Epigenetic Biomarkers and the Wnt/ß-Catenin Pathway in Opisthorchis viverrini-associated Cholangiocarcinoma: A Scoping Review on Therapeutic Opportunities.

BACKGROUND: Epigenetic modifications, such as DNA methylation and histone modifications, are pivotal in regulating gene expression pathways related to inflammation and cancer. While there is substantial research on epigenetic markers in cholangiocarcinoma (CCA), Opisthorchis viverrini-induced cholangiocarcinoma (Ov-CCA) is overlooked as a neglected tropical disease (NTD) with limited representation in the literature. Considering the distinct etiological agent, pathogenic mechanisms, and pathological manifestations, epigenetic research plays a pivotal role in uncovering markers and potential targets related to the cancer-promoting and morbidity-inducing liver fluke parasite prevalent in the Great Mekong Subregion (GMS). Emerging studies highlight a predominant hypermethylation phenotype in Opisthorchis viverrini (O. viverrini) tumor tissues, underscoring the significance of abnormal DNA methylation and histone modifications in genes and their promoters as reliable targets for Ov-CCA. PRINCIPAL FINDINGS: Relevant published literature was identified by searching major electronic databases using targeted search queries. This process retrieved a total of 81 peer-reviewed research articles deemed eligible for inclusion, as they partially or fully met the pre-defined selection criteria. These eligible articles underwent a qualitative synthesis and were included in the scoping review. Within these, 11 studies specifically explored Ov-CCA tissues to investigate potential epigenetic biomarkers and therapeutic targets. This subset of 11 articles provided a foundation for exploring the applications of epigenetics-based therapies and biomarkers for Ov-CCA. These articles delved into various epigenetic modifications, including DNA methylation and histone modifications, and examined genes with aberrant epigenetic changes linked to deregulated signalling pathways in Ov-CCA progression. CONCLUSIONS: This review identified epigenetic changes and Wnt/ß-catenin pathway deregulation as key drivers in Ov-CCA pathogenesis. Promoter hypermethylation of specific genes suggests potential diagnostic biomarkers and dysregulation of Wnt/ß-catenin-modulating genes contributes to pathway activation in Ov-CCA progression. Reversible epigenetic changes offer opportunities for dynamic disease monitoring and targeted interventions. Therefore, this study underscores the importance of these epigenetic modifications in Ov-CCA development, suggesting novel therapeutic targets within disrupted signalling networks. However, additional validation is crucial for translating these novel insights into clinically applicable strategies, enhancing personalised Ov-CCA management approaches.

Examining the Acceptability of Helminth Education Packages "Magic Glasses Lower Mekong" and "Magic Glasses Opisthorchiasis" and Their Impact on Knowledge, Attitudes, and Practices Among Schoolchildren in the Lower Mekong Basin: Protocol for a Cluster Randomized Controlled Trial.

BACKGROUND: Helminths are a major global health issue, impacting health, educational, and socioeconomic outcomes. Infections, often starting in childhood, are linked to anemia, malnutrition, cognitive deficit, and in chronic cases of Opisthorchis viverrini (OV), cholangiocarcinoma. The main control strategy for helminth infection is mass drug administration; however, this does not prevent reinfection. As such, prevention strategies are needed. The "Magic Glasses" is a school-based cartoon health education package that has demonstrated success in improving knowledge, attitudes, and practices (KAP) surrounding soil-transmitted helminths (STH) in China and the Philippines. This study is designed to assess the acceptability and impact of the 2 new versions of the Magic Glasses targeting STH and OV designed for the Lower Mekong audience in Cambodia, Lao People's Democratic Republic (PDR), and Thailand. OBJECTIVE: The objective of this study is to evaluate the acceptability of the "Magic Glasses Lower Mekong" and "Magic Glasses Opisthorchiasis" education packages among schoolchildren in the Lower Mekong Basin, and the impact of these education packages on students' KAP surrounding STH and OV, respectively. METHODS: Schoolchildren will be recruited into a cluster randomized controlled trial with intervention and control arms in rural schools in Cambodia, Lao PDR, and Thailand. Schoolchildren's initial acceptability of the intervention will be evaluated using an adapted questionnaire. Sustained acceptability will be assessed at 9-month follow-up through focus group discussions with students and interviews with teachers. Impact will be evaluated by KAP questionnaires on STH and OV. KAP questionnaires will be administered to children at baseline and at follow-up. Indirect impact on parents' KAP of OV and STH will be assessed through focus group discussions at follow-up. RESULTS: The trial is in progress in Lao PDR and Thailand and is expected to commence in Cambodia in January 2024. The results of the study are expected to be available 18 months from the start of recruitment. We hypothesize that participants enrolled in the intervention arm of the study will have higher KAP scores for STH and OV, compared with the participants in the control arm at follow-up. We expect that students will have initial and sustained acceptability of these intervention packages. CONCLUSIONS: This trial will examine the acceptability of the "Magic Glasses Opisthorchiasis" and "Magic Glasses Lower Mekong" interventions and provide evidence on the effectiveness of the "Magic Glasses" on KAP related to OV and STH among schoolchildren in the Lower Mekong Basin. Study results will provide insight on acceptability and impact indicators and inform a scaling up protocol for the "Magic Glasses" education packages in Cambodia, Lao PDR, and Thailand. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12623000271606; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=385315&isReview=true. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/55290.

