Abstract
For antifungal susceptibility testing of nonsporulating or poorly sporulating dermatophytes, a fragmented-mycelium inoculum preparation method was established and compared to broth microdilution testing according to CLSI and EUCAST guidelines. Moreover, the in vitro activity of new antifungal agents against dermatophytes was evaluated. Agreement between the mycelial inoculum method and the CLSI broth microdilution method was high (93% to 100%). Echinocandins (minimal effective concentration [MEC], ≤0.5 mg/liter) and posaconazole (MIC, ≤3.00 mg/liter) showed good activity against all tested dermatophytes.
TEXT
Antifungal susceptibility testing (AST) remains an open research field since the development of the first antifungal agent (AFA). Dermatomycosis and onychomycosis are infectious diseases which are associated with long-term therapy (1) and high relapse rates (2, 3). As treatment responses become evident only weeks or months after initiation of antifungal therapy, the choice of an effective antifungal therapy is essential; antifungal susceptibility testing simplifies decision making.
Several inoculum preparation methods exist, but they are all based on conidia, which are difficult to gain from poorly or slowly sporulating dermatophytes within a reasonable time. To overcome this limitation, fragmented mycelium could be used instead. The usefulness of this approach for susceptibility testing of dermatophytes was evaluated in a previous study (4, 5), but the comparison with the current gold standard of the Clinical and Laboratory Standards Institute (CLSI) (6) remains to be performed. So far, mycelium inoculum was compared for only Aspergillus spp. with European Committee on Antifungal Susceptibility Testing (EUCAST) broth microdilution (5).
The aim of this study was 2-fold: (a) to compare fragmented inoculum preparation as a modified EUCAST approach with the standard CLSI (6) and EUCAST (7) methods for antifungal susceptibility testing of dermatophytes and (b) to investigate the in vitro activity of new AFAs against common dermatophytes.
The following five strains from the German culture collection (DSMZ, Braunschweig, Germany) were used for quality control (QC) as they deliver stable and reproducible results: Microsporum canis ATCC 28327, Trichophyton mentagrophytes ATCC 18748 and ATCC 9533, Trichophyton verrucosum ATCC 38485, and Trichophyton rubrum DSM 4167. For mycelium inoculum preparation, all strains were cultured on Sabouraud (SAB)-2% glucose agar for 2 to 6 days at 30°C, while for conidial suspension preparation, strains had to be grown on average for 21 days (±7 days).
Susceptibility testing was performed according to CLSI (6) or EUCAST (7) guidelines for broth microdilution testing of molds. The fragmented inoculum preparation method was performed as previously published (4, 5). In short, NaCl was dropped on the colony surface and the colony was rubbed with a sterile scalpel to release hyphae for inoculum preparation. Hyphae were homogenized, checked microscopically, and diluted with 0.85% NaCl to a final concentration of 1.2 × 105 to 5 × 105 viable units (VU)/ml (viable units are the hyphal segment framed by two intact septa) using a Neubauer counting chamber.
Inoculum viability concentration was verified by plating 100 μl on SAB-2% glucose agar plates in duplicate and incubating it at 30°C for 72 h. The deviation between counted VU and grown colonies was approximately 5%, which is in line with the conidial inoculum. The ready-to-use microdilution panels based on RPMI 1640 manufactured by Merlin GmbH (Berlin, Germany) consisting of voriconazole (VRC), posaconazole (PSC), fluconazole (FLC), anidulafungin (ANI), caspofungin (CAS), micafungin (MCA), and amphotericin B (AMB) were used as described by Czaika (4) and Schmalreck et al. (5). Aspergillus fumigatus (ATCC 204305) and Aspergillus flavus (ATCC 204304) were used for quality control of AST (7). The results obtained for amphotericin B and echinocandins were also included in the study for the possible benefit they may provide for isolates refractory to standard treatment choices for dermatophyte infections. Microdilution plates were incubated at 30°C ± 1°C, until optimal growth in the control well was achieved. The MIC and minimal effective concentration (MEC) were determined visually with a magnification mirror and microscope, respectively, after 2 to 9 days and 1 day after the first reading, respectively. All calculations and statistical analyses were performed with log2 MIC values using SAS software (SAS Institute, North Carolina, USA). Nonparametric parameters (MIC values) were compared using the Mann-Whitney U test and the Wilcoxon signed-rank test. A P value of <0.05 was considered statistically significant.
