Educational Workshop

Educational Workshop EW08: 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) Derek Brown (Peterborough, UK)

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 1

Tasks

1. 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 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 2

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 regulatregulatoryory 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 Committees D, F, N, NL, S, UK,

EUCAST General Committee All European Countries + ISC/FESCI

EUCAST Steering Committee BSAC, CA‐SFM, CRG, DIN, NWGA, SRGA And 2 reps from the General Committee

Subcommittees Antifungals Anaerobes Expert groups Expert Rules

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 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)

• 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 – 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.org

requires no login

6 Kahlmeter - EUCAST Clinical Breakpoints

Q 3

Tools for determining CLINICAL BREAKPOINTS

1. Dose or doses 2. 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 species 4. Resistance mechanisms in target organisms 5. Clinical indications 6. 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

Wild type Clinical resistance Clinical resistance Clinical resistance Clinical resistance Phenotypically detectable resistance Genetically detectable 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 √ • Nhli(i)New cephalosporin (ongoing) • Glycopeptides (ongoing)

• Extensions of indications (none ongoing)

Topicals and less commonly used drugs

1. Mupirocin (Topical) 11.Cefoperazone 2. Polymyxin B (Topical) 12.Pefloxacin 3. Bacitracin (Topical) 13.Cefradine 4. Streptomycin (hlr for enterococci) 14.Cefamandole 5. Neomycin (Topical) 15.Sulfisoxazole 6. Sulfamethoxazole (UTI) 16.Pipemidic acid 7. Cephalothin (expert rules?) 17.Kanamycin 8. Sulfadiazine 18.Ceftizoxime 9. Spiramycin 19.Cefprozil 10.Nalidixic acid (screening)

+ 45 others

8 Kahlmeter - EUCAST Clinical Breakpoints

Microorganisms without breakpoints

Define relevant drugs, breakpoints, methodology and MIC-distributions.

• Helicobacter spp √ • Campylobacter spp √ • Clostridium difficile √ • Legionella spp • Pasteurella multocida • Listeria monocytogenes •… •…

EUCAST breakpoint tables

MIC (mg/L) brpts* S≤ 2 R>2 Zone (mm) brpts* S≥ 22 R<22 Insufficient evidence IE (Literature: ”not enough evidence for a Can not be substituted. breakpoint” or ”no indication”) Can be s upplemented with an MIC w ithou t 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-distributions Web-links to Zone diameter distributions Web-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

The MIC is the reference for all other phenotypic tests

E coli /Mecillinam 10 ug 969 isolates, of which 930 consecutive 512 256

150 128 64 125 32 16 100 8 tes a 4 75 2 1

No of isol 50 0.5 25 0.25 0.125 0 0.064 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 0.032 Inhibition zone diameter (mm) Breakpoints S R MIC ≤8>8 Zone diameter ≥15 <15

10 Kahlmeter - EUCAST Clinical Breakpoints

Q 4

On a national level:

National strategies and joint decisions on AST are needed! NAC National Antimicrobial Committee

National Antimicrobial Committees tasks • Subcommittee on Antimicrobial susceptibility testing – Strategy at national level – Implementation of breakpoints and methods – 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

EUCAST breakpoints

Decisions for 2010/11: Discussions ongoing:

Sweden Spain Denmark Greece Norway Italy Belgium Turkey The Netherlands Israel Austria Estonia Ireland Finland Scotland Wales Switzerland

ENAC European Network of Antimicrobial Committees

The EUCAST General Committee.

EUCAST April 2010 • 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. • 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. • 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.

Q5Q 5

Thank you!

13 Brown - Implementation of the EUCAST disk-diffusion method

Implementation of the EUCAST di sk diff us ion 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 breakpoints • Disk diff usi on me thod s calib ra te d 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 – 16-20h incubation – Most disk contents same – Most control strains same – Calibrated to EUCAST MIC breakpoints

Mueller-Hinton-fastidious (MH-F) Mueller-Hinton agar + 5% defibrinated horse blood and 20 mg/L β-NAD S. pneumoniae ATCC 49619 H. influenzae NCTC 8468

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

Figure 7: Ceftriaxone MIC v zone diameter for ceftriaxone withn=350 Enterobacteriaceae y=18.9 - 0.49x >=64 35 111122 4 33 r=0.95

32 11 1116 11

16 114 22 1 11

8 111122

4 33 g/ml) μ 2 1

MIC ( 1 1 1

0.5 2 4 1

0.25 4 5 3 2 11 Ceftriaxone 0.12 5 226 3 7 11

0.06 11 7 11 12 4 33 1 1

0.03 2 891012 13 19 1

<=0.015 1115558 13 12 15 3 6 54 11

6 9 12 15 18 21 24 27 30 33 36 39 42 45 Ceftriaxone Zone Diameter (mm)