Transcriptome changes of liver fluke Opisthorchis viverrini in diabetic hamsters.

A recent study in hamsters showed that infection with the liver fluke Opisthorchis viverrini in diabetic hosts worsens the severity of hepatobiliary disease. However, the effects of diabetes on the worm's phenotype and gene expression pattern remain unknown. This study investigated the impact of diabetes on the global gene expression and development of O. viverrini in diabetic hamsters. Parasitological parameters were assessed, and mRNA sequencing with bioinformatic analysis was performed. The study revealed that worm establishment rates in diabetic hamsters were directly correlated with fasting plasma glucose levels. Interestingly, worms collected from diabetic hosts exhibited stunted growth and reduced egg production. Transcriptomic analysis revealed significant alterations in gene expression, with 4314 and 567 differentially expressed genes at 21- and 35-days post-infection, respectively. Gene ontology enrichment analysis highlighted changes in biological processes related to stress response, metabolism, and cellular organization. Notably, genes associated with parasite virulence, including granulin, tetraspanins, and thioredoxins, showed significant upregulation in diabetic hosts. These findings demonstrate the profound impact of host diabetic status on O. viverrini development and gene expression, providing insights into the complex interplay between host metabolism and parasite biology, including molecular adaptations of O. viverrini in hosts. This study contributes to our understanding of opisthorchiasis in the context of metabolic disorders and may inform future strategies for disease management in diabetic human populations.

Title: Modifications du transcriptome de la douve du foie Opisthorchis viverrini chez les hamsters diabétiques. Abstract: Une étude récente sur les hamsters a montré que l'infection par la douve du foie Opisthorchis viverrini chez les hôtes diabétiques aggrave la gravité de la maladie hépatobiliaire. Cependant, les effets du diabète sur le phénotype et le profil d'expression génétique du ver restent inconnus. Cette étude a examiné l'impact du diabète sur l'expression génétique globale et le développement d'O. viverrini chez les hamsters diabétiques. Les paramètres parasitologiques ont été évalués et un séquençage de l'ARNm avec analyse bioinformatique a été effectué. L'étude a révélé que les taux d'établissement des vers chez les hamsters diabétiques étaient directement corrélés au taux de glucose plasmatique à jeun. Il est intéressant de noter que les vers récupérés auprès d'hôtes diabétiques présentaient une croissance retardée et une production d'œufs réduite. L'analyse transcriptomique a révélé des altérations significatives de l'expression génétique, avec 4 314 et 567 gènes exprimés de manière différentielle à 21 et 35 jours après l'infection, respectivement. L'analyse d'enrichissement de l'ontologie génétique a mis en évidence des changements dans les processus biologiques liés à la réponse au stress, au métabolisme et à l'organisation cellulaire. Notamment, les gènes associés à la virulence du parasite, en particulier la granuline, les tétraspanines et les thiorédoxines, ont montré une régulation positive significative chez les hôtes diabétiques. Ces résultats démontrent l'impact profond du statut diabétique de l'hôte sur le développement et l'expression génétique d'O. viverrini, offrant un aperçu de l'interaction complexe entre le métabolisme de l'hôte et la biologie du parasite, y compris les adaptations moléculaires d'O. viverrini chez les hôtes. Cette étude contribue à notre compréhension de l'opisthorchiase dans le contexte des troubles métaboliques et peut éclairer les futures stratégies de gestion de la maladie pour les populations humaines diabétiques.

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.

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.

Re- Infection Rate of Opisthorchis Viverrini Five Years After Treatment with Praziquantel in High-Risk Area: A Community-Based Study.

