Inflammatory responses to Opisthorchis viverrini infection in animal models: A comparison between susceptible and nonsusceptible hosts in different anatomical locations.

Background: Inflammation caused by Opisthorchis viverrini infection increases the risk of cholangitis, cholecystitis, and leads to bile duct cancer (cholangiocarcinoma or CCA). However, only certain infected individuals are susceptible to CCA, suggesting the involvement of host factors in cancer development. In addition, there are reports indicating differences in the locations of CCA. Aim: This study aims to investigate cellular inflammatory responses in the common bile duct (CB), intrahepatic bile duct (IHB), and gallbladder (GB) in susceptible and non-susceptible hosts following O. viverrini infection. Methods: Thirty Syrian golden hamsters (a susceptible host) and 30 BALB/c mice (a non-susceptible host) infected with O. viverrini were studied at six time points (five animals per group). Histopathological evaluations were conducted on samples from the IHB, CB, and GB. Inflammatory cell infiltration was quantitatively assessed and compared between groups and time points. Statistical analysis was performed using one-way ANOVA, with a significance level of p < 0.05. Results: Inflammation was significantly more pronounced in the IHB compared to the other two biliary locations. In comparison between susceptible and non-susceptible hosts, the intensity of inflammation was higher in the OV+H group than in the OV+M group (p < 0.05). Conclusion: This study highlights the association between host response to inflammation, tissue location, and host susceptibility, with the IHB showing particular susceptibility to inflammation and pathological changes. These findings contribute to our understanding of the increased risk of CCA in susceptible hosts.

Inflammatory cell responses in biliary mucosa during Opisthorchis viverrini infection: Insights into susceptibility differences among hosts.

Background: Individual host susceptibility is believed to be a risk factor in the interaction between the host and the parasite. Since studying time series in humans is limited, animal models are replaced. Aim: This study aims to explore and compare the pattern of inflammatory cell types along the biliary tract and their association with proliferative lesions in the early development of cholangiocarcinoma from susceptible and nonsusceptible animal models. Methods: Thirty male Syrian golden hamsters and 30 BALB/c mice, serving as the susceptible and nonsusceptible animal models, were used in this comparative study. The animals were infected with 50 Opisthorchis viverrini metacercariae via gastric intubation. At days 1, 2, 7, 14, 28, and 56 postinfection (p.i.), five animals were randomly selected from each group and humanely sacrificed. The hepatobiliary tissues were collected and processed for histopathological study. Histochemical and immunohistochemical staining were applied to differentiate the inflammatory cell types. Kruskal-Wallis and Mann-Whitney tests were applied to assess all semi-quantitative and quantitative variables. The correlation between each variable was also analyzed using Spearman rank at a p-value < 0.05. Results: The results demonstrated that mice had different patterns of infiltrating cell types when compared to hamsters. This suggested that the cellular response to the infection in mice occurred earlier than that in hamsters. The response in mice reached its peak at D7 to D14 and then rapidly declined at D28. In contrast, although the inflammatory response in hamsters started slowly, the response reached the peak at D28 and maintained a high level until D56. Significant differences in the number of inflammatory cells between mice and hamsters were seen at D1 (p = 0.047), D7 (p = 0.049), D28 (p = 0.040), and D56 (p < 0.040). Conclusion: The inflammatory responses to O. viverrini infection in the nonsusceptible animal model occurred and declined earlier while the response in the susceptible animal model occurred later in a gradual manner. Both rodents are suitable animal models for the studies of opisthorchiasis susceptibility.

Neutrophils form extracellular traps in response to Opisthorchis viverrini crude antigens, which are elevated in neutrophils from opisthorchiasis patients with hepatobiliary abnormalities.

