Educational Workshop EW01: Antimicrobial susceptibility testing with EUCAST breakpoints and methods Arranged with the European Committee on Antimicrobial Susceptibility Testing (EUCAST)

Convenors: Gunnar Kahlmeter (Växjö, SE) Derek F.J. Brown (Peterborough, UK)

Faculty: Rafael Canton Moreno (Madrid, ES) Derek F.J. Brown (Peterborough, UK) Gunnar Kahlmeter (Växjö, SE) Christian G. Giske (Stockholm, SE) Johan Willem Mouton (Nijmegen, NL) Alasdair P. MacGowan (Bristol, UK) Maiken Cavling Arendrup (Copenhagen, DK)

1

2 Canton - What is new in 2013: update on EUCAST breakpoints

www.EUCAST.org

WHAT IS NEW IN 2013: UPDATE ON EUCAST BREAKPOINTS

Departamento de Microbiología II Universidad Complutense. Madrid

2013 www.eucast.org

National Breakpoint Committees F, N, NL, S, UK

Contract 2011-14 NACs = National Antimicrobial Susceptibility Testing Committees EUCAST General Committee (GC) All European Countries + Countries from outside EUCAST Steering Committee BSAC, CA‐SFM, CRG, NWGA, SRGA + 3 reps from the GC ±2 “visiting” members from the GC

Subcommittees Experts Antifungals (ECDC Networks, Expert Rules ESCMID Study Groups) Anaerobes Industry Resistance mechanisms

EUCAST implementation

Q1

3 Canton - What is new in 2013: update on EUCAST breakpoints

. EUCAST General Committee - 35 countries, including Russia and Australia - New countries with interest of being represented (Brazil, US, Japan…) . National breakpoint committees and NACs - Continuous consultation on breakpoints - New countries already represented in the General Committee formed a NAC (Portugal, Germany, …) - New countries outside Europe have declared interest to form a NAC (Brazil, US, ..) . Four new Standard Operation Procedures (03-01-2013) - 3.0 Review and revision of breakpoints - 4.0 EUCAST committees - 5.0 EUCAST interaction with NACs - 6.0 maintenance of EUCAST web site (on going)

National AST Committees (NACs), January 2013

Yes Finland In the process of forming a NAC

Norway No Russia Sweden Estonia No information Latvia

Denmark Lithuania

Ireland Belarus Great Britain Nether- lands Poland

Germany Ukraine Belgium Luxembourg Czech Republic Slovakia Moldova

France Switzer- Austria Hungary land Romania Slovenia Croatia Serbia Bosnia- Herze- Bulgaria govina Monte- Portugal Italy negro Mace- donia Turkey Spain Albania

Greece

Malta

Countries not on the map: Australia Iceland Israel

Implementation of EUCAST breakpoints, January 2013

% Laboratories

>50% Finland

10-50% Norway Russia <10% Sweden Estonia

No information Latvia

Denmark Lithuania

Ireland Belarus Great Britain Nether- lands Poland

Germany Ukraine Belgium Czech Luxembourg Republic Slovakia Moldova

France Switzer- Austria Hungary land Romania Slovenia Croatia Serbia Bosnia- Herze- Bulgaria govina Monte- Portugal Italy negro Mace- donia Turkey Spain Albania

Greece

Malta

Countries not on the map: Australia Iceland Israel

4 Canton - What is new in 2013: update on EUCAST breakpoints

Guidelines followed in EARS-Net EQA 2012 (National representation is biased, as in UK NEQAS)

EARS-Net 2012 - Guidelines used by participants SRGA SFM 90 Year % NWGA CLSI 80 2009 22 EUCAST 70 2010 29 DIN BSAC 60 2011 47 50 2012 61 40 Country

30

20

10

0 AT BE BU CY CZ DE DK EE ES FI FR GR HR HU IE IS IT LT LU LV MT NL NO PL PT RO SE SI TR UK No. of participants

ECDC/EARS-Net and UKNEQAS (C. Walton)

EUCAST implementation

Q2

EUCAST breakpoints, 2013

5 Canton - What is new in 2013: update on EUCAST breakpoints

New version of EUCAST breakpoint tables v3.0 (Jan, 2013)

. New drugs - Ceftaroline breakpoints . New microorganisms - Pasteurella multocida, Campylobacter . Rewording of notes - S. pneumoniae and - HLGR in Enterococcus spp. . New footnotes for - Yersinia enterocolitica . New technical notes - specific reading instructions for correct interpretation of the disk diffusion tests - S. maltophila and SXT - Staphylococci and benzypenicillin - Enterococci and . New expressions of - -trometamol, now fosfomycin oral antimicrobials - , now cefuroxime i.v. - , now cefuroxime oral . New table of PK/PD (non-species related) breakpoints

EUCAST breakpoints, 2013

All new modifications highlighted in yellow

New version of EUCAST breakpoint tables v3.0 (Jan, 2013)

. New drugs: Ceftaroline breakpoints

6 Canton - What is new in 2013: update on EUCAST breakpoints

New version of EUCAST breakpoint tables v3.0 (Jan, 2013)

. New drugs: Ceftaroline QC ranges

On going Rationale Document

New version of EUCAST breakpoint tables v3.0 (Jan, 2013)

. New microorganisms - Pasteurella multocida MH-F CO2, 35ºC, 18h

Clinical brakpoints Disk diffusion based on ECOFFs in the breakpoints calibrated absence of MIC-clinical with MIC breakpoints correlations

New version of EUCAST breakpoint tables v3.0 (Jan, 2013)

7 Canton - What is new in 2013: update on EUCAST breakpoints

New version of EUCAST breakpoint tables v3.0 (Jan, 2013)

. PK/PD breakpoints listed separately

New version of EUCAST breakpoint tables v3.0 (Jan, 2013)

PK/PD breakpoints

. Based on pharmacokinetic and pharmacodynamic data only

. Used as the basis for determination of clinical breakpoints

. In determination of clinical breakpoints the PK/PD breakpoint may be modified in the light of microbiological or clinical data.

. They may be used with microorganisms for which there are no breakpoints

. These breakpoints should not be used when there are species specific breakpoints, such as values, Note or “–” in the tables

The EUCAST breakpoints philosophy

MIC (mg/L) brpts* S≤ 2 R>2 mg/L Zone (mm) brpts* S≥ 22 R<22 mm Insufficient evidence IE (Literature: ”not enough evidence for a Can not be substituted. breakpoint” 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”

*when numbers are the same = no intermediate category

8 Canton - What is new in 2013: update on EUCAST breakpoints

The EUCAST breakpoints philosophy

S R CLSI, 2010

S R EUCAST, 2010

ECOFF

The EUCAST breakpoints philosophy

. MIC by broth microdilution is the current reference method (ISO)

. If MIC testing is used, it should either be the standard reference method as described in the ISO document or am MIC method shown to give equivalent result to the ISO method

. If disk diffusion is used and validated following ISO guidelines, the categorization of S / I / R is fully equivalent to that of MIC testing.

. If there is a deviation in a local method (medium, incubation time, volume, inoculum, …) that local method should also be validated against the ISO doc. The same is true for commercial systems.

. Many labs state they use MICs but do deviate from the ISO protocol which may explain differences in the results

The EUCAST breakpoints philosophy

. Disk diffusion calibrated with MIC values

EUCAST Laboratory for Antimicrobial Susceptibility Testing

9 Canton - What is new in 2013: update on EUCAST breakpoints

EUCAST breakpoints: What is coming 2013-14?

. Ongoing breakpoints - New agents with EMA: - anti-MRSA - macrolides - anti-mycobacterials - glycopeptides - (in conjunction with CLSI and under TATFAR initiative) - Topical agents (if available based on systemic breakpoints, otherwise based on ECOFFs) - Fluoroquinolones and enterococci in UTI - Corynebacterium spp. - Co-amoxiclav and Enterobacteriaceae in UTI - Acinetobacter spp. and - Other organisms Legionella spp., non-aeruginosa spp. Actinomyces, Nocardia, Streptomyces, HACEK Aeromonas, Vibrio, Leuconostoc, Lactobacillus, Pediococcus

Transatlantic Taskforce on Antimicrobial Resistance

. Antimicrobial resistance is a significant and multifaceted public health problem

. Purpose of the taskforce

To identify urgent antimicrobial resistance issues that could be better addressed by intensified cooperation between the US and the EU within the following key areas:

1. Appropriate therapeutic use of antimicrobials in the medical and veterinary communities 2. Prevention of healthcare- and community- associated drug-resistant infections 3. Strategies for improving the pipeline of new antimicrobial drugs

Colistin / polymixin B breakpoints

. Initiative to set common EUCAST and CLSI breakpoints . Memorandum of Understanding (MOU) - signed Oct-12, 2012 and effective Nov-1, 2012 – Nov 1, 2013 . Creation of ad hoc Working Group - 2 co-chairs, 2+2 members representing EUCAST and CLSI - absence of members with commercial / industry affiliation - colistin and polymixin B breakpoints for - Enterobacteriaceae - - Acinetobacter spp.

10 Canton - What is new in 2013: update on EUCAST breakpoints

EUCAST breakpoints: What is coming 2013-14?

General consultation until 14 June 2013

. Breakpoints are based on epidemiological cut-off values (ECOFFs)

EUCAST breakpoints: What is in coming 2013-14?

2013 www.eucast.org

National Breakpoint Committees F, N, NL, S, UK

Contract 2011-14 NACs = National AntibiogramCommittees EUCAST General Committee All European Countries + Countries from outside

EUCAST Steering Committee BSAC, CA‐SFM, CRG, NWGA, SRGA And 3 reps from the General Committee

Subcommittees Experts Antifungals (ECDC Networks, Expert Rules ESCMID Study Anaerobes Groups) Resistance mechanisms

11 Canton - What is new in 2013: update on EUCAST breakpoints

www.eucast.org

EUCAST website changes

www.eucast.org

…all free, no charge!

12 Canton - What is new in 2013: update on EUCAST breakpoints

Acknowledgements

EUCAST Laboratory for Antimicrobial Susceptibility Testing and participant laboratories

13 Brown - What is new in 2013: update on EUCAST methods

What’s new in EUCAST methods?

Derek Brown EUCAST Scientific Secretary

MIC determination

• MH-F broth for broth for broth microdilution testing of fastidious microorganisms

• Gradient MIC testing on MH-F agar

EUCAST methodology for fastidious organisms

Disk diffusion Broth microdilution Media MH-F MH-F broth* Inoculum McFarland 0.5 5 x 105 cfu/mL 16-20 h 16-20 h 35±1°C Incubation 35±1°C Ambient air (sealed 5% CO 2 panels)

* MH-F broth M-H broth with 5% lysed horse blood and 20 mg/L β-NAD. Horse blood lysed by repeated freezing and thawing.

14 Brown - What is new in 2013: update on EUCAST methods

Validation of MH-F broth

• Comparison of distributions of MICs with MH-F broth with collated distributions on EUCAST MIC distribution website (http://mic.eucast.org/Eucast2/)

• Tests on H. influenzae and S. pneumoniae

• MH-F data courtesy of Ron Jones, JMI Laboratories, USA

Validation of MH-F broth H. Influenzae with

EUCAST database 13508 observations 21 data sources

90 80 70 60 Broth microdilution with 50 MH-F broth No of isolates 40 30 150 isolates tested in 20 10 duplicate 0 1 2 4 8 16 32 64 0.5 128 256 512 0.25 ≥ 0.008 0.016 0.032 0.064 0.125 0.004 ≤

15 Brown - What is new in 2013: update on EUCAST methods

Validation of MH-F broth H. Influenzae with ciprofloxacin

EUCAST database 12688 observations 21 data sources

200

150

Broth microdilution with 100

MH-F broth isolates of No 150 isolates tested in 50

duplicate 0 1 2 4 8 16 32 64 0.5 128 256 512 0.25 ≥ 0.004 0.008 0.016 0.032 0.064 0.125 ≤

Validation of MH-F broth S. pneumoniae with cefotaxime

EUCAST database 12800 observations 24 data sources

30

25

20

15 Broth microdilution with

No of isolates of No 10

MH-F broth 5

100 isolates 0 1 2 8 16 32 64 0.5 128 256 512 0.03 0.06 0.12 0.25 ≥ 0.008 0.015 ≤ MIC (mg/L)

Validation of MH-F broth S. pneumoniae with trimethoprim-sulfamethoxazole

EUCAST database 31592 observations 11 data sources

30

25

20

15 Broth microdilution with

No of isolates of No 10 MH-F broth 5 100 isolates 0 1 2 4 8 16 32 64 0.5 128 256 512 0.03 0.12 0.25 ≥ 0.002 0.004 0.008 0.015 MIC (mg/L)

16 Brown - What is new in 2013: update on EUCAST methods

Gradient tests on MH-F medium

• Details of tests validated by manufacturer listed on EUCAST website http://www.eucast.org/antimicrobial_susceptibility_testing /compliance_of_manufacturers Compliance of manufacturers of AST materials and devices with EUCAST guidelines

• Data are based on information from manufacturers

• The accuracy of data in these tables is not verified by EUCAST and the inclusion of any materials or devices does not indicate endorsement by EUCAST.

