M38-A2
`
`Vol. 28 No. 16
`
`Replaces M38-A
`Vol. 22 No. 16
`
`Reference Method for Broth Dilution
`
`Antifungal Susceptibility Testing of
`Filamentous Fungi; Approved Standard--
`Second Edition
`
`This document addresses 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.
`
`CLINICAL AND
`
`// LABORATORY
`
`STANDARDS
`INSTITUTE”
`{Farrnedy ~ccr.s)
`
`CFAD V. Anacor, |PR201 5-01776 ANACOR EX. 2083 - “I/50
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`

`
`i
`Reference Method for Broth Dilution Antifungal Susceptibility Testing
`of Filamentous Fungi; Approved Standard—Second Edition
`
`M38-A2
`
`ISBN 1-56238-668—9
`
`John H. Rex, MD
`Barbara D. Alexander, MD, MIIS
`David Andes, MD
`Beth Arthington~Skaggs, PhD
`Steven D. Brown, PhD
`Vishnu Chaturveli, PhD
`
`Ana Espinehlngroff, PhD
`Mahmoud A. Ghannoum, MSc, PhD
`Cynthia C. Knapp, MS
`Mary R. Motyl, Phl), D(ABMM)
`Luis Ostrosky-Zeichner, MD, FACP
`Michael Pfaller, MD
`Daniel J. Sheehan, Phi)
`Thomas J. Walsh, MD
`
`Abstract
`
`Clinical and laboratory Standards Institute document M38—A2-—Rt{{£?:-cm-c _lfe!J‘mc2’fl):' Bmfli z’)ii’tm'rm Arit{!i.trrgcJt' 5':r.sL‘e;JtihiIi{t'
`l'ie.s'{i:rg of'Fit'c.'me.'1:'rJr:.s' Fiorgi; App.='o1‘t,’d Sram."am’——ScctJr.=c."
`lfdirfoit describes 21 method For
`testing the stlsceptibility of
`tilamentous ftmgi (moulds) that cause invasive (.-ts-].r;crg:'t'a't:.s' spp.. Fm-.::r:'nm spp._ Rhizopm orjv;-cm (R. arr!n':t:.s'], Pmi.«Jalic.s-c-Em-ft:
`boydii [Sccdo.vpm-rim: u;n'o.s';7e:-iii:.rm,l. S. pi‘r):'{fic‘c:n.\-_ .S}ooro.':’m'.r .¥(T.iIe37L';(ii. and other opportunistic pathogenic moulds) and
`cutaneous (dcrmatopli)-‘tc. Tt‘ichoph_t-‘tort.
`.-‘l/;‘fcr‘o.vponmi. and !i;Jidcr'n:opti_t=tr;zi spp.)
`fungal
`infections to antiltingal agents.
`Selection of antitungal agents: preparation of antit'ung_al 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 responsibilities ol‘ the manufacturer and the user in quality control is also presented.
`
`i"‘c.m'ng of
`Clinical and Laboratory Standards Institute (CLSI). Re,ti3rence .-lIerhm!_,v‘é;r Broil: I)£!::ri::z2 Aiirifiriigcit S‘r:.w.'eprib:‘i":'r_1'
`F!'Iamcnrcm.t‘ Frrr1gi,' Appmtme’ Sta:idaI'(Jl—---Second Ectiliwi. Cl.Sl document M33-A2 (ISBN E56233-668-9). Clinical and
`Laborator}-' Standards Institute. 940 West Valley Road, Suite I400. Wayne. Penns}-'l\-'ania 19087-1893 USA. 2008.
`
`The Clinical and I.-ahordtoa)’ Standards Institute consensus process. which is the mechanism for 1'110\'lI1g a rlocuinent through
`two or more levels of review by the health care community. is an ongoing process. Users should expect revised editions ofan}-'
`giver: document, Because rapid changes in tceltnolog_\-' may atffcet the procedures. methods. and protocols in a standard or
`guideline. users should replace outdated editions with the current editions of’ CI_SL’l\"C‘('_‘l.S documents. Current editions are l
`listed in the CLSI catalog and posted on our \\-‘ebsitc at vwt-'w.clsi.org. If}-‘our 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: 6l0.688.tJ7t]t};
`ii-Mail:
`custtiinerscrttice-'§:i3elsiorg: Website: wwu-'.clsi.org
`
`/ CLINICAL AND
`
`LABORATORY
`STANDARDS
`I NSTITUTE"‘
`{Formerly NCCLS)
`
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`

`
`l
`
`Clinical and Laboratory_Standards Institute
`
`.-'ldvctncing QLtalt't_r in Ileciltfr (."urt’ Te.s‘t.*'ii-g
`
`[CLSL
`Institute
`C.‘linical and Laboratory Standards
`fonncrly NCCLS) is an international, interdisciplinary,
`nonprofit.
`standards—developing,
`and
`educational
`organization that promotes the development and use of
`voluntary consensus standards and guidelines within the
`health care conununity. It
`is recognized worldwide for
`the application of its unique consensus process in the
`development of standards and guidelines For patient
`testing and related health care issues. Our process is
`based on the principle that consensus is an et't'ectivc and
`cost-effective way to improve patient testing and health
`care services.
`
`In addition to developing and promoting the use of
`voluntary consensus
`standards
`and guidelines. we
`provide an open and unbiased forum to address critical
`issues affecting the quality of patient testing and health
`C2lI'C_
`
`PUBLICATIONS
`
`is published as :1 standard, guideline, or
`A document
`committee report.
`
`Standard A document developed through the consensus
`process
`that
`clearly
`identities
`specific,
`essential
`requirements for materials, methods, or practices for use
`in an unmodified Form. A standard may,
`in addition.
`contain discretionary
`elements. which are
`clearly
`identified.
`
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`for
`a general operating
`practice, procedure, or material
`for voluntary use. A
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`Most documents are subject to two levels of consensus
`“pi'oposcd" and “approvcd." Depending on the need For
`field evaluation or data collection, documents may also be
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`
`Proposed A consenstts document undergoes the first stage
`of review by the health care comnlunity as a proposed
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`and thorough technical review. including an overall review
`of its scope. approach. and utility. and a line—by-line review
`of its technical and editorial content.
`
`Approved An approved standard or guideline has achieved
`consensus within the health care cormnunity. It should be
`reviewed to assess the utility of the final document.
`to
`ensure attainment oiiconsensus {ie, that comments on earlier
`versions have been satisfactorily addressed), and to identify
`the need for additional consensus documents.
`
`Our standards and guidelines 1'cprescnt a consensus opinion
`on good practices and reflect the substantial agreement by
`materially affected.
`competent,
`and
`interested
`parties
`obtained
`by
`following CI.SI’s
`established consensus
`procedu1'es. Provisions in CLSI standards and guidelines
`may be more or less stringent than applicable regulations.
`Consequently. eontortiiance to this voluntary consensus
`document does not relieve the user of responsibility for
`compliance with applicable regulations.
`COIVIMENTS
`
`The eomrnents of‘ users are essential
`
`to the consensus
`
`process. Anyone may submit a comnient, and all comments
`are addressed, according to the consensus process. by the
`committee
`that wrote
`the document. All
`comments.
`
`including those that result in a change to the document when
`published at the next consensus level and those that do not
`result in a change. are responded to by the comtnittee in an
`appendix to the document. Readers are strongly encouraged
`to comment in any form and at any time on any document.
`Address comments to Clinical and Laboratory Standauds
`Institute, 940 West Valley Road, Suite til-(ll). Wayne. PA
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`
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`
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`
`0
`
`It
`
`the atithoiization ofa project
`
`VOLUNTEER PARTICIPATION
`
`the development and open review of documents
`
`the revision of documents in response to cotnments
`by users
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`
`a consensus
`
`Ilealth care pi‘olessioI1als in all specialties are urged to
`volunteer for participation in CLSI projects. Please contact
`us
`at
`costoinerset'vice(r_tgclsiorg or
`-+-6l().o88.t)1t)tl
`for
`additional intonnation on committee participation.
`
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`

`
`"Number 16
`
`M3 8-A3
`
`Copyright @2008 Clinical and Laboratory Standards Institute. Except as stated below, neither this
`publication nor any portion thereof may be adapted, copied, or otherwise reproduced, by any means
`(electronic, mechanical, photocopying, recording, or otherwise) without prior written permission from
`Clinical and Laboratory Standards Institute (“CLSI”).
