Confident identification of filamentous fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without subculture-based sample preparation (2015)

•We evaluated the Filamentous Fungi library version 1.0 of Bruker.•We compared the extraction method versus the direct method.•We identified fungi from human samples prospectively.•Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is ready for use in the mycology laboratory. The identification of moulds by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been evaluated repeatedly for use in clinical diagnostic settings.1Ling H. Yuan Z. Shen J. Wang Z. Xua Y. Accuracy of MALDI-TOF mass spectrometry for the identification of clinical pathogenic fungi: a meta-analysis.J Clin Microbiol. 2014; 52: 2573-2582Crossref PubMed Scopus (50) Google Scholar However, recommended protocols are based on the analysis of time-consuming subcultures rather than primary growth obtained on solid isolation media. In order to compare the identification performance of MALDI-TOF MS applied to primary colonies (protocol A) versus subculture samples (protocol B), 111 reference specimens representing 30 clinically relevant species were analyzed. Each of these species was identified, at least once, by internal transcribed spacer (ITS) sequence analysis, which was considered the gold standard in this study (Table 1). In addition, each of the 111 strains was identified by phenotype testing.Table 1Total number of reference strains and number of reference strains confirmed by ITS sequence analysis per species/groupSpecies/groupTotal number of reference strains included in this studyNumber of reference strains confirmed by ITS sequence analysisAspergillus flavus83Aspergillus fumigatus3711Aspergillus nidulans44Aspergillus niger138Aspergillus ochraceus11Aspergillus terreus54Aspergillus ustus21Aspergillus versicolor11Cladosporium herbarum11Curvularia verruculosa21Exophiala dermatitidis11Fusarium dimerum22Fusarium oxysporum11Fusarium solani44Lichtheimia corymbifera44Mucor circinelloides22Paecilomyces lilacinus11Paecilomyces variotii21Penicillium chrysogenum44Penicillium citrinum11Rhizomucor pusillus11Rhizopus microsporus21Rhizopus oryzae11Scedosporium apiospermum66Scedosporium prolificans11Scopulariopsis brevicaulis11ITS, internal transcribed spacer. Open table in a new tab ITS, internal transcribed spacer. In protocol A, primary growth on Sabouraud agar (Difco Sabouraud Agar, Modified) after 24 to 72 h of incubation (time required for the first visible mycelium growth) was collected carefully, avoiding agar, by means of a wooden toothpick. The biomass was deposited on an unpolished target plate, air-dried, and overlaid with 1 μl alpha-cyano-4-hydroxycinnamic acid. In protocol B, and in accordance with the Bruker protocol, the filamentous fungi were inoculated in 8 ml of Sabouraud liquid broth (Difco Sabouraud Maltose Broth), which was rotated overnight at room temperature. The sediments of overnight subcultures were washed thoroughly, extracted with acetonitrile–formic acid, and then 1 μl of supernatant was placed onto an unpolished target plate and processed further, as above. MALDI-TOF MS was performed using a Microflex LT instrument and Biotyper software in conjunction with Filamentous Fungi library version 1.0 (Bruker). As per the manufacturer's recommendation, a score of ≥2.0 indicates confident species-level (or species group-level) identification, a score of 1.7–1.99 indicates confident genus-level identification, and a score of <1.7 is considered unreliable.2Schulthess B. Ledermann R. Mouttet F. Zbinden A. Bloemberg G.V. Böttger E.C. Hombach M. Use of the Bruker MALDI Biotyper for identification of molds in the clinical mycology laboratory.J Clin Microbiol. 2014; 52: 2797-2803Crossref PubMed Scopus (85) Google Scholar The following species and species groups gave confident and comparable identification scores using either protocol A or B, and were identified correctly (designation according to Bruker): Aspergillus flavus (n = 8), A. fumigatus (n = 37), A. nidulans (n = 4), A. niger (n = 13), A. ochraceus (n = 1), A. terreus (n = 5), A. ustus (n = 2), A. versicolor (n = 1), Cladosporium herbarum (n = 1), Exophiala dermatitidis (n = 1), Fusarium dimerum (n = 2), F. oxysporum (n = 1), F. solani (n = 4), Lichtheimia corymbifera (n = 4), Mucor circinelloides (n = 2), Paecilomyces lilacinus (n = 1), P. variotii (n = 1), Penicillium chrysogenum (n = 4), P. citrinum (n = 1), Rhizomucor pusillus (n = 1), Rhizopus microsporus (n = 2), R. oryzae (n = 1), Scedosporium apiospermum (n = 6), S. prolificans (n = 1), S. brevicaulis (n = 1). However, Coprinellus marculentus, Neoscytalidium dimidiatum, Neurospora tetrasperma, Phialemonium obovatum, and Scytalidium hyalinum were not identified reliably due to the lack of corresponding reference entries in the database. Schulthess et al.2Schulthess B. Ledermann R. Mouttet F. Zbinden A. Bloemberg G.V. Böttger E.C. Hombach M. Use of the Bruker MALDI Biotyper for identification of molds in the clinical mycology laboratory.J Clin Microbiol. 