Opisthorchis viverrini (Ov) infection can cause several disease conditions of the bile duct including hepatobiliary abnormalities (HBAs) and the most severe, cholangiocarcinoma (CCA). Fibrosis occurs when tissues are damaged and normal wound-healing responses are dysregulated. Neutrophils are the first cells to migrate to an infection site to protect the host from intruding extracellular pathogens through a wide range of effector mechanisms such as phagocytosis, production of reactive oxygen species, proteases, or release of neutrophil extracellular traps (NETs). In this work, we used confocal microscopy to assess whether Ov crude antigens can cause release of NETs from neutrophils from Ov-free individuals. We demonstrated for the first time that these antigens could induce release of NETs ex vivo in a dose-dependent manner from neutrophils isolated from Ov-free individuals. Intriguingly, when we measured NETs from neutrophils isolated from Ov-infected patients, we found increased spontaneous production of NETs in patients with HBAs. Interestingly, exposure to Ov crude antigens lowered the level of NETs released by neutrophils from patients with active Ov infection regardless of HBA status. We propose that in the case of acute Ov infection, even when concentration of Ov antigens is relatively low, neutrophils can form NETs. However, when this infection becomes chronic, manifesting as a definite HBA, the levels of NET production are reduced when treated with Ov crude antigens. Excessive production of proinflammatory mediators from these NETs might have effects on the parasites, but may also lead to excessive injury of surrounding tissues resulting in HBAs and may lead eventually to the most severe complications such as CCA.

The effects of temperature and salinity on the longevity of Opisthorchis viverrini cercariae: a climate change concern.

Research on the effects of environmental factors influenced by climate change on parasite transmissibility is an area garnering recent attention worldwide. However, there is still a lack of studies on the life cycle of Opisthorchis viverrini, a carcinogenic trematode found in countries of the Lower Mekong subregion of Lao PDR, Cambodia, Myanmar, Vietnam and Thailand. To evaluate the influences of environmental factors water temperature and salinity on the transmissibility of the liver fluke O. viverrini through cercarial stage, longevity of O. viverrini cercaria was examined at different experimental temperatures (22°C, 30°C and 38°C) and salinities (2.5 parts per thousand (PPT), 3.75 PPT and 5 PPT). The results reveal that different temperatures have statistically significant effects on cercarial longevity. The cercariae exhibited a thermostability zone ranging between 22°C and 30°C. Cercarial longevity was significantly shortened when water temperatures reached 38°C. Salinity also plays a key role in cercarial longevity, with cercarial survival significantly shorter at a salinity of 3.75 PPT than at 2.5 PPT and 5 PPT. A combined analysis of salinity and temperature revealed unique trends in cercarial longevity. At all experimental salinities, cercarial longevity was lowest when incubated in 38°C, but statistically significant from cercarial longevity at temperatures of 22°C and 30°C, and salinities of 2.5 PPT and 5 PPT. The results suggest that higher temperatures negatively impact parasite longevity. This reflects that O. viverrini transmission patterns may be impacted by changes in water temperature and salinity resulting from climate change.

Current and Novel Therapies Against Helminthic Infections: The Potential of Antioxidants Combined with Drugs.

Infections caused by Schistosoma haematobium and Opisthorchisviverrini are classified as Group 1 biological carcinogen and it has been postulated that parasites produce oxysterol and estrogen-like metabolites that might be considered as initiators of infection-associated carcinogenesis. Chemotherapy for these helminthic infections relies on a single drug, praziquantel, (PZQ) that mainly targets the parasite. Additionally, PZQ has some major drawbacks as inefficacy against juvenile form and alone it is not capable to counteract pathologies associated to infections or prevent carcinogenesis. There is an urgent need to develop novel therapeutic approaches that not only target the parasite but also improve the pathologies associated to infection, and ultimately, counteract or/and prevent the carcinogenesis processes. Repurposing the drug in combination of compounds with different modes of action is a promising strategy to find novel therapeutics approaches against these helminthic infections and its pathologies. Here, we emphasized that using antioxidants either alone or combined with anthelmintic drugs could ameliorate tissue damage, infection-associated complications, moreover, could prevent the development of cancer associated to infections. Hence, antioxidants represent a potential adjuvant approach during treatment to reduce morbidity and mortality. Despite the success of some strategies, there is a long way to go to implement novel therapies for schistosomiasis.

