mycoses Diagnosis,Therapy and Prophylaxis of Fungal Diseases

Original article

Multiplex PCR assay for the detection of common dermatophyte

nail infections

I. Dhib,1 A. Fathallah,1 A.Yaacoub,1 F. Hadj Slama,2 M. B. Said1 and R. Zemni2

1
Parasitology-Mycology Laboratory, Farhat Hached Hospital, Sousse, Tunisia and 2Medicine Faculty, Immunology and Genetic Laboratory, Sousse, Tunisia

Summary Onychomycosis is one of the most prevalent dermatophytic diseases. Mycological

methods used in the conventional diagnosis may not be optimal. Multiplex (MX)
PCR was reported as a reliable alternative. Dermatophyte gene sequence records
were used to design a MX PCR for detection and identification of dermatophytes in
nail specimens. A MX PCR method based on the amplification of the chitin synthase
1 and internal transcribed spacer genes was developed. The study included 93
strains of dermatophytes and non-dermatophytic fungi, six dermatophytic reference
strains and 201 nail specimens from patients with dermatophytic onyxis. DNA
extraction directly from nail samples was carried out by using the QIAamp DNA
extraction kit (Quiagen). A set of primers was designed and their specificity was
assessed. MX PCR detected the causal agent in specimens from which Trichophyton
rubrum and T. interdigitale grew in culture and also identified a dermatophyte species
in an additional 32 specimens that were negative in microscopy and culture. None
of the investigated non-dermatophytic strains was positive. Sensitivity of MX PCR
was higher as compared to mycological examination (97% vs. 81.1%). MX PCR for
direct detection of dermatophytes from nail samples yielded mixed flora in 32.8% of
samples. MX PCR proved sensitive and adequate for the diagnosis of dermatophytic
onychomycosis. It is much adapted to cases where culture is negative or contami-
nated by overgrowing moulds, which makes the identification of the causal agent
problematic.

Key words: Multiplex PCR, chitine synthase 1 gene, ITS gene, onychomycosis, dermatophytes.

particularly in terms of whether or not it is a dermato-

Introduction
Onychomycosis is the most common nail disease. It is
mainly caused by dermatophytes of the genus Tricho-
phyton. However, various non-dermatophyte filamen-
tous fungi are often isolated from nails and usually
considered as transient contaminants and are not the
actual aetiological agent. Treatment of onychomycosis
is closely linked to the identity of the causative agent,
Correspondence: I. Dhib, Faculty of Medicine, Parasitology-Mycology Lab-
oratory, Mohamed El Karoui Street, 4002 Sousse, Tunisie.
Tel: +216 73 222 600. Fax: +216 73 224 899.
E-mail: dhib.imen@yahoo.fr
Submitted for publication 5 April 2013
Accepted for publication 7 May 2013
phyte.1 Onychomycosis is routinely diagnosed by
mycological examination, which includes direct exami-
nation and culture.2
Direct microscopic examination of infected nail
material may give false positive results as it is usually
enable to differentiate between dermatophyte and non-
dermatophyte filamentous fungi. Conventionally the
definitive diagnosis is based on culture isolation, but
culture of toenails is often associated with contami-
nants. Furthermore, morphological and physiological
characteristics may vary; the phenotypic features can
be influenced by factors such as temperature variation
and medium.3
Routine identification by microscopy and culture
requires considerable training of personnel and consid-
erable supervisory expertise. Molecular approaches

