GRAM NEGATIVE ORGANISMS: KEY AST CHALLENGES

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GRAM NEGATIVE ORGANISMS: KEY AST CHALLENGES Amanda T. Harrington, PhD, D(ABMM) Associate Professor, Pathology Director, Clinical Microbiology Laboratory Loyola University Medical Center

Transcript of GRAM NEGATIVE ORGANISMS: KEY AST CHALLENGES

Page 1: GRAM NEGATIVE ORGANISMS: KEY AST CHALLENGES

GRAM NEGATIVE ORGANISMS:

KEY AST CHALLENGES

Amanda T. Harrington, PhD, D(ABMM)

Associate Professor, Pathology

Director, Clinical Microbiology Laboratory

Loyola University Medical Center

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Discuss current challenges regarding

antimicrobial susceptibility testing for Gram

negative organisms

Discuss basic limitations to assessing

carbapenem resistance in GNRs

Discuss antimicrobial susceptibility testing

and reporting strategies for GNRs

OBJECTIVES

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ENTEROBACTERIACEAE

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Cefazolin testing to predict oral cephalosporins for

uUTI

ESBLs and AmpCs

Carbapenem Resistance

THE CHALLENGES

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Cefazolin:

Surrogate Agent to Predict Oral Cephs

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Infectious Diseases Society of America Practice Guideline

re: β-lactams for Acute uUTI in Women

http://www.idsociety.org

β-lactams including

amox-clav,

cefdinir,

cefaclor,

cefpodoxime

are choices when other agents (e.g.,

nitrofurantoin, trimeth-sulfa, fosfomycin,

fluoroquinolone), cannot be used

Other β-lactams (e.g., cephalexin) are

less well studied but may also be

appropriate in certain settings.

uUTI = uncomplicated urinary tract infection

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CEFAZOLIN % SUSCEPTIBLE

WITH 2010 AND PRE-2010 BREAKPOINTS

54

69

11

72

81 83

0

20

40

60

80

100

E. coli (n=581) K. pneumoniae

(n=313)

P. mirabilis

(n=169)

% S

usce

pti

ble

"New" Cefazolin <=2

"Old" Cefazolin <=8

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M100 -S25 TABLE 1 A

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Test/ Report

Group Agent

MIC Breakpoint (µg/ml) Comments

Susc Int Res

Cephems (Parenteral)

A Cefazolin ≤2 4 ≥8 based on dose of 2 g every 8 h

Cephems (Oral)

U Cefazolin* ≤16 - ≥32 Footnote (20)

ENTEROBACTERIACEAE CEFAZOLIN

* “surrogate” agent

CLSI M100-S25 Table 2A

• (20) Cefazolin predicts results for the oral agents - cefaclor, cefdinir,

cefpodoxime, cefprozil, cefuroxime axetil, cephalexin, and loracarbef when

used for therapy of uncomplicated UTIs due to E. coli, K. pneumoniae, and

P. mirabilis.

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PRESCRIBING CEFAZOLIN VS. MIC

INTERPRETATION

IM, IV administration

Introduced 1973; today, mostly used for:

MSSA

Prophylaxis for some surgical procedures

If GNR known “S” (only E. coli, K. pneumoniae, P. mirabilis): Uncomplicated UTIs; bacteremia

M100-S25. Table 2A.

Reason for Testing Breakpoints (µg/ml)

Dose S I R

Predict cefazolin use for uUTI ≤16 - ≥32 1 g every 12 h

(IM or IV)

Predict cefazolin use for systemic

infections ≤2 4 ≥8

2 g every 8 h (IM or IV)

Surrogate for oral cephalosporins to

use for uUTI ≤16 - ≥32 PO (various)

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ampicillin >32 R

cefazolin 8

ciprofloxacin >2 R

nitrofurantoin ≤16 S

trimeth-sulfa >4/76 R

SPECIMEN: URINE

DIAGNOSIS: CYSTITIS

E. COLI

MIC (µg/ml)

How should cefazolin MIC of 8 µg/ml be

interpreted and reported for treatment

of UTI?

