Supplemental Digital Content is available in the text This review aims to evaluate the effectiveness and safety of acupuncture treatment for reducing opioid consumption in patients with chronic pain.We will search the following electronic databases from their inception to November 2019: MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials, the Cumulative Index to Nursing and Allied Health Literature, the Allied and Complementary Medicine Database, the China National Knowledge Infrastructure (a Chinese database), the Japan Science and Technology Information Aggregator (a Japanese database), and five Korean databases . Randomized controlled trials comparing acupuncture to no treatment, sham acupuncture, and other active interventions for the reduction of opioid consumption in chronic pain patients will be included. The risk of bias will be assessed using the Cochrane risk of bias tool. The primary outcomes will include the prescribed or consumed dose of opioids and withdrawal symptoms related to opioid reduction. A meta-analysis will be performed to estimate a pooled effect, if possible.This study may provide important practical guidance for patients, practitioners, and health-policy makers regarding the use of acupuncture in opioid taper support programs.The results will be disseminated through a peer-reviewed journal or conference presentations.Trial registration number: PROSPERO 2019: CRD42019143486 As a result, the prescription of opioids has increased over the last 2 decades, especially in the United States, United Kingdom, and Australia, for patients with high-impact chronic pain as well as acute postoperate and cancer pain. Among the 25.3 million patients with chronic pain in the Unites States, opioids were prescribed for more than 240 million times in 2015. However, many studies have revealed limited evidence opioid use for long-term pain relief in chronic-pain conditions. Moreover, addiction and overuse of opioids leads to considerable adverse effects, such as bladder dysfunction, sedation, immune suppression, and even death.Chronic pain, defined as pain lasting more than 3 months, is one of the most common reasons for long-term medication prescription and affects about 20% to 50% of adults worldwide.1.2 In addition to pain reduction, acupuncture treatment is a promising adjunct to facilitate the reduction or tapering of pain medications, including opioids, in patients with chronic pain. It may also be used to treat opioid use disorder, which may involve opioid cravings, anxiety, depression, or insomnia.Acupuncture is defined as an intervention that stimulates specific points  using dry needles with some kind of manipulation . Recently, a rigorous meta-analysis of data from 20,827 individuals showed that acupuncture is effective for nonspecific chronic musculoskeletal pain when compared to a sham control, and the treatment effects persisted over time.1.3 Acupuncture may also facilitate opioid tapering by reducing adverse events related to opioid use or withdrawal.,10 In addition, it may reduce pain intensity and symptoms associated with comorbidities of chronic pain.Although the mechanism of acupuncture treatment for reducing opioid consumption remains unclear, some studies have shown that acupuncture increased the μ-opioid receptor binding ability, which could lead patients to demand lower doses of opioids.1.4 and avoid medication dependence. Although one Cochrane review focused on interventions for the reduction of prescribed opioid use, only 1 acupuncture study was included due to a limited search that did not include Korean or Chinese databases. A systematic review focusing on acupuncture was recently published, but it evaluated only clinical symptoms related to opioid use or withdrawal, not the reduction of prescribed opioids. Therefore, a review with an up-to-date systematic search is needed to evaluate acupuncture for the reduction of opioid consumption; this will help us determine whether acupuncture is an effective treatment option for opioid taper support programs.Long-term use of opioids in patients with chronic musculoskeletal pain is known to provide limited pain relief, and the potential side effects may lead to opioid-related death from addiction and overdose. Non-pharmacologic interventions, such as acupuncture have been proposed as alternatives to cope with these problems, address chronic pain,1.5The objective of this systematic review is to evaluate the benefits and harms of acupuncture treatment for the reduction of opioid consumption in patients with chronic pain in comparison to no treatment, sham acupuncture, or other therapies such as pharmacological, physiological, educational, or psychological interventions.22.1http://www.crd.york.ac.uk/ PROSPERO) on October 2019. If protocol amendments occur, we will update the changes in the PROSPERO and disclose them in the publications for the results of this study.The protocol of this review has been prospectively registered  of acupuncture treatment for the reduction of opioid use will be included in the review. Non-randomized controlled trials, observational studies, qualitative studies, and laboratory studies will be excluded. Language will not be a restriction for study eligibility.2.2.2Data from all adult patients taking opioid medication for non-cancer pain for more than 2 months will be included; there will be no dose limitation. The diagnosis criteria and classification of pain conditions will not be limited and will include various musculoskeletal, mono- or poly-neuropathic, visceral, or head pain.2.2.3Acupuncture treatment is defined in 2 components:(1)needling with various types of stimulation (2)specific points .Trials examining acupuncture treatment involving the above 2 components will be included in this review. However, trials including non-penetrating stimulation  will not be included. Only trials in which acupuncture treatment was compared with other forms of acupuncture methods will be excluded. If the acupuncture group simultaneously received acupuncture and other multidisciplinary treatments for pain management or opioid tapering, only trials in which the same program was administered to the both groups will be included.The control will be considered as no treatment, sham acupuncture, or other opioid taper support programs including pharmacological, physiological, educational, or psychological interventions.2.2.4Primary outcomes1.Opioid dose (prescribed or consumed): If possible, all opioid doses will be converted into morphine milligram equivalents.2.Withdrawal symptoms related to opioid reductionSecondary outcomes1.Pain intensity2.Psychological measurements3.Physical measurements4.Quality of life (QoL)5.Clinical global improvement in symptoms6.Retention of treatments7.Adverse events related to acupuncture treatment2.32.3.1The Cochrane Library, 2019, Issue 11), the Cumulative Index to Nursing and Allied Health Literature , the Allied and Complementary Medicine Database , the China National Knowledge Infrastructure , the Japan Science and Technology Information Aggregator Electronic , and five Korean databases .The following 12 databases will be searched from inception to November 2019: MEDLINE (1946 to November 2019), EMBASE (1980 to November 2019), the Cochrane Central Register of Controlled Trials , opioid use , and acupuncture . The special search strategies for MEDLINE are presented online in Appendix 1.2.42.4.1http://www.prisma-statement.org).Two researchers (SL and DHJ) will independently screen potentially eligible articles by reading the titles and abstracts. The researchers will independently select and check their decisions according to predefined criteria on a standard eligibility form. Any disagreement regarding the selection of article will be resolved through discussion, and if a disagreement remains, a third researcher will resolve the disagreement. The flow process will be summarized in a Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA)-compliant flow chart (2.4.2Two researchers (SL and DHJ) will independently read the full text of the selected articles and extract data using a standard data extraction form. This data will include author, year of publication, country, study design, sample size, participants, condition, acupuncture intervention, control intervention, outcome measures, main results, and adverse events. Discrepancies regarding the extracted data will be resolved through discussion or consultation between the researchers. When data in an article is insufficient or unclear, the researchers will contact the first or corresponding author via email to request additional information.2.4.3Two researchers (SL and DHJ) will independently conduct the quality assessment using the tool for assessing risk of bias based on the Cochrane Handbook for Systematic Reviews of Interventions. The following seven domains will be assessed:(1)random sequence generation;(2)allocation concealment;(3)blinding of participants;(4)blinding of outcome assessors;(5)incomplete outcome data;(6)selective outcome reporting; and(7)other sources of bias .The risk of bias will be categorized into 3 levels: low, high, and unclear risk of bias. Any discrepancies in the assessment will be resolved through discussion between the researchers.2.4.4For continuous data, the mean difference (MD) will be presented with 95% confidence intervals (CIs). If the scales measuring the continuous outcomes among the studies in the analysis do not correspond, the standardized mean difference (SMD) will be used. For dichotomous data, the risk ratio (RR) with 95% CIs will be used to measure the treatment effect. Ordinal data will be converted to dichotomous data if the data needs to be pooled. For example, assessments of global improvement graded as ‘recovery’, ‘markedly effective’ ‘effective’, and ‘ineffective’ will be dichotomized into ‘improved’ or ‘not improved’. A patient who achieves more than 50% reduction in opioid consumption will be defined as ‘responder’, and their data will be analyzed as dichotomous data.2.4.5When an outcome variable is assessed repeatedly (at more than 2 follow-up time points after post-treatment), only the last assessment will be chosen for the main analysis.2.4.6For missing or incomplete data, we will attempt to contact the first or corresponding authors to request the additional data. If this is not possible, only the available data will be analyzed, and the potential impact of the missing data will be addressed in the discussion.2.4.7P < .10 will be used to determine the presentation of heterogeneity. The I2 statistic will also be used to summarize heterogeneity among the trials; I2 of 0% to 40% might not be important, 30% to 60% may represent moderate heterogeneity, 50% to 90% may represent substantial heterogeneity, and 75% to 100% may represent considerable heterogeneity.First, heterogeneity will be judged by visual inspection of the forest plot; then, a chi-square test with a significance level of 2.4.8 If asymmetry is identified in the funnel plot, Egger linear regression method will be performed.When more than 10 trials are available, a funnel plot will be used to detect reporting bias.2.4.9 When a trial includes more than two acupuncture treatments with different stimulation methods  or points , meta-analysis could be performed carefully with consideration of whether the data of different acupuncture treatments could be combined into 1 merged acupuncture treatment. On the contrary, when a trial has more than 2 control interventions , the data of the acupuncture treatment will be split equally and compared with each control intervention separately.The meta-analysis will be performed using Review Manager software . A random effects model will be planned to pool data, as considerable clinical heterogeneity involving conditions, acupuncture regimens, and control interventions is expected in the studies included in the review. However, the data will not be pooled when considerable heterogeneity is detected without explanation for the clinical and methodological diversity.2.4.10When sufficient data are available, subgroup analysis will be performed according to the following:1.Style of acupuncture stimulation 2.Type of control intervention 2.4.11When appropriate, sensitivity analysis will be performed to identify whether the results of the review are robust after removing the trials with high risk of bias.2.4.12The main outcomes (primary outcomes and adverse events) will be summarized in the “Summary of findings” tables. The quality of evidence in the main outcomes will be assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach  with GRADE software. The quality of evidence will be categorized using the following four levels: high, moderate, low, and very low quality.2.5As this is a systematic review, ethical approval and informed consent were not necessary. The results of this review will be disseminated through conference presentations and peer-reviewed journal articles.3This is the study protocol of a systematic review and meta-analysis on the use of acupuncture treatment for reduction of opioid consumption in patients with chronic pain.,17 Furthermore, a recent systematic review showed that conventional acupuncture or transcutaneous electric acupoint stimulation reduced consumption of opioid analgesics, especially on the first day after surgery. Though, in the last several years, the long-term abuse of opioid therapy has been coined an opioid crisis and recognized as one of the most challenging problems of public health, research for reducing long-term opioid consumption is not well known. Recently, various federal regulatory and professional organizations have started to encourage the use of multidisciplinary approaches, including nonpharmacologic interventions, to reduce long-term opioid consumption and opioid use disorder. In this context, acupuncture treatment, which is already supported by rigorous evidence for chronic pain, has received attention as the most promising treatment option for this strategy.,21 Zheng and colleagues presented the possibility of using electroacupuncture to reduce opioid consumption in patients with chronic musculoskeletal pain through conducting sham-controlled clinical trials.,10 However, to determine the role of acupuncture in the multidisciplinary approach used in opioid taper support programs, there must be comprehensive evidence of acupuncture for the reduction of opioid consumption.Many studies have shown that perioperative acupuncture treatment effectively reduces acute opioid consumption after surgery.Therefore, we will conduct a systematic review searching all relevant literature, without any language restrictions, in the Korean, Chinese, and Japanese databases to include any relevant trials of acupuncture for the reduction of opioid consumption in patients with chronic pain. The results of this systematic review will be useful for patients, practitioners, and health policy-makers who wish to include acupuncture among the multidisciplinary treatment options available in opioid taper support programs. Moreover, this review will be constructive for researchers planning further clinical trials.The author would like to thank Professor Kun Hyung Kim and Jung Won Kang for encouraging suggestions and helpful comments.Conceptualization: Seunghoon LeeFunding acquisition: Seunghoon LeeMethodology and project administration: Seunghoon LeeWriting : Seunghoon LeeWriting (review and editing): Dae-Hyun Jo, Seunghoon LeeSeunghoon Lee orcid: 0000-0003-0088-2296.


