Amblyomma variegatum, Hyalomma truncatum, Hyalomma marginatum rufipes, Rhipicephalus (Boophilus) microplus, Rhipicephalus evertsi evertsi and Rhipicephalus sanguineus sl. Among those, 471 ticks were randomly selected for molecular and proteomic analyses. Tick legs submitted to MALDI-TOF MS revealed a concordant morpho/molecular identification of 99.6%. The inclusion in our MALDI-TOF MS arthropod database of MS reference spectra from ethanol-preserved tick leg specimens was required to obtain reliable identification. When tested by molecular tools, 76.6%, 37.6%, 20.8% and 1.1% of the specimens tested were positive for Rickettsia spp., Coxiella burnetii, Anaplasmataceae and Borrelia spp., respectively. These results support the fact that MALDI-TOF is a reliable tool for the identification of ticks conserved in alcohol and enhances knowledge about the diversity of tick species and pathogens transmitted by ticks circulating in Mali.Ticks are considered the second vector of human and animal diseases after mosquitoes. Therefore, identification of ticks and associated pathogens is an important step in the management of these vectors. In recent years, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising method for the identification of arthropods including ticks. The objective of this study was to improve the conditions for the preparation of tick samples for their identification by MALDI-TOF MS from field-collected ethanol-stored Malian samples and to evaluate the capacity of this technology to distinguish infected and uninfected ticks. A total of 1,333 ticks were collected from mammals in three distinct sites from Mali. Morphological identification allowed classification of ticks into 6 species including  Rickettsia spp., Coxiella burnetii, Borrelia spp. and Anaplasmataceae. These results support the use of MALDI-TOF MS in entomology, tick diseases epidemiology and improve the knowledge of tick species-diversity and tick-borne pathogens circulating in Mali.Ticks are among the most important vectors and reservoirs of several animal and human pathogens such as viruses, bacteria and protozoa. However, very few studies have been done on ticks in Mali. At present, little information is available about tick species infesting livestock or human tick-borne diseases transmitted in Mali. The identification of tick species and the determination of pathogens associated are essential to evaluate epidemiology and risks of human and animal diseases: the One Health approach. Current identification methods are time consuming, expensive and laborious. Previous studies have shown that MALDI-TOF mass spectrometry analyses may allow accurate tick species identification. A recent study suggested that it was possible to identify ticks preserved in alcohol by MALDI-TOF MS. The aim of the present study was to improve tick leg sample preparation conditions for their identification by MALDI-TOF MS from Malian ethanol-preserved specimens collected in the field. This study provided 99.4% concordance between morphological and MALDI-TOF identification. The detection of microorganisms was also performed by molecular biology revealing the presence of the presence of  Ticks are bloodsucking arthropods that parasitize most of the vertebrates in the world and occasionally bite humans . About 9Amblyomma (Am.) variegatum, Rhipicephalus (Rh.) spp. and Hyalomma (Hy.) spp. are the main ticks monitored by Malian veterinarians for their effects on livestock healthcare and productivity , an, an28], The goal of the present work was to determine tick population diversity and associated pathogens from alcohol stored specimens collected on cattle in Mali by using MALDI-TOF MS and molecular approaches with specimens collected in the field. First, optimized sample preparation conditions for ticks stored in ethanol for MALDI-TOF MS analysis were established. Second, based on morphological and molecular identification of ticks, an MS reference spectra database was created and tested blindly using new tick specimens. In addition, tick-associated bacterial pathogens were screened by molecular biology on half-tick body parts and leg MS spectra from ticks mono-infected or not by bacterial pathogenic agents, and they were compared to assess the efficiency of this proteomic tool for classification of ticks according to their infectious status.Tick collection protocols were developed as of a large study under the GIRAFE programme, UMI 3189 and MSHP-MRTC HFV project. The protocols were cleared by the FMPOS IRB in 2015 and 2016. Verbal informed consent was obtained from managers of the livestock selected for tick sampling directly on mammals. The collection of ticks on domestic animals did not involve national parks or other protected areas or endangered or protected species.Ticks were collected from three localities in Mali, including Bamako, Kollé and Bougoula Hameau, in September 2015 and August 2016 . Bamako,Ticks were collected from domestic animals and cattle. Examination of all body parts was conducted from the tail to the head of the animal to detect ticks on the skin. All ticks (engorged and non-engorged) were collected manually with forceps. The ticks of the same animal were counted, pooled in the same tube and stored at room temperature in 70% v/v ethanol (ticks collected in September 2015) or frozen at -20°C (ticks collected in August 2016) until morphological, molecular and MALDI-TOF MS analyses. Ticks were transferred from MRTC  to the URMITE laboratories  for analysis.Ticks were identified morphologically to the species level firstly by a PhD student and then checked by expert tick entomologists using previously established taxonomic identification keys . Tick idEach tick was dissected with a new sterile surgical blade to remove the legs, which were used for MALDI-TOF MS analyses. The rest of the tick was longitudinally cut in two equal parts. The half part with legs cut off was immediately used for molecular biology, and the second half was stored frozen as a backup sample for any additional analysis.Each half-tick without legs was transferred to a 1.5 mL tube containing 180 μL of G2 lysis buffer and 20 μL proteinase K , and incubated at 56°C overnight. DNA extraction from the half-tick was performed with an EZ1 DNA Tissue Kit (Qiagen) according to manufacturer recommendations. The DNA from each sample was eluted with 100 μL of Tris-EDTA (TE) buffer (Qiagen) and was either immediately used or stored at -20°C until use.Rhipicepalus (Boophilus) microplus identification. DNA from Am. variegatum specimens reared at the laboratory was used as positive control. PCR products of the positive samples were purified and sequenced as described previously [http://blast.ncbi.nlm.nih.gov).Standard PCR, using an automated DNA thermal cycler amplifying a 405-base pair fragment of the mitochondrial 12S RNA gene , was useeviously . The seq™ Real-Time (Bio-Rad) with the Eurogentec Takyon qPCR kit . The qPCR reaction contained 10 μl of Takyon Master Mix , 3.5 μl sterile distilled water, 0.5 μl of each of the primers and probe and 5 μl of the DNA extract. A total of 471 samples were screened using primers and probes, targeting specific sequences of the following bacterial pathogens: Rickettsia spp., Anaplasmataceae spp., Borrelia spp., Bartonella spp. and Coxiella burnetii  were considered positive. Positive samples for Rickettsia spp. were then submitted to a qPCR system specific for detecting R. africae [R. africae but positive for Rickettsia spp. were submitted to gltA gene sequencing to determine Rickettsia species [Anaplasmataceae spp. were submitted to amplification using standard PCR and sequencing to identify the bacteria species [Rickettsia montanensis, Bartonella elizabethae, Anaplasma phagocytophilum, Coxiella burnetii and Borrelia crocidurae was used as positive controls and mix as negative controls in PCR, respectively. All these bacteria come from the strains of culture of our laboratory and Borrelia crocidurae was cultured in Barbour-Stoenner-Kelly (BSK-H) liquid medium supplemented with rabbit serum. Only samples considered as negative , were submitted to 12 S tick gene amplification to control the correctness of DNA extraction.Quantitative PCR was performed according to the manufacturer's protocol using a PCR detection system; a CFX Connectburnetii . For Bor africae . Negativ species . All tic species ,35. Tick species . PCR tes species . The DNAThree protocols were tested for tick leg sample preparation from specimens stored in 70% ethanol to determine the one exhibiting the best MS results . For eacThe homogenized tick legs were centrifuged at 2000 g for 30 seconds and 1 μL of the supernatant from each sample was carefully dropped onto the MALDI-TOF target plate as previously described . Each spProtein mass profiles were obtained using a Microflex LT MALDI-TOF mass spectrometer  using parameters previously described . The speAm. variegatum specimens from Kollé per sample preparation protocol as previously described [The reproducibility of spectra was evaluated by analyzing ten escribed . The selescribed . CCI wasBased on the correlation of morphological and molecular results of tick identification, two to five specimens per species were used to assess MS spectra reproducibility from specimens of the same tick species, and the MS spectra specificity from specimens of distinct tick species. These analyses were performed with the average spectral profiles  obtained from the four spots of each individual tick specimen using Flex analysis v.3.3 and ClinProTools 2.2 softwares . Tick species exhibiting reproducible and specific MS spectra were then included in-house MS spectra reference database. To upgrade the database, MSP reference spectra were created using spectra from at least 2 specimens per species of both genders by the automated function of the MALDI-Biotyper software v3.0. . MS spectra were created with an unbiased algorithm using information on the peak position, intensity and frequency . The speAm. variegatum and 23 Rh (B) microplus were fully engorged. Database 1 was composed of specimens of fresh or frozen arthropods  and its upgraded version, which includes the 20 MS spectra from specimens of the 6 tick species collected in Mali and alcohol-preserved (Database 2). Among the 451 ticks tested 51 thropods  [16,24,3A. variegatum infected by R. africae were compared to those of 12 uninfected specimens from the same species. Moreover, MS spectra of 36 uninfected specimens of Hy. truncatum were also compared with the spectra of 23 specimens of Hy. truncatum infected by C. burnetii.These comparative analyses to determine the infectious status of ticks were made by ClinProTools v.2.2 software . Only tick leg MS spectra from species with at least five mono-infected or pathogen-free specimens were included in this analysis. The spectra of 30 specimens of Hyalomma genus (1.55% of ticks collected) were not morphologically identified to the species level. Morphologically, six distinct tick species belonging to three genera were identified among ticks collected in September 2015  was the overall predominant tick species collected from different sites, followed by two species of the Hyalomma genus, Hy. truncatum  and Hy. m. rufipes . The three other tick species, Rh. (Bo.) microplus, Rh. e. evertsi and Rh. sanguineus sensus lato, represented less than 3.10% (n = 38). The five Rh. sanguineus sl [Rh. sanguineus sl. Three hundred sixteen of the 1,222 ticks collected from three sites in 2015 and 111 ticks in 2016 had specimens of six species randomly selected for molecular and proteomic analyses  microplus, were randomly selected for molecular analysis. A GenBank query revealed that 12S gene sequences were available for the 6 tick species. BLAST analysis indicated high identity  of 12S rRNA gene sequences among specimens classified per species according to morphological identification (Rh (B) microplus had high sequence identity with their respective homolog species available in GenBank  (ompA fragment in the remaining ticks positive for Rickettsia spp. and negative for R. africae (n = 26) was used for identification of these Rickettsia spp. R. aeschlimannii and R. mongolitimonae were detected in 24 and 2 tick specimens, respectively  of the specimens were positive , 2 ticks were