Mycopathologia (2009) 167:133137
DOI 10.1007/s11046-008-9165-5
Dermatophyte Infections in Cairo, Egypt
S. M. Zaki N. Ibrahim K. Aoyama
Y. M. Shetaia K. Abdel-Ghany Y. Mikami
Received: 31 March 2008 / Accepted: 14 October 2008 / Published online: 30 October 2008
Springer Science+Business Media B.V. 2008
Abstract In this study, we examined dermatophyte
infections in patients referred to the Department of
Dermatology, EL-Houd El-Marsoud Hospital, Cairo,
during March 2004 to June 2005. Of 506 patients
enrolled in this investigation, 403 (79.6%) were
clinically diagnosed as having dermatophytoses (age
range 670 years; males 240; females 163). Species
identification determined by observation of their
macroscopic and microscopic characteristics was
complemented with sequencing of the internal tran-
scribed spacer ITS1-5.8S-ITS2 rDNA region. The
most common dermatophyte infection diagnosed was
tinea capitis (76.4%), followed by tinea corporis
(22.3%) and tinea unguium (1.2%). The most
frequently isolated dermatophyte species was Trich-
ophyton violaceum, which accounted for most
(71.1%) of all the recovered dermatophytes, followed
S. M. Zaki (&)
N. Ibrahim
Y. M. Shetaia

K. Abdel-Ghany
Microbiology Department, Faculty of Science, Ain Shams
University, Abbassia 11566, Cairo, Egypt
e-mail: shzaki@yahoo.com
K. Aoyama
Y. Mikami
Medical Mycology Research Center, Chiba University,
Chiba, Japan
by Microsporum canis (21.09%), Trichophyton ru-
brum (6.2%), and Microsporum boullardii (0.49%);
both Epidermophyton floccosum and Trichophyton
tonsurans were each only rarely isolated (0.24%).
Keywords Dermatophytes
Fungal infection

ITS1-5.8S-ITS2
Egypt
Introduction
Dermatophytes are a group of closely related fungi that
cause dermatophytoses in humans and animals [1].
Dermatophyte infections constitute a major public
health problem in different countries. Its prevalence is
much higher in developing than in industrialized
countries [24]. The most common factors affecting
the distribution and transmission of dermatophyte
infections are climatic conditions, general hygiene,
and animal contact [5, 6].
Epidemiological studies on dermatophyte infec-
tions have been carried out extensively in many
countries. Differences in the incidence of infection
and etiological agents have been reported [7].
In Egypt, the prevalence of dermatophyte infec-
tions has not been well clarified. This study was
aimed at investigating the prevalence of dermato-
phytoses and their etiological agents among patients
examined at the Department of Dermatology,
EL-Houd El-Marsoud Hospital, Cairo, during March
2004 to June 2005.
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Materials and Methods
Patients and Sampling
In all, 506 patients with suspected dermatophytoses
were referred to the Outpatient Clinic of Dermatol-
ogy, El-Houd El-Marsoud Hospital, Cairo, during
March 2004 to June 2005, were included in this
investigation. Gender, age, and anatomic sites of the
lesion(s) were recorded for each patient. Samples of
skin scrapings, hair fragments, and nail cuttings were
collected and subjected to direct microscopy and
culture. Microscopy of specimens was carried out
after addition of 23 drops of 20% KOH solution
with 36% dimethylsulfoxide. Cultures were per-
formed on Sabouraud dextrose agar containing
chloramphenicol and cycloheximide. Plates were
incubated at 2528C for 46 weeks and were Amplification reactions were performed in 25 ll
examined twice weekly.
Identification of the Isolated Dermatophyte
Species
Identification of dermatophytes was performed
through macro-morphological and micromorphologi-
cal examination of colonies. Molecular typing
methods were used by comparing the ITS1-5.8S-
ITS2 rDNA region sequence data of the isolated strains
with reference strain data deposited in GenBank.
Molecular Typing Methods
Extraction of DNA
A small amount of mycelium grown on Sabourauds
dextrose agar was suspended in 200 ll of TE buffer
(100 mM TrisHCl, pH 8.0, 1 mM EDTA) in an
Eppendorf tube (1.5 ml). The DNA extraction was
carried out according to the procedure described by
Sandhu et al. [8]. Briefly, 250 ll of GPT reagent
(6 M guanidine thiocyanate dissolved in 50 mM Tris
(pH 8.3)) and 700 ll of phenol-buffered in Tris
(pH 8.0) were added to a washed fungal inoculum in
a screw-cap tube and boiled for 15 min; 250 ll of
chloroform-isoamyl alcohol (25:24:1 v/v) was then
added. The aqueous phase was separated by centri-
fugation at 14,0009 g, mixed with an equal amount
of 100% isopropanol and 1/10 volume of 3 M
ammonium acetate, and placed at (-20C) for 1 h.