Enterobacteriaceae. We found that the deletion of either of the orf35 and orf36 genes, which are dispensable for conjugative transfer, increased the plasmid mobilization efficiency. Real-time quantitative PCR (RT-qPCR) analysis suggested the involvement of orf35 and orf36 in regulating the expression of transfer genes. We also revised the host range of pCTX-M3 by showing that its conjugative transfer system has a much broader host range than its replicon.Horizontal gene transfer is responsible for rapid changes in bacterial genomes, and the conjugative transfer of plasmids has a great impact on the plasticity of bacteria. Here, we present a deletion analysis of the conjugative transfer system genes of the pCTX-M3 plasmid of the IncM group, which is responsible for the dissemination of antibiotic resistance genes in  Citrobacter freundii, is classified in the IVB subfamily and in the MPFI group, as are the conjugation systems of IncI1 group plasmids. Although the majority of the conjugative genes of the IncM and IncI1 plasmids display conserved synteny, there are several differences. Here, we present a deletion analysis of 27 genes in the conjugative transfer regions of pCTX-M3. Notably, the deletion of either of two genes dispensable for conjugative transfer, namely, orf35 and orf36, resulted in an increased plasmid mobilization efficiency. Transcriptional analysis of the orf35 and orf36 deletion mutants suggested an involvement of these genes in regulating the expression of conjugative transfer genes. We also revised the host range of the pCTX-M3 replicon by finding that this replicon is unable to support replication in Agrobacterium tumefaciens, Ralstonia eutropha, and Pseudomonas putida, though its conjugation system is capable of introducing plasmids bearing oriTpCTX-M3 into these bacteria, which are representatives of Alpha-, Beta-, and Gammaproteobacteria, respectively. Thus, the conjugative transfer system of pCTX-M3 has a much broader host range than its replicon.Plasmid conjugative transfer systems comprise type IV secretion systems (T4SS) coupled to DNA processing and replication. The T4SSs are divided into two phylogenetic subfamilies, namely, IVA and IVB, or on the basis of the phylogeny of the VirB4 ATPase, into eight groups. The conjugation system of the IncM group plasmid pCTX-M3, from IMPORTANCE Horizontal gene transfer is responsible for rapid changes in bacterial genomes, and the conjugative transfer of plasmids has a great impact on the plasticity of bacteria. Here, we present a deletion analysis of the conjugative transfer system genes of the pCTX-M3 plasmid of the IncM group, which is responsible for the dissemination of antibiotic resistance genes in Enterobacteriaceae. We found that the deletion of either of the orf35 and orf36 genes, which are dispensable for conjugative transfer, increased the plasmid mobilization efficiency. Real-time quantitative PCR (RT-qPCR) analysis suggested the involvement of orf35 and orf36 in regulating the expression of transfer genes. We also revised the host range of pCTX-M3 by showing that its conjugative transfer system has a much broader host range than its replicon. The DNA processing functions are provided by the DNA transfer and replication (Dtr) system, also called the relaxosome complex (Agrobacterium tumefaciens VirB/D4 secretion system and the conjugation systems of the IncF and IncP plasmids are classified as type IVA (T4ASS) (Legionella pneumophila (Dot/Icm) and in other important pathogens  is transferred from a donor to a recipient cell after physical contact between the cells is established. The process may be regarded as a combination of DNA processing functions coupled to a type IV secretion system  , 3, wherathogens . The majAgrobacterium tumefaciens VirB/D4 secretion system responsible for transfer DNA (T-DNA) transfer to plant cells during infection. This T4SS consists of 11 proteins (VirB1 to VirB11) and the coupling protein VirD4 , a member of the IncM incompatibility group  (oriTColIb-P9 (from the IncI1 plasmid ColIb-P9), and plasmids with oriTpCTX-M3 can be mobilized by a ColIb-P9-derived plasmid  ; howeverolIb-P9) . The pCT plasmid ; both oftra and trb genes potentially involved in pCTX-M3 conjugative transfer. We found that the deletion of either of the orf35 and orf36 genes, both of which are dispensable for pCTX-M3 transfer, increases the mobilization efficiency of oriTpCTX-M3-bearing plasmids into E. coli and A. tumefaciens. The deletion of these genes also affected the transcription of other conjugative transfer genes. In addition, we verified the host range of the pCTX-M3 conjugation system and found that the host range of its replicon, reported previously to comprise Alpha-, Beta-, and Gammaproteobacteria . Ho. Hotra apensable . Moreovee operon  (14).tra and trb regions. For this purpose, a collection of 27 derivatives with a deletion in each of the genes in the tra and trb regions, as well as orf35 from the pCTX-M3 leading region and orf46, which is located downstream of trbC, was constructed  by replacing a given gene with the cat gene  from E. coli BW25113 cells to the recipient E. coli JE2571Rifr cells, both in liquid and on filters, was determined . For the majority of the pCTX-M3 derivatives, the complementation fully or at least partially restored the conjugation efficiency (10−4 to 10−1 per donor cell). However, for the nikB, traM, traN, traU, traW, and traY deletion derivatives, the presence of the complementing gene rescued the conjugation efficiency only to levels less than 10−5; for traP and pri, the complementation had no detectable effect , abolished conjugative transfer; however, nikB can be complemented well  transfer efficiency or, for traY, 10× lower than the WT transfer efficiency . The transcript levels of all these genes were at least 10-fold higher than those found in the strain bearing pCTX-M3  to undergo conjugative transfer was tested after complementation with a plasmid bearing the corresponding gene . The results obtained for the pri deletion in pCTX-M3 differ from those for the sog deletion in R64, where the deletion resulted in only a small drop in the transfer efficiency, from 10−2 to 10−3 , which generates RNA primers for plasmid replication, and the shorter SogS (844 aa), which is a product of translational reinitiation within the sog reading frame  and a putative 689-aa protein comprising the C-terminal moiety of the primase. The high level of the pri transcript  of L. pneumophila, which acts as a type IV coupling protein (T4CP)  . In the n (T4CP) . The DottraN gene of both R64 . The deletion of these genes completely abolishes the conjugative transfer of pCTX-M3 and R64  of L. pneumophila and with VirB8 of A. tumefaciens (traU gene encodes a putative ATPase that is a homologue of DotO (IcmB) of L. pneumophila . Surprisingly, pCTX-M3orf36::cat was approximately 5 times more effective than the WT plasmid . The protein product of orf35 of pCTX-M3 exhibits 44% amino acid sequence similarity with that of yggA, the first gene in the leading region of R64 , whose involvement in mobilization has not been studied.To identify the pCTX-M3 genes affecting the mobilization efficiency of plasmids bearing fr cells . The helA. tumefaciens as the recipient, E. coli carrying pCTX-M3orf35::cat or pCTX-M3orf36::cat was >100- or 10-fold more efficient, respectively, as a pToriT donor than was E. coli bearing pCTX-M3 or pCTX-M3orf46::cat .With f46::cat . Howeverorf35 and orf36 on plasmid mobilization, the deletions of these genes were complemented with appropriate plasmids, namely, pALorf35 and pALorf36, respectively. The pABBoriT plasmid was mobilized to an E. coli recipient from E. coli DH5α donors. The helper plasmids pCS, pC35S, and pC36S, which were derived from pCTX-M3, pCTX-M3orf35::cat, and pCTX-M3orf36::cat, respectively, and lack kanamycin resistance, were generated for use with the Kmr pABBoriT plasmid  plasmid pToriT, which contains oriTpCTX-M3, into A. tumefaciens (10−4 transconjugants per donor after 30 min of mating).Earlier studies  have demy mating . HoweverA. tumefaciens is inconsistent with previous results  as the donor and E. coli, A. tumefaciens, Ralstonia eutropha, and Pseudomonas putida as recipients. In such a system, the transfer of pCTX-M3 during mating reflects the host range of both its conjugation system and the IncM replicon, while the transfer of pToriT, the mobilizable broad-host-range plasmid containing oriTpCTX-M3, reflects the host range of the conjugation system of pCTX-M3 only. Transconjugants carrying pCTX-M3 were selected on plates containing gentamicin and rifampin, while those with pToriT were selected on plates with tetracycline and rifampin  or with kanamycin and rifampin (A. tumefaciens and P. putida). As shown in A. tumefaciens and R. eutropha  and, with a lower efficiency, also into P. putida (3 × 10−6 per donor cell). In contrast, pCTX-M3 transconjugants were obtained only in the E. coli recipient. Thus, pCTX-M3 itself, when transferred, cannot be established in A. tumefaciens, R. eutropha, or P. putida. These results indicate that the host ranges of the pCTX-M3 conjugative transfer system and its replicon differ markedly; the former shows a broad range comprising Alpha-, Beta-, and Gammaproteobacteria, and the latter is restricted to Enterobacteriaceae. A similar observation concerning the differences between the host ranges of the conjugation system and the replicon has been reported previously for the narrow-host-range mobilizable Klebsiella pneumoniae plasmids pIGMS31 and pIGMS32  conjugation system (P. putida plasmid NAH7 of the IncP-9 group (with the MPFT system) (The finding that pCTX-M3 is not transferred into  results  showing  pIGMS32 . These pn system . In addi system) .I group, it differs from the one encoded by IncI1 plasmids. Therefore, it would be valuable to reevaluate the mobilization host range of MPFI systems. It has been shown that ssDNA transiently generated during conjugative transfer triggers the SOS response in recipient cells unless the plasmid codes for an anti-SOS factor  or on agar-solidified LB plates (E. coli and P. putida) or 28°C (A. tumefaciens and R. eutropha). When required, antibiotics were added to the medium at the following final concentrations (μg/ml): ampicillin, 100; chloramphenicol, 20; gentamicin, 50; kanamycin, 50; rifampin, 100; tetracycline, 20 or 6 (for pToriT selection).The strains used in this work are listed in B plates  at eithePlasmid DNA was isolated by alkaline lysis using Plasmid Mini or Plasmid Midi kits  according to the manufacturer's instructions. DNA cloning was performed according to standard protocols . All thetra and trb regions were constructed through lambda Red-mediated recombination  cells were electrotransformed with these DpnI-treated PCR products and were selected on LB agar plates with chloramphenicol at 37°C . Single colonies were isolated by