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

机译：尿液PCR检测法对莱姆病的诊断的关键评估

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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 × 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.
机译：近年来，人们采取了许多方法来将尿液PCR建立为莱姆病的诊断工具，但结果却相矛盾。在本研究中，建立了标准化协议，该协议从健康供体中滴出尿液，并带有确定量的整个或 Borrelia DNA。靶向 ospA 的嵌套实时PCR的发展实现了对这些样品的高度灵敏和定量的分析。我们显示以下内容。 （i）加标尿液样品在20°C下最多可保存6个月，对加标回收率没有负面影响。 （ii）将10 ml尿液以40,000× g 的速度离心30分钟，导致两个刺突浓度，即整个 Borrelia 和DNA浓度升高。 （iii）48％（23个样品中的11个）尿液样品中DNA峰值恢复的抑制可归因于核酸酶活性。通过碱化尿液或在冰上处理样品来消除这种情况。尽管条件优化，但对12例红斑偏头痛患者的尿液样本进行了分析，该临床阶段被认为与最高细菌载量有关，但仅对一个样本进行了阳性结果。通过替代PCR靶向鞭毛蛋白，所有12个样品均为阴性。我们的研究结果支持人们怀疑尿液是否可作为诊断莱姆病的方法。

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