chinese patient was admitted to our hospital due to a 4-year history of recurrent upper and lower extremity pain with low-grade fever up to 37.5°C, as well as a 2-year history of chest distress and dyspnea after activity. He complained of an aggravation of chest distress and dyspnea 3 months before admission. No other medical history was reported. Physical examination revealed sporadic angiokeratomas on his waist and back (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref> C). His blood pressure was normal when he had no extremity pain and could rise to approximately 160/110 mm Hg, with a heart rate of 110 bpm, when the pain attacked. Twelve-lead electrocardiogram showed QT prolongation (QTc = 501 ms) and intraventricular conduction block (QRS = 152 ms) (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref> A and B). Transthoracic echocardiography (TTE) revealed dilated left and right ventricles, mild mitral and tricuspid valve insufficiency, and an ejection fraction of 61%, with no evidence of myocardial hypertrophy (Table <xref rid=\"T1\" ref-type=\"table\">1</xref>, Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref> D). Cardiovascular magnetic resonance (CMR) confirmed that the ventricles were dilated (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref> F and G, Table <xref rid=\"T2\" ref-type=\"table\">2</xref>). No significant abnormalities were found in first-pass perfusion or late gadolinium enhancement (LGE) CMR. Native T1 mapping of the left ventricle is shown in Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref> E. The mean T1 was 1262 ms. Routine blood, urine, and stool tests were all unremarkable, as were hepatic and renal function, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), joint X-ray and magnetic resonance imaging (MRI), 24-hour urine catecholamine assay, <sup>99m</sup>Tc-octreotide scan, and ophthalmologic examination. The patient was negative for human leukocyte antigen-B27 (HLA-B27). Leukocyte α-galactosidase A activity was 0.1 nmol/hour/mg protein, which is below the normal range of 29.0 to 64.4 nmol/hour/mg protein. Polymerase chain reaction amplification and Sanger sequencing of the <italic>GLA</italic> exons from genomic DNA of the patient revealed an insertion of 1 nucleotide between positions 76 and 77 in the cDNA (c.76_77insT; Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref> H). The present report is the first to describe this novel mutation, which can generate a premature termination codon at amino acid position 30. The patients mother was heterozygous for the mutation, as shown in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref> I.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>(A) I, II, III, aVR, aVL, aVF leads. (B) V1-V6 leads. (C) Sporadic angiokeratomas on the waist and back. (D) Transthoracic echocardiogram showing left ventricular dilation. LVIDs = left ventricular internal diameter in systole, LVIDd = left ventricular internal diameter in diastole, EDV = end-diastolic volume, ESV = end-systolic volume, FS = short-axis fractional shortening, EF = ejection fraction. (E) Native T1 mapping of the left ventricle. (F) Long-axis four-chamber CMR in diastole. (G) Short-axis two-chamber CMR in diastole. (H) Part of the electropherogram of exon 1 of the <italic>GLA</italic> gene in the patient. The mutation is indicated by the arrow. (I) The same part of the gene in the patients mother, in which the mutation is indicated by the arrow.</p></caption><graphic xlink:href=\"medi-99-e22326-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 1 (Continued)</label><caption><p>(A) I, II, III, aVR, aVL, aVF leads. (B) V1-V6 leads. (C) Sporadic angiokeratomas on the waist and back. (D) Transthoracic echocardiogram showing left ventricular dilation. LVIDs = left ventricular internal diameter in systole, LVIDd = left ventricular internal diameter in diastole, EDV = end-diastolic volume, ESV = end-systolic volume, FS = short-axis fractional shortening, EF = ejection fraction. (E) Native T1 mapping of the left ventricle. (F) Long-axis four-chamber CMR in diastole. (G) Short-axis two-chamber CMR in diastole. (H) Part of the electropherogram of exon 1 of the <italic>GLA</italic> gene in the patient. The mutation is indicated by the arrow. (I) The same part of the gene in the patients mother, in which the mutation is indicated by the arrow.</p></caption><graphic xlink:href=\"medi-99-e22326-g002\"/></fig><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Transthoracic echocardiogram results.</p></caption><graphic xlink:href=\"medi-99-e22326-g003\"/></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Cardiovascular magnetic resonance results.</p></caption><graphic xlink:href=\"medi-99-e22326-g004\"/></table-wrap><p>The patient was treated using a non-steroidal anti-inflammatory drug (loxoprofen sodium 60 mg q8 hour) after admission, but his extremity pain did not improve. After the definitive diagnosis of FD, his medication was changed to tramadol (100 mg prn), metoprolol (23.75 mg qd), and angiotensin-converting enzyme inhibitor (fosinopril sodium 5 mg qd). Metoprolol and fosinopril sodium were used to delay ventricular remodeling and the possible future decline of renal function. The patient refused enzyme replacement therapy for financial reasons. The tramadol treatment only partially relieved the patients extremity pain, and was discontinued 2 weeks later. The patient was followed up for 29 months. Twenty months after his discharge from the hospital, echocardiography, electrocardiography, renal function, and routine blood and urine tests showed no significant changes. Starting approximately 24 months after his discharge, the patient reported a slow and gradual decrease in the frequency and degree of extremity pain and chest distress, which corroborated previous findings that pain tends to diminish with age.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup></p></sec><sec><label>3</label><title>Discussion</title><p>α-Galactosidase A enzyme deficiency in FD results in an abnormal accumulation of glycosphingolipids in various organs. The typical clinical presentations of FD begin in childhood or adolescence and include acroparesthesia, angiokeratomas, gastrointestinal symptoms, corneal opacities, and renal manifestations.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> Cardiac and cerebrovascular involvement tends to occur in adulthood. The most common cardiac involvement is concentric LVH. Others include myocardial fibrosis, heart failure, coronary artery disease, aortic and mitral valve abnormalities, and conduction abnormalities.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup></p><p>The present patient was a 16-year-old boy who presented with acroparesthesia, angiokeratomas, and chest distress. The results of the enzyme assay and <italic>GLA</italic> gene sequencing confirmed the diagnosis of FD. Although we identified the same mutation in his mother, there was no family history of FD or related symptoms. Since the most common cardiac manifestation of FD is adult-onset LVH, the results of TTE and CMR showing ventricular dilation confused us before we performed the enzyme assay and gene sequencing. We considered spondyloarthropathy, but excluded this possibility when the ESR, CRP, HLA-B27, and joint X-ray and MRI were negative. Similarly, we excluded pheochromocytoma because the 24-hour urine catecholamine assay and <sup>99m</sup>Tc-octreotide scan were normal. Although some studies have reported that LVH can progress to a dilated phase in the late stage of the disease,<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>,<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> the present study constitutes the first reported case of early-onset ventricular dilation in FD. Moreover, we identified a novel mutation: c.76_77insT.</p><p>The mechanism underlying the formation of dilated ventricles is uncertain. Since the diagnosis was clear in the present case, we did not perform an endomyocardial biopsy for ethical reasons. However, electrocardiography revealed conduction abnormalities, and TTE showed mitral and tricuspid valve regurgitation, indicating that the ventricular dilation was caused by FD. Conduction abnormalities are thought to result from glycosphingolipid deposition in the conduction system of the heart.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> Valve regurgitation is also common in the disease.</p><p>Another noteworthy finding was the result of CMR T1 mapping. Sado et al reported that the mean T1 in FD patients was approximately 200 ms lower than that in healthy controls.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> However, in the present case, the mean T1 was only slightly lower than the normal value, which is 1300 ms in our hospital. Sado et al suggested that the T1 lowering is caused by glycosphingolipid deposition and water-lipid interaction. However, they also found that, in some segments with myocardial fibrosis, the local T1 could be normal or even raised because of regional fibrosis. Moreover, several studies have shown that, regardless of the LGE result, the mean T1 in patients with dilated cardiomyopathy is higher than that in normal controls, suggesting diffuse fibrosis.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>,<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup> Therefore, it may be that the mean T1 in the present patient was slightly reduced because his lesions had progressed to a myocardial scarring phase, in which diffuse fibrosis can elevate or pseudonormalize T1. Diffuse fibrosis may also explain the normal LGE-CMR result.</p><p>The natural course of FD cardiac variant progresses from glycosphingolipid deposition to myocardial fibrosis, with thinning of the affected walls.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> In the present case report, we further hypothesized that diffuse fibrosis itself causes ventricle dilation. It follows that, depending on the speed of progression of fibrosis, the dilated phase may occur in the early stages of the disease, not only in the late stages as other cases have suggested. Therefore, in patients with ventricular dilation of unknown causes, comprehensive history taking and thorough physical examination should be performed. If typical manifestations of FD exist, such as acroparesthesia, angiokeratomas, or renal involvement, then a diagnosis of FD should be considered, and an α-galactosidase A enzyme assay should be conducted to verify the diagnosis.</p></sec><sec><label>4</label><title>Informed consent</title><p>Written informed consent was obtained from the patient and his parents for publication of this case report and any accompanying images. The study was approved by the local ethics committee of Peking Union Medical College.</p></sec><sec><title>Author contributions</title><p><bold>Resources:</bold> Liang Wang.</p><p><bold>Supervision:</bold> Liang Wang.</p><p><bold>Visualization:</bold> Jian Cao.</p><p><bold>Writing – original draft:</bold> Jia Tang, Chao Wu.</p><p><bold>Writing – review & editing:</bold> Liang Wang.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: FD = Fabry disease, LVH = left ventricular hypertrophy, TTE = transthoracic echocardiogram, CMR = cardiovascular magnetic resonance, LGE = late gadolinium enhancement, ESR = erythrocyte sedimentation rate, CRP = C-reactive protein, MRI = magnetic resonance imaging, HLA-B27 = human leukocyte antigen-B27.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Tang J, Wu C, Cao J, Wang L. Fabry disease with early-onset ventricular dilation: A case report. <italic>Medicine</italic>. 2020;99:39(e22326).</p></fn><fn fn-type=\"financial-disclosure\"><p>There is no funding for this article.</p></fn><fn fn-type=\"equal\"><p>JT and CW are contributed equally to this work and should be considered co-first authors.</p></fn><fn fn-type=\"COI-statement\"><p>The authors have no conflicts of interest to disclose.</p></fn><fn fn-type=\"other\"><p>The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>[1]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Germain</surname><given-names>DP</given-names></name></person-group>\n<article-title>Fabry disease</article-title>. <source>Orphanet J Rare Dis</source>\n<year>2010</year>;<volume>5</volume>:<fpage>30</fpage>.<pub-id 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991464</article-id><article-id pub-id-type=\"pmc\">PMC7523755</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-08530</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022397</article-id><article-id pub-id-type=\"art-access-id\">22397</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>7100</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Clinical Trial</subject></subj-group></article-categories><title-group><article-title>Retrospective cohort trial protocol of screw fixation compared with hemiarthroplasty for displaced femoral neck fractures in elderly patients</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Qin</surname><given-names>Boquan</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Cui</surname><given-names>Linxian</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ren</surname><given-names>Yi</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0003-1754-7216</contrib-id><name><surname>Zhang</surname><given-names>Hui</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Department of Orthopaedic Surgery, West China Hospital, Sichuan University</aff><aff id=\"aff2\"><label>b</label>Department of Cardiology, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Sichuan, China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Hui Zhang, Department of Orthopaedic Surgery, West China Hospital, Sichuan University, Sichuan 610041, China (e-mail: <email>caesarzh@163.com)</email></corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22397</elocation-id><history><date date-type=\"received\"><day>27</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22397.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>There is limited evidence for the evaluation of the efficacy and safety of the hemiarthroplasty versus screw fixation in elderly patients with the displaced femoral neck fractures. Our current investigation aimed at assessing the complications, functional outcome, and revision rate of the patients (over 65 years old) who received internal fixation or hemiarthroplasty via a same senior surgeon.</p></sec><sec sec-type=\"methods\"><title>Methods:</title><p>A retrospective study was conducted on elderly patients with displaced femoral neck fractures from May 2014 to February 2018. The current study was carried out at our hospital and it was approved through our institutional review committee of West China Hospital. Inclusion criteria were as follows: the patients were 65 years or older, this is the anesthesia grade. The higher grade of the patients,the greater risk of surgery. level I–III, and the patients with displaced intracapsular fractures of the femoral neck, with the radiographic and clinical follow-up of 12 months or more. The major outcome was the revision rate between the 2 groups. And the secondary outcomes contained the life quality and functional outcome detected via utilizing the interview-administered and self-administered questionnaires, length of hospital stay, surgery time, and hip-related complications (such as hip dislocation, loosening or breakage of implant, wound problems, infection, osteolysis, neurovascular injury, and bone nonunion).</p></sec><sec sec-type=\"results\"><title>Results:</title><p>It was assumed that hemiarthroplasty would result in fewer revisions or complications and better functional scores in comparison with internal fixation technique.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>displaced femoral neck fractures</kwd><kwd>hemiarthroplasty</kwd><kwd>protocol</kwd><kwd>revision</kwd><kwd>screw fixation</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>Sichuan Science and Technology Department Project</funding-source><award-id>2019YFS0270</award-id><principal-award-recipient>Hui Zhang</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Displaced femoral neck fractures are the most familiar injuries in the elderly. With the increase of the elderly population in China, the number of displaced femoral neck fractures is also increasing.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> They can be challenging to treat, as the femoral neck lacks periosteum and therefore relies on direct bone healing, resulting in the rates of osteonecrosis and nonunion of femoral head is high. Although the incidence of injury is high, in the elderly patients, the surgical treatment of displaced femoral neck fractures is still uncertain.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>–<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup></p><p>Internal fixation is the optimism method to treat the elderly displaced femoral neck fractures patients. An alternative treatment option for the elderly patients with displaced femoral neck fractures is hemiarthroplasty, because the treatment allows the patient to move early.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> Nevertheless, the optimal treatment of a independent, removable patients with the femoral neck displaced intracapsular fracture is still controversial.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>–<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> Many former researches have conducted the comparison between internal fixation and hemiarthroplasty or either total hip arthroplasty, and the obtained results indicate that the internal fixation technique is less effective, and its reoperation rate is between 18% and 47%.<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>–<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</sup></p><p>Nevertheless, there is limited evidence for the evaluation of the efficacy and safety of the hemiarthroplasty versus screw fixation in elderly patients with the displaced femoral neck fractures. The superiority of the 2 approaches remains uncertain owing to the lack of a direct comparison of clinical effects between the 2 techniques in the present literature. Our current investigation aimed at assessing the complications, functional outcome, and revision rate of the patients (over 65 years old) who received internal fixation or hemiarthroplasty via a same senior surgeon. It was assumed that hemiarthroplasty would result in fewer revisions or complications and better functional scores in comparison with internal fixation technique.</p></sec><sec><label>2</label><title>Materials and methods</title><sec><label>2.1</label><title>Study design</title><p>A retrospective study was conducted on elderly patients with displaced femoral neck fractures from May 2014 to February 2018. All cases were performed by a surgeon. The current study was carried out at our hospital and it was approved through our institutional review committee of West China Hospital (SC001090). This current investigation also has been registered in Research Registry (researchregistry5956).</p></sec><sec><label>2.2</label><title>Study population</title><p>Inclusion criteria were as follows: the patients were 65 years or older, with the American Society of Anesthesiologists level I–III, and the patients with displaced intracapsular fractures of the femoral neck, with the radiographic and clinical follow-up of 12 months or more. And exclusion criteria contained the patients under the age of 65, preexisting hip abnormality (such as osteoarthritis), patients with the American Society of Anesthesiologists IV level, pathological fracture, and patients with physical or medical comorbidities that restricting walking, and patients with ongoing infectious disease.</p></sec><sec><label>2.3</label><title>Intervention and techniques</title><p>All the surgeries in our current study were implemented via a senior surgeons, or via a researcher under his direct supervision. Prior to incision, each patient was given the general anesthesia to prepare for the operation of hip joint, and then covered with a routine sterile manner. Here are the technical details of 2 groups:</p><sec><label>2.3.1</label><title>Hemiarthroplasty group</title><p>All the operations were conducted through the fellowship trained consultants with a posterior or lateral approach depending on the surgeon's preference. The standard 130 mm cementless porous coated femoral stems were utilized. The head size of the prosthesis is increased by 2 mm, which could accurately replicate the femoral head of the patient, and the femoral head size was determined using the hemispherical template during surgery.</p></sec><sec><label>2.3.2</label><title>Screw fixation group</title><p>Two 7.0 mm diameter partially threaded, cannulated, and cancellous screws (Hip Pins; Smith & Nephew) were inserted, with inferior screw as close to the medial cortex as possible. The 2 screws were positioned posteriorly and centrally and then they inserted as deep as possible in order to ensure that the screws can be fixed in subchondral bone. And if femoral head tilts backwards, the surgeon will attempt a closed reduction.</p></sec></sec><sec><label>2.4</label><title>Postoperative care</title><p>One day after operation, a medium volume hemovac drain was placed and then removed. Low-dose warfarin or aspirin were utilized to prevent thrombosis after operation. The patients took 5 mg of warfarin orally on the night of operation, and then adjusted daily as needed to keep the international standardized ratio of 1.8 to 2.2. All patients were given a same standardized multimodality pain regimen after operation, with 2 doses of 200 mg of celecoxib, 4 doses (1 g) of the acetaminophen, the morphine (first 48 hours) or the tramadol (after 48 hours) to relieve pain. All the patients adopted a same rehabilitation program after operation. On the first day after surgery, they received exercise training in the range of activities and used crutches to carry out the partial weight-bearing. At the same time, all the patients were given a same preoperative antibiotic regimen.</p></sec><sec><label>2.5</label><title>Outcome measures</title><p>The major outcome was the revision rate between the 2 groups. And the secondary outcomes contained the life quality and functional outcome detected via utilizing the interview-administered and self-administered questionnaires, length of hospital stay, surgery time, and hip-related complications (such as hip dislocation, loosening or breakage of implant, wound problems, infection, osteolysis, neurovascular injury, and bone nonunion). Functional outcome questionnaires include hip disability and osteoarthritis outcome score (HOOS) and the Harris hip score (HHS) (Table <xref rid=\"T1\" ref-type=\"table\">1</xref>).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Postoperative outcomes.</p></caption><graphic xlink:href=\"medi-99-e22397-g001\"/></table-wrap><p>HHS was developed to evacuate the hip surgery outcomes and to assess a variety of hip disabilities and the related treatments in adult population. The areas covered include function, pain, the range of motion, and loss of deformity. The highest score is 100 points (optimism possible outcome), including function (7 items, between 0 and 47 points), pain (1 item, between 0 and 44 points), the motion range (2 items, 5 points), and the loss of deformity (1 item, 4 points).</p><p>HOOS is a tool to evaluate the patients’ views on the hip and the related issues. It is designed to be utilized in adults with the hip disability without or with osteoarthritis. HOOS was composed of 5 subscales: pain, function of daily activities, other symptoms, function of recreation and exercise, and quality of life associated with hip joints.</p></sec><sec><label>2.6</label><title>Statistical analysis</title><p>All the analyses were implemented with the software of SAS (version 9.3 and SAS Enterprise Guide 6.1; SAS Institute, American NCSU). The error probability of type-I of all the analysis was set as <italic>P</italic> < .05. The medians and proportions were utilized to describe the baseline cohort characteristics, and then the were compared baseline cohort characteristics via applying chi-square tests for the categorical variables and Wilcoxon rank sum tests for the continuous variables between the 2 groups. For all the tests, <italic>P</italic> < .05 was considered as significance in statistics.</p></sec></sec><sec><label>3</label><title>Discussion</title><p>Intracapsular hip fracture is a most familiar reasons for elderly patients to enter the acute orthopedic ward. In the absence of displacement, almost all the intramedullary femoral neck fractures in China have been treated with the screw osteosynthesis, there is no consensus on the displaced femoral neck fractures treatment. Our present investigation aims at assessing the complications, functional outcome, and revision rate of the patients (over 65 years old) who received internal fixation or hemiarthroplasty via a same senior surgeon. It is assumed that hemiarthroplasty would result in fewer revisions or complications and better functional scores in comparison with internal fixation technique. The limitations of our research involved the use of a single surgeon, a single implant model and manufacturer, and the lack of patient randomization, as well as the lack of advanced imaging techniques (computed tomography) for the accurate measurements before and after the measurements. Furthermore, the limitations of our present investigation contain the inherent limitations in any retrospective cohort research, involving the observational bias or the possibility of selection.</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization:</bold> Boquan Qin, Yi Ren, Hui Zhang.</p><p><bold>Data curation:</bold> Boquan Qin, Linxian Cui, Yi Ren.</p><p><bold>Formal analysis:</bold> Boquan Qin, Linxian Cui, Yi Ren.</p><p><bold>Funding acquisition:</bold> Hui Zhang.</p><p><bold>Investigation:</bold> Boquan Qin, Yi Ren.</p><p><bold>Methodology:</bold> Boquan Qin, Linxian Cui.</p><p><bold>Resources:</bold> Hui Zhang.</p><p><bold>Software:</bold> Linxian Cui.</p><p><bold>Supervision:</bold> Hui Zhang.</p><p><bold>Validation:</bold> Linxian Cui, Yi Ren.</p><p><bold>Visualization:</bold> Linxian Cui, Yi Ren.</p><p><bold>Writing – original draft:</bold> Boquan Qin.</p><p><bold>Writing – review & editing:</bold> Hui Zhang, Linxian Cui.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: HHS = Harris hip score, HOOS = hip disability and osteoarthritis outcome score.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Qin B, Cui L, Ren Y, Zhang H. Retrospective cohort trial protocol of screw fixation compared with hemiarthroplasty for displaced femoral neck fractures in elderly patients. <italic>Medicine</italic>. 2020;99:39(e22397).</p></fn><fn fn-type=\"other\"><p>Trial registration: This study protocol was registered in Research Registry (researchregistry5956).</p></fn><fn fn-type=\"financial-disclosure\"><p>Funding is from Sichuan Science and Technology Department Project (2019YFS0270).</p></fn><fn fn-type=\"COI-statement\"><p>The authors have no conflicts of interest to disclose.</p></fn><fn fn-type=\"other\"><p>Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>[1]</label><mixed-citation publication-type=\"journal\"><person-group 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991462</article-id><article-id pub-id-type=\"pmc\">PMC7523756</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-08519</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022394</article-id><article-id pub-id-type=\"art-access-id\">22394</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3700</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Clinical Trial</subject></subj-group></article-categories><title-group><article-title>Perioperative analgesia after intrathecal morphine or local infiltration anesthesia for total knee replacement</article-title><subtitle>A protocol for randomized controlled trial</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Qi</surname><given-names>Zhengrong</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Guo</surname><given-names>Ai</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0001-5756-9810</contrib-id><name><surname>Ma</surname><given-names>Lifeng</given-names></name><degrees>MD</degrees><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Zhiyao</given-names></name><degrees>MM</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Bo</given-names></name><degrees>MM</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Jingxin</given-names></name><degrees>MM</degrees></contrib></contrib-group><aff>Department of Orthopedics, Beijing Friendship Hospital, Capital Medical University, Beijing, China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Lifeng Ma, Department of Orthopedics, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China (e-mail: <email>malifeng@ccmu.edu.cn</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22394</elocation-id><history><date date-type=\"received\"><day>27</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22394.pdf\"/><abstract><title>Abstract</title><sec><title>Objective:</title><p>We perform this protocol for randomized controlled trial to compare the efficacy of intrathecal morphine and local infiltration anesthesia (LIA) in the treatment of the postoperative pain after total knee replacement (TKR).</p></sec><sec sec-type=\"methods\"><title>Methods:</title><p>This is a randomized controlled, single center trial which was performed from March 2019 to March 2020. This trial is conducted according to the SPIRIT Checklist of randomized researches. It is authorized via the Ethics Committee of Beijing Friendship Hospital (2019-P2-050-01). Eighty participants who undergo TKR were randomized into 2 groups. Intrathecal morphine group: 0.1 mg of the morphine was intrathecally injected, and the spinal anesthetic was injected at the same time in the group LIA; In the LIA group: the knee joint was infiltrated with epinephrine, ketorologic acid and ropivacaine in the process of operation, and the identical mixture was injected 2 bolus through the intraarticular catheter after operation. The main outcome variables were the visual analog scale and the consumption amount of opioid every 6-hour interval within 2 days postoperatively. The secondary outcome variables were the side effects associated with opioid, the length of hospital stay, motion range, and the loss of blood collected by the closed suction drainage. All the required analyses were carried out via applying the SPSS for Windows Version 19.0.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>The clinical outcome variables between groups were shown in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>.</p></sec><sec sec-type=\"conclusion\"><title>Conclusion:</title><p>This protocol will provide the evidence on which technique can achieve better analgesia after TKR.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>intrathecal morphine</kwd><kwd>local infiltration anesthesia</kwd><kwd>protocol</kwd><kwd>total knee replacement</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>Beijing Municipal Science &amp; Technology Commission Project</funding-source><award-id>D171100003217001</award-id><principal-award-recipient>Ai Guo</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Total knee replacement (TKR) is a successful surgical approach for the treatment of end-stage knee osteoarthritis in terms of functional recovery and pain relief.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> The demand for primary TKR is estimated to grow by 3.5 million procedures in the United States by 2030.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> Although TKR is popular, approximately 20% of the patients are not satisfied. Published studies indicated that an estimated 40% to 60% of patients report severe pain following TKR, which is the common reasons for dissatisfaction.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>,<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> The aggravation of pain is associated with the proinflammatory states, pain regulation system damage and the tissue damage. Inadequate perioperative analgesia may be related to the increased costs, prolonged hospital stay and adverse clinical outcomes.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>,<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup> Many methods have been applied to reduce pain following TKR, including local infiltration analgesia (LIA), oral analgesics, peripheral nerve block, and intrathecal morphine.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>–<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup> However, the analgesic efficacy of these techniques and their effect on postoperative opioid consumption remains unclear.</p><p>LIA via an infusion catheter has been shown to be more effective than single bolus administration, is easy to perform and delivers analgesia directly to the source of pain. Wound infection is a major concern, which is disastrous for arthroplasties. Intrathecal morphine may be delivered at the same time as spinal anesthesia and therefore the additional time required is negligible. One disadvantage, however, is the potential risk of opiate toxicity. In addition, opioid can lead to many side effects, for instance, respiratory depression, gastrointestinal reactions, constipation and urine retention.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>,<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> Therefore, multimodal analgesia has been extensively used in perioperative period of TKR. Currently, perioperative analgesia after intrathecal morphine or LIA for TKR still controversial. We perform this protocol for randomized controlled trial to compare the efficacy of intrathecal morphine and LIA in the treatment of the postoperative pain after TKR.</p></sec><sec><label>2</label><title>Methods</title><sec><label>2.1</label><title>Study design</title><p>This is a randomized controlled, single center trial which was implemented from March 2019 to March 2020. This trial is conducted according to the SPIRIT Checklist of randomized researches. It was authorized via the Ethics Committee of Beijing Friendship Hospital (2019-P2-050-01), and it has been registered in the research registry (researchregistry5942).</p></sec><sec><label>2.2</label><title>Patients</title><p>Eighty participants who undergo TKR were analyzed. In the random envelope, all patients were assigned a random number via using the random number Table <xref rid=\"T1\" ref-type=\"table\">1</xref>, and the result of allocation was hidden. Patients were randomly divided into LIA group (with 40 patients) and the intrathecal morphine (with 40 patients). Inclusion criteria contains</p><list list-type=\"simple\"><list-item><label>1.</label><p>elective primary unilateral TKR;</p></list-item><list-item><label>2.</label><p>people between the ages of 50 and 70;</p></list-item><list-item><label>3.</label><p>BMI less than 40 kg/m<sup>2</sup>;</p></list-item><list-item><label>4.</label><p>the acceptance of patients to participate in this work.</p></list-item></list><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Outcome measures between intrathecal morphine and local infiltration anesthesia after total knee replacement.</p></caption><graphic xlink:href=\"medi-99-e22394-g001\"/></table-wrap><p>The exclusion criteria contains:</p><list list-type=\"simple\"><list-item><label>1.</label><p>patients with the history of severe renal and hepatic dysfunction;</p></list-item><list-item><label>2.</label><p>contraindication to spinal anesthesia (refusal, coagulopathy, sepsis, local infection, spinal defects, previous laminectomy);</p></list-item><list-item><label>3.</label><p>Contraindication to nonsteroidal antiinflammatory drugs;</p></list-item><list-item><label>4.</label><p>Patient has an emotional or neurological condition that would prevent their willingness to participate in the study.</p></list-item></list></sec><sec><label>2.3</label><title>Analgesic protocol</title><p>All the patients were given 10 mg of diazepam orally 1 hour before the planned operation, and all the surgeries were implemented under the spinal anesthesia, and a 27-G pencil-point spinal needle was utilized in the intervertebral space of L3/L4, with patient in sitting position. In the morphine group, 0.1 mg of the morphine (0.25 ml) was intrathecally injected, and the same volume of 0.9% normal saline and 17.5 mg of bupivacaine without glucose were injected into the LIA group. All the patients were given propofol intravenously or continued intraoperative infusion as required. If the patient experienced pain in the process of operation, the dosage of fentanyl intravenous injection is 25 to 50 μg, up to 300 μg.</p><p>In the group of LIA, epinephrine (0.5 mg), 30 mg of ketorolac acid and ropivacaine (300 mg) were infiltrated into the periarticular soft tissues via surgeons in the operation process. After cutting the bone and before prosthesis implantation, all the tissues injured during the operation were systematically injected by injecting 40 to 50 mL into the collateral ligament and posterior capsule. After implantation of the prosthesis, an injection of 50 to 70 mL through a capsule incision was performed. Ultimately, 50 mL of the ropivacaine (100 mg) without ketorolac or epinephrine infiltrated subcutaneous tissue before the skin is closed.</p></sec><sec><label>2.4</label><title>Outcomes</title><p>The main outcome variables were the consumption amount of opioids in the patient-controlled analgesia administered every 6-hour within 24 hours after the operation, and the degree of pain assessed postoperatively via the visual analog scores<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> every 6 hours until 24 hours. Visual analog scores for the pain is between 0 mm (representing no pain) and 100 mm (representing extreme pain). The secondary outcome variables were the side effects associated with opioid, the length of hospital stay, motion range, and the loss of blood collected by the closed suction drainage. The side effects of morphine included nausea and vomiting, retention of urinary, itching and the respiratory depression.</p></sec><sec><label>2.5</label><title>Statistical analysis</title><p>All the required analyses were carried out via applying the SPSS for Windows Version 19.0. All data were expressed with appropriate characteristics, for instance, mean, median, standard deviation as well as percentage. The comparison between the 2 groups was conducted through using Mann-Whitney <italic>U</italic> test or the independent samples <italic>t</italic> test. And the comparison of categorical variables between the groups was implemented via the Chi-square test. A value of <italic>P</italic> < .05 was considered as the significant in statistics.</p></sec></sec><sec><label>3</label><title>Results</title><p>The clinical outcome variables between groups were shown in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>.</p></sec><sec><label>4</label><title>Discussion</title><p>Many end-stage knee osteoarthritis patients have improved the mobility, life quality, and improved the pain after TKR.<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]</sup> Despite the superb results achieved by TKR, offering an adequate postoperative pain control and rehabilitation is a challenge for the providers, as good management of pain can improve patient outcomes. The lack of a definitive “gold standard” and various programs of pain management after operation indicate that there is much room for improving the standards of care.<sup>[<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</sup> LIA is effective in the knee surgery. It is on the basis of the systematic infiltration of a epinephrine, nonsteroidal antiinflammatory drugs and long-acting local anesthetics mixture into the tissues around surgical area in order to obtain satisfactory control of pain with slight physiological interference,<sup>[<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]</sup> but the outcomes are not generally optimistic.<sup>[<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</sup> Epidural analgesia has become a commonly used treatment option, which offers excellent pain relief, but there are also some risks, for instance, epidural hematoma, bradycardia, hypotension, and prolonged lower extremity motor block time and other side effects.<sup>[<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]</sup> Based on the above discussion, we perform this protocol to compare the effectiveness of LIA and intrathecal morphine in the treatment of the postoperative pain after TKR.</p></sec><sec><label>5</label><title>Conclusion</title><p>This protocol will provide the evidence on which technique can achieve better analgesia after TKR.</p></sec><sec><title>Author contributions</title><p>Lifeng Ma and Ai Guo plan the study design. Bo Yang reviewed the protocol. Jingxin Zhang will collect data. Zhengrong Qi and Zhiyao Li write the manuscript. All authors approve the submission.</p><p><bold>Conceptualization:</bold> Lifeng Ma.</p><p><bold>Formal analysis:</bold> Ai Guo.</p><p><bold>Funding acquisition:</bold> Zhiyao Li.</p><p><bold>Methodology:</bold> Bo Yang, Jingxin Zhang.</p><p><bold>Software:</bold> Bo Yang.</p><p><bold>Writing – original draft:</bold> Zhengrong Qi.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: LIA = local infiltration analgesia, TKR = total knee replacement, VAS = visual analog scale.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Qi Z, Guo A, Ma L, Li Z, Yang B, Zhang J. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991424</article-id><article-id pub-id-type=\"pmc\">PMC7523760</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-08291</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022262</article-id><article-id pub-id-type=\"art-access-id\">22262</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3800</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Systematic Review</subject></subj-group></article-categories><title-group><article-title>Effectiveness and safety of massage in the treatment of anxiety and depression in patients with cancer</article-title><subtitle>A protocol for systematic review and meta-analysis</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-5715-6909</contrib-id><name><surname>Qin</surname><given-names>Siyu</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0001-7947-6914</contrib-id><name><surname>Xiao</surname><given-names>Yuanyi</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Chi</surname><given-names>Zhenhai</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhu</surname><given-names>Daocheng</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Cheng</surname><given-names>Pan</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Yu</surname><given-names>Ting</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Haiyan</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-2860-8320</contrib-id><name><surname>Jiao</surname><given-names>Lin</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><aff id=\"aff1\"><label>a</label>College of Acupuncture-Moxibustion and Tuina, Jiangxi University of Traditional Chinese Medicine</aff><aff id=\"aff2\"><label>b</label>The Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang, China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Lin Jiao, Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang, China (e-mail: <email>jl0809@126.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22262</elocation-id><history><date date-type=\"received\"><day>19</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>20</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22262.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>Anxiety and depression, complications of cancer, are prevalent but often overlooked mental illnesses. Studies have demonstrated that massage therapy is useful in relieving anxiety and depression of cancer survivors. However, the mechanism is still unclear and no systematic review has provided sufficient evidence for the treatment. Therefore, this protocol is carried out to comprehensively evaluate the reliability of cancer patients with anxiety and depression treated by massage.</p></sec><sec sec-type=\"methods\"><title>Methods:</title><p>We will systematically search the relevant literature from PubMed, Cochrane Library, EMBASE, Web of Science, Wanfang, Chongqing VIP, CNKI and Chinese Biomedical Literature Database from the establishment of the databases to June 1, 2020. In addition, we will only include randomized controlled trials about massage for cancer survivors with anxiety and depression, regardless of language and publication status. Two experienced researchers will separately screen the literature, collect data, analyze data and synthesize data using RevMan V.5.3 software. The quality of the included trials in the study will be assessed by the Cochrane bias risk assessment tool.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>The protocol for the meta-analysis will systematically evaluate the reliability of massage therapy for cancer patients with anxiety and depression.</p></sec><sec sec-type=\"conclusion\"><title>Conclusion:</title><p>This conclusion will provide an important basis for evaluating whether massage is reliable in treating cancer survivors who feel anxious and depressed.</p></sec><sec><title>INPLASY registration number:</title><p>INPLASY202060101</p></sec></abstract><kwd-group><title>Keywords</title><kwd>anxiety</kwd><kwd>cancer</kwd><kwd>depression</kwd><kwd>massage</kwd><kwd>protocol</kwd><kwd>systematic review</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>National Natural Science Foundation of China</funding-source><award-id>81860877</award-id><principal-award-recipient>Lin Jiao</principal-award-recipient></award-group><award-group id=\"award2\" award-type=\"Fundref\"><funding-source>National Natural Science Foundation of China</funding-source><award-id>81660821</award-id><principal-award-recipient>Lin Jiao</principal-award-recipient></award-group><award-group id=\"award3\" award-type=\"Fundref\"><funding-source>Jiangxi Provincial Department of Science and Technology</funding-source><award-id>20181BBG70047</award-id><principal-award-recipient>Lin Jiao</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Cancer, a concerning public health problem, threatens the health of human beings all over the world. There are 18.1 million new cancer patients and 9.6 million cancer deaths in 2018.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> Not only the physical health of patients but also their mental health can be significantly affected by a diagnosis of cancer.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> Anxiety and depression, complications of cancer, are prevalent but often overlooked mental illnesses.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> Many research have also shown that the most common psychological states with cancer patients are anxiety and depression.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>–<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> Meanwhile, two-thirds of cancer survivors with depression are often associated with significant anxiety in the clinical.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> Currently, a series of studies have reported that anxiety and depression can produce some negative effects on patients’ quality of life (QOL), health expenditures, continuity of treatment, and cancer survival.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>,<xref rid=\"R11\" ref-type=\"bibr\">11</xref>,<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> Therefore, it is very important to find more effective treatments alleviating anxiety and depression symptoms of patients who have been diagnosed with cancer.</p><p>At present, pharmacotherapy and psychotherapy, the main treatment means for anxiety and depression of cancer survivors, play an important role in improving these distressing emotions.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R3\" ref-type=\"bibr\">3</xref>,<xref rid=\"R12\" ref-type=\"bibr\">12</xref>,<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> However, antidepressants and anxiolytics may bring a variety of adverse impacts such as headaches, addiction, seizures, suicide, and interactions with anticancer treatments.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>–<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]</sup> Besides, the psychological intervention available to patients is also limited due to the lack of providers and financial resources.<sup>[<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</sup> So, it is extremely necessary to seek an alternative treatment that is effective, cheaper, and safer. It is reported that Complementary and Alternative Medicine interventions are used by more than half of cancer patients to relieve related symptoms.<sup>[<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]</sup> Nowadays, massage which is 1 of the most widely used complementary and alternative medicine therapies can not only relieve cancer-related symptoms but also bring physical and mental pleasure.<sup>[<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</sup></p><p>Massage, defined as a method of manipulating body tissue by hand, has certain effects on the vessel, nerves, and muscle system of the body.<sup>[<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]</sup> Compared to pharmacotherapy and psychotherapy, massage has unique advantages because of its non-invasive, low-cost, and safety characteristics.<sup>[<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</sup> According to a report, in North American medical centers, massage treatment as a supportive treatment is gradually available for cancer survivors to improve comfort level, lessen symptoms and related side effects.<sup>[<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]</sup> Many studies have found that massage can reduce muscle fatigue, improve blood flow, relax mood as well as relieve cancer symptoms such as anxiety, depression, pain, and nausea.<sup>[<xref rid=\"R20\" ref-type=\"bibr\">20</xref>,<xref rid=\"R22\" ref-type=\"bibr\">22</xref>–<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]</sup>Moreover, the result which was published in the Journal of Clinical Oncology (JCO) also demonstrated that massage could relieve anxiety and depression of cancer survivors.<sup>[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]</sup></p><p>To our knowledge, there is no recent systematic review discussing whether massage therapy is safe and effective in treating anxiety and depression symptoms of patients who have been diagnosed with cancer. Therefore, we perform this protocol to comprehensively assess the effect of massage for cancer patients who feel anxious and depressed.</p></sec><sec><label>2</label><title>Methods</title><sec><label>2.1</label><title>Study registration</title><p>The registration number of this study is INPLASY202060101. We will strictly perform this protocol by following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocol (PRISMA-P) statement guidelines.<sup>[<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]</sup></p></sec><sec><label>2.2</label><title>Inclusion criteria for study selection</title><sec><label>2.2.1</label><title>Type of studies</title><p>Only randomized controlled trials (RCTs) about massage for cancer survivors with anxiety and depression will be included. There are no restrictions on language and publication status. In addition, non-RCTs, experience report, reviews will not be included.</p></sec><sec><label>2.2.2</label><title>Types of Participants</title><p>All cancer patients feeling anxious and depressed will be included. There are no limitations for age, ethnicity, and gender.</p></sec><sec><label>2.2.3</label><title>Types of interventions</title><sec><label>2.2.3.1</label><title>Experimental interventions</title><p>The experimental group will only include massage therapies, which include reflexology, acupressure, manual lymphatic drainage, tuina, general massage, etc. There is no restriction on the types of massage, treatment sites, treatment time, and the frequency.</p></sec><sec><label>2.2.3.2</label><title>Control interventions</title><p>The interventions of the control group will include any therapies without massage, such as drugs, psychotherapy, routine care, placebo, cupping therapy, acupuncture, etc.</p></sec></sec><sec><label>2.2.4</label><title>Types of outcome measures</title><sec><label>2.2.4.1</label><title>Primary outcomes</title><list list-type=\"simple\"><list-item><label>1.</label><p>The State Anxiety Inventory.</p></list-item><list-item><label>2.</label><p>The Center for Epidemiological Studies Depression scale.</p></list-item></list></sec><sec><label>2.2.4.2</label><title>Additional outcomes</title><list list-type=\"simple\"><list-item><label>1.</label><p>The Quality of Life Questionnaire Core 30 from the European Organization for Research on Treatment of Cancer.</p></list-item><list-item><label>2.</label><p>Any adverse events.</p></list-item></list></sec></sec></sec><sec><label>2.3</label><title>Search methods</title><p>RCTs relating to massage management for anxiety and depression of cancer survivors will be retrieved from PubMed, Cochrane Central Register of Controlled Trials, EMBASE, Web of Science, Chinese Biomedical Literature Database, Wanfang Database, Chongqing VIP Database and Chinese National Knowledge Infrastructure from the establishment of the databases to June 1, 2020. In addition, The Clinicaltrials.gov, Chinese Clinical Trial Registry will also be carefully retrieved to obtain unpublished or ongoing trial data. The detailed PubMed searching strategy is listed in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Search strategy used in PubMed database.</p></caption><graphic xlink:href=\"medi-99-e22262-g001\"/></table-wrap></sec><sec><label>2.4</label><title>Data collection and analysis</title><sec><label>2.4.1</label><title>Selection of studies</title><p>All searched literature will be imported into EndNote software (V.X9) for removing duplicate literature. The 2 researchers (SQ and YX) will independently read the title and abstract to exclude irrelevant literature. After preliminary screening, they will carefully read the full text to determine whether the related studies are eventually included in the protocol. Then, a cross-check will be conducted by 2 researchers (SQ and YX). Finally, if there is any disagreement when the 2 researchers perform the above operation, it will be discussed or resolved by the third researcher (LJ). The specific literature screening flow chart will be presented in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Flow diagram of study selection process.</p></caption><graphic xlink:href=\"medi-99-e22262-g002\"/></fig></sec><sec><label>2.4.2</label><title>Data extraction and management</title><p>Two researchers (SQ and ZC) will separately extract the following informations by using data extraction forms which have been prepared in advance:</p><list list-type=\"simple\"><list-item><label>(1)</label><p>Research Characteristics: Year of publication, Journal, title, information of the author.</p></list-item><list-item><label>(2)</label><p>Participants’ basic information: Gender, age, course of the disease, country, sample size, severity of anxiety, and depression.</p></list-item><list-item><label>(3)</label><p>Study methods: Randomization, allocation concealment, blinding, result analysis method.</p></list-item><list-item><label>(4)</label><p>Intervention: Operation name, treatment sites, treatment time, course of treatment, and frequency.</p></list-item><list-item><label>(5)</label><p>Outcomes measurement: Primary and secondary outcomes.</p></list-item></list></sec></sec><sec><label>2.5</label><title>Evaluation of bias risk in included studies</title><p>Two experienced researchers (SQ and ZC) will separately assess the quality of the trials using the Cochrane bias risk assessment tool.<sup>[<xref rid=\"R27\" ref-type=\"bibr\">27</xref>]</sup> It includes 7 aspects: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective outcome reporting, and other bias. Each item can be classified as high, low, and unclear risk bias levels. When the items related to studies are not clear, we will contact the authors to get the required information. It is necessary to consult the third researcher (LJ) to make a reliable decision if there is any controversy.</p></sec><sec><label>2.6</label><title>Data synthesis</title><p>The following data analysis will be performed using RevMan 5.3 software. When the measured outcomes are dichotomous data, the risk ratio (RR) with 95% confidence interval (CI) will be adopted. When the measured outcomes are continuous data, Weighted Mean Difference (WMD) with 95% CI will be adopted if we use the same measurement instrument. And the Standardized Mean Difference (SMD) with 95% CI will be applied if we use different measurement instruments. <italic>χ</italic><sup>2</sup> test and <italic>I</italic><sup>2</sup> test will be utilized to investigate the heterogeneity. When the heterogeneity is considered to be not obvious (<italic>P</italic> > 0.1 or <italic>I</italic><sup>2</sup> < 50%), we will choose the fixed-effect model. On the contrary, when the heterogeneity is considered to be obvious (<italic>P</italic> ≤ 0.1 or <italic>I</italic><sup>2</sup> ≥ 50%), the random-effect model will be chosen and the subgroup analysis or sensitivity analysis will be conducted to seek potential causes of intergroup heterogeneity. A descriptive analysis is necessary to be carried out if the heterogeneity is too significant.</p></sec><sec><label>2.7</label><title>Management of missing data</title><p>If the data of the included studies are unclear or missing, we will do our best to contact the related authors of the article to acquire complete data. If the above way of obtaining data information is unsuccessful, we will only use current data for the data analysis.</p></sec><sec><label>2.8</label><title>Subgroup analysis</title><p>If significant heterogeneity exists in the included trials, it is necessary to perform a subgroup analysis to reduce heterogeneity between groups based on differences in massage methods, course of the disease, sample size, the severity of anxiety and depression, and so on.</p></sec><sec><label>2.9</label><title>Sensitivity analysis</title><p>If necessary, sensitivity analysis will be performed to evaluate whether the conclusions of the meta-analysis are stable or reliable by excluding trials of low quality.</p></sec><sec><label>2.10</label><title>Assessment of reporting biases</title><p>Funnel plots will be adopted to detect the publication bias if the included trials exceed 10. Egger test will be performed to analyze the potential causes of publishing bias if the asymmetry exists in the funnel plots.</p></sec><sec><label>2.11</label><title>Quality of evidence</title><p>The quality of evidence will be independently evaluated by 2 researchers using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE),<sup>[<xref rid=\"R28\" ref-type=\"bibr\">28</xref>]</sup> and will be assessed into high, moderate, low, and very low levels.</p></sec><sec><label>2.12</label><title>Ethics and dissemination</title><p>In the study, patients’ personal information is not involved, so ethical approval is not necessary. Results from this protocol will be disseminated in a peer-reviewed journal.</p></sec></sec><sec><label>3</label><title>Discussion</title><p>Anxiety and depression, complications of cancer, seriously affect the mental health of cancer patients. Even though drug therapy and psychotherapy are effective in relieving anxiety and depression, these treatments may have some side effects. Massage as a complementary and alternative therapy has been widely used to alleviate anxiety and depression symptoms of patients who have been diagnosed with cancer, due to its non-invasive, safe, and inexpensive features.<sup>[<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</sup> Studies have shown that massage therapy can be effective in easing mood and reducing cancer-related symptoms, including depression, anxiety pain, fatigue, and so on.<sup>[<xref rid=\"R20\" ref-type=\"bibr\">20</xref>,<xref rid=\"R24\" ref-type=\"bibr\">24</xref>,<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]</sup> However, its clinical mechanism of action is still unclear and no systematic review has provided sufficient evidence for this treatment. We hope that the results of this study are useful to patients, clinicians, and health policymakers.</p><p>However, the study may have the following limitations: First, significant heterogeneity may exist due to different massage methods, different treatment sites, and time. Second, certain language bias may be caused due to the absence of language limitations.</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization</bold>: Siyu Qin, Yuanyi Xiao.</p><p><bold>Data curation</bold>: Siyu Qin, Zhenhai Chi.</p><p><bold>Formal analysis</bold>: Siyu Qin, Yuanyi Xiao.</p><p><bold>Funding acquisition</bold>: Lin Jiao.</p><p><bold>Methodology</bold>: Siyu Qin, Zhenhai Chi.</p><p><bold>Software</bold>: Siyu Qin, Yuanyi Xiao.</p><p><bold>Supervision</bold>: Lin Jiao.</p><p><bold>Writing – original draft</bold>: Siyu Qin, Yuanyi Xiao.</p><p><bold>Writing – review & editing</bold>: Lin Jiao.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: CI = confidence interval, RCTs = randomized controlled trials.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Qin S, Xiao Y, Chi Z, Zhu D, Cheng P, Yu T, Li H, Jiao L. Effectiveness and safety of massage in the treatment of anxiety and depression in patients with cancer: A protocol for systematic review and meta-analysis. <italic>Medicine</italic>. 2020;99:39(e22262).</p></fn><fn fn-type=\"equal\"><p>SQ and YX contributed equally to this work and should be considered as co-first authors.</p></fn><fn fn-type=\"other\"><p>The authors have registered this protocol in the INPLASY network (No. INPLASY202060101).</p></fn><fn fn-type=\"supported-by\"><p>This study was supported by The National Natural Science Foundation of China (Grant number: 81860877, 81660821); Jiangxi Provincial Science and Technology Department Major Project Innovation Fund Project (Grant number: 20181BBG70047).</p></fn><fn fn-type=\"COI-statement\"><p>The authors have no conflicts of interest to disclose.</p></fn><fn fn-type=\"other\"><p>The datasets generated during and/or analyzed during the current study are publicly 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991452</article-id><article-id pub-id-type=\"pmc\">PMC7523762</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-06743</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022358</article-id><article-id pub-id-type=\"art-access-id\">22358</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3800</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Systematic Review</subject></subj-group></article-categories><title-group><article-title>Herbal medicine <italic>Siho-sogan-san</italic> for functional dyspepsia</article-title><subtitle>A protocol for a systematic review and meta-analysis</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Ha</surname><given-names>Na-Yeon</given-names></name><degrees>KMD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Jeong</surname><given-names>Hae-in</given-names></name><degrees>KMD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Lee</surname><given-names>Ha-nul</given-names></name><degrees>KMD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Ko</surname><given-names>Seok-Jae</given-names></name><degrees>KMD, PhD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Park</surname><given-names>Jae-Woo</given-names></name><degrees>KMD, PhD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Kim</surname><given-names>Jinsung</given-names></name><degrees>KMD, PhD</degrees><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><aff>Department of Gastroenterology, College of Korean Medicine, Kyung Hee University, Seoul, Republic of Korea.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Jinsung Kim, Department of Gastroenterology, College of Korean Medicine, Kyung Hee University, 24 Kyungheedae-ro, Dongdaemun-gu, Seoul, Republic of Korea (e-mail: <email>oridoc@khu.ac.kr</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22358</elocation-id><history><date date-type=\"received\"><day>29</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>26</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22358.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>Functional dyspepsia (FD) is characterized by persistent and recurrent dyspeptic symptoms, such as postprandial fullness and epigastric pain. Although it is rarely severe or life-threatening, it can degrade the quality of life and cause social and economic issues. As symptoms often persist despite the treatment with conventional Western medicine, herbal medicine can be considered as an alternative for treating FD. <italic>Siho-sogan-san</italic> (SHS) is a traditional herbal formula prescribed for dyspepsia for hundreds of years. This protocol for a systematic review was designed to evaluate the safety and efficacy of SHS for the treatment of FD through a meta-analysis.</p></sec><sec sec-type=\"methods\"><title>Methods:</title><p>Studies will be searched from the following electronic databases up to March 2020: Embase, MEDLINE (via PubMED), Cochrane Central Register of Controlled Trials, Allied and Complementary Medicine Database, Korean Medical Database, KoreaMed, Korean Studies Information Service System, National Digital Science Library, Oriental Medicine Advanced Searching Integrated System, China National Knowledge Infrastructure Database, and Citation Information by Nii. Randomized controlled trials of SHS and herb-added SHS for treating FD will be selected in this review. The control groups of no-treatment, placebo, and conventional Western medicine will be compared with SHS for its efficacy. The synergetic effect of SHS with Western medicine will also be analyzed in comparison with conventional Western medicine alone. Two independent reviewers will collect the data and assess the risk of bias in individual studies. The total clinical effectiveness rate will be synthesized and evaluated as primary outcome.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>This systematic review will present an adequate clinical evidence of SHS for the treatment of FD based on specific parameters, including dyspepsia-related symptoms, gastric emptying, and adverse events.</p></sec><sec sec-type=\"conclusion\"><title>Conclusion:</title><p>This study will provide evidence for the safety and efficacy of SHS for the treatment of patients with FD.</p></sec><sec><title>Review Registry Unique Identifying Number:</title><p>reviewregistry952.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>functional dyspepsia</kwd><kwd>protocol</kwd><kwd>randomized controlled trials</kwd><kwd><italic>Siho-sogan-san</italic></kwd><kwd>systematic review</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>Korea Health Industry Development Institute</funding-source><award-id>HB16C0019</award-id><principal-award-recipient>Jinsung Kim</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>In general, patients with functional dyspepsia (FD) complain of postprandial fullness, early satiety, epigastric pain, and epigastric burning.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> According to the Rome IV diagnostic criteria, FD is diagnosed based on symptoms alone and does not require any evidence of systemic problems.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> The mechanisms underlying FD are unclear. FD can affect the patient's quality of life and accounts for 14% to 66% of the medical care burden in several countries.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> It is divided into the following 2 subgroups depending on the symptoms: postprandial distress syndrome and epigastric pain syndrome. Several underlying pathophysiologic mechanisms, such as impaired gastric motility, visceral hypersensitivity, and psychological factors, have been reported.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>,<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> Although <italic>Helicobacter pylori</italic> eradication, proton pump inhibitor therapy, and other additional treatment have been proposed to manage patients with FD, most of symptoms usually persist and recur. It explains in part why FD patients seek complementary and alternative medicine, including herbal medicine, despite their limited evidence.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup></p><p><italic>Siho-sogan-san</italic> (SHS) is a traditional herbal prescription, composed of 7 herbs, that has been widely used to improve dyspeptic symptoms similar with FD for hundreds of years. A review of meta-analysis on 13 randomized controlled trials (RCTs) showed that traditional Chinese medicine (TCM) was more effective in the treatment of FD with liver–stomach disharmony syndrome compared to prokinetics, without serious adverse effects. Among selected studies, <italic>Radix Paeoniae</italic>, <italic>Radix Bupleuri</italic>, <italic>Radix Glycyrrhizae</italic>, and <italic>Rhizoma Cyperi</italic> were frequently included and prescribed in the formula, in that order. In addition, these herbs make up SHS as well, functioning in soothing the liver and regulating <italic>qi</italic>.<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup> An animal study showed that <italic>Rhizoma Cyperi</italic> had an effect to improve gastric emptying and reduce gastric damage.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> Saikosaponin, a main component of <italic>Radix Bupleuri</italic>, has anti-inflammatory and antidepressant-like effects.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>,<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> From the perspective that there are some relations between functional gastrointestinal disorders (FGIDs) and psychosocial factors, such as anxiety disorder and depression, it is expected that SHS may be effective and safe for treating FD.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup></p><p>In a previous review of meta-analysis, SHS showed higher efficacy and safety than prokinetic drugs. However, this analysis was limited in quality of included studies, database resources, type of comparators, and outcome parameters.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> Therefore, the aim of this review is to provide proper evidence to support the clinical usage of SHS for the treatment of FD, through the procedure of searching and synthesizing RCTs of SHS on FD systematically.</p></sec><sec><label>2</label><title>Methods</title><sec><label>2.1</label><title>Eligibility criteria</title><sec><label>2.1.1</label><title>Types of studies</title><p>RCTs and quasi-RCTs will be included in this systematic review, whereas animal or cell research, non-RCTs, and review articles will be excluded.</p></sec><sec><label>2.1.2</label><title>Types of participants</title><p>Patients diagnosed with FD using the Rome criteria will be included, regardless of the sex, age, and race. The latest revised version, that is, version IV, of the Rome criteria was established in 2016 and is used as the standard for diagnosing FGIDs, including FD. Two reviewers (HJ and HL) will independently classify the study by the time of its publication based on 1991 and apply similar criteria as the Rome criteria upon the inter-agreement eligibility (e.g., persistent dyspeptic symptoms without any structural defects). Although secondary symptoms caused by other diseases should be excluded, some patients will be selected in this review because the possibility of accompanying morbidity of FD with gastroesophageal reflux disease or irritable bowel syndrome was raised in the Rome IV criteria.</p></sec><sec><label>2.1.3</label><title>Types of interventions</title><p>Studies on SHS, including modified SHS (herb-added formula) alone or in combination with Western medicine, will be selected. The following comparisons will be made with SHS: no other treatment, placebo (indistinguishable from real SHS), and Western drugs, including prokinetics and antidepressants. If possible, this review will also analyze the efficacy of SHS in combination with other Western drugs for the treatment of FD compared to conventional Western medicine alone.</p></sec><sec><label>2.1.4</label><title>Types of outcome measures</title><p>The total clinical effective rate will be analyzed as primary outcome. Secondary outcomes will include dyspepsia-related symptom score, gastric emptying, quality of life, adverse events, and so on.</p></sec></sec><sec><label>2.2</label><title>Search studies</title><sec><label>2.2.1</label><title>Database resources</title><p>The following electronic databases will be included in this study, regardless of the language, from its inception to March 2020:</p><list list-type=\"simple\"><list-item><label>1.</label><p>Embase</p></list-item><list-item><label>2.</label><p>MEDLINE (via PubMED)</p></list-item><list-item><label>3.</label><p>Cochrane Central Register of Controlled Trials</p></list-item><list-item><label>4.</label><p>Allied and Complementary Medicine Database</p></list-item><list-item><label>5.</label><p>Korean Medical Database</p></list-item><list-item><label>6.</label><p>KoreaMed</p></list-item><list-item><label>7.</label><p>Korean Studies Information Service System</p></list-item><list-item><label>8.</label><p>National Digital Science Library</p></list-item><list-item><label>9.</label><p>Oriental Medicine Advanced Searching Integrated System</p></list-item><list-item><label>10.</label><p>China National Knowledge Infrastructure Database</p></list-item><list-item><label>11.</label><p>Citation Information by Nii</p></list-item></list><p>In addition, appropriate data from Clinical Research Information Service and ClinicalTrials.gov will be included.</p></sec><sec><label>2.2.2</label><title>Search strategy</title><p>We will apply search strategies, including the terms of disease part (e.g., disturbance, illness, indigestion, and gut) and intervention part (e.g., herb, <italic>Siho, Saiko, and Chaihu</italic>) according to each database format (e.g., shown in Table <xref rid=\"T1\" ref-type=\"table\">1</xref> for PubMed).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Search strategy used in PubMed.</p></caption><graphic xlink:href=\"medi-99-e22358-g001\"/></table-wrap></sec></sec><sec><label>2.3</label><title>Data collection and assessment</title><sec><label>2.3.1</label><title>Study selection</title><p>Two reviewers (HJ and HL), well-educated in the search process, will independently select relevant articles, screening the titles and abstracts and excluding duplicates or inappropriate studies. The articles will be uploaded and arranged by Endnote X8 (Clarivate Analytics). For screening, the investigators (HJ and HL) will independently review the full text of articles to select suitable ones. Details of the study selection process are presented in a Preferred Reporting Items for Systematic Review and Meta-analysis flow diagram (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>). Disagreement between 2 reviewers will be resolved by adjustments of an arbiter (N-YH).