Liver Fluke Opisthorchis Viverrini Research Articles

Suppression of mRNAs encoding CD63 family tetraspanins from the carcinogenic liver fluke Opisthorchis viverrini results in distinct tegument phenotypes

The liver fluke Opisthorchis viverrini infects 10 million people in Southeast Asia and causes cholangiocarcinoma (CCA). Fluke secreted and tegumental proteins contribute to the generation of a tumorigenic environment and are targets for drug and vaccine-based control measures. Herein, we identified two tetraspanins belonging to the CD63 family (Ov-TSP-2 and Ov-TSP-3) that are abundantly expressed in the tegument proteome of O. viverrini. Ov-tsp-2 and tsp-3 transcripts were detected in all developmental stages of O. viverrini. Protein fragments corresponding to the large extracellular loop (LEL) of each TSP were produced in recombinant form and antibodies were raised in rabbits. Ov-TSP-2 and TSP-3 were detected in whole worm extracts and excretory/secretory products of O. viverrini and reacted with sera from infected hamsters and humans. Antibodies confirmed localization of Ov-TSP-2 and TSP-3 to the adult fluke tegument. Using RNA interference, Ov-tsp-2 and tsp-3 mRNA expression was significantly suppressed for up to 21 days in vitro. Ultrastructural observation of tsp-2 and tsp-3 dsRNA-treated flukes resulted in phenotypes with increased tegument thickness, increased vacuolation (tsp-2) and reduced electron density (tsp-3). These studies confirm the importance of CD63 family tegument tetraspanins in parasitic flukes and support efforts to target these proteins for vaccine development.

Development of a Potent Wound Healing Agent Based on the Liver Fluke Granulin Structural Fold.

Granulins are a family of protein growth factors that are involved in cell proliferation. An orthologue of granulin from the human parasitic liver fluke Opisthorchis viverrini, known as Ov-GRN-1, induces angiogenesis and accelerates wound repair. Recombinant Ov-GRN-1 production is complex and poses an obstacle for clinical development. To identify the bioactive region(s) of Ov-GRN-1, four truncated N-terminal analogues were synthesized and characterized structurally using NMR spectroscopy. Peptides that contained only two native disulfide bonds lack the characteristic granulin β-hairpin structure. Remarkably, the introduction of a non-native disulfide bond was critical for formation of β-hairpin structure. Despite this structural difference, both two and three disulfide-bonded peptides drove proliferation of a human cholangiocyte cell line and demonstrated potent wound healing in mice. Peptides derived from Ov-GRN-1 are leads for novel wound healing therapeutics, as they are likely less immunogenic than the full-length protein and more convenient to produce.

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.

Efficacy and safety of praziquantel against light infections of Opisthorchis viverrini : a randomised parallel single blind dose-ranging trial

The liver fluke Opisthorchis viverrini, highly prevalent in Southeast Asia, is an important public health burden, including a risk factor for developing an aggressive bile duct cancer, cholangiocarcinoma, in chronically infected patients. Praziquantel, administered at a single 40 mg/kg dose in preventive chemotherapy programs and 3 × 25 mg/kg for individual treatment, is the drug of choice, yet information on the nature of the dose-response relationship is lacking. We performed a randomized, parallel, single-blind dose-ranging phase 2 trial in the Lao People’s Democratic Republic in O. viverrini–infected adults. Patients were randomly assigned to 30 mg/kg, 40 mg/kg, 50 mg/kg, or 3 × 25 mg/kg praziquantel or placebo. Adverse events were recorded at baseline, 3 hours, and 24 hours posttreatment. Cure rates (CRs) and egg reduction rates (ERRs) were estimated 3 weeks after drug administration using available case analysis. Dose-response curves were predicted using Emax models. Two-hundred seventeen O. viverrini–infected patients were assigned to the 5 treatment arms. The majority (94.3%) of patients harbored light infections. The Emax model predicted a high efficacy among the observed dose range. We observed CRs ranging from 92.7% to 95.5% and ERRs >99.5% for all praziquantel treatment groups. Adverse events were mild but higher in the standard treatment group (3 × 25 mg/kg) than in the single-dose treatment arms. Single-dose praziquantel appears to be as efficacious as the standard 3 × 25 mg/kg regimen for the treatment of O. viverrini infections, while presenting fewer adverse events. Further studies are necessary in moderate and heavy O. viverrini infections. Randomized Controlled Trials (ISRCTN77186750).

