Orientia tsutsugamushi, obtained from patient blood, vector chiggers, and rodent reservoirs, particularly for the dominant 56-kD type-specific antigen gene (tsa56), and whole-genome sequences, which are increasing our knowledge of the diversity of this unique agent. We explore and discuss the potential of sequencing and other effective tools to geographically trace rickettsial disease agents, and develop control strategies to better mitigate the rickettsioses.The rickettsioses of the “Far East” or Asia–Australia–Pacific region include but are not limited to endemic typhus, scrub typhus, and more recently, tick typhus or spotted fever. These diseases embody the diversity of rickettsial disease worldwide and allow us to interconnect the various contributions to this special issue of Tropical Medicine and Infectious Disease. The impact of rickettsial diseases—particularly of scrub typhus—was substantial during the wars and “police actions” of the last 80 years. However, the post-World War II arrival of effective antibiotics reduced their impact, when recognized and adequately treated (chloramphenicol and tetracyclines). Presently, however, scrub typhus appears to be emerging and spreading into regions not previously reported. Better diagnostics, or higher population mobility, change in antimicrobial policies, even global warming, have been proposed as possible culprits of this phenomenon. Further, sporadic reports of possible antibiotic resistance have received the attention of clinicians and epidemiologists, raising interest in developing and testing novel diagnostics to facilitate medical diagnosis. We present a brief history of rickettsial diseases, their relative importance within the region, focusing on the so-called “tsutsugamushi triangle”, the past and present impact of these diseases within the region, and indicate how historically, these often-confused diseases were ingeniously distinguished from each another. Moreover, we will discuss the importance of DNA-sequencing efforts for  Tropical Medicine and Infectious Disease (TMID), remain of international public health concern [Of all infectious diseases the world has experienced, few have truly altered human history. In centuries past, diseases such as smallpox, plague, tuberculosis, and epidemic typhus  have been responsible for millions of deaths through uncontrolled outbreaks. The military impact has been dramatic. Hans Zinsser 1878–1940), bacteriologist and immunologist, famous for his significant work on typhus, stated that the explosive outbreaks of epidemic typhus influenced the outcome of more wars between the 16th and the 19th century than any soldier or general [878–1940, concern ,3.The Past and Present Threat of Rickettsial Diseases”, leads us to focus on one region of the world that is hyperendemic, both historically and presently, for a host of rickettsial diseases. This is a vast area we are calling the “Asia–Australia–Pacific” or AAP region. For a map of the region, see the paper in this special issue by Luce-Federow et al. [The discovery of antimicrobial agents in the last century altered human perceptions of epidemics of infectious diseases and the associated effects of large outbreaks. Our wartime experiences of the past 100 years led to improved recognition and treatments for many infectious diseases not previously defined. Ironically, the interest in these diseases rapidly declined when effective antimicrobials or vaccines became available and the once devastating diseases became controllable, even though wars themselves subside. This poses a problem for endemic areas, where the diseases may persist. Despite available treatment, the lack of clinical awareness, often coupled with diagnostic difficulties, can lead to under-recognition with considerable morbidity and preventable mortality. The history of “typhus” in the Far East and the Asia–Australia–Pacific region follows this pattern. The broad topic for this TMID special issue, “w et al. . Indeed,Rickettsia prowazekii-infected lice (Pediculus humanus corporis) has played a relatively minor role in modern times as a cause of fevers in the AAP. During the Korean conflict (1950–1953), epidemic typhus had a significant impact on the civilian population. This was due, in part, to growing resistance of the vector body louse to dichlorodiphenyltrichloroethane (DDT) [Orientia tsutsugamushi. Consistent with this finding, scrub typhus is part of the focus in 10 of the 19 papers within this special issue in which rickettsial diseases in the AAP are examined [Today the reported incidences of rickettsioses in the world and for our purposes within the AAP are increasing. This is especially true of scrub typhus , and to a lesser extent murine typhus  and spotted fever. Scrub and murine typhus represent the two most common forms of rickettsial diseases in these regions. Other rickettsioses such as tick-borne rickettsioses and spotted fever, though relatively infrequent in the AAP, are, nevertheless, endemic in the region . In trop 1950–195, epidemine (DDT) . The lowexamined ,13,14,15examined  reportedRickettsia honei, R. australis, R. japonica, R. felis) do occur in the region, and some have been gaining traction. Most of these rickettsial agents are transmitted by ticks or fleas. For example, Australian cases of spotted fever group rickettsiosis (SFGR), or Queensland tick typhus (QTT), were reported in the 1940s with agent isolation from ticks in the early 1970s and were identified as Rickettsia australis [The other rickettsioses, though relatively infrequent in the AAP, are, nevertheless, endemic in the region, and recent studies, including several in this special issue, suggest the influence of the other rickettsioses in the region ,18,19. Tustralis . In thisustralis  report tustralis .Rickettsia honei, was initially recovered in 1962 from an ixodid tick near Chiang Mai [Rickettsia japonica, briefly described above, was isolated from the blood of a patient in Japan and found to cause febrile illness [O. tsutsugamushi and R. japonica occurs in blood of infected patients—they found no cross-reaction. Tshokey, et al. [In northern Thailand, the Thai tick typhus agent, TT-118, now called iang Mai , but theiang Mai . A similiang Mai , and a miang Mai . Another illness . In this illness  test the, et al.  report t, et al.  deals wiThe occurrence of rare cases of epidemic typhus and sporadic occurrence of SFGR cases throughout the AAP, however, can be viewed in stark contrast to scrub typhus. Thus, we have chosen to place our primary focus on one disease in particular, scrub typhus, as it occurs in the AAP region. To understand best the status of scrub typhus in the AAP, it is useful to review the progression of diagnosis of rickettsial diseases.Because research on disease in the AAP region has historically played a major role in redefining rickettsial diseases, we present here a brief account of the rickettsial diseases that have been lumped originally under the misleading term “tropical typhus” and consider their relative importance within the region, focusing on the so-called “tsutsugamushi triangle.”  with epidemic character and often included bubonic pest, typhus, dysentery, yellow fever, cholera, meningococcal diseases, scurvy, syphilis, and importantly, variola . The works of Hippocrates, 460 BC , allow ius cases . Few manus cases . A varieus cases . Medicalth century and underscore its later association with times of crisis, wars and famine [th century on, and over a further three and a half centuries “typhus” was gradually redefined as a collection of distinctive diseases that affected specific populations [Typhus exanthématique” by Boisier de Sauvages in Montpellier (1760) was an important attempt to distinguish the disease epidemic typhus [The earliest concise accounts consistent with classical typhus arise from the end of the 15ulations ,34. The c typhus .As the confusion gradually lifted, the term typhus was increasingly associated with epidemic typhus fever, which over time received the appellation of “classic typhus”. However, the secrets of typhus were only unraveled a few years before World War I (WWI). The years from 1910 to 1915 proved to be important for rickettsia-associated typhus research, with a number of substantial discoveries made around the world to determine the causative agents, their vectors, and the identification of hosts and reservoirs. This resulted in the recognition of rickettsial diseases as distinct diseases and the creation of “Rickettsiology” as a discipline. The impact of this developing science on our appreciation of the impact of epidemic typhus in particular became pronounced. During the war and post war-time period between 1917 and 1923, 30 million cases and 3 million deaths were recorded in European Russia alone, where combatants were returning home [Investigations into typhus and its related forms were intense in Europe and America around WWI with the driving philosophy that typhus was contagious, epidemic and associated with overpopulation and poverty. Due to difficulties in culturing rickettsiae, the etiologic relationships between typhus and other similar diseases were not firmly established until the 1930s, but typhus fever was regarded as a unitary disease. Rocky Mountain spotted fever (RMSF) and Japanese spotted fever (JSF) were considered as belonging to different categories of disease, although they were known to be transmitted to humans by ticks, which may act as the reservoir of the agent, or which may acquire the agent from animals such as squirrels, chipmunks, rats, or related forms [Bacillus proteus, a strain that caused agglutination when mixed with sera from typhus patients. Subsequent tests showed cross-reaction agglutination with whole antigen from this bacillus, subsequently reclassified in the genus Proteus. The Proteus species used was termed OX19 .A new era of rickettsiology had been triggered by the discovery of an innovative diagnostic test for typhus by Weil and Felix in 1916 . In 1915R. prowazekii) and a form of typhus from Australia, described by Hone in 1922 [In most geographic regions, the Weil–Felix test successfully identified epidemic typhus from other fevers. Increasingly, however, the original Weil–Felix test suggested complexity for typhus diagnosis in the tropics. Fletcher ,38, in M in 1922 .B. proteus X19, was actually a strain of P. mirabilis rather than P. vulgaris, and termed OXK . The accidental use of the OXK strain subsequently became the cornerstone of “scrub typhus” diagnostics. From the background of “tropical typhus”, Fletcher in 1923 was the first to show that using the Weil–Felix test with antigens from X19 and