M24A2E

March 2011
M24-A2
Susceptibility Testing of Mycobacteria,
Nocardiae, and Other Aerobic Actinomycetes;
Approved Standard—Second Edition
This standard provides protocols and related quality control

parameters and interpretive criteria for the susceptibility testing
of mycobacteria, Nocardia spp., and other aerobic actinomycetes.
A standard for global application developed through the Clinical and Laboratory Standards Institute consensus process.
Licensed to: Clinical Diagnostics Clinical Diagnostics

This document is protected by copyright. CLSI order #Ord-61971, Downloaded on 1/14/2017.
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M24-A2
Vol. 31 No. 5
ISBN 1-56238-746-4 Replaces M24-A
ISSN 0273-3099 Vol. 23 No. 18
Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic
Actinomycetes; Approved Standard—Second Edition
Volume 31 Number 5
Gail L. Woods, MD Gaby E. Pfyffer, FAMH, PhD
Barbara A. Brown-Elliott, MS, MT(ASCP)SM John C. Ridderhof, DrPH
Patricia S. Conville, MS, MT(ASCP) Salman H. Siddiqi, PhD
Edward P. Desmond, PhD Richard J. Wallace, Jr, MD
Geraldine S. Hall, PhD Nancy G. Warren, PhD
Grace Lin, MS Frank G. Witebsky, MD
Abstract
This document addresses the susceptibility testing of Mycobacterium tuberculosis complex (MTBC), clinically significant slowly
and rapidly growing mycobacterial species, Nocardia spp., and other aerobic actinomycetes. Included in this standard are
recommendations for the selection of agents for primary and secondary testing, organism group–specific methodologies,
reporting recommendations, and quality control criteria for the above-listed organisms. Recommendations regarding the selection
of agents for testing mycobacteria are based primarily on guidelines from US agencies. For testing MTBC, M24 recognizes agar
proportion as the primary methodology on which all other methodologies are essentially based. This document also includes
recommendations for use of commercial broth susceptibility methods with shorter incubation times, which are now in widespread
use in the susceptibility testing of MTBC.
Clinical and Laboratory Standards Institute (CLSI). Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic
Actinomycetes; Approved Standard—Second Edition. CLSI document M24-A2 (ISBN 1-56238-746-4). Clinical and Laboratory
Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2011.
The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through
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guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in
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customerservice@clsi.org; Website: www.clsi.org.

Number 5 M24-A2
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Suggested Citation
CLSI. Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes; Approved
Standard—Second Edition. CLSI document M24-A2. Wayne, PA: Clinical and Laboratory Standards
Institute; 2011.
Proposed Standard
July 1990
Tentative Standard
December 1995
Tentative Standard—Second Edition

December 2000
Approved Standard
April 2003
Approved Standard—Second Edition

March 2011
ISBN 1-56238-746-4
ISSN 0273-3099
ii
Volume 31 M24-A2
Committee Membership
Area Committee on Microbiology

John H. Rex, MD, FACP John D. Turnidge, MD Jeffrey L. Watts, PhD,
Chairholder SA Pathology at Women’s and RM(AAM)
AstraZeneca Children’s Hospital Pfizer Animal Health
Cheshire, United Kingdom North Adelaide, Australia Kalamazoo, Michigan, USA
Mary Jane Ferraro, PhD, MPH Advisors Melvin P. Weinstein, MD

Vice-Chairholder Robert Wood Johnson
Massachusetts General Hospital Donald R. Callihan, PhD Medical School
Boston, Massachusetts, USA BD Diagnostic Systems New Brunswick, New Jersey,
Sparks, Maryland, USA USA
Nancy L. Anderson, MMSc,
MT(ASCP) James H. Jorgensen, PhD Nancy Wengenack, PhD
Centers for Disease Control and University of Texas Health Science Mayo Clinic
Prevention Center Rochester, Minnesota, USA
Atlanta, Georgia, USA San Antonio, Texas, USA
Matthew A. Wikler, MD,
Barbara Ann Body, PhD, Jean B. Patel, PhD, D(ABMM) MBA, FIDSA
D(ABMM) Centers for Disease Control and IASO Pharma, Inc.
Laboratory Corporation of America Prevention San Diego, California, USA
Burlington, North Carolina, USA Atlanta, Georgia, USA
Michael L. Wilson, MD
Betty A. Forbes, PhD, D(ABMM) Michael A. Pfaller, MD Denver Health Medical
Medical College of Virginia University of Iowa College of Center
Campus Medicine Denver, Colorado, USA
Richmond, Virginia, USA Iowa City, Iowa, USA
Gail L. Woods, MD
Thomas R. Fritsche, MD, PhD Thomas R. Shryock, PhD VA (Central Arkansas
Marshfield Clinic Elanco Animal Health Healthcare System)
Marshfield, Wisconsin, USA Greenfield, Indiana, USA Little Rock, Arkansas, USA
Freddie Mae Poole, MS, MT Jana M. Swenson, MMSc Barbara L. Zimmer, PhD
FDA Center for Devices and Centers for Disease Control and Siemens Healthcare
Radiological Health Prevention Diagnostics
Silver Spring, Maryland, USA Atlanta, Georgia, USA West Sacramento, California,
USA
Fred C. Tenover, PhD, D(ABMM)
Cepheid
Sunnyvale, California, USA
Subcommittee on Antimycobacterial Susceptibility Testing

Gail L. Woods, MD Edward P. Desmond, PhD John C. Ridderhof, DrPH
Chairholder California Department of Public Centers for Disease Control and
VA (Central Arkansas Veterans Health Prevention
Healthcare System) Richmond, California, USA Atlanta, Georgia, USA
Little Rock, Arkansas, USA
Geraldine S. Hall, PhD Frank G. Witebsky, MD
Marieann R. Brill, MBA Cleveland Clinic National Institutes of Health,
FDA Center for Devices and Cleveland, Ohio, USA Clinical Center
Radiological Health (retired) Bethesda, Maryland, USA
Rockville, Maryland, USA Scott B. Killian
Trek Diagnostic Systems Advisors
Barbara A. Brown-Elliott, MS, Cleveland, Ohio, USA
MT(ASCP)SM Patricia S. Conville, MS,
University of Texas Health Science Gaby E. Pfyffer, FAMH, PhD MT(ASCP)
Center at Tyler Kantonsspital Luzern National Institutes of Health
Tyler, Texas, USA Luzern, Switzerland Bethesda, Maryland, USA
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Number 5 M24-A2
Advisors (Continued) Beverly Metchock, DrPH, Staff

D(ABMM)
Leonid B. Heifets, MD, PhD Centers for Disease Control and Clinical and Laboratory Standards
National Jewish Medical and Prevention Institute
Research Center Atlanta, Georgia, USA Wayne, Pennsylvania, USA
Denver, Colorado, USA
Richard J. Wallace, Jr., MD Lois M. Schmidt, DA
Clark B. Inderlied, PhD University of Texas Health Science Vice President, Standards
Childrens Hospital Los Angeles Center at Tyler Development
Los Angeles, California, USA Tyler, Texas, USA
Tracy A. Dooley, BS, MLT(ASCP)
Kenneth C. Jost, Jr., M(ASCP) Audrey Wanger, PhD Staff Liaison
Texas Department of State Health University of Texas Health Science
Services Center Melissa A. Lewis, ELS
Austin, Texas, USA Houston, Texas, USA Editorial Manager
Grace Lin, MS Megan P. Larrisey, MA

California Department of Public Assistant Editor
Health
Richmond, California, USA
Acknowledgment
CLSI and the Area Committee on Microbiology gratefully acknowledge the following individuals for
their help in preparing the approved-level, second edition of this document:
Salman H. Siddiqi, PhD

BD Diagnostic Systems (retired)
Sparks, Maryland, USA
Nancy G. Warren, PhD

Pennsylvania Bureau of Laboratories (retired)
Lionville, Pennsylvania, USA
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Volume 31 M24-A2
Contents
Abstract ....................................................................................................................................................i
Committee Membership........................................................................................................................ iii
Foreword .............................................................................................................................................. vii
1 Scope .......................................................................................................................................... 1
2 Standard Precautions.................................................................................................................. 1
3 Terminology............................................................................................................................... 1
3.1 Definitions .................................................................................................................... 1
3.2 Abbreviations and Acronyms ....................................................................................... 3
4 Antimycobacterial Susceptibility Testing for Mycobacterium tuberculosis Complex .............. 3
4.1 Introduction ................................................................................................................... 3
4.2 Agar Proportion Method ............................................................................................... 6
4.3 Commercial Broth Systems With Shorter Incubation Time for Susceptibility
Testing of Mycobacterium tuberculosis Complex ...................................................... 13
4.4 Pyrazinamide Susceptibility Testing........................................................................... 14
4.5 Quality Control ........................................................................................................... 15
4.6 Implementing New Methods....................................................................................... 17
4.7 Molecular Detection of Drug Resistance .................................................................... 18
5 Susceptibility Testing of Nontuberculous Mycobacteria ......................................................... 19
5.1 Introduction ................................................................................................................. 19
5.2 Preparation of the Inoculum for the Microdilution Method ....................................... 20
5.3 Quality Control Procedures......................................................................................... 21
5.4 Susceptibility Testing of Mycobacterium avium Complex ......................................... 22
5.5 Susceptibility Testing of Mycobacterium kansasii ..................................................... 24
5.6 Susceptibility Testing of Mycobacterium marinum .................................................... 25
5.7 Susceptibility Testing of Miscellaneous Slowly Growing Nontuberculous
Mycobacteria .............................................................................................................. 25
5.8 Antimycobacterial Susceptibility Testing of Rapidly Growing Mycobacteria ........... 26
6 Susceptibility Testing of Aerobic Actinomycetes ................................................................... 29
6.1 Method ........................................................................................................................ 29
6.2 Antimicrobial Agents .................................................................................................. 29
6.3 Preparation of the Inoculum ....................................................................................... 30
6.4 Inoculum Suspensions ................................................................................................ 30
6.5 Susceptibility Test Method ......................................................................................... 30
6.6 Reporting of Results ................................................................................................... 33
6.7 Quality Control ........................................................................................................... 33
Table 1. Antituberculous Drugs and Their Recommended Concentrations in Middlebrook 7H10 and
7H11 Agar Medium .............................................................................................................................. 34
Table 2. Stock, Working, and Final Concentrations of Antituberculous Drug Solutions for Agar
Proportion ............................................................................................................................................. 35
Table 3. Dilution of Sputum Concentrate for Inoculation of the Susceptibility Test Medium for the
Direct Susceptibility Test ...................................................................................................................... 37
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Contents (Continued)
Table 4. Antimycobacterial Agents and Interpretive Criteria for Mycobacterium avium Complex ..... 38
Table 5. Antimycobacterial Agents and Minimal Inhibitory Concentration (MIC) Values Indicating
Resistance for Testing M. kansasii ....................................................................................................... 39
Table 6. Antimycobacterial Agents and Minimal Inhibitory Concentration (MIC) Values Indicating
Resistance for Susceptibility Testing of Mycobacterium marinum (Routine Testing Is Not
Recommended) ..................................................................................................................................... 40
Table 7. Broth Microdilution Interpretive Criteria for Rapidly Growing Mycobacteria ...................... 41
Table 8. Quality Control Ranges of Minimal Inhibitory Concentrations (MICs) (μg/mL) for
Mycobacterium peregrinum ATCC® 700686, Staphylococcus aureus ATCC® 29213, Pseudomonas
aeruginosa ATCC® 27853, and Enterococcus faecalis ATCC® 29212 When Testing Rapidly
Growing Mycobacteria ......................................................................................................................... 42
Table 9. Broth Microdilution Interpretive Criteria for Nocardia and Other Aerobic Actinomycetes .. 43
References ............................................................................................................................................. 44
Appendix A. Susceptibility Testing of Mycobacterium tuberculosis Complex to Second-line Drugs

Using Commercial Shorter Incubation Liquid Media Systems ............................................................ 48
Appendix B. Suggested Approach to Mycobacterium tuberculosis Complex Susceptibility Testing in

Resource-Limited Countries ................................................................................................................. 49
Appendix C. Example Illustrating Drug Calculation for Meropenem Trihydrate ................................ 50
Appendix D. Preparation and Plating of 7H10 and 7H11 Agar Medium (See Appendixes E and F) .. 51
Appendix E. Preparation of Media With Drug-Containing Disks ........................................................ 52
Appendix F. Preparing 7H10/H11 Agar With Liquid Drug ................................................................. 53
Appendix G. McFarland 0.5 Barium Sulfate Turbidity Standard ......................................................... 54
Appendix H. Determining Percentage of Resistance ............................................................................ 55
Appendix I. Drugs Available for Susceptibility Testing of Mycobacterium tuberculosis Complex

Using Commercial Shorter Incubation Liquid Media Systems Cleared for Use by the US Food and
Drug Administration and Their Equivalence in the Agar Proportion Method ...................................... 56
Appendix J. Agar Disk Elution Method for Mycobacterium haemophilum ......................................... 57
Appendix K. Expected Antimicrobial Susceptibility Patterns of the Most Commonly Isolated

Nocardia ............................................................................................................................................... 58
The Quality Management System Approach ........................................................................................ 60
Related CLSI Reference Materials ....................................................................................................... 61
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Volume 31 M24-A2
Foreword
CLSI document M24 was revised based on thoughtful comments from laboratorians involved with regular
testing of mycobacteria and/or aerobic actinomycetes, but further refinements are anticipated as more
relevant data become available. The document addresses Mycobacterium tuberculosis complex (MTBC),
certain nontuberculous mycobacteria (NTM), and Nocardia and other aerobic actinomycetes. Currently,
sufficient data exist to support recommendations for susceptibility testing of MTBC, Mycobacterium
avium complex (MAC), Mycobacterium kansasii, the rapidly growing mycobacteria, Nocardia spp., and
certain other aerobic actinomycetes. The breakpoints for Nocardia and other aerobic actinomycetes are
based on organism population distributions, clinical data, breakpoints used for other organisms, and the
experience of experts in the field.
In tuberculosis (TB), there is generally a mixture of intracellular and extracellular bacilli, as well as
actively growing and dormant, metabolically inactive ones. For this reason, pharmacokinetic
(PK)/pharmacodynamic (PD) studies have limited ability to predict which drugs will be effective in the
treatment of tuberculosis (TB). In fact, it is believed that PK studies would suggest that pyrazinamide
should be ineffective against MTBC, when in practice it is a key component of TB treatment.1 Due to
these limitations, experts developed a creative approach to MTBC susceptibility testing, ie, comparing
minimal inhibitory concentration (MIC) values of isolates from patients who failed treatment with those
of strains never exposed to the anti-TB drug. Clinically resistant strains have higher MIC values, and the
wild-type strains, for effective anti-TB drugs, have lower MIC values. The critical concentration is the
test concentration that best differentiates these two populations. Follow-up studies showed that testing
based on this concept predicted treatment success vs failure for a drug.1 If, in the future, clinically relevant
PK/PD data become available, the information will be included in the document, as appropriate.
Laboratory tests for evaluating the susceptibility of mycobacteria and aerobic actinomycetes can confirm
the choice of the initial course of chemotherapy and the emergence of drug resistance when a patient fails
to show a satisfactory bacteriological response to treatment. Additionally, they can guide the choice of
further treatment with different drugs. Susceptibility testing of MTBC can also be used to estimate the
prevalence of primary and acquired drug resistance (defined by the World Health Organization2 as “drug
resistance among new cases” and “drug resistance among previously treated patients,” respectively) in a
community. For each of these purposes, use of a reliable technique to perform the test is essential.
Currently, first-line therapy for TB includes isoniazid (INH), rifampin (RMP), ethambutol (EMB), and
pyrazinamide (PZA). To ensure that clinicians are provided with comprehensive information regarding
this multidrug regimen, initial isolates from all patients should be tested for susceptibility to all four
agents. This is a change from the previous edition, which stated that laboratories might consider testing
INH, RMP, and EMB only, if their pulmonary and/or infectious disease specialists and TB control officer
agree with the reduced panel. Susceptibility testing should be repeated if the patient is culture positive
after three months of appropriate therapy or earlier if the patient shows clinical evidence of failure to
respond to therapy or is unable to tolerate the initial drug regimen. To ensure the earliest possible
detection of resistance, a commercial, shorter incubation system should be used in conjunction with rapid
methods for primary culture and identification. In this way, first-line susceptibility test results for most
MTBC isolates should be reported within 15 to 30 days of receipt of the specimen in the laboratory.
Multidrug-resistant (MDR) TB, although not a major problem in most developed countries, is a serious
threat to TB control globally. Recently, the emergence of extensively drug-resistant TB (isolates resistant
to the two best first-line agents—INH and RMP—and the best second-line drugs—the fluoroquinolones
and either amikacin, kanamycin, or capreomycin), which is associated with extremely poor outcomes and
a high mortality rate in patients with concomitant human immunodeficiency virus (HIV) infection, has
heightened the concern of global TB control. Consequently, the importance of susceptibility testing to
secondary anti-TB drugs has grown. Moreover, drugs other than the traditional secondary agents—most
importantly the newer fluoroquinolones—have been evaluated and shown to be effective additions to the
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anti-TB regimen. Given the efficacy and ease of administration, it is anticipated that the use of the newer
fluoroquinolones to treat TB will increase in the next several years and, as a result, susceptibility testing
of these agents will become more important.
Susceptibility testing of NTM and aerobic actinomycetes should be performed on clinically significant
isolates3 (see the paragraph below) that exhibit variability in susceptibility to clinically useful
antimicrobial agents and/or significant risk of acquired mutational resistance to one or more of these
agents. Because the latter two criteria are not true for Mycobacterium marinum, routine susceptibility
testing of this species is not indicated.
To determine clinical significance of NTM recovered from respiratory cultures, the American Thoracic
Society (ATS) recommends the following criteria: cultures of at least two positive sputum specimens or
one bronchial wash or lavage sample are usually sufficient to confirm clinical significance. Alternatively,
a transbronchial or lung biopsy with mycobacterial histopathological features and positive culture for
NTM is sufficient to establish clinical significance. In addition, isolates from normally sterile sites (such
as blood, cerebrospinal fluid, or tissues) typically are considered clinically significant.
To facilitate further development of CLSI document M24, the subcommittee requests comments and
suggestions for improvement with regard to the methods included herein.
All authors of this document donated considerable time to its development; I would like to personally
thank all of them for their valuable contributions.
Gail L. Woods, MD
Chairholder, Subcommittee on Antimycobacterial Susceptibility Testing
Note that the trade names BACTEC™ 460 TB, BACTEC™ MGIT™ 960, and VersaTREK®
are included in either Appendix A and/or Appendix I. It is the Clinical and Laboratory
Standards Institute’s policy to avoid using a trade name unless the product identified is
the only one available, or it serves solely as an illustrative example of the procedure,
practice, or material described. In this case, the subcommittee and area committee believe
the trade names are used to provide interpretive criteria that are specific to the listed
system. These three systems were the only ones cleared by the US Food and Drug
Administration at the time this document was completed.
Key Words
Antimycobacterial drugs, antituberculous drugs, drug susceptibility
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Volume 31 M24-A2
Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic

Actinomycetes; Approved Standard—Second Edition
1 Scope
CLSI document M24-A2 contains protocols for the susceptibility testing of three major categories of
mycobacterial species (Mycobacterium tuberculosis complex [MTBC]; the slowly growing
nontuberculous mycobacteria [NTM]; and the rapidly growing mycobacteria) and recommendations for
susceptibility testing of Nocardia spp. and miscellaneous aerobic actinomycetes. This document also
provides guidance on the selection of primary and, for some organisms, secondary agents for testing and
reporting; instructions for performing the standard agar proportion method for MTBC and broth
microdilution for NTM; and quality control (QC) protocols for each organism category. Testing and
reporting recommendations and principles of QC procedures apply to use of commercial shorter-
incubation broth systems that have been cleared by the US Food and Drug Administration (FDA) for
testing MTBC, as well as the reference methods.
2 Standard Precautions
Because it is often impossible to know what isolates or specimens might be infectious, all patient and
laboratory specimens are treated as infectious and handled according to “standard precautions.” Standard
precautions are guidelines that combine the major features of “universal precautions and body substance
isolation” practices. Standard precautions cover the transmission of all known infectious agents and thus
are more comprehensive than universal precautions, which are intended to apply only to transmission of
blood-borne pathogens. Standard and universal precaution guidelines are available from the US Centers
for Disease Control and Prevention (CDC).4 For specific precautions for preventing the laboratory
transmission of all known infectious agents from laboratory instruments and materials and for
recommendations for the management of exposure to all known infectious diseases, refer to CLSI
document M29.5
The mycobacteriology laboratory presents a unique set of circumstances in terms of observance of

biosafety precautions. For more information, it is suggested that the reader refer to Biological Safety:
Principles and Practices by Fleming and Hunt.6 Also, the publication Biosafety in Microbiological and
Biomedical Laboratories is now in its fifth edition and is available online at
http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.7
3 Terminology
3.1 Definitions
accuracy (measurement) – closeness of agreement between a measured quantity value and a true
quantity value of a measurand (ISO/IEC Guide 99).8
antimicrobial susceptibility test interpretive category – a classification based on an in vitro response of

an organism to an antimicrobial agent; NOTE 1: For mycobacteria, two different categories, “critical
concentration” and “minimum inhibitory concentration,” have been used to categorize the in vitro results;
NOTE 2: For members of the MTBC, when tested against the lower concentration of some agents, the
“critical concentration” category is applied. Testing of an additional higher concentration may also be
recommended for some agents. However, there is no “intermediate” interpretive category when the
“critical concentration” category is applied, even when testing is performed both at the critical
concentration and the additional higher concentration; NOTE 3: For NTM and for the aerobic
actinomycetes, only the “minimum inhibitory concentration” category is applied.
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Clinical and Laboratory Standards Institute. All rights reserved. 1
Number 5 M24-A2
borderline antimicrobial susceptibility test interpretive category – an interpretive category applicable

only to certain results obtained with MTBC isolates tested against pyrazinamide by the radiometric
instrument method (refer to the manufacturer’s package insert); NOTE: Repeat testing may determine
whether the isolate in question is susceptible or resistant.
critical concentration – the “critical concentrations” of antituberculous drugs were adopted by

international convention9; NOTE: For each drug, the critical concentration is the lowest concentration
that inhibits 95% of “wild-type” strains of M. tuberculosis that have not been exposed to the drug, but that
simultaneously does not inhibit strains of M. tuberculosis considered resistant that are isolated from
patients who are not responding to therapy.
culture – 1) the intentional growing of microorganisms (such as bacteria or viruses) or tissues, in a

controlled environment, for purposes of identification or other scientific study, or for commercial and/or
medicinal use; 2) the product resulting from the intentional growth of microorganisms or tissue.
culture medium – a substance or preparation used for the cultivation and growth of microorganisms or
tissue.
direct susceptibility test – a procedure based on inoculation of drug-containing media directly with a
processed (concentrated after digestion and decontamination) specimen that is smear-positive for acid-fast
bacilli (AFB) to determine the proportion or percentage of resistant MTBC in the patient’s bacterial
population (see Section 4.2.4).
indirect susceptibility test – a procedure based on inoculation of drug-containing media using organisms
grown in culture (see Section 4.2.3).
intermediate antimicrobial susceptibility test interpretive category – for minimal inhibitory

concentration, an interpretive category that implies that an infection due to the isolate may be
appropriately treated in body sites where the drugs are physiologically concentrated or when a high
dosage of drug can be used; also indicates a “buffer zone” that should prevent small, uncontrolled,
technical factors from causing major discrepancies in interpretations.
measurand – quantity intended to be measured (ISO/IEC Guide 99).8
minimal inhibitory concentration (MIC) – the lowest concentration of an antimicrobial agent that
prevents visible growth of a microorganism in a broth dilution susceptibility test.
Mycobacterium avium complex (MAC) – includes the species M. avium (subspecies avium,
colombiense, silvaticum, hominissuis, paratuberculosis) and Mycobacterium intracellulare.
Mycobacterium tuberculosis complex (MTBC) – includes the species M. tuberculosis, Mycobacterium

bovis, M. bovis BCG, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium africanum,
Mycobacterium microti, and Mycobacterium canettii.
nontuberculous mycobacteria (NTM) – all species of mycobacteria other than those in the MTBC.
resistant antimicrobial susceptibility test interpretive category – 1) For critical concentration,

resistance is defined as diminished susceptibility of a strain that differs from wild-type strains from
patients who have not been treated with the drug, so that the strain is unlikely to show clinical
responsiveness to the drug; 2) For minimal inhibitory concentration, resistant isolates are not inhibited
by the usually achievable concentrations of the agent at the site of infection with normal dosage
schedules, and/or fall in the range where specific microbial resistance mechanisms are likely (eg, β-
lactamases), and clinical efficacy has not been reliable in treatment studies.
©
2 Clinical and Laboratory Standards Institute. All rights reserved.
Volume 31 M24-A2
susceptible antimicrobial susceptibility test interpretive category – 1) for critical concentration, a

level of susceptibility that is not significantly different from that of wild-type strains from patients who
have not been treated with the drug, so that the strain is likely to show clinical responsiveness to the drug;
2) for minimal inhibitory concentration, an interpretive category that implies that an infection due to the
isolate may be appropriately treated with the dosage of an antimicrobial agent recommended for that type
of infection and infecting species, unless otherwise indicated, given the levels attainable with the agent at
the site of infection.
validation – confirmation, through the provision of objective evidence, that requirements for a specific
intended use or application have been fulfilled (ISO 9000).10
verification – confirmation, through the provision of objective evidence, that specified requirements have
been fulfilled (ISO 9000).10
3.2 Abbreviations and Acronyms

