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Cholangiocarcinoma (CCA) is a life-threatening disease that impacts patients worldwide. In Southeast Asian countries, the liver fluke Opisthorchis viverrini plays a major role in inducing carcinogenesis of the bile ducts. Due to its asymptomatic nature, O. viverrini infections are rarely treated, consequently leading to the development of advanced stages of CCA before diagnosis. Despite the current use of exosomal microRNAs (miRNA) as diagnostic biomarkers for the early detection of many types of cancer, the applications for miRNA remain limited with CCA. Circulating exosomes, membranous vesicles essential for intercellular communication, were found to contain unique miRNA. In this study, we conducted next-generation sequencing (Ion Torrent PGM) and bioinformatics to characterize and compare the contents of exosomal miRNA derived from the plasma of CCA patients, O. viverrini-infected patients, and healthy individuals, as well as to identify and validate key molecules as markers for screening the diagnosis of CCA and O. viverrini infection. The obtained results showed the success of using NGS technology in discovering exosomal miRNAs, specifically miR-194-5p and miR-192-5p, both of which were upregulated in the O. viverrini-infected group. Interestingly, miR-192-5p was upregulated while miR-194-5p was downregulated in CCA, suggesting their potential use as biomarkers for screening CCA and O. viverrini infection, especially in O. viverrini-endemic areas., Competing Interests: Declaration of competing interest The authors declared no competing interests for this study, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Opisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis., Methods: The Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique., Results: The recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively., Conclusions: The high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces., (© 2024. The Author(s).)

Clonorchis sinensis , Opisthorchis viverrini , and Opisthorchis felineus are important liver flukes that cause a considerable public health burden in eastern Asia, southeastern Asia, and eastern Europe, respectively. The life cycles are complex, involving humans, animal reservoirs, and two kinds of intermediate hosts. An interplay of biological, cultural, ecological, economic, and social factors drives transmission. Chronic infections are associated with liver and biliary complications, most importantly cholangiocarcinoma. With regard to diagnosis, stool microscopy is widely used in epidemiologic surveys and for individual diagnosis. Immunologic techniques are employed for screening purposes, and molecular techniques facilitate species differentiation in reference laboratories. The mainstay of control is preventive chemotherapy with praziquantel, usually combined with behavioral change through information, education and communication, and environmental control. Tribendimidine, a drug registered in the People's Republic of China for soil-transmitted helminth infections, shows potential against both C. sinensis and O. viverrini and, hence, warrants further clinical development. Novel control approaches include fish vaccine and biological control. Considerable advances have been made using multi-omics which may trigger the development of new interventions. Pressing research needs include mapping the current distribution, disentangling the transmission, accurately estimating the disease burden, and developing new diagnostic and treatment tools, which would aid to optimize control and elimination measures., Competing Interests: The authors declare no conflict of interest.

Objectives: This study aimed to assess the diagnostic potential of cell-free DNA (cfDNA) and cell-free miRNA (cf-miRNA) for distinguishing between Healthy, asymptomatic opisthorchiasis viverrini and cholangiocarcinoma in a preliminary manner., Methods: In this study, 36 participants were enrolled into three health status groups: a healthy control group (HC), Opisthorchis viverrini-infected group (OV), and a cholangiocarcinoma group (CCA), each comprising 12 participants. Concentration measurements of cfDNA and cf-miRNA from plasma were conducted. Additionally, ultra-low-pass whole-genome sequencing (ULP-WGS) was employed to investigate DNA alterations., Results: The study revealed a significant elevation in plasma cfDNA concentration in the cholangiocarcinoma (CCA) group compared to healthy controls (HC) and Opisthorchis viverrini-infected (OV) groups (P < 0.001). The cfDNA concentration demonstrated a sensitivity of 75.00% and specificity of 95.83% for differentiating cholangiocarcinoma, with a cut-off of > 30.50 ng/ml plasma. Likewise, the concentration of cf-miRNA in the CCA group significantly differed from that in the HC and OV groups, demonstrating a sensitivity of 83.33% and specificity of 95.83% with a cut-off set at > 70.50 ng/ml plasma. Furthermore, a positive correlation between plasma concentrations of cfDNA and cf-miRNA suggests a potential relationship between these two biomarkers. These findings indicated the diagnostic potential of cfDNA and cf-miRNA in distinguishing cholangiocarcinoma, emphasizing their role as promising biomarkers for further investigation and clinical applications., Conclusion: Elevated plasma concentrations of cfDNA and cf-miRNA could serve as potential diagnostic tools for distinguishing cholangiocarcinoma from other conditions. cf-miRNA was superior to cfDNA in terms of sensitivity.

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., Competing Interests: The authors declare that they have no competing interests., (© 2024 Thanchomnang et al.)

Cholangiocarcinoma (CCA) is a prevalent cancer in Southeast Asia, with Opisthorchis viverrini (O.viverrini) infection being the primary risk factor. Most CCA cases in this region are diagnosed at advanced stages, leading to unfavorable prognoses. The development of stage-specific biomarkers for Opisthorchis viverrini-induced cholangiocarcinoma (Ov-CCA) holds crucial significance, as it facilitates early detection and timely administration of curative interventions, effectively mitigating the high morbidity and mortality rates associated with this disease in the Great Mekong region. Biomarkers are a promising approach for early detection, prognosis, and targeted treatment of CCA. Disease-specific biomarkers facilitate early detection and enable monitoring of therapy effectiveness, allowing for any necessary corrections. This review provides an overview of the potential O. viverrini-specific molecular biomarkers and important markers for diagnosing and monitoring Ov-CCA, discussing their prognostic, predictive, and diagnostic value. Despite the limited research in this domain, several potential biomarkers have been identified, encompassing both worm-induced and host-induced factors. This review offers a thorough examination of historical and contemporary progress in identifying biomarkers through multiomics techniques, along with their potential implications for early detection and treatment.

Raw or undercooked freshwater fish consumption contributes to persistent Opisthorchis viverrini infection in Northeast Thailand. This study aims to assess the relationship between misconceptions, unhealthy eating habits, and O. viverrini infection. Data were obtained from the Cholangiocarcinoma Screening and Care Program in Northeast Thailand from 2019 to 2021. Participants were screened for O. viverrini annually over the following 2 years using the Kato-Katz technique. Misconceptions and unhealthy eating habits were assessed through questionnaires. The relationship between these factors and O. viverrini infection was evaluated using adjusted risk ratios (ARRs) and 95% confidence intervals (CIs) from generalized estimating equations under binomial regression framework. Of 5375 participants screened for O. viverrini over 3 years, infection rates were 21.53%, 10.7%, and 4.6% each year, respectively. Out of those, 636 participants responded to questions regarding misconceptions. Results showed that participants who believed in the efficacy of putting lime or red ants in Koi pla (raw fish salad) or eating Koi pla with white whiskey to kill parasites, and early-stage cholangiocarcinoma can be cured, were 41% (ARR, 1.41; 95% CI, 1.03-1.94) and 57% (ARR, 1.57; 95% CI, 1.06-2.33), respectively, more likely to be infected with O. viverrini. Our study confirms that belief in using lime or red ants in Koi pla or eating Koi pla with white whiskey to make it cooked, or early-stage cholangiocarcinoma can be cured, increases O. viverrini infection risk in high-risk populations. Changing health beliefs and eating habits is necessary to reduce O. viverrini infection and its risk to cholangiocarcinoma., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Screening for opisthorchiasis, a parasitic worm infection affecting many millions of people in Southeast Asia, has traditionally relied on faecal egg examination such as the formalin-ethyl acetate concentration technique (FECT) and Kato-Katz method. Although the urinary enzyme-linked immunosorbent assay (ELISA) has been used more recently, we developed a urinary antigen-based rapid diagnostic test (RDT) to simplify diagnosis and as a point-of-care testing (POCT) and field applications for surveillance and control of opisthorchiasis., Methods: A urinary Opisthorchis viverrini (OV)-RDT was developed using immunochromatographic methodology with a specific monoclonal antibody against OV. The diagnostic performance of the urinary OV-RDT was compared to that of quantitative faecal FECT and urinary antigen ELISA (n = 493). Cross-reactivities of urinary OV-RDT with other helminthiases coexisted with O. viverrini were determined (n = 96). A field trial in the application of urinary OV-RDT was compared with urinary antigen ELISA at baseline screening and assessment of drug treatment outcomes in opisthorchiasis (n = 1629). The McNemar chi-square, Kruskal-Wallis and Cohen's kappa coefficient (κ-value) tests were used for statistical analyses., Results: Urinary OV-RDT had sensitivity of 94.2% and specificity of 93.2%, compared to faecal FECT. Urinary OV-RDT had high diagnostic agreement (Kappa = 0.842-0.874, P < 0.001) and quantitative correlation with urinary antigen ELISA (Kruskal-Wallis tests = 316.2, P < 0.0001) and faecal FECT (Kruskal-Wallis tests = 362.3, P < 0.0001). The positive rates by OV-RDT, ELISA and FECT were 48.9%, 52.5% and 49.3%, respectively. Cross-reactions of urinary OV-RDT with other helminthiases were few (2%). Field trials of urinary OV-RDT yielded comparable prevalence of O. viverrini between urinary OV-RDT (53.2%) and urinary antigen ELISA (54.0%). OV screening showed high diagnostic agreement (kappa > 0.8, P < 0.0001) between urinary OV-RDT and urinary antigen ELISA. The cure rates of opisthorchiasis at 1 month post-praziquantel treatment determined by urinary OV-RDT (86.6%) and urinary antigen ELISA (80.5%) were similar (P > 0.05)., Conclusions: The urinary OV-RDT test has high potential as a new tool for screening and evaluating treatment outcomes in opisthorchiasis. The ease of sample collection and simplicity of urinary OV-RDT may facilitate mass screening, control and elimination of opisthorchiasis, thereby contributing to a reduction in the disease burden in Southeast Asia., (© 2023. The Author(s).)