positive for Ehrlichia sp. urmitei TCI148 (GenBank ACCN KT 364334.1) and 1 tick for Ehrlichia sp. rustica TCI141 (GenBank ACCN KT 364330.1). A. marginale was detected in 3 ticks and A. sp. ivoriensis TCI50 (GenBank ACCN KT 364336.1) in 1 specimen (Borrelia spp. was detected in 1.1% (3/274) of ticks by qPCR. However, all standard PCR for determination of Borrelia species failed. No Bartonella spp. was detected in the ticks tested.Among the ticks tested, 41.8% (197/471) were negative for the six bacteria tested, 37.4% (176/471) were positive for one bacterium and 20.8% (98/471) were found co-infected by two or three of the screened bacteria. Among the 274 specimens found positive for at least one bacteria tested, 76.6% (210/274) were infected by 184/210) . The ampectively . Screenipositive . Fifty-sspecimen . BorreliAm. variegatum) were used. The first comparison concerned the “de-alcoholization” and “dry” protocols  with the “dry” and “direct” methods was performed . The besrotocols . The visrotocols . This rerotocols . Thus, t genders . The com genders , could n genders  for the  genders . These rRhipicephalus genus were not clustered in the same part of the dendrogram. The profile of the spectra of specimens preserved in alcohol was different from those of fresh specimens of the same species; this difference was also observed between manual sample homogenization and automated sample crushing using the TissueLyser apparatus.Twenty ticks, including several specimens per species coming from distinct localities, were identified by sequencing 12S tick gene. Their non-infected status was also controlled for the microorganisms tested in the present work by q PCR. These specimens were selected for evaluating intra-species reproducibility and inter-species specificity of MS spectra. Comparison of the MS spectra with Flex analysis software indicated reproducibility of the MS profiles between tick specimens from the same species . MoreoveAm. variegatum, 64 Hy. truncatum, 16 Hy. m. rufipes, 26 Rh. (Bo.) microplus, 7 Rh. e. evertsi and 5 Rh. sanguineus sl were queried against the DB 1 spectra database. The blind test against DB 1 revealed correct identification for some specimens of Hy. truncatum (n = 5) and Hy. m. rufipes (n = 5), with LSVs > 1.8 (Am. variegatum and 23 Rh (Bo.) microplus were fully engorged.To assess the efficacy of the in-house MS reference spectra database, named database 1 (DB 1) to correctly identify tick specimens preserved in alcohol, half of the MS spectra from ticks included in the present study were randomly selected. Then, MS spectra from 178 specimens including 60 Vs > 1.8 . For theVs > 1.8 . ThereafRh (Bo) microplus, Rh. e. evertsi and Rh. sanguineus sl stored in alcohol, with LSVs ranging from 1.89 to 2.71   showed 96.7% (325/340) concordance between morphological identification and MALDI-TOF MS identification. The percentage of concordant identification with morphology was 100% for  to 2.71 . A total to 2.71 . The rem449/451) .Am. variegatum uninfected and 12 infected by R. africae using the gel view tool and Principal Component Analysis by ClinProTools software (Bruker), revealed no differences between the two groups  microplus and Rh. e. evertsi that were collected from cattle and Rh. sanguineus sl from dogs. Rh. e. evertsi was found only in Bamako, while all other species of ticks were found on cattle in the three locations. In support of these morphological identification results, several studies have reported the presence of these tick species in Mali, except for Rh. (Bo) microplus [Rh. (Bo) microplus, which is a southeast Asian tick, was introduced in the southeast of Africa  by cattle from Madagascar [Rh. (Bo) microplus has only been found in three other countries of West Africa  [Rh (B) microplus represent 70% and 63.2% of ticks in Burkina Faso and Benin and Ivory Coast respectively [Am. variegatum was the most prevalent species in the three sites of the present study [In this study, the morphological identification of ticks revealed the presence of six species, including icroplus –9. Rh. (dagascar . The prena Faso) . Biguezoectively , 55. Ournt study .Am. variegatum, Hy. m. rufipes, Rh. (Bo) microplus, Rh. e. evertsi and Rh. sanguineus sl were 99–100% with sequences of the same species available in GenBank. Interestingly, lower sequence identities (96–97%) of Hy. truncatum compared to the corresponding reference sequence in GenBank were observed. It could be hypothesized that the sequence differences could correspond to genetic variation within ticks of the same species adapted to different geographic regions of a country or countries, as previously described [Hy. truncatum collected in Mali and that available on GenBank tick collected in Zimbabwe [To confirm the morphological identification of tick specimens that were used for creating the MALDI-TOF MS database, sequencing of the 12S rRNA gene was performed. The 12S rRNA gene was chosen to validate identification because this gene is known as a reliable tool for molecular identification of ixodid ticks . The covescribed . The difZimbabwe  could exZimbabwe .Rickettsia spp. was detected in 76.6% of infected ticks collected from cattle, among which R. africae was found in 87.6% (184/210). R. africae was detected in 92.2% of Am. variegatum, a cattle tick found throughout sub-Saharan Africa. Such high prevalence of R. africae in Am. variegatum has already been reported [R. africae was also detected in Rhipicephalus spp. and Hyalomma spp., respectively 7.9% and 9.2%. Other recent studies have detected R. africae in other tick genera, including Rhipicephalus and Hyalomma [R. africae is the etiological agent of African tick-bite fever in humans (ATBF) [R. aeschlimannii have been observed in Hyalomma spp., with 9% and 52.6% respectively in Hy. truncatum and Hy. m. rufipes. These data are comparable with those of previous studies that reported 45% to 55% of Hy. m. rufipes and 6% to 7% of Hy. truncatum were DNA carriers of R. aeschlimannii in Senegal [R. aeschlimannii identified in our work were identical to those of R. aeschlimannii, previously detected in Hy. truncatum collected in Senegal (GenBank accession number HM050276.1). R. aeschlimannii is an agent of spotted fever in humans [R. aeschlimannii is found in sub-Saharan Africa, North Africa, Europe and Asia [In this study, DNA from reported –61. R. aHyalomma ,62,63. Rs (ATBF) . R. aesc Senegal . The seqn humans . R. aescand Asia ,65. Our R. mongolitimonae was identified in Hy. truncatum from Mali. It had been previously detected in Hy. truncatum from the countries bordering Mali, including Niger [R. mongolitimonae 12S sequence of the present study were 99% identical with the same sequence fragment of a strain previously isolated from a patient from Algeria (GenBank DQ097081.1).For the first time, the presence of ng Niger  and Seneng Niger . R. mongBorrelia species have been identified in Mali, B. crocidurae in the soft tick (O. sonrai) and B. theileri in the hard tick (Rh. geigyi) [Borrelia spp. in 2 specimen of Am. variegatum and 1 of Hy. truncatum by qPCR using 16S Borrelia and ITS4 genes. Similarly, Ehounoud et al. previously reported the presence of Borrelia spp. in the same tick species in Ivory Coast [Until now, two  geigyi) ,13. Our ry Coast . Unfortury Coast .C. burnetii, the agent of Q fever, was detected for the first time in ticks in Mali, with a prevalence of 33.4% in the six tick species identified. These results differ from those of Ehounoud et al. in Ivory Coast, who found only one tick infected with C. burnetii [C. burnetii. It is poorly documented in Africa. A recent study conducted in febrile African patients found one male adult patient (0.3%) infected with C. burnetii in Algeria and six patients (0.5%) in Senegal [C. burnetii was detected in humans as well as in ticks [In the present work, burnetii . Q fever Senegal . Howeverin ticks .Anaplasmataceae bacteria family was previously considered to be pathogens of veterinary importance [A. marginale in 11.5% of Rh (Bo.) microplus. This is the first demonstration of the presence of A. marginale, the agent of bovine anaplasmosis [A. marginale is an intracellular bacterium responsible for bovine anaplasmosis which manifests with anemia and jaundice [E. ruminantium was found in Am. variegatum, Hy. truncatum, Rh (Bo.) microplus, and Rh. e. evertsi. The prevalence of E. ruminantium was 13.9% in ticks. Potential new species of Ehrlichia and Anaplasma  have been detected in Rh (Bo.) microplus and Hy. truncatum. These bacteria had already been detected in ticks from Ivory Coast [The portance . Howeverportance . Here, wlasmosis  in Mali.jaundice . Also, Ery Coast .R. africae [However, co-infections have been found in the ticks in this study. The percentage of co-infected ticks was 23.1% (109/471), and we describe for the first time multiple co-infections in ticks in Mali. Recently, multiple co-infections in ticks have been reported in Ivory Coast; these co-infections systematically involved  africae . The per africae .Hy. truncatum specimens were misidentified by MALDI-TOF MS. The reasons for the misidentification of the two specimens remain unknown. Additionally, all ticks frozenly stored were correctly identified by the blind test.To avoid bias, we choose to query the MS spectra of 178 specimens of ticks, including 6 species against DB 1 which includes several families of arthropods, including mosquitoes. We constantly improve it with new specimens collected in the field and find it more relevant to carry out a total interrogation without the knowledge without any filter on a specific family. The results of the blind test revealed correct identification in 10 specimens only with high log score values, even though this database contained the same tick species that were also preserved in alcohol. This misidentification could be attributed to several factors: (i) the method used for sample crushing  thThe results of this work show that MALDI-TOF MS is superior to morphological identification, as the correct identification percentage is 99.6% for all tested. It is also interesting because there are fewer entomologists able to identify ticks and the morphological identification keys are not always available. Another advantage of MALDI-TOF is that it can identify ticks that are completely engorged or damaged, for which morphological characteristics can be deformed or even disappear making morphological identification difficult or impossible. Conversely, the proteomic strategy proposed here, does not require specific skill or expertise, reagents are very cheap so the running cost is very low compared to a molecular biology. The current limiting factors of MALDI-TOF MS analysis are the small diversity of tick species included in the MS spectra reference database and the relative elevate cost to acquire the machine. Nevertheless, it high-throughput and large application for microorganisms identification either in research or medical diagnosis, do of this emerging tool a highly competitive method also for medical entomology studies. It is likely that MALDI-TOF MS will realize similar revolution in medical entomology as it was occurred in microbiology.Our results confirm those of previous studies, according to which MALDI-TOF MS could be used for identification of ticks preserved in alcohol, but it requires the creation of a database with specimens stored in the same condition .Borrelia spp. or spotted fever group rickettsiae [In our work, MALDI-TOF MS analysis was not able to differentiate ticks which were infected or not by the bacteria that were screened. However, preliminary studies from our laboratory seemed promising, as MALDI TOF analysis allowed differentiation of ticks infected or not by kettsiae ,27. The To conclude, the present work has confirmed that MALDI-TOF MS may represent a rapid and inexpensive alternative tool for accurate identification of ticks collected in the field and stored in alcohol. The recent demonstration of the use of MALDI-TOF MS for identification of ticks and associated pathogens requires further investigation.S1 FigAm. variegatum unifected (Red) and infected by R. africae (Green)  and Hy.truncatun unifected (Red), infected by C.burnetii (Green) .Representation of MS profiles of (TIF)Click here for additional data file.