AFB acid-fast bacilli
ATCC American Type Culture Collection
ATS American Thoracic Society
CAMHB cation-adjusted Mueller-Hinton broth
CAP College of American Pathologists
CDC Centers for Disease Control and Prevention
CFU colony-forming units
CLIA Clinical Laboratory Improvement Amendments
DMSO dimethyl sulfoxide
EMB ethambutol
FDA US Food and Drug Administration
HIV human immunodeficiency virus
HPLC high-performance liquid chromatography
INH isoniazid
LJ Lowenstein-Jensen
MAC Mycobacterium avium complex
MDR multidrug resistant
MIC minimal inhibitory concentration
MTBC Mycobacterium tuberculosis complex
NTM nontuberculous mycobacteria
OADC oleic acid–albumin–dextrose–catalase
PAS p-aminosalicylic acid
PZA pyrazinamide
QA quality assurance
QC quality control
qPCR real-time polymerase chain reaction
RMP rifampin
SDW sterile distilled water
TB tuberculosis
4 Antimycobacterial Susceptibility Testing for Mycobacterium tuberculosis

Complex
4.1 Introduction
Current methods for susceptibility testing of MTBC (see the definition for MTBC in Section 3.1 for
species) are based on proportion methods and are considered equivalent to the standard methods
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Number 5 M24-A2
established by Canetti and colleagues.9 The proportion methods used globally rely on a bacteriological
definition of drug resistance that was developed in recognition of the difficulties in defining clinical
resistance for mycobacteria. Resistance is defined as “a decrease in susceptibility of sufficient degree to
be reasonably certain that the strain concerned is different from a sample of wild strains of human type
that have never come into contact with the drug.”11 For several decades, the method of proportion using
Middlebrook 7H10 or 7H11 agar12 has been considered the standard method and is described in this
document.
The agar proportion and radiometric methods define resistance as growth of greater than 1% of an
inoculum of bacterial cells in the presence of a “critical” concentration of antituberculous drug. The
critical concentrations of antituberculous drugs were adopted by international convention and represent
the lowest concentrations of drugs that inhibit 95% of “wild strains” of M. tuberculosis that have never
been exposed to the drugs, while at the same time not inhibiting strains of M. tuberculosis that have been
isolated from patients who are not responding to therapy, and that are considered resistant. The
recommended critical concentrations of drug were originally determined in egg-based Lowenstein-Jensen
medium, which is still used in some countries outside the United States. Equivalent concentrations of
drugs were later established in Middlebrook 7H10 and 7H11 for the agar proportion method13 and in the
media used in commercial susceptibility test systems.14-16 Every laboratory should test the susceptibility
of MTBC to the critical concentration of drug for the test method it is using. The critical concentration is
the standard that allows interpretation of tests by any of the procedures. When greater than 1% of the
tested bacterial population in a clinical isolate becomes resistant to the critical concentration of a drug,
that drug is not (or soon will not be) useful for continued antituberculous chemotherapy.12
Using the critical concentrations of primary antituberculous drugs (ie, isoniazid [INH], rifampin [RMP],
ethambutol [EMB], and pyrazinamide [PZA]), the results of in vitro susceptibility testing of these agents
correlate well with clinical effectiveness in patients with tuberculosis (TB). However, data concerning
testing of secondary antituberculous drugs (see Table 1) are more limited.
Users of this document should be aware that the standardized agar proportion method for susceptibility
testing of MTBC described here is not a rapid method. To ensure the earliest possible detection of
resistant organisms, a broth method with a shorter incubation time is the recommended standard of
practice for drug susceptibility testing of MTBC in many industrialized nations.17 Using a commercial
broth susceptibility testing method with a shorter incubation time, in conjunction with primary culture and
identification approaches that reduce lengthy incubations, enables earlier reporting of results. The CDC
has recommended that mycobacteriology laboratories work toward the goal of reporting first-line
susceptibility results for MTBC within 15 to 30 days of receipt of the initial diagnostic specimen.18
Ideally, susceptibility results should be available within 7 to 14 days after isolation of MTBC. When
reporting susceptibility test results, the laboratory must meet current local, regional, or national
regulations.
Procedural instructions in this document for performing the reference agar proportion method do not
apply to commercial rapid broth systems for testing MTBC. Manufacturers of those systems determine
unique drug concentrations and specific procedural instructions to produce results equivalent to the agar
proportion method. Each laboratory using these systems is responsible for following those specific
procedural instructions. If changes are made to the manufacturer’s procedure, laboratories are required
under the Clinical Laboratory Improvement Amendments (CLIA) regulations to establish performance.
Commercial system manufacturers are obligated to ensure that system performance meets user needs and
performs reliably when testing is done in accordance with the product labeling.
At this time, there are three FDA-cleared broth systems for indirect testing of MTBC. These are
recommended in this document for initial susceptibility testing with primary antituberculous drugs. No
commercially available broth systems have been cleared by FDA for slow-growing NTM, rapidly
growing mycobacteria, Nocardia, or other aerobic actinomycetes. The agar proportion method, on the
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other hand, is used to confirm results from commercial broth systems (if necessary) and to test additional
drugs or concentrations of drugs that are not available in those commercial systems. The agar proportion
method is also used as the standard against which new methods are evaluated and for characterizing in
vitro susceptibility of new antimycobacterial drugs.
Susceptibility testing should be performed on the first isolate of MTBC obtained from every patient.17,19
Testing should be repeated if cultures fail to convert to negative after three months of therapy or earlier if
there is clinical evidence of failure to respond to therapy. Any commercial shorter incubation broth
system used should have been previously demonstrated to produce results that correlate with those
obtained with the standardized agar proportion method. (Refer to Section 4.3 for more recommendations
regarding use of commercial short-incubation broth systems.) If the results obtained for a patient’s isolate
by a commercial shorter incubation system are in any way ambiguous or problematic, testing should be
repeated to resolve the problem. If initial results indicate resistance to any agent and the resistance has
been verified by excluding the presence of contaminants or NTM, each laboratory may determine if
repeat testing of the agent or agents to confirm the resistance from the initial testing is necessary. Refer to
Section 4.3 for more recommendations. If repeat testing is planned, the initial resistance should be
reported without delay. This report should indicate that the drug resistance findings are preliminary and
confirmatory testing has been initiated. Testing secondary agents at the same time as any repeat testing is
strongly recommended, so that several drugs to which the isolate is susceptible can be identified and
results for secondary agents are not delayed.
Initial susceptibility testing of MTBC should include all primary drugs: INH (at the critical
concentration), RMP, EMB, and PZA. This combination of tests provides the clinician with
comprehensive information related to the four-drug therapy currently recommended for treatment of most
patients with TB in the United States.19 This is a change from the previous edition, which recommended
testing two concentrations of INH (the critical concentration and a higher one) and stated that laboratories
might consider testing INH, RMP, and EMB only, if their pulmonary and/or infectious disease specialists
and TB control officer agreed with the reduced panel. The reason for recommending that all first-line
drugs be tested initially is that resistance to these drugs is becoming more common, is not predictable, and
essentially always requires modification of treatment. If there is resistance to one or more primary agents,
the goal is to use as many remaining first-line drugs as possible. Therefore, it is essential to have
susceptibility test results for those remaining primary agents.
Although initial testing typically involves primary drugs only, primary and second-line drugs may be
tested simultaneously if mutations associated with INH and/or RMP resistance have been detected by
molecular assays, or epidemiological situations support the practice and resources are available. Second-
line drugs (Table 1), both traditional (like capreomycin and ethionamide) and newer agents such as
levofloxacin or moxifloxacin, should be tested whenever an isolate of MTBC is resistant to RMP or any
two of the primary drugs. Cycloserine is an option therapeutically, but testing is not recommended due to
technical issues. Isolates with monoresistance to the critical concentration of INH also should be tested
for susceptibility to second-line agents if the clinician is planning to include a fluoroquinolone in the
treatment regimen. Therefore, communication with the clinician caring for the patient is critical.
In resource-rich countries where laboratories have the capacity to perform more tests, when second-line
drug testing is indicated, at least one drug from each class and a higher concentration of both INH and
EMB should be tested (see Table 1 and Appendix A for the list of recommended drugs and
concentrations). Laboratories should test at least one fluoroquinolone drug; it is not necessary to test all
three listed in the table. The fluoroquinolone tested should be selected based on consultation with the
physician(s) managing treatment of most patients with drug-resistant TB. Two aminoglycosides—
amikacin and kanamycin—are listed in Table 1. Because resistance to kanamycin may not indicate
resistance to amikacin, it may be desirable to test both aminoglycosides. In resource-limited countries, on
the other hand, choices of antimicrobials must be prioritized (see Appendix B). In either case, a
“piecemeal” approach should be avoided to prevent delays in reporting susceptibility test results. This is
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of particular concern when second-line drug testing is performed using the slower agar proportion
method. If second-line testing is not done in-house, the isolate should immediately be forwarded to a
laboratory with expertise in second-line drug testing. Isolates should be retained until results are available
to complete reporting requirements. Additionally, for patients with resistant isolates, including resistance
only to the lower, critical concentration of INH, referral to or consultation with a specialist in treatment of
drug-resistant TB should be considered.
4.2 Agar Proportion Method
This procedure is performed by inoculating equal quantities of several dilutions of a standardized

inoculum onto agar-based medium with and without the test drug. Separate, countable colonies should be
observed on a control quadrant without any of the drug. The number of colony-forming units (CFU)
growing on the drug-containing medium compared with those growing on the drug-free medium are then
determined and expressed as a percentage. Strains of tubercle bacilli in which growth on drug-containing
media represents more than 1% of the number of colonies that develop on drug-free media are considered
to be resistant to that agent.12
4.2.1 Antituberculous Agents
4.2.1.1 Source
Antimicrobial standards or reference powders, for use with the agar proportion method, can be obtained
from the United States Pharmacopoeia (www.usp.org) or commercial sources.
Pharmacy stock or other clinical preparations should not be used. Acceptable drug standards bear a label
that states the generic name, its assay potency (usually expressed in micrograms of drug per milligram of
powder), and its expiration date. Store the antimicrobial powders as recommended by the manufacturer or
at –20 °C or below in a desiccator (preferably in a vacuum). When the desiccator is removed from the
freezer, allow it to come to room temperature before opening (to avoid condensation of water).
4.2.1.2 Weighing Antituberculous Drugs
All antimicrobial agents are assayed for standard units of activity. The assay units may differ widely from
the actual weight of the powder and often may differ between drug production lots. Thus, a laboratory
must standardize its antimicrobial solutions based on assays of the lots of antimicrobial powders that are
being used.
The value for potency supplied by the manufacturer should include consideration of measures of purity
(usually by high-performance liquid chromatography [HPLC] assay), water content (eg, by Karl Fischer
analysis or by weight loss on drying), and the salt/counter-ion fraction (if the compound is supplied as a
salt instead of free acid or base). The potency may be expressed as a percentage, or in units of
micrograms per milligrams (w/w).
In some cases, a certificate of analysis with values for each of these components may be provided with
antibiotic powders; in this case, an overall value for potency may not be provided but can be calculated
from HPLC purity, water content, and when applicable, the active fraction for drugs supplied as a salt (eg,
hydrochloride, mesylate). However, if when testing these calculations, any value is unknown or is not
clearly determined from the certificate of analysis, it is advisable that the factors used in this calculation
be confirmed with the supplier or the manufacturer. An example calculation can be found in Appendix C.
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4.2.1.3 Selection and Concentration of Antituberculous Drugs
The primary antituberculous drugs are INH, RMP, EMB, and PZA. Streptomycin is now considered a
second-line drug in the United States and many laboratories no longer test it with INH, RMP, EMB, and
PZA but include it during secondary testing. Table 1 lists the concentrations recommended for the first
three drugs for use with the agar proportion method, using 7H10 or 7H11 agar medium.20 See Section 4.4
of this document for a discussion of susceptibility testing of PZA. Testing of secondary antituberculous
drugs as discussed in Section 4.1 should be performed on all isolates of MTBC that are resistant to RMP
or resistant to any two of the primary drugs.
4.2.1.4 Preparation and Storage of Stock and Working Solutions
Examples of stock solution concentrations of antituberculous drugs made from drug powders or
lyophilized commercial products are noted in Table 2.
4.2.1.4.1 Stock Solutions
Prepare stock solutions of antituberculous agents available as powders at concentrations of at least

1000 μg/mL and preferably 10 000 μg/mL, except as noted in Table 2, footnote b. Approximately 100 mg
of drug (depending on the potency) dissolved in 10 mL of sterile distilled water would yield a stock
solution of 10 000 μg/mL.
Some drugs must be dissolved in solvents other than water. In such cases, it is necessary to use only
solvent sufficient to solubilize the antimicrobial powder, and then dilute to the final stock concentration
with sterile distilled water or appropriate buffer, as suggested in Table 2.
Because microbial contamination is extremely rare, solutions that have been prepared aseptically but not
filter sterilized are generally acceptable. However, if desired, solutions may be sterilized by membrane
filtration. Paper, asbestos, or sintered glass filters, which may adsorb appreciable amounts of certain
antituberculous agents, must not be used. Whenever filtration is used, it is important to document the
absence of adsorption by appropriate assay procedures.
Dispense small volumes of the sterile stock solutions into sterile polypropylene or polyethylene vials
appropriate for low-temperature storage, carefully seal, and store for six to 12 months at –60 °C or below.
Thaw to room temperature and use without delay, discard excess, and never refreeze.
4.2.2 Preparation of Drug Medium
4.2.2.1 Agar Medium
Either 7H10 or 7H11 agar medium is acceptable for susceptibility testing. 7H11 agar medium is
especially useful in the recovery of INH-resistant strains of MTBC. Those who do use 7H11 agar should
be aware that different concentrations of some antimycobacterial agents must be used with this medium
(see Table 1). Directions for preparation of 7H10 or 7H11 medium are found in Appendix D.
These guidelines are restricted to Middlebrook agar media only. Guidelines and drug concentrations for
inspissated egg media for susceptibility testing of MTBC are different and can be found in the literature.21
4.2.2.2 Agar Medium With Drug-Containing Disks
Many laboratories use the method of Wayne and Krasnow22 in which paper disks containing specified
amounts of antituberculous drugs are placed in individual quadrants of 100-mm plastic Petri plates.
Commercially prepared disks are available. A 5-mL amount of drug-free 7H10 or 7H11 agar medium is
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dispensed into each quadrant. This simplified procedure allows each laboratory to make just the number
of plates needed for immediate use and eliminates labeling error, because the disks carry identification
codes and antimicrobial contents. The reliability of this method has been established and results have
been shown to be comparable to those achieved by standard methods.23,24 Directions for preparation of
these media are found in Appendix E.
4.2.2.3 Agar Medium With Liquid Drug
Agar medium may be prepared with liquid drug instead of drug disks. Very close attention to exact
delivery of the drug to the medium is required, and laboratories that prepare their own media usually opt
to use the drug-containing disks. The method for preparing 7H10/7H11 agar with liquid drug is found in
Appendix F.
4.2.3 Indirect Susceptibility Test
An indirect susceptibility test uses organisms grown in culture, either in a liquid medium or on the surface
of a solid medium. The preparation of a standard inoculum is critical, because variations in the number of
bacilli in the inoculum can alter the interpretation of the test.
4.2.3.1 Preparation of the Inoculum
4.2.3.1.1 Inoculum From Solid Media for the Agar Proportion Method
Perform the following steps:
(1) The inoculum may be prepared by scraping freshly grown colonies (as early as adequate growth is
attained but not more than four to five weeks old) from the surface of the agar medium, taking care
to sample all parts of the growth. Care should also be taken not to scrape off any medium. Primary
cultures, rather than subcultures, should be used whenever possible. Subcultures grown in broth
may contain an insufficient number of slowly growing resistant tubercle bacilli in the culture, thus
giving a “false-susceptible” result.
(2) Transfer the selected bacterial mass to a sterile 16- × 125-mm screw-cap tube containing 6 to 10
glass or plastic beads in 3 to 5 mL of sterile water, sterile normal saline, or phosphate-buffered
water (pH 7.4; 0.067 M).
(3) Emulsify the growth along the inside wall of the tube using a spatula or applicator stick. After
tightly closing the cap, homogenize the contents of the tube by vigorous agitation on a vortex mixer
for one to two minutes, using sufficient speed to obtain only swirling, centrifugal mixing rather than
churning, which may result in increased aerosol production.
(4) Allow the tube to stand for 30 minutes or longer in a test tube rack to allow larger clumps of
organism to settle and to decrease the possibility of aerosol dispersion when the tube is opened.
(5) Withdraw the supernatant suspension, and transfer it to another sterile glass tube. Adjust the
absorbance by adding sterile water until the density is equivalent to that of a 0.5 to 1 McFarland
turbidity standard. McFarland turbidity standards may be purchased commercially or prepared in-
house using barium chloride and sulfuric acid (see Appendix G).
NOTE: Freshly grown primary cultures in broth, usually 7H9, may be used to prepare the inoculum.
After mixing well, allow the suspension to settle for 30 minutes to reduce any aerosol at the top of the
tube and allow larger clumps to settle. Then adjust the turbidity of the supernatant to be equivalent to that
of a 0.5 to 1 McFarland standard by adding sterile medium.
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4.2.3.2 Inoculation and Incubation of Media
4.2.3.2.1 7H10/7H11 Agar Plates
To inoculate the plates, perform the following steps:
(1) Prepare separate 10-2 and 10-4 dilutions of the standardized suspension in sterile normal saline,
sterile water, or phosphate-buffered water (pH 7.4; 0.067 M).
(2) Using a sterile safety pipette, inoculate 0.1 mL of the 10-2 dilution onto the control quadrant and
onto each of the drug-containing quadrants (this can be done by inoculating three drops at different
points on each quadrant of the agar plate).
(3) Similarly, inoculate 0.1 mL of the 10-4 dilution onto the control quadrant and onto each of the drug-
containing quadrants in a second series of drug-containing plates.
NOTE: If the culture to be tested is older than four to five weeks or scant growth is present, it may be
necessary to use lower dilutions of the inoculum, such as 1:10 and 1:1000, or to subculture the organism
first in broth. The broth is incubated at 35 to 37 °C, with daily observation and mixing until the turbidity
matches that of a 0.5 to 1 McFarland standard.
(4) Allow the inoculated plates to stand at room temperature (the agar side faces up with lids on) until
the inoculum liquid has absorbed into the agar (ie, until the spots are dry).
(5) Seal each plate in a CO2-permeable polyethylene bag or with a CO2-permeable tape.
(6) Incubate the plates agar side down, at 35 to 37 °C in an atmosphere of 5% to 10% CO2. Incubation
in 5% to 10% CO2 does not have a detrimental effect on the antimycobacterial drugs tested
routinely in 7H10/7H11 agar medium. To prevent production of formaldehyde gas, plates should
not be exposed to sunlight.
(7) To prevent visual interference from condensation, plates removed from the incubator should be
placed on the bench at room temperature for a few hours or overnight in the agar side–up position,
without removing them from the plastic bags. Examine the plates carefully, using a dissecting
microscope, each week for a period of no longer than three weeks. If there are at least 50 colonies
(1+) on the control medium and the colonies are mature, resistance may be reported before three
weeks. However, the interpretation “drug susceptible” should not be made until the third week to
provide adequate time for growth.
Alternately, a modified indirect susceptibility method that requires less medium can be considered.24
Prepare organism dilutions of 10-2 and 10-4 as described in the steps listed above. Inoculate one control
quadrant without drug and all drug-containing quadrants with 0.1 mL of the 10-2 dilution. Inoculate a
second drug-free control with 0.1 mL of the 10-4 dilution, which generally gives countable colonies (see
Appendix H, Example 3).
4.2.3.3 Interpretation and Result Reporting
The pathobiology of MTBC differs from that of many other bacteria. MTBC is an intracellular pathogen,
and in this regard, it is important to be aware that the intracellular drug concentration and activity may
differ considerably from the corresponding values in serum and/or other extracellular fluids. Also, the
infecting bacilli often are composed of differing mixtures of populations of actively growing, slowly
growing, and latent organisms at different sites and inside walled-off tubercles; drug effectiveness may
vary among these different populations. As a consequence of these aspects of MTBC infection, in vitro
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susceptibility testing of MTBC differs from that of aerobic and facultative bacteria in the following ways
that directly or indirectly impact the reporting of results:
• Testing with any antimycobacterial agent is performed at one or, for a few agents, two different
concentrations.
• “Critical” concentrations of certain drugs (the in vitro concentrations thought to be most relevant for
predicting clinical responsiveness) were established many years ago, and for some drugs, the values
for these concentrations differ depending on the testing medium used.
• There is no uniform consensus regarding the clinical relevance of the results of testing concentrations
other than the critical concentration.
• The reference agar proportion method employs a percentage calculation to determine resistance or
susceptibility.
• The reference radiometric method for PZA susceptibility testing employs a calculation procedure
unique to that drug to determine resistance or susceptibility.
Reports should include the name of each drug tested, as well as a clinically helpful interpretive comment,
such as “susceptible,” “resistant,” or “borderline,” the last for PZA only. If a laboratory wishes to report
the concentration at which each drug was tested, it should also specify the testing medium and/or testing
method used, and/or specify the equivalent reference method concentrations. (If the equivalent reference
method concentrations are given, then stating the actual concentrations tested and/or the testing method is
optional.) Laboratories using the reference agar proportion method also have the option of reporting
percent resistance, if they so choose. However, at this time, there is no evidence to suggest that a lower
percent resistance may indicate partial drug efficacy in the clinical management of the patient. To avoid
confusion, whenever testing is performed at concentrations in addition to the “critical” concentrations (or
their equivalents in methods other than the reference agar proportion method), the concentrations and
method, or the equivalent reference method concentrations, should be specified.
If an organism is resistant to the critical concentration of INH, the following comment should be
appended to the results: “This test result indicates the presence of at least low-level resistance to INH. A
specialist in the treatment of drug-resistant TB should be consulted concerning the appropriate therapeutic
regimen and dosages.”
In the case of an organism tested against two concentrations of INH, to the lower concentration of which
the organism is resistant and to the higher of which it is susceptible, the following comment should be
appended to the results: “These test results indicate low-level INH resistance. A specialist in the treatment
of drug-resistant TB should be consulted concerning the appropriate therapeutic regimen and dosages.”
In the case of an organism tested against two concentrations of INH, to both of which it is resistant, the
following comment should be appended to the results: “These test results indicate high-level resistance to
INH. A specialist in the treatment of drug-resistant TB should be consulted concerning the appropriate
therapeutic regimen and dosages.”
4.2.3.3.1 Interpretation of Growth Observed on 7H10/7H11 Agar Plates
The amount of growth in each quadrant is recorded as follows:
>500 colonies (confluent growth) 4+