Compliance of manufacturers with EUCAST guidelines, 11 March 2013 Gradient tests: Etest

Agents in EUCAST tables Manufacturer Validated for MH‐F β‐lactamase inhibitor format but not available bioMérieux Agents validated for MH‐F: Fixed 2:1 ratio: ‐clavulanate Amoxicillin‐clavulanate Ampicillin‐sulbactam Ampicillin‐sulbactam Roxithromycin Fixed inhibitor concentration (mg/L): Cefotaxime ‐ (4) Ticarcillin‐clavulanate (2) Cefuroxime Chloramphenicol Clindamycin Erythromycin Levofloxacin Moxifloxacin Ofloxacin Tetracycline Trimethoprim‐sulfamethoxazole (not for H. influenzae and M. catarrhalis) Vancomycin

The accuracy of data in these tables is not verified by EUCAST and the inclusion of any materials or devices does not indicate endorsement by EUCAST

Compliance of manufacturers with EUCAST guidelines, 11 March 2013 Gradient tests: MIC Test Strip

Agents in EUCAST tables Manufacturer Validated for MH‐F β‐lactamase inhibitor format but not available

Liofilchem Cefadroxil Amoxicillin Clindamycin Fixed 2:1 ratio: Cefalexin Amoxicillin‐clavulanate Doripenem Amoxicillin‐clavulanate Cefazolin Ampicillin Doxycycline Ampicillin‐sulbactam Ceftibuten Ampicillin‐sulbactam Ertapenem Roxithromycin Azithromycin Erythromycin Fixed inhibitor concentration Telithromycin Benzylpenicillin Imipenem (mg/L): Ticarcillin Cefaclor Levofloxacin Amoxicillin‐clavulanate (2) Ampicillin‐sulbactam (4) Meropenem Piperacillin‐tazobactam (4) Cefotaxime Minocycline Ticarcillin‐clavulanate (2) Moxifloxacin Ceftaroline Ofloxacin Ceftriaxone Rifampicin Cefuroxime Cephalothin Tetracycline Chloramphenicol Trimethoprim‐ Ciprofloxacin sulfamethoxazole Clarithromycin Vancomycin

The accuracy of data in these tables is not verified by EUCAST and the inclusion of any materials or devices does not indicate endorsement by EUCAST

17 Brown - What is new in 2013: update on EUCAST methods

Compliance of manufacturers with EUCAST guidelines, 11 March 2013 Gradient tests: M.I.C.Evaluator

β‐lactamase inhibitor Manufacturer Agents in EUCAST tables but not available Validated for MH‐F format

Thermo Fisher Ampicillin‐sulbactam Mupirocin No. Fixed 2:1 ratio: Scientific (Oxoid) Azithromycin Nalidixic acid Expected in 2013. Amoxicillin‐clavulanate Netilmicin Cefaclor Nitrofurantoin Cefadroxil Norfloxacin Cefalexin Ofloxacin Cefazolin Piperacillin Cefepime Piperacillin‐tazobactam Cefixime Quinupristin‐dalfopristin Rifampicin Cefpodoxime Roxithromycin Ceftibuten Spectinomycin Cefuroxime Streptomycin Chloramphenicol Clarithromycin Telithromycin Colistin Ticarcillin Doripenem Ticarcillin‐clavulanate Doxycycline Tobramycin Ertapenem Trimethoprim Fosfomycin Trimethoprim‐ Fusidic acid sulfamethoxazole Minocycline Moxifloxacin

The accuracy of data in these tables is not verified by EUCAST and the inclusion of any materials or devices does not indicate endorsement by EUCAST

Disk diffusion

• Reading instructions for difficult tests added to breakpoint table • Campylobacter method and zone diameter breakpoints • Pasteurella multocida method and zone diameter breakpoints • Zone diameter breakpoints for new agents • Data on correlation of disk diffusion breakpoints with MIC breakpoints • Revision of zone diameter breakpoints • Screening tests

Reading instructions for difficult disk diffusion tests in breakpoint tables http://www.eucast.org/antimicrobial_susceptibility_testing/breakpoints Stenotrophomonas maltophilia and trimethoprim‐sulfamethoxazole Outer zone ≥16mm = SNo trace of zone = R

18 Brown - What is new in 2013: update on EUCAST methods

Reading instructions for difficult disk diffusion tests in breakpoint tables http://www.eucast.org/antimicrobial_susceptibility_testing/breakpoints S. aureus and benzylpenicillin Zone diam ≥ 26 mm Fuzzy zone edge = S Sharp zone edge = R

Reading instructions for difficult disk diffusion tests in breakpoint tables http://www.eucast.org/antimicrobial_susceptibility_testing/breakpoints Enterococcus. spp. and vancomycin Zone diam ≥12 mm Sharp zone edge = S Fuzzy zone edge = R

EUCAST disk diffusion method for Campylobacter spp. (jejuni and coli) Media MH-F (pre-dried plates) Inoculum McFarland 0.5 Microaerobic environment Incubation 41°C 24 h* Reading EUCAST standard reading

* Campylobacter coli isolates with insufficient growth are reincubated immediately and read after a total of 40-48 h incubation

19 Brown - What is new in 2013: update on EUCAST methods

Calibration of disk diffusion method for Campyplobacter spp. http://www.eucast.org/antimicrobial_susceptibility_testing/calibration_and_validation

• 30 C. jejuni and 27 C. coli from two collections • MIC by ISO broth microdilution • Disk diffusion in duplicate by EUCAST method

• Study in collaboration between EUCAST, Central Veterinary Institute (CVI), Lelystad, the Netherlands and National Institute for Health and Welfare (THL), Turku, Finland.

Ciprofloxacin 5 µg vs. MIC, Campylobacter jejuni and coli 57 isolates tested in duplicate 40 (3 data sources)

35 MIC (mg/L) 30 ≥32 25 16 8 20 4 2 15 No of isolatesNo of 1 0.5 10 0.25 5 ≤0.12

0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Inhibition zone diameter (mm)

Breakpoints ECOFF MIC S≤0.5, R>0.5 mg/L WT≤0.5 mg/L Zone diameter S≥26, R<26 mm

Erythromycin 15 µg vs. MIC, Campylobacter jejuni 30 isolates tested in duplicate 12 (3 data sources)

10 MIC (mg/L) ≥128 8 64 32 6 16 8

No of isolatesNo of 4 4 2 1 2 0.5

0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm)

Breakpoints ECOFF MIC S≤4, R>4 mg/L WT≤4mg/L Zone diameter S≥20, R<20 mm

20 Brown - What is new in 2013: update on EUCAST methods

Erythromycin 15 µg vs. MIC, Campylobacter coli 27 isolates tested in duplicate 35 (3 data sources)

30 MIC 25 (mg/L) ≥64 20 32 16 15 8

No of isolatesNo of 4 10 2 1 5

0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm)

Breakpoints ECOFF MIC S≤8, R>8 mg/L WT≤8 mg/L Zone diameter S≥24, R<24 mm

Tetracycline 30 µg vs. MIC, Campylobacter jejuni and coli 29 isolates tested in duplicate 10 (2 data sources)

9 MIC (mg/L) 8 ≥128 7 64 32 6 16 5 8 4 4

No of isolatesNo of 2 3 1 2 0.5 0.25 1 0.12 0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Inhibition zone diameter (mm)

Breakpoints ECOFF MIC S≤2, R>2 mg/L WT≤1mg/L (C. jejuni), ≤2 mg/L (C. coli) Zone diameter S≥30, R<30 mm

EUCAST disk diffusion method for Pasteurella multocida

Medium MH-F Inoculum McFarland 0.5

Incubation 5% CO2, 35±1°C, 18 h ± 2 h Read zone edges at the point of complete inhibition of growth Reading viewed from the front of the plate with the lid removed and with reflected light

21 Brown - What is new in 2013: update on EUCAST methods

Calibration of disk diffusion method for Pasteurella multocida http://www.eucast.org/antimicrobial_susceptibility_testing/calibration_and_validation

• 131 P. multocida from two collections • MIC by gradient tests on MH-F agar • Disk diffusion in duplicate by EUCAST method

• Study in collaboration between EUCAST, Central Veterinary Institute (CVI), Lelystad, the Netherlands and JMI Laboratories, North Liberty, Iowa, USA

Benzylpenicillin 1 unit vs. MIC Pasteurella multocida, 131 isolates 45 (3 data sources)

40

35 MIC (mg/L) 30 4 25 2 1 20 0.5

No of isolatesNo of 0.25 15 0.12 10 0.06

5

0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm)

Breakpoints ECOFF MIC S≤0.5, R>0.5 mg/L WT≤0.5 mg/L Zone diameter S≥17, R<17 mm

Amoxicillin-clavulanate 2-1 µg vs. MIC Pasteurella multocida, 128 isolates 45 (3 data sources)

40

35

30 MIC 25 (mg/L) 0.5 20 0.25

No of isolatesNo of 0.12 15

10

5

0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm)

Breakpoints ECOFF MIC S≤1, R>1 mg/L WT≤1mg/L Zone diameter S≥15, R<15 mm

22 Brown - What is new in 2013: update on EUCAST methods

Ciprofloxacin 5 µg vs. MIC Pasteurella multocida, 131 isolates 25 (3 data sources)

20 MIC (mg/L) 15 0.12 0.06 0.03 10 0.015 No of isolatesNo of 0.008 0.004 5

0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Inhibition zone diameter (mm)

Breakpoints ECOFF MIC S≤0.06, R>0.06 mg/L WT≤0.06 mg/L Zone diameter S≥27, R<27 mm

Nalidixic acid 30 µg vs. Ciprofloxacin MIC Pasteurella multocida, 62 isolates 18 (2 data sources)

16

14 MIC 12 (mg/L) 0.12 10 0.06 8 0.03 0.015 No of isolatesNo of 6 0.008 0.004 4

2

0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm)

Breakpoints ECOFF Ciprofloxacin MIC S≤0.06, R>0.06 mg/L WT≤0.06 mg/L Nalidixic acid zone diameter (screen) S≥23 mm

Zone diameter breakpoints for new agents

• MIC breakpoints set as part of the marketing authorisation through EMA

• 2012 Ceftaroline

23 Brown - What is new in 2013: update on EUCAST methods

Zone diameter breakpoints for ceftaroline

MIC breakpoints Zone diameter Organisms (mg/L) breakpoints (mm) S ≤ R > S≥ R< Staphylococcus aureus 1 1 20 20 Streptococcus pneumoniae 0.25 0.25 Note2 Note2 Streptococcus Groups A, B, C, G Note1 Note1 Note1 Note1 Haemophilus influenzae 0.03 0.03 IP IP Enterobacteriaceae 0.5 0.5 23 23

1. Infer susceptibility from susceptibility to benzylpenicillin 2. Screen with disk IP = In preparation

Zone diameter breakpoints for new agents - ceftaroline Ceftaroline 5 µg vs. MIC S. aureus, 100 clinical isolates tested at 2 sites x2 50 45 40 35 4 30 2 25 1 20 0.5

No of isolates No of 0.25 15 0.12 10 0.06 5 0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm) Note in breakpoint table: For isolates with zone diameters 19-21 mm,

determine the MIC to confirm the susceptibility [poster 1602 Sunday 13.30-14.30]

Data on correlation of zone diameter breakpoints with EUCAST MIC breakpoints

Extensive new data added to EUCAST website

MIC-zone diameter distributions

Zone diameter distributions with MIC data identified in histograms

24 Brown - What is new in 2013: update on EUCAST methods

MIC-zone diameter distributions http://mic.eucast.org/Eucast2

Zone diameter distributions with MICs http://www.eucast.org/antimicrobial_susceptibility_testing/calibration_and_validation

Screening for beta-lactam resistance in S. pneumoniae

Oxacillin screen can give useful information on susceptibility to beta-lactam agents other than benzylpenicillin

Detailed data on EUCAST website http://www.eucast.org/antimicrobial_susceptibility_testing/calibration_and_validation

Poster 1578, Sunday 13.30-14.30

25 Brown - What is new in 2013: update on EUCAST methods

Oxacillin 1 µg vs. Benzylpenicillin MIC S. pneumoniae, 148 clinical isolates 80 MIC (mg/L) 70 Classical screen for beta‐lactam non‐susceptibility in S. penumoniae 32 16 60 CLSI and EUCAST 8 50 4 2 40 1 0.5 30 No of isolatesNo of 0.25 0.12 20 0.06 10 0.03 0.015 0 ≤0.008 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm)

Breakpoints ECOFF Benzylpenicillin MIC S≤0.06, R>2 mg/L WT≤0.06 mg/L Oxacillin zone diameter (screen) S≥20, R<20 mm

Oxacillin 1 µg vs. Ampicillin MIC S. pneumoniae, 153 clinical isolates 80 MIC 70 (mg/L) Isolates with oxacillin zone 16 60 diameter ≥8 mm are 8 50 susceptible to ampicillin 4 2 40 1 0.5 30 No of isolatesNo of 0.25 0.12 20 0.06 10 0.03 0.015 0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm)

Breakpoints Ampicillin MIC S≤0.5, R>2 mg/L Oxacillin zone diameter (screen) Ampicillin S ≥ 8 mm

Oxacillin 1 µg vs. Cefotaxime MIC S. pneumoniae, 147 clinical isolates 80 MIC (mg/L) 70 32 Isolates with oxacillin zone 16 60 diameter ≥8 mm are 8 50 susceptible to cefotaxime 4 2 40 1 0.5 30 No of isolatesNo of 0.25 0.12 20 0.06 10 0.03 0.015 0 ≤0.008 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm)

Breakpoints Cefotaxime MIC S≤0.5, R>2 mg/L Oxacillin zone diameter (screen) Cefotaxime S ≥ 8 mm

26 Brown - What is new in 2013: update on EUCAST methods

Screening for beta-lactam resistance in S. pneumoniae Flowchart Oxacillin 1 μg disk Antimicrobial agent Further testing and/or interpretation Zone diameter (mm) All beta-lactam agents for which Report susceptible irrespective of clinical ≥ 20 mm clinical breakpoints are listed indication. (inlcuding those with "Note") Benzylpenicillin (meningitis) and (all Report resistant. indications) Test by an MIC method for the agent Benzylpenicillin (for infections other considered for clinical use and interpret than meningitis) according to the clinical breakpoints. Oxacillin zone diameter ≥ 8 mm: Report susceptible. Oxacillin zone diameter < 8 mm: < 20 mm* Ampicillin and amoxicillin (without determine the MIC of the beta-lactam and with beta-lactamase inhibitor), agent intended for clinical use but for cefepime, cefotaxime and ceftriaxone ampicillin, amoxicillin and piperacillin (without and with beta-lactamase inhibitor) infer susceptibility from the MIC of ampicillin. Test by an MIC method for the agent Other beta-lactam agents considered for clinical use and interpret according to the clinical breakpoints.

