CLSI Performance Standards for Antifungal Susceptibility Testing of Yeasts

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M60

Performance Standards for Antifungal Susceptibility Testing of Yeasts

This document includes updated minimal inhibitory

concentration, zone diameter, and quality control tables for the Clinical and Laboratory Standards Institute antifungal susceptibility testing documents M27 and M44.

A CLSI supplement for global application.

Licensed to: Ms. Constanta Popa

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Clinical and Laboratory Standards Institute

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November 2017 Replaces M27-S4 and M44-S3

Performance Standards for Antifungal Susceptibility Testing of Yeasts

Barbara D. Alexander, MD, MHS Gary W. Procop, MD, MS

Philippe Dufresne, PhD (RMCCM) Jeff Fuller, PhD, FCCM, D(ABMM)

Annette W. Fothergill, MA, MBA, MT(ASCP) Mahmoud A. Ghannoum, PhD, EMBA, FIDSA Kimberly E. Hanson, MD, MHS

Denise Holliday, MT(ASCP) Nicole M. Holliday, BA

Luis Ostrosky-Zeichner, MD, FACP, FIDSA, FSHEA Audrey N. Schuetz, MD, MPH, D(ABMM)

Dee Shortridge, PhD

Nathan P. Wiederhold, PharmD Adrian M. Zelazny, PhD, D(ABMM)

Abstract

Clinical and Laboratory Standards Institute document M60Performance Standards for Antifungal Susceptibility Testing of Yeasts includes the minimal inhibitory concentration, zone diameter, and QC tables developed following the standards described in CLSI documents M27¹ and M44.² The tabular information in this document is valid only when the methodology is followed as described in the current editions of CLSI documents M27¹ and M44.²Any previously published tables should be replaced with these new tables. Changes since the last edition appear in boldface type.

Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antifungal Susceptibility Testing of Yeasts. 1st ed.

CLSI supplement M60 (ISBN 1-56238-828-2 [Print]; ISBN 1-56238-829-0 [Electronic]). Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2017.

The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in the CLSI catalog and posted on our website at www.clsi.org. If you or your organization is not a member and would like to become one, or to request a copy of the catalog, contact us at: Telephone: +1.610.688.0100; Fax: +1.610.688.0700; E-Mail:

[email protected]; Website: www.clsi.org.

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Copyright ^©2017 Clinical and Laboratory Standards Institute. Except as stated below, any reproduction of content from a CLSI copyrighted standard, guideline, companion product, or other material requires express written consent from CLSI. All rights reserved. Interested parties may send permission requests to [email protected].

CLSI hereby grants permission to each individual member or purchaser to make a single reproduction of this publication for use in its laboratory procedures manual at a single site. To request permission to use this publication in any other manner, e-mail [email protected].

Suggested Citation

CLSI. Performance Standards for Antifungal Susceptibility Testing of Yeasts. 1st ed. CLSI supplement M60. Wayne, PA: Clinical and Laboratory Standards Institute; 2017.

Previous Editions:

M27-S4: May 2004, April 2005, April 2008, December 2012 M44-S3: January 2006, August 2007, August 2009

ISBN 1-56238-828-2 (Print) ISBN 1-56238-829-0 (Electronic) ISSN 1558-6502 (Print)

ISSN 2162-2914 (Electronic) Volume 37, Number 14

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Committee Membership

Subcommittee on Antifungal Susceptibility Tests

Barbara D. Alexander, MD, MHS Chairholder

Duke Univeristy Medical Center USA

Gary W. Procop, MD, MS Vice-Chairholder

Cleveland Clinic USA

Philippe Dufresne, PhD (RMCCM) Institut national de santé publique du Québec

Canada

Jeff Fuller, PhD, FCCM, D(ABMM) London Health Sciences Centre Canada

Mahmoud A. Ghannoum, PhD, EMBA, FIDSA

Case Western Reserve University USA

Kimberly E. Hanson, MD, MHS University of Utah and ARUP Laboratories

USA

Denise Holliday, MT(ASCP) BD Diagnostic Systems USA

Nicole M. Holliday, BA Thermo Fisher Scientific USA

Luis Ostrosky-Zeichner, MD, FACP, FIDSA, FSHEA

Memorial Hermann Healthcare System USA

Audrey N. Schuetz, MD, MPH, D(ABMM)

Mayo Clinic USA

Nathan P. Wiederhold, PharmD University of Texas Health Science Center at San Antonio

USA

Adrian M. Zelazny, PhD, D(ABMM) USA

Staff

Clinical and Laboratory Standards Institute

USA

Marcy L. Hackenbrack, MCM, M(ASCP)

Project Manager

Megan L. Tertel, MA, ELS Editorial Manager

Catherine E.M. Jenkins Editor

Kristy L. Leirer, MS Editor

Laura Martin Editor

Acknowledgment

CLSI and the Subcommittee on Antifungal Susceptibility Tests gratefully acknowledge the following volunteers for their important contributions to the development of this document:

Barbara D. Alexander, MD, MHS Duke Univeristy Medical Center USA

Annette W. Fothergill, MA, MBA, MT(ASCP)

USA

Dee Shortridge, PhD JMI Laboratories USA

Acknowledgment

CLSI and the Subcommittee on Antifungal Susceptibility Tests gratefully acknowledge the following former subcommittee members for their review of this document during development:

Sharon K. Cullen, BS, PMP, RAC Beckman Coulter, INc.