BACKGROUND: Opisthorchis viverrini (OV)  is a significant public health problem in Thailand, and  OV reinfection poses a serious risk of cholangiocarcinoma with little evidence of a decrease.  While numerous studies have explored OV reinfection and prevalence, most have been limited to short study period. Therefore, there is a need for long-term investigations to gather sufficient evidence. OBJECTIVES: This study aimed to access the current status of re-infection rates in high- risk areas and to determine associations between factors of and re-infection of OV at 5 years after treatment  with praziquantel. MATERIALS AND METHODS: In total, this study included 457 participants from a cohort study. Each participant was required to answer a questionnaire and undergo stool examination using the formalin ethyl acetate concentration technique. Data were analyzed using descriptive statistics and multiple logistic regression. RESULT: Out of the total 457 participants, 418 (91.5%) successfully completed the questionnaires and underwent stool examinations. Among the participants, 10.8% showed re-infection with OV. Using multivariate analysis, factors significantly associated with reinfection OV included yearly income (OR adj = 2.14, 95%CI = 1.11- 4.12, p-value = 0.022) and past stool examination five years (OR adj = 2.47, 95%CI = 1.13-5.43, p-value = 0.023), respectively. CONCLUSIONS: Subjects who frequently experience OV reinfection should undergo CCA screening by experts while closely monitoring their raw fish consumption behavior. Moreover, it is essential to implement comprehensive programs aimed at promoting behavioral changes and provide preventive education, with a specific focus on high epidemic areas, to discourage the consumption of raw fish.

Investigation of Social Innovations for Handling Opisthorchis viverrini and Cholangiocarcinoma in Thailand's Highest-Risk Areas for Further Development of Successful Solution.

BACKGROUND: Despite the national campaigns in 1997 to control Opisthorchis viverrini (OV), a significant risk factor for cholangiocarcinoma (CCA) was accounted for in the northeast of Thailand, which is also considered the highest-risk area for CCA as per the latest national-wide survey in 2019. Social innovations were employed to address OV and CCA issues in this area, but they have yet to be thoroughly investigated. Investigating social innovations in areas where issues can already be solved is a particular approach for expanding on a broad basis. OBJECTIVE: This study aimed to search for social innovations used to handle OV and CCA in northeast of Thailand. METHODS: Researchers coordinated with the Office of Disease Prevention and Control Region-10 (1 of 12 regional offices under the Department of Disease Control, Ministry of Public Health) to specify the model areas for taking OV and CCA. In addition, researchers cooperated with local coordination units for data collection. The data were coded and classified for content analysis, and triangulation was performed for comparative analysis. Finally, a meeting was conducted to summarize the social innovations discovered in each study site area. RESULTS: Our findings have been presented according to the epidemiologic triad: agent (risk factors), host (individuals/communities), and environment. The researchers and experts addressed and proposed social innovations for dealing with OV and CCA, such as the "3 Health" (Health Behaviours, Health Hygiene, and Environmental Health), in order to achieve a CCA-free society. CONCLUSIONS: This is the first study to highlight the significance of social innovations in managing OV and CCA. The findings will be utilized to further create strategies for addressing these public health issues in this and other regions in a sustainable and successful manner.

Study of the population genetic structure of Opisthorchis-like eggs in northern Thailand using mitochondrial genes.

BACKGROUND: Opisthorchis-like eggs are a public health problem in northern and northeastern Thailand. However, the genetic epidemiology and structure of these parasites in northern Thailand are unknown. Thus, this study investigated their population genetic structure using cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) nucleotide sequences. METHODOLOGY/PRINCIPAL FINDINGS: A study was conducted in the hill tribe regions of Chiang Mai Province, northern Thailand. Internal transcribed spacer 2 polymerase chain reaction and restriction fragment length polymorphism were used to distinguish 205 positive feces samples for Opisthorchis-like eggs. The results showed that the prevalence of O. viverrini and Haplorchis taichui was 10.5% and 38.2%, respectively, and the co-infection rate was 37.2%. To determine the genetic structure of O. viverrini and H. taichui using cox1 and nad1 genes, genetic analysis was performed using 30 randomly chosen fecal samples for Opisthorchis-like eggs. Pairwise FST analysis indicated that O. viverrini and H. taichui displayed nonsignificant genetic differentiation within Chiang Mai Province and between interpopulations from different geographic areas. Moreover, within the intrapopulation in Chiang Mai Province, cox1 presented higher gene flow than nad1 in O. viverrini, while nad1 demonstrated higher gene flow than cox1 in H. taichui. The neutrality tests based on Fu's Fs indicated population expansion and selective sweep from bottleneck or hitchhiking in O. viverrini and H. taichui populations, supported by haplotype network patterns. Phylogenetic tree analysis based on cox1 and nad1 revealed the monophyly of O. viverrini and H. taichui and genetic relationships with other isolates collected from Thailand, Lao People's Democratic Republic (PDR), and Vietnam. CONCLUSIONS/SIGNIFICANCE: This study investigated the molecular discrimination and genetic structure of Opisthorchis-like eggs in northern Thailand. The genetic information derived from this study could be associated with the background, molecular epidemiology, and disease severity of these parasites.

