Korean J Parasitol.  2017 Dec;55(6):601-606. 10.3347/kjp.2017.55.6.601.

Genotypic Identification of Cystoisospora in Immunocompromised Patients Using T(m)-Variation Analysis

• Basyoni MM
• Elghobary

• KMID: 2403710
• DOI: http://doi.org/10.3347/kjp.2017.55.6.601

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(m)s) 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(m)s 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-T(m)s analysis which is useful for rapid detection and screening of Cystoisospora genotypes principally in high risk groups.

Keyword

Cystoisospora; melting curve analysis; qPCR

MeSH Terms

Diagnosis
Egypt
Freezing
Genetic Variation
Genotype
Hospitals, University
Humans
Immunocompromised Host*
Lymphoma
Mammary Glands, Human
Mass Screening
Medical Oncology
Microscopy
Nuclear Medicine
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
RNA, Ribosomal
Sarcoma, Kaposi
RNA, Ribosomal