Carbapenemases - Amazon S3

Carbapenemases:

Molecular resistance and diagnosis

Dr. Matthew J Ellington

Carbapenemase producing Gram-negative Microorganisms. Where are we now? 4th June 2015 © Crown Copyright

KPC-producing K. pneumoniae: mean MICs

(mg/L)

KPC

K.pneumoniae

Imipenem 50

IPM + EDTA 9

Meropenem 18

Ertapenem 10

Aztreonam 64

Cefotaxime >256

CTX + clav 17

Ceftazidime 120

CAZ + clav 9.7

Cefpirome >64

Pip/tazo >64

KPC

K.pneumoniae

Cipro 7

Gentamicin 8.7

Tobramycin 22

Amikacin 6.5

Minocycline 13.5

Tigecycline (1.2)

Colistin 0.9

a) -Lactams b) Non--lactams

Data: David M. Livermore

2 Carbapenemase producing Gram-negative Microorganisms. Where are we now? 4th June 2015 © Crown Copyright

Carbapenemases - the big five

KPC – Ambler class A – inhibited by Boronic acid & Avibactam

NDM – Ambler class B – inhibited by EDTA

VIM – Ambler class B – inhibited by EDTA

IMP – Ambler class B – inhibited by EDTA

OXA-48 – Ambler class D – inhibited by Avibactam

KPC NDM VIM OXA-48 IMP

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Phenotypic detection - issues

E. coli ATCC25922 MIC

(mg/L)

OXA-48 in E. coli

ATCC25922 MIC (mg/L)

Imipenem 0.125 1

Meropenem 0.016 0.25

Ertapenem 0.016 2

Ceftazidime 0.125 0.5

Cefepime 0.064 0.5


Studentova et al., Fol Microbiol. 2015, 60:119-29

Commercially available systems - characteristics

Check-Direct CPE

on ABI 7500

Check-Direct CPE

on BD MAX

eazyplex SuperBug

Complete Xpert Carba-R

Assay coverage KPC, OXA-48-like,

NDM/VIMa

KPC, OXA-48-like, NDM,

VIMa KPC, OXA-48, NDM, VIM

KPC, OXA-48, NDM, VIM,

IMP-1-like

‘Big 5’

carbapenemases

NOT detected

IMP family IMP family IMP family Some OXA-48-like; some

IMP subgroups

Hands on time per

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

Assay run time ~1.75 h ~2.5 h 20 min ~50 min

Sample throughput Up to 94 tests in a batch Up to 22 tests in a batch 1 or 2 independent tests 1 to 80

independent testsb

a - The Check-Direct CPE kit does not distinguish between NDM and VIM producers on the ABI 7500 platform, but does so on the BD MAX.

b - Capacity of GeneXpert machines varies with model.


Adapted from: Findlay et al., 2015, J Antimicrob Chemother

Commercially available systems - performance

Assay

performance

Check-Direct CPE

on ABI 7500

Check-Direct CPE

on BD MAX

eazyplex

SuperBug

Complete

Xpert Carba-R

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

OXA-48-like

(n=100) 100% 100% 83%a 83%a

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

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

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

NDM + OXA-48-

like (n=2)

2x NDM;

2x OXA-48

2x NDM;

2x OXA-48

2x NDM;

1x OXA-48a

2x NDM;

1x OXA-48a

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

a - Sequencing identified OXA-181 in 18 isolates where the OXA-48-like gene was not detected.

b - Sequencing identified closest matches with IMP-4, IMP-7, IMP-8, IMP-13 and IMP-14 in 7 isolates where the IMP gene was not detected


PHE AMRHAI laboratory comparison:

Findlay et al., 2015, J Antimicrob Chemother

Allele diversity, assay coverage and sensitivity

• >120 alleles in ‘big 5’ families (http://www.lahey.org/Studies/; 9th April 2014)

• KPC: 18 variants

• NDM: 11 variants

• VIM: 41 variants

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

• IMP: 48 variants

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

much harder for IMP

MS Senior Leaders, 25th June 2014 © Crown Copyright 7 Carbapenemase producing Gram-negative Microorganisms. Where are we now? 4th June 2015 © Crown Copyright

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



Binding site regions – simple vs. hard

1 SNP 1 SNP

8 MS Senior Leaders, 25th June 2014 © Crown Copyright 8 Carbapenemase producing Gram-negative Microorganisms. Where are we now? 4th June 2015 © Crown Copyright

Assay design - characteristics 5 channels on ABI7500

6 Channels on Rotorgene

Used on both instruments – same primer/probe sequences, dyes switched

5 channels:

1 channel each for KPC, NDM, VIM, OXA-48-like

Plus 1 channel for IPC


AMRHAI submissions

0

100

200

300

400

500

600

03 04 05 06 07 08 09 10 11 toNov

Co

un

t

Year

VIM

NDM

IMP

OXA-48

KPC

Updated from HPR June 17th 2011


KPC

100

NDM

100

VIM

100

OXA-48

100

NDM +

OXA-48

2

IMP

24

None

24

*Others = Citrobacter spp. (n=15), Raoutella spp. (n=3), Leclercia adecarboxylata (n=2), Serratia marcesens (n=2), Kluyvera georgina (n=1).

Validation panel of 450 isolates


KPC

100

NDM

100

VIM

100

OXA-48

100

NDM +

OXA-48

2

IMP

24

None

24

*Others = Citrobacter spp. (n=15), Raoutella spp. (n=3), Leclercia adecarboxylata (n=2), Serratia marcesens (n=2), Kluyvera georgina (n=1).

Sensitivity / Specificity


100% 100% 100% 100%

100%

100% 100%

Ring Trial Participants


Leeds – ABI7500

Birmingham – ABI7500

Cambridge – Rotorgene

Colindale – ABI7500

- RotorGene

Ring trial / Multi centre evaluation


KPC

22

NDM

22

VIM

22

OXA-48

22

IMP

6

None

6

*Others = Citrobacter spp. (n=6), Raoutella spp. (n=2), Leclercia adecarboxylata (n=2), Serratia marcesens (n=1), Kluyvera georgiana (n=1).

Ring trial / Multi centre evaluation


KPC

22

NDM

22

VIM

22

OXA-48

22

*Others = Citrobacter spp. (n=6), Raoutella spp. (n=2), Leclercia adecarboxylata (n=2), Serratia marcesens (n=1), Kluyvera georgiana (n=1).

IMP

6

None

6

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

1) ABI7500

1) R’gene

3) ABI7500

2) ABI7500

4) R’gene

1) ABI7500

1) R’gene

3) ABI7500

2) ABI7500

4) R’gene

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

Stage 2: Finding the <1%

IMP group carbapenemases

Most commercial kits do not detect IMP

One does detect a sequence group: IMP-1

Commercial kit trial missed:

IMP-4, -7 -8 -13 & -14

Reference test (MJE design) detected


Summary Pays your money, takes your choice

Knowing expected epidemiology informs choices

Mol’ results alongside phenotypic testing remains important

PHE assay at least as good as best commercial system for cultures

Validated assay robust, portable on RotorGene and ABI7500

Patent pending

Detection of IMPs complex but good progress so far


Acknowledgements Peter Hawkey

Neil Woodford

Katie Hopkins

Nicholas Brown

Jacqueline Findlay

Danielle Meunier

Li Xu McCrae

Adela Bullya

Akab Raza

Carolyne Horner

Andrew Dodgson

Malcolm Guiver

Emma Heasman

Martin Curran


Carbapenemases - Amazon S3

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