4th Edition

M27
Reference Method for Broth Dilution
Antifungal Susceptibility Testing of Yeasts

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.

A standard for global application developed through the Clinical and Laboratory Standards Institute consensus process.

Subcommittee on Antifungal Susceptibility Tests

This document is protected by copyright.
 Clinical and Laboratory Standards Institute
Setting the standard for quality in medical laboratory testing around the world.

The Clinical and Laboratory Standards Institute (CLSI) is a not-for-profit membership organization that brings
together the varied perspectives and expertise of the worldwide laboratory community for the advancement of a
common cause: to foster excellence in laboratory medicine by developing and implementing medical laboratory
standards and guidelines that help laboratories fulfill their responsibilities with efficiency, effectiveness, and global
applicability.

Consensus Process

Consensus—the substantial agreement by materially affected, competent, and interested parties—is core to the
development of all CLSI documents. It does not always connote unanimous agreement but does mean that the
participants in the development of a consensus document have considered and resolved all relevant objections
and accept the resulting agreement.

Commenting on Documents

CLSI documents undergo periodic evaluation and modification to keep pace with advances in technologies,
procedures, methods, and protocols affecting the laboratory or health care.

CLSI’s consensus process depends on experts who volunteer to serve as contributing authors and/or as participants
in the reviewing and commenting process. At the end of each comment period, the committee that developed
the document is obligated to review all comments, respond in writing to all substantive comments, and revise the
draft document as appropriate.

Comments on published CLSI documents are equally essential and may be submitted by anyone, at any time, on
any document. All comments are managed according to the consensus process by a committee of experts.

Appeal Process

When it is believed that an objection has not been adequately considered and responded to, the process for
appeal, documented in the CLSI Standards Development Policies and Processes, is followed.

All comments and responses submitted on draft and published documents are retained on file at CLSI and are
available upon request.

Get Involved—Volunteer!
Do you use CLSI documents in your workplace? Do you see room for improvement? Would you like to get
involved in the revision process? Or maybe you see a need to develop a new document for an emerging
technology? CLSI wants to hear from you. We are always looking for volunteers. By donating your time and talents
to improve the standards that affect your own work, you will play an active role in improving public health across
the globe.

For additional information on committee participation or to submit comments, contact CLSI.

Clinical and Laboratory Standards Institute

950 West Valley Road, Suite 2500
Wayne, PA 19087 USA
P: +1.610.688.0100
F: +1.610.688.0700
www.clsi.org
[email protected]
Subcommittee on Antifungal Susceptibility Tests
This document is protected by copyright.
 M27, 4th ed.
November 2017
Replaces M27-A3
Reference Method for Broth Dilution 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)
Mahmoud A. Ghannoum, PhD, EMBA, FIDSA
Kimberly E. Hanson, MD, MHS
Denise Holliday, MT(ASCP)
Nicole M. Holliday, BA
Laura Kovanda, PhD
Shawn R. Lockhart, PhD, D(ABMM)
Luis Ostrosky-Zeichner, MD, FACP, FIDSA, FSHEA
Audrey N. Schuetz, MD, MPH, D(ABMM)
Nathan P. Wiederhold, PharmD
Adrian M. Zelazny, PhD, D(ABMM)

Abstract
Clinical and Laboratory Standards Institute standard M27—Reference Method for Broth Dilution Antifungal Susceptibility Testing
of Yeasts describes a method for testing the susceptibility to antifungal agents of yeasts that cause invasive fungal infections,
including Candida spp. and Cryptococcus neoformans. Selection and preparation of antifungal agents, implementation and
interpretation of test procedures, and the purpose and implementation of QC procedures are discussed. A careful examination of
the responsibilities of the manufacturer and the user in QC is also presented.

Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of
Yeasts. 4th ed. CLSI standard M27 (ISBN 1-56238-826-6 [Print]; ISBN 1-56238-827-4 [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.

Subcommittee on Antifungal Susceptibility Tests

This document is protected by copyright.
 M27, 4th ed.

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

Previous Editions:
December 1992, October 1995, June 1997, August 2002, April 2008

ISBN 1-56238-826-6 (Print)

ISBN 1-56238-827-4 (Electronic)
ISSN 1558-6502 (Print)
ISSN 2162-2914 (Electronic) Volume 37, Number 13
Subcommittee
ii on Antifungal Susceptibility Tests
This document is protected by copyright.
 M27, 4th ed.

Committee Membership

Consensus Council
Carl D. Mottram, RRT, RPFT, Karen W. Dyer, MT(ASCP), DLM Ross J. Molinaro, PhD, MLS(ASCP)CM,
FAARC Centers for Medicare & Medicaid DABCC, FACB
Chairholder Services Siemens Healthcare Diagnostics, Inc.
Mayo Clinic USA USA
USA
Thomas R. Fritsche, MD, PhD, FCAP, Joseph Passarelli
Dennis J. Ernst, MT(ASCP), FIDSA Roche Diagnostics Corporation
NCPT(NCCT) Marshfield Clinic USA
Vice-Chairholder USA
Center for Phlebotomy Education Andrew Quintenz
USA Mary Lou Gantzer, PhD, FACB Bio-Rad Laboratories, Inc.
BioCore Diagnostics USA
J. Rex Astles, PhD, FACB, DABCC USA
Centers for Disease Control and Robert Rej, PhD
Prevention Loralie J. Langman, PhD, DABCC, New York State Department of Health –
USA FACB, F-ABFT Wadsworth Center
Mayo Clinic USA
Lucia M. Berte, MA, MT(ASCP)SBB, USA
DLM, CQA(ASQ)CMQ/OE Zivana Tezak, PhD
Laboratories Made Better! FDA Center for Devices and
USA Radiological Health
USA

Subcommittee on Antifungal Susceptibility Tests

Barbara D. Alexander, MD, MHS Mahmoud A. Ghannoum, PhD, EMBA, Luis Ostrosky-Zeichner, MD, FACP,
Chairholder FIDSA FIDSA, FSHEA
Duke Univeristy Medical Center Case Western Reserve University Memorial Hermann Healthcare System
USA USA USA

Gary W. Procop, MD, MS Kimberly E. Hanson, MD, MHS Audrey N. Schuetz, MD, MPH,
Vice-Chairholder University of Utah and ARUP D(ABMM)
Cleveland Clinic Laboratories Mayo Clinic
USA USA USA

Denise Holliday, MT(ASCP) Nathan P. Wiederhold, PharmD

Philippe Dufresne, PhD (RMCCM)
BD Diagnostic Systems University of Texas Health Science
Institut national de santé publique du
USA Center at San Antonio
Québec
USA
Canada
Nicole M. Holliday, BA
Thermo Fisher Scientific Adrian M. Zelazny, PhD, D(ABMM)
Jeff Fuller, PhD, FCCM, D(ABMM)
USA USA
London Health Sciences Centre
Canada

Staff

Clinical and Laboratory Standards Megan L. Tertel, MA, ELS Kristy L. Leirer, MS
Institute Editorial Manager Editor
USA
Catherine E.M. Jenkins Laura Martin
Marcy L. Hackenbrack, MCM, Editor Editor
M(ASCP)
Project Manager

Subcommittee on Antifungal Susceptibility Tests iii

This document is protected by copyright.
 M27, 4th ed.

Acknowledgment for the Expert Panel on Microbiology

CLSI, the Consensus Council, and the Subcommittee on Antifungal Susceptibility Tests gratefully
acknowledge the Expert Panel on Microbiology for serving as technical advisors and subject matter experts
during the development of this standard.

Expert Panel on Microbiology

Richard B. Thomson, Jr., PhD, Carey-Ann Burnham, PhD, D(ABMM) David H. Pincus, MS, RM/SM(NRCM),
D(ABMM), FAAM Washington University School of SM(ASCP)
Chairholder Medicine bioMérieux, Inc.
Evanston Hospital, NorthShore USA USA
University HealthSystem
USA German Esparza, BSc Audrey N. Schuetz, MD, MPH,
Proasecal LTD D(ABMM)
Mary Jane Ferraro, PhD, MPH Colombia Mayo Clinic
Vice-Chairholder USA
Massachusetts General Hospital Mark G. Papich, DVM, MS
USA College of Veterinary Medicine, Ribhi M. Shawar, PhD, D(ABMM)
North Carolina State University FDA Center for Devices and
Lynette Y. Berkeley, PhD, MT(ASCP) USA Radiological Health
FDA Center for Drug Evaluation and USA
Research Jean B. Patel, PhD, D(ABMM)
USA Centers for Disease Control and Barbara L. Zimmer, PhD
Prevention Beckman Coulter - West Sacramento
USA USA

Acknowledgment

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

Laura Kovanda, PhD Shawn R. Lockart, PhD, D(ABMM)

Astellas Pharma Centers for Disease Control and
USA Prevention
USA

Acknowledgment

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

Sharon K. Cullen, BS, PMP, RAC David S. Perlin, PhD Nancy L. Wengenack, PhD, D(ABMM)
Beckman Coulter - West Sacramento New Jersey Medical School-UMDNJ Mayo Clinic
USA USA USA

Shawn R. Lockhart, PhD, D(ABMM) Dee Shortridge, PhD

Centers for Disease Control and JMI Laboratories
Prevention USA
USA

Subcommittee
iv on Antifungal Susceptibility Tests
This document is protected by copyright.
 M27, 4th ed.