Screening for beta-lactam resistance in H. influenzae

Benzylpenicillin screen can give useful information on susceptibility to beta-lactam agents

Detailed data on EUCAST website http://www.eucast.org/antimicrobial_susceptibility_testing/calibration_and_validation

Benzylpenicillin 1 unit vs. β-lactamase H. influenzae, 148 clinical isolates 100

80 All beta-lactamase positive isolates give zone

60 diameters <12 mm with benzylpenicillin 1 unit disk β-lactamase Positive 40 Negative No of isolatesNo of

20

0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm) Breakpoints Benzylpenicillin zone diameter (screen) S≥12, R<12 mm

27 Brown - What is new in 2013: update on EUCAST methods

Benzylpenicillin 1 unit vs. PBP mutations H. influenzae, 104 β-lactamase negative clinical isolates 60

50 Isolates with PBP 40 mutations give zone diameters <12 mm with PBP mutations 30 benzylpenicillin 1 unit disk Positive Negative No of isolatesNo of 20

10

0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm)

Breakpoints Benzylpenicillin zone diameter (screen) S≥12, R<12 mm

Screening for beta-lactam resistance in H. influenzae Supplementary table

Further testing and/or interpretation Benzylpenicillin 1 unit Beta-lactamase disk, Zone diameter (mm)

Report susceptible to all beta-lactam ≥ 12 mm Do not test agents for which clinical breakpoints are listed (including those with “Note”).

Test susceptibility to the beta-lactam Beta-lactamase negative agent intended for clinical use.

For ampicillin, amoxicillin and < 12 mm piperacillin, report resistant. Beta-lactamase positive For other beta-lactam agents, test susceptibility to the beta-lactam agent intended for clinical use.

Revision of zone diameter breakpoints • Breakpoints may be revised in the light of new information and changes highlighted in breakpoint tables (http://www.eucast.org/clinical_breakpoints)

• e.g. Telithromycin and strep groups A,B,C,G • 2012 S ≥22 mm, R <19 mm • 2013 S ≥20 mm, R <17 mm • e.g. Teicoplanin and S. pneumoniae • 2012 S ≥18 mm, R <18 mm • 2013 S ≥17 mm, R <17 mm

28 Brown - What is new in 2013: update on EUCAST methods

EUCAST methods What is coming in 2013-14? •MIC – Further validation of gradient tests • Disk diffusion – Corynebacterium spp. – Pseudomonas non-aeruginosa – Neisseria gonorrhoeae – New agents authorised by EMA – Some rapidly-growing anaerobes • Guidance note on Pseudomonas cepacia

29 Kahlmeter - ECOFFs, ECOFFs, ECOFFs

ECOFFs ECOFFs ECOFFs MIC wild type distributions and epidemiological cut‐off values

Gunnar Kahlmeter EUCAST, ESCMID and ECDC Clinical microbiology, Växjö, Sweden

EUCAST workshop, ECCMID 2013

Organism with MIC above ECOFF: ‐ erroneous identification ‐ erroneous MIC‐value ‐ resistance mechanism

Clinical breakpoints for S and R

ECOFF

EUCAST workshop, ECCMID 2013

Breakpoints

Clinical breakpoints • MIC concentrations decided by man (based on clinical outcome data, MIC distributions, accepted dosing, PK/PD-data) to distinguish treatable from non-treatable organisms. • should not divide wild type MIC distributions of target organisms. • may render wild type organisms Susceptible (S), Intermediate (I) or Resistant (R) • may change with a change in circumstances (indications, new resistance mechanisms, new dosing)

EUCAST workshop, ECCMID 2013

30 Kahlmeter - ECOFFs, ECOFFs, ECOFFs

Breakpoints

ECOFF • The ECOFF distinguishes between organisms without and with phenotypically expressed resistance mechanisms for a species and a drug in a defined test system. • Within a species, it is the highest MIC for organisms lacking phenotypically expressed resistance. • Organisms without resistance mechanisms are not by default treatable and organisms with resistance mechanisms are not by default resistant. • In a species considered susceptible (S) to the agent, it is the lowest possible S breakpoint.

EUCAST workshop, ECCMID 2013

Establishing ECOFFs

• ECOFFs should only be determined on

– MICs determined with methods calibrated to the internationally agreed standard method for broth microdilution (ISO)

– large numbers of MICs (n>100, ideally >1000)

– MICs from different places (minimum 3)

– MICs performed by many investigators (minimum 3, ideally 10 or more )

EUCAST workshop, ECCMID 2013

S.pneumoniae vs ciprofloxacin

MIC-value 0.002 0.004 0.008 0.016 0.032 0.064 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256 512 Ciprofloxacin 0000001428521600000000 Ciprofloxacin 0000000055119167110121202300 Ciprofloxacin 000000044536345411911000000 Ciprofloxacin 000000022153220100000 Ciprofloxacin 000000380256611111000000 Ciprofloxacin 00000015641551740000000 Ciprofloxacin 00000014351305130000000 Ciprofloxacin 0000001197251411010100000 Ciprofloxacin 0 0 0 0 0 0 1 16 125 102 28 3 0 0 0 0 0 0 0 Ciprofloxacin 00000018962095912001000 Ciprofloxacin 00000032092691031000000 Ciprofloxacin 0000002516154464100210000 Ciprofloxacin 0 0 0 0 6 4 4 13 245 854 379 9 6 1 1 1 0 0 0 Ciprofloxacin 0 0 0 0 3 0 2 22 225 917 401 16 3 2 4 1 0 0 0 Ciprofloxacin 00000139426933138115210200 Ciprofloxacin 0 0 0 0 2 0 3 13 402 1193 222 19 10 0 6 0 0 0 0 Ciprofloxacin 0000000275366182304002000 Ciprofloxacin 0000000236409186292111000 Ciprofloxacin 0000000220710522252221001000 Ciprofloxacin 0 0 0 0 6 7 25 130 2195 10500 4618 144 67 0 0 0 0 0 0 Ciprofloxacin 0000008104717695211200000 Ciprofloxacin 0 0 0 0 0 0 0 2 302 1777 786 102 1 6 0 0 0 0 0 Ciprofloxacin 0000002103335581101000000 Ciprofloxacin 000000171002652650100000 Ciprofloxacin 00000114351305130000000 Ciprofloxacin 0000008376016100000000 Ciprofloxacin 00000320228280491111000000 Ciprofloxacin 000000015122992831000000 Ciprofloxacin 0 0 0 0 0 0 1 10 120 331 78 2 2 0 0 0 0 0 0 Ciprofloxacin 0 0 0 0 0 2 3 27 155 111 18 4 1 0 0 0 0 0 0 Ciprofloxacin 0000000050544149232012100 Ciprofloxacin 0 0 0 0 0 4 4 10 181 256 74 6 2 0 1 0 0 0 0 Ciprofloxacin 00813984347721076918100030 Ciprofloxacin 0 0 0 0 0 1 0 14 120 272 96 8 0 2 0 0 0 0 0 Ciprofloxacin 000000066853121400000228 Ciprofloxacin 00000000165150184010000 EUCAST workshop, ECCMID 2013 Ciprofloxacin 00000012419955111000000 Ciprofloxacin 0 0 0 0 0 0 0 422 2706 13072 3987 320 68 31 82 62 0 0 0

31 Kahlmeter - ECOFFs, ECOFFs, ECOFFs

Tentative rules for aggregating MIC distributions and determine ECOFF MIC Distributions Action (none, aggregate or aggregate distributions* which disagree** and determine ECOFF) 1 0 None*** 2 0None 3 0Aggregate 3 3 1None 4 0Aggregate 4 and determine ECOFF 4 1Aggregate 3 4 >1 None 5 0Aggregate 5 and determine ECOFF 5 1Aggregate 4 5 >1 None 6 0Aggregate 6 and determine ECOFF 6 1Aggregated 5 and determine ECOFF 6 >1 Aggregate 4 7 0Aggregate 7 and determine ECOFF 7 1Aggregate 6 and determine ECOFF 7 2Aggregate 5 and determine ECOFF 7 >2 None etc EUCAST workshop, ECCMID 2013

EUCAST workshop, ECCMID 2013

Distribution of MICs and inhibition zone diameters defined by agent, species and test systems are freely available on the internet (www.eucast.org)

EUCAST workshop, ECCMID 2013

32 Kahlmeter - ECOFFs, ECOFFs, ECOFFs

EUCAST workshop, ECCMID 2013

MIC distributions and ECOFFs on EUCAST website

• 25 000 MIC distributions • Up to 100 000 MIC values per distribution • Data from many investigators (1 – 100 per distrib.) • Data from many time periods (1950 ‐ ) • Data from many geographical areas and projects (USA, Europe, Australia, Far East, South America, Sentry, Mystic, etc) • Data of multiple origin (Human clinical data, Surveillance programs, Veterinarian data, Wild life, Food safety programs) • Database secure on three servers in different parts of Dusseldorf under the official responsibility of EUCAST and ESCMID. • Ownership: – Software and administration: ECDC / ESCMID – Database: individual ownership of original data

EUCAST workshop, ECCMID 2013

Factors influencing the median and width of WT distributions

Median

Width

EUCAST workshop, ECCMID 2013

33 Kahlmeter - ECOFFs, ECOFFs, ECOFFs

The median of the MIC distribution:

•The inherent susceptibility of the species to the drug

The same applies to zone diameter distributions

EUCAST workshop, ECCMID 2013

The median of the MIC distribution:

•The inherent susceptibility of the species to the drug • Anything systematically influencing the activity of the drug:

• Medium –variation in MICs depending on medium • Inoculum –increasing MICs with higher inocula • Incubation –increasing MICs with longer incubation • Atmosphere –affects the activity of some drugs • pH –some drugs are more active at high pH, others at low

EUCAST workshop, ECCMID 2013

The median of the MIC (or zone diameter) distribution:

•The inherent susceptibility of the species to the drug • Anything systematically influencing the activity of the drug –Medium, inoculum, pH, cations, incubation atmosphere and time,

The width of the MIC (or zone diameter) distribution:

• Inherent variation in susceptibility to the drug • Biological variation in other traits that influence the MIC – any biological characteristic such as generation time, nutrient dependency, atmosphere dependency etc • Exogenous variation randomly influencing the activity of the drug – pH, cations, incubation atmosphere and time, etc • Variation in reading (between days, between readers, between systems) • The stability of the molecule • …

EUCAST workshop, ECCMID 2013

34 Kahlmeter - ECOFFs, ECOFFs, ECOFFs

S.aureus - clinical isolates vs S.aureus ATCC 29213 125 clinical isolates vs. 125 determinations of one type strain 36 33 Linezolid 30 S.aureus - clinical isolates 27 S.aureus ATCC 29213 24 How much of the variation in the wild type is due 21 to general variation and how much pertains to variation 18 in sensitivity to the dug? 15 No of obs 12 9 6 3 0 0 5 10 15 20 25 30 35 40 EUCASTZone workshop, diameter ECCMID (mm) 2013

MIC distributions in zone diameter histograms Linezolid No. of observations 40 S.aureus E.faecalis S.pneumoniae 30

20

10

0

20 22 24 26 28 30 32 34 20 22 24 26 28 30 32 34 20 22 24 26 28 30 32 34 21 23 25 27 29 31 33 35 21 23 25 27 29 31 33 35 21 23 25 27 29 31 33 35 Inhibition zone (mm) 0.25 mg/L 0.5 mg/L 1 mg/L 2 mg/L EUCAST workshop, ECCMID 2013

Sjölund et al, CMI, 2009

EUCAST workshop, ECCMID 2013

35 Kahlmeter - ECOFFs, ECOFFs, ECOFFs

C.difficile vs.

How much of the variation in the wild type is due to general variation and how much pertains to variation in susceptibility to the drug?

EUCAST workshop, ECCMID 2013

E. coli wild type; generation times (min) (n=100) Expect ed Normal 35 Sundquist, M, Kahlmeter, G. Factors influencing mecillinam susceptibility. Submitted for publication.

30

25

20

15 No. of obs.

10

5

0 5 6 7 8 9 10111213141516171819202122232425 EUCAST workshop,min ECCMID 2013

Methods for defining the wild type distribution (determining the ECOFF)

• The “eyeball” method (Kahlmeter) • The 95% rule (Pfaller) • The Normalised Resistance Interpretation (Kronvall) • The iterative statistical method (Turnidge) • Multimodal analysis (Meletiadis)

From John Turnidge, CLSI Workshop, Tampa, January 2013

EUCAST workshop, ECCMID 2013

36 Kahlmeter - ECOFFs, ECOFFs, ECOFFs

The Normalized Resistance Interpretation

• Uses an adaptation of a method originally devised for “ECOFFs” for zone diameter distributions – Kronvall et al., Clin Micro Infect 2003

From John Turnidge, CLSI Workshop, Tampa, January 2013

Iterative Statistical Method

From John Turnidge, CLSI Workshop, Tampa, January 2013

Iterative Statistical Method ‐ COFinder

Step 1. Population Data CRGAT FLUC All REVIEW AREA

MIC Log2MIC Raw Count Cum. Count Fitted 0.001 ‐10 0 0.0 Modal MIC 4 400 0.002 ‐900.0LogMIC Mode 2 2 Raw Count 0.004 ‐800.0Max Log2MIC 7 0.008 ‐700.0 350 Fitted 0.016 ‐6 0 0.0 Selected Log2 Mean 1.6667 =3.17 0.03 ‐5 0 0.0 Selected Log2 SD 1.1145 0.06 ‐400.0 300 0.125 ‐300.0 0.25 ‐2220.5 0.5 ‐124267.5 250 1 0 64 90 56.5 2 1 151 241 198.7 4 2 357 598 328.2 200 8 3 258 856 255.3 Count 16 4 68 924 93.5 150 32 5 40 964 16.0

64 6 8 972 1.3Selected COWT Values %> 128 7 3 975 0.0 COWT 95% 16 5.2% 100 256 8 975 #N/A COWT 97.5% 16 5.2% 512 9 975 #N/A COWT 99% 32 1.1% 1024 10 975 #N/A COWT 99.9% 64 0.3% 50 ? Enter/paste data 0 1 2 4 8 16 32 64 0.5 128 256 512

in this column 0.03 0.06 0.25 Data from 10 laboratories 0.001 0.002 0.004 0.008 0.016 0.125 [MIC]

Selected CO Values %> WT From John Turnidge, CLSI COWT 95% 16 5.2% COWT 97.5% 16 5.2% Workshop, Tampa, COWT 99% 32 1.1% January 2013 COWT 99.9% 64 0.3%

37 Kahlmeter - ECOFFs, ECOFFs, ECOFFs

”A biological approach”

• Associated resistance – an organism resistant to one agent is very likely to carry resistance to other agents.