Differential Protein Expression in the Hemolymph of Bithynia siamensis goniomphalos Infected with Opisthorchis viverrini.

Bithynia siamensis goniomphalos is a freshwater snail that serves as the first intermediate host of the human liver fluke Opisthorchis viverrini. This parasite is a major public health problem in different countries throughout the Greater Mekong sub-region (Thailand, southern Vietnam, Lao PDR and Cambodia). Chronic O. viverrini infection also results in a gradual increase of fibrotic tissues in the biliary tract that are associated with hepatobiliary diseases and contribute to cholangiocarcinoma (a fatal type of bile duct cancer). Infectivity of the parasite in the snail host is strongly correlated with destruction of helminths by the snail’s innate immune system, composed of cellular (hemocyte) and humoral (plasma) defense factors. To better understand this important host-parasite interface we applied sequential window acquisition of all theoretical spectra mass spectrometry (SWATH-MS) to identify and quantify the proteins from the hemolymph of B. siamensis goniomphalos experimentally infected with O. viverrini and compare them to non-infected snails (control group). A total of 362 and 242 proteins were identified in the hemocytes and plasma, respectively. Of these, 145 and 117 proteins exhibited significant differences in expression upon fluke infection in hemocytes and plasma, respectively. Among the proteins with significantly different expression patterns, we found proteins related to immune response (up-regulated in both hemocyte and plasma of infected snails) and proteins belonging to the structural and motor group (mostly down-regulated in hemocytes but up-regulated in plasma of infected snails). The proteins identified and quantified in this work will provide important information for the understanding of the factors involved in snail defense against O. viverrini and might facilitate the development of new strategies to control O. viverrini infection in endemic areas.

The Lawa model: A sustainable, integrated opisthorchiasis control program using the EcoHealth approach in the Lawa Lake region of Thailand

Opisthorchiasis caused by the carcinogenic liver fluke Opisthorchis viverrini is a major foodborne parasitic zoonotic disease in Thailand and neighboring Mekong countries. The infection is associated with several hepatobiliary diseases including cholangiocarcinoma (CCA). The rates of CCA in regions where the parasite is endemic are unprecedented. Extensive research on various aspects of opisthorchiasis has been conducted in recent decades. However, the current status of O. viverrini infection in the country is approaching 85% prevalence in certain endemic areas even after more than 30years of control programs in Thailand. The complex life cycle of the fluke, which involves several hosts/environments, makes it difficult to control by conventional methods. Therefore, a new control strategy using the EcoHealth/One Health approach named the “Lawa model” was introduced into the liver fluke endemic Lawa Lake region in Khon Kaen Province. This program has been underway for over ten years. The program includes treatment with anthelmintic drugs, novel intensive health education methods both in the communities and in schools, ecosystem monitoring and active community participation. We developed the “Liver fluke-free school program” as a part of the Lawa model with successful results. All key stakeholders were empowered to obtain competency in their control activities for the sustainability of the program in the community. Nowadays, the infection rate in the 12 villages surrounding the lake has declined to less than 10% from an average of 60% at the start. The Cyprinid fish species now show less than 1% prevalence of infection compared with a maximum of 70% during the baseline survey. No infected Bithynia snails in the lake were found during recent field studies. Of the lessons learned from the Lawa model, the importance of community participation is one practical and essential component. The key to the success of the project is multi-stakeholder participation with the active local Health Promoting Hospitals and the village health volunteers. The idea of the Lawa model is on the national agenda against liver fluke infection and CCA and is being scaled up to work in larger areas in Thailand. Internationally, the “Lawa model” is one of two programs that are showcased as successful control programs for helminths by the WHO Neglected Zoonotic Diseases (NZD4). Several media outlets have broadcast news reports about the program.

Identification and characterization of protein 14-3-3 in carcinogenic liver fluke Opisthorchis viverrini

