TaqMan real-time PCR assay for specific detection of Opisthorchis viverrini DNA in Thai patients with hepatocellular carcinoma and cholangiocarcinoma

The aim of this study was to develop TaqMan real-time PCR assay that detected Opisthorchis viverrini DNA from 18 normal and 18 tumor tissue specimens from Thai patients with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), who underwent liver resection from October 2005 to May 2006. Control liver specimens were seven non-primary liver cancers. A conserved probe representing 100% sequence homology was used as a reference for O. viverrini-specific probe. Five of six tumors (83%) and all six normal tissues from CCA group; and seven of twelve tumors (58%) and ten of twelve normal tissues (83%) from HCC group were found to have O. viverrini DNA. The O. viverrini DNA detection among HCC and CCA patients were not associated (p = 0.193; 90%CI). This RT-PCR will be a useful tool for investigating the relationship between cancer type and presence of the parasite and also for conducting epidemiological surveys.     

Opisthorchis viverrini-antigen induces expression of MARCKS during inflammation-associated cholangiocarcinogenesis

Myristoylated alanine rich C kinase substrate (MARCKS) has been implicated in PKC-mediated membrane-cytoskeleton alterations that underlie lipopolysaccharide (LPS)-induced macrophage responses. MARCKS is postulated to be involved in inflammation-associated CCA based on its overexpression in cholangiocarcinoma (CCA) and inflammatory cells. The aims of this study were to investigate localization patterns of MARCKS in hamster and human tissue during cholangiocarcinogenesis and to examine the involvement of MARCKS in inflammation. MARCKS protein expression was found prominently in inflammatory cells of Opisthorchis viverrini-treated as well as O. viverrini plus N-nitrosodimethylamine (NDMA)-treated hamsters from week 2 to week 3 of treatment. The positive signal decreased during week 4 to week 12, then increased again at week 26 when CCA developed. At the last time point the expression of MARCKS was observed in both cancer and inflammatory cells. MARCKS protein expression was also found in inflammatory cells, including macrophages in human CCA tissues. O. viverrini excretory/secretory products or worm antigen induced MARCKS mRNA and protein expression in a dose- and time-dependent manner in the human U937 macrophage cell line. The relative mRNA expression of MARCKS in white blood cells of O. viverrini-infected patients was significantly higher than in healthy subjects (P = 0.02). Thus, MARCKS is significantly expressed in macrophages and plays a role in inflammation-related CCA induced by O. viverrini.     

Molecular expression and enzymatic characterization of thioredoxin from the carcinogenic human liver fluke Opisthorchis viverrini

The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for years. Defense against oxidative damage can be mediated through glutathione and/or thioredoxin utilizing systems. Here, we report the molecular expression and biochemical characterization of a thioredoxin (Trx) from O. viverrini. O. viverrini Trx cDNA encoded a polypeptide of 105 amino acid residues, of molecular mass 11.63 kDa. The predicted protein has similarity to previously characterized thioredoxins with 26-51% identity. Recombinant O. viverrini Trx (Ov-Trx-1) was expressed as soluble protein in E. coli. The recombinant protein showed insulin reduction activity and supported the enzymatic function of O. viverrini thioredoxin peroxidase. Expression of Ov-Trx-1 at mRNA and protein levels was observed in all obtainable developmental stages of the liver fluke. Ov-Trx-1 was also detected in excretory-secretory products released by adult O. viverrini. Immunohistochemistry, Ov-Trx-1 was expressed in nearly all parasite tissue excepted ovary and mature sperms. Interestingly, Ov-Trx-1 was observed in the infected biliary epithelium but not in normal bile ducts. These results suggest that Ov-Trx-1 is essential for the parasite throughout the life cycle. In the host-parasite interaction aspect, Ov-Trx-1 may support thioredoxin peroxidase in protecting the parasite against damage induced by reactive oxygen species from inflammation.     

