cro and cpe: epidemiology and diagnostic tests · oxa-48: international plasmid ‘epidemic’...

CRO and CPE: Epidemiology and

diagnostic tests

Scottish Microbiology and Virology Network Scientific Meeting

22nd April 2016

Katie Hopkins PhD Clinical Scientist, Antimicrobial Resistance and Healthcare Associated Infections

(AMRHAI) Reference Unit

Public Health England © Crown copyright

Walking through a minefield of acronyms

Acronym Better to spell it out ! ‘Translation’ Defined by

CRE Carbapenem-resistant

Enterobacteriaceae

Must be Enterobacteriaceae and resistant to

carbapenems. May or may not produce a

carbapenemase

AST

CRO Carbapenem-resistant

organisms

Any carbapenem-resistant species (strictly, also

those with intrinsic resistance). May or may not

produce a carbapenemase.

AST

CPE Carbapenemase-producing

Enterobacteriaceae

Must be Enterobacteriaceae and produce a

carbapenemase. May or may not be resistant to

carbapenems.

Mechanism

detection

CPO Carbapenemase-producing

organisms

Any carbapenemase-producing species (strictly,

also those with intrinsic carbapenemases). May

or may not be resistant to carbapenems.

Mechanism

detection

2 CRO and CPE: Epidemiology and diagnostic tests SMVN Meeting 2016 © Crown copyright

• So, “CPEs are always CPOs and may also be CREs or CROs (unless they have

low MICs); every CRE is a CRO, but not every CRE or CRO is a CPE or a CPO”

Gram-negative resistance ad infinitum

• 5 of 7 ‘ESKAPEEs’ are Gram-negative

•Increasing reliance on carbapenems

• detection of carbapenemases required for IPC and public health

Pathogen Established problems Emerging threats

E. faecium VRE, HLGR, Amp-R Lin-R, Dap-R, Tig-R

S. aureus MRSA (ha/ca) Van-R, Lin-R, Dap-R

Klebsiella ESBLs Carbapenemases, Col-R

Acinetobacter MDR, Carbapenemases Tig-R, Col-R

Pseudomonas MDR, except Col Carbapenemases, Col-R

Enterobacter AmpC, ESBLs Carba-R, Carbapenemases

E. coli Cip-R, ESBLs Carbapenemases


4

Health Protection Report Vol 9 No. 2

– 16 January 2015

The ‘nightmare’ bacteria

CRO and CPE: Epidemiology and diagnostic tests SMVN Meeting 2016 © Crown copyright

5

The “big 5” carbapenemases

KPC

OXA-48-like

NDM

VIM

IMP

(22 variants)

(>10 variants)

(14 variants)

(43 variants)

(48 variants)


Global perspective: KPC-producers


Nordmann & Poirel, 2014

KPC: international strain epidemic of KPC +ve

K. pneumoniae

ST258

ST258

ST258

Nordmann et al. TLID 2009; 9: 228–36

NW England KPC outbreak:

K. pneumoniae STs 11, 25,

27, 248, (258 - Col-R), 321,

468, 490 and 491

plus Enterobacter, E. coli +

others

Global perspective: OXA-48-producers



OXA-48: international plasmid ‘epidemic’

OXA-48 (c. 62 kb) OXA-48

OXA-181 (c. 7kb)

Dimou et al. JAC 2012 ; Poirel et al. AAC 2012

• OXA-48 found on related plasmids in clonally diverse isolates of E. coli, K.

pneumoniae and E. cloacae.

Global perspective: NDM-producers



• Multiple plasmid backbones in

multiple clones of different

genera

NDM epidemiology is diverse


Kumarasamy et al., 2010

Kumarasamy et al., 2010

Nordmann et al., 2011


Global perspective: VIM- and IMP-producers

Nordmann et al., 2011

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3310682/figure/F3/

EARS-Net data: Proportion of carbapenem

resistant (R+I) invasive E. coli and K. pneumoniae,

2014

• Most countries <1% non-susceptibility in E.coli

• 6 countries reported >5% non-susceptibility in K. pneumoniae as judged by surveys

• Bacteraemia isolates account for <10% carbapenemase producers in UK


E. coli K. pneumoniae

Overall European situation regarding occurrence of CPE

using an epidemiological scale of nationwide expansion


N.B. epidemiological stage might

not represent the true extent of the

spread of CPE as it is a subjective

judgment by national experts

Albiger et al. submitted

Limiting the impact of carbapenemases

• Prompt detection essential for

identifying infected/colonized

patients: 1.appropriate patient management

2.rapid implementation of infection

control procedures

3.Prevent onwards transmission

• But how?