Peronophythora litchii, is one of the major diseases of litchi and has caused severe economic losses. P. litchii has the unique ability to produce downy mildew like sporangiophores under artificial culture. The pathogen had been placed in a new family Peronophytophthoraceae by some authors. In this study, the whole transcriptome of P. litchii from mycelia, sporangia, and zoospores was sequenced for the first time. A set of 23637 transcripts with an average length of 1284 bp was assembled. Using six open reading frame (ORF) predictors, 19267 representative ORFs were identified and were annotated by searching against several public databases. There were 4666 conserved gene families and various sets of lineage-specific genes among P. litchii and other four closely related oomycetes. In silico analyses revealed 490 pathogen-related proteins including 128 RXLR and 22 CRN effector candidates. Based on the phylogenetic analysis of 164 single copy orthologs from 22 species, it is validated that P. litchii is in the genus Phytophthora. Our work provides valuable data to elucidate the pathogenicity basis and ascertain the taxonomic status of P. litchii.Litchi downy blight, caused by  Litchi chinensis Sonn) is a delicious subtropical fruit in Southern China, Thailand, India, Vietnam, Australia, and South Africa. Litchi downy blight, caused by Peronophythora litchii Chen ex Ko et al., is one of the major diseases of litchi[P. litchii exclusively infects litchi, but later research has indicated that it can also infect the fruits of longan, tomato, and pawpaw upon artificial inoculation[Litchi  and Hyaloperonospora arabidopsidis [P. litchii was first isolated by Chen and was described in a new genus[P. litchii resembles downy mildews in terms of sporangiophore morphology, while characteristics of the mycelium and sexual reproduction, as well as the fact that the species can be easily cultured, renders it similar to Phytophthora and Pythium species. Before the advent of molecular phylogenetic studies in oomycetes, the former had been placed in the Peronosporaceae, while the latter were placed in the Pythiaceae. Consequently, the family Peronophythoraceae had been introduced[et al. It was suggested that P. litchii should be transferred to Phytophthora[P. litchii are basically determinate and occasionally indeterminate. However, the traditional taxonomic status of Peronophythora is mainly based on its morphological and physiological characteristics. In recent years, DNA sequences have been used in the phylogenetic studies of P. litchii. Analysis of ribosomal 28S and ITS sequences have revealed that P. litchii should be included in Phytophthora because of its closer phylogenetic relationship to Phytophthora than to downy mildews[et al. insisted that the taxonomic status of P. litchii as a distinct transitional species should be retained[P. litchii has provided convincing evidence that the P. litchii belongs to Phytophthora[Phytophthora and downy mildews[thaliana)–12. P. lnew genus. The cauntroduced. Soon aftophthora. Furthery mildews–15. Afte retained. Most retophthora. But they mildews.There is an evolutionary arms race between host plants and their pathogens. PathogePhytophthora protein), CBEL  proteins, and protease inhibitors. Elicitins and elictin-like proteins harbor a 98-amino-acid domain, which include a core of six conserved cysteines in the C-terminal. These are a type of PAMPs and can trigger the PTI of hosts[Recent molecular studies on oomycete pathogens have focused on secreted effectors, which are ubiquitous in pathogens and manipulate the defense responses of host plants,23. The  of hosts. NPP1-li of hosts,26. Cyto of hosts. RXLR-ty of hosts,28. Mean of hosts–31. Thes of hosts,27,28. I of hosts,32.P. litchii has lagged far behind other oomycete species. High-throughput sequencing techniques pave the way to collect genetic data rapidly and cost effectively for functional studies in non-model species. To ascertain the taxonomic status and gain more insight into the molecular pathogenicity mechanisms of P. litchii, we report the Illumina sequencing, de novo assembly, annotation and analysis of the P. litchii transcriptome herein. We investigated the potential effector arsenal (including RXLR effectors) and confirmed the phylogenetic status of P. litchii. Overall, our results serve as a crucial foundation for further study.Despite its economic importance, molecular study on P. litchii strain was isolated from diseased fruit of litchi in Hainan, cultured routinely on 10% V8 agar media at 25°C in the dark. Two-week-old plates were flooded with 10 mL of sterile water and kept at 4°C for 2 hours to promote zoospores release. Mycelia, sporangia and zoospores were collected by centrifugation at 2,000 × g for 10 min and immediately preserved in liquid nitrogen for RNA isolation.Total RNA was extracted using TRIzol reagent  according to the manufacturer’s instructions. The quality and quantity of RNA was checked by gel electrophoresis and spectrophotometry. Furthermore, Agilent 2100 Bioanalyzer  was applied to assess the integrity of the RNA sample. Prior to cDNA synthesis, RNA samples were treated with DNase I (Promega) to remove contaminating genomic DNA. The purified RNA was dissolved in RNase-free water and stored at -80℃ until used.TM 2000 (2 × 100 bp read length). All the experiments were carried out in the Majorbio corporations .RNA-seq libraries were prepared following TruSeqTM RNA sample preparation Kit from Illumina , using 5 μg of total RNA. Shortly, mRNA was isolated with oligo (dT) magnetic beads and was fragmented into small pieces using fragmentation buffer. cDNA synthesis, end repair, A-base addition and ligation of the Illumina-indexed adaptors were performed according to Illumina’s protocol. The cDNA fragments of 300–500 bp on 2% Low Range Ultra Agarose were selected, then the products were enriched by PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by TBS380, cDNA library was sequenced using Illumina HiSeqde novo using Trinity program (v20140717) with the default settings[http://transdecoder.sourceforge.net/) with 180 bp set as the minimum ORF length. The longest ORF was extracted from each gene as the candidate protein sequence. The quality of the assembly was benchmarked against the core set of eukaryotic genes using the Core Eukaryotic Genes Mapping Approach (CEGMA) algorithm  and Benchmarking Universal Single-Copy Orthologs (BUSCO) referring to core eukaryote genes[The raw reads were cleaned by eliminating adapters, low-quality sequences and discarded reads with more than 10% of bases that had a q-value lower than 20. The filtered and trimmed reads were assembled  settings. To elimote genes,35.−5, NCBI non-redundant nucleotide sequences (Nt) using blastn with a cutoff e-value of 1e-5[Assembled genes were compared to the following database: NCBI non-redundant protein sequences (Nr), SwissProt, Eukaryotic Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) using blastx with a cutoff e-value of 1ee of 1e-5,37. The e of 1e-5,39. Basee of 1e-5.P. litchii consisted of the proteins with a D-score more than 0.45 and without predicted transmembrane (TM) domains. RXLR and CRN effectors were initially identified based on the function annotation. In a second approach, RXLR and CRN effectors were identified with hmmsearch using Hidden Markov Model (HMM) profile[The genes were translated to all possible ORFs that encoded at least 60 amino acids (aa) from both strands. SignalP was used to identify an amino terminal signal peptide. The sec) profile,32. For ) profile, which a) profile. To sear) profile. Hits wiP. litchii and other 21 oomycetes using blastp[Albugo laibachii, Hyaloperonospora arabidopsidis, Phaeodactylum tricornutum, Phytophthora infestans, Phytophthora kernoviae, Phytophthora lateralis, Phytophthora nicotianae, Phytophthora parasitica, Phytophthora ramorum, Phytophthora sojae, Plasmopara halstedii, Pythium aphanidermatum, Pythium arrhenomanes, Pythium irregulare, Pythium iwayamai, Pythium ultimum, Pythium vexans, Saprolegnia declina, Saprolegnia parasitica and Thalassiosira pseudonana obtained from the Ensembl Genomes database: ftp://ftp.ensemblgenomes.org/pub/protists/release-33/, and the sequences of Phytophthora capsici obtained from the DOE Joint Genome Institute (JGI) database: http://genome.jgi.doe.gov). The proteins of 22 oomycetes did self-blastp-self search with a cutoff e-value of 1e-10. The pairwise similarities were used to identify single-copy genes in each species with Markov cluster (MCL) algorithm at an inflation rate (I) of 2. The member of group with just one gene was identified as single-copy gene. On the other run, the predicted proteins of P. litchii were used as queries to search against other 21 species separately by reciprocal blast hit (RBH) approach. The best hits of each protein with a cutoff e-value of 1e–6 were retrieved. Secondly, the proteins of each 21 species were used as queries to search against P. litchii and the previous filters were carried out. The pair of genes was treated as orthologs if they met the “bidirectional best hit” law. Multiple orthologs-identify were conducted among the other 21 oomycetes. The orthologous group was ascertained if any two genes were orthologs among 22 species.Identification of single-copy orthologous genes are critical to comparative analysis. Here, we grouped the predicted proteins of ng blastp  was performed amongst predicted protein sequences of P. litchii and other four oomycete species: H. arabidopsidis, P. halstedii, P. infestans, and P. sojae[On the other hand, an all-against-all blastp  complete single-copy genes, 15 (3.5%) fragmented genes, and 61 (14.2%) genes missing in P. litchii.Illumina sequencing yielded about 52.5 million pair-end reads with an average length of 200 bp. After removing adaptor sequences, ambiguous reads, and low-quality reads, we obtained approximately 49.6 million cleaned high-quality reads . Using tP. sojae (51.6%) and P. infestans (37.0%)  were successfully annotated in at least one database , whereas (37.0%) .P. litchii (19267), H. arabidopsidis (14321), P. halstedii (15469), P. infestans (17787), and P. sojae (26489), all protein from five species were clustered into 15350 groups  genes were assigned to 50 subcategories belonging to three major functional categories  . Among tTo classify orthologous gene products, 6920 (35.9%) genes were functionally classified into 25 KOG categories , S4 Fig.To identify the biological functions and interactions of genes, the genes were mapped to the reference canonical pathways in KEGG. A total of 4,624 (24.0%) genes were assigned to 32 groups, including 260 specific pathways , S5 Fig.P. litchii presumably secretes a battery of virulence proteins to promote infection. A total of 978 secrete proteins were identified in P. litchii using Signal[P. litchii genes against public databases. A total of 490 pathogenicity-related genes with known or putative roles in virulence were