automazione ed eucast - f.i.te.la.b...dna-probe hybridization (microarrays/luminex xmap assays) next...

Automazione ed EUCAST

Stefania Stefani

Dipartimento di Scienze Biomediche

e Biotecnologiche

UNICT

Automazione - Palermo - Dicembre 2014

• formed in 1996 and reorganised in 2001

• Represents all European countries.

• Determines breakpoints for all antimicrobials with activity

against bacteria and fungi.

• Involves expert groups to determine breakpoints for fastidious • Involves expert groups to determine breakpoints for fastidious

bacteria (Neisseria, Helicobacter, Clostridium difficile, Listeria,

Mycobacteria).

• Disk diffusion test since December 2009.


EUCAST tasks

• To harmonise breakpoints for existing antimicrobials, to

determined breakpoints for new antimicrobials, to revise

breakpoints when needed.

• To define wild type MIC distributions (and ECOFFs) for all

common bacteria and fungi for all antimicrobialscommon bacteria and fungi for all antimicrobials

• To standardise AST methodology across Europe

• To recommend QA schemes (internal and external)

• To liaise with national and international organisations involved

in antimicrobial resistance

• To advise European authorities on issues related antimicrobial

resistance.


� Il cardine di tutti i Il cardine di tutti i saggi di saggi di

sensibilitàsensibilità (ovviamente dei metodi (ovviamente dei metodi

fenotipici, ma indirettamente anche fenotipici, ma indirettamente anche

degli altri) è la degli altri) è la MICMIC.

Saggi di sensibilità : MIC Saggi di sensibilità : MIC


� I I breakpointbreakpoint servono servono

per interpretare i per interpretare i saggi di saggi di

sensibilitàsensibilità..

� La La MICMIC è alla base è alla base

della determinazione della determinazione

dei dei breakpointbreakpoint.

MICMICMICMIC

N. isolatiN. isolati

S. aureusS. aureus

Basso Alto Livello

Resistenza microbiologicaResistenza microbiologica

BREAKPOINT

microbiologico


MICMIC

CIPROCIPROClassificazione

microbiologica

Basso

livello

Resistenza Resistenza

microbiologicamicrobiologica

Alto

livello

WildWild--typetype

selvaggiselvaggi

moderato

Resistenza batterica: definizione Resistenza batterica: definizione

clinicaclinica

N. isolati

S. aureus

BREAKPOINT

clinici

Definiti in base a:

- breakpoint microbiol.

- farmacocinetica

- efficacia clinica

BREAKPOINT

microbiol.


MIC

CIPRO

WT

ClassificazioneClassificazione

clinicaclinica

Nessun problema?

Il processo di semplificazione voluto

dalle agenzie (EUCAST e CLSI) ha

soddisfatto pienamente le nostre

aspettative?


Discrepanze tra sistemi

automatici e metodiche di

riferimentoriferimento

Glicopeptidi e S.aureus

Resistenze ai beta-lattamici, colistine

e tigeciclina in Gramneg


Glycopeptides

EUCAST

4.0/2014

CLSI

2010

MIC breakpoints

(mg/L)

MIC breakpoints

(mg/L)

Staphylococcus

aureus

≤S >R S I R

Vancomycin 2 2 ≤2 4-8 ≥16

≤ ≥Teicoplanin 2 2 ≤8 16 ≥32

Telavancin (MRSA) 1 1

CoNS MIC breakpoints

(mg/L)

MIC breakpoints

(mg/L)

