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

Abstract

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 louse-borne 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.

Keywords: Borrelia; real-time PCR; relapsing fever; tick-borne pathogens.

FIG 1

(A) Phylogenetic tree of RF Borrelia species. Whole genomes were downloaded from GenBank (see Table S1 in the supplemental material for accession numbers) and aligned using progressiveMauve (40). Alignments of the eight housekeeping genes used in Borrelia multilocus sequence typing (uvrA, rplB, recG, pyrG, pepX, clpX, nifS, and clpA) (41) were extracted and concatenated, and a tree was created using maximum likelihood methodology and 500 bootstrap replicates in MEGA X (42). (B) Sequence alignment of the amplified region of Bh_0509 and homologs in other RF borreliae. The strains included are the same as those in panel A, except that B. hermsii YOR and B. miyamotoi FR64b are not included. Nucleotides that differ from the consensus are highlighted (red, A; yellow, G; blue, C; green, T). Arrows indicate primer and probe binding regions (5′ to 3′) and are annotated on the sequence to which they match exactly.

FIG 2

(A to C) Standard curves for B. hermsii 95-0544 (HEX channel) (A), B. miyamotoi HT31 (FAM channel) (B), and B. recurrentis 99-0708 (Cy5 channel) (C). Serial 10-fold dilutions of bacterial DNA from 1 ng to 1 fg were tested in duplicate. ΔRn, fluorescent reporter signal normalized to baseline. (D) Linearity of standard curves. Data from panels A to C are plotted, along with data for B. turicatae 90-901 (amplification curves not shown). RF borreliae are indicated by different symbols.