1. 1 Antifungal Susceptibility Test Department of Laboratory Medicine, Chung-Ang University Lee Mi-Kyung Feb. 6, 2010

2. Introduction Antifungal Drugs Antifungal Susceptibility Test : CLSI AFST Summary 22

3. 3 Fungal infections are a serious & growing public health problem Major fungal pathogens are increasing in incidence (systemic fungal infections) New pathogens are emerging Growing number of antifungal agents Drug-resistant fungal pathogens are emerging

4. 44 Fungal infections  No. of antifungals  Fungal infections  No. of antifungals  Need for reproducible, clinically relevant antifungal susceptibility testing  ▪ Provide a reliable measure of the relative activities of two or more antifungal agents ▪ Correlate with in vivo activity ▪ Predict the likely outcome of therapy ▪ To monitor the development of resistance ▪ Predict the therapeutic potential of newly discovered investigational agents Resistance to antifungal agents Resistance to antifungal agents

5. 5 Antifungal agents

6. 6 Antifungal susceptibility testing (AFST) 1. Broth-based method (M27-A3, M38-A2) Macrobroth dilution Microbroth dilution Colorimetric VITEK-2 system 2. Agar-based method Agar dilution E-test Disk diffusion test (M44-A, M51-P) 3. Flow Cytometry (FCM) 4. Sterol Quantitation (SQM)

7. 777 Current CLSI methods for AFST M27-A3 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts 4/28/2008 M27-S3 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts 4/28/2008 M38-A2 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi 4/28/2008 M44-A Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts 5/20/2004 M44-S2 Zone Diameter Interpretive Standards, Corresponding Minimal Inhibitory Concentration (MIC) Interpretive Breakpoints, and Quality Control Limits for Antifungal Disk Diffusion Susceptibility Testing of Yeasts 8/15/2007 M51-P Method for Antifungal Disk Diffusion Susceptibility Testing of Filamentous Fungi 6/30/2009 M51-S1 Method for Antifungal Disk Diffusion Susceptibility Testing of Filamentous Fungi Informational Supplement 6/30/2009

8. 8 CLSI M27-A3: Foreword Candida spp. & C. neoformans In 1985  20% (>200 beds) performing antifungal testing  Broth dilution  C. albicans or other spp. of yeasts  Tested only a few isolates/yr  Agreement of MIC among labs: unacceptably low  it would be useful to work a more reproducible reference testing procedure

9. 9 CLSI M27-A3: Foreword Proposed Standard (Dec. 1992): M27-P  broth macrodilution Tentative Standard (Oct. 1995): M27-T  broth microdilution  reference MIC ranges (2 QC strains) Approved Standard (Jun. 1997): M27-A  breakpoints for available antifungal agents Approved Standard (Aug. 2002): M27-A2 Approved Standard (Apr. 2008): M27-A3 & S3  24- & 48-hour reference MIC ranges

10. 10 CLSI M27-A3: Advantage Paralleling the broth macrodilution procedure Reference MIC ranges were established Interlaboratory agreement Ease to performance Economy More rapid results

11. 11 CLSI M27-A3: Antifungal agents Powder: store at -20 ℃ Prep. stock solution: 10 times, store at -60 ℃ ↓ SolventsNo. of conc. tested Amphotericin BDMSO 0.0313  16  g/mL Fluconazole*Water 0.125  64  g/mL Itraconazole*DMSO 0.0313  16  g/mL Voriconazole*DMSO 0.0313  16  g/mL Anidulafungin*DMSO 0.015  8  g/mL Micafungin*DMSO 0.015  8  g/mL Caspofungin*Water 0.015  8  g/mL Flucytosine*Water 0.125  64  g/mL * Interpretive breakpoints are available for Candida spp.

12. 12 CLSI M27-A3: Guidelines for selective testing As part of periodic batch surveys To aid in the management of refractory oropharyngeal infections due to Candida spp. in pts. who appear to be experiencing therapeutic failure To aid in the management of invasive infections due to Candida spp. when the utility of azole antifungal agents is uncertain (non-C. albicans)

13. 13 CLSI M27-A3: Test procedures Broth medium : RPMI 1640, 0.165 mol/L MOPS, pH 6.9-7.1 Preparing diluted antifungal agents (2x serial) : microdilution plate (96 U-shaped wells) plastic test tubes GC (growth control) SC (sterility control) FCZ 0.125  64  g/mL 0.1 mL  64 32 16 8 4 2 1 ……. 0.125 GC SC stored at -70 ℃ for up to 6 months

14. 14 CLSI M27-A3: Test procedures Inoculum prepararion 1. subcultured onto SDA at 35 ℃ (purity & viability) 2. Candida spp.(24h), C. neoformans (48h) 3. 0.5 McF (1  5X10 6 cells/mL) 1:1000 dilution (0.5  2.5X10 3 cells/mL) Incubation at 35 ℃ Candida spp.(24  48h), C. neoformans (70  74h) 

15. 15 CLSI M27-A3: Reading results Amount of growth-compared visually with GC 0: optically clear 1: slightly hazy 2: prominent decrease (  50%) in turbidity 3: slight reduction in turbidity 4: no reduction in turbidity GC  Score of 0: AMB  Score of 2: 5-FC, Azole, Echinocandin

16. 16 Antifungal Agent Minimum inhibitory concentration ( ㎍ /mL) Susceptible (S) Susceptible- dose dependent (SDD) Intermediate (I) Resistant (R) Nonsusceptible (NS) Anidulafungin≤ 2 > 2 Caspofungin≤ 2 > 2 Fluconazole≤ 816-32≥ 64 Flucytosine≤ 48-16≥ 32 Itraconazole≤ 0.1250.25-0.5≥ 1 Micafungin≤ 2 > 2 Voriconazole≤ 12≥ 4 Interpretive guidelines for in vitro susceptibility testing of Candida spp.