identified (P. sojae and H. arabidopsidis), the families of glycosyl hydrolases were significantly reduced in P. litchii  and elicitin-like proteins were about half of these in P. sojae and more than two-fold of H. arabidopsidis  was off firstly and followed by the branch of Albuginaceae. After the six Pythium species were off in the evolutionary tree, the eleven members of Peronosporaceae clustered into a group  were nested within Phytophthora clade with high bootstrap support and were separated by P. litchii and P. capsici. The branch length of P. halstedii and H. arabidopsidis both represented 0.175 genetic distance and were much longer than other nine members of Peronosporaceae  of transcripts was longer than that (1116 bp) of Phytophthora cactorum obtained using a similar approach[P. litchii transcriptome was comparable to that of P. infestans[P. sojae[H. arabidopsidis[P. halstedii[P. litchii [P. litchii genes was probably underestimated in genome project. It could be more accurately predicted if the transcriptome was incorporated into genome annotation. In addition, 97.2% of conserved core eukaryotic genes were mapped against the P. litchii transcriptome. The high mapped rate was comparable to that (93.6%) in A. laibachii and (95.0%) in H. arabidopsidis genome assemblies[P. litchii. Our work provided a high-quality transcriptome, which could be advantageous for further gene function validation and functional genomics approaches.Despite the severe economic pre- and post-harvest losses caused by l species–50. Tran approach. The geninfestans, but les[P. sojae and morehan that 16 bp of halstedii. There w litchii . The tothan that 16 bp of ssemblies. The simP. litchii-specific members. Based on gene annotations and pathway analyses, the unigenes of P. litchii were predominantly involved in cellular and metabolic processes, and signal transduction. In addition, some important pathways such as “MAPK signaling pathway”, “calcium signaling pathway”, and “plant hormone signal transduction”, were identified and potentially involved in pathogenesis. The predicted pathways together with the gene annotations can be used to further investigate gene function in the future.The conserved genes globally were examined by OrthoMCL across other four oomycete species. A total of 4666 gene families shared by all five species could be defined as the core gene set of Peronosporomycetidae . On the P. litchii contained mass of genes encoding proteins that were potentially involved in pathogenicity. ABC transporters, which are found in all species and transport substrates across cellular membranes, is essential in cell viability, virulence, and pathogenicity in oomycetes. Compared to P. sojae and H.arabidopsidis, the expansion of ABC transporters was observed in P. litchii. It was implied that the transport activity between host and pathogen was more frequent than others. The families of glycosyl hydrolases were significantly reduced. Meanwhile, the families encoding degradative enzymes, polygalacturonases, pectin methylesterases, pectinesterases, and extracellular proteases, were less than those in P. sojae and more than those in H. arabidopsidis. In general, P. litchii contained a relatively fewer genes involved hydrolytic enzymes than P. sojae, in accordance with the fact that the fruit of litchi has a gap-peel structure and is penetrated through the crack of pericarp by P. litchii. Thus, it is not necessary to maintain mass hydrolases for cell wall collapse.The transcriptome of Phytophthora and downy mildews secrete a vast repertoire of effector proteins that modulate host defenses and enable pathogenicity[P. litchii. These genes were abundant in P. sojae and P. litchii. The NPP1-like proteins may have contributed to necrotrophy life style of P. litchii. CBEL-like proteins, another apoplastic effectors, were also detected but had a similar level in P. litchii and H. arabidopsidis  for all nodes Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 FigThe results are summarized in three main categories: biological process, cellular component and molecular function. A set of 17647 genes were assigned to GO term based on blastx matches to known proteins.(TIFF)Click here for additional data file.S4 FigP. litchii genes with Nr hits were grouped into 25 KOG classification.In total, 6920 of the 19627 (TIFF)Click here for additional data file.S5 FigPathway assignment was summarized for five main categories: Cellular Processes (C), Environmental Information Processing (E), Genetic Information Processing (G), Metabolism (M) and Organismal Systems (O). A total of 4624 genes were assigned to 32 groups, and abundant genes were involved signal transduction.(TIFF)Click here for additional data file.


Limnoperna fortunei, has aggressively invaded South American freshwaters, having travelled more than 5000 km upstream across 5 countries. Along the way, the golden mussel has outcompeted native species and economically harmed aquaculture, hydroelectric powers, and ship transit. We have sequenced the complete genome of the golden mussel to understand the molecular basis of its invasiveness and search for ways to control it.For more than 25 years, the golden mussel, L. fortunei.We assembled the 1.6-Gb genome into 20 548 scaffolds with an N50 length of 312 Kb using a hybrid and hierarchical assembly strategy from short and long DNA reads and transcriptomes. A total of 60 717 coding genes were inferred from a customized transcriptome-trained AUGUSTUS run. We also compared predicted protein sets with those of complete molluscan genomes, revealing an exacerbation of protein-binding domains in L. fortunei’s genome will contribute to the investigation of bivalve genetics, evolution, and invasiveness, as well as to the development of biotechnological tools for aquatic pest control.We built one of the best bivalve genome assemblies available using a cost-effective approach using Illumina paired-end, mate-paired, and PacBio long reads. We expect that the continuous and careful annotation of  Limnoperna fortunei is an Asian bivalve that arrived in the southern part of South America about 25 years ago . Th. Thde noAUGUSTUS  personalAUGUSTUS  was traiAUGUSTUS  parameteAUGUSTUS  once agaAUGUSTUS  with theAUGUSTUS  to generna reads  re-mappiL. fortunei proteins and the total protein set predicted for 9 other mollusks: the mussels M. galloprovincialis, M. philippinarum, and B. platifrons, the clam Ruditapes philippinarum, the scallop Patinopecten yessoensis, the pacific oyster C. gigas, the pearl oyster Pinctada fucata   between u et al. ), and thOf all the orthologs found for the total 10 species, 44 groups are composed of single-copy orthologs containing 1 representative protein sequence of each species. These sequences were used to reconstruct a phylogeny: the single-copy ortholog sequences were concatenated and aligned with CLUSTALW , with a M. galloprovincialis, M. philippinarum, B. platifrons, Ruditapes philippinarum, Patinopecten yessoensis, C. gigas, Pinctada fucata, Lottia gigantean, and Haliotis discus hannai  obtained from the other 9 mollusks, according to a model based on the Poisson cumulative distribution. Bonferroni correction (P ≤ 0.05) was applied for false discovery, and absolute frequencies of Pfam-assigned domains were initially normalized by the total count number of Pfam-assigned domains found in L. fortunei to compensate for discrepancies in genome size and annotation bias.We performed a quantitative comparison of protein domains predicted from whole-genome projects of 10 molluscan species. The complete protein sets of s hannai  were subL. fortunei, the annotation against Pfam-A classified 40 127 domains in 24 513 gene models, of which 83 and 67 were expanded or contracted, respectively, in comparison with the other mollusks , Collagen, Immunoglobulins , and Ankyrins . These repeats have a variety of binding properties and are involved in cell-cell, protein-protein, and receptor-ligand interactions driving the evolutionary improvement of complex tissues and the immune defense system in metazoans [L. fortunei gene models against Uniprot identified 2 types of TLRs whose prototypical architecture of N-terminal extracellular LRR motifs including either a single or multiple cysteine cluster domain, a C-terminal TIR domain spaced by a single transmembrane-spanning domain [Drosophila [L. fortunei; the scc TLRs exhibit a higher degree of amino acid changes, higher molecular evolution, and diversification than the mcc TLRs. Overall, the expansion of these gene families might suggest an improved resistance to infections. It is, however, equally curious that other immune-related gene families such as Fribinogen_C and C1q seem to be contracted  as BioProject PRJNA330677 and under the accession numbers SRR5188384, SRR5195098, SRR518800, SRR5195097, SRR5188315, SRR5181514. This Whole Genome Shotgun Project has been deposited in the DDBJ/ENA/GenBank under accession number NFUK00000000. The version described in this paper is version NFUK01000000. Supporting data, also including annotations and BUSCO results, are available via the GigaScience repository, GigaDB [, GigaDB .L. fortunei specimens, 4 tissues each.Supplementary Table S1. RNA raw reads sequenced for 3 L. fortunei genome.Supplementary Table S2. RepeatMasker classification of repeats predicted in the Supplementary Table S3. Details of the online availability of the data used for ortholog assignment and protein domain expansion analysis.L. fortunei genome.Supplementary Table S4. Expanded protein families in the fortunei genome.Supplementary Table S5. Contracted protein families in the L. L. fortunei genes and proteins from the backers that supported us through crowdfunding (www.catarse.me/genoma).Supplementary Table S6. Fantasy names given to L. fortunei genome.Supplementary Figure 1. Steps performed for the prediction and annotation of the L. fortunei genome.Supplementary Figure 2. Phylogenetic tree of Toll-like (TLRs) receptors found in the BUSCO: Benchmarking Universal Single-Copy Orthologs; KEGG: Kyoto Encyclopedia of Genes and Genomes; SRA: Sequence Read Archive.Limnoperna fortunei specimens used for DNA extraction and sequencing were collected in the Jacuí River (29°59΄29.3″S 51°16΄24.0″W), southern Brazil. This bivalve is an exotic species in Brazil and is not characterized as an endangered or protected species.The authors declare that they have no competing interests.www.catarse.me/genoma).This work was supported by the Brazilian Government agencies CAPES (PVE 71/2013), FAPERJ APQ1 (2014), and FAPERJ/DFG (39/2014). Also, this work was funded through crowdfunding with the support of 346 people  -- Marco Gerdol31 Jul 2017 ReviewedClick here for additional data file.Reviewer_1_Report_(Revision_1) -- Marco Gerdol08 Nov 2017 ReviewedClick here for additional data file.Reviewer_2_Report_ -- Kevin Kocot23 Aug 2017 ReviewedClick here for additional data file.Supplemental materialClick here for additional data file.


