Educational Workshop. EW08: Antimicrobial susceptibility testing practical implementation of the EUCAST breakpoints and methods

Transcription

1 Educational Workshop EW08: Antimicrobial susceptibility testing practical implementation of the EUCAST breakpoints and methods arranged with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) Convenors: Gunnar Kahlmeter (Växjö, SE) Derek Brown (Peterborough, UK) Faculty: Gunnar Kahlmeter (Växjö, SE) Derek Brown (Peterborough, UK) Rafael Canton (Madrid, ES) Fred Tenover (Sunnyvale, US) Roland Leclercq (Caen, FR)

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3 Kahlmeter - EUCAST Clinical Breakpoints EUCAST Clinical Breakpoints Gunnar Kahlmeter ECCMID 2010 Q 1 Tasks 1. Determine clinical breakpoints for existing and new antibacterials, antifungals, antimycobacterials. 2. Define wild type MIC distributions and epidemiological cut-off values for bacteria and fungi. 3. Develop susceptibility testing methods and systems for internal QC. 4. Liaise with EMEA, ECDC, EFSA, EARSS and others involved in antimicrobial resistance. 5. Liaise with national committees involved in antimicrobial resistance and susceptibility testing, to facilitate implementation of European breakpoints 3

4 Kahlmeter - EUCAST Clinical Breakpoints Q 2 Why European breakpoints in Europe? EUCAST breakpoints based on EMEA approved indications and outcome evaluation, Pk/Pd, multiple MIC distributions, and modern principles of determining breakpoints related to European minimum and maximum dosages accepted by European regulatory authorities (EMEA, ECDC) official breakpoints in European SPCs (EMEA) case definitions for antimicrobial resistance surveillance (ECDC) transparant and published rationale behind decisions independent of commercial interests reviewed at intervals: with new member of class or on initiative of the profession, EMEA, the Company, EUCAST. in the public domain and free of charge National Breakpoint Committees D, F, N, NL, S, UK, EUCAST General Committee All European Countries + ISC/FESCI EUCAST Steering Committee BSAC, CA SFM, CRG, DIN, NWGA, SRGA And 2 reps from the General Committee Subcommittees Antifungals Anaerobes Expert Rules Expert groups 4

5 Kahlmeter - EUCAST Clinical Breakpoints Steering Committee and General Committee General Committee Each European country invited to appoint 1 representative each ISC and FESCI 1 rep each One meeting per year Steering Committee (11 members) Chairman, Scientific Secretary and Clinical Data Coordinator appointed by ESCMID Each national breakpoint committee 1 rep General Committee 2 reps 5 meetings per year Decision process EUCAST Steering Committee National breakpoint committees EUCAST General Committees and Expert groups, Industry (pharmaceutical + AST) EUCAST Steering Committee in agreement with National breakpoint Committees New consultation (?) Decision EUCAST Subcommittees Antifungals Antifungal drugs in need of breakpoints Breakpoints and rationale documents Methods Anaerobe bacteria Antibiotics in need of breakpoints Breakpoints Methods Expert Rules Tables of intrinsic resistance Expert rules (IF/THEN) 5

6 Kahlmeter - EUCAST Clinical Breakpoints Consultation with expert groups Neisseria spp (finalised) Anaerobes (ESGARAB, ongoing) Helicobacter pylori (EHSG, ongoing) Clostridium difficile (ESGCD, ongoing) Campylobacter (VetCast, ongoing) Listeria monocytogenes EUCAST Website requires no login 6

7 Kahlmeter - EUCAST Clinical Breakpoints Q 3 Tools for determining CLINICAL BREAKPOINTS 1. Dose or doses 2. Target organisms 3. MIC-distributions for target organisms - breakpoints not to divide MIC-distributions of WT target organisms - MIC distribution and ECOFFs determined for each species 4. Resistance mechanisms in target organisms 5. Clinical indications 6. Pharmacokinetics (Cmax, AUC, T½, Protein binding, Vd..) 7. Pharmacodynamic properties (peak conc/mic, AUC/MIC, TA, MCs) 8. Clinical outcome (clinical outcome/mic) 9. Epidemiological cutoffs, Pk/Pd-breakpoints and clinical data together determine the CLINICAL BREAKPOINT Wild type Susceptible Clinical resistance Clinical resistance Clinical resistance Clinical resistance Phenotypically detectable resistance Genetically detectable resistance Resistant 7

8 Kahlmeter - EUCAST Clinical Breakpoints EUCAST and existing antimicrobials Aminoglycosides Carbapenems & aztreonam Cephalosporins iv Cephalosporins oral Fluoroquinolones Glycopetides Macrolides and lincosamides Penicillins Tetracyclines Miscellaneous antimicrobials Antifungal drugs (flu- and voriconzole) EUCAST breakpoints for new drugs through European Medicines Agency (EMEA) Daptomycin Tigecycline Doripenem New cephalosporin (ongoing) Glycopeptides (ongoing) Extensions of indications (none ongoing) Topicals and less commonly used drugs 1. Mupirocin (Topical) 2. Polymyxin B (Topical) 3. Bacitracin (Topical) 4. Streptomycin (hlr for enterococci) 5. Neomycin (Topical) 6. Sulfamethoxazole (UTI) 7. Cephalothin (expert rules?) 8. Sulfadiazine 9. Spiramycin 10.Nalidixic acid (screening) 11.Cefoperazone 12.Pefloxacin 13.Cefradine 14.Cefamandole 15.Sulfisoxazole 16.Pipemidic acid 17.Kanamycin 18.Ceftizoxime 19.Cefprozil + 45 others 8

9 Kahlmeter - EUCAST Clinical Breakpoints Microorganisms without breakpoints Define relevant drugs, breakpoints, methodology and MIC-distributions. Helicobacter spp Campylobacter spp Clostridium difficile Legionella spp Pasteurella multocida Listeria monocytogenes EUCAST breakpoint tables MIC (mg/l) brpts* S 2 R>2 Zone (mm) brpts* S 22 R<22 Insufficient evidence IE (Literature: not enough evidence for a breakpoint or no indication ) Inappropriate drug (Literature: poor drug don t use! Numbered footnotes Lettered footnotes Can not be substituted. Can be supplemented with an MIC without t interpretation. Can be substituted with an automatic R. MIC-breakpoints Zone diameter breakpoints *when numbers are the same = no intermediate category Web-links to MIC-distributions Web-links to Zone diameter distributions Web-links to EUCAST Rationale Documents 9

