Expert rules in antimicrobial susceptibility testing: State of the art

Expert rules in antimicrobial susceptibility testing: State of the art ESCMID Postgraduate Education Course Antimicrobial Susceptibility Testing and Surveillance: from Laboratory to Clinic Hospital Universitario Ramón y Cajal SERVICIO DE MICROBIOLOGÍA Y PARASITOLOGÍA Dr. Rafael Cantón Dpto. de Microbiología Universidad Complutense Madrid. Spain

Antimicrobial susceptibility testing Clinical categorization (S, I, R) Intrepretive reading Application of expert rules Based on clinical breakpoints Based on resistance mechanisms knowledge Based both on clinical evidence and resistance mechanisms knowledge

ANTIBIOGRAM: CLINICAL CATEGORIZATION CLSI (NCCLS) CLINICAL BREAKPOINTS EUCAST CLINICAL BREAKPOINTS EPIDEMIOLOGYCAL CUT-OFF

Cefalosporins Antibiogram clinical categorization Enterobacteriaceae and extended spectrum cephalosporins CLSI EUCAST S ( ) R ( ) S ( ) R (>) ECOFF ( ) Cefotaxime 8 1 64 4 1 2 0.25 (E. coli) 0.125 (K. pneumoniae) Ceftazidime 8 4 32 16 1 8 4 0.5 Cefepime 8 8 32 16 1 8 4 0.125 M100S19, CLSI 2009 M100S20, CLSI 2010 www.eucast.org Version 1.0 Dec-2009 www.eucast.org Version 1.1 Apr-2010

Interpretive reading of in-vitro antimicrobial test results 1.- To establish the susceptibility phenotype 2.- To infer the potential resistance mechanism 3.- To predict previously defined phenotype from the resistance mechanism Patrice Courvalin, ASM News, 1992

Antimicrobial Antibiogram interpretative reading Importance of bacterial identification MIC (mg/l) Ampicillin >64 Amox/clav >32/16 Ticarcillin >64 Piperacillin 32 Piper/Tazo 16/4 Cefuroxime >64 Cefoxitin >32 Cefotaxime 4 Ceftazidime 8 Cefepime 1 Organisms Potential phenoype E. coli AmpC hyperproduction plasmid AmpC ESBL + porin deficiency K. pneumoniae ESBL + porin deficiency E. cloacae ESBL

Antibiogram interpretative reading: the classical example Escherichia coli and ESBL

Stenotrophomonas maltophilia (L1+ L2 enzymes) CTX CPM AMC CAZ ATM

ATM CTX AMC AMP CAZ ATM CTX AMC AMP CAZ

Proteus vulgaris hiperproduction of chromosomal b-lactamase (Class A) AMP PIP CEF CXM CAZ FOX CTX AMC FEP ATM Proteus vulgaris IPM Ampicillin Ticarcillin Piperacillin Piper/Tazo Amox/clav Cefalotin Cefoxitin Cefuroxime Cefotaxime Ceftazidime Cefepime Imipenem MIC >256 8->256 8->256 8-32 4-8 >256 2-4 >256 2-8 0,12-0,5 0,5-2 0,5-2 Interp. R R S/R S/R S/I R S R R S S/I S

CTX-30 µg AMC (20:10 µg) CTX+CLAV 30+10 µg CLAV -10 µg CTX-30 µg CTX CTX+CLAV-4 µg

Integration of intepretive reading in a clinical microbiology lab Microorganism To indentify clinical transcendence of the resistance phenotype Identification + antibiogram Phenotype characterization Infer of the resistance mechanism Refining susceptibility clinical interpretation Deduction of susceptibility of non-tested antibiotics Interpretation of antibiogram (S/I/R) Report of the results R. Cantón. Enferm Infecc Microbiol Clin 2002; 20: 176-186 R. Cantón. Enferm Infecc Microbiol Clin 2008; 28:375-85

