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1 Eric DANNAOUI ESCMID Postgraduate Education Course June 2013, Sibiu Antifungal susceptibility testing and detection of resistance: principles and practices Unité de Parasitologie-Mycologie, Laboratoire de microbiologie, HEGP Université Paris 5
2 What is a resistance How to detect resistance When to detect resistance The way to define resistance: ECOFFs and CBs Revision of CBs Reference methods: EUCAST, CLSI Commercialized methods (e.g. Etest) Other methods Recommendations in the clinical laboratory
3 «Clinical resistance» depends on many factors Patient Immune status Site of infection Foreign materials Non compliance Clinical failure Antifungal Fungistatic nature Dosage Pharmacokinetics Drug-drug interactions Fungus Microbiological resistance Biofilm production Inoculum size «Clinical resistance» is different from microbiological resistance
4 From MICs to Clinical Breakpoints : three steps MIC ECOFF MIC determination: technical issues (EUCAST, CLSI) CB Determination of clinical breakpoints (CBs) Determination of WT population for each species (ECOFFs/ECVs) S I R
5 Determination and analysis of MIC distributions
6 EUCAST definitions of wild-type MIC distribution and epidemiological cut-off Values (ECOFFs) Wild Type (WT) A micro-organism is defined as wild type (WT) for a species by the absence of acquired and mutational resistance mechanisms to the drug in question A micro-organism is categorized as wild type (WT) for a species by applying the appropriate cutoff value in a defined phenotypic test system Wild type micro-organisms may or may not respond clinically to antimicrobial treatment The wild type is presented as WT<z mg/l and non-wild type as NWT >z mg/l
7 EUCAST wild-type (WT) MIC distribution and epidemiological cut-off Values (ECOFFs) 1. Set-up of MIC distribution Specific for a given species Must integrate all MIC values of the distribution (lowest and highest values not truncated) 2. Methods for ECOFFs determination Simple estimation by visual inspection Statistical analysis (important when overlap between WT and non-wt populations) Turnidge et al. Clin Microbiol Infect 2006; 12: Turnidge et al. CMR 2007; 20:
8 EUCAST wild-type MIC distribution and epidemiological cut-off Values (ECOFFs) % of isolates C. albicans/ fluconazole MIC (mg/l) Epidemiological cut-off: WT 1 mg/l 3756 observations (12 data sources) ECOFF
9 EUCAST wild-type MIC distribution and epidemiological cut-off Values (ECOFFs) % of isolates C. glabrata/ fluconazole Epidemiological cut-off: WT 32 mg/l 898 observations (12 data sources) MIC (mg/l) 4 8 ECOFF
10 ECOFFs main characteristics Can be determined for all species No change over time Useful for epidemiological surveillance Sensitive for detection of resistance Non WT may or may not respond clinically Useful when CBs not determined
11 CLSI wild-type MIC distribution and epidemiological cutoff Values (ECVs) ITZ VRZ ECV ECV ECV PSZ A. fumigatus / azoles Adapted from Espinel-Ingroff et al., JCM 2010, 48, 3251
12 Interpretation of MICs Definition of Clinical Breakpoints (CBs)
13 EUCAST definitions of clinical breakpoints Clinical resistance and clinical breakpoints Clinically Resistant (R) A micro-organism is defined as resistant by a level of antimicrobial activity associated with a high likelihood of therapeutic failure A micro-organism is categorized as resistant (R) by applying the appropriate breakpoint in a defined phenotypic test system This breakpoint may be altered with legitimate changes in circumstances Clinical breakpoints are presented as S<x mg/l; I>x, <y mg/l; R>y mg/l
14 % of isolates EUCAST wild-type MIC distribution and epidemiological cut-off Values (ECOFFs) Determination of CBs C. albicans/ fluconazole WT Epidemiological cut-off: WT 1 mg/l 3756 observations (12 data sources) 0.5 ECOFF 1 Clinical failure? MIC (mg/l) Clinical failure? 16 Clinical failure? Genotypically detectable resistance Phenotypically detectable resistance 32 Clinical failure? Adapted from G. Kahlmeter
15 Strategy for CBs determination: Complex problem Should integrate many parameters Historical data ATF pharmacokinetics MIC distributions Molecular mechanisms of resistance Animal models Clinical data
16 EUCAST Procedure for Harmonising and Defining Breakpoints Relevant factors in setting breakpoints for new antimicrobials and assessment of data: Dosing, formulations (oral, iv), indications and practices, target organisms MIC distributions and ECOFFs determined Dose-effect relationships in vitro studies, animal studies and humans (PK/PD data) evaluated National CBs compared and discussed Clinical data relating outcome to MIC-values Modelling processes, Monte Carlo simulation Consensus breakpoints are sought CBs tested against target species MIC distributions. CBs should not divide the WT distributions No CBs if species a poor target for the drug ( - ) or insufficient evidence that the species is a good target for the drug (IE)
17 Clinical Breakpoints (CBs) main characteristics Useful to predict therapeutic efficacy Not determined for all species No obvious relationships between CBs and ECOFFs May change (revised by committees) over time
18 CLSI wild-type MIC distribution and epidemiological cutoff Values (ECVs) Determination of CBs % of isolates C. albicans/ fluconazole ECV 1 2 MIC (mg/l) Epidemiological cut-off: WT 1 mg/l 7851 observations (12 data sources) S I R S I R New CLSI CBs Old CLSI CBs
