Journal of Dermatological Science 69 (2013) 229235

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Journal of Dermatological Science

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Rapid real-time diagnostic PCR for Trichophyton rubrum and Trichophyton

mentagrophytes in patients with tinea unguium and tinea pedis using specic
uorescent probes
Yoshiharu Miyajima a, Kazuo Satoh a,b, Takao Uchida c, Tsuyoshi Yamada a, Michiko Abe d,
Shin-ichi Watanabe e, Miho Makimura a, Koichi Makimura a,f,g,*
a
Teikyo University Institute of Medical Mycology and Genome Research Center, Tokyo, Japan
b
Faculty of Medical Technology, Teikyo University, Tokyo, Japan
c
Department of Dermatology, Showa University School of Medicine, Tokyo, Japan
d
Department of Clinical Chemistry, School of Allied Health Science, Kitasato University, Kanagawa, Japan
e
Department of Dermatology, Faculty of Medicine, Teikyo University, Tokyo, Japan
f
Laboratory of Space and Environmental Medicine, Graduate School of Medicine, Teikyo University, Tokyo, Japan
g
General Medical Education Center, Teikyo University, Tokyo, Japan

A R T I C L E I N F O A B S T R A C T

Article history: Background: Trichophyton rubrum and Trichophyton mentagrophytes human-type (synonym, Trichophyton
Received 28 July 2011 interdigitale (anthropophilic)) are major causative pathogens of tinea unguium. For suitable diagnosis
Received in revised form 24 November 2012 and treatment, rapid and accurate identication of etiologic agents in clinical samples using reliable
Accepted 27 November 2012
molecular based method is required.
Objective: For identication of organisms causing tinea unguium, we developed a new real-time
Keywords: polymerase chain reaction (PCR) with a pan-fungal primer set and probe, as well as specic primer sets
Real-time PCR
and probes for T. rubrum and T. mentagrophytes human-type.
Trichophyton rubrum, Trichophyton
Methods: We designed two sets of primers from the internal transcribed spacer 1 (ITS1) region of fungal
mentagrophytes
ribosomal DNA (rDNA) and three quadruple uorescent probes, one for detection wide range pathogenic
fungi and two for classication of T. rubrum and T. mentagrophytes by specic binding to different sites in
the ITS1 region. We investigated the specicity of these primer sets and probes using fungal genomic
DNA, and also examined 42 clinical specimens with our real-time PCR.
Results: The primers and probes specically detected T. rubrum, T. mentagrophytes, and a wide range of
pathogenic fungi. The causative pathogens were identied in 42 nail and skin samples from 32 patients.
The total time required for identication of fungal species in each clinical specimen was about 3 h. The
copy number of each fungal DNA in the clinical specimens was estimated from the intensity of
uorescence simultaneously.
Conclusion: This PCR system is one of the most rapid and sensitive methods available for diagnosing
dermatophytosis, including tinea unguium and tinea pedis.
2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights
reserved.

1. Introduction etiologic agents are Trichophyton rubrum and Trichophyton

Tinea unguium is a dermatophyte infection and is the most
common supercial mycosis, being detected in more than 10% of
the general population in all over the world [1,2]. About half of the
patients with tinea pedis develop tinea unguium. The main
* Corresponding author at: Laboratory of Space and Environmental Medicine,
Graduate School of Medicine, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo
173-8605, Japan. Tel.: +81 3 3964 2140; fax: +81 3 3964 8415.
E-mail address: makimura@med.teikyo-u.ac.jp (K. Makimura).
mentagrophytes human-type [3] (synonym, Trichophyton interdi-
gitale (anthropophilic) [4]), although some non-dermatophytes
have also been reported to cause onychomycosis, including
Candida spp. [5,6], Aspergillus spp. [6], Fusarium spp. [7,8], and
others. For effective antifungal treatment, it is important to know
whether the pathogen is a dermatophyte or not because of some
anti-dermatophytic agents are not effective to non-dermato-
phytes. Recently the incidence of onychomycosis has been
increasing, and it accounts for up to 90% of toenail disorders
and at least 50% of ngernail disorders [1]. Scales from the infected
skin and nails of untreated patients spread this infection to other

