Search

This study investigated the relationship between immune responses to infection with the liver fluke, Opisthorchis viverrini, and the synthesis of the carcinogen, N-nitrosodimethylamine (NDMA) in humans. it also examined associations between synthesis of nitric oxide (NO) and nitrosation of amines, in vivo. Antibody and T cell responses to fluke antigens and post-alcohol urinary NDMA excretion were assessed among three groups of 40-50 men with no, moderate and heavy liver fluke infection. Markers of NO synthesis (nitrate, nitrite) and nitrosation (nitrosamino acids) were also measured in biological fluids. Assessments were carried out under controlled conditions which minimised intake of exogenous nitrate and nitrite and were carried out at two time points, namely before and 4 months after elimination of the infection with praziquantel treatment. No statistically significant variation was observed in the amount of NDMA excreted between the 3 groups. However, during active infection, a strong negative association was observed between in vitro lymphoproliferative responses to some liver fluke antigens and NDMA excretion. After treatment this association was reduced. Multivariate statistical models revealed a highly significant relationship between NDMA levels and urinary nitrate, stimulation indices for two T cell responses to two parasite antigens (MW 37 kDa and 110 kDa) and gall bladder dimension. NDMA levels after treatment were best described by the ratio between parasite-specific IgG2 and IgE, background levels of T cell proliferation, a urinary marker of nitrosation (N-nitrosothioproline) and usual level of alcohol consumption. These results suggest that individual background immunologic activity, parasite-specific responses and/or parasite products and NO synthesis are important determinants of endogenous generation of nitrosamines in O.viverrini-infected humans. [ABSTRACT FROM PUBLISHER]

Objective: Chronic Opisthorchis viverrini (OV) infection is the cause of advanced periductal fibrosis (APF), subsequently leading to cholangiocarcinoma (CCA). Natural killer (NK) cells can kill hepatic stellate cells (HSCs), the initiating cells for fibrosis formation, by using the interaction between the natural killer group 2 member D (NKG2D) receptor and its ligand on the HSCs. This can inhibit the fibrosis formation. Major histocompatibility complex class I chain-related A (MICA) is the ligand of the NKG2D receptor and has highly polymorphic characteristics that are involved in NKG2D binding and NK cell activation. This study aimed to investigate the polymorphism of MICA in OV-induced fibrosis., Method: MICA typing was performed by polymerase chain reaction- sequence specific primer (PCR-SSP) and sequencing in two groups: OV infection without fibrosis (N = 99) and with fibrosis (N = 290)., Result: Six alleles were identified and the MICA*010 allele had the highest frequency in both groups. The MICA*00201-02 allele was a protective factor for fibrosis (OR= 0.508, 95%CI= 0.34-0.76, Pc <0.05), while the MICA*019 allele was suggested to be a risk allele for fibrosis (OR=1.95, 95%CI=1.25-3.03, Pc<0.005). In addition, two motifs, glycine (G) at position 14 and glutamine (Q) at position 251, were negatively associated with fibrosis (G14: OR=0.508, 95%CI=0.34-0.76, Pc <0.05 and Q251: OR=0.586, 95%CI=0.41-0.84, Pc <0.05). Moreover, the distribution of the MICA-129 genotype also showed the protective genotype (Pc<0.05, OR=0.319, 95%CI= 0.12-0.54) for fibrosis. The MICA*00201-02 allele encoded all these motifs, and this suggested that it might lead to strong NK cell activation to kill HSCs, subsequently preventing fibrosis formation., Conclusion: This study described initial evidence suggesting that the polymorphism of the MICA gene might be a marker for OV-derived periductal fibrosis.

Background: Liver fluke infection caused by Opisthorchis viverrini is associated with several hepatobiliary diseases including advanced periductal fibrosis (APF) and cholangiocarcinoma. Recently, we demonstrated a persistent APF in over one-third of opisthorchiasis patients after worm removal by praziquantel (PZQ) treatment. However, the underlying mechanism(s) of this phenomena is unclear. Given a co-infection with Helicobacter pylori (H. pylori) especially cagA-positive strain enhances APF, we hypothesized that H. pylori with CagA virulent factor contributes to persistent APF., Materials and Methods: Seventy-five opisthorchiasis patients who underwent ultrasonography and treatment with PZQ were recruited in the 2-year follow-up study. Helicobacter and its cagA in the feces were examined by conventional and qPCR. Correlations between prevalence or bacterial loads of Helicobacter spp., H. pylori, and cagA-positive H. pylori before and after PZQ treatment were analyzed among resolved, slowly resolved, relapsed, and persistent APF groups., Results: Overall, prevalence of Helicobacter spp., H. pylori, and cagA-positive H. pylori declined after PZQ treatment. However, only the prevalence and bacterial loads of cagA-positive H. pylori detected at 2-year post-treatment were significantly lower than those before treatment (p < .05). In addition, both prevalence and bacterial loads of cagA-positive H. pylori were significantly lower in the resolved APF group after PZQ treatment, while there were no significant changes in the slowly resolved, relapsed, and persistent APF groups. Among the APF subgroups, cagA-positive H. pylori prevalence in both relapsed and persistent APF groups were significantly higher than the resolved APF group., Conclusion: The results support our hypothesis that H. pylori, especially cagA-positive strain, contributes to the relapsed and persistent APF. A supplementary antibiotic treatment for H. pylori to reduce persistent APF and eventually CCA is warranted., (© 2022 John Wiley & Sons Ltd.)

