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Paragonimiasis is a harmful food-borne zoonosis caused by lung flukes of the genus Paragonimus. The disease is found on most continents, several million people are at risk of infection, and it is a re-emerging disease in developing countries. The gold standard for diagnosis of pulmonary paragonimiasis requires the finding of eggs in sputa and/or fecal samples. In ectopic paragonimiasis cases, eggs are typically not seen, and supportive information is required such as a history of eating freshwater crabs or crayfishes, radiographic findings and immunological tests. Here, we developed a proof of concept based on lateral flow assay, an immunochromatographic test kit, named the paragonimiasis whole-blood test kit, for detection of specific IgG antibody in simulated whole-blood samples (WBSs) using worm excretory-secretory antigens to diagnose human paragonimiasis. The laboratory diagnostic values of this kit were compared with the detected IgG in serum samples. In simulated WBSs, the diagnostic sensitivity and specificity were 97.8 % and 96.1 %, respectively, while for serum samples, these values were 100.0 % and 94.8 %, respectively. The comparative IgG antibody detections whether a result was positive or negative between simulated WBSs and serum samples did not differ significantly with a concordance of 97.8 % in laboratory conditions using a circumscribed set of samples. The tool is fast and easy to use. The next step involves observing and evaluating native whole blood samples and using specific recombinant antigens need to be evaluated for support diagnosis of paragonimiasis caused by P. heterotremus, P. westermani and P. miyazakii at the bedside or at local and remote hospitals with limited facilities. It will also be valuable for epidemiological surveys in Asia where paragonimiasis is endemic.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can stimulate a systemic inflammatory response with severe lung involvement, multisystem dysfunction, and death in some cases. Immunosuppressive treatments have been proposed for management of COVID-19 patients, but these bring the risk of flare-up of pre-existing infections. Strongyloidiasis can become severe or fatal in immunocompromised individuals. This cross-sectional study determined the prevalence of anti-Strongyloides IgG antibody in sera collected from SARS-CoV-2 infected persons in a tertiary-care Thai hospital from January 2021 to January 2022. The survey was conducted using a rapid immunochromatographic test (ICT) kit based on a recombinant antigen of Strongyloides stercoralis known to be IgG-immunoreactive. High prevalence of anti-Strongyloides IgG antibody was found. Out of 297 SARS-CoV-2-infected patients 117 (39.4 %, 95 % CI 33.8–45.2 %) were positive for S. stercoralis according to the ICT kit. In areas where strongyloidiasis is endemic, we suggest using this point-of-care ICT kit for routine rapid screening in seriously ill COVID-19 patients who will be subjected to immunosuppressive treatment. Prompt anthelminthic treatment should be administered to prevent serious systemic strongyloidiasis in at-risk patients.

Human cysticercosis is a life-threatening zoonotic disease caused by infection with larvae (cysticerci) of the pork tapeworm, Taenia solium. This can affect the nervous system causing chronic headache and intracranial hypertension, potentially leading to epileptic seizures and paralysis. The disease is found in developing countries, especially in Southeast and South Asia, Sub-Saharan Africa, and Central and South America where porcine cysticercosis is endemic and people have a habit of eating undercooked pork. An immunochromatography-based test (ICT) kit, using T. solium cyst fluid as antigen, was manufactured to detect anti-T. solium IgG antibodies in human serum. To evaluate the kit, we used 187 serum samples including 24 from proven/confirmed cysticercosis cases, 133 from cases with other parasitosis and 30 healthy controls. Diagnostic efficiencies were calculated. The sensitivity, specificity, and accuracy were 83.3%, 92.0%, and 90.9%, respectively. Moreover, the ICT was positive before treatment but became negative after treatment, implying that this kit is also useful for follow-up monitoring post-treatment. In conclusion, we have successfully developed and present preliminary evaluation of an easy-to-handle rapid diagnostic tool for human cysticercosis in the form of an ICT platform using as antigen fluid from T. solium cysticerci.