10 Kahlmeter - EUCAST Clinical Breakpoints The MIC is the reference for all other phenotypic tests The MIC is the reference for all other phenotypic tests The MIC is the reference for all other phenotypic tests ates No of isola E coli /Mecillinam 10 ug 969 isolates, of which 930 consecutive Inhibition zone diameter (mm) Breakpoints S R MIC 8 >8 Zone diameter 15 <

11 Kahlmeter - EUCAST Clinical Breakpoints Q 4 On a national level: National strategies and joint decisions on AST are needed! NAC National Antimicrobial Committee National Antimicrobial Committees tasks Subcommittee on Antimicrobial susceptibility testing Strategy at national level Implementation of breakpoints and methods Education Liaison and consultation with EUCAST Liaison with groups involved in AMR-surveillance (ECDC, EARSS,.). QA Antimicrobial Policies Antimicrobial Resistance Surveillance Antimicrobial Consumption and Policies 11

12 Kahlmeter - EUCAST Clinical Breakpoints EUCAST breakpoints Decisions for 2010/11: Sweden Denmark Norway Belgium The Netherlands Austria Estonia Ireland Finland Scotland Wales Switzerland Discussions ongoing: Spain Greece Italy Turkey Israel ENAC European Network of Antimicrobial Committees The EUCAST General Committee. EUCAST April 2010 Harmonised breakpoints for all major antibacterial and antifungal drugs. Orphan drugs and microorganisms identified and prioritized Breakpoints for new drugs as part of the approval process with EMEA (daptomycin, tigecycline, doripenem). Epidemiological cut off values determined for all drugs. SOPs to describe formal relationship with EMEA. EUCAST breakpoints mandatory in European SPCs. ISO-standardized MIC-determination. Software and database for MIC- and zone distributions. Breakpoints implemented in national (F, D, N, NL, S, UK) systems EUCAST disk diffusion test launched Breakpoint tables, QC-tables, methodology documents available on website. 12

13 Kahlmeter - EUCAST Clinical Breakpoints EUCAST April 2011 EUCAST disk diffusion method implemented in 5-6 countries NACs in countries. National Educational Workshops on European AST in several countries. EUCAST breakpoints in all major systems for AST (BSAC, CA-SFM; Commercial systems Phoenix, Vitek2, Microscan, BioMic). All Rational Documents available on website. SOPs to describe formal relationship with ECDC. ECDC decided on European breakpoints as mandatory in surveillance of antimicrobial resistance and HCAI. Breakpoints and methods for Campylobacter, Helicobacter, C.difficile, and others. Breakpoints and methods several topical antimicrobials and several less commonly used drugs. Formal decision on the future relationship between EUCAST, ECDC, EMEA and ESCMID. Q5 Thank you! 13

14 Brown - Implementation of the EUCAST disk-diffusion method Implementation of the EUCAST disk diffusion method Derek Brown EUCAST Scientific Secretary Question1 EUCAST disc diffusion method Why develop a EUCAST disk diffusion test? Development of the method Calibration of the method Implementation in individual laboratories 14

15 Brown - Implementation of the EUCAST disk-diffusion method EUCAST early 2007 No plans for EUCAST disk diffusion test MICs can be interpreted with EUCAST breakpoints Automated systems can be calibrated to EUCAST breakpoints Disk diffusion i methods calibrated to EUCAST breakpoints are available in France, Sweden and the UK....little enthusiasm for a new EUCAST disk diffusion method EUCAST consultation and questionnaire France and UK likely to maintain their national methods, calibrated to EUCAST breakpoints, for the immediate future. Other countries were not keen to take on other national methods with unfamiliar technique. Most countries were currently using Kirby-Bauer type methods (particularly CLSI ) and, if they adopted EUCAST breakpoints, would prefer to retain the same methodology calibrated to EUCAST breakpoints (...but get rid of HTM) Benefits of a EUCAST disk diffusion method (based on the Kirby-Bauer approach) Identified as EUCAST method calibrated to EUCAST MIC breakpoints Wide base of expertise throughout Europe (and worldwide) KB technique familiar to most laboratories in countries without their own method Extensive database of MIC v Zone diameters available for KB method? 15

16 Brown - Implementation of the EUCAST disk-diffusion method EUCAST decision to develop a disk diffusion method June 2008 Based on KB approach MH medium MH-F for fastidious organisms 0.5 MF inoculum 16-20h incubation Most disk contents same Most control strains same Calibrated to EUCAST MIC breakpoints Mueller-Hinton-fastidious (MH-F) Mueller-Hinton agar + 5% defibrinated horse blood and 20 mg/l β-nad S. pneumoniae ATCC H. influenzae NCTC

17 Brown - Implementation of the EUCAST disk-diffusion method EUCAST QC tables Targets NOT in italics are from ISO and/or CLSI recommendations Question 2 17

18 Brown - Implementation of the EUCAST disk-diffusion method Calibration of EUCAST disk diffusion method Existing distributions of MIC v zone diameter with the same technique New distributions of MIC v zone diameter Zone diameter distributions for routine isolates Targeting critical areas of the MIC and zone diameter distributions Targeting specific resistances MIC (μg/ml) Ceftriaxone Figure 7: Ceftriaxone MIC v zone diameter for ceftriaxone with n=350 Enterobacteriaceae y= x >= r= <= Ceftriaxone Zone Diameter (mm) 18

19 Brown - Implementation of the EUCAST disk-diffusion method Zone diameter correlates 19

20 Brown - Implementation of the EUCAST disk-diffusion method Development of EUCAST disk diffusion method Tentative breakpoints have been published for all agents with MIC breakpoints Continuing validation through Existing and new distributions of MIC v zone diameter Targeting critical areas of MIC v zone diameter distributions and specific resistances Zone diameter distributions for routine isolates Ongoing maintenance Implementation EUCAST action Documentation Method description Breakpoints QC limits Education Teaching material (slideshow) Meetings Practical workshops Laboratory visits The laboratory in Vaxjo is an ESCMID Collaborative Centre the ESCMID Observership program could be used for visit. Publications/website Inform media, disk, zone reader manufacturers Implementation at national level National antimicrobial committees Strategy at national level for implementation of breakpoints and methods Inform people of implications for their laboratory/country Education through meetings, publications, websites, practical workshops Liaison with media, disk, zone-reader manufacturers Liaison and consultation with EUCAST. Liaison with groups involved in AMR-surveillance External Quality assessment Staged introduction may be appropriate eg. large labs first 20

