Mycopathologia (2017) 182:183–192
DOI 10.1007/s11046-016-0080-x
MALDI-TOF-Based Dermatophyte Identification
Coralie L’Ollivier . Stéphane Ranque
Received: 22 June 2016 / Accepted: 6 October 2016 / Published online: 12 October 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract MALDI-TOF MS has become increas-
ingly popular for microorganism identification in the
routine laboratory. Compared with conventional mor-
phology-based techniques, MALDI-TOF is relatively
inexpensive (per-unit identification), involves a rapid
result turnaround time and yields more accurate results
without the need for highly qualified staff. However,
this technology has been technically difficult to
implement for filamentous fungi identification. Iden-
tification of dermatophytes, a type of filamentous
fungi, remains particularly challenging, partly due to
the lack of clear species definition for some taxa or
within some species complexes. Review of the ten
studies published between 2008 and 2015 shows that
the accuracy of MALDI-TOF MS-based identification
varied between 13.5 and 100 % for dermatophytes.
This variability was partly due to inconsistencies
concerning critical steps of the routine clinical labo-
ratory process. Use of both a complete formic acid-
C. L’Ollivier
S. Ranque
Aix-Marseille University, IP-TPT UMR MD3,
13885 Marseille, France
C. L’Ollivier
S. Ranque
Laboratory of Parasitology-Mycology, APHM CHU
Timone, 13005 Marseille, France
S. Ranque (&)
Laboratoire de Parasitologie-Mycologie, AP-HM, CHU
Timone, 264 rue Saint Pierre, 13385 Marseille, France
e-mail: stephane.ranque@ap-hm.fr
acetonitrile protein extraction step and a manufacturer
library supplemented with homemade reference spec-
tra is essential for an accurate species identification.
This technique is conversely unaffected by variations
in other routine clinical laboratory conditions such as
culture medium type, incubation time and type of mass
spectrometry instrument. Provided that a reference
spectra library is adequate for dermatophyte identifi-
cation, MALDI-TOF MS identification is more eco-
nomical and offers an accuracy comparable to that of
DNA sequencing. The technique also represents an
advantageous alternative to the protracted and labor-
intensive dermatophyte identification via macroscopic
and microscopic morphology in the routine clinical
laboratory.
Keywords Mass spectrometry
Dermatophyte

