Evaluation of a Real-Time PCR Assay for the Detection, Genotyping, and Quantification of Borrelia Burgdorferi Sensu Lato in Ixodes Ticks in a Routine Laboratory Setting

Background: Testing of saved Ixodes ticks for the presence of Borrelia burgdorferi after biting a patient may be helpful to assess the risk of bacterial transmission and to decide upon preventive antibiotic treatment. A commercially available real-time PCR assay for the identification and quantification of Borrelia burdorferi sensu lato species (LightMix Kit for the detection of Borrelia spp., TIB MOLBIOL) was evaluated. Methods: Clinical and laboratory specimens were analysed for Borrelia DNA, including seven official proficiency panels (INSTAND, Germany, 2004 to 2009)), kit standards and 1121 ticks saved from bitten patients from all over Germany. Results: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia spielmanii, and Borrelia valaisiana were reliably detected by the real-time PCR. The recently described Borrelia lusitaniae (not pathogenic for humans) was not detected. Melt curve analysis of the PCR products allowed for a reliable Borrelia genospecies differentiation. Dilution series of proficiency panel samples revealed a detection limit of approximately 10(1) genomes / mL. Based on CP values the mean interassay coefficient of variation of the assay was 3.6 %, while the in traassay coefficients of variation were 1.6 % and 0.8 % for a strong positive and a weak positive kit standard, respectively. 20.2 % of the clinical ticks revealed detectable Borrelia burgdorferi DNA, with Borrelia afzelii being the most prominent subspecies. The quantification of Borrelia genomes in individual ticks showed a broad distribution ranging from 0.5 x 10(1) to 2.6 x 10(7) Borrelia equivalents per tick (median 1.6 x 10(4)). Conclusions: The LightMix Kit for the detection of Borrelia spp. represents an excellent tool for the identification and quantification of Borrelia species in ticks. It can serve to further elucidate the relevance of preventive antibiotic treatment following tick bites in upcoming clinical studies. (Clin. Lab. 2011;57:67-73)