Spatial analysis of cholangiocarcinoma in relation to diabetes mellitus and Opisthorchis viverrini infection in Northeast Thailand.

Cholangiocarcinoma (CCA) exhibits a heightened incidence in regions with a high prevalence of Opisthorchis viverrini infection, with previous studies suggesting an association with diabetes mellitus (DM). Our study aimed to investigate the spatial distribution of CCA in relation to O. viverrini infection and DM within high-risk populations in Northeast Thailand. Participants from 20 provinces underwent CCA screening through the Cholangiocarcinoma Screening and Care Program between 2013 and 2019. Health questionnaires collected data on O. viverrini infection and DM, while ultrasonography confirmed CCA diagnoses through histopathology. Multiple zero-inflated Poisson regression, accounting for covariates like age and gender, assessed associations of O. viverrini infection and DM with CCA. Bayesian spatial analysis methods explored spatial relationships. Among 263,588 participants, O. viverrini infection, DM, and CCA prevalence were 32.37%, 8.22%, and 0.36%, respectively. The raw standardized morbidity ratios for CCA was notably elevated in the Northeast's lower and upper regions. Coexistence of O. viverrini infection and DM correlated with CCA, particularly in males and those aged over 60 years, with a distribution along the Chi, Mun, and Songkhram Rivers. Our findings emphasize the association of the spatial distribution of O. viverrini infection and DM with high-risk CCA areas in Northeast Thailand. Thus, prioritizing CCA screening in regions with elevated O. viverrini infection and DM prevalence is recommended.

Influence of misconceptions and inappropriate eating behaviors on Opisthorchis viverrini infection among at-risk populations undergoing cholangiocarcinoma screening in Northeastern Thailand.

Raw or undercooked freshwater fish consumption contributes to persistent Opisthorchis viverrini infection in Northeast Thailand. This study aims to assess the relationship between misconceptions, unhealthy eating habits, and O. viverrini infection. Data were obtained from the Cholangiocarcinoma Screening and Care Program in Northeast Thailand from 2019 to 2021. Participants were screened for O. viverrini annually over the following 2 years using the Kato-Katz technique. Misconceptions and unhealthy eating habits were assessed through questionnaires. The relationship between these factors and O. viverrini infection was evaluated using adjusted risk ratios (ARRs) and 95% confidence intervals (CIs) from generalized estimating equations under binomial regression framework. Of 5375 participants screened for O. viverrini over 3 years, infection rates were 21.53%, 10.7%, and 4.6% each year, respectively. Out of those, 636 participants responded to questions regarding misconceptions. Results showed that participants who believed in the efficacy of putting lime or red ants in Koi pla (raw fish salad) or eating Koi pla with white whiskey to kill parasites, and early-stage cholangiocarcinoma can be cured, were 41% (ARR, 1.41; 95% CI, 1.03-1.94) and 57% (ARR, 1.57; 95% CI, 1.06-2.33), respectively, more likely to be infected with O. viverrini. Our study confirms that belief in using lime or red ants in Koi pla or eating Koi pla with white whiskey to make it cooked, or early-stage cholangiocarcinoma can be cured, increases O. viverrini infection risk in high-risk populations. Changing health beliefs and eating habits is necessary to reduce O. viverrini infection and its risk to cholangiocarcinoma.

Introduction.