Contents

Abstract ....................................................................................................................................................i
Committee Membership........................................................................................................................ iii
Foreword .............................................................................................................................................. vii
Chapter 1: Introduction ....................................................................................................................... 1
1.1 Scope............................................................................................................................. 1
1.2 Background ...................................................................................................................1
1.3 Standard Precautions..................................................................................................... 2
1.4 Terminology..................................................................................................................2
Chapter 2: Preparing for Antifungal Susceptibility Testing ............................................................... 5
2.1 Indications for Performing Antifungal Susceptibility Tests ......................................... 5
2.2 Selecting Antifungal Agents for Routine Testing and Reporting ................................. 6
Chapter 3: Antifungal Broth Dilution Susceptibility Testing Process for Yeasts ............................... 7
3.1 Preparing Antifungal Agents ........................................................................................ 9
3.2 Testing Procedures ...................................................................................................... 11
3.3 Reading Minimal Inhibitory Concentration Results ................................................... 15
3.4 Interpreting Results ..................................................................................................... 17
Chapter 4: Quality System Essential: Process Management – Quality Control ............................... 19
4.1 Quality Control Purpose ............................................................................................. 19
4.2 Quality Control Responsibilities ................................................................................. 19
4.3 Selecting Reference Strains ........................................................................................ 20
4.4 Storing Reference Strains ........................................................................................... 20
4.5 Controlling Media Batches and Plasticware Lots ....................................................... 22
4.6 Quality Control Frequency ......................................................................................... 22
4.7 Other Quality Control Procedures .............................................................................. 23
Chapter 5: Conclusion....................................................................................................................... 24
Chapter 6: Supplemental Information ............................................................................................... 24
References ................................................................................................................................ 25
Appendix A. Preparing Dilution Series of Water-Insoluble Antifungal Agents to Be
Used in Broth Dilution Susceptibility Tests ........................................................................... 27
Appendix B. Composition of Roswell Park Memorial Institute 1640 Culture Medium
(With Glutamine and Phenol Red but Without Bicarbonate) .................................................. 28
Appendix C. Preparing Roswell Park Memorial Institute 1640 Culture Medium ................... 29
Appendix D. Preparing Dilution Series of Water-Soluble Antifungal Agents to Be Used
in Broth Dilution Susceptibility Tests...................................................................................... 30
Appendix E. 0.5 McFarland Barium Sulfate Turbidity Standard ............................................ 31
The Quality Management System Approach ........................................................................... 32
Related CLSI Reference Materials .......................................................................................... 33

Subcommittee on Antifungal Susceptibility Tests v

This document is protected by copyright.
 M27, 4th ed.

Subcommittee
vi on Antifungal Susceptibility Tests
This document is protected by copyright.
 M27, 4th ed.

Foreword
With the increased incidence of systemic fungal infections and the growing number of available antifungal
agents, laboratory guidance for selecting antifungal therapy has gained greater attention. In 1982, the CLSI
Area Committee for Microbiology formed the Subcommittee on Antifungal Susceptibility Tests. In 1985,
this subcommittee published its first report, in which the results of a questionnaire and a small collaborative
study were presented. Based on these findings, the subcommittee concluded that it would be useful to work
toward a more reproducible reference testing procedure.

Agreement already existed regarding several elements of the procedure. For example, to facilitate additional
analysis of various test conditions, it was agreed that the reference method should be a broth dilution
procedure. Because of examples of drug antagonism by some complex media for certain antifungal agents,
the subcommittee restricted its interest to fully defined synthetic media only. Drug stock solution
preparation and dilution procedures previously developed for antibacterial testing procedures were adopted
with minor modifications. Despite agreement in some areas, for other factors, additional data needed to be
resolved, including:

 Inoculum preparation
 Inoculum size
 Choice among several synthetic media
 Incubation temperature
 Incubation duration
 End-point definition

These factors were the focus of a series of collaborative studies.1-4 As a result, the subcommittee reached
agreement on all factors, which led to the publication of M27-P in 1992. In the next four years, reference
minimal inhibitory concentration (MIC) ranges were established for two QC strains for the available
antifungal agents,5,6 and broth microdilution procedures paralleling the broth macrodilution reference
procedure became available.4,7-9 This information was included in a revised standard in 1995 (M27-T). In
revising the standard, the subcommittee focused its attention on developing relevant breakpoints for
available antifungal agents,10 included in M27-A in 1997. Since then, the subcommittee has developed
24- and 48-hour reference MIC ranges for microdilution testing of both established and newly introduced
antifungal agents.11 The study results are included in this standard and CLSI document M60.12

Overview of Changes

This standard replaces the previous edition of the approved standard, M27-A3, published in 2008. Several
changes were made in this edition, including:

 General:
– Revised document format and organization to reflect the CLSI quality system essential and path of
workflow document templates and the updated CLSI style

– Updated references to the previous informational supplements (M27-S4 and M44-S3) to reflect
CLSI document M60,12 the new supplement for broth dilution and disk diffusion yeast
susceptibility testing

– Added references to epidemiological cutoff values and CLSI documents M5713 and M5914

Subcommittee on Antifungal Susceptibility Tests vii

This document is protected by copyright.
 M27, 4th ed.

 Subchapter 1.4.2, Definitions:

– Revised the breakpoint and interpretive category definitions for consistency with other CLSI
antimicrobial susceptibility testing documents

– Added definitions for “wild-type” and “non-wild-type”

– Deleted all uses of the phrase “interpretive criteria”

 Chapter 3, Antifungal Susceptibility Testing Process:

– Added an antifungal susceptibility testing process flow chart

– Replaced procedural text with step-action tables

– Added an explanation for deleting breakpoints for itraconazole and flucytosine

– Changed recommended reading time for broth microdilution to 24 hours only for clinical isolates
and QC strains (24 and/or 48 hours was accepted for some antifungal agents in M27-A3)

– Deleted results interpretation information for ketoconazole

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

Key Words

Antifungal agent, broth macrodilution, broth microdilution, susceptibility testing, yeasts

Subcommittee
viii on Antifungal Susceptibility Tests
This document is protected by copyright.
 M27, 4th ed.

Reference Method for Broth Dilution Antifungal Susceptibility Testing of

Yeasts
Chapter 1: Introduction

This chapter includes:

 Standard’s scope and applicable exclusions

 Background information pertinent to the standard’s content

 Standard precautions information

 “Note on Terminology” that highlights particular use and/or variation in use of terms
and/or definitions

 Terms and definitions used in the standard

 Abbreviations and acronyms used in the standard

Scope

This standard describes a reference method for testing susceptibility to antifungal agents of yeasts that cause
infections, including Candida spp. and Cryptococcus spp. The intended users are laboratory personnel who
perform antifungal susceptibility testing on yeasts. The focus is on developing relevant breakpoints for
available antifungal agents10 and reference minimal inhibitory concentration (MIC) ranges for broth dilution
testing of both established and newly introduced antifungal agents.11 For MIC breakpoints, interpretive
categories, and MIC ranges for QC isolates, refer to CLSI document M60.12

This method has not been extensively validated for the yeast forms of dimorphic fungi, such as Blastomyces
dermatitidis or Histoplasma capsulatum. Also, testing filamentous fungi (moulds) introduces several
additional standardization problems not covered by this procedure and is not included. For an antifungal
broth dilution susceptibility testing reference method for filamentous fungi, refer to CLSI document M38.15

Commercially available susceptibility test systems are out of scope for this standard. It is recommended
that users of these systems refer to the manufacturer’s instructions as outlined in the package insert.

Background

This standard provides a reference method developed through a consensus process to facilitate agreement
among laboratories in measuring yeast susceptibility to antifungal agents. An important use of a reference
method is to provide a standard basis from which other methods can be developed, which also results in
interlaboratory agreement within specified ranges. For example, broth microdilution methods using an
indicator dye to facilitate breakpoint determinations have been configured to produce results paralleling
those obtained by the broth microdilution reference method. To the extent that any method produces results
concordant with this reference method, it would be considered to conform with this standard.

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 1
This document is protected by copyright.
 M27, 4th ed.

Standard Precautions

Because it is often impossible to know what isolates or specimens might be infectious, all patient and
laboratory specimens are 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 all known infectious agents and thus
are more comprehensive than universal precautions, which are intended to apply only to transmission of
bloodborne pathogens. Published guidelines are available that discuss the daily operations of diagnostic
medicine in humans and animals while encouraging a culture of safety in the laboratory.16 For specific
precautions for preventing the laboratory transmission of all known infectious agents from laboratory
instruments and materials and for recommendations for the management of exposure to all known infectious
diseases, refer to CLSI document M29.17

Terminology

A Note on Terminology

CLSI, as a global leader in standardization, is firmly committed to achieving global harmonization

whenever possible. Harmonization is a process of recognizing, understanding, and explaining differences
while taking steps to achieve worldwide uniformity. CLSI recognizes that medical conventions in the global
metrological community have evolved differently in different countries and regions and that legally
required use of terms, regional usage, and different consensus timelines are all important considerations in
the harmonization process. CLSI recognizes its important role in these efforts, and its consensus process
focuses on harmonization of terms to facilitate the global application of standards and guidelines.

NOTE: Current fungal taxonomy is being revised. Many genera have both a teleomorph (sexual state) and
an anamorph (asexual state) name. In this standard, the traditional Candida spp. and Cryptococcus spp.
names are used to provide continuity to both past procedures and associated documents such as CLSI
document M60.12

Definitions

antibiogram – overall profile of antimicrobial susceptibility testing results of a microbial species to a

battery of antimicrobial agents.

breakpoint – minimal inhibitory concentration (MIC) or zone diameter value used to categorize an
organism as susceptible, susceptible-dose dependent, intermediate, or resistant; NOTE 1: MIC or zone
diameter values generated by a susceptibility test can be interpreted based upon established breakpoints;
NOTE 2: See interpretive category.

epidemiological cutoff value (ECV) – the minimal inhibitory concentration (MIC) or zone diameter value
that separates microbial populations into those with and without acquired and/or mutational resistance based
on their phenotypes (wild-type or non-wild-type). The ECV defines the upper limit of susceptibility for the
wild-type population of isolates.

EXAMPLE:

Interpretive ECVs
Category MIC, µg/mL Zone Diameter, mm
Wild-type ≤4 ≥ 20
Non-wild-type ≥8 ≤ 19

Subcommittee
2 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

 wild-type (WT) – an ECV interpretive category defined by an ECV that describes isolates with no
mechanisms of acquired resistance or reduced susceptibility for the antimicrobial (antifungal) agent
being evaluated.

 non-wild-type (NWT) – an ECV interpretive category defined by an ECV that describes isolates
with presumed or known mechanisms of acquired resistance and reduced susceptibility for the
antimicrobial (antifungal) agent being evaluated.

interpretive category – category derived from microbiological characteristics, pharmacokinetic-

pharmacodynamic parameters, and clinical outcome data, when available; NOTE 1: Minimal inhibitory
concentration (MIC) or zone diameter values generated by a susceptibility test can be interpreted based
upon established breakpoints; NOTE 2: See breakpoint.

EXAMPLE:

Breakpoints*
Interpretive Category MIC, µg/mL Zone Diameter, mm
Susceptible 4  20
Susceptible-dose dependent 8–16 15–19
Intermediate 8–16 15–19
Resistant  32  14
* Formerly “interpretive criteria.”

