Mycopathologia DOI 10.1007/s11046-015-9898-x
Evaluation of the Bruker Matrix-Assisted Laser Desorption–Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) System for the Identification of Clinically Important Dermatophyte Species Nilgu¨n Karabıc¸ak . Onur Karatuna . Macit I˙lkit . Is¸ ın Akyar
Received: 11 March 2015 / Accepted: 2 May 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Dermatophytes can invade the stratum corneum of the skin and other keratinized tissues and are responsible for a broad diversity of diseases of skin, nails and hair. Although the standard identification of dermatophytoses depends on macroscopic and microscopic characterization of the colonies grown on special media, there are a number of limitations owing to intraspecies morphological variability, atypical morphology or interspecies morphological similarity which entails improvement in the identification methods. Matrix-assisted laser desorption–ionization timeof-flight mass spectrometry (MALDI-TOF MS) is a novel method which proved to be effective for rapid and reliable identification of dermatophytes grown in cultures when compared to conventional methods. We evaluated the performance of Bruker MALDI-TOF MS System (Bruker Daltonics, Germany) for
N. Karabıc¸ak National Mycology Reference Laboratory, Public Health Institute of Turkey, Sag˘lık Mah. Adnan Saygun Cad. No:55, Sıhhıye, 06100 Ankara, Turkey O. Karatuna (&) I. Akyar Department of Medical Microbiology, School of Medicine, Acıbadem University, ˙Ic¸erenko¨y Mah. Kayıs¸ dag˘ı Cad. No:32, Atas¸ ehir, 34752 Istanbul, Turkey e-mail:
[email protected] M. I˙lkit Division of Mycology, Department of Microbiology, School of Medicine, C¸ukurova University, Balcalı Kampu¨su¨, Sarıc¸am, 01330 Adana, Turkey
identification of clinically relevant dermatophytes. In order to increase the identification capacity of the system, we created supplemental spectral database entries using ten reference dermatophyte strains (ten species in two genera). The utility of the generated database was then challenged using a total of 126 dermatophytes (115 clinical isolates and 11 additional reference strains). The results were evaluated by both manufacturer-recommended and lowered cutoff scores. MALDI-TOF MS provided correct identification in 122 (96.8 %) and 113 (89.7 %) of the isolates with the lowered scores and using the supplemented database, respectively, versus 65 (51.6 %) and 17 (13.5 %) correct identifications obtained by the unmodified database and recommended scores at the genus and species levels, respectively. Our results support the potential utility of MALDI-TOF MS as a routine tool for accurate and reliable identification of dermatophytes. Keywords Dermatophyte Dermatophytose Identification MALDI-TOF MS
Introduction Dermatophytes, involving species within the genera Epidermophyton, Microsporum and Trichophyton, particularly attack and infect keratinized tissues (skin, nails, hair) [1, 2]. In Turkey, T. rubrum and T.
123
Mycopathologia
interdigitale are the most frequent etiologic agents encountered in dermatophytosis [3]. These common species are almost easy to identify but others present more diagnostic confrontations. The standard methods which require experienced technicians have low specificity and are often labor-intensive and very time-consuming due to colony development, sporulation and additional tests which may take up to 6 weeks [1, 2]. There is a pressing need for novel approaches for identification of the common fungal pathogens since the current molecular methods are time-consuming and costly [4–8]. Over the past few years, matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the bacteria and yeast identification process in the laboratory setting [9–13]. This method which has already been validated for rapid and accurate identification of bacterial, yeast and mold species depends upon characteristic fingerprints of intact cells and directly detects the molecular weights of the structural proteins from cultures of the microorganisms without preselection and purification steps [14]. Several recent papers have demonstrated that identification of many mold species by MALDI-TOF MS is accurate and rapid, two key factors in optimizing the treatment [13, 15–18]. In this study, we compared the performance of MALDI-TOF MS to conventional morphology-based methods for the identification of clinical dermatophyte isolates. The MALDI Biotyper database library was used unmodified from the manufacturer and in combination with a laboratory-developed spectral database including supplemental dermatophyte spectra.
