Viverrini Infection Research Articles

Perspectives in the vaccine development against Opisthorchis viverrini liver fluke

AbstractLiver flukes, Opisthorchis viverrini, infect around 10 million individuals in Southeast Asia, alone, and cause 26,000 deaths in the region per year. Despite being classified as a Group 1 carcinogen, and presenting one of the leading causes of cholangiocarcinoma in epidemic areas, O. viverrini infection is a neglected tropical disease. Control measures were implemented in epidemic areas to limit the outspread of infection; however, prophylactic vaccines are urgently needed to protect against future reinfections. This holds especially true due to the limited curative efficacy of the approved anthelmintic drug. In this article, we have briefly summarized the recently reported information regarding hepatobiliary cancer pathogenesis, approved treatment, and control measures against infection. Further, we highlighted the progress in the identification of protective antigens against Opisthorchiasis and proposed the investigation of additional promising antigens relying on vaccine progress against related infectious parasites. We highlighted the relative efficacies of the developed preclinical vaccines, suggested alternative vaccine designs and combinations, and commented on the required immunological responses. Moreover, we also reviewed biomaterials used in vaccine delivery against O. viverrini infections, summarized all the reported vaccine design approaches against the disease, and provided future perspectives regarding vaccine development, the utilization of biomaterials, and the discovery of highly protective antigens.

Effectiveness of public health interventions in reducing the prevalence of Opisthorchis viverrini: a protocol for systematic review and network meta-analysis

IntroductionThe carcinogenic liver fluke Opisthorchis viverrini is a major public health problem in the Mekong basin region. The liver flukes can induce cholangiocarcinoma, a bile duct cancer that causes a...

Performance of Mini Parasep® SF stool concentrator kit, Kato-Katz, and formalin-ethyl acetate concentration methods for diagnosis of opisthorchiasis in Northeast Thailand

BackgroundControl and elimination of the liver fluke (Opisthorchis viverrini) is a primary preventive strategy against cholangiocarcinoma in Southeast Asia. A sensitive parasitological diagnostic method is required to facilitate a surveillance and control program. In this study, we evaluated the performance of Mini Parasep® SF stool concentrator kit (stool kit) compared with Kato-Katz (KK) and the quantitative formalin-ethyl acetate concentration technique (FECT) for detection of O. viverrini and co-endemic parasitic infections.MethodsA cross-sectional survey for parasitic infection in residents aged > 15 years in a community in Kalasin province, Northeast Thailand, was conducted in 2018. Fecal samples were collected and screened by KK method, and a subset of samples was further examined by the stool kit and FECT methods. The results were analyzed for prevalence of parasitic infections in addition to the diagnostic performance of the methods for qualitative and quantitative detection of helminthiases.ResultsThe initial survey of parasitic infection determined by the KK method (n = 567) showed the prevalence of O. viverrini was 32.63%, followed by Taenia 2.65%, echinostomes 1.76%, hookworms 1.41%, Trichuris trichiura 0.53% and Strongyloides stercoralis 0.53%. Within a subset of samples tested with multiple diagnostics (n = 150), the detection rates of O. viverrini by the stool kit, FECT and KK methods were 27.3%, 30.7% and 28.7%, respectively. The diagnostic sensitivity for opisthorchiasis was similar for FECT (75.5%), KK(66.0%) and the stool kit (67.3%). For other parasitic infections, FECT and stool kit methods performed better than KK, particularly in detecting minute intestinal flukes (MIF), S. stercoralis and coinfections. When measuring the intensity of O. viverrini infection (fecal egg counts), the stool kit results showed a significant positive correlation with KK and FECT (P < 0.05).ConclusionsAs the stool kit is simple to use and shows a comparable performance to FECT, it may serve as an alternative method of fecal examination for screening of helminthiasis including opisthorchiasis.Graphical abstract

Efficacy of drugs against clonorchiasis and opisthorchiasis: a systematic review and network meta-analysis

