OBJECTIVE: To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay. METHODS: Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples. RESULTS: Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ² = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ² = 11.20, P < 0.05) and fluorescent RAA assay (χ² = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05). CONCLUSIONS Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method.
Animals ; Clonorchis sinensis/genetics* ; Metacercariae/genetics* ; Recombinases ; Fresh Water ; Fishes ; DNA ; Fish Diseases/diagnosis*

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
Animals ; Cat Diseases/parasitology* ; Cats ; China/epidemiology* ; Clonorchiasis/veterinary* ; Clonorchis sinensis/genetics* ; Dog Diseases/parasitology* ; Dogs ; Fish Diseases/parasitology* ; Fishes ; Humans ; Incidence ; Prevalence ; Species Specificity

The infection status with Clonorchis sinensis metacercariae (CsMc) was examined in freshwater fishes from Yangcheon (a branch of Gyeongho-gang), which is located in Sancheong-gun, Gyeongsangnam-do, the Republic of Korea. Total 2,201 fishes in 26 species were examined by the artificial digestion method through 7 years. CsMc were detected in 1,171 (53.2%) fishes in 21 spp. (80.8%) and their density was 85 per fish infected. Total 532 (99.6%) out of 534 Pungtungia herzi (index fish) examined were infected with 147 CsMc per fish infected. Metacercarial densities in this fish were highest in 2015 (179 CsMc), followed by 2012 (168), 2013 (152), 2016 (145), 2014 (114), and 2017 (89) respectively. In the gobioninid fish group, i.e., P. herzi, Sarcocheilichthys spp., Squalidus spp., Pseudogobio esocinus, Hemibarbus longirostris, and Hemibarbus labeo, 841 (92.7%) fishes were infected with 117 CsMc per fish infected. Total 250 (54.7%) acheilognathinid fish (bitterlings), Acheilognathus spp. and Acanthorhodeus spp. were infected with 5.8 CsMc. In the rasborinid fish (chubs) group, i.e., Zacco platypus, Zacco temminckii, and Zacco koreanus, 77 (13.7%) out of 563 fish examined were infected with 2.4 CsMc in average. The susceptibility indices of CsMc were 49.09 in the overall positive fish group, 104.15 in the gobioninid group, 3.17 in the acheilognathinid group and 0.35 in the rasborinid fish group respectively. Only 1 CsMc was detected in 3 fish species, Coreoperca herzi, Channa argus, and Lepomis macrochirus, respectively. Conclusively, it was confirmed that CsMc are moderately prevalent in fishes from Yangcheon in Sancheon-gun, Gyeongsangnam-do, Korea.
Clonorchis sinensis ; Cyprinidae ; Digestion ; Fishes ; Fresh Water ; Gyeongsangnam-do ; Korea ; Metacercariae ; Methods ; Platypus ; 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.
Animals ; Anorexia ; Bile ; Bile Ducts ; Biopsy, Needle ; Cats ; Chungcheongbuk-do ; Clonorchiasis ; Clonorchis sinensis ; Ecosystem ; Eggs ; Fasciola hepatica ; Follow-Up Studies ; Gyeongsangnam-do ; Korea ; Microscopy ; Ovum ; Panthera ; Physical Examination ; Praziquantel ; Quarantine ; Republic of Korea ; Shoulder ; Ultrasonography ; Urinary Bladder

The infection status of digenetic trematode metacercariae (DTM) was investigated in fishes from 2 representative visiting sites of migratory birds in Gyeongsangnam-do, the Republic of Korea (Korea). A totaly 220 freshwater fishes (7 species) were collected from Junam-jeosuji (reservoir), and 127 fishes (7 species) were also collected from Woopo-neup (swamp) in June and October 2017. As the control group, total 312 fish (22 spp.) from Yangcheon in Sancheong-gun, Gyeongsangnam-do were also collected in June and October 2017. All fishes collected in 3 sites were examined with the artificial digestion method. In the fishes from Junam-jeosuji, more than 4 species, i.e., Clonorchis sinensis, Echinostoma spp., Diplostomum spp. and Cyathocotyle orientalis, of DTM were detected and their endemicy was very low, 0.70. More than 6 species, i.e., C. sinensis, Echinostoma spp., Metorchis orientalis, Clinostomum complanatum, Diplostomum spp. and C. orientalis, of DTM were found in the fishes from Woopo-neup, and their endemicy was low, 5.16. In the fishes from Yangcheon, more than 8 species, i.e., C. sinensis, Metagonimus spp., Centrocestus armatus, C. complanatum, C. orientalis, M. orientalis, Echinostoma spp., and Diplostomum spp., of DTM were detected, and their endemicity was relatively high, 95.48. The percentages of avian trematode metacercariae (ATM) were 99.6% and 94.7% in fishes from Junam-jeosuji and Woopo-neup whereas it was 74.1% in the control site, Yangcheon. The above findings suggested that migratory birds partly affect in endemicity of DTM in the fish in the 2 visiting sites in Gyeongsangnam-do, Korea.
Birds ; Clonorchis sinensis ; Digestion ; Echinostoma ; Fishes ; Fresh Water ; Gyeongsangnam-do ; Heterophyidae ; Korea ; Metacercariae ; Methods ; Republic of Korea

