Espectrometría de masas MALDI-TOF: evaluación de la etapa preanalítica para la identificación de hongos miceliales

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Abstract

Se evaluaron 3 metodologías de extracción de proteínas para la identificación de hongos miceliales por MALDI-TOF MS en 44 aislados: la extracción con agua-ácido fórmico (E. agua), la extracción con zirconio-etanol-acetonitrilo-ácido fórmico (E. zirconio) y la recomendada por el proveedor del equipo (E. tubo). Se compararon 2 bases de datos: Bruker (BK) y BK + National Institutes of Health. Los resultados obtenidos utilizando dichas bases fueron los siguientes (en el orden citado): identificación correcta (IC) a nivel de género, 10 y 16 con E. agua; 27 y 32 con E. zirconio y 18 y 23 con E. tubo; IC a nivel de especie, 5 y 7 con E. agua; 17 y 20 con E. zirconio y 11 y 14 con E. tubo; identificaciones no confiables, 18 y 12 con E. agua y 9 y 4, tanto con E. zirconio como con E. tubo; ausencia de pico, 16 con E. agua, 8 con E. zirconio y 17 con E. tubo. La extracción con zirconio mostró el mejor rendimiento (p < 0,05).

Translated abstract

In order to optimize the identification of molds with MALDI-TOF MS, three protein extraction-methodologies were evaluated against 44 isolates: water extraction (WE), zirconium extraction (ZE) and the provider's recommended method (PRM). Two data bases were compared, Bruker (BK) and Bruker + National Institutes of Health. Considering both databases, results were respectively as follows: correct identification (CI) at gender level, 10 and 16 by WE; 27 and 32 by ZE and 18 and 23 by PRM; CI at species level, 5 and 7 by WE; 17 and 20 by ZE and 11 and 14 by PRM; non-reliable identification, 18 and 12 by WE; 9 and 4 by ZE and by PRM. No peaks were observed in 16 by WE, 8 by ZE and 17 by PRM. ZE showed the best perfomance (p < 0.05).

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Phylogenetic species recognition and species concepts in fungi.

John Taylor, David J Jacobson, Scott Kroken … (2000)

The operational species concept, i.e., the one used to recognize species, is contrasted to the theoretical species concept. A phylogenetic approach to recognize fungal species based on concordance of multiple gene genealogies is compared to those based on morphology and reproductive behavior. Examples where Phylogenetic Species Recognition has been applied to fungi are reviewed and concerns regarding Phylogenetic Species Recognition are discussed.

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Mould Routine Identification in the Clinical Laboratory by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry

Carole Cassagne, Stéphane Ranque, Anne-Cécile Normand … (2011)

Background MALDI-TOF MS recently emerged as a valuable identification tool for bacteria and yeasts and revolutionized the daily clinical laboratory routine. But it has not been established for routine mould identification. This study aimed to validate a standardized procedure for MALDI-TOF MS-based mould identification in clinical laboratory. Materials and Methods First, pre-extraction and extraction procedures were optimized. With this standardized procedure, a 143 mould strains reference spectra library was built. Then, the mould isolates cultured from sequential clinical samples were prospectively subjected to this MALDI-TOF MS based-identification assay. MALDI-TOF MS-based identification was considered correct if it was concordant with the phenotypic identification; otherwise, the gold standard was DNA sequence comparison-based identification. Results The optimized procedure comprised a culture on sabouraud-gentamicin-chloramphenicol agar followed by a chemical extraction of the fungal colonies with formic acid and acetonitril. The identification was done using a reference database built with references from at least four culture replicates. For five months, 197 clinical isolates were analyzed; 20 were excluded because they were not identified at the species level. MALDI-TOF MS-based approach correctly identified 87% (154/177) of the isolates analyzed in a routine clinical laboratory activity. It failed in 12% (21/177), whose species were not represented in the reference library. MALDI-TOF MS-based identification was correct in 154 out of the remaining 156 isolates. One Beauveria bassiana was not identified and one Rhizopus oryzae was misidentified as Mucor circinelloides. Conclusions This work's seminal finding is that a standardized procedure can also be used for MALDI-TOF MS-based identification of a wide array of clinically relevant mould species. It thus makes it possible to identify moulds in the routine clinical laboratory setting and opens new avenues for the development of an integrated MALDI-TOF MS-based solution for the identification of any clinically relevant microorganism.

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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.

Anna F Lau, Steven K Drake, Leslie B Calhoun … (2013)

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.