Infection of the liver fluke, Opisthorchis viverrini (OV) is an important public health problem in northeast Thailand and adjacent countries, where people have a habit of eating raw or undercooked fish. A community case-control study was carried out with 8,936 participants from 89 villages, in Khon Kaen province, Thailand. There were 3,359 OV-infected participants all of whom underwent ultrasonography of upper abdomen for the evaluation of hepatobiliary morbidity. The participants with advanced periductal fibrosis (APF) by ultrasound (n = 785) were invited to undergo annual follow-up ultrasonography for five years after praziquantel treatment. The sonographer was blinded with respect to status of OV infection at each visit. The study findings revealed variability in the study population profile of the hepatobiliary morbidities before and after praziquantel treatment over the follow up interval. At the end of the study, 32 (30.8%) out of 104 participants showed no relapse of APF whereas, by contrast, 39 (37.5%) participants showed relapse or persistent APF since the outset of the study (≥ two consecutive visits). The APF in most follow-up visits was significantly associated with male sex, with intrahepatic duct stones, with the width of the gallbladder "pre" minus "post" fatty meal, and with the ratio of left lobe of the liver to aorta. Five cases of suspected cholangiocarcinoma were observed over the five years of follow-up. This long-term ultrasound follow-up study demonstrates a significant incidence of persistent APF in over one-third of opisthorchiasis cases after praziquantel treatment, findings that support the prospect of ongoing cholangiocarcinogenesis long after successful elimination of liver fluke infection among the population., (Copyright © 2021. Published by Elsevier B.V.)

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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Millions of people are infected with the liver fluke, Opisthorchis viverrini (OV), but only ~25% of those infected develop liver disease and even fewer develop cholangiocarcinoma. The reasons for these differential outcomes following infection are unknown but it has been proposed that differential immune responses to the parasite may play a role. We therefore measured granulocyte (neutrophil) function in OV-infected individuals, with and without advanced periductal fibrosis, to determine if these cells have a "pro-inflammatory" phenotype that may contribute to liver disease post-infection. A case-controlled study (n = 54 in each cohort) from endemic OV-infected areas of northeastern Thailand measured neutrophil functions in whole blood from non-infected (healthy controls) and OV-infected individuals with and without APF. We measured reactive oxygen species production, phagocytosis, receptor expression and apoptosis. Secreted products from OV cultures (obtained after in vitro culture of parasites) stimulated reactive oxygen species production in non-infected healthy controls, but levels were two-fold greater after OV infection (P < 0.0001); neutrophil reactive oxygen species production in individuals with APF was double that observed in those without APF (P < 0.0001). OV-infected neutrophils had elevated CD11b expression and greater phagocytic capacity, which was even three-fold higher in those with advanced periductal fibrosis (P < 0.0001). This "activated" phenotype of circulating neutrophils was further confirmed by the observation that isolated neutrophils had delayed apoptosis ex vivo. We believe this is the first study to show that circulating blood neutrophil function is enhanced following OV infection and is more activated in those with advanced periductal fibrosis. We propose that this activated phenotype could contribute to the pathology of liver disease. These data support the hypothesis of an activated innate inflammatory phenotype following OV infection and provide the first evidence for involvement of neutrophils in disease pathology., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Liver fluke infection caused by Opisthorchis viverrini induces several hepatobiliary conditions including advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA), but >25% of the infected population develops APF and 1% develop CCA. The innate immune response is the first line of defence, and macrophages are critical regulators of fibrosis. We hypothesized that macrophages from infected individuals have different capacities to either promote or suppress periductal fibrosis. We compared phagocytic activities of macrophages of healthy individuals and O viverrini-infected individuals ± APF, and found that macrophages from infected individuals with APF ingested significantly higher numbers of beads compared with healthy controls and O viverrini-infected individuals without APF. To further investigate proteolytic activity, we monitored real-time phagosomal proteolysis of beads conjugated to DQ-BODIPY-BSA using live cell imaging. We show that macrophages from O viverrini-infected individuals with APF also have elevated phagosomal proteolysis activity, which is consistent with their increased phagocytic activity. Additionally, stimulated ROS production by blood monocytes was higher in individuals with APF compared with healthy controls and infected individuals without APF. These results suggest that during O viverrini infection, macrophages with high phagocytic and proteolytic activities together with elevated ROS production are the phenotypes that can promote tissue damage, which results in periductal fibrosis., (© 2018 John Wiley & Sons Ltd.)

Previous studies demonstrated that urinary 8-oxodG is a predictive biomarker for Opisthorchis viverrini (OV)-associated hepatobiliary disease (HBD) and cholangiocarcinoma (CCA). This study examined the effects of praziquantel treatment on the profile of urinary 8-oxodG in relation to HBD status. Infection with OV, levels of urinary 8-oxodG, and HBD status in terms of periductal fibrosis (PDF) assessed by abdominal ultrasonography (US) were monitored and compared in cohorts of participants in Khon Kaen, Thailand, before and 1 year after praziquantel treatment. Urinary 8-oxodG levels significantly decreased after treatment compared with the baseline level in OV-infected participants who had no HBD (PDF negative; PDF-ve) ( N = 14). Levels of 8-oxodG were unchanged after treatment in OV-infected subjects (OV+ve) who had positive PDF ( N = 52). Within the positive PDF (PDF+ve) group who became PDF-ve after treatment, there was no significant change in 8-oxodG levels between pre-and posttreatment (reversible PDF = 65.3%). In those who had persistent PDF+ve at both ultrasound sampling points, there was no significant difference in urinary 8-oxodG levels between pre- and posttreatment (persistent PDF = 34.6%). Based on a logistic regression model and receiver operation curve analysis, the increase of 8-oxodG levels was found to be associated with increasing risk of PDF. Measurement of urinary 8-oxodG and US increased the likelihood of discovering persistent PDF, which is a predictable condition for the patients at risk of OV-associated CCA. To identify high-risk individuals for CCA, it is useful to perform US in combination with urinary 8-oxodG measurement.