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ampicillin >32 R

cefazolin 8 S

ciprofloxacin >2 R

nitrofurantoin ≤16 S

trimeth-sulfa >4/76 R

SPECIMEN: URINE

DIAGNOSIS: CYSTITIS

E. COLI

MIC (µg/ml)

“Cefazolin results should only be used to predict potential

effectiveness of oral cephalosporins (eg, cephalexin) for

treating uncomplicated urinary tract infections.”

Final Report with

Optional Comment

Example 1

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ampicillin >32 R

oral cephalosporins S

ciprofloxacin >2 R

nitrofurantoin ≤16 S

trimeth-sulfa >4/76 R

MIC (µg/ml)

“Oral cephalosporins include cephalexin,

cefpodoxime, cefdinir.”

Final Report with

Optional Comment

Example 2

SPECIMEN: URINE

DIAGNOSIS: CYSTITIS

E. COLI

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ampicillin >32 R

cefazolin 8 R

ceftriaxone ≤0.5 S

ciprofloxacin >2 R

gentamicin 1 S

piper-tazobactam ≤ 8 S

trimeth-sulfa >4/76 R

SPECIMEN: BLOOD

DIAGNOSIS: PYELONEPHRITIS

E. COLI

MIC (µg/ml)

“Cefazolin susceptible results are based on a dose of

2 g every 8 h.”

Final Report with

Optional Comment

Example 3

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Commercial Systems

Most not FDA cleared for new breakpoints (BPs)

Some panels do not have low dilutions for

systemic breakpoints

Verify new breakpoints? urine? systemic?

Determine when testing for systemic isolates

needed

LIS

Does your system have flexibility?

CEFAZOLIN – CHALLENGES

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Test urine isolates with commercial system

(ex. Vitek 2)

Test other isolates (blood) with broth

microdilution (low dilution wells)

E. coli, Klebsiella spp. and Proteus mirabilis

Report “oral cephalosporins” on urine isolates

Report “cefazolin” on special request

CEFAZOLIN – ONE POTENTIAL SOLUTION

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ESBLs and AmpC

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β-lactamases -

Enterobacteriaceae

AmpC and ESBL

ESBL (plasmid)

E. coli Klebsiella P. mirabilis

Other organisms

AmpC (chromosome)

Induction of AmpC β-lactamase

Selection of stably derepressed AmpC-R mutants

AmpC transfer to plasmid

No CLSI

endorsed test for

AmpC!

CLSI ESBL test

for E, K, P…

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Agent Old Breakpoints Current Breakpoints

Susc Int Res Susc Int Res

Cefazolin ≤8 16 ≥32 ≤2 4 ≥8

Cefotaxime ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftizoxime ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftriaxone ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftazidime ≤8 16 ≥32 ≤4 8 ≥16

Cefepime ≤8 16 ≥32 ≤2 4 ≥8

Aztreonam ≤8 16 ≥32 ≤4 8 ≥16

ENTEROBACTERIACEAE - CEPHALOSPORINS

CLSI BREAKPOINTS (MIC µG/ML)1

M100-S25 Table 2A.

1 CLSI also revised corresponding disk diffusion breakpoints

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REVISED CEPHALOSPORIN /

AZTREONAM BREAKPOINTS

If you are using the “old” breakpoints (pre -2010) you

must continue to test for ESBLs

Minimal impact on overall %S for each agent

Considerable reluctance to eliminate ESBL testing

Infection prevention groups still want the information

Still not much data for “ESBL” isolates treated with

susceptible cephalosporin

More controversial than carbapenem

breakpoint changes!

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AmpC

Hydrolyze ceftriaxone, cefotaxime,

ceftazidime, cephamycins (cefoxitin) NOT

cefepime

Not inhibited by -lactamase inhibitors

Chromosomal in SPICE or MYSPACE species

“S” isolates can become R during therapy

E. coli, Klebsiella, P. mirabilis and Salmonella

can acquire ampC on plasmid

AMPC -LACTAMASES

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“SPICE” BUGS*

Serratia marcescens

Pseudomonas aeruginosa

Indole positive Proteeae –

Morganella morganii & Providencia spp.

