Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Abstract

Accurate species discrimination of filamentous fungi is essential, because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification through direct surface analysis of the fungal culture. By use of culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurements and MALDI BioTyper 2.0 software. The profiles of young and mature colonies were analysed for each of the reference strains, and species-specific spectral fingerprints were obtained. To evaluate the database, 103 blind-coded fungal isolates collected in the routine clinical microbiology laboratory were tested. As a reference method for species designation, multilocus sequencing was used. Eighty-five isolates were unequivocally identified to the species level (≥99% sequence similarity); 18 isolates producing ambiguous results at this threshold were initially rated as identified to the genus level only. Further molecular analysis definitively assigned these isolates to the species Aspergillus oryzae (17 isolates) and Aspergillus flavus (one isolate), concordant with the MALDI-TOF MS results. Excluding nine isolates that belong to the fungal species not included in our reference database, 91 (96.8%) of 94 isolates were identified by MALDI-TOF MS to the species level, in agreement with the results of the reference method; three isolates were identified to the genus level. In conclusion, MALDI-TOF MS is suitable for the routine identification of filamentous fungi in a medical microbiology laboratory.

Identification of Uncommon Clinically Important Yeasts and Moulds by the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) System in a Global Antifungal Surveillance Program

“…The results of this study both confirm and extend the findings of other investigators who have assured the ability of the Bruker Biotyper to ID clinically relevant yeasts and moulds (Alanio et al, 2011;Buchan and Ledeboer, 2013;Cassagne et al, 2011;Cassagne et al, 2013;Chen et al, 2013;De Carolis et al, 2012). The collection of 80 different species of clinically relevant yet rare yeasts and moulds all IDed by reference DNA sequencing methods presented a rigorous challenge to the Bruker MS system.…”

“…The application of MALDI-TOF MS to the ID of moulds has been more limited to date with the greatest experience having to do with the ID of Aspergillus species (Alanio et al, 2011;Cassagne et al, 2011;De Carolis et al, 2012).…”

Differentiation of clinically relevant Mucorales Rhizopus microsporus and R. arrhizus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)

“…MALDI-TOF MS has primarily been used for identification of bacteria and yeasts, and is increasingly applied in identification of filamentous fungi such as Fusarium, Aspergillus, Penicillium and Ramularia (Alanio et al, 2011;Bader et al, 2011;Hettick et al, 2008;De Carolis et al, 2012;Videira et al, 2015). In the basal lineages of the fungal kingdom, a comprehensive report on MALDI-TOF MS identification of Lichtheimia species has appeared (Schrödl et al, 2012).…”

“…In the basal lineages of the fungal kingdom, a comprehensive report on MALDI-TOF MS identification of Lichtheimia species has appeared (Schrödl et al, 2012). Other studies investigated more diverse sets of strains, but the number of mucoralean fungi was either limited or not the focus of the study and lacked comparison with the phylogeny of the fungi (De Carolis et al, 2012;Becker et al, 2014;Ranque et al, 2014).…”

La espectrometría de masas MALDI-TOF en el laboratorio de microbiología clínica

“…Su implementación en el diagnóstico se ha retrasado debido a las dificultades para estandarizar un método de extracción de proteínas eficiente, así como a las limitaciones en las bases de datos disponibles. En consecuencia, algunos grupos de investigación han desarrollado bases de datos suplementarias obtenidas en condiciones experimentales [26][27][28] . El estudio realizado por Cassagne y colaboradores 26 evaluó cinco métodos para la preparación de aislamientos de mohos filamentosos a partir de cultivo y su posterior identificación por espectrometría de masas con el sistema de Bruker Daltonics Inc. de 143 aislamientos, combinando variables relativas a las condiciones de cultivo (caldo/agar), tratamiento con y sin etanol y uso de tratamientos térmicos y procedimientos de lisis mecánica.…”