Giardia intestinalis, a cosmopolitan zoonotic parasite, is one of the most common causes of protozoal diarrhea in both humans and animals worldwide. Although G. intestinalis has been detected in many animals, information regarding its prevalence and genotype in Chinese racehorses is scarce. In the present study, we investigated the prevalence of G. intestinalis in racehorses and performed molecular characterization of the pathogen to assess its zoonotic potential. Two hundred and sixty-four racehorse fecal samples from six equestrian clubs located in different regions of the Sichuan province of southwestern China were examined. Nested polymerase chain reaction (PCR) analysis of the gene encoding triose-phosphate isomerase (tpi) showed the prevalence of G. intestinalis to be 8.3% (22/264), and the prevalence in different clubs varied from 3.6% to 13.5%. Three assemblages were identified in the successfully sequenced samples, including the potentially zoonotic assemblages A (n = 5) and B (n = 14), the mouse-specific assemblage G (n = 3), and a mixed A and B assemblage. Sequence analysis of tpi, glutamate dehydrogenase (gdh), and beta giardin (bg) loci revealed that the majority of sequences isolated from assemblage A were identical to the subtype AIV and assemblage B isolates showed variability among the nucleotide sequences of the subtype BIV. Using the nomenclature for the multilocus genotype (MLG) model, one each of multilocus genotypes A (MLG1) and B (MLG2) were identified, with MLG2 being a novel genotype. To the best of our knowledge, this is the first study to investigate G. intestinalis in Chinese racehorses. The presence of both animal and human assemblages of G. intestinalis in racehorses indicated that these animals might constitute a potential zoonotic risk to human beings. Giardia intestinalis  is a common flagellate intestinal parasite that can infect a wide range of animals, including humans, domestic animals, and wildlife [G. intestinalis have been described based on molecular analysis of G. intestinalis isolates from different host species [wildlife . So far, species , 3. Amon species . General species . Assembl species –8.Giardia cysts [Giardia species could cause dehydration, abdominal pain, weight loss, and mal-absorption in children or young animals; it has also been reported to cause no or mild symptoms in healthy individuals [8 cases of human giardiasis are reported worldwide, and the prevalence rates are 8–30% in developing countries [Giardiasis is transmitted mainly through ingestion of food or water contaminated with ia cysts . Domestiia cysts . Giardiaividuals . Every yountries , 12. Theountries .Giardia was first reported in South African horses [Giardia causes diarrhea in horses [G. intestinalis have been detected in horses [G. intestinalis that can infect humans or other animals. However, there has been only one study on G. intestinalis in grazing horses in Xinjiang, China, and very little is known about its prevalence in Chinese racehorses. Therefore, the present study aimed to investigate the prevalence and assemblages of G. intestinalis in Chinese racehorses to assess their potential for zoonotic transmission.n horses ; since tn horses . Althougn horses , 16, mosn horses . Assembln horses , suggestThe present study protocol was reviewed and approved by the Research Ethics Committee and the Animal Ethical Committee of the Sichuan Agricultural University. All fecal specimens were collected from animals with the permission of the club owners.Two hundred and sixty-four fecal samples were collected from racehorses of six equestrian clubs located in different regions of the Sichuan province in southwestern China, between June 2016 and March 2017 . There aG. intestinalis were determined using nested PCR amplification of the gene encoding triose-phosphate isomerase (tpi). Furthermore, tpi-positive specimens were analyzed by PCR amplification of the genes encoding beta giardin (bg) and glutamate dehydrogenase (gdh) [Genomic DNA was extracted directly from each fecal sample using the Stool DNA kit  according to the manufacturer’s instructions. The genomic DNA was stored at -20°C until polymerase chain reaction (PCR) amplification. The prevalence and assemblages of se (gdh) –20. The G. intestinalis reference nucleotide sequences from GenBank and analyzed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and Clustal X 1.83.The secondary PCR products of the expected sizes were directly sequenced by Life Technologies  using a BigDye® Terminator v3.1 cycle sequencing kit . Bidirectional sequencing was performed to confirm the accuracy of these sequences. To identify the assemblages and subtypes, the nucleotide sequences obtained in the present study were aligned with the G. intestinalis-infected horses and different clubs was analyzed using the chi-square test of the SPSS version 22.0 software . Differences were considered significant when P < 0.05.The relationship between the prevalence of tpi, MF169193-MF169196 for bg, and MF169197-MF169199 for gdh.All nucleotide sequences were submitted to the National Center for Biotechnology Information (NCBI) GenBank database under the following accession numbers: MF169200-MF169206 for G. intestinalis was 8.3% (22/264). All the tested clubs were Giardia-positive, and the infection rate ranged from 3.6% to 13.5% for the different clubs. The highest prevalence was in Club 2 , followed by Club 5 , Club 1 , Club 4 , Club 6 , and Club 3   .G. intestinalis-positive samples were infected with assemblage B, whereas 22.7% (5/22) were infected with assemblage A and 13.6% (3/22) were infected with assemblage G. Assemblage B was the most common genotype identified in all tested clubs; assemblage A was found in three of six clubs , and assemblage G was found only in Club 5  of the n Club 5 .G. intestinalis-positive specimens were detected based on three loci, tpi, bg, and gdh. Twenty-two tpi, 10 bg, and 7 gdh sequences were obtained, and four samples were sequenced for all three genes  were revealed for the tpi locus; 14 were identified as assemblage B (three different sequences), 5 as assemblage A (two different sequences), and 3 as assemblage G (two different sequences). Of the 5 assemblage A tpi nucleotide sequences, three  were identical to the sequence of KP780964 and the sub-assemblage was AIV (prairie dog from USA); the other two assemblage A (AV) nucleotide sequences (E29 and E34) were identical and showed 100% similarity (509/509 base pairs) with the reference sequence (GenBank accession number KP780973). Of the 14 assemblage B tpi nucleotide sequences, genetic polymorphisms were observed at two nucleotide sites (positions 276 and 371) using KM926534 as a reference sequence  (All 22 ee genes . Three Gsequence . In the and 277) .bg locus sequences, two were assemblage A sequences (n = 6) and one was assemblage B sequence (n = 4). The assemblage A sequences  were identical to the sequence of KM190700 (from water in Canada), and the other two amplified products (A7 and A40) had two base variations compared to the sequence of KM190700  were identified as assemblage A; these sequences were identical to a reference sequence from a cat in Brazil (EF507600). Two of the other 5 amplified products were identified assemblage B sequences, which differed by two and three bases from the reference sequence KP687770 (from a beaver in Canada) (Among the 7 sequences of the  Canada) .G. intestinalis in racehorses in China; this was determined to be 8.3%. The prevalence of G. intestinalis in horses shows a wide country-specific variation (0–35%). The prevalence of this parasite has additionally been reported in horses in Brazil (0.5%), Germany (5.4%), grazing horses of Xinjiang (1.5%) [G. intestinalis in dairy cattle was affected by climate, with significantly higher prevalence in winter than in other seasons [G. intestinalis was associated with infection of children and foals [G. intestinalis obtained in this study and that in other countries may be affected by many factors, such as the examination method, animal age, sample size, management system, timing of specimen collection, and climate.To our knowledge, this study is the first to confirm the presence of g (1.5%) , 21, 22 g (1.5%) , 23–27. ermany 5.%, grazinnd foals , 28. TheG. intestinalis occurs in a variety of hosts worldwide, data regarding molecular characterization of the species of equine origin is limited. In previous studies, G. intestinalis was detected in horses mainly by microscopy or direct immunofluorescence microscopy (DFA) [tpi, gdh, and bg genetic loci have been used to characterize and classify the genotypes/assemblages of Giardia species [G. intestinalis, and assemblages A and B are the most commonly identified assemblages [G. intestinalis might be common in China, and horses might be a source of giardiasis outbreaks.Although py (DFA) , 29–31; py (DFA) . Specifi species , 33. So emblages , 17. Assemblages –38. ThesG. intestinalis isolates were amplified at all three loci, forming two multilocus genotypes (MLG1-2). MLG1 (n = 2) was identified as assemblage A and MLG2 was identified as assemblage B. These results indicated the genetic diversity of G. intestinalis assemblages A and B in Chinese horses.In the present study, assemblage B was found to be more prevalent than assemblage A. Similar results were reported in a previous study in New York State and Western Australia . In contG. intestinalis in racehorses of the Sichuan province of southwestern China. We detected the potentially zoonotic assemblages A and B of G. intestinalis, and identified the mouse-specific assemblage G in racehorses for the first time. Multilocus sequence analysis revealed genetic diversity in assemblages A and B. These results provide basic data on the molecular characterization of the parasite in this region, and further systematic studies are required to investigate the transmission of giardiasis between humans and animals. Moreover, the presence of both animal and human assemblages of G. intestinalis in racehorses indicated that racehorses might serve as a potential source of infection for human giardiasis, and effective strategies and measures should be implemented to control its transmission from racehorses to humans.In conclusion, the present study is the first to demonstrate the prevalence  of


We report a case of spontaneous abortion associated with Zika virus infection in a pregnant woman who traveled from Spain to the Dominican Republic and developed a rash. Maternal Zika viremia persisted at least 31 days after onset of symptoms and 21 days after uterine evacuation. Evidence regarding the association of Zika virus infection and pregnancy loss (spontaneous abortions and stillbirths) has been reported recently (t) value of 33, and a urine sample was negative by real time RT-PCR  and the supernatant of the karyotype cell culture (Ct = 12). Differences in real-time PCR Ct values can be explained by active viral replication in the karyotype cell culture. We used the supernatant of the karyotype cell culture to inoculate Vero cells, where we observed a cytopathic effect. We confirmed virus isolation by subsequent infection of new Vero cells, RT-PCR analysis, and sequencing of the Zika virus envelope gene. This analysis suggested active Zika virus replication in embryonic cells. We also detected Zika virus by real time RT-PCR in fresh placental tissue samples from vacuum aspiration .Formalin-fixed paraffin-embedded placental tissues were also analyzed at the Centers for Disease Control and Prevention . Histopathological analyses of these placental tissues revealed perivillous fibrinoid deposition, focal coarse calcifications, and moderate increase of Hofbauer cells. The histological sections of the placental tissue, which were stained with hematoxylin and eosin, showed a focus of villous necrosis associated with calcifications. A small portion of embryonic membranes was visible, showing no noteworthy inflammatory infiltrate. Immunohistochemical testing on placental tissue did not show presence of Zika virus-specific immunostaining. The histological findings were not relevant to the diagnosis. No specific changes were observed, neither associated inflammation was identified, and only nonspecific mild abnormalities were present. Nevertheless, Zika virus RT-PCR assays and sequencing performed on RNA extracted from placental tissues identified the presence of Zika virus in the sample (Our investigation found evidence of Zika virus infection in tissue samples from an early pregnancy loss in a mother infected with Zika virus in the first trimester of pregnancy. Testing of tissues from vacuum aspiration and from chorionic villi sampling revealed that placenta and chorion contained Zika virus RNA. Isolation of Zika virus from the karyotype cell culture confirmed active viral replication in embryonic cells. All the tests performed suggest that the spontaneous abortion in this woman was likely associated with a symptomatic Zika virus infection occurring early in pregnancy. These findings provide further evidence of the association between Zika virus infection early in pregnancy and transplacental infection, as well as embryonic damage, leading to poor pregnancy outcomes (Details of laboratory testing for a 22-year-old pregnant woman with suspected Zika virus infection, 2016.


