588 602-13-3SlidesPerPage

2013 Webinar Series
CLSI 2013 AST Update

2/7/2013
Speaker
Janet A. Hindler, MCLS MT(ASCP), Sr. Specialist, Clinical Microbiology, UCLA Medical Center, Los Angeles, CA
Ms. Hindler has worked as a clinical microbiologist for over 40 years, the past 35 at UCLA Medical Center, Los Angeles, CA. She
has written and taught extensively in the area of antimicrobial susceptibility testing. From 2000-2004 she held a contract with the
Centers for Disease Control and Prevention (CDC) where her focus was to develop and conduct training programs in antimicrobial
susceptibility testing. Ms. Hindler is now a consultant with the Association of Public Health Laboratories to continue in this role.
She is a fellow in the American Academy of Microbiology, member of the Clinical and Laboratory Standards Institute
Subcommittee on Antimicrobial Susceptibility testing, consultant to CAPs Microbiology Resource Committee, Past Chair of ASM
Division C and Past President of the Southern California Branch of ASM. Ms. Hindler was the 2006 recipient of ASMs bioMerieux
Sonnenwirth award for leadership in clinical microbiology and in 2007 she received an award from the Clinical and Laboratory
Standards Institute for Excellence in Standards Development. In 2011, she received the John V. Bergen award, one of CLSIs
highest honors. And in 2012, she was awarded an honorary doctoral degree from Albright College, her alma mater. Ms. Hindler
has served as a consultant to the World Health Organization and assisted in teaching individuals in developing countries about
antimicrobial susceptibility testing and antimicrobial resistance. One of Ms. Hindler's primary professional goals is to provide
antimicrobial susceptibility testing information to clinical laboratory scientists and clinicians.
Objectives
At the conclusion of this program, participants will be able to:
Identify the major changes found in the new CLSI M100-S23.
Design a strategy for implementing the new practice guidelines into their laboratory practices.
Develop a communication strategy for informing clinical staff of significant AST and reporting changes.
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2/1/2013
Evaluation: www.surveymonkey.com/s/588-602-13
Whats New in the 2013 CLSI

Standards for Antimicrobial
Susceptibility Testing (AST) ?
Janet A. Hindler, MCLS MT(ASCP)

UCLA Health System
jhindler@ucla.edu
and consultant with the Association of

Public Health Laboratories
Objectives
List the major changes found in the

new M100-S23.
Describe a strategy for
implementation of the new practice
guidelines in your laboratory, as
appropriate.
jhindler clsi update 2013 1

2/1/2013
CLSI AST Standards

January 2013
M100-S23 Tables (2013)1
M02-A11 Disk Diffusion Method (2012)2

M07-A9 MIC Method (2012)2
M11-A7 Anaerobe MIC Testing (2007)
1 M100 updated at least yearly

2 M02, M07 updated every 3 years
4
Summary of
Changes
M100-S23 Page 15.
Major Changes 2013 (1)

Enterobacteriaceae
Expanded recommendations for interpreting / reporting
fluoroquinolones on Salmonella spp.
Staphylococcus spp.
Eliminated breakpoints (interpretive criteria) for all -
lactams except oxacillin, cefoxitin, and penicillin; added
breakpoints for ceftaroline (cephalosporin with anti-
MRSA activity)
Eliminated oxacillin disk diffusion test for
Staphylococcus aureus Group

2/1/2013

Neisseria gonorrhoeae
Increased emphasis on susceptibility testing in select
settings to identify emerging cephalosporin resistance
Streptococcus pneumoniae
Added test for inducible clindamycin resistance
Added breakpoints for doxycycline; revised breakpoints
for tetracycline
Streptococcus spp. -hemolytic Group
Expanded testing / reporting recommendations for
inducible clindamycin resistance

Quality Control
Quality control vs. verification
New alternative recommendation for going from daily to
weekly QC testing schedule
Revised resistant QC strain testing frequency for
screening tests
Additional Intrinsic Resistance Tables
Non-Enterobacteriaceae
Staphylococcus spp.
Enterococcus spp.
CLSI Breakpoint
Additions / Revisions
Since 2010
Newer breakpoints
now referred to as
current!
Previous breakpoints can be

found in the version of M100
that precedes the document
listed here, eg, previous
breakpoints for aztreonam are
listed in M100-S19 (January
2009).
M100-S23. Page 23.

9

2/1/2013
Breakpoint Revision Reminders!

Both CLSI and FDA set breakpoints for USA
Criteria for establishing breakpoints differ
slightly between CLSI and FDA
Clinical laboratories can use either CLSI or
FDA breakpoints
If using FDA-cleared commercial AST system, clinical
laboratory must perform verification for CLSI
breakpoints on that system
Manufacturers of commercial AST devices

must use FDA breakpoints!
10
What is the status of FDA breakpoint

revisions?
CLSI and FDA breakpoints now the same
for:
Ceftriaxone Enterobacteriaceae (2010)
Ertapenem Enterobacteriaceae (2012)
Imipenem Enterobacteriaceae and
Pseudomonas aeruginosa (2012)
Check with manufacturer of your

commercial AST system for status!
11
Ceftaroline
12

2/1/2013
Ceftaroline (1)
Broad-spectrum cephalosporin (with anti-
MRSA activity)
IV only
FDA clinical indications
Acute bacterial skin and skin structure infections
(ABSSSIs)
Community-acquired bacterial pneumonia (CABP)
E. coli, Klebsiella pneumoniae, Klebsiella oxytoca
Bactericidal against MRSA due to its affinity
for PBP2a and against penicillin-non-
susceptible Streptococcus pneumoniae due to
its affinity for PBP2x
13
Ceftaroline (2)
Not active against ESBL, ampC (class C), or
carbapenemase (e.g., KPC) producing
Enterobacteriaceae
No significant activity against Pseudomonas
aeruginosa
Some activity against anaerobes (not
Bacteroides fragilis Group)
14
Ceftaroline Breakpoints (g/ml) (1)

