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

The immunodominant TSA 56 gene of Orientia tsutsugamushi, (scrub typhus agent) has four variable regions (VD-I to VD-IV) making it useful for genotyping. As of date the genotyping data from India is based on partial 56kDa gene sequence analysis. The complete TSA 56 gene sequence is important for knowing the circulating strains and for designing region specific diagnostics and vaccines. This study was undertaken to determine Orientia tsutsugamushi genotypes circulating in and around Vellore using complete and partial TSA 56 gene. Of the 379 whole blood samples from suspected scrub typhus patients, 162 were positive by 47 kDa qPCR. Long protocol to amplify the complete TSA 56 gene ({approx}1605 bp) was performed on 21 samples. On the same 21 samples the partial gene sequence was also amplified using the Horinouchi ({approx}650bp) and the Furuya ({approx}480 bp) protocol. Using a combination of Sanger and Nanopore technology complete sequence was obtained for 9 and near complete (1551 to 1596 bp) for 4 respectively. As Furuya protocol gave multiple bands we obtained 480 bp sequences from the 13 complete gene sequences by in silico analysis. In contrast, 650bp sequences were obtained for 11 samples while for the remaining two we derived the 650 bp sequences from the complete gene sequences (Long protocol). Phylogenetic 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. The limited analysis showed the commonest Orientia tsutsugamushi genotypes circulating in and around Vellore is TA763 and that the 650 bp (Sanger) sequencing could be a cost effective method for identifying the scrub typhus genotypes. However, these results need to be validated by larger prospective multi-centric studies.