the streak plate method at 37°C, and the correct integration of the cat gene into the target gene was verified by PCR (35 cycles) with the primer pairs listed in Table S3 and S4. The integration of cat in the four longest genes  was further analyzed by multiplex PCR with three primers (see Table S5): (i) catReVer, which anneals to the cat gene, (ii) a primer located upstream of the deleted gene , and (iii) a primer that anneals to the gene to be deleted . The primers were designed so that the expected products were smaller than 1 kb and enabled discrimination between the native pCTX-M3 and the appropriate mutant plasmid. All mutated plasmids were verified by sequencing with the catU142 primer. The loss of pKD46 was checked by multiplex PCR with the repKD46F and repKD46R primers, which were designed to amplify the repA101 (thermosensitive replication) gene fragment, and with the TEMfor and TnTEMrev primers  as an internal PCR control (see Table S4). The constructed plasmid derivatives are listed in Table S1.The plasmids that were constructed and used in this study are listed in bination . First, cat gene was eliminated from six pCTX-M3 derivatives, namely, pCTX-M3nikB::cat, pCTX-M3pri::cat, pCTX-M3traP::cat, pCTX-M3traU::cat, pCTX-M3traW::cat, and pCTX-M3traY::cat, using pCP20, which encodes FLP recombinase, as a helper plasmid. In this process, the strain DH5α(pCP20) was electrotransformed with the appropriate pCTX-M3 derivative, and transformants were selected on LB agar with gentamicin at 28°C. Then, the transformants were streaked in parallel on LB with both chloramphenicol and gentamicin and were grown at 37°C. After colony purification, the clones that were chloramphenicol sensitive and gentamicin resistant were verified by PCR with the primers listed in Tables S3 and S5. The loss of pCP20 was verified by PCR with the repKD46F and repKD46R primers. The plasmids were then introduced into the BW25113 strain.The tra and trb regions for use in the complementation experiments (Table S1), specific genes were amplified by PCR using Pfu DNA polymerase  with the primers listed in Table S6 and were cloned into the pMT5 or pAL3 vectors, as described in Table S1. Genes which, probably due to the harmful effects of high-level expression, were not able to be cloned into the multicopy plasmid pMT5 (oriVpMB1), were cloned into the low-copy-number vector pAL3 (oriVP15A). Only the pALpri plasmid was constructed without a PCR amplification step, as indicated in Table S1. The cloned genes were verified by sequencing (primers listed in Tables S3 and S4). The expression of the complementing genes cloned into the pAL3 and pMT5 vectors is driven by the lactose operon promoter (Plac).To construct the plasmids carrying individual genes from the oriTpCTX-M3 were obtained by cloning the oriT sequence into appropriate plasmids, as described in Plasmids bearing orf35::cat, and pCTX-M3orf36::cat, respectively, with SalI and the recircularization of the largest DNA fragment. Thus, these plasmids are devoid of all resistance genes except the blaTEM-1 and blaCTX-M-3 genes present in pCTX-M3.The plasmids pCS, pC35S, and pC36S  were obtTaq or Pfu DNA polymerase with the supplied buffers (Thermo Fisher Scientific), deoxynucleoside triphosphate (dNTP) mixture, template DNA , and the appropriate primer pairs listed in Tables S2 to S6, according to the manufacturer's recommendations.PCR was performed in a Veriti thermal cycler  using DreamThe sequencing was performed in the DNA Sequencing and Oligonucleotide Synthesis Laboratory at the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, using a dye terminator sequencing kit and an automated sequencer .600] of 0.8 to 1) using a GeneJET RNA purification kit (Thermo Fisher Scientific) according to the manufacturer's protocol. RNA quality and integrity were checked by agarose gel electrophoresis, and the concentration was estimated using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Three biological replicates were analyzed.RNA was isolated from cells of BW25113 strains bearing a specific pCTX-M3 deletion derivative alone or in combination with a plasmid carrying an appropriate complementing gene in the late exponential phase of growth  and target genes are listed in Table S7. Real-time PCR was carried out using Real-Time 2×HS-PCR master mix SYBR (A&A Biotechnology) in a final volume of 10 μl in the LightCycler 480 system  with an initial denaturation at 95°C for 5 min followed by 40 cycles of amplification (95°C for 10 s and 50°C for 10 s). The relative gene expression in the deletion strains was calculated and normalized to the value obtained for a strain carrying the native pCTX-M3 plasmid (Reverse transcription was performed with random hexamer primers using the Maxima First Strand cDNA synthesis kit for RT-qPCR with dsDNase (Thermo Fisher Scientific). The specific qPCR primers used to amplify the reference (the  plasmid .8 CFU ml−1) were grown in LB to stationary phase; the cultures were then washed twice with LB medium, and the donor strain was resuspended in a volume of LB equal to the initial volume of the culture, while the recipient strain was resuspended in one-fourth of the initial culture volume. Then, 0.5 ml of the donor and recipient suspensions was mixed and filtered through a sterile Millipore HA 0.45-μm filter . The filter was incubated on an LB plate for 30 min at 37°C (E. coli) or 28°C (A. tumefaciens and E. coli). For the pCTX-M3 host range tests, the filter was incubated on an LB plate for 24 h at 37°C (P. putida and E. coli) or 28°C (A. tumefaciens and R. eutropha). Bacteria were washed from the filter with 1 ml of a sterile 0.85% NaCl solution. Conjugation was stopped by vigorously vortexing the mating mixture for 30 s and then placing it on ice. Serial dilutions of the mixture of donor, recipient, and transconjugant cells were plated on LB agar supplemented with the appropriate selection antibiotics. As a control, the donor and recipient cells were plated on LB supplemented with antibiotics for transconjugant selection. Mating in liquid was performed as described above but without the use of the filter: the mating mix was incubated for 30 min at 37°C, and conjugation was stopped by vortexing for 30 s. The conjugative transfer efficiency is equivalent to the number of transconjugants per donor cell.Cultures of the donor and recipient strains (approximately 10http://blast.ncbi.nlm.nih.gov/Blast.cgi) to search against the NCBI “nr” (nonredundant) DNA or protein database with standard parameters; no filters or masks were applied. The molecular weight and theoretical pI were calculated with the use of the ProtParam tool of Expasy (http://web.expasy.org/protparam/) (http://myhits.isb-sib.ch/cgi-bin/motif_scan). SignalP v.2.0  was used to search for signal peptides, TMPred (http://www.ch.embnet.org/software/TMPRED_form.html) was used to determine the presence of transmembrane helices (TMH) (http://www.cbs.dtu.dk/services/LipoP/) was used to predict lipid attachment motifs  . Motifs es (TMH) , and Lipt motifs .t test . P values of <0.05 were considered statistically significant.Data concerning the plasmid conjugative transfer frequencies are presented as the means ± standard deviations (SDs). The differences between the mobilization efficiencies of pToriT by the different plasmids  were tes


Lactobacillusreuteri and the microbial composition were investigated. Basic and acidic hydrolysis were tested for an exhaustive recovery of bound phenols: the acidic hydrolysis gave best yields. Methyl ferulate and neocarlinoside were identified for the first time in wheat. The inhibitory power of the extracts of several batches were investigated on PTP1B enzyme. The product was not able to inhibit the enzyme, otherwise, for the first time, a complete inhibition was observed for schaftoside, a major C-flavonoid of wheat. The microbial composition was assessed identifying Lactobacillus, Enterococcus, and Pediococcus as the main bacterial species. The fermented wheat was a suitable substrate for the grown of L. reuteri, recognized for its health properties in the human gut. The proposed method for phenols is easier compared to those based on strong basic hydrolysis; our results assessed the bound phenols as the major fraction, differently from that suggested by the literature for fermented cereals.Fermented cereals, staple foods in Asia and Africa, are recently receiving a growing interest in Western countries. The object of this work is the characterization of a fermented wheat used as a food ingredient and dietary supplement. To this aim, the phenolic composition, the activity on protein tyrosine phosphatase 1B (PTP1B), an enzyme overexpressed in type-II diabetes, the in vitro prebiotic properties on  Cereal fermentation is an ancient technique applied to improve food texture, shelf-life, digestibility, and food preservation, as well as able to remove anti-nutritional factors and toxins. It is a widely applied practice in Asian and African cultures where cereals, as staple crops, are processed before consumption. In recent years, there has been renewed interest in fermented foods in Europe, especially for the supposed health benefits linked to these foods . The ferTriticum aestivum), used as food ingredient and food supplement, is Lisosan® G. The production is from the wholegrain, grounded, added with water and a sourdough starter, than mixed to initiate the fermentation process, the whole product is recovered after three days, and dried. Recently, Lisosan® G has been object of in vivo and ex vivo studies, but few data are available on its composition. A protective activity was observed on Wistar rats fed with different dosages of Lisosan® G before the use of cisplatin as nephrotoxic, neurotoxic and ototoxic agent − ion, showed again the losses of 90 and 120 mass units, respectively. From MS3 experiments it was possible to confirm that the common fragment ion at 353 m/z is originated from the 563 m/z (after two successive losses of 120 and 90 mass units). According to literature [11 as isoschaftoside and 13 as schaftoside.The profiles at 210 nm, a non-selective wavelength, confirm the exclusive presence of flavonoid compounds. The mass spectra of compounds 9 and 10  showed ti et al. , these lin wheat , as a plterature ,16, it wC-glycosilated flavonoids are reported in 15 was identified as ferulic acid as also confirmed by the MS2 spectrum of its deprotonated molecular ion showing the species at 178 m/z, corresponding to the loss of a methyl group, and 134 m/z from the successive loss of carbon dioxide (59 mass units).The spectral data in positive and negative ion mode for the pool of ® G were 38 mg/100 g (S) was firstly tested and compared with a softer condition with 0.1 M NaOH (Method B). HPLC-DAD analysis highlighted that the former procedure induced a partial degradation of the phenolic compounds when compared with the weaker basic