USA

Shawn R. Lockhart, PhD, D(ABMM) Centers for Disease Control and Prevention

USA

David S. Perlin, PhD

New Jersey Medical School-UMDNJ USA

Nancy L. Wengenack, PhD, D(ABMM) Mayo Clinic

USA

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Licensed to: Ms. Constanta Popa iv

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Foreword

Users of CLSI documents M27¹ and M44² and this document should recognize that the standard methods described in CLSI documents are reference methods. These methods may be used for:

 Routine antifungal testing of patient isolates to guide therapy

 Evaluating commercial devices that will be used in medical laboratories

 Testing new agents or systems by drug or device manufacturers

Results generated by reference methods, such as those described in CLSI documents, may be used by regulatory authorities to evaluate commercial susceptibility testing device performance as part of the approval process. Regulatory clearance indicates that the commercial susceptibility testing device provides results that are substantially equivalent to those generated using reference methods for the organisms and antimicrobial agents described in the device manufacturer’s approved package insert.

However, CLSI breakpoints may also differ from those approved by various regulatory organizations for many reasons, including:

 Database differences

 Data interpretation

 Dosage amounts used in different parts of the world

 Public health policies

Differences also exist because CLSI proactively evaluates the need for changing breakpoints. The reasons why breakpoints may change, and the manner in which CLSI evaluates data and determines breakpoints, are described in CLSI document M23.³

When CLSI decides to change an existing breakpoint, regulatory organizations may also review data to determine how changing breakpoints may affect antimicrobial agent safety and effectiveness for the approved indications. When a regulatory authority changes breakpoints, commercial device manufacturers may have to conduct a clinical trial, submit the data to the regulatory organization, and await review and approval. For these reasons, a delay of one or more years may be needed if a breakpoint change is to be implemented by a device manufacturer. Some regulatory and accreditation requirements allow laboratories using cleared or approved testing devices to use existing regulatory organization breakpoints. Either those or CLSI susceptibility breakpoints may be acceptable to laboratory accreditation organizations. Other regulatory and accreditation requirements may vary. Each laboratory should check with its antimicrobial susceptibility test system manufacturer for additional information on the breakpoints used in its system software. Laboratories should be aware of their specific regulatory and accreditation requirements for using CLSI breakpoints.

Once verified by the CLSI document development process, breakpoints may be implemented as soon as they are published in a supplement. However, medical laboratories should discuss this implementation with appropriate stakeholders, such as infectious disease practitioners and the pharmacy department, as well as the hospital pharmacy and therapeutics and infection control committees, before implementing newly approved or revised breakpoints. When a device includes antimicrobial test concentrations that are sufficient to interpret susceptibility and resistance to an agent using the CLSI breakpoints, a laboratory could, after appropriate validation as outlined in CLSI document M52,⁴ choose to interpret and report results from that device using CLSI breakpoints.

NOTE: Current fungal taxonomy is under revision. Many genera have both a teleomorph (sexual state) and an anamorph (asexual state) name. In this document, the traditional Candida anamorph names are used to provide continuity to both past procedures and associated documents such as CLSI document M27.¹

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Overview of Changes

This document replaces the previous editions of the approved supplements M27-S4, published in 2012, and M44-S3, published in 2009. Several changes were made in this edition, including:

 General:

– Combined information from M27-S4 (antifungal broth dilution susceptibility testing) and M44-S3 (antifungal disk diffusion testing) into one informational supplement, which has been recoded as M60. Revised the title to harmonize with other CLSI susceptibility testing informational supplements

– Moved tables showing schemes for preparing dilution series of water-soluble and water-insoluble antifungal agents to be used in broth dilution susceptibility testing (originally Tables 4 and 5 in M27-S4) to the broth dilution testing document (CLSI document M27¹)

– Moved tables showing the formulation for Roswell Park Memorial Institute (RPMI) 1640 culture medium (originally Table 8 in M27-S4) to the broth dilution testing document (CLSI document (M27¹)

– Deleted modifications for special circumstances of broth dilution testing (originally Table 9 in M27-S4)

– Deleted the glossary with abbreviations, routes of administration, and drug classes for antifungal agents

 Foreword:

– Added a NOTE regarding changing fungal nomenclature and use of traditional fungal names

 Table 1 – Breakpoints and Interpretive Categories for In Vitro Broth Dilution Susceptibility Testing of Candida spp. and Select Antifungal Agents:

– Revised the footnote regarding interpretation of the intermediate category

– Changed the voriconazole “susceptible-dose dependent” category to “intermediate”

– Combined breakpoints for all antifungal agents into a single table

– Added a footnote regarding variability in results for susceptibility testing with caspofungin – Added a footnote regarding the intrinsic resistance of Candida krusei to fluconazole

– Added a NOTE explaining the deletion of breakpoints for Candida spp. when testing against itraconazole and flucytosine

– Added seven new references

 Table 2 – Solvents and Diluents for Preparing Stock Antifungal Agent Solutions for Broth Dilution Testing:

– Deleted water as a solvent or diluent for fluconazole and flucytosine – Added solvent and diluent information for isavuconazole

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 Table 4 – Recommended 24-Hour Minimal Inhibitory Concentration Limits for Two Quality Control Strains for Broth Microdilution Procedures:

– Changed title to reflect reading QC results at 24 hours only – Deleted columns for 48-hour QC results

– Added QC ranges for isavuconazole with Candida parapsilosis and C. krusei

– Revised the footnote regarding the variation in caspofungin QC ranges when testing against C. krusei

– Added a NOTE regarding adoption of 24-hour MIC QC ranges

– Added a NOTE referring commercial test system users to manufacturer instructions for QC testing and ranges

– Added one new reference

 Table 5 – Zone Diameter Interpretive Categories for Select Antifungal Agents Against Candida spp.:

– Added species-specific zone diameter interpretive categories for caspofungin and micafungin against selected Candida spp.

