Genotype Analysis on Orientia tsutsugamushi Causing Scrub Typhus in Malaysia: A Re-Emerging Disease

Introduction: Scrub typhus is caused by Gram-negative bacteria, Orientia tsutsugamushi. Humans are the dead-end host of scrub typhus. Currently, there is no vaccine available. The disease can be fatal without appropriate treatment. Here, we present the circulating OT genotypes in Malaysia and a tsa56-based single PCR to detect and determine OT genotypes, which is an approach to replace the time-consuming traditional nested PCR. Methods: The patients' blood or tissue samples (n = 1200), received from all hospitals in Malaysia from December 2022 to November 2024, were screened for rickettsial infections. Both htrA qPCR and nested PCR were performed to detect the presence of OT DNA. Simultaneously, a selection of DNA was evaluated for the new single PCR protocol and confirmed with Sanger sequencing. Results: We report that Pahang state of Peninsular Malaysia presents the highest number of acute scrub typhus infections in Malaysia within the 24 months period. There are four genotypes circulating in the Malaysian population. OT genotype Gilliam (n = 31, 29.2%) and Karp (n = 31, 29.2%) are the predominant OT genotypes in Malaysia, followed by TA763 (n = 22, 20.8%) and Kato (n = 22, 20.8%). The single-run PCR presents longer sequence size and similar results with the nested PCR. Conclusions: Acute scrub typhus infection is not rare in Malaysia and should be considered for undifferentiated febrile illness. The single-run PCR protocol is time-saving and a promising approach for OT detection and genotype analysis in a single run to complement a clinical diagnostic setting and surveillance.