An immunochromatographic test using whole blood for rapid diagnosis of human paragonimiasis and its diagnostic usefulness.

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.

Development of a Machine Learning Model for the Classification of Enterobius vermicularis Egg.

Enterobius vermicularis (pinworm) infections are a significant global health issue, affecting children predominantly in environments like schools and daycares. Traditional diagnosis using the scotch tape technique involves examining E. vermicularis eggs under a microscope. This method is time-consuming and depends heavily on the examiner's expertise. To improve this, convolutional neural networks (CNNs) have been used to automate the detection of pinworm eggs from microscopic images. In our study, we enhanced E. vermicularis egg detection using a CNN benchmarked against leading models. We digitized and augmented 40,000 images of E. vermicularis eggs (class 1) and artifacts (class 0) for comprehensive training, using an 80:20 training-validation and a five-fold cross-validation. The proposed CNN model showed limited initial performance but achieved 90.0% accuracy, precision, recall, and F1-score after data augmentation. It also demonstrated improved stability with an ROC-AUC metric increase from 0.77 to 0.97. Despite its smaller file size, our CNN model performed comparably to larger models. Notably, the Xception model achieved 99.0% accuracy, precision, recall, and F1-score. These findings highlight the effectiveness of data augmentation and advanced CNN architectures in improving diagnostic accuracy and efficiency for E. vermicularis infections.

Automatic detection of Opisthorchis viverrini egg in stool examination using convolutional-based neural networks.

Background: Human opisthorchiasis is a dangerous infectious chronic disease distributed in many Asian areas in the water-basins of large rivers, Siberia, and Europe. The gold standard for human opisthorchiasis laboratory diagnosis is the routine examination of Opisthorchis spp. eggs under a microscope. Manual detection is laborious, time-consuming, and dependent on the microscopist's abilities and expertise. Automatic screening of Opisthorchis spp. eggs with deep learning techniques is a useful diagnostic aid. Methods: Herein, we propose a convolutional neural network (CNN) for classifying and automatically detecting O. viverrini eggs from digitized images. The image data acquisition was acquired from infected human feces and was processed using the gold standard formalin ethyl acetate concentration technique, and then captured under the microscope digital camera at 400x. Microscopic images containing artifacts and O.viverrini egg were augmented using image rotation, filtering, noising, and sharpening techniques. This augmentation increased the image dataset from 1 time to 36 times in preparation for the training and validation step. Furthermore, the overall dataset was subdivided into a training-validation and test set at an 80:20 ratio, trained with a five-fold cross-validation to test model stability. For model training, we customized a CNN for image classification. An object detection method was proposed using a patch search algorithm to detect eggs and their locations. A performance matrix was used to evaluate model efficiency after training and IoU analysis for object detection. Results: The proposed model, initially trained on non-augmented data of artifacts (class 0) and O. viverrini eggs (class 1), showed limited performance with 50.0% accuracy, 25.0% precision, 50.0% recall, and a 33.0% F1-score. After implementing data augmentation, the model significantly improved, reaching 100% accuracy, precision, recall, and F1-score. Stability assessments using 5-fold cross-validation indicated better stability with augmented data, evidenced by an ROC-AUC metric improvement from 0.5 to 1.00. Compared to other models such as ResNet50, InceptionV3, VGG16, DenseNet121, and Xception, the proposed model, with a smaller file size of 2.7 MB, showed comparable perfect performance. In object detection, the augmented data-trained model achieved an IoU score over 0.5 in 139 out of 148 images, with an average IoU of 0.6947. Conclusion: This study demonstrated the successful application of CNN in classifying and automating the detection of O. viverrini eggs in human stool samples. Our CNN model's performance metrics and true positive detection rates were outstanding. This innovative application of deep learning can automate and improve diagnostic precision, speed, and efficiency, particularly in regions where O. viverrini infections are prevalent, thereby possibly improving infection sustainable control and treatment program.

Molecular identification and genetic diversity of zoonotic hookworm infections in domestic dogs from northeastern, Thailand.

