A New Gilliam Genotypic Variant of Orientia tsutsugamushi in Human Scrub Typhus Cases from South India

Scrub typhus, caused by <i>Orientia tsutsugamushi</i> (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 <i>GroEL</i> and 56-kDa TSA genes. A total of 105 serum samples were subjected to PCR amplification and phylogenetic analysis for the <i>GroEL</i> 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 <i>56-kDa</i> gene from the 11 IG-v samples revealed phylogenetic incongruence between the <i>GroEL</i> and <i>56-kDa</i> 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 <i>56-kDa</i> 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.