Educational Workshop EW01: Antimicrobial Susceptibility Testing with EUCAST Breakpoints and Methods

Educational Workshop EW01: Antimicrobial susceptibility testing with EUCAST breakpoints and methods

Arranged with EUCAST (The European Committee on Antimicrobial Susceptibility Testing)

Convenors: Gunnar Kahlmeter, Växjö, SE Rafael Canton Moreno, Madrid, ES

Faculty: Rafael Canton Moreno, Madrid, ES Susan J. Howard, Manchester, UK Johan W. Mouton, Rotterdam, NL Iztok Strumbelj, Murska Sobota, SI Alasdair P. MacGowan, Bristol, UK Derek F.J. Brown, Peterborough, UK Sören G. Gatermann, Bochum, Germany Ronald N. Jones, North Liberty, IA, USA

Marked in red = no handouts available

Canton - Update on EUCAST methods and breakpoints

www.EUCAST.org Update on EUCAST methods and breakpoints, 2015

Departamento de Microbiología II Universidad Complutense. Madrid

www.eucast.org

Top countries % visits-Q2014 http://www.eucast.org Unknown 17,6 USA 10,6 Germany 10.1 Visits: UK 6.1 - 50.000-60.000 visitors/month (60% from EU) Netherlands 5.7 Switzerland 4.0 Pages more visited (each visitor may see Itally 3.9 each page more than once) Sweden 3.6 - home 45.9% Denmark 3.3 - clinical breakpoints 69.2% Belgium 2.8 - MIC/zone distributions 5.5% France 2.2 - expert rules 3.3% Norway 1.9 - resistance mechanisms 3.2% Spain 1.8 Austria 1.7 Finland 1.7 China 1.7 Australia 1.5 Japan 1.5 Canton - Update on EUCAST methods and breakpoints

http://www.eucast.org

. No. of visitors per month

60000

50000 Overall EU

40000

30000

20000

10000

0 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4

2012 2013 2014

EUCAST-related publications

. Yearly evolution of publications in PubMed including “EUCAST” in the title and/or abstract

120

100

20 No. of publications of No.

*January-March, 2015

2015 www.eucast.org

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

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, NAK + 2 reps from the GC ±1‐2 “visiting” members from the GC

Subcommittees Experts Antifungals (AFST) (ECDC Networks, VetCAST ESCMID Study Groups) Whole genome sequencing and AST Industry Canton - Update on EUCAST methods and breakpoints

EUCAST translations Canton - Update on EUCAST methods and breakpoints

National Antimicrobial Committees (NACs) outside Europe

Countries with a NAC operating under EUCAST standards Countries with interest to establish a NAC under EUCAST standards

EUCAST subcommittees

Subcommittees Subcommittee on Antifungal Susceptibility Testing (EUCAST AFST) Standing subcommittee established in 2002 Veterinary Subcommittee on Antimicrobial Susceptibility Testing (VetCAST). Standing Subcommittee established in 2015 Subcommittee on the role of whole genome sequencing in antimicrobial susceptibility testing. Established in 2015.

Former subcommittees Subcommittee on Expert Rules and Intrinsic Resistance. Established in 2007, disbanded 2011. Subcommittee on Anaerobe Susceptibility Testing Established in 2007, disbanded 2011. Subcommittee on the detection of resistance mechanisms of clinical and/or public health importance. Established in 2011, disbanded 2014. Canton - Update on EUCAST methods and breakpoints

Veterinary committee on AST (VetCAST)

. Formed in 2015, dealing with antimicrobial susceptibility testing (AST) of bacterial pathogens of animal origin and zoonotic bacteria . Remit… - To establish a science‐based committee to cooperate with EU professionals in veterinary medicine, EMA, ECDC and EFSA - To determine antimicrobial breakpoints specific to the veterinary field - To harmonize veterinary AST in the EU - To provide AST/antimicrobial therapy education in the veterinary field - To initiate and coordinate EU research aimed at filling the current gaps in veterinary AST – Missing or insufficient veterinary specific breakpoints – Optimized methods for AST of bacterial pathogens of animal origin - To ensure that AST protocols and interpretive criteria are freely accessible online through the EUCAST website . Chairman (Dik Mevious), Secretary (Peter Damborg), …

Subcommittee on the role of whole genome sequencing (WGS) in AST of bacteria

. Formed in 2015 with the following remit - perform a systematic literature review of the role of WGS in antimicrobial susceptibility testing (AST) of bacteria (excluding mycobacteria) - determine the sensitivity and specificity of WGS compared with standard phenotypic AST - determine how WGS may be applied in clinical laboratories and the likely implications for phenotypic and other genotypic methods in use - determine the epidemiological implications of using WGS - determine the clinical implications of WGS for the selection of antimicrobial therapy - determine the principles of how the result of WGS for AST would be best presented to clinical users - describe the drivers and barriers to routine use of WGS . Coordinator (Alasdair MacGowan), ...

EUCAST Development Laboratories (Nov 2014 …) . Development and maintenance of EUCAST antimicrobial susceptibility testing (AST) methods - Bacteria (Växjö, Sweden) - Fungi (Statens Serum Institut, Copenhagen, Denmark) . Coordination of he EUCAST Network Laboratories in the development and validation of EUCAST methods, training, education and technical support to other laboratories.

EUCAST network laboratories (Nov 2014 …) . Microbiology laboratories with particular expertise and training in EUCAST AST for bacteria and/or fungal isolates . Develop, validate and troubleshoot EUCAST methods and/or train and educate other laboratories . Assist clinical breakpoint development by providing species -specific MIC datasets Canton - Update on EUCAST methods and breakpoints

Templete for RDs, 40 documents (New: ceftaroline; Drafted: cetobiprol, macrolides, penicillins, cephalosporins, SXT, aztreonam, choramphenicol)

SOP 8.0. Format and updating of EUCAST documents SOP 9.0. Procedure for establishing zone diameter breakpoints and QC criteria for new antibiotics

. Implementation of EUCAST breakpoints for AST (Euro surveillance)

Euro Surveillance 2015;20:pii=21008

Uptake of EUCAST guidelines by participants in UKNEQAS (updated)

100 90 80 70 60 CLSI 50 All EUCAST 40 30 20 10 Percent of laboratories 0 Nov 08 Nov 09 Nov 10 Nov 11 Apr 12 Dez 12 Mrz 13 Sep 13 Feb 14 Mrz 15 Courtesy of Derek Brown and Christine Walton

. MIC vs zone diameters files . Disk diffusion manual and slide show . Breakpoint and QC tables v5.0 . Compliance of manufacturers

. Frequently Asked Questions March 23rd , 2015 Canton - Update on EUCAST methods and breakpoints

EUCAST breakpoints, 2015 (version 5.0)

. Links to different pages (microorganisms) and EUCAST documents: - guidance documents, expert rules - detection of resistance mechanisms - breakpoints for topical use of antimicrobial agents

EUCAST breakpoints, 2015 (version 5.0)

. Guidance on reading EUCAST breakpoint tables

New format for comments Canton - Update on EUCAST methods and breakpoints

EUCAST breakpoints, 2015 (version 5.0)

New and revised breakpoints . Enterobacteriaceae Amikacin (zone diameters) . Staphylococcus spp. Telavancin (new) . Moraxella catarrhalis Ceftaroline (change from dash to IE) . Neisseria menigitidis Ciprofloxacin (remove intermediate catergory) . Neisseria gonorrhoeae Cefpodoxime,ceftibuten(change from IE to dash) . Clostridium difficile Fidaxomicin (new) . M. tuberculosis Delamanid and bedaquiline (new)

New EUCAST breakpoints, April 2015 . Staphylococcus spp. Dalbavancin, Oritavancin, Tedizolid (new) . Strep. group A, B, C, G Dalbavancin, Oritavancin, Tedizolid (new) . Strep. anginosus group Dalbavancin, Oritavancin, Tedizolid (new)

EUCAST breakpoints, 2015 (version 5.0)

Rewording of notes, new notes . Telavancin, tigecycline, Information on testing conditions daptomycin, fosfomycin . Aztreonam Enterobacteriaceae, Pseudomonas spp. . Trimethoprim-sulfameth. Stenotrophomonas maltophilia Enterococcus spp. . Cephalosporins Staphylococcus spp. . Clindamycin Strept. groups A, B, C and D Streptococcus pneumoniae Viridans group streptococci Quality control data . Haemophillus influenzae . Moraxella catharralis . Pasterurella multocida

EUCAST breakpoints, 2015 (version 5.0)

Clindamycin induble phenotype (MLSB) . Inducible clindamycin resistance can be detected by antagonism of clindamycin activity by a macrolide agent . Place the erythromycin and clindamycin disks 12-20 mm apart (edge to edge) and look for antagonism (the D phenomenon)

Antagonisms Report Organisms Comment to the report clindamycin No detected S Staphylococcus spp. Detected R Staphylococcus spp. Clindamycin may still be used for short-term therapy of less serious skin and soft tissue infections as constitutive resistance is unlikely to develop during such therapy R Strept. group A,B,C,G S. pneumoniae Viridans g. streptococci Canton - Update on EUCAST methods and breakpoints

EUCAST breakpoints: Delamanid and Bedaquiline

. EUCAST Clinical Breakpoint Table v. 5.0, valid from 2015-01-01

Mycobacterium tuberculosis EUCAST Clinical Breakpoint Table v. 5.0, valid from 2015-01-01 EUCAST was tasked with suggesting to EMA Recommended methods for antimicrobial breakpoints for new agents but has so far susceptibility testing of mycobacteria are not addressed breakpoints for existing currently under discussion. agents.

