This survey study reports on the current logistics and challenges of ordering and coordinating insurance authorization for genetic testing for pediatric patients with vascular anomalies. What are the barriers to obtaining genetic information for patients with vascular malformation (VM)?In this survey study of 55 vascular anomaly specialists (primarily pediatric hematologists-oncologists), barriers to genetic testing for VM were identified across vascular anomaly centers of all sizes, including lack of administrative support; unclear institutional, insurance, and laboratory requirements; and lack of clinician education.Findings from this survey suggest that, despite being critical to the comprehensive care of patients with VM, obtaining genetic information is difficult; study results and recommendations should have broader application to clinicians caring for patients for whom molecular diagnosis is important to medical management. Vascular malformations (VMs) are rare disorders of vasculogenesis associated with substantial morbidity. Improved understanding of their genetic basis is increasingly guiding management, but logistical barriers to obtaining genetic testing in patients with VM may constrain treatment options.To examine the institutional mechanisms for and obstacles to obtaining genetic testing for VM.This survey study invited members of the Pediatric Hematology-Oncology Vascular Anomalies Interest Group, representing 81 vascular anomaly centers (VACs) serving individuals up to 18 years of age, to complete an electronic survey. Respondents were mostly pediatric hematologists-oncologists (PHOs) but included geneticists, genetic counselors, clinic administrators, and nurse practitioners. Responses that were received between March 1 and September 30, 2022, were analyzed with descriptive methods. Requirements for genetic testing by several genetics laboratories were also reviewed. Results were stratified by size of the VAC.Vascular anomaly center and associated clinician characteristics and practice patterns for ordering and obtaining insurance approval for genetic testing for VMs were collected.Responses were received from 55 of 81 clinicians, for a response rate of 67.9%. Most respondents were PHOs (50 [90.9%]). Most respondents (32 of 55 respondents [58.2%]) replied that they order genetic testing on 5 to 50 patients per year and reported a genetic testing volume increase of 2- to 10-fold over the past 3 years (38 of 53 respondents [71.7%]). Most testing was ordered by PHOs (35 of 53 respondents [66.0%]), followed by geneticists (28 [52.8%]) and genetic counselors (24 [45.3%]). In-house clinical testing was more common at large and medium-sized VACs. Small VACs were more likely to use oncology-based platforms, which potentially miss low-frequency allelic variants in VM. Logistics and barriers varied by size of the VAC. Obtaining prior authorization was the duty shared among PHOs, nurses, and administrative staff, but the burden of insurance denials and appeals were on PHOs (35 of 53 respondents [66.0%]). Lack of administrative support; unclear institutional, insurance, and laboratory requirements; and lack of clinician education were barriers to genetic testing at VACs of all sizes. The effort to obtain genetic testing for patients with VM, compared with patients with cancer, was perceived as excessive, despite genetic testing being considered standard of care for this population.Results of this survey study showed the barriers to genetic testing for VM across VACs, described differences between VACs based on size, and proposed multiple interventions to support clinicians ordering genetic testing for VM. The results and recommendations should have broader application to clinicians caring for patients for whom molecular diagnosis is important to medical management. Procedures are often performed on children, with concerns about painful diagnostic procedures and anesthetic risks. Compared with cancers, most VMs harbor somatic pathogenic variants at lower variant allele frequencies (often <5% to 10%), requiring sensitive sequencing methods.To examine the institutional mechanisms for and obstacles to obtaining genetic testing for VM, we surveyed members of the Pediatric Hematology-Oncology Vascular Anomalies (PHO VA) Interest Group and reviewed requirements for genetic testing from several laboratories and insurance companies. The results identified gaps in the ability to optimize care for patients with VM. Herein, we offered guidance for successful genetic testing while minimizing clinician and institutional effort.AAPOR) reporting guideline.In March 2022, we sent an electronic survey using REDCap, version 13.4.11 (Vanderbilt University) to members of the PHO VA Interest Group, including primarily pediatric hematologists-oncologists (PHOs) representing 81 vascular anomaly centers (VACs) across the US. The Children’s Hospital of Philadelphia deemed this study exempt from institutional review board approval and waived the informed consent requirement because the study collected no patient data or protected health information and involved only survey procedures. We followed the American Association for Public Opinion Research , medium (25-100 patients per year), or large (>100 patients per year). The survey focused on patients with somatic postzygotic disorders causing VM, but some respondents included notes about testing for germline conditions. Because most respondents also cared for pediatric patients with cancer, they were asked to subjectively compare the ease of obtaining genetic studies for patients with VM vs patients with cancer.The survey  were reviewed for information on ordering, preauthorization, and billing requirements. Managers in these laboratories were contacted for clarification, if needed. An effort was made to determine the demographic and disease-specific information required from the originating clinic, whether preauthorization was needed, and whether responses varied with patient insurance coverage.Data were stratified by VAC size. Data were analyzed using descriptive methods.There were 55 survey respondents from 81 requests (67.9% response rate). Most respondents were PHOs (50 of 55 respondents [90.9%]); other respondents included geneticists, genetic counselors, clinic administrators, and nurse practitioners involved in the care of patients with VM . Of the As a measure of institutional commitment to vascular anomalies, respondents were asked about their membership in the national Consortium of Investigators of Vascular Anomalies (CaNVAS) and the International Society for the Study of Vascular Anomalies (ISSVA). Most members of CaNVAS and ISSVA were from large VACs. Geographic distribution of respondents by institution zip code showed a concentration of VACs, regardless of size, on the East Coast, with fewer sites elsewhere.Respondents reported a wide range in the number of patients per year who underwent genetic testing. Of these respondents, less than a quarter (12 of 53 [22.6%]) ordered testing on fewer than 5 patients per year, more than half (32 of 53 [60.4%]) ordered testing on 5 to 50 patients per year, and only a few (5 of 53 [9.4%]) from the large VACs ordered testing on 50 or more patients per year. Programs of all sizes reported an increase in the number of genetic tests. Nearly half of the respondents (26 of 53 [49.1%]) reported that their volume of genetic testing for patients with vascular anomalies had increased by 2- to 5-fold in the past 3 years. Twelve of 53 respondents (22.6%), most of whom were at large centers, reported that testing had increased more than 10-fold. At 6 of 11 small centers (54.5%), genetic testing numbers had stayed about the same. Most respondents (42 of 53 [79.2%]) reported that more than half of testing was done for pediatric patients 18 years or younger.Respondents reported a wide variation in the type of clinician who placed orders for genetic testing . Those wPeripheral blood samples for genetic testing were commonly sent to Invitae (27 of 53 [50.9%]), Seattle Children’s Hospital Molecular Genetics Laboratory (16 of 53 [30.2%]), Washington University in St Louis NGS Laboratory (15 of 53 [28.3%]), in-house (respondent’s institution) clinical testing (15 of 53 [28.3%]), and Foundation Medicine (14 of 53 [26.4%]) . For genAt 36 of 53 VACs (67.9%), the person ordering the test decided which laboratory to use for testing blood and tissue specimens. Respondents who selected the other or combination option for these questions commented that the decision was shared between the ordering clinical physician and a geneticist or genetics counselor, with influence by insurance coverage. The institution dictated which laboratory was to be used for genetic testing at 9 of 53 VACs (17.0%). Paired testing with both blood and tissue samples was common (23 of 53 respondents [43.4%]) but varied with clinical diagnosis, requirements of the laboratory, or insurance. Most respondents (28 of 53 [52.8%]) reported needing to place separate orders for blood and tissue specimens. Among 53 respondents, biopsies for tissue testing were commonly performed by pediatric surgeons (37 [69.8%]); dermatologists (33 [62.3%]); interventional radiologists (30 [56.6%]); ear, nose, and throat surgeons (23 [43.4%]); and PHOs (3 [5.7%]). The other category included plastic and orthopedic surgeons.Most respondents (30 of 53 [56.6%]) requested a gene panel when ordering genetic testing. Others (17 of 53 [32.1%]), with concerns that insurance would not cover a somatic overgrowth panel, ordered sequencing of a single gene, with reflex to broader testing if the initial testing results were negative.Processes for insurance approval for genetic testing are summarized in Many respondents (19 of 53 [35.8%]) reported needing a follow-up insurance authorization if a panel was requested after failure of diagnostic single-gene testing. One-third of respondents (17 of 53 [32.1%]) reported that insurance approval was obtained between 50% and 74% of the time on the first attempt. One-quarter of respondents (13 of 53 [24.5%]) reported a higher rate of approval—between 75% and 99%—on the first attempt. Twelve of 53 respondents (22.6%) reported not knowing how often approval was obtained.Despite most respondents commenting that a discussion with patients and families about insurance approval and testing costs was important, not all were sure that this conversation was happening in their clinics. Only 33 of 53 respondents (62.3%) could confirm that a conversation about financing genetic testing was occurring with patients and families. Only 1 of 53 respondents (1.9%) from a large VAC reported that an institutional managed care coordinator held these conversations. Three of 53 respondents (5.7%) reported such conversations did not happen at all. Three respondents (5.7%) reported that in 3 VACs no conversations about financing were occurring prior to genetic testing. In 6 of 53 VACs (11.3%), the respondents stated they did not know who was responsible for discussing the financial implications of genetic testing with patients. Most of the patient and family conversations about the financing of genetic testing appeared to be led by the ordering physician or nurse (36 of 53 VACs [67.9%]) or a genetic counselor (26 [49.1%]). Several responses suggested that some families may not be receiving any financial guidance prior to undergoing testing. Five of 53 respondents (9.4%) cited difficulty finding accurate information about insurance approval and testing costs prior to testing. Respondents also reported interacting