what to expect of the ”future” in antimicrobial
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What to expect of the ”future” in antimicrobial susceptibility testing?
Gunnar Kahlmeter EUCAST
Clinical microbiology, Blekinge and Kronoberg, Sweden
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The Future
• Any new techniques in the foreseeable future? • Any promising ”rapid” methods around? • Global harmonisation of breakpoints? • Global harmonisation of ECOFFs? • Global harmonisation of methods? • Any promising true automation? • Is TATFAR (transatlantic taskforce on AR)
signalling progress? • EUCAST – the next step!
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What is ”future” and what is ”rapid”?
• In AST the future has been around the corner since I took up clinical microbiology in 1971. Rapid ID (Maldi-Tof) and harmonisation of breakpoints are the most prominent achievments. And for some of us, the future is shrinking rapidly.
• ”Rapid” could be defined as having an ID- and AST-result within one hour of receiving the sample.
• ”Slow” could be defined as having an ID + AST 40 - 48 hours after receiving the sample. This is often today´s (and yesterday´s) standard!
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Methods for susceptibility testing • Phenotypic test methods
based on antimicrobial activity (MIC) and breakpoints (S≤X/R>Y mg/L)
• Genotypic test methods based on the detection of a resistance gene or its product
• Deduction – ”expert rules”
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Methods for susceptibility testing • Phenotypic test methods
based on antimicrobial activity (MIC) and breakpoints (S≤X/R>Y mg/L) – MIC, disk diffusion, automated systems like Phoenix, Vitek2, Microscan – Predicts susceptibility and resistance – Quantifiable
• Genotypic test methods based on the detection of a resistance gene or its product – mecA, vanA, vanB, ….PBP2, … betalactamase detection…. – Predicts resistance, not sensitivity – Not quantifiable
• Deduction – ”expert rules” – If mecA-positive then report betalactam antibiotics R;
If ESBL-positive, then report betalactam antibiotics R – or maybe not!? If erythromycin-resistant, then report roxithro- and clarithromycin R;
– Some rules predict susceptibility, others resistance. – Rules change over time – Not quantifiable
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Limitations of current methods? • Phenotypic methods:
– Require breakpoints – which calls for agreement! – Current methods normally 16 – 20 hours – Require species ID – Semi-automated or not automated.
• Genotypic methods: – Predict resistance – not susceptibility. – Predict known resistance – not new resistance. – Requires species ID – Not fully automated
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EUCAST was formed in 1996 and reformed in 2001.
Committee Country Disk Diffusion test?
EUCAST Europe Yes CLSI USA Yes
*EUCAST is now an umbrella for national breakpoint committees BSAC, CA-SFM, CRG, DIN, NWGA & SRGA.
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EUCAST and the review process Reviewed 2002 – 2009: • Aminoglycosides • Carbapenems & aztreonam (2nd review) • Cephalosporins iv (2nd review) • Cephalosporins oral • Fluoroquinolones • Glycopetides (2nd review) • Macrolides and lincosamines • Miscellaneous antimicrobials • Penicillins • Tetracyclines
• Antifungal drugs – fluconazole, voriconzole,
posaconazole – anidulafungin, amfotericin B
Topical agents: Mupirocin (LLR/HLR) Retapamulin (ECOFF)
Drugs being addressed: Cefalothin (ECOFF) Cefazoline (ECOFF) Cefoperazone (ECOFF) Sulbactam (alone) Kanamycin Streptomycin Josamycin Spiramycin ….
Lack of data for older drugs!