Identification
Performance
Clinical laboratory
Introduction
Dermatophyte infections, i.e., dermatophytosis, in
humans are essentially superficial fungal infections;
dermatophytes invade and propagate in keratinized
tissues such as hair, skin and nails. Although accurate
diagnosis is central to effective management of this
mycosis, a study by the European onychomycosis
observatory showed that only 3.4 % of general physi-
cians and 39.6 % of dermatologists perform sampling
to diagnose dermatophytosis [1]. This might be due to
123
184
the relatively poor sensitivity and protracted result
turnaround time of conventional diagnostic methods.
Until recently, identification of dermatophyte species
has been essentially performed via in vitro culture to
examine macroscopic colony morphology and micro-
scopic features of conidia and other hyphal features
such as chlamydospores and spiral hyphae. Conven-
tional dermatophyte identification is often complicated
and laborious due to the morphological similarities
between some species; the process thus requires a high
level of training and expertise [2–4]. Finally, the result
turnaround time of these conventional identification
methods can sometimes exceed 5 weeks. Matrix-
assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-TOF MS), which was initially
reserved for research, has appeared in microbiology
laboratories approximately 10 years ago. The wide-
spread interest of this technology is due to the accuracy
and speed of microorganism identification, relatively
low cost and simple integration into routine laborato-
ries. However, only a few studies have evaluated the
use of MALDI-TOF MS for the identification of
dermatophytes. Following a brief historical perspec-
tive of the MALDI-TOF MS revolution for microor-
ganism identification, we review the studies published
over the last 10 years and outline the pros and cons of
MALDI-TOF MS for dermatophyte identification in
the clinical laboratory.
Using MALDI-TOF MS for Microorganism
Identification
Mass spectrometry, a technique that has been used
since the late nineteenth century, was applied for
microorganism identification in the 1970s [5, 6]. The
expansion of this technology was facilitated by the
development of soft ionization MALDI-TOF MS
techniques, which allowed for the analysis of a
comprehensive protein panel. As mass spectrometers
have become simpler to operate (using the linear
mode), smaller (bench-top size) and less expensive,
the widespread application has expanded from basic
research to the clinical laboratory. MALDI-TOF MS
detects highly abundant proteins in a mass range
between 2 and 20 kDa, representing primarily riboso-
mal proteins along with a few housekeeping proteins.
The characteristic protein fingerprint of a particular
microorganism is used to identify it by matching with
123
Mycopathologia (2017) 182:183–192
species-specific protein patterns included in a com-
prehensive reference spectra library [7]. Thus, the
identity of a microorganism can be established to the
genus level and in many cases to the species and strain
level [8, 9]. During MALDI-TOF analysis, two
parameters are evaluated for each ion: the mass-to-
charge ratio m/z, which is measured by determining
the time required for it to travel the length of the time-
of-flight (TOF) tube, and its relative intensity. Based
on the TOF output, a characteristic spectrum is
preprocessed to serve as a barcode. The identification
of microbes via MALDI-TOF MS is performed by
comparing the barcode of the unknown organism with
the barcodes contained in a library. Depending on the
degree of conformity, the microorganism is identified
as belonging to the genus, species or subspecies of the
reference spectrum. The current enthusiasm for
MALDI-TOF MS-based microorganism identification
in the routine laboratory is fueled by its increased
identification accuracy compared with conventional
phenotypic techniques, relatively reduced per-unit
cost and rapid result turnaround time of only a few
minutes. Several ready-to-use MALDI-TOF bench-
top identification platforms for routine identification
of fungi are now commercially available.
MALDI-TOF MS-Based Fungal Identification
Several researchers have described the use of MALDI-
TOF MS to identify various human fungal pathogens.
Amiri-Eliasi and Fenselau [10] have reported the use
of MALDI-TOF MS to identify and characterize a
single-celled fungus, the yeast Saccharomyces cere-
visiae. In the clinical laboratory, MALDI-TOF MS-
based identification of fungi has evolved at a slower
pace than bacterial identification. The seminal study of
fungi (yeast) identification in the clinical laboratory
setting was published in 2009 [11]. The development
of a standardized MALDI-TOF MS procedure for
filamentous fungi identification in the routine labora-
tory necessitated a few more years [12, 13]. Indeed,
the inherent biological complexity of filamentous
fungi, as exemplified by the concomitant presence of
different fungal structures (hyphae and/or conidia) in
the same culture, renders them difficult to identify
[14]. Dermatophyte fungi are no exception to the rule.
Moreover, the cultures grow relatively slow, are not
very proliferative and often produce pigment.
Mycopathologia (2017) 182:183–192
Using MALDI-TOF for Dermatophyte
Identification
Between 2008 and 2015, ten studies addressed the
question of identifying dermatophytes using MALDI-
TOF MS [4, 9, 15–22]. The study features are
summarized in Table 1. Identification performance
varied between 13.5 and 100 % depending on the
study. This variability in identification performance
was partly due to inconsistencies in critical steps of the
routine clinical laboratory process.
Critical Factors for MALDI-TOF Dermatophyte
Identification in the Clinical Laboratory
MALDI-TOF MS identification of dermatophytes
may predominantly be influenced by critical factors
involved in the routine laboratory process, including
culture medium type, incubation time, protein extrac-
tion procedure, mass spectrometry instrument and the
reference spectra library.
Culture Medium
Reliable identification of species via MALDI-TOF
MS should ideally function independently of fungal
growth conditions. However, culture conditions may
profoundly affect the fungal physiology and protein
expression profile, and it has been presumed that
dermatophyte MALDI-TOF spectra would be influ-
enced by the type of culture medium and incubation
time [20]. Nevertheless, some authors have reported
that culture conditions did not affect bacterial identi-
fication via MALDI-TOF MS [23, 24]. Several studies
have also demonstrated that dermatophyte species
identification is unaffected by cultivation conditions
[9, 18, 20, 22]. For example, in a clinical study which
analyzed a set of isolates (5–30) of 5 dermatophyte
species grown on 6 distinct culture media, including
chocolate agar, inhibitory mold agar, Mycosel agar
and Sabouraud agar, Theels et al. [22] found no
evidence that the type of medium affected species
identification.
Incubation Time
Dermatophytes are isolated and grown in culture
media. Over time, the fungi develop specific
185
macroscopic and microscopic characteristics. The
vegetative part of a fungus, the mycelium, grows in
first and consists of branching hyphae. Spores are then
produced at the tip of or alongside the hyphae. Due to
these different growth stages, which are accompanied
by modification in composition of the cell wall, there
is a risk of obtaining a variety of fingerprints via
MALDI-TOF depending on the age of the culture.
According to various studies, the incubation time
before protein extraction ranges from 3 days to
3 weeks, without yielding markedly different results.
De Respinis et al. [18] found that increasing the
culture incubation time to 10 days and/or repeating the
analyses did not improve identification results. In
contrast, Packeu et al. [21] showed that the accuracy of
dermatophyte species identification was enhanced
with a prolonged incubation time; 100 % correct
identifications were reached by increasing incubation
time from three to 14 days. In our experience at the
Mycology Laboratory of Marseille University Hospi-
tal, MALDI-TOF MS is capable of identifying
dermatophytes before characteristic morphological
features appear. MALDI-TOF MS thus accelerates
identification result turnaround time and, although
dermatophytosis is not a medical emergency, facili-
tates patient management.
Protein Extraction and Matrix
Numerous MALDI-TOF-based identification proce-
dures have been described for microorganisms [25].
The direct identification of intact cells or spores is the
simplest approach. However, an initial protein extrac-
tion step using acidic solvents or bead disruption prior
to MS analysis may be required for microorganisms
that are difficult to lyse such as filamentous fungi [26].
Fungal cells are larger in size than bacterial cells and
possess a rigid cell wall. The cell wall of fungi,
including dermatophytes, is primarily composed of
polysaccharides which may vary with the taxonomic
group (e.g, chitin, beta-glucans, mannans, galac-
tomannans, arabinogalactans, rhamnomannans),
together with a smaller proportion of proteins, lipids
and polyphosphates. This particular composition,
which differs from that of the bacterial wall, partly
explains the need for a pre-analytical protein extrac-
tion step, which frees the proteins of interest to
generate the MALDI-TOF MS barcode. Regarding
dermatophytes, the development of MALDI-TOF MS
123
 Table 1 Main features of the published studies evaluating the performance of MALDI-TOF MS for dermatophyte identification
186