Citrobacter freundii

Enterobacter spp.

Concern – de-repression of AmpC = resistance develops on therapy

*Also Hafnia alvei, Aeromonas spp., Yersinia, Acinetobacter baumannii and

certain non-fermenting gram-negative rods

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Testing vs. no testing for AmpC or ESBL

Use current breakpoints vs. old

breakpoints

SEVERAL APPROACHES

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ESBL IN MYSPACE BUG

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PLASMID MEDIATED AMPC IN E.COLI

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Selective Reporting:

ceftriaxone for E. coli / Klebsiella / P. mirabilis

cefepime for SPICE organisms (poor inducer of AmpC)

IF doctor calls, report ceftriaxone with disclaimer

“Due to the high likelihood of selecting resistant mutants,

ceftriaxone for serious infections caused by Enterobacter

spp. may result in high level resistance”

Additional Comments based on testing:

Inducible beta-lactamase

Plasmid mediated beta-lactamase

SEVERAL APPROACHES

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Carbapenemase Detection

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“WE HAVE A WEIRD-LOOKING

KLEBSIELLA PNEUMONIAE ON BLOODS”

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BMD AST RESULTS: KLEBSIELLA

PNEUMONIAE

Agent

Amikacin 32 I

Aztreonam, cefepime, cetazidime, ceftriaxone

>32 R

Ciprofloxacin >2 R

Ertapenem >16 R

Gentamicin, tobramycin >10 R

Imipenem >16 R

Meropenem >16 R

Minocycline >32 R

Piperacillin-tazobactam >128 R

Trimeth-sulfa >4/76 R

Tigecycline 2 S

Colistin >16 ?

Isolate MHT+

“This isolate has unusual

carbapenem resistance results.

Infectious Diseases consultation

strongly suggested”

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WHAT IS A CRE?

- Any Enterobacteriaceae or just Klebsiella

pneumoniae?

- Resistant to any carbapenem, all carbapenems,

select carbapenems?

- Resistant vs. not-susceptible?

- Must have a carbapenemase?

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WHAT IS A CRE?

Current CDC Surveillance Definition

Enterobacteriaceae that are:

Resistant to one or more of the

following: doripenem, ertapenem,

meropenem, or imipenem* OR

Production of a carbapenemase detected

by PCR. MHT, Carba-NP, metallo-beta-

lactamase test

31 *Proteus/Providencia/Morganella exceptions for imipenem

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Clinically important Often resistant to multiple classes

of antibiotics

Pan-resistant CRE have been described

Associated with high mortality rates (up to 70%) May be > 50% in ICU patients

Combination therapy appears to improve outcomes

Epidemiologically important Highly transmissible

Have spread throughout healthcare settings across the United States, (endemic in some areas)

Potential for CRE to become widespread if not contained

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WHY ARE CRE IMPORTANT?

PR

AK

HI

States with CRE confirmed by CDC, 2013

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Mechanism testing is not recommended for guiding therapeutic decisions

Not routinely performed in many U.S. clinical laboratories

However, carbapenemase-producing CRE (CP-CRE) are currently believed to be primarily responsible for the increasing spread of CRE in the United States

Phenotypic definitions based bacteria’s antibiotic susceptibility pattern primary way clinical laboratories and infection prevention teams attempt to identify CRE

No phenotypic definition will perform perfectly in distinguishing carbepenemase producers

Will l ikley increase measured CRE prevalence due to the fact that a larger number of non-CP-CRE will meet the current definition

CHALLENGES

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Between carbapenem-resistant Enterobacteriaceae (CRE) and carbapenemase-producing (CP)-CRE?

The production of carbapenemases (called CP-CRE), enzymes that break down carbapenems and related antimicrobials making them ineffective.

