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Educational WorkshopEW08: Antimicrobial susceptibility testing – practical implementation of the EUCAST breakpoints and methods
arranged with the European Committee on Antimicrobial ( )Susceptibility Testing (EUCAST)
Convenors: Gunnar Kahlmeter (Växjö, SE)( j , )Derek Brown (Peterborough, UK)
Faculty: Gunnar Kahlmeter (Växjö SE)Faculty: Gunnar Kahlmeter (Växjö, SE)Derek Brown (Peterborough, UK)Rafael Canton (Madrid, ES)Fred Tenover (Sunnyvale, US)Roland Leclercq (Caen, FR)
Kahlmeter - EUCAST Clinical Breakpoints
EUCAST Clinical Breakpoints
Gunnar Kahlmeter
ECCMID 2010
Q 1Q
Tasks1. Determine clinical breakpoints for existing and new
antibacterials, antifungals, antimycobacterials.
2. Define wild type MIC distributions and epidemiological cut-off values for bacteria and fungi.
3 Develop susceptibility testing methods and systems for3. Develop susceptibility testing methods and systems for internal QC.
4. Liaise with EMEA, ECDC, EFSA, EARSS and others involved in antimicrobial resistance.
5. Liaise with national committees involved in antimicrobial resistance and susceptibility testing, to facilitate implementation of European breakpoints
3
Kahlmeter - EUCAST Clinical Breakpoints
Q 2Q
Why European breakpoints in Europe?
EUCAST breakpoints …• based on EMEA approved indications and outcome evaluation,
Pk/Pd, multiple MIC distributions, and modern principles of determining breakpoints
• related to European minimum and maximum dosages• accepted by European regulatory authorities (EMEA ECDC)accepted by European regulatory authorities (EMEA, ECDC)• official breakpoints in European SPCs (EMEA)• ”case definitions” for antimicrobial resistance surveillance
(ECDC)• transparant and published rationale behind decisions• independent of commercial interests • reviewed at intervals: with new member of class or on initiative of
the profession, EMEA, the Company, EUCAST.• in the public domain and free of charge
National Breakpoint CommitteesD, F, N, NL, S, UK,
EUCAST General CommitteeAll European Countries + ISC/FESCI
EUCAST Steering CommitteeBSAC, CA‐SFM, CRG, DIN, NWGA, SRGAAnd 2 reps from the General Committee
SubcommitteesAntifungalsAnaerobesExpert Rules
Expert groups
4
Kahlmeter - EUCAST Clinical Breakpoints
Steering Committee and General Committee
• General Committee– Each European country invited to appoint 1
representative each– ISC and FESCI 1 rep each– One meeting per yearOne meeting per year
• Steering Committee (11 members)– Chairman, Scientific Secretary and Clinical Data
Coordinator appointed by ESCMID– Each national breakpoint committee 1 rep– General Committee 2 reps– 5 meetings per year
Decision process
• EUCAST Steering Committee• National breakpoint committees• EUCAST General Committees and Expert
groups, Industry (pharmaceutical + AST)groups, Industry (pharmaceutical AST)
• EUCAST Steering Committee in agreement with National breakpoint Committees
• New consultation (?)• Decision
EUCAST Subcommittees
• Antifungals– Antifungal drugs in need of breakpoints – Breakpoints and rationale documents – Methods
• Anaerobe bacteria• Anaerobe bacteria– Antibiotics in need of breakpoints– Breakpoints– Methods
• Expert Rules– Tables of intrinsic resistance– Expert rules (IF/THEN)
5
Kahlmeter - EUCAST Clinical Breakpoints
Consultation with expert groups
• Neisseria spp (finalised)• Anaerobes (ESGARAB, ongoing)• Helicobacter pylori (EHSG, ongoing)• Clostridium difficile (ESGCD, ongoing)• Campylobacter (VetCast, ongoing)• Listeria monocytogenes• …
EUCAST Website
www.eucast.orgg
requires no login
6
Kahlmeter - EUCAST Clinical Breakpoints
Q 3Q
Tools for determining CLINICAL BREAKPOINTS
1. Dose or doses2. Target organisms 3. MIC-distributions for target organisms
- breakpoints not to divide MIC-distributions of WT target organisms- MIC distribution and ECOFFs determined for each speciesp
4. Resistance mechanisms in target organisms5. Clinical indications6. Pharmacokinetics (Cmax, AUC, T½, Protein binding, Vd..)7. Pharmacodynamic properties (peak conc/MIC, AUC/MIC, TA, MCs)8. Clinical outcome (clinical outcome/MIC)9. Epidemiological cutoffs, Pk/Pd-breakpoints and clinical data together
determine the CLINICAL BREAKPOINT
Clinical resistance
Wild type
Clinical resistance
Clinical resistance
Phenotypically detectable resistance
Genetically detectable resistance
Clinical resistance
Susceptible Resistant
7
Kahlmeter - EUCAST Clinical Breakpoints
EUCAST and existing antimicrobials• Aminoglycosides √• Carbapenems & aztreonam √• Cephalosporins iv √• Cephalosporins oral √• Fluoroquinolones √
√• Glycopetides √• Macrolides and lincosamides √• Penicillins √• Tetracyclines √• Miscellaneous antimicrobials √
• Antifungal drugs (flu- and voriconzole) √
EUCAST breakpoints for new drugs through European Medicines Agency (EMEA)
• Daptomycin √• Tigecycline √• Doripenem √
N h l i ( i )• New cephalosporin (ongoing)• Glycopeptides (ongoing)
• Extensions of indications (none ongoing)
Topicals and less commonly used drugs
1. Mupirocin (Topical)2. Polymyxin B (Topical)3. Bacitracin (Topical)4. Streptomycin (hlr for enterococci)5. Neomycin (Topical)6 Sulfamethoxazole (UTI)
11.Cefoperazone12.Pefloxacin13.Cefradine14.Cefamandole15.Sulfisoxazole
6. Sulfamethoxazole (UTI)7. Cephalothin (expert rules?)8. Sulfadiazine9. Spiramycin 10.Nalidixic acid (screening)
16.Pipemidic acid17.Kanamycin18.Ceftizoxime19.Cefprozil
+ 45 others
8
Kahlmeter - EUCAST Clinical Breakpoints
Microorganisms without breakpointsDefine relevant drugs, breakpoints, methodology and MIC-distributions.
• Helicobacter spp √• Campylobacter spp √• Clostridium difficile √• Legionella sppLegionella spp• Pasteurella multocida• Listeria monocytogenes• …• …
EUCAST breakpoint tablesMIC (mg/L) brpts* S≤ 2 R>2
Zone (mm) brpts* S≥ 22 R<22
Insufficient evidence (Literature: ”not enough evidence for a
IECan not be substituted.
Can be s pplemented ith an MIC itho tbreakpoint” or ”no indication”) Can be supplemented with an MIC without interpretation.
Inappropriate drug(Literature: poor drug – don´t use!
—Can be substituted with an automatic ”R”.
Numbered footnotes MIC-breakpoints
Lettered footnotes Zone diameter breakpoints
*when numbers are the same = no intermediate category
Web-links to MIC-distributionsWeb-links to Zone diameter distributionsWeb-links to EUCAST Rationale Documents
9
Kahlmeter - EUCAST Clinical Breakpoints
The MIC is the reference for all other phenotypic tests
The MIC is the reference for all other phenotypic tests
E coli /Mecillinam 10 ug969 isolates, of which 930 consecutive
100
125
150
ates
512
256
128
64
32
16
8
The MIC is the reference for all other phenotypic tests
0
25
50
75
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
Inhibition zone diameter (mm)
No
of is
ola
4
2
1
0.5
0.25
0.125
0.064
0.032
Breakpoints S RMIC ≤8 >8Zone diameter ≥15 <15
10
Kahlmeter - EUCAST Clinical Breakpoints
Q 4
National strategies and joint decisions on AST are needed!
