5th Edition

M23
Development of In Vitro Susceptibility Testing
Criteria and Quality Control Parameters

This guideline discusses the necessary and recommended data for

selecting appropriate breakpoints and quality control ranges for
antimicrobial agents.

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

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 Clinical and Laboratory Standards Institute
Setting the standard for quality in medical laboratory testing around the world.

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 M23, 5th ed.
January 2018
Replaces M23, 4th ed.
Development of In Vitro Susceptibility Testing Criteria and Quality Control
Parameters
Matthew A. Wikler, MD, MBA, FIDSA
James S. Lewis II, PharmD, FIDSA
Greg Moeck, PhD
David P. Nicolau, PharmD, FCCP, FIDSA
Michael Satlin, MD, MS

Abstract
Clinical and Laboratory Standards Institute guideline M23—Development of In Vitro Susceptibility Testing Criteria and Quality
Control Parameters offers guidance for developing breakpoints and QC ranges for antimicrobial susceptibility tests against aerobic
and anaerobic bacteria, as well as selected fungi, according to CLSI antimicrobial susceptibility testing standards. It describes the data
used by the Subcommittees on Antimicrobial Susceptibility Testing and Antifungal Susceptibility Tests to establish these breakpoints
and QC ranges for antimicrobial agents, including microbiological data, pharmacokinetic and pharmacodynamic characteristics, and
clinical data. As antimicrobial agents are used in practice, additional experience accrued may be used to reassess breakpoints or QC
ranges. Users of these guidelines should understand that susceptibility test results cannot predict clinical outcomes with absolute
certainty. They should be used along with best clinical judgment and laboratory support to most effectively serve the patient.

Clinical and Laboratory Standards Institute (CLSI). Development of In Vitro Susceptibility Testing Criteria and Quality Control
Parameters. 5th ed. CLSI guideline M23 (ISBN 1-56238-842-8 [Print]; ISBN 1-56238-843-6 [Electronic]). Clinical and Laboratory
Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2018.

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

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 M23, 5th ed.

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Suggested Citation

CLSI. Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters. 5th ed.
CLSI guideline M23. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.

Previous Editions:
November 1986, October 1990, August 1992, July 1994, April 1998, May 2001, October 2008, January
2016

ISBN 1-56238-842-8 (Print)

ISBN 1-56238-843-6 (Electronic)
ISSN 1558-6502 (Print)
ISSN 2162-2914 (Electronic) Volume 38, Number 6

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 M23, 5th ed.

Committee Membership

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

Subcommittee on Antimicrobial Susceptibility Testing

Melvin P. Weinstein, MD Romney M. Humphries, PhD, D(ABMM) Tony Mazzulli, MD, FACP,
Chairholder Accelerate Diagnostics FRCP(C)
Rutgers Robert Wood Johnson USA Mount Sinai Hospital
Medical School Canada
USA Stephen G. Jenkins, PhD, D(ABMM),
F(AAM) Robin Patel, MD
Jean B. Patel, PhD, D(ABMM) Weill Cornell Medicine Mayo Clinic
Vice-Chairholder USA USA
Centers for Disease Control and
Prevention James S. Lewis II, PharmD, FIDSA Sandra S. Richter, MD, D(ABMM),
USA Oregon Health and Science University FCAP, FIDSA
USA Cleveland Clinic
George M. Eliopolous, MD USA
Beth Israel Deaconess Medical Center Brandi Limbago, PhD
USA Centers for Disease Control and Prevention Michael Satlin, MD, MS
USA New York Presbyterian Hospital
Marcelo F. Galas USA
Pan American Health Organization Amy J. Mathers, MD, D(ABMM)
USA University of Virginia Medical Center Barbara L. Zimmer, PhD
USA Beckman Coulter – West Sacramento
USA

Working Group on AST Criteria and QC Parameters

Matthew A. Wikler, MD, MBA, James S. Lewis II, PharmD, FIDSA David P. Nicolau, PharmD, FCCP,
FIDSA Oregon Health and Science University FIDSA
Chairholder USA Hartford Hospital
IDTD Consulting USA
USA Greg Moeck, PhD
The Medicines Company Michael Satlin, MD, MS
Canada New York Presbyterian Hospital
USA

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 M23, 5th ed.

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, M(ASCP) Editor Editor
Project Manager

Acknowledgment for the Expert Panel on Microbiology

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

Expert Panel on Microbiology

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

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Contents

Abstract ....................................................................................................................................................i
Committee Membership........................................................................................................................ iii
Foreword .............................................................................................................................................. vii
Subcommittee on Antimicrobial Susceptibility Testing Mission Statement .........................................ix
Introduction ....................................................................................................................... 1
Scope............................................................................................................................. 1
Background ................................................................................................................... 1
Terminology.................................................................................................................. 2
Development of Susceptibility Tests Methods.................................................................. 7
Establishing the Reference Method for an Antimicrobial Agent .................................. 7
Antibacterial Fixed Combination Studies (Such as -Lactam Combination
Agents) .......................................................................................................................... 8
Developing Disks for Disk Diffusion Tests .................................................................. 9
Validating Microbiological Data Derived From Sources Other Than
Reference Methods ....................................................................................................... 9
Quality Control................................................................................................................ 11
Selecting Quality Control Strains ............................................................................... 11
Procedure for Establishing or Revising Quality Control Strains or Ranges ............... 12
Quality Control Data Presentation and Interpretation................................................. 16
Procedures for Establishing Breakpoints ........................................................................ 19
New Breakpoints......................................................................................................... 20
Revision of Breakpoints.............................................................................................. 23
Provisional Breakpoints .............................................................................................. 27
Periodic Breakpoint Review ....................................................................................... 27
Surrogate Testing ........................................................................................................ 29
Minimal Inhibitory Concentration Breakpoints .............................................................. 31
Epidemiological Cutoff Value .................................................................................... 32
Nonclinical Pharmacokinetic-Pharmacodynamic Cutoff............................................ 36
Clinical Exposure-Response Cutoff ............................................................................ 42
Clinical Cutoff ............................................................................................................ 44
Consideration of the Cutoffs to Determine Breakpoints ............................................. 46
Disk Diffusion Breakpoints............................................................................................. 49
Selecting Isolates and Sample Size ............................................................................. 49
Reagent Disks ............................................................................................................. 49
Error-Rate Bounded Method for Selecting Disk Diffusion Breakpoints
Based on Comparison With Dilution Test Results ..................................................... 49
Conclusion....................................................................................................................... 52
Supplemental Information ............................................................................................... 52
References ................................................................................................................................ 53
Appendix A. Guidelines for Clinical Isolate Selection and Sample Size ................................ 55
Appendix B. Sample Data Presentations ................................................................................. 58

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Contents (Continued)
Appendix C. Statement of Policy of the Antimicrobial Susceptibility Testing
Subcommittees of the Clinical and Laboratory Standards Institute ......................................... 62
Appendix D. Suggested Information to Be Covered on the Submission Cover Page ............. 63
Appendix E. Drug “X” Minimal Inhibitory Concentration vs Zone Diameter ........................ 64
The Quality Management System Approach ........................................................................... 66
Related CLSI Reference Materials .......................................................................................... 68

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 M23, 5th ed.

Foreword
CLSI develops standardized reference methods that measure the susceptibility of bacteria and fungi to
antimicrobial agents in vitro. In this regard, the CLSI Subcommittee on Antimicrobial Susceptibility
Testing (AST) is responsible for developing and updating the following CLSI susceptibility testing
documents:

 M02—Performance Standards for Antimicrobial Disk Susceptibility Tests1

 M07—Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically2

 M45—Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or
Fastidious Bacteria3

 M11—Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria4

 M100—Performance Standards for Antimicrobial Susceptibility Testing5 (supplement for M02,1 M07,2
and M114)

The CLSI Subcommittee on Antifungal Susceptibility Tests is responsible for developing and updating the
following CLSI susceptibility testing documents:

 M27—Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts6

 M44—Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts7

 M60—Performance Standards for Antifungal Susceptibility Testing of Yeasts8 (supplement for M276
and M447)

 M38—Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi9

 M51—Method for Antifungal Disk Diffusion Susceptibility Testing of Nondermatophyte Filamentous

Fungi10

 M61—Performance Standards for Antifungal Susceptibility Testing of Filamentous Fungi11

(supplement for M389 and M5110)

M23 is an important foundation guideline that supports these susceptibility testing documents. M23’s
purpose is to provide guidance on the data submitted by sponsors and the procedures followed by the CLSI
Subcommittee on AST to establish or revise QC ranges and susceptibility testing breakpoints for inclusion
in CLSI documents. The process for determining breakpoints and QC ranges for antifungal agents
is broadly the same as for the antibacterial agents, and the principles described in M23 also apply to
antifungal agents.

This guideline recognizes that submissions may be made by a wide variety of organizations or individuals
and that it is important to ensure the same processes are followed regardless of the data source.
Nevertheless, it also recognizes that the extent of the data that can be provided to support new or revised
breakpoints may vary significantly due to factors that include, but are not limited to, the age of the
antimicrobial agent and whether the sponsor has access to raw data or only published data.

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Essential Information

Content in this guideline marked with an asterisk (*) describes essential information required for review by
the CLSI Subcommittee on AST. All chapters and subchapters without an asterisk describe additional
information that may be supplied if available and that may be useful in supporting the selection of QC
ranges and susceptibility testing breakpoints.

Overview of Changes

This guideline replaces the previous edition of the approved guideline, M23, 4th ed., published in 2016.
Several changes were made in this edition, including:

 Deleted Subchapter 4.1.3 on publication of breakpoints that are different from those approved by the
US Food and Drug Administration

 Added a new subchapter (Subchapter 4.4) that describes a new process for periodically reviewing
established breakpoints

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

Key Words

Antimicrobial agents, standard dilution methods for bacteria that grow aerobically, standard disk diffusion
test, standard reference method for anaerobes, susceptibility testing

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Subcommittee on Antimicrobial Susceptibility Testing Mission Statement

The Subcommittee on Antimicrobial Susceptibility Testing is composed of representatives from the
professions, government, and industry, including microbiology laboratories, government agencies, health
care providers and educators, and pharmaceutical and diagnostic microbiology industries. Using the CLSI
voluntary consensus process, the subcommittee develops standards that promote accurate antimicrobial
susceptibility testing and appropriate reporting.

The mission of the Subcommittee on Antimicrobial Susceptibility Testing is to:

 Develop standard reference methods for antimicrobial susceptibility tests.

 Provide quality control parameters for standard test methods.

 Establish breakpoints for the results of standard antimicrobial susceptibility tests and provide
epidemiological cutoff values when breakpoints are not available.

 Provide suggestions for testing and reporting strategies that are clinically relevant and cost-effective.

 Continually refine standards and optimize detection of emerging resistance mechanisms through
development of new or revised methods, breakpoints, and quality control parameters.

 Educate users through multimedia communication of standards and guidelines.

 Foster a dialogue with users of these methods and those who apply them.

The ultimate purpose of the subcommittee’s mission is to provide useful information to enable
laboratories to assist the clinician in the selection of appropriate antimicrobial therapy for patient care. The
standards and guidelines are meant to be comprehensive and to include all antimicrobial agents for which
the data meet established CLSI guidelines. The values that guide this mission are quality, accuracy, fairness,
timeliness, teamwork, consensus, and trust.

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Development of In Vitro Susceptibility Testing Criteria and Quality Control

Parameters
Introduction

This chapter includes:

 Guideline’s scope and applicable exclusions

 Background information pertinent to the guideline’s content

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

 Terms and definitions used in the guideline

 Abbreviations and acronyms used in the guideline

Scope

This guideline provides direction for determining breakpoints and QC parameters for antimicrobial agents
that have a direct action on microorganisms. The intended audience includes sponsors (eg, antimicrobial
agent manufacturers) planning to submit data to establish or revise QC ranges and susceptibility testing
breakpoints and interpretive categories for inclusion in CLSI susceptibility testing documents. The methods
described do not apply to:

 Slow-growing mycobacteria, for which specific guidance is available (see CLSI document M2412)

 Antimicrobial agents formulated for direct administration to skin or mucous membranes or for
inhalation

 Antimicrobial agents that are intended to exert activity within the gut lumen

Guidance presented in M23 applies only to CLSI procedures and documents.

Background

Susceptibility testing breakpoints, interpretive categories, and QC parameters are established by the CLSI
Subcommittee on Antimicrobial Susceptibility Testing (AST) after comprehensive review of all available
relevant data. This guideline describes the procedures to be followed by the CLSI Subcommittee on AST
and by sponsors intending to submit data to facilitate timely review and decision-making processes. Data
requirements to support setting new breakpoints and QC parameters and amendments to existing
breakpoints are described.

The CLSI Subcommittee on AST has developed standardized methods that make it possible for laboratories
to perform reliable and meaningful broth dilution and disk diffusion susceptibility testing of fungi (see
CLSI documents M27,6 M38,9 M44,7 and M5110). The process for determining breakpoints, interpretive
categories, and QC ranges for antifungal agents is broadly the same as for the antibacterial agents. Thus, it
may be assumed that the principles described in this guideline apply equally to antifungal agents. For this
reason, the guideline refers to antimicrobial agents throughout. Where reference is made to the CLSI
Subcommittee on AST, in most instances the same applies to the CLSI Subcommittee on Antifungal
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Susceptibility Tests. A CLSI document on the criteria for developing and using epidemiological cutoff
values (ECVs) for guiding clinical decisions when testing fungi against antifungal drug combinations for
which there are no breakpoints has been published (see CLSI documents M5713 and M5914). The principles
outlined in this guideline for establishing and revising ECVs apply to antibacterial agents as well.

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: Mandates are generally reserved for CLSI standards but are occasionally allowed in CLSI
guidelines. In CLSI guidelines, use of the term “must” is either 1) based on a requirement or 2) indicative
of a necessary step to ensure patient safety or proper fulfillment of a procedure. The subcommittee
evaluated use of the term “must” and deemed it appropriate.

Definitions

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

clinical cutoff – a cutoff that is derived from any correlation there may be between clinical outcome and
the minimal inhibitory concentrations of an antimicrobial agent(s) for the infecting pathogen(s).

clinical exposure-response (CER) cutoff – the highest minimal inhibitory concentration value at which
efficacy would be predicted in patients based on CER relationships for efficacy in infected patients and on
human pharmacokinetics.

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

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

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 non-wild-type – 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 agent being evaluated.

interpretive category – category derived from microbiology 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:

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

MIC or zone diameter value breakpoints and interpretive categories are established per M23 for
categories of susceptible, intermediate, and resistant (and susceptible-dose dependent and
nonsusceptible, 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 dosage 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
dosage 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,
which should prevent small, uncontrolled, technical factors from causing major discrepancies in
interpretations, especially for drugs with narrow pharmacotoxicity margins.

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 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.

 Nonsusceptible (NS) – a category used for isolates for which only a susceptible breakpoint is
designated because of the absence or rare occurrence of resistant strains. Isolates for which the
antimicrobial agent MICs are above or zone diameters below the value indicated for the susceptible
breakpoint should be reported as nonsusceptible; NOTE 1: An isolate that is interpreted as
nonsusceptible does not necessarily mean that the isolate has a resistance mechanism. It is possible
that isolates with MICs above the susceptible breakpoint that lack resistance mechanisms may be
encountered within the wild-type distribution subsequent to the time the susceptible-only
breakpoint was set; NOTE 2: The term “nonsusceptible” should not be used when describing an
organism/drug category with SDD or intermediate and resistant interpretive categories. Isolates that
are in the categories of “intermediate” or “resistant” could be called “not susceptible” rather than
“nonsusceptible.”

minimal inhibitory concentration (MIC) – the lowest concentration of an antimicrobial agent that
prevents visible growth of a microorganism in an agar or broth dilution susceptibility test.

nonclinical pharmacokinetic-pharmacodynamic (PK-PD) cutoff – the highest minimal inhibitory

concentration value at which efficacy would be predicted in patients based on nonclinically derived PK-PD
relationships and human PK exposures.

on scale – indicates that minimal inhibitory concentration (MIC) concentrations to be tested include at least
one dilution above and one dilution below the MIC end point.

pharmacodynamics (PD) – the relationship between the unbound drug concentration over time and the
resulting antimicrobial effect.

pharmacokinetics (PK) – the study of the time course of drug absorption, distribution, metabolism, and
excretion.

pharmacokinetic-pharmacodynamic (PK-PD) index – the quantitative relationship between a measure

of drug exposure such as area under the curve and a microbiological parameter such as minimal inhibitory
concentration.

pharmacokinetic-pharmacodynamic (PK-PD) magnitude – the numerical value of the PK-PD index.

pharmacokinetic-pharmacodynamic (PK-PD) target – a magnitude for a PK-PD index at which a

desired level of predicted response is achieved.

quality assurance (QA) – 1) part of quality management focused on providing confidence that quality
requirements will be fulfilled15; 2) a comprehensive set of policies, procedures, and practices used to
monitor the laboratory’s entire testing process and ensure that the testing site’s results are reliable; NOTE:
The practice that encompasses all procedures and activities directed toward ensuring that a specified quality
of product is achieved and maintained. In the testing environment, this includes monitoring all the raw
materials, supplies, instruments, procedures, sample collection/transport/storage/processing,
recordkeeping, calibrating and maintaining equipment, quality control, proficiency testing, training of
personnel, and all else involved in the production of the data reported.

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quality control (QC) – part of quality management focused on fulfilling quality requirements15; NOTE:
A system for ensuring maintenance of proper standards by periodic inspection of the results and the
operational techniques that are used to ensure accuracy and reproducibility.

Abbreviations and Acronyms

AST antimicrobial susceptibility testing

ATCC®a American Type Culture Collection
AUC area under the curve
CART classification and regression tree
CER clinical exposure-response
CSF cerebrospinal fluid
DNA deoxyribonucleic acid
ECV epidemiological cutoff value
ELF epithelial lining fluid
ESBL extended-spectrum β-lactamase
FDA US Food and Drug Administration
MIC minimal inhibitory concentration
PAE postantibiotic effect
PD pharmacodynamic
PK pharmacokinetic
PK-PD pharmacokinetic-pharmacodynamic
PTA probability of target attainment
QA quality assurance
QC quality control
RNA ribonucleic acid
SD standard deviation

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

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Development of Susceptibility Tests Methods

This chapter includes:

 Data needed to establish the reference methods for individual antimicrobial agents or fixed
combinations of agents

 Selection of appropriate disk contents

 Requirements to support the validity of data generated using an alternative to an established reference
method

CLSI develops standardized reference methods for in vitro tests that measure the susceptibility of
microorganisms to antimicrobial agents. These methods may be used for routine AST of patient isolates
and for evaluating commercial devices that will be used in laboratories for AST, including testing of new
agents or systems by drug or device manufacturers.

This chapter considers the data needed to establish the reference methods for individual antimicrobial
agents or fixed combinations of agents. For some antimicrobial agents, modifications of standard reference
methods might be necessary to establish a reproducible in vitro test. The data needed to justify these
modifications are also described.