OXK could subdivide “tropical typhus” [Proteus OX19, and negative for OXK, whereas Fletcher found two contrasting groups of cases of “tropical typhus”, one group which strongly agglutinated Proteus OX19 but not OXK and a second group showing the reverse. The mistake that allowed OX19 to be used for diagnosis resulted in Fletcher having the ability to distinguish two diagnostic groups associated with different epidemiologies. One group was associated with patients from rural jungle areas (hence “bush” or “scrub” typhus). This was similar to the Japanese tsutsugamushi disease, which had been considered until then to possibly represent a separate entity. The second group distinguished by Fletcher represented the discovery of the closely related “shop typhus” from urban areas . The deThe work by Fletcher and Lewthwaite and many others at the Institute for Medical Research (IMR) in Kuala Lumpur triggered numerous reports from around the world describing diseases, which, while resembling epidemic typhus, were milder diseases that occurred sporadically. In the British colonies within the AAP, expatriate military personnel, especially in India (Sir John Megaw) and the Malay states (Dr. William Fletcher), encountered a vast array of “tropical fevers” ,42,43,44“… We call this variant ‘Tropical Typhus’ because it appears to be more common in the tropics than the epidemic form. It is necessary to distinguish it by some name—to call it simply ‘typhus’ is to mislead and alarm the public who, though they may be quite ignorant of everything else about typhus know that it’s highly infectious and may spread like a wildfire.” [Due to the impressive diversity of vectors, animal reservoirs, and different geographical settings, a new era of confusion arose. In 1924, in the midst of this confusion, the term “tropical typhus” was coined in Malaya by William Fletcher. He states: ldfire.” . Fletcheldfire.” ,48. The Rickettsia typhi, and whose vectors are fleas) was ultimately recognized as a separate disease from the epidemic typhus by three groups working independently: Fletcher in Malaya [Murine typhus , the agent of scrub typhus [Diagnostic improvements included refinement of laboratory techniques, discussed below, leading to the important discovery of antigenic differences between strains of b typhus . PreventR. prowazekii and R. typhi, and scrub typhus and its agent O. tsutsugamushi representing the overwhelming proportion of cases in the AAP geographic region.Thus, from an ill-defined mixture of fevers, the typhus fevers became clearly defined, including epidemic and murine typhus, with agents The capability to diagnose rickettsial diseases has evolved dramatically since the early serendipitous discovery of the Weil–Felix agglutinations in 1916 for typhus, and in 1924–1925 for scrub typhus . In the Proteus, a member of the gamma proteobacteria. It is especially useful because it separates STGO from other rickettsiae. Although generally lacking sensitivity and specificity for diagnosis, this test has served its purpose well. Even with its weaknesses, the WF test is inexpensive, easy to perform, and readily accessible; which explains its continued use in certain regions of the AAP.The Weil–Felix (WF) test is the oldest assay used for the diagnosis of rickettsial diseases ,63,64. TThe complement fixation test (CF), also a test from the early rickettsial diagnostics era (1940s), could be used for both serodiagnosis and serotyping of the infecting strain of bacteria ,66. GuinO. tsutsugamushi- and R. typhi-specific antibodies [Rickettsia japonica and O. tsutsugamushi, as well as the cross-reactivity observed between different serotypes of O. tsutsugamushi [The indirect immunofluorescence assay (IFA) has been used for over 50 years to quantify anti-rickettsial antibodies including tibodies ,69. Eachtibodies , Selgadotibodies , and Yuhtibodies  in this tibodies , IFA protibodies ,70. In ougamushi . R. typhi and O. tsutsugamushi [Commercial immunochromatographic rapid diagnostic tests (RDT) and immunodot assays produce rapid results, follow a simple protocol with no need for sophisticated electrical equipment, and are relatively inexpensive. They are highly attractive for point-of-care use in rural areas where the use of IFA may not be available. Moreover, the rapidity of these tests facilitates more rapid treatment, especially in rural areas with limited resources. The test(s) are based upon an enzyme-linked dot blot immunoassay, in which specific native antigens are immobilized on a membrane. Incubation of the membrane with patient sera allows for detection of antibodies to the antigen of interest on the membrane/dipstick. In the 1990s, many of these rapid dipstick/immunodot tests were developed through collaborations with the Department of Defense research laboratories and included assays for the detection of ugamushi ,72.The basic enzyme-linked immunosorbent assay (ELISA) is a technique to detect and quantify biologic substances such as peptides, proteins, lipopolysaccharides, antibodies, and hormones. In the simplest form, the ELISA is used to detect and quantify specific patient or animal antibodies against rickettsiae. Rickettsial antigens are attached to a solid surface, a 96-well plate or membrane, for example. As with most of the earlier serological assays, IFA, IIP, CF, dot blot assays, the ELISA was initially accomplished using native antigens derived from growing rickettsiae in embryonated hen eggs, or cell culture ,74,75. DO. tsutsugamushi. Publication of the corresponding gene sequences introduced the field of rickettsiology to the molecular biology era and consequently to molecular diagnostics [The first successful molecular cloning and gene expression of the rickettsial proteins involved the 110-kD and 56-kD proteins products of gnostics ,77,78. Tgnostics ,80.O. tsutsugamushi [With the introduction of molecular cloning, recombinant proteins based on the published sequences of rickettsial antigen genes were developed. Ching et al. developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to ugamushi . The RFAO. tsutsugamushi strains Karp, Kato, Gilliam, and TA716 for scrub typhus IgM and IgG detection. When validated against the IFA, a sensitivity of 84% and specificity of 98% was noted in Northern Thailand, and in a subsequent evaluation, a sensitivity of 91.5% with specificity of 92.4% was reported for Bangladesh. Still, the InBios ELISA endpoints have not been firmly established [A most recent and promising point-of-care (POC) commercial serodiagnostic tool, the InBios Scrub Typhus Detect™ IgM ELISA, was evaluated in scrub typhus-endemic Northern Thailand  and Bangablished . The diaO. tsutsugamushi targeted infected murine blood samples using primers derived from the just published O. tsutsugamushi 56-kD gene tsa56 DNA sequences of Stover, et al. [R. typhi was detected in fleas by PCR in the same year [As noted above, PCR diagnostics were “off and running” in 1990 as gene sequences were being published. The initial PCR-based detection of , et al. ,80. The , et al. , while Rame year . Due to ame year .O. tsutsugamushi, multilocus sequence typing/analysis (MLST/MLSA), multispacer typing (MST), and whole-genome sequencing [O. tsutsugamushi presented by Kelly et al. [Associated with the products of PCR were various approaches to the characterization of strains for comparison with each other, comparison of strains within the different geographic regions, for example. The sensitive genotyping methods that became available included restriction fragment length polymorphism (RFLP), single-gene sequencing of highly variable genes, such as the 56-kD type-specific antigen (TSA56) of quencing . Gene sey et al.  in the sAnaplasma and Ehrlichia. Elementary protocol errors and inappropriate data analysis can occur, which can detract from the use of this powerful technique. Choice of source material is important. However, when using the specimen of choice, biopsy of rash and/or eschar, and strict adherence to guidelines, the sensitivity of qPCR assay approaches up to 100% [Anaplasma phagocytophilum.Quantitative real-time PCR, or qPCR ,87, is n to 100% . Quantit to 100% , in whicRickettsia felis. Similarly, in their broad review of rickettsioses in Taiwan, Minahan et al. [Orientia extend beyond the AAP geographic region.In addition to some previous comments above concerning specific diagnostic tools, many of the papers included in this special issue make use of such tools in their studies. A number of papers utilized one or more of the diagnostic tools, often focusing their use to study the occurrence of rickettsial agents in particular geographic areas. Legendre and Macaluso  reviewedn et al.  indicaten et al.  perform n et al.  discussen et al.  details The possible combinations of diagnostic techniques that are being utilized in the analysis of scrub typhus are illustrated well in the study detailed by Naoi et al. , emphasiThe choice of a single gold standard diagnostic tool remains unclear, due to the bi-phasic nature of early rickettsemia followed by a subsequent antibody response. The difficulty of diagnosis is illustrated in the paper by Salgado Lynn . ReportiThe elevated caseloads and mortality rates caused by the rickettsial diseases during WWII, particularly scrub typhus in the AAP, were mission-threatening. The end of the war reduced the general concern, but investigators continued research and control efforts. Before the age of effective antibiotic therapy, scrub typhus patients suffered mortality rates of around 6–10%, sometimes exceeding 50% ,95—oftenFurther research produced the bacteriostatic protein synthesis inhibitor, tetracycline (introduced in 1953), which proved even more effective than chloramphenicol in treating scrub typhus. Tetracycline showed fewer side effects and became the primary treatment . Later, The case for treatment failures in scrub typhus has gained much attention in recent years ,102, andO. tsutsugamushi isolates that were transported from Malaya to Britain [Exhaustive efforts to develop an effective, long-lasting, and broadly protective scrub typhus vaccine for humans have not proven successful. Those efforts extend from the WWII era when two vaccines were developed; one by the British based upon the Karp strain, and a second by US researchers based upon the Volner strain. Both were evaluated in the field and were found to not be significantly better than controls in preventing disease or death ,106. Imm Britain ,107. Eff Britain ,109. An O. tsutsugamushi [gltA, encoding rickettsial citrate synthase [rOmpA, encoding spotted fever group (SFG) rickettsiae-specific 190-kD outer membrane protein A [Rickettsia genus-specific 17-kD outer membrane antigen [Molecular approaches to the study of rickettsia now routinely use DNA sequencing as the gold standard for diagnosis and identification of rickettsial agents. These procedures often utilize DNA sequences of specific highly variable genes, such as the 56-kD type-specific antigen (TSA56) of ugamushi ,80, or rsynthase , rOmpA, rotein A , and the antigen , each of antigen . Analyse antigen , or to d antigen , or to b antigen .O. tsutsugamushi, genotypic groups, such as those identified using the tsa56 gene sequence, may reflect the phylogenetic histories of the isolates being compared [O. tsutsugamushi have suggested that the phylogenetic histories of different genes may not reflect a unified evolutionary history of the genes within the genome [Within compared . Howevere genome ,121,122.O. tsutsugamushi may facilitate significant horizontal gene transfer between isolates that would be reflected by incompatible phylogenetic histories for different genes. The characteristic of the genome that would potentially be most important in promoting substantial horizontal transfer is the large proportion of the O. tsutsugamushi genome that is made up of repeated sequences [O. tsutsugamushi, the genome of the Boryong strain, suggested that the sequence represented "the most highly repeated bacterial genome sequenced to date” [O. tsutsugamushi strains that have been determined contain a large number of copies of genes for components of conjugative type IV secretion systems, for signaling and host-cell interaction proteins, as well as more than 400 copies of transposases, 60 copies of phage integrases, and 70 copies of reverse transcriptases. These multi-copy sequences have been hypothesized to facilitate intragenomic recombination. There is also the possibility that additional acceleration of strain differentiation may be associated with population bottlenecks that are likely to be associated with the intracellular nature and transmission patterns of rickettsial bacteria [It has been suggested that some unique aspects of the genome of equences . The orito date” . The genbacteria ,127,128.O. tsutsugamushi evolves. Much remains to be determined concerning the population genetic dynamics of O. tsutsugamushi, and the comparison of those dynamics with patterns occurring in other rickettsiae that contain far fewer repeated components in their genomes. Studies of gene differentiation for multiple genes has been limited to a few studies of isolates from Southeast Asia [We are still at the early stages of understanding how ast Asia ,121,122 ast Asia  and Koreast Asia .O. tsutsugamushi in the AAP. The most comprehensive analyses of O. tsutsugamushi phylogenetics were presented by Batty et al. [tsa56 gene, the 47-kD htraA gene, and a seven-gene MLST panel. Differences between single-gene phylogenies and multigene phylogenies within the genome of a single species are expected to occur; the significance of this is more difficult to evaluate [In contrast, isolates that have been studied as part of genomic comparisons have been collected from a large part of the range of y et al.  and Flesy et al. . Batty ey et al. . Some dievaluate ,131,132.O. tsutsugamushi [Horizontal gene transfer has been invoked to explain differences between gene trees within ugamushi ,118. Howugamushi . Much woO. tsutsugamushi. The genome sequences of isolates of O. tsutsugamushi reveal a completely different picture of genome change from that seen for genomes from R. prowazekii or R. typhi. In Rickettsia, gene order is maintained over large parts of the genus. Gene order is reasonably similar in various spotted fever group species. Comparisons between typhus group species and SFG forms show slightly more structural differences, primarily because a larger number of putative genes exist in the Typhus Group (TG) species, but most of the genome shows gene order preserved, although a few large inversions exist. For rickettsial forms within the AAP, a revealing difference between genome changes in Rickettsia and in Orientia can be seen when R. typhi genomes and O. tsutsugamushi genomes are examined. Genome sequences are available for five isolates of R. typhi . Gene order and gene number differs between isolates [R. typhi genomes can easily be compared over the entire length of the genome, with gene order conserved.This is significantly different from the situation found for elements ,123. Theisolates ,118. PaiO. tsutsugamushi allow a different, but limited approach to the comparison of isolates. Much work remains to understand how the evolution of single genes corresponds to the genome evolution of isolates in O. tsutsugamushi. However, investigations of highly variable genes, especially the tsa56 gene, do allow the examination of geographic variation within the species range of O. tsutsugamushi.Single-gene sequences in tsa56 gene is considerable, both within and between geographic areas of the AAP [tsa56 gene may give us an insight only into the evolution of cell surface variation, and perhaps do not indicate how other genes would reflect evolution of the population. This caveat is inserted because it remains unclear whether the phylogenetic relationships among sequences of the tsa56 gene would accurately reflect the phylogenetic relationship of other loci in the O. tsutsugamushi genome, or whether natural selection acting on the tsa56 gene has altered the relationship among isolates in a different way compared with other loci in the genome. If natural selection acts specifically on the gene such as the tsa56, the phylogeny of the gene may not completely mirror the evolution of the organism, or the phylogeny of other genes in the genome.Variation of the  the AAP . Diversitsa56 gene. The differentiation appears to be very significant. However, these numbers reflect the reporting of genetic sequences within the international DNA sequence databases, and do not necessarily reflect an epidemiologically appropriate sampling of isolates. Numbers in the table reflect a mixture of isolates from human cases and from mite vectors and rodent secondary hosts. Further, it is important to consider that some geographic regions in which scrub typhus is endemic are noted for the lack of information concerning genetic variability. These regions include Indonesia, Malaysia, and the Philippines and the large islands  of the southwestern Pacific region. There is also minimal information concerning Myanmar, Pakistan, and associated regions at the western edge of the range of scrub typhus, and from northern Australia.Examination of the tsa56 gene has been used to study variation in O. tsutsugamushi carried by both mite vectors and in the rodent populations that represent the normal host for the mites. Evidence exists that different species of trombiculid mites may act as hosts to different genetic lineages of bacteria, and that different rodent hosts may also be nonrandomly associated with genetic lineages of O. tsutsugamushi [The ugamushi .O. tsutsugamushi and the two species in the typhus group of Rickettsia. Vertical transmission may be different for O. tsutsugamushi compared with the typhus group rickettsiae [orientiae and the vector for spread of scrub typhus [tsa56 genotype of O. tsutsugamushi [O. tsutsugamushi within single rodent or mite hosts in nature [O. tsutsugamushi also exist [The nature of transmission by vectors for scrub typhus may also contribute to the differences that are observed between kettsiae ,137,138.kettsiae ,127,128.kettsiae . It is kb typhus . There iugamushi . Studiesn nature . Furtherso exist ,142,143.Leptotrombidium species affecting the differentiation of bacterial lineages that they carry and transmit.Other ecological aspects of scrub typhus could also contribute to differentiation between intraspecific lineages of bacteria. The various mite vectors can demonstrate seasonal differences in their abundance ,146,147.Ultimately, continued research efforts are required for a better understanding about scrub typhus; currently, the focus should be on addressing entomological aspects in transmission, dissecting host–pathogen interactions in a clinical disease severity context, and in the development of point-of-care diagnostics and an effective vaccine.Leptotrombdium mites has enabled for the first time paired-matched morpho- and genotyping, which should enable broader characterization of mites globally and their endosymbionts , the molecular epidemiology of mites transmitting scrub typhus, as well as an improved understanding of adult mite stages [Entomology: The recent discovery of UV-based autofluorescent microscopy in morphotyping e stages .Host–pathogen interactions: The availability of whole-genome sequencing provides a good platform to determine the virulence mechanisms of orientiae, as host-mediated pathogenic mechanisms and mechanisms of tissue injury remain poorly understood—ideally, these virulence factors should contribute towards developing a disease severity score/prediction based on clinical features and pathophysiological markers .Diagnostics: A lot of effort has gone into improving the notoriously difficult diagnostics, but a universally useful point-of-care tool remains elusive. Ideally, this should comprise an antigen/nucleic acid-based component with an antibody detection step. Coupling this with the use of noninvasive or less–invasive sample specimens, such as sampling eschars or saliva, would enable earlier diagnosis, broader epidemiological coverage globally, and contribute substantially to improved awareness and better management of these easily treatable diseases . Joint eVaccines: While extensive studies on features of the natural immune response were performed in the 1970–80s, this area has not received much attention lately—especially research with a focus on immune memory and clinically relevant correlates of protection is lacking. The ongoing genome-sequencing efforts, the availability of a characterized non-human primate model and first vaccine candidates conferring sterile immunity against high-dose homologous challenge with orientiae are promising prerequisites to progress this work .AAP rickettsioses are a fascinating array of clinically relevant diseases—these endemic and underappreciated diseases are associated with a large burden of disease globally, and to date, there are still no licensed vaccines, or vector control efforts in place. Despite increasing awareness in endemic regions, the public health burden and global distribution of scrub typhus remains poorly understood. Opportunities are vast for the next generation of clinicians and scientists, there is still a lot to do.