MIC breakpoint Notes (mg/L) Numbers for comments on MIC breakpoints

S ?≤ R >

Delamanid 0,06 0,06 Bedaquiline 0.25 0.25

EUCAST breakpoints

. Reviewed breakpoints with no changes: - linezolid and staphylococci and enterococci - teicoplanin and coagulase negative staphylococci - fluoroquinolones and Corynebacterium spp. - metronidazole and anaerobes - daptomycin and enterococci

Guidance note on breakpoints for topical agents

. Superficial skin and external eye and ear infections but not for bowel decontamination or inhaled agents

. EUCAST has not found a consensus to resolve different opinions - use ECOFFs for agents when used topically - use clinical breakpoints when available and ECOFF when there are not clinical breakpoints

. Acceptable distributions are not available for all topical agents

. Only specific breakpoints for nasal decolonisation of S. aureus with mupirocin is supported with clinical data

. If tissue is involved the use of systemic treatment and systemic breakpoints should be considered Canton - Update on EUCAST methods and breakpoints

Guidance note on breakpoints for topical agents

. ECOFFs and systemic clinical breakpoints for antimicrobial agents that are used topically

- = inappropriate combination; IE = insufficient evidence to set a clinical breakpoint; ND = No ECOFF defined on EUCAST MIC distribution website 1Agents also available for systemic use 2Breakpoints for nasal decontamination S≤1, R>256 mg/l.

EUCAST: What is coming for 2015-16?

. New and ongoing breakpoints (BP) and methodology -EMA β-lactam-β-lactamase inhibitor combinations, cephalosporins aminoglycosides, and oxazolidinones, pleuromutillin guidelines for companies submitting anti-mycobacterial agents - CLSI colistin and methodology - NACs temocillin, nitroxoline, spiramycin, tigecycline, sulbactam - Antimicrobial groups: fluoroquinolones and carbapenems - Neisseria gonorrhoeae and different antimicrobials - Breakpoints for Actinomyces spp., Eikenella corrhodens

. New RD documents (new agents and new RD due to revised BP) . New documents, technical notes / guidance documents - new version of expert rules (v3) - SOPs (revision of breakpoints) . New EUCAST definition of the intermediate category and ECOFF

www.eucast.org Canton - Update on EUCAST methods and breakpoints

Acknowledgements

More information: - Saturday 16:30 – 18:30, Hall B Resurrecting old antimicrobial agents - Sunday 09.00 – 10.30, Meeting Room 19 EUCAST Subcommittee on Antifungal Susceptibility Testing (AFST) General Committee Meeting 11.30 – 12.30, Hall B Benefits and challenges of site-specific breakpoints - Monday 13.00 – 14.30, Hall N EUCAST General Committee meeting 17.30 – 18.30, Meeting Room 6 EUCAST EUCAST Veterinary Subcommittee on Development Antimicrobial Susceptibility Testing (VetCAST) Laboratory - Tuesday 07.45 – 08.45, Hall I and Meet the Experts. EUCAST: frequently asked EUCAST Network questions laboratories Howard ‐ Antifungal breakpoints and susceptibility testing

Anti-Fungal Susceptibility Testing Subcommittee

Antifungal breakpoints and susceptibility testing

Dr Susan J Howard Manchester, UK

Scientific Secretary EUCAST Antifungal Steering Committee

EUCAST AFST SC: ArendrupEUCAST‐ AFST(chair),: MC Howard Arendrup (secretary), (Chair), Meletiadis WW Hope (data (Secretary), manager), MMouton, Cuenca Cuenca‐Estrella‐Estrella, & C Lass Lass‐Flörl‐Flörl

Anti-Fungal Susceptibility Testing Subcommittee

Disclosures ‐ none

Anti-Fungal Susceptibility Testing Subcommittee Agenda

• Setting the scene

• Methodology outline

• ECOFF/breakpoint setting

• Current guidelines

Anti-Fungal Susceptibility Testing Subcommittee Agenda

• Setting the scene • Methodology outline • ECOFF/breakpoint setting • Current guidelines

Anti-Fungal Susceptibility Testing Subcommittee Background

• The mortality rate associated with fungal disease is dire • Susceptibility is just one factor affecting outcome • Limited therapeutic drug class options • Global antifungal drug budget is significant (est. $US 5.7 billion in 2014)

Anti-Fungal Susceptibility Testing Subcommittee Why Susceptibility Test?

• Intrinsic resistance • Acquired resistance (increasing in some settings) • Some identifications challenging/slow • Historically multiple/differing methodologies, with no/limited breakpoints to allow comparison/interpretation

Anti-Fungal Susceptibility Testing Subcommittee Agenda

• Setting the scene • Methodology outline • ECOFF/breakpoint setting • Current guidelines

Anti-Fungal Susceptibility Testing Subcommittee Methodology

• Commercially available tests – Including disc diffusion, Sensititre, Vitek and Etest

• Broth dilution reference methods – CLSI & EUCAST

Anti-Fungal Susceptibility Testing Subcommittee EUCAST Method

• Flat bottom plates • RPMI with 2% glucose and MOPS • Incubate at 35 ±2˚C

Drug dilutions P N

Anti-Fungal Susceptibility Testing Subcommittee Yeasts Fermentative yeasts E.DEF 7.2 (2012) – 0.5‐2.5 x 105 cells/mL – 24 h and OD ≥ 0.2 – Spectrophotometer – 90% endpoint (AMB) / 50% endpoint (all others)

Anti-Fungal Susceptibility Testing Subcommittee Moulds Conidia forming moulds E.DEF 9.2 (2014) 5 Amb Casp Vor Itra Flu Cnt – 1‐2.5 x 10 cells/mL 8‐0.06 4‐0.03 16‐0.125 (avoid clumps) – 48 h  adequate growth* (*24h may be sufficient for some zygomycetes, some moulds require 72h) – Visual endpoint reading  amphotericin & azoles = no growth endpoint (MIC)  echinocandins = aberrant growth (MEC) MIC/MEC 0.5 ≤0.06 0.5 0.5 >16

Anti-Fungal Susceptibility Testing Subcommittee MEC Aspergillus

Growth control Aberrant growth

Anti-Fungal Susceptibility Testing Subcommittee Agenda

• Setting the scene • Methodology outline • ECOFF/breakpoint setting • Current guidelines

Anti-Fungal Susceptibility Testing Subcommittee ECOFFs versus Breakpoints Epidemiological cut off value (ECOFFs) – Based on the wild‐type MIC distributions – Upper limit of WT isolates – Typically describes isolates with identical susceptibility – Typically describes isolates with no resistance mechanisms Clinical breakpoints – Take other factors (e.g. PK/PD and clinical data) into account – More later…

Anti-Fungal Susceptibility Testing Subcommittee EUCAST fluconazole MIC C. glabrata

MIC50 ECOFF: Non‐WT ≤32 µg/ml WT •May or may not population respond depending on host & drug exposure • Harbour resistance mechanisms

http://mic.eucast.org/Eucast2/ EUCAST AFST SC: ArendrupEUCAST‐ AFST(chair),: MC Howard Arendrup (secretary), (Chair), Meletiadis WW Hope (data (Secretary), manager), MMouton, Cuenca Cuenca‐Estrella‐Estrella, & C Lass Lass‐Flörl‐Flörl Howard ‐ Antifungal breakpoints and susceptibility testing

Anti-Fungal Susceptibility Testing Subcommittee Wild‐type MIC Distributions

20 34 different C. glabrata isolates

0 isolates

of ‐5 No. ‐10

‐15

‐20

‐25 0.25 0.5 1 2 4 8 16 >16

MIC (µg/ml)

Arendrup AAC 2009 EUCAST AFST SC: ArendrupEUCAST‐ AFST(chair),: MC Howard Arendrup (secretary), (Chair), Meletiadis WW Hope (data (Secretary), manager), MMouton, Cuenca Cuenca‐Estrella‐Estrella, & C Lass Lass‐Flörl‐Flörl

