C.I. MEDICINA DI LABORATORIO Microbiologia Clinica CL Medicina e Chirurgia AA

Transcript

1 C.I. MEDICINA DI LABORATORIO Microbiologia Clinica CL Medicina e Chirurgia AA Modulo 3: ANTIBIOGRAMMA: TECNICHE PER LA DETERMINAZIONE E INTERPRETAZIONE «CRITICA» DEL RISULTATO Giovanni Di Bonaventura, PhD Università G. d Annunzio di Chieti-Pescara Nuovo Polo Farmacia, corpo D, III livello (tel ) Centro Scienze dell Invecchiamento (Ce.S.I.), V livello (tel ) gdibonaventura@unich.it 1

2

3 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)

4 Pan-resistenza: New Delhi metallo beta-lactamase (NDM-1) Oltre all'aumento della percentuale di ceppi di K. pneumoniae ed E. coli resistenti ai carbapenemi, si assiste a un importante aumento della prevalenza dei ceppi di batteri pan-resistenti che presentano contemporanea resistenza a cefalosporine di terza generazione, fluorochinoloni e aminoglicosidi.

5 WHY PERFORM ANTIMICROBIAL SUSCEPTIBILITY TEST (AST)? Individual 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

6 WHEN SHOULD AST BE PERFORMED? Defining the etiologic role of a microorganism Synergy between Microbiologist and Clinician Susceptibility testing is indicated for microorganisms causing infections warranting antimicrobial therapy when the susceptibility cannot be reliably predicted based on the 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 but on pathogenic ones only. Establishing the need for susceptibility testing requires a close working relationship between Microbiologist and Clinician

7 IN VITRO AST METHODS Overview Phenotypic tests QUANTITATIVE methods (MIC, µg/ml) Broth dilution REFERENCE methods 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

8 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)

9 SUSCEPTIBILITY TESTING METHODS Standardization Detailed and updated information concerning susceptibility assay methods and results interpretation are provided by CLSI (Clinical Laboratory Standards Institute), already NCCLS (National Committee for Clinical Laboratory Standards), and by EUCAST (European Committee for Antimicrobial Susceptibility Testing).

10 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).

11 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).

12 Phenotypic tests broth/agar dilution tests Pros & Cons - generation of quantitative results (MICs) - broth dilution also allows MBCs and synergy studies - high reproducibility ( gold standard ) - confirm an equivocal AST result - economic (reagents, instruments) - time-consuming - possible errors in preparing antibiotic dilutions

13 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).

14 Phenotypic tests Gradient diffusion assay

15 Phenotypic tests gradient diffusion Pros & Cons - quantitative results (MIC) - synergy testing - good correlation with reference methods (aerobes, anaerobes, pneumococci, streptococci, Haemophilus spp, Neisseriaceae, mycobacteria, and fungi) - intrinsic flexibility: more than 100 antibiotics, among which vancomycin, and lipophilic compounds (amphotericin), that would otherwise be a limitation for disk diffusion - confirm an equivocal AST result - helpful in detecting resistance in organisms that are highly inoculum dependent, as they can be falsely reported as susceptible in some automated systems - expensive approach, if more than a few drugs are tested ; not used for first line testing - possible errors in reading - some systematic biases toward higher or lower MICs (vs broth dilution testing) when testing certain organism-antimicrobial combinations

16 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.

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

18 Sistemi automatizzati Esecuzione

19 Phenotypic tests Automated systems Pros & Cons - reduction in labor - provide faster reporting of susceptibility results, potentially leading to the earlier initiation of appropriate antibiotic therapy - require manual inoculum preparation: over- or underinoculating or mixing cultures can result in false or misleading results - only a limited range of organisms can be tested: slow-growing or fastidious organisms must still be tested by alternative methods (e.g. mucoid P. aeruginosa strains from CF patients) - limited testing space on the antibiotic susceptibility cards; in some cases, only the breakpoint is reported (e.g., MIC 2 μg/ml) - inflexibility of drug selections available in standard commercial panels

20 Phenotypic tests Disk diffusion (Kirby-Bauer) Images from: EUCAST 2012 Version 2.1

21 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 bacteriostatic or 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, Haemophilus influenzae, and N. meningitidis

22 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) epidemiological cut-off values (ECOFFs) (EUCAST) separate microorganisms without (wild type) and with acquired or mutational resistance (non-wild type) (WT X mg/l) 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 that clinical response is likely to be less than with a susceptible strain.

