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

(1)Successful Identification of Clinical Dermatophyte and Neoscytalidium Species by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Kinda Alshawa,a,b Jean-Luc Beretti,a Claire Lacroix,b Martine Feuilhade,b Brunhilde Dauphin,c Gilles Quesne,a Noura Hassouni,a Xavier Nassif,a and Marie-Elisabeth Bougnouxa Department of Microbiology, Hôpital Necker-Enfants Malades AP-HP, Université Paris-Descartes, Paris, Francea; Department of Mycology-Parasitology, Hôpital Saint-Louis AP-HP, Université Paris Diderot Paris 7, Paris, Franceb; and Andromas SAS, Paris, Francec. D. ermatophytes 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 specific media to stimulate conidiation or pigment production is sometimes necessary, further retarding the diagnosis by several weeks (28). Phenotypic identification can also be difficult because of intraspecies morphological polymorphism and phenotypic pleomorphism (6). In the Netherlands, between 1991 and 2002, only 56% of 1,171 dermatophyte. July 2012 Volume 50 Number 7. isolates were successfully identified in a quality control exercise involving 50 laboratories (5). Alternative molecular methods have been developed to provide rapid and accurate dermatophyte identification. They include gene-specific PCR, restriction fragment length polymorphism analysis, sequencing of the large-subunit rRNA gene or of the chitin synthase-encoding gene, PCR fingerprinting, DNA hybridization, and sequencing of the internal transcribed spacer regions (ITS1 and ITS2). However, these molecular methods are time-consuming and expensive for the identification of all isolates usually recovered from clinical samples, stressing the need for new strategies for identification of these common fungal pathogens (5, 21, 22, 25, 31). Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) represents a new approach to microbial identification and is based on characteristic fingerprints of intact cells (16, 30). This technique has already been validated for rapid and accurate identification of bacterial, yeast, and mold species (1, 3, 4, 9–11, 15, 23, 24, 29, 33). In contrast, few studies have focused on MALDI-TOF MS for identification of dermatophyte fungi (14, 32). Here we describe the creation of a new database for the identification of dermatophyte and Neoscytalidium species. We then evaluated this new database on a panel of 381 clinical isolates.. Received 11 December 2011 Returned for modification 9 January 2012 Accepted 16 April 2012 Published ahead of print 25 April 2012 Address correspondence to Marie-Elisabeth Bougnoux, marie Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.06634-11. Journal of Clinical Microbiology. p. 2277–2281. 2277. Downloaded from on May 16, 2020 by guest. 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..

(2) Alshawa et al.. RESULTS. Fungal isolates. All isolates were obtained by culture of skin, hair, and nail samples received for routine examination on Sabouraud’s dextrose agar slants containing antibiotics, with and without cycloheximide (Bio-Rad, Marne la Coquette, France), at 26°C for 3 weeks, in order to obtain typical morphological characteristics (13). The reference database was constructed from a set of 50 reference strains belonging to 12 clinically relevant species of dermatophytes and 2 species of Neoscytalidium, namely, Epidermophyton floccosum, Microsporum langeronii, M. canis, M. persicolor, Trichophyton rubrum, T. mentagrophytes var. interdigitale, T. tonsurans, T. soudanense, T. erinacei, T. equinum, T. simii, T. violaceum, N. dimidiatum, and N. dimidiatum var. hyalinum. All these strains were obtained from clinical samples at Saint Louis Hospital; the species identification was based on usual macroscopic and microscopic criteria (13, 28) and was confirmed by sequencing the internal transcribed spacer (ITS1 and ITS2) regions of ribosomal DNA (21). To evaluate the database as a tool for routine identification by MALDI-TOF MS, we performed a 6-month prospective evaluation study, during which 360 dermatophyte isolates belonging to 7 different species and 21 isolates of N. dimidiatum or N. dimidiatum var. hyalinum were collected from patients with dermatomycosis (tinea capitis, n ⫽ 87; onychomycosis, n ⫽ 124; tinea pedis, n ⫽ 92; tinea corporis, n ⫽ 69; tinea manuum, n ⫽ 9). These 381 isolates were identified by means of both conventional culture criteria and MALDI-TOF