Advances in yeast and mold monodrug and combination drug antifungal susceptibility testing

Abstract

Advances were made in both yeast and mold rapid susceptibility assay (RSA) testing. The yeast RSA was modified to facilitate amphotericin B (AMB), itraconazole (ITC), and voriconazole (VRC) testing of Aspergillus fumigatus, A. terreus, and A. flavus clinical isolates. 16 h mold RSA AMB, ITC, and VRC RSA minimum inhibitory concentration (MIC) values were equal to or within a single, two-fold dilution of MICs obtained in 48 h with the National Committee for Clinical Laboratory Standards (NCCLS) M38-A assay and in 24 or 48 h with the mold Etest. Preliminary testing with A. sydowii, Scedosporium angiospermum, and Fusarium oxysporum suggests that the mold RSA would also be a suitable AMB and ITC susceptibility testing format for these opportunistic filamentous fungi. The AMB, ITC, and VRC susceptibilities of A. fumigatus conidia and hyphae were compared by modifying the mold RSA, with conidia and hyphae demonstrating similar susceptibilities to drug. The yeast RSA was validated by testing clinical control strains that were obtained from patients with known clinical outcome. Yeast RSA conditions were also optimized to facilitate FLC testing of C. glabrata isolates. The effects of concurrent and sequential amphotericin B, itraconazole, and voriconazole two-drug combinations on the conidial and hyphal inocula of A. fumigatus were determined using a checkerboard testing format, with drug interaction effects interpreted by calculating fractional inhibitory concentration indices. Our interpretations of the in vitro effects of concurrent and sequential antifungal combinations on A. fumigatus closely matched the reported outcomes of invasive aspergillosis patients treated with concurrent and sequential antifungal therapies. Importantly, both concurrent and sequential antifungal combinations had differential effects on conidial and hyphal inocula, suggesting that all combination testing be performed with hyphal inocula.