This includes enzymes like Klebsiella pneumoniae carbapenemase (KPC)

The combination of mechanisms other than carbapenemase production (called non-CP-CRE), most commonly the production of beta-lactamases (e.g., AmpC) in combination with alterations in the bacteria’s cell membrane (e.g. , porin mutations).

WHAT IS THE DIFFERENCE…

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BMD AST RESULTS: ENTEROBACTER

CLOACAE

Agent

Amikacin 16 S

Aztreonam, cefepime, cetazidime, ceftriaxone

>32 R

Ciprofloxacin >2 R

Ertapenem 0.5 S

Gentamicin, tobramycin 4 S

Imipenem 1 S

Meropenem >16 R

Minocycline >32 R

Piperacillin-tazobactam >128 R

Trimeth-sulfa >4/76 R

Tigecycline

Colistin

Isolate MHT+ ?

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REVIEW: MECHANISMS OF BETA-LACTAM

RESISTANCE IN ENTEROBACTERIACEAE

Remember! Not all CRE

have a “carbapenemase”

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CARBAPENEMASES Class Examples Produced by: Notes

A

ESBLs [TEM, SHV, CTX-M] KPC carbapenemases

SME carbapenemases

K. pneumoniae and other Enterobacteriaceae

S. marcescens

Most inhibited by clavulanic acid Usually plasmid-mediated (not SME)

B

Metallo-β-lactamases (MBL) (e.g. NDM, VIM, IMP, GIM, SPM carbapenemases)

P. aeruginosa

Enterobacteriaceae

Acinetobacter

S. maltophilia

Inhibited by EDTA Do not hydrolyze aztreonam

C AmpC Enterobacteriaceae Some non-fermenters

Inducible in some genera Not inhibited by clavulanic acid

D

OXA carbapenemases (e.g. OXA-48, -181, -232)

Acinetobacter baumannii

Enterobacteriaceae

Hydrolyze carbapenems to some degree

Adapted from Queenan & Bush. 2007. Clin Microbiol Rev. 20:440.

Bush & Jacoby. 2010. AAC. 54:969; Bush, K. 2013. Ann NY Acad Sci 1277:84. Slide from Janet Hindler

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MHT Carba NP Molecular

Use Enterobacteriaceae Enterobacteriaceae

P. aeruginosa

Acinetobacter

Enterobacteriaceae

P. aeruginosa

Acinetobacter

Strengths Simple Rapid Determines type of

carbapenemase

Limitations Some false pos (eg,

ESBL/ampC + porin)

Some false neg

(eg NDM)

Enterobacteriaceae

only

Special “fresh”

reagents

Some invalid results

False neg for OXA-

type carbapenemase

Special reagents

Specific to targeted

gene

Introduction to Tables 3B and 3C. Tests for

Carbapenemases in Enterobacteriaceae, Pseudomonas

aeruginosa, and Acinetobacter spp.

CLSI M100-S27. --now includes mCIM; will discuss later today

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NO

Still MDR

Still risk for infection control

YES

Hospitals may have different practices for non-CP CRE

New antimicrobial agents designed to target certain mechanisms

Mixing CP CRE and non-CP CRE ‘confuses’ the epidemiological

landscape

IS DETECTION OF CRE THAT DO/DON’T

PRODUCE CARBAPENEMASE

IMPORTANT?

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DO LABORATORIES NEED TO DO

CARBAPENEMASE MECHANISM TESTING?

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Cefazolin can be used as a surrogate agent to predict the activity of the oral cephalosporins for the treatment of uUTI caused by E. coli, Klebsiella, P. mirabilis

ESBL testing is no longer required if current cephalosporin / aztreonam breakpoints are used

Strategic reporting of ESBLs and AmpCs may be beneficial to individual hospital systems; consult with colleagues before changing practice

CRE is challenging, but there are a variety of practices available to help optimize patient care