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 • Continuinggg validation through – 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) – 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, practica l wor ks hops • 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 • Plan well in a dvance – 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 level During 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 level After implementation

• Keep the method up-to-date • Continue to educate staff • Report problems to EUCAST – 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, 2010

Dr. Rafael Cantón

Hospital Universitario Ramón y Cajal SERVICIO DE MICROBIOLOGÍA Y PARASITOLOGÍA

Methods for antimicrobial susceptibility testing

ƒ 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, ...) ƒ Genotypic test methods : based on the detection of a resistance gene or its product - mecA, vanA, vanB… - PBP2a, β-lactamase detection ƒ By deduction – “expert rules” - If mecA-positive, then report beta-lactam antibiotics as R - If erythromycin-R, then report azithro- and clarithromycin as R

Antimicrobial susceptibility automatic systems

Main objectives

ƒ To produce antimicrobial susceptibility testing (AST) results in a automated (or semi-automated) mode

ƒ To standardize AST avoiding uncontrolled differences

ƒ To offer AST in a shorter period of time than manual methods

ƒ To interpret AST results (clinical categorization / interpretation)

23 Cantón - Implementation of EUCAST breakpoints with automated systems

MIC based automatic systems

Antimicrobial susceptibility automatic systems

ƒ None of the current automatic susceptibility testing devices can be considered fully automated …

- Automated system consist of devices with computer-assisted incubation, reading, interpretation and reporting functions

- Semi-automated systems require off-line incubation* . The panels are automatically 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 hand or manually entered into a computer for interpretation and reporting

ƒ All instruments have implemented computer programs

Antimicrobial susceptibility automatic systems

Most automatic susceptibility testing devices have incorporated …

ƒ Interface connections with laboratory information systems (LIS) ƒ Quality control computer programs ƒ 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 antimicrobial susceptibility test results “expert rules”

ƒ Programs to manage results for epidemiological purpose

24 Cantón - Implementation of EUCAST breakpoints with automated systems

Antimicrobial susceptibility automatic systems

Classification

ƒ MIC based systems - agar dilution (no longer exists!) - microdilution: MicroScan, Sensititre, Phoenix, … - growth curves: VITEK legacy, VITEK2

ƒ Disc diffusion based systems - BIOMIC System - SIRSCAN System - OSIRIS System ……

Antimicrobial susceptibility automatic systems

Sensititre ARIS System (Trek Diagnostic Systems)

ƒ Standard 96-well microdilution panels with 22-24 antibiotics/panel

ƒ Manual or semi-automatic inoculation of panels

ƒ Fluor escent measu re me nt o f bacte ria l g ro wt h after generating a fluorescent product from a non-fluorescent substrate - Susceptibility testing results in 18-24 h

ƒ Data manager system and expert software rules

Antimicrobial susceptibility automatic systems

MicroScan WalkAway (Siemens Healthcare Diagnostics)

ƒ Standard size 96-well microdilution panels with 18-22 antibiotics with or without substrates for bacterial identification

ƒ Panels are manually inoculated

ƒ Turbidimetric reading (overnight panels) - susceptibility testing results in 15-18 h

ƒ Fluorimetric reading (rapid panels with a fluorometric substrate) - susceptibility testing results in 3.5-7 h

ƒ Data manager system and expert software rules

25 Cantón - Implementation of EUCAST breakpoints with automated systems

Antimicrobial susceptibility automatic systems

Vitek 2 (BioMérieux)

ƒ Separate plastic cards for ID and AST - AST: 64-well plastic card with 19-20 agents (deduce additional agents with computer software)

ƒ Optical reading every 15 minutes with a multichannel fluorometer and photometer to record fluorescence and turbidity

ƒ Changes in fluorescence over time (growth curves) comparing a growth control well with wells with drug concentrations

ƒ Susceptibility results in 4-18 h

ƒ Expert system (Advanced Expert System, AES) based in MIC and phenotype analysis

Antimicrobial susceptibility automatic systems

Phoenix System (BD Diagnostic Systems)

ƒ Combination panels for bacterial ID and AST AST: 84-wells (18-25 antimicrobial agents)

ƒ Manually or semi-automatic inoculation of panels

ƒ Monitor reading every 20 minutes with a turbidometric and colorimetric (oxidation-reduction indicator) growth detection

ƒ Changes in fluorescence over time (growth curve), comparing a growth control well with wells with various drug concentrations

ƒ Susceptibility results in 4-16 h

ƒ Expert system (Advance expert System)

Antimicrobial susceptibility automatic systems

Wider System (Fco. Soria Melguizo)

ƒ Standard size 96-well microdilution panels with 18-22 antibiotics with or without substrates for bacterial identification (panels are prepared by MicroScan)

ƒ Pan el s m an uall y in oculated an d ex tern all y in cubated

ƒ Panels are manually introduced in a video image reader after 15-18 h of incubation

