Parasites, Hosts and Diseases

The Korea Society for Parasitology and Tropical Medicine (대한기생충학·열대의학회)
권호 표지
DOI QR코드

DOI QR Code

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

  • Kaewkong, Worasak (Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University) ;
  • Intapan, Pewpan M. (Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University) ;
  • Sanpool, Oranuch (Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University) ;
  • Janwan, Penchom (Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University) ;
  • Thanchomnang, Tongjit (Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University) ;
  • Laummaunwai, Porntip (Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University) ;
  • Lulitanond, Viraphong (Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University) ;
  • Doanh, Pham Ngoc (Department of Parasitology, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology) ;
  • Maleewong, Wanchai (Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University)
  • Received : 2013.04.30
  • Accepted : 2013.10.11
  • Published : 2013.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at $82.4{\pm}0.09^{\circ}C$ and $85.9{\pm}0.08^{\circ}C$ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

Keywords

References

  1. Sripa B, Kaewkes S, Intapan PM, Maleewong W, Brindley PJ. Foodborne trematodiases in Southeast Asia-epidemiology, pathology, clinical manifestation and control. Adv Parasitol 2010; 72: 305-350. https://doi.org/10.1016/S0065-308X(10)72011-X
  2. Keiser J, Utzinger J. Food-borne trematodiases. Clin Microbiol Rev 2009; 22: 466-483. https://doi.org/10.1128/CMR.00012-09
  3. World Health Organization. Control of foodborne trematode infections. Report of a WHO Study Group. World Health Organ Tech Rep Ser 1995; 849: 1-157.
  4. Fürst T, Duthaler U, Sripa B, Utzinger J, Keiser J. Trematode infections: liver and lung flukes. Infect Dis Clin North Am 2012; 26: 399-419. https://doi.org/10.1016/j.idc.2012.03.008
  5. International Agency for Research on Cancer. Infection with liver fluke (Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans 1994; 61: 121-175.
  6. Fried B, Reddy A, Mayer D. Helminths in human carcinogenesis. Cancer Lett 2011; 305: 239-249. https://doi.org/10.1016/j.canlet.2010.07.008
  7. Thu ND, Dalsgaard A, Loan LT, Murrell KD. Survey for zoonotic liver and intestinal trematode metacercariae in cultured and wild fish in An Giang Province, Vietnam. Korean J Parasitol 2007; 45: 45-54. https://doi.org/10.3347/kjp.2007.45.1.45
  8. Traub RJ, Macaranas J, Mungthin M, Leelayoova M, Cribb T, Murrell KD, Thompson RC. A new PCR-based approach indicates the range of Clonorchis sinensis now extends to Central Thailand. PLoS Negl Trop Dis 2009; 3: e367. https://doi.org/10.1371/journal.pntd.0000367
  9. Dorny P, Praet N, Deckers N, Gabriel S. Emerging food-borne parasites. Vet Parasitol 2009; 163: 196-206. https://doi.org/10.1016/j.vetpar.2009.05.026
  10. Hong ST, Fang Y. Clonorchis sinensis and clonorchiasis, an update. Parasitol Int 2009; 61: 17-24.
  11. Stauffer WM, Sellman JS, Walker PF. Biliary liver flukes (opisthorchiasis and clonorchiasis) in immigrants in the United States: often subtle and diagnosed years after arrival. J Travel Med 2004; 11: 157-159.
  12. Fried B, Abruzzi A. Food-borne trematode infections of humans in the United States of America. Parasitol Res 2010; 106: 1263-1280. https://doi.org/10.1007/s00436-010-1807-0
  13. Yossepowitch O, Gotesman T, Assous M, Marva E, Zimlichman R, Dan M. Opisthorchiasis from imported raw fish. Emerg Infect Dis 2004; 10: 2122-2126. https://doi.org/10.3201/eid1012.040410
  14. Kaewkes S. Taxonomy and biology of liver flukes. Acta Trop 2003; 88: 177-186. https://doi.org/10.1016/j.actatropica.2003.05.001
  15. Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 2003; 49: 853-860. https://doi.org/10.1373/49.6.853
  16. Reed GH, Kent JO, Wittwer CT. High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 2007; 8: 597-608. https://doi.org/10.2217/14622416.8.6.597
  17. Tong SY, Giffard PM. Microbiological applications of high-resolution melting analysis. J Clin Microbiol 2012; 50: 3418-3421. https://doi.org/10.1128/JCM.01709-12
  18. Ngui R, Lim YA, Chua KH. Rapid detection and identification of human hookworm infections through high resolution melting (HRM) analysis. PLoS One 2012; 7: e41996. https://doi.org/10.1371/journal.pone.0041996
  19. Wongkamchai S, Monkong N, Mahannol P, Taweethavonsawat P, Loymak S, Foongladda S. Rapid detection and identification of Brugia malayi, B. pahangi, and Dirofilaria immitis by high-resolu tion melting assay. Vector Borne Zoon Dis 2013; 13: 31-36. https://doi.org/10.1089/vbz.2012.0971
  20. Elkins DB, Haswell-Elkins M, Anderson RM. The epidemiology and control of intestinal helminths in the Pulicat Lake region of Southern India. I. Study design and pre- and post-treatment observations on Ascaris lumbricoides infection. Trans R Soc Trop Med Hyg 1986; 80: 774-792. https://doi.org/10.1016/0035-9203(86)90384-6
  21. Sanpool O, Intapan PM, Thanchomnang T, Janwan P, Lulitanond V, Doanh PN, Van Hien H, Dung do T, Maleewong W, Nawa Y. Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real- time fluorescence resonance energy transfer PCR and melting curve analysis. Parasitol Res 2012; 111: 89-96. https://doi.org/10.1007/s00436-011-2804-7
  22. Galen RS. Predictive value and efficiency of laboratory testing. Pediatr Clin North Am 1980; 27: 861-869.
  23. Intapan PM, Thanchomnang T, Lulitanond V, Pongsaskulchoti P, Maleewong W. Rapid molecular detection of Opisthorchis viverrini in human fecal samples by real-time polymerase chain reaction. Am J Trop Med Hyg 2009; 81: 917-920. https://doi.org/10.4269/ajtmh.2009.09-0275
  24. Cai XQ, Yu HQ, Bai JS, Tang JD, Hu XC, Chen DH, Zhang RL, Chen MX, Ai L, Zhu XQ. Development of a TaqMan based realtime PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes. Parasitol Int 2012; 61: 183-186. https://doi.org/10.1016/j.parint.2011.06.010
  25. Rahman SM, Bae YM, Hong ST, Choi MH. Early detection and estimation of infection burden by real-time PCR in rats experimentally infected with Clonorchis sinensis. Parasitol Res 2011; 109: 297-303. https://doi.org/10.1007/s00436-011-2253-3
  26. De NV, Murrell KD, Cong le D, Cam PD, Chau le V, Toan ND, Dalsgaard A. The food-borne trematode zoonoses of Vietnam. Southeast Asian J Trop Med Public Health 2003; 34: 12-34.