0923-1811/$36.00 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jdermsci.2012.11.589
230 Y. Miyajima et al. / Journal of Dermatological Science 69 (2013) 229235
members of their household. Therefore, early diagnosis and
treatment of individual patients is essential to prevent the spread
of these infections.
The discrimination of two major etiologic agents, T. rubrum and
T. mentagrophytes human-type is important to investigate the
pathogenesis and clinical features related in each dermatophyte,
also is essential for epidemiological survey for dermatophytoses.
The standard methods for diagnosis of onychomycosis are
microscopy and culture. Direct microscopic examination is a basic
method to detect typical fungal elements. This test is still the gold
standard for the diagnosis of fungal infection, but requires skill and
knowledge about the morphological features of fungi. Culture is
the most important method for mycological diagnosis, but the
culture-positive rate from nail specimens is often not high enough.
Generally, the identication achieved by culture of supercial
samples is less than 40% [9]. Since the isolates may show atypical
features, fungal culture takes several days longer.
In order to overcome these defects of the classical methods,
molecular biological techniques based on the polymerase chain
reaction (PCR) have been developed [2,1021]. PCR analysis
becomes popular as the most reliable method for identication
of pathogenic fungi from clinical specimens. And to establish more
sensitive, more rapid, cheaper, and simpler PCR system for
practical use, several improvements have been attempted
[9,20,21].
Recently, real-time PCR methods for identication of several
dermatophytes have been reported [11,12,21,24]. These methods
are useful for identication of part or most of dermatophytes but
not other non-dermatophytic fungal pathogens that cause tinea
unguium or tinea pedis [17]. Practically quantitative studies are
also required to determine the threshold between infection and
contamination of clinical specimens.
In this study, we designed two primers sets and three different
sets of probes, based on previous analyses [3,1316], to detect the
etiologic agents of tinea unguium by real-time PCR. Our primers
and probes targeted the internal transcribed spacer 1 (ITS1) region
in order to detect a wide range of pathogenic fungi and the two
major causative dermatophytes, T. rubrum and T. mentagrophytes
human-type. We compared the results of our new PCR analyses
with the ndings obtained by microscopic examination and
culture of 42 clinical specimens.
2. Materials and methods
2.1. Design of the probes and primers
The sequences of the primers and probes used in this study are
shown in Table 1. The pan-fungal primer set (forward: ITS1F;
Table 1
The primers and probes used for real-time PCR.
Primers and probes
Pan-fungal primers ITS1F
ITS1R
Trichophyton primers ITS1F2
ITS1R2
Pan-fungal probe PF-ITS1
T. rubrum probe TR-ITS1
T. mentagrophytes probe TM-ITS1
Internal control primers MS2-TM3-F
MS2-TM3-R
Internal control probe MS2-TM2-Cy5
BHQ-3: Black Hole Quencher 3; Cy5: indodicarbocyanine; FAM: 6-carboxyuorescein; MGB: minor groove binder; NED: 20 -chloro-50 -uoro-70 ,80 -fused phenyl-1,4-dichloro-
6-carboxyuorescein; NFQ: non-uorescent quencher; VIC: 4,7,2-trichloro-7-phenyl-6-carboxyuorescein. Nucleotides. M: A or C; S: C or G; Y: C or T.
reverse: ITS1R), Trichophyton primer set (forward: ITS1F2; reverse:
ITS1R2), specic T. rubrum probe (TR-ITS1V), specic T. menta-
grophytes probe (TM-ITS1F), and pan-fungal probe (PF-ITS1N) were
designed with MEGA4 alignment software [22] and Primer Express
(Applied Biosystems, CA, USA), based on the upstream and
downstream sequences of the ITS1 region of the ribosomal RNA
gene (rDNA) of major pathogenic fungi (Fig. 1). These primers and
uorescent probes were synthesized by Applied Biosystems. The
internal control primers and internal control probe were synthe-
sized by SigmaAldrich (MO, USA).
2.2. Preparation of genomic DNA
The 46 fungal strains used in this study are listed in Table 2. All
of the strains were maintained in the TIMM culture collection of
Teikyo University Institute of Medical Mycology, most of them
were isolated from clinical cases or environment in Japan and part
of them were obtained from international culture collections.
Dermatophytes were fully grown on Sabourauds dextrose agar,
and were prepared by methods introduced by Makimura et al. [3].
The DNA was quantitated with a Quant-iT dsDNA HS Assay Kit and
Qubit uorometer (Invitrogen, Carlsbad, CA, USA). An amount of
genomic DNA corresponding to one cell (0.3 pg or roughly 100
ribosomal RNA gene copies at 3040 Mb per genome) was used as
a template in the real-time detection system to conrm the
spectrum and specicity of each probe.
2.3. Plasmid DNA preparation
As the positive control for the PCR assay, three plasmids were
constructed by using a TOPO TA Cloning Kit (Invitrogen, Carlsbad,
CA, USA). The ITS1 regions of T. rubrum (T99), T. mentagrophytes
human-type (T7), and Aspergillus fumigatus (TIMM0108) were
cloned into the pCR2.1 plasmid. The copy number of each plasmid
was calculated from the total length of the bases and the DNA
concentration.
2.4. Clinical specimens
The clinical samples were 32 nail specimens and 10 skin
specimens collected from 32 patients of Kawasaki Social Insurance
Hospital (Kanagawa, Japan) between May 16 and August 17, 2005
with written informed consent based on the guideline and
agreement of the institutional ethical committee (Table 1). All of
the subjects visited the dermatological outpatients department
and were diagnosed as the dermatophytoses by direct microscopic
examinations. Nail specimens from 39 healthy volunteers were
used as negative controls for the PCR assay.
Sequences
50 -TAACAAGGTTTCCGTAGGTGAACCT-30
50 -TCGCTGCGTTCTTCATCGA-30
50 -SSCCCCATTCTTGTCTACMTYAC-30
50 -AACGCTCAGACTGACAGCTCTTC-30
50 -[NED]-TTYAACAAYGGATCTCT-[NFQ-MGB]-30
50 -[VIC]-CGCGCTCCCCCTGC-[NFQ-MGB]-30
50 -[FAM]-CTCTCTTTAGTGGCTAAAC-[NFQ-MGB]-30
50 -TGCTCGCGGATACCCG-30
50 -AACTTGCGTTCTCGAGCGAT-30
50 -[Cy5]-ACCTCGGGTTTCCGTCTTGCTCGT-[BHQ-3]-30
 Y. Miyajima et al. / Journal of Dermatological Science 69 (2013) 229235 231

