Abstract
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.
Highlights
Borrelia burgdorferi sensu lato (s.l.) is transmitted through the bite of infected ticks in the Ixodes family and is the causative agent of Lyme borreliosis (LB), the most common vector borne disease in Europe and North America
Demonstration of Borrelia in tissues is valuable for confirming a diagnosis based on less clear manifestations of LB, in regions endemic for the disease, where positive serology may be due to past exposure to B. burgdorferi s.l
Negative Specimens The number of Borrelia 16S rRNA targets detected by qPCR for PCR positive patients varied considerably, ranging from 0.2 to 321 Borrelia 16S rRNA target copies per 10,000 human genome equivalents with a median of 15 (IQR: 5 to 39.75)
Summary
Borrelia burgdorferi sensu lato (s.l.) is transmitted through the bite of infected ticks in the Ixodes family and is the causative agent of Lyme borreliosis (LB), the most common vector borne disease in Europe and North America. By co-amplification of the human RNAseP gene, the bacterial load can be expressed in relation to human genomic DNA, thereby compensating for variations arising from the DNA extraction procedure and/or the size and quality of the biopsy (e.g. variations in tissue composition due to differences in the site from which the biopsy was obtained) We used this assay together with culture, to evaluate skin biopsies from 121 patients presenting with a solitary EM at the University Medical Centre Ljubljana in Slovenia. These data enabled us to compare the ability of both methods to confirm a clinical diagnosis of EM as well as providing information on quantification of B. burgdorferi s.l. in skin biopsies. The results of this proof-of-concept study serve to demonstrate the benefit of this PCR assay to determine the efficacy of a LB vaccine in clinical trials
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