On a national level:
NACNational Antimicrobial Committee
National Antimicrobial Committeestasks
• Subcommittee on Antimicrobial susceptibility testing– Strategy at national level– Implementation of breakpoints and methods
Education– Education – Liaison and consultation with EUCAST – Liaison with groups involved in AMR-surveillance
(ECDC, EARSS, ….).– QA
• Antimicrobial Policies• Antimicrobial Resistance Surveillance• Antimicrobial Consumption and Policies
11
Kahlmeter - EUCAST Clinical Breakpoints
Decisions for 2010/11:
SwedenDenmarkNorwayBelgium
Discussions ongoing:
SpainGreeceItalyTurkey
EUCAST breakpoints
The NetherlandsAustriaEstoniaIrelandFinlandScotlandWalesSwitzerland
Israel
ENAC European Network of Antimicrobial
CommitteesCommittees
The EUCAST General Committee.
• Harmonised breakpoints for all major antibacterial and antifungal drugs.
• Orphan drugs and microorganisms identified and prioritized• Breakpoints for new drugs as part of the approval process with EMEA
(daptomycin, tigecycline, doripenem).• Epidemiological cut off values determined for all drugs.• SOPs to describe formal relationship with EMEA.• EUCAST breakpoints mandatory in European SPCs.
EUCAST April 2010
• ISO-standardized MIC-determination. • Software and database for MIC- and zone distributions.• Breakpoints implemented in national (F, D, N, NL, S, UK) systems
2007 – 2010.• EUCAST disk diffusion test launched 2009.• Breakpoint tables, QC-tables, methodology documents available on
website.
12
Kahlmeter - EUCAST Clinical Breakpoints
EUCAST April 2011
• EUCAST disk diffusion method implemented in 5 - 6 countries• NACs in 10 – 15 countries.• National Educational Workshops on European AST in several
countries.• EUCAST breakpoints in all major systems for AST (BSAC, CA-SFM;
Commercial systems Phoenix, Vitek2, Microscan, BioMic).• All Rational Documents available on website• All Rational Documents available on website.• SOPs to describe formal relationship with ECDC.• ECDC decided on European breakpoints as mandatory in
surveillance of antimicrobial resistance and HCAI.• Breakpoints and methods for Campylobacter, Helicobacter,
C.difficile, and others.• Breakpoints and methods several topical antimicrobials and several
less commonly used drugs.• Formal decision on the future relationship between EUCAST,
ECDC, EMEA and ESCMID.
Q 5Q 5
Thank you!
13
Brown - Implementation of the EUCAST disk-diffusion method
Implementation of the EUCAST di k diff iEUCAST disk diffusion
method Derek Brown
EUCAST Scientific Secretary
Question1
EUCAST disc diffusion method
• Why develop a EUCAST disk diffusion test?
• Development of the method
• Calibration of the method
• Implementation in individual laboratories
14
Brown - Implementation of the EUCAST disk-diffusion method
EUCAST early 2007• No plans for EUCAST disk diffusion test• MICs can be interpreted with EUCAST breakpoints• Automated systems can be calibrated to EUCAST
breakpointsDi k diff i th d lib t d t EUCAST• Disk diffusion methods calibrated to EUCAST breakpoints are available in France, Sweden and the UK.
......little enthusiasm for a new “EUCAST disk diffusion method”
EUCAST consultation and questionnaire 2007-8
• France and UK likely to maintain their national methods, calibrated to EUCAST breakpoints, for the immediate future.
• Other countries were not keen to take on other national methods with unfamiliar technique.
• Most countries were currently using Kirby-Bauer type methods (particularly “CLSI”) and, if they adopted EUCAST breakpoints, would prefer to retain the same methodology calibrated to EUCAST breakpoints (.....but get rid of HTM)
Benefits of a EUCAST disk diffusion method (based on the
Kirby-Bauer approach)• Identified as EUCAST method calibrated to
EUCAST MIC breakpoints• Wide base of expertise throughout Europe (and
worldwide)• KB technique familiar to most laboratories in
countries without their own method• Extensive database of MIC v Zone diameters
available for KB method?
15
Brown - Implementation of the EUCAST disk-diffusion method
EUCAST decision to develop a disk diffusion method June 2008 Based on KB approach
– MH medium– MH-F for fastidious organisms– 0 5 MF inoculum– 0.5 MF inoculum– 16-20h incubation– Most disk contents same – Most control strains same– Calibrated to EUCAST MIC breakpoints
S. pneumoniae ATCC 49619 H. influenzae NCTC 8468
Mueller-Hinton-fastidious (MH-F)Mueller-Hinton agar + 5% defibrinated horse blood
and 20 mg/L β-NAD
16
Brown - Implementation of the EUCAST disk-diffusion method
EUCAST QC tables
Targets NOT in italics are from ISO and/or CLSI recommendations
Question 2
17
Brown - Implementation of the EUCAST disk-diffusion method
Calibration of EUCAST disk diffusion method
• Existing distributions of MIC v zone diameter with the same technique
• New distributions of MIC v zone diameter• Zone diameter distributions for routine isolates• Targeting critical areas of the MIC and zone
diameter distributions • Targeting specific resistances
1
2
4
8
16
32
>=64
MIC
(μg/
ml)
1 1
1
1 1 1 1
1 1 1 1 1
1 1 1 1 1 1 1
1 1 1 1
2 2
2 2
2 2
3 3
3 3
4
4
6
35
Figure 7: Ceftriaxonen=350y=18.9 - 0.49xr=0.95
MIC v zone diameter for ceftriaxone with Enterobacteriaceae
6 9 12 15 18 21 24 27 30 33 36 39 42 45Ceftriaxone Zone Diameter (mm)
<=0.015
0.03
0.06
0.12
0.25
0.5
1
Cef
triax
one
1 1 1 1 1
1
1 1 1 1
1 1
1 1
1
1 1
2
2 2
2
2
3
3 3
3
3
4
4
4
4
5 5 5 5
5
5
6
6
7
7
8
8 9 10
11
12
12
12
13
13
15
19
18
Brown - Implementation of the EUCAST disk-diffusion method
Zone diameter correlates
19
Brown - Implementation of the EUCAST disk-diffusion method
Development of EUCAST disk diffusion method
• Tentative breakpoints have been published for all agents with MIC breakpoints
• Continuing validation throughg g– Existing and new distributions of MIC v zone diameter– Targeting critical areas of MIC v zone diameter
distributions and specific resistances– Zone diameter distributions for routine isolates
• Ongoing maintenance
Implementation – EUCAST action• Documentation
– Method description– Breakpoints– QC limits
• Education– Teaching material (slideshow)– Teaching material (slideshow)– Meetings– Practical workshops– Laboratory visits
• The laboratory in Vaxjo is an ESCMID Collaborative Centre –the ESCMID Observership program could be used for visit.
– Publications/website • Inform media, disk, zone reader manufacturers
Implementation at national level”National antimicrobial committees”
• Strategy at national level for implementation of breakpoints and methods
• Inform people of implications for their laboratory/country• Education through meetings, publications, websites,
ti l k hpractical workshops• Liaison with media, disk, zone-reader manufacturers• Liaison and consultation with EUCAST.• Liaison with groups involved in AMR-surveillance• External Quality assessment• Staged introduction may be appropriate – eg. large labs
first
20
Brown - Implementation of the EUCAST disk-diffusion method
Implementation at local level Before routine use
• Ensure all stakeholders are informed of implications for their laboratory/hospital
• Appoint a ”champion” to implement the method• Visit laboratories using the method
Pl ll i d• Plan well in advance– media, disk, supplies– templates, zone-reader setup– computing (breakpoints/QC ranges)– documentation
• Train staff in advance – demonstrations, practical experience, ensure that QC requirements are met
• Use contacts in other laboratories and at EUCAST directly or through NACs or national EUCAST GC rep
Implementation at local levelDuring introduction
• Ensure that adequate staffing is available – will take more time at first
• Ensure that senior staff are available to answer questions and deal with problems
• Use national contacts – National Antibiotic Committees– Use contacts in laboratories already using the method
• Use EUCAST contacts– Erika Matuschek (Swedish External Reference Laboratory for
Antimicrobial Susceptibility Testing; ”SERLAST”) [email protected]
– Gunnar Kahlmeter (EUCAST and SERLAST)– Derek Brown (EUCAST)
Implementation at local levelAfter implementation
• Keep the method up-to-date• Continue to educate staff• Report problems to EUCAST
– Erika Matuschek (SERLAST)Erika Matuschek (SERLAST)
• Be prepared to help other laboratories
21
Brown - Implementation of the EUCAST disk-diffusion method
Acknowledgements• Gunnar Kahlmeter• Erika Matuschek and Jenny Åhman
• EUCAST Steering Committee• National Committees• Collaborating laboratories• Individual experts
22
Cantón - Implementation of EUCAST breakpoints with automated systems
Implementation of the EUCAST
breakpoints with automatic systems
Antimicrobial susceptibility testing – practical implementation of the EUCAST breakpoints and methods
20th ECCMID Vienna Austria 10 13 April 201020th ECCMID, Vienna, Austria, 10 – 13 April, 2010
Hospital Universitario Ramón y CajalSERVICIO DE MICROBIOLOGÍA Y
PARASITOLOGÍA
Dr. Rafael Cantón
Methods for antimicrobial susceptibility testing
Phenotypic test methods: based on antimicrobial activity and breakpoints
- MIC determination (broth, agar, gradient diffusion)- Disk diffusion (BSAC, CA-SFM, CLSI, SRGA, EUCAST...)- Automated systems (Vitek, Phoenix, MicroScans, Sensititre, ...)
