Mycopathologia (2017) 182:193–202
DOI 10.1007/s11046-016-0038-z
Diagnosis of Dermatophytosis Using Molecular Biology
Julie Verrier . Michel Monod
Received: 15 June 2016 / Accepted: 7 July 2016 / Published online: 1 August 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract Identification of fungi in dermatological
samples using PCR is reliable and provides signifi-
cantly improved results in comparison with cultures. It
is possible to identify the infectious agent when
negative results are obtained from cultures. In addi-
tion, identification of the infectious agent can be
obtained in 1 day. Conventional and real-time PCR
methods used for direct fungus identification in
collected samples vary by DNA extraction methods,
targeted DNA and primers, and the way of analysing
the PCR products. The choice of a unique method in a
laboratory is complicated because the results expected
from skin and hair sample analysis are different from
those expected in cases of onychomycosis. In skin and
hair samples, one dermatophyte among about a dozen
possible species has to be identified. In onychomyco-
sis, the infectious agents are mainly Trichophyton
rubrum and, to a lesser extent, Trichophyton interdig-
itale, but also moulds insensitive to oral treatments
J. Verrier
Groupe d’Etude des Interactions Hôte-Pathogène
(GEIHP), Institut de Biologie en Santé (PBH-IRIS), CHU
Angers, Université d’Angers, 4 rue Larrey, 49933 Angers,
France
e-mail: julie.verrier@univ-angers.fr
M. Monod (&)
Laboratoire de Mycologie, Service de Dermatologie,
Centre Hospitalier Universitaire Vaudois, BT403,
1011 Lausanne, Switzerland
e-mail: michel.monod@chuv.ch
used for dermatophytes, which renders fungal identi-
fication mandatory. The benefits obtained with the use
of PCR methods for routine analysis of dermatological
samples have to be put in balance with the relative
importance of getting a result in a short time, the price
of molecular biology reagents and equipment, and
especially the time spent conducting laboratory
manipulations.
Keywords Dermatology  Dermatophytes  Non-
dermatophyte fungi  Tinea corporis  Tinea capitis 
Tinea unguium  Onychomycosis  PCR identification
Introduction
The diagnosis of mycosis in dermatology is currently
based in many laboratories on both direct microscopic
examination of skin, nail or hair samples and cultures.
Direct mycological examination is an essential step to
confirm the clinical diagnosis of fungus infection, but
does not allow the identification of the fungal agent.
Therefore, culture assays are performed in parallel by
seeding skin scales, hair or nail scrapings onto rich
agar medium. A frequently used medium is that of
Sabouraud plus chloramphenicol as an antibiotic. Two
cultures are generally made at the same time, one with
and one without cycloheximide (ActidioneÒ), a com-
pound which is used for dermatophyte selection. Three
recurrent problems occur with cultures: (1) in many
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cases, the difficulty of dermatophyte identification
with certainty, (2) negative results (the culture assays
remain sterile) and (3) the isolation of various non-
dermatophyte filamentous fungi (NDF) such as Fusar-
ium spp., Aspergillus spp., Penicillium spp. and
Alternaria spp., especially from abnormal nails.
Repeated isolations of the same NDF in onychomy-
cosis indicate its involvement in nail infection with
some certainty [1].
The various problems with cultures and the time
necessary to deliver results inherent to the slow growth
of dermatophytes led several groups to develop
reliable and suitable PCR methods to rapidly identify
dermatophytes, yeasts and NDF in skin, hair and nails.
The present communication aims to review various
techniques for the diagnosis of skin mycoses using
molecular biology and to present their advantages, but
also to discuss critical factors for their introduction
into routine use.
Identification of Dermatophyte Species in Cultures
Morphological identification of dermatophyte species
in cultures is sometimes difficult or uncertain because
there are variations from one isolate to another, and
overlapping characters between species. Therefore,
DNA sequences are very useful for accurate determi-
nation. The polymorphism of the internal transcribed
spacers ITS1 and ITS2 flanking the DNA sequence
encoding the 5.8S rDNA is very sensitive and reliable
for distinguishing different species [2–6]. The 28S
rDNA sequences were also found to be suitable for
dermatophyte species identification [7] but less dis-
criminating [8]. Genes encoding topoisomerase II and
chitin synthase 1 were also used as targets to identify
dermatophytes at the species level [9–11]. Dermato-
phyte species identification by sequence comparison is
often problematic using the publicly available NCBI
database. It is plethoric with identical sequences under
different names (especially ITS and 28S sequences) as
the nomenclature of species such as those of the
Trichophyton mentagrophytes complex was a matter
of debate [12, 13]. Strains were also misidentified by
sequence depositors. A way to circumvent this prob-
lem is to directly use the publicly available ITS
sequence database at the Centraalbureau voor Schim-
melcultures dermatophyte website (http://www.cbs.
knaw.nl/dermatophytes/) or to create a database for the
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Mycopathologia (2017) 182:193–202
laboratory. It has to be mentioned that matrix-assisted
laser desorption/ionisation–time-of-flight mass spec-
trometry (MALDI-TOF MS) can now be used for
rapid dermatophyte species identification [14–16]. As
with DNA sequencing, closely related species in the
Microsporum gypseum and T. mentagrophytes species
complexes can be discriminated.
Direct Fungal Identification in Collected Samples
PCR methods for direct identification of the fungus in
collected samples varied from one author to another by
DNA extraction methods, PCR primers and the way of
analysing the PCR products. Primers used were
specific primers to detect only one dermatophyte
species, pan-dermatophyte primers (dermatophyte-
specific primers to detect any but only dermatophyte
species) and pan-fungal primers (primers to detect any
fungal species). The ITS and 28S rDNA sequences and
genes encoding topoisomerase II and chitin synthase
were used as targets. The various molecular methods
developed for direct fungal identification in dermato-
logical samples can be divided into two groups: those
applying conventional PCR and those applying real-
time PCR techniques. Most described methods
focused on dermatophyte detection and species iden-
tification. Few methods for onychomycosis paid
attention to yeasts and NDF as possible infectious
agents [17–20].
DNA Extraction
Fungal DNA extraction from skin, hair and nails was
most often performed after disruption of the keratin
structure. In some protocols, skin scales, hair and nails
were pre-cut into small pieces or mechanically
disrupted [21–30]. Many authors proceeded with
overnight digestion of the dermatological samples
with proteinase K in a lysis buffer at 55 °C, with or
without dithiothreitol (DTT) as a reducing agent
[24, 26, 27, 29, 31–36]. A non-enzymatic disruption
of skin, hair and nail samples could also be efficiently
performed at room temperature in 1 h or overnight
using a Na2S dissolving solution [18, 19, 37]. Prelim-
inary mechanical disruption of the keratin structure
was not performed in this case. DNA was most often
extracted from the prepared dermatological samples
using various commercialised kits, either manually or
Mycopathologia (2017) 182:193–202
using a robot [26, 28, 33]. DNA extraction with
phenol/chloroform/isoamyl alcohol was performed in
some protocols [17, 22, 23, 31, 38, 39]. A fast patented
15-min two-step buffer method has been described by
Brillowska-Dabrowska et al. [40–42], and a Dermato-
phyte PCR Kit (SSI Diagnostica, Hillerød, Denmark)
based on this method is now commercially available.
DNA was extracted by a 10-min incubation at 95 °C in
a buffer made of 60 mM sodium bicarbonate
(NaHCO3), 250 mM potassium chloride (KCl) and
50 mM Tris (pH 9.5). After the addition of a second
buffer containing 2 % bovine serum albumin (anti-
inhibition buffer) and vortex mixing, the DNA-
containing supernatant was ready for PCR. Compar-
ison of the efficiency of the various methods described
in the literature for DNA extraction from dermatolog-
ical samples is lacking. However, time consumption
varies a lot from one protocol to another. This is a
limiting factor which has to be considered for routine
usage.
Conventional PCR Techniques
Several methods aimed to detect one particular species
with specific primers. The detection of an amplicon of
particular size in an agarose gel allowed identification
of Trichophyton rubrum in nails [41, 43] and Mi-
crosporum canis in human and animal skin without
further analysis [42]. Multiplex PCR was performed
