2
M02-A11 Disk Diffusion Method (2012)^
M100 -Summary of Major Changes
#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 document.
#Dates of the recent breakpoint additions/revisions are
listed in the front of M100-S22.
3
Today’s Review:
M100 2012 changes
#Enterobacteriaceae
#Pseudomonas aeruginosa
#Staphylococcus species
4
Enterobacteriaceae -2012
1. Clarification on when to perform ESBL testing -briefly
2. Clarification on when to perform MHT
3. Re-revised ertapenem breakpoints for 2012
#Changed from new ones published in 2011
4. Added ciprofloxacin breakpoints for use with S. typhi and extraintestinal
isolates of Salmonella spp.
5
1
Susan Sharp, Ph.D.
Director, Kaiser Permanente Laboratory
MHT: Classification of Carbapenemases
Class
Carbapenemase
Found in Notes
A KPC
Enterobacteriaceae Hydrolyze all ?-lactams.Inhibited by clavulanic acid.SME
S. marcescens B
Metallo beta-lactamases
(IMP, VIM, GIM, SPM, NDM )
P. aeruginosa Enterobacteriaceae Acinetobacter S. Maltophilia
Hydrolyze all ?-lactams except aztreonam.
Somewhat inhibited by clavulanic acid.
Require zinc for enzymatic activity; inhibited by EDTA.
7
Reference: Queenan & Bush. 2007. Clin Microbiol Rev. 20:440
2. Clarification on when to perform MHT
Value of Modified Hodge test (MHT) for
Carbapenemase Detection
MHT
Carbapenemase (53)
Positive (N=35)
Negative (N=18)a Positive 24 (69%)11 (61%)Negative 7(20%)7 (39%)Not interpretable
4 (11%)
-
8
Reference: Girlich et al. 2012. J Clin Microbiol. 50:477
Sensitivity 77.4%; Specificity 38.9%. Better for KPC; poor for NDM-1
a
AmpC overproducers or ESBL +/-membrane permeability defect
CRE Examples
Organism MIC (μg/ml) 1
MHT Resistance
mechanism Ertap Imip Mero E. coli >16 R 4 R 4 R Pos Plasmid amp C
K. pneumoniae >16 R ≤0.25 S 8 R Pos ESBL E. coli >16 R 8 R >16 R Neg NDM-1K. pneumoniae
2 R
1 S
2 I
Pos
IMP-4
9
References: Anderson, KF et al. 2009. ICAAC. D-719
Limbago, BM. CLSI Agenda book. January 2011
10
Until laboratories can implement the current carbapenem
breakpoints, the Modified Hodge Test (MHT) should be performed as described ...
After implementation of the current breakpoints, MHT does not need to be performed other than for epidemiological or infection control purposes.
Not all carbapenemase-producing isolates of Enterobacteriaceae are MHT positive, and MHT-positive results may be encountered in isolates with carbapenem resistance mechanisms other than carbapenemase production.
Clarification on when to perform MHT: “Modified Carbapenemase Comment”
Reference: M100-S22; Table 2A, pages 45, 53 & 57
Why did CLSI revise ertapenem breakpoints for
13
14
OLD 2011 <0.25/0.5/>1.0NEW 2012 <0.5/1.0/>2.0
15
CLSI Document
MIC (μg/ml)
Disk Diffusion (mm)Susc
Int Res Susc Int Res M100-S20 (Jan. 2010) ≤24≥8≥1916-18≤15M100-S20U (June 2010) ≤0.250.5≥1≥2320-22≤19M100-S22 (Jan 2012)**
≤0.5
1.0
≥2
≥22
19-21
≤18
Enterobacteriaceae -Ertapenem Breakpoint Review
The story….
#
#
If fluoroquinolone-S, test nalidixic acid to detect reduced
fluoroquinolone susceptibility #
If nalidixic acid-R…..
#
Inform clinician that…
#“The isolate may not be eradicated by fluoroquinolone treatment; infectious diseases consult suggested.”
