Supplemental Testing
Transcript of Supplemental Testing
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SUPPLEMENTAL
TESTING
Tan Thean Yen
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To Supplement
Definition:
add as a supplementto what seems
insufficient
"supplement your
diet"
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Why supplement?
Current methods dont work well
Additional information provided bysupplemental testing
Alternative approaches to current methods
More rapid testing results
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Why NOT supplement?
Current breakpoints are adequate
More work
Confusing
Slower testing results
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Three areas to cover
1. inducible resistancev.s. de-repressed resistance
2. Beta-lactamases& beta-lactamase inhibitors
3. Heterogenous populations
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INDUCIBLE V.S. DE-REPRESSED
Part One
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I
N
DU
C
T
I
O
N
Inducible resistance
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Inducible resistance
May be
OR
De-repressed or resistant
Always
I
N
DU
C
T
I
O
N
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STAPHYLOCOCCI
Inducible resistance
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Clindamycin & Erythromycin
Macrolide
Erythromycin
Clarithromycin
Azithromycin
Lincosamide
Clindamycin
Lincomycin
Streptogramin
Quinupristin-
Dalfopristin
Pristinamycin
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Erythromycin Clindamycin Interpretation
S SOrganism susceptible
to both
R ROrganism resistant to
both
R S May have inducibleresistance
E
R
Y
T
HR
O
M
Y
C
I
N
&
C
L
I
N
DA
M
Y
C
I
N
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E
R
Y
T
HR
O
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Y
C
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N
&
C
L
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DA
M
Y
C
I
N
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Inducible resistance
E
R
Y
T
HR
O
M
Y
C
I
N
&
C
L
I
N
DA
M
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C
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Recommendations for MLSb
Clindamycin & erythromycin discs
Staphylococci: 15-26mm apart
Streptococci: 12 mm apart
look for flattening of zone of inhibition (D-zone)
applicable to staphylococci, beta-haemolytic
streptococci, S. pneumoniae, oral streptococci
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HR
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C
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&
C
L
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DA
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C
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Leclercq R. Mechanisms of Resistance to Macrolides and Lincosamides: Nature of the Resistance Elements and Their ClinicalImplications. Clin Infect Dis 2002;34:48292.
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Recommendations for MLSb
broth dilution:
use combination of erythromycin 4 g/ml and
clindamycin 0.5 g/ml in a single well
growth = inducible resistance
OR
use disc testing methods, on purity plate.
E
R
Y
T
HR
O
M
Y
C
I
N
&
C
L
I
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DA
M
Y
C
I
N
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Other methods
Agar dilution
Sensitivity = 91%, specificity = 97%
Vitek card
Sensitivity = 91%, specificity = 100%
Lavallee, C., et al. (2010). Performance of an Agar Dilution Method and a Vitek 2 Card for Detection of Inducible Clindamycin
Resistance in Staphylococcus spp..J. Clin. Microbiol. 48: 1354-1357
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HR
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&
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DA
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BETA-LACTAMASES
Part Two
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Beta-lactamases
CLASS A:
extended-spectrum beta-lactamases
CLASS C:
ampC beta-lactamases
CLASS B:
metallo-beta-lactamases
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Chromosomal
(born with it)
G
E
N
E
A
C
Q
U
I
S
I
TI
O
N
Plasmid
(acquired)
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ampC
beta-lactamases
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Plasmid-mediated
Klebsiella spp.
Salmonella spp.
Proteus mirabilis
(E. coli)
Chromosomal
Enterobacterspp. & most
other Enterobacteriaceae
Pseudomonas aeruginosa
A
M
P
C
E
N
Z
Y
M
E
S
ampC
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ampC
A
M
P
C
E
N
Z
Y
M
E
S
chromosomal
inducible
de-repressed (always on)
plasmid genes
inducible
de-repressed (always on)
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A
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P
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N
Z
Y
M
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IMP
CAZ
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A
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N
Z
Y
M
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IMP
CAZ
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Inducible
ampC resistance
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ampC inducers
Antibiotic Inducer Hydrolysed Effect
Cefoxitin Strong Yes Resistant
Carbapenem Strong No Susceptible
Clavulanic
acidModerate - Resistant
Aztreonam Weak Yes Susceptible
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M
P
C
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N
Z
Y
M
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ampC inhibitors
Inhibitors
cloxacillin
oxacillin
boronic acid
No inhibitory effect
clavulanate
tazobactam
sulbactam
A
M
P
C
E
N
Z
Y
M
E
S
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Extended spectrum beta-lactamases (ESBL)
beta-lactamases
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Extended spectrum beta-lactamasesactually a family of
related beta-lactamases
three main groups:SHV
TEM
CTX
usually plasmid-borne
CTX
SHVTEM
E
S
B
L
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sulbactam
clavulanic acid
tazobactam
E
S
B
L
Betalactam
inhibitor
Betalactam
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ESBL & beta-lactamase inhibitors
ESBL enzyme breaks
down cephalosporin
antibiotic
E
S
B
L
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ESBL & beta-lactamase inhibitors
Inhibitor binds to
ESBL enzyme.
