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Abstract Background Opisthorchis viverrini (OV) and soil-transmitted helminths (STH) are two of the most common helminths contributing to the Neglected Tropical Disease (NTDs) burden in the Lower Mekong Basin. Although mass drug administration is the cornerstone of control programs to reduce morbidity caused by these infections, this approach has limitations in preventing re-infections. Elimination requires additional measures such as reservoir host treatment, improved hygiene and health education to reinforce MDA's impact. This study aims to examine the impact of a scalable multi-component One Health Helminth Elimination program in the Lower Mekong Basin (HELM) that combines human praziquantel (PZQ) and albendazole (ALB) treatment with a program that includes the “Magic Glasses” and the “Lawa Model” interventions with health promotion at their core. Methods This study will employ a cluster randomized controlled trial (cRCT) in 18 rural communities (with sub-district or villages as cluster units) across Cambodia, Laos and Thailand. The control arm will receive one round of PZQ/ALB treatment, while in the intervention arm, multi-component HELM program will be implemented, which includes PZQ/ALB treatment together with the Magic Glasses and Lawa Model interventions. OV and STH infections levels will be evaluated in individuals aged 5–75 years at baseline and will be repeated at follow-up (12 months after the HELM intervention), using modified formalin ethyl-acetate concentration technique and quantitative PCR. The primary outcome of the study will be cumulative incidence of human OV and STH infections. Outcomes between the study arms will be compared using generalized linear mixed models, accounting for clustering. Discussion Evidence from this trial will quantify the impact of a multi-component One Health control strategy in interrupting Ov and STH infections in the Lower Mekong Basin. Trial registration Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12622000353796. Prospectively registered 28 February 2022.

The article analyzes statistical data on the 2020 vs 2019 opisthorchiasis incidence rate on the territory of the Russian Federation and in foreign countries, provides absolute and intensive morbidity indicators across entire the Russian Federation and specifically in its subjects — republics and regions, general prevalence of helminthiasis, and opisthorchiasis particularly overall pattern of parasitic diseases, as well as contribution of opisthorchiasis to general pattern of biohelminthiasis, its dynamics, and prevalence of relevant causative agent. A comparative analysis for incidence rates in the regions where opisthorchiasis was recorded and mean country-wide statistical parameters was carried out. As a result, it was concluded that despite a decline in the Russia-wide overall parasitic morbidity, an excess of mean indicators, sometimes significant, was noted in some regions. Thus, compared to Russia-wide magnitude of intensive indicator, it was higher by 4-fold in the Komi Republic, whereas in the Kurgan, Vologda, Arkhangelsk Regions and the Nenets Autonomous Okrug — by 3-fold. The article provides an analysis of the population age groups as well as percentage of opisthorchis-affected urban and rural populations. In addition, the special clinical and epidemiological significance of opisthorchiasis remains urgent due to severity of its course, affected various body systems and organs, emergence of serious complications leading to disability, and in some cases to fatal outcomes, the presence of natural foci, most of which are located in the Russian Federation. Almost all territories adjacent to the basins of the Ob, Irtysh, Tom rivers and their tributaries pose special concern regarding opisthorchiasis. The data related to prevalence of helminthiasis larvae detected in fish in 2020 and 2019 since 2010 are presented. Based on the data shown above, strict adherence to preventive measures plays an important role in reducing opisthorchiasis incidence rate and preventing its spread.

Objective Methods Results Conclusion Opisthorchis viverrini and cholangiocarcinoma have been recognised by the World Health Organisation as critical public health concerns, particularly in northeastern Thailand, where Opisthorchis viverrini is a significant cause of cholangiocarcinoma. This study examines the factors influencing Opisthorchis viverrini and cholangiocarcinoma prevention within the One Health framework in Kanthararom District, Sisaket Province, Thailand.A cross‐sectional study was conducted among 327 residents aged 20 and above, utilising a structured questionnaire to assess knowledge, attitudes, perceived self‐efficacy and prevention behaviours. Data were analysed using descriptive statistics, Pearson's product–moment correlation coefficient and multiple regression analysis to identify relationships and predictors within the dataset.The results showed that 53.52% of participants had moderate knowledge and attitudes towards prevention, while perceived self‐efficacy was generally high. Positive correlations were found between income, knowledge, attitudes, self‐efficacy and prevention behaviours. Key predictors of Opisthorchis viverrini and cholangiocarcinoma prevention behaviours included attitudes towards human health (β = 0.212), perceived self‐efficacy in human health (β = 0.211), attitudes towards animal health (β = 0.205) and knowledge about human health (β = 0.138), accounting for 24.40% of the variance in prevention behaviours. These predictors were statistically significant at 0.05 (F = 25.95; p <0.001).The findings suggest that enhancing knowledge, attitudes and self‐efficacy is crucial to improving Opisthorchis viverrini and cholangiocarcinoma prevention. This study underscores the need for integrated public health strategies incorporating human, animal and environmental health to reduce the prevalence of Opisthorchis viverrini and cholangiocarcinoma in high‐risk regions. [ABSTRACT FROM AUTHOR]