with many different insurance carriers, and nearly all respondents (50 of 53 [94.3%]) reported working with state Medicaid entities. Health insurance carriers that were grouped under the other category included state-specific plans, Cigna, and a few smaller carriers.Multiple respondents described their efforts to obtain genetic testing as “an uphill battle,” “monumental effort,” or “huge burden of time.” Many respondents commented on needing to spend multiple hours per patient on the process, and several admitted that their own lack of knowledge about the system and process was a barrier. While a few respondents reported adequate support for the processes surrounding genetic testing on patients with vascular anomalies, most reported that support was insufficient. A few VACs, even those with good support, experienced delays in obtaining insurance approval or dealing with denials. Issues with genetic testing specifically associated with VM included lack of familiarity with the diagnosis codes used by insurance companies and lack of precise diagnostic coding for vascular anomalies. Many respondents noted that the entire process of obtaining genetic testing for patients with cancer was far easier than for patients with vascular anomalies.As shown in 14 Disproportionate to their prevalence, many patients with VM experience substantial morbidities that interfere with their quality of life, including functional impairment, chronic pain, infection, and increased mortality. These patients require a comprehensive approach to care that includes physicians from multiple specialties, diagnostic imaging, histopathology, access to surgery and interventional procedures, and molecular diagnostic measures to support targeted medical therapy.16The overall prevalence of VMs in the US has been estimated at 1.5% of the general population.17 The introduction of sirolimus for the management of VM represented a paradigm shift in the approach to medical management.8 While sirolimus remains a critical part of the therapeutic armamentarium, data increasingly suggest that more precise targeting of gene variants may result in better outcomes.23 Because many of the genetic drivers of VM are shared with those found in cancers, most drugs under investigation have been borrowed from cancer treatments. Alpelisib , a drug that targets the PIK3CA gene, was approved in April 2022 by the US Food and Drug Administration for the treatment of a specific group of VM disorders: PIK3CA-Related Overgrowth Spectrum.24 Other drugs, such as trametinib  and cobimetinib, which target MEK, and miransertib, which targets AKT, are in clinical trials or are in early development.30Medical management of VM has shifted from symptom care alone to targeted therapies focused on controlling the genetic drivers of these diseases.Despite growing evidence that targeted therapies are an important component of comprehensive care for patients with VM, the results of the present survey underscore the difficulties in accessing genetic testing for this group of disorders. Respondents, most of whom were PHOs, universally noted that the obstacles in getting genetic information were unique to VM and were not similar to issues they often faced in obtaining molecular testing for patients with cancer. Most respondents also noted seeing a substantial increase in genetic testing for VM and thus anticipating further increases. Because genetic testing for VM is rapidly becoming standard of care, it is critical to address barriers to testing.Highlighted concerns included lack of administrative support for ordering genetic testing, difficulties in obtaining insurance coverage, and specific limitations of genetic testing in this population . Insurance denials, multiple orders (separate for blood and tissue testing), additional approvals for second-line testing, and appeals with requirements for peer-to-peer interactions were universal issues. We also confirmed considerable variation in insurance coverage among laboratories.Many of the difficulties in obtaining genetic testing for VM were magnified at small and midsize VACs. In-house genetic testing was much more available at large and midsize centers, and in-house research testing was observed only at large centers. Small VACs sent a higher proportion of testing to Foundation Medicine and Tempus, which are genomic profiling companies designed for oncology. Small centers also reported fewer resources for genetic testing, including fewer team members available for administrative processes, less availability of clinical geneticists or genetic counselors, and more uncertainty about testing processes. Clinicians from small centers were also less likely to be a part of national consortia for vascular anomalies  and less likely to have true multidisciplinary teams. The results confirmed findings from an unpublished study by the PHO VA Interest Group on VAC resources across the US , which noted that relatively few centers care for this population. Presumably, many patients are not tested and therefore may not benefit from emerging drug treatment options.31 Standard sequencing techniques for oncological diagnosis can fail to identify low-frequency somatic variants in VM, which can result in missed identification of relevant variants.34 Of note, there were differences in the ordering practices for blood vs tissue specimens in this survey, possibly due to involvement of different clinicians involved with biopsy or specimen processing.The method of genetic testing for VM is critical, as most are due to somatic, postzygotic sequence variations and will not be identified using standard methods for germline conditions. While the focus of this survey was on somatic disorders, some respondents included notes about testing for germline disorders. Notably, VACs of all sizes sent specimens (including tissue) to Invitae, a laboratory that is not validated for variant allele frequencies less than 20%. Such sequencing methods, although adequate for cancer evaluation, may miss detection of sequence variations at the low variant allele frequencies seen in many VMs.A major theme from this survey was that many respondents believed they lacked knowledge about the process of genetic testing for VM. Most respondents were PHOs, who self-reported that they were one of the major groups responsible for ordering genetic testing for VM. Unlike geneticists and genetic counselors, PHOs receive little formal training in genetic testing methods and genetic counseling. Many respondents expressed that their uncertainty about their knowledge of genetic testing was a major barrier. This opinion was particularly striking in the questions about providing counseling prior to genetic testing, with less than half of the respondents reporting that conversations with a genetic counselor were occurring prior to testing and several respondents reporting that no such discussions were occurring. It is important for all patients and families to be counseled appropriately prior to genetic testing, and it is clear from this survey that this step is lacking. Ideally, geneticists and genetic counselors would be part of every multidisciplinary team for VM, but for VACs without these resources, additional educational support for clinicians who order genetic testing might be worthwhile.A small number of respondents noted that their institutions recognized the downstream benefits of genetic testing for vascular anomalies, both in providing appropriate care for this patient population and in generating revenue and cost savings. While genetic testing is expensive, the benefit to the institution of billing for the procedure for biopsy and ongoing patient care once a patient has started targeted medical therapy should not be overlooked. Financial details were not examined in this survey.International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes relevant to vascular anomalies was developed by the PHO VA Interest Group and could be linked to laboratory ordering forms and made more generally available. The Cleveland Clinic has previously reported substantial cost savings for genetic testing from implementing a restricted-use initiative that limits ordering of genetic tests to those clinicians who routinely do such ordering.35 While restricted use may not be feasible at small VACs, one can envision a role for a more centralized support or mentoring team to provide education and support to small centers. Currently, there is a national web-based forum through which PHOs can seek input from a rotating group of national experts. Updated educational and logistical materials on genetic testing for VM could be maintained centrally, including key aspects of counseling, important considerations for sequencing methods, diagnostic and billing procedure codes, and templates of insurance authorization letters. Educational sessions on genetic testing for VM could be incorporated into conferences targeted at PHOs treating vascular anomalies. Although not a replacement for dedicated personnel with genetic testing training, access to experts could serve as a stopgap measure for clinicians lacking such support. Ultimately, more widespread access to vascular anomalies experts, including geneticists and genetic counselors, would facilitate testing. The findings of this survey reflect barriers to genetic testing in the US, which has both unique benefits and inadequacies compared with systems in European and other countries.39We believe a change in approach to genetic testing across the national PHO VA Interest Group could be implemented to yield substantial benefit. This approach could include educational modules and logistical support for physicians caring for patients with VMs. An itemization of the There were several limitations to this study, including the recall bias inherent in most cross-sectional surveys. The surveys were completed primarily by 1 PHO from each institution, rather than vascular anomalists from other specialties at that institution, potentially leading to bias. In particular, the perspective of geneticists and genetic counselors who see and test these patients outside of a multidisciplinary setting may be missing. However, respondents were asked to solicit input from other team members to better reflect the practices for testing patients with VM at their institution. While the emphasis of this survey was on somatic testing for VM, some respondents included notes about the testing they performed for germline conditions. Although these comments were easily abstractable from the responses, the differences in responses could have affected the results. The focus of this study was on the logistics and effort of genetic testing in patients with VM, not on obtaining targeted medications based on genetic testing results. Although separate, these issues are tightly linked due to the need for confirmatory genetic testing to obtain drug approval.In this survey study, we identified barriers to genetic testing for VM across VACs, described differences between VACs based on size, and proposed multiple interventions to support clinicians ordering genetic testing for VM. Because genetic testing for VM is rapidly becoming the standard of care, it is critical to identify and address the barriers to such testing. Barriers to genetic testing in VM are likely to be similar in other rare diseases. The results and recommendations of this study should have broader application not only to clinicians caring for patients for whom molecular diagnosis is important to medical management but also to health care and genomic medicine.