Prevents ESBL from
breaking down
antibiotic.
antibiotic
E
S
B
L
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ESBL & beta-lactamase inhibitors
Beta-lactamase inhibitors
compete
with the
beta-lactamase enzyme
E
S
B
L
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ESBL & beta-lactamase inhibitors
ESBL > Inhibitor=
resistant
Inhibitor > ESBL=
susceptibleE
S
B
L
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Double disk approximation
Clavulanic
acid cephalosporin
E
S
B
L
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Cefotaxime
Cefotaxime& clavulanate
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S
B
L
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Ceftazidime + Clavulanate
MIC 0.25
Ceftazidime
MIC > 32
E
S
B
L
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Supplementary methods todetect beta-lactamases
ampC and ESBL
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2 main methods
Antibiotic interactions Effect of inhibitors
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Beta-lactamases & inhibitors
Beta-lactamase
ampC
MBL
KPC
Inhibitor
cloxacillin
boronic acid
EDTA
mercaptopropionic acid (MPA)
other chelating agents
boronic acid
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Same principle
Substrate Enzyme Inhibitor
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ampC
Substrate Enzyme Inhibitor
imipenem
Strong inducer..
but generally
not broken
down by ampC
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ampC
Substrate Enzyme Inhibitor
imipenem
cefoxitin ampC cloxacillin
strong inducer ofampC
AND
broken down by ampC
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ampC
cefoxitin = small zone (disc)
(by itself) high MIC (dilution)
cefoxitin = larger zone (disc)
& cloxacillin lower MIC (dilution)
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Antibiotic 1
Ceftazidime Ceftazidime Meropenem
21 mm 21 mm 17 mm
Antibiotic 2
Ceftazidime &
clavulanate
Ceftazidime &
clavulanate
Meropenem &
EDTA
27 mm 16 mm 28 mm
Interpret-ation
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HETEROGENEOUS POPULATION
Part Three
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Heterogeneous mostly susceptible
small number of resistant
strains
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Methicillin resistance
mediated by the mecA geneM
E
T
H
I
CI
L
L
I
N
(mostly)
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Methicillin testing (disc)
S. aureus & S. lugdunensis
Oxacillin(MIC testing only)
Cefoxitin
(disc testing)
coagulase negative staph
Cefoxitin
(disc testing)
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E
T
H
I
CI
L
L
I
N
Th idi d d t S ll
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Kahl B C et al. J. Clin. Microbiol. 2003;41:410-413
Thymidine-dependent SmallColonial Variant S. aureus
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MRSA and SCVs
slow-growing, atypical phenotype
often seen in: cystic fibrosis, foreign-body
infections &osteomyelitis
susceptibility may be difficult to test
best to use pbp2a or mecA detection
Frank Kipp, Karsten Becker, Georg Peters, and Christof von Eiff. Evaluation of Different Methods To Detect MethicillinResistance in Small-Colony Variants ofStaphylococcus aureus.J Clin Microbiol. 2004 March; 42(3): 12771279
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other methods
detection of pbp2a
latex agglutination kits
immunochromatographic kits (Binax)
detection ofmecA gene by hybridisation
Evigene
detection ofmecA gene by PCR BD Diagnostics, Cepheid, Roche Molecular
Diagnostics
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H
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CI
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Other resistant S. aureus
mecCgene
confers resistance to oxacillin and beta-lactams
reliably detected by phenotypic methods*
not detected by genotypic methods for mecA
may show cefoxitin (R), Oxacillin (S) phenotype
in Vitek
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T
H
I
CI
L
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N
Skov R, et al. Phenotypic detection of mecC-MRSA: cefoxitin is more reliable than oxacillin.