hydrolysis: the use of 4 M NaOH led to the degradation of methyl ferulate converted in ferulic acid, and of compounds 9 and 10  and methyl ferulate (19), while the acidic hydrolysis (19), a compound previously detected in rice [® G with Method B (0.1 M NaOH in methanol); as expected, the formation of methyl ferulate was not observed and it was possible to conclude that this ester is not an artefact of the extraction process, but is naturally present in Lisosan® G. Observing the profile obtained from the basic extraction (Method BF) of the solid residue of Lisosan® G remaining after recovery of the free phenols, it was possible to assess that C-glycosylated flavonoids (9–13) were only present in free form (S) led to a partial degradation of the compounds of interest (some minor C-flavonoids) and reduced the total phenolic recovery, in comparison to the other applied methods  guaranteed a better recovery (up to 60% higher) in terms of total phenols  maintained almost the same amount of the C-glycosylated flavonoids (compounds 9–11 and 13) extracted with the simple procedure for free phenols (® G batches for the following reasons: (i) the quantitative data from the methods A were almost the same obtained applying the basic procedure B, but in the latter case applying a four times higher extractive ratio, (ii) the phenolic compounds are chemically stable in acidic media differently to what can happen in the basic media, in which the more hydroxylated flavonoids can go toward a partial degradation, and (iii) the same acid procedure applied to other cereals (data not shown) gave again the highest recovery in terms of total phenols.The basic hydrolysis (B) and the acidic procedure (A) applied to LG1) was the richest in terms of total phenolic content; the LG2 and LG3 resulted very similar otherwise the LG4 was the poorest  at a final concentration of 125 mg/mL and schaftoside, as main flavonoid, at a final concentration of 0.15 mg/mL, were tested adding different amount of extracts . Analogously to what observed for the schaftoside, the results in ® G extracts reduced the inhibitory activity of the molecule alone. In light with these findings, we can conclude that Lisosan® G contains unknown molecules that impede the interaction with the enzyme. These results agrees with recent evidence that confirmed the presence of different allosteric sites on the enzyme surface involved in the regulation of the enzyme activity [The choice of testing schaftoside was also due to the inhibitory activity already showed by flavonoids on PTP1B enzyme as reported by literature . The resf 6.4 µM a. Considactivity . Furtheractivity . FurtherBacteriological analysis firstly excluded the presence of microbial contaminants typical of food specimens, such as coliforms and staphylococci .Next-generation sequencing was chosen as suitable tool to perform the qualitative characterization of bacterial composition. The 16S rRNA gene sequencing was applied to identify the microbial composition of Lisosan® G (Lactobacillus (45.4%), Enterococcus (28%), and Pediococcus (17%) as the main bacterial genera.Since the microbial composition of Lisosan G is unknown, quantification of specific bacterial species of interest by Real-Time PCR or q-PCR was not applied. sosan® G  with LacLactobacillus fermentum, Enterococcus faecium, and Pediococcus acidiliacti. Generally, numerous fermenting bacteria, especially lactic acid bacteria (LAB), have been identified in sourdoughs, including Lactobacillus and Pediococcus spp. In addition to acidification, the proteolytic activity of LAB allows release of compounds which can promote growth or inhibition and metabolic activities of other microorganisms, as well as organoleptic characteristics. Enterococcus genus includes known probiotic strains and was considered a good candidate for co-culture in food fermentation processes [By BLAST alignment we found that the best hits for species identification (99% of identity) were rocesses .Lactobacillus reuteri is a common component of the microbiota of human and animal intestine and it is widely used in probiotic formulations targeted to infants and adults to reduce the incidence and severity of diarrhea, prevent colics and necrotic enterocolitis, and maintain a functional mucosal barrier [® G was capable of stimulating the growth of L. reuteri DSM 17938 by more than 1 Log (from 6.7 to 7.8) after 48 h of incubation; even if growth was significantly lower (p < 0.05) with respect to that on glucose. The ability to increase the growth of this microorganism is certainly of interest for this fermented matrix characterized by the presence of probiotic strains and phenolic compounds potentially able to exert growth inhibition of harmful bacteria. The reason for the lack of difference in growth potential when Lisosan® G was tested at different concentrations (0.5 and 1%) may also be related to the copresence of different molecules and a broad spectrum of action of this complex product. The beneficial effects of Lisosan® G on human health have already been demonstrated, but prebiotic effects have not been described yet.  barrier . As show® G , a food supplement obtained from lysed fine bran and germ of organic wheat grains (Triticum aestivum), were provided by Agrisan Srl Company, Larciano . All solvents used were analytical HPLC grade from Sigma Aldrich . Water was ultrapure , ferulic acid (purity ≥ 99%), and apigenin (purity ≥ 95%) were purchased from Extrasynthese .Four batches of LisosanFree phenols. Lisosan® G, LG1, (250 mg) was extracted with EtOH/H2O 80:20 v/v under magnetic stirring in ultrasonic bath for about 15 min. The extract was centrifuged at 5000 rpm for 10 min. Ten mL of the supernatant were evaporated to dryness and the residue re-dissolved in 1.5 mL of acidified H2O (1% HCOOH). The same extraction was performed with H2O acidified with 1% HCOOH.Bound and total phenols. An acidic hydrolysis (method A) and three different basic hydrolyses  were applied on the fermented wheat as reported below. All the hydrolyses were carried out by the help of an ultrasonic bath (40 MHz). Method BF: 1 g of the sample was extracted with 25 mL of NaOH 0.1 M in MeOH/H2O 7:3 v/v, sonicated for 1 h at 60 °C. The pH was adjusted to neutrality with acetic acid and the solution centrifuged at 5000 rpm for 10 min to recover the supernatant. The same procedure was applied to the solid residue recovered after the extraction of free phenols. Method B was the same of method BF with only a difference on the solvent-dry sample ratio:1 g of Lisosan® G was extracted in 100 mL NaOH 0.1 M in MeOH/H2O 7:3 v/v. Method BS: 1 g of the sample was extracted in 25 mL of MeOH/H20 7:3 v/v with NaOH 4 M, sonicated for 1 h at 60 °C. The pH was adjusted to neutrality with CH3COOH and the solution diluted to a final volume of 50 mL with MeOH/H20 7:3 v/v; the sample was then centrifuged at 5000 rpm for 10 min to recover the supernatant. Method A was an acidic hydrolysis performed on the four batches (LG1–LG4) using the mixture MeOH/H2SO4 9:1 v/v: 1 g of Lisosan® G was extracted in 25 mL, sonicated for 2 h at 60 °C, and then centrifuged at 5000 rpm for 10 min to recover the supernatant.® G (LG1) were almost completely dissolved in 150 mL of HCOOH (1%) under magnetic stirring for 15 min at 60 °C. After centrifugation , the supernatant was dried under vacuum and re-dissolved in 6 mL of distilled water obtaining the total Lisosan® G extract. This extract was fractionated by semipreparative HPLC using a Hewlett Packard 1050 series and a Polaris RP-C18 Ether column ; 12 fractions were recovered after 30 injections of 100 µL. Elution was carried out at a flow rate of 4 mL min−1 with CH3CN as solvent A and H2O (0.1% HCOOH) as solvent B. A linear elution gradient was employed: solvent A was increased from 0% to 10% in 10 min, from 10% to 15% in 10 min, from 15% to 30% in 10 min, from 30% to 100% in 5 min with a final plateau of 10 min. Total time of analysis was 45 min, equilibration time 10 min. The collection was carried out monitoring the chromatogram at 280 nm up to 15 min, successively 350 nm was the wavelength selected to detect and recover the flavonoids. The fractions were dried, re-dissolved in 1 mL of water, and controlled by analytical HPLC-DAD and MSn analysis.About 700 mg of Lisosan® G (250 mg) was dissolved in 50 mL of acidified H2O (1% HCOOH), after a magnetic stirring of 15 min. The extracts were then evaporated to dryness and re-dissolved in 2 mL of water. These extracts (LG1–LG4), the pure schaftoside (at concentration 2.7 µM), and the pure myricetin (at concentration 0.5 µM) were evaluated as inhibitors of the enzyme PTP1B. Furthermore, the IC50 was also determined for the schaftoside. Enzymatic assays were carried out using human recombinant PTP1B and p-nitrophenylphosphate (pNPP) as reference substrate. According to Paoli et al. [−1 cm−1). The net hydrolysis rate was determined subtracting the value of spontaneous hydrolysis rate of pNPP from each sample. The inhibitory power of LG extracts and schaftoside standard was tested adding different amount of extracts (10 and 50 µL/mL) for each assay. Then, the percentage of inhibition was calculated normalizing the absorbance values obtained for assays carried out in the presence of inhibitor versus the control test. All the results were expressed as a mean of three independent experiments. All four batches were treated as follow: the maximum amount of Lisosan® G 0 mg was 1 batch was tested in McConkey III medium  and in Mannitol salt medium . Lisosan® G (500 mg) were directly plated on the two different media and, after incubation for 24–48 h, presence of potential microbial contaminants were evaluated.In order to exclude the presence of bacterial contaminants of food specimens, such as coliforms and staphylococci, LG1 batch, library of 16S rRNA gene amplicons was prepared by IGA Technology Services  through amplification of the V3–V4 hypervariable region. The standard protocol was followed according to the 16S metagenomic sequencing library preparation guide from Illumina . Pooled V3–V4 amplicon libraries were sequenced using the Illumina MiSeq platform. Sequence data are available at European Bioinformatics Insitute-EMBL-EBI database [http://compmetagen.github.io/micca/, San Diego, CA, USA), as reported by Di Paola et al. [E-value) was used to select significant BLAST hits, keeping only outcomes with the lowest E-value .By LGdatabase , under ta et al. . A totalL. reuteri DSM 17938). The lyophilized strain was re-vitalized in the Man Rogosa Sharpe (MRS) medium  supplemented with 0.05% cysteine and incubated in anaerobic chamber at 37 °C for 24 h. The MRS medium composition was modified to perform the growth experiment with Lisosan® G in order to eliminate glucose and reduce the concentration of potential growth factors, as described in Khatib et al. [® G, LG1 batch, at 0.5% and 1% (w/v) in m-MRS. A positive growth