In general, the fragmented inoculum preparation method was comparable to CLSI (6) and EUCAST (7) methods (P > 0.05) (Tables 1 to 3).
TABLE 1.
Comparison of conidial versus fragmented-mycelium inoculum for CLSI method using a set of clinical isolates
| Clinical strain | AFAa | nb | Concn range (mg/liter) for method: |
|||
|---|---|---|---|---|---|---|
| Conidiumc |
Fragmented-mycelium inoculum prepn methodd |
|||||
| MIC | MEC | MIC | MEC | |||
| Trichophyton mentagrophytes | AMP | 4 | 0.25–1.0 | 1.0 | ||
| TER | 4 | 0.03 | 0.03–0.06 | |||
| FLC | 4 | 16.0–32.0 | 16.0–32.0 | |||
| POS | 4 | 0.5–1.0 | 0.25–2.0 | |||
| VRC | 4 | 0.125–0.5 | 0.5 | |||
| ITR | 4 | 0.25–2.0 | 1.0 | |||
| MCA | 4 | 0.016 | 0.016–16.0 | |||
| ANI | 4 | 0.016–0.03 | 0.016–16.0 | |||
| CAS | 4 | 0.25–1.0 | 0.06–0.5 | |||
| Trichophyton tonsurans | AMP | 3 | 0.06–0.5 | 0.25–1.0 | ||
| TER | 3 | 0.03 | 0.03 | |||
| FLC | 3 | 0.5–32.0 | 8.0–32.0 | |||
| POS | 3 | 0.5–1.0 | 0.25–0.5 | |||
| VRC | 3 | 0.06–0.5 | 0.03–0.25 | |||
| ITR | 3 | 0.125–1.0 | 0.125–2.0 | |||
| MCA | 3 | 0.016–0.125 | 0.016–0.03 | |||
| ANI | 3 | 0.016–0.03 | 0.016–0.03 | |||
| CAS | 3 | 0.25–2.0 | 0.06–1.0 | |||
| Trichophyton rubrum | AMP | 7 | 0.125–0.5 | 0.5 | ||
| TER | 7 | 0.03 | 0.03 | |||
| FLC | 7 | 0.125–32.0 | 0.25–4.0 | |||
| POS | 7 | 0.06–1.0 | 0.06–0.5 | |||
| VRC | 7 | 0.03–2.0 | 0.03–0.06 | |||
| ITR | 7 | 0.06–1.0 | 0.06–1.0 | |||
| MCA | 7 | 0.016 | 0.016 | |||
| ANI | 7 | 0.016 | 0.016 | |||
| CAS | 7 | 0.125–1.0 | 0.5–1.0 | |||
| Microsporum canis | AMP | 4 | 0.06–0.25 | 0.25 | ||
| TER | 4 | 0.06 | 0.03–0.06 | |||
| FLC | 4 | 4.0–16.0 | 2.0–32.0 | |||
| POS | 4 | 1.0 | 0.5 | |||
| VRC | 4 | 0.06–0.125 | 0.03–0.125 | |||
| ITR | 4 | 0.25–1.0 | 0.125–1.0 | |||
| MCA | 4 | 0.016 | 0.016 | |||
| ANI | 4 | 0.016 | 0.016 | |||
| CAS | 4 | 0.25–0.5 | 0.125–0.5 | |||
Tested antifungal agents (AFAs): amphotericin B (AMP), terbinafine (TER), fluconazole (FLC), posaconazole (POS), voriconazole (VRC), itraconazole (ITR), micafungin (MCA), anidulafungin (ANI), and caspofungin (CAS).
Number of isolates; each isolate was tested once.
Microdilution testing according to Clinical and Laboratory Standards Institute (CLSI) method (6) using conidia for inoculum preparation.
Microdilution testing according to Clinical and Laboratory Standards Institute (CLSI) method using fragmented mycelium for inoculum preparation.
TABLE 3.