© 2013 Blackwell Verlag GmbH doi:10.1111/myc.12096

I. Dhib et al.

have been developed to provide more rapid and accu-
rate alternatives for dermatophyte identification. These
methods include restriction fragment length polymor-
phism analysis RFLP,1,4,5 PCR-ELISA,6 double-round
PCR,7 nested PCR,8,9 real-time PCR,10,11 PCR using
(GACA)4 primer,12 sequencing.13,14 The main targets
have been the following genes or DNA fragments: the
ribosomal DNA region,1,3,10,13,15,16 DNA topoisomer-
ase II genes,6 actin gene7 and the chitin synthase
gene.8,14,17,18
In recent years, the multiplex (MX) PCR assay has
been used to identify a great variety of fungi including
dermatophytes.15–21 However, Trichophyton mentagro-
phytes complex was not included in these studies
despite its high frequency in nail infection.14,22,23 The
primary aim of this study was to design and develop a
MX PCR assay to identify dermatophytes on one hand
and T. rubrum and T. mentagrophytes complex on the
other hand directly from nails samples. The second
aim was to evaluate the MX PCR in comparison with
current standard diagnostic methods in patients with
suspected onychomycosis.
of macroscopic and microscopic characteristics of the
colonies.
Fungal strains
To optimise the PCR protocol and the specificity of the
primers, 70 strains mycologically identified as derma-
tophytes including 23 T. rubrum (TR), 35 T. mentagro-
phytes (TM) and five other species obtained from nail
scrapings, skin and hair fragments from patients with
dermatophytosis were included. In addition, six refer-
ence strains, 30 non-dermatophyte fungal strains
(moulds and yeast) and 2 human DNA specimens
were tested (Table 1). Reference strains were pur-
chased from the Central Bureau voor Schimmelcul-
tures (CBS, Utrecht, the Netherlands).
DNA extraction
DNA was extracted from nail material by using the
QiaAmp DNA extraction Kit (Qiagen, Venlo (Pays Bas),
Germany). Prior to the extraction, whole nails or
relatively large nail fragments weighing between 3 and

Materials and methods Table 1 Micro-organisms from cultures used in the study.

Reference strains N of clinical

Samples investigated Microorganisms (N of strains tested) isolates

The study was conducted in the Parasitology–Mycol- Dermatophytes

ogy laboratory of Farhat Hached hospital (Sousse, Epidermophyton floccosum CBS 358.93(1) –
Tunisia). The investigated patients were addressed to Microsporum canis CBS 132.88(1) –
Trichophyton interdigitale CBS 165.66(1) 35
the laboratory by the service of Dermatology of the
Trichophyton mentagrophytes CBS 106.67(1) –
same hospital and to a lesser extent by private Trichophyton rubrum CBS 494.62(1) 23
practitioners. Trichophyton violaceum CBS 374.92(1) –
Two sample collections were investigated in this Trichophyton schoenleinii – 1
study: Trichophyton tonsurans – 1
Trichophyton verrucosum – 1
Microsporum ferrugineum – 1
Clinical nail samples Microsporum gypseum – 1
Two hundred and eighty-one nail specimens were Other fungi
investigated. They include the following: (i) 201 Alternaria sp. 2
samples collected from patients with suspected ony- Aspergillus sp. 4
Acremonium sp. 2
chomycosis and (ii) 80 nail specimens obtained from
Chrysosporium sp. 2
healthy individuals with no clinical nor mycological Cladosporium sp. 1
evidence of onychomycosis and considered as negative Fusarium sp. 1
controls. Paecilomyces sp. 1
All collected samples were divided into three Penicillium sp. 2
Scopulariopsis sp. 1
portions. The first portion was examined microscopi-
Scytalidium sp. 1
cally in 30% KOH for the presence of fungal elements. Candida albicans 4
The second was cultured on Sabouraud dextrose agar C. tropicalis 2
supplemented with chloramphenicol and/or cyclohexe- C. parapsilosis 4
mide at 27 °C for up to 4 weeks. The third portion of C. krusei 2
Trichosporon sp. 1
the nail specimen was used for PCR analysis. Clinical
Human DNA 2
isolates were identified at the species level on the basis

