EUCAST olorofim MICs for 3,550 Danish mold and dermatophyte isolates from 2020 to 2023.

The dihydroorotate dehydrogenase (DHODH) inhibitor olorofim has potent activity against most molds. Recent reports of acquired resistance to olorofim and an agrochemical fungicide DHODH inhibitor warrant continuous monitoring of activity of olorofim. We examined the activity of olorofim against a contemporary, large collection of molds and explored the impact of different dilution schemes used for the preparation of microdilution plates. In total, 3,550 isolates referred to Statens Serum Institut during 2020-2023 were analyzed. Isolates were identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and/or sequencing. MICs of olorofim, amphotericin B, itraconazole, posaconazole, voriconazole, isavuconazole, and terbinafine were determined using EUCAST E.Def 9.4 and E.Def 11.0 protocols. Drug dilutions were prepared either using serial dilutions directly in the medium (Serial, 2020-2023) or according to the ISO standard 20776-1, with predilutions in DMSO before dilution in medium (introduced during 2023). For isolates with elevated MICs to azoles and olorofim, , and were sequenced as deemed relevant. Serial dilutions generated MICs 1-2 dilutions higher than the ISO dilutions for olorofim, voriconazole, and isavuconazole. Olorofim MICs were <0.5 mg/L (modal MICs 0.03-0.5 mg/L) against most isolates of , , , , , , and dermatophytes. Exceptions included isolates belonging to to and species complexes, and some rare molds (MICs > 1 mg/L). Olorofim susceptibility was stable over the years and not affected by Cyp51A, Hmg1, or PyrE Q35L alterations. Olorofim maintained broad, potent activity over the years against a contemporary collection of molds.