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?
• 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
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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)
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“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.
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• 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)
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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
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CLSI M100 contains…..
M100
Updates in
this edition
Answers to
user questions
M2 Tables
Disk Diffusion
•Test/report
•Breakpoints
•QC
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Glossary
I & II
M7 Tables
MIC
•Test/report
•Breakpoints
•QC
8
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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
• 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
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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
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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
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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
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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
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-lactams
-lactam ring
penicillin
penicilloic acid
There are many different types of -lactams and
-lactamases!
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CLSI M100-S20
Glossary I (Part I)
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CHANGE
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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
• Staphylococcus spp.
– Explain limitations of -lactamase testing
– Define MRSA
– Expand comment for testing oxacillin and cefoxitin
with S. aureus and S. lugdunensis
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Enterobacteriaceae Changes
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Enterobacteriaceae
Revised… Breakpoints (MIC µg/ml)
Agent
Cefazolin
CLSI M100-S19
(2009)
Susc
Int
Res
≤8
16
≥32
CLSI M100-S20
(2010)
Susc
Int
Res
≤1
2
≥4
Cefotaxime
Ceftizoxime
≤8
≤8
16-32
16-32
≥64
≥64
≤1
≤1
2
2
≥4
≥4
Ceftriaxone
≤8
16-32
≥64
≤1
2
≥4
Ceftazidime
Aztreonam
≤8
≤8
16
16
≥32
≥32
≤4
≤4
8
8
≥16
≥16
CLSI M100-S20.
Table 2A.
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Enterobacteriaceae
Revised… Breakpoints (disk diffusion mm)
Cefazolin*
CLSI M100-S19
(2009)
Susc
Int
Res
≥18 15-17 ≤14
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
Aztreonam
≥18
≥22
15-17
16-21
≤14
≤15
≥21
≥21
18-20
18-20
≤17
≤17
Agent
CLSI M100-S20 (2010)
Susc
NA
Int
NA
Res
NA
*disk diffusion breakpoints not yet established
CLSI M100-S20. Table 2A.
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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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Detection of ESBLs (1)
• Initial recommendations:
• Perform ESBL screen and confirmatory
tests for E. 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
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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
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CLSI ESBL Testing Recommendations
Purpose
For Patient Management
Perform ESBL screen and
confirmatory tests
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
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If using
Old
Revised
Breakpoints Breakpoints
M100-S19
M100-S20
Yes
No
Yes
No
Yes, if
requested
Yes, if
requested
Yes
No
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Enterobacteriaceae
Revised… Carbapenem Breakpoints (MIC µg/ml)
Agent
Doripenem
CLSI M100-S19
(2009)
Susc
Int
Res
-
CLSI M100-S20
(2010) Supplement
Susc
Int
Res
≤1
2
≥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!
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Impact of Imipenem Breakpoint Changes
Sahm, D. Eurofins Medinet, Inc.
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Proteus mirabilis and Imipenem
Sahm, D. Eurofins Medinet, Inc.
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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
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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
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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
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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
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OPTIONS for In-House Validation
(test system demonstrates comparable
S, I, R results to 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)
Reference
Method
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Non-Enterobacteriaceae
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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.
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Staphylococcus species
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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.”
CLSI M100-S20. pp. 62.
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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
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Staphylococcus aureus
Penicillin MICs ≤0.12 µg/ml
N
blaZ
Pos
69
4
197
28
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 and this could lead to
treatment failures if using penicillin
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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
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Pos
Neg
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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
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http://www.cdc.gov/ncidod/dhqp/pdf/ar/VRSA_testing_algo09v4.pdf
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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
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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.
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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.
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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.
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S. aureus or S. lugdunensis
Testing Both OX and CX
Resistance
mechanism
None
mecA
OX CX
S
R
S
R
S
R
R
S
Relative Report
Prevalence as OX:
Common
S
Common
R
mecA (low level
Uncommo
expression)
n
PBP changes or hyperproduction of β-lactamase
Rare
(borderline MRSA)
R
R
Courtesy of Jean Patel
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Added… to Glossary
New Subclass for Cephems
Class
Subclass
Cephems
Cephalosporins
with anti-MRSA
activity
Agents
Ceftaroline*
Ceftobiprole*
*Not FDA approved as of April 2010
CLSI M100-S20.
pp 144.
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Enterococcus species
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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 M100-S20. pp. 77.
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Streptococcus species
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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.
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Streptococcus spp.
β-hemolytic Group
♦ Extrapolate penicillin
“S” result to other βlactams listed here
* drugs listed have clinical
indication for respective
β-hemolytic streptococcal
group (large colonyforming strains)
CLSI M100-S20. pp. 93.
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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, MCLS MT(ASCP)
UCLA Medical Center
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