ANTIBIOGRAMMA: TECNICHE PER LA DETERMINAZIONE E INTERPRETAZIONE DEL RISULTATO. Giovanni Di Bonaventura, PhD

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1 ANTIBIOGRAMMA: TECNICHE PER LA DETERMINAZIONE E INTERPRETAZIONE DEL RISULTATO Giovanni Di Bonaventura, PhD

2 ANTIBIOTIC RESISTANCE... a dark scenario VRE 1st CTX-M ESBL VRE in animals CR-AB clones emerge CTX-M ESBL explosion starts Carbapenemases Enterobacteria; NDM-1 discovered PCR EMRSA Genome sequence Lin-R enterococci Dap-R staphs & enterococci (from: Woodford N, HPA)

3 WHY PERFORM ANTIMICROBIAL SUSCEPTIBILITY TESTS? Individualand and epidemiological purposes The performance of antimicrobial susceptibility testing by the clinical microbiology laboratory is important to: guide physicians in selecting antimicrobial therapy for treatment of individual patients confirm susceptibility to chosen empirical antimicrobial agents choose alternative agents when patient experiences adverse reaction to the (empirical) agents detect resistance in individual bacterial isolates reveal the changing trends in the local (ward, healthcare establishment, region, country) isolates: a guide for empiric therapy choices and antibiotic formulary decisions help the local pattern of antibiotic prescribing detection of outbreaks, requiring the need for implementation/change of infection control practices Data from routine antimicrobial susceptibility testing performed in clinical microbiology laboratories influences the therapeutic decisions for current and future patients

4 WHEN SHOULD A SUSCEPTIBILITY TEST BE PERFORMED? Definingthe etiologic role of a microorganism Synergy between Microbiologistand Clinician Susceptibility testing is indicated for microorganisms causing infections warranting antimicrobial therapy when the susceptibility cannot be reliably predicted based on he known characteristics of the organism: natural vs acquired resistance AST detects acquired resistance only Susceptibility testing should not be performed on probable contaminants: S. epidermidis is occasionally isolated from sterile site cultures (e.g. blood, joint fluid, cerebrospinal fluid) due to inadequate decontamination of the skin during specimen collection S. epidermidis can cause a true bloodstream infection in an immunocompromised patient or an infection at a specific body site (e.g. prosthetic joint, cerebrospinal fluid shunt) in which case, susceptibility testing should be performed Need for clinical informations: clinical symptoms can also be a determining factor when deciding whether to perform susceptibility tests (e.g. diagnosis of urinary tract infection with a low bacterial count) Susceptibility testing should not be routinely performed on commensal microorganisms buton pathogeniconesonly.establishingthe needforsusceptibilitytestingrequiresa close working relationship between Microbiologist and Clinician

5 IN VITRO SUSCEPTIBILITY TESTING METHODS Overview Phenotypictests QUANTITATIVE methods(mic, µg/ml) Broth dilution Agar dilution Gradient methods Automated systems QUALITATIVE methods(s, I, R) Disk diffusion Agar-incorporation breakpoint methods Ancillary tests (to screen/confirm resistance patterns) Genotypic(molecular) tests REFERENCE methods

6 SUSCEPTIBILITY TESTING METHODS Phenotypic tests Commonly used Growth-based, involving: a pure culture, exposed to a range of concentrations of an antimicrobial agent observation of the presence or absence of microbial growth after a period of incubation They are strongly affect by conditions of testing: purity and density of bacterial inoculum medium composition incubation conditions reading method interpretative criteria It is, therefore, mandatory to use standardized methods, as recommended by: CLSI(Clinical and Laboratory Standards Institute) EUCAST(European Union Committee for Antimicrobial Susceptibility Testing)

7 Phenotypic tests broth microdilution Antibiotic is incorporated into broth in doubling concentrations The lowest concentration of antibiotic that prevented visible growth represents the minimal inhibitory concentration (MIC).