control was performed using m-MRS with 0.5% (w/v) glucose and a negative control in m-MRS with no added carbon source. The medium containing Lisosan® G as potential carbon source was prepared as follows: the m-MRS ingredients were weighed and the medium autoclaved at 120 °C for 15 min. Lisosan® G was then added, the solution stirred at 80 °C and then autoclaved again at 102 °C for 10 min. The strain was grown overnight in MRS, centrifuged, washed in saline (0.9% NaCl), and re-suspended in saline to obtain an absorbance of 0.7 mAu at 600 nm. This suspension was used to inoculate at 2% (v/v) the flasks containing the m-MRS medium plus Lisosan® G or glucose or the negative control with no carbon source. The tubes were incubated at 37 °C in anaerobic conditions for 48 h and a 1 mL culture was sampled from each flask, serially diluted, and inoculated on MRS agar plates for viable bacterial counts at pre-established times . Upon incubation, the number of colonies, corresponding to the number of viable cells, were counted and expressed as CFU mL−1. The number was transformed into a log10 value (log CFU mL−1).The strain used was obtained from the German Collection of Microorganisms and Cell Cultures  and analyzed using a HP 1200L liquid chromatograph equipped with a DAD detector  using a Poroshell 120, EC-C18 column . The solvents for the mobile phase were (A) CH3CN and (B) 0.1% formic acid/water; the multi-step linear solvent gradient was: 0–5 min 0–10% A; 5–10 min 10–15% A; 10–20 min 15–30% A; 20–25 min 30–35% A; 25–28 min 35–40% A; 28–31 min 40–45% A; 31–42 min 100% A; 42–47 min 100–0% A; equilibration time 5 min; flow rate 0.4 mL min−1; injection volume 10 μL. The following wavelengths were simultaneously selected: 240 nm, 280 nm, 330 nm, 350 nm.The extracts from Lisosann on a LTQ-Orbitrap . Each fraction was taken to dryness by evaporation under vacuum and re-suspended in a CH3CN/H2O mixture, containing 0.1% formic acid. This solution was infused by syringe into the ESI interface of the instrument. Sheath and auxiliary gas flow rates were 10 and 2 (arbitrary units), respectively; capillary voltage and tube lens voltages, as the collision energy and wideband activation voltage in MSn experiments, were optimized for each compound of interest during the infusion. The mass spectrometer was calibrated with the standard mixture indicated by the producer immediately before the acquisition of the samples, both in positive and in negative ion mode.The isolated fractions by semipreparative HPLC were analyzed by direct infusion in ESI-HRMS and MSR2 = 1, linearity range 0–0.21 µg), while the flavonoid content was determined using a five-point calibration curve of apigenin at 350 nm .The phenolic acids were evaluated using a five-point calibration curve of ferulic acid at 330 nm, . Statistical analysis of data from PTP1B was performed using the Student t-tests, using OriginPro 2018  The differences between the groups were considered significant when p < 0.05.Each experiment was performed in triplicate, and the results were expressed as the mean values ± SD; the EXCEL software  in-house routines were applied. Significance in the prebiotic properties experiment was calculated within each evaluation time with a ® G. Contrary to what reported in literature, the bound phenols remained high (more than 80%), although Lisosan® G was obtained after a fermentation process, usually described as able to increase hydrolytic processes and to strongly reduce the aliquot of bound phenols in cereals. Again, despite some data in the literature, the acidic hydrolysis was able to extract the highest amount of cinnamic derivatives, without degradation of the pool of C-glycosylated flavonoids. It was demonstrated that applying both soft basic and acidic hydrolysis on the whole flour it was possible to recover free and bound phenols through only one extractive step with higher yields compared to those obtained with the stronger basic hydrolysis suggested in the literature to date. Regarding the microbial characterization, it showed the presence of bacterial genera with fermentative capability such as Lactobacillus, Pediococcus, and Enterococcus, generally recognized as safe and used in the production of fermented food. The proteolytic activity of these bacteria can contribute to the release of compounds, as phenols, growth of beneficial bacteria as L. reuteri, and inhibition of harmful bacteria. For the first time, a prebiotic effect on L. reuteri strain, widely used in probiotic formulation targeted to infants, was highlighted. This fermented wheat resulted not able to inhibit the PTP1B enzyme in vitro, otherwise the pure schaftoside, a main C-flavonoid of Lisosan® G, showed a strong inhibition power. Schaftoside was tested for the first time on PTP1B enzyme and was active as inhibitor at µM concentration (13.5 µM). Our findings open new perspectives to investigate on the role played by this C-glycosylated flavonoid and its analogous, typically present in wheat and in other cereals. Further studies are desirable to clarify the mechanism linked to the action of schaftoside and to test also other similar C-flavonoids on PTP1B enzyme. The International Diabetes Federation announced that the global population of diabetics in 2015 was close to 400 million, and this number could rise to 600 million in 2040. In this context, research on new functional foods that can help stem the onset of this disease in the near future is recognized as strategic. Overall, our results provide further insights on the nutraceutical potential of this fermented food, whose beneficial effects were previously demonstrated by recent in vivo and ex vivo experiments.This work improves knowledge on the composition and properties of this fermented grain, and it is the first report to focus on the content of free and bound phenolic compounds in Lisosan


ObjectiveTo assess the efficacy of awareness campaign aimed at Hepatitis B and C in a rural community in Rawalpindi, Pakistan.MethodsThis is a cross-sectional study conducted on patients from a primary health care facility in Rawalpindi. The duration of the study was three months (April 2019 to June 2019). Patients who were registered at the facility were included after consent had been obtained. The data was collected through a self-constructed questionnaire. The data obtained was analyzed on IBM's statistical package for the social sciences (SPSS) version 21 .ResultsOut of 35 participants, 16 (46%) were males and 19 (54%) were females. The mean age of the participants was 43.82 ± 19.56 years. The mean number of people in the house was 7.54 ± 3.88. Twenty nine (84%) participants were married while six (16%) participants were not. A majority of the participants had good prior knowledge about Hepatitis B and C. However, a few confusions remained about the mode of transmission, the vector for transmission if any and vaccination protocols. The campaign proved to reinforce many concepts and clear potential confusions of the participants.ConclusionsThis attempt at improving awareness proved to be fruitful. There is a dire need to ensure that multiple activities are organized so that the burden of the disease may be reduced. There will be a strong network of communication for flow of information if the activities occur regularly and in a focused manner. Hepatitis B virus has infected over 240 million people worldwide with about half of the cases progressing to a chronic phase . SimilarHepatitis B virus (HBV) has a complex mechanism of liver injury in terms of liver damage and viral control and these factors are primarily responsible for the delayed diagnosis and subsequent progression to chronic illness. Hepatitis C virus (HCV) on the other hand has a multitude of genotypes . The mosThe route of transmission of both these viruses are more or less similar and therefore, awareness campaigns and public health awareness measures deal with both of the infections as a combined entity . The disA common misconception centers on the myth that these viruses are transmitted by insect vectors, such as mosquitoes. A few cases have been reported of drug abusers being infected with diseases due to insect vectors but hepatitis continues to be the exception.The objective of the study was to assess the efficacy of a public health awareness activity centered on a rural community at a Primary Health Care facility in Rawalpindi, Pakistan.We conducted a cross-sectional study on a small community who were enrolled at a primary health care facility from April 2019 to June 2019. Patients who were willing to participate in the study were included. Patients who were not present on the days of the study were not included in the study.The data collection was divided into two parts. The first part of data collection was the administration of a self-constructed questionnaire prior to providing the patients information about Hepatitis B and C. This questionnaire was developed with the help of a prior questionnaire and advice from public health education experts . There wThe responses, apart from demographic details, were all scored. Each correct answer scored one point and each wrong answer scored zero point. The "Do not know" response was considered a wrong answer and scored zero point.The questionnaire was distributed to all participants. All the participants were given 20 minutes to complete the questionnaire. After the data collection, all the students were given a seminar on Hepatitis B and C. A question and answer session was held after the seminar to address any queries regarding Hepatitis B and C. The same questionnaire was administered at the end of the study period for post-awareness responses.The data obtained was analyzed on IBM's statistical package for the social sciences (SPSS) version 21 . Descriptive statistics were used to analyze and describe the data. Frequencies and percentages were calculated for qualitative variables like gender. Mean and standard deviation (SD) were calculated for quantitative variables like age.Out of 35 participants, 16 (46%) were males and 19 (54%) were females. The mean age of the participants was 43.82±19.56 years. The mean monthly household income was 26114 ± 12538 rupees. The mean number of people in the house was 7.54 ± 3.88. Twenty nine (84%) participants were married while six (16%) participants were not. The pre-awareness and post-awareness scores were then calculated. Table Table Table This is the first study in which efficacy of an awareness activity has been investigated as opposed to prior knowledge of the people or the general prevalence of disease , 15. TheThe awareness campaign corrected the misconception that any kind of physical contact with a patient is responsible for transmitting the disease, as shown by the paired samples t-test. Many studies have detailed that exchanged of bodily fluids, for example blood, semen, vaginal secretions, as the only form of physical contact through which HBV and HCV can spread .Another misconception that the campaign corrected was the myth that carriers of the disease can marry each other, as shown by the results of the study. A study conducted in China  explainsBy the end of the activity, participants also realized that insects are responsible for spread of diseases, like dengue  and malaThere are a few limitations to the study. This is a survey and statistical analysis could not be done in detail as opposed to other study designs. The study population was focused on one community and so, has to include other communities as well. The format of the campaign was discussion based. Therefore, any participant who did not raise questions during the discussion, unfortunately, could not gain a lot from the activity.Several attempts should be made to organize multiple activities on different levels of health care setup to ensure that the objective could be met. Additionally, instead of a discussion based format, it would be more appropriate if the format included more pictorial aids.The campaign provided a great degree of information regarding HBV and HCV but there is a dire need to expand such attempts so that the vulnerable population making up the majority of the nation can be protected.


