Candida auris is an emerging antifungal resistant yeast species causing nosocomial and invasive infections, emphasising the need of improved diagnostics and epidemiological typing methods. We show that MALDI-TOF VITEK-MS followed by amplified length polymorphisms allows for accurate species identification and subsequent epidemiological characterisation of strains encountered during potential outbreaks.

A large spectral dataset obtained from in vitro mono- and bi-microbial samples allowed us to evaluate the performance of the method in a comprehensive way. Provided that the reference matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprints were sufficiently distinct for the individual species, the method automatically predicted which bacterial species were present in the sample. Only few samples (5.3%) were misidentified, and bi-microbial samples were correctly identified in up to 61.2% of the cases. This method could be used in routine clinical microbiology practice.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS) has been introduced in clinical routine microbiology laboratories. For the rapid diagnosis of urinary tract infections, culture-independent methods prior MALDI-mediated identification have been described. Here, we describe a comparison of three of these methods based on their performance of bacterial identification and their potential as a routine tool for microbiology labs : (i) differential centrifugation, (ii) urine filtration and (iii) a 5-h bacterial cultivation on solid culture media. For 19 urine samples, all methods were directly compared and correct bacterial species identification by MALDI was used as performance indicator. A higher percentage of correct MALDI identification was obtained after filtration (78.9 %) and the growth-based method (84.2 %) as compared to differential centrifugation (68.4 %). Additional testing of 76 mono-microbial specimens (bacteriuria > 10(5) CFU/mL) confirmed the good performance of short growth with a 90.8 % correct MALDI score, with a potentially better fit to the routine workflow of microbiology labs.

The identification of mycobacteria outside biocontainment facilities requires that the organisms first be rendered inactive. Exposure to 70% ethanol (EtOH) either before or after mechanical disruption was evaluated in order to establish a safe, effective, and rapid inactivation protocol that is compatible with identification of Mycobacterium and Nocardia species using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A combination of 5 min of bead beating in 70% EtOH followed by a 10-min room temperature incubation period was found to be rapidly bactericidal and provided high-quality spectra compared to spectra obtained directly from growth on solid media. The age of the culture, the stability of the refrigerated or frozen lysates, and freeze-thaw cycles did not adversely impact the quality of the spectra or the identification obtained. Different approaches have been developed to hasten the identification of clinically significant mycobacteria compared to traditional biochemical phenotyping. These include DNA probe hybridization-and nucleic acid amplification-based assays with or without sequencing (1-4) and high-performance liquid chromatography (HPLC) to evaluate the mycolic acid composition (1, 2). More recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a reliable and rapid means for the identification of bacterial species, including mycobacteria through the recognition of specific proteins (5), PCR amplification profiles (6), or protein spectral profiles (7,8). Advanced databases for use with the latter approach are currently being expanded. Regardless of which option is used, it is essential that cultures are rendered nonviable prior to processing outside appropriate biocontainment facilities for obvious safety reasons. Previous reports have identified a variety of ways in which mycobacteria, including Mycobacterium tuberculosis can be successfully inactivated prior to manipulation, including heat treatment (9), exposure to lethal irradiation with or without photosensitizing chemicals (10-12), alcohol exposure (13), or a combination of treatments (14-16). While most were successful, temperatures and/or exposure times varied in order to achieve uniform cell death. Treatment with disinfecting agents such as 2% glutaraldehyde was not considered an option as it can result in extensive cross-linking of the nucleic acids, proteins, and other cellular components that are necessary for the MALDI-TOF MS identification process. Within the past year, two publications described the successful adaptation of a mycobacterial inactivation/ extraction protocol for use with MALDI-TOF MS-based identification (17, 18). Herein, we report a detailed description of an even more convenient process that was first presented in part in 2013 (19). MATERIALS AND METHODSAll studies involving mycobacteria were performed in a biological safety level 3 (BSL-3) laboratory. For various aspects of this study, 28 strains encompass...

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