19 Candida glabrata Antifungals: MIC - CBs Revision of CLSI Clinical Breakpoints Antifungal Susceptible SDD/Interm Resistant Echinocandins 2 ND ND WT Mutant Garcia-Effron et al. AAC 2009;53 :3690
20 Antifungals: MIC - CBs Definition of CBs for Candida spp. (µg/ml) Revision of CLSI Clinical Breakpoints 1-6 Antifungal Susceptible SDD/Interm Resistant Echinocandins 2 ND ND Species Caspo Mica Anidula C. albicans C. tropicalis C. krusei S I R For C. glabrata: S 0.12, I 0,25, R ³ 0.5 for caspo and anidula and S 0.06, I 0,125, R ³ 0.25 for micafungin 1. CLSI M27-A3, Pfaller et al. JCM 2006;44 : Pfaller et al. JCM 2008;46 : Pfaller et al. ICAAC 2010, M Pfaller et al. Drug resist Updat 2011,14, CLSI M27-S4, 2013
21 ECOFFs and CBs take-home messages ECOFFs and CBs: two different parameters ECOFFs: microbiological resistance CBs: clinical failure No relationships between these 2 parameters ECOFFs stable, CBs may be revised
22 How to detect resistance in practice
23 Parameter CLSI (M27-A3) Format Microdilution Microdilution EUCAST (E.Def 7.2) Culture medium RPMI + MOPS, ph 7.0 RPMI + MOPS + 2% glucose, ph 7.0 Final inoculum 0.5 à 2.5 x10 3 CFU /ml 0.5 to 2.5x10 5 CFU /ml Incubation 48h (24h echino, +/- AMB, FCZ) 24h Reading Visual Spectrophotometric MIC endpoint Candida spp. Technique standardization Azoles, 5FC, echino 50% inhibition 50% inhibition AMB 100% inhibition 90% inhibition To note: trends towards harmonization
24 Antifungal Microdilution broth techniques: CLSI, EUCAST 5FC FCZ ITZ VRZ PSZ TER AMB CAS Concentration (mg/l) MIC MIC MIC MIC MIC MIC MIC Les techniques in vitro 5FC : > 128 µg/ml FCZ : 4 µg/ml ITZ : 0.12 µg/ml VRZ : 0.06 µg/ml PSZ : 0.06 µg/ml TER : 0.25 µg/ml AMB : 0.5 µg/ml CAS : 0.12 µg/ml
25 Etest method Commercialized Standardized Easy to perform Adapted to routine testing in clinical lab Sometime better than ref technique (AMB et Candida) Alternative methods But Expensive May be difficult to read Sometime less effective than ref technique (5FC and Crypto)
26 Etest method Exponential gradient of antifungal Inoculum preparation Alternative methods Inoculation Incubation reading
27 Acquired resistance to echinocandins in Candida spp. Isolate Caspofungin MIC (µg/ml) EUCAST Etest S R Baixench, M. T., et al J Antimicrob Chemother 59: Dannaoui, E., et al Emerg Infect Dis 18:86-90.
28 Screening of azole-resistance in Aspergillus spp. Azole-containing agar Drug-free ITZ VRZ S R Isolate EUCAST MIC (µg/ml) ITZ VRZ >8 4 Choukri et al. Unpublished
29 When to perform AFST for resistance detection in practice?
30 Candida spp. infections On a routine basis When to detect resistance Identification to species level for deep site Routine testing of FCZ and echino against C. glabrata (deep site) Routine testing of other species possibly helpful but susceptibility usually predictable by species Use CBPs or ECVs to interpret results as appropriate Consider cross-resistance between fluconazole and all other azoles to be complete for C. glabrata Adapted from Pfaller et al. J. Clin. Microbiol. 2012, 50(9):2846
31 Candida spp. infections Mucosal candidiasis Determination of azole susceptibility not routinely necessary Susceptibility testing of azoles may be useful for patients unresponsive to therapy Invasive disease with clinical failure of initial therapy When to detect resistance Consider susceptibility testing as an adjunct AMB, 5FC, FCZ, VRZ, and an echinocandin Consultation with an experienced microbiologist recommended Adapted from Pfaller et al. J. Clin. Microbiol. 2012, 50(9):2846
32 Candida spp. infections When to detect resistance Infection with species with high rates of intrinsic or acquired resistance Susceptibility testing not necessary when intrinsic resistance is known C. lusitaniae and amphotericin B C. krusei and fluconazole, flucytosine C. guilliermondii and echinocandins With high rates of acquired resistance, monitor closely for signs of failure and perform susceptibility testing C. glabrata and fluconazole, AMB, and echinocandins C. krusei and AMB C. guilliermondii and AMB C. rugosa and AMB, fluconazole, and echinocandins Adapted from Pfaller et al. J. Clin. Microbiol. 2012, 50(9):2846
33 Candida spp. infections When to detect resistance New treatment options (e.g., echinocandins, voriconazole, posaconazole) or unusual species Susceptibility testing warranted if prior exposure to echinocandins or fluconazole Susceptibility testing may be helpful when patient is not responding to what should be effective therapy Patients who respond to therapy despite being infected with an organism later found to be resistant Best approach not clear Take into account severity of infection, patient immune status, consequences of recurrent infection, etc. Consider alternative therapy for infections with isolates that appear to be highly resistant to initial therapy Adapted from Pfaller et al. J. Clin. Microbiol. 2012, 50(9):2846
34 Aspergillus spp. infections Identification to the species level Routine testing of triazoles against Aspergillus spp. : ITZ, VRZ, PSZ Other mold infections When to detect resistance On a case-by-case basis After discussion between clinician and microbiologist
35 Thank you!
36
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