17
Salmonella spp. Extraintestinal Isolates: Nalidixic Acid -Old Recommendations
Reference: M100-S21. Table 2A. Page 46.
Salmonella spp.
Fluoroquinolone Resistance
Genotype
Phenotype
Nalidixic Acid
Ciprofloxacin
MIC (μg/ml)
No resistance genes
Usually
Susceptible ≤0.06
gyr A (single mutation) 1 chromosomal Usually Resistant 0.12 -1.0 gyr A, gyr B (multiple mutations)chromosomal
Resistant ≥4 qnr +/-; aac(6’)-lb-cr 2plasmid (newer mechanism)
Often Susceptible
0.12 -1.0
18
1 Low level resistance (reduced susceptibility); not detected with “standard”Enterobacteriaceae ciprofloxacin
breakpoints
2 qnr genes encodes for proteins that protect DNA-gyrase; aac(6’)1b-cr proteins modify quinolones making
them ineffective. Reports of delayed response to ciprofloxacin therapy or treatment failure with S. typhi and extraintestinal infections with these mechanisms.
Salmonella spp. USA 2009% S to Ciprofloxacin at MICs
Organism N
Ciprofloxacin MIC (μg/ml)
(%)≤0.06
0.12-1.0≥2.0Salmonella spp.(non-typhoidal)219297.7 2.20.1Salmonella typhi
361
39.9
56.5
3.6
19
https://www.doczj.com/doc/723900374.html,/narms/
CDC’s National Antimicrobial Resistance Monitoring System (NARMS)
20
Reference: CLSI Agenda Book January 2011
Enterobacteriaceae :
2012 Ciprofloxacin Breakpoints
Organism
DD (mm)MIC (μg/ml)
Susc Int Res Susc Int Res Enterobacteriaceae other than S. typhi and extraintestinal Salmonella spp. (remains same as for other enterics)≥21
16-20
≤15
≤1
2
≥4
S. typhi and extraintestinal Salmonella spp.
≥3121-30≤20≤0.060.12-0.5≥1
21
Reference: M100-S22. Table 2A. Page 48
2012New
Eliminates the need to screen with Naladixic acid
S. typhi and Extraintestinal Salmonella spp:Ciprofloxacin Testing
#Optimal to do ciprofloxacin MIC
#
The low MIC ranges reflected in new 2012 breakpoints may not be available on some automated systems –consider Etest?
#
If doing disk diffusion, consider testing nalidixic acid and ciprofloxacin #Nalidixic acid does best for isolates with gyrase mutations (gyrA)#Ciprofloxacin does best for isolates with plasmid-encoded gene mutations #
Breakpoints for other fluoroquinolones (e.g., levofloxacin) under evaluation
22
S. typhi & Extraintestinal Salmonella spp:Nalidixic Acid
#Nalidixic acid test, although not optimal, remains in 2012 M100-S22#
NOTES:
#
“Strains of Salmonella that test resistant to nalidixic acid may be associated with clinical failure or delayed response in fluoroquinolone-treated patients with extraintestinal salmonellosis.”
#
“However, nalidixic acid may not detect all mechanisms of fluoroquinolone resistance. “
#
CLSI considered deleting nalidixic acid test, however, it was retained due to plea from Latin American countries and others where S. typhi is more common and sophisticated susceptibility testing is lacking.
