Journal of Medical Microbiology (2015), 64, 25–31 DOI 10.1099/jmm.0.

079962-0

Evaluation of a commercial PCR test for the

diagnosis of dermatophyte nail infections
Anastasia Spiliopoulou,1 Christina Bartzavali,1 Eleni Jelastopulu,2
Evangelos D. Anastassiou1 and Myrto Christofidou1
Correspondence 1
Department of Microbiology, School of Medicine, University of Patras, 26500 Patras, Greece
Myrto Christofidou 2
Department of Hygiene, School of Medicine, University of Patras, 26500 Patras, Greece
christof@med.upatras.gr

Tinea unguium, known as onychomycosis, is a dermatophyte infection of nails with worldwide

Received 19 June 2014
Accepted 18 November 2014
distribution. Conventional methods for detecting fungi in nail specimens are either non-specific
(microscopy) or insensitive (culture). PCR has been used to improve sensitivity in detecting the
causative fungi in nail specimens from patients with suspected onychomycosis. Results of a
commercial multiplex PCR for the detection of dermatophytes, especially Trichophyton rubrum (the
main dermatophyte implicated), as compared to conventional methods are presented. A total of 418
nail scrapings obtained from dermatological outpatients were handled in the Laboratory of
Microbiology between May 2010 and May 2013. Among them, multiplex PCR detected 126
(30.1 %) dermatophyte-positive samples, whereas culture revealed 44 (10.5 %). Direct microscopy
revealed 63 (15.1 %) positive specimens. T. rubrum was identified in 116 out of 126 (92 %) positive
PCR samples and 40 out of 44 (91 %) dermatophyte-positive cultures. Implementation of PCR
increased species-specific detection of dermatophytes by 21.1 %, leading to a threefold increase
as compared to culture alone. Multiplex PCR offers a time-saving diagnostic tool for tinea unguium
and augments laboratory assistance to clinical evaluation for proper treatment.

INTRODUCTION associated species are Epidermophyton floccosum and

Trichophyton verrucosum (Moriarty et al., 2012). In addition
Tinea refers to superficial infection of skin, hair and nails
to dermatophytes, Candida and non-dermatophyte moulds
due to one of three fungal genera, Microsporum, Epidermo-
may be recovered from clinically affected nails; however, their
phyton and Trichophyton, collectively known as dermato-
clinical significance is controversial (Brillowska-Dabrowska
phytes (Clayton & Midgley, 1989; Hay, 1995; Moriarty
et al., 2007). Conventional diagnosis is based on detection of
et al., 2012). These associated infections are among the most
fungal elements by direct microscopy of clinical specimens,
common diseases worldwide and cause serious chronic
followed by culture and morphological identification of the
morbidity. Tinea unguium, a dermatophyte infection of
fungus. The whole procedure is time-consuming, requiring
nails, also known as onychomycosis, has a prevalence of at
10 to 15 days, sometimes up to 3 to 4 weeks, and accuracy
least 12.4 % in the general population of Europe (Moriarty
depends on the expertise of the personnel. Introduction of
et al., 2012), whereas in older individuals it is as high as 50 %
PCR-based methodology could increase sensitivity, specifi-
(Salgo et al., 2003). The disease is often atypical and
city, and speed, and, potentially, even reduce cost in the
aggressive in patients with untreated human immunodefi-
diagnostic approach associated with additional visits to the
ciency virus infection. Patients with psoriasis not only have
clinician and additional sampling and diagnostic tests, and
an increased risk of onychomycosis but also require laboratory
inconvenience for the patient due to the delay in appropriate
confirmation as the two conditions may be clinically similar
treatment (Jensen & Arendrup, 2012). Herein, we pre-
(Moriarty et al., 2012). Trichophyton rubrum is the main
sent the performance of a commercial multiplex PCR
pathogen implicated (Brillowska-Dabrowska et al., 2007; as compared to conventional methods (direct micros-
Tsoumani et al., 2011), followed by Trichophyton inter- copy and culture of nail samples) for the diagnosis of
digitale, formerly Trichophyton mentagrophytes var. inter- onychomycosis.
digitale (Cafarchia et al., 2013; Clayton & Midgley, 1989;
Gräser et al., 1999; Nenoff et al., 2007). Less commonly