EUCAST expert rules An expert rule... Description of action to be taken, based on current clinical and/or microbiological evidence, in response to specific antimicrobial susceptibility test results Assist clinical microbiologists in the interpretation of antimicrobial susceptibility tests Contribute to quality assurance by highlighting anomalous or unlikely results www.eucast.org

EUCAST expert rules A classical example... S. aureus resistant to oxacillin or cefoxitin Acquisition of the meca gene Report resistant to all β-lactams! www.eucast.org

EUCAST expert rules A classical example... P E OX CN AK Rule 12.3 (version 2) IF resistant to gentamicin, THEN report as resistant to all aminoglycosides* *Resistance to tobramycin is generally due to the production of ANT(4 )(4 )I or bifunctional APH(2 )-AAC(6) enzymes that determine loss of synergism of kanamycin, tobramycin and amikacin with β-lactams and glycopeptides irrespective of MIC values. www.eucast.org

EUCAST expert rules The EUCAST expert rules in antimicrobial susceptibility testing are divided into: - intrinsic resistances - exceptional phenotypes - interpretive rules www.eucast.org

EUCAST expert rules Intrinsic resistance Opposed to acquired or mutional resistance Characteristic of all (or almost all) representatives of the species The antimicrobial activity of the drug is clinically insufficient or antimicrobial resistance innate as to render it clinically useless Antimicrobial susceptibility testing is normally unnecessary In these species, susceptible results should be viewed with caution (this indicates an error in identification or susceptibility testing) - if S is confirmed the drug should preferably not be used - R may be expressed at a low level (MIC close to the S breakpoint) although the antibiotic is not considered clinically active

Ampicillin Amoxicilinclavulanate Ticarcillin Piperacillin Cefazolin Cefoxitin Cefamandole Cefuroxime Aminoglycosides Tetracyclines/ tigecycline Polymyxin B/ Colistin Nitrofurantoin Rule no. Organisms EUCAST expert rules Intrinsic resistances affecting ß-lactams 1.1 Citrobacter koseri R R R 1.2 Citrobacter freundii R R R R 1.3 Enterobacter cloacae R R R R 1.4 Enterobacter aerogenes R R R R 1.5 Escherichia hermannii R R R 1.6 Hafnia alvei R R R 1.7 Klebsiella spp. R R 1.8 Morganella morganii R R R R R R R 1.9 Proteus mirabilis R R R 1.10 Proteus vulgaris R R R R R R R 1.11 Proteus penneri R R R R R R R 1.12 Providencia rettgeri R R R Note 2 R R R 1.13 Providencia stuartii R R R R R R 1.14 Serratia marcescens R R R R R Note 3 R R 1.15 Yersinia enterocolitica R R R R R R R 1.16 Yersinia pseudotuberculosis R www.eucast.org

EUCAST expert rules Exceptional phenotypes Resistances of some bacterial species to particular antimicrobial agents which have not yet been reported or are very rare They should be checked as they may also indicate an error in identification or susceptibility testing. If they are confirmed locally: - the isolate should be further studied - sent to a reference laboratory for independent confirmation The may change with time as resistance may develop and increase over time There may also be local or national differences. Very rare in one hospital, area or country, may be more common in another

.. Rule no. EUCAST expert rules Organisms Exceptional phenotypes Excepcional phenotype 5.1 Any Enterobacteriaceae Resistant to ertapenem, meropenem, imipenem (except Proteus spp.) 5.2 P. aeruginosa, Acinetobacter spp. Resistant to colistin 5.3 Haemophilus influenzae Resistant to any 3 rd -gen. cephalosporin, carbapenems, fluoroquinolones 5.4 Moraxella catarrhalis Resistant to ciprofloxacin, any 3 rd -gen. cephalosporin 5.5 Neisseria meningitidis Resistant to penicillin (MIC>1 mg/l), 3 rd -gen. cephalosporins, ciprofloxacin 5.6 Neisseria gonorrhoeae Resistant to 3 rd -gen. cephalosporins, spectinomycin www.eucast.org