In addition, the chapter considers data needed for selecting appropriate disk contents whenever disk
diffusion susceptibility testing is feasible (based on the physicochemical properties of the agent). Finally,
the requirements to support the validity of data submitted to the CLSI Subcommittee on AST that were
generated using an alternative to an established reference method are considered.

Establishing the reference method for an individual antimicrobial agent should occur before selecting
provisional breakpoints to be applied to isolates obtained from patients in clinical trials. Once the reference
method has been established, preliminary QC testing (Tier 1 studies) should commence. If more than one
susceptibility testing method is proposed, the sponsor should establish the equivalency of methods (eg, agar
dilution and broth microdilution) as described in Subchapter 3.2.1.

Establishing the Reference Method for an Antimicrobial Agent

* The performance of each antimicrobial agent should be assessed in susceptibility testing studies
conducted using the CLSI standard reference methods relevant to its spectrum of activity as described in
the applicable CLSI documents (eg, M02,1 M07,2 M11,4 M276). These studies should establish:

 Specifications for solvents, diluents, or special supplements for the antimicrobial agent and instructions
for preparing stock solutions

 Stability of appropriate concentrations of the antimicrobial agent at specified incubation and storage
temperatures

 Influence of the growth medium, such as variations in pH, divalent cations, and age of media

 Influence of inoculum density

 Influence of incubation conditions, such as atmospheric conditions (eg, variations in CO2) and the
duration of incubation

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 Special instructions for reading reference minimal inhibitory concentration (MIC) tests (eg, 50%, 80%,
or 100% inhibition) or disk reading (eg, lighting conditions, inner and outer zone edges)
 The inclusion of pictures is encouraged to assist the user with special instructions or difficult to
read end points.

 Comparison of broth microdilution with agar dilution whenever both methods are being proposed as
reference methods for a specific antimicrobial agent
 In this case, see Appendix A regarding the sample size of studies to compare the two methods.

 Also, see Subchapter 3.2.1 regarding Tier 1 QC studies when the sponsor proposes more than one
reference method.

For some antimicrobial agents, modifications of standard reference methods might be necessary.16 Some
of the more common reasons for modifications include:

 The agent sticks to laboratory ware (eg, the plastic in microdilution trays).
 In such cases, addition of polysorbate 80 to solvents, diluents, and media may be needed to
determine unbound drug MICs.

 The agent needs the presence of a specific concentration of a divalent cation (such as Ca++) for
activation, in which case media supplementation may be necessary.

 The agent is inactivated in the presence of oxygen, in which case susceptibility testing may have to be
conducted only with freshly prepared media.

* If a sponsor wishes to propose a modification to the standard reference method, a submission should be
made to the Working Group on Methods Development and Standardization that includes the information
listed below. If the modification is approved, the information will be included in the applicable CLSI
documents. Except for the antimicrobial agent itself, components used in the reference method should be
available from multiple manufacturers unless there is adequate justification and the proposal is approved
by CLSI.

 Data to demonstrate that the standard reference methods do not correctly measure the antimicrobial
activity of the agent
 The data should include drug stability and/or activity studies in the reference testing medium.

 Data on all the variables tested to identify the reason for the effect on antimicrobial activity

 Data to demonstrate improved antimicrobial activity when using the proposed method
 Comparisons of AST results generated by the standard reference method with results generated by
the modified method should be provided.

 The data must include a sufficient number of isolates for each genus and species that is associated
with the infection types of interest for the antimicrobial agent. See Appendix A for additional
guidance on selecting isolates and sample size.

Antibacterial Fixed Combination Studies (Such as -Lactam Combination Agents)17,18

* For antibacterial agents that will be used as a combination product, data must be provided to show the
rationale for the concentration of each agent that will be used in AST.

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 The AST results using various concentrations of the two agents in combination should be provided.
 The data should provide a clear rationale for the selected testing conditions.

 If organisms resistant to the combination are available (eg, they belong to a genus or species that is not
within the spectrum of the combination or express a particular resistance mechanism that is not covered
by the combination), a representative sample of these organisms should be included in the AST using
different concentrations of the two agents.
 Resistant mutants generated in the laboratory may also be included. These organisms will help
demonstrate that the selected testing conditions can adequately detect resistant isolates and separate
the resistant and susceptible populations.

 There should be an evaluation of the correlation between AST results and the antibacterial activity
observed in in vitro and/or in vivo pharmacodynamic (PD) models.

Developing Disks for Disk Diffusion Tests19

* Data must be provided to demonstrate that the mass of the drug substance in the disks has been optimized
for the individual antimicrobial agent. For new antimicrobial agents of an existing class, it is not appropriate
to simply select a disk content that is commonly used for others in the same class. Rather, a thorough
evaluation of the appropriate disk content specific to the new agent must be conducted by testing a wide
range against the relevant species.

 The disk content studies must include a sufficient number of isolates for each genus and species that is
associated with the infection types of interest for the antimicrobial agent.
 See Appendix A for additional guidance on selecting isolates and sample size.

 The data from the disk content studies should be analyzed using scattergrams to determine which disk
concentration provides the best correlation between zone diameters and MICs (determined using the
reference method).
 Guidance on data presentation and acceptable discrepancy rates is provided in Subchapter 6.3.

 The ideal disk content provides zone diameters greater than 15 mm and less than 35 mm for most
susceptible strains but no detectable or small zone diameters of inhibition for resistant strains.

When the final disk content is chosen, all subsequent studies should be conducted with reagent disks,
regardless of source (commercial or other), that meet published requirements.20

Validating Microbiological Data Derived From Sources Other Than Reference

Methods

If CLSI reference methods change or if new reference methods are created, data generated using previously
accepted methods are acceptable for consideration by the CLSI Subcommittee on AST when the
relationship between the methods can be demonstrated. Studies initiated after CLSI publication of modified
or new reference methods should use the modified or new reference method. When alternative methods to
the established reference methods for an antimicrobial agent are used to generate microbiological data that
are submitted to the CLSI Subcommittee on AST for consideration, validation studies should be conducted.

* For an alternative broth microdilution method (eg, dried-form panels) or for other alternative test methods
(eg, those that use colorimetric or chromogenic end points or gradient diffusion), the validation of an
alternative AST method for an antimicrobial agent should include the MIC results for an antimicrobial
agent that has been compared with those obtained using the CLSI reference methods (eg, dried-form panels
could be compared with frozen-form panels) when tested in parallel. When available, the alternative and

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reference methods should be similarly compared for another antimicrobial agent of the same class. The
methodology should closely follow the CLSI reference procedures, including inoculum concentration,
medium and supplements, total well volume, and incubation environment (atmosphere and temperature).
The method of interpretation must be manual visual inspection. Any differences compared with the CLSI
reference method must be stated with an evaluation of the possible effect on the results.

* At a minimum, it is recommended that 100 isolates be tested for each group of organisms that is relevant
to the clinical use of the antimicrobial agent and for which separate breakpoints are likely to apply (eg, per
the groups in Table 2 of CLSI document M1005: Enterobacteriaceae, various nonfermentative gram-
negative bacilli, Staphylococcus spp., Streptococcus pneumoniae, other streptococci, Haemophilus spp.,
Enterococcus spp.). See Appendix A for additional guidance on selecting isolates and sample size.

* The QC testing must be performed concurrently and should include a minimum of 20 replicates for each
relevant control strain over the course of the study (see Subchapter 3.2.1). The results should clearly
document variability between methods, any bias for each organism group, and potential resolution of
discord. The target intermethod correlation should be ≥ 90% essential agreement (ie, at least 90% of results
fall within ± 1 log2 dilution step) across all clinical isolates tested. For QC strains, ≥ 95% of results should
fall within the expected range and there should be no evidence of skewing of results. See Subchapter 6.3
for additional guidance on presentation of data and acceptable discrepancy rates.

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Quality Control

This chapter includes:

 Criteria and selection of QC strains

 Procedure for establishing or revising QC strains or ranges
 Presenting and interpreting QC data

QC includes the procedure to monitor the test system to ensure accurate and reproducible results. This goal
is achieved by but not limited to testing carefully selected QC strains with known susceptibility to the
antimicrobial agents tested. The goals of a QC program are to monitor:

 Precision (reproducibility) and accuracy of susceptibility test procedures

 Performance of reagents used in the tests
 Performance of persons who carry out the tests and report the results

Selecting Quality Control Strains

To monitor the performance of in vitro susceptibility tests, it is necessary to know the acceptable variability
in expected results. Using carefully selected QC strains enables the microbiologist to have confidence that
the test is performing within acceptable standards and that the test results of interest are likely to be reliable.

Selection Criteria

Each QC strain should be obtained from a recognized source (eg, American Type Culture Collection
[ATCC®]) to provide broad access for users. All CLSI-recommended QC strains appropriate for the
antimicrobial agent and reference method should initially be evaluated. Occasionally, the standard QC
strains may not be sufficient for an individual antimicrobial agent for routine QC and/or QA purposes (eg,
does not reliably detect a problem testing condition; does not accomplish troubleshooting, validation and/or
verification, or assessment objectives). To select and qualify a new QC strain, the guidance below should
be followed. Before publication in CLSI standards, the new QC strain should be deposited in a recognized
source, if it has not yet been deposited, to ensure availability. MIC results should ideally be on scale, ie,
within the range of concentrations likely to be tested (eg, close to the breakpoint or ECV), or MICs should
fall near the middle of the concentration range tested.

QC strains with resistance mechanisms to a specific antimicrobial agent should ideally be categorized as
resistant by the breakpoint, and QC strains categorized as susceptible by the breakpoint should not contain
mechanisms that confer resistance to the specific antimicrobial agent. However, the process and criteria for
setting breakpoints and QC ranges are independent of each other, so this ideal situation may not always
apply. When selecting potential QC strains, the laboratory should consider:

 Avoiding QC strains for which the disk diffusion zones are very large (eg, > 35 mm) or very small (ie,
not easily measurable)

 Minimizing the number of strains to maintain and test by selecting a QC strain that can be used for
testing several antimicrobial agents

 Using QC strains that are qualified as a current QC strain from a similar method (eg, E. coli ATCC®
35218 with β-lactam combination agents on media used to test fastidious organisms)

 Using QC strains with stable resistance mechanisms

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 Avoiding strains requiring biosafety level 3 practices, when possible

 Using QC strains that can detect problems that may be related to:
 Testing conditions and procedural variations (eg, inoculum, reading, or incubation
time/temperature)

 Manufacturing and shipping and/or storage (eg, ability to detect under- or overpotency, critical
growth factors, or effects of heat, humidity, or light)

 Personnel competence

Routine and Supplemental Quality Control Strains

QC strains and their characteristics are described in multiple CLSI documents (see M02,1 M07,2 M45,3
M11,4 M100,5 M27,6 M60,8 M38,9 M44,7 M51,10 and M6111). Some QC strains are listed as “routine” and
others as “supplemental.” These can be defined as:

 Routine QC strains are tested regularly (eg, daily, weekly) to ensure that the test system performs as
expected and produces results that fall within specified limits. The routine QC strains recommended in
CLSI documents should be included when a laboratory performs CLSI reference AST. For commercial
test systems, manufacturers’ recommendations for QC should be followed.

 Supplemental QC strains are used to assess a particular characteristic of a test or test system in select
situations or may represent alternative QC strains. For example, Haemophilus influenzae ATCC® 10211
is more fastidious than H. influenzae ATCC® 49247 or H. influenzae ATCC® 49766 and is used to
ensure Haemophilus test medium can adequately support the growth of clinical isolates of H. influenzae
and Haemophilus parainfluenzae.

 Supplemental QC strains can also be used to assess the performance of a new test in an individual
laboratory, for training new personnel, or for competence assessment. It is not necessary to include
supplemental QC strains in routine AST QC programs.

Procedure for Establishing or Revising Quality Control Strains or Ranges

Preliminary Quality Control Testing (Tier 1 Studies)

When the reference susceptibility testing method has been confirmed (or a modification proposed) as
described in Subchapter 2.1 and when potential QC strains have been identified, preliminary QC ranges are
established by testing a minimum of 20 to 30 replicates with each proposed QC strain. Each replicate
consists of individually prepared inoculum, and testing is conducted over multiple days. Testing more than
one lot of media at multiple laboratories is recommended but not required. Results of Tier 1 studies should
be presented together with any specific instructions for preparation and testing according to the reference
method.

When two variations of a reference method exist, in the absence of equivalency data, the QC ranges are
noted to apply only to the method used to obtain the data. To establish equivalency of methods (eg, agar
dilution and broth microdilution), testing may be performed at one laboratory following the guidelines in
Table 1. See Appendix A for additional guidance on selecting isolates and sample size.

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Table 1. Tier 1 Testing Guidelines: Establishing Equivalency Between Testing Methods

Broth MIC Agar Dilution MIC
Organisms* 100 100
Total replicates per QC strain 20 20
Days (minimum) 2 2
Media lots 1 1
Laboratories 1 1
*Within the clinical spectrum for the antimicrobial agent.
Abbreviations: MIC, minimal inhibitory concentration; QC, quality control.

Data may be presented in summary form unless significant variation is observed (eg, day-to-day or skewed
results with one or more methods). Guidance on data presentation and acceptable discrepancy rates is
provided in Subchapter 6.3.

If the equivalency of methods is not established by testing, as suggested above, or if this preliminary testing
is not performed, future QC development (eg, Tier 2 studies; see Subchapter 3.2.2) should include all testing
methods for which a QC range is desired. Alternatively, the tables and/or glossaries should clearly indicate
the methods for which the guidelines do not apply, eg “Agar dilution has not been validated for ‘X’ testing
and should be performed by broth dilution only. Performance has been established for broth dilution only.”

Establishing Quality Control Expected Ranges (Tier 2 Studies)

To monitor the performance of in vitro susceptibility tests, it is necessary to know the acceptable variability
in results using appropriate QC strains. A Tier 2 QC study is designed to provide adequate data to establish
expected ranges for QC. Tier 2 studies evaluate reproducibility of the method within a laboratory, among
laboratories, and among reagent lots. All testing must be performed using CLSI reference methods as
appropriate for each organism group and antimicrobial agent (see CLSI documents M02,1 M07,2 M11,4
M276). QC ranges established with Tier 2 QC studies are published in the applicable CLSI documents (see
CLSI documents M608 and M1005).

 To establish QC ranges for an antimicrobial agent, Tier 2 results from at least seven laboratories from
seven separate and distinct institutions must be analyzed.

 For disk diffusion tests, whenever there is more than one manufacturer, evaluation should include two
lots of disks from two different manufacturers.

 Each of the seven laboratories should determine MICs of the antimicrobial agent for 10 replicates of
each QC strain on each of three media lots.
 Each replicate must use individually prepared inoculum suspensions.
 If Mueller-Hinton is used, the lots should be qualified for use in AST.21

 Each of the seven laboratories should also test 10 replicates of each QC strain (as above) on each of
three media lots and using two disk lots for disk diffusion.

 Testing a single replicate of each QC strain per day provides the best opportunity to detect day-to-day
variability.
 A minimum of three days of testing is recommended for broth dilution and disk diffusion testing;
however, agar dilution studies may be performed in two days with a maximum of five replicates
per day.

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 A twofold dilution schedule should be used to provide on-scale QC results.

 Whenever possible, the low end of the QC range should include dilutions that can be “accurately”
prepared (ie, dilutions lower than 0.002 µg/mL should be avoided) and span no more than five
dilutions below the provisional breakpoint for susceptibility.

 A control antimicrobial agent of the same or similar class should be tested in the same way and in
parallel, except that testing on one lot of media is sufficient.
 If there is no such control, an antimicrobial agent with a similar spectrum of activity should be
used.

 The results for the control must be within the expected control limits each day of testing.

 If the results are not within the expected control limits each day of testing, an investigation into the
cause of the problem should be conducted and the day’s testing should be repeated.

 If the day’s testing is being repeated, the original data should be footnoted, and if the original results
are shown to be erroneous or invalid, the original data should be excluded from the data analysis.

 Each laboratory should determine the colony counts to document the inoculum for at least one QC
strain on each day of testing.
 A minimum of five colony counts should be presented for each QC strain.

 Data should be reported to assess the consistency of the inoculum preparation and, if needed, for
use in troubleshooting.

The requirements for a Tier 2 study, as described above, are summarized in Table 2.

Table 2. Tier 2 Study: Requirements to Establish QC Expected Range

Category Number Required
MIC Study Disk Diffusion Study
Laboratories* 7 7
Media lots (different manufacturers)† 3 3
‡
Replicates (individual inoculum, maximum 4 per day) 10 10
Disk lots (different manufacturers) N/A 2
Total data points 210 (7 • 3 • 10) 420 (7 • 3 • 10 • 2)
* All laboratories test all media lots and replicates.
† Control drug is tested with one media lot. If only two commercial manufacturers exist for a type of media, multiple lots should
be included from one of the manufacturers and this should be noted in the presentation.
‡ A single replicate per day provides the best opportunity to detect day-to-day variability. A minimum of three days of testing is

recommended for broth dilution and disk diffusion testing; however, agar dilution studies may be performed in two days with a
maximum of five replicates per day.
Abbreviations: MIC, minimal inhibitory concentration; N/A, not applicable; QC, quality control.

Reassessing or Revising Quality Control Ranges (Tier 3 Quality Control Monitoring or

Supplemental Studies)

Tier 3 Quality Control Monitoring

QC ranges should be monitored as additional data are collected and sources of variability increase, referred
to as Tier 3 monitoring. Additional QC data are generated as broader experience is gained with testing of
an antimicrobial agent (eg, during clinical trials, as part of the development of commercial diagnostic tests,
and in routine laboratory use). Existing QC ranges may be reconsidered when a high frequency of out-of-
range QC results is observed.

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 QC data derived from clinical trials of a new antimicrobial agent should be presented to the CLSI
Subcommittee on AST as additional information to confirm the appropriateness of the expected range
established during the Tier 2 QC study. Such data may also support the need to reassess the expected
QC range.

 Individuals or groups other than the manufacturer of an antimicrobial agent may present QC data to the
CLSI Subcommittee on AST and request a reassessment of expected QC ranges. Sources of additional
data may include:
– Performance data from proficiency testing surveys

– End-user feedback to CLSI

– Performance data obtained from various studies using CLSI reference methods (eg, commercial
AST manufacturer studies, surveillance studies, research studies)

– Feedback from CLSI working groups (eg, based on QC data generated during breakpoint
reassessment)

Tier 3 Supplemental Study

To reassess an existing QC expected range, data can be collected prospectively or compiled retrospectively.
When possible, the Tier 2 QC data used to establish the current expected range should be reviewed when
reassessing QC ranges. To reassess and revise a QC expected range, a new Tier 2 study or a Tier 3
supplemental study can be conducted.

