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.     

Control of clonorchiasis by repeated praziquantel treatment and low diagnostic efficacy of sonography

In Korea, Clonorchis sinensis infection is still highly prevalent because case detection in the field is difficult and the detected cases used to be incompletely cured due to treatment failure. The present study tried to control clonorchiasis in an endemic village by repeated treatments with praziquantel every 6 months and to evaluate sonography as a diagnostic measure. By stool examinations, the egg positive rate in the endemic village was 22.7%, but it decreased to 19.6% at 6 months, 15.1% at 12 months. 12.2% at 18 months, 6.3% at 24 months, 11.4% at 30 months, and 6.3% at 42 months after the beginning of repeated praziquantel administration. The sonography showed 61 (49.6%) positive cases of 123 screened residents: among egg-positives the sonography positive rate was 52.2% and among egg-negatives it was still 49%. The rate among cured cases was 64.3% after 6 months, 50.0% after 12 months, 50.0% after 18 months, and 66.7% after 24 months. In a non-endemic village, 64 residents were found egg-negative by fecal examination, but 20 (31.3%) of them were positive by sonography. The present findings indicate that control of clonorchiasis in an endemic village by repeated praziquantel treatment for 42 months is still insufficient and sonography is of little value for diagnosis of clonorchiasis.     

A Case of Probable Mixed-Infection with Clonorchis sinensis and Fasciola sp.: CT and Parasitological Findings

We report here a human case probably mixed-infected with Clonorchis sinensis and Fasciola sp. who was diagnosed by computed tomography (CT) scan, serological findings, and/or fecal examination. The patient was a 43-year-old Korean female and was admitted to Kyung Hee University Hospital with the complaints of fever and abdominal pain. On admission, marked eosinophilia was noted in her peripheral blood. CT scan showed specific lesions for clonorchiasis and fascioliasis in the liver, along with lesions suggestive of amebic abscess. Micro-ELISA revealed positive results for the 2 helminthic infections. Eggs of C. sinensis and trophozoites of Entamoeba histolytica were observed in the stool. Treatment with praziquantel followed by metronidazole and tinidazole reduced abnormalities in the liver and eosinophilia. This is the first case report of a possible co-infection with 2 kinds of liver flukes in the Republic of Korea.     

Genomics and molecular genetics of Clonorchis sinensis: current status and perspectives

Clonorchiasis caused by Clonorchis sinensis is an important foodborne parasitosis of humans and animals, and is predominantly a hepatobiliary disease. Globally, nearly 35 million people were infected with C. sinensis, with approximately 15 million being in China. Patients would chronically present fatigue, jaundice, abdominal discomfort, along with the increased risk of developing into a form of cholangiocarcinoma that is fatal to humans. Treatment of clonorchiasis by praziquantel has been very successful, but this is dependent on early accurate diagnosis and correct species identification. The present article reviews the current status of knowledge in genomics and functional genomics of C. sinensis, and summarizes the main DNA-based techniques for the specific diagnosis of C. sinensis infection and studies of genetic variation in C. sinensis, and provides perspectives for future studies. The advances in genomics and molecular genetics of C. sinensis shed new sight on our understanding of population structure of C. sinensis as well as the prevention and control of clonorchiasis.     

Infection Status of Freshwater Fish with Metacercariae of Clonorchis sinensis in Korea

This study investigated freshwater fish for their current infection status with metacercariae of Clonorchis sinensis in Korea. Twenty-one species of freshwater fish (n = 677) were collected from 34 regions nationwidely from February 2007 to June 2008. They were individually examined by digestion technique. Eight species of freshwater fish from 17 different regions were recognized positive for the metacercariae of C. sinensis. The positive rates (range of metacercariae number per fish) of fish by the species were as follows: 48% (1-1,142) in Pseudorasbora parva, 60% (1-412) in Pungtungia herzi, 15.7% (1-23) in Pseudogobio esocinus, 29% (1-7) in Acheilognathus intermedia, 21% (1-4) in Odontobutis interrupta, 33% (1-6) in Zacco temmincki, 3.6% (1-4) in Zacco platypus, and 26.3% (1) in Hemibarbus labeo. The two species, P. parva and P. herzi, are able to be the index fish for estimation of C. sinensis transmission in a certain locality. Still several species of freshwater fish are briskly transmitting C. sinensis infection in many riverside areas of southern Korea.     

