Abstract
ObjectivesThe first objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for antimycobacterial susceptibility testing (AMST), launched in 2016, was to set a reference method for determining the MICs of antituberculous agents, since many protocols are used worldwide and a consensus one is needed for the determination of microbiological breakpoints. MethodsDuring 2017 and 2018, MIC determination protocols were evaluated prospectively in a multicentre study within the four AMST laboratories. MIC results were obtained for isoniazid, levofloxacin and amikacin on the reference strain Mycobacterium tuberculosis H37Rv ATCC 27294. Broth microdilution (BMD) in Middlebrook 7H9 and solid medium dilution (SMD) in Middlebrook 7H10 were performed using two inoculum concentrations. MICs were interpreted with regard to visual and 99% inhibition after 7, 14 or 21 days of incubation for BMD and 21 days for SMD. ResultsFollowing the EUCAST reference protocol, intra- and inter-assay agreements were within ±1 MIC dilution for >95% of the observations for the three drugs in both methods. MIC values, presented as MIC mode (range) for BMD and SMD respectively, were: 0.03 (0.015–0.06) mg/L and 0.12 (0.06–0.25) mg/L for isoniazid, 0.25 mg/L (0.25–0.5) and 0.5 mg/L (0.12–0.5) for levofloxacin, and 0.5 mg/L (0.5–1.0) and 0.5 mg/L (0.5–1.0) for amikacin. ConclusionsBoth SMD and BMD were reproducible and eligible as a reference method for MIC determination of the Mycobacterium tuberculosis complex (MTBC). BMD was finally selected as the EUCAST reference method. From now on it will be used to set epidemiological cut-off values and clinical breakpoints of new and old antituberculous agents.
Highlights
Several methods have been described for the determination of MIC of antituberculous agents against the Mycobacterium tuberculosis complex (MTBC) [1]
A stable reference method for MIC testing of the MTBC is required to define epidemiological cut-off values and clinical breakpoints according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) strategies, and to facilitate studies on the relationship between resistance mutations predicted from sequencing data in relation to the MIC level of resistance
The solid medium (SMD) protocol was derived from Clinical and Laboratory Standards Institute (CLSI) [4] with modifications detailed below: the inoculum was prepared as described in the EUCAST reference method but adjusted to McF1.0, and 100 mL of 10À2 and 10À4 dilutions were plated in duplicate onto plates filled in with 7H10-10%OADC
Summary
Several methods have been described for the determination of MIC of antituberculous agents against the Mycobacterium tuberculosis complex (MTBC) [1]. A stable reference method for MIC testing of the MTBC is required to define epidemiological cut-off values and clinical breakpoints according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) strategies, and to facilitate studies on the relationship between resistance mutations predicted from sequencing data in relation to the MIC level of resistance. In 2016, the EUCAST subcommittee for antimycobacterial drug susceptibility testing (AMST) was launched with a primary goal of defining a reference method for MIC determination on the MTBC. This reference method was made publicly available on the EUCAST website in July 2019 (https://www.eucast.org/mycobacteria/methods_in_ mycobacteria/) and published [2] along with a comment [3]. We report the results obtained by the AMST laboratories when testing various protocols with the objective of agreeing on one protocol that produces MIC results with good reproducibility
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