Scrub typhus (St) is a re-emerging mite-transmitted public health problem in Southeast Asia with escalating case incidences in the endemic areas. Though, more than 40 genotypes of the causative agent Orientia tsutsugamushi (Ot) have been documented, the information on the circulating genotypes in India is scanty. A hospital-based retrospective screening was undertaken to map the circulating molecular subtypes of the etiological agent in serologically confirmed scrub typhus (St) human cases, by targeting the GroEL gene of O. tsutsugamushi using the nested polymerase chain reaction method. Nine out of 34 samples (26%) yielded positive results and DNA sequencing analysis of six positive samples out of nine revealed that the sequences were related to three major genotypes, such as Karp (HSB1, FAR1), Kato (Wuj/2014, UT76), and Kawasaki (Kuroki, Boryong, Gilliam, and Hwasung). Additionally, the St-positive samples exhibited 100% and 99.45%; 97.53% and 97.81%; 96.99% nucleotide identity with the closely related Karp, Kato, and Kawasaki-related sequences, respectively. Overall, 94% of the nucleotides were conserved, and the variable site was 20/365 (5.5%). The prevalence of multiple genotypes among human cases further stresses the need to conduct in-depth studies to map the genotypes and their clinical relevance, and the contributing risk factors for the emergence of St cases in this area.

Scrub typhus, caused by Orientia tsutsugamushi (Ot), is a re-emerging public health concern across Southeast Asia. Although multiple Ot strains have been identified in endemic regions, their genetic characterization in India remains limited. We analyzed Ot strains from humans by targeting the GroEL and 56-kDa TSA genes. A total of 105 serum samples were subjected to PCR amplification and phylogenetic analysis for the GroEL gene, of which 33 (31.4%) were positive. Phylogenetic reconstruction revealed four major clades: Karp, Kato, Ot-TJTN (novel Ot-Thanjavur-Tamil Nadu), and the Gilliam group. Among the 33 PCR positives, 11 sequences clustered into a distinct monophyletic clade within the Gilliam group but diverged significantly from known classical Gilliam strains. The overall mean nucleotide diversity (π) was 0.02 (2%), while the divergence between these 11 sequences and the Gilliam strain was 0.039 (3.9%). The observed divergence indicates that these sequences represent the first identified Indian Gilliam variant (IG-v), showing marked genetic distinction from classical Gilliam and other related strains. Further analysis of the 56-kDa gene from the 11 IG-v samples revealed phylogenetic incongruence between the GroEL and 56-kDa genes, indicating antigenic reassortment involving three clades: Karp-like (n = 7), Ot-TJTN-like (n = 3), and Gilliam (n = 1). Similarity plot and recombination analyses, using 56-kDa Ot-TJTN and Karp-like clades as queries, against Ot reference strains revealed preliminary evidence of genetic exchange. These findings highlight the possible role of recombination and antigenic shift in driving the evolutionary dynamics and genetic diversity of Ot in this region. Notably, the identification of an IG-v marks a significant advancement in our understanding of the circulating Ot strains. This finding holds important implications for refining molecular diagnostics, enhancing serological assays, and developing broadly protective vaccines targeting region-specific variants.

Scrub typhus, caused by Orientia tsutsugamushi (Ot), is a re‐emerging public health concern across Southeast Asia. Although multiple Ot strains have been identified in endemic regions, its genetic diversity in India remains limited. We analysed Ot strains from humans by targeting the conserved GroEL and variable 56‐kDa genes. A total of 105 serum samples were subjected to PCR amplification and phylogenetic analysis for the GroEL gene, of which 33 (31.4%) were positive. Phylogenetic reconstruction revealed four major clades: Karp, Kato, Ot‐TJTN, and Gilliam‐related. Among the 33‐PCR positive samples, 11 sequences formed a distinct monophyletic clade within the Gilliam‐related group, diverging significantly from classical Gilliam strains. The overall mean nucleotide diversity (π) was 0.02 (2%), while the divergence between these 11 sequences and Gilliam‐ related strains was 0.039 (3.9%). This level of divergence supports the identification of a novel Gilliam‐descendant lineage, designated INDv‐Gilliam. Further analysis of the 56‐kDa gene from the 11 INDv‐Gilliam samples revealed genotypic incongruence, indicating antigenic reassortment involving three clades: Karp‐like (n = 7), Ot‐TJTN‐like (n = 3), and Gilliam (n = 1). Similarity plot and recombination analyses, using 56‐kDa Ot‐TJTN and Karp‐like clades as queries against Gilliam‐type references, provided evidence of recombination involving TA716‐Kato and Ikeda‐Karp strains. These findings highlight the role of recombination and antigenic shift in driving the evolutionary dynamics and genetic diversity of Ot in the region. Accordingly, the isolates (11 nos.) were classified as mixed recombinant genotypes: Indian variant Gilliam (INDv‐Gilliam), INDv‐Gilliam/Karp‐like, INDv‐ Gilliam/Ot‐TJTN, and INDv‐Gilliam/Gilliam‐type.