Abbreviations: 95 % CI, 95 % confidence interval; DOR, diagnostic odds

METHODS
ratio; LR2, negative likelihood ratio; LR+, positive likelihood ratio; NND, Four hundred and eighteen nail scrapings from an equivalent number
number needed to diagnose; NPV, negative predictive value; PPV, of elderly (50–80 year old) outpatients with clinically suspected
positive predictive value. onychomycosis (nail discoloration, thickening and separation from

079962 G 2015 The Authors Printed in Great Britain 25

A. Spiliopoulou and others
the nail bed) were processed in our laboratory from May 2010 to May
2013. Patients who had used topical or systemic antifungal drugs
within the previous 2 weeks and 6 months, respectively, were
excluded from sampling. The only prerequisite was an amount of
clinical sample of at least 90–100 mg. Specimens were homogenized
and divided into three portions (30–33 mg each). Samples were
examined by direct microscopy of 30 % KOH-treated and lactophenol
cotton blue mounts. Cultures were performed on Sabouraud glucose
agar, Sabouraud agar containing 0.005 % chloramphenicol and 0.5 %
cycloheximide (Mycobiotic), and dermatophyte test medium agar.
Plates were incubated at 26 uC for 30 days before being assessed as
negative due to no growth. Positive cultures were evaluated and
identified at the species level based on the gross morphological
characteristics of the colonies, followed by microscopic examination.
In parallel, the DNA from 30–33 mg nail samples was extracted by a
10 min incubation at 95 uC in 100 ml extraction buffer (60 mM
sodium bicarbonate, 250 mM potassium chloride and 50 mM Tris,
pH 9.5) and, afterwards, 100 ml anti-inhibition buffer (2 % BSA) was
added (Brillowska-Dabrowska et al., 2007, 2010). A multiplex PCR
(Dermatophyte PCR kit; Statens Serum Institut Diagnostica) was
performed following the manufacturer’s instructions. The reagents
consisted of two pairs of primers; the first (panDerm1 59-GAAGAA-
GATTGTCGTTTGCATCGTCTC-39 and panDerm2 59-CTCGAGG-
TCAAAAGCACGCCAGAG-39) targets the chitin synthase-encoding
gene (chitin synthase 1 – chs1) and served for detection of dermatophytes
in general, whereas the second (Trubrum-for 59-TCTTTGAACGCA-
CATTGCGCC-39 and Trubrum-rev 59-CGGTCCTGAGGGCGCTG-
AA-39) targets internal transcribed spacer gene 2 (its2) for the specific
detection of T. rubrum (Brillowska-Dabrowska et al., 2007, 2010).
Amplification was performed, after initial denaturation for 10 min at
95 uC, by 45 cycles of 30 s at 94 uC, 30 s at 60 uC and 30 s at 72 uC.
Afterwards, amplicons (approx. 366 bp for pan-dermatophytes and
203 bp for T. rubrum) were subjected to electrophoresis in 1 % agarose
gel and stained with ethidium bromide. T. rubrum and pan-
dermatophyte DNA were used as positive controls, buffer was used
as a negative control, and the primer mix included an internal plasmid
control that serves as a template for the T. rubrum-specific primers. A
100 bp DNA ladder (Promega) was used as a molecular size marker. A
strong band at 203 bp and a weaker or no band at 366 bp indicated the
presence of T. rubrum, whereas a strong band at 366 bp indicated the
presence of a non-T. rubrum dermatophyte. Multiplex PCR was
completed in 5 h and was applied twice a week. Results of direct
microscopy were available within 1–2 h of handling the specimen.
PCR-negative samples were not considered negative until culture was
completed (30 days). Investigators who assigned the PCR results were
blinded to results of direct microscopy or culture and vice versa. This is
a retrospective study and the Standards for Reporting of Diagnostic
Accuracy (STARD) statement were taken into account (Bossuyt et al.,
2003).
