Development of multiplex PCR for diagnosis of scrub typhus, rickettsial disease and leptospirosis

Abstract

Scrub typhus, leptospirosis, and murine typhus are life-threatening zoonosis worldwide. The causative agent of these diseases is Orientia tsutsugamushi, Leptospira interrogans, and Rickettsia typhi, respectively. Transmission of these bacteria occurs through vector biting or directly bacteria contraction. Clinical features of these diseases are as acute undifferentiated fever such as high fever, headache, muscular pain, and anorexia, resulting to delay diagnosis and mortality. Although a serological laboratory test is gold standard for scrub typhus, leptospirosis, and murine typhus diagnosis, it has own limitation because inadequate antibody in the early phase. In this study, we aimed to develop the multiplex PCR on the causative agents of scrub typhus, leptospirosis, and murine typhus detection and evaluate the performance of multiplex PCR compared to the serological tests. In the experiment, we optimized the appropriate multiplex PCR for detection of tested bacteria. Subsequently, we evaluated the specificity, sensitivity, and performance of the developed multiplex PCR. Our results showed only positive detectable PCR product from tested bacteria. The sensitivity and specificity of the developed multiplex PCR was 100% and 71.67% compared to the serological tests. And also, the assay found the co-infection of scrub typhus and leptospirosis. The designed multiplex PCR assay is sensitive, specific and rapid. The assay can be used for identifying the causative agent in the early phase of these diseases.