Clonorchis sinensis, Opisthorchis viverrini, and Opisthorchis felineus are the three most important human liver fluke species in the Opisthorchiidae family, infecting approximately 25 million people worldwide. Drug treatment is needed to control morbidity and is also useful in lowering transmission. Several drugs used in various regimens are available to treat these infections, but their comparative efficacy is uncertain. We aimed to compare the efficacy in terms of cure rate and egg reduction rate of currently registered drugs against human liver fluke infection. We conducted a systematic review using readily available electronic databases (MEDLINE, Embase, Cochrane Central Register of Controlled Trials, KoreaMed, China National Knowledge Infrastructure, and Wanfang Data) without language restrictions from inception until June 29, 2021. Clinical trials with pairwise comparison of drugs (praziquantel, albendazole, mebendazole, tribendimidine, or combinations of these drugs) against C sinensis, O viverrini, and O felineus were eligible, including trials comparing these drugs or their combinations with placebo. We compared efficacy in terms of cure rate by network meta-analysis. We conducted mixed binomial regression analyses for each species to derive predicted median cure rates for each drug regimen. The models included treatment and infection intensity as fixed factors, year of publication as covariate, and random effects of the different studies assumed to be normally distributed. We also assessed the quality of the included studies. This study was registered with PROSPERO (CRD42018109232). Overall, 26 trials from 25 studies were included, of which 18 involved C sinensis, seven studied O viverrini, and one focused on O felineus. These trials included a total of 3340 participants. The two long-term treatment courses against C sinensis infection using 400 mg of albendazole (400 mg twice a day for 5 days and 400 mg twice a day for 7 days) resulted in cure rates of 100%, while two other multiple-dose regimens of albendazole resulted in high predicted cure rates: 300 mg twice a day for 5 days (93·9% [95% CI 49·6-99·6]) and 400 mg twice a day for 3 days (91·0% [50·9-99·0]). The WHO-recommended praziquantel regimen (25 mg/kg three times a day for 2 days) also showed a high predicted cure rate (98·5% [85·4-99·9]) in C sinensis infection, and predicted cure rates were above 90% for several other multiple-dose praziquantel regimens, including 20 mg/kg three times a day for 3 days (97·6% [74·7-99·8]), 14 mg/kg three times a day for 5 days (93·9% [44·8-99·7]), and 20 mg/kg twice a day for 3 days (91·0% [50·9-99·0]). In O viverrini infection, the regimen of 50 mg/kg and 25 mg/kg of praziquantel given in a single day showed the highest predicted cure rate (93·8% [85·7-97·5]), while a single dose of 50 mg/kg praziquantel also resulted in a high predicted cure rate (92·1% [64·9-98·6]). The single dose of 400 mg tribendimidine showed a high predicted cure rate of 89·8% (77·5-95·8). A low quality of evidence was demonstrated in most studies, especially those published before 2000. Selection bias due to poor random sequence generation and allocation concealment was high, and performance and detection biases were frequently unreported. Praziquantel shows high efficacy against clonorchiasis and opisthorchiasis. Tribendimidine might serve as a treatment alternative and warrants further investigation. Although albendazole is efficacious when long treatment schedules (5 days or 7 days) are applied, limited size of studies and high risk of bias affect the interpretation of results. More high-quality studies are needed to promote the establishment of treatment guidelines for human liver fluke infection. Fourth Round of Three-Year Public Health Action Plan (2015-2017; Shanghai, China) and Swiss National Science Foundation.

Opisthorchis viverrini infection induces metabolic disturbances in hamsters fed with high fat/high fructose diets: Implications for liver and kidney pathologies

A combination of Opisthorchis viverrini infection and high fat/high fructose diets (HFa/HFr) intake is likely to enhance fatty liver and kidney pathologies. Here we investigated the combined effects of chronic O. viverrini infection and HFa/HFr intake on liver and kidney pathologies, metabolism, and gut microbiome in hamsters. Animals were infected with O. viverrini and fed with either standard chow (OV group) or HFa/HFr diet (OH group) and non–infected hamsters were fed with either standard chow (NC) or HFa/HFr diet (HF) for 8 months. The OH group exhibited dyslipidemia and the highest severity of fatty liver. Tubular damage, inflammatory cell infiltration, and tubular fibrosis were the most prominently observed in this group, supported by increased expression of KIM-1, HMGB-1, and MCP-1. Urinary 1H NMR metabolic profiles revealed that tauro-β-muricholic acid level was increased in the OV and OH groups, whereas metabolites involved in the TCA cycle and gut microbiota-associated metabolites (phenylacetylglycine, trimethylamine, and trimethylamine-N-oxide) were lower in OV, HF and OH groups compared to the NC group. Gut microbial profiles of the OH group were also different from other groups. In conclusion, O. viverrini infection and HFa/HFr diet-induced disturbance of metabolites and gut microbiota associated with concurrent liver and kidney pathologies in hamsters.