ƒ Expert software rules

26 Cantón - Implementation of EUCAST breakpoints with automated systems

Antimicrobial susceptibility automatic systems

Disc diffusion based systems

ƒ Video assisted reader plate to read and interpret disc diffusion susceptibility agar plates

ƒ Quantitative zone diameters are calculated by digitalization and software interpreted (SIR) with deduction of MIC

ƒ Expert software rules

Antimicrobial susceptibility automatic systems

MIC based systems Reporting Device Inoculation Reading time (hours) Sensititre Manual or Manual read or 18-24 semiautomatic Fluorescence MicroScan Manual Turbidity and 15-18 fluorometer 353.5-7 Phoenix Manual or Turbidity and 4-16 Semiautomatic colorimetric Vitek2 Semiautomatic Fluorometer, 4-18 photometer

These system fulfill FDA and ISO accuracy performance

Data obtained from Evangelista & Truant. In: Commercial methods in Clinical Microbiology. 2002

Antimicrobial susceptibility automatic systems

Acceptable performance for the clinical data for automatic AST devices with reference method (FDA)

ƒ Essential agreement (± 1 dilution): >89.9% ƒ Category agreement (interpretive results, SIR) >89.9%

ƒ Major discrepancies (false resistance): ≤ 3%* *based on the no. of susceptible organisms tested

ƒ Very major discrepancies (false susceptibility): ≤ 1.5%** **based on the no. of resistant organisms tested

ƒ 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

Antimicrobial susceptibility automatic systems

Accuracy of automatic AST devices (ISO 20776-2:2007)

Data shall be analyzed by using the appropriate interpretive breakpoints

ƒ Essential agreement (± 1 dilution): ≥ 90% ƒ Category agreement (interpretive results, SIR) ≥ 90%

ƒ Majjpor discrepancies (()false resistance): ≤ 3%* *based on the no. of susceptible organisms tested

ƒ Very major discrepancies (false susceptibility): ≤ 3%** **based on the no. of resistant organisms tested

ƒ 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.

Antimicrobial susceptibility automatic systems

… most evaluations of automatic AST systems have been performed with CLSI (NCCLS) breakpoints, but ...

ƒ Automatic systems currently available in Europe are incorporating the EUCAST breakpoints

ƒ Different systems advertise that they operate with EUCAST breakpoints but evaluations have not yet been performed for all:

- MIC based systems: Phoenix Vitek 2 MicroScan

- Disc diffusion based systems: BioMIC

Antimicrobial susceptibility automatic systems

Issues with EUCAST breakpoint implementation

ƒ Lower ranges of concentrations are needed (EUCAST breakpoints are mostly lower than CLSI)

- instability of certain antibiotics might affect accuracy (essential and categorical agreements) - carbapenems, β-lactam-β-lactamase inhibitor combinations, …

- major discrepancies (false resistance) could be observed, particularly with isolates expressing low level resistance mechanisms

28 Cantón - Implementation of EUCAST breakpoints with automated systems

Antimicrobial susceptibility automatic systems

Issues with EUCAST breakpoint implementation

ƒ 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≤ 22/R>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 the wild 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.

Antimicrobial susceptibility automatic systems

Issues with EUCAST breakpoint implementation

ƒ A desirable attribute...

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

ƒ A philosophical and technical change...

... breakpoints are interpreted and expressed differently

SR EUCAST ≤ > CLSI ≤ ≥

Low level resistance or decreased susceptibility High level S R resistance ECOFF

www.eucast.org

29 Cantón - Implementation of EUCAST breakpoints with automated systems

S R

Antimicrobial susceptibility automatic systems

An example... assessment of the Phoenix system & EUCAST breakpoints

Centre A* Centre B** (393 isolates) (362 isolates)

Categorical agreement 96.0% 99.1% Interpretive 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

Antimicrobial susceptibility automatic systems

Issues with EUCAST breakpoint implementation

ƒ EUCAST expert rules must be implemented with EUCAST breakpoints and not with CLSI breakpoints! For 3rd/4th gen. cephalosporins and Enterobacteriaceae test results should be reported as found irrespective of ESBL production

CLSI (2010) EUCAST (2010)

SR S R Cefotaxime ≤1 ≥4 = ≤1>2 Ceftriaxone ≤1 ≥4 = ≤1>2 Ceftazidime ≤4 ≥16 ≤1>4 Cefepime ≤8 ≥32 ≤1>4

30 Cantón - Implementation of EUCAST breakpoints with automated systems

Antimicrobial susceptibility automatic systems

ƒ Antimicrobial susceptibility automatic systems with EUCAST breakpoints are being implemented and introduced in Europe

ƒ EUCAST breakpoint implementation does not represent any fundamental problem

ƒ Initial evaluations of Phoenix system fulfil ISO requirements (ISO 20776 -2:2007)