Statistical analysis was performed using IBM SPSS Statistics for
Windows, version 21.0 (released 2012), and MedCalc 2014 (MedCalc
Software). The sensitivity, specificity, negative predictive value
(NPV), positive predictive value (PPV), positive likelihood ratio
(LR+), negative likelihood ratio (LR2), diagnostic odds ratio (DOR)
and number needed to diagnose (NND) with their 95 % confidence
intervals (95 % CIs) for PCR, culture and direct microscopy were
estimated. Statistical significance was achieved if P,0.05. For an
appropriate diagnostic test, desirable values for LR+ and LR2
should be ¢10 and ¡0.1, respectively (Garg et al., 2007). A DOR .1
is indicative of useful tests and higher DORs are indicative of better
test performance. The NND is the number of tests required in order
to gain a positive diagnosis. All diagnostic indices were estimated by
two approaches. In the first approach, the ‘gold’ reference standard
for diagnosis of onychomycosis was defined as the combination of a
positive dermatophyte culture and/or the presence of fungal hyphae
assessed by direct microscopy. The last criterion was considered as
26
positive only if no non-dermatophyte filamentous fungus was
cultured from the sample. In the second approach, the combination
of positive culture and PCR results was considered as the gold
reference standard for diagnosis of onychomycosis.
RESULTS
A total of 418 specimens obtained from dermatological
outpatients with clinically suspected tinea unguium (one
sample per patient) were handled in our laboratory during
a 3 year period. The majority (72 %) originated from
females and 78 % involved toenails. All samples were
processed by conventional methods (direct microscopy and
culture) and multiplex PCR analysis.
Direct microscopy by two independent personnel members
identified 63 (15.1 %) positive samples. Growth of dermato-
phytes was observed in 44 (10.5 %) specimens. T. rubrum
was identified to be the main dermatophyte as it represented
91 % of isolates (40 cases). The remaining four (9 %)
specimens were characterized as T. interdigitale. Both direct
microscopy and culture were positive in 39 cases. Direct
microscopy was positive in 24 dermatophyte-negative
cultures. Direct microscopy was negative in five specimens
that grew dermatophytes in culture. Altogether, the combina-
tion of conventional methods revealed 68 (16.3 %) positive
specimens. Finally, both direct microscopy and culture were
negative for dermatophytes in 350 cases.
However, multiplex PCR was positive in 126 (30.1 %) of all
nail specimens tested. Results of a PCR run are presented in
Fig. 1. T. rubrum was identified in 116 (92 %) samples,
whereas species other than T. rubrum were recognized in
10 (8 %) samples. In total, the combination of culture and
PCR identified 132 (31.6 %) dermatophyte-positive sam-
ples, including 122 T. rubrum and 10 dermatophytes other
than T. rubrum. The variety of combinations between con-
ventional methods and PCR results are presented in Table 1.
The presented data show that implementation of PCR
increases detection of dermatophytes by 21.1 % and leads
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
660 bp
366 bp
203 bp
Fig. 1. T. rubrum-specific and pan-dermatophyte multiplex PCR
product analysis. The bands are: 660 bp, internal control band;
366 bp, pan-dermatophyte band; and 203 bp, T. rubrum-specific
band. Lanes: 1, molecular size marker (100 bp DNA ladder); 2, T.
rubrum-positive sample; 3–10 and 12, negative samples; 11, pan-
dermatophyte-positive sample; 13, positive T. rubrum control; 14,
positive pan-dermatophyte control; 15, negative control.
Journal of Medical Microbiology 64
 Molecular diagnosis of dermatophyte nail infections