by adding species-specific primer pairs to the same
PCR mix [30, 35, 40]. Pan-dermatophyte primers were
added for the detection of a dermatophyte of any
species.
One or several post-PCR steps were performed to
increase sensitivity of dermatophyte detection in
dermatological samples and to identify infectious
fungi at the species level from amplicons obtained
with pan-fungal primers. The disadvantages of post-
PCR analysis are more manipulation, the risk of
contamination associated with the processing of PCR
products and the prolonged time to get a diagnostic
response. A simple method to increase sensitivity of
fungal detection with the inconvenience of a high risk
of contamination was to perform nested or semi-
nested PCR [22, 44, 45].
Restriction fragment length polymorphism (RFLP)
was performed to discriminate and identify selected
infectious fungal species in dermatological samples.
This technique was used to identify Trichophyton spp.
195
and Scytalidium spp. [38, 46] or Trichophyton spp.,
Fusarium spp. and Aspergillus spp. in onychomycosis
[18, 37]. The identification of two different species in
mixed nail infections was possible by interpreting
band profiles of agarose gels. Alternatively, amplicon
sequencing was revealed to be very efficient for
identification of the infectious agent in onychomyco-
sis [37, 45]. The trace file was readable in 80–90 % of
the cases when direct mycological examination was
positive. DNA extracted from nails with mixed
infections generated trace files with superimposed
signals (more than one peak per position), and
sequencing results were not suitable for fungal iden-
tification (Fig. 1) [45]. In contrast, amplicon sequenc-
ing was revealed to be not sensitive enough for
dermatophyte identification in clinical samples of
tinea capitis and tinea corporis [8]. Therefore, a nested
PCR step with pan-dermatophyte primers had to be
performed for dermatophyte identification.
A PCR terminal restriction fragment length poly-
morphism (PCR-TRFLP) assay was developed to
identify Trichophyton spp. as well as 12 non-dermato-
phyte species in onychomycosis including cases with
mixed infections [19] (Figs. 2, 3). Mixed infections
were detected in roughly 10 % of onychomycoses. In
addition, rare or new infectious agents were revealed.
TRFLP is a DNA fingerprinting technique used to
investigate the composition of microbial communities
in different ecological systems such as soil and water
[47–50]. In medicine, this technique has been used to
characterise the oral bacterial flora in saliva from
healthy subjects and patients with periodontitis [51].
The PCR-TRFLP assay can be automated and is a
method of choice for the rapid diagnosis of a large
number of clinical specimens.
With the same objective to identify dermatophytes
and NDF in onychomycosis, a pan-fungal PCR
microarray assay was developed using a specific
probe for the detection of 26 clinical relevant fungi;
17 % of clinical samples were found to give signals for
more than one species [17]. The prevalence of mixed
infections was found in the range revealed using
TRFLPs [19]. The results of fungal identification in
onychomycosis obtained by microarrays and TRFLP
are representative of the infection at the time when the
nail sample was collected.
A PCR-ELISA method was developed to increase
sensitivity of dermatophyte detection in infected skin
and nails [27]. PCR amplifications were first performed
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Fig. 1 Identification of infectious fungi in onychomycosis
following sequencing trace files of 28S rDNA amplicons.
A Trichophyton sp. B Mixed infection with Trichophyton sp.
and Fusarium sp. A superimposition of two sequences was
observed, and fungi cannot be identified by basic local
with five species-specific digoxigenin-labelled primers
targeting the topoisomerase II gene of five common
dermatophytes (five different reactions). The PCR
products were subsequently hybridised with 50 -biotiny-
lated species-specific oligonucleotide probes. The
hybridised DNA was transferred into microwells coated
with streptavidin, and the amplified DNA was revealed
using anti-digoxigenin-horseradish peroxidase antibod-
ies and peroxidase substrate. ELISA-based detection
was found to be ten times more sensitive than
conventional gel electrophoresis. A commercial PCR-
ELISA kit (Onychodiag) using a similar protocol was
launched on the market and evaluated to detect
dermatophytes in onychomycosis [52], but is no longer
