testing for the daily routine?

Transcription

1 What is the role of in vitro antifungal susceptibility testing for the daily routine? ESCMID, Rome 2010 Cornelia Lass-Flörl Medical University Innsbruck

2 Faculty disclosure Invited speaker: Pfizer, Gilead, MSD, Schering-Plough g Consultant: Pfizer, Gilead, Schering-Plough Research Grants: Pfizer, Gilead, Schering- Plough

3 Outline Current status of susceptibility testing for yeast and moulds Pro and cons Antifungal breakpoints and susceptibility Epidemiological cut-offs Clinical breakpoints Relationship with clinical outcome?

4 A brief history of antifungal susceptibility testing ti standardization di ti 1982 Established subcommittee 20% hospitals performing testing for yeast; intra/inter-laboratory t t agreement poor Conidia forming filamentous fungi 1986 Develop reproducible method Disk-diffusion Method-yeast Fothergill et al. Infect DisClin N Amer 2006;20: M27-P method introduced Synthetic medium (RPMI) Broth-based method x10 3 Disk-diffusion method-moulds moulds 1997 M27-A method introduced Breakpoints EUCAST Higher glucose; 24 hour endpoint; spectrophotometer

5 Susceptibility Testing To provide information for therapy To T provide early warning To get epidemiological data To get information for species ID

6 Susceptibility Testing We lack clinically derived breakpoint for most fungi Technical factors influence MICs Which in vitro method reflects best outcome Host factors are more important for survival

7 Susceptibility testing Standards: CLSI, EUCAST E-test, Agar-diffusion Ready to use test assays s Others (FACS)

8 Characteristics CLSI M27-A3 EUCAST Def Suitability Yeasts Fermentative ti Yeast Inoculum x105x x105x10 5 CFU/ml CFU/ml Test medium RPMI ,2%G RPMI 1640 G2% Format Microdilution Microdiluation Temperature 35 C 35 C Duration of incubation 46-50h Endpoint 24 h for yeasts 80% inhibition M27-A2 50% inhibition M27-A3 (azole) 24h 80% amphotericin B 50% inhibition azole Reading Visually Plate reader

9 Control Increasing Drug concentrations Media Control AMB 5FC FLU ITC VOR CAS

10 Common Problems with Microdilution Methodology Visual determination of azole MIC endpoint (trailing growth) prominent reduction MEC: Aspergillus and Candins Dramatic increase in azole MIC from 24 to 48 hours Poor growth of some isolates in tray Overcalling resistance with QC and test isolates Clustering of AMB MICs Candins: Drug solubility/stability Batch to batch variations Lass-Flörl et al., Mycoses 2009

11 Agar-based MIC determination Often more friendly to the workflow of the clinical laboratory Better visualization of organisms response? Agar-based methods Agar dilution Semi-solid agar antifungal susceptibility testing (SAAS) Chromagar Disk diffusion (M44-P) Etest (AB Biodisk/BioMereaux)

12 MICs defined via E-test Growth of fungus MIC= zone of inhibition

13 Development of Fluconazole Resistance in C. albicans 1 3 4

14 Caspofungin A. flavus C. tropicalis A. fumigatus

15 Fluconazole 25 µg/ml Fluconazole 50 µg/ml Caspofungin 2 µg-a. fumigatus Kirkpatrick k et al. J Clin Micro1998:36: Arikan et al. Antimicrob Agent Chemother 2002;46:3084

16 The disk diffusion test using the MH agar plate can be performed quickly, simply, and cost-effectively, and is practicable method for the initial testing of the susceptibility of Candida spp. to voriconazole and fluconazole. l (Lee SC. et al, JMII 2009) In 2002, the CLSI issued formal fluconazole disk interpretive criteria of 19 mm = S, mm = SDD, and 14 mm = R In 2005, the tentative Voriconazole breakpoints are 17 mm = S, mm = SDD, and 13 mm = R

17 MICs Standardized methods reproducible MICs detect resistant strains resistant phenotyp and genotyp correlate clinical l breakpoints? helps to identify species / genus MIC reading is problematic: Candins and Aspergillus Amphotericin B The in vitro - in vivo correlation for antifungal drugs is poor S does not predict successful treatment R does not necessarily predict clinical failure Lass-Flörl, Expert Rev 2010

18 How are Susceptibility Breakpoints Established? #1 Population MICs #2 PK:PD Data #3 Clinical outcome Cumu ulative % Inh hibited Conc. Increasing MIC Peak: MIC AUC: MIC CTime >MIC Time

19 Interpretative Guidelines Breakpoints for Yeasts R Drug EUCAST CLSI Fluconazole > 2 µg/ml > 64 µg/ml Amphotericin B n.a. n.a. Itraconazole n.a. > 1 µg/ml Voriconazole E cut-off > 4 µg/ml Posaconazole n.a. n.a. Caspofungin n.a. > 2* µg/ml Anidulafungin i n.a. > 2* µg/ml Micafungin n.a. > 2* µg/ml * Non susceptible

20 Facing In Vitro/In Vivo Correlation with Fungi Candida albicans Candins Intrinsic Candida glabrata Azoles Intrinsic and acquired Candida krusei Azoles Intrinsic Candida lusitaniae Amphotericin B Intrinsic and acquired Aspergillus fumigatus Azoles Intrinsic and Aspergillus fumigatus Candins acquired Intrinsic and acquired Aspergillus terreus Amphotericin B Intrinsic

21 Caspofungin susceptibility of isolates from patients with invasive candidiasis Lack of correlation of MIC with outcome Isolate es # of favorable outcome unfavorable outcome Breakpoint? 0,008 0,016 0,03 0,06 0,125 0,25 0, MICs Kartsonis et al. Antimicrob Agent Chemother 2005;49:3616.

22 Proposed EUCAST breakpoints for A. fumigatus and azoles Azole MICs (µg/ml) Susceptible Intermediate* Resistant Itraconazole < 2 2 > 2 Voricionazole < 2 2 > 2 Posaconazole < > 0.5 Verweij et al., Drug Resist Rev 2009;12:

23 Emerg Infect Dis Jul;15(7):

24 ..no clinically breakpoints for candins and amphotericin B Echinocandin resistance.(arendrup et al., 2009)

25 In vitro susceptibility testing: local epidemiology ogy and patients immune status Yeasts Sterile body site plus non-c. albicans Azole (?) Non-responder Molds A. fumigatus (azole) Non A. fumigatus all: Non responder Long treatment tm & azole Rare species

26 Resistance? PK/PD & Drug dosing? Pathogen virulence? Where do MICs fit? Diagnostic certainty? Drug interactions? Prior therapies? Concomitant Infections? Infection Site involvement? Hepatic & renal function? GI function? Infected hardware or catheters? Immunosuppression & reconstitution? Dose, timing, intensity of Immunosuppressive therapy?

27 Thank you very much for your attention!