Multiplex PCR for detection of entamoeba histolytica, giardia intestinalis and cryptosporidium spp. in stool of diarrheic patients in Egypt / Salwa Morsy Hussien Morsy ; Supervised Olfat Mohamed Mahmoud Elmatrawy , Marwa Adel Hassan , Jose' Miguel Rubio

Thesis (Ph.D.) - Cairo University - Faculty of Medicine - Department of Parasitology

Entamoeba histolytica, giardia intestinalis and cryptosporidium spp. are the most common diarrhea-causing protozoa having the same clinical presentations. A cross sectional study was designed to develop a multiplex real-time PCR (MT-PCR) assay for concurrent detection of E.histolytica, G. intestinalis, and Cryptosporidium spp. in one reaction and compare these results with those of microscopy and nPCR. A second objective is to investigate the role of collected data variables for susceptibility to infection with these protozoa among the study individuals. Stool samples were collected from 150 diarrheic patients attending outpatient clinics of Kasr Alainy school of Medicine, Cairo University. Their relative data were recorded. Samples were examined microscopically (examination of direct wet mount and concentrated samples with and without staining) and then subjected to copro-PCR assays (nPCR and multiplex real-time PCR). Out of the 100 samples subjected to multiplex real-time PCR, 12 samples were positive for Cryptosporidium spp. (among them 2 and 9 samples positive by microscopy using AF stain and nPCR) and 25 samples positive for G. intestinalis (among them 15 and 24 samples were positive by microscopy using iodine and nPCR). It is noticeable that E.histolytica was not detected by neither nPCR nor MT-PCR. Microscopy and nPCR showed lower sensitivity compared to MT- real time PCR (60% and 96% for G. intestinalis and 17% and 75% for Cryptosporidium spp. repectively). Estimating the study variables as risk factors, the type of stool was a risk factor for cryptosporidiosis while clinical symptoms showed significant association with giardiasis

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