`
`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, contact the Executive Vice President, Clinical and Laboratory
`Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.
`
`Suggested Citation
`
`(CLSI. Reference Method for Broth Dilution Antifimgal Susceptibility Testing of Filamentous Fungi;
`Approved Standara'—Second Edition. CLSI document M38-A2. Wayne, PA: Clinical and Laboratory
`Standards Institute; 2008.)
`
`Proposed Standard
`November 1998
`
`Approved Standard
`August 2002
`
`Approved Standard—Second Edition
`April 2008
`
`ISBN 1-3623 8-668-9
`ISSN 0273-3099
`
`ii
`
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`

`
`Volume 28
`
`M3 8-A2
`
`Committee Membership
`
`Area Committee on Microbiology
`
`Mary Jane Ferraro. PhD, MPH
`Chairholder
`
`Massachusetts General Hospital
`Boston, Massachusetts
`
`John H. Rex, MD, FACP
`Viee»ChairhoIder
`Astrazcneea
`
`Cheshire, United Kingdom
`
`Barbara Ann Body, PhD, D(ABlV[M)
`LabCorp
`Burlington, North Carolina
`
`Betty (Bet7.} A. Forbes, PhD, D(ABMM)
`Medical College of Virginia CH.rnpus
`Richmond, Virginia
`
`Freddie Mae Poole
`
`FDA Center for Devices and Radiological
`Health
`Rockville, Maryland
`
`Daniel F. Sahm, PhD
`Eurofins Medinet
`
`Herndon. Virginia
`
`Fred C. Tenover, PhD, ABMM
`Centers for Disease Control and
`I’reventit):1
`Atlanta, Georgia
`
`John D. Turnidge, MD
`Women’s and Children’s Hospital
`North Adelaide, Australia
`
`Michael L. Wilson, MD
`Denver Health Medical Center
`Denver, Colorado
`
`Advisers
`
`Nancy L. Anderson, MMSc. MT(ASCl’)
`Centers for Disease Control and
`Prevention
`
`Atlanta, Georgia
`
`Ellen Jo Baron, PhD
`Stanford Univ. Hospital &
`Medical School
`Palo Alto, California
`
`Donald R. Callihan, PhD
`BI) Diagnostic Systems
`Sparks, Maryland
`
`Lynne S. Garcia, MS
`LSG & Associates
`Santa Monica, California
`
`Richard L. llodinka, PhD
`Children's Hospital of Philadelphia
`Philadelphia, Pennsylvania
`
`James H. Jorgensen, PhD
`University of Texas Health
`Science Center
`San Antonio, Texas
`
`Michael A. Pfaller, MD
`University of Iowa College of
`Medicine
`
`Iowa City, Iowa
`
`Robert P. Rennie, PhD
`University of Alberta Hospital
`1.7.dinonton. Alberta, Canada
`
`Thomas R. Shryock, PhD
`lilanco Animal Health
`Greenfield, Indiana
`
`Jana M. Swenson, IVEMSC
`Centers for Disease Control
`and Prevention
`Atlanta, Georgia
`
`Melvin P. Weinstein, MD
`Robert Wood Johnson Medical
`School
`
`New Brunswick, New Jersey
`
`Matthew A. Wilder, MD,
`MBA, FIDSA
`Pacific Beach BioSeiences, Inc.
`San Diego, California
`
`Gail L. Woods, MD
`Central Arkansas Veterans
`I-Iealthcare
`Little Rock, Arkansas
`
`Subcommittee on Antifungal Susceptibility Tests
`
`John H. Rex, MD, FACP
`Chairholder
`Astrazeneea
`
`Cheshire, United Kingdom
`
`Mahmoud A. Ghannourn, MSC,
`PhD
`Vice-Chairltolder
`
`Case Western Reserve University
`Cleveland, Ohio
`
`Barbara D. Alexander, MD, Ml-IS
`Duke University Medical Center
`Durham, North Carolina
`
`David Andes, MD
`University of Wisconsin
`Madison, Wisconsin
`
`Steven D. Brown, PhD
`The Clinical Microbiology Institute
`Wilsonville, Oregon
`
`Cynthia L. Fowler, MD
`BioMerieux., Inc.