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Flow chart of the search process. AMED = Allied and Complementary Medicine Database, CENTRAL = Cochrane Central Register of Controlled Trials, Cinii = Citation Information by Nii, CNKI = China National Knowledge Infrastructure Database, KISS = Korean Studies Information Service System, KMbase = Korean Medical Database, NDSL = National Digital Science Library, OASIS = Oriental Medicine Advanced Searching Integrated System, RCTs = randomized controlled trials.</p></caption><graphic xlink:href=\"medi-99-e22358-g002\"/></fig></sec><sec><label>2.3.2</label><title>Data extraction</title><p>Two authors (HJ and HL) will independently extract related data with this review and fill the standard data extraction form. It will include information, such as the first author, publication year, study design, intervention, comparator, administration period, outcomes, and adverse events. Differences between the reviewers will be discussed and resolved by the arbiter (N-YH).</p></sec><sec><label>2.3.3</label><title>Assessment of the risk of bias in included studies</title><p>The risk of bias will be assessed independently by 2 investigators (HJ and HL) according to the Cochrane Collaboration's tool. The following items will be included: random sequence generation (selection bias), allocation concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective outcome reporting (reporting bias), and other bias. Each assessment will be divided into 3 stages: low, unclear, and high. The reviewers will discuss all disagreements, and the arbiter (N-YH) will intervene the problem and obtain a consensus.</p></sec></sec><sec><label>2.4</label><title>Data analysis</title><sec><label>2.4.1</label><title>Statistical analysis</title><p>The outcomes will be reported as the mean difference with 95% confidence interval (CI) for continuous data and risk ratio or odds ratio with 95% CI for categorical data.</p></sec><sec><label>2.4.2</label><title>Handling missing data</title><p>We will contact the authors of original studies by sending an email to request for missing data. The outcomes will be assessed based on the intent-to-treat analysis.</p></sec><sec><label>2.4.3</label><title>Assessment of heterogeneity</title><p>Random-effects models will be included in the meta-analysis. Heterogeneity in the included studies will be assessed by the <italic>I</italic>-squared statistic and chi-squared (<italic>χ</italic><sup><italic>2</italic></sup>) test (<italic>I</italic><sup><italic>2</italic></sup> ≥ 50% and <italic>P</italic> value < .1 mean substantial heterogeneity).</p></sec><sec><label>2.4.4</label><title>Data synthesis</title><p>The Review Manager program (version 5.3. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014) will be used for data synthesis. To evaluate the efficacy of SHS, the outcomes will be synthesized and compared with several types of interventions, such as no-treatment, placebo, and conventional Western medicine. If sufficient studies are included, the effect of SHS combined with conventional Western medicine versus Western medicine alone will also be measured to confirm the synergetic effect of the combination therapy.</p></sec><sec><label>2.4.5</label><title>Subgroup analysis</title><p>If it is necessary to investigate the causes of high heterogeneity in included studies, we will conduct a subgroup analysis of the following items: pattern identification of TCM, types of herbal medicine, administration period, and so on.</p></sec><sec><label>2.4.6</label><title>Assessment of publication bias</title><p>If there are more than 10 studies for the analysis, a funnel plot will be presented to evaluate the publication bias and assess small study effects.</p></sec><sec><label>2.4.7</label><title>Sensitivity analysis</title><p>“The Consolidated Standards of Reporting Trials and Extension for Herbal Interventions” will be applied for assessing the quality of reports. We will conduct a sensitivity analysis to assess the robustness of the findings in the meta-analysis.</p></sec><sec><label>2.4.8</label><title>Grading the quality of evidence</title><p>“The Grading of Recommendations Assessment, Development and Evaluation” will be used for assessing the quality of evidence.</p></sec></sec><sec><label>2.5</label><title>Ethics and dissemination</title><p>In this study, ethical approval is not required because the included data are based on previously reported articles, and identifying information of participants will not be revealed. Results of this systematic review will be submitted for publication in a peer-reviewed journal and disseminated electronically and in print.</p></sec></sec><sec><label>3</label><title>Discussion</title><p><italic>Siho-sogan-san</italic> (SHS in Korea, <italic>Chaihu-Shugan-San</italic> [CSS] in China, and <italic>Saiko-sokan-to</italic> [SST] in Japan) is a traditional herbal formula that has been prescribed to alleviate dyspeptic symptoms for hundreds of years.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> SHS includes the following 7 crude herbs: <italic>Radix Bupleuri, Aurantii nobilis Pericarpium, Rhizoma Cnidii, Rhizoma Cyperi, Radix Paeoniae, Poncirus trifoliata Rafinesque,</italic> and <italic>Radix Glycyrrhizae</italic>.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> Animal studies showed that administration of CSS promoted gastric emptying and intestinal transit.<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup></p><p>According to TCM theories, SHS has been applied to patients with various symptoms of the syndrome of liver–stomach disharmony due to liver–<italic>qi</italic> stagnation, closely related to emotional instability. The liver–stomach disharmony can be induced by stagnation of <italic>qi</italic> due to depression. Thus, soothing the liver, regulating the depressed <italic>qi</italic> and stomach, and finally, relieving pain is the treatment principle of SHS to improve gastrointestinal dysfunction.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> Through a previous review of meta-analysis for 10 RCTs, the effectiveness and safety of CSS for the treatment of depression has been confirmed.<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]</sup> In an animal model study, CSS had an antidepressant effect on the repression of anger and distress.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> In another animal model, CSS regulated hyperactivity of the hypothalamic–pituitary–adrenal axis in rats resulting from chronic stress and exerted antidepressive effects.<sup>[<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</sup></p><p>The review of meta-analysis for 22 RCTs of modified <italic>Chaihu Shugan</italic> powder (MCSP) for treating FD showed that MCSP, used both alone and together with prokinetic drugs, was more effective than prokinetic drugs alone, without inducing any serious adverse events.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> There were some limitations to present adequate clinical evidence for proving MCSP's effectiveness. Above all, a high risk of bias and poor methodology were observed in the included studies. Language and databased publications were limited to only English and Chinese, and only total effective rates were evaluated and compared as outcome. Thus, in this systematic review of meta-analysis, we will use various database resources, including from Korea and Japan, in order to research the evidence of herbal medicine comprehensively. Moreover, herb-added formulas of SHS will be included and analyzed as the treatment group with no change in the primary prescription principles, reflecting variously modified form from the original composition. This study will update the safety and efficacy of SHS in monotherapy or in combination with Western medicine, including not only prokinetic drugs also acid-suppressive agents, antidepressants, based on the recent RCTs. The comparison groups will be set to no-treatment, placebo, and conventional Western medicine alone to provide clinical evidence. In case of enough studies to be analyzed, quality of life and objective parameters, such as gastric emptying, will be synthesized and evaluated for assessing outcomes. The results will be used to identify the effectiveness of SHS to improve other factors in addition to subjective dyspepsia symptoms, and conversely, to determine the pathophysiology of FD. We anticipate that this review will provide clinical information for the usage of SHS in detail.</p><p>In this protocol for a systematic review, ethics approval is not required because it is based on information gathered by searching already reported articles and no additional primary data collected. Results of this systematic review will be published in a peer-reviewed journal and provided as electronic dissemination.</p></sec><sec><title>Acknowledgments</title><p>This study was supported by a grant through the project “Development of Korean medicine clinical practice guidelines” of the Guideline Center for Korean Medicine, National Institute for Korean Medicine Development.</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization:</bold> Na-Yeon Ha, Hae-in Jeong.</p><p><bold>Data curation:</bold> Na-Yeon Ha, Hae-in Jeong, Ha-nul Lee, Seok-Jae Ko, Jae-Woo Park, Jinsung Kim.</p><p><bold>Formal analysis:</bold> Jinsung Kim.</p><p><bold>Investigation:</bold> Na-Yeon Ha, Hae-in Jeong, Ha-nul Lee.</p><p><bold>Methodology:</bold> Seok-Jae Ko, Jae-Woo Park, Jinsung Kim.</p><p><bold>Resources:</bold> Jinsung Kim.</p><p><bold>Writing – original draft:</bold> Na-Yeon Ha.</p><p><bold>Writing – review & editing:</bold> Jinsung Kim.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: CSS = <italic>Chaihu-Shugan-San</italic>, FD = functional dyspepsia, FGIDs = functional gastrointestinal disorders, MCSP = modified <italic>Chaihu Shugan</italic> powder, RCTs = randomized controlled trials, SHS = <italic>Siho-sogan-san</italic>, SST = <italic>Saiko-sokan-to</italic>, TCM = traditional Chinese medicine.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Ha NY, Jeong Hi, Lee Hn, Ko SJ, Park JW, Kim J. Herbal medicine <italic>Siho-sogan-san</italic> for functional dyspepsia: A protocol for a systematic review and meta-analysis. <italic>Medicine</italic>. 2020;99:39(e22358).</p></fn><fn fn-type=\"other\"><p>This study was first conceptualized by N-YH and HJ. The protocol was designed by S-JK, J-WP, and JK. N-YH, HJ, and HL will participate in searching-related articles, data extraction, and assessment of risk of bias. Data synthesis and analysis will be executed by JK and HJ. The original draft of this review was written by N-YH, and finally confirmed by JK. All authors have read and approved the final manuscript.</p></fn><fn fn-type=\"supported-by\"><p>This study was supported by a grant from the Korean Health Technology R&D Project through the Korean Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare of the Republic of Korea (project number: HB16C0019). 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991426</article-id><article-id pub-id-type=\"pmc\">PMC7523765</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-07946</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022273</article-id><article-id pub-id-type=\"art-access-id\">22273</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>4700</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Systematic Review</subject></subj-group></article-categories><title-group><article-title>The impact of COVID-19 on intestinal flora</article-title><subtitle>A protocol for systematic review and meta analysis</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Fangyuan</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Lu</surname><given-names>Hua</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Xinyun</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Xinxin</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Qi</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Mi</surname><given-names>Ling</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref></contrib></contrib-group><aff id=\"aff1\"><label>a</label>College of Clinical Medicine, Chengdu University of Traditional Chinese Medicine</aff><aff id=\"aff2\"><label>b</label>Chengdu University of Traditional Chinese Medicine Affiliated Hospital</aff><aff id=\"aff3\"><label>c</label>College of Acupuncture and Tuina, Chengdu University of Traditional Chinese Medicine</aff><aff id=\"aff4\"><label>d</label>Maternal and Child Reproductive Hospital Affiliated to Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, P.R. China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Hua Lu, Chengdu University of Traditional Chinese Medicine Affiliated Hospital, No.39 Shi-Er-Qiao Road, Chengdu 610075, Sichuan Province, P.R. China (e-mail: <email>lh18980880525@126.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22273</elocation-id><history><date date-type=\"received\"><day>18</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>20</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22273.pdf\"/><abstract abstract-type=\"toc\"><p>Supplemental Digital Content is available in the text</p></abstract><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background</title><p>Coronavirus disease (COVID-19) sparked global concern for its outbreak and pandemic. It caused severe respiratory tract infections and a significant proportion of patients with gastrointestinal symptoms. Several studies have investigated the intestinal flora of COVID-19. However, so far there has been no evidence demonstrating the evidence on the association of COVID-19 with intestinal flora through meta-analysis. A systematic and comprehensive understanding of their relationship is essential to provide public health prevention or treatment strategy.</p></sec><sec sec-type=\"methods\"><title>Methods and analysis</title><p>This systematic review and meta-analysis will be reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Observational studies (cohort studies, case-control, and cross-sectional studies) and clinical trials will be eligible. Studies eligible for inclusion must contain participants with COVID-19. Systematic searches will be conducted in PubMed, EMBASE, Cochrane Library, Ovid, EBSCO, World Health Organization COVID-19 database, China National Knowledge Internet, WanFang Data, Chinese Scientific and Technological Journal Database, and Chinese Biomedical Databases. A pre-designed search strategy of medical subject headings and free text terms for COVID-19 and intestinal flora will be used. Two reviewers will independently screen the titles and abstracts, followed by full-text screening. Discrepancies will be resolved by consensus with a third reviewer. The reviewers will then extract data from each eligible article based on PECOS (Population, Exposure, Comparator, Outcomes, and Study design). The risk of bias and quality of included studies will be assessed using an appropriate tool. A random-effects meta-analysis will be considered where there are sufficiently homogeneous studies; otherwise, a narrative synthesis will be conducted. Heterogeneity among studies will be assessed using I<sup>2</sup> statistics. If substantial heterogeneity detected, subgroup analyses and meta-regression will be conducted to look for the potential causes.</p></sec><sec><title>Ethics and dissemination</title><p>Ethical approval is not required as we will use data from published articles. Findings will be published in a peer-reviewed journal.</p><p>PROSPERO registration number: CRD42020191640</p></sec></abstract><kwd-group><title>Keywords</title><kwd>coronavirus disease 2019</kwd><kwd>intestinal flora</kwd><kwd>novel coronavirus</kwd><kwd>systematic review</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>Ministry of Science and Technology of the People's Republic of China</funding-source><award-id>2018YFC1704305</award-id><principal-award-recipient>Lu Hua</principal-award-recipient></award-group><award-group id=\"award2\" award-type=\"Fundref\"><funding-source>Collaborative Innovation Center for Modern Science and Technology and Industrial Development of Jiangxi Traditional Medicine (CN)</funding-source><award-id>2017-EL-16</award-id><principal-award-recipient>Ling Mi</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Since novel coronavirus was first discovered in December 2019, an extremely high potential for dissemination resulted in the global coronavirus disease 2019 (COVID-19) pandemic in 2020. The mortality rate of COVID-19 is much higher than that of any common influenza, affecting millions of people worldwide. As of July 25, 2020, 15,581,009 confirmed cases of COVID-19, including 635,173 deaths, were reported to World Health Organization.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> COVID-19 is caused by a beta coronavirus called SARS-CoV-2 and similar to severe acute respiratory syndrome and Middle East Respiratory Syndrome, which affects the lower respiratory tract and manifests as pneumonia in humans.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> The most common symptoms were fever, cough, expectoration, haemoptysis, headache, myalgia, diarrhea, and fatigue.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> A significant proportion of these patients had gastrointestinal (GI) symptoms.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> Some researches indicated that SARS-CoV-2 might be spread by fecal-oral transmission, and diarrhea could be a presenting feature in the incubation period.<sup>[<xref rid=\"R5\" ref-type=\"bibr\">5</xref>,<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> Unfortunately, there are no drugs or vaccines effective for the prevention or treatment of COVID-19 patients in large-scale studies.<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>,<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> As a public health emergency of international concern, global disease control of COVID-19 is challenging.</p><p>The gastrointestinal tract and respiratory tract are part of a shared mucosal immune system termed the gut-lung axis.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> An increasing amount of evidence supports the existence of the gut-lung axis.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> The gastrointestinal tract and the respiratory tract's microbiota participate in the gut-lung axis, influencing both local, and distant immune responses.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> The intestinal microbiota supports local mucosal immunity and is increasingly being recognized as an essential modulator of the systemic immune system.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup> Accumulating evidence indicates that intestinal flora influences lung immunity.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> However, it is essential to note that the gut-lung axis is bidirectional and is one means of communication between the gut microbiota and the lungs.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> Respiratory diseases have long been associated with lung-gut axis.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>,<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> Recent findings now highlighted the essential roles for gut microbiota in shaping lung inflammation.<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]</sup> Interestingly, there is a bidirectional interaction between respiratory tract infections and gut microbiome. The mutual interactions mostly focused on asthma, acute, and chronic respiratory infections, have received increasing attention.<sup>[<xref rid=\"R16\" ref-type=\"bibr\">16</xref>–<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]</sup> Influenza virus infection is believed to cause changes in intestinal flora.<sup>[<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</sup> A previous study demonstrated that the composition and diversity of gut microbiota were altered after viral lung infections. For instance, viral lung infection resulted in an increase in the phylum <italic>Bacteroidetes</italic> and a corresponding decrease in the <italic>Firmicutes phylum</italic>.<sup>[<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</sup> While the gut microbiome also shapes the adaptive immune responses against respiratory pathogens.<sup>[<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]</sup> Intestinal flora is closely related to respiratory virus infection and may influence the occurrence and development of diseases through the gut-lung axis.<sup>[<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</sup> Thus, the gut Microbiota may play a potential role in the treatment of lung diseases.</p><p>Novel coronavirus also has an impact on the intestinal flora. Compared with healthy controls, COVID-19 patients had significantly reduced bacterial diversity, a considerably higher relative abundance of opportunistic pathogens.<sup>[<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</sup> The gut microbiome of the COVID-19 group was dominated by <italic>Streptococcus</italic>, <italic>Rothia</italic>, <italic>Veillonella</italic>, <italic>Erysipelatoclostridium</italic>, and <italic>Actinomyces</italic>, whereas the microbiome of health group was dominated by the genera <italic>Romboutsia</italic>, <italic>Faecalibacterium</italic>, <italic>Fusicatenibacter</italic>, and <italic>Eubacterium hallii</italic> group.<sup>[<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]</sup> Concurrently, patients with COVID-19 were characterized by enrichment of opportunistic pathogens and depletion of beneficial commensals.<sup>[<xref rid=\"R22\" ref-type=\"bibr\">22</xref>]</sup> The baseline abundance of <italic>Clostridium ramosum</italic>, <italic>Coprobacillus</italic>, and Clostridium hathewayi correlated with COVID-19 severity, while the abundance of Faecalibacterium <italic>prausnitzii</italic> (an anti-inflammatory bacterium) was negatively correlated with disease severity.<sup>[<xref rid=\"R22\" ref-type=\"bibr\">22</xref>]</sup><italic>Bacteroides thetaiotaomicron</italic>, <italic>Bacteroides massiliensis</italic>, <italic>Bacteroides dorei</italic>, and <italic>Bacteroides ovatus</italic> correlated inversely with SARS-CoV-2 load in fecal samples from patients throughout hospitalization.<sup>[<xref rid=\"R22\" ref-type=\"bibr\">22</xref>]</sup> Indeed, accumulating evidence indicates that microbiota can modulate the immune response in the course of both bacterial and viral infections, becoming a potential target in the management of all these diseases.<sup>[<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]</sup> The recent pandemic induced by COVID-19 reminded us that the potential value of the gut microbiota may be as a therapeutic target for COVID-19. It may be possible to look in the gut for a solution or mitigation of SARS-CoV-2 infection.<sup>[<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]</sup></p><p>As of yet, there has been no systematic review and meta-analysis of studies reporting on the relationship between COVID-19 and intestinal flora. A better understanding of the relationship between gut microbiota and COVID-19 to derive appropriate targets for prevention or treatment is needed. Therefore, we aim to ascertain the association between COVID-19 and intestinal flora that will facilitate management or prevention strategies of COVID-19.</p></sec><sec><label>2</label><title>Methods and analysis</title><p>This protocol is registered in the International Prospective Register of Systematic Reviews (PROSPERO), registration number CRD42020191640. We designed this systematic review and meta-analysis following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocol statement<sup>[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]</sup> and meta-analysis of Observational Studies in Epidemiology.<sup>[<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]</sup></p><sec><label>2.1</label><title>Inclusion/exclusion criteria for study selection</title><sec><label>2.1.1</label><title>Study designs and characteristics</title><p>Studies concerning the association between COVID-19 and intestinal flora, which meet all inclusion criteria, will be included in the systematic review. We will include cohort studies (both prospective and retrospective cohort studies), case-control studies, cross-sectional studies, and clinical trials. Report of the studies in any language is eligible to be included. Reviews, commentaries, short surveys, case reports, and letters will be excluded. Review inclusion criteria are specified according to Participant, Intervention (or Exposure), Comparator and Outcome.</p></sec><sec><label>2.1.2</label><title>Participants</title><p>Participants with COVID-19; not have any severe primary GI disease that can affect intestinal flora or taking probiotics for a long time that affect intestinal flora; could be received anti-viral therapy.</p></sec><sec><label>2.1.3</label><title>Exposure</title><p>The exposures of interest are infection with COVID-19 (determined from throat swabs).</p></sec><sec><label>2.1.4</label><title>Comparators</title><p>The comparator will be healthy population that without COVID-19.</p></sec><sec><label>2.1.5</label><title>Outcome measures</title><p>The outcome will be the changes of gut microbiota, which explicitly reported at least 1 of the following: fecal mycobiome profiles, the composition of intestinal flora, changes in the fecal fungal, or bacterial microbiomes, the abundance of opportunistic pathogens, the abundance of beneficial commensal bacteria, and gut microbiota diversity.</p></sec><sec><label>2.1.6</label><title>Information sources and search strategy</title><p>The search strategies were performed by FYL and XXW, and differences were resolved by discussion with a third reviewer (XYL). We conducted searches in PubMed, EMBASE, Cochrane Library, Ovid, EBSCO, World Health Organization COVID-19 database, China National Knowledge Internet, WanFang Data, Chinese Scientific, and Technological Journal Database, and Chinese Biomedical Databases. These databases will be searched for relevant articles, from November 2019 until 2021/04/30.</p><p>The search will consist of searching medical subject heading(MeSH) terms and free text (in the title and abstract) for the concepts “COVID-19” and “intestinal flora” (combined with the Boolean logic operation “AND”). The following search strategy will be used for PubMed (see online supplementary appendix 1), which will then be adapted for other databases to be searched. At the same time, we will search for the clinical trial registries (i.e., <ext-link ext-link-type=\"uri\" xlink:href=\"https://clinicaltrials.gov/\">https://clinicaltrials.gov/</ext-link>) and gray literature about coronavirus infections and intestinal flora on the corresponding website to complete the electronic databases’ deficiencies. Conference proceedings and academic exchange summaries will be manually retrieved. To identify other relevant study data, we will contact the first author or correspondent author via email or telephone to obtain incomplete data. The whole process of study selection is summarized as flowchart in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>flowchart.</p></caption><graphic xlink:href=\"medi-99-e22273-g001\"/></fig></sec></sec><sec><label>2.2</label><title>Study selection</title><p>Articles from the database searches will be imported into Endnote X9 software. Duplicates will be removed. Two reviewers (XXW and QZ) will independently screen titles and abstracts in parallel to select studies for inclusion. Records that meet the specified inclusion criteria will then be taken forward to full-text screening, and records with little information available will be excluded. Potentially eligible full-text reports will be scrutinized carefully to decide whether to include or not. The reviewers will repeatedly cross-check the results and indicate the reasons for rejecting the article. Disagreement in the above process will be solved through discussion with a third reviewer (XYL) where necessary.</p></sec><sec><label>2.3</label><title>Data extraction</title><p>Data extraction will be performed by 2 authors independently (FYL and QZ). Any disagreements will be settled through discussion or the involvement of a third reviewer (XYL). Data from each eligible article will be extracted and compiled using a standardized Excel spreadsheet. Eligible extracted items will be obtained following the PECOS steps (Population, Exposure, Comparator, Outcomes, and Study design. The following data items will be extracted:</p><sec><label>2.3.1</label><title>Population</title><p>Participants’ demographic factors (e.g., mean age, ethnic distribution, proportion of gender, body mass index), inclusion and exclusion criteria, comorbidities (e.g., GI disease), medication intake.</p></sec><sec><label>2.3.2</label><title>Exposure</title><p>Diagnosis of COVID-19, number of exposed subjects, details of COVID-19 severity.</p></sec><sec><label>2.3.3</label><title>Comparators</title><p>Number of unexposed subjects.</p></sec><sec><label>2.3.4</label><title>Outcomes</title><p>Identification of intestinal flora outcomes (intestinal flora measurement method), changes in the gut microbiota (i.e., fecal mycobiome profiles, the composition of intestinal flora, changes in the fecal fungal, or bacterial microbiomes, the abundance of opportunistic pathogens, the abundance of beneficial commensal bacteria, and gut microbiota diversity), any association between COVID-19 and intestinal flora, any risk estimate between COVID-19, and changes in the intestinal flora.</p></sec><sec><label>2.3.5</label><title>Study characteristics</title><p>Title, objective, study design, country/region, journal, first author, sample size, observation, or follow-up time time.</p><p>Once the data is extracted, the above information should be cross-checked by 2 reviewers independently.</p></sec></sec><sec><label>2.4</label><title>Quality and bias assessment</title><p>The methodological quality of the included studies will be assessed by 2 reviewers (FYL and XYL) using the risk of bias (ROB). Two independent reviewers will be blinded to the titles, authors, and years of publication of the studies to evaluate the ROB of each included study. Any disagreements that cannot be resolved will be discussed with a third party (QZ). We will assess the ROB for each study following the Cochrane Collaboration approach for both randomized and non-randomized studies.<sup>[<xref rid=\"R27\" ref-type=\"bibr\">27</xref>,<xref rid=\"R28\" ref-type=\"bibr\">28</xref>]</sup> The methodological quality and ROB of any randomized controlled trials will be qualified using the Cochrane Collaboration's tool for ROB (ROB) assessment.<sup>[<xref rid=\"R29\" ref-type=\"bibr\">29</xref>]</sup> This tool assesses ROB include reporting bias, selection bias, detection bias, performance bias, and attrition bias. Each domain ROB will be assigned a ROB category as “low”, “high”, or “unclear”. The Critical Appraisal Checklist for Analytical Cross Sectional Studies from The Joanna Briggs Institute will be applied to assess the quality of cross-sectional studies.<sup>[<xref rid=\"R30\" ref-type=\"bibr\">30</xref>]</sup> This tool consists of 8 items that could be scored as “yes”, “no”, “unclear” or “not applicable”. The 9-point Newcastle-Ottawa Quality Assessment Scale<sup>[<xref rid=\"R31\" ref-type=\"bibr\">31</xref>]</sup> will be used to assess the quality of longitudinal studies, including case-control, and cohort studies. This tool includes 8 items grouped into 3 categories: selection, comparability, and exposure (case-control studies) / outcome (cohort studies). The NOS score ≥ 7 will be considered as high-quality. A summary ROB table will be produced, with an additional table briefly justifying each judgement included in the appendix. Publication bias will be assessed graphically by funnel plots. If the funnel plots show asymmetry, we will apply the Egger regression test.<sup>[<xref rid=\"R32\" ref-type=\"bibr\">32</xref>]</sup> We will use RevMan 5.3 to pool the data for analysis.</p></sec><sec><label>2.5</label><title>Statistical analysis</title><sec><label>2.5.1</label><title>Assessment of heterogeneity</title><p>The choice of whether to conduct a meta-analysis and which model to use (fixed or random effects) will depend on the level of statistical heterogeneity assessed by the I<sup>2</sup> index. An I<sup>2</sup> value less than 50% represents a non-substantial level of heterogeneity. Substantial heterogeneity was considered where I<sup>2</sup> was > 50%. If there is no evidence of heterogeneity, a fixed effect model<sup>[<xref rid=\"R33\" ref-type=\"bibr\">33</xref>]</sup> will be adopted for the meta-analysis; otherwise, a random-effects model<sup>[<xref rid=\"R34\" ref-type=\"bibr\">34</xref>]</sup> will be used.</p></sec><sec><label>2.5.2</label><title>Data synthesis and analysis</title><p>If there are sufficient data in the selected studies with the same design and sufficiently homogeneous populations, exposures, and outcomes to calculate pooled effect estimates, we will consider performing a meta-analysis. If meta-analysis is not feasible due to high heterogeneity of studies, we will conduct a narrative synthesis. We will summarize the evidence for the association between COVID-19 and changes in the intestinal flora.</p></sec><sec><label>2.5.3</label><title>Subgroup and sensitivity analyses</title><p>If substantial heterogeneity detected, subgroup analyses, and meta-regression will be conducted to look for the potential causes. If sufficient data is collected, a priori variables of interest for subgroup analyses to explore statistical heterogeneity will include:</p><list list-type=\"simple\"><list-item><label>(1)</label><p>type of study design;</p></list-item><list-item><label>(2)</label><p>country;</p></list-item><list-item><label>(3)</label><p>characteristics of population;</p></list-item><list-item><label>(4)</label><p>underlying disease or comorbidities;</p></list-item><list-item><label>(5)</label><p>sample size;</p></list-item><list-item><label>(6)</label><p>covariates included in the original studies (age, sex, weight, height, body mass index, among others);</p></list-item><list-item><label>(7)</label><p>medication usage (antiviral therapy or intestinal management). Sensitivity analysis will be conducted to assess the robustness of summary estimates by removing the study one by one.</p></list-item></list></sec></sec><sec><label>2.6</label><title>Ethics and dissemination</title><p>Ethical approval is not required for this study, as it is a systematic review. The results will be disseminated by publication of the manuscript in a peer-reviewed journal and for national and international presentations.</p></sec></sec><sec><label>3</label><title>Summary</title><p>To date, no review has comprehensively explored the association between COVID-19 and intestinal flora. The protocol will facilitate an understanding of the impact of COVID-19 on intestinal flora. An increased understanding of the relationship between COVID-19 and intestinal flora may be possible to inform methods of therapy or prevention. Given the high mortality and the public health burden, the conduct of the present systematic review is of high clinical, and practical relevance. The study will have broad representativeness by including individuals of all age groups and from all continents. However, potential limitations are inherent in conducting systematic reviews and meta-analyses. There may be poor method quality, publication bias, information bias, etc. Some strategies will be adopted to ensure the lack of ROB. Two independent reviewers will conduct the systematic review and meta-analysis, and a third researcher will be consulted when consensus is not reached or inconsistencies exist in data collection. Furthermore, existing guidelines, the meta-analysis of Observational Studies in Epidemiology statement, Preferred Reporting Items for Systematic Reviews and Meta-Analyses, and Cochrane Collaboration Handbook recommendations will be followed.</p></sec><sec><title>Author contributions</title><p>The study concept was developed by FYL, XYL, and QZ. The manuscript of the protocol was drafted by FYL and critically revised by XXW and LM. HL developed and provided feedback for all sections of the review protocol and approved the final manuscript. The search strategy was developed by FYL and XXW. Study selection will be performed by XXW and QZ. Data extraction and quality assessment will be performed by FYL and QZ, with XYL as a third party in case of disagreements. All authors have approved the final version of the manuscript.</p><p><bold>Investigation:</bold> Xinxin Wang, Qi Zhang.</p><p><bold>Methodology:</bold> Xinyun Li.</p><p><bold>Supervision:</bold> Ling Mi.</p><p><bold>Writing – original draft:</bold> Fangyuan Li.</p><p><bold>Writing – review & editing:</bold> Lu Hua.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary Material</title><supplementary-material content-type=\"local-data\" id=\"SD1\"><caption><title>Supplemental Digital Content</title></caption><media mimetype=\"application\" mime-subtype=\"pdf\" xlink:href=\"medi-99-e22273-s001.docx\" orientation=\"portrait\" id=\"d38e805\" position=\"anchor\"/></supplementary-material></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: COVID-19 = coronavirus disease 2019, GI = gastrointestinal, ROB = risk of bias.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Li F, Lu H, Li X, Wang X, Zhang Q, Mi L. The impact of COVID-19 on intestinal flora: A protocol for systematic review and meta analysis. <italic>Medicine</italic>. 2020;99:39(e22273).</p></fn><fn fn-type=\"supported-by\"><p>This study is financially supported by the Ministry of Science and Technology of the People's Republic of China (2018YFC1704305) and special fund for Women and Children Reproductive Hospital affiliated to Chengdu University of Traditional Chinese Medicine (2017-EL-16).</p></fn><fn fn-type=\"COI-statement\"><p>The authors have no conflicts of interest to disclose.</p></fn><fn fn-type=\"other\"><p>Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>[1]</label><mixed-citation publication-type=\"book\"><comment>World Health Organization, 2020. Coronavirus disease 2019 (COVID-2019) situation report–49. 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991474</article-id><article-id pub-id-type=\"pmc\">PMC7523766</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-03773</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022415</article-id><article-id pub-id-type=\"art-access-id\">22415</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3700</subject></subj-group><subj-group><subject>Research Article</subject><subject>Observational Study</subject></subj-group></article-categories><title-group><article-title>Admitted AIDS-associated Kaposi sarcoma patients</article-title><subtitle>Indications for admission and predictors of mortality</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Vally</surname><given-names>Faheema</given-names></name><degrees>MBcHb</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Selvaraj</surname><given-names>Wencilaus Margret Pious</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-0648-4128</contrib-id><name><surname>Ngalamika</surname><given-names>Owen</given-names></name><degrees>MMed</degrees><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Zhou.</surname><given-names>Bingrong</given-names></name></contrib></contrib-group><aff>Dermatology and Venereology Division, University Teaching Hospital, University of Zambia School of Medicine, Lusaka, Zambia.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Owen Ngalamika, Dermatology and Venereology Division, University Teaching Hospital, University of Zambia School of Medicine, Lusaka 10101, Zambia (e-mail: <email>owen_ngalamika@yahoo.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22415</elocation-id><history><date date-type=\"received\"><day>25</day><month>4</month><year>2020</year></date><date date-type=\"rev-recd\"><day>19</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22415.pdf\"/><abstract><title>Abstract</title><p>Kaposi sarcoma (KS) is an AIDS-defining angioproliferative malignancy associated with high morbidity and mortality. Most KS patients in regions with high incidence such as sub-Saharan Africa present late with advanced stage disease. Admitted KS patients have high mortality rates. Factors associated with mortality of admitted KS patients are poorly defined.</p><p>We conducted a retrospective file review to ascertain reasons for admission and identify factors associated with mortality of admitted HIV-associated (epidemic) KS patients in Zambia. Baseline study variables were collected, and patients were retrospectively followed from admission to time of discharge or death.</p><p>Mortality rate for admitted epidemic KS patients was high at 20%. The most common reasons for admission included advanced KS disease, severe anemia, respiratory tract infections, and sepsis. The majority (48%) of admitted patients had advanced clinical stage with visceral involvement on admission. Clinical predictors of mortality on univariate analysis included visceral KS [odds ratio (OR) = 13.74; 95% confidence interval (95% CI) = 1.68–113; <italic>P</italic> = 0.02), fever (OR = 26; 95% CI = 4.85–139; <italic>P</italic> = .001), and sepsis (OR = 35.56; 95% CI = 6.05–209; <italic>P</italic> = .001). Baseline hemoglobin levels (5.6 vs 8.2 g/dL; <italic>P</italic> = .001) and baseline platelet counts (63 x 10^9/L vs 205 x 10^9/L; <italic>P</italic> = .01) were significantly lower in mortalities vs discharges. Baseline white cell counts were higher in mortalities vs discharges (13.78 x 10^9/L vs 5.58 x 10^9/L; <italic>P</italic> = .01), and HIV-1 viral loads at the time of admission were higher in mortalities vs discharges (47,607 vs 40 copies/μL; <italic>P</italic> = .02). However, only sepsis (or signs and symptoms of sepsis) were independently associated with mortality after controlling for confounders.</p><p>In conclusion, common reasons for admission of epidemic KS patients include advanced disease, severe anemia, respiratory tract infections, and signs and symptoms of sepsis. Signs and symptoms of sepsis are independent predictors of mortality in these patients.</p></abstract><kwd-group><title>Keywords</title><kwd>Admitted</kwd><kwd>HIV-associated</kwd><kwd>Kaposi sarcoma</kwd><kwd>mortality</kwd><kwd>predictors</kwd><kwd>sepsis</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>National Institutes of Health</funding-source><award-id>K43TW011095</award-id><principal-award-recipient>Owen Ngalamika</principal-award-recipient></award-group><award-group id=\"award2\" award-type=\"Fundref\"><funding-source>National Institutes of Health</funding-source><award-id>D43TW010354</award-id><principal-award-recipient>Not Applicable</principal-award-recipient></award-group><award-group id=\"award3\" award-type=\"Fundref\"><funding-source>National Cancer Institute</funding-source><award-id>U54CA221204</award-id><principal-award-recipient>Not Applicable</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Kaposi sarcoma (KS) is an angioproliferative spindle cell tumor of endothelial origin.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> It is caused by the Kaposi sarcoma herpesvirus (KSHV).<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> KSHV seroprevalence is high in sub-Saharan Africa, and hence the high prevalence of KS in this region.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> KS commonly not only affects the skin, but can also affect other parts of the body, including the gastrointestinal tract, respiratory tract, lymph nodes, and conjunctiva of the eyes.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup></p><p>There are 4 main subtypes of KS. These include classic, endemic, iatrogenic, and epidemic KS.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> Classic KS mainly affects elderly men of Eastern European descent. Endemic KS, also known as African cutaneous KS, is more common in sub-Saharan Africa among HIV-seronegative individuals. Iatrogenic KS mainly affects patients on immunosuppressive drugs such as those undergoing organ transplants. Epidemic or AIDS-Associated KS is seen in HIV-infected individuals, is a WHO HIV stage 4 disease, is the most common type of KS worldwide, and has a rapidly progressive disease course.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup></p><p>The introduction of antiretroviral therapy (ART) has led to a reduced incidence and improved survival of epidemic KS globally.<sup>[<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> However, despite combination antiretroviral therapy, epidemic KS is still highly prevalent with high mortality rates world-wide, and especially in sub-Saharan Africa.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> Between 2008 and 2013, the University Teaching Hospital (UTH) in Zambia recorded at least 726 incident KS cases, majority of which were HIV-associated.<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup></p><p>Unlike other forms of KS, AIDS-associated KS has a rapidly progressing disease course, and may disseminate in a short period of time to involve the viscera, including lungs and gastrointestinal tract. <sup>[5]</sup> About 10% to 20% of patients present with advanced disseminated disease and/or complications such as anemia, and are admitted to the inpatient medical wards. Several factors including stage of KS at presentation, ART status, and CD4 counts have been reported to predict outcomes of KS patients.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> In addition, recurrence rates of chemotherapy-treated epidemic KS patients are high.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup></p><p>KS remains one of the most common AIDS-related malignancies in Zambia, with numerous admissions to our hospitals. KS patients make up about 70% of all dermatological admissions at UTH. There is paucity of data regarding the factors affecting outcomes of admitted epidemic KS patients. Most previous studies have focused on outcomes of epidemic KS in general, and not specifically on the admitted population of epidemic KS patients. Therefore, this study aimed at determining reasons for admission, and identifying clinical and laboratory parameters that predict mortality or discharge of admitted epidemic KS patients.</p></sec><sec><label>2</label><title>Methods</title><p>A retrospective file review of a series of HIV-associated/epidemic KS patients admitted in the period from April 2019 to November 2019 was carried out. We reviewed medical records of adult epidemic KS patients aged 18 years and older, who were admitted to the medical wards of the Adult hospital of the University Teaching Hospitals (UTH) in Lusaka, Zambia.</p><p>We used printed data collection forms to obtain information from patient files. The information was later entered into an excel spreadsheet and then exported to STATA for analysis. The clinical and sociodemographic information collected included age, gender, previous hospital admissions, smoking, alcohol use, ART status, adherence to ART, duration on ART, other comorbidities (e.g., tuberculosis, sepsis, diabetes, hypertension, hepatitis B), duration of KS, KS clinical stage, history of receiving chemotherapy, fever, palor, jaundice, weight loss, treatment given during admission (chemotherapy, blood transfusion, oxygen, antibiotics, ART). We also collected laboratory parameters, including full blood count, liver function test, renal function test, HIV viral loads, CD4 counts, and histopathology staging.</p><p>The main objective of the study was to identify reasons for admission and factors associated with outcomes (discharge or mortality) of admitted adult epidemic KS patients at UTH. Ethical approvals for a waiver of consent were obtained from the University of Zambia Biomedical Research Ethics Committee, and the National Health Research Authority. Obtaining informed consent from the actual participants was not possible, as this was a retrospective study and study participants were not available to give consent, as they were either discharged or had died.</p><p>All data were analyzed using STATA version 15 (StataCorp, TX). Baseline characteristics were analyzed using summary and descriptive statistics. Continuous variables are presented as median and interquartile range, while dichotomous or categorical variables are presented as percentages. The Mann–Whitney test was used for group comparisons of continuous variables. Univariate logistic regression was used to determine the association between the dichotomous outcome and the categorical and continuous predictors. Multivariate logistic regression was used to control for confounders. Predictors with <italic>P</italic> values < .25 on univariate logistic regression and predictors known to be associated with the outcome regardless of <italic>P</italic> values, were included on the multivariate logistic regression model. Among the highly correlated variables, only one was included in the multivariable model. The prediction of our model for outcome was determined by the AUC-ROC curve. The Hosmer–Lameshow goodness of fit test was used to determine how well our model fit the data. We also used the logistic regression model to test for interactions between variables that may possibly modify the observed effect.</p></sec><sec><label>3</label><title>Results</title><p>We collected and analyzed information of 54 epidemic KS patients admitted in the period from April 2019 to April 2020. Among these, 43 (80%) were discharged, while 11 (20%) were mortalities. Baseline characteristics of the study participants, by outcome, are summarized in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Baseline characteristics of admitted KS patients by outcome.</p></caption><graphic xlink:href=\"medi-99-e22415-g001\"/></table-wrap><p>The most common reason for admission for discharged patients was advanced/disseminated KS followed by severe anemia, and the most common reason for admission for individuals who died was severe anemia followed by sepsis (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>). Other reasons for admission in all the patients included respiratory tract infections, acute gastroenteritis, upper gastrointestinal bleeding, upper airway obstruction, dysphagia, hypertension, deep vein thrombosis, wet gangrene, and generalized lymphadenopathy.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Admission diagnoses by outcome. Advanced KS and severe anemia were the most common admission diagnoses among the patients who were discharged. Severe anemia and sepsis were the most common admission diagnoses among the patients who died in the hospital. GI = gastrointestinal, RTI = respiratory tract infections.</p></caption><graphic xlink:href=\"medi-99-e22415-g002\"/></fig><p>KS clinical staging at presentation was different between the discharged patients and mortalities. Among the discharged patients, 14% presented with localized cutaneous KS, 49% had disseminated cutaneous KS, while 37% had advanced KS (cutaneous along with visceral KS). Among the mortalities, 9% had disseminated cutaneous disease on admission, while 91% had advanced KS.</p><p>On univariate logistic regression, the factors significantly associated with mortality included history of previous hospital admissions, presence of visceral KS, fever, and an admission diagnosis of sepsis. In addition, hospital interventions including oxygen therapy, intravenous antibiotics, and blood transfusion were significantly associated with mortality (Table <xref rid=\"T2\" ref-type=\"table\">2</xref>).</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Univariate logistic regression analysis for clinical predictors of mortality.</p></caption><graphic xlink:href=\"medi-99-e22415-g003\"/></table-wrap><p>We also collected and compared results of routine laboratory tests done on admission. Individuals who died had statistically significantly low hemoglobin levels and low platelet counts as compared to individuals who were discharged. Those who died also had statistically significantly high white blood cell counts and median HIV viral load as compared to those who were discharged (Table <xref rid=\"T3\" ref-type=\"table\">3</xref>).</p><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>Association between laboratory parameters and outcomes.</p></caption><graphic xlink:href=\"medi-99-e22415-g004\"/></table-wrap><p>We built a multivariate logistic regression model with variables that were statistically significant on univariate logistic regression and the laboratory parameters that were significantly associated with the outcome on univariate analysis. After adjusting for age, gender, hemoglobin levels, platelet count, and creatinine, sepsis remained a significant independent predictor of mortality (Table <xref rid=\"T4\" ref-type=\"table\">4</xref>). The multivariable logistic regression model was a good predictor of mortality with an area under the curve of 0.98. The model was also a good fit according to the Hosmer–Lameshow goodness of fit test (<italic>P</italic> = .96). In addition, there was no multicollinearity of the variables in the model, with a mean variance inflation factor of 1.36 (highest = 1.57, lowest = 1.13).</p><table-wrap id=\"T4\" orientation=\"portrait\" position=\"float\"><label>Table 4</label><caption><p>Multivariate logistic regression for predictors of mortality.</p></caption><graphic xlink:href=\"medi-99-e22415-g005\"/></table-wrap><p>Testing for interactions between sepsis and intravenous antibiotics (<italic>P</italic> = .30), and between blood transfusion and anemia (<italic>P</italic> = .69) were not statistically significant.</p></sec><sec><label>4</label><title>Discussion</title><p>This study evaluated the factors associated with mortality of admitted adult epidemic KS patients at UTH, in Zambia. Twenty percent of all our admitted KS patients during the study period died in the hospital. This is similar to a prospective 10-year study conducted in our neighboring country, Tanzania, where a mortality of 24.2% was reported.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> However, the Tanzanian study included all KS patients regardless of hospital admission status.</p><p>Common reasons for admission among patients who were eventually discharged included disseminated/advanced KS and severe anemia, whereas the most common reasons for patients who eventually died were severe anemia and sepsis. Advanced HIV disease is in itself a risk factor for developing anemia and/or sepsis.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>,<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup> In addition, KS has been previously reported to be associated with severe anemia.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> It is therefore not surprising that anemia and sepsis were among the most common reasons for admission.</p><p>Late KS presentation with advanced disease on admission was a common feature in both the discharged patients and in those who died. However, those who died had a higher proportion of individuals with advanced disease than those who were discharged. This is similar to a study conducted in another sub-Saharan African country where a majority (69%) of AIDS-associated KS patients presented with advanced disease and had high mortality rates.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> These findings suggest that presentation of KS in the early clinical stages are likely to improve outcomes.</p><p>Clinical predictors of mortality on univariate logistic regression included visceral KS, fever, and sepsis. Advanced KS presents as multiple disseminated KS lesions with or without massive lymphedema and often accompanied with visceral involvement. When KS involves the viscera, commonly lungs and gastrointestinal tract, patients often have systemic life-threatening complications requiring admission and intensive care. Hence, the prognosis for advanced KS is usually poor.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> Fever is a common sign of sepsis; thus, most of our patients who were diagnosed with sepsis also had fever.</p><p>Medical interventions, including blood transfusion, oxygen, and intravenous antibiotics, were also associated with death on univariate logistic regression. As most of the patients who died were very ill, with advanced KS disease on admission, they were more likely to be commenced on these medical interventions. Therefore, these treatments were only predictors of mortality in the sense that they were likely to be initiated in very ill KS patients who had advanced disease.</p><p>Several laboratory parameters, including low hemoglobin, low platelets, high white cell counts, and high HIV-1 viral loads, were also significantly associated with mortality. Anemia is a risk factor for HIV infection, and has previously been associated with shorter survival in HIV-infected patients.<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> The severe anemia in the individuals who died may be a result of the more advanced HIV disease and advanced KS. Thrombocytopenia has previously been reported in KS patients and supposedly results from platelet sequestration in the abnormal KS tumor vessels.<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>,<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</sup> Therefore, advanced disease with numerous KS lesions possibly explains why individuals who died had lower platelet counts. At the time of admission, patients who eventually died also had higher white cell counts above the upper limit of normal. Our findings are consistent with previous studies that have reported high white cell and neutrophil counts to be associated with a poor prognosis in the form of death or KS disease progression.<sup>[<xref rid=\"R17\" ref-type=\"bibr\">17</xref>,<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</sup> Higher white cell counts may reflect a higher degree of chronic inflammation or may be a sign of sepsis in individuals who died. HIV viral load was 500-fold higher at baseline in individuals who eventually died than individuals who were discharged. HIV viral load is a marker of HIV disease control.<sup>[<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]</sup> In addition, poor control of HIV infection predisposes to KS development and progression.<sup>[<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</sup> In our study cohort, individuals who died had a history of poor compliance to ART compared to those who were discharged from hospital. This may have contributed to the advanced disease presentation, high HIV viral load, and poor outcome. However, CD4 counts were not predictive of mortality.</p><p>After controlling for other predictors of mortality, only sepsis (or signs and symptoms of sepsis) was independently associated with mortality. Sepsis is common in immunosuppressed HIV patients. It has been previously identified as a major cause of admission and mortality in HIV patients.<sup>[<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]</sup> Unfortunately, blood cultures were not done to confirm the diagnosis of sepsis in our patients due to unavailability of resources. It is possible that the signs and symptoms of sepsis were due to a phenomenon called Kaposi sarcoma inflammatory cytokine response (KICS). KICS is common in severely ill KS patients, mimics severe sepsis, is associated with high Interleukin 6 (IL-6) production, associated with high KSHV viral loads, and is also associated with a high mortality rate.<sup>[<xref rid=\"R22\" ref-type=\"bibr\">22</xref>–<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]</sup> It is more probable that our patients had KICS and not sepsis. This is because empirical intravenous broad-spectrum antibiotics that are known to be effective in septic HIV individuals in our setting were administered in our patients with signs of sepsis; however, these antibiotics had no effect and even seemed to be associated with poor outcomes. Furthermore, there was no interaction between treatment with intravenous antibiotics and a diagnosis of sepsis. Prospective cohort studies and more extensive laboratory testing need to be conducted to address this issue.</p></sec><sec><label>5</label><title>Study limitations</title><p>We could not definitely differentiate sepsis vs KICS because blood cultures, KSHV viral loads, and IL-6 levels were not assessed during admission. A prospective cohort study would be required to definitively address this. In addition, the sample size was not powered enough to determine statistical differences in marginally significant variables including hemoglobin levels and creatinine on multivariate regression.</p></sec><sec><label>6</label><title>Conclusion</title><p>Advanced disease with visceral involvement, severe anemia, and symptoms and signs of sepsis were among the most common reasons for admission of epidemic KS patients. Several factors, including advanced KS clinical stage, sepsis, low hemoglobin, low platelet count, high white cell count, and high HIV viral load, were associated with mortality. However, only signs and symptoms of sepsis were independent predictors of mortality for admitted KS patients.</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization:</bold> Owen Ngalamika.</p><p><bold>Data curation:</bold> Faheema Vally, Wencilaus Margret Pious Selvaraj.</p><p><bold>Formal analysis:</bold> Owen Ngalamika.</p><p><bold>Funding acquisition:</bold> Owen Ngalamika.</p><p><bold>Investigation:</bold> Faheema Vally.</p><p><bold>Methodology:</bold> Faheema Vally, Owen Ngalamika.</p><p><bold>Resources:</bold> Faheema Vally, Owen Ngalamika.</p><p><bold>Supervision:</bold> Owen Ngalamika.</p><p><bold>Validation:</bold> Wencilaus Margret Pious Selvaraj, Owen Ngalamika.</p><p><bold>Writing – original draft:</bold> Faheema Vally.</p><p><bold>Writing – review & editing:</bold> Faheema Vally, Wencilaus Margret Pious Selvaraj, Owen Ngalamika.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: AIDS = advanced immunodeficiency syndrome, ART = antiretroviral therapy, AUC-ROC = area under curve – receiver operating characteristic curve, CI = confidence interval, HIV = human immunodeficiency virus, KICS = Kaposi sarcoma inflammatory cytokine syndrome, KS = Kaposi sarcoma, KSHV = Kaposi sarcoma associated herpes virus, OR = odds ratio, UTH = University Teaching Hospital, WHO = World Health Organization.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Vally F, Selvaraj WM, Ngalamika O. Admitted AIDS-associated Kaposi sarcoma patients: Indications for admission and predictors of mortality. <italic>Medicine</italic>. 2020;99:39(e22415).</p></fn><fn fn-type=\"supported-by\"><p>This work was supported by the Fogarty International Center, National Cancer Institute, and National Institutes of Health (NIH) under award numbers K43TW011095, D43TW010354, and U54CA221204. ON is an NIH Fogarty Fellow. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991408</article-id><article-id pub-id-type=\"pmc\">PMC7523774</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-02651</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022146</article-id><article-id pub-id-type=\"art-access-id\">22146</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>7400</subject></subj-group><subj-group><subject>Research Article</subject><subject>Observational Study</subject></subj-group></article-categories><title-group><article-title>Germinal ovarian tumors in reproductive age women</article-title><subtitle>Fertility-sparing and outcome</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0003-3522-4648</contrib-id><name><surname>Dellino</surname><given-names>Miriam</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Silvestris</surname><given-names>Erica</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Loizzi</surname><given-names>Vera</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Paradiso</surname><given-names>Angelo</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Loiacono</surname><given-names>Rosalia</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Minoia</surname><given-names>Carla</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Daniele</surname><given-names>Antonella</given-names></name><degrees>BS</degrees><xref ref-type=\"aff\" rid=\"aff5\"><sup>e</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Cormio</surname><given-names>Gennaro</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Bush.</surname><given-names>Eric</given-names></name></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Gynecologic Oncology Unit, IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy</aff><aff id=\"aff2\"><label>b</label>Department of Biomedical Sciences and Human Oncology, Unit of Obstetrics and Gynaecology</aff><aff id=\"aff3\"><label>c</label>IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy</aff><aff id=\"aff4\"><label>d</label>Haematology Unit, IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy</aff><aff id=\"aff5\"><label>e</label>Biology Research, Department of Experimental Oncology, IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Miriam Dellino, Gynecologic Oncology Unit, IRCCS Istituto Tumori “Giovanni Paolo II”, Bari 70124, Italy (e-mail: <email>miriamdellino@hotmail.it</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22146</elocation-id><history><date date-type=\"received\"><day>3</day><month>4</month><year>2020</year></date><date date-type=\"rev-recd\"><day>31</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>8</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22146.pdf\"/><abstract><title>Abstract</title><p>MOGCTs (malignant ovarian germ cell tumors) are rare tumors that mainly affect patients of reproductive age. The aim of this study was to evaluate the fertility and survival outcomes in young women with MOCGTs treated with fertility-sparing surgery (FSS).</p><p>From 2000 to 2018, data from 28 patients of reproductive age with a diagnosis of MOGCT at the University of Bari were collected. Most received FSS, and in patients treated conservatively, the reproductive outcome and survival were investigated. Data of patient demographics, clinical presentation, oncology marker dosage, staging, type of surgery, histological examination, survival, and reproductive outcome were collected from hospital and office charts. All informed consent was obtained from all patients. The median age was 24 (range: 9–45 years). The majority of the patients had stage IIIC. Twenty-four woman received FSS consisting of unilateral ovariectomy and omentectomy, whereas only 4 women, based on their stage (IIIC), received a radical surgery (hysterectomy with bilateral adnexectomy, lymphadenectomy, and omentectomy). Our study shows that FSS in MOGCTs can produce good results both on reproductive outcomes and on survival. Indeed, in our group, there was only 1 case of exitus as result of recurrence. Furthermore, patients after FSS maintained normal ovarian function and 5 of 5 women who tried to get pregnant succeeded spontaneously. The median follow-up was 90 months (range 3–159).</p><p>Conservative surgery for MOGCTs should be considered for women of reproductive age who wish to preserve fertility.</p></abstract><kwd-group><title>Keywords</title><kwd>fertility-sparing surgery</kwd><kwd>malignant ovarian germ cell tumors</kwd><kwd>outcome</kwd><kwd>ovarian neoplasms</kwd><kwd>reproductive age</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Ovarian germ cell tumors (OGCTs) represent 20% to 25% of all ovarian neoplasms, whereas malignant ovarian germ cell tumors (MOGCTs) comprise only 5%.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> MOGCTs show a peak prevalence in young women and adolescents. Approximately 60% of ovarian tumors are germ cell tumors in patients younger than 20 years, and one-third of these cases are malignant.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> MOGCTs include several histotypes, all deriving from primordial germ cells of the ovary, and represent a heterogeneous group of tumors with variable biological behavior, clinical presentation and prognosis.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> There is a variety of histologic types: dysgerminoma, immature teratoma, endodermal sinus tumor, choriocarcinoma, polyembryoma, and mixed MOGCTs.<sup>[<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> These tumors differ from epithelial ovarian cancer (EOC) by incidence at an earlier age, for their almost unilateral localization (95% of cases), for their high rate of growth, a rare tendency to spread, and finally for their good prognosis.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>,<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup> This is explained by the numerous diagnoses at early stages and their high chemosensitivity.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> Therefore, the treatment of this pathology in early stages requires only surgery, while in the presence of high-risk factors or advanced disease, after surgical treatment, chemotherapy is highly recommended.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>,<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> The most-used chemotherapeutic protocol is the combination of Bleomycin, Etoposide, and Cisplatin (BEP) or Bleomycin, Vinblastine, Cisplatin (PVB) for 4 to 6 cycles, and this is based on histotype and stage.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup> Patients are followed up with abdominal-pelvic examination and ultrasound, complete blood count, and a biochemistry profile every 2 to 3 months for the first 2 years, at semi-annual intervals up to year 5, and annual intervals thereafter. Chest X-ray is ordered at annual intervals or in case of clinical suspicion. Computed tomography of the thorax and abdomen is performed every 6 months for the first 2 years, and at annual intervals up to year 5.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> A variable long-term survival rate of 82% to 100% is reported in the literature in the early stages and 75% in the advanced stages.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup></p><p>Considering the frequent incidence of MOGCTs in young women and the high survival rate, clinicians should consider fertility-sparing surgery (FSS).<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> Correct information about the risks of iatrogenic infertility and the strategies available to reduce the incidence of this effect (reproductive counseling) should be offered to young cancer patients immediately after diagnosis, at subsequent stages of the disease, and before the start of treatment.<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> The main techniques of fertility preservation in patients who need to undergo cancer treatments are cryopreservation of embryos or eggs, cryopreservation of ovarian tissue, gonadic suppression with similar gonadotropin-releasing hormone (GnRH) analogues, and conservative surgery. FSS is identified as the cornerstone of early-stage MOGCT treatment. On the contrary, for the advanced stages, the possibility of performing an FSS in young patients who desire a pregnancy should be assessed in a personalized manner and after counseling with the patient.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> Indeed, current literature presents reassuring data relating to favorable overall survival (OS) and reproductive outcome after FSS.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup> In this study, we analyzed survival outcomes in women of childbearing age diagnosed with MOGCTs and treated with FSS. We also evaluated changes in the menstrual cycle and post-treatment pregnancies.</p></sec><sec><label>2</label><title>Material and methods</title><p>From 2000 to 2018, data from all women of reproductive age with a diagnosis of MOCGTs at the University of Bari were collected. Data of patient demographics, clinical presentation, oncology marker dosage, staging, type of surgery, histological examination, survival, and reproductive outcome were collected from hospital and office charts. Each patient was given a descriptive form of the study and was formally invited to participate. After having agreed, informed consent was submitted to all patients. All procedures performed in this study were in accordance with the Helsinki Declaration as revised in 2013. In addition, patients were also informed that the data collected for this study are protected by the Privacy Act; therefore, they were collected and used after obtaining written authorization from each patient for the use of personal data for scientific purposes only. The evaluation of the disease stage was performed using the Federation of Gynecology and Obstetrics (FIGO) classification,<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> whereas the histopathological definition was evaluated according to the classification of germ cell tumors of the ovary of the World Health Organization (WHO).<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]</sup> A cytological analysis of the ascitic fluid or the peritoneal washing and an intraoperative histological examination was performed in all cases. In addition, all patients presented an extemporaneous germ cell tumor subsequently confirmed at the definitive histological examination. The “maximum” debulking was defined as a tumor residue = 0 after primary or recurrent surgery, “optimal” in the case of a 1 cm tumor residue, and “not optimal” >1 cm. Disease-free survival (DFS) was defined as the period between diagnosis and recurrence, whereas OS was identified as the period between diagnosis and the time of death or last follow up.<sup>[<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</sup> Adjuvant BEP or PVB (average of 4 cycles every 3 weeks) was indicated for patients with IC or higher disease, immature teratoma or high-grade tumors, and residual disease after cytoreductive surgery. Patients were followed up with abdominal and pelvic examination and ultrasound, blood count, and tumor marker dosage every 2 to 3 months for the first 2 years, at semi-annual intervals up to year 5, and at annual intervals thereafter. Computed tomography of the thorax and abdomen was performed every year for the first 5 years. Chest X-ray was indicated at annual intervals for the first 2 years. The limitations of this study are represented by the limited number of cases, and the retrospective analysis.