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Mycopathologia (2009) 167:133137
Samples were centrifuged at 14,0009 g for 20 min,
and the nucleic acid pellet was washed with ice-cold
70% ethanol, dried, and re-suspended in sterile
TE-buffer at a concentration of 5 lg/ml.
Oligonucleotides
The oligonucleotide primers used for amplification
and sequencing of the ITS regions were those
described by White et al. [9]. This study used
ITS5 (50 -GGAAGTAAAAGTCGTAACAAGG-30 )
and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 )
(Pharmacia Biotech Inc., Tokyo, Japan).
PCR and DNA Sequencing of ITS1-5.8S-ITS2 Region
rDNA of Fungal Strains
reactions containing 2.5 ll of each primer (20 pm),
2.5 ll of genomic DNA (5 lg/ml), and one PCR bead
(PuReTaq Ready-To-Go; Amersham Biosciences).
The bead contains buffers, dNTPs, enzyme, stabiliz-
ers, and BSA. Amplification was performed with a
PCR Thermal Cycler (TaKaRa Shuzo Co. Ltd.,
Tokyo, Japan) using the initial denaturation at 94C
for 4 min, followed by 35 cycles at 94C for 2 min,
55C for 2 min, and 72C for 2 min, with final
extension at 72C for 10 min. The PCR reaction
products were sequenced directly using a Big-Dye
terminator reagent kit including Taq polymerase and
the protocol recommended by the manufacturer
(Model 3100 automated DNA sequencer; PerkinEl-
mer Inc/Applied Biosystems, Japan).
Alignments and Phylogenetic Tree Construction
The DNA sequences were aligned using Clustal W
(Version 1.83) [10]; the alignment was visually
corrected. The phylogenetic tree was constructed
using the neighbor-joining method applied to DNA
distance matrices calculated according to Kimura two-
parameter model [11]. The confidence values of
branches were determined using bootstrap analysis
[12].
Nucleotide Sequence Accession Numbers
The nucleotide sequence data reported in this study
appear in the NCBI GenBank nucleotide sequence
Mycopathologia (2009) 167:133137
database with the following accession numbers:
Epidermophyton floccosum (EU590653), Microspo-
rum boullardii (EU590654), Microsporum canis
(EU590655), Trichophyton rubrum (EU590657),
Trichophyton tonsurans (EU590658), and Trichophy-
ton violaceum (EU590656). Other published rDNA
sequences used as reference strains in the phylogenetic
analysis are as follows: E. floccosum (AY213646),
M. boullardii (AJ970143), M. canis (AB193630),
T. rubrum (EU200400), T. tonsurans (EF043271),
T. violaceum (AB194246), and Rhizopus oryzae
(EU337012).
Results
Frequency of Dermatophytoses in Examined
Patients
Among the 506 patients examined, 403 (79.6%) were
clinically diagnosed as having dermatophytoses (aged
670 years; males 240; females 163), while 103
(20.3%) showed negative results. Out of the 403
positive samples, 40 (9.9%) samples were found to be
positive by direct microscopy only, 22 (5.4%) by
culture only, and 351 (87%) by both techniques. The
most commonly diagnosed dermatophyte infection
was tinea capitis (76.4%), followed by tinea corporis
(22.3%) and tinea unguium (1.2%).
Identification of Dermatophyte Species Isolated
From Infection Sites
The obtained dermatophyte isolates were examined
morphologically and identified according to the mac-
roscopic and microscopic features described below.
Six species belonging to three genera were identified:
Epidermophyton floccosum, Microsporum boullardii,
Microsporum canis, Trichophyton rubrum, Tricho-
phyton tonsurans, and Trichophyton violaceum.
Molecular Typing
All six species yielded a unique PCR amplification.
The sequences of the ITS1-5.8S-ITS2 rDNA region
for the six species were from 481 to 710 bp. The
NCBI GenBank was accessed to identify the isolated
species through BLAST homology search using the
obtained ITS data. The ITS data of the isolated
135
E. floccosum (654 bp), M. canis (696 bp), T. rubrum
(660 bp), T. tonsurans (481 bp), and T. violaceum
(710 bp) were, respectively, highly similar (99%
identical) to the ITS data of E. floccosum (GenBank
Acc. No. AY213646), M. canis (GenBank Acc. No.
AB193630), T. rubrum (GenBank Acc. No. EU200-
400), T. tonsurans (GenBank Acc. No. EF043271),
and T. violaceum (GenBank Acc. No. AB194246).
Isolated strain of M. boullardii (554 bp) showed
relatively high similarity (98.1% identical) to Arth-
roderma corniculatum (teleomorph of M. boullardii)
(GenBank Acc. No. AJ970143). A phylogenetic tree
was constructed based on ITS1-5.8S-ITS2 rDNA
region sequences for the isolated dermatophyte
strains and the reference strains (Fig. 1). The tree
shows the clear identity between the isolated strains
and the reference strains (100% bootstrap support).