Cholangiocarcinoma (CCA) is a lethal cancer arising in the bile ducts within and just outside the liver. It occurs worldwide and falls into two etiologically defined groups, one related to chronic liver fluke infection and the other not. Liver fluke-related CCA is found in continental Southeast Asia (caused by Opisthorchis viverrini with infection leading to opisthorchiasis), East Asia (Clonorchis sinensis), and Eastern Europe and Russia (Opisthorchis felineus). Both O. viverrini and C. sinensis are classified as group one carcinogens, while recent data from O. felineus suggest the same. In Southeast Asia, an estimated 67.3 million people are at risk of O. viverrini infection and subsequently developing CCA. When the three liver fluke species are considered, an estimated 700 million people are at risk of infection and developing CCA globally. The northeast of Thailand (Isan) is the world's hot spot of liver fluke infection and CCA. Early detection, diagnosis, and surgical intervention/curative treatment of CCA are critical to increase life expectancy and quality of life of people in the region and globally. Despite concentrated recent efforts focusing on a multidisciplinary approach to understand the ecology, epidemiology, biology, public health, and social significance of infection by cancer causing liver flukes, it remains an underestimated and under-resourced public health problem. In addition, it is still believed to be a regional problem without global significance-this is not the case. This book focuses on O. viverrini as the main causative agent of CCA in Southeast Asia, but many aspects detailed in the following chapters also relate to the two other liver fluke species. Our aim is to produce a holistic framework including the basic biology of O. viverrini and its relation to the epidemiology of the disease through diagnosis to treatment, including palliative methods, pathology, and control.

Opisthorchis viverrini Life Cycle, Distribution, Systematics, and Population Genetics.

Opisthorchis viverrini plays a key role as the carcinogenic liver fluke causing bile duct cancer in Southeast Asia. A comprehensive understanding of its life cycle, distribution, systematics, and population genetics is critically important as they underpin the effective development and establishment of future prevention and control programs that center on opisthorchiasis and cholangiocarcinoma. This chapter provides detailed information concerning the basic biology and updated information of O. viverrini related to its host life cycle, transmission route via raw, partially cooked or fermented freshwater cyprinid fish, endemic areas, and the discovery of new foci. Previous sequential studies over the last two decades on the phylogenetic and systematic relationships, genetic variation, and population genetics of O. viverrini as well as its snail intermediate host Bithynia spp. are presented and discussed, which have led to the currently known complex species level systematics and population genetics framework of this host-parasite system. Additionally, further directions for comprehensive research are suggested to provide a more complete understanding of liver fluke, O. viverrini-related cholangiocarcinoma.

Distinct antibody response in susceptible and non-susceptible hosts of the carcinogenic liver fluke Opisthorchis viverrini infection.

Opisthorchis viverrini is a carcinogenic parasite that can cause bile duct cancer called cholangiocarcinoma. A study of the immune response of this parasite in susceptible and non-susceptible hosts may provide a clue to develop vaccines and immunodiagnostic markers, which are currently not available. Here, we compared the antibody response in susceptible Golden Syrian hamsters and non-susceptible BALB/c mice infected by the liver fluke. In mice, the antibody was detected between 1 and 2 weeks post-infection, whereas it was positive between 2 and 4 weeks post-infection in hamsters. Immunolocalization revealed that the antibody from mice reacts strongly with the tegumental surface and gut epithelium of the worm, while hamster antibody showed a weak signal in the tegument and a comparable signal in the gut of the worm. Immunoblot of the tegumental proteins demonstrated that while hamster antibody showed a broad specificity, mice strongly reacted with a single protein band. Mass spectrometry revealed these immunogenic targets. Recombinant proteins of the reactive targets were produced in the bacterial expression system. The immunoblot of these recombinant proteins confirm the reactivity of their native form. In summary, there is a different antibody response against O. viverrini infection in susceptible and non-susceptible hosts. The non-susceptible host reacts quicker and stronger than the susceptible host.

Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini.

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis.

Comparison of Cholangiocarcinoma and Hepatocellular Carcinoma Incidence Trends from 1993 to 2012 in Lampang, Thailand.