Heterodontus quoyi) and single nucleotide polymorphisms (SNPs) to examine evolutionary processes and their consequences for genetic divergence and island biogeography in a shallow-water marine species without larval dispersal. The sequential separation of individual islands from a central island cluster gradually established different ocean depths between islands that pose barriers to dispersal in H. quoyi. Isolation by resistance analysis suggested that ocean bathymetry and historical sea level fluctuations modified genetic connectivity. These processes resulted in at least three genetic clusters that exhibit low genetic diversity and effective population sizes that scale with island size and the level of geographic isolation. Our results exemplify that island formation and climatic cycles shape genetic divergence and biogeography of coastal marine organisms with limited dispersal comparable to terrestrial taxa. Because similar scenarios exist in oceanic islands around the globe our research provides a new perspective on marine evolution and biogeography with implications for the conservation of island biodiversity.Oceanic islands play a central role in the study of evolution and island biogeography. The Galapagos Islands are one of the most studied oceanic archipelagos but research has almost exclusively focused on terrestrial organisms compared to marine species. Here we used the Galapagos bullhead shark ( Oceanic archipelagos are ideal model systems to study evolutionary processes and their consequences on genetic variation in the light of island formation Emerson . IndividIn volcanic archipelagos individual islands are formed sequentially through recurring volcanic activity as tectonic plates move across hotspots. The progression rule describes the sequential colonization and subsequent genetic divergence from older towards younger volcanic islands in terrestrial organisms  provide a unique model system for the study of evolutionary processes in oceanic islands. The volcanic islands are separated from the South American coast by approximately 1000 km of up to 2000 m deep ocean  have a generation time of 22.5 years, slow adult growth rates (31.4– 32.7 mm year−1) and females mature between 8 and 10 and males between 11 and 14 years  has a strictly benthic lifestyle, spending the majority of time on or close to the ocean floor often hiding in caves and crevices, particularly juveniles, and inhabiting rocky reefs in proximity to cold-water upwelling areas at less than 40 m depth . A total of 33 locations on seven islands were visited between 2015 and 2018 . Samples were processed using the proprietary DArT Pty Ltd analytical pipeline, which includes technical replicates from a subset of samples to assess genotyping reproducibility  calculated for neutral SNPs with the divMigrate function of the diveRsity R package  and Mantel tests with 1000 permutations with the R package DartR  analysis McRae  using dePeterman . We compPeterman . PaleogePeterman . To testPeterman  were calPeterman . ResistaPeterman . The lindiveRsity R package , observed heterozygosity (HO), expected heterozygosity (HE), and inbreeding coefficients (FIS). We estimated local contemporary genetic effective population size (Ne) for each genetically distinct unit with more than eight individuals identified by clustering analyses using the bias-corrected linkage disequilibrium (LD) method implemented in NeEstimator v.2.01  for each individual genetic cluster using the formula 1/(2S) < Pcrit < = 1/S (S=number of samples) as suggested by  using the formula Necor = Ne/(0.098 + 0.219 × lnChr), where Chr is the number of chromosomes  and on each of two islands in the central-southeastern bioregion (Española and Floreana Islands). Despite exploring eight locations on 20 dives between 2015 and 2018 only eight individuals were sampled on San Cristóbal Island. Only a single individual was captured after diving at six locations around Santiago Island, which was not revisited after. The DArT pipeline generated 33606 SNPs and after quality filtering we retained a total of 9223 neutral SNPs and 180 individuals (Supplementary Information Table FST: 0.0133–0.0169) with the western locations compared to Floreana Island (FST: 0.0139–0.0161) based on FST values, but higher differentiation using DST (0.00038–0.00047 vs. 0.0003–0.00034). All pairwise comparisons within the western region were low for FST (0.0003–0.0028) and DST (0.000094–0.00014). With the exception of Punta Vicente Roca, the northernmost location on Isabela Island, all pairwise comparisons in the western region were not significant before and after false discovery rate (FDR) correction. We found no significant directional gene flow among any pair of sampling locations based on relative migration rates of the divMigrate method.Pairwise comparisons using neutral SNPs showed increasing genetic differentiation with greater depth among sampling locations and older island emergence time Fig. . EspañolK) as three . However, the scatter plot revealed a large gap between western and southeastern locations, indicating the presence of barriers between those locations and pairwise genetic distances among southeastern locations were high despite their geographical proximity. Single surface optimization using ResistanceGA selected and applied reverse monomolecular transformation to the contemporary and paleogeographic bathymetry surfaces , compared to the IBD model. The scatter plot displays that the model partially accounts for depth barriers, providing a better fit for the data. Finally, the paleogeographic IBR model revealed the strongest correlation with genetic distances  by taking into account ocean depth and historical sea level oscillations  ranged from 1.31 to 1.39 and observed heterozygosity (HO) from 0.091 to 0.098 (Table FIS) close to zero indicate the absence of recent bottlenecks.Genomic diversity was similarly low for all sampling locations. Allelic richness (Ne) was corrected for a 4% downward bias (Necor = Ne/0.959) to account for the number of chromosomes based on the congeneric H. japonicus and H. francisci. This resulted in the lowest Necor for Española, the most isolated and smallest island, followed by Floreana islands, which is geographically less isolated and about three times the size  while suitable shallow-water habitats acted as connectivity corridors  and rays  with low dispersal capacity and shallow depth distributions can maintain connectivity along continuous coastlines but show genetic differences across deep water between islands and islands and continental coasts at less than 50 km distance (Gaida Heterodontus francisci) sampled from different islands within the Channel Island archipelago separated by up to 100 km and 800 m deep water, despite showing strong genetic differences between the coast of California and the archipelago across 200 m deep water and at a distance of less than 20 km, unprecedented for any elasmobranch species  migrate up to 1000 km along the Australian coast and manage to cross the Bass Strait to Tasmania, likely because the strait is relatively shallow (on average 60 m) and has several islands that can be used as stepping stones  of continuous coastlines and across less than 10 km of 100 m deep water, between Isabela and Fernandina Islands, but larger ocean depths between islands pose effective barriers at distances of only 50 km. Recently, IBR analyses were applied to identify ocean depth as barrier to gene flow at hundreds of kilometers in a reef-associated pelagic shark  repeatedly connected landmasses of the central and western archipelago and exposed seamounts between the central and southeastern islands  between San Cristóbal, compared to Floreana Island, and the western archipelago, indicate that San Cristóbal Island may have separated second in sequence but partially reconnected to Floreana and the western archipelago during lower sea levels. To reinforce the sequential vicariance viewpoint we consider four alternative scenarios that have shaped genetic patterns in Galapagos fauna: (1) High levels of inter-island dispersal, (2) recent arrival, (3) multiple colonization events, and (4) strong divergent selection , led to relatively high genetic diversity  that scale with habitat availability in oceanic archipelagos (Frankham Ne approximates the census size (Nc) because of their low fecundity, consistent reproductive success, long life spans and late age at maturity  that are orders of magnitude smaller than the true number of adults in the population (Nc) owing to the high fecundity and variability in reproductive success , but similar estimates (3500) than populations at some isolated offshore locations (Delaval et al. Ne between the Galapagos and the continental coast and between Ne and Nc are needed to corroborate these results (Ryman et al. Ne estimates for individual islands approximate census sizes. Low genetic diversity and population sizes that scale to the amount of available resources are common in terrestrial island biogeography but, to our knowledge, unprecedented in marine organisms (Dawson Genomic signatures of isolation in Galapagos bullhead sharks are consistent with the gradual formation of barriers to dispersal and resemble those typical of terrestrial island biogeography. In oceanic islands, species with low dispersal commonly have lower genetic diversity and smaller population sizes compared to mainland populations owing to the reduced genetic variation of few founding individuals and because limited resources in small and fragmented habitats sustain smaller populations Frankham , 1997. GFrankham . In somes Dawson .This study shows that Galapagos bullhead sharks produce genetic and biogeographic signatures comparable to many terrestrial organisms in oceanic archipelagos and contrasting those commonly found in pelagic elasmobranchs and marine taxa with larval dispersal. Isolated marine populations with small effective population sizes and low genetic diversity are at high risk of extinction because they have reduced adaptive potential and lack replenishment from adjacent locations (Frankham et al. The data used in this study, including genotyped and filtered SNP data, and details of data analyses are openly available through the SUPPLEMENTAL MATERIAL


Understanding germplasm’s genetic diversity is essential for developing new and improved cultivars with stable yields under diverse environments. The objective of this study was to determine the genetic diversity and population structure of 128 maize inbred lines sourced from the International Institute of Tropical Agriculture (IITA), the International Maize and Wheat Improvement Centre (CIMMYT), and the University of KwaZulu-Natal (UKZN) using 11,450 informative single nucleotide polymorphism (SNP) markers. The inbred lines revealed highly significant (p < 0.001) levels of variability for the key phenotypic traits. The SNP markers had a mean gene diversity (GD) and polymorphic information content (PIC) of 0.40 and 0.31, respectively, indicating the existence of substantial genetic variation across the germplasm panel. The model-based population structure analysis identified three subpopulations (K = 3) among the inbred lines. This corroborated the phylogenetic analysis using phenotypic traits and molecular markers which classified the inbred lines into three groups. The findings of this study identified considerable genetic diversity for the selection of inbred lines with favourable alleles for multiple traits and could be useful to initiate marker-assisted selection (MAS) to identify significant loci associated with agronomic performance and multiple-stress tolerance. Zea mays L.) crop is a significant employer and source of foreign currency due to its multiplier effects. The crop has strong linkages throughout the economy, upstream to the input industries and downstream into the milling, animal feed, and food processing industries2. The hectarage planted with maize in South Africa varies depending on weather and market conditions, but on average, 2.5 to 2.75 million hectares of hybrid maize are planted each year, yielding 12 million tons of grain3. This translates to surplus maize exported to Zimbabwe, Botswana, Lesotho, Namibia, Eswatini, and Mozambique. South Africa is the only country in Africa that is in the top 10 of maize producers in the world4. However, climate