Département de pathologie et microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada, C.P. 5000, J2S 7C6.
J Dairy Sci. 2012 Nov;95(11):6436-48. doi: 10.3168/jds.2012-5328. Epub 2012 Sep 12.
Bacteriological culture (BC) is the traditional method for intramammary infection diagnosis but lacks sensitivity and is time consuming. Multiplex real-time PCR (mr-PCR) enables testing the presence of several bacteria and reduces diagnosis time. Our objective was to estimate bacterial species-specific sensitivity (Se) and specificity of both BC and mr-PCR tests for detecting bacteria in milk samples from clinical mastitis cases and from apparently normal quarters, using a Bayesian latent class model. Milk samples from 1,014 clinical mastitis cases and 1,495 samples from apparently normal quarters were analyzed by BC and mr-PCR. Two positive culture definitions were used: ≥1 cfu/0.01 mL and ≥10 cfu/0.01 mL of the specified bacteria. The mr-PCR was designed to simultaneously detect Staphylococcus aureus, Streptococcus uberis, Escherichia coli, and Streptococcus agalactiae. The priors used in our Bayesian model were weakly informative, with BC priors using the best available error data. Results were compared with those obtained using uniform priors for mr-PCR to test robustness. Weak and uniform priors gave about the same posterior distributions except for Strep. uberis from normal quarters and Strep. agalactiae. Multiplex real-time PCR Se on milk from clinical mastitis were lower than mr-PCR Se on milk from normal quarters. Multiplex real-time PCR Se was higher than BC on milk from normal quarters. Multiplex real-time PCR Se was generally lower than BC on milk from clinical mastitis and it varied by clinical severity. The estimate specificities of detection for all pathogens were ≥99%, regardless of sample type. The effect of milk sample preservation before testing was evaluated and may have been a factor that affected our observed results. A significant association was observed between sample age and mr-PCR results leading to reduced detection of E. coli and Strep. agalactiae in nonclinical samples. Differences in sample age between conduct of BC and of mr-PCR did not concur with any apparent differences between Se estimates of the 2 tests. Further work should be done to extend these results to other PCR-based tests for detecting bacterial species in milk samples, for which presented results could be used as prior parameter distributions. Limits of sample handling and storage and the potential existence of substances in clinical case samples that may interfere with PCR reactions also are worth further investigation.
细菌培养（BC）是诊断乳腺炎感染的传统方法，但该方法敏感性差且耗时。多重实时 PCR（mr-PCR）可以同时检测几种细菌的存在情况，并且可以缩短诊断时间。我们的目的是使用贝叶斯潜在类别模型来评估 BC 和 mr-PCR 检测临床乳腺炎和正常乳区牛奶样本中细菌的细菌种特异性灵敏度（Se）和特异性。对 1014 例临床乳腺炎病例和 1495 例正常乳区的牛奶样本进行了 BC 和 mr-PCR 分析。使用了两种阳性培养定义：指定细菌的 0.01ml 中≥1cfu 和≥10cfu。mr-PCR 设计用于同时检测金黄色葡萄球菌、无乳链球菌、大肠杆菌和停乳链球菌。在我们的贝叶斯模型中使用的先验是弱信息性的，BC 先验使用了最佳可用错误数据。结果与使用 mr-PCR 的均匀先验进行比较，以检验稳健性。弱先验和均匀先验除了来自正常乳区的无乳链球菌和停乳链球菌之外，给出了几乎相同的后验分布。来自临床乳腺炎的牛奶的多重实时 PCR Se 低于来自正常乳区的 mr-PCR Se。来自正常乳区的牛奶的多重实时 PCR Se 高于 BC。来自临床乳腺炎的牛奶的多重实时 PCR Se 通常低于 BC，并且因临床严重程度而异。所有病原体检测的特异性估计值均≥99%，与样本类型无关。评估了检测前牛奶样本保存的影响，这可能是影响我们观察结果的因素。观察到样本年龄与 mr-PCR 结果之间存在显著关联，导致非临床样本中大肠杆菌和停乳链球菌的检测减少。BC 和 mr-PCR 之间的样本年龄差异与这两种检测方法的 Se 估计值之间没有明显差异不一致。应该进一步开展工作，将这些结果扩展到其他用于检测牛奶样本中细菌种的基于 PCR 的检测方法，所呈现的结果可以用作先验参数分布。进一步研究还应关注样本处理和储存的限制以及可能干扰 PCR 反应的临床病例样本中存在的物质。