Background: Cholangiocarcinoma (CCA), a fatal bile duct cancer, has a high incidence in Western Siberia, Russian Federation. In addition, Opisthorchis felineus, a bile duct-dwelling trematode liver fluke is highly endemic. Closely related species have been shown to be cancerogenic agents in Asia. We therefore examined the association between O felineus infection and CCA in Western Siberia., Methods: We conducted a hospital-based, individually matched case-control study between January 2017 and August 2020 in Tomsk Oblast and Khanty-Mansiysk Autonomous Okrug, Yugra, Russian Federation. Histologically confirmed CCA patients (cases) were compared with matched age, sex, and place of residence hospital controls. The examination of study participants included the diagnosis of current and past O felineus infection, abdominal ultrasonographical assessment, physical examination, and interview on exposures to potential risk factors., Results: We identified 40 patients with CCA and 160 controls. Exposures to O felineus infection was strongly associated with CCA (odds ratio [OR], 3.9; 95% confidence interval [CI], 1.4-10.8; P = .008). Also, cases reported more often that they were currently or in the past were infected by O felineus compared with controls (OR, 4.03; 95% CI, 1.7-9.5; P = .001). Furthermore, cases reported river fish consumption and fishing habits significantly more often than controls (OR, 5.5; 95% CI, 1.5-19.8; P = .009 and OR, 3.3; 95% CI, 1.4-7.7; P = .005)., Conclusions: The study results revealed a strong significantly increased risk for CCA development in O felineus-infected individuals. Elaboration of the guidelines on screening programs for early CCA diagnosis, prevention, and treatment is socially important in endemic regions., Competing Interests: Potential conflicts of interest. P. O. reports a grant from University of Geneva to plan and implement a collaborative course in epidemiology between Swiss TPH and the Siberian State Medical University outside of the submitted work. All other authors have no conflicts of interest to disclose., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Antigen detected in urine for the diagnosis of opisthorchiasis has a low daily variation; however, the longer term variability in antigen concentrations is unknown. In this study, we prospectively monitored Opisthorchis viverrini antigen concentrations for 30 consecutive days and at subsequent monthly intervals in a cohort of opisthorchiasis-positive individuals. On the basis of the monoclonal antibody-based ELISA, the profiles of antigen-positive rate and antigen concentration exhibited no significant change over 30 days with a mean proportion positive of 87.1% (range 73.7%-100%), and the average antigen concentration was 29.7 ± 2.2 ng/mL (mean ± SE). The urine antigen concentration at baseline was similar to the subsequent measurements at 2, 4, 6, and 10 months in the follow-up study (P > 0.05). The consistency and low daily and long-term fluctuation of O. viverrini antigen in urine demonstrates the reliability of urine assay for diagnosis of opisthorchiasis.

Opisthorchiasis and related infections are persistent and substantial public health burdens from eastern Europe to southeastern and northern Asia. Snails of the family Bithyniidae act as first intermediate hosts not only for the trematodes of the family Opisthorchiidae but also for those of the family Notocotylidae. There are certain morphological similarities between the aforementioned trematode cercariae. In order to find natural local foci of opisthorchiasis, which are established primarily according to the presence of infected bithyniid snails at the area under examination, it is crucial to correctly identify the rediae and cercariae of the trematodes. The aim of our investigation was to evaluate the role of bithyniid snails in the transmission of Opisthorchiidae and Notocotylidae in ecosystems in the south of Western Siberia. We have been studying the process of bithyniid snail dissemination in Western Siberia and examining their infection by trematodes from 1994 until now. A total of 16,213 bithyniid snails in 23 water bodies (in 28 localities) of four major basins situated in the Novosibirsk region were inspected for trematode infestation. Long-term research has been conducted in the Kargat river estuary and the Ob river floodplain for 15 and 25 years, respectively. In both water bodies, the prevalence of notocotylid and opisthorchiid trematodes was positively correlated with the global land-ocean temperature index. Trematode parthenitae were identified if there were mature cercariae capable of leaving the shell of the host snail on their own. Identification to the species of opisthorchiid cercariae was verified using molecular genetic analysis methods targeting ITS2 locus. Opisthorchis felineus and Metorchis bilis, two opisthorchiid species that are potentially perilous to human health, were found in bithyniids in the Novosibirsk region. The mean prevalence of infection with notocotylid trematodes in bithyniid snails was higher than the corresponding prevalence of infection with opisthorchiid trematodes. The results of this study can be used to identify and predict natural foci of epidemiologically and/or epizootically dangerous diseases., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Antigen detection in urine using an enzyme-linked immunosorbent assay (ELISA) is more sensitive than fecal examination for diagnosis of opisthorchiasis and for assessment of the effects of drug treatment. It is not known whether day-to-day variation of urine composition, including levels of Opisthorchis viverrini antigen, influences the urine assay. We investigated this topic with the cooperation of participants from two localities in Northeast Thailand. Project participants were screened for parasite infections for three consecutive days using the quantitative formalin-ethyl acetate concentration technique (FECT) to detect O. viverrini eggs and the urine ELISA for detection of O. viverrini antigen. A subset of participants (n = 801) with matched fecal and urine samples were analyzed for comparison of inter-day prevalence estimates and the performance of the urine assay compared against FECT for diagnosis of opisthorchiasis. The daily prevalence measured by the urine assay ranged between 29.0%-30.2% while those by FECT ranged between 11.9%-20.2%. The cumulative three-day prevalence estimate determined by the urine antigen assay was 30.3%, which was significantly higher than that by FECT (20.2%, p < 0.05). A significant positive correlation was found between the concentration of antigen in urine and fecal egg counts (p < 0.001). Overall, the urine assay had better diagnostic performance for opisthorchiasis than fecal examination by FECT. The high sensitivity plus negligible daily variation of O. viverrini antigen in urine indicates the utility of the urine assay for diagnosis, as well as population screening, of opisthorchiasis., Competing Interests: The authors have declared that no competing interests exist.

Background: Control and elimination of the liver fluke (Opisthorchis viverrini) is a primary preventive strategy against cholangiocarcinoma in Southeast Asia. A sensitive parasitological diagnostic method is required to facilitate a surveillance and control program. In this study, we evaluated the performance of Mini Parasep ® SF stool concentrator kit (stool kit) compared with Kato-Katz (KK) and the quantitative formalin-ethyl acetate concentration technique (FECT) for detection of O. viverrini and co-endemic parasitic infections., Methods: A cross-sectional survey for parasitic infection in residents aged > 15 years in a community in Kalasin province, Northeast Thailand, was conducted in 2018. Fecal samples were collected and screened by KK method, and a subset of samples was further examined by the stool kit and FECT methods. The results were analyzed for prevalence of parasitic infections in addition to the diagnostic performance of the methods for qualitative and quantitative detection of helminthiases., Results: The initial survey of parasitic infection determined by the KK method (n = 567) showed the prevalence of O. viverrini was 32.63%, followed by Taenia 2.65%, echinostomes 1.76%, hookworms 1.41%, Trichuris trichiura 0.53% and Strongyloides stercoralis 0.53%. Within a subset of samples tested with multiple diagnostics (n = 150), the detection rates of O. viverrini by the stool kit, FECT and KK methods were 27.3%, 30.7% and 28.7%, respectively. The diagnostic sensitivity for opisthorchiasis was similar for FECT (75.5%), KK(66.0%) and the stool kit (67.3%). For other parasitic infections, FECT and stool kit methods performed better than KK, particularly in detecting minute intestinal flukes (MIF), S. stercoralis and coinfections. When measuring the intensity of O. viverrini infection (fecal egg counts), the stool kit results showed a significant positive correlation with KK and FECT (P < 0.05)., Conclusions: As the stool kit is simple to use and shows a comparable performance to FECT, it may serve as an alternative method of fecal examination for screening of helminthiasis including opisthorchiasis., (© 2022. The Author(s).)