Anti-Fungal Susceptibility Testing Subcommittee Wild‐type MIC Distributions

20 34 different C. glabrata isolates

0 isolates

of ‐5 No. ‐10

‐15 A single C. glabrata isolate ‐20 tested 51 times ‐25 0.25 0.5 1 2 4 8 16 >16

MIC (µg/ml)

Anti-Fungal Susceptibility Testing Subcommittee Variability in Wild‐type Distributions

60 C. glabrata repetitions

50 40 Reflects the inherent 30 20 susceptibility of the No. of Isolates of No. 10 0 WT population 0,25 0,5 1 2 4 8 16 >16 60 MIC (µg/ml) 50

40 One step lower 30 One step lower C. glabrata repetitions One step higher 20 60 No. of Isolates of No. 10 50

0 40 0.25 0.5 1 2 4 8 16 >16 30 60 MIC (µg/ml) 20

50 No. of Isolates 10 40 0 30 One step higher 0.25 0.5 1 2 4 8 16 >16 20

No. of Isolates of No. MIC (µg/ml) 10

0 0.25 0.5 1 2 4 8 16 >16

MIC (µg/ml) Arendrup AAC 2009 EUCAST AFST SC: ArendrupEUCAST‐ AFST(chair),: MC Howard Arendrup (secretary), (Chair), Meletiadis WW Hope (data (Secretary), manager), MMouton, Cuenca Cuenca‐Estrella‐Estrella, & C Lass Lass‐Flörl‐Flörl Howard ‐ Antifungal breakpoints and susceptibility testing

Anti-Fungal Susceptibility Testing Subcommittee Caspofungin and Variability (EUCAST)

EUCAST MIC50 is highlighted

No. of C. albicans isolates at the individual EUCAST MICs (mg/L) No. 0.002 0.004 0.007 0.015 0.03 0.06 0.12 0.25 0.5 1 2 ≥ 4 Tested CFG Lab 1 ND ND ND 20 30 35 45 130 Lab 2 ND ND NDConsiderable ND ND 123variation 162 for209 caspofungin102 9 1 606 Lab 3 ND ND ND ND ND 10 161 219 851404 Lab 4 ND ND ND 2 105 264 182 54 11 3 1 4 626 Lab 5 ND ND 61212 53 38 Lab 6 ND ND NDEUCAST ND 25 abstained51 from setting cut offs 31 Lab 7 ND ND ND ND 1 2 14 330 373 1 721 MFG Lab 1 ND ND ND ND 107 107

Lab 2 ND 121 2 123

Lab 3 ND 34 19 12 30 4 1 100

Lab 4 ND 78 19 2 1 100

Lab 5 ND ND ND 520 35 2 1 2 560

Lab 6 487252 239 4 4 590

Lab 7 ND ND 87 2 89 Espinel‐Ingroff AAC 2013, Arendrup Mycoses in press EUCAST AFST SC: ArendrupEUCAST‐ AFST(chair),: MC Howard Arendrup (secretary), (Chair), Meletiadis WW Hope (data (Secretary), manager), MMouton, Cuenca Cuenca‐Estrella‐Estrella, & C Lass Lass‐Flörl‐Flörl

Anti-Fungal Susceptibility Testing Subcommittee Population with WT and non‐WT

60 R

50 One step higher Non‐WT C. glabrata repetitions with MIC 64 µg/ml 40 One step lower 50 (4 steps higher) 30

No. of isolates 20

0 1 2 4 8 16 32 64 0.5 128 256 0.25 MIC (μg/ml)

60 60 Non‐WT with MIC 16 µg/ml 50 Non‐WT with 50 50 (2 steps higher) 40 MIC50 32 µg/ml 40

30 (3 steps higher) 30

No. of isolates 20 20 No. of isolates of No.

10 10

0 0 1 2 4 8 1 2 4 8 16 32 64 16 32 64 0.5 0.5 128 256 0.25 0.25 MIC (μg/ml) MIC (μg/ml)

Anti-Fungal Susceptibility Testing Subcommittee Real Life Breakpoint Example A. fumigatus Itraconazole Posaconazole

Clear separation WT and non‐WT Overlap WT and non‐WT  further analysis needed to identify all non‐WT

Verweij Drug Res Updates 2009 EUCAST AFST SC: ArendrupEUCAST‐ AFST(chair),: MC Howard Arendrup (secretary), (Chair), Meletiadis WW Hope (data (Secretary), manager), MMouton, Cuenca Cuenca‐Estrella‐Estrella, & C Lass Lass‐Flörl‐Flörl Howard ‐ Antifungal breakpoints and susceptibility testing

Anti-Fungal Susceptibility Testing Subcommittee EUCAST BP Setting Process

• MIC distributions – Per species – Several data sets – Epidemiological Cut Off Value (ECOFF)

• Dosing regimens used

• PK/PD

• MIC‐clinical outcome relationships – Per species – For wild type and non‐wild type isolates

Arendrup Drug Res Updates 2014 EUCAST AFST SC: ArendrupEUCAST‐ AFST(chair),: MC Howard Arendrup (secretary), (Chair), Meletiadis WW Hope (data (Secretary), manager), MMouton, Cuenca Cuenca‐Estrella‐Estrella, & C Lass Lass‐Flörl‐Flörl

Anti-Fungal Susceptibility Testing Subcommittee ECOFF versus Breakpoint (flucon) 1750 1500 C. albicans BP (S: ≤x ; R: >y) 1250 ECOFF 1 mg/L ≤2 mg/L ; >4 mg/L 1000 C. albicans 750 Isolates 500 250 3000 250 C. glabrata BP 200 ECOFF 32 mg/L ≤0.002 mg/L ; >32 mg/L 150 C. glabrata

Isolates 100

1400 120 C. krusei BP 100 ECOFF 128 mg/L none –not a good target 80 60 Isolates 40 C. krusei 20 0 1 2 4 8 16 32 64 0.5 128 256 512 0.25 0.002 0.004 0.008 0.016 0.032 0.064 0.125 MIC www.eucast.org EUCAST AFST SC: ArendrupEUCAST‐ AFST(chair),: MC Howard Arendrup (secretary), (Chair), Meletiadis WW Hope (data (Secretary), manager), MMouton, Cuenca Cuenca‐Estrella‐Estrella, & C Lass Lass‐Flörl‐Flörl

Anti-Fungal Susceptibility Testing Subcommittee Agenda

• Setting the scene

• Methodology outline

• ECOFF/breakpoint setting

• Current guidelines

Anti-Fungal Susceptibility Testing Subcommittee Website

www.eucast.org

Anti-Fungal Susceptibility Testing Subcommittee ECOFF/Breakpoint Updates

• Recent updates (since ECCMID 2014): – Mould E.DEF 9.2 definitive document – Itraconazole Candida rationale document – BP table (itra Candida BP added, caspo BP comment revised) • Forthcoming documents to look out for in 2015: – Amphotericin Candida rationale document – Posaconazole Candida rationale document – Voriconazole Candida rationale document – Isavuconazole Aspergillus rationale document

Anti-Fungal Susceptibility Testing Subcommittee Acknowledgements

The EUCAST Steering Committee Maiken Arendrup Joseph Meletiadis Johan Mouton Manuel Cuenca‐Estrella Cornelia Lass‐Flörl

The EUCAST General (Sub) Committee

Thank you for your attention

National introduction of EUCAST breakpoints and methods

Iztok Štrumbelj

• National Laboratory of Health, Environment and Food

Maribor, Slovenia

• Slovenian National Antimicrobial Susceptibility Testing Committee

Disclosure.

Iztok Štrumbelj: no conflict of interest, nothing to disclose.

Purpose: to describe the introduction of European Committee on Antimicrobial Susceptibility Testing (EUCAST) bacteriology breakpoints and methods in Slovenia.

Slovenia • Population: 2 million • Independent since 1991

http://www.slovenia.info/ http://www.itis.si/zemljevid 3 Strumbelj ‐ National introduction of EUCAST breakpoints and methods

Hospitals in Slovenia

29 hospitals (26 public, 3 private), most samples from:

• 2 university clinical centres • 11 general hospitals

Source: Zdravstveni statistični letopis 2012, Nacionalni inštitut za javno zdravje (NIJZ). Medical microbiology laboratories are parts of different public institutions

• 1 university laboratory (Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana) • 8 laboratories are departments of Centre for Medical Microbiology of National Laboratory of Health, Environment and Food • 3 are hospital laboratories

Certified medical microbiology laboratories in Slovenia

Quality system (QS) and certification of diagnostic laboratories 1. Quality system (QS), published in Official Gazette of Republic of Slovenia 2. Audit of laboratory QS by independent Commission 3. If requirements met: “permission to work” issued by Ministry of Health for 5 years.

