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Additional file 1: Table S1. Snail species composition (%) in the surrounding water bodies of BN and BT villages. Table S2. Potential influences of perceptual and behavioral factors on consumption habits of BN and BT villagers. Consumption frequency was based on all three raw fish dishes consumed. Odds ratio for the analysis of willingness to avoid consumption and O. viverrini (O.v.) infection status was included for the relation that was statistically significant. Table S3. Potential influences of reasons of consumption (comparing five individual reasons vs. two reasons of ecosystem services), and perceptual and behavioral factors on consumption frequencies of BN and BT villagers. Consumption frequency was based on higher risk raw fish dishes, koi pla and pla som. Table S4. Potential influences of perceptual and behavioral factors, and network indices of degree centrality and betweenness centrality on consumption habits of BN and BT villagers. Consumption frequency wasbased on all three raw fish dishes consumed. Table S5. Potential influences of ecosystem services, perceptual and behavioral factors, and network indices of degree centrality and betweenness centrality on consumption habits of BN and BT villagers. Consumption frequency was based on all three raw fish dishes consumed.

Background Human infection with Opisthorchis viverrini, a carcinogenic liver fluke inhabiting the biliary tree, is endemic in Southeast Asia. Chronic infection is associated with a fatal complication, cholangiocarcinoma (CCA), a late-presenting and aggressive malignancy. Currently, annual mortality rates from CCA mirror trends in incidence, due in part to limited availability of efficient prognostic and early diagnostic biomarkers. With ability to detect thousands of urinary metabolites using metabonomics, the urine metabolome holds great potential in providing an insight into system-level alterations in carcinogenesis and in identifying metabolic markers altered in response to disturbed homoeostasis. Methods Global molecular profiling using reversed-phase ultraperformance liquid chromatography mass spectrometry was utilised to acquire the urinary spectral profile of 137 Thai subjects (48 at high risk of infection, 41 with O. viverrini infection, 34 periportal fibrosis and 14 CCA) from Khon Kaen, Thailand. Results Multivariate statistical analysis identified perturbation in several molecular classes related to purine metabolism and lipid metabolism in the CCA urine metabolome. These markers mainly reflect changes in energy metabolism to support proliferation (increased fatty acid oxidation and purine recycling), DNA methylation and hepatic injury. Conclusions Several metabolites of biological interest were discovered from this proof-of-principle dataset. Augmenting these findings is essential to accelerate the development of urinary metabolic markers in CCA.

Attapol Titapun,1,2 Anchalee Techasen,2,3 Prakasit Sa-Ngiamwibool,2,4 Paiboon Sithithaworn,2,5 Vor Luvira,1,2 Tharatip Srisuk,1,2 Apiwat Jareanrat,1,2 Hasaya Dokduang,2,6 Watcharin Loilome,2,6 Bandit Thinkhamrop,2,7 Narong Khuntikeo1,2 1Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 2Cholangiocarcinoma Research Institute (CARI), Khon Kaen University, Khon Kaen, Thailand; 3Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand; 4Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 5Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 6Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; 7Department of Epidemiology and Biostatistics, Faculty of Public Health, Khon Kaen University, Khon Kaen, ThailandCorrespondence: Attapol TitapunDepartment of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandTel +66-43-363252Email attati@kku.ac.thBackground: Serum antibody for Opisthorchis viverrini (OV) is strong evidence for a history of OV infection in people. Currently, no studies have examined whether varying cholangiocarcinoma (CCA) prevalence levels are linked to previous OV infection nor have they provided comprehensive assessment and characterization of OV-associated CCA.Objective: Our study examined the prevalence of serum IgG antibodies for OV-positive CCA cases and determined whether there were correlations of IgG antibodies with histopathologic features, HER2, PD-L1, and FGFR2 expression, as well as their roles on the patients’ survival.Methods: The study involved 221 CCA surgical patients at Khon Kaen University Hospital, Thailand, from 2005 to 2017. Serum specimens were tested for OV IgG by ELISA. CCA tissue microarrays were used to examined for HER2, PD-L1, and FGFR2 expression. Logistic regression was used to investigate an association between factors and IgG. Cox regression was used to determine factors that affected CCA patient survival.Results: IgG for OV were positive in 162 cases, and the prevalence was 73.3% (95% CI=68.0– 78.7). About three quarters (78.3%) had large duct type tumors and concomitant intraductal papillary neoplasm of bile ducts (IPNB) occurred in 92 (50%) cases. HER2 expression was positive in 94 (61.4%) cases. Positive PD-L1 and FGFR2 expression occurred in 125 (83.9%) and 100 (67.1%) cases. IgG for OV had no significant correlation to any histological feature but had significant correlation with HER2 overexpression with adjusted OR=2.32 (95% CI=1.09– 4.96, P=0.03). Cases of CCA with OV IgG positive had a significantly poor prognosis with adjusted HR=1.66 (95% CI=1.13– 2.43, P=0.01).Conclusion: We found a high prevalence of serum IgG for OV-positive CCA patients and a correlation with overexpression of HER2. Moreover, IgG for OV and HER2 expression indicated poor survival of CCA. Therefore, future clinical studies for anti-HER2 treatments should focus on OV-associated CCA.Keywords: survival, intraductal type, FGFR2, PD-L1, immunoglobulin G, fluke-associated CCA