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., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The human liver fluke Opisthorchis viverrini (Ov), the primary risk factor for cholangiocarcinoma (CHCA), is a parasite endemic to southeast Asian countries. With no effective treatments for CHCA currently available, early diagnosis and treatment of Ov infection remains the only practical method for the prevention of CHCA. In this study, plasma phosphoproteomes of patients in the non-Ov infection, non-cholangiocarcinoma subject group (non-OVCCA), the asymptomatic Ov infected group (OV), and the CHCA group (CCA), were investigated to identify potential biomarkers for Ov infection and CHCA. The AKT signalling pathway was found to be up-regulated. Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (PIK3CB), an upstream signalling molecule, was selected as a potential biomarker and evaluated using indirect enzyme-linked immunosorbent assay (ELISA). Results demonstrated evidence that levels of PIK3CB in both the OV group and CCA group was statistically different compared to the non-OVCCA group (P < 0.01). However, the levels of PIK3CB between the OV group and the CCA group were found not to be statistically different. Sensitivity and specificity for OV using OD450 cut-off at >1.570 was 76 and 72%, respectively. For CCA, sensitivity and specificity using OD450 cut-off at >1.398 was 68 and 76%, respectively. Application of indirect ELISA detecting plasma PIK3CB will be of great benefit for screening of opisthorchiasis and CHCA.

Introduction: Opisthorchis viverrini (OV) is a major cause of infection in Southeast Asia. Previous studies in mouse models have shown that OV infection can contribute to immune-complex glomerulonephritis (GN). However, OV infection in human kidney tissue has never been demonstrated. Herein, we evaluated the association of OV infection with biopsy-proven glomerular disease., Methods: This study was performed in adult patients who underwent kidney biopsy between July 2016 and February 2017. All kidney tissue samples were processed using the standard techniques for renal pathological diagnoses and immunohistochemistry techniques to detect OV antigen. Pre-implanted donor kidney tissue samples were used as controls. The participants were also assessed for OV infection by serum OV immunoglobulin G antibody (Ab) levels and/or presence of OV eggs in stool., Results: Forty-three renal tissue samples from glomerular disease patients and 50 from transplant donors were included in the study. Mean age in the GN group was 41.7 ± 15.9 years, estimated glomerular filtration rate (eGFR) was 70.65 ± 36.61 mL/min/1.73 m2, and median proteinuria was 3.17 (1.70-4.95) g/day. Lupus nephritis (LN) was the most common diagnosis (32.6%), followed by IgA nephropathy (23.3%), IgM nephropathy (18.6%), and primary membranous nephropathy (MN; 7%). The OV antigen was observed in kidney tissue from patients with IgA nephropathy, LN, primary MN, focal segmental glomerulosclerosis, and IgM nephropathy. By contrast, no OV antigen was detected in tissue samples from the control group. The presence of OV antigens was observed in glomerular endothelial cells, mesangial cells, tubular cells, and peritubular capillaries. The odds ratio of positive serum OV Ab to predict the presence of OV antigen in kidney tissues was 4.47 (p = 0.057), and there was a negative correlation between levels of serum OV Ab and eGFR (r = -0.31, p = 0.04)., Discussion/conclusion: This is the first study to demonstrate the presence of OV antigen in human kidney tissue, which indicates that OV infection may be associated with biopsy-proven glomerular diseases., (© 2022 S. Karger AG, Basel.)

Background: Patients infected with a parasite often develop opisthorchiasis viverrini, which often progresses into cholangiocarcinoma (CCA) due to the asymptomatic nature of the infection. Currently, there are no effective diagnostic methods for opisthorchiasis or cholangiocarcinoma., Objective: The aim of this study was to identify the host-responsive protein that can be developed as a diagnostic biomarker of opisthorchiasis and cholangiocarcinoma., Methods: Plasma samples were collected from non-OVCCA, OV, and CCA subjects, and the proteomes were investigated by LC-MS/MS. Venn diagrams and protein network prediction by STITCH were used to identify the potential biomarkers. The level of candidate protein, the plasma checkpoint protein 1 (Chk1), was measured by indirect enzyme-linked immunosorbent assay (ELISA)., Results: Chk1 was present in the center of the protein network analysis in both the OV and CCA groups. In addition, the plasma Chk1 levels were significantly increased in both groups (P< 0.05). The sensitivity of the opisthorchiasis viverrini and cholangiocarcinoma was 59.38% and 65.62%, respectively, while the specificity of both was 85.71%., Conclusion: Chk1 was identified by differential plasma proteomes and was increased in O. viverrini-infected and cholangiocarcinoma-derived plasma samples. Higher levels of plasma Chk1 levels may serve as a potential diagnostic biomarker for opisthorchiasis and cholangiocarcinoma.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Aim: To study the activity of neutrophilic granulocytes in patients with opisthorchiasis, depending on the severity of liver fibrosis., Materials and Methods: A total of 74 patients with chronic opisthorchiasis (39 men and 35 women, average age 42.3 years) and 32 practically healthy patients (17 men and 15 women, average age 41.5 years) aged 24 to 60 years were examined. Diagnosis of opisthorchiasis was carried out by two methods: coprooscopy and identification of eggs or bodies of adult parasites in duodenal content. Liver fibrosis was determined by the method of elastometry according to the METAVIR scale in all 74 patients with opisthorchiasis. The study of the functional activity of neutrophils in the blood was performed to all 74 patients with opisthorchiasis and 32 healthy individuals from the control group by chemiluminescent analysis with measurement of the reactive oxygen species (ROS) production intensity in a spontaneous and zymosan-induced reaction in lucigenin and luminol-dependent processes., Results: Liver fibrosis F2 by METAVIR was registered in 20.3% of the examined individuals, liver fibrosis F3F4 by METAVIR was detected in 17.6% of patients with opisthorchiasis. In patients with opisthorchiasis with liver fibrosis F3F4 by METAVIR, a significant decrease in the functional activity of neutrophilic granulocytes was registered in comparison with individuals with liver fibrosis F0F1 by METAVIR, as evidenced by a significant decrease in the maximum intensity of ROS production (Imax) and the area under the curve (S) chemiluminescence in lucigenin and luminol-dependent processes both in the spontaneous and zymosan-induced reaction., Conclusion: These results provide new information to explain the mechanisms of liver fibrosis in patients opisthorchiasis and create opportunities for the development of diagnostics and preventive technologies.