Microsurgery is a specialized area of surgery that demands training and research at every step. As a useful material for training, experimental methods are important for this purpose. However, ethical and economic considerations have forced scientists to seek alternative training materials. Therefore, in this study we aimed to use embalmed human ca-daver tissue, which are similar to live structures.The superior thyroid artery of a Modified Larssen Solution (MLS)-preserved cadaver was laid on saline-soaked gauze. Cannulas were fixed on both sides with silk sutures. One milliliter of saline was injected into the vessel lumen and allowed to drain from the other side. During this procedure, it was observed that the tissue caliber and turgor were suf-ficient to mimic the pulse. Subsequently, an incision was made in the middle of the artery. Adventisectomy was per-formed, and it was observed that the adventitia dissects and pierces identically to living tissues . AnastomThere is very limited information about the use of cadaver-embalming techniques in microsurgical education. One of the best representatives of this topic is a study by Wolff et al. , which uLarssen Solution was first introduced by Sampaio and modified by Guimaraes Da Silva.  It was pMLS has been reported as a feasible technique for laparoscopic and instrumentation training . Another


N = 137) showed that an adapted brief version of prolonged exposure (PE) to the fearful memory of the event and situations, provided in the immediate aftermath after trauma (<72 h after a traumatic event), was effective in reducing early PTSD symptoms in rape victims. The aims of the present study were to adapt the brief PE protocol to a Swedish context and investigate its feasibility and delivery in 10 executive patients recruited at the Emergency Clinic for Rape Victims in Stockholm. Ten participants were provided with three sessions of early PE with overall successful results in terms of session attendance, home-work compliance, and also symptom reduction of PTSD and depressive symptoms. However, only a fraction of the screened patients at the Emergency Clinic (5.2%) were eligible to be included in the study, where the majority (40%) were excluded due to the time criteria of 72 h. In this article, we will present detailed results of the intervention and elaborate on how to increase feasibility of preventive interventions for rape victims. In the current form, providing PE with the strict time criteria was not feasible in the clinical setting that constitutes the Emergency Department for rape.Rape is the most common trauma leading to post-traumatic stress disorder (PTSD) among women, with a conditioned prevalence of up to 50%. PTSD is considered to be a lethal condition associated with increased risk of suicide, drug- and alcohol dependence, neurological- and vascular problems, as well as sick leave. Given the scope of this problem, novel and swiftly delivered interventions for this large vulnerable population are clearly warranted. One previous trial conducted in the United States ( Sexual assault, including sexual abuse, rape, intimate partner, and sexual violence, is a global public health concern affecting an estimated 12% of women around the world . In a reAmong women who have been exposed to rape, as much as 19–50% develop PTSD . Rape maN = 124) indicated that this brief video intervention could significantly reduce both marijuana misuse at 6 weeks follow-up (N = 140) by the same research group found that women with a pre-assault history of high marijuana use who were randomized to the video intervention had lower marijuana misuse scores from baseline to the 6-months follow-up (N = 164) where women randomized to the video condition had fewer anxiety symptoms at the 2-month follow-up but no main effects were found on PTSD symptoms (N = 154) compared to treatment as usual and an active control condition (pleasant imagery and relaxation instruction). Results showed that the video intervention had some efficacy in reducing substance use in rape victims with a history of sexual assault . Results showed that PE was especially effective in reducing symptoms of post-traumatic stress among the subsample of rape victims  with a medium effect size (d = 0.7) at 12 weeks. A more recent study did not replicate these promising findings when comparing the three session protocol against one session and assessment only . The clinic is one of the largest sexual assault centers in Europe with an intake of about 800 patients each year. Medical care, forensic examination, and psychological help are offered to patients in Stockholm county over 13 years who seek help within 1 month after the rape. Neither medical nor psychological help render any fee for the patient but is solely financed by the Stockholm county. The study was approved by the Regional Ethical Review Board in Stockholm, Sweden (ID:2016/2194–31).Eligible participants were patients of at least 18 years of age attending at the Emergency Clinic for Rape Victims at Stockholm South Hospital, Sweden, within 72 h  after experiencing a traumatic event meeting the DSM-5 criterion A for PTSD  and who had memories from the event. Exclusion criteria were (i) ongoing suicidal ideation or attempted suicide within the last 2 months, (ii) ongoing self-harm behavior, (iii) ongoing intoxication, (iv) other serious psychiatric comorbidity (ongoing psychotic symptoms or manic episode), (v) low cognitive capacity, (vi) not fluent in Swedish, and (vii) subjected to ongoing violence or threat.Recruitment was conducted in two steps. Each morning, a clinical psychologist or licensed psychotherapist did a pre-selection screening in the medical records of newly arrived patients. Eligible patients were asked to participate in the study at a routine appointment with either a clinical psychologist or a licensed psychotherapist. Patients who did not come to the routine appointment were instead reached by telephone. Patients signed informed consent at the assessment appointment and also completed the baseline battery consisting of Immediate Stress Reaction Checklist ISRC;  and BeckThe Posttraumatic Stress Disorder Checklist PCL-5; , a 20-itIn addition to the outcome measures above, we exploratory investigated if it is possible for rape victims to daily record the number of intrusive memories during treatment. This was done using a daily pen and paper diary where the participant was instructed to tick a box for the day and corresponding time period (morning/afternoon/evening/night) or indicate zero in the absence of intrusive memories. The diary was translated into Swedish by the first author and has been used in previous trials testing early provided psychological interventions .Prolonged exposure is based on The PE treatment protocol was translated to Swedish by the first author in a previous trial after it was generously made available by the study authors of the The participants were provided with the first PE session within 72 h after the rape. During the first PE session, participants were provided with a rationale of the PE treatment and the function of avoidance behaviors as maintaining symptoms of post-traumatic stress. Subsequently imaginal exposure, where the patient was instructed to revisit the memory of the rape for 20–30 min  and recounting the rape in present tense, was conducted together with the therapist. In order to address erroneous trauma-related cognitions, open-ended questions were used after the imaginal exposure. A voice recorder was used to record the imaginal exposure on which the patient was instructed to listen to the recording each day as homework. A technique to decrease arousal symptoms in the patient’s daily life, breathing retraining, was also taught to the patient. Subsequently, two additional 60-min sessions were provided to the participants. The aim of these two sessions was to review the homework assignments and conduct additional therapist-led imaginal exposure.A total of 191 patients underwent the pre-selection screening for eligibility of which 118 (61.7%) were immediately excluded due to the following reasons: >72 h had passed since the rape , not fluent in Swedish , <18 years of age , and male gender .Seventy-three patients remained for the next phase of screening, using the digital medical records from the forensic examination. Nineteen patients  were not able to schedule an appointment with the therapist within the stipulated time frame for inclusion . Another nine patients  could not be scheduled within 72 h due to lack of available appointments in either the clinical psychologist or psychotherapists schedule. Ten additional patients  were excluded because they had no memory of the rape, and two patients (1%) were too physically ill and needed somatic care. Fourteen patients  had other serious psychiatric comorbidity and were thus excluded, and seven additional patients  did not want to be reached by the clinic personnel after the forensic examination. A total of 12 patients  were asked to participate in the study of which two patients declined further participation in the study.Mean time from the traumatic event (rape) to the start of PE was 52.1 h (SD = 11.1). Baseline characteristics for the included participants are presented in Three of the included participants dropped out from the study after the second PE session. No reason for dropping out was given in two cases. The third participant stated being exposed to another traumatic event as a reason. Seven participants were provided with the full treatment protocol. Adherence to the treatment protocol was excellent, and all PE sessions were provided