Reference Method for Broth Dilution Antifungal ...Filamentous Fungi This standard includes the selection of antifungal agents, preparation of antifungal stock solutions and dilutions

M38 Reference Method for Broth Dilution Antifungal Susceptibility Testing for Filamentous Fungi

This standard includes the selection of antifungal agents, preparation of antifungal stock solutions and dilutions for testing implementation and interpretation of test procedures, and quality control requirements for susceptibility testing of filamentous fungi (moulds) that cause invasive and cutaneous fungal infections. A standard for global application developed through the Clinical and Laboratory Standards Institute consensus process.

“DRAFT DOCUMENT. This draft CLSI document is not to be reproduced or circulated for any purpose other than review and comment. It is not to be considered either final or published and may not be quoted or referenced. 28 December 2015.”

Clinical and Laboratory Standards Institute Setting the standard for quality in clinical laboratory testing around the world. The Clinical and Laboratory Standards Institute (CLSI) is a not-for-profit membership organization that brings together the varied perspectives and expertise of the worldwide laboratory community for the advancement of a common cause: to foster excellence in laboratory medicine by developing and implementing clinical laboratory standards and guidelines that help laboratories fulfill their responsibilities with efficiency, effectiveness, and global applicability. Consensus Process Consensus—the substantial agreement by materially affected, competent, and interested parties—is core to the development of all CLSI documents. It does not always connote unanimous agreement, but does mean that the participants in the development of a consensus document have considered and resolved all relevant objections and accept the resulting agreement. Commenting on Documents CLSI documents undergo periodic evaluation and modification to keep pace with advancements in technologies, procedures, methods, and protocols affecting the laboratory or health care. CLSI’s consensus process depends on experts who volunteer to serve as contributing authors and/or as participants in the reviewing and commenting process. At the end of each comment period, the committee that developed the document is obligated to review all comments, respond in writing to all substantive comments, and revise the draft document as appropriate. Comments on published CLSI documents are equally essential, and may be submitted by anyone, at any time, on any document. All comments are addressed according to the consensus process by a committee of experts. Appeals Process If it is believed that an objection has not been adequately addressed, the process for appeals is documented in the CLSI Standards Development Policies and Process document. All comments and responses submitted on draft and published documents are retained on file at CLSI and are available upon request. Get Involved—Volunteer! Do you use CLSI documents in your workplace? Do you see room for improvement? Would you like to get involved in the revision process? Or maybe you see a need to develop a new document for an emerging technology? CLSI wants to hear from you. We are always looking for volunteers. By donating your time and talents to improve the standards that affect your own work, you will play an active role in improving public health across the globe. For further information on committee participation or to submit comments, contact CLSI. Clinical and Laboratory Standards Institute 950 West Valley Road, Suite 2500 Wayne, PA 19087 USA P: 610.688.0100 F: 610.688.0700 www.clsi.org [email protected]

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Reference Method for Broth Dilution Antifungal Susceptibility Testing for Filamentous Fungi

Barbara D. Alexander, MD, MHS Gary W. Procop, MD Sharon K. Cullen, BS, RAC Philippe Dufresne, PhD (RMCCM) Jeff Fuller, PhD, FCCM, ABMM Mahmoud A. Ghannoum, MSc, PhD Kimberly E. Hanson, MD, MHS

Shawn R. Lockhart, PhD, D(ABMM) Luis Ostrosky-Zeichner, MD, FACP David S. Perlin, PhD Dee Shortridge, PhD Nathan P Wiederhold, PharmD Nancy L. Wengenack, PhD, D(ABMM)

Abstract Clinical and Laboratory Standards Institute document M38—Reference Method for Broth Dilution Antifungal Susceptibility Testing for Filamentous Fungi describes a method for testing the susceptibility to antifungal agents of filamentous fungi (moulds) and dermatophytes that cause invasive and cutaneous fungal infections. Selection of antifungal agents, preparation of antifungal stock solutions and dilutions for testing, implementation and interpretation of test procedures, and the purpose and implementation of quality control procedures are discussed. A careful examination of the manufacturer and the user responsibilities in quality control is also presented. In addition, a brief discussion regarding newly defined epidemiological cutoff values for certain Aspergillus spp and species complexes has been introduced. Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing for Filamentous Fungi. 3rd ed., DATE. CLSI standard M38. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2015.

The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in the CLSI catalog and posted on our website at www.clsi.org. If you or your organization is not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax: 610.688.0700; E-Mail: [email protected]; Website: www.clsi.org.


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Copyright ©2016 Clinical and Laboratory Standards Institute. Except as stated below, any reproduction of content from a CLSI copyrighted standard, guideline, companion product, or other material requires express written consent from CLSI. All rights reserved. Interested parties may send permission requests to [email protected]. CLSI hereby grants permission to each individual member or purchaser to make a single reproduction of this publication for use in its laboratory procedure manual at a single site. To request permission to use this publication in any other manner, e-mail [email protected]. Suggested Citation CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing for Filamentous Fungi. 3rd ed. CLSI standard, M38. Wayne, PA: Clinical and Laboratory Standards Institute; 2016.

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Committee Membership Consensus Council Subcommittee on Antifungal Susceptibility Tests Barbara D. Alexander, MD, MHS Chairholder Duke Univeristy Medical Center USA Gary W. Procop, MD Cleveland Clinic USA Sharon K. Cullen, BS, RAC Siemens Healthcare Diagnostics Inc. USA Philippe Dufresne, PhD (RMCCM) Institut national de santé, publique du Québec Canada Jeff Fuller, PhD, FCCM, ABMM Alberta Health Services Capital Health Canada Mahmoud A. Ghannoum, MSc, PhD Case Western Reserve University USA

Kimberly E. Hanson, MD, MHS University of Utah and ARUP Laboratories USA Shawn R. Lockhart, PhD, D(ABMM) Centers for Disease Control and Prevention USA Luis Ostrosky-Zeichner, MD, FACP University of Texas Medical School at Houston USA David S. Perlin, PhD New Jersey Medical Center-UMDNJ USA Dee Shortridge, PhD bioMérieux, Inc. USA Nancy L. Wengenack, PhD, D(ABMM) Mayo Clinic USA

Nathan P. Wiederhold, PharmD University of Texas, Health Science Center USA Staff Clinical and Laboratory Standards Institute USA Marcy Hackenbrack, MCM, M(ASCP) Project Manager Megan L. Tertel, MA Editorial Manager Joanne P. Christopher, MA Editor Alexander B. Phucas Editor

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Acknowledgment CLSI, the Consensus Committee on Microbiology, and the Subcommittee on Antifungal Susceptibility Tests gratefully acknowledge the following volunteers for their important contributions to the development of this document: Ana Espinel-Ingroff, MS, PhD Virginia Commonwealth University USA

Phillippe Dufresne, PhD (RMCCM) Institut national de santé publique du Québec, Laboratoire de santé publique du Québec Canada

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Contents

Abstract ............................................................................................................................................. i

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

Foreword ....................................................................................................................................... viii

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

Overview of Changes ..................................................................................................................... viii

Chapter 1: Introduction ..................................................................................................................... 1

1.1 Scope ....................................................................................................................... 1 1.2 Background ............................................................................................................. 1 1.3 Standard Precautions ................................................................................................ 2 1.4 Terminology ............................................................................................................ 2

Chapter 2: Indications for Performing Susceptibility Tests ................................................................ 5

Chapter 3: Antifungal Broth Dilution Susceptibility Testing Process for Filamentous Fungi .............. 6

3.1 Selecting Antifungal Agents for Routine Testing and Reporting ............................... 7 3.2 Preparation of Antifungal Agents ............................................................................. 7 3.3 Testing Procedures ................................................................................................. 10 3.4 Reading Minimal Inhibitory Concentration and Minimal Effective Concentration Results 15 3.5 Interpreting Results ................................................................................................ 16

Chapter 4: Quality System Essentials – Quality Control .................................................................. 20

4.1 Purpose .................................................................................................................. 20 4.2 Quality Control Responsibilities ............................................................................. 20 4.3 Selecting Reference Strains .................................................................................... 21 4.4 Storing Reference Strains ....................................................................................... 21 4.5 Routine Use of Reference Strains ........................................................................... 22 4.6 Controlling Media Batches and Plasticware Lots .................................................... 23 4.7 Quality Control Frequency ..................................................................................... 23 4.8 Other Control Procedures ....................................................................................... 24

Chapter 5: Conclusion ..................................................................................................................... 25

Chapter 6: Supplemental Information .............................................................................................. 25

References ...................................................................................................................................... 26

Additional References ..................................................................................................................... 29

Appendix A. Scheme for Preparing Dilution Series of Antifungal Agents to Be Used in Broth Dilution Susceptibility Tests for Nondermatophytes ...................................................................................... 30

Appendix B. Scheme for Preparing Dilution Series of Water-Insoluble Antifungal Agents to Be Used in Broth Dilution Susceptibility Tests of Dermatophytes ..................................................................... 31

Appendix C. Scheme for Preparing Dilutions of Water-Soluble Antifungal Agents to Be Used in Broth Dilution Susceptibility Tests ........................................................................................................... 32

Appendix D. Composition of RPMI-1640 Medium ......................................................................... 33

Appendix E. Preparing RPMI-1640 Medium ................................................................................... 34

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Abbreviation: .................................................................................................................................. 34

Appendix F. Oatmeal Agar.............................................................................................................. 35

Appendix G. Minimal Effective Concentrations of Caspofungin and Anidulafungin ........................ 36

Appendix G. McFarland 0.5 Barium Sulfate Turbidity Standard ...................................................... 38

The Quality Management System Approach .................................................................................... 40

Related CLSI Reference Materials .................................................................................................. 41

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Foreword With the increased incidence of systemic fungal infections and the growing number of antifungal agents, laboratory methods guiding the selection of antifungal therapy have gained greater attention. The CLSI Consensus Committee on Microbiology formed the Subcommittee on Antifungal Susceptibility Tests and concluded that a reproducible reference procedure for the antifungal susceptibility testing of filamentous fungi (moulds) would be useful. Accordingly, several studies were conducted to refine the methodology for performing nondermatophyte mould susceptibility testing.1,2,3,4,5 The resulting consensus method was published in 2002 as CLSI document M38 and the revised version in 2008.1,2 In CLSI document M38-A2, supplemental material (QC data for mould isolates as well as echinocandin testing guidelines) was incorporated and guidelines for testing dermatophyte moulds were provided.5,6,7,8,9,10 Since then, and in the absence of breakpoints for mould testing, epidemiological cutoff values (ECVs) for distinguishing wild-type and non-wild-type (isolates with intrinsic or acquired known resistance mechanisms or gene mutations) have been defined for some species and species complexes of Aspergillus (see CLSI documents M57 and M57S) 11,12,13,14,15 Although a discussion regarding breakpoints was introduced in the previous edition of M38, breakpoints have not been established by CLSI for mould testing, ECV data and recommendations for the development and their are incorporated into the newly developed CLSI documents M5716 and M57S17. QC data for testing mould isolates, as well as other testing guidelines, have been omitted from this edition of M38 and incorporated into the newly created CLSI document M38/M5118 Informational Supplement which combines supplemental material for both CLSI documents M38 [M38] and M51[M51].18 NOTE: Current fungal taxonomy is under revision. Many genera have both a teleomorph (sexual state) and an anamorph (asexual state) name. In this document, the traditional anamorph names are used to provide continuity to both past procedures and associated documents such as the current edition of M27-S. Overview of Changes General Document formatting and organization was revised to reflect the new CLSI quality system essential/path-of-workflow document template. References to the information supplement were updated to reflect the new informational supplement for broth dilution and disk diffusion testing of moulds. Added references to epidemiological cut off values and CLSI documents M57 and the current edition of M57-S. Fungal taxonomy has undergone major changes in recent years with the dual (asexual and sexual stages) nomenclature having been abolished and the constant reclassification/renaming of species that results from improved molecular characterization of fungi. Species names listed in M38 were revised to reflect the most recent taxonomic changes based on classification according to DNA barcoding and internal transcribed spacer ribosomal DNA sequencing. For more information regarding updated reclassification and fungal species refer to publically available information. For clarity, a list which includes previous and current taxonomical names for species used in the different collaborative studies (including QC studies) where each non-dermatophyte species was evaluated for testing guidelines of M38 methodology is also provided in Appendix A.