Association between Opisthorchis viverrini Infection and Glomerular Disease in Thailand

Introduction: Opisthorchis viverrini (OV) is a major cause of infection in Southeast Asia. Previous studies in mouse models have shown that OV infection can contribute to immune-complex glomerulonephritis (GN). However, OV infection in human kidney tissue has never been demonstrated. Herein, we evaluated the association of OV infection with biopsy-proven glomerular disease. Methods: This study was performed in adult patients who underwent kidney biopsy between July 2016 and February 2017. All kidney tissue samples were processed using the standard techniques for renal pathological diagnoses and immunohistochemistry techniques to detect OV antigen. Pre-implanted donor kidney tissue samples were used as controls. The participants were also assessed for OV infection by serum OV immunoglobulin G antibody (Ab) levels and/or presence of OV eggs in stool. Results: Forty-three renal tissue samples from glomerular disease patients and 50 from transplant donors were included in the study. Mean age in the GN group was 41.7 ± 15.9 years, estimated glomerular filtration rate (eGFR) was 70.65 ± 36.61 mL/min/1.73 m², and median proteinuria was 3.17 (1.70–4.95) g/day. Lupus nephritis (LN) was the most common diagnosis (32.6%), followed by IgA nephropathy (23.3%), IgM nephropathy (18.6%), and primary membranous nephropathy (MN; 7%). The OV antigen was observed in kidney tissue from patients with IgA nephropathy, LN, primary MN, focal segmental glomerulosclerosis, and IgM nephropathy. By contrast, no OV antigen was detected in tissue samples from the control group. The presence of OV antigens was observed in glomerular endothelial cells, mesangial cells, tubular cells, and peritubular capillaries. The odds ratio of positive serum OV Ab to predict the presence of OV antigen in kidney tissues was 4.47 (p = 0.057), and there was a negative correlation between levels of serum OV Ab and eGFR (r = −0.31, p = 0.04). Discussion/Conclusion: This is the first study to demonstrate the presence of OV antigen in human kidney tissue, which indicates that OV infection may be associated with biopsy-proven glomerular diseases.

Efficiency of the Stool-PCR Test Targeting NADH Dehydrogenase (Nad) Subunits for Detection of Opisthorchis viverrini Eggs.

Opisthorchis viverrini infection is the major parasitic infection problem in Southeast Asian countries, and long-term infection will lead to cholangiocarcinoma (CCA), the bile duct cancer. The early diagnosis of O. viverrini infection may interrupt the progression of the opisthorchiasis and other related illnesses, especially CCA. The current diagnostic procedure is stool examination by microscope-based methods such as direct smear and concentration techniques but it is limited by low parasite egg numbers. The molecular diagnosis prompts the chance to evaluate the light infection with low number of parasite eggs but is currently inconvenient for routine use due to special equipment requirement and unstable sensitivities. Our present study aims to establish the efficiency of OvNad subunits, the mitochondrial gene, for introducing as a potential diagnostic target by conventional PCR, the cheapest and easiest molecular procedure. A total of 166 stool samples were investigated microscopically by the PBS-ethyl acetate concentration technique (PECT); 75 samples were O. viverrini positive with 28 samples that were positive with single parasite (hookworm, A. lumbricoides, S. stercoralis, Taenia spp., and T. trichiura), 11 samples were with mixed infection, and 52 samples were without parasite detection. The detection limits of OvNad subunits were evaluated in artificially spiked samples containing 0, 1, 5, 10, 20, 50, and 100 Ov-eggs. The result suggested that the best detection efficacy was of OvNad5 that had exact detection limits at only 5 eggs. In the PCR amplification of OvNad subunits, there exist 100% specificities with varied sensitivities from 64%, 88%, 80%, and 100% of OvNad1, OvNad2, OvNad4, and OvNad5, respectively. OvNad subunits were amplified specifically without cross reactivity with the other collected parasites. Our study established that OvNad subunits, especially OvNad5, are the potent candidates for PCR amplification of stool containing Ov-eggs with high confidential sensitivity, specificity, PPV, and NPV even in the light infection that would be a benefit for developing as a routine diagnosis of O. viverrini infection.

Acetaminophen Overdose Enhances Early Cholangiocarcinoma in Opisthorchiasis Hamsters.