Infection with the liver fluke, Opisthorchis viverrini (Ov), is an important public health problem in northeast Thailand where people have a habit of eating raw or inadequately cooked fish. Opisthorchiasis is associated with a number of asymptomatic hepatobiliary abnormalities and cholangiocarcinoma (CCA) that can be observed conveniently by ultrasonography. Based on our accumulated data, characteristic features of ultrasonographic findings of hepatobiliary pathology in Ov infection are reviewed. Also, the ultrasonographic classification of periductal fibrosis in Ov infection of opisthorchiasis will be proposed herein., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

It has been suggested that repeated infection of Opisthorchis viverrini followed by repeated treatment with praziquantel (PZQ) increases risk of development of cholangiocarcinoma (CCA). Evidence for the prediction has accumulated based on findings of indirect approaches involving molecular changes and epidemiological trends. By contrast, here we directly monitored the impact of repeated liver fluke infection and treatment with PZQ on cholangiocarcinogenesis in a rodent model of human opisthorchiasis, using magnetic resonance imaging (MRI) and histopathology. Twenty five Syrian golden hamsters were assigned to five treatment groups: 1) infection with O. viverrini (OV group), 2) treatment with the carcinogen N-nitrosodimethylamine (NDMA) at 12.5ppm (DMN), 3) O. viverrini infection in tandem with NDMA (OD), 4) O. viverrini infection, NDMA, and treatment with PZQ (ODP), and 5) uninfected, untreated control. The repeated infections were established by intragastric inoculation of 50 metacercariae of O. viverrini to the OV, OD and ODP hamsters at weeks 0, 5 and 10. PZQ at 300mg/kg body weight was given to each hamster of the ODP group on weeks 4, 9 and 13 (four weeks after each infection). Imaging by MRI was undertaken on weeks 5, 10 and 14 (i.e. one week after each PZQ treatment). MRI revealed that the ODP hamsters did not develop CCA, whereas necropsy at week 40 revealed CCA in hamsters of the OD and DMN groups. Findings for histopathology and for proliferating cell nuclear antigen index conformed to the MRI findings. In overview, and notwithstanding that the immune response of individual hosts may play roles in cholangiocarcinogenesis, three cycles of the infection with O. viverrini followed treatment of the infection with PZQ did not increase the risk of bile duct cancer in this hamster model of liver fluke infection-induced CCA., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Opisthorchis viverrini infection induces chronic inflammation, and a minor proportion of infected individuals develop advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA). Inflammatory cytokines and/or their gene polymorphisms may link to these biliary pathologies. We therefore investigated associations among cytokine gene polymorphisms and cytokine production in 510 Thai cases infected with O. viverrini who presented with APF+ or APF-, as established by abdominal ultrasonography as well as in patients diagnosed with CCA. Levels of pro-inflammatory and anti-inflammatory cytokines were determined in culture supernatants after stimulation of peripheral blood mononuclear cells (PBMCs) with O. viverrini excretory-secretory (ES) products. Pro-inflammatory cytokines, IL-1β, IL-6, IFN-γ, LT-α, and TNF-α were significantly increased in CCA patients compared with non-CCA (APF- and APF+) cases. Polymorphisms in genes encoding IL-1β-511C/T, IL-6-174G/C, IFN-γ +874T/A, LT-α +252A/G, and TNF-α -308G/A were then investigated by using PCR-RFLP or allele specific-PCR (AS-PCR) analyses. In the CCA cases, LT-α +252A/G and TNF-α -308G/A heterozygous and homozygous variants showed significantly higher levels of these cytokines than the wild type. By contrast, levels of cytokines in wild type of IFN-γ +874T/A were significantly higher than the variants in CCA cases. IFN-γ +874T/A polymorphisms were associated with advanced periductal fibrosis, whereas IL-6 -174G/C polymorphisms were associated with CCA. To our knowledge, these findings provide the first demonstration that O. viverrini infected individuals carrying several specific cytokine gene polymorphisms are susceptible to develop fibrosis and CCA.

Recent reports suggest that Opisthorchis viverrini serves as a reservoir of Helicobacter and implicate Helicobacter in pathogenesis of opisthorchiasis-associated cholangiocarcinoma (CCA). Here, 553 age-sex matched cases and controls, 293 and 260 positive and negative for liver fluke O. viverrini eggs, of residents in Northeastern Thailand were investigated for associations among infection with liver fluke, Helicobacter and hepatobiliary fibrosis. The prevalence of H. pylori infection was higher in O. viverrini-infected than uninfected participants. H. pylori bacterial load correlated positively with intensity of O. viverrini infection, and participants with opisthorchiasis exhibited higher frequency of virulent cagA-positive H. pylori than those free of fluke infection. Genotyping of cagA from feces of both infected and uninfected participants revealed that the AB genotype accounted for 78% and Western type 22%. Participants infected with O. viverrini exhibited higher prevalence of typical Western type (EPIYA ABC) and variant AB'C type (EPIYT B) CagA. Multivariate analyses among H. pylori virulence genes and severity of hepatobiliary disease revealed positive correlations between biliary periductal fibrosis during opisthorchiasis and CagA and CagA with CagA multimerization (CM) sequence-positive H. pylori. These findings support the hypothesis that H. pylori contributes to the pathogenesis of chronic opisthorchiasis and specifically to opisthorchiasis-associated CCA.

