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B. burgdorferi sensu stricto ,  B. afzelii ,  B. garinii  and  B. bavariensis  are the principal species which account for Lyme borreliosis (LB) globally. We have developed an internally controlled duplex quantitative real time PCR assay targeting the  Borrelia  16S rRNA and the human RNAseP genes. This assay is well-suited for laboratory confirmation of suspected cases of LB and will be used to assess the efficacy of a vaccine against LB in clinical trials. The assay is highly specific, successfully detecting DNA extracted from 83 diverse  B. burgdorferi  sensu lato strains representing all major species causing LB, while 21 unrelated microbial species and human genomic DNA tested negative. The assay was highly reproducible and sensitive, with a lower limit of detection of 6 copies per PCR reaction. Together with culture, the assay was used to evaluate paired 3 mm skin biopsy samples taken from 121 patients presenting with solitary erythema migrans (EM) lesion. PCR testing identified more positive biopsy samples than culture (77.7% PCR positive versus 55.1% culture positive) and correctly identified all specimens scored as culture positive. OspA-based typing identified the majority of isolates as  B. afzelii  (96.8%) and the bacterial load was significantly higher in culture positive biopsies than in culture negative biopsies ( P <0.001). The quantitative data also enabled relationships between  Borrelia  burden and patient symptoms to be evaluated. The bacterial load was significantly higher among patients with systemic symptoms than without ( P  = 0.02) and was significantly higher for biopsies retrieved from patients with EM lesions with central clearing ( P <0.001). 16S copy numbers were moderately lower in samples from patients reporting a history of LB ( P  = 0.10). This is the first quantitative PCR study of human skin biopsies predominantly infected with  B. afzelii  and the first study to demonstrate a clear relationship between clinical symptoms in  B. afzelii- infected patients and  Borrelia  burden.

Quantitative Detection of Borrelia burgdorferi sensu lato in Erythema Migrans Skin Lesions Using Internally Controlled Duplex Real Time PCR