The document discusses updated CLSI antimicrobial susceptibility testing challenges, particularly focusing on the differences between CLSI and FDA breakpoints for Enterobacteriaceae, which have been revised to better reflect current resistance mechanisms. Strategies for validating these new breakpoints in commercial AST systems are outlined, along with the need for collaboration among laboratory professionals. The presentation also emphasizes understanding pharmacokinetics and pharmacodynamics to improve patient treatment outcomes.

1. Coping with Updated CLSI Antimicrobial Susceptibility Testing (AST) Challenges Janet A. Hindler, MCLS MT(ASCP) Sr. Specialist, Clinical Microbiology UCLA Medical Center [email_address] “ and a consultant with the Association of Public Health Laboratories”

2. At the conclusion of this talk, you will be able to…… Discuss some differences between CLSI and FDA breakpoints . List criteria used by CLSI to set or revise breakpoints. Describe strategies for verifying newly revised CLSI breakpoints on a commercial AST system.

3. CLSI AST Standards November 2010 M100-S20 Tables (January 2010) Lowered cephalosporin & aztreonam breakpoints (Enterobacteriaceae) M100-S20-U (June 2010) Lowered carbapenem breakpoints (Enterobacteriaceae)

4. EUCAST EMEA CLSI FDA Sets breakpoints Sets breakpoints Sets breakpoints Manufacturers of AST systems must use FDA breakpoints CLSI = Clinical and Laboratory Standards Institute (formerly NCCLS) EUCAST = European Committee on Antimicrobial Susceptibility Testing EMEA = European Medicines Agency Standards Setting EMEA sets breakpoints through EUCAST Regulatory Reassesses breakpoints 2010 - developing mechanism to reassess breakpoints Standards Setting Regulatory

5. Ceftriaxone Prescribing Information - FDA Breakpoints http://media.pfizer.com/files/products/uspi_ceftriaxone.pdf

6. CLSI and FDA and EUCAST breakpoints may differ because .…. Different criteria used to set breakpoints Sometimes different dosing regimens; different clinical indications (USA vs. Europe) Some breakpoints set many years ago and not yet reassessed by respective agency Examples (Enterobacteriaceae): *new 2010 ≤ 4 ≤ 4 ≤ 8 FDA Agent CLSI EUCAST Ceftriaxone ≤ 1* ≤ 1 Gentamicin ≤ 4 ≤ 2 Meropenem ≤ 1* ≤ 2

7. Status of FDA Breakpoint Revisions FDA Amendments Act (FDAAA) of 2007 contains 200 provisions re: drug marketing/labeling requires FDA to update drug labels FDA Guidance Documents (2009) Will consider breakpoints of national or international standards organizations Describes approach for pharmaceutical companies to update breakpoints in prescribing information CLSI leadership maintaining close watch

8. Why did CLSI lower cephalosporin, aztreonam, and carbapenem breakpoints for Enterobacteriaceae? (1) Most of these breakpoints were established over 20 years ago (before ESBLs and when virtually all Enterobacteriaceae were “S” to carbapenems) Newer tools / data to establish breakpoints (e.g., PK/PD with Monte Carlo simulations) Increased knowledge of old and newer β -lactam resistance mechanisms Revised breakpoints will better detect isolates with a variety of resistance mechanisms

9. Why did CLSI lower cephalosporin, aztreonam, and carbapenem breakpoints for Enterobacteriaceae? (2) Re: cephalosporin and aztreonam breakpoints CLSI ESBL test only standardized for E. coli, Klebsiella spp. and Proteus mirabilis (mid 1990s) ESBLs occur in other species ESBLs not always detected if multiple R mechanisms present For ESBL positive isolates: Low MICs to selected cephalosporins associated with being poor substrates for specific ESBL Animal studies indicated % T > MIC did not differ for ESBL positive vs. ESBL negative Enterobacteriaceae Animal studies showed no inoculum effect w/ ESBL producers

10. Why did CLSI lower cephalosporin, aztreonam, and carbapenem breakpoints for Enterobacteriaceae? (3) Re: Carbapenem breakpoints Carbapenem resistance “issues” increasing rapidly Phenotypic test for carbapenemases [Modified Hodge test (MHT)] Subjective interpretation Does not detect all carbapenem “R” mechanisms

11. When applying revised cephalosporin, aztreonam and carbapenem breakpoints for…. Patient management – no need to perform testing (ESBL test, Modified Hodge test ) to detect resistance mechanism Surveillance, epidemiology, infection control – ESBL and MHT tests (or other tests to detect resistance mechanism) may be useful

12. www.eucast.org

13. Setting / Revising Breakpoints Data Used by CLSI MIC distributions of “wild type” or normal populations of bacteria Wild type = no acquired “R” mechanisms MICs associated with clinical outcome Very limited “new” data for older drugs Pharmacokinetic-pharmacodynamic (PK/PD) analysis CLSI M23-A3 (2008) “Development of In Vitro Susceptibility Testing Criteria and QC Parameters; Approved Guideline” describes CLSI process for setting / revising breakpoints

14. www.eucast.org MEROPENEM MIC distribution example Blue = wild type isolates Red = isolates w/ acquired “R” mechanism

15. What is PK/PD? PK: pharmacokinetics - the process by which a drug is absorbed, distributed, metabolized, and eliminated by the body Relates to drug concentration over time PD: pharmacodynamics - the relationship between concentration of drug and its antimicrobial effects over time in vivo PK/PD can project potential efficacy of antimicrobial agents in vivo Key reference = Craig WA. 1998. CID. 26:1-10.