15 Bioconnections Masterclass 14th April 2015 © Crown copyright 15 CRO and CPE: Epidemiology and diagnostic tests SMVN Meeting 2016 © Crown copyright

The problem with spotting carbapenemase

producers

Carbapenem MIC

N

0.5 16

Wild-type Carbapenemase

ESBL / AmpC + porin loss

or true carbapenemase ?

•Not all carbapenemase producers are resistant to the carbapenems

•ESBL/AmpC + porin loss = not transferable, may have fitness cost rarely cause outbreaks

Phenotypic detection of ESBLs and carbapenemases - BSAC workshop 2015 © Crown copyright 16 CRO and CPE: Epidemiology and diagnostic tests SMVN Meeting 2016 © Crown copyright

Chromogenic screening media

17

• Brilliance CRE, CHROMagar KPC, chromID CARBA,

chromID OXA-48, COLOREX KPC, chromID CARBA

SMART, COLOREX mSuperCARBA…

• stool/rectal swabs

• Not all detect the “big 5” carbapenemases

• Odd strains that ‘don’t play by the rules’

• Organisms resistant to carbapenems due to other

mechanisms may grow

• Non-fermenters appear as colourless colonies

• Be cautious in interpretation of study data!


How can we identify a

carbapenemase-producing organism?

18

Clinical specimen Bacterial isolate

-lactamase inhibitor tests

Detect carbapenem

hydrolysis

some assays

Detect carbapenemase

antigens

some of the “big 5” some of the “big 5”


genes


EUCAST guidance for resistance

mechanism detection


http://www.eucast.org/resistance_mechanisms/

How can we identify a

carbapenemase-producing organism?

20

Clinical specimen Bacterial isolate

-lactamase inhibitor tests

Detect carbapenem

hydrolysis

some assays


antigens

some of the “big 5” some of the “big 5”


genes


Cleavage of -lactam ring by carbapenemases

+18 Da - 44 Da

red

yellow

hydrolysis decarboxylation

pH change


Detecting hydrolysis: CarbaNP test

• CarbaNP: yes/no answer:

– ‘in-house’ (Nordmann et al. [2012], Dortet et al. [2012])

– Rosco Diagnostica Rapid CARB Screen kit

– Biomerièux Rapid-Ec CarbaNP

• CarbaNP-II: determination of carbapenemase type (Dortet et al. [2012])

• Results ≤2 hrs with good sensitivity and specificity

• False-negatives with mucoid strains, some OXA-48

producers and other carbapenemases.

CPE screening – where are we now? IBMS Congress 2015 © Crown copyright 22 CRO and CPE: Epidemiology and diagnostic tests SMVN Meeting 2016 © Crown copyright

CarbaNP for direct detection of carbapenemase producers

in blood cultures

Phenotypic detection of ESBLs and carbapenemases - BSAC workshop 2015 © Crown

copyright

• Identification of a carbapenemase producer reduced from 24-48 hrs to 3-5 hrs

Dortet et al. 2013


Phenotypic detection of ESBLs and carbapenemases - BSAC workshop 2015 © Crown

copyright

Detecting hydrolysis: MALDI-ToF

Meropenem solution

Negative control

NDM-1 K. pneumoniae

NDM-1 A. baumannii

• Potential for detection of resistance to carbapenems but

currently no standardized protocol

• Bruker detection kit and software in the pipeline


MALDI-ToF for direct detection of

carbapenemase producers in blood cultures

Carvalhaes et al. 2014


Detecting carbapenemase antigens:

immunochromatographic tests

• Coris RESIST K-SeT sets

– Currently only KPC and OXA-48

• 100% sensitivity/specificity from bacterial colonies (Meunier et al. in press)

and spiked blood cultures (Wareham et al. 2016)

CPE screening – where are we now? IBMS Congress 2015 © Crown copyright 26 CRO and CPE: Epidemiology and diagnostic tests SMVN Meeting 2016 © Crown copyright

Detection of carbapenemase genes

KPC OXA-48-like

NDM VIM

• RT-PCR validated vs. 450 isolates.