Author Year Mass spectrometer Culture medium Culture Protein Matrix Library databases Tested isolates Correct
instrument incubation extraction type dermatophyte

123
time procedure identification rate

Erhard 2008 Axima@Saramis Various media, 4 weeks Matrix DHB 18 Reference species 20 Clinical isolates 99.9 %
et al. [9] including SDA solution (number isolates/species
unknown)
Theel et al. 2011 MALDI Biotyper Various media agar 3 days Formic acid/ CCA ML ± supplemented with 9 100 Well- ML 4.7 %
[22] plate acetonitrile clinical isolates and 4 characterized S-ML 36.8 %
reference strains (9 archived
S-ML ? LS C 1.7
species) dermatophyte
59.6 %
isolates and 92
clinical isolates
Alshawa 2012 Andromas SDA ? antibiotics 3 weeks Formic acid CCA 50 Clinical isolates (12 381 Clinical isolates 91.9 %
et al. with or without species)
[15] cycloheximide
L’Ollivier 2013 MALDI Biotyper SDA 3 days Formic acid/ CCA 48 Clinical isolates (17 133 Clinical isolates 97.8 %
et al. acetonitrile species)
[20]
Nenoff 2013 Axima@Saramis SDA 1–3 weeks Matrix DHB 285 Dermatophyte strains 285 Dermatophyte 99.3 %
et al. [4] solution (21 species) strains of 21
distinct species
De 2013 Axima@Saramis SDA gentamicin 3 days Matrix CCA 108 Reference strains (18 141 Clinical isolates 95.8 %
Respinis chloramphenicol solution species) (9 species)
et al. 2 agar plates
[17]
Packeu 2014 MALDI Biotyper SDA 3–14 days With or CCA 195 Reference strains and 176 Clinical isolates 40–100 %
et al. chloramphenicol without 58 species (from the
[21] plate formic BCCM/IHEM fungal
acid/ collection and one clinical
acetonitrile laboratory)
Calderaro 2014 MALDI Biotyper SDA plates with 3 weeks Formic acid/ CCA ML ± 13 reference strains 64 Clinical isolates Unclear (improved
et al. chloramphenicol acetonitrile (10 species) and 11 with S-ML)
[16] isolated strains (8 species)
De 2014 Vitek MS and SDA plates and on 5–10 days Formic acid/ CCA Vitek MS supplemented 131 Clinical isolates 88.5 %
Respinis Axima@Saramis Sabouraud acetonitrile with 134 well- (13 taxa)
et al. gentamicin characterized strains (17
[18] chloramphenicol species)
2 agar plate
Mycopathologia (2017) 182:183–192
 Mycopathologia (2017) 182:183–192 187

