Detection of Carbapenemases From a Technologist’s Perspective

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A partnership of the Advocate and Aurora Health Care System Detection of Carbapenemases From a Technologist’s Perspective Courtney Fraser MLS (ASCP) Microbiology Supervisor ACL Laboratories

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Detection of Carbapenemases From a Technologist’s Perspective. Courtney Fraser MLS (ASCP) Microbiology Supervisor ACL Laboratories. Disclosure. - PowerPoint PPT Presentation

Transcript of Detection of Carbapenemases From a Technologist’s Perspective

Page 1: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Detection of Carbapenemases From a

Technologist’s Perspective

Courtney Fraser MLS (ASCP)Microbiology Supervisor ACL Laboratories

Page 2: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Disclosure

I will discuss specific brand names in this presentation. I do not have stock in, nor receive honoraria for any commercial product mentioned in this presentation.

Page 3: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Introduction

• Automation is not available for carbapenemase confirmatory testing.

• Resistance mechanisms are not typically discussed in the classroom for most MLS students.

• Seasoned Techs currently in the field are not used to resistance in organisms and are unfamiliar with the mechanisms related to resistance.

• Education needed on antibiotic classifications.

Page 4: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Purpose of Presentation

• Define types of carbapenemases and understand classifications

• When to test• How to test• How to incorporate instrumentation tools

Page 5: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

What is a Carbapenemase?

• First, need to understand what are the carbapenems:

• Ertapenem• Imipenem• Meropenem• Doripenem

• Second, the suffix –ase indicates an enzyme• A carbapenemase is an enzyme produced

by bacteria that hydrolyze (deactivate) carbapenems

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A partnership of the Advocate and Aurora Health Care System

Page 7: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Carbapenems

All share a common β-lactam ring, β-lactamases target the β-lactam ring)

• differences are in ring stabilization from hydrolysis • protection of ring from β-lactamase by addition of large groups blocking the substrate site

Imipenem

Ertapenem

Meropenem

Large groups

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Why Important?• Studies have shown that rapid detection of

carbapenemases have attributed to positive patient treatment outcomes.

• Infection Control emergency – These mechanisms can be plasma-mediated allowing for easy transmission to other organisms.

• Detection of these resistance mechanisms should initiate contact precautions for patients , thus reducing the spread of nosocomial related infections.

• Deter inappropriate antibiotic use thus prolonging the efficacy of available antibiotics

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A partnership of the Advocate and Aurora Health Care System

The Rise of Carbapenemases

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ACL LaboratoriesOverview

ACL Laboratories is defined as an operating management agreement

between Advocate Health Care and Aurora Health Care.

• Two Central Labs (Rosemont, IL., West Allis,

WI.)

•Total of 27 hospitals

Rosemont

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Wisconsin vs. Illinois 2011

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KPC Isolates 2009 and 2011 - Chicago

Urine Blood Respiratory Other

2009 52 18 20 16

2011 114 35 39 50

10

30

50

70

90

110

Advocate KPC Isolates/Specimen Site

KP

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Po

siti

ve S

amp

les

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Types of β-Lactamases/Carbapenemases

• Carbapenemases• Type A (Ambler) – KPC – 1st described 1998, endemic

in NYC since 2004. Moving west. 1st isolates in Chicago area in 2/08

• Type B – Metallo ß-lactamases• Found in Stenotrophomonas maltophilia• Enterobacteriaceae and P. aeruginosa - Reported

sporadically in U.S. • Watch out for NDM-1 which has spread to Europe

(Sweden and England) from India • Type D – Oxa-40 – Endemic in Acinetobacter

baumannii in the Chicago area since 2002• Hyper AmpC producers

• Mutations in AmpC promoter and attenuator/promoter regions• Poorly inhibited by lactam—lactamase inhibitor combinations• E. coli produces AmpC (poorly) and is unlike other bacteria with

AmpC the gene is not inducible• No ampR regulatory geneAll the above can give a positive Hodge Test!

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TYPES OF CARBAPENEMASES

Enzyme Type Ambler Class Activity Spectrum Organism(s)

KPC (1-10)(plasmid)

A All β-lactams Enterobacteriaceae Ps. aeruginosa

SME A Carbapenems and aztreonam, but not 3rd/4th Gen cephalosporins

S. marcescens , not plasmidAssociated.

NMC–A, IMIMNC = Not metallo carbapenemaseIMI = IMI hydrolyzingΒ-lactamase

A Same as for SME Enterobacter spp.

GESGES= Guiana extended spectrum (plasmid)

A Imipenem and 3rd/4th cephalosporins

Ps. Aeruginosa and Enterobacteriaceae

IMI, VIM, NDM-1VIM = Verona Integron encoded MBL) NDM-1 = New Delhi metallo β lactamase

B (metallo-β-lactamases)

All β-lactams; can test susceptible to aztreonam(NDM-1 variable AZT resistance)

Pseudomonas spp. Acinetobacter spp. Enterobacteriaceae

OXA(Oxacillin hydrolyzing)

D Weakly active against carbapenems

A. baumanii, P. Aeruginosa, and rare Enterobacteriaceae

Pediatr Infect Dis J. 2010;29(1):68-70. 

S. marcescensenzyme

Page 15: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Recommendations for Screening

• Any Enterobacteriaceae• At least one carbapenem with an increased

MIC (ertapenem is most sensitive, but not specific) and at least one resistant 3rd or 4th generation cephalosporins:

• Ceftriaxone• Ceftazidime• Cefotaxime• Ceftizoxime• Cefepime

• Screen for KPC, MBL, and AmpC• ESBL testing is performed by automated

instrumentations.