Vancomycin 2 2 ≤4 8-16 ≥32

Teicoplanin 4 4 ≤8 16 ≥32

Da CLSI ad EUCAST . maggio 2011

MRSA – EUCAST 2014


EUCAST guideline for the detection of resistance mechanisms

and specific resistances of clinical and/or epidemiological

importance

Glycopeptide non susceptible S.aureus

Importance of detection of resistance

Required for antimicrobial susceptibility

categorization

YES

categorization

Infection control YES

Public Health YES


1) Recommended method for MIC detection

2) Recommended methods for NS (GRSA, GISA

and hGISA) detection

UN ALTRO ESEMPIO


Ertapenem, imipenem and

meropenem distribution in Klebsiella

pneumoniae – EUCAST 2014


MICs and outcome

Type of

infection

Antibiotic

regimen

IMP

mg/L

MEM

mg/L

ERT

mg/L

Ak

mg/L

CIP

mg/L

Mechanism

of resistance

Outcome

Bacteremia Meropenem

+amikacin

1 0.12 1 1 0.03 VIM-1 Death

SSTIs Ciprofoxacin

+amikacin

0.25 0.06 0.25 32 0.03 VIM-1 Cure

EU

.AB

.20

14

.05

1 D

ate

of

pre

pa

rati

on

Ma

y 2

01

4

IAI Cefepime

+amikacin

2 2 8 0.5 32 VIM-1+SHV12 Relapse, change, cure

Bacteremia Ertapenem 1 2 4 0.5 32 VIM-1+SHV-

12

Breaktrough bacteremia

and death

cUTI Imipenem 0.06 0.06 0.25 1 0.03 VIM-1+TEM-1 Relapse, change, cure

cUTI Meropenem 0.06 0.5 0.5 4 0.03 VIM-1 Relapse, change, death

cUTI Imipenem

+levofloxacin

2 2 2 1 32 VIM-1+SHV-

12

Relapse, not changed,

cure

Falcone et al., J Clin Microbiol 2009;47:3514

• Modificazioni di permeabilità della membranaesterna (perdita di porine) associati aiperproduzione di cefalosporinasi o ESBL

• Up-regulation dei sistemi di efflusso associati a

Meccanismi di resistenza ai carbapenemi

Le carbapenemasi sono β-lattamasi che idrolizzano lepenicilline, nella maggior parte dei casi le cefalosporine,e in maniera variabile i carbapenemi e i monobattami.Questi ultimi non sono idrolizzati dalle metallo-β-lattamasi• Up-regulation dei sistemi di efflusso associati a

• iperproduzione di cefalosporinasi o ESBL

• Produzione di carbapenemasi:

– β-lattamasi inibite dall’ac. clavulanico (classe A)

– Metallo-β-lattamasi (classe B)

– Oxacillinasi a spettro esteso (classe D)

lattamasi

KPC in K. pneumoniaeand E. coli

MBLs (IMP and VIM) in P. aeruginosa

Carbapenemasi nei Gram-negativi

MBLs (VIM and NDM) negli Enterobatteri

OXA-23/24/58 in Acinetobacter

OXA-48 in K. pneumoniaeand E. coli

December 2013


Rapid antimicrobial susceptibility

testing methods

five different ways to accelerate susceptibility testing in clinical diagnostics:

1. bypassing conventional culture by direct detection of the pathogen or

resistance mechanism in the primary sample;

2. bypassing plate or broth culture dependent susceptibility testing

(secondary culture); (secondary culture);

3. avoiding time consuming work steps/methods;

4. increasing the sensitivity to the detection of the infectious agent; that

means detecting the infectious agent in earlier disease stages at lower

viral or microbial loads;

5. earlier detection of an evolving drug resistance during treatment in

spreading less susceptible quasispecies.Automazione - Palermo - Dicembre 2014

Molecular Antimicrobial Resistant

Detection Methods

1a Nucleic acid amplificationNucleic acid amplification

1b DNA hybridizationDNA hybridization--basedbased1b DNA hybridizationDNA hybridization--basedbased

1c Next generation sequencing Next generation sequencing

NGSNGS


1. Molecular testing methods:

Polymerase chain reaction (qPCR, multiplex-PCR, LAMP)

DNA-probe hybridization (microarrays/Luminex xMAP

assays)

Next generation sequencing (NGS, WGS)

Recently developed rapid AST methods.

2. Fluorescence in situ hybridization (FISH):

peptide nucleic acid (PNA) Probes

3. Mass spectrometry based methods:

mass spectrometric beta-lactamase assays,

PCR/electrospray ionization mass spectrometry (PCR/ESI

MS), minisequencing.


Frickmann H et al. 2014 BioMed Research InternAutomazione - Palermo - Dicembre 2014

1a. Nucleic Acid Amplification Methods.

quantitative real-time PCR (qPCR)

• rapidly and simultaneously identification of multiple pathogens in a clinical

specimen and accurately detection of resistance genes within a remarkably

shorter time (4–6 hours).

• most of the available commercial qPCR assays detect the presence :• most of the available commercial qPCR assays detect the presence :

mecA/mecC genes, which confer methicillin resistance in S. aureus;

vanA/vanB genes, which confer glycopeptide resistance;

ESβL genes that encode extended-spectrum β-lactamases

RIF/INH genes which confer rifampicin/isoniazide in M. tuberculosis

• affordable, sensitive, specific, user friendly, not space demanding, and

deliverable qPCR has found various applications in point-of-care testing (POCT).