16. PK/PD Goal (“Target”) for β -lactams = % of time during dosing interval that drug level in the patient exceeds MIC (%T > MIC) (hypothetical) Serum Concentration (µg/ml) Time (hours) MIC Time above MIC dose dose Cmax (peak concentration)

17. What PK/PD target value is desired for β -lactams (e.g., “target attainment”)? Bacteriostatic and bactericidal activity of β -lactams depends on duration of time that free (unbound) drug levels exceed MIC (% T > MIC) Drusano GL. 2004. Nat Rev Microbiol. 2:289. *3 log reduction in colony-forming units 30-40 20-30 Carbapenems 50 30 Penicillins 60-70 35-40 Cephalosporins Bactericidal* (%) Bacteriostatic (%) Free Drug % Time > MIC Antimicrobials

18. Factors that Affect PK/PD Target Attainment Distribution of pathogen MICs (Species that would be treated with drug) Drug protein binding (unbound drug is active) Drug dosages (various options for some drugs) Drug administration (e.g., frequency of dosing; length of infusion) Patient characteristics that affect drug distribution and clearance of drug (e.g., kidney function)

19. Monte Carlo Simulation Used to answer question… What percentage of patients are likely to attain the “target”? Target for carbapenems = %T > MIC 30-40% = bactericidal activity Want ≥ 90% of patients to attain target Examine PK/PD data by simulating dosing regimens and drug levels among a large sample of patients and various MICs for pathogens that would be treated with the drug Plug in various MICs, dosing and drug levels that might be encountered in many different patients (e.g., 1000 patients)

20. What PK/PD data were used to revise CLSI breakpoints? Monte Carlo simulations Studies in animal models and results extrapolated to humans Limited clinical data Goal - determine PK/PD MIC breakpoint that could predict the likelihood that a specific drug dose would be effective against an organism with a specific MIC

21. Probability of Target Attainment Imipenem 1g every 8h 4 2 Percent of patients likely to attain desired target 1 Breakpoints ( µ g/ml): Revised = ≤ 1 Old = ≤ 4 Sakka et al. 2007. AAC. 51:3304. S I R

22. Enterobacteriaceae - Carbapenems Revised… Breakpoints (MIC µ g/ml) Corresponding disk diffusion breakpoints also revised CLSI M100-S20-U CLSI 2010 (Old) CLSI June 2010 (Revised) Agent Susc Int Res Susc Int Res Doripenem - - - ≤ 1 2 ≥ 4 Ertapenem ≤ 2 4 ≥ 8 ≤ 0.25 0.5 ≥ 1 Imipenem ≤ 4 8 ≥ 16 ≤ 1 2 ≥ 4 Meropenem ≤ 4 8 ≥ 16 ≤ 1 2 ≥ 4

23. CLSI M100-S20-U Revised Carbapenem Breakpoints and Dosing Correlates S I R S I R

24. New CLSI Carbapenem Dosage Comment “ Because of limited treatment options for infections caused by organisms with carbapenem MICs or zone diameters in the intermediate range , clinicians may wish to design carbapenem dosage regimens that use maximum recommended doses and possibly prolonged intravenous infusion regimens as has been reported in the literature.” CLSI M100-S20 (June 2010)

25. Imipenem Probability of Target Attainment 2 4 Revised Breakpoints ( µ g/ml): ≤ 1S 2I ≥ 4R 1 2 4 1 Sakka et al. 2007. AAC. 51:3304. S S R I R I

26. Carbapenem MIC - Reporting Strategy 1 Perform MHT if MIC 2-4 µ g/ml and “R” to ≥ 1 3 rd gen cephalosporin 2 MHT not needed for routine patient reporting; may perform for Infection Control but DO NOT change “S” or “I” to “R” 3 Change from CLSI M100-S20 January 2010 Meropenem Breakpoints ( µ g/ml) CLSI M100-S20-U Int Res Res Res 3 Res 3 Res Report if MHT positive Susc Susc 2 Res Res 8 Revised BPs 2 Susc Susc 4 Int Int 8 Old BPs 1 Int Int 2 4 Meropenem MIC ( µ g/ml) Res Report if MHT negative Res Report if MHT not done Revised Old ≥ 4 2 ≤ 1 ≥ 16 8 ≤ 4 Res Int Susc