List of certified diagnostic laboratories in the field of clinical bacteriology published at the website of Ministry of Health. (accessed March 19th, 2015) 12 laboratories: ‐ 1 specialised in mycobacteriology ‐ 11 perform antimicrobial susceptibility testing of common bacteria

Slovenian National Antimicrobial Susceptibility Testing Committee Slovenian acronym: SKUOPZ Slovenska komisija za ugotavljanje občutljivosti za protimikrobna zdravila

• Established: at the end of 2010. • Members: at least one member from each certified laboratory and one member from National Institute of Public Health

• Two working areas:

a) Methods for antimicrobial susceptibility testing (AST) b) Surveillance of antimicrobial resistance (AMR) ‐ three annual national reports were published on web (2011, 2012, 2013).

6 Strumbelj ‐ National introduction of EUCAST breakpoints and methods

Example page from SKUOPZ annual national report

http://www.imi.si/strokovna‐zdruzenja/skuopz

Antimicrobial susceptiblity testing (AST) methods used in Slovenia

Disk diffusion method used as principal method.

For MIC determination, gradient diffusion method is mostly used. Always for anaerobes.

Standard microdilution method not used routinely.

Automated AST systems used in few laboratories.

Both, MICs and zones, entered and interpreted in LIS.

Transition from CLSI to EUCAST

Introduction, June 2013

• EUCAST presented in ISIS – official journal of Slovenian Medical Chamber • Symposium about EUCAST for microbiologists and clinicians

The process, June 2013 –April 2014

Three main points • Laboratory information system (LIS) upgrade. • Laboratory methods. • Information for clinicians.

Final control • Results of external quality assessment. ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Note: in this presentation, “agent” means “antimicrobial agent”.

9 Strumbelj ‐ National introduction of EUCAST breakpoints and methods

Introduction, June 2013. EUCAST presented in ISIS – official journal of Slovenian Medical Chamber. Symposium with international speakers for microbiologists and clinicians, round table. Final unanimous decision: EUCAST should be introduced.

Consensus

During the whole process of EUCAST implementation –all decisions of SKUOPZ were accepted by consensus.

The first major decision was critical. • Request of some clinicians: to supplement EUCAST breakpoints with breakpoints from other AST systems, if EUCAST has no breakpoint for an agent / species (e.g. ampicilin‐sulbactam and Acinetobacter baumannii) • SKUOPZ decision: unacceptable. “Mixing” breakpoints would lead to a confusion.

Strict use of EUCAST interpretations was followed: (‐) dash in the EUCAST breakpoint tables: no testing or resistant result. (IE) insufficient evidence in the breakpoint tables: if necessary (rarely) MIC can be determined, but without interpretation into SIR category.

12 Strumbelj ‐ National introduction of EUCAST breakpoints and methods

Laboratory information system (LIS) upgrade (1)

Review of processes:

Identification of need: what do we want from LIS (responsibility of SKUOPZ)

Dedicated clinical microbiologist and software expert Alenka Štorman communicated the need for changes to software provider who finally implemented all software changes.

Alenka Štorman personally performed amazing amount of extensive and difficult tasks:

. entered all breakpoint values (MICs and zones) into LIS . wrote all comments (prepared by SKUOPZ) into LIS . linked them to the proper AST result for different species / agents . validated LIS after changes were implemented.

Laboratory information system (LIS) upgrade (2)

All laboratories use the same LIS provided by a Slovenian software company. The software development was started by Jana Kolman, MD and Alenka Štorman, MD, clinical microbiologists, in mid‐nineties.

INTERPRETATION OF ZONES AND MICs (into S, I, R category).

Background: it is not rare that one MIC / disk zone is interpreted differently for different species or that interpretation of MIC /zone is limited only to few species within bacterial group.

Automatic interpretation of MICs / zones, linked to different species, makes life easier.

Result of upgrade: all zones (calliper is used) and MICs are entered into LIS, where automatically interpreted as S, I or R.

Very rarely, manual correction of the result is necessary.

LIS precludes S, I, R interpretation if there are no EUCAST breakpoints.

Laboratory information system (LIS) upgrade (3)

COMMENTS OF RESULTS appearing on the results form

Background: for proper interpretation of many EUCAST results (S, I or R), comments are common and essential.

Basic idea: to make comments as user friendly as possible for laboratories (automatic link of the comment with the specific result of an agent) and clinicians (as short and as clear as possible).

Considerable amount of work and discussions was necessary to integrate (or split) guidelines and rules from different EUCAST documents into software rules so that comments are added to laboratory reports automatically ‐ 99 different comments were entered into LIS.

Result of LIS upgrade: comments are linked to the results so that appropriate comment is automatically linked to the agent or agent result.

15 Strumbelj ‐ National introduction of EUCAST breakpoints and methods

Breakpoints may be different for different species within a group

Species specific breakpoints within a group.

Software precludes use of inappropriate breakpoints for the species tested.

Example: Staphylococcus spp. ‐ breakpoints for cefoxitin screen disk (surrogate disk for “methicillin “ susceptibility / resistance)

Species Zone – methicilin susceptible S. aureus, S. lugdunensis and S ≥ 22 S. saprophyticus S. pseudintermedius S ≥ 35

Other coagulase‐negative staphylococci S ≥ 25

Breakpoints may be limited to some species within a group There is no interpretation (no breakpoints) for other species in the group.

Software precludes interpretation for species without breakpoints.

Example 1. Systemic infections.

Group Agent Breakpoints limited to Enterobacteriaceae Cefuroxime iv E. coli, Klebsiella spp. and P. mirabilis Example 2. Uncomplicated urinary tract infections only.

Group Agent Breakpoints limited to Enterobacteriaceae Nitrofurantoin E. coli Enterococcus spp. Nitrofurantoin E. faecalis Staphylococcus spp. Nitrofurantoin S. saprophyticus Streptococcus spp. Nitrofurantoin S. agalactiae 17

Manual correction of results

Example 1. Staphylococcus spp., inducible clindamycin resistance. Agent, disk “Zone test result” Reason for Final result for change the agent Clindamycin Zone ≥ 22 mm, Positive D‐ R ‐ resistant S – susceptible. phenomenon.

Example 2. Staphylococcus aureus, penicillin.

Agent, disk “Zone test result” Reason for Final result for change the agent Penicillin Zone ≥ 26 mm, The zone edge R ‐ resistant S ‐ susceptible is sharp.

18 Strumbelj ‐ National introduction of EUCAST breakpoints and methods

Comments / notes in the report

• Few comments are added manually, e.g. when resistance mechanism is determined. • Most of comments appear automatically ‐ they are linked to an element. “Element” is isolate and:

1. the agent or 2. result of the agent or 3. MIC of the agent.

• Comment may be a translation of EUCAST note or synthesis of several data from EUCAST documents (e.g. several notes in breakpoint tables). • One comment may be used for several different elements.

Comments linked to the agent

Applicability of the result of an agent to other agents

• Isolate: Staphylococcus spp. Agent: erythromycin • Comment: Result of erythromycin applies also to azithromycin, clarithromycin and roxithromycin.

• Isolate: Streptococcus agalactiae. Agent: penicillin. • Comment: Streptococcus agalactiae ‐ result of penicillin applies also to other penicillins (with the exception of phenoxymethylpenicillin and isoxazolylpenicillins), to cephalosporins (with the exception of cefixime and ceftazidime) and to carbapenemes.

Explanation about the agent tested

• Isolate from urine: Enterococcus spp. Agent: norfloksacin disk is tested. • Comment: Enterococcus ‐ result of norfloksacin disk is valid for ciprofloxacin and levofloxacin, not for norfloxacin. Result applies to uncomplicated urinary tract infections only. 20

Comments linked to the dose of an agent

General comment about high dose. • Isolate: Pseudomonas spp. • Agent: ceftazidime • Comment: Result applies to high dose therapy.

Specific once‐daily application of high daily dose. • Isolate: Pseudomonas spp. • Agent: gentamicin. • Comment: Result applies to once‐daily administration of high aminoglycoside dosages.

Specific dose of an agent for the isolate; comment may be specific for S or I result. • Isolate: isolate without species specific breakpoint, PK/PD breakpoints used. • Agent: imipenem. • Comment: Breakpoints apply to imipenem 500 mg x 4 daily administered intravenously over 30 minutes as the lowest dose. 1 g x 4 daily was taken into consideration for severe infections and in setting the R breakpoint. 21 Strumbelj ‐ National introduction of EUCAST breakpoints and methods

Comments linked to the SIR result of an agent Agent : methicillin (tested with cefoxitin disk, reported as oxacillin result).

Isolate: any staphylococcus. Result: S. Comment: Oxacillin susceptible staphylococcal isolate is also susceptible to other antistaphyloccal penicillins (e.g. cloxacillin, flucloxacillin), to penicillins with betalactamase inhibitors, to carbapenemes and to cephalosporins ‐ except for ceftazidime, cefixime and ceftibuten, which should not be used for staphylococcal infections.