A new nonavalent human papillomavirus (HPV) vaccine that includes genotypes 6/11/16/18/31/33/45/52/58 has been recently approved in Spain. A previous study has shown that attributable fraction of HPV related diseases in Spain is consistent with that reported in European and global studies. The aim of the present study was to estimate the annual direct costs associated to the following HPV-related diseases: genital warts, high grade precancerous lesions and cancer of cervix, vulva, vagina, anus and penis and head and neck cancer, caused by genotypes included in the nonavalent (9vHPV) and quadrivalent vaccines (4vHPV), in Spanish men and women.Cancer registries and epidemiological studies were used to estimate the number of new annual cases of genital warts, anogenital precancerous lesions and cancer of cervix, vulva, vagina, anus, penis and head and neck, as well as the fraction attributable to HPV infection and to genotypes targeted by both vaccines in Spain. Costs per patient for each disease were obtained from the literature. In addition, 142 specialists were surveyed to estimate cost per patient of vulvar, vaginal, anal and penile precancerous lesions. The annual burden of diseases attributable to types targeted by both vaccines was estimated and compared. All results were validated by a panel of experts.In 2016, new genital warts, precancerous lesions and cancers attributable to types targeted by the 9vHPV were estimated at 49,251, 29,405 and 3381, respectively. Among them, 12,597 new precancerous lesions and 530 new cancers were related to the 5 additional types covered by the 9vHPV. Annual cost of new cases of these diseases associated to types targeted by the 4vHPV and 9vHPV were estimated at 116.7 and 150.9 million € for the Spanish National Health Service (NHS), respectively (2017 €).HPV-related diseases represent a major burden for the Spanish NHS. Annual new cases and costs related to the 5 additional types from the 9vHPV represent a significant burden compared with that associated to types included in the 4vHPV.The online version of this article (10.1186/s13027-018-0187-4) contains supplementary material, which is available to authorized users. The fact that human papillomavirus (HPV) was recognized as a necessary cause of cervical cancer led to the development of a first generation of HPV vaccines: a bivalent and a 4-valent (4vHPV), capable of preventing diseases associated to genotypes 16/18 and 6/11/16/18, respectively. In 2007, Spanish national vaccination program included HPV-immunization for all girls aged 14 years old .A HPV vaccine 9-valent (9vHPV) that includes the following genotypes: 6/11/16/18/31/33/45/52/58 has been approved by the European Medicines Agency and the Spanish Agency of Medicine and Medical Products (AEMPS). This vaccine has proved to prevent 97% of precancerous lesions of cervix, vulva and vagina caused by its 5 additional genotypes (31/33/45/52/58), while maintaining protection against the 4 genotypes targeted by the former 4vHPV [Attributable fraction of HPV related diseases to these new 5 types has been already reported at global and European level , 5. In SThe aim of the present study was to estimate the annual direct costs associated to the following HPV related diseases: genital warts, high grade precancerous lesions and cancer of cervix, vulva, vagina, anus and penis and head and neck cancer, caused by genotypes included in the 9vHPV and 4vHPV, in Spanish men and women.Incidence rate for genital warts was obtained from an epidemiological study published by Castellsague et al. in 2009 .In order to estimate incidence rates for each type of cancer, the Cancer Spanish Registry REDECAN was searched . REDECANLiterature reporting incidence rates for precancerous lesions other than cervix in Spanish population was not available. Only one study estimating the number of newly diagnosed cervical precancerous lesions in Spain was identified . In the The annual number of each type of lesion, together with the 95% confidence intervals were estimated using the latest definitive Spanish population reported by the Spanish Statistical Office (December 2016)  .Except for cervical cancer, for which a study describing HPV-genotype distribution in cervical cancer cases in Spain published in 2012 was available  . For thoFor those diseases for which no direct costs were identified through the literature review , a survey on use of resources for these type of lesions was formulated for which respondents were gynecologists, coloproctologists and urologists from all around Spain with expertise in the management of the aforementioned diseases. The survey was designed to retrieve annual use of resources in terms of general practitioner, specialists and emergency visits, days of hospitalization, diagnosis tests performed  and treatment . The survey was validated by a panel of experts including one gynecologist, one urologist, one coloproctologist and one oncologist who tested its feasibility. The survey consisted of 2 main sections. The first section included two screening questions to identify the medical specialty of the participant and the number of patients with VIN2/3, VaIN2/3, AIN2/3 and/or PIN2/3 diagnosed or treated during the last 18 months. Only those gynecologists who had treated or diagnosed a minimum of 3 VIN2/3 and/or 5 VaIN2/3, coloproctologists who had treated or diagnosed a minimum of 3 AIN2/3 and urologists who had treated or diagnosed at least 5 PIN2/3 during the previous 18 months were allowed to participate. The second section included questions about resources used for the diagnosis and management of patients with the aforementioned pathologies. For each type of lesion, only aggregated data was collected, no individual use of resources was retrieved. Questions included number of visits, length of hospital stay, emergency visits, diagnoses test and treatments . No indirect costs were included. The survey was sent by email to 180 gynecologists, 380 coloproctologists and 201 urologists from the Mebos IQVIA panel. This panel includes healthcare professionals from all medical specialties and all Spanish regions, who work in public and/or private setting. A copy of the survey is presented as Additional file In order to transform the use of resources into cost per patient for each type of lesion, the cost of each intervention, visit and procedure was searched in a Spanish costs database . All cosAnnual direct costs for each disease estimated through the surveys were validated by a multidisciplinary panel of experts.In order to estimate the burden of HPV-related diseases associated to types targeted by each vaccine, the number of new annual cases of each disease was multiplied by the fraction attributable to HPV infection and by the fraction attributable to the specific genotypes extracted from the aforementioned studies , 22, 23.Annual incidence rates and estimated number of new annual cases in 2016 in Spain for each disease are presented in Table New cases of genital warts, precancerous lesions and cancers attributable to types targeted by the 9vHPV was estimated at 49,251, 29,405 and 3381, respectively, in 2016 in Spain. Out of which, all 49,251 new genital warts were attributable to the types targeted by the 4vHPV, whereas for precancerous lesions and cancer 12,597  and 530  new cases were attributable to the 5 additional types from the 9vHPV, respectively.Studies identified through the literature review, resources considered and total direct costs for the Spanish NHS 2017 €) are reported as Additional file 7 € are rRegarding those costs associated to VIN2/3, VaIN2/3, AIN2/3 and PIN2/3 estimated through surveys to specialists, 33 and 44 gynecologists provided data on the management of 99 VIN and 230 VaIN patients, respectively, 24 coloproctologists provided data from 72 patients with AIN2/3 and 56 urologists reported data on the management of 280 patients with PIN2/3. Only aggregated data was collected.Figure The estimated cost per patient for managing VIN2/3, VaIN2/3, AIN2/3 and PIN2/3 based on the use of resources reported by surveyed specialists was 3924€, 3554€, 3985€ and 2722€, respectively.The number of cases potentially attributable to the 5 additional types and associated costs have been estimated through the information presented in Tables Overall, new annual cases of diseases attributable to types targeted by the 4vHPV and 9vHPV represent more than 116 and 150 million € for the Spanish NHS, respectively.The two type of lesions that represent the greatest burden are genital warts  and CIN2/3 . Within malignancies, cervical cancer is the tumor with the highest burden   are also related to HPV. These type of lesions are difficult to prevent through cervical screening, due to their location, but HPV vaccines have shown to be effective in its prevention; i.e. in the per protocol population, 9vVPH efficacy to prevent CIN2/3, adenocarcinoma in situ, and cervical cancer due to the 5 additional types was 96.3% (CI 79.5–99.8) .Furthermore, all costs have been transformed to 2017 € according to the CPI evolution, so that they were comparable across studies. It is worth noting these costs include pharmacological treatments whose prices don’t necessarily may have evolved in the same manner as the CPI. Nevertheless, this transformation should not entail a major bias in the cost estimation, as pharmacological treatments are expected to represent a small percentage of the total cost of managing these type of patients in comparison with the costs associated to hospitalizations, visits to specialists and surgical or laser interventions.In addition, surveys used to estimate the cost for managing VIN2/3, VaIN2/3, AIN 2/3 and PIN 2/3 also entail some limitations, for instance, the fact that costs are estimated based on the answers of a limited group of experts that were not chosen in order to ensure geographic representativeness, as well as the fact that data was collected aggregately, could constitute a bias.In spite of the aforementioned limitations, the present study highlights the significant epidemiologic and economic burden associated to HPV-related diseases for the Spanish National Health Service. In 2016, 49,251, 29,405 and 3381 cases of new genital warts, precancerous lesions and cancers, respectively, were estimated to be attributable to types targeted by the 9vHPV. Among which, 12,597  and 530 , precancerous lesions and cancers could be