Background: Opisthorchiasis is caused by an infection with fish-borne liver flukes of the genus Opisthorchis. Opisthorchiasis frequently leads to chronic inflammation in the biliary tract and is classified as a group 1 biological carcinogen by the International Agency for Research on Cancer: a definitive risk for cholangiocarcinoma (CCA). Methods: We used the rapid immunochromatographic test (ICT) to detect anti-Opisthorchis viverrini IgG and IgG4 subclass antibodies in sera of patients with CCA. The ICT kits were developed based on soluble antigens excreted and secreted by O. viverrini adult worms. Results: ICT indicated sera was positive for IgG and IgG4 antibodies, respectively, in 22 (61.1%) and 15 (41.6%) participants of the 36 study participants diagnosed with CCA (P > 0.05). Our study also included groups with other cancers and with liver cirrhosis, where the IgG ICT and IgG4 ICT kits were 27.7% (13/47) and 25.5% (12/47) positive, respectively (P > 0.05). Neither total the IgG ICT nor the IgG4 ICT yielded positive results in a control group of 20 healthy participants. Moreover, the percentage positivity rate using the ICT for total IgG between the CCA group and the other cancers and liver cirrhosis group was significantly different (P < 0.05). By contrast, no significant difference between these groups was apparent in the ICT for IgG4 antibody. The CCA group was 6.53 times more likely to have positive anti–O. viverrini IgG antibody (odds ratio 6.53, P < 0.001) and 3.27 times more likely to have positive anti–O. viverrini IgG4 antibody (odds ratio 3.27, P = 0.010) than the non-CCA group. Conclusion: This information is of potential value for the development of a diagnostic biomarker to predict risk for O. viverrini infection-associated CCA.

Objective: Trichinellosis is a globally distributed food-borne parasitic disease. Initially, diagnosis is usually made based on clinical signs, symptoms, and history of eating raw or undercooked meat. In the present study, an immunochromatographic test (ICT) kit was developed to diagnose trichinellosis by detecting IgG antibodies in sera of infected humans.Methods: Somatic extract from Trichinella spiralis larvae was used as the antigen for the ICT kit development. Diagnostic efficacy was evaluated using human serum samples from proven trichinellosis patients, healthy persons, and those with other parasitic infections.Results: The diagnostic sensitivity, specificity, positive and negative predictive values and accuracy of the ICT kit were 100.0% (95%; CI 88.8 to 100.0%), 92.5% (95% CI; 86.9 to 96.2%), 73.8% (95% CI; 58.0 to 86.1%), 100.0% (95% CI; 97.3 to 100.0%) and 93.8% (95% CI; 89.2 to 96.9%), respectively.Conclusions: This ICT diagnostic kit can be used as a testing tool for human trichinellosis. This test should permit rapid detection of infection to enable prompt anthelminthic treatment. It can also be used for retrospective diagnoses and field surveys based at laboratories where sophisticated equipment is lacking.

Background Amebic liver abscess (ALA) caused by Entamoeba histolytica is usually diagnosed based on its clinical symptoms, medical imaging abnormalities of the liver, and serological tests, the most common being the enzyme-linked immunosorbent assay (ELISA). For more than three decades, no investigation has evaluated the diagnostic performance of immunoglobulin G (IgG) subclasses in the serodiagnosis of ALA. Herein, we assessed the efficiencies of anti-amebic IgG and IgG subclasses for diagnosing ALA. Methods A serological ELISA-based test was performed to assess its diagnostic performance using a total of 330 serum samples from ALA patients (n = 14), healthy individuals (n = 40), and patients with other diseases (n = 276). Results ELISA targeting the total IgG antibody to E. histolytica antigen exhibited 100% sensitivity 95% CI [76.8–100.0] and 97.8% specificity 95% CI [95.5–99.1], whereas the assay targeting IgG1 showed the same sensitivity (100% 95% CI [76.8–100.0]) and a slightly higher specificity (99.1% 95% CI [97.3–99.8]). The other IgG subclasses (IgG2, IgG3, and IgG4) displayed a lower sensitivity and specificity. The sensitivity and specificity did not significantly differ between tests measuring total IgG and IgG1 (Exact McNemar’s test; p > 0.05), with a concordance of 98.2%, represented by a Cohen’s kappa of 0.83 (p < 0.001), indicating almost perfect agreement. Conclusion ELISA targeting IgG1 can provide valuable information to clinicians in differentiating ALA from other parasitic diseases, cancers, cirrhosis, and viral hepatitis. However, enzyme-conjugated anti-human total IgG is cheaper than anti-human IgG subclasses. Therefore, we suggest that total IgG-based ELISA is sufficient for the routine serodiagnosis of human ALA and possibly other clinical manifestations of invasive amebiasis.