Effects of Opisthorchis viverrini infection on glucose and lipid profiles in human hosts: A cross-sectional and prospective follow-up study from Thailand.

Opisthorchis viverrini (OV) infection is endemic to the Northeast Thailand where the prevalence of Type 2 Diabetes mellitus (T2DM) is higher whilst the incidence of cardiovascular diseases (CVDs) is lower than the rest of Thailand. Helminth infection has both nutritional and immunological impact on their definitive hosts. Thus, a cross-sectional study was performed to see the effects of OV infection on glucose and lipid profiles. For this purpose, 200 each of OV infected and uninfected residents were recruited and their glycated hemoglobin (HbA1c), total cholesterol, triglycerides, low- and high-density lipoproteins (LDL and HDL) levels and anthropometric measurements, including BMI were examined. Then, as the prospective follow- up study, changes of those metabolic parameters of OV positive subjects (n = 120) before and after Praziquantel (PZQ) treatment were monitored for six months. The results showed that OV infection has a protective effect against hyperglycemia (OR 0.482 and p = .04) and metabolic disease risk group (OR 0.478 and p = .03). OV positive participants had lower HbA1c (5.5% Vs. 6.01%, p = .001) but higher HDL (54.07 Vs. 49.46 mg/dL, p = .001) than OV negative participants that are statistically significant. After PZQ treatment for OV-positive subjects, their serum levels of HbA1c (p < .05) and HDL (p < .05) significantly rose during the follow up. Apparently, OV infection lowers HbA1c but increases HDL in definitive human hosts.

Opisthorchis felineus infection provokes time-dependent accumulation of oxidative hepatobiliary lesions in the injured hamster liver.

Opisthorchiasis caused by food-borne trematode Opisthorchis felineus is a substantial public health problem, with 17 million persons infected worldwide. This chronic disease is associated with hepatobiliary inflammation, cholangiocyte dysplasia, cholangiofibrosis, intraepithelial neoplasia, and even cholangiocarcinoma among chronically infected individuals. To provide first insights into the mechanism by which O. felineus infection causes precancerous liver lesions, we investigated the level of oxidative stress (lipid peroxidation byproducts and 8-hydroxy-2'-deoxyguanosine) as well as the time course profiles of chronic inflammation and fibrogenesis markers in the dynamics of opisthorchiasis from 1 month to 1.5 years postinfection in an experimental model based on golden hamsters Mesocricetus auratus. For the first time, we showed that O. felineus infection provokes time-dependent accumulation of oxidative hepatobiliary lesions in the injured liver of hamsters. In particular, over the course of infection, lipid peroxidation byproducts 4-hydroxynonenal and malondialdehyde were upregulated; these changes in general correlate with the dynamics of hepatic histopathological changes. We detected macrophages with various immunophenotypes and elevated levels of CD68, COX2, and CD163 in the O. felineus-infected animals. Meanwhile, there was direct time-dependent elevation of TNF-α (R = 0.79; p < 0.001) and CD163 protein levels (R = 0.58; p = 0.022). We also provide quantitative data about epithelial hyperplasia marker CK7 and a marker of myofibroblast activation (α smooth muscle actin). Our present data provide first insights into the histopathological mechanism by which O. felineus infection causes liver injuries. These findings support the inclusion of O. felineus in Group 1 of biological carcinogens.

Vaccination of hamsters with Opisthorchis viverrini extracellular vesicles and vesicle-derived recombinant tetraspanins induces antibodies that block vesicle uptake by cholangiocytes and reduce parasite burden after challenge infection.

