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M44-A Vol. 24 No. 15 Replaces M44-P Vol. 23 No. 6

Method for Antifungal Disk Diffusion

Susceptibility Testing of Yeasts; Approved Guideline

This document provides newly established methodology for disk diffusion testing of Candida spp., criteria for quality control testing, and interpretive criteria.

A guideline for global application developed through the NCCLS consensus process

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NCCLS...

Serving the World’s Medical Science Community Through Voluntary Consensus

NCCLS is an international, interdisciplinary, nonprofit, standards-developing, and educational organization that promotes the development and use of voluntary consensus standards and guidelines within the healthcare community. It is recognized worldwide for the application of its unique consensus process in the development of standards and guidelines for patient testing and related healthcare issues. NCCLS is based on the principle that consensus is an effective and cost- effective way to improve patient testing and healthcare services.

In addition to developing and promoting the use of voluntary consensus standards and guidelines, NCCLS provides an open and unbiased forum to address critical issues affecting the quality of patient testing and health care.

PUBLICATIONS

An NCCLS document is published as a standard, guideline, or committee report.

Standard A document developed through the consensus process that clearly identifies specific, essential requirements for materials, methods, or practices for use in an unmodified form. A standard may, in addition, contain discretionary elements, which are clearly identified.

Guideline A document developed through the consensus process describing criteria for a general operating practice, procedure, or material for voluntary use. A guideline may be used as written or modified by the user to fit specific needs.

Report A document that has not been subjected to consensus review and is released by the Board of Directors.

CONSENSUS PROCESS

The NCCLS voluntary consensus process is a protocol establishing formal criteria for:

• the authorization of a project

• the development and open review of documents

• the revision of documents in response to comments by users

• the acceptance of a document as a consensus standard or guideline.

Most NCCLS documents are subject to two levels of consensus—“proposed” and “approved.” Depending on

the need for field evaluation or data collection, documents may also be made available for review at an intermediate (i.e., “tentative”) consensus level.

Proposed An NCCLS consensus document undergoes the first stage of review by the healthcare community as a proposed standard or guideline. The document should receive a wide and thorough technical review, including an overall review of its scope, approach, and utility, and a line- by-line review of its technical and editorial content.

Tentative A tentative standard or guideline is made available for review and comment only when a recommended method has a well-defined need for a field evaluation or when a recommended protocol requires that specific data be collected. It should be reviewed to ensure its utility.

Approved An approved standard or guideline has achieved consensus within the healthcare community. It should be reviewed to assess the utility of the final document, to ensure attainment of consensus (i.e., that comments on earlier versions have been satisfactorily addressed), and to identify the need for additional consensus documents.

NCCLS standards and guidelines represent a consensus opinion on good practices and reflect the substantial agreement by materially affected, competent, and interested parties obtained by following NCCLS’s established consensus procedures. Provisions in NCCLS standards and guidelines may be more or less stringent than applicable regulations. Consequently, conformance to this voluntary consensus document does not relieve the user of responsibility for compliance with applicable regulations.

COMMENTS

The comments of users are essential to the consensus process. Anyone may submit a comment, and all comments are addressed, according to the consensus process, by the NCCLS committee that wrote the document. All comments, including those that result in a change to the document when published at the next consensus level and those that do not result in a change, are responded to by the committee in an appendix to the document. Readers are strongly encouraged to comment in any form and at any time on any NCCLS document. Address comments to the NCCLS Executive Offices, 940 West Valley Road, Suite 1400, Wayne, PA 19087, USA.

VOLUNTEER PARTICIPATION

Healthcare professionals in all specialties are urged to volunteer for participation in NCCLS projects. Please contact the NCCLS Executive Offices for additional information on committee participation.

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M44-A ISBN 1-56238-532-1

Volume 24 Number 15

ISSN 0273-3099

Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts;

Approved Guideline

Daniel J. Sheehan, Ph.D.

Steven D. Brown, Ph.D.

Michael A. Pfaller, M.D.

David W. Warnock, Ph.D., FRCPath.

John H. Rex, M.D., FACP Vishnu Chaturvedi, Ph.D.

Ana Espinel-Ingroff, Ph.D.

Mahmoud A. Ghannoum, M.Sc., Ph.D.

Lynn Steele Moore, M.T.