ƒ Other manufacturers have been slow to accept the need for recalibrating their systems to ranges needed for EUCAST breakpoints. The delay caused by any change (new antibiotic or a new breakpoint) is a source of major concerns with the automated 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, 2010

Dr. Rafael Cantón

Hospital Universitario Ramón y Cajal SERVICIO DE MICROBIOLOGÍA Y PARASITOLOGÍA

31 Tenover - Supplementary tests – when routine methods are not enough

Supplementary TestsTests--When Routine Methods are Not EnoughEnough

Fred C. Tenover, Ph.D., (D)ABMM Senior Director for Scientific Affairs CepheidCepheid

Consulting Professor of Pathology Stanford University

Adjunct Professor of Epidemiology Rollins School of Public Heath Emory University

Supplementary AST Testing

¾¾ Frustrating but important aspect of susceptibility testing. Includes: ¾¾ Optimizing results to insure therapeutic successsuccess ¾¾ Confirming unusual resistance patterns ¾¾ Uncovering “stealth” resistance mechanisms ¾¾ Determining results for infrequently used antimicrobial agents.

Question 11Question

32 Tenover - Supplementary tests – when routine methods are not enough

Why is Supplementary Testing Necessary? ¾¾ New resistance mechanisms that don’t always result in MICs in the resistant range (such as carbapenemases) ¾¾ Inducible resistance mechanisms in bacterial isolates that are not recognized by standard methods (such as beta-beta-lactamases)lactamases) ¾¾ Failures of automated systems to identify nonnon-- susceptible strains (vancomycin intermediate S. aureus strains) ¾¾ Some infrequently used or recently approved drugs are not tested on standard panels (colistin)

Potential List of Additional Susceptibility Tests ¾¾ BetaBeta--lactamaselactamase testing for staphylococcal resistanceresistance ¾¾ Cefoxitin disk test to confirm oxacillin resistance in staphylococci ¾¾ DD--zonezone test for inducible clindamycin resistance ¾¾ Macro Etest for VISA and VRSA ¾¾ Screen for highhigh--levellevel aminoglycoside resistance in enterococci ¾¾ ESBL tests: screening and confirmation (CLSI) ¾¾ Screening tests for carbapenemase producers ¾¾ Colistin for multidrug resistant Klebsiellas and Pseudomonas aeruginosa

Tests for Staphylococci

¾¾ DD--zonezone test for inducible clindamycin resistance (erythromycin(erythromycin--R/R/ClindamycinClindamycin--SS)) ¾¾ Macro Etest for heterohetero--vancomycinvancomycin resistance ONLY for invasive isolates with vancomycin MICs of 2 µg/ml and suggestion of clinical failure ¾¾ Mupirocin for nasal decolonization studies ¾¾ ββ--lactamaselactamase test for borderline susceptible S. aureusaureusS. strains (if penicillin is to be used)

33 Tenover - Supplementary tests – when routine methods are not enough

Detecting Inducible Clindamycin Resistance in Staphylococci

¾¾ Clindamycin is an oral agent that is being rediscovered for community MRSA infections ¾¾ Problem:Problem: Many erythromycinerythromycin--resistantresistant clindamycin-clindamycin- susceptible strains have inducible clindamycin resistance (ermA/B/CermA/B/C); others remain susceptible to clindamycin (msrAmsrA)) ¾¾ Need to identify those strains that can still be treated successfully with clindamycin ¾¾ Solution:Solution: “D“D--zone”zone” test (Er and CC disks placed 15-15-2626 mm apart) easy and cheap; adds 24 hrs

Examples of Clindamycin Resistance Induction: “D“D--Zone Test”Zone Test”

E CC E CC

15mm

ErmA ErmC Disks can be placed in inner ring of disk dispenser

Question 2Question 2

34 Tenover - Supplementary tests – when routine methods are not enough

Vancomycin MICs: S. aureus CLSI breakpoints

SR0.125 0.25 0.5 1.0 2.0 4.0 8.0 16.0 32.0 µg/ml

Genotypic and phenotypic changes vanAvanA

susceptibility heteroresistance Clinical resistance

CLSI and EUCAST Vancomycin Breakpoints for S. aureusS. aureus (in µg/ml) Susceptible Intermediate Resistant

CLSICLSI <<2244 >>88 MICMIC EUCASTEUCAST <<22---- >2 MICMIC CLSICLSI-- nana nana nana EUCASTEUCAST DiskDisk

Scattergram of S. aureusaureusS. and Vancomycin; VRSA detected but CLSI breakpoints deleted

vanA-VRSA Previous breakpoint: >15 mm susceptible

PAGE | 12

35 Tenover - Supplementary tests – when routine methods are not enough

Population Analysis of hVISA and VISA

1.0E+08 1.0E+07 1.0E+06 VISA Inoculum 1.0E+05 Suscept. 1.0E+04 hVISA 1.0E+03 VISA 1.0E+02 hVISA 1.0E+01 Suscept 1.0E+00 013579 Note scale MIC (ug/ml) Subpopulation of hVISA isolates, for which MIC=8 μg/ml, are below detection level

