Antifungal Susceptibility Testing

Birgit Willinger Division of© Clinical by Microbiologyauthor Department of Laboratory Medicine Medical University Vienna Austria ESCMID Online Lecture Library Medical Mycology: The last 50 years

# of drugs

L-AmB ABCD ABLC Terbinafine Itraconazole Fluconazole Ketoconazole Miconazole 5-FC© by author

ESCMID Online Lecture Library J. Rex 2003 Antifungal Acitivity against Yeasts and Moulds

Pilze AMB FCZ ITZ VZ PCZ RCZ CF MF AF Candida albicans Candida tropicalis Candida parapsilosis Candida krusei Candida glabrata Cryptococcus neoformans Aspergillus fumigatus Mucor spp Rhizopus spp Fusarium spp pilze Schimmel- © by author Polyene; AMB = AmBisome® Azole; FCZ = Fluconazole; ITZ = Itraconazole; VZ = Voriconazole; PCZ = Posaconazole; RCZ = Ravuconazole Echinocandine; CF = Caspofungin; MF = Micafungin; AF = Anidulafungin ESCMID Online Lecture Library JP Donnelly & Malcolm Richardson In vitro Activity of Antifungals

Candida spp. Ampho Keto Flu Itra Vori Candine C. albicans +++ ++ +++ +++ +++ +++ C. glabrata ++ + ++ ++ ++ +++ guilliermondii ++ + +++ +++ +++ ++ C. krusei ++ + 0 ++ ++ +++ C. lusitaniae ++ ++ +++ +++ +++ +++ parapsilosis +++ ++ +++ +++ +++ ++ C. tropicalis +++ ++ +++ +++ +++ +++ © by author

+++ reliable activity with occasional resistance ++ moderate activity but resistance is noted + occasional activity Arikan & Rex. Manual of Clinical 0 no meaningfulESCMID activity Online Lecture LibraryMicrobiology 2003, 8th ed., 1859

Species Identification First Step in AST

• C. krusei: do not use fluconazole • C. glabrata: fluconazole is no longer recommended (EUCAST) • C. parapsilosis - echinocandins

• C. rugosa • C. norvegensis • C. lipolytica Reduced susceptibility • C. valida to azoles • C. inconspicua • C. zeylanoides © by author

Trichosporon • Echinocandins – only little activity • Rhodotorula ESCMID Online Lecture Library

Be aware of newer Candida spp.

• C. parapsilosis • C. orthopsilosis • C. metapsilosis

ESCMID Online LectureTavanti Library et al. JCM 2005; 43: 284 -292 Torro et al. CMI 2010; epub ahead print AFST Problems for AST of Fungi

Different morphologies even for yeasts

Budding Yeast

C. albicans C. glabrata ESCMID Online Lecture Library Rodriguez – Tudela 2007 AFST Problems for AST of Fungi

Different morphologies even for yeasts

Budding Yeast Pseudohypha

C. albicans C. albicans

C. glabrata C. parapsilosis ESCMID Online Lecture LibraryRodriguez – Tudela 2007 AFST Problems for AST of Fungi

Different morphologies even for yeasts

Budding Yeast Hypha Pseudohypha

C. albicans C. albicans C. albicans C. glabrata C. parapsilosis ESCMID Online Lecture LibraryRodriguez – Tudela 2007 AFST Problems for AST of Fungi

Different rate of growth

Mucorales

18-24 h ESCMID Online Lecture Library Rodriguez – Tudela 2007 AFST Problems for AST of Fungi

Different rate of growth

Mucorales Yeasts

18-24 h 24-48 h

ESCMID Online Lecture LibraryRodriguez – Tudela 2007 AFST Problems for AST of Fungi

Different rate of growth

Mucorales Yeasts Aspergillus

18-24 h 24-48 h 48-72 h ESCMID Online Lecture Library Rodriguez – Tudela 2007 AFST Problems for AST of Fungi