Mycobacterium tuberculosis infection to tuberculosis (TB) disease and alter the clinical presentation of TB. We assessed the prevalence and determined the clinical relevance of helminth co-infection among TB patients and household contact controls in urban Tanzania.Helminth infections can negatively affect the immunologic host control, which may increase the risk of progression from latent Between November 2013 and October 2015, we enrolled adult (≥18 years) sputum smear-positive TB patients and household contact controls without TB during an ongoing TB cohort study in Dar es Salaam, Tanzania. We used Baermann, FLOTAC, Kato-Katz, point-of-care circulating cathodic antigen, and urine filtration to diagnose helminth infections. Multivariable logistic regression models with and without random effects for households were used to assess for associations between helminth infection and TB.Strongyloides stercoralis was the predominant helminth species , followed by hookworm  and Schistosoma mansoni . An infection with any helminth was not associated with TB (adjusted odds ratio (aOR) 1.26, 95% confidence interval (CI): 0.88–1.80, p = 0.22), but S. mansoni infection was . Moreover, S. mansoni infection was associated with lower sputum bacterial load  and tended to have fewer lung cavitations .A total of 597 TB patients and 375 household contact controls were included. The median age was 33 years and 60.2% (585/972) were men. The prevalence of any helminth infection among TB patients was 31.8% (190/597) and 25.9% (97/375) among controls. S. mansoni infection was an independent risk factor for active TB and altered the clinical presentation in TB patients. These findings suggest a role for schistosomiasis in modulating the pathogenesis of human TB. Treatment of helminths should be considered in clinical management of TB and TB control programs. Mycobacterium tuberculosis, and parasitic worm infections are typical diseases of poverty. They often overlap geographically, and can occur in the same individual. Parasitic worm infections contribute to the down-regulation of the essential immune response against TB, and therefore can increase progression from latent M. tuberculosis infection to active TB. We conducted a case-control study in Dar es Salaam, the economic capital of Tanzania, where TB and helminths constitute a considerable burden. We found that infection with the blood fluke Schistosoma mansoni was associated with active TB, while none of the other parasitic worms showed such an association. Interestingly, TB patients infected with S. mansoni had significantly lower sputum bacterial load at diagnosis and tended to have fewer lung cavitations compared with TB patients without any parasitic worm infection. Diagnosis and treatment of parasitic worm infections, particularly schistosomiasis, should be considered during the management of TB patients and in the context of TB control programs. This could help to reduce the TB burden in settings where TB and parasitic worms co-exist.Tuberculosis (TB), caused by the bacterium  Mycobacterium tuberculosis remains a challenging disease to control. Indeed, over two billion people are estimated to be infected with M. tuberculosis worldwide ) and household contact controls (Xpert MTB/RIF negative), who were matched by age (±5 years) and whenever possible by sex. We prospectively followed-up TB patients and assessed the clinical outcomes comparing TB patients with and without helminth infection at 6 and 12 months after recruitment.We consecutively enrolled study participants starting in November 2013 until October 2015 to reach the required sample size. Over this period, we included adult TB patients (≥18 years of age and sputum-smear positive) and household contact controls. Any individual living in the same household as the index TB patients enrolled in the study is referred to as a household contact control. Controls at recruitment were free of symptoms and signs suggestive of TB, healthy on physical examination, and had a negative Xpert MTB/RIF result .Assuming a helminth prevalence of 45% in TB patients and 26% in controls based on results from previous publications  and a poTB patients and household contact controls were interviewed and underwent physical examination during recruitment at the study site (see under “Data Collection and Definitions”). We collected skinfold measurements from four body sites  using the Harpenden skinfold caliper . The perA patient was considered as having TB when any of the two submitted sputum samples were positive for AFB by staining sputum smears using the Ziehl-Nielsen (ZN) method, and a positive mycobacterial culture. Sputum smear microscopy was done at the Temeke district hospital under continuous quality control by the central tuberculosis reference laboratory . AFB smear-positive results were graded according to World Health Organization/International Union Against Tuberculosis and Lung Disease (WHO/IUATLD) guidelines: “scanty” with 1–9 AFB per 100 oil immersion fields; “1+” with 10–99 AFB per 100 immersion fields; “2+” with 1–10 AFB per 1 immersion field, and “3+” with >10 AFB per immersion field ,24. To rAscaris lumbricoides, hookworm, S. mansoni, and Trichuris trichiura infections. The Baermann method was used to identify Strongyloides stercoralis infections ), HIV-positive (27.3% [163] vs. 9.3% [35]), and smokers (18.1% [108] vs. 8.8% [33]). TB patients also had a lower median BMI  and a lower median hemoglobin level . The patient characteristics, stratified by HIV status, are shown in A total of 597 TB patients and 375 household contact controls were included. S. stercoralis  was the predominant helminth species, followed by hookworm , S. mansoni  and S. haematobium . Overall, TB patients were more frequently co-infected with any helminth species compared with controls . Study participants with occupational risk for acquiring schistosomiasis, such as working in rice fields, sand harvesting, washing cars, and fishing had higher odds of being infected with any helminth species . HIV-positive patients were less likely to be infected with any helminth species , but there was no significant association between TB and S. stercoralis or hookworm infection , had higher median hemoglobin levels  and higher median eosinophil counts . Furthermore, we found that TB patients co-infected with S. mansoni tended to have fewer lung cavities, although this association lacked statistical significance  were cured at the end of TB treatment (at 6 months), 17.4% (58) completed treatment , and 0.9% (3) were treatment failures (positive AFB smear result at 6 months). We found no significant associations between helminth infection (at time of recruitment) and poor gain in absolute weight , BMI , and body fat percentage  after 6 months on TB treatment, as shown in S. mansoni infection was a risk factor for TB disease. This association remained significant after adjustment for other known risk factors for TB, such as HIV infection, smoking, and underweight [M. tuberculosis and S. mansoni antigens showed that S. mansoni-induced CD4+ T cells disrupt the control of M. tuberculosis in infected macrophages [We present findings on the prevalence and association of TB and helminth co-infection among adult TB patients and household contact controls in a highly-urbanized setting of Dar es Salaam, Tanzania. We found that erweight . None oferweight ,36,37, aerweight ,13,15. Irophages .M. tuberculosis infection to active TB [S. mansoni, but not for other helminth species [S. mansoni infection  may have masked an association between TB and schistosomiasis in that study [Several studies in humans suggested that helminth infections may increase the risk for progression of latent ctive TB ,21,37 asctive TB . However species . A case-at study .S. mansoni, but not other helminth species, was associated with the clinical presentation among TB patients. Patients co-infected with S. mansoni had lower sputum bacterial loads at the time of TB diagnosis than S. mansoni-negative TB patients. Similarly, a study in Ethiopia observed lower sputum bacterial loads at TB diagnosis in TB patients co-infected with any helminth species [S. mansoni resembles the paucibacillary disease in HIV-positive individuals with severe immunosuppression, who frequently have negative or low bacterial M. tuberculosis loads in the sputum compared with HIV-negative patients [M. tuberculosis bacilli being expectorated in the sputum [S. mansoni tended to present less frequently with lung cavitations compared with S. mansoni-negative TB patients. Any helminth co-infection did not appear to have an effect on clinical outcomes during follow-up. We found no evidence for an effect of helminth co-infection on the gain in the percentage of body fat and BMI after 6 months . This might be explained by the fact that the administration of anthelmintic treatment offered to the study participants after diagnosis might have reversed the Th1 immune response [S. mansoni eggs [We also found that  species . Interespatients ,41. Hence sputum ,41. Thisresponse , and thuresponse , which aoni eggs .M. tuberculosis infection to active TB. The higher prevalence of helminth co-infection in TB patients has also been noted in other studies from different settings [We found that TB patients had a higher crude prevalence of helminth infections, as compared with household contact controls. The higher prevalence of helminth infections among TB patients could be the result of the pathogenic role of helminth infection in the progression from settings ,43. For settings . Overallsettings . It is cWe also found that occupation exposing people to regular water contacts  were associated with helminth infections. Being exposed to freshwater bodies and being involved in water-related activities have previously been reported to increase the risk of helminth infections . In the Our research has several strengths and limitations that warrant consideration. An important strength of our study is the large sample size and the recruitment of both TB patients and household contact controls with similar socioeconomic profiles and exposure patterns to both TB and helminth infection. Our findings may well apply to other settings with a similar prevalence of TB, HIV, and helminth infections in sub-Saharan Africa. Furthermore, we used recommended TB diagnostics and a suite of standardized, quality-controlled helminth diagnostics, which have comparable diagnostic performance to resource-intensive molecular test assays .M. tuberculosis, which is a prerequisite to develop TB. However, because Dar es Salaam is a high-burden setting for TB with considerable risk of transmission, and because living with a TB patient is a strong risk factor for TB [M. tuberculosis. Third, we did not check the helminth infection status for TB patients during and after completion of TB treatment, which could influence the clinical outcomes. However, we do not expect a high helminth re-infection rate after 6 months in our study area [Study limitations include the following. First, this is an observational study which cannot establish a causal relationship between helminth infections and TB disease. Second, we could not fully verify whether or not the household contact controls were latently infected with r for TB , it is rudy area . Fourth,udy area . Hence, S. mansoni, but not other helminth species, was found to be an independent risk factor for active TB in our study and was associated with the clinical presentation in TB patients. These findings suggest a role for S. mansoni, or helminth infection in general, in immunomodulation of human TB. Treatment of helminth infections should be considered in the clinical management of TB patients, and helminthiasis control/elimination through preventive chemotherapy might prove to be useful as an additional component of TB control programs. Further research is needed to establish the underlying mechanisms, and compare helminth-induced immune regulation by different helminth species. Prospective cohort studies that evaluate the effect of preventive anthelmintic chemotherapy on the incidence of M. tuberculosis infection and active TB could further help to understand the interaction between these diseases at the population level. Helminthiasis control measures, in combination with traditional TB control strategies, could potentially contribute to the global efforts to reduce TB incidence by 80% until 2030, as stipulated in WHO’s ambitious End TB Strategy [In conclusion, co-infection with Strategy .S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 TableAssociations of TB disease with helminth infection and other patient characteristics comparing TB patients and household contact controls without TB.