Liver cancer is the most common cancer in Northern Thailand, mainly due to the dietary preference for raw fish, which can lead to infection by the parasite, O. viverrini, a causal agent of cholangiocarcinoma. We conducted a temporal trend analysis of cross-sectional incidence rates of liver cancer in Lampang, Northern Thailand. Liver cancer data from 1993-2012 were extracted from Lampang Cancer Registry. The multiple imputation by chained equations method was used to impute missing histology data. Imputed data were analyzed using Joinpoint and age-period-cohort (APC) models to characterize the incidence rates by gender, region, and histology, considering hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). We observed a significant annual increase in CCA incidence and a considerable decrease in HCC incidence for both genders in Lampang. The APC analysis suggested that CCA incidence rates were higher in older ages, younger cohorts, and later years of diagnosis. In contrast, HCC incidence rates were higher in older generations and earlier years of diagnosis. Further studies of potential risk factors of CCA are needed to better understand and address the increasing burden of CCA in Lampang. Our findings may help to draw public attention to cholangiocarcinoma prevention and control in Northern Thailand.

Cholangiocarcinoma protective factors in Greater Mekong Subregion: Critical issues for joint planning to sustainably solve regional public health problems.

Although Opisthorchis viverrini (OV), lifestyle, and diet co-factors have a relatively high prevalence in the Greater Mekong Subregion (GMS) population, cumulative (0-74) incidence rates of cholangiocarcinoma (CCA) do not reach 5% in this region. Other co-factors must influence, but in this study, we only highlighted positive factors for guiding joint planning to address public health problems at the regional level. Therefore, we aimed to study prevalence and factors associated with CCA incidence focusing only on protective factors. A cross-sectional analytic study was carried out from June to October 2017. Participants with informed consent completed the questionnaires. Descriptive statistics were used to analyze general information. Primary variables were classified into high and low levels by mean. Logistic regression was employed to investigate the correlation between interesting variables and the overall risk level of CCA. The overall prevalence of CCA protective factors of the whole region was knowledge (61.39%), health beliefs (42.32%), prevention behavior (31.93%), and community participation (14.53%). When considering the proportions at a high level, they were 49.53%, 53.72%, 35.37%, and 49.67%, respectively. Significant factors associated with CCA prevention were females with secondary or vocational education, a high level of perceived seriousness and benefits, and community participation. These findings are likely to be helpful for both the public and administrators. First, it can be information for people to be aware of CCA risk. Second, policy-driven authorities at the local or regional level should apply the critical issues from this study for joint planning to sustainably solve regional public health problems.

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.

Risk Communication Through Health Beliefs for Preventing Opisthorchiasis-Linked Cholangiocarcinoma: A Community- Based Intervention in Multicultural Areas of Thailand.

OBJECTIVE: This research aimed to study the effects of the risk communication program through the Cambodian folk song to prevent Opisthorchiasis-linked cholangiocarcinoma (OV-CCA). METHODS: We conducted the quasi-experimental research between August and December 2017 in the Cambodian communities, one-fourth of ethnic minorities residing in multicultural areas of Sisaket Province, Thailand. The samples consisted of 94 equally people divided into experimental group and control group. The experimental group included 47 people at-risk of OV-CCA who received the program for 12 weeks, while the control group received regular services. We collected data by using a questionnaire with a reliability of 0.93. Descriptive and inferential statistics were used for data analysis. RESULTS: The study indicated that the socioeconomic information of both groups was not different. The mean scores of all issues (health beliefs, social support, and prevention behavior in the experimental group were higher than those of the control group with statistical significance. Closer inspection showed that the mean difference of the health beliefs was 55.61 points (95%CI: 52.39-57.42, p<0.001), social support was 9.09 points (95%CI: 8.12-10.05, p<0.001), and prevention behavior was 6.38 points (95%CI: 5.43-7.33, p<0.001). CONCLUSION: Through the Cambodian folk song, the risk communication program by applying the health beliefs and social support to prevent OV-CCA is beneficial for behavior modification in areas with similar cultures.

OVCCA Web Application as Supplementary Material to Facilitate Health Literacy Regarding Carcinogenic Human Liver Fluke: A Randomized Controlled Trial in Thailand.