3 T MRI and (1)H MRS were useful for quantitative investigation of the serial development of hepatobiliary changes in Opisthorchis viverrini infection in hamsters, and the differential diagnosis of cholangiocacinoma (CCA) development from bile duct changes and normal condition is unclear. In this study, we investigated the serial development of hepatobiliary changes and CCAgenesis in O. viverrini-infected and N-nitrosodimethylamine (NDMA) treated hamsters (ON group) using 3 T MRI and (1)H MRS and the results were compared with those either in the O. viverrini-infected group (OV group) and uninfected normal controls. In the ON group, CCAs were first found at 9 weeks post-infection, with sizes of ~2 mm. The typical MR signal characteristics of CCA were hypo- and occasionally isointensity signal on T1-weighted images, and mild-moderate to hyper-intensity signal on T2-weighted images compared to the liver parenchyma. T2-weighted images with fat suppression revealed dilatation of the intra- and extrahepatic bile ducts, and often defined the anatomical level of biliary obstruction, cystic lesions, liver abscesses, and CCA which was starting seen of these noticeable abnormalities at 5 weeks onwards. The results of fibrosis grading using MR images showed a positive correlation (r=0.90, P<0.038 by Spearman's rank correlation test) with those of the histopathological grading. In addition, 3.0 T (1)H MRS showed elevated choline and decreased lipids levels in the liver tissues of the ON group. In conclusion, MRI and (1)H MRS are useful for the quantitative investigation of the serial development of hepatobilary changes and CCA in hamsters, and are potentially useful as early diagnostic tools for CCA., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Opisthorchis viverrini is distinct among helminth infections as it drives a chronic inflammatory response in the intrahepatic bile duct that progresses from advanced periductal fibrosis (APF) to cholangiocarcinoma (CCA). Extensive research shows that oxidative stress (OS) plays a critical role in the transition from chronic O. viverrini infection to CCA. OS also results in the excision of a modified DNA lesion (8-oxodG) into urine, the levels of which can be detected by immunoassay. Herein, we measured concentrations of urine 8-oxodG by immunoassay from the following four groups in the Khon Kaen Cancer Cohort study: (1) O. viverrini negative individuals, (2) O. viverrini positive individuals with no APF as determined by abdominal ultrasound, (3) O. viverrini positive individuals with APF as determined by abdominal ultrasound, and (4) O. viverrini induced cases of CCA. A logistic regression model was used to evaluate the utility of creatinine-adjusted urinary 8-oxodG among these groups, along with demographic, behavioral, and immunological risk factors. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive accuracy of urinary 8-oxodG for APF and CCA. Elevated concentrations of 8-oxodG in urine positively associated with APF and CCA in a strongly dose-dependent manner. Urinary 8-oxodG concentrations also accurately predicted whether an individual presented with APF or CCA compared to O. viverrini infected individuals without these pathologies. In conclusion, urinary 8-oxodG is a robust 'candidate' biomarker of the progression of APF and CCA from chronic opisthorchiasis, which is indicative of the critical role that OS plays in both of these advanced hepatobiliary pathologies. The findings also confirm our previous observations that severe liver pathology occurs early and asymptomatically in residents of O. viverrini endemic regions, where individuals are infected for years (often decades) with this food-borne pathogen. These findings also contribute to an expanding literature on 8-oxodG in an easily accessible bodily fluid (e.g., urine) as a biomarker in the multistage process of inflammation, fibrogenesis, and infection-induced cancer.

The cholangiocarcinoma (CCA) is a relatively rare cancer worldwide but it is highly prevalent in Thailand where the liver fluke, Opisthorchis viverrini is endemic. There are reports that interleukin 6 (IL-6) may play an important role in the pathogenesis of opisthorchiasis associated CCA. Functionally, IL-6 can act on target cells through its receptor, IL-6R, and IL-6R polymorphisms may affect the functional activity of IL-6 leading to susceptibility to cholangiocarcinogenesis. Therefore, we assessed the association of the 48892 A/C (Asp358Ala) polymorphism in exon 9 of the IL-6R gene in 79 CCA cases compared to 80 healthy controls using the PCR- RFLP technique. The results showed significant differences between CCA cases and controls in overall genotype (p=0.001) and allele frequencies (p=0.0002). Chi-square for trend test revealed a significant association between genotype and CCA susceptibility (p=0.0002). The odds ratios (ORs) for genotype were 0.283 (95% CI=0.131-0.605, AC vs. AA; p=0.0003) and 0.206 (95% CI=0.196-1.245, CC vs. AA; p=0.0416), the OR for alleles was 0.347 (95% CI=0.187-0.633, allele C vs. allele A; p=0.0002) and that for the carrier C variant was 0.272 (95% CI=0.130-0.564; p=0.0001). This study demonstrated a close association between an IL-6R polymorphism, specifically higher A allele, and cholangiocarcinoma.

A 3T MR scanner was used to investigate the relationship between the alteration of bile duct lesions and the hepatic metabolic changes in hamsters infected with Opisthorchis viverrini by using 3T MRI and (1)H MR spectroscopy. Animals were divided into control and infected groups. Five normal hamsters were used as control; fifty-five hamsters were infected with O. viverrini to induce bile duct lesions and hepatic metabolic changes. T2-weighted image sequence in three orthogonal planes were conducted by MRI scans. Single-voxel (1)H MRS was performed to obtain the relative choline-to-lipid ratios. The livers and bile ducts were excised for the histologic examination. The progression of bile duct changes by histology and metabolic changes in O. viverrini infected hamsters were co-investigated. In the O. viverrini-infected group, the T2-weighted images revealed the time-dependent intra- and extra-hepatic duct dilatations in the liver. The mean (±SD) choline-to-lipid ratios were 0.11±0.035 in the control group, whereas the ratio in the infected group increased significantly with the progression of time. Histologic grading of hepatic inflammation and fibrosis were correlated well with the MRI grading (Spearman rank correlation test; r=0.746 and p<0.001). The control group showed no dilatation of the bile ducts and showed normal liver patterns. Noninvasive technique, MRI and (1)H MRS can demonstrated and applied to evaluate not only the inflammation-related fibrosis in the small bile ducts but also the metabolic changes in the liver induced by O. viverrini infection. A significant increase in the choline-to-lipids ratios were observed in parallel with the time-course of infection. O. viverrini infected in human is detected by stool examination. Hepatobiliary morbidity is detected and followed up by ultrasonography. MRI and MRS can be used in conjunction with ultrasonography for evaluation of progression of the disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Approximately 680 million people are at risk of infection with Opisthorchis viverrini (OV) and Clonorchis sinensis, with an estimated 10 million infected with OV in Southeast Asia alone. While opisthorchiasis is associated with hepatobiliary pathologies, such as advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA), animal models of OV infection show that immune-complex glomerulonephritis is an important renal pathology that develops simultaneously with hepatobiliary pathologies. A cardinal sign of immune-complex glomerulonephritis is the urinary excretion of immunoglobulin G (IgG) (microproteinuria). In community-based studies in OV endemic areas along the Chi River in northeastern Thailand, we observed that over half of the participants had urine IgG against a crude OV antigen extract (OV antigen). We also observed that elevated levels of urine IgG to OV antigen were not associated with the intensity of OV infection, but were likely the result of immune-complex glomerulonephritis as seen in animal models of OV infection. Moreover, we observed that urine IgG to OV antigen was excreted at concentrations 21 times higher in individuals with APF and 158 times higher in individuals with CCA than controls. We also observed that elevated urine IgG to OV antigen could identify APF+ and CCA+ individuals from non-cases. Finally, individuals with urine IgG to OV antigen had a greater risk of APF as determined by Odds Ratios (OR = 6.69; 95%CI: 2.87, 15.58) and a greater risk of CCA (OR = 71.13; 95%CI: 15.13, 334.0) than individuals with no detectable level of urine IgG to OV antigen. Herein, we show for the first time the extensive burden of renal pathology in OV endemic areas and that a urine biomarker could serve to estimate risk for both renal and hepatobiliary pathologies during OV infection, i.e., serve as a "syndromic biomarker" of the advanced pathologies from opisthorchiasis.