Orientia species, have been around for a very long time. Historical reference to the rickettsial disease scrub typhus was first described in China (313 AD) by Hong Ge in a clinical manual (Zhouhofang) and in Japan (1810 AD) when Hakuju Hashimoto described tsutsuga, a noxious harmful disease in the Niigata prefecture. Other clinicians and scientists in Indonesia, Philippines, Taiwan, Australia, Vietnam, Malaysia, and India reported on diseases most likely to have been scrub typhus in the early 1900s. All of these initial reports about scrub typhus were from an area later designated as the Tsutsugamushi Triangle—an area encompassing Pakistan to the northwest, Japan to the northeast and northern Australia to the south. It was not until the 21st century that endemic scrub typhus occurring outside of the Tsutsugamushi Triangle was considered acceptable. This report describes the early history of scrub typhus, its distribution in and outside the Tsutsugamushi Triangle, and current knowledge of the causative agents, Orientia species.Scrub typhus and its etiological agents,  Scrub typhus, a febrile disease with mild to life-threatening manifestations, is characterized by rapid onset of fever, headache, chills, arthralgias and myalgias and often the presentation of eschar prior to and a macularpapular rash following initiation of disease ,3,4,5,6.Orientia DNA is in abundance, unaffected by prior antibiotic treatment, and maintained in the lesion for the life of the lesion [The lack of scrub typhus-specific signs and symptoms makes the clinical diagnosis very difficult ,3,5. More lesion .Historically, scrub typhus has been around for a very long time. Human historical reference to scrub typhus  was firsIn Japan, in 1810, Hakuju Hashimoto described “tsutsuga”, a noxious harmful disease in the Niigata prefecture of the main island of Japan . HoweverTheileria tsutsugamushi [Rickettsia orientalis, was reported in 1930 in the Japanese Journal of Experimental Medicine [Rickettsia tsutsugamushi and Rickettsia akamushi, respectively [R. tsutsugamushi and Nagayo’s R. orientalis [R. tsutsugamushi [O. tsutsugamushi. Though much controversy was associated with this name [R. tsutsugamushi was moved out of the genus Rickettsia and into its own genus Orientia, with the new species name, Orientia tsutsugamushi [Up until the 1920s, the etiology of tsutsugamushi and, therefore, scrub typhus was unknown. In addition, multiple diseases which were later believed to be synonymous with tsutsugamushi were known as Japanese river fever, flood fever, island fever, Kedani (mite) disease, akamushi disease, shimamushi disease yochubio, and shashitsu [ugamushi . Howeverugamushi , as descugamushi . Hayashiugamushi . In the ugamushi , as descugamushi . In 1929ugamushi . This waugamushi . Unfortuugamushi . Ogata, ugamushi . MoreoveMedicine , as descMedicine . Ogata aectively ,18, as dugamushi . In the ugamushi  , which was significantly different to the high mortality rate (~20–40%) seen with tsutsugamushi in Japan at this time . Scrub tFurther, also in 1908, two cases of tsutsugamushi disease were described for two US military personnel, stationed at Camp Connell, Samar, the Philippines, based upon clinical records ,38. One In 1910, Mossman fever, later described as endemic glandular fever and, subsequently, determined to be scrub typhus, was reported in North Queensland, Australia ,42,43,44In 1915, a fever of unknown etiology among two individuals was reported in Saigon, Vietnam  to be a In 1915, a “mild” rickettsial disease called paratyphus was reported among 15 patients (no deaths) in the spring of 1913–1914 from Jemulpo, Incheon, Korea by Weir, a medical missionary . BecauseTrombicula akamshi. In addition, rickettsial diseases with mild presentations and low OX19 titers may not have been murine typhus but scrub typhus [During the period of 1910–1945, there was some evidence of endemic scrub typhus in Korea. A study of mites attached to wild rats collected in Suwon were similar to b typhus . During b typhus . Followib typhus  and consb typhus ,61,62,63b typhus ,64.In 1900, the Institute of Medical Research (IMR) in Kuala Lumpur, Malaysia, was established as the Pathological Institute with the aim to promote the health status of the local population. In 1924, the institute began research on “tropical typhus” in the Federated Malay States. In the annual IMR Bulletin in 1925, Fletcher and Lesslar described tropical typhus as containing two components—an urban or shop typhus and a rural or scrub typhus .Bacillus proteus X.19 (Proteus vulgaris) that was isolated from the urine of a patient with epidemic typhus [P. vulgaris agent was not the cause of the disease but was found to be agglutinated by antibodies developed during epidemic typhus. The cross-reactivity of the antibodies to the P. vulgaris antigens (OX19) has been successfully used since 1916 to serologically diagnose epidemic typhus and murine typhus. Subsequently, another strain of P. vulgaris (OX2) was identified that reacted with the sera of spotted fever patients [Proteus mirabilis Kingsbury strain which reacted with sera from scrub typhus patients but not with sera from typhus or spotted fever patients [Rickettsia typhi [O. tsutsugamushi isolations, diagnostics, treatment/prophylaxis, vaccine and immunology research, and vector and ecology research [Due to the fortuitous change in the composition of the Weil–Felix test, “tropical typhus” could be divided into two unique diseases. The Weil–Felix test initially utilized the strain of c typhus . The P. patients . These ppatients . It was patients ,69. Withpatients ,71. Moreia typhi . Throughresearch . Scrub tresearch ,76,77,78Trombicula deliensis on rodents and shrews in the Simla Hills, suggesting that similar to the reports from Malaya, that these mites may be the vectors of scrub typhus [O. tsutsugamushi Gilliam, was contracted by Dr. Gilliam in 1944 on the equally famous Stillwell Road, in Burma [In 1932, Christian reported OXK-positive typhus cases that he believed were due to tick bites . Due to b typhus . Boyd reb typhus . Interesb typhus . In 1944b typhus . One casb typhus . Subsequb typhus . The famin Burma . Nichollin Burma . Similarin Burma . Scrub tin Burma . This isin Burma ,91,92,93All of these reports of scrub typhus or diseases very similar to them throughout the Asia–Pacific region prior to WWII led to the assumption of a single rickettsial disease for a very large endemic region where many people were at risk of disease. Unfortunately, this assumption of a very large endemic area of scrub typhus was reinforced during WWII, where approximately 18,000 cases occurred among the allied forces and a similar number among the Japanese forces in the islands of Ceylon, Maldives, New Britain, Goodenough, and the Schouten Islands, and in the countries of China, Thailand, Japan, Australia, Lao, Cambodia, Vietnam, and Taiwan ,37,38,83Orientia which was identified within human cases, vector mites and mammalian hosts [O. tsutsugamushi, has a diversity of antigenic phenotypes and genetic genotypes found not only between countries but within countries [Consistent within the Tsutsugamushi Triangle has been the presence of a single species of an hosts ,83,94,98ountries ,100. HowR. orientalis (O. tsutsugamushi) antigens, the authors included scrub typhus in the panel of rickettsial diseases to assess. Among the ill Africans, several were positive for the various rickettsial antigens, including two individuals who reacted to the R. orientalis antigens. To confirm the skin tests, blood from the two Orientia-positive patients were tested for complement-fixing antibodies to O. tsutsugamushi and were found to be positive, with titers of 80 and 320. To assess the reactivity to the scrub typhus assays in other populations who lived closely with native Africans in Musha Hill, healthy individuals, including nine people born in Muscat, Oman, five born in Bombay, India, and two born in Africa, with parents who were born in Bombay, were tested using the same skin and blood tests for evidence of previous O. tsutsugamushi infection. The authors considered Muscat and Bombay as scrub typhus-endemic regions and thought that people from those areas may be antibody positive to O. tsutsugamushi and may, therefore, act as positive controls. Of the nine individuals, originally from Muscat, three had strong, weak, and negative skin reactivity and antibodies against O. tsutsugamushi were detected with titers of 1280  and 640  in eight. Of the five people born in Bombay, three displayed positive skin reactivity to O. tsutsugamushi antigens and, interestingly, the two people whose parents were from Bombay, but who were born in and never traveled outside of eastern Africa, were also positive. These results suggested the presence of scrub typhus in eastern Africa. The authors indicated that a similar study they conducted among natives in western Africa showed that they were negative for evidence of scrub typhus [In 1951, Giroud and Jadin published a report that indicated that scrub typhus occurred outside the Tsutsugamushi Triangle. This report described an outbreak of a febrile disease among native Africans from Ruanda-Urundi working on constructing a factory building in Musha Hill, Belgian Congo (now Rwanda and Burundi) . To inveb typhus .O. tsutsugamushi. Unfortunately, no molecular or culture evidence confirmed the case of scrub typhus. The third case was of an individual who had visited Tanzania [O. tsutsugamushi antigens from <16 to 1024 by an indirect immunofluorescence assay (IFA). Regrettably, none of these three cases had produced a culture of Orientia or had molecular evidence of the causative agents, thus, they were considered presumptive scrub typhus cases.In the 1990s, three case reports insinuated, but could not confirm, the presence of scrub typhus in Africa. The first described an individual from Japan visiting the Republic of Congo, who presented with fever six days after his return from Africa . The disTanzania . She hadThese results collectively implied that scrub typhus was endemic to Africa  and the Orientia species other than O. tsutsugamushi . Similarly, molecular assays for orientiae were not used to investigate the presence in mammalian hosts and/or arthropod vectors of orientiae outside of the Tsutsugamushi Triangle. This paradigm was changed with the two reports of scrub typhus in the United Arab Emirates and Chile. Thus, scientists included scrub typhus serological and molecular assays to conduct surveillance studies of rickettsial diseases in areas of Africa, South America, and Europe (O. tsutsugamushi that was confirmed by Western blot assays [O. tsutsugamushi ELISA antigens and 10 of these children seroconverted to O. tsutsugamushi antigens (3.6%). The seroreactivity was confirmed by Western blot analysis [O. tsutsugamushi ELISA antigens and one individual who reported a history of a febrile disease during the period of the study seroconverted to O. tsutsugamushi antigens by ELISA, IFA and Western blot tests [Following these two astounding reports, investigators looked to confirm and expand upon these results, utilizing current serological and molecular assays for evidence of d Europe . This ret assays . In an uanalysis . In Djibot tests .Orientia DNA among tissues of rodents from West Africa and Europe [Orientia spp. [O. tsutusgamushi. Trombiculid mites were collected from the trapped rodents to assess them for molecular evidence of Orientia. DNA preparations provided evidence of Orientia species from sequences of gene fragments of the rrs and htrA that were most closely aligned to but not identical with Ca. O. chuto  were seropositive against d by IFA . The secd by IFA ,114. It b typhus .O. tsutsugamushi [O. tsutsugamushi found throughout the Tsutsugamushi Triangle [Herpetacarus from Chiloé Island found the same orientiae as that associated with scrub typhus cases [Candidiatus Orientia chiloensis  and serological and molecular assays based upon