• Testing daily all ?CPE and ?MBL NFs positive results within 48 hours

• Aiming for deployment in regional PHE labs (and beyond…? Used at GRI)

• Already in use in Birmingham and Leeds PHE labs

• +es sent to AMRHAI for WGS (MICs, typing); -ves for further workup

+ internal

positive control

ACB ASM 2015 - Antimicrobial drug resistance: current challenges and future threats © Crown copyright 27 CRO and CPE: Epidemiology and diagnostic tests SMVN Meeting 2016 © Crown copyright

Detection of carbapenemase genes

Multiplex PCR assay for genes encoding acquired metallo-β-lactamases.

[Ellington et al. 2007]

Testing for other

carbapenemases still dictated by:

• information on

submission form

• MICs for rarer genes

ACB ASM 2015 - Antimicrobial drug resistance: current challenges and future threats © Crown copyright ACB ASM 2015 - Antimicrobial drug resistance: current challenges and future threats © Crown copyright 28 CRO and CPE: Epidemiology and diagnostic tests SMVN Meeting 2016 © Crown copyright

Commercial molecular assays


Company Assay Basis Cultures Clinical

samples

Coverage* Platform Time

Amplex Eazyplex SuperBug

complete or

eazyplex SuperBug

CRE

(CE-marked)

LAMP yes “screening

swabs”

KPC, OXA-

48, VIM,

NDM

Proprietary 15 m

(isolates)

or 30 m

(swabs)

Cepheid GeneXpert Carba-R

(CE-marked)

RT-

PCR

yes Rectal swabs KPC, OXA-

48, NDM,

VIM, IMP-1

Proprietary 50 m

Check-

Points

Check-Direct CPE

(CE-marked)

RT-

PCR

Yes Rectal/perianal

swabs

KPC, OXA-

48, NDM,

VIM

Multiple RT-

PCR

machines

2 h

Nanosphere Verigene BC-GN PCR/

array

No(?) Blood cultures KPC, OXA-

48, NDM,

VIM, IMP

Proprietary <2 h

* Coverage within OXA-48, VIM and IMP families varies between kits

AMRHAI commercial kit evaluation [Findlay et al. 2015]

Amplex

Eazyplex SuperBug

Complete

Cepheid

GeneXpert Carba-R

Check-Points

Check-Direct CPE

on ABI 7500

Check-Points

Check-Direct CPE

on BD MAX™

Assay coverage

KPC, OXA-48*,

NDM, VIM

KPC, OXA-48*, NDM,

VIM, IMP-1

KPC, OXA-48-like,

NDM/VIM

KPC, OXA-48-like,

NDM, VIM

‘Big 5’

carbapenemases NOT

detected IMP family Some IMP variants IMP family IMP family

Hands on time per

sample <5 min <5 min <5 min <5 min

Assay runtime 20 min ~50 min ~1.75 hrs ~2.5 hrs

No of samples that can

be processed at once

1 or 2 independent

tests

Up to 80 independent

tests Up to 94 batched Up to 22 batched

• A commercial system may be an interim solution whilst PHE assay was

developed

• Tested against 450 pure bacterial cultures, mostly Enterobacteriaceae


The Equipment

Cepheid GeneXpert Amplex Genie II

BD MAX (Check-Points)


• 100% sensitivity for detecting KPC, NDM and VIM genes

• OXA-48-like: Amplex v.1 and Cepheid v.1 did not detect the OXA-181

variant • Both kits subsequently modified to detect this allele

• IMP: Cepheid detected IMP-1-like, but NOT other variants • more common in NFs

• AMRHAI starting to accept local lab results with these assays at face value

Assay Performance [Findlay et al. 2015]