Commented [MH1]: Sheryl, add references to M57 and M57S

Commented [MH2]: Sheryl, change to 2 separate references. One for M38 and one for M51

Commented [MH3]: This is text added to M27 by Shawn Lockhart. Perhaps this can be edited for moulds.

Commented [MH4R3]: Is this note being retained?

Commented [MH5]: Hoog et al, 2015

Commented [MH6]: Atlas of Clinical Fungi Guarro et al, 2015 www.mycobank.org

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Subchapter 1.4.2 (Definitions) Revised the interpretive breakpoint definitions and criteria for consistency with other CLSI antimicrobial susceptibility testing documents. Added definitions for: • Non-wild-type • Wild-type Chapter 2 (Indication for Performing Susceptibility Testing) New indications added for testing of filamentous fungi with a discussion of environmental resistance of A. fumigatus. Chapter 3 (Testing Procedure) An antifungal testing process flow-chart was added. List of relevant drug concentrations to be tested for echinocandins expanded. Procedural text converted to step/action tables.

Established guide for reading and interpretation of results for filamentous fungi including dermatophytes Modified text on reading results in Subchapter 3.4 to include new information on echinocandins and isavuconazole antifungal agents and minimal inhibitory concentration and minimal effective concentration comparison. Appendixes (Original Tables) The table providing the list of solvents has been updated, deleted from M38, and moved to the newly combined supplement, M38/M51S. The table providing the recommended minimal inhibitory concentration or minimal effective concentration limits for QC and reference strains for broth dilution procedures have been deleted from M38 and moved to the combined supplement, M38/M51S. A correction was made to Appendix E (Composition of RPMI-1640 Medium), to provide a single riboflavin concentration (0.0002 g /L) as found in CLSI document M27. Dilution schemes for dermatophytes and non-dermatophytes isolates were harmonized with those of CLSi document M27 and revised to encompas the full dilution ranges recommended A table which lists previous and current taxonomical names of species found in the different collaborative studies is included in Appendix E. NOTE: The findings and conclusions in this document are those of the authors and do not necessarily represent the views of the organizations they represent. Key Words Antifungal agent, broth microdilution, dermatophytes, epidemiologic cutoff value, filamentous fungi, moulds, non-wild type, susceptibility testing, wild type

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Reference Method for Broth Dilution Antifungal Susceptibility Testing for Filamentous Fungi

Chapter 1: Introduction

This chapter includes: • Document scope and applicable exclusions

• Background information pertinent to the document content

• Standard precautions information

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

definitions

• Terms and definitions used in the document

• Abbreviations and acronyms used in the document

1.1 Scope This document describes the standard broth microdilution testing method for antifungal susceptibility testing of filamentous fungi (moulds) that cause invasive and/or cutaneous fungal infections.1,2,3,4,5,6,7,8,9,10

This document also covers testing conditions including inoculum preparation and inoculum size, incubation time and temperature, medium formulation, and criteria end-point determination.1,2,3,4,7,8,9 QC reference ranges and specific epidemiologic cut off values (ECVs) are summarized in the current editions of CLSI documents M38/M51 and M57S16.5,8,9,10,11,12,13,14,15 The intended audience includes diagnostic laboratory personnel, clinicians, and microbiologists who routinely perform antifungal susceptibility testing, use interpretive criteria to select suitable antifungal therapy, and those involved emerging resistance surveillance. The reference method is also useful for establishing ECVs and developing and validating alternate commercial methods for determining antifungal susceptibility of filamentous fungi. Therefore, the document is also of interest for both diagnostic and pharmaceutical companies and their regulatory counterparts. This method has not been evaluated in studies of the yeast or mould forms of dimorphic fungi, such as Blastomyces dermatitidis, Coccidioides immitis/posadasii, Histoplasma capsulatum, or Talaromyces marneffei (Penicillium marneffei), and only has been evaluated for the mycelial form of Sporothrix schenckii spp. complex. This method also has not been used in studies of dermatophytes with the echinocandins nor nondermatophyte moulds with ciclopirox, griseofulvin, or terbinafine. 1.2 Background The method described in this document is intended for testing common filamentous fungi including the dermatophytes, Trichophyton spp., Microsporum spp, and Epidermophyton spp.,9 more prevalent species of Aspergillus spp. and species complex, Fusarium spp. and species complex, Rhizopus spp., and other Mucorales (Zygomycetes),19 Scedosporium spp.(Pseudallescheria/Lomentospora),20 and the mycelial form of species included in the S. schenckii spp. complex, and dematiaceous (phaeoid/black) moulds.21 Caution

Commented [MH7]: Sheryl, add reference to M57S

Commented [MH8]: Sheryl this should be a xref to reference 17

Commented [MH9]: Moulds don't have interpretive criteria (onlyh ECVs) so should this still be here?

Commented [MH10]: Sheryl, add references Espinel-Ingroff A, Dawson K, Pfaller M, et al. Comparative and collaborative evaluation of standardization of antifungal susceptibility testing for filamentous fungi. Antimicrob Agents Chemother.1995;39:314-319.

Espinel-Ingroff A, Kerkering TM. Spectrophotometric method of inoculum preparation for the in vitro susceptibility testing of filamentous fungi. J Clin Microbiol. 1991;29:393-394.

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should be used when interpreting the minimal inhibitory concentration (MIC) and minimal effective concentration (MEC) results for any mould/drug combination. This document discusses a “reference” method developed through a consensus process to facilitate agreement among laboratories in measuring mould susceptibility to antifungal agents. NOTE: The relationship between in vitro vs in vivo data has only been evaluated in animal models.11 An important use of a reference method is to provide a standard basis from which other methods can be developed and which also will result in interlaboratory agreement within specified ranges. Such methods might have particular advantages, such as ease of performance, economy, or production of more rapid results; therefore, their development could be highly desirable. To the extent that any method produces concordant results with this reference method, it would be considered to be in conformity with M38. 1.3 Standard Precautions Because it is often impossible to know what isolates or specimens might be infectious, all patient and laboratory specimens are treated as infectious and handled according to “standard precautions.” Standard precautions are guidelines that combine the major features of “universal precautions and body substance isolation” practices. Standard precautions cover the transmission of all known infectious agents and thus are more comprehensive than universal precautions, which are intended to apply only to transmission of bloodborne pathogens. The Centers for Disease Control and Prevention addresses this topic in published guidelines that address the daily operations of diagnostic medicine in humans and animals while encouraging a culture of safety in the laboratory.22 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 M29.23 1.4 Terminology 1.4.1 A Note on Terminology CLSI, as a global leader in standardization, is firmly committed to achieving global harmonization wherever possible. Harmonization is a process of recognizing, understanding, and explaining differences while taking steps to achieve worldwide uniformity. CLSI recognizes that medical conventions in the global metrological community have evolved differently in the United States, Europe, and elsewhere; that these differences are reflected in CLSI, ISO, and European Committee for Standardization (CEN) documents; and that legally required use of terms, regional usage, and different consensus timelines are all important considerations in the harmonization process. In light of this, CLSI’s consensus process focuses on harmonization of terms to facilitate the global application of standards and guidelines. NOTE: Although the breakpoints described below are not yet available for any antifungal agent/mould species at time of this document's publication, ECVs based solely on in vitro data have been established (see CLSI documents M57S and M38/51S). Currently, breakpoints only apply to some Candida spp. and antifungal agent combinations. Definitions for interpretive categories are provided below in the event that breakpoints are published for filamentous fungi in the future. 1.4.2 Definitions antimicrobial susceptibility test interpretive category – a classification based on an in vitro response of an organism to an antimicrobial agent (including antifungal agents) at levels corresponding to blood or tissue levels attainable with usually prescribed doses of that agent.

Commented [MH11]: Add references

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1) susceptible (S) – a category that implies that isolates are inhibited by the usually achievable concentrations of antimicrobial agent when the dosage recommended to treat the site of infection is used.

2) intermediate (I) – a category that includes isolates with antimicrobial agent MICs that approach

usually attainable blood and tissue levels and for which response rates may be lower than for susceptible isolates; NOTE: The intermediate category implies clinical efficacy in body sites where the drugs are physiologically concentrated or when a higher than normal dosage of a drug can be used. This category also includes a buffer zone, which should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations, especially for drugs with narrow pharmacotoxicity margins.

3) resistant (R) – a category that implies that isolates are not inhibited by the usually achievable

concentrations of the agent with normal dosage schedules and/or that demonstrate MICs that fall in the range in which specific microbial resistance mechanisms (eg, b-lactamases) are likely, and clinical efficacy of the agent against the isolate has not been reliably shown in treatment studies.

antibiogram – overall profile of antimicrobial susceptibility results of a microbial species to a battery of antimicrobial agents. breakpoint//interpretive criteria – minimal inhibitory concentration (MIC) value used to indicate susceptible, intermediate, and resistant, as defined above.

For example, Candida albicans with an MIC of 0.5 µg/mL for fluconazole with the interpretive criteria of:

MIC (µg/mL) Susceptible £ 4 Intermediate 8–16

Resistant ³ 32 The isolate would be categorized as susceptible (treatable): “Susceptible breakpoint” is 24 µg/mL. “Resistant breakpoint” is 8 µg/mL.

epidemiological cut-off value (ECV) – the highest susceptibility endpoint of the wild-type minimal inhibitory population that detects emergence of in vitro resistance or that separates wild-type from non-wild-type isolates. These values are based solely on in vitro data and do not predict the clinical outcome to therapy as breakpoints do. epidemiological cut-off value (ECV) categorization criteria – minimal inhibitory concentration (MIC)/minimal effective concentration values to indicate wild-type or non-wild-type.

For example, A .fumigatus with a MIC of 0.5 (µg/mL) for itraconazole with the ECV criteria of:

MIC (µg/mL) Wild-type £ 1

Non-wild-type > 1

The isolate would be categorized as wild-type (an MIC below the ECV of 1 µg/mL), which however does not indicate that the isolate susceptible.