CLSI1,2 FDA2,3
Organism
S I R S I R
Enterobacteriaceae 0.5 1 2 0.5 1 2
Clinical efficacy shown for
E. coli, K. pneumoniae,
and K. oxytoca
CABP and skin isolates
only
Staphylococcus aureus 1 2 4 1 - -
S. aureus only, including Includes MRSA
MRSA Skin isolates only
1 M100-S23 Tables 2A and 2C

2 Corresponding disk diffusion breakpoints also defined
3 Ceftaroline Prescribing Information - see Drugs@FDA.gov
15

2/1/2013
Ceftaroline Breakpoints (g/ml) (2)

CLSI1,2 FDA2,3
Organism
S I R S I R
Haemophilus 0.5 - - 0.12 - -
influenzae
H. Influenzae only CABP isolates only
Streptococcus 0.5 - - 0.25 - -
pneumoniae
Nonmeningitis CABP isolates only
pyogenes
Skin isolates only
agalactiae
Skin isolates only
1 M100-S23 Tables 2E, 2G, 2H-1
2 Corresponding disk diffusion breakpoints also defined
3 Ceftaroline Prescribing Information - see Drugs@FDA.gov
4 -hemolytic streptococci (Table 2H-1); no species qualifier
16
Why do CLSI breakpoints differ slightly

from those of FDA?
S. aureus
CLSI added I and R because of strains with MICs of
2, 4, and 8 g/ml reported outside the US
S only breakpoint will not allow for testing variability
and create difficulties for labs when strains with MICs >
1 g/ml are encountered
Global surveillance - ceftaroline %S ( 1 g/ml):
MRSA (n=2849) 98.4%
MSSA (n=5088) 100%
S. pneumoniae, -hemolytic streptococci,
Haemophilus spp.
PK-PD data support breakpoint of 0.5 g/ml and even
playing field for ceftaroline vs. other cephalosporins
17
MSSA
CLSI June 2012 Agenda Book

18

2/1/2013
MRSA
CLSI June 2012 Agenda Book

19
Staphylococcus spp. Table 2C
Comment (17) not intended for patient reports;

discuss with infectious diseases, pharmacy, etc. when
developing / revising AST /reporting protocols
M100-S23. Page 75.

20
Ceftaroline vs. Other -Lactams

%Susceptible
Organism N Ceftaroline Ceftriaxone Imipenem
MSSA 186 100 100 100
MRSA 215 100 0 0
E. coli 1075 93.6 94.8 99.9
Klebsiella
pneumoniae 596 86.1 86.7 94.6
Streptococcus
pneumoniae 894 100 90.8 NA
Haemophilus
influenzae 381 100 100 NA
NA, not available

Jones et al. 2011. J Antimicrob Chemother. 66 (Suppl 3):iii69iii80.
21

2/1/2013
Cetaroline Placement
in Tables 1A and 1B
AndGroup C for
H. influenzae
S. pneumoniae
-hemoltyic streptococci
Table 1A
Drugs to Test/Report
M100-S23. Page 34.

22
When should we test ceftaroline?

When requested, for species with breakpoints
Note: may be prescribed for MRSA and used
to cover other species in confirmed or
suspected mixed infection
How can we test ceftaroline
(FDA cleared as of 2/1/13)
Disk diffusion
Hardy Diagnostics
MIC
Sensititre TREK MIC panels
Etest FDA cleared for S. aureus only
23
Salmonella spp.
Testing / reporting recommendations
Fluoroquinolones
24

2/1/2013
Salmonella Current Taxonomy

Two major species
Salmonella bongori (uncommon in human infections)
Salmonella enterica
Six subspecies including Salmonella enterica subsp.
enterica
>2500 serovars
Salmonella enterica subspecies enterica serovar Typhi

Salmonella enterica serovar Typhi
Salmonella ser. Typhi
Salmonella Typhi or S. Typhi
Typhoidal Salmonella = S. Typhi and S. Paratyphi A-C
WHO Collaborating Centre for Reference and Research on Salmonella
25
Salmonella Infections
Typhoidal
Require antimicrobial therapy from any source
Usually ceftriaxone or fluoroquinolones in adults
Non-typhoidal
Systemic sources require antimicrobial therapy
Gastroenteritis
Usually self-limiting
Therapy NOT recommended due to prolongation of carrier state
Therapy often indicated for:
Severe diarrhea
Patients with underlying medical conditions (e.g.,
immunosuppression)
26
Salmonella spp.
AST and Reporting (1)
(2) When fecal isolates of Salmonella and Shigella spp. are
tested, only ampicillin, a fluoroquinolone, and
trimethoprim-sulfamethoxazole should be reported
routinely. In addition, for extraintestinal isolates of
Salmonella spp., a third-generation cephalosporin
should be tested and reported, and chloramphenicol
may be tested and reported if requested.
M100-S23. Page 44.
Also reminder WARNING: For Salmonella spp., first- and second-

generation cephalosporins, cephamycins and aminoglycosides may
appear active in vitro, but are not effective clinically and should not
be reported as susceptible.
M100-S23. Page 30.
27

