Detection and genotyping of cryptosporidium spp. in diarrheic stools by PCR/RFLP analyses

Abstract

Aim: To compare microscopy and polymerase chain reaction (PCR) as methods for diagnosing Cryptosporidium spp. and identifying the genotype of the parasite in diarrheic stool. Materials and methods: Stool samples from diarrheic patients admitted to Dokuz Eylül University Medical Faculty were collected on a specific day of the week. Stool samples sent to our laboratory from patients affected by a diarrhea epidemic in İzmir were also included in the study. A total of 162 stool samples were examined by microscopic and molecular methods, and the validity of the molecular method was investigated. Results: Using Kinyoun acid-fast dye, Cryptosporidium spp. was found in 18 stool samples. Using PCR, Cryptosporidium spp.-specific bands were found in 15 of these cases. In stool samples that showed no Cryptosporidium spp. by Kinyoun acid-fast dye, a parasite-specific band was obtained in 6 of 144 cases. Th e restriction fragment length polymorphism (RFLP) method was applied in the cases in which the parasite was found with PCR. Cryptosporidium meleagridis was found in the stool sample of 1 case, and Cryptosporidium parvum-specific bands were seen in the stool samples of 20 cases. Th e mean age of those who were diagnosed with Cryptosporidium spp. was significantly lower than the mean age of study participants who were not diagnosed with the parasite (P = 0.006). It was found that the families of 44.4% of those diagnosed with Cryptosporidium spp. had similar complaints. Th is rate was statistically significant (P = 0.01). It was found that 88.9% of those diagnosed with Cryptosporidium spp. drank artesian and well water, and 11.1% drank bottled water. Th e rate of Cryptosporidium spp. in those who drank artesian and well water was significantly higher than the rate of those who drank city or bottled water (P < 0.001). While the Kinyoun acid-fast method is considered the gold standard, the sensitivity of the PCR method is 83.3% and the selectivity is 95.8%. Conclusion: It can be concluded that Kinyoun acid-fast dye, which can be used as a referential diagnostic method, has some limitations and that PCR is more sensitive and allows for identification of the parasite genotype. © TÜBİTAK.