change-induced biotic and abiotic stresses threaten maize productivity in tropical areas. Maize is severely affected by extreme weather events such as high temperatures, and unpredictable rainfall patterns, resulting in heat and drought stress or flooding5. Approximately 90% of maize in South Africa is produced on dry land under rain-fed conditions6.In South Africa, the maize  Rane, Payak & Renfro) and northern leaf blight  with previously resistant cultivars being affected. Thus, the selection of germplasm with improved resistance or tolerance to biotic and abiotic stresses is critical in developing resilient maize production systems adapted to climate change-induced stresses. The sub-Saharan Africa is a centre of diversity for tropical and subtropical maize which has evolved stress adaptation genes for wide adaptation in the harsh savannah climates. Hence, this germplasm can expand the genetic diversity required for the development of climate-resilient maize cultivars in South Africa.Rainfall has decreased drastically over the past few years causing a dramatic decline in maize volumes due to the El Nino induced droughts. The variations in the frequency and amount of rainfall received have deleterious effects on maize grain yields mainly when water deficits occur during flowering (anthesis and silking)12. The CIMMYT program in Africa is divided into the East and Southern Africa regional hubs. Yield, drought, heat, low N, Low P, Striga, grey leafspot, maize streak virus, rusts, and ear rots have been the primary targets for selection at the two hubs13.The Consultative Group of International Agricultural Research (CGIAR) centers comprising the International Maize and Wheat Improvement Centre (CIMMYT) and International Institute of Tropical Agriculture (IITA) have contributed immensely to the genetic improvement of tropical maize in Africa. Several inbred lines with tolerance to a diverse range of biotic and abiotic stresses have been developed by CIMMYT and IITA14. The MLN Screening facility in East Africa, Kenya identified notable resistance to the virus, and integrated the resistance genes into African-adapted germplasm. Currently, maize is a principal cereal in West Africa because of the Central Africa Maize Collaborative Research Network (WECAMAN) established by IITA and all the national programs in that region. The IITA’s breeding strategy of combining Striga resistance, earliness, drought- and low-N tolerance enhanced maize yield production in multiple stress environments. Maize grown in West and Central Africa should mature early before the onset of terminal drought which triggers Striga parasitic infections. It is noteworthy that both programs integrate elite exotic germplasm to expand the genetic and phenotypic elasticity of tropical and subtropical maize in the region. For instance, some of CIMMYT’s inbred lines developed in Africa are derived from Tuxpeño Sequía, and La Posta Sequía populations14, with the CIMMYT heterotic group A coming from populations and lines including the Tuxpeno, Kitale, BSSS , B73 and Salisbury white (N), in contrast the CIMMYT group B contains populations and lines from ETO, Ecuador 573, Lancaster, Mo17 and Southern cross (SC)15. Elite exotic germplasm introduces valuable new alleles that broaden the genetic divergence required to exploit heterosis for hybrid breeding16.However, the recent fall armyworm outbreaks in Southern Africa and the maize lethal necrosis virus (MLN) in East Africa presented new challenges warranting immediate action. To date, several multiple-stress tolerant CIMMYT maize lines (CMLs), including CMLs 536–544 and 571–572, and 587–592, bred in Southern Africa are available for public and private maize breeding programs in Africa17, containing GM traits such as glyphosate and insect resistance, thus improving grain yield. While these traits have resulted in considerable yield gains compared to conventional maize production, introgression of these traits will potentially result in deleterious genetic ‘bottlenecks’. In addition, most commercial breeding programs developed new inbred lines by recycling elite lines via pedigree which also narrows genetic divergence. Furthermore, most maize hybrid breeding programs, particularly in southern and eastern Africa, have utilized elite maize inbred lines from nine heterotic groups15 and derived from the “P” heterotic group  maize21. Trait phenotyping can be used in conjunction with molecular makers in the designation of entries into heterotic groups, particularly for highly heritable traits22. While morphological and agronomic polymorphisms are highly amenable to the environment and developmental stage of the plant, molecular markers, on the other hand, are very stable. Single nucleotide polymorphisms (SNPs) are the predominant form of naturally occurring genetic variation and offer adequate variation to distinguish closely related individuals. As a result, they have become the preferred DNA marker of choice for diversity studies. Additionally, SNPs are useful for studying genetic variation due to their low cost per data point, lower genotyping error rates, locus-specificity, high genomic abundance, potential for high throughput analysis, and codominance25.Knowledge of genetic relationships between local maize germplasm, and tropical and sub-tropical maize inbred lines will guide parental selection and the devising of appropriate breeding designs. Genetic diversity analysis differentiates breeding populations and assists in classifying inbred lines into definite heterotic groups26. Gaining insight into how locally bred lines are related to lines developed by the CGIAR, suffices in the exploitation of heterosis in local breeding programs. Therefore, the objectives of this study were to assess genetic diversity and interrelationships present among locally bred maize inbred lines and the CGIAR tropical and sub-tropical maize lines adapted to SSA conditions, to determine heterotic groups and select unique genotypes for biotic and abiotic stress tolerance breeding. The set of local South African germplasm used in the study represents important genetic resources for use in public and private maize breeding programs in the country.Thus, SNPs provide an opportunity for the assessment of genetic diversity among local South African inbred lines and exotic tropical and subtropical maize germplasm developed for national maize breeding programs in SSAStriga and MSV, as well as tolerance to drought, including TZE-W Pop DT STR C0, TZE-Y Pop DT STR C0, and TZE Comp 5-Y C6. The description of the germplasm used in the study is summarised in Table One hundred and twenty-eight (128) diverse maize inbred lines obtained from the International Maize and Wheat Improvement Centre (42), the International Institute of Tropical Agriculture (50), and the University of KwaZulu-Natal (36) were used in this study. The CIMMYT and IITA inbred lines were selected for their nutritional content and resistance/tolerance to biotic and/or abiotic stresses. The UKZN inbred lines were chosen to represent a sample of locally developed inbred lines to be used in commercial hybrid maize breeding. The CIMMYT lines were developed in Kenya and Zimbabwe  and were derived from the Tuxpeño Sequía, La Posta Sequía, and the Drought-Tolerant Population (DTP) yellow and white gene pools. The IITA germplasm was bred in Nigeria from several broad-based germplasm sources with resistance to −1. Field management followed the recommended maize agricultural practices.The test genotypes were evaluated under field conditions to complement molecular maker data. Two field trials were established at Ukulinga Research farm  of UKZN and Cedara Research Station  during the 2020/2021 cropping seasons. The experiments were arranged in 8 × 16 alpha lattice design with 2 replicates. Each plot consisted of 1 row, 5 m long, with inter and intra-row spacing of 0.8 and 0.3 m, respectively. The plant population density was 41,666.6 plants ha−1 was obtained from grain weight per plot adjusted to 12.5% grain moisture following CIMMYT (1999).Plant count (PC) was recorded as the total number of plants per plot counted after thinning. Days-to-anthesis (DA) and days-to-silking (DS) were recorded as the number of days from emergence to the date when 50% of the plants in a plot had produced pollen and silks, respectively. Anthesis-silking interval (ASI) was calculated as days to silking minus days to anthesis. Plant height (PH) expressed in centimetres (cm) was measured from base to tassel branching to the soil surface to the top of the tassel of 10 representative plants selected from each plot. Scoring the severity of rust (RST) disease of maize was done using a scale of 1–9, where 1 = 0% of leaf surface diseased (no rust) and 9 = 81–100% of leaf surface diseased. Field weight (FW) was measured as the total weight of unshelled cobs in kilograms (kg). Cob count (CC) was counted as number of cobs per plot at harvest. The number of kernel rows per ear (KR) and kernels per row per ear (KRE) were determined as counts. Ear length (EL) was recorded (cm) as the length of a cob from the tip to the base using the ruler, while the diameter of the cob (ED) was measured using a vernier calliper (mm). Grain moisture (MOI) content was measured using a moisture tester during shelling. Shelling percentage (SHP) was calculated as field weight minus grain weight and the difference was expressed as a percentage. A sample of 100 kernels, grain weight per ear (GWE) from each plot was weighed. Grain yield (GY) expressed in t hahttps://www.deltagen.agr.nz). Furthermore, the association among traits was deduced using Pearson correlation coefficients on the corrplot package in R software27.Data generated were subjected to analysis of variance after testing for normality and homogeneity of variance using R software. Trait BLUPs were computed using DeltaGen (https://www.seqart.net) in Nairobi, Kenya for DNA extraction and genotyping by sequencing. The DArTseq protocol was used to genotype samples using a SNP chip covering the ten maize chromosomes. The initial 50,941 SNPs from the genotyping-by-sequencing (GBS) pipeline were filtered by imputation to remove SNPs with 20% missing data and 5% minor allele frequency (MAF) using the snpReady package on R28. A total of 11,450 informative SNP markers were used after data imputation. Individual genotypes with > 20% missing data were removed.Fresh leaf samples were collected from 3 weeks old seedlings within each genotype and was shipped to SEQART AFRICA  and genetic diversity was performed using GenAlex30 after grouping the inbred lines based on the source of collection. Population structure was determined using the STRUCTURE v2.3.4 software31. The burn-in period and Markov Chain Monte Carlo (MCMC) length were set at 10,000 iterations, and the model was run by varying the number of clusters (K) from 1 to 10 with 10 alterations for each K. The appropriate K value was estimated by implementing the Evanno method using the STRUCTURE Harvester program32. A joint analysis of phenotypic and genotypic data was conducted. A phenotypic distance matrix was generated based on Gower’s distance, while the genotypic distance matrix was generated using Jaccard’s coefficient on the R software27. The phenotypic, and genotypic matrices were used to generate hierarchical clusters using the package “cluster” in R software27.The polymorphic information content (PIC), major allele frequency, the number of alleles, heterozygosity, and gene diversity were estimated using the R package “adegenet”The combined analysis of variance for the 15 quantitative traits studied is shown in Table −1 with a mean performance of 2.8 t ha−1. The top performing genotypes in terms of grain yield comprised TZISTR1190, TZISTR1261, CML540, CML571, and TZISTR1119, recording 5.9, 5.8, 5.6, 5.6, and 5.5 t ha−1, respectively. The least performing genotypes were CML545, 18 UK1-54 and TZISTR1162 recording 1.3, 0.9 and 0.9 t ha−1 respectively. The mean performances for all the phenotypic traits of the genotypes are summarized in Table The mean performance of the genotypes showed that days to 50% anthesis and silking (DA and DS) ranged from approximately 83 to 91 days. Anthesis and silking interval range from − 0.4 to 1.8 with a mean of 0.3. Plant height ranged from 99 to 274 cm with a mean of 179 cm. Cob count exhibited a mean of 