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Real-time PCR method for detection of the microsporidian Enterocytozoon bieneusi from stool specimens

Real-time PCR method for detection of the microsporidian Enterocytozoon bieneusi from stool specimens

Abstracts of the General Meeting of the American Society for Microbiology 103: C-283

Infection of immunocompromised individuals with parasites of the order Microsporidia can cause chronic, profuse, and watery diarrhea. Microsporidia have also been detected in immunocompetent children and travelers with chronic diarrhea. The current gold standard for detection of microsporidia is microscopy using a stain such as the Ryan modified trichrome blue stain. Microscopic detection of microsporidia is difficult resulting in decreased sensitivity and requiring considerable expertise on the part of the reader. This most likely results in underreporting of this infection and a lack of knowledge about the true prevalence of this infection. Previously we have reported the development of a rapidcycle, real-time PCR assay for the detection of the microsporidian Encephalitozoon intestinalis from stools that provides a 2-4 log increase in sensitivity over detection using the trichrome stain (Wolk DM et al. 2002. J. Clin. Microbiol. 40:3922-3928). Another organism, Enterocytozoon bieneusi, is thought to be responsible for approximately 80% of microsporidia infections in humans. In this study, we have developed a real-time PCR assay using the LightCycler platform (Roche Applied Sciences, Indianapolis, IN) that detects E. bieneusi in stools. E. bieneusi genomic DNA was extracted from stool samples using STAR buffer and the MagNA Pure workstation (Roche). Primers and fluorescence hybridization probes were designed that amplified and detected a 268bp region of the 16S rRNA gene of E. bieneusi. No amplification was seen using the appropriate negative controls and no cross-reactivity with E. intestinalis, E. hellem, or E. cuniculi was detected. Melting curve analysis provides a measure of assay reproducibility between analyses and the TM for the E. bieneusi amplicon/probe hybrid was 61.8+-0.7degreeC (n=8). The assay is readily adaptable to the clinical laboratory and provides improved detection of E. bieneusi in stool specimens as compared with the traditional trichrome blue stain.

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Accession: 035615106

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