</p></sec><sec><label>3</label><title>Results</title><p>Twenty-eight patients with a diagnosis of MOGCTs were studied. The median age was 24 (range: 9–45 years). The majority of the patients had stage IIIC. Most received FSS consisting of unilateral ovariectomy and omentectomy, whereas only 4 women, based on their stage (IIIC), received a radical surgery (hysterectomy with bilateral adnexectomy, lymphadenectomy, and omentectomy). The median follow-up was 90 months (range 3–159) (Table <xref rid=\"T1\" ref-type=\"table\">1</xref>). The onset of the disease was pain in 18 (64%), abdominal distension in 26 (26%), ascites in 2 (7%), dyspareunia in 3 (10%), weight gain 2 (7%), vaginal bleeding 1 (3.5%), and symptomless in 3 women (10%). The average diameter of the neoplasms was 9.2 cm (range 5–20). An increase was also recorded in the following tumor markers: carbohydrate antigen (CA) -125 in 3 (10.7%), beta-human chorionic gonadotropin (β-HCG) in 6 (21.4%), alpha-fetoprotein (AFP) in 11 (39%), CA 19.9 in 2 (7%), and CA-15.3 in 1 patient (3%). None of our patients showed an increase in carcinoembryonic-antigen (CEA). Tumor histology included 4 (14%) teratomas, 11 (39%) dysgerminomas, 3 (10%) endodermal sinus tumors, and 10 (35%) mixed MOCGTs tumors. Ten women had I A (35%), 5 presented stage I C (32%), 5 stage II A (18%), and 4 (14%) stage IIIC disease. Therefore, a total of 18 patients were treated with adjuvant therapy: 16 with BEP and 2 with PVC. The chemotherapeutic regimen was administered for 4 cycles every 3 weeks (Table <xref rid=\"T1\" ref-type=\"table\">1</xref>). Three women (12%) treated with FSS with a histology different from dysgerminoma had a recurrence 6 years after treatment; therefore, a completion of surgery was performed. Of the 28 women, 2 women did not survive: 1 with a diagnosis of endodermal sinus tumor died due to disease progression despite radical surgery and chemotherapy, and another died due to severe fatal toxicity after the first cycle of PVB chemotherapy. With a median follow-up period of 90 months, 5-year OS rate was 85% and the record of recurrence rate was 12% with a DFS of 88% in our population. Moreover, fertility preservation techniques were used in 8 patients by performing GnRH analogues during chemotherapy, and in 2 cases through cryopreservation of ovarian tissue and cryopreservation of oocytes upon commencement of treatment, respectively. In these last 2 cases, both patients later had spontaneous pregnancies without having to resort to thawing. Of the 28 patients of child-bearing age diagnosed with MOCGTs, excluding the 4 patients who had a hysterectomy after diagnosis, 3 after recurrence, 1 patient with pure gonadal dysgenesis (Swyer syndrome -karyotype 46 XY), and 1 death after chemotoxicity, we investigated the regularity of the post-treatment menstrual cycle and reproductive status. Of 19 women, 15 (78%) reported regular menstrual cycles during and after chemotherapy; on the contrary, the remaining 4 (21%) presented amenorrhea during chemotherapy but reported regular cycles after the end of treatment. Regarding the reproductive outcome, 4 patients had a pregnancy before the disease without attempting further conception, 11 patients (average age 32 years) declared that they were not currently interested in childbearing, 1 women had a spontaneous abortion (SAB) at 7 weeks, 1 had a voluntary pregnancy interruption (IVP) at 22 weeks following a diagnosis of multiple fetal malformations, and the remaining 5 reported having achieved pregnancy through spontaneous conception and with vaginal delivery. Of these 5 women, the median time to achieve a pregnancy was 8 years from surgery (range 3–15 years) and 4 of them had a combination of BEP + GnRH analogues, whereas 1 only chemotherapy after surgery (Table <xref rid=\"T2\" ref-type=\"table\">2</xref>).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Patient characteristics and treatment data.</p></caption><graphic xlink:href=\"medi-99-e22146-g001\"/></table-wrap><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Pregnancy outcome.</p></caption><graphic xlink:href=\"medi-99-e22146-g002\"/></table-wrap><sec><label>3.1</label><title>Statistical analysis</title><p>In order to report the survival analysis of patients with MOCGTs, a nonparametric statistical analysis was used represented by the Kaplan–Meier estimator (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Kaplan–Meier estimates in patients with malignant ovarian germ cell tumors (MOCGTs).</p></caption><graphic xlink:href=\"medi-99-e22146-g003\"/></fig></sec></sec><sec><label>4</label><title>Discussion</title><p>About 60% of OGCTs are diagnosed in patients under the age of 20, and one-third of these are malignant.<sup>[<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</sup> Primary tumor site, histologic subtype, and metastasis could represent significant prognostic factors for survival.<sup>[<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]</sup> Patients in our study had demographic and clinical features similar to those reported in other series, as regards age, histologic subtype, and primary tumor site.<sup>[<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</sup> Moreover, the ovaries and dysgerminomas were, respectively, the most common primary site and histologic subtype in our research, also comparable to further reports.<sup>[<xref rid=\"R19\" ref-type=\"bibr\">19</xref>,<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</sup> Furthermore, in our research, we found a high survival rate in concordance with the results of additional series.<sup>[<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]</sup> There are some reasons to explain this, such as the early diagnosis, unilateral localization, rare metastases,<sup>[<xref rid=\"R22\" ref-type=\"bibr\">22</xref>]</sup> and high chemosensitivity<sup>[<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]</sup> typical of these rare tumors. Moreover, the recorded recurrence rate in our patient population is low (about 12%) with a corresponding DFS of 88% and is comparable to other reports series.<sup>[<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]</sup> Therefore, considering the favorable prognosis of patients with MOCGT in the early stages, FSS is considered the therapeutic standard in women who want to maintain their reproductive function.<sup>[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>,<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]</sup> Several studies report reassuring data even in advanced MOGCTs, both on OS (87.9% at 5 years) and on the absence of alterations of the menstrual cycle and reproductive function.<sup>[<xref rid=\"R27\" ref-type=\"bibr\">27</xref>]</sup> In literature, the increase in AFP levels and a different histology from the dysgerminoma have been correlated to a greater incidence of recurrences. Therefore, in these cases, a more extensive surgical treatment and a greater number of chemotherapy cycles could be indicated, after individualized evaluation of the risk factors.<sup>[<xref rid=\"R28\" ref-type=\"bibr\">28</xref>–<xref rid=\"R30\" ref-type=\"bibr\">30</xref>]</sup> This is also reflected in terms of OS, which in the literature is reported to be equal to 97% in cases of dysgerminoma, and 60% (<italic>P</italic> < .001) in nongerminomas.<sup>[<xref rid=\"R31\" ref-type=\"bibr\">31</xref>]</sup> In our experience, all cases of recurrence recorded showed a histology different from dysgerminoma, and high dosage of AFP in the only case evolved in exitus for disease. Surgical treatment of advanced states must be supplemented with chemotherapy, whose effects on fertility have been widely discussed in literature. Solheim et al<sup>[<xref rid=\"R31\" ref-type=\"bibr\">31</xref>]</sup> compared patients who received 3 cycles of platinum-based chemotherapy with those who received more than three cycles. In the latter group the rate of infertility was significantly higher (<italic>P</italic> = .040).<sup>[<xref rid=\"R32\" ref-type=\"bibr\">32</xref>–<xref rid=\"R34\" ref-type=\"bibr\">34</xref>]</sup> Some authors found a reduction of primordial follicles, and stromal fibrosis following chemotherapy, with a parallel increase in gonadotropins and reduced levels of estrogen.<sup>[<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]</sup> These effects seem to be closely related to the type of drugs, dose, and chemotherapy duration.<sup>[<xref rid=\"R36\" ref-type=\"bibr\">36</xref>]</sup> In our study, there is no evidence of any reproductive outcome reduction following multiple chemotherapy cycles. Similar data are reported in several papers that showed that PEB versus PVC chemotherapy for MOCGTs does not appear to affect reproduction or the menstrual cycle, which normalized within 6 months in 90% of cases.<sup>[<xref rid=\"R37\" ref-type=\"bibr\">37</xref>–<xref rid=\"R39\" ref-type=\"bibr\">39</xref>]</sup> Similarly, we reported that 78% of patients had regular menstrual cycles during and after chemotherapy. On the contrary, 22% of patients presented amenorrhea during chemotherapy but had regular cycles 5 months after treatment. As regards fertility preservation techniques, treatment with GnRH analogues concomitant with adjuvant chemotherapy was proposed in 8 of the 18 patients, and 4 of these had spontaneous pregnancies. In 1 case, chemotherapy was performed alone. Indeed, recent studies support that chemotherapy regimens used for these neoplasms do not show particular toxicity; therefore, the association with GnRH analogues would not change the effects on fertility compared with the execution of the isolated therapy.<sup>[<xref rid=\"R40\" ref-type=\"bibr\">40</xref>,<xref rid=\"R41\" ref-type=\"bibr\">41</xref>]</sup> In subjects with ovarian dysfunction at the starting stage, the association of the GnRH analogues may be indicated. Pre-treatment anti-Mullerian hormone (AMH) serum levels and the age of each patient appear to be reliable predictive factors of ovarian activity recovery after treatment.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>,<xref rid=\"R42\" ref-type=\"bibr\">42</xref>]</sup> Similarly, the literature reports that AMH dosage and a pre-treatment fertility evaluation can help in the identification of patients with deficient ovarian reserve and who could benefit from a fertility preservation technique.<sup>[<xref rid=\"R43\" ref-type=\"bibr\">43</xref>]</sup> In this study, there was a very small percentage of pregnancy failure: 1 had SAB and 1 had IVP; these percentages are typically reported in a general population<sup>[<xref rid=\"R44\" ref-type=\"bibr\">44</xref>]</sup>; therefore, it is difficult to demonstrate a relationship.<sup>[<xref rid=\"R45\" ref-type=\"bibr\">45</xref>]</sup> The literature on the possible dangerous effects of cancer treatments on pregnancy did not show an increased risk of genetic or other defects in births of women previously receiving antineoplastic treatment.<sup>[<xref rid=\"R46\" ref-type=\"bibr\">46</xref>]</sup> Nevertheless, in these women, considering the limited cases studied, it would be advisable to monitor the pregnancy more strictly.<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> Indeed, the limitations of this study are represented by the limited number of cases, and the retrospective analysis</p></sec><sec><label>5</label><title>Conclusion</title><p>MOGCTs are rare tumors, which mainly affect patients of reproductive age.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> The increase of therapeutic rates has moved the attention of recent studies to variations in the menstrual cycle and reproductive outcome in patients after cure. Several studies report reassuring data relating to FSS treatment of MOCGTs in early stages; therefore, in these cases conservative surgery is now consolidated as a safe procedure. Moreover, for MOGCTs in advanced stages treated conservatively, the literature reports a noncompromised DFS and OS.<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup> In this case, personalized follow-up should be proposed in order to consider the risk factors, timing, and nature of the relapse.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> Furthermore, our study shows that FSS in advanced stages can produce good results both on reproductive outcomes and on survival. Indeed, in our group, there was only 1 case of exitus as result of recurrence. What is more, patients after FSS maintained normal ovarian function and 5 of 5 women who attempted to get pregnant succeeded spontaneously. Also, literature studies performed on a larger population report that patients with MOGCTs undergoing FSS had reassuringly high conception rates and low premature ovarian failure rates; however, in pre-treatment counseling, the risks of this approach in such a young population should be discussed.<sup>[<xref rid=\"R41\" ref-type=\"bibr\">41</xref>]</sup> Nevertheless, considering the rarity of advanced stage MOGCTs (20–30%)<sup>[<xref rid=\"R22\" ref-type=\"bibr\">22</xref>]</sup> and the few cases treated conservatively, the safety of FSS is accepted but not yet fully clinically supported.<sup>[<xref rid=\"R47\" ref-type=\"bibr\">47</xref>]</sup> Therefore, conservative surgery for MOGCTs in advanced stages needs a greater number of cases and meta-analysis in order to obtain a higher grade of recommendation,<sup>[<xref rid=\"R27\" ref-type=\"bibr\">27</xref>]</sup> but currently may represent a therapeutic option in patients available for extended follow-up and who subscribe to informed consent.</p></sec><sec><title>Author contributions</title><p>MD: (Corresponding authors) has made substantial contributions to the conception of the work, approved the submitted version, and agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which she was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.</p><p>ES has made substantial contributions to design of the work, approved the submitted version, and agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which she was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.</p><p>VL has made substantial contributions to the acquisition and analysis of data, approved the submitted version, and agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which author she was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.</p><p>AP has made substantial contributions to drafting the paper and its subsequent revision. He has also approved the submitted version and agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which he was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature</p><p>RL has made substantial contributions to drafting the paper and to its subsequent revision. He has also approved the submitted version and agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which he was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature</p><p>CM has made substantial contributions to the acquisition and analysis of data, approved the submitted version, and agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which she was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.</p><p>AD has made substantial contributions to the design of the study, approved the submitted version, and agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which she was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.</p><p>GC has made substantial contributions to drafting the work and its subsequent revision. He has also approved the submitted version and agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which he was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: β-HCG = Beta-Human chorionic gonadotropin, AFP = Alpha-fetoprotein, AMH = anti-Mullerian-hormone, BEP = Bleomycin, Etoposide, and Cisplatin, CA = carbohydrate antigen, CEA = carcinoembryonic-antigen, DFS = disease-free survival, EOC = Epithelial ovarian cancer, FIGO = Federation of Gynecology and Obstetrics, FSS = fertility-sparing surgery, GnRH = gonadotropin-releasing hormone, IVP = voluntary pregnancy interruption, OGCTs = germ cell tumors, OS = overall survival, PVB = Bleomycin, Vinblastine, Cisplatin, SAB = spontaneous abortion, WHO = World Health Organization.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Dellino M, Silvestris E, Loizzi V, Paradiso A, Loiacono R, Minoia C, Daniele A, Cormio G. Germinal ovarian tumors in reproductive age women: Fertility-sparing and outcome. <italic>Medicine</italic>. 2020;99:39(e22146).</p></fn><fn fn-type=\"supported-by\"><p>This work has been supported by a grant (5 × 1000) from the IRCCS Istituto Tumori “Giovanni Paolo II” Bari, Italy (number 332/2019) in the context of a scientific program exploring ovarian stem cell development to prevent infertility in oncological patients. The APC will be funded by IRCCS Istituto Tumori “Giovanni Paolo II” Bari, Italy.</p></fn><fn fn-type=\"supported-by\"><p>This research project has been supported in part by the Apulian Regional Project “Medicina di precision.”</p></fn><fn fn-type=\"other\"><p>There is no formal approval of the Ethics Committee, but the procedures were carried out in accordance with the Helsinki Declaration, as revised in 2013.</p></fn><fn fn-type=\"other\"><p>Informed consent was obtained from the patients through a dedicated form containing study design.</p></fn><fn fn-type=\"other\"><p>Written informed consent for the anonymous publication of information relating to the disease was regularly obtained from all individual participants included in the study, during the medical interview with the patient before the surgical or chemotherapy treatment.</p></fn><fn fn-type=\"other\"><p>The datasets used during the current study are available from the corresponding author on reasonable request.</p></fn><fn fn-type=\"COI-statement\"><p>The authors declare that they have no competing interests</p></fn><fn fn-type=\"other\"><p>The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991484</article-id><article-id pub-id-type=\"pmc\">PMC7523776</article-id><article-id pub-id-type=\"publisher-id\">MD-D-19-09726</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022469</article-id><article-id pub-id-type=\"art-access-id\">22469</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3300</subject></subj-group><subj-group><subject>Research Article</subject><subject>Clinical Case Report</subject></subj-group></article-categories><title-group><article-title>Ultrasound-guided pulsed radiofrequency treatment for distal suprascapular neuropathy</article-title><subtitle>A case report</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Chung</surname><given-names>Yang-Hoon</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-6384-7263</contrib-id><name><surname>Lee</surname><given-names>Joon-Ho</given-names></name><degrees>MD, PhD</degrees><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Koo</surname><given-names>Bon-Sung</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Jung</surname><given-names>Jaewoong</given-names></name><degrees>MD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Lee</surname><given-names>So Jeong</given-names></name><degrees>MD</degrees></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Saranathan.</surname><given-names>Maya</given-names></name></contrib></contrib-group><aff>Department of Anesthesiology and Pain Medicine, Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, Bucheon, Republic of Korea.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Joon-Ho Lee, Department of Anesthesiology and Pain Medicine, Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, 170 Jomaru-ro, Bucheon 14584, Republic of Korea (e-mail: <email>anpjuno@schmc.ac.kr</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22469</elocation-id><history><date date-type=\"received\"><day>11</day><month>12</month><year>2019</year></date><date date-type=\"rev-recd\"><day>25</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>31</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22469.pdf\"/><abstract><title>Abstract</title><sec><title>Rationale:</title><p>Suprascapular neuropathy is a rare cause of shoulder pain, and patients usually presents with posterosuperior shoulder pain and weakness on forward flexion and external rotation. Suprascapular neuropathy associated with rotator cuff pathology has received attention as an emerging cause of this condition. Suprascapular nerve (SSN) block can be used in these patients, and pulsed radio frequency (PRF) can be applied to achieve a long-term effect. Several studies have reported on PRF treatment of the SSN for shoulder pain, but most applied treatment to the nerve trunk under the transverse scapular ligament. This report describes a patient with suprascapular neuropathy treated with selective application of PRF to the distal SSN under ultrasound guidance.</p></sec><sec><title>Patient concerns:</title><p>A 68-year-old woman suffered from right posterior shoulder pain after traumatic full thickness rotator cuff tear. Her pain was not diminished despite of 2 surgeries.</p></sec><sec><title>Diagnoses:</title><p>She was diagnosed with entrapment of the distal SSN in the spino-glenoid (SGN) notch and suprascapular neuropathy.</p></sec><sec><title>Interventions:</title><p>She underwent surgery to decompress the entrapped SSN in the SGN. After that, we applied PRF on the distal SSN under ultrasound guidance for persistent pain. This treatment was repeated 3 times.</p></sec><sec><title>Outcomes:</title><p>PRF treatment resulted in a slight reduction in the visual analogue scale (VAS) pain score from 7–8/10 to 5–6/10 at the 2 weeks follow-up, and to 2–3/10 at the 1 month follow-up. The reduction in pain was maintained at the 1 year follow-up.</p></sec><sec><title>Lessons:</title><p>PRF treatment of the SSN is typically approached from the main branch in the suprascapular notch. We selectively applied PRF to the distal SSN close to the SGN. This technique was safe and effective.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>brachial plexus block</kwd><kwd>case report</kwd><kwd>nerve block</kwd><kwd>pulsed radiofrequency treatment</kwd><kwd>rotator cuff injuries</kwd><kwd>shoulder pain</kwd><kwd>ultrasonography</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>Soonchunhyang University</funding-source><award-id>no</award-id><principal-award-recipient>Joon-Ho Lee</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>The suprascapular nerve (SSN) provides sensory and motor innervation to a large portion of the shoulder, usually originating from the fifth and sixth cervical nerves. The SSN is vulnerable to injury at several sites as it passes from the neck to the shoulder.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> Among various reasons for injury, compression or traction of the nerve in association with rotator cuff pathology is a likely cause of suprascapular neuropathy.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup></p><p>Nonoperative treatment is the first choice for most patients with suprascapular neuropathy. Similar to several other neuropathies, pulsed radio frequency (PRF) is an emerging technique for treating this kind of neuropathy when other conservative treatments are ineffective. Although the clinical evidence is limited regarding the effect PRF on peripheral nerves, several reports indicate that PRF of the SSN successfully relieves shoulder pain.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>–<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> However, PRF treatment of the SSN is typically approached from the main branch in the suprascapular notch. The introduction of ultrasound-guided nerve block techniques has enabled a more selective approach, targeting the peripheral nerves and surrounding structures, and can also be applied to the SSN.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>,<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup></p><p>We report successful use of ultrasound-guided PRF treatment of the distal SSN for intractable posterior shoulder pain due to suprascapular entrapment neuropathy.</p></sec><sec><label>2</label><title>Case report</title><p>A 68-year-old woman was referred to our clinic complaining of electric shock-like pain (visual analogue scale [VAS] pain score of 7–8/10) in the right posterior shoulder. She had no remarkable past medical history. She underwent arthroscopic surgery for a full thickness rotator cuff tear about 4 months previously. After surgery, pain on abduction of the shoulder was mostly relieved but she still complained of posterior shoulder pain at rest. No difference in pain intensity was observed between rest and during movement. Preoperative magnetic resonance imaging showed atrophic signal changes in the infraspinatus muscle (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>). She was referred to the department of orthopedic surgery to confirm whether entrapment of the distal SSN in the spino-glenoid notch (SGN) was the source of her pain. Although electromyography and nerve conduction velocity studies revealed lesions of the distal SSN, the surgeon wanted to confirm the lesions by nerve block at the SGN before surgery. The distal SNN was blocked under ultrasound with a 6 to 13 MHz linear array transducer (SonoSite S-nerve; SonoSite Inc., Bothell, WA) using the out-of-plane technique at the SGN with 1 mL of 0.25% ropivacaine (Fig. <xref ref-type=\"fig\" rid=\"F2\">2</xref>).<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> The nerve block relieved the pain temporarily (VAS pains score dropped to 2–3/10). She was diagnosed with entrapment of the distal SSN in the SGN and suprascapular neuropathy.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Axial T2-weighted magnetic resonance images of the right shoulder demonstrating fatty atrophy of the infraspinatus muscle (arrows).</p></caption><graphic xlink:href=\"medi-99-e22469-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Ultrasound imaging of applying radiofrequency probe on suprascapular nerve in the spinoglenoid notch. Arrow indicates the needle tip and arrow heads indicate spinoglenoid notch near the glenoid (asterisk). ISP = infraspinatus muscle, TPZ = trapezius muscle.</p></caption><graphic xlink:href=\"medi-99-e22469-g002\"/></fig><p>She underwent a second surgery to decompress the entrapped SSN in the SGN 1 week later. However, the pain was not diminished at all after surgery. Her medications at that time included, pregabalin, OxyContin/naloxone, and acetaminophen/tramadol; the dosages were adjusted but the pain persisted. At the second visit to our clinic, SSN block was performed 3 times, as described above, with 2 mL 0.25% ropivacaine and 10 mg triamcinolone with a 1 week interval. However, her pain was relieved for only 2 and 3 days. We decided to apply PRF on the distal SSN. The PRF was done with a radiofrequency lesion generator (Diros OWL URF-3AP; Diros Technology Inc., Markham, ON, Canada) using a straight radio frequency probe with a 5 cm long, 5 mm active tip (467–005-TCH-S). After applying local anesthesia to the skin, the cannula was inserted under ultrasound guidance close to the SGN. After confirming the suprascapular artery near the nerve, the cannula was advanced carefully, so as not to damage the nearby vessels, until paresthesia was reported at 50 Hz and 0.3 to 0.5 mA. The motor response was also checked with 2 Hz of stimulation. PRF was applied for 150 seconds at 45 V and a pulse duration of 20 ms, not exceeding 42 °C. This treatment was repeated 3 times and resulted in a slight reduction in the VAS pain score from 7–8/10 to 5–6/10 at the 2 weeks follow-up, and to 2–3/10 at the 1 month follow-up. The reduction in pain was maintained at the 1 year follow-up. She was satisfied with her conditions. She underwent a rehabilitation program to restore shoulder function. Written informed consent was obtained from the patient for publication of this case report and accompanying images</p></sec><sec><label>3</label><title>Discussion</title><p>Suprascapular neuropathy is a rare cause of shoulder pain and usually occurs due to compression or traction following trauma, space-occupying lesions, or repetitive overhead activities.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> SSN traction in the suprascapular notch or SGN after a massive rotator cuff tear is another cause of suprascapular neuropathy and constitutes a significant part of this condition.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R9\" ref-type=\"bibr\">9</xref>,<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> Patients typically present with posterosuperior shoulder pain and weakness on forward flexion and external rotation. Atrophy of the supraspinatus or infraspinatus muscles is often detected during inspection. Electrodiagnostic testing, magnetic resonance imaging, and selective nerve block are useful diagnostic tools.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup> Nonoperative treatments, including nonsteroidal anti-inflammatory drugs, physical therapy, and nerve blocks, are the typical first-choice treatments and surgery is offered if these treatments fail. However, there have been few established indications for surgery in patients with isolated suprascapular neuropathy related to rotator cuff tears until now. Furthermore, similar to other musculoskeletal disorders, there are few alternative treatment modalities available if surgical treatments fail.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R8\" ref-type=\"bibr\">8</xref>,<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> The natural history of this condition is not well known, so there is no recommended duration for non-surgical treatments, except when space-occupying lesions exist.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup></p><p>SSN blocks have been used to manage various kinds of pain around the shoulder.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> They can also be used to confirm the exact cause of pain when the results of electrodiagnostic testing are inconclusive. The most commonly used techniques involve superior and posterior approaches. Regardless of the technique, the majority of nerve block methods target the SSN at the suprascapular notch under the transverse scapular ligament.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> Messina et al<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> suggested approaching to the distal SSN nearby to the SGN. The efficacy of this technique has not yet been validated, but it can be useful in certain circumstances.</p><p>Applications of PRF to treat peripheral nerves have been increasing. Clinical evidence of the efficacy of PRF for treating pain originating from peripheral nerves remains limited, but several reports show the effectiveness of PRF on the SSN.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>–<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> However, even though PRF is a relatively safe procedure compared with conventional heat radio frequency, it is invasive and can affect cellular structures.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>,<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> Thus, a more selective approach to the peripheral nerve could make this technique safer and more effective, and high resolution ultrasound provides additional guidance.</p><p>In conclusion, we report our experience of selectively applying PRF to the distal SSN to treat posterior shoulder pain due to suprascapular neuropathy.</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization</bold>: Yang-Hoon Chung, Joon-Ho Lee.</p><p><bold>Investigation:</bold> Bon-Sung Koo, Jaewoong Jung.</p><p><bold>Methodology:</bold> So Jeong Lee.</p><p><bold>Resources:</bold> Bon-Sung Koo, Jaewoong Jung, So Jeong Lee.</p><p><bold>Visualization:</bold> Yang-Hoon Chung.</p><p><bold>Writing – original draft:</bold> Yang-Hoon Chung, Joon-Ho Lee.</p><p><bold>Writing – review & editing:</bold> Yang-Hoon Chung, Joon-Ho Lee, Bon-Sung Koo, Jaewoong Jung, So Jeong Lee.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: PRF = pulsed radiofrequency, SGN = spinoglenoid, SSN = suprascapular nerve, VAS = visual analogue scale.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Chung YH, Lee JH, Koo BS, Jung J, Lee SJ. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991456</article-id><article-id pub-id-type=\"pmc\">PMC7523778</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-07174</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022369</article-id><article-id pub-id-type=\"art-access-id\">22369</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3700</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Systematic Review</subject></subj-group></article-categories><title-group><article-title>Acupotomy for patients with tarsal tunnel syndrome</article-title><subtitle>A protocol for systematic review and meta analysis</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Sun</surname><given-names>Xiaojie</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhou</surname><given-names>Qiaoyin</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Shi</surname><given-names>Chong</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Lan</surname><given-names>Yangjing</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Jia</surname><given-names>Yan</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Qiu</surname><given-names>Zuyun</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Shen</surname><given-names>Yifeng</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff5\"><sup>e</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Shiliang</given-names></name><degrees>MD, PhD</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Beijing University of Chinese Medicine</aff><aff id=\"aff2\"><label>b</label>Department of Acupuncture-Moxibustion, China-Japan Friendship hospital, Beijing</aff><aff id=\"aff3\"><label>c</label>Fujian University of Traditional Chinese Medicine</aff><aff id=\"aff4\"><label>d</label>Key Laboratory of Orthopedics & Traumatology of Traditional Chinese Medicine and Rehabilitation (Fujian university of TCM), Ministry of Education, Fuzhou, Fujian</aff><aff id=\"aff5\"><label>e</label>Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province, P. R. China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Shiliang Li, Department of Acupuncture-Moxibustion, China-Japan Friendship Hospital, No. 4, Sakura Garden Road, Beijing 100029, China (Doctor of Medicine e-mail: <email>zrlishiliang@163.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22369</elocation-id><history><date date-type=\"received\"><day>22</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>26</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22369.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>Tarsal tunnel syndrome (TTS) is a painful condition of the ankle that affects patients’ quality of life and ability to work. Multiple clinical studies of nerve decompression by acupotomy have been published in China, and the results are encouraging. However, the efficacy and security of this treatment have not been evaluated scientifically and systematically. The purpose of this systematic review protocol is to evaluate the efficacy and security of acupotomy treatment in patients with TTS, which will be helpful to clinical acupotomy doctors.</p></sec><sec sec-type=\"methods\"><title>Methods:</title><p>Relevant randomized controlled trials will be identified by searching 9 databases (PubMed, Embase, Cochrane Library, Chinese literature databases, the Chinese Biomedical Literature Database, China National Knowledge Infrastructure, SinoMed, Technology Journal and the Wanfang Database. Randomized controlled trials examining the use of acupotomy for TTS patients will be identified independently by 2 reviewers by searching the databases from inception to March 2020. Clinical effects will be evaluated as the primary outcome. Visual analog scale scores will be assessed as a secondary outcome. Review Manager 5.3 will be used to perform a fixed effects meta-analysis, and the evidence level will be evaluated by using the Grading of Recommendations Assessment, Development, and Evaluation framework. Continuous outcomes will be presented as mean differences or standard mean differences, while dichotomous data will be expressed as relative risks.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>This study will evaluate the effectiveness and safety of acupotomy in the treatment of TTS in randomized controlled trials with high-quality visual analog scale and Roles and Maudsley score.</p></sec><sec sec-type=\"conclusion\"><title>Conclusion:</title><p>This systematic review will provide evidence to determine whether acupotomy is an effective intervention for patients with TTS.</p></sec><sec><title>Registration number:</title><p>DOI 10.17605/OSF. IO/9PYC2 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://osf.io/9pyc2/\">https://osf.io/9pyc2/</ext-link>)</p></sec></abstract><kwd-group><title>Keywords</title><kwd>acupotomy</kwd><kwd>protocol</kwd><kwd>systematic review</kwd><kwd>tarsal tunnel syndrome</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Tarsal tunnel syndrome (TTS) is a local entrapment neuropathy that is identified as a focal compressive neuropathy of the posterior tibial nerve or 1 of its associated branches (medial plantar, lateral plantar, calcaneal nerves and Baxter's nerve<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup>) individually or collectively under the flexor retinaculum (laciniate ligament) on the medial side of the ankle.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> It is also called posterior TTS (PTTS); PTTS is different from anterior tarsal syndrome, in which the deep peroneal nerve is compressed under the inferior extensor retinaculum (cruciate ligament) on the dorsum of the foot.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> In 1933, posttraumatic compression of the tibial nerve was described by Pollock and Davis; then, in 1960, Kopell and Thompson described the clinical manifestations of TTS.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> However, it was not until 1962 that Keck and Lam named the condition in medical literature finally and officially.<sup>[<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup></p><p>Characteristic clinical manifestations of TTS include poorly localized paraesthesia, localized or radiating pain, burning pain, dysesthesia and hyperesthesia, and feelings of coldness that radiate from the retro-malleolar region to either the sole, heel or digits of the forefoot, or a combination of these areas.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> Some patients even feel as though there is a tight band around the foot. TTS usually worsens with the progression of the day and improves after relaxation, but it may manifest as cramping of the symptomatic foot. Symptoms are typically unilateral and rarely present bilaterally.<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>–<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup></p><p>TTS is relatively uncommon. The incidence of PTTS is not known; however, its incidence is less than that of carpal tunnel syndrome and that of cubital tunnel syndrome. It is easy to overlook or misdiagnosis. There is no doubt that clinical diagnosis can be identified. Causes of TTS can be classified into either intrinsic, extrinsic, or combinations of the 2.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> TTS tends to be more common in athletes such as joggers, football players, and martial arts athletes, who are subjected to prolonged weight-bearing periods inclusive of standing, walking, running or intense physical activity.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>–<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup></p><p>It is important to take remedial measures promptly because the longer a patient has TTS, the greater the potential for lasting nerve damage. The management of TTS can involve a variety of therapeutic interventions.</p><p>Conservative management includes anti-inflammatory medication, activity modification combined with progressive mobilization exercises and naturopathy.<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> Aspiration of ganglia may provide temporary benefit. Local anesthetic or corticosteroid infiltrations are recommended mainly to treat the “algetic form” of TTS to reverse any intraneural edema, but these infiltrations may increase risk.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> It is possible to decrease the pressure on the nerve to control symptoms by using orthotic shoes, activity modification, immobilization with a night splint, immobilizing braces or removable boot walker,<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> but these treatments are said to worsen the symptoms in many cases. It is necessary to undergo orthopedic treatment if there is any deformity of the foot. Physiotherapy may include a variety of techniques, including bracing, stretching, icing, massage, tens and soft tissue manipulation, remedial massage therapy and ultrasound; however, evidence regarding the effectiveness of these treatments is lacking.</p><p>Surgical intervention is considered after failed nonoperative treatments, but reported success rates after tarsal tunnel decompression have varied in the literature, ranging from 44% to 96%.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> Related complications may be apparent after surgical intervention, and postoperative complications include impaired wound healing, infection, and keloid formation.<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]</sup></p><p>The acupotome is a new miniature surgery instrument consisting of a bladed needle with a flat head and a cylindrical needle body that evolved from an acupuncture needle developed by Zhu Hanzhang in 1976.<sup>[<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</sup> The method of utilizing acupotomes to treat abnormal, cicatricial and contractured soft tissue with microtrauma has been given the name acupotomy therapy. Acupotomy therapy is considered a minimally invasive operation in traditional Chinese medicine that combines Chinese acupuncture therapy and modern surgical principles.<sup>[<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]</sup> Acupotomy treats TTS by releasing the soft tissue to release pressure from the tarsal tunnel, which reduces risk, time, and cost by converting open surgery to minimally invasive surgery.</p><p>Acupotomy has been widely used clinically by practitioners of traditional Chinese medicine, orthopedics and pain departments to treat TTS in China for many years.<sup>[<xref rid=\"R18\" ref-type=\"bibr\">18</xref>–<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]</sup> However, from the perspective of evidence-based medicine, the safety and efficacy of acupotomy on TTS needs to be discussed. There is limited evidence in the form of systematic reviews and meta-analyses with regard to acupotomy treatment for TTS. This study will assess the effectiveness and safety of acupotomy therapy for TTS to provide evidence for further enhancing the clinical curative effects on patients with TTS.</p><p>In this study, evidence-based medicine will be used to analyze and evaluate clinical randomized controlled trials (RCTs) in patients with TTS.</p></sec><sec><label>2</label><title>Methods</title><sec><label>2.1</label><title>Inclusion criteria for study selection.</title><sec><label>2.1.1</label><title>Types of studies</title><p>All RCTs examining the use of acupotomy therapy in TTS that were published in English and Chinese will be included in this systematic review and meta-analysis. Any study with a sample size less than 10 patients will be excluded from this review. Review articles, animal studies, nonclinical studies, and case reports will also be excluded. The research literature will be screened according to the criteria of the review objectives and participants, interventions, comparisons, and outcomes.</p></sec><sec><label>2.1.2</label><title>Types of patients</title><p>Only studies in which patients have a confirmed clinical diagnosis of TTS will be included. Diagnosis requires radial ankle pain exacerbated by the Hoffmann-Tinel sign (percussing or tapping at the suspected site of compression) or the Dorsiflexion-Eversion Test (everting and dorsiflexing the ankle while maximally dorsiflexing the metatarsophalangeal joints).<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> To reflect the condition's widespread nature, no restrictions will be placed upon age, sex, race, or educational status. Fracture and dislocation, muscle injury, bone tuberculosis, bone tumors, neurological symptoms and neuromuscular diseases or any systematic diseases will be excluded.</p></sec><sec><label>2.1.3</label><title>Types of interventions.</title><sec><label>2.1.3.1</label><title>Experimental interventions</title><p>The review will involve clinical trials that focus on acupotomy treatment (there will be no limitations on the needle materials, treatment methods and treatment courses). Evaluations of acupotomy plus another treatment compared to the same treatment alone will also be included. However, studies that compare different acupotomy insertions or different forms of acupotomy will be excluded.</p></sec><sec><label>2.1.3.2</label><title>Control interventions</title><p>Control interventions that include placebo controls, steroid injections, drug therapy, block therapy, surgery, no treatment, and acupuncture will be eligible.</p></sec></sec><sec><label>2.1.4</label><title>Types of outcome measures.</title><sec><label>2.1.4.1</label><title>Primary outcomes</title><p>The primary outcome measure of this systematic review will include improvement rates, functional tests, and pain relief. Evaluation will be performed by the visual analog score.</p></sec><sec><label>2.1.4.2</label><title>Secondary outcomes</title><p>The Roles and Maudsley score will be considered as secondary outcomes.</p><list list-type=\"simple\"><list-item><label>(1)</label><p>Safety: Safety will be measured by the recurrence rates of TTS, quality of life and adverse events, such as hemorrhage, serious discomfort, abscess, subcutaneous nodules, and infection.</p></list-item><list-item><label>(2)</label><p>Acceptance of the measured treatment will be determined by trial exit.</p></list-item></list></sec></sec></sec><sec><label>2.2</label><title>Search methods for the identification of studies</title><sec><label>2.2.1</label><title>Electronic searches</title><p>The following electronic databases will be searched by 2 reviewers from database inception to March 2020: PubMed, Embase, Cochrane Library, Chinese literature databases, the Chinese Biomedical Literature Database, China National Knowledge Infrastructure, SinoMed, China Science and Technology Journal (VIP) and the Wanfang Database. Acupotomy RCTs examining TTS will be identified by searching these databases.</p></sec><sec><label>2.2.2</label><title>Searching other resources</title><p>We will search the tables of contents for studies related to TTS and acupotomy, we will search the reference lists of the relevant literature, and we will search for systematic reviews to identify additional RCTs. We will also manually search relevant conference papers and will search Clinical Trials.gov and the WHO International Clinical Trials Registry Platform for new trials relevant to the topic. The search keywords or combination subject terms will include “Posterior Tarsal Tunnel Syndrome,” “Posterior Tibial Nerve Neuralgia,” “Tarsal Tunnel Syndrome,” “Tarsal Tunnel Syndromes,” “Tibial nerve entrapment,” “Tarsal Tunnel Entrapment Neuropathy,” “Tarsal Tunnel Tibial Neuropathy,” “TTS,” “Acupotomy,” “Small acupotomy,” “The small acupotomy,” “The needle knife,” “Needle-knife,” “Small needle knife,” “Acpotomoloy,” “Acupotome,” “Randomized controlled trials,” “Randomized controlled,” “Randomized,” “Controlled,” “Controlled study,” “Clinical trial,” “Controlled clinical trials,” and “Comparative study.” The accurate Chinese translation of these search terms will be used in the Chinese database. The detailed strategies for searching the PubMed database are presented in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Details of the search strategy for PubMed.</p></caption><graphic xlink:href=\"medi-99-e22369-g001\"/></table-wrap></sec></sec><sec><label>2.3</label><title>Data collection and analysis</title><sec><label>2.3.1</label><title>Selection of studies</title><p>The retrieved literature will be imported into the Endnote library by researchers (CS and YJ), and duplicate studies will be eliminated. Two reviewers (CS and YJ) will independently exclude articles that are noticeably below standard by reading the title and abstract. Next, the researchers will independently read the full texts, discuss the trials as a group, and contact the author to obtain details about the research to determine the eligibility of each trial (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>). The final list of articles will be converted into a Microsoft Excel format. Then, two researchers (XS and YL) will independently conduct the literature search and screening. Finally, a third independent reviewer (SL) will serve as an arbitrator and ultimately make decisions regarding inclusion.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Flow diagram of the study selection process. RCT = randomized controlled trial.</p></caption><graphic xlink:href=\"medi-99-e22369-g002\"/></fig></sec><sec><label>2.3.2</label><title>Data extraction and management</title><p>Data from all the selected eligible articles will be extracted by 2 independent reviewers (QZ and YL) into an Excel form. Any differences found will be resolved through discussion and recommendations from the third reviewer (SL). These data collection forms will include the reference ID, author, time of publication, randomization, participant characteristics, country, interventions, blinding, treatment indicators, follow-up, outcome indicators, research results, adverse events, and other detailed information. If necessary, we will contact the author of the trial to obtain further information.</p></sec><sec><label>2.3.3</label><title>Assessment of the risk of bias in the included studies</title><p>Two independent reviewers (YS and ZQ) will use the Cochrane Collaboration tool to assess the risk of bias for each included trial. The following seven aspects will be assessed: random sequence generation; allocation concealment; the blinding method for patients, researchers and outcome assessors; incomplete outcome data; selective reporting; and other biases as necessary. The risk of bias will be classified as low risk, high risk and unclear.<sup>[<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]</sup> The third reviewer (SL) will cross-check and resolve disagreements through discussion and arbitration to obtain the results of the evaluation.</p></sec><sec><label>2.3.4</label><title>Measures for treatment effect</title><p>We will use the relative risk or odds ratio to evaluate the enumeration data. For continuous data, the mean difference and 95% confidence interval (95% CI) will be used to assess the measurement data. If specific outcome metrics are measured using different outcome measurement scales, standardized mean difference with 95% CI will be used.</p></sec><sec><label>2.3.5</label><title>Dealing with missing data</title><p>Researchers will contact the corresponding authors to obtain information if there are missing or incomplete data for the primary results. If the missing data are not available, we will perform the analysis based on the available data.</p></sec><sec><label>2.3.6</label><title>Assessment of heterogeneity</title><p>Review Manager 5.3 for Windows; the Nordic Cochrane Center, Copenhagen, Denmark, will be used to evaluate the curative effect and publication bias. We will assess heterogeneously with the I2 statistic and the χ2 test in accordance with the Cochrane Handbook for Systematic Reviews of Interventions.<sup>[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]</sup> We will decide whether to use a fixed effects model or a random effects model based on the heterogeneity levels of the included studies. Specifically, we will use the <italic>χ</italic>2 test (α = 0.1) to analyze the heterogeneity of the research results and use the <italic>I</italic><sup>2</sup> value to determine the significance. If <italic>I</italic><sup>2</sup> ≤ 50%, the statistical heterogeneity among the trials will be considered negligible, and the size of the effect will be estimated by using a fixed effects model. <italic>I</italic><sup>2</sup> values > 50% will be considered evidence of significant heterogeneity among the trials. After excluding the effects of significant clinical heterogeneity, we will adopt the random effects model with 95% CI for meta-analysis. If there is significant clinical heterogeneity, we will perform a subgroup or sensitivity analysis or only present descriptive statistics.</p></sec><sec><label>2.3.7</label><title>Assessment of reporting bias</title><p>If there are more than 10 trials in the study, we will use the funnel plot to assess publication biases. We will analyze the causes for this outcome if asymmetry is observed in the funnel plot.</p></sec><sec><label>2.3.8</label><title>Sensitivity analysis</title><p>If possible, a sensitivity analysis will be carried out to verify the robustness of the conclusions of the review. When sufficient trials are available, we will perform a sensitivity analysis to identify whether the review conclusions are robust according to the following:</p><list list-type=\"simple\"><list-item><label>(1)</label><p>sample size,</p></list-item><list-item><label>(2)</label><p>heterogeneity qualities, and</p></list-item><list-item><label>(3)</label><p>methodological quality.</p></list-item></list><p>In addition, the analysis will be repeated after the exclusion of studies with low methodological quality.<sup>[<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]</sup></p></sec><sec><label>2.3.9</label><title>Grading the quality of evidence</title><p>We will assess the quality of evidence with the Grading of Recommendations Assessment, Development and Evaluation framework; evidence quality will be categorized into very low, low, moderate, or high levels.<sup>[<xref rid=\"R27\" ref-type=\"bibr\">27</xref>]</sup></p></sec></sec></sec><sec><label>3</label><title>Discussion</title><p>Acupotomy treatment is worth considering for TTS patients, as it is a minimally invasive surgery with higher acceptability and less pain. Some trials have reported that acupotomy can effectively reduce the symptoms of TTS; however, its efficacy has not been evaluated scientifically or systematically. To the best of our knowledge, there are no systematic reviews or meta-analyses of the effectiveness and safety of acupotomy on TTS that have been published. The purpose of this study was to assess the efficacy and safety of acupotomy treatment in patients with TTS. We believe our systematic review and meta-analysis will be beneficial to patients with TTS, clinicians and practitioners by providing a deeper understanding of the effectiveness of acupotomy therapy on TTS. Because there may be a risk of heterogeneity in the severity of different types of acupotomy and TTS, and the measurements and outcome assessment tools of the included studies may be different, there are some potential limitations to this review.</p></sec><sec><title>Author contributions</title><p><bold>Investigation:</bold> Chong Shi, Yangjing Lan.</p><p><bold>Methodology:</bold> Yan Jia, Zuyun Qiu.</p><p><bold>Supervision:</bold> Shiliang Li.</p><p><bold>Writing – original draft:</bold> Xiaojie Sun, Qiaoyin Zhou.</p><p><bold>Writing – review & editing:</bold> Yifeng Shen.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: 95% CI = 95% confidence interval, PTTS = posterior tarsal tunnel syndrome, RCTs = randomized controlled trials, TTS = tarsal tunnel syndrome.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Sun X, Zhou Q, Shi C, Lan Y, Jia Y, Qiu Z, Shen Y, Li S. Acupotomy for patients with tarsal tunnel syndrome: A protocol for systematic review and meta analysis. <italic>Medicine</italic>. 2020;99:39(e22369).</p></fn><fn fn-type=\"equal\"><p>XS and QZ contributed equally to this work and are co-first authors.</p></fn><fn fn-type=\"other\"><p>SL is the guarantor of the article. The manuscript was drafted by XS and QZ. CS, and YJ developed the search strategy. XS and YL will independently screen the potential studies. QZ and YL extract the data. ZQ and YS will assess the risk of bias and finish data synthesis. SL will arbitrate any disagreement and ensure that no errors occur during the review. All review authors critically reviewed, revised, and approved the subsequent and final version of the protocol.</p></fn><fn fn-type=\"supported-by\"><p>This work was supported by the China-Japan Friendship Hospital 2010 Research Fund projects (No. 2010-MS-38).</p></fn><fn fn-type=\"other\"><p>Ethical approval is not necessary because individuals cannot be identified.</p></fn><fn fn-type=\"COI-statement\"><p>The authors have no conflicts of interest to disclose.</p></fn><fn fn-type=\"other\"><p>The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>[1]</label><mixed-citation publication-type=\"journal\"><person-group 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Pain Rep</journal-id><journal-id journal-id-type=\"coden\">PAIREP</journal-id><journal-id journal-id-type=\"publisher-id\">Painreports</journal-id><journal-title-group><journal-title>Pain Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2471-2531</issn><publisher><publisher-name>Wolters Kluwer</publisher-name><publisher-loc>Philadelphia, PA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33062901</article-id><article-id pub-id-type=\"pmc\">PMC7523781</article-id><article-id pub-id-type=\"publisher-id\">PAINREPORTS-D-19-0141</article-id><article-id pub-id-type=\"doi\">10.1097/PR9.0000000000000823</article-id><article-id pub-id-type=\"art-access-id\">00011</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>New Directions for Physical Rehabilitation of Musculoskeletal Pain Conditions</subject></subj-group></article-categories><title-group><article-title>Exercise-induced hypoalgesia after acute and regular exercise: experimental and clinical manifestations and possible mechanisms in individuals with and without pain</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Vaegter</surname><given-names>Henrik Bjarke</given-names></name><xref ref-type=\"aff\" rid=\"aff1\">a</xref><xref ref-type=\"aff\" rid=\"aff2\">b</xref><xref ref-type=\"corresp\" rid=\"cor1\">*</xref></contrib><contrib contrib-type=\"author\"><name><surname>Jones</surname><given-names>Matthew David</given-names></name><xref ref-type=\"aff\" rid=\"aff3\">c</xref><xref ref-type=\"aff\" rid=\"aff4\">d</xref></contrib><aff id=\"aff1\"><label>a</label>Pain Research Group, Pain Center, Odense University Hospital, Odense, <country>Denmark</country></aff><aff id=\"aff2\"><label>b</label>Department of Clinical Research, Faculty of Health Sciences, University of Southern Denmark, Odense, <country>Denmark</country></aff><aff id=\"aff3\"><label>c</label>School of Medical Sciences, University of New South Wales, Sydney, <country>Australia</country></aff><aff id=\"aff4\"><label>d</label>Neuroscience Research Australia, Sydney, <country>Australia</country></aff></contrib-group><author-notes><corresp id=\"cor1\"><label>*</label>Corresponding author. Address: Pain Research Group, Pain Center, University Hospital Odense, Denmark, Heden 7-9, Indgang 200, DK-5000 Odense C, Denmark. Tel.: +45 65413869; fax: +45 65415064. E-mail address: <email>hbv@rsyd.dk</email> (H.B. Vægter).</corresp></author-notes><pub-date pub-type=\"collection\"><season>Sep-Oct</season><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>23</day><month>9</month><year>2020</year></pub-date><volume>5</volume><issue>5</issue><elocation-id>e823</elocation-id><history><date date-type=\"received\"><day>06</day><month>12</month><year>2019</year></date><date date-type=\"rev-recd\"><day>02</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>21</day><month>4</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The International Association for the Study of Pain.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">Creative Commons Attribution License 4.0 (CCBY)</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:href=\"painreports-5-e823.pdf\"/><abstract abstract-type=\"toc\"><p>This review describes methodology used in the assessment of the manifestations of exercise-induced hypoalgesia in humans and previous findings in individuals with and without pain. Possible mechanisms and future directions are discussed.</p></abstract><abstract><title>Abstract</title><p>Exercise and physical activity is recommended treatment for a wide range of chronic pain conditions. In addition to several well-documented effects on physical and mental health, 8 to 12 weeks of exercise therapy can induce clinically relevant reductions in pain. However, exercise can also induce hypoalgesia after as little as 1 session, which is commonly referred to as exercise-induced hypoalgesia (EIH). In this review, we give a brief introduction to the methodology used in the assessment of EIH in humans followed by an overview of the findings from previous experimental studies investigating the pain response after acute and regular exercise in pain-free individuals and in individuals with different chronic pain conditions. Finally, we discuss potential mechanisms underlying the change in pain after exercise in pain-free individuals and in individuals with different chronic pain conditions, and how this may have implications for clinical exercise prescription as well as for future studies on EIH.</p></abstract><kwd-group><title>Keywords:</title><kwd>Exercise</kwd><kwd>Hypoalgesia</kwd><kwd>Pain sensitivity</kwd><kwd>Mechanisms</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>1. Introduction</title><p>Exercise is guideline recommended treatment for a range of chronic pain conditions.<sup><xref rid=\"R49\" ref-type=\"bibr\">49</xref></sup> Regular exercise and physical activity in general have well-documented positive effects on a range of physical and mental health domains including cardiovascular health, stress, mood, sleep, and sexual health.<sup><xref rid=\"R146\" ref-type=\"bibr\">146</xref></sup> In addition, clinically important reductions in pain are often observed after 8 to 12 weeks of exercise therapy<sup><xref rid=\"R163\" ref-type=\"bibr\">163</xref></sup>; however, as little as 1 session of exercise can induce hypoalgesia. This phenomenon is known as exercise-induced hypoalgesia (EIH).<sup><xref rid=\"R92\" ref-type=\"bibr\">92</xref>,<xref rid=\"R195\" ref-type=\"bibr\">195</xref></sup> The first observation of EIH was published 40 years ago by Black et al..<sup><xref rid=\"R14\" ref-type=\"bibr\">14</xref></sup> During the past few decades, the number of studies investigating the effect of exercise on pain has increased dramatically, likely reflecting the increasing burden of pain as well as the recognized role of exercise in the treatment of pain.</p><p>This article will begin with a brief introduction to the methodology used in the assessment of the manifestations and mechanisms of EIH in humans. The second part of the article will present an overview of the findings from previous experimental studies investigating changes in pain perception after acute and regular exercise in pain-free individuals and in individuals with different chronic pain conditions. Possible mechanisms underlying the response to exercise in pain-free individuals and in individuals with different chronic pain conditions will also be discussed. In the last part of the article, implications for exercise prescription and future EIH studies will be addressed.</p><sec id=\"s1-1\"><title>1.1. Assessment of exercise-induced hypoalgesia—methodological considerations</title><p>The effect of a single bout of exercise on pain perception in humans has primarily been investigated experimentally in laboratory settings. The methods used in these investigations are diverse, incorporating different study designs and methods of pain assessment. Most often, EIH has been investigated using a within-group pre-post design, whereby participants' pain is assessed at different exercising and nonexercising body sites before and during/after exercise.<sup><xref rid=\"R130\" ref-type=\"bibr\">130</xref></sup> Controlled studies using similar methodology but different designs (eg, crossover trials and parallel trials) have also been conducted.<sup><xref rid=\"R80\" ref-type=\"bibr\">80</xref>,<xref rid=\"R162\" ref-type=\"bibr\">162</xref>,<xref rid=\"R195\" ref-type=\"bibr\">195</xref>,<xref rid=\"R204\" ref-type=\"bibr\">204</xref></sup> The results of these studies, especially those where participants were randomized to exercise or control, or where the order of exercise and control were randomized and counterbalanced for crossover trials, give a less biased estimate of the effect of a single bout of exercise on pain.</p><sec id=\"s1-1-1\"><title>1.1.1. Pain threshold, intensity, and tolerance</title><p>Pain has been quantified in a variety of ways in studies of EIH, with quantitative sensory testing used most often. Quantitative sensory testing describes a series of tests that measure the perceptual responses to systematically applied and quantifiable sensory stimuli (usually pressure, thermal, or electrical).<sup><xref rid=\"R23\" ref-type=\"bibr\">23</xref></sup> These tests typically involve the assessment of a person's pain threshold or pain tolerance which are, respectively, the minimum intensity of a stimulus that is perceived as painful and the maximum intensity to a noxious stimulus that the participant is willing to tolerate.<sup><xref rid=\"R116\" ref-type=\"bibr\">116</xref></sup> Ratings of pain intensity and unpleasantness during exposure to various noxious stimuli might also be measured. As an example, pressure may be applied at an increasing intensity over the lower leg using an inflated cuff, with participants asked to rate the point at which this pressure becomes painful (threshold) and then endure it for as long as possible (tolerance) while rating its intensity or unpleasantness. Using this example, EIH could manifest as an increase in pain threshold, an increase in pain tolerance, and/or a reduction in ratings of pain intensity or unpleasantness. These measures are most commonly assessed in the immediate postexercise period (eg, 0–15 minutes), but some studies have measured pain 30 to 60 minutes after exercise cessation to investigate the persistence of EIH.<sup><xref rid=\"R69\" ref-type=\"bibr\">69</xref>,<xref rid=\"R103\" ref-type=\"bibr\">103</xref></sup></p></sec><sec id=\"s1-1-2\"><title>1.1.2. Pain modulatory mechanisms</title><p>Methods that assess an individual's ability to modulate pain have been increasingly used in recent studies of EIH. These include temporal summation, spatial summation, conditioned pain modulation, and offset analgesia. Of these paradigms, temporal summation and conditioned pain modulation have been used most often. Temporal summation refers to an increase in pain after repetitive stimulation at the same intensity<sup><xref rid=\"R137\" ref-type=\"bibr\">137</xref></sup> and is considered a behavioural correlate of wind-up—the frequency-dependent increase in C-fibre-evoked responses of dorsal horn neurons after repetitive stimulation at a constant intensity.<sup><xref rid=\"R63\" ref-type=\"bibr\">63</xref></sup> Temporal summation paradigms provide information mostly about facilitatory mechanisms underlying nociceptive processes.<sup><xref rid=\"R23\" ref-type=\"bibr\">23</xref></sup> By contrast, conditioned pain modulation provides an index of the strength of pain inhibition. Conditioned pain modulation (ie, “pain inhibits pain”) involves the application of 2 noxious stimuli over 2 different areas of the body, with the more pronounced noxious stimulus (conditioning stimulus) subsequently inhibiting the perception of the weaker noxious stimulus (test stimulus).<sup><xref rid=\"R211\" ref-type=\"bibr\">211</xref>,<xref rid=\"R212\" ref-type=\"bibr\">212</xref></sup> Using these paradigms, EIH would manifest as a reduction in temporal summation and/or an increase in conditioned pain modulation, although evidence for the latter is limited.<sup><xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R36\" ref-type=\"bibr\">36</xref>,<xref rid=\"R122\" ref-type=\"bibr\">122</xref></sup></p></sec><sec id=\"s1-1-3\"><title>1.1.3. Nociceptive processing</title><p>Although not an assessment of pain per se, techniques that assess the function of the nociceptive pathways have sometimes been used to investigate EIH.<sup><xref rid=\"R38\" ref-type=\"bibr\">38</xref>,<xref rid=\"R80\" ref-type=\"bibr\">80</xref></sup> These more complex methods, which include evoked potentials and neuroimaging, may provide greater insight into the mechanisms of EIH compared to more commonly used quantitative sensory tests. Evoked potentials are cortical responses recorded at the scalp using electroencephalography in response to brief and intense stimuli. Evoked potentials are described by their polarities (negative [N] and positive [P]), latencies, and amplitudes, and consist of early, late, and ultra-late components. When analysing pain-related evoked potentials, the peak-to-peak amplitude of the N2P2 is the component most related to nociception, whereby larger N2P2 amplitude is associated with more pain.<sup><xref rid=\"R71\" ref-type=\"bibr\">71</xref></sup> There is evidence that both the sensory-discriminative and affective aspects of pain are captured by this late component of the evoked potential, and studies have shown exercise to reduce the amplitude of this component.<sup><xref rid=\"R72\" ref-type=\"bibr\">72</xref>,<xref rid=\"R145\" ref-type=\"bibr\">145</xref></sup> Neuroimaging is widely used in the study of pain, but to the best of our knowledge, only 2 studies have used neuroimaging to investigate acute EIH.<sup><xref rid=\"R38\" ref-type=\"bibr\">38</xref>,<xref rid=\"R165\" ref-type=\"bibr\">165</xref></sup> In one study, brain responses to noxious thermal stimuli before and after rest and exercise were measured using functional magnetic resonance imaging in women with fibromyalgia and healthy pain-free controls. The results suggested that, in the women with fibromyalgia, exercise-stimulated brain regions involved in descending pain inhibition which, in turn, was associated with lower pain ratings to thermal stimuli.<sup><xref rid=\"R38\" ref-type=\"bibr\">38</xref></sup> In the second study, brain responses to noxious thermal stimuli before and after walking and running exercises were measured using functional magnetic resonance imaging in 20 athletes. The results suggested that running exercise reduced the pain-induced activation in the periaqueductal gray, a key area in descending pain inhibition which, in turn, was associated with lower pain unpleasantness ratings to thermal stimuli.<sup><xref rid=\"R165\" ref-type=\"bibr\">165</xref></sup> Taken together, these results provide evidence that a single bout of exercise can modulate pain-related areas of the nervous system.</p><p>In addition to the different study designs and techniques used to quantify pain in investigations of EIH, the exercise protocols have also varied considerably. Aerobic and isometric exercise have been studied most often,<sup><xref rid=\"R130\" ref-type=\"bibr\">130</xref></sup> whereas dynamic resistance exercise has not commonly been used. Within each mode of exercise, the prescription has varied too. For example, aerobic exercise has consisted of cycling, running, and stepping of various durations (30 seconds–30 minutes) and intensities (low to high).<sup><xref rid=\"R69\" ref-type=\"bibr\">69</xref>,<xref rid=\"R129\" ref-type=\"bibr\">129</xref>,<xref rid=\"R195\" ref-type=\"bibr\">195</xref></sup> The same is true of isometric exercise where upper-limb and lower-limb exercise of both short and long duration (<5 seconds—exhaustion) and varied intensity (10%–100% MVC) have been studied.<sup><xref rid=\"R64\" ref-type=\"bibr\">64</xref>,<xref rid=\"R195\" ref-type=\"bibr\">195</xref></sup> Studies of dynamic resistance exercise have typically used whole-body training at moderate intensities.<sup><xref rid=\"R17\" ref-type=\"bibr\">17</xref>,<xref rid=\"R93\" ref-type=\"bibr\">93</xref></sup> Interestingly, EIH is reproducible with each type of exercise, even when modest doses are used.<sup><xref rid=\"R129\" ref-type=\"bibr\">129</xref>,<xref rid=\"R162\" ref-type=\"bibr\">162</xref></sup> This is described in more detail below.</p></sec></sec></sec><sec id=\"s2\"><title>2. Pain outcomes after acute and regular exercise in pain-free individuals</title><p>As illustrated in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>, a single session of exercise has repeatedly been observed to reduce pain sensitivity in pain-free individuals. Hypoalgesia after aerobic exercises (eg, bicycling or running), dynamic resistance exercises (eg, circuit training), and isometric exercises (eg, a wall squat) often produces an increase in pressure pain thresholds at exercising body areas of 15% to 20% compared with a quiet rest control condition.<sup><xref rid=\"R192\" ref-type=\"bibr\">192</xref>,<xref rid=\"R200\" ref-type=\"bibr\">200</xref></sup> Increases in pain thresholds can also be observed at nonexercising body areas, although larger hypoalgesic responses are consistently observed in areas closer to the exercising muscles compared with nonexercising muscle areas. The observed EIH response is short-lasting, often with a duration lasting from 5 minutes after exercise<sup><xref rid=\"R69\" ref-type=\"bibr\">69</xref></sup> to 30 minutes after exercise<sup><xref rid=\"R88\" ref-type=\"bibr\">88</xref></sup> and may depend on the modality of the pain test stimulus.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Summary of studies investigating acute exercise-induced hypoalgesia in pain-free individuals.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\">Exercise type</th><th rowspan=\"1\" colspan=\"1\">Exercise form</th><th rowspan=\"1\" colspan=\"1\">Intensity</th><th rowspan=\"1\" colspan=\"1\">Duration</th><th rowspan=\"1\" colspan=\"1\"># of partici pants</th><th rowspan=\"1\" colspan=\"1\">Pain test modality</th><th rowspan=\"1\" colspan=\"1\">Pain outcome</th><th rowspan=\"1\" colspan=\"1\">Local site</th><th rowspan=\"1\" colspan=\"1\">Remote site</th><th rowspan=\"1\" colspan=\"1\">Findings</th><th rowspan=\"1\" colspan=\"1\">Year</th><th rowspan=\"1\" colspan=\"1\">Author</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">70% HRmax</td><td rowspan=\"1\" colspan=\"1\">30 min</td><td rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\">Chemical</td><td rowspan=\"1\" colspan=\"1\">Pain intensity</td><td rowspan=\"1\" colspan=\"1\">Thigh</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">↑Pain intensity (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\">1984</td><td rowspan=\"1\" colspan=\"1\">Vecchiet et al.<sup><xref rid=\"R205\" ref-type=\"bibr\">205</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">50%–70% HRmax</td><td rowspan=\"1\" colspan=\"1\">20 min</td><td rowspan=\"1\" colspan=\"1\">91</td><td rowspan=\"1\" colspan=\"1\">Cold</td><td rowspan=\"1\" colspan=\"1\">CPI</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Hand</td><td rowspan=\"1\" colspan=\"1\">No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\">1992</td><td rowspan=\"1\" colspan=\"1\">Padawer and Levine<sup><xref rid=\"R143\" ref-type=\"bibr\">143</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic<break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling<break/><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>70%–75% VO<sub>2</sub>max<break/><break/><break/><break/>VO<sub>2</sub>max test</td><td rowspan=\"1\" colspan=\"1\"><break/>6 min<break/><break/><break/><break/>8–12 min</td><td rowspan=\"1\" colspan=\"1\"><break/>41<break/><break/><break/><break/>25</td><td rowspan=\"1\" colspan=\"1\"><break/>Cold<break/><break/><break/><break/>Cold</td><td rowspan=\"1\" colspan=\"1\">CPT<break/>CPTol<break/><break/><break/><break/>CPI</td><td rowspan=\"1\" colspan=\"1\"><break/>—<break/><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand<break/><break/><break/><break/>Arm</td><td rowspan=\"1\" colspan=\"1\">↑CPT<break/>↑CPTol<break/><break/><break/><break/>↓CPI</td><td rowspan=\"1\" colspan=\"1\"><break/>2013<break/><break/><break/><break/>2018</td><td rowspan=\"1\" colspan=\"1\">Pokhrel et al.