• By comparing the MIC distributions of all isolates and isolates wild type to all other agents

EUCAST workshop, ECCMID 2013

The original distribution with all tested organisms E. coli vs. mecillinam 931 isolates from the ECOSENS II Study

256 120 E. coli from women with UTI: 180‐200 from NRI 128 each of Greece, Portugal, Austria, UK and 100 Sweden. 64 All isolates were tested against 14 . 32 80 16 8 60 4

40 2 No of isolates of No 1 20 0,5 0,25 0 0,125 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 0,064 Inhibition zone diameter (mm) 0,032

EUCAST workshop, ECCMID 2013

The distribution when all organisms with resistance to other agents were removed.

E. coli vs. mecillinam 560 isolates with MICs below ECOFF for all other tested agents

120

100 2 1 80 0,5 60 0,25 0,125 40 No of of No isolates 0,064 20 0,032

0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Inhibition zone diameter (mm)

EUCAST workshop, ECCMID 2013

38 Kahlmeter - ECOFFs, ECOFFs, ECOFFs

The use of ECOFFs (1)

1. As a tool in the determination of clinical breakpoints – To avoid dividing wild type MIC distributions of target organisms – As a surrogate clinical breakpoint when clinical data pertain only to wild type organisms (and when PK/PD data are incomplete).

2. For sensitive detection of (screening for) resistance – oxacillin to detect all ‐R in S. pneumoniae – cefoxitin to detect resistance in S. aureus – benzylpenicillin to detect all betalactam resistance in H.influenzae – pefloxacin to detect quinolone resistance in Salmonella spp – meropenem to screen for KPC in Enterobacteriaceae

EUCAST workshop, ECCMID 2013

The use of ECOFFs (2)

3. For surveillance of antimicrobial resistance when clinical breakpoints… – have not been determined – are not sensitive enough – change over time – differ between systems (CLSI, FDA, EUCAST etc) – differ between humans, cows, pigs, birds, fish and camels.

4. To exclude resistance – To exclude methicillin resistance, resistance etc – food safety – in the development of functional foods

5. For clinical susceptibility reports?

EUCAST workshop, ECCMID 2013

Is it possible to report the WT/NWT status of an isolate as supplementary information to S, I and R

• E.faecalis with MIC 16 mg/L to gentamicin would be reported: RWT • E.coli with MIC 0.5 mg/L to ciprofloxacin would be reported: SNWT • E.faecalis with MIC 1.0 mg/L to would be reported: IEWT • K.pneumoniae with MIC 1.0 mg/L (and a KPC) would be reported: SNWT

EUCAST workshop, ECCMID 2013

39 Kahlmeter - ECOFFs, ECOFFs, ECOFFs

EUCAST workshop, ECCMID 2013

40 Giske - Detection of resistance mechanisms. When, how and why?

EUCAST Subcommitee for Detection of Resistance Mechanisms

Christian G. Giske, MD/PhD Chairman of the subcommittee Karolinska University Hospital and EUCAST ECCMID, 27 April 2013

The background

• Guidance on methods of detection and characterization of resistance mechanisms are required to tie in with – The EUCAST MIC breakpoints – The EUCAST disk diffusion method – EUCAST Expert Rules – The ECDC requirements for update of the EARS-Net manual

The remit

• To develop practical guidelines for detection of specific antimicrobial resistance mechanisms of clinical and/or epidemiological importance. • The guidance will include: – Definition of the mechanisms. – Explanation of the clinical and/or public health need for detection of the mechanisms. – An outline description of recommended methods of detection. – References to detailed descriptions of the methods. • Draft guidelines that have been subject to wide consultation via established EUCAST procedures and ECDC focal points

41 Giske - Detection of resistance mechanisms. When, how and why?

Mechanisms and bacteria • Methicillin-resistant S. aureus • Vancomycin low-level resistance in S. aureus (VISA/heteroVISA) • Vancomycin-resistant enterococci • Penicillin non-susceptible S. pneumoniae • Extended-spectrum β-lactamase producing Enterobacteriaceae • Acquired AmpC-producing Enterobacteriaceae • Acquired carbapenemases in Enterobacteriaceae

• February 2012: recruiting of members • March 2012: preparation of template • April 2012 (at ECCMID): first (and only) meeting, sharing the work between members • May and June 2012: preparation of first draft • July 2012: preliminary report at EUCAST-meeting in Stockholm • September 2012: revision of the first draft • October 2012: revised draft presented to the steering committee • November 2012: feedback from the EUCAST steering committee and the EARS-Net co-ordination group • December 2012: open consultation (one month) • January 2013: consultation closes • February-March 2013: revision of the document • April 2013: final consultation • May-June 2013: revision • July 2013: approval by the steering committee

Members of the SC • Christian G. Giske (Chair; Sweden, EUCAST and EARS-Net) • Luis Martinez-Martinez (Spain) • Rafael Canton (Spain and EUCAST) • Stefania Stefani (Italy) • Robert Skov (Denmark) • Youri Glupczynski (Belgium) • Patrice Nordmann (France) • Mandy Wootton (UK) • Vivi Miriagou (Greece) • Gunnar Skov Simonsen (Norway and EARS-Net) • Helena Zemlickova (Czech republic and EARS-Net) • James Cohen-Stuart (Netherlands) • Marek Gniadkowski (Poland)

42 Giske - Detection of resistance mechanisms. When, how and why?

What have we achieved?

• First consultation was very fruitful – many useful comments • Document is out on the second consultation • Following the second consultation we expect to be able to produce a final version that can be approved by EUCAST SC in July 2013 • Highly probable that (annual?) revisions will be needed • Publication in CMI is planned (autumn 2013)

How has the work been carried out?

• Just one initial meeting at ECCMID • Mostly e-mail contacts with circulation of drafts • Systematic literature searches in addition to authors’ personal experience • Methods evaluated in multi-centre studies preferred • Gram-positive and Gram-negative subgroups, which have both reported to the chairman

The guidelines

43 Giske - Detection of resistance mechanisms. When, how and why?

When should screening be carried out?

Phenotypic confirmation methods

44 Giske - Detection of resistance mechanisms. When, how and why?

When should ESBL-testing be done?

ESBL SCREENING: No ESBL cefotaxime I/R and/or I/R using EUCAST breakpoints No

yes

species dependent ESBL confirmation

Group 1: Group 2: E.coli, Klebsiella spp., P. mirabilis, Salmonella spp., Enterobacteriaceae with inducible chromosomal AmpC: Shigella spp., Raoultella spp. Enterobacter spp., Citrobacter freundii, Morganella morganii, Providencia spp, Serratia spp., Hafnia alvei.

ESBL CONFIRMATION1 ESBL CONFIRMATION with ceftazidime and cefotaxime+/- with cefepime +/- clavulanic acid

Negative: No ESBL2 Off range Positive: ESBL Negative: no ESBL2 Off range Positive: ESBL

1: If cefoxitin MIC > 8 mg/L, perform cefepime+/- clavulanic acid confirmation test 2: Genotypic or phenotypic confirmation of acquired AmpC is recommended in group 1 Enterobacteriaceae isolates with negative ESBL confirmation test. .

45 Giske - Detection of resistance mechanisms. When, how and why?

Group I Enterobacteriaceae (see Table 1)

Method Disk/tablet load Confirmation is positive if

Cefotaxime +/- clavulanic MIC ratio1 ≥ 8 or deformation Etest ESBL - acid ellipse / phantom zone present Ceftazidime +/- clavulanic MIC ratio1 ≥ 8 or deformation - acid ellipse / phantom zone present Combination disk Cefotaxime +/- clavulanic Cefotaxime 30 ug +/- ≥ 5 mm increase in inhibition diffusion test (CDT) acid clavulanic acid 10 ug zone2 Ceftazidime +/- clavulanic Ceftazidime 30 ug+/- ≥ 5 mm increase in inhibition

acid clavulanic acid 10 ug zone2 Cefotaxime +/- clavulanic Broth microdilution - MIC ratio1 ≥ 8 acid Ceftazidime +/- clavulanic - MIC ratio1 ≥ 8 acid Cefepime +/- clavulanic acid - MIC ratio1 ≥ 8 Double disk synergy Expansion of indicator Cefotaxime, ceftazidime and test (DDST) inhibition zone cefepime - towards amoxicillin-clavulanic

acid disc

Group II Enterobacteriaceae (see Table 1)

Method Antibiotic Screening is positive if MIC ratio1 ≥ 8 or deformation Etest ESBL Cefepime +/- clavulanic acid - ellipse / phantom zone present

Combination disk Cefepime +/- clavulanic acid Cefepime 30 ug ≥ 5 mm increase in inhibition zone2 diffusion test Clavulanic acid

10 ug Broth microdilution Cefepime +/- clavulanic acid - MIC ratio1 ≥ 8 Double disk synergy Expansion of indicator test (DDST) Cefotaxime, ceftazidime, cephalosporin inhibition zone - cefepime towards amoxicillin-clavulanic acid disc

46 Giske - Detection of resistance mechanisms. When, how and why?

47 Giske - Detection of resistance mechanisms. When, how and why?

48 Giske - Detection of resistance mechanisms. When, how and why?

Conclusions

• Pan-European guidelines are soon to be available • Scrutiny and constructive feedback from national methodology committees is pivotal to ensure that the guidelines are improved over time • My prediction: there will be a need for this work also beyond July 2013 • European standardization may be helpful for EARS- Net, but hopefully even more so for laboratories, patients and infection control

49 Canton - Report as tested

REPORT AS TESTED

Departamento de Microbiología II Universidad Complutense. Madrid

Report as tested

. Strictly apply the recommended breakpoints for MIC or inhibition zone values for clinical categorization as susceptible (S), intermediate (I) or resistant (R)

- to achieve the goal of having breakpoints - to avoid implementing expert rules - to avoid delay in reporting

Although breakpoints are different, report “as tested” is currently recommended by both CLSI and EUCAST committees

Breakpoint definition (ISO 20776-1:2006)

… beyond CLSI and EUCAST

Values of parameters, such as MICs, on the basis of which bacteria can be assigned to the clinical categories “susceptible”, “intermediate” and “resistant”

Susceptible bacterial strain inhibited in vitro by a concentration of an antimicrobial agent that is associated with a high likelihood of therapeutic success

Intermediate bacterial strain inhibited in vitro by a concentration of an antimicrobial agent that is associated with uncertain therapeutic effect

Resistant bacterial strain inhibited in vitro by a concentration of an antimicrobial agent that is associated with a high likelihood of therapeutic failure

50 Canton - Report as tested

Microbiological and clinical breakpoints

S R CLSI

S R EUCAST

ECOFF

Clinical breakpoints: the philosophy

. The aim of clinical breakpoints is to use MIC values … - to separate strains where there is a high likelihood of treatment success from those where treatment is more likely to fail - to adequately treat patients but not to detect resistance mechanisms from a microbiological point of view

. They are ultimately derived from human clinical studies comparing outcomes with the MICs for the infecting pathogen

. If clinical breakpoints are well established no actions (expert rules) are needed beyond MIC interpretation (interpretive reading)

.. but this has not been the case in the past!

Interpretive reading of AST results

. During more than twenty years interpretive reading of the antibiogram has been used to: - infer resistance mechanisms behind resistant phenotypes - identify resistant organisms for infection control purposes - apply expert rules* and modify (when needed!) previous clinical categorization Courvalin P. ASM News 19921992;58:368-75 Livermore et al. J Antimicrob Chemother 2001;48(Suppl 1):87-102 Cantón R. Enferm Infecc Microbiol Clin 2002; 20: 176-86 Cantón R. Enferm Infecc Microbiol Clin 2010; 28:375-85 Leclercq et al. Clin Microbiol infect 2013; 19:141-60 This approach was partially needed due to inadequate breakpoints!