2/1/2013
Salmonella spp.
AST and Reporting (2)
(2) Susceptibility testing is indicated for typhoidal
Salmonella (S. Typhi and Salmonella Paratyphi
AC) isolated from extraintestinal and
intestinal sources. Routine susceptibility
testing is not indicated for nontyphoidal
Salmonella spp. isolated from intestinal
sources.
M100-S23. Page 44.
28
What is the issue for fluoroquinolones

(FQs) and Salmonella?
Clinical response rates to ciprofloxacin are
poorer for isolates with decreased
ciprofloxacin susceptibility
(MICs of 0.12 1.0 g/ml)
Crump et al. 2008. Antimicrob Agents Chemother. 52:1278.
Parry et al. 2010. Antimicrob Agents Chemother. 54:5201.
MICs of 0.12 1.0 g/ml are S with
standard Enterobacteriaceae breakpoints
29
Salmonella spp.
Fluoroquinolone AST and Reporting
CLSI Standard Fluoroquinolone Breakpoints
M100-S21 One set of breakpoints for all Enterobacteriaceae
(2011) including Salmonella spp.
Nalidixic acid screen for reduced ciprofloxacin
susceptibility in extraintestinal isolates of
Salmonella spp.
M100-S22 Lower ciprofloxacin breakpoints for S. Typhi and
(2012) extraintestinal Salmonella spp.
M100-S23 Lower ciprofloxacin, levofloxacin and ofloxacin
(2013) breakpoints for use with all Salmonella spp.
Details described in:

Humphries et al. 2012. CID 55:1107-13.
30

2/1/2013
Salmonella spp.
Fluoroquinolone Resistance
Phenotype
Genotype Ciprofloxacin
Nalidixic Acid
MIC (g/ml)
Wild type (No resistance) 0.008-0.06 Usually susceptible
Chromosomal gyrA (single mutation) 0.12 - 2.0 Usually resistant
Chromosomal gyrB (single mutation) 0.12 0.5 Usually susceptible
Chromosomal gyrA, gyrB (multiple
4.0 Resistant
mutations)
PMQR (e.g. qnr or aac(6)-lb-cr) 0.12 - 2.0 Often susceptible
PMQR, plasmid-mediated quinolone resistance - newer mechanism and

less common than chromosomal gyrase mutations
31
Enterobacteriaceae (Table 2A)
Salmonella spp. and

Fluoroquinolones
M100-S23. Pages 48-49. 32
Enterobacteriaceae (Table 2A)

Antimicrobial DD (mm) MIC (g/ml)
Comments
Agent Susc Int Res Susc Int Res
Enterobacteriaceae
21 16-20 15 1 2 4 other than
Salmonella spp.
Ciprofloxacin
Salmonella spp.
0.12-
31 21-30 20 0.06 1 (including S. Typhi
0.5
and Paratyphi A-C)
Enterobacteriaceae
17 14-16 13 2 4 8 other than
Salmonella spp.
Levofloxacin
Salmonella spp.
- - - 0.12 0.25-1 2 (including S. Typhi
and Paratyphi A-C)
Salmonella spp.
Ofloxacin - - - 0.12 0.25-1 2 (including S. Typhi
and Paratyphi A-C)
33

2/1/2013
Salmonella spp. - Nalidixic Acid Test

Nalidixic acid 31 21-30 20 0.06 0.12-0.5 1
(37) Until laboratories can implement the current interpretive criteria for
ciprofloxacin, levofloxacin, and/or ofloxacin, nalidixic acid may be
used to test for reduced fluoroquinolone susceptibility in
Salmonella. Strains of Salmonella that test resistant to nalidixic acid
may be associated with clinical failure or delayed response in
fluoroquinolone-treated patients with salmonellosis.
Note that nalidixic acid may not detect all mechanisms of

fluoroquinolone resistance.
M100-S23. Page 49.

34
Salmonella spp. USA 2010

Distribution of Ciprofloxacin MICs
Wild Type Decreased Res
Susc
USA Data
*National Antimicrobial Resistance Monitoring System (NARMS)
http://www.cdc.gov/narms/
35
Salmonella spp. % Susceptible

USA 2010
Non-typhoidal S. Paratyphi
Antimicrobial Breakpoint S. Typhi
Salmonella spp. A-C
Agent (g/ml) (n=444)
(n=2474) (n=146)
Ampicillin 8.0 90.9 87.6 97.9
Ceftriaxone 1.0 97.2 100 100
Ciprofloxacin 0.06 97.3 31.1 9.6
Trimeth-sulfa 2/38 98.4 88.1 97.9
*National Antimicrobial Resistance Monitoring System (NARMS)

http://www.cdc.gov/narms/
36

2/1/2013
When should we test Salmonella spp.?

