Optimization and evaluation of a multiplex quantitative PCR assay for detection of nucleic acids in human blood from patients with spotted fever rickettsiosis, typhus rickettsiosis, scrub typhus, monocytic ehrlichiosis, and granulocytic anaplasmosis

Spotted fever group (SFGR) and typhus group (TGR) rickettsioses, scrub typhus (caused by Orientia tsutsugamushi, [OT]), 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 assay to detect SFGR, TGR, OT, and infections caused by Anaplasma phagocytophilum (AP), and Ehrlichia chaffeensis (EC) with ompA, 17-kDa surface antigen gene, tsa56, msp2/p44, and vlpt gene targets, respectively. Analytical sensitivity was ≥2 copies/μL (linear range 2 to 2x10⁵) and specificity 100%. Clinical sensitivity for SFGR, TGR, and OT was 25%, 20%, and 27%, respectively, and specificity 98%, 99%, and 100%, respectively. Clinical sensitivity for AP and EC was 93% and 84%, respectively, and specificity 99% and 98%, respectively. This multiplex qPCR assay could support early clinical diagnosis and treatment, confirm acute infections in the absence of a convalescent 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.