*Action to be taken (normally S or I to R), based on current clinical or microbiological evidence, in response to specific AST results

51 Canton - Report as tested

Interpretive reading of AST results

. Interpretative reading: the classical example

ESBL positive isolate

expert rule

resistant to all cephalosporins and aztreonam (irrespective of MICs)

Report as tested

Q1

Report as tested

Q2

52 Canton - Report as tested

MIC testing versus detection of resistance

. Both CLSI and EUCAST decided in separate processes (2009-10) to modify breakpoints for extended spectrum cephalosporins, based on - harmonization process (only in EUCAST) - MIC distribution of isolates with and without ESBLs, pAmpC, … - animal infection models with isolates with and without ESBLs - PK/PD calculations (Monte Carlo simulation, …) - clinical results available at the time of setting breakpoints

MacGowan A. Clin Microbiol Infect 2008; 14 (Suppl 1):166-8 Kahlmeter G. Clin Microbiol Infect 2008; 14 (Suppl 1):169-74 Dudley et al. Clin Infect Dis 2013; 56:1301-9

NO ESBL CONFIRMATION IS NEEDED UNLESS FOR EPIDEMIOLOGICAL OR INFECTION CONTROL PURPOSES

3rd/4th gen. cephalosporin breakpoints in Enterobacteriaceae

CLSI (2010-13) EUCAST (2009-13) Cefalosporins SR S R Cefotaxime ≤1 (8)* ≥4 (64) = ≤1>2

Ceftriaxone ≤1 (8) ≥4 (64) = ≤1>2

Ceftazidime ≤4 (8) ≥16 (32) ≤1>4 (8)

Cefepime ≤8 ≥32 ≤1>4 (8)

Aztreonam ≤4 (8) ≥16 (32) ≤1>4 (8)

*2009

This approach was extended to breakpoints for in Enterobacteriaceae

3rd/4th gen. cephalosporin breakpoints in Enterobacteriaceae

. CLSI and EUCAST “new” breakpoints were supported by PK/PD data, animal models and clinical outcome data

Enterobacteriaciae in a murine thigh Monte-Carlo simulations and target infection model: Cephalosporin % attainment rate (TAR) for intravenous T>MIC and microbiological efficacy ceftriaxone 2 g every 24 h

Andes & Craig. CMI 2005; 11(Suppl 6):10-7 MacGowan. CMI 2008; 14(Suppl 1):166-8

53 Canton - Report as tested

3rd/4th gen. cephalosporin breakpoints in Enterobacteriaceae

. Probability of target attainment (PTA) for ceftazidime

ceftazidime 1000 mg x3

100 - 2 log drop in viable Gram-negatives

80 requires 50% fT>MIC

60 PTA achieved >MIC

T . dose for MIC of criteria

f 40 99% percentile 20 95% percentile 1 g x 3 IV 4 mg/L S Mean 0 2 g x 3 IV 8 mg/L R . 0.25 0.5 1 2 4 8 16 32 64 MIC mg/L Ceftazidime rationale document, 2010 . EUCAST decreased ceftazidime and cefepime breakpoints due to evidence on clinical and MIC correlations: ≤1 mg/L no difference ESBL and non-ESBL producers 2-4 mg/L variable successful outcomes >4 mg/L poor outcomes

Paterson et al. JCM 2001; 3; 9:2206-12; Andes & Craig. CMI 2005; 11 (Suppl. 6):10-7 Bin et al. DMID 2006; 56:351-7; Bhat et al. AAC 2007; 51:4390-5

3rd/4th gen. cephalosporin breakpoints in Enterobacteriaceae

. Clinical data for ESBL producers indicates that outcome success decrease when 3rd gen ceph. MICs are ≥2 mg/L

Clinical outcome in patients with ESBL-producing Klebsiella spp. or E. coli bacteraemia and treated with 3rd gen. cephalosporin monotherapy

80 80

60 60

40 40 % of success % of success 20 20

0 0 ≤124816 ≤12 4 8 CMI (mg/L) CMI (mg/L) Paterson et al. JCM 2001; 39:2206-12 Andes & Craig. CMI 2005; 11 (Suppl. 6):10-7

What has been the impact of “report as tested”?

. “Theoretical” calculations (mainly on ESBLs) calculated microbiological impact on % of S-R isolates using CLSI or EUCAST breakpoints Howser et al. AAC 2010; 54:3043-6; Hoban et al. AAC 2010; 54:3031-4 Howser et al. EJCMID 2011; 30:173-9; Rodriguez-Baño et al. CMI 2012; 18:894-900

. Critical voices alerting on negative consequence for no further detection and reporting of ESBLs and carbapenemases Livermore et al. JAC 2012; 67:1569-77: Nordmann, Poirel. JAC 2013; 487-9

. Analysis and meta-analysis of different impact on mortality for ESBL and carbapenemase producing organisms Bonten et al. JAC 2012; 67:1311-20; Falagas et al. AAC 2012; 4214-22

. New publications on clinical outcomes - carbapenemase producing organisms treated with carbapenems Quereshi et all. AAC 2011; 56: 2108-13; Tzouvelekis et al.CMR 2012; 25: 682-707 Tumbarello et al. CID 2012; 55: 943-50

54 Canton - Report as tested

What has been the impact of “report as tested”?

. “Theoretical” calculations (mainly on ESBLs) calculated microbiological impact on % of S-R isolates using CLSI and EUCAST breakpoints Howser et al. AAC 2010; 54:3043-6; Hoban et al. AAC 2010; 54:3031-4 Howser et al. EJCMID 2011; 30:173-9; Rodriguez-Baño et al. CMI 2012; 18:894-900

- major impact when using CLSI rather than of EUCAST - greater impact for ceftazidime and cefepime than for cefotaxime - geographic dependent impact (different ESBL epidemiology) - origin (hospital or community-onset) dependent impact

What has been the impact of these “new” breakpoints?

. % of ESBL-E. coli isolates susceptible to 3rd / 4th gen. ceph. when using CLSI and EUCAST breakpoints in different studies

60 52 50 CTX CAZ FEP 40 38

30

20 19 14,714,7

% S S isolates % 7,5 8,7 10 5 2,3 00 0 0 Rodriguez-Baño Hoban (CLSI) Chen (CLSI) Rodriguez-Baño (CLSI) (EUCAST) Spain USA Asia Spain

What has been the impact of “report as tested”?

. Impact of CLSI & EUCAST breakpoints in ESBL-E. coli blood isolates

CTX-M-9 CTX-M-1 group SHV group

MIC (mg/L) MIC (mg/L)

S-EUCAST S-EUCAST S-CLSI 14.7% 0% S-CLSI 35.1% Rodriguez-Baño et al. Clin Microbiol Infect 2012; 18:894-900

55 Canton - Report as tested

What has been the impact of “report as tested”?

. Ceftazidime susceptibility of prevalent CTX-M producing E. coli

% of CAZ-S isolates CLSI EUCAST CTX-M-14 93 74 CTX-M-15 11 2

Willamson et al. EJCMID 2012; 31:821-4

Critical voices …

1.- Similar number of clinical cases on record where cephalosporins and carbapenems have proved effective and ineffective against infections due to low-MIC ESBL and carbapenemase producers, respectively

2.- Routine susceptibility testing is less precise than in research: ESBL and carbapenemase producers with MICs of 1–8 mg/L will oscillate between susceptibility categories according to who tests them and how.

3.- Although breakpoint committees advocate ESBL and carbapenemase detection for epidemiological purposes, some laboratories will abandon seeking these enzymes for treatment purposes, leading to a loss of critical infection control information

Critical voices …

1.- Susceptibility to carbapenems is observed for several carbapenemase producers 2.- There is a paucity of clinical successes of carbapenem-containing regimens for treating infections due to carbapenemase producers that are susceptible to carbapenems in vitro.

Detection will be useful for treating patients and for preventing nosocomial outbreaks of carbapenemase producers (and therefore MDR isolates), whatever the carbapenem resistance level is.

56 Canton - Report as tested

Impact of antibiotic MIC on infection outcome in patients with susceptible Gram-negative bacteria

. a higher all cause-mortality was observed for patients infected with strains with high MICs (Risk ratio 2.03; 95% CI, 1.05-3.92)

. differences in mortality were not statistically significant in patients infected with ESBLs (Risk ratio 1.89; 95% CI, 0.94-3.92)

Falagas et al. Antimicrob Agents Chemother 2012; 56 4214-22

Bacteraemia caused by ESBL-producing Enterobacteriaceae

. ESBL production in Enterobacteriaceae causing bacteremia is associated with higher mortality (OR 2.35; 95% CI, 1.90-2.91), but is reduced after adjustment for inadequate empirical therapy

Rottier et al. J Antimicrob Chemother 2012; 67:1311-20

Carbapenem breakpoints in Enterobacteriaceae

FDA CLSI (2010) EUCAST (EMA) (2010) S SRSRECOFF Imipenem ≤4 ≤1 (4)* ≥4 (16) ≤2>8≤0.5; ≤1**

Meropenem ≤4 ≤1 (4) ≥4 (16) ≤2>8≤0.125

Ertapenem ≤2 ≤0.25 (2) ≥1 (8) ≤0.5 >1 ≤0.06

Doripenem ≤0.5 ≤1 (ND) ≥4 (ND) ≤1>4 ≤0.12 *2009; **E. coli y K. pneumoniae; ND: not defined

EUCAST breakpoint are higher than those of CLSI !

What is the clinical impact?

57 Canton - Report as tested

Efficacy of antimicrobial regimens used to treat infections caused by carbapenemase-producing Klebsiella pneumoniae

Carbapenemase producing Enterobacteriaceae

Carbapenem monotherapy: 50 patients from 15 studies

80 75.0% 70 P = 0.02 60 50 % of 28.6% failures 40 30 20 10 0 ≤12 4 8>8

Tzouvelekis et al.Clin Microbiol Rev 2012; 25: 682-707

Mortality in bloodstream infections and KPC-K. pneumoniae

. Higher 30-day mortality rate in patients treated with monotherapy (54.3%) that those with combination (34.1%) therapy (P=0.02) . Significant decreased of mortality in patients treated with combination therapy including meropenem

. Kaplan-Meier curves (survival) . Mortality (%): monotherapy 50 Combination therapy 45 40 35 30 25 Monotherapy 20 15 10 5 0 Colistin Tigecycline Aminoglycoside Carbapenem

Tumbarello et al. Clin Infect Dis 2012; 55: 943-50

58 Canton - Report as tested

Mortality in bloodstream infections and KPC-K. pneumoniae

. Higher 30-day mortality rate in patients treated with monotherapy (54.3%) that those with combination (34.1%) therapy (P=0.02) . Significant decreased of mortality in patients treated with combination therapy including meropenem

. Kaplan-Meier curves (survival) . Mortality (%): combination therapy

60 Combination therapy 50 40 30 Monotherapy 20 Mortality Mortality (%) 10 0 COL + TIG + TIG + TIG + TIG + GEN GEN COL GEN + COL + MER MER

Tumbarello et al. Clin Infect Dis 2012; 55: 943-50

What is the impact of carbapenem MIC values? Mortality in bloodstream infections and KPC-K. pneumoniae . 30-day mortality rate in patients treated with combination therapy including meropenem stratified by meropenem MIC values

100 90 2/10 1/10 6/17 Nonsurvivors 80 70 60 % 50 Survivors 40 1/1 4/4 8/10 3/4 11/17 30 20 10 0 1248≥16

MIC (mg/L) Tumbarello et al. Clin Infect Dis 2012; 55: 943-50

MIC testing versus detection of resistance

Susceptibility MIC

Mechanism of resistance

Mechanism of resistance

Susceptibility MIC

59 Canton - Report as tested

MIC testing versus detection of resistance

Some additional issue

. Hetero-resistance, particularly in carbapenemase producers

. Different expression of ESBL and carbapenemase resistance genes

. Presence of ESBL and carbapenemase resistance genes in isolates within the wild type population (silent expression)

. Still waiting additional MIC correlations with clinical outcomes

REPORT AS TESTED

Departamento de Microbiología II Universidad Complutense. Madrid

60 Mouton - What are PK/PD breakpoints?

What are PK/PD breakpoints?

Johan W. Mouton MD PhD FIDSA Professor and pharmacodynamics

JWM Berlin 27-04-13

Susceptibility Report LAB REPORT

• Provides Clinician/Consultant guidelines how to optimally treat a patient (Freely translated from EUCAST guideline)

JWM Berlin 27-04-13

Is susceptibility (MICs) related to (clinical) outcome? treatment failure

MIC mg/L If yes, which values (breakpoints) make the difference?

JWM Berlin 27-04-13

61 Mouton - What are PK/PD breakpoints?

Potency of a drug Exposure to the bug Dosing Regimen in vitro (MIC) in vivo (PK)

Antimicrobial Efficacy of the Drug (Microbiological Cure)

Effect on Host ( Clinical Cure)

Mouton et al., Drug Resistance Updates 2011 JWM Berlin 27-04-13

Lowest concentration MIC with no visible growth after 18 hour incubation

10

8 PK

6

4 X-acin 500 mg conc mg/lconc .25 .5 1 2 4 8 2

0 0 4 8 12 16 20 24 MIC = 2 mg/L time h

JWM Berlin 27-04-13

Pharmacokinetic parameters : Measures of Exposure

AUC is usually linearly related to Dose

AUC

JWM Berlin 27-04-13

62 Mouton - What are PK/PD breakpoints?

Pharmacokinetic parameter MIC

Pharmacodynamic index (AUC/MIC, Peak/MIC, T>MIC)

JWM Berlin 27-04-13

Probability of cure after treatment with fluconazole Oropharygeal Candidiasis n=132

1.0 •Prob cure correlates with 0.8 EC50 43.69 AUC/MIC R² 0.9938 0.6 •POSITIVE correlation with 0.4

prob cure EXPOSURE 0.2

0.0 •INVERSE correlation with MIC 0 1 2 3 log AUC/MIC Pharmacodynamic index (AUC/MIC)

Rodriguez- Tudela et al, AAC 2007 JWM Berlin 27-04-13

Probability of cure after treatment with fluconazole Oropharygeal Candidiasis n=132

•If AUC is known because of 1.0 the standard dose

0.8 EC50 43.69 e.g. 400 mg ~ 400 mg.h/L R² 0.9938 0.6 •And an AUC/MIC of 100 is 0.4

prob cure required

0.2 •It follows that the breakpoint 0.0 1 10 100 1000 is 400/100 = 4 mg/L AUC/MIC mg.h/L

Rodriguez- Tudela et al, AAC 2007 JWM Berlin 27-04-13

63 Mouton - What are PK/PD breakpoints?

Susceptible (S)

A micro-organism is defined as susceptible by a level of antmicrobial activity associated with a high likelihood of therapeutic success. A micro-organism is categorized as susceptible by applying the appropriate breakpoint in a defined phenotypic test system.