SUMMARY

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NONFERMENTING GRAM

NEGATIVE RODS

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P. aeruginosa and Acinetobacter baumannii are

common nosocomial pathogens

Can be extremely drug-resistant

Same mechanisms responsible for carbapenem

resistance in Enterobacteriaceae may be present in

nonfermenting GNRs (e.g. plasmid beta-lactamase)

however resistance is typically combinatorial

Quick to become multidrug-resistant given arsenal of

intrinsic resistance mechanisms

BACKGROUND

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Main contributors:

Chromosomal AmpC

Loss of porin OprD

Hyperexpression of efflux pump MexAB-OprM

In a recent study of isolates with reduced to no

susceptibility to ceftazidime, a total of 21

different combinations of resistance mechanisms

were found

Chromosomal and acquired beta-lactamases

Metallo-beta-lactamase most common

carbapenemase

BETA-LACTAM RESISTANCE IN

P. AERUGINOSA

Castanheira M et. al., AAC, 2014

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Main contributors:

Chromosomal AmpC-type beta-lactamase

Oxacillinases

Metallo-beta-lactamases

Carbapenem resistance is most often linked

to a carbapenemase

Carbapenemases (e.g. OXA) pose additional

threat because many are located on mobile

genetic elements

BETA-LACTAM RESISTANCE IN

ACINETOBACTER

Potron A et. al., Int J Antimicrob Agents, 2015

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MAIN MECHANISMS OF CARBAPENEM

RESISTANCE

Enterobacteriaceae Cephalosporinase + porin loss +/- ESBL

Carbapenemase

P. aeruginosa Porin loss

Up-regulated efflux

Carbapenemase

Acinetobacter spp. Cephalosporinase + porin loss

Carbapenemase

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Accuracy of automated systems for detection of

carbapenem resistance is varied

For Acinetobacter baumannii/calcoaceticus

complex, reported very major error rates against

imipenem

Vitek2 – 0.7-4% VME

MicroScan – 2.8-25% VME

Phoenix - 1.9% VME

Disk diffusion and Etest are typically reliable

when compared against broth microdilution

DIFFICULTIES WITH PHENOTYPIC TESTING FOR

DETECTION OF CARBAPENEM RESISTANCE

Markelz AE et. al., AAC, 2011

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Disk diffusion and Etest may

be difficult to interpret (fuzzy

zones or inner colonies)

P. aeruginosa may be

mucoid hindering inoculum

density measurement and

zone definition

DIFFICULTIES WITH PHENOTYPIC TESTING

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Traditional phenotypic tests (e.g. MHT)

demonstrate poor performance for detection

of carbapenemases in P. aeruginosa and A.

baumannii

E-test for MBL can be ‘false positive’

Is determination of carbapenem resistance

mechanisms in nonfermenting GNRs

important?

TESTS FOR CARBAPENEMASES

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Is determination of carbapenem resistance

mechanisms in nonfermenting GNRs be important?

Infection prevention and control

Many mechanisms are chromosomal (and/or

combinatorial)

Would not want MDR Acinetobacter to become

endemic even if no plasmid-mediated resistance

mechanisms absent

Epidemiology – better tracking of transmission

events

Unlikely to affect treatment decisions

TESTS FOR CARBAPENEMASES

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Prior to initiating a carbapenemase

testing protocol (especially for

nonfermenting GNRs), determine how

(and if) mechanistic information will be

used by infection control and prevention

and pharmacy

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M100-S26 (TABLE 3)

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Results in a large number

of indeterminate and

falsely negative results

Not recommended by

CLSI for organisms other

than Enterobacteriaceae

Performance is less than

desirable for MBLs, which

may be present in

Pseudomonas >

Acinetobacter

MODIFIED HODGE TEST

+

-

?

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Based on in vitro hydrolysis of imipenem by a

bacterial lysate

Endorsed by CLSI for Enterobacteriaceae, P.

aeruginosa , and Acinetobacter spp.

