Detecting Carbapenemase-Producing Enterobacteriaceae: why isn t there a single best method?
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1 Detecting Carbapenemase-Producing Enterobacteriaceae: why isn t there a single best method? Professor Neil Woodford Antimicrobial Resistance & Healthcare Associated Infections (AMRHAI) Reference Unit Crown copyright
2 International Consensus: Carbapenem Resistance is a Critical AMR Threat 2 BSMT, 16th May 2014 Crown Copyright
3 The resistance ratchet keeps turning 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 5 of 7 ESKAPEEs are Gram-negative Increasing reliance on carbapenems The resistance issue for the next 5-10 years Highlighted in all recent AMR documents 3 BSMT, 16th May 2014 Crown Copyright
4 Carbapenemases come in many varieties Class Carbapenemase Enterobacteriaceae Non-fermenters A (non-metallo) KPC BIC, GES, IMI, NMC, SME + +/- B (metallo) IMP*, VIM* NDM AIM, DIM, GIM, SIM, SPM, TMB - ++ D (non-metallo) OXA-48-like +++ +/- OXA-23, -40, -58, -143, /- +++ Among the big 5, IMP- & VIM- types are integron-associated IMP-types described first, but have been overtaken by other types 4 BSMT, 16th May 2014 Crown Copyright
5 The Big 5 carbapenemases KPC VIM IMP NDM OXA-48 5 BSMT, 16th May 2014 Crown Copyright
6 Carbapenemase-producing Enterobacteriaceae in the UK (n = 2794) Imported & home grown Early cases often imported Klebsiella spp. 79%; E. coli 12%, Enterobacter spp., 7%; others 2% 6 BSMT, 16th May 2014 Crown Copyright AMRHAI, Unpublished data
7 Limiting the impact of carbapenemases Detecting resistance in the clinical laboratory is essential for identifying infected / colonized patients: 1. appropriate patient management 2. rapid implementation of infection control procedures 3. Prevent onwards transmission But how? 7 BSMT, 16th May 2014 Crown Copyright
8 Wild-type The problem with spotting the carbapenemase producers Carbapenemase N ESBL / AmpC + porin loss or true carbapenemase? 0.25 Carbapenem MIC 16 Human experts, subjective : computer algorithms, poor specificity relative ease : E. coli > Klebsiella spp. >> Enterobacter spp. High index of suspicion; supplemental tests, locally or in Ref. Lab. 8 BSMT, 16th May 2014 Crown Copyright
9 Negotiating the 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 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 9 BSMT, 16th May 2014 Crown Copyright
10 What laboratory tests do we need? Group to be defined Acronym Suitable lab tests Carbapenem-resistant Enterobacteriaceae / organisms CRE (or CRO) Must identify resistance: Susceptibility tests vs. carbapenems Growth on media with carbapenems Carbapenemase-producing Enterobacteriaceae / organisms CPE (or CPO) Must detect (or infer) carbapenemase production: Detect carbapenem hydrolysis Detect carbapenemase genes Beta-lactamase inhibitor tests Suitable for use on isolated bacteria Suitable for use on isolated bacteria or directly on clinical specimens 10 BSMT, 16th May 2014 Crown Copyright
11 Antibiotic susceptibility testing Quantitative methods (MIC) - agar or broth dilution - gradient strips (Etests, MICE) Qualitative methods (S/I/R) - disc diffusion - agar incorporation breakpoint method Automated methods BSMT, 16th May 2014 Crown Copyright
12 Chromogenic agars Brilliance CRE CHROMagar KPC ChromID Carba ChromID OXA-48 ChromID Carba Smart* Colorex chromogenic KPC Expensive Not all are suitable for all of big five carbapenemases May need two agars for maximum sensitivity There will be problematic strains 12 BSMT, 16th May 2014 Crown Copyright
13 EUCAST algorithm for carbapenemase detection APBA = aminophenyl boronic acid; PBA = phenyl boronic acid; DPA = dipicolinic acid; EDTA = ethylenediaminetetraacetic acid (all β- lactamase inhibitors added to discs or tablets containing meropenem in combination disc assays) 1 Combination of KPC + VIM may not show synergy but isolates are normally highly resistant to carbapenems easiest to detect with molecular assays. 2 High-level temocillin resistance [MIC > 32 mg/l, zone diameter <11 mm with temocillin 30 μg disc] are indicators of OXA-48 production, which should be considered in the absence of synergy with inhibitors 13 BSMT, 16th May 2014 Crown Copyright
14 Confirming carbapenemase production: supplemental tests BSMT, 16th May 2014 Crown Copyright
15 Confirmation tests Double disc synergy test gradient strip tests Four discs are tested Carbapenem only Carbapenem + M L inhibitor Carbapenem + KPC inhibitor Carbapenem + AmpC inhibitor Combination disc tests 15 BSMT, 16th May 2014 Crown Copyright
16 Inhibitor-based tests: pros and cons Pros: Relatively cheap and easy to perform Most extensively evaluated, therefore recommended by EUCAST for labs without special expertise in -lactamase detection. Cons: Some MBL inhibitors act non-specifically (especially EDTA) Most effective for Enterobacteriaceae; more false +ves with NFs May lack sensitivity if carbapenemase expressed at low level Difficult to interpret when multiple resistance mechanisms present No inhibitors for OXA BSMT, 16th May 2014 Crown Copyright