8 Phenotypic tests agar dilution Antibiotic is incorporated into agar in doubling concentrations. The lowest concentration of antibiotic that prevented visible growth onto agar represents the minimal inhibitory concentration (MIC).

9 Phenotypic tests broth/agar dilution tests Pros& Cons

10 Phenotypic tests Gradient diffusion A preformed and predefined gradient of varying antibiotic concentrations is immobilized in a dry format onto the surface of a plastic strip. The point where the growth or inhibition margin of the organism intersects the edge of the calibrated strip corresponds to the minimal inhibitory concentration (MIC).

11 Phenotypic tests Gradient diffusion-based commercial tests Etest (biomerieux) M.I.C. Evaluator (M.I.C.E.; Oxoid) MIC test strip (Liofilchem)

12 Phenotypic tests gradient diffusion Pros& Cons

13 Phenotypic tests Automated Systems Before the 1970s, labor-intensive manual susceptibility testing was the dominant method. Use of instrumentation can standardize the reading of end points and often produce susceptibility test results in a shorter period than manual readings because sensitive optical detection systems allow detection of subtle changes in bacterial growth. In 1974, the first automated system known as the Autobac I disk elution system was introduced by Pfizer Diagnostics. Now, more than 80% of clinical laboratories report using an automated instrument for primary susceptibility testing.

14 Phenotypic tests Automated Systems VITEK 2 (biomerieux) Microdilution tray Panel inoculation Reading technology Results available in Data analysis miniaturized automated colorimetric 8h (4-12h) ++ MICROSCAN WalkAway standard size manual (Siemens) PHOENIX (BD Diagnostics) SENSITITRE (Trek Diagn. Sys.) photometric/ fluorescent 20h (17-28h) ++ miniaturized manual colorimetric 10h (7-16h) +++ standard size manual fluorescent 15h ++ modified from: Kuper et al., Pharmacotherapy 2009

15 Phenotypic tests Automated systems Pros& Cons

16 Phenotypic tests Disk diffusion(kirby-bauer) Images from: EUCAST 2012 Version 2.1

17 Phenotypic tests Disk diffusion Pros& Cons -test simplicity, not requiring any special equipment -the provision of categorical results easily interpreted by all clinicians - flexibility in selection of antibiotics for testing -it is the least costly of all susceptibility methods - the lack of mechanization/automation of the test -difficult reading with bacteriostaticor high molecular weight antibiotics (vancomycin, colistin, macrolides) -not all fastidious or slow growing bacteria can be accurately tested; test has been standardized for testing streptococci, Haemophilusinfluenzae, and N. meningitidis

18 MICs and zone sizes are meaningless unless you apply interpretative criteria CLSI and EUCAST develop and promulgate MIC breakpoints employing some combination of four criteria: MIC frequency distribution analysis MIC assessment in the context of the presence or absence of known mechanisms of resistance evaluation of MICs based on drug levels in patients receiving antibiotic therapy(i.e. PK/PD analysis) clinical correlation (response rates in patients with infection compared to the drug MICs associated with their infecting pathogens) clinical breakpoints(clsi, EUCAST)indicate likelihood of therapeutic success (S) or failure (R) of antibiotic treatment based on microbiological findings (S Y mg/l and R> Z mg/l)an intermediate result (Y < I< Z mg/l) indicates thatclinical response is likely to be less than with a susceptible strain. epidemiological cut-off values (ECOFFs) (EUCAST)separate microorganisms without (wild type) and with acquired or mutational resistance (non-wild type) (WT X mg/l)

19 CLSI vs EUCAST La transizione dai criteri CLSI a quelli dettati da EUCAST comporterà, in alcuni casi, un abbassamento dei breakpoints e, di conseguenza, per alcune specifiche combinazioni microrganismo/antibiotico verrà data una interpretazione dell antibiogramma leggermente più restrittiva : alcuni isolati che prima venivano refertati come S risulteranno I o R (Tabella 1). non viene più consigliato il saggio di sensibilità per alcune combinazioni microrganismo/antibiotico non ritenute opportune in ambito terapeutico (Tabella 2).