18S 5.8S 28S

ITS1 rDNA ITS2
rDNA rDNA

TM-ITS1F TR-ITS1V
dermaF
dermaF2 PF-ITS1N
18S 5.8S
ITS1
rDNA rDNA
dermaR2
dermaR
Fig. 1. Hybridization sites of the primers and probes used in this study. Location of the hybridization sites for the primers and probes are shown in the ITS1 region of ribosomal
RNA gene. Arrows; primers, bars with stars; uorescent probes.

Table 2
Specicity of the primers and probes.

Species Strains Pan-fungal primers Trichophyton primers

Pan-fungal T. rubrum T. mentagrophytes T. rubrum T. mentagrophytes

probe probe probe probe probe

Major causative fungi for tinea

Acremonium curvulum NBRC 32242 +    
Arthroderma benhamiae SM103 +    
Arthroderma vanbreuseghemii TIMM2789 +  +  +
Epidermophyton ocosum 514 (Hasegawa) +    
522 (Hasegawa) +    
Microsporum canis TDGS945 +    
TDGS0222 +    
TDGS0223 +    
TDGS0269 +    
Microsporum gypseum TDGS0009 +    
TDGS0012 +    
Scopulariopsis brumptii NBRC 6441 +    
Scopulariopsis brevicaulis NBRC 4843 +    
Scytalidium lignicola NBRC 104988 +    
Trichophyton rubrum T99 + +  + 
T108 + +  + 
421 (Hasegawa) + +  + 
423 (Hasegawa) + +  + 
425 (Hasegawa) + +  + 
426 (Hasegawa) + +  + 
Trichophyton mentagrophytes human-type T7 +  +  +
T9 +  +  +
Trichophyton tonsurans T117 +    
Other pathogenic fungi
Aspergillus avus TIMM0059 +    
TIMM2935 +    
Aspergillus fumigatus JCM10253 +    
TIMM0108 +    
TIMM2920 +    
Aspergillus niger TIMM0113 +    
TIMM2915 +    
Candida albicans ATCC10231 +    
ATCC90028 +    
TIMM1768 +    
Candida glabrata ATCC90030 +    
CBS138 +    
Candida parapsilosis ATCC2209 +    
ATCC90018 +    
Candida tropicalis ATCC750 +    
TLCS S 9/1 +    
Chaetomium globosum TSY-0369 +    
Cryptococcus neoformans ATCC90113 +    
Tjane +    
Exophiala jeanselmei TSY-0396 +    
Fusarium oxysporum TSY-0351 +    
Fusarium solani TSY-0403 +    
Fusarium verticillioides TSY-0219 +    
Paecilomyces variotii TIMM3182 +    
Pseudallescherichia boydii TIMM0886 +    
Rhizopus oryzae TIMM0921 +    
Trichosporon asahii CBS2479 +    