21 Brown - Implementation of the EUCAST disk-diffusion method Implementation at local level Before routine use Ensure all stakeholders are informed of implications for their laboratory/hospital Appoint a champion to implement the method Visit laboratories using the method Plan well in advance media, disk, supplies templates, zone-reader setup computing (breakpoints/qc ranges) documentation Train staff in advance demonstrations, practical experience, ensure that QC requirements are met Use contacts in other laboratories and at EUCAST directly or through NACs or national EUCAST GC rep Implementation at local level During introduction Ensure that adequate staffing is available will take more time at first Ensure that senior staff are available to answer questions and deal with problems Use national contacts National Antibiotic Committees Use contacts in laboratories already using the method Use EUCAST contacts Erika Matuschek (Swedish External Reference Laboratory for Antimicrobial Susceptibility Testing; SERLAST ) erika.matuschek@ltkronoberg.se Gunnar Kahlmeter (EUCAST and SERLAST) Derek Brown (EUCAST) Implementation at local level After implementation Keep the method up-to-date Continue to educate staff Report problems to EUCAST Erika Matuschek (SERLAST) Be prepared to help other laboratories 21

22 Brown - Implementation of the EUCAST disk-diffusion method Acknowledgements Gunnar Kahlmeter Erika Matuschek and Jenny Åhman EUCAST Steering Committee National Committees Collaborating laboratories Individual experts 22

23 Cantón - Implementation of EUCAST breakpoints with automated systems Implementation of the EUCAST breakpoints with automatic systems Antimicrobial susceptibility testing practical implementation of the EUCAST breakpoints and methods 20 th ECCMID, Vienna, Austria, April, 2010 Dr. Rafael Cantón Hospital Universitario Ramón y Cajal SERVICIO DE MICROBIOLOGÍA Y PARASITOLOGÍA Methods for antimicrobial susceptibility testing Phenotypic test methods: based on antimicrobial activity and breakpoints - MIC determination (broth, agar, gradient diffusion) - Disk diffusion (BSAC, CA-SFM, CLSI, SRGA, EUCAST...) - Automated systems (Vitek, Phoenix, MicroScans, Sensititre,...) Genotypic test methods: based on the detection of a resistance gene or its product - meca, vana, vanb - PBP2a, β-lactamase detection By deduction expert rules - If meca-positive, then report beta-lactam antibiotics as R - If erythromycin-r, then report azithro- and clarithromycin as R Antimicrobial susceptibility automatic systems Main objectives To produce antimicrobial susceptibility testing (AST) results in a automated (or semi-automated) mode To standardize AST avoiding uncontrolled differences To offer AST in a shorter period of time than manual methods To interpret AST results (clinical categorization / interpretation) 23

24 Cantón - Implementation of EUCAST breakpoints with automated systems MIC based automatic systems Antimicrobial susceptibility automatic systems None of the current automatic susceptibility testing devices can be considered fully automated - Automated system consist of devices with computer-assisted incubation, reading, interpretation and reporting functions - Semi-automated systems require off-line incubation*. The panels are automatically read with computer-assisted interpretation and reporting *manual loading of each panel into the system is required - Manual systems use commercial (eventually in-house) panels that are read by laboratory personnel. Results are either recorded by hand or manually entered into a computer for interpretation and reporting All instruments have implemented computer programs Antimicrobial susceptibility automatic systems Most automatic susceptibility testing devices have incorporated Interface connections with laboratory information systems (LIS) Quality control computer programs Computer programs or expert systems: - to interpret phenotypes and infer resistance phenotypes antibiogram interpretive reading - to perform actions based in clinical evidences and resistance mechanisms knowledge in response to specific antimicrobial susceptibility test results expert rules Programs to manage results for epidemiological purpose 24

25 Cantón - Implementation of EUCAST breakpoints with automated systems Antimicrobial susceptibility automatic systems Classification MIC based systems - agar dilution (no longer exists!) - microdilution: MicroScan, Sensititre, Phoenix, - growth curves: VITEK legacy, VITEK2 Disc diffusion based systems - BIOMIC System - SIRSCAN System - OSIRIS System Antimicrobial susceptibility automatic systems Sensititre ARIS System (Trek Diagnostic Systems) Standard 96-well microdilution panels with antibiotics/panel Manual or semi-automatic inoculation of panels Fluorescent measurement e e of bacterial a growth after generating a fluorescent product from a non-fluorescent substrate - Susceptibility testing results in h Data manager system and expert software rules Antimicrobial susceptibility automatic systems MicroScan WalkAway (Siemens Healthcare Diagnostics) Standard size 96-well microdilution panels with antibiotics with or without substrates for bacterial identification Panels are manually inoculated Turbidimetric reading (overnight panels) - susceptibility testing results in h Fluorimetric reading (rapid panels with a fluorometric substrate) - susceptibility testing results in h Data manager system and expert software rules 25

26 Cantón - Implementation of EUCAST breakpoints with automated systems Antimicrobial susceptibility automatic systems Vitek 2 (BioMérieux) Separate plastic cards for ID and AST - AST: 64-well plastic card with agents (deduce additional agents with computer software) Optical reading every 15 minutes with a multichannel fluorometer and photometer to record fluorescence and turbidity Changes in fluorescence over time (growth curves) comparing a growth control well with wells with drug concentrations Susceptibility results in 4-18 h Expert system (Advanced Expert System, AES) based in MIC and phenotype analysis Antimicrobial susceptibility automatic systems Phoenix System (BD Diagnostic Systems) Combination panels for bacterial ID and AST AST: 84-wells (18-25 antimicrobial agents) Manually or semi-automatic inoculation of panels Monitor reading every 20 minutes with a turbidometric and colorimetric (oxidation-reduction indicator) growth detection Changes in fluorescence over time (growth curve), comparing a growth control well with wells with various drug concentrations Susceptibility results in 4-16 h Expert system (Advance expert System) Antimicrobial susceptibility automatic systems Wider System (Fco. Soria Melguizo) Standard size 96-well microdilution panels with antibiotics with or without substrates for bacterial identification (panels are prepared by MicroScan) Panels manually inoculated and externally incubated Panels are manually introduced in a video image reader after h of incubation Expert software rules 26