<sup><xref rid=\"R154\" ref-type=\"bibr\">154</xref></sup><break/><break/><break/><break/>Chretien et al.<sup><xref rid=\"R18\" ref-type=\"bibr\">18</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic<break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Bicycling<break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\">50 W<break/>100 W<break/>150 W<break/>200 W<break/><break/><break/>Increasing to 300W</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Max 8 min/step<break/><break/><break/>15–30 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>6<break/><break/><break/>7</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Electrical<break/><break/><break/>Electrical</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>EPT<break/><break/><break/>EPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>—<break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Tooth<break/><break/><break/>Tooth</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>↑EPT<break/><break/><break/>↑EPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>1984<break/><break/><break/>1985</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pertovaara et al.<sup><xref rid=\"R149\" ref-type=\"bibr\">149</xref></sup><break/><break/><break/>Kemppainen et al.<sup><xref rid=\"R87\" ref-type=\"bibr\">87</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">HR = 150/min</td><td rowspan=\"1\" colspan=\"1\">20 min</td><td rowspan=\"1\" colspan=\"1\">11</td><td rowspan=\"1\" colspan=\"1\">Electrical</td><td rowspan=\"1\" colspan=\"1\">EPT</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Tooth</td><td rowspan=\"1\" colspan=\"1\">↑EPT</td><td rowspan=\"1\" colspan=\"1\">1986</td><td rowspan=\"1\" colspan=\"1\">Olausson et al.<sup><xref rid=\"R140\" ref-type=\"bibr\">140</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">Increasing to 300 W</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">Electrical</td><td rowspan=\"1\" colspan=\"1\">EPT</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Tooth</td><td rowspan=\"1\" colspan=\"1\">↑EPT</td><td rowspan=\"1\" colspan=\"1\">1986</td><td rowspan=\"1\" colspan=\"1\">Kemppainen et al.<sup><xref rid=\"R86\" ref-type=\"bibr\">86</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">Increasing to 200 W</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">Electrical</td><td rowspan=\"1\" colspan=\"1\">EPT</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Tooth</td><td rowspan=\"1\" colspan=\"1\">↑EPT</td><td rowspan=\"1\" colspan=\"1\">1990</td><td rowspan=\"1\" colspan=\"1\">Kemppainen et al.<sup><xref rid=\"R88\" ref-type=\"bibr\">88</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>Increasing to 250 W</td><td rowspan=\"1\" colspan=\"1\"><break/>Fatigue</td><td rowspan=\"1\" colspan=\"1\"><break/>10</td><td rowspan=\"1\" colspan=\"1\"><break/>Electrical</td><td rowspan=\"1\" colspan=\"1\"><break/>EPT</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\">Tooth<break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↑EPT tooth<break/>↑EPT hand</td><td rowspan=\"1\" colspan=\"1\"><break/>1991</td><td rowspan=\"1\" colspan=\"1\"><break/>Droste et al.<sup><xref rid=\"R30\" ref-type=\"bibr\">30</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>Increasing to VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/>Unknown</td><td rowspan=\"1\" colspan=\"1\"><break/>17</td><td rowspan=\"1\" colspan=\"1\"><break/>Electrical</td><td rowspan=\"1\" colspan=\"1\">EPT<break/>EPTol</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↑EPT<break/>↑EPTol</td><td rowspan=\"1\" colspan=\"1\"><break/>2005</td><td rowspan=\"1\" colspan=\"1\"><break/>Drury et al.<sup><xref rid=\"R33\" ref-type=\"bibr\">33</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic<break/><break/>Aerobic<break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Bicycling<break/><break/>Bicycling<break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>1 KP<break/><break/>60 W<break/><break/><break/>Increasing to 200 W</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>5 min<break/><break/>10 min<break/><break/><break/>Unknown</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>60<break/><break/>21<break/><break/><break/>28</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Heat<break/><break/>Heat<break/><break/><break/>Heat</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>HPI<break/>TSPh<break/><break/>HPI<break/><break/><break/>HPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>—<break/><break/>—<break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\">Foot<break/>Lower leg<break/>Hand<break/>Forearm<break/><break/>Hand<break/><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>↓HPI lower extremity<break/>↓TSPh (lower extremity)<break/><break/><break/>↓HPI<break/><break/><break/>↓HPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2006<break/><break/>2014<break/><break/><break/>2019</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>George et al.<sup><xref rid=\"R50\" ref-type=\"bibr\">50</xref></sup><break/><break/>Ellingson et al.<sup><xref rid=\"R36\" ref-type=\"bibr\">36</xref></sup><break/><break/>St-Aubin et al.<sup><xref rid=\"R175\" ref-type=\"bibr\">175</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">75% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\">30 min</td><td rowspan=\"1\" colspan=\"1\">16</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Hand</td><td rowspan=\"1\" colspan=\"1\">↑PPT<break/>↓PPI</td><td rowspan=\"1\" colspan=\"1\">1996</td><td rowspan=\"1\" colspan=\"1\">Koltyn et al.<sup><xref rid=\"R95\" ref-type=\"bibr\">95</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic<break/><break/><break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling<break/><break/><break/><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>75% VO<sub>2</sub>max<break/><break/><break/><break/><break/>1. 75% VO<sub>2</sub>max<break/>2. 50% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/>30 min<break/><break/><break/><break/><break/>1. 10 min<break/>1. 20 min<break/>2. 10 min<break/>2. 20 min</td><td rowspan=\"1\" colspan=\"1\"><break/>20<break/><break/><break/><break/><break/><break/>80</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/><break/><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPI<break/><break/><break/><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>—<break/><break/><break/><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand<break/><break/><break/><break/><break/>Arm<break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/>No hypoalgesia<break/><break/><break/><break/><break/>↑PPTs<break/>After 75% VO<sub>2</sub>max<break/>(10 and 20 min)</td><td rowspan=\"1\" colspan=\"1\"><break/>2006<break/><break/><break/><break/><break/><break/>2014</td><td rowspan=\"1\" colspan=\"1\">Monnier-Benoit and Groslambert<sup><xref rid=\"R128\" ref-type=\"bibr\">128</xref></sup><break/><break/><break/><break/><break/>Vaegter et al.<sup><xref rid=\"R195\" ref-type=\"bibr\">195</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">75% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\">15 min</td><td rowspan=\"1\" colspan=\"1\">56</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Thigh</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">↑PPTs</td><td rowspan=\"1\" colspan=\"1\">2015</td><td rowspan=\"1\" colspan=\"1\">Vaegter et al.<sup><xref rid=\"R198\" ref-type=\"bibr\">198</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">75% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\">15 min</td><td rowspan=\"1\" colspan=\"1\">56</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPTol<break/>TSPp</td><td rowspan=\"1\" colspan=\"1\">Lower leg</td><td rowspan=\"1\" colspan=\"1\">Arm</td><td rowspan=\"1\" colspan=\"1\">↑PPTol lower leg<break/>↓TSPp lower leg</td><td rowspan=\"1\" colspan=\"1\">2015</td><td rowspan=\"1\" colspan=\"1\">Vaegter et al.<sup><xref rid=\"R196\" ref-type=\"bibr\">196</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\">1. 75% VO<sub>2</sub>max<break/>2. 50% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/>20 min</td><td rowspan=\"1\" colspan=\"1\"><break/>80</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPTol<break/>TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\"><break/>Arm</td><td rowspan=\"1\" colspan=\"1\"><break/>No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/>2015</td><td rowspan=\"1\" colspan=\"1\">Vaegter et al.<sup><xref rid=\"R196\" ref-type=\"bibr\">196</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>1. 70% VO<sub>2</sub>max<break/>2. 30% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>30 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>10</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\">↑PPT thigh<break/>After 70% VO<sub>2</sub>max<break/>↓PPT thigh and arm<break/>After 30% VO<sub>2</sub>max (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2016</td><td rowspan=\"1\" colspan=\"1\"><break/>Micalos and Arendt-Nielsen<sup><xref rid=\"R124\" ref-type=\"bibr\">124</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Increasing to VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Fatigue</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>50</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Knee</td><td rowspan=\"1\" colspan=\"1\">Ankle<break/>Arm<break/>Chest<break/>Head</td><td rowspan=\"1\" colspan=\"1\">↓PPT Chest (hyperalgesia)<break/>↓PPT Head (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2016</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Kruger et al.<sup><xref rid=\"R103\" ref-type=\"bibr\">103</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>RPE = 14–15</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>20 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>40</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Shin<break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↑PPT thigh<break/>↑PPT shin<break/>↑PPT hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Jones et al.<sup><xref rid=\"R81\" ref-type=\"bibr\">81</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>RPE = 17</td><td rowspan=\"1\" colspan=\"1\"><break/>5 min</td><td rowspan=\"1\" colspan=\"1\"><break/>36</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPT thigh<break/>↑PPT hand</td><td rowspan=\"1\" colspan=\"1\"><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/>Jones et al.<sup><xref rid=\"R79\" ref-type=\"bibr\">79</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>RPE = 16</td><td rowspan=\"1\" colspan=\"1\"><break/>15 min</td><td rowspan=\"1\" colspan=\"1\"><break/>34</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\">↑PPT thigh<break/>↑PPT shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/>2018</td><td rowspan=\"1\" colspan=\"1\">Vaegter et al.<sup><xref rid=\"R193\" ref-type=\"bibr\">193</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>1. HIIT: 90%–100% of max workload<break/>2. MICT: 65%–75% of HR</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>1. 10 × 1 min<break/>2. 30 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>28</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Shin<break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>2018</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Hakansson et al.<sup><xref rid=\"R58\" ref-type=\"bibr\">58</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>75% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>15 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>31</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Back<break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↑PPT thigh<break/>↑PPT back<break/>↑PPT hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2018</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Gajsar et al.<sup><xref rid=\"R45\" ref-type=\"bibr\">45</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic<break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling<break/><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\">50 W<break/><break/><break/><break/>75% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\">12 min<break/><break/><break/><break/>15 min</td><td rowspan=\"1\" colspan=\"1\">20<break/><break/><break/><break/>30</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">TSPp<break/><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\">Thigh<break/><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\">Shoulder<break/><break/><break/>Back<break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↓TSPp trapezius<break/><break/><break/>↑PPT thigh<break/>↑PPT back</td><td rowspan=\"1\" colspan=\"1\">2018<break/><break/><break/><break/>2019</td><td rowspan=\"1\" colspan=\"1\">Malfliet et al.<sup><xref rid=\"R118\" ref-type=\"bibr\">118</xref></sup><break/><break/><break/>Gomolka et al.<sup><xref rid=\"R54\" ref-type=\"bibr\">54</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">Lactate threshold</td><td rowspan=\"1\" colspan=\"1\">15 min</td><td rowspan=\"1\" colspan=\"1\">34</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Thigh</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">↑PPT thigh</td><td rowspan=\"1\" colspan=\"1\">2019</td><td rowspan=\"1\" colspan=\"1\">Vaegter et al.<sup><xref rid=\"R192\" ref-type=\"bibr\">192</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>75%–88% HRmax</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>20 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>15</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure<break/>Electrical</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT<break/>EPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\">Shoulder<break/>Thoracic spine<break/>Hand<break/>Esophagus</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/>van Weerdenburg et al.<sup><xref rid=\"R204\" ref-type=\"bibr\">204</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>1. 70% HR max<break/>2. 86% HR max</td><td rowspan=\"1\" colspan=\"1\"><break/>1. 24 min<break/>2. 4 × 4 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>29</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>HPT<break/>HPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>↓HPI after interval condition</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2014</td><td rowspan=\"1\" colspan=\"1\"><break/>Kodesh and Weissman-Fogel<sup><xref rid=\"R91\" ref-type=\"bibr\">91</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>1. 70% HRR<break/>2. 50%–55% HRR</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>20 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>27</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI<break/>HPI<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPT after high intensity<break/>↓HPI<break/>↓TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2014</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Naugle et al.<sup><xref rid=\"R132\" ref-type=\"bibr\">132</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>Intensity = pain level 3/10</td><td rowspan=\"1\" colspan=\"1\"><break/>15 min</td><td rowspan=\"1\" colspan=\"1\"><break/>16</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>HPT</td><td rowspan=\"1\" colspan=\"1\"><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↑PPT<break/>↑HPT</td><td rowspan=\"1\" colspan=\"1\"><break/>2016</td><td rowspan=\"1\" colspan=\"1\"><break/>Black et al.<sup><xref rid=\"R11\" ref-type=\"bibr\">11</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>1. 75% VO<sub>2</sub>max<break/>2. 50% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>25 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>43</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI<break/>HPI<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>↑PPTs</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2016</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Naugle et al.<sup><xref rid=\"R133\" ref-type=\"bibr\">133</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>60–70 W</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>20 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>40</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>HPT<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Achilles</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2016</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Stackhouse<sup><xref rid=\"R176\" ref-type=\"bibr\">176</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>70% HRR</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>15 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>16</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>HPT<break/>HPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Shin<break/>Foot</td><td rowspan=\"1\" colspan=\"1\">↑PPT thigh<break/>↑PPT shin<break/>↓HPI foot</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2019</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Jones et al.<sup><xref rid=\"R78\" ref-type=\"bibr\">78</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">200 W</td><td rowspan=\"1\" colspan=\"1\">20 min</td><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">Reflex</td><td rowspan=\"1\" colspan=\"1\">NFR</td><td rowspan=\"1\" colspan=\"1\">Thigh</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">↑NFR</td><td rowspan=\"1\" colspan=\"1\">1992</td><td rowspan=\"1\" colspan=\"1\">Guieu et al.<sup><xref rid=\"R55\" ref-type=\"bibr\">55</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Repeated back movements</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Lifting 5 kg</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>7 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>18</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/>Heat<break/>Cold</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>HPT<break/>CPT<break/>TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Back</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>↑PPT back<break/>↑CPT hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2019</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Kuithan et al.<sup><xref rid=\"R104\" ref-type=\"bibr\">104</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Running</td><td rowspan=\"1\" colspan=\"1\">Near anaerobic threshold</td><td rowspan=\"1\" colspan=\"1\"><break/>30 min</td><td rowspan=\"1\" colspan=\"1\"><break/>27</td><td rowspan=\"1\" colspan=\"1\"><break/>Cold</td><td rowspan=\"1\" colspan=\"1\">CPT<break/>CPI</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>↑CPT</td><td rowspan=\"1\" colspan=\"1\"><break/>2011</td><td rowspan=\"1\" colspan=\"1\">Wonders and Drury<sup><xref rid=\"R210\" ref-type=\"bibr\">210</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Running</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">30 min</td><td rowspan=\"1\" colspan=\"1\">22</td><td rowspan=\"1\" colspan=\"1\">Heat</td><td rowspan=\"1\" colspan=\"1\">HPI</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Forearm</td><td rowspan=\"1\" colspan=\"1\">No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\">1993</td><td rowspan=\"1\" colspan=\"1\">Fuller and Robinson<sup><xref rid=\"R44\" ref-type=\"bibr\">44</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Running</td><td rowspan=\"1\" colspan=\"1\"><break/>Self-selected</td><td rowspan=\"1\" colspan=\"1\"><break/>40 min</td><td rowspan=\"1\" colspan=\"1\"><break/>1</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PTT</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Arm</td><td rowspan=\"1\" colspan=\"1\">↑PPT<break/>↑PTT</td><td rowspan=\"1\" colspan=\"1\"><break/>1979</td><td rowspan=\"1\" colspan=\"1\"><break/>Black et al.<sup><xref rid=\"R14\" ref-type=\"bibr\">14</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Running</td><td rowspan=\"1\" colspan=\"1\">Self-selected</td><td rowspan=\"1\" colspan=\"1\">1 mile</td><td rowspan=\"1\" colspan=\"1\">15</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Hand</td><td rowspan=\"1\" colspan=\"1\">↑PPT hand</td><td rowspan=\"1\" colspan=\"1\">1981</td><td rowspan=\"1\" colspan=\"1\">Haier et al.<sup><xref rid=\"R57\" ref-type=\"bibr\">57</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Running</td><td rowspan=\"1\" colspan=\"1\">VO<sub>2</sub>max test</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">29</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPI</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Arm</td><td rowspan=\"1\" colspan=\"1\">↓PPI</td><td rowspan=\"1\" colspan=\"1\">2001</td><td rowspan=\"1\" colspan=\"1\">Oktedalen et al.<sup><xref rid=\"R139\" ref-type=\"bibr\">139</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Running</td><td rowspan=\"1\" colspan=\"1\"><break/>1. 75% VO<sub>2</sub>max<break/>2. 75% VO<sub>2</sub>max<break/>3. 50% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/>1. 10 min<break/>2. 30 min<break/>3. 10 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>12</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>↓PPI after 30 min at 75% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2004</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hoffman et al.<sup><xref rid=\"R69\" ref-type=\"bibr\">69</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Running</td><td rowspan=\"1\" colspan=\"1\">65%–75% of HRR</td><td rowspan=\"1\" colspan=\"1\">7 min</td><td rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Forearm</td><td rowspan=\"1\" colspan=\"1\">↑PPT</td><td rowspan=\"1\" colspan=\"1\">2004</td><td rowspan=\"1\" colspan=\"1\">Drury et al.<sup><xref rid=\"R32\" ref-type=\"bibr\">32</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Running</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">100 mile</td><td rowspan=\"1\" colspan=\"1\">30</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPI</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Hand</td><td rowspan=\"1\" colspan=\"1\">↓PPI</td><td rowspan=\"1\" colspan=\"1\">2007</td><td rowspan=\"1\" colspan=\"1\">Hoffman et al.<sup><xref rid=\"R67\" ref-type=\"bibr\">67</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Running</td><td rowspan=\"1\" colspan=\"1\"><break/>VO<sub>2</sub>max test</td><td rowspan=\"1\" colspan=\"1\"><break/>Unknown</td><td rowspan=\"1\" colspan=\"1\"><break/>62</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\">Shoulder<break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>2015</td><td rowspan=\"1\" colspan=\"1\"><break/>Stolzman et al.<sup><xref rid=\"R179\" ref-type=\"bibr\">179</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Running</td><td rowspan=\"1\" colspan=\"1\">110% Gas exchange threshold</td><td rowspan=\"1\" colspan=\"1\"><break/>30 min</td><td rowspan=\"1\" colspan=\"1\"><break/>26</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\">↑PPT forearm<break/>↑PPT thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>2019</td><td rowspan=\"1\" colspan=\"1\">Peterson et al.<sup><xref rid=\"R151\" ref-type=\"bibr\">151</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Running</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>85% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>44 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>12</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/>Heat<break/>Cold</td><td rowspan=\"1\" colspan=\"1\">PPI<break/>HPI<break/>CPI<break/>CPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hand<break/>Arm</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>↓HPI<break/>↓PPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>1984</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Janal et al.<sup><xref rid=\"R74\" ref-type=\"bibr\">74</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Running</td><td rowspan=\"1\" colspan=\"1\"><break/>85% HRmax</td><td rowspan=\"1\" colspan=\"1\"><break/>10 min</td><td rowspan=\"1\" colspan=\"1\"><break/>63</td><td rowspan=\"1\" colspan=\"1\">Heat<break/>Cold</td><td rowspan=\"1\" colspan=\"1\">HPT<break/>CPI</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\">Hand<break/>Forearm</td><td rowspan=\"1\" colspan=\"1\">↓HPT (hyperalgesia)<break/>↓CPI</td><td rowspan=\"1\" colspan=\"1\"><break/>2001</td><td rowspan=\"1\" colspan=\"1\">Sternberg et al.<sup><xref rid=\"R178\" ref-type=\"bibr\">178</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Running</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>75% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>30 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>14</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Heat<break/>Cold</td><td rowspan=\"1\" colspan=\"1\">HPT<break/>CPT<break/>HPI<break/>CPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2005</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Ruble et al.<sup><xref rid=\"R159\" ref-type=\"bibr\">159</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Step</td><td rowspan=\"1\" colspan=\"1\"><break/>63% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/>12 min</td><td rowspan=\"1\" colspan=\"1\"><break/>60</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPI<break/>PTT</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↓PPI<break/>↑PTT</td><td rowspan=\"1\" colspan=\"1\"><break/>1994</td><td rowspan=\"1\" colspan=\"1\">Gurevich et al.<sup><xref rid=\"R56\" ref-type=\"bibr\">56</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Step</td><td rowspan=\"1\" colspan=\"1\">50% of maximum number of steps in 1 minute</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>5 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>30</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPI<break/>TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/>↓PPI<break/>↓TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2019</td><td rowspan=\"1\" colspan=\"1\"><break/>Nasri-Heir et al.<sup><xref rid=\"R129\" ref-type=\"bibr\">129</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic<break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Walking<break/><break/><break/>Walking</td><td rowspan=\"1\" colspan=\"1\"><break/>6.5 km/h<break/><break/><break/>Fast walking</td><td rowspan=\"1\" colspan=\"1\">10 min<break/>40 min<break/><break/>6 min</td><td rowspan=\"1\" colspan=\"1\"><break/>5<break/><break/><break/>35</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT<break/><break/><break/>PPTol</td><td rowspan=\"1\" colspan=\"1\"><break/>Thigh<break/><break/><break/>Calf</td><td rowspan=\"1\" colspan=\"1\"><break/>Shoulder<break/><break/><break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/>No hypoalgesia<break/><break/><break/>↑cPTT Calf</td><td rowspan=\"1\" colspan=\"1\"><break/>2014<break/><break/><break/>2019</td><td rowspan=\"1\" colspan=\"1\"><break/>Lee<sup><xref rid=\"R110\" ref-type=\"bibr\">110</xref></sup><break/><break/><break/>Hviid et al.<sup><xref rid=\"R73\" ref-type=\"bibr\">73</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Anaerobic</td><td rowspan=\"1\" colspan=\"1\">Wingate test</td><td rowspan=\"1\" colspan=\"1\"><break/>“All-out”</td><td rowspan=\"1\" colspan=\"1\"><break/>30 seconds</td><td rowspan=\"1\" colspan=\"1\"><break/>50</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\">Shoulder<break/>Jaw</td><td rowspan=\"1\" colspan=\"1\"><break/>↓PPTs (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/>2012</td><td rowspan=\"1\" colspan=\"1\">Arroyo-Morales et al.<sup><xref rid=\"R3\" ref-type=\"bibr\">3</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Anaerobic<break/><break/><break/><break/><break/><break/>Anaerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycle<break/>Sprint<break/><break/><break/><break/><break/><break/>Wingate test</td><td rowspan=\"1\" colspan=\"1\">“All-out”<break/><break/><break/><break/><break/><break/><break/>“All-out”</td><td rowspan=\"1\" colspan=\"1\">3 × 6 seconds<break/><break/><break/><break/><break/><break/><break/>30 seconds</td><td rowspan=\"1\" colspan=\"1\">12<break/><break/><break/><break/><break/><break/><break/>50</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/><break/><break/><break/><break/><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/><break/><break/><break/>PPT<break/>HPT<break/>TSPh<break/>TSPc</td><td rowspan=\"1\" colspan=\"1\">Thigh<break/><break/><break/><break/><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\">Lower leg<break/><break/><break/><break/><break/><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↓PPTs (hyperalgesia)<break/><break/><break/><break/>↑PPT thigh<break/>↑HPT hand<break/>↓TSPh hand<break/>↓TSPc hand</td><td rowspan=\"1\" colspan=\"1\">2018<break/><break/><break/><break/><break/><break/><break/>2018</td><td rowspan=\"1\" colspan=\"1\">Klich et al.<sup><xref rid=\"R89\" ref-type=\"bibr\">89</xref></sup><break/><break/><break/><break/><break/>Samuelly-Leichtag et al.<sup><xref rid=\"R162\" ref-type=\"bibr\">162</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\">Full-body circuit</td><td rowspan=\"1\" colspan=\"1\"><break/>Moderate</td><td rowspan=\"1\" colspan=\"1\"><break/>20 min</td><td rowspan=\"1\" colspan=\"1\"><break/>17</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPTol</td><td rowspan=\"1\" colspan=\"1\"><break/>Shin</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPTol</td><td rowspan=\"1\" colspan=\"1\"><break/>1996</td><td rowspan=\"1\" colspan=\"1\">Bartholomew et al.<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\">Full-body circuit</td><td rowspan=\"1\" colspan=\"1\"><break/>75% 1RM</td><td rowspan=\"1\" colspan=\"1\">4 exercises 3 × 10 repetitions (45 min)</td><td rowspan=\"1\" colspan=\"1\"><break/>13</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↑PPT<break/>↓PPI</td><td rowspan=\"1\" colspan=\"1\"><break/>1998</td><td rowspan=\"1\" colspan=\"1\">Koltyn and Arbogast<sup><xref rid=\"R93\" ref-type=\"bibr\">93</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\">Full-body circuit</td><td rowspan=\"1\" colspan=\"1\"><break/>75% 1RM</td><td rowspan=\"1\" colspan=\"1\">4 exercises 3 × 10 repetitions (45 min)</td><td rowspan=\"1\" colspan=\"1\"><break/>21</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↑PPT<break/>↓PPI</td><td rowspan=\"1\" colspan=\"1\"><break/>2009</td><td rowspan=\"1\" colspan=\"1\">Focht and Koltyn<sup><xref rid=\"R42\" ref-type=\"bibr\">42</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Dynamic resistance<break/><break/><break/><break/>Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\"><break/>Upper-body circuit<break/><break/><break/><break/>Full-body circuit</td><td rowspan=\"1\" colspan=\"1\"><break/>Unknown<break/><break/><break/><break/><break/>60% 1RM</td><td rowspan=\"1\" colspan=\"1\">10 min<break/>40 min<break/><break/><break/><break/>3 exercises 12 repetitions</td><td rowspan=\"1\" colspan=\"1\"><break/>5<break/><break/><break/><break/>24</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT<break/><break/><break/>PPT<break/>PPTol</td><td rowspan=\"1\" colspan=\"1\"><break/>Shoulder<break/><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>—<break/><break/><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>No hyperalgesia<break/><break/><break/><break/>↑PPTol</td><td rowspan=\"1\" colspan=\"1\"><break/>2014<break/><break/><break/><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/>Lee<sup><xref rid=\"R110\" ref-type=\"bibr\">110</xref></sup><break/><break/><break/>Baiamonte et al.<sup><xref rid=\"R4\" ref-type=\"bibr\">4</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dynamic resistance<break/><break/><break/><break/>Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\">Kettlebell swings<break/><break/><break/><break/>Full-body circuit</td><td rowspan=\"1\" colspan=\"1\">8–12 kg<break/><break/><break/><break/><break/>60% 1RM</td><td rowspan=\"1\" colspan=\"1\">8 × 20 seconds<break/><break/><break/><break/>9 exercises 12 repetitions</td><td rowspan=\"1\" colspan=\"1\">32<break/><break/><break/><break/><break/>10</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT<break/><break/><break/><break/><break/>PTT</td><td rowspan=\"1\" colspan=\"1\">Lower back<break/><break/><break/><break/>Buttock<break/>Hand</td><td rowspan=\"1\" colspan=\"1\">—<break/><break/><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\">↑PPTs<break/><break/><break/><break/><break/>↑PTT hand</td><td rowspan=\"1\" colspan=\"1\">2017<break/><break/><break/><break/><break/>2018</td><td rowspan=\"1\" colspan=\"1\">Keilman et al.<sup><xref rid=\"R84\" ref-type=\"bibr\">84</xref></sup><break/><break/><break/><break/>McKean et al.<sup><xref rid=\"R119\" ref-type=\"bibr\">119</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\">Handgrip</td><td rowspan=\"1\" colspan=\"1\">100% MVC</td><td rowspan=\"1\" colspan=\"1\">30 contractions in 1 minute</td><td rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Forearm</td><td rowspan=\"1\" colspan=\"1\">↑PPT</td><td rowspan=\"1\" colspan=\"1\">2004</td><td rowspan=\"1\" colspan=\"1\">Drury et al.<sup><xref rid=\"R32\" ref-type=\"bibr\">32</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\">Handgrip</td><td rowspan=\"1\" colspan=\"1\">Medium</td><td rowspan=\"1\" colspan=\"1\">Maximum of 40 contractions in 1 minute</td><td rowspan=\"1\" colspan=\"1\">48</td><td rowspan=\"1\" colspan=\"1\">Heat</td><td rowspan=\"1\" colspan=\"1\">HPI</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Hand</td><td rowspan=\"1\" colspan=\"1\">↓HPI</td><td rowspan=\"1\" colspan=\"1\">2008</td><td rowspan=\"1\" colspan=\"1\">Weissman-Fogel et al.<sup><xref rid=\"R207\" ref-type=\"bibr\">207</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Dynamic ``Resistance</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Back extensions</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Bodyweight</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>3 × 15 repetitions</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>20</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Heat</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>HPI<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\">Foot<break/>Lower leg<break/>Hand<break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>↓HPI (lower extremity)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2006</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>George et al.<sup><xref rid=\"R50\" ref-type=\"bibr\">50</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\"><break/>Cervical flexions</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Head weight</td><td rowspan=\"1\" colspan=\"1\"><break/>3 × 10 repetitions</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>30</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>HPI<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Foot<break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPT<break/>↓HPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2011</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Bishop et al.<sup><xref rid=\"R10\" ref-type=\"bibr\">10</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Eccentric<break/><break/><break/><break/>Eccentric<break/><break/><break/><break/>Eccentric</td><td rowspan=\"1\" colspan=\"1\">Wrist extension<break/><break/><break/>Elbow flexion<break/><break/><break/>Heel-raise</td><td rowspan=\"1\" colspan=\"1\">30% MVC<break/><break/><break/><break/>Max<break/><break/><break/><break/>Bodyweight</td><td rowspan=\"1\" colspan=\"1\">5 × 10 repetitions<break/><break/><break/>10 × 6 repetitions<break/><break/>4 × 15 contractions</td><td rowspan=\"1\" colspan=\"1\">13<break/><break/><break/><break/>10<break/><break/><break/><break/>40</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/><break/><break/><break/>Pressure<break/>Electrical<break/><break/><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/><break/><break/><break/>PPT<break/>EPT<break/><break/><break/>PPT<break/>HPT<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\">Forearm<break/><break/><break/><break/>Arm<break/><break/><break/><break/>Achilles</td><td rowspan=\"1\" colspan=\"1\">—<break/><break/><break/><break/>—<break/><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\">↑PPT<break/><break/><break/><break/>↓PPT<break/>↓EPT (hyperalgesia)<break/><break/><break/>PPT<break/>↓TSPh</td><td rowspan=\"1\" colspan=\"1\">2010<break/><break/><break/><break/>2015<break/><break/><break/><break/>2016</td><td rowspan=\"1\" colspan=\"1\">Slater et al.<sup><xref rid=\"R171\" ref-type=\"bibr\">171</xref></sup><break/><break/><break/><break/>Lau et al.<sup><xref rid=\"R108\" ref-type=\"bibr\">108</xref></sup><break/><break/><break/>Stackhouse et al.<sup><xref rid=\"R176\" ref-type=\"bibr\">176</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">1. Knee extension<break/>2. Elbow flexion</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>1. 30% MVC<break/>2. 60% MVC</td><td rowspan=\"1\" colspan=\"1\">1. 90 seconds<break/>1. 180 seconds<break/>2. 90 seconds<break/>2. 180 seconds</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>80</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\">Thigh (knee extension)<break/>Arm (elbow flexion)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\">↑PPTs<break/>After low and high intensity exercises</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2014</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Vaegter et al.<sup><xref rid=\"R195\" ref-type=\"bibr\">195</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">1. Knee extension<break/>2. Elbow flexion</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>1. 30% MVC<break/>2. 60% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>80</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPTol<break/>TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Arm</td><td rowspan=\"1\" colspan=\"1\">↑PPTol (after both elbow and knee exercises)<break/>↓TSPp arm and leg (after low and high intensity exercises)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2015</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Vaegter et al.<sup><xref rid=\"R196\" ref-type=\"bibr\">196</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">1. Knee extension<break/>2. Elbow flexion</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>20% of MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Fatigue</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>64</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>↑PPT after elbow flexion (women only)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2016</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Lemley et al.<sup><xref rid=\"R113\" ref-type=\"bibr\">113</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">1. Knee extension<break/>2. Shoulder rotation</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>1. 1 kg<break/>2. 0.5 kg</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Fatigue</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>24</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Thigh<break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Shoulder<break/>`Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPT thigh + shoulder both conditions</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2003</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Kosek and Lundberg<sup><xref rid=\"R101\" ref-type=\"bibr\">101</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">Back extension</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>2 min</td><td rowspan=\"1\" colspan=\"1\"><break/>29</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>Back</td><td rowspan=\"1\" colspan=\"1\">Thigh<break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↑PPT thigh<break/>↑PPT hand (women)</td><td rowspan=\"1\" colspan=\"1\"><break/>2017</td><td rowspan=\"1\" colspan=\"1\">Gajsar et al.<sup><xref rid=\"R46\" ref-type=\"bibr\">46</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Elbow flexion</td><td rowspan=\"1\" colspan=\"1\">1. Max contractions<break/>2. 25% MVC<break/>3. 25% MVC<break/>4. 80% MVC</td><td rowspan=\"1\" colspan=\"1\">1. 3 reps<break/>2. Fatigue<break/>3. 2 min<break/>4. Fatigue</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>40</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPT and ↓PPI after max and after 25% MVC until fatigue</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2008</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hoeger Bement et al.<sup><xref rid=\"R64\" ref-type=\"bibr\">64</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">Elbow flexion</td><td rowspan=\"1\" colspan=\"1\"><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/>Fatigue</td><td rowspan=\"1\" colspan=\"1\"><break/>20</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\">↑PPT<break/>↓PPI</td><td rowspan=\"1\" colspan=\"1\"><break/>2009</td><td rowspan=\"1\" colspan=\"1\">Hoeger Bement et al.<sup><xref rid=\"R65\" ref-type=\"bibr\">65</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">Elbow flexion</td><td rowspan=\"1\" colspan=\"1\"><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/>Fatigue</td><td rowspan=\"1\" colspan=\"1\"><break/>26</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\">↑PPT<break/>↓PPI (men only)</td><td rowspan=\"1\" colspan=\"1\"><break/>2014</td><td rowspan=\"1\" colspan=\"1\"><break/>Bement et al.<sup><xref rid=\"R7\" ref-type=\"bibr\">7</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/>Elbow flexion</td><td rowspan=\"1\" colspan=\"1\">1. Max contractions<break/>2. 25% MVC<break/>3. 25% MVC</td><td rowspan=\"1\" colspan=\"1\">1. 3 reps<break/>2. Fatigue<break/>3. 2 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>24</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPT<break/>↓PPI (women only)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2014</td><td rowspan=\"1\" colspan=\"1\"><break/>Lemley et al.<sup><xref rid=\"R111\" ref-type=\"bibr\">111</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Elbow flexion</td><td rowspan=\"1\" colspan=\"1\">25% MVC</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">39</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPI</td><td rowspan=\"1\" colspan=\"1\">Hand</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">↓PPI</td><td rowspan=\"1\" colspan=\"1\">2014</td><td rowspan=\"1\" colspan=\"1\">Lemley et al.<sup><xref rid=\"R112\" ref-type=\"bibr\">112</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">Elbow flexion</td><td rowspan=\"1\" colspan=\"1\"><break/>40% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/>26</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>HPT</td><td rowspan=\"1\" colspan=\"1\"><break/>Arm</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPTs</td><td rowspan=\"1\" colspan=\"1\"><break/>2016</td><td rowspan=\"1\" colspan=\"1\"><break/>Jones et al.<sup><xref rid=\"R80\" ref-type=\"bibr\">80</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Arm abduction</td><td rowspan=\"1\" colspan=\"1\">1 kg</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">25</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">↑PPTs</td><td rowspan=\"1\" colspan=\"1\">2000</td><td rowspan=\"1\" colspan=\"1\">Persson et al.<sup><xref rid=\"R147\" ref-type=\"bibr\">147</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/>2 min</td><td rowspan=\"1\" colspan=\"1\"><break/>134</td><td rowspan=\"1\" colspan=\"1\"><break/>Cold</td><td rowspan=\"1\" colspan=\"1\">CPT<break/>CPI</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>↑CPT hand</td><td rowspan=\"1\" colspan=\"1\"><break/>2017</td><td rowspan=\"1\" colspan=\"1\">Foxen-Craft and Dahlquist<sup><xref rid=\"R43\" ref-type=\"bibr\">43</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Handgrip</td><td rowspan=\"1\" colspan=\"1\">25% MVC</td><td rowspan=\"1\" colspan=\"1\">3 min</td><td rowspan=\"1\" colspan=\"1\">34</td><td rowspan=\"1\" colspan=\"1\">Electrical</td><td rowspan=\"1\" colspan=\"1\">EPI</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Lower leg</td><td rowspan=\"1\" colspan=\"1\">↓EPI</td><td rowspan=\"1\" colspan=\"1\">2016</td><td rowspan=\"1\" colspan=\"1\">Umeda et al.<sup><xref rid=\"R188\" ref-type=\"bibr\">188</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\">1. 40% MVC<break/>2. 25% MVC</td><td rowspan=\"1\" colspan=\"1\">1. Fatigue<break/>2. 3 min</td><td rowspan=\"1\" colspan=\"1\"><break/>88</td><td rowspan=\"1\" colspan=\"1\"><break/>Heat</td><td rowspan=\"1\" colspan=\"1\"><break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\">↓TSPh for both conditions</td><td rowspan=\"1\" colspan=\"1\"><break/>2013</td><td rowspan=\"1\" colspan=\"1\"><break/>Koltyn et al.<sup><xref rid=\"R96\" ref-type=\"bibr\">96</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\">1. Maximal<break/>2. 40%–50% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/>2 min</td><td rowspan=\"1\" colspan=\"1\"><break/>31</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\">↑PPT<break/>↓PPI</td><td rowspan=\"1\" colspan=\"1\"><break/>2001</td><td rowspan=\"1\" colspan=\"1\"><break/>Koltyn et al.<sup><xref rid=\"R97\" ref-type=\"bibr\">97</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/>40%–50% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/>2 min</td><td rowspan=\"1\" colspan=\"1\"><break/>40</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↑PPT both sites<break/>↓PPI both sites</td><td rowspan=\"1\" colspan=\"1\"><break/>2007</td><td rowspan=\"1\" colspan=\"1\">Koltyn and Umeda<sup><xref rid=\"R98\" ref-type=\"bibr\">98</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric<break/><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">Handgrip<break/><break/><break/><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\">33% MVC<break/><break/><break/><break/>1. 25% MVC<break/>2. 25% MVC</td><td rowspan=\"1\" colspan=\"1\">3 min<break/><break/><break/><break/>1. 1 minute<break/>2. 3 min</td><td rowspan=\"1\" colspan=\"1\">79<break/><break/><break/><break/>23</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPTol<break/><break/><break/><break/>PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\">Hand<break/><break/><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\">—<break/><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\">↑PPTol<break/><break/><break/><break/>No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\">2009<break/><break/><break/><break/>2009</td><td rowspan=\"1\" colspan=\"1\">Alghamdi and Al-Sheikh<sup><xref rid=\"R1\" ref-type=\"bibr\">1</xref></sup><break/><break/><break/>Umeda et al.<sup><xref rid=\"R190\" ref-type=\"bibr\">190</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\">1. 1 minute<break/>2. 3 min<break/>3. 5 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>50</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPT and ↓PPI after all durations</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2010</td><td rowspan=\"1\" colspan=\"1\"><break/>Umeda et al.<sup><xref rid=\"R189\" ref-type=\"bibr\">189</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Handgrip</td><td rowspan=\"1\" colspan=\"1\">50% MVC</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">50</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Forearm</td><td rowspan=\"1\" colspan=\"1\">Forearm</td><td rowspan=\"1\" colspan=\"1\">↑PPT</td><td rowspan=\"1\" colspan=\"1\">2017</td><td rowspan=\"1\" colspan=\"1\">Black et al.<sup><xref rid=\"R12\" ref-type=\"bibr\">12</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Handgrip</td><td rowspan=\"1\" colspan=\"1\">50% MVC</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">26</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Forearm</td><td rowspan=\"1\" colspan=\"1\">Thigh</td><td rowspan=\"1\" colspan=\"1\">↑PPT forearm<break/>↑PPT thigh</td><td rowspan=\"1\" colspan=\"1\">2019</td><td rowspan=\"1\" colspan=\"1\">Peterson et al.<sup><xref rid=\"R151\" ref-type=\"bibr\">151</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\">1. 1% MVC<break/>2. 15% MVC<break/>3. 25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Unknown</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2008</td><td rowspan=\"1\" colspan=\"1\"><break/>Electrical<break/>Reflex</td><td rowspan=\"1\" colspan=\"1\"><break/>EPI<break/>NFR</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\"><break/>↓EPI after 15% and 25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2008</td><td rowspan=\"1\" colspan=\"1\"><break/>Ring et al.<sup><xref rid=\"R157\" ref-type=\"bibr\">157</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>27</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI<break/>HPI<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPT<break/>↓HPI (women)<break/>↓TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2014</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Naugle et al.<sup><xref rid=\"R131\" ref-type=\"bibr\">131</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>58</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\">↑PPT<break/>↓PPI<break/>↓TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2014</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Koltyn et al.<sup><xref rid=\"R94\" ref-type=\"bibr\">94</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>43</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI<break/>HPI<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>↑PPT<break/>↓TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2016</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Naugle et al.<sup><xref rid=\"R133\" ref-type=\"bibr\">133</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>58</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\">↑PPT<break/>↓PPI<break/>↓TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/>Brellenthin et al.<sup><xref rid=\"R16\" ref-type=\"bibr\">16</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/>58</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPI<break/>HPI</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\">↓PPI hand<break/>↓HPI hand</td><td rowspan=\"1\" colspan=\"1\"><break/>2018</td><td rowspan=\"1\" colspan=\"1\"><break/>Crombie et al.<sup><xref rid=\"R22\" ref-type=\"bibr\">22</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/>52</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>HPI</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/>↓PPT (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/>2018</td><td rowspan=\"1\" colspan=\"1\">Ohlman et al.<sup><xref rid=\"R138\" ref-type=\"bibr\">138</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Knee extension</td><td rowspan=\"1\" colspan=\"1\">21% MVC</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Thigh</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">↑PPT</td><td rowspan=\"1\" colspan=\"1\">1995</td><td rowspan=\"1\" colspan=\"1\">Kosek and Ekholm<sup><xref rid=\"R99\" ref-type=\"bibr\">99</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Knee extension</td><td rowspan=\"1\" colspan=\"1\">30% MVC</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">134</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">↑PPT</td><td rowspan=\"1\" colspan=\"1\">2017</td><td rowspan=\"1\" colspan=\"1\">Tour et al.<sup><xref rid=\"R185\" ref-type=\"bibr\">185</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Knee extension</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>0.75 kg</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>12 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>15</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure<break/>Electrical</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPT<break/>EPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\">Shoulder<break/>Thoracic spine<break/>Hand<break/>Esophagus</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>van Weerdenburg et al.<sup><xref rid=\"R204\" ref-type=\"bibr\">204</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/>Knee extension</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>30% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>20</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPTol<break/>HPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>↑PPTol</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/>Vaegter et al.<sup><xref rid=\"R199\" ref-type=\"bibr\">199</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Knee extension</td><td rowspan=\"1\" colspan=\"1\">20%–25% MVC</td><td rowspan=\"1\" colspan=\"1\">5 min</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>PPI<break/>HPI</td><td rowspan=\"1\" colspan=\"1\">Shin</td><td rowspan=\"1\" colspan=\"1\">Neck</td><td rowspan=\"1\" colspan=\"1\">↑PPT shin</td><td rowspan=\"1\" colspan=\"1\">2018</td><td rowspan=\"1\" colspan=\"1\">Harris et al.<sup><xref rid=\"R61\" ref-type=\"bibr\">61</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Pinch grip</td><td rowspan=\"1\" colspan=\"1\">25% MVC</td><td rowspan=\"1\" colspan=\"1\">15 seconds</td><td rowspan=\"1\" colspan=\"1\">38</td><td rowspan=\"1\" colspan=\"1\">Heat</td><td rowspan=\"1\" colspan=\"1\">HPI</td><td rowspan=\"1\" colspan=\"1\">Hand</td><td rowspan=\"1\" colspan=\"1\">Hand</td><td rowspan=\"1\" colspan=\"1\">No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\">2013</td><td rowspan=\"1\" colspan=\"1\">Paris et al.<sup><xref rid=\"R144\" ref-type=\"bibr\">144</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pinch grip</td><td rowspan=\"1\" colspan=\"1\">1. 5% MVC<break/>2. 25% MVC<break/>3. 50% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>15 seconds</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>42</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Heat</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>HPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↓HPI with larger effects for higher intensity</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2014</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Misra et al.<sup><xref rid=\"R127\" ref-type=\"bibr\">127</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Teeth-clenching</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">33</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Jaw</td><td rowspan=\"1\" colspan=\"1\">Forearm</td><td rowspan=\"1\" colspan=\"1\">↑PPT jaw</td><td rowspan=\"1\" colspan=\"1\">2019</td><td rowspan=\"1\" colspan=\"1\">Lanefelt et al.<sup><xref rid=\"R106\" ref-type=\"bibr\">106</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Trunk flexion</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">70</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Abdomen</td><td rowspan=\"1\" colspan=\"1\">Nailbed</td><td rowspan=\"1\" colspan=\"1\">↑PPT Abdomen</td><td rowspan=\"1\" colspan=\"1\">2019</td><td rowspan=\"1\" colspan=\"1\">Deering et al.<sup><xref rid=\"R27\" ref-type=\"bibr\">27</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">Wall squat</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/>35</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\">↑PPT thigh<break/>↑PPT shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/>2019</td><td rowspan=\"1\" colspan=\"1\">Vaegter et al.<sup><xref rid=\"R200\" ref-type=\"bibr\">200</xref></sup></td></tr></tbody></table><table-wrap-foot><fn fn-type=\"other\"><p>The table is organized according to exercise type, exercise form, pain test modality, and year of publication.</p></fn><fn fn-type=\"other\"><p>CPI, cold pain intensity; CPT, cold pain threshold; EPI, electrical pain intensity; EPT, electrical pain threshold; EPTol, electrical pain tolerance; HIIT, high-intensity interval training; HPI, heat pain intensity; HPT, heat pain threshold; HRmax, maximum heart rate; HRR, heart rate reserve; MICT, moderate-intensity continuous training; MVC, maximal voluntary contraction; NFR, nociceptive flexion reflex; PPI, pressure pain intensity; PPT, pressure pain threshold; PPTol, pressure pain tolerance; RM, repetition maximum; RPE, rating of perceived exertion; TSPc, temporal summation of cold pain; TSPh, temporal summation of heat pain; TSPp, temporal summation of pressure pain; VO<sub>2</sub>max, maximal aerobic capacity.</p></fn></table-wrap-foot></table-wrap><sec id=\"s2-1\"><title>2.1. Exercise intensity and duration</title><p>The hypoalgesic responses seem to be similar between exercise types,<sup><xref rid=\"R133\" ref-type=\"bibr\">133</xref>,<xref rid=\"R195\" ref-type=\"bibr\">195</xref></sup> although EIH differences have been observed,<sup><xref rid=\"R32\" ref-type=\"bibr\">32</xref></sup> but exercise intensity and duration quite consistently affect the EIH response. Exercise intensity affects the EIH response after aerobic exercise.<sup><xref rid=\"R69\" ref-type=\"bibr\">69</xref>,<xref rid=\"R124\" ref-type=\"bibr\">124</xref>,<xref rid=\"R132\" ref-type=\"bibr\">132</xref>,<xref rid=\"R195\" ref-type=\"bibr\">195</xref></sup> For example, in 80 pain-free individuals, it was observed that a moderate-to-high intensity bicycling exercise produced significantly larger EIH responses at the exercising quadriceps muscle, as well as at the nonexercising biceps and trapezius muscles, compared with a low-intensity bicycling exercise.<sup><xref rid=\"R195\" ref-type=\"bibr\">195</xref></sup> Findings on the influence of aerobic exercise duration are more equivocal, with one study observing a dose-response with larger effects after bicycling for 30 minutes compared with 10 minutes,<sup><xref rid=\"R69\" ref-type=\"bibr\">69</xref></sup> and one study observing no difference between bicycling for 10 minutes compared with 20 minutes.<sup><xref rid=\"R195\" ref-type=\"bibr\">195</xref></sup> Moreover, the fact that very short-duration aerobic exercise can elicit EIH<sup><xref rid=\"R129\" ref-type=\"bibr\">129</xref>,<xref rid=\"R162\" ref-type=\"bibr\">162</xref></sup> implies that intensity, or the combination of intensity and duration, may be more important for determining the size of EIH after aerobic exercise than either variable alone.</p><p>Exercise intensity and duration may also affect the EIH response after isometric exercises,<sup><xref rid=\"R64\" ref-type=\"bibr\">64</xref>,<xref rid=\"R127\" ref-type=\"bibr\">127</xref>,<xref rid=\"R157\" ref-type=\"bibr\">157</xref></sup> although the results are more inconsistent. In 40 individuals, pressure pain thresholds at the hand were increased and pressure pain intensity was decreased after low-intensity (25% of maximal voluntary contraction [MVC]) isometric elbow flexion until exhaustion. However, no hypoalgesia was observed when the contraction was held for only 2 minutes.<sup><xref rid=\"R64\" ref-type=\"bibr\">64</xref></sup> By contrast, hypoalgesia was found after 90 and 180 seconds isometric knee extensions and elbow flexion exercises at 30% MVC and 60%, respectively, in 80 healthy individuals; however, the hypoalgesic responses were not different in magnitude between low-intensity and high-intensity contractions nor between shorter or longer durations.<sup><xref rid=\"R195\" ref-type=\"bibr\">195</xref></sup> The fact that very low doses of isometric exercise (eg, three maximal contractions of 5-second duration, totaling 15 seconds of exercise) can produce EIH<sup><xref rid=\"R64\" ref-type=\"bibr\">64</xref></sup> lends further support to the lack of clear dose-response, which is further evidenced by a study of 50 individuals where elevations in pain threshold were not different between isometric handgrip exercises at 25% MVC for 1, 3, or 5 minutes.<sup><xref rid=\"R189\" ref-type=\"bibr\">189</xref></sup></p></sec><sec id=\"s2-2\"><title>2.2. Effects on pain modulatory mechanisms</title><p>As described, robust increases in pressure pain thresholds are observed after exercise, but exercise can also affect spinal and supraspinal mechanisms of pain. Temporal summation of pressure and heat pain was reduced after submaximal isometric exercises at 25% to 40% of MVC for 3 minutes,<sup><xref rid=\"R94\" ref-type=\"bibr\">94</xref>,<xref rid=\"R96\" ref-type=\"bibr\">96</xref>,<xref rid=\"R131\" ref-type=\"bibr\">131</xref>,<xref rid=\"R196\" ref-type=\"bibr\">196</xref></sup> and 20 minutes of aerobic exercise at 55% to 70% of heart rate reserve reduced temporal summation of heat pain<sup><xref rid=\"R132\" ref-type=\"bibr\">132</xref></sup>; however, temporal summation of pressure pain was not affected by 15 to 20 minutes of aerobic exercise at 50% to 75% of VO2max.<sup><xref rid=\"R196\" ref-type=\"bibr\">196</xref></sup> However, not all studies have shown exercise to have positive effects on pain mechanisms. For example, Alsouhibani et al. observed a decrease in the CPM response after exercise.<sup><xref rid=\"R2\" ref-type=\"bibr\">2</xref></sup> By contrast, other studies have found exercise to have no effect on CPM<sup><xref rid=\"R122\" ref-type=\"bibr\">122</xref></sup> or offset analgesia,<sup><xref rid=\"R61\" ref-type=\"bibr\">61</xref></sup> suggesting that exercise can, but does not always, influence spinal and supraspinal mechanisms of pain. Exercise can also influence the ability to cope with pain. The perceived pain intensity of a suprathreshold stimulus is consistently reduced by aerobic, isometric, and dynamic resistance exercises,<sup><xref rid=\"R42\" ref-type=\"bibr\">42</xref>,<xref rid=\"R64\" ref-type=\"bibr\">64</xref>,<xref rid=\"R98\" ref-type=\"bibr\">98</xref></sup> and acute exercise can reduce ratings of pain unpleasantness even in the absence of a change in pain intensity.<sup><xref rid=\"R80\" ref-type=\"bibr\">80</xref></sup> In addition, low-intensity nonpainful aerobic and isometric exercises also increase the tolerance to a painful stimulus. A 20% increase in pain tolerance was observed by Vaegter et al.<sup><xref rid=\"R199\" ref-type=\"bibr\">199</xref></sup> after a 3-minute submaximal isometric knee extension exercise, and after a 6-minute walking exercise<sup><xref rid=\"R73\" ref-type=\"bibr\">73</xref></sup> compared with rest in 35 pain-free individuals.</p></sec><sec id=\"s2-3\"><title>2.3. Factors influencing exercise-induced hypoalgesia</title><p>Exercise that produces acute hypoalgesia is often perceived as moderately painful with peak pain intensity ratings around 5 or 6 on a 0 to 10 numerical rating scale,<sup><xref rid=\"R193\" ref-type=\"bibr\">193</xref>,<xref rid=\"R200\" ref-type=\"bibr\">200</xref></sup> and painful exercises seem to have larger hypoalgesic effects than nonpainful exercises, at least in pain-free individuals,<sup><xref rid=\"R36\" ref-type=\"bibr\">36</xref></sup> but perhaps not in individuals with chronic pain.<sup><xref rid=\"R20\" ref-type=\"bibr\">20</xref>,<xref rid=\"R173\" ref-type=\"bibr\">173</xref></sup></p><p>Treatment expectations are a well-recognized factor known to modulate treatment outcomes and the information about the effect of exercise given to individuals before exercise influences the magnitude of the EIH response. First, a randomized controlled trial by Jones et al.<sup><xref rid=\"R81\" ref-type=\"bibr\">81</xref></sup> observed that the hypoalgesic effect after bicycling was slightly increased if positive information about EIH was given before the exercise compared to when no EIH information was given before exercise. Second, a randomized controlled trial by Vaegter et al. comparing positive vs negative pre-exercise information observed a 22% increase in pain thresholds in the positive information group, whereas the negative information group had a 4% decrease (hyperalgesia) in pain threshold at the exercising muscle (Vaegter et al., in review). Both studies observed a positive correlation between expectations and hypoalgesia after exercise.</p><p>Despite robust hypoalgesia after exercise on a group level, the response to exercise is not identical across individuals and across days. Several studies have investigated the stability of the EIH response in pain-free individuals across different days using a number of aerobic<sup><xref rid=\"R54\" ref-type=\"bibr\">54</xref>,<xref rid=\"R73\" ref-type=\"bibr\">73</xref>,<xref rid=\"R192\" ref-type=\"bibr\">192</xref>,<xref rid=\"R193\" ref-type=\"bibr\">193</xref></sup> and isometric<sup><xref rid=\"R200\" ref-type=\"bibr\">200</xref></sup> exercise protocols. Across protocols, some individuals consistently show hypoalgesia after exercise, some individuals consistently showed hyperalgesia after exercise, and some individuals had a change in their response from hypoalgesic to hyperalgesic or vice versa between days. Interestingly, the majority of individuals showed hypoalgesia at some point.</p></sec><sec id=\"s2-4\"><title>2.4. Regular exercise and pain</title><p>The effect of regular exercise and physical activity on pain sensitivity has been investigated, albeit less than the effect of a single session of exercise. In pain-free individuals, there have been relatively few studies investigating whether those who are more physically active experience greater EIH. The results of these studies show that EIH is usually similar between inactive and active pain-free individuals irrespective of the type of exercise they regularly perform (ie, aerobic or strength training) and the methods used to assess physical activity (ie, self-report or objectively measured using accelerometry).<sup><xref rid=\"R12\" ref-type=\"bibr\">12</xref>,<xref rid=\"R188\" ref-type=\"bibr\">188</xref>,<xref rid=\"R198\" ref-type=\"bibr\">198</xref></sup> However, Ellingson et al.<sup><xref rid=\"R35\" ref-type=\"bibr\">35</xref></sup> observed lower pain intensity ratings and lower pain unpleasantness ratings to suprathreshold heat pain stimulations in pain-free women who were physically active as defined by the current public health recommendations compared with women who were less physically active than recommended. There is also some evidence that individuals who are more physically fit experience greater EIH.<sup><xref rid=\"R138\" ref-type=\"bibr\">138</xref>,<xref rid=\"R166\" ref-type=\"bibr\">166</xref></sup></p><p>Regarding the effect of a longer period of exercise training in pain-free individuals, Hakansson et al.<sup><xref rid=\"R58\" ref-type=\"bibr\">58</xref></sup> observed changes in PPT in the legs after 6 weeks of moderate bicycling exercises (3 times/week) but not after high-intensity interval exercise. In addition, Jones et al.<sup><xref rid=\"R76\" ref-type=\"bibr\">76</xref></sup> observed increases in pressure pain tolerance but not pain threshold after bicycling 30 minutes at 75% of VO<sub>2</sub> max 3 times/week for 6 weeks compared with a control condition. These findings suggest that regular exercise in pain-free individuals specifically influences the ability to cope with pain (ie, pain perception above the pain threshold) rather than the level at which pain is first perceived (pain threshold). Similar observations have been found in athletes compared with less active individuals. A systematic review with meta-analysis by Tesarz et al.<sup><xref rid=\"R182\" ref-type=\"bibr\">182</xref></sup> showed consistently higher pain tolerance across different pain modalities (ie, pressure, heat, cold, electrical, and ischemic) in athletes; however, for pain thresholds, the conclusion was less consistent.</p><p>In addition to the effect on pain tolerance, regular exercise may also affect the ability to inhibit pain as assessed by the CPM paradigm. Naugle et al. observed that pain-free individuals reporting more regular physical activity also had a larger CPM response compared with individuals reporting less regular physical activity.<sup><xref rid=\"R134\" ref-type=\"bibr\">134</xref>,<xref rid=\"R135\" ref-type=\"bibr\">135</xref></sup> Although previous investigations on CPM in athletes have been equivocal because increased CPM<sup><xref rid=\"R52\" ref-type=\"bibr\">52</xref></sup> as well as decreased CPM<sup><xref rid=\"R181\" ref-type=\"bibr\">181</xref></sup> has been observed, the positive effect of regular exercise on CPM may be a potential mechanism underlying the preventive effect of exercise on pain because better CPM capacity has been associated with a reduced risk of chronic pain.<sup><xref rid=\"R211\" ref-type=\"bibr\">211</xref></sup> The preventive effect of regular exercise is supported by a recent systematic review with meta-analysis concluding that regular exercise performed 2 to 3 times/week reduces the risk of low back pain by 33%.<sup><xref rid=\"R169\" ref-type=\"bibr\">169</xref></sup> This is true even in those who are at an increased risk of developing chronic pain.<sup><xref rid=\"R115\" ref-type=\"bibr\">115</xref></sup></p></sec></sec><sec id=\"s3\"><title>3. Pain outcomes after acute and regular exercise in individuals with chronic pain</title><p>In individuals with different chronic pain conditions, the response to a single session of exercise is less consistent as hypoalgesia, reduced hypoalgesia, or even hyperalgesia (ie, increased sensitivity to pain) has been observed. As illustrated in Table <xref rid=\"T2\" ref-type=\"table\">2</xref>, hypoalgesia after exercise has, eg, been observed in individuals with chronic musculoskeletal pain,<sup><xref rid=\"R123\" ref-type=\"bibr\">123</xref>,<xref rid=\"R197\" ref-type=\"bibr\">197</xref></sup> shoulder pain,<sup><xref rid=\"R105\" ref-type=\"bibr\">105</xref></sup> patella femoral pain,<sup><xref rid=\"R180\" ref-type=\"bibr\">180</xref></sup> knee osteoarthritis,<sup><xref rid=\"R59\" ref-type=\"bibr\">59</xref>,<xref rid=\"R194\" ref-type=\"bibr\">194</xref></sup> menstrual pain,<sup><xref rid=\"R186\" ref-type=\"bibr\">186</xref></sup> and rheumatoid arthritis.<sup><xref rid=\"R117\" ref-type=\"bibr\">117</xref></sup> However, reduced EIH responses or even hyperalgesia after exercise has often been demonstrated in individuals with whiplash-associated disorder,<sup><xref rid=\"R203\" ref-type=\"bibr\">203</xref></sup> ME/CFS,<sup><xref rid=\"R123\" ref-type=\"bibr\">123</xref>,<xref rid=\"R202\" ref-type=\"bibr\">202</xref></sup> fibromyalgia pain,<sup><xref rid=\"R100\" ref-type=\"bibr\">100</xref>,<xref rid=\"R107\" ref-type=\"bibr\">107</xref>,<xref rid=\"R177\" ref-type=\"bibr\">177</xref></sup> painful diabetic neuropathy,<sup><xref rid=\"R90\" ref-type=\"bibr\">90</xref></sup> chronic musculoskeletal pain,<sup><xref rid=\"R19\" ref-type=\"bibr\">19</xref></sup> and also in a delayed-onset muscular soreness pain model.<sup><xref rid=\"R25\" ref-type=\"bibr\">25</xref></sup> Hyperalgesia after exercise is often observed in individuals with more widespread chronic pain conditions. This was first observed by Kosek et al.<sup><xref rid=\"R100\" ref-type=\"bibr\">100</xref></sup> in 5 individuals with fibromyalgia who showed a decrease in pain thresholds during and after an isometric knee extension exercise. The observation of hypoalgesia after exercise in some groups with chronic pain conditions and the observation of hyperalgesia after exercise in other groups with chronic pain may be influenced by whether the exercise is performed using a painful or nonpainful body area. Lannersten and Kosek<sup><xref rid=\"R107\" ref-type=\"bibr\">107</xref></sup> observed hypoalgesia after a 5-minute submaximal (25% of MVC) isometric exercise in individuals with shoulder myalgia when the exercise was performed by a nonpainful leg muscle but when the exercise was performed by the painful shoulder muscle, no hypoalgesic response was observed. Similarly, Burrows et al.<sup><xref rid=\"R17\" ref-type=\"bibr\">17</xref></sup> observed increases in pressure pain threshold after upper-body but not lower-body resistance exercise in people with knee osteoarthritis. These findings suggest that hypoalgesia can be induced by exercising nonpainful muscles in subjects with chronic pain,<sup><xref rid=\"R191\" ref-type=\"bibr\">191</xref></sup> which may have important implications for exercise prescription in the clinical setting.</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Summary of studies investigating acute exercise-induced hypoalgesia in individuals with different pain conditions.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\">Exercise type</th><th rowspan=\"1\" colspan=\"1\">Exercise form</th><th rowspan=\"1\" colspan=\"1\">Intensity</th><th rowspan=\"1\" colspan=\"1\">Duration</th><th rowspan=\"1\" colspan=\"1\"># of participants</th><th rowspan=\"1\" colspan=\"1\">Pain condition</th><th rowspan=\"1\" colspan=\"1\">Pain test modality</th><th rowspan=\"1\" colspan=\"1\">Pain outcome</th><th rowspan=\"1\" colspan=\"1\">Local site</th><th rowspan=\"1\" colspan=\"1\">Remote site</th><th rowspan=\"1\" colspan=\"1\">Findings</th><th rowspan=\"1\" colspan=\"1\">Year</th><th rowspan=\"1\" colspan=\"1\">Author</th></tr></thead><tbody><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">Increasing to 75% HRmax</td><td rowspan=\"1\" colspan=\"1\">Unknown</td><td rowspan=\"1\" colspan=\"1\">20</td><td rowspan=\"1\" colspan=\"1\">ME/CFS</td><td rowspan=\"1\" colspan=\"1\">Clinical</td><td rowspan=\"1\" colspan=\"1\">Pain intensity</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\">2017</td><td rowspan=\"1\" colspan=\"1\">Oosterwijck et al.<sup><xref rid=\"R141\" ref-type=\"bibr\">141</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">VO<sub>2</sub>max test</td><td rowspan=\"1\" colspan=\"1\">8–12 min</td><td rowspan=\"1\" colspan=\"1\">25</td><td rowspan=\"1\" colspan=\"1\">Chronic pain</td><td rowspan=\"1\" colspan=\"1\">Cold</td><td rowspan=\"1\" colspan=\"1\">CPI</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Arm</td><td rowspan=\"1\" colspan=\"1\">No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\">2018</td><td rowspan=\"1\" colspan=\"1\">Chretien et al.<sup><xref rid=\"R18\" ref-type=\"bibr\">18</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>1 KPa</td><td rowspan=\"1\" colspan=\"1\"><break/>5 min</td><td rowspan=\"1\" colspan=\"1\"><break/>12</td><td rowspan=\"1\" colspan=\"1\">Chronic<break/>Low back pain</td><td rowspan=\"1\" colspan=\"1\"><break/>Heat</td><td rowspan=\"1\" colspan=\"1\">HPI<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\">Forearm<break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\"><break/>↓ TSPh forearm</td><td rowspan=\"1\" colspan=\"1\"><break/>2009</td><td rowspan=\"1\" colspan=\"1\"><break/>Bialosky et al.<sup><xref rid=\"R9\" ref-type=\"bibr\">9</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">80% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\">30 min</td><td rowspan=\"1\" colspan=\"1\">23</td><td rowspan=\"1\" colspan=\"1\">DOMS MODEL</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Arm</td><td rowspan=\"1\" colspan=\"1\">No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\">2002</td><td rowspan=\"1\" colspan=\"1\">Dannecker et al.<sup><xref rid=\"R25\" ref-type=\"bibr\">25</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">70% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\">20 min</td><td rowspan=\"1\" colspan=\"1\">8</td><td rowspan=\"1\" colspan=\"1\">Chronic low back pain</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPI</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Hand</td><td rowspan=\"1\" colspan=\"1\">↓PPI</td><td rowspan=\"1\" colspan=\"1\">2005</td><td rowspan=\"1\" colspan=\"1\">Hoffman et al.