(+): detected and (): not detected.

232 Y. Miyajima et al. / Journal of Dermatological Science 69 (2013) 229235
Fig. 2. Representative amplication curves obtained by real-time PCR. Curves for three independent PCR batches are shown in each of the four plots. Baseline data were
obtained with fewer than 25 cycles. RFU: relative uorescence unit.
2.5. Preparation of DNA templates from clinical specimens
The 42 nail and skin specimens from 32 patients and 39 nail
specimens from healthy volunteers were stored at 80 8C before
use. Part of each sample was treated with 15% KOH and 40% diethyl
sulfoxide for detection of fungal elements by microscopic
examination. Another part of each samples were cultured on
Sabourauds dextrose agar containing chloramphenicol (0.05%)
with cycloheximide (0.5%) at 25 8C for several days to 2 weeks, and
morphological examination and sequencing were performed.
For PCR and sequencing analysis, nail and skin samples (approx.
2 mm  2 mm  1 mm, ca. 10 mg) were frozen for 1 h at 150 8C
or 5 min in liquid nitrogen. Frozen samples were ground in a Multi-
beads Shocker (Yasui Kikai, Osaka, Japan) with metal cones for
3 min. Then the powdered samples were extracted with phenol
chloroform, as described elsewhere [13]. Ethachinmate copreci-
pitation reagent (3 mL, Nippon Gene, Tokyo, Japan) was applied to
each sample at the beginning of isopropanol precipitation for
maximum recovery of DNA.
2.6. Quantitative real-time PCR analysis
A class 100 clean work station equipped with HEPA lters and a
UV lamp (Airtech Japan, Tokyo, Japan) was used during preparation
for prevention of airborne fungal contamination. The reaction
mixture for real-time PCR consisted of 10 ml of Premix Ex Taq
(Perfect Real Time) (Takara Bio, Shiga, Japan), 0.2 mM each of the
ITS1F and ITS1R (or ITS1F2 and ITS1R2) primer pairs, 0.2 pmol/ml
of TaqMan MGB probes (TR-ITS1V, TM-ITS1F, PF-ITS1N), and 0.4 ml
of ROX (tetrapropano-6-carboxyrhodamine) Reference Dye II
(Takara Bio, Shiga, Japan). The reaction mixture was combined
with 2 ml of DNA extracted from a sample in each well of a 96-well
MicroAmp plate (Applied Biosystems, CA, USA) and was adjusted
to a nal volume of 20 ml with distilled water. Quantitative PCR
and monitoring were done with a 7500 Fast Real-Time PCR System
(Applied Biosystems, CA, USA). Initial denaturation at 95 8C for 30 s
was followed by 60 cycles of amplication at 95 8C for 6 s and 60 8C
for 30 s.
For each real-time PCR assay, we used an internal control
template and primers based on the MS2 bacteriophage, as
described by Dreier et al. [23].
3. Results
3.1. Specicity of the probes
Representative real-time PCR uorescence signal curves for
three specic probes and the internal control probe are shown in
Fig. 2. The target ITS1 region of rDNA was successfully amplied
and the amplied fragments could be used for DNA cloning or
sequencing.
The specicity of the probes for fungal isolates is shown in
Table 2. The expected fungi were detected successfully by
different combinations of primer sets and probes. The
combination of pan-fungal primers and pan-fungal probe
reacted with DNA from all fungi, but did not react with human
DNA (data not shown).
The specic T. rubrum probe reacted specically with T. rubrum,
while the T. mentagrophytes probe reacted specically with T.
mentagrophytes human-type [3]. Arthroderma vanbreuseghemii is a
teleomorph of T. mentagrophytes that has the almost same ITS1
rDNA region sequence as T. mentagrophytes including its human-
type [3], and it also reacted with the T. mentagrophytes probe.
3.2. Minimum detectable DNA copy number
To determine the detection limit of this system, dilutions of
plasmid DNA were prepared and measured four times by real-
time PCR (Table 3). The results indicated that 50 template rDNA
copies was the minimum number for reliable detection by this
system.
3.3. Quantitative real-time PCR analysis of clinical samples
Toe nail and skin specimens from 32 patients with tinea pedis
and toe nail samples from 39 healthy volunteers were tested by
 Y. Miyajima et al. / Journal of Dermatological Science 69 (2013) 229235 233