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minimal effective concentration (MEC) – the lowest concentration of an antimicrobial agent that leads to the growth of small, rounded, compact hyphal forms as compared to the hyphal growth seen in the growth control well; NOTE: This terminology is currently used only with respect to testing of the echinocandin antifungal agents (see Appendix A). minimal inhibitory concentration (MIC) – the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in an agar or broth dilution susceptibility test. non-wild type (NWT) – isolates with mechanisms of resistance and reduced susceptibility of the agent being evaluated. quality assurance (QA) – part of quality management focused on providing confidence that quality requirements will be fulfilled; NOTE: The practice that encompasses all procedures and activities directed toward ensuring that a specified quality of product is achieved and maintained. In the testing environment, this includes monitoring all the raw materials, supplies, instruments, procedures, sample collection/transport/storage/processing, recordkeeping, calibrating and maintaining of equipment, quality control, proficiency testing, training of personnel, and all else involved in the production of the data reported. quality control (QC) – the operational techniques and activities that are used to fulfill requirements for quality; NOTE 1: In health care testing, the set of procedures designed to monitor the test method and the results to ensure test system performance; NOTE 2: QC includes testing control materials, charting the results and analyzing them to identify sources of error, and evaluating and documenting any remedial action taken as a result of this analysis. wild-type (WT) – isolates without mechanisms of resistance. 1.4.3 Abbreviations and Acronyms ATCC© American Type Culture Collection CFU colony forming units DMSO dimethyl sulfoxide MIC minimal inhibitory concentration MEC minimal effective concentration NWT non-wild type RPMI Royal Park Memorial Institute (culture medium) QA quality assurance QC quality control WT wild type

Commented [MH12]: ISO 9000

Commented [MH13]: ISO 9000

Commented [MH14]: I will manage this list as per CLSI style

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Chapter 2: Indications for Performing Susceptibility Tests

This chapter includes: • References to information on the general indications for performing antifungal susceptibility tests

Although the general indications for performing susceptibility tests are provided in CLSI document M2724 (broth medium and all issues related to the antifungal agents), specific testing conditions for filamentous fungi that are different (eg, inoculum preparation and quantification, minimal inhibitory concentration (MIC) and minimal effective concentration (MEC) readings and especially results interpretation) from those used for yeasts are described in the following chapters. Antifungal resistance (other than intrinsic resistance of some species) is generally not widespread for prevalent fungal species and is frequently observed after exposure to antifungals and/or prolonged antifungal treatment. However, during the last ten years, an increase in triazole resistance among the complexes of Aspergillus, especially A. fumigatus spp. complex has been reported especially in Europe. Although it began as a single triazole resistance (mostly itraconazole), it is currently evident that cross resistance can be common in aspergillosis, especially in areas where environmental resistance can be as high as >10%. Resistant isolates have been recovered from patients not exposed to any azole with resistance being associated with the presence of gene mutations (cyp51A-gene). This is a concern given that the recommended therapy for aspergillosis is voriconazole and other triazoles and mutant isolates have been associated with treatment failure and high MICs. Although breakpoints are not currently available for any mould species as they have been for more prevalent Candida spp. and triazoles (only fluconazole and voriconazole) and echinocandins, it has been possible to calculate ECVs for Aspergillus spp. and triazoles (isavuconazole included).

Commented [MH15]: Verweij et al, 2015

Commented [MH16]: Sheryl, move references from Subchapter 3.5.5.1 Howard SJ, Cerar D, Anderson MJ, et al. Frequency and evolution of azole resistance in Aspergillus fumigatus associated with treatment failure. Emerg Infect Dis. 2009;15:1068-1076. ➝van der Linden JWM, Jansen RR, Bresters D, et al. Azole-resistant central nervous system aspergillosis. Clin Infect. 2009;48:1111-1113. ➝Mavridou E, Bruggemann RJM, Melchers WJG, et al. Efficacy of posaconazole against three clinical Aspergillus fumigatus isolates with mutations in the cyp51A gene. Antimicrob Agents Chemother. 2010;54:860–65. ➝Klaassen CHW, de Valk HA, Curfs-Breuker IM, et al. Novel mixed-format real-time PCR assay to detect mutations conferring resistance to triazoles in Aspergillus fumigatus and prevalence of multi-triazole resistance among clinical isolates in the Netherlands. J Antimicrob. Chemother. 2010;65:901–905. ➝Van der Linden JW, Snelders E, Kampinga GA. Clinical implications of azole resistance in Aspergillus fumigatus, the Netherlands, 2007–2009. Emerg Infec Dis. 2011;17:1846–1854. ➝Camps SMT, van der Linden JWM, Li Y, et al. Rapid Induction of multiple resistance mechanisms in Aspergillus fumigatus during azole therapy: a case study and review of the literature. Antimicrob Agents Chemother. 2012;56:1-16. ➝Krishnan-Natesan S, Chandrasekar PH, Alangaden GJ, et al. Molecular characterization of cyp51A and cyp51B genes coding for P450 14alpha-lanost eroldemethylases A (CYP51Ap) and B (CYP51Bp) from voriconazole-resistant laboratory isolates of Aspergillus flavus. Int J Antimicrob Agents. 2008;32:519–524. ➝Lepak AJ, Marchillo K, Vanhecker J, et al. Posaconazole pharmacodynamic target determination against wild-type and Cyp51 mutant isolates of Aspergillus fumigatus in an in vivo model of invasive pulmonary aspergillosis. Antimicrob Agents Chemother. 2013;57:579–585. ➝Lepak AJ, Marchillo K, VanHecker J, et al. Isavuconazole (BAL4815) Pharmacodynamic target determination in an in vivo murine model of invasive pulmonary aspergillosis against wild-Type and cyp51 mutant isolates of Aspergillus fumigatus. Antimicrob Agents Chemother. 2013;57:6284.

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Chapter 3: Antifungal Broth Dilution Susceptibility Testing Process for Filamentous Fungi

This chapter includes: • Overview of antifungal broth dilution susceptibility testing process • Selection of antifungal agents for routine testing and reporting • Preparing the antifungal agents • Testing procedures • Reading and interpreting the results

Figure 1 provides an overview of the antifungal susceptibility testing process.

Figure 1. Antifungal Broth Dilution Susceptibility Testing Process

Start

Need for susceptibility testing is identified

Antifungal agents for testing are selected

Isolates are prepared for testing

The inoculum is prepared

Tubes/microdilution trays are inoculated

Tubes/microdilution trays are incubated

Susceptibility results are read

Susceptibility results are interpreted

Susceptibility reporting process

End

Antifungal agents are prepared for testing

Chapter 2

Subchapter 3.1

Subchapter 3.2

Subchapter 3.3.5

Subchapter 3.4

Subchapter 3.5

Subchapter 3.6

Subchapter 3.7

Subchapter 3.3.5

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3.1 Selecting Antifungal Agents for Routine Testing and Reporting Routine fungal susceptibility testing is not recommended. At each institution, the decision to perform testing on any individual fungal isolate is best made as a collaborative effort of infectious disease practitioners, the pharmacy and therapeutics committee, diagnostic microbiology laboratory personnel, and the infection control committee. 3.1.1 Generic Names

To minimize confusion, all antifungal agents should be referred to by official nonproprietary (ie, generic) names. 3.1.2 Number of Agents Tested

To make routine susceptibility tests relevant and practical, the number of antimicrobial agents tested should be limited. Although this is not an immediate issue for antifungal agents, the same principals apply. 3.1.3 Guidelines for Selective Testing Testing may be warranted under certain selected circumstances including: • As part of periodic batch surveys that establish antibiograms for collections of pathogenic isolates

obtained within an institution • To aid in the management of invasive and cutaneous infections due to filamentous fungi when the utility

of the azole antifungal agents is uncertain. Interpretive breakpoints are not available for any species of filamentous fungi vs any antifungal agent, and the clinical relevance of testing any mould-drug combination remains uncertain. However, ECVs have been defined for the most common Aspergillus spp. vs amphotericin B, caspofungin, and the triazoles, including isavuconazole (see CLSI documents M5716 and M57S17).11,12,13,14 Specimens for culture and other procedures should be obtained before initiating antifungal therapy. 3.2 Preparation of Antifungal Agents 3.2.1 Source Antifungal standards or reference powders can be obtained commercially or directly from the drug manufacturer. NOTE: Pharmacy stock or other clinical preparations should not be used. Acceptable powders bear a label that states the drug’s generic name, its assay potency (usually expressed in micrograms [µg] or International Units per mg of powder), and its expiration date. The powders must be stored as recommended by the manufacturers, or at −20 °C or below (never in a self-defrosting freezer), in a desiccator and preferably in a vacuum. When the desiccator is removed from the freezer, it should come to room temperature before opening (to avoid condensation of water). 3.2.2 Weighing Antifungal Powders Assay all antifungal agents for standard units of activity. The assay units can differ widely from the actual weight of the powder and often differ within a drug production lot. Therefore, a laboratory needs to standardize its antifungal solutions based on assays of the lots of antifungal powders used. Some agents may be affected by using treated plasticware. One such agent is caspofungin. Because of this, glass-ware or untreated plastic-ware should be used for weighing powders, preparing drug dilutions, and for the preparation of caspofungin microtiter trays. Commented [MH17]:

Weiderhold reference to be added when published

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In some cases, a certificate of analysis with values for each of these components may be provided with antimicrobial agent powders. In this case, an overall value for potency may not be provided, but can be calculated from high performance liquid chromatography purity, water content, and, when applicable, the active fraction for drugs supplied as a salt (see CLSI document M0725 for examples). Either of the following formulas should be used to determine the amount of powder or diluent needed for a standard solution:

(1)

or

(2)

The antifungal powder should be weighed on an analytical balance that has been calibrated by approved reference weights from a national metrology organization. Usually, it is advisable to accurately weigh a portion of the antifungal agent in excess of the required amount and to calculate the diluent needed to obtain the concentration desired. Example: To prepare 100 mL of a stock solution containing 1280 µg of antifungal agent per mL with antifungal powder that has a potency of 750 µg/mg, the first formula should be used to establish the weight of powder needed:

(3)

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

(4)

Therefore, 182.6 mg of the antifungal powder should be dissolved in 107.0 mL of diluent. 3.2.3 Preparing Stock Solutions Antifungal stock solutions should be prepared at concentrations of at least 1280 µg/mL or 10 times the highest concentration tested, whichever is greater. However, some antifungal agents of limited solubility need lower concentrations. In all cases, information provided by the drug manufacturer should be considered when determining solubility.

g/mg)(Potency g/mL)(ion Concentrat • (mL) Volume

= (mg)Weight µ

µ

g/mL)(ion Concentratg/mg)(Potency (mg)Weight

= (mL) Volumeµ

µ•

mg 170.7 =

(Potency)йg/mg 750

Conc.) (Desiredg/mL 1280

Vol.)(Target

mL100

= (mg)

Weightμ

•

mL 107.0 =

ion)Concentrat (Desiredйg/mL 1280

(Potency)йg/mg 750

ight)(Powder We

mg 182.6

= (mL)

Volume•

Commented [MH18]: Sheryl, please check the formatting/remove the space in "concentration"

Commented [MH19]: Sheryl, check formatting

Commented [MH20]: Sheryl, delete the space in We ight and Concentrat ion

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3.2.3.1 Using Solvents Information on antifungal compound solubility should be included with the drug. Drugs should be dissolved at concentrations at least 100 times higher than the highest desired test concentration. The two most commonly used solvents are analytical grade dimethyl sulfoxide (DMSO) and water. Ethyl alcohol, polyethylene glycol, and carboxy methyl cellulose have also been used as solvents (see CLSI document M38/M51S). Because DMSO or other solvents should be used for all agents, a series of dilutions at 100 times the final concentration should be prepared from the antifungal stock solution in the same solvent. Each intermediate solution should then be further diluted to final strength in the test medium (see Appendixes A and B). This procedure avoids dilution artifacts resulting from precipitation of compounds with low solubility in aqueous media. For example, to prepare for a broth microdilution test series containing a drug dissolved in DMSO, for which the highest desired test concentration is 16 µg/mL:

Step Action Comment(s) 1. Weigh 4.8 mg (assuming 100% potency)

of the antifungal powder

2. Dissolve the antifungal powder in 3.0 mL DMSO

This will provide a stock solution at 1600 µg/mL.