commercially available. ELISA methods request too
many manipulations in comparison with other PCR
methods to be of real interest for routine use.
Real-Time PCR Techniques for Dermatophyte
Identification in Dermatological Samples
Various real-time assays were developed to detect
one or several dermatophyte species in clinical
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Mycopathologia (2017) 182:193–202
alignment search tool (BLAST) analysis. However, careful
manual analysis of the trace file allows the detection of F.
oxysporum (upper line; main peaks) and T. rubrum (bottom line;
smaller peaks)
samples. The advantages of real-time PCR assays
are the use of a closed-tube system with the
automation of the reaction results. In this way,
without post-PCR analysis, the risk of contamina-
tion is limited. The first developed assay only
targeted T. rubrum in onychomycosis [32]. To
identify both T. rubrum and T. mentagrophytes, but
also the presence of a possible NDF in onychomy-
cosis, an assay was developed with the use of
specific Trichophyton primers and pan-fungal pri-
mers [31]. Specific probes for T. rubrum and T.
mentagrophytes and a pan-fungal probe were used.
Otherwise, all assays were designed for the simul-
taneous detection of several dermatophyte species,
without considering NDF as a possible infectious
agent. Discrimination of 11 dermatophyte species
could be performed in a single-tube real-time PCR
by melt-curve analysis [24]. Other assays consisted
of either two multiplex PCRs [26, 33] or multi-tube
reactions (one reaction detects a species or a species
complex) [36]. A pan-dermatophyte reaction was
included to detect the presence of any dermatophyte
whatever the species.
Mycopathologia (2017) 182:193–202
Fig. 2 PCR-TRFLP assay for fungal identification in ony-
chomycosis. 28S rDNA extracted from nail samples was
amplified using pan-fungal primers. One of these primers was
labelled by a dye. The amplicons were subsequently digested by
Reliability and Advantages of PCR Methods
Identification of fungi in dermatological samples
using PCR is reliable and provides significantly
improved results in comparison with cultures. It was
possible to identify the infectious agent in many cases
where negative results were obtained from fungal
cultures, but direct nail mycological examination
showed fungal elements [19, 26, 36]. The identifica-
tion of a dermatophyte in almost all cases where T.
rubrum or Trichophyton interdigitale grew in cultures
validated the assays. In addition, moulds such as
Fusarium spp., Acremonium spp., Aspergillus spp. or
Scopulariopsis spp. were identified with certainty as
the infectious agents of onychomycosis and not as
transient contaminants. The specificity of culture
results relative to PCR techniques could be assessed
when these NDF grew from samples positive by direct
mycological examination [19]. Identification of the
infectious agent can be obtained on the day when
sample reaches the laboratory or the next day, whereas
results from fungal cultures can take as long as
1–3 weeks.
197
selected restriction enzymes so that an infectious fungus could
be identified by the length of the labelled fragment (fungal
signature). The PCR products were separated using a DNA
analyser
Importance of Fungal Identification
in Dermatological Samples
Identification of the fungus in situ revealed that either
Fusarium sp., Acremonium sp. or Aspergillus sp. was
the unique infectious agent in many cases of ony-
chomycosis which did not respond to multiple
systemic treatments with terbinafine and itraconazole
[53]. Neither T. rubrum nor T. interdigitale was
identified, and insensitivity to treatment could not be
attributed to resistant dermatophyte strains. Although
the efficacy of standard oral treatments with terbina-
fine and/or azoles is proven against dermatophytes in
onychomycosis, these treatments are useless when an
NDF is the infectious agent, and an alternative therapy
should be prescribed. For instance, topical ampho-
tericin B was revealed to be efficacious, safe, cheap
and easy to apply in cases of NDF onychomycosis
[54]. The efficiency of photodynamic therapy was
reported in a case of Fusarium onychomycosis [55].
Dermatophyte identification is also particularly
useful for tinea capitis, because effective treatment
depends on the incriminated dermatophytes [56, 57].
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Ladder PET 12 010 2010-10-19.fsa Ladder PET 12 None