Table 3
Detection rate for each primer set and probe combination.a

DNA copies Pan-fungal primers Trichophyton primers

Pan-fungal probe T. rubrum probe T. mentagrophytes probe T. rubrum probe T. mentagrophytes probe

400 100 100 100 100 100

200 100 100 100 100 100
100 100 100 100 100 100
50 100 100 100 100 100
25 100 25 100 100 100
13 100 50 100 75 100
6 75 25 75 25 100
3 75 0 75 0 75
a
Detection rate with four amplications.

microscopy and culture (Table 4). Both microscopic examination
and culture of all samples from the healthy volunteers were
negative (data not shown).
DNA extracted from clinical specimens was also tested by our
real-time PCR system with positive control plasmids. The
comparative CT method (DDCT method) was used to calculate
the number of rDNA copies in the clinical samples. The amount of
DNA found in each nail sample is shown in Table 4 along with the
results of other examinations.
In the nail samples collected from the 39 healthy individuals,
fewer than 1500 copies of rDNA were sometimes detected (data
not shown), suggesting that non-pathogenic fungi could possibly

Table 4
Detection of fungi from clinical specimens.

Pt. no. Specimen Microscopy Culture Pan-fungal primers Trichophyton primers

Pan-fungal T. rubrum T. mentagrophytes T. rubrum probe T. mentagrophytes

probe probe probe probe

1 Nail +  9270 19,800 4590 96,800

2 Nail +  167,000 2910 1,920,000 13,500,000
3 Nail +  22,100 68,100 167,000
4 Nail + Tr 635,000 14,900,000 3,890,000
Skin +  735 2930 11,700
5 Nail + Tr 42,200 5090 2360 606,000 40,400
6 Nail +  142,000 2,790,000 13,000,000
7 Nail + Ca 125,250 1,545,000 4,430,000
8 Nail + Tm 65,400 79,400 16,400 1,520,000 174,000
Skin + Tm 375,000 375,000 1,500,000
9 Nail +  20,600 76,800 1230 322,500
Skin +  46,875 750,000 3,000,000
10 Nail +  27,300 2280 119,100 2520 1,490,000
11 Nail +  13,800 14,700 69,500 35,100
Skin +  2930 2930 23,400
12 Nail + Tr 34,100 44,900 2490 2,490,000 719,000
Skin + Tm 93,750 93,750 375,000
13 Nail +  11,700 3500 28,100 11,700 72,800
Skin + Tm 23,400 23,400 375,000
14 Nail + Tr 10,400 21,000 50,100
15 Nail +  17,700 54,800 306,000
16 Nail +  180 3090 10,900
17 Nail + Tr 5840 125,000 321,000
18 Nail +  16,100 245,000 3,690,000
Skin +  180 735 2925
19 Nail +  501,000 2,790,000 26,100,000
Skin +  188,000 188,000 23,400 1,500,000 1,500,000
20 Nail +  390 690 5510 960
21 Nail + Tr 4,650,000 7,160,000 2,850,000 14,100,000 3,380,000
22 Nail +  429,000 30 5865 46,875
Skin + Tr 360 11,715
23 Nail + Tr 111,000,000 1,520,000,000 60 2,430,000,000
Skin + Tr 46,875 750,000 6,000,000
24 Nail + Tr 1,346,000 23,700,000 76,100,000
25 Nail + Tr 270 9870 180,000
26 Nail + Tr 570,000 14,800,000 45,500,000
27 Nail +  12,400 267,000 105 1,360,000
28 Nail +  1730 2240 44,700
29 Nail + Tr 44,700 1,070,000 13,300,000
30 Nail +  45 5270
31 Nail + Tr 67,500 1,500,000 9,800,000
32 Nail +  939,000 639,000 1,370,000

