Subtype distribution and genetic diversity of Blastocystis in asymptomatic and symptomatic individuals from Puducherry, India

Abstract

Background: Blastocystis, an intestinal parasite, consists of 17 subtypes (ST), of which nine are capable of inhabiting humans. Blastocystis pathogenicity is controversial as it harbors both symptomatic and asymptomatic individuals. In India, molecular studies on Blastocystis are meager. Hence, large scale molecular epidemiological studies are required to unveil genetic diversity and pathogenicity of Blastocystis. Methods & Materials: In this cross-sectional analytical study, a total of 752 stool samples (379 male; 373 female) were collected from asymptomatic (group I, n = 384) and symptomatic (group II, n = 368) individuals. Group II was subcategorized based on the specific clinical symptoms: diarrhea (n = 152), inflammatory bowel disorder (IBD, n = 83), dysentery (n = 75), irritable bowel syndrome (n = 31), and colorectal cancer (n = 27). 18SSU rDNA was targeted in PCR for the detection and sub-typing of Blastocystis. 83 representative samples were sequenced and phylogenetic analysis was performed using MEGA 7 software. The subtypes and allele types were determined using http://www.pubmlst.org/Blastocystis database. Fisher's exact test was employed for the comparison between the groups. At 95% CI, p-value <0.05 was considered statistically significant. Results: Asymptomatic individuals had higher occurrence of Blastocystis (n = 135; 33.7%) compared to the symptomatic patients (n = 117; 31%) but statistically insignificant (p = 0.33, OR = 0.8599). Within-group II, patients having diarrhea had the highest detection rate of Blastocystis (n = 57; 37.1%) and the least in patients having IBD (n = 18; 21.6%). A comparison between subgroup IBD and asymptomatic group yielded statistically significant results (p = 0.016, OR = 0.5114). Out of the 252 samples positive for Blastocystis, ST1 (n = 79), ST2 (n = 23), ST3 (n = 145), and ST1 + ST3 mixed infections (n = 5) were identified. ST3 was predominant subtype in both groups. Sequenced samples were deposited in NCBI GenBank (Accession No. MK719604-MK719686). Alleles 31, 34, and 36 of ST3, alleles 9 and 11 of ST2 and alleles 4 and 80 of ST1 were detected. The phylogenetic tree showed no distinct segregation of sequences of symptomatic and asymptomatic isolates. Conclusion: This study mainly contributes to the genetic diversity aspects of Blastocystis in India. For the first time, ST2 was identified from India. However, the existence of all three subtypes in both symptomatic and asymptomatic individuals infers that inter-subtype variation may not be a reliable marker to assess the pathogenicity.