Frequency of Isolation of Dermatophyte Species
The most frequent dermatophyte species isolated was
T. violaceum which accounted for 71.1% of all derma-
tophytes recovered, followed by M. canis (21.09),
T. rubrum (6.20%), and M. boullardii (0.49%); both
E. floccosum and T. tonsurans were only rarely isolated
(0.24%).
In fact, T. violaceum was the most frequently
isolated species in tinea capitis (75.0%), tinea corpo-
ris (61.1%), and tinea unguium (60%), followed by
M. canis, which was isolated from tinea corporis
(28.8%) and tinea capitis (19.1%). Furthermore,
T. rubrum was isolated from tinea unguium (40%),
tinea corporis (8.8%), and tinea capitis (4.8%). Also,
M. boullardii was isolated twice from tinea capitis
(0.64%), T. tonsurans was isolated once from tinea
capitis (0.32%), and E. floccosum was isolated once
from tinea corporis (1.1%).
Discussion
In this study, the dermatophytoses were clinically
diagnosed in 79.6% of all patients examined in
El-Houd El-Marsoud Hospital, Cairo, during March
2004 to June 2005. Six species belonging to three
genera were isolated from infection sites and identified
by morphological and molecular typing methods.
Similar molecular identification methods have been
used previously by many investigators [1317].
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136
Fig. 1 Phylogenetic tree 0.02
based on ITS1-5.8S-ITS2
region sequences for the
isolated dermatophytes
species with reference
strains. Numbers at the
respective nodes are
percentages of 1,000
bootstrap replicates. The bar
indicates two base changes
per 100-nucleotide position
Tinea capitis was the most common manifestation
of dermatophytosis in our study followed by tinea
corporis. Comparison of our data to those of other
studies revealed that tinea capitis is dominant in other
countries in Africa [18] and Asia [1921]. However,
in Europe tinea corporis and tinea pedis were the
most common clinical forms of dermatophytosis [6,
22, 23]. Tinea unguium was the least prevalent
clinical form of dermatophytosis in our study
accounted for only 1.2% of the total dermatophytic
infection. However, in Europe tinea unguium was
widespread and accounted for 13.527.5% [6, 7, 23].
Actually, T. violaceum was the most frequently
isolated dermatophyte, it was recovered from a high
percentage of patients with tinea capitis (75%), and
was the major etiologic agent of tinea corporis
(61.1%) and tinea unguium (60%). In fact, T. viola-
ceum has been detected as the most prevalent species
in several African and Asian countries: Ethiopia
(81.6%), Libya (50%), the West Bank of Palestine
(83%), and Pakistan (65%) [3, 18, 24, 25]. However,
in some other countries T. violaceum was an only
rarely isolated dermatophyte: the Ivory Coast (2.3%),
Nigeria (6%), and Kuwait (19.3%) [2628].
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Mycopathologia (2009) 167:133137
100 M. boullardii (Egyptian Isolate) EU590654
17 M. boullardii AJ970143
M. canis (Egyptian isolate) EU590655
40 100 M. canis AB193630
T. rubrum (Egyptian isolate) EU590657
100
T. rubrum EU200400
99
T. violaceum (Egyptian isolate) EU590656
100 100 T. violaceum AB194246
T. tonsurans (Egyptian isolate)
EU590658
100 T. tonsurans EF043271
E .floccosum (Egyptian isolate)
EU590653
100
E. floccosum AY213646
Rhizopus oryzae EU337012
In terms of isolation, M. canis is ranked second
from tinea corporis (28.8%) and tinea capitis
(19.1%). However, M. canis was the most commonly
isolated dermatophyte species recorded in Kuwait,
Saudi Arabia, and the United Arab Emirates [20, 21,
28]. The emergence of M. canis from two types of
tinea in Egypt might reflect contact with animals
among the population in Cairo and its environs. In
addition, T. rubrum was isolated from tinea unguium
(40%), tinea corporis (8.8%), and tinea capitis
(4.8%); T. tonsurans was isolated once from tinea
capitis (0.32%) and E. floccosum was isolated once
from tinea corporis (1.1%).
Interestingly, the present study revealed the first
detection of the geophilic species of M. boullardii
infection in tinea capitis in Egypt. The emergence of
M. boullardii might be attributed to the relatively
close contact of the population with the environment
and animals: this species had been previously isolated
from animals and soil in Egypt [29, 30]. Furthermore,
M. boullardii was isolated previously from a tinea
capitis infection in Madagascar [31].
In conclusion, tinea capitis was the most common
among the patients in Cairo. It was mainly caused by
Mycopathologia (2009) 167:133137
the anthropophilic T. violaceum. The Zoophilic der-
matophyte M. canis ranks next in frequency, which
reflects the degree of contact there between the
human and animal populations. The geophilic der-
matophyte M. boullardii was isolated, but only
rarely, from clinical specimens, confirming the
frequent contact with the environment.
Acknowledgment This study was supported by a Grant from
the Ministry of Higher Education, Egyptian Government.
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