AbstractBackgroundMethodsResultsConclusionEnvironmental contamination with Opisthorchis viverrini eggs significantly impacts on opisthorchiasis transmission and disease control. This study assessed O. viverrini egg contamination in underwater sediments from canals and aimed to reduce O. viverrini transmission in egg–positive and downstream communities by controlling human opisthorchiasis in endemic area of Northeast Thailand.Sediment samples from 14 sites in UdonThani municipality were determined for O. viverrini eggs. Human fecal samples and fish were examined for opisthorchiasis and metacercaria infection, respectively. Water samples were analysed for faecal coliforms bacteria. From 2017 to 2023, transmission control efforts were evaluated by monitoring changes in human and fish infection rates.In 2017 and 2018, O. viverrini eggs were found in canal sediments at 153 and 86 eggs/liter, respectively. Human opisthorchiasis prevalence was 5.3%, with cyprinoid fish showing a 25.0% infection rate (11.6 cysts/infected fish) and significant fecal contamination (>5,500 CFU/100 ml). Conversely, egg–negative sites showed a 0.5% human infection rate and no metacercariae were found. After health interventions (2019 to 2023), O. viverrini eggs were not detected in sediments from canals, resulting in a significant reduction in human opisthorchiasis prevalence to 0.9% at the previously egg–positive site and from 12.2% to 1.7% in downstream communities (p < 0.001). Additionally, the prevalence of metacercariae in cyprinoid fish decreased from 11.4% (7.6 cysts/infected fish) to 4.5% (2.4 cysts/infected fish), indicating reduced transmission.The presence of O. viverrini eggs in sediment was associated to human opisthorchiasis transmission and demonstrated the significant effectiveness of health interventions in controlling the disease. [ABSTRACT FROM AUTHOR]

Background: Epidemiologically important food-borne trematodes Opisthorchis viverrini and Clonorchis sinensis are recognized as biological carcinogens of Group 1A, while Opisthorchis felineus is in Group 3 as noncarcinogenic to humans. Mechanisms of the biological carcinogenesis are still elusive. Some studies highlight chronic inflammation as a key factor and common pathway for cancer initiation and progression. Nonetheless, the chronic inflammation alone does not explain why these three species differ in carcinogenicity. We focused this study on genome-wide landscapes of liver gene expression and activation of cellular pathways in Mesocricetus auratus golden hamsters infected with C. sinensis (South Korea), O. viverrini (Thailand), or O. felineus (Russia) at 1 and 3 months after infection initiation. Methodology/Principal findings: Liver transcriptomes of golden hamsters (HiSeq Illumina, 2X150 bp) were sequenced at 1 and 3 months postinfection. Data processing was carried out using the following bioinformatic and experimental approaches: analysis of differential gene expression, estimates of proportions of affected liver cell types, liver histopathology, and examination of weighted gene coexpression networks. All infections caused enrichment with inflammatory response signaling pathways, fibrogenesis and cell proliferation, and IL2–STAT5, TNF–NF-κB, TGF-β, Hippo, MAPK, and PI3K–Akt signaling pathways. Nevertheless, species-specific responses to each infection were noted too. We also identified species-specific responses of liver cell types, differentially expressed gene clusters, and cellular pathways associated with structural liver damage (such as periductal fibrosis, epithelial neoplasia, and inflammation). Conclusions/Significance: This is the first comparative analysis of gene expression landscapes in the liver of experimental animals infected with O. viverrini, O. felineus, or C. sinensis. The trematodes have species-specific effects on the hepatobiliary system by triggering signaling pathways, thereby leading to differences in the severity of hepatobiliary structural lesions and contributing to the pathogenicity of closely related foodborne trematodes. Author summary: Food-borne trematodiases constitute an important group of the neglected tropical diseases caused by Opisthorchis felineus, O. viverrini, and Clonorchis sinensis liver fluke infections. These parasites affect the hepatobiliary system of mammals, including humans, by inducing cholangitis, bile duct neoplasia, and even cholangiocarcinoma in chronically infected individuals. Two species, O. viverrini and C. sinensis, are recognized group 1A biological carcinogens to humans, whereas O. felineus is classified as a noncarcinogen. Mechanisms of formation of the cholangiocarcinoma associated with helminth infection remain unknown. We focused on the research into early events that lead to precancerous changes. Here we examined differences in genome-wide profiles of liver gene expression and activation of cellular pathways in Mesocricetus auratus hamsters infected with C. sinensis (South Korea), O. viverrini (Thailand), or O. felineus (Russia) at 1 and 3 months after infection initiation. Infections with C. sinensis and O. felineus mostly featured development of periductal fibrosis accompanied by a huge inflammatory response in the liver. O. viverrini infection, however, showed severer hyperplasia and biliary-intraepithelial-neoplasia lesions. Thus, these related food-borne trematodes have species-specific effects on the liver during infection, which likely contribute to the differences in carcinogenic potential among these trematodes. [ABSTRACT FROM AUTHOR]

Three food-borne trematodes—Opisthorchis felineus, Opisthorchis viverrini, and Clonorchis sinensis—are closely related epidemiologically important species. Despite the similarity of their life cycles, these liver flukes also have marked differences in the geographical range, helminth biology, and hepatobiliary disorders. O. viverrini and C. sinensis are classified as Group 1 biological carcinogens while O. felineus is not. Direct comparisons of systemic response to the liver fluke infections are unexplored aspects. This study was carried out to identify species-specific liver and kidney responses in the hamster models after the infection with one of the three liver flukes. Liver periductal-fibrosis development was similar between hamsters infected with O. felineus or C. sinensis, whereas biliary intraepithelial neoplasia development was noticed predominantly in O. viverrini–infected ones. Species-specific renal damage was detected, including progression of interstitial fibrosis and IgA deposition in glomeruli of O. felineus–infected hamsters and C. sinensis–infected ones. A strong correlation (R = 0.63; P = 0.0001) was found between periductal fibrosis in the liver and kidney interstitial fibrosis. Future comparative studies are needed to elucidate the development of serious complications during the long term of the infection, as well as under the influence of additional factors, including concomitant infections and the use of dimethylnitrosamine to clarify the mechanisms underlying the liver fluke-associated carcinogenesis. Thus, our findings may stimulate new comparative studies on the pathogenicity. [ABSTRACT FROM AUTHOR]