27 Cantón - Implementation of EUCAST breakpoints with automated systems Antimicrobial susceptibility automatic systems Disc diffusion based systems Video assisted reader plate to read and interpret disc diffusion susceptibility agar plates Quantitative zone diameters are calculated by digitalization and software interpreted (SIR) with deduction of MIC Expert software rules Antimicrobial susceptibility automatic systems MIC based systems Device Inoculation Reading Sensititre Manual or semiautomatic Manual read or Fluorescence Reporting time (hours) MicroScan Manual Turbidity and fluorometer Phoenix Manual or Semiautomatic Turbidity and colorimetric 4-16 Vitek2 Semiautomatic Fluorometer, photometer 4-18 These system fulfill FDA and ISO accuracy performance Data obtained from Evangelista & Truant. In: Commercial methods in Clinical Microbiology Antimicrobial susceptibility automatic systems Acceptable performance for the clinical data for automatic AST devices with reference method (FDA) Essential agreement (± 1 dilution): >89.9% Category agreement (interpretive results, SIR) >89.9% Major discrepancies (false resistance): 3%* *based on the no. of susceptible organisms tested Very major discrepancies (false susceptibility): 1.5%** **based on the no. of resistant organisms tested Growth failure rates: < 10%*** ***for any genus or species tested Antimicrobial Susceptibility Test (AST) Systems. Guidance for Industry and FDA Class II Special Controls Guidance Document:, August 28,

28 Cantón - Implementation of EUCAST breakpoints with automated systems Antimicrobial susceptibility automatic systems Accuracy of automatic AST devices (ISO :2007) Data shall be analyzed by using the appropriate interpretive breakpoints Essential agreement (± 1 dilution): 90% Category agreement (interpretive results, SIR) 90% Major discrepancies (false resistance): 3%* *based on the no. of susceptible organisms tested Very major discrepancies (false susceptibility): 3%** **based on the no. of resistant organisms tested Reproducibility (± 1 dilution and/or SIR results): 95% ***for any genus or species tested Clinical laboratory testing and in vitro diagnostic test systems - Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices - Part 2: Evaluation of performance of antimicrobial susceptibility test devices. International Standard ISO :2007, ISO, Geneva. Antimicrobial susceptibility automatic systems most evaluations of automatic AST systems have been performed with CLSI (NCCLS) breakpoints, but... Automatic systems currently available in Europe are incorporating the EUCAST breakpoints Different systems advertise that they operate with EUCAST breakpoints but evaluations have not yet been performed for all: - MIC based systems: Phoenix Vitek 2 MicroScan - Disc diffusion based systems: BioMIC Antimicrobial susceptibility automatic systems Issues with EUCAST breakpoint implementation Lower ranges of concentrations are needed (EUCAST breakpoints are mostly lower than CLSI) - instability of certain antibiotics might affect accuracy (essential and categorical agreements) - carbapenems, β-lactam-β-lactamase inhibitor combinations, - major discrepancies (false resistance) could be observed, particularly with isolates expressing low level resistance mechanisms 28

29 Cantón - Implementation of EUCAST breakpoints with automated systems Antimicrobial susceptibility automatic systems Issues with EUCAST breakpoint implementation S and R breakpoints can be... - too close (essential and categorical agreements can be affected) e.g. ciprofloxacin and enterobacteriaceae (S 0.5 / R >1) vancomycin and staphylococci (S 2/R >2) - too separate (a wider concentration range is needed in the panel) e.g. aztreonam and P. aeruginosa (S 1 / R >16)* *The R breakpoint was increased from 8 to 16 mg/l to avoid dividing the wild type MIC distribution. The R breakpoint relates to high dose therapy. The S breakpoint is set to ensure that wild type isolates are reported I. Antimicrobial susceptibility automatic systems Issues with EUCAST breakpoint implementation A desirable attribute to include drug concentrations equal to ECOFFs* allowing detection of wild type organisms (no-r mechanism) *epidemiological cut off values A philosophical and technical change breakpoints are interpreted and expressed differently S R EUCAST > CLSI Low level resistance or decreased susceptibility ECOFF S R High level resistance 29

30 Cantón - Implementation of EUCAST breakpoints with automated systems S R Antimicrobial susceptibility automatic systems An example... assessment of the Phoenix system & EUCAST breakpoints Centre A* (393 isolates) Centre B** (362 isolates) Categorical agreement 96.0% 99.1% Interpretive discrepancies - minor discrepancies 2.4% 2.8% - Major discrepancies 1.2% 0.8% - Very major discrepancies 1.1% 1.3% *Morosini, García-Castillo, Cantón. Ramón y Cajal University Hospital. Madrid (Spain) **Giani, Conte, D Andrea, Rossoloni. University of Siena (Italy) Posters presented at 20th ECCMID, 2010 Antimicrobial susceptibility automatic systems Issues with EUCAST breakpoint implementation EUCAST expert rules must be implemented with EUCAST breakpoints and not with CLSI breakpoints! For 3 rd /4 th gen. cephalosporins and Enterobacteriaceae test results should be reported as found irrespective of ESBL production CLSI (2010) EUCAST (2010) S R S R Cefotaxime 1 4 = 1 >2 Ceftriaxone 1 4 = 1 >2 Ceftazidime >4 Cefepime >4 30

31 Cantón - Implementation of EUCAST breakpoints with automated systems Antimicrobial susceptibility automatic systems Antimicrobial susceptibility automatic systems with EUCAST breakpoints are being implemented and introduced in Europe EUCAST breakpoint implementation does not represent any fundamental problem Initial evaluations of Phoenix system fulfil ISO requirements (ISO :2007) 2:2007) Other manufacturers have been slow to accept the need for recalibrating their systems to ranges needed for EUCAST breakpoints. The delay caused by any change (new antibiotic or a new breakpoint) is a source of major concerns with the automated systems. Automation and EUCAST: join it! Implementation of the EUCAST breakpoints with automatic systems Antimicrobial susceptibility testing practical implementation of the EUCAST breakpoints and methods 20 th ECCMID, Vienna, Austria, April, 2010 Dr. Rafael Cantón Hospital Universitario Ramón y Cajal SERVICIO DE MICROBIOLOGÍA Y PARASITOLOGÍA 31

