Multicenter evaluation of low inoculum and a colorimetric assay for antifungal susceptibility testing of <i>Aspergillus fumigatus</i> to echinocandins with the EUCAST E.Def 9.4

ABSTRACT Echinocandin minimal effective concentration (MEC) determination for Aspergillus is challenging with the EUCAST method. We investigated the applicability of a lower inoculum (10 3 CFU/mL) and a colorimetric assay for micafungin and anidulafungin against Aspergillus fumigatus with the EUCAST E.Def 9.4 protocol. Eighteen shared A. fumigatus isolates (12 wild-type [WT] and 6 non-WT isolates) together with five local A. fumigatus isolates were tested (blinded to the phenotype) in three laboratories for susceptibility to anidulafungin and micafungin following the EUCAST E.Def 9.4, except using a final inoculum of 10 3 CFU/mL. Endpoints were determined after 48 h of incubation macroscopically (MEC-0; no haziness, MEC-1; slight haziness) and colorimetrically after up to an additional 3 h of incubation with XTT/menadione (400 mg/L/6.25 μM) (MIC-1, MIC-2, and MIC-3 corresponding to 25%, 50%, and 75% color development compared to drug-free control). The inter-center reproducibility and agreement between macroscopic and colorimetric endpoints within one twofold dilution were calculated. MEC and MIC distributions were analyzed, and the WT upper limit (WT-UL) was determined. MEC-0 was reproducible (83%–100%) and best correlated with MIC-2 (67%–100%), provided that good metabolic activity was observed in the growth control (&gt;0.5 absorbance at 450/630 nm). The WT-UL was 0.03 mg/L for macroscopic MEC-0 and colorimetric MIC-2 endpoints for both anidulafungin and micafungin. Visual MEC-0s and XTT MIC-2s of non-WT isolates were &gt;0.125 mg/L. The low inoculum facilitated echinocandin MEC determination, and the XTT assay increased objectivity and enabled automation for the separation of WT and non-WT A. fumigatus isolates with the EUCAST method. IMPORTANCE Susceptibility testing of Aspergillus to echinocandins is challenging with the EUCAST method because of the difficulties associated with the visual determination of the minimal effective concentration (MEC) and the lack of susceptibility interpretative criteria. We therefore conducted a multicenter study in order to define the MEC distribution of micafungin and anidulafungin against Aspergillus fumigatus with the EUCAST E.Def 9.4 protocol using a low inoculum of 10 3 CFU/mL and a colorimetric assay. The MEC was reproducible (83%–100%) and best correlated with colorimetric MIC-2, corresponding to a 50% reduction of color development (67%–100%). The upper MEC and MIC-2 limits of wild-type (WT) isolates were 0.03 mg/L for both anidulafungin and micafungin, whereas non-WT isolates had MEC and MIC-2 &gt;0.125 mg/L. The low inoculum facilitated echinocandin MEC determination, and the XTT assay increased objectivity and enabled automation for the separation of WT and non-WT A. fumigatus isolates with the EUCAST method.