BACKGROUND: The liver fluke Opisthorchis viverrini infects several million people in Southeast Asia. Adult flukes live in the bile ducts of humans, where they cause hepatobiliary pathology, including cholangiocarcinoma. Here, we investigated the potential of extracellular vesicles (EVs) secreted by the fluke and defined recombinant proteins derived from EVs to generate protective immunity in a hamster vaccination-challenge model. METHODOLOGY/PRINCIPAL FINDINGS: EVs isolated from the excretory-secretory products of O. viverrini and two recombinant EV surface proteins encoding the large extracellular loops (LEL) of Ov-TSP-2 (rOv-TSP-2) and Ov-TSP-3 (rOv-TSP-3) were adjuvanted and used to vaccinate hamsters intraperitoneally followed by challenge infection with O. viverrini metacercariae. The number of adult flukes recovered from hamsters immunized with EVs, rOv-TSP-2, rOv-TSP-3 and rOv-TSP-2+rOv-TSP-3 were significantly reduced compared to control animals vaccinated with adjuvant alone. The number of eggs per gram feces was also significantly reduced in hamsters vaccinated with rOv-TSP-2 compared to controls, but no significant differences were found in the other groups. The average length of worms recovered from hamsters vaccinated with EVs, rOv-TSP-2 and rOv-TSP-3 was significantly shorter than that of worms recovered from the control group. Anti-EV IgG levels in serum and bile were significantly higher in hamsters vaccinated with EVs compared to control hamsters both pre- and post-challenge. In addition, levels of anti-rOv-TSP antibodies in the serum and bile were significantly higher than control hamsters both pre- and post-challenge. Finally, antibodies against rOv-TSP-2 and rOv-TSP-3 blocked uptake of EVs by human primary cholangiocyte in vitro, providing a plausible mechanism by which these vaccines exert partial efficacy and reduce the intensity of O. viverrini infection. CONCLUSION/SIGNIFICANCE: Liver fluke EVs and recombinant tetraspanins derived from the EV surface when administered to hamsters induce antibody responses that block EV uptake by target bile duct cells and exert partial efficacy and against O. viverrini challenge.

Effect of Temperature on the Killing of Opisthorchis viverrini Eggs In Vitro.

Contaminated liver fluke egg in the environment has led to the high prevalence of human opisthorchiasis associated with cholangiocarcinoma in Southeast Asia. To find the effective lessening methods of Opisthorchis viverrini eggs in the contaminated environment, we investigated the temperature conditions for killing of these trematode eggs in vitro. Numerous O. viverrini eggs were obtained in the proximal part of uteri of adult worms from experimental hamsters. Mature eggs with miracidium were allocated by experimental groups (2 control: positive and negative and 4 treatment: 50, 60, 70, and 80°C) with 0.85% saline, and treated by the experimental plan. Eggs in each experimental groups were observed under the confocal microscope after stain with Propidium Iodide (PI) to evaluate the effect of temperatures. Eggs in 70 and 80°C groups were all killed after over 10 min heated. Majority of eggs in 60°C (10, 15, and 30 min heated), 70 and 80°C (5 min heated) groups were inactivated. However in 50°C group, below half of eggs were to be killed in all time lapse (10, 15 and 30 min). In order to prevent O. viverrini infection and cholangiocarcinoma, direct treatment of sewage by heating at 70 or 80°C at least 10 min is essential. Therefore, treatment of O. viverrini eggs at a high temperature is a potential method for controlling egg contamination in sewage.

Opisthorchis viverrini Proteome and Host-Parasite Interactions.

The omics technologies have improved our understanding of the molecular events that underpin host-parasite interactions and the pathogenesis of parasitic diseases. In the last decade, proteomics and genomics in particular have been used to characterize the surface and secreted products of the carcinogenic liver fluke Opisthorchis viverrini and revealed important roles for proteins at the host-parasite interface to ensure that the flukes can migrate, feed and reproduce in a hostile environment. This review summarizes the advances made in this area, primarily focusing on discoveries enabled by the publication of the fluke secreted proteomes over the last decade. Protein families that will be covered include proteases, antioxidants, oncogenic proteins and the secretion of exosome-like extracellular vesicles. Roles of these proteins in host-parasite interactions and pathogenesis of fluke-induced hepatobiliary diseases, including cholangiocarcinogenesis, are discussed. Future directions for the application of this knowledge to control infection and disease will also be discussed.