MIC or zone diameter value breakpoints and interpretive categories are established per CLSI
document M2318 for categories of susceptible, intermediate, and resistant (and susceptible-dose
dependent when appropriate).

 Susceptible (S) – a category defined by a breakpoint that implies that isolates with an MIC at or
below or zone diameters at or above the susceptible breakpoint are inhibited by the usually
achievable concentrations of antimicrobial agent when the dosage recommended to treat the site of
infection is used, resulting in likely clinical efficacy.

 Susceptible-dose dependent (SDD) – a category defined by a breakpoint that implies that

susceptibility of an isolate is dependent on the dosing regimen that is used in the patient. In order
to achieve levels that are likely to be clinically effective against isolates for which the susceptibility
testing results (either MICs or zone diameters) are in the SDD category, it is necessary to use a
dosing regimen (ie, higher doses, more frequent doses, or both) that results in higher drug exposure
than the dose that was used to establish the susceptible breakpoint. Consideration should be given
to the maximum approved dosage regimen, because higher exposure gives the highest probability
of adequate coverage of an SDD isolate. The drug label should be consulted for recommended
doses and adjustment for organ function; NOTE: The concept of SDD has been included within
the intermediate category definition for antimicrobial agents. However, this is often overlooked or
not understood by clinicians and microbiologists when an intermediate result is reported. The SDD
category may be assigned when doses well above those used to calculate the susceptible breakpoint
are approved and used clinically and for which sufficient data to justify the designation exist and
have been reviewed. When the intermediate category is used, its definition remains unchanged.

 Intermediate (I) – a category defined by a breakpoint that includes isolates with MICs or zone
diameters within the intermediate range that approach usually attainable blood and tissue levels and
for which response rates may be lower than for susceptible isolates; NOTE: The intermediate
category implies clinical efficacy in body sites where the drugs are physiologically concentrated or
when a higher than normal dosage of a drug can be used. This category also includes a buffer zone,
Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 3
This document is protected by copyright.
 M27, 4th ed.

which should prevent small, uncontrolled, technical factors from causing major discrepancies in
interpretations, especially for drugs with narrow pharmacotoxicity margins.

 Resistant (R) – a category defined by a breakpoint that implies that isolates with an MIC at or
above or zone diameters at or below the resistant breakpoint are not inhibited by the usually
achievable concentrations of the agent with normal dosage schedules and/or that demonstrate MICs
or zone diameters that fall in the range in which specific microbial resistance mechanisms are likely,
and clinical efficacy of the agent against the isolate has not been reliably shown in treatment studies.

minimal inhibitory concentration (MIC) – the lowest concentration of an antimicrobial agent that causes
a specified reduction in visible growth of a microorganism in an agar or broth dilution susceptibility test;
NOTE: The magnitude of reduction in visible growth is assessed using the following numerical scale: 0,
optically clear; 1, slightly hazy; 2, prominent decrease (≈ 50%) in visible growth; 3, slight reduction in
visible growth; and 4, no reduction in visible growth.

quality control (QC) – part of quality management focused on fulfilling quality requirements19; NOTE 1:
This includes the operational techniques and activities that are used to fulfill requirements for quality;
NOTE 2: In health care testing, this includes the set of procedures designed to monitor the test method and
the results to ensure test system performance; NOTE 3: QC includes testing QC materials, charting the
results and analyzing them to identify sources of error, and evaluating and documenting any remedial action
taken as a result of this analysis.

trailing growth – reduced but persistent growth over an extended range of concentrations that increases
with incubation time.

Abbreviations and Acronyms

DMSO dimethyl sulfoxide

ECV epidemiological cutoff value
MIC minimal inhibitory concentration
MOPS 3-(N-morpholino) propanesulfonic acid
NWT non-wild-type
pH negative logarithm of hydrogen ion concentration
QC quality control
RPMI Roswell Park Memorial Institute
WT wild-type

Subcommittee
4 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

Chapter 2: Preparing for Antifungal Susceptibility Testing

This chapter includes:

 Indications for performing antifungal susceptibility tests

 Selecting antifungal agents for routine testing and reporting

Indications for Performing Antifungal Susceptibility Tests

Susceptibility testing is indicated for any organism contributing to an infectious process warranting
antimicrobial chemotherapy if its susceptibility cannot be reliably predicted from the organism’s identity.
Susceptibility tests are most often indicated when the causative organism is thought to belong to a species
capable of exhibiting resistance to commonly used antimicrobial agents. Mechanisms of resistance include:

 Altered drug targets

 Altered drug uptake or efflux
 Absence of microbial enzymes to metabolize drug to active form
 Presence of microbial enzymes that hydrolyze the drug to the inactive form

Some organisms have predictable susceptibility to antimicrobial agents, and empiric therapy for these
organisms is widely accepted. Susceptibility tests are also important in epidemiological studies of resistance
and in studies of new antimicrobial agents.

Isolated colonies of each organism type that may be pathogenic should be selected from primary agar plates
and tested for susceptibility. Identification procedures are often performed at the same time. Mixtures of
different microorganism types should not be tested on the same susceptibility test plate or panel. Performing
susceptibility tests directly with patient material (eg, normally sterile body fluids and urine) should be
avoided except in medical emergencies in which direct microscopic examination suggests a single
pathogen. When testing has been performed directly with patient material, results should be reported as
preliminary, and the susceptibility test must be repeated using standardized methodology.

When the nature of the infection is not clear, the specimen contains mixed growth or normal flora, and/or
the organisms probably have little relationship to the infectious process being treated, susceptibility tests
are often unnecessary, and the results may be misleading.

The MIC obtained using a dilution test may provide the antimicrobial agent concentration needed to inhibit
the infecting organism at the infection site. However, the MIC does not represent an absolute value. The
“true” in vitro MIC is somewhere between the lowest test concentration inhibiting the organism’s growth
(ie, the MIC reading) and the next lower test concentration. For example, if twofold dilutions were used and
the MIC is 16 µg/mL, the “true” MIC would be between 16 and 8 µg/mL. Even under controlled conditions,
a dilution test may not yield the same end point each time it is performed. Generally, the acceptable test
reproducibility is within one twofold dilution of the actual end point. To avoid greater variability, the
dilution test must be standardized and carefully controlled.20,21

Traditionally, MICs have been determined using concentrations derived from serial twofold dilutions
indexed to the base 2 (eg, 1, 2, 4, 8, 16 µg/mL). Other dilution schemes have also been used, including as
few as two widely separated (or “breakpoint”) concentrations or concentrations between the usual values
(eg, 4, 6, 8, 12, 16 µg/mL). The results from these alternative methods may be equally useful; however,
some are more difficult to control. When there is growth inhibition at the lowest concentration tested, the
true MIC value cannot be accurately determined and should be reported as equal to or less than the lowest
concentration tested. To determine the breakpoint when concentrations between the usual dilutions are

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 5
This document is protected by copyright.
 M27, 4th ed.

tested, results falling between serial twofold dilutions should be rounded up to the next highest
concentration (eg, an MIC of 6 µg/mL would become 8 µg/mL).

Whenever MIC results are reported for directing therapy, an interpretive category (ie, susceptible,
susceptible-dose dependent, intermediate, or resistant) should accompany the MIC result based on the
breakpoints provided in CLSI document M60.12 When four or fewer consecutive concentrations are tested
or when nonconsecutive concentrations are tested, an interpretive category result must be reported. The
MIC result may also be reported, if desired.

Selecting Antifungal Agents for Routine Testing and Reporting

Although breakpoints are now available for many fungal drug-microorganism combinations (see CLSI
document M6012), routine testing is not recommended. At each institution, the decision to perform
antifungal susceptibility testing on any individual fungal isolate is best made collaboratively by each
institution’s infectious diseases practitioners, the pharmacy and therapeutics committee, diagnostic
microbiology laboratory personnel, and the infection control committee. Testing may be warranted under
selected circumstances, including:

 As part of periodic batch surveys that establish antibiograms for pathogenic isolate collections obtained
within an institution

 To help manage refractory Candida spp. infections in patients who appear to be experiencing
therapeutic failure with standard antifungal agents at standard doses

 To manage invasive Candida spp. infections when the utility of the azole antifungal agents or other
classes are uncertain (eg, when the infection is due to a non–Candida albicans isolate or resistance
patterns change)

Breakpoints are available for some Candida spp. vs anidulafungin, caspofungin, fluconazole, micafungin,
and voriconazole. The clinical relevance of testing any other fungal organism–antifungal agent combination
remains uncertain. Specimens for culture and other procedures should be obtained before initiating
antifungal therapy.

Generic Names

To minimize confusion, all antifungal agents should be referred to by international nonproprietary

(ie, generic) names.

Number of Agents Tested

To make routine susceptibility tests relevant and practical, the number of antimicrobial agents tested should
be limited. Although this is not an immediate issue for antifungal agents, the same principles apply.

Subcommittee
6 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

Chapter 3: Antifungal Broth Dilution Susceptibility Testing Process for Yeasts

This chapter includes:

 Antifungal broth dilution susceptibility testing process for yeasts overview

 Preparing antifungal agents for testing
 Preparing stock solutions
 Antifungal susceptibility testing procedures
 Reading antifungal susceptibility testing results
 Interpreting antifungal susceptibility testing results

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 7
This document is protected by copyright.
 M27, 4th ed.

Figure 1 provides an overview of the antifungal broth dilution susceptibility testing process for yeasts.

Start

Need for susceptibility

Subchapter 2.1
testing is identified

Antifungal agents for testing

Subchapter 2.2
are selected

Antifungal agent stock solutions Subchapter 3.1.3

are prepared

Growth medium, buffers, and diluted

Subchapters 3.2.1, 3.2.2, 3.2.3
antifungal agents are prepared

The inoculum is prepared Subchapter 3.2.4

Tubes or panels are inoculated

Subchapter 3.2.5
and incubated

Susceptibility test results are read Subchapter 3.3

Susceptibility test results

Subchapter 3.4
are interpreted

Susceptibility
Reporting
Process

End

* Five basic symbols are used in process flow charts: oval (signifies the beginning or end of a process), arrow (connects process
activities), box (designates process activities), diamond (includes a question with alternative “Yes” and “No” responses), pentagon
(signifies another process).
Figure 1. Antifungal Broth Dilution Susceptibility Testing Process for Yeasts*

Subcommittee
8 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

Preparing Antifungal Agents

Source

Antifungal agent standards or reference powders can be obtained directly from the drug manufacturer or
commercial sources. Pharmacy stock or other clinical preparations should not be used. Acceptable powders
have a label that states the antifungal agent’s generic name, its assay potency (usually expressed in
micrograms [µg] or international units per mg of powder), and its expiration date. Powders should be stored
as the manufacturer recommends, including length of storage. Alternatively, when there are no
manufacturer’s instructions, powders should be stored at −20°C or below (NOTE: Powders should never
be stored in a self-defrosting freezer), in a desiccator, and preferably in a vacuum. When the desiccator is
removed from the freezer, it should be adjusted to room temperature before opening to avoid water
condensation.

