Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples
Mami Taniuchi1, Jaco J Verweij, Orntipa Sethabutr
1Department 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.
Insights
A new multiplex PCR assay offers a convenient method for detecting Cyclospora, Cystoisospora, and Microsporidia enteropathogens. This molecular test improves upon traditional microscopy for diagnosing these difficult-to-identify infections.
Area of Science:
- Molecular Biology
- Clinical Diagnostics
- Parasitology
Background:
- Cyclospora, Cystoisospora, and Microsporidia are eukaryotic enteropathogens.
- Current detection methods rely on special stains and microscopy, which can be challenging.
- Accurate diagnosis is crucial for effective treatment of these gastrointestinal infections.
Purpose of the Study:
- To develop a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of key enteropathogens.
- To establish a sensitive and specific molecular diagnostic tool for Cyclospora, Cystoisospora, and Microsporidia.
- To offer a more convenient alternative to traditional microscopic examination of stool samples.
Main Methods:
- A multiplex PCR assay was designed with four primer sets targeting Cyclospora cayetanensis, Cystoisospora belli, Enterocytozoon bieneusi, and Encephalitozoon intestinalis.
- Amplicons were detected using specific probes coupled to Luminex beads.
- Assay sensitivity was assessed using spiked Encephalitozoon intestinalis spores, and cross-reactivity was evaluated.
Main Results:
- The assay detected as few as 10(1) Encephalitozoon intestinalis spores spiked into stool samples.
- No cross-reactivity was observed between the different primer sets.
- Evaluation on diarrheal specimens showed 87-100% sensitivity and 88-100% specificity compared to microscopy.
Conclusions:
- The developed multiplex PCR assay provides a convenient and accurate method for detecting Cyclospora, Cystoisospora, and Microsporidia.
- This molecular approach bypasses the limitations of traditional microscopic analysis.
- The assay demonstrates high sensitivity and specificity across diverse geographical locations.