Thinking beyond Opisthorchis viverrini for risk of cholangiocarcinoma in the lower Mekong region: a systematic review and meta-analysis.

BACKGROUND: Cholangiocarcinoma (CCA) is a fatal bile duct cancer associated with infection by the liver fluke, Opisthorchis viverrini, in the lower Mekong region. Numerous public health interventions have focused on reducing exposure to O. viverrini, but incidence of CCA in the region remains high. While this may indicate the inefficacy of public health interventions due to complex social and cultural factors, it may further indicate other risk factors or interactions with the parasite are important in pathogenesis of CCA. This systematic review aims to provide a comprehensive analysis of described risk factors for CCA in addition to O. viverrini to guide future integrative interventions. MAIN BODY: We searched five international and seven Thai research databases to identify studies relevant to risk factors for CCA in the lower Mekong region. Selected studies were assessed for risk of bias and quality in terms of study design, population, CCA diagnostic methods, and statistical methods. The final 18 included studies reported numerous risk factors which were grouped into behaviors, socioeconomics, diet, genetics, gender, immune response, other infections, and treatment for O. viverrini. Seventeen risk factors were reported by two or more studies and were assessed with random effects models during meta-analysis. This meta-analysis indicates that the combination of alcohol and smoking (OR = 11.1, 95% CI: 5.63-21.92, P <  0.0001) is most significantly associated with increased risk for CCA and is an even greater risk factor than O. viverrini exposure. This analysis also suggests that family history of cancer, consumption of raw cyprinoid fish, consumption of high nitrate foods, and praziquantel treatment are associated with significantly increased risk. These risk factors may have complex relationships with the host, parasite, or pathogenesis of CCA, and many of these risk factors were found to interact with each other in one or more studies. CONCLUSIONS: Our findings suggest that a complex variety of risk factors in addition to O. viverrini infection should be addressed in future public health interventions to reduce CCA in affected regions. In particular, smoking and alcohol use, dietary patterns, and socioeconomic factors should be considered when developing intervention programs to reduce CCA.

Exploratory metabolomics study of the experimental opisthorchiasis in a laboratory animal model (golden hamster, Mesocricetus auratus).

BACKGROUND: Opisthorchiasis is a parasitic infection caused by the liver flukes of the Opisthorchiidae family. Both experimental and epidemiological data strongly support a role of these parasites in the etiology of the hepatobiliary pathologies and an increased risk of intrahepatic cholangiocarcinoma. Understanding a functional link between the infection and hepatobiliary pathologies requires a detailed description a host-parasite interaction on different levels of biological regulation including the metabolic response on the infection. The last one, however, remains practically undocumented. Here we are describing a host response on Opisthorchiidae infection using a metabolomics approach and present the first exploratory metabolomics study of an experimental model of O. felineus infection. METHODOLOGY AND PRINCIPAL FINDINGS: We conducted a Nuclear Magnetic Resonance (NMR) based longitudinal metabolomics study involving a cohort of 30 animals with two degrees of infection and a control group. An exploratory analysis shows that the most noticeable trend (30% of total variance) in the data was related to the gender differences. Therefore further analysis was done of each gender group separately applying a multivariate extension of the ANOVA-ASCA (ANOVA simultaneous component analysis). We show that in the males the infection specific time trends are present in the main component (43.5% variance), while in the females it is presented only in the second component and covers 24% of the variance. We have selected and annotated 24 metabolites associated with the observed effects and provided a physiological interpretation of the findings. CONCLUSIONS: The first exploratory metabolomics study an experimental model of O. felineus infection is presented. Our data show that at early stage of infection a response of an organism unfolds in a gender specific manner. Also main physiological mechanisms affected appear rather nonspecific (a status of the metabolic stress) the data provides a set of the hypothesis for a search of the more specific metabolic markers of the Opisthorchiidae infection.