Protein 14-3-3s are abundant phospho-serine/threonine binding proteins, which are highly conserved among eukaryotes. Members of this protein family mediate metabolism and signal transduction networks through binding to hundreds of other protein partners. Protein 14-3-3s have been studied in other species of parasitic helminthes, but little is known about this protein in the carcinogenic liver fluke Opisthorchis viverrini. In this study, we identified and characterized protein 14-3-3s of O. viverrini. Seven protein 14-3-3 encoded sequences were retrieved from the O. viverrini genome database. Multiple alignment and phylogenetic analysis were performed. Two isoforms (protein 14-3-3 zeta and protein 14-3-3 epsilon) that have been previously found in the excretory-secretory (ES) products of O. viverrini were produced as recombinant protein in E. coli and the proteins were then used to immunize mice to obtain specific antibodies. Western blot analysis showed that both proteins were detected in all obtainable developmental stages of O. viverrini and the ES products. Immunolocalization revealed that both isoforms were expressed throughout tissues and organs except the gut epithelium. The highest expression was observed in testes especially in developing spermatocytes, suggesting their role in spermatogenesis. Prominent expression was also detected on tegumental surface of the parasite and on epical surface of bile duct epithelium indicates their additional role in host-parasite interaction. These findings indicate that protein 14-3-3s play important role in the life cycle of the parasite and might be involved in the pathogenesis of O. viverrini infection.

Changing patterns of prevalence in Opisthorchis viverrini sensu lato infection in children and adolescents in northeast Thailand

Infection with the liver fluke Opisthorchis viverrini sensu lato (s.l.), a group 1 carcinogen, is the most important risk factor for developing cholangiocarcinoma (CCA) in Southeast Asia. Cholangiocarcinoma is a fatal disease with the world’s highest incidence being found in northeast Thailand. Liver fluke infection occurs through eating raw or partially cooked cyprinid fish containing metacercariae and, therefore, the control of O. viverrini s.l. infection should lead to a reduction in CCA incidence. In this report, we review and analyze the age-prevalence profile data of O. viverrini to reveal temporal changes in patterns of prevalence pre- and post-control programs in Thailand. The profiles of O. viverrini prevalence have transformed from high prevalence in school children prior to 1983 to low prevalences after 1994. This pattern strongly suggests the influence of the health education program on the likelihood of school children becoming infected. In conjunction with current developments in health and socioeconomic conditions, we predict that the incidence of CCA will be reduced with time as the population cohorts that experienced the education programs reach the age at which CCA is most likely to develop, i.e. >50 years. The lessons learned in Thailand may be applicable to other areas endemic for human liver flukes.

Cytokine profiles in Opisthorchis viverrini stimulated peripheral blood mononuclear cells from cholangiocarcinoma patients

The carcinogenic liver fluke Opisthorchis viverrini causes chronic inflammation in the bile duct and resulting in unremitting tissue damage that lead to hepatobiliary diseases, including cholangiocarcinoma (CCA). Despite inflammatory cytokine expression having been studied in the animal model, so far no studies have been carried out on cytokines in human CCA cases. Here we report the profile of cytokine production in peripheral blood mononuclear cells (PBMCs) collected from O. viverrini-associated human CCA and uninfected normal controls after stimulation with O. viverrini-excretory secretory (ES) product. Eleven cytokine profiles including IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-α and LT-α measured by flow cytometry revealed both pro-inflammatory and anti-inflammatory cytokines were increased in the O. viverrini-associated CCA compared to uninfected normal controls. Specifically, net production levels of IFN-γ, IL-10, and LT-α were 40 to >320 times higher in CCA than those of controls. These results suggest dysregulation of the immune response in the liver fluke associated CCA.

Liver Flukes and the Microbiota in Cancer

Liver Flukes and the Microbiota in Cancer

Pharmacokinetic Study of Praziquantel Enantiomers and Its Main Metabolite R-trans-4-OH-PZQ in Plasma, Blood and Dried Blood Spots in Opisthorchis viverrini-Infected Patients.

BackgroundPraziquantel (PZQ) is the treatment of choice for infections with the liver fluke Opisthorchis viverrini, a major health problem in Southeast Asia. However, pharmacokinetic (PK) studies investigating the disposition of PZQ enantiomers (R- and S-PZQ) and its main metabolite, R-trans-4-OH-PZQ, in diseased patients are lacking. The implementation of a dried blood spot (DBS) sampling technique would ease the performance of PK studies in remote areas without clinical facilities. The aim of the present study is to provide data on the disposition of PZQ enantiomers and R-trans-4-OH-PZQ in opisthorchiasis patients and to validate the use of DBS compared to plasma and blood sampling.Methodology/Principal FindingsPZQ was administered to nine O. viverrini-infected patients at 3 oral doses of 25 mg/kg in 4 h intervals. Plasma, blood and DBS were simultaneously collected at selected time points from 0 to 24 h post-treatment. PK parameters were determined using non-compartmental analysis. Drug concentrations and areas under the curve (AUC0–24h) measured in the 3 matrices were compared using Bland-Altman analysis. We observed plasma AUC0–24hs of 1.1, 9.0 and 188.7 μg/ml*h and half-lives of 1.1, 3.3 and 6.4 h for R-PZQ, S-PZQ and R-trans-4-OH, respectively. Maximal plasma concentrations (Cmax) of 0.2, 0.9 and 13.9 μg/ml for R-PZQ, S-PQZ and R-trans-4-OH peaked at 7 h for PZQ enantiomers and at 8.7 h for the metabolite. Individual drug concentration measurements and patient AUC0–24hs displayed ratios of blood or DBS versus plasma between 79–94% for R- and S-PZQ, and between 108–122% for R-trans-4-OH.Conclusions/SignificancePharmacodynamic (PD) in vitro studies on PZQ enantiomers and R-trans-4-OH-PZQ are necessary to be able to correlate PK parameters with efficacy. DBS appears to be a valid alternative to conventional venous sampling for PK studies in PZQ-treated patients.