Strongyloides stercoralis and Opisthorchis viverrini are helminth parasites responsible for significantly neglected tropical diseases. This study aimed to evaluate the prevalence of these parasites and the risk factors for S. stercoralis and O. viverrini infections in northern and northeastern Thailand where relevant epidemiological data are scarce and outdated. A cross-sectional study was conducted in 2016 in six sub-districts across five provinces. Urine samples were analyzed to detect S. stercoralis and O. viverrini infection using urine enzyme-linked immunosorbent assays (urine-ELISA). Demographic data of participants were collected using a questionnaire. Environmental data, including land-surface temperature (LST), normalized difference vegetation index (NDVI), normalized difference water index (NDWI), and soil pH, were obtained from remote-sensing sources. Multiple logistic regression analysis identified risk factors associated with infection. Complete data were obtained for 2613 individuals. The overall prevalence was 39.15% (95% CI: 37.27–41.02) for S. stercoralis and 37.46% (95% CI: 35.61–39.32) for O. viverrini. Male sex was significantly associated with S. stercoralis (AOR, 1.53; 95% CI, 1.29–1.80, p < 0.001) and O. viverrini infections (AOR, 1.69; 95% CI, 1.43–2.00, p < 0.001). The prevalence of S. stercoralis infection increased with age. Spatially, the odds of S. stercoralis infection decreased with higher LST and soil pH, while O. viverrini infection was associated with higher soil pH and proximity to water bodies. Strongyloides stercoralis and O. viverrini are highly prevalent in these regions, highlighting the need for surveillance. [ABSTRACT FROM AUTHOR]

Opisthorchiasis, caused by the liver fluke Opisthorchis viverrini, is endemic to Southeast Asian countries and constitutes a major health problem as it increases the risk of cholangiocarcinoma. However, owing to the complicated life cycle of O. viverrini, there is no rapid method for monitoring the risk of infection in the environment. The inability to identify water sources at risk of infection presents a challenge in implementing preventive measures in areas where fish are a significant component of the local diet. Using the environmental DNA (eDNA) approach to detect parasitic DNA in water samples, it is possible to understand the distribution of O. viverrini and the dynamics of infection with high accuracy. However, the sensitivity of environmental samples remains a bottleneck. In this study, we developed a highly sensitive method for detecting O. viverrini eDNA by designing a multi-marker assay targeting two mitochondrial and two nuclear genes. We compared its performance with single-marker assays using tissue-derived DNA. The multi-marker assay exhibited the highest detection sensitivity. We also collected data from 56 sites in Sakon Nakhon, Thailand, in October 2022, and tested them for eDNA detection of O. viverrini. Eleven sites were positive in the multi-marker assay, whereas five sites were positive in the single-marker assay. This multi-marker assay can be used in the field to detect small organisms and rare species, other than parasites, that are expected to have low levels of eDNA. [ABSTRACT FROM AUTHOR]

Macro-organisms such as fluke parasites can induce carcinogenesis. Opisthorchis viverrini and Clonorchis sinensis are known to cause liver cancer. Opisthorchis felineus holds potential as a carcinogen. The carcinogenic drivers are complex and not fully known, but recent advances such as CRISPR gene editing of key fluke virulence factors have proved informative. For example, Ov-GRN-1 granulin growth factor is a critical virulence factor that drives host cell proliferation and ultimately promotes carcinogenic transformation. Secretion of extracellular vesicles (EVs) by flukes is a critical means of host–parasite communication and likely contributes to carcinogenesis. The impact of fluke infection on the host microbiome, as well as the fluke's own microbiome, is revealing likely roles in pathogenesis and requires more investigation. Candidate fluke vaccine antigens are emerging, but an effective fluke vaccine likely needs a multivalent approach. Liver fluke infection is a major risk for cholangiocarcinoma (CCA). It has been established that the Asian liver flukes, Clonorchis sinensis and Opisthorchis viverrini secrete growth factors, digestive enzymes, and extracellular vesicles (EVs) which contribute to abnormal cell development in the bile ducts where the worms reside. These secretions – combined with aberrant inflammation and repeated cycles of chronic wounding at the site of parasite attachment and grazing on the epithelium – promote biliary hyperplasia and fibrosis and ultimately malignant transformation. Application of post-genomic and gene-editing tools to the study of liver fluke immunobiology and pathogenesis has accelerated the discovery of essential virulence factors to which targeted therapies and diagnostics can be directed. [ABSTRACT FROM AUTHOR]

Aims: 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. Methods and Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Background: Cholangiocarcinoma (CCA) is high in morbidity and mortality rates which may be due to asymptomatic and effective diagnostic methods not available. Therefore, an effective diagnosis is urgently needed. Methods: Investigation of plasma circulating miRNA (cir-miRNA) was divided into two phases, including the discovery phase (pooled 10 samples each from three pools in each group) and the validation phase (17, 16, and 35 subjects of healthy control (HC), O. viverrini (OV), and CCA groups, respectively). The plasma from healthy control subjects, O. viverrini infected subjects, and CCA subjects was used. In the discovery phase, plasma was pooled by adding an equal volume of plasma, and cir-miRNA was isolated and analyzed with the nCounter® SPRINT Profiler. The significantly different cir-miRNAs were selected for the validation phase. In the validation phase, cir-miRNA was isolated and analyzed using real time-quantitative polymerase chain reaction (RT-qPCR). Subsequently, statistical analysis was conducted, and diagnostic parameters were calculated. Results: Differential plasma cir-miRNA profile showed at least three candidates including miR-423-5p, miR-93-5p, and miR-4532 as potential biomarkers. From validation of these cir-miRNAs by RT-qPCR, the result showed that the satisfied sensitivity and specificity to differential CCA group from HC and OV group was obtained from miR-4532 (P < 0.05) while miR-423-5p and miR-93-5p can be used for differential CCA from OV and HC group (P < 0.05) with high specificity but limited the sensitivity. In conclusion, candidate cir-miRNAs have been identified as potential biomarkers including miR-423-5p, miR-93-5p and miR-4532. Screening by miR-4532 and confirmed with miR-423-5p, miR-93-5p were suggested for differential CCA patients in the endemic area of O. viverrini. [ABSTRACT FROM AUTHOR]