Objective: Schistosomiasis japonica is an important helminthic disease in Asia. Sensitive and accurate diagnostic tools are indispensable for clinical diagnosis, screening infection and monitoring its control. In this study, we developed an immunochromatographic test (Sj-ICT) to detect anti-Schistosoma japonicum immunoglobulin G antibodies in human sera. Methods: Somatic extract from adult S. japonicum was used as an antigen. The Sj-ICT was developed and optimized as a point-of-care test. All 214 human serum samples were evaluated for diagnostic usefulness and comparison with an enzyme-linked immunosorbent assay (ELISA). Results: The diagnostic sensitivity, specificity, positive and negative predictive values, and accuracy of the Sj-ICT were 90.8%, 87.9%, 86.4%, 91.9% and 89.3%, respectively. For ELISA the values were respectively 91.8%, 87.9%, 86.5%, 92.7% and 89.7%. The concordance between both methods was 86.4 % (Cohen’s kappa value = 0.729). Conclusions: The immunochromatographic test kit developed can support clinical diagnosis and large-scale surveys in endemic areas without requiring additional facilities or ancillary supplies.

Abstract Background Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro. Methods Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. Results Gene knockout manipulation induced gene-disrupting indels, frequently 1–2 bp insertions and/or 8–30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n = 12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01). Conclusions The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with the liver fluke is a major risk factor for cholangiocarcinoma (CCA). The liver fluke Opisthorchis viverrini secretes a growth factor termed liver fluke granulin, a homologue of the human progranulin, which contributes significantly to biliary tract fibrosis and morbidity. Here, extracellular vesicle (EV)-mediated transfer of mRNAs from human cholangiocytes to naïve recipient cells was investigated following exposure to liver fluke granulin. To minimize the influence of endogenous progranulin, its cognate gene was inactivated using CRISPR/Cas9-based gene knock-out. Several progranulin-depleted cell lines, termed ΔhuPGRN-H69, were established. These lines exhibited >80% reductions in levels of specific transcript and progranulin, both in gene-edited cells and within EVs released by these cells. Profiles of extracellular vesicle RNAs (evRNA) from ΔhuPGRN-H69 for CCA-associated characteristics revealed a paucity of transcripts for estrogen- and Wnt-signaling pathways, peptidase inhibitors and tyrosine phosphatase related to cellular processes including oncogenic transformation. Several CCA-specific evRNAs including MAPK/AKT pathway members were induced by exposure to liver fluke granulin. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 cells exposed to liver fluke granulin. Of these, CCA-associated evRNAs modified the CCA microenvironment in naïve cells co-cultured with EVs from ΔhuPGRN-H69 cells exposed to liver fluke granulin, and induced translation of MAPK phosphorylation related-protein in naïve recipient cells in comparison with control recipient cells. Exosome-mediated crosstalk in response to liver fluke granulin promoted a CCA-specific program through MAPK pathway which, in turn, established a CCA-conducive disposition. Keywords: Opisthorchis viverrini, Liver fluke granulin, Extracellular vesicle, Cellular crosstalk