20 Kirby-Bauer Interpretazione dei risultati CLSI breakpoints

21 Kirby-Bauer Interpretazione dei risultati Esempio di curva di regressione tra i valori di MIC (mcg/ml) ed i diametri degli aloni di inibizione (mm) ottenuti nel KB.

22 Kirby-Bauer Interpretazione dei risultati EUCAST breakpoints

23 Microdiluizione in brodo / E-test Interpretazione dei risultati CLSI breakpoints

24 Refertazione dell antibiogramma Il Laboratorio di Microbiologia è di fatto un forte induttore di terapie antibiotiche, alcune appropriate, altre meno. Il referto microbiologico può costituire uno Il referto microbiologico può costituire uno strumento formidabile di comunicazione per l orientamento nell interpretazione degli esiti, ma anche la formazione e l aggiornamento su specifiche problematiche.

25 Refertazione dell antibiogramma Informazioni desumibili Risposte alle domande del Clinico: Qual è il patogeno in causa? La terapia empirica impostata è efficace anche sul patogeno isolato? Quali sono i farmaci che posso utilizzare in alternativa alla terapia empirica? Meno tossici Per via orale In realtà molte altre informazioni possono essere desunte meccanismo di resistenza probabile (testsaggiuntivi per determinare genotipi/fenotipi di resistenza: ESBL, meca, VISA, VRSA, etc.) es. farmaci equivalenti (sia R che S)

26 Refertazione dell antibiogramma Informazioni desumibili Test aggiuntivi per stafilococchi: Nitrocefin ß lattamasi Se POS = inattività di Penicillina e di tutte le molecole rappresentate Lattice per PBP2a meca Se POS = inattività di tutti i ß lattamici D-test MLSB inducibile Se POS = inattività di Macrolidi e Clindamicina Vancomicina Screen Agar VISA e VRSA Se POS = ridotta attività dei glicopeptidi (?)

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28 Interpretazione critica dell antibiogramma: MIC Note interprertative MBC Killing quotient Expert rules

29 Interpretazione critica dell antibiogramma: MIC MIC = numero magico? NON SEMPRE! La maggior parte dei Clinici non ne comprende appieno il significato Inoltre, non è probabilmente il parametro più adeguato per descrivere la complessità dei meccanismi di resistenza E' compito del Microbiologo spiegarne il significato Se correttamente interpretata e utilizzata, la MIC è uno strumento di grande utilità per la scelta della migliore strategia terapeutica, soprattutto in caso di particolari infezioni (endocarditi, osteomieliti, etc.), la cui criticità è dovuta a: sede di infezione (sangue, cuore, sistema nervoso centrale, polmone, tessuti profondi); condizioni cliniche del paziente; microrganismi multi-resistenti (MDR).

30 Interpretazione critica dell antibiogramma: MIC Per interpretare il valore di MIC in maniera corretta è necessario considerare che: valori preceduti da segno indicano che la crescita del microrganismo è stata inibita dalla più bassa concentrazione di antibiotico saggiata; esprimono, quindi, una notevole sensibilità indipendentemente dall entità del valore numerico. Esempio: MIC antibiotico X 8 MIC antibiotico Y 0,5 Il microrganismo è sensibile tanto a X quanto a Y se non preceduto da tale segno, il valore della MIC dovrebbe essere valutato anche in relazione alla distanza del valore dal breakpoint di sensibilità, tenendo presente che Antibiotico X vengono testate concentrazioni al raddoppio. Esempio: MIC antibiotico X = 0.25 (con breakpoint = 0.5) MIC antibiotico Y = 1 (con breakpoint = 8) Y è l antibiotico con la MIC più favorevole Antibiotico Y

