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Activité Antifongique Dans Les Plaques Elisa 96 Puits
Activité Antifongique Dans Les Plaques Elisa 96 Puits
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0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.4.1917–1920.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Fifty clinical isolates of Trichophyton rubrum were selected to test with ketoconazole, fluconazole, itracon-
azole, griseofulvin, and terbinafine by following the National Committee for Clinical Laboratory Standards
The dermatophytes are a group of closely related fungi that standardized to facilitate the establishment of quality control
have the capacity to invade the keratinized tissue of humans parameters and interpretative breakpoints (10). In spite of the
and other animals and produce dermatophytoses (24). The lack of a standardized method for testing dermatophytes, sev-
incidence of dermatophytoses has increased over recent years, eral authors have published various articles wherein several
particularly in immunocompromised patients (7, 22, 23). The species of these fungi have been tested (6, 9, 18, 19). In these
treatment of these cutaneous infections is based on the use of publications, different adaptations or modifications of the
topical and systemic antifungal agents (19). For localized non- NCCLS method have been made (3, 6).
extensive lesions, topical therapies are generally used. For The purpose of this study was to evaluate the variability of
tinea unguium, scalp ringworm, extensive lesions, or skin le- different microdilution susceptibility testing conditions (me-
sions with foliculitis, systemic antimycotic agents are necessary dium, incubation time, and temperature) for the determination
and itraconazole and terbinafine are the oral drugs presently of MICs of five antifungal drugs presently available for the
used most to treat severe conditions (7). Trichophyton rubrum, treatment of dermatophytoses for 50 clinical isolates of T.
among other dermatophytes, is a major causative agent for rubrum.
superficial dermatomycosis like onychomycosis and tinea pedis
and is known to account for as many as 70% of all dermato-
phyte infections (24). Infections due to T. rubrum are often MATERIALS AND METHODS
associated with frequent relapses following cessation of anti- Study design. Each isolate was tested with ketoconazole, fluconazole, itracon-
fungal therapy (15). azole, griseofulvin, and terbinafine by following the NCCLS susceptibility testing
In vitro analysis of the antifungal activity enables the com- guidelines for filamentous fungi (M38-A) (16). In addition, other susceptibility
parison between different antimycotics, which in turn may clar- testing conditions were evaluated: (i) three medium formulations including
RPMI 1640 (standard medium), McVeigh & Morton (MVM), and Sabouraud
ify the reasons for the lack of clinical response and assist dextrose broth (SDB); (ii) two incubation temperatures (28 and 35°C); and (iii)
clinicians in choosing an effective therapy for their patients (9). three incubation periods (4, 7, and 10 days).
In the in vitro method proposed by the National Committee Isolates. We selected 50 clinical isolates of T. rubrum which were maintained
for Clinical Laboratory Standards (NCCLS) for testing fila- in sterile saline (0.9%) at 4°C until testing was performed. The strains Candida
parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), T. rubrum (ATCC
mentous fungi, dermatophytes are not included (7). However,
40051), and Trichophyton mentagrophytes (ATCC 40004) were included as quality
it is important that methodologies used for in vitro testing be controls.
Media. The standard RPMI 1640 medium was buffered with 0.165 M mor-
pholinepropanesulfunic acid (MOPS) at 34.54 g per liter at pH 7.0. MVM was
* Corresponding author. Mailing address: Universidade Federal de prepared as recommended by Shadomy et al. (21), and SDB was prepared at pH
Minas Gerais, Departamento de Microbiologia, Av. Antônio Carlos, 7.0. All drugs were tested in the three media mentioned above for all isolates.
6627, P. O. Box 486, Belo Horizonte, Minas Gerais, Brazil. Phone: Antifungal drugs. Antifungal drugs were donated as follows: ketoconazole by
5531 499 2758. Fax: 5531 499 2730. E-mail: handan@mono.icb Janssen-Cilag, fluconazole by Pfizer, terbinafine by Novartis, and griseofulvin by
.ufmg.br. Schering Plough. Itraconazole was used in its commercial formulation (Janssen-
1917
1918 SANTOS AND HAMDAN J. CLIN. MICROBIOL.
TABLE 1. Ketoconazole, fluconazole, griseofulvin, itraconazole, and terbinafine in vitro susceptibility data for T. rubrum (ATCC 40004) and
T. mentagrophytes (ATCC 40051) with standard RPMI medium
Geometric mean MIC (g/ml)
Incubation
time Ketoconazole Flucoconazole Griseofulvin Itraconazole Terbinafine
(days)
T. rubrum T. mentagophytes T. rubrum T. mentagophytes T. rubrum T. mentagophytes T. rubrum T. mentagophytes T. rubrum T. mentagophytes
7 0.5 0.5 32.0 16.0 1.0 0.5 0.031 0.125 ⬍0.031 ⬍0.031
10 0.5 0.5 32.0 32.0 1.0 0.5 0.031 0.125 ⬍0.031 ⬍0.031
Cilag). All drugs were dissolved in 100% dimethyl sulfoxide (Gibco) following were performed by Wilcoxon (Mann-Whitney) and Kruskal-Wallis tests. A P
the protocol of NCCLS and were prepared in stock solutions of 1,000 g/ml. value of ⬍0.05 was considered significant.