15 and ranged between 3 and 27 per plot. Grain weight per cob ranged from 0 to 0.2 g with a mean of 0.1 g. Considerable variation was observed for grain yield ranging from 0.9 to 5.9 t haThe association among the 16 measured phenotypic traits is depicted by the heat map of Pearson correlation coefficients. The blue and red squares are negative and positive correlations, respectively  ranged between 0.03 and 0.50, with a mean of 0.40. While the polymorphic information content (PIC) range was between 0.03 and 0.38, with a mean of 0.31. The minor allele frequency (MAF) ranged from 0.01 to 0.50, with an average value of 0.34. The observed heterozygosity had the mean average of 0.67 varied between 0.59 and 0.83. Fixation index (F) ranged from − 1.1 to − 0.5 with a mean of − 0.69 Table . The resThe population structure analysis identified three subpopulations among the inbred lines based on the optimal K = 3 determined according to Evanno’s method Figs. , 3. The −1.The dendrogram based on phenotypic data showed three clusters: I, II, and III, each consisting of 81%, 9%, and 10% membership, respectively Fig. . ClusterBased on the genetic data, the dendrogram showed that Clusters I, II, and III consisted of 23%, 26%, and 51% membership, respectively Fig. . The firGenetic diversity studies are critical in identifying the individual genotypes among closely and distantly related groups for the start of new breeding initiatives. To breed novel hybrids with outstanding agronomic performance, multiple stress tolerance and high-quality yield, there is a need to understand the genetic interrelationships among the inbred lines. Hence, the rational of this study was to gain genetic relationships among South African-bred maize inbred lines, tropical and subtropical inbred lines developed across the African continent.33. The significant differences observed in days to 50% anthesis and silking (DA and DS) among genotypes are essential for crop development in drought-prone environments. Early flowering and maturity are desired characteristics because they contribute to drought escape. This is particularly important in the SSA context, adversely affected by climate change resulting in poor rainfall35.The study revealed significant genetic variation among the inbred lines using phenotypic traits and SNP markers. Analysis of variance showed highly significant differences among genotypes for grain yield and its associated component traits, indicating substantial genetic variation among the genotypes. Grain yield and its associated yield components are quantitatively inherited and are of polygenic inheritance which can enable selection between genotypes within a population36. The high number of SNP markers utilized in this study enabled a more precise estimate of the population structure. Gene diversity ranged between 0.03 and 0.5, with a mean of 0.4. Reference37 reported similar findings with the genetic diversity varying from 0.01 to 0.5. The polymorphism information content (PIC) predicts the relevance of a genetic marker for linkage analysis39. In this current study, the PIC average value was 0.31, indicating that the SNP markers employed could effectively discriminate between the genotypes. These results are in support of those reported by Refs.40. Single nucleotide polymorphisms are bi-allelic; therefore, their PIC values are often lower than SSRs; however, they offer higher genetic resolution42. The mean observed heterozygosity (Ho) of 0.67 reported in this study was considerably higher than those reported for inbred lines by Refs.44. This suggest(s) that some of the inbred lines were not homozygous. Further selfing and substantial selection is strongly advised to fix these inbred lines45. The inbreeding coefficient (F) quantifies the likelihood that two alleles at every locus within an organism are identical by descent from the two parents’ shared ancestor(s)46. In this study, the F value ranged from − 1.1 to − 0.5, with an average of − 0.69. Reference47 similarly observed a negative F mean value of − 0.29. A negative F value in a population sample indicates the presence of excess heterozygotes48.The phenotypic diversity observed in the test population was supported by the analysis of molecular variance. Genomic data is utilized in determining phylogenetic relationships in germplasm collections49. Likewise, CIMMYT has been the major source of white maize germplasm for most SSA commercial seed companies because of the prominence of white maize in the region, particularly in Southern Africa15. This presents genetic bottlenecks that limit breeding gains. Reference50 reported a wide divergence between Southern Africa sub-tropical germplasm and temperate maize lines suggesting the potential utility of temperate germplasm in expanding the genetic base of African germplasm. The combination of stress tolerance and high-yield performance can produce broad adaptation in hybrids for South Africa. The Water Efficient Maize for Africa (WEMA) program is a good example of the efficacy of synergy between the private and public sector germplasm. Under the project, Monsanto, and CIMMYT developed germplasm combining drought tolerance and optimum yield performance. The resultant hybrids have a 20% yield advantage under drought conditions and outperform most commercial hybrids in optimum environments51.The panel of selected SNPs used in this study effectively revealed the polymorphisms existing among and within the inbred line populations. The within inbred line variation accounted for the most significant proportion of the variation observed compared to among population differences. A constant germplasm exchange between IITA and CIMMYT which may explain the lack of high genetic identity and low distance genetic divergence observed between the two centres. For example, parental germplasm used to initiate the CIMMYT program for drought and low soil N stress tolerance in Kenya was sourced from the CIMMYT Southern Africa regional centre in Zimbabwe and early maturing populations from the International Institute of Tropical Agriculture (IITA)37 who reported the existence of 3 subpopulation among 94 early maturing tropical maize inbred lines using SNP markers. Similarly, the dendrogram grouped the genotypes into three primary clusters showing partial existence of origin as a source of diversity. In genetic diversity studies, reports of the grouping of genotypes according to their geographic origins are common47. However, the clustering of most inbred lines was not based on ancestry suggesting that maize inbred lines derived from the same populations do not always have the same selection. This also indicates that the study panel consisted of unique inbred lines. The UKZN inbred lines were bred for performance in high-potential environments while the CGIAR material was developed for adaptation in stress environments.The SNP analysis revealed the existence of three subpopulations (K = 3). These results are in agreement with those reported by Ref.The results of this study revealed that the maize inbred lines displayed phenotypic variation supported by genetic diversity, which will enable the selection and breeding of stress-tolerant maize hybrids. The genotypes exhibited highly significant variation in key agronomic traits such as DA, DS, PC, PH, and GY. High genetic distances between paired inbred lines demonstrated the distinctiveness of the evaluated genotypes and the availability of substantial genetic variation that could be utilized in the maize breeding program. The inbred lines were partially classified into three heterotic groups based on source background. Superior hybrids can be developed from inbred lines exhibiting the most comprehensive genetic distance within populations identified. Combining ability studies are recommended to confirm the agronomic performance and breeding values of the most divergent parental lines.Supplementary Tables.


MC1R) gene, resulting in a new hypothesis on the function of the encoded protein in regulating the cascade signals and leading to the production of different pigmentation. This result showed that local untapped breeds can be the reservoir of interesting genetic variants useful to better understanding underlying basic biological functions in mammals.Autochthonous pig breeds are important genetic resources, well adapted to local climatic conditions, environments, and traditional production systems, where they are associated with local and niche markets. The Greek Black Pig breed is the only local pig breed recognized in Greece. In this study, we started a population genetic characterization of this breed by analyzing a few gene markers associated with morphological and production traits and that usually differentiate wild boars from domestic breeds. The obtained results showed that, in the past, this breed experienced genetic admixture from two sources, wild boars and cosmopolitan breeds. On the one hand, this situation might raise some concerns for the genetic integrity of this animal genetic resource. On the other hand, this might contribute to within-population genetic variability reducing the problem of inbreeding of the small breed population. In this breed, we also identified a novel allele in the melanocortin 1 receptor  and compared these data with population genetic information available in other Mediterranean and Western Balkan pig breeds and wild boars. None of the investigated gene markers were fixed for one allele, suggesting that, in the past, this breed experienced introgression from wild boars and admixture from cosmopolitan pig breeds, enriching the breed genetic pool that should be further investigated to design appropriate conservation genetic strategies. We identified a new MC1R allele, containing two missense mutations already reported in two other independent alleles, but here present in the same haplotype. This allele might be useful to disclose biological information that can lead to better understanding the cascade transmission of signals to produce melanin pigments. This study demonstrated that autochthonous genetic resources can be an interesting reservoir of unexpected genetic variants.The Greek Black Pig (or Greek Pig) is the only recognized autochthonous pig breed raised in Greece, usually in extensive or semi-extensive production systems. According to its name, the characteristic breed coat color is solid black. In this study, with the aim to start a systematic genetic characterization of the Greek Black Pig breed, we investigated polymorphisms in major genes well known to affect exterior and production traits ( Sus scrofa, artificial directional selection in pigs has been the main driving force that substantially shaped the domestic genetic pools and differentiated them from the original European and Asian wild boar genetic background  [MC1R haplotype carries the same nucleotide of the D2E (MC1R*3) allele, that means it carries nucleotide C (MC1R pig sequence has been deposited in the European Nucleotide Archive (ENA) with accession number PRJEB59417. According to the two other nomenclatures of the allele series at this gene [MC1R*10 and 0302 . The reticulate position of this new MC1R sequence in the phylogenetic tree is due to the uncertainty of its origin between two equally plausible hypotheses: a de novo but recurrent mutation  that occurred in an D2E haplotype sequence; a recombination between an D2E allele and an e allele, which constituted a new haplotype carrying two mutations (p.N124D and p.A243T) of the two original alleles  allele, which is common in white breeds and populations such as Large White and Landrace. The KIT allele associated with the belted phenotype (g.8:43597545T) was detected, in heterozygous state, only in two other pigs. All these pigs had a typical solid black coat color. All remaining pigs had the wild-type genotypes in the investigated KIT gene markers. The markers were in the Hardy–Weinberg equilibrium.Only one pig carried the duplication of the NR6A1 and VRTN, were not fixed for the domestic (or mutated) alleles, with some differences in allele frequencies. In the NR6A1 gene, the mutated g.265347265T allele was the prominent allele (83.9%) in the Black Greek Pig breed. The wild-type C allele was present in 11 pigs. This polymorphic site was not in the Hardy–Weinberg equilibrium due to the lack of heterozygous animals . The analyzed polymorphism in VRTN (the SINE insertion or deletion) showed a balanced allele frequency with the mutated Q allele (with the insertion) that had a frequency of 44.9% and the wild-type allele  that had a frequency of 55.1%. Again, at this polymorphic site, the investigated population was not in the Hardy–Weinberg equilibrium (p = 0.04) due to an excess of heterozygous animals .The two investigated genes affecting vertebral number and teat number, IGF2 gene marker (frequency: 0.856). The G allele, considered the ancestral allele [A prevalence of the g.3072A allele was detected for the l allele ,41, was Allele frequencies for the same markers analyzed above were obtained for other 22 pig breeds and one wild boar population using whole-genome sequencing data from DNA pools . Table SIn both analyses, the Black Greek Pig breed was not closely positioned or clustered together with the wild boar population, even if wild-type alleles at all markers were also reported in this domesticated population.