Molecular detection of Cystoisospora belli by single-run polymerase chain reaction in stool samples.

ABSTRACT

INTRODUCTION: Cystoisospora belli (C. belli) is the only pathogenic species of the Cystoisospora genus responsible for severe diarrhea in immunocompromised patients. Most common microscopic method of diagnosis is less sensitive due to intermittent shedding of oocysts. We developed a new single-run polymerase chain reaction (PCR)-based diagnostic assay for C. belli. METHODS: A new single-run PCR-based diagnostic assay was standardized for the detection of C. belli. Diagnostic reproducibility and repeatability of the PCR assay were evaluated. A cross-sectional analytical study was done on a total of 354 stool samples collected from 331 immunocompromised patients with diarrhea. All the stool samples were tested for the presence of oocysts of C. belli and were also tested by our new PCR assay for C. belli. Three of the representative PCR products were confirmed by sequencing. Fisher's exact test was used to compare the two proportions. RESULTS: Microscopy detected C. belli in 11/354 (3.1%) of stool samples, and the new PCR-based assay detected C. belli in 16/354 (4.5%). The new single-run PCR-based assay detected C. belli in all the stool samples which were tested positive by microscopy and additionally detected C. belli in five stool samples. The developed PCR assay detected statistically significant proportion of C. belli (p < 0.001) as compared to microscopy. The 795 base pair PCR product from one microscopy positive stool sample and two microscopy negative stool samples were confirmed by sequencing. CONCLUSION: Our newly developed single-run PCR-based detection assay for C. belli is robust and reproducible. It may be used for molecular diagnosis of cystoisosporiasis especially in transplant, pediatrics, and human immunodeficiency virus (HIV) positive patients.