Note: This breakpoint may be altered with legitimate changes in circumstances

Intermediate (I) A micro-organism is defined as intermediate by a level of antimicrobial activity associated with indetermiate therapeutic effect. A micro-organism is categorized as intermediate by applying the appropriate breakpoints in a defined phenotypic test system. Note: This breakpoints may be altered with legitimate changes in circumstances. Resistant (R) bacteria are defined as resistant by a level of antimicrobial activity associated with a high likelihood of therapeutic failure. A micro-organism is categorized as resistant by applying the appropriate breakpoint in a defined phenotypic test system. Note: This breakpoint may be altered with legitimate changes in circumstances

WWW.EUCAST.ORG JWM Berlin 27-04-13

WE AIM FOR:

A high likelyhood of success for every one (S)

Hitting the PK/PD target

SETTING A BREAKPOINT –PK/PD (example 1) DETERMINE THE PK/PD TARGET e.g. value of the PK/PD Index

ESTIMATE EXPOSURE from the dosing regimen and PK, including population variability

CALCULATE PK/PD BREAKPOINT from PK/PD target = PK/PD Index

JWM Berlin 27-04-13

64 Mouton - What are PK/PD breakpoints?

Relationship between fAUC/MIC and Effect 121 patients with S. pneumoniae respiratory infection

fAUC/MIC cut-off ~34

• Relationship between fAUC:MIC ratio & microbiological response from a total 121 patients with respiratory tract infection involving S. pneumoniae. • fAUC:MIC > 34 had 92.6% response rate. • fAUC:MIC < 34 had 66.7% response rate.

Ambrose PG, Bhavnani SM, Owens RC. Infect Dis Clin N Amer. 2003;17:529-543.

GOOD Clinical Practice

Be sure that the fAUC/MIC ratio is at least appr. 34 in every patient

AUC

MIC

10 Levofloxacin 500 mg 8 f 6 AUC = 30-50 mg/L

4 conc mg/l conc

2

0 0 4 8 12 16 20 24 time h

65 Mouton - What are PK/PD breakpoints?

Clinical practice :

When starting treatment, we do not know :

• the AUC in the individual patient

10

8

6

4 conc mg/lconc

2

0 0 4 8 12 16 20 24 time h

Pharmacokinetics

Some people are more equal than others…

fAUC distribution levofloxacin (monte carlo simulation)

0.3

0.2 rel freq rel 0.1

0.0 0 0 0 0 00 00 0 00 0 00 0 0 00 00 0 .0 .0 0 0 .0 6.0 8.0 0.0 4.0 24.00028. 32 3 40.00044 4 52.00056. 6 6 68. 72

fAUC

66 Mouton - What are PK/PD breakpoints?

fAUC distribution levofloxacin (monte carlo simulation)

0.3

0.2 rel freq rel 0.1

0.0 0 0 0 0 00 00 0 00 0 00 0 0 00 00 0 .0 .0 0 0 .0 6.0 8.0 0.0 4.0 24.00028. 32 3 40.00044 4 52.00056. 6 6 68. 72

fAUC

On the average, this duck is dead

The fAUC is calculated for 10.000 patients using MCS This results in a probability distribution of AUCs The fAUC/MIC is calculated for each MIC 200 180 levofloxacin 500 mg x 1 oral 160 140 120 95% percentile 100 99% percentile AUC/MIC

f 80 60 mean 40 20 0 0.25 0.5 1 2 4 8 MIC mg/L

Mouton et al., 2004

67 Mouton - What are PK/PD breakpoints?

The fAUC is calculated for 10.000 patients using MCS This results in a probability distribution of AUCs The fAUC/MIC is calculated for each MIC 200 180 levofloxacin 500 mg x 1 oral 160 140 120 95% percentile 100 99% percentile AUC/MIC

f 80 60 mean 40 20 0 0.25 0.5 1 2 4 8 MIC mg/L

Mouton et al., 2004

The fAUC is calculated for 10.000 patients using MCS This results in a probability distribution of AUCs The fAUC/MIC is calculated for each MIC 200 180 levofloxacin 500 mg x 1 oral 160 140 120 95% percentile 100 99% percentile AUC/MIC

f 80 60 mean 40 20 0 0.25 0.5 1 2 4 8 MIC mg/L

Mouton et al., 2004

The fAUC is calculated for 10.000 patients using MCS This results in a probability distribution of AUCs The fAUC/MIC is calculated for each MIC 200 180 levofloxacin 500 mg x 1 oral 160 140 120 95% percentile 100 99% percentile AUC/MIC

f 80 60 mean 40 20 0 0.25 0.5 1 2 4 8 MIC mg/L

Mouton et al., 2004

68 Mouton - What are PK/PD breakpoints?

The fAUC is calculated for 10.000 patients using MCS This results in a probability distribution of AUCs The fAUC/MIC is calculated for each MIC 200 180 levofloxacin 500 mg x 1 oral 160 140 120 95% percentile 100 99% percentile AUC/MIC

f 80 60 mean 40 20 0 0.25 0.5 1 2 4 8 MIC mg/L

Mouton et al., 2004

The fAUC is calculated for 10.000 patients using MCS This results in a probability distribution of AUCs The fAUC/MIC is calculated for each MIC 200 180 levofloxacin 500 mg x 1 oral 160 140 120 95% percentile 100 99% percentile AUC/MIC

f 80 60 mean 40 20 0 0.25 0.5 1 2 4 8 MIC mg/L

Mouton et al., 2004

The fAUC is calculated for 10.000 patients using MCS This results in a probability distribution of AUCs The fAUC/MIC is calculated for each MIC 200 180 levofloxacin 500 mg x 1 oral 160 140 120 10.000 pt 95% percentile 100 99% percentile AUC/MIC

f 80 60 mean 40 20 0 0.25 0.5 1 2 4 8 MIC mg/L

Mouton et al., 2004

69 Mouton - What are PK/PD breakpoints?

200 180 levofloxacin 500 mg x 1 oral 160 140 120 95% percentile 100 99% percentile AUC/MIC

f 80 60 mean 40 20 0 0.25 0.5 1 2 4 8 MIC mg/L S = 1 mg/L

EUCAST., 2004

High dose levofloxacin 2x 500 mg, or 750 mg AUC 70-80

target 35 = 70 /2

70 Mouton - What are PK/PD breakpoints?

200 180 ciprofloxacin 500 mg q12h oral 160 140 120 100 99% CI AUC/MIC

f 80 60 Average 40 20 0 0.25 0.5 1 2 4 8 MIC mg/L

Pk/Pd S = 0.5 mg/L

“He chose poorly” -Knight from Indiana Jones: The Last Crusade

GOOD Clinical Practice

Be sure that the fAUC/MIC ratio is at least appr. 34 in every patient

This includes patients with a high clearance

Bugs with MICs that can be expected

SETTING A BREAKPOINT –PK/PD (example 2) DETERMINE THE PK/PD TARGET e.g. value of the PK/PD Index

ESTIMATE EXPOSURE from the dosing regimen and PK, including population variability

CALCULATE PK/PD BREAKPOINT from PK/PD target = PK/PD Index

71 Mouton - What are PK/PD breakpoints?

Ceftazidime in patients with nosocomial pneumonia

• randomized, double-blind phase 3 (NCT00210964): • comparing the efficacy of with the combination CAZ and linezolid • Ceftazidime 3dd 2 gr 2h infusion • Extensive and sparse sampling of ceftazidime

N=390 patients included 220 without Gram negatives in cultures N=170 with MIC

16 without N=154 with MIC and PK-estimates PK estimates

Muller et al, JAC 2013 68:900-906

Exposure-response Emax model

• Individual exposures to CAZ • Categorised (%fT>MIC per 10%) • Eradication rate per group • 154 patients

Muller et al, JAC 2013 68:900-906

Ceftazidime in patients with nosocomial pneumonia

Muller et al, JAC 2013 68:900-906

72 Mouton - What are PK/PD breakpoints?

Probability of Target Attainment - Ceftazidime

ceftazidime 1000 mg x3

100

80

60 BP = 4 mg/L >MIC T

f 40 99% percentile 20 95% percentile Mean 0 0.25 0.5 1 2 4 8 16 32 64 MIC mg/L

EUCAST rationale document

Susceptible (S)

A micro-organism is defined as susceptible by a level of antmicrobial activity associated with a high likelihood of therapeutic success. A micro-organism is categorized as susceptible by applying the appropriate breakpoint in a defined phenotypic test system.

Note: This breakpoint may be altered with legitimate changes in circumstances

Intermediate (I) A micro-organism is defined as intermediate by a level of antimicrobial activity associated with indetermiate therapeutic effect. A micro-organism is categorized as intermediate by applying the appropriate breakpoints in a defined phenotypic test system. Note: This breakpoints may be altered with legitimate changes in circumstances. Resistant (R) bacteria are defined as resistant by a level of antimicrobial activity associated with a high likelihood of therapeutic failure. A micro-organism is categorized as resistant by applying the appropriate breakpoint in a defined phenotypic test system. Note: This breakpoint may be altered with legitimate changes in circumstances WWW.EUCAST.ORG

Mouton et al, CMI 2012

73 Mouton - What are PK/PD breakpoints?

Implications for breakpoints therapeutic success

PK/PD Index

Susceptibility (MICs) are related to (clinical) outcome

high

low therapeutic success

MIC mg/ L

Susceptibility (MICs) are related to (clinical) outcome?

high breakpoint

low therapeutic success

MIC mg/ L Breakpoint values make the difference –but include PK!!!

74 Mouton - What are PK/PD breakpoints?

Conclusions

• PK/PD breakpoints reflect the relationship between exposure and clinical outcome

• PK/PD breakpoints are dependent on dose (!), pharmacokinetic profile and pharmacodynamic target

• The pharmacodynamic target MAY differ by species (e.g. Gram- vs Gram+)

• EUCAST PK/PD breakpoints are based on clinical data if available and otherwise on animal data and other data. Rationale documents describe the background.

JWM Berlin 27-04-13

75 MacGowan - Are all S equal?

EW01: Antimicrobial susceptibility testing with EUCAST breakpoints and methods

Are all “S” Equal?

Alasdair MacGowan Bristol Centre for Antimicrobial Research & Evaluation University of Bristol, North Bristol NHS Trust Southmead Hospital Bristol UK

Disclosures • Consultancy, lecturing, Advisory Boards:

– The Medicines Company, Bayer-Schering Healthcare, Astellas, Phico Therapeutics, Polymedix, Cerexa, AstraZeneca, Cubist

• Research Grants:

– European Union (FP7 and IMI), NIHR (England); Department of Health (England); North Bristol NHS Trust, Western CLRN, European CDC, HTA (England); Welsh Assembly Government; Cerexa, Cubist, AstraZeneca, Micron Research, Jones Microbiology Institute, BSAC, Forest Laboratories

• Trustee of the following UK based medical charities:

– Showering Fund, British Society for Antimicrobial Chemotherapy (former President)

Phenotypic laboratory testing does not determine:-

1) Likely adverse events

2) Likely drug interactions

3) Cost

In addition to phenotypic susceptibility these factors will determine patient outcomes and drug acceptability.

76 MacGowan - Are all S equal?

Pharmacodynamics and Clinical Breakpoints

Target attainment rates for daptomycin 4mg/kg/day, target AUC24 total/MIC ratio ≥348 (static effect); multidose PK

MIC (mg/L) Percentage of Healthy Infected patients S.aureus strains volunteers (SSTI) 0.06 - 100 100 0.12 0.4% 100 100 0.25 15.3% 100 100 0.5 63.6% 100 99.7 1.0 26.7% 100 90.3 2.0 - 43 39.7 4.0 - 0 3.4 8.0 - 0 0

EUCAST clinical breakpoint S≤1mg/L

Assumptions in PK-PD approach to clinical breakpoints

 Based on serum pharmacokinetics ?what about other infection sites ?IV or oral switch if poor

 Based on standard patients (?critically ill, obese, infected, etc)

 Based on standard drug dosing (bolus injection vs continuous infusion or prolonged infusion - B.lactams)

 MIC accurately reflects susceptibility or resistance mechanism (VISA, hVISA, efflux pumps and fluoroquinolones).

Pharmacokinetic issues Intravenous/oral administration Amoxicillin 2g 8hrly IV switch to 500mg 8hrly po, bioavailability 75-92% fT>MIC 40% as target

Target attainment rate

MIC (mg/L) Percentage 2g 8hrly IV 0.5g 8hrly po of E.coli strains ≤0.5 0.2 100 100 1 1.8 100 100 2 4.0 100 90 4 31.6 100 20 8 12.7 91 1.0 ≥16 39.7 8 0

EUCAST clinical breakpoints M.catarrhalis S=≤1mg/L (+clavulanic acid) H.influenzae S= ≤2mg/L B.haemolytic streptococci S= ≤0.5mg/L S.pneumoniae S ≤ 2mg/L Enterobacteriaceae S= - or ≤8mg/L

77 MacGowan - Are all S equal?

Pharmacokinetics Site of infection: acute pneumococcal meningitis versus non- meningitic infection

Clinical breakpoint S≤Xmg/L Meningitis Other infection site

Penicillin S≤0.06 S≤2

Meropenem S≤0.25 S≤2

Peterson et al, 2004; Yu et al 2003; Weinstein et al, 2009

Critically ill patients: response by MIC in VAP

Ceftazidime Meropenem Piperacillin/Tazobactam EUCAST cb P.aeruginosa ≤8 ≤2 ≤16 Enterobacteriaceae ≤1 ≤2 ≤8 ≤0.5 - 77% (23/30) >0.5 - 29% (5/17)

≤2 62 (28/45) - 4 38 (5/13) - 76 (19/25) 8 14 (1/7) - 40 (6/15) 16 - - 17 (1/6)

Lorente et al, 2006; Lorente et al, 2007; Lorente et al, 2008.

