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Clinical and Diagnostic Laboratory Immunology, August 2005, p. 910-917, Vol. 12, No. 8
1071-412X/05/$08.00+0     doi:10.1128/CDLI.12.8.910-917.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Critical Evaluation of Urine-Based PCR Assay for Diagnosis of Lyme Borreliosis

Biochemical Pharmacology, Faculty of Biology, University of Konstanz, Germany,¹ Untere Hofstatt 3, Kraichtal, Germany²

Received 23 January 2005/ Returned for modification 15 March 2005/ Accepted 17 May 2005

Many approaches were made in recent years to establish urine PCR as a diagnostic tool for Lyme borreliosis, but results are contradictory. In the present study, a standardized protocol spiking urine from healthy donors with a defined amount of whole Borrelia or Borrelia DNA was established. The development of a nested real-time PCR targeting ospA enabled a highly sensitive and quantitative analysis of these samples. We show the following. (i) Storage of spiked urine samples for up to 6 months at –20°C had no negative effect on spike recovery. (ii) Centrifugation of 10 ml of urine at 40,000 x g for 30 min resulted in a concentration of both spikes, i.e., whole Borrelia and DNA. (iii) The inhibition of DNA spike recovery in 48% (11 of 23 samples) of urine samples tested could be attributed to nuclease activity. This was abrogated by alkalizing the urine or by working with the samples on ice. Despite optimized conditions, analysis of urine samples of 12 patients with erythema migrans, the clinical stage considered to be associated with the highest bacterial load, revealed a positive result in only one sample. All 12 samples were negative by an alternative PCR targeting flagellin. The results of our study support doubts that urine is a suitable material for diagnosis of Lyme borreliosis.

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