Thomas Schön1, Jim Werngren2, Diana Machado3, Emanuele Borroni4, Maria Wijkander2, Gerard Lina5, Johan Mouton6, Erika Matuschek7, Gunnar Kahlmeter7, Christian Giske8, Miguel Santin9, Daniela Maria Cirillo4, Miguel Viveiros3, Emmanuelle Cambau10. 1. Department of Clinical Microbiology and Infectious Diseases, Kalmar County Hospital, Linköping University, Linköping, Sweden. 2. Public Health Agency of Sweden, Department of Microbiology, Unit of Laboratory Surveillance of Bacterial Pathogens, Solna, Sweden. 3. Unit of Medical Microbiology of the Instituto de Higiene e Medicina Tropical and Global Health and Tropical Medicine from Universidade NOVA de Lisboa, Lisbon, Portugal. 4. Emerging Bacterial Pathogen, IRCCS San Raffaele Scientific Institute, Milan, Italy. 5. CIRI, Centre International de Recherche en Infectiologie, Université Lyon 1, Ecole Normale Supérieure de Lyon, France; Centre National de Référence des Staphylocoques, Institut des Agent infectieux, Hôpital de la Croix Rousse, Hospices Civils de Lyon, Lyon, France. 6. Department of Medical Microbiology and Infectious Diseases, Erasmus Medical Centre, Rotterdam, the Netherlands. 7. Department of Clinical Microbiology, Central Hospital, Växjö, Sweden; EUCAST Development Laboratory, Växjö, Sweden. 8. Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden. 9. Department of Infectious Diseases, Bellvitge University Hospital-IDIBELL, University of Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain. 10. APHP-GHU Nord, Mycobactériologie spécialisée et de référence, laboratoire associé du Centre National de référence des mycobactéries et de la résistance des mycobactéries aux antituberculeux (CNR-MyRMA), Paris, France; Université de Paris, INSERM, IAME UMR1137, Paris, France. Electronic address: emmanuelle.cambau@aphp.fr.
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 105 CFU/mL suspension, obtained from a 10-2 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 96-well 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. 103 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 0.03-0.12 for isoniazid, 0.12-0.5 for levofloxacin and 0.25-1 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 calibrated against this reference method to be used with EUCAST breakpoints.
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 105 CFU/mL suspension, obtained from a 10-2 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 96-well 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. 103 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 0.03-0.12 for isoniazid, 0.12-0.5 for levofloxacin and 0.25-1 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 calibrated against this reference method to be used with EUCAST breakpoints.
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