EUCAST and Clinical Breakpoints-ECCMID2010.ppt

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


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


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


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


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


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


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


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


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


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


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


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


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


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


EUCAST QC tables

Targets NOT in italics are from ISO and/or CLSI recommendations

Question 2

17


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

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5 5 5 5

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8 9 10

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Zone diameter correlates

19


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


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


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


MIC based 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


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


Classification

MIC based systems- agar dilution (no longer exists!)

- microdilution: MicroScan, Sensititre, Phoenix, …

- growth curves: VITEK legacy, VITEK2


Disc diffusion based systems

- BIOMIC System

- SIRSCAN System

- OSIRIS System

……


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


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



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


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)


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



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


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%*


*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


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%*


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


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)


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



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)


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.


A desirable attribute...

... to include drug concentrations equal to ECOFFs*allowing detection of wild type organisms (no-R mechanism)

*epidemiological cut off values


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


RS


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


EUCAST expert rules must be implemented with EUCAST breakpointsand not with CLSI breakpoints!


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



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


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


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


34


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


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


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


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


38


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


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


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


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


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


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


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


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


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


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


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


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


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


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)


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



– 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


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


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


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


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


S. pyogenes and S. pneumoniaeBenzylpenicillin

0

5

10

15

20

25

30

35

-3 -2 -1 0 1 2 3


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


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


We thank bioMérieux and Oxoid for supplying the gradient strips

60

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