2 © 2013 Blackwell Verlag GmbH


5 mg, were cut into small pieces with a surgical blade.
Subsequently, nucleic acid extraction was performed
according to the manufacturer’s instructions. At the
end of the procedure, the DNA pellet was dissolved in
50–70 ll hydration solution, depending on the amount
of the nail material used at the beginning. A quantity of
2 µl of DNA was added to the PCR mixture.
To extract DNA from fungal cultures, we used the
rapid mini preparation method described by Liu et al.
[24] In brief, a small lump of mycelia (1 cm2 of diame-
ter) was added to 500 ll of lysis buffer (Tris–HCl,
EDTA, NaCl, SDS) and 150 ll of potassium acetate. The
tube was vortexed and an equal volume of isopropyl
alcohol was added to the supernatant. Then, the mix-
ture was centrifuged and the resultant DNA pellet was
washed in 70% ethanol and dissolved in 50 ll Tris–
EDTA. Extracted DNA was kept at 20 °C until use. A
quantity of 2 µl of DNA was added in PCR mixture.
Primers design and specificity
The nucleotide sequences of the different dermatophytes
were selected from the NCBI nucleotide database. A set
of primers detecting a DNA fragment encoding chitin
synthase 1 gene was designed. The primers amplify a
432 bp DNA fragment. To specifically amplify T. rubrum
and T. mentagrophytes, we aligned the two reference
sequences (T. rubrum: Z97993, T. mentagrophytes:
Z98000) of the internal transcribed spacer ITS and we
chose two sets of specific primers in the site where the
sequences were divergent.
The selected primers and their PCR product size are
shown in Table 2. The primers consisted of the follow-
ing: Derm primers that amplify all dermatophyte spe-
cies, TR primer and TM primer that specifically
amplify T. rubrum and T. mentagrophytes respectively.
Before the MX assays were set up and to optimise the
specificity of the primers, 23 T. rubrum and 35 T.
Table 2 Primer sequences, priming regions, annealing temperatures and target amplicon size for multiplex PCR.
Primer Nucleotide sequence Annealing T
name 5′? 3′ Ta°
Derm F GAA GCC TGG AAG AAG ATT GTC G 60°
Derm R CCT TGA TTT CAC CGC AGG CAC
TRF CCC CCC ACG ATA GGG ACCG 62°
TRR GAC TGA CAG CTC TTC AGA GAA TT
TMF GCC CCC CAC GAT AGG GCC AA 64°
TMR CTC GCC GAA CGG CTC TCC TG
TR, Trichophyton rubrum; TM, Trichophyton mentagrophytes; ITS, internal transcribed spacer.
Derm F and Derm R amplify all dermatophyte species.
© 2013 Blackwell Verlag GmbH
Multiplex PCR for detection of dermatophytes
mentagrophytes strains were tested in a species-specific
PCR by using separately the TR and TM primers
amplifying 214 and 132 bp fragments respectively.
After verification of the specificity of each set, we per-
formed a MX PCR using the three primers in the same
reaction.
MX PCR amplification
Multiplex PCR was performed on DNA extracts from all
fungal isolates under the following conditions: the ampli-
fication reaction was performed in a total volume of
50 ll; the PCR mixture contained 10 ll of 59 reaction
buffer (GoTaq DNA buffer; Promega, Madison, WI,
USA), 0.5 ll of 25 mmol l 1 desoxynucleoside triphos-
phates containing an equimolar mixture of dATP, dCTP,
dGTP and dTTP (Promega), 1 ll (30 lmol l 1) of each
primer, 1.25 unit of GoTaq DNA polymerase (Promega)
and 50 ng of template DNA.
Samples were amplified through 30 cycles in a ther-
mocycler (Thermolyne Amplitron II Series 1091, Barn-
stead Thermolyne Corporation, Dubuque, IA, USA) as
follows: initial denaturation for 5 min at 95 °C, dena-
turation for 30 s at 94 °C, annealing for 30 s at
60 °C and extension for 30 s at 72 °C. This was fol-
lowed by a final extension step for 10 min at 72 °C.
PCR products were separated on 2% agarose gel,
stained with ethidium bromide and visualised under
an UV illumination. Appropriate positive and negative
controls were included in every amplification.
Sensitivity of the single-primer-set PCR and MX PCR
assays
Analytical sensitivity was determined using serial dilu-
tions (starting from 5 pg up to 50 pg per reaction) of
purified DNA extracted from the two reference targets:
T. rubrum CBS 494.62 and T. interdigitale CBS 165.66.
Gene PCR product Results of single- primer-set
region (bp) PCR from fungal cultures N (%)
Chitine 432 58/58 (100)
synthase
gene 1
ITS gene 214 23/23 (100)
ITS gene 132 35/35 (100)
3
I. Dhib et al.

