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.     

Rapid detection of Opisthorchis viverrini copro-DNA using loop-mediated isothermal amplification (LAMP)

Opisthorchis viverrini and other foodborne trematode infections are major health problem in Thailand, the Lao People's Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. We therefore established a rapid, sensitive and specific method for detecting O. viverrini infection from the stool samples using the loop-mediated isothermal amplification (LAMP) method. A total of five primers from seven regions were designed to target the internal transcribed spacer 1 (ITS1) in ribosomal DNA for specific amplification. Hydroxy naphthol blue (HNB) was more effective to detect the LAMP product compared to the Real-time LAMP and turbidity assay for its simple and distinct detection. The LAMP assay specifically amplified O. viverrini ITS1 but not C. sinensis and minute intestinal flukes with the limit of detection around 10^− 3 ng DNA/μL. The sensitivity of the LAMP was 100% compared to egg positive samples. While all microscopically positive samples were positive by LAMP, additionally 5 of 13 (38.5%) microscopically negative samples were also LAMP positive. The technique has great potential for differential diagnosis in endemic areas with mixed O. viverrini and intestinal fluke infections. As it is an easy and simple method, the LAMP is potentially applicable for point-of-care diagnosis.     

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.     

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.     

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.     

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.     

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.     

The zoonotic,fish-borne liver flukes Clonorchis sinensis,Opisthorchis felineus and Opisthorchis viverrini

Clonorchis sinensis, Opisthorchis felineus and Opisthorchis viverrini are the three most important liver flukes involved in human health, infecting more than 45 million people worldwide. Both C. sinensis and O. viverrini, and possibly O. felineus, can induce human cholangiocarcinoma as well as inducing other hepatobiliary pathology. Although the life cycles of all three species are similar, only that of O. felineus in Europe remains predominantly zoonotic, while O. felineus in Asia and C. sinensis have a stronger mixture of zoonotic and anthroponotic components in their life cycles. Opisthorchis viverrini from the Mekong area of southeastern Asia is predominantly anthroponotic. Here we discuss the comparative epidemiology of these three taxa comparing in detail the use of first, second and final animal hosts, and consider the potential role of humans in spreading these pathogens. In addition we discuss the genetic structure of all three species in relation to potentially cryptic species complexes.     

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.     

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.     

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1 pg of purified genomic DNA, 5 EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent.     

RNA interference targeting cathepsin B of the carcinogenic liver fluke, Opisthorchis viverrini

Functional genomics have not been reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis. Here we describe the introduction by square wave electroporation of Cy3-labeled small RNA into adult O. viverrini worms. Adult flukes were subjected to square wave electroporation employing a single pulse for 20 ms of 125 V in the presence of 50 μg/ml of Cy3-siRNA. The parasites tolerated this manipulation and, at 24 and 48 h after electroporation, fluorescence from the Cy3-siRNA was evident throughout the parenchyma of the worms, with strong fluorescence evident in the guts and reproductive organs of the adult worms. Second, other worms were treated using the same electroporation settings with double stranded RNA targeting an endogenous papain-like cysteine protease, cathepsin B. This manipulation resulted in a significant reduction in specific mRNA levels encoding cathepsin B, and a significant reduction in cathepsin B activity against the diagnostic peptide, Z-Arg-Arg-AMC. This appears to be the first report of introduction of reporter genes into O. viverrini and the first report of experimental RNA interference (RNAi) in this fluke. The findings indicated the presence of an intact RNAi pathway in these parasites which, in turn, provides an opportunity to probe gene functions in this neglected tropical disease pathogen.     

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.     

Prevalence of Opisthorchis viverrini-Like Fluke Infection in Ducks in Binh Dinh Province,Central Vietnam

Following the first report of Opisthorchis viverrini infection in a domestic duck in Phu My District of Binh Dinh Province, Central Vietnam, many other cases were observed in the province. We determined the infection rate and intensity of O. viverrini infection in ducks in 4 districts of the province. A total of 178 ducks were randomly selected from 34 farms for examination of flukes in the liver and gall bladder. An infection rate of 34.3% (range 20.7-40.4% among districts) was found; the intensity of infection was 13.8 worms per infected duck (range 1-100). These findings show the role of ducks as a host for O. viverrini, duck genotype, which is sympatric with the human O. viverrini genotype in this province. It also stresses the need for investigations on the zoonotic potential and the life cycle of this parasite.     

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.     

Oxidative and nitrative DNA damage: key events in opisthorchiasis-induced carcinogenesis

Chronic inflammation induced by liver fluke (Opisthorchis viverrini) infection is the major risk factor for cholangiocarcinoma (CCA) in Northeastern Thailand. Increased levels of proinflammatory cytokines and nuclear factor kappa B that control cyclooxygenase-2 and inducible nitric oxide activities, disturb the homeostasis of oxidants/anti-oxidants and DNA repair enzymes, all of which appear to be involved in O. viverrini-associated inflammatory processes and CCA. Consequently oxidative and nitrative stress-related cellular damage occurs due to the over production of reactive oxygen and nitrogen species in inflamed target cells. This is supported by the detection of high levels of oxidized DNA and DNA bases modified by lipid peroxidation products in both animal and human tissues affected by O. viverrini-infection. Treatment of opisthorchiasis patients with praziquantel, an anti- trematode drug was shown to reduce inflammation-mediated tissue damage and carcinogenesis. The principal mechanisms that govern the effects of inflammation and immunity in liver fluke-associated cholangiocarcinogenesis are reviewed. The validity of inflammation-related biomolecules and DNA damage products to serve as predictive biomarkers for disease risk evaluation and intervention is discussed.