60 100 140 180 220 260 300

24000
AsV AcA AsF AcS
18000
FO Al/Cu FS
T P S
12000
Ca
6000

3520 2010 036 2011-02-03.fsa 3520 2010 None

30000
60 100
* 140 180 220 260 300

Trichophyton sp.
24000

18000

12000

6000

2437 2009 031 2011-02-03.fsa 2437 2009 None

25000
60 100 140
* 180 220 260 300

Fusarium oxysporum
20000

15000

10000

5000

PCR E 044 2011-03-17.fsa PCR E None

60 100
* 140
* 180 220 260 300

27000 Trichophyton sp.

21000 + Fusarium oxysporum
15000

9000

3000

PCR 014 009 2011-03-01.fsa PCR 014 None

60 100
* 140 180 220
* 260 300

27000 Trichophyton sp.

21000
+ Scopulariopsis brevicaulis
15000

9000

3000

Fig. 3 Identification of nail infectious fungi in onychomycosis
by TRFLP analysis. Fragments of the reference ladder were
labelled with Red-ATTO565. Fragments used to discriminate
infectious fungi in onychomycosis by TRFLP analysis were
labelled by another dye (Yellow-ATTO550, printed in black). In
this example, 11 fungi were discriminated: Trichophyton spp.
(T), Aspergillus versicolor (AsV), Candida sp. (Ca), Fusarium
oxysporum (FO), Penicillium citrinum (P), Acremonium alter-
natum (AcA), Alternaria spp./Curvularia sp. (Al/Cu), Scopular-
iopsis brevicaulis (S), Aspergillus flavus (AsF), Acremonium
strictum (AcS) and F. solani (FS). Mixed infections were
highlighted by multiple peaks