31 Interpretazione critica dell antibiogramma: Note interpretative In alcuni casi, il referto può essere integrato da note o commenti utili perché il Clinico possa interpretare ed utilizzare al meglio i risultati analitici. ESEMPIO 1: per il riscontro di MIC delle cefalosporine inferiori o uguali al limite di sensibilità in ceppi produttori di β-lattamasi a spettro esteso (ESBL) viene aggiunto un commento che segnala la possibilità di un insuccesso terapeutico nella terapia delle infezioni gravi. ESEMPIO 2: P. aeruginosa da emocoltura Le infezioni da P. aeruginosain pazienti granulocitopenici e le infezioni gravi in altri pazienti dovrebbero essere trattate con dosi massime di una penicillina anti-pseudomonas(carbossi-oppure ureido-penicillina) oppure ceftazidime in associazione con un aminoglicoside (CLSI, 2010).

32 Interpretazione critica dell antibiogramma: Attività battericida E necessario considerare la attività battericida di un antibiotico, SOPRATTUTTO in questi casi particolari: infezioni gravi: osteomieliti, endocarditi, meningiti, polmoniti focolaio di infezione situato in distretti anatomici difficilmente accessibili all antibiotico Concentrazione Minima Battericida (MBC): La più bassa concentrazione di antibiotico in grado di eradicare la crescita batterica di almeno il 99.9% (1 germe su elude l azione antibiotica) rispetto alla popolazione iniziale.

33 Interpretazione critica dell antibiogramma: killingquotient Tasso di uccisione (KQ) = MBC / MIC 1 KQ 4 per antibiotici battericidi (beta-lattamici, aminoglicosidi, chinolonici, glicopeptidi, cotrimossazolo, etc.) KQ > 4 per antibiotici batteriostatici (macrolidi, sulfamidici, trimethoprim, tetracicline, cloramfenicolo, etc.)

34 Interpretazione critica dell antibiogramma: expert rules Nella valutazione della antibiotico-sensibilità, una expert rule (ER; regola esperta ) descrive un azione da intraprendere sulla base di specifici risultati ottenuti nei tests di antibiotico-sensibilità. ERs sono basate sui vigenti breakpoints clinici e sulla conoscenza dei meccanismi di resistenza. ERs possono essere di ausilio al Microbiologo ed al Clinico nella interpretazione dei tests di antibiotico-sensibilità. ERs sono dettate da EUCAST ( pubblicate per la prima volta nel 2008, vengono costantemente aggiornate. Attualmente, sono divise in: resistenza intrinseca fenotipi eccezionali regole interpretative

35 How well do the results of phenotypic AST predict therapeutic outcome? In general, resistance as determined by use of in vitrosusceptibility tests is nearly always an independent risk factor for therapeutic failure in patients with infection who are treated with antimicrobial agents. BUT Does resistance alwayspredict failure; does susceptible alwaysdenote Does resistance alwayspredict failure; does susceptible alwaysdenote favorable response to therapy? Murray et al, AAC 1983

36 How well do the results of antimicrobial susceptibility tests predict therapeutic outcome? Essentially the same observations were made in other studies examining the clinical predictive value of several antibiotic MICs (i.e. meropenem, cefoperazone, ciprofloxacin)in immunocompetent patients,with monomicrobic infections treated with a single antibiotic administered parenterally in circumstances in which the penetration of drug to the site of infection is predictable: Gerber A. U., and W. A. Craig Worldwide clinical experience with cefoperazone. Drugs 22: Weinstein et al The clinical significance of positive blood cultures: a comprehensive analysis of 500 episodes of bacteremia and fungemia in adults. Rev. Infect. Dis. 5: Washington, J. A Discrepancies between in vitro activity and in vivo responsetoantimicrobialagents. Diagn. Microbiol. Infect. Dis. 1: Forrest, A., etal Pharmacodynamicsofintravenousciprofloxacinin seriously ill patients. Antimicrob. Agents Chemother. 37: Doern, G. V Interpretive criteria for in vitro antimicrobial susceptibility tests. Rev. Med. Microbiol. 6: Nguyen, M. H., V. L. Yu, and A. J. Morris Antimicrobialresistanceand clinical outcome of Bacteroidesbacteremia: findings of a multicenter prospective observational trial. Clin. Infect. Dis. 30: Evans, M. R., et al Short-term and medium-term outcomes of quinolone-resistant Campylobacter infection. Clin. Infect. Dis. 48: the rule (Rex & Pfaller, 2002): a susceptible result is associated with a favorable therapeutic response in 90-95% of patients when the infecting bacterium has been determined to be resistant, notwithstanding this result, nearly 60% of patients can be expected to respond to therapy