DNA was extracted from pure cultures as described by
Liu et al. [15].
Specificity of the MX PCR assay
Common dermatophytes, reference strains, non-der-
matophytic moulds, yeast and human DNA were used
to determine the specificity of the MX PCR (Table 1).
Data from mycological test and MX PCR were com-
pared using analysis of chi-squared test as appropriate.
The level of statistical significance was set at
P < 0.05.
nails, non-dermatophytic species or human DNA with
the three sets of oligonucleotides (Derm, TR and TM).
However, the designed TR primers also detected the
closely related T. violaceum species (from reference
strain and culture). When primer pairs for TM were
used, a cross-amplification occurred with T. schoenlei-
nii, T. verrucosum and T. tonsurans DNA known to
include highly similar ITS sequences and related
taxonomic features (Fig. 2).
For Epidermophyton floccosum, Microsporum canis, M.
gypseum, M. ferrugineum the MX PCR yielded amplifi-
cation with only Derm primers (Fig. 2).

Results Evaluation of single-primer-set PCR and MX PCR from

Sensitivity and specificity of the single-primer-set PCR
and MX PCR
Figure 1 shows PCR results with Derm, TR and TM
primers by using serial dilution of extracted DNA;
starting from 5 pg up to 50 pg per reaction. The low-
est concentration of DNA that gave a positive MX PCR
result for all the investigated dermatophyte species
was 50 pg in a PCR volume of 50 ll. Regarding speci-
ficity, no amplification was obtained from healthy
fungal cultures
Extracted DNA from 58 clinical dermatophyte isolates
including 23 TR and 35 TM strains was used for the
evaluation of Derm, TR and TM primers separately
and then in a MX PCR assay (Fig. 3).
When tested separately in the TR specific PCR, all T.
rubrum DNA samples (100%) yielded the expected
214 bp product. No PCR product was detected when
the 23 T. rubrum samples were tested in the TM spe-
cific PCR.

M 1 2 3 4 5 6 7 8 9 10 11 12
Figure 1 Results of single-set-primer PCR
with serial dilutions of the DNA extracted
from Trichophyton rubrum TR CBS 494.62
and T. interdigitale TI CBS165.66. M, size
markers (50 bp DNA ladder); lanes 1–3,
Derm PCR with TR; 1, 50 pg; 2, 30 pg;
3, 10 pg; lanes 4–7, TR PCR; 4, 50 pg;
5, 30 pg; 6, 10 pg; 7, 5 pg; lanes 8–12,
TM PCR; 8, 50 pg; 9, 30 pg; 10, 10 pg;
11, 5 pg; 12, 1 pg.

(a) (b)
M 1 2 3 4 5 6 7 8

Figure 2 Multiplex PCR results for differ-

ent species of dermatophytes (reference
400pb strains and clinical isolates). (a) M,
100 pb; line 1, Trichophyton rubrum RS;
200pb
line 2, T. mentagrophytes; line 3, T. viola-
100pb ceum RS; line 4, Microsporum gypseum;
line 5, Candida albicans; line 6, Microspo-
rum ferrugineum; line 7, Microsporum
canis RS; line 8, T. verrucosum. (b) M,
100 pb; line 1, T. interdigitale RS; line 2,
T. schoenleinii; line 3, T. tonsurans; line 4,
Arthroderma benhamiae RS.

4 © 2013 Blackwell Verlag GmbH

 Multiplex PCR for detection of dermatophytes

M 1 2 3 4 5 6 7 8 9 10 11 12 C–

Figure 3 Results of multiplex (MX) PCR

and Derm PCR, species specific PCR
applied to Trichophyton rubrum (TR) and
T. interdigitale (TI) clinical isolates. M, size
markers (100 bp DNA ladder); lanes 1,
2, 3, 4, MX PCR results (TR1, TR2, TM1,
TM2); lanes 5, 6, 7, 8, Derm PCR (TR1,
TR2, TM1, TM2); lanes 9, 10, TR PCR
(TR1, TR2); lanes 11, 12, TM PCR (TM1,
TM2); C , negative control.