Isolate: Stapylococcus aureus * Result: R. Comment: Stapylococcus aureus, resistant to oxacillin, is resistant to all beta‐ lactam agents; exceptions are ceftaroline and ceftobiprole if their antimicrobial susceptibility result is susceptibility (S).

*Note: Comments for CNS are slightly different ‐ no ceftaroline and ceftobiprole breakpoint. 22

Comments linked to the MIC of an agent

Isolate: Streptococcus pneumoniae. Comments relate to treatment of pneumonia.

Result and comment for parenteral penicillin, non‐meningeal criteria:

Sources.

Links to following »basic« documents are provided on the EUCAST web site http://www.eucast.org (current versions, accessed 25th March 2015).

General information at: http://www.eucast.org •The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 5.0, 2015. •Leclercq R et al. EUCAST expert rules in antimicrobial susceptibility testing (2011) •Giske CG et al. EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance. (Version 1.0. December 2013) •Guidance Document on Stenotrophomonas maltophilia (1 Feb 2012) •Guidance Document on Burkholderia cepacia group (20 July, 2013) •Guidance Document on Breakpoints for topical use of antimicrobial agents (2014) •Check list to facilitate implementation of antimicrobial susceptibility testing with EUCAST breakpoints (2010) •EUCAST QC table v 5.0 (2015‐01‐11) •Preparation of plates and media for EUCAST AST (v 4.0, 19 June, 2014) •Compliance of manufacturers of susceptibility testing devices and materials (25 September, 2014)

24 Strumbelj ‐ National introduction of EUCAST breakpoints and methods

Disk‐diffusion documents at: http://www.eucast.org (current versions, accessed 25th March 2015). •EUCAST Disk Diffusion ‐ Manual (v 5.0, 26 January, 2015) •EUCAST Disk Diffusion ‐ Slide Show (v 5.0, 26 January, 2015) •EUCAST Disk Diffusion ‐ Reading Guide (v. 4.0, 19 June, 2014) •Matuschek E et al. Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories. (2014)

Whenever a clear answer can’t be found in other documents, solution can be frequently found following the link: •Frequently Asked Questions (2015‐03‐23)

Whenever this option failed, we sent an E‐mail to Erika Matuschek, Clinical Scientist, responsible for the EUCAST Development Laboratory, Växjö, Sweden. Answers were extremely fast and helpful. Thanks!

Implementation of EUCAST – laboratory work

• Basic guide: this "check list". • "General issues" were discussed by SKUOPZ • Practical work was done in each laboratory • SKUOPZ member was "a champion among laboratory staff" in each laboratory –i.e. responsible for the process • About 6 months of work was necessary before Quality Control results were fully compliant and EUCAST implemented.

Laboratory methods ‐ training.

A lot of practice and “tuning” of procedures and staff was needed, most difficult parts were:

• searching for suitable MHF agar (home made or ready to use plates) • proper inoculum preparation, inoculation and reading of MHF plates.

Practical advice (provided by E. Matuschek): ‐ When inoculating haemophilus, plates must be sufficiently dry and swabs before inoculation extremely dry.

Procedures on Mueller Hinton were technically relatively simple, EUCAST disk diffusion reading guide and slide show very useful, however, considerable amount of time still needed for details.

27 Strumbelj ‐ National introduction of EUCAST breakpoints and methods

Laboratory methods ‐ materials

Some new quality control strains. ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐

Disk‐diffusion method • Some disks have different contents in CLSI and EUCAST. • New agar: Mueller Hinton Fastidious agar.

‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ MIC determination

Note: • SKUOPZ policy: use of commercial methods (including gradient diffusion) is the responsibility of each laboratory. Essential requirement –MIC materials (machines and consumables must be according to EUCAST).

Laboratory methods – materials, MICs

For EUCAST susceptibility testing purposes, the concentration of sulbactam is fixed at 4 mg/L, of clavulanic acid is fixed at 2 mg/L, of tazobactam is fixed at 4 mg/L.

Optimally, range of MIC values should be suitable for different purposes. Example: Enterobacteriaceae, meropenem

Purpose Lower MIC Higher MIC

Clinical breakpoints S ≤ 2 mg/L R > 8 mg/L

Screening for carbapenemase Negative screen Positive screen ≤ 0.12 mg/L > 0.12 mg/L EUCAST quality control range Minimum Maximum Escherichia coli ATCC 25922 0.008 0.06

Detailed Slovenian guidelines for Enterobacteriaceae – ESBL, AmpC and carbapenemases.

Guideline 001: Carbapenem resistance, Enterobacteriaceae.

Guideline 002: ESBL, AmpC, Enterobacteriaceae.

30 Strumbelj ‐ National introduction of EUCAST breakpoints and methods

Slovenian guidelines for carbapenemase ‐ producing Enterobacteriaceae .

Fully compliant with “EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance”, Version 1.0, December 2013. Details were specified, epidemiological definitions for CRE and CRE‐CPE were added. Screening cut –off: ‐ EUCAST guideline offers two possibilities for meropenem disk screening cut‐off. <25 mm (“best balance of sensitivity and specificity”) <27 mm (“may be used as a screening cut‐off in countries where OXA‐48 is endemic, but at the expense of lower specificity”). ‐ meropenem cut‐off < 27 mm was chosen (other combinations of carbapenems can be used)

Phenotypic methods for detection of carbapenemases:

Two methods with different mechanisms are used for each “suspicious” isolate: ‐ Carbapenem hydrolisis test (Carba NP test or Blue‐Carba). ‐ Combined disks (tablets) with carbapenemase inhibitors (including “triple “ disk + temocillin + cloxacillin)

Information for clinicians.

April 2014. Transition to EUCAST implemented. When possible, personal contacts with clinicians were used.

One page long information about EUCAST, supplemented with table of major differences between CLSI and EUCAST, was distributed to users when we changed to EUCAST. This information included link to SKUOPZ web home page, where concise but quite comprehensive information (15 pages) for clinicians were published: “Short explanation on transition to EUCAST “(in Slovene) http://www.imi.si/strokovna‐zdruzenja/skuopz Every day on each report. Comments available when needed.

“Short explanation on transition to EUCAST”

33 Strumbelj ‐ National introduction of EUCAST breakpoints and methods

Results of the process

April 2014: all but one diagnostic medical microbiology laboratories in Slovenia implemented EUCAST. Results of external quality assessment (EQA)

All SKUOPZ laboratories that have implemented EUCAST were asked to report all scored results of EQA since April 2014 to the end of 2014.

Cumulative results for 1154 bacteria/antibiotic results:

• Categorical agreement: 1149 (99,6 %) • Major discrepancy: 4 (0,3%) • Very major discrepancy: 1 (0,1%)

Conclusions

Introduction of EUCAST was a demanding national project: • coordinated by the national AST committee – SKUOPZ • activities “at the bench” were performed by each laboratory.

Three key points were: • Changes in laboratory information system • Changes in laboratory methods • Information for clinicians.

System was well accepted by both laboratories and clinicians.

Acknowledgments

EUCAST Secretariat, EUCAST Development Laboratory and speakers at EUCAST symposium in Ljubljana Rafael Cantón, Derek Brown, Gunnar Kahlmeter, Erika Matuschek, Arjana Tambić Andrašević, Iva Butić, Katja Seme

SKUOPZ members Ingrid Berce, Jerneja Fišer, Andrej Golle, Tatjana Harlander, Martina Kavčič, Jana Kolman, Slavica Lorenčič‐Robnik, Tadeja Matos, Verica Mioč, Manica Mueller‐Premru, Irena Piltaver‐Vajdec, Mateja Pirš, Katja Seme, Helena Ribič, Alenka Štorman, Viktorija Tomič, Barbara Zdolšek, Manca Žolnir Dovč.