Drug dilutions. Serial twofold dilutions were prepared according to the
NCCLS approved document (M38-A) at 100 times the strength of the final
TABLE 2. Ketoconazole, fluconazole, griseofulvin, itraconazole, and terbinafine in vitro susceptibility data for 50 T. rubrum isolates under
different broth microdilution testing conditions
MIC (g/ml) of:
Incubation
Medium time Ketoconazole Fluconazole Griseofulvin Itraconazole Terbinafine
(days)
Range 50% 90% Range 50% 90% Range 50% 90% Range 50% 90% Range 50% 90%
RPMI 7 0.0625–2.0 0.5 1.0 1.0–⬎64.0 ⬎64.0 ⬎64.0 0.25–2.0 1.0 1.0 ⬍0.031–1.0 0.125 0.25 ⬍0.031 ⬍0.031 ⬍0.031
10 0.0625–2.0 1.0 2.0 8.0–⬎64.0 ⬎64.0 ⬎64.0 0.25–2.0 1.0 1.0 ⬍0.031–1.0 0.25 0.5 ⬍0.031 ⬍0.031 ⬍0.031
SDB 7 0.031–2.0 0.25 1.0 2.0–32.0 16 32.0 0.25–2.0 0.5 1.0 ⬍0.031–0.5 0.0625 0.25 ⬍0.031 ⬍0.031 ⬍0.031
10 0.0625–2.0 0.5 1.0 4.0–32.0 32 32.0 0.25–4.0 1.0 2.0 ⬍0.031–0.5 0.125 0.25 ⬍0.031 ⬍0.031 ⬍0.031
MVM 7 0.125–2.0 0.5 1.0 4.0–⬎64.0 32 ⬎64.0 0.25–1.0 0.5 1.0 ⬍0.031–1.0 0.125 0.25 ⬍0.031 ⬍0.031 ⬍0.031
10 0.125–2.0 0.5 1.0 4.0–⬎64.0 64 64.0 0.25–2.0 1.0 1.0 0.031–1.0 0.125 0.5 ⬍0.031 ⬍0.031 ⬍0.031
VOL. 43, 2005 SUSCEPTIBILITY TESTING CONDITIONS FOR T. RUBRUM 1919
than when using MVM or SDB (P ⬍ 0.05). When terbinafine able visible growth of filamentous fungi, including dermato-
was tested, no parameter had any influence on MICs (P ⬍ phytes (4, 18). Between MVM and SDB, the first was the
0.05). medium which was more similar in comparison to RPMI. Al-
though MVM is a chemically defined medium (20) often used
in MIC determination for Paracoccidioides brasiliensis (11) by
DISCUSSION
the broth macrodilution method, MIC reading in microdilution
Although a standard method for the susceptibility testing of plates is harder because of the transparency of this medium
dermatophytes is lacking, there are several reports where the compared to the yellow color of the other media, which con-
antimicrobial susceptibility testing of dermatophytes has been fuses during visualization. There is no report about the use of
conducted by using either agar macrodilution or broth mac- MVM medium in MIC determination for dermatophytic fungi.
rodilution and microdilution tests; these reports have generally There is a scarcity of reports about the use of SDB for MIC
been extensions of either M27-A2 or M38-A methodologies. determination for any fungi, including dermatophytes (17). We
Results obtained by some authors (3) have shown great vari- tested SDB medium because its cost is considerably lower than
ability, probably due to the lack of standardization of different that of other tested media mentioned above and it is used in all
8. Fernández-Torres, B., I. Inza, and J. Guarro. 2003. Comparison of in vitro method for broth dilution antifungal susceptibility testing of filamentous
antifungal susceptibilities of conidia and hyphae of dermatophytes with fungi. Approved standard M38-A. National Committee for Clinical Labora-
thick-wall macroconidia. Antimicrob. Agents Chemother. 47:3371–3372. tory Standards, Wayne, Pa.
9. Ghannoum, M. A., V. Chaturvedi, A. Espinel-Ingroff, M. A. Pfaller, M. G. 17. Nimura, K., Y. Niwano, S. Ishiduka, and R. Fukumoto. 2001. Comparison of
Rinaldi, W. Lee-Yang, and D. W. Warnock. 2004. Intra- and interlaboratory in vitro antifungal activities of topical antimycotics launched in 1990s in
study of a method for testing antifungal susceptibilities of dermatophytes. Japan. Int. J. Antimicrob. Agents 18:173–178.
J. Clin. Microbiol. 42:2977–2979. 18. Norris, H. A., B. E. Elewski, and M. A. Ghannoum. 1999. Optimal growth
10. Gupta, A. K., and Y. Kohli. 2003. In vitro susceptibility testing of ciclopirox, conditions for determination of the antifungal susceptibility of three species
terbinafine, ketoconazole and itraconazole against dermatophytes and non- of dermatophytes with the use of a microdilution method. J. Am. Acad.
dermatophytes, and in vitro evaluation of combination antifungal activity. Dermatol. 40:S9–S13.
Br. J. Dermatol. 149:296–305. 19. Perea, S., A. W. Fothergill, D. A. Sutton, and M. G. Rinaldi. 2001. Compar-
11. Hahn, R. C., and J. S. Hamdan. 2000. Effects of amphotericin B and three ison of in vitro activities of voriconazole and five established antifungal
azole derivates on the lipids of yeasts cells of Paracoccidioides brasiliensis. agents against different species of dermatophytes using a broth macrodilu-
Antimicrob. Agents Chemother. 44:1997–2000. tion method. J. Clin. Microbiol. 39:385–388.
12. Jessup, C. J., J. Warner, N. Isham, I. Hasan, and M. A. Ghannoum. 2000.
20. Restrepo, A. M., and B. E. Jimenez. 1980. Growth of Paracoccidioides
Antifungal susceptibility testing of dermatophytes: establishing a medium for
brasiliensis yeast phase on a chemically defined culture medium. J. Clin.
inducing conidial growth and evaluation of susceptibility of clinical isolates.
Microbiol. 12:279–281.
J. Clin. Microbiol. 38:341–344.
21. Shadomy, S., A. Espinel-Ingroff, and R. Y. Cartwright. 1987. Estudios de