Statistical comparisons between the conidial (standard) and fragmented-mycelium inoculum using CLSI and EUCAST guidelinesa
| Guideline | AFAb | nc | Concn range (mg/liter) for method: |
P value | |||
|---|---|---|---|---|---|---|---|
| Conidium |
Fragmented mycelium |
||||||
| MIC | MEC | MIC | MEC | ||||
| CLSId | AMP | 18 | 0.06–1.0 | 0.25–1.0 | 0.011 | ||
| TER | 18 | 0.03–0.06 | 0.03–0.06 | 1.000 | |||
| FLC | 18 | 0.125–32.0 | 0.25–32.0 | 1.000 | |||
| POS | 18 | 0.06–1.0 | 0.06–2.0 | 0.929 | |||
| VRC | 18 | 0.03–2.0 | 0.03–0.5 | 0.404 | |||
| ITR | 18 | 0.06–2.0 | 0.06–2.0 | 0.674 | |||
| MCA | 18 | 0.016–0.125 | 0.016–16.0 | 0.472 | |||
| ANI | 18 | 0.016–0.03 | 0.016–16.0 | 0.461 | |||
| CAS | 18 | 0.125–2.0 | 0.06–1.0 | 0.660 | |||
| EUCASTe | AMP | 18 | 0.25–2.0 | 0.125–2.0 | 0.149 | ||
| TER | 18 | 0.03–0.125 | 0.03–0.125 | 0.180 | |||
| FLC | 18 | 1.0–64.0 | 0.06–64.0 | 0.674 | |||
| POS | 18 | 0.25–2.0 | 0.03–1.0 | 0.440 | |||
| VRC | 18 | 0.03–2.0 | 0.03–0.5 | 0.141 | |||
| ITR | 18 | 0.25–8.0 | 0.03–1.0 | 0.306 | |||
| MCA | 18 | 0.03–1.0 | 0.03–0.125 | 1.000 | |||
| ANI | 18 | 0.016–1.0 | 0.016–0.25 | 0.483 | |||
| CAS | 18 | 0.06–0.5 | 0.03–0.5 | 0.905 | |||
Tested clinical strains included Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton tonsurans, and Microsporum canis.
Tested antifungal agents (AFAs): amphotericin B (AMP), terbinafine (TER), fluconazole (FLC), posaconazole (POS), voriconazole (VRC), itraconazole (ITR), micafungin (MCA), anidulafungin (ANI), and caspofungin (CAS).
Number of isolates; each isolate was tested once.
Microdilution testing according to Clinical and Laboratory Standards Institute (CLSI) guidelines (6).
Microdilution testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (7).
TABLE 2.
Comparison of conidial versus fragmented-mycelium inoculum for EUCAST method using a set of clinical isolates
| Clinical strain | AFAa | nb | Concn range (mg/liter) for method: |
|||
|---|---|---|---|---|---|---|
| Conidiumc |
Fragmented-mycelium inoculum prepn methodd |
|||||
| MIC | MEC | MIC | MEC | |||
| Trichophyton mentagrophytes | AMP | 4 | 0.5–1.0 | 0.5–1.0 | ||
| TER | 4 | 0.03–0.06 | 0.03–0.06 | |||
| FLC | 4 | 16.0–64.0 | 64.0 | |||
| POS | 4 | 0.25–1.0 | 0.25–1.0 | |||
| VRC | 4 | 0.125–0.5 | 0.25–0.5 | |||
| ITR | 4 | 0.25–1.0 | 0.5–1.0 | |||
| MCA | 4 | 0.03 | 0.03–0.125 | |||
| ANI | 4 | 0.016 | 0.016–0.25 | |||
| CAS | 4 | 0.25 | 0.06–0.5 | |||
| Trichophyton tonsurans | AMP | 3 | 0.5–1.0 | 0.5–1.0 | ||
