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Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
ABSTRACT
Fifty clinical isolates of Trichophyton rubrum were selected to test with ketoconazole, fluconazole, itraconazole, griseofulvin, and terbinafine by following the National Committee for Clinical Laboratory Standards susceptibility testing guidelines for filamentous fungi (M38-A). In addition, other susceptibility testing conditions were evaluated: (i) three medium formulations including RPMI 1640 (standard medium), McVeigh & Morton (MVM), and Sabouraud dextrose broth (SDB); (ii) two incubation temperatures (28 and 35°C); and (iii) three incubation periods (4, 7, and 10 days). The strains Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), T. rubrum (ATCC 40051), and Trichophyton mentagrophytes (ATCC 40004) were included as quality controls. All isolates produced clearly detectable growth only after 7 days of incubation. MICs were significantly independent of the incubation temperature (28 or 35°C) (P < 0.05). Different incubation periods resulted in MICs which were consistently different for each medium when azoles and griseofulvin were tested (P < 0.05). MICs obtained from different media at the same incubation time for the same isolate were significantly different when azoles and griseofulvin were tested (P < 0.05). MICs were consistently higher (usually 1 to 2 dilutions) with RPMI than with MVM or SDB (P < 0.05). When terbinafine was tested, no parameter had any influence on MICs (P < 0.05). RPMI standard medium appears to be a suitable testing medium for determining the MICs for T. rubrum. MICs obtained at different incubation times need to be correlated with clinical outcome to demonstrate which time has better reliability.
INTRODUCTION
The dermatophytes are a group of closely related fungi that have the capacity to invade the keratinized tissue of humans and other animals and produce dermatophytoses (24). The incidence of dermatophytoses has increased over recent years, particularly in immunocompromised patients (7, 22, 23). The treatment of these cutaneous infections is based on the use of topical and systemic antifungal agents (19). For localized nonextensive lesions, topical therapies are generally used. For tinea unguium, scalp ringworm, extensive lesions, or skin lesions with foliculitis, systemic antimycotic agents are necessary and itraconazole and terbinafine are the oral drugs presently used most to treat severe conditions (7). Trichophyton rubrum, among other dermatophytes, is a major causative agent for superficial dermatomycosis like onychomycosis and tinea pedis and is known to account for as many as 70% of all dermatophyte infections (24). Infections due to T. rubrum are often associated with frequent relapses following cessation of antifungal therapy (15).
In vitro analysis of the antifungal activity enables the comparison between different antimycotics, which in turn may clarify the reasons for the lack of clinical response and assist clinicians in choosing an effective therapy for their patients (9). In the in vitro method proposed by the National Committee for Clinical Laboratory Standards (NCCLS) for testing filamentous fungi, dermatophytes are not included (7). However, it is important that methodologies used for in vitro testing be standardized to facilitate the establishment of quality control parameters and interpretative breakpoints (10). In spite of the lack of a standardized method for testing dermatophytes, several authors have published various articles wherein several species of these fungi have been tested (6, 9, 18, 19). In these publications, different adaptations or modifications of the NCCLS method have been made (3, 6).
The purpose of this study was to evaluate the variability of different microdilution susceptibility testing conditions (medium, incubation time, and temperature) for the determination of MICs of five antifungal drugs presently available for the treatment of dermatophytoses for 50 clinical isolates of T. rubrum.
MATERIALS AND METHODS
Study design. Each isolate was tested with ketoconazole, fluconazole, itraconazole, griseofulvin, and terbinafine by following the NCCLS susceptibility testing guidelines for filamentous fungi (M38-A) (16). In addition, other susceptibility testing conditions were evaluated: (i) three medium formulations including RPMI 1640 (standard medium), McVeigh & Morton (MVM), and Sabouraud dextrose broth (SDB); (ii) two incubation temperatures (28 and 35°C); and (iii) three incubation periods (4, 7, and 10 days).
Isolates. We selected 50 clinical isolates of T. rubrum which were maintained 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 40051), and Trichophyton mentagrophytes (ATCC 40004) were included as quality controls.
Media. The standard RPMI 1640 medium was buffered with 0.165 M morpholinepropanesulfunic acid (MOPS) at 34.54 g per liter at pH 7.0. MVM was prepared as recommended by Shadomy et al. (21), and SDB was prepared at pH 7.0. All drugs were tested in the three media mentioned above for all isolates.
Antifungal drugs. Antifungal drugs were donated as follows: ketoconazole by Janssen-Cilag, fluconazole by Pfizer, terbinafine by Novartis, and griseofulvin by Schering Plough. Itraconazole was used in its commercial formulation (Janssen-Cilag). All drugs were dissolved in 100% dimethyl sulfoxide (Gibco) following the protocol of NCCLS and were prepared in stock solutions of 1,000 μg/ml.