Background: Steatotic liver disease (SLD) prevalence is rising worldwide, linked to insulin resistance and obesity. SLD prevalence can surpass 10% even among those with normal weight. In Lao People's Democratic Republic (Lao PDR), where Opisthorchis viverrini (OV) trematode infection and type 2 diabetes mellitus (T2DM) are common, infection related liver morbidity such as cholangiocarcinoma (CCA) is high, but data on SLD prevalence is lacking. The objective of this study was to estimate the prevalence and explore determinants of SLD in rural southern Lao PDR for lean and non-lean populations. Method: A cross-sectional community-based study assessed SLD prevalence using abdominal ultrasonography (US). Factors investigated for association with SLD were identified by interview, serological tests (Hepatitis B surface antigen (HBsAg); lipids and HbA1c), anthropometrical measurements, and parasitological assessments (OV infection). Uni- and multivariable logistic regression analyses with SLD as endpoint were conducted separately for lean (body mass index (BMI) <23.0kg/m²) and non-lean (BMI ≥ 23.0kg/m²) participants. Result: 2,826 participants were included. SLD prevalence was 27.1% (95% confidence interval (95% CI) 24.0%-30.4%), higher among non-lean (39.8%) than lean individuals (17.4%). Lean individuals with OV infection had a statistically significant association with lower odds of SLD (adjusted odds ratio (aOR) 0.49, 95% CI 0.33-0.73). T2DM showed a significant positive association with SLD in both lean (aOR 3.58, 95% CI 2.28-5.63) and non-lean individuals (aOR 3.31, 95% CI 2.31-4.74) while dyslipidemia was significantly associated only in the non-lean group (aOR 1.83, 95% CI 1.09-3.07). Females participants exhibited elevated odds of SLD in both lean (aOR 1.43, 95% CI 1.02-2.01) and non-lean SLD (aOR 1.50, 95% CI 1.12-2.01). Conclusion: SLD prevalence is notably high among Laotian adults in rural areas, particularly in females and in non-lean individuals. Lean individuals with OV infection exhibited lower SLD prevalence. SLD was more prevalent in individuals with T2DM, independent of BMI. SLD adds to the burden of infection-related liver morbidity in Lao PDR. [ABSTRACT FROM AUTHOR]

Background: Opisthorchis viverrini (OV) and soil-transmitted helminths (STH) are two of the most common helminths contributing to the Neglected Tropical Disease (NTDs) burden in the Lower Mekong Basin. Although mass drug administration is the cornerstone of control programs to reduce morbidity caused by these infections, this approach has limitations in preventing re-infections. Elimination requires additional measures such as reservoir host treatment, improved hygiene and health education to reinforce MDA's impact. This study aims to examine the impact of a scalable multi-component One Health Helminth Elimination program in the Lower Mekong Basin (HELM) that combines human praziquantel (PZQ) and albendazole (ALB) treatment with a program that includes the "Magic Glasses" and the "Lawa Model" interventions with health promotion at their core. Methods: This study will employ a cluster randomized controlled trial (cRCT) in 18 rural communities (with sub-district or villages as cluster units) across Cambodia, Laos and Thailand. The control arm will receive one round of PZQ/ALB treatment, while in the intervention arm, multi-component HELM program will be implemented, which includes PZQ/ALB treatment together with the Magic Glasses and Lawa Model interventions. OV and STH infections levels will be evaluated in individuals aged 5–75 years at baseline and will be repeated at follow-up (12 months after the HELM intervention), using modified formalin ethyl-acetate concentration technique and quantitative PCR. The primary outcome of the study will be cumulative incidence of human OV and STH infections. Outcomes between the study arms will be compared using generalized linear mixed models, accounting for clustering. Discussion: Evidence from this trial will quantify the impact of a multi-component One Health control strategy in interrupting Ov and STH infections in the Lower Mekong Basin. Trial registration: Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12622000353796. Prospectively registered 28 February 2022. [ABSTRACT FROM AUTHOR]

Introduction: Cholangiocarcinoma (CCA) is a major contributor to hepatobiliary mortality in the Lao People's Democratic Republic (Lao PDR). Infection with the carcinogenic trematode Opisthorchis viverrini (OV), acquired through consumption of insufficiently-cooked river fish, is a known risk factor for the development of CCA. Together with OV, other risk factors contribute to the pathogenesis of CCA. We conducted this study to identify the burden of CCA and identify risk factors in high-risk communities in Lao PDR. Method: A cross-sectional study was performed in Champasack and Savannakhet provinces, southern Lao PDR, where OV infection is highly endemic. We assessed hepatobiliary morbidity with abdominal ultrasound (US). In addition, multiple risk factors known or suspected to be associated with CCA were assessed such as OV infection (examined by Kato-Katz technique for stool examination), lifestyle risks (e.g. smoking and alcohol consumption by face-to-face questionnaire), co-morbidity (e.g. diabetes mellitus) and hepatitis B infection status, both serologically tested. Results: In 3,400 participants, the overall prevalence of suspected CCA was 7.2% (95% confidence interval [95% CI] 5.4−9.6). The suspected CCA prevalence increased with age, and was higher in men at all ages. Almost all participants (88.3%) were infected with OV. In the multivariate regression analysis, suspected CCA was positively associated with OV infection (adjusted odds ratio [aOR] 3.4, 95% CI 1.7−6.5), and a history of cholecystectomy (aOR 2.7, 95% CI 1.5−4.9). Conclusion: Our CCA screening in high OV prevalence rural areas of Lao PDR uncovers a high public health burden, primarily driven by elevated OV infection rates. Urgent interventions are needed to curb OV infection in these communities. Age and gender disparities in suspected CCA prevalence highlight the need for targeted efforts. Beyond OV, notable factors like a history of cholecystectomy offer valuable insights for preventive strategies. This research enhances our understanding of hepatobiliary morbidity and informs public health initiatives in Lao PDR. Author summary: Evidence has long pointed to a significant burden of liver diseases in certain regions of the Lao People's Democratic Republic (Lao PDR), primarily attributed to the Opisthorchis viverrini (OV) liver fluke. This parasite is transmitted to humans through the consumption of raw or undercooked fish and fish products. Chronic OV infection can lead to cholangiocarcinoma (CCA), a fatal bile duct cancer. The objective of this research was to assess the prevalence of suspected CCA in rural OV-endemic communities in the southern part of Lao PDR. We conducted a cross-sectional study in selected communities within Savannakhet and Champasack provinces. Adults aged 35 years and older were enrolled and underwent liver disease assessment through abdominal ultrasound. OV infection was diagnosed via fecal examination, while lifestyle risk factors like smoking and alcohol consumption were evaluated through face-to-face interviews. Additionally, diabetes mellitus and hepatitis B infection status were determined serologically. We found a high prevalence of suspected bile duct cancer (CCA) at 7.2% overall. This rate was found higher in men compared to women, and to increase with age. Most participants (88.3%) were infected with OV, posing the highest risk for suspected CCA (adjusted odds ratio [aOR] 3.4). Additionally, a history of cholecystectomy (aOR 2.7) was a significant predictor of bile duct cancer. These results provide crucial insights for controlling liver morbidity in Lao PDR, emphasizing the urgent need to address OV infection and associated risk factors for CCA in affected communities. [ABSTRACT FROM AUTHOR]