The oxidized alpha-1 antitrypsin (ox-A1AT) is one modified form of A1AT, generated via oxidation at its active site by free radicals released from inflammatory cells which subsequently are unable to inhibit protease enzymes. The presence of ox-A1AT in human serum has been used as oxidative stress indicator in many diseases. As oxidative/nitrative damage is one major contributor in opisthorchiasis-driven cholangiocarcinogenesis, we determined A1AT and ox-A1AT expression in human cholangiocarcinoma (CCA) tissue using immunohistochemical staining and measured serum ox-A1AT levels by ELISA. A1AT and ox-A1AT were found to be expressed in the tumor of CCA patients. The group with high expression has a significant poor prognosis. Serum levels of ox-A1AT were also significantly higher in groups of patients with heavy Opisthorchis viverrini infection, advanced periductal fibrosis (APF) and CCA when compared with healthy controls (P < 0.001). Odds ratio (OR) analysis implicated high ox-A1AT levels as a risk predictor for APF and CCA (P < 0.001; OR = 140.5 and 22.0, respectively). In conclusion, as APF may lead to hepatobiliary diseases and an increased risk of CCA development, our results identified ox-A1AT as a potential risk indicator for opisthorchiasis-associated CCA. This marker could now be explored for screening of subjects living in endemic areas where the prevalence of opisthorchiasis still remains high.

Liver fluke infection caused by Opisthorchis viverrini is a major public health problem in Thailand and adjacent countries. In addition to infection-associated morbidity, infection with O. viverrini and the related Clonorchis sinensis are unarguable risk factors for cholangiocarcinoma (CAA, bile-duct cancer). Here we review the pathogenesis of opisthorchiasis and the association between O. viverrini infection and bile-duct cancer, focusing on the molecular parallels between wound healing, chronic inflammation, and cancer development. We review a schema for human disease progression from fluke infection, chronic opisthorchiasis, advanced periductal fibrosis, and cholangiocarcinogenesis, and present a rationale for biomarker discovery to facilitate early intervention. We conclude by addressing post-genomic advances with a view to developing new control strategies to combat this infectious cancer., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Chronic opisthorchiasis caused by Opisthorchis viverrini infection is characterized by advanced periductal fibrosis leading to hepatobiliary diseases (HBD), including cholangiocarcinoma (CCA). We aimed to determine fibrotic markers to differentiate HBD status including opisthorchiasis, benign biliary disease (BBD) and CCA. Candidate fibrotic markers in plasma of healthy individuals (n = 14) and patients with opisthorchiasis (n = 32, pre- and post-treatment with praziquantel), BBD (n = 31), CCA (n = 37) and other types of tumors (n = 14) were measured by ELISA and zymography. Plasma levels of hydroxyproline (HYP), collagen I, MMP-7 and TIMP2 in opisthorchiasis patients were significantly higher than those in healthy individuals, and MMP9/TIMP2 balance may be associated with tissue resorption after praziquantel treatment. HYP and TIMP-2 levels were significantly correlated with periductal fibrosis status evaluated by ultrasonography. Plasma HYP level of CCA patients was the highest among HBD patients (p < 0.05). ROC curves revealed HYP, MMP-7 and collagen I levels significantly distinguished opisthorchiasis, BBD and CCA (p < 0.001). Odd ratio (OR) analysis demonstrated these markers in opisthorchiasis were predictable for BBD risk (p < 0.05; OR = 28.50, 10.12 and 4.63 for collagen I, MMP-7 and HYP, respectively), and the risk was reduced by praziquantel treatment. Interestingly, only plasma HYP level in BBD was predictable for CCA risk (OR = 3.69; p = 0.020). In conclusion, plasma HYP, collagen I and MMP-7 may be useful as novel predictive markers of opisthorchiasis-related BBD, and HYP may be used as a diagnostic marker for CCA., (Copyright © 2011 UICC.)

