2bis MED LAB MICRO

ANTIBIOGRAMMA:
TECNICHE PER LA DETERMINAZIONE E
INTERPRETAZIONE DEL RISULTATO
Giovanni Di Bonaventura, PhD
ANTIBIOTIC RESISTANCE
... a “dark scenario”
CR-AB clones
emerge
VRE
1st CTX-M
ESBL
CTX-M ESBL
“explosion” starts
Carbapenemases –
Enterobacteria;
NDM-1 discovered
VRE in animals
Lin-R
enterococci
Dap-R staphs &
enterococci
EMRSA
Genome
sequence
PCR
1985
1990
1995
2000
2005
2010
2015
(from: Woodford N, HPA)
WHY PERFORM ANTIMICROBIAL SUSCEPTIBILITY TESTS ?
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
WHEN SHOULD A SUSCEPTIBILITY TEST 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 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
but on pathogenic ones only. Establishing the need for susceptibility testing requires a
close working relationship between Microbiologist and Clinician
IN VITRO SUSCEPTIBILITY TESTING METHODS
Overview
Phenotypic tests
QUANTITATIVE methods (MIC, µg/ml)
Broth dilution
Agar dilution
Gradient methods
Automated systems
REFERENCE methods
QUALITATIVE methods (S, I, R)
Disk diffusion
Agar-incorporation breakpoint methods
Ancillary tests (to screen/confirm resistance patterns)
Genotypic (molecular) tests
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)
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).
+
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).
Phenotypic tests – broth/agar dilution tests
Pros & Cons
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).
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 diffusion
Pros & Cons
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.
Phenotypic tests – Automated Systems
Microdilution
tray
Panel
inoculation
Reading
technology
Results
available in
Data
analysis
VITEK 2
(bioMerieux)
miniaturized
automated
colorimetric
8h (4-12h)
++
MICROSCAN
WalkAway
(Siemens)
standard size
manual
photometric/
fluorescent
20h (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 systems
Pros & Cons
Phenotypic tests – Disk diffusion (Kirby-Bauer)
Images from: EUCAST 2012 Version 2.1
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
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 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)
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).
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
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 ?
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
• 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 quotient
“Expert rules”
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).
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
Antibiotico X
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 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
(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).
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 1.000 elude l’azione antibiotica) rispetto alla popolazione
iniziale.
Interpretazione critica dell’antibiogramma:
killing quotient
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.)
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 (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
favorable response to therapy?”
Murray et al, AAC 1983
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. 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.
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
“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
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 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
SUSCEPTIBILITY TESTING METHODS
Genotypic tests - Detection of antimicrobial resistance determinants
TECHNIQUES
• Single and multiplex PCR
• Real-time PCR
• DNA sequencing
• Hybridisation-based techniques
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
Simple sample
preparation
“Black box”
approach:
molecular biology
steps hidden
Simple end-product
detection
Detection of resistance determinants requires technologies capable of
high--throughput multiplexing
high
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
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
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
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)
WHAT’S NEXT FOR AST ?
MALDI--TOF ... a “significant
MALDI
“significant departure
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.
WHAT’S NEXT FOR AST ?
MALDI--TOF ... a “significant
MALDI
“significant departure
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).
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
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.
“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)