| TER | 3 | 0.03–0.06 | 0.03 | |||
| FLC | 3 | 4.0–64.0 | 64.0 | |||
| POS | 3 | 0.125–16.0 | 0.06–0.5 | |||
| VRC | 3 | 0.06–16.0 | 0.06–0.125 | |||
| ITR | 3 | 0.125–8.0 | 0.5 | |||
| MCA | 3 | 0.03–1.0 | 0.03 | |||
| ANI | 3 | 0.016–1.0 | 0.016–0.03 | |||
| CAS | 3 | 0.125–0.5 | 0.03–0.25 | |||
| Trichophyton rubrum | AMP | 7 | 0.5–2.0 | 0.25–2.0 | ||
| TER | 7 | 0.03–0.06 | 0.03 | |||
| FLC | 7 | 1.0–64.0 | 0.06–8.0 | |||
| POS | 7 | 0.5–1.0 | 0.25–1.0 | |||
| VRC | 7 | 0.03–0.5 | 0.03–0.06 | |||
| ITR | 7 | 0.25–0.5 | 0.25–1.0 | |||
| MCA | 7 | 0.03 | 0.03 | |||
| ANI | 7 | 0.016–0.03 | 0.016–0.03 | |||
| CAS | 7 | 0.06–0.5 | 0.03–0.25 | |||
| Microsporum canis | AMP | 4 | 0.25–0.5 | 0.125–0.25 | ||
| TER | 4 | 0.06–0.125 | 0.03–0.125 | |||
| FLC | 4 | 32.0 | 16.0–32.0 | |||
| POS | 4 | 0.25–0.5 | 0.03–1.0 | |||
| VRC | 4 | 0.06–0.125 | 0.03–0.125 | |||
| ITR | 4 | 0.25–0.5 | 0.03–1.0 | |||
| MCA | 4 | 0.03 | 0.03 | |||
| ANI | 4 | 0.016–0.03 | 0.003 | |||
| CAS | 4 | 0.06–0.25 | 0.06–0.25 | |||
Tested antifungal agents (AFAs): amphotericin B (AMP), terbinafine (TER), fluconazole (FLC), posaconazole (POS), voriconazole (VRC), itraconazole (ITR), micafungin (MCA), anidulafungin (ANI), and caspofungin (CAS).
Number of isolates; each isolate was tested once.
Microdilution testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) method using conidia for inoculum preparation (7).
Microdilution testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) method using fragmented mycelium for inoculum preparation.
For the QC strains, no significant difference between the CLSI method (Table 3) and modified EUCAST method (=fragmented-mycelium inoculum) was observed. The best results were gained for T. mentagrophytes (P = 0.8) (Table 4). The agreement between the modified EUCAST method and CLSI method (6) was 88.9 to 100% (Table 5). Eighty clinical isolates and seven AFAs (Table 6) were tested with the modified EUCAST method, finding echinocandins in vitro as the most effective agents against dermatophytes (MEC at which 90% of the isolates tested are inhibited [MEC90], ≤0.5 mg/liter). Echinocandins (MEC90, ≤0.031 mg/liter) and AMB (MIC at which 90% of the isolates tested are inhibited [MIC90], ≤0.4 mg/liter) were the only compounds active against M. canis, while azoles had only limited activity. All tested Trichophyton spp. were susceptible against a wide range of AFAs, including AMB (MIC90 of ≤0.5 mg/liter), VRC, and PSC.
TABLE 4.