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EUCAST - breakpoints for new drugs with EMA
• Daptomycin √ • Tigecycline √ • Doripenem √ • Glycopeptides (two ongoing) • Cefalosporine (ongoing) • Anti-Tb (to be started)
• Glycopeptide (withdrawn) • Cefalosporine (withdrawn) • Fluoroquinolone (withdrawn) • Diaminopyrimidine (withdrawn)
• Extensions of indications (currently none)
EMA = European Medicines Agency
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Miscellaneous organisms Consultation with expert groups on breakpoints and
methods • Neisseria meningitidis (review) - 2011 • Moraxella catarrhalis (revised) - 2011 • Helicobacter pylori (finalized) - 2011 • Clostridium difficile (finalized) - 2011 • Campylobacter (ongoing) - 2011 • Listeria monocytogenes (consult) - 2011 • Pasteurella multocida (ongoing) - 2011 • Burkholderia cepacia (started) - 2012 • Corynebacteria (ongoing) - 2012 • M.tuberculosis – new agents - 2011/12 • …
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Common practices • Standardised phenotypic AST 16 – 20 hours
- EUCAST or CLSI or BSAC or CA-SFM.
• ”Direct” testing – shaves 20-24 h off the standard practice of culture (20h) + AST (16 – 20h). – Urine – direct inoculation
• Of all urines • Of positive urines (microscopi, rapid tests, flowcytometri)
– Blood cultures – direct (or semidirect) inoculation from a positive bottle
• ”Rapid AST” with automated methods – 8 h from pure culture
• ”Rapid AST” with MIC- or Disk Diffusion tests – Results 8 h from pure culture – Results 8 h from ……..?????
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Automation of AST – to… • Increase speed – rapid AST reports • Reduce need for hands-on work • Reduce the need for know how
– In running the test – In interpreting the result
• Increase standardisation • Simplify quality control • Reduce costs
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…will current trends in automation deal with the future? Very few hits on: • ”Automation in AST” - except evaluations of Phoenix, Vitek2,
Microscan • ”Rapid methods in AST” – basically a plethora of gene detection
protocols – some commercially available, others not (NDM-1, CTXinases, mecA and on variations to cover new genes, etc)
• Maldi-Tof – for the detection of ESBLs etc ?? • Flowcytometri, microcalorimeter detection of growth ??
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MIC • MIC – the minimum inhibitory concentration (mg/L
or µg/mL)
• MIC - the lowest concentration in a series of twofold concentrations that will inhibit the growth of a microorganism, as measured by the naked eye.
• Convention: The concentration series shall contain the concentration 1 mg/L 0.002, 0.004, 0.008, 0.015, 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512
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Methods for antimicrobial susceptibility testing MIC
• Broth dilution • Broth microdilution (BMD) • Agar dilution • Gradient tests (several)
Broth microdilution
(BMD)
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Methods for antimicrobial susceptibility testing Disk diffusion (DD)
• Disk diffusion – Correlated to MIC – Standardised DD: CLSI, EUCAST, BSAC, CA-SFM – MH (CLSI, EUCAST, CA-SFM), ISA (BSAC)
AST by Disk diffusion
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If you perform MIC determinations on many isolates of a well defined species using a standardized method,
this is what you get:
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…and if you do the same with disk diffusion you get exactly the same and the match is perfect
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E. coli with ciprofloxacin 5 ug223 clinical isolates
0
5
10
15
20
25
30
35
40
456 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Inhibition zone diameter (mm)
No o
f iso
late
s
321684210.50.250.1250.0320.0160.0080.004
Breakpoints S R MIC ≤0.5 >1 Zone diameter ≥22 <19
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Automated systems
• MIC-based (semiautomated) – Microdilution (Phoenix, Microscan, Sensititre) – Growth curve (Vitek2)
• Disk Diffusion (semiautomated)
– Biomic, Sirscan, Osiris
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Automated AST typically offer
• Interface with LIS (Laboratory Information Systems) • Quality Control programs • Interpretative reading and Expert Rules:
– Intrinsic susceptibility (warn against reporting resistant) – Intrinsic resistance (warn against reporting susceptible) – Expert rules: IF – THEN (deduce susceptibility and
resistance)
• Resistance epidemiology features
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Essential agreement
• ...can not be assessed when dilution series are truncated
Expected .06 .12 .25 .5 1 2 4 8 16 Obtained ≤.25 .5 1 2 >2 MICs (n) 24 52 31 3 2 22 31 12 45
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Categorical agreement (CA) • Number of categorical results (S, I and R) in agreement
Agreement Expected/Obtained CA S/S CA I/I CA R/R minor errors S/I, I/S, I/R, R/I ME S/R (false resistance) VME R/S (false susceptibility)
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Automated AST
Pros • Combined AST and ID – but are we interested? • Speed (6, 8 – 18h) • Labour – saving - ?? • Cost – savings - ??