A Tier 3 supplemental study (whether prospective or retrospective) should have sufficient sample size to
determine whether the out-of-range results are unusual or can be expected to occur routinely when the test
is performed correctly. A detailed investigation of outlying results is recommended to detect potential
causes for the variability observed. Adequate QC should be performed (eg, use of one or more control
antimicrobial agents and alternative QC strains) when collecting study data.

Collection of additional data is recommended when significant variability has been observed and should
focus on areas where the greatest variability was observed (eg, within laboratory, among laboratories, and
among media). Studies may also be conducted to assess performance within the range specified for the
reference method (eg, pH, incubation times, inoculum preparation) or with other potential sources of
variability (eg, reading end points, age of culture). Testing and manufacturing conditions should be
documented and conclusions included in the troubleshooting guides (see CLSI document M1005), as
appropriate. The requirements for a Tier 3 supplemental study, as described above, are summarized in Table
3.

Table 3. Tier 3 Supplemental Study: Minimum Requirements to Reassess an Existing QC Expected

Range
Category Tier 3 Guidelines
Laboratories or independent evaluations* 3
Media lots 2 (from different manufacturers)
Replicates (individual inoculum, maximum 4 per 10 per laboratory
day) 50 per media
Disk lots 2 (from different manufacturers)
Total data points needed 250 (MIC)
500 (disk)
*Prospective or retrospective study focused on areas of variability. Each laboratory is not required to test all lots.
Abbreviations: MIC, minimal inhibitory concentration; QC, quality control.

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Data from the two studies (ie, the original Tier 2 study and the repeat Tier 2 study or Tier 3 supplemental
study) should be analyzed separately and then compared. If the new data are significantly different from
the original data (eg, greater than a one-dilution shift), additional investigation may be necessary to identify
the cause of the difference or assess the effect of the allowable tolerances of the standard method. As a
result of these investigations and analyses, the QC range may be unchanged, changed (shifted), or expanded
(eg, four-dilution range), as appropriate.

Quality Control Data Presentation and Interpretation

QC study results should be presented with data organized by:

 Media lot
 Disk lot (for disk diffusion)
 Testing laboratory

QC study sponsors should record in full the manufacturing and/or testing conditions and results and retain
records for comparison with future studies, if needed. The results obtained with the control antimicrobial
agent should be reviewed to determine whether the study was performed appropriately.

The raw data should be examined for differences among laboratories, media lots, or disk lots. If significant
overall variability or variation exists within one of the above categories, sponsors should investigate
potential sources of variability and/or potential for errors (refer to the Tier 1 studies in Subchapter 3.2.1 and
the troubleshooting guide in CLSI document M1005 or to potential sources of errors from published
studies). Unless traced back to a manufacturing or laboratory error, detecting a significant difference among
laboratories, media lots, and/or disk lots usually means that QC ranges cannot be set for a specific QC strain
and antimicrobial agent. Similarly, QC ranges cannot be set when a significant proportion of the results are
off scale.

It is important to apply appropriate statistical tools to analyze the final set of QC data. Data from outlier
laboratories should be rejected from the final dataset based only on predefined statistical criteria, such as at
least two of three central tendency statistics for data from a potential outlier laboratory falling outside a
preset range. The RangeFinderb tool has the ability to analyze outliers and is available from CLSI.

The approaches to be taken to select the MIC and disk diffusion QC ranges for an antimicrobial agent are
described in Subchapters 3.3.1 and 3.3.2, respectively. Sample data presentations are provided in Appendix
B.

Quality Control Ranges for Minimal Inhibitory Concentrations

For MICs, the initial QC range proposal should be defined by the 95% confidence interval calculated around
the mode ± one doubling dilution for a three-dilution range. A three-dilution range is preferred. However,
the initial proposed range may need to be adjusted to a four-dilution range to include at least 95% of the
observed MIC values, to accommodate variability expected in routine testing, and/or when a bimodal
distribution is observed (see examples in Table 4). Ranges greater than four doubling dilutions suggest a
notable degree of variation, and consideration should be given to not using the QC strain in question for the
specific antimicrobial agent.

b Refer to CLSI Microbiology Communities webpage at http://clsi.org/standards/micro/rangefinder/.

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Table 4. Examples: Selecting Appropriate MIC QC Ranges

Example 1 Example 2 Example 3
0.03 µg/mL 0 0 0
0.06 µg/mL 5 15 0
0.12 µg/mL 100 73 30
0.25 µg/mL 104 110 170
0.50 µg/mL 1 12 10
1.00 µg/mL 0 0 0
Range, µg/mL 0.06–0.5 0.06–0.5 0.12–0.5
Abbreviations: MIC, minimal inhibitory concentration; QC, quality control.

Example 1 illustrates results where the MIC determinations for the antimicrobial agent against the QC strain
are distributed evenly across two dilutions. A four-dilution QC range of 0.06 to 0.5 µg/mL captures the
entire range of observed MICs.

Example 2 shows a mode of 0.25 µg/mL. However, there are many data points at 0.12 µg/mL. These data
points represent 66% of the values observed at the mode. With a “shoulder” off the modal value, which is
60% or more of the data points at the mode, the population is, by convention, considered to have a mode
spread across two dilutions, and the QC range is extended to four dilutions. In addition, using a three-
dilution range would not include at least 95% of the results. A four-dilution range may also be considered
when there is variability of the mode between lots and/or laboratories.

In Example 3, the distribution of MIC values has a clear mode of 0.25 µg/mL, and 95% of the results fall
within one dilution on either side of the mode.

The RangeFinder tool can be used for statistical analysis of QC results for MICs and to analyze outliers.
The mean, SD, and range of dilutions are calculated after conversion of the raw data to logarithms and
adjusting down to the midpoint of the MIC. For example, observations at an MIC of 4 µg/mL are converted
to logarithms to the base 2 (ie, log2 = 2) and adjusted down to halfway between this and the next lowest
concentration (ie, 2 µg/mL, log2 = 1), giving working values for this concentration of [2 − 1] / 2 = 0.5. The
expected range of MIC values is usually the 95% confidence interval calculated around the logarithmic
mean, converted back to MIC values, and adjusted upward to the nearest twofold dilutions. Occasionally
(eg, for Aspergillus) the 90% confidence interval may be used to determine the range, and guidance may
be included on actions to consider when the QC result is out of range during routine testing. If these
calculations result in a single dilution range, the QC range is set to a three-dilution range as one dilution
below to one dilution above the calculated concentration. If the calculations result in a two-dilution range,
the QC range is adjusted to three dilutions by adding a concentration to the range on the opposite side of
the mode to that seen in the raw data.

Quality Control Ranges for Disk Diffusion

For disk diffusion, the mean, SD, and range of zone diameters observed should be calculated. The
RangeFinder tool can be used for this purpose and to analyze outliers. Historically, the Gavan statistic22 has
been used for these purposes. Both methods are acceptable and can be complementary. The initial proposed
QC range of zone diameters should be defined by the 95% confidence interval calculated around the mean,
rounded down at the lower end and up at the upper end to the nearest whole millimeter. Ideally, the QC
range should be ≤ 12 mm. Occasionally (eg, for Aspergillus), the 90% confidence interval may be used to
determine the range, and guidance may be included on actions to consider when the QC result is out of
range during routine testing.

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Procedures for Establishing Breakpoints

This chapter includes:

 The procedure for submitting proposals for new or revised breakpoints

 Handling of proposals within the CLSI Subcommittee on AST, including the creation of ad hoc
working groups, to make the process more efficient and provide a high level of scientific dialogue
between sponsors and the CLSI Subcommittee on AST

 The criteria used to determine whether revision of a breakpoint should be considered

This chapter outlines the procedures to be followed by both sponsors and the CLSI Subcommittee on AST
to add new breakpoints and to revise existing breakpoints. The chapter should be read in conjunction with
Appendix C. CLSI breakpoints may differ from those approved by various regulatory authorities for many
reasons, including:

 The use of different databases

 Differences in data interpretation
 Differences in doses used in different parts of the world
 Different public health policies

Differences also exist because CLSI proactively evaluates the need for changing breakpoints. The reasons
why breakpoints may change and the manner in which CLSI evaluates data and determines breakpoints are
outlined in this chapter.

In the United States, it is acceptable for laboratories that use US Food and Drug Administration (FDA)–
cleared susceptibility testing devices to use existing FDA breakpoints. Either FDA or CLSI breakpoints are
acceptable to laboratory accrediting organizations in the United States. Policies in other countries may vary.
Each laboratory should check with the manufacturer of its antimicrobial susceptibility test system for
additional information on the breakpoints used in its system’s software.

When a device includes antimicrobial test concentrations sufficient to allow interpretation of susceptibility
and resistance to an agent using the CLSI breakpoints, a laboratory could choose, after appropriate
verification, to report results using CLSI breakpoints. Following discussions with appropriate stakeholders,
such as infectious diseases and pharmacy practitioners, the pharmacy and therapeutics and infection control
committees of the medical staff, and the antimicrobial stewardship team, newly approved or revised CLSI
breakpoints may be implemented by laboratories as soon as they are published in CLSI document M100.5

For determining new or revised breakpoints, the data described in Chapters 5 and 6 can be submitted at any
time. However, reference susceptibility testing methods and QC ranges should be established before
selecting breakpoints for an antimicrobial agent.

For a new antimicrobial agent, the sponsor should notify the CLSI Subcommittee on AST of the
antimicrobial agent abbreviation (up to three letters) to be used by diagnostic companies for all commercial
products used for susceptibility testing. This abbreviation will be added to Glossary II of CLSI document
M100.5 Information for glossaries and tables (eg, antimicrobial class, subclass, troubleshooting guidelines)
may be submitted as it becomes available.

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New Breakpoints

Requests for adding breakpoints for a new or an established antimicrobial agent should be submitted to the
Chairholder of the CLSI Subcommittee on AST and to the Chairholders of the Working Group on AST
Breakpoints in parallel (see Appendix D). The steps in the following subchapters are then taken.

Handling of Requests Received

Step Action Comment

1. Within 30 days after a request is received To expedite consideration of the request, the
from the sponsor, the Chairholder of the process can include meetings and/or
CLSI Subcommittee on AST, the teleconferences of the Working Group on AST
Chairholders of the Working Group on AST Breakpoints, with or without the sponsor,
Breakpoints, and the sponsor agree to: outside of the general meetings of the CLSI
 The process (within the confines of the Subcommittee on AST.
process described in this subchapter)
 The timing for evaluation of the request Formal presentations before the CLSI
 The timing for formal presentations to Subcommittee on AST and the Working Group
the CLSI Subcommittee on AST and the on AST Breakpoints should occur within 13
Working Group on AST Breakpoints months of receipt of the sponsor’s request.

Formal presentations to the Working Group on

AST Breakpoints are usually* made by
sponsors and/or members of the ad hoc
working group.

Formal presentations to the CLSI

Subcommittee on AST are usually* made by
Chairholders of the Working Group on AST
Breakpoints or by other persons appointed by
the Chairholders, who may be members of the
working group and/or the ad hoc working
group.
2. Within 45 days after a request is received The purpose of the ad hoc working group is
from the sponsor, the Chairholders of the threefold:
Working Group on AST Breakpoints, in 1. Provide an iterative process for in-depth
consultation with the Chairholder of the analysis of the sponsor’s proposal for
CLSI Subcommittee on AST, shall decide breakpoints by a small group of selected
whether to appoint an ad hoc working group experts who can request additional data to
for the purpose of evaluating the sponsor’s support or clarify the proposal of the
request.† sponsor if needed, anticipating questions or
concerns that might arise during formal
presentation to the full CLSI
Subcommittee on AST.
2. Focus on potentially problematic issues in
the data package relative to the breakpoints
being requested.
3. Provide a recommendation to the CLSI
Subcommittee on AST.

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Step Action Comment

3a. The ad hoc working group should consist of
at least one individual with expertise in each
type of data used to establish breakpoints, as
appropriate to the material provided by the
sponsor.

3b. Members of the ad hoc working group Members of the ad hoc working group may
review sections of data packets appropriate request additional information from the
to their areas of expertise and confer to sponsor for the purpose of making their
determine recommendations of the ad hoc assessments and share with the sponsor any
working group to the Working Group on concerns with the proposed breakpoints.
AST Breakpoints.

3c. After conferring, the decision of the ad hoc Representatives of the ad hoc working group,
working group to support the original or at the presentation before the Working Group
modified proposals of the sponsor or to make on AST Breakpoints, provide their consensus
alternative proposals are presented to the opinion for or against the sponsor’s final
Working Group on AST Breakpoints. proposals and present alternative
recommendations, if necessary.
* The Chairholders of the Working Group on AST Breakpoints, in consultation with the Chairholder of the CLSI Subcommittee on

AST, after discussion with the sponsor, may decide on alternative plans for presentations.
† On occasion there may be reasons why the Chairholders of the Working Group on AST Breakpoints, in consultation with the

Chairholder of the CLSI Subcommittee on AST, decide not to appoint an ad hoc working group. In these rare cases, a decision is
made on how to progress the application at the time the timelines are discussed and agreed on with the sponsor. The sponsor is
notified accordingly. The text above and below assumes that an ad hoc working group is appointed.
Abbreviation: AST, antimicrobial susceptibility testing.

Establishing and Publishing Breakpoints

Step Action Comment

1a. At the formal presentation to the Working Group on Requests for breakpoints that have not
AST Breakpoints, a sponsor may elect to request been reviewed by the ad hoc working
breakpoints for all or part of the categories of group are not considered.
organisms reviewed by the ad hoc working group.
The sponsor’s final request is presented to the CLSI The CLSI Subcommittee on AST may
Subcommittee on AST as described in Subchapter establish breakpoints for all or part of
4.1.1. the organism categories requested by
the sponsor.

1b. The CLSI Subcommittee on AST publishes The breakpoints acceptable to the CLSI
breakpoints for only the categories of organisms Subcommittee on AST for the
requested by the sponsor. categories of organisms requested by
the sponsor may differ from those
proposed by the sponsor.

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Step Action Comment

2a. If the CLSI Subcommittee on AST votes to establish The deferral period is no greater than
breakpoints for categories of organisms formally 13 months.
requested by the sponsor and the sponsor does not
agree with that determination (for all or part of the
original proposal), the sponsor may request that the
decision pertaining to the disputed elements of the
determination be deferred.

2b. Breakpoints for which there is agreement or for

which the sponsor has not requested a deferral will
be published at the next update of the applicable
supplement and/or relevant breakpoints tables unless
the CLSI Subcommittee on AST decides to delay
such publication.
3. If the sponsor requests deferral of a breakpoint The ad hoc working group reviews the
determination, the sponsor may provide additional materials in the same manner as
information to the ad hoc working group that described in Subchapter 4.1.1.
reviewed the initial submission to support the
sponsor’s position.
4. At a meeting of the Working Group on AST
Breakpoints within the deferral period, the sponsor
has the opportunity to present its data to support its
request, and representatives of the ad hoc working
group provide their consensus opinion for or against
the sponsor’s proposal and present alternative
recommendations, if necessary. Presentation of the
additional data and opinions of the ad hoc working
group and Working Group on AST Breakpoints is
then made to the CLSI Subcommittee on AST, as
described in Subchapter 4.1.1.

Whether or not additional material is provided to

support the sponsor’s proposal, the CLSI
Subcommittee on AST may take the following
actions at the end of the deferral period (ie, after 13
months) on the previously deferred portions of the
original proposal:
 Reconfirm its original decision; the breakpoints
will be published at the next update of the
applicable supplement and/or relevant
breakpoint tables unless the CLSI Subcommittee
on AST decides to delay such publication.
 Revise its original decision; the breakpoints will
be published at the next update of the applicable
supplement and/or relevant breakpoint tables
unless the CLSI Subcommittee on AST decides
to delay such publication.
 Decline to assign breakpoints for the originally
deferred requests.
Abbreviation: AST, antimicrobial susceptibility testing.

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Revision of Breakpoints

Breakpoint reassessment and revision may become necessary as new information becomes available.
Requests for breakpoint reassessment can be submitted from sponsors; members, advisors, and/or reviewers
of the CLSI Subcommittee on AST; regulatory or public health agencies; or other stakeholders, including
individuals.

The procedure for breakpoint reassessment is applicable only when CLSI breakpoints already exist for the
antimicrobial agent for one or more organisms or organism categories. All other requests for breakpoints
must follow the procedures outlined in Subchapter 4.1.

Requests for breakpoint reassessment and revision should be submitted to the Chairholder of the CLSI
Subcommittee on AST and to the Chairholders of the Working Group on AST Breakpoints. The Working
Group on AST Breakpoints reviews the submitted information, considering the criteria outlined in
Subchapter 4.2.1, to determine whether breakpoints reassessment is warranted. The Working Group on
AST Breakpoints makes a recommendation regarding the need for such breakpoints reassessment to the
CLSI Subcommittee on AST.

Situations in Which Breakpoints Reassessment May Be Considered

The following situations represent conditions under which reassessment might be considered:

 For some antimicrobial agents, susceptible-only breakpoints are initially established using wild-type
MIC distributions, because isolates with reduced susceptibility have not been detected. Susceptibility
criteria may be reviewed when any of the following occur:
– Newly recognized resistance mechanisms emerge within categories of organisms for which
breakpoints already exist.

– A population with an MIC distribution distinct from that of the wild-type population emerges
among clinical isolates, even if the resistance mechanisms or decreased susceptibility are not
known.

– The distribution of MICs has shifted rightward (higher MICs) so that significant numbers of isolates
consistently test in the nonsusceptible range. The exact number or proportion of organisms that test
nonsusceptible organisms that should prompt reconsideration of breakpoints has not been set
definitively, and judgment would be needed on this point.

 As new resistance mechanisms emerge (eg, new -lactamases) or are newly recognized (eg, by
molecular methods), resistance may not be reliably detected using established breakpoints and any
associated methods. The appearance of such resistance mechanisms may prompt review of breakpoints
when it seems possible or when it has been reported that they may adversely affect clinical response.

 Breakpoints may have been initially determined using collections of organisms from several species.
With increasing clinical experience, improved understanding of resistance mechanisms, and differences
in pharmacokinetic-pharmacodynamic (PK-PD) relationships predicting efficacy in different species,
evidence may emerge that susceptibility test performance would be improved by introducing separate
criteria for specific genera and species.
 For example, with the availability of tests for the mecA gene, it became clear that oxacillin
breakpoints for coagulase-negative staphylococci should be lower than those for Staphylococcus
aureus and Staphylococcus lugdunensis. In this case, providing one set of oxacillin breakpoints for
S. aureus and S. lugdunensis and different breakpoints for all other staphylococci enabled the
susceptibility test to better predict the presence of the mecA resistance gene.

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 When new PK-PD data indicate that existing breakpoints may have been set inappropriately high or
low.

 When breakpoints were set before the introduction of current analytical methods used to determine
relationships among drug exposure, organism susceptibility, and clinical response.