123
Mycopathologia (2017) 182:193–202
In cases of highly inflammatory dermatophytosis in
humans, it is important to identify with certainty the
precise etiologic agent and identify the possible source
of infection, which is often a pet. Adequately treating
an affected pet and its environment can help in the
prevention of recurrence or new infections, especially
in children. In contrast, dermatophyte identification is
not imperative in most cases of tinea corporis, as these
mycoses respond well to standard topical treatments,
regardless of the incriminated species.
Critical Points for Routine Use of PCR Techniques
PCR methods to identify fungi in dermatological
samples are definitely attractive. However, various
points have to be considered before their introduction
in a laboratory.
Heterogeneity of the Collected Samples
and Expected Results
PCR assays are suitable for direct fungal identification
in dermatological samples, provided that enough skin,
hair or nail material is collected for analysis. This is
not always the case. Only a small amount of material is
generally collected for mycological analysis of skin
and hair. In the case of onychomycosis, the collected
material varies a lot, and automation becomes prob-
lematic for this reason. When it is abundant or highly
positive by direct mycological examination, a larger
amount of DNA can be extracted and fungal identi-
fication can be routinely performed using a single-step
PCR assay [19, 26]. It also has to be taken in account
that the expected results of PCR fungal identification
in skin and hair mycoses differ from that in ony-
chomycosis. In skin and hair samples, one dermato-
phyte species among about a dozen has to be
identified. In nail samples, either T. rubrum or T.
interdigitale (or in rare cases other anthropophilic
species such as T. soudanense) has to be identified.
However, the infectious agent can be an NDF.
Risk of Contamination
One should be aware of possible laboratory contam-
ination risk generating false-positive results. In par-
ticular, precautions have to be taken when methods
involve the processing of PCR products, especially if a
199
PCR product is re-amplified by nested PCR or semi-
nested PCR. All manipulations of PCR products using
post-PCR techniques should be performed in a sepa-
rate room. The risk of contamination is more limited
using real-time PCR methods.
Result Interpretation with Sensitive Methods
PCR methods can be too sensitive and detect a
transient contaminant rather than an infectious agent.
For instance, in some cases only a T. rubrum sequence
was detected by sequencing an amplicon obtained
using pan-fungal primers, while Trichophyton sp. and
Fusarium sp. were detected by specific direct PCR
[45]. One should also be aware of the existence of T.
rubrum asymptomatic carriers [58]. Therefore, diag-
nosis of dermatophytosis should always be made
taking in account direct mycological examination,
cultures and PCR results.
Relative Importance of Getting a Result in a Short
Time
There is unanimity for claiming that identification of
the infecting agent in dermatological samples can be
obtained in 24 h or less using PCR, whereas results
from fungal cultures can take 1–3 weeks. However, it
is necessary to stress that there is no emergency to
initiate treatment in nails. In any case, the minimum
time reported by authors to get a PCR result is 5 h [40],
and the patient will not wait this time in the hospital or
the doctor’s consulting room to get a prescription (!).
In most cases of skin dermatophytosis, a topical
treatment can be administrated simply based on results
of direct mycological examination which takes 5 min.
In cases of hair dermatophytoses, clinical signs and
knowledge of the patient’s history and environment
(pet owner, rural location) can be helpful for the
orientation of a diagnosis. Highly inflamed lesions in
humans are caused by zoophilic and geophilic species
of dermatophytes [e.g. Trichophyton benhamiae (for-
mally Arthroderma benhamiae), T. mentagrophytes
(also named Arthroderma vanbreuseghemii), Tri-
chophyton erinacei, Trichophyton verrucosum,
M. canis and M. gypseum]. In contrast, anthropophilic
species (e.g. T. rubrum, T. interdigitale and T.
violaceum) tend to be associated with more chronic
or persistent less inflammatory infections. However, in
some exceptional cases of highly inflammatory
123
200
dermatophytosis in humans, especially in cases of
tinea capitis, the medical doctor can request that the
laboratory obtains the precise etiologic agent in order
to prescribe an adequate treatment.
Cost and Importance of the Number of Samples
Analysed per Week/Day
The cost for a PCR result including DNA extraction,
PCR and PCR product analysis remains affordable (in
the range of 10–15 euros). However, this amount does
not comprise the time spent by the technician to process
the material. Time consumption, in particular for DNA
extraction, has to be considered. To gain efficiency,
robots for DNA extraction can be used. Using manual
kits, 20 nail DNA extractions can be simultaneously
and easily made in 1 h by the same person. Post-PCR
techniques such as amplicon sequencing and RFLP
require few manipulations and are less time-consuming
than techniques using ELISA and microarrays.
Although suitable for clinical studies, the two latter
techniques are difficult to apply for routine use.
Economy can be achieved by establishing a molecular
diagnostic platform and/or by grouping the analyses to
be performed (for instance, once a week) as the results
are not urgent. It is not practically possible to recruit one
technician especially for one or few analyses a day.
The Choice of Methods and Procedures in Routine
Analysis
The choice of a PCR method to analyse dermatolog-
ical samples depends on the expected results and, in
particular, on the importance given to the identifica-
tion of NDF in onychomycosis. The benefits have to be
put in balance with the relative importance of getting a
result in a short time, the price of molecular biology
reagents and equipment, and the time spent conducting
laboratory manipulations. Numerous methods were
described for identifying the infectious agent in skin,
hair and nails, but many were tested on a small number
(N \ 100) of clinical samples. It is generally not
known which techniques are really routinely used, if
these techniques were preliminary investigations or if
they were used only for a particular clinical study.
The introduction of an ‘all PCR’ procedure as
routine is tempting, but such a procedure is difficult in
practice because of the heterogeneity of collected
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Mycopathologia (2017) 182:193–202
samples. For instance, 830 received samples out of
2267 were not retained due to a lack of material by
Wisselink et al. [26] in testing such a procedure where
real-time PCR results were compared to cultures. The
large ratio of negative PCR results was in the range of
50 % since the samples were not selected by direct
mycological examination. Fluorescence microscope
techniques are very sensitive for detecting rare hyphae
and spores in dermatological samples (Fig. 4) [59–62],
and the percentage of false-negative results is low in
comparison with culture results [45]. The number of
negative PCR results could be strongly reduced by
analysing samples that are previously shown to be
positive by direct mycological examination. In contrast
to ‘all PCR’ procedures, PCR can be used in comple-
ment to direct mycological examination and cultures.
Specific detection of a dermatophyte species and
amplicon sequencing after pan-fungal PCR are suit-
able for routine application for a large number of
samples. With the introduction of a strict ‘all PCR’
procedure as routine at the expense of cultures,
diagnostics could be performed by laboratory practi-
tioners unable to recognise common dermatophytes and
NDF in cultures. This education problem should not be
underestimated. It is also useful for the laboratory to
inform the clinician that a sufficient amount of material
(give informative data for the different types of
samples) should be sent allowing to perform a complete
and pertinent diagnostic procedure including direct
examination, culture and PCR if needed.
Few laboratories use PCR to identify fungal infec-
tious agents in dermatological samples. Laboratories
Fig. 4 Direct mycological examinations of clinical samples
using fluorescence microscopy showing hyphae in infected nails
Mycopathologia (2017) 182:193–202
using PCR techniques as routine are pioneers. There-
fore, it is important that the experience acquired in
these laboratories is shared to offer a choice of methods
and procedures in other laboratories.
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