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.

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.

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.

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.

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.

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.

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.

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.

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

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

Steatotic liver disease among lean and non-lean individuals in Southern Lao PDR: a cross-sectional study of risk factors.

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) <23.0 kg/m2) and non-lean (BMI ≥ 23.0 kg/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.

Natural variables separate the endemic areas of Clonorchis sinensis and Opisthorchis viverrini along a continuous, straight zone in Southeast Asia.

BACKGROUND: Clonorchiasis and opisthorchiasis, caused by the liver flukes Clonorchis sinensis and Opisthorchis viverrini respectively, represent significant neglected tropical diseases (NTDs) in Asia. The co-existence of these pathogens in overlapping regions complicates effective disease control strategies. This study aimed to clarify the distribution and interaction of these diseases within Southeast Asia. METHODS: We systematically collated occurrence records of human clonorchiasis (n = 1809) and opisthorchiasis (n = 731) across the Southeast Asia countries. Utilizing species distribution models incorporating environmental and climatic data, coupled machine learning algorithms with boosted regression trees, we predicted and distinguished endemic areas for each fluke species. Machine learning techniques, including geospatial analysis, were employed to delineate the boundaries between these flukes. RESULTS: Our analysis revealed that the endemic range of C. sinensis and O. viverrini in Southeast Asia primarily spans across part of China, Vietnam, Thailand, Laos, and Cambodia. During the period from 2000 to 2018, we identified C. sinensis infections in 84 distinct locations, predominantly in southern China (Guangxi Zhuang Autonomous Region) and northern Vietnam. In a stark contrast, O. viverrini was more widely distributed, with infections documented in 721 locations across Thailand, Laos, Cambodia, and Vietnam. Critical environmental determinants were quantitatively analyzed, revealing annual mean temperatures ranging between 14 and 20 °C in clonorchiasis-endemic areas and 24-30 °C in opisthorchiasis regions (P < 0.05). The machine learning model effectively mapped a distinct demarcation zone, demonstrating a clear separation between the endemic areas of these two liver flukes with AUC from 0.9 to1. The study in Vietnam delineates the coexistence and geographical boundaries of C. sinensis and O. viverrini, revealing distinct endemic zones and a transitional area where both liver fluke species overlap. CONCLUSIONS: Our findings highlight the critical role of specific climatic and environmental factors in influencing the geographical distribution of C. sinensis and O. viverrini. This spatial delineation offers valuable insights for integrated surveillance and control strategies, particularly in regions with sympatric transmission. The results underscore the need for tailored interventions, considering regional epidemiological variations. Future collaborations integrating eco-epidemiology, molecular epidemiology, and parasitology are essential to further elucidate the complex interplay of liver fluke distributions in Asia.

Opisthorchis viverrini excretory-secretory products suppress GLUT8 of cholangiocytes.

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

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

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

Temperature dependence of Opisthorchis felineus infection in the first intermediate host snail, Bithynia troschelii.

Opisthorchiasis is one of the most serious trematodiases in Russia, where the world's largest focus is located in the Ob basin. Temperature is an important factor affecting the metabolism of cold-blooded animals. It determines the development of the causative agent of opisthorchiasis, Opisthorchis felineus, and the success of infection of an intermediate host, the snail Bithynia troschelii. In the present study, the effect of water temperature on the development of the liver fluke O. felineus in the host snail was assessed, as was the temperature threshold at which B. troschelii hibernation initiates. Adult uninfected B. troschelii individuals collected from natural bodies of water were infected with O. felineus and maintained at different temperatures of water (18-30 °C, intervals of 3 °C) in the laboratory. Each snail was fed with embryonated uterine eggs of O. felineus at 24 °C. O. felineus infection in snails was detected using polymerase chain reaction (PCR) using specific primers. The prevalence of O. felineus infection in B. troschelii depends on the water temperature in which the snails are maintained. The highest infection rate of 45.2 % ± 12.1 % was observed at 27 °C (p ≥ 0.1). The longest lifespan of infected and uninfected B. troschelii was recorded at water temperatures of 24 and 27 °C. The snails were more successfully infected at the beginning of the warm season. Among the infected individuals, the majority (up to 85 %) were large snails. Cercarial shedding was not detected in experimentally infected snails. Apparently, this is due to the natural physiological state of Bithynia snails during the autumn-winter diapause, when opisthorchiids development in snails stops. At 10 °C, complete hibernation of all B. troschelii snails was observed, and infection by the trematodes became impossible. The highest prevalence of infection was recorded at 27 °C, suggesting that during climate warming, an increase in opisthorchiid infection of snails may occur, which must be considered when epidemiological measures are planned.

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

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

Modeling the persistence of Opisthorchis viverrini worm burden after mass-drug administration and education campaigns with systematic adherence.

Opisthorchis viverrini is a parasitic liver fluke contracted by consumption of raw fish, which affects over 10 million people in Southeast Asia despite sustained control efforts. Chronic infections are a risk factor for the often fatal bile duct cancer, cholangiocarcinoma. Previous modeling predicted rapid elimination of O. viverrini following yearly mass drug administration (MDA) campaigns. However, field data collected in affected populations shows persistence of infection, including heavy worm burden, after many years of repeated interventions. A plausible explanation for this observation is systematic adherence of individuals in health campaigns, such as MDA and education, with some individuals consistently missing treatment. We developed an agent-based model of O. viverrini which allows us to introduce various heterogeneities including systematic adherence to MDA and education campaigns at the individual level. We validate the agent-based model by comparing it to a previously published population-based model. We estimate the degree of systematic adherence to MDA and education campaigns indirectly, using epidemiological data collected in Lao PDR before and after 5 years of repeated MDA, education and sanitation improvement campaigns. We predict the impact of interventions deployed singly and in combination, with and without the estimated systematic adherence. We show how systematic adherence can substantially increase the time required to achieve reductions in worm burden. However, we predict that yearly MDA campaigns alone can result in a strong reduction of moderate and heavy worm burden, even under systematic adherence. We predict latrines and education campaigns to be particularly important for the reduction in overall prevalence, and therefore, ultimately, elimination. Our findings show how systematic adherence can explain the observed persistence of worm burden; while emphasizing the benefit of interventions for the entire population, even under systematic adherence. At the same time, the results highlight the substantial opportunity to further reduce worm burden if patterns of systematic adherence can be overcome.

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

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

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

Background: Human opisthorchiasis is a dangerous infectious chronic disease distributed in many Asian areas in the water-basins of large rivers, Siberia, and Europe. The gold standard for human opisthorchiasis laboratory diagnosis is the routine examination of Opisthorchis spp. eggs under a microscope. Manual detection is laborious, time-consuming, and dependent on the microscopist's abilities and expertise. Automatic screening of Opisthorchis spp. eggs with deep learning techniques is a useful diagnostic aid. Methods: Herein, we propose a convolutional neural network (CNN) for classifying and automatically detecting O. viverrini eggs from digitized images. The image data acquisition was acquired from infected human feces and was processed using the gold standard formalin ethyl acetate concentration technique, and then captured under the microscope digital camera at 400x. Microscopic images containing artifacts and O.viverrini egg were augmented using image rotation, filtering, noising, and sharpening techniques. This augmentation increased the image dataset from 1 time to 36 times in preparation for the training and validation step. Furthermore, the overall dataset was subdivided into a training-validation and test set at an 80:20 ratio, trained with a five-fold cross-validation to test model stability. For model training, we customized a CNN for image classification. An object detection method was proposed using a patch search algorithm to detect eggs and their locations. A performance matrix was used to evaluate model efficiency after training and IoU analysis for object detection. Results: The proposed model, initially trained on non-augmented data of artifacts (class 0) and O. viverrini eggs (class 1), showed limited performance with 50.0% accuracy, 25.0% precision, 50.0% recall, and a 33.0% F1-score. After implementing data augmentation, the model significantly improved, reaching 100% accuracy, precision, recall, and F1-score. Stability assessments using 5-fold cross-validation indicated better stability with augmented data, evidenced by an ROC-AUC metric improvement from 0.5 to 1.00. Compared to other models such as ResNet50, InceptionV3, VGG16, DenseNet121, and Xception, the proposed model, with a smaller file size of 2.7 MB, showed comparable perfect performance. In object detection, the augmented data-trained model achieved an IoU score over 0.5 in 139 out of 148 images, with an average IoU of 0.6947. Conclusion: This study demonstrated the successful application of CNN in classifying and automating the detection of O. viverrini eggs in human stool samples. Our CNN model's performance metrics and true positive detection rates were outstanding. This innovative application of deep learning can automate and improve diagnostic precision, speed, and efficiency, particularly in regions where O. viverrini infections are prevalent, thereby possibly improving infection sustainable control and treatment program.