Specific serum IgG, but not IgA, antibody against purified Opisthorchis viverrini antigen associated with hepatobiliary disease and cholangiocarcinoma

Opisthorchiasis caused by Opisthorchis viverrini infection induces hepatobiliary disease (HBD)-associated cholangiocarcinoma (CCA) via a chronic inflammatory immune response. Here, we evaluated specific IgG and IgA antibodies against different fractions of O. viverrini antigen in residents from an endemic community in Northeast Thailand with varying hepatobiliary abnormalities. Crude somatic O. viverrini antigen was purified into three fractions (viz., P1, P2 and P3) by gel infiltration chromatography and these served as antigens for detection of fluke-specific IgG and IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The results revealed fluke-specific IgG and IgA antibody levels—against these antigens from subjects with O. viverrini-positive HBD—higher than in subjects with O. viverrini-negative HBD. Interestingly, the rank of fluke-specific IgG (and not IgA) antibody levels against crude extract and P1 antigens was CCA > severe HBD > mild HBD > healthy individuals. Purified antigens reduced cross-reactivity with other parasites compared to the crude antigen. Multiple linear regression analysis showed that HBD status was significantly associated with the liver fluke-specific IgG antibody against purified antigens. These results suggest that purified O. viverrini-antigen improves serodiagnosis for the evaluation of opisthorchiasis-associated HBD, and may be useful in the screening of opisthorchiasis in subjects at risk of developing CCA.     

Levels of 8-OxodG Predict Hepatobiliary Pathology in Opisthorchis viverrini Endemic Settings in Thailand

Opisthorchis viverrini is distinct among helminth infections as it drives a chronic inflammatory response in the intrahepatic bile duct that progresses from advanced periductal fibrosis (APF) to cholangiocarcinoma (CCA). Extensive research shows that oxidative stress (OS) plays a critical role in the transition from chronic O. viverrini infection to CCA. OS also results in the excision of a modified DNA lesion (8-oxodG) into urine, the levels of which can be detected by immunoassay. Herein, we measured concentrations of urine 8-oxodG by immunoassay from the following four groups in the Khon Kaen Cancer Cohort study: (1) O. viverrini negative individuals, (2) O. viverrini positive individuals with no APF as determined by abdominal ultrasound, (3) O. viverrini positive individuals with APF as determined by abdominal ultrasound, and (4) O. viverrini induced cases of CCA. A logistic regression model was used to evaluate the utility of creatinine-adjusted urinary 8-oxodG among these groups, along with demographic, behavioral, and immunological risk factors. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive accuracy of urinary 8-oxodG for APF and CCA. Elevated concentrations of 8-oxodG in urine positively associated with APF and CCA in a strongly dose-dependent manner. Urinary 8-oxodG concentrations also accurately predicted whether an individual presented with APF or CCA compared to O. viverrini infected individuals without these pathologies. In conclusion, urinary 8-oxodG is a robust ‘candidate’ biomarker of the progression of APF and CCA from chronic opisthorchiasis, which is indicative of the critical role that OS plays in both of these advanced hepatobiliary pathologies. The findings also confirm our previous observations that severe liver pathology occurs early and asymptomatically in residents of O. viverrini endemic regions, where individuals are infected for years (often decades) with this food-borne pathogen. These findings also contribute to an expanding literature on 8-oxodG in an easily accessible bodily fluid (e.g., urine) as a biomarker in the multistage process of inflammation, fibrogenesis, and infection-induced cancer.     

Comparative histopathology of Opisthorchis felineus and Opisthorchis viverrini in a hamster model: an implication of high pathogenicity of the European liver fluke

