Antibiotics 102: Reading and Interpreting CLSI Antimicrobial Susceptibility Performance Documents Dave Warshauer, PhD,

1. Antibiotics 102: Reading and Interpreting CLSI Antimicrobial Susceptibility Performance Documents Dave Warshauer, PhD, D(ABMM) Deputy Director, Communicable Diseases

3. How Religious are We? • Washington State • Only 40% used current CLSI standards for S.pneumoniae AST • Only 29-69% accurate responses for 3 different case studies Counts, JM et al. JCM 45:2230-34, 2007

4. CLSI “Standards” and “Guidelines” for AST • Standards: • M2-A10 Disk Diffusion (2009) • M7-A8 MIC (2009) • M100-S20 Tables (2010) • Guidelines: • M39-A3 Cumulative Antibiograms (2009) • M45-A Infrequently Isolated / Fastidious Bacteria (2006)

5. “Standard” vs. “Guideline” • Standard – a document developed through the consensus process thatclearly identifies specific, essential requirements for material, methods, or practices for use in an unmodified form.A standard may, in addition, contain discretionary elements, which are clearly identified. • Guideline – a document developed through the consensus process describing criteria for a general operating practice, procedure, or material for voluntary use. A guideline may be used as written or modified by the user to fit specific needs.

6. M2, M7, and M100 describe standard consensus“reference methods”and may be used: By clinical labs forroutine testing Toevaluate commercial devices By drug or device manufacturers fortesting new agents or systems US clinical labs can use: CLSI test methodas written Methods thatperform comparablyto CLSI “reference method” (e.g. FDA-cleared diagnostic AST devices)

7. M7 and M2 Contents • Summary of Major Changes • Definitions of S, I, R • Indications for Performing AST • Antimicrobial agent descriptions • Agents for Routine Testing and Reporting • Procedures for testing • Fastidious and Problem Organisms • Quality Control Procedures • Limitations • References • Summary of Comments and Responses • Related CLSI Publications

8. CLSI M100 contains….. M100 Answers to user questions Updates in this edition M2 Tables Disk Diffusion Glossary I & II M7 Tables MIC • Test/report • Breakpoints • QC • Test/report • Breakpoints • QC

10. Antimicrobial Selection Guidelines for Testing and Reporting---Table 1 • Group A • Agents for inclusion in a routine, primary testing panel and for routine reporting for the specific organism groups

11. Antimicrobial Selection Guidelines for Testing and Reporting • Group B • Agents that warrant primary testing, but reported only selectively • Selected source---e.g. 3rd generation ceph. for an enteric gnb from CSF • A polymicrobial infection • Infection involving multiple sites • Case of patient with allergy • Purposes of infection control

12. Antimicrobial Selection Guidelines for Testing and Reporting • Group C • Alternative or supplemental antimicrobials that may require testing in institutions that harbor endemic or epidemic strains resistant to multiple primary drugs • For treatment of unusual situations e.g. chloramphenicol for extraintestinal Salmonella spp. • Infection control purposes

13. Antimicrobial Selection Guidelines for Testing and Reporting • Group U • Agents for treating UTIs • Note: Cephalothin now in Group U for Enterobacteriaceae • Group O • Agents have a clinical indication for the organism group but are generally not routinely tested and reported in the U.S. • Group Inv. • Investigational agents

14. Azithromycinorclarithromycinorerythromycin In a box, agents connected with“or”includes those for which… Cross-resistance and cross-susceptibility are nearly complete Clinical efficacy is similar Results of one agent can be used to predict results for the others Box with“ors”Example: Staphylococcus spp. CLSI M100-S20; Table 1

15. Mezlocillin Ticarcillin Piperacillin Box includes agents for which… Testing of one agent cannot be used to predict results for another Box without“ors”Example: Pseudomonas aeruginosa CLSI M100-S20; Table 1

16. -lactams -lactam ring penicilloic acid penicillin There are many different types of -lactams and -lactamases!

