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Objective:To observe the expression changes of microRNA(miR)-122 in liver tissue of rats infected with Clonorchis sinensis and its correlation with expression level of inflammatory cytokines. Methods:Totally 24 SPF grade Wistar male rats were selected and randomly divided into a control group (100 μl physiological saline gavage), a 4-week infection group (100 Clonorchis sinensis metacercariae gavage), and an 8-week infection group (100 Clonorchis sinensis metacercariae gavage) based on body weight (100-120 g) using a random number table method, with 8 rats in each group. Starting from the third week of infection, rat feces were collected and directly smeared with physiological saline for identification of Wistar rat animal models infected with Clonorchis sinensis. After 4 and 8 weeks of infection, the rats in the 4- and 8-week infection groups were euthanized, while 4 rats in the control group were euthanized, respectively. The heart blood and left lobe liver tissue and serum samples were collected from each group of rats. Using hematoxylin-eosin (HE) staining to observe liver pathological damage under the light microscope, real-time fluorescence quantitative PCR to detect the expression level of miR-122 in liver tissue, and Luminex 200 liquid suspension chip to detect the expression levels of serum inflammatory cytokines [tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6 (IL-6)]. The correlation between miR-122 and inflammatory cytokines was analyzed using Pearson correlation. Results:Under the light microscope, the morphology of hepatocytes in control group was normal, and no inflammatory cell infiltration was observed. There was inflammatory cells such as lymphocyte, eosinophil and other inflammatory cell infiltration around the portal area in the 4-week infection group. The hepatocytes of the 8-week infected rats were arranged in a disordered manner, with varying degrees of swelling, loose and lightly stained cytoplasm, and some hepatocytes showed watery degeneration; additionally, bile duct dilation and thickening of the bile duct wall were observed in the liver tissue. There were statistically significant differences of liver miR-122 (1.00 ± 0.32, 2.57 ± 0.60, 3.63 ± 1.63), serum TNF-α [(0.14 ± 0.06), (0.43 ± 0.09), (0.61 ± 0.10) ng/ml], and IL-6 expression levels [(0.03 ± 0.01), (1.06 ± 0.24), (1.48 ± 0.33) ng/ml] in control group, 4- and 8-week infection groups ( F = 13.36, 69.99, 82.23, P < 0.001). There was no statistically significant difference in expression level of IL-1β between different groups ( F = 2.15, P = 0.141). The Pearson correlation analysis showed that the expression level of miR-122 was positively correlated with the expression levels of inflammatory cytokines TNF-α and IL-6 ( r = 0.67, 0.80, P < 0.001). Conclusion:Clonorchis sinensis infection can increase the expression of miR-122 in the host liver tissue, and the miR-122 is closely related to the expression levels of inflammatory cytokines TNF-α and IL-6.
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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.
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Clonorchis sinensis is considered a class I carcinogen for cholangiocarcinoma, but an increasing number of studies have found that it is also closely associated with hepatocellular carcinoma. This paper reviews the discovery and prevalence, historical studies, key regional studies, animal models and complications of Clonorchis sinensis, and summarizes the possible molecular mechanisms of Clonorchis sinensis contributing to hepatocellular carcinoma development, in order to gain insight into the correlation between Clonorchis sinensis and hepatocellular carcinoma, and thus to provide new ideas for the study of the effects of Clonorchis sinensis infection on hepatocellular carcinoma.