Genotypic test methods:Genotypic test methods: based on the detection of a resistance gene or its product
- mecA, vanA, vanB… - PBP2a, β-lactamase detection
By deduction – “expert rules”- If mecA-positive, then report beta-lactam antibiotics as R- If erythromycin-R, then report azithro- and clarithromycin as R
Main objectives
To produce antimicrobial susceptibility testing (AST) resultsin a automated (or semi-automated) mode
To standardize AST avoiding uncontrolled differences
Antimicrobial susceptibility automatic systems
To offer AST in a shorter period of time than manual methods
To interpret AST results (clinical categorization / interpretation)
23
Cantón - Implementation of EUCAST breakpoints with automated systems
MIC based automatic systems
Antimicrobial susceptibility automatic systems
None of the current automatic susceptibility testing devicescan be considered fully automated …
- Automated system consist of devices with computer-assistedincubation, reading, interpretation and reporting functions
- Semi-automated systems require off-line incubation*. The panels areSemi automated systems require off line incubation . The panels areautomatically read with computer-assisted interpretation and reporting
*manual loading of each panel into the system is required
- Manual systems use commercial (eventually in-house) panels that are read by laboratory personnel. Results are either recorded by handor manually entered into a computer for interpretation and reporting
All instruments have implemented computer programs
Antimicrobial susceptibility automatic systems
Most automatic susceptibility testing devices have incorporated …
Interface connections with laboratory information systems (LIS)Quality control computer programs Computer programs or expert systems:
- to interpret phenotypes and infer resistance phenotypes
“antibiogram interpretive reading”
- to perform actions based in clinical evidences and resistance mechanisms knowledge in response to specific antimicrobialsusceptibility test results
“expert rules”
Programs to manage results for epidemiological purpose
24
Cantón - Implementation of EUCAST breakpoints with automated systems
Classification
MIC based systems- agar dilution (no longer exists!)
- microdilution: MicroScan, Sensititre, Phoenix, …
- growth curves: VITEK legacy, VITEK2
Antimicrobial susceptibility automatic systems
Disc diffusion based systems
- BIOMIC System
- SIRSCAN System
- OSIRIS System
……
Antimicrobial susceptibility automatic systems
Sensititre ARIS System (Trek Diagnostic Systems)
Standard 96-well microdilution panels with 22-24 antibiotics/panel
Manual or semi-automatic inoculation of panels
Fluorescent measurement of bacterial growth uo esce t easu e e t o bacte a g o tafter generating a fluorescent product from anon-fluorescent substrate
- Susceptibility testing results in 18-24 h
Data manager system and expert software rules
Antimicrobial susceptibility automatic systems
MicroScan WalkAway (Siemens Healthcare Diagnostics)
Standard size 96-well microdilution panels with 18-22 antibioticswith or without substrates for bacterial identification
Panels are manually inoculated
Turbidimetric reading (overnight panels) - susceptibility testing results in 15-18 h
Fluorimetric reading (rapid panels with a fluorometric substrate)- susceptibility testing results in 3.5-7 h
Data manager system and expert software rules
25
Cantón - Implementation of EUCAST breakpoints with automated systems
Antimicrobial susceptibility automatic systems
Vitek 2 (BioMérieux)
Separate plastic cards for ID and AST- AST: 64-well plastic card with 19-20 agents (deduce additional
agents with computer software)
Optical reading every 15 minutes with a multichannel fluorometerand photometer to record fluorescence and turbidityand photometer to record fluorescence and turbidity
Changes in fluorescence over time (growth curves) comparing agrowth control well with wells with drug concentrations
Susceptibility results in 4-18 h
Expert system (Advanced Expert System, AES)based in MIC and phenotype analysis
Antimicrobial susceptibility automatic systems
Phoenix System (BD Diagnostic Systems)
Combination panels for bacterial ID and ASTAST: 84-wells (18-25 antimicrobial agents)
Manually or semi-automatic inoculation of panels
Monitor reading every 20 minutes with a turbidometric andcolorimetric (oxidation-reduction indicator) growth detection
Changes in fluorescence over time (growth curve), comparing agrowth control well with wells with various drug concentrations
Susceptibility results in 4-16 h
Expert system (Advance expert System)
Antimicrobial susceptibility automatic systems
Wider System (Fco. Soria Melguizo)
Standard size 96-well microdilution panels with 18-22 antibioticswith or without substrates for bacterial identification (panels areprepared by MicroScan)
Panels manually inoculated and externally incubateda e s a ua y ocu ated a d e te a y cubated
Panels are manually introduced in a video image reader after15-18 h of incubation
Expert software rules
26
Cantón - Implementation of EUCAST breakpoints with automated systems
Antimicrobial susceptibility automatic systems
Disc diffusion based systems
Video assisted reader plate to read and interpret discdiffusion susceptibility agar plates
Quantitative zone diameters arecalculated by digitalizationy gand software interpreted (SIR) withdeduction of MIC
Expert software rules
Antimicrobial susceptibility automatic systems
MIC based systems
Device Inoculation ReadingReporting time (hours)
Sensititre Manual orsemiautomatic
Manual read orFluorescence
18-24
MicroScan Manual Turbidity and fluorometer
15-183 5-7fluorometer 3.5-7
Phoenix Manual orSemiautomatic
Turbidity andcolorimetric
4-16
Vitek2 Semiautomatic Fluorometer,photometer
4-18
Data obtained from Evangelista & Truant. In: Commercial methods in Clinical Microbiology. 2002
These system fulfill FDA and ISO accuracy performance
Acceptable performance for the clinical data for automatic ASTdevices with reference method (FDA)
Essential agreement (± 1 dilution): >89.9%Category agreement (interpretive results, SIR) >89.9%
Major discrepancies (false resistance): ≤ 3%*
Antimicrobial susceptibility automatic systems
*based on the no. of susceptible organisms tested
Very major discrepancies (false susceptibility): ≤ 1.5%****based on the no. of resistant organisms tested
Growth failure rates: < 10%*** ***for any genus or species tested
Antimicrobial Susceptibility Test (AST) Systems. Guidance for Industry and FDA Class II Special Controls Guidance Document:, August 28, 2009
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm080564.htm
27
Cantón - Implementation of EUCAST breakpoints with automated systems
Accuracy of automatic AST devices (ISO 20776-2:2007)
Data shall be analyzed by using the appropriate interpretive breakpoints
Essential agreement (± 1 dilution): ≥ 90%Category agreement (interpretive results, SIR) ≥ 90%
Major discrepancies (false resistance): ≤ 3%*
Antimicrobial susceptibility automatic systems
j p ( )*based on the no. of susceptible organisms tested
Very major discrepancies (false susceptibility): ≤ 3%****based on the no. of resistant organisms tested
Reproducibility (± 1 dilution and/or SIR results): ≥ 95%***for any genus or species tested
Clinical laboratory testing and in vitro diagnostic test systems - Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices - Part 2: Evaluation of performance of
antimicrobial susceptibility test devices. International Standard ISO 20776-2:2007, ISO, Geneva.