Prospects and Challenges towards Sustainable Liver Fluke Control.

The liver fluke Opisthorchis viverrini (Ov) is endemic in Southeast Asia where more than 10 million people are estimated to be infected. The infection is associated with several hepatobiliary diseases, including cholangiocarcinoma (CCA). Northeast Thailand is a hotspot for Ov transmission, and, despite extensive public health prevention campaigns led by the government, the prevalence of Ov infection is still high. High infection rates result from cultural and ecological complexities where wet-rice agrarian habitats, centuries-old raw-food culture, and the parasite's complex biology combine to create an ideal transmission arena. Here we review the state of our knowledge regarding the social-ecological determinants underlying Ov transmission. We also describe an integrative research rationale for liver fluke control better aligned with sustainable health development.

Differential Protein Expression Marks the Transition From Infection With Opisthorchis viverrini to Cholangiocarcinoma.

Parts of Southeast Asia have the highest incidence of intrahepatic cholangiocarcinoma (CCA) in the world because of infection by the liver fluke Opisthorchis viverrini (Ov). Ov-associated CCA is the culmination of chronic Ov-infection, with the persistent production of the growth factors and cytokines associated with persistent inflammation, which can endure for years in Ov-infected individuals prior to transitioning to CCA. Isobaric labeling and tandem mass spectrometry of liver tissue from a hamster model of CCA was used to compare protein expression profiles from inflammed tissue (Ovinfected but not cancerous) versus cancerous tissue (Ov-induced CCA). Immunohistochemistry and immunoblotting were used to verify dysregulated proteins in the animal model and in human tissue. We identified 154 dysregulated proteins that marked the transition from Ov-infection to Ov-induced CCA, i.e. proteins dysregulated during carcinogenesis but not Ov-infection. The verification of dysregulated proteins in resected liver tissue from humans with Ov-associated CCA showed the numerous parallels in protein dysregulation between human and animal models of Ov-induced CCA. To identify potential circulating markers for CCA, dysregulated proteins were compared with proteins isolated from exosomes secreted by a human CCA cell line (KKU055) and 27 proteins were identified as dysregulated in CCA and present in exosomes. These data form the basis of potential diagnostic biomarkers for human Ov-associated CCA. The profile of protein dysregulation observed during chronic Ovinfection and then in Ov-induced CCA provides insight into the etiology of an infection-induced inflammation-related cancer.

[Control of Opisthorchis viverrini infection for cholangiocarcinoma prevention]. / Vaincre la distomatose à Opisthorchis viverrini pour prévenir le cholangiocarcinome.