– Added zone diameter breakpoints and interpretive categories for Candida glabrata and micafungin and revised the footnote accordingly

– Changed voriconazole “susceptible-dose dependent” category to “intermediate”

– Added footnote regarding insufficient data to correlate C. glabrata voriconazole in vitro results with clinical outcome

– Added footnote regarding using breakpoints read at 48 hours if the growth control shows insufficient growth

– Added footnote regarding the absence of zone sizes for C. glabrata

– Added species-specific zone diameter interpretive categories for fluconazole and voriconazole against selected Candida spp.

– Moved voriconazole to Table 5, with the “susceptible-dose dependent” category, and changed voriconazole breakpoints originally listed in the “susceptible-dose dependent” category to

“intermediate.” Combined the breakpoint table for azole antifungal agents with the breakpoint table for echinocandins

– Added three new references

 Table 6 – Recommended Quality Control Zone Diameter (mm) Ranges:

– Added QC zone diameter ranges for micafungin

NOTE: The content of this document is supported by the CLSI consensus process and does not necessarily reflect the views of any single individual or organization.

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Key Words

Antifungal agent, azole, breakpoint, broth dilution, disk diffusion, echinocandin, interpretive category, minimal inhibitory concentration, quality control, susceptibility testing, yeasts, zone diameter

Abbreviations and Acronyms

ATCC^®^a American Type Culture Collection DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

I intermediate

MIC minimal inhibitory concentration QC quality control

R resistant

S susceptible

SDD susceptible-dose dependent

a ATCC^® is a registered trademark of the American Type Culture Collection.

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**Table 1. Minimal Inhibitory Concentration Breakpoints for In Vitro Broth Dilution Susceptibility Testing of Candida spp. and Select Antifungal Agents After 24-Hour Incubation**

Antifungal Agent Species

MIC Breakpoints and Interpretive Categories, µg/mL

S I^* SDD^† R

Anidulafungin^1,‡ C. albicans ≤0.25 0.5 – ≥1

C. glabrata ≤0.12 0.25 – ≥0.5

C. guilliermondii ≤2 4 – ≥8

C. krusei ≤0.25 0.5 – ≥1

C. parapsilosis ≤2 4 – ≥8

C. tropicalis ≤0.25 0.5 – ≥1

Caspofungin^1,‡,§ C. albicans ≤0.25 0.5 – ≥1

C. glabrata ≤0.12 0.25 – ≥0.5

C. krusei ≤0.25 0.5 – ≥1

C. tropicalis ≤0.25 0.5 – ≥1

Micafungin^1,‡ C. albicans ≤0.25 0.5 – ≥1

C. glabrata ≤0.06 0.12 – ≥0.25

C. krusei ≤0.25 0.5 – ≥1

C. tropicalis ≤0.25 0.5 – ≥1

Voriconazole^2,‡,¶ C. albicans ≤0.12 0.25–0.5 – ≥1

C. glabrata^# – – – –

C. krusei ≤0.5 1 – ≥2

C. parapsilosis ≤0.12 0.25–0.5 – ≥1 C. tropicalis ≤0.12 0.25–0.5 – ≥1

Fluconazole^3,† C. albicans ≤2 – 4 ≥8

C. glabrata^** – – ≤32 ≥64

C. krusei^†† – – – –

C. parapsilosis ≤2 – 4 ≥8

C. tropicalis ≤2 – 4 ≥8

* The “I” category provides a buffer zone for antimicrobial susceptibility testing that is necessary to avoid major and very major errors that may occur given the inherent variability of the in vitro testing method. Available data do not permit isolates with MIC results in the “I” range to be clearly categorized as either “S” or “R.” Strains with “I” MICs may respond clinically to a higher-than-standard dose of drug or in situations in which drug penetration is maximized.

† Susceptibility depends on achieving the maximum possible blood level. For fluconazole, doses higher than the standard dosing amount (6 mg/kg/day) may be needed in adults with normal renal function and body habitus.

‡ For these antifungal agents, the data are based substantially on experience with non-neutropenic patients with candidemia; their clinical relevance in other settings is uncertain.

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Table 1. (Continued)

§ Caspofungin susceptibility testing in vitro has been associated with significant interlaboratory variability, contributing to reports of false resistance when using the reference method described in CLSI document M27.^4,5 The cause of the variability is unclear. When testing caspofungin, susceptible results may be reported as “susceptible”; however, laboratories should confirm “I” or “R” results by a) additional susceptibility testing with micafungin⁶ or anidulafungin,⁷ b) DNA sequence analysis of FKS genes to identify resistance hot spot mutations in FKS1 (all Candida spp.) and FKS2 (C. glabrata only),^8,9 or c) sending to a referral laboratory for confirmation. Candida spp. resistant to anidulafungin or micafungin, or possessing characteristic FKS hot spot mutations are considered resistant to all echinocandins, including caspofungin, and should be reported as such.^6,7

¶ Breakpoints may also be used for 48-hour readings if 24-hour growth control shows insufficient growth.