 When prevailing antimicrobial dosage regimens differ substantially from dosage regimens used to
establish initial breakpoints or when a new formulation of an antimicrobial agent is introduced that
results in different pharmacokinetics (PK) characteristics.
– For example, registration of less frequent dosing schedules for -lactams may result in suboptimal
target attainment rates for critical pathogens using breakpoints established when more frequent
drug dosage regimens were used.

 When new data show that previously established breakpoints are not optimal for common uses of an
antimicrobial agent.
– For example, susceptibility breakpoints for ceftriaxone and cefotaxime against S. pneumoniae,
which had been set conservatively based on drug concentrations needed for treatment of meningitis,
were adjusted upward for treatment of nonmeningeal pneumococcal infections.

 When clinicians, laboratory practitioners, or public health agencies suggest poor prediction of clinical
response using existing susceptibility breakpoints or when a specific public health need is identified.
In some cases, existing breakpoints may be too liberal, resulting in inadequate clinical responses for
infections caused by organisms categorized as susceptible. In other situations, breakpoints may be too
stringent, with clinical response expected against infections caused by organisms not categorized as
susceptible by existing criteria.
– For example, published literature from several sources suggested that patients with invasive
salmonellosis responded suboptimally to standard fluoroquinolone regimens when MICs were
above those of wild-type organisms, even if below the breakpoint for susceptibility. In this case,
some breakpoints were revised to detect isolates likely to respond less well to standard
fluoroquinolone dosage regimens.

 When new knowledge and techniques applied to establish new or revised breakpoints for a more
recently studied antimicrobial agent may suggest that breakpoints for older agents of the same class are
potentially erroneous and should be re-evaluated.

 When significant rates of discordance are documented between reference MIC and disk diffusion test
results when testing recent clinical isolates.

 When changes are made to CLSI-approved reference methods that may have an effect on breakpoints.
– For example, a decision to alter previously established testing conditions by incubation of
inoculated media in increased concentrations of CO2 or for longer periods to improve growth of
fastidious organisms may necessitate re-evaluation of breakpoints for antimicrobial agents, the
activity of which might be affected by these modifications.

 When selecting alternative breakpoints will simplify testing and eliminate the need for additional tests
to detect specific resistance mechanisms.

 When differences exist between breakpoints established by CLSI and those of other regulatory
organizations responsible for determining breakpoints, the CLSI Subcommittee on AST may re-
examine its breakpoints in an effort to understand the reasons for such differences and to minimize
discrepancies between published breakpoints of CLSI and other organizations whenever possible.

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 If a sponsor submits a request to any regulatory agency or other organization responsible for
determining breakpoints, the sponsor is encouraged to submit such data to the CLSI Subcommittee on
AST for consideration.

Process for Handling a Reassessment

Considering the criteria outlined in Subchapter 4.2.1, the Working Group on AST Breakpoints makes a
recommendation regarding the need for a reassessment of existing breakpoints to the CLSI Subcommittee
on AST. When a reassessment could potentially affect and/or apply to other antimicrobial agents of the
same class, the Working Group on AST Breakpoints recommends that those agents be considered for
reassessment at the same time. After reviewing the recommendation of the Working Group on AST
Breakpoints, if the CLSI Subcommittee on AST decides to proceed with the reassessment, the following
steps occur:

Step Action Comment

1. Within 30 days, a timeline for this
reassessment is agreed upon between the
Chairholders of the Working Group on AST
Breakpoints and the Chairholder of the CLSI
Subcommittee on AST.
2a. Within 45 days, the Chairholders of the The data needed for the reassessment will be
Working Group on AST Breakpoints, in defined by the Working Group on AST
consultation with the Chairholder of the CLSI Breakpoints and/or by the ad hoc working
Subcommittee on AST, appoint an ad hoc group.
working group.* The make-up and purpose of
this ad hoc working group is as discussed in
Subchapter 4.1.1.

2b. The parties with a potential interest in revision For established antimicrobial agents, there
of breakpoints for the antimicrobial agent(s) in may be one or several manufacturers that hold
question are identified by the Working Group a license in the United States. Some or all of
on AST Breakpoints and/or the ad hoc the manufacturers may have an interest in or
working group. may have relevant data for the reassessment.
In addition, the sponsor proposing the
reassessment may not hold any license for the
antimicrobial agent(s) in question.

2c. Within 30 days of a decision to conduct a CLSI staff notifies the sponsor and all other
reassessment, the sponsor and any other interested parties.
parties with a potential interest that can be
identified are notified of the intent to conduct
such a reassessment, as well as the timeline for
the reassessment.

2d. Notification is provided electronically through If confirmation of receipt cannot be obtained,

an e-mail with a confirmed delivered and read CLSI staff makes all reasonable efforts to
receipt, and/or a written letter is delivered by contact the interested parties, documenting
mail or equivalent service with a such efforts and results in CLSI files.
documentation of receipt, which is
documented in the CLSI files. This procedure
is followed for all interested parties.

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Step Action Comment

3a. In the correspondence to the sponsor and all
interested parties and in the minutes of CLSI
Subcommittee on AST meetings, the
reassessment decision, rationale for such a
reassessment, data being sought, and timeline
for the reassessment process are clearly
outlined. This information is also posted on
the CLSI website.

3b. The CLSI Subcommittee on AST provides a Any submissions must be provided in
minimum of six months for any interested accordance with the recommendations for
parties to prepare a submission relevant to the data submission as described in Chapters 5
reassessment of the breakpoints if they wish to and 6.
do so.

3c. If an interested party chooses not to provide

data in response to this notification, the review
will proceed using all available data, including
the published literature.
* On occasion, there may be reasons why the Chairholders of the Working Group on AST Breakpoints, in consultation with the

Chairholder of the CLSI Subcommittee on AST, decide not to appoint an ad hoc working group. In these rare cases, a decision is
made on how to progress the application at the time the timelines are discussed and agreed on with the sponsor. The sponsor is
notified accordingly. The text above and below assumes that an ad hoc working group is appointed.
Abbreviation: AST, antimicrobial susceptibility testing.

Individual members of the ad hoc working group review sections of the submissions that correspond to
their areas of expertise. The ad hoc working group then confers to determine the recommendations that are
to be made to the CLSI Subcommittee on AST. Members of the ad hoc working group may request
additional information from and share any concerns with the sponsor and/or other interested parties. When
using other sources of data for the review, including the published literature, the ad hoc working group
directly obtains the information and/or works with CLSI staff to obtain such data.

Changing breakpoints can potentially lead to short-term disruption, confusion, regulatory difficulties,
reporting challenges, and unexpected costs. When making the reassessment evaluation, the ad hoc working
group considers the proposed changes’ effect on the users of CLSI documents by evaluating the following
questions:

 Will a change in breakpoints result in substantial changes in the categorization of organisms as

susceptible, intermediate, or resistant?

 Will the change in breakpoints drive increased or decreased use of the index agent or alternative
antimicrobial agents? What would be the ramifications of such changes in usage?

 Will it be feasible to fully implement the proposed changes with respect to laboratory personnel, testing
devices, and reporting systems?

 Will the changes result in greater or lesser discordance with breakpoints of other organizations that
determine breakpoints?

 Are the changes likely to create undue confusion among clinicians?

If revised breakpoints, although scientifically correct, are judged to have a minimal effect on the appropriate
use of the antimicrobial agent but likely to pose considerable challenges to implementation or other
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difficulties outlined above, the ad hoc working group and/or CLSI Subcommittee on AST may elect to
retain existing breakpoints. After reviewing the available data and conferring, the decision of the ad hoc
working group is presented to the CLSI Subcommittee on AST as outlined below:

Step Action Comment

1. The ad hoc working group presents to the During this presentation, the sponsor and any
CLSI Subcommittee on AST a summary of other interested parties have the opportunity to
relevant data, any other relevant present data or other relevant information.
considerations, and its recommendation.
2. The CLSI Subcommittee on AST may: The breakpoints are published at the next
 Agree to one or more changes in the update of the applicable supplement and/or
breakpoints. relevant breakpoints tables unless the CLSI
 Decide that no change in the breakpoints Subcommittee on AST decides to delay such
is required. publication.
 Decide that additional data are needed or
additional evaluation is required. The CLSI Subcommittee on AST clearly
outlines the additional data required and/or the
additional evaluations needed. In this case, the
procedure outlined in Subchapter 4.1.2 is
followed.
3. When reassessment leads to revision of CLSI
breakpoints that are no longer consistent with
those of the FDA, the CLSI Subcommittee
on AST notifies the FDA of such changes
and the reasoning behind them, so the FDA
can decide whether it should conduct its own
re-evaluation of breakpoints.*
* Following a decision by CLSI to revise existing breakpoints, the FDA and other regulatory authorities may also review data to
determine how changing the breakpoints may affect the safety and effectiveness of the antimicrobial agent for the approved
indications. If a regulatory authority changes the breakpoints, commercial device manufacturers may have to conduct a clinical
laboratory trial, submit the data to the regulatory authority, and await review and approval. For these reasons, a delay of one or
more years may be required if a change in breakpoints is to be implemented by a device manufacturer.
Abbreviations: AST, antimicrobial susceptibility testing; FDA, US Food and Drug Administration.

When the procedures outlined in Subchapters 4.1 and 4.2 to establish breakpoints are not followed
completely due to a well-documented public health need and/or inability to obtain all the data considered
likely to be relevant, it should be noted in published documents showing the revised breakpoints.

Provisional Breakpoints

For antimicrobial agents that are in development (ie, for which registration has not yet occurred for any
indication), information on zone diameter and MIC relationships, distributions of MICs for organisms
relevant to the intended clinical uses, and PK-PD indices may be submitted to the Chairholder of the CLSI
Subcommittee on AST and to the Chairholders of the Working Group on AST Breakpoints at any time. The
CLSI Subcommittee on AST can then assist in the selection of “provisional” breakpoints to be used by
clinical investigators during clinical trials that assess efficacy. Provisional breakpoints are not published in
CLSI documents.

Periodic Breakpoint Review

In order to ensure that breakpoints remain current and relevant over time, a new process for periodic reviews
has been implemented. Breakpoints for each antimicrobial class are reviewed periodically for potential
modification. Classes of antimicrobial agents routinely used clinically may receive priority and/or be more

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frequently reviewed. These periodic reviews are not meant to supplant the breakpoint review that may be
needed as outlined in Subchapter 4.2.

On an annual basis, the Chairholders of the Working Group on AST Breakpoints and the Chairholder of
the CLSI Subcommittee on AST create a schedule of breakpoints to be reviewed over the subsequent two-
year period. This schedule is presented at one of the public meetings of the CLSI Subcommittee on AST
and published in the summary minutes for that meeting. A request is made for any data available concerning
the breakpoints to be reviewed, along with a deadline for submission. In addition, pharmaceutical sponsors
of the antimicrobial agents to be reviewed are notified according to the current procedures. This periodic
review is not intended to result in a full reassessment of breakpoints unless initial review of available
information indicates that a need exists for such a full reassessment based on the criteria outlined in
Subchapter 4.2.1.

The following steps are taken with the goal of determining whether a reassessment of breakpoints is
warranted.

Step Action Comment

1. The Chairholders of the Working Group on AST Breakpoints
assign one or more experts for each class of breakpoints to be
reviewed.
2. The designated expert(s) works with CLSI staff to perform a
literature search for any information published and/or
presented that is relevant for review of the breakpoints for the
class of antimicrobial agents to be reviewed. In addition,
CLSI staff provides copies of published and/or presented
information that the designated expert(s) believes needs to be
reviewed.
3. CLSI staff provides the designated expert(s) with any
additional information that has been submitted relevant to the
review of the breakpoints being reviewed.
4. The designated expert(s) reviews and summarizes all relevant
information. This summary is submitted to the Chairholders
of the Working Group on AST Breakpoints and included in
the agenda materials for the relevant AST meeting.
5a. The review’s results are presented to the Working Group on
AST Breakpoints, who determines whether one or more of
the conditions outlined in Subchapter 4.2.1 have been met.

5b. If none of the conditions listed in Subchapter 4.2.1 have been

met, the Working Group on AST Breakpoints recommends to
the CLSI Subcommittee on AST that no changes be made to
the current breakpoints, with the year of review noted in
Tables 2 in CLSI document M100.5

5c. If one or more of the conditions listed in Subchapter 4.2.1

have been met, the Working Group on AST Breakpoints
determines whether a full reassessment of any one or more
breakpoints should be recommended to the CLSI
Subcommittee on AST.

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Step Action Comment

6a. The CLSI Subcommittee on AST considers the
recommendations of the Working Group on AST Breakpoints
and determines whether any one or more breakpoints will
undergo full reassessment.

6b. Any breakpoints that will not undergo full reassessment

continue to be published without change and are considered
“reviewed,” with the year of review noted in Tables 2 in
CLSI document M100.5

6c. For any breakpoints that will undergo a full reassessment, the
procedure outlined in Subchapter 4.2.2 is followed.
Abbreviation: AST, antimicrobial susceptibility testing.

Surrogate Testing

When new antimicrobial agents are approved by regulatory authorities, susceptibility testing of such agents
may present a challenge for microbiology laboratories that depend on automated testing devices and that
do not have the capability to conduct susceptibility testing using alternative methods. It is not unusual for
availability of automated testing devices for the new agent to be delayed by one or more years after initial
regulatory approval. To provide clinicians with the susceptibility information they need to treat patients, it
may be possible to use the susceptibility testing results of another antimicrobial agent in the same class to
predict susceptibility to the new antimicrobial agent. Specific criteria have yet to be defined to evaluate
surrogate testing proposals; however, sponsors are encouraged to submit and present data to the CLSI
Subcommittee on AST to support surrogate testing when there are compelling data and a clinical need.

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Minimal Inhibitory Concentration Breakpoints

This chapter includes:

 Data used for establishing breakpoints

 Additional considerations for the drug sponsor
 Elements to provide in the clinical trial summary

Four categories of data, ECV, nonclinical PK-PD cutoff, CER cutoff, and clinical cutoff, are useful in
establishing breakpoints. The typical use(s) of these data are detailed in Table 5. Each of these data sources
has distinct strengths and limitations, and the identification of breakpoints depends on consideration of all
four data types, as described in Subchapter 5.5.

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Table 5. Categories of Data Useful for Establishing Breakpoints

Category Typical Considerations
ECV  What is the typical distribution of MICs for the target pathogens?
 What is the performance of the in vitro susceptibility devices used to measure
MICs?
 Is a disk diffusion device available? If so, how do zone diameters relate to MICs?
 How do the MICs of isolates collected in clinical trials compare with the wild-type
distribution?
 What is the ECV?
Nonclinical  Based on nonclinical models, what is the PK-PD index most closely linked with
PK-PD antimicrobial effect?
cutoff  Based on nonclinical models, what magnitude of this PD index is needed for an
antimicrobial effect?
 What are the PKs of the test antimicrobial agent in patients with the type(s) of
infections for which it is intended to be used?
 In these patients, which covariates (eg, age or renal function) influence the PKs?
Do dosage adjustments for these covariates permit consistent exposure for treated
patients?
 What is the nonclinical PK-PD cutoff?
CER cutoff  What are the PKs of the test antimicrobial agent in patients with the type(s) of
infections for which it is intended to be used?
 In these patients, which covariates (eg, age or renal function) influence the PKs?
Do dosage adjustments for these covariates permit consistent exposure for treated
patients?
 What is the correlation between MIC values and likely efficacy predicted in
patients based on CER relationships for efficacy (exposure-response relationship)
in infected patients and using human PKs?
 What is the CER cutoff?
Clinical  What outcomes were observed in clinical trials?
cutoff  Do these outcomes predict a cutoff value that is consistent with the cutoffs
predicted by the nonclinical PK-PD cutoffs and the CER cutoffs?
 Is there a correlation of patient outcomes with the pathogens isolated in clinical
trials?
 Is there a correlation of patient outcomes with the MICs of the pathogens isolated in
clinical trials (simple correlation of outcomes to MIC), ie, is it possible to identify a
clinical cutoff?
 What is the clinical cutoff?
Abbreviations: CER, clinical exposure-response; ECV, epidemiological cutoff value; MIC, minimal inhibitory concentration; PD,
pharmacodynamic; PK, pharmacokinetic; PK-PD, pharmacokinetic-pharmacodynamic.

With respect to determination of MIC breakpoints, Subchapters 5.1, 5.2, 5.3, and 5.4 consider each of these
data sources in turn and indicate the types of data that are considered essential elements of submissions to
the CLSI Subcommittee on AST. Subchapter 5.5 discusses approaches to balancing the information
obtained from these different sources in determining MIC breakpoints. Similarly, Chapter 6 considers the
data needed to determine disk diffusion breakpoints.

Epidemiological Cutoff Value

This subchapter considers the microbiological data that should be assembled to support ECV selection as
defined in Subchapter 1.3.2, as well as the methods for selecting ECVs.

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* Mechanism of Action Studies

Sponsors should report what is known about a new antimicrobial agent’s mechanism of action, particularly
study results that demonstrate the mechanism of action (eg, inhibition of cell wall synthesis, lysis of cell
membrane, protein synthesis, and inhibition of DNA or RNA replication). These reports should provide
data to substantiate both physiological and morphological effects on the microorganisms. Understanding
the mechanism of action can provide a basis for understanding the development of resistance.

The report should include the chemical structure and a description of any structural or biological similarities
to known antimicrobial agents together with a discussion of any differences there may be in the mechanism
of action (eg, for β-lactam agents there should be a description of any differences there may be in the affinity
for penicillin-binding proteins in relevant organisms for the new and closely related existing agents).

Reports of studies evaluating microbial killing (eg, microbial kill curves) should be included along with
reports of mechanism of action studies. A discussion of the antimicrobial agent’s intracellular activity
should also be provided where appropriate.

* Mechanism of Resistance Studies

Resistance mechanisms may limit an antimicrobial agent’s clinical effectiveness. Therefore,

characterization of the mechanisms mediating resistance and their distribution within the proposed target
pathogens is important for defining the antimicrobial agent’s potential clinical usefulness. Common
resistance mechanisms include alterations of the antimicrobial agent by enzymes (eg, β-lactamases or
aminoglycoside-modifying enzymes), impermeability or efflux mechanisms that prevent access of the
antimicrobial agent to target sites, and changes in the target site that result in reduced affinity of the
antimicrobial agent. Whenever possible, it is recommended that sponsors provide the genotypic
characteristics of resistance mechanisms.

For some organisms and antimicrobial agents, resistance occurs in only a proportion of the overall
population (ie, heteroresistance). Testing should be done to detect the presence of heteroresistance. In
addition, studies on the frequency of spontaneous resistance to the new agent should be provided. When
resistance mechanisms against agents of the same antimicrobial class as the antimicrobial agent under
consideration are known, a sample of organisms with MICs or zone sizes in various parts of the population
distribution should be tested for the presence of that resistance trait. The results should demonstrate that
these resistance mechanisms can be recognized by the susceptibility testing methodology.