Weighing Antifungal Powders

All antifungal agents are assayed for standard units of activity. The assay units can differ widely from the
actual weight of the powder and often differ within a drug production lot. Thus, laboratories need to
standardize the antifungal solutions based on assays of the antifungal powder lots that are being used.

Either formula (1) or (2) can be used to determine the amount of powder or diluent needed for a standard
solution:

mL ∙ μg/mL
mg (1)
μg/mg

or

mg ∙ μg/mg (2)
mL
μg/mL

The antifungal agent powder should be weighed on an analytical balance that has been calibrated with
approved reference weights from a national metrology organization. Usually, it is advisable to accurately
weigh a portion of the antifungal agent in excess of the amount needed and to calculate the diluent volume
needed to obtain the concentration desired.

Example: To prepare a 100-mL stock solution containing 1280 µg of antifungal agent per mL with
antifungal powder that has a potency of 750 µg/mg, the first formula (1) should be used to establish the
weight of powder needed, as shown in formula (3):

100 mL ⋅ 1280 μg/mg

(3)
mg 170.7 mg
750 μg/mg

Because it is advisable to weigh a portion of the powder in excess of the amount needed, the powder should
be deposited on the balance until approximately 180 mg is reached. With that amount of powder weighed,
formula (2) should be used to determine the diluent amount to be measured, as shown in formula (4):

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 9
This document is protected by copyright.
 M27, 4th ed.

182.6 mg ⋅ 750 μg/mg

mL 107.0 mL (4)
1280 μg/mL

Therefore, 182.6 mg of the antifungal agent powder should be dissolved in 107 mL of diluent.

Preparing Stock Solutions

Antifungal agent stock solutions should be prepared at concentrations of at least 1280 µg/mL or 10 times
the highest concentration to be tested, whichever is greater. However, some antifungal agents with limited
solubility may need lower concentrations. NOTE: In all cases, information provided by the drug
manufacturer should be considered part of determining solubility.

Using Solvents Other Than Water

Some antifungal agents must be dissolved in solvents other than water (see CLSI document M6012).
Antifungal agent compound solubility information should be included with the agent. Such agents should
be dissolved at concentrations at least 100 times higher than the highest desired test concentration.
Commonly used solvents include:

 Analytical-grade dimethyl sulfoxide (DMSO)

 Ethyl alcohol
 Polyethylene glycol
 Carboxymethyl cellulose

When these solvents are used, a series of dilutions at 100 times the final concentration should be prepared
from the antifungal stock solution in the same solvent. Each intermediate solution should then be diluted
again to final strength in the test medium. This procedure avoids dilution artifacts resulting from
precipitation of low-solubility compounds in aqueous media. For example, to prepare for a broth dilution
test series containing a water-insoluble drug that can be dissolved in DMSO, for which the highest desired
test concentration is 16 µg/mL, the steps below should be followed.

Step Action Comment

1. Weigh 4.8 mg of the antifungal agent Assumes 100% potency of the antifungal agent
powder. powder. If the manufacturer indicates the powder
is not at 100% potency, adjust the weight upward
to compensate for the decrease in potency. See
Subchapter 3.1.2 for the appropriate calculations.
2. Dissolve the antifungal agent powder in Provides a stock solution at 1600 µg/mL.
3 mL DMSO.
3. Prepare additional dilutions of this stock See Appendix A.
solution in DMSO.
4. Dilute the solutions in DMSO 10-fold in See Subchapter 3.1.3.1.
test medium and an additional 10-fold
when inoculated. This step reduces the final solvent concentration to
1%.
5. Use DMSO at this concentration (without
drug) in the test as a dilution control.
Abbreviation: DMSO, dimethyl sulfoxide.

Subcommittee
10 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

Filtration

Normally, stock solutions do not support microorganism growth and can be assumed to be sterile. For
additional sterility assurance, the stock solutions should be passed through a membrane filter. Paper,
asbestos, or sintered glass filters, which may adsorb appreciable amounts of certain antifungal agents,
should not be used. Whenever filtration is used, the absence of adsorption should be confirmed by checking
and then documenting the results of the appropriate QC assays.

Storage

Sterile stock solutions should be dispensed in small volumes into sterile polypropylene or polyethylene
vials, carefully sealed, and stored (preferably at −60°C or below, but never at a temperature greater than
−20°C). Vials should be removed from storage as needed and used the same day. Any unused antifungal
agent should be discarded at the end of the day. Most antifungal agent stock solutions can be stored at or
below −60°C for six months or more without significant activity loss.9 In all cases, any instructions provided
by the drug manufacturer need to be considered and supersede these general recommendations. Any
significant antifungal agent deterioration may be determined by reviewing the susceptibility testing results
using QC strains (see CLSI document M6012).

Number of Concentrations Tested

The concentrations tested should include the breakpoints and the expected results for the QC strains. Based
on previous studies, the drug concentration ranges listed in Table 1 should be used.

Table 1. Recommended Antifungal Agent Concentrations

Antifungal Agent Concentration, µg/mL
Amphotericin B 0.0313–16
Anidulafungin 0.015–8
Caspofungin 0.015–8
Fluconazole 0.125–64
Flucytosine 0.125–64
Isavuconazole 0.0313–16
Itraconazole 0.0313–16
Ketoconazole 0.0313–16
Micafungin 0.015–8
Posaconazole 0.0313–16
Ravuconazole 0.0313–16
Voriconazole 0.0313–16

Testing Procedures

Preparing Growth Medium

A completely synthetic medium should be used for antifungal susceptibility testing. Roswell Park Memorial
Institute (RPMI) 1640 culture medium, with glutamine, without bicarbonate, and with phenol red as a pH
indicator, has been found to be at least as satisfactory as several other synthetic media and has been used to
develop the standard.2,3 The medium’s formula is provided in Appendix B, and the steps for its preparation
from powder are provided in Appendix C. Alternative media may be advantageous for some organisms and
some drugs.

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 11
This document is protected by copyright.
 M27, 4th ed.

Preparing Buffers

Media should be buffered to pH 7  0.1 at room temperature (25°C). A buffer that does not antagonize
antifungal agents should be selected. Tris buffer is unsatisfactory, because it antagonizes flucytosine
activity. Zwitterion buffers are preferable to buffers that readily traverse the cell membrane (eg, phosphate
buffers), because theoretically, the latter can produce unexpected interactions with antifungal agents. A
buffer satisfactory for antifungal susceptibility testing is 3-(N-morpholino) propanesulfonic acid (MOPS)
(final concentration 0.165 mol/L for pH 7). The pH of each medium batch should be checked with a pH
meter immediately after preparation and should be between 6.9 and 7.1 at room temperature (25°C). MIC
performance characteristics of each broth batch should be evaluated using a standard QC organism set (see
Subchapter 4.3 and CLSI document M6012).

Preparing Diluted Antifungal Agents

The conditions for preparing and storing diluted antifungal agents include:

 Using sterile 12- × 75-mm plastic test tubes to prepare drug dilutions and sterile, disposable, multiwell
microdilution plates (96 U-shaped wells made with untreated polystyrene) to perform the tests22

 Using a growth control tube containing RPMI 1640 culture medium without antifungal agents (but with
nonaqueous solvent when necessary) for each organism tested

NOTE: Some antifungal agents may be affected by using treated plasticware; therefore, glassware or
untreated plasticware should be used for preparing drug dilutions and microtiter trays.22

Water-Soluble Antifungal Agents

For water-soluble antifungal agents, twofold dilutions may be prepared volumetrically in broth (see
Appendix D). The procedure for water-insoluble antifungal agents differs from water-soluble agents and is
described in Subchapter 3.2.3.2. When running a small number of tests, the schedule in Appendix D should
be consulted.

The total volume of each prepared dilution depends on the number of tests performed. Because 0.1 mL of
each antifungal drug dilution is used for each test, 1 mL is adequate for about nine tests, with consideration
for pipetting. A single pipette should be used for measuring all diluents and for adding the stock antifungal
agent solution to the first tube. A separate pipette should be used for each remaining dilution in that set.
Because the drugs will be diluted 1:10 when combined with the inoculum, the working antifungal agent
solutions are 10 times more concentrated than the final concentrations.

Many users find working with 1:10 dilutions (see Appendix D) easy and convenient. However, some
automated pipettes deliver only 1- or 0.1-mL volumes; therefore, a ratio of 1:11 is preferable. If 1:11
dilutions are prepared, the dilution scheme should be altered so the same final antifungal agent
concentrations are obtained.

Water-Insoluble Antifungal Agents

For water-insoluble antifungal agents (refer to CLSI document M6012 for a current list), an initial dilution
series of the agent should be prepared at 100 times the final strength in an appropriate solvent (see
Subchapter 3.1.3.1). Each nonaqueous solution should then be diluted 10-fold in RPMI 1640 culture
medium. For example, if a dilution series with final concentrations in the range 16 to 0.0313 µg/mL is
desired, a concentration series from 1600 to 3.13 µg/mL should be prepared first in DMSO (see Subchapter
3.1.3.1).

Subcommittee
12 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

To prepare 1-mL volumes of diluted antifungal agent (sufficient for 10 tests):

1. Pipette 0.9-mL volumes of RPMI 1640 culture medium into each of 11 sterile test tubes.

2. Using a single pipette, add 0.1 mL of DMSO alone to one 0.9-mL lot of broth (control medium), then
0.1 mL of the lowest (3.13 µg/mL) drug concentration in DMSO, then 0.1 mL of the 6.25-µg/mL
concentration.