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@#Abstract: Objective To analyze the changes of coagulation indicators in patients with hepatitis B complicated with Clonorchis sinensis (C. Sinensis), and provide reference value for diagnosis, drug using and prognosis monitoring. Methods The patient samples were collected from the Third Affiliated Hospital of Sun Yat-sen University from June 2018 to February 2022 and divided into six groups. They were 40 healthy patients, 47 patients with simple chronic hepatitis B, 47 patients with post-hepatitis B liver cirrhosis, 40 patients with C. Sinensis mono-infection, 30 patients with chronic hepatitis B patients co-infected with C. Sinensis and 27 patients with post-hepatitis B liver cirrhosis patients coinfected with C. Sinensis. Four coagulation indexes, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB), were detected and compared among the groups. Results Compared with the healthy group, C. Sinensis mono-infection group had higher level of FIB and delayed PT, APTT; chronic hepatitis B group and chronic hepatitis B patients co-infected with C. Sinensis group had delayed PT, APTT, TT and significant lower FIB, these differences were statistically significant (all P<0.05). Compared with simple chronic hepatitis B group, post-hepatitis B liver cirrhosis and chronic hepatitis B patients co-infected with C. Sinensis had significant delayed PT, APTT, TT and lower FIB (all P<0.05). Compared with the post-hepatitis B liver cirrhosis and chronic hepatitis B patients co-infected with C. Sinensis, post-hepatitis B liver cirrhosis patients coinfected with C. Sinensis group had significant delayed PT, APTT, TT and lower FIB (all P<0.05). Conclusions The coinfection of C. Sinensis will further aggravate the coagulation dysfunction of HBV patients, leading to poor treatment and prognosis. HBV patients will have worse coagulation function in the process to post-hepatitis B liver cirrhosis; Therefore, it is important to pay attention to C. Sinensis co-infection when treating HBV patients, so that further guidance on clinical use and monitoring of prognosis can be provided.
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【Objective】 To analyze the polymorphism of erythrocyte membrane blood group glycoprotein A (GPA) related gene GYPA in high and low endemic population for clonidia sinensis infection, aimed at investigating the correlation between erythrocyte transmembrane glycoproteins and clonorchis sinensis infection. 【Methods】 From Dec 2019 to Jun 2020, anticoagulant blood samples were randomly collected in WuMing district (n=700) and GuiGang district (n=500 ) of Nanning city, and the IgG antibody to clonorchis sinensis in plasma was detected, and the DNA of leukocyte was extracted. The full-length exon and partial intron of GYPA gene were sequenced, mutations were characterized by gene cloning, and the risk of infection was calculated by chi-square test. 【Results】 The yield rate of IgG antibody was 62.7% (439/700) in WuMing district and 3.4% (17/500) in GuiGang district(P<0.05). The insertion of base C at the 54th position of intron-2 in GYPA gene caused the reading frame shift. The mutation was presented in 23.9% (105/439) and 17.6% (3/17) of the population with clonorchis sinensis exposure in WuMing and GuiGang area, respectively, while 49.4% (129/261) and 54.7% (264/483) in the negative population, respectively (P<0.05). 【Conclusion】 The infection rate of clonorchis sinensis in WuMing district was higher than that in GuiGang district. The mutation rate of reading frame shift caused by the insertion of base C at the 54th position of GYPA intron-2 was much lower in the positive population of clonorchis sinensis infection than the negative population, suggesting that the mutation is a protective gene in the negative population of clonorchis sinensis infection. It is necessary to study the mechanism of clonorchis sinensis infection and the mutation point of this gene in order to facilitate the early diagnosis of disease, blood transfusion management, treatment and prevention.
