Identification des dermatophytes par spectrométrie de masse MALDI-TOF

Introduction L’identification des dermatophytes par les méthodes microbiologiques conventionnelles est souvent longue et fastidieuse. La technique de spectrométrie de masse et sa variante MALDI-TOF (Matrix Assisted Laser Desorption Ionisation-Time of Flight) est un nouvel outil utilisé pour l’identification des bactéries et des levures dans les laboratoires d’analyses médicales. Nous avons récemment développé une méthode standardisée pour l’identification en routine des champignons filamenteux à partir de culture en milieu solide. L’objectif de cette étude est d’étendre cette méthode standardisée à l’identification des dermatophytes dans l’activité de routine du laboratoire. Matériel et méthode Une banque de référence contenant les spectres de masse de 44 souches parfaitement caractérisées correspondants à 13 espèces de dermatophytes a été générée sur un UltraFlex (BruckerDaltonics, Allemagne) couplé au logiciel MaldiBiotyper v2.1. Par la suite, 133 souches isolées de prélèvements cliniques ont été identifiées en comparant leur spectre à ceux inclus dans la banque de référence : l’identification d’espèce a été retenue si le Log Score (LS) obtenu était supérieur ou égal à 1,7. Enfin, l’identification par MALDI-TOF a été considérée comme correcte en cas de concordance avec l’identification morphologique ou moléculaire des isolats cliniques. Résultats L’identification par spectrométrie de masse(SM) a été correcte pour 130 (97,8 %) des isolats. Pour 2 isolats identifiés conventionnellement comme Microsporum canis, l’identification par SM n’a pas pu générer de spectre avec un LS valide. Pour un isolat correspondant à Microsporum audouinii, la SM a généré une mauvaise identification. Tous les isolats ont pu être identifiés après seulement 3 à 6 jours de culture avant l’apparition des caractères morphologiques conventionnels d’identification. Conclusion Le protocole de SM utilisé pour l’identification des champignons filamenteux au laboratoire est applicable aux dermatophytes. Une identification d’espèce peut être obtenue en 3 à 6 jours alors qu’une identification conventionnelle qui nécessite notamment des milieux de cultures complémentaires demande 2 à 3 semaines

Fast and accurate identification of dermatophytes by matrix-assisted laser desorption ionization-time of flight mass spectrometry: validation in the clinical laboratory.

&lt;p&gt;The performance of a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) workflow using an extensive reference database for dermatophyte identification was evaluated on 176 clinical strains. Using a direct-deposit procedure after 3 incubation days yielded 40% correct identification. Both increasing incubation time and using an extraction procedure resulted in 100% correct identification.&lt;/p&gt;</p

Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi

BACKGROUND: The poor reproducibility of matrix-assisted desorption/ionization time-of-flight (MALDI-TOF) spectra limits the effectiveness of the MALDI-TOF MS-based identification of filamentous fungi with highly heterogeneous phenotypes in routine clinical laboratories. This study aimed to enhance the MALDI-TOF MS-based identification of filamentous fungi by assessing several architectures of reference spectrum libraries. RESULTS: We established reference spectrum libraries that included 30 filamentous fungus species with various architectures characterized by distinct combinations of the following: i) technical replicates, i.e., the number of analyzed deposits for each culture used to build a reference meta-spectrum (RMS); ii) biological replicates, i.e., the number of RMS derived from the distinct subculture of each strain; and iii) the number of distinct strains of a given species. We then compared the effectiveness of each library in the identification of 200 prospectively collected clinical isolates, including 38 species in 28 genera.Identification effectiveness was improved by increasing the number of both RMS per strain (p&lt;10-4) and strains for a given species (p&lt;10-4) in a multivariate analysis. CONCLUSION: Addressing the heterogeneity of MALDI-TOF spectra derived from filamentous fungi by increasing the number of RMS obtained from distinct subcultures of strains included in the reference spectra library markedly improved the effectiveness of the MALDI-TOF MS-based identification of clinical filamentous fungi</p

MALDI-TOF mass spectrometry: revolutionising clinical laboratory diagnosis of mould infections

&lt;p&gt;Clinical diagnosis of mould infections currently involves complex species identification based on morphological criteria, which is often prone to error. Employing an extensive mould species reference spectral library (up to 2,832 reference spectra, corresponding to 708 strains from 347 species), we assessed the extent to which MALDI-TOF mass spectrometry (MALDI-TOF MS) enhanced the accuracy of species identification. MALDI-TOF MS data were validated against morphology- and DNA sequencing-based results using 262 clinical isolates collected over a four-month period in 2013. The implementation of MALDI-TOF MS displayed a dramatic improvement in mould identification at the species level (increased from 78.2% to 98.1%) and a marked reduction in misidentification rate (i.e., 9.8% to 1.2%). We then compared the mould identification results obtained before (i.e., 2011) and after (i.e., 2013) the implementation of MALDI-TOF MS in routine identification procedures, which increased from 64.57% to 100%. Re-assessment of a set of isolates from 2011 using this procedure, including MALDI-TOF MS, yielded an increase in species diversity from 16 to 42 species. Finally, applying this procedure during a 16-month period (2012-2013) enabled the identification of 1,094/1,107 (98.8%) clinical mould isolates corresponding to 107 distinct species. MALDI-TOF MS-based mould species identification may soon challenge traditional techniques in the clinical laboratory, as patient prognosis is largely contingent on rapid and accurate diagnosis. This article is protected by copyright. All rights reserved&lt;/p&gt;</p

A MALDI-TOF MS procedure for clinical dermatophyte species identification in the routine laboratory2160

The conventional identification of dermatophytes requires a long turnaround time and highly skilled mycologists. We have recently developed a standardized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay to routinely identify molds of potential clinical significance. This study objective was to determine if this same assay could also be employed to identify clinical dermatophytes in the routine laboratory setting. The effects of the inclusion of cycloheximide in the culture medium and incubation time were tested after building a reference spectra library that included 48 well-characterized isolates of 17 dermatophyte species. Then these same isolates were prospectively identified using this library. MALDI-TOF MS-based identification was effective regardless of the presence of cycloheximide or incubation time as 130/133 (97.8%) of the clinical isolates were appropriately identified. Two Microsporum canis isolates yielded uninformative spectra and one M. audouinii isolate was misidentified. Since one only requires a small colony for MALDI-TOF MS analysis, accurate identifications were obtained in 3-6 days and, specifically, before the appearance of their characteristic morphological features. Consequently, identification turnaround time was dramatically reduced as compared to that needed for conventional morphological identification. In conclusion, this standardized MALDI-TOF MS-based identification procedure for filamentous fungi effectively identifies clinical dermatophyte isolates and drastically reduces the response times in the routine clinical laboratory</p