potentially associated to the 5 additional types from the 9vHPV.In addition, a total cost of 150.9 million € has been estimated for managing new annual cases of diseases associated to the types targeted by the 9vHPV (34.24 million € more than those associated to the 4vHPV). Except for genital warts and pharynx, the 5 additional types included in the 9vHPV are responsible for a significant fraction of HPV-related diseases in Spain. Particularly, CIN2/3 and cervical cancer are the type of lesions for which a greater contribution of these 5 types and the consequent costs associated may be expected.The epidemiologic burden reported herein is expected to be reduced in the future, as a consequence of the current national vaccination program against HPV. In 2016, vaccination coverage rate achieved through this program was estimated at 77.8% for 12-year-old girls . In addiOur results clearly show the great burden of diseases and costs related to the types included in the 9vHPV. Overall, the total annual cost of these diseases is estimated at 150.9 million €, and affect not only females, but also males, which reinforces the need for considering the primary prevention of HPV related diseases in both genders.Additional file 1:Table S1. Full list of search strategies executed on the 25th of June of 2017 in Medline, Embase and Cochrane through the OVID platform: Medline Search strategy. Table S2. Use of resource survey for specialists. Table S3. Studies estimating direct costs of genital warts, precancerous lesions and cancer of cervical and vaginal, vulvar, anal, penile and head and neck cancers in Spain. Table S4. Costs of the interventions and resources, assumptions and sources applied to transform the use of resources from surveys to specialists to cost per patient with VIN2/3, VaIN2/3, AIN2/3 and PIN2/3. (PDF 682 kb)


Giardia intestinalis, the only causative agent of human giardiasis, can infect a wide range of animals. As no information concerning the prevalence and genotyping of G. intestinalis in raccoon dogs in China is available, examination of 305 faecal samples from raccoon dogs in Jilin Province (n = 110), Heilongjiang Province (n = 40), Liaoning Province (n = 72), Hebei Province (n = 54) and Shandong Province (n = 29) was conducted to estimate the prevalence of G. intestinalis in raccoon dogs in northern China and identify their genotypes using a genetic approach.G. intestinalis-positive by nested PCR amplification of the triosephosphate isomerase (tpi) gene. The prevalence of G. intestinalis was strongly related to the region and season of sampling. All 22 samples were analysed at the tpi, the glutamate dehydrogenase (gdh) and the beta giardin (bg) gene loci, showing 13, 3, 2 subtypes, respectively. The results also demonstrated that two raccoon dogs harboured mixed infections of assemblage C and assemblage D (or mixed C/D), whereas only assemblage C was detected in the remaining 20 samples. Moreover, five new multilocus genotypes, named as MLGs C1-C5, were observed in the assemblage C in the present study.Of 305 faecal samples from farmed raccoon dogs, 22 (7.21 %) were detected G. intestinalis infection in raccoon dogs in China. DNA sequence analysis of the tpi, gdh and bg gene indicated that 13, 3, 2 subtypes were found at these loci, respectively. Furthermore, this is also the first report of five new multilocus genotypes (MLGs C1-C5) in farmed raccoon dogs, which provides baseline data for further studies of the distribution of G. duodenalis in different hosts.This is the first report of  Giardia, comprised of six known species, is a protozoan genus with veterinary and public health importance .The study population comprised of 305 raccoon dogs collected from 5 provinces in northern China, where nearly 25,100,000 raccoon dogs represented the breeding stock in 2015. According to the fact that prevalence of n = 10), Liaoning Province (n = 72), Heilongjiang Province (n = 40), Hebei Province (n = 54), and Shandong Province (n = 29) in northern China in 2015. Samples were collected three times per year by seasons  from each of the eight farms, but probably not exactly from the same animals. Each of the samples was collected into a sterile disposal latex glove immediately after its defecation onto the bolster plates, and then transported to the laboratory. Information concerning region, season, gender and age were acquired.A total of 305 faecal samples were randomly collected from farmed raccoon dogs in Jilin Province  gene. Furthermore, tpi-positive specimens were also analysed by PCR amplification of the glutamate dehydrogenase (gdh) and the beta giardin (bg) gene. The PCR amplification primers and their annealing temperatures for the three genes are listed in Table Genomic DNA was extracted from each of fecal samples using the Stool DNA kit  according to the manufacturer’s instructions and stored at -20 °C until PCR test. Moreover, distilled water controls were included in each test to prevent/minimize cross-contamination at the DNA isolation or PCR phase. tpi, gdh and bg gene sequences of G. intestinalis available in the GenBank database using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and computer program Clustal X 1.83.Positive secondary PCR products were sequenced by Sangon Biotech Company . All products were sequenced bidirectionally to confirm the accuracy of sequence. Meanwhile, genotypes that produced sequences with mutations, including single nucleotide substitutions, deletions or insertions, were confirmed by DNA sequencing of at least two PCR products. Assemblages and subtypes were identified by alignment of the nucleotide sequences with known reference G. intestinalis prevalence (у) of farmed raccoon dogs in relation to region, age, gender and season were analysed by χ2 test using SAS version 9.1 . Results were considered statistically significant at P < 0.05. Odds ratios (ORs) and their 95 % confidence intervals (95 % CIs) were also calculated.The variation in G. intestinalis-positive in the present study, with 6.00 % in females and 8.39 % in males   , a value much higher than that found in foxes in Croatia (4.5 %) [Of 305 raccoon dogs, 7.21 % 22/305) were tested  (4.5 %)  and Norw (4.5 %) . The pre (4.5 %) , 0.6 % i (4.5 %) , but sli/305 were (4.5 %) . The difG. intestinalis transmission [G. intestinalis prevalence than those from other provinces (P = 0.0198)  Table . A prevismission . This isdy Table .G. intestinalis assemblages, namely assemblages A, B, C, D and E, have been found in canids worldwide [G. intestinalis infections and determine whether mixed infections exist in the raccoon dogs examined here, we applied multilocus genotyping . However, probably due to the smaller sample size, only assemblages C and D were found based on three loci and 22 tpi, 13 bg and 11 gdh gene sequences were acquired. These results suggest that the raccoon dog population studied exhibits a lower risk for transmission of zoonotic G. intestinalis to humans in the investigation sites. The fact that only two raccoon dogs were identified with mixed infections (assemblages C and D) whereas the remaining samples were infected only with assemblage C further confirm that C and D are the most prevalent G. intestinalis assemblages in canids.A total of five orldwide –26, and G. intestinalis were characterized at the gdh, tpi and bg gene loci, and high genetic polymorphism of G. intestinalis was observed at the three loci  previously reported from a pig in China (accession no. KJ668133), each of the remaining 12 sub-assemblage sequences (C1–C3 and C5–C13) had 99 % similarity with the reference sequence of assemblage C, which have not been recorded previously. Moreover, six and ten SNPs were observed at gdh and bg loci, respectively. At gdh locus, three sub-assemblages (C1–C3) were detected, with two known sub-assemblages (C1 and C3) and one novel sub-assemblage (C3). Two  of them were identical to the known sequences of assemblage C: accession no. JN587353  and D1 (KX014789), were reported in Canis lupus familiaris in Belgium previously [Canis lupus familiaris in Belgium [Canis lupus familiaris in Belgium [G. intestinalis isolates were successfully sequenced at all three loci, forming five new multilocus genotypes (MLGs) in assemblage C, namely MLGs C1–C5  and acc Croatia ). Assemb Croatia ). Furtheeviously ; our seq Belgium ), and as Belgium ), respecent MLGs .Table 4VG. intestinalis in farmed raccoon dogs in China. This study also found G. intestinalis assemblages C and D in farmed raccoon dogs by MLG model for the first time, with 13, three and one genotypes of sub-assemblage C at the tpi gdh and bg loci, respectively. Moreover, five new MLGs (MLGs C1–C5) were found in the present study. These findings have implications for further studies of the distribution of G. intestinalis in different hosts.The present study demonstrated the existence  of