When tested separately in the TM specific PCR, all
T. mentagrophytes strains (100%) showed the specific
132 bp product. No PCR product was detected when
the 35 T. mentagrophytes samples were tested in the
TR specific PCR.
Hence, all of the 23 T. rubrum and 35 T. mentagro-
phytes samples were positive in both Derm PCR and
MX PCR.
Conventional identification of fungi from toenail
samples
The results of the mycological examination of the 201
toenail samples are shown in Table 3. Direct examina-
tion was positive in 151 (71.1%) and culture in 132
(65.6%) of them respectively. Out of the 151 samples
found positive on direct examination, 112 were identi-
fied by culture as T. rubrum, four as T. mentagrophytes
and four as T. violaceum. For the 31 remaining sam-
ples, culture was either negative or contaminated.
For the 132 culture positive specimens, the causal
agent was T. rubrum in 122 cases, T. mentagrophytes
in six cases and T. violaceum in four cases. Culture was
negative or contaminated for 69 specimens including
the 31 samples found positive on direct examination.
All specimens taken from non-infected (healthy)
nails were negative on both direct examination and
culture. No mixed dermatophyte infections were
detected in culture.
Analysis of nail samples by MX PCR
The MX PCR was positive in 195 (97%) specimens out
of the 201 investigated nail samples. It identified T. ru-
brum in 109 (54.2%) samples, T. mentagrophytes in 12
(5.9%) samples and another dermatophyte species in 8
(3.9%) samples (Fig. 4). Sixty-six samples yielded three
bands (32.8%) and six specimens were negative. Fur-
thermore, 63 of 69 (91.3%) of culture negative or
contaminated specimens gave positive MX PCR results.
Thirty-one of them were positive on direct examina-
tion. Out of the 63 specimens, 8 yielded positive PCR
results with only Derm primers; 20 were positive with
both Derm and TR primers; 10 were positive with

Table 3 Summary of microscopy test, fungal culture and multiplex PCR in suspected nail cases with onychomycosis.

Direct
examination Culture MX PCR

Specimens in culture + + /c Derm1 TR2 TM3 3B Total

TR 112 10 122 0 0 0 84 0 38 122

TM 4 2 6 0 0 0 1 2 3 6
TV 4 0 4 0 0 0 4 0 0 4
Cont 15 0 – 15 0 3 4 3 5 15
Neg 16 38 – 54 6 5 16 7 20 54
Total 151 50 132 69 6 8 109 12 66 201

TR, Trichophyton rubrum; TM, T. mentagrophytes; TV, T. verrucosum; MX, multiplex; 3B, Derm+TR+TM specific bands; Con,
contaminated.
1
Dermatophyte band.
2
Derm +TR specific bands.
3
Derm +TM specific bands.

