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.     

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.     

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.     

Field survey focused on Opisthorchis viverrini infection in five provinces of Cambodia

BackgroundOpisthorchiasis is endemic in Thailand and Lao People's Democratic Republic and constitutes a major public health problem throughout the Mekong Basin. Although Cambodia is located in the Mekong Basin, the status of O. viverrini infection in that country was not previously clarified. This research was conducted to document the extent and distribution of O. viverrini infection in Cambodia.MethodsSurveillance was conducted in 55 villages in five Cambodian provinces. Research tools included stool examination using the Kato–Katz thick-smear technique, identification of intermediate hosts, and interviews covering factors related to O. viverrini infection. Some larvae and egg-positive stool samples were examined using PCR to detect O. viverrini DNA.ResultsA total of 16,082 stool samples from the 55 villages were examined, of which 1232 were egg positive. In 15 villages with egg-positive rates of greater than 10%, eggs were found in 998 of 3585 stool samples, for an egg-positive rate of 27.8%. PCR analysis showed that 30 of 33 samples were positive for O. viverrini DNA from five villages in Kampong Cham and Kampong Thom provinces. The first intermediate host Bithynia siamensis siamensis was identified in the target areas of Takaev, Kandal, and Kampong Cham provinces. Cercariae were identified morphologically as O. viverrini and some were confirmed using PCR. Metacercariae of O. viverrini were identified by morphologic observations, animal experiments, or PCR in six species of fish in the target areas.Discussion and conclusionsFour Cambodian provinces were identified as endemic areas of O. viverrini infection. Careful planning is necessary for effective field surveys, because complex environmental factors might be involved in the distribution of O. viverrini infection-endemic areas in Cambodia. Many problems remain to be resolved regarding the status of O. viverrini infection in Cambodia, and a nationwide baseline survey is necessary.     

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.     

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.     

Genome size estimation of liver fluke Opisthorchis viverrini by real-time polymerase chain reaction based method

The human liver fluke, Opisthorchis viverrini, has been categorized as a class one carcinogenic organism according to its strong association with cholangiocarcinoma, bile duct cancer which has high incidence in the northeast of Thailand. The lack of genome database of this parasite limited the studies aimed to understand the basic molecular biology of this carcinogenic liver fluke. The determination of the genome size is an initial step prior to the full genome sequencing. In this study, we applied an absolute quantitative real-time polymerase chain reaction for this aspect. Our results indicated the genome size of O. viverrini is 75.95 Mb or C value 0.083. The information of O. viverrini genome size is useful for estimation of sequence coverage and the cost of the parasite's whole genome sequencing using next-generation sequencing technologies.     

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.     

Monophyly of Opisthorchis viverrini populations in the lower Mekong Basin,using mitochondrial DNA nad1 gene as the marker

The liver fluke, Opisthorchis viverrini, causes serious public-health problems in the Lower Mekong Basin. This study aimed to clarify whether O. viverrini populations may be genetically divided into sub-specific taxa. We collected 6 populations of O. viverrini from different places in Cambodia, Lao PDR, and Thailand, along both sides of the Mekong River, and analyzed the population structure of these using the mitochondrial nad1 gene as a marker. The results of the DNA polymorphism measurements, by theta-w (θw) and -π (θπ) values, neutrality tests, and mismatch distribution, suggested that the population of O. viverrini has expanded under the influence of purifying selection and selective sweep. The analysis of molecular variance (AMOVA) test revealed no significant genetic differences among the O. viverrini populations on opposite sides of the Mekong River. O. viverrini haplotypes occurred in multiple populations, and no distinct geographical clade. The star-like haplotype network confirmed a demographic expansion of the O. viverrini population. Overall, the genetic data from these populations suggested that the postulated existence of an O. viverrini species complex should be rejected. The bio-geographical diversity of O. viverrini populations should be explored further, using other appropriate markers and a wider range of samples from geographically different areas.     

Hyperendemicity of Haplorchis taichui Infection among Riparian People in Saravane and Champasak Province,Lao PDR

In this study, we found that Haplorchis taichui, a heterophyid intestinal fluke, is highly prevalent, with heavy worm loads, among riparian people in Saravane and Champasak province, Lao PDR. Fecal specimens were collected from 1,460 people (717 men and 743 women) in 12 riparian (Mekong river) districts and were examined by the Kato-Katz fecal smear technique. The overall helminth egg positive rate was 78.8% and 66.4% in Saravane and Champasak province, respectively. The positive rate for small trematode eggs (STE), which included H. taichui and other heterophyids, Opisthorchis viverrini, and lecithodendriids, was 69.9% and 46.3% in Saravane and Champasak province, respectively. To obtain adult flukes, 30 STE-positive people were treated with 40 mg/kg praziquantel and then purged. Whole diarrheic stools were collected 4-5 times for each person and searched for fluke specimens using a stereomicroscope. Mixed infections with various species of trematodes (H. taichui, Haplorchis pumilio, O. viverrini, Prosthodendrium molenkampi, Centrocestus formosanus, and Echinochasmus japonicus) and a species of cestode (Taenia saginata) were found. However, the worm load was exceptionally high for H. taichui compared with other trematode species, with an average of 21,565 and 12,079 specimens per infected person in Saravane and Champasak province, respectively, followed by H. pumilio (41.9 and 22.5, respectively) and O. viverrini (9.4 and 1.5, respectively). These results show that diverse species of intestinal and liver flukes are prevalent among riparian people in Saravane and Champasak province, Lao PDR, with H. taichui being the exceptionally dominant species.     