Extraintestinal isolates
Typhoidal Salmonella from all sources
Other when requested (select patient
populations?)
How can we test Salmonella spp. and
fluoroquinolones?
Of commercial AST systems, only Etest currently
encompasses new low MIC breakpoints
Ciprofloxacin disk diffusion
Nalidixic acid but doesnt capture all isolates with
reduced fluoroquinolone susceptibility
37
Staphylococcus spp.
Eliminate all -lactam breakpoints except
oxacillin, cefoxitin, penicillin, ceftaroline
Eliminate oxacillin disk test for S. aureus Group
38
Staphylococcus spp. M100-S22. Table 2C.
Eliminate breakpoints for:

Penicillins
-lac inhibitor combos
Cephems (oral)
Cephems (parenteral)
Carbapenems
39

2/1/2013
Staphylococcus spp.
-Lactam Breakpoints Remaining
Penicillin
Represents penicillinase-labile penicillins
Oxacillin
Represents penicillinase-stable penicillins
Cefoxitin
Surrogate for oxacillin
Ceftaroline (added 2013)
Cephem with anti-MRSA activity
40
Why did CLSI remove other -lactam

breakpoints for Staphylococcus spp.?
Can predict results for -lactams with
established clinical efficacy for staphylococcal
infections by testing penicillin and oxacillin /
cefoxitin (CLSI recommendation since 1991)
Need to distinguish new cephems with activity
against MRSA from cephems with MSSA
activity only (eg, ceftaroline)
Breakpoints deleted had never been
extensively evaluated specifically for
Staphylococcus spp.
41
Staphylococcus spp. - -Lactams

Test Results
Oxacillin Predicts
Penicillin
(cefoxitin)
Susceptible to:
All penicillins
S S -lac / -lactamase inhibitor combos
Cephems
Carbapenems
Resistant to:
Penicillinase-labile penicillins
Susceptible to:
R S Penicillinase-stable penicillins
-lac / -lactamase inhibitor combos
Cephems
Carbapenems
Resistant to:
R R All -lactams (except cephems with anti-
MRSA activity, e.g., ceftaroline)
42

2/1/2013
-Lactams with
Antistaphylococcal Activity (1)
Penicillinase-labile Penicillinase-stable
Penicillins Penicillins
Amoxicillin Cloxacillin
Ampicillin Dicloxacillin
Carbenicillin Flucloxacillin
Mezlocillin Methicillin
Penicillin Nafcillin
Piperacillin Oxacillin
Ticarcillin
43
-Lactams with
Antistaphylococcal Activity (2)
-lactam/-lactamase
Cephems Carbapenems
Inhibitor Combinations
Cefaclor Cefoxitin Doripenem Amoxicillin-clavulanate
Cefamandole Cefpodoxime Ertapenem Ampicillin-sulbactam
Cephalexin Cefprozil Imipenem Ticarcillin-clavulanate
Cefazolin Ceftazidime Meropenem Piperacillin-tazobactam
Cefdinir Ceftizoxime
Cefepime Ceftriaxone
Cefmetazole Cefuroxime
Cefonicid Cephalothin
Cefoperazone Loracarbef
Cefotaxime Moxalactam
Cefotetan
44
Cefoxitin Disk Diffusion for S. aureus

and S. lugdunensis
Cefoxitin detects mecA-

mediated MRSA better than
oxacillin
Cefoxitin (R) Test cefoxitin as a surrogate
10 mm zone
- Report OXACILLIN, not
Zone (mm) cefoxitin
Drug
R I S
Cefoxitin 21* - 22**
Eliminate oxacillin disk diffusion
Oxacillin 10 11-12 13
test for S. aureus and S.
lugdunensis
* Report as oxacillin resistant
** Report as oxacillin susceptible
45

2/1/2013
Staphylococcus spp.
M100-S23. Page 75. 46
Staphylococcus spp.
(24) For staphylococci that test susceptible, aminoglycosides
are used only in combination with other active agents that test
susceptible.
Gentamicin Test / Report Group C - Supplemental (report on special

request)
Not widely recommended to use gentamicin in combination therapysee
Liu et al. 2011. Clin Infect Dis. 52:1
If gentamicin reported, consider adding comment to patient report re: not
to use gentamicin alone
M100-S23. Page 77. 47
What -lactams should we test / report against

staphylococci?
Oxacillin / cefoxitin
Penicillin
Test / report routinely?
Report if R; suppress if S and add comment
Contact laboratory if penicillin results needed?
Perform -lactamase test if penicillin S prior to
reporting penicillin result
Ceftaroline as requested
How can we test -lactams against staphylococci?
Oxacillin MIC only
Cefoxitin disk diffusion; MIC (S. aureus only)
Penicillin disk diffusion or MIC
48

2/1/2013
Emerging cephalosporin resistance
49
August 12, 2012
50
Antimicrobial Resistance
Therapeutic Detection of Mechanism of No Longer
Agent Resistance Resistance Recommended
1979 lactamase
CMRNG
Penicillin (chromosomally- 1987
mediated resistant
1983
N. gonorrhoeae);
altered PBP,
permeability
Mutations in gyrA
Fluoroquinolone Early 1990s 2007
and parC
2012 (cefixime
Cephalosporins 2009 penA mosaic
alone)
51

2/1/2013
M100-S23. Page 38.
i. Culture and susceptibility testing of N.

gonorrhoeae should be considered in
cases of treatment failure. Antimicrobial
agents recommended for testing
include, at a minimum, those agents
listed in Group A. The most recent CDC
guidelines for treatment and testing are
available at
http://www.cdc.gov/std/Gonorrhea/.
Routine testing not necessary
52
Uncomplicated GC Treatment
Recommendations
Ceftriaxone 250 mg IM in a single dose
(or if not an option, cefixime 400 mg PO in
a single dose)
PLUS
Azithromycin 1 g PO in a single dose
Or Doxycycline 100 mg PO 2x daily for 7
days
Note: two drugs with different mechanisms of action may improve

treatment and delay emergence of ceftriaxone resistance.
MMWR August 10, 2012.

