In silico identification and field validation of diagnostic marker gene targets for the improved detection of scrub typhus

Scrub typhus, a vector-borne zoonosis prevalent in the Asia-Pacific region, poses diagnostic challenges due to the pathogen's complex genome and diverse rodent and shrew hosts. The scarcity of reliable diagnostic tests hinders effective sentinel surveillance. This study aims to identify novel diagnostic gene targets using a bioinformatics approach to develop a highly sensitive and specific PCR assay for scrub typhus detection. Genome sequences of Orientia tsutsugamushi, the causative agent, were analyzed, leading to the selection of 11 potential diagnostic biomarkers. In-house conventional PCR assays targeting these biomarkers and published nested PCR assays, were tested on blood and tissue (spleen) samples of 150 field rodent and shrew. Among the tested genes, the tsa56 gene consistently demonstrated the highest detection rate in both conventional (55.6 %, n = 15) and nested (74 %, n = 20) assays, indicating it to be the most reliable diagnostic marker for scrub typhus. A novel nested PCR was designed targeting a unique tsa56 gene segment, which showed an analytical sensitivity of 4.7 (95 % CI: 2–7.4) copies/μL, and was able to detect O. tsutsugamushi in multiple hosts including human and mite samples. Moreover, fewer primer-template mismatches and no mismatches at the critical 3′ terminus with O. tsutsugamushi strains were observed. The assay did not show any cross-reaction with available non-target organisms. Thus, the developed nested PCR assay demonstrates enhanced sensitivity, specificity, and broader strain inclusivity. Overall, the study presents a promising tool for scrub typhus detection, which will aid in improved disease surveillance, outbreak prediction, and timely implementation of control measures.