Tissue Microbiome Profiling Identifies an Enrichment of Specific Enteric Bacteria in Opisthorchis viverrini Associated Cholangiocarcinoma

Cholangiocarcinoma (CCA) is the primary cancer of the bile duct system. The role of bile duct tissue microbiomes in CCA tumorigenesis is unestablished. To address this, sixty primary CCA tumors and matched normals, from both liver fluke (Opisthorchis viverrini) associated (OVa, n=28) and non-O. viverrini associated (non-OVa, n=32) cancers, were profiled using high-throughput 16S rRNA sequencing. A distinct, tissue-specific microbiome dominated by the bacterial families Dietziaceae, Pseudomonadaceae and Oxalobacteraceae was observed in bile duct tissues. Systemic perturbation of the microbiome was noted in tumor and paired normal samples (vs non-cancer normals) for several bacterial families with a significant increase in Stenotrophomonas species distinguishing tumors vs paired normals. Comparison of parasite associated (OVa) vs non-associated (non-OVa) groups identified enrichment for specific enteric bacteria (Bifidobacteriaceae, Enterobacteriaceae and Enterococcaceae). One of the enriched families, Bifidobacteriaceae, was found to be dominant in the O. viverrini microbiome, providing a mechanistic link to the parasite. Functional analysis and comparison of CCA microbiomes revealed higher potential for producing bile acids and ammonia in OVa tissues, linking the altered microbiota to carcinogenesis. These results define how the unique microbial communities resident in the bile duct, parasitic infections and the tissue microenvironment can influence each other, and contribute to cancer.

Similarity of a 16.5 kDa tegumental protein of the human liver fluke Opisthorchis viverrini to nematode cytoplasmic motility protein

Opisthorchis viverrini is the causative agent of human opisthorchiasis in Thailand and long lasting infection with the parasite has been correlated with the development of cholangiocarcinoma. In this work we have molecularly characterized the first member of a protein family carrying two DM9 repeats in this parasite (OvDM9-1). InterPro and other protein family databases describe the DM9 repeat as a protein domain of unknown function that has been first noted in Drosophila melanogaster. Two paralogous proteins have been partially characterized in the genus Fasciola, Fasciola hepatica TP16.5, a novel tegumental antigen in human fascioliasis and, recently F. gigantica DM9-1, a parenchymal protein with structural similarity to nematode cytoplasmic motility protein (MFP2). In this study, we show further evidence that this family of trematode proteins is related to MFP2 in sequence and structure. Soluble recombinant OvDM9-1 was used for structural analyses and for production of specific antisera. The native protein was detected in soluble and insoluble crude worm extracts and in seemingly various oligomeric forms in the latter. The potential for oligomerization was supported by cross-linking experiments of recombinant OvDM9-1. Structure prediction suggested a β-rich secondary structure of the protein and this was supported by a circular dichroism analysis. Molecular modeling in Phyre2 identified both MFP2 domains as distant homologs of OvDM9-1. The protein was located in tegumental type tissue and the cecal epithelium in the mature parasite. Recombinant OvDM9-1 was used as target in indirect ELISA but sera from infected hamsters showed only marginal reactivity towards it. It is proposed that OvDM9-1 and other members of this protein family have a role in cellular transport through functions on the cytoskeleton.

Pipeline for the identification and classification of ion channels in parasitic flatworms.