Opisthorchiasis which exerted by infection of Opisthorchis viverrini is strongly related to the incident of cholangiocarcinoma (CCA) in many Southeast Asian countries northeastern of Thailand. The O. viverrini infection is primarily caused by raw fish consumption, and repeated exposure to liver fluke. Meanwhile, acetaminophen is usually medicated to relieve pain in particularly people in northeast Thailand. Objective:This study therefore aimed at investigating effects of acetaminophen on pathogenesis in hamsters for opisthorchiasis. Methods:There were 4 groups of hamsters: i) uninfected hamster (N); ii) sole acetaminophen administration (N-Ac); iii) sole O. viverrini infection (OV); and iv) combination of O. viverrini infection and acetaminophen (OV-Ac) on pathology of hamsters for 1 month post infection. For analysis of histopathological changes through hematoxylin and eosin, Sirius red and immunohistostaining for Cytokeratin 19 (CK-19), Proliferating cell nuclear antigen (PCNA) and CA 19-9, serum’s hamsters were used detected for liver function tests and tumor-related genes expression. Results:After 1 month under these treatments, the OV-Ac showed significantly higher CCA risk, including inflammatory cells were aggregations around bile duct, new bile duct and fibrosis in subcapsular hepatic tissues, than other treatments. These pathological parameters were positively correlated with immunohistochemical staining derived from CK-19, PCNA and CA 19-9. In addition, OV-Ac had significantly higher liver function tests (ALT). Conclusion:Combined intake of liver fluke-contaminated raw fishes and acetaminophen rendered more severity of CCA than sole consumption of the contaminated raw fishes.

Infection rate of Opisthorchis viverrini metacercariae in cyprinoid fish from the markets and its association to human opisthorchiasis in the local community in the Northeast Thailand

The prevalence of Opisthorchis viverrini, a fish-borne zoonotic trematode that can provoke cholangiocarcinoma, is high in the Northeast Thailand. The aim of this study was to determine the prevalence of O. viverrini metacercariae in the cyprinid fish and determine its association of O. viverrini infection among the consumers who regularly buy fish in the markets. A cross-sectional study was conducted in nine provinces covering 20 districts of Thailand, and we examined 778 cyprinoid fish specimens belonging to five species purchased from local markets. Pepsin-HCl digestion method was used to recover O. viverrini metacercariae from fish. In all districts surveyed, O. viverrini metacercariae-positive fish were found with the infection rates ranging from 3.9 to 21.1%. All five fish species studied were positive for O. viverrini metacercariae: Henicorhynchus siamensis (13.7%), Cyclocheilichtys spp. (12.7%), Hampala spp. (8.1%), Systomus spp. (6.9%) and Barbonymus goniatus (5.0%). An average prevalence of O. viverrini infection was 7.1% in the fish consumers surveyed in the markets. The source of fish was determined and our results showed that parasitized fish are sold in markets up to 100 km away from the point of capture, which contributes to the dispersion and maintenance of this helminthiasis. Our results point to the transmission of liver flukes via markets, in spite of many active programs of health education, elimination, prevention and control aimed to reduce O. viverrini infection and subsequent cholangiocarcinoma in the endemic areas of Thailand.

Detection of Human Cholangiocarcinoma Markers in Serum Using Infrared Spectroscopy.

Simple SummaryCholangiocarcinoma is a form of liver cancer that is found, predominantly, in Thailand. Due to the non-specific symptoms and laboratory investigation, it is difficult to rule out cholangiocarcinoma from other liver conditions. Here, we demonstrate the development of a diagnostic tool for cholangiocarcinoma, based on the ATR-FTIR analyses of sera, coupled with multivariate analyses and machine learning tools to obtain a better specificity. The innovative approach that shows highly promising results for this otherwise difficult to diagnose cancer.Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium. Opisthorchis viverrini infection is a known high-risk factor for CCA and in found, predominantly, in Northeast Thailand. The silent disease development and ineffective diagnosis have led to late-stage detection and reduction in the survival rate. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) is currently being explored as a diagnostic tool in medicine. In this study, we apply ATR-FTIR to discriminate CCA sera from hepatocellular carcinoma (HCC), biliary disease (BD) and healthy donors using a multivariate analysis. Spectral markers differing from healthy ones are observed in the collagen band at 1284, 1339 and 1035 cm−1, the phosphate band () at 1073 cm−1, the polysaccharides band at 1152 cm−1 and 1747 cm−1 of lipid ester carbonyl. A Principal Component Analysis (PCA) shows discrimination between CCA and healthy sera using the 1400–1000 cm−1 region and the combined 1800—1700 + 1400–1000 cm−1 region. Partial Least Square-Discriminant Analysis (PLS-DA) scores plots in four of five regions investigated, namely, the 1400–1000 cm−1, 1800–1000 cm−1, 3000–2800 + 1800–1000 cm−1 and 1800–1700 + 1400–1000 cm−1 regions, show discrimination between sera from CCA and healthy volunteers. It was not possible to separate CCA from HCC and BD by PCA and PLS-DA. CCA spectral modelling is established using the PLS-DA, Support Vector Machine (SVM), Random Forest (RF) and Neural Network (NN). The best model is the NN, which achieved a sensitivity of 80–100% and a specificity between 83 and 100% for CCA, depending on the spectral window used to model the spectra. This study demonstrates the potential of ATR-FTIR spectroscopy and spectral modelling as an additional tool to discriminate CCA from other conditions.