Hookworm infections remain a significant public health concern in tropical and subtropical regions, including Thailand. This study investigated the species and genetic diversity of hookworm infections in domestic dogs from northeastern Thailand. The molecular analysis focused on amplifying and sequencing specific regions of ribosomal RNA genes (ITS1-5.8S-ITS2 region) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene in hookworm larvae recovered from 21 domestic dog stool samples. Among 21 larvae (one larva per infected dog) analyzed, 14 had sequences identical to Ancylostoma caninum, and 7 showed sequences almost identical to Ancylostoma ceylanicum. Phylogenetic analysis of cox1 sequences placed A. caninum and A. ceylanicum in separate clades. The median-joining network of A. caninum cox1 sequences from Thailand showed high haplotype diversity and belonged to the same cluster as sequences from Australia while forming separate clusters from those of A. caninum samples from the USA. The available published A. ceylanicum cox1 sequences (n = 33), in combination with seven sequences in the present study, represented 15 haplotypes distributed among three clusters. Interestingly, A. ceylanicum sequences from dogs and humans shared the same haplotypes. These findings are crucial for recognizing the potential for zoonotic transmission, highlighting the necessity for targeted control measures, and increasing awareness among pet owners and healthcare professionals to mitigate the risk of hookworm transmission to humans.

Improved diagnostic sensitivity of human strongyloidiasis using point-of-care mixed recombinant antigen-based immunochromatography.

Strongyloidiasis is a neglected tropical disease that can cause fatal complications due to hyperinfection and disseminated strongyloidiasis in immunocompromised patients. We used two Strongyloides stercoralis recombinant antigenic proteins, L3NieAg.01 (NIE) and IgG-immunoreactive antigen (SsIR), to develop the recombinant antigen-based immunochromatography test (ICT) kit. We constructed and compared kits using either the NIE (NIE ICT kit) or the SsIR (SsIR ICT kit) antigens and a kit using a mixture of both (NIE-SsIR ICT kit) for detection of anti-Strongyloides IgG antibody in human serum samples. Serum samples from normal healthy individuals (Group I, n = 40), proven strongyloidiasis patients (Group II, n = 100), and those with other parasitic infections (Group III, n = 154) were evaluated. Sensitivity and specificity were 81.0% and 84.0% for the NIE ICT kit, 89.0% and 83.5% for the SsIR ICT kit, and 95.0% and 90.2% for the NIE-SsIR ICT kit, respectively. The NIE-SsIR ICT kit provided the best diagnostic results; it can supplement stool examination for clinical diagnosis and can be used to screen for asymptomatic S. stercoralis infection in people at risk in endemic areas. The NIE-SsIR ICT kit can also be used in large-scale sero-epidemiological investigations in endemic areas without the need for additional facilities or ancillary supplies.

Title: Amélioration de la sensibilité diagnostique de l'anguillulose humaine grâce à l'immunochromatographie à base d'antigènes recombinants mixtes sur le lieu d'intervention. Abstract: La strongyloïdose est une maladie tropicale négligée qui peut entraîner des complications mortelles dues à une hyperinfection et à une strongyloïdose disséminée chez les patients immunodéprimés. Nous avons utilisé deux protéines antigéniques recombinantes de Strongyloides stercoralis, L3NieAg.01 (NIE) et l'antigène immunoréactif IgG (SsIR), pour développer un kit de test d'immunochromatographie (TIC) basé sur les antigènes recombinants. Nous avons construit et comparé des kits utilisant les antigènes NIE (kit NIE ICT), SsIR (kit SsIR ICT) ou un mélange des deux (kit NIE-SsIR ICT) pour la détection des anticorps IgG anti-Strongyloides dans des échantillons de sérum humain. Des échantillons de sérum provenant d'individus normaux en bonne santé (groupe I, n = 40), de patients atteints d'anguillulose avérée (groupe II, n = 100) et de patients atteints d'autres infections parasitaires (groupe III, n = 154) ont été évalués. La sensibilité et la spécificité étaient respectivement de 81,0 % et 84,0 % pour le kit NIE ICT, 89,0 % et 83,5 % pour le kit SsIR ICT, et 95,0 % et 90,2 % pour le kit NIE-SsIR ICT. Le kit NIE-SsIR ICT a fourni les meilleurs résultats de diagnostic ; il peut compléter l'examen des selles pour le diagnostic clinique et peut être utilisé pour dépister une infection asymptomatique à S. stercoralis chez les personnes à risque dans les zones d'endémie. Le kit NIE-SsIR ICT peut également être utilisé dans des enquêtes séro-épidémiologiques à grande échelle dans les zones endémiques sans nécessiter d'installations supplémentaires ou de fournitures auxiliaires.