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KPC

• Klebsiella pneumoniae Carbapenemase• 1st reported on the East Coast of the United

States in the late 1990’s• Currently found worldwide. Can be

associated with other Enterobacteriaceae.• KPC gene is plasmid-mediated which has

contributed to the rapid dissemination across the globe.

• Hydrolyze all beta-lactam antibiotics.• Can be detected by

• Modified Hodge Test• Boronic Acid inhibition

Page 17: Detection of Carbapenemases From a Technologist’s Perspective

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Modified Hodge Test (MHT)

• Used for the detection of KPC producing isolates

• Carbapenem disk: meropenem – gold standard – most specific

• Increased MIC for imipenem seen is Proteus, Providencia, and Morganella

• Indentation of the inhibition zone indicates that the test strain is hydrolyzing the carbapenem.• Not specific for KPC

• AmpC enzymes can give weak false positive results that can be accentuated by porin loss

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Modified Hodge Test (MHT)

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Alternative Test

• Rosco kit for carbapenemase detection• 4 disks all on one plate:

• Meropenem (MRP10) • Meropenem + Boronic Acid (MRPBO) *KPC• Meropenem + Cloxacillin (MR+CX) *AmpC• Meropenem + DPA Dipicolinic acid (MR+DP) *MBL

• Easier to read compared to the MHT• Reduced the risk of false positives for KPC• Detects true hyperproduction of AmpC by

utilizing a carbapenem instead of a cephamycin

Page 20: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Rosco Kit

Page 21: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Positive for KPC

Page 22: Detection of Carbapenemases From a Technologist’s Perspective

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Metallo Beta-Lactamase (MBL)

• Uses a zinc cation for the hydrolysis of the beta-lactam ring

• Activity is inhibited by EDTA (similar to clavulanic acid and ESBL)

• Hydrolyze all beta-lactam antibiotics except aztreonam.

• Chromosomal presence found in Stenotrophomonas, Aeromonas, and Chryseobacterium.

• Clinically significant MBLs have transmitted to other bacterial pathogens.

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A partnership of the Advocate and Aurora Health Care System

Detection of MBL

Ratio MP/MPI >8 (or 3 fold)

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AmpC

• Chromosomal = MY SPACE bugs• (Morgenella, Y. enterocolitica, Serratia, Providencia,

Aeromonas, Citrobacter, Enterobacter)

• Inducible = Any MYSPACE or Enterobacteriaceae containing AmpC plasmid

• Organism may develop resistance during prolonged therapy with 3rd generation cephalosporins.

• Identified in organisms exhibiting the following:• Resistant to cephamycins

• Cefoxitin• Cefotetan

• Sensitive to Cefepime

• Hyperproduction = Any Enterobacteriaceae• Caused by a mutation in the AmpC gene leading to

permanent hyperproduction or derepression.

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A partnership of the Advocate and Aurora Health Care System

The AmpC gene is now found on a plasmid• We can no longer predict which bacteria are

AmpC positive• Amp C is upregulated by treatment with β-

lactam drugs• Amount of enzyme produced is dependant on the

selection of stable mutants with upregulated genes.

Page 26: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

AmpC + Porin Loss = “Carbapenemase”

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AmpC

Many different AmpC enzymes

• C. freundii cluster • CMY-2

• Enterobacter cluster• MIR-1, ACT-1

• M. morganii cluster**• DHA-1

• H. alvei cluster• ACC-1

• Aeromonas cluster• CMY-1 and FOX-1

Degree of carbapenemase resistance is dependant on type of AmpC in a porin deficient isolate

Page 28: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Detection of AmpC Hyperproducer

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A partnership of the Advocate and Aurora Health Care System

Education and Training

• In the past, technologists did not need to use antibiotic classification in everyday use.

• Education may be needed to help technologists understand the importance of classifications.

• Tools are available in automated instrumentation that can help technologists on the bench.

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A partnership of the Advocate and Aurora Health Care System

MicroScan Report

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A partnership of the Advocate and Aurora Health Care System

Vitek Observa Report

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A partnership of the Advocate and Aurora Health Care System

Tools in Instrumentation

• Custom comments/alerts can be printed on the patient report generated from the instrument.

• Activation of these tools can significantly increase the technologist’s awareness on when to appropriately screen for carbapenemases.

• Can also be used for many other difficulties in AST reporting.

Page 33: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Creating a MicroScan Comment

Page 34: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

MicroScan Alert Rules

Page 35: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

MicroScan Patient Report

Page 36: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

How to Build a BIOART Rule

Page 37: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Page 38: Detection of Carbapenemases From a Technologist’s Perspective

A partnership of the Advocate and Aurora Health Care System

Vitek Patient Report

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A partnership of the Advocate and Aurora Health Care System

References Bush, Karen, Jacoby, George, Antimicrobial Agents and

Chemotherapy,Minireview Updated Functional Classification of Beta-Lactamases, Mar. 2010, Vol.54, No. 3, p.969-976.

Fernando, Pasteran, Tania Mendez, Melina Rapoport, Leonor Guerriero, and Alejandra Corso, Controlling False Positive Results Obtained with the Hodge…J. Clin. Microbiol. 2010, 48(4):1323 (2010).

Gazin, Muriel, Fabienne Paasch, Herman Goossens and Surbhi Malhotra-Kumar, Current Trends in Culture-Based and Molecular Detection of ESBL Harboring and Carbapenem Resistant Enterobacteriaceae, J. Clin. Microbiol. 2012, 50(4).

Rosco Diagnotica www.rosco.dk Siemens Microscan Biomerieux Vitek 2 Observa

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A partnership of the Advocate and Aurora Health Care System

A big THANK YOU to the ACL Sensitivity Training Team!!! Your passion and dedication to AST testing has served both patients and your fellow coworkers.

Questions?

THANK YOU!!!