CCA alpha-cyano-4-hydroxycinnamic acid, DHB 2.5-dihydroxybenzoic acid, LS log score, ML manufacturer reference spectra library, S-ML supplemented manufacturer
identification of intact cells began around the year

S-ML ? LS C 1.7
identification rate
dermatophyte 2000 with seminal studies using MALDI-TOF MS and

S-ML 31 %
a bacterial whole-cell protocol to identify Penicillium
ML 13.5 %

89.7 %
spp., Scytalidium dimidiatum and Trichophyton
Correct

rubrum [27]. Packeu et al. [21] achieved less than

40 % identification rates using the direct deposit
method with or without formic acid extraction after
reference strains

3 days of culture. If no correct identification was

isolates ? 11
Tested isolates

achieved, the isolates were reanalyzed after seven and

115 Clinical

14 days following a formic acid-acetonitrile extrac-

tion procedure, which yielded a 100 % reliable
identification.
Compared with previous MS techniques, laser
ML ± supplemented with

ionization in MALDI-TOF MS is assisted by a matrix,

10 reference strains (9

which allows for a softer separation of the molecules;

therefore, large size, low volatility and heat-sensitive
Library databases

molecules can be ionized without degradation.

MALDI-TOF thus detects fragile biomolecules such
species)

as peptides, proteins and glycoproteins. The sample is

embedded in the matrix prior to being ionized. A large
number of matrix substances have been used in
Matrix

various MALDI-TOF MS applications. Ionic matrices

CCA
type

are largely used in microorganism identification

protocols and consist of equimolar mixtures of con-
acetonitrile
Formic acid/

ventional MALDI matrix compounds, such as 2,5-

extraction
procedure

dihydroxybenzoic acid (DHB), alpha-cyano-4-hy-

Protein

droxycinnamic acid (CCA) or sinapinic acid (SA)

together with organic bases [e.g., pyridine (Py),
incubation

tributylamine (TBA) or N,N-dimethylethylenedi-

Culture

amine (DMED)] [28]. Although the two seminal

3 days
time

studies used DHB [9], CCA is now the most widely

used and recommended matrix for dermatophyte
identification [25].
Culture medium

reference spectra library, SDA Sabouraud dextrose agar

Mass Spectrometer Instrument

SDA plate

Three different MALDI-TOF identification platforms,

which are ready to use for routine identification of
Mass spectrometer

MALDI Biotyper

fungi, are currently available on the market: the

MALDI Biotyper (Bruker Daltonics, Bremen, Ger-
instrument

many), the Vitek MS (BioMerieux, Craponne, France)

and the Andromas (Andromas SAS, Paris, France).
Additionally, a fourth database, the Axima@Saramis
database which was initially developed by Anag-
Table 1 continued

2015
Year

nosTec (Potsdam, Germany), is also provided with the

Vitek MS equipment to analyze the spectra.
Karabicak

The analytical methodologies of these platforms are

Author

et al.
[19]