The prevalence of cholangiocarcinoma (CCA) in Southeast Asia is much higher than other areas of the world. Eating raw, fermented, or undercooked cyprinid fish, infected with the liver fluke, Opisthorchis viverrini sensu lato (sl), results in chronic biliary inflammation, periductal fibrosis, and increased cancer risk. There may be associated glomerulonephritis. The process of infection is difficult to disrupt because eating practices have proven extremely difficult to change, and the life cycle of the fluke cannot be broken due to high prevalence in canine and feline reservoir hosts. Fecal analysis and enzyme-linked immunosorbent assay tests can be used to diagnose opisthorchiasis. Diagnosis of CCA is complex, partly due to the lack of definitive imaging characteristics but also due to the difficulty of obtaining samples for cytology or histology. This cancer has proven to be resistant to common chemotherapy treatments and so the two avenues of treatment available are surgical resection and liver transplantation, both requiring early detection of the tumor for the best chances of success. Late presentation of symptoms reduces the chances of successful surgical intervention. While liver fluke infections can be treated with praziquantel, individuals will often become reinfected, and multiple reinfections can be more harmful than a singular, long-term infection. A key research on the detection and characterization of novel biomarkers in all parts of the carcinogenic pathway for early diagnosis is needed.

Munirah Alsaleh,1 Zoe Leftley,1 Thomas A Barbera,1 Paiboon Sithithaworn,2 Narong Khuntikeo,2 Watcharin Loilome,2 Puangrat Yongvanit,2 I Jane Cox,3 Nittaya Chamodol,2 Richard RA Syms,4 Ross H Andrews,1,2 Simon D Taylor-Robinson1 1Division of Surgery and Cancer, Imperial College London, London W2 INY, UK; 2Cholangiocarcinoma Research Centre, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; 3Faculty of Life Sciences & Medicine, King’s College London, London SE5 9NT, UK; 4Department of Electrical and Electronic Engineering, Imperial College London, London SW7 2AZ, UK Abstract: Cholangiocarcinoma (CCA) is a tumor with increasing prevalence around the world. The prevalence of CCA is highest in East Asia and most significantly in the countries through which the Mekong River flows, owing to the presence of liver flukes, which are consumed in raw fish dishes. Outside Asia, the causes of bile duct cancers for the most part are unknown. In this review, we assess the current state of knowledge in both fluke-associated and sporadic CCA, from etiological, diagnostic, and treatment perspectives. Keywords: cholangiocarcinoma, etiology, diagnosis, treatment, bile ducts

The liver fluke Opisthorchis viverrini sensu lato causes serious public-health problems in northeast Thailand and Southeast Asian countries. A hypothesis has been proposed that O. viverrini represents a species complex with varying levels of genetic differentiation in Thailand and Lao PDR. This study aimed to clarify whether O. viverrini populations can be genetically divided into separate taxa. We collected O. viverrini s.l. from eight different locations in Lao PDR and Thailand. The results of nad1, cox1, CF-int6, Pm-int9, ITS2 and 28S rDNA sequence analysis revealed that sub-structuring occurred between the eight populations. We found that O. viverrini s.l. from Sakon Nakhon (SK), Thailand, shows significant genetic differentiation (P

