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In vitro susceptibility testing of dermatophytes: comparison of disk diffusion and reference broth dilution methods
Diagnostic Microbiology and Infectious Disease 48 (2004) 259 –264
www.elsevier.com/locate/diagmicrobio
Mycology
In vitro susceptibility testing of dermatophytes: comparison of disk diffusion and reference broth dilution methods Nuriye Karaca, A. Nedret Koc¸* Department of Microbiology, Erciyes University, School of Medicine, Kayseri, Turkey Received 6 August 2003; accepted 13 October 2003
Abstract A total of 56 strains belonging to 4 species of dermatophytes were tested against 10 antifungal drugs by using a modification of the NCCLS (M38-P) standard for filamentous fungi. The minimum inhibitory concentration (MIC) values obtained using the dilution method were compared with the diameters of growth inhibition zones using the disk diffusion method. The antifungals used were itraconazole, fluconazole, ketoconazole, miconazole, sulconazole, oxiconazole, bifonazole, griseofulvin, ciclopiroxolamine, and terbinafine. Relative to the other agents tested, terbinafine possessed the highest antifungal activity against all of the dermatophytes. In contrast, fluconazole was the least active drug. An increase of MIC values was accompanied by a decrease of growth inhibition zone diameter. The disk diffusion method of fungal susceptibility assessment yields data consistent with results obtained from the dilution method. The study suggests the potential value of the disk diffusion method as a convenient alternative method for testing the susceptibilities of dermatophytes. © 2004 Elsevier Inc. All rights reserved. Keywords: Dermatophytes; Antifungals; Disk diffusion; Broth dilution
1. Introduction Dermatophytes affect keratinous tissue of humans and of other vertebrates, causing superficial infections. The organisms belong to three genera, Trichophyton, Epidermaphyton, and Microsporum (Weitzman & Summerbell, 1995; Kane & Summerbell, 1999; Hay, 2000). Dermatophytoses generally respond well to topical antifungal therapy, although local therapy may be inappropriate for extensive infections or for infections affecting the nail or scalp. In recent years, a number of safe and highly effective antifungal agents have been introduced into clinical practice. Among them, terbinafine, itraconazole, fluconazole, and more recently voriconaole and other azoles, still under investigation (Weitzman & Summerbell, 1995; McGinnis & Rinaldi, 1996; Norris et al., 1999). However, their activity against significant number of strains, representing a wide spectrum of dermatophyte species and following standard procedures, has not yet been investigated. The NCCLS published a standard method, M38-P (NCCLS, 1998), for
determining the MICs of several antifungal agents against conidium-forming filamentous fungi. For use in clinical laboratories to perform susceptibility testing, a simple, inexpensive, and reliable method is needed. A simplified disk diffusion test has the same important advantages. For practical reasons, that disk technique should be similar to be disk procedure that is being used to test antibacterial agents. Unfortunately, the procedure for antifungal agents remains in its infancy. The aim of this study has been to evaluate the in vitro activity of the traditionally available antifungal agents against a significant number of strains of dermatophytes by following the NCCLS guidelines for testing filamentous fungi, and compare the two methods of evaluating the efficacy of antifungal agents: a dilution method yielding the MIC value and disk diffusion method.
2. Materials and methods 2.1. Isolates
* Corresponding author. Tel.: ⫹90-352-437-4901-23381; fax: ⫹90352-437-96-55. E-mail address: [email protected] (A. Koc¸). 0732-8893/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2003.10.012
Fifty-six strains were isolated from infected skin, nail, and hair in the Microbiology and Clinical Microbiology
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Department of the Gevher Nesibe Hospital of Erciyes University. Isolates were collected over a 2-year period in the Mycology Laboratory. They included 50 strains of T. rubrum, 3 strains of T. mentagrophytes, 2 strains of T. tonsurans, and one strain of T. verrucosum. All isolates were identified by standard methods, which included identification based on the macroscopic and microscopic characteristics of the culture strains (Larone, 1993; Kane & Summerbell, 1999; Tu¨ mbay, 1983). Additional tests included those for the ability to produce a red pigment when the strains were grown on Potato Dextrose Agar (PDA) and for the ability to produce urease, as well as the hair perforation test (Larone, 1993; Tu¨ mbay, 1983). Isolates were stored at ⫺70°C on Brucella Broth (Acumedia) with 5% glycerine until the time of use, and prior to testing were subcultured on PDA at 28°C for 15 days to ensure optimal growth characteristics. The strain C. albicans ATCC 90028 was included as quality control. 2.2. Medium RPMI 1640 broth medium (Sigma Chemical Company, Madrid, Spain) with L-glutamine but without sodium bicarbonate and buffered at pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma, Madrid) was the medium used for broth microdilution susceptibility testing. Agar formulations used for the disk diffusion test were RPMI 1640 broth supplemented with 1.5% Bacto Agar (Difco Laboratories, Detroid, Mich.), and 2% glucose, and buffered to MOPS (Barry et al., 1996). The 15-cm diameter Petri plates contain RPMI at a depth of 4.0 mm. 2.3. Antifungal agents Ten antifungal drugs, supplied by the manufacturers as powders, were used: fluconazole (Fako Co., Istanbul, Turkey), ketoconazole (Bilim Co., Istanbul, Turkey), itraconazole (Nobel Co., Istanbul, Turkey), bifonazole (Sigma, Madrid), sulconazole (Sigma, Madrid), oxiconazole (Roche), miconazole (Sigma, Madrid), terbinafine (Novartis-Pharma), griseofulvin (Acros Chemical Company), and ciclopiroxolamine (Sigma, Madrid). Fluconazole was dissolved in sterile water; ketoconazole, itraconazole, sulconazole, oxiconazole, miconazole, bifonazole, griseofulvin and terbinafine were dissolved in 100% dimethyl sulfoxide; ciclopiroxolamine was dissolved in 20% methanol. All drugs were prepared as stock solutions of 1280 mcg/ml and stored at ⫺70°C until use. Serial twofold drug dilutions were prepared according to the NCCLS reference method (NCCLS 1998). Final drug concentrations were 0.006 to 32 mcg/ml for fluconazole, 0.008 to 4 mcg/ml for miconazole, sulconazole, oxiconazole, and ketoconazole, 0.016 to 8 mcg/ml for bifonazole and griseofulvin, 0.001 to 1 mcg/ml for terbinafine and itraconazole, and 0.03 to 16 mcg/ml for ciclopiroxolamine. 10-fold drug concentrations prepared in