(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S11 Table(DOCX)Click here for additional data file.S1 FigAscaris lumbricoides, Enterobius vermicularis, Trichuris trichiura, and Hymenolepis diminuta.(A) The prevalence of helminth infection summarized at the ward level. (B) The helminth species distribution at the study area. Other helminth infections include: (DOCX)Click here for additional data file.S1 Checklist(DOCX)Click here for additional data file.


DNA Verity Test, DVT) for the characterization and confirmation of the species or taxa present in herbal infusions. As a model plant for this protocol, Camellia sinensis, a traditional tea plant, was selected due to the following reasons: its historical popularity as a  beverage, its high selling value, the importation of barely recognizable raw product , and the scarcity of studies concerning adulterants or contamination. The DNA Verity Test includes both the sequencing of DNA barcoding markers and genotyping of labeled-PCR DNA barcoding fragments for each sample analyzed. This protocol (DVT) was successively applied to verify the authenticity of 32 commercial teas (simple or admixture), and the main results can be summarized as follows: (1) the DVT protocol is suitable to detect adulteration in tea matrices (contaminations or absence of certified ingredients), and the method can be exported for the study of other similar systems; (2) based on the BLAST analysis of the sequences of rbcL+matK±rps7-trnV(GAC) chloroplast markers, C. sinensis can be taxonomically characterized; (3) rps7-trnV(GAC) can be employed to discriminate C. sinensis from C. pubicosta; (4) ITS2 is not an ideal DNA barcode for tea samples, reflecting potential incomplete lineage sorting and hybridization/introgression phenomena in C. sinensis taxa; (5) the genotyping approach is an easy, inexpensive and rapid pre-screening method to detect anomalies in the tea templates using the trnH(GUG)-psbA barcoding marker; (6) two herbal companies provided no authentic products with a contaminant or without some of the listed ingredients; and (7) the leaf matrices present in some teabags could be constituted using an admixture of different C. sinensis haplotypes and/or allied species (C. pubicosta).In this study, we used several molecular techniques to develop a fast and reliable protocol ( The path to heaven passes through a teapot”, and this proverb is sadly true in recent years. In fact, considering the latest dramatic news concerning food fraud and soil contamination , it n Italy; , 4). Curn Italy; , 9, 10).n Italy; . Moreoven Italy; , 13, 14)n Italy; ; in factn Italy; , 17). Spn Italy; , and an n Italy; . Becausen Italy; , 21. Thun Italy; , 23), ben Italy; , 25.DNA Verity Test, DVT) for the taxonomic characterization and confirmation of herbal infusions. As a model plant for this protocol, Camellia sinensis (L.) Kuntze, the traditional tea plant, was selected for (1) its historical popularity as a  beverage . Some of these taxa resulted in incomplete sequences, resulting in incorrect comparison outputs . Notably, a polyAn sequence was detected in trnH(GUG)-psbA, representing a potentially diagnostic marker to discriminate species similar to C. sinensis  but showing the intrinsic problematic nature of a polyN . Thus, this marker was not used for taxonomical discrimination in the present study.Another molecular marker discarded as an ideal sequencing barcode was rbcL marker was used, reflecting both good discrimination power and universal primer amplification . According to these results, we hypothesized the contaminant was a tropical taxon belonging to Urticaceae family and was likely a Pouzolzia taxon. Briefly, Pouzolzia Gaudich.  is a pantropical genus of shrubs or herbs,  Friis & Wilmot-Dear), P. mixta Solms, and P. zeylanica. Moreover, matK sequences are listed for only the following four species: P. guineensis, P. mixta, P. sanguinea (both for var. sanguinea and var. elegans), and P. zeylanica, indicating incomplete DNA barcoding characterization. Pouzolzia species are well known in the Old Tropical World as sources of fiber [P. zeylanica (= P. indica Linn.) is also commonly used as an officinal plant  for some medical remedies  [P. hirta (L.) Hassk. var. hirta (= P. bennettiana Wight) is largely employed as a medicinal brew, referred to as “Oyik” in India [Pouzolzia zeylanica or allied taxa are frequent in the area where C. sinensis is cultivated [In the simple tea templates , N11 (a black decaffeinated sample) showed the presence of r herbs, , 68, 69 r herbs, ). Currenlnerary) , 73, 74.in India . PouzolztrnH(GUG)-psbA clones = Pimpinella anisum/aromatic/flabellifolia 100% identity; GenBank accession: KY928309}, while the other two are lacking (cinnamon and licorice). In contrast, V9 and V11 showed lower additional peaks at ca. 430 bp for Mentha sp. and ca. 552–577 bp for Citrus sp., respectively. These admixture templates were amplified using specific primers for the plant species identified in the ingredient list but were not clearly assessed in the genotyping and sequencing/cloning data.In the admixture templates , V10 had an obvious contaminated sequence, but only one certified additional ingredient was identified as anise {Citrus and Mentha sp., respectively, confirming the presence of these plants but not in the quantity listed on these green tea templates .According to the PCR results, in the template V10, no amplification fragments were obtained using the primers specific for cinnamon and licorice, confirming the absence of these two certified ingredients. According to the amplification and cloning results, anise was well represented in this tea green template and did not correspond with the quantity reported on the labels (2%). Considering the two other green templates, V9 and V11, positive amplifications were obtained using the primers specific for C. sinensis in the tea templates through the joint use of rbcL+matK±rps7-trnV(GAC) molecular markers. Rps7-trnV(GAC) is an additional and exclusive marker, which has been fundamental to discriminate C. sinensis from C. pubicosta, where the rbcL sequence has the nucleotide cytosine at 68 bp (SNP-C)  .rbcL(SNP-A)+matK sequences can identify C. sinensis and C. leptophylla; in contrast, rbcL(SNP-C)+matK+rps7-trnV(GAC) matches C. sinensis and C. grandibracteata. This species and C. leptophylla are critical taxa, as these species are also regarded as mere varieties of C. sinensis or as not distinct from it and, thus, as potentially only two wild crop relatives of the cultivated tea [C. leptophylla and C. grandibracteata were characterized at an obscure or limited native range. Indeed, C. leptophylla has been identified only at the top of Daqing Mountain  [C. sinensis [C. grandibracteata is exclusively identified from tea gardens along the Mekong River in Yunxian (south-west China) [In summary, ated tea , 77. Morn China) , while pt China) , and it t China) ).rbcL sequence (SNP-A/C) and both fragments of the rps7-trnV(GAC) sequence (haplo-CS 239 bp/haplo-CP 226 bp)  an admixture of C. sinensis and hybrids of C. pubicosta (♀)/C. sinensis (♂). These speculations are based on the presence of maternal inheritance in the chloroplast of C. sinensis according to Kaundun and Matsumoto [Based on the presence of the heterozygosis peaks in the  bp) see , the leaica, see ), (2) a atsumoto .C. pubicosta is a critical species, strictly allied to C. sinensis. The first specimens collected from Mount Bavi (currently in Vietnam) were tentatively included in C. sinensis. However, later, Merrill [C. sinensis on account of relevant features in his opinion. Indeed, some of the vegetative features reported in the protologue [C. sinensis. For example, the acuminate-caudate leaves also occur in C. sinensis var. assamica. However, C. pubicosta appears distinct from C. sinensis s.l. based on other relevant traits, chiefly due to its three styles, which are free and not connate as in C. sinensis. Thus, C. pubicosta was placed in the sect. Corallinae Sealy, and C. sinensis belongs to the sect. Thea (L.) Benth. & Hook. [C. pubicosta in the sect. Thea. According to the recent chloroplast genome phylogeny of Huang et al. [C. pubicosta was identified as a sister group to both C. sinensis var. assamica and C. grandibracteata terminal and therefore is better placed in the sect. Thea. According to Huang et al. [Camellia , some species falling in the C. sinensis varieties clade included C. pubicosta and C. grandibracteata  but as DNA Verity Test, DVT) was developed to increase both efficiency and rapidity and to become current with the new molecular data submitted to GenBank. After the optimization of the DVT protocol , the 32 green/black tea samples of C. sinensis were analyzed in a blind test. The DVT protocol comprises two approaches used jointly, i.e., (1) a rapid pre-screening analysis through a genotyping approach using trnH(GUG)-psbA and rps7-trnV(GAC) for individuating any trace of contamination by other plants  and (2) a Sanger sequencing approach using the rbcL+matK±rps7-trnV(GAC) to confirm both the presence of C. sinensis and identify food frauds .After packaging, the species from which the tea samples are obtained are morphologically unrecognizable. Therefore, we cannot know with certainty using traditional methods if the source plants are actually those reported in the listed ingredients. For this reason, in the present study, a standardized protocol of DNA barcoding characterization (We can summarize some of the main results as follows:DVT protocol is suitable for detecting adulteration in tea matrices (contamination or the absence of certified ingredients) and can be applied to study other similar systems.The rbcL+matK±rps7-trnV(GAC) chloroplast markers, C. sinensis can be taxonomically characterized.Using BLAST analysis of the sequences of Rps7-trnV(GAC) can be employed to discriminate C. sinensis from C. pubicosta.C. sinensis taxa.ITS2 is not an ideal DNA barcode for tea samples because of potential incomplete lineage sorting and hybridization/introgression phenomena in This genotyping approach is an easy, inexpensive and rapid pre-screening method to detect anomalies in the tea templates.Two herbal companies provided no authentic products with a contaminant (N11) or without some of the listed ingredients (V10).C. sinensis haplotypes and/or allied species (C. pubicosta).The leaf matrices present in some teabags could comprise an admixture of different