23
Reference: M100-S22. Table 2A. Page 48
Pseudomonas aeruginosa
1. Beta-lactam antibiotics:#Lowered breakpoints for:
#piperacillin
#piperacillin-tazobactam #ticarcillin
#
ticarcillin-clavulanic acid
2. Carbapenems:
#Lowered breakpoints for:
#imipenem #
meropenem
#
Added breakpoints for doripenem
24
#
Reference: Tam et al. 2008. Clin Infect Dis. 46:862
22.2%
85.7%
30.0%
20.5%
“Susceptible”
Example: Piperacillin -tazobactam MIC distribution Blue = wild type isolates
Red = isolates with acquired “R ”mechanism
Reference: https://www.doczj.com/doc/723900374.html,
P.aeruginosa
Old Breakpoints S <64 R >128
28
Pseudomonas aeruginosa:
Breakpoint Revisions
Agent Old M100-S21New M100-S22
Susc Int Res Susc Int Res Piperacillin ≤64-≥128≤1632-64≥128Piperacillin-tazobactam ≤64/4-≥128/4≤16/432/4-64/4≥128/4Ticarcillin ≤64-≥128≤1632-64≥128Ticarcillin-clavulanate
≤64/2
-≥128/2
≤16/2
32/2-64/2
≥128/2
Reference: M100-S22. Table 2B-1. Page 63
Corresponding DD breakpoints also revised.
1. Lowered breakpoints for Beta-lactam antibiotics:
#
P. aeruginosa MIC breakpoints are now the same as those for Enterobacteriaceae (with slight differences in disk diffusion breakpoints).
#
No more necessity for combination therapy when organisms are within the new, lowered “S”range. #
Deleted :
#
Comment from Table 2B-1 -“Rx:The susceptible category for ?-lactam implies the need for high-dose therapy for serious infections caused by P. aeruginosa . For these infections, monotherapy has
been associated with clinical failure.”
% MIC (N=7,846 Sentry 2005-09)
30
Reference: CLSI Agenda Book June 2011
Susc
Int
Res
OLD <4/8/>16NEW <2/4/>8
P. aeruginosa -% S
Old vs. New Carbapenem Breakpoints
(N=7,846 Sentry Surv. 2005-09)
Agent
% S
M100-S21 2011<4 / 8 / >16M100-S22 2012<2 / 4 / >8
Doripenem -76 Imipenem 7470Meropenem
79
72
31
32
Pseudomonas aeruginosa :Breakpoint Revisions
Agent
Old (M100-S21)
New M100-S22
Susc
Int Res
Susc Int Res Doripenem None ≤24≥8Imipenem ≤48≥16≤24≥8Meropenem
≤4
8
≥16≤2
4
≥8
Reference: M100-S22. Table 2B-1. Page 63
Corresponding DD breakpoints also revised.
Staphylococcus species
#
Penicillin testing
33
Staphylococcus spp. –Penicillin
The story…..
#
> 90% of staphylococci are penicillin “R”
#
Penicillin rarely considered for treatment of staphylococcal
infections #…BUT -Penicillin might be considered for infections
requiring lengthy therapy (e.g., endocarditis, osteomyelitis) IF penicillin were known to be “S”#
Some Staphylococcus spp. that test “S”by MIC or disk diffusion may possess a ?-lactamase (BL) and may fail penicillin therapy
34
36
-Sub isolate to blood agar
-Induction: Drop disk to induce BL production (e.g., oxacillin or cefoxitin)Pos Neg
Staphylococcus aureus
?-lactamase (BL)
#Induced nitrocefin BL test usually, but not always, detects
staphylococcal BL
#Other BL tests are more sensitive for BL:
#Cloverleaf test
#Penicillin disk zone edge test
#bla Z gene PCR not optimal for BL
#bla Z codes for BL production
#Several types of bla Z genes
37
Staphylococcus aureus
?-lactamase (BL) Study
#348 MSSA (low penicillin MICs) characterized for bla Z by PCR:
#303 PCR negative
#45 PCR positive
#Methods:
#Penicillin MICs
#Phenotypic BL tests
#Nitrocefin -Cefinase
#Nitrocefin -Dryslide
#Cloverleaf assay
#Penicillin disk zone edge
38
*Statens Serum Institut (Denmark), CDC (Atlanta), MGH (Boston)
Staphylococcus aureus
?-lactamase (BL) Study
Pen MIC
(μg/ml)
bla Z functional
Neg Pos
0.0082
0.01615
0.0321801
0.06905
0.121517
0.25114
0.54
1.0
2.02
4.01
30345
?1 bla Z neg and penicillin “R”
?23 bla Z pos and penicillin “S”
Reference: CLSI Agenda Book January 2011.