Table 1. Combinations of direct microscopy, culture and PCR results in the detection of dermatophytes in 418 nail samples

Direct microscopy Dermatophyte culture PCR [N positive5126 (30.14 %)] Frequency [N (%)]
[N positive563 (15.07 %)] [N positive544 (10.52 %)]

+ + + 38 (9.09)
2 + + 0 (0)
+ 2 + 20 (4.78)
2 2 + 68 (16.27)
+ + 2 1 (0.24)
2 + 2 5 (1.20)
+ 2 2 4 (0.96)
2 2 2 282 (67.46)

to a threefold increase as compared to culture alone
(10.5 % by culture vs 30.1 % by PCR, P,0.001; and 10.5 %
by culture vs 31.6 % by culture and/or PCR, P,0.001).
Similarly, culture by itself identified 33.3 % (44/132) of
laboratory-verified cases (by culture and/or PCR) of tinea
unguium, whereas PCR alone identified 95.5 % (126/132).
As shown in Table 2, T. rubrum was isolated by culture in
six cases where PCR was negative. In these cases, the
internal control was positive excluding the presence of PCR
inhibitors.
The sensitivity, specificity, NPV, PPV, LR+, LR2, DOR
and NND, with their 95 % CIs for PCR, culture and direct
microscopy, are shown in Table 3. By using conventional
methods as the gold standard, PCR demonstrated sensitiv-
ity up to 85.3 %, higher than culture (64.7 %), whereas
specificity was 80.6 %. The NPV was excellent (96.6 %) and
1.52 samples were adequate to achieve onychomycosis
diagnosis. However, the PPV was low (46 %), as positive
PCR results were considered as false positives in cases
where dermatophytes did not grow in culture. By using the
combination of PCR and culture as the gold standard, PCR
was superior exhibiting sensitivity values up to 95.4 %,
whereas culture and direct microscopy reached 33.3 and
44.7 %, respectively. Specificity values and PPVs were 100 %
for PCR and culture as these two tests were considered as the
gold standard, while the respective values were 98.6 and
93.65 % for direct microscopy. PCR performance was
excellent in all diagnostic indices tested.
Finally, growth of yeasts and non-dermatophyte moulds was
observed in 47 (11.2 %) samples (Table 2). In four (1 %)
additional cases, mixed dermatophyte–non-dermatophyte
cultures were obtained (in three cases T. rubrum and non-
albicans Candida and in one case T. rubrum and Peni-
cillium). Candida spp., predominantly non-albicans Candida
(81.5 %), were isolated from 27 (6.5 %) samples, whereas
non-dermatophyte moulds, such as Acremonium, Alternaria,
Aspergillus, Penicillium and Scopulariopsis spp., were recov-
ered in 24 (5.7 %) of them. PCR was negative in 38 out of 51
(74.5 %) non-dermatophyte mould, yeast and mixed posi-
tive cultures (Table 2). Among the remaining 13 (25.5 %)

Table 2. Fungal culture and PCR in 418 cases of suspected onychomycosis

No. of cases (%) Culture PCR

[Total5418 (100 %)]
T. rubrum T. interdigitale Non-dermatophytes T. rubrum Pan-dermatophytes

82 (19.6) 2 2 + (7 cases of 82*) + +/2D

34 (8.1) + 2 + (3 cases of 34d) + +/2D
6 (1.4) + 2 + (1 case of 6§) 2 2
6 (1.4) 2 2 + (3 cases of 6||) 2 +
4 (1.0) 2 + 2 2 +
37 (8.9) 2 2 + 2 2
249 (59.6) 2 2 2 2 2

*Four non-albicans Candida, two Aspergillus, one Penicillium.

DA T. rubrum-positive specimen may give a strong band of 203 bp and a weaker or no band at 366 bp (PanDerm) due to the relatively higher copy
number of its2 as compared to chs1.
dThree non-albicans Candida mixed infection.
§One Penicillium-mixed infection.
||One non-albicans Candida, two Aspergillus.
One Acremonium, two Alternaria, eleven Aspergillus, two Penicillium, two Scopulariopsis and nineteen Candida (fourteen non-albicans).

http://jmm.sgmjournals.org 27
A. Spiliopoulou and others

Table 3. Diagnostic indices of direct microscopy, culture and PCR for onychomycosis evaluation based on (a) direct microscopy and
culture as the gold standard, and (b) PCR and culture as the gold standard