Prevalence of clonorchiasis in southern endemic areas of Korea in 2006

This study was performed to investigate prevalence of clonorchiasis among the inhabitants living in villages along the 4 major rivers, Nakdong-gang (=river), Seomjin-gang, Youngsan-gang, and Guem-gang in southern Korea. From January to December 2006, a total of 24,075 stool samples (1 sample per an inhabitant) were collected in 23 localities and examined by the formalin-ether sedimentation technique. Of the inhabitants examined, 3,441 (14.3%) were found to harbor various types of intestinal parasite eggs, cysts or larvae. Numbers of infected people were 2,661 (11.1%) for Clonorchis sinensis, 431 (1.8%) for heterophyids, 226 (0.9%) for Entamoeba spp., 57 (0.2%) for Giardia lamblia, 30 (0.1%) for Trichuris trichiura, and 18 (0.07%) for echinostomes. Prevalence rates of clonorchiasis according to the river basin were 17.1% in Nakdong-gang, 11.2% in Seomjin-gang, 5.5% in Youngsan-gang and 4.6% in Guem-gang. Of the 2,661 C. sinensis egg-positive cases, 57.7% was male. The present findings suggest that clonorchiasis is still highly prevalent among inhabitants in the riverside areas of southern Korea, and it is necessary to implement a systematic control program in the endemic areas.     

Phylogeny and divergence time estimation of Coreoleuciscus splendidus populations (Teleostei: Cypriniformes) endemic to Korea based on complete mitogenome sequences

We determined the complete mitogenome sequences for four Coreoleuciscus splendidus strains (Teleostei: Cypriniformes) inhabiting the Han and Geum river drainages in the West Korea Subdistrict, and the Seomjin and Nakdong river drainages in the South Korea Subdistrict in the Korean peninsula. The gene contents and arrangement of C. splendidus were homogeneous to those of typical vertebrates. Phylogenetic analysis with the partitioned nucleotide matrix of concatenated mitochondrial genes revealed that C. splendidus formed a monophyletic group with gobionine species in the cyprinid lineage. The four river strains separated into two phylogenetically distinct groups, which were in accordance with the biogeographical distribution pattern in Korea according to subdistrict. The estimated divergence time among western and southern subdistrict populations was 37.9 ± 6.5 million years ago (Mya), whereas those between the two river strains in each subdistrict were estimated to be 3.2?C3.4 Mya. The presence of such distinct historical lineages has great implications for biogeography of ichthyofauna in Korea and future management and conservation plans of C. splendidus populations.     

Identification and Molecular Characterization of Parkin in Clonorchis sinensis

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn²⁺ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.     

Risk Factors of Clonorchis sinensis Human Infections in Endemic Areas,Haman-Gun,Republic of Korea: A Case-Control Study

Clonorchis sinensis is the most common fish-borne intestinal parasite in Korea. The aim of the present investigation was to survey the status of C. sinensis infection and analyze associated risk factors in residents of Haman-gun, Gyeongsangnam-do. A total of 5,114 residents from 10 administrative towns/villages voluntarily agreed to participate in the study, which comprised fecal examination, a questionnaire survey for risk factors, ultrasonography, and enzyme-linked immunosorbent assay for cancer biomarker detection in the blood. We detected C. sinensis eggs in 5.3% of the subjects. By region, Gunbuk-myeon had the highest number of residents with C. sinensis eggs. The infection rate and intensity were higher in male than in female residents. Based on the risk factor questionnaire, infection was highly associated with drinking, a history of C. sinensis infection, and the practice of eating of raw freshwater fish. Extension of the bile duct, infection intensity, and cancer biomarker detection significantly correlated with the presence of eggs in the study population. In conclusion, the development of feasible, long-term control policies and strategies for the elimination of C. sinensis in Korea is still required.     

