Genotyping of Orientia tsutsugamushi circulating in and around Vellore (South India) using TSA 56 gene

Abstract

PurposeThe immunodominant TSA 56 gene of Orientia tsutsugamushi, (scrub typhus agent) has four variable regions (VD-I to VD-IV) making it useful for genotyping. This study was undertaken to determine Orientia tsutsugamushi genotypes circulating in and around Vellore using complete and partial TSA 56 gene. MethodsOf the 162 patients positive by 47 ​kDa qPCR, on 21 samples PCR to amplify the complete TSA 56 gene (≈1605 bp: Long protocol) and the partial gene sequence using the Horinouchi (≈650bp) and the Furuya (≈480 bp) protocol was performed. Sanger and Nanopore sequencing was performed to obtain sequence data for assigning genotype. For 13 amplicons partial and complete gene data was obtained. ResultsPhylogenetic analysis of the complete gene (Long protocol) which includes VD-I to VD-IV region and partial gene (Horinouchi) which amplifies the VD-I to VD-III regions showed identical genotypes. Twelve belonged to TA763 genotype and one belongs to Karp genotype. The Furuya sequence (in silico) correctly identified the Karp genotype and 10 of the TA763 genotypes. Two TA763 genotypes (identified by complete and 650 bp partial gene analysis) were misidentified by Furuya sequence analysis as Karp genotype. ConclusionsAnalysis of the 13 complete 56 ​kDa gene sequences suggests that TA763 is the commonest genotype in Vellore. Sanger sequencing of the 650 bp fragment gives similar results. However, these results need to be validated by larger prospective multi-centric studies.