Development of a multiplex assay for the detection of antibodies to Borrelia burgdorferi in horses and its validation using Bayesian and conventional statistical methods
Development of a multiplex assay for the detection of antibodies to Borrelia burgdorferi in horses and its validation using Bayesian and conventional statistical methods
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DOI：
10.1016/j.vetimm.2011.08.005
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发表时间：
2011-12-15
影响因子：
1.8
通讯作者：
Groehn, Yrjo
中科院分区：
文献类型：
--
作者：
Wagner, Bettina;Freer, Heather;Groehn, Yrjo
莱姆病是一种人畜共患病媒传播疾病，发生在包括马在内的哺乳动物中。该疾病是由感应伯氏疏螺旋体组的螺旋体感染引起的。哺乳动物宿主的感染需要被感染的蜱在蜱叮咬时传播螺旋体。莱姆病的诊断是基于临床症状、可能接触受感染蜱虫以及抗体检测（传统上是通过ELISA和Western blotting （WB）进行的）。本报告描述了一种新的基于荧光珠的多重检测法的开发和验证，用于检测马血清中伯氏疏螺旋体外表面蛋白a （OspA）、OspC和OspF抗原的抗体。采用WB和新的多重检测法对562种马血清进行盲法平行检测。由于莱姆病抗体检测缺少真正的金标准，我们执行并比较了不同的统计方法来验证新的莱姆病多重检测。一种方法是使用WB结果作为新测试的roc曲线和似然比分析的“相对金标准”。通过分析建立了多重分析的临界值和解释范围。第二种统计方法使用贝叶斯模型计算多重检测的诊断敏感性和特异性。贝叶斯分析考虑到不存在检测伯氏疏螺旋体抗体的真正金标准，以及比较两种测试的估计灵敏度和特异性。因此，贝叶斯分析还对WB的诊断敏感性和特异性进行了评价。总体而言，该方法具有低背景值和宽动态定量范围的特点，可用于检测伯氏疏螺旋体OspA、OspC和OspF抗原的抗体。由于贝叶斯模型不能在该参数上运行，因此通过标准ROC曲线分析计算，基于OspA珠的检测方法的诊断敏感性和特异性分别为49%和85%。OspC和OspF检测的贝叶斯衍生诊断灵敏度分别为80%和86%。相比之下，贝叶斯估计WB对OspC的敏感性为72%，对OspF的敏感性为80%。多重检测对OspC和OspF的贝叶斯诊断特异性分别为79%和69%。WB分析对OspC的特异性为92%，对OspF的特异性为77%。虽然在没有真正金标准的情况下分析一种新的分析方法仍然具有挑战性，但当新技术和传统使用的测试标准在分析灵敏度上存在显着差异时，本文使用的方法可以帮助解决这一问题，从而导致新分析方法的诊断灵敏度和灵敏度值的计算出现问题。综上所述，新的多重检测法检测马血清中伯氏疏螺旋体OspA、OspC和OspF抗原的抗体，与WB分析相比，提高了分析和诊断的敏感性。多重分析是一种有价值的定量工具，可同时检测指示自然感染和/或针对莱姆病原接种疫苗的抗体。(C) 2011 Elsevier B.V.版权所有
Lyme disease is a zoonotic, vector-borne disease and occurs in mammals including horses. The disease is induced by infection with spirochetes of the Borrelia burgdorferi sensu lato group. Infection of mammalian hosts requires transmission of spirochetes by infected ticks during tick bites. Lyme disease diagnosis is based on clinical signs, possible exposure to infected ticks, and antibody testing which is traditionally performed by ELISA and Western blotting (WB). This report describes the development and validation of a new fluorescent bead-based multiplex assay for the detection of antibodies to B. burgdorferi outer surface protein A (OspA), OspC and OspF antigens in horse serum. Testing of 562 equine sera was performed blindly and in parallel by using WB and the new multiplex assay. Because a true gold standard is missing for Lyme antibody testing, we performed and compared different statistical approaches to validate the new Lyme multiplex assay. One approach was to use WB results as a 'relative gold standard' in ROC-curve and likelihood-ratio analyses of the new test. Cut-off values and interpretation ranges of the multiplex assay were established by the analysis. The second statistical approach used a Bayesian model for the calculation of diagnostic sensitivities and specificities of the multiplex assay. The Bayesian analysis takes into consideration that no true gold standard exists for detecting antibodies to B. burgdorferi and estimated sensitivities and specificities of both tests that were compared. Therefore, the Bayesian analysis also resulted in an evaluation of diagnostic sensitivity and specificity of WB. Overall, the new assay was characterized by low background values and a wide dynamic quantification range for the detection of antibodies to OspA, OspC and OspF antigens of B. burgdorferi. The diagnostic sensitivity and specificity for the OspA bead-based assay were calculated as 49% and 85%, respectively, and by a standard ROC curve analysis only because the Bayesian model could not be run on this parameter. The Bayesian-derived diagnostic sensitivities of the OspC and OspF assays were 80% and 86%, respectively. For comparison, the Bayesian-derived estimates for WB resulted in sensitivities of 72% for OspC and 80% for OspF. The Bayesian diagnostic specificities of the multiplex assay were 79% and 69% for OspC and OspF, respectively. WB analysis had specificities of 92% for OspC and 77% for OspF. Although the analysis of a new assay in the absence of a true gold standard remains challenging, the approach used here can help to address this problem when new technologies and traditionally used test standards differ significantly in their analytical sensitivities, which consequently causes problems in the calculation of diagnostic sensitivity and sensitivity values for the new assay. In summary, the new multiplex assay for the detection of antibodies to B. burgdorferi OspA, OspC and OspF antigens in horse serum has improved analytical and diagnostic sensitivities compared to WB analysis. Multiplex analysis is a valuable quantitative tool that simultaneously detects antibodies indicative for natural infection with and/or vaccination against the Lyme pathogen. (C) 2011 Elsevier B.V. All rights reserved.