200–500 colonies (almost confluent growth) 3+
100–200 colonies 2+
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50–100 colonies 1+
<50 colonies Record the actual number of colonies.
At least one of the control quadrants of the set of two dilutions should have a minimum of 50 colonies;
otherwise, the results are not valid, and the test must be repeated. If the plate with ≥50 colonies on the
control is not the same as the dilution with ≥50 colonies for the drug-containing quadrants, one can use
the number of countable colonies from the higher dilution plates, multiply this number by the dilution
difference between the two plates, and use this count as the denominator when calculating the percent
resistance (see Example 2 in Appendix H).
If the control quadrant has at least 1+ growth and there is no growth in the drug-containing quadrant, the
results can be reported as susceptible. In most cases, it will be possible to estimate the proportion of
resistant colonies as greater or less than 1% of the control population. Most of the culture results will be
obviously susceptible or resistant (see examples in Appendix H). When the control quadrant has confluent
growth (>500 colonies) and there is less than confluent growth on the drug-containing quadrant, the
percent resistance cannot be determined accurately and the test must be repeated.
Interpretation of the modified indirect susceptibility method follows guidelines for interpretation of the
standard indirect method. See Example 3 in Appendix H for a sample calculation and interpretation.
The presence of microcolonies may represent true resistance or partial resistance, or it may be a result of
drug degradation, followed by an overgrowth of susceptible organisms. One study reported that most
strains that had microcolonies with EMB in the agar proportion method had EMB-susceptible results with
the radiometric method.25 The significance of microcolonies is unknown. Because the frequency of
microcolonies may vary from one laboratory to another, each laboratory should determine how to best
report results. One approach is always to note the presence of microcolonies with a statement that their
significance is unknown. If a laboratory opts not to report microcolonies associated with a specific drug
(such as EMB), this decision should be based on its own experience with microcolonies (eg,
reproducibility on repeat testing) and consultation with its specialist in the treatment of TB.
The first reading at seven days is for the purpose of detecting the growth of contaminating bacteria or
fungi, and for the detection of any rapidly growing mycobacteria. Susceptibility test results should not be
reported on readings before three weeks of incubation, with the exception of a strain that is obviously
resistant to drugs, because drug-resistant tubercle bacilli can grow more slowly than susceptible strains. If
the culture in the drug-free control has not grown at the three-week reading, the test should be repeated,
preferably with a broth method.
4.2.4 Direct Susceptibility Test
4.2.4.1 Principle
The direct drug susceptibility test is a procedure based on inoculation of drug-containing media directly
with a processed (concentrated after digestion and decontamination) sputum specimen that is smear-
positive for acid-fast bacilli (AFB) to determine the proportion or percentage of resistant MTBC in the
patient’s bacterial population. The test should be performed only on smear-positive specimens, and only
by the agar proportion method or by a commercial broth system that has been cleared by FDA for direct
susceptibility testing. Unless manufacturers obtain FDA clearance for these indications, individual
laboratories testing direct specimens will need to conduct a study to establish, rather than to simply verify,
test performance characteristics to satisfy CLIA requirements.
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The advantages of the direct test are:
• Results can be reported within three weeks (from the time of specimen receipt in the laboratory) for a
majority of smear-positive specimens.
• The proportion of resistant bacteria recovered better represents the patient’s bacterial population.
• It is cost-efficient because the materials are less expensive than those for rapid broth methods.
The disadvantages of the direct test are:
• It may not be possible to calibrate the inoculum accurately, which may result in insufficient or
excessive growth on drug-free control quadrants.
• Possible growth of normal respiratory flora that may survive digestion and decontamination, making
results uninterpretable.
• The results of the test are valid only if the isolate is MTBC or M. kansasii (RMP only). Results for
other species should not be reported.
The total rate of failure for the direct method can reach 10% to 15% or more, which results in retesting by
one of the indirect methods.
4.2.4.2 Agar Plates
The direct susceptibility test is performed using 7H10 or 7H11 agar. The number of drug-containing
plates to be inoculated varies depending on whether only primary or both primary and secondary agents
are to be tested.
4.2.4.3 Inoculation and Incubation
After the digestion and decontamination steps, and confirmation that the specimen is AFB smear-positive,
the initial or processed specimen is inoculated onto drug-free (control) and drug-containing quadrants of
the agar medium. The inoculum used is based on the results of the AFB smear, performed using a
fluorochrome stain, as shown in Table 3. Each quadrant is inoculated with 0.1 mL of inoculum, except for
specimens containing less than five AFB per field, in which case the inoculum is increased to 0.2 mL.
After inoculation, the plates are treated as described in steps 4 to 7 in Section 4.2.3.2.1.
The plates are examined microscopically, using a dissecting microscope, without removing the plates
from the polyethylene bags, at one, two, and three weeks of incubation. The results observed at one and
two weeks of incubation are noted but not reported. The purpose of this examination is to evaluate for
growth of contaminating normal respiratory flora and to determine (at two weeks) if a sufficient number
of microcolonies are present on the drug-free medium. Contamination or insufficient growth suggests that
the direct test will fail. In such cases, an indirect test (preferably a commercial rapid broth method with a
short incubation time) can be initiated using primary growth from the initially inoculated media.
4.2.4.4 Reporting and Interpretation
Results are reported after three weeks of incubation, if there is sufficient growth on the drug-free medium,
as described in Section 4.2.3.3. Some isolates may not grow, or may not have sufficient growth for
adequate interpretation after three weeks of incubation. If this occurs, the test should be repeated.
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4.3 Commercial Broth Systems With Shorter Incubation Time for Susceptibility Testing
of Mycobacterium tuberculosis Complex
As stated previously, the standardized agar proportion method for the MTBC described here for
susceptibility testing of MTBC is not a rapid method. To ensure the early detection of resistance to
primary drugs, a commercial susceptibility test system with a shorter incubation should be used for
testing isolates of MTBC obtained from any patient.17 Any such commercial system used (those cleared
by the FDA [as of the time of publication of this document] for use in the United States are listed in
Appendix I) should have been previously demonstrated to produce results that correlate with those
obtained with the standardized agar proportion method. In addition, laboratories should choose a broth-
based system from a manufacturer that performs regular quality assurance (QA) testing to ensure that the
media and drug performance are consistent from one lot to the next, and that the media and drug lots
provide consistent results over long periods of time (eg, years) without drift. When deciding which
shorter incubation broth system to use, it is reasonable to ask the manufacturers to describe the measures
they take to ensure lot-to-lot consistency in drug susceptibility results, and to consider this information in
the decision process. Moreover, when a new broth system is implemented or testing personnel are newly
trained to use the system, reproducibility should be documented as part of the QA process.
It is important to ensure that susceptibility testing is performed on a pure culture. Indirect drug
susceptibility testing is performed after a culture is determined to be positive for MTBC and purity of the
culture is confirmed. When a commercial broth system flags a bottle/tube as positive, the presence of
AFB must be verified before identification or drug susceptibility testing is attempted. To do this, staining
a smear of the broth for AFB and subculturing an aliquot of the broth onto a 7H10 or 7H11 plate is
recommended. Subculture to a blood or chocolate agar plate is an acceptable alternative. The acid-fast
stained smear confirms the presence or absence of AFB and also allows assessment of cellular
morphology, which may differentiate MTBC from NTM (described in step 2 below). The Middlebrook
plate, which can be examined microscopically after incubation for a few days, provides a purity check,
and allows microscopic and gross examination of colonial morphology. When the liquid culture is
identified as positive for MTBC and the purity is confirmed, the broth can be used for indirect drug
susceptibility testing, if listed as an option by the manufacturer of the system. However, if growth on a
solid medium occurs before a broth culture is recognized as positive, drug susceptibility may be set up
from the solid medium.
If a culture is mixed with NTM or contaminated with other bacteria, reisolation of MTBC must be
attempted. The physician caring for the patient should be notified that drug susceptibility testing will be
delayed due to impurity of the culture. Reisolation of MTBC may take several weeks and sometimes may
not be successful. For these reasons, a molecular method for detecting drug resistance mutations may be
considered while attempting to reisolate MTBC. See Section 4.7.
Although drug susceptibility testing is performed on (reasonably checked) pure cultures, contamination
may be introduced during drug testing setup or an initially undetected mixed culture may be realized later.
It is critical to confirm absence of contamination or NTM for any drug showing resistant results before
reporting. The following steps are helpful to verify the initial resistant results:
(1) Examine the vial/tube showing resistance. Growth of MTBC usually forms clumps, and the
medium tends to remain clear. However, this may vary depending on the liquid medium used.
Before inverting or mixing, if the medium appears to be turbid, the presence of contaminants or
NTM may be suspected.
(2) Prepare a smear from the vial/tube, and perform acid-fast stain. Verify that cellular morphology is
compatible with MTBC (eg, cording) and that NTM or other bacteria are absent. Although the
MTBC may not show typical cording morphology due to certain effects imposed on MTBC cells by
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antituberculous agents, a dispersed distribution of AFB or random loose clumps throughout the
entire smear may be an indication of NTM.
(3) If there is a difficulty in discerning MTBC from NTM, make a subculture from the drug-resistant
vial/tube onto 7H10/7H11, and examine the colonial morphology.
If a laboratory has determined through its quality system that repeat testing to confirm resistance is not
required, a final report should be issued after verifying the drug resistance from the initial testing.
However, if repeat testing is planned, a preliminary report should be released, indicating that testing to
confirm the drug resistance has been initiated and a final report will follow. Once the initial resistance has
been confirmed, a final report should be issued as soon as possible. If the repeat test results do not
confirm the initial resistant result, a second preliminary report should be issued, and the attending
physician should be informed of the conflicting results as early as possible, preferably by phone. Sending
the isolate to a reference laboratory should be considered to avoid further delay in obtaining correct
results while investigating the problem or retesting a third time.
If any result obtained for a patient isolate with a commercial shorter incubation system is questionable,
repeat testing of the isolate using the same system or the standard agar proportion method should be done.
Repeat testing of an isolate to confirm rarely occurring resistant results such as mono-RMP resistance or
mono-EMB resistance from the initial testing also should be considered.
As of the time of publication of this document, commercial susceptibility test systems are not FDA-
cleared for testing drugs other than INH, RMP, EMB, PZA, and streptomycin. Laboratories adopting
commercial susceptibility systems for testing drugs other than these five should conduct studies
comparing results to the standardized agar proportion method using recommended test concentrations (see
Table 1 and Appendix A. NOTE: The critical concentration for testing linezolid by the standardized agar
proportion method has not been established).
4.4 Pyrazinamide Susceptibility Testing
PZA is a first-line drug for the treatment of TB. All initial patient isolates of MTBC should be tested for
susceptibility to this agent. Agar-based methods such as the agar proportion method described elsewhere
in this document have not proven to be satisfactory for PZA susceptibility testing because many isolates
fail to grow when the agar has been acidified to the degree necessary for PZA susceptibility testing.
Susceptibility testing using a radiometric method has proven to be satisfactory and is presently considered
the reference method for PZA susceptibility testing.26
The procedures for PZA susceptibility testing using the radiometric method are described in the
manufacturer’s instructions and should be followed scrupulously. The same considerations apply to the
reporting of PZA results as for other antimycobacterial agents. However, when using the radiometric
method, unlike the case for any other antimycobacterial agent, a “borderline” category exists for the
reporting of PZA results (see the manufacturer’s instructions for use).
PZA susceptibility testing using commercial nonradiometric rapid broth systems is now available. The
performance of these systems has been shown to be equivalent to that of the radiometric system.27,28
However, with these systems the “borderline” category for reporting of PZA results does not exist. Refer
to Section 4.3 for verification of resistant results. Repeat testing to confirm the initial resistant result
should be considered.
If an isolate is resistant only to PZA, determining the species of MTBC (eg, Mycobacterium bovis is
resistant to PZA, whereas most isolates of M. tuberculosis [in the strict sense] are susceptible to PZA)
should be considered.
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4.5 Quality Control
4.5.1 Purpose
The goals of a QC program are to assist in monitoring the:
• Reproducibility and accuracy of the susceptibility test procedure

• Performance of reagents used in the test
• Performance of persons who carry out the tests and read the results
The goals are achieved by, but are not limited to, the testing of QC strains with known susceptibility to
the antimicrobial agents to be tested.
4.5.2 Quality Control Responsibilities
Some laboratories rely heavily on pharmaceutical and diagnostic product manufacturers for provision of
reagents, media, or test systems for the performance of antimicrobial susceptibility tests. Although this
section is intended to apply to only the standard reference methods, it may be applicable to certain
commercially available test systems that are based primarily, or in part, on these methods.
An example of division of responsibility and accountability may be described as follows:
(1) Manufacturers (In-house or Commercial Products):
• Antimicrobial stability
• Antimicrobial labeling
• Potency of antimicrobial stock solutions
• Compliance with FDA quality system regulations
• Integrity of product and providing adequate directions for use
• Accountability and traceability to consignee
(2) Laboratory (User):
• Storage under the environmental conditions recommended by the manufacturer (to prevent drug
deterioration)
• Proficiency of personnel performing tests
• Adherence to the established procedure, eg, inoculum preparation, incubation conditions,

interpretation of end points
• Proper investigation of all unexpected results
Manufacturers should design and recommend a QC program that allows users to evaluate those variables
(eg, inoculum density, storage/shipping conditions) that most likely could cause user performance
problems and to determine that the test is performing correctly when used according to established
protocols.
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Multiple test parameters are monitored by following the QA guidelines described in this standard.
Acceptable results derived from testing QC strains do not guarantee accurate (true) results with all patient
isolates. When atypical or inconsistent results are encountered with patient isolates, repeat testing and/or
repeat identification procedures should be performed in an effort to ensure accurate (true) results, and
patient results should not be reported. Each laboratory should develop its own policies regarding
verification of atypical antimicrobial susceptibility testing results.
4.5.3 Selecting Quality Control Strains
QC of MTBC susceptibility testing should include an isolate that is completely susceptible to the
antimicrobial agents being tested. The strain M. tuberculosis H37Rv ATCC®a 27294 is well suited for use as
a “pansusceptible” control organism, and its performance is well documented. Alternately, M. tuberculosis
H37Ra, which is believed to be avirulent and therefore is less likely to cause laboratory-acquired
infections in the event of an accident, could be used if a laboratory has documentation that this strain
performs as expected. An advantage of using this strain is that its unique HPLC pattern is easily
detectable should evidence of the possibility of cross-contamination become an issue.
Some investigators suggest that laboratories also consider testing a resistant strain. In particular, those
testing both the critical concentration and a higher concentration of INH should consider testing a strain
(or isolate) of MTBC or an NTM that consistently demonstrates resistance to the low concentration of
INH but is susceptible to the higher concentration. ATCC® BAA-812 meets the criteria to be used for this
purpose.29 Single-drug–resistant strains also are available, but these strains are resistant to high
concentrations (50 to 1000 μg/mL) of drug and are not good candidates for QC purposes. Multidrug-
resistant (MDR) strains exist, but they pose an undesirable potential safety risk associated with frequent
use and, therefore, are not suitable for use in most clinical laboratories. Use of a single MTBC strain that
is resistant to more than two drugs is not recommended because of the serious, potentially life-threatening
consequences of a laboratory-acquired infection.
4.5.4 Source of Quality Control Strains
QC strains may be purchased from the American Type Culture Collection (ATCC; PO Box 1549,
Manassas, VA 20108 USA; www.atcc.org).
For those laboratories that choose to adopt QC methods that include using drug-resistant strains, there are
at least three possible sources for these isolates. In-house isolates with verified, reproducible low levels of
resistance to one or two drugs may be maintained for use in testing; resistant strain BAA-812 may be
purchased from ATCC®. Another source is through participation in an external proficiency or
performance testing program, such as the programs developed by the CDC and the College of American
Pathologists (CAP).
4.5.5 Storing and Testing Quality Control Strains
Store QC strains in a way that minimizes the possibility of mutation in the organism resulting from
repeated subculturing. Stock vials of strains can be prepared as suspensions (from solid or liquid cultures)
in a suitable stabilizer, distributed in small aliquots in multiple vials, and maintained at −20 °C or below
(preferably at –60 °C or below) and never in a self-defrosting freezer.
4.5.6 Frequency of Testing
Lot numbers of all media components should be recorded. Each new lot of drug or media component,
especially OADC and 7H10 powder, should be tested with the QC strains before use for patient testing
a
ATCC is a registered trademark of the American Type Culture Collection.
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(refer to CLSI document M2230 for a listing of exempt and nonexempt media QC recommendations).
Also, QC strains should be used each time a new batch of test medium is prepared, and at least once a
week, or with each run, both for QC purposes and to ensure QC strain stability during storage.
Three detailed assays that measure the comparative quality of media components are described in the
literature.31 They are as follows: a comparative resistance assay to monitor drug stability in solution and
in agar; a disk-potency assay to monitor the potency of disks impregnated with antimycobacterial agents;
and a standard concentrations assay to monitor changes in antibiograms caused by changes in the test
medium.
QC testing using M. tuberculosis H37Rv (ATCC® 27294) must be performed once each week that patient
isolates are tested. If a laboratory chooses to test a resistant strain (eg, ATCC® BAA-812) in addition to
H37Rv, both QC strains could be tested simultaneously or the resistant strain could be tested less
frequently (eg, monthly), unless a problem was identified, in which case more frequent testing would be
indicated. Results for drugs for which the QC result is not as expected should not be reported.
4.6 Implementing New Methods
When a new test method is implemented, its performance characteristics must be evaluated and show that
the test system is functioning appropriately. The first step in validation of a new method is to review the
literature for publications of studies of the new method. In cases in which the new test method is under
consideration for replacing an existing test method, the new method should be as accurate as the method
now in use. The easiest way to do this is to find a literature reference in which the new method, the
present method, and the reference method are all tested in parallel with a set of susceptible and resistant
cultures. Agreement between the new method and the reference method should be assessed for both
susceptible and resistant isolates. Agreement is not expected to be 100% but should be at least as good for
the new method as for the method previously in use. It may not be possible to find a literature reference in
which both the new method and the method currently in use are compared with the reference method. In
that case, it may be useful to estimate the predictive values for test results in the laboratory based on
sensitivity and specificity data in the available literature references. The frequency of false-positive (false-
resistant) results for each drug with the new method can be calculated from the specificity reported in the
literature reference. The specificity for each drug will give an estimate of the percentage of false-resistant
results. The frequency of false-resistant results is compared with the prevalence of true resistance to the
drug in the test population. Whether the predictive value of a resistant result will be satisfactory, or
whether resistant results with some drugs will require repeat testing should be determined. For a
discussion of predictive values and their calculation, refer to CLSI guideline I/LA18.32 Likewise, the
frequency of false-negative (false-susceptible) results will be apparent from the sensitivity reported in the
literature reference. False-susceptible results are considered a serious problem, because they can lead a
physician to rely on a drug to which the organism is resistant. Adoption of a new method in the laboratory
should be considered only if the frequency of false-susceptible results is very low (ie, no greater than that
of the previous method).
At times, considerations other than accuracy may prompt laboratorians to choose a new method over their
existing method. Such considerations could include cost, personnel time, elimination of radioactive
materials from the laboratory, and so on. However, it is important to assess the relative accuracy of the
new method so that:
• Test results by the new method that have reduced accuracy can be repeated, preferably by a different
method.
• Clinicians who use results of drug susceptibility testing to make patient care decisions can be made
aware of the diminished reliability of the new test results. When the new method is adopted, all of the
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laboratory’s physician clients and the local TB control program should be notified of any changes
expected in the accuracy of results.
A new testing method could be developed that is more accurate than the method currently in use and even
more accurate than the reference method. In this case, agreement of results with previously used methods
might not be the best way to validate the new procedure. Correlation of test results with performance of
the drugs in vivo (ie, treatment success or failure) could be used to validate the new method in this
circumstance. However, correlation of test results with in vivo performance of a drug usually is beyond
the scope of a laboratory that is planning to implement a new testing method. It generally is necessary to
rely on data from clinical trials published in the microbiology literature. For a general discussion of
comparisons among new methods, old methods, and clinical diagnosis, refer to CLSI document EP12.33
Once a new method has been chosen, the validation protocol should include performing the current test
method and a new method in parallel for a series of patient isolates. For testing of MTBC, the protocol
should assess whether the new method can reliably detect resistance for each antimycobacterial agent that
is tested. For some drugs, such as INH, the new method should be capable of detecting low-level resistant
strains that are most commonly encountered. Validation of minimal inhibitory concentration (MIC)
methods for NTM should assess the precision of each drug result with known patient isolates and QC
strains exhibiting MICs within the range of drug concentrations tested. With a new method, the laboratory
should further validate test results for several months by confirming results with selected isolates (eg,
drug-resistant MTBC) by another method or in another laboratory. For most laboratories, this involves
checking the agreement of test results when resistant isolates are sent to a referral laboratory for further
testing.
4.7 Molecular Detection of Drug Resistance
Several methods have been developed for the detection of mutations associated with drug resistance of
MTBC, but at the time of completing this document, none of these have been FDA-cleared. These include
real-time polymerase chain reaction (qPCR) methods34 and commercially available PCR/line probe
assays.35,36 These methods can be used to test positive cultures or smear-positive concentrated specimens
for identification of MTBC and detection of mutations associated with resistance to INH and RMP. When
a mutation associated with INH or RMP is detected, there is a high probability that the culture will be
resistant to the corresponding drug. Because drug resistance may be caused by mutations other than those
detectable by these methods, failure to detect such a mutation must be interpreted with
caution. Consideration must be given to the performance characteristics of the method used, as described
in the microbiology literature, as well as to the prevalence of various mutations associated with drug
resistance in the population being served, which are critical for proper interpretation of results from these
molecular methods. Laboratories should provide this information to the health care providers.
The use of molecular methods for rapid detection of drug resistance directly in acid-fast smear positive
concentrated specimens may be considered under the following circumstances:
(1) Patients with a wide range of contacts who may spread infection to many others
(2) Patients who are suspected of having a drug-resistant disease when a) there is a history of previous
treatment, b) patients are from countries or ethnic groups with increased drug resistance, c) patients
are not responding well to treatment, and d) patients have been exposed to MDR-TB
Note that detection of MTBC DNA in a treated patient does not necessarily indicate persistence of viable
mycobacteria. However, detection of a mutation or mutations associated with drug resistance may help to
explain a failure to respond to therapy. In addition, molecular methods may be performed when MTBC
cultures are mixed with other mycobacteria or contaminated with other bacteria.
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Molecular methods may reduce the time required for detection of drug resistance from approximately one
month using culture-based methods to approximately one day. Rapid detection of drug-resistant TB and
early initiation of effective regimens can reduce further spread of drug-resistant disease, decrease the
duration of illness, and increase the cure rate.37 At present, however, molecular methods must be
considered an adjunct to culture-based methods, and phenotypic culture-based drug susceptibility testing
must be performed when a pure culture becomes available. When mutations associated with MDR-TB are
detected, culture-based drug susceptibility testing for both primary and secondary drugs should be
performed as soon as possible to accelerate the availability of those drug results that are needed for
formulating effective regimens.
5 Susceptibility Testing of Nontuberculous Mycobacteria
5.1 Introduction
The standard recommended for susceptibility testing of NTM is broth microdilution. The MIC obtained
using a dilution test may tell a physician the concentration of antimicrobial agent needed at the site of
infection to inhibit the infecting organism. The MIC, however, does not represent an absolute value. The
“true” MIC is somewhere between the lowest test concentration that inhibits the organism’s growth (that
is, the MIC reading) and the next lower test concentration. If, for example, twofold dilutions were used
and the MIC was determined to be 16 μg/mL, the “true” MIC would be between 16 and 8 μg/mL. Even
under the best of controlled conditions, a dilution test may not yield the same end point each time it is
performed. Generally, the acceptable reproducibility of the test is within one twofold dilution of the actual
end point. To avoid greater variability, the dilution test must be standardized and carefully controlled as
described herein.
MICs have been determined using concentrations derived traditionally from serial twofold dilutions
indexed to the base 2 (eg, 1, 2, 4, 8, 16 μg/mL). Other dilution schemes have also been used, including
use of as few as two widely separated or “breakpoint” concentrations. The results from these alternative
methods may be equally useful clinically. When there is inhibition of growth at the lowest concentration
tested, the true MIC value cannot be accurately determined and should be reported as equal to or less than
the lowest concentration tested.
General recommendations concerning the test method and QC that apply to all NTM are described in
Sections 5.2 and 5.3. Detailed guidelines are provided for MAC, M. kansasii, M. marinum, and the
rapidly growing mycobacteria. These mycobacteria were selected because sufficient data exist on which
to base general recommendations. Several other slowly growing mycobacteria may cause human disease,
but there is little information on the correlation of in vitro susceptibility testing results and clinical
outcome for them. The recommendations concerning selection of antimicrobial agents generally reflect
the opinions of the ATS concerning appropriate therapy.3 Similarly, suggested indications for
antimicrobial susceptibility testing reflect the ATS guidelines.
In general, susceptibility testing of NTM should be considered only for those isolates believed to be
clinically significant. Isolates from blood, other sterile body fluids, or tissues are generally considered
clinically significant; however, determining the clinical significance of respiratory isolates is more
problematic. Many NTM may colonize the respiratory tract, and their isolation from respiratory
specimens such as sputum does not correlate with clinical disease. Currently, the ATS recommends the
following criteria for determining the clinical significance of NTM from respiratory specimens3: at least
two positive sputa or one bronchial wash or lavage sample are usually sufficient to confirm clinical
significance. Alternatively, a transbronchial or lung biopsy with mycobacterial histopathological features
and positive culture for NTM is sufficient to establish clinical significance. Single positive sputum
cultures that are AFB smear negative and/or contain low numbers of organisms are likely to be clinically
insignificant.
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Broth microdilution testing of pathogenic NTM requires skill acquired through experience with the test
method and knowledge of the expected susceptibility patterns of different species. Therefore, for
laboratories that encounter these organisms infrequently, referring those isolates for which susceptibility
testing is indicated to an established reference laboratory is recommended. For laboratories that choose to
do susceptibility testing in-house, test performance must be evaluated. Currently, no commercial
proficiency testing program regularly includes the NTM. The best alternative at present is comparison of
test results with those from an experienced reference laboratory. This should be done on initial validation
and at regular intervals thereafter to demonstrate continued proficiency.
5.2 Preparation of the Inoculum for the Microdilution Method
To standardize the inoculum density for a susceptibility test, a BaSO4 turbidity standard equivalent to a
0.5 McFarland standard or its optical equivalent (eg, a latex particle suspension that is commercially
available) should be used (see Appendix G).
5.2.1 Inoculum Suspensions
The inoculum should be prepared when there is adequate growth on an agar medium.
(1) Prepare suspensions by sweeping the confluent portion of growth on an agar medium with a sterile
cotton swab. Transfer the growth on the swab to 4.5 mL of sterile water containing sterile glass
beads (eg, 7 to 10 3-mm beads) until the turbidity matches the density of a 0.5 McFarland standard
(eg, by visual examination or by using a nephelometer). The suspension could also be prepared
directly from a broth culture.
(2) Vigorously mix suspensions on a vortex mixer for 15 to 20 seconds. If large clumps remain, allow
them to settle and use the supernatant for the inoculum suspension. Some organisms may also need
to be broken up using a sterile micropestle in a microcentrifuge tube, as described for aerobic
actinomycetes in Section 6.4.
(3) If inoculating MIC plates without the broth already added (ie, freeze-dried), prepare the final
inoculum (with an organism density of approximately 5 × 105 CFU/mL) by transferring 50 μL of
the suspension to 10 mL of cation-adjusted Mueller-Hinton broth (CAMHB) for rapidly growing
mycobacteria or 10 mL of CAMHB broth plus 5% OADC for slowly growing NTM. Invert the
tubes 8 to 10 times to mix the suspension thoroughly.
(4) Mix final inoculum suspensions well (by inverting the tube several times or vortexing) and pour
into sterile plastic troughs. Transfer 100 μL to each well of the microdilution tray.
Alternatively, if inoculating prepared plates containing antimicrobials in 100 μL of broth, calculate the
volume of standardized suspension to be added to 36 mL of water diluent to obtain a final organism
concentration of 1 × 105 to 5 × 105 CFU/mL (1 × 104 to 5 × 104 CFU per well in 0.1 mL volume). The
volume depends on the delivery system that is being used. For example, when using a disposable plastic
multipronged inoculating device that delivers 0.01 mL per well, prepare the inoculum as follows:
(1) Prepare a suspension equivalent to a 0.5 McFarland standard and mix by inverting the tube or using
a vortex mixer.
(2) Add 0.5 mL of this suspension to 4.5 mL of water diluent (1:10 dilution), and mix.
(3) Add 4 mL from step (2) to 36 mL of water diluent (1:10 dilution).
(4) Prongs deliver 0.01 mL into 0.1 mL in well (1:10 dilution).