Introduction: Infection with liver flukes (Opisthorchis viverrini) is partly attributed to their ability to thrive in sub-basin habitats, causing the intermediate host to remain within the watershed system throughout the year. It is crucial to conduct spatial monitoring of fluke infection at a small basin analysis scale as it helps in studying the spatial factors influencing these infections. The number of infected individuals was obtained from local authorities, converted into a percentage, and visually represented as raster data through a heat map. This approach generates continuous data with dependent variables. Methods: The independent set comprises nine variables, including both vector and raster data, that establish a connection between the location of an infected person and their village. Design spatial units optimized for geo-weighted modeling by utilizing a clustering and overlay approach, thereby facilitating the optimal prediction of alternative models for infection. Results and discussion: The Model-3 demonstrated the strongest correlation between the variables X5 (stream) and X7 (ndmi), which are associated with the percentage of infected individuals. The statistical analysis showed t-statistics values of −2.045 and 0.784, with corresponding p -values of 0.016 and 0.085. The RMSE was determined to be 2.571%, and the AUC was 0.659, providing support for these findings. Several alternative models were tested, and a generalized mathematical model was developed to incorporate the independent variables. This new model improved the accuracy of the GWR model by 5.75% and increased the R 2 value from 0.754 to 0.800. Additionally, spatial autocorrelation confirmed the difference in predictions between the modeled and actual infection values. This study demonstrates that when using GWR to create spatial models at the sub-basin level, it is possible to identify variables that are associated with liver fluke infection. [ABSTRACT FROM AUTHOR]

This study involved two series of independent trials that investigated the storage temperature and time of raw fish to eliminate Opisthorchis viverrini metacercarial infection in Thai pickled fish. A total of 330 healthy silver barb fishes (30‐day posthatch) were infected with O. viverrini cercariae at an inoculation ratio of 50 cercariae/fish. After infection, the fish was reared for 3 months. The first trial was to evaluate the effect of fish storage temperature in the recovery from O. viverrini metacercariae. The infected fishes were randomly divided into five groups and stored at −20°C, 2°C, 4°C, and 8°C or room temperature (as a control) for 24 h. The results showed that at a storage temperature of −20°C, the fish initially had a reduced recovery rate from O. viverrini metacercariae. The second trial investigated the storage time for the recovery of infected fish stored at −20°C for 18, 24, 36, 48, or 60 h and used room temperature as a control group. The results revealed that storing infected fish at −20°C for 48 and 60 h had the lowest recovery rate from O. viverrini metacercariae at 0.00%. In conclusion, storing infected fish at a temperature of −20°C for a period of 48 h or longer could restrict the O. viverrini metacercaria recovery rate. These results were recommended as suitable conditions for the preparation of Pla‐som to avoid O. viverrini metacercaria contamination. [ABSTRACT FROM AUTHOR]

Functional genomics are needed for helminths to facilitate basic and transformational studies to improve global public health. This review charts the recent advances in CRISPR genetic engineering for flatworm parasites. Schistosomes were genome edited with Cas9 and Cas12a for knockout (KO) mutation and transgene knock-in (KI). Approaches for Schistosoma mansoni were adapted for Schistosoma japonicum and the liver fluke, Opisthorchis viverrini. KO of a liver fluke growth factor confirmed a role in biliary disease and malignancy. Cas12a- and Cas13a-based diagnosis was established in regions endemic for schistosomiasis and opisthorchiasis. Genome safe harbor sites have been predicted in S. mansoni using computational genome annotation and chromatin structure analysis and targeted for KI with GFP. Derivation of heritably transgenic loss- or gain-of function lines is the next milestone. CRISPR genome editing is actively used for schistosomes and other flukes. The ability to genetically manipulate these flatworms enables deeper investigation of their (patho)biological nature. CRISPR gene knockout (KO) demonstrated that a liver fluke growth mediator contributes to disease progression. Genome safe harbor sites have been predicted in Schistosoma mansoni and targeted for transgene insertion. CRISPR-based diagnosis has been demonstrated for infection with schistosomes and Opisthorchis viverrini. This review charts the progress, and the state of play, and posits salient questions for the field to address. Derivation of heritably transgenic loss-of-function or gain-of-function lines is the next milestone. [ABSTRACT FROM AUTHOR]

We present a genome assembly from a specimen of Opisthorchis viverrini (liver fluke; Platyhelminthes; Trematoda; Opisthorchiida; Opisthorchiidae). The genome sequence has a total length of 627.20 megabases. Most of the assembly (97.89%) is scaffolded into 6 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 18.04 kilobases in length.