Intestinal parasitic infections (IPIs) remain a public-health problem worldwide, including in countries of the Lower Mekong subregion. Increases in human migration from neighboring countries might cause reemerging parasitic infections, leading to spread of parasites in the landscape. Here, we conducted a cross-sectional study to identify the prevalence of IPIs in migrant workers from Myanmar, Lao PDR, and Cambodia who were dwelling in Nakhon Ratchasima Province, northeastern Thailand. The identification of Strongyloides species and genetic differentiation of worms from migrant workers with different countries of origin was also assessed. Fresh stool samples were collected from 338 migrant workers and examined for evidence of IPIs using agar plate culture (APC) and the formalin-ethyl acetate concentration technique (FECT). Among those nine samples positive for nematodes by APC, the Strongyloides or hookworm species present was confirmed using the polymerase chain reaction (PCR) followed by DNA sequencing. This revealed eight cases of Strongyloides stercoralis infection and one of Necator americanus. Fifty-one out of 338 individuals (15.09%) were positive for IPIs using FECT and APC. Eggs of Opisthorchis-like flukes were the most common parasite (11.83% of samples), followed by S. stercoralis (2.37%), Entamoeba coli (1.50%), hookworm (0.89%), Taenia sp. (0.60%) and Hymenolepis nana (0.30%). The genetic differentiation of S. stercoralis recovered from migrant workers with different countries of origin was analyzed. Specimens of S. stercoralis isolated from workers from Lao PDR, Cambodia and Myanmar were genetically similar to those sequenced from Thailand. However, there were population-genetic differences between S. stercoralis from these Southeast Asian countries and other regions of the world. This study demonstrated that IPIs were prevalent in migrant workers in the northeastern region of Thailand. Our findings provided molecular confirmation of the presence of S. stercoralis and explored the genetic differentiation of S. stercoralis from those infected migrant workers. An effective anti-parasitic drug should be provided for migrant workers and its administration enforced.

Human gastrointestinal helminthic infections have a direct and/or indirect effect on the composition of the host gut microbial flora. Here, we investigated the effect of infection with a soil-transmitted intestinal nematode, Strongyloides stercoralis, on the gut microbiota of the human host. We also investigated whether composition of the microbiota in infected persons might vary across endemic regions. Fecal samples were obtained from volunteers from two areas endemic for strongyloidiasis, Khon Kaen Province in northeastern Thailand and Nakhon Si Thammarat Province in southern Thailand. Samples from Khon Kaen were from infected (SsNE) and uninfected (NegNE) individuals. Similarly, samples from the latter province were from infected (SsST) and uninfected (NegST) individuals. DNA sequences of the V3-V4 regions of the bacterial 16S rRNA gene were obtained from the fecal samples. No statistical difference in alpha diversity between groups in terms of richness or diversity were found. Statistical difference in beta diversity was observed only between NegNE and NegST. Some significant differences in species abundance were noted between geographical isolates. The SsNE group had a higher abundance of Tetragenococcus holophilus than did the SsST group, whereas Bradyrhizobium sp. was less abundant in the SsNE than the SsST group. For the uninfected groups, the NegNE had a higher abundance of T. holophilus than the NegST group. Our data showed that S. stercoralis infection leads to only minor alterations in the relative abundance of individual bacterial species in the human gut: no detectable effect was observed on community structure and diversity.

Cholangiocarcinoma (CCA) is highly prevalent in the northeastern region of Thailand. Current diagnostic methods for CCA are often expensive, time-consuming, and require medical professionals. Thus, there is a need for a simple and low-cost CCA screening method. This work developed a rapid label-free technique by Raman spectroscopy combined with the multivariate statistical methods of principal component analysis and linear discriminant analysis (PCA-LDA), aiming to analyze and classify between CCA (n = 30) and healthy (n = 30) serum specimens. The model's classification performance was validated using k-fold cross validation (k = 5). Serum levels of cholesterol (548, 700 cm-1), tryptophan (878 cm-1), and amide III (1248,1265 cm-1) were found to be statistically significantly higher in the CCA patients, whereas serum beta-carotene (1158, 1524 cm-1) levels were significantly lower. The peak heights of these identified Raman marker bands were input into an LDA model, achieving a cross-validated diagnostic sensitivity and specificity of 71.33% and 90.00% in distinguishing the CCA from healthy specimens. The PCA-LDA technique provided a higher cross-validated sensitivity and specificity of 86.67% and 96.67%. To conclude, this work demonstrated the feasibility of using Raman spectroscopy combined with PCA-LDA as a helpful tool for cholangiocarcinoma serum-based screening.

