Spotted fever group rickettsioses (SFGR),
typhus group
rickettsioses (TGR),
scrub typhus (caused by Orientia tsutsugamushi),
ehrlichiosis, and
anaplasmosis often present as undifferentiated
fever but are not treated by agents (
penicillins and
cephalosporins) typically used for acute febrile illness. Inability to diagnose these
infections when the patient is acutely ill leads to excess morbidity and mortality. Failure to confirm these
infections retrospectively if a convalescent blood sample is not obtained also impairs epidemiologic and clinical research. We designed a multiplex real-time quantitative PCR (qPCR) assay to detect SFGR, TGR, O. tsutsugamushi, and
infections caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis with the ompA, 17-kDa
surface antigen gene, tsa56, msp2 (p44), and vlpt gene targets, respectively. Analytical sensitivity was ≥2 copies/μl (linear range, 2 to 2 × 105) and specificity was 100%. Clinical sensitivities for SFGR, TGR, and O. tsutsugamushi were 25%, 20%, and 27%, respectively, and specificities were 98%, 99%, and 100%, respectively. Clinical sensitivities for A. phagocytophilum and E. chaffeensis were 93% and 84%, respectively, and specificities were 99% and 98%, respectively. This multiplex qPCR assay could support early clinical diagnosis and treatment, confirm acute
infections in the absence of a convalescent-phase serum sample, and provide the high-throughput testing required to support large clinical and epidemiologic studies. Because replication of SFGR and TGR in endothelial cells results in very low
bacteremia, optimal sensitivity of qPCR for these
rickettsioses will require use of larger volumes of input
DNA, which could be achieved by improved extraction of
DNA from blood and/or extraction of
DNA from a larger initial volume of blood.