Asian liver fluke infections, namely infection with the trematode helminths Opisthorchis viverrini, O. felineus and Clonorchis sinensis, are highly endemic in many settings in Asia and Eastern Europe. Deeply culturally rooted habits of raw and undercooked fish dishes consumption are the origin of the infection. Asian liver fluke infections are typically prevalent in rural, resource-poor settings with little access to quality health services. The presence of adult fluke worms in the biliary ducts in combination with other socio-culturally, behavioural and geographically rooted risk factors such as nitrosamine rich diets, smoking, alcohol consumption, concomitant viral hepatitis infections and other factors determine the severity of developing morbidity. Chronic infection may induce cholangiocarcinoma (CCA), a fatal bile duct cancer. In most Asian liver fluke endemic settings, the true extent of liver fluke induced liver morbidity is unknown and underestimated, resulting in an inadequate allocation of resources for prevention and control. The lack of easily measurable morbidity markers are a fundamental problem. Also lacking are detailed risk pattern assessments for the hepatobiliary morbidity. In Lao PDR, about half of the population is infected with O. viverrini.Objectives: Our project pursues the following objectives: (i) to assess the prevalence of different hepatobiliary pathologies associated with O. viverrini infection in adult residents living in central and southern Lao PDR; (ii) to identify factors associated with the risk and presence of moderate (periductal fibrosis) and severe morbidity (advanced periductal fibrosis and precancerous lesions) in O. viverrini-infected adults in central and southern Lao PDR, with particular emphasis on PZQ treatment history; (iii) to assess the predictive capacity for the presence of O. viverrini-induced (advanced) periductal fibrosis and CCA as assessed by ultrasound in a new O. viverrini-endemic setting of candidate, CCA-related biomarkers (i.e. CA19-9, alpha 1-fetoprotein mucin-5AC, CYFRA21-1, serum IL-6, serum ES exosomes, urinary 8-oxodG, urinary miR-21 and miR-192) and to investigate how the concentration level of these biomarkers is influenced by behavioural and PZQ treatment factors associated with severe hepatobiliary morbidity (see Objective 2); (iv) to establish a population-based biobank consisting of urine, serum and stool aliquots from an O. viverrini-infected (Ov+) case-control sample (Ov+ cases: persons with different severity stages of hepatobiliary pathology; Ov+ controls: persons without evidence hepatobiliary pathology) for future research on (i) the validation of novel CCA-related candidate biomarkers and (ii) the identification of novel diagnostic biomarkers through agnostic or targeted -omics approaches.Methods: The study consists of three parts: (i) a cross-sectional survey in two provinces of Lao PDR (Champasack and Saravane) in which randomly selected adults (>25 years) will be enrolled and examined on the presence of O. viverrini infection (Ov+) with a parasitological stool examination and on liver morbidity (healthy, PF+, APF+, CCA-like lesions) by using ultrasonography and on risk factors by using a questionnaire. A total of 2,625 participants will be enrolled; (ii) a case-control study will be embedded in the cross-sectional study. A case-group 1 with severe morbidity (APF+, Ov+), a case-group 2 with moderate morbidity (PF+, Ov+) will be compared to age, sex and village of residence matched controls which have no morbidity (PF-, Ov+). All participants are O. viverrini positive (Ov+). In cases and controls the concentration of the new biomarkers, ES exosomes of O. viverrini and 8-oxodG will be measured as well as other known factors contributing to morbidity development. In total 945 patients will be enrolled in the case-control study, 315 patients per group; and (iii) a biobank containing aliquots (full blood, serum, stool and urine) of each participant in the case-control study, will be established.Expected results: The study will provide detailed information on the amount of hepatobiliary morbidity present in two provinces of Lao PDR. Furthermore, it will elucidate the risk pattern responsible for severe morbidity development in addition to O. viverrini infection and therefore provide new mechanistic insights. In particular, it will show whether history of treatment may results in morbidity increase, which has direct implications for control in Lao PDR and other Asian liver fluke endemic settings and will inform treatment policy.