twofold serial dilutions 0.1-ml drug volume) were maintained at ⫺70°C until needed. Disks of fluconazole, itraconazole, and ketoconazole for disk diffusion test were obtained (Rosco) in the potence of 15 mcg/disk, 8 mcg/disk, and 15 mcg/disk, respectively. Disks for other drugs were prepared considering the potence in the Neo-Sensitabs Susceptibility Testing Catalog (Rosko 1998), 25 mcg/disk for griseofulvin, bifonazole, sulconazole and oxiconazole, 10 mcg/disk for miconazole, 5 mcg/ disk for terbinafine, and 50 mcg/disk for ciclopiroxolamine (Munro 1992). Disks were stored at ⫺70°C until used. 2.4. In vitro susceptibility testing methods The broth microdilution method was performed according to NCCLS M38-P guidelines (NCCLS, 1998) and as described by Espinel-Ingroff et al. (Espinel-Ingroff et al., 1997; Espinel-Ingroff et al., 1999). Each well was inoculated on the day of the test with 100 l of the 2X conidial inoculum suspension mentioned above. This step diluted the drug concentrations and inoculum densities to the final desired test concentration. The growth control wells contained 100 l of sterile drug-free medium and were inoculated with 100 l of the corresponding diluted inoculum suspension. Sterility control was performed by including 100 l of uninoculated, drug-free medium. The disk diffusion method was performed in accordance with used the agar diffusion used test in clinical bacteriology (NCCLS 1999). 2.5. Preparation of both test inocula Stock inoculum suspensions of the dematophytes were prepared from 7- to 15-day old cultures grown on PDA at 28°C. Mature colonies were covered with approximately 10 ml of sterile 85% saline, and the suspensions were made by gently probing the colony with the tip of a Pasteur pipette. The resulting mixture of conidia and hyphal fragments was with drawn and transferred to sterile tubes. Heavy particles of the suspension were allowed to settle for 10 to 15 min at room temperature, and the upper homogeneous suspension was used for further testing. The optical densities of the suspensions were read at 530 nm and adjusted to 0.15 to 0.17 to yield 0.6 ⫻ 106 to 1.4 ⫻ 106 to spores/ml of strains. The suspensions containing conidia and hyphal fragments were diluted with RPMI 1640 medium to obtain the final desired inoculum size of approximately 0.4 ⫻ 104 to 5 ⫻ 104 spores/ml. The microdilution plates were incubated at 28°C for 10 to 15 days. The MICs endpoint for fluconazole, ketoconazole, itraconazole, sulconazole, oxiconazole, bifonazole, and miconazole were defined as the lowest concentration that produced prominent inhibition of growth (approximately 50% inhibition) relative to the drug-free growth control, and the MICs of terbinafine, griseofulvin, and ciclopiroxolamine were 80% (Espinel-Ingroff et al., 1997; NCCLS 1998). Spore suspensions adjusted to a con-
N. Karaca, A.N. Koc¸ / Diagnostic Microbiology and Infectious Disease 48 (2004) 259 –264
261
Table 1 In vitro activities of 10 antifungal agents against 56 strains of dermatophytes Species (no. of strains tested)
Antifungal agents
MIC ranges (mcg/ml)
MICs GMa
MIC50sc (mcg/ml)
Fluconazole Ketoconazole Itraconazole Sulconazole Oxiconazole Bifonazole Miconazole Terbinafine Griseofulvin Ciclopiroxolamine
⬍0.06–⬎32 ⬍0.008–⬎4 ⬍0.001–1 ⬍0.008–4 ⬍0.008–2 ⬍0.016–1 0.008–4 ⱕ0.001–1 0.016–8 ⬍0.03–8
8 0.5 0.1 0.5 0.2 0.2 0.8 0.02 0.6 0.5
4 0.25 0.008 0.25 0.125 0.25 0.25 0.002 0.5 0.25
Fluconazole Ketoconazole Itraconazole Sulconazole Oxiconazole Bifonazole Miconazole Terbinafine Griseofulvin Ciclopiroxolamine
16–32 0.008–4 0.008–0.06 0.016–0.125 0.06–0.5 0.125–0.25 1 ⱕ0.001 0.016–0.125 0.5–1
26.7 1.3 0.03 0.06 0.4 0.2 1 0.001 0.09 0.08
32 0.016 0.016 0.03 0.5 0.25 1 0.001 0.125 1
Fluconazole Ketoconazole Itraconazole Sulconazole Oxiconazole Bifonazole Miconazole Terbinafine Griseofulvin Ciclopiroxolamine
0.25–8 0.016–0.125 ⱕ0.001–0.5 ⱕ0.008–0.03 ⱕ0.008–2 ⱕ0.016–0.5 0.016–0.25 ⱕ0.001–0.008 0.5–1 0.125–1
4 0.07 0.25 0.02 1 0.25 0.1 0.005 0.75 0.6
Fluconazole Ketoconazole Itraconazole Sulconazole Oxiconazole Bifonazole Miconazole Terbinafine Griseofulvin Ciclopiroxolamine
16 4 1 4 4 8 4 1 1 4
MIC90sc (mcg/ml)
Mean inhibition zone diameter ⫾ SEb (cm)
32 1 0.5 1 0.5 0.5 2 0.008 1 1
25.32 ⫾ 12.07 39.4 ⫾ 6.57 42.68 ⫾ 9.59 57.26 ⫾ 11.96 59.4 ⫾ 7.97 42.26 ⫾ 7.03 35.48 ⫾ 9.08 95.92 ⫾ 12.52 47.4 ⫾ 14.24 46.06 ⫾ 7.06
Trichophyton rubrum (50)
Trichophyton mentagrophytes (3) 13.33 ⫾ 5.77 33.33 ⫾ 9.45 41.67 ⫾ 7.23 54.0 ⫾ 8.72 54.0 ⫾ 17.44 50.0 ⫾ 12.49 28.33 ⫾ 10.41 92.33 ⫾ 7.51 45.0 ⫾ 11.36 51.33 ⫾ 18.48
Trichophyton tonsurans (2) 44.5 ⫾ 7.78 53.0 ⫾ 24.04 57.5 ⫾ 9.12 68.0 ⫾ 16.97 66.0 ⫾ 25.46 58.0 ⫾ 16.97 57.5 ⫾ 3.54 101.0 ⫾ 1.41 52.5 ⫾ 24.75 39.5 ⫾ 6.36
Trichophyton verrucosum (1) 10 30 24 24 25 12 11 16 32 20
a
Geometric mean. Standard error. c The MICs at which 50% and 90% of the isolates tested were inhibited were determined for each drug (MIC50s and MIC90s, respectively). b
centration of 106 spores/ml for disk diffusion test. Each solidified medium was inoculated by dipping a sterile swab into the cell suspension and streaking it across the surface of the agar. The plates were dried at room temperature for 15 min before applying the antifungal agents disks. The agar plates were incubated at 28°C for 10 days. Zone diameter end points were read at 50% growth inhibition for fluconazole, ketoconazole, itraconazole, sulconazole, oxiconazole, bifonazole, and miconazole. The endpoints of terbinafine, griseofulvin, and ciclopiroxolamine were defined as 80%
inhibition compared with the growth control. Microcolonies within the diameter were ignored. 2.6. Statistics SPSS/PC for IBM package (version 9.0) was used for the statistical analysis of data. P values of ⬍0.05 were considered statistically significant. The correlation between microdilution method and disk diffusion method was analyzed by means of regression analysis.