# For C. glabrata and voriconazole, current data are insufficient to demonstrate a correlation between in vitro susceptibility testing and clinical outcome.

** For fluconazole, these guidelines are based on extensive experience with mucosal and invasive infections due to Candida spp.

When an isolate is identified as C. glabrata and the MIC is ≤32 µg/mL, it should be determined whether fluconazole is appropriate in the specific clinical context. If so, patients should receive a maximum dosage regimen of fluconazole. Expert consultation on selecting a maximum dosage regimen may be useful.

†† Isolates of C. krusei are assumed to be intrinsically resistant to fluconazole, so their MICs should not be interpreted using this scale.

Abbreviations: DNA, deoxyribonucleic acid; I, intermediate; MIC, minimal inhibitory concentration; R, resistant; S, susceptible;

SDD, susceptible-dose dependent.

NOTE 1: Information in boldface type is new or modified since the previous edition.

NOTE 2: The selected breakpoints have been established to distinguish resistant mutants from susceptible isolates, and differences in breakpoints reflect methodological issues. Due to in vitro methodological issues, the breakpoint for micafungin against C. glabrata is lower than that of other echinocandins, which does not reflect any inherent clinical differences in efficacy. True differences in antifungal activity among the echinocandins are rare.¹⁰

NOTE 3: The MIC breakpoints (µg/mL) for Candida spp. are shown against the indicated agents. If MICs are measured using a scale yielding results that fall between the categories, the next highest category is implied. Thus, an isolate for which the fluconazole MIC equals 3 µg/mL would be placed in the SDD category.

NOTE 4: Previous breakpoints for itraconazole and flucytosine were established with minimal clinical data; emerging data now suggest the previous breakpoints were not correct and should not be used. For Candida spp. and itraconazole, epidemiological cutoff values that define the limit of the wild-type distribution are established and may be useful for distinguishing between wild-type and non-wild-type isolates (those with intrinsic or acquired known resistance mechanisms) (see CLSI documents M57¹¹ and M59¹²).

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References for Table 1

1 Pfaller MA, Diekema DJ, Andes D, et al.; CLSI Subcommittee for Antifungal Testing. Clinical breakpoints for the echinocandins and Candida revisited: integration of molecular, clinical, and microbiological data to arrive at species-specific interpretive criteria. Drug Resist Updat.

2011;14(3):164-176.

2 Pfaller MA, Andes D, Arendrup MC, et al. Clinical breakpoints for voriconazole and Candida spp.

revisited: review of microbiologic, molecular, pharmacodynamic, and clinical data as they pertain to the development of species-specific interpretive criteria. Diagn Microbiol Infect Dis. 2011;70(3):330- 343.

3 Pfaller MA, Andes D, Diekema DJ, Espinel-Ingroff A, Sheehan D; CLSI Subcommittee for Antifungal Susceptibility Testing. Wild-type MIC distributions, epidemiological cutoff values and species-specific clinical breakpoints for fluconazole and Candida: time for harmonization of CLSI and EUCAST broth microdilution methods. Drug Resist Updat. 2010;13(6):180-195.

4 CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 4th ed. CLSI standard M27. Wayne, PA: Clinical and Laboratory Standards Institute; 2017.

5 Espinel-Ingroff A, Arendrup MC, Pfaller MA, et al. Interlaboratory variability of caspofungin MICs for Candida spp. using CLSI and EUCAST methods: should the clinical laboratory be testing this agent? Antimicrob Agents Chemother. 2013;57(12):5836-5842.

6 Pfaller MA, Messer SA, Diekema DJ, Jones RN, Castanheira M. Use of micafungin as a surrogate marker to predict susceptibility and resistance to caspofungin among 3,764 clinical isolates of Candida by use of CLSI methods and interpretive criteria. J Clin Microbiol. 2014;52(1):108-114.

7 Pfaller MA, Diekema DJ, Jones RN, Castanheira M. Use of anidulafungin as a surrogate marker to predict susceptibility and resistance to caspofungin among 4,290 clinical isolates of Candida by using CLSI methods and interpretive criteria. J Clin Microbiol. 2014;52(9):3223-3229.

8 Arendrup MC, Perlin DS. Echinocandin resistance: an emerging clinical problem? Curr Opin Infect Dis. 2014;27(6):484-492.

9 Garcia-Effron G, Lee S, Park S, Cleary JD, Perlin DS. Effect of Candida glabrata FKS1 and FKS2 mutations on echinocandin sensitivity and kinetics of 1,3-beta-D-glucan synthase: implication for the existing susceptibility breakpoint. Antimicrob Agents Chemother. 2009;53(9):3690-3699.

10 Arendrup MC, Perlin DS, Jensen RH, Howard SJ, Goodwin J, Hope W. Differential in vivo activities of anidulafungin, caspofungin, and micafungin against Candida glabrata with and without FKS resistance mutations. Antimicrob Agents Chemother. 2012;56(5):2435-2442.

