ANTIBIOGRAMMA:

TECNICHE PER LA DETERMINAZIONE E

INTERPRETAZIONE DEL RISULTATO

Giovanni Di Bonaventura, PhD

CR-AB clones emerge

VRECarbapenemases –Enterobacteria;

NDM-1 discovered

CTX-M ESBL “explosion” starts

VRE in animals

1st CTX-M ESBL

ANTIBIOTIC RESISTANCE ... a “dark scenario”... a “dark scenario”

1985 1990 1995 20052000 20152010

Lin-R enterococci

VRE in animals

Dap-R staphs & enterococci

EMRSA

PCR

Genome

sequence

(from: Woodford N, HPA)

WHY PERFORM ANTIMICROBIAL SUSCEPTIBILITY TESTS ?IndividualIndividual and and epidemiologicalepidemiological purposespurposes

� 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 � 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

WHEN SHOULD A SUSCEPTIBILITY TEST BE PERFORMED ?DefiningDefining the the etiologicetiologic rolerole ofof a a microorganismmicroorganism

SynergySynergy betweenbetween MicrobiologistMicrobiologist and and ClinicianClinician

� 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, � 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

IN VITRO SUSCEPTIBILITY TESTING METHODSOverviewOverview

� Phenotypic tests

� QUANTITATIVE methods (MIC, µg/ml)� Broth dilution

� Agar dilution

� Gradient methods

REFERENCE methods

� Automated systems

� QUALITATIVE methods (S, I, R)� Disk diffusion

� Agar-incorporation breakpoint methods

� Ancillary tests (to screen/confirm resistance patterns)

� Genotypic (molecular) tests

SUSCEPTIBILITY TESTING METHODSPhenotypicPhenotypic teststests

� 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� 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)

Phenotypic tests – broth microdilution

Antibiotic is incorporated into broth in doubling concentrations.

64 32 16 8 4 2 1 0.5 0.25 0.12 - +

The lowest concentration of antibiotic that prevented visible growth represents the

minimal inhibitory concentration (MIC).

Antibiotic is incorporated into agar in doubling concentrations.

Phenotypic tests – agar dilution

The lowest concentration of antibiotic that prevented visible growth onto agar

represents the minimal inhibitory concentration (MIC).

Phenotypic tests – broth/agar dilution testsProsPros & & ConsCons

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).

Etest (bioMerieux)

Phenotypic tests – Gradient diffusion-based commercial tests

Etest (bioMerieux)

M.I.C. Evaluator (M.I.C.E.; Oxoid)

MIC test strip (Liofilchem)

Phenotypic tests – gradient diffusionProsPros & & ConsCons

• 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.

Phenotypic tests – Automated Systems

was introduced by Pfizer Diagnostics.

• Now, more than 80% of clinical laboratories report using an automated instrument

for primary susceptibility testing.

Microdilution

tray

Panel

inoculation

Reading

technology

Results

available in

Data

analysis

VITEK 2

(bioMerieux)miniaturized automated colorimetric 8h (4-12h) ++

MICROSCAN

WalkAway standard size manualphotometric/

20h (17-28h) ++

Phenotypic tests – Automated Systems

WalkAway

(Siemens)

standard size manualphotometric/

fluorescent20h (17-28h) ++

PHOENIX

(BD Diagnostics)miniaturized manual colorimetric 10h (7-16h) +++

SENSITITRE

(Trek Diagn. Sys.)standard size manual fluorescent 15h ++

modified from: Kuper et al., Pharmacotherapy 2009

Phenotypic tests – Automated systemsProsPros & & ConsCons

Phenotypic tests – Disk diffusion (Kirby-Bauer)

Images from: EUCAST 2012 Version 2.1

Phenotypic tests – Disk diffusionProsPros & & ConsCons

- 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

MICs and zone sizes are meaningless… unless you apply interpretative criteria… 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

� 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.

� epidemiological cut-off values (ECOFFs) (EUCAST) separate microorganisms without (wild type)

and with acquired or mutational resistance (non-wild type) (WT ≤ X mg/L)

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)

CLSI vs EUCAST

� alcuni isolati che prima venivano refertati

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).

Kirby-Bauer

Interpretazione dei risultati – CLSI breakpoints

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.

Kirby-Bauer

Interpretazione dei risultati – EUCAST breakpoints

Microdiluizione in brodo / E-test

Interpretazione dei risultati – CLSI breakpoints

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.

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 ?terapia empirica ?� Meno tossici

� 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)

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– 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 (?)

Interpretazione “critica” dell’antibiogramma:

MIC

Note interprertative

MBC

Killing quotientKilling quotient

“Expert rules”

� 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

Interpretazione critica dell’antibiogramma:

MIC

� 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).

