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

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          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.

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          Author and article information

          Contributors
          Role: Editor
          Journal
          PLoS One
          PLoS ONE
          plos
          plosone
          PLoS ONE
          Public Library of Science (San Francisco, USA )
          1932-6203
          2013
          16 May 2013
          : 8
          : 5
          Affiliations
          [1 ]Vaccine R&D, Baxter Bioscience, Orth/Donau, Austria
          [2 ]Faculty of Medicine, Institute for Biostatistics and Medical Informatics, Ljubljana, Slovenia
          [3 ]Department of Infectious Diseases, University Medical Centre Ljubljana, Ljubljana, Slovenia
          [4 ]Paracelsus Medical University, Spinal Cord Injury and Tissue Regeneration Center Salzburg, Institute of Tendon and Bone Regeneration, Salzburg, Austria
          The Johns Hopkins University School of Medicine, United States of America
          Author notes

          Competing Interests: A. Traweger is a former employee of Baxter. M. O’Rourke, P.N. Barrett and I. Livey are salaried employees of Baxter, holding stocks/shares. M. O’Rourke and I. Livey hold a patent for a vaccine against LB. L. Lusa, D. Stupica, V. Maraspin, and F. Strle report no conflicts of interest. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

          Conceived and designed the experiments: MO AT FS IL. Performed the experiments: MO AT DS VM. Analyzed the data: LL MO AT PNB FS IL. Contributed reagents/materials/analysis tools: DS VM PNB FS. Wrote the paper: MO. Critically revised the manuscript and approved the final version to be published: MO AT LL DS VM PNB FS IL.

          Article
          PONE-D-12-36117
          10.1371/journal.pone.0063968
          3655952
          23696863
          f729bf6b-1ae8-498f-85f2-d18a2c428c71

          This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

          Page count
          Pages: 9
          Funding
          This study was funded by Baxter and supported in part by the Slovenian Research Agency (Grant Numbers P3-0296 and J3-3636). Baxter employees were involved in study design, data collection and analysis, the decision to publish, and preparation of the manuscript. The Slovenian Research Agency had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
          Categories
          Research Article
          Medicine
          Clinical Research Design
          Clinical Trials
          Epidemiology
          Diagnostic Medicine
          Clinical Laboratory Sciences
          Test Evaluation
          Epidemiology
          Clinical Epidemiology
          Epidemiological Methods
          Infectious Diseases
          Bacterial Diseases
          Lyme Disease
          Vectors and Hosts
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