Successful Identification of Clinical Dermatophyte and Neoscytalidium Species by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

c Dermatophytes are keratinolytic fungi responsible for a wide variety of diseases of glabrous skin, nails, and hair. Their identification, currently based on morphological criteria, is hindered by intraspecies morphological variability and the atypical morphology of some clinical isolates. The aim of this study was to evaluate matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a routine tool for identifying dermatophyte and Neoscytalidium species, both of which cause dermatomycoses. We first developed a spectral database of 12 different species of common and unusual dermatophytes and two molds responsible for dermatomycoses (Neoscytalidium dimidiatum and N. dimidiatum var. hyalinum). We then prospectively tested the performance of the database on 381 clinical dermatophyte and Neoscytalidium isolates. Correct identification of the species was obtained for 331/360 dermatophytes (91.9%) and 18/21 Neoscytalidium isolates (85.7%). The results of MALDI-TOF MS and standard identification disagreed for only 2 isolates. These results suggest that MALDI-TOF MS could be a useful tool for routine and fast identification of dermatophytes and Neoscytalidium spp. in clinical mycology laboratories. Dermatophytes are a unique group of closely related filamentous fungi that invade keratinized cutaneous structures, including the stratum corneum, nails, and hair, of humans and animals, resulting in an infection referred to as dermatophytosis, ringworm, or tinea (34). These superficial mycoses affect 20% to 25% of the world's population (17). Accurate etiologic diagnosis of dermatophytoses is important, not only to guide clinical management but also for epidemiological purposes. In addition, knowledge of the zoophilic or anthropophilic origin of a dermatophyte may assist with prophylactic measures (28). Neoscytalidium dimidiatum and its hyaline mutant, N. dimidiatum var. hyalinum, can cause clinical lesions resembling those induced by Trichophyton rubrum. Neoscytalidium spp. are molds responsible for foot dermatomycoses in tropical and subtropical areas, and an increasing number of cases are being reported in temperate countries among immigrants from tropical areas (18,20). As there is no effective oral or topical treatment for skin and nail infections due to Neoscytalidium spp., accurate etiologic diagnosis is required to discriminate these infections from those due to dermatophyte species (18,27). Thus, improper antifungal treatments, often expensive and sometimes associated with drug toxicity, are avoided.Diagnosis of dermatomycoses is based on the detection of septate hyphae by direct microscopic examination of clinical samples. Direct microscopic examination is rapid and inexpensive but does not provide genus or species identification, and results are falsely negative in 5% to 15% of cases (26,34). Species identification is based on macroscopic and microscopic examination of cultures. Culture-based identification is more specific but often takes 2 to 3 weeks. Subculture or the use of...

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

“…Nonetheless, the risk of spore aerosolization and potential laboratory contamination will likely result in the continued use of protein extraction for filamentous fungi, with the added advantage of better quality spectra and enhanced sensitivity and specificity. While many different extraction procedures have been developed (14,17,28), our alternate extraction procedure is not restrictive to specific portions of mycelia or growth in liquid media. Future comparative versatility studies are needed to help standardize the application of MALDI-TOF MS for mold identification in a clinical setting.…”

“…However, its clinical application for the identification of filamentous fungi has lagged due to challenges in developing an efficient protein extraction method and the limited databases available. Consequently, many groups have developed in-house supplementary databases that target only select pathogens that are most prominent in their patient population (13)(14)(15)(16)(17).…”

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

“…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).…”

“…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)(9)(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)(19)(20)(21)(22).…”

Genotyping and Molecular Characterization of Dermatophytes Isolates Collected from Clinical Samples

“…In the last couple of years, the fi eld of fungal characterizations by matrix-assisted laser desorption/ ionization time-of-fl ight mass spectrometry (MALDI-TOF) has been explored [18]. PCR technology is simple, rapid and, in the absence of specifi c nucleotide sequence information for the many dermatophyte species, able to generate speciesspecifi c or strain-specifi c DNA polymorphisms on the basis of characteristic band patterns detected by agarose gel electrophoresis [13].…”