Bacterial content in mosquito larvae and adults is altered by dynamic interactions during life and varies substantially in variety and composition depending on mosquito biology and ecology. This study aimed to identify the microbiota in Aedes aegypti and Aedes albopictus and in water from their breeding sites in northeastern Thailand, a dengue-endemic area. Bacterial diversity in field-collected aquatic larvae and subsequently emerged adults of both species from several locations were examined. The microbiota was characterized based on analysis of DNA sequences from the V3-V4 region of the 16S rRNA gene and exhibited changes during development, from the mosquito larval stage to the adult stage. Aedes aegypti contained a significantly higher number of bacterial genera than did Ae. albopictus, except for the genus Wolbachia, which was present at significantly higher frequencies in male Ae. albopictus (p < 0.05). Our findings also indicate likely transstadial transmission from larva to adult and give better understanding of the microbial diversity in these mosquitoes, informing future control programs against mosquito-borne diseases.

Chronic human liver fluke infections caused by Opisthorchis viverrini and Clonorchis sinensis can last for decades and cause liver and biliary diseases, including life-threatening pathology prior to cholangiocarcinoma (CCA). CCA generally has a poor prognosis. Serological diagnosis can support parasitological examination in diagnosing disease and screening for the risk of CCA. Here, we present an improved and innovative lateral flow immunochromatographic test (ICT) kit that uses whole-blood samples (WBS) rather than serum to diagnose human opisthorchiasis, which also successfully diagnosed human clonorchiasis. This ICT includes a soluble worm extract of O. viverrini adults and colloidal-gold-labeled conjugates of the IgG antibody to evaluate the diagnostic values with simulated WBS (n = 347). Simulated WBS were obtained by the spiking infection sera with red blood cells. The diagnostic sensitivity, specificity, positive and negative predictive values, and accuracy for detecting opisthorchiasis were 95.5%, 87.0%, 80.5%, 97.2%, and 90.1%, respectively. For clonorchiasis, these findings were 85.7%, 87.0%, 53.6%, 97.2%, and 86.8%, respectively. Combined for both diseases, they were 93.2%, 87.0%, 84.0%, 94.6%, and 89.6%, respectively. The ICT kit can possibly replace the ICT platforms for antibody detection in serum samples in field surveys in remote areas where sophisticated equipment is not available.

DNA-sequencing was performed on the V3-V4 regions of 16S rRNA genes to investigate the microbial diversity of five samples of fermented freshwater fish (pla-ra) from three provinces in northeastern Thailand. The samples had salt concentrations ranging from 7 to 10%, pH values from 4.83 to 7.15, and D-/L-lactic acid concentrations of 90 to 450 mg/l. A total of 598 operational taxonomic units were annotated at various taxonomic ranks based on the SILVA Database. The lactic-acid and halophilic genera Tetragenococcus, Halanaerobium and Lactobacillus were among the dominant taxa of bacteria. The top 20 non-redundant taxa were considered in more detail. In two pla-ra samples, Tetragenococcus muriaticus was commonly identified. Halanaerobium fermentans was the most abundant species in a third sample and co-dominant in another sample. Lactobacillus rennini was dominant in the pla-ra sample from Roi Et Province. Additionally, other beneficial bacteria were detected including Staphylococcus nepalensis, Lactobacillus sakei, Lactobacillus pentosus, Weissella confusa, and Bifidobacterium bifidum. Differences between samples may be due to use of different raw materials, salt concentrations, recipes, processes and fermentation periods. The microbial communities in pla-ra provide a better understanding of the production outcomes of traditional products. Further optimization of the fermentation process, for example by using dominant bacterial taxa in starter cultures, may improve processes of food fermentation, food quality and flavor control, providing useful guidelines for industrial applications.