Comparison of conidial and fragmented-mycelium inoculum methods for CLSI using a set of quality control strains
| Control strain | AFAa | nb | Concn range (mg/liter) for method: |
P value | |||
|---|---|---|---|---|---|---|---|
| CLSIc |
Fragmented-mycelium inoculum prepn methodd |
||||||
| MIC | MEC | MIC | MEC | ||||
| Trichophyton mentagrophytes ATCC 9533 | VRC | 23 | 0.032–0.5 | 0.064–0.5 | 0.3 | ||
| POS | 3 | 0.125–0.5 | 0.125–0.5 | NTe | |||
| FLC | 23 | 8.0–125.0 | 2.0–32.0 | 0.1 | |||
| ANI | 3 | 0.001–0.016 | 0.004–0.008 | NT | |||
| CAS | 3 | 0.008–0.125 | 0.032–0.064 | NT | |||
| MCA | 3 | 0.001–0.016 | 0.004–0.008 | NT | |||
| Trichophyton mentagrophytes ATCC 18748 | VRC | 18 | 0.032–0.5 | 0.032–0.25 | 0.8 | ||
| POS | 18 | 0.064–1.0 | 0.064–0.5 | 0.3 | |||
| FLC | 18 | 16.0–256.0 | 8.0–64.0 | 0.2 | |||
| ANI | 3 | 0.004–0.016 | 0.004–0.032 | NT | |||
| CAS | 3 | 0.032–0.125 | 0.064–0.125 | NT | |||
| MCA | 3 | 0.004–0.016 | 0.004–0.025 | NT | |||
| Trichophyton tonsurans DSM 12285 | VRC | 12 | 0.016–0.25 | 0.064–0.25 | 0.4 | ||
| POS | 3 | 0.25–1.0 | 0.25–0.25 | NT | |||
| FLC | 12 | 16.0–256.0 | 8.0–64.0 | 0.3 | |||
| ANI | 3 | 0.002–0.008 | 0.004–0.004 | NT | |||
| CAS | 3 | 0.004–0.016 | 0.008–0.008 | NT | |||
| MCA | 3 | 0.002–0.008 | 0.004–0.004 | NT | |||
| Trichophyton rubrum DSM 4167 | VRC | 18 | 0.064–0.25 | 0.064–0.25 | 0.2 | ||
| POS | 18 | 0.064–0.25 | 0.25–0.5 | 0.1 | |||
| FLC | 18 | 4.0–64.0 | 2.0–64.0 | 0.3 | |||
| ANI | 4 | 0.016–0.064 | 0.032–0.064 | NT | |||
| CAS | 4 | 0.064–0.25 | 0.125–0.125 | NT | |||
| MCA | 4 | 0.016–0.064 | 0.032–0.064 | NT | |||
| Microsporum canis ATCC 28327 | VRC | 9 | 0.032–0.5 | 0.125–0.5 | NT | ||
| POS | 3 | 0.25–1.0 | 0.5–0.5 | NT | |||
| FLC | 9 | 8.0–128.0 | 8.0–32.0 | NT | |||
| ANI | 3 | 0.064–0.125 | 0.008–0.008 | NT | |||
| CAS | 3 | 0.008–0.032 | 0.016–0.016 | NT | |||
| MCA | 3 | 0.004–0.016 | 0.008–0.008 | NT | |||
Tested antifungal agents (AFAs): voriconazole (VRC), posaconazole (POS), fluconazole (FLC), anidulafungin (ANI), caspofungin (CAS), and micafungin (MCA).
Number of replicates tested with the same quality control strain.
Microdilution testing according to Clinical and Laboratory Standards Institute (CLSI) guidelines (6).
Microdilution testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (7) using fragmented mycelium for inoculum preparation.
NT, insufficient sample size for statistical analysis.
TABLE 5.
MICs of quality control strains and cumulative percentages of agreement within log2 dilutions for the standard CLSI and modified EUCAST fragmented-mycelium inoculum methods
| Organism | AFAa | nb | % of isolates with MIC and MEC difference: |
Agreementc (%) |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| >+2 | +2 | +1 | 0 | −1 | −2 | >−2 | ±1 | ±2 | |||
| T. mentagrophytes ATCC 9533 | VRC | 23 | 8.7 | 13.0 | 78.3 | 91.3 | 100.0 | ||||
| POS | 3 | 33.3 | 66.6 | 100.0 | 100.0 | ||||||
| FLC | 23 | 13.0 | 21.7 | 26.1 | 17.4 | 17.4 | 4.3 | 65.2 | 95.7 | ||
| ANI | 3 | 33.3 | 66.6 | 66.6 | 100.0 | ||||||
| CAS | 3 | 33.3 | 66.6 | 66.6 | 100.0 | ||||||
| MCA | 3 | 33.3 | 33.3 | 33.3 | 66.6 | 100.0 | |||||
| T. mentagrophytes ATCC 18748 | VRC | 18 | 5.6 | 66.7 | 27.8 | 100.0 | 100.0 | ||||