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Fig. 1. Terbinafine disk zone diameters and NCCLS Reference Method log10 MICs. The regression statistic is y ⫽ 46.184 –18.906 log10 (terbMIC), r ⫽ 0.744.
Fig. 2. Ciclopiroxolamine disk zone diameters and NCCLS Reference Method log10 MICs. The regression statistic is y ⫽ 44.952–1.265 log10 (ciclopiroxMIC), r ⫽ 0.081.
3. Results
4. Discussion
All isolates of dermatophytes tested produced detectable growth at time points ranging from 7 to 15 days. The range of MICs for the microdilution test and mean inhibition zone for the disk diffusion test of 10 antifungal agents are summarized in Table 1. When all the strains were considered together, the geometric mean MICs of terbinafine and itraconazole were apparently the lowest, 0.002 mcg/ml and 0.1 mcg/ml, respectively. Relative to other agents tested terbinafine possessed the highest antifungal activity against dermatophytes. The geometric mean MICs for ketoconazole, sulconazole, oxiconazole, bifonazole, miconazole, griseofulvin and ciclopiroxolamine were 0.5, 0.5, 0.2, 0.2, 0.8, 0.6, and 0.5 mcg/ml, respectively. Application of two methods of testing correlated. An increase of MIC values was accompanied by a decrease of inhibition zone diameters. The observed MICs of all the drugs tested showed a broad range of variability against the species of Trichophyton. The correlation between MIC values and diameter of growth inhibition zone was analyzed by means of regression analysis. A favorable correlation was seen between MIC and size of inhibition zone around the disk for fluconazole, ketoconazole, itraconazole, oxiconazole, miconazole, terbinafine and griseofulvin, and the correlation coefficients r ⫽ ⫺0.694, ⫺0.298, ⫺0.314, ⫺0.504, ⫺0.439, ⫺0.744, and ⫺0.313 (p ⬍ 0.05), respectively. However, correlation was not seen between two methods for bifonazole, sulconazole and ciclopiroxolamine, and the correlation coefficients r ⫽ ⫺0.245, ⫺0.099, and ⫺0.081 (p ⬎ 0.05), respectively. Figs. 1 and 2 have been presented to show the examples of the drugs which have a favorable and no correlation between zone sizes and MICs, respectively.
A reference method for the antifungal susceptibility testing of dermatophytes is not available (Ghannoum, 2001). In recent years, several studies on the in vitro susceptibility of dermatophytes to antifungal drugs have been done and the results have shown considerable variation (Jessup et al., 2000; Mock et al., 1998). Results of antifungal susceptibility tests are influenced by type of medium, pH, inoculum size, incubation temperature and time and endpoid definition on interlaboratory variability (Espinel-Ingroff et al., 1991; Espinel-Ingroff et al., 1992; Ghannoum et al., 1996). In this study, the NCCLS method of testing filamentous fungi (NCCLS 1998) has been adapted for testing 56 strains of dermatophytes. In this study, the MICs values of the strains varied in a wide range as ⬍0.06-⬎32 mcg/ml, 0.016-8 mcg/ml, and ⱕ0.03-8 mcg/ml for fluconazole, griseofulvin, and ciclopiroxolamine, respectively. Similar results were published by other authors (Korting & Rosenkrans, 1989; Korting et al.,1995; Kibar et al., 2001). Our results showed the lowest activity of all the antifungals tested against dermatophytes, previous studies (Korting et al.,1995; Ferna´ ndez-Torres et al., 2001). Terbinafine and itraconazole demonstrated the high in vitro and in vivo activities against dermatophytes (Grant & Clissold, 1989; Balfour & Faulds, 1992). Terbinafine and itraconazole were the most effective antifungal agents, which agrees with a number of studies carried out by other authors (Korting et al.,1995; Ferna´ ndez-Torres et al., 2000; Ferna´ ndez-Torres et al., 2001), and these drugs provided larger inhibition areas in the disk diffusion method (Mock et al., 1998). The effectiveness of the other imidazoles were similar. To be easly integrated into the work flow of clinical laboratory, an antifungal susceptibility test should be as
N. Karaca, A.N. Koc¸ / Diagnostic Microbiology and Infectious Disease 48 (2004) 259 –264
close as possible to the method that is being used to test antibacterial agents. The disk diffusion procedure we used diffused from the standard method only in the use of RPMI agar in place of Mueller-Hinton agar. The inocula are adjusted by using a spectrophotometer to compare turbidity to 0.15 to 0.17 at 530 nm. Dermatophytes inocula have been adjusted as Jessup et al. (2000). Zone size limits for quality control strains have not yet to be defined by a multilaboratory collaborative study. Selection of broth dilution MIC endpoints presents a similar source of variability that must be controlled, but that is not unique to tests of antifungal agents. Barry and Brown (1996) showed that the disk test seems to be appropriate for tests of most Candida species to fluconazole. Similarly, Meis et al.(2000), reported that the disk diffusion method, which was facile and economical to perform, offers a reproducible and accurate means to assess the in vitro susceptibility of Candida species to fluconazole. Macura (1993), has reported that the disk diffusion method of fungal susceptibility assessment yields data consistent with results obtained from the dilution method and recommended for routine use because of its simplicity, which is in agreement with previous reports we found. The correlation between microdilution method and disk diffusion method was superior to those of fluconazole, ketoconazole, itraconazole, oxiconazole, miconazole, terbinafine, and griseofulvin in the susceptibility testing of dermatophytes. There were, however, some discrepancies in ciclopiroxolamine, bifonazole, and sulconazole. To treat successfully the superficial mycoses tending to be chronic infection, isolation of the agent, species identification, and susceptibility to antifungal drugs by microdulition method or disk diffusion method is simple and rapid enough to be applied in routine mycologic diagnostics. However, additional studies are needed to further validate the correlation between the disk test and NCCLS reference micro/macro broth dilution method and may lead to wide acceptance and standardization of fungal disk testing and interpretive criteria. (National Committee for Clinical Laboratory Standards, 1998 and 1999)
References Balfour, J. A., & Faulds, D. (1992). Terbinafine: a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in superficial mycoses. Drugs 43, 259 –284. Barry, A. L., & Brown, S. D. (1996). Fluconazole disk diffusion procedure for determining susceptibility of Candida species. J Clin Microbiol 34, 2154 –2157. Espinel-Ingroff, A., Barlett, M., Bowden, R., et al (1997). Multicenter evaluation of proposed standardized procedure for antifungal susceptibility testing of filamentous fungi. J Clin Microbiol 35, 139 –143. Espinel-Ingroff, A., Kerkering, T. M., Goldson, P. R., & Shadomy, S. (1991). Comparison study of broth macrodilution and microdilution antifungal susceptibility test. J Clin Microbiol 29, 1089 –1094.