EUCAST expert rules Interpretive rules: IF...THEN approach Simple rules - IF S. aureus is R to oxacillin or cefoxitin THEN report R to all β-lactams - IF not all carbapenems are tested, THEN test results regarding one carbapenem (imipenem, meropenem, ertapenem, doripenem) cannot be inferred to the other carbapenems. Complicated rules - IF Enterobacteriaceae I to tobramycin, R to gentamicin and S to amikacin THEN report R to tobramycin They are evidence supported, but often not conclusive with different opinions regarding the most appropriate clinical action Based on current published evidence (the quality of evidence should be assessed and exceptions to any rules should be noted)

EUCAST expert rules Evidences A. There is clinical evidence that reporting the test result as susceptible leads to clinical failures B. Evidence is weak and based only on a few case reports or on experimental models. It is presumed that reporting the test result as susceptible may lead to clinical failures C. There is no clinical evidence, but microbiological data suggest that clinical use of the agent should be discouraged

EUCAST expert rules Evidences A. There is clinical evidence that reporting the test result as susceptible leads to clinical failures

EUCAST expert rules Evidences B. Evidence is weak and based only on a few case reports or on experimental models. It is presumed that reporting the test result as susceptible may lead to clinical failures

EUCAST expert rules Evidences C. There is no clinical evidence, but microbiological data suggest that clinical use of the agent should be discouraged

EUCAST expert rules Interpretive rules can be modified over time...!

Rule No. EUCAST expert rules Interpretive rules can be modified over time... Rule Exceptions Scientific basis Grade* References 9.1* If R or I to any 3 rd or 4 th gen. oxyimino-ceph. (i.e. cefepime, cefotaxime, cefpirome, cefpodoxime, ceftazidime or ceftriaxone) or aztreonam, test for ESBL. If positive, edit the S result for any of these cephalosporins, including 4 th gen. agents and for aztreonam, to I and edit the I result to R. ESBL producers may appear susceptible to penicillin/ ß- lactamase inhibitor combinations. The use of these combinations against ESBL producers remains controversial, and should be approached with caution. If ESBL negative see rule 9.2. * Enterobacteriaceae (for Klebsiella oxytoca and Citrobacter koseri see 9.3) A few ESB producers may appear S in vitro to some 3 rd or 4 th gen. oxyiminocephalosporin or aztreonam. Efficacy of cefotaxime, ceftazidime and ceftriaxone against ESBL-producing isolates with MICs lower than 2 mg/l remains to be fully documented A. There is clinical evidence that reporting the test result as susceptible leads to clinical failures B. Evidence is weak based only on a few case reports or on experimental models. It is presumed that reporting the test as susceptible my lead to clinical failures C. There is currently no clinical evidence, but microbiological data suggest that clinical use of the agent should be discouraged C Burn-Buisson et al. 1987 Jarlier V et al., 1988. Livermore DM and Brown DF, 2001. Wong- Beringer A et al., 2002. Paterson D and Bonomo R, 2005 Paterson DL et al. 2004 Bhavnani SM et al. 2006

EUCAST expert rules 2010 Ceftazidime and cefepime R breakpoint has been reduced from >8 mg/l to >2 mg/l Cefalosporins EUCAST S ( ) R (>) Cefotaxime 1 2 Ceftazidime 1 4 Cefepime 1 4 Report as found regardless of the presence of a resistance mechanism (ESBL or other)

EUCAST expert rules EUCAST expert rules should be applied with EUCAST breakpoints!...