53
GC Treatment Failure
Test of cure is recommended for treatment
failure:
Symptoms persisting after completing
recommended therapy
Positive culture 72 hours after
recommended therapy
Positive NAAT 7 days after recommended
therapy
Recent report of cefixime treatment
failures associated elevated cefixime MICs
Allen et al. 2013. JAMA. 309:163.
54

2/1/2013
Ceftriaxone MIC Distributions
0.03 g/ml www.eucast.org

55
Percentage of urethral Neisseria gonorrhoeae isolates (n = 32,794) with

elevated cefixime MICs (0.25 g/mL) and ceftriaxone MICs (0.125 g/mL) -
Gonococcal Isolate Surveillance Project, United States, 2006August 2011.
*cefixime not tested 2007-2008 Jan August 2011
MMWR August 10, 2012.

56
AST Methods
CLSI Reference Methods
Agar dilution MIC
Disk diffusion
Media & Incubation Conditions
GC agar base + 1% defined growth supplement
36C (do not exceed 37C), 5% CO2, 20-24 h
Interpret (see M100-S23 Table 2F for other drugs)
1
Cefixime 31 - - 0.25 - -
1
Ceftriaxone 35 - - 0.25 - -
1CDC uses 0.125 g/mL for non-susceptibility

Etest
57

2/1/2013
What should we do about testing /

reporting for GC today?
NAAT for routine diagnosis
Maintain capability of performing C&S when
treatment failure suspected (reference lab
option?)
Decreased susceptibility to cephalosporins
and/or cephalosporin therapeutic failures should
be reported to public health department
58
Inducible Clindamycin Resistance

-hemolytic Streptococcus spp.
59
Some Indications for Clindamycin

Therapy in 2013
Penicillin-allergic patients
E.g. dental infections, aspiration pneumonia, lung
abscess, head and neck infections, pelvic infections
Intrapartum prophylaxis for highly penicillin-allergic
women colonized with Group B Streptococcus
Strep throat penicillin therapy failures
Bone and joint infections
Notes:
Often more use in peds than adults
Concern Clostridium difficile
60

2/1/2013

Staphylococcus aureus
Known poor outcome with clindamycin therapy for
isolates with inducible clindamycin resistance
Lewis et al. 2005. Clin Infect Dis. 40:280.
-hemolytic streptococci
New data (J Jorgensen. June 2012. CLSI meeting presentation)
Four human cases of infection (inducible clindamycin resistance)
did not resolve on clindamycin therapy
2 Streptococcus agalactiae, 1 Group A streptococcus,
1 Group C streptococcus
No isolate had constitutive clindamycin resistance
after failed clindamycin therapy
-hemolytic streptococci and S. pneumoniae
Recent animal model data suggest inducible clindamycin
resistance is clinically significant
61
-hemolytic Streptococci & S. pneumoniae

Resistance Mechanism Determinant Erythro Clinda
Efflux mef R S
Ribosome modification1 erm R S2
R
Ribosome modification erm R
constitutive
1 known as MLSB resistance phenotype (M, macrolide; L, lincosamide; S,
streptogramin B)
2 requires induction to show resistance
mef = macrolide efflux
erm = erythromycin ribosome methylase
62
Neutropenic Murine Thigh Model
#1 GBS Isolate #1
Constitutive Clindamycin R
#2 GBS Isolate #2
Inducible Clindamycin R
(early killing then regrowth)
GBS Isolate #3
#3
Clindamycin Susceptible
CLSI Agenda book, June 2012 (WA Craig) 63

2/1/2013
Neutropenic Murine Thigh Model
#1 Spneu Isolate #1
Constitutive Clindamycin R
#2
Spneu Isolates #2 & #3
Inducible Clindamycin R
#3 (early killing then regrowth)
#4 Spneu Isolate #4
Clindamycin Susceptible
CLSI Agenda book, June 2012 (WA Craig) 64
-hemolytic Streptococci & S. pneumoniae

Inducible Clindamycin Resistance Testing
Only if Erythromycin-R and Clindamycin-S
D-Zone Test:
Routine disk diffusion method 12 mm
Place 2 g clindamycin disk 12 E CC
mm from edge of 15 g
erythromycin disk
No induction
Broth microdilution test 1 well
1 g/mL erythromycin and 0.5
g/mL clindamycin in same well E CC
Induction
65
What should we do about testing /

reporting for inducible clindamycin
resistance?
Add
Organism Test1
Comment 2
Staphylococcus aureus Yes No
Coag-neg staphylococci If requested Optional
-hemolytic streptococci If requested Optional
Streptococcus pneumoniae If requested Optional
1 Report clindamycin R if inducible clindamycin test is positive

2 This isolate is presumed to be clindamycin resistant based on
detection of inducible clindamycin resistance
66

2/1/2013
-hemolytic Streptococcus spp.

Clindamycin & Group B Streptococcus from
Prenatal Screens
67
Many
footnotes!
Routine testing not necessary
M100-S23. Page 38.