European liver fluke (Opisthorchis felineus) and Asian liver fluke (Opisthorchis viverrini) are similar in morphology but comparative pathology of the infections has not been described. We therefore did comparative histopathology of both parasites in an experimental animal model. The study was conducted in 3 groups of 105 Syrian golden hamsters; the first and second groups fed with 50 metacercariae of O. felineus (OF) or O. viverrini (OV) and the last group was uninfected controls. Five hamsters in each group were euthanized on weeks 1, 2, 4, 8, 12 and 24 post-infection. The liver tissue was fixed and processed for routine histopathology and immunohistochemistry for proliferation markers (BrdU or PCNA). Overall, the liver histopathology of O. felineus and O. viverrini infection was generally similar. However, various histopathogical features including intense inflammation, fibrosis, biliary and goblet cell hyperplasia and dysplasia occurred earlier in the OF group. In addition, the existence of precancerous lesions such as cholangiofibrosis in a long-term infection was observed only in this group. O. felineus is larger in size than O. viverrini which, together with its excreted and secreted antigens, likely is crucial in the induction of liver fluke induced disease. The differences in nature and timing of the histopathological profile indicate that opisthorchiasis caused by the European liver fluke O. felineus is more pathogenic than its Asian relative O. viverrini.     

Establishment of an Allo-Transplantable Hamster Cholangiocarcinoma Cell Line and Its Application for In Vivo Screening of Anti-Cancer Drugs

Opisthorchis viverrini (O. viverrini) is a well-known causative agent of cholangiocarcinoma (CCA) in humans. CCA is very resistant to chemotherapy and is frequently fatal. To understand the pathogenesis of CCA in humans, a rodent model was developed. However, the development of CCA in rodents is time-consuming and the xenograft-transplantation model of human CCA in immunodeficient mice is costly. Therefore, the establishment of an in vivo screening model for O. viverrini-associated CCA treatment was of interest. We developed a hamster CCA cell line, Ham-1, derived from the CCA tissue of O. viverrini-infected and N-nitrosodimethylamine-treated Syrian golden hamsters. Ham-1 has been maintained in Dulbecco''s Modified Essential Medium supplemented with 10% fetal bovine serum for more than 30 subcultures. These cells are mostly diploid (2n=44) with some being polyploid. Tumorigenic properties of Ham-1 were demonstrated by allograft transplantation in hamsters. The transplanted tissues were highly proliferative and exhibited a glandular-like structure retaining a bile duct marker, cytokeratin 19. The usefulness of this for in vivo model was demonstrated by berberine treatment, a traditional medicine that is active against various cancers. Growth inhibitory effects of berberine, mainly by an induction of G1 cell cycle arrest, were observed in vitro and in vivo. In summary, we developed the allo-transplantable hamster CCA cell line, which can be used for chemotherapeutic drug testing in vitro and in vivo.     

The liver fluke Opisthorchis viverrini expresses nitric oxide synthase but not gelatinases

Host-parasite interaction during infection with the liver fluke Opisthorchis viverrini plays an important role in opisthorchiasis-associated cholangiocarcinoma via nitric oxide (NO) production. Host cells induce nitric oxide synthase (NOS)-dependent DNA damage and secrete Ras-related C3 botulinum toxin substrate (Rac)1, heme oxygenase (HO)-1, and gelatinases (matrix metalloproteinase (MMP)2 and MMP9). We evaluated whether these enzymes are expressed in O. viverrini. Colocalization of NOS and Rac1 was most prominently detected on day 30 post-infection (p.i.) in the gut, reproductive organ, eggs, acetabular and tegument. Expression of HO-1, an antioxidative enzyme, increased in a similar pattern to NOS, but was not present in the tegument. The levels of nitrate/nitrite, end products of NO, and ferric reducing antioxidant capacity, an indicator of antioxidant enzyme capacity, in parasite homogenates were highest on day 30 p.i. and then decreased on day 90 p.i. In contrast, zymography revealed that MMP2 and MMP9 were not present in parasite homogenates at all time points. In conclusion, O. viverrini induces NOS expression and NO production, but does not express gelatinases. The study may provide basic information and an insight into drug design for prevention and/or intervention approaches against O. viverrini infection.     

