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

Joseph Meletiadis, Maria Siopi, Lamprini Kanioura, Karin Meinike Jørgensen, David S Perlin, Johan W Mouton, Maiken Cavling Arendrup

Abstract

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.

Original languageEnglish
JournalThe Journal of antimicrobial chemotherapy
Volume75
Issue number7
Pages (from-to)1799-1806
Number of pages8
ISSN0305-7453
DOIs
Publication statusPublished - 1 Jul 2020

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