We propose an image-based class retrieval system for ancient Roman Republican coins that can be instrumental in various archaeological applications such as museums, Numismatics study, and even online auctions websites. For such applications, the aim is not only classification of a given coin, but also the retrieval of its information from standard reference book. Such classification and information retrieval is performed by our proposed system via a user friendly graphical user interface (GUI). The query coin image gets matched with exemplar images of each coin class stored in the database. The retrieved coin classes are then displayed in the GUI along with their descriptions from a reference book. However, it is highly impractical to match a query image with each of the class exemplar images as there are 10 exemplar images for each of the 60 coin classes. Similarly, displaying all the retrieved coin classes and their respective information in the GUI will cause user inconvenience. Consequently, to avoid such brute-force matching, we incrementally vary the number of matches per class to find the least matches attaining the maximum classification accuracy. In a similar manner, we also extend the search space for coin class to find the minimal number of retrieved classes that achieve maximum classification accuracy. On the current dataset, our system successfully attains a classification accuracy of 99% for five matches per class such that the top ten retrieved classes are considered. As a result, the computational complexity is reduced by matching the query image with only half of the exemplar images per class. In addition, displaying the top 10 retrieved classes is far more convenient than displaying all 60 classes. For a given coin class, this information includes the issuer, date of issuance, information about the material of coin, and the complete description of obverse and reverse motifs. An example of such is shown in We propose a holistic image-based class retrieval system for ancient Roman Republican coins that can be instrumental for a number of non-expert audience such as museum visitors, Numismatics students, hobbyists, auctions Websites, as well as the art market in general. For instance, most of the museums visitors are completely unaware about the historic context of the displayed coins. Such scholarly information about the ancient coins is only available in standard reference books , where tNonetheless, the classification of ancient coins based on their images is a challenging task due to variations caused by several factors. A main problem is the high intra-class and low inter-class variations due to large number of coin classes. In case of Roman Republican coins, there are 550 classes with more than 1000 subclasses [Due to the aforementioned challenges, the image-based methods proposed for the classification of modern day coins are not adaptable for ancient coins . The metNonetheless, all these methods use one or the other form of image classification where the task is to classify a given coin image into one of the coin classes. We extend this concept a step further by showing the class relevant information from the expert sources in a user friendly manner. This is due to the fact that the knowledge about an ancient coin is also of utmost importance when it comes to particular communities such as archaeologist, students and researchers. Consequently, the proposed system has a potential to be used as an assisting or educational tool at a number of places such as museums, educational institutes, or even online auction websites.The database consists of 60 coin classes where each class is represented by 10 exemplar images of their reverse side motifs. The dimensions of all the images are The exemplar coin images of any given class differ from one another mainly due to the non-rigid deformations caused by wear and tear on the coin.All the coins are imaged with a homogeneous background.The coin is depicted at the image center in almost all of the images.As the coins belong to the Roman Republican era, the scale variations in the coin images remains negligible.All the coins are imaged with their canonical orientations resulting in negligible variations caused by rotations.The graphical user interface (GUI) of our proposed holistic system for image-based classification of Roman Republican coins is shown in We use automatic coin segmentation  to extraThe local image patches are densely sampled and then represented with local feature descriptors. In the proposed framework, we use the Local Image Descriptor Robust to Illumination Changes (LIDRIC) , which iFor a query image, the segmentation and features extraction are performed as stated previously. The next step is to match the local features of the query image with those of the exemplar images in the database. A depicted in max” operation is then performed on these 10 values, where the maximum score is considered for that particular coin class. Consequently, for a query image, the number of similarity scores reduces to 60; one per coin class.The matching assigns similarity scores to each exemplar image of all the coin classes where a higher score shows a high similarity. In this way, for each coin class, similarity scores are determined from its exemplar images. However, a “Finally, all the 60 classes are ranked in a descending order based on their similarity values where the one with highest similarity is assigned to the query image. However, our system aims at the depiction of candidate classes for the query image along with their information from the standard Numismatics source leading to the depiction of top “N” classes in the GUI.For a query image, the relevant classes are found by matching it with exemplar images of each class. As it is an online image matching process, the cost of computations must be optimized by evaluating the number of matches per class. For instance, in the worst case, each class has 10 exemplar images and there are a total of 60 classes resulting in 600 image matchings. This will ultimately increase waiting time in our proposed framework due to which, we evaluate the number of matches per class. Furthermore, for the query image, all the 60 classes will be ranked from top to bottom with respect to their similarity scores and consequently will be displayed in the GUI. However, displaying all the classes along with their information will cause inconvenience for the users. Therefore, another parameter that is worth investigating is the number of retrieved classes to be displayed in the GUI.For the number of matches, we start by comparing the query image with two randomly selected images of each class. We repeat this experiment by increasing the number of randomly selected images to three, four and up to nine. Through this procedure, we find the optimal number of matches both in terms of classification accuracy and time. For instance, in case of experiment for comparing with two random images from each class, we have a total of A few examples of such ranking with query images from classes “1”, “2”, and “7” are shown in M and the top most relevant retrieved classes denoted by N. The number of matches are empirically selected as M, the experiments are performed 5 times and an average classification accuracy is reported. Similarly, for a given query image, all the 60 coin classes are ranked based on their achieved similarity scores from high to low. The correctly predicted label for a query image can be at any position among those 60 labels. However, for displaying, we search for least number of retrieval positions N where highest possible classification accuracy is also achieved. For instance, the value M and N are shown in We carried out elaborated experiments on the relationship of the number of matches denoted by M and N.An overall increase in classification accuracy is observed by increasing both The lowest accuracy is achieved by considering The highest classification accuracy of 99% is achieved for M should be 5 and that of N should be 10.In order to reduce the computational time, the value of To summarize, our proposed framework retrieves most relevant classes for a given query image by matching it with five randomly selected class exemplar images stored in the database. This results in a total of 300 (® Core™ i7-8700K CPU of 3.70GHz with 12 threads in Matlab® R2018b. The experiments are repeated 20 times and the average time taken by each setting along with the standard deviation is shown in We also performed experiments to show the time taken by matching a query image with various number of class exemplar images. The matching is performed using the parallel architecture of an IntelWe proposed an image-based framework for class retrieval of the Roman Republican coins. It takes a query image as an input, classifies using exemplar-based image classification, and then displays the resulting candidate top 10 classes in the GUI along with their information from the standard reference book. Our proposed framework can become an interesting application tool for a wide range of users such as museum visitors, Numismatics students and researchers, hobbyists, and online auction websites. We thoroughly evaluated various parameters of the framework such as number of exemplar images per class to which the query image will be matched and the number of top relevant classes to be displayed in the GUI. From our experiments, we found that the five images per class and a search space for the relevant retrieved classes extended up to 10 position successfully classifies the query image with 99% accuracy.In future, we plan to extend our database to span more coin classes, shift the current matching algorithm to GPU-based computations in order to meet the real-time matching, and include more state-of-the-art methods for image matching.