Diagnostic index* Direct microscopy Culture PCR

Evaluation based on direct microscopy and culture as the gold standard

Se (95 % CI) 92.65 (83.66–97.54) 64.71 (52.17–75.91) 85.29 (74.61–92.70)
Sp (95 % CI) 100.00 (98.95–100.00) 100.00 (98.94–100.00) 80.57 (76.03–84.59)
PPV (95 % CI) 100.00 (94.26–100.00) 100.00 (91.88–100.00) 46.03 (37.12–55.13)
NPV (95 % CI) 98.59 (96.74–99.54) 93.58 (90.60–95.84) 96.58 (93.79–98.34)
LR+(95 % CI) +D +D 4.39 (3.47–5.55)
LR2 (95 % CI) 0.07 (0.03–0.17) 0.35 (0.26–0.49) 0.18 (0.1–0.32)
DOR (95 % CI) 8093.36 (442.02–148 186.44) 1273.24 (76.09–21 303.85) 24.05 (11.69–49.48)
NND (95 % CI) 1.08 (1.03–1.19) 1.55 (1.33–1.89) 1.52 (1.36–1.82)
Evaluation based on PCR and culture as the gold standard
Se (95 % CI) 44.70 (36.04–53.59) 33.33 (25.37–42.06) 95.45 (90.36–98.30)
Sp (95 % CI) 98.60 (96.45–99.61) 100.00 (98.71–100.00) 100.00 (98.71–100.00)
PPV (95 % CI) 93.65 (84.52–98.20) 100.00 (91.88–100.00) 100.00 (97.09–100.00)
NPV (95 % CI) 79.44 (74.85–83.52) 76.47 (71.84–80.68) 97.95 (95.58–99.24)
LR+ (95 % CI) 31.96 (11.86–86.13) +D +D
LR2 (95 % CI) 0.56 (0.48–0.65) 0.67 (0.59–0.75) 0.05 (0.02–0.1)
DOR (95 % CI) 56.97 (20.04–161.99) 288.11 (17.56–4726.46) 11 151.46 (623.42–199 469.94)
NND (95 % CI) 2.31 (2.68–2.02) 3.00 (3.60–2.58) 1.05 (1.08–1.02)

Se, Sensitivity; Sp, specificity.

*Sensitivity, specificity, PPV and NPV results represent values per cent.
DCannot be estimated/infinity.

PCR-positive samples, non-dermatophyte cultures were
obtained in 10 (19.6 %), whereas 3 cases concerned mixed
T. rubrum–non-dermatophyte-positive cultures.
DISCUSSION
Results indicate that incorporation of multiplex PCR
techniques in routine laboratory processing of nail scrapings
not only augments detection of dermatophytes, but also, in
the vast majority of cases, identifies the causative agent. In
another study, employment of PCR increased detection of T.
rubrum in nail scrapings by approximately 20 %, whereas
positive T. rubrum culture but negative PCR was found in
3 % of samples (Brasch et al., 2011). In addition, the specific
commercial PCR, used in the present study, was evaluated in
four other studies also (Brillowska-Dabrowska et al., 2007,
2010; Chandran et al., 2013; Kondori et al., 2010). These
studies outline the importance of the dermatophyte PCR kit
use as it increases detection rates by 10 to 19.5 %, as
compared to cultures alone. The amount of extracted DNA
is also an important parameter determining sensitivity of
PCR and has been investigated (Bontems et al., 2009). A
technical difficulty of PCR is the determination of the
appropriate quantity of sample that will reassure a positive
result. Different recommended amounts varying from 1–2
to 30–100 mg are mentioned in the literature (Bontems
et al., 2009; Litz & Cavagnolo, 2010). In our study, the use of
the dermatophyte PCR kit was evaluated in a larger number
of samples (418 specimens), with a fixed large amount of
sample (30–33 mg per sample). PCR performed better as
compared to culture alone or the combination of conven-
tional methods by detecting 19.6 and 13.8 % additional
dermatophyte-positive samples, respectively.
Diagnostic indices for PCR were estimated by two
approaches. The rationale for the above was that despite
the fact that conventional methods are generally acknowl-
edged as the gold standard for the diagnosis of dermato-
phytosis, this approach has raised scepticism among
researchers (Gräser et al., 2012; Jensen & Arendrup, 2012).
PCR assays have demonstrated higher sensitivity and/or
specificity than conventional microscopy and culture.
Although a false-positive PCR result cannot be excluded, it
is more likely that these were false-negative results of the
conventional diagnostic methods (Effendy et al., 2005;
Paugam et al., 2013). In the present study, PCR was found to
be far more sensitive for detection of dermatophytes than
conventional methods. Thus, an alternative approach by use
of a combination of PCR and culture dermatophyte-positive
results as the gold standard stands for a more realistic
assessment of true onychomycosis cases. Similar approaches
have been implemented by other researchers (Garg et al.,
2007; Rothmund et al., 2013). Low PCR diagnostic indices
such as PPV, likelihood ratios and specificity, at a lesser
extent, by the use of conventional methods as the gold
standard can be explained by the lower number of onycho-
mycosis cases identified by culture and/or direct microscopy