<sup><xref rid=\"R68\" ref-type=\"bibr\">68</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Increasing to 130 W</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>37 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>26</td><td rowspan=\"1\" colspan=\"1\"><break/>Chronic fatigue syndrome</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\">Hand<break/>Lower back<break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>↓PPTs (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2010</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Meeus et al.<sup><xref rid=\"R123\" ref-type=\"bibr\">123</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Increasing to 130 W</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>37 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>21</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Chronic low back pain</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\">Hand<break/>Lower back<break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>↑PPTs</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2010</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Meeus et al.<sup><xref rid=\"R123\" ref-type=\"bibr\">123</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/>1. 75% HRmax<break/>2. Self-paced</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>Unknown</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>22</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/>hronic fatigue syndrome</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/>Hand<break/>Lower back</td><td rowspan=\"1\" colspan=\"1\">↑PPT lower back (after self-paced)<break/>↓PPTs calf/hand (after self-paced) (hyperalgesia)<break/>↓PPTs (after 75% HRmax) (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>2010</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/>Van Oosterwijck et al.<sup><xref rid=\"R202\" ref-type=\"bibr\">202</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\">1. Increasing to 75% HRmax<break/>2. Self-paced</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>1. Unknown<break/>2. Individual</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>20</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>ME/CFS</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand<break/>Lower back</td><td rowspan=\"1\" colspan=\"1\">No hypoalgesia/some hyperalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2010</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Van Oosterwijck et al.<sup><xref rid=\"R202\" ref-type=\"bibr\">202</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>1. 62% HRmax<break/>2. Self-paced</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>20 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>21</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Fibromyalgia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT<break/>PPI<break/>PPTol</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↑PPT and PPTol (both conditions)<break/>↓PPI (both conditions)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>2011</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Newcomb et al.<sup><xref rid=\"R136\" ref-type=\"bibr\">136</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/>1. 75% HRmax<break/>2. Self-paced</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>Unknown</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>22</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>WAD</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/>Hand<break/>Lower back</td><td rowspan=\"1\" colspan=\"1\">↑PPT lower back (after self-paced)<break/>↓PPTs calf/hand (after self-paced) (hyperalgesia)<break/>↓PPTs (after 75% HRmax) (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/><break/>2012</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/><break/><break/><break/><break/>Van Oosterwijck et al.<sup><xref rid=\"R203\" ref-type=\"bibr\">203</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">Increasing to 75% HRmax</td><td rowspan=\"1\" colspan=\"1\">Maximum of 15 min</td><td rowspan=\"1\" colspan=\"1\">19</td><td rowspan=\"1\" colspan=\"1\">Fibromyalgia with chronic fatigue</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">TSPp</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Shoulder<break/>Hand</td><td rowspan=\"1\" colspan=\"1\">No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\">2015</td><td rowspan=\"1\" colspan=\"1\">Meeus et al.<sup><xref rid=\"R122\" ref-type=\"bibr\">122</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Aerobic</td><td rowspan=\"1\" colspan=\"1\">Bicycling</td><td rowspan=\"1\" colspan=\"1\">Increasing to 75% HRmax</td><td rowspan=\"1\" colspan=\"1\">Maximum of 15 min</td><td rowspan=\"1\" colspan=\"1\">16</td><td rowspan=\"1\" colspan=\"1\">RA</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">TSPp</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Shoulder<break/>Hand</td><td rowspan=\"1\" colspan=\"1\">No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\">2015</td><td rowspan=\"1\" colspan=\"1\">Meeus et al.<sup><xref rid=\"R122\" ref-type=\"bibr\">122</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>75% of VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>15 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>61</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Chronic MSK pain</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT<break/>PTTol<break/>TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Arm<break/>Shoulder<break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\">↑PPTs<break/>↑PPTol<break/>↑TSPp (in high pain sensitive patients)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>2016</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Vaegter et al.<sup><xref rid=\"R197\" ref-type=\"bibr\">197</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic<break/><break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling<break/><break/><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\">1. 70% HRmax<break/><break/><break/><break/><break/>2. 75%–85% HRmax<break/>75% of VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\">1. Continuous 20 min<break/><break/><break/><break/><break/>2. Interval 5 × 4 min 15 min</td><td rowspan=\"1\" colspan=\"1\"><break/>15<break/><break/><break/><break/><break/><break/>14</td><td rowspan=\"1\" colspan=\"1\">Chronic fatigue syndrome<break/><break/><break/><break/><break/>Knee OA</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT<break/><break/><break/><break/><break/>PTTol</td><td rowspan=\"1\" colspan=\"1\">Thigh<break/><break/><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\">Shoulder<break/>Hand<break/><break/><break/><break/>Arm<break/>Shoulder<break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\">↑PPT thigh after interval<break/><break/><break/><break/><break/>↑PPTs</td><td rowspan=\"1\" colspan=\"1\">2016<break/><break/><break/><break/><break/>2017</td><td rowspan=\"1\" colspan=\"1\">Sandler et al.<sup><xref rid=\"R164\" ref-type=\"bibr\">164</xref></sup><break/><break/><break/><break/><break/>Vaegter et al.<sup><xref rid=\"R194\" ref-type=\"bibr\">194</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\">75% of HRmax</td><td rowspan=\"1\" colspan=\"1\"><break/>30 min</td><td rowspan=\"1\" colspan=\"1\"><break/>21</td><td rowspan=\"1\" colspan=\"1\"><break/>WAD</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\">Neck<break/>Shin</td><td rowspan=\"1\" colspan=\"1\"><break/>No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/>Smith et al.<sup><xref rid=\"R172\" ref-type=\"bibr\">172</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic<break/><break/><break/><break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling<break/><break/><break/><break/><break/><break/><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>Increasing to 75% HRmax<break/><break/><break/><break/><break/><break/>50 W</td><td rowspan=\"1\" colspan=\"1\"><break/>Unknown<break/><break/><break/><break/><break/><break/><break/>12 min</td><td rowspan=\"1\" colspan=\"1\"><break/>40<break/><break/><break/><break/><break/><break/><break/>20</td><td rowspan=\"1\" colspan=\"1\"><break/>Knee OA<break/><break/><break/><break/><break/>Chronic fatigue syndrome</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/><break/><break/><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT<break/><break/><break/><break/><break/><break/><break/>TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/>Thigh<break/>Knee<break/><break/><break/><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Forearm<break/><break/><break/><break/><break/><break/><break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPTs (if normal CPM)<break/><break/>↓PPTs (if abnormal CPM)<break/>No hyperalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/>2017<break/><break/><break/><break/><break/><break/><break/>2018</td><td rowspan=\"1\" colspan=\"1\"><break/>Fingleton et al.<sup><xref rid=\"R40\" ref-type=\"bibr\">40</xref></sup><break/><break/><break/><break/><break/><break/><break/>Malfliet et al.<sup><xref rid=\"R118\" ref-type=\"bibr\">118</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Bicycling</td><td rowspan=\"1\" colspan=\"1\"><break/>70% VO<sub>2</sub>max</td><td rowspan=\"1\" colspan=\"1\"><break/>30 min</td><td rowspan=\"1\" colspan=\"1\"><break/>27</td><td rowspan=\"1\" colspan=\"1\"><break/>Gulf veterans</td><td rowspan=\"1\" colspan=\"1\">Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\">PPT<break/>HPI</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↑HPI (if pain) (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/>2010</td><td rowspan=\"1\" colspan=\"1\"><break/>Cook et al.<sup><xref rid=\"R19\" ref-type=\"bibr\">19</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/>Running</td><td rowspan=\"1\" colspan=\"1\"><break/>Bruce test</td><td rowspan=\"1\" colspan=\"1\"><break/>Fatigue</td><td rowspan=\"1\" colspan=\"1\"><break/>10</td><td rowspan=\"1\" colspan=\"1\"><break/>Fibromyalgia</td><td rowspan=\"1\" colspan=\"1\"><break/>Heat</td><td rowspan=\"1\" colspan=\"1\"><break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Hands</td><td rowspan=\"1\" colspan=\"1\">↑TSPh (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/>2001</td><td rowspan=\"1\" colspan=\"1\"><break/>Vierck et al.<sup><xref rid=\"R206\" ref-type=\"bibr\">206</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Running</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>5 km/hour</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>3 × 5min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>5</td><td rowspan=\"1\" colspan=\"1\">Chronic fatigue syndrome</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hands</td><td rowspan=\"1\" colspan=\"1\"><break/>↓PPTs (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2004</td><td rowspan=\"1\" colspan=\"1\"><break/>Whiteside et al.<sup><xref rid=\"R208\" ref-type=\"bibr\">208</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Aerobic<break/><break/><break/><break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Walking<break/><break/><break/><break/><break/><break/><break/>Walking</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Self-selected<break/><break/><break/>1. Continuous<break/>1.3 m/second<break/>2. Interval 1.3 m/second</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>4 min<break/><break/>1. 45 min<break/>2. 3 × 15 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>20<break/><break/><break/><break/><break/><break/><break/>27</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Plantar fasciopathy<break/><break/><break/><break/><break/><break/><break/><break/>Knee OA</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Clinical pain PPT<break/><break/><break/><break/><break/><break/>Clinical pain</td><td rowspan=\"1\" colspan=\"1\">Pain intensity during test<break/><break/><break/><break/>PPT<break/>Pain intensity</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Heel<break/><break/><break/><break/><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>—<break/><break/><break/><break/><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>No hypoalgesia<break/><break/><break/>↑Pain intensity continuous walking (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>2018<break/><break/><break/><break/><break/><break/><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Riel et al.<sup><xref rid=\"R156\" ref-type=\"bibr\">156</xref></sup><break/><break/><break/><break/><break/><break/><break/><break/>Farrokhi et al.<sup><xref rid=\"R39\" ref-type=\"bibr\">39</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Aerobic</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Stepping</td><td rowspan=\"1\" colspan=\"1\">50% of maximum number of steps in 1 minute</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>5 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>30</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>TMD</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPI<break/>TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>↓TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>2019</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Nasri-Heir et al.<sup><xref rid=\"R129\" ref-type=\"bibr\">129</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\">Leg exercises</td><td rowspan=\"1\" colspan=\"1\">1. 60% 1RM<break/>2. Self-selected</td><td rowspan=\"1\" colspan=\"1\">2 exercises 6 × 10 repetitions</td><td rowspan=\"1\" colspan=\"1\"><break/>32</td><td rowspan=\"1\" colspan=\"1\"><break/>Fibromyalgia</td><td rowspan=\"1\" colspan=\"1\"><break/>Clinical</td><td rowspan=\"1\" colspan=\"1\">Pain intensity</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/>Hyperalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/>2018</td><td rowspan=\"1\" colspan=\"1\">da Cunha Ribeiro et al.<sup><xref rid=\"R24\" ref-type=\"bibr\">24</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Dynamic resistance<break/><break/><break/><break/>Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Knee extensions<break/><break/><break/><break/>Knee extensions</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>1RM<break/><break/><break/><break/><break/>8RM</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>6 × 10 repetitions<break/><break/><break/><break/>1 exercise 3 × 8 repetitions</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>20<break/><break/><break/><break/><break/>21</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Knee OA<break/><break/><break/><break/>Patellar tendinopathy</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Clinical<break/><break/><break/><break/>Clinical pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>Pain intensity DOMS<break/><break/>Pain intensity during SLS PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Knee<break/><break/><break/><break/>Knee shin</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>—<break/><break/><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\">No change in pain intensity<break/>More DOMS than controls<break/><break/><break/>↓Pain intensity ↑PPT shin</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2013<break/><break/><break/><break/><break/>2019</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Germanou et al.<sup><xref rid=\"R51\" ref-type=\"bibr\">51</xref></sup><break/><break/><break/><break/><break/>Holden et al.<sup><xref rid=\"R70\" ref-type=\"bibr\">70</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\">Arm-raises</td><td rowspan=\"1\" colspan=\"1\">Fast</td><td rowspan=\"1\" colspan=\"1\">6 min</td><td rowspan=\"1\" colspan=\"1\">24</td><td rowspan=\"1\" colspan=\"1\">Knee OA</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">Thigh</td><td rowspan=\"1\" colspan=\"1\">↑PPT shoulder</td><td rowspan=\"1\" colspan=\"1\">2020</td><td rowspan=\"1\" colspan=\"1\">Hansen et al.<sup><xref rid=\"R59\" ref-type=\"bibr\">59</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\"><break/>1. Hip abductions<break/>2. Knee extensions</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Load = 12RM</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>3 exercises 12 repetitions</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>30</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>PFP</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT<break/>PTTol<break/>TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Knee<break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Elbow (PPT)</td><td rowspan=\"1\" colspan=\"1\">↑PPT (lower leg)<break/>↑PPTol (after knee exercises)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>2019</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Straszek et al.<sup><xref rid=\"R180\" ref-type=\"bibr\">180</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Lower-body circuit</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>60% 1RM</td><td rowspan=\"1\" colspan=\"1\"><break/>3 exercises 10 repetitions</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>11</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Knee OA</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT<break/>PPTol</td><td rowspan=\"1\" colspan=\"1\"><break/>Thigh<break/>Knee<break/>Shin</td><td rowspan=\"1\" colspan=\"1\">Shoulder<break/>Arm<break/>Forearm<break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2014</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Burrows et al.<sup><xref rid=\"R17\" ref-type=\"bibr\">17</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Dynamic Resistance</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Upper-body circuit</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>60% 1RM</td><td rowspan=\"1\" colspan=\"1\"><break/>3 exercises<break/>10 repetitions</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>11</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Knee OA</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT<break/>PPTol</td><td rowspan=\"1\" colspan=\"1\">Shoulder<break/>Arm<break/>Forearm<break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>Thigh<break/>Knee<break/>Shin</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>↑PPTs (across sites)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2014</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Burrows et al.<sup><xref rid=\"R17\" ref-type=\"bibr\">17</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Dynamic resistance<break/><break/>Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\">Back extensions<break/><break/>Repeated back movements</td><td rowspan=\"1\" colspan=\"1\"><break/>Bodyweight<break/><break/><break/><break/><break/><break/><break/>Lifting 5 kg</td><td rowspan=\"1\" colspan=\"1\">3 × 15 repetitions<break/><break/><break/><break/><break/><break/><break/>7 min</td><td rowspan=\"1\" colspan=\"1\"><break/>12<break/><break/><break/><break/><break/><break/><break/>18</td><td rowspan=\"1\" colspan=\"1\">Chronic<break/>low back pain<break/><break/><break/><break/><break/>Chronic<break/>low back pain</td><td rowspan=\"1\" colspan=\"1\"><break/>Heat<break/><break/><break/><break/>Pressure<break/>Heat<break/>Cold</td><td rowspan=\"1\" colspan=\"1\">HPI<break/>TSPh<break/><break/><break/>PPT<break/>HPT<break/>CPT<break/>TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/>—<break/><break/><break/><break/><break/><break/><break/>Back</td><td rowspan=\"1\" colspan=\"1\">Forearm<break/>Lower <break/><break/><break/><break/><break/><break/>Leg<break/>Hand</td><td rowspan=\"1\" colspan=\"1\">↓TSPb forearm<break/><break/><break/><break/><break/><break/><break/>↑CPT hand</td><td rowspan=\"1\" colspan=\"1\"><break/>2009<break/><break/><break/><break/><break/><break/><break/>2019</td><td rowspan=\"1\" colspan=\"1\"><break/>Bialosky et al.<sup><xref rid=\"R9\" ref-type=\"bibr\">9</xref></sup><break/><break/><break/><break/><break/><break/><break/>Kuithan et al.<sup><xref rid=\"R104\" ref-type=\"bibr\">104</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Dynamic resistance</td><td rowspan=\"1\" colspan=\"1\"><break/>Cervical<break/>flexion</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Head weight</td><td rowspan=\"1\" colspan=\"1\"><break/>10 × 10 seconds</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>13</td><td rowspan=\"1\" colspan=\"1\"><break/>Chronic neck pain</td><td rowspan=\"1\" colspan=\"1\">Clinical pain<break/>Pressure</td><td rowspan=\"1\" colspan=\"1\">Pain intensity<break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Neck</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\">↓Pain intensity<break/>↑PPTs</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2018</td><td rowspan=\"1\" colspan=\"1\">Galindez-Ibarbengoetxea et al.<sup><xref rid=\"R47\" ref-type=\"bibr\">47</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/>Elbow flexion</td><td rowspan=\"1\" colspan=\"1\">1. 25% MVC<break/>2. 25% MVC<break/>3. 100% MVC</td><td rowspan=\"1\" colspan=\"1\">1. 2 min<break/>2. Fatigue<break/>3. 3 reps</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>15</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Fibromyalgia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT<break/>PPI</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Hand</td><td rowspan=\"1\" colspan=\"1\"><break/>No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2011</td><td rowspan=\"1\" colspan=\"1\">Hoeger Bement et al.<sup><xref rid=\"R66\" ref-type=\"bibr\">66</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>18</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Diabetic neuropathy</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Heat</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>HPI<break/>TSPh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Hand<break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>—</td><td rowspan=\"1\" colspan=\"1\">↓HPI and TSPh (if no pain)<break/>No changes (if pain)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>2014</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Knauf and Koltyn<sup><xref rid=\"R90\" ref-type=\"bibr\">90</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/>25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/>64</td><td rowspan=\"1\" colspan=\"1\">Menstrual pain</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\">Forearm<break/>Shin</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPTs</td><td rowspan=\"1\" colspan=\"1\"><break/>2018</td><td rowspan=\"1\" colspan=\"1\"><break/>Travers et al.<sup><xref rid=\"R186\" ref-type=\"bibr\">186</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Handgrip</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>30% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>90 seconds</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>12</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Fibromyalgia</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/>Heat</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT<break/>HPI</td><td rowspan=\"1\" colspan=\"1\"><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\">↓PPTs<break/>↑HPI (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>2005</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Staud et al.<sup><xref rid=\"R177\" ref-type=\"bibr\">177</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Knee extension</td><td rowspan=\"1\" colspan=\"1\">20%–25% MVC</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">14</td><td rowspan=\"1\" colspan=\"1\">Fibromyalgia</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Thigh</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">↓PPT (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\">1996</td><td rowspan=\"1\" colspan=\"1\">Kosek et al.<sup><xref rid=\"R100\" ref-type=\"bibr\">100</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Knee extension</td><td rowspan=\"1\" colspan=\"1\">10%–15% MVC</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">17</td><td rowspan=\"1\" colspan=\"1\">Fibromyalgia</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Thigh</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">↑PPT (shoulder)</td><td rowspan=\"1\" colspan=\"1\">2007</td><td rowspan=\"1\" colspan=\"1\">Kadetoff and Kosek<sup><xref rid=\"R82\" ref-type=\"bibr\">82</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Knee extension</td><td rowspan=\"1\" colspan=\"1\">50% MVC</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">66</td><td rowspan=\"1\" colspan=\"1\">Knee OA</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Thigh</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">↑PPTs</td><td rowspan=\"1\" colspan=\"1\">2013</td><td rowspan=\"1\" colspan=\"1\">Kosek et al.<sup><xref rid=\"R102\" ref-type=\"bibr\">102</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Knee extension</td><td rowspan=\"1\" colspan=\"1\">50% MVC</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">47</td><td rowspan=\"1\" colspan=\"1\">Hip OA</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Thigh</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">↑PPTs</td><td rowspan=\"1\" colspan=\"1\">2013</td><td rowspan=\"1\" colspan=\"1\">Kosek et al.<sup><xref rid=\"R102\" ref-type=\"bibr\">102</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Knee<break/>extension</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>30% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>90 seconds</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>61</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Chronic<break/>MSK pain</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT<break/>PTTol<break/>TSPp</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\">Arm<break/>Shoulder<break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>↑PPTs<break/>↑PPTol</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2016</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Vaegter et al.<sup><xref rid=\"R197\" ref-type=\"bibr\">197</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Knee extension</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>30% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>90 seconds</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>14</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Knee OA</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>PPT<break/>PTTol</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\">Arm<break/>Shoulder<break/>Lower leg</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>↑PPTs</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Vaegter et al.<sup><xref rid=\"R194\" ref-type=\"bibr\">194</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Knee extension</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>10% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>5 min</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>40</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Knee OA</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Thigh<break/>Knee</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\">↑PPTs (if normal CPM)<break/>↓PPTs (if abnormal CPM)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Fingleton et al.<sup><xref rid=\"R40\" ref-type=\"bibr\">40</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric<break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">Knee extension<break/><break/>Knee extension</td><td rowspan=\"1\" colspan=\"1\"><break/>30% MVC<break/><break/><break/>30% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/>Fatigue<break/><break/><break/>5 min</td><td rowspan=\"1\" colspan=\"1\"><break/>130<break/><break/><break/>46</td><td rowspan=\"1\" colspan=\"1\"><break/>Fibromyalgia<break/><break/><break/>RA</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure<break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT<break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>—<break/><break/><break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>Shoulder<break/><break/><break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPT<break/><break/><break/>↑PPTs</td><td rowspan=\"1\" colspan=\"1\"><break/>2017<break/><break/><break/>2018</td><td rowspan=\"1\" colspan=\"1\"><break/>Tour et al.<sup><xref rid=\"R185\" ref-type=\"bibr\">185</xref></sup><break/><break/><break/>Lofgren et al.<sup><xref rid=\"R117\" ref-type=\"bibr\">117</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Knee extension</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>70% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>5 × 45 seconds</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>21</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Patellar tendinopathy</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure clinical</td><td rowspan=\"1\" colspan=\"1\">Pain intensity during SLS<break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Knee<break/>Shin</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Forearm</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>↓Pain intensity<break/>↑PPT shin</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>2019</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/><break/>Holden et al.<sup><xref rid=\"R70\" ref-type=\"bibr\">70</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">1. Knee extension<break/>2. Shoulder rotation</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>20%–25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Fatigue</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>20</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Shoulder pain</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Thigh<break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Shoulder<break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPTs (during knee extension)</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2010</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Lannersten and Kosek<sup><xref rid=\"R107\" ref-type=\"bibr\">107</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\">1. Knee extension<break/>2. Shoulder rotation</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>20%–25% MVC</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Fatigue</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>20</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Fibromyalgia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Thigh<break/>Shoulder</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Shoulder<break/>Thigh</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>No hypoalgesia</td><td rowspan=\"1\" colspan=\"1\"><break/><break/><break/>2010</td><td rowspan=\"1\" colspan=\"1\"><break/><break/>Lannersten and Kosek<sup><xref rid=\"R107\" ref-type=\"bibr\">107</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Shoulder abduction</td><td rowspan=\"1\" colspan=\"1\">1 kg</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">19</td><td rowspan=\"1\" colspan=\"1\">Chronic shoulder pain</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">↑PPT</td><td rowspan=\"1\" colspan=\"1\">2003</td><td rowspan=\"1\" colspan=\"1\">Persson et al.<sup><xref rid=\"R148\" ref-type=\"bibr\">148</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Shoulder abduction</td><td rowspan=\"1\" colspan=\"1\">Weight of arms</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">22</td><td rowspan=\"1\" colspan=\"1\">Fibromyalgia</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">Shin</td><td rowspan=\"1\" colspan=\"1\">↓PPT shin (hyperalgesia)</td><td rowspan=\"1\" colspan=\"1\">2012</td><td rowspan=\"1\" colspan=\"1\">Ge et al.<sup><xref rid=\"R48\" ref-type=\"bibr\">48</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Shoulder abduction</td><td rowspan=\"1\" colspan=\"1\">20%–25% MVC</td><td rowspan=\"1\" colspan=\"1\">5 min</td><td rowspan=\"1\" colspan=\"1\">24</td><td rowspan=\"1\" colspan=\"1\">Shoulder pain</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Shoulder</td><td rowspan=\"1\" colspan=\"1\">Thigh<break/>Shin</td><td rowspan=\"1\" colspan=\"1\">↑PPTs</td><td rowspan=\"1\" colspan=\"1\">2016</td><td rowspan=\"1\" colspan=\"1\">Kuppens et al.<sup><xref rid=\"R105\" ref-type=\"bibr\">105</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Squat</td><td rowspan=\"1\" colspan=\"1\">70% MVC</td><td rowspan=\"1\" colspan=\"1\">1 exercise 5 × 45 sec repetitions</td><td rowspan=\"1\" colspan=\"1\">6</td><td rowspan=\"1\" colspan=\"1\">Patella tendinopathy</td><td rowspan=\"1\" colspan=\"1\">Clinical</td><td rowspan=\"1\" colspan=\"1\">Pain intensity during SLS</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">↓Pain intensity</td><td rowspan=\"1\" colspan=\"1\">2015</td><td rowspan=\"1\" colspan=\"1\">Rio et al.<sup><xref rid=\"R158\" ref-type=\"bibr\">158</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\">Isometric</td><td rowspan=\"1\" colspan=\"1\">Tooth clenching</td><td rowspan=\"1\" colspan=\"1\">—</td><td rowspan=\"1\" colspan=\"1\">Fatigue</td><td rowspan=\"1\" colspan=\"1\">20</td><td rowspan=\"1\" colspan=\"1\">TMD</td><td rowspan=\"1\" colspan=\"1\">Pressure</td><td rowspan=\"1\" colspan=\"1\">PPT</td><td rowspan=\"1\" colspan=\"1\">Jaw</td><td rowspan=\"1\" colspan=\"1\">Forearm</td><td rowspan=\"1\" colspan=\"1\">↑PPT jaw</td><td rowspan=\"1\" colspan=\"1\">2019</td><td rowspan=\"1\" colspan=\"1\">Lanefelt et al.<sup><xref rid=\"R106\" ref-type=\"bibr\">106</xref></sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"><break/>Isometric</td><td rowspan=\"1\" colspan=\"1\"><break/>Wall squat</td><td rowspan=\"1\" colspan=\"1\"><break/>Bodyweight</td><td rowspan=\"1\" colspan=\"1\"><break/>3 min</td><td rowspan=\"1\" colspan=\"1\"><break/>21</td><td rowspan=\"1\" colspan=\"1\"><break/>WAD</td><td rowspan=\"1\" colspan=\"1\"><break/>Pressure</td><td rowspan=\"1\" colspan=\"1\"><break/>PPT</td><td rowspan=\"1\" colspan=\"1\"><break/>—</td><td rowspan=\"1\" colspan=\"1\">Neck<break/>Shin</td><td rowspan=\"1\" colspan=\"1\"><break/>↑PPTs</td><td rowspan=\"1\" colspan=\"1\"><break/>2017</td><td rowspan=\"1\" colspan=\"1\"><break/>Smith et al.<sup><xref rid=\"R172\" ref-type=\"bibr\">172</xref></sup></td></tr></tbody></table><table-wrap-foot><fn fn-type=\"other\"><p>The table is organized according to exercise type, exercise form, pain test modality, and year of publication.</p></fn><fn fn-type=\"other\"><p>DOMS, delayed-onset muscle soreness; HPI, heat pain intensity; HPT, heat pain threshold; HRmax, maximum heart rate; MVC, maximal voluntary contraction; PPI, pressure pain intensity; PPT, pressure pain threshold; PPTol, pressure pain tolerance; RM, repetition maximum; RPE, rating of perceived exertion; SLS, single-leg stand; TSPh, temporal summation of heat pain; TSPp, temporal summation of pressure pain; VO<sub>2</sub>max, maximal aerobic capacity.</p></fn></table-wrap-foot></table-wrap><sec id=\"s3-1\"><title>3.1. Factors related to lack of exercise-induced hypoalgesia</title><p>Individuals with facilitated central pain mechanisms, which are commonly observed in several chronic musculoskeletal pain conditions,<sup><xref rid=\"R121\" ref-type=\"bibr\">121</xref></sup> often report reduced hypoalgesia after exercise. Vaegter et al.<sup><xref rid=\"R197\" ref-type=\"bibr\">197</xref></sup> observed reduced EIH after submaximal isometric exercise and after bicycling exercise in chronic pain patients with high widespread pain sensitivity compared with patients with low pain sensitivity. In addition, in high pain-sensitive patients, an increase in temporal summation of pain was observed after aerobic exercise<sup><xref rid=\"R177\" ref-type=\"bibr\">177</xref>,<xref rid=\"R197\" ref-type=\"bibr\">197</xref></sup> possibly mimicking the pain flare-up after exercise reported in clinical practice by some individuals with widespread chronic pain.<sup><xref rid=\"R24\" ref-type=\"bibr\">24</xref></sup> Also, Fingleton et al.<sup><xref rid=\"R40\" ref-type=\"bibr\">40</xref></sup> observed reduced pressure pain thresholds (hyperalgesia) after both aerobic and isometric exercises in individuals with knee osteoarthritis who demonstrated an impaired CPM response. By contrast, pain thresholds increased in knee osteoarthritis individuals with a normal CPM response suggesting that patients with impaired CPM, which is also a common finding in individuals with chronic pain,<sup><xref rid=\"R114\" ref-type=\"bibr\">114</xref>,<xref rid=\"R121\" ref-type=\"bibr\">121</xref></sup> may have less acute hypoalgesic effect from exercise.</p><p>Another possible explanation for the lack of hypoalgesia after exercise often observed in individuals with chronic pain is that the exercise dose–response relationship is different in individuals with chronic pain compared with pain-free subjects. Newcomb et al.<sup><xref rid=\"R136\" ref-type=\"bibr\">136</xref></sup> observed a larger EIH response in individuals with fibromyalgia after 20 minutes of aerobic exercise at a preferred intensity (45% of maximal heart rate) compared with a prescribed and higher-intensity aerobic exercise (60%–75% of maximal heart rate). Similarly, Coombes et al.<sup><xref rid=\"R20\" ref-type=\"bibr\">20</xref></sup> showed that isometric exercise above but not below an individual's pain threshold increased pain responses to exercise in people with lateral epicondylalgia. These results could indicate that lower-intensity exercise creates less input to facilitated central pain mechanisms resulting in a net balance of pain inhibition and a reduction in the pain sensitivity after exercise. This may be different for chronic exercise, however, where a small benefit of painful over nonpainful exercise has been observed, albeit for clinical pain at baseline as opposed to experimental pain in the immediate post-exercise period.<sup><xref rid=\"R173\" ref-type=\"bibr\">173</xref></sup> Other possible explanations for reduced EIH include use of opioids and negative expectations about the effect of exercise. Interactions between EIH mechanisms and the use of analgesics may affect the response to exercise. Individuals treated with opioids report less CPM,<sup><xref rid=\"R155\" ref-type=\"bibr\">155</xref></sup> and reduced effects of opioids have been reported in animals after long-term exercise.<sup><xref rid=\"R174\" ref-type=\"bibr\">174</xref></sup> As observed in pain-free individuals, negative expectations are associated with the hypoalgesic response after exercise. Interestingly, most patients with chronic pain referred to multidisciplinary pain treatment do not expect exercises to cause less pain; on the contrary, the majority expects more pain after exercise (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1.</label><caption><p>Expectations about the effects of low-intensity exercise, moderate-intensity exercise, and vigorous-intensity exercise on pain reported by patients (n = 500) referred for interdisciplinary pain treatment at a University Hospital Pain Center in Denmark (unpublished data from the clinical pain registry, PainData).</p></caption><graphic xlink:href=\"painreports-5-e823-g001\"/></fig></sec><sec id=\"s3-2\"><title>3.2. Regular exercise and pain</title><p>Regular exercise is guideline recommended treatment for a wide range of chronic pain conditions.<sup><xref rid=\"R49\" ref-type=\"bibr\">49</xref>,<xref rid=\"R146\" ref-type=\"bibr\">146</xref></sup> Regular exercise is safe and generally well accepted by individuals with mild to moderate chronic pain; however, the effects on pain and pain sensitivity are somewhat conflicting, and the level of evidence for a positive effect is generally low.<sup><xref rid=\"R49\" ref-type=\"bibr\">49</xref></sup> Clinically relevant reductions in pain and pain sensitivity are often observed after 8 to 12 weeks of exercise therapy in individuals with knee or hip osteoarthritis,<sup><xref rid=\"R170\" ref-type=\"bibr\">170</xref></sup> but randomized controlled trials often observe smaller effects with pain reductions of less than 10 on a 100-point numerical rating scale<sup><xref rid=\"R62\" ref-type=\"bibr\">62</xref></sup> or even no change in pain after exercise therapy compared with passive sham therapy.<sup><xref rid=\"R8\" ref-type=\"bibr\">8</xref></sup></p><p>To the best of our knowledge, only 2 studies have investigated whether habitual physical activity levels predict pain responses to acute exercise in individuals with chronic pain. Coriolano et al.<sup><xref rid=\"R21\" ref-type=\"bibr\">21</xref></sup> found that people with knee osteoarthritis who self-reported more physical activity experienced less exacerbation in pain after completing performance-based tests and a physiological test (submaximal arm ergometer test). In people with fibromyalgia, Umeda et al.<sup><xref rid=\"R187\" ref-type=\"bibr\">187</xref></sup> showed that participants who were more physically active reported a smaller increase in ratings of muscle pain intensity during isometric handgrip exercise. Taken together, these results suggest that being more physically active is associated with reduced pain responses to acute exercise in individuals with chronic pain. These results are consistent with cross-sectional data showing negative associations between fitness and pain (ie, more fitness, less pain) in people with fibromyalgia<sup><xref rid=\"R77\" ref-type=\"bibr\">77</xref></sup> and knee osteoarthritis (Jones et al., in review) as well as longitudinal data showing benefit of longer periods of regular exercise training on reducing pain in individuals with chronic pain.<sup><xref rid=\"R49\" ref-type=\"bibr\">49</xref></sup></p></sec></sec><sec id=\"s4\"><title>4. Underlying mechanisms of exercise-induced hypoalgesia in humans</title><p>There are numerous biological and cognitive factors that contribute to pain, so changes in any one or more of these by acute exercise could account for EIH. It is not clear, however, what these mechanisms are or whether the mechanisms are similar or distinct between healthy individuals and individuals with chronic pain. The contrasting magnitude of EIH between pain-free individuals and individuals with chronic pain<sup><xref rid=\"R130\" ref-type=\"bibr\">130</xref></sup> suggests that the mechanisms of EIH are disrupted in individuals with chronic pain. That is, some aspect of chronic pain (eg, inflammation, sensitization, and fear of movement) interferes with the normal hypoalgesic effect of acute exercise. These potential mechanisms will be described in more detail hereafter.</p><sec id=\"s4-1\"><title>4.1. Opioid and cannabinoid systems</title><p>The most commonly proposed mechanism of EIH is enhanced descending inhibition by activation of the opioid and cannabinoid systems. The contraction of skeletal muscle increases the discharge of mechanosensitive afferents (ie, A-delta and C-fibres) which, in turn, activates central descending opioid pain pathways.<sup><xref rid=\"R29\" ref-type=\"bibr\">29</xref>,<xref rid=\"R184\" ref-type=\"bibr\">184</xref></sup> Exercise also increases the release of endogenous cannabinoids. These opioid and cannabinoid pathways have receptors throughout the peripheral and central nervous systems that can produce analgesia when stimulated.<sup><xref rid=\"R29\" ref-type=\"bibr\">29</xref>,<xref rid=\"R184\" ref-type=\"bibr\">184</xref></sup></p><p>Human studies investigating the role of opioids and cannabinoids in EIH have yielded equivocal findings. For example, opioid antagonists such as naloxone and naltrexone have been shown to increase, decrease, or have no effect on EIH.<sup><xref rid=\"R30\" ref-type=\"bibr\">30</xref>,<xref rid=\"R31\" ref-type=\"bibr\">31</xref>,<xref rid=\"R74\" ref-type=\"bibr\">74</xref>,<xref rid=\"R94\" ref-type=\"bibr\">94</xref>,<xref rid=\"R140\" ref-type=\"bibr\">140</xref></sup> Moreover, correlations between EIH and exercise-induced changes in plasma concentrations of beta-endorphins and endocannabinoids are not always observed.<sup><xref rid=\"R94\" ref-type=\"bibr\">94</xref>,<xref rid=\"R139\" ref-type=\"bibr\">139</xref>,<xref rid=\"R165\" ref-type=\"bibr\">165</xref></sup> A limitation of these human investigations is that they are more constrained than rodent studies in their ability to investigate whether opioids and cannabinoids are acting through peripheral and/or central actions to influence pain after exercise; however, there is some evidence that blocking blood flow to a limb during exercise attenuates EIH in pain-free individuals, suggesting that peripheral factors are important.<sup><xref rid=\"R79\" ref-type=\"bibr\">79</xref></sup></p></sec><sec id=\"s4-2\"><title>4.2. Stress-induced hypoalgesia</title><p>Exercise-induced hypoalgesia might also be a form of stress-induced analgesia, related to the release of various stress hormones during exercise. However, evidence to support this in humans is mixed. For example, EIH is related to increases in growth hormone during exercise,<sup><xref rid=\"R149\" ref-type=\"bibr\">149</xref></sup> but another study found that the suppression of exercise-induced growth hormone release by cyproheptadine had no effect on EIH.<sup><xref rid=\"R86\" ref-type=\"bibr\">86</xref></sup> Dexamethasone, a steroid medication, has been shown to attenuate EIH by reducing secretion of adrenocorticotropin<sup><xref rid=\"R88\" ref-type=\"bibr\">88</xref></sup>; however, other studies have found no effect of dexamethasone on pain in healthy individuals.<sup><xref rid=\"R209\" ref-type=\"bibr\">209</xref></sup> A small pilot study of 7 healthy individuals showed that exercise-induced changes in neuropeptide Y, allopregnanolone, pregnenolone, and dehydroepiandrosterone were related to EIH.<sup><xref rid=\"R167\" ref-type=\"bibr\">167</xref></sup> However, because concentrations of these substances were only measured in the plasma, it is not clear whether they were acting through peripheral or central mechanisms to influence pain. Moreover, because this was only a small pilot study, more studies are needed to confirm the findings.</p></sec><sec id=\"s4-3\"><title>4.3. Cardiovascular systems</title><p>Exercise-induced changes in the cardiovascular system have also been proposed as a mechanism of EIH. That is, elevations in blood pressure by exercise are thought to attenuate pain through baroreceptor-related mechanisms (ie, the activation of arterial baroreceptors by exercise subsequently activates pain-related brain areas involved in pain modulation). Although it is true that people with high blood pressure are less sensitive to pain (ie, hypertension-associated hypoalgesia),<sup><xref rid=\"R161\" ref-type=\"bibr\">161</xref></sup> there is currently little evidence that acute changes in blood pressure by exercise are related to EIH.<sup><xref rid=\"R28\" ref-type=\"bibr\">28</xref>,<xref rid=\"R157\" ref-type=\"bibr\">157</xref>,<xref rid=\"R189\" ref-type=\"bibr\">189</xref>,<xref rid=\"R190\" ref-type=\"bibr\">190</xref></sup> Moreover, acute increases in blood pressure by exercise could not account for the persistence of EIH after exercise (eg, 15 minutes after exercise cessation<sup><xref rid=\"R210\" ref-type=\"bibr\">210</xref></sup> because blood pressure would have presumably returned to baseline, or indeed be lower, by this time).</p></sec><sec id=\"s4-4\"><title>4.4. Central pain modulatory systems</title><p>The influence of exercise on reducing the sensitivity of the central nervous system has also been explored as a mechanism of EIH. These studies show that acute exercise can reduce temporal summation<sup><xref rid=\"R96\" ref-type=\"bibr\">96</xref>,<xref rid=\"R131\" ref-type=\"bibr\">131</xref>,<xref rid=\"R196\" ref-type=\"bibr\">196</xref>,<xref rid=\"R206\" ref-type=\"bibr\">206</xref></sup> and increase thresholds to elicit the nociceptive withdrawal reflex,<sup><xref rid=\"R55\" ref-type=\"bibr\">55</xref></sup> although there is some evidence contrary to the latter observation.<sup><xref rid=\"R125\" ref-type=\"bibr\">125</xref></sup> These results imply that exercise can reduce pain through reductions in central nervous system sensitivity at spinal and supraspinal levels, but exactly where in the nociceptive pathway these changes occur is not known. Improved efficacy of descending inhibitory pathways by exercise has been studied as a mechanism of EIH as well, but there is little direct evidence to support this. For example, Alsouhibani et al.<sup><xref rid=\"R2\" ref-type=\"bibr\">2</xref></sup> observed a decrease in the CPM response after exercise, Meeus et al. found no effect of aerobic exercise on CPM in healthy individuals,<sup><xref rid=\"R122\" ref-type=\"bibr\">122</xref></sup> and Ellingson et al.<sup><xref rid=\"R36\" ref-type=\"bibr\">36</xref></sup> showed that EIH was comparable for nonpainful and painful exercise, although the latter should have evoked a larger “pain inhibits pain” effect. A few studies have found small positive correlations between conditioned pain modulation and EIH<sup><xref rid=\"R13\" ref-type=\"bibr\">13</xref>,<xref rid=\"R112\" ref-type=\"bibr\">112</xref>,<xref rid=\"R198\" ref-type=\"bibr\">198</xref></sup> suggesting that the 2 may share similar mechanisms; however, EIH is usually somewhat smaller in magnitude but more enduring than conditioned pain modulation so the 2 are likely distinct.<sup><xref rid=\"R112\" ref-type=\"bibr\">112</xref>,<xref rid=\"R195\" ref-type=\"bibr\">195</xref></sup></p></sec><sec id=\"s4-5\"><title>4.5. Psychological contributing factors</title><p>Changes in pain cognition might also account for some of the effect of acute exercise on pain. It has been shown that exercise can reduce ratings of pain unpleasantness in the absence of a change in ratings of pain intensity,<sup><xref rid=\"R80\" ref-type=\"bibr\">80</xref></sup> suggesting that alterations in the appraisal of noxious stimuli contribute to EIH. Cognitive and psychosocial factors including pain self-efficacy, coping strategies, fear of pain, and stress are known to underlie some of the difference in pain between athletes and nonathletes,<sup><xref rid=\"R53\" ref-type=\"bibr\">53</xref>,<xref rid=\"R75\" ref-type=\"bibr\">75</xref>,<xref rid=\"R142\" ref-type=\"bibr\">142</xref></sup> but their relation to EIH is less clear. For example, several studies have shown that individuals with higher levels of catastrophizing experience less EIH,<sup><xref rid=\"R16\" ref-type=\"bibr\">16</xref>,<xref rid=\"R131\" ref-type=\"bibr\">131</xref>,<xref rid=\"R207\" ref-type=\"bibr\">207</xref></sup> although this is not always observed and correlations between EIH and other psychosocial factors (eg, fear of pain, pain attitudes, and anxiety) seem negligible.<sup><xref rid=\"R112\" ref-type=\"bibr\">112</xref>,<xref rid=\"R201\" ref-type=\"bibr\">201</xref></sup> Therefore, the contribution of cognitive factors to EIH remains poorly understood but seems limited. More studies are needed to investigate whether these cognitive factors are related to EIH and, more importantly, whether they can be manipulated to augment it.<sup><xref rid=\"R81\" ref-type=\"bibr\">81</xref></sup></p></sec><sec id=\"s4-6\"><title>4.6. Impaired EIH: disrupted or distinct mechanisms</title><p>The mechanisms of EIH in individuals with chronic pain are equally if not more unclear. Because exercise has such varying effects on pain within and between individuals with chronic pain, it is difficult to determine whether there is a consistent mechanism that contributes to changes in pain with acute exercise. Moreover, it is not clear if the mechanisms of EIH in individuals with chronic pain are the same as pain-free individuals and are just disrupted, or whether separate mechanisms related to the presence of chronic pain are involved as well.</p><p>The fact that EIH can occur at exercised and remote sites in individuals with chronic pain shows that EIH is not always disrupted in these individuals.<sup><xref rid=\"R38\" ref-type=\"bibr\">38</xref>,<xref rid=\"R136\" ref-type=\"bibr\">136</xref>,<xref rid=\"R197\" ref-type=\"bibr\">197</xref></sup> However, there are also several demonstrations that exercise with a painful joint or muscle can either diminish EIH compared to when a nonpainful body part is exercised (ie, exercise of the upper limb in people with knee osteoarthritis, but pain measurement in the lower limb)<sup><xref rid=\"R17\" ref-type=\"bibr\">17</xref></sup> or, worse, can increase pain.<sup><xref rid=\"R19\" ref-type=\"bibr\">19</xref>,<xref rid=\"R20\" ref-type=\"bibr\">20</xref>,<xref rid=\"R107\" ref-type=\"bibr\">107</xref>,<xref rid=\"R177\" ref-type=\"bibr\">177</xref></sup> These results are both opposite to what is normally seen in pain-free individuals where EIH is usually greatest for the exercised body part. Therefore, the results of the above studies provide some evidence that compared to healthy individuals, the mechanisms of EIH in individuals with chronic pain are both similar and distinct. However, because the mechanisms of EIH are still poorly understood in both groups, there is little direct evidence to support this.</p><p>Regarding mechanisms of EIH that may be similar, but disrupted, in individuals with chronic pain compared to pain-free individuals, altered excitability of the central nervous system after exercise is perhaps the most obvious. In pain-free individuals, acute exercise reliably reduces temporal summation,<sup><xref rid=\"R96\" ref-type=\"bibr\">96</xref>,<xref rid=\"R196\" ref-type=\"bibr\">196</xref>,<xref rid=\"R206\" ref-type=\"bibr\">206</xref></sup> whereas the opposite effect has been observed in individuals with chronic pain.<sup><xref rid=\"R197\" ref-type=\"bibr\">197</xref>,<xref rid=\"R206\" ref-type=\"bibr\">206</xref></sup> By contrast, one of the few studies to combine acute exercise with analgesic medication showed that paracetamol and placebo had comparable effects on temporal summation and conditioned pain modulation after exercise in pain-free individuals and individuals with chronic pain.<sup><xref rid=\"R122\" ref-type=\"bibr\">122</xref></sup> Because paracetamol is a predominantly central acting agent that can affect opioids, cannabinoid and serotonergic pathways,<sup><xref rid=\"R168\" ref-type=\"bibr\">168</xref></sup> this finding provides little support to the notion that exercise reduces pain through central changes in these pathways or that differences in the sensitivity of these pathways through exercise accounts for the greater EIH in pain-free individuals compared to individuals with chronic pain. More studies using drugs with less ubiquitous effects would be useful to further investigate how different substances are involved in EIH in humans and whether these differ between pain-free individuals and individuals with chronic pain.</p><p>As for mechanisms of EIH that might be distinct between pain-free individuals and individuals with chronic pain, reductions in inflammation by acute exercise are one such possibility. Inflammation plays a key role in the pathogenesis of several chronic pain states, so it is possible that reductions in inflammation by exercise may reduce pain in these individuals. However, the results of studies examining the effect of acute exercise on inflammation in individuals with chronic pain are mixed and the relation between the changes in inflammatory markers and pain has seldom been explored. Moreover, differences in the exercise-induced changes in inflammatory markers between individuals with chronic pain and pain-free individuals were only sometimes, but not always, observed. Therefore, it remains unclear to what extent EIH is related to acute changes in inflammation by exercise in individuals with chronic pain or whether this is a distinct mechanism of EIH in these populations. Another possibility is opioid-induced hyperalgesia. As already mentioned, interactions between EIH mechanisms and the use of analgesics may affect the response to exercise. Individuals treated with opioids report less CPM,<sup><xref rid=\"R155\" ref-type=\"bibr\">155</xref></sup> and reduced effects of opioids have been reported in animals after long-term exercise.<sup><xref rid=\"R174\" ref-type=\"bibr\">174</xref></sup> This may be explained by opioid-induced hyperalgesia which, paradoxically, leads to a reduction in central opioid receptor availability<sup><xref rid=\"R60\" ref-type=\"bibr\">60</xref></sup> and hence less potential to modulate pain through opioidergic mechanisms (as shown in pain-free individuals.<sup><xref rid=\"R152\" ref-type=\"bibr\">152</xref></sup></p><p>Psychosocial and cognitive factors are heavily implicated in the development and persistence of chronic pain.<sup><xref rid=\"R34\" ref-type=\"bibr\">34</xref></sup> These same cognitive factors influence responses to experimental noxious stimuli in pain-free individuals as well,<sup><xref rid=\"R150\" ref-type=\"bibr\">150</xref></sup> but their relation to EIH has seldom been examined, particularly in individuals with chronic pain. Accordingly, it is still not known whether cognitive factors are directly involved in EIH, or, perhaps more importantly, whether they can be manipulated to influence pain responses to exercise. Although there is some evidence to support this in pain-free individuals,<sup><xref rid=\"R81\" ref-type=\"bibr\">81</xref></sup> it remains to be determined whether preceding exercise with education can also influence EIH in individuals with chronic pain in whom negative expectations about pain and exercise are more prevalent and therefore likely harder to change. It may be that, because of their more entrenched negative beliefs about pain and exercise, more intensive education is required in individuals with chronic pain to produce the same effect. Some combination of pain neuroscience education and EIH education might also be required. Nonetheless, if the effect can be replicated in individuals with chronic pain, it could have important applications for exercise prescription in clinical practice.</p><p>Regarding regular exercise, despite the large number of studies that have shown exercise training to reduce pain in people with chronic pain,<sup><xref rid=\"R49\" ref-type=\"bibr\">49</xref></sup> the mechanisms by which it does this is poorly understood. This is largely because many of the studies did not analyze which changes occurring with exercise (biological and/or psychological changes) were associated with the observed improvements in pain. Moreover, few of the studies investigated where in the nociceptive pathways (ie, peripheral, spinal, and/or supraspinal pathways) changes might be occurring due to exercise, which could account for the observed reductions in pain. As a result, the precise mechanisms of pain attenuation by exercise training are not known, but several possibilities exist that are likely common to individuals with chronic pain.</p><p>Improved structure and function of the musculoskeletal system is one such possibility. In people with knee osteoarthritis, chronic exercise can improve several musculoskeletal factors important in the development and progression of the disease including body mass, joint alignment, proprioception, cartilage structure and function, inflammation, and muscle strength.<sup><xref rid=\"R6\" ref-type=\"bibr\">6</xref>,<xref rid=\"R160\" ref-type=\"bibr\">160</xref></sup> Of these possible mediators, improvements in muscle strength are the strongest contributor to the positive effect of physical exercise on improved osteoarthritis symptoms.<sup><xref rid=\"R160\" ref-type=\"bibr\">160</xref></sup></p><p>Desensitization of the nervous system is another possibility. In humans, exercise-induced changes in biomarkers associated with nociceptive pathways have been reported (eg, inflammatory factors and neurotransmitters),<sup><xref rid=\"R83\" ref-type=\"bibr\">83</xref></sup> but again it is not clear whether these changes reduce pain due to the peripheral or central actions of these factors. Preliminary evidence shows that exercise can normalise aberrant brain activity in people with fibromyalgia.<sup><xref rid=\"R41\" ref-type=\"bibr\">41</xref></sup> This finding is in agreement with the results of a few cross-sectional studies showing that people with fibromyalgia who are more physically active have more typical brain responses to pain compared to less active individuals.<sup><xref rid=\"R37\" ref-type=\"bibr\">37</xref>,<xref rid=\"R120\" ref-type=\"bibr\">120</xref></sup> However, not all studies have shown chronic exercise to attenuate aberrant brain responses in individuals with chronic pain,<sup><xref rid=\"R126\" ref-type=\"bibr\">126</xref></sup> so the role of changes in brain activity as a mechanism of pain relief by regular exercise remains unclear.</p><p>Finally, exercise-induced improvements in mood could be another shared mediator of the positive effect of exercise on pain in individuals with chronic pain. The role of both general (eg, depression and anxiety) and pain-specific (eg, catastrophizing and self-efficacy) psychosocial processes in the development and maintenance of chronic pain is clear.<sup><xref rid=\"R34\" ref-type=\"bibr\">34</xref></sup> Many of these psychosocial factors are positively influenced by exercise,<sup><xref rid=\"R85\" ref-type=\"bibr\">85</xref>,<xref rid=\"R183\" ref-type=\"bibr\">183</xref></sup> so it is plausible that this could result in improvements in pain either directly or indirectly through changes in both the sensory and emotional aspects of pain.</p></sec></sec><sec id=\"s5\"><title>5. Implications and future perspectives</title><sec id=\"s5-1\"><title>5.1. Clinical implications</title><p>Most types of exercise can reduce pain sensitivity at exercising and nonexercising muscles in pain-free individuals, with a larger hypoalgesic response at the exercising muscles. In individuals with chronic pain, the hypoalgesic response after exercise is less consistent; however, in addition to other well-documented physical and mental health benefits related to exercise, exercise can sometimes induce hypoalgesia in individuals with chronic pain. Regarding exercise prescription in clinical settings, it may be worth considering: (1) that exercising nonpainful body areas if possible as well as using low-intensity exercises such as walking may be useful as a first step, (2) that individuals' beliefs, expectations, and exercise preference should be assessed before exercise prescription to minimize the risk of a poor outcome, and (3) that these beliefs and expectations could be modified through education or other interventions to improve pain responses to exercise in people with chronic pain. There is some evidence that combining exercise training and education has superior effects compared to exercise alone in individuals with chronic pain,<sup><xref rid=\"R15\" ref-type=\"bibr\">15</xref>,<xref rid=\"R153\" ref-type=\"bibr\">153</xref></sup> but this is yet to be properly explored in the context of pain responses to a single bout of acute exercise in individuals with chronic pain.</p></sec><sec id=\"s5-2\"><title>5.2. Implications for future exercise-induced hypoalgesia studies</title><p>In addition to the above-mentioned implications, we also propose several methodological recommendations for future studies of EIH. First, studies should use a randomized controlled design (parallel or crossover), or at the very least include a control group/condition. This is because the causal effects of exercise on pain are best inferred from randomized controlled trials. As shown in Tables <xref rid=\"T1\" ref-type=\"table\">1</xref> and <xref rid=\"T2\" ref-type=\"table\">2</xref>, there have been well over 150 studies of EIH in pain-free individuals and individuals with chronic pain. However, the minority of these used a randomized controlled design or a nonrandomized controlled design. Instead, EIH was often investigated using a single-arm pre-post design. A major limitation of this type of study design is that the effects of habituation to noxious stimuli, as well as statistical phenomena such as regression to the mean, are not accounted for. To truly determine whether a single bout of exercise causes a reduction in pain, randomized controlled trials are needed. Second, it is important that these randomized controlled trials use large(r) sample sizes. The majority of EIH studies are small (n ≤ 50), and it is well documented that small studies are inherently biased to find larger effects.<sup><xref rid=\"R26\" ref-type=\"bibr\">26</xref></sup> Hence, most studies of EIH probably overestimate the effect of exercise on pain. Consequently, despite the enormous amount of EIH studies to date, the true effect of a single bout of exercise on pain is still unknown. Larger randomized controlled trials, of which there are currently very few, are clearly needed to determine this.</p><p>As evident in Tables <xref rid=\"T1\" ref-type=\"table\">1</xref> and <xref rid=\"T2\" ref-type=\"table\">2</xref>, there is substantial heterogeneity in methodology used in EIH studies, making it difficult to synthesise the results of this vast literature. Therefore, we also recommend that future EIH studies share a somewhat common methodology so that the results between studies can be more easily compared. To this end, it may be useful for future studies to share a common method of pain assessment. Pressure pain thresholds at local and remote sites may be the most appropriate because these have been studied most often and do not require expensive equipment (although they may be more prone to experimenter bias if using handheld algometry). It would also be of benefit to include assessment of both experimental and clinical pain in individuals with chronic pain to better understand the effects of exercise on “real life” pain. Moreover, it may be useful to prescribe and report exercise using a common index so that the amount of work performed can be quantified. This would help clarify the dose–response effect of exercise on pain, a result that may have important clinical implications such as determining the minimal effective dose with respect to hypoalgesia for each mode of exercise as well as identifying volumes and/or intensities of exercise that may be more likely to exacerbate pain in individuals with chronic pain. Finally, Lee et al.<sup><xref rid=\"R109\" ref-type=\"bibr\">109</xref></sup> recently outlined several issues in clinical pain research including transparency, underpowered studies, and researcher degrees of freedom. The use of preregistration and registered reports, data sharing, and greater adherence to reporting guidelines were suggested as areas for improvement and we believe that EIH studies would benefit from adopting these recommendations.</p></sec></sec><sec sec-type=\"COI-statement\"><title>Disclosures</title><p>The authors have no conflicts of interest to declare.</p></sec></body><back><fn-group><fn fn-type=\"COI-statement\"><p>Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>[1]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alghamdi</surname><given-names>KS</given-names></name><name><surname>Al-Sheikh</surname><given-names>MH</given-names></name></person-group>\n<article-title>Effect of stress on pain perception in young women</article-title>. <source>Saudi Med J</source>\n<year>2009</year>;<volume>30</volume>:<fpage>478</fpage>–<lpage>84</lpage>.<pub-id pub-id-type=\"pmid\">19370271</pub-id></mixed-citation></ref><ref id=\"R2\"><label>[2]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alsouhibani</surname><given-names>A</given-names></name><name><surname>Vaegter</surname><given-names>HB</given-names></name><name><surname>Hoeger Bement</surname><given-names>M</given-names></name></person-group>\n<article-title>Systemic exercise-induced hypoalgesia following isometric exercise reduces conditioned pain modulation</article-title>. <source>Pain Med</source>\n<year>2019</year>;<volume>20</volume>:<fpage>180</fpage>–<lpage>90</lpage>.<pub-id pub-id-type=\"pmid\">29618132</pub-id></mixed-citation></ref><ref id=\"R3\"><label>[3]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arroyo-Morales</surname><given-names>M</given-names></name><name><surname>Rodriguez</surname><given-names>LD</given-names></name><name><surname>Rubio-Ruiz</surname><given-names>B</given-names></name><name><surname>Olea</surname><given-names>N</given-names></name></person-group>\n<article-title>Influence of gender in the psychoneuroimmunological response to therapeutic interval exercise</article-title>. <source>Biol Res Nurs</source>\n<year>2012</year>;<volume>14</volume>:<fpage>357</fpage>–<lpage>63</lpage>.<pub-id pub-id-type=\"pmid\">22661642</pub-id></mixed-citation></ref><ref id=\"R4\"><label>[4]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baiamonte</surname><given-names>BA</given-names></name><name><surname>Kraemer</surname><given-names>RR</given-names></name><name><surname>Chabreck</surname><given-names>CN</given-names></name><name><surname>Reynolds</surname><given-names>ML</given-names></name><name><surname>McCaleb</surname><given-names>KM</given-names></name><name><surname>Shaheen</surname><given-names>GL</given-names></name><name><surname>Hollander</surname><given-names>DB</given-names></name></person-group>\n<article-title>Exercise-induced hypoalgesia: pain tolerance, preference and tolerance for exercise intensity, and physiological correlates following dynamic circuit resistance exercise</article-title>. <source>J Sports Sci</source>\n<year>2017</year>;<volume>35</volume>:<fpage>1</fpage>–<lpage>7</lpage>.</mixed-citation></ref><ref id=\"R5\"><label>[5]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bartholomew</surname><given-names>JB</given-names></name><name><surname>Lewis</surname><given-names>BP</given-names></name><name><surname>Linder</surname><given-names>DE</given-names></name><name><surname>Cook</surname><given-names>DB</given-names></name></person-group>\n<article-title>Post-exercise analgesia: replication and extension</article-title>. <source>J Sports Sci</source>\n<year>1996</year>;<volume>14</volume>:<fpage>329</fpage>–<lpage>34</lpage>.<pub-id pub-id-type=\"pmid\">8887212</pub-id></mixed-citation></ref><ref id=\"R6\"><label>[6]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beckwee</surname><given-names>D</given-names></name><name><surname>Vaes</surname><given-names>P</given-names></name><name><surname>Cnudde</surname><given-names>M</given-names></name><name><surname>Swinnen</surname><given-names>E</given-names></name><name><surname>Bautmans</surname><given-names>I</given-names></name></person-group>\n<article-title>Osteoarthritis of the knee: why does exercise work? 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991413</article-id><article-id pub-id-type=\"pmc\">PMC7523783</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-08129</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022212</article-id><article-id pub-id-type=\"art-access-id\">22212</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>5600</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Systematic Review</subject></subj-group></article-categories><title-group><article-title>Auricular acupuncture for premature ovarian insufficiency</article-title><subtitle>A protocol for systematic review and meta-analysis</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Luo</surname><given-names>Yehao</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Donghan</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Tang</surname><given-names>Xiusong</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wei</surname><given-names>Luqiu</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Lizhen</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Pang</surname><given-names>Yuzhou</given-names></name><degrees>Prof</degrees><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0001-9836-4571</contrib-id><name><surname>Fang</surname><given-names>Gang</given-names></name><degrees>Prof</degrees><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Guangxi university of Traditional Chinese Medicine, Nanning, Guangxi Province</aff><aff id=\"aff2\"><label>b</label>Macau University of Science and Technology, Macau</aff><aff id=\"aff3\"><label>c</label>Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan Province</aff><aff id=\"aff4\"><label>d</label>Guangxi Zhuang Yao Medicine Center of Engineering and Technology, Guangxi University of Chinese Medicinee, Nanning, Guangxi Province, China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Gang Fang, Guangxi university of Traditional Chinese Medicine, Nanning 530200, Guangxi Province, PR China (e-mail: <email>fglzyznn@sina.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22212</elocation-id><history><date date-type=\"received\"><day>14</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>18</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22212.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>A lot of attention has been given to premature ovarian insufficiency (POI) as it poses considerable health risks to women. It is characterized by oligomenorrhea, amenorrhea, infertility, autoimmune disorders, and ischemic heart disease, with increased mortality. Previous research indicates that auricular acupuncture is proven effective in treating POI in clinical practice. However, systematic review has not been carried out. Therefore, this study aims at evaluating the curative effect and safety of auricular acupuncture treatment for POI through systematic review and meta-analysis.</p></sec><sec sec-type=\"methods\"><title>Methods and analysis:</title><p>The following databases will be searched for relevant information before August 2020: PubMed, Embase, Cochrane Library, Web of Science, and CNKI. Major results: levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estrogen (E2). Secondary results: modified Kupperman Index, imaging results including ovarian size, antral follicle count, and blood flow changes in the ovary using color Doppler ultrasound; total effective rate, adverse event and intervention, and hospitalization expenses. Data will be collected independently by 2 researchers, and the risk of bias in meta-analysis will be evaluated according to “Cochrane Handbook for Systematic Reviews of Interventions”. All data analysis will be conducted using Review Manager V.5.3. and Stata V.12.0.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>The curative effect and safety of auricular acupuncture treatment for POI patients will be evaluated systematically.</p></sec><sec sec-type=\"conclusion\"><title>Conclusion:</title><p>In the systematic review, the published evidence of auricular acupuncture treatment for POI will be summarized to provide guidance for promotion and application.</p></sec><sec><title>Ethics and dissemination:</title><p>The private information from individuals will not be published. This systematic review also will not involve endangering participant rights. Ethical approval is not required. The results may be published in a peer-reviewed journal or disseminated in relevant conferences.