11 CLSI. Principles and Procedures for the Development of Epidemiological Cutoff Values for Antifungal Susceptibility Testing. 1st ed. CLSI guideline M57. Wayne, PA: Clinical and Laboratory Standards Institute; 2016.

12 CLSI. Epidemiological Cutoff Values for Antifungal Susceptibility Testing. 1st ed. CLSI supplement M59. Wayne, PA: Clinical and Laboratory Standards Institute; 2016.

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Table 2. Solvents and Diluents for Preparing Stock Antifungal Agent Solutions for Broth Dilution Testing

Antifungal Agent

Solvent^*,†

(Full-Strength and Intermediate Solutions)

Diluent

(Final Concentration)

Amphotericin B DMSO Medium

Anidulafungin DMSO Medium

Caspofungin DMSO Medium

Fluconazole DMSO Medium

Flucytosine DMSO Medium

Isavuconazole DMSO Medium

Itraconazole DMSO Medium

Ketoconazole DMSO Medium

Micafungin DMSO Medium

Posaconazole DMSO Medium

Voriconazole DMSO Medium

* DMSO can be toxic and also allows other drugs to be absorbed through the skin. Before using DMSO, consult the DMSO safety data sheet.

† Follow the manufacturer’s recommendations when selecting a solvent.

Abbreviation: DMSO, dimethyl sulfoxide.

NOTE: Information in boldface type is new or modified since the previous edition.

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Table 3. Recommended 48-Hour Minimal Inhibitory Concentration Limits for Two Quality Control and Four Reference Strains for Broth Macrodilution Procedures

^1,2

Organism Purpose Antifungal Agent

MIC Range, µg/mL

% of MICs Within Range Candida

parapsilosis ATCC^®* 22019

QC Amphotericin B

Fluconazole Itraconazole Ketoconazole Flucytosine

0.25–1 2–8 0.06–0.25 0.06–0.25 0.12–0.5

99.1 99.1 99 99 98.6 Candida krusei

ATCC^® 6258

QC Amphotericin B

Fluconazole Itraconazole Ketoconazole Flucytosine

0.25–2 16–64 0.12–0.5 0.12–0.5 4–16

99.5 99.1 94 100 96.8 Candida albicans

ATCC^® 90028

Reference Amphotericin B Fluconazole Flucytosine

0.5–2 0.25–1

0.5–2

91.9 97.3 95 C. albicans ATCC^®

24433

0.25–1 0.25–1 1–4

99.5 95.9 91.9 C. parapsilosis

ATCC^® 90018

0.5–2 0.25–1

0.12–0.25

96.4 98.2 99.5 Candida tropicalis

ATCC^® 750

0.5–2 1–4

0.12–0.25

93.7 95.5 99.5

* ATCC^® is a registered trademark of the American Type Culture Collection.

Abbreviations: ATCC^®, American Type Culture Collection; MIC, minimal inhibitory concentration; QC, quality control.

1 Pfaller MA, Bale M, Buschelman B, et al. Quality control guidelines for National Committee for Clinical Laboratory Standards recommended broth macrodilution testing of amphotericin B, fluconazole, and flucytosine. J Clin Microbiol. 1995;33(5):1104-1107.

2 Rex JH, Pfaller MA, Lancaster M, Odds FC, Bolmström A, Rinaldi MG. Quality control guidelines for National Committee for Clinical Laboratory Standards–recommended broth macrodilution testing of ketoconazole and itraconazole. J Clin Microbiol. 1996;34(4):816-817.

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Table 4. Recommended 24-Hour Minimal Inhibitory Concentration Limits for Two Quality Control Strains for Broth Microdilution Procedures

^1,2

Organism Antifungal Agent

MIC Range,

µg/mL Mode

% of MICs Within Range Candida parapsilosis

ATCC^®* 22019

Amphotericin B 0.25–2 0.5 97.1

Anidulafungin 0.25–2 1 95

Caspofungin 0.25–1 0.5 96.7

Fluconazole 0.5–4 2 98.2

Flucytosine 0.06–0.25 0.12 99.2

Isavuconazole 0.015–0.06 0.06 90.5

Itraconazole 0.06–0.5 0.25 95.8

Ketoconazole 0.03–0.25 0.06/0.12 97.5

Micafungin 0.5–2 1 100

Posaconazole 0.03–0.25 0.12 96.7

Voriconazole 0.016–0.12 0.06 100

Candida krusei ATCC^® 6258

Amphotericin B 0.5–2 1 100

Anidulafungin 0.03–0.12 0.06 97.9

Caspofungin^† 0.12–1^† 0.5^† 98.8

Fluconazole 8–64 16 100

Flucytosine 4–16 8 97.5

Isavuconazole 0.06–0.5 0.25 95.2

Itraconazole 0.12–1 0.5 95.8

Ketoconazole 0.12–1 0.5 95.4

Micafungin 0.12–0.5 0.25 99.6

Posaconazole 0.06–0.5 0.25 100

Voriconazole 0.06–0.5 0.25 98.3

* ATCC^® is a registered trademark of the American Type Culture Collection.

† The QC ranges were established using data generated in 2010 from 15 reference laboratories. Since then, caspofungin susceptibility testing has been associated with significant variation. Therefore, misclassification of susceptible isolates may occur despite acceptable performance of the QC strains according to the range above (see CLSI document M27³).^4-6

Abbreviations: ATCC^®, American Type Culture Collection; MIC, minimal inhibitory concentration; QC, quality control.