Immunodiagnosis of clonorchiasis using a recombinant antigen

A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected in view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. sinensis antigen with 28 kDa as a β-galactosidase fusion protein produced in Escherichia coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies of a 30 base pair repeat and an additional 320 bases. The deduced amino acid sequence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis. C. sinensis adult worm RNA showed a positive reaction with the cloned gene. Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed high specificity for diagnosis of clonorchiasis.     

Decrease of Metagonimus yokogawai Endemicity along the Tamjin River Basin

The Tamjin River which flows from Jangheung-gun via Gangjin-gun to the South Sea was reported to be a highly endemic area of Metagonimus yokogawai infection in 1977 and 1985. However, there were no recent studies demonstrating how much change occurred in the endemicity, in terms of prevalence and worm burden, of metagonimiasis in this river basin. Thus, a small-scale epidemiological survey was carried out on some residents along the Tamjin River basin in order to determine the current status of M. yokogawai infection. A total of 48 fecal samples were collected and examined by the Kato-Katz thick smear and formalin-ether sedimentation techniques. The egg positive rate of all helminths was 50.0%, and that of M. yokogawai was 37.5%, followed by C. sinensis 22.9% and G. seoi 4.2%. To obtain the adult flukes of M. yokogawai, 6 egg positive cases were treated with praziquantel 10 mg/kg in a single dose and purged with magnesium sulfate. A total of 5,225 adult flukes (average 871 specimens per person) of M. yokogawai were collected from their diarrheic stools. Compared with the data reported in 1977 and 1985, the individual worm burdens appeared to have decreased remarkably, although the prevalence did not decrease at all. It is suggested that the endemicity of M. yokogawai infection along the Tamjin River has been reduced. To confirm this suggestion, the status of infection in snail and fish intermediate hosts should be investigated.     

Clonorchiasis--a historical review of contributions of Japanese parasitologists

In the early stage of research on Clonorchis and clonorchiasis, Japanese parasitologists made a tremendous contribution on the elucidation of the life cycle of this parasite and on the epidemiology of the disease. Harujiro Kobayashi first identified cyprinoid fish as the second intermediate hosts for Clonorchis sinensis in 1912, Subsequently Parafossarulus snails were identified as the first intermediate host by Masatomo Muto in 1918. Kenso Ishisaka recorded the first human case of clonorchiasis in Japan in 1877, and two Japanese clinicians, Shigeru Matsumoto and Tsukasa Ohi, recorded the endemic nature of this disease in Korea and Taiwan respectively, in the same year, 1915.     

Improved genomic resources and new bioinformatic workflow for the carcinogenic parasite Clonorchis sinensis: Biotechnological implications

Clonorchis sinensis (family Opisthorchiidae) is an important foodborne parasite that has a major socioeconomic impact on ~35 million people predominantly in China, Vietnam, Korea and the Russian Far East. In humans, infection with C. sinensis causes clonorchiasis, a complex hepatobiliary disease that can induce cholangiocarcinoma (CCA), a malignant cancer of the bile ducts. Central to understanding the epidemiology of this disease is knowledge of genetic variation within and among populations of this parasite. Although most published molecular studies seem to suggest that C. sinensis represents a single species, evidence of karyotypic variation within C. sinensis and cryptic species within a related opisthorchiid fluke (Opisthorchis viverrini) emphasise the importance of studying and comparing the genes and genomes of geographically distinct isolates of C. sinensis. Recently, we sequenced, assembled and characterised a draft nuclear genome of a C. sinensis isolate from Korea and compared it with a published draft genome of a Chinese isolate of this species using a bioinformatic workflow established for comparing draft genome assemblies and their gene annotations. We identified that 50.6% and 51.3% of the Korean and Chinese C. sinensis genomic scaffolds were syntenic, respectively. Within aligned syntenic blocks, the genomes had a high level of nucleotide identity (99.1%) and encoded 15 variable proteins likely to be involved in diverse biological processes. Here, we review current technical challenges of using draft genome assemblies to undertake comparative genomic analyses to quantify genetic variation between isolates of the same species. Using a workflow that overcomes these challenges, we report on a high-quality draft genome for C. sinensis from Korea and comparative genomic analyses, as a basis for future investigations of the genetic structures of C. sinensis populations, and discuss the biotechnological implications of these explorations.     