17. CLSI M100-S20 Glossary I (Part I)

18. CHANGE

19. CLSI AST Standards Major Changes 2010 Enterobacteriaceae Revised disk diffusion and MIC breakpoints for: cefazolin, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, aztreonam Eliminate need for ESBL screen and confirmatory tests when using revised breakpoints Staphylococcus spp. Explain limitations of -lactamase testing Define MRSA Expand comment for testing oxacillin and cefoxitin with S. aureus and S. lugdunensis

20. Enterobacteriaceae Changes

21. EnterobacteriaceaeRevised…Breakpoints (MIC µg/ml) CLSI M100-S20. Table 2A.

22. Enterobacteriaceae Revised…Breakpoints (disk diffusion mm) *disk diffusion breakpoints not yet established CLSI M100-S20. Table 2A.

23. Why did CLSI lower breakpoints? • Previous breakpoints established over 20 years ago • Increased knowledge of β-lactam resistance mechanisms • Increased knowledge of pharmokinetics and pharmacodynamics (PK/PD)

24. Detection of ESBLs (1) • Initial recommendations: • Based on: • Some isolates had elevated MICs in “S” range • Some (limited) data showing poor outcomes in patients with ESBL-producing isolates • Perform ESBL screen and confirmatory tests for E. coli, Klebsiella spp., and Proteus mirabilis

25. Detection of ESBLs (2) • Now we know! • ESBL phenotypic tests not optimal • Presence of multiple resistance mechanisms may mask ESBL in confirmatory test • ESBL + AmpC • ESBL + porin mutation • ESBLs are present in species of Enterobacteriaceae other than E.coli, Klebsiella spp., P. mirabilis where confirmatory test is more problematic • Some labs not doing • MIC correlates better with outcome than knowledge of “R” mechanism

26. CLSI ESBL Testing Recommendations

27. Enterobacteriaceae Revised…Carbapenem Breakpoints (MIC µg/ml) There will be a special CLSI M100-S20 Supplement to be published Spring 2010 with Enterobacteriaceae Tables only with these breakpoints!

28. Impact of Imipenem Breakpoint Changes Sahm, D. Eurofins Medinet, Inc.

29. Proteus mirabilis and Imipenem Sahm, D. Eurofins Medinet, Inc.

30. Will tests for carbapenemases (e.g., Modified Hodge test) be needed with the new carbapenem breakpoints for Enterobacteriaceae? • NO----- For patient management, tests for carbapenemases are not necessary • YES-----If requested, tests for carbapenemases may be done forInfection Control purposes

31. What steps should be included in a plan to implement revised breakpoints? • Determine if AST system can accommodate revised breakpoints • Contains low concentrations of drug? • Have a mechanism to interpret MICs with revised breakpoints (might be done with LIS)? • Discuss with Infectious Diseases, Pharmacy, Infection Control Manufacturers of commercial test systems are required by law to use FDA breakpoints Currently, NO commercial AST system is FDA-cleared with the new breakpoints

32. AST Methods Used in Clinical Labs • 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 • Laboratories can use CLSI breakpoints

33. OPTIONS Laboratory director must determine what is best for his/her laboratory and patients Implement Now? Implement when revised breakpoints are available on laboratory’s commercial AST system? Perform validation

34. OPTIONS for In-House Validation (test system demonstrates comparable S, I, R results to reference method)

35. Non-Enterobacteriaceae

36. Acinetobacter spp. • Deleted colistin / polymyxin from Table 1 • No FDA clinical indication for Acinetobacter spp. • No changes in breakpoints in Table 2B-2 CLSI M100-S20. pp. 29.