Drug dilutions. Serial twofold dilutions were prepared according to the NCCLS approved document (M38-A) at 100 times the strength of the final concentration, followed by further dilutions (1:50) in RPMI, MVM, or SDB medium to yield twice the final strength required for the test. Ketoconazole, itraconazole, and terbinafine were prepared in ranges from 0.031 to 16.0 μg/ml. Fluconazole and griseofulvin were prepared in ranges from 0.125 to 64.0 μg/ml.
Preparation of inocula. The isolates were transferred from sterile saline (0.9%) to potato dextrose agar at 28°C for 7 days to produce conidia. The fungal colonies were covered with 5 ml of sterile saline (0.9%), and the suspensions were made by gently probing the surface with the tip of a Pasteur pipette. The mixture of conidia and hyphae fragments was filtered with a Whatman filter model 40 (pore size, 8 μm), which retains hyphae fragments and permits passage of only T. rubrum microconidia. The densities of these suspensions were adjusted with a spectrophotometer at a wavelength of 520 nm to a transmittance of 70 to 72%. The inoculum sizes ranged from 2 x 106 to 4 x 106 CFU/ml. Inoculum quantification was made by counting microconidia in a hematocytometer and by plating 0.01 ml of suspensions in SDA. The plates were incubated at 28°C and were examined daily for the presence of fungal colonies. The inoculum suspensions were diluted (1:50) in RPMI, MVM, or SDB medium to obtain a cell number ranging from 2 x 104 to 4 x 104 CFU.
Test procedure. Flat-bottomed microdilution plates (96 wells) were set up in accordance with the NCCLS reference method (16). Each microdilution well containing 100 μl of the twofold drug concentration was inoculated with 100 μl of the diluted inoculum suspension. For each test plate, two drug-free controls were included, one with the medium alone (sterile control) and the other with 100 μl of medium plus 100 μl of inoculum suspension (growth control). The microdilution plates were incubated at 28 and 35°C and were read visually after 4, 7, and 10 days of incubation.
Reading and interpretation of MICs. Endpoint determination readings were performed visually based on comparison of the growth in wells containing the drug with that of the growth control. For azole agents and for griseofulvin, the MIC was defined as the lowest concentration showing prominent growth inhibition (a drop in growth corresponding to approximately 80% of the growth control. For terbinafine, the MIC was defined as the lowest concentration showing 100% growth inhibition. MIC ranges of each drug were obtained to facilitate comparisons of the activities of tested drugs, as well as readings of the MIC at which 50% of the isolates were inhibited (MIC50); similarly, MIC90 is the MIC at which 90% of the isolates were inhibited.
Data analysis. Determinations of all the MICs were repeated twice. Comparisons of influence of incubation temperature, incubation time, and tested media were performed by Wilcoxon (Mann-Whitney) and Kruskal-Wallis tests. A P value of <0.05 was considered significant.
RESULTS
All isolates produced clearly detectable growth only after 7 days of incubation. Therefore, the first determination of MIC endpoints was possible only after that time for all testing conditions. Tables 1 and 2 summarize the susceptibility data for reference dermatophyte isolates and for 50 T. rubrum isolates, respectively.
Quality control and reference isolates. Fluconazole and itraconazole MICs for the strains C. parapsilosis (ATCC 22019) and C. krusei (ATCC 6258) were within established ranges (2), and similar values were obtained by using MVM and SDB media. The MICs for T. rubrum (ATCC 40051) and T. mentagrophytes (ATCC 40004) are summarized in Table 1.
Effect of the incubation temperature on MICs. The incubation temperature did not influence MICs significantly (P < 0.05). This was true for all tested media and all incubation times and for all tested drugs (with no dilution interval). MICs obtained when plates were incubated at 28°C (Table 2) were similar to or even the same as MICs obtained when incubating plates at 35°C.
Effect of the incubation time and media on MICs. Growth was insufficient at 4 days of incubation for 6 isolates with RPMI, for 9 isolates with SDB, and for 19 isolates with MVM, but MICs were obtained with other testing conditions. The statistical analyses revealed that different incubation periods resulted in MICs which were consistently different for each medium when azoles and griseofulvin were tested (P < 0.05). MICs obtained with different media at the same incubation time for the same isolate were significantly different when azoles and griseofulvin were tested (P < 0.05). MICs were consistently higher (usually 1 to 2 dilutions) when using RPMI than when using MVM or SDB (P < 0.05). When terbinafine was tested, no parameter had any influence on MICs (P < 0.05).
