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

Tsutsugamushi disease and Japanese spotted fever are representative rickettsioses in Japan, and are caused by infection with Orientia tsutsugamushi and Rickettsia japonica, respectively. For molecular-based diagnosis, conventional PCR assays, which independently amplify respective rickettsial DNA, are usually used; however, this approach is time-consuming. Here, we describe a new duplex real-time PCR assay for the simultaneous detection of O. tsutsugamushi and spotted fever group rickettsiae, and its evaluation using several PCR conditions in 6 public health laboratories. The detection limit of the assay was estimated to be 10² copies and the sensitivity was almost identical to that of 3 conventional PCR methods. A total of 317 febrile patients were selected as clinically suspected or confirmed cases of rickettsioses. The detection efficiency of this assay for O. tsutsugamushi from blood or skin (eschar) specimens appeared to be almost the same as that of the conventional PCR method, even when performed in different laboratories, whereas the efficiency for spotted fever group rickettsiae tended to be higher than that of the 2 traditional double PCR assays. Our duplex real-time PCR is thus a powerful tool for the rapid diagnosis of rickettsioses, especially at the acute stage of infection.