Viverrini Infection Research Articles

Enhanced detection of Opisthorchis viverrini infection: A comparative evaluation of modified one-step FECT and conventional diagnostic methods in low-intensity setting

The formalin-ethyl acetate concentration technique (FECT) is one of the most sensitive diagnostic method not only for all helminths, but also for Opisthorchis viverrini infections in stool examinations. However, it remains a diagnostic problem for light infections. We modified the one-step FECT to determine the low-intensity of O. viverrini infection and compared with various conventional detection methods. The study utilized 160 egg-positive and 160 randomly negative stool samples for O. viverrini eggs by conventional FECT (cFECT) to compare the methods, including the simple smear, the Kato-Katz method, the two commercial stool examination kits, and the one-step FECT. Our results showed that the one-step FECT method had the highest sensitivity (95.6 %), followed by cFECT (87.9 %), the Kato-Katz (55.5 %), Aquisfek SF-FIX (48.3 %), simple smear (42.3 %), and Mini Parasep® SF (35.1 %). The ability of one-step FECT exhibited better ability to detect low parasite intensities compared to the cFECT (18 eggs per gram (e.p.g.) versus 34 e.p.g.) and the other conventional diagnostic methods. In addition, the investigation of O. viverrini infection in endemic regions in northeastern Thailand based on 3900 fecal samples revealed that the one-step FECT with an intensity of 66.8 e.p.g. (range 18–226) was significantly higher in sensitivity than cFECT, which had an intensity of 58.0 e.p.g. (range 34–214). Interestingly, fecal samples with less than 50 e.p.g. could not be detected by cFECT in 67 % of cases, and 69 out of 3900 samples were negative. In conclusion, one-step FECT improves the detection of low-intensity O. viverrini infection, which is suitable for parasites screening, especially for low-intensity infections in the community.

Risk factors of cholangiocarcinoma in areas not endemic for liver fluke infection.

Thailand has the world's highest prevalence of cholangiocarcinoma (CCA), especially in the endemic area of liver fluke Opisthorchis viverrini infection. However, other regions of Thailand still have relatively high CCA prevalence. We aimed to determine CCA risk factors in areas not endemic for OV infection. A case--control study was performed at a referral center during December 2016-December 2017. We collected blood samples and information from CCA patients and identified them as cases. The control group comprised patients who visited a gastrointestinal clinic for colorectal cancer screening colonoscopy. Logistic regression analysis was used to determine risk factors for CCA. Of 138 participants, O. viverrini infection rate was higher in the case than in the control group (57.1% vs. 36.1%, P = 0.023). Male, O. viverrini infection, smoking, alcohol consumption, and biliary tract diseases were independent risk factors, whereas diabetes, obesity, and cirrhosis were not associated with CCA. By age and sex-adjusted analysis, chronic biliary tract diseases, especially choledochal cysts and smoking, were risk factors for CCA, with adjusted odds ratio (aOR) of 12.7 (95% confidence interval [CI]: 1.4-116.9) and 3.8 (95% CI: 1.3-11.8), respectively, while O. viverrini infection became insignificant risk for CCA (aOR 1.8, 95% CI: 0.8-4.1). In contrast with endemic areas for O. viverrini infection, chronic biliary tract diseases and smoking are major risk factors, whereas O. viverrini infection has trivial contribution to the development of CCA.

Detection of the carcinogenic liver fluke, Opisthorchis viverrini: comparison of two coprological methods versus the automatic feces analyzer.

