Simultaneous Detection and Differentiation of Clinically Relevant Relapsing Fever Borrelia with Semimultiplex Real-Time PCR

Bacterial vector-borne diseases, including Borrelia species, present a significant diagnostic, clinical, and public health challenge due to their overlapping symptoms and the breadth of causative agents and arthropod vectors. The relapsing fever (RF) borreliae encompass both established and emerging pathogens and are transmitted to humans by soft ticks, hard ticks, or lice. We developed a real-time semimultiplex PCR assay that detects multiple RF borreliae causing human illness and classifies them into one of three groups. The groups are based on genetic similarity and include agents of soft-tick relapsing fever (Borrelia hermsii and others), the emerging hard-tick-transmitted pathogen B. miyamotoi, and the agent of louseborne relapsing fever (B. recurrentis). The real-time PCR assay uses a single primer pair designed to amplify all known pathogenic RF borreliae and multiple TaqMan probes to allow the detection of and differentiation among the three groups. The assay detects all RF borreliae tested, with an analytical limit of detection below 15 genome equivalents per reaction. Thirty isolates of RF borreliae encompassing six species were accurately identified. Thirty-nine of 41 residual specimens (EDTA whole blood, serum, or plasma) from patients with RF were detected and correctly classified. None of 42 clinical samples from patients with other infections and 46 culture specimens from non-RF bacteria were detected. The development of a single-assay real-time PCR approach will help to improve the diagnosis of RF by simplifying the selection of tests to aid in the clinical management of acutely ill RF patients.