68
Streptococcus spp. -hemolytic Group

Table 1B
p. ..When Group B Streptococcus is isolated

from a pregnant woman with severe penicillin
allergy (high risk for anaphylaxis), erythromycin
and clindamycin, (including inducible
clindamycin resistance) should be tested, and
only clindamycin should be reported.
M100-S23. Page 40.

69

2/1/2013
http://www.cdc.gov/groupbstrep/about/prevention.html
70
Check for inducible

clindamycin R if
erythromycin-R and
clindamycin-S
71
More on GBS Prenatal Screen

(notes from bottom of algorithm)
.and testing for inducible clindamycin

resistance has been performed and is negative
(no inducible resistance), then clindamycin can
be used for GBS intrapartum prophylaxis
instead of vancomycin.
72

2/1/2013
Table 2H-1 Supplemental

Table 1. Testing for Inducible
Clindamycin Resistance in -
hemolytic Streptococci
AST of -hemolytic Streptococci

not needed routinely
If clindamycin needed, must test for
inducible clindamycin resistance
Test / report clindamycin on
prenatal screens (penicillin allergic)
M100-S23. Page 116.

73
When should we perform AST on -

hemolytic Streptococci? What drugs
should we report?
Group A Streptococcus
When requested (penicillin allergic patient)
Report erythromycin, clindamycin
Group B Streptococcus
Prenatal screen isolates from penicillin
allergic patients
Report clindamycin
74
Inducible clindamycin resistance
Tetracycline and doxycycline
Penicillin results and other -lactams
75

2/1/2013
Table 2G Supplemental Table 1.

Testing for Inducible
Clindamycin Resistance in
If clindamycin tested, should

screen for inducible clindamycin
resistance
M100-S23. Page 109.

76
Streptococcus pneumoniae (n=1021)

Phenotypic Tests vs. ermB PCR
ermB PCR N D-zone pos Broth pos2
positive 66 653,4 643,5
negative 36 0 0
1 erythromycin-R and clindamycin-S

2 1 g/ml erythromycin and 0.5 g/ml clindamycin
3 1 isolate ermB pos but negative in both phenotypic tests
4 Sensitivity: 98.5% Specificity: 100%
5 Sensitivity: 97% Specificity: 100%
Jorgensen et al. 2011. J Clin Microbiol. 49:3332.

77
Why add breakpoints for doxycycline

and S. pneumoniae?
Doxycycline is an option for treating
community-acquired pneumonia
Mandell et al. 2007. Clin Infect Dis. 44:S27
Note: some isolates may be susceptible to
doxycycline but resistant to tetracycline
Doxycycline 28 25-27 24 0.25 0.5 1 New
Tetracycline 28 25-27 24 1 2 4 Revised
MIC data and PK/PD data used to set breakpoints

78

2/1/2013
CLSI Rationale Document

Doxycycline and
Tetracycline Breakpoints for
S. pneumoniae
http://www.clsi.org/wp-content/uploads/2013/01/Doxycycline-
and-Tetracycline-Breakpoints-for-Streptococcus-pneumoniae.pdf
79

Doxycycline MIC (g/ml) vs. tetM
CLSI Doxy-Tet Rationale Doc.

80

Tetracycline MIC (g/ml) vs. tetM
CLSI Doxy-Tet Rationale Doc.

81

2/1/2013
Extrapolating Other -Lactams from Penicillin
(5) For nonmeningitis isolates, a penicillin MIC of 0.06 g/mL (or oxacillin
zone 20 mm) can predict susceptibility to the following -lactams:
Ampicillin Cefepime Cefuroxime
Ampicillin-sulbactam Cefotaxime Doripenem
Amoxicillin Cefpodoxime Ertapenem
Amoxicillin-clavulanate Cefprozil Imipenem
Cefaclor Ceftaroline Loracarbef
Cefdinir Ceftizoxime Meropenem
Cefditoren Ceftriaxone Penicillin
Deleted: Penicillin MICs 2 g/mL indicate susceptibility to parenteral

penicillin, amoxicillin, amoxicillin-clavulanic acid, cefepime, cefotaxime,
ceftriaxone, and ertapenem.
Only extrapolate if penicillin MIC is 0.06 g/ml

or oxacillin zone is 20 mm
M100-S23. Page 105.
82
Penicillin vs. Cefotaxime or
Amoxicillin MICs
Strains with mosaic PBP genes can
have
Low penicillin MICs (<1 g/ml) and high
cefotaxime MICs (2-32 g/ml)
Low penicillin MICs (1-2 g/ml) and higher
amoxicillin MICs (4-8 g/ml)
McDougal et al. 1995. Antimicrob Agents Chemother. 39:2282.
Ruiz et al. 1998. Antimicrob Agents Chemother. 42:2768
Doit et al. 1999. Antimicrob Agents Chemother. 43:1480
83
When should we report doxycycline on S.

pneumoniae?
When AST is performed, if drug is on panel,
and stakeholders agree it should be reported
routinely; non-CSF isolates.
When can we extrapolate penicillin S
results to other -lactam agents?
For non-meningitis, if penicillin MIC is 0.06
g/mL and drug of interest is listed in CLSI
M100-S23 Table 2G (5).
84

2/1/2013
Quality Control
QC vs. verification
Daily to Weekly QC
QC for screening tests
85
M100-S23. Page 32.
QC
Verification
To ensure AST quality, lab must perform:

QC - routine
Verification when new AST system introduced or
new drug added to existent AST system
CLIA requirement per CLIA 42 CFR 493.1253[b]
Reference - ASM Cumitech 31A (2009) 86
QC Recommendations
Previously.
in Tables 2A-2G Minimum QC
now Routine QC
M100-S23. Page 44.