Liver fluke infection, particularly Opisthorchis viverrini, poses a significant public health risk in Thailand, where it is closely associated with cholangiocarcinoma and contributes to substantial mortality in the northeastern region. Diagnosis of this condition employs various parasitological approaches. This research aims to compare the diagnostic accuracy of three parasitological techniques: the Kato Katz technique (KKT), the formalin-ethyl acetate concentration technique (FECT), and the Fully Automatic Feces Analyzer (FAFA) for O. viverrini identification. A total of 455 fecal specimens were collected from rural areas across five provinces in northeastern Thailand. The specimens were processed according to each method and examined through microscopy for KKT and FECT, and by utilizing an artificial intelligence-based machine for FAFA. Data analysis was conducted to assess parasitic infection rates and observe diagnostic accuracy. The results revealed a parasitic infection rate of 19.34%, with the majority of infections attributed to O. viverrini (18.02%), followed by Strongyloides stercoralis (0.88%). FECT exhibited the highest positive detection of O. viverrini eggs (16.48%), followed by FAFA (10.55%), and KKT (8.57%), respectively. Statistical analysis indicated sensitivity and specificity values for O. viverrini detection by KKT (100% and 89.21%), FECT (98.67% and 97.63%), and FAFA (97.92% and 91.15%). The positive predictive value, negative predictive value, and kappa were reported for FECT (89.16%, 99.73%, 0.92), FAFA (56.63%, 99.73%, 0.67), and KKT (45.78%, 100%, 0.58). Additionally, the preparation time for KKT, FECT, and FAFA was 30, 15, and 10 min, respectively. In conclusion, this study highlights FECT, KKT, and FAFA as comparably sensitive in diagnosing O. viverrini. The FAFA machine emerges as a potentially valuable tool for detecting O. viverrini and other parasitic infections, showcasing promise for clinical use. The findings provide valuable insights into the diagnostic landscape and underscore the potential of FAFA in enhancing efficiency and accuracy in parasitological assessments.

Unveiling the Transmission Potential of Opisthorchis viverrini and Intestinal Helminths Along the Thailand-Laos Border in Thailand.

In pursuit of enhancing prevention efforts for the notably high endemic Opisthorchis viverrini infection in lower Mekong sub-region countries, particularly Thailand and Laos, this cross-sectional study investigated the transmission potential of O. viverrini and other intestinal helminths along the Thailand-Laos border in seven Thai villages. Human and pet faecal samples, Bithynia snails and cyprinid fish were analysed for helminth infections. Additionally, a questionnaire survey assessed relevant risk factors among the human population. Two groups of helminth infections were detected in humans: foodborne infections (FBIs) including O. viverrini, minute intestinal flukes (MIFs), and Taenia spp., and soil-transmitted infections (STIs) including hookworm and Strongyloides stercoralis, with prevalence rates of 7.4%, 0.5%, 2.5%, 0.5%, 2.5% and 3%, respectively. Smoking was identified as a risk factor for O. viverrini infection [adjusted odds ratios (ORa) = 3.12, 95% confidence intervals (CI): 1.33-7.30, p = 0.009] and FBIs (ORa = 2.47, 95% CI: 1.14-5.33, p = 0.022), while male was a risk factor for FBIs (ORa = 2.62, 95% CI: 1.16-5.94, p = 0.021). In dogs, hookworm, Toxocara spp., Spirometra mansoni, Trichuris vulpis and Hymenolepis diminuta were identified with prevalence rates of 35.6%, 8.1%, 8.1%, 1.2% and 1.2%, respectively. In cats, hookworm, Toxocara spp., S. mansoni, Strongyloides spp., Platynosomum fastosum, MIFs and H. diminuta were identified with prevalence rates of 50%, 17.9%, 10.7%, 7.1%, 3.6%, 3.6% and 3.6%, respectively. Bithynia snails showed 2% virgulate and 0.7% unknown cercariae infections, while among 19 cyprinid fish species, only unknown metacercariae were found. Our findings underscore the necessity of an integrated approach following the One Health concept to effectively combat these parasitic diseases while addressing human, animal and environmental health.

MicroRNA profiling of exosomes derived from plasma and their potential as biomarkers for Opisthorchis viverrini-associated cholangiocarcinoma

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.

Characteristics of SARS-CoV-2 and Opisthorchis viverrini coinfections: insights into immune responses and clinical outcomes.

The effects of co-infections with SARS-CoV-2 and parasitic diseases have been little investigated in terms of immune response, disease dynamics, and clinical outcomes. This study aimed to explore the impact of co-infection with Opisthorchis viverrini and SARS-CoV-2 on the immune response concerning clinical symptoms and the severity of pulmonary abnormalities. A cross-sectional study was conducted, including healthy participants as controls, participants with opisthorchiasis, SARS-CoV-2 infection, and a co-infection group with both diseases. Characteristics of SARS-CoV-2 infection were assessed based on clinical parameters and severity of pulmonary abnormalities, whereas opisthorchiasis burden was evaluated by eggs-per-gram (EPG) counts. Immune responses were assessed by measuring levels of interferon-γ (IFN-γ), SARS-CoV-2 anti-spike receptor binding domain (RBD) IgG, and neutralizing antibody against SARS-CoV-2. In the co-infected group, clinical parameters and hospitalization rates were lower than in the SARS-CoV-2 group. Pulmonary abnormalities, such as bronchial fibrosis, were commonly observed in the SARS-CoV-2 group, leading to hospitalization in some cases. Participants with opisthorchiasis had higher IFN-γ levels than healthy individuals. IFN-γ levels were significantly lower in the co-infection group compared with the SARS-CoV-2 group (P = 0.002). There was a significant (P = 0.044) positive correlation between RBD-specific IgG and percent neutralization levels in the SARS-CoV-2 group. Levels of both were somewhat lower (not statistically significant) in the co-infection group. A negative correlation was observed between opisthorchiasis burden (EPG counts) and IFN-γ and RBD-specific IgG levels in the co-infected group. Following vaccination, the increase in IgG levels against the RBD protein was significantly lower in the co-infected group than in the SARS-CoV-2 group. These results suggest that O. viverrini infection suppresses immune responses and may lead to a reduction in severity in cases of SARS-CoV-2 co-infection.