MS. MALDI-TOF MS. (i) Sample preparation. After a 3-week incubation period, the colony surface was scraped with a sterile scalpel and harvested in 10 ␮l of formic acid (70%, vol/vol). Then, 1 ␮l of this mixture was deposited on the target plate (Andromas, Paris, France) and allowed to dry at room temperature. One microliter of matrix solution (saturated solution of ␣-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid) was then added and allowed to cocrystallize with the sample. The strains used for database construction were deposited in 10 replicates, while isolates used to test the database were deposited in 2 replicates. (ii) Instrument type. All the samples were processed using the Andromas system (Paris, France), including a mass spectrometry apparatus combined with control software and various databases for identification of microorganisms. Before this study, no database was available for identification of dermatophyte and Neoscytalidium species. For each sample, positive ions were extracted with an accelerating voltage of 20 kV in linear mode. The spectra were analyzed and compared over an m/z range of 3,000 to 20,000 with Andromas software (Paris, France). (iii) Data analysis. The Andromas software identified the number of common peaks between the spectra of the tested isolate and the speciesspecific spectral fingerprints of the reference strains in the database (i.e., dermatophyte and Neoscytalidium database). For each isolate, all peaks with an intensity greater than 0.1 were retained and were compared with the peaks for the species-specific spectral fingerprints of each reference strain, taking into account possible variations in the m/z value of ⫾10. Then, the percentage of common peaks was obtained with the following formula: 100 ⫻ (number of peaks common between the peaks of the tested isolate and the peaks of the species-specific spectral fingerprint/ total number of peaks specific to the species-specific spectral fingerprint). Acceptable identification of a tested strain corresponds to the species having ⱖ66% of peaks in common with the reference strains in the database. Only the first- and second-best matches were retained. A difference of at least 10% between the first and the second match was required. When the identification was not acceptable (i.e., ⬍66% of peaks are in common with those for the reference strain or a ⬍10% difference exists between the first and second matches), a second run was performed and the results were analyzed in a similar manner. An external control (Pseudomonas aeruginosa) was used to validate the calibration for each experiment (12).. Reference database. The database for dermatophyte and Neoscytalidium identification was engineered using a previously described strategy (8, 12). A set of reference strains selected as belonging to clinically relevant dermatophyte and Neoscytalidium species and described in Materials and Methods was used to construct the mass spectral database. The MALDI-TOF MS spectra obtained in 10 different runs were analyzed for each reference strain. The majority of ions detected were in the range 4,000 to 14,000 Da. For each reference strain, we retained only the peaks with a relative intensity of greater than 0.1 that were constantly present in all 10 spectra obtained for a given strain. The standard deviation for each conserved peak did not exceed a 6 m/z value. The set of peaks, so-called spectral fingerprints, was specific for each selected reference strain. Figure 1 shows a selection of the specific spectral fingerprints for the 7 most common dermatophyte species, N. dimidiatum, and N. dimidiatum var. hyalinum. Validation of the database. In order to determine whether this database could be used for the identification of dermatophytes and Neoscytalidium species, we compared the identities of the clinical isolates obtained by the conventional methods with those obtained by MALDI-TOF MS. The database was tested blindly with the 381 clinical isolates described in Materials and Methods. The two methods agreed for 331 (91.9%) of the 360 dermatophyte isolates (Table 1). Twenty-seven isolates (7.5%) could not be identified, because spectral acquisition failed after 2 runs. The results of MALDI-TOF MS and conventional criteria disagreed for 2 isolates (0.5%): two T. mentagrophytes var. interdigitale isolates were identified by MALDI-TOF MS as T. rubrum and T. tonsurans. Eighteen (85.7%) of the 21 Neoscytalidium isolates (91.7% of N. dimidiatum var. hyalinum and 77.8% of N. dimidiatum isolates) were correctly identified by MALDI-TOF MS. None of the isolates was incorrectly identified, but spectral acquisition failed after 2 runs for 3 Neoscytalidium isolates.. 2278. DISCUSSION. We have developed a MALDI-TOF MS database allowing species identification of commonly encountered dermatophytes. The three dermatophytes usually responsible for tinea pedis and onychomycosis (T. rubrum, T. mentagrophytes var. interdigitale, and E. floccosum) were successfully identified in, respectively, 97.5% (154/158), 97.6% (80/82), and 100% (5/5) of cases. The four dermatophyte species usually responsible for tinea capitis (M. canis, M. langeronii, T. tonsurans, and T. soudanense) were successfully identified in, respectively, 91.7% (11/12), 80.8% (21/26), 88.5% (23/26), and 72% (36/50) of cases, and none was misidentified. Spectral acquisition failed for 19% (5/26) and 28% (14/50) of M. langeronii and T. soudanense isolates, respectively, possibly owing to difficulties in harvesting fungal material from the colony surface, which was very dense or dry. This technical problem was not mentioned in the two previous reports of dermatophyte identification by MALDI-TOF MS, as no M. langeronii or T. soudanense isolates were tested (14, 32). Concerning Neoscytalidium spp., our database performed better for N. dimidiatum var. hyalinum identification than for N. dimidiatum identification. N. dimidiatum is a pigmented dematiaceous fungus producing a melanin-like pigment in vitro and in vivo, whereas N. dimidiatum var. hyalinum is unable to produce melanin in vitro (27). Recently, Buskirk et al. demonstrated that synthetic melanin and dark fungal pigments can inhibit the de-. Journal of Clinical Microbiology. Downloaded from on May 16, 2020 by guest. MATERIALS AND METHODS.

(3) Identification of Dermatophytes by MALDI-TOF MS. Downloaded from on May 16, 2020 by guest FIG 1 MALDI-TOF MS spectra of 7 species of dermatophytes, 1 Neoscytalidium dimidiatum strain, and 1 Neoscytalidium dimidiatum var. hyalinum reference strain.. July 2012 Volume 50 Number 7. 2279.

(4) Alshawa et al.. TABLE 1 Identification of the 381 clinical dermatophyte and Neoscytalidium isolates by MALDI-TOF mass spectrometry with the Andromas system No. (%) of isolates Group and species. Neoscytalidium spp. N. dimidiatum N. dimidiatum var. hyalinum Total a. Accurately identified. No spectral acquisitiona. Misidentified. 5 158 82. 5 (100) 154 (97.5) 80 (97.6). 0 4 (2.5) 0. 0 0 2 (2.4). 50 26 1 26 12 360. 36 (72) 23 (88.5) 1 21 (80.8) 11 (91.7) 331 (91.9). 14 (28) 3 (11.5) 0 5 (19.2) 1 (8.3) 27 (7.5). 0 0 0 0 0 2 (0.5). 9 12. 7 (77.8) 11 (91.7). 2 (22.2) 1 (8.3). 0 0. 21. 18 (85.7). 3 (14.3). 0. Corresponding to the isolates for which spectral acquisition failed after 2 runs.. sorption/ionization process during MALDI-TOF MS, leading to signal suppression (7). This difference in black pigment synthesis between these two closely related molds could explain the poor performance of MALDI-TOF MS for N. dimidiatum identification in our study. We have developed a very simple and rapid experimental protocol that does not require subculture or microscopic examination of the colony, thus avoiding laborious sample manipulations. This is a major advantage over the fastidious and time-consuming conventional phenotypic methods used to identify this group of filamentous fungi. However, this study has some limitations. While our database includes fingerprints of the more relevant species of dermatophytes involved in cases of human infections, some important species are missing, such as Microsporum audouinii, M. gypseum, M. praecox, M. ferrugineum, Trichophyton mentagrophytes, T. schoenleinii, and T. verrucosum. Indeed, we have chosen to develop an identification system including the species most prevalent in our clinical practice, such as T. soudanense and M. langeronii, which are species frequently isolated from patients with tinea capitis in France (2, 18, 19). However, this database can be easily updated according to the local epidemiology. Another limitation is that among the five uncommon dermatophyte species included in the database (M. persicolor, T. erinacei, T. equinum, T. simii, and T. violaceum), only one isolate of T. erinacei has been tested and correctly identified. None of the four other uncommon species could be evaluated, as none was collected during the study period. Last, the database has been validated using only 3-week-old cultures in order to allow full development of the morphological characteristics of the dermatophytes which are needed for identification on the basis of morphological criteria. It is possible that younger cultures can be used for identification using MALDI-TOF MS, but this has not been evaluated in a systematic manner. Currently, only two studies have evaluated performances of MALDI-TOF MS for identification of dermatophytes (13, 32).. 2280. ACKNOWLEDGMENTS Kinda Alshawa is supported by a research fellowship from the Syrian Ministry of Higher Education, Damascus University. J.-L.B., X.N., and M.-E.B. are shareholders of Andromas SAS.. REFERENCES 1. Alanio A, et al. 2011. Matrix-assisted laser desorption ionization timeof-flight mass spectrometry for fast and accurate identification of clinically relevant Aspergillus species. Clin. Microbiol. Infect. 17:750 –755. 2. Alshawa K, et al. 2012. Increasing incidence of Trichophyton tonsurans in Paris, France: a 15-year retrospective study. Br. J. Dermatol. 166:1149 – 1150. 3. Bader O, et al. 2011. Improved clinical laboratory identification of human pathogenic yeasts by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin. Microbiol. Infect. 17:1359 –1365. 4. Benagli C, Rossi V, Dolina M, Tonolla M, Petrini O. 2011. Matrixassisted laser desorption ionization-time of flight mass spectrometry for the identification of clinically relevant bacteria. PLoS One 6:e16424. doi: 10.1371/journal.pone.0016424. 5. Bergmans AM, et al. 2008. Validation of PCR-reverse line blot, a method for rapid detection and identification of nine dermatophyte species in nail, skin and hair samples. Clin. Microbiol. Infect. 14:778 –788. 6. Bistis GN. 1959. Pleomorphisms in the dermatophytes. Mycologia 51: 440 – 444. 7. Buskirk AD, et al. 2011. Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi. Anal. Biochem. 411:122–128. 8. Carbonnelle E, et al. 2007. Rapid identification of staphylococci isolated in clinical microbiology laboratories by matrix-assisted laser desorption ionization–time of flight mass spectrometry. J. Clin. Microbiol. 45:2156 – 2161. 9. Carbonnelle E, et al. 2011. MALDI-TOF mass spectrometry tools for bacterial identification in clinical microbiology laboratory. Clin. Biochem. 44:104 –109. 10. Coulibaly O, et al. 2011. Pseudallescheria/Scedosporium complex species. Journal of Clinical Microbiology. Downloaded from on May 16, 2020 by guest. Dermatophytes E. floccosum T. rubrum T. mentagrophytes var. interdigitale T. soudanense T. tonsurans T. erinacei M. langeronii M. canis Total. Total. Using the Voyager DE Pro MS system (Applied Biosystems, Darmstadt, Germany) and the SARAMIS spectral database (AgnosTec, Potsdam, Germany), Erhard and coworkers succeeded in identifying five dermatophyte species (T. rubrum, T. interdigitale, T. tonsurans, M. canis, and Arthroderma benhamiae) (14). They achieved a high confidence level since 19 out 20 isolates were correctly identified. However, this collection was limited. Recently, Theel and coworkers tested a larger collection of isolates to evaluate performances of the Bruker Biotyper MALDI-TOF MS system (32). Using the MicroFlex LT system (Bruker Daltonics, Bremen, Germany) and the MALDI Biotyper library (version 3.0) in combination with a supplemented library containing an additional 20 dermatophyte spectra, the authors succeeded in identifying isolates to the genus level but not the species level. Indeed, from a total of 171 dermatophyte isolates, 159 (93%) were correctly identified to the genus level and only 102 (59.6%) were correctly identified to the species level. In comparison, using the Andromas system, we obtained a correct identification to the species level for 331/360 (91.9%) of dermatophyte isolates. These results showed that the Andromas system represents a powerful tool to discriminate dermatophyte species and is suited for use on the vast majority of routine samples that are commonly processed in mycology laboratories. Our results show that this technique could replace conventional methods for dermatophyte identification in the near future, not only in specialized research facilities but also in clinical laboratories. This system will become more powerful as the database is expanded by including other dermatophyte species and other fungi with similar morphologies..

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