TY - JOUR
T1 - A multicentre study to optimize echinocandin susceptibility testing of Aspergillus species with the EUCAST methodology and a broth microdilution colorimetric method
AU - Meletiadis, Joseph
AU - Siopi, Maria
AU - Kanioura, Lamprini
AU - Jørgensen, Karin Meinike
AU - Perlin, David S.
AU - Mouton, Johan W.
AU - Arendrup, Maiken Cavling
N1 - Funding Information:
This study was supported by unrestricted grants from Astellas (GR-21-RG-88) and MSD (53591).
Publisher Copyright:
© 2020 The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
PY - 2020/7/1
Y1 - 2020/7/1
N2 - 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.
AB - 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.
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U2 - 10.1093/jac/dkaa102
DO - 10.1093/jac/dkaa102
M3 - Article
C2 - 32330938
AN - SCOPUS:85086747717
SN - 0305-7453
VL - 75
SP - 1799
EP - 1806
JO - Journal of Antimicrobial Chemotherapy
JF - Journal of Antimicrobial Chemotherapy
IS - 7
ER -