ABSTRACT There is strong epidemiological evidence that development of various cancer types is linked to infection with flukes (Platyhelminthes: Trematoda) in Africa, Asia and the Middle East. The exact nature of the mechanism by which cancer is induced by these parasites is unknown. Here, we provide a new hypothesis suggesting that flukes are not the primary cause of cancer but act as vectors of cancer‐inducing microbial pathogens. These pathogens adaptively induce tumours to attract and help flukes to feed on blood from the tumour. Pathogen take‐up by fluke vectors also takes place in the tumour; therefore, tumour formation in this case is the result of a mutualistic and adaptive relationship between the microbe and the helminth parasite. The suggested mechanism for cancer induction provided here may help us gain deeper understanding about cancer in general and its relationship with microbes and parasites. By further elaborating the unique nexus between flukes, carcinogenic microbes and cancer, in the future it will also help us to broaden our oncological perspective to reduce human death and suffering from this serious disease group.

Abstract Background Helminth 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. Methods We 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. Results Eco-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

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 marks 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 systematic 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/β-catenin pathway deregulation as key drivers in Ov-CCA pathogenesis. Promoter hypermethylation of specific genes suggests potential diagnostic biomarkers and dysregulation of Wnt/β-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. Author summary: This review examines the role of epigenetic changes and the Wnt/β-catenin signalling pathway in Opisthorchis viverrini-induced cholangiocarcinoma (Ov-CCA). Through an exhaustive analysis of relevant studies, the primary epigenetic alteration identified in Ov-CCA pathogenesis is hypermethylation of tumor suppressor gene promoters. This epigenetic dysregulation shows several affected genes involved in the Wnt/β-catenin signalling pathway, which plays a central role in opisthorchiasis progression. Network analysis revealed an interaction between these genes and β-catenin, suggesting Wnt/β-catenin pathway dysregulation contributes to Ov-CCA development and progression. Most methylated genes act as tumor suppressors by normally inhibiting the Wnt/β-catenin pathway. Targeting this pathway offers therapeutic potential for Ov-CCA. Epigenetic markers hold promise as biomarkers, emphasizing the therapeutic potential of targeting disrupted gene and signalling networks through epigenetic modulators. This approach presents an innovative strategy for developing personalized therapies tailored to the molecular profiles of Ov-CCA patients. However, translating these findings into clinical practice requires rigorous validation and further in-depth exploration. Nonetheless, the insights obtained highlight the crucial necessity for ongoing research on epigenetic-based interventions, intending to enhance both diagnostic accuracy and therapeutic effectiveness in managing Ov-CCA. [ABSTRACT FROM AUTHOR]

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Objectives: The current study was performed to determine the antioxidant, anti-inflammatory, and anti-cholangiocarcinoma (CCA) properties of Antidesma thwaitesianum, also known as "MAO," whole plant extract on Opisthorchiasis in animal models and CCA cell lines. Materials and Methods: Ethanol was used to extract compounds from the whole ripe fruit. The phytochemical investigation of MAO extract was done to evaluate antioxidant activity, and high-performance liquid chromatography was used to identify the active compounds. The efficacy of MAO extract against OV was evaluated in vivo. The anti-CCA activity was evaluated using superoxide dismutase (SRB), cell cycle arrest, apoptosis, and western blot analyses. Results: MAO extract possessed flavonoid and phenolic contents, antioxidant activity, and an expressed cyanidin-3-O-glycosides content of 0.08 µg/mg extract. MAO extract demonstrated hepatoprotective effects through raised alanine transaminase and alkaline phosphatase levels, as well as an influence on oxidative stress via decreased MDA and increased glutathione, superoxide dismutase, and catalase levels. MAO extract significantly inhibited the migration of CCA cells in a concentration- and time-dependent manner, as well as triggered cell cycle arrest on G1 and activated apoptosis pathways via upregulation of C3, downregulation of cyclin-dependent kinase inhibitor p21, cyclin D, and cyclin-dependent kinases 2 expression. MAO extract inhibited inflammation, which in turn decreased fibrosis in hamsters. It also increased hepatoprotective activity. Conclusion: Our findings demonstrate the potential benefits of MAO extract in both in vitro and animal studies of hamster opisthorchiasis. However, more research should be done to ascertain the mechanisms of activity of MAO extracts and elute bioactive components in order to confirm their safety and examine their clinical applications. [ABSTRACT FROM AUTHOR]

[Display omitted] • Previous population genetic analysis of Opisthorchis viverrini eggs revealed two populations. • Cat- and human-specific populations use different intermediate fish hosts. • There are distinct morphologies in egg, metacercaria, and adult worm stages of O. viverrini. • Our study confirms distinct human- and cat-specific O. viverrini populations. Infection by the zoonotic fish-borne trematode, Opisthorchis viverrini, remains a crucial health issue in Thailand and neighboring countries. Recently, molecular analysis revealed two populations of putative O. viverrini : one found primarily in human hosts ("human-specific" population) and the other primarily in cats ("cat-specific" population). It is unclear how the infective stages (metacercariae) of these different populations circulate among definitive and reservoir hosts in nature. To gain an insight into this, mitochondrial cox 1 and nad 1 gene sequences of metacercariae from fish intermediate hosts were examined. None of 192 metacercariae from cyprinid fish in Lao PDR and Thailand had sequences typical of "cat-specific" O. viverrini , suggesting that cyprinid fish are not the main second intermediate hosts of this population. Interestingly, all 20 O. viverrini -like metacercariae from snakehead fish (Channa striata) shared 99.51–100% sequence identity with eggs from cats naturally infected in a previous study. Hence, we propose a modification of the known transmission dynamics of O. viverrini : consumption of metacercariae within snakehead fish provides another pathway for cats and (occasionally) humans to acquire infection. We also performed morphological comparisons of eggs, metacercariae, and adult flukes (raised in hamsters) of both Opisthorchis populations. The "cat-specific" population has eggs that are narrower and adults that are shorter and wider than in the human-specific population. The metacercaria of the "cat-specific" population is elliptical, while that of the "human-specific" population is oval, occasionally rounded. Our results confirmed that O. viverrini -like metacercariae from snakehead fish are the infective stages of the "cat-specific" fluke. This provides a new insight into the dissemination and transmission of each population in the second intermediate host. The identity of the cat-specific population is discussed. [ABSTRACT FROM AUTHOR]