© 2013 Blackwell Verlag GmbH 5

I. Dhib et al.
M 1 2 3 4 5
400
200
100
Figure 4 Results of multiplex PCR for DNA extracted directly
from nail specimen. M, size markers (50 bp DNA ladder); lane 1
and 2, specimen from Trichophyton rubrum onychomycosis; lanes
3 and 4, specimen from T. interdigitale onychomycosis; lane 5,
negative control.
M 1 2 3
400
200
130
Figure 5 Results of multiplex (MX) PCR from nail specimen with
mixed infection. M, size markers (100 bp DNA ladder); lane 1,
amplification with all primer pairs: Derm, TR and TM; lanes 2
and 3, specimen from Trichophyton rubrum onychomycosis.
both Derm and TM primers. The 25 remaining sam-
ples yielded three bands in MX PCR (Table 3).
In 66 (32.8%) of 201 specimens, MX PCR showed
mixed infections as revealed by the production of three
bands (Fig. 5). To check this result, these 66 samples
were tested in species-specific PCR. Fifty-nine of the 66
(89.4%) specimens were positive in both PCR assays,
six were confirmed as T. mentagrophytes and one as T.
rubrum.
From the 59 cases, we randomly sequenced 10 PCR
products obtained with TR and TM specific primers
(ABI PRISM 310 genetic analyser, Applied Biosystems,
Foster City, CA, USA). All the TR products were identi-
cal to the Z97993 reference sequence of T. rubrum.
Similarly, TM sequences were identical to the
FM986758 reference sequence of T. interdigitale.
Conventional vs. molecular identification
The concordance between culture isolation and MX
PCR ranged from 0% for mixed infections to 89.34%
6 © 2013 Blackwell Verlag GmbH
140
MX PCR Culture
120
100
80
60
40
20
0
TR(89.34%) TM(33.33%) Cul-/C(8.69%) Mixed(0%)
Figure 6 Comparison of multiplex PCR and culture results.
for TR isolates (Fig. 6). MX PCR positivity was found
to be significantly higher than that found by direct
microscopy (P < 0.001) and culture (P  0.001).
PCR detected fungal material in all 163 specimens
shown to be positive in microscopy and culture. Of the
66 mixed infections detected by MX PCR, the culture
was negative in 20 and contaminated in 5 of them.
The culture yield T. rubrum in 38 cases and T. mentag-
rophytes in 3 cases.
Discussion
Correct diagnosis of dermatophytic onychomycosis and
identification of the causal agent are of a major impor-
tance as they allow appropriate antifungal treatment
to be promptly instituted.
Diagnosis of onychomycosis is currently performed by
direct mycological examination and culture on Sabou-
raud dextrose agar medium. The precise identification
of the dermatophyte in cause is based on the macro-
scopic and microscopic characters of the grown colo-
nies. However, false negative results of direct
examination occur in 5–15% of cases, depending on the
skill of the observer and the quality of sampling.6
Furthermore, dermatophyte hyphae are very difficult to
distinguish from those of non-dermatophytic fungi-like
moulds, which often only occur as transient contami-
nants and are not as the actual aetiological agent of the
disease.17 On the other hand, culture is time-consuming
and overgrowing of moulds in the culture medium can
prevent the development of the pathogen. Last, the sen-
sitivity of culture is often suboptimal or low.6,7,25
Molecular techniques are much beneficial for derma-
tophyte identification as they are rapid and sensitive.
Moreover, these methods rely on genetic characters,
which are more constant than phenotypic ones and