Effect of light intensity on Opisthorchis viverrini cercarial shedding levels from Bithynia snails--a preliminary study

Opisthorchis viverrini requires Bithynia snails as the first intermediate host and cyprinid fish as the second intermediate host. Very low natural infection rates have been reported in Bithynia snails, but very high rates have been found in cyprinid fish in the same endemic region. This study investigated the effect of light intensity, the most important stimulus, on the quantity of O. viverrini cercariae shed from naturally infected Bithynia (Digoniostoma) siamensis goniomphalos snails. Snails were evaluated for cercariae output every hour after exposure to various light intensities for a total period of 7 h. The same infected snail was tested under different intensities of light: in the dark, and at 1000, 3000 and 5000 lx. The data showed that under exposure to 1000 and 3000 lx of light, the average percentage and number of cercariae released were higher than that exposed to 5000 lx during the first 2 h of the experiment. In contrast, under higher illumination (5000 lx) a longer time (6 h) was required to stimulate the peak emergence of cercariae. Darkness was not able to induce O. viverrini cercariae emergence. Among the three intensities of light, exposure at 1000 lx induced the highest average number of released cercariae per snail and the highest percentage of cercarial emergence within the first 2 h (125, 54.86%), followed by exposure at 3000 lx (69, 25.58%) and 5000 lx (12, 7.78%). The results suggest that the light intensity of 1000 lx for 2 h would be optimal for O. viverrini cercarial shedding from naturally infected B. (D.) siamensis goniomphalos snails.     

Fecal bacterial contamination in natural water reservoirs as an indicator of seasonal infection by Opisthorchis viverrini in snail intermediate hosts

Opisthorchis viverrini, a carcinogenic liver fluke, requires Bithynia snails as the first intermediate host, which release cercariae after ingesting fluke eggs from contaminated water. Fecal bacterial contamination and O. viverrini-infected Bithynia snails were investigated in samples collected from natural water reservoirs in Ban Phai, Chonnabot and Muang Districts (Ban Lerngpeuy) in Khon Kaen Province, northeast Thailand, where there is a high incidence of cholangiocarcinoma. Water was sampled and examined six times (February, April, June, August, October and December 2006). The most probable number (MPN) index and coliform counts were utilized to evaluate fecal contamination; the cercarial shedding method was conducted for detecting infected snails. The data revealed that all water samples had a high MPN index number, and fecal coliform levels above the WHO standard. This indicated that water in these reservoirs was contaminated with feces or manure constituents. Water sampling from Ban Lerngpeuy showed full-scale bacterial contamination (> 1609 MPN index) throughout the year. This finding was correlated with the highest prevalence of O. viverrini-infected snails, which were found nearly all year round in this area. Slightly lower fecal contamination levels were detected in water samples from Chonnabot and Ban Phai, with high MPN index numbers and coliform counts from April to October. This corresponded with the higher recovery of infected snails in June and August, but with relatively lower prevalence than those found in Ban Lerngpeuy. Among the sampling sites, the people in Ban Lerngpeuy live nearer to the reservoir than do those in Ban Phai and Chonnabot. These results indicate that fecal bacterial contamination in natural water reservoirs is an important indicator of seasonal transmission of O. viverrini eggs to snail intermediate hosts. Sanitation improvement is essential and future investigations on the sources of contamination are needed.     

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.     

High Prevalence of Haplorchis taichui, Phaneropsolus molenkampi, and Other Helminth Infections among People in Khammouane Province, Lao PDR

The prevalence of liver and intestinal helminth infections, including Opisthorchis, Haplorchis, Phaneropsolus, hookworms, Enterobius, and Taenia, was surveyed in Khammouane province, Lao PDR. Fecal specimens were collected from 1,242 people (590 men and 652 women) in 3 Mekong riverside villages and were examined by the Kato-Katz thick smear technique. The overall helminth egg positive rate was 81.1%. The positive rate for small trematode eggs, including Opisthorchis viverrini, heterophyids, and lecithodendriids, was 81.1% and the positive rate for hookworms was 6.7%. To obtain adult worms, 35 people who were positive for small trematode eggs were treated with 20-30 mg/kg praziquantel and 10-15 mg/kg pyrantel pamoate, and then purged. Diarrheic stools were collected from 33 of these people and searched for helminth parasites using a stereomicroscope. Mixed infections with various helminths (Haplorchis taichui, Haplorchis yokogawai, Prosthodendrium molenkampi, Phaneropsolus bonnei, echinostomes, hookworms, Trichostrongylus spp., Trichuris trichiura, Enterobius vermicularis, and/or Taenia saginata) were found. The total number of helminth specimens collected was 20,907 (approximately 634 per person). The most common species was H. taichui, followed by P. molenkampi, O. viverrini, P. bonnei, E. vermicularis, hookworms, and Trichostrongylus spp. These results show that diverse species of intestinal nematodes, trematodes, and cestodes are infecting humans in Khammouane province, Lao PDR.     

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.     

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 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.     

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.     

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.