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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
Subject(s)
Animals , Cats , Dogs , Humans , Cat Diseases/parasitology , China/epidemiology , Clonorchiasis/veterinary , Clonorchis sinensis/genetics , Dog Diseases/parasitology , Fish Diseases/parasitology , Fishes , Incidence , Prevalence , Species SpecificityABSTRACT
Objective To understand the real prevalence of Clonorchis sinensis infections in the freshwater fish in mainland China, so as to provide insights into clonorchiasis control and detection of freshwater fish. Methods All literatures reporting the prevalence of C. sinensis infections in the freshwater fish, the second intermediate host of the parasite, were jointly retrieved in Chinese and English electronic databases from January 1, 2010 to December 31, 2020, including Wanfang Data, CNKI, PubMed, Web of Science, Embase and Cochrane Library. All studies were screened based on inclusion and exclusion criteria, and the quality of all enrolled literatures was evaluated. The pooled prevalence of C. sinensis infections in freshwater fish and its 95% confidence interval (CI) were estimated using the software Stata version 15.0, and subgroup analyses were performed to investigate the region-, season- and sample source-specific pooled prevalence of C. sinensis infections in freshwater fish. In addition, the sensitivity and publication bias of all included studies were analyzed. Results A total of 40 eligible literatures were included in this study, including 37 Chinese literatures and 3 English literatures, and there were 10 high-quality literatures, 27 moderate-quality literatures and 3 low-quality literatures. A total of 53 species containing 37 959 freshwater fish were reported in these 40 studies, and 73.58% (39/53) of freshwater fish species were identified with C. sinensis infections. Meta-analysis showed 23.5% [95% CI: (0.19, 0.28)] pooled prevalence of C. sinensis infections in freshwater fish in mainland China, and subgroup analyses higher prevalence of C. sinensis infections in freshwater fish in northeastern China [35.7%, 95% CI: (0.22, 0.50)] than in central [25.9%, 95% CI: (0.04, 0.48)] and southern China [20.6%, 95% CI: (0.09, 0.32)], higher prevalence of C. sinensis infections in freshwater fish sampled in spring [44.1%, 95% CI: (0.35, 0.53)] than in autumn [6.7%, 95% CI: (0.05, 0.08)] and summer [3.3%, 95% CI: (−0.01, 0.07)], and higher prevalence of C. sinensis infections in freshwater fish sampled from natural water [25.2%, 95% CI: (0.17, 0.33)] than from retail trades [22.2%, 95% CI: (0.17, 0.28)] and breeding chain [12.3%, 95% CI: (0.03, 0.22)]. However, all included studies had a publication bias with a low sensitivity. Conclusions The prevalence of C. sinensis infections is high in freshwater fish in mainland China, and there are still challenges for clonorchiasis control. Reinforcement of health education, diagnostics development and food safety supervision is recommended in future clonorchiasis control programs.
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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.
Subject(s)
Animals , Humans , Rats , Aspartic Acid Endopeptidases , Biology , Cathepsin D , Cathepsins , Clonorchiasis , Clonorchis sinensis , Eggs , Helminths , Intestines , Metacercariae , Ovum , Parasites , Peptide Hydrolases , ReproductionABSTRACT
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.
Subject(s)
Clonorchis sinensis , Cytochromes c , Cytochromes , Databases, Nucleic Acid , Diagnosis , DNA , Electron Transport Complex IV , Hydrogen , Mummies , NAD , Niacinamide , Oxidoreductases , Parasites , Republic of KoreaABSTRACT
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.
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Humans , Carrier Proteins , Cholangiocarcinoma , Cholangitis , Clonorchis sinensis , Cytokines , Fasciola hepatica , Fibrosis , Interleukin-6 , Liver CirrhosisABSTRACT
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.
Subject(s)
Clonorchis sinensis , Cyprinidae , Digestion , Fishes , Fresh Water , Korea , Metacercariae , Methods , Platypus , Republic of KoreaABSTRACT
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.