`Durham, North Carolina
`
`Elizabeth M. Johnson
`The HPA Centre for Infections
`
`Bristol, United Kingdom
`
`Cynthia C. Knapp, MS
`Trek Diagnostic Systems
`Cleveland, Ohio
`
`Mary R. Motyl, P111), D{ABl\/[NI]
`Merck & Company. Inc.
`Rahway, New Jersey
`
`Luis ('}strosky-Zeichner, MD, FACP
`University of Texas Medical School
`at Houston
`Houston, Texas
`
`Michael A. Pfaller, MD
`University of Iowa College of
`Medicine
`Iowa City, Iowa
`
`Daniel J. Sheehan, l’hD
`Pfizer Inc
`New York, New York
`
`Thomas J. Walsh, MD
`National Cancer Institute
`
`Betliesda, Maryland
`
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`iii
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`

`
`"I
`
`M38-A2
`
`Staff
`
`Clinical and Laboratory Standards
`Institute
`
`Wayne, Pennsylvania
`
`Lois M. Schmidt, DA
`Vice President, Standards‘
`Development and .-Markeririg
`
`Tracy A. Dooley. BS, lV[L'I'{ASCP)
`SIafi'Liaf.roI:*
`
`Ron Quicho
`Pirojecf M'cmager
`
`Melissa A. Lewis
`Editor
`
`Annette W. Fothergill, MA. MBA,
`M’I‘(ASCP]
`University of Texas Health Science
`Center
`San Antonio, Texas
`
`Thomas R. F-ritsche, PIMD
`JMI Laboratories
`
`North Liberty, Iowa
`
`Freddie Mae Poole
`FDA Ctr. for DevicesfRad. Health
`
`Rockville, Maryland
`
`Michael G. Rinaldi, PhD
`University of Texas Health Science
`Center
`
`San Antonio, Texas
`
`Guy St. Germain
`lnstitut National de Santé Publique
`Du Quebec Centre de Doc.-INSPQ
`St.-Anne—dc—Bellev11e, Canada
`
`Number 16
`
`Advisers
`
`Beth Arthington-Skaggs, Phl)
`Centers for Disease Control and
`Prevention
`
`Atlanta, Georgia
`
`Shukal Bala
`Food and Drug Administration
`Rockville, Maryland
`
`Ozlem Bclen, MD. MPH, MSC
`FDA CDER
`Silver Spring, Maryland
`
`Vishnu Chaturvedi, PhD
`New York State Dept. of Health
`Albany. New York
`
`Daniel J. Diekcma, MD, FACP
`University of Iowa College of
`Medicine
`
`Iowa City, Iowa
`
`Ana Espinel-Ingroff, PhD
`Medical College of Virginiafll CU
`Richmond, Virginia
`
`Acknowledgment
`
`The Subcommittee on Antifungal Susceptibility Tests gratefully acknowledges its working group for their
`help in preparing the approved-level, second edition of this standard:
`
`Ana Espinel-Ingroff, PhD
`Mahmoud A. Ghannoum, MSc, PhD
`Mary R. Motyl, PhD, D(ABMM)
`Thomas J . Walsh, MD
`
`Medical College ofVirginia!\/CU
`Case Western Reserve University
`Merck & Company, Inc.
`National Cancer Institute
`
`iv
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`

`
`M38"A2
`
`iii
`
`vii
`
`viii
`
`i.._.n
`
`Volume 28
`
`Contents
`
`Committee
`
`Updated Information in This
`
`Standard
`
`I 2 3
`
`4 Definitions M
`
`U:
`
`Antifungal
`
`5.1 Source
`
`Weighing Antifungal
`Preparing Stock
`Number ofConcentrations
`
`5.2
`5.3
`5.4
`
`5.5
`
`Selection ofAr1tifungal Agents for Routine Testing and Reporting.............................
