ARTICLE
Auteur(s) :, Zafer
Cetinkaya1,*, Nuri Kiraz2, Semsettin
Karaca3, Mustafa Kulac3, Ihsan H
Ciftci1, Orhan C Aktepe1, Mustafa
Altindis1, Nilay Kiyildi1, Meltem
Piyade1
1Afyon Kocatepe University, Faculty of Medicine,
Department of Microbiology, Afyon, Turkey.
2Osmangazi University, Faculty of Medicine, Department
of Microbiology, Eskisehir, Turkey
3Afyon Kocatepe University, Faculty of Medicine,
Department of Dermotology, Afyon, Turkey
accepté le 6 Avril 2005
The possibilities for the treatment of superficial fungal
infections have improved enormously over the last 30 years.
Nevertheless, there remains room for new antifungals with superior
efficacy and safety profiles. Dermatomycosis is an infection with
fungi related to the skin: glabrous skin, hair and/or nails. Oral
treatment of fungal infections in dermatology has become a
preferred modality for the management of these very common
conditions [1, 2]. In recent years, the number of infections caused
by these fungi has considerably increased, causing particular
concern when immunocompromised patients are infected [3]. Although,
there are increasing numbers of antifungals available for treatment
of dermatophytes, some cases and relapses have been unresponsive to
treatment. In such cases, the effects of treatment have been
determined by influencing the target fungus, as well as the
pharmacokinetic properties of the drug. The determination of fungus
in vitro antifungal susceptibility has been reported to be
important for the ability to eradicate dermatophytes [4].In order
to predict the ability of a given antimycotic agent to eradicate
dermatophytes, determination of the in vitro susceptibility of
dermatophytes may prove helpful. Various tests, such as agar
diffusion, agar dilution, and broth dilution tests can be used for
the determination of MICs. With dermatophytes, a correlation
between the in vitro data and clinical outcome has been
demonstrated for the micro dilution test [4, 5].The aim of this
study was to evaluate the in vitro activity of the ketoconazole,
itraconazole, terbinafine and fluconazole against strains of
dermatophyte by following the NCCLS guidelines for testing
filamentous fungi.
Materials and methods
Microscopic examination and cultures of samples from 561 patients
from the Dermatology Clinic were performed in the Mycology
laboratory between June 2002 and January 2004. The samples included
toe nail, foot, inguinal region, trunk, hands and head. Microscopic
examinations of samples were performed with potassium hydroxide 15%
(Antibioticos S.p.A, Rodano, Italy) and 0.1% calcoflour white
solution (Sigma, Poole, United Kingdom). The method of urea
hydrolysis, in vitro hair perforation tests, growth on polished
rice grains, colony characteristics and microscopic morphology were
used for identification [6].
Preparation of inocula
The isolates were subcultured onto potato dextrose agar (PDA)
(Oxoid, Basingstoke, Hampshire, United Kingdom) plates at 28 °C.
Stock inoculum suspensions of each isolate were prepared for each
experiment from 7 to 14 day-old cultures grown on PDA. The fungal
colonies were covered with ca. 10 mL of distilled water, and
suspensions were made by gently probing the surface with the tip of
a Pasteur pipette. The resulting mixture of conidia and hyphal
fragments was withdrawn and transferred to a sterile tube. Heavy
particles of the suspension (when they were present) were allowed
to settle for 3 to 5 min, and the upper homogeneous suspension
was used for further testing. The suspensions were mixed for 15
seconds with a vortex mixer, and their densities were read using a
spectrophotometer at a wavelength of 530 nm and adjusted to 95%
transmittance [3].
The suspensions containing conidia and hyphal fragments were
diluted 1:10 RPMI 1640 medium (with L-glutamine without
bicarbonate) (Sigma, Steinheim, Germany), (pH 7.0, with 0.165 M
morpholinepropanesulfonic acid) (Merc, Darmstadt, Germany) to
obtain the final desired inoculum size of approximately 0.5 ×
104 to 5 × 104 CFU/mL. Inoculum
quantification was performed in the laboratory by plating
0.01 mL of a 1:100 dilution of the adjusted inoculum on PDA
plates. The plates were incubated at 28 °C and examined for the
presence of fungal colonies. Inoculum colonies were counted as
CFU/milliliter when growth became visible.