BACKGROUND: Liver fluke, Opisthorchis viverrini, is associated to cholangiocarcinoma which is found frequently in some areas of Southeast Asian countries particularly in Thailand, Lao People Republic Democratic, Cambodia.  This study sought to investigate the effects of an O. viverrini and cholangiocarcinoma (OVCCA) web application to facilitate health literacy regarding O. viverrini in Northeast Thailand. METHODS: A randomized controlled trial study was performed among an intervention group (n=63) and a control group (n=63) during a one-year period from July 2019 to May 2020. The intervention group received the health literacy promotion program of O. viverrini information through the OVCCA web application for 6 weeks. The control group received an activity package from the public health department. The success of the program was evaluated at week 24 after the groups finished the last activity. ANCOVA, t-test and multiple logistic regression were used for data analysis for both groups. RESULTS: The scores for knowledge; ability to access, understand, appraise, and apply information; motivation for protection; and practice of O. viverrini prevention were significantly higher for the intervention group than for the control group. The results indicated that a health literacy promotion program through an OVCCA web application could be advantageous for preventing and controlling O. viverrini infection. CONCLUSION: This intervention may be used as a potential strategy and guideline for self-care and health promotion in other endemic areas.

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.

Therapeutic challenges at the preclinical level for targeted drug development for Opisthorchis viverrini-associated cholangiocarcinoma.

INTRODUCTION: Cholangiocarcinoma (CCA) is a malignant tumor of bile duct epithelium with the highest incidence found in Thailand. Some patients are considered suitable for adjuvant therapy and surgical resection is currently the curative treatment for CCA patients. Tumor recurrence is still a hurdle after treatment; hence, finding novel therapeutic strategies to combat CCA is necessary for improving outcome for patients. AREAS COVERED: We discuss targeted therapies and other novel treatment approaches which include protein kinase inhibitors, natural products, amino acid transporter-based inhibitors, immunotherapy, and drug repurposing. We also examine the challenges of tumor heterogeneity, cancer stem cells (CSCs), the tumor microenvironment, exosomes, multiomics studies, and the potential of precision medicine. EXPERT OPINION: Because CCA is difficult to diagnose at the early stage, the traditional treatment approaches are not effective for many patients and most tumors recur. Consequently, researchers are exploring multi-aspect molecular carcinogenesis to uncover molecular targets for further development of novel targeted drugs.

Hepatobiliary morbidities detected by ultrasonography in Opisthorchis viverrini-infected patients before and after praziquantel treatment: a five-year follow up study.

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.

Genetic Differentiation of Opisthorchis-Like Eggs in Northern Thailand Using Stool Specimens Under National Strategic Plan to Control Liver Fluke Infection and Cholangiocarcinoma.

Liver fluke infection caused by Opisthorchis viverrini is recognized as a potential risk factor for cholangiocarcinoma (CCA). The National Strategic Plan to Control Liver Fluke Infection and Cholangiocarcinoma has implemented microscopic-based stool examination screening. However, eggs of O. viverrini and minute intestinal flukes (MIFs) are nearly morphologically similar and could result in inaccurate O. viverrini diagnosis. Stool specimens were collected from eight districts of Chiang Mai Province in northern Thailand. Opisthorchis-like eggs were identified with the Kato-Katz technique and differentiated for O. viverrini and MIFs using molecular study by PCR and PCR-restriction fragment length polymorphism targeting the internal transcribed spacer 2 (ITS2) gene. Prevalence of Opisthorchis-like eggs was 5.9% from a total of 9,570 specimens. From PCR assays, all liver flukes were O. viverrini and all MIFs were Haplorchis taichui. The distribution of species was H. taichui (38.2%), O. viverrini (10.5%), coinfection of H. taichui and O. viverrini (37.2%), and 14.1% were negative from PCR. Totally, H. taichui was found in 75.4% of infections from Opisthorchis-like specimens. ITS2 nucleotide sequencing analysis showed a single variant of O. viverrini with no variation and two variants of H. taichui. This study first revealed the genetic background of Opisthorchis-like eggs in northern Thailand. Minute intestinal flukes are occasionally misdiagnosed as O. viverrini leading to misinterpretation and overestimation of the burden of O. viverrini infection. Molecular diagnosis such as PCR could effectively discriminate species of Opisthorchis-like eggs and help shape the robustness of epidemiological data to control liver fluke infection and raise awareness of other risk factors for CCA.

