An improved in-house lysis-filtration protocol for bacterial identification from positive blood culture bottles with high identification rates by MALDI-TOF MS

作者：Tsuchida Sachio; Murata Syota; Miyabe Akiko; Satoh Mamoru; Takiwaki Masaki; Matsushita Kazuyuki; Nomura Fumio^*

来源：Journal of Microbiological Methods, 2018, 148: 40-45.

DOI：10.1016/j.mimet.2018.03.014

• Bacterial identification
• Blood culture
• Matrix-assisted laser desorption ionization
• time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is now a well established method for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial proteins are a prerequisite for successful identification, and a variety of protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We developed a new, in-house lysis-filtration protocol and prospectively evaluated its performance compared to the Sepsityper kit. The in-house protocol consists of three simple steps: lysis by ammonium chloride, aspiration with a syringe fitted with a 0.45-mu m membrane, and centrifugation to collect microbes. The novel protocol requires only 20 min.
Performance of the in-house protocol was evaluated using a total of 117 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the in-house protocol or the commercial kit, and isolated cells were subjected to direct identification by mass spectrometry fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with score > 1.7 and > 2.0 obtained using the in-house protocol were 99.2% and 85.5%, respectively, whereas those obtained using the Sepsityper Kit were 85.4% and 61.5%, respectively. For Gram-positive cases, the in-house protocol yielded scores > 1.7 and > 2.0 at 98.5% and 76.1%, respectively, whereas the commercial kit yielded these scores at 76.1% and 43.3%, respectively. Although these are preliminary results, these values suggest that this easy lysis-filtration protocol deserves assessment in a larger-scale test.

• 出版日期2018-5