Effect of information-knowledge-attitude-practice theory-based health education on clonorchiasis control among community residents and primary school students in Zhongshan City / 中国血吸虫病防治杂志

OBJECTIVE@#To evaluate the effect of information-knowledge-attitude-practice (IKAP) theory-based health education interventions on clonorchiasis control among community residents and primary school students in Torch High-tech Development Zone of Zhongshan City, so as to provide insights into formulation of clonorchiasis control strategies among different types of populations.@*METHODS@#Residents were randomly sampled from two communities and students from two primary schools using a random cluster sampling method in Torch High-tech Development Zone, Zhongshan City from 2019 to 2021, and health education pertaining to clonorchiasis control was performed based on the IKAP theory. The changes in the awareness of basic clonorchiasis control knowledge and belief, self-reported risk of Clonorchis sinensis infections and dietary behaviors were compared among community residents and primary school students before and after health education interventions.@*RESULTS@#The participants included 146 male and 151 female community residents and 158 male and 153 female primary school students, with no significant difference detected in gender distribution (χ2 = 0.16, P > 0.05). The mean awareness of basic clonorchiasis control knowledge increased by 44.71% and 43.28% among primary school students and community residents 6 months following health education, and there were significant differences in the awareness of each item of basic clonorchiasis control knowledge before and after health education (χ2 = 41.53 to 284.44, all P values < 0.05). The proportions of primary school students and community residents thinking very high and high risks of C. sinensis infections increased from 9.35% and 6.71% before health education to 22.15% and 37.75% after health education, but only the difference of the attitudes in community residents achieve statistical significance (χ2 = 92.18, P < 0.05). The frequency of separation of raw and cooked foods with chopping board and knife significantly increased among community residents and primary school students following health education (χ2 = 16.04 to 62.65, all P values < 0.05). The frequency of eating raw freshwater fish (χ2 = 32.85, P < 0.05), fish congee (χ2 = 7.02, P < 0.05) and fish fillet hot pot (χ2 = 4.88, P < 0.05) significantly reduced among primary school students following health education, while only the frequency and proportions of eating raw freshwater fish have significantly reduced in community residents (χ2 = 11.77, P < 0.05).@*CONCLUSIONS@#Health education interventions based on the IKAP theory are effective to increase the awareness of clonorchiasis control knowledge and improve dietary behaviors associated with C. sinensis infections among community residents and primary school students in Zhongshan City.

Interspecies Phylogenetic Analysis of / 生物医学与环境科学（英文）

Objective@#This study aims to investigate the infection of @*Method@#Infection of the definitive human host and intermediate fish host by @*Results@#In 2016-2020, the average population infection rate of Hunan was 1.38%, while in Tongdao County the rate was up to 26.90%, and the highest fish infection rate was detected in Qiyang County (99.44% in the dorsal fin of @*Conclusion@#The systematically study of

Status of common parasitic diseases in Korea in 2019

This study aimed to determine the status of common parasitic disease in Korea in 2019. Twelve parasitic diseases were selected: toxocariasis, anisakiasis, paragonimiasis, sparganosis, cysticercosis, toxoplasmosis, clonorchiasis, enterobiasis, trichuriasis, trichomoniasis, cryptosporidiosis, and malaria. Their biology, epidemiology, pathogenesis, symptoms and signs, diagnosis, treatment, and prognosis were evaluated. Of the parasitic diseases, toxocariasis was the most prevalent according to serological results. Anisakiasis should be considered when acute gastrointestinal symptoms occur with a recent past history of raw seafood ingestion. Paragonimiasis, sparganosis, and cysticercosis can be diagnosed using an enzyme-linked immunosorbent assay; thus, enzyme-linked immunosorbent assay needs to be performed for suspected cases. Toxoplasmosis and cryptosporidiosis are opportunistic infections. The symptoms and signs are aggravated under immunocompromised conditions. Although the egg positivity rate of Clonorchis sinensis is higher than that of other intestinal parasitic diseases, encountering patients with complaints of symptoms caused by clonorchiasis is rare because the worm burden is low. Trichomoniasis is usually managed by gynecologists; therefore, it should be included in the differential diagnoses of vaginal diseases. The annual number of malaria cases has decreased, although it remains at approximately 500 cases per year. Malaria should be suspected when symptoms such as intermittent fever, headache, and splenomegaly are noted especially when the patients reside near demilitarized zones. Although the prevalence and number of reported cases of parasitic diseases have decreased in Korea, we should consider parasitic diseases in the list of differential diagnoses.