`
`6 Test
`
`6.1 Broth
`
`
`
`.:_.._.._-.::::E:._.._..:_-,:;uo-—.:|--.1-.:I:::\u1L.nU1-l=~4b~L.».:r\JtxJM
`
`Preparing Diluted Antifiingal Agents
`lnoculum
`Inoculating RPMI-1640
`
`6.2
`6.3
`6.4
`6.5
`
`6.6
`6.7
`6.8
`6.9
`
`7
`
`Qc......_ ..
`
`MIC and MEC ReadingResults
`Interpretation
`Broth Macrodilution Modifications
`OtherModifications
`
`..
`
`7.1 Purpose
`7.2
`QCResponsibilities
`7.3
`Selecting Reference Strains
`7.4
`Storing Reference Strains
`7.5
`Routine Use ofReferenceStrains
`7.6
`Batch ofMediun1 and Lot ofP1asticwareControl
`
`7.7
`7.8
`
`QC
`Other Control Procedures
`
`n—sp—Aa—Au—In—Ii—In—-u—-n—n
`
`lJ‘ILi‘I-5'-‘-lib-JI‘Jl\J'—"—‘
`
`Table 1. Solvents and Diiuents for Preparation of Stock Solutions of Antifungal Agents 5
`
`Table 2. Scheme for Preparing Dilution Series of Water-Insoluble Antifungal Agents to Be
`Used in Broth Dilution Susceptibility Tests for Nondermatophyte Isolates
`
`a—- "--3
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`
`Number 16
`
`Contents (Continued)
`
`Table 2A. Scheme for Preparing Dilution S
`cries of Water-Insoluble Antifungal Agents to
`Be Used in Broth Dilution Susceptibility Tests for Derrnatophyte Isolates
`
`Table 3. Scheme for Preparing Dilutions of
`Broth Dilution Susceptibility Tests..,..........
`
`Water-Soluble Antifungal Agents to Be Used in
`
`Table 4. Recommended MIC or MEC Limits for QC and Reference Strains for Broth Dilution
`
`Table 5. Composition ofRPMI-1640Medium
`
`M3 8-A3
`
`18
`
`u—l \D
`
`(NJ 5
`
`Ix.) tr.)
`
`Appendix A. Ml5Cs ofCaspofungin and
`
`Appendix B. RPMI-1640
`
`l\.J
`
`...28
`
`Appendix C. McFarland 0.5 Barium Sulfate Turbidity Standard
`
`Appendix D. Oatmeal
`
`Summary ofDelegate Comments and Subcommittee
`
`The Quality Management System
`
`..29
`
`..30
`
`Related CLSI ReferenceMaterials
`
`vi
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`
`Volume 23
`
`Foreword
`
`' M38-/X2
`
`With the increased incidence of systemic fungal infections and the growing number of antifungal agents,
`laboratory methods to guide the selection of antifungal therapy have gained greater attention. The CLSI
`Area Committee on Microbiology formed the Subcommittee on Aritifungal Susceptibility Testing, and
`data for testing filamentous fungi were collected in a series of collaborative studies. As a result, CLSI
`document M2?‘ was published with the establishment of quality control MIC ranges and the development
`of breakpoints.
`
`Based on these achievements, the subcommittee concluded that it would be useful to work toward a
`reproducible reference testing procedure for the antifungal susceptibility testing of filamentous fungi
`(moulds). A working group on filamentous fungi was formed and charged with the responsibility of
`carrying out studies to collect data and to refine the methodology to perform susceptibility testing of these
`fungal species. As a result of several collaborative studies, agreement within the subcommittee was
`achieved regarding testing conditions
`for
`the nondennatophyte moulds
`that
`included inoculurn
`preparation and inoculum size, incubation time and temperature, medium formulation, and criteria for
`MIC dcterminatiori.3‘5 This consensus method was published in 2002 as M38-A.
`
`(QC data for mould isolates as well as echinocandin testing
`In M38-A2, supplemental material
`guidelines) has been incorporated.” In addition, methods for testing dermatophyte moulds are provided,
`based on a series of consensus studies.m’”
`
`Because of its suitability for antifungal susceptibility testing of yeasts, synthetic RPM]-1640 medium was
`the test medium that the subcommittee evaluated as the potential reference medium for moulds including
`the dermatophytes.2‘3"w‘” The subcommittee has evaluated other media formulations, but the standard
`RPMI medium facilitated more consistent identification of itraconazole resistance in Aspergiiius spp.
