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

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How Religious are We?

• Standards:

– Only 40% used current CLSI standards for S. pneumoniae AST – Only 29-69% accurate responses for 3 different case studies

– 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)

Counts, JM et al. JCM 45:2230-34, 2007 3

“Standard” vs. “Guideline”

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• M2, M7, and M100 describe standard consensus “reference methods” and may be used:

• 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.

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CLSI “Standards” and “Guidelines” for AST

• Washington State

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– 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. FDAFDA-cleared diagnostic AST devices) 5

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M7 and M2 Contents • • • • • • • • • • • •

CLSI M100 contains…..

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

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M100 Updates in this edition

Answers to user questions

M2 Tables Disk Diffusion •Test/report •Breakpoints •QC 7

M7 Tables MIC

Glossary I & II

•Test/report •Breakpoints •QC

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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

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Antimicrobial Selection Guidelines for Testing and Reporting

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Antimicrobial Selection Guidelines for Testing and Reporting • Group C

• Group B

– 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

– 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 WISCONSIN STATE LABORATORY OF HYGIENE

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Antimicrobial Selection Guidelines for Testing and Reporting

Box with “ors” Example: Staphylococcus spp.

• Group U – Agents for treating UTIs

Azithromycin or clarithromycin or erythromycin

• 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

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 M100M100-S20; Table 1 13

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Box without “ors” Example: Pseudomonas aeruginosa Mezlocillin Ticarcillin Piperacillin

-lactams

Box includes agents for which… – Testing of one agent cannot be used to predict results for another

-lactam ring

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penicilloic acid

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CHANGE

CLSI M100M100-S20 Glossary I (Part I)

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penicillin

There are many different types of -lactams and -lactamases!

CLSI M100M100-S20; Table 1 WISCONSIN STATE LABORATORY OF HYGIENE

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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

Enterobacteriaceae Changes

• Staphylococcus spp. – Explain limitations of -lactamase testing – Define MRSA – Expand comment for testing oxacillin and cefoxitin with S. aureus and S. lugdunensis 19

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Enterobacteriaceae Revised… Breakpoints (MIC µg/ml) Agent Cefazolin Cefotaxime Ceftizoxime

CLSI M100-S19 (2009) Susc Int Res ≤8 16 ≥32 ≤8 ≤8

16-32 16-32

≥64 ≥64

Enterobacteriaceae Revised… Breakpoints (disk diffusion mm)

CLSI M100-S20 (2010) Susc Int Res ≤1 2 ≥4 ≤1 ≤1

2 2

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≥4 ≥4

Cefazolin*

CLSI M100-S19 (2009) Susc Int Res ≥18 15-17 ≤14

Cefotaxime Ceftizoxime

≥23 ≥20

Agent

15-22 15-19

≤14 ≤14

CLSI M100-S20 (2010) Susc NA

Int NA

Res NA

≥26 ≥25

23-25 22-24

≤22 ≤21

Ceftriaxone

≤8

16-32

≥64

≤1

2

≥4

Ceftriaxone

≥21

14-20

≤13

≥23

20-22

≤19

Ceftazidime

≤8

16

≥32

≤4

8

≥16

Ceftazidime

≥18

15-17

≤14

≥21

18-20

≤17

Aztreonam

≤8

16

≥32

≤4

8

≥16

Aztreonam

≥22

16-21

≤15

≥21

18-20

≤17

*disk diffusion breakpoints not yet established

CLSI M100M100-S20. Table 2A. WISCONSIN STATE LABORATORY OF HYGIENE

CLSI M100M100-S20. Table 2A. 21

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Detection of ESBLs (1)

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)

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• Initial recommendations: • Perform ESBL screen and confirmatory tests for E. coli, coli, Klebsiella spp., and Proteus mirabilis

• Based on: – Some isolates had elevated MICs in “S” range – Some (limited) data showing poor outcomes in patients with ESBL-producing isolates WISCONSIN STATE LABORATORY OF HYGIENE

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CLSI ESBL Testing Recommendations

Detection of ESBLs (2) • Now we know!

Purpose

– ESBL phenotypic tests not optimal • Presence of multiple resistance mechanisms may mask ESBL in confirmatory test

For Patient Management Perform ESBL screen and confirmatory tests

– 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 25

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Enterobacteriaceae

Revised… Carbapenem Breakpoints (MIC µg/ml) Agent

CLSI M100-S19 (2009)

Edit “S” to “R” for cephalosporins, penicillins, aztreonam For Infection Control Perform ESBL screen and confirmatory tests Edit “S” to “R” for cephalosporins, penicillins, aztreonam

If using Revised Old Breakpoints Breakpoints M100-S20 M100-S19 Yes

No

Yes

No

Yes, if requested

Yes, if requested

Yes

No 26

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Impact of Imipenem Breakpoint Changes

CLSI M100-S20 (2010) Supplement

Doripenem

Susc -

Int -

Res -

Susc ≤1

Int 2

Res ≥4

Ertapenem Imipenem

≤2 ≤4

4 8

≥8 ≥16

≤0.25 ≤1

0.5 2

≥1 ≥4

Meropenem

≤4

8

≥16

≤1

2

≥4

There will be a special CLSI M100-S20 Supplement to be published Spring 2010 with Enterobacteriaceae Tables only with these breakpoints!