Characterization of the bacterial microbiota of cattle ticks in northeastern Thailand through 16S rRNA amplicon sequencing.

Ticks are vectors of a variety of pathogens that can infect humans and animals. Ticks also harbor non-pathogenic microbiota. This study characterized the microbiota of the ticks infesting beef cattle in Thailand. Two species of ticks; Rhipicephalus microplus (n = 15) and Haemaphysalis bispinosa (n = 5), were collected in seven provinces in northeastern Thailand. Microbial community profile of ticks was examined based on sequences of the V3-V4 region of 16S rRNA gene. Proteobacteria (Pseudomonadota) was the most abundant phylum, followed by Firmicutes (Bacillota), and Actinobacteriota. Coxiella-like endosymbiont was the most abundant bacterial taxon overall (49% of sequence reads), followed by Anaplasma (8.5%), Corynebacterium (5.5%), Ehrlichia (3.9%), and Castellaniella (3.4%). Co-infections of the pathogenic bacteria Ehrlichia and Anaplasma were detected in 19/20 (95%) female ticks. The tick with the lowest number of bacteria had the lowest abundance of the Coxiella-like endosymbiont, and the pathogenic bacteria Anaplasma and Ehrlichia were absent. This study provides baseline information of the microbiota of cattle ticks in northeastern Thailand, suggesting that ticks carry a few dominant bacterial taxa that are primarily non-pathogenic but can co-occur with pathogenic microorganisms. The information obtained is useful for monitoring disease outbreaks in the future and informing prevention and control strategies against cattle tick-borne diseases.

Development and evaluation of an immunochromatography-based point-of-care test kit for a rapid diagnosis of human cysticercosis.

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.

Bacterial community structure of Anopheles hyrcanus group, Anopheles nivipes, Anopheles philippinensis, and Anopheles vagus from a malaria-endemic area in Thailand.

Bacterial content of mosquitoes has given rise to the development of innovative tools that influence and seek to control malaria transmission. This study identified the bacterial microbiota in field-collected female adults of the Anopheles hyrcanus group and three Anopheles species, Anopheles nivipes, Anopheles philippinensis, and Anopheles vagus, from an endemic area in the southeastern part of Ubon Ratchathani Province, northeastern Thailand, near the Lao PDR-Cambodia-Thailand border. A total of 17 DNA libraries were generated from pooled female Anopheles abdomen samples (10 abdomens/ sample). The mosquito microbiota was characterized through the analysis of DNA sequences from the V3-V4 regions of the 16S rRNA gene, and data were analyzed in QIIME2. A total of 3,442 bacterial ASVs were obtained, revealing differences in the microbiota both within the same species/group and between different species/group. Statistical difference in alpha diversity was observed between An. hyrcanus group and An. vagus and between An. nivipes and An. vagus, and beta diversity analyses showed that the bacterial community of An. vagus was the most dissimilar from other species. The most abundant bacteria belonged to the Proteobacteria phylum (48%-75%) in which Pseudomonas, Serratia, and Pantoea were predominant genera among four Anopheles species/group. However, the most significantly abundant genus observed in each Anopheles species/group was as follows: Staphylococcus in the An. hyrcanus group, Pantoea in the An. nivipes, Rosenbergiella in An. philippinensis, and Pseudomonas in An. vagus. Particularly, Pseudomonas sp. was highly abundant in all Anopheles species except An. nivipes. The present study provides the first study on the microbiota of four potential malaria vectors as a starting step towards understanding the role of the microbiota on mosquito biology and ultimately the development of potential tools for malaria control.

Microbiome Composition and Microbial Community Structure in Mosquito Vectors Aedes aegypti and Aedes albopictus in Northeastern Thailand, a Dengue-Endemic Area.

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.

Gut microbiota diversity in human strongyloidiasis differs little in two different regions in endemic areas of Thailand.

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.

Prevalence of intestinal parasitic infections and genetic differentiation of Strongyloides stercoralis among migrant workers from Myanmar, Lao PDR and Cambodia in northeastern Thailand.

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.

Evaluation of total immunoglobulin G and subclass antibodies in an enzyme-linked immunosorbent assay for serodiagnosis of human amebic liver abscess.