NOTE 1: Information in boldface type is new or modified since the previous edition.

NOTE 2: The MIC for anidulafungin, caspofungin, and micafungin is the lowest concentration at which a score of 2 (prominent decrease in turbidity [>50% inhibition of growth compared with the growth control]; see CLSI document M27³) is observed after 24 hours of incubation.

NOTE 3: The 24-hour MIC QC ranges in boldface type were adopted during a Subcommittee on Antifungal Susceptibility Tests Web conference held January 11, 2014. These ranges are considered tentative for one year from the publication date of M60 and are open for comment.

NOTE 4: When a commercial test system is used for susceptibility testing, refer to the manufacturer’s instructions for QC test recommendations and QC ranges.

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1 Barry AL, Pfaller MA, Brown SD, et al. Quality control limits for broth microdilution susceptibility tests of ten antifungal agents. J Clin Microbiol. 2000;38(9):3457-3459.

2 Krisher K, Brown SD, Traczewski MM. Quality control parameters for broth microdilution tests of anidulafungin. J Clin Microbiol. 2004;42(1):490.

3 CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 4th ed. CLSI standard M27. Wayne, PA: Clinical and Laboratory Standards Institute; 2017.

4 Shields RK, Nguyen MH, Press EG, et al. The presence of an FKS mutation rather than MIC is an independent risk factor for failure of echinocandin therapy among patients with invasive candidiasis due to Candida glabrata. Antimicrob Agents Chemother. 2012;56(9):4862-4869.

5 Arendrup MC, Pfaller MA; Danish Fungaemia Study Group. Caspofungin Etest susceptibility testing of Candida species: risk of misclassification of susceptible isolates of C. glabrata and C. krusei when adopting the revised CLSI caspofungin breakpoints. Antimicrob Agents Chemother. 2012;56(7):3965- 3968.

6 Espinel-Ingroff A, Arendrup MC, Pfaller MA, et al. Interlaboratory variability of caspofungin MICs for Candida spp. using CLSI and EUCAST methods: should the clinical laboratory be testing this agent? Antimicrob Agents Chemother. 2013:57(12):5836-5842.

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8

Table 5. Zone Diameter and Equivalent Minimal Inhibitory Concentration Breakpoints for Select Antifungal Agents Against Candida spp. After 24-Hour Incubation

^1-9

Antifungal

Agent Species

Zone Diameter Breakpoints and

Interpretive Categories, mm^* Equivalent MIC Breakpoints and Interpretive Categories, µg/mL

S I SDD R S I SDD R

Caspofungin

C. albicans ≥17 15–16 – ≤14 ≤0.25 0.5 – ≥1

C. glabrata – – – – ≤0.12 0.25 – ≥0.5

C.

guilliermondii ≥13 11–12 – ≤10 ≤2 4 – ≥8

C. krusei ≥17 15–16 – ≤14 ≤0.25 0.5 – >1

C. parapsilosis ≥13 11–12 – ≤10 ≤2 4 – ≥8

C. tropicalis ≥17 15–16 – ≤14 ≤0.25 0.5 – >1

Micafungin

C. albicans ≥22 20–21 – ≤19 ≤0.25 0.5 – ≥1

C. glabrata^† ≥30 28–29 – ≤27 ≤0.06 0.12 – ≥0.25

C.

guilliermondii ≥16 14–15 – ≤13 ≤2 4 – ≥8

C. krusei ≥22 20–21 – ≤19 ≤0.25 0.5 – ≥1

C. parapsilosis ≥ 16 14–15 – ≤13 ≤2 4 – ≥8

C. tropicalis ≥22 20–21 – ≤19 ≤0.25 0.5 – ≥1

Voriconazole^‡

C. albicans ≥17 15–16 – ≤14 ≤0.12 0.25–0.5 – ≥1

C. glabrata^§ – – – – – – – –

C. krusei ≥15 13–14 – ≤12 ≤0.5 1 – ≥2

C. parapsilosis ≥17 15–16 – ≤14 ≤0.12 0.25–0.5 – ≥1

C. tropicalis ≥17 15–16 – ≤14 ≤0.12 0.25–0.5 – ≥1

Fluconazole

C. albicans ≥17 – 14–16 ≤13 ≤2 – 4 ≥8

C. glabrata – – ≥15 ≤14 – – ≤32 ≥64

C. krusei^¶ – – – – – – – –

C. parapsilosis ≥17 – 14–16 ≤13 ≤2 – 4 ≥8

C. tropicalis ≥17 – 14–16 ≤13 ≤2 – 4 ≥8

* The zone diameter interpretive categories for micafungin, caspofungin, and voriconazole were adopted at a meeting of the Subcommittee on Antifungal Susceptibility Tests held January 8, 2011. The zone diameter interpretive categories for fluconazole in boldface were adopted at a meeting of the Subcommittee on Antifungal Susceptibility Tests held June 12, 2010. These zone diameters and interpretive categories are considered tentative for one year from the publication date of M60 and are open for comment.

† Zone diameter interpretive categories for C. glabrata and micafungin were adopted during an electronic vote by the Subcommittee on Antifungal Susceptibility Tests held in March 2017. The zone diameters are considered tentative for one year from the publication of M60 and are open for comment.