32 Tenover - Supplementary tests when routine methods are not enough Supplementary Tests- When Routine Methods are Not Enough Fred C. Tenover, Ph.D., (D)ABMM Senior Director for Scientific Affairs Cepheid Consulting Professor of Pathology Stanford University Adjunct Professor of Epidemiology Rollins School of Public Heath Emory University Supplementary AST Testing Frustrating but important aspect of susceptibility testing. Includes: Optimizing results to insure therapeutic success Confirming unusual resistance patterns Uncovering stealth resistance mechanisms Determining results for infrequently used antimicrobial agents. Question 1 32

33 Tenover - Supplementary tests when routine methods are not enough Why is Supplementary Testing Necessary? New resistance mechanisms that don t always result in MICs in the resistant range (such as carbapenemases) Inducible resistance mechanisms in bacterial isolates that are not recognized by standard methods (such as beta-lactamases) Failures of automated systems to identify non- susceptible strains (vancomycin intermediate S. aureus strains) Some infrequently used or recently approved drugs are not tested on standard panels (colistin) Potential List of Additional Susceptibility Tests Beta-lactamase testing for staphylococcal resistance Cefoxitin disk test to confirm oxacillin resistance in staphylococci D-zone test for inducible clindamycin resistance Macro Etest for VISA and VRSA Screen for high-level aminoglycoside resistance in enterococci ESBL tests: screening and confirmation (CLSI) Screening tests for carbapenemase producers Colistin for multidrug resistant Klebsiellas and Pseudomonas aeruginosa Tests for Staphylococci D-zone test for inducible clindamycin resistance (erythromycin-r/ R/Clindamycin-S) Macro Etest for hetero-vancomycin resistance ONLY for invasive isolates with vancomycin MICs of 2 µg/ml and suggestion of clinical failure Mupirocin for nasal decolonization studies β-lactamase test for borderline susceptible S. aureus strains (if penicillin is to be used) 33

34 Tenover - Supplementary tests when routine methods are not enough Detecting Inducible Clindamycin Resistance in Staphylococci Clindamycin is an oral agent that is being rediscovered for community MRSA infections Problem: Many erythromycin-resistant resistant clindamycin- susceptible strains have inducible clindamycin resistance (erma/b/c); others remain susceptible to clindamycin (msra) Need to identify those strains that can still be treated successfully with clindamycin Solution: D-zone test (Er and CC disks placed mm apart) easy and cheap; adds 24 hrs Examples of Clindamycin Resistance Induction: D-Zone Test E CC E CC 15mm ErmA ErmC Disks can be placed in inner ring of disk dispenser Question 2 34

35 Tenover - Supplementary tests when routine methods are not enough S Vancomycin MICs: S. aureus CLSI breakpoints R µg/ml Genotypic and phenotypic changes vana susceptibility heteroresistance Clinical resistance CLSI and EUCAST Vancomycin Breakpoints for S. aureus (in µg/ml) Susceptible Intermediate Resistant CLSI MIC EUCAST MIC CLSI- EUCAST Disk <2 4 >8 <2 -- >2 na na na Scattergram of S. aureus and Vancomycin; VRSA detected but CLSI breakpoints deleted vana-vrsa Previous breakpoint: >15 mm susceptible PAGE 12 35

36 Tenover - Supplementary tests when routine methods are not enough Population Analysis of hvisa and VISA 1.0E E E+06 VISA 1.0E E E E E+01 Suscept hvisa 1.0E MIC (ug/ml) Inoculum Note scale Subpopulation of hvisa isolates, for which MIC=8 μg/ml, are below detection level Suscept. hvisa VISA Fig 1. Population Analysis for Strain 9AJ57 on BHI vs. MHA 10 9 (CFU/mL) Log 10 ( Subpopulation apparent only on BHI ATCC AJ57-MHA 9AJ57-BHI Vancomycin Concentrations (mcg/ml) Mueller-Hinton MIC= 1 µg/ml BHI MIC = 4 µg/ml hvisa: Clinical Relevance Population Analysis Shows Increasing MICs Log 10 (CF/ml) VISA control ATCC VBHI VBHI VBHI VBHI VBHI Vancomycin Concentrations (µg/ml) Gradual increase in vancomycin MICs during 10 weeks of therapy with vancomycin for endocarditis 36

37 Tenover - Supplementary tests when routine methods are not enough Detecting hvisa via Macro Etest Macro Etest Criteria: Vanco + Ti Teico >8 ug/ml OR Teico Alone >12 ug/ml 2 McFarland, BHI agar, incubation for 48 hours Mupirocin Breakpoints CLSI has approved both MIC and disk diffusion breakpoints for high-level mupirocin resistance in Staphylococcus aureus (No EUCAST breakpoints yet) High-level mupirocin resistance is indicated by: MIC 512 µg /ml No zone of inhibition (6 mm) around a 200 µg disk PAGE 18 37

38 Tenover - Supplementary tests when routine methods are not enough Tests for Enterococci Test for high-level gentamicin and streptomycin resistance to establish synergy with cell wall active agents (penicillin or vancomycin) Use only for sterile site isolates Motility and pigment production tests for low-level level vancomycin resistance Distinguish vanb from vanc (E. gallinarum and E.casseliflavus) Important for infection control View of Beta-Lactamases (2010) The Road to Carbapenem Resistance Class A TEM, SHV, CTX-M, KPCs others Class B Metalloenzymes, VIM others Class C AmpCs and others Class D OXA ESBLS; Carbapenemases Most are carbapenemases AmpC + porin change = carbapenem resistance Some are carbapenemases PAGE 20 Question 3 38

