1. Dave Warshauer, PhD, D(ABMM)
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: , 2007

4. 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)

5. “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.

6. 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)

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 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

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. 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

15. 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

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

22. 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.

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
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

27. 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!

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
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

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)
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 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)

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) 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.

39. 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

40. 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 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

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

44. 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 Antimicrob Agents Chemother. 52:2244

45. 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.

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 Clin Microbiol Infect. Epub. 10/09
Chambers, H Clin Microbiol Rev. 10:781.

47. 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.

48. 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

49. 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.

50. Enterococcus species

51. 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.

52. Streptococcus species

53. 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.

54. 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.

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