Antibacterial Spectrum of Activity

* Susceptibility Test Methods

Sponsors should describe the methods used for generating susceptibility data in nonclinical studies and in
clinical trials (eg, broth microdilution, agar dilution, disk diffusion). See Chapter 2 regarding establishing
reference methods for an antimicrobial agent and the validation of alternative methods (ie, methods
different from the reference methods). For example, if sponsors use freeze-dried panels to assess the MIC
of the drug during clinical trials, they should conduct a comparative study to demonstrate comparability of
MIC results for the frozen and freeze-dried panels.

* Spectrum of Activity

Sponsors should evaluate the antimicrobial agent’s activity against a test panel of organisms that are
relevant to the intended clinical uses, including individual species of interest and species that may be
associated with group-level descriptors (eg, Enterobacteriaceae or viridans group streptococci). The

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number of isolates tested and their diversity (eg, geographic distribution, relevant clinical genera and
species, relevant resistance mechanisms) are important.

Dilution and disk diffusion tests should be performed on a large collection of isolates, which should include
examples of clinically relevant isolates appropriate both for the class of the agent being evaluated and for
its anticipated clinical use. The collection should include isolates showing important resistance
mechanisms, including resistance to antimicrobial therapies commonly used for indications sought by the
new agent. Refer to Appendix A for additional guidance on organism selection and sample size.

Sponsors should identify whether there are subtypes (eg, genotypes, serotypes, or multilocus sequencing
types) that correlate with a resistance phenotype and include these in the test panel. Organisms that
demonstrate heteroresistance to the antimicrobial agent under study should also be included, if applicable.
The presentation of the data on the spectrum of activity should include:

 * A description of the susceptibility testing method(s) used to determine the activity of the antimicrobial
agent
– If an experimental method is used, the details of the method and its performance characteristics in
the laboratory where such testing was performed should be included.

 * A description of all susceptibility testing QC measures

– All susceptibility test results should be accompanied by a summary of QC data obtained in MIC
tests performed after QC parameters were set.

 * The MICs of the antimicrobial agent for each organism (by genus- or species-level identification)
presented in tabular form to display the numbers tested, MIC range, and the MIC50 and MIC90

 * Frequency distribution of number of isolates at each MIC in the form of histograms showing the
number of isolates (y-axis) with MICs at each concentration of the antimicrobial agent (x-axis),
spanning a range of concentrations that encompass all MICs measured or reaching the upper limit of
concentrations feasible to test
– The x-axis is customarily drawn in intervals of twofold increasing antimicrobial concentrations
(see Figure 1).

 * Additional histograms (designed as above) for each bacterial pathogen relevant to the intended
clinical use
– These histograms should include:
o A presentation of isolates obtained in clinical trials compared with all other isolates tested by
MIC (see Figure 1).
o A presentation of MICs according to the presence or absence of phenotypic resistance, specific
resistance mechanisms (if known), and virulence markers (if applicable to individual
pathogens). In addition, MICs should be displayed for organisms that are commonly grouped
together for the purposes of determining breakpoints (eg, Enterobacteriaceae).

 The number of isolates tested for MIC determination in each laboratory if multiple laboratories
generated the datasets

 A description of the MIC distributions by individual geographic region and all regions combined with
a discussion of any variability observed in light of prevalent resistance mechanisms

 Minimum bactericidal concentrations when appropriate based on the drug’s mechanism of action

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900 875
Preclinical
800 Clinical
700
Number of Isolates

600 530
500
400 333
276
300
184 190
200 141

100 6 30 46
9 11 26 2 10 3 5
0
0.03 0.06 0.12 0.25 0.5 1 2 4 8
MIC, µg/mL

Abbreviation: MIC, minimal inhibitory concentration.

Figure 1. Distribution by MIC of Isolates Collected in Surveillance Studies and Clinical Trials

Cross-Resistance Between Antimicrobial Agents

Sponsors should compare the activity of a new antimicrobial agent product to the activity profile of other
antimicrobial agents with the same or a very similar mechanism of action to assess the possibility of cross-
resistance. Sponsors should present and evaluate the potential for cross-resistance.

Under some circumstances, tentative inferences can be drawn about cross-resistance between antimicrobial
agents within a specific population of isolates from regression analyses of MICs for one antimicrobial agent
compared with another. If cross-resistance exists, a strong correlation between the MICs of the two agents
being compared would be expected, and a majority of the MICs will be clustered on a 45-degree diagonal.
If resistance affects the activity of one agent more than the other, the regression line will fall above or below
the 45-degree diagonal (see Figure 2).
Existing Drug, µg/mL

New Drug, µg/mL

Figure 2. Scattergram Comparing New Drug With Existing Drug. Vertical lines are presumptive
susceptible and resistant breakpoints for a new drug, and horizontal lines are susceptible and resistant
breakpoints for the existing drug. MIC correlates in the upper right and lower left boxes are in categorical
agreement. The diagonal line marks equivalent MICs for the two drugs (absolute agreement).

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* MICs should be tabulated for the antimicrobial agent under study compared with one or more established
agents according to the phenotypic and/or genotypic characteristics of the isolates tested. For example, if
the antimicrobial agent under study is a new gyrase inhibitor, the MICs should be compared with those for
established fluoroquinolones against fluoroquinolone-resistant isolates. The methodology and the criteria
used to characterize isolates as resistant should be described.

* Determination of Epidemiological Cutoff Values

ECVs should be determined for each organism or group of organisms for which breakpoints are proposed.
ECVs are determined by collecting and merging MIC distribution data from a range of sources and applying
techniques for estimating the upper end of the wild-type distribution. To be reliable, ECVs are estimated
by accounting for both biological (strain-to-strain) variation and MIC assay variation within and among
laboratories. They are based on the assumption that the wild-type distribution of a particular antimicrobial
agent–organism combination does not vary geographically or over time. A number of conditions must be
fulfilled to generate reliable ECVs. The most important are:

 An ECV can be determined only within a single species because of the genetic diversity among species
within a genus.

 MIC values included in the merged dataset must have been determined using a recognized reference
method such as the CLSI MIC broth dilution method (refer to CLSI document M072), which is also the
international reference standard.23

 Data must be sourced from at least three separate laboratories, and there should be at least 100 unique
strains included in the merged dataset and weighted before pooling if more than 50% of MICs were
generated in a single laboratory.

 As much as possible, the MIC values included in an individual laboratory’s data must be on scale. This
condition applies particularly to MICs of the presumptive wild-type strains.
 Before merging data for ECV estimation, the MIC distribution from each individual laboratory is
inspected, and if the lowest concentration tested is also a mode, these data cannot be included in
the merged dataset.

Once acceptable data are merged, a number of methods can be used to estimate the ECV. The method used
to determine ECVs should be described. The simplest method is visual inspection, which generally works
for MIC distributions when there is clear separation of wild-type and non-wild-type. When there is no clear
separation between wild-type and non-wild-type strains, visual inspection becomes subjective and recourse
to statistical methods should be considered to remove any potential observer bias from the estimation.
Several methods have been proposed and published.24-29 The sponsor should explain and justify the
methodology that is used.

Estimation of ECVs from MIC distributions may be supplemented with molecular tests for known
resistance mechanisms as a form a confirmation. The detection of a resistance gene per se in organisms
with MICs at or below the ECV does not necessarily invalidate the choice of ECV, unless it can be
accompanied by evidence that the gene is being expressed.

Nonclinical Pharmacokinetic-Pharmacodynamic Cutoff

The nonclinical PK-PD cutoff is the highest MIC value at which efficacy would be predicted in patients
based on nonclinically derived PK-PD relationships and human PK exposures. The nonclinical PK-PD
cutoff is derived from identifying the PK-PD target (which is defined by the PK-PD index best describing
the exposure-response relationship and the magnitude of the PK-PD index associated with efficacy) in

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nonclinical studies, then applying it in Monte Carlo simulation using human PK exposures to predict the
percentage of patients likely to achieve the PK-PD target at a given MIC.

Analyses describing the relationship between exposure and response for efficacy of antimicrobial agents in
nonclinical studies must be included in the submission. Standardized PK-PD terminology for anti-infective
agents must be used.30

 PK-PD index refers to the quantitative relationship between a measure of drug exposure, such as area
under the curve (AUC) and a microbiological parameter such as MIC.

 PK-PD magnitude refers to the numerical value of the PK-PD index.

 PK-PD target is a magnitude for a PK-PD index at which a desired level of predicted response is
achieved.

The science of PK-PD relationships is an evolving one. Therefore, submissions to the CLSI Subcommittee
on AST should clearly describe the sources of data, assumptions, and details of the models and simulations
used to support the proposed breakpoints. This information will enable comparisons with other methods
and facilitate re-examination of nonclinical PK-PD cutoffs if it becomes necessary to do so in the future.

Static In Vitro Studies

Static in vitro experiments, such as time-kill studies and postantibiotic effect (PAE) studies, serve as an
initial step in the process of identifying the PK-PD index or indices most closely associated with efficacy.
Static in vitro experiments can serve to support nonclinical and clinical PK-PD study results.

For antimicrobial agents that display a concentration-dependent pattern of bactericidal activity in time-kill
studies, the ratio of area under the concentration-time curve over 24 hours to the MIC of the pathogen
(AUC0-24:MIC ratio) and/or the ratio of the maximal drug concentration to the MIC of the pathogen
(Cmax:MIC ratio) is usually predictive of efficacy in PK-PD model systems. For antimicrobial agents that
display a time-dependent pattern of bactericidal activity in time-kill studies without PAE, the percentage of
time during a dosing interval that drug concentrations remain above the MIC of the antimicrobial agent for
the pathogen (%Time > MIC) is usually predictive of efficacy in PK-PD model systems. For antimicrobial
agents that display a time-dependent pattern of bactericidal activity in time-kill studies and demonstrate
PAE, the AUC0-24:MIC ratio is usually predictive of efficacy in PK-PD model systems. For agents that have
intracellular activity, information on intracellular concentration and activity against target pathogens may
be appropriate. Sponsors may provide:

 Time-kill study results, when such studies have been conducted

 At a minimum, studies should be conducted using isolates relevant to the intended clinical uses
(representative of the predominant pathogens and/or the major resistance mechanisms) over a range
of clinically relevant drug concentrations. Using well-characterized strains, such as ATCC® strains,
is encouraged to allow comparison with historical data for other antimicrobial agents.

 Studies should be conducted at least in duplicate. The sponsor should follow standard procedures
described in CLSI document M26.31 If other methods are used, details of the procedures must be
provided.

 PAE study results, when such studies have been conducted

 PAE studies should be conducted with clinically relevant isolates.
 Studies should be conducted at least in duplicate. Detailed methods must be provided.32

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Nonclinical Pharmacokinetic-Pharmacodynamic Studies

* Nonclinical PK-PD infection model systems are used to identify the PK-PD index or indices that are most
closely associated with efficacy. Additionally, nonclinical PK-PD model systems provide insight into the
magnitude of the PK-PD index or indices necessary to achieve relevant clinical end points in patients. Data
from these studies are used to ultimately derive the nonclinical PK-PD cutoff. Nonclinical PK-PD studies
can serve to support clinical PK-PD study results when such data are available.

* The sponsor should conduct studies (or provide existing data) to identify the PK-PD index or indices most
closely related to efficacy. Indices typically evaluated include %Time > MIC, AUC0-24:MIC ratio, and
Cmax:MIC ratio. Other indices may also be evaluated, as appropriate. Typically, these data are generated
from adequately controlled and well-designed nonclinical PK-PD infection models (animal or in vitro).
Examples of acceptable models include the neutropenic mouse thigh and lung infection models33,34 and in
vitro PD models35 (eg, chemostat or hollow-fiber systems). Studies should be designed with treated and
untreated control arms, and PKs should be assessed. PK-PD data from these studies should be of high
quality with well-defined model conditions and study design variables. Detailed methods and pertinent
experimental data should be provided for appropriate interpretation of PK-PD results.

Other nonclinical models (eg, non-neutropenic mouse model) may be considered if sufficient data are
submitted to demonstrate correlation of the proposed model to an acceptable model or to the clinical
indication. Ideally, nonclinical models should mimic the human indication as closely as possible. This type
of model is of particular interest for indications when drug penetration to the infection site is an issue (eg,
endocarditis, meningitis, and pneumonia).36 Whenever possible, assessment of exposure to the
antimicrobial agent at the infection site (eg, epithelial lining fluid [ELF] for agents intended to treat
pneumonia) in addition to plasma or serum is recommended. Site-specific infection models may be
considered supplementary to the acceptable PK-PD models mentioned above.

* When conducting nonclinical PK-PD studies, a range of suitable isolates of species representative of those
most relevant to the intended clinical uses and exhibiting an MIC range across the wild-type distribution
should be examined. A minimum of three strains should be tested for each bacterial species or group that
has its own interpretative criteria (see CLSI document M1005). In the case of Enterobacteriaceae, testing
≥ 3 strains of each species most relevant to the clinical uses is appropriate. Strains with different phenotypes
(eg, methicillin-resistant S. aureus vs methicillin-susceptible S. aureus, extended-spectrum β-lactamase
[ESBL] vs non-ESBL) should be evaluated, if relevant. Including a few strains that have been previously
characterized in PK-PD models (eg, ATCC® strains) is also recommended to allow comparison with
historical data generated with other antimicrobial agents. Testing of additional isolates beyond the
minimum requirement may be necessary to assess for interstrain PD variability that is not explained by
MIC alone.37

* When undertaking analyses of PK-PD relationships, model parameters and closeness of fit should be
stated. For example, when a Hill-type function is fit to PK-PD data, sponsors should provide E0, EC50, Emax,
and the Hill coefficient. Reporting of the 95% confidence interval around the mean parameter values is
encouraged. Details of the analysis methods used should be provided.

* Each PK-PD index should be expressed as a function of free drug concentrations. If the PK-PD index or
indices are not presented as free drug, the sponsor must provide scientific reasoning to justify why this is
appropriate. The magnitude of the PK-PD index or indices necessary to achieve selected end points should
be provided. These may include EC80; net bacterial stasis; or 1-, 2-, and/or 3-log10 reductions in bacterial
densities. Although no specific end point is consistently most relevant, general guidance can be provided
using the neutropenic mouse thigh model, for which there is evidence demonstrating the translation to
humans.33,34 Some example scenarios include:

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 For severe, life-threatening infections of high bacterial burden, such as nosocomial pneumonia,
achieving the magnitude of the PK-PD index associated with a minimum of a 1-log10 reduction using
the neutropenic mouse thigh or lung model has shown correlation with favorable clinical outcomes in
patients.34

 For low-density infections that are treated in part by surgical interventions, such as complicated skin
and skin-structure infections or complicated intra-abdominal infections, achieving the magnitude of the
PK-PD index associated with a minimum of net stasis using the neutropenic mouse thigh model has
correlated well with favorable clinical outcomes in patients.34

* The sponsor should provide justification for the nonclinical PK-PD target selected to derive the
nonclinical PK-PD cutoff. The rationale should describe the magnitude of the PK-PD index that is being
targeted for the selected efficacy end point (ie, the PK-PD target) and how it relates to the clinical indication.

When breakpoints are being proposed for a species for which robust nonclinical models have not been
validated, it may be acceptable to extrapolate the nonclinical PK-PD target from one species to another.
The sponsor should provide evidence to support the relevance of the proposed nonclinical PK-PD target to
the species. Advice can also be requested from the CLSI Working Group on AST Breakpoints.

Human Pharmacokinetic Data

* Human PK data are critical for development of breakpoints for in vitro susceptibility testing. These data:

 Provide an understanding of exposure to the antimicrobial agent achieved following administration of

the selected dosage regimens in target patient populations (ie, the types of infected patients in whom
the antimicrobial agent is likely to be used).

 Enable assessment of interpatient variability.

 Enable analysis of the covariates that influence drug exposure.

* Population PK models should be developed to predict human drug exposure for Monte Carlo simulation
and for analyses exploring exposure-response relationships in the target patient population.

* The sponsor should provide PK information derived from the target patient population. These data should
be sufficient (eg, derived from limited detailed and more extensive sparse sampling) to provide robust point
estimates of PK parameter values, assess interpatient variability, and identify clinically significant
covariates. If PK data from the target patient population are not available, the sponsor must provide
information from relevant volunteer and special population (eg, renal impairment) PK studies along with
an explanation as to why these data are adequate in place of target patient population data.

* The sponsor must provide information on human protein binding to estimate free drug concentrations.
Human protein binding should be evaluated at clinically relevant concentrations and over a range to assess
for concentration-dependent binding. Detailed methods must be provided.

If available, total and free drug concentration-time data in relevant nonhomogenate tissues and body fluids
should be presented to help understand drug penetration to the infection site (see examples below). Drug
penetration should be evaluated by the rate and extent of drug exposure at the body site relative to plasma
or serum. Although the science around PK-PD using body site PKs is evolving, the difference in
the tissue:plasma or tissue:serum exposure ratio between humans and animals (from which the nonclinical
PK-PD target is derived) should at least be considered.38

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Some examples of data showing drug penetration to the infection site include:

 Data showing the PKs of the free drug in urine, which should be provided if the drug will be used to
treat urinary tract infection.

 Free drug concentration data in ELF, which may be important in the evaluation if antimicrobial agents
are intended to treat pneumonia.36 Studies to assess ELF penetration often use a population approach
with a single bronchoalveolar lavage assessment per subject for a compilation of multiple assessments
at different time points relative to the time of drug administration.

 Free drug concentration data in CSF, which must be presented if the antimicrobial agent is intended to
treat meningitis. CSF penetration studies often use a population approach for sampling.

 Free drug concentration data in vegetations, abscesses, and skin, which may be helpful for drugs that
are to be used to treat endocarditis, abdominal abscess, and skin infection, respectively. For skin
infection, the data can be obtained with a skin blister fluid or tissue microdialysis study.

Monte Carlo Simulation and Probability of Target Attainment

Monte Carlo Simulation

* Monte Carlo simulation is useful for evaluating potential nonclinical PK-PD cutoffs.39 Typically,
simulation inputs include measures of central tendency statistics for PK parameters and their associated
variance. Through this statistical algorithm, it is possible to estimate the probability of attaining the target
drug exposure consistent with efficacy (derived from nonclinical PK-PD studies) in the context of the MIC
of the pathogen(s) of interest, which is more formally known as the probability of target attainment (PTA).

A large variety of software programs can be used to conduct Monte Carlo simulation analysis. Monte Carlo
simulation has been the technique most commonly used to provide data for evaluation by the CLSI
Subcommittee on AST in determining nonclinical PK-PD cutoffs, and the information needed to support
these simulations is provided below. If alternative methods are used by the sponsor, corresponding elements
should be assumed necessary.

* It is preferred that the PK inputs for Monte Carlo simulations be based on a target patient population PK
model. The validity of this tool is based on an assumption that the population PK model provides an
adequate description of drug exposure in the target patient population. Although there is no set number of
individuals that should be included in developing a patient population PK model, generally the dataset
should be sufficient to provide precise estimates of the main PK parameters and their associated variances
and allow for assessment of the covariates’ effect.