39 Tenover - Supplementary tests when routine methods are not enough Supplementary Tests for Gram Negative Bacilli Critical issues irrespective of breakpoint system you use (EUCAST or CLSI) If you use CLSI and the new breakpoints have not been implemented in your lab ESBL Testing Carbapenemase Testing If you use EUCAST be aware of changes Report cephalosporin and carbapenem results as they test (no further testing needed except for colistin or other drugs not currently on automated panels) Why Detect ESBL-Producing Strains? To improve the accuracy of susceptibility test reports by indicating which beta- lactam drugs may not be effective clinically How? Change the interpretations of extended- spectrum cephalosporins and penicillins from S to R for ESBL producing strains But is this still necessary? Proficiency Testing Results for ESBL- Producing Klebsiella pnemoniae Number of labs (%) Reported as ESBL 83 (31%) Yes 8 (3%) Yes 68 (25%) No 112 (41%) No Changed penicillins and cephalosporins from S to R Yes No Yes No 44% of labs under-reported resistance according to CLSI guidelines 39

40 Tenover - Supplementary tests when routine methods are not enough Confusion About How to Interpret the Test ESBL Disk Diffusion Confirmation Method: K. pneumoniae Zone Diameter (mm) CAZ + CLAV CAZ Ceftazidime 14 Ceftazidime + Clav 23 CTX + CLAV CTX Cefotaxime 20 Cefotaxime + Clav 24* Increase in zone diameter by >5 mm with Clavulanic Acid for EITHER drug = ESBL Organisms with Both AmpCs and ESBLs Organism β-lactamases ESBL AmpC Cefepime MIC/disk PAGE 26 E coli E coli E coli E coli E coli E coli E coli E coli CTXM-5, CTXM-28 28, ACT-1 CTXM-2, ACT-1 CTXM-5, ACT CTXM-2, CTX-M-28, ACT-1 CTXM-15, ACT-1 TEM-1, SHV-7, CMY-2 CTXM-3, ACT-1 SHV-5, CTXM-2, CMY K. pneumoniae SHV, CTXM-2, DHA K. pneumoniae TEM-1, TEM-10, SHV-7, ACT-1 P. mirabilis CTXM-15, DHA Yes Yes Yes Yes Yes Yes No No No No No CRO MIC No R R No R R No R R No R R No R R Yes No R/S R/I R R Yes S I Yes S S (4) Yes S R Yes S R CLSI New Cephalosporin Breakpoints to be Published in 2010 PAGE 27 Use ESBL testing only for infection control 40

41 Tenover - Supplementary tests when routine methods are not enough EUCAST New Cephalosporine Breakpoints to be Published in 2010 Cefazoline Cefuroxime Cefotaxime Ceftriaxone Ceftazidime Cefepime Aztreonam -/ -mg/l 8 / >8 mg/l 1 1 / >2 mg/l 1 1 / >2 mg/l 1 1 / >4 mg/l 1 1 / >4 mg/l 1 / >4 mg/l A Word About KPC β-lactamases Klebsiellas Producing Chaos PAGE 29 Carbapenem Test Results for 15 K. pneumoniae by Method IMIPENEM MEROPENEM Method R I S R I S Broth Disk MScan Phoenix Sensititre Vitek Vitek2* *Meropenem phenotype is deduced by Vitek2; no reports for 2 isolates 41

42 Tenover - Supplementary tests when routine methods are not enough CLSI Screening Criteria for Identifying Carbapenemase Producers (including KPCs) Standard CLSI susceptible breakpoints MIC (µg/ml) Disk (mm) Values suggesting carbapenemase activity* MIC (µg/ml) Disk (mm) Ertapenem <2 > Imipenem <4 > N/A Meropenem <4 > PAGE 31 Perform a Modified Hodge Test N/A, not applicable (poor test performance) Carbapenem Inactivation Assay (Modified Hodge Test) E. coli ATCC PAGE Figure 1. The MHT performed on a small MHA plate. (1) K. pneumoniae ATCC BAA-1705, positive result; (2) K. pneumoniae ATCC BAA-1706, negative result; and (3) a clinical isolate, positive result Inhibition of E. coli ATCC by ertapenem Enhanced growth of E. coli ATCC Carbapenemase produced by K. pneumoniae ATCC BAA-1705 inactivated ertapenem that diffused into the media. Thus, there is no longer sufficient ertapenem here to inhibit E. coli ATCC and an indentation of the zone is noted. New QC strains CLSI Has Lowered Breakpoints Based on PK/PD and MIC Distributions to Increase Testing Accuracy (Delayed implementation) Former CLSI breakpoints New Breakpoints (2010) MIC (µg/ml) Disk (mm) MIC (µg/ml) Disk (mm) Ertapenem <2 - >8 >19- <15 < >1 >23 - <19 Imipenem <4 - >16 >16 - <13 <1 - >4 >23 - <19 Meropenem <4 - >16 >16 - <13 <1 - >4 >23 - <19 Doripenem <1 - >4 >23 - <19 PAGE 33 42

43 Tenover - Supplementary tests when routine methods are not enough EUCAST Clinical Breakpoints* *Reviewed 2009 Carbapenems MIC breakpoint (mg/l) Disk content (µg) Zone diameter breakpoint (mm) S R> S R< Doripenem Ertapenem Imipenem Meropenem Polymyxin B Disk Diffusion Test Doesn t Work for All Species False susceptible by disk Gales, AC, et al. JCM 39:183-90, 2001 Polymyxin Testing of P. aeruginosa CLSI breakpoints: EUCAST breakpoints: MIC <2, S; 4, I; >8 R Disk <11 R; >12 S MIC <2 S; >2 R 43

44 Tenover - Supplementary tests when routine methods are not enough CONCLUSIONS Supplementary tests are necessary to insure accurate susceptibility test reports. Not every isolate requires supplementary tests; in many cases, you can reserve testing for isolates from sterile sites New breakpoints will reduce the need for much of this testing on gram-negative bacilli but implementation on automated systems may be slow Pay attention to changes in whichever system you use (CLSI or EUCAST) THANK YOU 44

45 Leclercq Expert rules in susceptibility testing Expert rules in susceptibility testing Roland Leclercq, Caen, France Expert rules V1 (V2 in a near future..) Intrinsic resistance Exceptional phenotypes (mainly resistance) Interpretive reading Basis for rules Rules Intrinsic resistance Exceptional phenotypes (mainly resistance) are based on analysis of in vitro data (MICs/breakpoints, frequencies of resistance..) 45