</p><p>Open Science Framework (OSF) registration number: <ext-link ext-link-type=\"uri\" xlink:href=\"http://osf.io/tg9mw\">http://osf.io/tg9mw</ext-link></p></sec></abstract><kwd-group><title>Keywords</title><kwd>auricular acupuncture</kwd><kwd>premature ovarian insufficiency</kwd><kwd>protocol</kwd><kwd>systematic review</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>National Natural Science Foundation of China</funding-source><award-id>No.81660830</award-id><principal-award-recipient>Gang Fang</principal-award-recipient></award-group><award-group id=\"award2\" award-type=\"Fundref\"><funding-source>Guangxi Key Laboratory of Disaster Prevention and Engineering Safety (CN)</funding-source><award-id>No.15-140-32-06</award-id><principal-award-recipient>Gang Fang</principal-award-recipient></award-group><award-group id=\"award3\" award-type=\"Fundref\"><funding-source>Guangxi First-class Discipline Construction of Guangxi University of Chinese Medicine</funding-source><award-id>No.2019XK038</award-id><principal-award-recipient>Gang Fang</principal-award-recipient></award-group><award-group id=\"award4\" award-type=\"Fundref\"><funding-source>Development Program of High-level Talent Team under Qihuang Project of Guangxi University of Chinese Medicine</funding-source><award-id>No.2018005</award-id><principal-award-recipient>Gang Fang</principal-award-recipient></award-group><award-group id=\"award5\" award-type=\"Fundref\"><funding-source>Guangxi first-class discipline construction project</funding-source><award-id>No. Gui Jiao Ke Yan [2018]12</award-id><principal-award-recipient>Gang Fang</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Premature ovarian insufficiency (POI), previously referred to as premature ovarian failure or premature menopause, is a common disease with the global incidence of 1% to 3%. Its most significant clinical manifestations include dysfunction of ovaries, amenorrhea and infertility before age 40.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> Twenty five percent to 30% of POI cases are caused by known hereditary causes, but the cause of 50% to 90% of POF cases is unknown (idiopathic).<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> Currently, there is no therapeutic intervention that is able to effectively recover the fertility of POI patients, and this severely affects the reproduction and life quality of women of childbearing age.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup></p><p>Currently, hormone replacement therapy (HRT) is used as a major approach to treat POI in Western Medicine,<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> and stem cell transplantation is developing.<sup>[<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> A great deal of clinical and research has indicated that it has definite therapeutic effect, high safety and high patient acceptance to treat POI using auricular acupuncture alone or in combination with traditional Chinese medicine or Western medicine, however the mechanism of action is not clear.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> Auricular acupuncture is a traditional Chinese collateral meridian therapy, including auricular embedding therapy, auricular plaster therapy, bloodletting at auricular points and automatic auricular therapeutic device. Clinical research has indicated that auricular acupuncture is a simple, inexpensive, and manageable non-pharmaceutical therapy.<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup> Previous reviews have indicated the result of auricular acupuncture treatment for POI.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>,<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> Compared with HRT, it is a simple and safe therapy for POI that is able to significantly improve symptoms.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> However, there is a lack of evidence of results of auricular acupuncture in treating POI. Therefore, the paper will evaluate the effectiveness and safety of auricular acupuncture treatment for POI. This review will be the first evaluation of the impact of auricular acupuncture.</p></sec><sec><label>2</label><title>Objectives</title><p>In a randomized controlled trial (RCT), the efficacy and side effects of auricular acupuncture in treating POI have been evaluated systematically. We expect to provide reference for POI treatment in the field of traditional Chinese medicine.</p></sec><sec><label>3</label><title>Methods</title><sec><label>3.1</label><title>Study registration</title><p>The protocol of the systematic review has been registered.</p><p>Registration: OSF Preregisration.2020, Aug.14. osf.io/tg9 mw. This systematic review protocol will be conducted and reported strictly according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement guidelines, and the important protocol amendments will be documented in the full review.</p></sec><sec><label>3.2</label><title>Criteria for considering studies for this review</title><p>We will strictly screen studies that meet the following inclusion criteria.</p><sec><label>3.2.1</label><title>Type of included studies</title><p>Only RCTs (except Quasi-RCTs and cluster RCTs) will be included. Animal mechanism studies and non-randomized clinical trials will be excluded. Article that substantially overlaps with another published article in print or electronic media will be excluded. Duplicate publications produced by a single experiment and published as separate papers with different criteria for.</p><p>Measuring results, priority will be given to original publications and other publications will be excluded. The language and time of publication will not be restricted.</p></sec><sec><label>3.2.2</label><title>Participants</title><p>We will include RCTs of participants of 18 years or older, of any sex, race/ethnicity, and diagnosed with POI (diagnosis as defined by the individual trial). We will accept RCTs in which participants had any duration and severity of the disease. Due to different pathways and mechanisms, we will exclude the trials of patients with POF caused by ovariectomy, congenital developmental dysplasia of reproductive organ, acquired organic diseases (e.g., malignant tumors of reproductive organ), endocrine system diseases, iatrogenic injuries (e.g., surgery, chemoradiotherapy).</p></sec><sec><label>3.2.3</label><title>Interventions and controls</title><p>The interventions included auricular point alone or in combination with other conventional treatments. The control group received only conventional treatment. The choice of conventional treatment for each RCT may not be entirely consistent, but auricular point alone or in combination with conventional treatment should be the only difference between intervention and control.</p></sec><sec><label>3.2.4</label><title>Type of outcome measures</title><p>Major results: levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estrogen (E2). Secondary results: modified Kupperman Index, imaging results including ovarian size, antral follicle count and blood flow changes in the ovary using color Doppler ultrasound; total effective rate, adverse event and intervention, and hospitalization expenses.</p></sec></sec><sec><label>3.3</label><title>Search methods</title><sec><label>3.3.1</label><title>Search resources</title><p>This review will include the following electronic databases from their inception to Aug 2020: Electronic database includes PubMed, Embase, Cochrane Library, Web of Science, CNKI (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>.) The research flowchart.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>The research flowchart. This figure shows the Identification, Screening, Eligibility and Included when we searching articles.</p></caption><graphic xlink:href=\"medi-99-e22212-g001\"/></fig></sec><sec><label>3.3.2</label><title>Search strategies</title><p>The following MeSH terms and their combinations will be searched:</p><list list-type=\"simple\"><list-item><label>1.</label><p>auricular acupuncture OR auricular needle OR ear acupuncture OR auricular point sticking OR auricular point;</p></list-item><list-item><label>2.</label><p>RCT OR RCTs;</p></list-item><list-item><label>3.</label><p>premature ovarian failure OR premature ovarian insufficiency OR primary ovarian insufficiency OR decreased ovarian reserve function.</p></list-item></list><p>The search strategy for PubMed is shown in (Table <xref rid=\"T1\" ref-type=\"table\">1</xref>). Other electronic data bases will be used the same strategy.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Search strategy in PubMed database.</p></caption><graphic xlink:href=\"medi-99-e22212-g002\"/></table-wrap></sec></sec><sec><label>3.4</label><title>Data collection and analysis</title><sec><label>3.4.1</label><title>Studies selection</title><p>There will be 2 researchers (YL and DX) carry out the selection of research literature independently using endnote x<sup>9</sup> software. We will first make the preliminary selection by screening titles and abstracts. Secondly, we will download full text of the relevant studies for further selection according to the inclusion criteria. If there is any different opinion, 2 researchers will discuss and reach an agreement. If a consensus could not be reached, there will be a third researcher (FG) who make the final decision. The details of selection process will be displayed in the PRISMA flow chart.</p></sec><sec><label>3.4.2</label><title>Assessment of risk of bias</title><p>The assessment of risk of bias will be carried out by 2 independent reviewers (LYH and WLQ), using the Cochrane Collaborations “Risk of bias” tool. Study bias will be conducted as either: “unclear,” “low,” or “high” risk for the following criteria: random sequence generation, allocation concealment, blinding, incomplete data, selective outcome reporting, and other bias. The assessment of the bias has caused controversy, there is a need for discussion with a third reviewer (PYZ). The graphic representations of potential bias within and across studies using Rev Man V.5.3.5.</p></sec><sec><label>3.4.3</label><title>Measures of treatment effect</title><p>Statistical analyses will be conducted using the risk ratio with 95% confidence intervals (CIs). Odds ratio (OR) and relative risk (RR) are commonly used for dichotomous outcomes data. For continuous outcomes, the weighted mean difference (WMD), or the standard mean difference (SMD) will be analyzed.</p></sec><sec><label>3.4.4</label><title>Unit of analysis issues</title><p>The unit of analysis will be the individual participant.</p></sec><sec><label>3.4.5</label><title>Dealing with missing data</title><p>Among the results of several studies with insufficient data or missing data, the corresponding author will be contacted to complement the contents. If the corresponding author cannot be contacted, the data alone will be conducted.</p></sec><sec><label>3.4.6</label><title>Assessment of heterogeneity</title><p>The assessment of heterogeneity will be conducted by Review Manager (V.5.3.5). Chi-Squared test and <italic>I</italic><sup>2</sup>value of the forest, plot will be calculated to assess heterogeneity, according to the Cochrane Handbook. The <italic>I</italic><sup>2</sup>value is classified into 4 levels: little or no heterogeneity (0%–40%), moderate heterogeneity (30%–60%), substantial heterogeneity (50%–90%), and considerable heterogeneity (75%–100%).</p></sec><sec><label>3.4.7</label><title>Assessment of reporting biases</title><p>If the numbers of available studies are sufficient, funnel plots will be assessed reporting biases.</p></sec><sec><label>3.4.8</label><title>Data synthesis</title><p>Review Manager (V.5.3.5) will be used to analyze. The test indicated little or no heterogeneity; a fixed effect model will be used for data. The random effect model will be adopted when there is considerable heterogeneity (I2% ≥ 50%). If there is considerable variation in results (I2% ≥ 75%), the meta-analysis will not be performed. The narrative and qualitative summary will be available.</p></sec><sec><label>3.4.9</label><title>Subgroup analysis and investigation of heterogeneity</title><p>Subgroup analysis will be conducted to assess heterogeneity. The different types of auricular acupuncture (embedding therapy on-ear point, auricular point seeds pressure, bloodletting at auricular points, auricular point on auto chemotherapy) may be affected heterogeneity.</p></sec><sec><label>3.4.10</label><title>Sensitivity analysis</title><p>Sensitivity analysis will be used to assess the robustness of the results. It is possible to determine according to methodological quality, sample size, and analysis-related issues. The studies that follow a sequence will be removed from all the inclusion reviews. The Chi-Squared test and <italic>I</italic><sup>2</sup> value will be used to quantify statistical heterogeneity.</p></sec><sec><label>3.4.11</label><title>Summary of evidence</title><p>The assessment of evidence for all outcomes will be summarized using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. The quality of evidence will be rated as high, moderate, low, and very low quality.</p></sec></sec></sec><sec><label>4</label><title>Discussion</title><p>Premature ovarian insufficiency has many causes, including genetic, immune and metabolic factors, and enzyme defect. They can cause premature ovarian insufficiency by reducing follicles in the follicular pool or follicular dysfunction.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>,<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> From the perspective of channels and collaterals, all channels and collaterals converge at ears, and auricular points are distributed like an upside-down infant. By stimulating particular auricular points, the physiological functions of particular parts of the body can be strengthened, the circulation of qi and blood can be promoted, and the reproductive function of hypothalamus-pituitary-ovary axis can be regulated.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> According to research on clinical treatment, it has good therapeutic effect to treat POI using auricular points. As POF is in the final stage of ovarian function failure, treatment is often delayed when the disease is diagnosed. Therefore in 2016, “premature ovarian failure” was replaced by “premature ovarian failure” in “Guideline on Management of Women with Premature Ovarian Insufficiency” by ESHRE. Early detection and safe treatment with lasting therapeutic effect are crucial. Different from HRT, auricular acupuncture is not hormone but it has hormone-like effects. The goal of recovering ovarian function can be achieved by overall regulation of patients hormone levels. Its advantages include simple operation, high safety and reliability, lasting therapeutic effect and high treatment acceptance. It has prominent advantages in reducing complications of POI and relieving clinical symptoms of patients with POI, including irregular menstruation, infertility, menopausal syndrome and hormonal regulation. However, the mechanism and standards of treating POI using auricular points are not expounded systematically. In short, this systematic review and meta-analysis can help identify the potential value of auricular points in treating POI and improving amenorrhea, infertility and life quality. This study can provide a foundation for the release of POI treatment guidelines and treatment options of POI patients, and thus benefit more patients.</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization:</bold> Yehao Luo, Donghan Xu.</p><p><bold>Data curation:</bold> Donghan Xu, Luqiu Wei, Fang Gang.</p><p><bold>Formal analysis:</bold> Yehao Luo, Xiusong Tang, Lizhen Wang.</p><p><bold>Funding acquisition:</bold> Yuzhou Pang, Fang Gang.</p><p><bold>Investigation:</bold> Donghan Xu, Luqiu Wei.</p><p><bold>Project administration:</bold> Yuzhou Pang.</p><p><bold>Quality assessment:</bold> Fang Gang, Yehao Luo.</p><p><bold>Software:</bold> Luqiu Wei, Donghan Xu, Lizhen Wang.</p><p><bold>Supervision:</bold> Yehao Luo.</p><p><bold>Validation:</bold> Yehao Luo, Lizhen Wang.</p><p><bold>Writing – original draft:</bold> Yehao Luo, Donghan Xu, Xiusong Tang.</p><p><bold>Writing – review & editing:</bold> Luqiu Wei, Fang Gang, Yuzhou Pang.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: CI = confidence interval, CNKI = China National Knowledge Infrastructure, EBM = evidence-based medicine, RCT = randomized controlled trial, RR = relative risk, SR = systermatic review, WMD = weight mean difference.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Luo Y, Xu D, Tang X, Wei L, Wang L, Pang Y, Fang G. Auricular acupuncture for premature ovarian insufficiency: A protocol for systematic review and meta-analysis. <italic>Medicine</italic>. 2020;99:39(e22212).</p></fn><fn fn-type=\"other\"><p>YL and DX contributed equal to this work and are co-first authors.</p></fn><fn fn-type=\"other\"><p>If amendments are needed, the authors will update their protocol to include any changes in the whole process of research.</p></fn><fn fn-type=\"other\"><p>This project is funded by National Natural Science Foundation of China (No.81660830); Open Project for Guangxi Key Laboratory of Chinese Medicine Foundation Research of Guangxi (No.15-140-32-06); Open Project for Guangxi First-class Discipline Construction of Guangxi University of Chinese Medicine (No.2019XK038); Development Program of High-level Talent Team under Qihuang Project of Guangxi University of Chinese Medicine (No.2018005); Guangxi first-class discipline construction project (No. Gui Jiao Ke Yan [2018] 12). 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991422</article-id><article-id pub-id-type=\"pmc\">PMC7523784</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-03397</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022256</article-id><article-id pub-id-type=\"art-access-id\">22256</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>4000</subject></subj-group><subj-group><subject>Research Article</subject><subject>Systematic Review and Meta-Analysis</subject></subj-group></article-categories><title-group><article-title>Comparative effectiveness of different therapies for treating striae distensae</article-title><subtitle>A systematic review and network meta-analysis</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Lu</surname><given-names>Haishan</given-names></name><degrees>Medical Master</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Guo</surname><given-names>Jian</given-names></name><degrees>Medical Master</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Hong</surname><given-names>Xudong</given-names></name><degrees>Medical Doctor</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Aifen</given-names></name><degrees>Medical Master</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Xudong</given-names></name><degrees>PhD</degrees></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0001-6312-8245</contrib-id><name><surname>Shen</surname><given-names>Shengxian</given-names></name><degrees>Medical Doctor</degrees><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Zhou.</surname><given-names>Bingrong</given-names></name></contrib></contrib-group><aff>Department of Dermatology, PLA 903 Hospital, Hangzhou, Zhejiang, China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Shengxian Shen, Department of Dermatology, PLA 903 Hospital, 14 Lingyin Road, Hangzhou 31000, Zhejiang, China (e-mail: <email>345625207@qq.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22256</elocation-id><history><date date-type=\"received\"><day>16</day><month>4</month><year>2020</year></date><date date-type=\"rev-recd\"><day>3</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>16</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22256.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>Striae distensae (SD) are common and aesthetically undesirable dermal lesions. The aim of this study is to comprehensively evaluate the effectiveness of different therapies in treating striae distensae using network meta-analysis.</p></sec><sec sec-type=\"methods\"><title>Methods:</title><p>A systematic search of electronic databases up to December 1, 2019 was conducted. Randomized controlled trails (RCTs) examining the effectiveness of different methods in treating striae distensae were included. The primary outcomes are clinical effective rate and patient's satisfaction degree. Risk of bias was assessed by the Cochrane risk of bias tool. Network meta-analysis was based on Bayesian framework.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>Fourteen trails that met the criteria with 651 subjects were included. The results of the network meta-analysis show that topical tretinoin combined bipolar radiofrequency showed the highest probability of being the best method to improve the clinical effectiveness and patient satisfaction rate of treating SD (84.5% and 95.7% respectively), closely followed by bipolar radiofrequency (75.3% and 84.3% respectively). Among laser treatment, CO<sub>2</sub> fractional laser is superior to other lasers in the clinical effectiveness and patient satisfaction (72.0% and 58.1% respectively). Statistics showed the topical tretinoin was the worst-performing option in improving the clinical effectiveness and patient satisfaction rate of SD treatment (5.4% and 5.1% respectively).</p></sec><sec sec-type=\"conclusion\"><title>Conclusion:</title><p>Based on the results of network meta-analysis, we recommend treating striae distensae with bipolar radio frequency combined topical tretinoin. The commonly used CO<sub>2</sub> fractional laser can be considered as alternative treatment candidate. Additional large-scale RCTs are necessary to obtain more precise estimates of their relative efficacy.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>clinic treatment</kwd><kwd>meta-analysis</kwd><kwd>randomized controlled trail</kwd><kwd>striae distensae</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>Zhejiang Province Public Welfare Technology Application Research Project</funding-source><award-id>LGF18H110003</award-id><principal-award-recipient>Haishan LU</principal-award-recipient></award-group><award-group id=\"award2\" award-type=\"Fundref\"><funding-source>Zhejiang Province Medicine and Health Technology Plan Project</funding-source><award-id>2018KY631</award-id><principal-award-recipient>Jinfang Wu</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Striae distensae (SD) or stretch marks are common and aesthetically undesirable dermal lesions, which closely related to rapid skin stretching, hormonal changes, and genetic factors.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> Manifested as atrophic linear plaques, SD initially present as flattened or slightly raised pink or red scars (striae rubra) before subsequently turning into paler, flat, and permanent (striae alba).<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> SD generally appears along the cleavage lines in the abdomen, hips, thighs, and breasts, commonly developing during pubertal growth spurts and pregnancy, and associated with Cushing syndrome and oral or topical corticosteroid use.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> The pathophysiological mechanism of SD involved in an inflammatory reaction and elastolysis arising from the release of elastase from mast cells.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> Similar to scars, SD histologically is characterized by thinning of the overlying epidermis, loss of rete ridges, densely packed collagen bundles aligned parallel to the reticular dermis and atrophic skin.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> Although not regarded as a disease, SD results in substantial psychological discomfort and cosmetic concern for women.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup></p><p>Several treatments methods, such as topical agents, microdermabrasion, laser therapies, light therapies, needling therapy, radio frequency (RF) devices, platelet-rich plasma (PRP) injection, have been advocated with variable efficacy.<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup> However, there are no clear ranking of these therapies in terms of their clinical effectiveness and patient satisfaction. With technological advances in aesthetic medicine, an updated quantitative comparative research was needed to aid in clinical decision making.</p><p>To tackle this problem, network meta-analysis that is a statistical tool of quantifying evidence from a network of multiple randomized controlled trails (RCTs) can be employed.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> This method can simultaneously compare multiple competing interventions that have no head-to-head trails available in a single statistical model.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> The aims of our study are to conduct a systematic review and network meta-analysis of RCTs to comprehensively assess the clinical effectiveness and patient satisfaction of different methods for treating striae distensae.</p></sec><sec><label>2</label><title>Methods</title><p>According to Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement and PRISMA Extension Statement for Network Meta-Analysis, a systematic review and network meta-analysis was performed.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> The data and information used in our study was from previously published clinical trials; therefore, ethical approval from Ethics Committee and Institutional Review Board was not required.</p><sec><label>2.1</label><title>Search strategy and selection criteria</title><p>We searched PubMed, Cochrane Library, and EMBASE online databases by 2 independent authors (JG and DX). The literature search was performed until December 1, 2019, and all published RCTs were included in the review. The following key terms were selected under Medical Subject Headings for the search: “striae distensae” or “stretch marks” or “striae rubra” or “striae alba” or “striae gravidarum” and “randomized controlled trails.” To avoid the potential omission of studies, we searched additional databases, such as opengrey.eu for gray literature. We also manually screened reference lists of previous systematic reviews. Two reviewers (AC and XZ) independently reviewed the titles and abstracts of all the studies, and the studies that satisfied the inclusion criteria were retrieved for assessments. Disagreements between the reviewers were resolved by consensus. Duplicates were removed using Endnote X9 (Thomson Reuters Co, New York).</p></sec><sec><label>2.2</label><title>Eligibility criteria</title><sec><label>2.2.1</label><title>Types of studies</title><p>Randomized controlled clinical trials for treating striae distensae were included. Studies should evaluate at least 2 therapies. Trials were excluded if they did not provide validated therapeutic protocols and did not report the results of the clinical effectiveness and patient satisfaction.</p></sec><sec><label>2.2.2</label><title>Types of interventions</title><p>Interventions included topical agents, microdermabrasion, laser therapies, light therapies, needling therapy, radio frequency RF devices, PRP injection, and combined therapies. The therapeutic methods were compared with each other.</p></sec><sec><label>2.2.3</label><title>Types of outcomes</title><p>We selected the clinical effectiveness and patient satisfaction rate as the outcomes of this study. Interventions included topical agents, microdermabrasion, laser therapies, light therapies, needling therapy, radio frequency RF devices, PRP injection, and combined therapies. The therapeutic methods were compared with each other. The clinical effectiveness was analyzed using photographic materials by 2 dermatologists blinded to the study group. Evaluators used a quartile grading scale of 0 = no improvement, 1 = 1% to 25% (mild) improvement, 2 = 26% to 50% (moderate) improvement, 3 = 51% to 75% (good) improvement, and 4 = 76% to 100% (excellent) improvement. Each participant was asked to rate overall satisfaction as 0 = unsatisfied, 1 = slightly satisfied, 2 = moderately satisfied, 3 = satisfied, or 4 = very satisfied.</p></sec></sec><sec><label>2.3</label><title>Data extraction and quality assessment</title><p>Two reviewers (AC and XZ) independently extracted the data using a predesigned strategy. The extracted information included the participant characteristics, interventions, intervention time, outcome measures, funding, and conflicts of interests. Then, the data were integrated. Disagreements within the included study were resolved through discussion. If an agreement could not be reached, a third reviewer was consulted. Two reviewers (JG and XDH) independently assessed the risk of bias using the Cochrane Collaboration Risk of Bias Tool (Review Manager, V.5.2), which involving random sequence generation, allocation concealment, blinding, incomplete outcome data, selective reporting, and other biases. We also assessed the risk of bias across trials. If more than 50% of the information was from trials at a low risk of bias, the domain was judged to be at a low risk of bias. Similarly, if most information was from RCTs with an unclear/high risk of bias, the domain was considered to be at an unclear/high risk of bias.</p></sec><sec><label>2.4</label><title>Statistical analysis</title><p>Network meta-analysis using Bayesian-network method was applied to synthesize evidence for the primary outcome. This approach estimated relative effects of multiple treatments by fitting generalized linear model under Bayesian framework. Heterogeneity (<italic>I</italic><sup>2</sup>) was evaluated to determine variability between the included studies. <italic>I</italic><sup>2</sup> value of 25% was defined as low heterogeneity, 50% as moderate heterogeneity, and 75% as high heterogeneity. Consistency of results from direct and indirect evidence was analyzed using the node-splitting analysis of inconsistency. All statistical analyses were conducted using STATA version 16.0.</p></sec><sec><label>2.5</label><title>Inconsistency analysis and sensitivity analysis</title><p>Model inconsistency was assessed using the node-splitting method. If the <italic>P</italic> value was smaller than .05, then an inconsistency was considered as detected. The node-splitting models were generated via the STATA 14.0. A sensitivity analysis was conducted to test the influence of low-quality studies. If the result of rank probability changes slightly, indicating that the results were credible.</p></sec></sec><sec><label>3</label><title>Result</title><sec><label>3.1</label><title>Search results</title><p>Our search strategy yielded a total of 1350 potentially relevant articles. Of these, 59 trials were included based on the titles and abstracts. After careful full-text screening, 45 articles were discarded for the reasons listed in Figure <xref ref-type=\"fig\" rid=\"F1\">1</xref>. Fourteen RCTs<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>–<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]</sup> met the inclusion criteria, involving nonablative laser, CO<sub>2</sub> fractional laser, Nd:YAG laser, intense pulsed light, topical tretinoin, carboxytherapy, platelet-rich plasma injection, microdermabrasion, bipolar radiofrequency, microneedling, and combined treatment methods. The characteristics of the included trials are presented in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>. The total numbers of participants in these studies were 651.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Flowchart for literature search result. RCT = randomized controlled trial.</p></caption><graphic xlink:href=\"medi-99-e22256-g001\"/></fig><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Characteristics of the included studies.</p></caption><graphic xlink:href=\"medi-99-e22256-g002\"/></table-wrap></sec><sec><label>3.2</label><title>Risk of bias and quality assessment</title><p>Quantification of the risk of bias assessment is presented in Figure <xref ref-type=\"fig\" rid=\"F2\">2</xref>. A random sequence was generated in 13 trials, suggesting the risk of bias in randomization was low. All the studies had unclear information about the methods used to conceal the allocation, therefore, we considered that the risk of bias was unclear for the domain of allocation concealment. The outcomes evaluators were successfully blinded in all included trials, and the risk of bias in this domain was judged to be low. However, the participants and personnel were unclearly blinded in most trials (n = 12, 85.7%). As for the incomplete outcome data element, there was a low risk of bias because most studies reported complete data (n = 11, 78.6%). There was also a low risk of selective outcome reporting because 12 of the studies had a low risk of bias in this domain (85.7%). In addition, the risk of other biases was also low (n = 10, 71.4%). Overall, the certainty evidence of study was moderate.</p><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>Risk of bias graph (upper) and summary (lower).</p></caption><graphic xlink:href=\"medi-99-e22256-g003\"/></fig></sec><sec><label>3.3</label><title>Results of the network meta-analysis</title><p>The network graph was built via STATA shown in Figure <xref ref-type=\"fig\" rid=\"F3\">3</xref>. The size of the circle represents the number of participants, and the thickness of the edge represents the number of studies. All potential comparisons were calculated and presented as ORs and 95% CrIs. The results are listed in Table <xref rid=\"T2\" ref-type=\"table\">2</xref>, and the significant differences are shaded.</p><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Network of comparisons of nonablative laser, CO<sub>2</sub> fractional laser, Nd:YAG laser, intense pulsed light, topical tretinoin, carboxytherapy, platelet-rich plasma (PRP) injection, microdermabrasion, bipolar radiofrequency, microneedling, and combined treatment methods for treating SD. Note: The size of the circle represents the number of participants, and the thickness of the edge represents the number of studies.</p></caption><graphic xlink:href=\"medi-99-e22256-g004\"/></fig><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Results (odd radio, with 95% confidence interval) of the network meta-analysis.</p></caption><graphic xlink:href=\"medi-99-e22256-g005\"/></table-wrap><p>In the clinical effectiveness aspect, CO<sub>2</sub> fractional laser, bipolar radiofrequency, topical tretinoin + bipolar radiofrequency significantly yielded better outcomes than microdermabrasion; CO<sub>2</sub> fractional laser, PRP injection, PRP injection + microdermabrasion, carboxytherapy, bipolar radiofrequency, topical tretinoin + bipolar radiofrequency were significantly more superior than topical tretinoin; CO<sub>2</sub> fractional laser was significantly better than intense pulsed light. No significant differences were found in other comparisons (Table <xref rid=\"T2\" ref-type=\"table\">2</xref>, lower-left triangle).</p><p>With regard to patient satisfaction rate, CO<sub>2</sub> fractional laser, PRP injection, PRP injection + microdermabrasion, microneedling, carboxytherapy, bipolar radiofrequency, topical tretinoin + bipolar radiofrequency significantly yielded better outcomes than microdermabrasion and topical tretinoin; nonablative Er: glass laser, CO<sub>2</sub> fractional laser, PRP injection, PRP injection + microdermabrasion, microneedling, carboxytherapy, bipolar radiofrequency, topical tretinoin + bipolar radiofrequency were significantly more superior than intense pulsed light; topical tretinoin + bipolar radiofrequency was significantly better than PRP injection. No significant differences were found in other comparisons (Table <xref rid=\"T2\" ref-type=\"table\">2</xref>, upper-left triangle).</p></sec><sec><label>3.4</label><title>Rank probability based on SUCRA</title><p>The ranking probability in terms of the clinical effectiveness and patient satisfaction rate is illustrated in Figure <xref ref-type=\"fig\" rid=\"F4\">4</xref>. Larger areas under the surface under cumulative ranking curve represent better effectiveness. Topical tretinoin combined bipolar radiofrequency showed the highest probability of being the best method to improve the clinical effectiveness and patient satisfaction rate of treating SD (84.5% and 95.7% respectively), closely followed by bipolar radiofrequency (75.3% and 84.3% respectively). Among laser treatment, CO<sub>2</sub> fractional laser is superior to other lasers in the clinical effectiveness and patient satisfaction (72.0% and 58.1% respectively). Statistics showed the topical tretinoin was the worst-performing option in improving the clinical effectiveness and patient satisfaction rate of SD treatment (5.4% and 5.1% respectively).</p><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>SUCRA of nonablative laser, CO<sub>2</sub> fractional laser, Nd:YAG laser, intense pulsed light, topical tretinoin, carboxytherapy, platelet-rich plasma (PRP) injection, microdermabrasion, bipolar radiofrequency, microneedling, and combined treatment methods for improving the clinical effectiveness (A) and patient satisfaction rate (B) of treating SD. Note: The area under the curve represents the cumulative rank probability of each treatment. The larger the area, the better the cumulative rank probability. SUCRA = surface under the cumulative ranking curve.</p></caption><graphic xlink:href=\"medi-99-e22256-g006\"/></fig></sec><sec><label>3.5</label><title>Inconsistency analysis</title><p>Inconsistency in network meta-analysis denotes the consistency between direct and indirect evidence for each intervention. The node splitting analysis showed all clinical trials were consistent between direct and indirect comparisons (<italic>P</italic> > .05).</p></sec><sec><label>3.6</label><title>Sensitivity analysis</title><p>After excluding 1 low-quality study (Soliman et al), the SUCRA changed slightly, although no change occurred in the rank probabilities, indicating that the results of the network meta-analysis are robust.</p></sec></sec><sec><label>4</label><title>Discussion</title><sec><label>4.1</label><title>Main findings</title><p>In this systematic review and network meta-analysis, we combined direct and indirect evidence from 14 studies including 651 participants. Our network meta-analysis compared the clinical effectiveness and patient satisfaction of microdermabrasion, topical tretinoin, nonablative Er: glass laser, CO<sub>2</sub> fractional laser, PRP injection, PRP injection combined microdermabrasion, IPL, microneedling, carboxytherapy, bipolar radiofrequency, topical tretinoin combined bipolar radiofrequency, Nd:YAG laser therapy for the treatment of SD. With moderate certainty, our study indicated that topical tretinoin combined bipolar radiofrequency and bipolar radiofrequency had the highest probability of providing the best outcome in terms of clinical effectiveness and patient satisfaction. Topical tretinoin was the worst-performing option in improving the clinical effectiveness and patient satisfaction rate of SD treatment.</p></sec><sec><label>4.2</label><title>Agreements and disagreements with other studies</title><p>A previous review reported that 1540-nm nonablative fractionated laser was a worthy first-line modality for the treatment of SD,<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> based on clinical and anecdotal experience, which was inconsistent with our meta-analysis. On the one hand, our study indicated that bipolar radiofrequency was superior to laser treatment. On the other hand, CO<sub>2</sub> ablative fractional laser provided better outcome than nonablative Er: glass laser in the clinical effectiveness and patient satisfaction of SD treatment.</p><p>Bipolar radiofrequency can pass through the skin, and generate heat due to the resistance of the skin, acting on the deep dermis and fibrosis septum, and sometimes fascia, causing the contraction of existing collagen and synthesis of new collagen.<sup>[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]</sup> It accelerates the healing by means of fractional mode, so as to improve the safety of the treatment, similar to the mechanism of fractional lasers.<sup>[<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]</sup> The bipolar radiofrequency system used in the included study can deliver energy in a fractional manner, and generates an array of micro-thermal zones of thermal injury in the epidermis and dermis. We assumed that the bipolar fractional radiofrequency system disrupts the stratum corneum, causing microchannels in the epidermis to alter the skin permeability so as to promote topical tretinoin penetration. The combined therapy is indicated to have a synergistic effect, which accelerates collagen synthesis and fibroblast activity.<sup>[<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]</sup> Consistent with our study, the majority of trials evaluating RF for the treatment of SD have reported significant improvements, with few side effects including erythema and edema. In addition, some studies have also demonstrated subjective improvement of SD via bipolar RF combined with and ablative CO2 laser and bipolar RF combined with PRP injection.<sup>[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>–<xref rid=\"R27\" ref-type=\"bibr\">27</xref>]</sup> The participants with Fitz-partrick Skin Types IV and V reported on adverse event, indicating that bipolar RF may be considered as a potential and safe therapeutic option for SD patients with skin of color.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup></p><p>A variety of laser parameters have been studied either alone or in combination with other modalities for the treatment of SD, among which the effectiveness of nonablative fractional lasers versus ablative fractional lasers was controversial.<sup>[<xref rid=\"R5\" ref-type=\"bibr\">5</xref>,<xref rid=\"R28\" ref-type=\"bibr\">28</xref>]</sup> Our study found that CO<sub>2</sub> ablative fractional laser was significantly superior to nonablative Er: glass laser. However, it was based on only 1 RCT directly comparing CO<sub>2</sub> ablative fractional laser and nonablative Er: glass laser.</p><p>Numerous studies reported that tretinoins was able to increase tissue collagen I levels through stimulation of fibroblasts so as to improve the appearance of early SD.<sup>[<xref rid=\"R29\" ref-type=\"bibr\">29</xref>,<xref rid=\"R30\" ref-type=\"bibr\">30</xref>]</sup> In our study, compared with other methods, the topical tretinoin was the worst-performing option in improving the clinical effectiveness and patient satisfaction rate of SD treatment. However, when topical tretinoin combined with bipolar RF, this strategy showed the highest probability of being the best method to improve the clinical effectiveness and patient satisfaction rate of treating SD. Our study suggested that topical tretinoin can be used to treat SD combined with other treatment methods.</p></sec><sec><label>4.3</label><title>Strengths and limitations</title><p>There were several strengths in our net-work meta-analysis. Firstly, our study was the first network meta-analysis of RCTs to comprehensively assess the clinical effectiveness and patient satisfaction of different methods for treating striae distensae. Secondly, we conducted a comprehensively searched for literature, with 2 independent reviewers assessing quality, to reduce any potential bias. Thirdly, our network model consisted of a closed loop, which allowed for the assessment of inconsistency for both direct and indirect evidence. More importantly, all the trials included were RCTs, which were the highest level of evidence, thus our results should strongly reflect the true clinical effectiveness of these methods. In addition, the node-splitting analysis showed all clinical trials were consistent between direct and indirect comparisons, indicating our analysis was based on consistent evidence.</p><p>Nevertheless, this study also has limitations. First, some treatments were presented in only 1 study. Thus, more high-quality RCTs were needed in the future to corroborate these results. Second, the participants were unclearly blinded in most trials, which may result in participant bias. Third, the English restriction during the search can possibly affect the comprehensiveness of the search strategy. Finally, there may be a publication bias because all the articles reported positive results of their clinical trials. But our funnel plots showed the publication bias was low (Fig. <xref ref-type=\"fig\" rid=\"F5\">5</xref>).</p><fig id=\"F5\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><p>Funnel plots of the included studies involving the clinical effectiveness (A) and patient satisfaction rate (B) of treating SD.</p></caption><graphic xlink:href=\"medi-99-e22256-g007\"/></fig></sec></sec><sec><label>5</label><title>Conclusion</title><p>Our network meta-analysis compared the clinical effectiveness and patient satisfaction of 14 modalities for treating striae distensae. With moderate certainty, our study indicated that topical tretinoin combined bipolar radiofrequency and bipolar radiofrequency had higher probability of providing the better outcome in terms of clinical effectiveness and patient satisfaction. However, topical tretinoin only was the worst-performing option in improving the clinical effectiveness and patient satisfaction rate of SD treatment. Additional large-scale RCTs are necessary to obtain more precise estimates of their relative efficacy.</p></sec><sec><title>Acknowledgments</title><p>The authors gratefully acknowledge the financial support from “Zhejiang Provincial Natural Science Foundation Basic Public Welfare Research Project”. (LGF18H110003).</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization:</bold> Haishan Lu, Jian Guo, Shengxian Shen.</p><p><bold>Formal analysis:</bold> Haishan Lu, Jian Guo, Xudong Hong, Aifen Chen.</p><p><bold>Funding acquisition:</bold> Haishan Lu, Aifen Chen, Xudong Zhang.</p><p><bold>Investigation:</bold> Haishan Lu, Jian Guo, Aifen Chen.</p><p><bold>Resources:</bold> Jian Guo, Shengxian Shen.</p><p><bold>Supervision:</bold> Shengxian Shen, Xudong Zhang.</p><p><bold>Writing – original draft:</bold> Haishan Lu, Jian Guo.</p><p><bold>Writing – review & editing:</bold> Haishan Lu, Xudong Hong, Shengxian Shen.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: CI = confidence interval, CO<sub>2</sub> = carbon dioxide, OR = odd radio, PRISMA = Preferred Reporting Items for Systematic Reviews and Meta-Analysis, PRP = platelet-rich plasma, RCTs = randomized controlled trails, RF = radio frequency, SD = striae distensae, SUCRA = surface under the cumulative ranking curve.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Lu H, Guo J, Hong X, Chen A, Zhang X, Shen S. Comparative effectiveness of different therapies for treating striae distensae: A systematic review and network meta-analysis. <italic>Medicine</italic>. 2020;99:39(e22256).</p></fn><fn fn-type=\"financial-disclosure\"><p>Funding Source: Zhejiang Province Medicine and Health Technology Plan Project; Award ID: 2018KY631.</p></fn><fn fn-type=\"equal\"><p>HL and JG contributed equally and co-first authors to this work.</p></fn><fn fn-type=\"COI-statement\"><p>The authors have no conflicts of interest to disclose.</p></fn><fn fn-type=\"other\"><p>The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>[1]</label><mixed-citation publication-type=\"journal\"><person-group 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991439</article-id><article-id pub-id-type=\"pmc\">PMC7523786</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-08158</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022317</article-id><article-id pub-id-type=\"art-access-id\">22317</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>4300</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Systematic Review</subject></subj-group></article-categories><title-group><article-title>Effects of Guizhi decoction for diabetic cardiac autonomic neuropathy</article-title><subtitle>A protocol for a systematic review and meta-analysis</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-0086-1607</contrib-id><name><surname>Chen</surname><given-names>Junmin</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Cai</surname><given-names>Jiawei</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wei</surname><given-names>Mengya</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Xiaoran</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhong</surname><given-names>Min</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Min</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Yu</surname><given-names>Yang</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Qiu</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu</aff><aff id=\"aff2\"><label>b</label>College of acupuncture and massage, Chengdu University of Traditional Chinese Medicine , No 37 Shi-er-qiao Road, Chengdu, Sichuan Province, PR China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Qiu Chen, Medical Department of Endocrinology, Hospital of Chengdu University of Traditional Chinese Medicine, No 39 Shi-er-qiao Road, Chengdu 610072, Sichuan Province, PR China (e-mail: <email>chenqiu1005@cdutcm.edu.cn</email>); Yang Yu, College of acupuncture and massage, Chengdu University of Traditional Chinese Medicine , No 37 Shi-er-qiao Road, Chengdu 610075, Sichuan Province, China (e-mail: <email>zj110110@163.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22317</elocation-id><history><date date-type=\"received\"><day>19</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>20</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22317.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>Diabetic cardiac autonomic neuropathy (DCAN) is one of the serious complications of diabetes. The pathogenesis of DCAN has not been fully elucidated. There is currently no effective treatment for such chronic disease. Traditional Chinese medicine has a long clinical history for the prevention and treatment of diabetes and chronic complications, and it also shows certain advantages in the treatment of DCAN. Many clinical studies have confirmed that Chinese medicine Guizhi decoction can reduce the clinical symptoms and improve neuronal function of patients with DCAN. So we intend to conduct a systematic review further clarified the effectiveness and safety of Guizhi decoction for DCAN.</p></sec><sec sec-type=\"methods\"><title>Methods:</title><p>We will search each database from the built-in until July 2020. The English literature mainly searches Cochrane Library, PubMed, EMBASE, and Web of Science, while the Chinese literature comes from CNKI, CBM, VIP, and Wangfang database. Simultaneously we will retrieval clinical registration tests and grey literatures. In this study, only the clinical randomized controlled trials (RCTs) were selected to evaluate the efficacy and safety of Guizhi decoction in the treatment of DCAN. The 2 researchers independently conducted literature selection, data extraction, and quality assessment. Statistical heterogeneity among studies will be evaluated using the Cochran Q test (x<sup>2</sup>) and the <italic>I</italic><sup>2</sup> statistical value. We will utilize the Review Manage software V5.3.0 (The Nordic Cochrane Center, The Cochrane Collaboration, 2014, Copenhagen, Denmark) to statistically analyze all data.</p></sec><sec><title>Ethics and dissemination:</title><p>This study is a protocol for a systematic review of Guizhi decoction as a treatment of DCAN patients.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>This study will provide high-quality synthesis of effectiveness and safety of Guizhi decoction for DCAN.</p></sec><sec sec-type=\"conclusion\"><title>Conclusion:</title><p>This systematic review aims to provide new options for Guizhi decoction treatment of DCAN in terms of its efficacy and safety.</p></sec><sec><title>Registration number:</title><p>INPLASY202080018.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>diabetic cardiac autonomic neuropathy</kwd><kwd>Guizhi decoction</kwd><kwd>meta-analysis</kwd><kwd>protocol</kwd><kwd>systematic review</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>the Key R &amp; D plan of the Ministry of science and technology of the people's Republic of China</funding-source><award-id>2018YFC1704104</award-id><principal-award-recipient>Qiu Chen</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Cardiac autonomic nerves (CANs) include the sympathetic nerve and the vagus nerve, which check and balance each other and play important roles in the regulation of heart rate, conduction, and cardiac contractility. Diabetic cardiac autonomic neuropathy (DCAN) is one of the severe complications of diabetes mellitus; damage of CANs in a high glucose environment causes sympathetic-vagal imbalance,<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> which thus causes abnormal neurotransmitter signaling and increased asynchronization of cardiac electrophysiology.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>,<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> About 2.5% to 50.0% of diabetic patients have cardiac autonomic neuropathy.<sup>[<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> It is reported that diabetic cardiac autonomic neuropathy is easy to be ignored in many complications of diabetes. Because the balance of cardiac autonomic nerves is broken, the incidence of myocardial ischemia, painless myocardial infarction and sudden cardiac death is increased,<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> and the mortality rate within 5 years is as high as 50%, of which sudden cardiac death accounts for 28%.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R7\" ref-type=\"bibr\">7</xref>,<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> Compared with the patients without DCAN, the mortality increased 3 to 4 times.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>,<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> The time variability of left ventricular repolarization in DCAN patients increased, and the proportion of malignant arrhythmia caused by it in the cause analysis of sudden death was 50% to75%.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup> According to a research, strict blood sugar control could slow the progression of CAN diseases but could not reverse CAN pathological changes.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> Therefore, in addition to protecting the sympathetic and vagus nerves from further damage, regulation of the imbalance of these 2 nerves is even more important for the treatment of diabetic CAN diseases. Because the pathogenesis of the disease has not been fully elucidated, western medicine lacks the corresponding treatment plan, some drugs interfere with glycolipid metabolism, drug target is single, the safety, and effectiveness need to be further confirmed.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> Our previous studies have shown that based on the treatment methods of balancing yin and yang and harmonizing Ying and Wei in Chinese medicine theory, taking Xinhe granule and Tiaoxin decoction containing Guizhi decoction can improve the symptoms of autonomic neuropathy of coronary heart disease.<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>,<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]</sup> Traditional Chinese medicine suggests that diabetic cardiac autonomic neuropathy belongs to the category of “chest obstruction” and “palpitation”. Guizhi decoction can effectively play the role of regulating Ying Wei, reverse the remodeling of myocardial collagen, inhibit inflammatory factors, inhibit the abnormal remodeling of sympathetic nerve, and maintain the balance of autonomic nerve.<sup>[<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</sup> Studies also confirmed that Guizhi decoction decreased the accumulation of inflammatory factors including nuclear factor κ B (NF-κ B), interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and endothelin (ET-1) in the heart of streptozocin (STZ) rats; reversed spontaneous myocardial collagen remodeling in diabetic rats; significantly reduced myocardial basement membrane thickness; prevented damage and thickening of the myocardial basement membrane; and improved oxygen diffusion barriers,<sup>[<xref rid=\"R17\" ref-type=\"bibr\">17</xref>,<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</sup> thus to prevent and treat diabetic myocardial damage.</p><p>In recent years, the advantages of traditional Chinese medicine in the prevention and treatment of this kind of chronic diseases have been widely recognized around the world.<sup>[<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]</sup> The prescription of Guizhi decoction firstly recorded in the book of Shanghan Lun written by Zhang Zhongjing, is composed of Ramulus Cinnamomi (Guizhi), Paeonia lactiflora (Shaoyao), Rhizoma Zingiberis Recens (Shengjiang), Licorice (Gancao), and Jujube (Dazao). We intend to collect randomized controlled trials (RCTs) about Guizhi decoction for DCAN based on the basis of evidence-based medicine, and conduct a meta-analysis of its efficacy and safety to provide higher quality clinical evidence for Chinese medicine treatment of DCAN.</p></sec><sec><label>2</label><title>Methods</title><sec><label>2.1</label><title>Protocol registration</title><p>The systematic review protocol has been registered on the LNPLASY website as INPLASY202080018. (<ext-link ext-link-type=\"uri\" xlink:href=\"https://inplasy.com/inplasy-2020-8-0018/\">https://inplasy.com/inplasy-2020-8-0018/</ext-link>). It is reported following the guidelines of Cochrane Handbook for Systematic Reviews of Interventions and the Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocol (PRISM).<sup>[<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</sup></p></sec><sec><label>2.2</label><title>Eligibility criteria</title><sec><label>2.2.1</label><title>Study design</title><p>This study only included RCTs of Guizhi decoction for DCAN. However, animal experiments, reviews, case reports, and non-randomized controlled trials are excluded.</p></sec><sec><label>2.2.2</label><title>Participants</title><p>DCAN patients must meet the World Health Organization (WHO) “diabetes” diagnosis standard in 1999.<sup>[<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]</sup> The diagnosis standard of cardiac autonomic neuropathy shall refer to Toronto standard and European cardiovascular disease prevention guidelines,<sup>[<xref rid=\"R22\" ref-type=\"bibr\">22</xref>,<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]</sup> regardless of race, gender, and age. Special patients with severe diabetic complications, severe cardiac insufficiency, pregnancy, etc. are not included.</p></sec><sec><label>2.2.3</label><title>Interventions</title><p>Both groups were cured with conventional diabetes treatments recommended by the American Diabetes Association (ADA) guidelines, including diet, exercise, and hypoglycemic, and lipid-lowering therapies.<sup>[<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]</sup> The experiment group used Guizhi decoction or modified Guizhi decoction, while the control group applied for placebo, nutritional neurological drugs, or no treatment. In addition, the 2 groups did not take any drugs that interfered with the outcome indicators. Trials will be included at least 4 weeks of treatment.</p></sec><sec><label>2.2.4</label><title>Outcomes</title><p>The primary outcomes include patient before and after treatment: markedly effective: symptoms improved significantly >70%; effective: symptoms reduced by 30% to 70%; ineffective: symptom improvement is <30% or no improvement, or even worse. In addition to Heart rate variability (HRV) recognized as the most accurate and sensitive index to judge whether diabetic patients have autonomic nervous system damage, fasting blood glucose (FBG) and 2 hours Postprandial Blood Glucose (2 hours PBG) were also included. Secondary outcomes included glycosylated hemoglobin and adverse events.</p></sec></sec><sec><label>2.3</label><title>Study search</title><p>We will search each database from the built-in until July 2020. The English literature mainly searches Cochrane Library, PubMed, EMBASE, and Web of Science, while the Chinese literature comes from CNKI, CBM, VIP, and Wangfang database. Simultaneously we will retrieval clinical registration tests and grey literatures. According to the PICOS principle, the keywords of our search terms were: (“Guizhi Tang” OR “Guizhi Decoction”) AND (“diabetic cardiac autonomic neuropathy” OR “DCAN”).</p></sec><sec><label>2.4</label><title>Study selection</title><p>We will manage the electronic citations we downloaded from the above databases in Endnote X8 for Mac (Thomson Reuters, USA). First of all, 2 independent reviewers initially screened the literatures that did not meet the pre-established standards of the study by reading the title and abstract. Secondly, download the remaining literatures and read the full text carefully to further decide whether to include or not. Finally, the results were cross-checked repeatedly by reviewers. A final decision will be made through consensus when there were discrepancies. Details of the selection process were shown in the flow chart (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Flow diagram of study selection.</p></caption><graphic xlink:href=\"medi-99-e22317-g001\"/></fig></sec><sec><label>2.5</label><title>Data extraction and management</title><p>According to the characteristics of the study, we prepared excel table for data acquisition before data extraction. The results of the qualified study were extracted independently by 2 reviewers and the data extraction form was filled in. Duplicate literature will be removed. If there is any dispute, the 2 reviewers can discuss or consult the third party to reach an agreement. The main data extracted are as follows: first author, year of publication, source of funds, intervention measures in experimental group, intervention measures in control group, treatment time, course of disease, number of patients in each group, age, gender, prognosis, and safety of patients. If you find something unclear in the study, you can contact the author of the communication directly for more detailed information. The above information was finally cross checked by 2 reviewers.</p></sec><sec><label>2.6</label><title>Risk of bias assessment</title><p>Two review authors will independently evaluate the design, execution, and reporting of the included RCTs based on the Cochrane risk of bias tool. The following 7 items affiliated with bias risk, including random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective outcome reporting, and other biases, will be evaluated by 2 reviewers. Each item is classified as “Low risk”, “High risk” or “Unclear risk”. The discrepancies will get a consistent conclusion by discussing between both reviewers or seeking the third-party consultation.</p></sec><sec><label>2.7</label><title>Statistical analysis</title><p>The risk ratio (RR) for dichotomous data will be calculated, respectively, along with 95% CI, the weighted mean difference (WMD). For continuous data, the mean difference (MD) or standardized mean difference (SMD) with 95% CI will be estimated. If we use the same scale to measure an outcome in different studies, we will use MD. Similarly, if we use different scales to measure the same outcome, we will use SMD. If an outcome measure contains less than 2 trials, we will summarize the results descriptively. Statistical heterogeneity among studies will be evaluated using the Cochran Q test (x<sup>2</sup>) and the <italic>I</italic><sup>2</sup> statistical value. We will categorize the heterogeneity using the following rules. <italic>I</italic><sup>2</sup> of 0% to 25% indicates low heterogeneity. <italic>I</italic><sup>2</sup> of 25% to 50% represents moderate heterogeneity. And <italic>I</italic><sup>2</sup> of 75% to 100% represents high heterogeneity. When the P value from a x<sup>2</sup> test is more than .10 or <italic>I</italic><sup>2</sup> 50%, we will adopt the fixed-effects model. Otherwise, there will be perceptible differences between the studies. Subgroup analysis will be performed to identify possible explanations for statistical heterogeneity, taking into account pre-specified factors.</p><p>We will utilize the Review Manage software V5.3.0 (The Nordic Cochrane Center, The Cochrane Collaboration, 2014, Copenhagen, Denmark) to statistically analyze all data. The overall RR with its 95% CI for dichotomous data will be estimated. The MD or SMD with 95% CI will be calculated for continuous data in different situations. The fixed-effects model will be employed as appropriate for analysis. If the heterogeneity in the study is significant, subgroup analysis will be conducted to investigate possible sources of statistical heterogeneity. When a meta-analysis is not available, descriptive summaries of individual studies will be provided.</p></sec><sec><label>2.8</label><title>Additional analysis</title><sec><label>2.8.1</label><title>Subgroup analysis</title><p>If the results are heterogeneous, we will conduct subgroup analysis based on different reasons. Heterogeneity is manifested in race, age, gender, different intervention methods, drug dosage, course of treatment and so on.</p></sec><sec><label>2.8.2</label><title>Sensitivity analysis</title><p>To identify the robustness of the meta-analysis results, we will conduct a sensitivity analysis by omitting each of the RCT, or excluding the RCTs with high risk of bias, or excluding the RCTs with missing data.</p></sec><sec><label>2.8.3</label><title>Reporting bias</title><p>If there are more than 10 studies in meta-analysis, the symmetry of the funnel plot will be evaluated to check for publication bias and to interpret the results carefully, grading the quality of evidence. In this systematic review, the evidence quality of the whole study was assessed by the “recommended assessment, formulation and evaluation (grade)” standard developed by the World Health Organization and international organizations to achieve transparency and simplification. The quality of evidence was divided into 4 levels: high, medium, low, and very low. Slope profiler 3.2 will be used for analysis.</p></sec></sec></sec><sec><label>3</label><title>Discussion</title><p>DCAN is a common chronic complication of diabetes. At present, drug treatment is universally used in the treatment of this disease, and there are some shortcomings, such as little effect or large side effects. Therefore, both clinicians and patients hope to seek a new treatment to improve symptoms with low adverse reactions. Traditional Chinese medicine has been used to treat diabetes and diabetic complications in China for many years.<sup>[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>,<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]</sup> It has not only outstanding efficacy, but also has few side effects and low economic costs. Clinical studies have shown that Guizhi Decoction can alleviate the symptoms of DCAN and improve the overall clinical efficacy.<sup>[<xref rid=\"R27\" ref-type=\"bibr\">27</xref>–<xref rid=\"R29\" ref-type=\"bibr\">29</xref>]</sup> However, there is no evidence-based medicine to confirm the efficacy of Guizhi Decoction for DCAN. So we attempt to perform this meta-analysis to provide high-quality evidence for the clinical efficacy and safety of Guizhi Decoction. Finally, we will classify the existing evidence to provide a better guide for clinical use.</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization:</bold> Junmin Chen, Jiawei Cai, Qiu Chen, Yang Yu.</p><p><bold>Data curation:</bold> Mengya Wei, Xiaoran Zhang.</p><p><bold>Funding acquisition:</bold> Qiu Chen, Yang Yu.</p><p><bold>Investigation:</bold> Min Zhong, Min Liu.</p><p><bold>Methodology:</bold> Junmin Chen, Jiawei Cai, Qiu Chen.</p><p><bold>Project administration:</bold> Qiu Chen, Yang Yu.</p><p><bold>Software:</bold> Junmin Chen, Jiawei Cai.</p><p><bold>Supervision:</bold> Xiaoran Zhang.</p><p><bold>Validation:</bold> Junmin Chen.</p><p><bold>Writing – original draft:</bold> Junmin Chen, Jiawei Cai.</p><p><bold>Writing – review & editing:</bold> Qiu Chen, Yang Yu.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: CANs = Cardiac autonomic nerves, CI = confidence interval, DCAN = diabetic cardiac autonomic neuropathy, MD = mean difference, RCT = randomized controlled trial, RR = risk ratio, SMD = standardized mean difference.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Chen J, Cai J, Wei M, Zhang X, Zhong M, Liu M, Yu Y, Chen Q. Effects of Guizhi decoction for diabetic cardiac autonomic neuropathy: A protocol for a systematic review and meta-analysis. <italic>Medicine</italic>. 2020;99:39(e22317).</p></fn><fn fn-type=\"equal\"><p>JC and JC contributed equally to this work as co-first authors.</p></fn><fn fn-type=\"supported-by\"><p>This study was supported by the Key R & D plan of the Ministry of science and technology of the people's Republic of China: Research on the Academic Viewpoints, Unique Diagnostic and Treatment Methods and Major Diseases Prevention and Treatment Experience of Illustrious Senior Traditional Chinese Medicine Practitioners in Western China (No. 2018YFC1704104).</p></fn><fn fn-type=\"other\"><p>Taking into account the systematic review of this protocol, ethical ratification is not required. In this study, participants were not recruited and data were not collected from participants. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991416</article-id><article-id pub-id-type=\"pmc\">PMC7523790</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-08141</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022224</article-id><article-id pub-id-type=\"art-access-id\">22224</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3600</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Systematic Review</subject></subj-group></article-categories><title-group><article-title>Efficacy and safety of total glucosides of paeony for rheumatoid arthritis</article-title><subtitle>A protocol for systematic review and meta-analysis</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Tang</surname><given-names>Ce</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ye</surname><given-names>Lianghong</given-names></name><degrees>MS</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Hu</surname><given-names>Zhipeng</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Wenxiang</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>e</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Kuang</surname><given-names>Tingting</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>e</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Fan</surname><given-names>Gang</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>e</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Yi</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>e</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>XiuHua</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Maoyi</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu</aff><aff id=\"aff2\"><label>b</label>Traditional Chinese Medicine hospital. TongLiang. ChongQing, Chongqing</aff><aff id=\"aff3\"><label>c</label>Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan</aff><aff id=\"aff4\"><label>d</label>School of Ethnic Medicine.</aff><aff id=\"aff5\"><label>e</label>School of basic medical sciences, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Maoyi Yang, Hospital of Chengdu University of Traditional Chinese Medicine, No.39 Shi-er-qiao Road, Chengdu, 610072, Sichuan Province, P.R. China (e-mail: <email>3406673658@qq.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22224</elocation-id><history><date date-type=\"received\"><day>17</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>19</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22224.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by erosion of joints and surrounding tissues. RA not only causes the decline of patients’ physical function and quality of life, but also brings huge economic burden to patients’ families and society. Total glucosides of paeony (TGP) is commonly used in treating RA in China. At present, there are many clinical reports about this medicine, but these reports have their own flaws. Therefore, there is an urgent need for systematic review and meta-analysis of the existing clinical evidence.</p></sec><sec sec-type=\"methods\"><title>Methods and analysis:</title><p>Literature search will be carried out in 6 databases, and the literatures will be screened according to the inclusion and exclusion criteria. The clinical effective rate will be taken as primary outcome. Serum rheumatoid factor, C-reactive protein, erythrocyte sedimentation rate, Western Ontario and McMaster before and after treatment and adverse effects will be secondary outcomes. The heterogeneity of the study will be examined by <italic>χ</italic><sup>2</sup> and <italic>I</italic><sup>2</sup> test. To identify the source of heterogeneity, subgroup analysis will be carried out. The sensitivity test will be conducted investigate the stability of results. Funnel plot and Egger test will be used to evaluate publication bias. Finally, the quality of evidence will be summarized.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>The results will be published in peer-reviewed journals.</p></sec><sec sec-type=\"conclusion\"><title>Conclusions:</title><p>This study will systematically evaluate the efficacy of TGP in the treatment of RA. The results of this study can better guide clinical practice.</p></sec><sec><title>OSF registration number:</title><p>DOI 10.17605/OSF.IO/85QVF.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>meta-analysis</kwd><kwd>protocol</kwd><kwd>Rheumatoid arthritis</kwd><kwd>systematic review</kwd><kwd>total glucosides of paeony</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>National Natural Science Foundation of China</funding-source><award-id>81903922</award-id><principal-award-recipient>Tang Ce</principal-award-recipient></award-group><award-group id=\"award2\" award-type=\"Fundref\"><funding-source>National Key Research and Development Program of China</funding-source><award-id>2017YFC1703900</award-id><principal-award-recipient>Tang Ce</principal-award-recipient></award-group><award-group id=\"award3\" award-type=\"Fundref\"><funding-source>Xinglin Scholars Research Promotion Project of Chengdu University of Traditional Chinese Medicine</funding-source><award-id>BSH2019002</award-id><principal-award-recipient>Tang Ce</principal-award-recipient></award-group><award-group id=\"award4\" award-type=\"Fundref\"><funding-source>Chongqing Science and Technology Bureau and the Health Commission joined the Chinese medicine science and technology project</funding-source><award-id>ZY201802041</award-id><principal-award-recipient>Tang Ce</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by erosion of joints and surrounding tissues.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> The main pathological changes of RA are chronic synovial inflammation.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> Inflammation invades articular cartilage, subchondral bone, ligaments, tendons, among others, causing destruction of articular cartilage, bone and joint capsule, and various degrees of systemic symptoms.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>,<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> The disease is characterized by progressive exacerbation, incurable, and easy recurrence. In China, the disability rates of RA patients in 1 to 5 years, 5 to 10 years, 10 to 15 years, and ≥15 years was 18.6%, 43.5%, 48.1%, and 61.3%, respectively, and the disability rate increases with the duration of the disease.