WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis and Section of Epidemiology, Department of Biomedical Research, Royal Tropical Institute (KIT), Amsterdam, The Netherlands.
PLoS One. 2009 Sep 18;4(9):e7093. doi: 10.1371/journal.pone.0007093.
Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first--for treatment important--4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance.
现有的血清学诊断方法无法在疾病的早期急性阶段确认临床疑似的钩端螺旋体病。已经描述了几种用于早期诊断钩端螺旋体病的常规和实时 PCR，但这些方法尚未得到充分评估。我们开发了一种针对 secY 的基于 SYBR Green 的实时 PCR，并根据国际指南进行了验证。为了确定分析特异性，本研究纳入了来自致病性、非致病性和中间型钩端螺旋体属的 56 株钩端螺旋体菌株以及其他 46 种微生物的 DNA。所有致病性钩端螺旋体均呈阳性反应。我们没有发现与腐生性钩端螺旋体和其他微生物的交叉反应，表明具有很高的分析特异性。PCR 的分析灵敏度从同源培养菌株 M 20 的每个反应 1 个拷贝，以及异源菌株 1342 K 和 Sarmin 的 1.2 和 1.5 个拷贝。在加标血清和血液以及肾脏组织中，M 20 的灵敏度为 10 和 20 个拷贝，1342 K 为 15 和 30 个拷贝，Sarmin 为 30 和 50 个拷贝。为了确定诊断灵敏度（DSe）和特异性（DSp），我们纳入了 26 例经实验室确认的和 107 例疑似钩端螺旋体病的临床血样作为前瞻性连续队列。基于培养作为金标准，我们发现 DSe 和 DSp 分别为 100%和 93%。所有 8 个 PCR 阳性样本在培养结果为阴性后均发生血清学转换，这意味着实际 DSp 更高。当将培养和血清学作为金标准时，DSe 较低（89%），而 DSp 较高（100%）。在发病后第 1 天至第 4 天（对治疗重要）采集的样本中，DSe 为 100%。通过对 20 例确诊阳性和 20 例阴性啮齿动物的肾脏样本进行盲法检测，确定了该检测方法的重复性和可重复性均为 100%。总之，我们首次描述了一种用于检测致病性钩端螺旋体的稳健的 SYBR Green 实时 PCR 的开发，并对其临床准确性进行了详细评估，从而提供了一种具有明确令人满意性能的钩端螺旋体病早期诊断方法。