Prevalence and Intensity of Opisthorchis viverrini Metacercarial Infection in Fish from Phnom Penh, Takeo, and Kandal Provinces, Cambodia.

The prevalence and intensity of Opisthorchis viverrini metacercariae (OvMc) were investigated in fish from 3 southern administrative regions along the Mekong River in Cambodia, i.e., Phnom Penh, Takeo, and Kandal Provinces from 2017 to 2020. A total of 295 freshwater fish (24 species) were transported to our laboratory with ice and examined using the artificial digestion method. In Phnom Penh, among 4 fish species positive for OvMc, 9 (23.7%) of 38 specimens examined were infected, and their intensity of infection averaged 4.3 metacercariae per infected fish. In Takeo Province, among 10 fish species positive for OvMc, 24 (38.1%) out of 63 fish examined were infected, and their intensity of infection was av. 14.4 metacercariae per infected fish. In particular, all of 3 Osteochilus schlegelii fish examined were infected, and their infection intensity was high, 34.7 metacercariae per fish. In Kandal Province, among 6 fish species positive for OvMc, 46 (90.2%) out of 51 specimens examined were infected, and their infection intensity was 24.0 metacercaraie per infected fish. All fish of Systomus orphoides (n=17), Barbonymus altus (n=14), and Rasbora aurotaenia (n=2) were infected, and their intensity of infection averaged 37.7, 21.6, and 18.5 metacercariae per fish, respectively. Metacercariae of Haplochis yokogawai, Haplorchis taichui, and Centrocestus formosanus were detected in fish from Takeo and Kandal Provinces. From these results, it has been confirmed that a variety of fish species from Phnom Penh, Takeo, and Kandal Provinces are commonly infected with OvMc, and preventive measures to avoid human O. viverrini infection should be performed in Cambodia.

Association of comorbidity between Opisthorchis viverrini infection and diabetes mellitus in the development of cholangiocarcinoma among a high-risk population, northeastern Thailand.

Cholangiocarcinoma (CCA) is a category of lethal hepatobiliary malignancies. Previous studies have found that Opisthorchis viverrini infection and diabetes mellitus (DM) are closely correlated with CCA. However, few studies have discussed the association of CCA with a combination of both O. viverrini infection and DM. This study aimed to assess the correlation of CCA with various combinations of O. viverrini infection and DM among a high-risk population in northeastern Thailand. This study included participants from 20 provinces in northeastern Thailand who had been screened for CCA in the Cholangiocarcinoma Screening and Care Program (CASCAP) between 2013 and 2019. Histories of O. viverrini infection and DM diagnosis were obtained using a health questionnaire. CCA screening used ultrasonography with a definitive diagnosis based on histopathology. Multilevel mixed-effects logistic regression was performed to quantify the association, which is presented as adjusted odds ratios (aOR) and their 95% confidence intervals (CI). Overall, 263,776 participants were included, of whom 32.4% were infected with O. viverrini, 8.2% were diagnosed with DM, and 2.9% had a history of both O. viverrini infection and DM. The overall rate of CCA was 0.36%. Of those infected with O. viverrini, 0.47% had CCA; among those with DM, 0.59% had CCA and among those infected with O. viverrini and had DM, 0.73% had CCA. Compared with participants who were not infected with O. viverrini and were non-DM, the aOR for those infected with O. viverrini and with DM was 2.36 (95% CI: 1.74-3.21; p-value <0.001). The combination of O. viverrini infection and DM was highly associated with CCA, and these two conditions had a combined effect on this association that was greater than that of either alone. These findings suggest that CCA screening should have a strong focus on people with a combination of O. viverrini infection and DM.