Integrated Eco-Health approach significantly reduces helminth infections in endemic Khong islands with emphasis on Schistosoma mekongi

BackgroundHelminth infections, including Opisthorchis viverrini, hookworm, and Trichuris trichiura, are prevalent in Khong district, Champasack province, southern Lao People’s Democratic Republic (PDR). Schistosomiasis caused by Schistosoma mekongi is of public health concern on the islands of the Khong district. This study aimed to assess the impact of an Eco-Health/One-Health approach in combination with mass drug administration (MDA) to reduce these helminth infections.MethodsWe conducted a community intervention using a stepped-wedge trial approach on two endemic islands (Donsom and Donkhone) of the Khong district, Champasack province, Lao PDR, between April 2012 and March 2013. In each study village, 30–40 households were randomly selected. All members of selected households, who were at home during the study period were invited to participate in the study. A baseline study was conducted to assess helminth infections, knowledge attitudes and practices toward Schistosoma mekongi infection, behavior of open defecation and availability of latrine at home. After the baseline (T0), the Eco-Health/One-Health approach was implemented on Donsom (intervention) and Donkhone island (control). An assessment was conducted in 2014 (T1), one year after the completion of intervention implementation, to assess the short-term impact of the Eco-Health/One-Health approach on helminth infections and compare intervention and control islands. Later in 2015, the Eco-Health/One-Health approach was implemented on control island (Donkhone). After the implementation of intervention, the parasitological assessments were conducted annually in humans in 2015 (T2), in 2016 (T3) and in 2017 (T4), and in dogs in 2017 (T4) to evaluate the long-term impact of the intervention on helminth infections. Frequency was used to describe the prevalence of helminth infections. Logistic regression was applied to associate the KAP (knowledge, attitudes, and practices and open defecation behavior) and the reduction of helminth infections between intervention and control islands. The reduction in prevalence pre- and post-intervention was associated using a McNemar test. A two-independent sample t-test was applied to compare the mean eggs per gram (EPG) of helminth infections between control and intervention islands. A paired t-test test was used to compare the mean EPG of stool samples before (baseline) and after (follow-up) interventions for the two islands separately. A P-value lower than 0.05 was considered statistically significant.ResultsEco-Health/One-Health approach appears to be associated with reduction in prevalence of S. mekongi by 9.0% [odds ratio (OR) = 0.49, P = 0.003] compared to the use of mass drug administration alone (control island). Additionally, this intervention package significantly reduced O. viverrini infection by 20.3% (OR = 1.92, P < 0.001) and hookworm by 17.9% (OR = 0.71, P = 0.045), respectively. Annual parasitological assessments between 2012 and 2017 showed that the Eco-Health/One-Health approach, coupled with MDA, steadily reduced the prevalence of S. mekongi on the intervention island from 29.1% to 1.8% and on the control island from 28.4% to 3.1%, respectively.ConclusionsThe study findings suggest that the Eco-Health/One-Health approach appears to be associated with a significant reduction in prevalence of S. mekongi and helminth co-infections, particularly hookworm and T. trichiura. Therefore, implementing the Eco-Health/One-Health approach in schistosomiasis-endemic areas could accelerate the achievement of national goals for transmission interruption by 2025 and elimination by 2030.

Integrated One-Health approach for prevention and control of Opisthorchis viverrini infection in rural Thailand: a 3-year study.

