Scrub typhus is a mite-borne, re-emerging public health problem in India, particularly in Tamil Nadu, South India. More than 40 serotypes of Orientia tsutsugamushi have been documented worldwide. However, the information on the circulation of its molecular sub-types in India is scanty. 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 the 56 kDa type-specific antigen (TSA) genes. Out of 59 samples, 14 partial fragments of GroEL and the 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-Tamil Nadu (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 56 kDa variable domains (VDΙ-ΙΙΙ) of TSA gene sequences revealed a distinctive new genotypic cluster of eight samples (66.6%), and the remaining four (33.4%) were Karp-like genotypes. The Simplot analysis for the similarity and event of recombination testing elucidated the existence of the new genotype of the Ot-TJTN cluster, which was undescribed so far, in the Kato and TA716 lineages. The significant findings recommend further studies to understand the ongoing transmission dynamics of different O. tsutsugamushi strains in vector mites, rodent hosts, and humans in this region.

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 (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.

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.