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

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

CLSI “Standards” and “Guidelines” for AST 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)

“Standard” vs. “Guideline” Standard – a document developed through the consensus process that clearly 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.

US clinical labs can use: M2, M7, and M100 describe standard consensus “reference methods” and may be used: By clinical labs for routine testing To evaluate commercial devices By drug or device manufacturers for testing new agents or systems US clinical labs can use: CLSI test method as written Methods that perform comparably to CLSI “reference method” (e.g. FDA-cleared diagnostic AST devices)

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

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

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

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

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

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

Box with “ors” Example: Staphylococcus spp. Azithromycin or clarithromycin or erythromycin 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 CLSI M100-S20; Table 1

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

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

CLSI M100-S20 Glossary I (Part I)

CHANGE

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

Enterobacteriaceae Changes

Enterobacteriaceae Revised… Breakpoints (MIC µg/ml) Agent CLSI M100-S19 (2009) CLSI M100-S20 (2010) Susc Int Res Cefazolin ≤8 16 ≥32 ≤1 2 ≥4 Cefotaxime 16-32 ≥64 Ceftizoxime Ceftriaxone Ceftazidime ≤4 8 ≥16 Aztreonam CLSI M100-S20. Table 2A.

Enterobacteriaceae Revised… Breakpoints (disk diffusion mm) Agent CLSI M100-S19 (2009) CLSI M100-S20 (2010) Susc Int Res Cefazolin* ≥18 15-17 ≤14 NA Cefotaxime ≥23 15-22 ≥26 23-25 ≤22 Ceftizoxime ≥20 15-19 ≥25 22-24 ≤21 Ceftriaxone ≥21 14-20 ≤13 20-22 ≤19 Ceftazidime 18-20 ≤17 Aztreonam ≥22 16-21 ≤15 *disk diffusion breakpoints not yet established CLSI M100-S20. Table 2A.

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)

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

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

CLSI ESBL Testing Recommendations Purpose If using Old Breakpoints M100-S19 Revised Breakpoints M100-S20 For Patient Management Perform ESBL screen and confirmatory tests Yes No Edit “S” to “R” for cephalosporins, penicillins, aztreonam For Infection Control Yes, if requested

Enterobacteriaceae Revised… Carbapenem Breakpoints (MIC µg/ml) Agent CLSI M100-S19 (2009) CLSI M100-S20 (2010) Supplement Susc Int Res Doripenem - ≤1 2 ≥4 Ertapenem ≤2 4 ≥8 ≤0.25 0.5 ≥1 Imipenem ≤4 8 ≥16 Meropenem There will be a special CLSI M100-S20 Supplement to be published Spring 2010 with Enterobacteriaceae Tables only with these breakpoints!

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

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

Will tests for carbapenemases (e. g 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 for Infection Control purposes

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

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

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

OPTIONS for In-House Validation (test system demonstrates comparable S, I, R results to reference method) Reference Method Disk diffusion CLSI reference broth or agar dilution Other Isolates 5 ESBL (+) 5 ESBL (-) and ESBL screen positive 20 other Enterobacteriaceae (preferably with MICs 0.5 - 8 µg/ml range) Acceptance Criteria ≥90% category (S, I, R) agreement ≤3% very major errors?? ≤7% combined major and minor errors ?? (establish prior to commencing validation)

Non-Enterobacteriaceae

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.

Staphylococcus species

Staphylococcus spp. Penicillin Susceptible “(11) An induced -lactamase test 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.

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

Staphylococcus aureus Penicillin MICs ≤0.12 µg/ml blaZ Pos Of the blaZ Pos, No.(%) Induced -lactamase Pos Reference 69 4 1/4 (25) CLSI Agenda Book 6/09 197 28 11/28 (39) Kaase et al. 2008. Clin Microbiol Infect. 14:614 Conclusion: induced β-lactamase test may not detect staphylococci that have blaZ and this could lead to treatment failures if using penicillin

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

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

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

Staphylococcus spp. - Linezolid Added… “R” Breakpoint CLSI M100-S19 (2009) CLSI M100-S20 (2010) Susc Int Res MIC (µg/ml) ≤4 - ≥8 Zone (mm) ≥21 ≤20 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

MRSA = S. aureus with mecA and/or oxacillin MIC >2 µg/ml Definition of MRSA “(2) MRSA are those strains of S. aureus that express mecA or another 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.

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.

S. aureus or S. lugdunensis Testing 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.

S. aureus or S. lugdunensis Testing Both OX and CX Resistance mechanism Relative Prevalence Report as OX: S None Common R mecA mecA (low level expression) Uncommon PBP changes or hyper-production of β-lactamase (borderline MRSA) Rare Courtesy of Jean Patel

Added… to Glossary New Subclass for Cephems Agents Cephems Cephalosporins with anti-MRSA activity Ceftaroline* Ceftobiprole* *Not FDA approved as of April 2010 CLSI M100-S20. pp 144.

Enterococcus species

Revised… Enterococcus spp. β-lactamase Testing “(8) Penicillin or ampicillin resistance among enterococci due to -lactamase production has been reported very rarely. Penicillin or ampicillin resistance due to -lactamase production is not reliably detected with routine disk or dilution methods but is detected using a direct, nitrocefin-based -lactamase test. Because of the rarity of -lactamase–positive enterococci, this test need not be performed routinely, but can be used in selected cases. A positive -lactamase test predicts resistance to penicillin, as well as amino- and ureidopenicillins.” CLSI M100-S20. pp. 77.

Streptococcus species

Revised… Streptococcus spp Revised… Streptococcus spp. β-hemolytic Group Extrapolation of Penicillin Results “(6) For the following organism groups, an organism that is susceptible to penicillin can be considered susceptible to the listed antimicrobial agents when used for approved indications and need not be tested against those agents. For β-hemolytic streptococci (Groups A, B, C, G): ampicillin, amoxicillin, amoxicillin-clavulanic acid, ampicillin-sulbactam, cefazolin, cefepime, cephradine, cephalothin, cefotaxime, ceftriaxone, ceftizoxime, imipenem, ertapenem, and meropenem. In addition, for group A streptococci only: cefaclor, cefdinir, cefprozil, ceftibuten, cefuroxime, cefpodoxime, and cephapirin.” CLSI M100-S20. pp. 93.

Streptococcus spp. β-hemolytic Group Groups A, B, C, G Plus these for Group A only Ampicillin Cefaclor Amoxicillin Cefdinir Amox-clav Cefprozil Amp-sulb Ceftibuten Cefazolin Cefuroxime Cefepime Cefpodoxime Cephalothin Cephapirin Cephradine Cefotaxime Ceftizoxime Ceftriaxone Ertapenem Imipenem Meropenem Extrapolate penicillin “S” result to other β-lactams listed here * drugs listed have clinical indication for respective β-hemolytic streptococcal group (large colony-forming strains) CLSI M100-S20. pp. 93.

Janet Hindler, MCLS MT(ASCP) Acknowledgements Janet Hindler, MCLS MT(ASCP) UCLA Medical Center