Multi-epitope protein production and its application in the diagnosis of opisthorchiasis.

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.

Investigation of Plasma Cell-Free DNA and MiRNA in Cholangiocarcinoma and Opisthorchiasis Viverrini Patients.

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.

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.

Current State of Knowledge on Blood and Tissue-Based Biomarkers for Opisthorchis viverrini-induced Cholangiocarcinoma: A Review of Prognostic, Predictive, and Diagnostic Markers.

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.

Clonorchiasis and opisthorchiasis: epidemiology, transmission, clinical features, morbidity, diagnosis, treatment, and control.

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.

Accuracy of a new rapid diagnostic test for urinary antigen detection and assessment of drug treatment in opisthorchiasis.

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.

Influence of misconceptions and inappropriate eating behaviors on Opisthorchis viverrini infection among at-risk populations undergoing cholangiocarcinoma screening in Northeastern Thailand.

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.

Long-term Persistence of Opisthorchis viverrini Antigen in Urine: A Prospective Study in Northeast Thailand.

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.

Opisthorchis Felineus Infection is a Risk Factor for Cholangiocarcinoma in Western Siberia: A Hospital-based Case-control Study.

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.

Effects of day-to-day variation of Opisthorchis viverrini antigen in urine on the accuracy of diagnosing opisthorchiasis in Northeast Thailand.

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.

Bithyniid snails as hosts of Opisthorchiidae and Notocotylidae in the south of Western Siberia, Russia.

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.

Performance of Mini Parasep® SF stool concentrator kit, Kato-Katz, and formalin-ethyl acetate concentration methods for diagnosis of opisthorchiasis in Northeast Thailand.

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.

Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta as a potential biomarker for Opisthorchis viverrini infection and cholangiocarcinoma.

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.

Association between Opisthorchis viverrini Infection and Glomerular Disease in Thailand.

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.

Plasma checkpoint protein 1 (Chk1) as a potential diagnostic biomarker for opisthorchiasis and cholangiocarcinoma.

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.

Rapid assessment of Opisthorchis viverrini IgG antibody in serum: A potential diagnostic biomarker to predict risk of cholangiocarcinoma in regions endemic for opisthorchiasis.

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.

Comparative assessment of immunochromatographic test kits using somatic antigens from adult Opisthorchis viverrini and IgG and IgG4 conjugates for serodiagnosis of human opisthorchiasis.

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.

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.

A MULTIPLEX PCR ASSAY FOR THE DIFFERENTIAL DETECTION OF OPISTHORCHIS FELINEUS AND METORCHIS BILIS.

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.

[Functional activity of blood neutrophilic granulocytes in patients with opisthorchiasis, depending on the severity of liver fibrosis].

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.