Thomas Hughes,1,* Thomas O’Connor,1,* Anchalee Techasen,2,3 Nisana Namwat,2,3 Watcharin Loilome,2,3 Ross H Andrews,2–4 Narong Khuntikeo,3,5 Puangrat Yongvanit,3,6 Paiboon Sithithaworn,3,7 Simon D Taylor-Robinson1 1Division of Digestive Health, Department of Surgery and Cancer, Imperial College London, London, UK; 2Department of Biochemistry, Faculty of Medicine, Liver Fluke and Cholangiocarcinoma Centre, 3Cholangiocarcinoma Screening and Care Program (CASCAP), Khon Kaen University, Khon Kaen, Thailand; 4Faculty of Medicine, St Mary’s Campus, Imperial College, London, UK; 5Department of Surgery, 6Department of Biochemistry, 7Department of Parasitology, Faculty of Medicine, Liver Fluke and Cholangiocarcinoma Centre, Khon Kaen University, Khon Kaen, Thailand *These authors contributed equally to this work Abstract: The prevalence of cholangiocarcinoma (CCA) in Southeast Asia is much higher than other areas of the world. Eating raw, fermented, or undercooked cyprinid fish, infected with the liver fluke, Opisthorchis viverrini sensu lato (sl), results in chronic biliary inflammation, periductal fibrosis, and increased cancer risk. There may be associated glomerulonephritis. The process of infection is difficult to disrupt because eating practices have proven extremely difficult to change, and the life cycle of the fluke cannot be broken due to high prevalence in canine and feline reservoir hosts. Fecal analysis and enzyme-linked immunosorbent assay tests can be used to diagnose opisthorchiasis. Diagnosis of CCA is complex, partly due to the lack of definitive imaging characteristics but also due to the difficulty of obtaining samples for cytology or histology. This cancer has proven to be resistant to common chemotherapy treatments and so the two avenues of treatment available are surgical resection and liver transplantation, both requiring early detection of the tumor for the best chances of success. Late presentation of symptoms reduces the chances of successful surgical intervention. While liver fluke infections can be treated with praziquantel, individuals will often become reinfected, and multiple reinfections can be more harmful than a singular, long-term infection. A key research on the detection and characterization of novel biomarkers in all parts of the carcinogenic pathway for early diagnosis is needed. Keywords: Opisthorchis viverrini, CCA, Thailand, Laos, treatment, parasite, carcinogen, public health, helminth

Background: An enhanced understanding of the hookworm genome and its resident mobile genetic elements should facilitate understanding of the genome evolution, genome organization, possibly host-parasite co-evolution and horizontal gene transfer, and from a practical perspective, development of transposon-based transgenesis for hookworms and other parasitic nematodes. Methodology/Principal Findings: A novel mariner-like element (MLE) was characterized from the genome of the dog hookworm, Ancylostoma caninum, and termed bandit. The consensus sequence of the bandit transposon was 1,285 base pairs (bp) in length. The new transposon was flanked by perfect terminal inverted repeats of 32 nucleotides in length with a common target site duplication TA, and it encoded an open reading frame (ORF) of 342 deduced amino acid residues. Phylogenetic comparisons confirmed that the ORF encoded a mariner-like transposase, which included conserved catalytic domains, and that the bandit transposon belonged to the cecropia subfamily of MLEs. The phylogenetic analysis also indicated that the Hsmar1 transposon from humans was the closest known relative of bandit, and that bandit and Hsmar1 constituted a clade discrete from the Tc1 subfamily of MLEs from the nematode Caenorhabditis elegans. Moreover, homology models based on the crystal structure of Mos1 from Drosophila mauritiana revealed closer identity in active site residues of the catalytic domain including Ser281, Lys289 and Asp293 between bandit and Hsmar1 than between Mos1 and either bandit or Hsmar1. The entire bandit ORF was amplified from genomic DNA and a fragment of the bandit ORF was amplified from RNA, indicating that this transposon is actively transcribed in hookworms. Conclusions/Significance: A mariner-like transposon termed bandit has colonized the genome of the hookworm A. caninum. Although MLEs exhibit a broad host range, and are identified in other nematodes, the closest phylogenetic relative of bandit is the Hsmar1 element of humans. This surprising finding suggests that bandit was transferred horizontally between hookworm parasites and their mammalian hosts.