3. Continue in sequence through the concentration series, each time adding 0.1-mL volumes to 0.9 mL
broth.

These volumes can be adjusted according to the total number of tests needed. Because there will be a 1:10
dilution of the antifungal agents when combined with the inoculum, the working antifungal agent solutions
are 10-fold more concentrated than the final concentrations.

Preparing the Inoculum

All organisms should be subcultured at least twice onto antimicrobial-free growth media (eg, Sabouraud
dextrose agar or potato dextrose agar) and passaged to ensure purity and viability. The incubation
temperature throughout must be 35°C. The steps below should be used to prepare the inoculum.

Step Action Comment

1. Choose 5 colonies that are at least 1 mm in Select colonies from 24-hour–old cultures
diameter. of Candida spp. or 48-hour–old cultures
of C. neoformans.
2. Suspend the colonies in 5 mL sterile 0.145-mol/L
saline (8.5 g/L NaCl; 0.85% saline) or sterile
water.
3. Vortex the resulting suspension for 15 seconds.
4. Adjust the cell density with a spectrophotometer This procedure will yield a yeast stock
by adding sufficient sterile saline or sterile water suspension containing 1 × 106 to 5 × 106
to increase the transmittance to that produced by a cells per mL.
0.5 McFarland standard (see Appendix E) at
530 nm wavelength.
5. Make a working suspension by preparing a 1:100 This procedure results in 5 × 102 to 2.5 ×
dilution followed by a 1:20 dilution of the stock 103 cells per mL.2
suspension with RPMI 1640 culture medium.
Abbreviation: RPMI, Roswell Park Memorial Institute.

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 13
This document is protected by copyright.
 M27, 4th ed.

Inoculating and Incubating Susceptibility Tests

Inoculating Broth Macrodilution Tubes

Before adjusting the inoculum, the following steps should be used to inoculate the tubes.

Step Action Comment

1. Place 0.1 mL of the various antifungal agent
concentrations in 12- × 75-mm tubes.
2. Add 0.1 mL of diluent without antifungal
agent to the growth control tube.
3. Add 0.9 mL of the adjusted inoculum to each Should be completed within 15 minutes after
tube in the dilution series. the inoculum has been standardized or within
two hours if the inoculum is kept at 4°C.
4. Mix each tube. Results in a 1:10 dilution of each antifungal
agent concentration and a 10% dilution of the
inoculum.

Inoculating Broth Microdilution Plates

The 10-fold antifungal agent dilutions described for the broth macrodilution procedure should be diluted
again 1:5 with RPMI 1640 culture medium to achieve the twofold strength needed for the broth
microdilution test. The following steps should be used to prepare the dilutions.

Step Action Comment

1. Prepare and adjust the stock inoculum suspensions, See Subchapter 3.1.3.
as described for the broth macrodilution test.
2. Mix the stock yeast suspension by vortexing for
15 seconds.
3. Dilute the suspension 1:50, then to 1:20 with medium Should result in an inoculum
to obtain the twofold test inoculum. containing 1 × 103 to 5 × 103 CFU/mL.
4. Inoculate the wells. When the wells are inoculated, the
(twofold) inoculum 1:1 is diluted, and
the desired final inoculum size is
achieved (0.5 × 103 to 2.5 × 103
CFU/mL).
Abbreviation: CFU, colony-forming unit.

Subcommittee
14 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

To perform the broth microdilution test, sterile, disposable, multiwell microdilution plates (96 U-shaped
wells) and the following steps should be used.

Step Action Comment

1. Using a multichannel pipette, dispense Dispense in order of concentration with either the
the twofold drug concentrations into rows lightest or lowest concentration in the first well
1 to 10 in the microdilution plate wells in and then a gradient going up or down across the
100-µL volumes. next row of wells. The trays may be sealed in
plastic bags and stored frozen at −70°C for up to 6
months without the drug potency deteriorating.
2. On testing day, inoculate each Brings the drug dilutions and inoculum densities
microdilution tray well with 100 µL of to the final concentrations (0.5 × 103 to 2.5 × 103
the corresponding twofold diluted CFU/mL).
inoculum suspension.
3. Inoculate the growth control wells with Growth control wells contain 100 µL of sterile,
100 µL of the corresponding diluted drug-free medium.
(twofold) inoculum suspensions.
Test the QC organisms in the same manner each
time an isolate is tested (see CLSI document
M6012).

One cell in the last column of the microdilution

plate may be used to perform the sterility control
(drug-free medium only).
Abbreviations: CFU, colony-forming unit; QC, quality control.

Microdilution plates and macrodilution tubes should be incubated (without agitation) at 35°C in ambient
air for 24 hours (or up to 48 hours for isolates that are not growing well at 24 hours). When testing
C. neoformans, tubes should be incubated for a total of 70 to 74 hours before determining results.

Reading Minimal Inhibitory Concentration Results

After the appropriate incubation time, the tubes and plates should be observed for the presence or absence
of visible growth using the same lighting conditions and read method. Microtiter plates should be viewed
under normal laboratory lighting using a manual mirror viewer or reader.

Reading Time for Broth Macrodilution

For broth macrodilution, QC limits are established for all antifungal agents at 48 hours (see CLSI document
M6012); therefore, broth macrodilution MICs should be read at 48 hours. The amount of growth in the tubes
containing the antifungal agent should be compared visually with the amount of growth in the growth-
control tubes (no antifungal agent) used in each set of tests and scored using the following numerical scale:

 0 – Optically clear
 1 – Slightly hazy
 2 – Prominent decrease ( 50%) in turbidity
 3 – Slight reduction in turbidity
 4 – No reduction in turbidity

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 15
This document is protected by copyright.
 M27, 4th ed.

Reading Time for Broth Microdilution

For broth microdilution, QC limits are established for all antifungal agents at 24 hours (see CLSI document
M6012). Broth microdilution MICs should be read at23-25:

 24 hours for the echinocandins, amphotericin B, fluconazole, flucytosine, itraconazole, isavuconazole,

posaconazole, ravuconazole, and voriconazole

 72 hours for Cryptococcus spp. isolates against all antifungal agents referenced above

NOTE: Current breakpoints are defined only for the 24-hour readings.

Agitating the plates is optional and may simplify reading the end points. Microdilution wells should be
scored using a reading mirror and the growth in each well compared with the growth control (antifungal
agent–free) well.

Pipetting, vortexing, or other mixing techniques should be tried when clumping of an isolate hinders
scoring.24,26 The MIC for amphotericin B is defined as the lowest concentration in which there is no growth
(optically clear). For the azoles, echinocandins, and flucytosine, the MIC is defined as the lowest
concentration at which there is a 50% decrease in growth (prominent decrease in turbidity).

Some Candida spp. isolates, typically C. albicans and C. tropicalis, exhibit significant trailing growth
(partial growth inhibition over an extended antifungal concentration range) for azoles, especially
fluconazole and voriconazole.27 Estimated as occurring for fluconazole in about 5% of isolates,28 this
trailing growth can be so large that it makes a susceptible isolate appear completely resistant. Two
independent in vivo investigations of this phenomenon using murine models of disseminated candidiasis
showed that isolates with this behavior should be categorized as “susceptible” rather than “resistant.”28,29
This concept has been corroborated by a clinical demonstration in which oropharyngeal candidiasis caused
by such isolates responds to low doses of fluconazole typically used to treat susceptible isolates.30

Amphotericin B

For amphotericin B, end points are typically well defined, and the MIC is easily read as the lowest antifungal
agent concentration that prevents any discernible growth (score of 0). Trailing growth end points with
amphotericin B are not usually encountered.

Flucytosine and Azole Antifungal Agents

For flucytosine and especially for azoles, end points are typically less well defined than those described for
amphotericin B, which may contribute to a significant source of variability. Applying a less stringent end
point, an approximately 50% reduction in growth relative to the antifungal agent–free growth control, has
improved interlaboratory agreement and distinguishes between presumed susceptible and resistant isolates.
When turbidity persists, it is often identical for all antifungal agent concentrations above the MIC. Even
dispersing clumps that can become evident after incubation can make end-point determination more
reproducible. Reference strains with defined susceptibility can also be used when training new personnel.

Echinocandin Antifungal Agents

For echinocandins, MIC end points should be determined after 24 hours incubation and read as the lowest
drug concentration to produce a prominent decrease in turbidity (≈ 50% reduction). This translates to an
approximately 50% growth reduction relative to the antifungal agent–free growth control.

Subcommittee
16 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

Interpreting Results

Breakpoints have been established for only some organism-drug combinations (see CLSI document M6012).
The clinical relevance of testing other organism–antifungal agent combinations remains uncertain, but the
relevant information can be summarized as outlined in the following subchapters.

Amphotericin B

Experience using the procedures described in this standard indicates that amphotericin B MICs for Candida
spp. isolates are tightly clustered between 0.25 and 1 µg/mL. Some research has suggested that
commercially available methods may provide a more accurate interpretation of the in vitro MIC.31

NOTE: For Candida spp. without breakpoints, epidemiological cutoff values (ECVs) that define the wild-
type (WT) distribution limit have been established for common Candida spp. and amphotericin B and may
be useful for distinguishing between WT and non-wild-type (NWT) isolates (ie, those with intrinsic or
acquired known resistance mechanisms) (see CLSI document M5914).

Flucytosine

Previously published CLSI breakpoints for flucytosine were established with minimal clinical data, and
emerging data now suggest those breakpoints were not correct. Therefore, the previously published
breakpoints should not be used.

NOTE: For Candida spp. without breakpoints, ECVs that define the WT distribution limit have been
established for Candida spp. and some antifungal agents and may be useful for distinguishing between WT
and NWT isolates (see CLSI document M5914).

Fluconazole

Species-specific breakpoints for C. albicans, Candida glabrata, Candida parapsilosis, and Candida
tropicalis and fluconazole have been established (see CLSI document M6012).10 These breakpoints are not
applicable to Candida krusei; therefore, identification to the species level is necessary to accurately
interpret and report MICs. In addition, when an isolate is identified as C. glabrata and the MIC is
≤ 32 µg/mL, patients should receive the maximum fluconazole dosage regimen.32 Expert consultation on
selecting a maximum dosage regimen may be useful. The utility of testing isolates of C. neoformans is
unknown, but data suggest a correlation between elevated MIC and clinical failure.33

Itraconazole

Previously published CLSI breakpoints for itraconazole were established with minimal clinical data, and
emerging data now suggest those breakpoints were not correct. Therefore, the previously published
breakpoints should not be used.