Chronic infections of humans with Opisthorchis viverrini and Clonorchis sinensis spanning decades may lead to life-threatening pathology prior to cholangiocarcinoma (CCA), which usually has a poor prognosis. Serological tools can support the parasitological examination in clinical diagnosis and support screening for risk of CCA. We developed novel immunochromatographic test kits using a soluble, somatic tissue extract of adult O. viverrini worms as an antigen and colloidal gold-labeled conjugates of IgG and IgG4 antibodies, and evaluated the diagnostic values of both the OvSO-IgG and OvSO-IgG4 kits. For diagnosis of human opisthorchiasis individually, the diagnostic sensitivity, specificity, and positive and negative predictive values with 95% confidence intervals in the OvSO-IgG kit were 86.6% (78.9-92.3), 89.5% (84.2-93.5), 82.9% (74.8-89.2), and 91.9% (87.0-95.4), respectively, while the 75% (65.9-82.7), 98.4% (95.5-99.7), 96.6% (90.3-99.3), and 87% (81.7-91.2), respectively, for the OvSO-IgG4 kit at the prevalence of infection of 37.1%. Twenty-three (76.7%) and 14 (46.7%) of 30 clonorchiasis sera showed positive reactivity with the OvSO-IgG and OvSO-IgG4 kits, respectively. There was 84.1% (κ-value = 0.649) concordance between the two kits, which was statistically significant (p < 0.001). Both ICT kits can be employed as quick and easy point-of-care diagnostic tools, and hence, the OvSO-IgG and OvSO-IgG4 kits can support expanded capacity for clinical diagnosis of human opisthorchiasis and clonorchiasis. These kits may find utility in large-scale surveys in endemic areas where there are limited sophisticated medical facilities or capacity., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Opisthorchis felineus and Metorchis bilis are two common small worms that parasitize in the gallbladder and bile ducts of the liver of humans and carnivores. These parasites have a severe impact on health and are considered pathogens of serious diseases worldwide, such as cholangiocarcinoma. However, there are still no commercially available molecular diagnostic kits capable of simultaneously detecting these parasites in humans. Therefore, the study aimed to develop a multiplex PCR analysis that will differentially determine these two opisthorchiasis infections in one reaction. Two specific primer pairs for a multiplex polymerase chain reaction (PCR) were designed based on corresponding mitochondrial genome sequences. The multiplex assay detection limit was assessed by serial dilutions of the genomic DNAs of trematode worms examined. Naturally, infected samples of human bile and feces were tested using the developed assay. A multiplex PCR assay was developed based on mitochondrial DNA that accurately and simultaneously identifies two trematode species in one reaction using specific fragment sizes of 307 and 252 bp for O. felineus and M. bilis, respectively. The optimal reaction conditions, specificity, and sensitivity of the multiplex PCR assay were investigated. The lowest DNA concentration detected was 100 pg for M. bilis and O. felineus in a 25μl reaction system. This study provides an efficient tool for the simultaneous detection of O. felineus and M. bilis. The proposed multiplex PCR assay will be potentially useful in epidemiological studies, diagnosis, and treatment of this mixed opisthorchiasis infection.

Cholangiocarcinoma (CCA) is a common hepatobiliary cancer in East and Southeast Asia. The data of microbiota contribution in CCA are still unclear. Current available reports have demonstrated that an Opisthorchis viverrini (OV) infection leads to dysbiosis in the bile duct. An increase in the commensal bacteria Helicobacter spp. in OV-infected CCA patients is associated with bile duct inflammation, severity of bile duct fibrosis, and cholangiocyte proliferation. In addition, secondary bile acids, major microbial metabolites, can mediate cholangiocyte inflammation and proliferation in the liver. A range of samples from CCA patients (stool, bile, and tumor) showed different degrees of dysbiosis. The evidence from these samples suggests that OV infection is associated with alterations in microbiota and could potentially have a role in CCA. In this comprehensive review, reports from in vitro, in vivo, and clinical studies that demonstrate possible links between OV infection, microbiota, and CCA pathogenesis are summarized and discussed. Understanding these associations may pave ways for novel potential adjunct intervention in gut microbiota in CCA patients., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Opisthorchis viverrini and other foodborne trematode infections are major health concerns in the Greater Mekong Subregion. Currently, the gold-standard diagnostic method for opisthorchiasis is conventional stool examination for the presence of parasite eggs. This method lacks sensitivity and needs an experienced technician. We therefore produced monoclonal antibodies to highly immunogenic O. viverrini proteins aiming at detecting specific antigens in the feces. In this study, BALB/C mice were immunized using semi-purified somatic antigens and spleen cells were fused with a Sp2/0 myeloma cell line. Four hybridomas (1A2, 1E12, 2C7 and 8D6) were selected and cloned due to their strong reaction against O. viverrini somatic protein, resulting in three IgM clones and one IgG2 clone. Immunohistochemistry showed that 1A2, 1E12, 2C7 and 8D6 stained the parenchyma cells, gut, tegument and muscles, respectively. Western-blot analysis revealed that only antibody 1A2 could detect coproantigen (approx. 73 kDa protein) in feces of hamsters infected with O. viverrini. The 1A2 monoclonal antibody may be of value in the diagnosis of opisthorchiasis by coproantigen detection., (Copyright © 2020. Published by Elsevier B.V.)

Background: A urine antigen assay was applied to evaluate chemotherapeutic outcomes and reinfection patterns of opisthorchiasis in Thailand., Methods: We used a prospective study design by following opisthorchiasis subjects at baseline and post-treatment using a urine antigen assay and faecal examination by the formalin-ethyl acetate concentration technique (FECT)., Results: The antigen of Opisthorchis viverrini in urine diminished within 4 weeks after praziquantel treatment. Concurrent faecal examinations by FECT showed that faecal eggs were negative at 4 weeks after treatment. In a subsequent study, reinfection rates and intensity patterns of O. viverrini were evaluated at 48 weeks after praziquantel treatment. Within a group of subjects with curative treatment (n=137), 16.8% became reinfected according to FECT and 27.7% according to the urine antigen assay (p<0.05). There were significant correlations in intensity of infection between pretreatment and at 48 weeks post-treatment in both faecal egg counts and antigen levels in urine., Conclusions: The results suggested that in addition to screening, the urine antigen assay is an efficient tool for monitoring outcomes of drug treatment and reinfection in opisthorchiasis. Due to the ease of urine sample collection and handling, the urine assay becomes an alternative method to faecal examination for diagnosis and monitoring of opisthorchiasis., (© The Author(s) 2020. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Tegumental and excretory-secretory proteins are reported as diagnostic antigens for human opisthorchiasis. Rhophilin associated tail protein1-like (OvROPN1L) protein of Opisthorchis viverrini sperm tail showed potential as a diagnostic antigen. The OvROPN1L recombinant fragments were assayed for diagnostic antigenicity for human opisthorchiasis using indirect ELISA. The strongest antigenic region was a N-terminus peptide of M1 - P56. One synthetic peptide (P1, L3-Q13) of this region showed the highest antigenicity to opisthorchiasis. Sera from other parasitic infections including Strongyloides stercoralis, hookworm, Taenia spp, minute intestinal flukes, Paragonimus spp showed lower reactivity to P1. Peptide P1 is located in the disordered N-terminus of ROPN1L supporting its suitability as linear epitope. In the Platyhelminthes the N-terminal sequence of ROPN1L is diverging with taxonomic distance further suggesting that peptide P1 has potential as diagnostic tool in the genus Opisthorchis/Clonorchis. It should be further evaluated in combination with peptides derived from other O. viverrini antigens to increase its diagnostic power.

Background: The liver fluke, Opisthorchis felineus, is widely distributed throughout Europe and large parts of the Russian Federation. In Western Siberia, information about opisthorchiasis is lacking although infection may lead to severe liver and bile duct diseases. We aimed to assess the current prevalence of O. felineus infection along with associated risk factors and morbidity in rural Western Siberia., Methods: We conducted a community-based, cross-sectional study in the rural Shegarskiy district, Tomsk Oblast, Russian Federation. All household members (≥ 7 years) present on the survey day were enrolled (n = 600). Two stool samples per person were examined for helminth eggs, using PARASEP (DiaSys Ltd, UK). The number of eggs per gram (EPG) of feces was recorded. Each study participant was interviewed to determine risk factors, using a pre-tested questionnaire. An abdominal ultrasonography examination of liver and bile ducts was performed with a mobile, high resolution ultrasound device. In total, 488 persons completed assessments (two stool samples, completed questionnaires); of those, 436 individuals had an ultrasonography (US) examination., Results: We observed a prevalence of O. felineus infection of 60.2%. Significant risk factors for infection were the consumption of river fish (odds ratio from adjusted analysis [aOR] 2.4, 95% CI 1.52-3.95, p<0.001), particularly stock fish (OR from multivariable analysis [mOR] 3.2, 95% CI 2.63-3.80, p<0.001), smoked fish (mOR 1.5, 95% CI 1.24-1.72, p<0.001), frozen fish (mOR 1.6, 95% CI 1.29-2.02, p<0.001), and raw fish (mOR 1.4, 95% CI 1.05-1.84, p = 0.02); and fishing activities (mOR 1.2, 95% CI 1.03-1.43, p = 0.019). Women had a higher risk of infection than men. Infection was associated positively with age and negatively with socio-economic status. The respondents' general awareness of opisthorchiasis was quite high (93.2%), but their knowledge about infection transmission and prevention was insufficient. Children aged 7-18 years old had a lower level of awareness compared to adults. The abdominal ultrasonography results demonstrated a strong association between O. felineus infection and gallbladder stones (mOR 2.8, 95% CI 1.33-6.04, p = 0.007) and periductal fibrosis of intrahepatic bile ducts (mOR 1.9, 95% CI 1.08-3.46, p = 0.026)., Conclusion: O. felineus infection is highly prevalent in rural regions of Western Siberia, and associated with severe hepatobiliary pathology. Identified risk factors will be used to develop a comprehensive targeted O. felineus infection control program., Competing Interests: The authors have declared that no competing interests exist.