Opisthorchis viverrini infection is a pressing health issue in rural Southeast Asia and is associated with the risk of cholangiocarcinoma. Despite control efforts, high infection rates persist, including evidence of reinfection post-treatment. This study aimed to address this public health concern through an integrated One-Health approach in endemic areas in rural Thailand over a 3-year period. The study included data from 3600 participants from Udon Thani Province, Thailand, during the years 2020 to 2022 and involved integrated epidemiological data collection and risk factor analysis to understand the impact of various interventions on disease transmission in the community. The efficacy of interventions was assessed by monitoring the incidence of O. viverrini reinfection in 2021 and 2022. In 2020, 218 cases of O. viverrini infection (6.0%) were identified. Significant risk factors included proximity to water bodies and consumption of raw fish. Variables contributing to infection risk among participants (P < 0.001) were education level, engagement in traditional ceremonies, poor sanitation, absence of ducks in nearby water bodies, self-medication for parasitic conditions, and multiple infections within a household. Dogs, cats, and cyprinoid fish showed prevalence rates of 5.4%, 6.3%, and 11.5%, respectively. Geographic analysis revealed clusters of infected households around water bodies. Interventions, including in-depth interviews, focus-group discussions, health education, anthelminthic treatment, and biological control using local free-range ducks, were implemented, resulting in no human reinfections in the second year and a minimal 0.3% prevalence rate in the third year. This study offers valuable insights into the dynamic changes in infection prevalence, making a significant contribution to effective disease control and community health promotion. This integrated One-Health approach proved to be an effective strategy for the prevention and control of opisthorchiasis.

Anthelmintic activity and pathophysiological effect of anthelmintic drugs against carcinogenic liver fluke, Opisthorchis viverrini.

Human liver fluke, Opisthorchis viverrini poses a significant risk for development of cholangiocarcinoma (CCA) in Thailand, primarily attributed to consumption of undercooked cyprinoid fishes. The current use of anthelmintic drug treatment such as praziquantel (PZQ), as the main therapeutic agent against O. viverrini. There is a need to explore the efficacy of alternative anthelmintic drugs for O. viverrini treatment. This study aimed to assess the efficacy of anthelmintic drugs, which are commonly use in endemic areas of Southeast Asian countries; PZQ, albendazole (AL), niclosamide (NI), and mebendazole (ME) at concentrations of 600, 400, 500, and 500 mg/ml. The study included a negative and positive control group treated with roswell park memorial institute (RPMI) and PZQ. Reactive oxygen species (ROS) levels, indicative of oxidative stress, were quantified using 2',7'-dichlorofluorescein diacetate staining. Morphological changes were observed using scanning electron microscopy. Furthermore, motility assessments were conducted at various time points (0, 5, 30 minutes, 1, 3, 6, 12, and 24 hours), calculating relative motility (RM) and survival index (SI). The results revealed a significant increase of ROS levels with the intensity and corrected total worm fluorescence (CTWF) mostly observed in order of PZQ, followed by NI, ME, and AL, respectively. Morphological damage was presented the tegumental swelling, papillae changes, and disruption of microvilli (Mv), particularly in the group treated with the most effective anthelmintics PZQ, NI, ME, and AL, while negative control group did not exhibit such alterations. Also, the most efficacy for suppressing the motility of adult worms were displayed in PZQ treatment group, followed by NI, ME, and AL, respectively. Overall, first novel findings suggest that apart from NI, ME, and AL demonstrate potential as alternative therapeutic options for O. viverrini infection. Furthermore, animal model is needed to investigate the efficacy of NI, ME, and AL compare with standard treatment.

Spatial analysis of cholangiocarcinoma in relation to diabetes mellitus and Opisthorchis viverrini infection in Northeast Thailand

Cholangiocarcinoma (CCA) exhibits a heightened incidence in regions with a high prevalence of Opisthorchis viverrini infection, with previous studies suggesting an association with diabetes mellitus (DM). Our study aimed to investigate the spatial distribution of CCA in relation to O. viverrini infection and DM within high-risk populations in Northeast Thailand. Participants from 20 provinces underwent CCA screening through the Cholangiocarcinoma Screening and Care Program between 2013 and 2019. Health questionnaires collected data on O. viverrini infection and DM, while ultrasonography confirmed CCA diagnoses through histopathology. Multiple zero-inflated Poisson regression, accounting for covariates like age and gender, assessed associations of O. viverrini infection and DM with CCA. Bayesian spatial analysis methods explored spatial relationships. Among 263,588 participants, O. viverrini infection, DM, and CCA prevalence were 32.37%, 8.22%, and 0.36%, respectively. The raw standardized morbidity ratios for CCA was notably elevated in the Northeast’s lower and upper regions. Coexistence of O. viverrini infection and DM correlated with CCA, particularly in males and those aged over 60 years, with a distribution along the Chi, Mun, and Songkhram Rivers. Our findings emphasize the association of the spatial distribution of O. viverrini infection and DM with high-risk CCA areas in Northeast Thailand. Thus, prioritizing CCA screening in regions with elevated O. viverrini infection and DM prevalence is recommended.