NOTE: For Candida spp. without breakpoints, ECVs that define the WT distribution limit have been
established for Candida spp. and itraconazole and may be useful for distinguishing between WT and NWT
isolates (see CLSI document M5914).

Voriconazole

Breakpoints for C. albicans, C. parapsilosis, C. tropicalis, and C. krusei and voriconazole have been
established (see CLSI document M6012). These data are drawn from in vitro tests, animal models, and six
clinical trials, with the majority taken from a clinical trial in non-neutropenic patients with candidemia.34
In other settings, the clinical relevance of breakpoints is uncertain.
Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 17
This document is protected by copyright.
 M27, 4th ed.

Posaconazole, Ravuconazole, and Isavuconazole

Experience testing posaconazole and ravuconazole with broth dilution methodology indicates that Candida
and Cryptococcus spp. MICs vary between 0.03 and 16 µg/mL, with the majority of isolates inhibited by ≤
1 µg/mL for both antifungal agents. For isavuconazole, yeast MICs range between 0.008 to 8 µg/mL, with
the majority (90% of tested isolates having an equivalent or lower MIC) inhibited by 0.5 µg/mL.35 However,
data are not yet available to indicate a correlation between MIC and treatment outcome with these agents.

Echinocandins (Anidulafungin, Caspofungin, and Micafungin)

Using procedures described in this standard, MIC survey data for > 2500 patient isolates of Candida spp.
indicate that MICs vary between 0.007 and 8 µg/mL, with ≥ 99% of isolates inhibited by ≤ 2 µg/mL.36,37
However, isolates harboring FKS mutations, the major resistance mechanism to echinocandins, have been
identified with MICs that fall below 2 µg/mL. Therefore, species-specific breakpoints have been established
that enable discrimination between potentially resistant mutants and susceptible strains (see CLSI document
M6012).

NOTE: For Candida spp. without breakpoints, ECVs that define the WT distribution limit have been
established for Candida spp. and anidulafungin and micafungin and may be useful for distinguishing
between WT and NWT isolates (see CLSI document M5914). 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 Subchapter 3.2. The cause of the variability is unclear. For this
reason, no ECVs have been established.

Subcommittee
18 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

Chapter 4: Quality System Essential: Process Management – Quality Control

This chapter includes:

 QC purpose
 QC responsibilities
 QC strain selection
 QC strain storage
 Routine uses for QC strains
 Media batches and plasticware lot control
 QC frequency
 Other QC procedures

Quality Control Purpose

The goals of a QC program are to monitor:

 Susceptibility test procedure’s precision (repeatability) and accuracy

 Performance of reagents, testing conditions, and instructions used in the test
 Performance of persons conducting the tests and reading the results

The goals are best realized by, but are not limited to, using QC and reference strains selected for their
genetic stability and usefulness in the particular method being controlled.5,6,11,38-40

Quality Control Responsibilities

Antifungal susceptibility test manufacturers and users share responsibility for quality. The primary purpose
of QC testing performed by manufacturers (ie, for laboratory-developed reference methods or commercial
methods) is to ensure the testing materials and reagents have been appropriately manufactured. The primary
purpose of QC testing performed by laboratories (ie, users) is to ensure the testing materials and reagents
are maintained properly and testing is performed according to established procedures. Manufacturers (of
commercial and/or laboratory-developed products) are responsible for:

 Antifungal agent stability

 Antifungal agent labeling
 Antifungal agent stock solution potency
 Compliance with good manufacturing practices
 Product integrity
 Accountability and traceability to the consignee

Manufacturers should design and recommend a QC program that aids users in evaluating the variables
(eg, inoculum levels, storage, shipping conditions) that will most likely cause user performance problems
and to determine the assay is performing as expected when directions for use are followed. The laboratory
is responsible for:

 Correct storage (to prevent antifungal agent deterioration)

 Operator proficiency
 Adherence to procedure (eg, inoculum effect, incubation conditions [time and temperature])

Laboratories should familiarize themselves with applicable regulatory and accreditation requirements for
QC.
Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 19
This document is protected by copyright.
 M27, 4th ed.

Selecting Reference Strains

Ideal reference strains for dilution method QC have MICs that fall near the midrange of the concentration
for all antifungal agents tested. An ideal QC strain is inhibited at the fifth dilution of a nine-dilution log2
series; however, strains for which the antifungal agent MICs are between the third and seventh dilution are
acceptable. Before a strain is accepted as a reference strain, it should be tested for as long as necessary to
demonstrate that its antifungal susceptibility pattern is genetically stable. CLSI document M2318 provides
guidelines for selecting appropriate QC strains and determining acceptable MIC ranges. The QC strains
listed in CLSI document M6012 were selected according to these criteria.5,6,11

Storing Reference Strains

Methods for Prolonged and Short-Term Storage

Reference strains must be stored so that the possibility of organism mutation is minimized. There are two
preferred methods for prolonged reference strain storage. Yeasts may be grown on potato dextrose agar and
then frozen at −70°C.41 Alternatively, reference strains can be preserved by suspending fungal cells in 15%
glycerol solution in small vials and freezing and storing them at −70°C.

For short-term storage, working stock cultures can be grown on Sabouraud dextrose agar or potato dextrose
agar slants or plates until sufficient growth is observed and stored at 2 to 8°C. Fresh slants or plates should
be prepared at two-week intervals by serial transfer from frozen stock. To avoid mixed cultures, no more
than three passages should be made after removal from frozen stock culture. Whenever aberrant results
occur, a new stock culture should be obtained.

Sources for Reference Strains

Reference strains should be obtained from a source that can provide information on the culture’s origin
(eg, from commercial sources with documented culture history or from reference institutions with
demonstrated ability to store and use the organisms consistently with minimal contamination). A new stock
culture should be obtained whenever a significant deviation from the expected end point is observed.

Subcommittee
20 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

Preparing Strains for Storage

To prepare strains for storage, the steps below should be followed.

Step Action Comment

1. Grow the organisms overnight on Sabouraud
dextrose agar or potato dextrose agar plates.
2. Select growth from several colonies and perform See CLSI document M6012 for expected
the appropriate susceptibility tests to demonstrate MICs of some reference strains.
that they produce the expected MIC results.
3. Subculture strains yielding expected results onto the
same medium as the primary culture.
4. Incubate subcultures long enough for sufficient Usually from 1 to 3 days
growth to occur.
5. Examine the resulting growth carefully to ensure
the culture is pure.
6. Suspend the growth from the plate in 15% glycerol
to make a heavy suspension.
7. Distribute the turbid suspension in small volumes
(1 to 2 drops) into several small containers.
8. Place the containers in a −70°C freezer or in liquid
nitrogen.
Abbreviation: MIC, minimal inhibitory concentration.

Stocks prepared using this procedure can be stored indefinitely without significant risk of altering antifungal
susceptibility patterns. When the supply of containers is nearly exhausted, this process should be repeated
to prepare a new supply.

Routine Use of Reference Strains

For routine reference strain use, the steps below should be followed.

Step Action Comment

1. Remove a container of the culture from the
−70°C freezer or obtain a lyophilized vial.
2. Thaw the frozen mixture or rehydrate the
lyophilized culture.
3. Subculture the mixture onto potato dextrose agar  24 hours for Candida spp.
plates and incubate at 35°C.  48 hours for C. neoformans
4. Remove 4–5 colonies and subculture them to the
appropriate susceptibility test medium.
5. Subculture the colonies onto potato dextrose
agar slants.
6. Incubate the agar slants overnight.
7. Store the resulting cultures on agar slants at The agar slants may be used as working
2 to 8°C. stock cultures. At least every two weeks,
replace them with new slants prepared from
the freezer supply.
8. Subculture growth from the stored slant to an Always perform susceptibility testing on
agar plate for testing. colonies from overnight plates.

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 21
This document is protected by copyright.
 M27, 4th ed.

Controlling Media Batches and Plasticware Lots

For batch or lot control:

 Each new batch of medium, macrodilution tube lot, or microdilution plate lot must be tested with one
of the QC strains listed in CLSI document M6012 to determine whether MICs fall within the expected
range. If the MICs do not fall within the expected range, the batch or lot must be rejected. Only batches
of medium, tube, or plate lots that have been tested according to the acceptance limits described in this
standard and that meet those limits should be used for testing patient isolates.

 To ensure the medium’s sterility, at least one uninoculated tube or microdilution plate from each batch
should be incubated for the same amount of time as needed to complete the test.

 New lots of RPMI 1640 culture medium should be tested for acceptable performance before being used
to test patient isolates. It must be confirmed that the pH falls between 6.9 and 7.1 (see Subchapter 3.2.2).

 Lot numbers of all materials and reagents used in these tests must be documented.

Quality Control Frequency

Minimal Inhibitory Concentration Ranges

CLSI document M6012 lists MIC QC limits for a single control test.5,6,11 In general, one of every 20 MIC
values in a series of 20 consecutive tests might be out of control (ie, outside the stated range) due to random
test variation. Two consecutive out-of-control results or any more than two out-of-control results in 20
consecutive control tests need corrective action. Any time corrective action is taken, the count of 20 begins
again.

NOTE: This procedure should not be confused with the procedure for establishing satisfactory performance
of MIC tests for the purpose of performing QC tests weekly instead of daily (see Subchapter 4.6.2).

Testing Frequency

The overall test system performance should be monitored by testing appropriate reference strains each day
the test is performed. However, test monitoring frequency may be reduced if the laboratory can document
satisfactory performance with daily control tests. For this purpose, satisfactory performance is defined as:

 Testing all reference strains and documenting results for 30 consecutive test days

 Ensuring that for each drug-microorganism combination, no more than three of the 30 MIC values
(ie, MIC values obtained from one drug-microorganism combination for 30 consecutive test days) are
outside the accuracy ranges stated in CLSI document M6012

NOTE: This procedure is only for establishing satisfactory performance of MIC tests for the purpose of
performing QC tests weekly instead of daily. This procedure should not be confused with the steps that
must be taken for corrective action defined in the bullets below.