Carbapenemases Detected

Isolates Tested Amplex v.1 Cepheid v.1 Check-Points ABI

7500

Check-Points BD

MAX™

KPC (n=100) 100% 100% 100% 100%

OXA-48-like (n=100) 83% 83% 100% 100%

NDM (n=100) 100% 100% 100% 100%

VIM (n=100) 100% 100% 100% 100%

IMP (n=24) n/a 71% (17/24) n/a n/a

NDM+OXA-48-like (n=2) 2x NDM; 1x OXA-

48-like

2x NDM; 1x OXA-

48-like

2x NDM; 2x OXA-

48-like

2x NDM; 2x OXA-

48-like

Non-carba (n=24) 0% 0% 0% 0%


NICE medtech innovation briefing

• Based on AMRHAI evaluation and two others:

• Tenover et al (2013) – Xpert MDRO = earlier

version

• Anandan et al (2015) – no explanation of

discrepancies

• Provides objective information to aid local

decision making

• Is NOT NICE guidance or a recommendation


https://www.nice.org.uk/advice/mib52

Molecular diagnostics to guide empiric

therapy

• Current molecular tests to detect/infer resistance mechanisms are

surrogates for rapid susceptibility testing

• Absence of a resistance mechanism doesn’t confirm susceptibility

• cannot indicate appropriate empiric therapy

• Presence of a resistance mechanism used to infer likely resistance

• false resistance (unexpressed/partial genes)

• Indicates potentially inappropriate empiric therapy

• carbapenemase detected: carbapenem NOT suitable as sole agent

• Still need to culture organism for :

– AST – still essential to confirm susceptibility for patient treatment

– Typing – needed to investigate transmission


Allele diversity, assay coverage and

sensitivity

• For maximum sensitivity, molecular assays must include probes/primers to

detect all known gene variants

• >150 alleles in ‘big 5’ families (http://www.lahey.org/Studies/; 16th April ‘16)

• KPC: 24 variants

• NDM: 16 variants

• IMP: 53 variants

• VIM: 46 variants

• OXA-48-like: -48, -181, -232 (at least 10 variants)

• Family coverage ‘simple’ for KPC, NDM; harder for OXA-48-like; much

harder for IMP and VIM

• Solutions usually include compromises and won’t detect rarer or new

enzymes


http://www.lahey.org/Studies/



Carbapenemases come in many varieties

36

Enzyme Type Classification by

Ambler Class

Activity Spectrum Organism(s)

KPC A All β-lactams Enterobacteriaceae,

P. aeruginosa,

A. baumannii

SME A Carbapenems and aztreonam,

but not 3rd/4th G

cephalosporins

S. marcescens

NMC–A

IMI

A Carbapenems and aztreonam,

but not 3rd/4th G

cephalosporins

Enterobacter species

GES A Imipenem and 3rd/4th

cephalosporins

P. aeruginosa and

Enterobacteriaceae

IMP

VIM

NDM

AIM, GIM, SIM, (not detected

in the UK yet)

DIM, SPM

B (metallo-β-lactamases) All β-lactams except

monobactams (aztreonam)

Pseudomonas species

Acinetobacter species

Enterobacteriaceae

OXA D Weakly active against

carbapenems

A. baumannii,

Enterobacteriaceae

and rarely P. aeruginosa


Other new carbapenemases

• FR1-1: class A carbapenemase [Dortet et al. 2015]

• Enterobacter cloacae

• Patient hospitalised in France; previous travel to Switzerland

• Plasmid-mediated

• BKC-1: class A carbapenemase [Nicoletti et al. 2015]

• Klebsiella pneumoniae

• Brazil

• Plasmid-mediated

• VCC-1: class A carbapenemase [Mangat et al. 2016]

• Vibrio cholerae

• Canada

• LMB-1: class B carbapenemase [Lange et al. 26th ECCMID #EP0187]

• Enterobacter cloacae

• Austria


Filling the gaps in carbapenem

resistance surveillance

• Need better understanding of patient-related risk factors

other than travel or hospitalisation abroad

• Who are ‘high-risk’ patients?


ERS data collection


Two-stage submission process

Summary

• CRO and CPE continue to be a problem

• Epidemiology of CPE diverse depending on carbapenemase family

• Clonal vs. plasmid spread

• Whole genome sequencing

• Limiting the impact of carbapenemases requires rapid detection

• Phenotypic vs. genotypic

• In-house vs. commercial

• But need to think beyond the “big 5”

• Enhance surveillance needed to improve our understanding of

local and national epidemiology


cro and cpe: epidemiology and diagnostic tests · oxa-48: international plasmid ‘epidemic’...

Documents