1 Antibacterial Susceptibility Testing Basics Sandra S. Richter, M.D. Cleveland Clinic 10/19/18 Disclosures: Research funding from bioMerieux, BD Diagnostics Objectives • Describe antimicrobial susceptibility testing (AST) methods available for bacteria • Discuss use of special phenotypic and molecular methods to detect antimicrobial resistance • Determine when AST should be performed and how to troubleshoot unusual results Antimicrobial Susceptibility Testing • A principle function of clinical microbiology lab • Predict outcome of treatment with agents tested • 90-60 rule • 90% success when organism is “Susceptible” • 60% success when organism is “Resistant” • Intermediate • Higher doses required to ensure efficacy • Buffer zone that prevents borderline susceptibility from incorrect categorization as R or S • Guide selection of most appropriate agent • Most narrow spectrum • Least expensive Susceptibility Test Methods • Qualitative (S, I, R) • Disk diffusion • Quantitative (MIC) • Broth macrodilution • Broth microdilution • Agar dilution (single concentration per plate) • Agar gradient diffusion (Etest) • Automated instrument Antimicrobial Susceptibility Testing • MIC = Minimum inhibitory concentration that inhibits growth over defined interval (usually 18-24 h) • MIC is compared to levels of drug achieved in human body fluids to determine breakpoints
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1
Antibacterial
Susceptibility Testing
Basics
Sandra S. Richter, M.D.
Cleveland Clinic
10/19/18
Disclosures: Research funding from bioMerieux, BD
Diagnostics
Objectives
• Describe antimicrobial susceptibility testing
(AST) methods available for bacteria
• Discuss use of special phenotypic and
molecular methods to detect antimicrobial
resistance
• Determine when AST should be performed
and how to troubleshoot unusual results
Antimicrobial Susceptibility Testing
• A principle function of clinical microbiology lab
• Predict outcome of treatment with agents tested
• 90-60 rule• 90% success when organism is “Susceptible”
• 60% success when organism is “Resistant”
• Intermediate• Higher doses required to ensure efficacy
• Buffer zone that prevents borderline susceptibility from incorrect categorization as R or S
• Guide selection of most appropriate agent • Most narrow spectrum
• Least expensive
Susceptibility Test Methods
• Qualitative (S, I, R)
• Disk diffusion
• Quantitative (MIC)
• Broth macrodilution
• Broth microdilution
• Agar dilution (single concentration per plate)
• Agar gradient diffusion (Etest)
• Automated instrument
Antimicrobial Susceptibility Testing
• MIC = Minimum inhibitory concentration
that inhibits growth over defined interval
(usually 18-24 h)
• MIC is compared to levels of drug
achieved in human body fluids to
determine breakpoints
2
Broth microdilution method M11: Reference Agar Dilution• 2-fold dilutions of
antimicrobial agents added to molten agar (cooled to 48-50ºC), mixed, poured into Petri dish, allowed to solidify
• 0.5 McFarland suspension of isolate inoculated onto each plate in concentration series using replicating device (105
CFU/spot)
• Read after 42-48 h anaerobic incubation
• MIC = lowest concentration inhibiting growth
Commercial AST Systems
• Introduced in 1980s; in most labs since 1990s
• Manual or semiautomated broth microdilution (small
volume)
• Automated broth microdilution (large volume)
• Semiautomated disk diffusion - popular outside of U.S.
• DMS with LIS interface and expert system
analysis
• Epidemiology software
Acceptable performance for FDA
clearance of AST system
• Multicenter comparison of system to CLSI
reference method
• >90% categorical agreement (CA)
• >90% essential agreement (EA)
• <1.5% very major errors (VME)
• <3% major errors (ME)
• >95% reproducibility
• Sufficient no. resistant organisms tested
• <10% growth failure for each organism group
11
FDA clearance of AST systems
• Antimicrobial/organisms without acceptable
performance listed as limitation in the device PI
(alternative method recommended)
• Reporting of results for agents without known clinical
efficacy against organism is discouraged (consult
drug label, CLSI M100 Table 1A, Sanford Guide to
Antimicrobial Therapy)
• Manufacturers required to apply FDA interpretive
standards to AST system results12
FDA clearance of AST systems
• When breakpoints change a comparative study is required
• If new breakpoints affect device performance a new 501(k) submission is required
• If validated, labs may report result using interpretive
criteria other than those published in device label