– Counting exceptions and limited capacity and the need for running a supplementary test, this needs careful consideration.
• Standardisation • QC • Expert rules to suggest resistance mechanism.
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Automated AST
Cons • Many exceptions – supplementary testing needed.
– Missing agents – Missing organisms
• Do not respond well to rapid development – New drugs – New breakpoints – New resistance mechanisms – Approval procedures take too long – Updates take too long
• Limited capacity • Limitations caused by short dilutions series • Limitations caused by availability of panels
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Approval process The process is too time consuming!
• The machines are ”susceptibility testing machines” and are approved as such.
• Performance and approval focus on categorical agreement. • Every breakpoint change needs new FDA approval.
• The machines should have been developed as ”MIC determination machines”:
• Approval should have been focused on essential agreement only – ”get the MIC right”.
• ….in which case breakpoint changes could be implemented without new FDA approval.
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Future phenotypic AST (6 - 8 h)
• Automated MIC-determination – ID known through Maldi-Tof – machines dedicated to MIC determination – complete dilution series – approval on MIC performance (not on categorical
agreement) – 1 – 4 - 6 – 8 h (!?) – new techniques for measuring
growth may reduce time further. – Output: WT or NWT, Clinical S, I o R
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Future phenotypic AST (6 – 8 h)
• Automated disk diffusion testing – Automated
• plate inoculation, • disk application, • incubation • and zone diameter reading against variable protocols.
Database of zone diameter distributions related to incubation time and adjusted breakpoints.
– Output: WT or NWT, Clinical S, I or R
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If you perform MIC determinations on many isolates of a well defined species using a standardized method,
this is what you get:
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…and if you do the same with disk diffusion you get exactly the same and the match is perfect
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Future susceptibility report? Clinical breakpoints and assessment of resistance mechanisms
• Escherichia coli – Ampicillin R – Cefotaxime SNWT – Trimethoprim R – Nitrofurantoin S – Ciprofloxacin SNWT
• Enterococcus faecalis – Ampicillin S – Gentamicin RWT
– Ciprofloxacin IEWT
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Back to the beginning!
……….
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The Future • Any new techniques on the horizon?
– Maldi-Tof, – Alternative ways of measuring growth for early phenotypic detection of R?
• Any promising ”rapid” methods around? – Automatic reading of disk diffusion? 8 hour AST – There should have been an MIC-determination machine around but alas!
• Global harmonisation of breakpoints? – CLSI and EUCAST getting ready to harmonize? NO – EUCAST is strong and making headway in many countries!
• Global harmonisation of methods? – Yes – to some extent.
• TATFAR (transatlantic taskforce on AR) – progress? – CDC and ECDC have been tasked with organising common ECOFFs!
• EUCAST beyond 2011?
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EUCAST and the future • 2012 new contract with ECDC • External expert committee financed by ECDC and peopled by the
profession • Developing standards for ECDC surveillance (AMR, HCAI) • The breakpoint committee of EMA for new, including new anti-Tb
agents, and existing antimicrobials – breakpoints and ECOFFs.
• Standardizing AST methods and IQC and advising on EQC. • Communicating with national AST committees (NAC) and the
profession through public consultation and the website (www.eucast.org)
• Education • Address issues with locally acting agents and with the activity of
agents i biofilms.
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Thank you
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