3. Prepare further dilutions of this stock solution in DMSO.

See Appendixes A and B

4. Dilute the solutions in DMSO 1:50 in the test medium

See Subchapter 3.2. When inoculated, a further 2× (twofold) dilution will result (see Subchapter 3.4), reducing the final solvent concentration to 1% DMSO at each drug concentration, as well as in the growth control (drug-free medium) used in the test as a solvent control.

The example above assumes 100% potency of the antifungal powder. If the potency is different, the calculations in Subchapter 3.2.2 should be applied. 3.2.3.2 Filtration Normally, stock solutions do not support contaminating microorganisms and can be assumed to be sterile. If additional sterility assurance is desired, solutions should be passed through a membrane filter. Paper, asbestos, or sintered glass filters, which may adsorb appreciable amounts of certain antifungal agents should not be used. Whenever filtration is used, it is important to document the absence of adsorption by results of appropriate assay procedures.

3.2.3.3 Storage Small volumes of the sterile stock solutions should be dispensed into sterile polypropylene or polyethylene vials, carefully sealed, and stored (preferably at −60 °C or below, but never at a temperature greater than −20 °C). Vials should be removed as needed and used the same day. Any unused drug should be discarded at the end of the day. Most antifungal agent stock solutions can be stored at −60 °C or below for six months or more without significant loss of activity.26 In all cases, the instructions provided by the drug manufacturer need to be considered and supersede these general recommendations provided here. Any significant antifungal agent deterioration/loss of potency must be determined. This should be reflected in the susceptibility testing results using QC strains or reference strains such as those listed in CLSI document M38/M51S18.

Commented [MH21]: Add reference to M38/M51S

Commented [MH22]: There is no refrence to Appendix C (Scheme for preparing Antifungal Agents in Water). A reference needs to be added.

Commented [MH23]: Please verify that this is the correct subchapter to reference

Commented [PD24]: Glass could be an option for caspofungin to avoid plastic, binding.

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3.2.4 Number of Concentrations Tested 3.2.4.1 Nondermatophyte Moulds The concentrations tested should encompass the expected results for the available QC strains. Based on previous studies for nondermatophyte moulds, the following drug concentration ranges may be relevant: • Amphotericin B, itraconazole, ketoconazole, posaconazole, voriconazole, and isavuconazole: 0.0313

to 16 µg/mL

• Flucytosine and fluconazole: 0.125 to 64 µg/mL • Echinocandins (anidulafungin, caspofungin, and micafungin): 0.008 to 4 µg/mL or lower, especially

for anidulafungin and micafungin. 3.2.4.2 Dermatophyte Moulds Suitable drug concentration ranges for testing dermatophytes are: • Ciclopirox: 0.06 to 32 µg/mL • Fluconazole and griseofulvin: 0.125 to 64 µg/mL • Itraconazole, voriconazole, and terbinafine: 0.001 to 0.5 µg/mL • Posaconazole: 0.004 to 8 µg/mL.9 3.3 Testing Procedures 3.3.1 Growth Medium The completely synthetic medium, Royal Park Memorial Institute (culture medium) (RPMI)-1640 (with glutamine, without bicarbonate, and with phenol red as a pH indicator), is satisfactory for testing filamentous fungi, and has been used to develop the reference method.1,2,9

The formula for this medium is provided in Appendix D and directions for medium preparation from powder is provided in Appendix E. 3.3.2 Buffers

Media should be buffered to a pH of 7.0 ± 0.1 at 25 °C using 3-[N-morpholino] propanesulfonic acid (MOPS) at a final concentration of 0.165 mol/L at pH 7.0. The pH of each medium batch should be checked with a pH meter immediately after preparation. The pH should be between 6.9 and 7.1 at room temperature (25 °C). MIC performance characteristics of each batch of broth should be evaluated using a standard set of QC organisms (see Subchapter 4.3 and CLSI document M38/M5118). 3.3.3 Preparing Diluted Antifungal Agents The conditions for preparing and storing diluted antifungal agents are as follows: • Sterile, plastic test tubes should be used to prepare drug dilutions and sterile, disposable, multiwell

microdilution plates (96 U-shaped wells, untreated polystyrene) should be used to perform the tests (see Subchapter 3.2.2 for potential interlaboratory variability caused by plastic-ware and the use of untreated plastic ware to avoid this problem).

Commented [PD25]: Provided as a table in M27. Should we harmonize. Ranges are also slightly different for

Commented [MH26]: 5/12: add reference to Nathan Weiderholds article if published before M38 or add note regarding observations by members of the SC

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• A growth control well containing RPMI-1640 medium without antifungal agents (but with nonaqueous solvent) should be used for each organism tested.

The total volume of each dilution prepared depends on the number of tests performed. Because 0.1 mL of each antifungal drug dilution is used for each test, 1.0 mL is adequate for approximately eight tests (one microdilution tray), allowing for pipetting. A single pipette should be used for measuring all diluents and then for adding the stock antifungal solution to the first tube. A separate pipette for each remaining dilution in that set should be used. Because there will be a 1:2 dilution of the drugs when combined with the inoculum, the working antifungal solutions are two times more concentrated than the final concentrations. An initial dilution series of the agent should be prepared at 100 times the final strength in an appropriate solvent (see Subchapter 3.2.3.1). Then, each of these nonaqueous solutions should be diluted 1:50 in RPMI-1640 medium. For example, if a dilution series with final concentrations in the range 16 µg/mL to 0.0313 µg/mL is desired, a concentration series from 1600 to 3.13 µg/mL should have been prepared first in DMSO (see Subchapter 3.2.3.1 and Appendix A or B ). To prepare 5-mL volumes of diluted antifungal agent (sufficient for 45 tests): 1. Pipette 4.9-mL volumes of RPMI-1640 medium into each of 10 sterile test tubes. 2. Using a single pipette, add 0.1 mL of DMSO alone to one 4.9-mL lot of medium (control medium),

then 0.1 mL of the lowest (3.13 µg/mL) drug concentration in DMSO, then 0.1 mL of the 6.25-µg/mL concentration

3. Continue in sequence up the concentration series, each time adding 0.1-mL volumes to 4.9 mL medium. These volumes can be adjusted according to the total number of tests needed. Because there will be a 1:2 dilution of the drugs when combined with the inoculum, the working antifungal solutions are twofold more concentrated than the final concentrations. 3.3.4 Broth Macrodilution Modifications Published data show good concordance between results obtained using the broth microdilution methodology described above and a broth macrodilution adaptation.1,2 Some laboratories may choose to implement broth macrodilution rather than the broth microdilution method, primarily for safety issues. The steps and testing conditions relevant to the broth macrodilution test are provided below. This method has not been evaluated with the echinocandins or for the dermatophytes. The 100 times final strength drug dilutions described for the broth microdilution procedure should be diluted 1:10 with RPMI-1640 to achieve the 10 × (10-fold) strength needed for the broth macrodilution test The steps are listed below. .

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Step Action Comment(s) 1. Prepare and adjust the stock inoculum

suspensions, as described for the broth microdilution test

2. Mix the stock conidia or sporangiospore suspension by vortexing for 15 seconds

3. Dilute 1:100 with medium to obtain the test inoculum

0.4 × 104 to 5 × 10 4 CFU/mL

4. Dispense the 10× drug concentrations into 12 × 75-mm sterile tubes in 0.1 mL volumes

Macrodilution tubes may be sealed in plastic bags and stored frozen at –70 ºC for up to six months without deterioration of drug potency.

Abbreviations: CFU, colony forming units 3.3.5 Preparing the Inoculum When there is a risk of substantial spatter or aerosolization, the manipulation should be performed in a Class IIA or IIB biological safety cabinet. See CLSI document M29Error! Bookmark not defined. for additional information and Subchapter 1.3. 3.3.5.1 Nondermatophyte Moulds Reliable nongerminated conidial or sporangiospore suspensions can be prepared by a spectrophotometric procedure.27,28,29,30 Concentrations of viable conidial or sporangiospore test inocula in a range of approximately 0.4 ×104 to 5 ×104 CFU/mL provide the most reproducible MIC data.1,28 The steps for preparing the inoculum are listed below.

Nondermatophyte Moulds Step Action Comment(s) 1. Induce conidium and sporagiospore

formation. Grow most fungi on sporulation media (eg, potato dextrose agar, carrot juice agar, etc) for seven days at 35 °C or until good sporulation is obtained. Good sporulation may be obtained after 48 hours of incubation for some isolates (eg, Mucorales and Aspergillus spp. and species complex). Fusarium spp. and species complex may need to be incubated for 48 to 72 hours at 35 °C and then until day seven at 25 °C to 28 °C.

2. Cover sporulating colonies with approximately 1 mL of sterile 0.85% saline.

3. Prepare a suspension by gently probing the colonies with the tip of a transfer pipette.

Adding one drop (approximately 0.01 mL) of polysorbate 20 will facilitate the preparation of Aspergillus spp. inocula.

4. Withdraw and transfer the resulting mixture of conidia or sporangiospores and hyphal fragments to a sterile tube.

5. Allow heavy particles to settle for 3 to 5 minutes.

6. Transfer the upper homogeneous suspension to a sterile tube and tighten the cap.

Commented [MH27]: Note: Tween is a trademarked name so we can't use it. Hence the change to polysorbate 20

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7. Mix by vortexing for 15 seconds CAUTION: Remove the cap carefully, as liquid adhering to the cap may produce aerosols when opened.

8. Read and adjust the densities of the conidial or sporangiospore suspensions to an OD at 530 nm

0.09 to 0.13: Aspergillus spp. and species complex, Exophiala dermatitidis, Purpureocillium lilacinum, Paecilomycs variotii, S. schenckii and species complex. 0.15 to 0.17: Cladophialophora bantiana, Fusarium spp. and species complex, Ochroconis gallapova, Rhizopus spp. and Mucorales,Scedosporium (Lomentospora/Pseudallescheria)* 0.25 to 0.30: Alternaria spp.*, Bipolaris spp.*

9. Dilute these suspensions 1:50 in the standard medium.

Inoculum suspensions of Scedosporium sp. (Lomentospora/Pseudallescheria), Bipolaris hawaiensis and spicifera, and Alternaria spp. and other less prevalent dematiaceaous (black) moulds may require a lower (50%) dilution factor. The 1:50 inoculum dilutions will be 2 × (twofold) more concentrated than the density needed or approximately 0.4 × 104 to 5 × 104 CFU/mL.