Clonorchis sinensis is a carcinogenic human liver fluke that promotes hepatic inflammatory environments via direct contact or through their excretory-secretory products (ESPs), subsequently leading to cholangitis, periductal fibrosis, liver cirrhosis, and even cholangiocarcinoma (CCA). This study was conducted to examine the host inflammatory responses to C. sinensis ESPs and their putative protein components selected from C. sinensis expressed sequenced tag (EST) pool databases, including TGF-β receptor interacting protein 1(CsTRIP1), legumain (CsLeg), and growth factor binding protein 2 (CsGrb2). Treatment of CCA cells (HuCCT1) with the ESPs or bacterial recombinant C. sinensis proteins differentially promoted the secretion of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) as well as anti-inflammatory cytokines (IL-10, TGF-β1, and TGF-β2) in a time-dependent manner. In particular, recombinant C. sinensis protein treatment resulted in increase (at maximum) of ~7-fold in TGF-β1, ~30-fold in TGF-β2, and ~3-fold in TNF-α compared with the increase produced by ESPs, indicating that CsTrip1, CsLeg, and CsGrb2 function as strong inducers for secretion of these cytokines in host cells. These results suggest that C. sinensis ESPs contribute to the immunopathological response in host cells, leading to clonorchiasis-associated hepatobiliary abnormalities of greater severity.
Carrier Proteins ; Cholangiocarcinoma ; Cholangitis ; Clonorchis sinensis ; Cytokines ; Fasciola hepatica ; Fibrosis ; Humans ; Interleukin-6 ; Liver Cirrhosis

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.
Animals ; Aspartic Acid Endopeptidases ; Biology ; Cathepsin D ; Cathepsins ; Clonorchiasis ; Clonorchis sinensis ; Eggs ; Helminths ; Humans ; Intestines ; Metacercariae ; Ovum ; Parasites ; Peptide Hydrolases ; Rats ; Reproduction

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.
Animals ; Anisakiasis ; Biology ; Clonorchiasis ; Clonorchis sinensis ; Cryptosporidiosis ; Cysticercosis ; Diagnosis ; Diagnosis, Differential ; Eating ; Enterobiasis ; Enzyme-Linked Immunosorbent Assay ; Epidemiology ; Fever ; Headache ; Humans ; Korea ; Malaria ; Opportunistic Infections ; Ovum ; Paragonimiasis ; Parasitic Diseases ; Prevalence ; Prognosis ; Republic of Korea ; Seafood ; Sparganosis ; Splenomegaly ; Toxocariasis ; Toxoplasmosis ; Trichuriasis ; Vaginal Diseases

A 59-year-old woman presented with abdominal pain. Abdominal computerized tomography was suggestive of biliary stones. During endoscopic retrograde cholangiopancreatography, adult worms resembling Clonorchis sinensis (C. sinensis) were drained. Eggs were detected in stool using the formalin-ether concentration method and C. sinensis-specific antibody was detected in the serum. A diagnosis of C. sinensis infection was made. The symptoms of the patient gradually resolved after treatment with anti-parasite medication. The patient lived in a non-endemic region for C. sinensis infection and had no history of intake of raw or undercooked freshwater fishes. South Korea is one of the endemic countries for C. sinensis infection and people can be infected via indirect routes of transmission such as cooking utensils. Therefore, the possibility of C. sinensis infection should be considered in patients presenting with biliary diseases in South Korea. We describe the clinical findings of this case with a review of literature.
Abdominal Pain ; Adult ; Cholangiopancreatography, Endoscopic Retrograde ; Cholangitis ; Cholecystitis ; Cholecystitis, Acute ; Clonorchis sinensis ; Cooking and Eating Utensils ; Diagnosis ; Eggs ; Female ; Fishes ; Fresh Water ; Humans ; Korea ; Methods ; Middle Aged ; Ovum

We analyzed Clonorchis sinensis ancient DNA (aDNA) acquired from the specimens of the Joseon mummies. The target regions were cytochrome C oxidase subunit 1 (CO1), internal transcribed spacer 1 (ITS1), nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase subunits 2 (NAD2) and 5 (NAD5). The sequences of C. sinensis aDNA was completely or almost identical to modern C. sinensis sequences in GenBank. We also found that ITS1, NAD2 and NAD5 could be good markers for molecular diagnosis between C. sinensis and the other trematode parasite species. The current result could improve our knowledge about genetic history of C. sinensis.
Clonorchis sinensis ; Cytochromes c ; Cytochromes ; Databases, Nucleic Acid ; Diagnosis ; DNA ; Electron Transport Complex IV ; Hydrogen ; Mummies ; NAD ; Niacinamide ; Oxidoreductases ; Parasites ; Republic of Korea