Laboratory staff of Slovenian clinical microbiology laboratories

Review of this presentation Katja Seme, Mateja Pirš, Jernej Štrumbelj

36 Brown ‐ Clindamycin susceptibility testing and reporting

Clindamycin susceptibility testing and reporting

Derek Brown EUCAST Scientific Secretary

EUCAST Educational Workshop, ECCMID 2015

Clindamycin

• Lincosamide agent classified in the macrolide, lincosamide and streptogramin group

• Used to treat staphylococcal and streptococcal skin and soft tissue infections, particularly in general practice and outpatients

Clindamycin resistance mechanisms in staphylococci and streptococci • Most resistance to macrolide, lincosamide and streptogramin type B (MLSB) antimicrobial agents is mediated by the erm genes (ribosomal methylation) and is induced by erythromycin, clarithromycin and azithromycin, but not by clindamycin (dissociated resistance or MLSB inducible resistance). • Strains with MLSB constitutive resistance are resistant to clindamycin. •Efflux • Ribosomal mutation Brown ‐ Clindamycin susceptibility testing and reporting

Clindamycin MIC distribution for S. aureus

S R

Clindamycin MIC distribution for coagulase-negative staphylococci

S R

Clindamycin MIC distribution for Streptococcus pyogenes

S R Brown ‐ Clindamycin susceptibility testing and reporting

Clindamycin MIC distribution for Streptococcus agalactiae

S R

Detection of inducible clindamycin resistance in staphylococci and streptococci by disk diffusion • Disk diffusion –the “D test” • Erythromycin 15 µg and clindamycin 2 µg disks placed 12-20 mm apart (edge to edge) for staphylococci, 12-16 mm apart for streptococci • “Antagonism” of clindamycin by erythromycin indicates inducible resistance

Detection of inducible clindamycin resistance in staphylococci and streptococci by broth microdilution • Test clindamycin MIC in presence of fixed concentration of erythromycin – Staphylococci 4 mg/L erythromycin – Streptococci 1 mg/L erythromycin • Staphylococci – Clindamycin MIC >0.25 mg/L in presence of erythromycin and ≤0.25 mg/L without erythromycin indicates inducible resistance (Swenson et al, JCM, 2007) • Streptococci – Clindamycin MIC >0.5 mg/L in presence of erythromycin and ≤0.5 mg/L without erythromycin indicates inducible resistance (Bowling et al, JCM, 2010) Brown ‐ Clindamycin susceptibility testing and reporting

Detection of inducible clindamycin resistance in staphylococci by automated systems • Vitek 2 (Sensitivity 80-95%) – Bobenchik et al, JCM 2014. Inducible R detected in 24/30 isolates – Gardiner et al, Pathology 2013. Inducible R detected in 191/201 isolates – Lavallee et al, JCM 2010. Inducible R detected in 124/134 isolates – Buchan et al, DMID 2012. Inducible R detected in 51/56 isolates • Phoenix – Buchan et al, DMID 2012. Inducible R detected in 56/56 isolates • Microscan – Ji et al, Korean J Lab Med 2010. Inducible R detected in 58/58 isolates

Detection of inducible clindamycin resistance in streptococci by automated systems • Vitek 2 (Sensitivity 36-95%) – Tazi et al, JAC 2007. Inducible R detected in 9/25 of Group B isolates – Tang et al, JCM 2004. Inducible R detected in 17/18 of Group B isolates

• Phoenix – Buchan et al, DMID 2012. Inducible R detected in 100% of Group B isolates (not clear how many tested) – Richter et al, JCM 2007 Inducible R detected in 23/23 of Group B isolates

Reporting inducible clindamycin resistance in staphylococci and streptococci • Should staphylococci and streptococci with inducible clindamycin resistance be reported resistant or susceptible?

• Inducible strains segregate clindamycin resistant mutants, which may be selected during treatment, possibly leading to treatment failure Brown ‐ Clindamycin susceptibility testing and reporting

Reporting inducible clindamycin resistance in staphylococci and streptococci • Guidance on reporting has been inconsistent

Susceptible with warning that resistance may develop during treatment OR Resistant with note that less serious infections may still be treatable

EARSNet External Quality Assessment S. aureus with inducible clindamycin resistance (MIC 0.12 mg/L, but resistance induced by erythromycin) • Reports from participants

Percent reporting • Specimen N SI R 0275 (2011) 775 24.0 1.8 74.2 1377 (2012) 705 7.5 1.4 91.1

• Reports of susceptible not related to guideline or method • Different recommendations from EUCAST and CLSI – EUCAST expert rules recommend reporting resistant, or susceptible with warning of possible failure due to selection of resistant mutants. Avoid use in serious infections – CLSI report resistant with note that some may respond

Evidence for clinical significance of inducible resistance to clindamycin in staphylococci and streptococci? • In vitro studies

• Animal models

• Clinical outcome data Brown ‐ Clindamycin susceptibility testing and reporting

In vitro studies (S. aureus)

• Constitutive resistant mutants can be selected by culture in the presence of clindamycin. Panagea et al. JAC 1999: 44; 581-2 Daurel et al. JCM 2008: 46; 546-55

• Time-kill studies indicate initial killing but rapid regrowth Leplante et al. AAC 2008: 52; 2156-62

Animal models of infection with MLSBi S. aureus

• Mouse thigh model with low inoculum (105) – Clindamycin reduced CFU by 0.45 and1.3 logs for two isolates at 72h. – Constitutive resistant mutants detected at 72h.

• Mouse thigh model with high inoculum (107) – Clindamycin showed bacteriostatic activity at 24h and growth of 0.39 and 1.28 logs for two isolates at 72h. – Constitutive resistant mutants detected at 24h.

Leplante et al. AAC 2008: 52; 2156-62

Animal models of infection with MLSBi streptococci

• Neutropenic mouse thigh model with inoculum (107) – Clindamycin reduced CFU over 12h for two isolates (one Group A and one Group B) but then regrowth to similar level to constitutive resistant isolates by 72h.

Lewis et al. AAC 2014: 58; 1327-31 Brown ‐ Clindamycin susceptibility testing and reporting

Issues in assessing clinical data on significance of MLSBi resistance • Anecdotal cases of failures more likely to be reported than successes • Non-severe cases may have spontaneous favourable outcome • Initial treatment often with a β-lactam agent or vancomycin, the clindamycin • Treatment of non-severe cases in general practice or as outpatients so often no follow up unless treatment fails • Contribution of treatment when there is surgical intervention is unclear

Clinical data on outcome of infection with MLSBi S. aureus

Reference No. of Outcome Susceptibility patients McGehee et al. 3 2 failed 2 S pre-treatment AAC 1968:13;392 1 success 3 R post-treatment Not tested for MLSBi Drinkovic et al. 3 1 failed 1 MLSBc JAC 2001:48:315 2 success Frank . 10 2 failed 1 MLSBc Ped Inf Dis J 2002:21;530 Rao. JAC 2000:45;715 3 1 failed 1 MLSBc 2 success Watanakunacorn. 1 1 failed 1 S pre-treatment Am J Med 1976:60;419 1 R post-treatment Not tested for MLSBi Siberry et al. 1 1 failed 1 MLSBc Clin Inf Dis 2003:37;1257

Clinical data on outcome of infection with MLSBi streptococci

Reference No. of Outcome Post therapy patients susceptibility Lewis et al. 8 with Gp B 8 failed AAC 2014:58;1327 Brown ‐ Clindamycin susceptibility testing and reporting

Inducible clindamycin resistance in staphylococci and streptococci

• There is a significant risk of failure of therapy in treatment of more severe infections • There is not strong evidence relating to treatment of less serious infections

• EUCAST guidelines for staphylococci and streptococci in breakpoint tables v. 5.0 2015 If MLSBi detected, then report as resistant Consider adding this comment to the report: "Clindamycin may still be used for short-term therapy of less serious skin and soft tissue infections as constitutive resistance is unlikely to develop during such therapy". Gatermann, Expert Rules ECCMID 2015

Expert rules in antimicrobial susceptibility testing

Sören Gatermann Bochum, Germany [email protected]

Objectives

● improve therapeutic relevance of susceptibility testing results

● recognition of the unusual

● should have consequences

● therapy ● infection control ● satisfy curiosity

Recognition of the unusual

● natural resistance

● e.g. clindamycin or cefalosporins in enterococci ● typical susceptibility

● penicillin in ß-haemolytic streptococci ● carbapenems in enterobacteria – in some places rare in others frequent

Gatermann, Expert Rules ECCMID 2015

Improve therapeutic relevance

● staphylococci

● cefoxitin/oxacillin predicts utility of ß-lactams ● D-phenomenom predicts utility of clindamycin ● enterobacteria

● carbapenemases ● ESBL ● AmpC

Improve therapeutic relevance

● pneumococci

● in pneumonia ● in meningits ● H. influenzae

● detect ß-Lactamase ● BLNAR

Jones ‐ Susceptibility testing of new and revived antimicrobial agents

Susceptibility Testing of New and Revived Antimicrobial Agents

Ronald N. Jones, MD, FAAM, FASCP, FCAP, FIDSA* JMI Laboratories North Liberty, Iowa USA

*Scientific Secretary of the US Committee on Antimicrobial Susceptibility Testing (USCAST)1

Disclosure and Transparency Statement

JMI Laboratories, Inc., received additional research and educational grants in 2012 to 2014 from Achaogen, Actelion, Affinium, American Proficiency Institute (API), AmpliPhi Bio, Anacor, Astellas, AstraZeneca, Basilea, BioVersys, Cardeas, Cempra, Cerexa, Daiichi, Dipexium, Durata, Fedora, Forest Research Institute, Furiex, Genentech, GlaxoSmithKline, Janssen, Johnson & Johnson, Medpace, Meiji Seika Kaisha, Melinta, Merck, MethylGene, Nabriva, Nanosphere, Novartis, Pfizer, Polyphor, Rempex, Roche, Seachaid, Shionogi, Synthes, The Medicines Co., Theravance,Thermo Fisher Scientific, VenatoRx, Vertex, Waterloo, and some other corporations. Some JMI employees are advisors/consultants for Astellas,Cubist Pharmaceuticals, Pfizer, Cempra, Cerexa‐Forest, and Theravance.

