Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates – the EUCAST broth microdilution reference method for MIC determination

Abstract

Scope: Several methods are used worldwide for antibiotic susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC). The variability in the results obtained with these methods hampers setting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines. Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs) need to be standardized for MTBC isolates for old and new agents. Our objective was to establish a standardized reference method for MIC determination for MTBC. Methods: The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared protocols of MIC determination with regard to medium, inoculum preparation, antituberculous agent preparation, incubation, reading of the results and interpretation. Recommendations: The EUCAST reference method of MIC determination for MTBC is the broth microdilution method in Middlebrook 7H9-10% OADC medium. The final inoculum is a 10 5 CFU/mL suspension, obtained from a 10 À2 dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonies with glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96well polystyrene microtitre sterile plate with a lid incubated at 36 ± 1 C. Reading is done using an inverted mirror as soon as the 1:100 diluted control (i.e. 10 3 CFU/mL suspension) shows visual growth. The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth. Mycobacterium tuberculosis H37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within the range 0.03e0.12 for isoniazid, 0.12e0.5 for levofloxacin and 0.25e1 mg/L for amikacin. Conclusions: The EUCAST reference method for MTBC was endorsed by EUCAST after public consultation and will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference method is not primarily intended to be used under routine conditions and the AST methods will need to be

What is the role of the EUCAST reference method for MIC testing of the Mycobacterium tuberculosis complex?

“…This was made possible by the strong involvement of the ESCMID study group of mycobacterial infections (ESGMYC). In 2019, AMST proposed a new protocol for broth microdilution (BMD) testing in Middlebrook 7H9 using a carefully standardised inoculum [11]. Following a public consultation, this protocol was validated in the four AMST laboratories.…”

“…The reference method is described on the EUCAST website and is detailed in an accompanying article [11]. To ensure standardization, a QC target/range for H37Rv ATCC 27294 will be defined for each drug, as it is routinely done for other pathogens [9].…”

Multicentre testing of the EUCAST broth microdilution reference method for MIC determination on Mycobacterium tuberculosis

“…Middlebrook 7H9 broth microdilution (BMD) was tested with a high and low inoculum (1:10 and 1:100 dilutions of a controlled McFarland (McF) 0.5 suspension) in comparison with the proportion method onto Middlebrook 7H10 solid medium (SMD). BMD was performed according to the EUCAST reference method [2]. The 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 10 À2 and 10 À4 dilutions were plated in duplicate onto plates filled in with 7H10-10%OADC.…”

“…SMD was read using both 1:1 and 1:100 diluted controls according to the CLSI. BMD was read for visual growth when the 1:100 dilution growth control was positive, according to the EUCAST reference method [2]. Intra-and inter-laboratory agreement for BMD and SMD were calculated for MIC values ± 1 MIC dilution of the mode, measured in quadruplicates and repeated in at least four separate experiments in each laboratory.…”

“…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]. Herein, 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.…”