according to treatment protocol. Homework compliance was high in these seven participants, ranging from 85% completion between session 2 and 3 and 100% between session 1 and 2.Data attrition was high at the 2-month follow-up; only six of the ten (60%) enrolled participants completed the CAPS-5 interview with the independent clinician. Data attrition was also high on the daily intrusion diary from session 1 to 3 where only three of ten (30%) participants had daily registrations the full treatment period.The mean score on the PCL-5 was 49.9 points (SD = 9.5) at post-treatment (end of session 3) which had reduced to 38.8 points (SD = 14.5) at the 2-month follow-up. The corresponding number for the BDI was 21.6 points (SD = 7.1) at post-treatment and 18.3 points (SD = 9.9) at the 2-month follow-up.The mean score on the CAPS-5 was 28.3 points (SD = 13.7) at the 2-month follow-up where three participants also received a diagnosis of PTSD and were offered standard PE treatment for PTSD. The mean ISI score was 12 points (SD = 4.48), the mean MSPSS point was 68 points (SD = 11.11), and the mean WHODAS score was 19.5 points (SD = 3.70) at the 2-month follow-up. No serious adverse events that could be attributed to the treatment were reported during the treatment or at the 2-month follow-up.In this study, we hypothesized that a brief PE intervention provided in the early aftermath of rape would be a feasible and acceptable early psychological treatment for PTSD in regular Swedish care. Our hypotheses were partly supported: Of the 10 included individuals, seven participants completed the full PE protocol, and these individuals had high degree of treatment adherence. Additionally, results indicated clinically meaningful reductions in PTSD symptoms in these individuals. However, it is important to point out that the treated sample consisted of only a fraction (5.2%) of the total intake at the clinic during the study period.As much as 40% of the screened sample was excluded due to the time criteria of 72 h. One well-reported factor in the literature is that only a small proportion of rape victims seek help from mental health professionals in the early aftermath of trauma  and therTen percent of the screened sample was not fluent in Swedish and therefore excluded. Translator assisted trauma focused CBT for PTSD has proven to be effective , and oneAbout one-quarter of the screened sample was excluded due to the age criteria (at least 18 years old). Previous research indicate female adolescents as victims of sexual assault in a higher, disproportionately degree . AdolescThe Emergency Clinic for Rape Victims did only provide care to female rape victims during the main execution phase of this study which unfortunately excluded an additional 10% of the screened sample. About 4–10% of all reported rapes include a male victim , and stuTwelve (6.2%) patients were finally asked to participate in the study, and two of these individuals declined participation. Of the 10 included participants, three participants dropped out of treatment. Homework assignment completion rate was high among the seven participants that underwent the full treatment, between 85 and 100%. One important challenge for future research is how to overcome the degree of data loss found in this study (40% of the included participants did not show up at the 2-month follow-up assessment). As previously discussed, digital innovations  may provide solutions to some of these problems. Several studies have shown promising results of smart phone assessments for several mental health problems  as well The study comes with limitations. One major limitation is that the lack of a control group to control for spontaneous fluctuations. Although the treated participants on average had a clinical meaningful decline in PTSD symptoms, this symptom reduction may as well be explained by natural recovery. However, it is important to stress that the aim of this study was merely to assess feasibility and delivery of the PE protocol. Future studies should consequently use a parallel group design to investigate efficacy of this treatment with extended time criteria. Another limitation in this study was that the independent assessor was not blinded to the aim study hypothesis and time-point, and thus, the mean scores on the CAPS-5 should be interpreted with caution. We also used a “supervision on demand” approach which means that we did not make a systematic control for treatment fidelity of the intervention. Future research may want to investigate this issue in more detail and to investigate possible therapist drifts when providing early PE interventions. The low rate of eligible patients to recruit also poses a major limitation. Comparing these figures with previous studies on early interventions after trauma shows that a low rate of eligibility and recruitment is unfortunately common stressing the need for the field overall of finding ways around it .To summarize, early provided PE seems to be an acceptable and deliverable intervention for rape victims. However, of the 191 screened patients, we were only able to include 10 participants (5.2% of the screened sample) in the study. The main reason for this was an inability to recruit participants in the stipulated time window for this study of 72 h. We suggest an alternative approach which includes offering the first intervention session when the patients present irrespective of time since rape and also to develop remotely delivered interventions. Complementary interventions, delivered through other formats , that are easily accessible – irrespective of gender, language barriers, and geographical distances – are clearly needed in order to swiftly reach the millions of rape victims around the globe.The raw data supporting the conclusions of this article will be made available by the authors upon request given that the request comply with Swedish and EU laws regulating protection of identifiable data.The studies involving human participants were reviewed and approved by the Regional Ethical Review Board in Stockholm, Sweden (ID:2016/2194–31). The patients/participants provided their written informed consent to participate in this study.KL and LN conducted the data collection. MB and EA performed the data analysis and interpretation, and first draft of the article. All authors contributed to the study design and writing of the article, and read and approved the final manuscript.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 coronavirus disease 2019 (COVID-19) pandemic is likely to lead to a significant increase in mental health disorders among healthcare workers (HCW).We evaluated the rates of anxiety, depressive and post-traumatic stress disorder (PTSD) symptoms in a population of HCW in the UK.An electronic survey was conducted between the 5 June 2020 and 31 July 2020 of all hospital HCW in the West Midlands, UK using clinically validated questionnaires: the 4-item Patient Health Questionnaire(PHQ-4) and the Impact of Event Scale-Revised (IES-R). Univariate analyses and adjusted logistic regression analyses were performed to estimate the strengths in associations between 24 independent variables and anxiety, depressive or PTSD symptoms.P < 0.001), depression  and PTSD . The availability of adequate personal protective equipment (PPE), well-being support and lower exposure to moral dilemmas at work demonstrated significant negative associations with these symptoms (P ≤ 0.001).There were 2638 eligible participants who completed the survey . The rates of clinically significant symptoms of anxiety, depression and PTSD were 34.3%, 31.2% and 24.5%, respectively. In adjusted analysis a history of mental health conditions was associated with clinically significant symptoms of anxiety (odds ratio (OR) = 2.3, 95% CI 1.9–2.7, We report higher rates of clinically significant mental health symptoms among hospital HCW following the initial COVID-19 pandemic peak in the UK. Those with a history of mental health conditions were most at risk. Adequate PPE availability, access to well-being support and reduced exposure to moral dilemmas may protect hospital HCW from mental health symptoms. As of August 2020, the UK had recorded the fifth highest number of deaths from COVID-19 worldwide.19 This region also observed a high incidence of COVID-19 cases and a high mortality rate.20 The study was approved by the UK Health Research Authority . Research Ethics Committee approval was not required for this study and this was confirmed by the HRA. Site-specific approval was obtained from each of the research and development departments of all participating acute general (n = 7) and mental health (n = 3) NHS hospital Trusts. Informed consent was obtained from all participants and recorded electronically at the start of the study.A cross-sectional survey of HCW employed in ten NHS acute general and mental health hospitals set in the West Midlands, UK was conducted between the 5 June 2020 and the 31 July 2020. The county has an ethnically and socioeconomically diverse population.Eligible participants included all staff who worked or volunteered on-site at one of the participating hospitals for over 50% of their working week during the initial peak of the COVID-19 pandemic in the UK. For this study, the latter was defined as the 23 March 2020 to the 23 May 2020. Staff who were on any type of leave for over 50% of the time during this period and those working from home or based in the community were not eligible to take part in this study. Eligible participants were invited to complete a confidential, voluntary electronic survey using the SurveyMonkey  online survey administration and management platform. The survey was approximately 15 minutes long. It was distributed to participants via email, newsletters, posters, flyers and social media platforms to maximise reach and encourage participation.The survey collected self-reported data on 24 independent variables . These i22 The Impact of Event Scale-Revised (IES-R) score was used to assess symptoms of PTSD.23The 4-item Patient Health Questionnaire (PHQ-4) was used to assess symptoms of anxiety and depression.21 The corresponding figures for the depression subscale were 83% and 90%, respectively, for major depressive disorder.22 The sensitivity and specificity of the IES-R has been reported as 91% and 82%, respectively, for a diagnosis of PTSD, where the cut-off score was ≥33.23 In this study, we used a score of ≥3 for each subscale of the PHQ-4 as the threshold score to detect the presence of clinically significant symptoms of anxiety and depression, and a score of  ≥33 on the IES-R as the threshold score to detect the presence of clinically significant symptoms of PTSD.The psychometric properties of the PHQ-4 were acceptable as a screening tool. Studies have reported a sensitivity of 86% and specificity of 83% for generalised anxiety disorder on the anxiety subscale