Fig 1. Population Analysis for Strain 9AJ57 on BHI vs. MHA

10

9

8 Subpopulation apparent only 7 on BHI

6 ATCC 29213

CFU/mL) 99J5AJ57-MHA ( 5 9AJ57-BHI 10

Log 4

3

2

1 0 0.25 0.5 0.75 1 2 3 4 6 8 10 12 14 16 32 Vancomycin Concentrations (mcg/mL)

Mueller-Hinton MIC= 1 µg/ml BHI MIC = 4 µg/ml

hVISA: Clinical Relevance Population Analysis Shows Increasing MICsMICs

9

8 Gradual

7 increase 12 in vancomycin 6 34 VISA control 5 MICs during (CF/ml)

10 ATCC29213 491-0.5VBHI 4 10 weeks of Log 492-0.5VBHI 493-0.5VBHI 494-0.5VBHI 3 therapy with 522-0.5VBHI

2 vancomycin

1 for 00.250.50.7512346810 endocarditis Vancomycin Concentrations (µg/ml)

36 Tenover - Supplementary tests – when routine methods are not enough

Detecting hVISA via Macro Etest

Macro Etest Criteria: Vanco + TiTeico >8 ug/ml OR Teico Alone >12 ug/ml

2 McFarland, BHI agar, incubation for 48 hours

Mupirocin Breakpoints

¾¾CLSI has approved both MIC and disk diffusion breakpoints for highhigh--levellevel mupirocin resistance in Staphylococcus aureusaureusaureus (No EUCAST breakpoints yet) ¾¾HighHigh--levellevel mupirocin resistance is indicated by:

zz MIC ≥512 µg /ml

zz No zone of inhibition (6 mm) around a 200 µg disk

PAGE | 18

37 Tenover - Supplementary tests – when routine methods are not enough

Tests for Enterococci

¾¾ Test for highhigh--levellevel gentamicin and streptomycin resistance to establish synergy with cell wall active agents (penicillin or vancomycin)

zz Use only for sterile site isolates ¾¾ Motility and pigment production tests for lowlow--levellevel vancomycin resistance

zz Distinguish vanBvanB from vanCvanC ((E. gallinarum and E.casseliflavus))

zz Important for infection control

View of Beta-Lactamases (2010) The Road to Carbapenem Resistance

Class A Class B Class C Class D TEM, SHV, Metallo- AmpCs OXA CTX-M, KPCs enzymes, VIM and others others others

ESBLS; Most are AmpC + porin change Some Carba- carba- = carbapenem are resistance penemases penemases carba- penemases

PAGE | 20

Question 33Question

38 Tenover - Supplementary tests – when routine methods are not enough

Supplementary Tests for Gram Negative Bacilli ¾¾ Critical issues irrespective of breakpoint system you use (EUCAST or CLSI) ¾¾ If you use CLSIand the new breakpoints have not been implemented in your lab

zz ESBL Testing

zz Carbapenemase Testing ¾¾ If you use EUCAST be aware of changes

zz Report cephalosporin and carbapenem results as they test (no further testing needed except for colistin or other drugs not currently on automated panels)panels)

Why Detect ESBLESBL--ProducingProducing Strains?Strains? ¾¾ To improve the accuracy of susceptibility test reports by indicating which betabeta-- lactam drugs may not be effective clinicallyyy ¾¾ How? Change the interpretations of extendedextended--spectrum cephalosporins and penicillins from “S” to “R” for ESBL producing strains ¾¾ But is this still necessary?

Proficiency Testing Results for ESBL-ESBL- Producing Klebsiella pnemoniae Number of Reported as Changed labs (%)(%)labs ESBLESBL penicillins and cephalosporins from S to R 83 (31%) Yes YesYes 8 (3%) YesYes No 68 (25%)(25%)68 NoNo Yes 112 (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 Test ESBL Disk Diffusion Confirmation Method: K. pneumoniae 700603700603

Zone Diameter (mm)