Diagnostic values of parasite-specific antibody detections in saliva and urine in comparison with serum in opisthorchiasis

Infection by the liver fluke (Opisthorchis viverrini) causes hepatobiliary disease and bile duct cancer (cholangiocarcinoma, CCA) in endemic areas in Southeast Asia. Measurements of humoral immune response particularly parasite-specific antibodies are useful not only for serodiagnosis but they have been implicated as risk factors of CCA. In this study, we used indirect Enzyme Immunosorbent Assay (ELISA) to measure O. viverrini-specific immunoglobulins in serum, urine and saliva and assessed efficacies in diagnosis of opisthorchiasis and evaluated the relationship of antibodies among clinical specimens in a sample population in endemic areas in Khon Kaen, Thailand. By employing the Receiver Operation Characteristics (ROC) analysis, diagnostic efficacy based upon the area under the curve (AUC) revealed that serum, salivary IgG and IgA performed better than urine for diagnosis of opisthorchiasis. Seropositive cases were found in both parasite egg-negative as well as O. viverrini egg-positive groups. The levels of serum IgG correlated with intensity of O. viverrini infection (P < 0.05). Diagnostic sensitivities based on serum and salivary IgG, IgA also positively associated with the intensity of infection. Correlations between serum antibodies and those in saliva were found to be greater in egg-negative than egg-positive individuals for O. viverrini. Our findings indicated a complex interrelation between antibody responses in different clinical specimens triggered by liver fluke infection. More comprehensive examinations are needed to determine the potential utility of salivary antibody detection which, in combination with the conventional fecal examination method, may better assist in the identification of individuals with opisthorchiasis. Furthermore, it may provide a better indicator of the risk of disease, particularly CCA.     

The current status of opisthorchiasis and clonorchiasis in the Mekong Basin

This review highlights the current status and control of liver fluke infections in the Mekong Basin countries where Opisthorchis and Clonorchis are highly endemic. Updated data on prevalence and distribution have been summarized from presentations in the “96 Years of Opisthorchiasis. International Congress of Liver Flukes”. It is disturbing that despite treatment and control programs have been in place for decades, all countries of the Lower Mekong Basin are still highly endemic with O. viverrini and/or C. sinensis as well as alarmingly high levels of CCA incidence. A common pattern that is emerging in each country is the difference in transmission of O. viverrini between lowlands which have high prevalence versus highlands which have low prevalence. This seems to be associated with wetlands, flooding patterns and human movement and settlement. A more concerted effort from all community, educational, public health and government sectors is necessary to successfully combat this fatal liver disease of the poor.     

Overexpression of PDGFA and its receptor during carcinogenesis of Opisthorchis viverrini-associated cholangiocarcinoma

Cholangiocarcinoma (CCA) is a crucial health problem in northeastern part of Thailand, which is caused by a combination of Opisthorchis viverrini infection and nitrosamine. A better understanding of its molecular mechanism is an important step to discover and develop the new diagnostics and therapies for CCA. To reveal the involvement of potential genes in the development of CCA, the present study investigated the expression kinetics of platelet-derived growth factor alpha (Pdgfa) and its receptor (Pdgfra) during the tumorigenesis of CCA induced by O. viverrini infection with quantitative RT-PCR, and confirmed the expression with immunohistological staining. The results showed that in the hamster model of opisthorchiasis-associated CCA, the expression of Pdgfa was increased after infection plus N-nitrosodimethylamine (NDMA) administration, reached its peak at 2 months post infection, and remained at the high level until 6 months. Similarly, the expression of Pdgfra was increased time-dependently. The positive immunostaining for PDGFA proteins was observed in the cytoplasm of epithelial tumor cells of hamster CCA. Moreover, the analysis of the expression of these genes in 10 cases of human opisthorchiasis-associated CCA showed that Pdgfa was overexpressed in 80%, and Pdgfra was overexpressed in 40% cases (> 3.0 folds, compared with the expressions of adjacent normal tissues). This result suggests that PDGFA is likely involved in the tumorigenesis of opisthorchiasis-associated CCA, and may be a promising candidate biomarker for diagnosis and treatment strategies of CCA.     