Background: Cholangiocarcinoma (CCA) is an extremely aggressive cancer that is usually fatal. Although globally morbidity and mortality are increasing, knowledge of the disease remains limited. The Mekong region of Southeast Asia, and particularly the northeast of Thailand, has by far the highest incidence of CCA worldwide with 135.4 per 100,000 among males and 43.0 per 100,000 among females being reported in Khon Kaen Province. Most patients are first seen during late stage disease with 5-year survival being less than 10 %. Starting in 1984, control and prevention strategies have been focused on health education. Although early detection can substantially increase 5-year survival, there are currently no strategies to increase early diagnosis. Methods/design: The Cholangiocarcinoma Screening and Care Program (CASCAP) is a prospective cohort study comprising two cohorts- the screening and the patient cohorts. For the screening cohort, ultrasound examination will be carried out regularly at least annually to determine whether there is current bile duct and/or liver pathology so that the optimal screening program for early diagnosis can be established. This cohort is expected to include at least 150,000 individuals coming from high-risk areas for CCA. For the patient cohort, it is estimated that about 25,000 CCA patients will be included during the 5-year recruitment period. All CCA patients will be treated according to routine clinical care and followed so that effective surgical treatment can be formulated. This cohort is indeed a conventional cancer registry. Thus, CASCAP is an ongoing project in which the number of participants changes dynamically. Discussions: This is the first project on CCA that involves screening the at risk population at the community level. At the time of preparing this report, a total of 85,927 individuals have been enrolled in the screening cohort, 55.0 % of whom have already undergone ultrasound screening, and 2661 CCA cases have been enrolled in the patient cohort. Among the participants of the screening, whose mean age was 53.8 ± 9.8 years, 55.6 % were female, 77.5 % attained primary school as the highest level of education, 79.9 % were farmers, 29.9 %, reported having relatives with CCA, 89.1 % had eaten uncooked fish, and 42.2 % of those who had been tested for liver fluke were found to be infected.

Prevalences and intensity of infection with Angiostrongylus cantonensis third stage larvae were examined in mollusks to determine whether they are potential intermediate hosts in eight provinces, northeast Thailand. Mollusk samples were collected from 24 reservoirs (3 reservoirs/province) in close to human cases during the previous year. Six out of 24 localities and 9 (3 new record species) out of 27 species were found with the infection. The highest intensity in infected species was found to be only one or two snails, whereas the majority had very low or no infection. The highest density was found in Pila pesmei and the lowest in Pila polita. The edible snails, P. polita, P. pesmei, and Hemiplecta distincta have the potential to transmit A. cantonensis to man. The varying density levels of larvae in infected snails may reflect observed variation in symptoms of people who traditionally eat a raw snail dish. Refereed/Peer-reviewed

The food-borne trematodes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis, have long been recognized as the cause of major human health problems, with an estimated 40 million infected persons. Of the three species of liver fluke, only O. viverrini is classified as a type 1 carcinogen because of its role as an initiator of chronic inflammation and the subsequent development of cholangiocarcinoma. At present, there are no techniques for the early diagnosis of cholangiocarcinoma and it is fatal for most patients. There is considerable variation in parasite prevalence and disease presentation in different geographical areas, the latter of which may be associated with genetic differences among parasites. In the present study, multilocus enzyme electrophoresis was used to provide a comprehensive genetic characterization of O. viverrini from different geographical localities in Thailand and the Peoples’ Democratic Republic of Laos. Parasites from different localities were compared genetically at 32 enzyme loci. The results of the genetic analyses are sufficient to reject the null hypothesis that O. viverrini represents a single species. Therefore, O. viverrini consists of at least two genetically distinct, yet morphologically similar (i.e. cryptic) species. Moreover, there was also separation of the different populations of snails (i.e. the first intermediate hosts) into two distinct genetic groups that corresponded with the delineation of O. viverrini into two species. This suggests that there may be a history of co-evolution in this host–parasite lineage. Additionally, five distinct genetic groups of parasites were detected, each of which occurred within a different and independent river wetland system. Our findings have major implications for the implementation of effective control and surveillance programs targeted to these medically important food-borne parasites.