for scores ≥3.2-tests to assess the significance of association. Adjusted analysis using binary logistic regression modelling was performed to obtain odds ratios (ORs) and 95% CIs.Data were collated using Microsoft Excel  and summarised as median (interquartile range) for non-normal data, and as proportions (percentage) for categorical data. Statistical analysis was performed using SPSS V.25 . The rates of mental health symptoms were calculated. For the categorical survey responses that were returned as ‘prefer not to say’ or ‘unsure,’ where combined analysis with the other responses was not possible, these were treated as missing data during statistical analysis. Univariate (unadjusted) analysis of measurements was performed for 24 predetermined, independent exposure variables using χP-value of less than 0.05 was assigned as the level of statistical significance.The regression analysis was conducted separately for each dependent variable , depressive  and PTSD (IES-R ≥ 33) symptoms), against all 24 measured independent variables to identify factors demonstrating a significant association for each symptom group. Multicollinearity of the 24 independent variables was assessed by calculating their variance inflation factors (VIFs). A n = 2096, 79.5%) were women and 19.9% (n = 524) were men  respondents did not disclose their gender. Nearly a fifth  belonged to Black, Asian and minority ethnic (BAME) groups. The majority  were in a relationship and nearly half  had dependents. In total, 84.0% (n = 2216) of participants were UK residents since childhood, the remainder emigrated as adults.A total of 2706 participants completed the survey of whom only 2638 met the eligibility criteria. Their median age was 42 years (interquartile range (IQR) = 32–51). The majority  reported a history of mental health conditions and 78.2% (n = 769) of these participants were prescribed medication or psychological therapies. Approximately a quarter  reported a history of physical illness. Smokers accounted for 11.4% (n = 301) of the sample and 65.4% (n = 1725) consumed alcohol.Around two-fifths , followed by doctors . Many staff  performing various other roles within the hospitals also took part in the survey (Supplementary Table 1 available at https://doi.org/10.1192/bjo.2020.150).Most respondents were nurses  had worked in healthcare for 10 years or less. The majority worked in acute general hospitals  and the remainder at mental health hospitals . A minority  were uncertain of their hospital type. Staff were based on in-patient wards , on intensive therapy units , in emergency departments  and other locations within their respective hospitals . The majority of participants  reported that they undertook patient-facing duties .A third of participants  reported that adequate PPE was available at their workplace , were unsure  or preferred not to comment . The majority were aware of well-being measures implemented by their employer  but only 15.4% (n = 405) accessed any form of psychological support during the study period. A third  of participants were redeployed as a result of the pandemic and 38.5% (n = 1015) reported increased working hours. In addition, 51.2% (n = 1351) reported morally uncomfortable changes in the way they worked.Just over a half , this was either confirmed on polymerase chain reaction testing of nasopharyngeal swabs (n = 277), diagnosed by a clinician (n = 47) or self-diagnosed based on symptoms and Public Health England guidance (n = 396). Approximately a fifth of staff  also reported that a cohabitant had developed COVID-19 during the period in question. Only 17.1% (n = 452) reported an admission to hospital with COVID-19 for self, or a close family member or friend.Of those who reported a diagnosis of COVID-19 (n = 906) scored ≥3; on the depression subscale of the PHQ-4, 31.2% (n = 824) scored ≥3 and 24.5% (n = 647) scored ≥33 on the IES-R .On the anxiety subscale of the PHQ-4, 34.3%  for each of the dependent variables: anxiety, depression and PTSD symptoms using the clinically significant thresholds previously stated. All 24 independent variables considered in the univariate analysis were included in the regression analysis. An assessment of multicollinearity of the 24 independent variables revealed all the VIFs to be less than 2 (maximum 1.6), indicating the validity of including all these independent variables in the logistic regression modelling.P ≤ 0.001)  .Table 3P < 0.001). Doctors and nurses were 20% less likely to report anxiety compared with other hospital HCW . Furthermore, those who reported adequate PPE availability, well-being support availability and where no morally uncomfortable changes took place, were around 50% less likely to have anxiety symptoms . A history of mental health conditions had the strongest association, with two and half times greater odds of hospital HCW reporting depressive symptoms . There was an almost twofold increase in odds of depressive symptoms when the participant was based in an acute general hospital compared with a mental health setting . On the other hand, alcohol consumers were 20% less likely to experience these symptoms . Furthermore, staff with adequate PPE availability, adequate well-being support and those who did not report morally uncomfortable changes in the way they worked were up to 50% less likely to report depressive symptoms . Several exposures were associated with increased likelihood of clinically significant PTSD symptoms: female gender, history of physical illness, smoking, being based on in-patient wards, in emergency departments or on ITUs, redeployment and admission to hospital for self, a close family or friend with COVID-19 . There were no significant associations between the remaining exposures and PTSD symptoms (Supplementary Table 2).There were 30–50% less odds of clinically significant PTSD symptoms when there were adequate PPE and well-being support available  reported a baseline rate of clinically significant PTSD symptoms  among 15–16% of HCW from the UK.7 Differences could be attributed to a multitude of factors including cultural, political and socioeconomic variations across the two study populations.The higher rates of mental health symptoms identified in the current study might be associated with working in a hospital setting during the COVID-19 pandemic. Nevertheless, the rates of symptoms of anxiety and depression among HCW in this study is lower compared with data from China that reported rates of anxiety of 40.6%; of depression of 50.4%, of sleep disturbance of 34% and of distress of 71.5%.In our adjusted analysis a previous history of mental health conditions consistently demonstrated odds of greater than two in participants reporting clinically significant symptoms of anxiety, depression and PTSD. In contrast, the availability of adequate PPE, access to well-being support and not experiencing morally uncomfortable changes in the way they worked were all significantly negatively associated with participants reporting these symptoms.Younger participants aged 40 or under demonstrated greater odds of reporting clinically significant anxiety symptoms. Smoking was associated with depression and PTSD symptoms but not anxiety. Female gender and a hospital admission for self, or a close family member or a friend were associated with anxiety and PTSD symptoms but not depression. Several other exposures were associated with PTSD symptoms but not anxiety or depression .Our findings may prompt healthcare employers to focus their attention on the provision of specific interventions that may protect HCW against an adverse impact on mental health during crises such as the COVID-19 pandemic. These may include the provision of greater access to well-being support for staff, ensuring the availability of adequate PPE and protection from exposure to moral dilemmas in the workplace.Furthermore, careful workforce planning to mitigate the adverse effects of redeployment and minimising the risk of viral infection may reduce the risk of staff experiencing PTSD symptoms. Attention should also be given to staff at greater risk such as those with a history of mental health conditions, female staff and smokers. Special consideration and additional support in the workplace could also be considered for younger employees, redeployed staff members and those working in potentially high-risk areas There were some unexpected findings in this analysis. One such finding was the protective effect working as a doctor or a nurse had on participants reporting clinically significant symptoms of anxiety and PTSD. This could be attributed to factors such as training, experience and coping mechanisms or resilience from previous working practices in stressful healthcare environments. Furthermore, there was no statistically significant increase in odds of self-reported mental health symptoms among staff undertaking patient-facing duties compared with staff in other roles. Therefore, it is important to ensure support is available to staff in all job roles who may potentially be at risk and not just front-line clinical staff.We also observed reduced odds of clinically significant symptoms of depression and PTSD reported by participants who consumed any amount of alcohol. The clinical significance and relevance of the latter association are unknown.There were some limitations in our study. The time elapsed between traumatic exposure and the onset of symptoms is key to making a diagnosis of PTSD. However, the aim of the study was not to make diagnoses of mental health disorders but to screen the target population for the presence of clinically concerning symptoms. The survey was conducted relatively close to the duration of the UK's COVID-19 pandemic peak. The elevated scores on the IES-R may be representative of an acute stress reaction that usually resolves within a few months. Further follow-up of these participants is required to ascertain the persistence of symptoms – a planned analysis by our study group. Our data is from a cross-sectional survey. Therefore, causal inferences cannot be made. Furthermore, the data were collected through a self-report questionnaire, which is at risk of responder bias.There were also several strengths. This study is one of the first in the UK to report on the mental health impact on hospital staff of working during the COVID-19 pandemic. These findings may be generalisable to the wider UK population of hospital employees given the relatively large sample size and representative demographic sample (Supplementary Table 3).In conclusion, during the COVID-19 pandemic, there were higher rates of common mental health symptoms in hospital HCW in the UK, especially among those with a history of mental health conditions. Occupational interventions such as adequate PPE and well-being support availability, and reduced exposure to moral dilemmas appear to protect hospital HCW against these symptoms.