Materials and Methods Fungal Isolates and Initial Phenotypic Identification All isolates were acquired by culture of skin, hair and nail specimens collected for routine examination on Sabouraud dextrose agar (SDA) slants containing chloramphenicol, with and without cycloheximide (Oxoid, Cheshire, UK), at 25 °C for 3 weeks. The clinical dermatophyte isolates were identified and stored in the National Mycology Reference Laboratory (NMRL), Public Health Institute of Turkey (PHIT).
123
The initial identification of clinical strains was accomplished by phenotypic (macroscopic and microscopic) characterization. Biochemical methods and different growth media such as Trichophyton agars 1–4 containing supplements along with the classical Sabouraud agar plates were used [19, 20]. The reference strains used in the study were obtained from the CBS-KNAW Fungal Biodiversity Centre (Utrecht, The Netherlands) and the ATCC (Manassas, VA, USA) (Table 1). A total of ten reference strains (ten species in two genera) were used to construct the supplemental dermatophyte database library (Table 1). The utility of the database was then tested using a total of 126 dermatophytes (115 clinical isolates and 11 additional reference strains (Table 2). Extraction of Fungal Material and MALDI-TOF MS Analysis Extraction of fungal material and preparation for MALDI-TOF MS analysis were performed as described in [21] using fungal colony grown on SDA plates which were incubated for 72 h at 27 °C. The MALDI target plate (MTP 384 polished steel target, Bruker Daltonics, Bremen, Germany) was loaded into Bruker Autoflex III MALDI-TOF MS instrument (Bruker Daltonics), and the spectra were automatically recorded in the linear positive ion mode with delayed extraction at a laser frequency of 20 Hz within a mass range between 2000 and 20,000 Da. Only peaks with a signal/noise ratio C10 were considered. This led to an automatically calculated score, from 0 to 1000, which then is converted into a log(score) from 0 (no spectra) to 3 (perfect match). The log(score) values were acquired by comparing unknown sample spectra by using the MALDI Biotyper software, first employing the MALDI Biotyper Library (MBL) (database version 3.1.2.0) alone and then employing the MALDI Biotyper Support Library (MBSL) in addition to the MBL which includes the in-house-generated spectral profiles of reference dermatophyte strains. The cutoff scores recommended by the manufacturer for genusand species-level identification (C1.7 and C2.0, respectively) and in addition lowered log(score) values (C1.5 and C1.7 for genus- and species-level identification, respectively) were used in this study. These lowered criteria were chosen based on previous publications that analyzed the effect of lowered scores on bacterial, yeast and dermatophyte identification
Mycopathologia Table 1 Reference dermatophyte strains used in the study to build up the database and challenge the created database Species
Strain no
Clinical origin
Geographic area
Species represented in the MALDI Biotyper Library database version 3.1.2.0
Reference strains used to supplement the database (n = 10) Microsporum audouinii
CBS 732.88
Hair, skin
Egypt
No
Microsporum ferrugineum
CBS 118548
Hair
China
No
Trichophyton mentagrophytes
a
ATCC 9533
Tinea pedis
Unknown
Yes
Trichophyton raubitschekii
CBS 125605
Hand
Turkey
No
Trichophyton rubrum
CBS 127.447
Unknown
Unknown
Yes
Trichophyton schoenleinii
CBS 564.94
Skin scales of head
Unknown
No
Trichophyton soudanense
CBS 440.63
Scalp
Germany (patient from Ghana)
No
Trichophyton verrucosum
CBS 282.82
Hair, skin
Norway
No
Trichophyton violaceum
CBS 119446
Tinea capitis
Gabon
No
Trichophyton yaoundeib
CBS 677.82
Scalp
Netherlands (patient from Morocco)
No
Reference strains used to challenge the created database (n = 11) Epidermophyton floccosum
ATCC 26072
Foot
Unknown
Yes
Microsporum audouinii
CBS 545.93
Hair
Netherlands
No
Microsporum ferrugineum
CBS 449.61
Unknown
Zaire
No
Microsporum ferrugineum
CBS 457.80
Head
Kenya
No
Trichophyton mentagrophytesa
ATCC MYA-4439
Toenail
USA
Yes
Trichophyton rubrum Trichophyton soudanensec
ATCC MYA-4438 CBS 463.63
Toenail Skin, head
USA Africa (country unknown)
Yes No
Trichophyton tonsurans
ATCC 28942
Human
England
Yes
Trichophyton verrucosum
CBS 134.66
Hair
Netherlands
No
Trichophyton violaceum
CSB 253.88
Skin, head
Netherlands
No
Trichophyton violaceum
CBS 555.84
Skin, head
Netherlands
No
a
T. mentagrophytes ATCC 9533 and T. mentagrophytes ATCC MYA-4439’s synonym is T. interdigitale according to the new taxonomy
b
Trichophyton yaoundei
c
T. soudanense isolates were deposited as T. violaceum at the CBS until January 2001
efficiencies [21–23]. Log(score)-based identification results were acknowledged correct when they agreed with those of phenotypic identification. Each run included a bacterial test standard with a characteristic peptide and protein profile, provided by the manufacturer for calibration, a negative extraction control and the reference QC strains. Failures were repeated using the same methods. Reference Spectra Creation To supplement the MBL, main spectrum profiles (MSPs) of ten reference strains representative of ten
taxonomically distinct species in two genera of dermatophytes were created (Table 1). Fungal extracts were used to create the MSPs as described in [21].