S
R
40
?5% sheep blood agar
?S. aureus ATCC 25923 as the
indicator organism
Isolates A-D are all
BL positive
A
B
C
D
BL negative
41
?-lactamase
positive
?-lactamase
negative
aureus
Disk Zone Edge Test (10 U penicillin disk and standard disk diffusion method)
?-lactamase negative,
S. aureus QC:
Neg -ATCC 25923
Pos -ATCC 29213
Reference: M100-S22. Table 2C Supplemental Table 1. Page 83
Staphylococcus aureus
3 Lab BL Study Results (N=348)
Test Sensitivity Specificity
Cefinase77%100%
Dryslide88%100%
Cloverleaf100%100%
Penicillin disk zone edge96%100%
43 Reference: CLSI Agenda Book January, 2011
Staphylococcus spp. –Penicillin
Optional Strategy
#Report penicillin if “R”
#Suppress penicillin if “S”and add note “Contact laboratory
if penicillin results needed”
#If penicillin “S”and penicillin results needed, perform:
#S. aureus
#Nitrocefin BL test , and if negative%
#Penicillin zone edge test
#CoNS (including S. lugdunensis)
#Induced nitrocefin BL test
45
Summary
#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-S22.
#Minutes of CLSI AST Subcommittee meetings and other materials
are available at https://www.doczj.com/doc/723900374.html,.
46
Action Items -1
#Doripenem breakpoints have been added to M100-S22. --Results
from testing other carbapenems cannot be used to predict results
for doripenem.
#QC ranges for P. aeruginosa ATCC 27853 with cefepime have
been revised.
# E. coli ATCC 25922 with colistin have been revised.
#Ertapenem breakpoints for Enterobacteriaceae have been revised.
47
#Clinical laboratories that have not implemented current CLSI
breakpoints for cephalosporins and aztreonam for
Enterobacteriaceae should continue to perform ESBL testing.
#Clinical laboratories that have not yet implemented current
CLSI breakpoints for carbapenems and Enterobacteriaceae
should continue to perform Modified Hodge test.
#New ciprofloxacin breakpoints have been added to
Enterobacteriaceae tables be used for S. typhi and Salmonella
spp. from extraintestinal sources.
48
Action Items -2
#
Applying new breakpoints for S. typhi and Salmonella spp. from extraintestinal sources is preferred to nalidixic acid testing to detect reduced fluoroquinolone susceptibility.
#Breakpoints for piperacillin, piperacillin-tazobactam, ticarcillin,
ticarcillin-clavulanic acid, imipenem, and meropenem have been revised for P. aeruginosa .
#
S. aureus isolates where penicillin zones are ≥29 mm or penicillin MICs are ≤0.12 μg/ml, a penicillin ‘disk zone edge test’should be performed before reporting penicillin susceptible.
49
Action Items -3
What are some of the issues currently being addressed by CLSI AST Subcommittee?
#Fluoroquinolone breakpoints for several organism groups #
Quality control
#Frequency for MIC and disk diffusion tests; frequency for screen tests #
QC ranges for colistin / polymyxin B; optimal medium for testing
#
Intrinsic resistance tables for non-Enterobacteriaceae , for gram-+ bacteria #
Enterobacteriaceae
#Levofloxacin breakpoints for S. typhi and extraintestinal Salmonella spp.#Cefepime breakpoints
#
Colistin / polymyxin B breakpoints
#
Staphylococcus spp.
#
Eliminate oxacillin disk diffusion test for S. aureus #
Streptococcus pneumoniae
#Doxycycline and tetracycline breakpoints #
Inducible clindamycin resistance
50
谢谢。
51