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.