Detection of Microsporidium spp. and Cryptosporidium spp. in diarrheic stool samples, ıdentification of species by PCR

Aim: Our objective was to use Weber?s trichrome staining method in detection of Microsporidium spp. of which convenient staining method for diagnosis yet to be standardized in our country, and to compare effectiveness of microscopy and PCR methods in detection of Microsporidium spp. and Cryptosporidium spp. in diarrheic stool samples and species identification of both parasites.Materials and Methods: Stool samples of 162 diarrheic patients who attended to various clinics of Hospital of Faculty of Medicine, University of Dokuz Eylul between October 2005 and January 2009 and of patients in an outbreak of diarrhea which took place in a village in Buca district of İzmir, were included in our study. All samples were stained by Kinyoun acid-fast method to search Cryptosporidium spp. and by Weber?s trichrome staining method to search Microsporidium spp. and were examined by microscopy. Additionally, PCR was used for both parasites to test its validity as a diagnostic tool. In succeeding step, RFLP was used in species identification of both, Cryptosporidium spp. and Microspordium spp.Results: Oocysts of Cryptosporidium spp. were detected in 18 cases by Kinyoun acid fast staining method. PCR revealed Cryptosporidium spp. spesific bands in 15 of 18 patients who were staining positive and 6 of staining negative patients. RFLP method was applied to PCR positive stool samples and one sample had Cryptosporidium meleagridis spesific band while remaining 20 samples had Cryptosporidium parvum spesific bands.We observed spores of Microsporidium spp. in stool samples of 4 cases stained by Weber?s trichrome method and only one of them was positive in PCR. Panmicrosporidian primers were used in amplification and PCR positive patient revealed Encephalitozoon intestinalis spesific bands.Conclusion: PCR is an important diagnotic tool in diagnosis of Cryptosporidium spp., especially in cases with high parasite density. In diagnosing Microsporidium spp., staining procedure sustains its validity and more molecular studies are needed to supply convenient DNA extraction procedure.Keywords: Cryptosporidium spp., Microsporidium spp., Kinyoun acid fast, Weber?s trichrome, PCR-RFLP.