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Diagn Microbiol Infect Dis. 2011 Dec;71(4):386-90. doi: 10.1016/j.diagmicrobio.2011.08.012. Epub 2011 Oct 6.

Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples.

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1
Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908, USA. mt2f@virginia.edu

Abstract

Cyclospora, Cystoisospora, and Microsporidia are eukaryotic enteropathogens that are difficult to detect in stool samples because they require special stains and microscopy. We developed a multiplex polymerase chain reaction (PCR) reaction with 4 primer sets to amplify Cyclospora cayetanensis, Cystoisospora belli, Enterocytozoon bieneusi, and Encephalitozoon intestinalis. Detection of the amplicon is through specific probes coupled to Luminex beads. Sensitivity of the assay was evaluated using Encephalitozoon intestinalis spores and revealed detection of 10(1) spores spiked into stool. No cross-reactivity was observed. We evaluated the assay on diarrheal specimens from Thailand, Tanzania, Indonesia, and the Netherlands that had been previously tested by microscopy, and the assay yielded 87-100% sensitivity and 88-100% specificity. Microscopy-negative/PCR-positive samples had lower Luminex values, suggesting they were true but with lower burden infections. In summary, this is a convenient single PCR reaction that can detect Cyclospora, Cystoisospora, and Microsporidia without the need for cumbersome microscopic analysis.

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