Human liver fluke infection caused by Opisthorchis viverrini increases the risk of cholangiocarcinoma (CCA) reported along the Mekong basin including Thailand, Lao People's Democratic Republic (PDR), Cambodia, and Vietnam. The highest incidence of CCA has been reported in northeastern Thailand where liver fluke infection is prevalent. This study aimed to investigate the prevalence of O. viverrini infection in a northeastern-descendent community in rural Sa Kaeo Province, eastern Thailand, using stool examination and molecular technique. The Kato-Katz method was performed to determine eggs per gram (EPG) for infection intensity. Phosphate-buffered saline-ethyl acetate concentration was used to prepare specimens for polymerase chain reaction (PCR) and restriction fragment length polymorphism of the internal transcribed spacer 2 (ITS2) region of the ribosomal RNA. From 1,245 specimens, 105 (8.4%) samples were identified as Opisthorchis -like eggs from stool examination, and all positive specimens indicated light infection (< 1,000 EPG). From positive Opisthorchis -like egg samples, 55.2% (58/105) were identified as O. viverrini eggs from ITS2-PCR assay for which low infection intensity might result in a negative PCR result (44.8%). Using multiple logistic regression analysis, males were at 3.1 times higher risk of acquiring O. viverrini infection than females. From phylogenetic analysis, in eastern Thailand, nucleotide sequences of O. viverrini were grouped as a monoclade as those isolated from Greater Mekong, Vietnam, Myanmar, and west Siberia. The results revealed that the surveyed community is a low-grade endemic area of O. viverrini infection. Thus, data from this study can be used to improve health-promoting programs and activities to control the infection and its subsequent CCA.

Chronic infections with the food-borne liver flukes, Opisthorchis viverrini or Clonorchis sinensis , associate with cholangiocarcinoma, bile duct cancer, which generally has a poor prognosis. We have produced a rapid and simple immunochromatographic test (ICT) kit for the diagnosis of opisthorchiasis and clonorchiasis by the detection of IgG antibodies in human infection sera. Sera from volunteers with proven opisthorchiasis and several other parasitic diseases and from healthy controls were evaluated for the presence of liver fluke infection-specific antibodies using a preparation of excretory-secretory antigen from adult stage O. viverrini absorbed onto ICT strips. Diagnostic values were compared with an ELISA. The diagnostic sensitivity, specificity, and positive and negative predictive values of the ELISA were 100%, 98.3%, 97.9%, and 100%, whereas those for the ICT were 94.6%, 91.2%, 89.7%, and 95.4%, respectively. There was 91.7% concordance between the ICT with ELISA, and differences in performance between the tests were not statistically significant ( P > 0.05). Twenty-seven of 30 (90%) of the clonorchiasis sera also were positive by ICT. This new ICT provides a facile, rapid test for point-of-care testing tool, which can be used at the bedside without the need for sophisticated equipment. Moreover, the ICT can be anticipated to supplement stool examination as a screening tool in the clinic for the diagnosis of opisthorchiasis and clonorchiasis, and in addition, it may be useful in screens of populations at risk of liver fluke infection-associated cholangiocarcinoma.

Objectives: To assess associations between periductal fibrosis (PDF) and bile duct dilatation (BDD) in ultrasonography (US) screening of population at risk of cholangiocarcinoma (CCA) due to residence in an endemic area for Opisthorchis viverrini. CCA survival rates are low, and early identification of risk factors is essential. BDD is one symptom that can identify patients at risk of CCA. Detection of PDF by US can also identify at-risk patients, at an earlier stage of CCA development. Identification of association between PDF and BDD will inform screening practices for CCA risk, by increasing the viability of PDF screening for CCA risk., Setting: Nine tertiary care hospitals in Northeast Thailand., Design: Cross-sectional study., Participants: Study subjects in the Cholangiocarcinoma Screening and Care Program (CASCAP) in Northeast Thailand. CASCAP inclusion criteria are all residents of Northeast Thailand aged ≥40 years. Participants are recruited through CCA screening centres and through primary healthcare units. So far, 394 026 have been enrolled., Methods: PDF and BDD were identified through US. PDF was categorised into three groups, PDF1, 2 and 3, depending on their high echo locality in the peripheral, segmental and main bile duct, respectively. Associations between PDF and BDD were determined by adjusted OR and 95% CI using multiple logistic regression., Results: BDD was found in 6.6% of PDF3, 1.7% of PDF2 and 1.4% of PDF1 cases. Among PDF cases, especially in PDF3, BDD was found in men more than in women (8.9% and 4.6%, respectively). Compared with non-PDF, the association between PDF3 and BDD was highly significant (adjusted OR=5.74, 95% CI 4.57 to 7.21, p<0.001)., Conclusions: Our findings reveal that there is a relationship between PDF and BDD, which is associated with CCA. Therefore, PDF can also be an indicator for suspected CCA diagnosis through US., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Opisthorchiasis, caused by Opisthorchis viverrini, remains the public health significance in Thailand, particularly in the northeastern region. Number of parasitological techniques is available for diagnosis. However, the detection the parasite's eggs in stool still referred as gold standard. Today, most people living in the endemic areas harbored the light infection. In this study, we compared the performance of formalin-ethyl acetate concentration technique (FECT), Kato-Katz technique, fecal parasite concentrator kit (FPCK) and direct simple smear technique for O. viverrini egg examination in stool. The results revealed that the FECT gave the best sensitivity (91.0%) followed by Kato-Katz technique, FPCK and direct simple smear techniques. Interestingly, the FECT showed the highest sensitivity in both groups of egg per gram (EPG) <50 (94.3%) and EPG ≥ 50 (100%). The FPCK and direct simple smear technique exhibited the higher sensitivity in EPG ≥ 50 group compared with EPG < 50 group (p <  0.05). However, the sensitivity of FPCK was not statistically differed from that direct simple smear in EPG <50 and EPG ≥50 group. In conclusion, the FECT showed the highest efficiency to detect O. viverrini egg in stool, followed by Kato-Katz technique, FPCK and direct simple smear technique. Substituting FPCK (Mini Parasep® Kit) for FECT and Kato-Katz method is not recommended for low intensity infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cholangiocarcinoma (CCA) is a cancer with high mortality owing to its aggressiveness and resistance to therapy. The liver flukes of the Opisthorchiidae family have been recognized as risk factors of CCA. Opisthorchis felineus infection occurs in Western Siberia, the biggest endemic area in the Russian Federation, and is associated with chronic inflammation of the bile ducts, which may be linked to severe hepatobiliary morbidity. We report two cases of confirmed CCA who had a chronic O. felineus infection. Both cases presented unspecific symptoms at the onset of the disease, a stage when severe pathological changes already had occurred. Both patients were living in endemic areas but did not receive any antihelminthic treatment. This report underlines the need for assessment of O. felineus infection as a causative factor of CCA. The results will provide further arguments for control of O. felineus in the Russian Federation.

To combat and eventually eliminate the transmission of the liver fluke Opisthorchis viverrini, an accurate and practical diagnostic test is required. A recently established urine antigen detection test using monoclonal antibody-based enzyme-linked-immunosorbent assay (mAb-ELISA) has shown promise due to its high diagnostic accuracy and the use of urine in place of fecal samples. To further test the utility of this urine assay, we performed a cross sectional study of 1,043 people in 3 opisthorchiasis endemic communities in northeast Thailand by applying urine antigen detection together with copro-antigen detection methods. The quantitative formalin-ethyl acetate concentration technique (FECT) was concurrently performed as a reference method. The prevalence of O. viverrini determined by urine antigen detection correlated well with that by copro-antigen detection and both methods showed 10-15% higher prevalence than FECT. Within the fecal negative cases by FECT, 29% and 43% were positive by urine and copro-antigen detection, respectively. The prevalence and intensity profiles determined by antigen detection and FECT showed similar patterns of increasing trends of infection with age. The concentration of antigen measured in urine showed a positive relationship with the concentration of copro-antigen, both of which were positively correlated with fecal egg counts. The data observed in this study indicate that urine antigen detection had high diagnostic accuracy and was in concordance with copro-antigen detection. Due to the ease and noninvasiveness of sample collection, the urine assay has high potential for clinical diagnosis as well as population screening in the program for the control and elimination of opisthorchiasis., Competing Interests: The authors have declared that no competing interests exist.