DISCUSSION
Although a standard method for the susceptibility testing of dermatophytes is lacking, there are several reports where the antimicrobial susceptibility testing of dermatophytes has been conducted by using either agar macrodilution or broth macrodilution and microdilution tests; these reports have generally been extensions of either M27-A2 or M38-A methodologies. Results obtained by some authors (3) have shown great variability, probably due to the lack of standardization of different parameters that can influence the MIC determination, such as method of inoculum preparation (8) and length and temperature of incubation (4, 7, 14). Ghannoum et al. (9) demonstrated, in a multicenter study, a high level of intra- and interlaboratory agreement for the determination of MICs undertaken in RPMI medium at a temperature of 35°C and using a 4-day incubation period.
In our study, in general, we followed recommendations of the NCCLS for testing filamentous fungi with some adaptations. First of all, we used Whatman's filter model 40 to separate hyphae from conidia, resulting in homogenous inocula comprised of only microconidia. We think that the separation of these structures is an important step in MIC determination because antifungal susceptibilities of hyphae and microconidia are probably different.
The ideal incubation temperature, 28 or 35°C, is still a matter of debate. It is well known that the majority of dermatophyte species show an optimal growth when incubation temperature is between 28 and 30°C (7). However, various authors have proposed higher temperatures, such as 35 or 37°C (1, 3, 9, 18, 19). In our experience, this parameter did not significantly influence (P < 0.05) the MICs for a specific incubation time in all tested media. This result is directly comparable with that reported by Norris et al. (18) and is different from that reported by Fernández-Torres et al. (7), who demonstrated better growth when fungi were incubated at 28°C.
A number of authors have proposed different incubation times, ranging from 3 to 20 days, for testing dermatophytes (1, 3, 9, 18, 19). After 4 days, T. rubrum was observed to grow poorly; however, 7 days was found to be sufficient to observe prominent growth in control wells. Fernández-Torres et al. (7, 8) obtained similar results. Different findings were obtained by Jessup et al. (12), Ghannoum et al. (9), and Favre et al. (5), who found 4 days of incubation sufficient to observe prominent growth in control wells. Statistical analysis revealed that an increased incubation time of not only 10 days compared to 7 days increases MICs by 1 to 2 dilutions with the same medium but also 7 days relative to 4 days (data not shown). The exception was for terbinafine, where the incubation time did not influence MICs.
All tested media (standard RPMI broth, MVM, and SDB) presented statistically different results (P < 0.05) with no dilution interval, except for terbinafine. Greater MICs were obtained with buffered RPMI relative to other tested media. The medium proposed by NCCLS allowed adequate growth of all isolates, confirming reports that this medium produces a suitable visible growth of filamentous fungi, including dermatophytes (4, 18). Between MVM and SDB, the first was the medium which was more similar in comparison to RPMI. Although MVM is a chemically defined medium (20) often used in MIC determination for Paracoccidioides brasiliensis (11) by the broth macrodilution method, MIC reading in microdilution plates is harder because of the transparency of this medium compared to the yellow color of the other media, which confuses during visualization. There is no report about the use of MVM medium in MIC determination for dermatophytic fungi. There is a scarcity of reports about the use of SDB for MIC determination for any fungi, including dermatophytes (17). We tested SDB medium because its cost is considerably lower than that of other tested media mentioned above and it is used in all mycology laboratories; however, SDB presented lower MICs and had high discrepancy compared to MVM and RPMI (P < 0.05).
The evaluation of in vitro activities of tested drugs revealed that terbinafine was the most potent active drug, confirming reports by Korting et al. (13) and Fernández-Torres et al. (6). Between the azoles, itraconazole was the most active, followed by ketoconazole and fluconazole. Similar results were obtained by Korting et al. (13), who tested numerous isolates of T. rubrum by a RPMI microdilution method. With respect to griseofulvin, the great majority of tested isolates presented MICs of 2 μg/ml and this result is comparable with that reported by Jessup et al. (12).
In conclusion, this investigation has demonstrated that microdilution assay for dermatophytes is convenient and reproducible. The results of this study show that the maintenance of the isolates in sterile saline before transfer to potato dextrose agar promotes conidial production and enables the use of inoculum containing only this structure. RPMI standard medium appears to be a suitable testing medium for the determination MICs for Trichophyton rubrum, strengthening the data from authors that recommend RPMI for testing dermatophytes. In our experiments, temperature did not consistently influence MICs, revealing that tests could be performed at 28 or 35°C. However, MICs obtained at different incubation times need to be correlated with clinical outcome to demonstrate which time has better reliability.
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