17 Limiting the impact of carbapenemases Detecting resistance in the clinical laboratory is too slow if using traditional AST methods and supplemental tests RAPID diagnostics are essential for identifying infected / colonized patients: 1. appropriate patient management 2. rapid implementation of infection control procedures 3. Prevent onwards transmission 17 BSMT, 16th May 2014 Crown Copyright
18 Rapid Diagnostics to Guide Empiric Therapy Rapid 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 indicates potentially inappropriate empiric therapy Carbapenemase detected: carbapenem NOT suitable as sole agent (Confirming susceptibility = prime criterion for appropriate therapy) 18 BSMT, 16th May 2014 Crown Copyright
19 BSMT, 16th May 2014 Crown Copyright
20 Detecting hydrolysis: CarbaNP test CarbaNP: yes/no answer: Enterobacteriaceae (Nordmann et al. [2012]) Pseudomonas aeruginosa (Dortet et al. [2012]) Rosco Diagnostica Rapid CARB Screen kit CarbaNP-II: determination of carbapenemase type (Dortet et al. [2012]) Results 2 hrs Reported good sensitivity and specificity (but most papers by inventors!) Tijet et al. (2013): false-negatives with mucoid strains and OXA-48 producers 20 BSMT, 16th May 2014 Crown Copyright
21 Detecting hydrolysis: MALDI-ToF MS Meropenem solution Negative control NDM-1 K. pneumoniae NDM-1 A. baumannii (Ledeboer, N. A. et al. 2011) (Hrabák J et al. 2012) Positive evaluations for detection of resistance to carbapenems and other -lactams (Burckhardt & Zimmermann 2011; Hrabák et al. 2011; Sparbler et al. 2012; Hrabák et al BSMT, 16th May 2014 Crown Copyright
22 MALDI-ToF for direct detection of carbapenemase producers in blood cultures Carvalhaes JAC 2014 Apr 9 Epub 22 BSMT, 16th May 2014 Crown Copyright
23 Commercial molecular systems Increasing numbers of products for a growing market Amplex Hyplex Superbug ID Becton Dickinson BDMax CRE biomerieux - NucliSens Cepheid GeneXpert MDRO / Carba-R Check-Points Check MDR Carba / Check-Direct CPE / microarrays Coverage of big five carbapenemases varies None would find novel carbapenemases Range from yes/no tests to full group differentiation 23 BSMT, 16th May 2014 Crown Copyright
24 Examples of commercial real time solutions Company Kit Basis Isolates Samples Amplex eazyplex SuperBug complete or eazyplex SuperBug CRE (CE-marked) RT PCR Yes screening swabs Family Coverage * KPC, OXA- 48, VIM, NDM Platform Proprietary Time 15 mins (isolates) or 30 min (swabs) Becton Dickinson BD MAX CRE (RUO) RT PCR Yes clinical samples, screening swabs KPC, OXA- 48, NDM Proprietary (Open) 2 h biomerieux NucliSENS EasyQ KPC (RUO) NASBA Yes stool samples, rectal swabs KPC Proprietary 3-4 h Cepheid GenXpert Carba-R (RUO) RT PCR Yes rectal swabs KPC, NDM, OXA-48, VIM, IMP Proprietary 50 min Check- Points Check-Direct CPE (CE-marked) RT PCR Yes rectal/perianal swabs KPC, OXA- 48, VIM, NDM Multiple RT machines 2 h N.B. Coverage of alleles within the IMP, VIM and OXA-48 families varies between kits 24 BSMT, 16th May 2014 Crown Copyright
25 Allele diversity, assay coverage and sensitivity For maximum sensitivity, molecular assays must include probes / primers to detect all known gene variants >120 alleles in big 5 families ( 9 th April 2014) KPC: 18 variants NDM: 11 variants IMP: 48 variants VIM: 41 variants OXA-48-like: -48, -181 (at least 10 variants) Solutions usually include compromises and won t detect rarer enzymes Family coverage simple for KPC, NDM; harder for OXA-48-like; much harder for IMP and VIM 25 BSMT, 16th May 2014 Crown Copyright
26 So, there isn t a single best method because of the diversity of carbapenemases their different molecular classes the variation between genes within major familes all hydrolyze carbapenems, but have no other shared properties because of the diversity of host strains level of resistance is contigent on e.g. porin status etc. 26 BSMT, 16th May 2014 Crown Copyright
27 , but at least we can have a standard approach 27 BSMT, 16th May 2014 Crown Copyright
28 , but at least we can have a standard approach 28 BSMT, 16th May 2014 Crown Copyright
29 , but at least we can have a standard approach 29 BSMT, 16th May 2014 Crown Copyright
30 , but at least we can have a standard approach 30 BSMT, 16th May 2014 Crown Copyright
31 , but at least we can have a standard approach 31 BSMT, 16th May 2014 Crown Copyright
32 Coming soon, SMI v. 2.0 Revision of SMI in 2014 Gathering evidence Comparison of commercial assays Evaluations of media Seek to make it a NICE-approved guideline 32 BSMT, 16th May 2014 Crown Copyright
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