| POS | 18 | 5.6 | 11.1 | 55.6 | 27.8 | 94.4 | 100.0 | ||||
| FLC | 18 | 77.8 | 11.1 | 11.1 | 88.9 | 88.9 | |||||
| ANI | 3 | 33.3 | 66.6 | 66.6 | 100.0 | ||||||
| CAS | 3 | 33.3 | 66.6 | 100.0 | 100.0 | ||||||
| MCA | 3 | 33.3 | 33.3 | 33.3 | 66.6 | 100.0 | |||||
| Trichophyton tonsurans DSM 12285 | VRC | 12 | 8.3 | 16.7 | 66.7 | 8.3 | 91.7 | 100.0 | |||
| POS | 3 | 33.3 | 33.3 | 33.3 | 66.7 | 100.0 | |||||
| FLC | 12 | 8.3 | 41.7 | 50.0 | 50.0 | 100.0 | |||||
| ANI | 3 | 33.3 | 66.6 | 100.0 | 100.0 | ||||||
| CAS | 3 | 66.6 | 33.3 | 100.0 | 100.0 | ||||||
| MCA | 3 | 33.3 | 66.6 | 100.0 | 100.0 | ||||||
| Microsporum canis ATCC 28327 | VRC | 9 | 66.7 | 11.1 | 22.2 | 33.3 | 100.0 | ||||
| POS | 3 | 33.3 | 33.3 | 33.3 | 100.0 | 100.0 | |||||
| FLC | 9 | 11.1 | 88.9 | 11.1 | 100.0 | ||||||
| ANI | 3 | 100.0 | 0.0 | 0.0 | |||||||
| CAS | 3 | 33.3 | 66.6 | 100.0 | 100.0 | ||||||
| MCA | 3 | 33.3 | 66.6 | 100.0 | 100.0 | ||||||
| T. rubrum DSM 4167 | VRC | 18 | 27.8 | 72.2 | 100.0 | 100.0 | |||||
| POS | 18 | 5.6 | 61.1 | 27.8 | 5.6 | 33.3 | 94.4 | ||||
| FLC | 18 | 5.6 | 94.4 | 100.0 | 100.0 | ||||||
| ANI | 4 | 25.0 | 75.0 | 100.0 | 100.0 | ||||||
| CAS | 4 | 25.0 | 25.0 | 50.0 | 100.0 | 100.0 | |||||
| MCA | 4 | 25.0 | 75.0 | 100.0 | 100.0 | ||||||
Tested antifungal agents (AFAs): voriconazole (VRC), posaconazole (POS), fluconazole (FLC), anidulafungin (ANI), caspofungin (CAS), and micafungin (MCA).
Number of replicates tested with the same quality control strain.
Cumulative percentage of agreement between the results is defined as the proportion of CLSI (6) MIC results that were within ±1 log2 and ±2 log2 dilutions of the modified EUCAST fragmented-mycelium inoculum method MIC results.
TABLE 6.
Antifungal activities of antifungal agents against clinical isolates of Microsporum canis, T. mentagrophytes, T. rubrum, and Trichophyton tonsuransc
| Organism (n = 20b) | AFAa | Concn range (mg/liter) |
Characteristic MIC or MEC value (mg/liter) |
||||
|---|---|---|---|---|---|---|---|
| MIC | MEC | MIC50 | MEC50 | MIC90 | MEC90 | ||
| Microsporum canis | VRC | 0.06–32.00 | 0.25 | 32.00 | |||
| PSC | 0.13–3.00 | 1.00 | 2.00 | ||||
| FLC | 2.00–256.00 | 32.00 | 256.00 | ||||
| AMB | 0.06–0.50 | 0.25 | 0.40 | ||||
| ANI | 0.00–0.03 | 0.01 | 0.03 | ||||
| CAS | 0.00–0.02 | 0.02 | 0.02 | ||||
| MCA | 0.00–0.03 | 0.01 | 0.03 | ||||
| Trichophyton mentagrophytes | VRC | 0.01–1.00 | 0.06 | 0.25 | |||
| PSC | 0.02–1.00 | 0.06 | 0.25 | ||||
| FLC | 2.00–128.00 | 16.00 | 64.00 | ||||
| AMB | 0.13–1.00 | 0.25 | 0.50 | ||||
| ANI | 0.00–0.06 | 0.01 | 0.02 | ||||
| CAS | 0.00–0.25 | 0.03 | 0.06 | ||||
| MCA | 0.00–1.00 | 0.13 | 0.50 | ||||
| Trichophyton rubrum | VRC | 0.01–32.00 | 0.06 | 0.13 | |||
| PSC | 0.02–0.50 | 0.05 | 0.38 | ||||
| FLC | 1.00–256.00 | 16.00 | 32.00 | ||||
| AMB | 0.13–0.25 | 0.13 | 0.25 | ||||
| ANI | 0.00–0.06 | 0.01 | 0.06 | ||||
| CAS | 0.03–0.13 | 0.06 | 0.13 | ||||
| MCA | 0.00–0.06 | 0.01 | 0.50 | ||||
| Trichophyton tonsurans | VRC | 0.02–1.00 | 0.25 | 0.55 | |||
| PSC | 0.03–0.25 | 0.06 | 0.25 | ||||
| FLC | 2.00–64.00 | 24.00 | 64.00 | ||||
| AMB | 0.13–0.25 | 0.13 | 0.25 | ||||
| ANI | 0.00–0.03 | 0.02 | 0.06 | ||||
| CAS | 0.01–0.02 | 0.02 | 0.06 | ||||