27. Specimen: Nasal wash Diagnosis: Respiratory distress Klebsiella pneumoniae amikacin >32 R aztreonam >32 R cefepime >32 R ceftazidime >32 R ceftriaxone >32 R ciprofloxacin >2 R colistin 1* ertapenem >8 R** gentamicin >10 R imipenem 4 R** meropenem 8 R** piper-tazobactam >128 R tigecycline 1 S tobramycin >10 R trimeth-sulfa > 4/76 R MIC ( µ g/ml) Modified Hodge Test Negative CDC subsequently confirmed (by PCR) NDM-1 carbapenemase UCLA September 2010 * no interpretive criteria ** interpreted with Revised CLSI Breakpoints

28. Brief CLSI and FDA Breakpoint (BP) Story Might be delay in addition of new BPs to commercial system Use of CLSI or FDA BPs is acceptable to accrediting bodies ; thus can use “Old” (same as FDA BPs) or “New” CLSI BPs Labs can validate (verify) new BPs on commercial system CLSI M100-S20

29. What criteria are used by FDA to determine if commercial AST system results are acceptable?* http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm080564.htm * must also meet criteria for very major, major and minor errors. >89.9 % Test S, I, and R agree with REF S, I, R Category Agreement (CA) >89.9 % Test MIC within +/- 1 doubling dilution of the REF MIC Essential Agreement (EA) FDA Acceptable Limits Defined as… Criteria

30. Manufacturers of commercial AST systems… Cannot submit performance data for revised CLSI breakpoints until FDA revises breakpoints in each drug’s “Prescribing Information” For Disk diffusion Manufacturer does not have to submit data to FDA Cannot include revised breakpoints in package insert until FDA revises breakpoints in Prescribing Information

31. Steps to Implementing Revised CLSI Cephalosporin, Aztreonam and Carbapenem Breakpoints Determine if low concentrations are available on panel Discuss with stakeholders (lab director, infectious diseases, pharmacy, infection control, others) Perform “verification” Controversial Make computer / protocol changes Inform stakeholders of changes

32. Verification of Revised Breakpoints Possible Isolate Selection Isolates to Test for Verification Revised Breakpoints 5 ESBL (+) 5 ESBL (-) and ESBL screen positive 20 other Enterobacteriaceae (preferably with MICs within S range using old BPs) Cephalosporins and Aztreonam 5 carbapenemase (+) 5 Modified Hodge Test (-) and elevated carbapenem MICs 20 other Enterobacteriaceae Carbapenems

33. S, I, R Test MIC AST (revised breakpoints) S, I, R Reference AST* (revised breakpoints) * Disk diffusion, reference broth or agar dilution MIC, other MIC? Acceptable = ≥ 90% overall categoric agreement No very major errors (false “S”) ≤ 7% combined major (false “R”) and minor errors (I/R or I/S) Verify Revised Breakpoints Determine Categoric Agreement vs JH Suggestion (not CLSI recommendation)

34. Ceftriaxone MIC ( µ g/ml) vs. Zone Diameter (mm) CLSI Previous (MIC ≤ 8; zone ≥21) vs. Revised (MIC ≤ 1; zone ≥23) Breakpoints for Enterobacteriaceae Jones et al. 2005. Diagn Microbiol Infect Dis. 52:235-246. previous Susc Res revised

35. Example: Categoric (S, I, R) Agreement Test MIC Panels vs. Reference Disk Diffusion Old vs. Revised Breakpoints - Ceftriaxone Old Breakpoints: 1 minor error Revised Breakpoints: 1 major errors (false R) 1 minor error Revised Breakpoints (S, I, R) OLD Breakpoints (S, I, R) 5 4 3 2 1 Strain R I S S S ≥ 21, 14-20, ≤ 13 Reference DD 12 19 21 23 25 Ref DD (mm) ≥ 23, 20-22, ≤ 19 ≤ 1, 2, ≥ 4 ≤ 8, 16-32, ≥ 64 R R I S S Reference DD R R R R S Test MIC 32 8 4 4 1 Test MIC ( µ g/ml) R S S S S Test MIC

36. UCLA) Decisions re: Revised Enterobacteriaceae Breakpoints Adopted revised carbapenem breakpoints Perform MHT on request (note: meropenem concentrations on urine panels not low enough; will test isolates “R” to ceftriaxone and piperacillin-tazobactam by disk diffusion method) Adopt revised cephalosporin breakpoints Continue ESBL testing ( E. coli, Klebsiella, P. mirabilis ) Edit “S” results to “R” for cephalosporins, penicillins, and aztreonam for ESBL producers Inform Infectious Diseases of ESBL-producing isolates that have “S” MICs to one or more cephalosporins or aztreonam for follow up Cefazolin Use old breakpoints (S, MIC ≤ 8 µ g/ml) for urine isolates (limitations understood by medical staff) Use revised 2011 breakpoints (S, MIC ≤ 2 µ g/ml) for other isolates

37. www.clsi.org

38. Thank you!