Disk Diffusion
Disk-based susceptibility testing is convenient and economical and several commercial disk systems are available; however, the results are somewhat contradictory and the choice of growth medium appears critical; some investigators use RPMI-1640 agar supplemented with 0.2% glucose while others apply Mueller–Hinton (MH) agar supplemented with 2% glucose and 0.5 µg/ml methylene blue so the zone sizes are easy to read. A large number of antifungals are available in tablets.
The method was applied to determine the MIC of amphotericin B, fluconazole and ketoconazole against ocular fungal isolates.[72] Using YNB medium the authors concluded the technique to be reliable, cost effective and easy to perform with consistent results. Recently, a multicenter evaluation was performed to correlate inhibition zone diameters in millimeters to broth dilution MICs of five antifungal agents.[73] Based on these results, the optimal testing conditions for Aspergillus disk-diffusion testing were: plain MH agar; incubation times of 24 h for A. fumigatus, A. flavus and A. niger and 48 h for other species; and posaconazole 5 µg, voriconazole 1 µg, itraconazole 10 µg, caspofungin 5 µg and amphotericin B 5 µg disks. Agar-based methods hold promise as simple and reliable methods for determining susceptibilities of filamentous fungi.[74] For amphotericin B, the best correlation between reference MICs and zone diameters was observed at 16 to 48 h on MH agar.[75]
By contrast, Ozkutuk et al. compared the efficacy of CLSI -M 38-A with disk diffusion and Etest in determining the susceptibility of Aspergillus spp. to amphotericin B, itraconazole and voriconazole.[76] The disk diffusion test demonstrated good correlation with the Etest but poor correlation with the broth microdilution test for the three antifungal agents tested. New investigational disk-diffusion data for candins against Aspergillus are available.[77]