Molecular detection and genotyping of Orientia tsutsugamushi in human serum samples from a scrub typhus outbreak in Vellore and adjacent districts, Tamil Nadu, India.

Scrub typhus, caused by Orientia tsutsugamushi, is a clinically important vector-borne disease in the Asia-Pacific region. This study aimed to detect O. tsutsugamushi DNA in human serum samples collected during a scrub typhus outbreak in Vellore and adjacent districts of Tamil Nadu, India, and to genotype the pathogen based on the tsa56 gene sequence. A total of 40 serum samples were analyzed, including nine IgM ELISA-positive samples and 31 IgM-negative samples. Nested PCR targeting the 47-kDa htrA gene detected O. tsutsugamushi DNA in all nine IgM-positive samples and in 10 of the 31 IgM-negative samples, confirming the presence of O. tsutsugamushi DNA in serum. This study demonstrates the detection of Orientia tsutsugamushi DNA in human serum using a 47-kDa htrA gene-based nested PCR assay and highlights the value of integrating this molecular approach with IgM ELISA for early and accurate diagnosis of scrub typhus. From one serum sample, the 1571-bp tsa56 gene was amplified. The tsa56 gene based phylogenetic analysis revealed three distinct clusters within this geography and the isolate from this study was closely related to Cluster A. Amino acid sequence alignment of the TSA56 variable domains indicated distinct variations in VD-I, II and III, while VD-IV remained conserved in Clusters B and C. These findings highlight the importance of integrating molecular diagnostics with serology and emphasize the need for improved molecular surveillance and diagnostics to better characterize strain diversity, improve disease management and vaccine development.