J Antimicrob Chemother. Sep 2013.
Cartwright EJP, et al. Use of Vitek 2 antimicrobial susceptibility profile to identify mecC in methicillin-resistant Staphylococcus aureus. J ClinMicrobiol 2013;51:27324.
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Moderately resistant S. aureus
dont have the mecA gene
altered pbp (penicillin binding proteins)
cefoxitin (S), oxacillin (R)
rare phenotypeshould respond to drugs like augmentin,
cephalexin
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T
H
I
CI
L
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Massidda, Orietta, et al. Borderline methicillin-susceptible Staphylococcus aureus strains have more in common than reduced susceptibility topenicillinase-resistant penicillins. Antimicrobial Agents and Chemotherapy 40.12 (1996): 2769-2774.
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Vancomycin
large molecule, diffuses slowly in agar
no disc diffusion criteria for S. aureus
resistance to vancomycin:
low-level (intermediate-resistance)
high-level (vanA mediated) heterogenous (spectrum)
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A
N
C
O
MY
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hVISA: Heterogenous resistance to vancomycin
Laboratory
S. aureus isolate with
susceptible vancomycinMIC but with proportion
of cells with reduced
vancomycin susceptibility
Clinical
exposure to vancomycin
(prolonged)high bacterial load
Howden, BP., et al. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous
vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications.Clinical Microbiology Reviews 23.1 (2010): 99-139.
V
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C
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MY
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hVISA
V
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N
C
O
MY
C
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hVISA
slow growing
phenotypic variation
MIC = S
unstable phenotype
V
A
N
C
O
MY
C
I
N
Howden, BP., et al. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous
vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications.Clinical Microbiology Reviews 23.1 (2010): 99-139.
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hVISA
GRD strip
Screening plates with Va
Etest macro method
Population analysis
varying sensitivity and
specificity
? gold standard
V
A
N
C
O
MY
C
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hVISA
Susceptibility Definition Clinical Laboratory
VSSA MIC 2
VSSA with
increased MIC
(!) Potential clinical
failure
hVISA MIC 2
Screening (+)
PAP 0.9
Potential clinical
failure
Perform screening
if risk factors
present
VISA / VRSA MIC 4 Avoid vancomycin
Holmes NE, et al. Relationship between vancomycin-resistant Staphylococcus aureus, vancomycin-intermediate S. aureus, high vancomycin MIC,
and outcome in serious S. aureus infections. J Clin Microbiol 2012;50:2548
52.
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SUPPLEMENTAL TESTINGOR MIC?
No Through Road
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Traditional way
Tested result
Piperacillin
-tazobactam SCefotaxime R
Ceftazidime S
Cefepime S
key-hole effect present
Reported result
Piperacillin
-tazobactam ?Cefotaxime R
Ceftazidime R
Cefepime R
ESBL present
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Its the MIC way
Tested result
Piperacillin
-tazobactam SCefotaxime R
Ceftazidime S
Cefepime S
key-hole effect present
Reported result
Piperacillin
-tazobactam SCefotaxime R
Ceftazidime S
Cefepime S
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Why?
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For the gene
The presence of a resistance gene
makes a difference to whether or
not a particular antibiotic willwork.
Its not just the MIC.
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For the gene
Look for particular resistanceenzymes or phenotype:
ampC
ESBLMBL
Modify the susceptibilityaccording to the presence of
resistance enzymes.
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For the M.I.C.
The breakpoint for each
antibiotic determines
whether patient willrespond to that antibiotic.
It doesnt matter what the
resistance gene is.
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FOR
Life is much simpler!
Simple = more consistent
lab results
AGAINST
Is it **really** true?
Is it true for **every**
enzyme?
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Opinion!
inducible clindamycin
resistance
(blood & bone)
Enterobacterspp. and
cephalosporin susceptible(blood)
plasmid ampC
ESBL
KPC
MBL
(probably dont changetested result)
Change susceptibility ifpresent
Dont know
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Conclusions
Standard susceptibility methods work for most
organism / antibiotic combinations.
Some resistance phenotypes may need
supplemental methods to detect.
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Conclusions
Supplement or MIC alone?
(need more clinical evidence)
Its complicated.
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Tan Thean Yen