they can characterise atypical dermatophytes that are
difficult to identify by mycological examination
techniques.12
For many years, efforts have been made to establish
fast, highly sensitive and specific molecular-based tech-
niques for species or even strain identification of der-
matophytes, to use them as possible alternatives for
routine identification of fungi.8,21,25 All these tech-
niques are still based on the time-consuming primary
culture and many of them have a poor reproducibility.
In particular, RFLP analysis is quite complex and labo-
rious and, therefore, is not easily employed for routine
diagnostic purposes.1,4,5 Sequencing of PCR products
is a very powerful method for the correct typing of
dermatophytes but, unfortunately, it is not convenient
for the processing large numbers of samples.13,14 Real-
time PCR proved valuable in the identification of der-
matophytes because of its high sensitivity and rapidity,
but it is costly.10,11
This study aimed at evaluating a MX PCR technique
based on the amplification of the CHSI gene and the
ITS region which are the most widely used targets in
the molecular diagnosis of dermatophytic onychomy-
cosis in humans.8,17,21,23 On the other hand, MX PCR
was shown to be a powerful tool for the characterisa-
tion of dermatophytes when DNA extracted from clini-
cal specimens is used.1,6,7,9,17
We were only interested in T. rubrum and T. mentag-
rophytes complex because they are the most frequent
among the species isolated in our region.14,23 In addi-
tion, previous reports on PCR assays that allow distin-
guishing TR and TM are very few.9
In this study, MX PCR was applied to a collection of
culture samples (standards and controls) of dermato-
phytes and non-dermatophytes fungi, and to nail spec-
imens obtained from patients with dermatophytic
onychomycosis previously confirmed by mycological
examination.
The analysis of our results showed that the specific-
ity of the technique was excellent as none of the
non-dermatophytic fungal specimens and none of the
uninfected nails yielded positive results in MX PCR. Our
results are in agreement with most previously stud-
ies.4,6,7,11,16,17,25 As far as sensitivity is considered, MX
PCR may be considered very satisfactory as 100% of
controls and 97% of nail specimens yielded positive
results. Sensitivity values reported in previous studies
using different PCR methods and primers ranged
between 51% and 94.8%.1,4,6,7,11,17,19,21 On the other
hand, our results showed the PCR to be more sensitive
than mycological examination (97% vs. 81.1%). This
finding is in accordance with most previously reported
© 2013 Blackwell Verlag GmbH
Multiplex PCR for detection of dermatophytes
studies.5,7,9,13,19,20,25 In contrast, in some reports,
results of PCR and mycological examination were
nearly similar in terms of sensitivity.3,12,15
The threshold of DNA detection in MX PCR was
50 pg of DNA per reaction. This value is similar to
that reported in Candida and Aspergillus systemic infec-
tions.15,16 In contrast, it is much higher than the
threshold reported in MX PCR for the detection of
other non-dermatophyte fungi.19
The limited existing genomic data on dermatophytes
and the close ITS gene sequence similarity between
related dermatophyte species (e.g. T. rubrum and T. vio-
laceum on one hand, and T. mentagrophytes, T. schoen-
leinii and T. tonsurans on the other hand) impede
designing specific primers for all known dermatophyte
species. Indeed, ITS region primer pair TR was found
to cross-react with T. violaceum, and TM with T. tonsu-
rans, T. equinum and T. schoenleinii. This is not unex-
pected since there is a high degree of genetic similarity
in the ITS region between T. rubrum and T. violace-
um7,11,12 and between T. mentagrophytes complex, T.
tonsurans and T. equinum.6,11 However, a PCR assay
based on the amplification of the T1 microsatellite
marker that distinguishes between T. rubrum and T. vi-
olaceum when a high-resolving acrylamid gel is used
was recently reported.1
The primers described by Beifuss B et al. [6] and
Brillowska-Dabrowska A et al. [17] supposed to be spe-
cific for TR-ITS gene were shown to also amplify the
TM Z98000 sequence, after alignment of both primers
through BLAST in the GenBank sequences database.
When MX PCR was applied to non-dermatophyte
fungi including Candida, Aspergillus and other moulds,
no cross reactivity was detected between DNA of the
investigated species making the MX PCR easier to
interpret as compared to MX PCR applied to
dermatophytes.15,16,19
In our 69 patients with a negative or contaminated
culture including the 31 positive cases on direct exam-
ination, MX PCR was positive in 63 (91.3%) of them.
The failure of mycological techniques may be
explained by the presence of undetected hyphae on
microscopic examination and/or the treatment of the
patients prior to the examination. In addition, we can-
not rule out the presence of moulds, which are known
to inhibit dermatophyte growth in culture.
Hence, when the identity of the causal agent cannot
be ascertained by culture, PCR is very useful and
appropriate. This finding is in agreement with previous
reports where it has been shown that the rate of posi-
tivity of PCR in culture negative specimens ranged
between 55.8% and 78.3%.1,8,17
7
I. Dhib et al.
Our MX PCR results revealed the high frequency of
mixed infections (i.e. association of two dermatophyte
species in the same clinical specimen). This finding is
somewhat unexpected and is usually very rare when
specimens are examined by conventional mycological
techniques. Similar results were, however, previously
reported in some studies using various PCR meth-
ods.4,6,9 The scarcity of mixed infections when only
mycological techniques are used might be explained
by the competition of species that ultimately favours
one species at the expense of another. Contamination
of samples and cross reactivity of some of the primers
when MX PCR is used, is at first sight unlikely, but
cannot be definitely ruled out. Further investigations
on mixed infections are needed. It is worth mentioning
that of the 66 mixed infections revealed by MX PCR,
seven of them may actually be considered falsely
mixed as six were only positive for T. mentagrophytes
and one only positive for T. rubrum when specimens
were tested with species-specific primers. This finding
is not surprising because the specificity of a single pri-
mer PCR is higher as the technical conditions of MX
PCR are suboptimal comparatively to species-specific
PCR. The remaining 59 cases are very likely true
mixed infection.
In summary, we developed a PCR method for the
appropriate identification of clinical dermatophyte iso-
lates which is sensitive, rapid and specific for all der-
matophyte specimens and could be incorporated into
clinical laboratory diagnostic procedures.
Acknowledgment
This work was supported by the 04/UR/08-05
Research Unit, from the Ministry of Health, Tunisia.
Disclosure
The authors declare that they have no conflict of
interest.
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