Helicobacter pylori GroEL Seropositivity Is Associated with an Increased Risk of Opisthorchis viverrini-Associated Hepatobiliary Abnormalities and Cholangiocarcinoma

Despite the synergistic effect of Opisthorchis viverrini and Helicobacter pylori co-infection on pathogenesis of severe hepatobiliary abnormalities (HBA) including advanced periductal fibrosis and replace with cholangiocarcinoma (CCA) have been established, the immune response to H. pylori in O. viverrini infected population has never been explored. Hence, this study aimed to investigate the antibody responses to 2 immunogenic H. pylori proteins in O. viverrini-infected patients with HBA and CCA. The risk analysis by multinomial logistic regression revealed that GroEL seropositivity was associated with higher risks of hepatobiliary abnormalities and CCA with adjusted odds ratios (95% confidence intervals) of 2.11 (95% CI=1.20–3.71, P=0.008) and 2.13 (95% CI=1.21–3.75, P=0.009), respectively. These findings indicate that GroEL seropositivity might be a biomarker for early detection of O. viverrini associated HBA and CCA.     

Co-infection with Opisthorchis viverrini and Haplorchis taichui detected by human fecal examination in Chomtong district, Chiang Mai Province, Thailand

Diseases caused by the liver fluke, Opisthorchis viverrini and the minute intestinal fluke, Haplorchis taichui, are clinically important, especially in the Northeast and North regions of Thailand. It is often difficult to distinguish between these trematode species using morphological methods due to the similarity of their eggs and larval stages both in mixed and co-infections. A sensitive, accurate, and specific detection method of these flukes is required for an effective epidemiological control program. This study aimed to determine the prevalence of O. viverrini and H. taichui infections in human feces by using formalin-ether sedimentation and high annealing temperature random amplified polymorphic DNA (HAT-RAPD) PCR methods. Fecal specimens of people living along the Mae Ping River, Chomtong district were examined seasonally for trematode eggs using a compound microscope. Positive cases were analyzed in HAT-RAPD, DNA profiles were compared with adult stages to determine the actual species infected, and specific DNA markers of each fluke were also screened. Our results showed that out of 316 specimens, 62 were positive for fluke eggs which were pre-identified as O. viverrini and H. taichui. In addition, co-infection among these two fluke species was observed from only two specimens. The prevalence of H. taichui infections peaked in the hot-dry (19.62%), gradually decreased in the rainy (18.18%), and cool-dry seasons (14.54%), respectively. O. viverrini was found only in the hot-dry season (6.54%). For molecular studies, 5 arbitrary primers (Operon Technologies, USA) were individually performed in HAT-RAPD-PCR for the generation of polymorphic DNA profiles. The DNA profiles in all 62 positives cases were the same as those of the adult stage which confirmed our identifications. This study demonstrates the mixed infection of O. viverrini and H. taichui and confirms the extended distribution of O. viverrini in Northern Thailand.     

Hepatic cytochrome P450 2A6 and 2E1 status in peri-tumor tissues of patients with Opisthorchis viverrini-associated cholangiocarcinoma

Endogenous nitrosation due to chronic inflammation is enhanced in opisthorchiasis and plays a crucial role in the development of cholangiocarcinoma (CCA). Hepatic cytochrome P450 (CYP) family enzymes, especially CYP2A6 and CYP2E1, are involved in the metabolism of procarcinogens; these two enzymes metabolize endogenous nitrosamines to carcinogenic N-dimethylnitrosamine (NDMA). CYP2A6 activity is increased in patients infected with Opisthorchis viverrini. Our aim was to determine whether the expression and function of CYP2A6 and 2E1 in the livers of patients with O. viverrini-associated cholangiocarcinoma (CCA) was altered compared to livers without CCA. Livers of CCA patients (n = 13 cases) showed increased enzyme activities, protein and mRNA levels of CYP2A6 whereas the enzyme activity and protein levels of CYP2E1 were markedly decreased (P < 0.05). CYP2E1 mRNA levels were not altered. Large numbers of inflammatory cells and increased iNOS expression was found in areas adjacent to the tumor. The data provide evidence to support the concept that enhanced CYP2A6 activity and diminished CYP2E1 activity probably involve to the progression of CCA.     