Different rate of growth

Mucorales Yeasts Aspergillus Black Yeasts

18-24 h 24-48 h 48-72 h Several days ESCMID Online Lecture Library Rodriguez – Tudela 2007 Standardized assays for antifungal susceptibility testing

• EUCAST - yeasts • EUCAST – moulds (Aspergillus) • CLSI M27-A3- macro-/microdilution – yeasts (including Candida and Cryptococcus) • CLSI M38-A2 – microdilution - moulds (Aspergillus, Fusarium, Rhizopus, Pseudallescheria boydii, mycelial form of Sporothrix© by schenckiiauthor • CLSI M 44-A – agardiffusion/ yeasts • CLSIESCMID M51-A – agardiffusion/ Online Lecture filamentous Library fungi • DIN 58940-84 – microdilution/yeasts

Epidemiological Cutoff Values (ECV)

• ECV define upper limit of „wild type“ distributions

• Establish cutoffs to help detect emergence of reduced susceptibility in the absence of clinical breakpoints – ... or „in addition ©to „clinicalby author breakpoints“ • Helps identify organisms requiring further characterization ESCMID Online Lecture Library Antifungal Susceptibility Testing - Yeasts

ESCMID Online Lecture Library Differences between ComparisonCLSI M27-A2 of CLSI &and E. EUCASTDis 7.1

CLSI EUCAST-AFST M-27A2 E. Dis 7.1

Glucose 10 times more! 0.2 % 2% Shape of well Round bottom Flat bottom 100 times 0.5 - 2.5 x 103 0.5 - 2.5 x 105 Inoculum (cfu/mL)bigger! Incubation (h)Quicker! 48 24 Reading visual spectrophotometric Endpoint optically© by clear author and lowest concentration prominent decrease that inhibits growth by at least a 90% or 50% ESCMID Online Lecture Library

Rodriguez Tudela 2007 Pitfalls: Trailing growth

• Azoles produce incomplete and partial inhibition • Some Candida strains show reduced but persistent growth over an extended range of the concentrations used • Difficulties in interpretation of the visual endpoint • Mainly C. albicans, C. tropicalis • Important: incubation period – 24h strains being susceptible, resistant after 48 h – Therapeutic outcome correlates© by withauthor 24h results! • Slight trailing in echinocandins possible ESCMID Online Lecture Library AFST Problems for AST of Fungi Trailing effect or partial inhibition of growth

The trailing effect is not universal Only for some drugsDepending & bugs of endpoint

Both 50% susceptible % Growth% 1 susceptible 80% 1 resistant © by author

Drug concentration ESCMID Online Lecture Library

ESCMID Online Lecture Library 3 strains with: High MICs by EUCAST Low MICs by CLSI No data ?????

ESCMID Online Lecture Library 5 strains with: High MICs by CLSI Low MICs by EUCAST

ESCMID Online Lecture Library Similar MICs at 24 h

ESCMID Online Lecture Library Strains with trailing effect at 48 h

ESCMID Online Lecture Library Breakpoints for Fluconazole established by EUCAST

Susceptible Intermediate Resistant

EUCAST < 2 mg/L 4 mg/L > 4 mg/L • Valid for C. albicans, ©C. parapsilosisby author and C. tropicalis • invalid for – C. krusei intrinsically resistent – C. glabrata

JL Rodriguez Tudela,ESCMID 17th ECCMID 2007 Online Lecture Library Breakpoints according to CLSI and EUCAST for Candida spp.