Infection with liver flukes (Opisthorchis viverrini) is partly due to their ability to thrive in habitats in sub-basin areas, causing the intermediate host to remain in the watershed system throughout the year. Spatial modeling is used to predict water source infections, which involves designing appropriate area units with hexagonal grids. This allows for the creation of a set of independent variables, which are then covered using machine learning techniques such as forest-based classification regression methods. The independent variable set was obtained from the local public health agency and used to establish a relationship with a mathematical model. The ordinary least (OLS) model approach was used to screen the variables, and the most consistent set was selected to create a new set of variables using the principal of component analysis (PCA) method. The results showed that the forest classification and regression (FCR) model was able to accurately predict the infection rates, with the PCA factor yielding a reliability value of 0.915. This was followed by values of 0.794, 0.741, and 0.632, respectively. This article provides detailed information on the factors related to water body infection, including the length and density of water flow lines in hexagonal form, and traces the depth of each process. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Helminth 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. Methods: We 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. Results: Eco-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. Conclusions: The 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. [ABSTRACT FROM AUTHOR]

Few researches have explored the production of pharmaceuticals from aquatic plants. Therefore, this study explored, for the first time, the phytochemical composition and bioactivities of ten aquatic plants. Aquatic plant shoots from various Nile River canals were collected, dried, and ground for aqueous extract preparation. Phytochemical composition and antioxidant capacity were assessed using DPPH assays. Extracts were tested for antiparasitic, antibacterial, anti-biofilm, and anticancer activities through standard in vitro assays, measuring IC50 values, and evaluating mechanisms of action, including cell viability and high-content screening assays. The results showed that the aquatic plants were rich in pharmaceutical compounds. The antioxidant capacity of these extracts exceeded that of vitamin C. The extracts showed promising antiparasitic activity against pathogens like Opisthorchis viverrini and Plasmodium falciparum, with IC50 values between 0.7 and 2.5 µg/mL. They also demonstrated low MICs against various pathogenic bacteria, causing DNA damage, increased plasma membrane permeability, and 90% biofilm inhibition. In terms of anticancer activity, extracts were effective against a panel of cancer cell lines, with Ludwigia stolonifera exhibiting the highest efficacy. Its IC50 ranged from 0.5 µg/mL for pancreatic, esophageal, and colon cancer cells to 1.5 µg/mL for gastric cancer cells. Overall, IC50 values for all extracts were below 6 µg/mL, showing significant apoptotic activity, increased nuclear intensity, plasma membrane permeability, mitochondrial membrane permeability, and cytochrome c release, and outperforming doxorubicin. This study highlights the potential of aquatic plants as sources for new, safe, and effective drugs with strong antiparasitic, antibacterial, and anticancer properties. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors (serpins) participate in the regulation of inflammation, blood coagulation, and complement activation in humans. This research aimed to identify and characterize such inhibitors of the human liver fluke Opisthorchis viverrini. Parasite proteins that might contribute to the modulation of host physiology are of particular interest, especially as chronic opisthorchiasis increases the risk of developing biliary cancer. BLAST was used to find hypothetical serpins predicted from the parasite genome data. RNA extraction and reverse transcriptase PCR were used to isolate a serpin cDNA and to determine developmental transcript abundance. The evolutionary relation to other trematode serpins was revealed by phylogenetic analysis. Recombinant serpin was expressed in Escherichia coli and used to test the immunoreactivity of human opisthorchiasis sera and the inhibition of human serine proteases. A substantial serpin family with high sequence divergence among the members was found in the genus Opisthorchis. A serpin, different from previously analyzed trematode serpins, was cloned. The transcript was only detected in metacercariae and newly excysted juveniles. Human opisthorchiasis sera showed statistically significant reactivity to recombinant serpin. The serpin caused moderate inhibition of thrombin and low inhibition of kallikrein and chymotrypsin. This parasite serpin could be further evaluated as a diagnostic tool for early infection. Kallikrein and thrombin are involved in fibrinolysis; therefore, further research should explore the effects of the parasite serpin on this process. [ABSTRACT FROM AUTHOR]

The liver fluke O. viverrini (Opisthorchis viverrini), a neglected tropical disease (NTD), endemic to the Great Mekong Subregion (GMS), mainly afflicts the northeastern region of Thailand. It is a leading cause of cholangiocarcinoma (CCA) in humans. Presently, the treatment modalities for opisthorchiasis incorporate the use of the antihelminthic drug praziquantel, the rapid occurrence of reinfection, and the looming threat of drug resistance highlight the urgent need for vaccine development. Recent advances in "omics" technologies have proven to be a powerful tool for such studies. Utilizing candidate proteins identified through proteomics and refined via immunoproteomics, reverse vaccinology (RV) offers promising prospects for designing vaccines targeting essential antibody responses to eliminate parasite. Machine learning-based computational tools can predict epitopes of candidate protein/antigens exhibiting high binding affinities for B cells, MHC classes I and II, indicating strong potential for triggering both humoral and cellmediated immune responses. Subsequently, these vaccine designs can undergo population-specific testing and docking/dynamics studies to assess efficacy and synergistic immunogenicity. Hence, refining proteomics data through immunoinformatics and employing computational tools to generate antigenspecific targets for trials offers a targeted and efficient approach to vaccine development that applies to all domains of parasite infections. In this review, we delve into the strategic antigen selection process using omics modalities for the O. viverrini parasite and propose an innovative framework for vaccine design. We harness omics technologies to revolutionize vaccine development, promising accelerated discoveries and streamlined preclinical and clinical evaluations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical Abstract

