Antibiotics 102: Reading and Interpreting CLSI Antimicrobial Susceptibility Performance Documents

1WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE

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

• 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)

“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.

• M2, M7, and M100 describe standard M2, M7, and M100 describe standard consensusconsensus “reference methods”“reference methods” and may and may be used:be used:– By clinical labs forBy clinical labs for routine testingroutine testing

• ToTo evaluate commercial devicesevaluate commercial devices

– By drug or device manufacturers forBy drug or device manufacturers for testing testing new agents or systemsnew agents or systems

• US clinical labs can use:US clinical labs can use:– CLSI test methodCLSI test method as writtenas written– Methods thatMethods that perform comparablyperform comparably to CLSI to CLSI

“reference method” (e.g. FDA-cleared “reference method” (e.g. FDA-cleared diagnostic AST devices)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

Updates in Updates in this editionthis edition

M2 Tables M2 Tables Disk DiffusionDisk Diffusion

M7 TablesM7 TablesMICMIC

GlossaryGlossaryI & III & II

Answers to Answers to user questionsuser questions

CLSI M100 contains…..CLSI M100 contains…..

•Test/reportTest/report•BreakpointsBreakpoints•QCQC

M100M100

•Test/reportTest/report•BreakpointsBreakpoints•QCQC

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

• 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

• 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 CLSI M100-S20; Table 1

Box without “ors” Example: Pseudomonas aeruginosa

MezlocillinTicarcillinPiperacillin

Box includes agents for which… – Testing of one agent

cannot be used to predict results for another

CLSI M100-S20; Table 1CLSI M100-S20; Table 1

penicillinpenicillin penicilloic acidpenicilloic acid

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

-lactams

-lactam ring-lactam ring

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

AgentCLSI M100-S19

(2009)CLSI M100-S20

(2010)

Susc Int Res Susc Int Res

Cefazolin ≤8 16 ≥32 ≤1 2 ≥4

Cefotaxime ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftizoxime ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftriaxone ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftazidime ≤8 16 ≥32 ≤4 8 ≥16

Aztreonam ≤8 16 ≥32 ≤4 8 ≥16

CLSI M100-S20. CLSI M100-S20. Table 2A. Table 2A.

Enterobacteriaceae Revised… Breakpoints (disk diffusion mm)

AgentCLSI M100-S19

(2009)CLSI M100-S20 (2010)

Cefazolin* ≥18 15-17 ≤14 NA NA NA

Cefotaxime ≥23 15-22 ≤14 ≥26 23-25 ≤22

Ceftizoxime ≥20 15-19 ≤14 ≥25 22-24 ≤21

Ceftriaxone ≥21 14-20 ≤13 ≥23 20-22 ≤19

Ceftazidime ≥18 15-17 ≤14 ≥21 18-20 ≤17

Aztreonam ≥22 16-21 ≤15 ≥21 18-20 ≤17

*disk diffusion breakpoints not yet establishedCLSI M100-S20. Table 2A. 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 Perform ESBL screen and confirmatory tests for tests for E. coliE. coli, , KlebsiellaKlebsiella spp., and spp., and Proteus mirabilisProteus 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 BreakpointsM100-S19

Revised BreakpointsM100-S20

For Patient Management

Perform ESBL screen and confirmatory tests Yes No

Edit “S” to “R” for cephalosporins, penicillins, aztreonam Yes No

For Infection ControlPerform ESBL screen and confirmatory tests

Yes, if requested

Edit “S” to “R” for cephalosporins, penicillins, aztreonam Yes No

Enterobacteriaceae Revised… Carbapenem Breakpoints (MIC µg/ml)

AgentCLSI M100-S19

(2009)CLSI M100-S20

(2010) Supplement

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

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., 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 Manufacturers of commercial test systems are required by law to use FDA breakpointsare required by law to use FDA breakpoints

Currently, NO commercial AST system isCurrently, NO commercial AST system is FDA-cleared with the new breakpointsFDA-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

Implement Now?Implement Now? Implement when revised Implement when revised breakpoints are available on breakpoints are available on

laboratory’s commercial laboratory’s commercial AST system? AST system?

