Evaluation of Diagnostic Assay for Rickettsioses Using Duplex Real-Time PCR in Multiple Laboratories in Japan

Abstract

Tsutsugamushi disease (TD) and Japanese spotted fever (JSF) are known as representative rickettsioses in Japan, and caused by infection with Orientia tsutsugamushi and Rickettsia japonica, respectively. For molecular-based diagnosis, conventional PCRs which independently amplify respective rickettsial DNAs are usually used, but it’s time-consuming. Here, we describe a new duplex real-time PCR assay for simultaneous detection of O. tsutsugamushi and spotted fever group rickettsiae, and evaluated under several PCR conditions in 6 public health laboratories. The detection limit of the assay was estimated 10² copies and the sensitivity was almost same as that of 3 conventional PCRs. Total of 317 febrile patients were selected as clinically suspected or confirmed cases for rickettsioses. The detection efficiency of this assay for O. tsutsugamushi from specimens of blood or skin (eschar) seems to be almost same as that of conventional nested PCR even in different laboratory tests, while the efficiency for SFG rickettsiae tends to be higher than that of 2 traditional double PCRs. Our duplex real-time PCR is a powerful tool for rapid diagnosis of rickettsioses, especially at the acute stage of infection.