Mucin-producing hamster cholangiocarcinoma cell line, Ham-2, possesses the aggressive cancer phenotypes with liver and lung metastases.

Cholangiocarcinoma (CCA) is an aggressive bile duct cancer. Opisthorchis viverrini (O. viverrini) infection is a significant cause of CCA in the Greater Mekong subregion. Currently, there is no standard chemotherapeutic regimen for CCA. A unique hamster carcinogenesis model of O. viverrini-associated CCA was established. Molecular targets identified from the hamster CCA-comparative model are valuable for target identification and validation. Hamster CCA was induced by the administration of O. viverrini metacercariae and N-nitrosodimethylamine. Hamster-derived cancer cells were isolated and continuously cultured for more than 6 months. Ham-2 cell line was established and characterized in vitro and in vivo. Ham-2 exhibited chromosome hyperploidy. A comparative study with previously established cell line, Ham-1, demonstrated that Ham-2 acquired slower growth, higher adhesion, higher migration, and resistance to doxorubicin and 5-fluorouracil (5-FU). In BALB/c Rag-2/Jak3 double-deficient (BRJ) mice, Ham-2 subcutaneous transplantation formed mucin-producing cancers, which morphologically resemble human tubular cholangiocarcinoma. Intravenous-injected Ham-2 established the metastatic nodules in the lungs and livers of BRJ mice. Altogether, a new hamster cholangiocarcinoma cell line, Ham-2, which acquired more aggressive phenotypes in vitro and in vivo, was established. This cell line might be a valuable tool for comparative drug target identification and validation.

Biliary Migration, Colonization, and Pathogenesis of O. viverrini Co-Infected with CagA+ Helicobacter pylori.

Co-infection with the cagA strain of Helicobacter pylori exacerbates the pathology of human liver fluke Opisthorchis viverrini (OV) infection leading to cholangiocarcinoma. However, underlying mechanisms remain unclear. We report a significant increase in cagA-positive and cagA-negative H. pylori in the stomach, blood, bile, and in the OV worms of co-infected Syrian golden hamsters at one hour, three hours, and one month, post-infection, compared to hamsters infected with either OV or H. pylori alone. Except in the worms, H. pylori numbers declined at three months post-infection, particularly in the bile fluid of co-infected animals. Both strains of H. pylori were immunohistochemically detected in the tegument of the worm, as well as in the bile duct epithelium when co-infected with O. viverrine, but not in H. pylori infection alone. Interestingly, only the cagA-positive strain was detected in the gut of the worm. Co-infection between cagA-positive H. pylori and O. viverrini resulted in a more severe biliary pathology and decreased E-cadherin expression in vivo and in vitro than those of the cagA-negative strain. These data suggest that O. viverrini acts as a carrier of cagA-positive H. pylori and co-migrates to the bile ducts, whereas O. viverrini facilitates H. pylori colonization and enhances the biliary pathogenesis and carcinogenesis.

Opisthorchis viverrini Infection Induces Metabolic and Fecal Microbial Disturbances in Association with Liver and Kidney Pathologies in Hamsters

Opisthorchis viverrini (Ov) infection causes hepatobiliary diseases and is a major risk factor for cholangiocarcinoma. While several omics approaches have been employed to understand the pathogenesis of opisthorchiasis, effects of Ov infection on the host systemic metabolism and fecal microbiota have not been fully explored. Here, we used a 1H NMR spectroscopy-based metabolic phenotyping approach to investigate Ov infection-induced metabolic disturbances at both the acute (1 month postinfection, 1 mpi) and chronic (4 mpi) stages in hamsters. A total of 22, 3, and 4 metabolites were found to be significantly different in the liver, serum, and urine, respectively, between Ov+ and Ov- groups. Elevated levels of hepatic amino acids and tricarboxylic acid (TCA)-cycle intermediates (fumarate and malate) were co-observed with liver injury in acute infection, whereas fibrosis-associated metabolites (e.g., glycine and glutamate) increased at the chronic infection stage. Lower levels of lipid signals ((CH2)n and CH2CH2CO) and higher levels of lysine and scyllo-inositol were observed in serum from Ov+ hamsters at 1 mpi compared to Ov- controls. Urinary levels of phenylacetylglycine (a host-bacterial cometabolite) and tauro-β-muricholic acid were higher in the Ov+ group, which coexisted with hepatic and mild kidney fibrosis. Furthermore, Ov+ animals showed higher relative abundances of fecal Methanobrevibacter (Archaea), Akkermansia, and Burkholderia-Paraburkholderia compared to the noninfected controls. In conclusion, along with liver and kidney pathologies, O. viverrini infection resulted in hepatic and mild renal pathologies, disturbed hepatic amino acid metabolism and the TCA cycle, and induced changes in the fecal microbial composition and urinary host-microbial cometabolism. This study provides the initial step toward an understanding of local and systemic metabolic responses of the host to O. viverrini infection.