Survival of Patients with Cholangiocarcinoma Receiving Surgical Treatment in an O. viverrini Endemic Area in Thailand: A Retrospective Cohort Study.

OBJECTIVE: To investigate risk factors associated with mortality in cholangiocarcinoma patients receiving surgical treatment in Thailand's endemic area and their survival rate. MATERIALS AND METHODS: Medical records of patients with histologically confirmed cholangiocarcinoma, who underwent surgical treatment at Sanpasitthiprasong Regional Hospital from  October 1, 2013 to  October, 31 2015, were retrospectively included. Patients' vital status (death/alive) and date of death were obtained from the Interior Ministry's death certificate. Cox proportional hazard regression was used to examine factors associated with mortality. RESULTS: Out of 295 patients with cholangiocarcinoma (CCA), 180(58%) were intrahepatic CCA, 86(28%) were perihilar CCA, and 29 (9%) were distal CCA. Three groups were homogenous in terms of age and gender. Most of our patients referred with abdominal pain (63%), especially those who were intrahepatic CCA (77%). However, almost 80% of the perihilar CCA and distal CCA patients came with jaundice. Tumor markers (CEA and CA19-9) were not different between groups p=0.74 and p=0.43 respectively. Median survival of patients with intrahepatic CCA, perihilar CCA, and distal CCA patients was 14.6, 14.2, and 14.0 months, respectively. Factors independently associated with mortality in intrahepatic CCA patients were number and size of tumors and presence of perineural invasion (Hazard ratio (HR) 1.09[1.03 - 1.15], 1.07[1.02 - 1.13], and 2.09 [1.28 - 3.39], respectively). In perihilar CCA patients, having positive lymph nodes and resection status were independently associated with mortality. Compared to R0 resection, R1, R2, and no resection of perihilar CCA were associated with a 2-, 8- and 4-fold increase in the risk of mortality (HR 2.17 (0.99 - 4.78), 7.97 (3.22 - 19.71), and 4.21 (0.51 - 34.82), respectively). CONCLUSION: CCA patients in this endemic area had fairly poor survival. Factors associated with mortality in intrahepatic CCA were number and size of tumors and perineural invasion. However, risk factors for perihilar CCA included positive lymph nodes and resection status.

Oxysterols of helminth parasites and pathogenesis of foodborne hepatic trematodiasis caused by Opisthorchis and Fasciola species.

The foodborne trematodiases refer to a cluster of zoonotic neglected tropical diseases caused by trematodes, with transmission involving ingestion of contaminated plants, fishes, and crustaceans. Over 40 million people are infected with foodborne trematodes and 750 million are at risk of infection. From a public health point of view, important species include Clonorchis sinensis, Opisthorchis viverrini, Opisthorchis felineus, Fasciola hepatica, and Fasciola gigantica. Infection with C. sinensis and O. viverrini is classified as a group 1 biological carcinogen and a major risk factor for cholangiocarcinoma. The carcinogenic potential of the infection with O. felineus is less clear but recent biochemical and histopathological findings revealed that opisthorchiasis felinea also fits this pattern. By contrast, evidence of carcinogenic potential of infection with F. hepatica or F. gigantica, close phylogenetics relatives of Opisthorchis, is less certain. Oxysterols have been essentially described in animal model of opisthorchiasis and associated cholangiocarcinoma. Several oxysterol-like metabolites have been detected not only on developmental stages of O. viverrini and O. felineus but also on biofluids from experimentally infected hamsters as products of the activities of the liver flukes. These sterol derivatives are metabolized to active quinones that can modify host DNA. We have postulated that helminth parasite-associated sterols might induce tumor-like phenotypes in biliary epithelia, the cells of origin of liver fluke infection-associated cholangiocarcinoma, through the formation of DNA adducts, dysregulation of apoptosis, and other homeostatic pathways. Here we review, interpret, and discuss findings of oxysterol-like metabolites detected in liver flukes and their role in carcinogenesis, aiming to enhance understanding the pathogenesis of foodborne trematodiasis caused by Opisthorchis and Fasciola species. In future, further investigations will be necessary in order to comprehend relationship between liver flukes' oxysterols and their role in infection-associated diseases in humans.