Subcommittee
22 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

When these conditions are fulfilled, each reference strain must be tested at least once per week and
whenever any reagent component is changed. Whenever an MIC value outside the accuracy range is
observed using the weekly accuracy monitoring system, daily QC tests must be reinstated long enough to
define the aberrant result’s source and to document the problem’s resolution by:

 Testing with appropriate reference strains and documenting the results for five consecutive test days

 Ensuring that for each drug-microorganism combination, all five MIC values (ie, MIC values obtained
from one drug-microorganism combination for five consecutive test days) are within the accuracy
ranges stated in CLSI document M6012

 Continuing daily control testing if resolution of the problem cannot be documented (ie, at least one of
the five MIC values is observed to be outside the accuracy range)
– To return to weekly testing, documenting satisfactory performance for another 30 consecutive test
days is needed. For some antifungal agents, QC tests need to be performed more frequently than
once per week due to relatively rapid degradation of the agent.

Other Quality Control Procedures

Growth Control

Each broth macrodilution or microdilution series should include a growth control of RPMI 1640 culture
medium without antifungal agent plus 1% of the solvent used (eg, DMSO) to assess test organism viability.
With broth tests, the growth control also serves as a turbidity control for reading end points.

Purity Control

A sample of each inoculum should be streaked onto a suitable agar plate and incubated until there is
sufficient visible growth to detect mixed cultures and to provide freshly isolated colonies if retesting is
necessary.

End-Point Interpretation Control

End-point interpretation should be monitored periodically to minimize MIC end-point variation among
observers. Each laboratory worker performing these tests should independently read a selected set of
dilution tests. The results should be recorded and compared with the results obtained by an experienced
reader. All laboratory personnel should read tests using the same lighting conditions and read method
(eg, manual mirror viewer). QC and reference strains with predetermined MICs are particularly useful for
this purpose, especially with fluconazole.5,6,11

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 23
This document is protected by copyright.
 M27, 4th ed.

Chapter 5: Conclusion
This standard outlines the steps necessary for performing antifungal susceptibility testing of yeasts. It
describes preparation of media and materials, testing procedures, QC performance, and results
interpretation. It also describes the role of QC performed during antifungal susceptibility testing. With this
standard as a guide, a laboratory should be able to implement an antifungal susceptibility testing program
that meets quality standards.

Chapter 6: Supplemental Information

This chapter includes:

 References
 Appendixes
 The Quality Management System Approach
 Related CLSI Reference Materials

Subcommittee
24 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

References
1
Pfaller MA, Burmeister L, Bartlett MS, Rinaldi MG. Multicenter evaluation of four methods of yeast inoculum preparation. J Clin
Microbiol. 1988;26(8):1437-1441.
2
Pfaller MA, Rinaldi MG, Galgiani JN, et al. Collaborative investigation of variables in susceptibility testing of yeasts. Antimicrob
Agents Chemother. 1990;34(9):1648-1654.
3
Fromtling RA, Galgiani JN, Pfaller MA, et al. Multicenter evaluation of a broth macrodilution antifungal susceptibility test for yeasts.
Antimicrob Agents Chemother. 1993;37(1):39-45.
4
Espinel-Ingroff A, Kish CW, Kerkering TM, et al. Collaborative comparison of broth macrodilution and microdilution antifungal
susceptibility tests. J Clin Microbiol. 1992;30(12):3138-3145.
5
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.
6
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.
7
Barchiesi F, Colombo AL, McGough DA, Rinaldi MG. Comparative study of broth macrodilution and microdilution techniques for
in vitro antifungal susceptibility testing of yeasts by using the National Committee for Clinical Laboratory Standards’ proposed
standard. J Clin Microbiol. 1994;32(10):2494-2500.
8
Espinel-Ingroff A, Kerkering TM, Goldson PR, Shadomy S. Comparison study of broth macrodilution and microdilution antifungal
susceptibility tests. J Clin Microbiol. 1991;29(6):1089-1094.
9
Jorgensen JH, Turnidge JD. Susceptibility test methods: dilution and disk diffusion methods. In: Jorgensen JH, Pfaller MA, Carroll
KC, et al., eds. Manual of Clinical Microbiology. 11th ed. Washington, DC: ASM Press; 2015:1253-1273.
10
Rex JH, Pfaller MA, Galgiani JN, et al. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual
framework and analysis of in vitro-in vivo data for fluconazole, itraconazole, and Candida infections. Clin Infect Dis.
1997;24(2):235-247.
11
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.
12
CLSI. Performance Standards for Antifungal Susceptibility Testing of Yeasts. 1st ed. CLSI supplement M60. Wayne, PA; Clinical and
Laboratory Standards Institute; 2017.
13
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.
14
CLSI. Epidemiological Cutoff Values for Antifungal Susceptibility Testing. 1st ed. CLSI supplement M59. Wayne, PA: Clinical and
Laboratory Standards Institute; 2016.
15
CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi. 3rd ed. CLSI standard M38. Wayne,
PA: Clinical and Laboratory Standards Institute; 2017.
16
Miller JM, Astles R, Baszler T, et al. Biosafety Blue Ribbon Panel, Centers for Disease Control and Prevention (CDC). Guidelines
for safe work practices in human and animal medical diagnostic laboratories. MMWR Suppl. 2012;61(1):1-102.
17
CLSI. Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline—Fourth Edition.
CLSI document M29-A4. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.
18
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.
19
ISO. Quality management systems – Fundamentals and vocabulary. ISO 9000. Geneva, Switzerland: International Organization for
Standardization; 2015.
20
Pfaller MA, Jones RN; College of American Pathologists Microbiology Resource Committee. Performance accuracy of antibacterial
and antifungal susceptibility test methods: report from the College of American Pathologists Microbiology Surveys Program (2001-
2003). Arch Pathol Lab Med. 2006;130(6):767-778.
21
Rex JH, Pfaller MA. Has antifungal susceptibility testing come of age? Clin Infect Dis. 2002;35(8):982-989.

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 25
This document is protected by copyright.
 M27, 4th ed.

22
Fothergill AW, McCarthy DI, Albataineh MT, Sanders C, McElmeel M, Wiederhold NP. The effects of treated vs untreated
polystyrene on caspofungin in vitro activity against Candida species. J Clin Microbiol. 2016;54(3):734-738.
23
Ostrosky-Zeichner L, Rex JH, Pfaller MA, et al. Rationale for reading fluconazole MICs at 24 hours rather than 48 hours when testing
Candida spp. by the CLSI M27-A2 standard method. Antimicrob Agents Chemother. 2008;52(11):4175-4177.
24
Espinel-Ingroff A, Canton E, Peman J, Rinaldi MG, Fothergill AW. Comparison of 24-hour and 48-hour voriconazole MICs as
determined by the Clinical and Laboratory Standards Institute broth microdilution method (M27-A3 document) in three laboratories:
results obtained with 2,162 clinical isolates of Candida spp. and other yeasts. J Clin Microbiol. 2009;47(9):2766-2771.
25
Pfaller MA, Messer SA, Boyken L, et al. Further standardization of broth microdilution methodology for in vitro susceptibility testing
of caspofungin against Candida species by use of an international collection of more than 3,000 clinical isolates. J Clin Microbiol.
2004;42(7):3117-3119.
26
Anaissie E, Paetznick V, Bodey GP. Fluconazole susceptibility testing of Candida albicans: microtiter method that is independent of
inoculum size, temperature, and time of reading. Antimicrob Agents Chemother. 1991;35(8):1641-1646.
27
Arthington-Skaggs BA, Lee-Yang W, Ciblak MA, et al. Comparison of visual and spectrophotometric methods of broth microdilution
MIC end point determination and evaluation of a sterol quantitation method for in vitro susceptibility testing of fluconazole and
itraconazole against trailing and nontrailing Candida isolates. Antimicrob Agents Chemother. 2002;46(8):2477-2481.
28
Arthington-Skaggs BA, Warnock DW, Morrison CJ. Quantitation of Candida albicans ergosterol content improves the correlation
between in vitro antifungal susceptibility test results and in vivo outcome after fluconazole treatment in a murine model of invasive
candidiasis. Antimicrob Agents Chemother. 2000;44(8):2081-2085.
29
Rex JH, Nelson PW, Paetznick VL, Lozano-Chiu M, Espinel-Ingroff A, Anaissie EJ. Optimizing the correlation between results of
testing in vitro and therapeutic outcome in vivo for fluconazole by testing critical isolates in a murine model of invasive candidiasis.
Antimicrob Agents Chemother. 1998;42(1):129-134.
30
Revankar SG, Kirkpatrick WR, McAtee RK, et al. Interpretation of trailing endpoints in antifungal susceptibility testing by the
National Committee for Clinical Laboratory Standards method. J Clin Microbiol. 1998;36(1):153-156.
31
Wanger A, Mills K, Nelson PW, Rex JH. Comparison of Etest and National Committee for Clinical Laboratory Standards broth
macrodilution method for antifungal susceptibility testing: enhanced ability to detect amphotericin B-resistant Candida isolates.
Antimicrob Agents Chemother. 1995;39(11):2520-2522.
32
Pappas PG, Kauffman CA, Andes DR, et al. Clinical practice guideline for the management of candidiasis: 2016 update by the
Infectious Diseases Society of America. Clin Infect Dis. 2016;62(4):e1-e50.
33
Aller AI, Martin-Mazuelos E, Lozano F, et al. Correlation of fluconazole MICs with clinical outcome in cryptococcal infection.
Antimicrob Agents Chemother. 2000;44(6):1544-1548.
34
Kullberg BJ, Sobel JD, Ruhnke M, et al. Voriconazole versus a regimen of amphotericin B followed by fluconazole for candidemia
in non-neutropenic patients: a randomized non-inferiority trial. Lancet. 2005;366(9495):1435-1442.
35
Pfaller MA, Messer SA, Rhomberg PR, Jones RN, Castanheira M. In vitro activities of isavuconazole and comparator antifungal
agents tested against a global collection of opportunistic yeasts and molds. J Clin Microbiol. 2013;51(8):2608-2616.
36
Pfaller MA, Boyken L, Hollis RJ, Messer SA, Tendolkar S, Diekema DJ. In vitro activities of anidulafungin against more than 2,500
clinical isolates of Candida spp., including 315 isolates resistant to fluconazole. J Clin Microbiol. 2005;43(11):5425-5427.
37
Pfaller MA, Boyken L, Hollis RJ, Messer SA, Tendolkar S, Diekema DJ. Global surveillance of in vitro activity of micafungin against
Candida: a comparison with caspofungin by CLSI-recommended methods. J Clin Microbiol. 2006;44(10):3533-3538.
38
Espinel-Ingroff et al. Quality control and reference guidelines for CLSI broth microdilution method (M38-A document) for
susceptibility testing of an anidulafungin against molds. J Clin Microbiol. 2007;45(7):2180-2182.
39
Ghannoum et al. Interlaboratory study of quality control isolates for a broth microdilution method (modified CLSI M38-A) for testing
susceptibilities of dermatophytes to antifungals. J Clin Microbiol. 2006;44(12):4353-4356.
40
Krisher et al. Quality control parameters for broth microdilution tests of anidulafungin. J Clin Microbiol. 2004;42(1):490.
41
Pasarell L, McGinnis MR. Viability of fungal cultures maintained at -70°C. J Clin Microbiol. 1992;30(4):1000-1004.