A Clonorchiasis Case of a Leopard Cat, Prionailurus bengalensis euptilurus, Diagnosed by Ultrasonography and Egg Detection in Republic of Korea

A clonorchiasis case in a captive leopard cat, Prionailurus bengalensis euptilurus, was confirmed by ultrasonographic findings and egg morphologies found in the bile juice sample in the Korea. The leopard cat was introduced from the wild habitat of Gyeongsangnam-do, to Cheongju Zoo in Cheongju-si, Chungcheongbuk-do, Korea in August 2014. Physical examinations were basically performed for quarantine and check-up health. The cat was comparatively good in health except anorexia. The cyst-like bile duct dilation and the increased echogenicity of gall bladder wall and hepatic parenchyma were observed by ultrasonography. Ultrasound-guided needle biopsy was conducted for collecting bile juice and the specimens were observed under light microscope. The numerous small trematode eggs were detected in the bile juice sample of the light microscopy. The eggs were 25–33 (28±3) μm by 18–22 (20±1) μm in size and showed typical characteristics of Clonorchis sinensis egg, i.e., a dominantly developed operculum, shoulder rim and dust-like wrinkles in surface. To treat the liver fluke infection, 20 mg/kg of praziquantel was orally administered only once to the case. Follow-up studies including fecal examinations were conducted during 2 years after treatment. But no more eggs were detected from the case. In the present study, we described the first clonorchiasis case of leopard cat, which was confirmed by ultrasonographic findings and egg morphologies from the bile juice sample in Korea.

Partial Characterization of Two Cathepsin D Family Aspartic Peptidases of Clonorchis sinensis

Cathepsin D (CatD, EC 3.4.23.5) is a member belonging to the subfamily of aspartic endopeptidases, which are classified into the MEROPS clan AA, family A1. Helminth parasites express a large set of different peptidases that play pivotal roles in parasite biology and pathophysiology. However, CatD is less well known than the other classes of peptidases in terms of biochemical properties and biological functions. In this study, we identified 2 novel CatDs (CsCatD1 and CsCatD2) of Clonorchis sinensis and partially characterized their properties. Both CsCatDs represent typical enzymes sharing amino acid residues and motifs that are tightly conserved in the CatD superfamily of proteins. Both CsCatDs showed similar patterns of expression in different developmental stages of C. sinensis, but CsCatD2 was also expressed in metacercariae. CsCatD2 was mainly expressed in the intestines and eggs of C. sinensis. Sera obtained from rats experimentally infected with C. sinensis reacted with recombinant CsCatD2 beginning 2 weeks after infection and the antibody titers were gradually increased by maturation of the parasite. Structural analysis of CsCatD2 revealed a bilobed enzyme structure consisting of 2 antiparallel β-sheet domains packed against each other forming a homodimeric structure. These results suggested a plausible biological role of CsCatD2 in the nutrition and reproduction of parasite and its potential utility as a serodiagnostic antigen in clonorchiasis.

Liver Fluke-Associated Biliary Tract Cancer

Cholangiocarcinoma (CCA) is an aggressive cancer arising from epithelial cells of the bile duct. Most patients with CCA have an unresectable tumor at the time of diagnosis. In Western countries, the risk of CCA increases in patients with primary sclerosing cholangitis, whereas liver fluke infection appears to be the major risk factor for CCA in Asian countries. A diagnosis of liver fluke infection often relies on stool samples, including microscopic examination, polymerase chain reaction-based assays, and fluke antigen detection. Tests of serum, saliva and urine samples are also potentially diagnostic. The presence of liver fluke along with exogenous carcinogens magnifies the risk of CCA in people living in endemic areas. The “liver fluke-cholangiocarcinoma” carcinogenesis pathways consist of mechanical damage to the bile duct epithelium, immunopathologic and cellular reactions to the liver fluke's antigens and excretory/secretory products, liver fluke-induced changes in the biliary tract microbiome and the effects of repeated treatment for liver fluke. A vaccine and novel biomarkers are needed for the primary and secondary prevention of CCA in endemic areas. Importantly, climate change exerts an effect on vector-borne parasitic diseases, and awareness of liver fluke should be enhanced in potentially migrated habitat areas.

