In this study we evaluated the capacity of MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) to identify clinical mould isolates. We focused on two aspects of MALDI-TOF MS identification: the sample processing and the database. Direct smearing of the sample was compared with a simplified processing consisting of mechanical lysis of the moulds followed by a protein extraction step. Both methods were applied to all isolates and the Filamentous Fungi Library 1.0 (Bruker Daltonics) was used for their identification. This approach allowed the correct species-level identification of 25/34 Fusarium spp. and 10/10 Mucor circinelloides isolates using the simplified sample processing. In addition, 7/34 Fusarium spp. and 1/21 Pseudallescheria/Scedosporium spp. isolates were correctly identified at the genus level. The remaining isolates-60-could not be identified using the commercial database, mainly because of the low number of references for some species and the absence of others. Thus, an in-house library was built with 63 local isolates previously characterized using DNA sequence analysis. Its implementation allowed the accurate identification at the species level of 94 isolates (91.3%) and the remaining nine isolates (8.7%) were correctly identified at the genus level. No misidentifications at genus level were detected. In conclusion, with improvements of both the sample preparation and the feeding of the database, MALDI-TOF MS is a reliable, ready to use method to identify moulds of clinical origin in an accurate, rapid, and cost-effective manner.

Use of MALDI-TOF MS plus the sonication-based extraction method enabled direct and accurate identification of microorganisms in liquid culture media in 15 minutes, in contrast to the 24 hours of subculture required for conventional identification, allowing the administration of a targeted antimicrobial therapy.

Matrix-assisted laser desorption–ionization/time of flight mass spectrometry (MALDI-TOF MS) has been widely implemented for the rapid identification of microorganisms. Although most bacteria, yeasts and filamentous fungi can be accurately identified with this method, some closely related species still represent a challenge for MALDI-TOF MS. In this study, two MALDI-TOF-based approaches were applied for discrimination at the species-level of isolates belonging to the Cryptococcus neoformans complex, previously characterized by Amplified Fragment Length Polymorphism (AFLP) and sequencing of the ITS1-5.8S-ITS2 region: (i) an expanded database was built with 26 isolates from the main Cryptococcus species found in our setting (C. neoformans, C. deneoformans and AFLP3 interspecies hybrids) and (ii) peak analysis and data modeling were applied to the protein spectra of the analyzed Cryptococcus isolates. The implementation of the in-house database did not allow for the discrimination of the interspecies hybrids. However, the performance of peak analysis with the application of supervised classifiers (partial least squares-discriminant analysis and support vector machine) in a two-step analysis allowed for the 96.95% and 96.55% correct discrimination of C. neoformans from the interspecies hybrids, respectively. In addition, PCA analysis prior to support vector machine (SVM) provided 98.45% correct discrimination of the three analyzed species in a one-step analysis. This novel method is cost-efficient, rapid and user-friendly. The procedure can also be automatized for an optimized implementation in the laboratory routine.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Identification of Nocardia and Mycobacterium species by matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS) is still a challenging task that requires both suitable protein extraction procedures and extensive databases. This study aimed to evaluate the VITEK MS Plus system coupled with updated RUO (v4.17) and IVD (v3.2) databases for the identification of Nocardia spp. and Mycobacterium spp. clinical isolates. Sample preparation was carried out using the VITEK MS Mycobacterium/Nocardia kit for protein extraction. From 90 Nocardia spp. isolates analysed, 86 (95.6%) were correctly identified at species or complex level using IVD and 78 (86.7%) using RUO. Only two strains were misidentified as other species pertaining to the same complex. Among the 106 non‐tuberculous Mycobacterium clinical isolates tested from a liquid culture medium, VITEK MS identified correctly at species or complex level 96 (90.6%) isolates in the IVD mode and 89 (84.0%) isolates in the RUO mode. No misidentifications were detected. Although the IVD mode was unable to differentiate members of the M. fortuitum complex, the RUO mode correctly discriminated M. peregrinum and M. septicum . The robustness and accuracy showed by this system allow its implementation for routine identification of these microorganisms in clinical laboratories.