28 Journal of Medical Microbiology 64


as compared to culture. The only way to increase the posi-
tivity rate of a low sensitivity test, such as culture, is to
increase the number of analysed samples (Paugam et al.,
2013).
Despite positive PCR, negative culture results (88 cases or
21 % of nail samples) may be explained because of
entrapment of fungus in the keratin; the PCR extraction
step facilitates overcoming this handicap. In addition,
previous treatments, such as local antifungal agents, may
lead to non-viable fungi unable to grow on culture media
(Brasch et al., 2011; Mehlig et al., 2014). In our study, use
of antifungals was an exclusion criterion and cannot explain
the low sensitivity of culture. Nevertheless, PCR will detect
non-viable cells with intact nucleic acid (Bergman et al.,
2013). Finally, overgrowth of non-dermatophyte moulds in
the culture medium may prevent or obscure development of
a pathogen (Bontems et al., 2009). Negative PCR results
despite a positive culture, although rare (6 cases, 1.4 % of
nail samples), may be explained by an imbalanced distri-
bution of fungal elements within different parts of a sample
leading to an insufficient amount of DNA within the material
used (Paugam et al., 2013). Alternatively, the presence of
PCR-inhibitory substances may be involved (Bergman et al.,
2013; Brasch et al., 2011; Mehlig et al., 2014). The presence
of PCR-inhibitory substances is excluded in our study since
internal control was positive in all cases. A limitation inherent
to this multiplex PCR assay is that mixed dermatophyte
infections including T. rubrum cannot be differentiated
from infections with T. rubrum alone. Mixed infections are
relatively rare, but may occur in ~3 % of cases (Paugam et al.,
2013) and this might have negative consequences on the
assessment of dermatophyte epidemiology. The collection of
at least 90–100 mg of nail sample from each patient as an
inclusion criterion requires an advanced stage onycho-
mycosis and results could be different in patients at an early
stage and/or with a limited form of the disease.
Although non-dermatophyte filamentous fungi can be the
aetiological agent of onychomycosis, detection in nail
specimens may be attributed to contamination, transient
colonization and infection of a traumatized or otherwise
diseased nail, mixed infection and persistence after cure of
the dermatophyte or even contamination in the laboratory;
thus, repeated recovery is often required before a pathogenic
role is considered (Bontems et al., 2009; Brillowska-
Dabrowska et al., 2007; Mehlig et al., 2014). The low
number of colonies in cases where Aspergillus, Penicillium,
Scopulariopsis, Acremonium and Alternaria were observed,
along with the fact that direct microscopy was negative and
they were not isolated in a subsequent sample, enhanced
their role as contaminants. Our data show that in ten
samples where culture yielded growth of non-dermatophyte
moulds and yeasts, PCR was positive for T. rubrum (seven)
and other dermatophytes (three). In a previous study, the
specificity of PCR was assessed by using 21 strains of non-
dermatophyte fungi, including Aspergillus niger and yeasts.
In all cases, no PCR products were detected (Brillowska-
Dabrowska et al., 2007). This is reinforced by our study,
http://jmm.sgmjournals.org
Molecular diagnosis of dermatophyte nail infections
where PCR was negative in 38 non-dermatophyte mould
and yeast positive cultures.
Indisputably, culture is necessary for identification of
dermatophytes other than T. rubrum or even non-derma-
tophyte moulds that may be involved in nail infections
(Paugam et al., 2013). However, the low sensitivity of
culture in the diagnosis of onychomycosis is commonly
accepted (Litz & Cavagnolo, 2010; Mehlig et al., 2014).
However, direct microscopy, especially with the addition of
fluorochrome and periodic acid–Schiff staining (Gupta
et al., 2008; Idriss et al., 2013; Litz & Cavagnolo, 2010), has
higher sensitivity but lacks specificity as it cannot provide