� 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

Interpretazione critica dell’antibiogramma:

MIC

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

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 X

Antibiotico Y

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 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. 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).

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

Interpretazione critica dell’antibiogramma:Attività battericida

antibiotico in grado di eradicare la crescita batterica di almeno il 99.9% (1

germe su 1.000 elude l’azione antibiotica) rispetto alla popolazione

iniziale.

� Tasso di uccisione (KQ) = MBC / MIC

1 ≤ KQ ≤ 4 per antibiotici battericidi

Interpretazione critica dell’antibiogramma:

killing quotient

1 ≤ KQ ≤ 4 per antibiotici battericidi

(beta-lattamici, aminoglicosidi, chinolonici,

glicopeptidi, cotrimossazolo, etc.)

KQ > 4 per antibiotici batteriostatici

(macrolidi, sulfamidici, trimethoprim, tetracicline,

cloramfenicolo, etc.)

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 • ERs possono essere di ausilio al Microbiologo ed al Clinico nella

interpretazione dei tests di antibiotico-sensibilità.

• ERs sono dettate da EUCAST (http://www.eucast.org): pubblicate per la

prima volta nel 2008, vengono costantemente aggiornate. Attualmente,

sono divise in:

– resistenza intrinseca

– fenotipi eccezionali

– regole interpretative

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 “Does resistance always predict failure; does susceptible always denote

favorable response to therapy?” Murray et al, AAC 1983

How well do the results of antimicrobial susceptibility tests

predict therapeutic outcome ?

� Gerber A. U., and W. A. Craig. 1981. Worldwide clinical experience with cefoperazone. Drugs 22:108–118.

� Weinstein et al. 1983. The clinical significance of positive blood cultures: a comprehensive analysis of 500 episodes of bacteremia

and fungemia in adults. Rev. Infect. Dis. 5:54–70.

� Washington, J. A. 1983. Discrepancies between in vitro activity and in vivo response to antimicrobial agents. Diagn. Microbiol.

Infect. Dis. 1:25–31.

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:

“the 90-60 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

Infect. Dis. 1:25–31.

� Forrest, A., et al. 1993. Pharmacodynamics of intravenous ciprofloxacin in seriously ill patients. Antimicrob. Agents Chemother.

37:1073–1081.

� Doern, G. V. 1995. Interpretive criteria for in vitro antimicrobial susceptibility tests. Rev. Med. Microbiol. 6:126–136.

� Nguyen, M. H., V. L. Yu, and A. J. Morris. 2000. Antimicrobial resistance and clinical outcome of Bacteroides bacteremia: findings

of a multicenter prospective observational trial. Clin. Infect. Dis. 30:870–876.

�Evans, M. R., et al. 2009. Short-term and medium-term outcomes of quinolone-resistant Campylobacter infection. Clin. Infect. Dis.

48:1500–1506

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

IN VITRO SUSCEPTIBILITY TESTING METHODSOverviewOverview

� 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

SUSCEPTIBILITY TESTING METHODS GenotypicGenotypic teststests -- Detection Detection ofof antimicrobialantimicrobial resistanceresistance determinantsdeterminants

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

Detection Detection ofof resistanceresistance determinantsdeterminants requiresrequires technologiestechnologies capablecapable ofof

highhigh--throughputthroughput multiplexingmultiplexing

Liquid-phase microarrays:

� Luminex XTAG technology (Luminex, Austin, TX): microspheres

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)

� 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

Using molecular assays to: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 beta-

lactams, 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

Clinical significance of molecular testsClinical 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

MOLECULAR DETECTION OF RESISTANCE DETERMINANTSInherent Inherent technical challengestechnical 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)

WHAT’S NEXT FOR AST ? MALDIMALDI--TOFTOF ... ... a “a “significantsignificant departuredeparture”” fromfrom traditionaltraditional molecularmolecular techniquestechniques

� 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.

� 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

WHAT’S NEXT FOR AST ? MALDIMALDI--TOFTOF ... ... a “a “significantsignificant departuredeparture”” fromfrom traditionaltraditional molecularmolecular techniquestechniques

that do not involve direct

metabolism of the

antibacterial cannot be

detected using this method).

Array technology-based TOTAL PROFILING

(more cost-effective than PCR)

WHAT’S NEXT FOR AST ? CHIPS ... with everything you desireCHIPS ... with everything you desire

(more cost-effective than PCR)

� species identification

� resistance genes

� virulence genes

� epidemicity predictors

� strain-specific markers

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)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.

“The distance between the clinical microbiology laboratory and the ill

patient’s bed is only as long as you, Microbiologist and Clinician, choose patient’s bed is only as long as you, Microbiologist and Clinician, choose

to make it”

(Silas G. Farmer, 1977, personal communication)