This cohort study evaluates the suitability of including pathogenic copy number variants associated with neuropsychiatric disorders in population screening by determining their prevalence and penetrance and exploring the personal utility of disclosing results. Are neuropsychiatric disorders that are associated with pathogenic copy number variants suitable for inclusion in population-based genomic screening programs?In this health care system population of more than 90 595 participants, copy number variants associated with neuropsychiatric disorders were prevalent and penetrant. Participant responses to receiving these genomic results were overall positive, suggesting personal utility.Neuropsychiatric disorders associated with genetic variants should be considered in the design of population-based genomic screening programs. Population screening for medically relevant genomic variants that cause diseases such as hereditary cancer and cardiovascular disorders is increasing to facilitate early disease detection or prevention. Neuropsychiatric disorders (NPDs) are common, complex disorders with clear genetic causes; yet, access to genetic diagnosis is limited. We explored whether inclusion of NPD in population-based genomic screening programs is warranted by assessing 3 key factors: prevalence, penetrance, and personal utility.To evaluate the suitability of including pathogenic copy number variants (CNVs) associated with NPD in population screening by determining their prevalence and penetrance and exploring the personal utility of disclosing results.In this cohort study, the frequency of 31 NPD CNVs was determined in patient-participants via exome data. Associated clinical phenotypes were assessed using linked electronic health records. Nine CNVs were selected for disclosure by licensed genetic counselors, and participants’ psychosocial reactions were evaluated using a mixed-methods approach. A primarily adult population receiving medical care at Geisinger, a large integrated health care system in the United States with the only population-based genomic screening program approved for medically relevant results disclosure, was included. The cohort was identified from the Geisinger MyCode Community Health Initiative. Exome and linked electronic health record data were available for this cohort, which was recruited from February 2007 to April 2017. Data were collected for the qualitative analysis April 2017 through February 2018. Analysis began February 2018 and ended December 2019.The planned outcomes of this study include (1) prevalence estimate of NPD-associated CNVs in an unselected health care system population; (2) penetrance estimate of NPD diagnoses in CNV-positive individuals; and (3) qualitative themes that describe participants’ responses to receiving NPD-associated genomic results.P < .001); 66.4% (470) of CNV-positive individuals had a history of depression and anxiety compared with 54.6% (49 118 of 89 887) of CNV-negative individuals . 16p13.11 (71 [0.078%]) and 22q11.2 (108 [0.119%]) were the most prevalent deletions and duplications, respectively. Only 5.8% of individuals (41 of 708) had a previously known genetic diagnosis. Results disclosure was completed for 141 individuals. Positive participant responses included poignant reactions to learning a medical reason for lifelong cognitive and psychiatric disabilities.Of 90 595 participants with CNV data, a pathogenic CNV was identified in 708 . Seventy percent (n = 494) had at least 1 associated clinical symptom. Of these, 28.8% (204) of CNV-positive individuals had an NPD code in their electronic health record, compared with 13.3% (11 835 of 89 887) of CNV-negative individuals (odds ratio, 2.21; 95% CI, 1.86-2.61; This study informs critical factors central to the development of population-based genomic screening programs and supports the inclusion of NPD in future designs to promote equitable access to clinically useful genomic information. However, comprehensive, population-based genomic screening programs, such as that carried out within the Geisinger MyCode Community Health Initiative, are quickly emerging as promising complementary public health options to proactively affect patient care.12 Several key factors have been described as critical to implement these programs, including results that inform disease prevention, early detection, or management; affect access to social services; and benefit family members.13 In addition, personal utility, equitable access to personally and medically relevant genomic information, and health care utilization effects are important factors.13 Furthermore, there is public interest for inclusion of genomic disorders that are often not designated as medically actionable, such as neuropsychiatric disorders (NPDs).15Genome-wide testing technologies, such as exome sequencing and chromosomal microarray, are being increasingly used across clinical settings as standard of care for identifying medically relevant genomic variants that cause a variety of conditions, including hereditary cancer syndromes, cardiovascular disorders, and neurodevelopmental disorders.17 Lifetime prevalence for common psychiatric disorders, including depression and anxiety, has been estimated globally at 29%.18 Individuals with NPDs have higher than expected rates of chronic medical conditions and associated early mortality and account for a disproportionately large share of health care costs, including emergency department and inpatient visits and prescription drugs.20 Collectively, rare pathogenic copy number variants (CNVs) and single-gene sequence–level variants represent the largest proportion of known NPD causes to date, yet research into precision health strategies has lagged behind other conditions, such as cancer and cardiovascular disease.27Neuropsychiatric disorders, such as autism spectrum disorder, schizophrenia, and bipolar disorder, comprise an etiologically heterogeneous group of conditions affecting at least 14% to 18% of children and adults in the US.28 Additionally, subclinical learning or social differences are often present, which may affect an individual’s well-being.29 Many NPD-associated genes and CNVs share biochemical and neurobiological pathways with other brain disorders, such as epilepsy and intellectual disability, that are frequent comorbidities in people with NPDs.31 The phenotypic variability of these large effects is modulated by family genomic background through the aggregate contribution of common variants of small effect size, quantified by polygenic risk scores,37 and may be further influenced by environmental experiences.Rare CNVs have large, primary effects on neuronal pathways and are causative of brain dysfunction, manifesting as clinically distinct disorders that exhibit a high degree of variable expressivity .28 Addit43 Hundreds of distinct genetic NPD causes are now known, representing a growing subset of pediatric- and adult-onset developmental and psychiatric diagnoses. However, clinical genetic testing in adult NPD populations is much less common, raising concerns about equitable access to highly relevant information for these individuals, and the penetrance of NPD-associated CNVs from an unselected population is less well known.Genomic testing for NPDs and other brain disorders is increasingly embraced in the pediatric setting, with diagnostic yields for sequence variants and CNVs approaching 40%, and observations of NPD-associated CNVs in variably affected adults have informed our understanding of the clinical variability and natural history of these disorders.46 Previous population-based studies have reported an estimated prevalence of approximately 1% for NPD-associated CNVs,48 indicating they may be as, or more, common than hereditary cancer and cardiovascular disorders currently included in population-based genomic screening programs. While these initiatives have allowed an unbiased assessment of population prevalence for genomic variants, they often lack detailed clinical correlation, particularly for NPD phenotypes. Furthermore, because these research initiatives do not have approval to return clinically confirmed and medically relevant information back to participants, there are very limited data on the perceived utility of this information from program participants.Our understanding of genomic contributors to human disease has gained momentum in recent years through analyses of large-scale research population data sets that provide a crucial counterbalance to clinically ascertained cohorts.To explore the suitability of including NPD-associated CNVs in population-based genomic screening programs, we assessed key factors incorporated into development and implementation decision-making frameworks: prevalence, penetrance, and early indications of participant response and personal utility. We report evidence from the first study, to our knowledge, of a large-scale, health care system–based population, using a linked electronic health record (EHR) and genomic data set, that supports inclusion of NPD-associated CNVs in population-based genomic screening and provides experience on the feasibility and logistical components for implementation.49 We evaluated the frequency of 31 pathogenic, recurrent CNVs  spanned at least 90% of the defined region. CNVs called from exome data were confirmed by an array-based method using PennCNV or manual inspection of the signal intensity data.54DNA sample preparation and exome sequencing were performed in collaboration with the Regeneron Genetics Center as previously described.International Classification of Diseases, Ninth Revision and International Statistical Classification of Diseases and Related Health Problems, Tenth Revision billing codes . A mixed-method approach was used to evaluate participants’ responses to receiving results, with written consent provided for audio recording of in-person disclosure sessions. Age was calculated based on the last encounter in EHR. Tests of association were performed with logistic regression adjusted for age and sex. Reported P values were 2-sided, and statistical significance was set at .005. Reported P values are Bonferroni corrected for the 9 tests of association performed in the study. All statistical analyses were performed using R statistical software version 3.4.1 . Analysis began February 2018 and ended December 2019. A detailed description is provided in the eMethods in the To assess the frequency of relevant CNV-related phenotypes in CNV-positive individuals compared with CNV-negative individuals, 48 Estonian Genome Center of the University of Tartu (0.7%),46 and deCODE (1.16%).47 However, when a direct comparison was done across cohorts limiting the analysis to our 31 CNVs of interest, the DiscovEHR prevalence rates were notably higher for some CNVs , possibly due to ascertainment bias or technical differences with previous studies . When we broadened our NPD definition to include 2 common psychiatric disorders, depression and anxiety, we observed 66.4% (470 of 708) of CNV-positive individuals with this history compared with 54.6% (49 118 of 89 887) of CNV-negative individuals .We estimated the penetrance of NPD and congenital malformations in individuals with CNVs. We found that 28.8% (204 of 708) of CNV-positive individuals had an NPD-associated code in their EHR (eTable 3 in the P < .001). Cardiac defects were the most common congenital malformations observed in 6.5% (46 of 708) of CNV-positive individuals followed by congenital malformations affecting the urinary system in 2.9% (21 of 708), central nervous system in 2.3% (16 of 708), and genital organs in 2.1% (15 of 708). Cleft lip and/or palate were observed in 0.99% (7 of 