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

BackgroundOpisthorchiasis 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.MethodsThe 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.ResultsThe 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.ConclusionsThe 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.Graphical

Production and immunological characterization of the novel single-chain variable fragment (scFv) antibodies against the epitopes on Opisthorchis viverrini cathepsin F (OvCatF)

BackgroundOpisthorchis viverrini infection is a significant health problem in several countries, especially Southeast Asia. The infection causes acute gastro-hepatic symptoms and also long-term infection leading to carcinogenesis of an aggressive bile duct cancer (cholangiocarcinoma; CCA). Hence, the early diagnosis of O. viverrini infection could be the way out of this situation. Still, stool examination by microscopic-based methods, the current diagnostic procedure is restricted by low parasite egg numbers in the specimen and unprofessional laboratorians. The immunological procedure provides a better chance for diagnosis of the infection. Hence, this study aims to produce single-chain variable fragment (scFv) antibodies for use as a diagnostic tool for O. viverrini infection. MethodsThis study uses phage display technologies to develop the scFv antibodies against O. viverrini cathepsin F (OvCatF). The OvCatF-deduced amino acid sequence was analyzed and predicted for B-cell epitopes used for short peptide synthesis. The synthetic peptides were used to screen the phage library simultaneously with OvCatF recombinant protein (rOvCatF). The potentiated phages were collected, rescued, and reassembled in XL1-blue Escherichia coli (E. coli) as a propagative host. The positive clones of phagemids were isolated, and the single-chain variable (scFv) fragments were sequenced, computationally predicted, and molecular docked. The complete scFv fragments were digested from the phagemid, subcloned into the pOPE101 expression vector, and expressed in XL1-blue E. coli. Indirect ELISA and Western analysis were used to verify the detection efficiency. ResultsThe scFv phages specific to OvCatF were successfully isolated, subcloned, and produced as a recombinant protein. The recombinant scFv antibodies were purified and refolded to make functional scFv. The evaluation of specific recognition of the particular epitopes and detection limit results by both computational and laboratory performances demonstrated that all three recombinant scFv antibodies against OvCatF could bind specifically to rOvCatF, and the lowest detection concentration in this study was only one hundred nanograms. ConclusionOur produced scFv antibodies will be the potential candidates for developing a practical diagnostic procedure for O. viverrini infection in humans in the future.

The Effect of a High Monosodium Glutamate Diet in Inducing Changes in Microbiota Diversity and Kidney Pathology in Hamsters Infected with Opisthorchis viverrini

OBJECTIVE: To investigate the effect of a combination of a high dose of monosodium glutamate (MSG) and chronic Opisthorchis viverrini (O. viverrini) infection on kidney pathology and microbiota changes compared to either factor alone.METHODS: Forty male golden hamsters were divided into four groups (10 hamsters per group): non-infected hamsters fed with standard diet (NC), O. viverrini infected hamsters fed with standard diet (OV), non-infected hamsters fed with high doses of MSG in drinking water (MS), and O. viverrini infected hamsters fed with high doses of MSG in drinking water (OM). After 8 months, fecal samples were collected, DNA extracted and subjected to 16S-rRNA sequencing analysis to determine microbial diversity. Kidneys were also collected for histopathological study.RESULTS: Kidney histopathology showed tubular damages and tubular fibrosis were significantly prominent in the OM group, which showed higher pathology changes than in the OV group or MS groups. Next generation sequencing indicated that the levels of Firmicutes to Bacteroides ratio decreased in the OV group (0.28), MS group (0.43) and OM group (0.43) respectively when compared to control group (0.52). In genus levels, Methanobrevibacter, Ruminococcus_1, Escherichia Shigella, Bacteroides, Akkermansia and Oligella were abundance in the OM group.CONCLUSION: The changing of gut microbiota distribution and kidney pathology changes were more severe in the cases of O. viverrini infection together with MSG consumption. This study provides a first step towards focusing on diet and parasitic infections.