A cross sectional study on hepatobiliary abnormalities in opisthorchiasis was performed in 8936 males and females aged from 20 to 60 years from 90 villages of Khon Kaen province, Northeast Thailand. All were stool-examined for Opisthorchis viverrini infection by standard quantitative formalin/ethyl acetate concentration technique. Of these, 3359 participants with stool egg positive underwent ultrasonography of the upper abdomen. The hepatobiliary abnormalities detected by ultrasound are described here. This study found a significantly higher frequency of advanced periductal fibrosis in persons with chronic opisthorchiasis (23.6%), particularly in males. Risks of the fibrosis included intensity of infection, and age younger than 30 years. Height of left lobe of the liver, cross-section of the gallbladder dimensions post fatty meal, sludge, and, interestingly, intrahepatic duct stones were significantly associated with the advanced periductal fibrosis. Eleven suspected cholangiocarcinoma (CCA) cases were observed. This study emphasizes the current status of high O. viverrini infection rate and the existence of hepatobiliary abnormalities including suspected CCA in opisthorchiasis endemic areas of Thailand., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Opisthorchiasis caused by Opisthorchis viverrini infection induces hepatobiliary disease (HBD)-associated cholangiocarcinoma (CCA) via a chronic inflammatory immune response. Here, we evaluated specific IgG and IgA antibodies against different fractions of O. viverrini antigen in residents from an endemic community in Northeast Thailand with varying hepatobiliary abnormalities. Crude somatic O. viverrini antigen was purified into three fractions (viz., P1, P2 and P3) by gel infiltration chromatography and these served as antigens for detection of fluke-specific IgG and IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The results revealed fluke-specific IgG and IgA antibody levels-against these antigens from subjects with O. viverrini-positive HBD-higher than in subjects with O. viverrini-negative HBD. Interestingly, the rank of fluke-specific IgG (and not IgA) antibody levels against crude extract and P1 antigens was CCA>severe HBD>mild HBD>healthy individuals. Purified antigens reduced cross-reactivity with other parasites compared to the crude antigen. Multiple linear regression analysis showed that HBD status was significantly associated with the liver fluke-specific IgG antibody against purified antigens. These results suggest that purified O. viverrini-antigen improves serodiagnosis for the evaluation of opisthorchiasis-associated HBD, and may be useful in the screening of opisthorchiasis in subjects at risk of developing CCA., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Infection by the liver fluke (Opisthorchis viverrini) causes hepatobiliary disease and bile duct cancer (cholangiocarcinoma, CCA) in endemic areas in Southeast Asia. Measurements of humoral immune response particularly parasite-specific antibodies are useful not only for serodiagnosis but they have been implicated as risk factors of CCA. In this study, we used indirect Enzyme Immunosorbent Assay (ELISA) to measure O. viverrini-specific immunoglobulins in serum, urine and saliva and assessed efficacies in diagnosis of opisthorchiasis and evaluated the relationship of antibodies among clinical specimens in a sample population in endemic areas in Khon Kaen, Thailand. By employing the Receiver Operation Characteristics (ROC) analysis, diagnostic efficacy based upon the area under the curve (AUC) revealed that serum, salivary IgG and IgA performed better than urine for diagnosis of opisthorchiasis. Seropositive cases were found in both parasite egg-negative as well as O. viverrini egg-positive groups. The levels of serum IgG correlated with intensity of O. viverrini infection (P<0.05). Diagnostic sensitivities based on serum and salivary IgG, IgA also positively associated with the intensity of infection. Correlations between serum antibodies and those in saliva were found to be greater in egg-negative than egg-positive individuals for O. viverrini. Our findings indicated a complex interrelation between antibody responses in different clinical specimens triggered by liver fluke infection. More comprehensive examinations are needed to determine the potential utility of salivary antibody detection which, in combination with the conventional fecal examination method, may better assist in the identification of individuals with opisthorchiasis. Furthermore, it may provide a better indicator of the risk of disease, particularly CCA., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Opisthorchiasis caused by Opisthorchis viverrini induces periductal fibrosis via host immune/inflammatory responses. Plasma protein alteration during host-parasite interaction-mediated inflammation may provide potential diagnostic and/or prognostic biomarkers. To search for target protein changes in O. viverrini-infected hamsters, a 1-D PAGE gel band was trypsin-digested and analyzed by a LC-MS/MS-based proteomics approach in the plasma profile of infected hamsters, and applied to humans. Sixty seven proteins were selected for further analysis based on at least two unique tryptic peptides with protein ID score >10 and increased expression at least two times across time points. These proteins have not been previously identified in O. viverrini-associated infection. Among those, proteins involved in structural (19%), immune response (13%), cell cycle (10%) and transcription (10%) were highly expressed. Western blots revealed an expression level of protein tyrosine phosphatase alpha (PTPα) which reached a peak at 1 month and subsequently tended to decrease. Fibronectin significantly increased at 1 month and tended to increase with time, supporting proteomic analysis. PTPα was expressed in the cytoplasm of inflammatory cells, while fibronectin was observed mainly in the cytoplasm of fibroblasts and the extracellular matrix at periductal fibrosis areas. In addition, these protein levels significantly increased in the plasma of O. viverrini-infected patients compared to healthy individuals, and significantly decreased at 2-months post-treatment, indicating their potential as disease markers. In conclusion, our results suggest that plasma PTPα and fibronectin may be associated with opisthorchiasis and the hamster model provides the basis for development of novel diagnostic markers in the future.

Opisthorchis viverrini is considered among the most important of the food-borne trematodes due to its strong association with advanced periductal fibrosis and bile duct cancer (cholangiocarcinoma). We investigated the relationship between plasma levels of Interleukin (IL)-6 and the risk of developing advanced fibrosis and bile duct cancer from chronic Opisthorchis infection. We show that IL-6 circulates in plasma at concentrations 58 times higher in individuals with advanced fibrosis than age, sex, and nearest-neighbor matched controls and 221 times higher in individuals with bile duct cancer than controls. We also observed a dose-response relationship between increasing levels of plasma IL-6 and increasing risk of advanced fibrosis and bile duct cancer; for example, in age and sex adjusted analyses, individuals with the highest quartiles of plasma IL-6 had a 19 times greater risk of developing advanced periductal fibrosis and a 150 times greater risk of developing of bile duct cancer than individuals with no detectable level of plasma IL-6. Finally, we show that a single plasma IL-6 measurement has excellent positive predictive value for the detection of both advanced bile duct fibrosis and bile duct cancer in regions with high O. viverrini transmission. These data support our hypothesis that common mechanisms drive bile duct fibrosis and bile duct tumorogenesis from chronic O. viverrini infection. Our study also adds a unique aspect to the literature on circulating levels of IL-6 as an immune marker of hepatobiliary pathology by showing that high levels of circulating IL-6 in plasma are not related to infection with O. viverrini, but to the development of the advanced and often lethal pathologies resulting from chronic O. viverrini infection.

