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

Authors: SELMA USLUCA, ÜMİT AKSOY

Abstract: 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. The 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. The 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. This 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. The 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.

Keywords: Cryptosporidium spp., Kinyoun acid-fast, PCR-RFLP

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