title: Real-time PCR and flow cytometry in detection of Cyclospora oocysts in fecal samples of symptomatic and asymptomatic pediatrics patients.
abstract: The magnitude of Cyclospora oocysts excretion in relation to infection intensity among cyclosporiasis patients was assessed using flow cytometry and quantitative real-time PCR (RT-PCR). Oocysts from stool samples of 25 (14.8%) gastro-intestinal symptomatic pediatrics patients (169) and of 10 (2.8%) asymptomatic gastrointestinal ones (350) were identified by modified Ziehl-Neelsen (MZN) and modified Acid Fast Trichrome (MAFT) staining methods and confirmed by its auto-fluorescent characterizations. Also, 10 infants with negative stool samples were selected as controls. The intensity of infection was calculated as number of oocysts/200 microscopic filed with immersion 400. Flow cytometry and RT-PCR assessed relation between symptoms and oocysts excretions compared to MZN & MAFT. The infection severity in symptomatic patients were identified by MZN & MAFT as mild (16%), moderate (24%) and severe (60%) All asymptomatic patients had mild infection. Flow cytometry was done for stool samples and 100% Cyclospora oocysts were in symptomatic and asymptomatic patients. None was detected in controls. RT-PCR was done for stools with both a species-specific primer set and dual fluorescent labeled Cyclospora cayetanensis hybridization probe by unique regions of 18S rRNA gene sequence. DNA of C. cayetanensis was in 100% of symptomatic and asymptomatic patients and in 20% of controls. In repetitive examination of stools Cyclospora oocysts were neither detected by staining nor flow cytometry. Based on oocysts counts, no differences were found between flow cytometry and RT-PCR in compared to staining methods.
authors.affiliation: Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia 41152, Egypt.
authors.forename: Eman M
authors.initials: EM
authors.lastname: Hussein
authors.forename: Amal A
authors.initials: AA
authors.lastname: El-Moamly
authors.forename: Hamdy A
authors.initials: HA
authors.lastname: Dawoud
authors.forename: Hanaa
authors.initials: H
authors.lastname: Fahmy
authors.forename: Hesham E
authors.initials: HE
authors.lastname: El-Shal
authors.forename: Nagwan A
authors.initials: NA
authors.lastname: Sabek
mesh_terms: D000818:Animals; D002648:Child; D002675:Child, Preschool; D021744:Cyclospora / Q000302:isolation & purification*; D021866:Cyclosporiasis / Q000175:diagnosis* / Q000453:epidemiology; D005243:Feces / Q000469:parasitology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008297:Male; D033661:Oocysts; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity