Skip to main content
Log in

Early detection and estimation of infection burden by real-time PCR in rats experimentally infected with Clonorchis sinensis

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

A real-time PCR assay targeting the cytochrome c oxidase gene of Clonorchis sinensis was developed and its diagnostic value was investigated in experimentally infected rats. The PCR assay amplified as little as 0.1 pg of C. sinensis genomic DNA and a single egg from experimentally spiked negative feces. The assay could detect C. sinensis DNA in feces 2 weeks post-infection (PI), whereas the Kato–Katz method and formalin–ether concentration techniques could detect the eggs in the feces from 3 weeks PI. Inverse correlations between eggs per gram of feces (EPG) and Ct values were observed 4, 6, and 8 weeks PI. Worm burden was positively correlated with EPG but negatively correlated with Ct values in different groups at 8 weeks PI. Fecal real-time PCR provides a potential tool for the diagnosis of clonorchiosis before eggs can be detected by conventional methods, and may be used to estimate the worm burden in the host.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Bouvard V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, Benbrahim-Tallaa L, Guha N, Freeman C, Galichet L, Cogliano V (2009) A review of human carcinogens—part B: biological agents. Lancet Oncol 10(4):321–322. doi:10.1016/S1470-2045(09)70096-8

    Article  PubMed  Google Scholar 

  • Choi MH, Park IC, Li S, Hong ST (2003) Excretory–secretory antigen is better than crude antigen for the serodiagnosis of clonorchiasis by ELISA. Korean J Parasitol 41(1):35–39. doi:10.3347/kjp.2003.41.1.35

    Article  PubMed  Google Scholar 

  • Choi D, Lim JH, Lee KT, Lee JK, Choi SH, Heo JS, Jang KT, Lee NY, Kim S, Hong ST (2006) Cholangiocarcinoma and Clonorchis sinensis infection: a case–control study in Korea. J Hepatol 44(6):1066–1073. doi:10.1016/j.jhep.2005.11.040

    Article  PubMed  Google Scholar 

  • Chung DI, Choi DW (1988) Intensity of infection and development of adult Clonorchis sinensis in hamsters. Korean J Parasitol 26(1):9–14

    Article  Google Scholar 

  • Guy RA, Payment P, Krull UJ, Horgen PA (2003) Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage. Appl Environ Microbiol 69(9):5178–5185. doi:10.1128/AEM.69.9.5178-5185.2003

    Article  PubMed  CAS  Google Scholar 

  • Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166(4):557–580. doi:10.1016/S0022-2836(83)80284-8

    Article  PubMed  CAS  Google Scholar 

  • Hong ST (2003) Clonorchis sinensis. In: Miliotis MD, Bier JW (eds) International handbook of foodborne pathogens. Marcel Dekker, New York, pp 581–592

    Google Scholar 

  • Hong ST, Rim HJ, Min DY, Li X, Xu J, Feng Z, Lee SH (2001) Control of clonorchiasis by repeated treatments with praziquantel. Korean J Parasitol 39(4):285–292. doi:10.3347/kjp.2001.39.4.285

    Article  PubMed  CAS  Google Scholar 

  • Hong ST, Choi MH, Kim CH, Chung BS, Ji Z (2003) The Kato–Katz method is reliable for diagnosis of Clonorchis sinensis infection. Diagn Microbiol Infect Dis 47(1):345–347

    Article  PubMed  Google Scholar 

  • Kim EM, Verweij JJ, Jalili A, van Lieshout L, Choi MH, Bae YM, Lim MK, Hong ST (2009) Detection of Clonorchis sinensis in stool samples using real-time PCR. Ann Trop Med Parasitol 103(6):513–518. doi:10.1179/136485909X451834

    Article  PubMed  CAS  Google Scholar 

  • Korea Association of Health Promotion (2004) Prevalence of intestinal parasitic infections in Korea—the seventh report. Korea Association of Health Promotion, Korea

    Google Scholar 

  • Le TH, Van De N, Blair D, Sithithaworn P, McManus DP (2006) Clonorchis sinensis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination. Exp Parasitol 112(2):109–114. doi:10.1016/j.exppara.2005.09.012

    Article  PubMed  CAS  Google Scholar 

  • Li S, Kang HW, Choi MH, Hong ST (2006) Long-term storage of Clonorchis sinensis metacercariae in vitro. Parasitol Res 100(1):25–29. doi:10.1007/s00436-006-0242-8

    Article  PubMed  Google Scholar 

  • Lim MK, Ju YH, Franceschi S, Oh JK, Kong HJ, Hwang SS, Park SK, Cho SI, Sohn WM, Kim DI, Yoo KY, Hong ST, Shin HR (2006) Clonorchis sinensis infection and increasing risk of cholangiocarcinoma in the Republic of Korea. Am J Trop Med Hyg 75(1):93–96