Frank C. Odds, Ph.D., FRCPath.

Michael G. Rinaldi, Ph.D.

Thomas J. Walsh, M.D.

Abstract

NCCLS broth dilution reference methods are available for susceptibility testing of yeasts (see NCCLS document M27—

Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts) and moulds (see NCCLS document M38—

Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi). There still remains, however, a need for an alternative simple, rapid, and cost-effective approach to determine susceptibility of fungal organisms to various classes of antifungal agents that would make antifungal susceptibility testing more readily available to the clinical microbiology laboratory. The NCCLS Subcommittee on Antifungal Susceptibility Testing has developed a disk diffusion method for testing Candida species to fluconazole and voriconazole. Zone interpretive criteria (breakpoints) have been approved for fluconazole as well as quality control parameters for both fluconazole and voriconazole. One significant advantage of this method is that qualitative results can usually be determined after only 20 to 24 hours incubation as opposed to 48 hours with NCCLS document M27. There are currently more than ten systemically active antifungal agents and it is expected that this document will further encourage the development of disk diffusion testing for at least some of these agents.

NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline. NCCLS document M44-A (ISBN 1-56238-532-1). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.

THE NCCLS consensus process, which is the mechanism for moving a document through two or more levels of review by the healthcare 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 NCCLS documents. Current editions are listed in the NCCLS Catalog, which is distributed to member organizations, and to nonmembers on request. If your organization is not a member and would like to become one, and to request a copy of the NCCLS Catalog, contact the NCCLS Executive Offices. Telephone: 610.688.0100;

Fax: 610.688.0700; E-Mail: [email protected]; Website: www.nccls.org

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Number 15 NCCLS

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This publication is protected by copyright. No part of it may be reproduced, stored in a retrieval system, transmitted, or made available in any form or by any means (electronic, mechanical, photocopying, recording, or otherwise) without prior written permission from NCCLS, except as stated below.

NCCLS hereby grants permission to reproduce limited portions of this publication for use in laboratory procedure manuals at a single site, for interlibrary loan, or for use in educational programs provided that multiple copies of such reproduction shall include the following notice, be distributed without charge, and, in no event, contain more than 20% of the document’s text.

Reproduced with permission, from NCCLS publication M44-A—Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline (ISBN 1-56238-532- 1). Copies of the current edition may be obtained from NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.

Permission to reproduce or otherwise use the text of this document to an extent that exceeds the exemptions granted here or under the Copyright Law must be obtained from NCCLS by written request.

To request such permission, address inquiries to the Executive Director, NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.

Suggested Citation

(NCCLS. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline.

NCCLS document M44-A [ISBN 1-56238-532-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2004.)

Proposed Guideline April 2003

Approved Guideline May 2004

ISBN 1-56238-532-1 ISSN 0273-3099

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Committee Membership

Area Committee on Microbiology

Mary Jane Ferraro, Ph.D., M.P.H.

Chairholder

Massachusetts General Hospital Boston, Massachusetts

James H. Jorgensen, Ph.D.

Vice-Chairholder

University of Texas Health Science Center

San Antonio, Texas Donald R. Callihan, Ph.D.

BD Diagnostic Systems Sparks, Maryland David L. Sewell, Ph.D.

Veterans Affairs Medical Center Portland, Oregon

Thomas R. Shryock, Ph.D.

Lilly Research Laboratories

Jana M. Swenson, M.M.Sc.

Centers for Disease Control and Prevention

Atlanta, Georgia Michael L. Wilson, M.D.

Denver Health Medical Center Denver, Colorado

Advisors

Ellen Jo Baron, Ph.D.

Stanford University Hospital &

Medical School Stanford, California Lynne S. Garcia, M.S.

LSG & Associates Santa Monica, California

Richard L. Hodinka, Ph.D.

Children’s Hospital of Philadelphia Philadelphia, Pennsylvania Michael A. Pfaller, M.D.

University of Iowa College of Medicine

Iowa City, Iowa Robert P. Rennie, Ph.D.

Provincial Laboratory for Public Health

Edmonton, AB, Canada Melvin P. Weinstein, M.D.

Robert Wood Johnson Medical School

New Brunswick, New Jersey Gail L. Woods, M.D.