37 Why is that? PROBABLY BECAUSE OF EXPERIMENTAL SETTING: Drugs are tested in the laboratory as single agents against pure cultures of planktonic putative pathogens NOT REPRESENTATIVE FOR PATIENTS: with polymicrobial infections with biofilm-associated infections receiving combination therapy receiving non-standardized dosage amounts of drug having an infection in sites where drug concentrations are different what would be predicted based on plasma pharmacokinetic determinants (e.g. urinary tract infections) infected with microorganisms more/less virulent (virulence determinants expression) MOREOVER : ASTs are performed in the absence of host factors (complement, cytokines, white blood cells, antibodies) that mitigate for or against improvement or disease progression in patients with infections

38 IN VITRO SUSCEPTIBILITY TESTING METHODS Overview Phenotypictests QUANTITATIVE methods(mic, µg/ml) Broth dilution Agar dilution Gradient methods Automated systems QUALITATIVE methods(s, I, R) Disk diffusion Agar-incorporation breakpoint methods Ancillary tests (to screen/confirm resistance patterns) Genotypic(molecular) tests

39 SUSCEPTIBILITY TESTING METHODS Genotypic tests-detection of antimicrobial resistance determinants TECHNIQUES Single and multiplex PCR Real-time PCR DNA sequencing Hybridisation-based techniques Simple sample preparation REQUIREMENTS Must be rapid (TATs), inexpensive, accurate, and easy! - directly from the specimens -rapid (i.e., less than 30 min test for ESBL detection) Platform must be sufficiently versatile to justify investment - target several key species by multiplex approached - several targets for Gram-negative resistance (e.g. carbapenemases) Relatively hands-free, with scope for automation Black box approach: molecular biology steps hidden Simple end-product detection

40 Detection of resistance determinants requires technologies capableof high-throughput multiplexing Real-time PCR is affected by the limited number of unique fluorophores that can be used for simultaneous detection of multiple targets (max 6 detection channels): GeneXpertSystem (Cepheid, Sunnyvale, CA): C. difficile, MRSA, Enterovirus, vana, GBS, Flu(not simultaneously) Liquid-phase microarrays: LuminexXTAG technology (Luminex, Austin, TX): microspheres labeled with red dye to simultaneously detect up to 100 targets in a single reaction tube. BeadExpress(Illumina, San Diego, CA): holographic beads to label up to 300 targets simultaneously, but it has not been tested in a clinical laboratory or with antimicrobial resistance targets. Solid-phase microarrays: NanosphereInc. (Northbrook, IL): simultaneously identifys. aureus, CoNS, Streptococcus spp. (-anginosus, -pneumoniae, -pyogenes, - agalactiae), and Micrococcusspp., in addition to detecting meca, vana, and vanb directly from positive bloodcultures

