ESBL Laboratory Detection Problems

1
ESBL Laboratory Detection Problems

• Hugo Donaldson
• SpR Medical Microbiology
• Royal Victoria Hospital Belfast

2
HPA National Standard Method

• Recommends
• Screening followed by confirmation
• Screening with either cefpodoxime or cefotaxime
  and ceftazidime
• Automated methods

3
Confirmatory Tests 1

• Double disc tests (not recommended)

4
Confirmatory Tests 2

• Combination disc methods

5
Confirmatory Tests 3

• Etest ESBL strips

6
Introduction

• VITEK2 AST N-054 card introduced in 2007.
  Replaced AST N-037 card.
• ESBL isolates detected by combination disc
  method loaded onto VITEK2 for identification and
  further sensitivities using GN id card and AST
  N-054 card.
• Discrepant results noted. Evaluation of each
  method carried out.

7
Method 1

• ESBL producing E. coli isolates (n137) recovered
  from clinical specimens from patients in
  community care homes were evaluated.
• MC OLeary, AC Loughrey, PJ Rooney et al. An
  investigation into the Importance of Nursing
  Homes in the Epidemiology of ESBL producing E.
  coli.
• These were phenotypically characterised by the UK
  Health Protection Agency (HPA) Antibiotic
  Resistance Monitoring and Reference Laboratory as
  CTX-M producing E. coli.

8
Method 2

• PCR for a characteristic IS26-blaCTX-M-15 link
  showed seventy-three belonged to strain A, which
  typically produces a low level of CTX-M-15
  enzyme 64 were non-strain A and their CTX-M
  enzymes remain to be fully identified.
• Woodford N, Ward ME, Kaufmann ME, et al.
  Community and hospital spread of Escherichia coli
  producing CTX-M extended-spectrum ß-Lactamases in
  the UK. J Antimicrob Chemother 2004 54 735-743
• Karisik E, Ellington MJ, Pike R et al. Molecular
  characterization of plasmids encoding CTX-M-15 ß
  -lactamases from Escherichia coli strains in the
  United Kingdom. J Antimicrob Chemother 2006 58
  665-668

9
Method 3

• Each isolate was tested using
• the AST N-054 card for the VITEK2
• MASTDISCS? ID ESBL detection combination disc
  diffusion (DD) test

10
Method 4

• All VITEK2 advanced expert system (AES)
  beta-lactam phenotypic summary interpretations
  that included ESBL were categorised as
  successful ESBL detection
• MAST DD result was categorised as successful for
  ESBL detection if one or more cephalosporin zone
  size was enhanced by ?5mm in the presence of
  clavulanic acid

11
Results
Table 1.
a 95 confidence interval 59.7-75.1 b 95
confidence interval 94.5-99.9 c plt0.0001
12
Results
ß lactam sensitivity data
13
HPA Reporting Guidance

• Organisms inferred to have ESBLs should be
  reported resistant to ALL penicillins (except
  temocillin), cephalosporins (except cefoxitin),
  and to aztreonam, irrespective of routine
  susceptibility results

14
Discussion 1

• The ability of the VITEK2 AES to detect ESBL
  production in E. coli with the AST-N054 card
  (sensitivity 67.9) was poorer than the 74-100
  previously reported by other investigators, who
  mostly used a heterogeneous mix of ESBL producers
  and other AST cards with different combinations
  of cephalosporins, or AST cards with dedicated
  ESBL confirmatory tests.

15
Discussion 2

• Concerns have been raised previously about the
  ability of the VITEK2 AES to detect
  ESBL-producing organisms with low MICs when the
  investigators used AST cards containing neither
  cefpodoxime nor a specific ESBL test.
• Sanders CC, Peyret M, Moland ES et al. Ability
  of the VITEK 2 Advanced Expert System To Identify
  ß-Lactam Phenotypes in Isolates of
  Enterobacteriaceae and Pseudomonas aeruginosa. J
  Clin Microbiol 2000 38 570-574.
• Sturenberg E, Sobottka I, Feucht HH et al.
  Comparison of BD Phoenix and VITEK2 automated
  antimicrobial susceptibility test systems for
  extended-spectrum beta-lactamase detection in
  Escherichia coli and Klebsiella species clinical
  isolates. Diagn Microbiol Infect Dis 2003 45
  29-34.

16
Discussion 3

• Why?
• Antibiotic panel on AST N-054 card changed from
  AST N-037 (cefpodoxime replaced with ertapenem)
• VITEK2 AES version (software version
  WSVT2-R04.03 current at time of testing)

17
Discussion 4

• In those areas of the UK, where E. coli strain A
  are the dominant producers of CTX-M-15
  ?-lactamase, it seems prudent to use an
  alternative method to screen E. coli for ESBL
  production this might include disc diffusion
  testing or an alternative VITEK2 AST card.

18
Acknowledgements
M. McCalmont, D.M. Livermore, P.J. Rooney, G.
Ong, E. McHenry, R. Campbell, R. McMullan, AC
Loughrey.