Critical illness: Blood Stream Infection due to E.coli or K.pneumoniae producing ESBLs: ertapenem

Ertapenem Sepsis related mortality MIC (mg/L) ≤0.25 5.3% (3/57) p=0.002 >0.25 33.3% (6/18)

≤0.5 11.6% (8/69) p=0.8 >0.5 20% (1/5)

Lee et al, 2012

EUCAST clinical breakpoint S≤0.5mg/L

78 MacGowan - Are all S equal?

Ertapenem pharmacokinetics in critically ill patients

Healthy volunteers Critically Ill Cmax (mg/L) 253 53

817 188 AUC(0 - ∞) (mg/L.h)

Vss(L) 5.7 26.8

Unbound fT>MIC ≤30% in 25% of patients

Brink et al, 2009

Critical illness: Blood stream infection caused by Gram-negative organisms; Levofloxacin (n=275)

Levofloxacin MIC(mg/L) ≤0.25 mg/L 0.5 mg/L ≥1mg/L P value Mortality 12.5% 11.5% 14.3% 0.91

Length of stay post 7.3 7.9 16.4 0.02 culture

Length of levofloxacin 4.8 6.2 13.3 0.01 therapy post culture

Duration of infection 1.0 1.2 2.1 <0.001 (days)

De Fife et al, 2009 EUCAST clinical breakpoint S≤1mg/L

Cmax, Cmin, t½, AUC24 all significantly different to healthy volunteers in critical care for levofloxacin Rebuck et al, 2002

Non-standard dosing: continuous infusion(CI)/prolonged infusion (PI)

Doripenem 500mg IV by 4hr infusion (PI) , 6hr vs imipenem 500mg 30 min infusion (II) 6hr in VAP patients. MIC ≤4mg/L - doripenem 97% isolates - imipenem 91% isolates For E.coli, Klebsiella sp, P.aeuruginosa and Enterobacter cloacae

Doripenem Imipenem PI II Clinical cure 73% (46/63) 55% (28/51)

Microbiologic cure 73% (46/63) 55% (28/51)

Chastre et al, 2008

79 MacGowan - Are all S equal?

Non-standard dosing: continuous infusion B.lactam therapy in VAP

%Cure MIC Ceftazidime Meropenem Piperacillin/ (mg/L) tazobactam CI II CI II CI II (n=56) (n=65) (n=42) (n=42) (n=49) (n=46) ≤0.5 100 23 >0.5 80 29

≤2 92 62 4 90 38 90 76 8 75 14 88 40 16 87 17

EUCAST Clinical Bp Enterobacteriaceae ≤1 ≤2 ≤8 P.aeruginosa ≤8 ≤2 ≤16

Lorente et al 2006, 2007, 2008

MIC reflects susceptibility?

Intra-abdominal infection treated with moxifloxicin

Anaerobe MIC (mg/L) Clinical Cure

≤2 91.2% (31/34) 4 82.4% (14/17) 8 83.3% (5/6) 16 66.7% (16/24) 32 83.3% (5/6)

Goldstein et al 2001

FDA/CLSI clinical breakpoint S≤2mg/L Confounders: mixed infection; surgery; non-challenging indication-acute appendicitis, altered pharmacodynamics

Vancomycin MIC and S.aureus susceptibility: present status

EUCAST clinical breakpoint for S.aureus is sensitive ≤2mg/L for vancomycin

However:- “based primarily on clinical evidence, those strains of S.aureus with vancomycin MICs values of 2mg/L, which are on the border of the wild type MIC distribution, including hVISA phenotype are likely to have impaired clinical responses to vancomycin”

EUCAST vancomycin rationale document 2.1, 17th June, 2010

80 MacGowan - Are all S equal?

MICs and outcome – new data (1)

Systematic review and meta-analysis of vancomycin MICs and outcomes Van Hal et al 2012, CID 54, 755  22 studies included: 2383 MRSA and 507 MSSA BSI

Conclusions:  Vancomycin MIC significantly associated with mortality in MRSA infection (OR 1.64, p <0.01)

 E.test most common method of determining MIC

 8 studies where E.test result stratified MIC as ≤1.0, ≥1.5 or ≥2mg/L. MIC ≥2mg/L associated with increased mortality in MRSA infection (OR 1.7 p<0.01). MIC ≥1.5mg/L not associated with increased mortality versus MIC ≤1.0mg/L

MICs and outcome – new data (2)

Teicoplanin Chang et al 2012; JAC 67, 736-41

> MRSA bacteraemia, hospital based retrospective observational study (n=101)

> teicoplanin MIC>1.5mg/L associated with higher mortality (48.9% deaths vs 26%)

> EUCAST clinical breakpoint for S.aureus S≤2mg/L

Predicting raised vancomycin MICs in MRSA Lubin et al 2011, CID 52, 997

Scoring System:

Score Age >50 years 3 Vancomycin for >48hr in last week 2 Chronic liver disease 2 History of MRSA bacteraemia 2 Non-tunneled central line 1

Score ≥4 - negative predictive rate 91% positive predictive value 30%

81 MacGowan - Are all S equal?

Vancomycin MICs and MSSA outcomes

Holmes et al 2011, CID 204, 340

8 Australian hospitals 532 patients with S.aureus bacteraemia

Increasing vancomycin MIC associated with mortality in vancomycin treated patients but also MSSA patients treated with (mortality 26.8% MIC >1.5mg/L, 12.2% <1.5mg/L: by E.test) and also:- Han et al 2012, AAC, 56, 5164-5170

The problem with definitions/MICs

Vancomycin Vancomycin MIC, µg/ml status by PAP–AUC ≤0.5 1 2 4 8 16 Susceptible 11914000 (n=106) hVISA 0 3 126 28 0 0 (n=157) VISA 0001190 (n=20)

Wootton M. Antimicrob Agents Chemother 2005;49:3982–3983

Potential clinical approaches – when to test Vancomycin susceptibility (MIC and/or PAP)

 Perform MIC on all MRSA from serious infection (blood, respiratory, IE, bone & joint) assess therapy on basis of result: maybe method dependent

 Perform MIC on all MRSA isolates: the clinical significance is not always obvious

 Perform occasional surveys of Vancomycin susceptibility on local MRSA isolates – see Bowker et al, P1582 this ECCMID

 Perform MIC testing on patients responding poorly to Vancomycin for MRSA infections i.e. persistently positive blood cultures after >5days `

 Use other agents to vancomycin for severe infections, use combination therapy with vancomycin for severe infections

82 MacGowan - Are all S equal?

Conclusions:

 Categorical susceptibility testing has to be evaluated in a clinical context: risk of adverse events, potential interactions; requirement for combination therapy

 Pathogens reported as susceptible may not all respond the same way to different drugs or to the same drug because of patient factors or drug dosing factors

 Not all “S” are equal

83 Arendrup - Antifungal susceptibility testing in the routine lab?

Antifungal Susceptibility Testing (AFST) in the routine lab?

Maiken Cavling Arendrup [email protected] Unit of Mycology Statens Serum Institute Denmark

Disclosures: Research grants & Speaker: Astellas, Gilead, MSD & Pfizer; Advisory board: MSD, Pcovery, Pfizer; Acted as consultant for: Alcimed, Astellas, Gilead & Pfizer Chair(wo)man for EUCAST-AFST Advisor for CLSI-AFST

Question slides 1-2 Please

Q1 Which of the following statements is false? 1. Correct species identification predicts susceptibility and resistance 2. For Candida infections, AFST is most important in patients exposed to antifungal treatment 3. For Aspergillus infections, AFST is always important as azole resistant isolates are encountered in azole naïve patients 4. Susceptibility testing of Aspergillus should be done in a mycology reference laboratory

M. Cavling Arendrup

Motivation behind AFST

Why? – Detect (unexpected) Resistance – Acquired - not predicted by the “name” – Intrinsic - isolates difficult to identify

When? – Candida: particularly AF exposed patients – Aspergillus: even azole naïve patients

How? – Reference methods – Commercial methods M. Cavling Arendrup

84 Arendrup - Antifungal susceptibility testing in the routine lab?

Reference methods

 CLSI – M27-A3 Yeast broth dilution 120 $ – M27-S4 QC and BPs 35 $ – M38-A2 Mould broth dilution 120 $ – M44-A2 Yeast disk diffusion 200 $ 880 $ – M44-S3 QC and BPs 35 $ – M51-A Mould disk diffusion 170 $ – M51-S1 QC and ECVs 200 $

 EUCAST – Methods EDef 7.2 (yeast) and EDef 9.1 (mould) – Rationale documents (breakpoints) – Candida: amphotericin; anidula-, micafungin;flu-, vori- and posaconazole Free – Aspergillus: amphotericin, itra-, posa- and voriconazole – Technical notes in Clin Microbiol Infect www.clsi.org, www.eucast.org M. Cavling Arendrup

Candida Testing: E.DEF 7.2 (2012)

 Flat bottom plates  Incubation at 35 ± 2˚C

 RPMI with 2% glucose & MOPS  24h and  OD ↑ by ≥ 0.2 5  0.5-2.5 x 10 cells/mL  50% inhibition, Spec. reading

Drug dilutions P N 0.8

0.7 PSC

0.6 ANI

0.5 VOR value 0.4 ITR OD 0.3 FLU

0.2 Pos contr

0.1 50% inhibition 0.0

Concentration mg/L M. Cavling Arendrup

AMB & Azole breakpoints for Candida spp Breakpoints (BPs): S: ≤X; R: >Y Revised BPs CLSI CLSI EUCAST M27-S3 M27-S4 AMB ≤1 ≤1 ≤1; >1

Fluco ≤8; >32 ≤2; >4 (alb, para, trop) ≤2; >4 (alb, para, trop) SDD ≤32; R >32 (glab) S ≤0.002; R >32 (glab) (krus poor target) (krus poor target) Itra ≤0.125; >0.5 ≤0.125; >0.5 -

Posa - - ≤0.06; >0.06 (alb, para, trop) (glab/krus IE) Vori ≤1; >2 ≤0.125; >0.5(alb, para, trop) ≤0.125; >0.125 (alb, para, trop) ≤0.5; >1 (krus) (glab/krus IE) (glab IE) www.eucast.org; Pfaller Drug Resist Updat. 2010 & 2011; www.clsi.org M. Cavling Arendrup

85 Arendrup - Antifungal susceptibility testing in the routine lab?

EUCAST vs. CLSI MICs fluconazole C. albicans C. glabrata C. krusei

2000 400 150 300 100 1000 200 100 50 0 0 -100 0 -1000 -200 -50 -2000 -300 -400 -100 -500 -3000 -150 -600 -4000 -700 -200 1 1 1 2 4 8 2 4 8 2 4 8 16 16 16 32 64 32 64 32 64 0.5 0.5 0.5 128 512 128 512 128 512 256 256 256 0.25 0.25 0.25 0.016 0.125 0.016 0.125 0.016 0.125 0.002 0.004 0.008 0.032 0.064 0.002 0.004 0.008 0.032 0.064 0.002 0.004 0.008 0.032 0.064 C. parapsilosis C. tropicalis 600 200

400 100 0 200 EUCAST MIC distribution -100 above the X-axis 0 -200

‐200 -300 CLSI MIC distribution -400 below the X-axis ‐400 -500 ‐600 -600

‐800 -700 1 2 4 8 1 2 4 8 16 32 64 16 32 64 0.5 0.5 128 512 256 128 256 512 0.25 0.25 0.016 0.125

0.002 0.004 0.008 0.016 0.032 0.064 0.125 0.002 0.004 0.008 0.032 0.064 M. Cavling Arendrup

Echinocandin breakpoints for Candida spp Breakpoints (BPs): S: ≤X; R: >Y Revised BPs CLSI CLSI Revised 2011 EUCAST M27-S3 (M27-S4) www.eucast.org/clinical_breakpoints/ ≤0.032; >0.032 (alb) ≤0.25; >0.5 (alb, krus, trop) ≤0.06; >0.06 (glab, krus, trop) ANF ≤2 ≤0.002; >4 (para) ≤0.125; >0.25 (glab) (guillier IE) ≤2; >4 (para, guillier) CSF ≤2 -

≤0.016; >0.016 (alb) ≤0.25; >0.5 (alb, krus, trop) ≤0.03; >0.03 (glab, krus, trop) MFG ≤2 ≤0.06; >0.125 (glab) ≤0.002; >2 (para) (guillier IE) ≤2; >4 (para, guillier)

www.eucast.org; Pfaller Drug Resist Updat. 2010 & 2011; www.clsi.org M. Cavling Arendrup

EUCAST & CLSI Anidulafungin MICs 0.002 0.004 0.008 0.016 0.032 0.064 0.125 >=16 0.25 0.5 Candida sp 1 2 4 8 ECOFF BP

albicans EUCAST 284 576 314 427* 703* 142543400040.03 0.03

albicans CLSI 338 1278 1542 896 216 12 1 0.125 0.25

glabrata EUCAST 55 43 64 177* 441* 111 28115331200.06 0.06

glabrata CLSI 7 161 715 320 26 2 2 2 1 0.25 0.125

krusei EUCAST 2113 26* 83* 34 8 40000000.06 0.06

krusei CLSI 4 159 91 14 1 1 0.125 0.25

parapsilosis 0310462367817196 13 7 2 4 (4) EUCAST parapsilosis CLSI 1 2 1 1 14 49 319 765 86 4 2

tropicalis EUCAST 18 34 17 47* 175* 24 7 63100020.06 0.06

tropicalis CLSI 41 254 493 173 24 7 1 3 0.125 0.25

Arendrup CMI 2011, www.eucast.org and www.clsi.org M. Cavling Arendrup

86 Arendrup - Antifungal susceptibility testing in the routine lab?

EUCAST vs. CLSI MICs anidulafungin

C. albicans C. glabrata C. krusei C. parapsilosis C. tropicalis

EU 10 C. albicans (S) 10 C. glabrata (S) 12 C. krusei (S) 10 C. tropicalis (S) 9 8 C. parapsilosis (S) C. albicans (R) 9 9 CA C. glabrata (R) C. krusei (R) 7 8 10 C. tropicalis (R) 8 8 7 6 7 7 ST 6 8 6 5 6 5 5 6 4 5 4 4 3 4 No. of of isolates No.