37. Staphylococcus species

38. Staphylococcus spp. Penicillin Susceptible “(11) Aninduced -lactamasetest should be performed on staphylococcal isolates with penicillin MICs ≤ 0.12 µg/mL or zone diameters ≥ 29 mm before reporting the isolate as penicillin susceptible. However, the prevalence of penicillin-susceptible S. aureus strains is low. Isolates that test as susceptible to penicillin may still produce β-lactamase, which is usually detected by an induced β-lactamase test. Occasional isolates are not detected by induced β-lactamase testing.Thus, for serious infections, laboratories should consider performing MIC tests for penicillin and testing for induced β-lactamase production on subsequent isolates from the same patient.” CLSI M100-S20. pp. 62.

39. Staphylococcus spp. Penicillin Susceptible (2) • Perform aninduced -lactamasetest on staphylococcal isolates if penicillin… • MIC ≤0.12 µg/ml • Zone diameter ≥29 mm ….before reporting penicillin “S” • Several studies demonstrated an induced -lactamase testusually but not alwaysdetects S. aureus capable of producing -lactamase • blaZgene codes for -lactamase production NOT detected by -lactamase test

40. StaphylococcusaureusPenicillin MICs ≤0.12 µg/ml Conclusion:induced β-lactamase test may not detect staphylococci that have blaZ and this could lead to treatment failures if using penicillin

41. Induced ß-lactamase Test Oxacillin (inducer) • Sub isolate to agar (e.g., BAP, MHA) • Drop ß-lactam disk (e.g., oxacillin, cefoxitin) • Incubate overnight • Test cells from periphery of zone • If β-lactamase positive, report penicillin R Pos Neg

42. Staphylococci and Vancomycin Revised recommendation…Re: vancomycin MIC, when should staphylococci be sent to a public health or reference laboratory for further testing? • S. aureus • MIC 4 µg/ml – maybe • MIC ≥8 µg/ml – yes • Coagulase-negative staphylococci (CoNS) • MIC ≥32 µg/ml – yes

43. http://www.cdc.gov/ncidod/dhqp/pdf/ar/VRSA_testing_algo09v4.pdfhttp://www.cdc.gov/ncidod/dhqp/pdf/ar/VRSA_testing_algo09v4.pdf

44. Staphylococcus spp. - Linezolid Added…“R” Breakpoint • Linezolid non-susceptible S. aureus rare 0.05% (7 / 15,280 isolates) CLSI agenda book June 2009. • Resistance mechanisms have been identified • rRNA mutations and cfr-mediated resistance (which can be plasmid encoded) Mendes et al. 2008. Antimicrob Agents Chemother. 52:2244

45. Definition of MRSA “(2) MRSA are those strains of S. aureus that expressmecAoranother mechanism of methicillin resistance, such as changes in affinity of penicillin binding proteins for oxacillin (modified S. aureus [MOD-SA] strains)” MRSA = S. aureus with mecA and/or oxacillin MIC >2 µg/ml CLSI M100-S20. pp. 60.

46. What about mecA negative MRSA? • Mechanisms: • Modifications in penicillin-binding proteins (PBPs) 1,2,4 (MOD-SA) • Hyperproduction of blaZ-encoded penicillinase • Methicillinase • Infrequently encountered • Limited clinical information in literature re: therapy with β-lactams Croes, S et al. 2009. Clin Microbiol Infect. Epub. 10/09 Chambers, H. 1997. Clin Microbiol Rev. 10:781.

47. S. aureus or S. lugdunensisTesting Both Oxacillin (OX) and Cefoxitin (CX) “(12) Cefoxitin is used as a surrogate for oxacillin resistance; report oxacillin susceptible or resistant based on the cefoxitin result. If both cefoxitin and oxacillin are tested against S. aureus or S. lugdunensis and either result is resistant, the organism should be reported as oxacillin resistant.” CLSI M100-S20. pp. 62.

48. S. aureus or S. lugdunensisTesting Both OX and CX Courtesy of Jean Patel

49. Added…to Glossary New Subclass for Cephems *Not FDA approved as of April 2010 CLSI M100-S20. pp 144.

50. Enterococcus species