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Item does not contain fulltext We here show adequate species identification for bacterial isolates of the genus Nocardia spp. through VITEK mass spectrometry. Application of a specific sample preparation method in combination with a robust matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) database leads to 94% accurate identification to the species level on a set of 164 isolates. The possibility to identify Nocardia spp. using MALDI-TOF MS will be available in the next release of VITEK MS update (IVD Version 3.0).

Identification of microorganisms by MALDI-TOF MS has been widely accepted in clinical microbiology. However, for Mycobacterium spp. and Nocardia spp. such identification has not yet reached the optimal level of routine testing. Here we describe the development of an identification tool for 49 and 15 species of Mycobacterium spp. and Nocardia spp., respectively. During database construction, a number of ambiguous reference identifications were revealed and corrected via molecular analyses. Eventually, more than 2000 individual mass spectra acquired from 494 strains were included in a reference database and subjected to bio-statistical analyses. This led to correct species identification and correct combination of species into several complexes or groups, such as the Mycobacterium tuberculosis complex. With the Advanced Spectrum Classifier algorithm, class-specific bin weights were determined and tested by cross-validation experiments with good results. When challenged with independent isolates, overall identification performance was 90% for identification of Mycobacterium spp. and 88% for Nocardia spp. However, for a number of Mycobacterium sp. isolates, no identification could be achieved and in most cases, this could be attributed to the production of polymers that masked the species-specific protein peak patterns. For the species where >20 isolates were tested, correct identification reached 95% or higher. With the current spectral database, the identification of Mycobacterium spp. and Nocardia spp. by MALDI-TOF MS can be performed in routine clinical diagnostics although in some complicated cases verification by sequencing remains mandatory.

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.

The development of reliable and rapid methods for the identification of patients colonized with vancomycin-resistant enterococci (VRE) is central to the containment of this agent within a hospital environment. To this end, we evaluated a prototype chromogenic agar medium (VRE-BMX; bioMérieux, Marcy l'Etoile, France) used to recover VRE from clinical specimens. This medium can also identify isolated colonies as either vancomycin-resistant Enterococcus faecium or Enterococcus faecalis , based on distinct colony colors. We compared the performance of VRE-BMX with bile esculin azide agar supplemented with vancomycin (BEAV). For this study, 147 stool samples were plated on each test medium and examined after 24 and 48 h of incubation. At 24 h, the sensitivity and specificity of each medium were as follows: BEAV, 90.9% and 89.9%, respectively; VRE-BMX, 96.4% and 96.6%, respectively. The positive predictive values (PPV) of VRE-BMX and BEAV at 24 h were 89.8% and 80.7%, respectively. VRE-BMX provided the identification of 10 isolates of vancomycin-resistant E. faecalis and 4 isolates of vancomycin-resistant E. faecium that were not recovered by BEAV. Further, VRE-BMX was capable of identifying patients colonized with both E. faecium and E. faecalis , a feature useful for infection control purposes that is not a function of BEAV. In terms of the recovery of vancomycin-resistant E. faecium and E. faecalis , the sensitivity and PPV were as follows: BEAV, 75.7% and 74.6%, respectively; VRE-BMX, 95.5% and 91.3%, respectively. In this initial evaluation, we found that VRE-BMX provided improved recovery of VRE from stool specimens, with the added advantage of being able to differentiate between vancomycin-resistant E. faecalis and E. faecium . Extending the incubation period beyond 24 h did not significantly improve the recovery of VRE and resulted in decreased specificity.

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.

Motivation: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been broadly adopted by routine clinical microbiology laboratories for bacterial species identification. An isolated colony of the targeted microorganism is the single prerequisite. Currently, MS-based microbial identification directly from clinical specimens can not be routinely performed, as it raises two main challenges: (i) the nature of the sample itself may increase the level of technical variability and bring heterogeneity with respect to the reference database and (ii) the possibility of encountering polymicrobial samples that will yield a ‘mixed’ MS fingerprint. In this article, we introduce a new method to infer the composition of polymicrobial samples on the basis of a single mass spectrum. Our approach relies on a penalized non-negative linear regression framework making use of species-specific prototypes, which can be derived directly from the routine reference database of pure spectra. Results: 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. Availability and implementation: The complete dataset including both the reference database and the mock-up mixture spectra is available at http://archive.ics.uci.edu/ml/datasets/MicroMass. Contact: pierre.mahe@biomerieux.com Supplementary information: Supplementary data are available at Bioinformatics online.