Optimal testing conditions for determining MICs and minimum fungicidal concentrations of new and established antifungal agents for uncommon molds: NCCLS collaborative study

This collaborative three-center study evaluated NCCLS M38-A document testing conditions and other testing conditions for the antifungal susceptibility testing of 35 isolates of Aspergillus nidulans, A. terreus, Bipolaris hawaiiensis, B. spicifiera, Cladophialophora bantiana, Dactylaria constricta, Fusarium solani, Paecilomyces lilacinus, Scedosporium prolificans, Trichoderma longibrachiatum, and Wangiella dermatitidis for itraconazole, three new triazoles (voriconazole, posaconazole, and ravuconazole), and amphotericin B. MICs and minimum fungicidal concentrations (MFCs) were determined in each center by using four media (standard RPMI-1640 [RPMI], RPMI with 2% dextrose [RPMI-2%], antibiotic medium 3 [M3], and M3 with 2% dextrose [M3-2%]) and two criteria of MIC determination (complete growth inhibition [MICs-0] and prominent growth inhibition [MICs-2]) at 24, 48 and 72 h. MFCs were defined as the lowest drug concentrations that yielded <3 colonies (approximately 99 to 99.5% killing activity). The reproducibility (within three wells) was higher among MICs-0 (93 to 99%) with either RPMI or M3 media than among all MICs-2 (86 to 95%) for the five agents at 48 to 72 h. The agreement for MFCs was lower (86 to 94%). Based on interlaboratory agreement, the optimal testing conditions were RFMI broth, 48 to 72 h of incubation and 100% growth inhibition (MIC-0); MFCs can be obtained after MIC determination with the above optimal testing parameters. These results warrant consideration for inclusion in the future version of the NCCLS M38 document. However, the role of these in vitro values as predictors of clinical outcome remains to be established in clinical trials.