+E in the MC1R gene and of the wild-type allele of the NR6A1 gene in several pig breeds, including the Greek Black Pig breed. Gene flow might be also derived from other cosmopolitan breeds, again as also evidenced by the presence of several domestic alleles in the MC1R and KIT genes. This signature of reverse gene flow is not only evident in the Greek Black Pig breed but also in several other autochthonous breeds , that is associated with the dominant black coat color and that is expected to produce a constitutive activation of the receptor with a downstream signal transmission, mediated by the cAMP pathway, that leads to eumelanin production in the melanocytes [e allele at the Extension locus in pig [OCA2), encoding the homolog of the mouse p (pink-eyed dilution) gene, located on SSC15, might play a relevant role in affecting coat color in Duroc pigs [The most interesting results that we obtained was the identification of a novel  lineage ,20,22; M GenBank ,37,38,39e effect ,36. Thisanocytes ,48; p.A2s in pig . Accordis in pig . More deroc pigs .MC1R allele was originated. According to the obtained MC1R*10 nucleotide sequence, two equal probable hypotheses, as mentioned above, can be constructed. The first hypothesis assumes that a de novo and recurrent missense mutation (c.727G > A) causing the amino acid substitution p.A243T occurred in the MC1R*3 sequence, producing a new haplotype. This hypothesis might be supported by the fact that in another informative position (c.491C > T), allele MC1R*10 had the same nucleotide of allele MC1R*3. However, as the c.727G > A is downstream from the other two informative nucleotide substitutions that distinguish ED2 from e, it is not possible to completely exclude that MC1R*10 could have been produced by a rare recombination event involving both D2E and e right in the middle between the c.491C > T and the c.727G > A polymorphic sites to obtain the new haplotype that contained both missense mutations in the same allelic form. To lead these hypotheses in one or the other direction, other informative nucleotide sites should be obtained by sequencing downstream flanking regions and reconstruct a larger haplotype structure of the new MC1R*10 allele.It remains to infer the way in which this novel MC1R*10 allele in the breed population can also suggest that this novel allele probably emerged quite recently in the Greek Black Pig breed, through a new genetic event that could have good chances to be occurred in this breed. In this context, it is interesting to note that alleles D2E and e  allele, are the two most frequent alleles in the Greek Black Pig breed. Therefore, a recombination event between these two alleles  would be possible in this breed. It also seems more plausible that this allele is a new allele that emerged in this breed as no other studies, as far as we know, described this form in any other pig populations .The low frequency of the Q allele in the VRTN gene, which has been associated with a larger number of vertebrae and teats than the wild-type allele in many pig breeds  h,50 hQ ale allele , is the The investigation of DNA markers in untapped pig breeds can disclose the genetic history of these autochthonous animal genetic resources, usually well adapted to the local production systems. The quite high genetic variability detected in the five genes investigated in the Greek Black Pig breed indicated that admixture with both wild boar populations and other pig breeds occurred quite frequently (probably before the beginning of the conservation program), suggesting that it would be important to establish a well-defined plan able to maintain the genetic integrity of this pig breed. Other studies, including the genotyping of additional candidate gene markers and the analysis of thousands of SNPs covering the whole genome of the animals are needed to obtain a more detailed and improved picture of the two directions of gene flow (from wild boars and from cosmopolitan breeds) that might have shaped the current population genomic structure of the Greek Black Pig breed.MC1R allele in this breed, not previously reported in any other pig populations, indicates that local animal genetic resources can be the reservoir of interesting genetic variants that can be useful to understand basic biological mechanisms underlying relevant processes and phenotypic traits, with potential applications in other fields, including the use of unique gene markers for local breed product authentication.The identification of a novel


Genetic testing can facilitate the diagnosis and subsequent therapeutic management of rare diseases. However, there is a lack of data on the use of genetic testing for rare diseases. This study aims to describe the utilization rate and troubles encountered by clinicians in treating rare diseases with genetic testing.A cross-sectional electronic questionnaire survey was conducted between June and October 2022 among the medical staff from the hospitals covering all provinces, municipalities, and autonomous regions of China. The survey on genetic testing focused on whether genetic testing was used in the diagnosis and treatment of rare diseases, the specific methods of genetic testing, and the problems encountered when using genetic testing.A total of 20,132 physicians who had treated rare diseases were included, of whom 35.5% were from the central region, 36.7% were from the eastern region, and 27.8% were from the western region. The total utilization rate of genetic testing for rare diseases was 76.0% (95%CI: 75.4–76.6). The use of genetic testing was highest in the Eastern region (79.2% [95% CI: 78.3–80.1]), followed by the Central (75.9% [95% CI: 74.9–76.9]) and Western regions (71.9% [95% CI: 70.7–73.1]). More than 90% (94.1% [95%CI: 93.4–94.8]) of pediatricians had used genetic testing to treat rare diseases, with surgeons having the lowest use of genetic testing (58.3% [95% CI: 56.6–60.0]). Physicians’ departments and education levels affect the use of genetic testing. Most physicians have used a variety of genetic tests in the management of rare diseases, the most popular methods were “Whole-exome sequencing (Proband)” and “Whole-exome sequencing (families of three or more)”. Doctors have encountered many problems with the use of genetic testing in the diagnosis and treatment of rare diseases, among which the high price was the main concern of medical workers.Three-quarters of physicians used genetic testing in rare disease practice, and there were regional differences in the use of genetic testing. Recognition of the utilization of genetic testing can help identify patterns of resource utilization in different regions and provide a more comprehensive picture of the epidemiology of rare diseases in jurisdictions.The online version contains supplementary material available at 10.1186/s13023-023-02847-7. Rare diseases are a general term for a group of diseases with extremely low incidence and prevalence, and there are more than 7,000 known rare diseases  in the wAccurate genetic testing facilitates not only the diagnosis of genetic disorders but also the subsequent treatment and management of rare diseases , 8. A prClarifying the use of genetic testing in rare diseases can help rationalize the allocation of healthcare resources and improve the diagnosis and treatment of rare diseases . HoweverTherefore, this study surveyed whether there were differences in the utilization rate and troubles encountered by doctors in the diagnosis and treatment of rare diseases in different regions with different economic levels. The aim of this study is to describe the use of genetic testing for rare diseases among clinicians and to provide a basis for the rational provision and allocation of medical resources for rare diseases in the future.This study is a national cross-sectional survey. From June and October 2022, a baseline electronic survey was conducted among medical staff in the China collaboration network . The colA total of 21,323 individuals who had been treating rare diseases were included in this study. Six were excluded due to missing key information, resulting in the inclusion of 21,317 healthcare workers. This study aimed to describe the use and trouble of genetic testing among physicians in the diagnosis of rare diseases, so 1,185 individuals from functional or other non-clinical departments were excluded, resulting in the inclusion of 20,132 physicians for analysis.The structure and items of the questionnaire designing using literature review and expert consultation. The distribution of questionnaires mainly relied on the China Alliance for Rare Diseases. All clinicians could fill in the electronic questionnaire by scanning the two-dimensional code on the official website via their mobile phones or computers, and the unique identifier of each questionnaire was confirmed by the hospital department and the name abbreviation of the physician. And all participating physicians were deidentified to preserve the anonymity of the questionnaire. Any questions that clinicians may have about the questionnaire could be answered by consulting the researcher online.The contents of the survey included the province, hospital, education, title, length of service, and department in which they worked. This study divided the geographical area into the eastern, central, and western regions according to the provinces where the doctors worked; the education levels were divided into bachelor’s degree or below, master’s degree and doctor’s degree; the professional titles were divided into the resident physician, attending physician, associate chief physician, and chief physician; the department was classified as pediatrics, surgical, non-surgical, diagnosis-related; and the length of service was classified as ≤ 5 years, 6–10 years, 11–20 years, 21–30 years and > 30 years.The questionnaire on the diagnosis and treatment of rare diseases was mainly concerned with whether there had been any previous experience in diagnosing or treating rare diseases. The definition of rare diseases was mainly derived from the First List of Rare Diseases  and rareThe section on genetic testing in the questionnaire included whether genetic testing had been used in the diagnosis and treatment of rare diseases, the specific method of genetic testing ”, “Whole-exome sequencing (families of three or more)”, etc.) and problems with the use of genetic testing .All results of this study were analyzed according to subgroups in the central, eastern, and western regions of China. Quantitative data were described by mean and standard deviation, and differences between groups were analyzed by analysis of variance. The qualitative data were described by frequency and percentage, and the differences between groups were compared by the chi-square test. As the main objective of this study was to describe the use of genetic testing by physicians in the diagnosis and treatment of rare diseases, the use of genetic testing was described using 95% confidence intervals (CI). Physician characteristics regarding influences on the use of genetic testing were analyzed using multifactorial logistic regression, with results presented using odds ratios (ORs) and 95% CI. All data were cleaned and analyzed with the use of SAS 9.4 software, and two-sided validation was performed by two people. P < 0.05 was considered statistically significant.A total of 20,132 