Results The performance of the MBL, alone and in combination with the MBSL supplemented with spectral profile entries belonging to ten reference dermatophyte strains, was evaluated for identification of dermatophytes by testing a set of 115 clinical isolates
123
Mycopathologia Table 2 Performance of the supplemented MALDI Biotyper Library using manufacturer-recommended and lowered spectral score criteria for challenge reference strains (n = 11) and clinical isolates (n = 115) Reference strains (n = 11)
Genus level (spectral score) C1.5
a
MBL
C1.7 MBSL
b
MBL
Species level (spectral score) C1.7a
MBSL
MBL
C2.0b MBSL
MBL
MBSL
Epidermophyton floccosum (n = 1) ATCC 26072
1
1
1
1
1
1
0
Microsporum audouinii (n = 1) CBS 545.93
NA
1
NA
1
NA
1
NA
0 0
Microsporum ferrugineum (n = 2) CBS 449.61, CBS 457.80
NA
2
NA
2
NA
2
NA
0
Trichophyton mentagrophytes (n = 1) ATCC MYA-4438
1
1
1
1
1
1
1
1
Trichophyton rubrum (n = 1) ATCC-4438
1
1
0
1
0
1
0
0
Trichophyton soudanense (n = 1) CBS 436.63 Trichophyton tonsurans (n = 1) ATCC 28942
NA 1
1 1
NA 1
1 1
NA 1
1 1
NA 0
0 0
Trichophyton verrucosum (n = 1) CBS 134.66
NA
1
NA
1
NA
1
NA
1
Trichophyton violaceum (n = 2) CBS 253.88, CBS 555.84
NA
1
NA
0
NA
0
NA
0
Clinical isolates (n = 115) Epidermophyton floccosum (n = 1)
1
1
1
1
1
1
0
0
Microsporum canis (n = 19)
19
19
19
19
19
19
8
10
Trichophyton interdigitale (n = 21)
20
21
20
21
20
21
3
7
Trichophyton rubrum (n = 68)
41
67
18
59
18
59
4
18
Trichophyton tonsurans (n = 6)
4
4
4
4
4
4
1
2
Total (including repeats) (n)
89
122
65
113
65
113
17
39
Correct identification (%)
70.6
96.8
51.6
89.7
51.6
89.7
13.5
31.0
a
Lowered spectral score;
b
manufacturer-recommended spectral score
MBL MALDI Biotyper Library, MBSL MALDI Biotyper Support Library, NA not applicable (this species is not represented in MALDI Biotyper Library database version 3.1.2.0)
(five species in three genera) and eleven additional reference strains (nine species in three genera) (Table 1). We evaluated both the manufacturer-recommended and the lowered spectral scores for identification at genus and species levels. All tested clinical isolates were included in the evaluated MBL database version 3.1.2.0. Using the recommended spectral scores and MBL, correct identifications at the genus and species levels were obtained in 65 (51.6 %) and 17 (13.5 %) of the challenge isolates (n = 126), respectively. When MBSL was used in combination with MBL, the performance was increased to achieve correct identifications in 113 (89.7 %) and 39 (31.0 %) of the isolates at the genus and species levels, respectively (Table 2). Using the lowered spectral scores, however, resulted in substantial improvement in identification. For the genus level, spectral score of C1.5 instead of C1.7 as a threshold resulted in correct identification for 89 (70.6 %) and 122 (96.8 %) of the isolates, using the MBL and MBSL, respectively. At the species
123
level, the contribution of the lowered spectral scores was also significant, and spectral score of C1.7 instead of C2.0 as a threshold resulted in correct identification for 65 (51.6 %) and 113 (89.7 %) of the isolates, using the MBL and MBSL, respectively. The five species which were not represented in the MBL database version 3.1.2.0 (Microsporum audouinii, M. ferrugineum, Trichophyton soudanense, T. verrucosum, T. violaceum) were added to the MBSL by the in-house-generated entries and challenged by testing of additional reference strains (n = 7) of the same species (Table 1). Using the recommended spectral scores yielded correct identification in 5/7 and 1/7 of the isolates for genus- and species-level identification, respectively. The lowered spectral scores improved the results by achieving correct identification in 6/7 and 5/7 of the isolates for genusand species-level identification, respectively. The two T. violaceum reference strains (CBS 253.88 and CBS 555.84) could not be identified at the species level with the lowered spectral scores.