Ligularia species and the hybridization direction was ascertained by three chloroplast DNA fragments. There were two hybridization groups at the study site, and they both showed bidirectional but asymmetric hybridization. Although natural hybridization occurs in three Ligularia species, their hybrids seemed to be limited to the F1 generation and therefore species boundaries might be maintained.Natural hybridization is common in plants and results in different evolutionary consequences for hybridizing species. Here, we used three low-copy nuclear loci to test the natural hybridization hypothesis among three  Ligularia species  and four types of morphologically intermediate individuals were discovered growing together in an area subject to human disturbance. In this study, we used three low-copy nuclear loci to test the natural hybridization hypothesis and the hybridization direction was ascertained by three chloroplast DNA fragments. The results indicated there were two hybridization groups at the study site, L. cyathiceps × L. duciformis and L. duciformis × L. yunnanensis, and two types of morphologically intermediate individuals were produced by L. cyathiceps and L. duciformis, and another two types were produced by L. duciformis and L. yunnanensis, while no hybrids between L. cyathiceps and L. yunnanensis were observed. Both hybridizing groups showed bidirectional but asymmetric hybridization and the factors influencing the symmetry are discussed. Most hybrids produced by the two hybridization groups seemed to be F1 generation. Hybrids with different morphologies within the same hybridization group showed similar genetic components. The results suggest that although human disturbance may promote natural hybridization among the three Ligularia species bringing them together, hybrids are limited to F1s and therefore species boundaries might be maintained.Natural hybridization is common in plants and results in different evolutionary consequences to hybridizing species. Pre- and post-zygotic reproductive isolating barriers can impede hybridization between closely related species to maintain their species integrity. In Northwest Yunnan, three  Natural hybridization is common across plants, particularly in rapidly radiating groups , and canLigularia, a highly diversified genus belonging to Senecioneae (Asteraceae), is comprised of about 140 species distributing in Asia and Europe  region of the nuclear ribosomal DNA and chloroplast DNA (cpDNA) fragments. Moreover, studies on chemical compounds combined with nuclear ribosome ITS sequence also confirmed natural hybridization of L. nelumbifolia and L. subspicata, and of L. cymbulifera and L. tongolensis  were found growing together and four types of morphologically intermediate individuals  were discovered. According to morphological distinction, it is assumed that there are two hybridization groups, i.e. Type A and B individuals are presumed to be hybrids of L. cyathiceps and L. duciformis, while Type C and D individuals are supposed to be hybrids of L. duciformis and L. yunnanensis. In spite of frequently reported studies on natural hybridization of Ligularia in recent years, there is no report on the complicated relationships at the morphologically diverse hybrid zone described above involving three putative parents.During the field investigation in Tianchi , a place severely disturbed by farming, deforestation and tourism, three psbA–trnH, trnL–rpl32 and trnQ–5′rps16) were used to explore the relationships among L. cyathiceps, L. duciformis, L. yunnanensis and all the morphologically intermediate individuals observed in the contact zone. Our aims were to (i) identify if morphologically intermediate individuals are produced by hybridization between the three coexisting species and decouple the occurrence of two natural hybridization groups suggested by the morphologically intermediate individuals between L. cyathiceps and L. duciformis by one side and between L. duciformis and L. yunnanensis by the other side; (ii) if hybridization is confirmed, assess the direction of natural hybridization; and (iii) compare the consequences of two putative hybridization groups and their influence to three putative parental species.Natural hybrids can show various morphological characteristics, such as parental-like, intermediate or novel traits, and morphological evidence alone is inadequate in the identification of hybrids  and amplL. cyathiceps, L. duciformis and L. duciformis distribute sympatrically. Ligularia cyathiceps and Ligularia yunnanensis are two alpine species endemic to Northwest Yunnan with altitudes from 3000 m to 4000 m a.s.l. , where m a.s.l. . Ligularm a.s.l. . Ligularigularia . These t , 1.0 μL dNTPs (10 mM), 1.0 μL BSA (20 g/L), 0.2 μL Taq DNA polymerase (5 U/μL) , 0.5 μL of each primer and 12.3 μL double-distilled water. PCR was conducted in a thermocycler with the following conditions: an initial 5 min denaturation at 80 °C, followed by 30 cycles of 45 s at 94 °C, 45 s annealing at 53 °C, 50 s extension at 65 °C and a final extension for 7 min at 65 °C. All PCR products were purified by electrophoresis with a 1.2 % agarose gel and then a Pearl Gel Extraction Kit  was used. Then, they were sequenced in both directions with the amplification primers using an ABI 3730 DNA automated sequencer with the BigDye Terminator Cycle Sequencing Ready Reaction Kit .Three chloroplast intergenic spacers P > 0.5) by PAUP*4.0b10 . Multiple alignments were performed manually with Geneious Pro version 4.8.2 . For three low-copy nuclear loci, haplotype inference was implemented with PHASE in DnaSP version 5.0 , two (dA1–2) and two (yA1) haplotypes, respectively  in haplotype network analysis, in which L. cyathiceps (cluster I) and L. duciformis (cluster II) were separated by six nucleotide substitutions and L. duciformis (cluster II) and L. yunnanensis (cluster III) were separated by five nucleotide substitutions , all individuals but one (T7) showed two divergent haplotypes (cA1/dA1 and cA2/dA1) originated from L. cyathiceps and L. duciformis, and the haplotype of individual T7 was a combination of two haplotypes (cA1/UN1) found in L. cyathiceps cluster (L. duciformis and L. yunnanensis (Type C and D), all individuals but six  had combined haplotypes (dA1/yA1 and dA2/yA1) nested in clusters of L. duciformis and L. yunnanensis (L. duciformis haplotype (dA1/dA1), while the other four individuals H12, S1, S4 and S32 were homozygous for a L. yunnanensis haplotype (yA1/yA1).For the putative hybrids of  cluster . For thenanensis . IndividL. duciformis from L. yunnanensis  and L. duciformis (cluster II) were separated by six nucleotide substitutions and L. duciformis (cluster II) and L. yunnanensis (cluster III) were separated by three nucleotide substitutions , all individuals but one (F14) had two divergent haplotypes  identified from L. duciformis and L. cyathiceps clusters, respectively (L. duciformis (dB1/dB1). Haplotypes for the putative hybrids of L. duciformis and L. yunnanensis (Type C and D) showed higher polymorphism with four types of haplotype composition as follows  Eight individuals  were homozygous for a L. duciformis haplotype (dB1/dB1). (iii) Six individuals  were homozygous for one of L. yunnanensis haplotypes (yB1/yB1 and yB2/yB2). (iv) Nine individuals  showed mixed haplotypes  originated from L. yunnanensis cluster.For the putative hybrids of ectively . Individ follows : (i) TheL. cyathiceps from L. duciformis and one 2-bp indel differing L. duciformis from L. yunnanensis  and L. duciformis (cluster II) were separated by seven nucleotide substitutions and L. duciformis (cluster II) and L. yunnanensis (cluster III) were separated by 13 nucleotide substitutions , all individuals had two divergent haplotypes  identified from L. cyathiceps and L. duciformis clusters, respectively (L. duciformis and L. yunnanensis (Type C and D), all individuals but seven  possessed combined haplotypes of L. duciformis and L. yunnanensis (dD1/yD1) (L. duciformis haplotype (dD1/dD1), whereas individuals H12, H13, S1 and S32 were homozygous for the L. yunnanensis haplotype (yD1/yD1).For the putative hybrids of ectively . For thedD1/yD1) . IndividpsbA–trnH, trnL–rpl32 and trnQ–5′rps16) was 2214 bp with 27 polymorphic sites and seven indels [see]. Nine haplotypes were inferred in total, of which three (cP1–3), one (dP1) and three (yP1–3) haplotypes were from L. cyathiceps, L. duciformis and L. yunnanensis, respectively (L. cyathiceps haplotypes (cP1–3) grouped into one cluster (cluster I), whereas two L. yunnanensis haplotypes (yP2–3) and the L. duciformis haplotype (dP1) formed into another cluster (cluster II) and another L. yunnanensis haplotype (yP1) was in the third cluster (cluster III) , most (17 of 24) individuals had the same L. duciformis haplotype (dP1), and six individuals  had haplotypes consistent with L. cyathiceps  (L. cyathiceps haplotypes (cP1 and cP2). For the putative hybrids of L. duciformis and L. yunnanensis (Type C and D), most (53 of 64) individuals possessed haplotypes of L. yunnanensis (yP1) and five individuals  had haplotypes of L. duciformis (dP1) (L. yunnanensis (yP1) with one mutation step. Genotypes at three low-copy nuclear loci and combined cpDNA fragments for all the investigated individuals are listed in For the putative hybrids of and cP3) . Individis (dP1) . The othL. cyathiceps, L. duciformis and L. yunnanensis in an area of contact in Tianchi, Shangri-La, Yunnan where the three species occur together. Our results suggested that the endemic L. cyathiceps showed relatively higher haplotype diversity than widely distributing L. duciformis, indicating high genetic diversity of L. cyathiceps at the study site. In addition, L. duciformis and L. yunnanensis showed closer genetic distance in two nuclear loci (A12 and B14) and combined cpDNA data, particularly in cpDNA data where two L. yunnanensis haplotypes grouped with the L. duciformis haplotype. These observations are consistent with the morphological classification  are the major pollinators  complex  putative hybrids, thus natural hybridization between L. cyathiceps and L. duciformis is bidirectional but asymmetric, and L. duciformis is the primary maternal parent. However, for the hybridization L. duciformis × L. yunnanensis, cpDNA results suggest that L. yunnanensis is the maternal parent of most (82.81 %) putative hybrids. Two hybridization groups show distinctive asymmetry in natural hybridization and different factors may be responsible for their asymmetry.As L. duciformis and L. cyathiceps. Ligularia duciformis have larger compound corymb inflorescences than the racemose inflorescences of L. cyathiceps; therefore, it would be likely that L. duciformis is more attractive to pollinators and acts as the maternal parent to accept pollen transferred from L. cyathiceps.Differences in floral traits could drive differences in floral preferences and floral constancy of pollinators, which may affect the levels and direction of hybridization .X.G. and Y.-H.W. conceived and designed the experiments. X.G. and J.-J.Y. collected plant materials. N.-N.Z. performed the experiments, analysed the data and wrote the manuscript. X.G. and J.-J.Y. revised the manuscript. All authors read and approved the final manuscript.None declared.The following additional information is available in the online version of this article—Table S1. Variation sites of A12 locus for six haplotypes in all investigated individuals.Table S2. Variation sites and indels of B14 locus for 21 haplotypes in all investigated individuals.Table S3. Variation sites and indels of D30 locus for nine haplotypes in all investigated individuals.Table S4. Variation sites and indels of three chloroplast fragments for nine haplotypes in all investigated individuals.Table S5. Genotypes at three low-copy nuclear loci and combined cpDNA fragments for all the investigated individuals.Supporting InformationClick here for additional data file.