Opisthorchis viverrini antigens up-regulates the expression of CD80 and MHC class II in JAWSII mouse dendritic cells and promotes IL-10 and TGF-β secretions.

Dendritic cells (DCs) are antigen-presenting cells (APC) involved in the initiation of immune responses. Maturation of DCs is characterized by the high expression of major histocompatibility complex (MHC) class II and co-stimulatory clusters of differentiation (CD) 40, CD80, and CD86 molecules. Matured DCs are required for T cell differentiation and proliferation. However, the response of DCs to Opisthorchis viverrini antigens has not yet been understood. Therefore, this study sought to determine the expression of surface molecules of JAWSII mouse DCs stimulated by crude somatic (CS) and excretory-secretory (ES) antigens of O. viverrini. ES antigen significantly induced only mRNA expression of CD80 and MHC class II in JAWSII mouse DCs, while CS antigen promoted up-regulation of both mRNA and protein levels of CD80 and MHC class II, indicating relative maturation of JAWII mouse DCs. Moreover, the secreted cytokines from the co-cultures of O. viverrini antigens stimulated JAWSII DC with naïve CD4+ T cells was determined. Significantly increased levels of immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-β) were found. The up-regulation of these cytokines may indicate the response of regulatory T cells (Treg) to CS antigen-stimulated JAWSII DC. These findings may lead to a better understanding of the role that DCs play in O. viverrini infection.

CagA+ Helicobacter pylori infection and N-nitrosodimethylamine administration induce cholangiocarcinoma development in hamsters.

Helicobacter pylori (HP) has been detected in the hepatobiliary tract of cholangiocarcinoma (CCA) patients in regions both endemic and non-endemic for Opisthorchis viverrini (OV) infection. However, whether H. pylori infection promotes CCA development remains unknown. We investigated CCA development in hamsters induced by a combination of infection with H. pylori and administration of N-nitrosodimethylamine (NDMA) and compared findings with those in an OV plus NDMA group. Eighty-five hamsters were divided into four groups: (1) normal, (2) administered NDMA, (3) infected with cagA+ H. pylori and administered NDMA (HN group), and (4) infected with OV and administered NDMA (ON group). Animals were euthanized at 3 and 6months post-infection. Histopathological changes of liver and the expression of markers associated with carcinogenesis were studied. At 3months post-infection (p.i.), cholangitis and lymphoid follicles without tumor appearance were noted in the HN group, whereas extensive fibrosis was seen in members of the ON group, 10% of which had developed tumors. At 6months p.i., 10% of hamsters administered NDMA alone had developed CCA, whereas in the HN and ON groups, 20% and 60% of hamsters, respectively, had developed CCA. Cytokeratin-19 (CK19) expression was observed in the CCA tissues of both the HN and the ON groups, confirming the bile duct origin of the CCA cells. CCA development in the HN group might be inflammation-mediated, as suggested by overexpression of HMGB1, PCNA, IL-8, and 8-OxodG in CCA tissues. cagA+ H. pylori infection and carcinogen intake can induce CCA development with slow progression.