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.

An Innovative Test for the Rapid Detection of Specific IgG Antibodies in Human Whole-Blood for the Diagnosis of Opisthorchis viverrini Infection.

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.

Development and Accuracy Evaluation of Lateral Flow Immunoassay for Rapid Diagnosis of Schistosomiasis Mekongi in Humans.

Schistosoma mekongi infection is endemic in countries along the Mekong River and certain of its tributaries in the lower Mekong basin, especially in Lao People's Democratic Republic and Cambodia. Diagnosis of schistosomiasis is crucial before treatment and epidemiological surveys before and/or after an intervention, such as a mass drug administration. A newly developed immunochromatographic test (ICT) for the diagnosis of schistosomiasis mekongi, based on antiparasite antibody detection in human sera, was evaluated. The schistosomiasis mekongi-ICT (Smk-ICT) strip was developed using somatic antigen from adult S. mekongi. In total, 209 serum samples were examined, including 14 from parasitologically proven schistosomiasis mekongi patients, 30 from schistosomiasis japonica patients, other parasitosis (n = 135), and healthy volunteers (n = 30) from areas not endemic for S. mekongi. Eleven schistosomiasis mekongi samples were positive according to the Smk-ICT, whereas all healthy control samples were negative. Cross-reactions with paragonimiasis heterotremus, sparganosis, trichinellosis, and taeniasis saginata samples were observed at 2.4% (4/165). The diagnostic sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 78.6% (95% confidence interval [CI] 49.2-95.3), 97.6% (95% CI 93.9-99.3), 73.3% (95% CI 44.9-92.2), 98.2% (95% CI 94.7-99.6), and 96.1% (95% CI 92.1-98.4), respectively. The Smk-ICT kit might be useful to assess the prevalence of disease before establishing transmission control and mass deworming campaigns in countries in the Mekong River subregion.

Promoter polymorphism of TNF-α (rs1800629) is associated with ischemic stroke susceptibility in a southern Thai population.

Stroke represents the leading cause of disability and mortality amongst the elderly worldwide. Multiple risk factors, including both genetic and non-genetic components, as well as their interactions, are proposed as etiological factors involved in the development of ischemic stroke (IS). Promoter polymorphisms of the IL-6-174G/C (rs1800795) and TNF-α-308G/A (rs1800629) genes have been considered as predictive risk factors of IS; however, these have not yet been evaluated in a Thai population. The aims of this study were to investigate the association of IL-6-174G/C and TNF-α-308G/A polymorphisms with IS. Genomic DNA from 200 patients with IS and 200 controls were genotyped for IL-6-174G/C and TNF-α-308G/A polymorphisms using TaqMan™ SNP genotyping and quantitative PCR-high resolution melting analysis, respectively. It was found that the TNF-α-308 A allele was significantly associated with an increased risk of IS development compared with the G allele [odds ratio (OR)=2.044; 95% CI=1.154-3.620; P=0.014]. Moreover, the IS risk was significantly higher in the presence of TNF-α-308 GA or AA genotypes compared with that in the presence of GG genotypes with a dominant inheritance (OR=1.971; 95% CI=1.080-3.599; P=0.027). However, there was no association between IL-6-174G/C and the risk of IS development. The interaction study demonstrated that IL-6-174 GG and TNF-α-308 GG genotypes enhanced IS susceptibility when combined with hypertension, hyperlipidemia and alcohol consumption. Hypertensive and hyperlipidemic subjects with the TNF-α-308 GA and AA genotypes were more likely to develop IS compared with those who did not have these two conditions and had the GG genotype. In a matched study design (1:1), the IL-6-174 GC genotype was associated with higher IL-6 levels in the control group. Collectively, the present results highlight the utility of the TNF-α-308G/A polymorphism as a predictive genetic risk factor for development of IS.

Development of an immunochromatographic point-of-care test for detection of IgG antibody in serodiagnosis of human trichinellosis.

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.

Development of Immunochromatographic Test Kit for Rapid Detection of Specific IgG4 Antibody in Whole-Blood Samples for Diagnosis of Human Gnathostomiasis.

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.

High prevalence of opisthorchiasis in rural populations from Khammouane Province, central Lao PDR: serological screening using total IgG- and IgG4-based ELISA.