23 Categorie terapeutiche: definizioni Per un dato antibiotico, in accordo con le linee guida CLSI/EUCAST, un ceppo batterico verrà refertato quale sensibile, intermedio o resistente: Sensibile Il ceppo viene inibito nella crescita da concentrazioni di antibiotico raggiungibili in vivo (sieriche, tessutali). Un infezione sostenuta da un ceppo batterico isolato può essere trattata appropriatamente con il dosaggio usuale dell antibiotico testato e raccomandato per il tipo di infezione clinica. Indica una elevata probabilità di successo terapeutico. Intermedio (a sensibilità intermedia) Il ceppo mostra una MIC borderline rispetto ai livelli raggiungibili in vivo (sierici e tessutali) di antibiotico la cui efficacia potrebbe dunque essere minore di quella registrata per gli isolati sensibili. Tuttavia, questa categoria suggerisce l efficacia clinica nei siti corporei dove gli antibiotici sono fisiologicamente concentrati (chinolonici e -lattamici nelle urine) o quando l antibiotico può essere utilizzato a concentrazioni più alte di quelle normali in assenza di significativi effetti collaterali ( lattamici). Rappresenta una buffer zone (zona cuscinetto) che dovrebbe evitare/ridurre rilevanti errori interpretativi (falsa sensibilità) a seguito di errori di natura tecnica, soprattutto nel caso di molecole con un ristretto margine di farmacotossicità. Indica un effetto terapeutico incerto. Resistente Il ceppo non viene inibito nella crescita dalle normali concentrazioni sistemiche raggiunte in vivo (sieriche e tessutali) dall antibiotico in seguito a somministrazione di dosi normali. Questa categoria predice una elevata probabilità di fallimento terapeutico.

24 CLSI vs EUCAST more stringent interpretation Transition from CLSI to EUCAST criteria generally determined lower breakpoints and, therefore, a more stringent AST interpretation: some isolates, previously considered as S, will be I or R (Table 1). for some not clinically relevant antibiotic/microorganism combinations, AST is no more long suggested (Table 2).

25 Kirby-Bauer Results interpretation CLSI breakpoints

26 Kirby-Bauer Results interpretation CLSI breakpoints Example of regression curve between MIC values (mcg/ml) and diameters (mm) of the inhibition halos measured at KB.

27 Kirby-Bauer Results interpretation EUCAST breakpoints

28 Broth microdilution / E-test Results interpretation CLSI breakpoints

29 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 strumento formidabile di comunicazione per l orientamento nell interpretazione degli esiti, ma anche la formazione e l aggiornamento su specifiche problematiche.

30 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? minore tossicità somministrazione per via orale In realtà molte altre informazioni possono essere desunte meccanismo di resistenza probabile (tests aggiuntivi per determinare genotipi/fenotipi di resistenza: ESBL, meca, VISA, VRSA, etc.) es. farmaci equivalenti (sia R che S)

31 Refertazione dell antibiogramma Informazioni desumibili Test aggiuntivi per stafilococchi: Nitrocefin ß lattamasi se POS = resistenza a Penicillina ed altre beta-lattamine Lattice per PBP2a meca se POS = resistenza verso tutti i ß lattamici D-test MLSB inducibile se POS = resistenza a Macrolidi e Clindamicina Vancomicina Screen Agar VISA e VRSA se POS = ridotta attività dei glicopeptidi

32

33 Interpretazione critica dell antibiogramma MIC Note interpretative MBC Killing quotient Expert rules

34 Interpretazione critica dell antibiogramma: MIC Non è corretto selezionare una molecola per la terapia sulla base del valore assoluto di MIC. Molti clinici non comprendono a pieno che l interpretazione clinica del valore di MIC è strettamente correlata al suo breakpoint-s. Per interpretare il valore di MIC in maniera corretta è necessario infatti considerare che esiste un parametro, chiamato Quoziente MIC-Breakpoint (MBQ). Tale quoziente, riferito ad un particolare ceppo patogeno, si calcola dividendo il suo valore di breakpoint di sensibilità per la esatta MIC misurata sperimentalmente. In particolare questo valore è molto utile con i farmaci battericidi concentrazionedipendenti (es. fluorochinoloni). Supponendo che nella definizione dei breakpoints siano stati considerati parametri importanti quale la tossicità, il valore di MBQ sarà direttamente proporzionale alla appropriatezza ed alla efficacia del farmaco, nonché alla capacità di poter regolare le dosi terapeutiche (margine di intervento terapeutico).

35 Interpretazione critica dell antibiogramma: MBQ In particolare: medesimi valori di MIC per due farmaci diversi nei confronti di uno stesso patogeno possono essere interpretati clinicamente in modo diverso, qualora abbiano differenti valori di breakpoints. un valore di MIC più basso di un antibiotico potrebbe rappresentare un valore di resistenza, mentre un valore più alto di MIC di un altro antibiotico potrebbe indicare un valore di sensibilità, semplicemente a causa dei differenti breakpoints.

36 Interpretazione critica dell antibiogramma: MBQ Antibiotico X Antibiotico Y 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

37 Interpretazione critica dell antibiogramma: MIC Tuttavia: si sottolinea come la disponibilità della MIC non debba far trascurare gli aspetti di farmacocinetica e farmacodinamica e le considerazioni sull'impatto sull'ecologia microbica del paziente e dell'ospedale che dovrebbero sempre guidare il processo di scelta ragionata dell antibiotico-terapia più appropriata.

38 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 breakpoint 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. aeruginosa in 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).

39 Interpretazione critica dell antibiogramma: Attività battericida: MBC 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.