Subject(s)
Adult , Female , Humans , Middle Aged , Abdominal Pain , Cholangiopancreatography, Endoscopic Retrograde , Cholangitis , Cholecystitis , Cholecystitis, Acute , Clonorchis sinensis , Cooking and Eating Utensils , Diagnosis , Eggs , Fishes , Fresh Water , Korea , Methods , OvumABSTRACT
Objective To establish a recombinase aided isothermal amplification (RAA) assay for detection of Clonorchis sinensis. Methods The 18S ribosomal RNA (18S rRNA) sequence of C. sinensis was used as the target sequence, and specific primers and probes were designed, synthesized and screened to establish a rapid fluorescent RAA assay for the detection of C. sinensis. Then, the sensitivity of the fluorescent RAA assay was evaluated using the recombinant plasmids containing various copy numbers of DNA fragments and C. sinensis genomic DNA at various concentrations, and the specificity of the fluorescent RAA as say was evaluated using the genomic DNA of Ascaris lumbricoides, Echinococcus granulosus, Schistosoma japonicum, Ancylostoma duodenale and S. mansoni as templates. DNA samples were extracted from the feces containing C. sinensis eggs and freshwater fish containing metacercaria for the fluorescent RAA assay, and the performance for detection of C. sinensis-infected samples was preliminarily assessed in the field. Results A fluorescent RAA assay for detection of C. sinensis was successfully established, which was feasible for specific amplification of C. sinensis genomic DNA at 39 °C within 20 min. The lowest detection limit was 10 copies/μL if the recombinant plasmid containing various copy numbers of DNA fragments was used as a template, and the lowest detection limit was 3 pg/μL if the C. sinensis genomic DNA at various concentrations served as a template. All detections were negative if the genomic DNA of A. lumbricoides, E. granulosus, S. japonicum, A. duodenale, and S. mansoni was used as templates. In addition, the fluorescent RAA assay showed a high performance for the detection of C. sinensis-infected samples in the field, which successfully detected C. sinensis-infected human and rat fecal samples and Pseudorasbora parva samples. Conclusion A fluorescent RAA assay is successfully established, which is simple, rapid, sensitivity and specific for detection of C. sinensis.
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Objective To understand Clonorchis sinensis infections in cats in Nanning City, so as to provide evidence for the control of the reservoir host of C. sinensis. Methods The cat livers were purchased from cat slaughterhouses in Nanning City. The cat gallbladder and liver were dissected, and liver flukes were collected and counted. Then, the worms were subjected to morphological observation, amplification of the ITS2 gene and sequencing. The species of the worms were identified using BLAST. Results A total of 105 cat livers were collected from two cat slaughterhouses, and 68 were detected with C. sinensis infections, with an infection rate of 64.76%. The highest burden was 980 worms in a single liver, and the mean burden was 72 worms in a liver. There were 3 types of liver flukes with various size and morphology, and all were identified as C. sinensis by means of morphological observation, ITS2 gene amplification, sequencing and sequence alignment. Conclusion There is a high infection rate of C. sinensi in marketed cats in Nanning City, and it is therefore suggested that targeted interventions should be intensified for the management of C. sinensis infections in cats.
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Objective To compare the performance of modified Kato-Katz thick smear method (KK method) and PCR assay in field detection of Clonorchis sinensis in human fecal samples, which provides insight into the selection of tools for detecting C. sinensis. Methods Based on the epidemiological investigation of human C. sinensis infections in Tengxian County of Guangxi Zhuang Autonomous Region in 2016, a total of 133 fecal samples were randomly selected and stored at -20 ℃. All fecal samples were detected for C. sinensis infection using KK method and PCR assay, and the detection rate was compared between the two techniques. In addition, Kappa test was used to evaluate the consistency between the two methods. Results Among all fecal samples, the overall detection rate of C. sinensis was 77.44% (103/133), and the detection rate was significantly higher by PCR assay (70.68%, 93/133) than by KK method (57.14%, 76/133) (χ2 = 26.15, P < 0.01). There were 88.16% (67/76) of the microscopy-positive fecal samples positive for PCR assay, and 47.37% (27/57) of the microscopy-negative fecal samples positive for PCR assay. The detection rate of C. sinensis by PCR assay (94.74%, 18/19) was higher in fecal samples with EPG of > 1 000 than in samples with EPG of < 1 000 (85.96%, 49/57) (χ2 = 1.05, P = 0.436). The consistency of the detection rate of C. sinensis was moderate between the KK method and PCR assay (Kappa = 0.73). Conclusions The detection rate of C. sinensis by PCR assay is significantly higher than by KK method. In low-endemic areas of C. sinensis infections, the combination of KK method and PCR assay is suggested, so as to improve the detection rate.