46 Leclercq Expert rules in susceptibility testing Interpretive reading is more complex What is interpretive reading? Inference of resistance mechanisms from susceptibility test results and interpretation of clinical susceptibility on the basis of the resistance mechanism Interpretive reading: the process Process Test susceptibility Infer resistance mechanism Interpret clinical susceptibility on the basis of the resistance mechanism Example S. aureus resistant to cefoxitin (oxacillin) Acquisition of the meca gene Report resistant to all β-lactams Expert rules should be evidence based In particular for the interpretive rules since a «S» report may be changed to «I» or «R» Decreases the number of available antibiotics Rules should be based on current evidence (microbiology, experimental models, clinical l data) Evidence should be published Quality of evidence should be assessed Exceptions are possible and should be noted 46

47 Leclercq Expert rules in susceptibility testing Grading of evidence base for EUCAST Expert rules A B C Clinical evidence that reporting the test result as susceptible leads to clinical failures. Evidence is weak and based only on a few case reports or on experimental models No clinical evidence, but microbiological data suggest that clinical use of the agent should be discouraged The objective of this presentation is to review evidence for some examples of rules Activity of aminoglycosides against S. aureus 1. Gentamicin activity is better predicted by the test of kanamycin 2. Bacteriostatic activity of amikacin is better predicted by the test of kanamycin 3. Bactericidal activity of amikacin is better predicted by the test of kanamycin 4. Bactericidal activity of amikacin may be maintained against isolates resistant to gentamicin 47

48 Leclercq Expert rules in susceptibility testing EUCAST rule MICs of aminoglycosides for staphylococci with various aminoglycoside-resistance phenotypes Phenotype (enzyme) MIC (mg/l) Amikacin Tobramycin Gentamicin Susceptible K [APH(3 )] KT [ANT(4 )(4 )] KTG [AAC(6 )-APH(2 )] S R Asseray N et al. Antimicrob Agents Chemother, 2002, 46: Effect of amikacin in a rabbit Staphylococcus aureus endocarditis infection model 10 Control S K KT KTG Gentamicin S K KT KTG Amikacin S K KT KTG NI P< P< Asseray N et al. Antimicrob Agents Chemother, 2002, 46:

49 Leclercq Expert rules in susceptibility testing Basis for evidence? Microbiological data Experimental data (one study in an endocarditis model) No clinical data: Effect of aminoglycosides difficult to assess since they are always used in combination with an active cell-wall inhibitor (β-lactam, glycopeptide) Evidence graded C Phenotypes of resistance to macrolides and clindamycin in S. aureus E C MLS B constitutive E C MLS B inducible E C RIBOSOMAL METHYLATION (A2058) (erm gene) D-zone test EFFLUX (MsrA pump) E C Clindamycin may select resistant mutants in MLS Bi S. aureus E C Positive D-zone test: Selection of MLS B constitutive mutants with clindamycin [for erm(c)frequency: 10-7 ] E C Negative D-zone test: No selection of mutants with clindamycin (not substrate for the pump) 49

50 Leclercq Expert rules in susceptibility testing Interpretive reading 1. If D-test positive: not enough evidence for clinical failure with clindamycin therapy report as «S» to clindamycin 2. If D-test positive, evidence for clinical failure with clindamycin therapy: grade A report as «R» to clindamycin 3. If D-test positive, evidence for clinical failure with clindamycin therapy: grade C report as «S» to clindamycin with a warning «Clinical failure during treatment with clindamycin may occur by selection of resistant mutants. 4. If D-test positive, you need a molecular test to report EUCAST rule If D-test positive, Either report as resistant to clindamycin and lincomycin or report as susceptible with a warning: Clinical failure during treatment with clindamycin n or lincomycin may occur by selection of constitutively resistant mutants. The use of clindamycin/ lincomycin is probably best avoided in severe infections. Mutation frequencies to clindamycin resistance ermc erma Susceptible and efflux Daurel et al., J Clin Microbiol

51 Leclercq Expert rules in susceptibility testing Inducibly MLS B resistant isolates: clindamycin therapy failures No of patients treated with clindamycin No of failures No of MLSB constitutive isolates selected Reference 3 2 1/3 Rao (2000) 2 2 2/2 McGehee (1968) 3 1 1/3 Drinkovic (2002) 2 2 1/2 Frank (2001) 1 1 1/1 Siberry (2003) 1 1 1/1 Levin (2005) /12 Grade B E. coli producing CTX-M-15 AMX CF CTX FEP Synergism between 3GC/aztreonam and clavulanic acid TIC Amox-clav Aztreonam Ceftazidime PIP TCC IPM MOX TZP CPO FOX CXM ( Cattoir V.) EUCAST interpretive rule 9.1 (Enterobacteriaceae) If R or I to any 3rd or 4th gen. oxyiminocephalosporin or aztreonam, and positive for ESBL edit the S result for any of the oxyiminoceph. and aztreonam as I and the I result as R 1. Evidence for this rule is A (in vitro data + experimental models + clinical data) 2. Evidence for this rule is B (in vitro data + experimental models) 3. Evidence for this rule is C (weak evidence) 4. The rule has been set up only to justify the job of microbiologists 5. You do not trust this rule and you never use it 51

52 Leclercq Expert rules in susceptibility testing MICs of beta-lactams for selected ESBL ESBL MIC (mg/l) Cefotaxime Ceftazidime Cefepime Aztreonam CTX-M CTX-M CTX-M TEM SHV (Breakpoints cefotaxime: 1, 2 mg/l) R, I, S A murine thigh infection model for evaluation of activity of 3rd GC according to MIC The % T>MIC was predictive of activity of 3rd/4thgen. cephalosporins against ESBL and non ESBL groups The ß-lactam MIC of an ESBL-producing isolate can be used to predict likely human outcomes from PK/PD models Andes & Craig. Clin Microbiol Infect 2005; 11 (Supp. 6): 10-7 Monte-Carlo simulations and target attainment rates (TAR) for intravenous ceftriaxone 2 g every 24 h S I R TAR at T>MIC (30% for ceftriaxone) for a static to one log pathogen kill at 24 h is taken to be most predictive of outcomes in humans EUCAST Breakpoints MacGowan A. Clin Microbiol Infect 2008; 14 (Suppl 1):

