MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Christophe Mérieux Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Front Cell Infect Microbiol. 2018 Apr 5;8:107. doi: 10.3389/fcimb.2018.00107. eCollection 2018.
Pneumonia and meningitis continue to present an enormous public health burden and pose a major threat to young children. Among the causative organisms of pneumonia and meningitis, bacteria are the most common causes of serious disease and deaths. It is challenging to accurately and rapidly identify these agents. To solve this problem, we developed and validated a 12-plex PCR coupled with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method (bacterial pathogen-mass spectrometry, BP-MS) that can be used to simultaneously screen for 11 key bacterial pathogens related to pneumonia and meningitis. Forty-six nasopharyngeal swabs and 12 isolates were used to determine the specificity of the method. The results showed that, using the BP-MS method, we could accurately identify the expected bacteria without cross-reactivity with other pathogens. For the 11 target bacterial pathogens, the analytical sensitivity of the BP-MS method was as low as 10 copies/reaction. To further evaluate the clinical effectiveness of this method, 204 nasopharyngeal swabs from hospitalized children with suspected pneumonia were tested using this method. In total, 81.9% (167/204) of the samples were positive for at least one of the 11 target pathogens. Among the 167 bacteria-positive samples, the rate of multiple infections was 55.7% (93/167), and the most frequent combination was with , representing 46.2% (43/93) two-pathogen mixed infections. We used real-time PCR and nested PCR to confirm positive results, with identical results obtained for 81.4% (136/167) of the samples. The BP-MS method is a sensitive and specific molecular detection technique in a multiplex format and with high sample throughput. Therefore, it will be a powerful tool for pathogen screening and antibiotic selection at an early stage of disease.
肺炎和脑膜炎仍然是巨大的公共卫生负担，对幼儿构成重大威胁。在引起肺炎和脑膜炎的病原体中，细菌是导致严重疾病和死亡的最常见原因。准确、快速地识别这些病原体具有挑战性。为了解决这个问题，我们开发并验证了一种 12 重聚合酶链反应（PCR）结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）（细菌病原体-质谱，BP-MS）方法，可用于同时筛查与肺炎和脑膜炎相关的 11 种关键细菌病原体。我们使用 46 个鼻咽拭子和 12 个分离株来确定该方法的特异性。结果表明，使用 BP-MS 方法，我们可以准确识别预期的细菌，而不会与其他病原体发生交叉反应。对于 11 种目标细菌病原体，BP-MS 方法的分析灵敏度低至 10 拷贝/反应。为了进一步评估该方法的临床效果，我们使用该方法检测了 204 份来自住院疑似肺炎患儿的鼻咽拭子。总共有 81.9%（167/204）的样本至少有一种 11 种目标病原体呈阳性。在 167 个细菌阳性样本中，混合感染率为 55.7%（93/167），最常见的组合是 与 ，占 46.2%（43/93）的两种病原体混合感染。我们使用实时 PCR 和巢式 PCR 对阳性结果进行了确认，81.4%（136/167）的样本结果一致。BP-MS 方法是一种敏感、特异的分子检测技术，可在多重格式和高样本通量下进行。因此，它将成为疾病早期病原体筛查和抗生素选择的有力工具。