Perform Perform validationvalidation

OPTIONSOPTIONS

Laboratory director must determine what Laboratory director must determine what is best for his/her laboratory and patientsis best for his/her laboratory and patients

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

CLSI M100-S20. pp. 29.CLSI M100-S20. pp. 29.

AcinetobacterAcinetobacter spp. spp.

• Deleted Deleted colistin / polymyxincolistin / polymyxin from Table 1 from Table 1•No FDA clinical indication for No FDA clinical indication for AcinetobacterAcinetobacter spp. spp.•No changes in breakpoints in Table 2B-2No changes in breakpoints in Table 2B-2

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.”

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

NblaZ 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:Conclusion: induced induced ββ-lactamase test may not detect -lactamase test may not detect staphylococci that have staphylococci that have blablaZ and this could lead to Z and this could lead to treatment failures if using penicillin treatment failures if using penicillin

Induced ß-lactamase Test

OxacillinOxacillin(inducer)(inducer)

-Sub isolate to agar (e.g., BAP, Sub isolate to agar (e.g., BAP, MHA) MHA)

-Drop ß-lactam disk (e.g., Drop ß-lactam disk (e.g., oxacillin, cefoxitin)oxacillin, cefoxitin)

-Incubate overnightIncubate overnight-Test cells from periphery of Test cells from periphery of zonezone

-If If ββ-lactamase positive, report -lactamase positive, report penicillin Rpenicillin R PosPos NegNeg

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

Staphylococci and Vancomycin

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

Staphylococcus spp. - Linezolid Added… “R” BreakpointCLSI M100-S19

(2009)CLSI M100-S20

(2010)Susc Int Res Susc Int Res

MIC (µg/ml)

≤4 - - ≤4 - ≥8

Zone (mm) ≥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

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 = MRSA = S. aureusS. aureus with with mecmecA A and/or and/or

oxacillin MIC >2 oxacillin MIC >2 µµg/mlg/ml

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-lactamsCroes, S et al. 2009. Clin Microbiol Infect. Epub. 10/09Chambers, 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. CLSI M100-S20. pp. 62.

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

OX CX Resistance mechanism

Relative Prevalence

Report as OX:

S S None Common SR R mecA Common R

S R mecA (low level expression)

Uncommon R

R SPBP changes or hyper-

production of β-lactamase (borderline MRSA)

Rare R

Courtesy of Jean Patel Courtesy of Jean Patel

Added… to Glossary New Subclass for Cephems

Class Subclass Agents

Cephems Cephalosporins with anti-MRSA activity

Ceftaroline*

Ceftobiprole*

CLSI M100-S20. CLSI M100-S20. pp 144.pp 144.

*Not FDA approved as of April 2010

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.”

Streptococcus species

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.”

Streptococcus spp. β-hemolytic Group

Groups A, B, C, G

Plus these for Group A only

Ampicillin CefaclorAmoxicillin CefdinirAmox-clav CefprozilAmp-sulb CeftibutenCefazolin CefuroximeCefepime CefpodoximeCephalothin CephapirinCephradine

Cefotaxime

Ceftizoxime

Ceftriaxone

Ertapenem

Imipenem

Meropenem

♦ Extrapolate penicillin Extrapolate penicillin “S” result to other “S” result to other ββ--lactams listed herelactams listed here

** drugs listed have clinical drugs listed have clinical indication for respective indication for respective ββ-hemolytic streptococcal -hemolytic streptococcal group (large colony-group (large colony-forming strains)forming strains)

Acknowledgements

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

Antibiotics 102: Reading and Interpreting CLSI Antimicrobial Susceptibility Performance Documents

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WISCONSIN STATE LABORATORY OF HYGIENE 1 Antibiotics 102: Reading and Interpreting CLSI Antimicrobial Susceptibility Performance Documents Dave Warshauer,