41 Using molecular assays to: confirm phenotypic assays Several reports have described the use of PCR to confirm the presence of KPCs in members of the family Enterobacteriaceaefollowing identification of resistance by phenotypic assays. The modified Hodge test (MHT) is replaced by PCR, eliminating the subjectivity of MHT and confirming the presence of the KPC resistance determinant. predict treatment failure better than phenotypic assays Enterobacteriaceae bacteria are often found to have low MICs for many betalactams, but patients frequently fail therapy with these agents because ESBLs and AmpC resistance genes are expressed at high levels only when induced by an environmental stimulus, absent in the experimental setting of a phenotypic assay. The presence/absence of mecais a much better predictor of failure in patients with S. aureus infections treated with beta-lactamsthan is any in vitroast Marschall J, et al. J Clin Microbiol 2009;47:239 Tenover. Ann. N. Y. Acad. Sci. 2010;1213:70

42 Clinical significance of molecular tests rapid PCR (GeneXpert system; Cepheid, Sunnyvale, CA) differentiation between S.aureusand CoNS, and assessment of methicillin resistance from positive blood cultures combining this system with an effective antimicrobial stewardship program, vancomycin treatment was reduced of 1.7 days, length of stay in ICU of 6.2 days, reaching an overall savings of $21,000 per patient per septic episode

43 MOLECULAR DETECTION OF RESISTANCE DETERMINANTS Inherent technical challenges Adequate clinical specificity meca(also found in methicillin-resistant CoNS) vana(also associated with vancomycin-resistant S. aureus) vanb (also found in Streptococcus mitis, Streptococcus bovis, Eggerthella lenta, Clostridium spp., and Ruminococcus lactaris) genes in commensals Adequate clinical sensitivity to reveal low level of expression, without detecting contaminating organisms Differentiation between plasmid and chromosomal carriage of genes KPC genes: plasmidic(high expression) vs chromosomal(may not be expressed) Identification of subtle single nucleotide polymorphisms (SNPs) TEM10 differs from TEM12 by a single aminoacid(but differs by 100-fold in resistance) Detection of known mechanisms only (availability of sequence data) - resistant isolates with known genes identified (new variants, if sufficient homology) - many, but not all (more than 200 unique ESBLs described) Finding a genetic resistance determinant is not sufficient - false-resistance (no or partial expression; partial gene)

44 WHAT S NEXT FOR AST? MALDI-TOF... a significant departure from traditional molecular techniques direct detection of resistance determinants by MALDI-TOF has remained elusive because many proteins involved in drug resistance, such as the beta-lactamases, are frequently not expressed at high levels compared to other bacterial proteins. a solution to this issue may involve using a MALDI-TOF mass spectrometer to detect the metabolites produced as a result of the beta-lactamase hydrolysis reaction rather than the beta-lactamase itself.

45 WHAT S NEXT FOR AST? MALDI-TOF... a significant departure from traditional molecular techniques this method has significant potential but may not replace all ASTs due to: the multiple manipulations required; the variability of antimicrobial targets (targets that do not involve direct metabolism of the antibacterial cannot be detected using this method).

46 WHAT S NEXT FOR AST? CHIPS... with everything you desire Array technology-based TOTAL PROFILING (more cost-effective than PCR) species identification resistance genes virulence genes epidemicitypredictors strain-specific markers

47 TO SUM UP AST is not an exact science. The clinical predictive value of in vitroast is currently often limited. For this reason, care should be exercised in deciding when to perform AST on bacteria recovered from patients with infection. What can be done about enhancing the clinical predictive value of in vitro AST? establishing MIC breakpoints on the bases of correlation of MICs with outcome in patients with infection (need for carefully structured clinical studies) detecting bacterial resistance determinants, better if directly in clinical material, as a surrogate for, or replacement of, in vitro tests for antibacterial activity Although molecular assays have significant potential, they cannot replace phenotypic testsbecause of inherent technical limitations to be solved. Until we have better in vitropredictors ofoutcome, itismore importantthanever thatmicrobiologistextends their scope of activities to include extensive interaction with Clinician in trying to optimize the use of the AST results.

48 The distance between the clinical microbiology laboratory and the ill patient s bed is only as long as you, Microbiologist and Clinician,choose to make it (Silas G. Farmer, 1977, personal communication)