Clonorchis sinensis, Opisthorchis viverrini, and O. felineus are liver flukes of human and animal pathogens occurring across much of Europe and Asia. Nevertheless, they are often underestimated compared to other, better known neglected diseases in spite of the fact that many millions of people are infected and hundreds of millions are at risk. This is possibly because of the chronic nature of the infection and disease and that it takes several decades prior to a life-threatening pathology to develop. Several studies in the past decade have provided more information on the molecular biology of the liver flukes which clearly lead to better understanding of parasite biology, systematics, and population genetics. Clonorchiasis and opisthorchiasis are characterized by a chronic infection that induces hepatobiliary inflammation, especially periductal fibrosis, which can be detected by ultrasonography. These chronic inflammations eventually lead to cholangiocarcinoma (CCA), a usually fatal bile duct cancer that develops in some infected individuals. In Thailand alone, opisthorchiasis-associated CCA kills up to 20,000 people every year and is therefore of substantial public health importance. Its socioeconomic impacts on impoverished families and communities are considerable. To reduce hepatobiliary morbidity and CCA, the primary intervention measures focus on control and elimination of the liver fluke. Accurate diagnosis of liver fluke infections in both human and other mammalian, snail and fish intermediate hosts, are important for achieving these goals. While the short-term goal of liver fluke control can be achieved by praziquantel chemotherapy, a comprehensive health education package targeting school children is believed to be more beneficial for a long-term goal/solution. It is recommended that a transdisciplinary research or multisectoral control approach including one health and/or eco health intervention strategy should be applied to combat the liver flukes, and hence contribute to reduction of cholangiocarcinoma in endemic areas.

The present study describes a small liver fluke recovered at post-mortem and the pathological alteration produced by the parasite in the liver of a street dog from Assam, India. The spatulate parasite measured 3-4 mm in length and 1.0-1.25 mm in width with spiny body surface and showed a grossly visible peduncle projecting from the ventral surface at the anterior portion. The eggs of the parasite contained well developed miracidia. The parasite was identified as Paropisthorchis caninus. Identity of the parasite is discussed in the light of available literature and found to be morphologically different from other reported species of dog Opisthorchis. Pathological lesions observed in the liver included degeneration of hepatic parenchyma, hyperplasia of bile duct epithelium, fibrous tissue proliferation and formation of pseudo lobule which were the characteristics of chronic proliferative inflammation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objectives: To determine the potential for immunodiagnostic application of two recombinant forms of Clonorchis sinensis omega-class glutathione transferases (rCsGSTo1 and rCsGSTo2) against human small liver-fluke C. sinensis and Opisthorchis viverrini infections., Methods: Specific antibody levels against rCsGSTo1 and rCsGSTo2 in patients' sera of egg-positive opisthorchiasis (n = 87) and clonorchiasis (n = 120), as well as those in sera from patients with other helminthic infections (n = 252) and healthy controls (n = 40) were retrospectively analysed by ELISA., Results: We observed highly positive correlation coefficients between specific antibody levels against rCsGSTo1 and rCsGSTo2 and egg counts per gramme of faeces (EPG) of patients with opisthorchiasis (n = 87; r = 0.88 for rCsGSTo1 and r = 0.90 for rCsGSTo2). Sera from opisthorchiasis patients whose EPG counts >100 (n = 43) revealed high antibody titres against both antigens. Patients' sera with low EPG counts (<100, n = 44) also exhibited reliable sensitivities of 93.2% and 97.7% for rCsGSTo1 and rCsGSTo2, respectively. Sera from clonorchiasis patients showed sensitivities of 90% (108/120 samples) and 89.2% (107/120 sera) for rCsGSTo1 and rCsGSTo2. Overall diagnostic sensitivities for liver-fluke infections were 92.3% for rCsGSTo1 (191/207 samples) and 93.2% for rCsGSTo2 (193/207 samples). Specificities were 89.7% (rCsGSTo1) and 97.6% (rCsGSTo2)., Conclusions: Detection of specific antibody levels against rCsGSTo1 or rCsGSTo2 might be promising for the serodiagnosis of patients infected with these two phylogenetically close carcinogenic liver-flukes., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Parasitic diseases are relatively rarely diagnosed and treated in Europe. Therefore, European clinicians are not familiar with their clinical and imaging features. In an era of increased human migration, it is fundamental for clinicians to be able to identify such diseases. We have recently described the features of cystic echinococcosis, schistosomiasis, fascioliasis and ascariasis. Here, we report on the clinical and imaging features as well as on the current therapy options of infections by the small liver flukes: Clonorchis sinensis , Opisthorchis viverrini (Southeast Asian liver fluke) and Opisthorchis felineus (cat liver fluke) and other Opisthorchis species prevalent in South Asia., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (© Georg Thieme Verlag KG Stuttgart · New York.)

The carcinogenic liver fluke, Opisthorchis viverrini, is a serious health problem in Southeast Asia where infection is associated with cholangiocarcinoma, a major cause of death in Thailand. This cross-sectional study aimed to screen for O. viverrini infection among a Thai rural population of 560 individuals from Nakhon Ratchasima, Khonkaen, and Chaiyaphum provinces with a verbal screening test with a mobile application (OvApp). Faecal samples were also processed with a mini-parasep sf parasite faecal concentrator. The infection rate of O. viverrini was found to be 2.86%. The majority of infections were detected in males aged 41–50, with primary school being their highest education level, and who were engaged in agricultural occupations. In screening for O. viverrini infection, the OvApp had a high sensitivity (87.5%), specificity (94.6%), negative predictive value (98.9%), and accuracy (98.6%). The positive predictive value was 70.0% for the OvApp. The observed agreement was substantial for this application (k-value = 0.64) indicated that it is a potentially useful tool for decreasing the cost of large-scale O. viverrini screening., (Creative Commons Attribution License)

Previous studies demonstrated that urinary 8-oxodG is a predictive biomarker for Opisthorchis viverrini (OV)-associated hepatobiliary disease (HBD) and cholangiocarcinoma (CCA). This study examined the effects of praziquantel treatment on the profile of urinary 8-oxodG in relation to HBD status. Infection with OV, levels of urinary 8-oxodG, and HBD status in terms of periductal fibrosis (PDF) assessed by abdominal ultrasonography (US) were monitored and compared in cohorts of participants in Khon Kaen, Thailand, before and 1 year after praziquantel treatment. Urinary 8-oxodG levels significantly decreased after treatment compared with the baseline level in OV-infected participants who had no HBD (PDF negative; PDF-ve) ( N = 14). Levels of 8-oxodG were unchanged after treatment in OV-infected subjects (OV+ve) who had positive PDF ( N = 52). Within the positive PDF (PDF+ve) group who became PDF-ve after treatment, there was no significant change in 8-oxodG levels between pre-and posttreatment (reversible PDF = 65.3%). In those who had persistent PDF+ve at both ultrasound sampling points, there was no significant difference in urinary 8-oxodG levels between pre- and posttreatment (persistent PDF = 34.6%). Based on a logistic regression model and receiver operation curve analysis, the increase of 8-oxodG levels was found to be associated with increasing risk of PDF. Measurement of urinary 8-oxodG and US increased the likelihood of discovering persistent PDF, which is a predictable condition for the patients at risk of OV-associated CCA. To identify high-risk individuals for CCA, it is useful to perform US in combination with urinary 8-oxodG measurement.

Intestinal parasitic infection rate among school-aged children in Thailand has been decreasing. However, certain intestinal parasites remain problematic in some regions. This cross-sectional study was conducted between February and September 2016 in three suburban government primary schools (KK, BR, and HK), Saraburi, Thailand. Stool was collected from 263 asymptomatic subjects (4-15 years old), using simple direct smear, formalin-ether concentration, Boeck and Drbohlav's Locke-Egg-Serum (LES) medium culture, and agar plate culture. A self-administered questionnaire was used to collect data about lifestyle and socioeconomic status. The overall rate of intestinal parasites was 22.1% (15.6% single infection and 6.5% multiple infections). The helminths involving the digestive system found were Strongyloides stercoralis (1.5%) and Opisthorchis viverrini (0.4%). For protozoan infection, the major cause was Blastocystis hominis (17.5%). The other protozoa included Endolimax nana (4.6%), Entamoeba coli (3.4%), Entamoeba histolytica / Entamoeba dispar (1.1%), and Giardia intestinalis (0.8%). The sensitivity for the detection of B. hominis increased with the LES culture technique. The infection rate of each organism was not significantly different among the three schools except for B. hominis which showed the highest prevalence in the HK school ( P = 0.001). This was correlated with the questionnaire results in which the HK school showed the highest risk of drinking contaminated water ( P = 0.004). The present study emphasized the persistent problems of protozoan infections among suburban school-aged children. Lifestyle was still an important factor for intestinal parasitic infections among suburban school-aged Thai children in this study. Health education as well as routine surveillance was necessary to control the infections.