BACKGROUND: Human opisthorchiasis, caused by Opisthorchis viverrini, is a public health problem in Southeast Asia and a major risk factor for cholangiocarcinoma. In Lao PDR, seroprevalence and the relationship between the number of O. viverrini eggs in infected people and specific antibody responses are still unknown. We evaluated and compared parasitological and serological screening methods in the community in an endemic area of opisthorchiasis in Lao PDR. METHODS: Seroprevalence of O. viverrini-specific total IgG and IgG4 antibodies and their relationships with O. viverrini egg intensities were evaluated in Khammouane Province, central Lao PDR, using ELISA and a modified formalin ethyl-acetate concentration technique (FECT). RESULTS: FECT stool examination revealed O. viverrini eggs in 70.3% (90/128) of individuals (95% CI 61.6 to 78.1%) while ELISA (based on total IgG and on IgG4 antibodies to O. viverrini) found 98.4% (95% CI 94.5 to 99.8%) and 89.8% (95% CI 83.3 to 94.5%) of sera, respectively. There was a positive and significant correlation between numbers of O. viverrini eggs per gram and levels of both IgG (R2=0.168, p<0.001) and IgG4 (R2=0.219, p<0.001) antibodies. CONCLUSIONS: A high prevalence of human opisthorchiasis in Lao PDR was found using a new platform, serological screening in the community. This points to a need for sustainable control of this liver fluke infection.

An Unusual Case of Gastric Gnathostomiasis Caused by Gnathostoma spinigerum Confirmed by Video Gastroscopy and Morphological and Molecular Identification.

Human gnathostomiasis is a harmful foodborne parasitic infection caused by nematodes of the genus Gnathostoma. Here, we report an unusual case of gastric gnathostomiasis seen in a hospital in Thailand along with the clinical characteristics, treatment, and outcome. A 39-year-old man presented with complaints of epigastric pain, dizziness, and history of passing dark, tarry stools for 2 days. The patient had a history of consuming raw freshwater fish. Supplementary differential diagnosis was performed via rapid serological testing, and presence of the causative agent was confirmed based on video gastroscopy, morphology of the removed parasite, and molecular identification. After its surgical removal from the stomach, the parasite was morphologically identified as Gnathostoma species. Molecular identification was performed via DNA extraction from the recovered worm, and amplification and sequencing of the second internal transcribed spacer (ITS2) region and partial cytochrome c oxidase subunit I (cox1) gene. The ITS2 and cox1 sequences were consistent with those of Gnathostoma spinigerum. Clinicians in endemic areas should therefore be aware of the rare clinical manifestations and use of supplementary serological tests to facilitate early diagnosis and treatment of gastric gnathostomiasis.

Prevalence of Soil-Transmitted Helminth Infections and Associated Risk Factors among Schoolchildren in Nakhon Si Thammarat, Thailand.

BACKGROUND: Soil-transmitted helminth infections constitute a public health problem in the rural areas of tropical and subtropical regions, including Thailand. We aimed to determine the prevalence of soil-transmitted helminth infections and underlying risk factors among the schoolchildren living in the rural areas of southern Thailand. METHODS: A cross-sectional survey was conducted between Sep and Nov 2018 in the district of Thasala, Nakhon Si Thammarat, Thailand. A total of 192 children, aged 6-12 yr were enrolled. Each child provided a single stool sample that was subjected to a suite of microscopic diagnoses for soil-transmitted helminth. A questionnaire was administered to determine risk factors of the infections. Logistic regression models were applied to investigate associations. RESULTS: The overall prevalence of soil-transmitted helminth infections was 3.13%; Strongyloides stercoralis 2.08%; hookworm 1.04% and Trichuris trichiura 0.52%. Children who cutting fingernails short can prevent soil-transmitted helminth infections highly up to 90% (crude OR = 0.1; 95% confidence interval = 0 - 0.8; P = 0.020). CONCLUSION: The finding of the study shows a sharp decrease in the prevalence of soil-transmitted helminth among schoolchildren in the southern Thailand in the past two decades with prevalence dropping below 5% for soil-transmitted helminth. However, the prevalence of S. stercoralis remained stable over time. These results suggest that the culture method should be used to access strongyloidiasis situation in the older age group who greater contact with soil for agriculturists.