Bell pepper endornavirus (BPEV), Southern tomato virus (STV) and Pepper cryptic virus 2 (PCV-2). Phylogenetic analysis showed that the Dominican Republic TCSV isolate has a close relationship with other TCSV isolates and a reassortant isolate between TCSV and Groundnut ringspot virus (GRSV), all found in USA. BPEV, STV and PCV-2 isolates from Dominican Republic were close related to corresponding American isolates. The possible biological implications of these virus-mixed infections are discussed.The Tomato chlorotic spot virus (TCSV) was first reported in the 1980s, having its occurrence limited to Brazil and Argentina. Due to an apparent mild severity in the past, molecular studies concerning TCSV were neglected. However, TCSV has disseminated over the USA and Caribbean countries. In Dominican Republic TCSV has been recently reported on important cultivated crops such as pepper and beans. In this work, we provide the first complete genome of a TCSV isolate from symptomatic plants in Dominican Republic, which was obtained by high-throughput sequencing. In addition, three dsRNA viruses from different virus families were identified coinfecting these plants  Tospovirus, family Bunyaviridae , 2.5 μl 10X buffer, 0.5 μl primer forward [10 μM], 0.5 μl primer reverse [10 μM], 0.5 μl dNTPs solution [10 mM], 0.1 μl Taq DNA polymerase, 1 μl cDNA, and water up to 25 μL. The amplification program consisted of a primary denaturation at 94 °C for 2 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, and one step final extension at 72 °C for 5 min. PCR products were sent for Sanger sequencing at Macrogen Inc. (South Korea). The sequences were then compared with those deposited in the GenBank database via BLAST.Specific primers were designed to determine by RT-PCR which plants were infected with the viruses found in the deep sequencing analysis Table . The totFor phylogenetic analysis, multiple alignments were performed by MUSCLE implemented in Seaview v.4.5.4 and the phylogenetic trees were built by PhyML software –15. MaxiTomato chlorotic spot virus (TCSV) was assembled   Fig.  19].Fig.FigTomatThe sequencing also revealed RNA genomes of dsRNA viruses from three different genera, apart from the complete genome of TCSV and partial sequences of TSWV. All virus sequences matched with species previously reported infecting crops Table .Table 2VBell pepper endornavirus (BPEV-DR) isolate (Family Endornaviridae) was found and traced back in chilli pepper (C. frutescens) (GenBank accession no. KX525267). The viruses from family Endornaviridae present a dsRNA genome that range in length from about 14kbp to 17.6kbp [A contig corresponding to a complete sequence of a  17.6kbp . The gen 17.6kbp –23, 24. Southern tomato virus (STV-DR) isolate was found infecting tomato (GenBank accession no. KX525266). The STV is classified within the genus Amalgavirus , usually presenting a dsRNA genome of about 3.5 kb that encodes two overlapping ORFs [A Pepper cryptic virus 2 (PCV-2-DR) isolate was identified in sweet pepper . The PCV-2 belongs to the genus Deltapartitivirus (family Partitiviridae), presenting a bipartite dsRNA genome [A A genome . The RNAIn this paper, we present the detection of viruses in symptomatic plants from important crops in Dominican Republic by deep sequencing. Viruses from four different genera were identified in a RNA pool isolated from tomato, potato, chilli pepper, sweet pepper and long beans samples. RT-PCR reactions confirmed the presence of those viruses in each plant host. A PCV-2 isolate (PCV-2-DR) was identified in sweet pepper, coexisting with the tospovirus TSWV. A STV isolate (STV-DR) was mix-infecting tomato together with TSWV as well. A BPEV isolate (BPEV-DR) was co-infecting chilli pepper together with TCSV.Capsicum spp. species [Capsicum frutescens plants showing chlorotic and necrotic spots, mild curling on leafs and warped fruits  and Pepper mild mottle virus (PMMoV) [C. frutescens) from Dominican Republic do not seem to be associated with one factor only, since there are more than the usual chlorotic rings induced by tospoviruses in the leaves. It might be possible that the symptoms are due to the mixed infection and/or a virus-synergistic effect between BPEV and TCSV.The endornaviruses do not show significant nucleotide identity between species, which evidences host/virus co-evolution , 28. BPE species , 21, 23.Capsicum annuum in mixed or single infections, being isolated from symptomatic and asymptomatic plants [Cuncumber mosaic virus (CMV), TSWV, PVY and PMMoV. Single infection of PCV-2 has not caused any visible symptoms [PCV-2 has been basically detected in cultivars of c plants , 30. PCVsymptoms , 29–33. Tobbaco mosaic virus (TMV), PVY, TSWV, Pepino mosaic virus (PepMV), Tomato leaf curl virus (TYLCV), CMV and Tomato chlorosis virus (ToCV) [STV has been reported infecting different tomatoes cultivars worldwide , 34, 35.s (ToCV) , 34–38. s (ToCV) , 26. In Tomato chlorotic spot virus (TCSV) was first reported in tomatoes plants from Brazil in the 1980s [Capsicum species are one of the main crop product traded by Dominican Republic. As solanaceous plants, which have been important tospoviruses hosts, these crops became an important virus source.he 1980s , 40. Unthe 1980s –45. Howehe 1980s , 46–49. he 1980s . This rehe 1980s . Besideshe 1980s . Among the 1980s . AccordiThe recent Dominican Republic (DR) scenario represents an important model to study plant virus interaction and evolution in a restricted area, since multiple plant viruses infections seem to be a common feature and not the exception.Bell pepper endornavirus (BPEV), Southern tomato virus (STV) and Pepper cryptic virus 2 (PCV-2). The NGS and PCR data showed viruses that have not been described before in DR coexisting in the same host with other important plant viruses, recently reported in the country, as the tospoviruses TCSV and TSWV. The nature and the implications of such interactions are still unknown.This work provided the first complete genome of a TCSV isolate from symptomatic plants in Dominican Republic, which was obtained by high-throughput sequencing. Coinfections with three viruses from different virus families were also identified,


Sequence-specific PCR is the most common approach for virus identification in diagnostic laboratories. However, as specific PCR only detects pre-defined targets, novel virus strains or viruses not included in routine test panels will be missed. Recently, advances in high-throughput sequencing allow for virus-sequence-independent identification of entire virus populations in clinical samples, yet standardized protocols are needed to allow broad application in clinical diagnostics. Here, we describe a comprehensive sample preparation protocol for high-throughput metagenomic virus sequencing using random amplification of total nucleic acids from clinical samples.In order to optimize metagenomic sequencing for application in virus diagnostics, we tested different enrichment and amplification procedures on plasma samples spiked with RNA and DNA viruses. A protocol including filtration, nuclease digestion, and random amplification of RNA and DNA in separate reactions provided the best results, allowing reliable recovery of viral genomes and a good correlation of the relative number of sequencing reads with the virus input. We further validated our method by sequencing a multiplexed viral pathogen reagent containing a range of human viruses from different virus families. Our method proved successful in detecting the majority of the included viruses with high read numbers and compared well to other protocols in the field validated against the same reference reagent. Our sequencing protocol does work not only with plasma but also with other clinical samples such as urine and throat swabs.The workflow for virus metagenomic sequencing that we established proved successful in detecting a variety of viruses in different clinical samples. Our protocol supplements existing virus-specific detection strategies providing opportunities to identify atypical and novel viruses commonly not accounted for in routine diagnostic panels.The online version of this article (doi:10.1186/s40168-017-0317-z) contains supplementary material, which is available to authorized users. To date, sequence-specific PCR is the most common approach for virus identification and quantification in diagnostic laboratories as it is highly sensitive, rapid, and cost effective. However, specific PCR requires prior knowledge of the virus sequence, and a separate assay needs to be designed for each individual virus or virus type. Recently, high-throughput or next generation sequencing (NGS) technologies enabled metagenomic-based identification of viruses by sequencing random fragments of all genomes present in a clinical or environmental sample –3. As viDespite the numerous promising attempts to apply metagenomics to virology, direct sequencing of nucleic acids obtained from biological samples results in a high background of genetic material mainly derived from the host and bacteria hampering the detection of viruses , 23. SamVarious virus enrichment methods for clinical samples have been suggested. Commonly employed methods include filtration, ultracentrifugation, and nuclease treatment , 24–31. Here, we tested various conditions and methods for virus enrichment, nucleic acid extraction, and unbiased amplification with the goal to create a sensitive and robust workflow for metagenomic sequencing of clinical samples in virus diagnostics. We spiked several virus types into plasma from healthy donors to cover different classes of viruses: small non-enveloped DNA (Human adenovirus), large enveloped DNA (Human herpesvirus 4/EBV or Human herpesvirus 5/CMV), small non-enveloped RNA virus (Poliovirus), and enveloped RNA virus , and Human herpesvirus 5  were propagated on MRC-5 cells  obtained from the European Collection of Cell Cultures . Viruses were cultivated for 14 days, cells were sonicated for 5 s, centrifuged (5 min at 1200 rpm), and the supernatant harvested and filtered (0.45 μm). Human herpesvirus 4 (HHV-4) was propagated on B-95 cells (marmoset B-lymphoblastoid cell line) centrifuged (5 min at 1200 rpm), and the supernatant harvested and filtered (0.45 μm). Influenzavirus A/WSN/33 was propagated on A549 cells  and Influenzavirus A/H1N1/PR8 on MDCK cells  for up to 4 days, and the supernatants were centrifuged (10 min at 1200 rpm). Aliquots were stored at −80 °C.Viral loads in spiked samples and amplicon concentrations