Liver fluke infection caused by Opisthorchis viverrini is a major public health problem in Thailand and the Lao People's Democratic Republic (Lao PDR; Laos). Currently, more than 600 million people are at risk of infection with these fish-borne trematodes and/or their close relatives. Opisthorchiasis has been studied extensively in Thailand, where about 8 million people are infected with the liver fluke. Here we review the pathogenesis, control and re-emergence of O. viverrini infection, in particular in Thailand and, to a lesser extent in Lao PDR given the contiguous geographical range of O. viverrini through these two regions. We also review the association of O. viverrini infection and cholangiocarcinoma, bile duct cancer, and highlight new findings on pathogenesis of liver fluke-induced cholangiocarcinogenesis. Last, we comment on national control strategies in Thailand for the control of O. viverrini infection aimed at reduction in the prevalence of O. viverrini-associated liver cancer in the longer term., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Unlabelled: More than 750 million people are at risk of infection with foodborne liver flukes. Opisthorchis viverrini is considered among the most important of these parasites, due to its strong association with cholangiocarcinoma (CCA). O. viverrini infection results in a chronic inflammatory challenge to the host, which can lead to advanced, pathogen-specific disease sequelae including obstructive jaundice, hepatomegaly, cholecystitis, as well as CCA. However, before disease sequelae are apparent, important inflammatory changes to the liver can be detected early during O. viverrini infection. In a case-control study involving 328 men and women with O. viverrini infection, we determined the presence of advanced periductal fibrosis in asymptomatic, O. viverrini-infected individuals and then measured cytokine responses to O. viverrini excretory/secretory products (ES). In the 200 participants with advanced periductal fibrosis (cases), levels of interleukin-6 (IL-6) to O. viverrini ES were 8 times higher than levels of the 128 O. viverrini-infected individuals without advanced periductal fibrosis (controls). Moreover, elevated IL-6 to parasite ES was associated with increased risk of advanced periductal fibrosis by 63% in a model adjusted for sex and age. The risk of advanced periductal fibrosis was also found to increase with higher levels of IL-6: individuals in the third quartile of IL-6-ES production had a 127% higher risk of developing advanced periductal fibrosis than individuals in the first quartile of IL-6 production. O. viverrini-infected individuals with advanced periductal fibrosis showed other hepatobiliary abnormalities, including reduced gallbladder contractility and the presence of gallbladder sludge., Conclusion: These data strongly implicate a role for parasite-specific IL-6 in the pathogenesis of advanced periductal fibrosis in opisthorchiasis, with possible links to other hepatobiliary abnormalities, including CCA.

Parasitic infection of the biliary tree is caused by liver flukes, namely Clonorchis sinensis and Opisthorchis viverrini. These flukes reside in the peripheral small bile ducts of the liver and produce chronic inflammation of the bile duct, bile duct dilatation, mechanical obstruction, and bile duct wall thickening. On imaging, peripheral small intrahepatic bile ducts are dilated, but the large bile ducts and extrahepatic bile ducts are not dilated or slightly dilated. There is no visible caused of obstruction. Sometimes, in heavy infection, adult flukes are demonstrated on sonography, CT or MR cholangiography as small intraluminal lesions. The flukes in the gallbladder may appear as floating, small objects on sonography. Chronic infection may result in cholangiocarcinoma of the liver parenchyma or along the bile ducts. Human infection of Fasciola hepatica, a cattle flukes, may occur inadvertently, and the flukes migrate in the liver (hepatic phase) and reside the bile ducts (biliary phase). Image findings in the hepatic phase present with multiple, small, clustered, necrotic cavities or abscesses in the peripheral parts of the liver, showing "tunnels and caves" sign, reflecting parasite migration in the liver parenchyma. In the biliary phase, the flukes are demonstrated in the intra- and extrahepatic bile ducts and the gallbladder as small intraluminal flat objects, sometimes moving spontaneously. Bile ducts are dilated.

In Thailand, liver cancer is the most common malignancy in males and the third most common among females. In the Northeast region, cholangiocarcinoma (CHCA) is the prevalent type, with Opisthorchis viverrini (OV), an endemic liver fluke, being considered the cause. We evaluated the role of ultrasound (U/S) for cholangiocarcinoma screening as part of a larger cohort to characterize the linkage between liver fluke infestation and CHCA in Khon Kaen (Northeast Thailand). Most people (77%) had normal U/S findings while only 0.5% had suspected CHCA; thus, U/S should be used primarily for those with the highest risk, presenting symptoms and/or being OV positive.

Objective: To evaluate the diagnostic value of clinical findings, electromyography and magnetic resonance imaging of root lesions in traumatic brachial plexus injuries, Material and Method: The authors clinically evaluated 175 cervical roots (C5-8, T1) in patients with traumatic brachial plexus injuries then with electromyography and magnetic resonance imaging (MRI). Each finding, taken alone and in combination with each other, was compared with the findings of exploratory surgery., Results: The mean correlations of the clinical findings, electromyography and MRI in relation to the surgical findings were 60, 87 and 70 percent, respectively. The mean correlation was highest (90%) when the positive findings from the clinical, electromyographic or MRI investigations were combined. The correlation of the C5-6 root injuries was improved to 100% though the mean correlation was only 90%., Conclusion: The combination of clinical findings and electromyography correlated well with the surgical findings of root lesions caused by traumatic brachial plexus injury. MRI markedly improved identification only of C5-6 root injuries.

Low potassium and magnesium status and decreased Na, K-pump activity is an endemic condition among rural Northeast Thais. The authors examined the effect of supplementing potassium and magnesium on erythrocyte potassium, sodium and magnesium content and on Na, K-pump activity. Rural Northeast Thai renal stone patients (62) were recruited, divided into four groups and supplemented for one month with potassium chloride (Group1, n = 16), potassium-sodium citrate (Group2, n = 15), chelated magnesium (Group 3, n =16) and potassium-magnesium citrate (Group 4, n =15) in order to achieve 40 mmol potassium, 10 mmol magnesium and 60 mmol citrate daily. After supplementation with potassium (Groups 1, 2 and 4), plasma potassium and Na, K-pump activity rose significantly in Groups 1, 2 and 4, but erythrocyte potassium rose only in Groups 2 and 4. When supplementing elemental magnesium (Groups 3 and 4), the chelated magnesium caused a significant increase in plasma potassium, erythrocyte potassium, sodium and magnesium without a significant increase in Na, K-pump activity. By contrast, potassium-magnesium citrate caused a significant increase in erythrocyte potassium and magnesium and Na, K-pump activity, but depressed erythrocyte sodium. These results suggest the forms of potassium and /or magnesium salts being supplemented should be considered because they affect erythrocyte potassium, sodium and magnesium content and Na, K-pump activity differently.

