Automazione ed EUCAST. Stefania Stefani Dipartimento di Scienze Biomediche e Biotecnologiche UNICT

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1 Automazione ed EUCAST Stefania Stefani Dipartimento di Scienze Biomediche e Biotecnologiche UNICT

2 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 bacteria (Neisseria, Helicobacter, Clostridium difficile, Listeria, Mycobacteria). Disk diffusion test since December 2009.

3 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 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.

4 Saggi di sensibilità : MIC Il cardine di tutti i saggi di sensibilità (ovviamente dei metodi fenotipici, ma indirettamente anche degli altri) è la MIC. MIC La MIC è alla base della determinazione dei breakpoint. I breakpoint servono per interpretare i saggi di sensibilità.

5 Resistenza microbiologica N. isolati S. aureus BREAKPOINT microbiologico Livello Basso moderato livello Alto livello Classificazione microbiologica Wild-type selvaggi Resistenza microbiologica MIC CIPRO

6 Resistenza batterica: definizione clinica N. isolati S. aureus Definiti in base a: - breakpoint microbiol. - farmacocinetica - efficacia clinica BREAKPOINT microbiol. BREAKPOINT clinici WT Classificazione clinica MIC CIPRO

7 Nessun problema? Il processo di semplificazione voluto dalle agenzie (EUCAST e CLSI) ha soddisfatto pienamente le nostre aspettative?

8 Discrepanze tra sistemi automatici e metodiche di riferimento Glicopeptidi e S.aureus Resistenze ai beta-lattamici, colistine e tigeciclina in Gramneg

9 Glycopeptides EUCAST 4.0/2014 MIC breakpoints (mg/l) CLSI 2010 MIC breakpoints (mg/l) Staphylococcus aureus S >R S I R Vancomycin Teicoplanin Telavancin (MRSA) 1 1 CoNS MIC breakpoints (mg/l) MIC breakpoints (mg/l) Vancomycin Teicoplanin

10 Da CLSI ad EUCAST. maggio 2011

11

12 MRSA EUCAST 2014

13 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 Infection control Public Health YES YES YES 1) Recommended method for MIC detection 2) Recommended methods for NS (GRSA, GISA and hgisa) detection

14 UN ALTRO ESEMPIO

15

16 Ertapenem, imipenem and meropenem distribution in Klebsiella pneumoniae EUCAST 2014

17 MICs and outcome Type of infection Antibiotic regimen Bacteremia Meropenem +amikacin IMP mg/l MEM mg/l ERT mg/l Ak mg/l CIP mg/l Mechanism of resistance Outcome VIM-1 Death SSTIs IAI Ciprofoxacin +amikacin Cefepime +amikacin VIM-1 Cure VIM-1+SHV12 Relapse, change, cure Bacteremia Ertapenem VIM-1+SHV- 12 Breaktrough bacteremia and death cuti Imipenem VIM-1+TEM-1 Relapse, change, cure cuti Meropenem VIM-1 Relapse, change, death cuti Imipenem +levofloxacin Falcone et al., J Clin Microbiol 2009;47: VIM-1+SHV- 12 Relapse, not changed, cure EU.AB Date of preparation May 2014

18 Meccanismi di resistenza ai carbapenemi Le carbapenemasi sono β-lattamasi che idrolizzano le Modificazioni di permeabilità della membrana penicilline, esterna nella (perdita maggior parte di dei porine) casi le cefalosporine, associati a e iperproduzione in maniera variabile di cefalosporinasi i carbapenemioeesbl 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)

19 Carbapenemasi nei Gram-negativi KPC in K. pneumoniae and E. coli MBLs (IMP and VIM) in P. aeruginosa MBLs (VIM and NDM) negli Enterobatteri OXA-23/24/58 in Acinetobacter OXA-48 in K. pneumoniae and E. coli

20 December 2013

21

22 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); 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.

23 Molecular Antimicrobial Resistant Detection Methods 1a Nucleic acid amplification 1b DNA hybridization-based based 1c Next generation sequencing NGS

24 Recently developed rapid AST methods. 1. Molecular testing methods: Polymerase chain reaction (qpcr, multiplex-pcr, LAMP) DNA-probe hybridization (microarrays/luminex xmap assays) Next generation sequencing (NGS, WGS) 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.

25 Frickmann H et al BioMed Research Intern

26 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 : 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).

27 Roche Molecular Systems Inc. 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): identify vana, vanb, vanb2/3 in VRE (req. DNA extraction).

28 Becton,Dickinson U.K. Ltd./ Cepheid SmartCycler 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, wound and nasal swabs. 3.BD GeneOhm MRSA: direct detection of MRSA from nasal swab.

29 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. Microfluidic cartridge Total hands-on time < 1 minute

30 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.

31 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 and resistance to rifampicin (RIF) by nucleic acid amplification technique. In december 2010, the WHO endorsed the test for use in TB endemic countries

32 4. Xpert Carba-R detects and differentiates the most prevalent carbapenemases (KPC, NDM, VIM, OXA-48 and IMP-1) in 48 minutes

33 1a. Nucleic Acid Amplification Methods. multiplex PCR

34 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/).

35 Filmarray System The panel includes 19 bacteria, five yeasts, and three antibiotic resistance genes: meca, vana/vanb, and the KPC gene. 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.

36 1b. DNA probe-based hybridisation assays DNA Microarray Technology

37 3. Next Generation Sequencing (NGS).

38 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. 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.

39 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.

40 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 evaluation in multicentre studies. Several choices for MRSA, VRE, ESBLs. For new resistance genes and mechanisms reference laboratories are mandatory.

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