1.LightCycler® MRSA Advanced Test: identify MRSA direct from nasal swabs.

2.LightCycler® SeptiFast MecA Test: identify MRSA direct from blood samples.

3.LightCycler® VRE Detection Kit (RUO):

Roche Molecular Systems Inc.

3.LightCycler® VRE Detection Kit (RUO): identify vanA, vanB, vanB2/3 in VRE (req. DNA extraction).


1.BD GeneOhm™ VanR: ID of VRE direct from perianal and/or rectal swabs.

2.BD GeneOhm™ StaphSR: detection and differentiation of MRSA/SA from blood culture,

Becton,Dickinson U.K. Ltd./Cepheid SmartCycler®

detection and differentiation of MRSA/SA from blood culture, wound and nasal swabs.

3.BD GeneOhm™ MRSA: direct detection of MRSA from nasal swab.


GeneXpert System

•Fully integrated and automated sample preparation,

RT-PCR and detection.

• Specimens don’t need to be batched.

• <2 mins hands-on time.

• Results in <1hr

•6 targets per sample.•6 targets per sample.

•Microfluidic cartridge

Total hands-on time < 1 minute


1. Xpert® MRSA

A comprehensive approach to MRSA infection control, capable of detecting

strains with all SCCmec types found in both healthcare-acquired and

community-acquired MRSA.


2. Xpert® vanA/vanB

allows an immediate identification of VRE carriers from non-carriers taken from perianal and rectal swab

3. Xpert® MTB/RIF

identify Mycobacterium tuberculosis (MTB) DNA andresistance to rifampicin (RIF) by nucleic acid amplificationtechnique.

In december 2010, the WHO endorsed the test for usein TB endemic countries


detects and differentiates the most prevalent

carbapenemases (KPC, NDM, VIM, OXA-48 and

IMP-1) in 48 minutes

4. Xpert® Carba-R


1a. Nucleic Acid Amplification Methods.

multiplex PCRmultiplex PCR


Unyvero Pneumonia Application (UPA) assay

multiplex end-point PCR with amplicon detection

by array hybridization developed to rapidly and

simultaneously detect from respiratory specimens:

18 bacterial species, Pneumocystis jirovecii and

22 resistance markers

(http://www.curetis.com/).


Filmarray System

a closed diagnostic system that combines nucleic acid extraction from clinical

specimens, high-order nested multiplex PCR analysis and post-PCR DNA melting

curve analysis to identify several pathogens and susceptibility markers directly

from positive blood culture bottles in 1 h (CE IVD) marking in June 2013.

The panel includes 19 bacteria, five yeasts, and three antibiotic resistance

genes: mecA, vanA/vanB, and the KPC gene .

from positive blood culture bottles in 1 h (CE IVD) marking in June 2013.


DNA Microarray Technology

1b. DNA probe-based hybridisation assays


3. Next Generation Sequencing (NGS).


• To date costs and scarcely available user-friendly bioinformatics

platforms make difficult the use of NGS technologies in diagnostic

microbiology.

• NGS technologies provide high-resolution genotyping in a short

time frame of only two to five days .time frame of only two to five days .

• Therefore, NGS/WGS in the microbiological laboratory will be the

logical next step for the routine diagnosis of infection and the

prediction of antimicrobial susceptibility, potentially replacing

traditional cultural approaches on the intermediate or long term.


Phenotypic vs. genotypic: advantages

•Can be performed direct from clinical specimens:- Rapid.- Good for difficult to culture organisms or slow-growers.- May reduce biohazard risk.

•Potential for automation.

•Simple yes/no answer - not dependent on S/I/R categories.

•Sort out ambiguous phenotypic results.

•Good for resistance mechanisms that encode low-level resistance.

•Inform epidemiological studies.Automazione - Palermo - Dicembre 2014

In conclusion

• Molecular assays for detection of AMR have yielded a

wealth of information.

• Unlikely to replace, but instead augment, phenotypic

susceptibility testing.

• Commercial kits seem to be promising but thorough • Commercial kits seem to be promising but thorough

evaluation in multicentre studies.

• Several choices for MRSA, VRE, ESBLs.

• For new resistance genes and mechanisms reference

laboratories are mandatory.


automazione ed eucast - f.i.te.la.b...dna-probe hybridization (microarrays/luminex xmap assays) next...

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