CAZ + CLAV CAZ Ceftazidime 14 Ceftazidime + Clav 23

Cefotaxime 20

CTX + CLAV CTX Cefotaxime + Clav 24*

Increase in zone diameter by >5 mm with Clavulanic Acid for EITHER drug = ESBL

Organisms with Both AmpCs and ESBLs

Organism β-lactamases ESBL AmpC Cefepime CRO MIC/disk MIC E colicoliE CTXMCTXM--5,5, CTXM-CTXM-2828,, YesYes NoNo RRRR ACTACT--11 E coliE coli CTXMCTXM--2,2,ACT- ACT-11YesYes NoNo RRRR E coliE coli CTXMCTXM--5,5, ACT Yes NoNo RRRR E coliE coli CTXMCTXM--2,2, CTXCTX--MM--28,28, YesYes NoNo RRRR ACTACT--11 E coliE coli CTXMCTXM--15,15, ACTACT--11YesYes NoNo RRRR E coliE coli TEMTEM--1,1, SHV-SHV-77,, CMYCMY--22YesYes YesYes R/SR/S RR E coliE coli CTXMCTXM--3,3, ACTACT--11 NoNo NoNo R/IR/I RR E coliE coli SHVSHV--55,, CTXM-CTXM-2,2,CMY NoNo YesYesS II K. pneumoniae SHVSHV,, CTXM-CTXM-2,2, DHA NoNo YesYesS S (4)(4)S K. pneumoniae TEMTEM--1,1, TEM-TEM-10,10, SHV-SHV-77,, NoNo YesYesS RR ACTACT--11 P. mirabilis CTXMCTXM--15,15, DHA NoNo YesYesS R

PAGE | 26

CLSI New Cephalosporin Breakpoints to 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 Breakpoints to be Published in 2010

¾¾ CefazolineCefazoline--// -mg/L- mg/L ¾¾ Cefuroxime ≤8 / >8 mg/L ¾¾ Cefotaxime ≤≤1 / >2 mg/L ¾¾ Ceftriaxone ≤≤1 / >2 mg/L ¾¾ Ceftazidime ≤≤1 / >4 mg/L ¾¾ CefepimeCefepime ≤≤1 / >4 mg/L ¾¾ AztreonamAztreonam ≤1 / >4 mg/L

A Word About KPC ββ--lactamaseslactamases ¾¾KKlebsiellaslebsiellas ¾¾PProducingroducing ¾¾CChaoshaos

PAGE | 29

Carbapenem Test Results for 15 K. pneumoniae by Methodby Method IMIPENEM MEROPENEM MethodMethodRI R ISSRRIISS BrothBroth13 20 2 01414 1100 DiskDisk311 3 11110 1 10 5500 MScanMScan 7777111313 1111 PhoenixPhoenix5588221212 1122 SensititreSensititre 0022131303 0 31212 VitekVitek 5500101033 3 399 Vitek2*Vitek2* 446655444455

*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 Carbapenemase Producers (including KPCs)

Standard CLSI Values suggesting susceptible carbapenemase breakpoints activity*activity* MIC Disk MIC Disk ((µµg/mL)g/mL) (mm) ((µµg/mL)g/mL)(mm) Ertapenem < 22>>1919219 2 19--2121

ImipenemImipenem <44>>1616 22--44N/A†N/A†

Meropenem <44>>1616 22--441616--2121

Perform a Modified Hodge Test

PAGE | 31 † N/A, not applicable (poor test performance)

Carbapenem Inactivation Assay (Modified Hodge Test)

E. coli ATCC ® 25922

3 Inhibition of E. coli ATCC ® 25922 by ertapenem + 1 2 - 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.

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; New QC strains

PAGE | 32and (3) a clinical isolate, positive result

CLSI Has Lowered Breakpoints Based on PK/PD and MIC Distributions to Increase Testing Accuracy (Delayed implementation) Former CLSI New Breakpoints (2010) breakpoints

MIC Disk MIC Disk ((µµg/mL)g/mL) (mm) ((µµg/mL)g/mL)(mm) ErtapenemErtapenem <<2 -->>88>>1919--<<1515 <0.25 ->- >1 >23 -<- <1919

ImipenemImipenem <<4 -->>1616 >16 -<- <1313 <1 -->>44>>23 -<- <1919

Meropenem <<4 -->>1616 >>16 -<- <1313 <<1 -->>44 >>23 -<- <1919

------<<1 -->>44 >>23 -<- <1919 DoripenemDoripenem

PAGE | 33

42 Tenover - Supplementary tests – when routine methods are not enough

EUCAST Clinical Breakpoints* *Reviewed 2009

Carbapenems MIC breakpoint Disk Zone diameter (mg/L) content breakpoint (µg) (mm)

S ≤ R> S ≥ R<

Doripenem 1 4 10 24 18 Ertapenem 0.5 1 10 25 22 Imipenem 2 8 10 21 15 Meropenem 2 8 10 22 16

Polymyxin B Disk Diffusion Test Doesn’t Work for All Species

False susceptible by disk

Gales, AC, et al. JCM 39:183-90, 2001

Polymyxin Testing of P. aeruginosa

CLSI breakpoints: MIC <2, S; 4, I; >8 R Disk <11 R; >12 S EUCAST breakpoints: MIC <2 S; >2 R