708) of CNV-positive individuals, a 9.84 higher odds  than CNV-negative individuals (0.08% [68 of 89 887]). The prevalence estimates of other congenital malformations were approximately 2- to 3-fold higher in the CNV-positive group when compared with CNV-negative individuals, including cardiac , central nervous system , and genital malformations . In contrast, congenital malformations affecting the urinary system were observed at an equal prevalence of 3.0% , in CNV-positive (21 of 708) and CNV-negative (2716 of 89 887) individuals. Overall, 69.8% (494 of 708) of CNV-positive individuals had NPD, including depression and anxiety, or a congenital malformation compared with 57.5% (51 645 of 89 887) of CNV-negative individuals .Congenital malformations in 1 of 5 categories  were observed in 13.3% (94 of 708) of CNV-positive individuals  had an EHR-documented genetic diagnosis. The mean (SD) age of participants with a genetic diagnosis was 20.33 (14.53) years , reflecting a strong bias toward offering clinical genetic testing to younger individuals. For the subset of individuals with 1 of 9 CNVs prioritized for disclosure, 77.1% (216 of 280) had a relevant NPD or congenital malformation code (49.6% [139 of 280] after excluding depression and/or anxiety) and only 8.6% (24 of 280) had a known genetic diagnosis.The results disclosure process was completed for 141 DiscovEHR patient-participants with pathogenic NPD-associated CNVs. Participant perspectives on receiving CNV diagnoses were collected via genetic counselor postsession assessments . Many participants revealed learning issues, social difficulties, or hospitalizations for mental health issues that were not captured in the EHR. It was common for participants to have a perceived explanation for their personal and/or family history of NPD, often attributing it to social circumstances . Participants expressed relief and satisfaction at finally having a medical explanation for a lifelong history of learning and behavioral struggles. The CNV result reassured them that their NPD history was not their fault, and several expressed that they wished they had known this information earlier in life. Most participants  observed that their genetic diagnosis fit or made sense with their personal or medical histories.Participants typically stated that they planned to share their genetic test results with family members and often brought a support person to the disclosure session. Concerns about the potential for NPD in their children and grandchildren were a motivation for communicating results within a family, especially given the 50% chance of inheriting the CNV. However, a subset  indicated that they may not share the information broadly with family members who are less supportive of them. To date, cascade testing has been completed for 26 family members resulting in 16 newly detected CNV diagnoses, primarily in participants’ adolescent and young adult offspring, 94% of whom had clinically diagnosed NPD of previously unknown origin.55 These programs have the potential to have broad reach and ensure individuals with medically relevant genomic variants are identified proactively, avoiding the selection bias inherent in current clinical referral-based systems, which fail to identify a significant proportion (reported estimates up to 50%) of at-risk individuals.58 In addition to the value that population screening can have on disease management, several other concepts have been recognized as critical outcomes, including access to social services, personal utility, health care utilization optimization, equal access, and communication of results within health care systems and families.13Population-based genomic screening programs are a promising element to the cost-effective implementation of genomic medicine.59 Lynch syndrome (1 in 440 [0.2%]),60 or hypertrophic cardiomyopathies (1 in 500 [0.2%]).61 This population CNV prevalence is consistent with previous estimates from European populations,48 and additional study of more diverse populations is warranted. Differences between our data and previous estimates, most notably for 15q13.3 deletion, could be explained by ascertainment bias because individuals with more severe NPD may be more likely to be included in our population.62 Furthermore, because our CNV detection used both exome and microarray methods, with secondary clinical confirmation, our true positive rate could be increased. Research exploring the inclusion of NPD-associated single-gene disorders is also recommended to more completely represent all genetic causes of NPD.This study illustrates that NPD-associated CNVs are both prevalent and penetrant in a health care system–based population. Nearly 1% of MyCode participants have an NPD-associated CNV, collectively exceeding the prevalence estimates of disorders regularly included in population-based screening programs, such as familial hypercholesterolemia (1 in 200 to 250 [0.4%-0.5%]),63 These penetrance estimates (35%-70%) are comparable with some of the highest health risks associated with hereditary cancer and cardiovascular disorders, including BRCA1/2 (38%-87% lifetime risk for breast cancer), Lynch syndrome , and familial hypercholesterolemia (30%-50% risk for coronary event).64 Genetic causes will increasingly inform NPD care by hastening diagnosis and treatment of developmental or psychiatric concerns  and by monitoring for known medical risks .65The medical histories of MyCode participants with NPD-associated CNVs reflect the elevated risk for NPD and congenital malformation. Approximately 35% of all CNV-positive individuals, and 77% of those with a pathogenic CNV prioritized for participant disclosure, had documented NPD or congenital malformation in their medical records. Additionally, our data are consistent with previous observations that individuals with rare CNVs are at increased risk for depression.67 A genomic result in a given patient could have relatively few medical management implications but be perceived as highly valuable in personal or social ways.70 These benefits can influence decisions to adhere to medical recommendations, one’s ability to seek or obtain social or educational support services, family communication about NPD, and self-understanding.75 Participants in this study expressed several indications of personal utility, including the value of having a medical explanation for their disabilities. This notion was often expressed as a sense of reassurance that they are not at fault for their NPD, a validation that their learning and/or psychiatric difficulties were real, and an indication of enhanced understanding of themselves and their families. The personal utility of finding an etiological diagnosis may prove to be among the most important benefits of genomic testing for NPD.There is growing recognition of the broader benefits to patients learning genomic results, beyond the conservative and cost-centric requirement of medical actionability, including consideration of the personal utility of genomic information, which requires that genomic information be “used for decisions, actions, or self-understanding which are personal in nature.”28 However, only 5.8% of individuals in our cohort had previously received a genetic diagnosis, demonstrating that the care received by most adults with NPD-associated CNVs has not been informed by a genetic cause. Instead, most individuals live with symptom-based developmental, psychiatric, and medical diagnoses without knowing the underlying genetic cause that ties these findings together. The majority of study participants were already adults when submicroscopic CNVs were first discovered, and, because clinical testing for NPD-associated CNVs is not often offered to adults outside of parental testing in a pediatric setting, were unlikely to have had access to diagnostic testing that is now considered standard of care for pediatric developmental disorders and congenital malformations. Thus, adults with NPD are unlikely to have access to genomic information of high clinical and personal relevance to them and their family members. Importantly, 94% of participants’ family members who were identified as having a CNV by cascade testing had clinically diagnosed NPD of unknown cause, further highlighting the unmet need for genomic testing in this population.Clinically defined NPD, such as autism spectrum disorder, schizophrenia, and bipolar disorder, are intertwined at a biological level by shared genomic underpinnings that can be readily diagnosed through clinically available genetic testing.76 The medical community, including psychiatry, is also beginning to respond to these public views, moving away from a historically paternalistic approach to genomic result disclosure.77 Psychosocial outcomes data also indicate that patient-participants adjust to genomic information with little long-term negative consequences.78 Identifying genetic diagnoses for individuals with NPD allows us to move beyond vague discussions about multifactorial risk to more targeted, medical explanations.Public views about receiving NPD-associated genomic results, as well as genomic results associated with nonactionable diseases, generally lean toward ensuring access and disclosure.Ongoing research is needed to further document clinical, psychological, and family outcomes of CNV results disclosure and to inform development of disease-specific clinical models and support tools to complement population-based genomic screening. Additionally, research that incorporates polygenic risk and other factors could inform CNV-specific risk estimates for particular NPD and congenital malformation diagnoses. We anticipate that medicalizing NPD through the identification of specific genetic causes, paired with targeted genetic counseling and family cascade testing, may decrease stigma, increase self-advocacy, and lead to closer engagement of NPD patients with health care clinicians. These improvements could ultimately translate into more cost-effective utilization of health care resources and improved compliance with treatment recommendations.Our prevalence and penetrance estimates of pathogenic CNVs in DiscovEHR likely underestimate the true prevalence in the general population and represent a milder range of observed clinical phenotypes. While we did not exclude individuals with more severe cognitive and psychiatric disabilities from this study, they are likely underascertained in the DiscovEHR cohort owing to difficulty ensuring informed research consent or use of specialized medical care outside the general health care system, such as residential psychiatric facilities. Furthermore, our penetrance estimates relied on structured EHR data; many participants shared additional NPD histories during disclosure sessions that were not formally documented in the EHR. The advanced age of our participants also likely introduced a survival bias due to underrepresentation of individuals with severe medical conditions, such as CNV-related congenital cardiac anomalies, that reduce life expectancy.79This study of a large, health care system population demonstrates that there is a significant proportion of our population that could benefit from including NPD in population-based genomic screening programs. We have established that NPD-associated CNVs have sufficient prevalence and penetrance to be considered in the development and clinical implementation of such programs. Furthermore, we conclude that identification and disclosure of causative genomic variants is clinically and personally valuable for individuals with NPD and their families, who are likely to be underserved and have more limited access to this meaningful information.