very similar [29]. These systems primarily differ from

each other with regard to spectra preprocessing,

123
188
algorithms used to create references and algorithms
used to compare and express similarity between an
unknown spectrum and reference spectra (interpreta-
tion criteria). For dermatophyte identification, these
commercialized libraries are too deficient for routine
use. The high identification rates obtained in several
studies were possible due to the implementation of the
databases with in-house reference spectra libraries
(see below). These different platforms are flexible (to
varying degrees) to construct an accurate reference
spectra library. The two most flexible systems,
MALDI Biotyper and Andromas, offer the possibility
to use the average of spectral information due to
replicated spectra issued from the same strain [29].
Reference Spectra Library
In 2008, Erhard et al. [9] have tested MALDI-TOF MS
to identify clinical dermatophytes in a blind compar-
ative study validated using conventional morpholog-
ical identification and DNA sequencing of the internal
transcribed spacer (ITS) regions of the rRNA genes.
For all identifications via MS, a 99.9 % degree of
confidence was obtained with the major clinical
dermatophyte species, i.e., T. rubrum, T. interdigitale,
T. tonsurans and Arthroderma benhamiae. These
results were obtained using a homemade reference
spectra library exported to the SARAMIS software
package (AnagnosTec). Nenoff et al. [4] then evalu-
ated the SARAMIS reference spectra library and
software package to identify 285 dermatophyte iso-
lates. The encouraging results showed 99.3 % agree-
ment with DNA-based identification results; however,
all tested isolates had been used to construct the
library. Similarly, MALDI-TOF MS identification of
360 dermatophyte isolates belonging to seven distinct
species yielded 91.9 % identification accuracy, using
the Andromas system [15]. However, it became clear
that one of the main limitations of MALDI-TOF MS
identification is that accuracy depends on the quality
of the reference spectra library. The results differ
significantly depending on whether they were obtained
from the standard manufacturer library (ML) alone or
the supplemented manufacturer library (S-ML).
Library supplementation is therefore necessary for
accurate and efficient MALDI-TOF MS-based der-
matophyte identification. In fact, all studies evaluating
MALDI-TOF MS for dermatophyte identification
used their own reference library, thereby highlighting
123
Mycopathologia (2017) 182:183–192
the severe limitation of commercialized reference
spectra libraries. Moreover, successful species identi-
fication is dependent on the number of strain entries in
the reference spectra library for a given species. For
example, Theel et al. [22] correctly identified only 4.7
and 36.8 % of 171 isolates using the ML and S-ML,
respectively. Karabiçak et al. [19] aimed to identify
115 isolates and 11 reference strains and obtained a
successful identification rate of 13.5 versus 31 %
using the Bruker ML and S-ML, respectively. De
Respinis et al. [18] evaluated the performance of the
Vitek MS Plus system for dermatophyte identification.
A total of 134 dermatophyte isolates were included to
expand the manufacturer’s reference spectra library,
which was then validated using 131 clinical dermato-
phyte isolates. Using the S-ML library, the authors
accurately identified 95.4 % of strains. The efficiency
of the MALDI-TOF MS identification system can be
improved with an appropriate log score threshold. For
example, Theel et al. [22] correctly identified 36.8 and
59.6 % of 171 isolates using the manufacturer’s log
score threshold (i.e., 2.0) and a reduced log score
threshold of 1.7, respectively. Likewise, Karabiçak
et al. [19] improved the level of identification from 31
to 89.7 % by lowering the log score threshold from 2.0
to 1.7.
Spectra Comparison Algorithm
Literature concerning the architecture of in-house
reference spectra libraries for dermatophyte identifi-
cation via MALDI-TOF MS is scarce. According to
Theel et al. [22] and Karabiçak et al. [19], each extract
from reference strains was spotted onto eight individ-
ual wells, and spectra were collected in triplicate,
yielding a total of 24 spectra per isolate. To enhance
MALDI-TOF MS-based identification of filamentous
fungi, it has been necessary to assess the architecture
of several reference spectrum libraries. For example,
Normand et al. [30] compared the effectiveness of
different architectures of distinct reference spectra
libraries. The standard recommendation to address
problems associated with the heterogeneity of
microorganism species is merely to increase the
number of strains per species in the library. This study
