supplemental testing

7/29/2019 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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May be

OR

De-repressed or resistant

Always

I

N

DU

C

T

I

O

N


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STAPHYLOCOCCI



10/72

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


12/72

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

M

Y

C

I

N

&

C

L

I

N

DA

M

Y

C

I

N


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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

E

R

Y

T

HR

O

M

Y

C

I

N

&

C

L

I

N

DA

M

Y

C

I

N

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

N

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

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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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

M

P

C

E

N

Z

Y

M

E

S

IMP

CAZ


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A

M

P

C

E

N

Z

Y

M

E

S

IMP

CAZ


25/72

A

M

P

C

E

N

Z

Y

M

E

S

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

A

M

P

C

E

N

Z

Y

M

E

S


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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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Inhibitor binds to

ESBL enzyme.

Prevents ESBL from

breaking down

antibiotic.

antibiotic

E

S

B

L


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Beta-lactamase inhibitors

compete

with the

beta-lactamase enzyme

E

S

B

L


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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

E

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


imipenem

Strong inducer..

but generally

not broken

down by ampC


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ampC


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)

M

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

M

E

T

H

I

CI

L

L

I

N


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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

M

E

T

H

I

CI

L

L

I

N


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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

M

E

T

H

I

CI

L

L

I

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

M

E

T

H

I

CI

L

L

I

N

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)

V

A

N

C

O

MY

C

I

N


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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

A

N

C

O

MY

C

I

N


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hVISA

V

A

N

C

O

MY

C

I

N


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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

I

N


60/72

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


Ceftazidime S

Cefepime S

key-hole effect present

Reported result

Piperacillin


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

supplemental testing

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