Genotypic Identification of Cystoisospora in Immunocompromised Patients Using Tm-Variation Analysis
2
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
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 (Tms) 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 Tms at 85.8˚C and 88.6˚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-Tms analysis which is useful for rapid detection and screening of Cystoisospora genotypes principally in high risk groups.
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 (Tms) 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 Tms at 85.8˚C and 88.6˚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-Tms analysis which is useful for rapid detection and screening of Cystoisospora genotypes principally in high risk groups.
참고문헌 (18)
- Lindsay DS, "Biology of Isospora spp. from humans, nonhuman primates, and domestic animals", Clin Microbiol Rev, 10, pp.19~34 (1997)
- Jongwutiwes S, "Morphologic and molecular characterization of Isospora belli oocysts from patients in Thailand", Am J Trop Med Hyg, 77, pp.107~112 (2007)
- Murphy SC, "Molecular diagnosis of cystoisosporiasis using extended-range PCR screening", J Mol Diagn, 13, pp.359~362 (2011)
- Bialek R, "Comparison of autofluorescence and iodine staining for detection of Isospora belli in feces", Am J Trop Med Hyg, 67, pp.304~305 (2002)
- Ahmed NH, "Comparison of different methods of detection of enteric pathogenic protozoa", Indian J Med Microbiol, 31, pp.154~160 (2013)
- Bruijnesteijn van Coppenraet LE, "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, 15, pp.869~874 (2009)
- Müller A, "Detection of Isospora belli by polymerase chain reaction using primers based on small-subunit ribosomal RNA sequences", Eur J Clin Microbiol Infect Dis, 19, pp.631~634 (2000)
- Taniuchi M, "Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and microsporidia in stool samples", Diagn Microbiol Infect Dis, 71, pp.386~390 (2011)
- ten Hove RJ, "Real-time polymerase chain reaction for detection of Isospora belli in stool samples", Diagn Microbiol Infect Dis, 61, pp.280~283 (2008)
- Lalonde LF, "Detection and differentiation of coccidian oocysts by real-time PCR and melting curve analysis", J Parasitol, 97, pp.725~730 (2001)
- Taniuchi M, "Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples", Diagn Microbiol Inf Dis, 71, pp.386~390 (2011)
- Wittwer CT, "High-resolution genotyping by amplicon melting analysis using LC Green", Clin Chem, 49, pp.853~860 (2003)
- El-Shazly AM, "Intestinal parasites in Dakahlia governorate with different techniques in diagnosing protozoa", J Egypt Soc Parasitol, 36, pp.1023~1034 (2006)
- Samarasinghe B, "Phylogenetic analysis of Cystoisospora species at the rRNA ITS1 locus and development of a PCR-RFLP assay", Exp Parasitol, 118, pp.592~595 (2008)
- Yao H, "Use of ITS2 region as the universal DNA barcode for plants and animals", PLoS One, 5, pp.e13102~ (2010)
- Read SJ, "Light cycler multiplex PCR for the laboratory diagnosis of common viral infections of the central nervous system", J Clin Microbiol, 39, pp.3056~3059 (2001)
- Ririe KM, "Product differentiation by analysis of DNA melting curves during the polymerase chain reaction", Anal Biochem, 245, pp.154~160 (1997)
- Yakovchuk P, "Basestacking and base-pairing contributions into thermal stability of the DNA double helix", Nucleic Acids Res, 34, pp.564~574 (2006)