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(5) Cover inoculated trays with an adhesive seal, and incubate in ambient air. Inoculate the nutrient
agar plate (eg, 5% sheep blood, trypticase soy agar) with a loopful of the final inoculum to check
for purity. Incubation temperature and time are discussed for each species or group in the following
sections.
5.3 Quality Control Procedures
See Section 4.5 for details on the purpose of a QC program and responsibilities, as well as
recommendations for selecting, storing, and frequency of testing QC organisms.
5.3.1 Strains Used for Quality Control
Ideal strains for QC of dilution tests have MICs that fall near the middle of the concentration range tested
for all antimicrobial agents; eg, an ideal control strain would be inhibited at the fourth dilution of a seven-
dilution series, but strains with MICs at either the third or fifth dilution would also be acceptable. In
certain instances with newer, more potent antimicrobial agents, it may be necessary to test additional QC
strains not normally tested to provide on-scale values. QC strains recommended for MAC, M. kansasii,
and the rapidly growing mycobacteria are listed in Sections 5.4, 5.5, and 5.8, respectively.
5.3.2 Storing Quality Control Strains
(1) Working control cultures of mycobacteria should be subcultured weekly (or with each test run if
done less often than weekly) from frozen stocks. These stock cultures should be stored at –20 °C or
below (preferably –60 °C or below) in an appropriate stabilizer (eg, tryptic soy broth with 15%
glycerol), and never in a self-defrosting freezer. This should minimize the risk of altering their
antimicrobial susceptibility.
(2) Before testing, the strains should be subcultured onto appropriate agar plates to obtain isolated
colonies.
(3) Colonies are grown or suspended for testing according to the recommended inoculum-preparation
procedures.
(4) A QC culture may be used to monitor precision and accuracy of dilution tests as long as there is no
significant change in the MICs that cannot be attributed to faulty methodology. If an unexplained
result suggests a change in the control organism’s inherent susceptibility, a fresh culture of the
frozen stock control strain should be obtained.
5.3.3 Batch or Lot Quality Control
(1) Each new batch or lot of microdilution trays should be tested with the appropriate QC strains to
determine if MICs obtained with the batch fall within the expected range; if they do not, the batch
must be rejected.
(2) At least one uninoculated microdilution tray from each batch should be incubated overnight to
verify sterility of the medium.
(3) Records should be kept of the lot numbers of all materials and reagents used in performing
susceptibility tests.
(4) Results for drugs for which the QC result is not in range should not be reported.
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5.3.4 Frequency of Testing
The overall performance of the susceptibility test system must be monitored by testing appropriate QC
strains weekly or each day the test is performed (if testing is done less often than weekly).
5.3.5 Other Control Procedures
Each microdilution broth tray should include a growth control of basal medium without antimicrobial
agent to assess viability of the test organisms. The growth control also serves as a turbidity control for
reading end points.
5.3.6 Purity Control
For broth dilutions that may be mixed with regular bacteria, testing for purity is suggested. Following
inoculation of broth dilution tests, streak a sample from each inoculum on a suitable agar plate (eg,
chocolate agar or trypticase soy agar) and incubate overnight to detect mixed cultures. Broth dilutions
should also be subcultured to appropriate solid media to detect mixed cultures of mycobacteria. These
media could include Middlebrook 7H10 or 7H11 agar for mycobacteria, or chocolate agar if M.
haemophilum is suspected. These subcultures should be incubated long enough for the morphology of the
organism or organisms to become apparent to check for mixed growth and to provide freshly isolated
colonies in case retesting proves necessary.
5.3.7 End-Point Interpretation Control
Monitor end-point interpretation periodically to minimize variation in the interpretation of MIC end
points among observers. All laboratory personnel who perform these tests should independently read a
selected set of dilution tests. The results are recorded and compared with the results obtained by an
experienced reader. All readers should agree within ±1 concentration of one another.38 If a lack of
agreement exists, retraining of personnel should be considered.
5.4 Susceptibility Testing of Mycobacterium avium Complex
5.4.1 Indications for Performing Susceptibility Tests
Currently, uniform agreement concerning the indications for susceptibility testing of isolates of MAC
does not exist. However, investigators who have extensively studied MAC disease have recommended
that isolates be tested in the following situations3,39:
• Clinically significant isolates from patients on prior macrolide therapy
• Isolates from patients who develop bacteremia while on macrolide prophylaxis
• Isolates from patients who relapse while on macrolide therapy
• Initial isolates from blood or tissue (eg, from patients with disseminated disease) or from clinically
significant respiratory samples (eg, sputum or bronchoalveolar lavage fluid) to establish baseline
values. If baseline testing is not performed, saving the isolate for future testing (if necessary) is
strongly recommended
Susceptibility testing should be repeated after three months of treatment for patients with disseminated
disease and after six months of treatment for patients with chronic pulmonary disease, if the patient shows
no clinical improvement or clinical deterioration and is still culture positive. If baseline testing was not
performed, both initial and recent isolates should be tested concurrently.
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5.4.2 Antimicrobial Agents
The only antimicrobial agents for which a correlation between in vitro susceptibility tests for MAC and
clinical response has been demonstrated in controlled clinical trials are the macrolides (azithromycin and
clarithromycin). Therefore, these are the primary drugs to which susceptibility of MAC isolates should be
evaluated.3 The mechanism of acquired mutational resistance in isolates of MAC (ie, 23S rRNA gene
mutation) is the same for both clarithromycin and azithromycin. For this reason, to be most cost-effective,
only one macrolide need be tested. Because of the technical difficulties associated with testing
azithromycin (ie, poor solubility at the high concentrations of drug that must be tested), clarithromycin is
the most appropriate class drug for the macrolides. Susceptibility testing of azithromycin is not
recommended.
For macrolide-resistant MAC isolates and/or isolates from patients who cannot tolerate macrolide
therapy, susceptibility testing of the secondary agents moxifloxacinb and linezolidb may also be
considered. Although there are insufficient data using linezolid and moxifloxacin to establish the
correlation of in vitro results with clinical response, tentative breakpoints for these two agents have been
proposed (see Table 4). In vitro MIC data for EMB, RMP, and rifabutin have shown poor correlation with
clinical response, and alternate agents such as amikacinb and streptomycin have not been adequately
studied. This prohibits the selection of breakpoints for these agents that separate susceptible from resistant
strains.
5.4.3 Susceptibility Test Method
Susceptibility testing should be performed using a broth-based method, either macrodilution or

microdilution. However, for azithromycin, macrodilution is preferred, because solubility is less
problematic when using larger volumes of broth. For either method, the inoculum should be prepared
using transparent colonies, if they are present. With regard to macrodilution, there is some consensus in
the United States that the radiometric method using the 12B medium is accurate and reliable.40 For broth
microdilution testing, cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 5% OADC is
recommended.41 Microdilution trays are incubated at 35 to 37 °C in ambient air and examined after seven
days. If growth is poor, trays should be reincubated and read again at 10 to 14 days.
The optimal pH at which to test the macrolides has been debated.39-42 The macrolides are more active in
vitro in mildly alkaline conditions (pH 7.3 to 7.4) than under mildly acidic conditions (pH 6.8, which is
the pH of the commercially available radiometric 12B medium), which more closely reflects the internal
environment of the macrophage. However, it has been shown that testing susceptibility of MAC isolates
to macrolides in broth at either pH 6.8 or pH 7.4 is acceptable, providing that recommendations for
interpretation of the breakpoints are followed (see Table 4).43
5.4.4 Reporting Results
MIC values and an interpretation should be reported as shown in Table 4.
The interpretive criteria are based, in part, on a monotherapy trial of disseminated MAC disease in
humans.44 Wild-type MAC isolates are uniformly susceptible to macrolides, but macrolide resistance
develops within a few months with monotherapy and may develop with combination therapy. Molecular
analysis of MAC isolates that have developed resistance to macrolides in vitro has shown that more than
95% of isolates have acquired a point mutation in the V-domain of the 23S rRNA gene.45-47 In vitro
susceptibility testing of these isolates has shown a clarithromycin MIC of ≥32 μg/mL at pH 6.8 (≥16
μg/mL at pH 7.3 to 7.4). Therefore, these breakpoints have been selected to define clinically significant
resistance.
b
Amikacin, linezolid, and moxifloxacin are not approved by the FDA for use in the treatment of MAC.
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Untreated wild strains of MAC are unlikely to be intermediate or resistant to macrolides. Therefore, it is
recommended that such results should not be reported until confirmed by repeat testing, and/or the
identity of the isolate is confirmed. A confirmed result of intermediate susceptibility may suggest that the
patient has a mixed population of MAC organisms; these patients should be followed for emerging
resistance.
5.4.5 Quality Control
M. avium (ATCC® 700898) is recommended for QC of broth dilution testing of MAC. The range of
acceptable results is 1 to 4 μg/mL for clarithromycin at pH 6.8 (0.5 to 2 at pH 7.3 to 7.4). When testing by
microbroth dilution, M. marinum ATCC® 927 is an acceptable alternative QC organism; the acceptable
range for clarithromycin is 0.25 to 1 μg/mL.
When testing secondary drugs, there are sufficient data to support QC recommendations only for
microbroth dilution testing. For moxifloxacin and linezolid, the acceptable ranges are 0.25 to 2.0 μg/mL
and 4 to 16 μg/mL, respectively, when testing M. avium ATCC® 700898. When testing M. marinum
ATCC® 927, acceptable ranges are 1 to 4 μg/mL for both moxifloxacin and linezolid. Additionally,
P. aeruginosa ATCC® 27853 can be used for QC testing of moxifloxacin (acceptable ranges are found in
CLSI document M100).48
Working control cultures of MAC should be subcultured weekly or each time susceptibility testing is
performed (if done less often than weekly). For storage, these stock cultures should be maintained as
described in Section 5.3.2, although the working stock culture may be stored at room temperature for up
to one month.
The overall performance of the susceptibility test system should be monitored by testing the appropriate
QC strain weekly or each time the test is performed, if it is done less often than weekly.
5.5 Susceptibility Testing of Mycobacterium kansasii
The drugs clinically active against M. kansasii that are used routinely for therapy include INH, RMP, and
EMB. An alternative drug regimen that includes RMP, EMB, and clarithromycinc has also been shown to
be effective.49 Rifabutin is used in place of RMP in HIV-infected patients being treated with protease
inhibitors.3 MICs for these drugs for untreated strains will fall within a narrow range, and routine
susceptibility testing is generally not needed. Treatment failure can occur and is associated with increases
in MIC values (resistance) to RMP and occasionally to INH and/or EMB, as well.50 However, such
resistance invariably includes resistance to RMP.50 Thus, susceptibility testing is important in patients
who failed therapy or had a poor response to initial therapy.
The commercial radiometric system,51,52 agar proportion,50,52 and broth microdilution50,53,54 have been used
for testing with the primary treatment drugs. However, broth microdilution using CAMHB supplemented
with 5% OADC is recommended. The critical concentrations of both RMP (1 μg/mL) and EMB (5 μg/mL)
used for testing MTBC inhibit wild strains of M. kansasii, with good correlation between Middlebrook
7H10 agar and broth methods.50-54 However, the INH MICs for wild strains of M. kansasii have ranged
from 0.5 to 5.0 μg/mL,53,55 so the standard MTBC critical concentration of 0.2 μg/mL in Middlebrook
7H10 agar invariably shows resistance, and the 1.0-μg/mL concentration gives variable results, even with
multiple cultures of the same patient strain. For that reason, testing of INH for M. kansasii is not
recommended with either of these concentrations.3 Given that treatment failure is associated with RMP
resistance and drug treatment histories are generally unavailable to the laboratory, susceptibility to RMP
and clarithromycin (used in the alternative regimen) are the currently recommended drugs for primary
testing. Isolates susceptible to RMP are all susceptible to rifabutin, and special testing is not required for
c
Clarithromycin is not approved by the FDA for use in the treatment of M. kansasii.
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patients being treated with protease inhibitors. Susceptibility testing should be repeated if patients remain
culture positive after three months of appropriate therapy.
Secondary drug testing of M. kansasii isolates resistant to the 1.0-μg/mL concentration of RMP is
recommended. The proposed drugs and the MIC values indicating resistance are given in Table 5;
however, experience with these drug concentrations for isolates of M. kansasii is limited.53,54,56-58
Incubation should be for 7 to 14 days at 35 to 37 °C in 5% to 10% CO2 or ambient air, although when
testing macrolides, incubation in CO2 should be avoided. MIC values and the interpretations (as shown in
Table 5) should be reported.
5.5.1 Quality Control
M. kansasii ATCC® 12478, M. marinum ATCC® 927, and/or Enterococcus faecalis ATCC® 29212 may
be used for QC of susceptibility testing of clinical isolates of M. kansasii to RMP. Expected results for the
QC strains are M. kansasii ATCC® 12478, ≤ 1 μg/mL; M. marinum ATCC® 927, ≤0.25 to 1.0 μg/mL; and
E. faecalis ATCC® 29212, 0.5 to 4.0 μg/mL. The selected QC strain should be tested weekly or each time
the test is performed, if less often than weekly.
5.6 Susceptibility Testing of Mycobacterium marinum
The major indications for susceptibility testing of NTM are variability in susceptibility to clinically useful
antimicrobial agents and/or a significant risk of acquired mutational resistance to one or more of these
agents. In general, neither of these applies to isolates of M. marinum; therefore, routine susceptibility
testing of this species is not recommended. Drugs that have been used successfully as single agents for
therapy of clinical disease caused by M. marinum are RMP, doxycycline, minocycline, trimethoprim-
sulfamethoxazole, and clarithromycin; the combination of RMP and EMB has also been used.3 Isolates of
M. marinum have a very narrow range of MICs for each of these agents, and because disease due to M.
marinum is usually localized and the number of organisms is low (most tissue biopsies are AFB smear
negative), single-drug therapy is widely used and acquired mutational resistance is rare.3
Susceptibility testing of M. marinum may be considered for patients in whom several months of therapy
have failed and who remain culture positive. Broth microdilution using CAMHB supplemented with 5%
OADC is the recommended method of testing susceptibility of M. marinum, although other methods have
been evaluated.52,55,59 Microdilution trays should be incubated at 28 to 30 °C for seven days. Drugs
recommended for susceptibility testing are listed in Table 6. Incubation in 5% to 10% CO2 or ambient air
is acceptable except when testing a macrolide, in which case CO2 should be avoided. MIC values and the
interpretations (as shown in Table 6) should be reported.
5.7 Susceptibility Testing of Miscellaneous Slowly Growing Nontuberculous

Mycobacteria
5.7.1 Nonfastidious Species
A number of other slowly growing NTM exist that may cause human disease and for which susceptibility
testing results in agar and/or broth have been reported (eg, isolates of M. terrae/nonchromogenicum,
M. xenopi,60 M. malmoense,59 and M. simiae). The same primary and secondary drugs listed for M. kansasii
in Table 5 should be tested, with the use of the same interpretive criteria. Although too few isolates have
been tested to recommend a specific method for these species, broth dilution using CAMHB with OADC
is suggested. M. xenopi does not grow well in CAMHB, and some isolates grow better at 42 to 45 °C than
at 35 to 37 °C. However, the optimal medium to use for M. xenopi has not been determined, and
incubation at the higher temperatures is often problematic due to dehydration of the broth before the end
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of the incubation period. Mycobacterium gordonae rarely is clinically significant; therefore, susceptibility
testing is rarely required and should be discouraged.
5.7.2 Fastidious Species
A number of fastidious slowly growing NTM that have been associated with clinical disease exist, and for
some of these species, susceptibility testing has been done. This includes M. haemophilum (requires ferric
ammonium citrate or hemin to grow), M. genavense (requires incubation for more than six weeks using
the radiometric system),61 and M. ulcerans (requires incubation for four to six weeks and grows best at
30 °C).62 Too little is known about these species to recommend a standard method for susceptibility
testing. One option for testing M. haemophilum is described in Appendix J. Each laboratory should
establish its own in-house validation for these organisms.
5.8 Antimycobacterial Susceptibility Testing of Rapidly Growing Mycobacteria
5.8.1 Introduction
The recommendations described in this section are based predominantly on data from studies that have
included the Mycobacterium fortuitum group (M. fortuitum, M. peregrinum, and the species included in
the former M. fortuitum third biovariant complex), M. chelonae, and M. abscessus; however, they also
apply to other clinically significant rapid growers. The method described is the standard broth dilution
method (see CLSI document M0763 for details on performance of broth microdilution for rapidly growing
aerobic bacteria).3,64-71
Because susceptibility test results for some drugs (eg, tobramycin and imipenem) pertain to certain
species or groups, identification of isolates to the species level or, at a minimum, differentiation of
M. fortuitum group from M. chelonae-abscessus group, is recommended.
5.8.2 Indications for Performing Susceptibility Tests
Susceptibility testing is indicated for any rapidly growing mycobacteria that are considered clinically
significant (eg, isolates from blood, tissue, and skin and soft tissue lesions).3 These organisms (especially
M. abscessus) may cause pulmonary disease, but they also may transiently colonize the respiratory tract.
Therefore, not all rapidly growing mycobacteria recovered from sputum or other respiratory specimens
are clinically significant. Diagnostic microbiological criteria developed by the ATS for symptomatic
patients with radiographic opacities or high-resolution computed tomography that shows multifocal
bronchiectasis with multiple small nodules are summarized in Section 5.1.3 Clinical failure to eradicate
rapidly growing mycobacteria from almost any site (except respiratory tract) after six months of
appropriate antimicrobial therapy necessitates the need to confirm species identity and repeat
susceptibility testing.
5.8.3 Antimicrobial Agents
Agents that should be tested against the rapidly growing mycobacteria are amikacin, cefoxitin,
ciprofloxacin, clarithromycin, doxycycline (or minocycline), imipenem, linezolid,72,73 moxifloxacin,
trimethoprim-sulfamethoxazole, and tobramycin.
5.8.4 Susceptibility Test Method
Prepare the inoculum in CAMHB and inoculate the trays as described in Section 5.2, and incubate at 28 to
30 °C. Trays should be examined after 72 hours. If growth (appearing as turbidity or a deposit of cells at
the bottom of the well) in the growth control well is sufficient (ie, at least 2+, based on the scale
illustrated in Figure 1), record the MIC. Otherwise, reincubate the tray and read on day 4, and if growth is
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Volume 31 M24-A2
still insufficient in the growth control well, read again on day 5. If growth remains insufficient on day 5,
the test should be repeated. For all drugs except clarithromycin, the final reading should be at no more
than five days. For some isolates from patients who have been on antimicrobial therapy, repeated passage
onto solid media may be required in order to obtain sufficient growth for interpretation of MIC values.
Recently, the erm gene, which causes inducible resistance to macrolides, has been identified in several
species of rapidly growing mycobacteria, although the clinical significance is not fully understood.74,75 To
ensure detection of inducible macrolide resistance in rapidly growing mycobacteria, it is currently
recommended that the final reading for clarithromycin be at least 14 days, unless resistance (MIC ≥8
µg/mL) is recognized earlier, at which time the report should be finalized.74-76 However, further in vitro
studies with large numbers of isolates are warranted to determine optimal reading times.
Of the infections due to species of rapidly growing mycobacteria that exhibit inducible resistance to
clarithromycin (ie, susceptible at day 3 but resistant at day 14, confirming the presence of the erm gene),
those caused by M. abscessus are especially problematic because the isolates generally are resistant to all
other oral antimicrobials that are tested against rapidly growing mycobacteria. A recent study showed that
the presence of a functioning erm gene in isolates of M. abscessus is associated with delayed treatment
response and possible treatment failure in patients with M. abscessus lung disease on macrolide-
containing regimens.77 However, including a macrolide (clarithromycin or azithromycin) in the multidrug
regimen should still be considered in such situations, as no other effective oral drugs are available.
Additionally, before to the recent discovery of the presence of the erm gene, clarithromycin was used for
many years in this setting with clinical response in some patients.3
For all but one of the drugs recommended in Section 5.8.3, the MIC is the lowest concentration of drug
that inhibits visible growth. The exception is trimethoprim-sulfamethoxazole, for which the end point is
the well with approximately 80% inhibition of growth compared with the growth in the control well with
no drug (eg, well 1B if 1E is the growth control as shown in Figure 1).
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Figure 1. Guidelines for Interpretation of Broth Microdilution MIC End Points When Testing
Rapidly Growing Mycobacteria. The ± sign denotes one to five countable colonies/flakes and or slight
but definite turbidity. Care must be taken to read known negative wells because a slight precipitate related
to the inoculum may be seen.
1E Control growth (4+)