10. Inoculate each well with 0.1 mL of the corresponding diluted inoculum suspension.

Prepare the test inoculum in a sufficient volume to inoculate all test wells.

*May require a lower (50%) dilution factor Abbreviations: CFU, colony forming unit; OD, optical density 3.3.5.2 Dermatophyte Moulds Most dermatophyte isolates produce sufficient conidia on potato dextrose agar. However, conidium formation by Trichophyton rubrum is very poor on standard fungal media including potato dextrose agar. Therefore, oatmeal agar (see Appendix F for preparation instructions) is recommended as the optimal growth medium for inducing conidium formation in T. rubrum isolates.9,31 The steps for preparing the inoculum are listed below.

Dermatophyte Moulds Step Action Comment(s)

1. Inoculate dermatophytes onto potato dextrose or oatmeal agar (T. rubrum isolates only)

2. Incubate at 30 °C for 4 to 5 days or until good conidial growth is present.

3. Cover colonies with approximately 1 mL of sterile 0.85% saline

4. Prepare a suspension by gently probing the colonies with the tip of a transfer pipette or sterile swab.

5. Let the resulting suspension settle for 5 to 10 minutes

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6. Count the conidia using a hemacytometer The final suspension should be made 2× more concentrated than the density needed for testing (1 × 103 to 3 × 103 CFU/mL).9

Abbreviations: CFU, colony forming units 3.3.6 Quantifying the Inoculum The final inoculum's accuracy may be verified as outlined in the following subchapters. 3.3.6.1 Quantifying the Inoculum of Nondermatophyte Moulds This step can be performed by plating 0.01 mL of a 1:10 dilution of the adjusted inoculum on Sabouraud glucose (dextrose) agar to determine the viable number of CFUs/mL.1,2,3 The plates should be incubated at 28 °C to 30 °C and observed daily for the presence of fungal colonies. Colonies should be counted as soon as possible after visible growth appears, especially for isolates Rhizopus spp. and other Mucorales. Incubation times will range from 24 hours or less (Rhizopus spp.) to five days Scedosporium (Lomentospora/Pseudallescheria). 3.3.6.2 Quantifying the Inoculum of Dermatophyte Moulds This step can be performed by plating 0.01 mL dilution of the adjusted inoculum on Sabouraud glucose agar to determine the viable number of CFU/mL. The plates should be incubated at 28 °C to 30 °C and observed daily for the presence of fungal colonies. 3.3.7 Inoculating the Wells For microbroth dilution, each well should be inoculated on the day of the test with 0.1 mL of the 2 × conidial or sporangiospore inoculum suspension. This step dilutes the drug concentrations, inoculum densities, and solvent, if used, to the final desired test concentrations. The growth control wells contain 0.1 mL of the corresponding diluted inoculum suspension and 0.1 mL of the drug diluent (2%) without antifungal agent (see Subchapter 3.3.3). QC and reference organisms should be tested in the same manner and included each time an isolate is tested. For macrobroth dilution, each tube should be inoculated on the day of the test with 0.9 mL of the corresponding diluted inoculum suspension, bringing the drug dilutions and inoculum densities to the final concentrations listed for the microdilution method. The growth control receives 0.1 mL of 10× the drug diluent without antifungal agent and is inoculated with 0.9 mL of the corresponding diluted inoculum suspensions. QC organisms should be tested in the same manner and included each time an isolate is tested. 3.3.8 Incubating the Trays All microdilution trays and macrobroth dilution tubes should be incubated at 35 °C without agitation and observed for the presence or absence of visible growth. 3.3.8.1 Non-dermatophyte Moulds Some mould isolates may not grow at 35 °C and incubation at 30 °C is more suitable. See Table 2 for recommended incubation times for determining MICs/MECs.

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Table 2. Recommended Incubation Times for Determing Minimal Inhibitory Concentrations or Minimal Effective Concentrations of Non-dermatophyte Moulds.

Antifungal Agent Genus/species Recommended Incubation (hrs)

All except echinocandins (amphotericin B,

fluconazole, triazoles)

Rhizopus spp. Mucorales 21–26

Aspergillus spp. and species complex Fusarium spp. and species complex

S. schenckii species complex C. bantiana

E. dermatitidis P. lilacinum P. variotii

46–50

Scedosporium spp. L. prolificans 70–74

Echinocandins*

Aspergillus spp. and species complex Paecilomyces variotti 21–26

Scedosporium spp. (Lomentospora / Pseudallescheria)

46–72

*Alternative: Read on first day when sufficient growth (ie, confluent growth covering the bottom of the well) is present in the growth control well (ie, contained drug-free medium). 3.3.8.2 Dermatophyte Moulds The dermatophyte mould isolate MICs should be determined after incubating for four days.9 3.4 Reading Minimal Inhibitory Concentration and Minimal Effective Concentration

Results 3.4.1 General The MIC is the lowest concentration of an antifungal agent that substantially inhibits organism growth, as detected visually when testing most antifungal agents. For the conventional microdilution procedure, the growth in each MIC well should be compared to the growth control with the aid of a reading mirror. For broth macrodilution, the tubes should be scored and the MICs determined as described for the broth microdilution procedure. When testing echinocandin antifungal agents, the MEC evaluation provides more consistent and reproducible susceptibility data than the conventional MIC reading.6,7,8 The MEC is the lowest drug concentration that leads to the growth of small, rounded, compact hyphal forms as compared to the hyphal growth seen in the growth control well (see Appendix G). For evaluating the MEC, the growth in each well should be compared to the growth control (drug-free medium) using a reading mirror. 3.4.2 Amphotericin B For amphotericin B, end points are typically well defined and the MIC is easily read as the lowest drug concentration that prevents any discernible growth (100% inhibition). Trailing end points with amphotericin B are usually not encountered. Such a pattern may reflect clinically relevant drug resistance. 3.4.3 Fluconazole, Flucytosine, and Ketoconazole For fluconazole, flucytosine, and ketoconazole, end points are typically less well defined than for amphotericin B, a problem which may be a significant source of variability. Applying a less stringent end

Commented [MH28]: Should this be defined in the terminology subchapter?

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point (allowing some turbidity above the MIC) has improved interlaboratory agreement. For this drug class, the turbidity allowed corresponds to approximately 50% or more (nondermatophyte mould isolates) to 80% or more (dermatophyte isolates) reduction in growth compared to the control well growth (drug-free medium). When this turbidity persists, it is often identical for all drug concentrations above the MIC. 3.4.4 Itraconazole, Posaconazole, Voriconazole, and Isavuconazole For non-dermatophyte mould isolates, the MIC is read as the lowest drug concentration that prevents any discernible growth (100% inhibition). Trailing end points with these agents against Aspergillus spp. and most other opportunistic mould pathogens are not usually encountered. Such a pattern could reflect clinically relevant drug resistance as it has been demonstrated for Aspergillus fumigatus strains that have been clinically resistant to itraconazole.3,27 However, when testing dermatophyte isolates against voriconazole, posaconazole, and itraconazole, the turbidity allowed corresponds to approximately 80% or more reduction in growth compared to the growth in the control well (drug-free medium). 3.4.5 Echinocandins (anidulafungin, caspofungin, micafungin) For echinocandins, end points are also typically less well defined than that described for amphotericin B, and application of the MEC end point improves reproducibility.6,7,8 The MEC is read as the lowest drug concentration that leads to the growth of small, rounded, compact hyphal forms as compared to the hyphal growth seen in the growth control well (see Appendix G). 3.4.6 Ciclopirox For ciclopirox, end points are typically less well defined than that described for amphotericin B. Applying a less stringent end point (allowing some turbidity above the MIC) has improved interlaboratory agreement. For this drug class, the turbidity allowed corresponds to approximately 80% or more reduction in growth compared to the growth in the control well (drug-free medium). 3.4.7 Griseofulvin For griseofulvin, end points are typically less well defined than that described for amphotericin B. Applying a less stringent end point (allowing some turbidity above the MIC) has improved interlaboratory agreement. For this drug class, the turbidity allowed corresponds to approximately 80% or more reduction in growth compared to the growth in the control well (drug-free medium). 3.4.8 Terbinafine For terbinafine, end points are typically less well defined than that described for amphotericin B. Applying a less stringent end point (allowing some turbidity above the MIC) has improved interlaboratory agreement. For this drug class, the turbidity allowed corresponds to approximately 80% or more reduction in growth compared to the growth in the control well (drug-free medium). 3.5 Interpreting Results Although breakpoints for mould testing have not been established or approved by CLSI or any regulatory organization, ECVs have been defined for some Aspergillus spp. and species complex vs amphotericin B, caspofungin, and four triazoles (isavuconazole, itraconazole, posaconazole and voriconazole). These ECVs may help identify NWT Aspergillus spp. isolates (ie, isolates for which the antifungal agent being evaluated appears to have reduced activity and may harbor resistance mechanisms).11,12,13,14,15 For information on ECVs, see CLSI documents M5716 and M57S17.

Commented [PD29]: From Ana: Another MEC definition. Multiple thoroughout M38.

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For non-Aspergillus spp. and for analytical purposes, susceptibility endpoints were assigned by the error bounding method during the collaborative study evaluating agar disk diffusion method performance.30 MICs below 1 µg /mL are usually reported for S. apiospermum and Purpureocillium lilacinum (Paecilomyces lilacinus) with posaconazole and voriconazole; for Alternaria spp. and Bipolaris spicifera with the triazoles. For some Mucorales with posaconazole and amphotericin B, isolates were grouped arbitrarily as “susceptible” (MIC or MEC < 1 µg/mL) and “resistant” (MIC or MEC > 4 µg /mL). Note: Although these endpoints and ECVs would help identify certain mould isolates as potentially resistant to triazoles, amphotericin B, and/or caspofungin (see the subchapters below), these endpoints are based solely on in vitro data. Therefore, the clinical relevance of testing this group of fungal pathogens remains uncertain. 3.5.1 Amphotericin B Experience using the procedures described in Subchapter 3.3 indicates that amphotericin B MICs for most nondermatophyte, opportunistic mould isolates are clustered between 0.5 and 2.0 µg/mL with modes (most frequent MIC) for Aspergillus spp. ranging between 0.5 and 1 µg/mL, except for A. terreus section complex (mode, 2 µg/mL).12 However, amphotericin B MICs for some other species (eg, species complexes of A. flavus, A. nidulans and A. versicolor, some members of the Aspergillus section Fumigati, Acremonium strictum, P. lilacinum, S. apiospermum, and L. prolificans) can be above 2 µg/mL and as high as 16 µg/mL).12,27,32,33 Although limited data are available regarding correlation between MIC and amphotericin B treatment outcomes for the filamentous fungi, MICs above 2 µg/mL (or above the ECV) have been associated with treatment failures. MICs below 2 µg/mL have been associated with clinical cure among 29 patients treated with amphotericin B for invasive aspergillosis caused by A. fumigatus species complex (eight cases), A. terreus species complex (nine cases) and Aspergillus flavus species complex (12 cases). Poor response to different formulations of amphotericin B was reported in patients with infections caused by A. terreus species complex (MICs > 1 µg/ml).34,35

3.5.2 Flucytosine Filamentous fungi are not usually susceptible to flucytosine and most MICs are > 64 µg/mL for these isolates. The exceptions are some Aspergillus spp. and dematiaceous (phaeoid/black) fungal isolates. 3.5.3 Fluconazole Filamentous fungi are usually not susceptible to fluconazole and most MICs are > 64 µg/mL for these isolates. The exceptions are some dimorphic and dermatophyte fungi isolates. 3.5.4 Ketoconazole Experience using the procedures described in this document indicates that nondermatophyte mould MICs vary between 0.0313 and 16 µg/mL. However, data are not yet available to confirm a correlation between MIC and treatment outcome with ketoconazole. ECVs have not been defined for this agent. 3.5.5 Itraconazole, Posaconazole, Voriconazole, and Isavuconazole The importance of proper drug dilution preparation for these water-insoluble compounds cannot be over-emphasized (see CLSI document M2724).3,4 Using incorrect solvents or deviating from the dilution scheme suggested in Appendixes A and B can lead to substantial errors due to dilution artifacts.