The International Agency for Research on Cancer (IARC) has classified two liver flukes as carcinogenic to humans (Group 1): Opisthorchis viverrini in 1994 and Clonorchis sinensis in 2009. This review is focused on O. viverrini, the most studied of these two trematodes, which infects nearly 10 million people in Southeast Asia. The life cycle involves two intermediate hosts living in fresh water: a snail of the genus Bithynia and a ciprinid fish. The definitive hosts (human, cat, dog) become infected by ingesting raw fish containing metacercariae, the infective stage of the parasite. Adult flukes attach to the epithelium of the bile ducts where they feed for as long as 10 to 30 years, resulting in chronic inflammation, epithelial hyperplasia, periductal fibrosis and formation of granuloma. For a long asymptomatic, the distomatosis is revealed by a chronic cholangitis when the parasite load becomes high. Complications can occur with time: gallstones, cholangitis, liver abscess, pancreatitis and, after a few decades, cholangiocarcinoma (CCA). The epidemiological correlation between the prevalence of O. viverrini infection and the incidence of CCA has been demonstrated in the northeast of Thailand. Specifically, the Khon Kaen province has the highest incidence rate in the world. The CCA can develop asymptomatically for a long time, especially in intrahepatic locations. It is often discovered at a late stage, unresectable. Its prognosis is dreadful with a survival rate less than 5% at 5 years. The phenomenon of carcinogenesis induced by O. viverrini is multifactorial. It has been specially studied using experimental infection on the Syrian golden hamster. Three intricated mechanisms are involved: (i) the direct damage caused by adult worms on the bile duct epithelium, (ii) the immunopathologic processes related to chronic inflammation (oxidative stress) and (iii) the mitogenic and anti-apoptotic effects of the proteins secreted by the parasite. Exogenous cofactors are also involved, such as nitrosamines in fish-based dishes undercoocked or fermented, very popular in these endemic regions. Despite the effectiveness of praziquantel to successfully cure this distomatose, opisthorchiasis persists endemic in areas where the incidence of CCA tends to progress. Mass deworming campaigns are ineffective due to the frequency of reinfection in the exposed population. Repeating alternatively cures and reinfections may promote carcinogenesis. The failure of prevention programs reflects the difficulty of changing the traditional habits of consuming raw or fermented fish. Pending a vaccine prophylaxis, control strategies are based on integrated measures involving the treatment of reservoir hosts, sanitation and efforts of continuing information and education to deter the consumption of uncooked fish and to improve the sanitation in rural areas.

Urinary microRNA-192 and microRNA-21 as potential indicators for liver fluke-associated cholangiocarcinoma risk group.

Opisthorchis viverrini infection induces chronic inflammation in the bile ducts, leading to periductal fibrosis (PDF), which possibly associates to cholangiocarcinoma (CCA). Patients with CCA have a poor prognosis, which is linked to asymptomatic disease and late diagnosis. Hence, detecting early stage CCA is essential. Secretory miRNAs have been promoted as biomarkers for pathological changes associated with parasitic infections, fibrosis and/or cancer. We aimed to determine levels of miR-192 and miR-21 in the urine of O. viverrini infected, periductal fibrosis (PDF) and CCA groups using qRT-PCR. We found that miR-192 was significantly higher in O. viverrini infected, PDF and also CCA groups (p<0.05) than in healthy controls. By utilizing the Receiver Operation Characteristics (ROC) analysis, miR-192 differentiated patients with opisthorchiasis (the area under the curve; AUC=0.766), PDF subjects (AUC=0.781) and CCA patients (AUC=0.682) from healthy controls. MiR-21 was significantly higher in PDF and CCA groups (p<0.05) than in healthy controls. MiR-21 discriminated PDF subjects (AUC=0.735) and CCA patients (AUC=0.682) from healthy controls. Combined levels of these two miRNAs revealed an increased AUC of 0.812 for separating opisthorchiasis, AUC of 0.815 in discriminating PDF subjects, and AUC of 0.849 in differentiating CCA from healthy controls. Odds ratios (OR) indicated high levels of miR-192/miR-21 as risk predictors for opisthorchiasis, PDF and CCA. Levels of these miRNAs declined significantly for patients following praziquantel treatment. In conclusion, urinary miR-192/miR-21 have potential as risk indicators for opisthorchiasis and PDF-associated CCA in the endemic region.

Genomics of worms, with an emphasis on Opisthorchis viverrini - opportunities for fundamental discovery and biomedical outcomes.

