A high-resolution nested PCR assay for genotyping Orientia tsutsugamushi based on a defined 700 bp segment of the TSA56 gene.

OBJECTIVES: To define a reproducibly specified TSA56 fragment that preserves genotype assignment relative to the full-length gene and to establish a standardized sequence-based genotyping workflow using nested PCR and phylogenetic assignment. METHODS: We screened 355 complete TSA56 sequences across the gene and designed nested primers to generate an ∼800-bp amplicon covering the selected segment. Analytical performance was assessed across 17 genotypes, clinical applicability was evaluated in 216 acute-phase blood samples, and interlaboratory agreement was assessed across three laboratories. RESULTS: The nt 151-850 segment achieved complete genotype concordance and the lowest RF distance to the full-length reference topology. The assay amplified all 17 genotypes, showed no cross-reactivity with four non-target pathogens, yielded genotype-specific LOD95 values of 2.40-102.17 copies/μL, and had total repeatability CVs below 5%. Nested PCR detected 45/47 composite-positive specimens and none of 50 composite-negative specimens, enabling assignment of seven circulating genotypes. Genotype assignments were fully concordant across the three laboratories. CONCLUSIONS: This standardized amplicon-and-analysis workflow supports reproducible TSA56 genotyping and more comparable sequence-based surveillance of scrub typhus.