BackgroundIon channels are well characterised in model organisms, principally because of the availability of functional genomic tools and datasets for these species. This contrasts the situation, for example, for parasites of humans and animals, whose genomic and biological uniqueness means that many genes and their products cannot be annotated. As ion channels are recognised as important drug targets in mammals, the accurate identification and classification of parasite channels could provide major prospects for defining unique targets for designing novel and specific anti-parasite therapies. Here, we established a reliable bioinformatic pipeline for the identification and classification of ion channels encoded in the genome of the cancer-causing liver fluke Opisthorchis viverrini, and extended its application to related flatworms affecting humans.MethodsWe built an ion channel identification + classification pipeline (called MuSICC), employing an optimised support vector machine (SVM) model and using the Kyoto Encyclopaedia of Genes and Genomes (KEGG) classification system. Ion channel proteins were first identified and grouped according to amino acid sequence similarity to classified ion channels and the presence and number of ion channel-like conserved and transmembrane domains. Predicted ion channels were then classified to sub-family using a SVM model, trained using ion channel features.ResultsFollowing an evaluation of this pipeline (MuSICC), which demonstrated a classification sensitivity of 95.2 % and accuracy of 70.5 % for known ion channels, we applied it to effectively identify and classify ion channels in selected parasitic flatworms.ConclusionsMuSICC provides a practical and effective tool for the identification and classification of ion channels of parasitic flatworms, and should be applicable to a broad range of organisms that are evolutionarily distant from taxa whose ion channels are functionally characterised.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1428-2) contains supplementary material, which is available to authorized users.

Xanthohumol inhibits STAT3 activation pathway leading to growth suppression and apoptosis induction in human cholangiocarcinoma cells.

STAT3 plays a significant role in the development of cholangiocarcinoma (CCA) associated with the liver fluke (Opisthorchis viverrini; Ov). Xanthohumol (XN), a prenylated flavonoid extracted from hops, has known anticancer activity and could potentially target STAT3. The present study determined the effect of XN on STAT3, as well as ascertained its usefulness against CCA. The CCA cell proliferation at 20 µM and 50 µM of XN was shown to inhibited, while 20 µM partially inhibited IL-6-induced STAT3 activation. At 50 µM, the inhibition was complete. The reduction in STAT3 activity at 20 and 50 µM was associated with a significant reduction of CCA cell growth and apoptosis. We also found that the administration of 50 µM XN orally in drinking water to nude mice inoculated with CCA led to a reduction in tumor growth in comparison with controls. In addition, apoptosis of cancer cells increased although there was no visible toxicity. The present study shows that XN can inhibit STAT3 activation both in vivo and in vitro due to suppression of the Akt-NFκB signaling pathway. XN should be considered as a possible therapeutic agent against CCA.

Development and validation of an enantioselective LC–MS/MS method for the analysis of the anthelmintic drug praziquantel and its main metabolite in human plasma, blood and dried blood spots

Praziquantel (PZQ) is the treatment of choice against various trematode and cestode infections. To study the pharmacokinetics of PZQ in patients infected with the liver fluke Opisthorchis viverrini, we developed and validated an enantioselective liquid chromatography coupled to tandem mass spectrometry method for the analysis of R - and S -PZQ and its R -trans-4-OH-PZQ metabolite in human plasma, blood and dried blood spots (DBS). The analytes were detected in the positive mode using selected reaction monitoring (R- and S-PZQ: m/z 312.2→202.2; R-trans -4-OH-PZQ: m/z 328.0→202.0). Prior to the chiral separation with a cellulose tris(3-chloro-4-methylphenylcarbamate) column, the analytes were purified from matrix contaminants and concentrated on a C-18 trapping column. The analytical range for each PZQ enantiomer was 0.01–2.5μg/mL, and 0.1–25μg/mL for the metabolite. The method met the requirements regarding precision (±15%, ±20% at the lower limit of quantification-LLOQ), intra- and inter-assay accuracy (85–115%, 80–120% at LLOQ), and linearity (R2≥0.998). The analytes were stable in stock solutions as well as in plasma, blood and DBS. For DBS, the influences of hematocrit and blood spot size were considered as minor. Our validation results show that the method presented here is precise, accurate and selective, and can be used for pharmacokinetic studies. Moreover, the enantioselective separation was achieved with a run time of 11.5min and a simple sample processing method.

Carcinogenic Parasite Secretes Growth Factor That Accelerates Wound Healing and Potentially Promotes Neoplasia.

Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world.

Plasma IgG autoantibody against actin-related protein 3 in liver fluke Opisthorchis viverrini infection.