3A ± Few countable colonies; no turbidity (also, wells 5B and 2D are ±)
5C ± Few countable colonies; hazy turbidity (also, well 3E is ±)
5D 1+ Definite haze or turbidity
5E 2+ Definite turbidity and “clumpy growth”
1B 2+ Moderate countable colonies; slight turbidity
1D 3+ Heavy colonial growth
4E 3+ Heavy growth; turbidity
1C 4+ Heavy confluent growth comparable to control (also, wells 2A and 2E are 4+)
Wells 4A, 5A, 2B, 4B, 4C, 3D, and 4D are all negative.
5.8.5 Reporting of Minimal Inhibitory Concentration Results
MIC values and the interpretations should be reported for all isolates. Breakpoints for the rapidly growing
mycobacteria are listed in Table 7.
The MIC values determined as described in this document may be reported directly to clinicians for
patient care purposes. However, it is essential for an understanding of the data by all clinicians that an
interpretive category result (ie, “susceptible,” “intermediate,” and “resistant” as defined in Section 3.1)
also be provided routinely. For most agents, these categories were developed by determining MICs of a
large number of isolates, including those with known mechanisms of resistance relevant to the particular
class of drug. In addition, whenever possible, the in vitro interpretive criteria were analyzed in relation to
studies of clinical efficacy in the treatment of specific pathogens.
5.8.6 Recommended Strains for Quality Control
Mycobacterium peregrinum ATCC® 700686 (see Table 8 for QC ranges), incubated for three days at
30 ± 2 °C, is recommended for controlling dilution testing of rapidly growing mycobacteria. Alternatively,
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Staphylococcus aureus ATCC® 29213, P. aeruginosa ATCC® 27853, and/or E. faecalis ATCC® 29212
may be used for QC.
6 Susceptibility Testing of Aerobic Actinomycetes78-85
6.1 Method
The recommended method for susceptibility testing of the commonly encountered aerobic actinomycetes
(eg, Nocardia spp., Rhodococcus equi and other Rhodococcus spp., Gordonia spp., Tsukamurella spp.,
Streptomyces spp.) is broth microdilution. However, some technical issues have been noted with various
antimicrobials especially when testing the Nocardiae. Ceftriaxone results have proven difficult to interpret
consistently when performed by broth microdilution (unpublished data). There may be a particular
problem with false-resistant results for ceftriaxone, primarily when testing N. brasiliensis. There may also
be a problem with false-resistant results for imipenem when testing N. farcinica using the broth
microdilution methodology. Further studies are needed to investigate this issue. Breakpoints have been
established for interpretation of MICs for Nocardia spp. only (see Table 9). See Section 6.6 for reporting
results from other genera.
Sulfonamide results have also been shown to be particularly difficult to interpret consistently using the
broth microdilution method. A disk diffusion test using a sulfisoxazole disk may be performed
concurrently with broth microdilution testing in order to confirm the susceptibility or resistance of an
isolate to the sulfonamide drugs (unpublished data). Alternatively, disk diffusion may be performed
following broth microdilution if the MIC result is questionable.
R. equi is unique among the aerobic actinomycetes due to its ability to grow rapidly in most microdilution
susceptibility panels. R. equi can be set up in microdilution panels used to test rapidly growing aerobic
gram-positive bacteria, following the procedure for broth microdilution testing described in CLSI
document M07.63 Additionally, microdilution gram-positive panels often include vancomycin and RMP,
which are key drugs for treatment of R. equi infections.
The inoculum suspension for microdilution testing in CAMHB (see Sections 6.4 and 6.5) may be
prepared from a blood or trypticase soy agar plate that has been incubated at 35 ± 2 °C in ambient air until
growth is sufficient for testing, up to seven days. Incubation conditions for microdilution panels are 35 ±
2 °C in ambient air. Incubation time is three days for most aerobic actinomycetes, although some species
(eg, Nocardia nova) may require incubation for up to five days, whereas results for R. equi and some
isolates of Tsukamurella spp. generally can be read at 24 to 48 hours.
6.2 Antimicrobial Agents
Drugs that are recommended for primary testing are amikacin, amoxicillin-clavulanic acid, ceftriaxone,
ciprofloxacin, clarithromycin, imipenem, linezolid, minocycline, moxifloxacin, trimethoprim-
sulfamethoxazole, and tobramycin. Secondary drugs that may be tested include cefepime, cefotaxime, and
doxycycline.
For R. equi testing, the above listed primary drugs plus rifampin and vancomycin are recommended. Of
these, vancomycin and rifampin are particularly useful therapeutically. If rifampin and vancomycin are
not included in the gram-positive microdilution panels in use, the isolate should be sent to a reference
laboratory for susceptibility testing of these drugs.
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6.3 Preparation of the Inoculum
A nephelometer should be used to standardize the inoculum for susceptibility testing. The reading
corresponding to a 0.5 McFarland standard should be determined for the instrument in use.
6.4 Inoculum Suspensions
For R. equi, susceptibility tests should be performed using procedures outlined for gram-positive
organisms as described in CLSI document M07.63
For Nocardia and aerobic actinomycetes other than R. equi, the inoculum should be made from growth on
blood or trypticase soy agar medium after sufficient incubation at 35 ± 2 °C in ambient air to allow
preparation of a heavy suspension of organism (usually three to seven days).
(1) Prepare a heavy suspension of the organism in a small volume of sterile, deionized water or saline
in a microcentrifuge tube, using a loop or sterile applicator stick to sweep the area of confluent
growth on the agar. Break up organism clumps using a micropestle in the microcentrifuge tube.
Alternatively, prepare the suspension in a small culture tube using 7 to 10 3-mm glass beads,
mixing vigorously with a vortex mixer until clumps are broken up.
(2) Allow the suspension to sit for approximately 15 minutes to allow any remaining large clumps to
settle to the bottom of the tube.
(3) Using a Pasteur pipette, add several drops of the supernatant of the heavy suspension to 2 mL of
water or saline in a tube compatible with the nephelometer; check the turbidity of the suspension
with the nephelometer and adjust to make a suspension equal to that of a 0.5 McFarland standard. If
there is a range of readings that correspond to a 0.5 McFarland suspension, the turbidity of the
Nocardia suspension should be in the middle of that range. This will provide sufficient inoculum
for those organisms that tend to form tighter clumps.
NOTE: The suspension approximating the 0.5 McFarland standard can also be made from a broth culture;
large clumps of organism should be broken up by vortexing with glass beads, as described in step 1. Allow
the broth suspension to sit for approximately 15 minutes to allow any remaining large clumps to settle to
the bottom of the tube.
6.5 Susceptibility Test Method
The preparation of the final inoculum depends on the type of susceptibility panel being used.
For panels with dried antibiotics (no broth added):
(1) Add 50 μL of the adjusted 0.5 McFarland suspension to 10 mL of CAMHB.
(2) Invert the tube 8 to 10 times, or vortex to mix thoroughly.
(3) Add 100 μL of the suspension to each well of the panel.
(4) Add a drop of the suspension to a 5% sheep blood or trypticase soy agar plate as a purity check,
streaking in a manner to obtain isolated colonies.
For panels to which broth has already been added (frozen panels):
(1) Add 1 mL of the adjusted 0.5 McFarland suspension to 29 mL of water.
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Volume 31 M24-A2
(2) Invert the tube 8 to 10 times, or vortex to mix thoroughly.
(3) Pour the entire suspension into an inoculating trough.
(4) Add 10 μL of the final suspension to each well of the panel using a multipronged inoculator.
(5) Add a drop of the suspension to a 5% sheep blood or trypticase soy agar plate as a purity check,
streaking in a manner to obtain isolated colonies.
Disk diffusion testing for confirmation of sulfonamide results:
(1) Using the adjusted 0.5 McFarland suspension, perform an antimicrobial disk susceptibility test
according to procedures outlined in CLSI document M02.86
(2) Use a 250-μg sulfisoxazole disk as the representative sulfonamide.
(3) Incubate in ambient air at 35 ± 2 °C for 72 hours.
Ideally, the final organism concentration in the inoculum should be approximately 1.0 × 105 to 5.0 × 105
CFU/mL (1.0 × 104 to 5.0 × 104 CFU per well). For Nocardia spp., this organism density may be difficult
to achieve, especially with isolates that form tight clumps. If the inoculum is carefully prepared, colony
count plates need not be routinely inoculated. Instead, interpretation of MICs should include evaluation of
organism density in the control well; growth that is very light or very heavy may give results different
from those expected for that species (see Appendix K); these tests should be repeated, or the isolates
should be referred to a reference laboratory for repeat testing. Evaluation of the quantity of organism
growth on the sulfisoxazole disk diffusion test after appropriate incubation will indicate the adequacy of
the inoculum. Growth should not be confluent; streak marks should be obvious and clear areas between
the streaks should be apparent (unpublished data).
Cover inoculated trays with an adhesive seal, and place panels in a plastic bag to further prevent drying.
Do not place more than three panels in a stack. Incubate the trays at 35 ± 2 °C in ambient air. Examine the
trays after 72 hours (24 hours for R. equi, 24 and 48 hours for some strains of Tsukamurella). If growth
(appearing as turbidity or a deposit of cells at the bottom of the well) in the growth control well is
sufficient (ie, at least 2+, as illustrated in Figure 2), record the MIC. Otherwise, reincubate the tray and
read daily thereafter (for up to five days total) until growth is sufficient. For all but one of the drugs
recommended in Section 6.2, the MIC is the lowest concentration of drug that inhibits visible growth. The
exception is trimethoprim-sulfamethoxazole, for which the MIC is difficult to determine accurately.
General recommendations state that the end point is the well with approximately 80% inhibition of
growth compared with the growth in the positive control well (or the well with the lowest concentration
of trimethoprim-sulfamethoxazole, if that well shows more growth than the positive control well). A more
practical approach may be to interpret the MIC as being the dilution that shows a significant difference in
the amount of growth as compared with the positive control well or to an adjacent well with a higher
antibiotic concentration.
The result of disk diffusion testing with sulfisoxazole should be compared to the result obtained for
sulfonamides using broth microdilution if the latter is questionable. A disk diffusion zone of inhibition
≥35 mm indicates susceptibility to sulfonamides, while zones ≤15 mm indicate resistance.87 Zone sizes of
16 to 34 mm are uninterpretable due to lack of sufficient data. Isolates showing discrepant results between
microdilution and disk diffusion testing should be retested or sent to a reference laboratory for repeat
testing. Caution should be taken when reporting a Nocardia species as resistant to sulfonamides, as most
species are susceptible to these agents.
Examples of interpretation of broth microdilution tests are shown in Figure 2.

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Number 5 M24-A2
• Antibiotic concentrations and growth amount estimates are indicated to the right of the wells. Arrows
indicate the MIC.
• Abbreviations: POS, positive control; NEG, negative control; 4+, heavy growth; 3+, heavy growth or
turbidity; 2+, moderate growth or turbidity; 1+, haze, slight turbidity, moderate countable colonies;
+/-, few countable colonies or minimal turbidity.
A Ciprofloxacin MIC = 4 µg/mL

B Tobramycin MIC = >32 µg/mL
C Trimethoprim/sulfamethoxazole MIC = 2/38 µg/mL (MIC at approximately 80% inhibition)
D Amoxicillin/clavulanic acid MIC = 16/8 µg/mL
E Minocycline MIC = 4 µg/mL
Figure 2. Examples of Growth and End-point Determination for Nocardia spp. in Microdilution
Panels
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Volume 31 M24-A2
6.6 Reporting of Results
For Nocardia spp., both an MIC value and an interpretation (ie, “susceptible,” “intermediate,” “resistant”)
should be reported for each drug tested, as indicated in Table 9. The results for Nocardia spp. should be
compared with those expected for the species being tested (see Appendix K). If the MIC value results
change in interpretation (S, I, R) from what would be expected, testing should be repeated and/or the
organism should be sent to a reference laboratory experienced in testing aerobic actinomycetes for
confirmation.
For R. equi, the breakpoints for Staphylococcus aureus should be used as provided in CLSI document
M100.48 These breakpoints should be considered tentative pending accumulation of more information.
For other genera of aerobic actinomycetes (other than R. equi), provide the MICs and refer to footnote “a”
in Table 9.
6.7 Quality Control
The overall performance of the susceptibility test system should be monitored by testing appropriate QC
strains weekly or each day the test is performed (if testing is done less often than weekly). The
recommended strains for QC are S. aureus ATCC® 29213 and P. aeruginosa ATCC® 27853. Escherichia
coli ATCC® 35218 may be used for amoxicillin-clavulanic acid. Acceptable ranges for these strains are
those listed in CLSI document M100.48
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Table 1. Antituberculous Drugs and Their Recommended Concentrations in

12,20,88-90
Middlebrook 7H10 and 7H11 Agar Medium
Concentrations (μg/mL)a
Primary Drugs 7H10 agar 7H11 agar
Isoniazidb 0.2 0.2

1.0 1.0
Rifampin 1.0 1.0
Ethambutolb 5.0 7.5c

10.0 NRd
Pyrazinamidee NRd NRd
Second-line Drugsf
Amikacing 4.0 -h
Capreomycin 10.0 10.0
Ethionamide 5.0 10.0
Kanamycing 5.0 6.0
Levofloxacini 1.0 -h
Moxifloxacinj 0.591 0.592
Ofloxacin 2.0i 2.0
p-Aminosalicylic
acid 2.0 8.0
Rifabutin 0.5 0.5
Streptomycin 2.0 and 10 2.0 and 10
NOTE: Information in boldface is new or modified since the previous edition.

a
The concentrations listed are interpretive criteria.
b
The higher concentration of EMB and INH should be tested as second-line drugs after resistance at the critical
concentration is detected.
c
Data supporting equivalency with 7H10 (5.0 μg/mL) are limited.
d
NR = not recommended.
e
For PZA testing, the manufacturer’s directions for the radiometric or nonradiometric methods should be followed (see Section
4.4).
f
Second-line drugs, at least one drug from each class, should be tested on isolates of M. tuberculosis that are resistant to
RMP or any two primary drugs.
g
Amikacin and kanamycin are aminoglycosides, but resistance to kanamycin may not indicate resistance to amikacin. It
may be desirable to test both aminoglycosides.
h
Breakpoints for establishing susceptibility have not been determined.
i
Testing levofloxacin at 1.0 μg/mL is equivalent to testing ofloxacin at 2.0 μg/mL.
j
The number of studies for moxifloxacin susceptibility testing by agar proportion method is limited; further studies are
required.
©
Licensed to: Clinical
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Volume 31
Table 2. Stock, Working, and Final Concentrations of Antituberculous Drug Solutions for Agar Proportion
Clinical
Working Volume of
Diagnostics
Concentration Working Final

and Laboratory
Stockb (μg/mL) from Stock Concentration (mL) Concentration

Antimicrobial Potencya Concentration Concentration for to Add to 200 mL of Drug in
ClinicalStandards
Agent (μg/mg) Solvent (μg/mL) 7H10 Agar 7H10 Agar 7H10 Agar
Amikacin Varies SDW 10 000 1000 0.8 4.0
with lot
Diagnostics
Capreomycin Varies SDW 10 000 1000 2.0 10.0

Institute. All rights reserved.
with lot
Ethambutol 1000 SDW 10 000 1000 1.0 5.0

2.0 10.0
Ethionamide 1000 DMSO or 10 000 1000 1.0 5.0

ethylene glycol
Isoniazid 1000 SDW 10 000 200 0.2 0.2

1.0 1.0
Kanamycin Varies SDW 10 000 1000 1.0 5.0

with lot
Levofloxacin 1000 ½ 10 000 200 1.0 1.0

volume of
water
then 0.1 mol/L
NaOH dropwise
to dissolve
Moxifloxacin Varies SDW 10 000 100 0.5 0.5

with lot
M24-A2
35
Licensed to: Clinical
Table 2. (Continued)
Number 5
Working Volume of
36Diagnostics Clinical Diagnostics
Concentration Working Final

Stockb (μg/mL) from Stock Concentration (mL) Concentration
Antimicrobial Potencya Concentration Concentration for to Add to 200 mL of Drug in
Agent (μg/mg) Solvent (μg/mL) 7H10 Agar 7H10 Agar 7H10 Agar
Ofloxacin 1000 ½ 10000 1000 0.4 2.0

volume of
water
then 0.1 mol/L
NaOH dropwise to
dissolve
p-Amino- 1000 SDW 10 000 1000 0.4 2.0

salicylic acid
Rifabutin Varies Methanol 10 000 100 1.0 0.5

with lot
Rifampin 1000 DMSOc 10 000c 1000 0.2 1.0

©
Clinical and Laboratory Standards Institute. All rights reserved.
Streptomycin Varies SDW 10 000 1000 0.4 2.0

with lot 2.0 10.0
Abbreviations: SDW, sterile distilled water; DMSO, dimethyl sulfoxide.

a
Calculate the weight based on potency if less than 100% (described in Section 4.2.1.2).
b
100 mg of active drug (depending on the potency) dissolved in 10 mL of sterile distilled water will yield a stock solution of 10 000 μg/mL. Sterilize stock solutions by
filtration through 0.22-μm pore membrane, dispense into sterile vials, and store up to 12 months at –70 °C. Thaw to room temperature and use without delay, discard
excess, and never refreeze. Lower concentration stock solutions and higher storage temperatures have also demonstrated satisfactory stability for 12 months (ie,
capreomycin 1000 μg/mL at –20 °C, EMB HCl 5000 μg/mL at 3 to 7 °C, INH 200 μg/mL at 3 to 7 °C, kanamycin 500 μg/mL at –20 °C, streptomycin 2000 μg/mL at 3
to 7 °C, and PAS 2000 μg/mL at 3 to 7 °C). (Reference: Griffith ME, Bodily HW. Stability of antimycobacterial drugs in susceptibility testing. Antimicrob Agents
Chemother. 1992;36:2398-2402.)
c
An alternative solvent for RMP is N,N-dimethylformamide at 50 000 μg/mL, followed by dilution to working concentrations in tepid SDW; however, these RMP
M24-A2
preparations have not been successfully stored. Another alternative solvent for RMP is methanol.
Volume 31 M24-A2
Table 3. Dilution of Sputum Concentrate for Inoculation of the Susceptibility Test

Medium for the Direct Susceptibility Testa
No. of AFB/field (at 200× to 400× magnification) observed using:
Fluorochrome Stain Dilutions
<25b Undiluted
25–50 Undiluted, 1:10
50–250 Undiluted, 1:100
>250 1:100, 1:1000
a
Inoculation of two sets of 7H10 or 7H11 plates is recommended, if sufficient inoculation material exists.
b
Increase inoculum to 0.2 mL for specimens with less than 5 AFB seen per field at 200× to 400× magnification.
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Number 5 M24-A2
Table 4. Antimycobacterial Agents and Interpretive Criteria for Mycobacterium

avium Complex
MIC (μg/mL) for category
a
Antimicrobial Agent Method (pH) S Ib R
Primary
Clarithromycinc Broth microdilutiond ≤8 16 ≥32

(pH 7.3–7.4)
Radiometric method ≤16 32 ≥64

(pH 6.8)e
Secondary
Moxifloxacinf Broth microdilutiond ≤1 2 ≥4

(pH 7.3–7.4)
Linezolidf Broth microdilutiond ≤8 16 ≥32

(pH 7.3–7.4)
NOTE: Information in boldface is new or modified since the previous edition.

a
Although EMB, RMP, rifabutin, streptomycin, and amikacin are useful clinically, breakpoints for
determining susceptibility and resistance have not been established.
b
For an isolate with a result of intermediate to a macrolide, including the following comment in the
report is suggested: An intermediate macrolide test result may indicate emerging resistance. The patient
should be monitored carefully, and subsequent MAC isolates should be tested for possible development
of macrolide resistance.
c
Clarithromycin is the class drug for macrolides, and is the only drug that need be tested.
d
CAMHB (pH 7.3–7.4) with 5% OADC.
e
If radiometric method pH 7.3 to 7.4 is used, breakpoints are ≤4 µg/mL (susceptible), 8–16 µg/mL
(intermediate), and ≥32 µg/mL (resistant).
f
These breakpoints are considered tentative and should be reported as such pending the
accumulation of further information.
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Volume 31 M24-A2
Table 5. Antimycobacterial Agents and Minimal Inhibitory Concentration (MIC)

Values Indicating Resistance for Testing M. kansasii
MIC Indicating
Resistance
Antimycobacterial Agent (μg/mL)
Primary agents
Clarithromycina >16
>1
Rifampinb
Secondary agents
Amikacin >32
Ciprofloxacinc >2
>4
Ethambutol
Isoniazid -d
Linezolid >16
Moxifloxacin >2
Rifabutin >2
Streptomycin -d
Trimethoprim-sulfamethoxazole >2/38
a
Clarithromycin is considered a primary drug in patients receiving the short-course and/or intermittent
therapeutic regimens consisting of RMP, EMB, and clarithromycin. In patients receiving the classic
regimen of RMP, EMB, and INH, clarithromycin is a secondary agent. Clarithromycin is the class
representative for the “newer” macrolides (clarithromycin, azithromycin, and roxithromycin).
b
For patients who are taking protease inhibitors, isolates susceptible to RMP can be assumed to be
susceptible to rifabutin.
c
Ciprofloxacin and levofloxacin are interchangeable, but both are less active in vitro than moxifloxacin.
d
INH and streptomycin may be useful clinically, but breakpoints to establish susceptibility and resistance
for NTM have not been established. Report the MIC value only, with no interpretation, for these drugs.
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Number 5 M24-A2
Table 6. Antimycobacterial Agents and Minimal Inhibitory Concentration (MIC)

Values Indicating Resistance for Susceptibility Testing of Mycobacterium marinum
(Routine Testing Is Not Recommended)
MIC Indicating
Resistance
Antimycobacterial Agent (μg/mL)
Amikacin >32
Ciprofloxacin >2
Clarithromycina >16
Doxycycline/minocycline >4
Ethambutol >4
Moxifloxacin >2
Rifabutin >2
Rifampin >1
Trimethoprim- >2/38
sulfamethoxazole
a
Class representative for macrolides (eg, clarithromycin, azithromycin, and roxithromycin).
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Volume 31 M24-A2
Table 7. Broth Microdilution Interpretive Criteria for Rapidly Growing

Mycobacteria
MIC (μg/mL) for category:
____________________________________________
Antimicrobial
Agent Susceptible Intermediate Resistant
a
Amikacin ≤16 32 ≥64
Cefoxitin ≤16 32–64 ≥128
Ciprofloxacinb ≤1 2 ≥4
Clarithromycinc ≤2 4 ≥8
Doxycyclined ≤1 2–4 ≥8
Imipeneme ≤4 8–16 ≥32
Linezolid ≤8 16 ≥32
Meropenem ≤4 8–16 ≥32
Moxifloxacin ≤1 2 ≥4
Trimethoprim- ≤2/38 – ≥4/76

sulfamethoxazolef
Tobramycing ≤2 4 ≥8
NOTE 1: These breakpoints, with the exception of cefoxitin, doxycycline, imipenem, linezolid, and moxifloxacin, are derived
from aerobic dilution interpretive criteria found in CLSI document M100.48 Cefoxitin interpretive criteria are derived from data
found in reference 55; linezolid interpretive criteria are derived from data found in references 67 and 68.
NOTE 2: Information in boldface is new or modified since the previous edition.