… most evaluations of automatic AST systems have beenperformed with CLSI (NCCLS) breakpoints, but ...
Automatic systems currently available in Europe are incorporatingthe EUCAST breakpoints
Different systems advertise that they operate with EUCAST
Antimicrobial susceptibility automatic systems
breakpoints but evaluations have not yet been performed for all:
- MIC based systems: Phoenix Vitek 2MicroScan
- Disc diffusion based systems: BioMIC
Issues with EUCAST breakpoint implementation
Lower ranges of concentrations are needed (EUCAST breakpointsare mostly lower than CLSI)
- instability of certain antibiotics might affect accuracy (essentialand categorical agreements)
Antimicrobial susceptibility automatic systems
and categorical agreements) - carbapenems, β-lactam-β-lactamase inhibitor combinations, …
- major discrepancies (false resistance) could be observed, particularlywith isolates expressing low level resistance mechanisms
28
Cantón - Implementation of EUCAST breakpoints with automated systems
Issues with EUCAST breakpoint implementation
S and R breakpoints can be ...
- too close (essential and categorical agreements can be affected)
e.g. ciprofloxacin and enterobacteriaceae (S≤ 0.5 / R >1)vancomycin and staphylococci (S≤ 2 / R >2)
Antimicrobial susceptibility automatic systems
vancomycin and staphylococci (S≤ 2 / R >2)
- too separate (a wider concentration range is needed in the panel)
e.g. aztreonam and P. aeruginosa (S≤ 1 / R >16)*
*The R breakpoint was increased from 8 to 16 mg/L to avoid dividing thewild type MIC distribution. The R breakpoint relates to high dose therapy.The S breakpoint is set to ensure that wild type isolates are reported I.
Issues with EUCAST breakpoint implementation
A desirable attribute...
... to include drug concentrations equal to ECOFFs*allowing detection of wild type organisms (no-R mechanism)
*epidemiological cut off values
Antimicrobial susceptibility automatic systems
epidemiological cut off values
A philosophical and technical change...
... breakpoints are interpreted and expressed differently
S REUCAST ≤ >CLSI ≤ ≥
Low level resistance or decreased susceptibility
High level
www.eucast.org
High level resistanceRS
ECOFF
29
Cantón - Implementation of EUCAST breakpoints with automated systems
RS
Antimicrobial susceptibility automatic systems
An example... assessment of the Phoenix system & EUCAST breakpoints
Centre A*(393 isolates)
Centre B**(362 isolates)
Categorical agreement 96.0% 99.1%
Interpretive discrepanciesInterpretive discrepancies
- minor discrepancies 2.4% 2.8%
- Major discrepancies 1.2% 0.8%
- Very major discrepancies 1.1% 1.3%
*Morosini, García-Castillo, Cantón. Ramón y Cajal University Hospital. Madrid (Spain)**Giani, Conte, D’Andrea, Rossoloni. University of Siena (Italy)
Posters presented at 20th ECCMID, 2010
Issues with EUCAST breakpoint implementation
EUCAST expert rules must be implemented with EUCAST breakpointsand not with CLSI breakpoints!
Antimicrobial susceptibility automatic systems
For 3rd/4th gen. cephalosporins and Enterobacteriaceaetest results should be reported as found irrespective of ESBL production
CLSI (2010) EUCAST (2010)
S R S RCefotaxime ≤1 ≥4 ≤1 >2
Ceftriaxone ≤1 ≥4 ≤1 >2Ceftazidime ≤4 ≥16 ≤1 >4Cefepime ≤8 ≥32 ≤1 >4
==
30
Cantón - Implementation of EUCAST breakpoints with automated systems
Antimicrobial susceptibility automatic systems
Antimicrobial susceptibility automatic systems with EUCASTbreakpoints are being implemented and introduced in Europe
EUCAST breakpoint implementation does not represent anyfundamental problem
Initial evaluations of Phoenix system fulfil ISO requirements(ISO 20776 2:2007)(ISO 20776-2:2007)
Other manufacturers have been slow to accept the need forrecalibrating their systems to ranges needed for EUCASTbreakpoints. The delay caused by any change (new antibioticor a new breakpoint) is a source of major concerns with theautomated systems.
Automation and EUCAST: join it!
Implementation of the EUCAST
breakpoints with automatic systems
Antimicrobial susceptibility testing – practical implementation of the EUCAST breakpoints and methods
20th ECCMID Vienna Austria 10 13 April 201020th ECCMID, Vienna, Austria, 10 – 13 April, 2010
Hospital Universitario Ramón y CajalSERVICIO DE MICROBIOLOGÍA Y
PARASITOLOGÍA
Dr. Rafael Cantón
31
Tenover - Supplementary tests – when routine methods are not enough
Supplementary TestsSupplementary Tests-- When When Routine Methods are Not Routine Methods are Not
EnoughEnoughFred C. Tenover, Ph.D., (D)ABMM Fred C. Tenover, Ph.D., (D)ABMM
Senior Director for Scientific AffairsSenior Director for Scientific AffairsSenior Director for Scientific AffairsSenior Director for Scientific AffairsCepheidCepheid
Consulting Professor of PathologyConsulting Professor of PathologyStanford UniversityStanford University
Adjunct Professor of EpidemiologyAdjunct Professor of EpidemiologyRollins School of Public HeathRollins School of Public Heath
Emory UniversityEmory University
Supplementary AST TestingSupplementary AST Testing
Frustrating but important aspect of Frustrating but important aspect of susceptibility testing. Includes:susceptibility testing. Includes:Optimizing results to insure therapeutic Optimizing results to insure therapeutic successsuccessConfirming unusual resistance patternsConfirming unusual resistance patternsUncovering “stealth” resistance Uncovering “stealth” resistance mechanismsmechanismsDetermining results for infrequently used Determining results for infrequently used antimicrobial agents. antimicrobial agents.
Question 1Question 1
32
Tenover - Supplementary tests – when routine methods are not enough
Why is Supplementary Testing Why is Supplementary Testing Necessary?Necessary?