ARUP Research Institute Salt Lake City, Utah

Subcommittee on Antifungal Susceptibility Tests

John H. Rex, M.D., FACP Chairholder

AstraZeneca

Cheshire, United Kingdom Vishnu Chaturvedi, Ph.D.

New York State Dept. of Health Albany, New York

Ana Espinel-Ingroff, Ph.D.

Medical College of Virginia/VCU Richmond, Virginia

Mahmoud A. Ghannoum, M.Sc., Ph.D.

Case Western Reserve University Cleveland, Ohio

Frank C. Odds, Ph.D., FRCPath.

University of Aberdeen Aberdeen, Scotland, United Kingdom

University of Iowa College of Medicine

Iowa City, Iowa

Michael G. Rinaldi, Ph.D.

University of Texas Health Science Center

San Antonio, Texas Daniel J. Sheehan, Ph.D.

Pfizer Inc

New York, New York

Lynn Steele-Moore, M.T.

FDA Center for Drug Evaluation &

Research

Rockville, Maryland Thomas J. Walsh, M.D.

National Cancer Institute Bethesda, Maryland David W. Warnock, Ph.D., FRCPath.

Atlanta, Georgia Advisor

Arthur L. Barry, Ph.D.

The Clinical Microbiology Institute Wilsonville, Oregon

Working Group on Antifungal Disk Diffusion Susceptibility Testing of Yeasts

Daniel J. Sheehan, Ph.D.

Chairholder Pfizer Inc

New York, New York Steven D. Brown, Ph.D.

The Clinical Microbiology Institute Wilsonville, Oregon

University of Iowa College of Medicine Iowa City, Iowa

David W. Warnock, Ph.D., FRCPath.

Atlanta, Georgia Staff

Tracy A. Dooley, M.L.T.(ASCP) Staff Liaison

NCCLS

Wayne, Pennsylvania

Donna M. Wilhelm Editor NCCLS

Wayne, Pennsylvania Melissa A. Lewis Assistant Editor NCCLS

Wayne, Pennsylvania

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Contents (Continued)

Table 1. Zone Diameter Interpretive Standards and Corresponding Minimal Inhibitory

Concentrations (MIC) Breakpoints for Candida spp. ...11

Table 2. Recommended Quality Control Zone Diameter (mm) Ranges...11

References...12

Appendix A. Preparation of Mueller-Hinton Agar + 2% Glucose and 0.5 µg/mL Methylene Blue Dye ...13

Appendix B. McFarland 0.5 Barium Sulfate Turbidity Standard ...15

Appendix C. Quality Control Protocol Flow Charts...16

Summary of Comments and Working Group Responses ...18

The Quality System Approach...20

Related NCCLS Publications...21

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Foreword

Due to the increased incidence of systemic fungal infections and number of antifungal agents available for systemic administration, antifungal susceptibility testing has gained greater recognition. Today, antifungal susceptibility testing has come of age in guiding physicians in the selection of antifungal therapy. Broth macrodilution and microdilution reference methods are now available for susceptibility testing of both yeasts (see NCCLS document M27—Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts) and moulds (see NCCLS document M38—Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi). A commercial broth microdilution panel has been cleared for clinical use by the FDA. To make antifungal susceptibility testing more readily available to clinical microbiology laboratories, there still remains a need for alternative, simple, rapid, and cost-effective approaches. Disk diffusion testing has served as such an example for antibacterial testing (see NCCLS document M2—Performance Standards for Antimicrobial Disk Susceptibility Tests) and, therefore, the NCCLS Subcommittee on Antifungal Susceptibility Tests has developed recommendations for disk diffusion testing for antifungal agents.

A disk diffusion method for testing Candida species against the triazoles (fluconazole and voriconazole) has been developed. This method often provides qualitative results 24 hours sooner than the standard method for testing yeasts used in NCCLS document M27—Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. In addition, the use of supplemented Mueller-Hinton agar in lieu of RPMI 1640 medium should make antifungal susceptibility testing more readily available to at least some clinical laboratories, and at reduced cost. Zone interpretive criteria (breakpoints) for fluconazole and quality control parameters for both fluconazole and voriconazole have been established according to standard NCCLS procedures. NCCLS expects that this document will encourage the development of disk diffusion testing for other antifungal agents and fungal genera.