Cited by

  1. Molecular characterization of the unique Mesostephanus appendiculatus (Trematoda: Cyathocotylidae) by small ribosomal RNA from Egypt vol.116, pp.4, 2013, https://doi.org/10.1007/s00436-016-5342-5
  2. LAMPhimerus: A novel LAMP assay for detecting Amphimerus sp. DNA in human stool samples vol.11, pp.6, 2017, https://doi.org/10.1371/journal.pntd.0005672
  3. Molecular discrimination of Opisthorchis -like eggs from residents in a rural community of central Thailand vol.11, pp.11, 2017, https://doi.org/10.1371/journal.pntd.0006030
  4. Performance evaluation of existing immunoassays for Clonorchis sinensis infection in China vol.11, pp.1, 2013, https://doi.org/10.1186/s13071-018-2612-3
  5. Development of Conventional Multiplex PCR: A Rapid Technique for Simultaneous Detection of Soil-Transmitted Helminths vol.8, pp.3, 2013, https://doi.org/10.3390/pathogens8030152
  6. Clonorchiasis sinensis detected by laparoscopic exploration of biliary tracts in two patients with obstructive jaundice vol.19, pp.None, 2019, https://doi.org/10.1186/s12879-019-3679-y
  7. Are Melanoides tuberculata and Tarebia granifera (Gastropoda, Thiaridae), suitable first intermediate hosts of Clonorchis sinensis in Vietnam? vol.15, pp.1, 2021, https://doi.org/10.1371/journal.pntd.0009093