Opisthorchiasis secondary to Opisthorchis viverrini infection leads to cholangiocellular carcinoma through chronic inflammation of the bile ducts and possibly inducing autoimmunity. It was hypothesized that plasma autoantibodies directed against self-proteins are biomarkers for opisthorchiasis. Plasma from patients with opisthorchiasis was tested using proteins derived from immortalized cholangiocyte cell lines, and spots reacting with plasma were excised and subjected to LC-MS/MS. Seven protein spots were recognized by IgG autoantibodies, and the highest matching scored protein was actin-related protein 3 (ARP3). The antibody against ARP3 was tested in plasma from 55 O. viverrini-infected patients, 24 patients with others endemic parasitic infections and 17 healthy controls using Western blot and ELISA. Immunoreactivity against recombinant ARP3 was significantly more prevalent in opisthorchiasis compared to healthy controls at Western blotting and ELISA (P < 0.05). Plasma ARP3 autoantibody titres were also higher in opisthorchiasis compared to healthy individuals (P < 0.01) and other parasitic infections including Strongyloides stercoralis (P < 0.001), echinostome (P < 0.05), hookworms (P < 0.001) and Taenia spp. (P < 0.05). It was further characterized in that the ARP3 autoantibody titre had a sensitivity of 78.18% and specificity of 100% for opisthorchiasis. In conclusion, it may be suggested that plasma anti-ARP3 might represent a new diagnostic antibody for opisthorchiasis.

Carcinogenic Liver Fluke Secretes Extracellular Vesicles That Promote Cholangiocytes to Adopt a Tumorigenic Phenotype.

Background. Throughout Asia, there is an unprecedented link between cholangiocarcinoma and infection with the liver fluke Opisthorchis viverrini. Multiple processes, including chronic inflammation and secretion of parasite proteins into the biliary epithelium, drive infection toward cancer. Until now, the mechanism and effects of parasite protein entry into cholangiocytes was unknown.Methods. Various microscopy techniques were used to identify O. viverrini extracellular vesicles (EVs) and their internalization by human cholangiocytes. Using mass spectrometry we characterized the EV proteome and associated changes in cholangiocytes after EV uptake, and we detected EV proteins in bile of infected hamsters and humans. Cholangiocyte proliferation and interleukin 6 (IL-6) secretion was measured to assess the impact of EV internalization.Results. EVs were identified in fluke culture medium and bile specimens from infected hosts. EVs internalized by cholangiocytes drove cell proliferation and IL-6 secretion and induced changes in protein expression associated with endocytosis, wound repair, and cancer. Antibodies to an O. viverrini tetraspanin blocked EV uptake and IL-6 secretion by cholangiocytes.Conclusions. This is the first time that EVs from a multicellular pathogen have been identified in host tissues. Our findings imply a role for O. viverrini EVs in pathogenesis and highlight an approach to vaccine development for this infectious cancer.

Trematode diversity in the freshwater snail Bithynia siamensis goniomphalos sensu lato from Thailand and Lao PDR.

In order to obtain a comprehensive understanding of trematode diversity in Bithynia siamensis goniomphalos sensu lato, the first intermediate host of the liver fluke Opisthorchis viverrini s.l., the prevalence of larval trematode species was investigated in different localities in Thailand and Lao People's Democratic Republic (Lao PDR). In Thailand, snail samples were collected from 29 localities in the nine provinces: Buri Ram, Surin, Chaiya Phum, Maha Sarakham, Khon Kaen, Kalasin, Mukdahan, Sakon Nakhon and Nakhon Phanom. In Lao PDR, snail samples were collected from 21 localities in Vientiane Province and six localities in Savannakhet Province. Snails were identified by standard morphological criteria and then examined for trematode infection using the cercarial shedding method. Twenty different types of cercariae were detected and identified, based on morphological criteria. Virgulate type 1 emerged as the most common cercaria, with an average prevalence of 10.90% (range 0.26-54.22%) in Thailand and 6.58% (range 1.15-89.77%) in Lao PDR. Opisthorchis viverrini s.l. cercariae were the fourth most common in Thailand, with an average prevalence of 1.59% (0.15-6.93), while in Lao PDR their prevalence was 0.96% (0.08-8.37). The high diversity of trematode cercariae observed in this study indicates that B. s. goniomphalos s.l. is highly susceptible to infection with a variety of trematode species. However, the role of non-opisthorchiid trematodes as fish-borne parasites in human health is not fully known and further molecular identification is required.