Development of a Clinically Comprehensive Database and a Simple Procedure for Identification of Molds from Solid Media by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

b Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the rapid and highly accurate identification of clinical pathogens but has not been utilized extensively in clinical mycology due to challenges in developing an effective protein extraction method and the limited databases available. Here, we developed an alternate extraction procedure and constructed a highly stringent database comprising 294 individual isolates representing 76 genera and 152 species. To our knowledge, this is the most comprehensive clinically relevant mold database developed to date. When challenged with 421 blinded clinical isolates from our institution, by use of the BioTyper software, accurate species-level (score of >2.0) and genus-level (score of >1.7) identifications were obtained for 370 (88.9%) and 18 (4.3%) isolates, respectively. No isolates were misidentified. Of the 33 isolates (7.8%) for which there was no identification (score of <1.7), 25 were basidiomycetes not associated with clinical disease and 8 were Penicillium species that were not represented in the database. Our library clearly outperformed the manufacturer's database that was obtained with the instrument, which identified only 3 (0.7%) and 26 (6.2%) isolates at species and genus levels, respectively. Identification was not affected by different culture conditions. Implementation into our routine workflow has revolutionized our mycology laboratory efficiency, with improved accuracy and decreased time for mold identification, eliminating reliance on traditional phenotypic features.

Insights into the Evolution of the Mutational Resistome of Pseudomonas Aeruginosa in Cystic Fibrosis

“…On the contrary, uniform growth without any pigment production was observed in liquid media. As the pigments may interfere with MALDI mass spectra [18], this may be the reason of maximum sensitivity of protocol D. In addition, Lau et al (2013) also reported cross identification among database, while using solid media [11]. The difference in protein extraction protocol and database diversity may be the possible reason for the difference in sensitivity between protocols C and D [24].…”

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.…”

Transplantation of human skin microbiota in models of atopic dermatitis

“…Colonies were then taken for species identification by mass spectrometry using MALDI-TOF analysis. Bacterial protein extraction for MALDI-TOF mass spectrometry using the BioTyper (v3.1, Bruker Daltonics Inc.) was performed by the NIH Clinical Center microbiology lab using previously described methods (46), instrument settings, and calibration (47,48). BioTyper identification was supplemented by additional mass spectra profiles provided by several NIH-developed databases (46,49,50).…”