after random amplification were determined by qPCR as described previously for HHV-5 , polioviFor experiments using different ratios of virus input, virus stocks were quantified by digital PCR using the QuantStudio 3D Digital PCR System (Life Technologies/Thermo Fisher Scientific), which allows for absolute quantification without the need of a standard. Reactions were performed in a final volume of 15 μl with 7.5 μl 2× QuantStudio 3D Digital PCR Master Mix, 0.25 μM of each primer and 0.125-μM probe (same primer and probes as used in the qPCR assays described above). The template was diluted as necessary for optimal digital PCR readout. For RNA virus quantification, the reaction additionally contained 0.4 μl 40× TaqMan RT Enzyme Mix and an initial reverse transcription step for 15 min at 48 °C followed by 10 min at 96 °C. Cycling conditions were 50 cycles of 2 min at 60 °C, 30 s at 98 °C, and 60 °C for 2 min. After read-out on the QuantStudio 3D Digital PCR instrument, the raw data was imported into QuantStudio 3D AnalysisSuite Cloud Software (version 3.0.2.2) to calculate absolute copy numbers.Human clinical samples were obtained from healthy blood donors  or from diagnostic samples  and stored at −20 °C. Samples were centrifuged at 2000 rpm for 10 min , spiked with viruses to achieve quantitative PCR threshold cycles  of 22–25, which are considered as high positive samples in diagnostic tests for most viruses. Filtration was done using a 0.45-μm PES filter . Freeze-thaw cycles, if applicable, were performed by freezing the samples at −20 °C.Hilden, Germany), 130 μl 10× nuclease buffer , 30 μl PBS, and 10-μl water was added. The reaction was incubated for 1 h at 37 °C in a thermoshaker at 1400 rpm. Samples were treated with protease  for 30 min at 37 °C to remove nuclease activity.Nuclease treatment with DNase and RNase was performed as previously described  with reaQIAamp Viral RNA Mini Kit (Qiagen), PureLink Viral RNA/DNA Mini Kit (Life Technologies/Thermo Fisher Scientific), and NucliSENS EasyMAG system  were used according to the manufacturer’s instructions. Two input volumes of spiked plasma were tested, 500 and 1000 μl, and eluted into 25 μl to achieve a high sample concentration. Large starting volumes were loaded into the extraction columns in multiple steps according to the manufacturer’s instructions, if necessary.In a first protocol for unbiased nucleic acid amplification , we procFor the RNA workflow, cDNA was generated by reverse transcription with a primer containing a random octamer linked to an anchor sequence ATCGTCGTCGTAGGCTGCTCNNNNNNNN , 36, 37.The DNA workflow started at the denaturation step at 94 °C for 2 min and was performed with the same random primer as used in the RNA workflow prior to second strand synthesis. Second strand synthesis was performed using the same conditions as described for the RNA workflow. Further amplification with the anchor primer and AmpliTaq Gold (Thermo Fisher Scientific) was performed as previously described , but sephttps://github.com/ozagordi/VirMet/releases/tag/v0.3.3) [e value was reported, given the identity was higher than 75%. Reads which did not match genomes used in the cleaning step and did not match viral genomes included in the database were reported as of unknown origin.The quality and size of the anchor PCR products were assessed by capillary gel electrophoresis . PCR products were quantified with PicoGreen (Invitrogen/Thermo Fisher Scientific) and diluted to 0.2 ng/μl. DNA and RNA preparations were pooled in equal concentrations for constructing sequencing libraries with the NexteraXT protocol . Individual samples were dual indexed during the library preparation and pooled for sequencing. Libraries were sequenced on a MiSeq (Illumina) for 1 × 150 cycles with version 3 reagents and the “FASTQ only” workflow. Samples were demultiplexed using MiSeq Reporter v2.4.60. Raw sequencing reads are available from the Zenodo repository (10.5281/zenodo.814807). Reads were processed with a dedicated bioinformatic pipeline “VirMet” version 0.3.3 developed in our laboratory (/v0.3.3) . Briefly/v0.3.3)  against,http://www.sanger.ac.uk/science/tools/smalt-0, default thresholds) against the following reference genomes: Human adenovirus 7 (GenBank AY495969.1), Human poliovirus 1 Mahoney (V01149.1), and HHV-5 strain AD169 (X17403.1).Optimization of the protocol was assessed by comparing the virus amplicon concentrations after random amplification by qPCR, by evaluating the fractions of reads of different taxonomic categories after sequencing, and by counting the absolute number of reads and the fraction of the total quality filtered reads for each individual spiked virus. All sequencing experiments were performed in duplicates or triplicates. Statistical analysis was done in R version 3.3.2 using linear models . CoveragFirst, three different methods of total nucleic acid extraction were tested: QIAamp Viral RNA Mini Kit, PureLink Viral RNA/DNA Mini Kit, and the automated NucliSENS EasyMAG system. Plasma from healthy donors was spiked with different viruses  and extracted, and the concentration in the eluate determined by qPCR. EasyMAG extraction was most efficient for both RNA viruses, while virus concentrations for DNA viruses were similar to PureLink extraction. The Qiagen extraction kit led to the lowest recovery of viral genomes for all tested viruses . All viruses were spiked at a qPCR threshold cycle  in the range of 22–25. Different orders of sample processing corresponding to different pre-analytical situations were then tested using the virus-spiked plasma: the condition “same day” extraction comprised filtration, extraction, and short-term storage before further processing at −20 °C; “pre-processed” comprised filtration, storage at −80 °C, and later extraction; “archived” samples comprised storage at −20 °C, filtration, and extraction. In all three conditions, each sample was processed filtered as well as non-filtered.In all conditions, the fraction of virus reads significantly increased as a result of filtration. Same day extraction with filtration and extraction followed by freezing showed the highest enrichment of virus reads Fig. a.Fig. 1FConsidering individual viruses used for spiking, for all three viruses, significantly more reads were reported for filtered samples, both in numbers and in the fraction of total reads passing quality filtering Fig. , althougNext, we tested the effect of nuclease treatment, which takes advantage of the presence of a stable virus capsid that protects the viral genome from digestion, in metagenomic sequencing of plasma spiked with two RNA viruses (poliovirus and influenzavirus A) and two DNA viruses (adenovirus and HHV-4). Spiked plasma incubated at 4 °C without reaction buffer and nucleases served as a control.In samples that were treated with nucleases, we observed an increase in the fraction of reads passing quality filtering . Viral amplicon concentrations after the anchor PCR were higher for input strategy 2  containing 25 different human viruses from different virus families   and a DNA virus (adenovirus). First, we spiked a concentration of 50,000 copies/μl of both viruses; we then spiked ten times more and ten times less of one virus while keeping the other virus constant at 50,000 copies/μl, and vice versa for the other virus.After extraction, viral amplicons were quantified in the eluate and the ratio of spiked viruses was perfectly maintained  sequences and important for maximizing the amount of quality reads to increase sensitivity. Nuclease treatment enriched for all viruses, except HHV-5, which might, probably due to its size or envelope, not be as stable as smaller viruses. While the combination of low-speed centrifugation, filtration, and nuclease-treatment also showed the greatest increase in the proportion of viral sequences in other studies  and filtPoliovirus reads were recovered in highest numbers in our experiments, although the amount of spiked viruses was adjusted to similar levels . This might be due to the fact that poliovirus has the smallest virion and genome size among the spiked viruses, what might have facilitated its efficient enrichment and amplification. In contrast, HHV-4 virus is a large virus in respect to virion and genome size and often only single reads could be detected. It was suggested that filtration might actually have an adverse effect on large virus genomes such as herpesvirus and mimivirus . In otheSeparating the random amplification into two separate workflows for RNA and DNA was more advantageous for the DNA viruses than for the RNA viruses. This could be either a result of concomitant amplification of DNA viruses in the RNA workflow or an indication that DNA genome amplification was negatively affected during the reverse transcription and second strand synthesis in the previous combined workflow. Yet, for both the combined and separate workflow, reads were uniformly distributed across the reference genomes and we did not observe differences.In order to validate our method, we sequenced a multiplexed viral pathogen reagent that was expected to contain 25 viruses across different families, genome types, and sizes . At mostTo assess how robust our sample preparation is, we correlated different ratios of spiked viruses with the number of corresponding sequencing reads. In both virus combinations and experiments performed, there was a good correlation between the ratio of spiked viruses and the ratio of the resulting reads Fig. . Direct Finally, spiking the same amount of viruses in different samples showed that the protocol presented here could be applied not only for plasma, but also for other clinical samples such as urine and swabs. A more stringent virus enrichment protocol is needed to sequence stool samples or biopsies, as those contain a lot of bacterial or human background reads, respectively , 54.A metagenomic virus sequencing protocol, as presented here, allows diagnostic laboratories to potentially identify any virus present in clinical samples with a single analysis. Such an approach reduces the time and cost spent today on multiple tests performed for each distinct virus and allows detecting rare or novel viruses not accounted for in routine test panels. Characterization of the virome and its alterations in specific disease settings might help to better understand and manage infectious diseases. Finally, our analysis highlights the need for validation methods and standards for metagenomic sequencing approaches in clinical diagnostics.