* If a target patient population PK model is not available, the sponsor may use a relevant volunteer-derived
PK model. When using a population PK model constructed from healthy volunteer data, it is imperative to
make adjustments to the model so the Monte Carlo simulation results more accurately reflect drug exposure
and its associated variability in the target patient population. An explanation as to why the volunteer-derived
PK model is adequate in place of a target patient population model must be provided along with sensitivity
analyses. Examples of adjustments to the population PK model include:

 In healthy volunteers, the mean point estimate of drug clearance is often somewhat similar, but the data
dispersion around the mean point estimate is considerably less variable than that observed in a patient
population. In such a circumstance, the variability around the point estimate of drug clearance to a
magnitude similar to that expected in the target patient population should be artificially inflated.

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 The inflated variance approach is crucial for any drug that may be used in patients with severe
infections associated with considerable systemic upset because of the large PK variability observed
in this population.

 The level of variance applied for simulation should be justified by the sponsor.

 Healthy volunteers often have normal renal function, whereas patients often have some degree of renal
impairment.
 In this scenario, a representative distribution for creatinine clearance to better reflect that seen in
the target patient population should be imposed.

 The distribution of renal function applied for simulation should be justified by the sponsor.

* Monte Carlo simulation should use the same PK model from which the PK parameter and dispersion
estimates were obtained. Exceptions are model adjustments, as previously described, intended to better
estimate the PKs and associated variability in the target patient population.

* Monte Carlo simulation can use PK-PD relationships derived from nonclinical infection models or from
the target patient population. When the nonclinical PK-PD target is used, differences in protein binding
between the nonclinical and human systems must be considered.

* Simulation results must be presented stratified by MIC value and in the context of the MIC distribution
for the species of interest. It must be noted whether any of the MIC values were censored (eg, values ≤ 0.25
µg/mL changed to 0.125 µg/mL). For Monte Carlo analyses performed using censored MIC populations,
simulations should be performed with and without those values.

* The underlying population distributions (eg, normal, log normal) and/or assumptions used for the various
inputs (PK data, MIC data), as well as the number of simulations performed, must be described. The total
number of iterates (ie, simulated patients) conducted in the Monte Carlo simulation depends on the
variability of the data and the complexity of the analyses. Generally, 1000 to 5000 iterates are sufficient for
the majority of Monte Carlo simulations.

Probability of Target Attainment

* PTA is the most commonly reported output for Monte Carlo simulation and is the final step in determining
nonclinical PK-PD cutoff.

* For the purpose of identifying nonclinical PK-PD cutoffs, 90% PTA at a given MIC is considered
acceptable by the CLSI Subcommittee on AST. However, there are circumstances in which higher or lower
percentages may be acceptable. For example, if the consequence of not attaining the target PK-PD threshold
is a high probability of a patient’s death (eg, suboptimal drug exposure in the context of a patient with
pulmonary anthrax), higher PTA thresholds may be judged appropriate. Conversely, a lower PTA may be
acceptable if the infection is less severe.

Additional Considerations

* The sponsor must provide details of the methods used for measurements of total and active drug
concentration in animal and human plasma, serum, or body fluids. This material should provide a
description of any microbiologically active metabolites of the antimicrobial agent.

The relative performance of assays for drug concentration measurement, such as high-performance liquid
chromatography, should be provided. If bioassay methods are used for antimicrobial agents with active

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metabolites, relevant information about the differences in metabolite formation in humans and animals
should be provided.

Clinical Exposure-Response Cutoff

The CER cutoff is the highest MIC value at which efficacy would be predicted in patients based on CER
relationships for efficacy in infected patients and on human PKs. In this guideline, “exposure-response
relationship” refers to the exposure-response relationship for efficacy; it does not include the exposure-
response relationship for safety.

The CER cutoff is derived by using a population PD model based on data from humans to describe drug
disposition, Monte Carlo simulation to generate an exposure distribution, and an exposure-response
relationship based on clinical data. Acceptable approaches for analyses that can be conducted to identify
exposure-response relationships based on clinical data and the application of such data to identify a CER
cutoff are described in Subchapter 5.3.1.

As discussed in Subchapter 5.3.1, deriving a CER cutoff may not be possible using data from the clinical
trial program, for various reasons. Streamlined programs studying limited numbers of patients may provide
insufficient data to support the identification of such a relationship. Additionally, if high success rates are
observed in trials in conjunction with a dosage regimen that effectively provided all (or nearly all) patients
with an exposure that was high relative to the MIC of the antimicrobial agent for their pathogens, it will
reduce the likelihood of being able to identify a CER cutoff.

That limitation understood, analyses describing the exposure-response relationships of antimicrobial agents
in infected patients should be included where feasible in submissions to the CLSI Subcommittee on AST
to support requests for breakpoints. Standardized PK-PD terminology for anti-infective agents must be used
when reporting such analyses, ie, PK-PD index, PK-PD magnitude, and PK-PD target (see Subchapter
5.2).30,39 However, for determining a CER cutoff, where the exposure-response relationships will be
determined in infected patients vs animal models of infection, these parameters are referred to as “clinically
derived.”

NOTE: The science of determining exposure-response relationships in infected patients is evolving.

Therefore, submissions to the CLSI Subcommittee on AST should clearly describe the sources of data,
assumptions, and details of the models and simulations used to support the identification of the CER cutoff.
This information allows comparisons to be made with other methods and facilitates re-evaluation of CER
cutoffs should it become necessary to do so in the future.

Analyses to Identify Exposure-Response Relationships for Efficacy

CER relationships are established based on data from an evaluable population of infected patients enrolled
in clinical trials in which:

 Clinical outcomes are assessed.

 Adequate PK data are collected.
 Baseline MIC values of the antimicrobial agent for the pathogens of interest are determined.

Sponsors need to provide justification for efficacy end points chosen for evaluation (see Subchapter 5.3).

Statistical approaches for evaluating univariable exposure-response relationships are based on the nature of
the variables for the efficacy end point and the PK-PD index to be evaluated. Acceptable approaches that
can be undertaken include those described in Table 6. Sponsors should provide justification for alternative
methods.

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Table 6. Evaluating Univariable Exposure-Response Relationships

Efficacy End-Point Category
PK-PD Index Dichotomous
Category Variables Continuous Variables Time-to-Event
Continuous variables Logistic regression and Linear regression, Cox proportional
Hill-type/Emax Spearman correlation, hazards and/or
and Hill-type/Emax parametric regression
models
Categorical variables Chi-square or Fisher Analysis of variance Log-rank tests
exact test and Wilcoxon rank sum
test
Abbreviation: PK-PD, pharmacokinetic-pharmacodynamic.

As shown in Table 6, PK-PD indices can be evaluated as continuous or categorical variables. The
assessment of PK-PD indices as categorical variables enables characterization of potential nonlinearity or
nonmonotonicity. To this end, a tool such as classification and regression tree (CART) analysis can be used
to provide guidance on how to construct two-group or multigroup categorical variables. For a dichotomous
efficacy end point, the resulting splits of a classification tree can be used; for a continuous efficacy end
point, the resulting splits of a regression tree can be used.

If other patient factors in addition to PK-PD index are found to be predictive of the efficacy end point based
on the results of univariable analyses, multivariable analyses should be undertaken to evaluate predictors
of outcome. In such cases, it may be more appropriate to consider distributions for such patient factors in
addition to those for PK parameters when conducting Monte Carlo simulations to assess model-predicted
percent probabilities of response.

Application of Exposure-Response Relationships to Identify the Clinical Exposure-Response

Cutoff

The exposure-response relationship identified, as described in Subchapter 5.3.1, can then be used to identify
the CER cutoff. Two typical methods are described in Subchapters 5.3.2.1 and 5.3.2.2.39

Determining a Clinical Exposure-Response Cutoff Based on the Model-Predicted Probability of

Response by Minimal Inhibitory Concentration

Using a population PK model based on data from humans to describe drug disposition, Monte Carlo
simulation to generate an exposure distribution, and the CER relationship, model-predicted percent
probabilities of response can be determined for a dichotomous efficacy end point at given MIC values. For
a continuous end point, mean (or median) response can be evaluated by MIC. In the case of a dichotomous
end point, the MIC chosen as the CER cutoff is determined by the selected summary statistic (eg, median),
which provides a percent probability of model-predicted response consistent with that desired for the
disease state.

Determining a Clinical Exposure-Response Cutoff Based on the Probability of Pharmacokinetic-

Pharmacodynamic Target Attainment

Using a population PK model based on data from humans to describe drug disposition, Monte Carlo
simulation to generate an exposure distribution, and a clinically derived PK-PD target (eg, a CART-derived
value that is highly predictive of response or a model-derived value associated with a high probability or
level of response), the percent probability of PK-PD target attainment can be determined at given (or
various) MIC values. For dichotomous efficacy end points, the MIC chosen as the CER cutoff is determined
as the highest MIC at which target attainment of at least 90% is achieved.

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An important limitation of this approach is that failure to achieve a PK-PD target as evidenced by low
percent probabilities of PK-PD target attainment at a given MIC value does not necessarily predict that the
percentage of successful responses will be equally low at that same MIC value. Thus, the evaluation of
model-predicted percent probabilities of response described above, when possible to carry out, is expected
to provide predictions that are more aligned with observed response by MIC value than assessments of
percent probabilities of PK-PD target attainment.

Additional Considerations

Sponsors are strongly encouraged to collect adequate PK data in phase 2 and 3 clinical trials for the purpose
of attempting to establish CER relationship(s) for efficacy so that a CER cutoff can be determined. The
exposure-response analyses should consider the nature of the clinical end point and whether univariable or
multivariable analyses are more appropriate. The sponsor should fully justify why a particular analytical
approach was used to identify a CER cutoff. Bayesian analysis of preclinical and clinical data may be
considered an appropriate option.40 Exposure-response relationships derived from clinical data may not be
sufficiently optimal to set an initial CER cutoff. This outcome is commonly due to the inability to
adequately characterize exposure-response relationships for efficacy based on data from phase 2 and 3
clinical trials in which too few failures occurred or there were too few patients with exposure in a critical
range. However, some recommendations to enhance the likelihood of generating sufficient data to support
a CER cutoff are made in this subchapter.

The number of patients needed for a meaningful exposure-response analysis is influenced by the dose range
under study, the sample size, and characteristics and quality of the data. For example, for a dichotomous
efficacy end point, if the numbers of failures or successes are too high, it would be difficult to identify an
exposure-response relationship. For a time-to-event efficacy end point, the statistical approaches are more
sensitive so that more can be done with data from fewer patients as long as the percentages of censored data
are within reasonable limits. Specific guidance on numbers of patients needs to be provided in relation to
the actual study design. Time-to-event efficacy end points (eg, time to fever reduction) that have been
demonstrated to be clinically relevant as well as relevant to the drug exposure should be considered in study
designs.

Sponsors should report the diagnostics of fitting exposure-response data to statistical models (model
building) and evaluation of the predictability of the model (model validation). These diagnostics lend
confidence to the strength of the underlying exposure-response relationship, which has an important role in
Monte Carlo simulation.

Although CER cutoff is an attractive concept due to its integration of data from several sources and obvious
clinical relevance, it is not necessarily more reliable than the methods determining a cutoff based on
nonclinically derived PK-PD targets (eg, from animal models) and human PK exposures. Nevertheless, if
clinical data are sufficient to support a significant CER relationship, CER cutoff can be useful for predicting
efficacy.

Clinical Cutoff

For the purpose of setting breakpoints, clinical cutoff is based on a simple evaluation of any correlation
between clinical outcome and the MIC(s) of an antimicrobial agent(s) for the infecting pathogen(s). See
Subchapter 5.3 for a description of the CER cutoff, which is defined as the highest MIC value at which
likely efficacy would be predicted in patients based on CER relationships for efficacy in infected patients
and on human PKs.

During clinical evaluation of new antimicrobial agents or in trials that evaluate an existing antimicrobial
agent for the treatment of new types of infections, sponsors should investigate any correlation that might
be discernible between in vitro susceptibility test results and therapeutic outcomes (clinical and

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microbiological). For established antimicrobial agents, sponsors proposing new or revised breakpoints may
not have access to the raw data from clinical trials. Thus, sponsors and the CLSI Subcommittee on AST
may have to rely on the limited information available from the literature and published regulatory reviews.

A clinical cutoff based on a simple correlation of outcome to MIC has significant limitations, which the
CER cutoff attempts to overcome. Although it is still considered useful to review a simple outcome from
MIC data, it is not usually possible to determine clinical cutoffs from clinical trial data due to several factors,
which may include:

 The inherent structure of clinical trials, with exclusion of patients with resistant organisms

 The rarity of pathogens resistant to the antimicrobial agent when preregistration trials are conducted

 The limited range of infection types studied, which limits the microbial species treated

 The contribution of other intrinsic and extrinsic factors (eg, host immune systems, adjunctive
treatments, adjunctive surgery) that contribute to between-patient variability

 Limited interpretation of outcome analyses by patient or pathogen due to the lack of straightforward
identification of the major pathogen for some infections (eg, in polymicrobial intra-abdominal
infections).

 The absence of significant numbers of resistant organisms with very high MICs. Usually, the dosage
regimen(s) studied will have been chosen based on PK-PD analyses to ensure coverage of organisms
with MICs across the entire wild-type range. When significant numbers of resistant organisms with
very high MICs are absent, the clinical outcome data (simple correlation of outcomes to MIC) may not
be able to provide meaningful evidence to either support or reject the nonclinical PK-PD cutoffs and/or
CER cutoffs.

* Nevertheless, subject to availability of sufficient relevant data to the sponsor, submissions to the CLSI
Subcommittee on AST to support breakpoints should include a summary of the clinical data that describes
clinical and microbiological outcomes according to the MICs of the antimicrobial agent under study that
were documented for the infecting pathogens. Presentations of outcomes by MIC should be separated by
the dosage regimen administered when clinical dose-finding studies were conducted.

In many cases, especially for new antimicrobial agents, the same data will have been submitted or will be
submitted to regulatory agencies. The sponsor should note that if the data presented to the CLSI
Subcommittee on AST differ from those presented to the regulatory agencies, the differences should be
described and explained. Presentation of the clinical outcome data in the submission should include the
elements described in Subchapters 5.4.1 and 5.4.2.

Description of Clinical Trials

* The sponsor should provide a summary of each clinical trial, with attention to the following elements:

 Description of the study population

 Types of infection treated, with details of diagnostic criteria

 Dosage regimens for the antimicrobial agent under study and all comparative regimens, including any
switches that were allowed, with details of issues such as infusion times and total therapy duration

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 Timing of all visits at which clinical and microbiological data were collected, noting at which visits
outcomes were assessed

 Definitions of the analysis populations (eg, modified intent to treat, clinically evaluable,
microbiologically evaluable)

 Definitions of response

 Primary and secondary end points

 Statistical approach and sample size determination

Presentation of Outcomes

* Whenever the sponsor has access to the data, the presentation should include:

 Correlations between clinical and microbiological outcomes (categorized as in the protocol), drug
exposures, and actual MICs of the pathogens, analyzed by patient and by pathogen for individual
clinical trials and after pooling across trials
– For the outcomes by pathogen, an analysis by genus (Escherichia, Klebsiella, Proteus) and by
individual species, for the more commonly encountered pathogens, should be included in addition
to analyses by broad category (eg, Enterobacteriaceae).

 Results, which should be obtained using established CLSI reference methods for the antimicrobial agent
– If any results are obtained using other methods or systems, data must be presented to demonstrate
the comparability of such methods with CLSI reference methods (see Chapter 2).

 QC data to support the reliability of the MIC data

 Data described in the first bullet repeated for each of the predefined patient populations for the analysis
of efficacy

 The summarized approach to data handling, with special regard to the handling of missing data and
default of indeterminate outcomes to failures in some analysis populations

 Separate analyses, if applicable, for patients with infections at specific body sites and for patients with
concurrent bacteremia

 A detailed consideration of the data from patients with clinical and/or microbiological failure

 A separate presentation of any data on outcomes from patients who were treated despite having an
infection due to an organism that would be designated resistant based on the proposed breakpoints

Consideration of the Cutoffs to Determine Breakpoints

Subchapters 5.1, 5.2, 5.3, and 5.4 describe the:

 ECV
 Nonclinical PK-PD cutoff
 CER cutoff
 Clinical cutoff

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For the purposes of determining susceptibility test breakpoints, it is necessary to take into account and
weight the various sets of data and outputs of the relevant analyses that may be available. It is unlikely that
a submission to the CLSI Subcommittee on AST to support breakpoint determination will include all the
above elements and cutoffs. For example, available data may be inadequate to support derivation of a CER
cutoff. In addition, it is very unusual for analyses of clinical outcomes by MIC to yield a clear clinical
cutoff.

Depending on the available data and the cutoffs that have been identified, the combinations of information
relevant to determining breakpoints will vary considerably. Some approaches to consideration of the
available data include the following scenarios, in which it is assumed that any differences there may be
between available cutoffs are ≤ 2-fold. If the difference between any available cutoffs is > 4-fold, the
possible reasons for the discrepancy must be explored before any attempt is made to determine breakpoints
from the cutoff values.

 If the ECV for a particular species or group of species is significantly greater than the nonclinical PK-
PD and the CER and/or clinical cutoffs, it can be safely assumed that the species is intrinsically resistant
to the antimicrobial agent when used at the dosage regimen on which the cutoffs are based.

 If the ECV is less than the other cutoffs, it is clear that the species is intrinsically susceptible, and it is
only necessary to choose among the available cutoffs, if they are not the same, to select breakpoints.

 If the available cutoffs are not the same, the relative weighting of the cutoffs depends on their perceived
robustness. For example, if a clinical cutoff was established, it is likely to be less reliable than the
nonclinical PK-PD cutoff. If a CER cutoff was established using a full pharmacometric analysis of
clinical trials but differs from the nonclinical PK-PD cutoff, it may be more difficult to give one more
weight over the other. The value of the data supporting each cutoff would need to be evaluated on a
case-by-case basis.

 There is more uncertainty when one or more of the available cutoffs fall within the wild-type MIC
distributions. In general, it is considered undesirable to set breakpoints that fall within the wild-type
MIC distribution, because the interpretation of the test result will be greatly influenced by normal test
variation. When this situation occurs, the potential clinical effect needs to be considered. For example,
if the intended use of the antimicrobial agent is for nonsevere infections caused by the species or group
of organisms being examined and the preferred breakpoints based on available cutoffs fall near the high
end of the wild-type distribution, there may not be a significant effect on appropriate clinical use.

 If there are only an ECV and a nonclinical PK-PD cutoff, the considerations are similar to those outlined
above in the third and fourth bullet points.

 It is considered very unusual when a submission includes only an ECV and a clinical cutoff. On
occasion, this situation may occur if a breakpoint revision is proposed for an antimicrobial agent that
has been in use for many years and signals from clinical studies or during routine use suggest that the
current breakpoints are inappropriate and if there is perceived to be sufficient urgency to revise the
breakpoints without generating data to support a nonclinical PK-PD cutoff.