263
Espinel-Ingroff, A., Kish, C. W., Kerkering, T. M., et al (1992). Collaborative comparison of broth macrodilution and microdilution antifungal susceptibility tests. J Clin Microbiol 30, 3138 –3145. Espinel-Ingroff, A., White, T., & Pfaller, M. A. (1999). Antifungal agents and susceptibility tests. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, & R. H. Yolken (Eds.). Manual of clinical microbiology, 7th ed, (pp. 1640 –1652). Washington DC: ASM Press. Ferna´ ndez-Torres, B., Carrillo, A. J., Martin, E., Del Palacio, A., Moore, M. K., Valverde, A., Serrano, M., & Guarro, J. (2001). In vitro activities of 10 antifungal drugs against 508 dermatophyte strains. Antimicrob Agents Chemother 45, 2524 –2528. Ferna´ ndez-Torres, B., Va´ zquez-Veiga, H., Llova, X., Pereiro, M., & Guarro, J. (2000). In vitro susceptibility to itraconazole, clotrimazole, ketoconazole and terbinafine of 100 isolates of Trichophyton rubrum. Chemotherapy 46, 390 –394. Ghannoum, M. A. (2001). Antifungal susceptibility testing of dermatophytes. Dermatol Online J 7, 23C. Ghannoum, M. A., Rex, J. H., & Galgiani, J. N. (1996). Susceptibility testing of fungi: current status of correlation of in vitro data with clinical outcome. J Clin Microbiol 34, 489 – 495. Grant, S. M., & Clissold, S. P. (1989). Itraconazole. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in superficial and systemic mycoses. Drugs 37, 310 –344. Hay, R. J. (2000). Dermatophytosis and other superficial mycoses. In G. L. Mandell, J. E. Bennett, & R. Dolin (Eds.). Mandell, Douglas and Bennett’s principles and practice of infections diseases, 5th ed, (pp. 2757–2768). Philadelphia: Churchill Livingstone. Jessup, C. J., Warner, J., Isham, N., Hasan, I., & Ghannoum, M. A. (2000). Antifungal susceptibility testing of dermatophytes: establishing a medium for inducing conidial growth and evaluation of susceptibility of clinical isolates. J Clin Microbiol 38, 341–344. Kane, J., & Summerbell, R. C. (1999). Trichophyton, Microsporum, Epidermophyton, and agents of superficial mycoses. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, & R. H. Yolken (Eds.). Manual of clinical microbiology, 7th ed, (pp. 1275–1294). Washington DC: ASM Press. Kibar, F., Ko¨ ksal, F., Acar, A., Akbaba, M., & Yoldascan, E. (2001). C¸ esitli dermatofitlerin griseofulvin, flukonazol ve itrakonazol duyarlılıklarının in vitro arastırılması. Mikrobiyol Bu¨ lt 35, 299 –305. Korting, H. C., & Rosenkrans, S. (1989). In vitro susceptibility of dermatophytes from Munich to griseofulvin, miconazole and ketoconazole. Mycoses 33, 136 –139. Korting, H. C., Ollert, M., Abeck, D., Abeck, D., et al (1995). Results of German multicenter study of antimicrobial susceptibilities of Trichophyton rubrum and Trichophyton mentagrophytes strains causing tinea unguium. Antimicrob Agents Chemother 39, 1206 –1208. Larone, D. H. (1993). Medically important fungi: a guide to identification, 2nd ed, (pp. 117–137). Washington DC. Macura, A. B. (1993). In vitro susceptibility of dermatophtes to antifungal drugs: a comparison of two methods. Int J Dermatol 32, 533–536. McGinnis, M. R., & Rinaldi, M. G. (1996). Antifungal drugs: Mechanisms of action, drug resistance, susceptibilitiy testing, and assays of activity in biologic fluids. In V Lorian (Ed). Antibiotics in laboratory medicine, 4th ed, (pp. 176 –211). Philadelphia: Williams & Wilkins. Meis, J., Petrou, M., Bille, J., Ellis, D., Gibbs, D., & The Global Antifungal Surveillance Group (2000). A. global evaluation of the susceptibility of Candida species to fluconazole by disk diffusion. Diagn Microbiol Infect Dis 36, 215–223. Mock, M., Monod, M., Baudraz-Rosselet, F., & Panizzon, R. G. (1998). Tinea capitis dermatophytes: susceptibility to antifungal drugs tested in vitro and in vivo. Dermatology 197, 361–367. Munro, S. (1992). Disk diffusion susceptibility testing. In: H. D. Isenberg (Ed.). Clinical microbiolgy procedures handbook, vol: 1, (pp. 5.1–5.2). Washington DC: ASM Press. National Committee for Clinical Laboratory Standards. (1998). Reference method for broth dilution antifungal susceptibility testing of conidium-
264
N. Karaca, A.N. Koc¸ / Diagnostic Microbiology and Infectious Disease 48 (2004) 259 –264
forming filamentous fungi; proposed standard. NCCLS document M38-P. Wayne, PA: NCCLS. National Committee for Clinical Laboratory Standards. (1999). Performance standards for antimicrobial susceptibility testing. Ninth supplement to the M2-A6 M100-S9. Villanova, PA: NCCLS. Norris, H. A., Elewski, B. E., & Ghannoum, M. A. (1999). Optimal growth conditions for the determination of the antifungal susceptibility of three
species of dermatophytes with the use of a microdilution method. J Am Acad Dermatol 40, S9 –S13. Rosko, A. S. (1998). User’s Guide Neo-Sensitabs susceptibility testing, 10th ed. Tu¨ mbay, E. (1983). Pratik tıp mikolojisi, ss 69. Bilgehan Basımevi, I˙zmir. Weitzman, I., & Summerbell, R. C. (1995). The dermatophytes. Clin Microbiol Rev 8, 240 –259.