3 rd & 4 th gen. cephalosporin breakpoints in Enterobacteriaceae CLSI (2010) EUCAST (2010) Cefalosporins S R S R Cefotaxime 1 4 1 >2 Ceftriaxone 1 4 1 >2 Ceftazidime 4 16 1 >4 Cefepime 8 32 1 >4 Aztreonam 4 16 1 >4 Remember! R category is in CLSI while > in EUCAST = =

Cephalosporin breakpoints in Enterobacteriaceae Current EUCAST approach is aimed - To provide breakpoints consistent with new Pk/Pd knowledge - To balance the clinical significance of ESBL resistance mechanism - To relate MIC values to clinical response (nevertheless more clinical evidence are still needed!) - To avoid the delay of reporting for ESBL-producing organisms

Cephalosporin breakpoints in Enterobacteriaceae E. coli with reduced susceptibility to 3 rd & 4 th gen. cephalosporins Antibiotic MIC (mg/l) CLSI EUCAST 2009 2010 2009 2010 Amoxicillin >16 R R R R Amox/clav 4/2 a ; 8/2 b S S S S Cefazolin >32 R R R R Cefoxitin 4 S R S NA c NA c Ceftotaxime >16 R R R R Ceftazidime 4 S R S I R I Cefepime 2 S R S I R I Aztreonam 4 S R S I R I Imipenem 0.25 S S S S Meropenem 0.03 S S S S a CLSI: 2:1 ratio; b EUCAST: fixed concentration (2 mg/l); c NA: not available

Cephalosporin breakpoints in Enterobacteriaceae The murine thigh infection model showed that the % T>CMI was similar in therapy (3 rd /4 th gen. cephalosporins) against ESBL and non-esbl groups - PK/PD breakpoints should be independent of the resistance mechanisms - The MIC of ß-lactam in an ESBL-producing isolate can be used to predict likely human outcomes from PK/PD models Andes & Craig. Clin Microbiol Infect 2005; 11 (Supp. 6): 10-7

% of clinical failure cephalosporin treat Cephalosporin breakpoints in Enterobacteriaceae Clinical outcome of 3 er /4 th gen. cephalosporins treatment in patients with serious infections due to ESBL-organisms % of clinical failure with cephalosporin treatment 100 80 60 40 20 0 <1 2 4 8 16 MIC MIC (mg/l), 3rd of 3/ rd 4th /4 gen. cephalosporins Paterson et al. J Clin Microbiol 2001; 39:2206-12

% of success Cephalosporin breakpoints in Enterobacteriaceae Clinical outcome in 42 patients with ESBL-producing Klebsiella spp. or E. coli bacteraemia and treated with cephalosporin monotherapy 80 60 40 20 0 1 2 4 8 CMI (mg/l) Andes & Craig. Clin Microbiol Infect 2005; 11 (Supp. 6): 10-7

Cephalosporin breakpoints in Enterobacteriaceae The EUCAST approach Cefotaxime and ceftriaxone breakpoints: S 1 / R >2 mg/l - consistent with Pk/Pd breakpoints - clinical data: outcome is poor when MICs are >2 mg/l no differences with non-esbl producers with MICs 1 mg/l S R MacGowan A. Clin Microbiol Infect 2008; 14 (Suppl 1):166-8

Cephalosporin breakpoints in Enterobacteriaceae The EUCAST approach Ceftazidime, cefepime, aztreonam: S 1 / R > 4 mg/l - Pk/Pd: - probability of target attainment (PTA), ft/mic = 60% - Clinical data: - with doses of 1 g x 3, PTA is achieved for MICs of 4 mg/l S 2 g x 3, PTA is achieved for MICs of 8 mg/l R - no differences with non-esbl producers with MICs 1 mg/l - the S breakpoint was reduced from 4 to 1 mg/l due to uncertain reports of clinical outcome for isolates with MICs 2-4 mg/l - outcome decrease with MICs >4 mg/l R breakpoint

Cephalosporin breakpoints in Enterobacteriaceae When using the new EUCAST breakpoints. - it is no longer necessary to edit results for cephalosporins or aztreonam from susceptible to resistant (report as found ) NO CLINICAL CATEGORIZATION MODIFICATION IS NEEDED - ESBL testing is no longer necessary before reporting the results, but still be useful for epidemiological or infection control purposes NO ESBL CONFIRMATION IS NEED UNLESS FOR EPIDEMIOLOGY