Development of a subunit vaccine against the cholangiocarcinoma causing Opisthorchis viverrini: a computational approach.

Opisthorchis viverrini is the etiological agent of the disease opisthorchiasis and related cholangiocarcinoma (CCA). It infects fish-eating mammals and more than 10 million people in Southeast Asia suffered from opisthorchiasis with a high fatality rate. The only effective drug against this parasite is Praziquantel, which has significant side effects. Due to the lack of appropriate treatment options and the high death rate, there is a dire need to develop novel therapies against this pathogen. In this study, we designed a multi-epitope chimeric vaccine design against O. viverrini by using immunoinformatics approaches. Non-allergenic and immunogenic MHC-1, MHC-2, and B cell epitopes of three candidate proteins thioredoxin peroxidase (Ov-TPx-1), cathepsin F1 (Ov-CF-1) and calreticulin (Ov-CALR) of O. viverrini, were predicted to construct a potent multiepitope vaccine. The coverage of the HLA-alleles of these selected epitopes was determined globally. Four vaccine constructs made by different adjuvants and linkers were evaluated in the context of their physicochemical properties, antigenicity, and allergenicity. Protein-protein docking and MD simulation found that vaccines 3 was more stable and had a higher binding affinity for TLR2 and TLR4 immune receptors. In-silico restriction cloning of vaccine model led to the formation of plasmid constructs for expression in a suitable host. Finally, the immune simulation showed strong immunological reactions to the engineered vaccine. These findings suggest that the final vaccine construct has the potential to be validated by in vivo and in vitro experiments to confirm its efficacy against the CCA causing O. viverrini.

Liver Flukes: Clonorchis and Opisthorchis.

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 is 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 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 CCA in endemic areas.

Prevalence of opisthorchiid and other endoparasitic infections among cats and dogs in Novosibirsk oblast (Western Siberia, Russia).

To determine the current reservoir status and prevalence of Opisthorchis felineus infection in opisthorchiosis-endemic areas, feces samples were collected from 103 cats and 101 dogs admitted to shelters or discount sterilization centers from various districts of Novosibirsk city and from villages and towns along the Ob River (Novosibirsk Oblast). The feces samples were evaluated using two methods simultaneously: the formalin-ether sedimentation assay and a modified flotation assay. The prevalence of opisthorchiid infection was higher among cats (12.6%; 95% confidence interval 6.9-20.6%) than among dogs (4.0%; 95% confidence interval 1.1-9.8%). Overall prevalence of endoparasites among cats was 42.7% (five helminthic and four protozoan species) and among dogs 51.5% (eight helminthic and five protozoan species). Among the parasites found in the dogs, the prevalence of Sarcocystis sp. was the highest (16.8%), and in cats, the highest prevalence was shown by Giardia sp. and Toxocara cati (14.6% for each species). These results confirm the considerable contribution of stray cats and dogs to the maintenance of opisthorchiid eggs in the environment; these data should be taken into account for the development of effective control and education programs.

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.

Misclassification of Opisthorchis viverrini and Minute Intestinal Fluke Eggs by Routine Laboratory Staff Using Images from the Kato-Katz Method.

BACKGROUND: The Kato-Katz method is a commonly used diagnostic tool for helminth infections, particularly in field studies. This method can yield inaccurate results when samples contain eggs that are similar in appearance, such as Minute Intestinal Fluke (MIF) and Opisthorchis viverrini (OV) eggs. The close resemblance of eggs can be problematic and raises the possibility of false diagnoses. The objectives were to compare the diagnostic performance of the Kato-Katz method for accurately identifying MIF and OV and to provide evidence of possible misclassification.  Methods: Based on questionnaire responses from 15 (young parasitologists and public health staff), the test comprised 50 MIF egg images and 50 OV egg images, for a total of 100 Google Form questionnaires. RESULTS: The morphology of MIF and OV eggs found size and shape similarity and found that the shoulder rims were small, while the OV egg found the knobs had disappeared. The opercular conjunction was apparent, the shoulder rims and miricidium were prominent. The average percentage of correctly classified infections was 61.6 ± 12.1%. The accuracy percentages for both public health staff and young parasitologists in identifying were found to be 59.0 ± 14.8 and 66.8 ± 2.8, respectively. There was no significant difference observed in both groups. CONCLUSION: These findings highlight the need for improving the accuracy of parasite identification. Preserving stool samples before the Kato-Katz method can help mitigate the potential degradation or distortion of parasite eggs. The incorrect classification of both eggs had an impact on treatment plans and the policy of parasite control programs.