New resistance mechanisms that don’t always New resistance mechanisms that don’t always result in MICs in the resistant range (such as result in MICs in the resistant range (such as carbapenemases) carbapenemases) Inducible resistance mechanisms in bacterial Inducible resistance mechanisms in bacterial isolates that are not recognized by standard isolates that are not recognized by standard methods (such as betamethods (such as beta--lactamases)lactamases)Failures of automated systems to identify nonFailures of automated systems to identify non--susceptible strains (vancomycin intermediate S. susceptible strains (vancomycin intermediate S. aureus strains)aureus strains)Some infrequently used Some infrequently used or recently approvedor recently approveddrugs are not tested on standard panels (colistin)drugs are not tested on standard panels (colistin)
Potential List of Additional Potential List of Additional Susceptibility TestsSusceptibility Tests
BetaBeta--lactamase testing for staphylococcal lactamase testing for staphylococcal resistanceresistanceCefoxitin disk test to confirm oxacillin resistance in Cefoxitin disk test to confirm oxacillin resistance in staphylococcistaphylococciDD--zone test for inducible clindamycin resistancezone test for inducible clindamycin resistanceMacro Etest for VISA and VRSAMacro Etest for VISA and VRSAScreen for highScreen for high--level aminoglycoside resistance in level aminoglycoside resistance in enterococcienterococciESBL tests: screening and confirmation (CLSI)ESBL tests: screening and confirmation (CLSI)Screening tests for carbapenemase producersScreening tests for carbapenemase producersColistin for multidrug resistant Colistin for multidrug resistant Klebsiellas Klebsiellas and and Pseudomonas aeruginosaPseudomonas aeruginosa
Tests for Staphylococci Tests for Staphylococci
DD--zone test for inducible clindamycin zone test for inducible clindamycin resistance (erythromycinresistance (erythromycin--R/R/ClindamycinClindamycin--SS) ) Macro Etest for heteroMacro Etest for hetero--vancomycin vancomycin resistance resistance ONLYONLY for invasive isolates with for invasive isolates with vancomycin MICs of 2 vancomycin MICs of 2 µg/ml and µg/ml and suggestion of clinical failuresuggestion of clinical failureMupirocin for nasal decolonization studiesMupirocin for nasal decolonization studiesββ--lactamase test for borderline susceptible lactamase test for borderline susceptible S. aureusS. aureus strains (if penicillin is to be used) strains (if penicillin is to be used)
33
Tenover - Supplementary tests – when routine methods are not enough
Detecting Inducible Clindamycin Detecting Inducible Clindamycin Resistance in StaphylococciResistance in Staphylococci
Clindamycin is an oral agent that is being rediscovered Clindamycin is an oral agent that is being rediscovered for community MRSA infectionsfor community MRSA infectionsProblem:Problem: Many erythromycinMany erythromycin--resistant clindamycinresistant clindamycin--susceptible strains have inducible clindamycinsusceptible strains have inducible clindamycinsusceptible strains have inducible clindamycin susceptible strains have inducible clindamycin resistance (resistance (ermA/B/CermA/B/C); others remain susceptible to ); others remain susceptible to clindamycin (clindamycin (msrAmsrA))Need to identify those strains that can still be treated Need to identify those strains that can still be treated successfully with clindamycin successfully with clindamycin Solution:Solution: “D“D--zone” test (Er and CC disks placed 15zone” test (Er and CC disks placed 15--26 26 mm apart) easy and cheap; adds 24 hrsmm apart) easy and cheap; adds 24 hrs
Examples of Clindamycin Examples of Clindamycin Resistance Induction:Resistance Induction:
“D“D--Zone Test”Zone Test”
ErmA ErmC
E ECC CC
Disks can be placed in inner ring of disk dispenser
15mm
Question 2Question 2
34
Tenover - Supplementary tests – when routine methods are not enough
Vancomycin MICs: Vancomycin MICs: S. aureus S. aureus CLSI breakpointsCLSI breakpoints
Genotypic and phenotypic
0.125 0.25 0.5 1.0 2.0 4.0 8.0 16.0 32.0µg/ml
S R
Genotypic and phenotypic changes vanAvanA
heteroresistancesusceptibility Clinicalresistance
CLSI and EUCAST CLSI and EUCAST VancomycinVancomycinBreakpoints for Breakpoints for S. aureusS. aureus (in (in µg/ml)µg/ml)
SusceptibleSusceptible IntermediateIntermediate ResistantResistant
CLSICLSIMICMIC
<<22 44 >>88MICMIC
EUCASTEUCASTMICMIC
<<22 ---- >2>2
CLSICLSI--EUCASTEUCAST
DiskDisk
nana nana nana
Scattergram of Scattergram of S. aureusS. aureus and Vancomycin; and Vancomycin; VRSA detected but CLSI breakpoints deletedVRSA detected but CLSI breakpoints deleted
Previous breakpoint:>15 mm susceptible
vanA-VRSA
PAGE | 12
35
Tenover - Supplementary tests – when routine methods are not enough
Population Analysis of hVISA and VISAPopulation Analysis of hVISA and VISA
1.0E+041.0E+051.0E+061.0E+071.0E+08
Suscept.hVISA
VISA Inoculum
1.0E+001.0E+011.0E+021.0E+03
0 1 3 5 7 9
MIC (ug/ml)
VISA
Suscept hVISA
Note scale
Subpopulation of hVISA isolates, for whichMIC=8 μg/ml, are below detection level
Fig 1. Population Analysis for Strain 9AJ57 on BHI vs. M HA
(CFU
/mL)
6
7
8
9
10
ATCC 29213 9AJ57-MHA
Subpopulation apparent only on BHI
Vancomycin Concentrations (mcg/mL)
0 0.25 0.5 0.75 1 2 3 4 6 8 10 12 14 16 32
Log 10
(
1
2
3
4
59 J59AJ57-BHI
Mueller-Hinton MIC= 1 µg/mlBHI MIC = 4 µg/ml
hVISA: Clinical RelevancehVISA: Clinical RelevancePopulation Analysis Shows Increasing Population Analysis Shows Increasing
MICsMICs
7
8
9
Gradual increase
in vancomycin1 2
Vancomycin Concentrations
0 0.25 0.5 0.75 1 2 3 4 6 8 10
Log 1
0 (C
F/m
l)
1
2
3
4
5
6
ATCC29213 491-0.5VBHI 492-0.5VBHI 493-0.5VBHI 494-0.5VBHI 522-0.5VBHI
(µg/ml)
in vancomycinMICs during10 weeks oftherapy with vancomycin
for endocarditis
3 4 VISA control
36
Tenover - Supplementary tests – when routine methods are not enough
Detecting hVISA via Detecting hVISA via Macro EtestMacro Etest
MacroEtest
Criteria:Vanco +
T i
2 McFarland, BHI agar, incubation for 48 hours
Teico>8 ug/ml
ORTeico Alone
>12 ug/ml
Mupirocin BreakpointsMupirocin Breakpoints
CLSI has approved both MIC and disk CLSI has approved both MIC and disk diffusion breakpoints for highdiffusion breakpoints for high--level level mupirocin resistance in mupirocin resistance in Staphylococcus Staphylococcus aureusaureus (No EUCAST breakpoints yet)(No EUCAST breakpoints yet)aureus aureus (No EUCAST breakpoints yet)(No EUCAST breakpoints yet)HighHigh--level mupirocin resistance is level mupirocin resistance is indicated by:indicated by:
MIC ≥512 µg /mlMIC ≥512 µg /mlNo zone of inhibition (6 mm) around a No zone of inhibition (6 mm) around a 200 µg disk200 µg disk
PAGE | 18
37
Tenover - Supplementary tests – when routine methods are not enough
Tests for EnterococciTests for Enterococci
Test for highTest for high--level gentamicin and level gentamicin and streptomycin resistance to establish streptomycin resistance to establish synergy with cell wall active agents synergy with cell wall active agents (penicillin or vancomycin) (penicillin or vancomycin)
Use only for sterile site isolatesUse only for sterile site isolatesMotility and pigment production tests for Motility and pigment production tests for lowlow--level vancomycin resistancelevel vancomycin resistance
Distinguish Distinguish vanBvanB from from vanCvanC ((E. gallinarum and E. gallinarum and E.casseliflavusE.casseliflavus))Important for infection controlImportant for infection control
View of Beta-Lactamases (2010)The Road to Carbapenem Resistance
Class ATEM, SHV,
CTX-M, KPCsothers
Class BMetallo-
enzymes, VIMothers
Class CAmpCs
and others
Class DOXA
PAGE | 20
ESBLS; Carba-
penemases
Some are
carba-penemases
Most are carba-
penemases
AmpC + porin change = carbapenem
resistance
Question 3Question 3
38
Tenover - Supplementary tests – when routine methods are not enough
Supplementary Tests for Gram Supplementary Tests for Gram Negative BacilliNegative Bacilli
Critical issues irrespective of breakpoint Critical issues irrespective of breakpoint system you use (EUCAST or CLSI)system you use (EUCAST or CLSI)If you use If you use CLSICLSI and the new breakpoints and the new breakpoints have not been implemented in your labhave not been implemented in your lab
ESBL TestingESBL TestingCarbapenemase TestingCarbapenemase Testing
If you use If you use EUCAST EUCAST be aware ofbe aware of changeschangesReport cephalosporin and carbapenem results Report cephalosporin and carbapenem results as they test (no further testing needed as they test (no further testing needed except except for for colistin or other drugs not currently on automated colistin or other drugs not currently on automated panels)panels)
Why Detect ESBLWhy Detect ESBL--Producing Producing Strains?Strains?
To To improve the accuracyimprove the accuracy of susceptibility of susceptibility test reports by indicating which betatest reports by indicating which beta--lactam drugs may not be effective lactam drugs may not be effective clinicallyclinicallyyyHow? Change the interpretations of How? Change the interpretations of extendedextended-- spectrum cephalosporins and spectrum cephalosporins and penicillins from “S” to “R” for ESBL penicillins from “S” to “R” for ESBL producing strainsproducing strainsBut is this still necessary?But is this still necessary?