Dear Editor,Hepatitis A virus (HAV) is highly contagious and causes liver disease that is symptomatic in more than 80% of adults besides children. The virus is widespread in all parts of the world and transmitted by the fecal-oral route . The infRural areas of Egypt have a high prevalence of HAV, reaching to 100% . Consump


AbstractDryopterisblanfordii (C.Hope) C.Chr. is a member of the Dryopteridaceae, growing in high altitude Picea or Abies forests (2900–3500 m) in China and India. Phylogenetic relationships between D.blanfordiisubsp.nigrosquamosa and closely related species of Dryopteris were investigated using a combined analysis of multiple molecular data sets . An assumption about the position of D.blanfordiisubsp.nigrosquamosa within Dryopteris was made by using the Maximum Likelihood and Bayesian Inference approach and chloroplast marker sequences of Dryopteris species from GenBank. The results demonstrated that Asian taxa D.blanfordiisubsp.nigrosquamosa and D.laeta as well as two American species D.arguta and D.marginalis belong to the same clade, all four of them being part of DryopterissectionDryopteris. Dryopteris is a large fern genus of some 225–230 species belonging to the Dryopteridaceae (Dryopterisblanfordii (C.Hope) C.Chr. grows in Picea or Abies forests at 2900–3500 m in China , Afghanistan, India, Kashmir, Nepal and Pakistan (nigrosquamosa (Ching) Fraser-Jenkins and blanfordii. Dryopterisblanfordiisubsp.nigrosquamosa is abundant in India   , China  .D.blanfordii are still unresolved. More than a quarter of a century ago, Dryopteris into several sections based on the comparison of macro- and micro-morphological traits. He established Sect. Remotae with a single species (D.blanfordii) in the Indian subcontinent and with two European species (Dryopterisremota Hayek. and D.corleyi Fraser-Jenk.). Fraser-Jenkins suggested that all species from Sect. Remotae are allopolyploids resulting from hybridization between species from different sections  (The current taxonomy and infrageneric position of ginatae) .Dryopteris was reconstructed and 13 phylogenetic clades (or major evolutionary lineages) were identified using DNA sequences of four plastid loci   and clade Lophodium (D.remota)  and a single nuclear marker pgiC were used to analyse 97 Dryopteris species. The data of these phylogenies, although valuable, were far from complete and the taxonomic position of D.blanfordiisubsp.nigrosquamosa remained unclear.In a recent study, a phylogeny including 100 species of  spacer) . Two of remota) . Similarremota) , 2012b, D.blanfordiisubsp.nigrosquamosa, adult plants from the Botanical Garden of Moscow State University were used. The parent plant of Dryopterisblanfordii(C.Hope)C.Chr.subsp.nigrosquamosa (Ching) Fraser-Jenk was collected in 2003 in Uttar Pradesh State, India, at 3000 m. Spores of the specimen were germinated under artificial conditions in the greenhouse complex of MSU Botanical Garden. Subsequently, developed sporophytes were transplanted to the outdoor section of the Botanical Garden. The adult specimens were used for DNA sampling. The voucher specimen was deposited at Herbarium MW. Reference morphological charPageBreakacters for D.blanfordiisubsp.nigrosquamosa were scored from a type specimen  and from descriptions of Dryopterisblanfordii(C.Hope)C.Chr.subsp.nigrosquamosa  of fronds using a slightly modified cpDNA extraction protocol (PE) sequences (2×300bp) with a double number of each library reads about 1.2–1.97M were made. After quality trimming by Trimmomatic  in BEAST  of 84 Dryopteris species (including D.blanfordiisubsp.nigrosquamosa) was analysed.To determine the phylogenetic position of in BEAST . A combiPageBreakmethod to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach  for heuristic search was obtained by applying the Neighbour-Joining approach . All posBI analyses, optimal models of molecular evolution for combined matrices were identified using jModelTest2 (BIC).Bayesian analyses were run for 20,000,000 generations with four MCMC chains in two independent runs. The first 2,000,000 samples from each run were discarded as burn-in. Convergence was assessed by comparing the standard deviation of split frequencies between different runs  version v1.6.0 . For ML delTest2  through D.blanfordiisubsp.nigrosquamosa and other Dryopteris species, i.e. protein-coding regions of rbcL and matK genes and intergenic spacers psbA-trnH, trnP-petG, rps4-trnS, trnL-trnF and rbcL-accD. These markers were assembled into a single data matrix consisting of 3734 total bases. The optimal model of molecular evolution for combined matrices was TPM1uf+G+I with BIC = 36592.7258. The phylogenetic tree is shown in Fig. D.blanfordiisubsp.nigrosquamosa, D.laeta, D.marginalis and D.arguta. The clades containing D.blanfordiisubsp.nigrosquamosa were well-supported (≥80% bootstrap support). Dryopterisblanfordiisubsp.nigrosquamosa is close to D.laeta (bootstrap=100/PP=100%), D.arguta (bootstrap=87/PP=99.6%) and D.marginalis (bootstrap=96/PP=100%). The results of Bayesian Inference analysis based on the combined matrix were highly congruent with the strict consensus tree from ML analysis. The clade that included D.arguta, D.marginalis, D.laeta and D.blanfordiisubsp.nigrosquamosa in the combined matrix of seven markers had the posterior probability (PP) value of 100%.Seven marker regions of the assembled cp genome were used for determining phylogenetic relationships between D.blanfordii analysed in the present work have 55–57×30–35cm fronds. The frond dissection is 2-pinnate with symmetrical pinnae and pinnules , 1-pinnate-pinnatifid to 2-pinnate-pinnatifid fronds  with very few lanceolate scales; deciduous, ovate-oblong or deltoid-ovate, 3-pinnate-pinnatifid, 25–50×15–40cm, herbaceous to thinly papyraceous laminae; pinnules with toothed margins ending in an acute apex; sori in 1 or 2 rows on each side of pinnule costa; indusia orbicular-reniform, membranaceous, margin eroded  and the D.laeta specimen is from a population located in Iwaizumi . According to some recent studies (Dryopterisgoeringiana (Kunze) Koidz. is a synonym of D.laeta, growing in Japan. Our analyses rather showed that D.goeringiana proves to be related to Dryopterisstewartii Fraser-Jenk., Dryopterislacera (Thunb.) Kunze and Dryopterissieboldii (Van Houtte ex Mett.) Kuntze D.blanfoDryopterisarguta, D.marginalis, D.laeta and D.blanfordiisubsp.nigrosquamosa differ from each other in many morphological characters (Table rs Table , but shaD.blanfordiisubsp.nigrosquamosa within the Dryopteris genus was defined using phylogenetic analyses based on chloroplast markers. Our results demonstrate that Asian species D.blanfordiisubsp.nigrosquamosa and D.laeta belong to the same clade as two North American species D.arguta and D.marginalis, with all four species being part of the DryopterissectionDryopteris.The relationship of


Puerto Rico has been heavily impacted by Zika virus, a mosquitoborne flavivirus that emerged in the Americas during 2015. Although most persons with Zika virus show no symptoms, the virus can cause neurologic and other complications, including fetal microcephaly. Local Zika virus transmission in Puerto Rico has been reported since December 2015. To prevent transfusion-associated transmission, local blood collection ceased in March 2016 but resumed in April 2016 after Zika virus screening of blood donations became available. Using data from screening of blood donations collected by the 2 largest blood centers in Puerto Rico during April 3–August 12, 2016, and assuming a 9.9-day duration of viremia, we estimated that 469,321 persons in Puerto Rico were infected during this period, for an estimated cumulative incidence of 12.9%. Results from blood donation screening during arboviral outbreaks can supplement routine clinical and surveillance data for improved targeting of prevention efforts. Aedes aegypti mosquitoes, has rapidly spread in the Americas since it emerged in the region in 2015  recommended that all US areas with active Zika virus transmission cease blood collections unless donations are screened by NAT or treated with approved pathogen-reduction technology . In this study, we used data on PVDs to estimate Zika virus incidence.For these analyses, we used data from blood donations collected by the Banco de Sangre de Servicios Mutuos  during April 3–August 12, 2016, and by the Banco de Sangre del Centro Médico de la Administración de Servicios Médicos  during April 4–July 31, 2016. These organizations collect most blood donations in Puerto Rico  by using purpose-written routines, and we used StatXact version Eleven (http://www.cytel.com) for Fisher exact test.We used US Census estimates for 2014 for population totals by stratum  were included in the analysis; 175 were excluded because of invalid data or residence outside of Puerto Rico. Included donors made 22,028 total blood donations during the study period. Of all included donors, 190 (153 BSIS and 37 ASEM) were PVDs; 21,278 had cobas Zika-nonreactive screening test results; 20,912 were first-time donors; and 14,407 (67%) were men . ReporteCombining donation data from all health regions, we found no statistically significant difference in cobas Zika test reactivity by age group (p = 0.32), but the proportion of reactivity (number of reactive donations/number of donations) significantly differed by donor sex  and by health region (p<0.001). By health region, the association between reactivity and sex was significant in only 1 .Based on the 2014 US Census Puerto Rico population estimate of 3,639,000 residents and using a mean viremia duration of 9.9 days (SD ± 3.94 days) and stratifying by health region and sex, we estimated the number of incident Zika virus infections for April 3–August 12, 2016, to be 469,321 . This number represents a Zika virus cumulative incidence of 12.9% (95% CI 11.0%–15.4%) for Puerto Rico for the 5-month period , 2. The Estimates of the total number and percentage of the population infected with Zika virus during the study period are given using mean viremia durations of 7–21 days  EstimateIn this analysis of routine blood donation screening data from the 2 largest blood collection centers in Puerto Rico, we estimated that 469,321 persons were infected with Zika virus during April–August 2016, assuming a mean viremia duration of 9.9 days. The estimated cumulative incidence of Zika virus infection for the study period was 12.9%.Among the parameters used in this estimation, mean duration of Zika virus viremia is most influential because it is inversely related to the overall estimate of the number of persons infected with Zika virus in Puerto Rico. To our knowledge, the mean duration of viremia in serum is still unknown but has been shown to range from 4–10 weeks in gravid women   might differ from the 2014 US Census population estimate of 3.6 million in our model. Second, the demographic composition of blood donors, specifically sex and age, does not match that of the general population. Data from this study show that men represented >67% of blood donors. Furthermore, data from persons <16 years of age were unavailable because of blood donor age restrictions, so the estimates we give for the whole population include an extrapolation to this age group. Although the data do not indicate a substantial difference in Zika virus incidence by age, whether the lack of data from the 0–15-year age group substantially affected our population incidence estimates is unknown. Alternatively, with regard to sex and infectivity, few data are available to support a predisposition for Zika virus infection in men; nevertheless, the statistically significant study finding of a male-to-female ratio of infectivity of 1.41 among donors suggests the need for further exploration of any possible interplay between sex and the length of viremia from Zika virus infection or Zika virus susceptibility. Third, blood donors are subjected to a medical examination and questionnaire to ascertain signs and symptoms of illness, and potential donors who are feeling ill are excluded from donation. Consequently, blood donor screening data might underestimate infection incidence because of the exclusion of symptomatic persons. Because our model adjusted for the exclusion of these persons, this limitation should not affect our analysis; however, this factor is an important consideration when blood screening data are used as a surveillance tool. Last, the duration of Zika virus viremia is unknown, and assumptions made for this model were based on limited data. Important research priorities will be to determine viremia duration through longitudinal follow-up of infected blood donors and studies of acute infection in animal models, resulting in more precise calculation of viral kinetics.In summary, the findings of this study suggest that a much larger proportion of the population in Puerto Rico was infected with Zika virus during April–August 2016 than reported through surveillance. Although Puerto Rico mandates reporting of Zika virus infections, the conveyance of arboviral surveillance data across local, state, and national levels is often delayed and can affect strategic planning and interventions. Blood donation screening data can augment clinical Zika virus surveillance data to provide real-time communication of Zika virus incidence estimates to enable better ascertainment of the extent of outbreaks and improved targeting of prevention and response efforts.Additional statistical methods for study of Zika virus infection incidence in Puerto Rico using blood donor data.