identification of species or even at the genus level and does
not differentiate unquestionably between dermatophytes
and other moulds (Bontems et al., 2009; Brillowska-Dabrowska
et al., 2007; Garg et al., 2007; Jensen & Arendrup, 2012;
Mehlig et al., 2014). Taking into account the low sensitivity
of culture-based diagnosis in tinea unguium in combination
with the long turnaround time for growth, as well as the
requirement of experienced personnel, we strongly recom-
mend application of complementary methods. PCR tech-
nology offers rapid results, as well as high sensitivity and
specificity. Hence, a number of PCR-based strategies have
been developed to identify dermatophytes directly from
clinical specimens. Along with conventional or end-point
PCR methods, real-time PCR assays, as well as the imple-
mentation of post-PCR-techniques, have become available.
Conventional PCRs benefit from simplicity and lower cost,
and target the identification of the most common derma-
tophyte, T. rubrum (Brasch et al., 2011), or pan-dermato-
phytes (Dhib et al., 2012; Luk et al., 2012). By means of a
multiplex PCR, simultaneous identification of T. rubrum
and pan-dermatophytes (Brillowska-Dabrowska et al. 2007,
2010; Kondori et al., 2010; Chandran et al., 2013; the present
study) or T. rubrum, T. mentagrophytes and pan-dermato-
phytes (Dhib et al., 2014) is achieved. Moreover, Kim et al.
(2011) identified nine different dermatophyte species, and
Mehlig et al. (2014) identified an assortment of dermato-
phytes along with non-dermatophyte moulds (Scopulariopsis
brevicaulis, Aspergillus spp.) and yeasts (Candida spp.),
whereas Li et al. (2011) distinguished the groups of pathogens
implicated in onychomycosis (dermatophytes, yeasts and
moulds). In a recent work, the primers adopted by the
commercial PCR kit that is evaluated in the present study
were used in two separate PCRs for the detection of pan-
dermatophytes and specific detection of T. rubrum, supple-
mented with a third PCR for T. interdigitale (Dubljanin et al.,
2014). However, real-time PCR is less laborious, may enable a
broader spectrum of simultaneous species detection and as a
closed system reduces contamination risk (Alexander et al.,
2011; Bergman et al., 2013; Bergmans et al., 2010; Miyajima
et al., 2013; Paugam et al., 2013; Wisselink et al., 2011).
Finally, post-PCR strategies, e.g. nested PCR (Verrier et al.,
2013), PCR-ELISA (Beifuss et al., 2011; Pankewitz et al., 2013),
PCR-RFLP (Elavarashi et al., 2013; Ghannoum et al., 2013;
Verrier et al., 2012) and DNA microarray assays (Sato et al.,
2010), may increase the number of species identified;
29
A. Spiliopoulou and others
however, they prolong the turnaround time (Jensen &
Arendrup, 2012). In our settings, specific detection of T.
rubrum in parallel with pan-dermatophyte primers is
sufficient as the vast majority of positive samples were
identified as T. rubrum [40 out of 44 (91 %) by culture, and
122 out of 132 (92.4 %) by the combination of culture and
PCR]. Similar results were obtained in recent studies
(Bergman et al., 2013; Verrier et al., 2012). Nevertheless,
dermatophytes belonging to the less terbinafine-susceptible
genus Microsporum are unanimously reported to be very
rare agents of onychomycosis. Therefore, detection of DNA
of dermatophytes other than T. rubrum in a nail specimen
by the pan-dermatophyte primers will probably represent
infection with a terbinafine-susceptible dermatophyte and
will provide sufficient information to guide the clinician,
despite a lack of species identification (Brillowska-
Dabrowska et al., 2007).
In conclusion, by using a large number of samples, and a
fixed amount of specimen, we have demonstrated that
application of multiplex PCR in clinical settings provides
rapid results with high sensitivity and specificity, augment-
ing laboratory assistance to clinical evaluation of tinea
unguium and saving the patient from delay in initiating an
appropriate treatment.
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