17. 17 CLSI M27-A3: Time of reading 24 h48h Amphotericin BYes FluconazoleYes ItraconazoleNoYes PosaconazoleNoYes RavuconazoleNoYes VoriconazoleNoYes FlucytosineNoYes EchinocandinsYesNo AMB: No approved interpretive breakpoints are available

18. 18 CLSI M27-A3: Problems Trailing phenomenon with some strains of Candida spp. / azoles (5%) Unreliable detection of resistance to AMB Poor growth of some organisms Labour-intensive Hard to determine objectively through visual reading

19. 19 CLSI M38-A2: Foreword & Scope Filamentous fungi (Aspergillus spp., Fusarium spp., Rhizopus spp., Pseudallescheria boydii, Sporothrix schenckii, & other pathogenic molds) Dermatophytes (Trichophyton, Microsporum, & Epidermophyton spp.)  Proposed Standard (Nov. 1998) ): M38-P  Approved Standard (Aug. 2002): M38-A  Approved Standard (Apr. 2008): M38-A2

20. 20 CLSI M38-A2: Guidelines for selective testing As part of periodic batch surveys To aid in the management of invasive & cutaneous infections due to filamentous fungi when the utility of the azole is uncertain Interpretive breakpoints are not available Definition  Minimal effective concentration (MBC) : the lowest conc. of an antimicrobial agent that leads to the growth of small, rounded, compact hyphal forms as compared to the hyphal growth seen in the GC - echinocandin

21. 21 CLSI M38-A2: Test procedures Inoculum prepararion  Final conc. Nondermatophytes: 0.4  5X10 4 CFU/mL Dermatophytes: 1  3X10 3 CFU/mL Incubation  at 35 ℃ or 30 ℃ (some of Alternaria spp.)  21  26 hrs, 46  50 hrs, 46  72hrs

22. 22 CLSI M38-A2: Reading results Amount of growth-compared visually with GC  AMB: 100% inhibition  5-FC, FCZ, Ketokonazole 50% or more inhibition (nondermatophytes) 80% or more inhibition (dermatophytes)  other azole: 100% inhibition Interpretation : The clinical relevance of testing ramains uncertain, and breakpoints with proven relevance have yet to identified or approved by CLSI or any regulatory agency.

23. 23 CLSI M44-A: Disk diffusion of Yeasts Mueller-Hinton agar + 2% Glucose (fungal growth) & 0.5  g/mL Methylene Blue dye (zone edge definition) 0.5 McF standard (1  5X10 6 cells/mL) Reading: after 20-24 hrs of incubation

24. 24 Zone diameter interpretive standards for Candida spp. Inhibition zone diameter (mm) Susceptible (S) Dose-dependent susceptible (S-DD) Resistant (R) Fluconazole> 1915 - 18 < 14 Voriconazole> 1714 - 16 < 13

25. 25  3227 Candida spp. performed by 47 centers  The overall categoric agreement between participant disk diffusion test results and reference MIC results was 87% for fluconazole and 95.2% for voriconazole.

26. 26 CLSI M51-P: Disk diffusion of Filamentous fungi Nonsupplemented Mueller-Hinton agar 0.4  5X10 6 CFU/mL Reading: after 24-72 hrs of incubation Zone diameter : prominent reduction in growth (80%) Microcolonies or Slight trailing ignored: azole, caspofungin not ignored for AMB

27. 27 Zone diameter epidemiological cutoff values (ECV) and corresponding MIC or minimal effective concentration (MEC) for filamentous fungi a Antifungal agentDisk Content Zone diameter, Nearest whole (mm) Equivalent MIC or MEC b ECV (μg/mL) ECV Amphotericin B10 μg151 Caspofungin5 μg171 Itraconazole10 μg171 Posaconazole5 μg171 Voriconazole1 μg171 a ECV: The value shown the highest MIC/MEC (lowest zone diameter) of isolates belonging to the wild-type (WT) distribution. The ECV is expressed as WT Xmm). b MEC applies to caspofungin only.

28. 28 Vitek 2 Yeast AST card (bioMerieux, France) The first automatic AFST More rapid results Eliminates the difficulties in reading of other methods (trailing zone) AMB, 5-FC, FCZ, VOR (Oct 2006: licence for FCZ)

29. 29 If the Vitek 2 system is used as a routine test for fluconazole MIC measurement, it is recommended that Candida spp. where the MIC is above 4 μg/mL be examined using a different method.

30. 30 Quantitative agar diffusion method using antifungal gradient strips: direct reading of MICs. Available for all systemic AFST, excellent alternative to the CLSI standard. E-Test (AB Biodisk, Sweden)

31. 31 Sterol quantitation method (SQM) Susceptible Resistant

32. 32 Flu 0 μg /mL Flu 64 μg /mL Heating ATCC22019 MCF 63 MCF 173 MCF 250 Flowcytometry method: PI (Propidium iodide)

33. 33 C. tropicalis M27-A2 (0.5 μg /mL) FCM (1 μg /mL) (A) Flu 0 (B) Flu 1 (C) Flu 2 MCF 107 MCF 199 MCF 216 (D) Flu 4 (E) Flu 8 (F) Flu 16 MCF 191 MCF 200 MCF 216 (G) Flu 32 (H) Flu 64 MCF 202 MCF 165

34. 34 Future Direction of AFST Improved clinical correlation Decreased subjectivity of endpoint determination Decreased time to result (rapid) Personalized tests (simple) Cost-effective Automation