How land use shapes biodiversity and functional trait composition of animal communities is an important question and frequently addressed. Land-use intensification is associated with changes in abiotic and biotic conditions including environmental homogenization and may act as an environmental filter to shape the composition of species communities. Here, we investigated the responses of land snail assemblages to land-use intensity and abiotic soil conditions , and analyzed their trait composition . We characterized the species’ responses to land use to identify ‘winners’ (species that were more common on sites with high land-use intensity than expected) or ‘losers’ of land-use intensity (more common on plots with low land-use intensity) and their niche breadth. As a proxy for the environmental ‘niche breadth’ of each snail species, based on the conditions of the sites in which it occurred, we defined a 5-dimensional niche hypervolume. We then tested whether land-use responses and niches contribute to the species’ potential vulnerability suggested by the Red List status.Our results confirmed that the trait composition of snail communities was significantly altered by land-use intensity and abiotic conditions in both forests and grasslands. While only 4% of the species that occurred in forests were significant losers of intensive forest management, the proportion of losers in grasslands was much higher (21%). However, the species’ response to land-use intensity and soil conditions was largely independent of specific traits and the species’ Red List status (vulnerability). Instead, vulnerability was only mirrored in the species’ rarity and its niche hypervolume: threatened species were characterized by low occurrence in forests and low occurrence and abundance in grasslands and by a narrow niche quantified by land-use components and abiotic factors.Land use and environmental responses of land snails were poorly predicted by specific traits or the species’ vulnerability, suggesting that it is important to consider complementary risks and multiple niche dimensions. Land use disturbs natural environments, changes local geographical landscape structure and alters local biotic and abiotic conditions, e.g. microclimate –6. ReducLand snails are an important macroinvertebrate group that is directly and indirectly involved in ecosystem processes such as litter decomposition or nutrient cycling , 11. TheStudies on trait composition of snail communities in Sweden pointed to the importance of the species’ niche-width and the importance of local environmental conditions over spatial variables . While tIn the present study, we investigated land snail communities at forest and grassland sites in different regions of Germany, which were characterized by different land-use types and intensities. We aimed to test whether the trait composition of the snail community is influenced by land-use intensity (and soil conditions). We then tested the responses of each snail species to land-use intensity; ‘winners’ significantly increase in abundance and occurrence with land-use intensity, whereas ‘losers’ significantly decrease compared to the null model , 39. We The trait composition of land snail communities differed strongly between forests and grasslands within regions, indicated by a strong differentiation of community-weighted mean trait values (CWMs). Assemblages of forest species consisted of larger species, consistently showed lower light and higher humidity preference, lower drought resistance and mostly lower inundation tolerance than grassland assemblages; differences in the number of offspring were inconsistent among forest and grassland habitats Fig. .Fig. 1TrIn forests, land-use intensity and abiotic conditions significantly influenced the CWMs of all traits investigated, although often in a different way across regions , whereas 12% were ‘winners’ and thus increased with forest management intensity Table . The proMonacha cartusiana profited from high LUI  and fertilization (4% losers and 4% winners) had a very little impact compared to the combined LUI.In grasslands, many species were predominantly found at low land-use intensities (LUI); 21% of all species were significant losers and only UI Table . HoweverHowever, in both forests and grasslands, species’ land-use responses (i.e. their ‘winner/loser’ status) were independent of their traits; i.e. losers in forests or grasslands were neither characterized by a smaller or larger shell size nor by lower or higher numbers of offspring nor by lower or higher light preference etc.  were associated with higher pH values  as compared to forest soils, and many snail species   is associated with a high vulnerability of the species, neither in forests nor in grasslands Table . A betteLand snail species are slow-dispersing organisms, and historical influences are of general importance for their distribution . Their dOur study showed that snail assemblages varied consistently in their trait composition  across regions and among the two habitats, forests and grasslands. The variation between regions is consistent with a biogeographic gradient of increasing land snail diversity from the north to south caused by historical and ecological factors  , 22 and In our study, 4% of all forest and 21% of all grassland snail species were significant losers concerning the compound indices of land-use intensity, including three land-use components in the forests or in the grasslands, respectively. The proportion of losers among grassland snail species was lower than the level found for grasshoppers (about 52%)  and planWhile increasing land-use intensity in open habitats is known to trigger a decline of pollinator species, and such losses were associated with species-specific trait attributes such as a narrow diet breadth, climate specialization, a large body-size and low fecundity –39, we dCochlicopa lubricella is a xerophilic land snail [Note that single land-use parameters and abiotic conditions are often confounded in real landscapes as in our study, and thus responses of some snail species may not always correspond to single environmental dimensions as known from their global distribution or other sources. For example, nd snail  whereas The range of resources and the ecological conditions generally define the niche breadth and determine the geographical area of a species at the small or large scale . SpecialOnly a few snails in our study across managed forests and grasslands are considered threatened or endangered according to the national Red List. Consistent with the expectation based on their environmental niche breadth, the species’ vulnerability status was significantly predicted by a particularly narrow niche hypervolume—an index that includes single land-use components as well as pH and soil moisture in each habitat. The smaller the hypervolume of a species, the higher its vulnerability according to the Red List. In addition, rarity was important: in forests, the most important predictor for their vulnerable status was a low number of sites in which they occurred. In grasslands, both their restricted occurrence and low total abundance predicted the species’ vulnerability.In summary, our results indicate that the trait composition of snail communities was significantly altered by land-use intensities and abiotic conditions, and several species especially in grasslands were losers of intensive land use. These land-use and environmental responses were largely independent of specific traits and the species’ Red List status—this suggests that complementary risks may be important for predicting a species’ vulnerability. Instead, species vulnerability was mirrored in the species’ rarity and its overall niche hypervolume including single land-use components and abiotic factors.https://www.bexis.uni-jena.de/PublicData/PublicDataSet.aspx?DatasetId=24986. Wehner et al. [http://www.biodiversity-exploratories.de) [Data for this study were already part of a previous analysis of biodiversity and community composition, i.e. Wehner et al.  and are r et al.  collecteIn each region, 100 experimental plots (50 in forests and 50 in grasslands) were setup in 2008 along a land-use gradient covering different management types and intensities including mowing, grazing and fertilization in grasslands and the proportion of non-native trees, the proportion of dead-wood with saw cuts and the proportion of wood harvested in forests Table . Forest In June 2017, Wehner et al.  took fivAs our current study focuses on species-level responses, only those individuals that could be assigned to the species level were used . Grassland plots  in one region (Schorfheide) harbored large numbers of aquatic and semi-aquatic snails. In contrast to our previous analysis that covered all snails recorded , we exclAll statistical analyses were performed in R 3.5.2  using thMorphological and life-history trait values for all snail species were obtained from an established trait database by Falkner et al.  and comppTi is the trait value of species i, ai,p is the abundance of species i in plot p and Ap the total abundance of all snails in plot p .For comparing snail communities among habitats and regions, the community weighted mean (CWM) of each trait was calculated as CWM per plot We characterized the environmental conditions of each forest or grassland plot by its land-use intensity and two abiotic soil parameters , we calculated each species’ “environmental niche”. The method has been established in the context of the Biodiversity Exploratories and was applied to several taxa such as grasshoppers , cicadasNi sites with the same probability, with Ni being the number of sites in which species i was found. The null model thus chooses values of the focal land-use parameter  of Ni sites and calculates a distribution of predicted AWMs and AWSDs values for each species based on 10,000 iterations. The null model was restricted to the one, two, or three regions in which the species was recorded to consider potential distribution boundaries of each species in Germany that may not be related to plot conditions [In addition to the AWM as a niche optimum, we also characterized the “niche breadth” of each species to a single environmental variable using the abundance-weighted standard deviation (AWSD) . To testnditions .p value for the significance of the deviation between observed and expected values. A ‘winner’ is defined as a species with an observed AWM larger than the upper 5% of the distribution of AWMs obtained by the null models (i.e. adapted on higher-than average land-use intensity), a ‘loser’ shows an observed AWM smaller than the lower 5% . For species which could be classified neither as ‘losers’ nor as ‘winners’, we tested whether they are specialized on intermediate land-use or abiotic levels, that is, whether they have an intermediate AWM with a narrower niche than expected. We standardized the niche breadth as weighted coefficient of variation (CV = AWSD/AWM) to account for the increase in SD with increasing mean, and compared observed CV and expected CV from the null models. This comparison allows us to distinguish ‘opportunists’ (observed CV ≥ expected CV) from species that are ‘specialized’ on intermediate land-use intensities  [As in any randomization model, the proportion of AWMs or AWSDs from 10,000 null models with greater or smaller expected values respectively than the observed value, provides the  CV ≠ 0) . The envVulnerability  of land snail species was obtained from the Red List 2011  and its relation to abiotic soil conditions, we used a general linearized model with Poisson distribution including vulnerability as response factor and the respective land-use parameter or abiotic factor, the number of plots where the species occurred and its total abundance as explanatory factors. Values for grazing and fertilization were square-root transformed prior to statistical analyses and data on abundances and occurrence were log transformed because of data structure.n‐dimensional niche concept) as a proxy for the total ‘niche breadth’ of each snail species by multiplying the abundance-weighted standard deviations (AWSD) of all three single land-use components as well as of pH and soil moisture, respectively. The hypervolume was defined for forests and grasslands separately.Finally, we calculated a five-dimensional niche hypervolume  and grasslands  on the community weighted mean of the maximum shell size, the number of offspring, light preference, humidity preference, drought resistance and inundation tolerance. * p < 0.05, ** p < 0.01, *** p < 0.001. ↓ negative effect, ↑ positive effect.Additional file 2: Appendix 2. Influence of the abundance-weighted mean (AWM) of the forest management index on the maximum shell size, number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in forests. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 3: Appendix 3. Influence of the abundance-weighted mean (AWM) of the proportion of non-native trees on the maximum shell size, number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in forests. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 4: Appendix 4. Influence of the abundance-weighted mean (AWM) of the proportion of deadwood with saw cuts on the maximum shell size, number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in forests. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 5: Appendix 5. Influence of the abundance-weighted mean (AWM) of the proportion of wood harvested on the maximum shell size, number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in forests. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 6: Appendix 6. Influence of the abundance-weighted mean (AWM) of soil pH on the maximum shell size, number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in forests. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 7: Appendix 7. Influence of the abundance-weighted mean (AWM) of soil moisture on the maximum shell, size number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in forests. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 8: Appendix 8. Influence of the abundance-weighted mean (AWM) of land-use intensity (LUI) on the maximum shell size, number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in grasslands. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 9: Appendix 9. Influence of the abundance-weighted mean (AWM) of mowing on the maximum shell size, number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in grasslands. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 10: Appendix 10. Influence of the abundance-weighted mean (AWM) of grazing on the maximum shell size, number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in grasslands. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 11: Appendix 11. Influence of the abundance-weighted mean (AWM) of fertilization on the maximum shell size, number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in grasslands. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 12: Appendix 12. Influence of the abundance-weighted mean (AWM) of soil pH on the maximum shell size, number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in grasslands. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 13: Appendix 13. Influence of the abundance-weighted mean (AWM) of soil moisture on the maximum shell size, number of offspring, light preference, humidity preference, drought resistance and inundation tolerance in grasslands. Species in italics are land-use “winners”, species in bold are land-use “losers”.Additional file 14: Appendix 14. Relation of the abundance-weighted means (AWM) of the forest management index, proportion of non-native trees, proportion of dead wood with saw cuts, proportion of wood harvested, pH and soil moisture and the proportional occurrence of a certain species in forests.Additional file 15: Appendix 15. Relation of the abundance-weighted means (AWM) of the land-use intensity, mowing, grazing, fertilization, pH and soil moisture and the proportional occurrence of a certain species in forests.