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

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

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.

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.

Prevalence estimates of Opisthorchis viverrini and Clonorchis sinensis infection in the Greater Mekong subregion: a systematic review and meta-analysis.

BACKGROUND: Opisthorchiasis and clonorchiasis, caused by Opisthorchis viverrini and Clonorchis sinensis, respectively, are significant yet neglected foodborne trematodiases in the Great Mekong Subregion (GMS). Despite the reporting of the prevalence of these human liver flukes in the region over the past decades, there has been a lack of a comprehensive and systematic consolidation of this data. Therefore, we aimed to conduct a thorough systematic review and meta-analysis to synthesize and analyze time-trend prevalence estimates of both O. viverrini and C. sinensis across the GMS for the past 30 years. METHODS: This study undertakes a systematic review using a comprehensive search for published articles in PubMed, EMBASE, Scopus, Cochrane and Thai Journal Online databases until early 2023. The pooled prevalence of O. viverrini and C. sinensis infection was analyzed through a random-effects meta-analysis, with meta-regression analysis used to quantify associations with study characteristics. Sub-group analysis was conducted, whenever comparison data were available, to assess the risk of O. viverrini and C. sinensis infection in each GMS country. Heterogeneity among studies was assessed using the Q statistic and quantified by using the I 2 Index. RESULTS: From a total of 2997 articles, 155 articles comprising 218 datasets and 751,108 participants were included for review. The GMS prevalence of O. viverrini was 21.11% [45,083/260,237; 95% confidence interval (CI): 17.74-24.47%]. Pooled prevalence estimates were highly observed in Laos (34.06%, 95% CI: 26.85-41.26%), followed by Thailand (18.19%, 95% CI: 13.86-22.51%), and Cambodia (10.48%, 95% CI: 5.52-15.45%). Myanmar and Vietnam had limited data sources for calculation. Clonorchis sinensis infection in GMS was 25.33% (95% CI: 18.32-32.34%), with Guangxi, China, exhibiting the highest prevalence rates at 26.89% (95% CI: 18.34-35.43%), while Vietnam had a prevalence rate of 20.30% (95% CI: 9.13-31.47%). O. viverrini prevalence decreased significantly over time, whereas C. sinensis infection appeared to be stable consistently over time in both China and Vietnam. CONCLUSIONS: This comprehensive study, drawing from the largest datasets to date, offers an in-depth systematic prevalence review of human liver flukes in the Greater Mekong Subregion. It underscores the imperative for systematic surveillance, data collection, and the implementation of intervention and control measures for these infectious diseases of poverty.

Identification and functional analysis of mitogenic miRNA of the carcinogenic liver fluke Opisthorchis viverrini.

Opisthorchis viverrini is a pathogenic liver fluke that is known to cause cholangiocarcinoma in chronic infections. The underlying mechanism for this carcinogenesis is believed to be multifactorial, with parasite-derived excretory-secretory (ES) products potentially playing major roles. A recent study on these ES products has identified microRNAs (miRNA) that originate from O. viverrini but their influence on carcinogenesis remains understudied. Hence, we aimed to investigate the role of these miRNAs in the carcinogenesis of O. viverrini-associated cholangiocarcinoma. The mature miRNA sequences were retrieved from published data. Bioinformatics analysis was employed to identify miRNA targets and to identify potentially mitogenic miRNAs. An in vitro study was conducted to test the effects of miRNA on the bile duct epithelial cell lines. The miRNA target prediction analysis revealed that Ov_miRNA_EV_36/ovi-miR-3479a targets cancer-associated pathways. Hence, it was selected and used to assess its effect on the cell proliferation rate of H69 and MMNK-1 cholangiocyte cell lines. The results showed that Ov_miRNA_EV_36/ovi-miR-3479a induced significant cell proliferation in both cell lines when compared to negative controls. These results indicate that Ov_miRNA_EV_36/ovi-miR-3479a may play an essential role in the carcinogenesis of O. viverrini and therefore warrant further investigations.