www.elsevier.com/locate/diagmicrobio
Mycology
In vitro susceptibility testing of dermatophytes: comparison of disk diffusion and reference broth dilution methods Nuriye Karaca, A. Nedret Koc¸* Department of Microbiology, Erciyes University, School of Medicine, Kayseri, Turkey Received 6 August 2003; accepted 13 October 2003
Abstract A total of 56 strains belonging to 4 species of dermatophytes were tested against 10 antifungal drugs by using a modification of the NCCLS (M38-P) standard for filamentous fungi. The minimum inhibitory concentration (MIC) values obtained using the dilution method were compared with the diameters of growth inhibition zones using the disk diffusion method. The antifungals used were itraconazole, fluconazole, ketoconazole, miconazole, sulconazole, oxiconazole, bifonazole, griseofulvin, ciclopiroxolamine, and terbinafine. Relative to the other agents tested, terbinafine possessed the highest antifungal activity against all of the dermatophytes. In contrast, fluconazole was the least active drug. An increase of MIC values was accompanied by a decrease of growth inhibition zone diameter. The disk diffusion method of fungal susceptibility assessment yields data consistent with results obtained from the dilution method. The study suggests the potential value of the disk diffusion method as a convenient alternative method for testing the susceptibilities of dermatophytes. © 2004 Elsevier Inc. All rights reserved. Keywords: Dermatophytes; Antifungals; Disk diffusion; Broth dilution
1. Introduction Dermatophytes affect keratinous tissue of humans and of other vertebrates, causing superficial infections. The organisms belong to three genera, Trichophyton, Epidermaphyton, and Microsporum (Weitzman & Summerbell, 1995; Kane & Summerbell, 1999; Hay, 2000). Dermatophytoses generally respond well to topical antifungal therapy, although local therapy may be inappropriate for extensive infections or for infections affecting the nail or scalp. In recent years, a number of safe and highly effective antifungal agents have been introduced into clinical practice. Among them, terbinafine, itraconazole, fluconazole, and more recently voriconaole and other azoles, still under investigation (Weitzman & Summerbell, 1995; McGinnis & Rinaldi, 1996; Norris et al., 1999). However, their activity against significant number of strains, representing a wide spectrum of dermatophyte species and following standard procedures, has not yet been investigated. The NCCLS published a standard method, M38-P (NCCLS, 1998), for
determining the MICs of several antifungal agents against conidium-forming filamentous fungi. For use in clinical laboratories to perform susceptibility testing, a simple, inexpensive, and reliable method is needed. A simplified disk diffusion test has the same important advantages. For practical reasons, that disk technique should be similar to be disk procedure that is being used to test antibacterial agents. Unfortunately, the procedure for antifungal agents remains in its infancy. The aim of this study has been to evaluate the in vitro activity of the traditionally available antifungal agents against a significant number of strains of dermatophytes by following the NCCLS guidelines for testing filamentous fungi, and compare the two methods of evaluating the efficacy of antifungal agents: a dilution method yielding the MIC value and disk diffusion method.
2. Materials and methods 2.1. Isolates
* Corresponding author. Tel.: ⫹90-352-437-4901-23381; fax: ⫹90352-437-96-55. E-mail address: [email protected] (A. Koc¸). 0732-8893/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2003.10.012
Fifty-six strains were isolated from infected skin, nail, and hair in the Microbiology and Clinical Microbiology
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Department of the Gevher Nesibe Hospital of Erciyes University. Isolates were collected over a 2-year period in the Mycology Laboratory. They included 50 strains of T. rubrum, 3 strains of T. mentagrophytes, 2 strains of T. tonsurans, and one strain of T. verrucosum. All isolates were identified by standard methods, which included identification based on the macroscopic and microscopic characteristics of the culture strains (Larone, 1993; Kane & Summerbell, 1999; Tu¨ mbay, 1983). Additional tests included those for the ability to produce a red pigment when the strains were grown on Potato Dextrose Agar (PDA) and for the ability to produce urease, as well as the hair perforation test (Larone, 1993; Tu¨ mbay, 1983). Isolates were stored at ⫺70°C on Brucella Broth (Acumedia) with 5% glycerine until the time of use, and prior to testing were subcultured on PDA at 28°C for 15 days to ensure optimal growth characteristics. The strain C. albicans ATCC 90028 was included as quality control. 2.2. Medium RPMI 1640 broth medium (Sigma Chemical Company, Madrid, Spain) with L-glutamine but without sodium bicarbonate and buffered at pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma, Madrid) was the medium used for broth microdilution susceptibility testing. Agar formulations used for the disk diffusion test were RPMI 1640 broth supplemented with 1.5% Bacto Agar (Difco Laboratories, Detroid, Mich.), and 2% glucose, and buffered to MOPS (Barry et al., 1996). The 15-cm diameter Petri plates contain RPMI at a depth of 4.0 mm. 2.3. Antifungal agents Ten antifungal drugs, supplied by the manufacturers as powders, were used: fluconazole (Fako Co., Istanbul, Turkey), ketoconazole (Bilim Co., Istanbul, Turkey), itraconazole (Nobel Co., Istanbul, Turkey), bifonazole (Sigma, Madrid), sulconazole (Sigma, Madrid), oxiconazole (Roche), miconazole (Sigma, Madrid), terbinafine (Novartis-Pharma), griseofulvin (Acros Chemical Company), and ciclopiroxolamine (Sigma, Madrid). Fluconazole was dissolved in sterile water; ketoconazole, itraconazole, sulconazole, oxiconazole, miconazole, bifonazole, griseofulvin and terbinafine were dissolved in 100% dimethyl sulfoxide; ciclopiroxolamine was dissolved in 20% methanol. All drugs were prepared as stock solutions of 1280 mcg/ml and stored at ⫺70°C until use. Serial twofold drug dilutions were prepared according to the NCCLS reference method (NCCLS 1998). Final drug concentrations were 0.006 to 32 mcg/ml for fluconazole, 0.008 to 4 mcg/ml for miconazole, sulconazole, oxiconazole, and ketoconazole, 0.016 to 8 mcg/ml for bifonazole and griseofulvin, 0.001 to 1 mcg/ml for terbinafine and itraconazole, and 0.03 to 16 mcg/ml for ciclopiroxolamine. 10-fold drug concentrations prepared in