‡ Breakpoints may also be used for 48-hour readings if the 24-hour growth control shows insufficient growth.

§ For C. glabrata and voriconazole, current data are insufficient to demonstrate a correlation between in vitro susceptibility testing and clinical outcome.

¶ Isolates of C. krusei are assumed to be intrinsically resistant to fluconazole and their MICs should not be interpreted using this scale.

Abbreviations: I, intermediate; MIC, minimal inhibitory concentration; R, resistant; S, susceptible; SDD, susceptible-dose dependent.

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1 Arendrup MC, Park S, Brown S, Pfaller M, Perlin DS. Evaluation of CLSI M44-A2 disk diffusion and associated breakpoint testing of caspofungin and micafungin using a well-characterized panel of wild-type and fks hot spot mutant Candida isolates. Antimicrob Agents Chemother.

2011;55(5):1891-1895.

2 Brown SD, Tracezewski MM. Caspofungin disk diffusion breakpoints and quality control. J Clin Microbiol. 2008;46(6):1927-1929.

3 Pfaller MA, Diekema DJ, Ostrosky-Zeichner L, et al. Correlation of MIC with outcome for Candida species tested against caspofungin, anidulafungin, and micafungin: analysis and proposal for interpretive MIC breakpoints. J Clin Microbiol. 2008;46(8):2620-2629.

4 Pfaller MA, Andes D, Arendrup MC, et al. Clinical breakpoints for voriconazole and Candida spp. revisited: review of microbiologic, molecular, pharmacodynamic, and clinical data as they pertain to the development of species-specific interpretive criteria. Diagn Microbiol Infect Dis.

2011;70(3):330-343.

5 Pfaller MA, Diekema DJ, Rex JH, et al. Correlation of MIC with outcome for Candida species tested against voriconazole: analysis and proposal for interpretive breakpoints. J Clin Microbiol.

2006;44(3):819-826.

6 Barry AL, Pfaller MA, Rennie RP, Fuchs PC, Brown SD. Precision and accuracy of fluconazole susceptibility testing by broth microdilution, Etest, and disk diffusion methods. Antimicrob Agents Chemother. 2002;46(6):1781-1784.

7 Pfaller MA, Diekema DJ, Sheehan DJ. Interpretive breakpoints for fluconazole and Candida revisited:

a blueprint for the future of antifungal susceptibility testing. Clin Microbiol Rev. 2006;19(2):435-447.

8 Pfaller MA, Hazen KC, Messer SA, et al. Comparison of results of fluconazole disk diffusion testing for Candida species with results from a central reference laboratory in the ARTEMIS global antifungal surveillance program. J Clin Microbiol. 2004;42(8):3607-3612.

9 Rex JH, Pfaller MA, Galgiani JN, et al.; Subcommittee on Antifungal Susceptibility Testing of the National Committee for Clinical Laboratory Standards. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole, and Candida infections. Clin Infect Dis. 1997;24(2):235-247.

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10

Table 6. Recommended Quality Control Zone Diameter (mm) Ranges After 24-Hour Incubation

^1-5

Antifungal Agent

Disk Content,

µg

Candida albicans ATCC^® 90028

Candida parapsilosis ATCC^® 22019

Candida tropicalis ATCC^® 750

Candida krusei ATCC^® 6258

Caspofungin 5 18–27 14–23 20–27 19–26

Fluconazole 25 28–39 22–33 26–37 –^†

Micafungin^‡ 10 24–31 14–23 24–30 23–29

Posaconazole 5 24–34 25–36 23–33 23–31

Voriconazole 1 31–42 28–37 –^† 16–25

 ATCC^® is a registered trademark of the American Type Culture Collection.

† QC ranges have not been established for these fungal strains and antimicrobial agent combinations due to their extensive interlaboratory variation during initial QC studies.

‡ The zone diameter QC ranges in boldface were adopted at a meeting of the Subcommittee on Antifungal Susceptibility Tests held January 23, 2010. These zone diameters and interpretative categories are considered tentative for one year from the publication date of M60 and are open for comment.

Abbreviations: ATCC^®, American Type Culture Collection; QC, quality control.

1 Arendrup MC, Park S, Brown S, Pfaller M, Perlin DS. Evaluation of CLSI M44-A2 disk diffusion and associated breakpoint testing of caspofungin and micafungin using a well-characterized panel of wild- type and fks hot spot mutant Candida isolates. Antimicrob Agents Chemother. 2011;55(5):1891-1895.

2 Barry A, Bille J, Brown S, et al. Quality control limits for fluconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue. J Clin Microbiol. 2003;41(7):3410-3412.

3 Brown SD, Traczewski M. Caspofungin disk diffusion breakpoints and quality control. J Clin Microbiol. 2008;46(6):1927-1929.

4 Brown S, Traczewski M. Quality control limits for posaconazole disk susceptibility tests on Mueller- Hinton agar with glucose and methylene blue. J Clin Microbiol. 2007;45(1):222-223.

5 Pfaller MA, Barry A, Bille J, et al. Quality control limits for voriconazole disk susceptibility test on Mueller-Hinton agar with glucose and methylene blue. J Clin Microbiol. 2004;42(4):1716-1718.

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References

1 CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 4th ed. CLSI standard M27. Wayne, PA:

Clinical and Laboratory Standards Institute; 2017.