Hyperinfection and disseminated infection by the parasitic nematode Strongyloides stercoralis can be induced by iatrogenic administration of steroids and immunosuppression and lead to an elevated risk of mortality. Responses of free-living stages of S. stercoralis to the therapeutic corticosteroid dexamethasone (DXM) were investigated using RNA-seq transcriptomes of DXM-treated female and male worms. A total of 17,950 genes representing the transcriptome of these free-living adult stages were obtained, among which 199 and 263 were differentially expressed between DXM-treated females and DXM-treated males, respectively, compared with controls. According to Gene Ontology analysis, differentially expressed genes from DXM-treated females participate in developmental process, multicellular organismal process, cell differentiation, carbohydrate metabolic process and embryonic morphogenesis. Others are involved in signaling and signal transduction, including cAMP, cGMP-dependent protein kinase pathway, endocrine system, and thyroid hormone pathway, as based on Kyoto Encyclopedia of Genes and Genomes analysis. The novel findings warrant deeper investigation of the influence of DXM on growth and other pathways in this neglected tropical disease pathogen, particularly in a setting of autoimmune and/or allergic disease, which may require the clinical use of steroid-like hormones during latent or covert strongyloidiasis.

Abstract Background Strongyloidiasis is prevalent in northeast Thailand. This study aimed to evaluate the impact of the Health Education and Preventive Equipment Package (HEPEP), a package we developed to improve awareness and aid in the prevention of Strongyloides stercoralis infection among rural communities in northeast Thailand. Methods This was an intervention trial conducted in 12 villages (six interventions and six controls) in rural areas of northeast Thailand from March 2016 to September 2017. Single stool sample was collected from each participant and examined using agar plate culture (APC) technique. Each participant was interviewed using a pre-tested questionnaire, treated with single dose of ivermectin (200 μg/Kg), and allocated to either the intervention or control group. Members of the intervention group were given “Practices to Prevent Strongyloidiasis” poster and vinyl boards containing information aimed at raising awareness of S. stercoralis and strongyloidiasis. In addition, they were given a poster lecture regarding the lifecycle of S. stercoralis before being treated with ivermectin. Aside from that, they were also given a protective equipment package. Monthly refresher courses were provided by village health volunteers (VHVs) regarding the health information they had received and proper equipment usage. The control group, on the other hand, was only provided with a five-minute lecture regarding strongyloidiasis. Assessment of new infection was conducted 3 months later in 327 and 318 participants in the intervention group and control group, respectively. Results The HEPEP had 41% greater efficacy in preventing S. stercoralis infection in the intervention group than the measures taken in the control group (adjusted Odds Ratio (aOR) = 0.59; 95%CI: 0.41 to 0.85, P-value = 0.005). The intervention group also scored significantly higher on all aspects of a test of S. stercoralis knowledge compared with the control group (mean difference (mean dif.) = 2.89, P-value =

Abstract Background Two important helminths, Strongyloides stercoralis (an intestinal roundworm) and Opisthorchis viverrini (a liver fluke), are endemic in northeast Thailand. There have been national campaigns in place aimed at the control and eradication of soil-transmitted helminthiasis and opisthorchiasis in Thailand for several decades. However, these helminths still exist and raise concerns regarding public health. This study aimed to evaluate the current prevalence of S. stercoralis and O. viverrini infections in rural communities in northeast Thailand. The data from this study will be useful to improve strategies for future helminth prevention and control. Methods A cross-sectional study was conducted from December 2016 to June 2017 in Mueang Khon Kaen district in Khon Kaen, Thailand. The participants were selected using a simple random sampling method. Demographic data were collected using a questionnaire. Stool samples were collected and processed using agar plate culture to determine the presence of S. stercoralis infection and an in-house formalin-ethyl acetate concentration technique to determine the presence of O. viverrini and other intestinal parasite infections (IPIs). Results In total, 602 persons were enrolled. However, only 526 were analyzed for S. stercoralis and 387 for O. viverrini risk factors. The overall prevalence of S. stercoralis infection was 23.0% (95% confidence interval [95%CI]: 19.4 to 26.6). The prevalence of O. viverrini infection and IPIs other than S. stercoralis was 20.4% (95%CI: 16.5 to 24.8). The prevalence of O. viverrini infection was 19.4% (95%CI: 15.6 to 23.7). Male sex was significantly associated with S. stercoralis infection [Adjusted Odds Ratio (aOR) 4.0; 95%CI: 2.5 to 6.2; P-value