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> RA not only causes the decline of patients’ physical function and quality of life, but also brings huge economic burden to patients’ families and society. Therefore, how to control RA more effectively and delay its progression has become a hot topic in medical research.</p><p>Traditional Chinese medicine (TCM) has a long history and rich experience in the treatment of RA. White peony is a TCM herbal medicine widely used by Chinese medicine doctors in treating RA. It was found that the main active ingredient of paeony is a group of glycosides called total glucosides of white paeony (TGP). At present, TGP has been processed into Chinese patent medicine and widely used in clinical practice. In animal experiments, TGP was found to be able to inhibit inflammation, analgesia, and autoimmune response. These pharmacological effects may be through the inhibition of interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α) and joint synovial cell proliferation, bidirectional regulation of IL-2, and regulation of NF- κB signaling pathway.<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>–<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> In clinical trials, TGP can downregulate serum rheumatoid factor (RF), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) levels, reduce inflammatory reaction, and relieve arthritis symptoms. In clinical trials, combination treatments of TGP and other medicines have better efficacy than other drugs alone.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup></p><p>However, there are some defects in these clinical trials, such as small sample size, single center research, and low quality. The systematic review of TGP in the treatment of RA did not provide a comprehensive summary and evaluation of the evidence.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> Therefore, this research will conduct a meta-analysis on the clinical efficacy of TGP in the treatment of RA to provide high-quality evidence for clinical practice.</p></sec><sec><label>2</label><title>Methods</title><sec><label>2.1</label><title>Study registration</title><p>We have completed the registration of this study on the Open Science Framework (OSF)(<ext-link ext-link-type=\"uri\" xlink:href=\"https://osf.io/\">https://osf.io/</ext-link>) and the DOI is 10.17605/OSF.IO/85QVF. The report of this system review protocol is based on the Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocols (PRISMA-P) checklist.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup></p></sec><sec><label>2.2</label><title>Inclusion and exclusion criteria</title><sec><label>2.2.1</label><title>Study design</title><p>Only randomized controlled trials (RCTs) will be included in this study. Non-TCTs, due to their high risk of bias, will not be included in this study.</p></sec><sec><label>2.2.2</label><title>Participants</title><p>Patients with a clear diagnosis of RA will be included in this study. Ideally, the diagnostic criteria should be clearly reported in articles. If the author did not report the diagnostic criteria, we will exclude this research in the main analysis and include it in the sensitivity analysis to assess the impact of the presence or not of the diagnostic criteria on the results. We will not limit other demographic characteristics of participants.</p></sec><sec><label>2.2.3</label><title>Interventions and comparators</title><p>TGP is a kind of Chinese patent medicine, which is made of total glucosides extracted from white peony. At present, the dosage form of TGP on the market is capsule, the standard is 0.3 g, and each capsule contains no less than 104 mg paeoniflorin. We will not limit the usage and dosage of TGP. In addition to TGP, white peony also contains paeonol, volatile oil, and other components. Therefore, the research on the treatment of RA with water extract or ethanol extract of white peony will not be included in this study, whether it is used alone or in combination with other drugs. The control group can be placebo, conventional treatment, and care or no treatment. Studies that compare TGP with other traditional Chinese herbal medicine will not be included.</p></sec><sec><label>2.2.4</label><title>Outcomes</title><p>Primary outcome: clinical effective rate.</p><p>Secondary outcomes: serum rheumatoid factor (RF), CRP, ESR, Western Ontario and McMaster before and after treatment; adverse effects.</p></sec><sec><label>2.2.5</label><title>Other criteria</title><p>Conference articles and abstract articles will not be included in this study because there are no data to be analyzed. For these articles, we will classify them as awaiting classification, and contact the authors for detailed research data.</p></sec></sec><sec><label>2.3</label><title>Study search</title><p>The following databases will be searched from its inception to August 2020: PubMed, Embase, Cochrane Library Central Register of Controlled Trials, and 4 Chinese databases: China National Knowledge Infrastructure database, Wanfang Data Knowledge Service Platform, and the VIP information resource integration service platform. There will be no language restriction. We will also search the Chinese clinical trial registry (ChiCTR) and ClinicalTrials.gov to find ongoing research. In addition, we will search the references of the included articles to find more citations. The search strategy of PubMed can be viewed in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Search strategy in PubMed.</p></caption><graphic xlink:href=\"medi-99-e22224-g001\"/></table-wrap></sec><sec><label>2.4</label><title>Study selection</title><p>Two authors will screen the literature by reading the title and abstract independently. We will download the full text of all possible eligible literatures. Any differences in the research process will be resolved through consultation with a third researcher. If it is still unable to decide whether to include a study or not after negotiation, the study will be classified as awaiting classification. We will draw a flow chart and provide a list of excluded literature and reasons for exclusion (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>).</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Flow chart of study selection.</p></caption><graphic xlink:href=\"medi-99-e22224-g002\"/></fig></sec><sec><label>2.5</label><title>Data extraction</title><p>We will extract the data from the literature by using a pre-specified form. The following data will be extracted: first author name, publication time, country, type of study, number of trial group, number of control group, main outcome, secondary outcomes, and adverse events. We will contact the author for more information if necessary. Data extraction will be carried out independently by 2 authors.</p></sec><sec><label>2.6</label><title>Risk of bias assessment</title><p>The risk of bias of included studies will be assessed by using the latest version 2 of the Cochrane risk-of-bias tool for randomized trials (RoB2).<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>,<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> We will assess the risk of bias of primary outcome of the research. The nature of the effect of interest was “intention-to-treat” effect. The bias risk assessment will be conducted by MY and ZH independently, and inconsistencies will be solved by consensus. This tool has 5 domains: bias arising from the randomization process, bias due to deviations from intended interventions, bias due to missing outcome data, bias in measurement of the outcome, and bias in selection of the reported result. Each domain has 3 judgments: low risk of bias, some concerns, and high risk of bias. The results will be represented as “traffic light” plots and weighted bar plots.</p></sec><sec><label>2.7</label><title>Data analysis</title><p>R studio Version 1.2.1335 will be used for data analysis. We will pool data that do not have significant clinical heterogeneity. Binary variables will be represented by risk ratio (RR) and 95% confidence interval (CI) and continuous variables will be represented by mean difference (MD) and 95% CI.<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> The statistical heterogeneity will be identified by using <italic>χ</italic><sup>2</sup> test with significance level of α = .1. At the same time, we will use <italic>I</italic><sup><italic>2</italic></sup> statistics to quantify the size of heterogeneity. <italic>I</italic><sup><italic>2</italic></sup> ≥50% will be considered as significant heterogeneity.<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]</sup> If quantitative synthesis is not appropriate, the results will be presented as tables.</p></sec><sec><label>2.8</label><title>Subgroup analysis</title><p>We will conduct subgroup analysis based on pre-set subgroup hypotheses to explore the heterogeneity between studies. Subgroup hypotheses include the following: gender (male/female), control group (no treatment or placebo/conventional treatment), treatment period (depending on data).</p></sec><sec><label>2.9</label><title>Sensitivity analysis</title><p>We will conduct sensitivity analysis to explore whether the results are robust. We will carry out sensitivity analysis by using different effect measures (RR or OR) and statistical models (fixed effect model or random effects model). In addition, we also included studies without clear diagnostic criteria into the analysis to explore the impact of the presence or not of diagnostic criteria on the research results.</p></sec><sec><label>2.10</label><title>Publication bias</title><p>If >10 studies are included, we will conduct publication bias assessment. We will assess publication bias by funnel chart and quantify publication bias by Egger test. Funnel plot is asymmetric or <italic>P</italic> < .05 in Egger test indicates the existence of publication bias.</p></sec><sec><label>2.11</label><title>Certainty of evidence</title><p>Finally, we will evaluate the certainty of the evidence by using Grading of Recommendations Assessment, Development and Evaluate system (GRADE). The quality of the evidence will be rated as high, medium, low, or very low. Finally, the main results of this result will be presented as a summary of table.</p></sec><sec><label>2.12</label><title>Patient and public involvement</title><p>No patients and public will be involved in the study.</p></sec><sec><label>2.13</label><title>Ethics and dissemination</title><p>Ethical approval is not needed. Our findings will also be published in peer-reviewed journals.</p></sec></sec><sec><label>3</label><title>Discussion</title><p>RA is a chronic and recurrent rheumatic immune disease. In China, TCM is often used as an adjuvant treatment for RA and TGP is one of the representative drugs. The current systematic review of the drug in the treatment of RA has flaws. Therefore, this study will comprehensively summarize and evaluate the evidence for the treatment of RA with GLP. To ensure the quality of the methodology, this study will use the latest version of the risk of bias assessment tool. In addition, we will evaluate the certainty of the evidence and present the results as summary of table, which will be more conducive to the clinical application of the results of this study.</p><sec><label>3.1</label><title>Amendments</title><p>In the process of research, if there is any need to modify our plan, we will update our plan in time.</p></sec></sec><sec><title>Author contributions</title><p><bold>Conceptualization:</bold> Ce Tang , Lianghong Ye.</p><p><bold>Data curation:</bold> Zhipeng Hu, Maoyi Yang.</p><p><bold>Formal analysis:</bold> Wenxiang Wang, Tingting Kuang.</p><p><bold>Investigation:</bold> Gang Fan, Yi Zhang.</p><p><bold>Methodology:</bold> Maoyi Yang, Zhipeng Hu.</p><p><bold>Project administration:</bold> XiuHua Liu.</p><p><bold>Software:</bold> Ce Tang.</p><p><bold>Visualization:</bold> Lianghong Ye, Zhipeng Hu.</p><p><bold>Writing – original draft:</bold> Ce Tang.</p><p><bold>Writing – review & editing:</bold> Maoyi Yang.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: CI = confidence interval, CRP = C-reactive protein, ESR = erythrocyte sedimentation rate, GRADE = Grading of Recommendations Assessment, Development and Evaluate system, IL-1 = Interleukin-1, MD = mean difference, RA = Rheumatoid arthritis, RCTs = randomized controlled trials, RF = rheumatoid factor, RR = risk ratio, TGP = Total glucosides of paeony, TNF-α = tumor necrosis factor-α, WOMAC = Western Ontario and McMaster.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Tang C, Ye L, Hu Z, Wang W, Kuang T, Fan G, Zhang Y, Liu X, Yang M. Efficacy and safety of total glucosides of paeony for rheumatoid arthritis: A protocol for systematic review and meta-analysis. <italic>Medicine</italic>. 2020;99:39(e22224).</p></fn><fn fn-type=\"equal\"><p>CT, LY, and ZH contributed equally to this work and are co-first authors.</p></fn><fn fn-type=\"supported-by\"><p>Funding: This work will be supported by the National Natural Science Foundation of China (No. 81903922), the National Key Research and Development Program of China (No. 2017YFC1703900), Xinglin Scholars Research Promotion Project of Chengdu University of Traditional Chinese Medicine (BSH2019002), Chongqing Science and Technology Bureau and the Health Commission joined the Chinese medicine science and technology project (ZY201802041).</p></fn><fn fn-type=\"COI-statement\"><p>The authors report no conflicts of interest in this research.</p></fn><fn fn-type=\"other\"><p>The datasets generated during and/or analyzed during the current study are available from the 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991405</article-id><article-id pub-id-type=\"pmc\">PMC7523792</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-07598</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022025</article-id><article-id pub-id-type=\"art-access-id\">22025</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3700</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Systematic Review</subject></subj-group></article-categories><title-group><article-title>Arthroscopic capsular release for the treatment of post-stroke frozen shoulder</article-title><subtitle>A protocol for systematic review</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Zong</surname><given-names>Long-ze</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ma</surname><given-names>Li</given-names></name><degrees>MB</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-6178-6663</contrib-id><name><surname>Liu</surname><given-names>Ying-ying</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Department of Joint Surgery, Yanan University Affiliated Hospital, Yan’an, China</aff><aff id=\"aff2\"><label>b</label>Department of Neurology, Yanan University Affiliated Hospital, Yan’an, China</aff><aff id=\"aff3\"><label>c</label>Third Ward of Neurology Department, Cardiology and Cerebrovascular Specialty Section, Yanan University Affiliated Hospital, Yan’an, China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Ying-ying Liu, Third Ward of Neurology Department, Cardiology and Cerebrovascular Specialty Section, Yanan University Affiliated Hospital, Intersection of Shuangyong Avenue and Yongxiang Road, Yan’an, Shaanxi 716000, China (e-mail: <email>1650477908@qq.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22025</elocation-id><history><date date-type=\"received\"><day>31</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>3</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22025.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>This study will assess the efficacy and safety of arthroscopic capsular release (ACR) for the treatment of post-stroke frozen shoulder (PSFS).</p></sec><sec sec-type=\"methods\"><title>Methods:</title><p>We will carry out a systematic study of randomized controlled trials that assess the efficacy and safety of ACR for PSFS. We will search all potential records for any eligible trials from selected electronic databases (MEDLINE, EMBASE, Cochrane Library, Web of Science, Chinese Biomedical Literature Database, WANGFANG, and China National Knowledge Infrastructure) and grey literature sources from inception to the present. Two authors will independently perform study selection, data extraction, and study quality assessment. Any disagreement will be solved by a third author via consultation. Statistical analysis will be carried out by RevMan 5.3 software.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>This study will comprehensively summarize current eligible studies to systematically assess the efficacy and safety of ACR for PSFS.</p></sec><sec sec-type=\"conclusion\"><title>Conclusion:</title><p>This study will provide evidence to determine whether ACR is an effective management for patients with PSFS.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>arthroscopic capsular release</kwd><kwd>efficacy</kwd><kwd>frozen shoulder</kwd><kwd>stroke</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>Yan’an Diabetes Prevention Research and Technology Innovation Team</funding-source><award-id>2015CXTD-09</award-id><principal-award-recipient>Not Applicable</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Stroke is a major health problem worldwide.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> It is also one of the leading causes of serious long-term disability, such as difficulty in swallow, speech problem, urinary or bowel incontinence, depression, anxiety, emotional problems, limbs paralysis, numbness, and pain (including shoulder pain).<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>–<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> Several studies report that frozen shoulder (FS) may be one of the most substantial reasons of post-stroke FS (PSFS).<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>–<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> It is reported that about 56.6% stroke patients affect PSFS.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> In addition, PSFS also can be identified in 77% stroke patients with hemiplegic shoulder pain.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>,<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> If such disorder cannot be treated effectively, it greatly affects quality of life in those patients.</p><p>Arthroscopic capsular release (ACR) is reported to manage PSFS.<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>–<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]</sup> However, evidence from previous studies has been conflicting, and their results are inconsistent.<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>–<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]</sup> In addition, no existing systematic review examines the efficacy and safety of ACR for the treatment of PSFS. Thus, this is the first systematic review to evaluate the efficacy and safety of ACR for the treatment of PSFS.</p></sec><sec><label>2</label><title>Methods and analysis</title><sec><label>2.1</label><title>Study registration</title><p>This study protocol has been registered through INPLASY202070128. We organized it based on the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISRMA) Protocol statement.<sup>[<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</sup></p></sec><sec><label>2.2</label><title>Inclusion criteria</title><sec><label>2.2.1</label><title>Types of studies</title><p>In this study, we will only consider randomized controlled trials (RCTs) for inclusion, which evaluate the efficacy and safety of ACR for PSFS. Besides RCTs, all other studies will be excluded.</p></sec><sec><label>2.2.2</label><title>Types of interventions</title><p>Patients in the treatment group were treated with ACR alone. Control treatments can be any intervention, such as conventional medication. We will exclude comparators involving ACR.</p></sec><sec><label>2.2.3</label><title>Types of participants</title><p>All participants with a confirmed diagnosis of PSFS will be included. There will be no restrictions regarding the age, sex, country, and other factors.</p></sec><sec><label>2.2.4</label><title>Types of outcome measurements</title><p>The primary outcome is shoulder pain, as measured by any pain scale, such as Numeric Rating Scale.</p><p>The secondary outcomes are shoulder function (as evaluated by associated indexes, such as Shoulder Pain and Disability Index), shoulder motion range (as examined by relevant tool, such as Range of Joint Motion Evaluation Chart), shoulder muscle strength (as identified by any tool, such as Cybex Norm isokinetic dynamometer), health-related quality of life (as appraised by any connected questionnaire, such as 36-Item Short Form Survey), and adverse events.</p></sec></sec><sec><label>2.3</label><title>Search strategy</title><p>To identify all relevant articles, we will undertake literature search from both electronic databases and grey literature sources to avoid missing potential studies. We will not limit language and publication status. First, we will search the following electronic databases from inception to the present in MEDLINE, EMBASE, Cochrane Library, Web of Science, Chinese Biomedical Literature Database, WANGFANG, and China National Knowledge Infrastructure. We will create search strategy sample of MEDLINE in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>. Similar search strategy for other electronic databases will be modified and adapted. Second, we will examine grey literature sources, such as conference proceedings, reference list of included studies, and ongoing trials from websites of clinical trial registry.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Search strategy of MEDLINE.</p></caption><graphic xlink:href=\"medi-99-e22025-g001\"/></table-wrap></sec><sec><label>2.4</label><title>Data collection and analysis</title><sec><label>2.4.1</label><title>Selection of studies</title><p>Two authors will independently carry out study selection based on the predefined eligibility criteria. Any division will be solved by a third author through discussion. First, all searched citations will be imported to EndNote X9, and all duplicates will be removed. Second, we will check titles/abstracts of the potential studies, and will eliminate any irrelevant one. Third, we will read full-text of the remaining articles against all inclusion criteria, and all fulfilled studies will be included. We will record all excluded studies with reasons. The results of study selection will be presented in a PRISMA flowchart.</p></sec><sec><label>2.4.2</label><title>Data extraction and management</title><p>Two independent authors will extract data using a pre-designed data extraction form in all eligible trials. Any divergences will be resolved by a third author through consultation. The extracted data comprise of title, first author, publication time, patient characteristics, trial design, trial setting, sample size, details of interventions and controls, outcome indicators, results, conclusion, follow-up information, conflict of interest, and other essential data.</p></sec><sec><label>2.4.3</label><title>Missing data dealing with</title><p>We will contact original trial authors to obtain any unclear or missing data if it occurs. Otherwise, we will analyze available data and will discuss its potential affects to this study.</p></sec></sec><sec><label>2.5</label><title>Study quality assessment</title><p>Two authors will independently assess study quality of each eligible trial using Cochrane Risk of Bias Tool. We will appraise each study through 7 aspects, and each one will be valued as low, unclear, or high risk of bias. Any different views will be figured out with the help of a third author through discussion.</p></sec><sec><label>2.6</label><title>Statistical analysis</title><p>We will perform statistical analysis using RevMan 5.3 software. All continuous outcome indicators will be expressed using weighted mean difference (MD) or standard MD with 95% confidence intervals (95% CIs), and all dichotomous outcome indicators will be estimated using risk ratio with 95% CIs. We will check heterogeneity across included trials using <italic>I</italic><sup><italic>2</italic></sup> statistic. <italic>I</italic><sup><italic>2</italic></sup> ≤50% indicates acceptable heterogeneity, and we will use a fixed-effects model. <italic>I</italic><sup><italic>2</italic></sup> > 50% suggests remarkable heterogeneity, and we will employ a random-effects model. Whenever necessary under acceptable heterogeneity, we will carry out a meta-analysis based on the sufficient similarity in study information, patient characteristics, details of intervention and control, and study quality. Otherwise, if we identify considerable heterogeneity, we will conduct a subgroup analysis to explore its sources. If a meta-analysis is deemed not to be undertaken, we will report study results using a narrative summary.</p></sec><sec><label>2.7</label><title>Additional analysis</title><sec><label>2.7.1</label><title>Subgroup analysis</title><p>We will undertake a subgroup analysis according to the different study information, participant patient characteristics, variations of intervention and control, and study quality.</p></sec><sec><label>2.7.2</label><title>Sensitivity analysis</title><p>We will conduct a sensitivity analysis to test the robustness of the merged outcomes by excluding trials with low quality.</p></sec><sec><label>2.7.3</label><title>Reporting bias</title><p>We will examine reporting bias using funnel plot and Egger regression test when over 10 RCTs are eligible on the same outcome indicator.<sup>[<xref rid=\"R21\" ref-type=\"bibr\">21</xref>,<xref rid=\"R22\" ref-type=\"bibr\">22</xref>]</sup></p></sec></sec><sec><label>2.8</label><title>Grading the quality of evidence</title><p>The quality of evidence for all outcome indicators will be appraised using the Grading of Recommendations Assessment, Development, and Evaluation.<sup>[<xref rid=\"R23\" ref-type=\"bibr\">23</xref>]</sup> Each outcome indicator will be graded into 4 levels: high, moderate, low, and very low quality.</p></sec><sec><label>2.9</label><title>Ethics and dissemination</title><p>This study will not need ethical approval, because it will not collect individual patient data. We expect to publish this study on a peer-reviewed journal or a relevant conference or meeting.</p></sec></sec><sec><label>3</label><title>Discussion</title><p>PSFS is one of the most common complications in stroke survivors, which greatly affect quality of life for them. Therefore, effective managements are needed to treat PSFS. Numerous studies reported that ACR has been used for treating PSFS effectively. However, there is no systematic review specifically relevant to ACR for PSFS, which may restrict its clinical application. Thus, this study will first investigate the efficacy and safety of ACR for PSFS. Its results may provide robust evidence for both clinical practice and patients</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization:</bold> Long-ze Zong, Ying-ying Liu.</p><p><bold>Data curation:</bold> Long-ze Zong, Li Ma, Ying-ying Liu.</p><p><bold>Formal analysis:</bold> Long-ze Zong, Li Ma, Ying-ying Liu.</p><p><bold>Investigation:</bold> Ying-ying Liu.</p><p><bold>Methodology:</bold> Li Ma.</p><p><bold>Project administration:</bold> Ying-ying Liu.</p><p><bold>Resources:</bold> Long-ze Zong, Li Ma.</p><p><bold>Software:</bold> Long-ze Zong, Li Ma.</p><p><bold>Supervision:</bold> Ying-ying Liu.</p><p><bold>Validation:</bold> Long-ze Zong, Li Ma, Ying-ying Liu.</p><p><bold>Visualization:</bold> Long-ze Zong, Li Ma, Ying-ying Liu.</p><p><bold>Writing – original draft:</bold> Long-ze Zong, Li Ma, Ying-ying Liu.</p><p><bold>Writing – review & editing:</bold> Long-ze Zong, Ying-ying Liu.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: ACR = arthroscopic capsular release, CIs = confidence intervals, PRISRMA = Preferred Reporting Items for Systematic Reviews and Meta-Analysis, PSFS = post-stroke frozen shoulder, RCTs = randomized controlled trials.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Zong Lz, Ma L, Liu Yy. Arthroscopic capsular release for the treatment of post-stroke frozen shoulder: A protocol for systematic review. <italic>Medicine</italic>. 2020;99:39(e22025).</p></fn><fn fn-type=\"supported-by\"><p>This study was supported by Yan’an Diabetes Prevention Research and Technology Innovation Team (2015CXTD-09). 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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991472</article-id><article-id pub-id-type=\"pmc\">PMC7523793</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-03237</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022413</article-id><article-id pub-id-type=\"art-access-id\">22413</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>5300</subject></subj-group><subj-group><subject>Research Article</subject><subject>Observational Study</subject></subj-group></article-categories><title-group><article-title>Nomogram to predict risk for early ischemic stroke by non-invasive method</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Shuliang</given-names></name><degrees>Master Degree</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ma</surname><given-names>Chunye</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0001-7350-4187</contrib-id><name><surname>Zhang</surname><given-names>Ce</given-names></name><degrees>Master Degree</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Shi</surname><given-names>Rui</given-names></name><degrees>Bachelor's Degree</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Schaller.</surname><given-names>Bernhard</given-names></name></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Department of Neurology</aff><aff id=\"aff2\"><label>b</label>Clinical Drug Trial Institution, The Second Hospital of Dalian Medical University, Dalian, China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Ce Zhang, The Second Hospital of Dalian Medical University, Dalian, China (e-mail: <email>zhangce88@126.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22413</elocation-id><history><date date-type=\"received\"><day>20</day><month>4</month><year>2020</year></date><date date-type=\"rev-recd\"><day>20</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22413.pdf\"/><abstract><title>Abstract</title><p>Stroke is the acute onset of neurological deficits and is associated with high morbidity, mortality, and disease burden. In the present study, we aimed to develop a scientific, nomogram for non-invasive predicting risk for early ischemic stroke, in order to improve stroke prevention efforts among high-risk groups. Data were obtained from a total of 2151 patients with early ischemic stroke from October 2017 to September 2018 and from 1527 healthy controls. Risk factors were examined using logistic regression analyses. Nomogram and receiver operating characteristic (ROC) curves were drawn, cutoff values were established. Significant risk factors for early ischemic stroke included age, sex, blood pressure, history of diabetes, history of genetic, history of coronary heart disease, history of smoking. A nomogram predicting ischemic stroke for all patients had an internally validated concordance index of 0.911. The area under the ROC curve for the logistic regression model was 0.782 (95% confidence interval [CI]: 0.766–0.799, <italic>P</italic> < .001), with a cutoff value of 2.5. The nomogram developed in this study can be used as a primary non-invasive prevention tool for early ischemic stroke and is expected to provide data support for the revision of current guidelines.</p></abstract><kwd-group><title>Keywords</title><kwd>early ischemic stroke</kwd><kwd>non-invasive factors</kwd><kwd>risk prediction</kwd><kwd>nomogram</kwd><kwd>screening</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Stroke refers to the acute onset of neurological deficits due to impairments in local blood circulation, with symptoms lasting at least 24 hours.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> The incidence of ischemic stroke is higher than that of hemorrhagic stroke, accounting for 60% to 70% of all stroke cases.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> Stroke is associated with high morbidity, mortality, and disease burden, significantly impacting both individual families and society.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> The disease burden of stroke continues to increase worldwide, further exacerbating the adverse socio-economic effects of the disease.<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> The World Health Organization has predicted that if the mortality rate remains unchanged, nearly 4 million people will die from stroke each year in China by 2030. Thus, stroke prevention is critical for promoting human health and reducing social burden. Non-invasive methods for actively assessing stroke risk are required to ensure appropriate intervention and stroke control in high-risk groups.<sup>[<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup></p><p>The 2018 Guidelines for Early Treatment of Acute Ischemic Stroke issued by the American Heart Association/American Stroke Association,<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> and the 2018 Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke issued by the Chinese Medical Association<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup> have highlighted the need for active control of vascular risk factors for effective stroke prevention. Despite a thorough review, the authors of these guidelines failed to identify a scientific, non-invasive scale for assessing early ischemic stroke risk. Given the urgent need for a practical, non-invasive system for evaluating stroke risk, we developed a nomogram based on the characteristics of ischemic stroke and related factors.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> Self-assessments can be performed at home, providing a basis for the prevention of ischemic stroke in high-risk individuals. In the present study, we used this nomogram to compare data between patients with early ischemic stroke and healthy controls.</p></sec><sec><label>2</label><title>Methods and materials</title><sec><label>2.1</label><title>Date collection</title><p>We collected all patients data from 2151 patients with early ischemic stroke treated at our neurology department from October 1, 2017 to September 30, 2018. The patient sample included 1289 men (59.92%) and 862 women (40.08%), with an average age of 66.9 ± 11.9 years. A simple random sampling method was used to collect data from 1527 healthy controls, including 787 men (51.53%) and 740 women. The average age of the controls was 59.6 ± 13.9 years.</p></sec><sec><label>2.2</label><title>Investigation method</title><p>We collected data regarding the following input variables: sex (male/female), age (years), height (cm), weight (kg), history of smoking (yes/no), history of alcohol use (yes/no), history of hypertension (yes/no), history of diabetes (years), family history (yes/no), history of coronary heart disease (yes/no), bilateral arterial blood pressure (mmHg), and pulse (beats/min). The occurrence of ischemic stroke was regarded as the output variable. All data were desensitized and encrypted to remove identifying information. Thus, the requirement for informed consent was waived by the hospital ethics committee. Ethics committee approval no. 90, 2017, review by ethics committee of the Second Affiliated Hospital of Dalian Medical University.</p></sec><sec><label>2.3</label><title>Inclusion and exclusion criteria</title><p>All included patients were diagnosed with early ischemic stroke in accordance with 2018 American Heart Association/American Stroke Association guidelines.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> Patients were diagnosed based on acute onset, the presence of focal neurological deficits, imaging lesions, or symptoms/physical signs persisting for >24 hours. Non-vascular causes and cerebral hemorrhage were excluded based on brain computed tomography (CT)/magnetic resonance imaging (MRI) findings. Additional inclusion criteria were as follows: first initial ischemic stroke and complete information regarding the factors investigated.</p><p>Patients with incomplete data or data that could not be effectively desensitized were excluded, along with those who had cerebral infarction in combination with other diseases, except for diabetes, high blood pressure, and high blood lipid levels. Patients with a National Institutes of Health Stroke Scale (NIHSS) score ≥16 points were also excluded.</p><p>Inclusion criteria for the healthy control group were as follows: no abnormalities during routine health examinations and complete information regarding all factors investigated. Those with incomplete information, information that could not be effectively desensitized, or a history of disease based on hospital medical records were excluded.</p><p>In accordance with these inclusion and exclusion criteria, data were extracted from the Hospital Information System using the SQL language.</p></sec><sec><label>2.4</label><title>Statistical analysis</title><p>SPSS version 13.0 (Chicago, IL, USA) was used for analyses. The measurement data were expressed as the mean ± standard deviation. Two independent samples <italic>t</italic> tests were used to compare data between the groups. Count data were expressed as quantities, and the relevant comparisons were performed using Chi-square tests. The level of statistical significance was set at <italic>P</italic> < .05. Modeling analyses were performed using the Backward LR method in SPSS Modeler 14.1. To calculate the accuracy of the model, the inclusion and exclusion criteria were set to 0.05 and 0.1, respectively. Receiver operating characteristic (ROC) curves were then drawn, and the cutoff value was calculated.</p><p>We used R3.6.1 software to draw the nomogram diagram and calculate the c-index.</p></sec></sec><sec><label>3</label><title>Results</title><sec><label>3.1</label><title>General characteristics</title><p>The general characteristics of the patient and control groups are presented in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>. Non-invasive factors exhibiting significant differences between the groups were included in the multivariate model (<italic>P</italic> < .05).</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Screening for non-invasive risk factors for early ischemic stroke.</p></caption><graphic xlink:href=\"medi-99-e22413-g001\"/></table-wrap></sec><sec><label>3.2</label><title>Results of logistic regression analysis</title><p>The 13 non-invasive variables identified in Section 3.1 were included in the model for assessing ischemic stroke. A binomial modeling procedure was adopted, and main effects were examined while considering the significance threshold. The significance threshold was set to 0.05, the maximum iteration number was 20, and the maximum stepwise dichotomy was 5. Following Backward LR analysis, the following 6 factors were excluded: sex, blood pressure, genetic history, coronary heart disease history, smoking history, and diabetes history. Age was entered into the final model and was found to be statistically significant. The regression model fits good, and the results of the analysis are shown in Table <xref rid=\"T2\" ref-type=\"table\">2</xref>.</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Results of multivariate logistic regression analysis.</p></caption><graphic xlink:href=\"medi-99-e22413-g002\"/></table-wrap></sec><sec><label>3.3</label><title>Non-invasive nomogram for risk prediction</title><p>Based on the results of the logistic regression analysis, a non-invasive nomogram was developed using data for all patients based on 7 standard parameters. The internally validated c-index of this nomogram was 0.911 (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>). In leave-one-out cross-validation, the c-index for models based on data ranged from 0.869 to 0.967, suggesting similar model performance across different patient groups.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Nomogram non-invasive predicting risk for early ischemic stroke. Non-invasive factor including gender, history of diabetes, genetic history, history of coronary heart disease, history of smoking, age. After the scores of all factors were calculated and the total score was added up, the risk value of ischemic stroke was obtained by the total score corresponding to the risk score.</p></caption><graphic xlink:href=\"medi-99-e22413-g003\"/></fig><p>The area under curve of ROC for early ischemic stroke risk predicted model is 0.782 (95% confidence interval [CI]: 0.766–0.799, <italic>P</italic> < .001), and the ROC curve is shown in Fig. <xref ref-type=\"fig\" rid=\"F2\">2</xref>. Based on the largest Youden index, the cutoff value of the final scale was 2.5. High model accuracy was observed in model, ranging from 78.91% to 80.76% (Table <xref rid=\"T3\" ref-type=\"table\">3</xref>).</p><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>ROC curve of early ischemic stroke predicts model. The area under curve of ROC for early ischemic stroke risk predicted model is 0.782 (95% CI: 0.766–0.799, <italic>P</italic> < .001). CI = confidence interval; ROC = receiver operating characteristic.</p></caption><graphic xlink:href=\"medi-99-e22413-g004\"/></fig><table-wrap id=\"T3\" orientation=\"portrait\" position=\"float\"><label>Table 3</label><caption><p>Predictive accuracy (n = 3678).</p></caption><graphic xlink:href=\"medi-99-e22413-g005\"/></table-wrap></sec></sec><sec><label>4</label><title>Discussion</title><p>In the present study, we analyzed real-world clinical data to develop a non-invasive screening tool for early ischemic stroke, which achieved a prediction accuracy of up to 80.76%. Our nomogram could be used to non-invasive screening for early ischemic stroke patients to determine the need for deep therapy. Patient at low risk of early ischemic stroke may be managed expectantly to avoid the potential therapy. By contrast these at high risk of ischemic stroke may be candidates for early therapy. Thus, this nomogram can be used to aid in level 1 prevention of ischemic stroke, and will undoubtedly provide useful information for reducing morbidity and disease burden. In addition to its non-invasive nature and high degree of accuracy, our scale is advantageous in that it is convenient, easy to use, and associated with lower costs than more invasive screening measures. Big data mining may also aid in making monogram more accurate and effective. Furthermore, nomogram can be disseminated to non-professionals via the media or internet within a short period of time, increasing public awareness of risk factors for ischemic stroke.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup></p><p>In accordance with previous findings, our study supports the notion that identifying patients at high risk for early ischemic stroke and taking active and effective preventive measures is critical for controlling stroke incidence.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> Such preventive measures can reduce the incidence of ischemic stroke and decrease morbidity, disability, and mortality rates.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> For example, modifying lifestyle factors may aid in preventing stroke in high-risk populations. As >76% of strokes are first-episode cases, effective primary prevention is especially important for reducing the incidence of stroke.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup></p><p>Our results indicated that advanced age is among the most important factors influencing the development of ischemic stroke. Age ranging from 31 to 50 years or 51 to 70 years was negatively correlated with the occurrence of early ischemic stroke, while a positive correlation was observed in patients over 70 years of age. Indeed, recent studies have indicated that young and middle-aged patients account for only 9.5% to 17.4% of all cases of cerebrovascular disease.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> However, our analysis indicated that smoking exerted the greatest impact on the onset of ischemic stroke. Although the dangers of smoking are well known and the benefits of smoking cessation have been widely confirmed, smoking is not well controlled among Chinese patients with stroke, especially among patients over 40 years of age: although the smoking age has increased, the average smoking rate has increased as well.<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup></p><p>In accordance with previous research, our data indicated that hypertension is a key risk factor for early ischemic stroke.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>–<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</sup> Effective control of high blood pressure may therefore reduce the risk of early ischemic stroke. We also observed that coronary heart disease significantly increased the risk of ischemic stroke. Given that previous studies<sup>[<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]</sup> have reported that patients with ischemic stroke exhibit coronary artery lesions with obvious clinical symptoms, treatment and prevention of coronary heart disease may reduce ischemic stroke risk as well. In addition, studies have revealed that hyperglycemia leads to lipid condensation in the vascular endothelium and that atheroma plaque cleavage leads to the occurrence of ischemic stroke.<sup>[<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</sup> Epidemiological studies have further indicated that ischemic stroke tends to occur within families.<sup>[<xref rid=\"R19\" ref-type=\"bibr\">19</xref>]</sup> In accordance with this finding, we observed an increased risk of ischemic stroke in patients with a family history of ischemic stroke. Thus, monitoring and prevention efforts should also focus on relevant family history.</p><p>Although the present study included a large sample of patients and controls, further large-scale, multicenter studies are required to improve the accuracy of the nomogram and the robustness/universality of the model. Such studies will provide a more reliable and comprehensive basis for controlling early ischemic stroke.</p><p>In this study, we developed a non-invasive screening tool for ischemic stroke using clinical data, the prediction accuracy ranged from 78.91% to 80.76%. Our findings may support the revision of current guidelines for stroke prevention. But the study also had several limitations. First, the study was a single-centered observational design. Second, given the nature of the study, plausible mechanisms for Ischemic Stroke were not addressed. Third, the accuracy of the model needs to be improved. Further studies are needed to improve the prediction effect of the model. Make better improvement on the effect of the nomogram.</p></sec><sec><label>5</label><title>Conclusion</title><p>The nomogram developed in this study can be used as a primary non-invasive prevention tool for early ischemic stroke and is expected to provide data support for the revision of current guidelines.</p></sec><sec><title>Acknowledgments</title><p>The authors would like to thank Editage [<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.editage.cn/\">www.editage.cn</ext-link>] for English language editing.</p></sec><sec><title>Author contributions</title><p><bold>Formal analysis:</bold> Ce Zhang.</p><p><bold>Investigation:</bold> Shulaing Chen, Ce Zhang.</p><p><bold>Project administration:</bold> Ce Zhang.</p><p><bold>Software:</bold> Rui Shi.</p><p><bold>Validation:</bold> Chunye Ma, Shuliang Chen.</p><p><bold>Visualization:</bold> Rui Shi, Ce Zhang.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: CT = computed tomography, MRI = magnetic resonance imaging, NIHSS = National Institutes of Health Stroke Scale, OR = odds ratio, ROC = receiver operating characteristic.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Chen S, Ma C, Zhang C, Shi R. Nomogram to predict risk for early ischemic stroke by non-invasive method. <italic>Medicine</italic>. 2020;99:39(e22413).</p></fn><fn fn-type=\"other\"><p>SC is the first author.</p></fn><fn fn-type=\"COI-statement\"><p>There is no funding for this study and no funding conflicts between authors.</p></fn><fn fn-type=\"other\"><p>The authors declare that there is no duality of interest associated with this manuscript.</p></fn><fn fn-type=\"con\"><p>Contribution statement: All authors were responsible for drafting and revising this review. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991445</article-id><article-id pub-id-type=\"pmc\">PMC7523795</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-02093</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022335</article-id><article-id pub-id-type=\"art-access-id\">22335</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>5700</subject></subj-group><subj-group><subject>Research Article</subject><subject>Clinical Case Report</subject></subj-group></article-categories><title-group><article-title>Oral plasmablastic lymphoma</article-title><subtitle>A case report</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0001-9841-7856</contrib-id><name><surname>Zizzo</surname><given-names>Maurizio</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zanelli</surname><given-names>Magda</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Martiniani</surname><given-names>Roberta</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Sanguedolce</surname><given-names>Francesca</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff5\"><sup>e</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>De Marco</surname><given-names>Loredana</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Martino</surname><given-names>Giovanni</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff6\"><sup>f</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Parente</surname><given-names>Paola</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff7\"><sup>g</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Annessi</surname><given-names>Valerio</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Manzini</surname><given-names>Lorenzo</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ascani</surname><given-names>Stefano</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff8\"><sup>h</sup></xref></contrib></contrib-group><contrib-group><contrib contrib-type=\"editor\"><name><surname>Saranathan.</surname><given-names>Maya</given-names></name></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Surgical Oncology Unit, Azienda Unità Sanitaria Locale-IRCCS di Reggio Emilia</aff><aff id=\"aff2\"><label>b</label>Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, Modena</aff><aff id=\"aff3\"><label>c</label>Pathology Unit, Azienda Unità Sanitaria Locale-IRCCS di Reggio Emilia, Reggio Emilia</aff><aff id=\"aff4\"><label>d</label>Onco-Hematology Unit, Azienda Ospedaliera Santa Maria di Terni, University of Perugia, Terni</aff><aff id=\"aff5\"><label>e</label>Pathology Unit, Azienda Ospedaliero-Universitaria, Ospedali Riuniti di Foggia, Foggia</aff><aff id=\"aff6\"><label>f</label>Hematology Unit, CREO, Azienda Ospedaliera di Perugia, University of Perugia, Perugia</aff><aff id=\"aff7\"><label>g</label>Pathology Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Foggia</aff><aff id=\"aff8\"><label>h</label>Pathology Unit, Azienda Ospedaliera Santa Maria di Terni, University of Perugia, Terni, Italy.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Maurizio Zizzo, Surgical Oncology Unit, Azienda Unità Sanitaria Locale-IRCCS di Reggio Emilia, Viale Risorgimento, 80, 42123 Reggio Emilia, Italy (e-mail: <email>zizzomaurizio@gmail.com</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22335</elocation-id><history><date date-type=\"received\"><day>7</day><month>3</month><year>2020</year></date><date date-type=\"rev-recd\"><day>2</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22335.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"intro\"><title>Introduction:</title><p>Plasmablastic lymphoma (PBL) is an uncommon and aggressive large B-cell lymphoma commonly diagnosed in human immunodeficiency viruses -positive patients. Oral cavity is the most commonly PBL affected site. Most oral PBLs presented as asymptomatic swellings, frequently associated with ulcerations and bleeding. Most cases lacked B-symptoms, suggesting a more local involvement of the disease. No standard treatment is yet for oral PBL. Five-year survival rate recorded no more than 33.5%.</p></sec><sec><title>Patient concerns:</title><p>A 39-year-old male presented to Dental Clinic with 1 month swelling of the oral cavity, in absence of any other symptoms or signs. He followed antibiotic therapy just on suspicion of an oral abscess and later oral surgical treatment on suspicion of bone neoplasm.</p></sec><sec><title>Diagnosis:</title><p>Surgical specimen analysis highlighted a diffuse infiltrate of large-sized atypical cells with plasmablastic appearance and plasma cell phenotype. Oral cavity PBL was diagnosed. Blood tests recorded mild lymphopenia and positive human immunodeficiency viruses serology.</p></sec><sec><title>Interventions:</title><p>Patient underwent chemotherapy including intrathecal methotrexate prophylaxis, in addition to a highly active antiretroviral therapy.</p></sec><sec><title>Outcomes:</title><p>At 12 months from diagnosis, patient recorded complete hematological remission.</p></sec><sec sec-type=\"conclusion\"><title>Conclusions:</title><p>Oral PBL diagnosis requires a high level of suspicion and awareness both by physicians and pathologists. They should be aware of the extent of such disease which is often mistaken as oral abscess or infected tooth, thus leading to delay the most appropriate diagnostic evaluation. As PBL is an aggressive non-Hodgkin lymphoma, a delayed diagnosis might negatively impact on both treatment and survival.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>B-cells</kwd><kwd>Epstein-Barr virus</kwd><kwd>human immunodeficiency viruses</kwd><kwd>oral cavity</kwd><kwd>plasmablastic lymphoma</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Plasmablastic lymphoma (PBL) is an uncommon and aggressive large B-cell lymphoma showing an immunoblastic and plasmablastic morphology with plasmacytic immunophenotype.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> PBL is commonly diagnosed in human immunodeficiency viruses (HIV)-positive patients, but it can also be detected in HIV-negative patients and those affected by immunosuppressive conditions.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> PBL etiology is unclear, although the role of Epstein-Barr virus (EBV) is frequently assumed, as it is detected in 78% cases.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> Moreover, due to its low incidence, PBL prognostic features are rarely understood, especially when PBL affects oral and maxillofacial regions, thus impairing an appropriate therapeutic management.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup></p><p>Our case corroborates the paramount role that early clinical suspicion and correct diagnosis play in choosing the most appropriate treatment. Our patient underwent antibiotic therapy just on suspicion of an oral abscess. Later, on suspicion of bone neoplasm, a complete surgical removal of his lesion was carried out.</p></sec><sec><label>2</label><title>Case Presentation</title><p>A 39-year-old male presented to Dental Clinic with 1 month swelling of the oral cavity. Following administration of oral antibiotics due to suspected oral abscess, the patient worsened (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>). His clinical records included juvenile adenoidectomy, cigarette smoking and recurrent varicella-zoster virus infections occurred in the previous year. Panoramic dental x-rays showed a lesion of left upper dental arch (Fig. <xref ref-type=\"fig\" rid=\"F2\">2</xref>). On suspicion of primitive bone tumor, radical excision was carried out, including the removal of a 40 mm x 18 mm soft tissue area, in addition to removal of 15 mm x 8 mm x 7 mm maxillary bone segment and 2 teeth (Fig. <xref ref-type=\"fig\" rid=\"F3\">3</xref>). Histology highlighted a diffuse infiltrate of large-sized atypical cells with plasmablastic appearance (Fig. <xref ref-type=\"fig\" rid=\"F4\">4</xref>). Neoplastic cells expressed a plasma cell phenotype which was CD138 (Fig. <xref ref-type=\"fig\" rid=\"F5\">5</xref>, <italic>left</italic>) and IRF4/MUM1 positive while being CD45, CD20 and PAX5 negative. A high proliferative index was recorded. In situ hybridization for EBV encoded small RNA showed positive results in neoplastic cells (Fig. <xref ref-type=\"fig\" rid=\"F5\">5</xref>, <italic>right</italic>). Oral cavity PBL was diagnosed. Subsequent blood tests recorded mild lymphopenia and positive HIV serology, revealing a previously unknown HIV infection.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Clinical image at diagnosis highlights the lesion of the oral cavity.</p></caption><graphic xlink:href=\"medi-99-e22335-g001\"/></fig><fig id=\"F2\" orientation=\"portrait\" position=\"float\"><label>Figure 2</label><caption><p>A dental panoramic radiography revealed a lesion of the upper left dental arch.</p></caption><graphic xlink:href=\"medi-99-e22335-g002\"/></fig><fig id=\"F3\" orientation=\"portrait\" position=\"float\"><label>Figure 3</label><caption><p>Clinical image at follow-up highlights the outcome of oral surgical treatment.</p></caption><graphic xlink:href=\"medi-99-e22335-g003\"/></fig><fig id=\"F4\" orientation=\"portrait\" position=\"float\"><label>Figure 4</label><caption><p>Histology highlights a diffuse infiltrate of large-sized atypical cells with a plasmablastic appearance.</p></caption><graphic xlink:href=\"medi-99-e22335-g004\"/></fig><fig id=\"F5\" orientation=\"portrait\" position=\"float\"><label>Figure 5</label><caption><p><italic>Left</italic> The neoplastic cells expressed a plasma cell phenotype, being positive for CD138. <italic>Right</italic> In situ hybridization for Epstein-Barr virus (EBV)-encoded small RNA (EBER) yielded a positive result in the neoplastic cells.</p></caption><graphic xlink:href=\"medi-99-e22335-g005\"/></fig><p>Patient was referred to a health facility specialized in lymphoma associated with immunodeficiency, where he underwent intensive chemotherapy including intrathecal methotrexate prophylaxis, in addition to a highly active antiretroviral therapy (HAART). At 12 months from diagnosis, patient recorded complete hematological remission and he is at present in good clinical condition.</p></sec><sec><label>3</label><title>Discussion</title><p>Following Kaposi's sarcoma, non-Hodgkin lymphoma (NHL) is the most common HIV-related neoplasm.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> Being approximately 2% of all primary extranodal lymphomas, many cases affect extranodal sites, including oral cavity and jaw bones.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> During pre-HAART era, NHL incidence was 60–200 times higher in HIV-infected patients than in HIV-non-infected ones.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> However, since mid-1990 s, when HAART was first introduced, NHL incidence has seemingly been declining.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> Currently, NHL risk is 80 to 100 times greater in HIV-positive patients than in HIV-negative ones.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> Acquired immune deficiency syndrome related NHLs are mainly aggressive high-grade B-cell lymphomas: among them, Burkitt lymphoma, diffuse large B-cell lymphoma, primary effusion lymphoma, being PBL the most common type.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup></p><p>In HIV-infected patients, PBL and NHL mainly happen at a young age, recording male predominance (5.7:1).<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref>,<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> PBL median age is 39 years (range 7–86).<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> On the contrary, a review including more than 400 diffuse large B-cell lymphoma HIV-non-infected patients recorded a 64-year median age (range 14–98 years).<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup></p><p>Although being relatively rare, oral cavity is the most commonly PBL affected site, followed by gastrointestinal tract.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> Lymph nodes, skin, bone and genitourinary tract are less frequently affected.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> As regards oral cavity, gingiva is the most commonly affected area, followed by palate, where PBL usually appears as a soft tissue lesion.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> Most oral PBLs presented as asymptomatic swellings, frequently associated with ulcerations and bleeding. Most cases lacked B-symptoms, suggesting a more local involvement of the disease.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup></p><p>A statistically significant association between HIV-positive patients and EBV-positive PBL ones emerged, thus suggesting that HIV infection might represent a permissive environment for chronic EBV infection and allow subsequent latency that would lead EBV-infected B-cells to malignancy.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup></p><p>Differential diagnosis for an expanding oral lesion includes primarily infectious diseases (viridans and other streptococci, <italic>Peptostreptococcus</italic> spp, <italic>Bacteroides</italic> spp, <italic>Actinomyces israelii</italic>, and <italic>Actinobacillus actinomycetemcomitans</italic>) in addition to malignant processes as primary squamous cell tumour, metastatic tumour, Kaposi's sarcoma, and other forms of lymphoma that may occur in the oral cavity (DLBCL not otherwise specified, ALK-positive large B-cell lymphoma, primary effusion lymphoma, plasmacytomas, Burkitt's lymphoma, and multiple myeloma).<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref>,<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> Neoplastic cells express plasma cell markers such as CD38, MUM1, CD138, VS38c, while showing negativity for typical B-cell antigens (eg, CD20, CD79α).<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup></p><p>No standard treatment is yet for oral PBL.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup> Different chemotherapy regimens have led to different results. HIV-infected patients who had been treated with HAART and chemotherapy showed better survival rates.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> However, many patients died in a very short follow-up time.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> Five-year survival rate recorded no more than 33.5%.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> EBV, B-symptoms and chemotherapy alone (without HAART) may contribute to such poor prognosis.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup></p></sec><sec><label>4</label><title>Conclusion</title><p>Oral PBL diagnosis requires a high level of suspicion and awareness both by physicians and pathologists. In particular, infectious disease clinicians, dentists, stomatologists, oral and maxillofacial surgeons should be aware of the extent of such disease which is often mistaken as oral abscess or infected tooth, thus leading to delay the most appropriate diagnostic evaluation. As PBL is an aggressive NHL, a delayed diagnosis might negatively impact on both treatment and survival.</p></sec><sec><title>Acknowledgments</title><p>We thank Dr Daniela Masi (AUSL-IRCCS di Reggio Emilia) for English editing.</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization:</bold> Maurizio Zizzo, Magda Zanelli, Stefano Ascani.</p><p><bold>Data curation:</bold> Maurizio Zizzo, Magda Zanelli, Roberta Martiniani, Francesca Sanguedolce, Loredana De Marco, Giovanni Martino, Paola Parente, Valerio Annessi, Lorenzo Manzini, Stefano Ascani.</p><p><bold>Formal analysis:</bold> Maurizio Zizzo, Magda Zanelli, Roberta Martiniani, Stefano Ascani.</p><p><bold>Investigation:</bold> Roberta Martiniani, Giovanni Martino, Stefano Ascani.</p><p><bold>Methodology:</bold> Maurizio Zizzo, Magda Zanelli, Roberta Martiniani, Giovanni Martino, Stefano Ascani.</p><p><bold>Project administration:</bold> Maurizio Zizzo, Magda Zanelli, Stefano Ascani.</p><p><bold>Resources:</bold> Roberta Martiniani, Giovanni Martino, Stefano Ascani.</p><p><bold>Supervision:</bold> Maurizio Zizzo, Magda Zanelli, Stefano Ascani.</p><p><bold>Writing – original draft:</bold> Maurizio Zizzo.</p><p><bold>Writing – review & editing:</bold> Maurizio Zizzo, Magda Zanelli.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: EBV = Epstein-Barr virus, HAART = highly active antiretroviral therapy, HIV = human immunodeficiency viruses, NHL = non-Hodgkin lymphoma, PBL = plasmablastic lymphoma.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Zizzo M, Zanelli M, Martiniani R, Sanguedolce F, De Marco L, Martino G, Parente P, Annessi V, Manzini L, Ascani S. Oral plasmablastic lymphoma: A case report. <italic>Medicine</italic>. 2020;99:39(e22335).</p></fn><fn fn-type=\"other\"><p>Ethical approval - Local ethics committee (Comitato Etico dell’Area Vasta Emilia Nord, Italy) ruled out any formal ethics approval for this particular case.</p></fn><fn fn-type=\"other\"><p>Patient Consent Statement - Written informed consent was obtained from patient for publication of this case report and all related images. A copy of the written consent is available at Editor-in-Chief of this journal.</p></fn><fn fn-type=\"COI-statement\"><p>The authors have no funding and conflicts of interest to disclose.</p></fn><fn fn-type=\"other\"><p>All data generated or analyzed during this study are included in this published article and its supplementary information files.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>[1]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rodrigues-Fernandes</surname><given-names>CI</given-names></name><name><surname>de Souza</surname><given-names>LL</given-names></name><name><surname>Santos-Costa</surname><given-names>SFD</given-names></name><etal/></person-group>\n<article-title>Clinicopathological analysis of oral plasmablastic lymphoma: a systematic review</article-title>. <source>J Oral Pathol Med</source>\n<year>2018</year>;<volume>47</volume>:<fpage>915</fpage>–<lpage>22</lpage>.<pub-id pub-id-type=\"pmid\">29917262</pub-id></mixed-citation></ref><ref id=\"R2\"><label>[2]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Riedel</surname><given-names>DJ</given-names></name><name><surname>Gonzalez-Cuyar</surname><given-names>LF</given-names></name><name><surname>Zhao</surname><given-names>XF</given-names></name><etal/></person-group>\n<article-title>Plasmablastic lymphoma of the oral cavity: a rapidly progressive lymphoma associated with HIV infection</article-title>. <source>Lancet Infect Dis</source>\n<year>2008</year>;<volume>8</volume>:<fpage>261</fpage>–<lpage>7</lpage>.<pub-id pub-id-type=\"pmid\">18353267</pub-id></mixed-citation></ref><ref id=\"R3\"><label>[3]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sarode</surname><given-names>SC</given-names></name><name><surname>Sarode</surname><given-names>GS</given-names></name><name><surname>Patil</surname><given-names>A</given-names></name></person-group>\n<article-title>Plasmablastic lymphoma of the oral cavity: a review</article-title>. <source>Oral Oncol</source>\n<year>2010</year>;<volume>46</volume>:<fpage>146</fpage>–<lpage>53</lpage>.<pub-id pub-id-type=\"pmid\">20138565</pub-id></mixed-citation></ref><ref id=\"R4\"><label>[4]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wagner</surname><given-names>VP</given-names></name><name><surname>Ortiz</surname><given-names>L</given-names></name><name><surname>da Silva</surname><given-names>HP</given-names></name><etal/></person-group>\n<article-title>Impact of highly active antiretroviral therapy in the development and remission of oral plasmablastic lymphoma</article-title>. <source>Indian J Dent Res</source>\n<year>2016</year>;<volume>27</volume>:<fpage>559</fpage>–<lpage>62</lpage>.<pub-id pub-id-type=\"pmid\">27966518</pub-id></mixed-citation></ref><ref id=\"R5\"><label>[5]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhattacharyya</surname><given-names>S</given-names></name><name><surname>Bains</surname><given-names>APS</given-names></name><name><surname>Sykes</surname><given-names>DL</given-names></name><etal/></person-group>\n<article-title>Lymphoid neoplasms of the oral cavity with plasmablastic morphology-a case series and review of the literature</article-title>. <source>Oral Surg Oral Med Oral Pathol Oral Radiol</source>\n<year>2019</year>;<volume>128</volume>:<fpage>651</fpage>–<lpage>9</lpage>.<pub-id pub-id-type=\"pmid\">31494113</pub-id></mixed-citation></ref></ref-list></back></article>\n"