Understanding the Biliary Dyspepsia / 대한췌담도학회지

Functional dyspepsia is a very common disease and there are two types of dyspepsia. One is functional dyspepsia in the gastrointestinal tract and the other is pancreatobiliary dyspepsia. Biliary dyspepsia is caused by biliary tract disease and can even cause biliary pain. Acalculous biliary pain (ABP) is biliary colic without gallstones, it is caused by functional biliary disorder or structural disorders such as microlithiasis, sludges or parasitic infestation like Clonorchiasis. The endoscopic ultrasonography is helpful tool for differential diagnosis of ABP. Although sphincter of Oddi manometry (SOM) is performed for the confirmative diagnosis of sphincter of Oddi dysfunction (SOD), several non-invasive tests have been studied because of some practical limitations and invasiveness of SOM itself. In fact, the most clinically used easy test to diagnose functional biliary disorder is quantitative hepatobiliary scintigraphy and it can distinguish gallbladder dyskinesia, SOD, or combined type. Initial treatment of functional biliary disorder is adequate dietary control and medication, but if the symptoms worsened or recurred frequently, laparoscopic cholecystectomy could be performed with gallbladder dyskinesia. If SOD is suspected, additional SOM should be considered and endoscopic sphincterotomy (EST) can be done according to the outcome. If the SOM is not available, the patient could be diagnosed by stimulated ultrasound.

A Case of Papular Eruption Associated with Clonorchiasis / 대한피부과학회지

No abstract available.

Stratified sampling survey of major human parasitic diseases in Henan province / 中华流行病学杂志

Objective: To understand the prevalence of major human parasitic diseases and related factors in Henan province. Methods: This stratified sampling survey was carried out according to the requirement of national survey protocol of major human parasitic diseases, 2014-2015. The prevalence of soil-transmitted helminths infection, taeniasis and intestinal protozoiasis were surveyed in 104 sites selected from 35 counties (districts) and the prevalence of clonorchiasis was surveyed in 62 sites selected from 37 townships. In each survey spot, 250 persons were surveyed. A total of 26 866 persons and 15 893 persons were surveyed. Modified Kato-Katz thick smear was used to detect the eggs of intestinal helminthes. Tube fecal culture was used to identify the species of hookworm. The Enterobius eggs were detected in children aged 3 to 6 years by using adhesive tape. The cyst and trophozoite of intestinal protozoa were examined with physiological saline direct smear method and iodine stain method. Results: The overall infestation rate of intestinal parasites was2.02% in Henan, and the worm infection rate was higher than protozoa infection rate. Fourteen kinds of intestinal parasites were found, including nematode (5 species), trematode (2 species), and protozoan (7 species). The infection rate of Enterobius vermicularis was highest, and Qinba Mountain ecological area had the highest infestation rate of intestinal parasites in 4 ecological areas of Henan. There was no significant difference in intestinal parasite infection rate between males and females (χ(2)=3.630, P=0.057), and the differences in intestinal parasite infection rate among different age groups had significance (χ(2)=124.783, P=0.000 1). The infection rate reached the peak in age group ≤9 years and the major parasite was Enterobius vermicularis. Furthermore the overall human infection rate of parasite showed a downward trend with the increase of educational level of the people (χ(2)=70.969, P=0.000 1), the differences had significance (χ(2)=120.118, P=0.000 1). For different populations, the infection rate of intestinal parasites was highest among preschool children. The infection of intestinal helminth was mainly mild, only 2 severe cases were detected. The infection rate of Clonorchis sinensis in urban residents was only 0.006%. Logistic regression analysis showed that being preschool children (χ(2)=15.765, P=0.000 1) and drinking well water (χ(2)=45.589, P=0.000 1) were the risk factors for intestinal parasite infection, and annual income per capita of farmers was the protective factor against intestinal parasite infection. The infection rates of protozoa and intestinal parasites decreased sharply compared with the results of previous two surveys, and the rate of intestinal helminth infection also dropped sharply compared with the second survey. The numbers of protozoa, helminth and intestinal parasites detected in this survey were all less than the numbers found in the previous two surveys. Conclusions: Compared the results of three surveys in Henan, the infection rate of protozoa and intestinal parasites showed a downward trend. The prevention and treatment of Enterobius vermicularis infection in children should be the key point of parasitic disease control in the future.

Establishment and role of national clonorchiasis surveillance system in China / 中华流行病学杂志

Clonorchiasis is one key food-borne parasitic disease in China. Owing to several years'efforts and preparation, the national clonorchiasis surveillance system in China has been established preliminarily since 2016. In this article, the necessity to establish the national clonorchiasis surveillance system is explained. Then, the structure, content and corresponding methods of the surveillance system are briefly introduced. Key points in the surveillance are summarized and the development of surveillance in future is discussed. Furthermore, the contribution of clonorchiasis surveillance in China to the world is also analyzed.

Infections of Soil-Transmitted Helminth in Refugees from North Korea

Soil-transmitted helminthiases (STH) are now no longer public health problems in the Republic of Korea (South Korea), but their status are unavailable in the residents of North Korea (NK) despite the expectation of large scale traffic and future reunification of the Korean Peninsula. A total of 20 female refugees from NK who had been admitted to the Division of Gastroenterology, Dankook University Hospital, were subjected in this study. Among them, 15 refugees were examined by the colonoscopy and 10 ones were examined with the stool examination (formalin-ether sedimentation). Both diagnostic methods were commonly adopted in 5 patients. Eggs of Trichuris trichiura were detected in 7 out of 10 refugees in the stool examination. In the colonoscopy, T. trichiura worms were found in 6 (40.0%) out of 15 refugees. Total 9 (45.0%) peoples were confirmed to be infected with human whipworms. Additionally, 1 case of clonorchiasis was diagnosed in the stool examination and a worm of Ascaris lumbricoides was discovered from a trichuriasis case. These findings suggested that STH is highly prevalent in NO, in which living conditions are not so good in the aspect of general hygiene and medical care.

Significance of Serology by Multi-Antigen ELISA for Tissue Helminthiases in Korea

It is clinically important to differentiate tissue-invading helminthiasis. The purpose of this study was to assess the specific immunoglobulin G (IgG) antibody positive rates for clonorchiasis, paragonimiasis, cysticercosis, and sparganosis 4 helminthiases from 1996 to 2006 using multi-antigen enzyme-linked immunosorbent assay (ELISA) in Korea. Results of 6,017 samples, which were referred to our institute for serodiagnosis, were analyzed. The subjects with positive serum IgG antibodies were 1,502 (25.0%) for any of the 4 helminthiases. The overall positive numbers for clonorchiasis, paragonimiasis, cysticercosis, and sparganosis were 728 (12.1%), 166 (2.8%), 729 (12.1%), and 263 (4.4%), respectively. The positive serologic reaction to multi-antigens was determined in 309 (20.6%) of the 1,502 total seropositive subjects. Those with multi-antigen positivity were regarded as positive for the antigen of strongest reaction but cross-reaction to others with weak positive reaction. Annual seropositive rates for those 4 tissue helminthiases ranged from 12.1% to 35.7%. The highest rate was observed in age from 60 to 69 years old and prevalence of men (27.4%; 1,030/3,763) was significantly higher than of women (19.1%; 332/1,741). Hospital records of 165 ELISA positive patients were reviewed to confirm correlation with their clinical diagnosis. Paragonimiasis was highly correlated as 81.8% (9/11), cysticercosis 29.9% (20/67), clonorchiasis 29.0% (20/69), and sparganosis 11.1% (2/18). In conclusion, the multi-antigen ELISA using 4 helminth antigens is useful to differentiate suspected tissue-invading helminthiases, especially ELISA diagnosis of paragonimiasis is reliable. The seropositivity is still high among suspected patients in Korea.

Prevalence of Clonorchis sinensis Infection among Residents along 5 Major Rivers in the Republic of Korea

Clonorchis sinensis is currently the most important parasite affecting public health problems in the Republic of Korea. We investigated the prevalence of C. sinensis infection among residents living along 5 major rivers in Korea. A total of 42,562 individual stool samples were collected from 37 localities and examined using the formalin-ether sedimentation technique. Helminth eggs were detected in 4,052 (9.5%) residents and 3,586 (8.4%) were infected with C. sinensis. The egg positive rate of C. sinensis in Nakdong, Seomjin, Geum, Yeongsan, and Han River was 11.7%, 9.9%, 6.5%, 3.1%, and 1.0%, respectively. The overall prevalence of clonorchiasis by sex was 11.2% in males and 6.2% in females. The age-prevalence was the highest in the 50-59 years band. It has been reconfirmed that the endemicity of clonorchiasis is higher in southern areas of Korea, especially along Nakdong and Seomjin Rivers. A combination of continuous control programs with health education initiatives is urgently required in these highly endemic areas of clonorchiasis in Korea.

Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells

Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.

Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells

Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.

Prevalence and Related Factors of Clonorchiasis among Five Major Riverside Residents in South Korea

PURPOSE: This study attempted to investigate the prevalence and related factors of Clonorchiasis among five major riverside residents in South Korea. METHODS: This study is descriptive research, nationwide survey, and the subjects are 23,492 residents selected by convenience sampling. Data collection was conducted between March 1 and June 30, 2011, and stool collection and questionnaire survey were conducted by affiliated public health centers in 38 cities and Gun's. RESULTS: The prevalence rates of Clonorchiasis in the five major riversides were as follows: the Guem River 15.2%; the Nakdong River 11.9%; the Seomjin River 10.9%; the Han River 5.7%; and the Yeongsan River 3.9%. The prevalence rates were shown to be significantly high among people who had highly frequent experiences of eating and cooking freshwater raw fish, were diagnosed with liver and/or biliary tract diseases, and drank less than once a month. CONCLUSION: These results suggest that it is necessary to improve awareness of Clonorchiasis and provide intensive public health education for the riverside residents. And the target groups should be set up by reflecting the characteristics of at-risk groups, and it is necessary to prepare customized strategies for prevention and management of Clonorchiasis.

Pancreatic mucinous cystadenoma of borderline malignancy associated with Clonorchis sinensis

No abstract available.

Pathological Lesions and Inducible Nitric Oxide Synthase Expressions in the Liver of Mice Experimentally Infected with Clonorchis sinensis

The nitric oxide (NO) formation and intrinsic nitrosation may be involved in the possible mechanisms of liver fluke-associated carcinogenesis. We still do not know much about the responses of inducible NO synthase (iNOS) induced by Clonorchis sinensis infection. This study was conducted to explore the pathological lesions and iNOS expressions in the liver of mice with different infection intensity levels of C. sinensis. Extensive periductal inflammatory cell infiltration, bile duct hyperplasia, and fibrosis were commonly observed during the infection. The different pathological responses in liver tissues strongly correlated with the infection intensity of C. sinensis. Massive acute spotty necrosis occurred in the liver parenchyma after a severe infection. The iNOS activity in liver tissues increased, and iNOS-expressing cells with morphological differences were observed after a moderate or severe infection. The iNOS-expressing cells in liver tissues had multiple origins.

Characterization of a Clonorchis sinensis antigen, calmodulin, and its relationship with liver fibrosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis.</p><p><b>METHODS</b>The full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein.</p><p><b>RESULTS</b>The recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1: 51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis.</p><p><b>CONCLUSION</b>The pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis- associated hepatic fibrosis.</p>

Prevalence and Risk Factors of Clonorchiasis among Residents of Riverside Areas in Muju-gun, Jeollabuk-do, Korea

We evaluated the status of Clonorchis sinensis infection and potential risk factors among residents of riverside areas (Geumgang) in Muju-gun, Jeollabuk-do (Province), Korea. From January to February 2010, a total of 349 (171 males, 178 females) stool samples were collected and examined by the formalin-ether concentration technique. Also, village residents were interviewed using questionnaires to obtain information about C. sinensis infection-related risk factors. Overall egg-positive rate of C. sinensis was 13.2%. Egg-positive rates were significantly higher in males, farmers, and residents who had lived there more than 20 years, and in residents who had eaten raw freshwater fish than in opposite groups, respectively. However, there was no significant difference between age groups, education levels, cigarette smoking, alcohol drinking, health status, past history of infection, and experience of clonorchiasis medication and examination. Logistic regression analysis was performed to determine risk factors for clonorchiasis. On univariate analysis, the odds ratios for males, farmers, those who had lived there more than 20 years, and who had eaten raw freshwater fish were 2.41, 4.44, 3.16, and 4.88 times higher than those of the opposites, respectively. On multivariate analysis, the odds ratio of residents who had eaten raw freshwater fish was 3.2-fold higher than that of those who had not. These results indicate that residents living in Muju-gun, along the Geum River, Korea, have relatively high C. sinensis egg-positive rates, and the habit of eating raw freshwater fish was the major factor for the maintenance of clonorchiasis.