The systematics and population genetics of Opisthorchis viverrini sensu lato: implications in parasite epidemiology and bile duct cancer

Together with host and environmental factors, the systematics and population genetic variation of Opisthorchis viverrini may contribute to recorded local and regional differences in epidemiology and host morbidity in opisthorchiasis and cholangiocarcinoma (CCA). In this review, we address recent findings that O. viverrini comprises a species complex with varying degrees of population genetic variation which are associated with specific river wetland systems within Thailand as well as the Lao PDR. Having an accurate understanding of systematics is a prerequisite for a meaningful assessment of the population structure of each species within the O. viverrini complex in nature, as well as a better understanding of the magnitude of genetic variation that occurs within different species of hosts in its life cycle. Whether specific genotypes are related to habitat type(s) and/or specific intermediate host species are discussed based on current available data. Most importantly, we focus on whether there is a correlation between incidence of CCA and genotype(s) of O. viverrini. This will provide a solid basis for further comprehensive investigations of the role of genetic variation within each species of O. viverrini sensu lato in human epidemiology and genotype related morbidity as well as co-evolution of parasites with primary and secondary intermediate species of host.     

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.     

Ultrasonography assessment of hepatobiliary abnormalities in 3359 subjects with Opisthorchis viverrini infection in endemic areas of Thailand

A cross sectional study on hepatobiliary abnormalities in opisthorchiasis was performed in 8936 males and females aged from 20 to 60 years from 90 villages of Khon Kaen province, Northeast Thailand. All were stool-examined for Opisthorchis viverrini infection by standard quantitative formalin/ethyl acetate concentration technique. Of these, 3359 participants with stool egg positive underwent ultrasonography of the upper abdomen. The hepatobiliary abnormalities detected by ultrasound are described here. This study found a significantly higher frequency of advanced periductal fibrosis in persons with chronic opisthorchiasis (23.6%), particularly in males. Risks of the fibrosis included intensity of infection, and age younger than 30 years. Height of left lobe of the liver, cross-section of the gallbladder dimensions post fatty meal, sludge, and, interestingly, intrahepatic duct stones were significantly associated with the advanced periductal fibrosis. Eleven suspected cholangiocarcinoma (CCA) cases were observed. This study emphasizes the current status of high O. viverrini infection rate and the existence of hepatobiliary abnormalities including suspected CCA in opisthorchiasis endemic areas of Thailand.     

A Granulin-Like Growth Factor Secreted by the Carcinogenic Liver Fluke,Opisthorchis viverrini,Promotes Proliferation of Host Cells

The human liver fluke, Opisthorchis viverrini, infects millions of people throughout south-east Asia and is a major cause of cholangiocarcinoma, or cancer of the bile ducts. The mechanisms by which chronic infection with O. viverrini results in cholangiocarcinogenesis are multi-factorial, but one such mechanism is the secretion of parasite proteins with mitogenic properties into the bile ducts, driving cell proliferation and creating a tumorigenic environment. Using a proteomic approach, we identified a homologue of human granulin, a potent growth factor involved in cell proliferation and wound healing, in the excretory/secretory (ES) products of the parasite. O. viverrini granulin, termed Ov-GRN-1, was expressed in most parasite tissues, particularly the gut and tegument. Furthermore, Ov-GRN-1 was detected in situ on the surface of biliary epithelial cells of hamsters experimentally infected with O. viverrini. Recombinant Ov-GRN-1 was expressed in E. coli and refolded from inclusion bodies. Refolded protein stimulated proliferation of murine fibroblasts at nanomolar concentrations, and proliferation was inhibited by the MAPK kinase inhibitor, U0126. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of murine fibroblasts and a human cholangiocarcinoma cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumorigenic environment that may ultimately manifest as cholangiocarcinoma.