twofold serial dilutions 0.1-ml drug volume) were maintained at ⫺70°C until needed. Disks of fluconazole, itraconazole, and ketoconazole for disk diffusion test were obtained (Rosco) in the potence of 15 mcg/disk, 8 mcg/disk, and 15 mcg/disk, respectively. Disks for other drugs were prepared considering the potence in the Neo-Sensitabs Susceptibility Testing Catalog (Rosko 1998), 25 mcg/disk for griseofulvin, bifonazole, sulconazole and oxiconazole, 10 mcg/disk for miconazole, 5 mcg/ disk for terbinafine, and 50 mcg/disk for ciclopiroxolamine (Munro 1992). Disks were stored at ⫺70°C until used. 2.4. In vitro susceptibility testing methods The broth microdilution method was performed according to NCCLS M38-P guidelines (NCCLS, 1998) and as described by Espinel-Ingroff et al. (Espinel-Ingroff et al., 1997; Espinel-Ingroff et al., 1999). Each well was inoculated on the day of the test with 100 l of the 2X conidial inoculum suspension mentioned above. This step diluted the drug concentrations and inoculum densities to the final desired test concentration. The growth control wells contained 100 l of sterile drug-free medium and were inoculated with 100 l of the corresponding diluted inoculum suspension. Sterility control was performed by including 100 l of uninoculated, drug-free medium. The disk diffusion method was performed in accordance with used the agar diffusion used test in clinical bacteriology (NCCLS 1999). 2.5. Preparation of both test inocula Stock inoculum suspensions of the dematophytes were prepared from 7- to 15-day old cultures grown on PDA at 28°C. Mature colonies were covered with approximately 10 ml of sterile 85% saline, and the suspensions were made by gently probing the colony with the tip of a Pasteur pipette. The resulting mixture of conidia and hyphal fragments was with drawn and transferred to sterile tubes. Heavy particles of the suspension were allowed to settle for 10 to 15 min at room temperature, and the upper homogeneous suspension was used for further testing. The optical densities of the suspensions were read at 530 nm and adjusted to 0.15 to 0.17 to yield 0.6 ⫻ 106 to 1.4 ⫻ 106 to spores/ml of strains. The suspensions containing conidia and hyphal fragments were diluted with RPMI 1640 medium to obtain the final desired inoculum size of approximately 0.4 ⫻ 104 to 5 ⫻ 104 spores/ml. The microdilution plates were incubated at 28°C for 10 to 15 days. The MICs endpoint for fluconazole, ketoconazole, itraconazole, sulconazole, oxiconazole, bifonazole, and miconazole were defined as the lowest concentration that produced prominent inhibition of growth (approximately 50% inhibition) relative to the drug-free growth control, and the MICs of terbinafine, griseofulvin, and ciclopiroxolamine were 80% (Espinel-Ingroff et al., 1997; NCCLS 1998). Spore suspensions adjusted to a con-
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Table 1 In vitro activities of 10 antifungal agents against 56 strains of dermatophytes Species (no. of strains tested)
Antifungal agents
MIC ranges (mcg/ml)
MICs GMa
MIC50sc (mcg/ml)
Fluconazole Ketoconazole Itraconazole Sulconazole Oxiconazole Bifonazole Miconazole Terbinafine Griseofulvin Ciclopiroxolamine
⬍0.06–⬎32 ⬍0.008–⬎4 ⬍0.001–1 ⬍0.008–4 ⬍0.008–2 ⬍0.016–1 0.008–4 ⱕ0.001–1 0.016–8 ⬍0.03–8
8 0.5 0.1 0.5 0.2 0.2 0.8 0.02 0.6 0.5
4 0.25 0.008 0.25 0.125 0.25 0.25 0.002 0.5 0.25
Fluconazole Ketoconazole Itraconazole Sulconazole Oxiconazole Bifonazole Miconazole Terbinafine Griseofulvin Ciclopiroxolamine
16–32 0.008–4 0.008–0.06 0.016–0.125 0.06–0.5 0.125–0.25 1 ⱕ0.001 0.016–0.125 0.5–1
26.7 1.3 0.03 0.06 0.4 0.2 1 0.001 0.09 0.08
32 0.016 0.016 0.03 0.5 0.25 1 0.001 0.125 1
Fluconazole Ketoconazole Itraconazole Sulconazole Oxiconazole Bifonazole Miconazole Terbinafine Griseofulvin Ciclopiroxolamine
0.25–8 0.016–0.125 ⱕ0.001–0.5 ⱕ0.008–0.03 ⱕ0.008–2 ⱕ0.016–0.5 0.016–0.25 ⱕ0.001–0.008 0.5–1 0.125–1
4 0.07 0.25 0.02 1 0.25 0.1 0.005 0.75 0.6
Fluconazole Ketoconazole Itraconazole Sulconazole Oxiconazole Bifonazole Miconazole Terbinafine Griseofulvin Ciclopiroxolamine
16 4 1 4 4 8 4 1 1 4
MIC90sc (mcg/ml)
Mean inhibition zone diameter ⫾ SEb (cm)
32 1 0.5 1 0.5 0.5 2 0.008 1 1
25.32 ⫾ 12.07 39.4 ⫾ 6.57 42.68 ⫾ 9.59 57.26 ⫾ 11.96 59.4 ⫾ 7.97 42.26 ⫾ 7.03 35.48 ⫾ 9.08 95.92 ⫾ 12.52 47.4 ⫾ 14.24 46.06 ⫾ 7.06
Trichophyton rubrum (50)
Trichophyton mentagrophytes (3) 13.33 ⫾ 5.77 33.33 ⫾ 9.45 41.67 ⫾ 7.23 54.0 ⫾ 8.72 54.0 ⫾ 17.44 50.0 ⫾ 12.49 28.33 ⫾ 10.41 92.33 ⫾ 7.51 45.0 ⫾ 11.36 51.33 ⫾ 18.48
Trichophyton tonsurans (2) 44.5 ⫾ 7.78 53.0 ⫾ 24.04 57.5 ⫾ 9.12 68.0 ⫾ 16.97 66.0 ⫾ 25.46 58.0 ⫾ 16.97 57.5 ⫾ 3.54 101.0 ⫾ 1.41 52.5 ⫾ 24.75 39.5 ⫾ 6.36
Trichophyton verrucosum (1) 10 30 24 24 25 12 11 16 32 20
a
Geometric mean. Standard error. c The MICs at which 50% and 90% of the isolates tested were inhibited were determined for each drug (MIC50s and MIC90s, respectively). b
centration of 106 spores/ml for disk diffusion test. Each solidified medium was inoculated by dipping a sterile swab into the cell suspension and streaking it across the surface of the agar. The plates were dried at room temperature for 15 min before applying the antifungal agents disks. The agar plates were incubated at 28°C for 10 days. Zone diameter end points were read at 50% growth inhibition for fluconazole, ketoconazole, itraconazole, sulconazole, oxiconazole, bifonazole, and miconazole. The endpoints of terbinafine, griseofulvin, and ciclopiroxolamine were defined as 80%
inhibition compared with the growth control. Microcolonies within the diameter were ignored. 2.6. Statistics SPSS/PC for IBM package (version 9.0) was used for the statistical analysis of data. P values of ⬍0.05 were considered statistically significant. The correlation between microdilution method and disk diffusion method was analyzed by means of regression analysis.
