neil woodford antibiotic resistance monitoring & reference laboratory, centre for infections...
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Neil Woodford
Antibiotic Resistance Monitoring & Reference Laboratory, Centre for Infections
Antibiotic susceptibility testing: then, now and hereafter
AST Why?
Cannot assume susceptibility or resistance Guidance for treatment of the individual
patient Background information for empirical
treatment To set local and national prescribing policies To monitor epidemiological trends For surveillance of resistance To test the activity of new agents Means of detecting new resistances
CR-AB clones emerge
Resistance mechanisms
VRE
1985 1990 1995 20052000 20152010
Carbapenemases –
Enterobacteria;
NDM-1 discovered
CTX-M ESBL ‘explosion’
starts
Lin-R enterococci
VRE in animals
Dap-R staphs & enterococci
1st CTX-M ESBL
EMRSA
Woodford graduated
PCRGenome
sequence
AST How?
Quantitative methods (MIC, mg/L) Agar dilution Broth dilution Gradient methods
Qualitative methods (S I R) Disk diffusion Agar-incorporation breakpoint methods
Automated methods
1940s1940s 1946 Garrett:1946 Garrett:
multiple replication devicemultiple replication device concept of critical dilutionsconcept of critical dilutions
forerunner of agar-incorporation forerunner of agar-incorporation breakpointsbreakpoints
Modified by Steers et al 1959
1950s1950s
Comparative methodComparative method Joan Stokes 1955Joan Stokes 1955 Stokes & Waterworth 1972Stokes & Waterworth 1972 Stokes & Ridgway 1980Stokes & Ridgway 1980
Humphrey & Lightbown 1952
r2 = 9.21 Dt (logM – log 4πhDtc) r radius of the inhibitory zone t time from start c MIC D diffusion constant M disc potency H depth of agar
Zone size is: directly proportional to the diffusion
constant directly proportional to the log of the disc
potency inversely proportional to the log of the MIC
MICs and zone sizes are meaningless
…unless you apply interpretative criteria
clinical breakpoints indicate likelihood of therapeutic success (S) or failure (R ) of antibiotic treatment based on microbiological findings (S≤ Y mg/L and R> Z mg/L)
epidemiological cut-off values (ECOFFs) separate microorganisms without (wild type) and with acquired or mutational resistance (non-wild type) (WT≤ X mg/L)
European CommitteesEuropean Committees
BSACUnited Kingdom
CA-SFMFrance
CLSI (NCCLS) USA
DINGermany
SRGASweden
CRGNetherlands
NWGANorway
StandardisationStandardisation 1959 Ericsson & Steers – evaluation of 1959 Ericsson & Steers – evaluation of
methodsmethods 1961 WHO – standardisation1961 WHO – standardisation 1964 Isenberg – comparison of methods in 1964 Isenberg – comparison of methods in
USAUSA 1964 Truant – standardised tube dilution MICs1964 Truant – standardised tube dilution MICs 1966 Bauer-Kirby1966 Bauer-Kirby
1975 NCCLS 1975 NCCLS CLSI CLSI 1998 BSAC Standardised Method 1998 BSAC Standardised Method 2009 EUCAST Standardised Method2009 EUCAST Standardised Method
EUCAST clinical MIC breakpoints
• Dosing, formulations
• Wild-type MIC distribution
• Existing national breakpoints
• PK/ PD data & Monte Carlo simulations
• Clinical data - outcome studies
• Tentative breakpoints are set for target species so as to avoid splitting the WT MIC distribution
• Consultation on proposed values
• Approval / publication on EUCAST website
”Dashed” – laboratories are recommended not to test
against this species
Click on name to directly access
MIC distributions
Insufficient evidence
Breakpoint tablesBreakpoint tablesavailable at http://www.eucast.orgavailable at http://www.eucast.org
”Wild type”
EUCAST determines epidemiological cut-off values for early detection of resistance
ECOFF: WT ≤ 0.032 mg/L
Adding value to AST…
• Interpretative reading
– Infer mechanisms from patterns (antibiograms)
– Recognise grossly unusual
– Edit susceptibilities / identify further drugs to test
– Tentative surveillance of resistance mechanisms
• it’s not an exact science
– there are always exceptions and anomalies
Interpretative reading
Examine the whole phenotype Apply “expert” knowledge …, but you must
Identify to species Test a large panel of antibiotics
All in a box: automated AST +/- ‘expert’ interpretation
What’s more important for appropriate therapy ?
MIC
Mechanism
Supplemental tests for Supplemental tests for mechanismsmechanisms
Hetero-resistance
• small sub-population of cells
• not easily detected
• some MRSA• h-VISA• colistin resistance
• may be distinct from full resistance
GISA
Hetero-GISA
GSSA
Molecular detection: where and why ?
• In the Reference Laboratory
– confirmation of unusual resistance
– surveillance of resistance mechanisms
– monitoring spread of resistance genes / strains
– identify strains likely to contain novel resistance mechanisms
• In the clinical diagnostic laboratory
– rapid detection for patient management
– infection control
Molecular detection: different needs
• In the Reference Laboratory,
– testing “pure” cultures
– myriad assays and formats
– numerous bug-drug combinations
• In the clinical diagnostic laboratory
– directly from specimens
– need to target key species
– format must be simple, rapid and cost-effective
– problems with genes in commensals
Molecular detection
Simple and multiplex PCR Real-time PCR DNA sequencing Hybridisation-based techniques
Molecular tests in clinical labs
Black box approach:
molecular biology steps hidden
Simple end-product detection
Simple samplepreparation
• Must be rapid (TATs), inexpensive, reliable !• Platform must be sufficiently versatile to justify investment• Relatively hands-free, with scope for automation• On-going – e.g. <30 min test for ESBL detection
Chips with everything…going beyond AST!
Total profiling; more cost-effective than PCR
• species identification• resistance genes• virulence genes• epidemicity predictors• strain-specific markers
Molecular detection: the inherent problem
• Molecular methods only detect known mechanisms• only as good as available sequence data• resistant isolates with known genes identified
• & new variants, if sufficient homology• false-resistance (unexpressed / partial genes)
• Susceptibility must always be confirmed• can’t base treatment on a negative molecular result • can’t detect genuinely new resistance mechanisms• will never (?) replace cheap phenotypic methods
What next for AST ?
• in the Reference Laboratory
– increased use of arrays, especially to support surveys, neural networks, web-based tools ?
• in the clinical diagnostic laboratory
– ↑ automated systems
– simple molecular methodologies
– tailored systems
– competitive market niche
Acknowledgements
Charles Easmon
Cathy Ison
Trevor Winstanley
Derek Brown
Robert George
David Livermore
ARMRL staff, 1988-present
Collaborators, 1985-present
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