The final concentrations of the antifungal agents were 0.03-16
μg mL–L for ketoconazole (Ilsan-Iltas, Istanbul,
Turkey), 0.03-16 μg mL–L for terbinafine (Novartis,
Istanbul, Turkey), 0.03-16 μg mL–L for itraconazole
(Neuland laboratories limited, Jinnararn, India) and 0.125-64 μg
mL–L for fluconazole (Pfizer, Istanbul, Turkey). Serial
drug dilutions were performed according to the NCCLS reference
method, beginning at 100 times the test concentration followed by a
further 1:50 dilution in RPMI medium to yield twice the final
concentration required for testing [3, 7, 8].
The microplates were incubated at 28 °C and were read at 3, 7,
and 14 days of incubation. The minimum inhibitory concentrations
(MICs) were determined by visual inspection of the growth
inhibition of each well compared with that of the growth control
(drug-free) well. Following incubation, the MICs of ketoconazole,
terbinafine, itraconazole and fluconazole were read as the lowest
concentration at which 80% inhibition of growth. MIC quantification
was performed by plating 10 μL samples on PDA plates. These
samples were obtained from first well above and third well under of
MICs values. PDA plates were evaluated at the seventh day ( (figure 1) ).
Quality control
Quality control was ensured by testing the NCCLS recommended strain
Candida parapsilosis ATCC 22019.
Statistical analysis
Statistical analysis of the collected data was performed by means
of SPSS Program (Statistical Software Package of Social Sciences,
version 10). Statistical analysis was performed using chi-square
test and one way ANOVA tests.
Results
Hypha was microscopically in 293/561 (52.2%) of samples. Growth
rate was found as 123/293 (42.0%) in samples showing hypha. Growth
was found as 5/268 (1.7%) in samples that did not show hypha. The
isolated specimens of dermatophytes strains were obtained from the
toe nail 62 (48.4%), foot 40 (31.3%), inguinal region 16 (12.5%),
trunk 5 (3.9%), hands 3 (2.3%) and head 2 (1.6%). The frequency of
dermatophytes localization in the nail and foot was found higher
than in other regions (p < 0.01) (table 1)( Table 1 ).
The distribution of isolated species, 128 dermatophytes strains
were Trichophyton rubrum 108 (84.4%), Trichophyton mentagrophytes
11 (8.6%), Epidermophyton floccosum 5 (3.9%), Microsporum canis 2
(1.5%), and Trichophyton tonsurans 2 (1.5%). Detectable growth
could be clearly visualized for all remaining isolates within seven
days. No difference was found between visual evaluation and by
control culture examination ( (figure 1) ).
Mean MIC values (± SE) of ketoconazole, terbinafine,
itraconazole and fluconazole for dermatophytes by the microdilution
methods are shown in table 2( Tableau 2
).
The MICs for five strains of dermatophytes ranged between 0.03-8
μg/mL for ketoconazole, 0.03-4 μg/mL for terbinafine, 0.03-8 μg/mL
for itraconazole and 0.25-64 μg/mL for fluconazole. The mean
minimum inhibitory concentrations (MICs) of fluconazole were
consistently higher for dermatophytes (p < 0.001).