40 Interpretazione critica dell antibiogramma: Attività battericida: killing quotient La informazione sulla natura battericida di un antibiotico nei confronti di un isolato può essere desunta anche dal calcolo del Killing Quotient: Tasso di uccisione (KQ) = MBC / MIC 1 KQ 4 per antibiotici battericidi (β-lattamici, aminoglicosidi, chinolonici, glicopeptidi, cotrimossazolo, etc.) KQ > 4 per antibiotici batteriostatici (macrolidi, sulfamidici, trimethoprim, tetracicline, cloramfenicolo, etc.)

41 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 attuale 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

42 Interpretazione critica dell antibiogramma: EUCAST expert rules - resistenza intrinseca

43 Interpretazione critica dell antibiogramma: EUCAST expert rules - resistenza intrinseca

44 Interpretazione critica dell antibiogramma: EUCAST expert rules - resistenza intrinseca

45 Interpretazione critica dell antibiogramma: FENOTIPI ECCEZIONALI EUCAST expert rules Fenotipi di resistenza esibiti da alcune specie batteriche in maniera infrequente o per la prima volta. Debbono essere monitorati, potendo indicare un errore identificativo e/o tecnico. Se confermato localmente, il ceppo batterico in esame dovrebbe essere inviato ad un Centro di riferimento per una conferma indipendente. Anche questi fenotipi possono evolvere nel tempo. Pertanto, alcuni di essi potrebbero essere straordinari in un area, ma più frequenti in altre. Esempi: Streptococcus pyogenes resistente a penicillina Staphylococcus aureus resistente a vancomicina Enterococcus faecium sensibile ad ampicillina Enterobacteriaceae resistenti a carbapenemici (rari, ma in aumento) anaerobi resistenti a metronidazolo

46 Interpretazione critica dell antibiogramma: EUCAST expert rules fenotipi eccezionali

47 Interpretazione critica dell antibiogramma: EUCAST expert rules fenotipi eccezionali

48 Interpretazione critica dell antibiogramma: EUCAST expert rules REGOLE INTERPRETATIVE ( interpretative rules, IR) Consistono nella: Deduzione dei meccanismi di resistenza sulla base dei risultati dei tests di sensibilità Interpretazione della sensibilità clinica sulla base dei meccanismi di resistenza Le evidenze alla base delle IR sono spesso non conclusive, e potrebbero dunque prevedere differenze di opinione riguardo alla azione clinica più appropriata. Classificazione della evidenza: A. Forte evidenza clinica che la interpretazione di un risultato come sensibile CAUSA insuccesso clinico. B. Debole evidenza clinica, basata solo su pochi case reports su modelli sperimentali, che la interpretazione di un risultato come sensibile POTREBBE CAUSARE un insuccesso clinico. C. Nessuna evidenza clinica, sebbene i risultati microbiologici indichino che l utilizzo clinico della molecola DOVREBBE essere fortemente sconsigliato. Azioni correttive : Raccomandazioni Editing risultati (da S ad I/R, da I a R) sulla base dei meccanismi di resistenza dedotti Commenti (warning per particolari resistenze; spiegazione azioni correttive) Consigli (esecuzione di tests aggiuntivi)

49 Interpretazione critica dell antibiogramma: EUCAST expert rules regole interpretative

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

51 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 response to antimicrobial agents. Diagn. Microbiol. Infect. Dis. 1: Forrest, A., et al Pharmacodynamics of intravenous ciprofloxacin in 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 Antimicrobial resistance and clinical outcome of Bacteroides bacteremia: 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

52 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

53 IN VITRO SUSCEPTIBILITY TESTING METHODS Overview Phenotypic tests 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

54 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

55 Detection of resistance determinants requires technologies capable of 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): GeneXpert System (Cepheid, Sunnyvale, CA): C. difficile, MRSA, Enterovirus, vana, GBS, Flu (not simultaneously) Liquid-phase microarrays: Luminex XTAG 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: Nanosphere Inc. (Northbrook, IL): simultaneously identify S. aureus, CoNS, Streptococcus spp. (-anginosus, -pneumoniae, -pyogenes, - agalactiae), and Micrococcus spp., in addition to detecting meca, vana, and vanb directly from positive bloodcultures

56 Luminex XTAG technology

57 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 Enterobacteriaceae following 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 meca is a much better predictor of failure in patients with S. aureus infections treated with beta-lactams than is any in vitro AST. Marschall J, et al. J Clin Microbiol 2009;47:239 Tenover. Ann. N. Y. Acad. Sci. 2010;1213:70

58 Clinical significance of molecular tests rapid PCR (GeneXpert system; Cepheid, Sunnyvale, CA) differentiation between S.aureus and 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

59 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 (none or partial expression; partial gene)

60 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.

61 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).

62 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 epidemicity predictors strain-specific markers

63 TO SUM UP AST is not an exact science. The clinical predictive value of in vitro AST 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 tests because of inherent technical limitations to be solved. Until we have better in vitro predictors of outcome, it is more important than ever that Microbiologist extends their scope of activities to include extensive interaction with Clinician in trying to optimize the use of the AST results.

64 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)