Enhanced neutrophil functions during Opisthorchis viverrini infections and correlation with advanced periductal fibrosis.

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.

Animation as Supplementary Learning Material About Carcinogenic Liver Fluke in Classes for Primary Schoolchildren.

Carcinogenic liver fluke is still an issue of great concern in some countries of Southeast Asia, particularly in Thailand, Cambodia, the Lao People's Democratic Republic, and Vietnam. The infection, caused by Opisthorchis viverrini, is associated to cholangiocarcinoma and is endemic among human populations for whom raw fish is frequently consumed. Prevention and health education are required. Therefore, this study aimed to evaluate the effectiveness of educational intervention to improve knowledge among primary schoolchildren based on animation-assisted education. In this study, 80 participants (40 participants in the experimental group and 40 participants in the comparison group) were selected in 2018. The effectiveness of an interactive animation program in improving the knowledge of students studying liver fluke was determined based on scores on tests given before and immediately after completion of a 4.29-min animated program on the liver fluke life cycle, risk factors, disease, diagnosis, treatment, prevention, and control. Data were analyzed using SPSS-22 via paired t tests and independent samples t tests at a significance level of 0.05. A marked and significant improvement was observed in the immediate posttest compared with the pretest scores. More importantly, the students who had used the animated program achieved a significantly higher score on the final test than the comparison group. The results offered in the first report show that the use of the animated program facilitated education about liver fluke. It is strongly believed that animations are good supplementary learning materials for students, particularly for learning about serious concepts.

Clonorchis sinensis and clonorchiasis.

Clonorchis sinensis is a fish-borne trematode that inhabits the bile duct of mammals including humans. Clonorchiasis is prevalent in China, Korea, and Vietnam, and 15-20 million people are estimated to be infected by this fluke. Freshwater snails act as the first intermediate host for the proliferation of C. sinensis larvae and shed the cercariae into water. The cercariae penetrate the skin of freshwater fish and transform to metacercariae. Humans are infected by eating raw or undercooked freshwater fish as dishes of filet, "sashimi," or congee, which contain C. sinensis metacercariae. In humans, the C. sinensis metacercariae excyst in the duodenum, and juvenile flukes migrate up via bile chemotaxis into bile ducts. Once there, C. sinensis provokes hyperplasia of the bile duct epithelium, obstructive jaundice, ascites, liver enlargement and cirrhosis, and infrequent cholangiocarcinoma (CCA). Although the association between C. sinensis infection and CCA has been firmly established in past decades, the underlying mechanisms are not elucidated in detail. In the context of chronic clonorchiasis-associated hepatobiliary aberrations, the constitutive disruption of redox homeostasis and dysregulation of physiological signaling pathways may promote the malignant transformation of cholangiocytes, thus leading to substantial acquisition of a more aggressive phenotype by these cells: CCA. With advances of genomic and molecular biological approaches, diverse C. sinensis proteins that are essential for parasite physiology and pathogenicity have been identified and characterized. Some of the proteins have been considered as attractive targets for development of vaccines and chemotherapeutics. Candidate antigens for reliable serodiagnosis of clonorchiasis have been studied.