Development of multiplex PCR for neglected infectious diseases

Scrub typhus, murine typhus, and leptospirosis are widely neglected infectious diseases caused by Orientia tsutsugamushi, Rickettsia typhi, and pathogenic Leptospira spp., respectively. Patients usually present with non-specific symptoms and therefore are commonly diagnosed with acute undifferentiated febrile illness. Consequently, patients face delayed treatment and increased mortality. Antibody-based serological test currently used as gold standard has limitations due to insufficient antibody titers, especially in the early phase of infection. In this study, we aimed to develop multiplex PCR to combine 3 primer pairs that target specific genes encoding 56-kDa TSA of O. tsutsugamushi, 17-kDa antigen of R. typhi, and LipL32 of L. Interrogans and evaluate its performance in comparison to the standard serological tests. Using EDTA blood samples of known patients, the sensitivity and specificity of our multiplex PCR was 100% and 70%, respectively. In addition, the assay was able to diagnose the co-infection of scrub typhus and leptospirosis. The assay may be useful in identifying causative agents during the early phase of these diseases, enabling prompt and appropriate treatment. Author summary Scrub typhus, murine typhus, and leptospirosis are diagnosed as acute undifferentiated febrile illness. Diagnostic tests for these diseases depend on antibody detection. However, antibody detection is still limited by its tendency to return negative results during the early phase of aforementioned diseases. In this study, a novel multiplex PCR has been developed for detecting Orientia tsutsugamushi, Rickettsia typhi, and Leptospira interrogans that are simultaneously amplified in a single tube. The results have shown that multiplex PCR could be used as a diagnostic tool for detecting bacteria during the early phase of scrub typhus, murine typhus, and leptospirosis, allowing for administration of appropriate treatment.