Opisthorchis viverrini, a human liver fluke, is highly prevalent in Southeast Asia. Definitive diagnosis of infection is usually achieved parasitologically through the discovery of fluke eggs in feces. However, the eggs of O. viverrini are difficult to differentiate morphologically from those of other minute intestinal flukes in fecal samples, even for experienced technicians. The present study developed a label-free optical methodology for analysis of O. viverrini eggs using Raman spectroscopy. Raman features of O. viverrini eggs were reported that can be used as marker bands for the efficient analysis of O. viverrini eggs in fecal specimens. The methodology presented here allows for the rapid detection of O. viverrini egg infection and can be readily and practicably applied in any clinical setting, even those in which a trained parasitologist is not available.

CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.

Human gnathostomiasis is a harmful food-borne zoonosis caused by roundworms of the genus Gnathostoma. The parasite can occasionally migrate to the central nervous system, causing life-threatening disease and death. Here, we report a new point-of-care (POC) test kit, the gnathostomiasis blood immunochromatographic test (GB-ICT) kit. The kit is based on recombinant Gnathostoma spinigerum antigen and detects specific IgG4 antibody in whole-blood samples (WBSs). The GB-ICT kit showed potentially high diagnostic values with simulated WBSs (n = 248), which were obtained by spiking patients’ sera with red blood cells. The accuracy, sensitivity, specificity, and positive and negative predictive values were 95.2%, 100%, 93.8%, 81.5%, and 100%, respectively. Ten WBSs from clinically suspected gnathostomiasis patients were all positive according to the GB-ICT kit, while 10 WBSs from healthy volunteers were negative. The GB-ICT kit is a simple and convenient POC testing tool using finger-prick blood samples: venous blood sampling and serum separation processes are not required. The GB-ICT kit can support clinical diagnosis in remote areas and field settings without sophisticated equipment facilities.

Human strongyloidiasis is an important soil-transmitted helminthiasis that affects millions worldwide and can develop into fatal systemic strongyloidiasis in immunosuppressed patients. We have developed two new rapid and simple-to-use immunochromatographic test (ICT) kits for rapid serodiagnosis that support stool examination for clinical diagnosis. Strongyloides stercoralis recombinant IgG immunoreactive antigen (GenBank: AAB97359.1; rSsIR-based ICT kit) was used for detection of IgG and IgG4 antibodies. The diagnostic efficacy of both kits was evaluated using human serum samples from strongyloidiasis patients, healthy individuals, and those with other parasitosis. At a prevalence of infection of 36.4%, the diagnostic sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the rSsIR-based IgG ICT kit were 91.7%, 83.8%, 76.4%, 94.6%, and 86.7%, respectively, and those of the rSsIR-based IgG4 ICT kit were 78.3%, 84.8%, 74.6%, 87.3%, and 82.4% respectively. The concordance between the two kits was 89.7%. The recombinant antigen can be produced to an unlimited extent and the kits can be used as point-of-care diagnostic tools and in large-scale surveys in endemic areas throughout tropical regions without necessitating additional facilities or ancillary supplies.