Commented [MH30]: From PDufresne: Should we remove as ECV will eventually replace those arbitrary breakpoints? Could add confusion as they are not breakpoints and from what I understand were done with disk diffusion method.

Commented [MH31]: Should a reference to M57 and M57S be added here?

Commented [MH32]: Sheryl, update the reference for Manual of clinical microbiology with this Johnson EM, Cavling-Arendrup M. Susceptibility Test Methods: Yeasts and Filamentous Fungi. In: Jorgensen JH, Pfaller MA, Carroll KC, et al, eds. Manual of Clinical Microbiology, 11th ed. Washington, DC: ASM Press; 2015: 2255-2281

Commented [PD33]: From Ana: do we need to include this agent?

Commented [MH34]: Are these references correct?

Commented [PD35R34]: Yes but do we need both? The principle is the same.

Commented [MH36R34]: There are actually A, B, and C

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3.5.5.1 Nondermatophyte Moulds. Experience using the recommended procedures indicates that nondermatophyte mould MICs for triazoles vary between 0.0313 and 16 µg/mL (most Aspergillus spp. modes between 0.06 and 0.5 µg/mL).11,14,32,33,36,37,38,39 Reduced triazole susceptibility as well as cross phenotypic resistance has been documented for some members of the Aspergillus section Nigri, Aspergillus section Fumigati, and Fusarium species complex.11,14,32,36,37 MICs below 4 µg/mL are usually reported for S. apiospermum and P. lilacinum with posaconazole and voriconazole and among some dematiceous moulds (eg, Alternaria spp. and Bipolaris spp.).40,41,42,43,44,45,46

The pharmacodynamics of isavuconazole and posaconazole were studied in a murine neutropenic invasive aspergillosis model using both WT and non-WT (cyp51 mutants) isolates of A. fumigatus species complex. A predictor of favorable treatment response was an MIC of < 0.5 µg/mL with isavuconazole and an MIC of 0.25 or 0.5 µg/mL with posaconazole.47,48 Voriconazole effectively prolonged survival and reduced the fungal load in a murine model of invasive infection caused by strains of A. terreus species complex for which the MICs were ≤ 1 µg/mL, or the ECV for this species vs voriconazole.11,49 These results support the potential for using ECVs in the clinical setting for detecting azole resistance (NWT MIC) due to cyp51A mutations, a resistance mechanism in some Aspergillus spp. Limited data are available to indicate a correlation between MIC and treatment outcome with these triazoles and other moulds. ECVs have only been defined for certain Aspergillus spp. and species complex (see CLSI documents M57 and M57S ). 3.5.5.2 Dermatophyte Moulds Azole MICs, including fluconazole, itraconazole, posaconazole, and voriconazole are usually low against dermatophytes, but high fluconazole MICs (> 16 µg/mL) have been reported.50 Correlation between in vitro triazole MICs for dermatophyte isolates with clinical outcome remains to be determined. 3.5.6 Echinocandins (anidulafungin, caspofungin, micafungin) Work to date has focused principally on testing Aspergillus isolates and little information exists to guide work with other moulds; however, non-Aspergillus mould MECs are usually high (> 4 µg/mL).51,52 Caspofungin MECs for Aspergillus spp. vary between 0.016 and 16 µg/mL (most modes are between 0.06 and 0.25 µg/mL) with frequently lower values when testing anidulafungin and micafungin.13,33,36,53,54 The frequency of caspofungin MECs above the ECV for Aspergillus spp. vary according to the species and it can be high for the species complexes of A. nidulans, A. terreus and A. versicolor (6.7 to 14.1% MECs above ECVs of 0.25 to 0.5 µg/mL).13 ECVs have not been defined for the other two echinocandins or any other moulds. Correlation of high echinocandin MECs with clinical outcome and FKS1 gene mutations remains to be determined. Although caspofungin MECs of ≥ 1 µg/mL for A. fumigatus were obtained from breakthrough infections in patients receiving this agent, genetic information was not available.55 3.5.7 Ciclopirox Most ciclopirox MICs are ≥ 1 µg/mL for the dermatophytes. Correlation of MIC with clinical outcome has yet to be determined.9 3.5.8 Griseofulvin Most griseofulvin MICs are ≤ 1 µg/mL for the dermatophytes. Correlation of MIC with clinical outcome has yet to be determined.9

Commented [MH37]: Sheryl, these references should be moved to the beginning of chapter 2

Commented [MH38]: Does this mean the ECV was ≤ 1

Commented [MH39]: Sheryl, add reference to M57

Commented [MH40]: Add reference to M57S

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3.5.9 Terbinafine Most terbinafine MICs are ≤ 0.25 µg/mL for the dermatophytes, but MICs ≥ 0.5 µg/mL have been reported for T. rubrum.56 Correlation of MIC with clinical outcome has yet to be determined.

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Chapter 4: Quality System Essentials – Quality Control

This chapter includes: • The purpose of quality control • Responsibilities for quality control • Selecting and storing quality control strains • Routine uses for reference strains • Controlling media batches and plasticware lots • Quality control frequency • Other control procedures

4.1 Purpose The goals of a QC program are to monitor: • The precision (repeatability) and accuracy of the susceptibility test procedures • The performance of reagents, testing conditions, and instructions used in the test • The performance of persons performing the tests and reading the results The goals are best accomplished by, but not limited to, using QC and reference strains selected for their genetic stability and usefulness in the particular method being controlled.8,10,57,58,59,60

4.2 Quality Control Responsibilities Antifungal susceptibility test manufacturers and users have a shared responsibility for quality. The primary purpose of QC testing performed by manufacturers (in-house reference methods or commercial methods) is to ensure that the testing materials and reagents have been appropriately manufactured. The primary purpose of QC testing performed by laboratories (users) is to ensure that the testing materials and reagents are maintained properly and testing is performed according to established protocols.

Manufacturers (commercial and/or laboratory developed products) are responsible for: • Antifungal stability • Antifungal labeling • Antifungal agent stock solution potency • Compliance with good manufacturing practices • Product integrity • Accountability and traceability to the consignee Manufacturers of commercial products should design and recommend a QC program that enables users to evaluate the variables (eg, inoculum levels, storage/shipping conditions) that most likely will cause user performance problems and to determine that the assay is performing correctly when carried out according to directions for use. Laboratories (user) is responsible for: • Storage (drug deterioration) • Operator proficiency • Adherence to procedure (eg, inoculum effect, incubation conditions [time and temperature])

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Laboratories should familiarize themselves with the regulatory and accreditation requirements for QC in their specific location. 4.3 Selecting Reference Strains Ideal reference strains for QC of dilution methods have MICs that fall near the mid-range of the concentration for all antifungal agents tested. An ideal control strain is inhibited at the fifth dilution of a nine-dilution-log2 series; however, strains with MICs between the third and seventh dilution are acceptable. Before a strain is accepted as a reference, it should be tested for as long as necessary to demonstrate that its antifungal susceptibility pattern is genetically stable. CLSI document M2361 provides guidelines for selecting appropriate QC strains and determining acceptable MIC or MEC ranges. The QC strains listed in CLSI document M38/M51S18 were selected in accordance with the criteria in CLSI document M2361 and can be used as controls for the antifungal susceptibility testing of moulds until mould isolates are selected. In addition, the reference mould isolates listed in CLSI document M38/M51S also can be used. Also shown are additional strains that can be useful for conducting reference studies.1,5,8,10,57,58,59 4.4 Storing Reference Strains 4.4.1 Methods for Prolonged and Short-term Storage Reference strains must be stored in a way that minimizes the possibility of mutation in the organisms. • There are two preferred methods for prolonged storage of reference strains. The fungal isolates may be

grown on potato dextrose agar and then frozen at −70 °C.62 Alternatively, cells can be preserved in 10% to 15% glycerol solution in small vials and storing them at at either −70 °C, in liquid nitrogen, or in the vapor of liquid nitrogen.63,64

• For short-term storage, working stock cultures can be grown on Sabouraud dextrose agar until sufficient

growth is observed and stored at 2 °C to 8 °C. Prepare fresh slants at two-week intervals by serial transfer from frozen stock. To avoid mixed cultures, no more than three passages should be made after removal from frozen stock culture. Whenever aberrant results occur, a new stock should be obtained.

4.4.2 Sources for Reference Strains Reference strains should be obtained from a source that can provide information on the culture's origin (eg, from commercial sources with documented culture history, or from reference institutions with demonstrated ability to store and use the organisms consistently with minimal contamination). A new stock culture should be obtained whenever a significant deviation from the expected end point is observed. 4.4.3 Preparing Strains for Storage Use the following steps to prepare strains for storage.

Step Action Comment(s) 1. Inoculate moulds onto an appropriate solid

medium. For most moulds, use potato dextrose agar. For dermatophytes, use Sabouraud dextrose agar or oatmeal agar (T. rubrum isolates).

2. Incubate the plates for 7 days at 28-32 °C. 3. Select growth from several colonies and perform

the appropriate susceptibility tests to See CLSI document M38/M5118 for expected MICs for QC and/or reference strains

Commented [MH41]: Sheryl, add a reference to M38/M57S

Commented [MH42]: Is this information in M38/M51 as well?

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demonstrate that they give the expected MIC or MEC results

4. Subculture strains yielding expected results onto the same medium that was used for the primary culture.

5. Incubate the plates long enough for sufficient growth to occur.

Usually 1–7 days

6. Examine the resulting growth carefully Ensure the growth is a pure culture. 7. Suspend the growth from the plate in 10-15% (or

other suitable stabilizing fluid) to make a heavy suspension (or if lyophilizing, suspend the growth in the appropriate medium).

See Subchapter 4.4.1?

8. Distribute the turbid suspension in small volumes (1 or 2 drops/approximately 100 to 25 µL) into suitable sterile containers.

9. Place these containers in a freezer at appropriate temperature or in liquid nitrogen.

See Subchapter 4.4.1

Abbreviations: MEC, minimal effective concentration; MIC, minimal inhibitory concentration; QC, quality control Stocks prepared using this procedure can stored indefinitely without significant risk of alteration in antifungal susceptibility patterns. When the supply of containers is nearly exhausted, repeat this process to make a new supply. 4.5 Routine Use of Reference Strains Use the following steps to prepare reference strains for routine use.

Step Action Comment(s) 1. Remove a container of the culture from the

freezer or obtain a lyophilized vial.