Neglected tropical diseases cause substantial morbidity and mortality in animals and people globally. Opisthorchiasis is one such disease, caused by the carcinogenic, Asian liver fluke, Opisthorchis viverrini. This hepatobiliary disease is known to be associated with malignant cancer (cholangiocarcinoma, CCA) and affects millions of people in Asia, including Thailand, Lao People's Democratic Republic (PDR) and Cambodia. No vaccine is available, and only one drug (praziquantel) is routinely employed against the parasite. Relatively little is known about the molecular biology of the fluke itself and the disease complex that it causes in humans. With the advent of high-throughput nucleic acid sequencing and bioinformatic technologies, it has now become possible to gain global insights into the molecular biology of parasites. The purpose of this minireview is (i) to discuss recent progress on the genomics of parasitic worms, with an emphasis on the draft genome and transcriptome of O. viverrini; (ii) to use results from an integrated, global analysis of the genomic and transcriptomic data, to explain how we believe that this carcinogenic fluke establishes in the biliary system, how it feeds, survives and protects itself in such a hostile, microaerobic environment within the liver, and to propose how this parasite evades or modulates host attack; and (iii) to indicate some of the challenges, and, more importantly, the exciting opportunities that the 'omic resources for O. viverrini now provide for a plethora of fundamental and applied research areas. Looking ahead, we hope that this genomic resource stimulates vibrant and productive collaborations within a consortium context, focused on the effective control of opisthorchiasis.

Molecular and Biochemical Characterization of Opisthorchis viverrini Calreticulin.

Calreticulin (CALR), a multifunctional protein thoroughly researched in mammals, comprises N-, P-, and C-domain and has roles in calcium homeostasis, chaperoning, clearance of apoptotic cells, cell adhesion, and also angiogenesis. In this study, the spatial and temporal expression patterns of the Opisthorchis viverrini CALR gene were analyzed, and calcium-binding and chaperoning properties of recombinant O. viverrini CALR (OvCALR) investigated. OvCALR mRNA was detected from the newly excysted juvenile to the mature parasite by RT-PCR while specific antibodies showed a wide distribution of the protein. OvCALR was localized in tegumental cell bodies, testes, ovary, eggs, Mehlis' gland, prostate gland, and vitelline cells of the mature parasite. Recombinant OvCALR showed an in vitro suppressive effect on the thermal aggregation of citrate synthase. The recombinant OvCALR C-domain showed a mobility shift in native gel electrophoresis in the presence of calcium. The results imply that OvCALR has comparable function to the mammalian homolog as a calcium-binding molecular chaperone. Inferred from the observed strong immunostaining of the reproductive tissues, OvCALR should be important for reproduction and might be an interesting target to disrupt parasite fecundity. Transacetylase activity of OvCALR as reported for calreticulin of Haemonchus contortus could not be observed.

Suspected cases of cholangiocarcinoma seen in reference hospitals in Lao People's Democratic Republic.

Cholangiocarcinoma (CCA) poses a significant public health problem in Southeast Asia, with chronic Opisthorchis viverrini infection being the main risk factor. Epidemiological data on CCA is not available for Lao People Democratic Republic (Lao PDR), though there is a high prevalence of O. viverrini infection in the country. A hospital record-based retrospective study was carried out in six referral hospitals (four in the capital city, one in Savannakhet, one in Pakse) to assess the number of suspected CCA cases admitted between January 2006 and December 2010. In total, we identified 274 patients with liver lesions suggesting CCA, as diagnosed by ultrasound and CT scan. Male gender and age 50years and above were main characteristics of those identified. The majority were admitted to hospitals in the central (58.0%) and southern (29.6%) parts of Lao PDR. Abdominal pain (48.5%) and jaundice (14.2%) were the main symptoms leading to hospitalisation. Some 267 (97.4%) patients had dilated bile ducts; 40 (74.1%) had gallstone or intrahepatic stone, and 33 (12.0%) had a parasitologically confirmed O. viverrini infection. This study provides the first estimated numbers of suspected CCA cases admitted to selected hospitals in Lao PDR. Definitive diagnosis of CCA was not possible. Therefore, case diagnosis and registration of suspected CCA cases are warranted to gather evidence for a national burden estimate of CCA, to develop large-scale studies and adequate preventive and curative health services. A cancer registry would be a most useful tool.