Sahm, D. Eurofins Medinet, Inc. 27

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Proteus mirabilis and Imipenem

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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

Sahm, D. Eurofins Medinet, Inc. WISCONSIN STATE LABORATORY OF HYGIENE

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What steps should be included in a plan to implement revised breakpoints? ♦ Determine if AST system can accommodate revised breakpoints

AST Methods Used in Clinical Labs • Disk diffusion

- Contains low concentrations of drug? - Have a mechanism to interpret MICs with revised breakpoints (might be done with LIS)?

– 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

♦ 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 FDAFDA-cleared with the new breakpoints 31

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

OPTIONS

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 • ≥90% category (S, I, R) agreement Criteria • ≤3% very major errors?? • ≤7% combined major and minor errors ?? (establish prior to commencing validation)

Laboratory director must determine what is best for his/her laboratory and patients Implement Now?

Implement when revised breakpoints are available on laboratory’ laboratory’s commercial AST system?

Perform validation WISCONSIN STATE LABORATORY OF HYGIENE

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Acinetobacter spp.

Non-Enterobacteriaceae

• Deleted colistin / polymyxin from Table 1 •No FDA clinical indication for Acinetobacter spp. •No changes in breakpoints in Table 2B2B-2 CLSI M100M100-S20. pp. 29. WISCONSIN STATE LABORATORY OF HYGIENE

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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.”

Staphylococcus species

CLSI M100M100-S20. pp. 62.

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Staphylococcus spp. Penicillin Susceptible (2)

Staphylococcus aureus Penicillin MICs ≤0.12 µg/ml

• 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 39

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blaZ Pos

69

4

197

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Of the blaZ Pos, No.(%) Reference Induced -lactamase Pos 1/4 (25) CLSI Agenda Book 6/09 11/28 (39) Kaase et al. 2008. Clin Microbiol Infect. 14:614

Conclusion: induced β-lactamase test may not detect staphylococci that have blaZ blaZ and this could lead to treatment failures if using penicillin WISCONSIN STATE LABORATORY OF HYGIENE

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Revised recommendation… Re: vancomycin MIC, when should staphylococci be sent to a public health or reference laboratory for further testing?

Oxacillin (inducer)

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N

Staphylococci and Vancomycin

Induced ß-lactamase Test

-Sub isolate to agar (e.g., BAP, MHA) -Drop ßß-lactam disk (e.g., oxacillin, cefoxitin) -Incubate overnight -Test cells from periphery of zone -If β-lactamase positive, report penicillin R

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• S. aureus – MIC 4 µg/ml – maybe – MIC ≥8 µg/ml – yes • Coagulase-negative staphylococci (CoNS) – MIC ≥32 µg/ml – yes Pos

Neg 41

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Staphylococcus spp. - Linezolid Added… “R” Breakpoint

MIC (µg/ml) Zone (mm)

CLSI M100-S19 (2009) Susc Int Res ≤4 -

CLSI M100-S20 (2010) Susc Int Res ≤4 ≥8

≥21

≥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

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

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Definition of MRSA

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What about mecA negative 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 mecA and/or oxacillin MIC >2 µg/ml

• 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.

CLSI M100M100-S20. pp. 60. 45

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S. aureus or S. lugdunensis Testing Both OX and CX

S. aureus or S. lugdunensis Testing Both Oxacillin (OX) and Cefoxitin (CX)

Resistance mechanism None mecA

OX 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.”

S R

S R

S

R

R

S

Relative Report Prevalence as OX: S Common R Common

Uncommo mecA (low level n expression) PBP changes or hyperRare production of β-lactamase (borderline MRSA)

CLSI M100M100-S20. pp. 62. WISCONSIN STATE LABORATORY OF HYGIENE

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R R

Courtesy of Jean Patel 47

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Added… to Glossary New Subclass for Cephems Class

Subclass

Cephems

Cephalosporins with anti-MRSA activity

Agents Ceftaroline* Ceftobiprole*

Enterococcus species

*Not FDA approved as of April 2010 CLSI M100M100-S20. pp 144. WISCONSIN STATE LABORATORY OF HYGIENE

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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, nitrocefinbased -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 M100M100-S20. pp. 77. WISCONSIN STATE LABORATORY OF HYGIENE

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Revised… Streptococcus spp. β-hemolytic Group Extrapolation of Penicillin Results

♦ Extrapolate penicillin “S” result to other βlactams listed here * drugs listed have clinical indication for respective β-hemolytic streptococcal group (large colonycolonyforming strains) CLSI M100M100-S20. pp. 93. 53

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Streptococcus spp. β-hemolytic Group

“(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 M100M100-S20. pp. 93. WISCONSIN STATE LABORATORY OF HYGIENE

Streptococcus species

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Groups A, B, C, G Ampicillin Amoxicillin Amox-clav Amp-sulb Cefazolin Cefepime Cephalothin Cephradine Cefotaxime Ceftizoxime Ceftriaxone Ertapenem Imipenem Meropenem

Plus these for Group A only Cefaclor Cefdinir Cefprozil Ceftibuten Cefuroxime Cefpodoxime Cephapirin

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

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