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Disk Diffusion Breakpoints

This chapter includes:

 Selecting isolates for disk diffusion testing

 Selecting disk breakpoints

Selecting Isolates and Sample Size

In general, studies should be performed with isolates representing all species that are likely to be tested by
the disk diffusion procedure, the majority of which should belong to species that are most relevant to the
intended clinical uses. Additional details on recommended sample sizes are provided in Appendix A.

For analysis of data by the error-rate bounded method (see Subchapter 6.3), the nature of the culture
collection studied is critically important. When reporting the results of such studies, the type of culture
collection used must be specified.

Reagent Disks

See Subchapter 2.3 regarding the development of disks. All studies should be conducted with reagent disks,
regardless of source (commercial or other), that meet the requirements stipulated in published regulations.20

Error-Rate Bounded Method for Selecting Disk Diffusion Breakpoints Based on

Comparison With Dilution Test Results

* Breakpoints and Discrepancy Rates

The error-rate bounded method41 may be used to select zone diameter breakpoints and to calculate
discrepancy rates between MICs and zone diameter test results. The error-rate bounded method usually
needs to be modified42 because two MIC values are normally described to define an “intermediate” category
additional to the “susceptible” and “resistant” categories. Data should be displayed as a scattergram, with
zone diameters on the x-axis and MICs on the y-axis and horizontal and vertical lines showing the proposed
breakpoints.

In practice, the proposed zone diameter breakpoints are simply adjusted until the number of false-
susceptible disk diffusion test results (very major discrepancies) and false-resistant disk tests (major
discrepancies) are held to a minimum. Minor discrepancies (ie, when one of the test results is intermediate
and the other is susceptible or resistant) should also be considered in these determinations. When data points
for a large proportion of isolates are close to the proposed or approved breakpoints for an antimicrobial
agent, a high percentage of minor discrepancies may be expected. Software to undertake the error-rate
bounded method is available online at https://dbets.shinyapps.io/dBETS/. The software also provides two
model-based analyses that may be used for comparison and validation purposes.

For initial determination or reassessment of breakpoints, a table should be provided showing the total
number of isolates for each species tested and the number of minor, major, or very major discrepancies
recorded for each species or organism group. Table 7, or Table 8 when there is no “intermediate” range,
can be used as a template. When selecting zone diameter breakpoints, consideration given to the species
tested and whether they belong to the same group of organisms as one of the QC strains will help determine
the range of the “intermediate” category, when it exists. The minimum “intermediate” range should equal
half or be 1 mm wider than half of the relevant QC range, preferably a little wider to accommodate
interlaboratory variation. The maximum “intermediate” range should be no wider than the relevant QC

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range. When there is no “intermediate” category, there should be a single interpretive zone diameter 2 mm
lower than the lowest observed zone diameter in the “susceptible” category.

Acceptable Discrepancy Rates for Challenge Sets of Organisms (see Appendix A)

Because of the inherent variation in MIC end points, discrepancy rates (defined in Subchapter 6.3.1) will
be directly proportional to the percentage of isolates with antimicrobial agent MICs in the range of one
twofold concentration above the intermediate MIC (I + 1) and one twofold concentration below the
intermediate MIC (I − 1). Thus, when an entire population is used as the denominator for calculating
discrepancy rates, the rate will be determined largely by the population of MICs in the I + 1 to I − 1 range.
For example, when 90% of the isolates have highly susceptible drug MICs (as is common with newer
antimicrobial agents), the discrepancy rate will be considerably less than that of a population in which 40%
of the MICs fall in the I + 1 to I − 1 range. Using the total I + 1 to I − 1 subpopulation as the denominator for
calculating discrepancies provides a more accurate assessment of the discrepancy by accounting for normal
technical variability in the testing method.

Of greater concern are discrepancies that occur with MICs ≥ 2 twofold concentrations above (≥ I + 2) or
below (≤ I − 2) the intermediate MIC. For such nonconcordant MIC–zone diameter pair results, the data
should be checked for obvious analytical or transcriptional errors (transcriptional errors should be
corrected). If there has been contamination, the isolate(s) can be retested on a single occasion, with the
original data point replaced with the retest results. If a review reveals no obvious explanation for the
nonconcordant MIC–zone diameter results, additional evaluation is needed to investigate the discrepancies.
The test should be repeated at least twice, using separate inocula. If at least two of the three results are
identical, the original data point should be replaced with this test result in the scatterplot. If all results
(original and repeat results) are different, the original results should be retained. In a separate table, detailed
results of all investigations of discordant pairs should be recorded; all results obtained on repeat testing
should be shown.

Based in part on the data from the scattergrams of antimicrobial agents for which breakpoints have been
approved by CLSI, Table 7 is provided as a guideline for acceptable discrepancy rates using specific MIC
subpopulations as the denominator. These guidelines, along with other factors, will be weighed in assessing
the appropriateness of proposed breakpoints.

When there is a two-dilution intermediate range, the process for determining discrepancy rates remains the
same, with one modification. The MIC range of I + 1 to I − 1 will include both intermediate MICs plus one
dilution above the higher intermediate MIC and one dilution below the lower intermediate MIC. For
example, if the intermediate range is 2 to 4 µg/mL, the MIC range of I + 1 to I − 1 will include 8, 4, 2, and
1 µg/mL (see Table 7).

Table 7. Guideline for Acceptable Discrepancy Rates (With Intermediate Ranges)*

MIC Range Discrepancy Rates
1-Dilution 2-Dilution
Intermediate Range Intermediate Range Very Major Major Minor
I+2  IHigh + 2 < 2% N/A < 5%
I + 1 to I − 1 IHigh + 1 to ILow − 1 < 10% < 10% < 40%
I−2  ILow − 2 N/A < 2% < 5%
*See example in Appendix E.
Abbreviations: I, intermediate; MIC, minimal inhibitory concentration; N/A, not applicable.

IHigh and ILow are the higher and lower MICs in a two-dilution intermediate range.

When there is no intermediate range (ie, when the breakpoints distinguish only susceptible and resistant
populations), the process for determining discrepancy rates is similar (see Table 8). If there are no

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intermediate ranges for both MICs and disk diffusion breakpoints, minor discrepancies are not a
consideration.

Table 8. Guideline Acceptable Discrepancy Rates (Without Intermediate Range)

MIC Range Discrepancy Rates
No Intermediate Range Very Major Major Minor
R+1 < 2% N/A < 5%
R+S < 10% < 10% < 40%
S−1 N/A < 2% < 5%
Abbreviations: MIC, minimal inhibitory concentration; N/A, not applicable; R, resistant; S, susceptible.

* Acceptable Discrepancy Rates for Unselected Clinical Isolates (see Appendix A)

Ideally, when evaluating a large collection of unselected clinical isolates, very major discrepancy rates
should be less than 1.5% and major discrepancies should be less than 3% when calculated based on all
isolates.

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Conclusion
This guideline outlines the data that should be assembled and provided to the CLSI Subcommittee on AST
and its working groups to support recommendations for QC strains and ranges and susceptibility testing
breakpoints for inclusion in CLSI documents.

For new antimicrobial agents, the process begins with identification of reference susceptibility testing
methods as described in Chapter 2, followed by initial studies to identify QC strains and ranges as described
in Chapter 3. During the development of a new antimicrobial agent, depending on its properties and
intended uses, it may be possible and appropriate to assemble much of the data described in Chapters 5 and
6 to support identification of both MIC and disk diffusion breakpoints.

Additional information that points to the need to add to or revise existing QC strains, ranges, and
breakpoints may become available. In particular, Chapters 5 and 6 recognize that it is not always possible
or appropriate to provide all the types of information that could be useful to support additions and changes
in breakpoints. Nevertheless, updating breakpoints as new evidence emerges is an important part of the life
cycle of an antimicrobial agent, and sponsors are encouraged to submit whatever information can be
assembled to the CLSI Subcommittee on AST.

Chapter 4 provides detailed guidance to sponsors regarding the processes that are usually followed once a
sponsor comes forward with a submission. It also provides the framework, including timelines, for the
actions to be taken by the CLSI Subcommittee on AST and Working Group on AST Breakpoints and
outlines procedures to be followed depending on the existence of breakpoints recommended by the FDA
and when they were published.

This guideline provides direction while maintaining a degree of flexibility that should facilitate the
development of submissions to the CLSI Subcommittee on AST and ensure that all submissions are handled
within an appropriate timeframe.

Supplemental Information

This chapter includes:

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

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References
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CLSI. Performance Standards for Antimicrobial Disk Susceptibility Tests. 13th ed. CLSI standard M02. Wayne, PA: Clinical and
Laboratory Standards Institute; 2018.
2
CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 11th ed. CLSI standard M07.
Wayne, PA: Clinical and Laboratory Standards Institute; 2018.
3
CLSI. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria. 3rd ed. CLSI
guideline M45. Wayne, PA: Clinical and Laboratory Standards Institute; 2016.
4
CLSI. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard—Eighth Edition. CLSI document
M11-A8. Wayne, PA: Clinical and Laboratory Standards Institute; 2012.
5
CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 28th ed. CLSI supplement M100. Wayne, PA: Clinical and
Laboratory Standards Institute; 2018.
6
CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 4th ed. CLSI standard M27. Wayne, PA:
Clinical and Laboratory Standards Institute; 2017.
7
CLSI. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline—Second Edition. CLSI document
M44-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.
8
CLSI. Performance Standards for Antifungal Susceptibility Testing of Yeasts. 1st ed. CLSI supplement M60. Wayne, PA: Clinical and
Laboratory Standards Institute; 2017.
9
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.
10
CLSI. Method for Antifungal Disk Diffusion Susceptibility Testing of Nondermatophyte Filamentous Fungi; Approved Guideline. CLSI
document M51-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2010.
11
CLSI. Performance Standards for Antifungal Susceptibility Testing of Filamentous Fungi. 1st ed. CLSI supplement M61. Wayne, PA:
Clinical and Laboratory Standards Institute; 2017.
12
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CLSI document M24-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2011.
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. 2nd ed. CLSI supplement M59. Wayne, PA: Clinical and
Laboratory Standards Institute; 2018.
15
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Standardization; 2015.
16
Bradford PA, Petersen PJ, Young M, Jones CH, Tischler M, O‘Connell J. Tigecycline MIC testing by broth dilution requires use of
fresh medium or addition of the biocatalytic oxygen-reducing reagent oxyrase to standardize the test method. Antimicrob Agents
Chemother. 2005;49(9):3903-3909.
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Bradford PA, Sanders CC. Use of a predictor panel to evaluate susceptibility test methods proposed for piperacillin-tazobactam.
Antimicrob Agents Chemother. 1993;37(12):2578-2583.
18
Petersen PJ, Jones CH, Venkatesan AM, Mansour TS, Projan SJ, Bradford PA. Establishment of in vitro susceptibility testing
methodologies and comparative activities of piperacillin in combination with the penem β-lactamase inhibitor BLI-489. Antimicrob
Agents Chemother. 2009;53(2):370-384.
19
Bradford PA, Sanders CC. Use of a predictor panel for development of a new disk for diffusion tests with cefoperazone-sulbactam.
Antimicrob Agents Chemother. 1992;36(2):394-400.
20
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Antimicrobial susceptibility test disc (Codified at 21 CFR §866.1620). US Government Publishing Office; published annually.
21
ISO. Clinical laboratory testing – Criteria for acceptable lots of dehydrated Mueller-Hinton agar and broth for antimicrobial
susceptibility testing. ISO/TS 16782. Geneva, Switzerland: International Organization for Standardization; 2016.

©Clinical and Laboratory Standards Institute. All rights reserved. 53

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 M23, 5th ed.

22
Gavan TL, Jones RN, Barry AL, et al. Quality control limits for ampicillin, carbenicillin, mezlocillin, and piperacillin disk diffusion
susceptibility tests: a collaborative study. J Clin Microbiol. 1981;14(1):67-72.
23
ISO. Clinical laboratory testing and in vitro diagnostic test systems – Susceptibility testing of infectious agents and evaluation of
performance of antimicrobial susceptibility test devices – Part 1: Reference method for testing the in vitro activity of antimicrobial
agents against rapidly growing aerobic bacteria involved in infectious diseases. ISO 20776-1. Geneva, Switzerland: International
Organization for Standardization; 2006.
24
Kronvall G, Kahlmeter G, Myhre E, Galas MF. A new method for normalized interpretation of antimicrobial resistance from disk test
results for comparative purposes. Clin Microbiol Infect. 2003;9(2):120-132.
25
Meletiadis J, Mavridou E, Melchers WJ, Mouton JW, Verweij PE. Epidemiological cutoff values for azoles and Aspergillus fumigatus
based on a novel mathematical approach incorporating cyp51A sequence analysis. Antimicrob Agents Chemother. 2012;56(5):2524-
2529.
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Cantón E, Pemán J, Hervás D, et al; FUNGEMYCA Study Group. Comparison of three statistical methods for establishing tentative
wild-type population and epidemiological cutoff values for echinocandins, amphotericin B, flucytosine, and six Candida species as
determined by the colorimetric Sensititre YeastOne method. J Clin Microbiol. 2012;50(12):3921-3926.
27
Kahlmeter G, Brown DF, Goldstein FW, et al. European harmonization of MIC breakpoints for antimicrobial susceptibility testing of
bacteria. J Antimicrob Chemother. 2003;52(2):145-148.
28
Pfaller MA, Diekema DJ, Ghannoum MA, et al.; Clinical and Laboratory Standards Institute Antifungal Testing Subcommittee. Wild-
type MIC distribution and epidemiological cutoff values for Aspergillus fumigatus and three triazoles as determined by the Clinical
and Laboratory Standards Institute broth microdilution methods. J Clin Microbiol. 2009;47(10):3142-3146.
29
Turnidge J, Kahlmeter G, Kronvall G. Statistical characterisation of bacterial wild-type MIC value distributions and the determination
of epidemiological cut-off values. Clin Microbiol Infect. 2006;12(5):418-425.
30
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terminology for anti-infective drugs: an update. J Antimicrob Chemother. 2005;55(5):601-607.
31
CLSI. Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline. CLSI document M26-A. Wayne,
PA: Clinical and Laboratory Standards Institute; 1999.
32
Craig WA, Gudmundsson S. Postantibiotic effect. In: Lorian V, ed. Antibiotics in Laboratory Medicine. 4th ed. Baltimore, MD:
Williams & Wilkins; 1996:296-329.
33
Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis.
1998;26(1):1-10.
34
Ambrose PG, Bhavnani SM, Rubino CM, et al. Pharmacokinetics-pharmacodynamics of antimicrobial therapy: it’s not just for mice
anymore. Clin Infect Dis. 2007;44(1):79-86.
35
Gloede J, Scheerans C, Derendorf H, Kloft C. In vitro pharmacodynamic models to determine the effect of antibacterial drugs.
J Antimicrob Chemother. 2010;65(2):186-201.
36
Silverman JA, Mortin LI, Vanpraagh AD, Li T, Alder J. Inhibition of daptomycin by pulmonary surfactant: in vitro modeling and
clinical impact. J Infect Dis. 2005;191(12):2149-2152.
37
Soon RL, Ly NS, Rao G, et al. Pharmacodynamic variability beyond that explained by MICs. Antimicrob Agents Chemother.
2013;57(4):1730-1735.
38
Rodvold KA, Nicolau DP, Lodise TP, et al. Identifying exposure targets for treatment of staphylococcal pneumonia with ceftobiprole.
Antimicrob Agents Chemother. 2009;53(8):3294-3301.
39
Ambrose PG, Meagher AK, Passarell JA, et al. Use of a clinically derived exposure-response relationship to evaluate potential
tigecycline-Enterobacteriaceae susceptibility breakpoints. Diagn Microbiol Infect Dis. 2009;63(1):38-42.
40
Ambrose PG, Hammel JP, Bhavnani SM, Rubino CM, Ellis-Grosse EJ, Drusano GL. Frequentist and Bayesian pharmacometric-based
approaches to facilitate critically needed new antibiotic development: overcoming lies, damn lies, and statistics. Antimicrob Agents
Chemother. 2012;56(3):1466-1470.
41
Metzler DM, DeHaan RM. Susceptibility tests of anaerobic bacteria: statistical and clinical considerations. J Infect Dis.
1974;130(6):588-594.
42
Brunden MN, Zurenko GE, Kapik B. Modification of the error-rate bounded classification scheme for use with two MIC break points.
Diagn Microbiol Infect Dis. 1992;15(2):135-140.

54 ©Clinical and Laboratory Standards Institute. All rights reserved.

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 M23, 5th ed.

Appendix A. Guidelines for Clinical Isolate Selection and Sample Size

Abbreviation for Appendix A

MIC minimal inhibitory concentration

This appendix provides guidelines for clinical isolate selection and sample size for the purposes of:

 Describing the antimicrobial activity of an antimicrobial agent (see Subchapters 5.1.3.2 and 5.1.4 of
this guideline)

 Assessing the correlation between minimal inhibitory concentrations (MICs) and zone diameters
obtained from disk diffusion tests (see Subchapter 6.3 of this guideline)

In general, it is recommended that studies of broad-spectrum antimicrobial agents include at least 500
isolates, the majority of which should belong to species that are most relevant to the intended clinical uses.
Fewer isolates (eg, 100) may suffice for narrow-spectrum agents or those developed for treatment of a
narrow range of organisms (eg, a single genus or species).

When the spectrum of activity of the antimicrobial agent includes several organisms or groups of organisms
that have separate breakpoints (eg, Streptococcus pneumoniae, Haemophilus spp., Enterococcus spp.)
and/or the antimicrobial agent is targeting organisms of a specific subgroup within a species, such as
methicillin-resistant Staphylococcus spp., data for such organism subgroups should be obtained.

The following list indicates the number of isolates that should be studied in specific organism groups.

 Enterobacteriaceae (≥ 100 isolates; > 300 preferred)

 Pseudomonas aeruginosa (≥ 100 isolates)

 Acinetobacter spp. (≥ 100 isolates)

 Staphylococcus (≥ 100 isolates; ≥ 300 strains, including Staphylococcus aureus and coagulase-negative
staphylococci, preferred)
 If applicable, 100 methicillin-resistant and 100 methicillin-susceptible S. aureus

 Enterococcus spp. (≥ 100 isolates)

 Haemophilus influenzae and Haemophilus parainfluenzae (≥ 100 isolates)

 Neisseria gonorrhoeae (≥ 100 isolates)

 S. pneumoniae (≥ 100 strains; ≥ 300 isolates preferred)

 Streptococcus spp. other than S. pneumoniae (≥ 100 isolates; ≥ 300 isolates with both β- and α-hemolytic
species preferred)

 Other single species (≥ 100 isolates)

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 M23, 5th ed.