A Note on Terminology

NCCLS, as a global leader in standardization, is firmly committed to achieving global harmonization wherever possible. Harmonization is a process of recognizing, understanding, and explaining differences while taking steps to achieve worldwide uniformity. NCCLS recognizes that medical conventions in the global metrological community have evolved differently in the United States, Europe, and elsewhere; that these differences are reflected in NCCLS, International Organization for Standardization (ISO), and European Committee for Standardization (CEN) documents; and that legally required use of terms, regional usage, and different consensus timelines are all obstacles to harmonization. In light of this, NCCLS recognizes that harmonization of terms facilitates the global application of standards and is an area of immediate attention. Implementation of this policy must be an evolutionary and educational process that begins with new projects and revisions of existing documents.

Of particular note in NCCLS document M44-A are two terms whereby NCCLS intends to eliminate confusion, over time, through its commitment to harmonization. For the most part, in this guideline, the term “accuracy” is used correctly in its metrological sense, to refer to the closeness of the agreement between the result of a (single) measurement and a true value of a measurand, thus comprising both random and systematic effects. But there are several instances in this document where accuracy is defined the way ISO defines “trueness,” i.e., the closeness of the agreement between the average value from a large series of measurements and true value of a measurand. To facilitate understanding, when used this way, “trueness” has been inserted parenthetically. Also, the terms are defined in the guideline’s Definitions section along with explanatory notes. During the next scheduled revision of this document, they will be reviewed for consistency with international use, and revised appropriately.

Key Words

Antifungal, antimicrobial, disk, disk diffusion, Kirby-Bauer method, susceptibility testing

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Volume 24 M44-A

Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts;

Approved Guideline 1 Scope

With a need to make antifungal susceptibility testing more readily available to the clinical laboratory, NCCLS document M44—Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts provides an established methodology for disk diffusion testing of Candida spp., zone interpretive criteria for fluconazole, and recommended quality control ranges for fluconazole and voriconazole.

2 Introduction

The method described in this document is intended for testing Candida species. This method does not currently encompass any other genera and has not been used in studies of the yeast form of dimorphic fungi, such as Blastomyces dermatitidis or Histoplasma capsulatum. Moreover, testing of filamentous fungi (i.e., moulds) is not addressed in the current procedure.

The method described herein must be followed exactly to obtain reproducible results. When new problems are recognized or improvements in these criteria are developed, changes will be incorporated into future editions of this guideline and also distributed in periodic informational supplements.

3 Standard Precautions

Because it is often impossible to know what might be infectious, all human specimens are to be treated as infectious and handled according to “standard precautions.” Standard precautions are guidelines that combine the major features of “universal precautions” and “body substance isolation” practices. Standard precautions cover the transmission of any pathogen and thus are more comprehensive than universal precautions, which are intended to apply only to transmission of blood-borne pathogens. Standard precaution and universal precaution guidelines are available from the U.S. Centers for Disease Control and Prevention (Guideline for Isolation Precautions in Hospitals. Infection Control and Hospital Epidemiology. CDC. 1996;Vol 17;1:53-80), (MMWR 1987;36[suppl 2S]2S-18S), and (MMWR 1988;37:377-382, 387-388). For specific precautions for preventing the laboratory transmission of blood- borne infection from laboratory instruments and materials and for recommendations for the management of blood-borne exposure, refer to the most current edition of NCCLS document M29—Protection of Laboratory Workers from Occupationally Acquired Infections.

4 Definitions

Antimicrobial Susceptibility Test Interpretive Category – 1) A classification based on an in vitro response of an organism to an antimicrobial agent at levels of that agent corresponding to blood or tissue levels attainable with usually prescribed doses of that agent; 2) Susceptible Antimicrobial Susceptibility Test Interpretive Category – A category that implies that an infection due to the isolate may be appropriately treated with the dosage of an antimicrobial agent recommended for that type of infection and infecting species, unless otherwise contraindicated; 3) Susceptible-Dose Dependent (S-DD) Antimicrobial Susceptibility Test Interpretive Category – A category that includes isolates with antimicrobial agent MICs that approach usually attainable blood and tissue levels and for which response rates may be lower than for susceptible isolates; 4) Resistant Antimicrobial Susceptibility Test Interpretive Category – Resistant isolates that are not inhibited by the usually achievable concentrations of the agent with normal dosage schedules or where clinical efficacy has not been reliable in treatment studies.