| MCA | 0.00–0.03 | 0.00 | 0.06 | ||||
Tested antifungal agents (AFAs): voriconazole (VRC), posaconazole (PSC), fluconazole (FLC), amphotericin B (AMB), anidulafungin (ANI), caspofungin (CAS), and micafungin (MCA).
Number of replicates tested with the same quality control strain.
Activity was evaluated using the fragmented-mycelium inoculum preparation (modified EUCAST) method.
The major finding of this study was that the modified EUCAST method is highly comparable to the well-established EUCAST (7) and CLSI (6) methods. Moreover, susceptibility testing of poorly sporulating dermatophytes was faster and could be conducted even with sterile dermatophytes. The limitation of the modified EUCAST method was a delayed growth as a consequence of slower proliferation of hyphae than conidia. Our in vitro data suggest that AMB, PSC, and all echinocandins are AFAs that merit further evaluation in respect to their activity against dermatophytes. The activity of echinocandins against dermatophytes was previously reported (1, 2). Only M. canis (ATCC 28327) showed a high degree of variability between modified EUCAST and CLSI methods (6) for anidulafungin (−4 dilution steps compared to CLSI method). The modified EUCAST method was compared with the conidium method and already applied for susceptibility testing of Penicillium notatum and Penicillium chrysogenum (8). The MICs and MECs generated with the modified EUCAST method and classical EUCAST method (7) were in line with previous studies by Bezjak (9). Even though MICs and MECs generated with the modified EUCAST method tended to be lower (due to the lower growth rate) than those with conidial inoculum, overall agreement was sufficient to make the mycelium preparation method a potential alternative for AST of dermatophytes.
The suitability of mycelium inoculum was previously found sufficient for broth micro-and macrodilution and for agar-based (e.g., disc, tablet, or strip test) susceptibility assays of filamentous fungi (5) and dermatophytes (4).
In conclusion, fragmented-mycelium inoculum preparation is a reliable modification of the EUCAST broth microdilution method and enables AST within a reasonable time. The role of echinocandins as possible effective AFAs against dermatophytes deserves further investigation.
ACKNOWLEDGMENTS
This work was not financially supported by internal funding.
We thank Sandra Leitner, Caroline Hörtnagl, and Bettina Sartori for technical assistance.
M. Lackner has received honoraria for invited talks by the pharmaceutical company Forest Pharmaceuticals. In the past 5 years, C. Lass-Flörl has received grant support from the Austrian Science Fund (FWF), MFF Tirol, Astellas Pharma, Gilead Sciences, Pfizer, Schering Plough, and Merck Sharp & Dohme. She has been an advisor/consultant to Gilead Sciences, Merck Sharp & Dohme, Pfizer, and Schering Plough. She has received travel/accommodation expenses from Gilead Sciences, Merck Sharp & Dohme, Pfizer, Astellas, and Schering Plough and has been paid for talks on behalf of Gilead Sciences, Merck Sharp & Dohme, Pfizer, Astellas, and Schering Plough.
B. Risslegger and G. Blum have no potential conflicts of interests to declare.
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