a
Isolates of M. abscessus with an MIC of ≥64 μg/mL should be retested. If the repeat result is ≥64 μg/mL, the MIC should be
reported with the comment: 1) The MIC is greater than expected for this species; and 2) if the drug is being considered for
therapy, the laboratory should be notified so the isolate can be sent to a reference laboratory for confirmation of resistance.
b
Ciprofloxacin and levofloxacin are interchangeable. Both are less active in vitro than the newer 8-methoxy fluoroquinolones.
c
Pulmonary infections caused by any of the rapidly growing mycobacteria should not be treated with clarithromycin alone. The
final reading for nonpigmented rapidly growing mycobacteria should be at 14 days to ensure detection of inducible macrolide
resistance, unless the isolate is resistant at an earlier reading.74,75 Clarithromycin is the class representative for the newer
macrolides (ie, azithromycin and roxithromycin).
d
Minocycline can be substituted.
e
These breakpoints are considered tentative and should be reported as such pending the accumulation of further
information. If the MIC is >8 μg/mL for the M. fortuitum group, the M. smegmatis group, and M. mucogenicum, the test
should be repeated with an incubation period of no more than three days. If the repeat result is >8 μg/mL, the MIC should be
reported with the comment: The MIC is greater than expected for this species; and if the drug is being considered for therapy,
the laboratory should be notified so the isolate can be sent to a reference laboratory for confirmation of resistance. Imipenem
results do not predict results for meropenem or ertapenem. Activity against rapidly growing mycobacteria is greater for
imipenem than for meropenem or ertapenem.
f
MIC is indicated by 80% inhibition of growth.
g
Tobramycin is used predominantly for treatment of M. chelonae infections. If the MIC to tobramycin is >4 μg/mL for an
isolate of M. chelonae, the test should be repeated. If the repeat result is >4 μg/mL, the MIC should be reported with the
comment: The MIC is greater than expected for this species; and if the drug is being considered for therapy, the laboratory
should be notified so the isolate can be sent to a reference laboratory for confirmation of resistance. Tobramycin should not be
used to treat infections with M. abscessus or the M. fortuitum group. The efficacy of inhaled tobramycin for treatment of M.
abscessus pulmonary infection is unknown.
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Number 5 M24-A2
Table 8. Quality Control Ranges of Minimal Inhibitory Concentrations (MICs)

(μg/mL) for Mycobacterium peregrinum ATCC® 700686, Staphylococcus aureus
ATCC® 29213, Pseudomonas aeruginosa ATCC® 27853, and Enterococcus faecalis
ATCC® 29212 When Testing Rapidly Growing Mycobacteria
MIC range
(μg/mL) for MIC range MIC range MIC range
Mycobacterium (μg/mL) for (μg/mL) for (μg/mL) for
peregrinum S. aureus P. aeruginosa E. faecalis
ATCC® 700686 ATCC® 29213 ATCC® 27853 ATCC® 29212
Antimicrobial (preferred (alternate (alternate (alternate
Agent organism) organism) organism) organism)
Amikacin ≤1–4 1–4 1–4 64–256
Cefoxitin 4–32 1–4 −a −a
Ciprofloxacin ≤0.12–0.5 0.12–0.5 0.25–1 0.25–2
Clarithromycin ≤0.06–0.5 0.12–0.5 −a −a
Doxycycline 0.12–0.5 0.12–0.5 −a 2–8
Imipenem 2–16 0.015–0.06 1–4 0.5–2
Linezolid 1–8 1–4 −a 1–4
Meropenemb 2–16 0.03–0.12 0.25−1 2−8
Minocycline 0.12–0.5 0.06–0.5 −a 1–4
Moxifloxacin ≤0.06–0.25 4–16 1–8 0.06–0.5
Tobramycin 2–8 0.12–1 0.25–1 8–32
Trimethoprim- ≤0.25/4.8–2/38 ≤0.5/9.5 8/152–32/608 ≤0.5/9.5

sulfamethoxazolec
a
A dash indicates no studies have been performed by current recommended methods.
b
Preliminary MIC studies (unpublished data) show meropenem has in vitro activity against some species of rapidly growing
mycobacteria including the M. fortuitum group, the M. mucogenicum group, and some pigmented rapidly growing species.
c
MIC is indicated by 80% inhibition of growth.
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Volume 31 M24-A2
Table 9. Broth Microdilution Interpretive Criteria for Nocardia and Other Aerobic
Actinomycetesa,b
Minimal Inhibitory Concentration (μg/mL) for category
Antimicrobial Agent Susceptible Intermediate Resistant
Primary
Amikacinc ≤8 – ≥16
Amoxicillin-clavulanic acid ≤8/4 16/8 ≥32/16
Ceftriaxone ≤8 16–32 ≥64
Ciprofloxacind ≤1 2 ≥4
Clarithromycine ≤2 4 ≥8
Imipenem ≤4 8 ≥16
Linezolidf ≤8 – –
Minocyclinec ≤1 2–4 ≥8
Moxifloxacin ≤1 2 ≥4
Trimethoprim-sulfamethoxazole ≤2/38 – ≥4/76
Tobramycin ≤4 8 ≥16
Secondary
Cefepime ≤8 16 ≥32
Cefotaxime ≤8 16–32 ≥64
Doxycycline ≤1 2–4 ≥8
a
Breakpoints in this table apply to Nocardia and can tentatively be used for other aerobic actinomycetes. Use of these
breakpoints for other aerobic actinomycetes is based on organism population distributions, clinical data, breakpoints used for
other organisms, and the experience of experts in the field. These breakpoints are considered tentative and should be reported
as such pending the accumulation of further information.
b
For R. equi, use the interpretive criteria as indicated in CLSI document M10048 for Staphylococcus aureus, with the inclusion
of results for vancomycin and rifampin. The interpretive categories should be considered and reported as tentative pending
accumulation of further information.
c
The following antimicrobial breakpoints differ from current recommendations from CLSI document M10048 for organisms that
grow aerobically: amikacin, minocycline, and moxifloxacin.
d
Ciprofloxacin and levofloxacin are interchangeable. Both are less active in vitro than the newer 8-methoxy fluoroquinolones.
e
Class representative for newer macrolides.
f
No Nocardia isolates with linezolid MIC values >8 μg/mL have been reported.93
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Number 5 M24-A2
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Nash KA, Brown-Elliott BA, Wallace RJ Jr. A novel gene, erm (41), confers inducible macrolide resistance to clinical isolates of
Mycobacterium abscessus but is absent from Mycobacterium chelonae. Antimicrob Agents Chemother. 2009;53:1367-1376.
77
Koh WJ, Jeon K, Lee NY, et al. Clinical significance of differentiation of Mycobacterium massiliense from Mycobacterium abscessus. Am
J Respir Crit Care Med. 2011;183(3):405-410.
78
Brown BA, Wallace RJ Jr. Broth microdilution MIC test for Nocardia spp. In: Isenberg HD, ed. Clinical Microbiology Procedures
Handbook. 2nd ed. Section 5: Antimicrobial Susceptibility Testing. Washington, DC: American Society of Microbiology; 1992:5.12.1.
79
Shapiro CL, Haft RF, Gantz NM, et al. Tsukamurella paurometabolum: A novel pathogen causing catheter-related bacteremia in patients
with cancer. Clin Infect Dis. 1992;14:200-203.
80
Steingrube VA, Brown BA, Gibson JL, et al. DNA amplification and restriction endonuclease analysis for differentiation of 12 species and
taxa of Nocardia, including recognition of four new taxa within the Nocardia asteroides complex. J Clin Microbiol. 1995;33:3096-3101.
81
Wallace RJ Jr, Brown BA, Blacklock Z, et al. New Nocardia taxon among isolates of Nocardia brasiliensis associated with invasive
disease. J Clin Microbiol. 1995;33:1528-1533.
82
Wallace RJ Jr, Brown BA, Tsukamura M, Brown JM, Onyi GO. Clinical and laboratory features of Nocardia nova. J Clin Microbiol.
1991;29:2407-2411.
83
Wallace RJ Jr, Steele LC, Sumter G, Smith JM. Antimicrobial susceptibility patterns of Nocardia asteroides. Antimicrob Agents
Chemother. 1988;32:1776-1779.
84
Wallace RJ Jr, Tsukamura M, Brown BA, et al. Cefotaxime-resistant Nocardia asteroides strains are isolates of the controversial species
Nocardia farcinica. J Clin Microbiol. 1990;28:2726-2732.
85
Wilson RW, Steingrube VA, Brown BA, et al. Recognition of a Nocardia transvalensis complex by resistance to aminoglycosides,
including amikacin, and PCR-restriction fragment length polymorphism analysis. J Clin Microbiol. 1997;35:2235-2242.
86
CLSI. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Tenth Edition. CLSI document M02-A10.
Wayne, PA: Clinical and Laboratory Standards Institute; 2009.
87
Wallace RJ Jr, Septimus EJ, Musher DM, Martin RR. Disk diffusion testing of Nocardia species. J Infect Dis. 1977;135(4):568-576.
88
Pfyffer GE, Bonato DA, Ebrahimzadeh A, et al. Multicenter laboratory validation of susceptibility testing of Mycobacterium tuberculosis
against classical second-line and newer antimicrobial drugs by using the radiometric BACTEC 460TB technique and the proportion method
with solid media. J Clin Microbiol. 1999;37:3179-3186.
89
Sanders CA, Nieda RR, Desmond EP. Validation of the use of Middlebrook 7H10 agar, BACTEC MGIT 960, and BACTEC 460 12B
media for testing the susceptibility of Mycobacterium tuberculosis to levofloxacin. J Clin Microbiol. 2004;42:5225-5228.
90
Piersimoni C, Lacchini C, Penati V, et al. Validation of the agar proportion and 2 liquid systems for testing the susceptibility of
Mycobacterium tuberculosis to moxifloxacin. Diag Microbiol and Infect Dis. 2007;57:283-287.
91
Angeby KA, Jureen P, Giske CG, et al. Wild-type MIC distribution of four fluoroquinolones active against Mycobacterium tuberculosis in
relation to current critical concentrations and available pharmacokinetic and pharmacodynamic data. J Antimicrob Chemother.
2010;65(5):946-952.
92
Somasundaram S, Paramasivan NC. Susceptibility of Mycobacterium tuberculosis strains to gatifloxacin and moxifloxacin by different
methods. Chemotherapy. 2006;52:190-195.
93
Brown-Elliott BA, Ward SC, Crist CJ, Mann LB, Wilson RW, Wallace RJ Jr. In vitro activities of linezolid against multiple Nocardia
species. Antimicrob Agents Chemother. 2001;45:1295-1297.
©
Number 5 M24-A2
Appendix A. Susceptibility Testing of Mycobacterium tuberculosis Complex to

Second-line Drugs Using Commercial Shorter Incubation Liquid Media Systemsa,b
BACTEC™ 460 BACTEC™ MGIT™ 960
Amikacinc 1.01,2 1.02,3,4

Capreomycin 1.251,2 2.52,3,4
Ethionamide 2.52,4 5.02,4,6
Kanamycinc 5.01 2.54
Levofloxacind 2.05 1.56
Linezolid 1.02 1.02
Moxifloxacind,e 0.57 0.257,8
Ofloxacind 2.01,2,4 2.02,4
Rifabutin 0.51,2 0.52
a
The concentrations (µg/mL) listed are interpretive criteria.
b
Most concentrations listed are based on multicenter studies. Systems are not cleared by the FDA for testing second-line anti-
TB drugs.
c
Amikacin and kanamycin are aminoglycosides, but resistance to kanamycin may not indicate resistance to amikacin. It may be
desirable to test both aminoglycosides.
d
Three fluoroquinolone drugs are listed: ofloxacin is the class representative for fluoroquinolones. Laboratories should test at
least one fluoroquinolone drug; it is not necessary to test all three fluoroquinolones listed.
e
Susceptibility to moxifloxacin (MOX) at 0.25 µg/mL by MGIT 960 predicts susceptibility to levofloxacin (LEV). Due to
higher potency of MOX than LEV and older quinolones, MOX may have clinical efficacy when it demonstrates resistance at
this concentration but susceptibility at a higher concentration. However, clinical data are not available at this time. It may be
advisable to implement reflex testing of MOX at 0.5, 1, 2, and 4 µg/mL for quantitative determination of the level of
resistance. Future clinical studies are warranted to fully investigate the clinical efficacy of MOX for strains with MIC between
0.5–4 µg/mL.
References for Appendix A

1
Pfyffer GE, Bonato DA, Ebrahimzadeh A, et al. Multicenter laboratory validation of susceptibility testing of Mycobacterium
tuberculosis against classical second-line and newer antimicrobial drugs by using the radiometric BACTEC 460 technique and the
proportion method with solid media. J Clin Microbiol. 1999;37:3179-3186.
2
Rüsch-Gerdes S, Pfyffer GE, Casal M, Chadwich M, Siddiqi S. Multicenter laboratory validation of the BACTEC MGIT 960
technique for testing susceptibilities of Mycobacterium tuberculosis to classical second-line drugs and newer antimicrobials. J Clin
Microbiol. 2006;44:688-692.
3
Krüüner A, Yates MD, Drobniewski FA. Evaluation of MGIT 960-based antimicrobial testing and determination of critical
concentrations of first- and second-line antimicrobial drugs with drug-resistant clinical strains of Mycobacterium tuberculosis. J
Clin Microbiol. 2006;44:811-818.
4
Rodrigues C, Jani J, Shenai S, Thakkar P, Siddiqi S, Mehta A. Drug susceptibility testing of Mycobacterium tuberculosis against
second-line drugs using the Bactec MGIT 960 system. Int J Tuberc Lung Dis. 2008;12:1449-1455.
5
Sanders CA, Nieda RR, Desmond EP. Validation of the use of Middlebrook 7H10 agar, BACTEC MGIT 960, and BACTEC 460
12B media for testing the susceptibility of Mycobacterium tuberculosis to levofloxacin. J Clin Microbiol. 2004;42:5225-5228.
6
Lin S-Y G, Desmond E, Bonato D, Gross W, Siddiqi S. Multicenter evaluation of BACTEC MGIT 960 system for second-line drug
susceptibility testing of Mycobacterium tuberculosis complex. J Clin Microbiol. 2009;47:3630-3634.
7
World Health Organization. 2008. Policy guidance on drug-susceptibility testing (DST) of second-line antituberculosis drugs.
WHO/HTM/TB/2008.392. Geneva, Switzerland: WHO.
8
Lin S-Y G, Desmond E, Schecter GF, Jost KC Jr, Ortiz, E. Moxifloxacin susceptibility testing of Mycobacterium tuberculosis with
MGIT 960 system. Abstract. 2010. ASM General Meeting at San Diego.
©
Volume 31 M24-A2
Appendix B. Suggested Approach to Mycobacterium tuberculosis Complex

Susceptibility Testing in Resource-Limited Countries1
Stepa,b Recommended drugs for testing
1 Isoniazid, rifampin
2 Ethambutol, streptomycin, pyrazinamide
3 Amikacin, kanamycin, capreomycin, fluoroquinolonec of choice
a
If resources allow, steps 1 and 2 may be combined when indicated based on epidemiological conditions
and/or treatment modalities (eg, standardized or individualized MDR-TB regimens involving first-line
drugs).
b
Steps 1 and 3 may be merged in settings where extremely drug-resistant TB is a concern to allow more
rapid detection of patients with such isolates.
c
Selection of the most appropriate fluoroquinolone for treatment should be based on surveillance data.
Only one fluoroquinolone needs to be tested due to extensive cross-resistance.
Reference for Appendix B

1
Policy guidance on drug-susceptibility testing (DST) of second-line antituberculosis drugs. Geneva,
Switzerland: World Health Organization; 2008.
http://www.who.int/tb/publications/2008/who_htm_tb_2008_392.pdf. Accessed February 21, 2011.
©
Number 5 M24-A2
Appendix C. Example Illustrating Drug Calculation for Meropenem Trihydrate

Example: meropenem trihydrate
Certificate of analysis data:
Assay purity (by HPLC): 99.8%

Measured water content (by Karl Fischer analysis): 12.1% (w/w)
Active fraction: 100% (supplied as the free acid and not a salt)
Potency calculation from above data:
Potency = (Assay purity) × (Active fraction) × (1 − Water Content)

Potency = (998) × (1.0) × (1 – 0.121)
Potency = 877 μg/mg or 87.7%
Use either of the following formulas to determine the amount of powder or diluent needed for a standard
solution:
Weight (mg) = Volume (mL) • Concentration (μg/mL) (C1)

Potency (μg/mg)
or
Volume (mL) = Weight (mg) • Potency (μg/mg)

Concentration (μg/mL) (C2)
Weigh the antimicrobial powder on an analytical balance that has been calibrated by approved reference
weights from a national metrology organization. If possible, weigh more than 100 mg of powder. It is
advisable to accurately weigh a portion of the antimicrobial agent in excess of that required and to
calculate the volume of diluent needed to obtain the final concentration desired as in formula C2 above.
Example: To prepare 100 mL of a stock solution containing 1280 μg/mL of antimicrobial agent with
antimicrobial powder that has a potency of 750 μg/mg, 170 to 200 mg of the antimicrobial powder should
be accurately weighed. If the actual weight is 182.6 mg, the volume of diluent needed is then as follows:
182.6 mg • 750 μg/mg

Volume (mL) = (Actual Weight) (Potency) = 107.0 mL
1280 μg/mL
(Desired Concentration) (C3)
Therefore, the 182.6 mg of antimicrobial powder is to be dissolved in 107.0 mL of diluent.
©
Volume 31 M24-A2
Appendix D. Preparation and Plating of 7H10 and 7H11 Agar Medium (See
Appendixes E and F)
(1) Prepare 7H10 or 7H11 agar medium from a dehydrated base as recommended by the manufacturer.
(2) After the agar is autoclaved, allow it to cool to 50 to 56 °C in a water bath before adding the
required oleic acid–albumin–dextrose–catalase (OADC) supplement (warmed to room temperature,
22 to 25 °C) and the appropriate antimycobacterial agent. This medium is usually prepared in lots
of 200 mL.
(3) Incorporate the antituberculous agent into the agar medium contained in plastic quadrant Petri
dishes. There are two methods for preparing the drug medium:
a. Drug is delivered to the medium via a disk.

b. Liquid drug is added to the medium.
Either a disk of the antituberculous agent is placed in the quadrant and the agar medium is poured
over it (method a), or each of the antituberculous agents is incorporated into the agar medium and
poured into one of three quadrants of a Petri dish (method b).
(4) Fill one of the quadrants of the Petri dish with 7H10 or 7H11 agar medium that does not contain
any antituberculous agents. This quadrant serves as a positive growth control. The other three
quadrants contain agents to be tested.
©
Number 5 M24-A2
Appendix E. Preparation of Media With Drug-Containing Disks

In preparing disk elution plates, it is necessary to perform the following steps:
(1) Dispense the disks into individual quadrants of sterile Petri dishes using aseptic technique.
(2) Prepare complete OADC-enriched 7H10 or 7H11 agar medium (180 mL of 7H10 agar plus 20 mL
OADC, as described in Appendix D). Dispense 5.0 mL of this medium into each quadrant,
overlaying the disk while keeping it approximately centered in the quadrant. Accurate delivery of
5.0 mL of medium into each quadrant is essential to ensure the correct final concentration of drug.
Allow the agar to solidify at room temperature.
(3) Before use or storage, plates can be thoroughly dried and the drug allowed to diffuse from the disk
by placing the plates with lids partially removed in a laminar flow hood for several hours and then
incubating the plates, with lids on, at room temperature overnight. Alternately, if a laminar flow
hood is unavailable, the plates can be incubated media side up, lids closed, at room temperature
overnight.
(4) The susceptibility plates may then be used immediately or stored in sealed plastic bags at 4 to 8 °C
for no more than 28 days. Protect all plates from light during storage.
(5) Test samples of each batch of plates for sterility by incubating at 35 °C for 48 hours; discard these
samples.
©
Volume 31 M24-A2
Appendix F. Preparing 7H10/H11 Agar With Liquid Drug

(1) Thaw a tube of the frozen stock of the drug and dilute with sterile distilled water to yield a working
concentration (usually 200 to 10 000 µg/mL).
(2) To achieve the desired final concentration (see Table 2), add the appropriate volume of working
solution as shown in Table 2 to 180 mL of sterile 7H10 or 7H11 agar tempered in a water bath at 50
to 56 °C. Tighten the cap and mix by inversion of the tube.
(3) Add OADC (20 mL for a 200-mL total volume) and mix again.
(4) Dispense 5-mL amounts into labeled quadrants of a series of sterile plastic Petri plates. One
quadrant of each plate should be reserved for agar medium without any added drug to serve as a
growth control.
(5) Dispense the media onto the plates as quickly as possible after mixing the component parts to
prevent partial solidification of the agar in the mixing container. Avoid production of bubbles. The
agar in each quadrant should be 3 to 4 mm deep. Allow the agar to solidify at room temperature.
(6) Before use or storage, plates should be thoroughly dried by placing the plates, with lids partially
removed, in a laminar flow hood for several hours until the surface is dry. Avoid overdrying
because this will alter the final drug concentration.
(7) After drying, use the plates immediately, or store them in sealed plastic bags at 4 to 8 °C for no
more than 28 days. Protect all plates from light during storage.
(8) Test one or more plates from each batch of plates for sterility by incubating at 35 °C for 48 hours;
discard these samples.
©
Number 5 M24-A2
Appendix G. McFarland 0.5 Barium Sulfate Turbidity Standard

To standardize the inoculum density, a BaSO4 turbidity standard is used and may be prepared as follows:
(1) A 0.5-mL aliquot of 0.048 mol/L BaCl2 (1.175% w/v BaCl2 • 2H2O) is added to 99.5 mL of 0.18
mol/L (0.36 N) H2SO4 (1% v/v) with constant stirring to maintain a suspension.
(2) The correct density of the turbidity standard should be verified by using a spectrophotometer with a
1-cm light path and matched cuvettes to determine the absorbance. The absorbance at 625 nm
should be 0.08 to 0.10 for the 0.5 McFarland standard.
(3) The barium sulfate suspension should be transferred in 4- to 6-mL aliquots into screw-cap tubes of
the same size as those used for growing or diluting the bacterial inoculum.
(4) These tubes should be tightly sealed and stored in the dark at room temperature.
(5) The barium sulfate turbidity standard should be vigorously agitated on a vortex mixer before each
use and inspected for a uniformly turbid appearance. If large particles appear, the standard should
be replaced.
NOTE: McFarland standards made from latex particle suspensions are available commercially.
Latex particle suspensions should be mixed by inverting gently, not on a vortex mixer, immediately
before use.
(6) Monthly replacement of the barium sulfate standards or verification of their densities should be
considered.
©
Volume 31 M24-A2
Appendix H. Determining Percentage of Resistance

The formula for determining percentage of resistance and some examples are:
% Resistant = number of colonies on drug-containing quadrant • 100

number of colonies on control quadrant
Example 1. Sample Calculation and Interpretation
Growth on
Antimycobacterial Agent/Concentration 10-2 10-4 % Resistant
Control 4+ 100 colonies —
INH (0.2 μg/mL) 2+ 10 colonies 10
RMP (1.0 μg/mL) 0 0 colonies 0
EMB (5.0 μg/mL) 0 0 colonies 0
% Resistant to INH = 10 colonies on drug-containing quadrant • 100 = 10%

100 colonies on control quadrant
Interpretation based on the calculation above: susceptible to RMP and EMB; 10% resistance to INH.
Example 2. Sample Calculation and Interpretation
Growth on
-2
Antimycobacterial Agent/Concentration 10 10-4 % Resistant
INH (0.2 μg/mL) 100 colonies 0 colonies 2
RMP (1.0 μg/mL) 0 0 colonies 0
EMB (5.0 μg/mL) 0 0 colonies 0
% Resistant in INH = 100 colonies on drug-containing quadrant • 100 = 2%