Presentation Outline

1. Background of typical AST development 2. Contemporary challenges to AST development ‐ New agents (lipoglycopeptides, oxazolidinones) with specific technical issues ‐ Revived older agents or combinations 3. Integrating AST challenges with emerging understanding of drug exposure (PK/PD) optimization 4. Conclusions

3 Jones ‐ Susceptibility testing of new and revived antimicrobial agents

Background of AST Development (“The Past”) 1. Mode of actions, breadth of spectrum, and cidality 2. MIC distributions and establishing the treatable wildtype (WT) distribution 3. Pharmacokinetics (dosing ranges) 4. Establishing reference method (broth or agar) and required supplements 5. Standardizing disk test correlate testing details 6. Assigning clinical breakpoints driven by Phase 3 trial response rates

Background of AST Development (“Now”)

1. Mode of actions, breadth of spectrum, and cidality 2. MIC distributions and establishing the treatable wildtype (WT) distribution 3. Pharmacokinetics (dosing ranges), with greater emphasis on pharmacodynamics. 4. Establishing reference method (broth or agar) and required supplements, with greater technical challenges of media quality, supplements and method variations. 5. Standardizing disk test correlate testing details, but drug disk contents may differ by geographic region or the diffusion methods may be compromised. 6. Assigning clinical breakpoints driven by Phase 3 trial response rates, but pharmacodynamic modeling and TA data have become very helpful. 5

Examples of Recent AST Development and Breakpoint Issues • Lipoglycopeptide development, a long painful history – Dalbavancin* – Oritavancin* – Telavancin* • Oxazolidinones – Linezolid – Tedizolid* • Combination drugs – Ceftazidime‐Avibactam (CAZ‐AVI)* – Ceftolozane‐Tazobactam (TOL‐TAZ)* • Polymyxins among other agents – Colistin and Polymyxin B

*Six new drug breakpoints evaluated in the last year A1 Jones ‐ Susceptibility testing of new and revived antimicrobial agents

Glycopeptides (Vancomycin, Teicoplanin)

Lipoglycopeptides (Dalbavancin, Oritavancin, Telavancin)

Greater potency Heavy molecular weight Extended serum elimination half‐life Less soluble “Sticky”

Structure of Dalbavancin

Dalbavancin Activity Trends from Key Publicationsa

a MIC50/MIC90 (% >0.12 µg/ml)

Pathogen Jones (1999‐2000) Biedenbach (2002‐2007) Jones (2012) Staphylococci

S. aureus (no.) (155) (46,773) (1,000) 0.12/0.25 (45) 0.06/0.06 (1) 0.06/0.06 (<1) CoNS (no.) (67) (12,308) (122) 0.12/0.25 (30) ≤0.03/0.06 (1) 0.06/0.06 (<1)

a. From Jones et. al. J. Chemother. 2001; 13: 244-254, and Biedenbach et. al. Antimicrob. Agents Chemother. 2009; 53: 1260-1263, and Jones et. al. Diagn. Microbiol. Inf. Dis. 2013; 76: 122-124.

A4 Jones ‐ Susceptibility testing of new and revived antimicrobial agents

FIG 1. Influence of various P-80 concentrations (0.000002 to 2%) on the dalbavancin MIC results for S. aureus (11 strains) and beta-hemolytic streptococci (4 strains)

*From Rennie et. al. J. Clin. Microbiol. 2007; 45: 3151‐3154 A5

Dry-form MIC Panel Validation (Sensititre®)*

*From Jones et. al. Int. J. Antimicrob. Agents 2004; 23:197‐199.

Surrogate Testing (2006)

99.8% predictive

A7 *From Jones et. al. J. Clin. Microbiol. 2006; 44: 2622‐2625. Jones ‐ Susceptibility testing of new and revived antimicrobial agents

Surrogate Testing

Figure 1. S. aureus (33,688 strains) isolated from the USA and Europe in 2011-2013a.

4 2 (µg/ml)

1 MIC 0.5 4 1

0.25 1 1 32 10 0.12 2 195 2895 119

Dalbavancin 0.06 5 3193 17946 325 0.03 1 36 2888 6000 34 0.120.250.5124816 Vancomycin MIC (µg/ml)

a. Broken horizontal line shows USA-FDA breakpoint (≤0.12 µg/ml) having acceptable predictive values (99.9%); and the solid vertical line is the vancomycin breakpoint.

*From Jones et. al. (in press) A8

Surrogate Testing

Table 2. Summary of vancomycin test result accuracy for predicting dalbavancin susceptibility using two breakpoint concentrations (≤0.12 and ≤0.25 µg/ml) when tested against eight Gram-positive pathogen/groups in 2011-2013.

Surrogate accuracy for breakpoint at: Pathogen or species group (no. tested) ≤0.12 ≤0.25

S. aureus (33,688) 99.86a 99.99

β‐haemolytic streptococci (5,722) 98.97 100.00 S. pyogenes (2,297) 100.00a 100.00 S. agalactiae (2,495) 97.72a 100.00

Viridans group streptococci (2,800) 100.00 100.00 S. anginosus group (758) 100.00a 100.00

CoNS (4,576) 97.55 99.87

Enterococci (6,515) 98.43 99.94

a. Underlined percentage shows USA-FDA approved breakpoint for clinical use versus indicated species/groups. A9 *From Jones et. al. (in press)

Oritavancin MIC results (across 14 years)

Organism/Group Oritavancin MIC90 values (g/mL) by reference Nicas et al. Schwalbe et al. Jones et al. Biavasco et al. Zeckel et al. Noviello et al. Arhin et al. Resistant subsets (1996) (1996) (1996) (1997) (2000) (2001) (2009) S. aureus Methicillin-susceptible 0.5 − 24 240.12 Methicillin-resistant 0.5 1 4 4 2 4 0.12 CoNS Methicillin-susceptible −−44 2− 0.25 Methicillin-resistant 0.5 1 8 4 2 − 0.25 S. haemolyticus − 22 4 −−0.12 E. faecalis Vancomycin-susceptible − 12 − 110.06 Vancomycin-resistant − 22 −−−1 E. faecium Vancomycin-susceptible − 0.5 0.25 − 0.5 0.25 0.03 Vancomycin-resistant − 14 − 220.25 S. pneumoniae Penicillin-susceptible −−≤0.015 ≤0.03 0.008 ≤0.03 0.004 Penicillin-resistant −−0.06 ≤0.03 0.015 ≤0.03 0.008 β-hemolytic streptococci Group A −−0.5 0.25 −−0.25 Group B −−0.25 −−−0.25 Group C 1 −− −−- Group G 1 −− −−- Nicas et al., AAC, 1996 Schwalbe et al., AAC, 1996 Zeckelet al., AAC, 2000 A10 Jones et al., AAC, 1996 Noviello et al., JAC, 2001 Biavasco et al., AAC, 1997 Arhin et al., AAC, 2009 Jones ‐ Susceptibility testing of new and revived antimicrobial agents

A11 *From Arhin et. al. Agents Chemo. 2008; 52: 1597-1603

Oritavancin activity as tested against staphylococcal species/groups and resistant subsets submitted to the International Oritavancin Resistance Surveillance follow application of P-80 (first three years)

Organism / MIC (g/ml) Cumulative % of isolates inhibited at each MIC (g/ml) Year/Group (no tested) 50% 90% ≤0.008 0.015 0.03 0.06 0.12 0.25 S. aureus 2008 All (6,664) 0.03 0.06 1.8 21.5 77.1 97.5 99.8 99.9

2009 All (4,522) 0.015 0.03 7.0 52.5 92.4 99.2 99.9 100.0

2010 All (5,438) 0.03 0.06 4.3 42.2 85.2 96.9 99.5 100.0 OXA-S (3,269) 0.03 0.06 4.5 41.1 84.8 96.9 99.4 100.0 VAN MIC, ≤1 g/ml (3,248) 0.03 0.06 4.6 41.1 84.9 96.9 99.4 100.0 VAN MIC, 2 g/ml (21) 0.03 0.06 0.0 42.9 76.2 100.0 OXA-R (2,169) 0.03 0.06 3.9 43.9 85.8 96.8 99.6 100.0 VAN MIC, ≤1 g/ml (2,136) 0.03 0.06 4.0 44.5 86.5 97.2 99.7 100.0 VAN MIC, 2 g/ml (33) 0.06 0.12 0.0 3.0 39.4 72.7 93.9 100.0

