Parasites, Hosts and Diseases

The Korea Society for Parasitology and Tropical Medicine (대한기생충학·열대의학회)
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Genotypic Identification of Cystoisospora in Immunocompromised Patients Using Tm-Variation Analysis

  • Basyoni, Maha M.A. (Parasitology Department, Faculty of Medicine, Cairo University) ;
  • Elghobary, Hany Ahmed Fouad (Clinical and Chemical Pathology Department, Faculty of Medicine, Cairo University)
  • Received : 2017.06.12
  • Accepted : 2017.11.24
  • Published : 2017.12.31
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Abstract

Cystoisospora is responsible for morbidity in immunocompromised patients. PCR is sensitive for diagnosing Cystoisospora; however, it needs reevaluation for differential molecular diagnosis of cystoisosporiasis. We aimed at evaluating melting curve analysis (MCA) after real-time PCR (qPCR) in diagnosis and genotyping of Cystoisospora as an alternative to conventional PCR. We included 293 diarrheic stool samples of patients attending the Department of Clinical Oncology and Nuclear Medicine of Cairo University Hospitals, Egypt. Samples were subjected to microscopy, nested PCR (nPCR), and qPCR targeting the internal transcribed spacer 2 region (ITS2) of the ribosomal RNA (r RNA) gene followed by melting temperatures ($T_ms$) analysis and comparing the results to PCR-RFLP banding patterns. Using microscopy and ITS2-nPCR, 3.1% and 5.8% of cases were Cystoisospora positive, respectively, while 10.9% were positive using qPCR. Genotyping of Cystoisospora by qPCR-MCA revealed 2 genotypes. These genotypes matched with 2 distinct melting peaks with specified $T_ms$ at $85.8^{\circ}C$ and $88.6^{\circ}C$, which indicated genetic variation among Cystoisospora isolates in Egypt. Genotype II proved to be more prevalent (65.6%). HIV-related Kaposi sarcoma and leukemic patients harbored both genotypes with a tendency to genotype II. Genotype I was more prevalent in lymphomas and mammary gland tumors while colorectal and hepatocellular tumors harbored genotype II suggesting that this genotype might be responsible for the development of cystoisosporiasis in immunocompromised patients. Direct reliable identification and differentiation of Cystoisospora species could be established using $qPCR-T_ms$ analysis which is useful for rapid detection and screening of Cystoisospora genotypes principally in high risk groups.

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References

  1. Lindsay DS, Dubey JP, Blagburn BL. Biology of Isospora spp. from humans, nonhuman primates, and domestic animals. Clin Microbiol Rev 1997; 10: 19-34.
  2. Jongwutiwes S, Putaporntip C, Charoenkorn M, Iwasaki T, Endo T. Morphologic and molecular characterization of Isospora belli oocysts from patients in Thailand. Am J Trop Med Hyg 2007; 77: 107-112.
  3. Murphy SC, Hoogestraat DR, Sen Gupta DJ, Prentice J, Chakrapani A, Cookson BT. Molecular diagnosis of cystoisosporiasis using extended-range PCR screening. J Mol Diagn 2011; 13: 359-362. https://doi.org/10.1016/j.jmoldx.2011.01.007
  4. Bialek R, Binder N, Dietz K, Knobloch J, Zelck UE. Comparison of autofluorescence and iodine staining for detection of Isospora belli in feces. Am J Trop Med Hyg 2002; 67: 304-305. https://doi.org/10.4269/ajtmh.2002.67.304
  5. Ahmed NH, Chowdhary A. Comparison of different methods of detection of enteric pathogenic protozoa. Indian J Med Microbiol 2013; 31: 154-160.
  6. Bruijnesteijn van Coppenraet LE, Wallinga JA, Ruijs GJ, Bruins MJ, Verweij JJ. Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy. Clin Microbiol Infect 2009; 15: 869-874. https://doi.org/10.1111/j.1469-0691.2009.02894.x
  7. Muller A, Bialek R, Fatkenheuer G, Salzberger B, Diehl V, Franzen C. Detection of Isospora belli by polymerase chain reaction using primers based on small-subunit ribosomal RNA sequences. Eur J Clin Microbiol Infect Dis 2000; 19: 631-634. https://doi.org/10.1007/s100960000322
  8. Taniuchi M, Verweij JJ, Sethabutr O, Bodhidatta L, Garcia L, Maro A, Kumburu H, Gratz J, Kibiki G, Houpt ER. Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and microsporidia in stool samples. Diagn Microbiol Infect Dis 2011; 71: 386-390. https://doi.org/10.1016/j.diagmicrobio.2011.08.012
  9. ten Hove RJ, van Lieshout L, Brienen EA, Perez MA, Verweij JJ. Real-time polymerase chain reaction for detection of Isospora belli in stool samples. Diagn Microbiol Infect Dis 2008; 61: 280-283. https://doi.org/10.1016/j.diagmicrobio.2008.03.003
  10. Lalonde LF, Gajadhar AA. Detection and differentiation of coccidian oocysts by real-time PCR and melting curve analysis. J Parasitol 2001; 97: 725-730.
  11. Taniuchi M, Verweij JJ, Sethabutr O, Bodhidatta L, Garcia L, Maro A, Kumburu H, Gratz J, Kibiki G, Houpt ER. Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples. Diagn Microbiol Inf Dis 2011; 71: 386-390. https://doi.org/10.1016/j.diagmicrobio.2011.08.012
  12. Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ. High-resolution genotyping by amplicon melting analysis using LC Green. Clin Chem 2003; 49: 853-860. https://doi.org/10.1373/49.6.853
  13. El-Shazly AM, Awad SE, Sultan DM, Sadek GS, Khalil HH, Morsy TA. Intestinal parasites in Dakahlia governorate with different techniques in diagnosing protozoa. J Egypt Soc Parasitol 2006; 36: 1023-1034.
  14. Samarasinghe B, Johnson J, Ryan U. Phylogenetic analysis of Cystoisospora species at the rRNA ITS1 locus and development of a PCR-RFLP assay. Exp Parasitol 2008; 118: 592-595. https://doi.org/10.1016/j.exppara.2007.10.015
  15. Yao H, Song J, Liu C, Luo K, Han J, Li Y, Pang X, Xu H, Zhu Y, Xiao P, Chen S. Use of ITS2 region as the universal DNA barcode for plants and animals. PLoS One 2010; 5: e13102. https://doi.org/10.1371/journal.pone.0013102
  16. Read SJ, Mitchell JL, Fink CG. Light cycler multiplex PCR for the laboratory diagnosis of common viral infections of the central nervous system. J Clin Microbiol 2001; 39: 3056-3059. https://doi.org/10.1128/JCM.39.9.3056-3059.2001
  17. Ririe KM, Rasmussen RP, Wittwer CT. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 1997; 245: 154-160. https://doi.org/10.1006/abio.1996.9916
  18. Yakovchuk P, Protozanova E, Frank-Kamenetskii MD. Basestacking and base-pairing contributions into thermal stability of the DNA double helix. Nucleic Acids Res 2006; 34: 564-574. https://doi.org/10.1093/nar/gkj454