Objectives: The current study was performed to determine the antioxidant, anti-inflammatory, and anti-cholangiocarcinoma (CCA) properties of Antidesma thwaitesianum, also known as "MAO," whole plant extract on Opisthorchiasis in animal models and CCA cell lines. Materials and Methods: Ethanol was used to extract compounds from the whole ripe fruit. The phytochemical investigation of MAO extract was done to evaluate antioxidant activity, and high-performance liquid chromatography was used to identify the active compounds. The efficacy of MAO extract against OV was evaluated in vivo. The anti- CCA activity was evaluated using superoxide dismutase (SRB), cell cycle arrest, apoptosis, and western blot analyses. Results: MAO extract possessed flavonoid and phenolic contents, antioxidant activity, and an expressed cyanidin-3-O-glycosides content of 0.08 μg/mg extract. MAO extract demonstrated hepatoprotective effects through raised alanine transaminase and alkaline phosphatase levels, as well as an influence on oxidative stress via decreased MDA and increased glutathione, superoxide dismutase, and catalase levels. MAO extract significantly inhibited the migration of CCA cells in a concentration- and time-dependent manner, as well as triggered cell cycle arrest on G1 and activated apoptosis pathways via upregulation of C3, downregulation of cyclin-dependent kinase inhibitor p21, cyclin D, and cyclin-dependent kinases 2 expression. MAO extract inhibited inflammation, which in turn decreased fibrosis in hamsters. It also increased hepatoprotective activity. Conclusion: Our findings demonstrate the potential benefits of MAO extract in both in vitro and animal studies of hamster opisthorchiasis. However, more research should be done to ascertain the mechanisms of activity of MAO extracts and elute bioactive components in order to confirm their safety and examine their clinical applications. [J Adv Vet Anim Res 2024; 11(3.000): 637-647]

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. [ABSTRACT FROM AUTHOR]

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 I2 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. [ABSTRACT FROM AUTHOR]

Background: Cholangiocarcinoma (CCA) caused by Opisthorchis viverrini is a well‐known and significant public health issue in northeastern Thailand; however, a link between pesticide exposure (PE) and CCA risk has not yet been established. Therefore, our research objective was to investigate the relationship between PE and CCA risk. Methods: A hospital‐based matched case–control study was carried out. All cases (in‐patients) and controls (out‐patients) were volunteers at a tertiary hospital in northeast Thailand. Between 2015 and 2019, 178 incident cases of pathologically‐confirmed CCA and 356 controls were selected from the check‐up clinic from the Srinagarind Hospital outpatient database (two controls per case). The recruited controls were individually‐matched to the CCA cases based on sex, age (±5 years) and admission date (±3 months). During face‐to‐face interviews, a standardised pre‐tested questionnaire was used to collect data. Multivariable conditional logistic regression was used to analyse the data. Results: The respective frequency of PE between the 178 CCA cases and 356 controls was 77.0% versus 87.6% for never used, 14.6% versus 5.3% for have used but stopped and 8.4% versus 7.0% for currently using. After adjusting for the highest educational attainment, smoking behaviour, alcohol use and family history of cancer, PE was not significantly associated with CCA (p‐value = 0.086). Using volunteers who have never used PE as the reference group, the respective odds of developing CCA for those who have ever used but have since stopped and are currently using was 2.04 (adjusted OR = 2.04; 95% CI: 1.03–4.04) versus 0.83 (adjusted OR = 0.83; 95% CI: 0.39–1.76) times more likely to develop CCA than those who had never used PE. Conclusion: There is no association between PE and the risk of CCA. Notwithstanding the finding, future research should focus on enhancing PE assessment methods that consider complex chemical mixtures, chemicals of interest, historical exposure and exposure pathways. Moreover, there is need for more extensive and longer population‐based cohort studies that include younger, non‐occupationally exposed individuals during periods of developmental susceptibility. [ABSTRACT FROM AUTHOR]

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.

Abstract Hepatitis B and C virus, Opisthorchis viverrini and Clonorchis sinensis, are all individually known to put a person at increased risk for cholangiocarcinoma and hepatocellular carcinoma. This paper seeks to determine if there is any interaction between liver flukes and hepatitis virus infection that are known to put a person at an increased risk for cholangiocarcinoma and hepatocellular carcinoma collectively. This paper seeks to determine whether there is any publicly available articles in English that determine if having a hepatitis viral co-infection along with liver flukes would influence the risk of developing liver cancer. We followed PRISMA systematic review guidelines to conduct a literature review. Three manuscripts fit the search criteria. Two presented evidence in support of a synergistic relationship between liver fluke and viral hepatitis infection while the other found no relationship. One manuscript determined that the interaction between hepatitis B and C. sinensis did not have any significant risk of liver cancer. Studies found that HBV affected progression of co-infection to liver cancer but may have its own disease state worsened by presence of liver flukes. Only one paper was found that presented data on HCV, therefore no conclusion can be drawn due to the lack of evidence discovered. Of the studies, the conclusions and strength of the data were mixed. However, the stronger studies suggested a synergistic relationship between liver flukes and HBV to increase the risk of progressing to liver cancer.

Abstract Background 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. Methods 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. Results 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 10−1 ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1 ng. 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. Conclusions 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. Graphical abstract

Background: Inflammation caused by Opisthorchis viverrini infection increases the risk of cholangitis, cholecystitis, and lead 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. Additionally, there are reports indicating differences in the locations CCA. Aim: 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. Methods: Thirty Syrian golden hamsters (a susceptible host) and 30 BALB/c mice (a non-susceptible host) infected with O. viverrini were studied at 6 time points (5 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 [Open Vet J 2024; 14(2.000): 664-673]