demonstrated that increasing the number of mass
spectra generated from distinct subcultures of a given
strain yields a significant improvement in filamentous
fungi identification. This adjustment can also partially
Mycopathologia (2017) 182:183–192
Fig. 1 MALDI-TOF mass spectra obtained from isolates of the
Trichophyton rubrum complex showing the relative hetero-
geneity of protein profiles within the T. rubrum isolates and the
overcome the issue of relatively rare species, for
which the number of available strains is insufficient to
construct reference spectra libraries.
Taxonomy and Species Resolution
Recurrent misidentification issues have been fre-
quently reported. In the study of Theel et al. [22],
one Microsporum canis isolate was identified as M.
audouinii, one Trichophyton mentagrophytes isolate
was identified as T. tonsurans, and 16 T. rubrum
isolates were identified as T. soudanense. Erhard et al.
[9] found one T. rubrum isolate that also showed
several mass signals that were typical of T. violaceum.
Difficulties in the identification of T. violaceum strains
via MALDI-TOF MS were also observed in other
studies; the researchers associated this complication
with the close relationship between T. rubrum and T.
violaceum. De Respinis et al. [18] showed that only
189
relative similarity between isolates of the closely related (if not
synonymous) T. violaceum and T. soudanense species
47 % (18/38 strains) of the African T. rubrum
population (i.e., T. soudanense) were accurately
recognized; many strains were misidentified as T.
violaceum.
The partial lack of relationship between fungal
genotypes and corresponding phenotypes (i.e., colony
morphology/microscopic morphology, sexuality and
genetic makeup) has caused a debate among mycol-
ogists, especially concerning the ‘‘dermatophytes
species’’ definition and the taxonomic classification
proposed by Gräser et al. [2]. Some biotypes that were
unambiguously considered as distinct species, based
on profound differences in morphology and pattern of
infection, are now consistently considered synony-
mous according to DNA-based phylogeny and popu-
lation genetic analyses. In particular, the cosmopolitan
Trichophyton rubrum and the endemic African agent
of childhood tinea capitis, Trichophyton soudanense,
are effectively indistinguishable using the ITS
123
190
Fig. 2 MALDI-TOF protein profiles of isolates of the Tri-
chophyton mentagrophytes complex revealing the relatively
heterogeneity within this species complex, and the clear
differences with the mass spectra obtained from species of the
ribosomal DNA barcode [2]. The T. rubrum complex
consists of two anthropophilic species, T. rubrum and
T. violaceum. Misidentification between T. rubrum, T.
soudanense and T. violaceum in different studies using
distinct MALDI-TOF MS reference spectra libraries
indicates that MALDI-TOF MS identification is
limited with the current identification gold standard
for a given taxon. In particular, the limited genetic
distance observed by ITS sequencing between T.
soudanense and T. violaceum raises the question of
their recognition as separate species. In Fig. 1, anal-
ysis of the MALDI-TOF mass spectra of dermatophyte
isolates of the T. rubrum complex highlights the
heterogeneity of spectra among the T. rubrum isolates
and the relative similarity between T. violaceum and T
soudanense isolates. The current relatively low taxo-
nomic resolution concerning the Trichophyton men-
tagrophytes species complex also hampers accurate
MALDI-TOF-based species identification. In Fig. 2,
the MALDI-TOF protein profiles of isolates in this
complex are distinct from that of a T. rubrum isolate
but relatively heterogeneous among species. However,
123
Mycopathologia (2017) 182:183–192
T. rubrum complex. Trichophyton mentagrophytes and T.
interdigitale spectra share numerous peaks. The major peak in
the T. tonsurans IHEM1936 mass spectrum is also present in the
mass spectra obtained for the two T. interdigitale isolates
major peaks in T. tonsurans mass spectra are also
present in those of T. interdigitale.
Taxonomic errors or imprecisions in the reference
spectra library will automatically yield inaccurate
MALDI-TOF identification results. Improved
MALDI-TOF MS-based dermatophyte identification
thus requires reliable and curated reference libraries
including spectra collected from isolates or strains
unambiguously identified at the species level. The
current gold standard approach for the identification of
dermatophyte isolates is analysis of the ITS sequence
barcode [31]. However, it is important to update the
databases with the new dermatophyte DNA sequences
according to the current dermatophyte species
definition.
Conclusion and Perspectives
MALDI-TOF MS represents a major technical step