Opisthorchis viverrini excretory-secretory products suppress GLUT8 of cholangiocytes.

Opisthorchis viverrini infection and the subsequent bile duct cancer it induces remains a significant public health problem in Southeast Asia. Opisthorchiasis has been reported to cause reduced plasma glucose levels among infected patients. The underlying mechanism for this phenomenon is unclear. In the present study, evidence is presented to support the hypothesis that O. viverrini exploits host cholangiocyte glucose transporters (GLUTs) in a similar manner to that of rodent intestinal nematodes, to feed on unabsorbed glucose in the bile for survival. GLUT levels in a cholangiocyte H69 cell line co-cultured with excretory-secretory products of O. viverrini were examined using qPCR and immunoblotting. GLUT 8 mRNA and expressed proteins were found to be downregulated in H69 cells in the presence of O. viverrini. This suggests that O. viverrini alters glucose metabolism in cells within its vicinity by limiting transporter expression resulting in increased bile glucose that it can utilize and potentially explains the previously reported anti-insulin effect of opisthorchiasis.

Ov-RPA-CRISPR/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces.

Opisthorchis viverrini infection is traditionally diagnosed using the Kato-Katz method and formalin ethyl-acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O.viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA-CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA-CRISPR/Cas assay to detect O.viverrini infection in field-collected human feces. To detect O.viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA-CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA-CRISPR/Cas12a assay for detecting O.viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA-CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods. The Ov-RPA-CRISPR/Cas12a assay exhibited high specificity for detecting O.viverrini DNA. On the basis of the detection limit, the assay could detect O.viverrini DNA at concentrations as low as 10-1ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1ng. To validate the Ov-RPA-CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O.viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA-CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections. The Ov-RPA-CRISPR/Cas12a assay developed in this study can successfully detect O.viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O.viverrini infection, marking an initial step in the development of point-of-care diagnosis.

T helper cell responses to Opisthorchis viverrini infection associate with host susceptibility.

Opisthorchis viverrini infection is endemic in the lower Mekong subregion. The liver is an organ that worms are drawn to and cause damage. However, the immune-related susceptibility in the liver is poorly understood. In this study, we investigated T helper (Th) cell responses in the liver of BALB/c mice and golden Syrian hamsters during 2-28days post-infection (DPI). We found that Th cell responses were distinct between mice and hamsters in terms of dynamics and polarization. Mice exhibited the early induction of Th1, Th2, Th17, and regulatory T (Treg) cells responses after the presence of O. viverrini worms at 2 DPI. In hamsters, the late induction of Th1/Th17, downregulation of Th2/Treg responses and early elevation of suppressive cytokine interleukin (IL)-10 were found together with swift reduction of Th cell numbers. Interestingly, expressions of IL-4 (Th2 functional cytokine) and Foxp3 (Treg lineage) were completely different between mice and hamsters which elevated in mice but suppressed in hamsters. These results suggest that early induction and well-regulation are related to host resistance. In contrast, late induction of Th cell response might allow immature worms to develop in the host. Our findings provide a greater understanding in Th cell response-related susceptibility in O. viverrini infection which would be targeting immunity for the development of immune-based intervention such as vaccine.

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

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. 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. 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. 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.

Metagenomics and metaproteomics alterations are associated with kidney disease in opisthorchiasis hamsters fed a high-fat and high-fructose diet.