    PubMed  Google Scholar 

  • McManus DP, Le TH, Blair D (2004) Genomics of parasitic flatworms. Int J Parasitol 34(2):153–158. doi:10.1016/j.ijpara.2003.11.003

    Article  PubMed  CAS  Google Scholar 

  • Monteiro L, Bonnemaison D, Vekris A, Petry KG, Bonnet J, Vidal R, Cabrita J, Megraud F (1997) Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. J Clin Microbiol 35(4):995–998

    PubMed  CAS  Google Scholar 

  • Müller B, Schmidt J, Mehlhorn H (2007) PCR diagnosis of infections with different species of Opisthorchiidae using a rapid clean-up procedure for stool samples and specific primers. Parasitol Res 100(4):905–909. doi:10.1007/s00436-006-0321-x

    Article  PubMed  Google Scholar 

  • Park GM (2007) Genetic comparison of liver flukes, Clonorchis sinensis and Opisthorchis viverrini, based on rDNA and mtDNA gene sequences. Parasitol Res 100(2):351–357. doi:10.1007/s00436-006-0269-x

    Article  PubMed  Google Scholar 

  • Rim HJ (2005) Clonorchiasis: an update. J Helminthol 79(3):269–281

    Article  PubMed  Google Scholar 

  • Sandoval N, Siles-Lucas M, Lopez Aban J, Perez-Arellano JL, Garate T, Muro A (2006) Schistosoma mansoni: a diagnostic approach to detect acute schistosomiasis infection in a murine model by PCR. Exp Parasitol 114(2):84–88. doi:10.1016/j.exppara.2006.02.012

    Article  PubMed  CAS  Google Scholar 

  • Sato M, Thaenkham U, Dekumyoy P, Waikagul J (2009) Discrimination of O. viverrini, C. sinensis, H. pumilio and H. taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions. Acta Trop 109(1):81–83. doi:10.1016/j.actatropica.2008.09.015

    Article  PubMed  CAS  Google Scholar 

  • Shen C, Kim J, Lee JK, Bae YM, Choi MH, Oh JK, Lim MK, Shin HR, Hong ST (2007) Collection of Clonorchis sinensis adult worms from infected humans after praziquantel treatment. Korean J Parasitol 45(2):149–152

    Article  PubMed  Google Scholar 

  • ten Hove RJ, Verweij JJ, Vereecken K, Polman K, Dieye L, van Lieshout L (2008) Multiplex real-time PCR for the detection and quantification of Schistosoma mansoni and S. haematobium infection in stool samples collected in northern Senegal. Trans R Soc Trop Med Hyg 102(2):179–185. doi:10.1016/j.trstmh.2007.10.011

    Article  PubMed  Google Scholar 

  • Thaenkham U, Visetsuk K, do Dung T, Waikagul J (2007) Discrimination of Opisthorchis viverrini from Haplorchis taichui using COI sequence marker. Acta Trop 103(1):26–32. doi:10.1016/j.actatropica.2007.05.006

    Article  PubMed  CAS  Google Scholar 

  • Traub RJ, Macaranas J, Mungthin M, Leelayoova S, Cribb T, Murrell KD, Thompson RC (2009) A new PCR-based approach indicates the range of Clonorchis sinensis now extends to Central Thailand. PLoS Negl Trop Dis 3(1):e367. doi:10.1371/journal.pntd.0000367

    Article  PubMed  Google Scholar 

  • Verweij JJ, Pit DS, van Lieshout L, Baeta SM, Dery GD, Gasser RB, Polderman AM (2001) Determining the prevalence of Oesophagostomum bifurcum and Necator americanus infections using specific PCR amplification of DNA from faecal samples. Trop Med Int Health 6(9):726–731

    Article  PubMed  CAS  Google Scholar 

  • Verweij JJ, Brienen EA, Ziem J, Yelifari L, Polderman AM, Van Lieshout L (2007) Simultaneous detection and quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples using multiplex real-time PCR. Am J Trop Med Hyg 77(4):685–690

    PubMed  CAS  Google Scholar 

  • Wykoff DE (1959) Studies on Clonorchis sinensis. IV. Production of eggs in experimentally infected rabbits. J Parasitol 45(1):91–94

    Article  PubMed  CAS  Google Scholar 

  • Xia CM, Rong R, Lu ZX, Shi CJ, Xu J, Zhang HQ, Gong W, Luo W (2009) Schistosoma japonicum: a PCR assay for the early detection and evaluation of treatment in a rabbit model. Exp Parasitol 121(2):175–179. doi:10.1016/j.exppara.2008.10.017

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by grant from the Seoul National University College of Medicine Research Fund (800-20050250).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Min-Ho Choi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rahman, S.M.M., Bae, Y.M., Hong, ST. et al. Early detection and estimation of infection burden by real-time PCR in rats experimentally infected with Clonorchis sinensis . Parasitol Res 109, 297–303 (2011). https://doi.org/10.1007/s00436-011-2253-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-011-2253-3

Keywords

Navigation