forward and an alternative to time-consuming and
labor-intensive dermatophyte identification via
Mycopathologia (2017) 182:183–192
macroscopic and microscopic morphology or DNA
sequencing. The technique has proven to be unaffected
by variations in the routine clinical laboratory proce-
dure such as culture medium, incubation time and MS
instrument. Complete formic acid-acetonitrile protein
extraction yields higher-quality spectra in the routine
laboratory workflow than the rapid ‘‘direct deposit’’
procedures. MALDI-TOF MS-based identification
can be impossible if the sample is contaminated with
culture medium, notably when fungal colonies are
included and cannot be separated from the agar.
Nevertheless, the main limitation remains the inade-
quate representation of dermatophyte species in the
reference spectra libraries of current commercialized
MALDI-TOF identification systems. The laboratories
are required to expand the manufacturer reference
spectra library by using an in-house reference library
to encompass inter- and intra-specific dermatophyte
diversity, which however requires skilled mycologists
and is only possible in few centers with access to this
technology. MALDI-TOF MS networking between
clinical laboratories has proven useful for laboratories
that are not equipped with this technology [32]. As the
commercialized MALDI-TOF identification solutions
currently available are inadequate and with no
prospect of substantial improvement in the near future,
development of a web-based reference MALDI-TOF
spectra library specialized in fungal identification and
curated by expert mycologists would represent an
interesting venture. Such a platform could enable
scientists and physicians worldwide to query species
online in a similar manner that is currently available
for nucleic acid sequence identification.
Currently, MALDI-TOF MS-based identification
requires successful growth of the fungus in culture.
However, even when sampling is adequate, dermato-
phyte cultures show relatively poor sensitivity, with
approximately 30 % false-negative culture results
[33, 34]. Another exciting future application would
be direct MALDI-TOF MS-based identification of
dermatophytosis from clinical samples, as described
by Hollemeyer et al. [35]. This method is rapid and
does not require a cultivation step. The mass spectra of
tryptic digests of nail samples infected with T. rubrum
were found to significantly differ from those of healthy
individuals and patients suffering from psoriasis.
According to Hollemeyer et al. [35], the specific
fingerprint of samples infected with T. rubrum can be
191
explained by gradual degradation of structural pro-
teins during the progression of the fungal infection.
Acknowledgments We thank Sandra Moore for proofreading
the manuscript.
References
1. Effendy I, Lecha M, Feuilhade de Chauvin M, Di Chiacchio
N, Baran R. European onychomycosis observatory. Epi-
demiology and clinical classification of onychomycosis.
J Eur Acad Dermatol Venereol. 2005;19(Suppl 1):8–12.
2. Gräser Y, Scott J, Summerbell R. The new species concept
in dermatophytes-a polyphasic approach. Mycopathologia.
2008;166:239–56.
3. Kanbe T. Molecular approaches in the diagnosis of der-
matophytosis. Mycopathologia. 2008;166:307–17.
4. Nenoff P, Erhard M, Simon JC, et al. MALDI-TOF mass
spectrometry—a rapid method for the identification of
dermatophyte species. Med Mycol. 2013;51:17–24.
5. Aluyi HA, Drucker DB. Fingerprinting of carbohydrates of
Streptococcus mutans by combined gas–liquid chromatog-
raphy–mass spectrometry. J Chromatogr. 1979;78:209–18.
6. Fisher-Hoch S, Hudson MJ, Thompson MH. Identification
of a clinical isolate as Legionella pneumophila by gas
chromatography and mass spectrometry of cellular fatty
acids. Lancet. 1979;2:323–5.
7. Murray PR. What is new in clinical microbiology-microbial
identification by MALDI-TOF mass spectrometry: a paper
from the 2011 William Beaumont hospital symposium on
molecular pathology. J Mol Diagn. 2012;14:419–23.
8. Fagerquist CK, Garbus BR, Miller WG, et al. Rapid iden-
tification of protein biomarkers of Escherichia coli
O157:H7 by matrix-assisted laser desorption ionization-
time-of-flight-time-of-flight mass spectrometry and top-
down proteomics. Anal Chem. 2010;82:2717–25.
9. Erhard M, Hipler UC, Burmester A, Brakhage AA,
Wöstemeyer J. Identification of dermatophyte species
causing onychomycosis and tinea pedis by MALDI-TOF
mass spectrometry. Exp Dermatol. 2008;17:356–61.
10. Amiri-Eliasi B, Fenselau C. Characterization of protein
biomarkers desorbed by MALDI from whole fungal cells.
Anal Chem. 2001;73:5228–31.
11. Marklein G, Josten M, Klanke U, et al. Matrix-assisted laser
desorption ionization-time of flight mass spectrometry for
fast and reliable identification of clinical yeast isolates.