Appendix A. (Continued)
For some less common organism groups, fewer isolates can be used (≥ 50). For less common organisms
that have similar characteristics, ≥ 20 isolates can be used. For example:

 Stenotrophomonas maltophilia
 Vibrio cholerae

For the purposes of:

 Modifying a reference method (see Subchapter 2.1 of this guideline)

 Establishing equivalency between broth and agar dilution (see Subchapter 2.1 of this guideline)

 Establishing the validity of data generated using an alternative to a reference method (see Subchapter
2.4 of this guideline)

At a minimum, it is recommended that 100 isolates should be tested for each group of organisms that is
relevant to the clinical use of the antimicrobial agent and for which separate breakpoints are likely to apply
(eg, per the groups in Tables 2 of CLSI document M1001: Enterobacteriaceae, various nonfermentative
gram-negative bacilli, Staphylococcus spp., S. pneumoniae, other streptococci, Haemophilus spp.,
Enterococcus spp.).

For the purposes of:

 Selecting an appropriate disk content (see Subchapter 2.3 of this guideline)

At a minimum, it is recommended that 30 isolates be tested for each group of organisms that is relevant to
the clinical use of the antimicrobial agent and for which separate breakpoints are likely to apply (eg, per
the groups in Tables 2 of CLSI document M1001: Enterobacteriaceae, various nonfermentative gram-
negative bacilli, Staphylococcus spp., S. pneumoniae, other streptococci, Haemophilus spp., Enterococcus
spp.).

Collections should include both susceptible isolates and isolates with known resistance mechanisms that
are relevant to the type of antimicrobial agent studied (if available). In the absence of naturally occurring
resistant isolates, laboratory mutants with high MICs may be used and should be identified as such in the
presentation of the data. If possible, it is preferable to include a subset of isolates for which MICs are around
the proposed breakpoint for susceptibility or near the epidemiological cutoff value for the antimicrobial
agent.

Two major types of culture collections are listed below. For studies that compare MICs and zone diameters
with the aim of identifying disk diffusion breakpoints, data generated using both types of culture collection
should be available for review by the CLSI Subcommittee on Antimicrobial Susceptibility Testing, in which
case results should be presented separately for each (see Subchapters 6.3.2 and 6.3.3 of this guideline).

 A carefully chosen challenge set of organisms should be gathered to include isolates with all known
resistance mechanisms relevant to the antimicrobial agent evaluated. A similar number of wild-type
susceptible isolates with no known resistance mechanisms should be included, and all relevant species
should be represented.

56 ©Clinical and Laboratory Standards Institute. All rights reserved.

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 M23, 5th ed.

Appendix A. (Continued)

 A large collection of isolates should be gathered from at least three geographically separate study sites.
The distribution of the sites should be explained. Wherever possible, the numbers suggested for study
should be recent clinical isolates (eg, within the previous three years), although the use of banked
isolates may be necessary for less commonly encountered genera and/or species. The organisms studied
should include wild-type and resistant phenotypes or genotypes (if detected) obtained from infected
patients and identified by referenced procedures. If mechanisms of resistance to the agent are
understood, the numbers of isolates in the collection with each type of resistance mechanism should be
presented. Except for antimicrobial agents with a narrow spectrum of activity or those being developed
for limited indications, no more than 20% to 30% of the total isolates tested should be of the same
species.

Reference for Appendix A

1
CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 28th ed. CLSI supplement
M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.

©Clinical and Laboratory Standards Institute. All rights reserved. 57

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 M23, 5th ed.

Appendix B. Sample Data Presentations

Abbreviations for Appendix B

MIC minimal inhibitory concentration

QC quality control
SD standard deviation

RangeFinder MIC or RangeFinder Disk spreadsheet calculators may assist in providing initial estimates of
QC ranges. Refer to the CLSI website at http://clsi.org/standards/micro/rangefinder/ for information
regarding the use of these tools in studies that follow M23 for the determination of appropriate QC ranges.
Tables B1 to B4 provide examples of RangeFinder data analysis.

Table B1. Example of the RangeFinder Analysis of QC Disk Diffusion Range From an Eight-
Laboratory Study
All
ZD Laboratory 1 Laboratory 2 Laboratory 3 Laboratory 4 Laboratory 5 Laboratory 6 Laboratory 7 Laboratory 8 Laboratories
20 0
21 0
22 0
23 0
24 0
25 0
26 1 1
27 9 9
28 1 22 1 24
29 3 8 3 10 1 25
30 6 15 11 22 24 8 86
31 21 27 5 19 19 3 11 7 112
32 21 10 2 15 6 13 11 19 97
33 10 4 1 1 22 18 11 67
34 2 1 18 10 11 42
35 4 7 3 14
36 3 3
37 0
38 0
39 0
40 0
Abbreviations: QC, quality control; ZD, zone diameter.

Calculations:

 There are no obvious outlier laboratories.

 The calculated mean of the zone diameters is 31.373 mm.

 The calculated SD of the zone diameters is 1.797 mm.

 The 95% confidence interval of the zone diameters is 31.373 ± 1.96 • 1.797 = 31.373 ± 3.522 or 27.851
to 34.895, which when rounded down and up, respectively, to the nearest whole millimeter becomes
the range 27 to 35 mm.

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Appendix B. (Continued)
©Clinical and Laboratory Standards Institute. All rights reserved.

Table B2. Example of the RangeFinder Analysis of QC MIC Range From an Eight-Laboratory Study
Midpoint Midpoint Log2 Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory All
MIC MIC MIC 1 2 3 4 5 6 7 8 Laboratories
0.002 0.001381 −9.5 0
0.004 0.002762 −8.5 0
0.008 0.005524 −7.5 1 1
0.016 0.011049 −6.5 1 1 1 4 7
0.03 0.022097 −5.5 7 13 7 7 3 23 1 2 63
0.06 0.044194 −4.5 23 16 23 22 27 5 29 24 169
0.13 0.088388 −3.5 0
0.25 0.176777 −2.5 0
0.5 0.353553 −1.5 0
1 0.707107 −0.5 0
2 1.414214 0.5 0
4 2.828427 1.5 0
8 5.656854 2.5 0
Abbreviations: MIC, minimal inhibitory concentration; QC, quality control.

Calculations:

 There are no obvious outlier laboratories.

 The calculated mean of midpoint log2 converted observations is −4.833; the (geometric) mean is therefore 2−4.833 or 0.035 µg/mL.

 The calculated SD of midpoint log2 converted observations is 0.554.

 The 95% confidence interval of midpoint log2 converted observations around the geometric mean is −4.833 ± 1.96 • 0.554 = −4.833 ± 1.086 or
−5.913 to −3.747. Converting these values back to MIC values, they become 2−5.913 to 2−3.747 or 0.017 to 0.074, which when rounded up to the
standard twofold dilution series becomes the range 0.03 to 0.125 µg/mL.

M23, 5th ed.

59
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Appendix B. (Continued)

M23, 5th ed.

60

Table B3. Example of Traditional Analysis of QC MIC Range From an Eight-Laboratory Study

MIC, Lot Lot Lot Lab Lab Lab Lab Lab Lab Lab Lab All
g/mL 1 2 3 1 2 3 4 5 6 7 8 Labs
0.015
0.03
0.06
0.12
0.25

0.5
1 31 96 71 16 32 24 32 46 16 32 198
2 97 32 57 32 16 48 24 16 2 32 16 186
4

8
©Clinical

16
32
64
128
and Laboratory Standards Institute. All rights reserved.

256
512

N 128 128 128 48 48 48 48 48 48 48 48 384

GEOMEAN 1.691 1.189 1.362 1.587 1.260 2.000 1.414 1.260 1.029 1.587 1.260 1.399
MODE 2 1 1 2 1 2 1 1 1 2 1 1
MIN 1 1 1 1 1 2 1 1 1 1 1 1
MAX 2 2 2 2 2 2 2 2 2 2 2 2
RANGE 2 2 2 2 2 1 2 2 2 2 2 2

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

Abbreviations: ATCC®, American Type Culture Collection; Lab, laboratory; MAX, maximum; MIC, minimal inhibitory concentration; MIN, minimum; QC, quality control.
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Appendix B. (Continued)
©Clinical

Table B4. Example of Traditional Analysis of QC Disk Diffusion Range From an Eight-Laboratory Study
and Laboratory Standards Institute. All rights reserved.

Escherichia coli ATCC® 35218 on Mueller-Hinton Agar

Disk Manufacturer: “A” and “B” Data Combined

Zone, Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory Laboratory All
mm 1 2 3 4 5 6 7 8 Laboratories
Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot Lot
A B C A B C A B C A B C A B C A B C A B C A B C A B C
20
21
22
23
24
25 1 2 1 1 1 1 3
26 8 2 2 4 1 3 1 3 13 7 4
27 6 15 8 5 5 8 4 6 4 1 5 3 5 3 1 5 4 1 25 40 24
28 4 3 7 8 7 8 10 7 8 3 1 4 4 2 11 6 9 4 12 15 2 1 6 46 40 56
29 1 3 5 2 3 6 6 7 7 9 10 8 9 7 6 9 8 13 6 5 5 7 7 51 48 50
30 1 1 1 7 7 6 6 1 7 2 2 2 6 2 17 18 16
31 2 4 3 2 1 2 1 4 6 5
32 2 1 1 2
33 1 2 2 1
34 1 1
35
36
37
38
20 20 20 20 20 20 20 20 20 20 20 20 20 20 22 20 20 20 20 20 20 20 20 20 160 160 162
60 60 60 60 62 60 60 60 482

Mean 27.1 27.6 28.2 29.5 29.3 28.2 28.5 28.5 28.4
SD 0.933 1.010 0.820 0.892 1.386 0.732 0.651 1.682 1.299
Median 27 28 28 29 29 28 28 29 28
Range 5 6 4 5 8 3 4 9 10

M23, 5th ed.

All Laboratory Mean 28.4 Rounded out All Laboratory Median 28 Median + 1/2 R
Average SD 1.05 Median of Ranges (MR) 5
± 2 SDs 26.27 30.45 26 31 1/2 MR Rounded up (R) 3.00 25 31
61

Abbreviations: ATCC®, American Type Culture Collection; QC, quality control; SD, standard deviation.
 M23, 5th ed.

Appendix C. Statement of Policy of the Antimicrobial Susceptibility Testing

Subcommittees of the Clinical and Laboratory Standards Institute

Abbreviations for Appendix C

FDA US Food and Drug Administration

SC subcommittee

1. Initial breakpoints for drugs approved or under review by the US Food and Drug Administration
(FDA): For antimicrobial agents with newly FDA-approved breakpoints, the relevant CLSI
susceptibility testing subcommittees (SCs)—Subcommittee on Antimicrobial Susceptibility Testing
and Subcommittee on Antifungal Susceptibility Tests—will use the principles outlined in CLSI
guideline M23, Development of In Vitro Susceptibility Testing Criteria and Quality Control
Parameters, as listed below.

 Susceptibility testing criteria and quality control parameters to review

 The FDA-specified breakpoints for the organisms listed in the clinical indications and in vitro
microbiology sections of the product label

 The publicly available portions of the registration package submitted to the FDA and publicly
available materials that discuss FDA review of the materials

 Any supplemental information the manufacturer wishes to provide to CLSI through an

informational presentation based on M23

After completing this review, as described in M23, the relevant SC will publish breakpoints based on
its overall analysis of the information. If the data are believed to be supportive, the SC can set
breakpoints for organisms that are not covered by the FDA-specified breakpoints.

2. Initial breakpoints for drugs that lack FDA-approved breakpoints: For antimicrobial agents that
lack FDA-approved breakpoints, manufacturers or other interested parties may submit information on
breakpoints approved by other regulatory agencies, along with any other information of the types
enumerated in Section 1. Upon receipt and review of such information, the relevant SC will publish
breakpoints based on its overall analysis of the information. CLSI guideline M23 provides guidance on
the principles used by CLSI subcommittees to determine suitable breakpoints.

3. Revision of breakpoints: The relevant SC considers proposals to update its published breakpoints for
existing drugs when issues of emerging resistance or other compelling evidence arise. Data
demonstrating a need for a change should be presented as described in CLSI guideline M23 and are
analyzed following the principles outlined in that guideline.

4. Periodic breakpoint review: The review and potential modification of breakpoints for each
antimicrobial class will occur periodically. The purpose of these periodic reviews is to ensure that
breakpoints remain current and relevant over time. These periodic reviews are not meant to supplant
the breakpoint review that may be needed as noted in Section 3.

5. Publication of breakpoints: Because breakpoints can directly affect patient care, CLSI understands
the importance of communicating any new or modified breakpoints in a timely manner. To accomplish
this goal, CLSI will publish any new or modified breakpoints on the CLSI website (www.clsi.org)
within 90 days of their approval by the relevant subcommittee. Breakpoints for antimicrobial agents
that are under review and awaiting regulatory approval will be included.

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 M23, 5th ed.

Appendix D. Suggested Information to Be Covered on the Submission Cover Pagea

Abbreviation for Appendix D

FDA US Food and Drug Administration

Suggested information to be covered on the submission cover page includes:

 Executive summary describing the specific request being made by the sponsor (eg, inclusion in Table 1
of CLSI document M100,1 breakpoints, quality control data)

 US Food and Drug Administration (FDA) status (package insert attached if approved) and status in
other countries

 FDA-approved and/or -proposed indications for use (include organisms)

 Clinical conditions for which the drug is targeted

 Dosage of drug to be used for indications/data being evaluated

 If a request is being made for the addition of the drug to CLSI document M07,2 solvents and diluents
needed for stock solution preparation (for Table 6 of CLSI document M1001)

 Antimicrobial agent abbreviation to be used by diagnostic manufacturers

References for Appendix D

1
CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 28th ed. CLSI supplement
M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.
2
CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically.
11th ed. CLSI standard M07. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.

a Only information relevant for the request being made needs to be included.

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Appendix E. Drug “X” Minimal Inhibitory Concentration vs Zone Diameter

64

M23, 5th ed.

Examples of scattergram presentation and tabular presentation of error rates, based on the error-rate bounded method (see Subchapter 6.3 of this
guideline) are presented in Figure E1 and Table E1.

I+3  16 13 1 2 1 3 2 1

I+2 8.0 1 1 3 1 2 1 1 2 1

I+1 4.0 1 4 2 3 1 1

I 2.0 1 2 1 5 2 1 4 1 1 1

I− 1 1.0 3 3 5 7 3 3 3 1 2 1 3 1
I− 2 0.5 1 2 3 1 2 3 5 8 13 4 4 1 2 1 1 1

I− 3 0.25 1 1 2 3 5 6 11 13 9 6 5 3 1 4 1

I− 4 0.12 1 1 6 13 9 6 7 6 1 3 1

I− 5 0.06 1 1 4 5 6 5 4 3 8 10 5 7 3 1

I− 6  0.03 3 7 17 20 20 9 15 12 10 10 1 1 6
©Clinical

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 > 35
Abbreviations: I, intermediate; MIC, minimal inhibitory concentration.
Figure E1. Example of a Scattergram of MICs vs Corresponding Zone Diameters (495 Challenge Organisms)
and Laboratory Standards Institute. All rights reserved.
 M23, 5th ed.

Appendix E. (Continued)
Table E1. Discrepancy Rates for Drug “X”
MIC Range Number Very Major (%) Major (%) Minor (%)
≥I + 2 36 1 (2.8)* N/A 2 (5.6)
I + 1 to I − 1 66 1 (1.5) 0 18 (27.3)
≤I−2 371 N/A 1 (0.3) 2 (0.5)
Total 473 2 (0.4) 1 (0.2) 22 (4.7)
* This figure exceeds the proposed limit. However, because only 36 MICs fell in the  I + 2 range (and at least 50 would be needed
for one discrepancy to equal 2%), this should be considered acceptable, particularly because the overall discrepancy rates are so
low. Furthermore, it appears this discrepant result was not repeated and that it very likely would not be discrepant on repeat testing.
Abbreviations: I, intermediate; MIC, minimal inhibitory concentration; N/A, not applicable.

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 M23, 5th 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

M23 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

Continual
Personnel

Inventory

Records
Process
Safety

Event
X
M02
M07
M11
M24
M26
M27
M38
M44
M45
M51
M57
M59
M60
M61
M100

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 M23, 5th ed.

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.

M23 does not cover any of the medical laboratory path of workflow processes. For a description of the 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
M02 M02 M02 M02
M07 M07 M07 M07
M11 M11 M11 M11
M24 M24 M24 M24
M26 M26 M26
M27 M27 M27 M27 M27
M38 M38 M38 M38 M38
M44 M44 M44 M44
M45 M45 M45
M51 M51 M51 M51 M51
M59 M59 M59
M60 M60 M60
M100 M100 M100

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 M23, 5th ed.

Related CLSI Reference Materials

M02 Performance Standards for Antimicrobial Disk Susceptibility Tests. 13th ed., 2018. This standard covers
the current recommended methods for disk susceptibility testing and criteria for quality control testing.

M07 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 11th ed.,
2018. This standard covers reference methods for determining minimal inhibitory concentrations of aerobic
bacteria by broth macrodilution, broth microdilution, and agar dilution.

M11 Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria. 8th ed., 2012. This standard
provides reference methods for the determination of minimal inhibitory concentrations of anaerobic bacteria by
agar dilution and broth microdilution.

M24 Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes. 2nd ed., 2011. This
standard provides protocols and related quality control parameters and interpretive criteria for the susceptibility
testing of mycobacteria, Nocardia spp., and other aerobic actinomycetes.

M26 Methods for Determining Bactericidal Activity of Antimicrobial Agents. 1st ed., 1999. This document
provides procedures for determining the lethal activity of antimicrobial agents.

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

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.

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

M45 Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or
Fastidious Bacteria. 3rd ed., 2016. This guideline informs clinical, public health, and research laboratories on
susceptibility testing of infrequently isolated or fastidious bacteria that are not included in CLSI documents
M02, M07, or M100. Antimicrobial agent selection, test interpretation, and quality control are addressed.

M51 Method for Antifungal Disk Diffusion Susceptibility Testing of Nondermatophyte Filamentous Fungi. 1st
ed., 2010. This document describes the guidelines for antifungal susceptibility testing by the disk diffusion
method of nondermatophyte filamentous fungi (moulds) that cause invasive disease.

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. 2nd ed., 2018. 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.

M61 Performance Standards for Antifungal Susceptibility Testing of Filamentous Fungi. 1st ed., 2017. This
document provides updated quality control tables for the Clinical and Laboratory Standards Institute antifungal
susceptibility testing documents M38 and M51.

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

68 ©Clinical and Laboratory Standards Institute. All rights reserved.

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 M23, 5th ed.

Related CLSI Reference Materials (Continued)

M100 Performance Standards for Antimicrobial Susceptibility Testing. 28th ed., 2018. This document includes
updated tables for the Clinical and Laboratory Standards Institute antimicrobial susceptibility testing standards
M02, M07, and M11.

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 M23, 5th ed.

NOTES

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