Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting

Hettick, Justin M.; Green, Brett J.; Buskirk, Amanda D.; Kashon, Michael L.; Slaven, James E.; Janotka, Erika; Blachère, Françoise M.; Schmechel, Detlef; Beezhold, Donald H.

doi:10.1016/j.ab.2008.05.051

Cited by 113 publications

(92 citation statements)

References 23 publications

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“…D ermatophytes in the genera Epidermophyton, Microsporum, and Trichophyton are usually characterized and identified by cultural and morphological characters and physiological tests or, more recently, by sequencing (1). Morphological identification is time-consuming and complex, usually requiring expert mycological knowledge, while sequencing is comparatively expensive and at least 2 to 3 days elapse before sequencing results are available.Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a reliable technique for the identification and typing of microbial pathogens such as bacteria (2-7), yeasts (8-10), and filamentous fungi, including dermatophytes (7,(11)(12)(13)(14)(15)(16)(17). Recent studies confirm that this technique may be very attractive for dermatophyte identification (18-22).…”

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confidence: 99%

“…Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a reliable technique for the identification and typing of microbial pathogens such as bacteria (2-7), yeasts (8-10), and filamentous fungi, including dermatophytes (7,(11)(12)(13)(14)(15)(16)(17). Recent studies confirm that this technique may be very attractive for dermatophyte identification (18-22).…”

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confidence: 99%

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Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) Mass Spectrometry Using the Vitek MS System for Rapid and Accurate Identification of Dermatophytes on Solid Cultures

et al. 2014

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d; POLE Pharma Consulting, Breganzona, Switzerland e The objective of this research was to extend the Vitek MS fungal knowledge base version 2.0.0 to allow the robust identification of clinically relevant dermatophytes, using a variety of strains, incubation times, and growth conditions. First, we established a quick and reliable method for sample preparation to obtain a reliable and reproducible identification independently of the growth conditions. The Vitek MS V2.0.0 fungal knowledge base was then expanded using 134 well-characterized strains belonging to 17 species in the genera Epidermophyton, Microsporum, and Trichophyton. Cluster analysis based on mass spectrum similarity indicated good species discrimination independently of the culture conditions. We achieved a good separation of the subpopulations of the Trichophyton anamorph of Arthroderma benhamiae and of anthropophilic and zoophilic strains of Trichophyton interdigitale. Overall, the 1,130 mass spectra obtained for dermatophytes gave an estimated identification performance of 98.4%. The expanded fungal knowledge base was then validated using 131 clinical isolates of dermatophytes belonging to 13 taxa. For 8 taxa all strains were correctly identified, and for 3 the rate of successful identification was >90%; 75% (6/8) of the M. gypseum strains were correctly identified, whereas only 47% (18/38) of the African T. rubrum population (also called T. soudanense) were recognized accurately, with a large quantity of strains misidentified as T. violaceum, demonstrating the close relationship of these two taxa. The method of sample preparation was fast and efficient and the expanded Vitek MS fungal knowledge base reliable and robust, allowing reproducible dermatophyte identifications in the routine laboratory. D ermatophytes in the genera Epidermophyton, Microsporum, and Trichophyton are usually characterized and identified by cultural and morphological characters and physiological tests or, more recently, by sequencing (1). Morphological identification is time-consuming and complex, usually requiring expert mycological knowledge, while sequencing is comparatively expensive and at least 2 to 3 days elapse before sequencing results are available.Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a reliable technique for the identification and typing of microbial pathogens such as bacteria (2-7), yeasts (8-10), and filamentous fungi, including dermatophytes (7,(11)(12)(13)(14)(15)(16)(17). Recent studies confirm that this technique may be very attractive for dermatophyte identification (18-22). In one study, the rate of correct identification of isolates belonging to the T. mentagrophytes complex was 89% (19), and in others, the overall rates of successful identification of dermatophyte species reached 95.8% (18), 97.8% (21), and 99.3% (20), demonstrating the potential of MALDI-TOF MS to replace classical identification methods. The technique has now also been used for the direct identification of dermatophytes in c...

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Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) Mass Spectrometry Using the Vitek MS System for Rapid and Accurate Identification of Dermatophytes on Solid Cultures

et al. 2014

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“…Many initial fungal MALDI-TOF MS studies focused on isolate identification using purified fungal spores (3,13,15,28). Only more recently has this technology been applied directly to unfractionated fungal colonies (10,11,17). Studies examining the application of MADLI-TOF MS specifically for dermatophyte identification are limited (6), and to our knowledge, there have been no published studies to date on the performance of the Bruker Biotyper system (Bruker Daltonics, Billerica, MA) for the identification of dermatophytes.…”

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Dermatophyte Identification Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Theel¹,