Objective: Introduction: Among the cases of biohelminthosis, which are registered in Ukraine, opisthorchiasis is the most common and constitutes the second largest centre in Europe. Due to the growth of migration processes and global tourism, opisthorchiasis is becoming increasingly relevant for the countries of the European Union and the United States. Under modern conditions, the clinical course of many infectious and parasitic diseases has changed. The aim: The present paper discusses and analyzes the cases of opisthorchiasis and hepatitis B virus which are challenging in terms of diagnostics and choice of treatment tactics., Conclusion: Conclusion: The course of acute infections, the formation of results and the effectiveness of treatment are influenced by a number of factors, among which mixed infections are of particular interest.

Opisthorchiasis is a neglected tropical disease, caused by infection with the fish-borne trematode Opisthorchis viverrini sensu lato that afflicts more than 10million people in Southeast Asia, including Thailand, Lao PDR, Vietnam and Cambodia. The disease is characterized by a chronic infection that induces hepatobiliary inflammation, especially periductal fibrosis, which can be detected by ultrasonography. This chronic inflammation eventually leads to cholangiocarcinoma (CCA), a usually fatal bile duct cancer that develops in approximately 1% of O. viverrini-infected individuals. In Thailand alone, CCA kills up to 20,000 people every year and is therefore of substantial public health importance. Its socioeconomic impacts on impoverished families and communities are considerable. To reduce O. viverrini-associated morbidity and CCA, the primary intervention measures focus on opisthorchiasis control and elimination. Accurate diagnoses of O. viverrini infection, in both mammalian, snail and fish intermediate hosts, are important for achieving these goals. Despite extensive efforts over several decades to find sensitive and specific diagnostics for opisthorchiasis, a simple and robust diagnostic method is still required. Here we review earlier and current developments in the search for new diagnostics for opisthorchiasis, with practical applications in the research laboratory, the clinic and the field. Of the methods currently available, the urine antigen assay shows considerable potential for the diagnosis and screening of opisthorchiasis. Nevertheless, these new assays require validation, determination of their cost-effectiveness when applied for mass screening in an endemic setting in support of policy decisions for national public health programs aimed at the control and elimination of opisthorchiasis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Opisthorchis viverrini infection is a major public health problem in northern and northeastern Thailand. The chronic infection of O. viverrini is related to cholangiocarcinoma which causes high mortality in endemic areas. Therefore, the diagnosis, treatment, control and prevention of O. viverrini infection are necessary. The morphology of the egg is very similar to that of other species of human liver flukes (Opisthorchis felineus and Clonorchis sinensis) as well as that of small intestinal flukes in the family Heterophyidae. Thus, molecular characterization is crucially required to discriminate species of Opisthorchis-like eggs in fecal examination., Methodology/principal Findings: We aimed to determine the prevalence of O. viverrini infection among villagers living in Sanamchaikate District, Chachoengsao Province, in central Thailand, where O. viverrini infection has previously been reported. A total of 2,609 fecal samples were examined for Opisthorchis-like eggs using microscopic examination. PCR-RFLP analysis of the ITS2 region was used to discriminate Opisthorchis-like eggs. The genetic structure of O. viverrini infection was demonstrated using nucleotide sequencing of cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 1 (nad1). Testing of evolutionary neutrality of the cox1 and nad1 sequences of O. viverrini was performed using Tajima's D tests and Fu's Fs tests. Moreover, the haplotype networks and phylogenetic trees were constructed to study the relationships of O. viverrini isolated from different endemic areas. A high prevalence of O. viverrini infection is still observed in a rural community of Chachoengsao Province, central Thailand. The overall prevalence of Opisthorchis-like eggs using microscopic examination was 16.8%. PCR-RFLP profiles showed the predominant infection of O. viverrini (9.6%) including very low infections of other small intestinal flukes, Haplorchis taichui (0.08%) and Euparyphium albuferensis (0.08%). The genetic structure of O. viverrini populations in central Thailand was also described and revealed a non-significant difference in genetic diversity. In addition, the genetic background of the O. viverrini populations was closely related to the isolate from Lao PDR., Conclusions/significance: Our study highlighted the prevalence of O. viverrini infection in central Thailand indicating that control programs and health education regarding opisthorchiasis is still required in this endemic area. Additionally, the study demonstrated the genetic structure of O. viverrini, in central Thailand which could provide information on the molecular epidemiology of this parasite.

Opisthorchis viverrini infection induces chronic inflammation in the bile ducts, leading to periductal fibrosis (PDF), which possibly associates to cholangiocarcinoma (CCA). Patients with CCA have a poor prognosis, which is linked to asymptomatic disease and late diagnosis. Hence, detecting early stage CCA is essential. Secretory miRNAs have been promoted as biomarkers for pathological changes associated with parasitic infections, fibrosis and/or cancer. We aimed to determine levels of miR-192 and miR-21 in the urine of O. viverrini infected, periductal fibrosis (PDF) and CCA groups using qRT-PCR. We found that miR-192 was significantly higher in O. viverrini infected, PDF and also CCA groups (p<0.05) than in healthy controls. By utilizing the Receiver Operation Characteristics (ROC) analysis, miR-192 differentiated patients with opisthorchiasis (the area under the curve; AUC=0.766), PDF subjects (AUC=0.781) and CCA patients (AUC=0.682) from healthy controls. MiR-21 was significantly higher in PDF and CCA groups (p<0.05) than in healthy controls. MiR-21 discriminated PDF subjects (AUC=0.735) and CCA patients (AUC=0.682) from healthy controls. Combined levels of these two miRNAs revealed an increased AUC of 0.812 for separating opisthorchiasis, AUC of 0.815 in discriminating PDF subjects, and AUC of 0.849 in differentiating CCA from healthy controls. Odds ratios (OR) indicated high levels of miR-192/miR-21 as risk predictors for opisthorchiasis, PDF and CCA. Levels of these miRNAs declined significantly for patients following praziquantel treatment. In conclusion, urinary miR-192/miR-21 have potential as risk indicators for opisthorchiasis and PDF-associated CCA in the endemic region., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Diagnosis of Opisthorchis viverrini infection by conventional stool examination is increasingly difficult due to the low intensity of the infection after several rounds of control programmes in endemic regions as well as coinfections with intestinal flukes. Therefore sensitive and specific diagnostic test is needed. In this study, a coproantigen sandwich ELISA using recombinant O. viverrini cathepsin F (rOv-CF) was developed. This sandwich ELISA employing chicken IgY raised against rOv-CF in combination with rabbit IgG antibody to the somatic O. viverrini antigens showed a lower detection limit (LLD) of 70ng native O. viverrini somatic antigens by spiking the parasite antigens into control feces. When applied to the diagnosis, the IgY-based sandwich ELISA exhibited sensitivity and specificity of 93.3% and 76.7%, respectively, in an investigation of 90 human cases positive or negative for opisthorchiasis. The positive predictive value (PPV) and negative predictive value (NPV) for this coproantigen detection were 66.7% and 95.2%, respectively. This IgY-based sandwich ELISA using parasite cathepsin F detection shows a promising immunodiagnostic alternative for human opisthorchiasis in endemic regions., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