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Fig. 1. Terbinafine disk zone diameters and NCCLS Reference Method log10 MICs. The regression statistic is y ⫽ 46.184 –18.906 log10 (terbMIC), r ⫽ 0.744.
Fig. 2. Ciclopiroxolamine disk zone diameters and NCCLS Reference Method log10 MICs. The regression statistic is y ⫽ 44.952–1.265 log10 (ciclopiroxMIC), r ⫽ 0.081.
3. Results
4. Discussion
All isolates of dermatophytes tested produced detectable growth at time points ranging from 7 to 15 days. The range of MICs for the microdilution test and mean inhibition zone for the disk diffusion test of 10 antifungal agents are summarized in Table 1. When all the strains were considered together, the geometric mean MICs of terbinafine and itraconazole were apparently the lowest, 0.002 mcg/ml and 0.1 mcg/ml, respectively. Relative to other agents tested terbinafine possessed the highest antifungal activity against dermatophytes. The geometric mean MICs for ketoconazole, sulconazole, oxiconazole, bifonazole, miconazole, griseofulvin and ciclopiroxolamine were 0.5, 0.5, 0.2, 0.2, 0.8, 0.6, and 0.5 mcg/ml, respectively. Application of two methods of testing correlated. An increase of MIC values was accompanied by a decrease of inhibition zone diameters. The observed MICs of all the drugs tested showed a broad range of variability against the species of Trichophyton. The correlation between MIC values and diameter of growth inhibition zone was analyzed by means of regression analysis. A favorable correlation was seen between MIC and size of inhibition zone around the disk for fluconazole, ketoconazole, itraconazole, oxiconazole, miconazole, terbinafine and griseofulvin, and the correlation coefficients r ⫽ ⫺0.694, ⫺0.298, ⫺0.314, ⫺0.504, ⫺0.439, ⫺0.744, and ⫺0.313 (p ⬍ 0.05), respectively. However, correlation was not seen between two methods for bifonazole, sulconazole and ciclopiroxolamine, and the correlation coefficients r ⫽ ⫺0.245, ⫺0.099, and ⫺0.081 (p ⬎ 0.05), respectively. Figs. 1 and 2 have been presented to show the examples of the drugs which have a favorable and no correlation between zone sizes and MICs, respectively.
A reference method for the antifungal susceptibility testing of dermatophytes is not available (Ghannoum, 2001). In recent years, several studies on the in vitro susceptibility of dermatophytes to antifungal drugs have been done and the results have shown considerable variation (Jessup et al., 2000; Mock et al., 1998). Results of antifungal susceptibility tests are influenced by type of medium, pH, inoculum size, incubation temperature and time and endpoid definition on interlaboratory variability (Espinel-Ingroff et al., 1991; Espinel-Ingroff et al., 1992; Ghannoum et al., 1996). In this study, the NCCLS method of testing filamentous fungi (NCCLS 1998) has been adapted for testing 56 strains of dermatophytes. In this study, the MICs values of the strains varied in a wide range as ⬍0.06-⬎32 mcg/ml, 0.016-8 mcg/ml, and ⱕ0.03-8 mcg/ml for fluconazole, griseofulvin, and ciclopiroxolamine, respectively. Similar results were published by other authors (Korting & Rosenkrans, 1989; Korting et al.,1995; Kibar et al., 2001). Our results showed the lowest activity of all the antifungals tested against dermatophytes, previous studies (Korting et al.,1995; Ferna´ ndez-Torres et al., 2001). Terbinafine and itraconazole demonstrated the high in vitro and in vivo activities against dermatophytes (Grant & Clissold, 1989; Balfour & Faulds, 1992). Terbinafine and itraconazole were the most effective antifungal agents, which agrees with a number of studies carried out by other authors (Korting et al.,1995; Ferna´ ndez-Torres et al., 2000; Ferna´ ndez-Torres et al., 2001), and these drugs provided larger inhibition areas in the disk diffusion method (Mock et al., 1998). The effectiveness of the other imidazoles were similar. To be easly integrated into the work flow of clinical laboratory, an antifungal susceptibility test should be as
N. Karaca, A.N. Koc¸ / Diagnostic Microbiology and Infectious Disease 48 (2004) 259 –264
close as possible to the method that is being used to test antibacterial agents. The disk diffusion procedure we used diffused from the standard method only in the use of RPMI agar in place of Mueller-Hinton agar. The inocula are adjusted by using a spectrophotometer to compare turbidity to 0.15 to 0.17 at 530 nm. Dermatophytes inocula have been adjusted as Jessup et al. (2000). Zone size limits for quality control strains have not yet to be defined by a multilaboratory collaborative study. Selection of broth dilution MIC endpoints presents a similar source of variability that must be controlled, but that is not unique to tests of antifungal agents. Barry and Brown (1996) showed that the disk test seems to be appropriate for tests of most Candida species to fluconazole. Similarly, Meis et al.(2000), reported that the disk diffusion method, which was facile and economical to perform, offers a reproducible and accurate means to assess the in vitro susceptibility of Candida species to fluconazole. Macura (1993), has reported that the disk diffusion method of fungal susceptibility assessment yields data consistent with results obtained from the dilution method and recommended for routine use because of its simplicity, which is in agreement with previous reports we found. The correlation between microdilution method and disk diffusion method was superior to those of fluconazole, ketoconazole, itraconazole, oxiconazole, miconazole, terbinafine, and griseofulvin in the susceptibility testing of dermatophytes. There were, however, some discrepancies in ciclopiroxolamine, bifonazole, and sulconazole. To treat successfully the superficial mycoses tending to be chronic infection, isolation of the agent, species identification, and susceptibility to antifungal drugs by microdulition method or disk diffusion method is simple and rapid enough to be applied in routine mycologic diagnostics. However, additional studies are needed to further validate the correlation between the disk test and NCCLS reference micro/macro broth dilution method and may lead to wide acceptance and standardization of fungal disk testing and interpretive criteria. (National Committee for Clinical Laboratory Standards, 1998 and 1999)
References Balfour, J. A., & Faulds, D. (1992). Terbinafine: a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in superficial mycoses. Drugs 43, 259 –284. Barry, A. L., & Brown, S. D. (1996). Fluconazole disk diffusion procedure for determining susceptibility of Candida species. J Clin Microbiol 34, 2154 –2157. Espinel-Ingroff, A., Barlett, M., Bowden, R., et al (1997). Multicenter evaluation of proposed standardized procedure for antifungal susceptibility testing of filamentous fungi. J Clin Microbiol 35, 139 –143. Espinel-Ingroff, A., Kerkering, T. M., Goldson, P. R., & Shadomy, S. (1991). Comparison study of broth macrodilution and microdilution antifungal susceptibility test. J Clin Microbiol 29, 1089 –1094.