Renal stone patients in rural northeast Thailand have a low potassium and magnesium status and low urinary excretion of citrate. We measured the changes of urinary citrate excretion and assessed in vivo skeletal muscle metabolism for intracellular-pH, cytosolic-[Mg(2+)] and phosphorylation potential (using the phosphorus magnetic resonance spectroscopy (31)P-MRS) after oral supplementation to hypokaliuric renal stone patients with oral potassium and magnesium salts. The patients comprised four groups: Group 1 (n = 10) control, Group 2 (n = 3), Group 3 (n = 5) and Group 4 (n = 6) supplemented for a month with potassium citrate, potassium citrate plus amino acid chelated magnesium, and potassium-magnesium citrate, respectively. Though urinary citrate excretion was increased in all three supplemented groups, the increases in intracellular-pH, cytosolic-[Mg(2+)] and phosphocreatine (PCr)/beta-ATP were prominent only in Group 3. The increase in PCr/beta-ATP was also observed in Group 4.

This paper presents an overview of recent progress in the clinical study of opisthorchiasis and treatment. Details of clinical manifestations of opisthorchiasis, hepatobiliary diseases and liver functions, community-based ultrasound study, treatment efficacy, and future study are discussed.

Previous studies of renal stone disease (RSD) in Thailand indicated abnormal urinary aggregator and inhibitor composition among farmers with excessive sweat loss. Our aim was to compare the proton MR spectra obtained from the kidneys of 32 proven cases of RSD (aged 38 to 65 yrs) with nine age-matched normal control subjects. We used the STEAM sequence with TE = 15 ms and TR = 2,000 ms. The spectra at 3.25, 3.6 and 3.9 ppm were analyzed. The results showed a correlation between the three peaks (p < 0.001), however, there was no significant difference between the RSD group and the normal control subjects. We therefore concluded that there was no overloading of these osmolytes among the renal stone patients.

Eosinophilic meningoencephalitis is prevalent and widely distributed in Thailand, especially in the northeastern and central parts of the country. Angiostrongylus cantonensis is one of the causative agents of fatal eosinophilic meningoencephalitis. The nematodes produce extensive tissue damage by moving through the brain and inducing an inflammatory reaction. We report the clinical features and the findings revealed by MR imaging and MR spectroscopy in six patients with eosinophilic meningoencephalitis. The clinical presentation included severe headache, clouded consciousness, and meningeal irritation. Abnormal findings on MR images included prominence of the Virchow-Robin spaces, subcortical enhancing lesions, and abnormal high T2 signal lesions in the periventricular regions. Proton brain MR spectroscopy was performed in three patients and was abnormal in one severe case, showing decreased choline in a lesion. Small hemorrhagic tracts were found in one case. Lesions thought to be due to microcavities and migratory tracts were found in only one case. We believe the MR imaging and MR spectroscopy findings are of diagnostic value and helpful in understanding the pathogenetic mechanisms of the disease.

The purpose of this study was to examine in vivo the activity of cytochrome P450 (CYP) 2A6, an enzyme capable of activating carcinogens, including N-nitrosodimethylamine, in humans with the carcinogenic liver fluke infection, opisthorchiasis viverrini, before and after treatment with the antiparasitic agent, praziquantel. Coumarin hydroxylase activity of CYP 2A6 was assessed by administering a probe drug, coumarin, and measuring its metabolite, 7-hydroxycoumarin, in urines collected between 0-2 h and 2-4 h of 106 people with varying intensities of Opisthorchis viverrini infection. Five individuals who did not excrete any detectable 7-hydroxy coumarin (and have a genetic defect probably leading to an absence of catalytic activity of the CYP 2A6 protein) were excluded from analysis. Infected people excreted an average of 22.7 mumol of 7-hydroxycoumarin in the first 2 h after taking the drug, whereas the mean of the uninfected group was 19.4 mumol; this difference did not reach statistical significance (P = 0.10). However, a highly significant increase in CYP 2A6-related activity was observed in infected individuals who also had radiological evidence of biliary fibrosis (28.1 mumol) compared to those without (19.4 mumol; P = 0.01). Reassessments of coumarin hydroxylase activity of CYP 2A6 made 2 months after praziquantel treatment showed highly significant reductions in the amount of 7-hydroxycoumarin excreted among the infected groups but no difference in the uninfected group. These results suggest that expression of CYP 2A6 is induced among chronically infected people who also have fibrosis of the intrahepatic bile duct. As already demonstrated in an animal model and now observed in humans for the first time, this increase in CYP 2A6-related enzyme activity may represent an important mechanistic link between inflammatory products of chronic liver fluke infection (e.g., DNA alkylation damage from endogenously formed N-nitrosamines) and the high risk of cholangiocarcinoma faced by infected individuals.

The liver fluke, Opisthorchis viverrini, is both highly prevalent and closely associated with cholangio-carcinoma in northeast Thailand. This study measured associations between intensity of liver fluke infection and nonmalignant hepatobiliary disease diagnosed by ultrasonography among 1, 807 largely asymptomatic adult residents drawn from endemic communities. Abnormalities significantly associated with intensity of infection included gallbladder enlargerment in all dimensions, presence of sludge, irregular gallbladder wall, liver enlargement, and enhanced portal vein radicle echoes. While gallbladder enlargement was not sex-specific, the prevalence odds of the other abnormalities were 2-3 times higher among males compared with females. Those recently treated with the anthelmintic praziquantel had higher odds of these abnormalities compared with others with the same infection status who were untreated. The low prevalence of gallstones suggests that this impairment of gallbladder structure and function does not frequently stimulate gallstone formation. However, gallbladder disturbances, together with chronic inflammation and fibrosis of the bile ducts, which are visualized as enhanced portal vein radicle echoes, may contribute to the strikingly enhanced susceptibility to cholangiocarcinoma among people, especially males, with heavy liver fluke infection.