87

2/1/2013
Frequency of Routine QC Testing

Options
Each day test is performed
Weekly following documentation of
successful daily QC
20-30 day plan
New 3 x 5 day plan
For weekly QC, documentation needed when:

Add New AST system
Add new drug
Modify certain test variables see M100-S23 Tables 3C & 4F
88
Routine AST QC New Option

Convert from Daily to Weekly QC Schedule
(for each drug / organism combination)
3 x 5 day plan
Perform 3 tests each day for 5 days (15 results)
Use 3 separate inoculum preparations for each
replicate / day
Statistically comparable to current plan (20-30
day) for identifying problems
Advantages
Possibly identify problems quicker
Complete QC testing quicker
Utilize fewer resources
89
3 x 5 Day QC Plan 2 Phases

Phase 1 test 3 replicates for 5 days (15 results)
PASS if 0-1 of 15 results out of range
FAIL if 4 of 15 results out of range
Go to Phase 2 if 2-3 of 15 results out of range
Phase 2 test another 3 replicates for 5 days (15

more results)
PASS if 2-3 of 30 results out of range
FAIL if 4 of 30 results out of range
90

2/1/2013
3 x 5 Day
QC Plan
Pass Fail
M100-S23. Pages
136 & 152.
Statistical summary here.

http://www.clsi.org/wp-content/uploads/2012/11/June-2012-Attachment-2.pdf
91
Have accrediting agencies acknowledged

the new 3 x 5 day QC plan?
Not at this time but CMS is considering
adoption of the plan. Other accrediting
agencies (e.g., CAP) could accept the new
plan following publication in CLIA
regulations.
92
AST Screening Tests 1

QC Frequency
QC Strain OLD (M100-S22) New (M100-S23)
Negative
Test routinely Test routinely
(susceptible)
Positive Test with each new

Test routinely batch / lot / shipment of
(resistant) testing materials
1 Applies to disks, or agar plates used for agar dilution, or

single wells or tubes used with broth dilution methods
93

2/1/2013
Staphylococcus aureus Group M100-S23.

Page 85.
Table 2C Supplemental Table 1
Susceptible strain
QC routine
Resistant strain
QC Lot/shipment
94
Disk Diffusion QC Range Changes

Pseudomonas aeruginosa ATCC 27853
Acceptable Range
Antimicrobial Agent
(mm)
Gentamicin 17-23
Tobramycin 20-26
M100-S23. Pages 130-131.

95
Intrinsic Resistance Tables

Enterobacteriaceae a few changes / additions
Non-Enterobacteriaceae new
Staphylococcus spp. - new
Enterococcus spp. - new
M100-S23. Page 176-179.
96

2/1/2013
Check Q&A more on:

QC
Verification
M100-S23. Page 196.

97
Additional CLSI Changes and Topics

Under Evaluation (1)
Continuing reevaluation of breakpoints
Enterobacteriaceae cefepime
Acinetobacter and other non-Enterobacteriaceae -
carbapenems
Fluoroquinolones
and others
Colistin / Polymyxin B
Reexamine best method for testing these difficult
agents (international collaboration)
98
Additional CLSI Changes and Topics

Under Evaluation (2)
Improve QC recommendations
Expand availability / user friendliness of concise
Rationale Documents behind new/modified
recommendations
Reformat M100 booklet
CLSI AST Subcommittee welcomes new volunteers!
99

2/1/2013
An online interactive searchable version of

M100-S23 enables you to easily
Access M100-S23 all you need is a web connection
Dynamically filter by tables, organisms, and agents
View only the information you want to see
Customize for your institutions formulary
Easily find additional CLSI AST standards and
guidelines within the eM100 Resource Center
Available at www.CLSI.org
100
CLSI
Website
Find information
from AST meetings
Order CLSI AST
products
http://www.clsi.org/standards-
development/microbiology/
subcommittee-on-ast/
101
The following summary slides will

not be discussed and are
presented for participants
review.
102

2/1/2013
Summary (1)
CLSI updates AST tables (M100) each January.
CLSI updates documents that describe how to perform
reference disk diffusion (M02) and reference MIC (M07)
tests every 3 years.
Changes to CLSI documents are summarized in the front
of each document.
Information listed in boldface type is new or modified
since the previous edition of M100.
Recent breakpoint addition/revision dates are listed in the
front of M100-S23.
Manufacturers of commercial AST systems, by law, MUST
use FDA breakpoints.
103
Summary (2)
FDA breakpoints have recently been revised for ceftriaxone
and ertapenem (Enterobacteriaceae) and for imipenem
(Enterobacteriaceae and Pseudomonas aeruginosa).
M100-S23 contains breakpoints for ceftaroline, a broad-
spectrum cephalosporin with anti-MRSA activity, for
several organism groups.
Routine susceptibility testing is not indicated for
nontyphoidal Salmonella spp. isolated from intestinal
sources.
M100-S23 includes unique breakpoints for ciprofloxacin,
levofloxacin and ofloxacin for use when testing any
Salmonella spp.
104
Summary (3)
Reduced susceptibility to ciprofloxacin is most
commonly found in typhoidal strains of Salmonella spp.
The only -lactams with breakpoints for Staphylococcus
spp. in M100-S23 are penicillin, oxacillin, cefoxitin, and
ceftaroline.
When testing Staphylococcus spp., results for anti-
staphylococcal -lactams other than penicillin, oxacillin,
cefoxitin, and ceftaroline can be deduced from testing
penicillin and oxacillin (or cefoxitin).
The oxacillin disk diffusion test is no longer considered
reliable for any Staphylococcus spp. Cefoxitin disks are
used to detect methicillin-resistant S. aureus and
coagulase-negative staphylococcus.
105