A12

Telavancin AST History

• No P‐80 supplement in microdilution broth until 2014 • Testing re‐evaluated post USA‐FDA and EUCAST/EMA approvals • Re‐evaluated QC guidelines, testing detail and breakpoints are now published (EUCAST/EMA, USA‐FDA) with P‐80 supplemented broth • Potency of telavancin like those of other lipoglycopeptides (S. aureus MIC 50/90 at 0.06 µg/mL)

A13 Jones ‐ Susceptibility testing of new and revived antimicrobial agents

Conclusions for Lipoglycopeptide AST

• Requires modified reference MIC tests – Broth microdilution with 0.002% polysorbate‐80 • Agar dilution and diffusion methods are compromised • Limited availability of commercial methods, especially automated systems – Surrogate testing strategies have been suggested

A14

Examples of Recent AST Development and Breakpoint Issues

• Lipoglycopeptide development, a long painful history – Dalbavancin* – Oritavancin* – Telavancin* • Oxazolidinones – Linezolid – Tedizolid* • Combination drugs – Ceftazidime‐Avibactam (CAZ‐AVI)* – Ceftolozane‐Tazobactam (TOL‐TAZ)* • Polymyxins among other agents – Colistin and Polymyxin B

*Six new drug breakpoints evaluated in the last year

Endpoint Reading Exceptions for Linezolid

B2 *From CLSI M07-A10 2015 Jones ‐ Susceptibility testing of new and revived antimicrobial agents

B3 *From CLSI M07-A10 2015

Summary of non-susceptibility rates documented for linezolid when tested against a total of 52,082 clinical isolates included in the 9 year ZAAPS Program (2004-2012)

B4 *From Mendes et. al. J. Antimicrob. Chemo. 2014; 69: 1582-1588

Activity of Tedizolid and Linezolid against Pathogens with Important Resistant Phenotypes in the 2011- 2012 STAR Program

Cum. percentage of isolates inhibited at MIC

Organism (N) Antimicrobial MIC50 MIC90 (µg/mL) of: ≤0.25 0.5 1 2 4 MRSA (1770) Tedizolid 0.25 0.5 65.9 99.7 99.8 99.9 100.0 Linezolid 2 2 0.2 0.5 31.3 97.0 99.7 MSSA (2729) Tedizolid 0.25 0.5 66.0 100.0 ‐‐‐ Linezolid 2 2 0.2 0.5 25.3 94.5 99.7 VRE (163) Tedizolid 0.25 0.5 62.6 98.8 99.4 100.0 ‐ Linezolid 2 2 0.0 1.2 41.7 98.8 99.4 VSE (705) Tedizolid 0.25 0.5 87.4 100.0 ‐‐‐ Linezolid 1 2 0.6 6.5 58.4 99.7 100.0

B5 *From Sahm et. al. Diagn. Microb. Inf. Dis. 2015; 81: 112-118 Jones ‐ Susceptibility testing of new and revived antimicrobial agents

Direct comparison of Thermo Fisher tedizolid MIC testing device (Sensititre®) results to that of reference frozen-form panel values (285 strains)

Variation in log2 dilution steps: Organism (no. tested) ‐2 ‐1 Exact +1 +2 % Essential Agreement

S. aureus (110) 0 6 89 15 0 100.0 CoNS (20) 0 1 16 3 0 100.0 Enterococci (40) 0 0 28 12 0 100.0 S. pneumoniae (30) 0 2 26 2 0 100.0 β‐hemolytic streptococci (70) 0 21 47 2 0 100.0 S. anginosus group (15) 0 5 7 3 0 100.0

All strains (285) 0 35 213 37 0 100.0

B6 *From Jones et. al. J. Clin. Microbiol. 2015; 53: 657-659

Conclusions for Oxazolidinone AST

• Class having potent Gram‐positive spectrum but not considered as cidal agents • Tedizolid QC range for S. aureus spans the susceptible breakpoint • Some breakpoint organizations and regulator‐imposed post‐marketing global surveillance programs apply modified endpoint interpretations (80% inhibition, disregard trailing growth) • Correlate disk diffusion and commercial methods/systems may be delayed and surrogate strategies could be an option (data pending)

Examples of Recent AST Development and Breakpoint Issues

*Six new drug breakpoints evaluated in the last year

C1 Jones ‐ Susceptibility testing of new and revived antimicrobial agents

Combination Drug Testing Strategies

• Active agent tested on a log2 dilution schedule with a fixed concentration of the inhibitor for reference MIC method • Active agent and a validated amount of inhibitor in disks for diffusion method – Active drug traditional disk loading for geographic region (30‐µg/ml or 10‐µg/ml) adjusted to PK‐PD breakpoint – Inhibitor concentration generally approximating to dosing ratio at 2 or 3: 1 (30‐20, 30‐10, 10‐6 µg proposed) • Correlative accuracy between breakpoints for the methods shall be predictably acceptable because of the long history of cephalosporin AST methods

50% fT > MIC for Ceftazidime and 50% f T > CT of 1 mg/L Avibactam Following IV Administration of 2.5g CAZ-AVI q8h (2 hour infusion) Overlaid on a Histogram of MIC Distributions for Enterobacteriaceae collected during 2012 INFORM

C3 *From USA‐FDA AIDAC Presentation, 2014

Figure 1. Cumulative percentage activity curves for CAZ-AVI and four comparison agents tested against 9,261 Enterobacteriaceae from USA SENTRY Program (2013)

100 98.2% 99.9% susceptible to CAZ‐AVI* 92.5% 90 89.7% 92.2%

60 Piperacillin‐tazobactam 50 Ceftazidime inhibited Cefepime % 40 Meropenem 30 CAZ‐AVI

0 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 MIC (µg/ml)

C4 *From INFORM Program Jones ‐ Susceptibility testing of new and revived antimicrobial agents

Example of Ceftolozane-Tazobactam Activity against P. aeruginosa (USA, 2011-2012)a

Cum. % inhibited at MIC (mg/L): Organism subsets (no. tested) ≤124 816

All strains (1971) 82.6 90.4 96.1b 98.5 99.0 MDR (310) 29.0 55.2 79.0 90.3 93.5 XDR (175) 17.1 45.7 70.9 85.7 90.3 MEM-NS (388) 53.1 71.6 85.3 92.8 95.1

a. From Farrell et al. Antimicrob. Agents Chemother. 57: 6305-6310 (2013) b. USA-FDA approved breakpoint, Enterobacteriaceae coverage = 93.3% at ≤ 2 mg/L (7,071 strains)

Conclusions for β-lactamase Inhibitor Combination AST

• In vitro methods for ceftazidime‐avibactam and ceftolozane‐tazobactam appear sound for MIC reference and disk diffusion testing • Recommended breakpoints will be consistent with PK‐PD validated high‐dose regimens for a limited number of indications • Expanded spectrums appear very broad against Gram‐negative pathogens, many with MDR profiles

C7 Jones ‐ Susceptibility testing of new and revived antimicrobial agents

Examples of Recent AST Development and Breakpoint Issues

*Six new drug breakpoints evaluated in the last year D1

Correlation between polymyxin B and colistin MIC values when testing 15,377 Gram-negative bacilli collected worldwide in 2013

Scattergram showing polymyxin B MIC results obtained with versus without the addition of P-80 to the MHB (all species: N=247)

D3 *From Sader et. al. Diagn. Microb. Inf. Dis. 2012; 74: 412-414 Jones ‐ Susceptibility testing of new and revived antimicrobial agents

Re-evaluation of Polymyxin AST: Report of the Joint EUCAST/CLSI ad hoc Polymyxin Breakpoint Working Group

• Agents stick to plastics via an electrostatic interaction (concentration dependent) • PK‐PD data is limited for polymyxin B • Polymyxin components may differ in potency by pathogen (debated) • Colistin breakpoints will appear in late 2015 • Potencies of colistin and polymyxin B are “not quite” comparable • AST methods – Agar dilution and disk/gradient diffusion are suboptimal – Broth microdilution is reference test, using polystrnene trays not P‐80 • Attend lecture by Professor Turnidge on other revived drugs in Hall B (5:00PM)

* Minutes of CLSI Subcommittee, 2015 D4

Stable-Functional Components of EUCAST System EMA/CHMP “Ultimate regulatory approval function for antimicrobial agents including breakpoints”

EUCAST* ECDC “Authority to develop and “Support function” suggest AST breakpoints”

Has regulated process for interactions with drug sponsors ESCMID and agencies (SOP/H/3043) “Support function”

*recommends/endorses methods of choice available worldwide (ISO, CLSI etc), and modifies per need of EU region and member NACs; method listed in regulatory documents E1

Assuming the Development Objectives are Achieved, Do We Still Have a Problem? • “YES! Getting the breakpoints to the end‐user to favorably influence patient care. Improve implementation of breakpoint updates found in the EUCAST documents via diagnostic systems”

• “Delays of greater than one year would clearly be excessive, if the breakpoint determining process was effective.” ‐R.N. Jones (2015)