J Clin Microbiol. 2009;47:2912–7.
12. Cassagne C, Ranque S, Normand AC, et al. Mould routine
identification in the clinical laboratory by matrix-assisted
laser desorption ionization time-of-flight mass spectrome-
try. PLoS One. 2011;6:e28425.
13. Ranque S, Normand AC, Cassagne C, et al. MALDI-TOF
mass spectrometry identification of filamentous fungi in the
clinical laboratory. Mycoses. 2014;57:135–40.
14. Santos C, Paterson RR, Venâncio A, Lima N. Filamentous
fungal characterizations by matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry. J Appl Micro-
biol. 2010;108:375–85.
123
192
15. Alshawa K, Beretti JL, Lacroix C, et al. Successful identi-
fication of clinical dermatophyte and Neoscytalidium spe-
cies by matrix-assisted laser desorption ionization-time of
flight mass spectrometry. J Clin Microbiol.
2012;50:2277–81.
16. Calderaro A, Motta F, Montecchini S, et al. Identification of
dermatophyte species after implementation of the in-house
MALDI-TOF MS database. Int J Mol Sci.
2014;15:16012–24.
17. De Respinis S, Tonolla M, Pranghofer S, et al. Identification
of dermatophytes by matrix-assisted laser desorption/ion-
ization time-of-flight mass spectrometry. Med Mycol.
2013;51:514–21.
18. De Respinis S, Monnin V, Girard V, et al. 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.
19. Karabıçak N, Karatuna O, İlkit M, Akyar I. 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. Mycopathologia. 2015;180:165–71.
20. L’Ollivier C, Cassagne C, Normand AC, et al. A MALDI-
TOF MS procedure for clinical dermatophyte species
identification in the routine laboratory. Med Mycol.
2013;51:713–20.
21. Packeu A, De Bel A, l’Ollivier C, et al. Fast and accurate
identification of dermatophytes by matrix-assisted laser
desorption ionization-time of flight mass spectrometry:
validation in the clinical laboratory. J Clin Microbiol.
2014;52:3440–3.
22. Theel ES, Hall L, Mandrekar J, Wengenack NL. Dermato-
phyte identification using matrix-assisted laser desorption
ionization-time of flight mass spectrometry. J Clin Micro-
biol. 2011;49:4067–71.
23. Carbonnelle E, Beretti JL, Cottyn S, et al. Rapid identifi-
cation of staphylococci isolated in clinical microbiology
laboratories by matrix-assisted laser desorption ionization-
time of flight mass spectrometry. J Clin Microbiol.
2007;45:2156–61.
24. Pennanec X, Dufour A, Haras D, Réhel K. A quick and easy
method to identify bacteria by matrix-assisted laser des-
orption/ionisation time-of-flight mass spectrometry. Rapid
Commun Mass Spectrom. 2010;24:384–92.
123
Mycopathologia (2017) 182:183–192
25. Chalupová J, Raus M, Sedlářová M, Sebela M. Identifica-
tion of fungal microorganisms by MALDI-TOF mass
spectrometry. Biotechnol Adv. 2014;32:230–41.
26. Hettick JM, Green BJ, Buskirk AD, et al. Discrimination of
Penicillium isolates by matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry fingerprinting.
Rapid Commun Mass Spectrom. 2008;22:2555–60.
27. Welham KJ, Domin MA, Johnson K, Jones L, Ashton DS.
Characterization of fungal spores by laser desorption/ion-
ization time-of-flight mass spectrometry. Rapid Commun
Mass Spectrom. 2000;14:307–10.
28. Tholey A, Heinzle E. Ionic (liquid) matrices for matrix-
assisted laser desorption/ionization mass spectrometry-ap-
plications and perspectives. Anal Bioanal Chem.
2006;386:24–37.
29. Cassagne C, Normand AC, L’Ollivier C, Ranque S, Piar-
roux R. Performance of MALDI-TOF MS platforms for
fungal identification. Mycoses. 2016 Apr. 8. doi: 10.1111/
myc.12506. [Epub ahead of print] Review. PMID:
27061755.
30. Normand AC, Cassagne C, Ranque S, et al. Assessment of
various parameters to improve MALDI-TOF MS reference
spectra libraries constructed for the routine identification of
filamentous fungi. BMC Microbiol. 2013;13:76.
31. Irinyi L, Serena C, Garcia-Hermoso D, et al. International
Society of Human and Animal Mycology (ISHAM)-ITS
reference DNA barcoding database—the quality controlled
standard tool for routine identification of human and animal
pathogenic fungi. Med Mycol. 2015;53:313–37.
32. Martiny D, Cremagnani P, Gaillard A, et al. Feasibility of
matrix-assisted laser desorption/ionisation time-of-flight
mass spectrometry (MALDI-TOF MS) networking in uni-
versity hospitals in Brussels. Eur J Clin Microbiol Infect
Dis. 2014;33:745–54.
33. Robert R, Pihet M. Conventional methods for the diagnosis
of dermatophytosis. Mycopathologia. 2008;166:295–306.
34. Hay R. Literature review. Onychomycosis. J Eur Acad
Dermatol Venereol. 2005;19(Suppl 1):1–7.
35. Hollemeyer K, Jager S, Altmeyer W, Heinzle E. Proteolytic
peptide patterns as indicators for fungal infections and
nonfungal affections of human nails measured by matrix-
assisted laser desorption/ionization time-of-flight mass
spectrometry. Anal Biochem. 2005;338:326–31.