2/1/2013
Summary (4)
Emerging resistance to cephalosporins in Neisseria
gonorrhoeae is a significant public health concern.
Laboratories should maintain the ability to provide culture
and susceptibility testing of N. gonorrhoeae for patients
who are considered treatment failures. This might include
reference laboratory alignment as an alternative to in
house testing.
Inducible clindamycin resistance has been reported to be
clinically significant for S. aureus.
More recently, studies have shown inducible clindamycin
resistance can be clinically significant for Streptococcus
pneumoniae and -hemolytic streptococci.
106
Summary (5)
Testing for inducible clindamycin resistance in
erythromycin-R and clindamycin-S isolates of S. aureus,
S. pneumoniae and -hemolytic streptococci should be
performed if clindamycin is reported.
AST should be performed on isolates of Group B
streptococci from highly penicillin-allergic women. This
should include testing for inducible clindamycin
resistance, and only clindamycin (not erythromycin)
should be reported.
Doxycycline can now be tested against S. pneumoniae,
when indicated.
Results for -lactams other than penicillin (nonmeningitis
isolates) can be deduced from S. pneumoniae that have
penicillin MICs 0.06 g/ml or oxacillin zones 20 mm.
107
Summary (6)
To ensure AST quality, laboratories must perform routine
QC testing. In addition, verification studies are required
when a new AST system is added, a new drug is added to
an existent AST system, or there is a change in certain
testing variables.
Routine QC testing can be done daily or weekly; the latter
providing there is documentation that daily QC has been
satisfactorily performed.
An alternative to documenting daily QC by use of the 20-
30 day QC plan is the new 3 x 5 day QC plan.
For screening tests, QC of the susceptible strain must be
performed routinely but the resistant QC strain need only
be tested with each new lot/shipment of testing materials.
108

2/1/2013
Summary (7)
M100-S23 now contains tables that define intrinsic
resistance profiles for Enterobacteriaceae and also non-
Enterobacteriaceae, Staphylococcus spp. and
Enterococcus spp.
Minutes of CLSI AST Subcommittee meetings and other
materials are available at www.clsi.org.
CLSI and other groups welcome help with improving
susceptibility testing!
109
And thanks to:

APHL Staff
CLSI Staff
CLSI Subcommittee on AST especially:
Jean Patel
Jim Jorgensen
Susan Munro
110

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2013 Webinar Series

CLSI 2013 AST Update

Whats New in the 2013 CLSI

Janet A. Hindler, MCLS MT(ASCP)

and consultant with the Association of

List the major changes found in the

jhindler clsi update 2013 1

CLSI AST Standards

M100-S23 Tables (2013)1

M02-A11 Disk Diffusion Method (2012)2

1 M100 updated at least yearly

M100-S23 Page 15.

Major Changes 2013 (1)

jhindler clsi update 2013 2

Major Changes 2013 (2)

Major Changes 2013 (2)

Previous breakpoints can be

M100-S23. Page 23.

jhindler clsi update 2013 3

Breakpoint Revision Reminders!

Manufacturers of commercial AST devices

What is the status of FDA breakpoint

Check with manufacturer of your

jhindler clsi update 2013 4

Ceftaroline Breakpoints (g/ml) (1)

1 M100-S23 Tables 2A and 2C

jhindler clsi update 2013 5

Ceftaroline Breakpoints (g/ml) (2)

Why do CLSI breakpoints differ slightly

CLSI June 2012 Agenda Book

jhindler clsi update 2013 6

CLSI June 2012 Agenda Book

Staphylococcus spp. Table 2C

Comment (17) not intended for patient reports;

M100-S23. Page 75.

Ceftaroline vs. Other -Lactams

NA, not available

jhindler clsi update 2013 7

M100-S23. Page 34.

When should we test ceftaroline?

jhindler clsi update 2013 8

Salmonella Current Taxonomy

Salmonella enterica subspecies enterica serovar Typhi

Also reminder WARNING: For Salmonella spp., first- and second-

jhindler clsi update 2013 9

M100-S23. Page 44.

What is the issue for fluoroquinolones

Details described in:

jhindler clsi update 2013 10

PMQR, plasmid-mediated quinolone resistance - newer mechanism and

Enterobacteriaceae (Table 2A)

Salmonella spp. and

Enterobacteriaceae (Table 2A)

jhindler clsi update 2013 11

Salmonella spp. - Nalidixic Acid Test

Antimicrobial DD (mm) MIC (g/ml)

Note that nalidixic acid may not detect all mechanisms of

M100-S23. Page 49.

Salmonella spp. USA 2010

Salmonella spp. % Susceptible

Ceftriaxone 1.0 97.2 100 100

Ciprofloxacin 0.06 97.3 31.1 9.6

Trimeth-sulfa 2/38 98.4 88.1 97.9

*National Antimicrobial Resistance Monitoring System (NARMS)