Combatting AMR: diagnostics
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1 Combatting AMR: diagnostics Professor Neil Woodford Antimicrobial Resistance & Healthcare Associated Infections (AMRHAI) Reference Unit Crown copyright
2 Gonorrhoea: a paradigm for better diagnostics International approach to treatment Recommendation changes when resistance rate exceeds 5% Many patients are over-treated to prevent under-treating a few Gonococci in UK (2013): >80% are PEN-susceptible >70% are CIP-susceptible Neither drug is used Can t detect AMR at presentation Efficacy Stewardship 2 Sydney, 20th November 2014 Crown Copyright
3 Stages in treatment Suspected severe bacterial infection Take samples for microbiology Start smart then focus Empiric broad-spectrum therapy (broad spectrum) Review Clinical diagnosis / continuing need for antibiotics Prescribing decisions: Stop, Switch IV to Oral, Change, Continue, Outpatient Parenteral Antibiotic Therapy (OPAT) Narrow spectrum therapy guided by AST data after h Faster diagnostics can reduce time on empiric therapy Reduce inappropriate antibiotic use (improve outcomes & stewardship) 3 Sydney, 20th November 2014 Crown Copyright
4 Carbapenemase producers: low chance that empiric therapy will be appropriate Metallo-enzyme Producers (IMP, NDM or VIM) 90% 4 Sydney, 20th November 2014 Crown Copyright HPR, 2011; 5: issue 24 (17/06/11; Woodford & Livermore)
5 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 5 Sydney, 20th November 2014 Crown Copyright
6 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. 6 Sydney, 20th November 2014 Crown Copyright
7 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 7 Sydney, 20th November 2014 Crown Copyright
8 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 88 Sydney, 20th November 2014 Crown Copyright
9 Confirming carbapenemase production: supplemental tests 9 9 Sydney, 20th November 2014 Crown Copyright
10 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 10 Sydney, 20th November 2014 Crown Copyright
11 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 11 Sydney, 20th November 2014 Crown Copyright
12 Commercial systems Increasing numbers of products for a growing market Phenotypic detection of hydrolysis Colorimetric e.g. Rosco Diagnostica Rapid CARB Screen kit; biomerieux Rapidec MALDI-ToF Molecular detection of resistance genes Coverage of big five carbapenemases varies Range from yes/no tests to full group differentiation None will find novel carbapenemases 12 Sydney, 20th November 2014 Crown Copyright
13 Examples of commercial real time solutions Company Kit Basis Isolates Samples Amplex eazyplex SuperBug complete or eazyplex SuperBug CRE (CE-marked) LAMP 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 13 Sydney, 20th November 2014 Crown Copyright
14 The Equipment BD MAX (Check-Points) Cepheid GeneXpert Amplex Genie II 14 Sydney, 20th November 2014 Crown Copyright
15 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 15 Sydney, 20th November 2014 Crown Copyright
16 There isn t a single best detection method because of the diversity of carbapenemases their different molecular classes the variation between genes within major families 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. 16 Sydney, 20th November 2014 Crown Copyright
17 Rapid Diagnostics to Guide Empiric Therapy Rapid tests for mechanisms = surrogates for rapid AST 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) 17 Sydney, 20th November 2014 Crown Copyright
18 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. 2012) No false-positives or false-negatives 18 Sydney, 20th November 2014 Crown Copyright
19 MALDI-ToF for direct detection of carbapenemase producers in blood cultures Carvalhaes JAC 2014 Apr 9 Epub 19 Sydney, 20th November 2014 Crown Copyright
20 Current processes for bacteria Nature Reviews Genetics 13, (September 2012) 20 Sydney, 20th November 2014 Crown Copyright
21 WGS-based future practice In one step generate the complete diagnostic, typing and surveillance information Nature Reviews Genetics 13, (September 2012) 21 Sydney, 20th November 2014 Crown Copyright
22 For the future: WGS-informed diagnostics? 22 Sydney, 20th November 2014 Crown Copyright
23 LN(Clone Intensity) LN(Clone Intensity) Accelerate: AST from sample in 5 h Time-lapse imaging and analysis Response over time of individual cells to single antibiotic concentration used to infer MIC E. coli vs. 4 μg/ml Piperacillin-Tazobactam MIC=8(S) Hours MIC=128(R) 23 Sydney, 20th November 2014 Crown Copyright Hours
24 Become an Antibiotic Guardian As an Antibiotic Guardian, choose a simple action based pledge and encourage others to join you in protecting antibiotics against the growing threat of antibiotic resistance at: The Antibiotic Guardian campaign was established by PHE to improve public and professional knowledge and stimulate engagement on tackling antibiotic resistance 24 Sydney, 20th November 2014 Crown Copyright
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