Oviaño Marina, Ramírez Cecilia de la Luna, Barbeyto Luis Pedro, Bou Germán
J Antimicrob Chemother. 2017 May 1;72(5):1350-1354. doi: 10.1093/jac/dkw579.
Development of an automated MALDI-TOF MS-based method for the rapid, direct detection of carbapenemase-producing Enterobacteriaceae in clinical urine samples within 90 min of sample reception.
A total of 3041 urine samples were processed by flow cytometry, and a cut-off value of ≥1.5 × 10 5 bacteria/mL was used to select samples for the study. Following these criteria, 608 samples were selected for direct bacterial identification. Detection of carbapenemase activity by MALDI-TOF MS analysis was only performed after reliable direct identification of Gram-negative bacilli. A novel protocol was developed for extracting bacteria from urine samples by using the Sepsityper Kit (Bruker Daltonik, Germany). Carbapenem resistance was detected with imipenem as an antibiotic marker and the results were automatically interpreted using the STAR-BL module of MALDI-TOF Biotyper Compass software (Bruker Daltonik, Germany).
The MALDI-TOF MS-based assay yielded direct reliable identification of 91% (503/553) of the samples. The assay showed 100% sensitivity (30/30) and specificity (454/454) for detecting carbapenemase activity within 90 min of sample reception. Isolates included in the study were further characterized by PCR and sequencing, and bla OXA-48 was detected from all isolates that tested positive in the MALDI-TOF MS-based resistance assay.
The proposed protocol for the direct analysis of urine samples by MALDI-TOF MS is suitable for use in clinical laboratories to identify bacteria and detect carbapenemase activity, thus saving at least 24-48 h relative to current routine methods.
开发一种基于基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）的自动化方法，用于在接收临床尿液样本后90分钟内快速、直接检测产碳青霉烯酶肠杆菌科细菌。
通过流式细胞术对总共3041份尿液样本进行处理，并使用≥1.5×10⁵ 个细菌/mL的截断值来选择用于研究的样本。按照这些标准，选择了608份样本进行直接细菌鉴定。仅在可靠地直接鉴定出革兰氏阴性杆菌后，才通过MALDI-TOF MS分析检测碳青霉烯酶活性。开发了一种新方案，使用Sepsityper试剂盒（德国布鲁克道尔顿公司）从尿液样本中提取细菌。以亚胺培南作为抗生素标志物检测碳青霉烯耐药性，并使用MALDI-TOF Biotyper Compass软件（德国布鲁克道尔顿公司）的STAR-BL模块自动解读结果。
基于MALDI-TOF MS的检测方法对91%（503/553）的样本进行了直接可靠的鉴定。该检测方法在接收样本后90分钟内检测碳青霉烯酶活性的灵敏度为100%（30/30），特异性为100%（454/454）。研究中纳入的分离株通过聚合酶链反应（PCR）和测序进一步鉴定，在基于MALDI-TOF MS的耐药性检测中检测为阳性的所有分离株均检测到blaOXA-48。
所提出的通过MALDI-TOF MS直接分析尿液样本的方案适用于临床实验室鉴定细菌和检测碳青霉烯酶活性，相对于当前的常规方法至少节省24 - 48小时。