Table 1 Isolated dermatophyte strains in relation to
localization
|
|
|
Localization
|
|
|
|
Toe nail
|
Foot
|
Inguinal region
|
Trunk
|
Hands
|
Head
|
|
Dermatophytes
|
n
|
%
|
n
|
%
|
n
|
%
|
n
|
%
|
n
|
%
|
n
|
%
|
n
|
%
|
|
Trichophyton rubrum
|
108
|
84.4
|
55
|
88.7
|
34
|
85.0
|
12
|
75.0
|
4
|
80.0
|
3
|
100.0
|
0
|
0.0
|
|
Trichophyton mentagrophytes
|
11
|
8.6
|
5
|
8.1
|
4
|
10.0
|
1
|
6.3
|
0
|
0.0
|
0
|
0.0
|
1
|
50.0
|
|
Epidermophyton floccosum
|
5
|
3.9
|
1
|
1.6
|
0
|
0.0
|
3
|
18.7
|
1
|
20.0
|
0
|
0.0
|
0
|
0.0
|
|
Microsporum canis
|
2
|
1.5
|
0
|
0.0
|
2
|
5.0
|
0
|
0.0
|
0
|
0.0
|
0
|
0.0
|
0
|
0.0
|
|
Trichophyton tonsurans
|
2
|
1.5
|
1
|
1.6
|
0
|
0.0
|
0
|
0.0
|
0
|
0.0
|
0
|
0.0
|
1
|
50.0
|
|
Total
|
128
|
100.0
|
62
|
100.0
|
40
|
100.0
|
16
|
100.0
|
5
|
100.0
|
3
|
100.0
|
2
|
100.0
|
Tableau 2 MICs of the for drugs against the different
species of dermatophytes
|
Dermatophytes
|
n
|
Ketocozole
|
Terbinafine
|
|
Range µg/mL
|
Mean
|
MIC 50
|
MIC 90
|
Range µg/mL
|
Mean
|
MIC 50
|
MIC 90
|
|
Trichophyton rubrum
|
108(%84.4)
|
0.03-8
|
0.39 ± 0.92
|
0.25
|
0.5
|
0.03-4
|
0.17 ± 0.43
|
0.6
|
0.25
|
|
Trichophyton mentogrophytes
|
11(%8.6)
|
0.25-2
|
0.75 ± 0.49
|
0.5
|
1
|
0.03-1
|
0.27 ± 0.36
|
0.125
|
1
|
|
Epidermophyton floccosum
|
5(%3.9)
|
0.125-4
|
1.12 ± 1.64
|
0.25
|
4
|
0.3-0.06
|
0.05 ± 0.02
|
0.06
|
0.06
|
|
Microsporum canis
|
2(%1.5)
|
0.06-0.125
|
0.09 ± 0.04
|
0.06
|
0.125
|
0.06
|
0.06 ± 0.0
|
0.06
|
0.06
|
|
Trichophyton tonsurans
|
2(%1.5)
|
0.125-0.25
|
0.19 ± 0.09
|
0.125
|
0.25
|
0.3-0.06
|
0.04 ± 0.02
|
0.03
|
0.06
|
|
Dermatophytes
|
n
|
Itraconozole
|
Fluconazole
|
|
Range µg/mL
|
Mean
|
MIC 50
|
MIC 90
|
Range µg/mL
|
Mean
|
MIC 50
|
MIC 90
|
|
Trichophyton rubrum
|
108(%84.4)
|
0.06-8
|
0.43 ± 0.92
|
0.25
|
0.5
|
0.25-64
|
6.67 ± 9.94
|
4
|
16
|
|
Trichophyton mentogrophytes
|
11(%8.6)
|
0.06-1
|
0.37 ± 0.33
|
0.25
|
1
|
2-64
|
16.18 ± 19.02
|
8
|
32
|
|
Epidermophyton floccosum
|
5(%3.9)
|
0.03-0.25
|
0.11 ± 0.09
|
0.125
|
0.25
|
1-64
|
19.6 ± 25.31
|
8
|
64
|
|
Microsporum canis
|
2(%1.5)
|
0.03-0.125
|
0.08 ± 0.07
|
0.03
|
0.125
|
16-32
|
24.0 ± 11.31
|
16
|
32
|
|
Trichophyton tonsurans
|
2(%1.5)
|
0.125-0.5
|
0.31 ± 0.26
|
0.125
|
0.5
|
8-32
|
20.0 ± 16.97
|
8
|
32
|
Discussion
Dermatomycosis is caused by a series of Dermatophytes, with
worldwide predominance of T. rubrum as the main causal agent [9,
10]. Most superficial infections caused by dermatophytes can be
rapidly eradicated with topical and systemic antifungals. Numerous
topical agents and several systemic ones are available, but
comparison of their in vitro activity against dermatophytes has
been hampered by the lack of a well accepted MIC assay for these
fungi [11]. Fernandez-torres et al. [3] recommended NCCLS for
testing filamentous fungi. The same methods were used in our study.