Taxonomic profiling of ribosomal RNA (rRNA) sequences has been the accepted norm for inferring the composition of complex microbial ecosystems. Quantitative Insights Into Microbial Ecology (QIIME) and mothur have been the most widely used taxonomic analysis tools for this purpose, with MAPseq and QIIME 2 being two recently released alternatives. However, no independent and direct comparison between these four main tools has been performed. Here, we compared the default classifiers of MAPseq, mothur, QIIME, and QIIME 2 using synthetic simulated datasets comprised of some of the most abundant genera found in the human gut, ocean, and soil environments. We evaluate their accuracy when paired with both different reference databases and variable sub-regions of the 16S rRNA gene.We show that QIIME 2 provided the best recall and F-scores at genus and family levels, together with the lowest distance estimates between the observed and simulated samples. However, MAPseq showed the highest precision, with miscall rates consistently <2%. Notably, QIIME 2 was the most computationally expensive tool, with CPU time and memory usage almost 2 and 30 times higher than MAPseq, respectively. Using the SILVA database generally yielded a higher recall than using Greengenes, while assignment results of different 16S rRNA variable sub-regions varied up to 40% between samples analysed with the same pipeline.Our results support the use of either QIIME 2 or MAPseq for optimal 16S rRNA gene profiling, and we suggest that the choice between the two should be based on the level of recall, precision, and/or computational performance required. Genome sequencing has provided an unprecedented view of the microbial diversity of ecosystems from wide-ranging environments. For example, the commensal flora of the human gut has been extensively explored for potential associations with the onset of many human diseases . Similark-mer search approach that outputs confidence estimates at different taxonomic ranks.One of the crucial steps in almost all microbiome-based analyses is inference of community composition through taxonomic classification. For a few decades now , the comActinobacteria group, has prompted the development of more specialized primers  (ELIXIR-EXCELERATE)A.A., A.L.M., A.T., and R.D.F. performed the analyses; A.A., A.L.M., and R.D.F. conceived the study and wrote the manuscript. All authors have read and approved the final manuscript.GIGA-D-18-00043.pdfClick here for additional data file.GIGA-D-18-00043_R1.pdfClick here for additional data file.Response_to_Reviewer_Comments_Original_Submission.pdfClick here for additional data file.Reviewer_1_Report_ -- Nicholas Bokulich2/20/2018 ReviewedClick here for additional data file.Reviewer_2_Report_ -- Finlay Maguire3/19/2018 ReviewedClick here for additional data file.Supplemental materialClick here for additional data file.


The use of electronic cigarettes (ECs) has increased drastically over the past five years, primarily as an alternative to smoking tobacco cigarettes. However, the adverse effects of acute and long-term use of ECs on the microbiota have not been explored. In this pilot study, we sought to determine if ECs or tobacco smoking alter the oral and gut microbiota in comparison to non-smoking controls.We examined a human cohort consisting of 30 individuals: 10 EC users, 10 tobacco smokers, and 10 controls. We collected cross-sectional fecal, buccal swabs, and saliva samples from each participant. All samples underwent V4 16S rRNA gene sequencing.Prevotella (P = 0.006) and decreased Bacteroides (P = 0.036) in tobacco smokers. The Shannon diversity was also significantly reduced (P = 0.009) in fecal samples collected from tobacco smokers compared to controls. No significant difference was found in the alpha diversity, beta-diversity or taxonomic relative abundances between EC users and controls.Tobacco smoking had a significant effect on the bacterial profiles in all sample types when compared to controls, and in feces and buccal swabs when compared to EC users. The most significant associations were found in the gut, with an increased relative abundance of From a microbial ecology perspective, the current pilot data demonstrate that the use of ECs may represent a safer alternative compared to tobacco smoking. However, validation in larger cohorts and greater understanding of the short and long-term impact of EC use on microbiota composition and function is warranted. Tobacco cigarettes are the leading cause of preventable diseases in the world . SmokingPorphyromonas, Neisseria, and Gemella in tobacco smokers, but the lung communities were not affected  and gut bacterial communities.The study was approved by the Baylor College of Medicine Institutional Review Board (IRB H-38043). Written informed consent was obtained prior to collection of data and samples.n = 10), tobacco smokers (n = 10), and matched controls (n = 10). All participants were recruited from the Houston area. Inclusion criteria for EC users included daily use of ECs for at least six months. Inclusion criteria for tobacco smokers included an Fagerstrom test for nicotine dependence ≥4 and smoked a minimum of 10 cigarettes per day. Subject variables between the three exposure groups were comparable, with no significant difference in the sex, age, diet, height/weight, or race  as per the manufacturers’ protocol. Entire buccal swabs and 500 μl saliva samples were extracted using the PowerMicrobiome RNA isolation kit  as per the manufacturers’ protocol, omitting the necessary steps for co-elution of DNA and RNA, and with elution of nucleic acids in 50 μl.The bacterial 16S rRNA gene V4 region was amplified by PCR using barcoded Illumina adapter-containing primers 515F and 806R  and sequP < 0.05, pairwise significance was determined based on the Mann–Whitney test  algorithm  and feces (P < 0.001) in comparison to buccal swab samples (P < 0.001) between the three sample types  . The Sha samples . Dominanle types . Thus, aP = 0.009), but the number of observed OTUs was comparable between all groups  and EC users (P = 0.009), but controls and EC users were not significantly different (P = 0.261) (P = 0.049) and EC users (P = 0.033), but controls and EC users were comparable (P = 0.886) (P = 0.046) and EC users were comparable to both tobacco smokers and controls  . This wa= 0.886) . In salicontrols .Prevotella (P = 0.006) and decreased Bacteroides (P = 0.036) in tobacco smokers (Prevotella had significantly increased relative abundance in tobacco smokers compared to controls (P = 0.008) and EC users (P = 0.003), but no difference between EC users and controls (P = 0.99). Whereas Bacteroides showed significantly decreased relative abundance in tobacco smokers compared to controls (P = 0.017) and EC users (P = 0.003), but no difference between EC users and controls (P = 0.684). No significant difference in any bacterial genera was observed between the different groups in saliva or buccal swab samples (Bacteroides was negatively correlated with CO level (P = 0.042) and Prevotella was positively correlated with CO levels (P = 0.011)  ; Fig. S3This pilot study aimed to characterize the effects of EC vapor and tobacco smoke exposure on the bacterial profiles at multiple distinct and relevant body sites in a human cohort. To our knowledge this work represents the first study to concurrently explore the effect of EC vapor and tobacco smoke exposure on the microbiota. With users of ECs increasing at an unprecedented rate, it is imperative to understand the potential influences on host well-being, for which the oral and gut microbiota may have important consequences.We report, for the first time, that regular use of ECs does not measurably influence oral or gut bacterial communities. However, compared to controls, tobacco smoking had a significant effect on the bacterial profiles in all samples analyzed, with the most significant associations found in the gut. This is in accordance with existing data showing the gut microbiota changes following smoking cessation , 2014. TPrevotella, in accordance with existing data  microbiota. These results were surprising given the immediate proximity of the oral environment, relative to the gut, in smoke/vapor exposure. Indeed, smoking tobacco cigarettes has previously been shown to significantly alter the bacterial community in oral and lung samples . Convers samples ; Fig. S3This study has several potential limitations. First, the cohort information was collected by questionnaire and while one EC user reported occasional use of tobacco cigarettes (one per week maximum), it is possible other participants used tobacco cigarettes and did not report this. However, to control for this we tested the CO levels  in all individuals and found tobacco smokers had higher CO ppm compared to EC users and controls, which would be expected . Second,Prevotella and decreased relative abundance of Bacteroides. From a microbial ecology perspective, this study supports the perception that ECs represent a safer alternative to tobacco smoking. However, other end points besides the microbiota will be important to consider when determining the impact of ECs on human health and disease. At a time when EC use continues to rise, we highlight the need for greater understanding on the direct short and long-term impact of exposure to vapor on the microbiome composition and function.In summary, we found that tobacco smoking significantly alters the bacterial profiles in feces, buccal, and saliva samples. Compared to controls, exposure to ECs had no effect on the oral or gut communities. Changes in the gut microbiota of tobacco smokers were associated with increased relative abundance of 10.7717/peerj.4693/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.4693/supp-2Supplemental Information 2Con, control; EC, electronic cigarette user, TS, tobacco smoker.Click here for additional data file.10.7717/peerj.4693/supp-3Supplemental Information 3(A) Weighted UniFrac PCoA. (B) Alpha diversity. (C) Boxplot of most significant bacterial genera. All genera in the box plot were significantly different by Kruskal-Wallis with a P < 0.001.Click here for additional data file.10.7717/peerj.4693/supp-4Supplemental Information 4Genera ordered based on P value by reverse numerical order. All genera with >1% mean abundance included. No genera were found to be significantly different by exposure in either (A) Buccal swab or (B) Saliva.Click here for additional data file.10.7717/peerj.4693/supp-5Supplemental Information 5Correlations are based on linear regression.Click here for additional data file.10.7717/peerj.4693/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.4693/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.4693/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.4693/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.4693/supp-10Supplemental Information 10Click here for additional data file.