Proficiency Testing Results for ESBLProficiency Testing Results for ESBL--Producing Producing Klebsiella pnemoniaeKlebsiella pnemoniae
Number of Number of labs (%)labs (%)
Reported as Reported as ESBLESBL
Changed Changed penicillins and penicillins and cephalosporins cephalosporins
from S to Rfrom S to R83 (31%)83 (31%) YesYes YesYes
8 8 (3%)(3%) YesYes NoNo68 (25%)68 (25%) NoNo YesYes112 112 (41%)(41%) NoNo NoNo
44% of labs under-reported resistance according to CLSI guidelines
39
Tenover - Supplementary tests – when routine methods are not enough
Confusion About How to Interpret the TestConfusion About How to Interpret the TestESBL Disk Diffusion Confirmation Method: ESBL Disk Diffusion Confirmation Method:
K. pneumoniaeK. pneumoniae 700603700603
Zone Diameter (mm)
Ceftazidime 14CAZ + CLAV CAZ Ceftazidime 14Ceftazidime + Clav 23
Cefotaxime 20Cefotaxime + Clav 24*
Increase in zone diameter by >5 mm with Clavulanic Acid for EITHER drug = ESBL
CAZ + CLAV
CTX + CLAV
CAZ
CTX
Organisms with Both AmpCs and ESBLs Organisms with Both AmpCs and ESBLs Organism β-lactamases ESBL AmpC Cefepime
MIC/diskCRO MIC
E coliE coli CTXMCTXM--5, CTXM5, CTXM--2828, , ACTACT--11
YesYes NoNo RR RR
E coliE coli CTXMCTXM--2,2, ACTACT--11 YesYes NoNo RR RRE coliE coli CTXMCTXM--5,5, ACTACT YesYes NoNo RR RRE coliE coli CTXMCTXM--2, CTX2, CTX--MM--28,28,
ACTACT--11YesYes NoNo RR RR
PAGE | 26
ACTACT 11E coliE coli CTXMCTXM--15,15, ACTACT--11 YesYes NoNo RR RRE coliE coli TEMTEM--1, 1, SHVSHV--77, , CMYCMY--22 YesYes YesYes R/SR/S RRE coliE coli CTXMCTXM--3,3, ACTACT--11 NoNo NoNo R/IR/I RRE coliE coli SHVSHV--55, , CTXMCTXM--2,2, CMYCMY NoNo YesYes SS II
K. pneumoniaeK. pneumoniae SHVSHV, , CTXMCTXM--2,2, DHADHA NoNo YesYes SS S (4)S (4)K. pneumoniaeK. pneumoniae TEMTEM--1, TEM1, TEM--10, 10, SHVSHV--77, ,
ACTACT--11NoNo YesYes SS RR
P. mirabilisP. mirabilis CTXMCTXM--15,15, DHADHA NoNo YesYes SS RR
CLSI New Cephalosporin Breakpoints CLSI New Cephalosporin Breakpoints to be Published in 2010to be Published in 2010
PAGE | 27
Use ESBL testing only for infection control
40
Tenover - Supplementary tests – when routine methods are not enough
EUCAST New Cephalosporine EUCAST New Cephalosporine Breakpoints to be Published in 2010Breakpoints to be Published in 2010CefazolineCefazoline -- / / -- mg/Lmg/LCefuroximeCefuroxime ≤8 / >8 mg/L≤8 / >8 mg/LCefotaximeCefotaxime ≤≤1 / >2 mg/L1 / >2 mg/LCeftriaxoneCeftriaxone ≤≤1 / >2 mg/L1 / >2 mg/LCeftazidimeCeftazidime ≤≤1 / >4 mg/L1 / >4 mg/LCefepimeCefepime ≤≤1 / >4 mg/L1 / >4 mg/LAztreonamAztreonam ≤1 / >4 mg/L≤1 / >4 mg/L
A Word About KPC A Word About KPC ββ--lactamaseslactamases
KKlebsiellaslebsiellasPProducingroducingPProducingroducingCChaoshaos
PAGE | 29
Carbapenem Test Results for 15 Carbapenem Test Results for 15 K. K. pneumoniaepneumoniae by Methodby Method
MethodMethod RR II SS RR II SSBrothBroth 1313 22 00 1414 11 00DiskDisk 33 1111 11 1010 55 00
IMIPENEM MEROPENEM
MScanMScan 77 77 11 1313 11 11PhoenixPhoenix 55 88 22 1212 11 22SensititreSensititre 00 22 1313 00 33 1212VitekVitek 55 00 1010 33 33 99Vitek2*Vitek2* 44 66 55 44 44 55
*Meropenem phenotype is deduced by Vitek2; no reports for 2 isolates
41
Tenover - Supplementary tests – when routine methods are not enough
CLSI Screening Criteria for Identifying CLSI Screening Criteria for Identifying Carbapenemase Producers Carbapenemase Producers (including KPCs)(including KPCs)
Standard CLSI Standard CLSI susceptible susceptible breakpoints breakpoints
Values suggesting Values suggesting carbapenemase carbapenemase
activity*activity*MIC MIC DiskDisk MIC MIC Disk Disk
PAGE | 31
((µµg/mL)g/mL) (mm)(mm) ((µµg/mL)g/mL) (mm)(mm)ErtapenemErtapenem <<22 >>1919 22 1919--2121
ImipenemImipenem <<44 >>1616 22--44 N/A†N/A†
MeropenemMeropenem <<44 >>1616 22--44 1616--2121
† N/A, not applicable (poor test performance)Perform a Modified Hodge Test
Carbapenem Inactivation AssayCarbapenem Inactivation Assay(Modified Hodge Test)(Modified Hodge Test)
3
E. coli ATCC ® 25922
Inhibition of E. coli ATCC ®
PAGE | 32
1 2
Figure 1. The MHT performed on a small MHA plate. (1) K. pneumoniae ATCC® BAA-1705, positive result; (2) K. pneumoniae ATCC® BAA-1706, negative result; and (3) a clinical isolate, positive result
25922 by ertapenem
Enhanced growth of E. coli ATCC ®
25922. Carbapenemase produced by K. pneumoniae ATCC ® BAA-1705 inactivated ertapenem that diffused into the media. Thus, there is no longer sufficient ertapenem here to inhibit E. coli ATCC® 25922 and an indentation of the zone is noted.
New QC strains
+ -
CLSI Has Lowered Breakpoints Based on CLSI Has Lowered Breakpoints Based on PK/PD and MIC Distributions to Increase PK/PD and MIC Distributions to Increase
Testing Accuracy (Delayed implementation)Testing Accuracy (Delayed implementation)Former CLSI Former CLSI breakpointsbreakpoints
New Breakpoints (2010)New Breakpoints (2010)
MIC MIC DiskDisk MIC MIC Disk Disk
PAGE | 33
((µµg/mL)g/mL) (mm)(mm) ((µµg/mL)g/mL) (mm)(mm)ErtapenemErtapenem <<2 2 -- >>88 >>1919-- <<1515 <<0.25 0.25 -- >>1 1 >>23 23 -- <<1919
ImipenemImipenem <<4 4 -- >>1616 >>16 16 -- <<1313 <<1 1 -- >>44 >>23 23 -- <<1919
MeropenemMeropenem
DoripenemDoripenem
<<4 4 -- >>1616
----
>>16 16 -- <<1313
----
<<1 1 -- >>44
<<1 1 -- >>44
>>23 23 -- <<1919
>>23 23 -- <<1919
42
Tenover - Supplementary tests – when routine methods are not enough
EUCAST Clinical Breakpoints*EUCAST Clinical Breakpoints**Reviewed 2009*Reviewed 2009
Carbapenems MIC breakpoint (mg/L)
Disk content
(µg)
Zone diameter breakpoint
(mm)
S ≤ R> S ≥ R<S ≤ R> S ≥ R<
Doripenem 1 4 10 24 18Ertapenem 0.5 1 10 25 22Imipenem 2 8 10 21 15
Meropenem 2 8 10 22 16
Polymyxin B Disk Diffusion Test Polymyxin B Disk Diffusion Test Doesn’t Work for All SpeciesDoesn’t Work for All Species
False susceptibleby disk
Gales, AC, et al. JCM 39:183-90, 2001
Polymyxin Testing of Polymyxin Testing of P. aeruginosaP. aeruginosa
CLSI breakpoints: MIC <2, S; 4, I; >8 RDisk <11 R; >12 S
EUCAST breakpoints: MIC <2 S; >2 R
43
Tenover - Supplementary tests – when routine methods are not enough
CONCLUSIONSCONCLUSIONSSupplementary tests are necessary to Supplementary tests are necessary to insure accurate susceptibility test reports.insure accurate susceptibility test reports.Not every isolate requires supplementary Not every isolate requires supplementary tests; in many cases, you can reserve tests; in many cases, you can reserve testing for isolates from sterile sitestesting for isolates from sterile sitestesting for isolates from sterile sitestesting for isolates from sterile sitesNew breakpoints will reduce the need for New breakpoints will reduce the need for much of this testing on grammuch of this testing on gram--negative negative bacilli but implementation on automated bacilli but implementation on automated systems may be slowsystems may be slowPay attention to changes in whichever Pay attention to changes in whichever system you use (CLSI or EUCAST)system you use (CLSI or EUCAST)
THANK YOUTHANK YOU
44
Leclercq – Expert rules in susceptibility testing
Expert rules in susceptibility testing
Roland Leclercq, Caen, France
Expert rules V1(V2 in a near future..)
• Intrinsic resistance
• Exceptional phenotypes (mainly resistance)
• Interpretive reading
Basis for rules
• Rules– Intrinsic resistance– Exceptional phenotypes (mainly Exceptional phenotypes (mainly
resistance)
are based on analysis of in vitro data (MICs/breakpoints, frequencies of resistance..)
45
Leclercq – Expert rules in susceptibility testing
Interpretive reading is more complex
What is interpretive reading? Inference of resistance mechanisms from susceptibility test results and from susceptibility test results and interpretation of clinical susceptibility on the basis of the resistance mechanism
Interpretive reading: the process
Process• Test susceptibility
Example• S. aureus resistant to
cefoxitin (oxacillin)
• Infer resistance mechanism
• Interpret clinical susceptibility on the basis of the resistance mechanism
• Acquisition of the mecA gene
• Report resistant to all β-lactams
Expert rules should be evidence based
• In particular for the interpretive rules since a « S » report may be changed to « I » or « R »
Decreases the number of available antibiotics
• Rules should be based on current evidence(mi bi l xp im nt l m d ls lini l d t )(microbiology, experimental models, clinical data)
• Evidence should be published
• Quality of evidence should be assessed
• Exceptions are possible and should be noted
46
Leclercq – Expert rules in susceptibility testing
Grading of evidence base forEUCAST Expert rules
AClinical evidence that reporting the test result as susceptible leads to clinical failures.
B Evidence is weak and based only on a few case reports or on experimental
No clinical evidence, but microbiological data suggest that clinical use of the agent should be discouraged
C
few case reports or on experimental models
The objective of this presentation is to review evidence
for some examples of rulesfor some examples of rules
Activity of aminoglycosides against S. aureus
1. Gentamicin activity is better predicted by the test of kanamycin
2. Bacteriostatic activity of amikacin is better predicted by the test of kanamycin predicted by the test of kanamycin
3. Bactericidal activity of amikacin is better predicted by the test of kanamycin
4. Bactericidal activity of amikacin may be maintained against isolates resistant to gentamicin
47
Leclercq – Expert rules in susceptibility testing
EUCAST rule
Phenotype (enzyme)
MIC (mg/L)
Amikacin Tobramycin Gentamicin
Susceptible 1 0.5 0.5
MICs of aminoglycosides for staphylococci with variousaminoglycoside-resistance phenotypes
K[APH(3’)]
1 0.25 0.25
KT [ANT(4’)(4’’)] 8 16 0.5
KTG[AAC(6’)-APH(2’’)]
8 64 64
S RAsseray N et al. Antimicrob Agents Chemother, 2002, 46:1591-1593
10
9
8
7
S K KTAmikacin
S K KTControl
KTG S K KTGentamicin
KTG KTG
Effect of amikacin in a rabbit Staphylococcus aureus endocarditis infection model
P<0.0001P<0.0001
6
5
4
32
NI
Asseray N et al. Antimicrob Agents Chemother, 2002, 46:1591-1593
48
Leclercq – Expert rules in susceptibility testing
Basis for evidence?
• Microbiological data• Experimental data (one study in an
endocarditis model)• No clinical data:
– Effect of aminoglycosides difficult to assess since they are always used in combination with an active cell-wall inhibitor (β-lactam, glycopeptide)
Evidence graded C
Phenotypes of resistance to macrolides and clindamycin in S. aureus
E C
MLSB constitutive
E C
E C
MLSB constitutive
MLSB inducible
RIBOSOMAL METHYLATION(A2058)(erm gene)D-zone test
EFFLUX (MsrA pump)
E
C
Clindamycin may select resistantmutants in MLSBi S. aureus
E CPositive D-zone test:Selection of MLSB constitutive mutantswith clindamycin [for erm(C)frequency: 10-7]
Negative D-zone test: No selection of mutants with clindamycin(not substrate for the pump)
E
C
49
Leclercq – Expert rules in susceptibility testing
Interpretive reading 1. If D-test positive: not enough evidence for clinical
failure with clindamycin therapy report as « S » to clindamycin
2. If D-test positive, evidence for clinical failure with clindamycin therapy: grade A report as « R » to clindamycin
3. If D-test positive, evidence for clinical failure with clindamycin therapy: grade C report as « S » to clindamycin with a warning « Clinical failure during treatment with clindamycin may occur by selection of resistant mutants”.
4. If D-test positive, you need a molecular test to report
EUCAST ruleIf D-test positive, Either report as resistant to clindamycin and
lincomycin or report as susceptible with a warning: “Clinical
failure during treatment with clindamycin or fa ur ur ng tr atm nt w th c n amyc n r lincomycin may occur by selection of constitutively resistant mutants”.
The use of clindamycin/ lincomycin is probably bestavoided in severe infections.
Mutation frequencies to clindamycin resistance
Susceptible and effluxermC ermA
Daurel et al., J Clin Microbiol 2008
50
Leclercq – Expert rules in susceptibility testing
Inducibly MLSB resistant isolates: clindamycin therapy failures
No of patientstreated withclindamycin
No of failures No of MLSBconstitutiveisolatesselected
Reference
3 2 1/3 Rao (2000)
2 2 2/2 McGehee (1968)
3 1 1/3 Drinkovic (2002)
2 2 1/2 Frank (2001)
1 1 1/1 Siberry (2003)
1
12
1
9
1/1
7/12
Levin (2005)
Grade B
E. coli producing CTX-M-15
Amox-clav
AMX
TIC
CF CTX
Aztreonam
FEP
Synergism between3GC/aztreonamand clavulanic acid
Synergism between3GC/aztreonamand clavulanic acid
( Cattoir V.)
TZP FOXCPO
PIP TCC IPM
CXM
MOX
Ceftazidime
EUCAST interpretive rule 9.1 (Enterobacteriaceae)
If R or I to any 3rd or 4th gen. oxyimino-cephalosporin or aztreonam, and positive for ESBL edit the S result for any of the oxy-iminoceph. and aztreonam as I and the I result as R
1. Evidence for this rule is A (in vitro data + f (experimental models + clinical data)
2. Evidence for this rule is B (in vitro data + experimental models)
3. Evidence for this rule is C (weak evidence)4. The rule has been set up only to justify the job
of microbiologists5. You do not trust this rule and you never use it
51
Leclercq – Expert rules in susceptibility testing
MICs of beta-lactams for selected ESBL
ESBL MIC (mg/L) Cefotaxime Ceftazidime Cefepime Aztreonam
CTX-M-1 64 0.5 16 16
CTX-M-15
CTX-M-16
TEM-3
SHV-2
256
16
2
1
128
8
8
8
32
2
0.5
0.5
128
8
1
0.5
R, I, S(Breakpoints cefotaxime: 1, 2 mg/L)
A murine thigh infection model for evaluationof activity of 3rd GC according to MIC
The % T>MIC was predictiveof activity of 3rd/4thgen. cephalosporins against ESBL and non ESBL groups
The ß-lactam MIC of an ESBL-producingisolate can be used to predict likely humanoutcomes from PK/PD models
Andes & Craig. Clin Microbiol Infect 2005; 11 (Supp. 6): 10-7
Monte-Carlo simulations and target attainment rates (TAR) for intravenous ceftriaxone 2 g every 24 h
TAR at T>MIC (30% for ceftriaxone) for a staticto one log pathogen kill atto one log pathogen kill at24 h is taken to be mostpredictive of outcomes in humans
MacGowan A. Clin Microbiol Infect 2008; 14 (Suppl 1):166-8
S
IR
EUCAST Breakpoints
52
Leclercq – Expert rules in susceptibility testing
60
80
100
al fa
ilure
≤1 mg/L EUCAST susceptible
Clinical failures with 3rd generation cephalosporins against ESBL producing organisms
S I R
0
20
40
<=1 2 4 8 16
MIC (mg/L)
% C
linic
a breakpoint
Paterson et al. J Clin Microbiol 2001; 39:2206
Evidence for rule 9.1• Weak evidence for this rule
• Both in vitro, experimental and clinical data rather suggest: « report as found ». However, only few clinical data are availableclinical data are available
• Are we ready to leave interpretation for ESBL?