Echinostomes are common intestinal parasites causing zoonotic disease, which are endemic worldwide. Of the four species of medically important echinostomes reported in Thailand, two species, Echinostoma revolutum and Hypodereaum conoideum, have been detected in poultry. These two parasites are morphologically similar and are sometimes difficult to distinguish. In the present study, multilocus enzyme electrophoresis was used to differentiate E. revolutum from H. conoideum collected from domestic ducks in Thailand. The parasites were compared using 22 enzymes with 30 presumptive enzyme loci. The two species of echinostome could be distinguished at 17 of the 30 enzyme loci. Several loci were polymorphic within each species, suggesting that these can be used to examine their population genetics.

To the Editor: The emergence and reemergence of infectious diseases are major problems for healthcare systems worldwide. Unfortunately, because accurate prediction of the occurrence of such diseases is difficult, if not impossible, surveillance and control can be carried out only after the outbreak has occurred. Predicting the likelihood of a disease outbreak should make it possible to start surveillance programs before outbreaks occur and to initiate control programs before the population has become seriously affected. We used data on changes in land use patterns to predict the likelihood of malaria reemergence in northeastern Thailand. Because natural rubber is of major economic importance and cannot be replaced by synthetic alternatives, the demand for and production of this commodity has consistently increased (1). This situation has led to changes in agricultural practices in various countries in Southeast Asia; rubber production has increased in Myanmar, Laos, Thailand, and Vietnam (1,2). Northeastern Thailand (Isaan) is a relatively poor area, and most rubber plantations belong to smallholders and provide them with a large source of income (3). In 1993, ≈284 km2 of northeastern Thailand were covered by rubber plantations; this area increased to 422 km2 in 1998 and to 948 km2 by 2003 (3). Since then, planting has increased exponentially so that, by 2006, the total area planted with rubber was >2,463 km2; new plantings expanded another ≈1,345 km2 from 2004 to 2006 and increased to a total of 5,029 km2 in 2007 (3). The plants mature ≈6 years after planting; at that stage, the trees can reach 10–12 m in height, although the growth rate depends on the physical and biotic environment (4). Deforestation in northeastern Thailand early in the last century led to an extreme reduction in the incidence of malaria (5) because the main vector mosquito in this area, Anopheles dirus sensu stricto, is forest dwelling and requires a shaded environment for its survival and reproduction (6). Currently, the northeastern part of the country is relatively free of autochthonous malaria cases except for 3 provinces that border Cambodia and Laos (5), Srisaket, Ubon Ratchathani, and Surin. In Srisaket and Ubon Ratchathani, 25% and 31%, respectively, of malaria cases are imported, particularly from Cambodia (7). Mosquitoes are sensitive to changes in environmental conditions, such as shade, temperature, and humidity. These conditions are often influenced by land use change, such as conversion of rice paddies to rubber plantations (8). In addition to providing economic benefits for the population, rubber plantations also provide suitable habitats for A. dirus s.s., perhaps even better habitats than those found in the original rain forest; new plantations lead to increased mosquito density and disease incidence (8). Thus, planting large tracts of rubber potentially increases the likelihood of the reemergence of malaria in northeastern Thailand, although a malaria vector such as A. dirus s.s. could return without reemergence of the disease (9). Should malaria return, the greatly reduced contact between the local Isaan population and Plasmodium spp. over the past ≈50 years suggests that malaria would enter a highly susceptible population, potentially leading to major health problems at the individual and regional levels. This possibility is of particular concern because several strains of Plasmodium in Thailand and surrounding countries are multidrug resistant, which leads to treatment difficulties (5). Each land use change creates different microclimatic conditions, which directly and indirectly affect the occurrence and distribution of malaria (10). Whether malaria will return as a major health threat likely depends on the size and fragmentation of the individual plantation areas. The required size of a plantation for the survival of the vector population is unclear, but large areas of plantation tend to offer dense vegetation and, therefore, high humidity and shade, which provide suitable environmental conditions for larval habitats, even during the dry season (8). Conversely, during the rainy season, conditions at the edges of fragmented forests, where human settlements are often located, become favorable for larval habitats, rendering villagers susceptible to the disease (6). In addition to changes in habitat and microclimate, social or political changes in the region may affect the transborder movement of malaria into Thailand with consequences for potential reemergence (7). Although the association between rubber plantations and malaria is well known in Southeast Asia, the potential for reemergence should receive substantially more attention from economic, agricultural, and environmental planning bodies. Changes in land use and land cover have the potential to facilitate the transmission of disease to humans. Understanding the influence of land use change on malaria occurrence is critical for shaping future surveillance and control strategies.

