RESUMO
BACKGROUND: Blastocystis is one of the most common parasites, reported from both human and animals. This parasite is more prevalent in regions with low levels of hygiene, close contact with animal and unsuitable disposal systems. The aim of the study was to subtype Blastocystis sp., isolated from diarrheic and non-diarrheic patients using sequencing of 18S ribosomal DNA. METHODS: Totally, 300 stool samples were collected from diarrheic and nondiarrheic patients referred to Imam Reza Hospital, Tehran from Apr to Aug 2015. All samples were concentrated using conventional Formalin - ether technique and recognized under light microscope. The fresh stool samples were also cultivated in clotted fetal bovine medium and examined for growing of Blastocystis every 48 h with direct smear slides for 10 d. DNA extraction was performed on all positive samples. Amplified DNA fragment of 18S rDNA was sequenced and compared with reference genes, previously deposited in Genbank database. RESULTS: The number of diarrheic and non-diarrheic patients participated in the study was 134 (44.66%) and 166 (55.34%), respectively. Three subtypes 1, 2, 3 were identified from positive samples. Subtype 2 was the most prevalent (36.5%) followed by subtype 1 (33.3%) and subtype 3 (30.2%). There were no mixed subtypes. Furthermore, the most prevalent subtypes in diarrheic and non-diarrheic patients were subtype 2 (39.28%) and subtype 1 (37.14%), respectively. CONCLUSION: Blastocystis sp., is one of the most prevalent unicellular parasites among diarrheic and non-diarrheic patients. Indeed, ST2 was the most prevalent subtype particularly in those samples collected from diarrheic patients.
RESUMO
Blastocystis is a common intestinal parasitic protist infecting birds and mammals. Blastocystis comprises at least 17 subtypes (ST), of which ST1-ST9 have been detected in humans. Significant correlation between certain subtypes and pathogenicity remains to be established. Nevertheless, some studies suggest a potential linkage between subtypes (inter- and intra-subtype variation) and clinical manifestations. The aim of this study was to identify intra-subtype genetic variation of subtypes of Blastocystis in stools samples submitted by diarrheic and non-diarrheic patients. A 550-bp fragment of the nuclear small subunit ribosomal rRNA gene was amplified from 58 culture-positive samples isolated from diarrheic and non-diarrheic Iranian patients. PCR products were sequenced and sequences subjected to phylogenetic analysis. Intra-and inter-subtype variation was calculated. Based on comparison with reference sequences in GenBank, ST1, ST2 and ST3 were found in 18 (31.03%), 21 (36.22%), and 19 (32.75%) of the samples, respectively. Diarrheic stools were observed in eight (44.44%), 10 (47.61%), and nine (47.36%) patients with ST1, ST2, and ST3, respectively. No statistically significant correlation was found between subtypes and diarrhea (P=1.000). Multiple sequence alignment exhibited a within-subtype similarity of 98.76%, 97.17%, and 99.78% in ST1, ST2, and ST3, respectively. Highest similarity was seen among ST3 isolates, while lowest similarity was seen among ST2 isolates. Phylogenetic analysis did not suggest any correlation between diarrhea and intra-subtype variation. Inter- and intra-subtype variation in SSU rRNA gene appears not to reflect differences in the clinical outcome of Blastocystis carriage.