Subcommittee
26 on Antifungal Susceptibility©Tests
Clinical and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

Appendix A. Preparing Dilution Series of Water-Insoluble Antifungal Agents to Be

Used in Broth Dilution Susceptibility Tests
Solvent, Final
mL Intermediate Concentration
Concentration, Volume, (eg, Concentration, at 1:100,
Step µg/mL Source mL + DMSO) = µg/mL = µg/mL Log2
1 1600 Stock – – 1600 16 4
2 1600 Stock 0.5 0.5 800 8 3
3 1600 Stock 0.5 1.5 400 4 2
4 1600 Stock 0.5 3.5 200 2 1
5 200 Step 4 0.5 0.5 100 1 0
6 200 Step 4 0.5 1.5 50 0.5 −1
7 200 Step 4 0.5 3.5 25 0.25 −2
8 25 Step 7 0.5 0.5 12.5 0.125 −3
9 25 Step 7 0.5 1.5 6.25 0.0625 −4
10 25 Step 7 0.5 3.5 3.13 0.0313 −5
Abbreviation: DMSO, dimethyl sulfoxide.

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 27
This document is protected by copyright.
 M27, 4th ed.

Appendix B. Composition of Roswell Park Memorial Institute 1640 Culture Medium

(With Glutamine and Phenol Red but Without Bicarbonate)
Constituent Water, g/L Constituent Water, g/L

L-arginine (free base) 0.2 Biotin 0.0002

L-asparagine (anhydrous) 0.05 D-pantothenic acid 0.00025

L-aspartic acid 0.02 Choline chloride 0.003

L-cystine • 2HCl 0.0652 Folic acid 0.001

L-glutamic acid 0.02 Myo-inositol 0.035

L-glutamine 0.3 Niacinamide 0.001

Glycine 0.01 PABA 0.001

L-histidine (free base) 0.015 Pyridoxine HCl 0.001

L-hydroxyproline 0.02 Riboflavin 0.0002

L-isoleucine 0.05 Thiamine HCl 0.001

L-leucine 0.05 Vitamin B12 0.000005

L-lysine • HCl 0.04 Calcium nitrate  H2O 0.1

L-methionine 0.015 Potassium chloride 0.4

L-phenylalanine 0.015 Magnesium sulfate (anhydrous) 0.04884

L-proline 0.02 Sodium chloride 6

L-serine 0.03 Sodium phosphate, dibasic 0.8

(anhydrous)

L-threonine 0.02 D-glucose 2

L-tryptophan 0.005 Glutathione, reduced 0.001

L-tyrosine • 2Na 0.02883 Phenol red, Na 0.0053

L-valine 0.02
Abbreviation: PABA, para-aminobenzoic acid.

Subcommittee
28 on Antifungal Susceptibility©Clinical
Tests and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

Appendix C. Preparing Roswell Park Memorial Institute 1640 Culture Medium

Roswell Park Memorial Institute (RPMI) 1640 culture medium buffered with 0.165 mol/L
3-(N-morpholino) propanesulfonic acid (MOPS), 1 L (see Appendix B)

To prepare 10.4 g powdered RPMI 1640 culture medium (with glutamine and phenol red, without
bicarbonate) 34.53 g MOPS buffer, follow the steps below.

Step Action Comment

1. Dissolve powdered medium in 900 mL distilled water.
2. Add MOPS and stir until dissolved. MOPS is at a final concentration
of 0.165 mol/L.
3. While stirring, adjust the pH to 7 at 25°C using 1 mol/L
sodium hydroxide.
4. Add more water to bring medium to a final volume of 1 L.
5. Filter, sterilize, and store at 4°C until use.
Abbreviations: MOPS, 3-(N-morpholino) propanesulfonic acid; pH, negative logarithm of hydrogen ion concentration.

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 29
This document is protected by copyright.
 M27, 4th ed.

Appendix D. Preparing Dilution Series of Water-Soluble Antifungal Agents to Be

Used in Broth Dilution Susceptibility Tests
Final
Intermediate Concentration
Concentration, Volume, Medium, Concentration, at 1:10,
Step µg/mL Source mL + mL = µg/mL = µg/mL Log2
1 5120 Stock 1 7 640 64 6
2 640 Step 1 1 1 320 32 5
3 640 Step 1 1 3 160 16 4
4 160 Step 3 1 1 80 8 3
5 160 Step 3 0.5 1.5 40 4 2
6 160 Step 3 0.5 3.5 20 2 1
7 20 Step 6 1 1 10 1 0
8 20 Step 6 0.5 1.5 5 0.5 −1
9 20 Step 6 0.5 3.5 2.5 0.25 −2
10 2.5 Step 9 1 1 1.25 0.125 −3
11 2.5 Step 9 0.5 1.5 0.625 0.0625 −4
12 2.5 Step 9 0.5 3.5 0.3125 0.03125 −5

Subcommittee
30 on Antifungal Susceptibility©Clinical
Tests and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

Appendix E. 0.5 McFarland Barium Sulfate Turbidity Standard

To standardize the inoculum density, a barium sulfate turbidity standard (0.5 McFarland standard) should
be used. The procedure for preparing a barium sulfate turbidity standard (0.5 McFarland standard) is listed
below.

Step Action Comment

1. Add 0.5 mL of 0.048 mol/L BaCl2 (1.175%
w/v BaCl2 × 2H2O) to 99.5 mL of 0.18 mol/L
H2SO4 (1% v/v).
2. Verify the correct density of the turbidity The absorbance at 625 nm should be
standard by using a spectrophotometer with a 0.08–0.13 for the 0.5 McFarland standard.
1-cm light path.
3. Distribute 4–6 mL into screw-cap tubes. Screw-cap tubes should be the same size as
those used in growing or diluting the broth
culture inoculum.
4. Tightly seal the tubes. Store the prepared tubes in the dark at room
temperature.
5. Vortex this turbidity standard immediately
before use.
6. Replace standards or recheck their densities
monthly after preparation.

Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 31
This document is protected by copyright.
 M27, 4th ed.

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

M27 covers the QSE indicated by an “X.” For a description of the other documents listed in the grid, please refer to
the Related CLSI Reference Materials section.
Customer Focus

Nonconforming
Documents and
Purchasing and

Improvement
Facilities and
Organization

Management

Management

Management

Assessments
Information
Equipment
Personnel

Continual
Inventory

Records
Process
Safety

Event
X
M23
M29
M38
M57
M59
M60

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.

M27 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

Sample collection

Results reporting
Sample transport

and processing
Sample receipt

Results review
and follow-up

and archiving
Interpretation
Examination

Examination

management
ordering

Sample

X X X X X
M38 M38 M38 M38 M38
M59 M59 M59
M60 M60 M60

Subcommittee
32 on Antifungal Susceptibility©Clinical
Tests and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 M27, 4th ed.

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 selecting appropriate breakpoints and quality
control ranges for antimicrobial agents.

M29 Protection of Laboratory Workers From Occupationally Acquired Infections. 4th ed., 2014. Based on US
regulations, this document provides guidance on the risk of transmission of infectious agents by aerosols,
droplets, blood, and body substances in a laboratory setting; specific precautions for preventing the laboratory
transmission of microbial infection from laboratory instruments and materials; and recommendations for the
management of exposure to infectious agents.

M38 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi. 3rd ed.,
2017. This standard includes antifungal agent selection, preparation of antifungal stock solutions and dilutions
for testing, test procedure implementation and interpretation, and quality control requirements for susceptibility
testing of filamentous fungi (moulds) that cause invasive and cutaneous fungal infections.

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.

M60 Performance Standards for Antifungal Susceptibility Testing of Yeasts. 1st ed., 2017. 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.

CLSI documents are continually reviewed and revised through the CLSI consensus process; therefore, readers should refer to the
most current editions.
Subcommittee on Antifungal
©Clinical and Laboratory Susceptibility
Standards Institute. All rights reserved.Tests 33
This document is protected by copyright.
 M27, 4th ed.

NOTES

Subcommittee
34 on Antifungal Susceptibility©Clinical
Tests and Laboratory Standards Institute. All rights reserved.
This document is protected by copyright.
 Explore the Latest Offerings From CLSI!
As we continue to set the global standard for quality in laboratory testing, we are adding products and
programs to bring even more value to our members and customers.

By becoming a CLSI member, your laboratory will join 1,600+ other

influential organizations all working together to further CLSI’s efforts
to improve health care outcomes. You can play an active role in
raising global laboratory testing standards—in your laboratory, and
around the world.

Find out which membership option is best for you at www.clsi.org/membership.

Find what your laboratory needs to succeed! CLSI U provides

convenient, cost-effective continuing education and training
resources to help you advance your professional development. We
have a variety of easy-to-use, online educational resources that make
eLearning stress-free and convenient for you and your staff.

See our current educational offerings at www.clsi.org/education.

When laboratory testing quality is critical, standards are needed and

there is no time to waste. eCLIPSE™ Ultimate Access, our cloud-based
online portal of the complete library of CLSI standards, makes it easy
to quickly find the CLSI resources you need.

Learn more and purchase eCLIPSE at clsi.org/eCLIPSE.

Subcommittee on Antifungal Susceptibility Tests

This document is protected byFor more information, visit www.clsi.org today.
copyright.
 950 West Valley Road, Suite 2500, Wayne, PA 19087 USA PRINT ISBN 1-56238-826-6
P: +1.610.688.0100 Toll Free (US): 877.447.1888 F: +1.610.688.0700 ELECTRONIC ISBN 1-56238-827-4
E: [email protected] www.clsi.org

Subcommittee on Antifungal Susceptibility Tests

This document is protected by copyright.