53 Leclercq Expert rules in susceptibility testing Clinical failures with 3rd generation cephalosporins against ESBL producing organisms % Clinica al failure S I R 1 mg/l EUCAST susceptible breakpoint <= MIC (mg/l) Paterson et al. J Clin Microbiol 2001; 39:2206 Evidence for rule 9.1 Weak evidence for this rule Both in vitro, experimental and clinical data rather suggest: «report as found». However, only few clinical data are available Are we ready to leave interpretation for ESBL?. Revision of the rule in the next version: «report as found» (detection of ESBL still required for infection control and epidemiological reasons) Grading of evidence base for EUCAST Expert rules 50 rules are graded A,B or C 1. 2/3 are graded C 2. 90% are graded C 3. 1/2 are graded A 4. 1/3 are graded A, 1/3 are graded B and 1/3 are graded C 53

54 Leclercq Expert rules in susceptibility testing Grading of evidence base for EUCAST Expert rules A 8 (16%) B 9 (18%) C 33 (66%, 2/3) Conclusion Interpretive reading of susceptibility tests is recognized as a major process to report reliable results of AST to clinicians 2/3 of rules based on in vitro evidence Need for more clinical studies to assess the clinical impact of recommendations 54

55 Kahlmeter - Gradient tests on EUCAST media Can MH-F be used for gradient MIC determination for streptococci and Haemophilus influenzae? Gunnar Kahlmeter Charlotta Karlsson, Erika Matuschek, Jenny Åhman Växjö, Sweden EUCAST disk diffusion test recommends two media: Unsupplemented Mueller- Hinton agar for nonfastidious organisms Mueller-Hinton agar with 5% defibrinated horse blood and 20 mg/l β-nad for fastidoius organisms (MH-F) 55

56 Kahlmeter - Gradient tests on EUCAST media Aim It would be practical if routine microbiological laboratories could use MH and MH-F for both disk diffusion testing and gradient MIC-testing. To evaluate whether the MH-F medium used for disk diffusion testing of streptococci and Haemophilus influenzae in the EUCAST method could be used for gradient tests To evaluate whether there was any systematic difference between MH-F and the two media recommended for gradient tests by the manufacturers. Gradient tests for MIC determination More user friendly than microdilution and agar dilution assays. Instantly available for MIC determination of single isolates Several studies show good agreement with reference methods; however for some antibiotics systematically high or low results obtained. Gradient tests investigated: Etest (biomérieux) M.I.C.Evaulator (Oxoid) Manufacturers recommendations Etest (biomérieux) M.I.C.Evaulator, M.I.C.E. (Oxoid) Non-fastidious organisms: Mueller-Hinton Mueller-Hinton with lyzed sheep blood Streptococcus pneumoniae Streptococcus A, B, C and G HTM-medium Haemophilus influenzae 56

57 Kahlmeter - Gradient tests on EUCAST media Manufacturer recommendations compared with EUCAST disk method Haemophilus influenzae biomérieux Oxoid EUCAST Medium HTM a) HTM b) ISA + sheep blood and NAD MH-F Inoculum McF 0.5 in broth (McF 1 if mucoid) McF 0.5 in suitable media McF 0.5 in saline Incubation 35ºC, 5% CO h 35-37ºC, 5% CO h 35ºC, 5% CO 2, h Manufacturer recommendations compared with EUCAST disk method Streptococcus pneumoniae biomérieux Oxoid EUCAST Medium MH + 5% sheep blood a) HTM b) ISA + sheep blood and NAD MH-F Inoculum McF 0.5 in broth (McF 1 if mucoid) McF in suitable media McF 0.5 in saline Incubation 35ºC, 5% CO h 35-37ºC, 5% CO 2 a) h b) h 35ºC, 5% CO 2, h Methodology H. influenzae, S. pneumoniae and S. pyogenes QC strains and clinical isolates were tested MH-F vs. recommended media for Etest and M.I.C.Evaluator HTM for H. influenzae MH + 5% sheep blood for streptococci Incubation in 5% CO 2 and 35ºC for 20 h. 57

58 Kahlmeter - Gradient tests on EUCAST media H. influenzae* MH-F compared with reference medium MH-F < MH-Str MH-F = MH-Str MH-F > MH-Str 3 dil 2 dil 1 dil 0 1 dil 2 dil 3 dil No of readings Etest (Number of readings) Amoxicillin/clavulanic acid Ampicillin Ciprofloxacin Tetracycline SXT * MICE (Number of readings) Amoxicillin/clavulanic acid Ampicillin Ciprofloxacin Tetracycline * Trimethoprim-sulfamethoxazole (SXT) was not available for MICE H. Influenzae ATCC 49247, NCTC 8468 and three clinical isolates H. influenzae Ampicillin 35 0 MH-F = Reference medium - 30 MH-F < Reference medium + MH-F > Reference medium 25 No of readings E-test MICE Dilution steps' difference Tetracycline No of readings E-test MICE Dilution steps' difference S. pyogenes* and S. pneumoniae* MH-F compared with reference medium MH-F < MH-Str MH-F = MH-Str MH-F > MH-Str 3 dil 2 dil 1 dil 0 1 dil 2 dil 3 dil No of readings Etest (Number of readings) Benzylpenicillin Erythromycin Moxifloxacin* Tetracycline SXT * MICE (Number of readings) Benzylpenicillin Erythromycin Tetracycline * Moxifloxacin and Trimethoprim-sulfamethoxazole (SXT) were not available for MICE *S. pyogenes CCUG 25571, S. pneumoniae ATCC and three clinical S. pneumoniae isolates 58

59 Kahlmeter - Gradient tests on EUCAST media S. pyogenes and S. pneumoniae Benzylpenicillin No of readings E-test MICE Dilution steps' difference Tetracycline No of readings E-test MICE Dilution steps' difference 0 MH-F = Reference medium - MH-F < Reference medium + MH-F > Reference medium Conclusions I The correlation between MICs obtained on MH-F and reference media was good for Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae Results were reproducible and easily read Our data suggest that MH-F may be a suitable substrate for gradient test MIC determination. Conclusions II We call on manufacturers and others to extend our trials and validate the use of MH-F Ffor gradient MIC-testing. 59

60 Kahlmeter - Gradient tests on EUCAST media We thank biomérieux and Oxoid for supplying the gradient strips 60