ESCMID Online Lecture Library Lass-Flörl et al. 2010; Mycoses 53: 1-11 EUCAST Breakpoints

Antifungal Species related breakpoints (S)

C. albicans C. glabrata C. krusei C. parapsilosis C. tropicalis

Amphotericin B 1.0/1.0 1.0/1.0 1.0/1.0 1.0/1.0 1.0/1.0

Anidulafungin 0.03/0.03 0.06/0.06 0.06/0.06 -- 0.06/0.06

Fluconazole 2.0/4.0 IE -- 2.0/4.0 2.0/4.0

Posaconazole 0.06/0.06 IE © byIE author0.06/0.06 0.06/0.06

Voriconazole 0.125/0.125 IE IE 0.125/0.125 0.125/0.125

ESCMID Online Lecture Librarywww.eucast.org Voriconazole Activity Against Fluconazole R Isolates

ESCMID Online Lecture Library Pfaller & Diekema, JCM 2007 Posaconazole Activity Against Fluconazole R Isolates

ESCMID Online Lecture Library Candida and Echinocandins

• Most recently introduced anti Candida-agents

– Target glucan synthase complex – Resistance associated with mutations in 2 highly conserved regions of fks1/fks2 – Mutants have reduced sensitivity of glucan synthase and poor response in© murine by author candidiasis models

Effron et al., AAC 2009; 3. 112 ESCMID Online LecturePerlin DS, Drug Library Resist Update 2007; 10: 121 Baixench MT et al., JAC, 2007; 59: 107 Pitfalls: Paradoxical Effect

• Growth of fungal strain in vitro at high concentrations > MIC • More often seen with caspofungin than with other echinocandins C. tropicalis • Never seen with C. glabrata • Mechanism: unknown, decrease in ß- 1,3 and ß-1,6-glucan and an increase of chitin synthesis © by author

• Porbably no clinical significance A. fumigatus ESCMID Online Lecture Library Chamilos et al. 2007, AAC epub Echinocandin Wild Type MIC Distribution and Epidemiological Cutoff Values (ECVs) for C. albicans

ESCMID Online Lecture Library Pfaller, ECCMID 2010 Candida and Echinocandins

• New IDSA guidelines emphasize echinocandins for severely ill patients with invasive candidiasis, or for those with prior azole exposure • CLSI established clinical breakpoint of < 2 µg/ml • Does this breakpoint© by encompass author isolates with fks mutations?

ESCMID Online Lecture LibraryEffron et al., AAC 2009; 3. 112 Pfaller, ECCMID 2010 C. parapsilosis and Echinocandins

• C. parapsilosis isolated in 53% of cancer pts who developed candidemia while receiving caspofungin therapy (Cancer 2009; 115: 4745-52)

• Strong correlation between caspofungin usage and a 400% increase in C. parapsilosis and a 400% increase in C. parasilosis BSI (J Infect 2008; 56: 126-129)

• Species-specific incidence of C. parasilosis BSI has doubled in the US between 1993 and 2009 (CDC 2009)

• Improved response in treating© ptsby with author C. parapsilosis BSI with high- dose caspofungin (150 mg/d) vs standard dose (70 mg load/50 mg daily): 81% vs 61%; not statistically significant (CID 2009; 48: 1676- 84)

ESCMID Online Lecture LibraryPfaller, ECCMID 2010

Candida Summary

• Greatest concern is still C. glabrata • Potential emergence of less common

species inherent of aquired azole resistance • Most Candida remain highly susceptible to echinocandins, but the current clinical breakpoint is insufficient© by author for detection of resistant isolates – CBP will be lowered substantially ESCMID Online Lecture Library Antifungal Susceptibility Testing - Moulds

ESCMID Online Lecture Library Antifungal susceptibility testing for filamentous fungi

ESCMID Online Lecture Library Lass-Flörl et al., 2010 Mycoses, 1 - 11 Aspergillus spp. and Azoles

• In vitro „reduced susceptibility“ remains 0- 5% in most large surveys, often using itraconazole – Most data for Aspergillus section

• Case reports and case series suggest that multiply-azole resistant Aspergillus could © by author emerge – ? Association with agricultural azole use

Rodriguez-Tudela et al. AAC 2008; 52: 2468 ESCMID Online LectureVerweij et al.Library N Engl J Med 2007; 356: 1481 Arendrup et al. AAC 2008; 52: 3504 Epidemiological Cutoff Values for Azoles and Aspergillus spp.