Hall²,

Mandrekar

et al. 2011

J Clin Microbiol

109

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The performance of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (MS) for the identification of dermatophytes from clinical cultures was compared to that of dermatophyte identification using 28S rRNA gene sequencing. The MALDI Biotyper library (MBL; version 3.0) was used alone and in combination with a supplemented library containing an additional 20 dermatophyte spectra (S-MBL). Acquired spectra were interpreted using both the manufacturer-recommended scores (genus, >1.7; species, >2.0) and adjusted cutoff values established by this study (genus, >1.5; species, >1.7); identifications required a minimum 10% difference in scores between the top two different organisms to be considered correct. One hundred well-characterized, archived dermatophyte isolates and 71 fresh dermatophyte cultures were evaluated using both libraries and both sets of cutoff criteria. Collectively, the S-MBL significantly outperformed the MBL at both the genus (93% versus 37.4%; P < 0,0001) and species (59.6% versus 20.5%; P < 0.0001) levels when using the adjusted score criteria. Importantly, application of the lowered cutoff values significantly improved genus (P ‫؍‬ 0.005)-and species (P < 0.0001)-level identification for the S-MBL, without leading to an increase in misidentifications. MALDI-TOF MS is a cost-effective and rapid alternative to traditional or molecular methods for dermatophyte identification, provided that the reference library is supplemented to sufficiently encompass clinically relevant, intraspecies strain diversity.Dermatophytes, comprising species within the genera Epidermophyton, Microsporum, and Trichophyton, exclusively invade and infect keratinized tissues (i.e., hair, skin, and nails). These organisms are among the most common fungal infections in humans, accounting for over 5 million physician visits annually in the United States (7, 26). Dermatophytes primarily cause superficial dermal mycoses and rarely progress to more invasive disease (8). In the United States, Trichophyton rubrum remains the most common cause of tinea infections and the most frequently isolated dermatophytic pathogen (7,24).Laboratory identification of dermatophytes is typically based on macroscopic observation of colony morphology (i.e., pigmentation, growth rate, texture, etc.) on selective media, followed by microscopic examination of conidia. In addition, specialized Trichophyton agars may be used for differentiation of species within the genera (14). These phenotypic techniques require experienced technologists and are often labor-intensive with a prolonged turnaround time. With the advancement of modern molecular techniques, some laboratories have turned to dermatophyte identification schemes involving sequencing of specific ribosomal DNA regions or real-time PCR amplification of discrete genes (2, 20). While these methods are highly accurate and rapid, they are costly and can be complex to implement.Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) m...

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“…Matrix-assisted laser desorption ionizationϪtime of flight mass spectrometry (MALDI-TOF MS) has proven useful, but mold identification remains challenged by the limited access to validated purpose-built databases that are necessary because of small species and strain representations in commercial libraries (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).…”

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Performance of Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectrometry for Identification of Aspergillus, Scedosporium, and Fusarium spp. in the Australian Clinical Setting

et al. 2016

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dWe developed an Australian database for the identification of Aspergillus, Scedosporium, and Fusarium species (n ‫؍‬ 28) by matrix-assisted laser desorption ionization؊time of flight mass spectrometry (MALDI-TOF MS). In a challenge against 117 isolates, species identification significantly improved when the in-house-built database was combined with the Bruker Filamentous Fungi Library compared with that for the Bruker library alone (Aspergillus, 93% versus 69%; Fusarium, 84% versus 42%; and Scedosporium, 94% versus 18%, respectively). Rapid, accurate mold identification is important due to the widening spectrum of pathogens and species-specific differences in antifungal susceptibility (1-3). Matrix-assisted laser desorption ionizationϪtime of flight mass spectrometry (MALDI-TOF MS) has proven useful, but mold identification remains challenged by the limited access to validated purpose-built databases that are necessary because of small species and strain representations in commercial libraries (4-16).Given the prior poor performance of the Bruker Filamentous Fungi Library v1.0 (Bruker Daltonics, Bremen, Germany) for mold identification using the manufacturer-recommended broth-based protein extraction methods (in our laboratory Ͼ50% of isolates were not identified; internal data) and because the geographic generalizability of in-house-built databases is not yet known, we hypothesized that a MS library of molds relevant to our region (17-21) will improve identification. Here, we constructed an in-house database containing 117 strains (see Table S1 in the supplemental material) covering 28 species of Aspergillus, Scedosporium, and Fusarium encountered in Australia. Challenge isolates (also n ϭ 117; 21 species) comprising 55 Aspergillus, 45 Fusarium, and 17 Scedosporium clinical strains (Table 1) were then used to assess the performance of the Bruker library alone versus that of the Bruker library supplemented with the in-house library for species identification.All isolates were identified using phenotypic methods (22) with definitive identification by DNA sequencing of the internal transcribed spacer (ITS) (all isolates), ␤-tubulin (Aspergillus and Scedosporium spp.), and partial elongation factor-1alpha (EF-1␣) (to identify Fusarium to the species complex level) gene regions (23)(24)(25)(26). Sequence data were analyzed against the Centraalbureau voor Schimmelkultures (http://www.cbs.knaw.nl/Collections /BioloMICSSequences.aspx?fileϭall), International Society for Human and Animal Mycology ITS (http://its.mycologylab.org/), and Fusarium-ID (http://www.fusariumdb.org/index.php) databases, and species were assigned using published criteria (27).Protein extraction for MALDI-TOF MS was performed as previously described (11). The Bruker bacterial test standard (Bruker Daltonics) was used for calibration and Aspergillus ustus CBS 261.67T scoring of Ն2.00 was required for quality extraction and spectra acceptability (11). The in-house database was constructed using published protocols (11, 28) with 20 to 25 quality spe...

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Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting

Cited by 113 publications

References 23 publications

Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) Mass Spectrometry Using the Vitek MS System for Rapid and Accurate Identification of Dermatophytes on Solid Cultures

Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) Mass Spectrometry Using the Vitek MS System for Rapid and Accurate Identification of Dermatophytes on Solid Cultures

Dermatophyte Identification Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Performance of Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectrometry for Identification of Aspergillus, Scedosporium, and Fusarium spp. in the Australian Clinical Setting

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