L-type amino acid transporter 1 (LAT1) is highly expressed in various human cancers, including cholangiocarcinoma (CCA), the most common cancer in Northeast Thailand. Chronic inflammation and oxidative stress induced by liver fluke, Opisthorchis viverrini, infection has been recognized as the major cause of CCA in this area. We show here that an increased expression of LAT1 and its co-functional protein CD98 are found during carcinogenesis induced by Ov in hamster CCA tissues. We also demonstrate that oxidative stress induced by H 2 O 2 is time-dependent and dramatically activates LAT1 and CD98 expression in immortal cholangiocytes (MMNK1). In addition, H 2 O 2 treatment increased LAT1 and CD98 expression, as well as an activated form of AKT and mTOR in MMNK1 and CCA cell lines (KKU-M055 and KKU-M213). We also show that suppression of PI3K/AKT pathway activity with a dual PI3K/mTOR inhibitor, BEZ235, causes a reduction in LAT1 and CD98 expression in KKU-M055 and KKU-M213 in parallel with a reduction of activated AKT and mTOR. Interestingly, high expression of LAT1 in human CCA tissues is a significant prognostic factor for shorter survival. Taken together, our data show that LAT1 expression is significantly associated with CCA progression and cholangiocarcinogenesis induced by oxidative stress. Moreover, the expression of LAT1 and CD98 in CCA is possibly regulated by the PI3K/AKT signaling pathway., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Opisthorchis felineus (family Opisthorchiidae) is a parasitic flatworm representing a serious threat to humans in some countries. Opisthorchiasis occurs after consumption of raw or undercooked cyprinid fish infected by the metacercarial stage of the parasite. Due to its small size, detection of the parasite in fish fillet is time-consuming and difficult. Furthermore, isolated metacercariae can be identified to genus but not to species level using morphological features and molecular techniques are necessary. In this work, we describe the development of primers for a diagnostic PCR amplification of a 254-bp fragment of the cytochrome c oxidase I in the mitochondrion of Opisthorchis felineus metacercariae isolated from fish fillet, together with a validation protocol for this method., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Treatment of the liver fluke infection Opisthorchis viverrini relies exclusively on praziquantel. Tribendimidine could be an alternative treatment option. We aimed to assess the efficacy and safety of ascending single, oral doses of tribendimidine in patients with O viverrini infection., Methods: We did two randomised, parallel-group, single-blind, dose-ranging, phase 2 trials in children (aged 8-14 years) and adults and adolescents (≥15 years) in three O viverrini endemic villages in Champasack province, southern Laos. Patients with O viverrini infection were randomly assigned, via a computer-generated central block-randomisation procedure, with block sizes of three (study 1) and four, eight, and 12 (study 2), to receive oral tribendimidine at doses of 200 mg, 400 mg, or 600 mg in a 1:1:1 ratio (adults and adolescents in study 1); 25 mg, 50 mg, 100 mg, or 200 mg (four 50 mg tablets) in a 1:1:1:1 ratio (adults and adolescents in study 2); or 100 mg, 200 mg, or 400 mg in a 1:1:1 ratio (children in study 1). One non-randomised group of children received tribendimidine 50 mg (study 2). Participants, investigators, and laboratory technicians doing the diagnostic assessments were masked to group assignment, but the investigator administering treatment could have recognised the treatment group based on the number of tablets. The primary objective was to estimate the dose-response relation in terms of cure rate and egg reduction rate. We did available-case analysis of all patients with primary endpoint data. We predicted dose-response associations with Emax models. This trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN96948551., Findings: Between Oct 25, 2012, and Nov 5, 2013, 318 adolescents and adults were randomly assigned to seven tribendimidine dose groups: 200 mg (n=51), 400 mg (n=49), or 600 mg (n=47) in study 1, and 25 mg (n=39), 50 mg (n=47), 100 mg (n=44), or 200 mg (four 50 mg tablets; n=41) in study 2. 128 children were randomly assigned to receive tribendimidine 100 mg (n=44), 200 mg (n=40), or 400 mg (n=44) in study 1; 39 children were enrolled and received tribendimidine 50 mg in study 2. In adolescents and adults, the number of patients cured increased with increasing tribendimidine doses up to 100 mg: ten of 39 patients (25·6%, 95% CI 13·0-42·1) were cured in the 25 mg group, 20 of 47 patients (42·6%, 28·3-57·8) were cured in the 50 mg group, and 34 of 44 patients (77·3%, 62·2-88·5) were cured in the 100 mg group; geometric mean egg reduction rates were 86·9% (95% CI 74·8-93·4), 95·9% (92·7-97·7), and 99·1% (98·2-99·7), respectively. The 200 mg dose resulted in cure in 40 of 47 (83·0%, 69·2-92·5) adolescents and adults given the 200 mg tablet and 25 of 41 (61·0%, 95% CI 44·5-75·8) of those given four 50 mg tablets; the 400 mg dose resulted in cure in 43 of 47 patients (91·5%, 79·6-97·6) and the 600 mg dose resulted in cure in 36 of 45 patients (80·0%, 65·4-90·4). Corresponding egg reduction rates were 99·8% (95% CI 99·7-100·0) with one 200 mg tablet, 97·9% (95·9-99·2) with four 50 mg tablets, 99·9% (99·8-100·0) with 400 mg, and 99·8% (99·6-99·9) with 600 mg. The Emax model predicted an egg reduction rate of 99·0% (95% CI 95·7-99·8) at 111 mg in adolescents and adults. 50 mg tribendimidine had moderate efficacy in children, with cure recorded in 16 of 39 patients (41·0%, 95% CI 25·6-57·9). The 100 mg dose resulted in cure in 40 of 44 children (98·9%, 95% CI 78·3-97·5) and an egg reduction rate of 99·7% (95% CI 99·0-100·0), with no increased efficacy at higher doses. The Emax model predicted an egg reduction rate of 99·0% (95% CI 92·2-99·9) at 215 mg. Few adverse events were reported and were mostly mild, with few moderate and no serious events. The most common adverse events 3 h after treatment in adolescents and adults were vertigo (n=35 [11%]), headache (n=9 [3%]), nausea (n=6 [2%]), and fatigue (n=4 [1%]), and in children were headache (n=3 [2%]), vertigo (n=2 [1%]), and fatigue (n=2 [1%])., Interpretation: Tribendimidine has excellent efficacy and tolerability at doses of 100 mg and above. Our study included mainly adults and children with low-intensity O viverrini infection; future studies should assess the efficacy of tribendimidine in patients with infections of moderate and high intensity., Funding: Department for International Development, Medical Research Council, Wellcome Trust Joint Global Health Trials Scheme., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Opisthorchis viverrini is endemic in the Lower Mekong Basin, including Thailand, Lao People’s Democratic Republic, Cambodia, and central Vietnam. This figure indicates that it is a serious public health problem in Thailand, particularly in northeastern and northern region., Objective: To survey the nutritional status among schoolchildren including the association between nutritional status and related O. viverrini infection that is reported in their living areas., Material and Method: A cross-sectional survey of nutritional status among schoolchildren was conducted in the rural communities of Surin province, Thailand, between September 2013 and July 2014. Nutritional evaluations, Socio-economic, and family information were collected through questionnaires. Four hundred sixty two children aged between 6 to 14 years were enrolled. Pearson correlation test and multiple logistic regression were used in this study., Results: Most respondents who answered the questionnaire were mother (37.23%), and followed by children (34.41), female (51.73%), and age rank older than 9 to 12 years old (41.99%). Schoolchildren were evaluated for weigh for age, which resulted in normal weight (60.82%), wasting (8.01%), underweight (7.58%), obesity (2.38%), and overweight (1.95%). Height for age was divided into normal height (60.17%), stunting (0.74%), slightly short (4.76%), tall (3.46%), and slightly tall (2.6%). Weight for height was divided into appropriate (54.11%), thin (10.82%), slightly thin (8.02%), obesity (2.38%), and morbid obesity (1.73%). Children were not infected with O. viverrini in this study even if 15.8% of them consumed undercooked particularly cyprinoid fish. Children with slightly underweight was statistically significant associated with O. viverrini infection (Adjust R square = 0.445, p<0.05). The equation for prediction of liver fluke infection among schoolchildren who had slightly underweight is Y = -0.508 + 0.517 X(12) and Z = 0.694 X(12)., Conclusion: The present study indicates that stunting, underweight and wasting children were found in high infectious areas of O. viverrini. Slightly underweight was associated with infection. Therefore, implementation of nutrient in this group is urgently required. Furthermore, health education for children and relative family should be encouraged for further campaign in this province.

Opisthorchiasis and cholangiocarcinoma (CCA) are major public health problems in Thailand and countries in the lower Mekong Subregion. Elimination of opisthorchiasis will be an important step toward the prevention, control and reduction of CCA. In order to achieve this goal, a sensitive and robust diagnostic method is required to identify people with current Opisthorchis viverrini sensu lato infection as the parasite is a group 1 carcinogen believed to be an etiology of CCA. To date, sensitive parasitological methods, such as formalin-ethyl acetate concentration technique (FECT) is preferred, but it is not practical in a remote primary care setting. In this study, we evaluated the diagnostic accuracy of a commercial stool concentrator kit with that of a direct simple smear method and a modified FECT. In diagnosing parasite infection and opisthorchiasis, the commercial kit had greater sensitivity (43.8-58.5%) than direct smear method (12.5-31.7%), but was less sensitive than FECT (73.2-75%). In a separate sample population, similar results were obtained when comparing the diagnostic accuracy of the commercial kit and FECT. However, the commercial kit was more effective in a field setting than FECT, and had better accuracy than direct smear method, which suggests that the kit could have potential utility in epidemiological studies and control programs of opisthorchiasis, as well as other parasitic infections. The design of the self-contained one-tube kit plus its long storage time after sample preparation provides a considerable advantage over other methods, such as direct or Kato thick smear method, under similar field conditions.