50 colonies on control quadrant, multiplied
by the dilution factor of 100, which is the
difference between 10-4 and 10-2
Interpretation based on the calculation above: susceptible to EMB and RMP; 2% resistance to INH.
Example 3. Sample Calculation and Interpretation of the Modified Indirect Proportion Methodd
Growth on:
Antimycobacterial Agent/Concentration 10-2 10-4 % Resistant
INH (0.2 µg/mL) 2+ NI* >1
INH (1.0 µg/mL) 0 NI 0
RMP (1.0 µg/mL) 0 NI 0
EMB (5.0 µg/mL) 25 colonies NI <1
* NI – not inoculated (see Section 4.2.3.2).
Interpretation: Resistant to INH (0.2 µg/mL) (because colony counts [2+] are greater than the 10-4 control
[50 colonies]); and susceptible to EMB (because colony counts [25 colonies] are less than the 10-4 control
[50 colonies]), INH (1.0 µg/mL), and RMP.
d
Hawkins JE. Drug susceptibility testing. In: Kubica GP, Wayne LG, eds. The Mycobacteria: a Sourcebook. New York, NY:
Marcel Dekker, Inc.; 1984. Reprinted with permission from Marcel Dekker, Inc.
©
Number 5 M24-A2
Appendix I. Drugs Available for Susceptibility Testing of Mycobacterium

tuberculosis Complex Using Commercial Shorter Incubation Liquid Media Systems
Cleared for Use by the US Food and Drug Administrationa and Their Equivalence
in the Agar Proportion Method
System and Concentration (μg/mL)

BACTEC™
™
BACTEC MGIT™ 7H10 Agar
Antimicrobial Agent 460 TB 960 VersaTREK® Equivalent
Isoniazid 0.1 0.1 0.1 0.2
Isoniazid 0.4 0.4 0.4 1.0
Rifampin 2.0 1.0 1.0 1.0
Ethambutol hydrochloride 2.5 5.0 5.0 5.0
Ethambutol hydrochloride 7.5 7.5b 8.0 10.0
Pyrazinamide 100 100 300 –c
b
Streptomycin 2.0 1.0 – 2.0
Streptomycin 6.0 4.0 –b 10.0
a
Cleared for use by the FDA at the time this document was completed.
b
Not available for sale in the United States.
c
Not available or not recommended.
BACTEC™ 460 TB and BACTEC™ MGIT™ 960 = BD (Sparks, Maryland, USA).
VersaTREK® = Trek Diagnostics Systems (Cleveland, Ohio, USA).
©
Volume 31 M24-A2
Appendix J. Agar Disk Elution Method for Mycobacterium haemophilum1,2

The agar disk elution method for testing Mycobacterium haemophilum described here is similar to the agar
proportion method for testing MTBC as described in Section 4.2.
(1) Add 0.5 mL of OADC enrichment to the bottom of each well in six-well culture plates. Round six-well
culture plates are recommended for optimal distribution of the drug throughout the media.
(2) Place the appropriate antimicrobial disks (as listed in the following table) to be tested in the bottom of the
well so that the OADC soaks into the disks. Add one hemin disk to each well. One well should be left without
antimicrobial disks (but with hemin disk) as a growth control.
Agar Disk Elution of M. haemophilum

Antimycobacterial Agent/ Number of Disks in 5 mL Final Concentration in
Concentration (μg) Media Well (μg/mL)
Amikacin 30 2 12
Ciprofloxacin 5 2 2
Clarithromycin 15 5 15
Doxycycline 30 1 6
Linezolid 30 1 6
Minocycline 30 1 6
Rifampin 5 1 1
Trimethoprim-sulfamethoxazole 23.75/1.25 2 0.5/9.5
(3) Allow plates to stand for 15 minutes at room temperature for the drug to elute from the disks into the
enrichment.
(4) Add 4.5 mL of melted and cooled or Middlebrook 7H10 agar (without OADC) to each well and gently swirl
to mix with a wooden applicator stick making sure that the disks are centered. Use a separate stick for each
well.
(5) Allow the agar to solidify with lids ajar for 30 to 60 minutes in a laminar flow hood so that any accumulated
moisture can evaporate. The susceptibility plates may then be used immediately or stored in sealed plastic
bags for up to one week.
(6) Test samples of batches of plates for sterility as described in the procedure for the agar proportion method for
testing MTBC (see Appendix E).
(7) Prepare a suspension of the test organism in broth or sterile water until it matches the 0.5 McFarland turbidity
standard. Dilute this suspension 1:100 with broth or sterile water and inoculate the wells with 100 μL of
this suspension.
(8) Incubate at 28 to 30 °C for 14 to 21 days.
(9) No growth in drug-containing well is susceptible, except for trimethoprim-sulfamethoxazole, for which 80%
inhibition of growth is susceptible.
(10) Any growth in drug-containing well is resistant.
References for Appendix J

1
McBride ME, Rudolph AH, Tschen JA, et al. Diagnostic and therapeutic considerations for cutaneous
Mycobacterium haemophilum infections. Arch Dermatol. 1991;127:276-277.
2
Vadney FS, Hawkins JE. Evaluation of a simple method for growing Mycobacterium haemophilum. J Clin
Microbiol. 1985;28:884-885.
©
Licensed to: Clinical58
Appendix K. Expected Antimicrobial Susceptibility Patterns of the Most Commonly Isolated Nocardia*
Number 5
Diagnostics Clinical Diagnostics
Species/Complex
Amox/Clav Ceftriax Imi Cipro Mino Linez Sulfa Amik Tobra Clar
N. cyriacigeorgica R S S R V S S S ND R
N. abscessus S S R R V S S S ND R
N. nova complex† R S S R V S S S ND S
N. transvalensis complex‡ S/R S V S V S S R R R
N. farcinica S R V S V S S S R R
N. brasiliensis S S/R R R S S S S S R
©
N. pseudobrasiliensis R S/R R S R S S S S S
N. otitidiscaviarum R R R S V S S S ND V
*
Abbreviations: Amik, amikacin; Amox/Clav, amoxicillin clavulanic acid; Ceftriax, ceftriaxone; Cipro, ciprofloxacin; Clar, clarithromycin; Imi,
imipenem; Linez, linezolid; Mino, minocycline; ND, no data; R, resistant; S, susceptible; Sulfa, sulfonamides including trimethoprim-sulfamethoxazole;
Tobra, tobramycin; V, variable.
†
Members of the N. nova complex include N. africana, N. elegans, N. kruczakiae, N. nova, and N. veterana.
‡
Members of the N. transvalensis complex include N. blacklockiae, N. transvalensis, and N. wallacei.
M24-A2
Volume 31 M24-A2
This page is intentionally left blank.
©
Number 5 M24-A2
The Quality Management System Approach

Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system approach in the
development of standards and guidelines, which facilitates project management; defines a document structure via a
template; and provides a process to identify needed documents. The approach is based on the model presented in
document HS01—A Quality Management System Model for Health Care. The quality management system 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
quality system essentials (QSEs) are as follows:
Documents and Records Equipment Information Management Process Improvement

Organization Purchasing and Inventory Occurrence Management Customer Service
Personnel Process Control Assessments―External and Facilities and Safety
Internal
M24-A2 addresses the quality system essentials (QSEs) indicated by an “X.” For a description of the other
documents listed in the grid, please refer to the Related CLSI Reference Materials section on the following page.
and Inventory
Improvement
Facilities and
Organization
Management
Management
Assessments
and Records
and Internal
Information
Occurrence
Documents
Purchasing
Equipment
—External
Personnel
Customer
Control
Process
Process
Service
Safety
X
EP12
I/LA18 I/LA18
M02
M07 M07
M22
M29
Adapted from CLSI document HS01—A Quality Management System Model for Health Care.
Path of Workflow
A path of workflow is the description of the necessary steps to deliver the particular product or service that the
organization or entity provides. For example, CLSI document GP26⎯Application of a Quality Management System
Model for Laboratory Services defines a clinical laboratory path of workflow that consists of three sequential
processes: preexamination, examination, and postexamination. All clinical laboratories follow these processes to
deliver the laboratory’s services, namely quality laboratory information.
M24-A2 addresses the clinical laboratory path of workflow steps indicated by an “X.” For a description of the other
documents listed in the grid, please refer to the Related CLSI Reference Materials section on the following page.
Preexamination Examination Postexamination

receipt/processing
Sample collection
Results reporting
Sample transport
Results review
and follow-up
and archiving
Interpretation
Examination
Examination
management
ordering
Sample
Sample
X X X X
I/LA18 I/LA18 I/LA18 I/LA18 I/LA18
M02 M02 M02 M02
M07 M07 M07 M07
M100 M100 M100
Adapted from CLSI document HS01—A Quality Management System Model for Health Care.
60 ©
Volume 31 M24-A2
Related CLSI Reference Materials∗

EP12-A2 User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline—Second Edition
(2008). This document provides a consistent approach for protocol design and data analysis when evaluating
qualitative diagnostic tests. Guidance is provided for both precision and method-comparison studies.
I/LA18-A2 Specifications for Immunological Testing for Infectious Diseases; Approved Guideline—Second Edition
(2001). This document addresses specimen collection, handling, and storage, as well as performance criteria
for the comparison of immunological test kits and specifications for reference materials.
M02-A10 Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Tenth
Edition (2009). This document contains the current Clinical and Laboratory Standards Institute-recommended
methods for disk susceptibility testing, criteria for quality control testing, and updated tables for interpretive
zone diameters.
M07-A8 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved
Standard—Eighth Edition (2009). This document addresses reference methods for the determination of
minimal inhibitory concentrations (MICs) of aerobic bacteria by broth macrodilution, broth microdilution, and
agar dilution.
M22-A3 Quality Control for Commercially Prepared Microbiological Culture Media; Approved Standard—
Third Edition (2004). This document contains quality assurance procedures for manufacturers and users of
prepared, ready-to-use microbiological culture media.
M29-A3 Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline—
Third Edition (2005). Based on US regulations, this document provides guidance on the risk of transmission
of infectious agents by aerosols, droplets, blood, and body substances in a laboratory setting; specific
precautions for preventing the laboratory transmission of microbial infection from laboratory instruments and
materials; and recommendations for the management of exposure to infectious agents.
M100-S21 Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational

Supplement (2011). This document provides updated tables for the Clinical and Laboratory Standards
Institute antimicrobial susceptibility testing standards M02-A10 and M07-A8.
∗
CLSI documents are continually reviewed and revised through the CLSI consensus process; therefore, readers should refer to
the most current editions.
©
Active Membership
(as of 1 January 2011)
Sustaining Members Health Canada Gen-Probe 48th Medical Group/MDSS RAF
Institut Pasteur de Côte d’Ivoire Genzyme Diagnostics Lakenheath (AE)
Abbott Institute of Tropical Medicine Dept. of GlaxoSmithKline 55th Medical Group/SGSAL (NE)
American Association for Clinical Clinical Sciences Greiner Bio-One Inc. 59th MDW/859th MDTS/MTL Wilford
Chemistry Laboratoire National de Santé Publique Habig Regulatory Consulting Hall Medical Center (TX)
AstraZeneca Pharmaceuticals MA Dept. of Public Health Laboratories HandyLab Inc. 81st MDSS/SGSAL (MS)
BD Malaria Research Training Center Himedia Labs Ltd Academisch Ziekenhuis-VUB UZ Brussel
Beckman Coulter, Inc. Meuhedet Central Lab HistoGenex N.V. (Belgium)
bioMérieux, Inc. Ministry of Health and Social Welfare - Icon Laboratories, Inc. ACL Laboratories (IL)
College of American Pathologists Tanzania Innovotech, Inc. ACL Laboratories (WI)
Diagnostica Stago Namibia Institute of Pathology Instrumentation Laboratory Adams County Hospital (OH)
GlaxoSmithKline National Cancer Institute, OBBR, NIH Integrated BioBank Adena Regional Medical Center Hospital
National Institute of Standards and National Center for Disease Control and IntelligentMDx, Inc. (OH)
Technology Public Health Intuity Medical Affiliated Laboratory, Inc. (ME)
Ortho-Clinical Diagnostics, Inc. National Health Laboratory Service C/O ITC Corp Akron Children’s Hospital (OH)
Roche Diagnostics, Inc. F&M Import & Export Services Japan Assn. of Clinical Reagents Industries Al Noor Hospital (United Arab Emirates)
National HIV & Retrovirology Lab Public Johnson & Johnson Pharmaceutical Al Rahba Hospital (United Arab Emirates)
Professional Members Health Agency of Canada Research & Develop., L.L.C. Alameda County Medical Center (CA)
National Institute of Standards and Kaiser Permanente Albany Medical Center Hospital (NY)
AAMI Technology KoreaBIO Albemarle Hospital (NC)
American Association for Clinical National Pathology Accreditation Advisory Krouwer Consulting Alberta Health Services (Alberta, Canada)
Chemistry Council Lab PMM All Children’s Hospital (FL)
American Association for Laboratory New York State Dept. of Health Laboratory Specialists, Inc. Allegiance Health (MI)
Accreditation NJ State Department of Health and Senior LifeLabs Alpena Regional Medical Center (MI)
American Association for Respiratory Care Services LifeScan, Inc. Alta Bates Summit Medical Center (CA)
American Medical Technologists Ontario Agency for Health Protection and Liofilchem SRL Alverno Clinical Laboratories, Inc. (IN)
American Society for Clinical Laboratory Promotion LipoScience, Inc. American University of Beirut Medical
Science Pennsylvania Dept. of Health Maine Standards Company, LLC Center (NJ)
American Society for Clinical Pathology SA Pathology Masimo Corp. Anand Diagnostic Laboratory (India)
American Society for Microbiology Saskatchewan Health-Provincial Mbio Diagnostics, Inc. Anne Arundel Medical Center (MD)
American Type Culture Collection Laboratory Medical Device Consultants, Inc. Antech Diagnostics (CA)
Association of Public Health Laboratories Scientific Institute of Public Health Merck & Company, Inc. Antelope Valley Hospital District CA)
Associazione Microbiologi Clinici Italiani State of Alabama Merial Limited APP - Unipath (CO)
(AMCLI) The Nathan S. Kline Institute Micromyx, LLC Appalachian Regional Healthcare System
British Society for Antimicrobial University of Iowa, Hygienic Lab Nanosphere, Inc. (NC)
Chemotherapy US Naval Medical Research Unit #3 Nihon Kohden Corporation Arkansas Children’s Hospital (AR)
Canadian Society for Medical Laboratory USAMC - AFRIMS Nissui Pharmaceutical Co., Ltd. Arkansas Dept of Health Public Health
Science Virginia Department of Agriculture - NJK & Associates, Inc. Laboratory (AR)
COLA Animal Health Laboratories NorDx - Scarborough Campus Arkansas Methodist Medical Center (AR)
College of American Pathologists NovaBiotics Arnot Ogden Medical Center Laboratory
College of Medical Laboratory Industry Members Novartis Institutes for Biomedical Research (NY)
Technologists of Ontario Optimer Pharmaceuticals, Inc. Artemis Health, Inc. (CA)
College of Physicians and Surgeons of 3M Medical Division Ortho-Clinical Diagnostics, Inc. Asan Medical Center (Korea, Republic Of)
Saskatchewan Abbott Ortho-McNeil, Inc. Asante Health System (OR)
Critical Path Institute Abbott Diabetes Care Paratek Pharmaceuticals, Inc. Asiri Group of Hospitals Ltd. (Sri Lanka)
ESCMID Abbott Point of Care Inc. PathCare Pathology Laboratory Aspen Valley Hospital (CO)
Family Health International Access Genetics PerkinElmer Genetics, Inc Aspirus Wausau Hospital (WI)
Hong Kong Accreditation Service AdvaMed Pfizer Animal Health Associated Regional & University
Innovation and Technology Commission Akonni Biosystems Pfizer Inc Pathologists (UT)
International Federation of Biomedical Ammirati Regulatory Consulting Pfizer Italia Srl Atlantic City Regional Medical Center (NJ)
Laboratory Science Anapharm, Inc. Phadia AB Atrium Medical Center (OH)
International Federation of Clinical AspenBio Pharma, Inc. Philips Healthcare Incubator Auburn Regional Medical Center WA)
Chemistry Astellas Pharma PPD Augusta Health (VA)
Italian Society of Clin. Biochem. and Clin. AstraZeneca Pharmaceuticals ProteoGenix, Inc. Aultman Hospital (OH)
Molec. Biology Astute Medical, Inc. QML Pathology Avera McKennan Hospital (SD)
JCCLS Ativa Medical Quality Regulatory Solutions AZ Sint-Jan (Belgium)
National Society for Histotechnology, Inc. Axis-Shield PoC AS Quotient Bioresearch Ltd. Azienda Ospedale Di Lecco (Italy)
Nova Scotia Association of Clinical Bayer Healthcare, LLC Diagnostic Radiometer America, Inc. Azienda Ospedaliera di Padova (Italy)
Laboratory Managers Division Roche Diagnostics GmbH Azienda Ospedaliera di Verona (Italy)
Ontario Medical Association Quality BD Roche Diagnostics, Inc. Azienda Policlinico Umberto I Di Roma
Management Program-Laboratory BD Biosciences - San Jose, CA Roche Molecular Systems (Italy)
Service BD Diagnostic Systems RPL Laboratory Solutions, Inc. Baptist Hospital for Women (TN)
RCPA Quality Assurance Programs Pty BD Vacutainer Systems DBA RPL Compliance Solutions Baptist Hospital of Miami (FL)
Limited Beaufort Advisors, LLC Sanofi Pasteur Baptist Memorial Hospital (MS)
SIMeL Beckman Coulter Cellular Analysis Sarstedt, Inc. Barnes-Jewish Hospital (MO)
Sociedad Española de Bioquímica Clínica y Business Center Seventh Sense Biosystems Baton Rouge General (LA)
Patología Molec. Beckman Coulter, Inc. Siemens Healthcare Diagnostics, Inc. Baxter Regional Medical Center (AR)
Sociedade Brasileira de Patologia Clínica Beth Goldstein Consultant Siemens Healthcare Diagnostics Products BayCare Health System (FL)
The Joint Commission Bio-Rad Laboratories, Inc. GmbH Baylor Health Care System (TX)
The Korean Society for Laboratory Bio-Rad Laboratories, Inc. Soloy Laboratory Consulting Services, Llc Bayou Pathology, APMC (LA)
Medicine Bio-Rad Laboratories, Inc. - France SomaLogic Baystate Medical Center (MA)
World Health Organization Bioanalyse, Ltd. Sphere Medical Holding Limited BC Biomedical Laboratories (BC, Canada)
BioDevelopment S.r.l. Streck Laboratories, Inc. Beloit Memorial Hospital (WI)
Government Members Biohit Oyj. Super Religare Laboratories Ltd. Berg Diagnostics (MA)
Biomedia Laboratories SDN BHD Sysmex America, Inc. Bio-Reference Laboratories (NJ)
Armed Forces Institute of Pathology bioMérieux, Inc. Sysmex Corporation - Japan Blanchard Valley Hospital (OH)
BC Centre for Disease Control Blaine Healthcare Associates, Inc. The Clinical Microbiology Institute Blanchfield Army Community Hospital
Centers for Disease Control and Prevention BRI Consultants Limited The Medicines Company (KY)
Centers for Disease Control and Prevention Calloway Laboratories TheraDoc Bon Secours Health Partners (VA)
- Namibia Canon U.S. Life Sciences, Inc. Therapeutic Monitoring Services, LLC Bonnyville Health Center (AB, Canada)
Centers for Disease Control and Prevention Cempra Pharmaceuticals, Inc. Theravance Inc. Boston Medical Center (MA)
- Tanzania Cepheid Thermo Fisher Scientific Boulder Community Hospital (CO)
Centers for Disease Control and Prevention Cerilliant Corp. Thermo Fisher Scientific, Oxoid Products Boyce & Bynum Pathology Labs (MO)
RETRO-CI CDC/PEPFAR Compliance Insight, Inc. Thermo Fisher Scientific, Remel Brant Community Healthcare System/Brant
Centers for Medicare & Medicaid Services Constitution Medical Inc Transasia Bio-Medicals Limited General Hospital (ON, Canada)
Centers for Medicare & Medicaid Controllab Trek Diagnostic Systems Bremerton Naval Hospital (WA)
Services/CLIA Program Copan Diagnostics Inc. Tulip Group Bridgeport Hospital (CT)
Chinese Committee for Clinical Laboratory Crescendo Bioscience Ventana Medical Systems Inc. Brooke Army Medical Center (TX)
Standards Cubist Pharmaceuticals, Inc. Veracyte, Inc. Broward General Medical Center (FL)
Chinese Medical Association (CMA) Dahl-Chase Pathology Associates PA Vivacta Cadham Provincial Laboratory-MB Health
Danish Institute for Food and Veterinary Diagnostica Stago Watson Pharmaceuticals (MB, Canada)
Research DX Assays Pte Ltd. Wellstat Diagnostics, LLC Calgary Laboratory Services (AB, Canada)
Department of Veterans Affairs Eiken Chemical Company, Ltd. XDx, Inc. California Department of Public Health
DFS/CLIA Certification Elanco Animal Health (CA)
Diagnostic Accreditation Program Elkin Simson Consulting Services Associate Active Members California Pacific Medical Center (CA)
FDA Center for Biologics Evaluation and Emika Consulting Cambridge Health Alliance (MA)
Research Enigma Diagnostics 31st Medical Group SGSL (AE) Canadian Science Center for Human and
FDA Center for Veterinary Medicine Eurofins Medinet 3rd Medical Group (AK) Animal Health (MB, Canada)
FDA Ctr. for Devices/Rad. Health Evidia Biosciences Inc.
Cape Fear Valley Medical Center DynaLIFE (AB, Canada) International Health Management Memorial Regional Hospital (FL)
Laboratory (NC) E. A. Conway Medical Center (LA) Associates, Inc. (IL) Mercy Franciscan Mt. Airy (OH)
Capital Coast Health (New Zealand) East Georgia Regional Medical Center Jackson County Memorial Hospital (OK) Mercy Hospital & Medical Center (IL)
Capital Health - Regional Laboratory (GA) Jackson Memorial Hospital (FL) Methodist Dallas Medical Center (TX)
Services (AB, Canada) Eastern Health - Health Sciences Centre Jackson Purchase Medical Center (KY) Methodist Hospital (PA)
Capital Health System Mercer Campus (NL, Canada) Jessa Ziekenhuis VZW (Belgium) Methodist Hospital (TX)
(NJ) Eastern Health Pathology (Victoria, John C. Lincoln Hospital - N.MT. (AZ) Methodist Hospital Park Nicollet Health
Carilion Labs Charlotte (NC) Australia) John F. Kennedy Medical Center (NJ) Services (MN)
Carl R. Darnall Army Medical Center Easton Hospital (PA) John H. Stroger, Jr. Hospital of Cook Methodist Hospital Pathology (NE)
Department of Pathology (TX) Edward Hospital (IL) County (IL) MetroHealth Medical Center (OH)
Carolinas Healthcare System (NC) Effingham Hospital (GA) John Muir Health (CA) Metropolitan Hospital Center (NY)
Carpermor S.A. de C.V. (Mexico) Elmhurst Hospital Center (NY) Johns Hopkins Medical Institutions (MD) Metropolitan Medical Laboratory, PLC
Catholic Health Initiatives (KY) Emory University Hospital (GA) Johns Hopkins University (MD) (IA)
Cedars-Sinai Medical Center (CA) Evangelical Community Hospital (PA) Johnson City Medical Center Hospital (TN) Miami Children’s Hospital (FL)
Central Baptist Hospital (KY) Evans Army Community Hospital (CO) JPS Health Network (TX) Mid Michigan Medical Center - Midland
Centre Hospitalier Anna-Laberge (Quebec, Exeter Hospital (NH) Kailos Genetics (AL) (MI)
Canada) Exosome Diagnostics, Inc. (MN) Kaiser Permanente (MD) Middelheim General Hospital (Belgium)
Chaleur Regional Hospital (NB, Canada) Federal Medical Center (MN) Kaiser Permanente Medical Care (CA) Middlesex Hospital (CT)
Chang Gung Memorial Hospital (Taiwan) Fletcher Allen Health Care (VT) Kenora-Rainy River Reg. Lab. Program Mike O’Callaghan Federal Hospital (NV)
Changhua Christian Hospital (Taiwan) Florida Hospital (FL) (ON, Canada) Minneapolis Medical Research Foundation
CHC Labs (FL) Fort Loudoun Medical Center (TN) King Abdulaziz Hospital, Al Ahsa Dept. of (MN)
Chester County Hospital (PA) Fort St. John General Hospital (BC, Pathology & Laboratory Medicine (Al- Mississippi Baptist Medical Center (MS)
Children’s Healthcare of Atlanta (GA) Canada) hasa, Saudi Arabia) Mississippi Public Health Lab (MS)
Childrens Hosp.- Kings Daughters (VA) Forum Health Northside Medical Center King Fahad National Guard Hospital Monongalia General Hospital (WV)
Children’s Hospital & Research Center At (OH) KAMC - NGHA (Saudi Arabia) Montreal General Hospital (Quebec,
Oakland (CA) Fox Chase Cancer Center (PA) King Fahad Specialist Hospital-Dammam, Canada)
Childrens Hospital Los Angeles (CA) Gamma-Dynacare Laboratories (ON, K.S.A. (Eastern Region, Saudi Arabia) Mt. Carmel Health System (OH)
Children’s Hospital Medical Center (OH) Canada) King Faisal Specialist Hospital (MD) Mt. Sinai Hospital (ON, Canada)
Children’s Hospital of Central California Garden City Hospital (MI) King Hussein Cancer Center (Jordan) Naples Community Hospital (FL)
(CA) Garfield Medical Center (CA) Kings County Hospital Center (NY) Nassau County Medical Center (NY)
Children’s Hospital of Philadelphia (PA) Gaston Memorial Hospital (NC) King’s Daughters Medical Center (KY) National B Virus Resource Laboratory
Childrens Hospital of Wisconsin (WI) Geisinger Medical Center (PA) Kingsbrook Jewish Medical Center (NY) (GA)
Children’s Hospitals and Clinics (MN) Genesis Healthcare System (OH) Kingston General Hospital (ON, Canada) National Cancer Center (Korea, Republic
Children’s Medical Center (OH) George Washington University Hospital Laboratória Médico Santa Luzia Ltda Of)
Children’s Medical Center (TX) (DC) (Brazil) National Institutes of Health, Clinical
Children’s Memorial Hospital (IL) Ghent University Hospital (Belgium) Labette Health (KS) Center (MD)
Christiana Care Health Services (DE) Golden Valley Memorial Hospital (MO) Laboratory Alliance of Central New York National Naval Medical Center (MD)
CHU - Saint Pierre (Belgium) Good Shepherd Medical Center (TX) (NY) National University Hospital Department of
CHU Sainte-Justine (Quebec, Canada) Grana S.A. (TX) Laboratory Corporation of America (NJ) Laboratory Medicine (Singapore)
CHUM Hopital Saint-Luc (Quebec, Grand River Hospital (ON, Canada) LabPlus Auckland District Health Board National University of Ireland, Galway
Canada) Grey Bruce Regional Health Center (ON, (New Zealand) (NUIG) (Ireland)
CHW-St. Mary’s Medical Center (CA) Canada) LAC/USC Medical Center (CA) Nationwide Children’s Hospital (OH)
City of Hope National Medical Center Gundersen Lutheran Medical Center (WI) Lafayette General Medical Center (LA) Nationwide Laboratory Services (FL)
(CA) Guthrie Clinic Laboratories (PA) Lakeland Regional Medical Center (FL) Naval Hospital Great Lakes (IL)
Clarian Health - Clarian Pathology Haga Teaching Hospital (Netherlands) Lancaster General Hospital (PA) Naval Medical Center Portsmouth (VA)
Laboratory (IN) Halton Healthcare Services (ON, Canada) Landstuhl Regional Medical Center Naval Medical Clinic Hawaii (HI)
Clearstone Central Laboratories (ON, Hamad Medical Corporation (Qatar) (Germany) NB Department of Health (NB, Canada)
Canada) Hamilton Regional Laboratory Medicine Langley Air Force Base (VA) New England Baptist Hospital (MA)
Cleveland Clinic (OH) Program - St. Joseph’s (ON, Canada) Laredo Medical Center (TX) New Lexington Clinic (KY)
Cleveland Heartlab, LLC (OH) Hanover General Hospital (PA) LeBonheur Children’s Medical Center New York City Department of Health and
Clinical Hospital Merkur (Croatia) Harford Memorial Hospital (MD) (TN) Mental Hygiene (NY)
Clinical Labs of Hawaii (HI) Harris Methodist Fort Worth (TX) Legacy Laboratory Services (OR) New York Presbyterian Hospital (NY)
Clinton Memorial Hospital (OH) Harrison Medical Center (WA) Lewis-Gale Medical Center (VA) New York University Medical Center (NY)
Colegio de Tecnólogos Médicos de Puerto Hartford Hospital (CT) L’Hôtel-Dieu de Québec (PQ, Canada) Newark Beth Israel Medical Center (NJ)
Rico (PR) Health Network Lab (PA) Licking Memorial Hospital (OH) Nor-Lea General Hospital (NM)
College of Physicians and Surgeons of Health Sciences Research Institute (Japan) LifeLabs Medical Laboratory Services North Carolina Baptist Hospital (NC)
Alberta (AB, Canada) Health Waikato (New Zealand) (BC, Canada) North District Hospital (China)
Collingwood General & Marine Hospital Heartland Health (MO) Loma Linda University Medical Center North Mississippi Medical Center (MS)
(ON, Canada) Heidelberg Army Hospital (AE) (LLUMC) (CA) North Shore Hospital Laboratory (New
Columbia Regional Hospital (MO) Helen Hayes Hospital (NY) Long Beach Memorial Medical Center- Zealand)
Commonwealth of Kentucky (KY) Henry Ford Hospital (MI) LBMMC (CA) North Shore-Long Island Jewish Health
Commonwealth of Virginia (DCLS) (VA) Henry M. Jackson Foundation for the Long Island Jewish Medical Center (NY) System Laboratories (NY)
Community Hospital (IN) Advancement of Military Medicine-MD Louisiana Office of Public Health Northridge Hospital Medical Center (CA)
Community Hospital of the Monterey (MD) Laboratory (LA) Northside Hospital (GA)
Peninsula (CA) Hi-Desert Medical Center (CA) Louisiana State University Medical Ctr. Northwest Texas Hospital (TX)
Community Medical Center (NJ) Highlands Medical Center (AL) (LA) Northwestern Memorial Hospital (IL)
Community Memorial Hospital (WI) Hoag Memorial Hospital Presbyterian (CA) Lourdes Hospital (KY) Norton Healthcare (KY)
Complexe Hospitalier de la Sagamie Hoboken University Medical Center (NJ) Lower Columbia Pathologists, P.S. (WA) Ocean County Medical Laboratories (NJ)
(Quebec, Canada) Holy Cross Hospital-Lab (MD) Lyndon B. Johnson General Hospital (TX) Ochsner Clinic Foundation (LA)
Consultants Laboratory of WI LLC (WI) Holy Name Hospital (NJ) Maccabi Medical Care and Health Fund Ohio State University Hospitals (OH)
Contra Costa Regional Medical Center Holy Spirit Hospital (PA) (Israel) Ohio Valley Medical Center (WV)
(CA) Hôpital du Haut-Richelieu (PQ, Canada) Madigan Army Medical Center (WA) Onze Lieve Vrouwziekenhuis (Belgium)
Cook Children’s Medical Center (TX) Hôpital du Sacré-Coeur de Montréal Mafraq Hospital (United Arab Emirates) Ordre Professionnel Des Technologistes
Cookeville Regional Medical Center (TN) (Quebec, Canada) Magnolia Regional Health Center (MS) Médicaux Du Québec (Quebec, Canada)
Cornwall Community Hospital (ON, Hôpital Maisonneuve-Rosemont (PQ, Main Line Clinical Laboratories, Inc. (PA) Orebro University Hospital (Sweden)
Canada) Canada) Makerere University Walter Reed Project Orlando Regional Healthcare System (FL)
Corona Regional Medical Center (CA) Hôpital Santa Cabrini Ospedale (PQ, Makerere University Medical School Ospedale Casa Sollievo Della Sofferenza -
Covance CLS (IN) Canada) (Uganda) IRCCS (Italy)
Creighton Medical Lab (NE) Horizon Health Network (NB, Canada) Marquette General Hospital (MI) Our Lady’s Hospital For Sick Children
Creighton University Medical Center (NE) Hospital of St. Raphael (CT) Martha Jefferson Hospital (VA) (Ireland)
Crozer-Chester Medical Center (PA) Hôtel-Dieu Grace Hospital Library (ON, Martin Luther King, Jr./Drew Medical Palmetto Baptist Medical Center (SC)
Cumberland Medical Center (TN) Canada) Center (CA) Pathlab (IA)
Darwin Library NT Territory Health Hôtel-Diu de Lévis (PQ, Canada) Martin Memorial Health Systems (FL) Pathology and Cytology Laboratories, Inc.
Services (NT, Australia) Hudson HealthCare Inc (NJ) Mary Hitchcock Memorial Hospital (NH) (KY)
David Grant Medical Center (CA) Hunter Area Pathology Service (Australia) Mary Imogene Bassett Hospital (NY) Pathology Associates Medical Lab. (WA)
Daviess Community Hospital (IN) Hunterdon Medical Center (NJ) Massachusetts General Hospital (MA) Peace River Regional Health Center (FL)
Deaconess Hospital Laboratory (IN) Imelda Hospital (Belgium) Mater Health Services - Pathology Penn State Hershey Medical Center (PA)
Dean Medical Center (WI) Indian River Memorial Hospital (FL) (Australia) Pennsylvania Hospital (PA)
DHHS NC State Lab of Public Health (NC) Inova Central Laboratory (VA) Maxwell Air Force Base (AL) Peterborough Regional Health Centre (ON,
DiagnoSearch Life Sciences Inc. Institut fur Stand. und Dok. im Med. Lab. Mayo Clinic (MN) Canada)
(Maharashtra, India) (Germany) MCG Health (GA) Piedmont Hospital (GA)
Diagnostic Laboratories (CA) Institut National de Santé Publique Du Meadows Regional Medical Center (GA) Pitt County Memorial Hospital (NC)
Diagnostic Laboratory Services, Inc. (HI) Quebec Centre de Doc. - INSPQ (PQ, Medecin Microbiologiste (Quebec, Canada) Potomac Hospital (VA)
Diagnostic Services of Manitoba (MB, Canada) Medical Center Hospital (TX) Prairie Lakes Hospital (SD)
Canada) Institute of Clinical Pathology and Medical Medical Center of Louisiana At NO- Presbyterian Hospital - Laboratory (NC)
Dimensions Healthcare System Prince Research (Australia) Charity (LA) Presbyterian/St. Luke’s Medical Center
George’s Hospital Center (MD) Institute of Laboratory Medicine Medical Centre Ljubljana (Slovenia) (CO)
DMC University Laboratories (MI) Landspitali Univ. Hospital (Iceland) Medical College of Virginia Hospital (VA) Princess Margaret Hospital (Hong Kong,
Drake Center (OH) Institute of Medical & Veterinary Science Medical University of South Carolina (SC) China)
Driscoll Children’s Hospital (TX) (SA, Australia) Memorial Hermann Healthcare System Providence Alaska Medical Center (AK)
DUHS Clinical Laboratories Franklin Site Integrated Regional Laboratories South (TX)
(NC) Florida (HCA) (VA) Memorial Hospital at Gulfport (MS)
Dynacare Laboratory (WI) Intermountain Health Care Lab Services Memorial Medical Center (IL)
Dynacare NW, Inc - Seattle (WA) (UT) Memorial Medical Center (PA)

