Fast and Accurate Identification of Dermatophytes by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Validation in the Clinical Laboratory

Abstract

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. Dermatophytes, which invade and infect keratinized tissues, affect a large proportion of the population, and over $500,000,000 per year is spent on antifungal drugs (1). This specific group of filamentous fungi can be separated in three anamorphic genera, Epidermophyton, Microsporum, and Trichophyton, and divided into geophilic, zoophilic, and anthropophilic species. Though resistance is uncommon (2), a proper identification of the infection agent is essential from an epidemiological point of view as well as for the choice of the antifungal treatment regimen and determining the source of infection (1).Conventional identification of dermatophyte species relies mainly on the morphological characteristics of the strains, but it can take several weeks before the discriminative characteristics appear. In recent decades, however, molecular methods have been employed for dermatophyte species identification. Although DNA sequencing has become the gold standard for identifications, it is quite costly and time-consuming. Nowadays, matrixassisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) represents a powerful tool for rapid and accurate identification of microorganisms (3-9) and has also proven to be promising for identification of filamentous fungi (10-17) and dermatophytes (18-24). The approach is based on the acquisition of a protein profile (between 2 and 20 kDa) and its comparison to a database with reference spectra (25). Since current databases for dermatophytes are quite limited and generally contain only the most common species, the robustness of a homemade, extensive database was evaluated using a MALDI-TOF MSbased workflow.The database was constructed with a total of 195 reference strains and 58 species of Arthroderma, Epidermophyton, Microsporum, and Trichophyton, originating from the BCCM/IHEM fungal collection and the mycology laboratory of the CHU Timone in Marseille, France, and their identity was determined by DNA sequencing (see the full list in Table S1 in the supplemental material). Reference spectra included in the library were obtained after 3 days and for some slow-growing strains after 7 and/or 14 days of culture at 25°C on solid Sabouraud medium supplemented with chloramphenicol. In order to enhance the effectiveness and diversity of the library, 10 raw mass spectra from 4 different subcultures from each strain were generated (26). For the acquisition of the mass spectra, a formic acid/acetonitrile extraction protocol described for molds (27) was applied and main spectra (MSP) were created using the MSP creation function of the Maldi Biotyper...

Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Clinically Relevant Filamentous Fungi

“…Prior studies have evaluated the accuracy of MALDI-TOF MS for the identification of only a few genera or groups of organisms, such as Aspergillus spp. or the dermatophytes (12,13,(19)(20)(21)(22)(23)(24)(25)(26). In our study, which examined 319 isolates from 43 different genera, the Vitek MS version 3.0 library was able to correctly identify 66.8% of isolates and 76.8% of the isolates after additional SARAMIS analysis.…”

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

“…Recent studies confirm that this technique may be very attractive for dermatophyte identification (18)(19)(20)(21)(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.…”

Fast identification of dermatophytes by MALDI‐TOF/MS using direct transfer of fungal cells on ground steel target plates

“…This process reduced the time to result and the risk of error since the fungal material from the primary culture is directly deposited on the target plate without further manipulations. A similar approach was described by Packeu et al but less than 40% identification rates were obtained from 3 day old subcultures. Here we achieved an 87% identification rate with the direct deposit method using an unpolished target plate.…”

“…This method has been validated for rapid and accurate identification of bacteria, yeasts and most recently, of filamentous fungi . However, the identification of dermatophytes by MALDI‐TOF MS is not as straightforward as described for bacteria and yeasts and to date, only a few studies have addressed the relevance and performance of this technique to identify dermatophytes . The overall performance of MALDI‐TOF MS to identify dermatophytes at species level varies from 60% to 100% and is critically dependent on the quality of the database used .…”

“…The overall performance of MALDI‐TOF MS to identify dermatophytes at species level varies from 60% to 100% and is critically dependent on the quality of the database used . Moreover, it is not yet clear whether a strong standardisation of procedures such as culture media, incubation time, quantity and type of colony is required for correct identification . Some authors reported that the accuracy of species identification was markedly improved by a prolonged incubation time of the cultures from 3 to 14 days, while others found that the MALDI‐TOF MS is able to provide early identification using young colonies, even before morphological characteristics are expressed …”