High level of sensitivity and specificity (>90% for

both) in early reports, reevaluation indicates my be

less for sensitive depending upon user

Poor sensitivity for OXA-48-type carbapenemases

(can adjust method), GES in P. aeruginosa

Labor intensive (requires imipenem reagent

preparation at time of use)

CARBA NP

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CARBA-NP: COMMERCIAL VERSIONS

Rosco Rapid CARB Screen

Biomerieux RAPIDEC CARBA NP

A B

CLSI M100-S25 Method

• Few studies of commercial

assay performance

• CARBA NP method

outperforms CARB Screen

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Several tests utilize inhibitors that allow for

detection and differentiation of carbapenemases

DOUBLE DISK/INDIRECT/COMBINED/

INHIBITION TESTS

1 Mathers AJ et. al. , JCM 2013

1

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Imipenem disk compared to

imipenem + EDTA disk (commonly

look for ≥5 mm difference in zone of

inhibition with addition of EDTA)

Inhibition by EDTA is a characteristic

used to distinguish MBLs from other

beta-lactamases

Carbapenemases other than MBLs

may be responsible for carbapenem

resistance – this is not a standalone

test

COMBINED DISK TEST

Imipenem

Imipenem +

EDTA

MBLs

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Take home message: no phenotypic test can

be used as a standalone test for detection and

differentiation of carbapenemases in

nonfermenting GNRs

So what about molecular tests…

PHENOTYPIC TESTS

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As with all molecular methods, dependent upon

knowledge about target sequence

Carbapenem resistance may result from

mechanisms other than carbapenemases

Would correctly indicate no carbapenemases

present

Stewardship: could use result and initiate use of

carbapenem while awaiting susceptibility results

Knowledge of local mechanisms are beneficial

MOLECULAR METHODS

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Oxacillinase genes in Acinetobacter may be detected but may not result in phenotypic resistance

Probably one of the best methods for detection of oxacillinases in Acinetobacter

Will not detect all OXA types

Commercially -available assays are a great method for detection of some carbapenemases (NDM, VIM, IMP, OXA, KPC) but many others would be missed

P. aeruginosa may be challenging

MOLECULAR METHODS

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Emergence of resistance in P. aeruginosa against

imipenem is common, especially with prolonged

treatment

0.3% of the genome is devoted to antimicrobial resistance

genes

10% genes organized in pathogenicity islands that can be

easily mobilized

Genes may be up or downregulated based on antibiotic

pressure

Differing opinions on frequency of susceptibility

testing for GNRs (range 1 – 3 days)

FREQUENCY OF SUSCEPTIBILITY TESTING

Mesaros N et. al., Clin Microbiol Infect, 2006

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Plug in intrinsic resistance first

Check for updates

REPORTING STRATEGY

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P. AERUGINOSA

aztreonam >32 R

ceftazidime <8 S

imipenem ≤2 S

ciprofloxacin >4 R

gentamicin >16 R

pip-tazo ≤ 8 S

ertapenem R

Consider reporting

some intrinsically

resistant agents

(e.g. ertapenem)

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ACINETOBACTER

amp/sulbact <8S

ceftazidime <8 S

imipenem ≤2 S

ciprofloxacin >4 R

gentamicin >16 R

pip-tazo ≤ 16 S

ertapenem R

Consider reporting

some intrinsically

resistant agents

(e.g. ertapenem)

*sulbactam is the

active component

*

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Current commercial panels should have appropriate concentrations to differentiate susceptibility and resistance

Detection of mechanism(s) responsible for carbapenem resistance is not necessary for treatment but may aid in identification of resident resistance mechanisms

REPORTING STRATEGY

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No standard definition for reporting of

carbapenem resistance in nonfermenting

GNRs (e.g. carbapenem resistant organism,

multi-drug resistant organism)

Many public health laboratories focus on CRE,

so guidance is somewhat lacking

Develop protocol with infection control for

notification of carbapenem-resistant

organisms

REPORTING CARBAPENEM RESISTANCE

IN NON-ENTEROBACTERIACEAE

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Accurate detection of carbepenemases in non-

Enterobacteriacea is challenging with currently available

methods

Detection of carbepenemases may not be clinically relevant to

providers

Proceed with caution

Strategic reporting and frequency of testing may be effective

strategies for antimicrobial stewardship

SUMMARY