title: Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples.
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.
authors: Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA 22908, USA. mt2f@virginia.edu
authors: Mami
authors: M
authors: Taniuchi
authors: Jaco J
authors: JJ
authors: Verweij
authors: Orntipa
authors: O
authors: Sethabutr
authors: Ladaporn
authors: L
authors: Bodhidatta
authors: Lynne
authors: L
authors: Garcia
authors: Athanasia
authors: A
authors: Maro
authors: Happiness
authors: H
authors: Kumburu
authors: Jean
authors: J
authors: Gratz
authors: Gibson
authors: G
authors: Kibiki
authors: Eric R
authors: ER
authors: Houpt
mesh_terms: D003429:Cross Reactions; D021744:Cyclospora / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D016819:Encephalitozoon; D021862:Enterocytozoon; D005243:Feces / Q000382:microbiology* / Q000469:parasitology*; D005767:Gastrointestinal Diseases / Q000175:diagnosis / Q000382:microbiology / Q000469:parasitology; D006801:Humans; D016814:Microsporidia / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009181:Mycoses / Q000175:diagnosis / Q000382:microbiology; D015345:Oligonucleotide Probes / Q000235:genetics; D011528:Protozoan Infections / Q000175:diagnosis / Q000469:parasitology; D021745:Sarcocystidae / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes