Molecular identification and genotyping of Blastocystis spp. in children with clinical symptoms in southeast Iran using PCR-sequencing method

Blastocystis spp. is a zoonotic anaerobic parasite in the large intestine of humans and many vertebrates. It is mostly found in people who are in contact with animals. Current study aimed at identifying the prevalence of Blastocystis spp. and its common genotypes in children with clinical symptoms of diarrhea in the city of Zahedan, Southeast of Iran. A cross-sectional descriptive study was conducted on ۶۰ children under ten years of age with gastrointestinal symptoms, especially diarrhea. After sample collection, stool samples were subjected to direct stool testing for initial diagnosis. After microscopic diagnosis, DNA extraction and Polymerase chain reaction (PCR) test with small subunit ribosomal RNA (SSU rRNA) gene target were performed, and PCR products were purified and sequenced. Nucleotide sequences were revised using Chromas biotechnology software version ۲.۴ and CLC genomic work bench software ۱۱. Nucleotide sequences were aligned using BLAST database and compared with reference genotypes of Blastocystis spp. in gene bank. Genotyping of sequences was done using CLC genomic work bench software ۱۱ and phylogenetic tree drawing was done using MEGA۷ software with Neighbor-Joining statistical method applying Kimura ۲-parameter method. Out of ۶۰ examined cases, ۵ children (۸.۳۳%) were positive by direct microscopic and PCR tests, where a ۵۰۰ (۴۷۹) bp fragment in the SSU-rRNA target was detected. Genetic analysis identified ۴ subtypes, including subtypes ۱, ۲, ۳, and ۵. The percentage of nucleotide identity with the sequences in the gene bank was found to be between ۹۳ and ۱۰۰%. Considering the presence of subtypes ۳ and ۵ in the study and the evidence of their zoonotic, examining the parasite dynamics and epidemiological principles can be effective in the control strategy.

کلیدواژه ها:

Blastocystis spp ، Children ، PCR-sequencing ، southeast Iran