43 Tenover - Supplementary tests – when routine methods are not enough

CONCLUSIONS ¾¾ Supplementary tests are necessary to insure accurate susceptibility test reports. ¾¾ Not every isolate requires supplementary tests; in many cases, you can reserve testing for isolates from sterile sites ¾¾ New breakpoints will reduce the need for much of this testing on gramgram--negativenegative bacilli but implementation on automated systems may be slow ¾¾ Pay attention to changes in whichever system you use (CLSI or EUCAST)

THANK 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 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 interpretation of clinical susceptibility on the basis of the resistance mechanism

Interpretive reading: the process

Process Example • Test susceptibility • S. aureus resistant to cefoxitin (oxacillin)

• Infer resistance • Acquisition of the mechanism mecA gene

• Interpret clinical • Report resistant to all susceptibility on the β-lactams basis of the resistance mechanism

Expert rules should be evidence based

• In particular for the interpretive rules since a « S » report may be changed to « I » or « R » Æ Decreases the number of available antibiotics • Rules should be based on current evidence (micro bio logy, exper ime nta l mdmode ls, clinica l dtdata )

• 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 for EUCAST Expert rules Clinical evidence that reporting A the test result as susceptible leads to clinical failures. Evidence is weak and based only on a B few case reports or on experimental models No clinical evidence, but C microbiological data suggest that clinical use of the agent should be discouraged

The objective of this presentation is to review evidence for 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 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

MICs of aminoglycosides for staphylococci with various aminoglycoside-resistance phenotypes

Phenotype MIC (mg/L) (enzyme) Amikacin Tobramycin Gentamicin

Susceptible 1 0.5 0.5

K 1 0.25 0.25 [APH(3’)] KT [ANT(4’)(4’’)] 8 16 0.5

KTG 86464 [AAC(6’)-APH(2’’)]

S R Asseray N et al. Antimicrob Agents Chemother, 2002, 46:1591-1593

Effect of amikacin in a rabbit Staphylococcus aureus endocarditis infection model

Control Gentamicin Amikacin S K KT KTG S K KT 10 KTG S K KT KTG 9 8 7 6 NI 5 4 3 2 P<0.0001 P<0.0001

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

EC

MLSB constitutive

EC EFFLUX (MsrA pump) RIBOSOMAL METHYLATION E MLS inducible B (A2058) E C (erm gene) C D-zone test

Clindamycin may select resistant

mutants in MLSBi S. aureus

Positive D-zone test:

ECSelection of MLSB constitutive mutants with clindamycin [for erm(C)frequency: 10-7]

E

Negative D-zone test: No selection of mutants with clindamycin C (not substrate for the pump)

49 Leclercq – Expert rules in susceptibility testing

Interpretive reading

1. If D-test positive: not enough evidence for clinical failure with clindamycin therapy Æ report as « S » to clindamycin

2. If D-test positive, evidence for clinical failure with clindamycin therapy: grade A Æ report as « R » to clindamycin

3. If D-test positive, evidence for clinical failure with clindamycin therapy: grade C Æ 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 rule

If D-test positive, Either report as resistant to clindamycin and lincomycin or report as susceptible with a warning: “Clinical failur e duri ng tratmtreatme nt wthwith cnclind amyci n or lincomycin may occur by selection of constitutively resistant mutants”. The use of clindamycin/ lincomycin is probably best avoided in severe infections.

Mutation frequencies to clindamycin resistance

Š ermC Š ermA Š Susceptible and efflux

Daurel et al., J Clin Microbiol 2008

50 Leclercq – Expert rules in susceptibility testing

Inducibly MLSB resistant isolates: clindamycin therapy failures

No of patients No of failures No of MLSB Reference treated with constitutive clindamycin isolates selected

321/3Rao(2000)

2 2 2/2 McGehee (1968)

31 1/3Drinkovic(2002)

221/2Frank(2001)

1 1 1/1 Siberry (2003)

1 1 1/1 Levin (2005)

12 9 7/12

Grade B

E. coli producing CTX-M-15

AMX CF CTX FEP Synergism between 3GC/aztreonam Amox-clav Aztreonam and clavulanic acid TIC Ceftazidime

PIP TCC IPM MOX

CXM TZP CPO FOX

( Cattoir V.)

