A Simple Genotyping Method for Rapid Differentiation of Blastocystis Subtypes and Subtype Distribution of Blastocystis spp. in Thailand

摘要

Blastocystis spp. is one of the most common protozoa of humans and animals worldwide. The genetic diversity of Blastocystis spp. might be associated with a wide range of symptoms. However, the prevalence of each subtype is different in each country. Until now, there is no standard method for subtyping of Blastocystis spp. We developed a sequential restriction fragment length polymorphism (RFLP) analysis for the rapid differentiation of human Blastocystis subtypes. A large-scale study was also conducted to determine the subtype distribution of Blastocystis spp. in Thailand. Stool samples were collected from 1025 school-age students in four regions of Thailand. Blastocystis infections were identified by direct smear, formalin ethyl-acetate concentration technique (FECT), Boeck and Drbohlav’s Locke-Egg-Serum (LES) medium culture, and polymerase chain reaction (PCR) of small-subunit ribosomal DNA (SSU rDNA). Subtypes of Blastocystis spp. were determined by RFLP. Phylogenetic tree of partial SSU rDNA sequences of Blastocystis spp. was constructed using the Maximum Likelihood (ML) method. Out of 1025 students, 416 (40.6%) were positive for Blastocystis spp. Using two steps of RFLP reactions, we could determine subtype one–three among these students. Subtype 3 was the most common subtype (58.72%) in Thai students, followed by subtype 1 (31.2%), and subtype 2 (10.1%). Blastocystis subtype 3 was the most prevalent in all regions of Thailand. The subtype distribution of Blastocystis spp. in Thailand was different from other countries.