Prevalence of Zoonotic Trematode Metacercariae in Freshwater Fish from Gangwon-do,Korea

The infection status of zoonotic trematode metacercariae was investigated in a total of 2,293 freshwater fish collected from 11 rivers or streams in 9 administrative regions of Gangwon-do, Korea for 5 years (2009-2013). All fish were collected by netting methods and examined using the artificial digestion methods. Clonorchis sinensis metacercariae were detected in 4 fish species, i.e., Pungtungia herzi, Squalidus japonicus coreanus, Acheilognathus rhombeus, and Ladislabia taczanowskii, from only Hantangang in Cheorwon-gun. Metagonimus spp. metacercariae were found in 1,154 (50.3%) fish and their average number per infected fish was 55.8. Among the positive fish species, especially Tribolodon hakonensis from Namdaecheon in Yangyang-gun and Plecoglossus altivelis from Osipcheon in Samcheok-si were most heavily infected. Centrocestus armatus metacercariae were detected in 611 (26.7%) fish and the average metacercarial burden per infected fish was 1,032. Two chub species, Zacco platypus and Zacco temminckii were highly and heavily infected with C. armatus metacercariae in almost all regions surveyed. Echinostoma spp. metacercariae were also found in 24 fish from a few localities, but their numbers per fish infected were very low. From the above results, it is confirmed that the metacercariae of intestinal flukes, especially Metagonimus spp. and C. armatus, were heavily infected, while C. sinensis metacercariae were rarely found in fish from Gangwon-do, Korea.     

High-quality reference genome for Clonorchis sinensis

The Chinese liver fluke, Clonorchis sinensis, causes the disease clonorchiasis, affecting ~35 million people in regions of China, Vietnam, Korea and the Russian Far East. Chronic clonorchiasis causes cholangitis and can induce a malignant cancer, called cholangiocarcinoma, in the biliary system. Control in endemic regions is challenging, and often relies largely on chemotherapy with one anthelmintic, called praziquantel. Routine treatment carries a significant risk of inducing resistance to this anthelmintic in the fluke, such that the discovery of new interventions is considered important. It is hoped that the use of molecular technologies will assist this endeavour by enabling the identification of drug or vaccine targets involved in crucial biological processes and/or pathways in the parasite. Although draft genomes of C. sinensis have been published, their assemblies are fragmented. In the present study, we tackle this genome fragmentation issue by utilising, in an integrated way, advanced (second- and third-generation) DNA sequencing and informatic approaches to build a high-quality reference genome for C. sinensis, with chromosome-level contiguity and curated gene models. This substantially-enhanced genome provides a resource that could accelerate fundamental and applied molecular investigations of C. sinensis, clonorchiasis and/or cholangiocarcinoma, and assist in the discovery of new interventions against what is a highly significant, but neglected disease-complex.     

Epidemiological Survey on the Infection of Intestinal Flukes in Residents of Muan-gun, Jeollanam-do, the Republic of Korea

Infection status of intestinal flukes was investigated in residents of Muan-gun, Jeollanam-do, the Republic of Korea. Total 1,257 fecal samples of residents were examined by formalin-ether sedimentation technique and Kato-Katz thick smear method. Helminth eggs were detected from 95 (7.6%) residents, and eggs of heterophyid flukes and Clonorchis sinensis were found from 62 (4.9%) and 40 (3.2%) cases, respectively. The larger heterophyid eggs, somewhat dark-brown in color and 37.7 × 21.5 µm in average size, and found in 32 (2.6%) out of 62 egg positive cases of heterophyid flukes. To confirm the adult flukes, we performed worm recovery from 12 cases after praziquantel treatment and purgation with MgSO₄. A total of 1,281 adult flukes, assigned to 7 species, were recovered from 9 cooperative cases. Heterophyes nocens (total 981 specimens) was collected from 9 cases, Stictodora fuscata (80) from 7, Gymnophalloides seoi (75) from 5, Pygidiopsis summa (140) from 3, Stellantchasmus falcatus (3) from 2, and Stictodora lari and Acanthotrema felis (each 1 worm) from 1 case each. The intrauterine eggs of S. fuscata collected from the recovered worm were identical with the larger heterophyid eggs detected in the stool examination. By the present study, it was confirmed that A. felis is a new intestinal fluke infecting humans, and residents in Muan-gun, Jeollanam-do are infected with variable species of intestinal trematodes.     

