A multicentre study to optimize echinocandin susceptibility testing of Aspergillus species with the EUCAST methodology and a broth microdilution colorimetric method

Background The determination of the minimal effective concentration (MEC) of echinocandins against Aspergillus species is subjective, time consuming and has been associated with very major errors. Methods The MECs/MICs of 40 WT [10 each of Aspergillus fumigatus species complex (SC), Aspergillus flavus SC, Aspergillus terreus SC and Aspergillus niger SC] and 4 non-WT A. fumigatus isolates were determined with EUCAST E.Def 9.3.1 read microscopically, macroscopically, spectrophotometrically and colorimetrically in three centres. The optimal conditions for spectrophotometric (single- versus multi-point readings) and colorimetric (XTT/menadione concentration and stability, incubation time) methods were evaluated in preliminary studies using different cut-offs for the determination of macroscopic, spectrophotometric and colorimetric MIC endpoints compared with the microscopically determined MEC. Inter-centre and inter-method essential (within one 2-fold dilution) agreement (EA) and categorical agreement (CA) were determined. Results Both macroscopic and spectrophotometric endpoint readings showed poor inter-centre EA (53%–66%) and low CA (41%–88%) in distinguishing WT from non-WT A. fumigatus SC isolates, while significant differences compared with the microscopic MECs were observed for all echinocandins (EA 6%–54%). For the colorimetric method, the optimal conditions were 400 mg/L XTT/6.25 μΜ menadione, incubation for 1–2 h until the drug-free control reached an absorbance at 450/630 nm of >0.8 and use of 50% inhibition of XTT conversion as a cut-off for all species and echinocandins. All non-WT isolates had high XTT MICs >1 mg/L, whereas the overall inter-centre EA and CA were 72%–89% and 100%, respectively. Conclusions The XTT colorimetric assay improved the antifungal susceptibility testing of echinocandins against Aspergillus spp., reliably detecting non-WT isolates.