Sequence Analysis of the TSA 56 kDa and GroEL Gene Reveals the Circulation of a Novel Genotype of Orientia tsutsugamushi in Human Cases of Scrub Typhus in Thanjavur District, Tamil Nadu, Southeastern India

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Abstract
Scrub typhus (St) is a mite-borne, re-emerging public health problem in India, particularly in Tamil Nadu, South India. More than 40 serotypes of Orientia tsutsugamishi (Ot) have been documented worldwide. However, the information on the circulation of its molecular sub-types in India is scanty and has not been completely understood. A retrospective study was conducted among serologically confirmed cases of scrub typhus. DNA isolated from blood, was screened by a nested polymerase chain reaction (nPCR), targeting the GroEL and 56 kDa TSA genes. Out of 59 samples, 14 partial fragments of GoEL and twelve 56 kDa genes were PCR amplified and DNA sequenced. The neighbor-joining (NJ) analysis indicated three distinct phylogenetic clades, including a novel genotype, designated as Ot-Thanjavur-Tamilnadu (Ot-TJTN, 9 nos. 64.3%); Karp-like (4 nos. 28.6%); and Kuroki-Gilliam type (1 no. 7.1%). Also, phylogenetic analysis of twelve sequences of the 56 kDa gene targeting variable domains (VDI-VDIII) revealed a distinctive new genotypic cluster of 8 samples (66.6%), and the remaining four (33.4%) were Karp-like genotypes. The Simplot analysis for the similarity and event of recombination testing with prototype reference sequences elucidated the existence of the new genotype of the Ot-TJTN cluster, which was undescribed so far, in the Kato and TA716 lineages. These isolates are genetically related to strains from Southeast Asian countries. The results of a phylogenetic analysis strongly suggest that the Ot-TJTN isolate is undescribed and probably region-specific. The significant findings recommend further studies on understanding the ongoing transmission dynamics of different Ot stains locally circulating in vector mites, rodent hosts, and humans in this region, to implement one health approach in the future.

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