Human Infections with Liver and Minute Intestinal Flukes in Guangxi, China: Analysis by DNA Sequencing, Ultrasonography, and Immunoaffinity Chromatography

The prevalence of liver and intestinal fluke infections was determined by surveying inhabitants of Hengxuan, Fusui, and Shanglin villages which were known to be endemic for liver flukes in Guangxi, China in May 2010. A total of 718 people were examined for helminth eggs by the Kato-Katz thick smear technique, ultrasonography, immunoaffinity chromatography, and DNA sequencing. The overall egg positive rate was found to be 59.6% (28.0-70.6%) that included mixed infections with liver and intestinal flukes. Cases showing higher than 20,000 eggs per gram of feces (EPG) were detected between 1.3% and 16.2%. Ultrasonographic findings exhibited overall 28.2% (72 of 255 cases) dilatation rate of the intrahepatic bile duct. Clonorchis sinensis infection was detected serologically in 88.3% (38 of 43 cases) among C. sinensis egg positive subjects by the immunoaffinity chromatography using a specific antigen for C. sinensis. For differential diagnosis of the liver and intestinal flukes, more precise PCR and nucleotide sequencing for copro-DNA were performed for 46 egg positive cases. Mixed infections with C. sinensis and Metagonimus yokogawai were detected in 8 of 46 egg positive cases, whereas 29 specimens were positive for Haplorchis taichui. Ultrasonographic findings and immunoaffinity chromatography results showed usefulness, even in a limited way, in figuring out of the liver fluke endemicity.     

Serologic Survey of Toxoplasmosis in Seoul and Jeju-do, and a Brief Review of Its Seroprevalence in Korea

Knowledge of the prevalence of human Toxoplasma gondii infection is required in the Republic of Korea. In this study, we surveyed the seroprevalence of T. gondii infection and analyzed the risk factors associated with seropositivity among residents in 2 administrative districts; Seoul and the island of Jeju-do, which have contrasting epidemiologic characteristics. Sera and blood collected from 2,150 residents (1,114 in Seoul and 1,036 in Jeju-do) were checked for IgG antibody titers using ELISA and for the T. gondii B1 gene using PCR. In addition, participants completed a questionnaire that solicited information on gender, age, occupation, eating habits, history of contact with animals, and travel abroad. The T. gondii B1 gene was not detected in all residents examined. However, ELISA showed 8.0% (89 of 1,114 sera) positive for IgG antibodies against T. gondii in Seoul and 11.3% (117 of 1,036 sera) in Jeju-do. In both districts, the positive rates were higher in males than in females, and those 40-79 years of age showed higher rates than other ages. In Seoul, residents older than 70 years of age showed the highest positive rate, 14.9%, whereas in Jeju-do the highest prevalence, 15.6%, was in those in their sixties. The higher seropositive rate in Jeju-do than in Seoul may be related to eating habits and occupations. The present results and a review of related literature are indicative of an increased seroprevalence of T. gondii in Korea in recent years.     

Painless Jaundice Caused by Clonorchis sinensis Infection: A Case Report

A man with only yellowing of the skin and eye sclera was diagnosed with clonorchiasis, which rarely manifested jaundice as the initial symptom. However, because of a lack of evidence for a diagnostic gold standard, the time until definitive diagnosis was more than a week. The diagnostic process relied on inquiring about the patient’s history, including the place of residence, dietary habits, and symptoms, as well as on serological findings, an imaging examination, and pathological findings. MRCP and CT results showed mild dilatation of intrahepatic ducts and increased periductal echogenicity. The eggs were ultimately found in stool by water sedimentation method after the negative report through direct smear. DNA sequencing of PCR production of the eggs demonstrated 98-100% homology with ITS2 of Clonorchis sinensis. After anti-parasite medical treatment, the patient’s symptoms were gradually relieved. Throughout the diagnostic procedure, besides routine examinations, the sedimentation method or concentration method could be used as a sensitive way for both light and heavy C. sinensis infection in the definite diagnosis.