2. Let the frozen mixture thaw or rehydrate the lyophilized culture.

3. For Candida spp.: • Transfer a portion of the mixture onto

Sabouraud dextrose agar • Incubate at 35 °C for 24 hours. For moulds: • Subculture on potato dextrose agar or

oatmeal agar (T. rubrum isolates only)

4. Incubate 4 to 5 days (dermatophyte isolates) or 7 days (nondermatophyte isolates) or until good conidial growth is present.

Incubate at 35 °C

5. Remove 4 to 5 colonies and subculture them to medium for the appropriate susceptibility tests potato dextrose agar or Sabouraud dextrose agar

Incubate at 35 °C

6. Store cultures at 2 to 8 °C. 7. Subculture from the slant to an agar plate.

• Sabouraud dextrose for Candida spp. • Potato dextrose agar for filamentous fungi

or oatmeal agar (for T. rubrum isolates only).

Always perform susceptibility tests on colonies from: • Overnight plates (Candida spp.) • 7-day cultures (nondermatophyte

isolates)

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• 4- to 5-day cultures (dermatophyte isolates).

The agar slants may be used as working stock cultures. Agar slants should be replaced regularly with new slants prepared from the freezer supply at least every two weeks. 4.6 Controlling Media Batches and Plasticware Lots Control batches and plasticware lots by using the following criteria. • Each new batch of medium or lot of microdilution trays or macrodilution tubes should be tested with

one of the QC strains listed in CLSI document M38/M5118 to determine if MICs or MECs fall within the QC expected range. If they do not, the batch or lot shuld be rejected.

• At least one uninoculated tube from each batch should be incubated for the same amount of time as

needed to complete the test to ensure the medium’s sterility. • New lots of RPMI-1640 medium should be tested for acceptable performance before being used to test

clinical isolates, because recent studies have demonstrated that some lots do not perform adequately. The pH should be 6.9 to 7.1 (see Subchapter 6.1.2).

• The lot numbers of all materials and reagents used in these tests should be recorded. 4.7 Quality Control Frequency 4.7.1 Minimal Inhibitory Concentration or Minimal Effective Concentration Ranges MIC or MEC accuracy ranges for a single control test are listed in CLSI document M38/M51S18. In general, 1 out of every 20 MIC values in a series of 20 consecutive tests might be out of control (ie, outside the stated range) due to random test variation. Two consecutive out-of-control results or any more than 2 out-of-control results in 20 consecutive control tests need corrective action. Any time corrective action is taken, the count of 20 begins again. NOTE: Do not confuse this procedure with the procedure for establishing satisfactory performance of MIC tests for the purpose of performing weekly instead of daily QC tests (see Subchapter 4.7.2). 4.7.2 Frequency of Testing The overall test system performance should be monitored by testing appropriate reference strainseach day the test is performed. However, the frequency of test monitoring may be reduced if the laboratory can document satisfactory performance with daily control tests. For this purpose, satisfactory performance is defined as: • Documenting that all reference strains were tested for 30 consecutive test days.

• For each drug-microorganism combination, no more than 3 of the 30 MIC or MEC values (ie, MIC or

MEC values obtained from one drug-microorganism combination for 30 consecutive test days) were outside the accuracy ranges stated in CLSI document M38/M51S18 .

NOTE: This procedure is only for establishing satisfactory performance of MIC or MEC tests for the purpose of performing weekly instead of daily QC tests. Do not confuse this procedure with the steps that must be taken for corrective action defined in Subchapter 4.7.1.

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• The overall test system performance evaluation (as outlined above) must be restarted (ie, monitored for 30 consecutive test days) each time a reagent component (new batch of stock drug or new batch of frozen QC organisms) is changed.

• When these conditions are fulfilled, each reference strain must be tested at least once per week.

Whenever an MIC or MEC value outside the accuracy range is observed using the weekly accuracy monitoring system, daily control tests must be reinstated long enough to define the source of the aberrant result and to document resolution of the problem. Resolution of the problem may be documented as follows:

- Testing with appropriate reference strains for five consecutive test days should be performed.

- For each drug-microorganism combination, all of the five MIC or MEC values (ie, MIC or MEC

values obtained from one drug-microorganism combination for five consecutive test days) must be within the accuracy ranges stated in the current edition of CLSI document M38/M51.18

• If resolution of the problem cannot be documented (ie, at least one of the five MIC or MEC values is observed to be outside the accuracy range), daily control testing must be continued. To return to weekly testing, documenting satisfactory performance for another 30 consecutive test days as described in this Subchapter is necessary.

4.8 Other Control Procedures 4.8.1 Growth Control To assess test organism viability, each broth microdilution or macrodilution series should include a growth control composed of RPMI-1640 medium without antifungal agent plus 1% of the solvent used (eg, DMSO). With broth tests, the growth control also serves as a turbidity control for reading end points. 4.8.2 Purity Control A sample of each inoculum should be streaked onto a suitable agar medium and incubated until there is sufficient visible growth to detect mixed cultures and to provide freshly isolated colonies in the event retesting is needed. 4.8.3 Controlling End-point Interpretation End-point interpretation should be monitored periodically to minimize variation in the interpretation of MIC or MEC end points among observers. All laboratory personnel performing these tests should independently read a selected set of dilution tests. The results should be recorded and compared to the results obtained by an experienced reader. Specific reference strains with predetermined MICs are particularly helpful for this purpose, especially with itraconazole and other triazoles.

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Chapter 5: Conclusion This document describes a broth dilution method to evaluate the susceptibility of filamentous fungi (including dermatophytes) to antifungal agents (eg, amphotericin B, triazoles, echinocandins, etc). Detailed information regarding preparation of antifungal stock solutions, inoculum densities, and susceptibility endpoint interpretation (MICs and MECs) are provided. QC procedures are also provided. With the proposal of species-specific ECVs for certain Aspergillus spp. vs the triazoles including isavuconazole, amphotericin B, and caspofungin, MICs/MECs obtained using this methodology should have some clinical relevance. The additional data establishing interpretive breakpoints for moulds is not available. ECVs could identify non-WT Aspergillus isolates with reduced susceptibility to the antifungal agent under consideration for clinical use. This is important since infections caused by Aspergillus spp. are the most common mould infections. For more information on developing and using ECVs, see CLSI documents M57[M57] and M57S. Chapter 6: Supplemental Information

This chapter includes: • References • Appendixes • The Quality Management System Approach • Related CLSI Reference Materials

Commented [MH43]: Sheryl, add reference to M57

Commented [MH44]: Sheryl, add a reference to M57S

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2004;42:490. 61 CLSI. Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters; Approved Guideline—Third Edition. CLSI

document M23-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2008. 62 Pasarell L, McGinnis MR. Viability of fungal cultures maintained at –70 °C. J Clin Microbiol. 1992;30:1000-1004. 63 Espinel-Ingroff A, Montero O, Morrillo D. Long-term preservation of fungal isolates in commercially prepared Microbank cryogenic vials. J

Clin Microbiol. 2004;42:1257-1259. 64 Baker M, Jeffries P. Use of commercially available cryogenic vials for long-term preservation of dermatophyte fungi. J Clin Microbiol.

2006;44:617-618.

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Additional References Taxonomic References Aspergillus spp Nomenclature: Samson RA, Visagie CM, Houbraken J, et al. Phylogeny, identification and nomenclature of the genus Aspergillus. Stud Mycol. 2014;78:141–173. Talaromyces and Penicillium Nomeclature: Yilmaz N, Visagie CM, Houbraken J, Frisvad JC, Samson RA. Polyphasic taxonomy of the genus Talaromyces. Stud Mycol. 2014;78:175-341. Changing Nomenclature: de Hoog GS, Chaturvedi V, Denning DW, et al. ISHAM Working Group on Nomenclature of Medical Fungi. Name changes in medically important fungi and their implications for clinical practice. J Clin Microbiol. 2015;53(4):1056-62. Purpureocillium lilacinum Nomenclature: Luangsa-Ard J, Houbraken J, van Doorn T, et al. Purpureocillium, a new genus for the medically important Paecilomyces lilacinus. FEMS Microbiol Lett. 2011;321(2):141-9. Scedosporium Nomenclature: Lackner M, de Hoog GS, Yang L, et al. Proposed nomenclature for Pseudallescheria, Scedosporium and related genera. Fungal Diversity. 2014;67(1):1-10 Verweij PE, Ananda-Rajah M, Andes D, et al. International expert opinion on the management of infection caused by azole-resistant Aspergillus fumigatus. Drug Resist. Updat. (2015),

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Appendix A. Scheme for Preparing Dilution Series of Antifungal Agents to Be Used in Broth Dilution Susceptibility Tests for Nondermatophytes

Antifungal Solution

Step

Concentration (µg/mL)

Source

Volume

(mL)

+

Solvent (mL) (eg,

DMSO)

=

Intermediate


=

Final

Concentration at 1:100 (µg/mL)

Log2

1 1600 Stock - - 1600 16 4 2 1600 Stock 0.5 0.5 800 8.0 3 3 1600 Stock 0.5 1.5 400 4.0 2 4 1600 Stock 0.5 3.5 200 2.0 1 5 200 Step 4 0.5 0.5 100 1.0 0 6 200 Step 4 0.5 1.5 50 0.5 −1 7 200 Step 4 0.5 3.5 25 0.25 −2 8 25 Step 7 0.5 0.5 12.5 0.125 −3 9 25 Step 7 0.5 1.5 6.25 0.0625 −4 10 25 Step 7 0.5 3.5 3.13 0.0313 −5 11 3.13 Step 10 0.5 0.5 1.56 0.0156 −6 12 3.13 Step 10 0.5 1.5 0.78 0.0078 −7

Abbreviation: DMSO, dimethyl sulfoxide

Commented [PD45]: Covers the full range of dilutions recommended. 1X final concentration rather that 2X. Harmonized with M27A4 draft

Commented [MH46]: Should this be water insoluable as in App B?

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Appendix B. Scheme for Preparing Dilution Series of Water-Insoluble Antifungal Agents to Be Used in Broth Dilution Susceptibility Tests of Dermatophytes

Antifungal Solution

Step


Source

Volume

(mL)

+

Solvent (mL) (eg,

DMSO)

=

Intermediate


=

Final

Concentration at 1:100 (µg/mL)

Log2

1 6400 Stock - - 6400 64 6 2 6400 Stock 0.5 0.5 3200 32 5 3 6400 Stock 0.5 1.5 1600 16 4 4 6400 Stock 0.5 3.5 800 8 3 5 800 Step 4 0.5 0.5 400 4 2 6 800 Step 4 0.5 1.5 200 2 1 7 800 Step 4 0.5 3.5 100 1.0 0 8 100 Step 7 0.5 0.5 50 0.5 −1 9 100 Step 7 0.5 1.5 25 0.25 −2 10 100 Step 7 0.5 3.5 12.5 0.125 −3 11 12.5 Step 10 0.5 0.5 6.25 0.0625 −4 12 12.5 Step 10 0.5 1.5 3.13 0.0313 −5 13 12.5 Step 10 0.5 3.5 1.56 0.0156 −6 14 1.56 Step 13 0.5 0.5 0.78 0.0078 −7 15 1.56 Step 13 0.5 1.5 0.39 0.0039 −8 16 1.56 Step 13 0.5 3.5 0.195 0.00195 −9 17 0.195 Step 16 0.5 0.5 0.0975 0.000975 −10

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Appendix C. Scheme for Preparing Dilutions of Water-Soluble Antifungal Agents to Be Used in Broth Dilution Susceptibility Tests

Antifungal Solution

Step

Concentration

(µg/mL)

Source

Volume

(mL)

+

Medium

(mL)

=

Intermediate


=

Final

Concentration at 1:10

(µg/mL)

Log2 1 5120 Stock 1.0 7.0 640 64 6 2 640 Step 1 1.0 1.0 320 32 5 3 640 Step 1 1.0 3.0 160 16 4 4 160 Step 3 1.0 1.0 80 8 3 5 160 Step 3 0.5 1.5 40 4 2 6 160 Step 3 0.5 3.5 20 2 1 7 20 Step 6 1.0 1.0 10 1 0 8 20 Step 6 0.5 1.5 5 0.5 −1 9 20 Step 6 0.5 3.5 2.5 0.25 −2 10 2.5 Step 9 1.0 1.0 1.25 0.125 −3 11 2.5 Step 9 0.5 1.5 0.625 0.0625 −4 12 2.5 Step 9 0.5 3.5 0.3125 0.03125 −5

Commented [MH47]: There is still no reference in the main body of the text to this appendix. Where should it be added.