physicians who had treated rare diseases were included, of whom 35.5% were from the central region, 36.7% were from the eastern region, and 27.8% were from the western region. Among the interviewed physicians, 21.9% came from pediatrics, 16.1% from surgical departments, 59.4% from non-surgical departments, and 2.6% from diagnosis-related departments. The junior title accounted for 14.3%, the intermediate title for 35.5%, the associate senior title for 28.3%, and the senior title for 21.9%. Nearly 70% of the respondents had graduate education or above, and more than 80% had worked for more than 5 years. There were significant differences (P < 0.001) in the departments, professional titles, education levels, and length of service of the interviewed doctors in different regions (Table trend =0.033), but the trend was not statistically significant in the central and eastern regions. The higher the education level of doctors, the higher the utilization rate of genetic testing in the treatment of rare diseases (Ptrend <0.001), which was consistent across different regions. The use of genetic testing in the diagnosis of rare diseases did not increase with the years of the doctors’ experience (Ptrend =0.537) . The use of genetic testing was highest in the Eastern region (79.2% [95% CI: 78.3–80.1]), followed by the Central (75.9% [95% CI: 74.9–76.9]) and Western regions (71.9% [95% CI: 70.7–73.1]) ” and “Whole-exome sequencing (families of three or more) testing techniques. “Chromosomal Microarray analysis” was used by relatively few physicians Fig. . There wIn terms of genetic testing, the majority of doctors chose " too expensive” 73.6%), followed by “the report is too complicated to confirm the results” (44.7%) and " too many genetic companies on the market to know how to choose” (44.5%). Nearly 30% of the doctors were " unsure about the results of genetic testing”, “did not understand the scope of genetic testing” and “did not know how to advise patients on genetic aspects” ” and “Whole-exome sequencing (families of three or more)”. Whole-exome sequencing is the high-throughput sequencing of only 1% of the genome using sequence capture technology, detecting approximately 20,000 exons and 10 bp of introns upstream and downstream of the exons, and is the best option for patients with multiple symptoms, difficult diagnoses, and suspected genetic factors . ComparePhysicians have encountered many problems with the use of genetic testing in the diagnosis and treatment of rare diseases, among which the high price was the main concern of medical workers. Although advances in technology have led to a reduction in the price of genetic tests, they are still expensive for the majority of patients compared to conventional diagnostics. Especially in most developing countries, genetic testing is not included in routine health insurance , and patThe main advantages of this study are that the sample size is relatively large, the survey scope is wide, and the findings cover different regions of China with different economic levels, which can provide relevant data support for the formulation of health policies. This study also has the following limitations. First of all, this study was only conducted in China, and the national policies in other countries should be considered when generalizing the results to other settings. Second, whether a doctor uses genetic testing technology in the diagnosis and treatment of rare diseases may be related to whether a rare disease is related to genetic factors. However, the field of rare diseases progresses slowly compared with common diseases, and it is not yet possible to determine that a particular rare disease is not related to genetic factors. And more and more diseases are finding breakthroughs at the genetic level. In addition, the experience with genetic testing for rare diseases is self-reported and cannot be verified. However, this is an inherent flaw of questionnaires. Moreover, our study was conducted with doctors, whose use of genetic testing is not driven by profit compared to other parties.In conclusion, our study showed that three-quarters of Chinese clinicians used genetic testing in the diagnosis and treatment of rare diseases, and there were regional differences in the utilization of genetic testing. Physicians have encountered many problems in the application of genetic testing technology in the diagnosis and treatment of rare diseases, with high prices being a major concern for medical workers. Our data contribute to identifying resource utilization patterns across different regions and providing a more comprehensive picture of the epidemiology of RDs in jurisdictions.Below is the link to the electronic supplementary material.Supplementary Material 1: Utilization rate of different genetic testing methods and problems encountered in genetic testing


Strongyloides IgG in urine by enzyme-linked immunosorbent assay (ELISA) for diagnosis of strongyloidiasis reportedly has comparable performance to conventional serum assays. Initial comparisons of urine assays using commercial ELISA kits designated for serology have shown its diagnostic potential but sub-optimal accuracy. In the present study, we optimized urine ELISA protocols based on different antigen types and evaluated their accuracies in determining the epidemiology of strongyloidiasis in Northeast Thailand. Paired urine and fecal samples of 966 individuals from the study community were collected for three consecutive days and tested for strongyloidiasis. We compared three ELISA protocols using different antigens including crude S. stercoralis antigen (Ss-ELISA), crude S. ratti antigen (Sr-ELISA) and recombinant NIE antigen (NIE-ELISA) and fecal examination by agar plate-culture (APCT) technique and formalin-ethyl acetate concentration technique (FECT). The optimized ELISA protocols using three different antigen sources yielded significantly higher prevalence rates of strongyloidiasis (58.9–65.1%) than those by fecal examination methods (19.7%). The prevalence of strongyloidiasis determined by ELISA protocols significantly increased with age  and males had higher prevalence than females . Diagnostic agreements between ELISA protocols were moderate (κ = 0.461–0.586) and the agreement between each ELISA protocol and fecal examinations were slight (κ = 0.139–0.210). The results obtained by urine ELISA protocols using three different antigens showed comparable diagnostic performances, provided further supports for the utility of urine as an alternative clinical specimen for diagnosis of strongyloidiasis.Detection of anti- Strongyloidiasis is a neglected tropical disease (NTDs), with transmission occurring globally in tropical and subtropical regions , 2. It oStrongyloides stercoralis invades the host through the skin and travels by various pathways to the small intestine of the host to reach patent infection. One unique characteristic of S. stercoralis is its autoinfection cycle whereby the parasite re-enters the enteral circulation or through the perianal skin without shedding larvae into the external environment [S. stercoralis in human stool tends to be intermittent, which leads to conventional parasitological method having low sensitivity to detect chronic asymptomatic infection [As a soil transmitted helminth, ironment , 6. Larvnfection . Examinanfection , 9. As snfection , 11.S. stercoralis third stage larvae as a homologous antigen [S. ratti or S. venezuelensis as heterologous antigens [S. ratti or S. stercoralis larval antigens are comparable in their accuracy for serodiagnosis of strongyloidiasis in endemic area in northeast Thailand [S. stercoralis antigens, particularly a 31-kDa recombinant antigen (NIE) which was derived from a S. stercoralis third stage larva cDNA library [Strongyloides IgG in serum [Several serological tests have been reported that have increased diagnostic accuracy for strongyloidiasis , 13. The antigen , 15, or antigens , 17. ELIThailand . Recombi library , have alin serum , 20, 21.S. stercoralis endemic areas.Generally, the serological methods provide greater diagnostic accuracies than coprological methods, serving as the preferred detection methods for individuals at increased risk of lethal complications from chronic strongyloidiasis and patients awaiting transplantation or in immune therapy , 6. HoweS. ratti as antigen in a nation-wide mapping of strongyloidiasis in Cambodia, was recently reported [S. stercoralis crude and recombinant protein (NIE) antigens had lower diagnostic performance than ELISA using S. ratti antigen [Strongyloides antigen as well as recombinant NIE antigens from S. stercoralis should have similar performances in diagnosis of strongyloidiasis.In a search of clinical specimens other than blood, a urine-based ELISA was shown to have similar diagnostic performance to serum IgG for immunodiagnosis of strongyloidiasis . The appreported . An asse antigen . HoweverStrongyloides NIE recombinant antigen (NIE-ELISA), crude S. stercoralis (Ss-ELISA) and crude S. ratti antigen (Sr-ELISA). The prevalence of strongyloidiasis over three consecutive days in previous study showed that the variation of prevalence by urine ELISA was low while there were significant variations by parasitological examination [In this study, we examined the community-based epidemiological patterns of strongyloidiasis regarding effects of age and sex of the local residents based on parasitological and immunological diagnoses in northeast Thailand. We also assessed and compared the detection rate and agreement of ELISA protocols using three different antigen sources i.e. the mination . CompariThe study protocol concerning the participant enrollments as well as clinical sample collection and examination were approved by the Ethics Committee of Khon Kaen University, Khon Kaen, Thailand (HE654013) in compliance with the Declaration of Helsinki. The project activity was explained and written informed consent was obtained from each participant. The study included residents of all sexes aged > 18 years. All parasite-infected participants discovered during the project operation were given specific anthelmintic treatments. All sample analyses were anonymized, and blinding was applied during sample analysis and result interpretations.S. ratti parasite antigen was approved by the Institutional Animal Ethical Committee, Khon Kaen University (IACUC-KKU-100/61). The experiment animals were handled in accordance with good animal practice according to the guidelines for the Care and Use of Laboratory Animals of the National Research Council of Thailand.The protocol for laboratory animal experiments required for production of Strongyloides sp. infection were excluded from the study. The original invitations for enrollment included 1,025 people and 966 individuals enrolled, consisted of 463 were men and 503 were women with a median age of 53 (range 18–86). Each study participant was requested to provide paired specimens of feces and urine samples for three consecutive days. Urine samples were collected in the morning and were transferred into sample tubes containing Sodium azide as preservative (0.1%) and kept in insulated ice box. Fecal samples were collected in wide-mouthed containers and kept in an insulated box at ambient temperature. Any vehicle or person having contact with the sample box and accessories was cleaned with 1% Lugol’s iodine or 70% isopropyl alcohol solution to prevent transmission of pathogens. Within the same day, urine and fecal samples were transported to the laboratory at Khon Kaen University for processing.The sample population participating in this study resided in Nong Krung Sri District (NKS), Kalasin Province, Northeast Thailand. The study area in this study was in nearby sub districts (Tambon) in NKS reported previously . This stPositive and negative control sera of strongyloidiasis kept in the Department Biobank were collected from our previous study . NegativFecal samples were examined for parasitic infections using both the agar plate-culture technique (APCT) and the formalin-ethyl acetate concentration technique (FECT). The procedure for APCT was as described elsewhere . BrieflyFor FECT, the method was described previously . BrieflyS. ratti and S. stercoralis antigens was performed as previously described [S. ratti, feces of Wistar rats previously infected with S. ratti were cultured to