ESCMID Online Lecture LibraryEspinell Ingroff et al. JCM 2010 Aspergillus in General • Azole resistance among Aspergillus remains uncommon, but is emerging

• Clinical significance of elevated MICs requires further study

• Clinical breakpoints unavailable: ECVs will have to suffice © by author – VRC/ITR: 1-2 µg/ml – PSC: 0.25 – 0.5 µg/ml – Echinocandines:ESCMID Online 0.06 – 0.12 Lecture µg/ml Library Easier Methods

ESCMID Online Lecture Library CLSI M 44-A – agardiffusion for yeasts • Candida vs. fluconazole and voriconazole • Reproducible method and good correlation with the reference method • Mueller-Hinton agar supplemented with 2% glucose and methylene blue as the test medium • Advantages in comparison to RPMI – Clear inhibition zone edges – Less intrazonal growth – Easy interpretation of inhibition© by zoneauthor diameters

R S-DD S Fluconazole 25 µg < 14 mm 15 - 18 mm > 19 mm VoriconazoleESCMID 1 µg Online< 13 Lecturemm 14 – 16Library mm > 17 mm Disk Diffusion Assay - Voriconazole

RPMI + 2% Glucose and MH-agar + 2% Glucose and MethyleneESCMID blue Online LectureMethylene Library blue CLSI 51-A – agardiffusion for filamentous, non dermatophyte fungi • Moulds vs amphotericin B, caspofungin, itraconazole, posaconazole, and voriconazole • This method often provides qualitative results 8 to 24 hours sooner than the standard CLSI document M381 method. • use of nonsupplemented Mueller-Hinton agar should make antifungal susceptibility testing more readily available to clinical laboratories at a reduced cost. • Although clinical breakpoints have not been assigned, tentative epidemiological© cutoffby author values (ECVs) have been developed. The ECVs are used to detect those isolates with reduced susceptibility. ESCMID Online Lecture Library E-Test®

• Agar based diffusion method (AB BioDisk, Sweden  bioMerieux, France) • Available for • Amphotericin B • Fluconazole, ketoconazole, itraconazole, voriconazole, posaconazole • Caspofungin, anidulafungin, micafungin • Flucytosine © by author • agreement with CLSI method usually fairly good • more efficacious then reference method to detect AmB resistance in Candida ESCMID Online Lecture Library Multicenter Comparison of the VITEK 2 AST with CLSI

• 426 Candida species, comparison • Determination of endpoints spectrophotometrically after an incubation period of 9.1 -2.1 h (mean 14 h) • Excellent agreement for all tested antifungals ( AmB, 5-FC, voriconazole) • Discrepancies: C. glabrata – Vori (24 h CLSI) C. krusei – Vori (48 h CLSI)

AmB 99,1 Flucytosin 98,2 0 0.9 0.9 Voriconazole 98,6 0 0.2 1.2 ESCMID Online Lecture Library Pfaller et al. 2007 JCM 45: 3522-3528 Important issues when performing AST • Identify the isolate at least to the genus level, better to species level • For Candida species from sterile sites, perform routine susceptibility testing for fluconazole and according to the local epidemiology include other azoles. • Perform susceptibility testing as an adjunctive to treatment for patients© withby author invasive disease and clinical failure of initial therapy, or with break- through infection. ESCMID Online Lecture Library Important issues when performing AST

• Do not test isolates with a high rate of intrinsic resistance – C. krusei and fluconazole – A. terreus and amphotericin B • Perform susceptibility testing as an adjunctive to treatment for patients with invasive disease, long-term azole treatment and ⁄or recurrent cultivation of a fungus • Perform susceptibility testing as an adjunctive to treatment for patients with© by invasive author disease and infection with rare moulds or other fungi • Use for antimycotic stewardship – deescalation therapy (e.g. fluconazole  echinocandine)! ESCMID Online Lecture Library

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