Numbers represent estimated rDNA copies per reaction. The threshold of this system is 1500 copies/reaction. Identied fungal species; Tr: Trichophyton rubrum, Tm:
Trichophyton mentagrophytes, and Ca: Candida albicans. (+) Fungal elements detected and () culture negative or no PCR amplication.
234 Y. Miyajima et al. / Journal of Dermatological Science 69 (2013) 229235
be present in clinical samples at this level. Accordingly, we set
1500 copies as the threshold for detection by this PCR method.
4. Discussion
Our three probes with different uorescent dyes allowed
simultaneous detection of two major pathogenic dermato-
phytes and a wide range of fungi in only 3 h. One hour was
required for preparation of DNA from clinical samples, 1 h for
setting up the PCR reaction, and 1 h for PCR amplication. We
designed specic uorescence probes and primers for T.
rubrum and T. mentagrophytes human-type, which allowed
rapid diagnosis of the pathogens causing tinea unguium and
tinea pedis. Some patients are infected by non-dermatophyte
fungi, as reported by Ebihara et al. [17]. Our pan-fungal probe
made it possible to detect a wide spectrum of fungi, as well as
the major pathogenic dermatophytes. If further analysis is
considered necessary, DNA cloning and sequencing could be
done by using amplied fragments obtained from our
real-time PCR.
With the present PCR system, 50 copies of rDNA are sufcient
for identication of the target pathogen, so the sensitivity is high
enough to nd fungi in a small clinical specimen. For example, to
obtain 50 copies of rDNA for A. fumigatus would require only one or
two cells. Accordingly, quantication of real-time PCR data is
important for appropriate evaluation of clinical samples contain-
ing both pathogenic and non-pathogenic fungi. In a few nail
samples obtained from healthy volunteers, fewer than 1500 copies
of fungal DNA were detected, suggesting that this represented
commensal fungi living on the nails and skin or environmental
contamination.
Our results also indicated that the primer set for Trichophyton
was more sensitive for detection of T. rubrum and T. mentagro-
phytes human type than the pan-fungal primer set (Table 4). Seven
samples were negative with pan-fungal primers, but were positive
with the Trichophyton-specic primers, indicating that both primer
sets should be used for accurate detection. As also reported by
Ebihara et al. [17], infection with multiple fungi was detected in
nail samples from nine patients (patients 1, 5, 8, 10, 11, 12, 13, 19,
and 21).
In the present study, we used 42 clinical samples in which
fungal elements were detected by microscopic examination.
However, deep infection of the nails is sometimes missed by
microscopic analysis and identication of specic fungi by
microscopy requires much experience.
The success rate of culture in this study was 45.2% (19/42
samples), being 43.8% for nails (14/32) and 50.0% for skin (5/10).
Such results are typical at our laboratory. Sato et al. [9] also
reported a low success rate for culture of fungi from clinical
specimens (34%). PCR can detect all pathogenic fungi, whether
viable or not, and is more sensitive than culture methods. Also, our
PCR detection system can specically identify T. rubrum and T.
mentagrophytes human type after only a few hours. There are some
discrepancies of the results of species identication between
culture and the PCR system. As reported by Ebihara et al. [17], co-
infection by T. rubrum, T. mentagrophytes human-type, or other
fungi, that are not rare in nail samples. If multi-fungi contained in a
specimen, it is possible to detect one of them by chance. One
possible reason is the sensitivity of T. rubrum probe is slightly low
than T. mentagrophytes probe as shown in Table 3, however, we
need to have father evaluations to clear the clinical state and the
pathogens.
The real-time PCR described here should be useful in the clinical
setting. Once the system is established, it is easy to employ for in
routine examination. From 96 to 384 samples (with negative and
positive controls) can be processed in a single reaction, and testing
is completed after 3 h.
In conclusion, this real-time PCR system is a sensitive, reliable,
and rapid method for specic identication of T. rubrum and T.
mentagrophytes. Our system may be useful for large-scale
epidemiological studies and for clinical trials of antifungal
therapy.
Acknowledgement
This study was partly supported by Hisamitsu Pharmaceutical
Co., Inc. (Tokyo, Japan) via a grant (Koichi Makimura).
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