produce third-stage filariform larvae using a filter-paper culture method [S. stercoralis, fecal samples from S. stercoralis-positive subjects in endemic area from our previous study [S. stercoralis and S. ratti were separately pooled and stored as sediments at -20°C until required for antigen preparation.Preparation of crude escribed , 18. Fore method . In the S. ratti or S. stercoralis) was thawed and suspended in phosphate-buffered saline (PBS) pH 7.4 containing protease inhibitor mix  and then disrupted by sonication. The larval homogenate was allowed to further solubilize at 4°C overnight and then was centrifuged at 15,000 rpm for 30 minutes at 4°C. The protein concentration of the supernatant was measured using the Bradford protein assay , the solution aliquoted and stored at −20°C until used as crude S. ratti and S. stercoralis antigens in the ELISA. The NIE recombinant protein prepared as described elsewhere [The larval sediment (lsewhere  was kindS. ratti (Sr-ELISA) or S. stercoralis (Ss-ELISA) crude antigens were similar to those in our previous report [S. stercoralis NIE recombinant protein, the protocol (NIE-ELISA) used in this trial were modified from previous study [S. stercoralis-positive and normal human urine samples. The optimal conditions were subsequently used for sample analyses.The protocols for urine ELISA using s report , 24. In us study . For optS. ratti antigen (5 μg/mL), S. stercoralis antigen (1 μg/mL) or NIE antigen (0.3 μg/mL) at 4°C overnight. Ninety-six-well microtiter plates  were used. The plates were washed with washing buffer  twice before blocking by 3% skimmed milk  and 0.5% Tween-20 in PBS for about 2 hours at ambient temperature. Undiluted urine samples (100 μL/well) were added in duplicate and incubated 1 hour at 37°C. A triplicate washing was performed and then 100 μL/well of 1:4,000 dilution of horseradish peroxidase conjugate goat anti-human IgG  was added. After 1 hour at 37°C, the plates were washed 3 times with washing buffer. Subsequently, 100 μL/well of substrate solution was added and the plates incubated (Sr-ELISA and Ss-ELISA) for 1 hour at room temperature in a humidified dark box using o-phenylenediamine solution . In the case of NIE-ELISA, 100 μL/well of 3,3′,5,5′-tetramethylbenzidine solution  was used and the plates incubated for 30 minutes. The final step was to stop the reaction by adding 4N sulfuric acid (50 μL/well), then to measure the optical density (OD) of each well using an ELISA reader  at 492 nm for Sr-ELISA and Ss-ELISA and 450/620 nm for NIE-ELISA.The ELISA procedure began by coating the plates with S. stercoralis were assessed to determine the specificity of the ELISA tests. In addition to the previous reports for Ss-ELISA and Sr-ELISA [O. viverrini (n = 21), Taenia spp. (n = 4), Trichuris trichiura (n = 1), Echinostoma sp. (n = 3), Minute intestinal fluke (n = 1), and hookworms (n = 1).In order to improve the accuracy of ELISA, the OD values were used to calculate the arbitrary antibody unit in the ELISA protocol reported previously , 29, 30.Sr-ELISA , 22, a sStrongyloides IgG between in individuals with and without co-infecting O. viverrini were assessed by Mann–Whitney U test and chi-square tests. MedCalc version 11.6.1.0 software  was used to calculate the cut-off values based on ROC curves to obtain the area under the curve (AUC) value for each ELISA protocol. The cut-off values for Sr-ELISA (101 units/mL), Ss-ELISA (283 units/mL) and NIE-ELISA (428 units/mL) were used to determine the negative and positive diagnostic results. Analytical graphs were constructed using GraphPad Prism version 7.0 . All statistical tests were considered significant at p values less than 0.05.The data generated in this study were entered into an Excel spread sheet and analyzed using SPSS version 21.0 software . Data on prevalence of strongyloidiasis obtained from daily as well as cumulative test results were compared using McNemar’s chi-square tests. Chi-square tests were also used to assess the association of prevalence with age and sex of the study participants. The diagnostic performance of each ELISA protocol was calculated as detection rate. Diagnostic agreements between diagnostic tests were determined based on Cohen’s kappa value (κ) that is interpreted as follows: almost perfect agreement (0.81–1.00), substantial (0.61–0.80), moderate (0.41–0.60), fair (0.21–0.40), slight (0.00–0.20), and poor agreement (< 0.00) . The corS. stercoralis (19.7%), O. viverrini (12.6%), hookworms (2.6%), Blastocystis hominis (0.4%) and Giardia duodenalis (0.4%).Prevalence rates of parasitic infection determined by fecal examinations (APCT and FECT) in 966 individuals were as follows: S. stercoralis, daily and cumulative prevalence rates over three consecutive days determined by fecal examinations and urine ELISA are compared in For S. stercoralis infection according to fecal examinations increased significantly between examinations over three consecutive days from 11.8% to 19.7%  of S. stercoralis for diagnosis of strongyloidiasis are shown in Detection rates by the urine ELISA protocols based on antigens prepared from Comparisons between each pair of ELISA protocols yielded kappa values (κ = 0.461–0.586) in the range of moderate agreement . The agrStrongyloides IgG levels among individuals with various parasitic infections determined by fecal examination. Of those individuals positive for S. stercoralis according to fecal examination, Sr-ELISA also identified 70.7% as positive. Corresponding values for Ss-ELISA and NIE-ELISA were 65.7% and 67.1%, respectively. Of those positive for O. viverrini, urine ELISAs yielded values above the cut-off for 46.9% (NIE-ELISA) and 39.5% (for both Sr-ELISA and Ss-ELISA). In individuals with hookworm infection, 3 of 6 cases (50%) yielded values above the cut-off in NIE-ELISA and 5 of 6 cases (83.30%) by Ss-ELISA or Sr-ELISA. No positive test was seen for B. hominis (n = 4) and G. doudenalis (n = 4) by any of the 3 ELISAs.O. viverrini on levels of anti-Strongyloides IgG were analyzed and results are shown in S1 Fig.Since opisthorchiasis was the second most common infection identified in the study population, effects of co infection with Strongyloides IgG determined by each ELISA protocol in individuals with and without opisthorchiasis . The levels of anti-Strongyloides IgG in the participants with and without opisthorchiasis (mean ± standard error) by Sr-ELISA were 298.3 ± 97.4 unit/mL and 310.6 ± 33.9 unit/mL, by Ss-ELISA were 524.7 ± 114.5 unit/mL and 548.9 ± 40.4 unit/mL, and by NIE-ELISA were 749.7 ± 141.6 unit/mL and 772.7 ± 47.8 unit/mL.There were no significant differences for the level of anti-Strongyloides IgG, no differences were found between the participants with and without O. viverrini infection determined by fecal examination . In the participants with and without opisthorchiasis, the positive rates were 39.5% v.s.40.3% by Sr-ELISA, 39.5% v.s. 42.4% by Ss-ELISA and 43.2% v.s. 41.1% by NIE-ELISA.For positive rates of anti-O. viverrini were seen in most protocol in serum Ss-ELISA [Taenia sp. and hookworms for NIE ELISA.The reactivity of ELISA protocols to other parasitic infection based on previous publications , 22 and Ss-ELISA , urine SStrongyloides IgG in urine using three different preparations of Strongyloides antigen were similar. Urine ELISA yielded significantly higher prevalence rates of strongyloidiasis than those estimated by standard fecal examinations. Agreements between ELISA protocols in diagnosis for strongyloidiasis were moderate (κ = 0.461–0.586) while diagnostic agreements between each ELISA protocol and fecal examinations were slight (κ = 0.139–0.210). The Sr-ELISA and NIE-ELISA protocols had slightly better detection rate in diagnosis of strongyloidiasis than Ss-ELISA. The results that different Strongyloides antigen based ELISAs achieved similar diagnosis provided further support for the utility and reliability of urine for diagnosis of strongyloidiasis and future control program. In Thailand, currently no specific community-based control program targeting strongyloidiasis is being implemented. Specific laboratory diagnostic services for serodiagnosis i.e. parasitological method (APCT) and serodiagnosis are available in tertiary care health centers or provincial hospital settings.The age-prevalence profiles of strongyloidiasis determined by detection of The unit-based ELISA protocol similar to those reported previously from our group used in this study , 29, 30.Strongyloides antigens for serodiagnosis of strongyloidiasis in serum ELISA found that S. stercoralis and S. ratti antigens were more sensitive than the NIE antigen (in studies mostly from USA and Latin America) [S. stercoralis [S. ratti [S. stercoralis recombinant protein [Previous comparisons between different America)  or were America) . Previourcoralis , 36, S. S. ratti , 24 and  protein . We have protein .S. stercoralis and the recombinant NIE antigen exhibit considerable sensitivity for serodiagnosis [S. ratti, S. stercoralis and the recombinant protein (NIE) of S. stercoralis showed comparable results. The levels of antigen-specific IgG in urine were significantly and quantitatively related across the three forms of antigen. Moreover, the ongoing study from our group showed significant reductions of S.ratti-specific IgG in urine at 3 months post treatment (unpublished data). The evaluation of antibody level in urine by these ELISA protocols required further study.In the case of urine analysis, Sr-ELISA was used in a mass population screening in Cambodia to assess risk factors and geographical distribution of strongyloidiasis . Althougiagnosis , 24, 38,It is interesting to note that when using the standard reference fecal examination, the detection rates (88%) of Sr-ELISA were high in the present study. Previous studies in Northeast Thailand found a sensitivity of 93% and specificity of 41%  and sensStrongyloides antigens in the ELISA protocols showed no difference between co-infection with and without opisthorchiasis. The results in this study suggested that co-infection with O. viverrini has no effect on the Strongyloides-specific IgG in urine for diagnosis of strongyloidiasis. Since other intestinal or blood, and tissue nematodes i.e. Trichuris, and Ascaris and filariasis are currently not present in the study community, tests for cross reactions with these parasites have not been performed.Similar to previous reports in which urine ELISA as well as serum ELISA was used for diagnosis of strongyloidiasis, we found cross reactivity 3 ELISAs with opisthorchiasis and also hookworms. Since the study community is an endemic area for opisthorchiasis, observed positive ELISA for strongyloidiasis in parasitologically proven opisthorchiasis cases is not unexpected. Comparisons of urine IgG by three different The association between age of the participants and prevalence of strongyloidiasis observed in this study is comparable with previous reports , 39–42. There were several limitations we acknowledge in this study. First is the false negative diagnosis by urine ELISA in parasitological positive cases. Similar observations have been reported in previous serum-based diagnostic studies and were believed to be associated with low immunity  or excesS. stercoralis, S. ratti and NIE antigen for ELISA were comparable. These results strengthen the utility of urine ELISA for diagnosis of strongyloidiasis.In conclusion, the results in this study indicated that urine ELISA protocols were more sensitive than fecal examination. Concordance was observed between ELISA protocols using different sources of antigens for diagnosis of strongyloidiasis. The quantitative and qualitative diagnostic parameters by ELISA protocols using S1 FigStrongyloides IgG by Sr-ELISA (A), Ss-ELISA (B), and NIE-ELISA (C) among individuals with and without parasitologically confirmed opisthorchiasis. The gray horizontal lines represent the cutoff values. Short black lines indicate the mean with lower and upper standard error. Ov- means negative O. viverrini egg; Ov+ means positive O. viverrini egg.Distribution of anti-(TIF)Click here for additional data file.S1 TableData shown are correlation coefficients between antibody levels (IgG antibody unit/mL) from each pair of ELISA protocols according to Spearman correlation tests and p values .(DOCX)Click here for additional data file.