. Revision of the rule in the next version:« report as found » (detection of ESBL still required for infection control and epidemiological reasons)
Grading of evidence base forEUCAST Expert rules
50 rules are graded A,B or C1. 2/3 are graded C2. 90% are graded C3. 1/2 are graded A4. 1/3 are graded A, 1/3 are graded B
and 1/3 are graded C
53
Leclercq – Expert rules in susceptibility testing
Grading of evidence base forEUCAST Expert rules
– A 8 (16%)– B 9 (18%)B 9 (18%)– C 33 (66%, 2/3)
Conclusion
• Interpretive reading of susceptibility tests is recognized as a major process to report reliable results of AST to clinicians
• 2/3 of rules based on in vitro evidence
• Need for more clinical studies to assess the clinical impact of recommendations
54
Kahlmeter - Gradient tests on EUCAST media
Can MH-F be used for gradient MIC determination for streptococci and
Haemophilus influenzae?
Gunnar Kahlmeter Ch l tt K l E ik M t h kCharlotta Karlsson, Erika Matuschek,
Jenny Åhman
Växjö, Sweden
EUCAST disk diffusion test recommends two media:
• Unsupplemented Mueller-Hinton agar for non-fastidious organisms
• Mueller-Hinton agar with 5% defibrinated horse blood and 20 mg/L β-NAD for fastidoius organisms (MH-F)
55
Kahlmeter - Gradient tests on EUCAST media
AimIt would be practical if routine microbiological laboratories could use
MH and MH-F for both disk diffusion testing and gradient MIC-testing.
• To evaluate whether the MH-F medium used for disk diffusion testing of streptococci and Haemophilus influenzae in the EUCAST method could be used for gradient testscould be used for gradient tests
• To evaluate whether there was any systematic difference between MH-F and the two media recommended for gradient tests by the manufacturers.
Gradient tests for MIC determination
• More user friendly than microdilution and agar dilution assays.
• Instantly available for MIC determination of single isolatesisolates
• Several studies show good agreement with reference methods; however for some antibiotics systematically high or low results obtained.
• Gradient tests investigated:– Etest (bioMérieux)– M.I.C.Evaulator (Oxoid)
Manufacturers recommendationsEtest (bioMérieux)
M.I.C.Evaulator, “M.I.C.E.” (Oxoid)
• Non-fastidious organisms: Mueller-Hinton• Mueller Hinton with lyzed sheep blood• Mueller-Hinton with lyzed sheep blood
– Streptococcus pneumoniae– Streptococcus A, B, C and G
• HTM-medium– Haemophilus influenzae
56
Kahlmeter - Gradient tests on EUCAST media
Manufacturer recommendations compared with EUCAST disk method
bioMérieux Oxoid EUCAST
Haemophilus influenzae
Medium HTM
a) HTM b) ISA + sheep
blood and NAD
MH-F
Inoculum McF 0.5 in broth (McF 1 if mucoid)
McF 0.5 in suitable media
McF 0.5 in saline
Incubation 35ºC, 5% CO2
20-24 h35-37ºC, 5% CO2
18-20 h35ºC, 5% CO2, 16-20 h
Manufacturer recommendations compared with EUCAST disk method
bioMérieux Oxoid EUCASTa) HTM
Streptococcus pneumoniae
Medium MH + 5% sheep blood
a) HTM b) ISA + sheep
blood and NAD
MH-F
Inoculum McF 0.5 in broth(McF 1 if mucoid)
McF 0.5-1 in suitable media McF 0.5 in saline
Incubation 35ºC, 5% CO2
20-24 h
35-37ºC, 5% CO2
a) 20-24 h b) 18-20 h
35ºC, 5% CO2, 16-20 h
Methodology
• H. influenzae, S. pneumoniae and S. pyogenes– QC strains and clinical isolates were tested
• MH-F vs. recommended media for Etest andMH F vs. recommended media for Etest and M.I.C.Evaluator– HTM for H. influenzae– MH + 5% sheep blood for streptococci
• Incubation in 5% CO2 and 35ºC for 20 h.
57
Kahlmeter - Gradient tests on EUCAST media
H. influenzae*MH-F compared with reference medium
3 dil 2 dil 1 dil 0 1 dil 2 dil 3 dil No of readingsEtest (Number of readings)Amoxicillin/clavulanic acid 2 33 35Ampicillin 5 21 9 35Ciprofloxacin 5 29 1 35Tetracycline 2 19 13 1 35SXT * 15 20 35
MH-F < MH-Str MH-F = MH-Str MH-F > MH-Str
H. Influenzae ATCC 49247, NCTC 8468 and three clinical isolates
MICE (Number of readings)Amoxicillin/clavulanic acid 7 25 3 35Ampicillin 3 30 1 1 35Ciprofloxacin 7 26 2 35Tetracycline 3 15 17 35
* Trimethoprim-sulfamethoxazole (SXT) was not available for MICE
H. influenzaeAmpicillin
0
5
10
15
20
25
30
35
-3 -2 -1 0 1 2 3
Dilution steps' difference
No o
f rea
ding
s
E-testMICE
0 • MH-F = Reference medium- • MH-F < Reference medium+ • MH-F > Reference medium
Tetracycline
0
5
10
15
20
25
30
35
-3 -2 -1 0 1 2 3
Dilution steps' difference
No
of r
eadi
ngs
E-testMICE
S. pyogenes* and S. pneumoniae* MH-F compared with reference medium
MH-F = MH-Str MH-F > MH-StrMH-F < MH-Str3 dil 2 dil 1 dil 0 1 dil 2 dil 3 dil No of readings
Etest (Number of readings)Benzylpenicillin 10 23 2 35Erythromycin 5 21 9 35Moxifloxacin* 1 1 20 13 35Tetracycline 3 27 5 35SXT * 6 21 5 3 35
*S. pyogenes CCUG 25571, S. pneumoniae ATCC 49619 and three clinical S. pneumoniae isolates
MICE (Number of readings)Benzylpenicillin 4 28 3 35Erythromycin 3 22 10 35Tetracycline 1 30 4 35
* Moxifloxacin and Trimethoprim-sulfamethoxazole (SXT) were not available for MICE
58
Kahlmeter - Gradient tests on EUCAST media
S. pyogenes and S. pneumoniaeBenzylpenicillin
0
5
10
15
20
25
30
35
-3 -2 -1 0 1 2 3
Dilution steps' difference
No
of r
eadi
ngs
E-testMICE
T t li
0 • MH-F = Reference medium- • MH-F < Reference medium+ • MH-F > Reference medium
Tetracycline
0
5
10
15
20
25
30
35
-3 -2 -1 0 1 2 3
Dilution steps' difference
No
of re
adin
gs
E-testMICE
Conclusions I• The correlation between MICs obtained on MH-F and
reference media was good for Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae
• Results were reproducible and easily read
• Our data suggest that MH-F may be a suitable substrate for gradient test MIC determination.
We call on manufacturers and others to extend our trials and validate the use of MH F for
Conclusions II
validate the use of MH-F for gradient MIC-testing.
59
Kahlmeter - Gradient tests on EUCAST media
We thank bioMérieux and Oxoid for supplying the gradient strips
60