263
Espinel-Ingroff, A., Kish, C. W., Kerkering, T. M., et al (1992). Collaborative comparison of broth macrodilution and microdilution antifungal susceptibility tests. J Clin Microbiol 30, 3138 –3145. Espinel-Ingroff, A., White, T., & Pfaller, M. A. (1999). Antifungal agents and susceptibility tests. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, & R. H. Yolken (Eds.). Manual of clinical microbiology, 7th ed, (pp. 1640 –1652). Washington DC: ASM Press. Ferna´ ndez-Torres, B., Carrillo, A. J., Martin, E., Del Palacio, A., Moore, M. K., Valverde, A., Serrano, M., & Guarro, J. (2001). In vitro activities of 10 antifungal drugs against 508 dermatophyte strains. Antimicrob Agents Chemother 45, 2524 –2528. Ferna´ ndez-Torres, B., Va´ zquez-Veiga, H., Llova, X., Pereiro, M., & Guarro, J. (2000). In vitro susceptibility to itraconazole, clotrimazole, ketoconazole and terbinafine of 100 isolates of Trichophyton rubrum. Chemotherapy 46, 390 –394. Ghannoum, M. A. (2001). Antifungal susceptibility testing of dermatophytes. Dermatol Online J 7, 23C. Ghannoum, M. A., Rex, J. H., & Galgiani, J. N. (1996). Susceptibility testing of fungi: current status of correlation of in vitro data with clinical outcome. J Clin Microbiol 34, 489 – 495. Grant, S. M., & Clissold, S. P. (1989). Itraconazole. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in superficial and systemic mycoses. Drugs 37, 310 –344. Hay, R. J. (2000). Dermatophytosis and other superficial mycoses. In G. L. Mandell, J. E. Bennett, & R. Dolin (Eds.). Mandell, Douglas and Bennett’s principles and practice of infections diseases, 5th ed, (pp. 2757–2768). Philadelphia: Churchill Livingstone. Jessup, C. J., Warner, J., Isham, N., Hasan, I., & Ghannoum, M. A. (2000). Antifungal susceptibility testing of dermatophytes: establishing a medium for inducing conidial growth and evaluation of susceptibility of clinical isolates. J Clin Microbiol 38, 341–344. Kane, J., & Summerbell, R. C. (1999). Trichophyton, Microsporum, Epidermophyton, and agents of superficial mycoses. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, & R. H. Yolken (Eds.). Manual of clinical microbiology, 7th ed, (pp. 1275–1294). Washington DC: ASM Press. Kibar, F., Ko¨ ksal, F., Acar, A., Akbaba, M., & Yoldascan, E. (2001). C¸ esitli dermatofitlerin griseofulvin, flukonazol ve itrakonazol duyarlılıklarının in vitro arastırılması. Mikrobiyol Bu¨ lt 35, 299 –305. Korting, H. C., & Rosenkrans, S. (1989). In vitro susceptibility of dermatophytes from Munich to griseofulvin, miconazole and ketoconazole. Mycoses 33, 136 –139. Korting, H. C., Ollert, M., Abeck, D., Abeck, D., et al (1995). Results of German multicenter study of antimicrobial susceptibilities of Trichophyton rubrum and Trichophyton mentagrophytes strains causing tinea unguium. Antimicrob Agents Chemother 39, 1206 –1208. Larone, D. H. (1993). Medically important fungi: a guide to identification, 2nd ed, (pp. 117–137). Washington DC. Macura, A. B. (1993). In vitro susceptibility of dermatophtes to antifungal drugs: a comparison of two methods. Int J Dermatol 32, 533–536. McGinnis, M. R., & Rinaldi, M. G. (1996). Antifungal drugs: Mechanisms of action, drug resistance, susceptibilitiy testing, and assays of activity in biologic fluids. In V Lorian (Ed). Antibiotics in laboratory medicine, 4th ed, (pp. 176 –211). Philadelphia: Williams & Wilkins. Meis, J., Petrou, M., Bille, J., Ellis, D., Gibbs, D., & The Global Antifungal Surveillance Group (2000). A. global evaluation of the susceptibility of Candida species to fluconazole by disk diffusion. Diagn Microbiol Infect Dis 36, 215–223. Mock, M., Monod, M., Baudraz-Rosselet, F., & Panizzon, R. G. (1998). Tinea capitis dermatophytes: susceptibility to antifungal drugs tested in vitro and in vivo. Dermatology 197, 361–367. Munro, S. (1992). Disk diffusion susceptibility testing. In: H. D. Isenberg (Ed.). Clinical microbiolgy procedures handbook, vol: 1, (pp. 5.1–5.2). Washington DC: ASM Press. National Committee for Clinical Laboratory Standards. (1998). Reference method for broth dilution antifungal susceptibility testing of conidium-
264
N. Karaca, A.N. Koc¸ / Diagnostic Microbiology and Infectious Disease 48 (2004) 259 –264
forming filamentous fungi; proposed standard. NCCLS document M38-P. Wayne, PA: NCCLS. National Committee for Clinical Laboratory Standards. (1999). Performance standards for antimicrobial susceptibility testing. Ninth supplement to the M2-A6 M100-S9. Villanova, PA: NCCLS. Norris, H. A., Elewski, B. E., & Ghannoum, M. A. (1999). Optimal growth conditions for the determination of the antifungal susceptibility of three
species of dermatophytes with the use of a microdilution method. J Am Acad Dermatol 40, S9 –S13. Rosko, A. S. (1998). User’s Guide Neo-Sensitabs susceptibility testing, 10th ed. Tu¨ mbay, E. (1983). Pratik tıp mikolojisi, ss 69. Bilgehan Basımevi, I˙zmir. Weitzman, I., & Summerbell, R. C. (1995). The dermatophytes. Clin Microbiol Rev 8, 240 –259.