No. of isolates of No. 4 3 isolates of No. No. of isolates of No.

3 isolates of No. 3 2 2 2 2 2 1 1 1 1 0 0 0 0 1 1

1 0 2 4 8 2 4 8 2 4 8 1 2 4 8 16 16 16 1 16 2 4 8 0.5 0.5 0.5 0.5 16 0.01 0.13 0.01 0.13 0.01 0.13 0.02 0.03 0.06 0.25 0.02 0.03 0.06 0.25 0.02 0.03 0.06 0.25 0.01 0.13 0.5 0.02 0.03 0.06 0.25 0.01 0.13 0.02 0.03 0.06 0.25 MIC (μg/ml) MIC (μg/ml) MIC (μg/ml) MIC (μg/ml) MIC (μg/ml)

C. albicans C. glabrata C. krusei C. parapsilosis C. tropicalis C. albicans (S) C. glabrata (S) C. krusei (S) 7 6 9 10 C. parapsilosis (S) 7 C. tropicalis (S) C. albicans (R) C. glabrata (R) C. krusei (R) 8 9 C. tropicalis (R) 6 5 6 7 8 5 7 5 4 6 CL 6 4 5 4 3 5 3 4 4 3

SI isolates of No. 2 3 2 No. of isolates No. of isolates No. of isolates 3 No. of isolates 2 2 2 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 4 4 4 4 4 16 16 16 16 16 0.02 0.06 0.25 0.02 0.06 0.25 0.02 0.06 0.25 0.02 0.06 0.25 0.02 0.06 0.25 MIC (μg/ml) MIC (μg/ml) MIC (μg/ml) MIC (μg/ml) MIC (μg/ml)

Arendrup et al AAC 2010, Pfaller Drug res updates 2011, M27-S4 M. Cavling Arendrup

Commercial testing methods

Test performance

– Eg. variability in zone interpretation Interpretation of endpoints – Caveats adopting CLSI /EUCAST BPs – Echinocandins as an example – Etest – Vitek

M. Cavling Arendrup

C. albicans. Etest.

Amphotericin B Fluconazole M. Cavling Arendrup

87 Arendrup - Antifungal susceptibility testing in the routine lab?

C. albicans. Etest.

Amphotericin B Fluconazole M. Cavling Arendrup

Adopting BPs: CLSI & Echinocandin Etest

 Are Test MIC50s for each species = reference MIC? C. albicans

60,00% 60,00% 80,00% CLSI-ANF CLSI-CSF CLSI-MFG 40,00% 60,00% 40,00% Anidulafungin Micafungin Caspofungin 40,00% 20,00% 20,00% 20,00% 0,00% 0,00% 0,00%

-20,00% -20,00% -20,00%

-40,00% -40,00% -40,00% Etest Etest -60,00% Etest

-60,00% <=0.002 0,004 <=0.007 0,015 0,03 0,06 0,12 0,25 0,5 1 2 4 >=8

<=0.002 0,004 <=0.007 0,015 0,03 0,06 0,12 0,25 0,5 1 2 4 >=8 -60,00% <=0.002 0,004 <=0.007 0,015 0,03 0,06 0,12 0,25 0,5 1 2 4 >=8

MIC (µg/ml) MIC (µg/ml) MIC (µg/ml)

Pfaller JCM 2010 and Axner-Elings JCM 2011 M. Cavling Arendrup

Echinocandin Etest MICs & CLSI BP 0.002 0.004 0.008 0.016 0.032 0.064 0.125 0.25 0.5 1 2 4 8 <16 C. albicans* 80 43 31 5 1 C. glabrata 81 8 67 6 C. dublinensis 14 2 11 1 C. tropicalis 8 2 5 1 C. krusei 5 5 C. parapsilosis 36 11411 6 13 C. guillermondii 2 1 1 C. lusitaniae 8 7 1

C. albicans* 80 17 29 26 6 2 C. glabrata 81 23 58 C. dublinensis 14 2 7 5 C. tropicalis 8 2 5 1 C. krusei 5 3 2 C. parapsilosis 36 16 20 C. guillermondii 2 11 C. lusitaniae 8 6 2

C. albicans* 80 36 38 6 C. glabrata 81 4 74 3 C. dublinensis 14 6 7 1 C. tropicalis 8 2 5 1 S80S/P C. krusei 5 1 4 C. parapsilosis 36 2824 2 Micafungin Caspofungin Anidulafungin C. guillermondii 2 2 C. lusitaniae 8 1 6 1 Axner-Elings JCM 2011 M. Cavling Arendrup

88 Arendrup - Antifungal susceptibility testing in the routine lab?

Echinocandin Etest MICs & EUCAST BP 0.002 0.004 0.008 0.016 0.032 0.064 0.125 0.25 0.5 1 2 4 8 <16 C. albicans* 80 43 31 5 1 C. glabrata 81 8 67 6 C. dublinensis 14 2 11 1 C. tropicalis 8 2 5 1 C. krusei 5 5 C. parapsilosis 36 1 14 11 6 13 C. guillermondii 2 11 C. lusitaniae 8 7 1

C. albicans* 80 17 29 26 6 2 C. glabrata 81 23 58 C. dublinensis 14 2 7 5 C. tropicalis 8 2 5 1 C. krusei 5 3 2 C. parapsilosis 36 16 20 C. guillermondii 2 11 C. lusitaniae 8 6 2

C. albicans* 80 36 38 6 C. glabrata 81 4 74 3 C. dublinensis 14 6 7 1 S80S/P C. tropicalis 825 1 C. krusei 514 C. parapsilosis 36 2 8 24 2 Micafungin Caspofungin Anidulafungin C. guillermondii 2 2 C. lusitaniae 816 1 Axner-Elings JCM 2011 M. Cavling Arendrup

Etest: Anidulafungin & EUCAST BP C. albicans C. glabrata FKS mutants: S 10 10 7% (2/29) 9 9 8 8 A 7 7 6 6 FKS mutants: I N 5 5 4 4 0% (0/29) No. of isolates I 3 of isolates No. 3 2 2 1 1 WT as I/R 0 0 1 1 2 4 8 2 4 8 16 16 13% (6/47) 0.5 0.5 >32 >32 0.25 0.25 0.015 0.125 0.015 0.125 0.002 0.004 0.008 0.032 0.064 0.002 0.004 0.008 0.032 0.064 <0.002 <0.002 MIC (μg/ml) MIC (μg/ml) C. krusei C. tropicalis C. parapsilosis 10 10 10 9 9 9 8 8 8 7 7 7 6 6 6 5 5 5 4 4 4 No. of of No. isolates No. of isolates of No. 3 isolates of No. 3 3 2 2 2 1 1 1 0 0 0 1 1 2 4 8 2 4 8 1 2 4 8 16 16 16 0.5 0.5 >32 >32 0.5 >32 0.25 0.25 0.25 0.015 0.125 0.015 0.125 0.002 0.004 0.008 0.032 0.064 0.002 0.004 0.008 0.032 0.064 0.015 0.125 0.002 0.004 0.008 0.032 0.064 <0.002 <0.002 <0.002 MIC (μg/ml) MIC (μg/ml) MIC (μg/ml) Arendrup et al AAC 2010 and EUCAST-AFST M. Cavling Arendrup

Echinocandin Etest MICs & CLSI BP 0.002 0.004 0.008 0.016 0.032 0.064 0.125 0.25 0.5 1 2 4 8 <16 C. albicans* 80 43 31 5 1 C. glabrata 81 8 67 6 C. dublinensis 14 2 11 1 C. tropicalis 8 2 5 1 C. krusei 5 5 C. parapsilosis 36 11411 6 13 C. guillermondii 2 1 1 C. lusitaniae 8 7 1

C. albicans* 80 17 29 26 6 2 C. glabrata 81 23 58 C. dublinensis 14 2 7 5 C. tropicalis 8 2 5 1 C. krusei 5 3 2 C. parapsilosis 36 16 20 C. guillermondii 2 11 C. lusitaniae 8 6 2

C. albicans* 80 36 38 6 C. glabrata 81 4 74 3 C. dublinensis 14 6 7 1 C. tropicalis 8 2 5 1 S80S/P C. krusei 5 1 4 C. parapsilosis 36 2824 2 Micafungin Caspofungin Anidulafungin C. guillermondii 2 2 C. lusitaniae 8 1 6 1 Axner-Elings JCM 2011 M. Cavling Arendrup

89 Arendrup - Antifungal susceptibility testing in the routine lab?

Etest: Caspofungin & CLSI BP

10 C. albicans 10 C. glabrata 9 9 FKS mutants: S 8 8 7% (2/29) 7 7 6 6 5 5 FKS mutants: I 4 4 7% (2/29) No. ofNo. isolates 3 of No. isolates 3 2 2 1 1 WT as I/R 0 0 1 1 2 4 8 2 4 8

16 13% (6/47) 16 0.5 >32 0.5 >32 0.25 0.25 0.015 0.125 0.015 0.125 0.002 0.004 0.008 0.032 0.064 0.002 0.004 0.008 0.032 0.064 <0.002 <0.002 MIC (μg/ml) MIC (μg/ml) C. tropicalis C. krusei C. parapsilosis 10 10 10 9 9 9 8 8 8 7 7 7 6 6 6 5 5 5 4 4 4

No. of No. isolates 3 ofNo. isolates 3 ofNo. isolates 3 2 2 2 1 1 1 0 0 0 1 1 1 2 4 8 2 4 8 2 4 8 16 16 16 0.5 0.5 0.5 >32 >32 >32 0.25 0.25 0.25 0.015 0.125 0.015 0.125 0.015 0.125 0.002 0.004 0.008 0.032 0.064 0.002 0.004 0.008 0.032 0.064 0.002 0.004 0.008 0.032 0.064 <0.002 <0.002 <0.002 MIC (μg/ml) MIC (μg/ml) MIC (μg/ml) Arendrup et al AAC 2010 M. Cavling Arendrup

Etest: Caspofungin and CLSI BP C. glabrata (136) 50% C. albicans (278) 60%

Etest 40% 30% Etest 20% 10% 0% ‐10% ‐20% ‐30% CLSI ‐40% CLSI (4283) ‐50% (1236) ‐60% 0.070.0160.030.060.1250.250.5124816>32 0.070.0160.030.060.1250.250.5124816>32 MIC (mg/L) Titel

C. krusei (26) 50% C. tropicalis (24) 65% 40% Etest Etest 30% 45% 20% 10% 25% 0% 5% ‐10% ‐20% ‐15% ‐30% CLSI ‐35% CLSI ‐40% (996) (270) ‐50% ‐55% 0.070.0160.030.060.1250.250.5124816>32 0.07 0.016 0.03 0.06 0.125 0.25 0.5 1 2 4 8 16 >32 Titel Titel

Arendrup & Pfaller AAC 2012 M. Cavling Arendrup

Caspofungin and Vitek

Vitek 2

Breakpoint

MIC range Antifungal compound EUCAST CLSI Card conc. reported 1, 4, 16, 32 11 1, 4, 8, 16 22 4, 8, 16, 64 -4 0.5, 1, 4, 8 0.12 0.12 1, 4, 8 0.25/0.125

Astvad AAC 2013 M. Cavling Arendrup

90 Arendrup - Antifungal susceptibility testing in the routine lab?

Vitek Caspofungin

14 C. albicans 14 C. glabrata FKS mutants  “S” 12 S I R 12 S I R Vitek: 19.4% (6/31)* 10 10

8 8 FKS mutants  “I”

6 6 Vitek: 0% (0/30)

4 4 WT as I/R 2 2 Vitek: C. glabrata !! 0

0 0.063 0.125 0.25 0.5 1 2 4 8 16 ≤ ≥ 0.064 0.125 0.5 1 2 8 16 NG 0.25 4 * 2 C. glabrata FKS mutants with MIC ≤0.25 mg/L counted as S 20 14 18 C. tropicalis C. krusei 12 16 S I R S I R 14 10 12 8 10 8 6 6 4 4 2 2 0 0.064 0.125 ≤ 0.5 1 2 ≥ 8 16 NG 0 0.25 4 0.064 0.125 ≤ 0.5 1 2 ≥ 8 16 NG 0.25 4

Astvad AAC 2013 M. Cavling Arendrup

Take home messages Commercial Tests

You have to check for each drug-bug combination

BPs can only be adopted if your MICs mirror those for the ref. method!

Recommendations for echinocandins – Echinocandins: – Anidulafungin Etest with EUCAST BPs – VITEK2 problematic: MIC range doesn’t cover the BP for C. glabrata

M. Cavling Arendrup

UK Clin Mycology Network

Local Specialised* Regional Mycology microbiol lab microbiol lab mycology lab reference lab Tests Primary fungal  AND:  AND:  AND: cultures Cryp Ag test Fungal serology ID all fungi C. albicans >< Asp GM test ID unusual fungi Typing non-albicans AFST techniques Dermatophyte TDM National ID standards and Microscopy Training training programme fungal elements Surveillance & PCP Molecular techniques Strain collection Research Health care scientists 0 ≥ 1 ≥ 3 dedicated mycol *This level of service should be available at all teaching hospitals and specialist cancer, haematology, transplant & HIV centres

M. Cavling Arendrup

91 Arendrup - Antifungal susceptibility testing in the routine lab?

11-14 October 2013, Copenhagen, Denmark M. Cavling Arendrup

Acknowledgements STATENS SERUM (in alphabetic order): INSTITUT

The EUCAST Steering Committee M Cuenca-Estrella W Hope C Lass-Flörl The EUCAST General Committee

Collaborators on MIC studies SJ Howard DS Perlin M Pfaller P Verweij

The Danish Fungaemia Study Group

Thank you for your attention M. Cavling Arendrup

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