Providence Health Care (BC, Canada) South Bend Medical Foundation (IN) The Charlotte Hungerford Hospital (CT) University of Medicine & Dentistry of New
Providence Health Services, Regional South County Hospital (RI) The Children’s Mercy Hospital (MO) Jersey (UMDNJ) (NJ)
Laboratory (OR) South Miami Hospital (FL) The Cooley Dickinson Hospital, Inc. (MA) University of Minnesota Medical Center-
Providence Medford Medical Center (OR) Southern Community Laboratories The Credit Valley Hospital (ON, Canada) Fairview (MN)
Provincial Health Services Authority (BC, (Canterbury, New Zealand) The Hospital for Sick Children (ON, University of Missouri Hospital (MO)
Canada) Southern Health Care Network (Australia) Canada) University of MS Medical Center (MS)
Provincial Laboratory for Public Health Spectra East (NJ) The Michener Inst. for Applied Health University of Pittsburgh Medical Center
(AB, Canada) St. Agnes Healthcare (MD) Sciences (ON, Canada) (PA)
Queen Elizabeth Hospital (China) St. Anthony Hospital (OK) The Naval Hospital of Jacksonville (FL) University of Texas Health Center (TX)
Queen Elizabeth Hospital (P.E.I., Canada) St. Barnabas Medical Center (NJ) The Nebraska Medical Center (NE) University of the Ryukyus (Japan)
Queensland Health Pathology Services St. Christopher’s Hospital for Children The Ottawa Hospital (ON, Canada) University of Virginia Medical Center
(Australia) (PA) The Permanente Medical Group (CA) (VA)
Queensway Carleton Hospital (ON, St. Elizabeth Community Hospital (CA) The Toledo Hospital (OH) UPMC Bedford Memorial (PA)
Canada) St. Eustache Hospital (Quebec, Canada) The University of Texas Medical Branch UZ-KUL Medical Center (Belgium)
Quest Diagnostics JV (OH) St. Francis Hospital (SC) (TX) VA (Asheville) Medical Center (NC)
Quest Diagnostics, Incorporated (CA) St. John Hospital and Medical Center (MI) Timmins and District Hospital (ON, VA (Bay Pines) Medical Center (FL)
Quintiles Laboratories, Ltd. (GA) St. John’s Mercy Medical Center (MO) Canada) VA (Central Texas) Veterans Health Care
Rady Children’s Hospital San Diego (CA) St. John’s Regional Health Center (MO) Tokyo Metro. Res. Lab of Public Health System (TX)
Ramathibodi Hospital (Thailand) St. Joseph Hospital (IN) (Japan) VA (Chillicothe) Medical Center (OH)
Redington-Fairview General Hospital (ME) St. Joseph Mercy Hospital (MI) Touro Infirmary (LA) VA (Cincinnati) Medical Center (OH)
Regions Hospital (MN) St. Joseph’s Medical Center (CA) Trident Medical Center (SC) VA (Dayton) Medical Center (OH)
Reid Hospital & Health Care Services (IN) St. Jude Children’s Research Hospital (TN) Trinity Medical Center (AL) VA (Decatur) Medical Center (GA)
Renown Regional Medical Center (NV) St. Luke’s Hospital (IA) Tripler Army Medical Center (HI) VA (Durham) Medical Center (NC)
Research Medical Center (MO) St. Luke’s Hospital (PA) Tuen Mun Hospital, Hospital Authority VA (Indianapolis) Medical Center (IN)
Response Genetics, Inc. (CA) St. Mary Medical Center (CA) (China) VA (Miami) Medical Center (FL)
Rex Healthcare (NC) St. Mary of Nazareth Hospital (IL) Tufts Medical Center Hospital (MA) VA (San Diego) Medical Center (CA)
River Valley Health-Chalmers Regional St. Mary’s Hospital (WI) Tulane Medical Center Hospital & Clinic VA (Tampa) Hospital (FL)
Hospital (NB, Canada) St. Tammany Parish Hospital (LA) (LA) Valley Health / Winchester Medical Center
Riverside County Regional Medical Center Stanford Hospital and Clinics (CA) Turku University Central Hospital (VA)
(CA) Stanton Territorial Health Authority (NT, (Finland) Vancouver Coastal Health Regional
Riverside Health System (VA) Canada) Twin Lakes Regional Medical Center (KY) Laboratory (BC, Canada)
Riverside Methodist Hospital (OH) State of Connecticut Department of Public UCI Medical Center (CA) Vancouver Island Health Authority (SI)
Riyadh Armed Forces Hospital, Health (CT) UCLA Medical Center Clinical (BC, Canada)
Sulaymainia (Saudi Arabia) State of Ohio/Corrections Medical Center Laboratories (CA) Vanderbilt University Medical Center (TN)
Riyadh National Hospital (Saudi Arabia) Laboratory (OH) UCSD Medical Center (CA) Via Christi Regional Medical Center (KS)
Rockford Memorial Hospital (IL) State of Washington Public Health Labs UCSF Medical Center China Basin (CA) Viracor-IBT Reference Laboratory (MO)
Royal Victoria Hospital (ON, Canada) (WA) UMC of El Paso - Laboratory (TX) Virginia Beach General Hospital (VA)
Sacred Heart Hospital (FL) Stillwater Medical Center (OK) UMC of Southern Nevada (NV) Virginia Regional Medical Center (MN)
Sacred Heart Hospital (WI) Stony Brook University Hospital (NY) UNC Hospitals (NC) Virtua - West Jersey Hospital (NJ)
Sahlgrenska Universitetssjukhuset Stormont-Vail Regional Medical Ctr. (KS) Union Clinical Laboratory (Taiwan) WakeMed (NC)
(Sweden) Strong Memorial Hospital (NY) United Christian Hospital (Kowloon, Hong Walter Reed Army Medical Center (DC)
Saint Francis Hospital & Medical Center Sudbury Regional Hospital (ON, Canada) Kong) Warren Hospital (NJ)
(CT) Sunnybrook Health Sciences Centre (ON, United Clinical Laboratories (IA) Washington Hospital Center (DC)
Saint Mary’s Regional Medical Center Canada) United States Air Force School of Waterbury Hospital (CT)
(NV) Sunrise Hospital and Medical Center (NV) Aerospace Medicine / PHE (TX) Waterford Regional Hospital (Ireland)
Saints Memorial Medical Center (MA) Sutter Roseville Medical Center (CA) Unity HealthCare (IA) Wayne Memorial Hospital (NC)
Salem Memorial District Hospital (MO) Swedish Edmonds Hospital (WA) Univ. of Pennsylvania Health System (PA) Weirton Medical Center (WV)
Sampson Regional Medical Center (NC) Swedish Medical Center (CO) Universitá Campus Bio-Medico di Roma West China Second University Hospital,
Samsung Medical Center (Korea, Republic Sydney South West Pathology Service (Italy) Sichuan University (China)
Of) Liverpool Hospital (NSW, Australia) Universitair Ziekenhuis Antwerpen West Jefferson Medical Center (LA)
San Francisco General Hospital-University T.J. Samson Community Hospital (KY) (Belgium) West Penn Allegheny Health System-
of California San Francisco (CA) Taichung Veterans General Hospital University College Hospital (Ireland) Allegheny General Hospital (PA)
Sanford USD Medical Center (SD) (Taiwan) University Hospital (GA) West Shore Medical Center (MI)
SARL Laboratoire Caron (France) Taipei Veterans General Hospital (Taiwan) University Hospital Center Sherbrooke West Valley Medical Center Laboratory
Scott & White Memorial Hospital (TX) Taiwan Society of Laboratory Medicine (CHUS) (Quebec, Canada) (ID)
Seattle Children’s Hospital/Children's (Taiwan) University of Alabama Hospital Laboratory Westchester Medical Center (NY)
Hospital and Regional Medical Center Tallaght Hospital (Ireland) (AL) Western Baptist Hospital (KY)
(WA) Tartu University Clinics (Estonia) University of Chicago Hospitals Western Healthcare Corporation (NL,
Sebastian River Medical Center (FL) Temple Univ. Hospital - Parkinson Pav. Laboratories (IL) Canada)
Seoul National University Hospital (Korea, (PA) University of Colorado Health Sciences Wheaton Franciscan Laboratories (WI)
Republic Of) Texas Children’s Hospital (TX) Center (CO) Wheeling Hospital (WV)
Seoul St. Mary’s Hospital (Korea, Republic Texas Department of State Health Services University of Colorado Hospital (CO) Whitehorse General Hospital (YT, Canada)
Of) (TX) University of Illinois Medical Center (IL) William Beaumont Army Medical Center
Sheik Kalifa Medical City (United Arab Texas Health Presbyterian Hospital Dallas University of Iowa Hospitals and Clinics (TX)
Emirates) (TX) (IA) William Beaumont Hospital (MI)
Shiel Medical Laboratory Inc. (NY) The AGA Khan University Hospital University of Kentucky Med. Ctr. (KY) William Osler Health Centre (ON, Canada)
Shore Memorial Hospital (NJ) (Pakistan) University of Maryland Medical System Winchester Hospital (MA)
Singapore General Hospital (Singapore) The Brooklyn Hospital Center (NY) (MD) Winn Army Community Hospital (GA)
Wishard Health Sciences (IN)
Womack Army Medical Center Department
of Pathology (NC)
York Hospital (PA)

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M24A2E

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March 2011

This standard provides protocols and related quality control

Licensed to: Clinical Diagnostics Clinical Diagnostics

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

Clinical and Laboratory Standards Institute

Licensed to: Clinical Diagnostics Clinical Diagnostics

Tentative Standard—Second Edition

Approved Standard—Second Edition

Area Committee on Microbiology

Mary Jane Ferraro, PhD, MPH Advisors Melvin P. Weinstein, MD

Subcommittee on Antimycobacterial Susceptibility Testing

Advisors (Continued) Beverly Metchock, DrPH, Staff

Grace Lin, MS Megan P. Larrisey, MA

Salman H. Siddiqi, PhD

Nancy G. Warren, PhD

Committee Membership........................................................................................................................ iii

Foreword .............................................................................................................................................. vii

Appendix A. Susceptibility Testing of Mycobacterium tuberculosis Complex to Second-line Drugs

Appendix B. Suggested Approach to Mycobacterium tuberculosis Complex Susceptibility Testing in

Appendix C. Example Illustrating Drug Calculation for Meropenem Trihydrate ................................ 50

Appendix E. Preparation of Media With Drug-Containing Disks ........................................................ 52

Appendix F. Preparing 7H10/H11 Agar With Liquid Drug ................................................................. 53

Appendix G. McFarland 0.5 Barium Sulfate Turbidity Standard ......................................................... 54

Appendix H. Determining Percentage of Resistance ............................................................................ 55

Appendix I. Drugs Available for Susceptibility Testing of Mycobacterium tuberculosis Complex

Appendix J. Agar Disk Elution Method for Mycobacterium haemophilum ......................................... 57

Appendix K. Expected Antimicrobial Susceptibility Patterns of the Most Commonly Isolated

The Quality Management System Approach ........................................................................................ 60

Related CLSI Reference Materials ....................................................................................................... 61

Antimycobacterial drugs, antituberculous drugs, drug susceptibility

Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic

The mycobacteriology laboratory presents a unique set of circumstances in terms of observance of

antimicrobial susceptibility test interpretive category – a classification based on an in vitro response of

borderline antimicrobial susceptibility test interpretive category – an interpretive category applicable

critical concentration – the “critical concentrations” of antituberculous drugs were adopted by

culture – 1) the intentional growing of microorganisms (such as bacteria or viruses) or tissues, in a

intermediate antimicrobial susceptibility test interpretive category – for minimal inhibitory

measurand – quantity intended to be measured (ISO/IEC Guide 99).8

Mycobacterium tuberculosis complex (MTBC) – includes the species M. tuberculosis, Mycobacterium

resistant antimicrobial susceptibility test interpretive category – 1) For critical concentration,

susceptible antimicrobial susceptibility test interpretive category – 1) for critical concentration, a

3.2 Abbreviations and Acronyms

4 Antimycobacterial Susceptibility Testing for Mycobacterium tuberculosis

4.2 Agar Proportion Method

This procedure is performed by inoculating equal quantities of several dilutions of a standardized

4.2.1 Antituberculous Agents

4.2.1.2 Weighing Antituberculous Drugs

4.2.1.3 Selection and Concentration of Antituberculous Drugs

4.2.1.4 Preparation and Storage of Stock and Working Solutions

4.2.1.4.1 Stock Solutions

Prepare stock solutions of antituberculous agents available as powders at concentrations of at least

4.2.2 Preparation of Drug Medium

4.2.2.1 Agar Medium

4.2.2.2 Agar Medium With Drug-Containing Disks

4.2.2.3 Agar Medium With Liquid Drug

4.2.3 Indirect Susceptibility Test

4.2.3.1 Preparation of the Inoculum

Perform the following steps: