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.

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.

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.

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.

Association between Opisthorchis viverrini infection in cats and humans: Non-spatial and spatial analyses.

Cats are recognized as significant reservoir hosts for human opisthorchiasis, particularly in areas with a high prevalence of infection. Despite this, the precise role of cats in the transmission of Opisthorchis viverrini between humans and felines remains unclear. This study investigates the association between these two hosts through both spatial and non-spatial analyses in the endemic Thanya sub-district of Thailand. A total of 105 owned cats were randomly sampled from 15 villages within the sub-district for stool examination. A questionnaire was administered to 66 cat owners to explore the human-pet relationship. Household locations were collected using GPS devices. Non-spatial analyses revealed a positive association between the two hosts (P= 0.011; OR 7, 95% CI: 1.6-30.9), highlighting two independent significant risk factors: cat owners consuming raw fish (P = 0.028; OR = 4.52, 95% CI: 1.25-19.45) and feeding cats raw fish (P = 0.011; OR = 16.41, 95% CI: 2.78-317.04) according to multivariate analysis. Spatial analysis provided further support to the non-spatial findings (p = 0.0123; OR = 3.45, 95% CI = 0.88-13.61). Multiple autologistic regression confirmed two significant risk factors: cat owners consuming raw fish (p = 0.054; OR = 3.37, 95% CI: 0.98-11.59) and feeding cats raw fish (p = 0.014; OR = 7.43, 95% CI: 1.49-37.05). Risk mapping identified the western part of the study site as a hotspot for O. viverrini infection. Hyper-endemic focusing revealed a union of human and cat buffers at 0.46 km², with an overlapping area of 0.22 km² (47.83%). This study underscores the impact of owners' behaviors, specifically consuming and feeding raw fish to cats, on the increased probability of infection in cats. It emphasizes the need for effective opisthorchiasis control through health education targeting cat owners in endemic areas.

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.

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

Clonorchis sinensis, Opisthorchis viverrini, and Opisthorchis felineus are important liver flukes that cause a considerable public health burden in eastern Asia, southeastern Asia, and eastern Europe, respectively. The life cycles are complex, involving humans, animal reservoirs, and two kinds of intermediate hosts. An interplay of biological, cultural, ecological, economic, and social factors drives transmission. Chronic infections are associated with liver and biliary complications, most importantly cholangiocarcinoma. With regard to diagnosis, stool microscopy is widely used in epidemiologic surveys and for individual diagnosis. Immunologic techniques are employed for screening purposes, and molecular techniques facilitate species differentiation in reference laboratories. The mainstay of control is preventive chemotherapy with praziquantel, usually combined with behavioral change through information, education and communication, and environmental control. Tribendimidine, a drug registered in the People's Republic of China for soil-transmitted helminth infections, shows potential against both C. sinensis and O. viverrini and, hence, warrants further clinical development. Novel control approaches include fish vaccine and biological control. Considerable advances have been made using multi-omics which may trigger the development of new interventions. Pressing research needs include mapping the current distribution, disentangling the transmission, accurately estimating the disease burden, and developing new diagnostic and treatment tools, which would aid to optimize control and elimination measures.

Hepatic vascular changes associated with Opisthorchis felineus infection in Syrian hamsters and humans.

The liver fluke Opisthorchis felineus is a foodborne zoonotic pathogen endemic to Russia, Kazakhstan, and several European countries. The adult flukes affect the hepatobiliary system of piscivorous mammals and humans, thereby causing numerous complications, including liver fibrosis. Detailing the mechanisms of progression of the fibrotic complications is a hot topic in the field of research on opisthorchiasis pathogenesis. Pathologic angiogenesis appears to be associated with the fibrogenic progression due to active participation in the recruitment of inflammatory cells and many factors involved in the modulation of the extracellular matrix. The aim of the study was to evaluate neoangiogenesis and amyloid deposits in liver tissues of model animals and patients with confirmed chronic opisthorchiasis. In addition, we assessed a possible correlation of neoangiogenesis with liver fibrosis. We found a significant increase in the number of newly formed vessels and amyloid deposits in the liver of people with chronic opisthorchiasis compared to that of uninfected ones. Thus, for the first time we have demonstrated neoangiogenesis and amyloid deposits during O. felineus infection in a Mesocricetus auratus model. Regression analysis showed that CD34+ newly formed vessels correlate with fibrosis severity in the course of the infection. Our results indicate the potential contribution of angiogenesis to the progression of liver fibrosis, associated with O. felineus infection.

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

BACKGROUND: Screening for opisthorchiasis, a parasitic worm infection affecting many millions of people in Southeast Asia, has traditionally relied on faecal egg examination such as the formalin-ethyl acetate concentration technique (FECT) and Kato-Katz method. Although the urinary enzyme-linked immunosorbent assay (ELISA) has been used more recently, we developed a urinary antigen-based rapid diagnostic test (RDT) to simplify diagnosis and as a point-of-care testing (POCT) and field applications for surveillance and control of opisthorchiasis. METHODS: A urinary Opisthorchis viverrini (OV)-RDT was developed using immunochromatographic methodology with a specific monoclonal antibody against OV. The diagnostic performance of the urinary OV-RDT was compared to that of quantitative faecal FECT and urinary antigen ELISA (n = 493). Cross-reactivities of urinary OV-RDT with other helminthiases coexisted with O. viverrini were determined (n = 96). A field trial in the application of urinary OV-RDT was compared with urinary antigen ELISA at baseline screening and assessment of drug treatment outcomes in opisthorchiasis (n = 1629). The McNemar chi-square, Kruskal-Wallis and Cohen's kappa coefficient (κ-value) tests were used for statistical analyses. RESULTS: Urinary OV-RDT had sensitivity of 94.2% and specificity of 93.2%, compared to faecal FECT. Urinary OV-RDT had high diagnostic agreement (Kappa = 0.842-0.874, P < 0.001) and quantitative correlation with urinary antigen ELISA (Kruskal-Wallis tests = 316.2, P < 0.0001) and faecal FECT (Kruskal-Wallis tests = 362.3, P < 0.0001). The positive rates by OV-RDT, ELISA and FECT were 48.9%, 52.5% and 49.3%, respectively. Cross-reactions of urinary OV-RDT with other helminthiases were few (2%). Field trials of urinary OV-RDT yielded comparable prevalence of O. viverrini between urinary OV-RDT (53.2%) and urinary antigen ELISA (54.0%). OV screening showed high diagnostic agreement (kappa > 0.8, P < 0.0001) between urinary OV-RDT and urinary antigen ELISA. The cure rates of opisthorchiasis at 1 month post-praziquantel treatment determined by urinary OV-RDT (86.6%) and urinary antigen ELISA (80.5%) were similar (P > 0.05). CONCLUSIONS: The urinary OV-RDT test has high potential as a new tool for screening and evaluating treatment outcomes in opisthorchiasis. The ease of sample collection and simplicity of urinary OV-RDT may facilitate mass screening, control and elimination of opisthorchiasis, thereby contributing to a reduction in the disease burden in Southeast Asia.

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

Raw or undercooked freshwater fish consumption contributes to persistent Opisthorchis viverrini infection in Northeast Thailand. This study aims to assess the relationship between misconceptions, unhealthy eating habits, and O. viverrini infection. Data were obtained from the Cholangiocarcinoma Screening and Care Program in Northeast Thailand from 2019 to 2021. Participants were screened for O. viverrini annually over the following 2 years using the Kato-Katz technique. Misconceptions and unhealthy eating habits were assessed through questionnaires. The relationship between these factors and O. viverrini infection was evaluated using adjusted risk ratios (ARRs) and 95% confidence intervals (CIs) from generalized estimating equations under binomial regression framework. Of 5375 participants screened for O. viverrini over 3 years, infection rates were 21.53%, 10.7%, and 4.6% each year, respectively. Out of those, 636 participants responded to questions regarding misconceptions. Results showed that participants who believed in the efficacy of putting lime or red ants in Koi pla (raw fish salad) or eating Koi pla with white whiskey to kill parasites, and early-stage cholangiocarcinoma can be cured, were 41% (ARR, 1.41; 95% CI, 1.03-1.94) and 57% (ARR, 1.57; 95% CI, 1.06-2.33), respectively, more likely to be infected with O. viverrini. Our study confirms that belief in using lime or red ants in Koi pla or eating Koi pla with white whiskey to make it cooked, or early-stage cholangiocarcinoma can be cured, increases O. viverrini infection risk in high-risk populations. Changing health beliefs and eating habits is necessary to reduce O. viverrini infection and its risk to cholangiocarcinoma.

Microsatellite markers for Opisthorchis felineus to understand its genetic diversity and transmission patterns of opisthorchiosis.

Opisthorchis felineus is a food-borne trematode which causes opisthorchiosis and affects mainly the liver and bile ducts of the liver with a possible risk of bile duct carcinogenesis resulting in cholangiocarcinoma. In Russia, O. felineus is mainly endemic in Western Siberia (Ob and Irtysh river basins) and occurs throughout the Volga, Kama, Don, and Dnepr river basins. The prevalence, intensity, and clinical significance of human infections and the incidence of cholangiocarcinoma vary geographically in endemic regions. Currently, there is substantial evidence on genetic variation of O. felineus, but information on the population genetic structure is so far very scarce. Because microsatellite DNA of this parasite is not available, we for the first time isolated sufficient microsatellite loci to examine the genetic diversity and population structure of O. felineus, using multiple nuclear loci approach. A total of ten highly polymorphic microsatellite loci from a constructed enriched genomic DNA library were characterized, using 29 samples representing huge O. felineus metapopulation extended in latitude over 5000 km from Middle Europe to Western Siberia. At least three populations can be discerned as result of analysis of the microsatellite loci genetic diversity. Based on the results for the first time, a hypothesis was put forward about the formation of a modern habitat of O. felineus.

Epidemiology and Control of Opisthorchis viverrini Infection: Implications for Cholangiocarcinoma Prevention.

It is known that Opisthorchis viverrini (OV) is the most significant risk factor for the development of cholangiocarcinoma (CCA); hence, it is also known as carcinogenic parasite. Effective control and elimination of OV infection should significantly reduce O. viverrini-related CCA. This chapter includes details of the three recently developed innovative tools, namely the Isan cohort database software, an OV-RDT for screening of O. viverrini, and an ultrasound telecommunication system. Past and current control programs, i.e., education, medication, and sanitation were discussed and stressed the need for a comprehensive control program which encompasses primary, secondary, and tertiary patient care programs for confirmation and management of suspected CCA cases. The approach of mathematical modeling for control of OV and CCA was also briefly described. Additionally, we highlighted the current progress toward control of OV and CCA in Thailand and potential for expansion into nearby countries in Southeast Asia.

Roaming behavior of the owned domestic cats (Felis catus) with possible roles in the transmission of Opisthorchis viverrini in the endemic area in Khon Kaen, Thailand.

In the endemic area of the liver fluke (Opisthorchis viverrini) infection, effective control of opisthorchiasis using One Health approaches requires transmission interruption in all hosts in the parasite life cycle. Owned domestic cats (Felis catus) are considered an important species among mammalian reservoirs. Similar to humans, household cats become infected by eating raw, contaminated fish and spread the O. viverrini eggs to the environment by defecation. In rural areas, cat owners usually feed their cats with human food or food waste, especially fish dishes. There is a high risk for cats to become infected and deposit eggs in the peri-household environment. The lack of research on domestic cats led us to study their roaming behavior, roaming range, habitat preference and risk of disease transmission. GPS loggers were harnessed to 28 domestic cats in 7 villages around Lawa Lake, the endemic area of human opisthorchiasis. All cats were examined for the O. viverrini infection status using modified formalin-ether concentration technique (MFECT) before application of the GPS device used to observe and record their roaming distance and range area. Then, composition analysis for habitat selection was performed using the location data. Additionally, cat owners were interviewed using participatory epidemiologic methods for their perceptions and knowledge about the reservoir hosts of this trematode. Our study suggested that the prevalence of O. viverrini in cats in this area has not declined appreciably in comparison with our previous study four years ago, although the treatment and control has been applied intensively in humans. The longest distance that individual cats roamed ranged from 51 to 363 meters (mean±SD = 154.48±90.71). The cats traveled tracks around their home. There was no significant difference in the average distance that cats ranged between O. viverrini infected group (154.13±72.80 meters) and the non-infected group (154.67±101.39 meters) was found. Compositional analysis suggested that the domestic cats preferred urban habitats where food and safety were probably more available. It appears that the main transmission route for infection of cats was contaminated fish fed to cats or fish that remained in their houses. Therefore, owners should refrain from feeding their cats potentially contaminated foods, food scraps and raw fish remains and prevent their cats from accessing them. Moreover, the participatory epidemiology results suggest that awareness of villagers is not enough for the effective and sustainable control of the disease.

A socio-ecological framework for examining foodborne parasitic infection risk.

Human liver fluke infection through the consumption of raw freshwater fish is one of the foodborne parasitic infections of global concern. Despite decades of health campaign efforts, high prevalence of infection remains in different areas of the Lower Mekong Basin. This necessitates the consideration of the infection differences between places and the human-environment complexities of disease transmission. This paper used the socio-ecological model as a framework to unraveled the social science dimensions of liver fluke infection. We conducted questionnaire surveys in Northeast Thailand to gather participants' knowledge on liver fluke infection and reasons for raw fish consumption. We synthesized our findings with prior work to identify factors influencing liver fluke infection at four socio-ecological levels. At the individual level, gender and age differences in food consumption habits and personal hygiene of open defection presented the behavioral risks. At the interpersonal level, family tradition and social gathering affected the disease risk. At the community level, physical-social-economic environments of land use and modernization, community health infrastructure and health volunteer support accounted for the varying degree of infection. At the policy level, impacts of regional and national regulations on disease control, health system organization structure, and government development projects were of concerned. The findings provide insights into how infection risks are shaped by people's behavior, social connectedness, interactions with places, and the interplay of these multi-level socio-ecological influences. The framework therefore allows a more comprehensive understanding of liver fluke infection risks to inform a culturally sensitive and sustainable disease control program.

High endemicity of Opisthorchis viverrini infection among people in northern Cambodia confirmed by adult worm expulsion.

Opisthorchis viverrini infection is an emerging disease in Cambodia, especially in central and southeastern areas. However, its status in northern areas bordering Lao PDR has been relatively unknown. The present study was performed to investigate the status of O. viverrini infection among people in Preah Vihear and Stung Treng provinces through fecal examination to detect eggs and recovery of adult flukes from some of the egg-positive cases. Fecal examinations were performed on a total of 1101 people from 10 villages in the 2 provinces using the Kato-Katz thick smear technique. For recovery of adult flukes and other helminth parasites 10 volunteers positive for eggs of Opisthorchis viverrini and/or minute intestinal flukes (Ov/MIF), in Kampong Sangkae village, Preah Vihear province, were administered a single oral dose of 40 mg/kg praziquantel plus 5-10 mg/kg of pyrantel pamoate and purged with 40-50 g magnesium salts. Adult trematodes, together with nematodes and cestodes expelled in diarrheic stools were collected under a stereomicroscope or with the naked eye. The proportion of egg-positive cases for overall liver and intestinal helminths was high but not notably different between the 2 provinces, 65.5% in Preah Vihear versus 64.7% in Stung Treng. The average proportion of Ov/MIF egg-positive cases was 59.8%. A total of 315 adult specimens of O. viverrini were recovered from the 10 volunteers (4-98 specimens per individual; mean, 32). A smaller number of Haplorchis taichui adults, an intestinal fluke, were found mixed-infected in 7 (103 specimens in total; 1-31 per individual; mean, 15) of the 10 volunteers. Adult specimens of hookworms, Enterobius vermicularis, Trichostrongylus sp., and a Taenia tapeworm strobila were recovered in some cases. Based on the results, it has been confirmed that the surveyed areas in Preah Vihear and Stung Treng provinces, Cambodia, are highly endemic areas of O. viverrini infection with a low-grade mixed infection with H. taichui.

Comparison of a Urine Antigen Assay and Multiple Examinations with the Formalin-Ethyl Acetate Concentration Technique for Diagnosis of Opisthorchiasis.

Detection of worm antigen in urine is a sensitive diagnostic method for opisthorchiasis, particularly for light-intensity infections; however, the presence of eggs in feces is essential for validating results from the antigen assay. To address the issue of low sensitivity of fecal examination, we modified the protocol for the formalin-ethyl acetate concentration technique (FECT) and compared it against urine antigen measurements for detection of the parasite Opisthorchis viverrini. First, we optimized the FECT protocol by increasing the number of drops for examinations from the standard two drops to a maximum of eight. We were able to detect additional cases after examination of ≥ 3 drops, and the prevalence of O. viverrini saturated after examination of ≥ 5 drops. We then compared the optimized FECT protocol (examining five drops of suspension) against urine antigen detection for the diagnosis of opisthorchiasis in field-collected samples. The optimized FECT protocol detected O. viverrini eggs in 25 of 82 individuals (30.5%) who had positive urine antigen tests but were fecal egg negative by the standard FECT protocol. The optimized protocol also retrieved O. viverrini eggs in 2 of 80 antigen-negative cases (2.5%). In comparison with the composite reference standard (combined FECT and urine antigen detection), the diagnostic sensitivity of examining two and five drops of FECT and the urine assay was 58.2, 67, and 98.8%, respectively. Our results show that multiple examinations of fecal sediment increase the diagnostic sensitivity of FECT and thus provide further support for the reliability and utility of the antigen assay for diagnosis and screening of opisthorchiasis.

Opisthorchis viverrini, Clonorchis sinensis and Opisthorchis felineus liver flukes affect mammalian host microbiome in a species-specific manner.

BACKGROUND: Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis are epidemiologically significant food-borne trematodes endemic to diverse climatic areas. O. viverrini and C. sinensis are both recognized to be 1A group of biological carcinogens to human, whereas O. felineus is not. The mechanisms of carcinogenesis by the liver flukes are studied fragmentarily, the role of host and parasite microbiome is an unexplored aspect. METHODOLOGY/PRINCIPAL FINDINGS: Specific pathogen free Mesocricetus auratus hamsters were infected with C. sinensis, O. viverrini and O. felineus. The microbiota of the adult worms, colon feces and bile from the hamsters was investigated using Illumina-based sequencing targeting the prokaryotic 16S rRNA gene. The analysis of 43 libraries revealed 18,830,015 sequences, the bacterial super-kingdom, 16 different phyla, 39 classes, 63 orders, 107 families, 187 genera-level phylotypes. O. viverrini, a fluke with the most pronounced carcinogenic potential, has the strongest impact on the host bile microbiome, changing the abundance of 92 features, including Bifidobacteriaceae, Erysipelotrichaceae, [Paraprevotellaceae], Acetobacteraceae, Coriobacteraceae and Corynebacteriaceae bacterial species. All three infections significantly increased Enterobacteriaceae abundance in host bile, reduced the level of commensal bacteria in the gut microbiome (Parabacteroides, Roseburia, and AF12). CONCLUSIONS/SIGNIFICANCE: O. felineus, O. viverrini, and C. sinensis infections cause both general and species-specific qualitative and quantitative changes in the composition of microbiota of bile and colon feces of experimental animals infected with these trematodes. The alterations primarily concern the abundance of individual features and the phylogenetic diversity of microbiomes of infected hamsters.

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

Antigen detected in urine for the diagnosis of opisthorchiasis has a low daily variation; however, the longer term variability in antigen concentrations is unknown. In this study, we prospectively monitored Opisthorchis viverrini antigen concentrations for 30 consecutive days and at subsequent monthly intervals in a cohort of opisthorchiasis-positive individuals. On the basis of the monoclonal antibody-based ELISA, the profiles of antigen-positive rate and antigen concentration exhibited no significant change over 30 days with a mean proportion positive of 87.1% (range 73.7%-100%), and the average antigen concentration was 29.7 ± 2.2 ng/mL (mean ± SE). The urine antigen concentration at baseline was similar to the subsequent measurements at 2, 4, 6, and 10 months in the follow-up study (P > 0.05). The consistency and low daily and long-term fluctuation of O. viverrini antigen in urine demonstrates the reliability of urine assay for diagnosis of opisthorchiasis.