We compared antifungal activities of dermatophyte strains
isolated from the studied groups against ketoconazole, terbinafine,
itraconazole, and fluconazole. Ketoconazole can induce hepatitis
due to idiosyncrasy, with fatal outcome. For that reason
ketoconazole is no longer used for onychomycosis. However, it can
still be used for skin and hair disease if topical treatment is not
effective. The allylamine terbinafine belongs to the newer
antifungal agents as well. In vitro activity is directed against a
broad range of dermatophytes and moulds as well, but has a lower
activity against yeasts [1]. Itraconazole is a triazole antifungal
agent. The advantage of itraconazole is its activity spectrum.
Itraconazole concentration was significantly higher in pathological
skin than non-pathological skin. The itraconazole concentration in
the lesional tissues was higher in the central sites than in the
marginal sites [12]. However, absorption problems of itraconazole
limit its clinical usefulness [4, 13, 14]. Fluconazole, a
bis-triazole antifungal agent characterized by good
bioavailability, low protein binding, and a long half-life of about
30 hours in serum [4].
Favre et al. [11] reported that allylamine terbinafine was the
most potent agent against some dermotophytes spp. Nimura et al.
[15] observed that terbinafine was an extremely potent antifungal
activity against Trichophyton spp. Fernandez-Torres et al. [16]
reported the results of the in vitro activities of 10 antifungal
agents against 24 species of dermatophytes represented by 508
strains. In general, the three drugs were very active against all
the species tested. Overall, terbinafine was the most active,
showing the lowest geometric mean MIC (0.04 μg/mL). Itraconazole
showed good antifungal activity, with its geometric mean MICs being
similar (0.21 and 0.42 μg/mL, respectively). Terbinafine was very
active against all the species. However, it was ineffective against
Microsporum cookei and Microsporum racemosum.
In a study by Perea et al. [8] the calculated MICs of the
controls were within an acceptable range for the six drugs tested.
The comparison of the in vitro susceptibilities to voriconazole and
other agents showed that voriconazole was more active than
ketoconazole, griseofulvin, and fluconazole against all species and
was less active than itraconazole and terbinafine. Korting et al.
[4] reported that all isolates could be attributed to only three
closely related concentration steps in the cases of terbinafine
(0.001 to 0.05 μg/mL) and ketoconazole (0.5 to 2.0 μg/mL), with
terbinafine also exhibiting the lowest MICs among all the tested
antimycotic agents. The highest MICs were measured for fluconazole,
with two isolates requiring 1.024 μg/mL for complete growth
prevention. Düver et al. [17] studied antifungal susceptibility in
T. rubrum specimens and found MIC intervals of fluconazole,
ketoconazole, itraconazole and terbinafine as < 0.125-4 μg/mL,
< 0.031-2 μg/mL, < 0.031-l μg/mL and < 0.031-0.5 μg/mL,
respectively. Terbinafine was reported to be the most effective
antifungal agent against T. rubrum specimens. Terbinafine was
reported to be an extremely potent agent against dermatophytes
[18].
In our study, we observed that terbinafine had the lowest MIC
values compared to ketoconazole, itraconazole and fluconazole (p
< 0.001). The mean minimum inhibitory concentration of
fluconazole was consistently higher for dermatophyte species. The
mean MICs for the five species of dermatophytes ranged between
0.09-1.12 μg/mL for ketoconazole, 0.04-0.27 μg/mL for terbinafine,
0.08-0.43 μg/mL for itraconazole, and 16.18- 24.0 μg/mL for
fluconazole. Our results correlated with the results of other
investigators.
In conclusion, it may be useful to undertake periodical
screening programs to detect a possible species of, and to have the
affected subjects under control for early diagnosis or treatment of
fungal infections. Our data on the prevalence and susceptibility of
dermatophyte isolates may contribute to a rational choice of
antifungal treatment. We consider that studies on the antifungal
susceptibility of dermatophytes can be beneficial for investigation
of development of in vitro resistance, which has not yet been
encountered in dermatophyte species, and for management of cases
clinically unresponsive to treatment.
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