Mycopathologia
MALDI-TOF MS provided correct identification in 122 (96.8 %) and 113 (89.7 %) of the isolates with the lowered scores and using the MBSL at the genus and species levels, respectively. The performance of the lowered spectral scores significantly exceeded the manufacturer’s recommended cutoff scores at both the genus (96.8 vs. 89.7 %) and species (89.7 vs. 31.0 %) levels. Misidentifications and Disagreements A total of 11 isolates (8.7 %) (nine T. rubrum and two T. violaceum) could not be identified at the species level by the lowered log(score) of 1.7 with MBSL. The results of MALDI-TOF MS and morphology-based identification disagreed for two isolates (1.6 %). The two T. tonsurans isolates that were identified as T. interdigitale (formerly T. mentagrophytes) by MALDI-TOF MS were resolved in favor with MALDI-TOF MS with 16S rRNA gene sequencing.
Discussion In this study, identification of clinically important dermatophytes by Bruker MALDI-TOF MS system was evaluated using the commercially available reference spectra library (MBL) and the in-housecreated support library (MBSL) in comparison with traditional morphology-based methods. Bruker MALDI Biotyper performed poorly with the database version installed (version 3.1.2.0) and the manufacturer-recommended cutoff scores (51.6 % and 13.5 % identification accuracy at the genus and species levels, respectively). However, supplementation of the database with the in-house-created spectral profiles belonging to reference dermatophyte strains and using the lowered cutoff scores yielded 96.8 and 89.7 % identification accuracy at the genus and species levels, respectively. The three dermatophyte genera which are commonly isolated in the clinical laboratory, i.e., Epidermophyton spp., Microsporum spp. and Trichophyton spp., are largely represented in the reference spectra library. In order to increase the database content, we have included ten additional reference dermatophyte spectra (Table 1). Presumably, the supplemental library entries improved the identification performance by providing variable spectra representing intraspecies
variance. The results of our study supported the findings of previous studies in which supplemental database was used to enhance the accuracy of identification [17, 24]. Supplementation of the database did not only enable the identification of species which were not previously represented in the database but also resulted in improved identification of clinical isolates which were already represented in the database (Table 2). In this study, all challenge strains of Microsporum spp. (n = 22) and Epidermophyton floccosum (n = 2) were accurately identified to the species level by MALDI-TOF MS. However, two T. violaceum reference strains and nine clinical T. rubrum isolates could not be identified at the species level by MALDI-TOF MS (Table 2). Difficulties in identification of T. violaceum strains by MALDI-TOF MS were also observed in other studies; the researchers linked this disability to the closeness between T. rubrum and T. violaceum [21, 25, 26]. It is likely that MALDI-TOF MS can perform more efficiently if species that are endemic to a region are added to spectral libraries. Previous research on identification of dermatophytes with MALDI-TOF MS yielded successful results. In another study with Bruker MALDI Biotyper, 171 dermatophyte isolates were evaluated and 159 (93 %) were correctly identified at the genus level; however, correct identification at the species level was achieved in only 102 (59.6 %) of the isolates [21]. Studies with other commercially available MALDI-TOF MS systems resulted with promising results, as well. Evaluation of the MALDI-TOF MS spectra with SARAMIS database and software package (AnagnosTec, Potsdam, Germany) for 285 dermatophytes showed 78.2 and 99.3 % agreement with the conventional and molecular identification results, respectively [25]. Similarly, examination of 360 dermatophyte isolates belonging to seven different species by MALDI-TOF MS in Andromas system (Paris, France) resulted in 91.9 % identification accuracy [14]. Vitek MS Plus System (bioMe´rieux, France) was also evaluated for the performance of dermatophyte identification. A total of 134 dermatophytes were used to expand the fungal knowledge database which was then validated using 131 clinical isolates of dermatophytes belonging to 13 taxa, and 125 (95.4 %) strains were accurately identified [18]. Using the Bruker MALDI-TOF MS Autoflex III instrument with MBL and the in-house-generated