Opisthorchis viverrini (O. viverrini, Ov) infection and consumption of high-fat and high-fructose (HFF) diet exacerbate liver and kidney disease. Here, we investigated the effects of a combination of O. viverrini infection and HFF diet on kidney pathology via changes in the gut microbiome and host proteome in hamsters. Twenty animals were divided into four groups; 1) fed a normal diet not infected with O. viverrini (normal group), 2) fed an HFF diet and not infected with O. viverrini (HFF), 3) fed a normal diet and infected with O. viverrini (Ov), and 4) fed an HFF diet and infected with O. viverrini (HFFOv). DNA was extracted from fecal samples and the V3-V4 region of the bacterial 16S rRNA gene sequenced on an Illumina MiSeq sequencing platform. In addition, LC/MS-MS analysis was done. Histopathological studies and biochemical assays were also conducted. The results indicated that the HFFOv group exhibited the most severe kidney injury, manifested as elevated KIM-1 expression and accumulation of fibrosis in kidney tissue. The microbiome of the HFFOv group was more diverse than in the HFF group: there were increased numbers of Ruminococcaceae, Lachnospiraceae, Desulfovibrionaceae and Akkermansiaceae, but fewer Eggerthellaceae. In total, 243 host proteins were identified across all groups. Analysis using STITCH predicted that host proteome changes may lead to leaking of the gut, allowing molecules such as soluble CD14 and p-cresol to pass through to promote kidney disease. In addition, differential expression of TGF-beta-activated kinase 1 and MAP3K7-binding protein 2 (Tab2, involving renal inflammation and injury) are predicted to be associated with kidney disease. The combination of HFF diet and O. viverrini infection may promote kidney injury through alterations in the gut microbiome and host proteome. This knowledge may suggest an effective strategy to prevent kidney disease beyond the early stages.

Inflammatory responses to Opisthorchis viverrini infection in animal models: A comparison between susceptible and nonsusceptible hosts in different anatomical locations.

Inflammation caused by Opisthorchis viverrini infection increases the risk of cholangitis, cholecystitis, and leads to bile duct cancer (cholangiocarcinoma or CCA). However, only certain infected individuals are susceptible to CCA, suggesting the involvement of host factors in cancer development. In addition, there are reports indicating differences in the locations of CCA. This study aims to investigate cellular inflammatory responses in the common bile duct (CB), intrahepatic bile duct (IHB), and gallbladder (GB) in susceptible and non-susceptible hosts following O. viverrini infection. Thirty Syrian golden hamsters (a susceptible host) and 30 BALB/c mice (a non-susceptible host) infected with O. viverrini were studied at six time points (five animals per group). Histopathological evaluations were conducted on samples from the IHB, CB, and GB. Inflammatory cell infiltration was quantitatively assessed and compared between groups and time points. Statistical analysis was performed using one-way ANOVA, with a significance level of p < 0.05. Inflammation was significantly more pronounced in the IHB compared to the other two biliary locations. In comparison between susceptible and non-susceptible hosts, the intensity of inflammation was higher in the OV+H group than in the OV+M group (p < 0.05). This study highlights the association between host response to inflammation, tissue location, and host susceptibility, with the IHB showing particular susceptibility to inflammation and pathological changes. These findings contribute to our understanding of the increased risk of CCA in susceptible hosts.

Association of Cholangiocarcinoma among People with Periductal Fibrosis.

Cholangiocarcinoma (CCA) is a highly fatal tumor, and the most favorable chance for long-term survival lies in curative resection. Periductal fibrosis (PDF), a precancerous condition associated with chronic inflammation of the bile ducts, can serve as a screening marker for CCA using hepatobiliary ultrasonography (US). However, limited studies have explored the relationship between PDF and CCA. This study aimed to investigate the association between PDF and CCA in a population at risk in Northeast Thailand. The study included participants enrolled in the Cholangiocarcinoma Screening and Care Program (CASCAP) between 2013 and 2021 who underwent US. Histological evaluations were conducted following the standard protocol of the tertiary hospital at Khon Kaen University, Thailand. PDF was defined as the presence of fibrosis in the peripheral (PDF1), segmental (PDF2), or main bile duct (PDF3), diagnosed by well-trained general practitioners or radiologists. The association between PDF and CCA was assessed using multiple logistic regression, calculating adjusted odds ratios (AORs) and 95% confidence intervals (CIs). Out of 751,061 participants, the overall prevalence of PDF was 115,267 (15.35%), with an overall CCA rate of 0.11%. The rates of CCA were 0.1%, 0.15%, and 0.27% in participants with PDF1, PDF2, and PDF3, respectively. After adjusting for gender, age at enrollment, education levels, history of O. viverrini infection, smoking, and alcohol consumption, the AORs for CCA were 0.94 (95% CI: 0.74 - 1.20), 1.4 (95% CI: 1.03 - 1.91), and 2.52 (95% CI: 1.38 - 4.58) for participants with PDF1, PDF2, and PDF3, respectively. Our findings demonstrate a significant association between fibrosis of the segmental and main bile ducts (PDF2 and PDF3) and CCA, with the strongest association observed in participants with PDF3. Hepatobiliary US screening could serve as a valuable tool for early detection of CCA, enabling timely curative treatment.