EA 3882-Laboratoire de Biodiversité et d'Ecologie Microbienne (LUBEM), SFR 148 ScInBioS, Faculté de Médecine, Université de Brest, Brest F-29200, France.
BMC Microbiol. 2013 Jun 21;13:143. doi: 10.1186/1471-2180-13-143.
The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients.
We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes.
Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did.
Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies.
囊性纤维化（CF）患者的肺部对铜绿假单胞菌特别敏感。这种细菌在 CF 患者的不良预后中起着重要作用。在疾病进展过程中，CF 患者首次获得铜绿假单胞菌是管理的关键步骤。定量 PCR（qPCR）提供了一个机会，可以更早地检测到 CF 患者首次获得铜绿假单胞菌。由于缺乏经过验证的方案，我们的目标是找到一种用于检测 CF 患者中铜绿假单胞菌的最佳分子方案。
我们比较了两种以前描述的 qPCR 格式，用于早期检测 CF 痰样本中的铜绿假单胞菌：靶向 oprL 基因的 qPCR 和靶向 gyrB 和 ecfX 基因的多重 PCR。
在体外对大量铜绿假单胞菌分离株和其他革兰氏阴性杆菌进行测试后，oprL qPCR 表现出更好的灵敏度（阈值为 10 CFU/mL 与 730 CFU/mL 相比），而 gyrB/ecfX qPCR 表现出更好的特异性（90%与 73%相比）。这些结果在 46 份经培养证实为铜绿假单胞菌阳性的 CF 痰样本中得到了验证。体外试验显示，qPCR 检测到的细菌细胞数量比基于培养的方法多 100 倍。
基于这些结果，我们提出了一种参考分子方案，该方案结合了两种 qPCR，其灵敏度为 100%，阈值为 10 CFU/mL，特异性为 100%。这种基于组合 qPCR 的方案可以进行调整并用于其他未来的前瞻性研究。