2014; 52: 2797-2803Crossref PubMed Scopus (85) Google Scholar showed that lowering the cut-off from 2 to >1.7 for a species-level identification resulted in no misidentifications. Applying that condition to our results, and in agreement with the findings of Schulthess et al., the overall percentage of correct identification increased from 68.7% to 76.4% and from 63.6% to 75.15% for protocols A and B, respectively (Table 2).Table 2Fungal reference strains analyzed using protocols A and B and the percentage of correct identification at the species- or group-level by MALDI-TOF MSSpecies/groupNumber of strainsProtocol AProtocol B% correct species IDscore >2% correct species IDscore >1.7% correct species IDscore >2% correct species IDscore >1.7Aspergillus flavus810010085.785.7Aspergillus fumigatus3794.694.697.3100Aspergillus nidulans410010075100Aspergillus niger1392.310076.992.3Aspergillus ochraceus101000100Aspergillus terreus5100100100100Aspergillus nidulans410010075100Aspergillus ustus21001005050Aspergillus versicolor1100100100100Cladosporium herbarum1100100100100Exophiala dermatitidis11001000100Fusarium dimerum250100100100Fusarium oxysporum1100100100100Fusarium solani47510075100Lichtheimia corymbifera4100100100100Mucor circinelloides2100100100100Paecilomyces lilacinus1100100100100Paecilomyces variotii21001005050Penicillium chrysogenum41001005075Penicillium citrinum1100100100100Rhizomucor pusillus1100100100100Rhizopus microsporus210010050100Rhizopus oryzae100100100Scedosporium apiospermum650100100100Scedosporium prolificans1100100100100Scopulariopsis brevicaulis1100100100100MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Open table in a new tab MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protocol A was then applied prospectively for primary growth derived from 49 clinical samples, which included respiratory specimens (n = 36), swabs (n = 10), and other specimens (n = 3), using a cut-off value of >1.7 for confident identification. Forty-four specimens exhibited a score >1.7 (71.6%) and were identified correctly by MALDI-TOF MS. Among these 44 filamentous fungi, three were confirmed by ITS sequence analysis, as they had not been identified previously in clinical specimens in our laboratory (Alternaria alternata, Chaetomium globosum, and Fusarium proliferatum). The remaining five specimens gave unreliable scores. Nevertheless, for four of them, the identification suggested by MALDI-TOF MS (Aspergillus niger, score 1.60; Aspergillus unguis, score 1.51; Scedosporium apiospermum, score 1.50; Rhizopus oryzae, score 1.56) was confirmed by ITS sequence analysis. One specimen without a reliable score could not be identified by MALDI-TOF MS because of the lack of reference data for Uncinocarpus queenslandicus, the agent present (Table 3).Table 3Prospective analysis of 48 clinical isolates using protocol A and the percentage of correct identification at the species- or group-level by MALDI-TOF MSSpecies/groupNumber of strains% correct speciesID score >1.7% unreliableID score <1.7Aspergillus flavus41000Aspergillus fumigatus271000Aspergillus niger47525Aspergillus unguis10100Alternaria alternata11000Chaetomium globosum11000Fusarium dimerum11000Fusarium oxysporum11000Fusarium proliferatum21000Fusarium solani11000Penicillium citrinum11000Paecilomyces variotii21000Scedosporium apiospermum10100Rhizopus oryzae10100MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Open table in a new tab MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In conclusion, confident identification of filamentous fungi by MALDI-TOF MS is feasible without subculture of primary growth. We emphasize that this approach requires biomass free of culture medium, which may negatively impact MALDI-TOF MS identification. These findings may further support the implementation of MALDI-TOF MS in the clinical setting in order to achieve rapid and reliable diagnosis of fungal agents. Conflict of interest/funding: None.