2 CLSI. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline—Second Edition. CLSI document M44-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.

3 CLSI. Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters. 4th ed. CLSI guideline M23.

Wayne, PA: Clinical and Laboratory Standards Institute; 2016.

4 CLSI. Verification of Commercial Microbial Identification and Antimicrobial Susceptibility Testing Systems. 1st ed. CLSI guideline M52. Wayne, PA: Clinical and Laboratory Standards Institute; 2015.

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12

The Quality Management System Approach

Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system (QMS) approach in the development of standards and guidelines that facilitates project management, defines a document structure using a template, and provides a process to identify needed documents. The QMS approach applies a core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any health care service’s path of workflow (ie, operational aspects that define how a particular product or service is provided). The QSEs provide the framework for delivery of any type of product or service, serving as a manager’s guide. The QSEs are:

Organization Personnel Process Management Nonconforming Event Management

Customer Focus Purchasing and Inventory Documents and Records Assessments

Facilities and Safety Equipment Information Management Continual Improvement

M60 covers the QSE indicated by an “X.” For a description of the documents listed in the grid, please refer to the Related CLSI Reference Materials section.

Organization Customer Focus Facilities and Safety Personnel Purchasing and Inventory Equipment Process Management Documents and Records Information Management Nonconforming Event Management Assessments Continual Improvement

X M23 M27 M44 M52 M57 M59

Path of Workflow

A path of workflow is the description of the necessary processes to deliver the particular product or service that the organization or entity provides. A laboratory path of workflow consists of the sequential processes: preexamination, examination, and postexamination and their respective sequential subprocesses. All laboratories follow these processes to deliver their services, namely quality laboratory information.

M60 covers the medical laboratory path of workflow processes indicated by an “X.” For a description of the other documents listed in the grid, please refer to the Related CLSI Reference Materials section.

Preexamination Examination Postexamination

Examination ordering Sample collection Sample transport Sample receipt and processing Examination Results review and follow-up Interpretation Results reporting and archiving Sample management

X X X

M27 M27 M27 M27 M27

M44 M44 M44

M59 M59 M59

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Related CLSI Reference Materials

^

M23 Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters. 4th ed., 2016.

This guideline discusses the necessary and recommended data for the selection of appropriate interpretive criteria and quality control ranges for antimicrobial agents.

M27 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 4th ed., 2017. This standard covers antifungal agent selection and preparation; test procedure implementation and interpretation; and quality control requirements for susceptibility testing of yeasts that cause invasive fungal infections.

M44 Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts. 2nd ed., 2009. This document provides newly established methodology for disk diffusion testing of Candida spp., criteria for quality control testing, and interpretive criteria.

M52 Verification of Commercial Microbial Identification and Antimicrobial Susceptibility Testing Systems.

1st ed., 2015. This guideline includes recommendations for verification of commercial US Food and Drug Administration–cleared microbial identification and antimicrobial susceptibility testing systems by clinical laboratory professionals to fulfill regulatory or quality assurance requirements for the use of these systems for diagnostic testing.

M57 Principles and Procedures for the Development of Epidemiological Cutoff Values for Antifungal Susceptibility Testing. 1st ed., 2016. This guideline includes the criteria for developing and using epidemiological cutoff values for guiding clinical decisions when testing fungal species and antifungal agent combinations for which there are no breakpoints.

M59 Epidemiological Cutoff Values for Antifungal Susceptibility Testing. 1st ed., 2016. This document includes the epidemiological cutoff value and quality control tables developed according to criteria provided in the Clinical and Laboratory Standards Institute guideline M57.

 CLSI documents are continually reviewed and revised through the CLSI consensus process; therefore, readers should refer to the most current editions.

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14

NOTES

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ELECTRONIC ISBN 1-56238-829-0

CLSI Performance Standards for Antifungal Susceptibility Testing of Yeasts

M60

Performance Standards for Antifungal Susceptibility Testing of Yeasts

Clinical and Laboratory Standards Institute

Performance Standards for Antifungal Susceptibility Testing of Yeasts

Abstract

Committee Membership

Contents

Foreword

Abbreviations and Acronyms

Table 1. Minimal Inhibitory Concentration Breakpoints for In Vitro Broth Dilution Susceptibility Testing of Candida spp. and Select Antifungal Agents After 24-Hour Incubation

Table 2. Solvents and Diluents for Preparing Stock Antifungal Agent Solutions for Broth Dilution Testing

Table 3. Recommended 48-Hour Minimal Inhibitory Concentration Limits for Two Quality Control and Four Reference Strains for Broth Macrodilution Procedures

Table 4. Recommended 24-Hour Minimal Inhibitory Concentration Limits for Two Quality Control Strains for Broth Microdilution Procedures

Table 5. Zone Diameter and Equivalent Minimal Inhibitory Concentration Breakpoints for Select Antifungal Agents Against Candida spp. After 24-Hour Incubation

Table 6. Recommended Quality Control Zone Diameter (mm) Ranges After 24-Hour Incubation

References

The Quality Management System Approach

Related CLSI Reference Materials

For more information, visit www.clsi.org today.

Explore the Latest Offerings From CLSI!

**Table 1. Minimal Inhibitory Concentration Breakpoints for In Vitro Broth Dilution Susceptibility Testing of Candida spp. and Select Antifungal Agents After 24-Hour Incubation**