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"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"letter\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Hemasphere</journal-id><journal-id journal-id-type=\"publisher-id\">HS9</journal-id><journal-title-group><journal-title>HemaSphere</journal-title></journal-title-group><issn pub-type=\"epub\">2572-9241</issn><publisher><publisher-name>Wolters Kluwer Health</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">33062948</article-id><article-id pub-id-type=\"pmc\">PMC7523798</article-id><article-id pub-id-type=\"publisher-id\">HemaSphere-2020-0148</article-id><article-id pub-id-type=\"doi\">10.1097/HS9.0000000000000483</article-id><article-id pub-id-type=\"art-access-id\">00016</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Letter</subject></subj-group><subj-group subj-group-type=\"hwp-journal-coll\"><subject>001</subject></subj-group></article-categories><title-group><article-title>SARS-CoV-2 in Myelodysplastic Syndromes: A Snapshot From Early Italian Experience</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Mossuto</surname><given-names>Sandra</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Attardi</surname><given-names>Enrico</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Alesiani</surname><given-names>Francesco</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Angelucci</surname><given-names>Emanuele</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Balleari</surname><given-names>Enrico</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Bernardi</surname><given-names>Massimo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Binotto</surname><given-names>Gianni</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Bosi</surname><given-names>Costanza</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff8\"><sup>8</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Calvisi</surname><given-names>Anna</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff9\"><sup>9</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Capodanno</surname><given-names>Isabella</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff10\"><sup>10</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Carbone</surname><given-names>Antonella</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><xref ref-type=\"aff\" rid=\"aff12\"><sup>12</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Castelli</surname><given-names>Andrea</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff13\"><sup>13</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Cerrano</surname><given-names>Marco</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff14\"><sup>14</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ciancia</surname><given-names>Rosanna</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff15\"><sup>15</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Cilloni</surname><given-names>Daniela</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff16\"><sup>16</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Clavio</surname><given-names>Marino</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff17\"><sup>17</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Clissa</surname><given-names>Cristina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff18\"><sup>18</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Crisà</surname><given-names>Elena</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff19\"><sup>19</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Crugnola</surname><given-names>Monica</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff20\"><sup>20</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Della Porta</surname><given-names>Matteo G.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff21\"><sup>21</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Di Renzo</surname><given-names>Nicola</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff22\"><sup>22</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Di Veroli</surname><given-names>Ambra</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><xref ref-type=\"aff\" rid=\"aff23\"><sup>23</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Fattizzo</surname><given-names>Roberto</given-names></name><xref ref-type=\"aff\" rid=\"aff24\"><sup>24</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Fava</surname><given-names>Carmen</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff25\"><sup>25</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Fenu</surname><given-names>Susanna</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><xref ref-type=\"aff\" rid=\"aff26\"><sup>26</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ferrara</surname><given-names>Ida L.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff27\"><sup>27</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Fianchi</surname><given-names>Luana</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><xref ref-type=\"aff\" rid=\"aff28\"><sup>28</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Filì</surname><given-names>Carla</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff29\"><sup>29</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Finelli</surname><given-names>Carlo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff30\"><sup>30</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Giai</surname><given-names>Valentina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff31\"><sup>31</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Frattini</surname><given-names>Francesco</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff32\"><sup>32</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Gaidano</surname><given-names>Valentina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff33\"><sup>33</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Guaragna</surname><given-names>Gianluca</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff34\"><sup>34</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Gumenyuk</surname><given-names>Svitlana</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><xref ref-type=\"aff\" rid=\"aff35\"><sup>35</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Latagliata</surname><given-names>Roberto</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><xref ref-type=\"aff\" rid=\"aff36\"><sup>36</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Mancini</surname><given-names>Stefano</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><xref ref-type=\"aff\" rid=\"aff37\"><sup>37</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Messa</surname><given-names>Emanuela</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff38\"><sup>38</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Molteni</surname><given-names>Alfredo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff39\"><sup>39</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Musto</surname><given-names>Pellegrino</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff40\"><sup>40</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Niscola</surname><given-names>Pasquale</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff41\"><sup>41</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Oliva</surname><given-names>Esther</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff42\"><sup>42</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Palumbo</surname><given-names>Giuseppe A.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff43\"><sup>43</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Pelizzari</surname><given-names>Annamaria</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff44\"><sup>44</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Pilo</surname><given-names>Federica</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff45\"><sup>45</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Poloni</surname><given-names>Antonella</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff46\"><sup>46</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Riva</surname><given-names>Marta</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff47\"><sup>47</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Rivellini</surname><given-names>Flavia</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff48\"><sup>48</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Sarlo</surname><given-names>Chiara</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><xref ref-type=\"aff\" rid=\"aff49\"><sup>49</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Sciumé</surname><given-names>Mariarita</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff24\"><sup>24</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Secchi</surname><given-names>Roberto</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><xref ref-type=\"aff\" rid=\"aff50\"><sup>50</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Selleri</surname><given-names>Carmine</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff27\"><sup>27</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Tafuri</surname><given-names>Agostino</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><xref ref-type=\"aff\" rid=\"aff51\"><sup>51</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Santini</surname><given-names>Valeria</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib></contrib-group><aff id=\"aff1\"><label>1</label>Italian MDS Foundation - ETS (FISIM - ETS), Bologna, Italy</aff><aff id=\"aff2\"><label>2</label>Hematology, University of Florence, AOU Careggi, Florence, Italy</aff><aff id=\"aff3\"><label>3</label>Hematology and Transplant Center, Area Vasta 3 Macerata-ASUR Marche, Ospedale di Civitanova Marche, Italy</aff><aff id=\"aff4\"><label>4</label>Hematology and Transplant Center, IRCCS Policlino San Martino Hospital, Genova, Italy</aff><aff id=\"aff5\"><label>5</label>Internal Medicine- Azienda Sanitaria locale 1 Imperiese- Imperia, Italy</aff><aff id=\"aff6\"><label>6</label>Hematology and Bone Marrow Transplantation, IRCCS San Raffaele Scientific Institute, Milan, Italy</aff><aff id=\"aff7\"><label>7</label>Unit of Hematology and Clinical Immunology, University of Padova, Padova, Italy</aff><aff id=\"aff8\"><label>8</label>Division of Hematology, AUSL di Piacenza, Piacenza, Italy</aff><aff id=\"aff9\"><label>9</label>Hematology Division and Bone Marrow Transplantation Unit, San Francesco Hospital, Nuoro, Italy</aff><aff id=\"aff10\"><label>10</label>Hematology Unit, Azienda Unità Sanitaria Locale-IRCCS, Reggio Emilia, Italy</aff><aff id=\"aff11\"><label>11</label>GROM-L (Gruppo Romano-Laziale MDS), Italy</aff><aff id=\"aff12\"><label>12</label>Hematology Unit, Presidio Ospedaliero di Frosinone, Italy</aff><aff id=\"aff13\"><label>13</label>Division of Hematology, Ospedale Degli Infermi, Biella, Italy</aff><aff id=\"aff14\"><label>14</label>Division of Hematology, University of Torino, AOU Città della Salute e Della Scienza, Torino, Italy</aff><aff id=\"aff15\"><label>15</label>Unit of Onco-hematology, Hematopoietic Transplants and Cell Therapies, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Italy</aff><aff id=\"aff16\"><label>16</label>Department of Clinical and Biological Sciences of the University of Turin, San Luigi Hospital, Orbassano, Turin, Italy</aff><aff id=\"aff17\"><label>17</label>Clinic of Hematology, Department of Internal Medicine (DiMI), University of Genoa, Genoa, Italy</aff><aff id=\"aff18\"><label>18</label>Hematology and Hematopoietic Stem Cell Transplant Center, AORMN (Azienda Ospedaliera Ospedali Riuniti Marche Nord), Pesaro, Italy</aff><aff id=\"aff19\"><label>19</label>Division of Hematology, Department of Translational Medicine, Università del Piemonte Orientale and Ospedale Maggiore della Carità, Novara, Italy</aff><aff id=\"aff20\"><label>20</label>Hematology Unit and BMT Center, Azienda Ospedaliero Universitaria di Parma, Parma, Italy</aff><aff id=\"aff21\"><label>21</label>Cancer Center, Humanitas Research Hospital and Humanitas University, Milan, Italy</aff><aff id=\"aff22\"><label>22</label>Hematology and BMT Unit, Ospedale Vito Fazzi, Lecce, Italy</aff><aff id=\"aff23\"><label>23</label>Hematology Unit Ospedale. Bel Colle-Viterbo, Italy</aff><aff id=\"aff24\"><label>24</label>Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy</aff><aff id=\"aff25\"><label>25</label>Department of Clinical and Biological Sciences of the University of Turin, Mauriziano Hospital, Italy</aff><aff id=\"aff26\"><label>26</label>Hematology Department, AO, San Giovanni-Addolorata, Rome, Italy</aff><aff id=\"aff27\"><label>27</label>Department of Medicine and Surgery, University of Salerno, Salerno, Italy</aff><aff id=\"aff28\"><label>28</label>Hematology Unit, Università Cattolica del Sacro Cuore (UCSC) Roma, Italy</aff><aff id=\"aff29\"><label>29</label>Clinical Hematology, Transplant Center and Cell Therapy, Azienda Sanitaria Universitaria Integrata di Udine, S. Maria della Misericordia, Udine, Italy</aff><aff id=\"aff30\"><label>30</label>UO Hematology, AOU Policlinico Sant’Orsola-Malpighi, University of Bologna, Bologna, Italy</aff><aff id=\"aff31\"><label>31</label>Division of Hematology, Department of Oncology, AOU Città della Salute e della Scienza, Turin, Italy</aff><aff id=\"aff32\"><label>32</label>Department of Hematology and Transfusion Medicine, Carlo Poma Hospital, Mantova, Italy</aff><aff id=\"aff33\"><label>33</label>Hematology, SS.Antonio, Biagio e Cesare Arrigo Hospital, Alessandria, Italy</aff><aff id=\"aff34\"><label>34</label>Hematology and BMT Unit-“Antonio Perrino” Hospital, 72100 Brindisi, Italy</aff><aff id=\"aff35\"><label>35</label>Hematology and Stem Cell Transplantation Unit, Regina Elena National Cancer Institute IRCCS-IFO - Rome, Italy</aff><aff id=\"aff36\"><label>36</label>Hematology Department, University La Sapienza, Rome, Italy</aff><aff id=\"aff37\"><label>37</label>Hematology Unit, AO San Camillo-Forlanini, Rome, Italy.</aff><aff id=\"aff38\"><label>38</label>UO Internal Medicine, ASLTo4, Carmagnola, Italy</aff><aff id=\"aff39\"><label>39</label>Hematology Unit, ASST Cremona, Cremona, Italy</aff><aff id=\"aff40\"><label>40</label>Chair of Hematology and Unit of Hematology and Stem Cell Transplantation, “Aldo Moro” University School of Medicine, AOU Consorziale Policlinico, Bari, Italy.</aff><aff id=\"aff41\"><label>41</label>Hematology Unit, Sant’Eugenio Hospital, Rome, Italy</aff><aff id=\"aff42\"><label>42</label>UO Hematology, Grande Ospedale Metropolitano, “Bianchi Melacrino Morelli”, Reggio Calabria, Italy</aff><aff id=\"aff43\"><label>43</label>Department of Scienze Mediche Chirurgiche e Tecnologie Avanzate, “G.F. Ingrassia”, University of Catania, Italy</aff><aff id=\"aff44\"><label>44</label>Hematology, ASST-Spedali Civili, Brescia, Italy</aff><aff id=\"aff45\"><label>45</label>Hematology and Transplant Center, Ospedale Oncologico “Armando Businco” Cagliari, Italy</aff><aff id=\"aff46\"><label>46</label>Hematology, Polytechnic University of Marche, AUO Ospedali Riuniti, Ancona, Italy</aff><aff id=\"aff47\"><label>47</label>Hematology, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy</aff><aff id=\"aff48\"><label>48</label>Onco-Hematology, “A. Tortora” Hospital, Pagani (Sa), Italy</aff><aff id=\"aff49\"><label>49</label>Hematology and Stem Cell Transplantation Unit, University Campus Bio-Medico, Rome, Italy</aff><aff id=\"aff50\"><label>50</label>Hematology Division, University of Tor Vergata, Rome, Italy.</aff><aff id=\"aff51\"><label>51</label>Hematology Institute, La Sapienza University of Rome, S. Andrea Hospital, Rome, Italy.</aff><author-notes><corresp>Correspondence: Valeria Santini (e-mail: <email>valeria.santini@unifi.it</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><month>10</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>23</day><month>9</month><year>2020</year></pub-date><volume>4</volume><issue>5</issue><elocation-id>e483</elocation-id><history><date date-type=\"received\"><day>13</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>10</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"hs9-4-e483.pdf\"/><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><p>Myelodysplastic syndrome patients are subjects of advanced age, vulnerable and frail, whose outcome is heavily influenced by pre-existing comorbidities worsening the hematologic condition. Infections are a rather common cause of death (around 30%), especially, but not only, for IPSS-R higher risk patients.<sup><xref rid=\"R1\" ref-type=\"bibr\">1</xref>–<xref rid=\"R3\" ref-type=\"bibr\">3</xref></sup> In MDS there is a significant impairment of lymphopoiesis, resulting in lymphopenia (ALC < 1.0 × 10<sup>9</sup>/l) in around 38% of MDS patients and poor prognosis.<sup><xref rid=\"R4\" ref-type=\"bibr\">4</xref></sup> Data on innate and adoptive immune systems (either disease related or due to immunosenescence) and the subsequent supposed susceptibility and incidence of viral infections in MDS are scarce.<sup><xref rid=\"R5\" ref-type=\"bibr\">5</xref></sup></p><p>With all these considerations in mind, at Coronavirus outbreak in Italy, the spread of the COVID-19 pandemic was so extremely rapid that we were expecting to face in short times a very large number of severely symptomatic MDS patients and tried to rapidly learn from the earlier and more severely hit areas.<sup><xref rid=\"R6\" ref-type=\"bibr\">6</xref></sup> As per April 28, date until which we collected our data, and in full emergency, the number of diagnosed cases of SARS-Cov2 in Italy was 199.470 with 25.215 deaths.<sup><xref rid=\"R7\" ref-type=\"bibr\">7</xref></sup> In the same period, in the general population around 10% of tested cases were actually infected.<sup><xref rid=\"R8\" ref-type=\"bibr\">8</xref></sup></p><p>Collection of data in the national MDS Registry (FISiM) and the regional Registry for Rome and surrounding area (GROM) has been approved by local Ethical Committees. Through these 2 networks we collected data regarding laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in MDS patients symptomatic and tested from February 24 to April 28, 2020. Data have been obtained from 50 Centers. Total number of MDS patients followed up in that period was 5326 as per April 28, median age 73 years. As per national guidelines, oropharingeal and nasal swab with PCR was performed in regional laboratories for all suspected cases: 305/5326, tested irrespective of gravity of symptoms, and the presence of true SARS-CoV-2 infection sent for confirmation in the national reference central laboratory (Istituto superiore di sanità -ISS). Confirmed SARS-CoV-2 was diagnosed in 63/305 tested cases (20.6%), globally in 63/5326 (1.18%) MDS patients, in the time frame indicated above. Median age of affected MDS patients was 78 years. We evaluated the distribution of SARS-CoV-2 cases dividing the Country into three macro-regions, considering adhering Centers and the epidemiology and cumulative incidence of COVID-19 in Italy:<sup><xref rid=\"R7\" ref-type=\"bibr\">7</xref></sup> 3 Regions of Northern Italy (Lombardia, Piemonte and Emilia Romagna, SARS-CoV-2 > 500 cases /100.000 inhabitants, as per April 28), Rome and surroundings (specific GROM Registry), and Rest of Italy (Table <xref rid=\"T1\" ref-type=\"table\">1</xref>). The majority of SARS-CoV-2 cases and cumulative incidence among MDS patients was localized in the 3 Regions of Northern Italy (LPE) (1.6%), consistent with the data of COVID-19 epidemic in the general population of the area (62.4% of total Italian cases),<sup><xref rid=\"R7\" ref-type=\"bibr\">7</xref></sup> while in Rome and Rest of Italy it was < 1%, (0.3 and 0.85% respectively). Median age of affected MDS patients in LPE Regions was 81 years, in Rome 71 years and in the rest of Italy 77 years.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Distribution of SARS-CoV-2 Positive MDS Patients Diagnosed from February 24th to April 28th 2020, Respect to Number of MDS Patients in Treatment in 50 Italian Hematology Centers.</p></caption><graphic xlink:href=\"hs9-4-e483-g001\"/></table-wrap><p>At the time of analysis, only 33/63 patients were alive, indicating a lethality rate significantly higher than that of non–MDS population (same age range 70–79 years: 28.9% deceased, lethality 24%).<sup><xref rid=\"R7\" ref-type=\"bibr\">7</xref></sup></p><p>Available details on demographics, clinical characteristics and treatment of 63 MDS patients with SARS-CoV-2 are indicated in Table <xref rid=\"T2\" ref-type=\"table\">2</xref>. It is evident that SARS-CoV-2 affected prevalently male subjects, confirming the observation in non-MDS Italian affected population aged 70 to 79 yrs. In particular, although numbers are extremely small, the lethality rate was higher in male MDS patients (73% of total deaths). To note, the same trend was noted for male patient in the general population infected (lethality for male 29.5% vs 16.7% for female aged 70–79 years),<sup><xref rid=\"R7\" ref-type=\"bibr\">7</xref></sup> while survival of MDS patients was not apparently influenced by age (median age 78 years in both groups). Reported cause of death for all 30 cases was respiratory failure, in 82% of cases COVID-19 was complicated by bacterial pneumonia and 5% cardiac failure. ARDS was indicated in 50% of deceased cases. Regarding IPSS-R risk categories, the majority of patients who recovered were lower risk ones (62%), while deceased patients were in the great majority IPSS-R higher risk ones (17/30). There is no statistically significant difference for infection, gravity of infection or survival according to the type of treatment received, in part due to the small figures when we come to the granularity of therapies. A higher proportion of patients was in treatment with azacitidine, consistent with their diagnosis of IPSS-R higher risk MDS. The totality of the MDS patients who were diagnosed with SARS-CoV-2 had multiple severe comorbidities (> 3 comorbidities 80% of cases).</p><table-wrap id=\"T2\" orientation=\"portrait\" position=\"float\"><label>Table 2</label><caption><p>Demographics and Clinical Characteristics of MDS Patients Diagnosed with SARS-CoV-2 and Available Data on MDS and COVID-19 Treatment.</p></caption><graphic xlink:href=\"hs9-4-e483-g002\"/></table-wrap><p>A few patients received only supportive care for COVID-19 infection, either for milder clinical presentation (3/33) or, on the contrary, for a rapid and extremely aggressive onset leading to early death (3/30). In the majority of cases, MDS specific therapies were suspended.</p><p>The impact of SARS-CoV-2 on the frail MDS population was evaluated in a limited time frame during the peak of the pandemic in Italy and the strict national lockdown. Incidence of symptomatic infection was not as relevant as expected in MDS patients for whom neutropenia, lymphopenia, stress erythropoiesis and iron overload could have determined a substantial susceptibility to and gravity of SARS-CoV-2. Similar observations were recently reported, in a much younger population of beta thalassemic patients.<sup><xref rid=\"R9\" ref-type=\"bibr\">9</xref></sup> Median age of SARS-CoV-2 MDS patients was higher than that of the affected Italian MDS population, and this, together with comorbidities, may account for the high lethality rate observed.<sup><xref rid=\"R10\" ref-type=\"bibr\">10</xref></sup> This report is limited and preliminary (early landmark date), produced during the health emergency. Here we share the international problem of general epidemiology of SARS-CoV-2. In fact, we do not have data for asymptomatic infected MDS patients, for whom diagnostic procedures were not performed, and still complete data are lacking. At present, after resolution of the health emergency, routine serology evaluation of COVID-19 antibodies is ongoing for MDS patients managed in our Centers.</p></body><back><fn-group><fn fn-type=\"COI-statement\"><p>The authors have no conflicts of interest to disclose.</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dayyani</surname><given-names>F</given-names></name><name><surname>Conley</surname><given-names>AP</given-names></name><name><surname>Strom</surname><given-names>SS</given-names></name><etal/></person-group>\n<article-title>Cause of death in patients with lower-risk myelodysplastic syndrome</article-title>. <source><italic toggle=\"yes\">Cancer.</italic></source>\n<year>2010</year>;<volume>116</volume>:<fpage>2174</fpage>–<lpage>2179</lpage>.<pub-id pub-id-type=\"pmid\">20162709</pub-id></mixed-citation></ref><ref id=\"R2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nachtkamp</surname><given-names>K</given-names></name><name><surname>Stark</surname><given-names>R</given-names></name><name><surname>Strupp</surname><given-names>C</given-names></name><etal/></person-group>\n<article-title>Causes of death in 2877 patients with myelodysplastic syndromes</article-title>. <source><italic toggle=\"yes\">Ann Hematol.</italic></source>\n<year>2016</year>;<volume>95</volume>:<fpage>937</fpage>–<lpage>944</lpage>.<pub-id pub-id-type=\"pmid\">27025507</pub-id></mixed-citation></ref><ref id=\"R3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Latagliata</surname><given-names>R</given-names></name><name><surname>Niscola</surname><given-names>P</given-names></name><name><surname>Fianchi</surname><given-names>L</given-names></name><etal/></person-group>\n<article-title>Pulmonary infections in patients with myelodysplastic syndromes receiving frontline azacytidine treatment</article-title>. <source><italic toggle=\"yes\">Hematol Oncol.</italic></source>\n<year>2020</year>;<volume>38</volume>:<fpage>189</fpage>–<lpage>196</lpage>.<pub-id pub-id-type=\"pmid\">31891213</pub-id></mixed-citation></ref><ref id=\"R4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Silzle</surname><given-names>T</given-names></name><name><surname>Blum</surname><given-names>S</given-names></name><name><surname>Schuler</surname><given-names>E</given-names></name><etal/></person-group>\n<article-title>Lymphopenia at diagnosis is highly prevalent in myelodysplastic syndromes and has an independent negative prognostic value in IPSS-R-low-risk patients</article-title>. <source><italic toggle=\"yes\">Blood Cancer J.</italic></source>\n<year>2019</year>;<volume>9</volume>:<fpage>63</fpage>.<pub-id pub-id-type=\"pmid\">31399557</pub-id></mixed-citation></ref><ref id=\"R5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Girmenia</surname><given-names>C</given-names></name><name><surname>Candoni</surname><given-names>A</given-names></name><name><surname>Delia</surname><given-names>M</given-names></name><etal/></person-group>\n<article-title>Infection control in patients with myelodysplastic syndromes who are candidates for active treatment: Expert panel consensus-based recommendations</article-title>. <source><italic toggle=\"yes\">Blood Rev.</italic></source>\n<year>2019</year>;<volume>34</volume>:<fpage>16</fpage>–<lpage>25</lpage>.<pub-id pub-id-type=\"pmid\">30448050</pub-id></mixed-citation></ref><ref id=\"R6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fagiuoli</surname><given-names>S</given-names></name><name><surname>Lorini</surname><given-names>FL</given-names></name><name><surname>Remuzzi</surname><given-names>G</given-names></name></person-group>\n<article-title>Adaptations and lessons in the province of Bergamo</article-title>. <source><italic toggle=\"yes\">N Engl J Med.</italic></source>\n<year>2020</year>;<volume>382</volume>:<fpage>e71</fpage>.<pub-id pub-id-type=\"pmid\">32369276</pub-id></mixed-citation></ref><ref id=\"R7\"><label>7.</label><mixed-citation publication-type=\"book\"><comment>Istituto Superiore Sanità. <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.epicentro.iss.it/\">https://www.epicentro.iss.it/</ext-link>. 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991453</article-id><article-id pub-id-type=\"pmc\">PMC7523799</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-08316</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022359</article-id><article-id pub-id-type=\"art-access-id\">22359</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3800</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Systematic Review</subject></subj-group></article-categories><title-group><article-title>Safety and efficacy of <italic>Soyo-san</italic> for the treatment of functional dyspepsia</article-title><subtitle>A protocol for systematic review and meta-analysis</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Ha</surname><given-names>Na-Yeon</given-names></name><degrees>KMD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Lee</surname><given-names>Ha-nul</given-names></name><degrees>KMD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Jeong</surname><given-names>Hae-in</given-names></name><degrees>KMD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Ko</surname><given-names>Seok-Jae</given-names></name><degrees>KMD, PhD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Park</surname><given-names>Jae-Woo</given-names></name><degrees>KMD, PhD</degrees></contrib><contrib contrib-type=\"author\"><name><surname>Kim</surname><given-names>Jinsung</given-names></name><degrees>KMD, PhD</degrees><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib></contrib-group><aff>Department of Gastroenterology, College of Korean Medicine, Kyung Hee University, Seoul, Republic of Korea.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Jinsung Kim, Department of Gastroenterology, College of Korean Medicine, Kyung Hee University, 24 Kyungheedae-ro, Dongdaemun-gu, Seoul, Republic of Korea (e-mail: <email>oridoc@khu.ac.kr</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22359</elocation-id><history><date date-type=\"received\"><day>20</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>26</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22359.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>Functional dyspepsia (FD) is a common condition characterized by gastrointestinal symptoms, such as abdominal fullness and epigastric pain. With the limitations of conventional Western medical treatments, symptoms often recur and lead to poor quality of life. <italic>Soyo-san</italic> (SYS) is a traditional herbal medicine that has been frequently used to treat indigestion. This protocol was designed to investigate the safety and efficacy of SYS for treating FD through a systematic review and meta-analysis.</p></sec><sec sec-type=\"methods\"><title>Methods:</title><p>Trials will be searched from the following 11 electronic databases, up to March 2020: EMBASE, Medline (via PubMED), the Cochrane Central Register of Controlled Trials (CENTRAL), Allied and Complementary Medicine Database (AMED), Korean Medical Database (KMbase), KoreaMed, Korean Studies Information Service System (KISS), National Digital Science Library (NDSL), Oriental Medicine Advanced Searching Integrated System (OASIS), China National Knowledge Infrastructure Database (CNKI), and Citation Information by Nii (CiNii). Randomized controlled trials (RCTs) of SYS or modified SYS for FD will be included in this systematic review. The effects of control interventions such as placebo, no-treatment, and conventional Western medicine will be compared with those of SYS. RCTs investigating the synergetic effect of SYS and Western medicine compared with conventional Western medicine alone will also be evaluated. Two investigators will independently extract the data and assess the risk of bias in the included studies. The total clinical effective rate will be measured as the main outcome.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>This systematic review will provide data on the use of SYS in the treatment of FD, based on indicators such as dyspepsia-related symptom score, recurrence rate, and adverse events.</p></sec><sec sec-type=\"conclusion\"><title>Conclusion:</title><p>This study will determine the safety and efficacy of SYS for the treatment of FD.</p></sec><sec><title>Review Registry Unique Identifying Number:</title><p>reviewregistry969.</p></sec></abstract><kwd-group><title>Keywords</title><kwd>functional dyspepsia</kwd><kwd>meta-analysis</kwd><kwd>protocol</kwd><kwd><italic>Soyo-san</italic></kwd><kwd>systematic review</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>Korea Health Industry Development Institute</funding-source><award-id>HB16C0019</award-id><principal-award-recipient>Jinsung Kim</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Functional dyspepsia (FD) is a functional gastrointestinal disorder (FGID) characterized by the following symptoms: early satiation, postprandial fullness, epigastric pain, or epigastric burning.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> The prevalence of FD is between 5% and 11% globally.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> These symptoms frequently relapse and chronically affect patients, resulting in decreased quality of life and productivity, and increased economic burden.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>,<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> In FD patients, structural problems related to the symptoms are not identified on upper gastrointestinal examinations, such as endoscopy.<sup>[<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> It is known that the pathophysiology of FD may involve slow gastric emptying, insufficient gastric relaxation, and gastric hypersensitivity.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> Acid-suppression therapy, prokinetic agents, helicobacter pylori eradication therapy, and antidepressants are currently prescribed as standard treatment for FD. Nevertheless, due to the inefficacy of conventional medical therapies, up to 50% of FD patients resort to complementary and alternative medicine.<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup> As the pathophysiology of FD remains unclear and complex etiologies have been associated with various symptoms, a multi-target treatment like herbal medication may be useful.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup></p><p>Soyo-san (SYS; Soyo-san in Korean, Xiaoyao-san in Chinese, or Shoyo-san in Japanese) is a traditional herbal formula that consists of the following crude herbs: Atractylodis Rhizoma Alba, Paeoniae Radix Alba, Poria, Bupleuri Radix, Angelicae Gigantis Radix, Liriopis Tuber, Glycyrrhizae Radix, Menthae Herba, and Zingiberis Rhizoma Recens.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> It has been prescribed for centuries to treat various conditions of the gastrointestinal tract, including abdominal pain, and abdominal distention after eating.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> Although a definite mechanism has not been established, it is known that some factors, including delayed gastric emptying, and visceral hypersensitivity, influence the manifestation of several FD symptoms. Among them, SYS is expected to relieve indigestion symptoms by acting on gastrointestinal motility disorders associated with emotional disorders such as depression and anxiety.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>,<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup></p><p>A previous study systematically assessed the effects of SYS; however, the analysis compared SYS with only prokinetic medicines, and the outcomes could not be generalized.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> Thus, other conventional Western medicines and various parameters need to be reviewed to establish reliable evidence and formulate clinical practice guidelines for use in the treatment of FD. The purpose of this systematic review of randomized controlled trials (RCTs) is to confirm the safety and efficacy of SYS in the treatment of FD.</p></sec><sec><label>2</label><title>Methods</title><sec><label>2.1</label><title>Inclusion criteria for study selection</title><sec><label>2.1.1</label><title>Types of studies</title><p>RCTs and quasi-RCTs will be included in this systematic review. Animal studies, cell experiments, case reports, non-RCTs, and commentaries will be excluded.</p></sec><sec><label>2.1.2</label><title>Types of participants</title><p>The FD patients diagnosed with the Rome criteria will be included, and there are no region, sex, and age limitations. The Rome criteria are diagnostic criteria for the FGIDs. They were first announced in 1992 and last revised in 2016. For this reason, the studies conducted before 1991 will be selected after the agreement of 2 independent reviewers (HL and HJ), if their criteria are similar to the Rome criteria (e.g., no evidence of organic disease). Secondary symptoms, induced by conditions, such as gastric ulcer, gastroesophageal reflux disease (GERD), and irritable bowel syndrome (IBS), will be excluded. However, patients with GERD/IBS and FD diagnosed by Rome IV criteria will be exceptionally included, since Rome IV criteria demonstrate that FD may coexist with GERD or IBS.</p></sec><sec><label>2.1.3</label><title>Types of interventions</title><p>RCTs of SYS will be included; SYS alone and modified SYS (herbs-added prescription). These interventions will be compared with SYS: placebo SYS, no treatment at all, and conventional Western drugs, including prokinetics, digestive enzymes, and antidepressants, among others. This systematic review will also compare the effects of combination therapy (SYS plus Western medicine) and Western medicine alone on FD.</p></sec><sec><label>2.1.4</label><title>Types of outcome measures</title><p>The total clinical effective rate will be measured for the primary outcome. Secondary outcomes will include dyspepsia-related symptom score, functional dyspepsia symptoms rating, self-rating depression scale, self-rating anxiety scale, recurrence after treatment, and adverse events.</p></sec></sec><sec><label>2.2</label><title>Search studies</title><sec><label>2.2.1</label><title>Database resources</title><p>We will search through the following 11 electronic databases from their beginning dates up to March 2020: EMBASE, Medline (via PubMED), the Cochrane Central Register of Controlled Trials (CENTRAL), Allied and Complementary Medicine Database (AMED), Korean Medical Database (KMbase), KoreaMed, Korean Studies Information Service System (KISS), National Digital Science Library (NDSL), Oriental Medicine Advanced Searching Integrated System (OASIS), China National Knowledge Infrastructure Database (CNKI) in Chinese, and Citation Information by Nii (CiNii) in Japanese. The fifth to ninth (v–ix) are Korean databases. The data from the Clinical Research Information Service and ClinicalTrials.gov will also be searched.</p></sec><sec><label>2.2.2</label><title>Search strategy</title><p>The search terms, including symptom-related (e.g., indigestion, dyspepsia, disorder, discomfort, and stomach) and intervention-related (e.g., herbal medicine, <italic>Soyo</italic>, <italic>Xiaoyao</italic>, <italic>Shoyo</italic>), will be used independently or together in each database. For example, the specific search strategies for Medline (via PubMed) are presented in Table <xref rid=\"T1\" ref-type=\"table\">1</xref>. Modified search strategies will be designed for other databases without language restrictions.</p><table-wrap id=\"T1\" orientation=\"portrait\" position=\"float\"><label>Table 1</label><caption><p>Search strategy used in PubMed.</p></caption><graphic xlink:href=\"medi-99-e22359-g001\"/></table-wrap></sec></sec><sec><label>2.3</label><title>Data collection and assessment</title><sec><label>2.3.1</label><title>Study selection</title><p>After training on the selection procedure, 2 investigators (HL and HJ) will use Endnote X8 (Clarivate Analytics, Philadelphia) to upload the literature and independently review the titles and abstracts to exclude duplicate and irrelevant articles. During the screening, the independent reviewers (HL and HJ) will read the full-text articles and select studies that are appropriate for analysis. The details of the study selection process are summarized in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram (Fig. <xref ref-type=\"fig\" rid=\"F1\">1</xref>). They will discuss all disagreements and reach a consensus unless the arbiter (NH) intervenes and makes adjustments.</p><fig id=\"F1\" orientation=\"portrait\" position=\"float\"><label>Figure 1</label><caption><p>Flow chart of the search process. AMED = Allied and Complementary Medicine Database, CENTRAL = Cochrane Central Register of Controlled Trials, Cinii = Citation Information by Nii, CNKI = China National Knowledge Infrastructure Database, KISS = Korean Studies Information Service System, KMbase = Korean Medical Database, NDSL = National Digital Science Library, OASIS = Oriental Medicine Advanced Searching Integrated System, RCTs = randomized controlled trials.</p></caption><graphic xlink:href=\"medi-99-e22359-g002\"/></fig></sec><sec><label>2.3.2</label><title>Data extraction</title><p>The standard form will be filled with the data, which would have been independently extracted by the 2 reviewers (HL and HJ). It will include the following information: the first author, year of publication, study design, interventions, treatment period, outcomes, and adverse events, among others. Any differences between the reviewers will be discussed and resolved by the arbiter (NH).</p></sec><sec><label>2.3.3</label><title>Assessment of the risk of bias in included studies</title><p>Two reviewers (HL and HJ) will independently assess the risk of bias using the Cochrane collaboration's tool. It consists of the following items: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias. Each result will be one of the following: low, unclear, and high. The reviewers will resolve all disagreements through discussion, and if necessary, the third researcher (NH) will intervene and settle the disagreement.</p></sec></sec><sec><label>2.4</label><title>Data analysis</title><sec><label>2.4.1</label><title>Statistical analysis</title><p>The Review Manager program (version 5.3. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014) will be used for data synthesis. We will use the mean difference with 95% confidence interval (CI) for evaluating continuous data, and risk ratio or odds ratio with 95% CI for dichotomous data. A random-effects model will be included in the meta-analysis. The heterogeneity of included studies will be assessed by the <italic>I</italic>-squared statistic (<italic>I</italic><sup><italic>2</italic></sup> ≥ 50% means substantial heterogeneity) and the Chi-squared (<italic>χ</italic><sup>2</sup>) test (<italic>P</italic> < .1 means statistical significance).</p></sec><sec><label>2.4.2</label><title>Data synthesis</title><p>The efficacy of SYS will be synthesized and compared with the controls, including placebo, no-treatment, and conventional Western medicine. The effect of combined SYS and Western medicine will be compared with Western medicine alone to confirm the synergetic effect of combination therapy.</p></sec><sec><label>2.4.3</label><title>Subgroup analysis</title><p>If there is high heterogeneity among selected studies, we will perform subgroup analysis. The cause of heterogeneity will be evaluated in terms of pattern identification in traditional Chinese medicine (TCM), species of added herbs and types of Western medicine, among others.</p></sec><sec><label>2.4.4</label><title>Reporting bias analysis</title><p>If there are >10 articles for analysis, we will generate a funnel plot to assess publication bias.</p></sec><sec><label>2.4.5</label><title>Sensitivity analysis</title><p>A sensitivity analysis will be conducted to confirm the robustness of the findings. The Consolidated Standards of Reporting Trials (CONSORT) and Extension for Herbal Interventions will be applied for assessment of methods and reporting quality of each trial.</p></sec><sec><label>2.4.6</label><title>Grading the quality of evidence</title><p>The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) will be used for assessing the quality of evidence.</p></sec><sec><label>2.4.7</label><title>Handling missing data</title><p>We will try contact the authors of original research articles to obtain additional missing data (e.g., sending an e-mail). The intent-to-treat analysis will be used for assessing the data.</p></sec></sec><sec><label>2.5</label><title>Ethics and dissemination</title><p>Identifying information of each participant will not be published, and this protocol does not need ethical approval. The systematic review will be published in a peer-reviewed journal and disseminated electronically.</p></sec></sec><sec><label>3</label><title>Discussion</title><p>SYS is a well-known and widely used herbal medicine in Korea, China, and Japan. In a clinical study on 20 patients with FD using an electrogastrogram (EGG), <italic>Jia</italic>-<italic>Wei</italic>-<italic>Xiao</italic>-<italic>Yao</italic>-<italic>San</italic> decoction improved dyspeptic symptoms and normalized gastric motility.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> SYS has also been used to treat dyspeptic symptoms with psychological disorders such as depression. In TCM theories, dysfunction of the liver and spleen may cause abnormal digestion and can be cured by soothing liver-<italic>qi</italic> stagnation and promoting spleen transportation, interpreted as an effect of modulating neurotransmitter or hormone levels.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> In an animal study that used chronic multi-stressed rats, the administration of <italic>Xiaoyao Powder</italic> regulated serum corticosterone and gastrointestinal hormones.<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup> In a randomized, placebo-controlled trial of 180 perimenopausal women with FD and depression, <italic>Xiaoyao pill</italic> reduced depression, and improved the plasma levels of motilin and gastrin, and gastric emptying rate.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> A clinical study on FD patients with <italic>Gan-qi</italic> stagnation of <italic>Pi-</italic>deficiency syndrome type (FD-GP) showed that Modified Xiaoyao Powder alleviated clinical symptoms and regulated some parameters of EGG within normal range, elevating pharmacokinetic characteristics of ferulic acid, one of its active constituents.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>,<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]</sup> As mentioned above, SYS is often prescribed for FD patients with GP (“liver stagnation” and “spleen deficiency”), one of the syndromes of pattern identification in TCM. According to the TCM standard for diagnosing syndromes of FD, the “liver depression and spleen deficiency” type is characterized by symptoms such as stomach pain or discomfort, belching and acid reflux.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> Meanwhile, a meta-analysis of 14 RCTs of Modified Xiaoyao-san (MXS) for treatment of FD found that MXS, alone or combined with other prokinetic agents, was more effective than prokinetic drugs for treating FD. Although the high risk of bias makes the evidence weak, it is remarkable that a drug overdose can be managed with an alternative herbal medicine without causing any serious side-effects. Thus, more controlled trials must be conducted to evaluate the safety and efficacy of MXS in the treatment of FD.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup></p><p>Although previous meta-analyses on the effects of SYS on FD have been performed, synergetic effects with only prokinetic drugs, as controls, were investigated and the evaluation of effectiveness was confined to symptom improvement.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> Most of the reviewed literature was Chinese studies, and the quality of the research methodologies was weak. In this systematic review of randomized controlled trials, we intend to review databases outside China to establish outcomes that can be generalized for clinical practice in various countries including Korea. In addition, control groups, including placebo and no-treatment groups, will be set and synergistic effects with drugs such as antidepressants, PPIs, and prokinetics will be analyzed. In the outcomes, detailed subjective symptoms and, if possible, effects on objective variables such as gastric emptying rate will be evaluated. Accordingly, it will be a more reliable study; it is designed to reflect the recent RCT trends on SYS, modified SYS, and SYS with Western medicine for the treatment of FD, and establish evidence for use in clinical practice.</p><p>Ethics approval is not required, because this systematic review is a literature-based study that will use already published data and no additional data will be collected. The results of the review will be published in a peer-reviewed journal and disseminated electronically or in print.</p></sec><sec><title>Acknowledgments</title><p>This study was supported by a grant through the project “Development of Korean medicine clinical practice guidelines” of the Guideline Center for Korean Medicine, National Institute for Korean Medicine Development.</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization:</bold> Na-Yeon Ha, Ha-nul Lee.</p><p><bold>Data curation:</bold> Na-Yeon Ha, Ha-nul Lee, Hae-in Jeong, Seok-Jae Ko, Jae-Woo Park, Jinsung Kim.</p><p><bold>Formal analysis:</bold> Jinsung Kim.</p><p><bold>Investigation:</bold> Na-Yeon Ha, Ha-nul Lee, Hae-in Jeong.</p><p><bold>Methodology:</bold> Seok-Jae Ko, Jae-Woo Park, Jinsung Kim.</p><p><bold>Resources:</bold> Jinsung Kim.</p><p><bold>Writing – original draft:</bold> Na-Yeon Ha.</p><p><bold>Writing – review & editing:</bold> Jinsung Kim.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: EGG = electrogastrogram, FD = functional dyspepsia, FGID = functional gastrointestinal disorder, GERD = gastroesophageal reflux disease, GP = Gan-qi stagnation of Pi-deficiency syndrome type, IBS = irritable bowel syndrome, MXS = Modified Xiaoyao-san, RCTs = randomized controlled trials, SYS = Soyo-san, TCM = traditional Chinese medicine.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Ha NY, Lee Hn, Jeong Hi, Ko SJ, Park JW, Kim J. Safety and efficacy of <italic>Soyo-san</italic> for the treatment of functional dyspepsia: A protocol for systematic review and meta-analysis. <italic>Medicine</italic>. 2020;99:39(e22359).</p></fn><fn fn-type=\"supported-by\"><p>This study was supported by a grant from the Korean Health Technology R&D Project through the Korean Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare of the Republic of Korea (project number: HB16C0019). 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[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Medicine (Baltimore)</journal-id><journal-id journal-id-type=\"publisher-id\">MEDI</journal-id><journal-title-group><journal-title>Medicine</journal-title></journal-title-group><issn pub-type=\"ppub\">0025-7974</issn><issn pub-type=\"epub\">1536-5964</issn><publisher><publisher-name>Lippincott Williams & Wilkins</publisher-name><publisher-loc>Hagerstown, MD</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32991463</article-id><article-id pub-id-type=\"pmc\">PMC7523800</article-id><article-id pub-id-type=\"publisher-id\">MD-D-20-08362</article-id><article-id pub-id-type=\"doi\">10.1097/MD.0000000000022396</article-id><article-id pub-id-type=\"art-access-id\">22396</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>3800</subject></subj-group><subj-group><subject>Research Article</subject><subject>Study Protocol Systematic Review</subject></subj-group></article-categories><title-group><article-title>Multiple Traditional Chinese Medicine interventions for idiopathic pulmonary fibrosis</article-title><subtitle>A protocol for systematic review and meta-analysis of overview</subtitle></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\" authenticated=\"false\">http://orcid.org/0000-0002-7026-8307</contrib-id><name><surname>Zhang</surname><given-names>Hao-Yang</given-names></name><degrees>MD</degrees><xref ref-type=\"aff\" rid=\"aff1\"><sup>a</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Pang</surname><given-names>Li-Jian</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"aff2\"><sup>b</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Lv</surname><given-names>Xiao-Dong</given-names></name><degrees>PhD</degrees><xref ref-type=\"aff\" rid=\"aff3\"><sup>c</sup></xref><xref rid=\"cor1\" ref-type=\"corresp\"><sup>∗</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Chuang</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff4\"><sup>d</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Nan</surname><given-names>Ming-Hua</given-names></name><degrees>MM</degrees><xref ref-type=\"aff\" rid=\"aff5\"><sup>e</sup></xref></contrib></contrib-group><aff id=\"aff1\"><label>a</label>Graduate School, Liaoning University of Traditional Chinese Medicine</aff><aff id=\"aff2\"><label>b</label>Respiratory department, Affiliated Hospital of Liaoning University of Traditional Chinese Medicine</aff><aff id=\"aff3\"><label>c</label>Liaoning University of Traditional Chinese Medicine</aff><aff id=\"aff4\"><label>d</label>Emergency Department, Affiliated Hospital of Liaoning University of Traditional Chinese Medicine</aff><aff id=\"aff5\"><label>e</label>Cardiovascular department, The Second Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning, China.</aff><author-notes id=\"cor1\"><corresp><label>∗</label>Correspondence: Xiao-Dong Lv, Liaoning University of Traditional Chinese Medicine, Shenyang 110033, Liaoning, China (e-mail: <email>lnzyxdl@yeah.net</email>).</corresp></author-notes><pub-date pub-type=\"collection\"><day>25</day><month>9</month><year>2020</year></pub-date><pub-date pub-type=\"epub\"><day>25</day><month>9</month><year>2020</year></pub-date><volume>99</volume><issue>39</issue><elocation-id>e22396</elocation-id><history><date date-type=\"received\"><day>27</day><month>8</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.</copyright-statement><copyright-year>2020</copyright-year><license license-type=\"open-access\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\" specific-use=\"CC-BY\"><license-p>This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0\">http://creativecommons.org/licenses/by/4.0</ext-link></license-p></license></permissions><self-uri xlink:href=\"medi-99-e22396.pdf\"/><abstract><title>Abstract</title><sec sec-type=\"background\"><title>Background:</title><p>The therapeutic strategies of idiopathic pulmonary fibrosis (IPF) tend to be comprehensive. Improving the major symptoms and quality of life (QoL) is as important as postponing the process of fibrosis. However, only pirfenidone and nintedanib conditionally recommended by guidelines and no definite proof indicate that they can significantly ameliorate the main symptoms and QoL of IPF sufferers. At present, multiple types of Traditional Chinese Medicine (TCM) interventions alone or in combination with conventional western medicine managements are widespreadly applied in IPF treatment, which seemingly have a promising clinical effect, especially in ameliorating the main symptoms and improving QoL. Subsequently, the number of relevant studies in systematic reviews(SRs) and meta-analyses of randomized controlled trials(RCTs) increased significantly. Hence, we plan to implement an overview to collect, evaluate, and summarize the results of these SRs.</p></sec><sec sec-type=\"methods\"><title>Methods:</title><p>An all-round literature retrieval will be conducted in 9 electronic databases, including PubMed, EMBASE, CINAHL, Cochrane Library, Epistemonikos, CNKI, CBM, Wanfang, and VIP. We will focus on the systematic review and meta-analysis of RCTs for multiple TCM interventions alone or in combination with routine western medicine measures in IPF treatment. The main outcomes we follow with interest include the improvement of major symptoms (cough, dyspnea) and QoL. Secondary outcomes will consist of minor symptoms improvement, clinical total effective rate, lung function, blood gas analysis, 6-minute walk text, adverse events, acute exacerbation, all-cause mortality, and IPF-related mortality. Two reviewers will independently select the SRs satisfactory with the enrolling criteria, extract key characteristics, and datas on predefined form, evaluate methodological quality by AMSTAR-2, ROBIS and PRISMA tools, and the quality of evidences adopting GRADE method. In case of any divergence will be reached an agreement by discussion or adjudicated by a third senior reviewer. We will perform a narrative synthesis of the proofs from SRs included.</p></sec><sec sec-type=\"results\"><title>Results:</title><p>The findings of this overvew will be presented at relevant conferences and submitted for peer-review publication.</p></sec><sec sec-type=\"conclusion\"><title>Conclusions:</title><p>We expect to obtain comprehensive and reliable evidence of IPF treated by diversified TCM interventions from the potential standard SRs, which may provide suggestions for future RCTs and SRs.</p></sec><sec><title>Registration number:</title><p>INPLASY 202080110</p></sec></abstract><kwd-group><title>Keywords</title><kwd>idiopathic pulmonary fibrosis</kwd><kwd>overview</kwd><kwd>protocol</kwd><kwd>systematic review</kwd><kwd>traditional chinese medicine</kwd></kwd-group><funding-group><award-group id=\"award1\" award-type=\"Fundref\"><funding-source>Natural Science Foundation of China</funding-source><award-id>NO:81373579</award-id><principal-award-recipient>Xiao-Dong Lv</principal-award-recipient></award-group><award-group id=\"award2\" award-type=\"Fundref\"><funding-source>Natural Science Foundation of China</funding-source><award-id>NO:81403290</award-id><principal-award-recipient>Li-Jian Pang</principal-award-recipient></award-group><award-group id=\"award3\" award-type=\"Fundref\"><funding-source>Department of Science and Technology of Liaoning Province</funding-source><award-id>XLYC1808011</award-id><principal-award-recipient>Xiao-Dong Lv</principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>OPEN-ACCESS</meta-name><meta-value>TRUE</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec><label>1</label><title>Introduction</title><p>Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fibrotic interstitial lung disease (ILD) characterized by extensive pulmonary remodeling caused by abnormal deposition of extracellular matrix.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>]</sup> The etiology of IPF is still unclear, and it often occurs in middle-aged and elderly people.<sup>[<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> IPF is the most common idiopathic interstitial pneumonia (IIP), accounting for about 60% of cases.<sup>[<xref rid=\"R3\" ref-type=\"bibr\">3</xref>]</sup></p><p>It is estimated that the annual incidence of IPF is 6.8 to 16.3/100,000,<sup>[<xref rid=\"R4\" ref-type=\"bibr\">4</xref>]</sup> and about 40,000 new cases are diagnosed each year in Europe.<sup>[<xref rid=\"R5\" ref-type=\"bibr\">5</xref>]</sup> There is a lack of large-scale epidemiological study in China. Nevertheless, the ILD epidemiological document indicates that its morbidity in China have an increasing tendency, what's more, with the surging of domestic population aging, the number of patients will show continuously increasing trend accompanied by growth of disease burden.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup> IPF has a poor prognosis, with a median survival of 2 to 3 years after diagnosis.<sup>[<xref rid=\"R1\" ref-type=\"bibr\">1</xref>,<xref rid=\"R2\" ref-type=\"bibr\">2</xref>]</sup> The recent study suggests that IPF survival has not improved significantly and mortality seems to be rising, although this may partly reflect improved recognition and diagnosis.<sup>[<xref rid=\"R7\" ref-type=\"bibr\">7</xref>]</sup></p><p>The pathogenesis of IPF is complex and complicated, but there are sufficient proofs bear out that its mechanism is closely related to immunity and inflammation, involving a variety of cytokines and signaling pathways.<sup>[<xref rid=\"R8\" ref-type=\"bibr\">8</xref>]</sup> Transforming growth factor-β (TGF-β) is considered to be probably the principal profibrotic cytokine within them.<sup>[<xref rid=\"R9\" ref-type=\"bibr\">9</xref>]</sup> Additionally, connective tissue growth factor, platelet-derived growth factor, vascular endothelial growth factor, interleukin-1α, tumor necrosis factor-α, and interferon-γ are closely related to IPF development. The involved pathways mainly include wnt/β-catenin, shh (sonic hedgehog) and notch signaling pathway and so on.<sup>[<xref rid=\"R10\" ref-type=\"bibr\">10</xref>]</sup> Recent literatures suggest that epigenetic regulation mechanism may be interrelated with occurrence and development of the disease, in which the DNA methylation, histone modification and micro RNA changes are probably the key factors in triggering IPF.<sup>[<xref rid=\"R11\" ref-type=\"bibr\">11</xref>]</sup></p><p>Dry cough and dyspnea are the main clinical manifestations of IPF, within which the nonproductive cough is an irritating symptom presenting in 73% to 86% of patients.<sup>[<xref rid=\"R12\" ref-type=\"bibr\">12</xref>]</sup> Besides, breathless has been proven to be bound up with survival and tend to impaired lung function as the condition progresses.<sup>[<xref rid=\"R13\" ref-type=\"bibr\">13</xref>]</sup> Owing to progressive worsening of symptoms and irreversible deterioration in lung function, patients will become more debilitated and progressively restricted in activity, which probably bring about a lower quality of life (QoL).<sup>[<xref rid=\"R14\" ref-type=\"bibr\">14</xref>]</sup></p><p>Therefore, the therapeutic strategies of IPF should be comprehensive. Alleviating symptoms, improving QoL, and postponing disease progression are of equal importance for cases.<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>]</sup> However, currently available options for conventional drugs in western medicine are limited, only pirfenidone and nintedanib are conditionally recommended by evidence-based guidelines for IPF therapy.<sup>[<xref rid=\"R16\" ref-type=\"bibr\">16</xref>]</sup> Pirfenidone is an oral multi-target small molecule therapeutic drug with effects of anti-inflammatory, antioxidant, and anti-fibrotic.<sup>[<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]</sup> Nintedanib, a small molecule tyrosine kinase inhibitor (TKI), plays an antifibrotic role by blocking intracellular signal transduction, fibroblast proliferation, migration, and transformation.<sup>[<xref rid=\"R18\" ref-type=\"bibr\">18</xref>]</sup> However, neither drug can take a turn for the worse in fibrosis progression,<sup>[<xref rid=\"R15\" ref-type=\"bibr\">15</xref>–<xref rid=\"R17\" ref-type=\"bibr\">17</xref>]</sup> mild to moderate adverse events such as gastrointestinal symptoms and abnormal liver function often occur during the application of both drugs.<sup>[<xref rid=\"R19\" ref-type=\"bibr\">19</xref>,<xref rid=\"R20\" ref-type=\"bibr\">20</xref>]</sup> Furthermore, there are insufficient proofs corroborate that the 2 medications can significantly improve the major symptoms and QoL of sufferers, and the high price seriously hinders the application of patients in China.<sup>[<xref rid=\"R6\" ref-type=\"bibr\">6</xref>]</sup></p><p>Therefore, some deficiencies and gaps need to be further filled. Traditional Chinese Medicine (TCM) has been used for thousands of years to treat respiratory diseases in China and some other countries in Asia. Although TCM is not the mainstream treatment for IPF, it has been increasingly accepted as a form of complementary and alternative medicine in western countries.<sup>[<xref rid=\"R21\" ref-type=\"bibr\">21</xref>]</sup> There are many types of TCM interventions, including Chinese herb formulas (CHFs), acupuncture, moxibustion, acupoint application, and so on. They have been well authenticated that many CHFs or extracts possess the effects on regulating cytokines, signal transduction pathways, and oxidative stress, as well as inhibiting extracellular matrix synthesis.<sup>[<xref rid=\"R22\" ref-type=\"bibr\">22</xref>–<xref rid=\"R24\" ref-type=\"bibr\">24</xref>]</sup> What is more exhilarating is that the latest researches provide additional evidence that some CHFs are inclined to affect epigenetic mechanism to achieve the therapeutic purpose.<sup>[<xref rid=\"R25\" ref-type=\"bibr\">25</xref>]</sup> Due to the diversity of active ingredients in CHF compositions and the potential synergistic effect among them, which enable them to have a wide-ranging targets and multiple therapeutic mechanisms.<sup>[<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]</sup> Moreover, acupuncture, moxibustion, and acupoint application also have the functions of regulating and improving inflammation and immunity.<sup>[<xref rid=\"R27\" ref-type=\"bibr\">27</xref>–<xref rid=\"R29\" ref-type=\"bibr\">29</xref>]</sup></p><p>In recent years, a great quantity of clinical trials has been carried out in the treatment of IPF by multiple types of TCM interventions alone or in combination with conventional western medicine measures. Many trials have shown that these TCM interventions seem to have positive significance for IPF treatment, especially for the improvement of symptoms and QoL.<sup>[<xref rid=\"R26\" ref-type=\"bibr\">26</xref>]</sup> Subsequently, the number of SRs and meta-analyses pooling these results increased significantly.<sup>[<xref rid=\"R30\" ref-type=\"bibr\">30</xref>–<xref rid=\"R32\" ref-type=\"bibr\">32</xref>]</sup> Currently, there is still no overview of systematic review (OoSR) to synthesize the evidences of effectiveness of multiple TCM interventions, either alone or combined with routine western medicine measures, on the main symptoms and QoL of IPF patients in these SRs. Only 1 OoSR protocol has been published, which mainly focuses on CHFs as intervention in the therapy of pulmonary fibrosis, and the indicator of symptoms improvement is limited to TCM symptom score.<sup>[<xref rid=\"R33\" ref-type=\"bibr\">33</xref>]</sup> Consequently, for the sake of systematically collecting, evaluating, and summarizing the proofs in these SRs, we project to perform an OoSR and drafted this protocol. This OoSR will officially assess the methodological quality of SRs included and the certainty of evidence in SRs, and we will implement a narrative synthesis of the evidence-based of our interest from the selected SRs.</p></sec><sec><label>2</label><title>Objective</title><p>The purpose of this OoSR is to summarize SRs and meta-analyses that assess the effects of multiple TCM interventions alone or combined with conventional western medicine treatment measures for the improvement of main symptoms and QoL in IPF patients. We will include potential proofs pooled in all relevant SRs, present the latest evidence body, and report our findings in a descriptive way.</p></sec><sec><label>3</label><title>Methods</title><sec><label>3.1</label><title>Study protocol and registration</title><p>This protocol was designed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) 2015 checklist.<sup>[<xref rid=\"R34\" ref-type=\"bibr\">34</xref>]</sup> It is registered on the International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY no. 202080110, <ext-link ext-link-type=\"uri\" xlink:href=\"https://inplasy.com/\">https://inplasy.com/</ext-link>)</p></sec><sec><label>3.2</label><title>Eligibility criteria</title><sec><label>3.2.1</label><title>Types of reviews</title><p>We will include published and peer-reviewed SRs based on RCTs, and provide meta-estimates of the indicators of main symptoms (cough, dyspnea) and/or QoL. SRs published only in abstract, without meta-analyses, non-SRs, or other overviews will be excluded. We will not be place restrictions on publication time of SRs and RCTs included.</p></sec><sec><label>3.2.2</label><title>Participants</title><p>We will restrict our overview to studies of human patients with IPF in stable stage. We will exclude meta-analyses of trials exclusively populations in IPF with acute exacerbation or IPF in stable period with other respiratory diseases.</p></sec><sec><label>3.2.3</label><title>Interventions</title><p>TCM therapy alone or combined with routine western medicine measures should be applied in the treatment group. TCM interventions comprise CHFs, acupuncture, moxibustion, and acupoint application.</p></sec><sec><label>3.2.4</label><title>Comparisons</title><p>Intervention measures in the control group we defined consist of conventional pharmacotherapy, placebo, oxygen therapy, and no treatment.</p></sec><sec><label>3.2.5</label><title>Outcomes</title><sec><label>3.2.5.1</label><title>Primary outcomes</title><p>We are interested in indicators of major symptoms (dry cough, dyspnea) and QoL improvement, and therefore all reliable measurements of them will meet the criteria, such as TCM symptom score (dry cough, dyspnea), the St.George's respiratory questionnaire, Leicester cough questionnaire, the breathing problems questionnaire, the MOS item short from health survey, a tool to asses QoL in IPF, and so on.</p></sec><sec><label>3.2.5.2</label><title>Secondary outcomes</title><p>The types of secondary outcome measurements contain improvement of minor symptoms, total clinical effective rate, pulmonary function, blood gas analysis, 6-minute walking test, adverse events, acute exacerbation, all-cause mortality, IPF-related mortality.</p></sec></sec></sec><sec><label>3.3</label><title>Information sources</title><p>An overall retrieval will be performed in the following digital databases: PubMed, EMBASE, CINAHL (Cumulative Index to Nursing and Allied Health Literature), Cochrane Library and Epistemonikos, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, WangFang Database, and Chinese Scientific Journal Database. The range of search time is from their inceptions to June 2020. The language of published SRs will be restricted in English and Chinese. The bibliographies of identified articles and gray literature will also be searched.</p><p>We will seek help from experts in the IPF domain to confirm other potential SRs. A medical librarian will establish and run a retrieval formula to identify relevant studies. The search strategy will undergo internal peer review. The document retrieval strategy in PubMed is as follows and we will adapt it for each database.</p><fig orientation=\"portrait\" id=\"d38e547\" position=\"float\"><graphic xlink:href=\"medi-99-e22396-g001\"/></fig></sec><sec><label>3.4</label><title>Studies selection and data collection process</title><p>The selection of SRs and also the extraction of key characteristics will progress in duplicate. We will use NoteExpress V3.0 software to manage retrieved studies and eliminate duplications. First, 2 investigators (ZHY and PLJ) will independently and separately screen the titles and abstracts, and further select eligible literatures by reading the full texts. The consistency of screening and selection will be measured by Kappa statistics, and any disagreements will be resolved by discussion or arbitration by a third senior reviewer (LXD). The reviewers will document all studies that do not meet the criteria and provide reasonable reasons for exclusion in the process. Inconformity studies with justifications will also be reported in the table.</p><p>Two reviewers (ZHY and LC) will independently extract key characteristics and data from all standard-compliant systematic reviews and meta-analyses, and use predefined form designed to summarize the informations of each SR. Any divergence will be adopted by discussion or adjudicated by a third senior reviewer (LXD). If any important information elements are missing, inadequate or inconsistent reporting in SRs, we will attempt to contact the authors for desired data or obtain directly from the original study.</p><p>The reviewers will collect following data items from each SR included: basic informations of SR (title, author, country, publication time, funding, conflicts of interest); basic informations of primary study (author, country, publication time, study design); search strategy (database, time range, retrieval date); population characteristics (age, sex, race, course of disease, diagnostic criteria, setting); interventions in treatment group (type, dosage form, dose, intensity, frequency, course of treatment); interventions in control group (type, dosage form, dose, intensity, frequency, course of treatment); primary and secondary outcome measures; results (risk of bias in original studies, total number of studies and cases, meta-estimate, detection and reporting of subgroups).</p></sec><sec><label>3.5</label><title>Assessment of methodological quality of included reviews</title><p>Assessment of multiple systematic reviews-2 (AMSTAR-2) is a tool used to measure the methodological quality, which has been demonstrated relatively simple, reliable, and effective for methodological evaluation of SRs.<sup>[<xref rid=\"R35\" ref-type=\"bibr\">35</xref>]</sup> The AMSTAR-2 tool comprises 16 items, covering the whole process of SR in topic selection, design, registration, information extraction, data statistical analysis, and discussion. According to the guidance document of AMSTAR-2, the overall methodological quality of each SR may be classified as “high,” “moderate,” “low,” and “very low.” Scores will be completed and calculated through the online AMSTAR-2 checklist (<ext-link ext-link-type=\"uri\" xlink:href=\"https://amstar.ca/Amstar_Checklist.php\">https://amstar.ca/Amstar_Checklist.php</ext-link>) in this study.</p><p>Risk of bias in systematic reviews (ROBIS) is a new tool for assessing the bias risk in the design, execution, and analysis processes of SRs,<sup>[<xref rid=\"R36\" ref-type=\"bibr\">36</xref>]</sup> which mainly consist of 3 phases: evaluating the goodness of fit between the issue to be solved in SRs and the target one, identifying the extent of risk of bias in production process, and judging the overall bias risk of SRs. The responses to each iconic question in ROBIS tool mainly including “yes” “probably yes” “no”“probably no” and “no information.”</p><p>We will also appraise reporting quality of each SR included by referring to criteria specified in Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA),<sup>[<xref rid=\"R37\" ref-type=\"bibr\">37</xref>]</sup> and further embody the integrity and transparency of this overview.</p><p>The quality assessment of methodology will be separately completed by 2 reviewers (ZHY and LC), and in case of any disagreements in the process will be settled through discussion or judged by a third senior reviewer (LXD) if necessary. We will calculate kappa statistics to understand the consistency of assessments by AMSTAR-2, ROBIS, and PRISMA between 2 reviewers. Kappa <0.2 is considered as “poor agreement,” 0.2 to 0.4 as “fair agreement,” 0.4 to 0.6 as “moderate agreement,” 0.6 to 0.8 as “substantial agreement,” and 0.8 to 1.0 as “almost perfect agreement.” Based on the evaluation results, we will not exclude any SR during this phase. This overview will not reassess the risk of bias of primary studies in SRs included; instead, we will collect their results of individual study as well as evaluating methods, and report any findings in the review.</p></sec><sec><label>3.6</label><title>Assessment of quality of evidence in included reviews</title><p>The quality of evidence pooled within the SRs and meta-analyses will be assessed independently by 2 overview authors (ZHY and NMH) using Grading of Recommendations Assessment, Development and Evaluation (GRADE) method, which is an algorithm developed to assign levels of meta-analysis evidence for OoSR study types.<sup>[<xref rid=\"R38\" ref-type=\"bibr\">38</xref>]</sup> According to this approach, RCTs begin to be identified as high-quality evidence to support the estimation of intervention effects. Five factors including study limitations/risk of bias, publication bias, imprecision, inconsistency and indirectness that may lead to reduce the level of evidence, and 3 factors including large magnitude of an effect, dose–response gradient, and effect of plausible residual confounding tend to increase the quality of evidence. The overall quality of proof will be judged as “high,” “moderate,” “low,” or “very low”. Differences over the rating quality of evidence will be reached an agreement through consultation or adjudication by a third senior researcher (LXD).</p></sec><sec><label>3.7</label><title>Data synthesis</title><p>This overview is designed to collect and present the current evidence body of improvement in symptoms and QoL of IPF treated with various TCM interventions alone or combined conventional western medicine measures, and data from primary studies may be pooled to estimate for several times in relevant SRs. Thus, this study will not consider the overlap of original studies between SRs, and we will not perform meta-analysis. However, we will assess it to understand the overall extent of coverage, describe the number and scale of overlapping primary studies with their weight in the analysis through a narrative way, and then create a table to visually demonstrate it.</p><p>We will generate and present a summary of the results of primary and secondary outcome measures in all included SRs. When a meta-analysis was performed, we will report the relative risk, odds ratio or hazard ratio for dichotomous outcomes, and weighted mean difference or standard mean difference (SMD) for continuous outcomes with their 95% confidence intervals and heterogeneity estimates.</p><p>We will employ the PRISMA flow chart to summarize the screening process of the studies, report the key characteristics extracted from the SRs using a predesigned table, present the results of methodological quality assessed by AMSTAR-2, ROBIS, and PRISMA in tabular form, demonstrate the pool effect estimates with their confidence of evidence adopting forms and forest plots.</p></sec><sec><label>3.8</label><title>Strengths and limitations of this study</title><list list-type=\"order\"><list-item><p>This study will be the first OoSR to integrate and summarize the relevant evidence of treating IPF with TCM intervention measures.</p></list-item><list-item><p>We will use multiple tools to formally assess the quality of methodology and evidence included in the SRs to reflect the integrity and transparency of this article.</p></list-item><list-item><p>The language of literature retrieval is limited to Chinese and English, which may lead to omission.</p></list-item></list></sec></sec><sec><label>4</label><title>Discussion</title><p>We intend to carry out a formal OoSR and have drafted this manuscript of protocol. The overview will be summarized evidence based on the effectiveness of multiple TCM interventions alone or in combination with conventional western medicine measures to alleviate the main symptoms and QoL of patients with IPF; in addition, other proofs of efficacy and safety will also be generalized. This study will systematically collect, evaluate, and synthesize these results, which may benefit clinicians, policy deciders, and clinical guideline makers. We expect that the results of this overview will highlight the gaps in current evidence, which will provide suggestions for future RCTs and SRs.</p></sec><sec><title>Author contributions</title><p><bold>Conceptualization:</bold> Hao-yang Zhang, Xiao-Dong Lv, Li-Jian Pang.</p><p><bold>Investigation:</bold> Hao-yang Zhang, Xiao-Dong Lv, Li-Jian Pang, Chuang Liu, Ming-Hua Nan.</p><p><bold>Methodology:</bold> Hao-yang Zhang, Xiao-Dong Lv, Li-Jian Pang, Chuang Liu, Ming-Hua Nan.</p><p><bold>Project administration:</bold> Hao-yang Zhang, Li-Jian Pang.</p><p><bold>Resources:</bold> Ming-Hua Nan.</p><p><bold>Writing – original draft:</bold> Hao-yang Zhang.</p><p><bold>Writing – review & editing:</bold> Hao-yang Zhang, Xiao-Dong Lv, Li-Jian Pang, Chuang Liu, Ming-Hua Nan.</p></sec></body><back><fn-group><fn fn-type=\"abbr\"><p>Abbreviations: AMSTAR-2 = assessment of multiple systematic reviews-2, GRADE = grading of recommendations assessment, development and evaluation, INPLASY = International Platform of Registered Systematic Review and Meta-analysis Protocols, IPF = idiopathic pulmonary fibrosis, PRISMA = preferred reporting items for systematic reviews and meta-analyses, RCTs = randomized controlled trials, ROBIS = risk of bias in systematic review, SRs = systematic reviews.</p></fn><fn fn-type=\"other\"><p>How to cite this article: Zhang HY, Pang LJ, Lv XD, Liu C, Nan MH. Multiple Traditional Chinese Medicine interventions for idiopathic pulmonary fibrosis: A protocol for systematic review and meta-analysis of overview. <italic>Medicine</italic>. 2020;99:39(e22396).</p></fn><fn fn-type=\"other\"><p>H-YZ and L-JP contribute equally to this paper and are co-first authors of this paper.</p></fn><fn fn-type=\"supported-by\"><p>This work is supported by Natural Science Foundation of China (NO:81373579, NO:81403290) and High Level Innovation Team of Liaoning Province's “plan of rejuvenating Liaoning talents” (XLYC1808011).</p></fn><fn fn-type=\"other\"><p>Formal ethics approval and the informed consent are not required for this overview as we will only analyze published literature and no primary data will be collected.</p></fn><fn fn-type=\"COI-statement\"><p>The authors report no conflicts of interest.</p></fn><fn fn-type=\"other\"><p>All data generated or analyzed during this study are included in this published article [and its supplementary information files].</p></fn></fn-group><ref-list><title>References</title><ref id=\"R1\"><label>[1]</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raghu</surname><given-names>G</given-names></name><name><surname>Remy-Jardin</surname><given-names>M</given-names></name><name><surname>Myers</surname><given-names>JL</given-names></name><etal/></person-group>\n<article-title>Diagnosis of Idiopathic Pulmonary Fibrosis. 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