Identification of Cryptosporidium species by real time PCR method

Cryptosporidium spp. are protozoa which only live in a host cell and may cause an infection that may result in the death of people with immune deficiency, in healthy individuals and children. Resistant to acid staining technique is often used in the diagnosis of cryptosporidiosis. Today, thanks to the development of molecular methods , in order to obtain epidemiological data in asymptomatic carrier detection and identification of Cryptosporidium species level was possible. The aim of the present study was to determine Cryptosporidium species/genotypes in humans in Adana, Turkey using melting curve analysis after real-time PCR and sequencing based on the small subunit rRNA gene. A total of 320 faecal samples were collected from humans with diarrhea applying to Parasitology Department of Medicine Fakulty in Cukurova University between April 2011 and Fabruary 2014. This stool samples were examined with direct smears, concentration, modified acid- fast staining, ELISA (Enzyme Linked Immunoabsorbent Assay), conventional Polymerase Chain Reaction (PCR) and based on the SSU rRNA genes by real time PCR method. Moreover, genomic DNA of each fecal sample was screened for the presence of Cryptosporidium with real-time PCR using primers and hibridization probes, Fluorescence Resonance Energy Transfer (FRET), which was genotyped by analyzing the DNA sequences of small subunit rRNA (SSU rRNA). In the results of working, 22 positive with staining method, 26 positive with ELISA, 27 positive with standart PCR and 31 positive with real time PCR were found Cryptosporidium spp. Molecular and genotype characterization found the following protozoan species; Cryptosporidium hominis (12), Cryptosporidium parvum (7), Cryptosporidium meleagridis (7) and Cryptodporidium andersoni (5). The results obtained in this study support previous findings showing that Cryptosporidium hominis infections are more prevalent than Cryptosporidium parvum infections in Adana and they demonstrate that real time PCR procedure is a simple, fast and voluable tool for the detection and differention of Cryptosporidium species.