Genetic variation among 231 individuals of Opisthorchis viverrini from the Ban Phai District, Khon Kaen Province (Thailand) was examined at three polymorphic enzymes: enolase (ENOL), phosphoglucomutase (PGM), and triose phosphate isomerase (TPI). Four alleles were detected for TPI and PGM, whereas only two alleles were detected for ENOL. The inferred genotype frequencies for both TPI and ENOL were not significantly different from Hardy–Weinberg equilibrium. In contrast, the inferred genotype frequencies for PGM showed a significant departure from Hardy–Weinberg equilibrium, with a lack of heterozygous individuals. This heterozygote deficiency suggests non-random mating and/or potentially high self fertilization.

Infection with Opisthorchis viverrini and its associated cholangiocarcinoma (CCA) is an underestimated problem in the Mekong region of Southeast Asia, despite the widespread use of praziquantel and health education measures for parasite control. Although data from Cambodia, Laos and Vietnam are rare, data from Thailand often show wide-ranging variability in epidemiological parameters, including human morbidity and the prevalence and incidence of CCA. The recent discovery of high levels of population genetic variability in O. viverrini in different wetlands in Thailand and Laos, which indicates the presence of sibling species, suggests that we have underestimated the complexity of this epidemiological situation. Future research should determine the relationship between the genetic variability of O. viverrini and patterns of opisthorchiasis-related disease.

SUMMARYThe present study compared the genetic variation among 14 different geographical isolates of Opisthorchis viverrini sensu lato from Thailand and Lao PDR using sequence data for 2 mitochondrial DNA genes, the subunit 1 of NADH dehydrogenase gene (nad1) and cytochrome c oxidase gene (cox1). Four different nad1 haplotypes were detected among isolates, all of which were identical at the amino acid sequence level. Nucleotide sequence variation among 14 isolates ranged from 0 to 0·3% for nad1. Two different cox1 haplotypes were detected among isolates. These two haplotypes differed at 2 nucleotide positions, one of which resulted in a change in the amino acid sequence. Nucleotide sequence variation among isolates for cox1 ranged from 0 to 0·5%. Comparison of cox1 sequences of O. viverrini to those of other trematodes revealed nucleotide differences of 13–31%. A phylogenetic analysis of the cox1 sequence data revealed strong statistical support for a clade containing O. viverrini and 2 other species of opisthorchid trematodes; O. felineus and Clonorchis sinsensis.

The relationship between pregnancy and both the susceptibility and pathogenicity of parasite infections is disputed. This study compares the prevalence and intensity (as measured by density of eggs in stool samples) of intestinal helminth infections in pregnant and control groups of women from Khon Kaen Province in the northeast of Thailand. Stool samples were taken at the end of the first, second and third trimesters of pregnancy as well as 2 months after parturition and compared for the two groups. There were no significant changes in the prevalence of any of the common helminth species during the course of pregnancy or between the pregnant and control groups. Nor was there any evidence that the density of helminth eggs in the stool samples differed between sample times or between the pregnant and control groups. Our study therefore supports the hypothesis that pregnancy does not influence the course of human infection with helminths.

Genetic variation in Opisthorchis viverrini adults originating from different locations in northeast Thailand and Laos, People’s Democratic Republic (PDR), was examined using random amplified polymorphic DNA (RAPD) analyses. In an initial analysis, the genomic DNA of one fluke from each of ten localities was amplified using 15 random primers (10-mers); however, genetic variation among O. viverrini specimens was detected reliably for only four primers. A more detailed RAPD analysis using these four primers was conducted on ten individuals from nine localities. Considerable genetic variation was detected among O. viverrini from different geographical areas and among some individuals from the same collecting locality. Comparison of the RAPD profiles revealed that O. viverrini adults from Laos PDR were genetically distinct from those from northeast Thailand. The taxonomic significance of this finding needs to be explored in more detail. The RAPD markers established in the present study provide opportunities to examine the biology and epidemiology of O. viverrini and fish-borne trematodes within the region. Additionally, application of these markers in such studies could have important implications in relation to the prevalence of cholangiocarcinoma in different regions of Asia.