National Program of Opisthorchiasis in Thailand; Situation and Policy Strategy

Opisthorchiasisis, a chronic parasitic inflammatory disease of the bile ducts, caused by liver fluke or Opisthorchis viverrini infections is a crucial significant public health problem in Thailand. Most evidence of Opisthorchiasis is reported from national programs. Thus the magnitude of liver fluke infection at the national level required to assess the effectiveness of the national opisthorchiasis control program. In Thailand, infections with Opisthorchis viverrini are associated with a high mortality secondary to Cholangiocarcinoma (CCA). A systematic countrywide national survey in Thailand already reported from the past more than half-century (59 years) in 1957 to date in 2019, revealed the declining trend of the overall helminthiasis infection from 62.9% (in 1957) to 8.13% (in 2009). The National Opisthorchiasis control program managers have initiated and established a national plan to prevent and control these eradicate diseases through the interaction and responses of the provincial public health sectors. However, the national control program managers need to have information determined by evidence-based needs. These intensive data are representative of all ages of the population of Thailand and are derived from the national household survey. The national prevalence of Opisthorchis viverrini in this study was carried out in 77 provinces. A total of 9,904 samples of stool specimens were obtained from the population by using a cluster random sampling under WHO guidelines. The results of this large scale survey showed that the overall prevalence of Opisthorchaisis among Thai people was 4.0%. The highest prevalence of O. viverrini was 11.9 %, in the health region 5 in the Northeast region. During this national survey of Opisthorchiasis, geographic information is also conducted to investigate the prevalence of O. viverrini. The spatial analysis would be the trends and analyze the risk factors that correlate with infections among rural Thai people. The recommendation that already showed the impact of National program useful for every country especially in Mae King basin for elimination and control for opisthorchiasis.

Investigation of possible alternate animal reservoir hosts of Opisthorchis viverrini

Chronic opisthorchiasis caused by Opisthorchis viverrini (O. viverrini) adversely affects human health and is associated with a fatal bile duct cancer (cholangiocarcinoma). Although cats and dogs are known animal reservoir hosts of opisthorchiasis, there is limited information about whether other fish-eating animals are fluke reservoirs. Wetlands along Chi River of Thailand have abundant intermediate host snails and fish for O. viverrini and diverse other animal species. This led to our investigation into whether other fish-eating animals can also become infected and be alternate reservoir hosts for human opisthorchiasis. Our preliminary study focused on the O. viverrini infection status of crab-eating or long-tailed macaques roaming in Kosumpi National Forest Park in Chi River Basin, Kosumpisai District of Mahasarakam Province, and rodents, small residential mammals and fish-eating birds living in Lawa wetland complex of Khon Kaen Province. Fecal samples of each animal were collected and modified formalin ether concentration technique was applied to identify infections. Additionally, participatory epidemiology was used to retrieve information from local communities on opisthorchiasis transmission in these animals. No O. viverrini infection was found in any fecal samples suggesting that monkeys, rodents, small residential mammals and birds in these two wetlands probably do not serve as alternate reservoir hosts of O. viverrini.

Improvement of a PCR-based method for the detection of Opisthorchis viverrini eggs in human stool samples by targeting internal transcribed spacer-2 (ITS-2), cytochrome oxidase subunit 1 (cox1), and cytochrome b (cyb).

Opisthorchis viverrini infection causes various complications in patients, ranging from asymptomatic to severe chronic disease including cholangiocarcinoma (CCA). O. viverrini is endemic in Southeast Asia and acting as a risk for CCA. Early diagnosis of O. viverrini infection can reduce the number of CCA cases. The routine diagnosis for opisthorchiasis is direct wet smear, sometimes coupled with concentration techniques, which has limitations when investigating light infection or if done by laboratorians with lack of experiences. PCR-based methods have been established for the detection of O. viverrini egg DNA from stool samples, but have never fully succeeded for light infections when compared to wet smear concentration techniques. This study aims to improve the PCR-based method for detection of O. viverrini eggs in stool samples by targeting the genes ITS-2, cox1, and cyb. The results reveal higher sensitivity than conventional concentration techniques, with all newly designed primers. ITS-2 has an overall sensitivity of 76.9% with 66.7% in the samples with < 50 EPG, while cox1 has shown 96.2% overall sensitivity and 94.1% in the same EPG intervals. Interestingly, the new pointing target, cyb, has shown 100% sensitivity in all egg intervals in this study, particularly for light infections (EPG less than 100). No cross-reactivity was found in Taenia spp., Trichuris trichiura, Ascaris lumbricoides, Capillaria philippinensis, and hookworm. The procedure is convenient, with shorter steps compared to previous reports, and it appears appropriate for use in the diagnosis of light infection with O. viverrini. These three genes are good candidates for use in PCR-based detection of the parasite eggs. Further testing with a larger cluster of samples is however necessary.