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Appendix D. Composition of RPMI-1640 Medium Constituent

g/L Water

Constituent

g/L Water

L-arginine (free base)

0.200

Biotin

0.0002

L-aspargine (anhydrous)

0.050

D-pantothenic

0.00025

L-aspartic acid

0.020

Choline chloride

0.003

L-cystine • 2HCl

0.0652

Folic acid

0.001

L-glutamic acid

0.020

Myo-inositol

0.035

L-glutamine

0.300

Niacinamide

0.001

Glycine

0.010

PABA

0.001

L-histidine (free base)

0.015

Pyridoxine HCl

0.001

L-hydroxyproline

0.020

Riboflavin

0.0002 0.000005

L-isoleucine

0.050

Thiamine HCl

0.001

L-leucine

0.050

Vitamin B12

0.000005

L-lysine • HCl

0.040

Calcium nitrate • H2O

0.100

L-methionine

0.015

Potassium chloride

0.400

L-phenylalanine

0.015

Magnesium sulfate (anhydrous)

0.04884

L-proline

0.020

Sodium chloride

6.000

L-serine

0.030

Sodium phosphate, dibasic (anhydrous)

0.800

L-threonine

0.020

D-glucose

2.000

L-tryptophan

0.005

Glutathione, reduced

0.001

L-tyrosine • 2Na

0.02883

Phenol red, Na

0.0053

L-valine

0.020

Commented [PD48]: Why 2 concentrations. Only 0.000005 in M27-S4

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Appendix E. Preparing RPMI-1640 Medium

RPMI-1640 medium buffered with 0.165 mol/L 3-[N-morpholino] propanesulfonic acid (MOPS), 1 L (see Appendix D) To prepare 10.4 g powdered RPMI-1640 medium (with glutamine and phenol red, without bicarbonate) 34.53 g MOPS buffer, use the steps below.

Step Action Comment(s) 1. Dissolve powdered medium in 900 mL

distilled water

2. Add MOPS and stir until dissolved MOPS is at a final concentration of 0.165 mol/L 3. While stirring, adjust the pH to 7.0 at 25 °C

using 1 mol/L sodium hydroxide

4. Add additional water to bring medium to a final volume of 1 L

5. Filter, sterilize, and store at 4 °C until use Abbreviation: Reference for Appendix E 1. CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard—Fourth Edition. CLSI document

M27-A4. Wayne, PA: Clinical and Laboratory Standards Institute; 2016.

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Appendix F. Oatmeal Agar To prepare oatmeal agar, use the steps below.

Step Action Comment(s) 1. To 1 L of distilled water, add:

• 100 g baby oatmeal cereal • 15 g granulated agar • 0.03 g gentamicin

2. Mix well. 3. Dispense 500 mL into liter autoclavable

beakers. Note: The mixture tends to boil over.

4. Autoclave at 121 °C for 20 minutes. 5. Immediately pour into petri plates and allow

to cool

6. Store at 4 °C to 6 °C. QC: Positive: Trichophyton rubrum – conidia formation Negative: None Sterility: No growth Reference 1. Jessup CJ, Warner J, Isham N, Hasan I, Ghannoum MA. Antifungal susceptibility testing of dermatophytes: establishing a medium for

inducing conidial growth and evaluation of susceptibility of clinical isolates. J Clin Microbiol. 2000; 38: 341-344.

Commented [MH49]: Should this be "no conidia formed"?

Commented [PD50]: What is meant is that there’s no strain to be used as a negative control .

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Appendix G. Minimal Effective Concentrations of Caspofungin and Anidulafungin G1. Minimal Effective Concentrations Caspofungin (Figure below reprinted with permission from the American Society for Microbiology and the author.)

Figure G1. Example of Caspofungin MEC Results. Shown are dilution series of caspofungin (column 12 is the drug-free growth control, and columns 11 to 1 contain drug concentrations that ascend in two-fold steps from 0.007 in column 11 to 8 µg/mL in column 1) vs eight Aspergillus isolates after 24 hours of incubation. The MECs are the lowest concentrations of caspofungin that led to the growth of small, rounded, compact hyphal forms as compared to the hyphal growth seen in the growth control well (column 12).1 MECs for rows A and B (Aspergillus niger) are the wells of column 7 (0.12 µg/mL), and for rows C to H (A. flavus, A. terreus and A. fumigatus) are the wells of column 6 (0.25 µg/mL). G2. Minimal Effective Concentrations of Anidulafungin

Isolate MEC

0.015 0.03 0.06 0.13 0.25 0.5 1 2 4 8 16 32 Growth control

S. apiospermum ATCC® MYA-3634

4

F. solani ATCC® MYA-3636

>32

A. fumigatus ATCC® MYA-3727

< 0.015

A. flavus ATCC® MYA-3626

<0.015

Figure G2. Example of Anidulafugin MEC Results. Shown are dilution series of anidulafungin vs different mould isolates after 24 hours (Aspergillus isolates), 48 hours (Fusarium solani isolate), and 72

Commented [MH51]: Reference to espinel-Ingroff, JCM, 2003: 4.3-409

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hours (S. apiospermum) of incubation. The MECs are the lowest concentrations of anidulafungin that led to the growth of small, rounded, compact hyphal forms as compared to the hyphal growth seen in the growth control wells.2 The Scedosporium isolate’s MEC might be read as 2 µg/mL, but at 4 µg/mL, the change in morphology is more defined and all wells have the same trailing. References for Appendix G 1 Espinel-Ingroff A. Evaluation of broth microdilution testing parameters and agar diffusion Etest procedure for testing

susceptibilities of Aspergillus spp. to caspofungin acetate (MK-0991). J Clin Microbiol. 2003;41:403-409. 2 Espinel-Ingroff A, Fothergill A, Ghannoum MA, et al. Broth microdilution guidelines for susceptibility testing of anidulafungin

against filamentous fungi. J Clin Microbiol. 2007;45:1811-1820.

Commented [MH52]: Are these references still relevant?

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Appendix G. McFarland 0.5 Barium Sulfate Turbidity Standard To standardize the inoculum density, use a BaSO4 turbidity standard (0.5 McFarland standard). The procedure for preparing this turbidity standard consists of the steps below.

Step Action Comment(s) 1. Add 0.5 mL of 0.048 mol/L BaCl2 (1.175%

w/v BaCl2•2H2O) to 99.5 mL of 0.18 mol/L (0.36 N) H2SO4 (1% v/v).

2. Use a spectrophotometer with a 1-cm light path and matched cuvette to determine the absorbance and verify the correct density of the turbidity standard

The absorbance at 625 nm should be 0.08 to 0.13 for the 0.5 McFarland standard.

3. Distribute 4 to 6 mL into screw-cap tubes Tubes should be the same size as those used in growing or diluting the broth culture inoculum.

4. Tightly seal the tubes 5. Store the tubes in the dark at room

temperature. Vigorously agitate this turbidity standard on a mechanical vortex mixer just before use. Replace standards or recheck their densities monthly after preparation.

Commented [MH53]: There is no reference to this appendix in the text and no mention of a McFarland standard. If it is not discussed in M38, the appendix should be deleted. 5/12: Ana will check if this is still needs to be in procedure or should be moved to the supplement or deleted

Commented [MH54R53]: Can this be deleted?

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M38



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

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M38 addresses the 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.

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Path of Workflow A path of workflow is the description of the necessary processes to deliver the particular product or service that the organization or entity provides. A laboratory path of workflow consists of the sequential processes: preexamination, examination, and postexamination and their respective sequential subprocesses. All laboratories follow these processes to deliver the laboratory’s services, namely quality laboratory information. M38 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

Exam

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orde

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colle

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M38



Related CLSI Reference Materials* EP23™ Laboratory Quality Control Based on Risk Management; Approved Guideline. 1st ed., 2011. This document

provides guidance based on risk management for laboratories to develop quality control plans tailored to the particular combination of measuring system, laboratory setting, and clinical application of the test.

M07 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 10th ed.,

2015. This standard addresses reference methods for the determination of minimal inhibitory concentrations of aerobic bacteria by broth macrodilution, broth microdilution, and agar dilution.

M23 Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters. 3rd ed., 2008.

This document addresses the required and recommended data needed for the selection of appropriate interpretive criteria and quality control ranges for antimicrobial agents.

M27 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. 4th ed., 2016. This

document addresses the selection and preparation of antifungal agents; implementation and interpretation of test procedures; and quality control requirements for susceptibility testing of yeasts that cause invasive fungal infections.

M29 Protection of Laboratory Workers From Occupationally Acquired Infections. 4th ed., 2004. Based on US

regulations, this document provides guidance on the risk of transmission of infectious agents by aerosols, droplets, blood, and body substances in a laboratory setting; specific precautions for preventing the laboratory transmission of microbial infection from laboratory instruments and materials; and recommendations for the management of exposure to infectious agents.

M38/M51 Performance Standards for Antifungal Susceptibility Testing of Filamentous Fungi; Draft Informational

Supplement. 1st ed., 2016. This supplement provides updated tables for the Clinical and Laboratory Standards Institute antifungal susceptibility testing documents M38-A3 and M51-A.

M51 Method for Antifungal Disk Diffusion Susceptibility Testing of Nondermatophyte Filamentous Fungi. 1st

ed., 2010. This document describes the guidelines for antifungal susceptibility testing by the disk diffusion method of nondermatophyte filamentous fungi (moulds) that cause invasive disease.

M57 Principles and Procedures for the Development of Epidemiological Cutoff Values for Antifungal

Susceptibility Testing; Draft Guideline. 1st ed., 2016. This document describes the criteria for development and use of epidemiological cutoff values for guiding clinical decisions for fungi/antifungal combinations for which there are no breakpoints.

M57S Principles and Procedures for the Development of Epidemiological Cutoff Values for Antifungal

Susceptibility Testing; Draft Informational Supplement. 1st ed. 2016. This document provides the epidemiological cutoff value tables for the Clinical and Laboratory Standards Institute antifungal susceptibility testing document M57.

* Proposed-level documents are being advanced through the Clinical and Laboratory Standards Institute consensus process; therefore, readers should refer to the most current editions.

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