123
Mycopathologia
supplemental spectral entries, we managed to identify the most clinically relevant human dermatophytic pathogens, T. rubrum, T. interdigitale, T. tonsurans and M. canis. In addition, species rarely encountered in the clinical setting, such as E. floccosum, M. audouinii, M. ferrugineum, T. soudanense and T. verrucosum, could also be successfully identified. However, satisfactory performance in accurate identification could only be obtained by using the lowered cutoff values. Favorably, this increased performance in identification accuracy obtained with the lower score values was not accompanied by an increase in misidentifications. Based on our data, use of the MALDI-TOF MS system supplemented with reference spectra of additional strains and lowered cutoff values is a successful approach for the identification of dermatophytes. However, development of softwares relying on an enhanced spectral reference library and optimization of cutoff values are necessary to further implement this technology into routine laboratories. Even though the technique provides many advantages, such as being rapid, cost-effective and labor-saving, the method is culture-based and the non-growth of microscopically positive clinical materials in culture (30–50 %, particularly for nails) remains as an intractable problem [25]. In conclusion, MALDI-TOF MS provided highly accurate and reproducible results for the identification of dermatophytes and reduced the identification time from up to 2 weeks to approximately 20 min. The supplemented library enhanced the identification capacity for dermatophyte species. The MALDI-TOF MS technology has the potential to provide new insights into the epidemiology of dermatophytes through rapid and accurate identification of dermatophytes isolated in clinical mycology laboratories.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
References 14. 1. Padhye AA, Summerbell RC. The dermatophytes. In: Merz W, Hay R, editors. Topley and Wilson’s microbiology and microbial infections, medical mycology, vol. 5. 10th ed. London: Hodder Arnold; 2005. p. 220–43. 2. Summerbell R. Trichophyton, Microsporum, Epidermophyton, and agents of superficial mycoses. In: Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D, editors. Manual of clinical microbiology. 10th ed.
123
15.
Washington: American Society for Microbiology Press; 2011. p. 1919–42. Ilkit M. Onychomycosis in Adana, Turkey: a 5-year study. Int J Dermatol. 2005;44:851–4. Gra¨ser Y, El Fari M, Vilgalys R, Kuijpers AF, De Hoog GS, Presber W, Tietz H. Phylogeny and taxonomy of the family Arthrodermataceae (dermatophytes) using sequence analysis of the ribosomal ITS region. Med Mycol. 1999;37: 105–14. Bergmans AM, Schouls LM, van der Ent M, Klaassen A, Bo¨hm N, Wintermans RG. 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. 2008;14:778–88. Li HC, Bouchara JP, Hsu MM, Barton R, Su S, Chang TC. Identification of dermatophytes by sequence analysis of the rRNA gene internal transcribed spacer regions. J Med Microbiol. 2008;57:592–600. Li XF, Tian W, Wang H, Chen H, Shen YN, Lv GX, Liu WD. Direct detection and differentiation of causative fungi of onychomycosis by multiplex polymerase chain reactionbased assay. Eur J Dermatol. 2011;21:37–42. Menotti J, Machouart M, Benderdouche M, Cetre-Sossah C, Morel P, Dubertret L, Derouin F, Feuilhade De Chauvin M, Lacroix C. Polymerase chain reaction for diagnosis of dermatophyte and Scytalidium spp. onychomycosis. Br J Dermatol. 2004;151:518–9. Saleeb PG, Drake SK, Murray PR, Zelazny AM. Identification of mycobacteria in solid-culture media by matrixassisted laser desorption ionization–time of flight mass spectrometry. J Clin Microbiol. 2011;49:1790–4. Carbonnelle E, Mesquita C, Bille E, Day N, Dauphin B, Beretti JL, Ferroni A, Gutmann L, Nassif X. MALDI-TOF mass spectrometry tools for bacterial identification in clinical microbiology laboratory. Clin Biochem. 2011;44: 104–9. Benagli C, Rossi V, Dolina M, Tonolla M, Petrini O. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the identification of clinically relevant bacteria. PLoS One. 2011. doi:10.1371/journal.pone. 0016424. Stevenson LG, Drake SK, Shea YR, Zelazny AM, Murray PR. Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of clinically important yeast species. J Clin Microbiol. 2010;48:3482–6. Gautier M, Ranque S, Normand AC, Becker P, Packeu A, Cassagne C, L’Ollivier C, Hendrickx M, Piarroux R. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: revolutionizing clinical laboratory diagnosis of mould infections. Clin Microbiol Infect. 2014;20:1366– 71. Alshawa K, Beretti JL, Lacroix C, Feuilhade M, Dauphin B, Quesne G, Hassouni N, Nassif X, Bougnoux ME. Successful identification of clinical dermatophyte and Neoscytalidium species by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2012;50:2277–81. De Carolis E, Posteraro B, Lass-Flo¨rl C, Vella A, Florio AR, Torelli R, Girmenia C, Colozza C, Tortorano AM, Sanguinetti M, Fadda G. Species identification of Aspergillus,
Mycopathologia
16.
17.
18.
19.
20. 21.
Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Microbiol Infect. 2012;18:475–84. Hettick JM, Green BJ, Buskirk AD, Kashon ML, Slaven JE, Janotka E, Blachere FM, Schmechel D, Beezhold DH. Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting. Anal Biochem. 2008;380:276–81. Normand AC, Cassagne C, Ranque S, L’Ollivier C, Fourquet P, Hendrickx M, Piarroux R. Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi. BMC Microbiol. 2013. doi:10.1186/ 1471-2180-13-76. De Respinis S, Monnin V, Girard V, Welker M, Arsac M, Cellie`re B, Durand G, Bosshard PP, Farina C, Passera M, Van Belkum A, Petrini O, Tonolla M. 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. J Clin Microbiol. 2014;52:4286–92. Larone DH. Medically important fungi: a guide to identification. 3rd ed. Washington: American Society for Microbiology Press; 2002. Sutton DA, Fothergill AW, Rinaldi MG. Clinically significant fungi. Baltimore: Williams & Wilkins; 1998. Theel ES, Hall L, Mandrekar J, Wengenack NL. Dermatophyte identification using matrix-assisted laser desorption
22.
23.
24.
25.
26.
ionization-time of flight mass spectrometry. J Clin Microbiol. 2011;49:4067–71. Alatoom AA, Cunningham SA, Ihde SM, Mandrekar J, Patel R. Comparison of direct colony method versus extraction method for identification of gram-positive cocci by use of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2011;49:2868–73. Dhiman N, Hall L, Wohlfiel SL, Buckwalter SP, Wengenack NL. Performance and cost analysis of matrix-assisted laser desorption ionization-time of flight mass spectrometry for routine identification of yeast. J Clin Microbiol. 2011;49:1614–6. Lau AF, Drake SF, Calhoun LB, Henderson CM, Zelazny AM. Development of a clinically comprehensive database and a simple procedure for identification of molds from solid media by matrix-assisted laser desorption ionizationtime of flight mass spectrometry. J Clin Microbiol. 2013;51:828–34. Nenoff P, Erhard M, Simon JC, Muylowa GK, Herrmann J, Rataj W, Gra¨ser Y. MALDI-TOF mass spectrometry—a rapid method for the identification of dermatophyte species. Med Mycol. 2013;51:17–24. Erhard M, Hipler UC, Burmester A, Brakhage AA, Wo¨stemeyer J. Identification of dermatophyte species causing onychomycosis and tinea pedis by MALDI-TOF mass spectrometry. Exp Dermatol. 2008;17:356–61.
123