Opisthorchis viverrini is endemic in the South East Asian region, especially in Cambodia, Lao People's Democratic Republic, Vietnam and Thailand, but there have been no previous records from Myanmar. During stool surveys of rural populations in three regions of Lower Myanmar, Opisthorchis-like eggs were found in 34 out of 364 (9.3%) participants by stool microscopy after using the modified formalin-ether concentration technique. DNA was extracted from these positive stool samples and a portion of the mitochondrial cytochrome c oxidase subunit I (cox1) gene was amplified using the polymerase chain reaction and then sequenced. DNA sequences, successfully obtained from 18 of 34 positive samples (Bago Region, n = 13; Mon State, n = 3; Yangon Region, n = 2), confirmed that the eggs were of O. viverrini. Sequences showed 99.7% identity with O. viverrini mitochondrial cox1 (GenBank accession no. JF739555) but 95%, 88.7%, 82.6% and 81.4% identities with those of Opisthorchis lobatus from Lao People's Democratic Republic (GenBank accession nos. HQ328539-HQ328541), Metorchis orientalis from China (KT239342), Clonorchis sinensis from China (JF729303) and Opisthorchis felineus from Russia (EU921260), respectively. When alignement with other Opisthorchiidae trematodes, 81% similarity with Metorchis bilis from Czech Republic (GenBank accession nos. KT740966, KT740969, KT740970) and Slovakia (GenBank accession nos. KT740971-KT740973), 84.6% similarity with Metorchis xanthosomus from Czech Republic (GenBank accession no. KT740974), 78.6% similarity with M. xanthosomus from Poland (GenBank accession no. KT740968) and 82.2% similarity with Euamphimerus pancreaticus from Czech Republic (GenBank accession no. KT740975) were revealed. This study demonstrated, for the first time, O. viverrini from rural people in Myanmar using molecular methods and is an urgent call for surveillance and control activities against opisthorchiasis in Myanmar.

Angiostrongylus cantonensis is a zoonotic nematode parasite causing human eosinophilic meningitis (or meningoencephalitis) worldwide. A closely related species, Angiostrongylus malaysiensis, might also be a human pathogen. Larvae were obtained from land snails in Lao PDR, Cambodia, Myanmar and Thailand. We sequenced two nuclear gene regions (nuclear ribosomal ITS2 and SSU rRNA) and a portion of one mitochondrial gene (COI) from these larvae. Angiostrongylus cantonensis and A. malaysiensis were identified. This is the first report of the molecular identification of the two Angiostrongylus species in Lao PDR, Cambodia and Myanmar. The regional distributions of the two species broadly overlap. Phylogenetic relationships were inferred including data from Angiostrongylus species deposited in public databases. All the gene regions we sequenced have potential value in distinguishing between species of Angiostrongylus. The COI gene exhibited the greatest intraspecific variation in the study region (five haplotypes in A. cantonensis and four in A. malaysiensis) and might be suitable for more detailed phylogeographic studies.

Background: Taenia saginata is a tapeworm found in cattle worldwide. Analysis of genetic diversity in different geographical populations of T. saginata not only helps to understand the origin, transmission and spread of this organism, but also to evaluate the selection pressures acting on T. saginata and how it is responding to them. However, there are few reports of the genetic variability of T. saginata populations in different regions of the world, including Lao PDR and Thailand. We report the genetic diversity of T. saginata populations in Lao PDR and northeastern Thailand together with sequences of T. saginata from other countries deposited in GenBank. Results: Mitochondrial cox1 sequence analysis revealed that 15 and 8 haplotypes were identified in 30 and 21 T. saginata isolates from Lao PDR and northeastern Thailand, respectively. Fifty-three haplotypes were identified from 98 sequences. Phylogenetic tree and haplotype network analyses revealed that global isolates of T. saginata were genetically divided into five groups (A, B, C1, C2 and D). Taenia saginata isolates from Lao PDR and northeastern Thailand belonged to either Group A or B. Taenia saginata from western Thailand clustered in groups C1, C2 and D, and populations from the northeast and western Thailand were found to be genetically distinct. Taenia saginata isolates in Lao PDR and Thailand were also found to be genetically diverse but the degree of genetic differentiation was low. Conclusions: Taenia saginata populations from Lao PDR and northeastern Thailand are genetically distinct from the population in western Thailand and it is proposed that T. saginata has been dispersed by different transmission routes in Southeast Asia. [ABSTRACT FROM AUTHOR]