Dyspnea on exertion for two years, progressing to low cardiac output and syncopelately, treated with dobutamine and usual drugs for congestive heart disease.Physical examination: Good general condition, eupneic, acyanotic, normal pulse ratein the four limbs. Weight: 70 Kg, Height: 160 cm, blood pressure (right arm): 90/60mmHg, HR: 94 bpm.Precordium: Apex beat was not palpable, without systolic impulses. Low heart sounds,and low intensity heart murmur heard in the left lower sternal border. Liver was notpalpable and lungs were clear.Electrocardiography: Sinus rhythm, conduction abnormality seen in theleft branch with long QRS duration , negative T-wave in I,aVL and V6 (AT = +155º), biatrial overload, and enlarged, peaked p-wave(AP+77º). (AP+77º). .Chest radiography:Enlarged heart due to round-shaped left ventriculararch and double-density left atrium with elevation of left bronchus. Congestion ofpulmonary vessels, enlarged descending aorta, dilation of mid-aortic arch.Cardiothoracic index 0.61  and dilatedatriums. Systolic dysfunction and diffuse hypokinesis of hypokinesis of rightventricle, TAPSE = 0.7 CM. Significant left ventricular dysfunction .Computed tomography coronary angiography: Left-dominant coronarycirculation. The right ventricle was perfused by an arterial branch originating atthe posterior sinus of Valsalva and bifurcating into posterior circumflex artery,ventricular artery and marginal artery. It was also perfused by the anteriorventricular artery, a coronary artery branch that arises from the anterior Valsalvasinus. Left ventricle was perfused at right by the artery that arises from anteriorValsalva sinus as a thin branch and travels towards the anterior surface .Preserved right atrial function and enlarged left atrium. Delayed-enhancement inanterior and lower junction and in both ventricular outflow tracts. Significanttricuspid regurgitation.Ergospirometry: Peak oxygen consumption of 16.4 ml/kg/min, 76% of peakVO2 (56% of peak VO2 predicted for age); respiratory compensation point was notreached. Slope VE/VCO2 of 31.Clinical diagnosis: Corrected transposition of the great arteries withsevere biventricular dysfunction and no associated defects.Clinical reasoning: There were clinical evidence of correctedtransposition of the great arteries, particularly a late ventricular dysfunctiondetected few years ago due to tiredness. This is corroborated byelectrocardiographic signs, especially the orientation of ventricular repolarizationcharacterized by orientation of the T-wave to the right. The diagnosis was wellestablished by echocardiography and magnetic resonance imaging. The late ventriculardysfunction was probably caused by relative coronary insufficiency caused bysystemic right ventricular hypertrophy, despite good irrigation seen in computedtomography coronary angiography.Differential diagnosis: In adult patients, all other causes ofventricular dysfunction may be considered, including ischemic cardiomyopathy anddilated cardiomyopathy of other causes.Management: Heart transplantation was indicated due to significantbiventricular dysfunction.Comments: Corrected transposition of the great arteries with noassociated defects has an incidence of 10-15%. Both patients with naturalprogression of the disease and those who undergo surgical techniques for functionalcorrection progress to different degrees of systemic right ventricular dysfunctionin adult age.1,2 It becomes even worse with advanced age and occurs in 50-80% ofthese cases. From eight more advanced age cases reported in the literature, five ofthem had congestive heart failure.3The congestive syndrome may be explained by relative coronary insufficiency relatedto the hypertrophied systemic right ventricle. In this regard, the decreasedcoronary flow has been well documented in the literature and recognized as aconsequence of right ventricular dysfunction, and the main long-term sequela of thiscondition. Decreased coronary flow after vasodilation with adenosine, resulting inaltered vasoreactivity and possible microcirculation was previouslyreported,4 which mayexplain the ventricular dysfunction. Therefore, the best option for these patientsmay be atrial and arterial anatomic repair by double switch operation in some stageof the disease.1,2


Introduction: Data on the association of bisphenol A (BPA) exposure and autoimmunity in humans is unclear. Objective: To elucidate the influence of BPA on thyroid autoimmunity, in the present study we assessed the association between serum BPA and thyroid autoantibodies. Methods: Serum samples from 2361 subjects, aged ≥15 years, from the Thai 4th National Health Examination Survey were measured for BPA, antithyroglobulin (TgAb), antithyroperoxidase (TPOAb) and antithyrotrophin receptor (TRAb) antibodies. Results: The proportion of subjects positive for TgAb, TPOAb and TRAb were 11.1%, 14.9% and 1.9%, respectively. With regard to BPA, 51.9% had serum BPA levels exceeding the detection limit of the assay (0.3). There was a significant increasing trend for subjects with TgAb (p < 0.05) and TPOAb (p < 0.001) positivity as BPA quartiles increased, particularly in the highest quartile. In contrast, no relationship between BPA quartiles and TRAb was found. Logistic regression analysis showed that age, gender and BPA quartiles were determinants of TPOAb or TgAb positivity, independent of BMI. However, only the association between BPA and TPOAb positivity was consistent in both men and women. Conclusions: BPA was independently associated with TPOAb positivity. However, its mechanism related to TPOAb positivity, subsequently leading to autoimmune thyroid disease, needs further investigation. Bisphenol A (BPA), the monomeric form of polycarbonate plastic, is an endocrine disruptor that has been implicated in a number of disorders. BPA can affect autoimmunity both directly and indirectly . For exaAlthough the effects of BPA on autoimmunity in experimental animals and cell culture systems appear to be more or less consistent, the association of BPA exposure and autoimmunity in humans is much less clear. In the U.S. National Health and Nutrition Examination Survey (NHANES), BPA and triclosan were shown to be likely to negatively affect human immune function, as assessed by serum cytomegalovirus antibody and allergy or hay fever diagnosis . We prevThis study used data and a subsample of sera from the 4th National Health Examination Survey, a representative cross-sectional survey of the Thai population. The survey was conducted in 2009 and included a total sample size of 21,960 Thais aged 15 years and over. Details of the sampling method have been described elsewhere [Data collection included a face-to-face interview conducted in the community and a subsequent health examination and blood sample collection. Weight and height were measured by trained field staff using standard procedures. Venous blood samples were obtained from participants in the morning after fasting overnight. Serum samples were transferred and stored at −80 °C.Serum BPA levels were determined by competitive ELISA  with a detection limit of 0.3 ng/mL. Intra- and inter-assay precision were 7.0% and 13.6%, respectively. Serum thyroglobulin antibody (TgAb), thyroid peroxidase antibody (TPOAb) and thyroid receptor antibody (TRAb) levels were measured by electrochemiluminescence immunoassay on a Cobas e 411 analyzer . The assays had intra-assay precision of 6.1%, 9.2% and 11.5%, respectively. In the present study, levels of TgAb, TPOAb and TRAb above 115 IU/mL, 34 IU/mL and 1.75 IU/L, respectively, were considered positive.U-test for comparison of two groups. Multivariate analyses were performed using a logistic regression model to identify independent predictors of positive thyroid autoantibodies. Statistical significance was established at p < 0.05. All statistical analyses were performed using SPSS for Windows version 20.0 .After excluding those with serum thyroid-stimulating hormone (TSH) levels lower or higher than 0.34 and 5.11 IU/mL, respectively . On the p < 0.05) and TPOAb (p < 0.001) positivity as BPA quartiles increased, particularly in the highest quartile. In contrast, no relationship between BPA quartiles and TRAb was found. In logistic regression analysis, age, gender and BPA quartiles were determinants of TgAb or TPOAb positivity, whereas BMI was not an independent predictor - and non-MHC-related. Probable environmental factors include pregnancy, smoking, and iodine and selenium intake. Xenobiotics such as substances in tobacco smoke  and siliThere are a number of limitations of the present study. As mentioned previously, causal inference cannot be readily obtained due to the cross-sectional design of the present study. With regard to BPA, the ELISA method used possesses lower sensitivity compared with liquid chromatography-mass spectrometry (LC-MS) technique ,27. MoreBPA was independently associated with TPOAb positivity in both men and women. The finding may suggest a role of BPA exposure in autoimmune thyroid disease. However, its mechanism related to TPOAb positivity, and probable subsequent autoimmune thyroid disease, warrants further investigation.