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 ff(or this rule is A (in vitro data + 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 microbiologists 5. 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-15 256 128 32 128

CTX-M-16 16 8 2 8

TEM-3 2 8 0.5 1

SHV-2 1 8 0.5 0.5

(Breakpoints cefotaxime: 1, 2 mg/L) R, I, S

A murine thigh infection model for evaluation of activity of 3rd GC according to MIC

The % T>MIC was predictive of activity of 3rd/4thgen. cephalosporins against ESBL and non ESBL groups

The ß-lactam MIC of an ESBL-producing isolate can be used to predict likely human outcomes 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 static to one log pathogen kill at 24 h is taken to be most S predictive of outcomes in humans I R

EUCAST Breakpoints

MacGowan A. Clin Microbiol Infect 2008; 14 (Suppl 1):166-8

52 Leclercq – Expert rules in susceptibility testing

Clinical failures with 3rd generation cephalosporins against ESBL producing organisms

100 SI R 80 ≤1 mg/L EUCAST 60

l failure susceptible

a breakpoint 40

% Clinic % 20

0 <=1 2 4 8 16 MIC (mg/L)

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 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 for EUCAST Expert rules

50 rules are graded A,B or C 1. 2/3 are graded C 2. 90% are graded C 3. 1/2 are graded A 4. 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 for EUCAST Expert rules

– A 8 (16%) – 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 Charl ott a K ar lsson, Er ika Ma tusc he k, 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

Aim It 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 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 isolates

• 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 recommendations Etest (bioMérieux) M.I.C.Evaulator, “M.I.C.E.” (Oxoid)

• Non-fastidious organisms: Mueller-Hinton • 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

Haemophilus influenzae

bioMérieux Oxoid EUCAST

a) HTM Medium HTM b) ISA + sheep MH-F blood and NAD McF 0.5 in broth McF 0.5 in McF 0.5 in Inoculum (McF 1 if mucoid) suitable media saline

35ºC, 5% CO 35-37ºC, 5% CO 35ºC, 5% CO , Incubation 2 2 2 20-24 h 18-20 h 16-20 h

Manufacturer recommendations compared with EUCAST disk method Streptococcus pneumoniae

bioMérieux Oxoid EUCAST

a) HTM MH + 5% sheep Medium b) ISA + sheep MH-F blood blood and NAD McF 0.5 in broth McF 0.5-1 in Inoculum McF 0.5 in saline (McF 1 if mucoid) suitable media

35-37ºC, 5% CO2 35ºC, 5% CO 35ºC, 5% CO , Incubation 2 a) 20-24 h 2 20-24 h 16-20 h b) 18-20 h

Methodology

• H. influenzae, S. pneumoniae and S. pyogenes – QC strains and clinical isolates were tested

• MH-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

MH-F < MH-Str MH-F = MH-Str MH-F > MH-Str

3 dil 2 dil 1 dil 0 1 dil 2 dil 3 dil No of readings Etest (Number of readings) Amoxicillin/clavulanic acid 2 33 35 Ampicillin 5 21 935 Ciprofloxacin 5 29 135 Tetracycline 2 19 13 1 35 SXT * 15 20 35

MICE (Number of readings) Amoxicillin/clavulanic acid 7 25 335 Ampicillin 3 30 11 35 Ciprofloxacin 7 26 235 Tetracycline 3 15 17 35

* Trimethoprim-sulfamethoxazole (SXT) was not available for MICE

H. Influenzae ATCC 49247, NCTC 8468 and three clinical isolates

H. influenzae

Ampicillin

035 • MH-F = Reference medium

-30• MH-F < Reference medium + • MH-F > Reference medium 25

20 E-test MICE 15 No of readings of No 10

5

0 -3-2-10123 Dilution steps' difference

Tetracycline

35

30

25

20 E-test MICE 15 No of readings 10

5

0 -3 -2 -1 0 1 2 3 Dilution steps' difference

S. pyogenes* and S. pneumoniae* MH-F compared with reference medium

MH-F < MH-Str MH-F = MH-Str MH-F > MH-Str 3 dil 2 dil 1 dil 0 1 dil 2 dil 3 dil No of readings Etest (Number of readings) Benzylpenicillin 10 23 235 Erythromycin 5 21 935 Moxifloxacin* 1120 13 35 Tetracycline 3 27 535 SXT * 6 21 53 35

MICE (Number of readings) Benzylpenicillin 4 28 335 Erythromycin 3 22 10 35 Tetracycline 1 30 435 * Moxifloxacin and Trimethoprim-sulfamethoxazole (SXT) were not available for MICE

*S. pyogenes CCUG 25571, S. pneumoniae ATCC 49619 and three clinical S. pneumoniae isolates

58 Kahlmeter - Gradient tests on EUCAST media

S. pyogenes and S. pneumoniae

Benzylpenicillin

35

30

25

20 E-test MICE 15 No of readings of No 10

5

0 -3-2-10123 Dilution steps' difference

TtTetracycli ne

35

30

25

20 E-test MICE 15 No of readings of No 10

5

0 -3-2-10123 Dilution steps' difference

0 • MH-F = Reference medium - • MH-F < Reference medium + • MH-F > Reference medium

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.

Conclusions II

We call on manufacturers and others to extend our trials and validate the use of MH-FforF for gradient MIC-testing.

59 Kahlmeter - Gradient tests on EUCAST media

We thank bioMérieux and Oxoid for supplying the gradient strips

60