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Objective To compare the performance of modified Kato-Katz thick smear method (KK method) and PCR assay in field detection of Clonorchis sinensis in human fecal samples, which provides insight into the selection of tools for detecting C. sinensis. Methods Based on the epidemiological investigation of human C. sinensis infections in Tengxian County of Guangxi Zhuang Autonomous Region in 2016, a total of 133 fecal samples were randomly selected and stored at -20 ℃. All fecal samples were detected for C. sinensis infection using KK method and PCR assay, and the detection rate was compared between the two techniques. In addition, Kappa test was used to evaluate the consistency between the two methods. Results Among all fecal samples, the overall detection rate of C. sinensis was 77.44% (103/133), and the detection rate was significantly higher by PCR assay (70.68%, 93/133) than by KK method (57.14%, 76/133) (χ2 = 26.15, P < 0.01). There were 88.16% (67/76) of the microscopy-positive fecal samples positive for PCR assay, and 47.37% (27/57) of the microscopy-negative fecal samples positive for PCR assay. The detection rate of C. sinensis by PCR assay (94.74%, 18/19) was higher in fecal samples with EPG of > 1 000 than in samples with EPG of < 1 000 (85.96%, 49/57) (χ2 = 1.05, P = 0.436). The consistency of the detection rate of C. sinensis was moderate between the KK method and PCR assay (Kappa = 0.73). Conclusions The detection rate of C. sinensis by PCR assay is significantly higher than by KK method. In low-endemic areas of C. sinensis infections, the combination of KK method and PCR assay is suggested, so as to improve the detection rate.
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Objective To establish a recombinase aided isothermal amplification (RAA) assay for detection of Clonorchis sinensis. Methods The 18S ribosomal RNA (18S rRNA) sequence of C. sinensis was used as the target sequence, and specific primers and probes were designed, synthesized and screened to establish a rapid fluorescent RAA assay for the detection of C. sinensis. Then, the sensitivity of the fluorescent RAA assay was evaluated using the recombinant plasmids containing various copy numbers of DNA fragments and C. sinensis genomic DNA at various concentrations, and the specificity of the fluorescent RAA as say was evaluated using the genomic DNA of Ascaris lumbricoides, Echinococcus granulosus, Schistosoma japonicum, Ancylostoma duodenale and S. mansoni as templates. DNA samples were extracted from the feces containing C. sinensis eggs and freshwater fish containing metacercaria for the fluorescent RAA assay, and the performance for detection of C. sinensis-infected samples was preliminarily assessed in the field. Results A fluorescent RAA assay for detection of C. sinensis was successfully established, which was feasible for specific amplification of C. sinensis genomic DNA at 39 °C within 20 min. The lowest detection limit was 10 copies/μL if the recombinant plasmid containing various copy numbers of DNA fragments was used as a template, and the lowest detection limit was 3 pg/μL if the C. sinensis genomic DNA at various concentrations served as a template. All detections were negative if the genomic DNA of A. lumbricoides, E. granulosus, S. japonicum, A. duodenale, and S. mansoni was used as templates. In addition, the fluorescent RAA assay showed a high performance for the detection of C. sinensis-infected samples in the field, which successfully detected C. sinensis-infected human and rat fecal samples and Pseudorasbora parva samples. Conclusion A fluorescent RAA assay is successfully established, which is simple, rapid, sensitivity and specific for detection of C. sinensis.
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Objective To understand Clonorchis sinensis infections in cats in Nanning City, so as to provide evidence for the control of the reservoir host of C. sinensis. Methods The cat livers were purchased from cat slaughterhouses in Nanning City. The cat gallbladder and liver were dissected, and liver flukes were collected and counted. Then, the worms were subjected to morphological observation, amplification of the ITS2 gene and sequencing. The species of the worms were identified using BLAST. Results A total of 105 cat livers were collected from two cat slaughterhouses, and 68 were detected with C. sinensis infections, with an infection rate of 64.76%. The highest burden was 980 worms in a single liver, and the mean burden was 72 worms in a liver. There were 3 types of liver flukes with various size and morphology, and all were identified as C. sinensis by means of morphological observation, ITS2 gene amplification, sequencing and sequence alignment. Conclusion There is a high infection rate of C. sinensi in marketed cats in Nanning City, and it is therefore suggested that targeted interventions should be intensified for the management of C. sinensis infections in cats.
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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.