CYP2D6 has the potential to predict differences in metabolism of nortriptyline. This information could optimize pharmacotherapy. We determined the costs and effects of routine genotyping for old aged Dutch depressed inpatients.Genotyping for With a decision-tree, we modelled the first 12 weeks of nortriptyline therapy. Direct costs of genotyping, hospitalization, therapeutic drug monitoring and drugs were included. Based on genotype, patients could be correctly, sub-, or supratherapeutically dosed. Improvement from sub- or supratherapeutically dosed patients to correctly dosed patients was simulated, assuming that genotyping would prevent under- or overdosing of patients. In the base case, this improvement was assumed to be 35%. A probabilistic sensitivity analysis (PSA) was performed to determine uncertainty around the incremental cost-effectiveness ratio (ICER).In the base case analysis, costs for genotyping were assumed €200 per test with a corresponding ICER at €1 333 000 per QALY. To reach a €50 000 per QALY cut-off, genotyping costs should be decreased towards €40 per test. At genotyping test costs < €35 per test, genotyping was dominant. At test costs of €17 per test there was a 95% probability that genotyping was cost-effective at €50 000 per QALY.CYP2D6 genotyping was not cost-effective at current genotyping costs at a €50 000 per QALY threshold, however at test costs below €40, genotyping could be costs-effective. Major depressive disorder (MDD) is a disease with a significant burden of disease in the aging European population . AntidepCYP2D6) enzyme .,25.24,25CYP2D6 genotype information would only optimize dose finding of nortriptyline during the titration phase. Therefore, effects were limited to the timespan of the titration phase. According to expert opinion, this phase is usually completed within three evaluations. As such, we chose a time horizon of maximally three evaluations. To assess the time it would take for three evaluations to occur, we used guideline recommendations and retrospective (2009–2014) anonymous TDM data from the Clinical Pharmacy of the Diaconessen Hospital Meppel/Hoogeveen, the Netherlands  depends on the number and type of variant alleles which are analyzed. In the model, genetic variation in the CYP2D6*2, *3, *4, *5, alleles, and duplications of the gene were used for the clinical effects. These effects were based on the prediction of the PM and UM phenotype among a European Caucasian population. In a pharmacokinetic model it has been reported that when CYP2D6 phenotype predictions are based on genetic variations in these alleles 78.5% of PMs would have supratherapeutic nortriptyline plasma concentrations at a normal daily dose [CYP2D6 alleles As a result, a 51.1% instead of 78.5% of the patients had supratherapeutic plasma concentrations in the PM group. This corresponded to a reduction of ~35% (1- (51.1/78.5) = 0.35) of inadequately dosed patients [For economic evaluations of pharmacogenomics the analytical and clinical validity of the genetic test has to be defined . The anaily dose . In the patients . This reDepending on their genotype, PMs and UMs in the ‘genotyping’ cohort received a different start dosage compared to patients in the ‘care as usual’ cohort. Dose adaptations were based on the advices given in the Dutch guidelines: 40% of standard dose for PMs and 160% of standard dose for UMs . A detaiBetter efficacy as well as a reduction of ADRs can be expected in patients who have nortriptyline plasma concentrations within the therapeutic range as compared to outside . Both afFor the first evaluation we assumed correctly dosed patients were patients with a nortriptyline plasma concentration within the therapeutic range. Proportions of therapeutic, sub-, and supratherapeutic plasma concentrations were extracted from literature . After tCYP2D6 metabolic capacity was found to be 13% longer than the average (7.8 instead of 6.9 days) [In addition to an improved quality of life, a reduction in hospitalization days was included in the model. Hospitalization of US patients with MDD and a decreased .9 days) . Among t.9 days) . To tranCYP2D6 were based on costs for qualitative DNA amplification at €38 per amplification [CYP2D6 test. Note that test costs can be different between labs, due to different methods used for genotyping . Furthermore, we assumed genotype information would be available before or at the moment of start of nortriptyline treatment.Our evaluation was performed from a health-care insurance payers perspective and only direct medical costs were included in the model . This pefication . AssuminThe incremental cost-effectiveness ratio (ICER) was calculated by comparing costs and quality adjusted life years (QALYs) between the two cohorts. In addition break even test costs were calculated.Input variables for the deterministic sensitivity analysis are shown in Threshold analysis was performed on all variables to calculate break even test costs and maximal test costs to reach the €50 000 per QALY cut off . For furCYP2D6 IMs are false positive [Two scenario analysis were performed. First, a scenario in which the proportions of incorrectly dosed patients at the second evaluation did differ per genotype was included. For this differentiation, the same estimates as for the first evaluation were used . In the second scenario analysis dose adaptations for IMs (60% of standard dose) were included . No datapositive . This meIn the base case scenario an ICER of €1 333 000 per QALY was found. If test costs were €40, an ICER of €50 000 per QALY was found and when test costs were €35 per test, test costs equaled costs savings (i.e. break even). At lower costs, genotyping would become the dominant strategy. A summary of the model outcomes is shown in The results of the deterministic sensitivity analyses are shown in CYP2D6 test costs to obtain an ICER of €50 000 per QALY and breakeven test costs are shown in The PSA revealed that in the base case scenario (i.e. at current genotyping costs) a probability applied of <1% for genotyping to be cost-effective (€50 000 per QALY). If genotyping costs were €17 a probability of >95% genotyping to be cost-effective applied (€50 000 per QALY). Cost-effectiveness acceptability curves of the maximal The first scenario analysis indicated that when proportions of incorrectly dosed patients at the second evaluation equaled those of the first evaluation, this would increase the test costs to reach the €50 000 per QALY cut-off with €3 (i.e. €43 vs. €40 per test in the base case). This scenario analysis had no influence on break even test costs. In the scenario analysis which included dose adaptations for IM, test costs to reach the €50 000 per QALY cut-off or break even were identified at €68 or €66, respectively, due to more costs savings by reduced inpatient care of correctly dosed IMs. Although the costs in the genotyping cohort were lower in this scenario, a QALY loss among false positive IMs was introduced by genotyping. This was due to a larger utility loss when dosed sub- instead of supratherapeutic. As a result, incremental utility gain in the genotyping cohorts was reduced with 0.07 towards a remaining QALY gain of 0.05 in the genotyping cohort. Consequently, a higher ICER of €2 400 000 was found. If the proportion of falls positive IMs was decreased towards 26% (instead of 38%) an ICER, similar to the base case scenario was found.CYP2D6 among older hospitalized and depressed patients who start nortriptyline therapy. The results were mainly dependent on genotyping costs. At a price of €190 per test, we found genotyping for CYP2D6 unlikely to be cost-effective. In the PSA, a probability of <1% was found at the €50 000 per QALY threshold. However, when genotyping costs were <€35 per test, genotyping was found to be dominant. Within countries costs for CYP2D6 genotyping can be different between labs. For example, in the UK costs of £30 (€39) and £500 (€645) are reported whereas in US costs of $439 (€363) are found, respectively [CYP2C19 and CYP2C9. For such packages, even higher test costs of $914 (€1106) are reported [Our cost-effectiveness study assessed possible economic implications of genotyping for d £500 €6 are reporeported . Neverthreported ,48. BesiOur analysis has some limitations. Firstly, the model was based on some assumptions of which the most important one was the genotyping-facilitated effect in 35% of patients transferring from sub- or supra-therapeutically dosed to therapeutically dosed. Except for the pharmacokinetic modeling study, a description of such estimates was not found . It is iTMPT) in combination with azathioprine treatment it was found that clinicians continued to give a low dosing although the genotype information suggested otherwise [Similar approaches as sketched here for depressed patients can be found in other disease areas. In a trial based analysis of genotyping for thiopurine methyltransferase  was indicated [CYP2D6 polymorphisms. Similar to our model, the authors had to make substantial assumptions. In addition, they used an expert opinion to predict the clinical effects of genotyping in relation to treatment response. As a consequence, one of their main recommendations was to conduct a prospective study to assess the real clinical effects of genotyping. The same holds true for our model, however our assumptions were based on a thorough pharmacokinetic study [Matchar ndicated . They usic study . In addiCurrently some of the authors are performing a prospective trial  to evaluate outcomes of CYP2D6 genotyping among patients who will be initiated on nortriptyline therapy. Patient inclusion already started when we conducted this cost-effectiveness analysis, so we were unable to use these outcomes for the trial design. However the trial gave insight in current processes in psychiatry in The Netherlands, which was necessary to build a realistic cost-effectiveness model.CYP2D6 enzyme in older Dutch inpatients treated with nortriptyline was performed. The effects of genotyping were estimated with a decision tree. At current costs, genotyping was not cost-effective. Our finding suggests genotyping would be cost-effective (i.e. €50 000 per QALY) when genotyping costs were decreased towards €40 per test. When genotyping costs were decreased towards €35 per test, genotyping was found to be the dominant treatment option.A cost-effectiveness analysis on effects of genotyping for the S1 FigBased on retrospective (2009–2014) collected TDM data from the Clinical Pharmacy of the Diaconessen Hospital Meppel/Hoogeveen, the Netherlands, 100 out of 264 (38%) patients aged ≥60 years received TDM for the second time and 43 out of these 100 (43%) patients received TDM for a third time.(PDF)Click here for additional data file.S1 TableDose adjustments were based on guidelines from the Dutch pharmacogenetics working group.(PDF)Click here for additional data file.