Molecular Probes Detect Enteropathogens in Stool

A multiplex polymerase chain reaction (PCR) assay has been developed to identify four eukaryotic enteropathogens in stool samples.

Cyclospora, Cystoisospora, and Microsporidia are eukaryotic enteropathogens that are difficult to detect in stool samples because they require special stains and microscopy.

Scientists collaborating with the University of Virginia (Charlottesville, VA, USA) developed a multiplex PCR with four primer sets to amplify two protozoan parasites Cyclospora cayetanensis and Cystoisospora belli, and two microsporidian parasites, Enterocytozoon bieneusi, and Encephalitozoon intestinalis. The amplicons were detected through specific probes coupled to Luminex beads. Sensitivity of the assay was evaluated using E. intestinalis spores and revealed detection of 101 spores spiked into stool. No cross-reactivity was observed with samples containing a variety of other pathogenic protozoa, helminths, bacteria or viruses.

The assay was evaluated on 236 diarrheal fecal specimens from Thailand, Tanzania, Indonesia, and The Netherlands that had been previously tested by microscopy. Detection of the amplicons was performed by the BioPlex 200 (Bio-Rad; Hercules, CA, USA) or the Luminex 100 (Luminex Corporation; Austin, TX, USA). The multiplex assay yielded a sensitivity of 87% to 100% and a specificity of 88% to 100%. Microscopy-negative/PCR-positive samples had lower Luminex values, suggesting they were true positives, but with a lower burden of infections. The limit of detection was 102 plasmid copies for Cystoisospora and Enterocytozoon bieneusi and 103 plasmid copies for Cyclospora.

The authors concluded that the assay is robust, with field-testing for this work having been successfully performed on-site in diverse locations including Thailand, Tanzania, Virginia, and The Netherlands. The molecular assay was validated on a large number of specimens for important eukaryotic enteropathogens that can be used in isolation or in the context of additional enteropathogen screening algorithms. The study was published on October 6, 2011, in the journal Diagnostic Microbiology and Infectious Disease.

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University of Virginia
Bio-Rad
Luminex Corporation