`in
`eight
`laboratories.5 Drug stock solution preparation and dilution previously developed for antifungal
`testing of yeasts procedures (CLSI document M2?)1 also were adopted.
`
`Key Words
`
`antifungal, broth niicrodihition, dermatophytes, filamentous fungi or moulds, susceptibility testing
`
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`
`Number 16
`
`M33-A2
`
`Updated Information in This Edition
`
`Definitions {Section 41
`
`Added definition for:
`
`Minimal effective concentration (MEC)
`
`Quality control
`
`Additional Sections and Appendix
`
`Inoculnrn quantitation (Section 6.3.1)
`
`MIC and MEC reading results for:
`Echinocandins (Section 6.6.5)
`Ciclopirox (Section 6.6.6)
`Grieseofulvin (Section 6.6.7)
`Terbinatine (Section 6.6.8)
`
`Interpretation of results for:
`Ecliinocandins (Section 6.7.6)
`Ciclopirox (Section 6.1?)
`Griseofulvin (Section 6.18)
`Terbinafine ( Section 6.7.9)
`
`Appendix A. MEGS of Caspofungin and Anidulafungin:
`Al: MECS for Caspofungin
`A2: MECS for Anidulafungin
`
`Data lnclusionflslxclusion
`
`Expanded list of relevant drug concentration to include echinocandins and other suitable drug
`concentration ranges for testing dermatophytes (Section 5.4)
`
`Expanded recomrnendations on inoculum preparation of dermatophyte species (Section 6.3)
`
`Additional recommendations on incubation of Alter-nczria spp. and echinocandins (Section 6.5)
`
`Modified section on reading results to include new information on echinocandins antifungal agents and
`MIC and MEC comparison (Section 6.6.1)
`
`Expanded reconnnendations on reading results of itraconazole and new triazoles (Section 6.6.4)
`
`Tables
`
`All related tables are incorporated at the end of the document. Updates on each table include:
`
`Table 1: Solvents and Diluents for Preparation of Stock Solutions of Antifungal Agents
`Added solvents and diluent recornniendations for the following:
`Anidulafmigiii
`Griseofulvin
`Caspofungin
`Micafuligin
`Ciclopirox
`'I‘erbinafine
`
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`Volume 28
`
`M38-A2
`
`Updated Information in This Edition (Continued)
`
`Tables 2 and 2A: Scheme for Preparing Dilution Series of Water-Insoluble Antifungal Agents to Be
`Used in Broth Dilution Susceptibility Tests
`Expansion of the table to separate scheme for preparing dilution series of water insoluble antifungal
`agents for nondermatopbyte isolates {Table 2) and dermatophyte isolates (Table 2A)
`
`Deletion of Log; column
`
`Table 3: Scheme for Preparing Dilutions of Water-Soluble Antifungal Agents to Be Used in Broth
`Dilution Susceptibility Tests
`Deletion of Log; column
`
`Table 4: Recommended MIC or MEC Limits for QC and Reference Strains for Broth Dilution
`Procedures
`Addition of Mode and Incubation Time column
`
`Addition of/updated data on the following organisms:
`Paecii’0n:yce.s variotfi ATCC® MYA-3630
`Candida parap.s‘i!0sr'.s‘ ATCC® 22019: Micafungin
`Candida kmsei ATCCQC 6258: Micafungin
`ASpe.!'gfNit.s' flaws A'rcc"" 204304
`AspeJ‘gf1.-’usfi:rr:igarus ATcc® MYA-362?
`A3peJ'gflJ.'u.9fla1*u3 A'I‘CC°° MYA-3631
`Aspergflfus .ferreu.-I AT('.‘.C® MYA-3633
`Fusarium m()m1'ifi)I'.Ji:e ATCC® MYA-3629
`F‘z;.s‘arium soiam'ATCC""' 3636
`Scedosporimi-: apio.9pe:'mzmz ATCC“ MYA-3 635
`Scedosporimii api0.s'perm Ion ATCC/‘E MYA-3 63 4
`Trichophyton mentagrophyfes MRI. 1957 ATCCPD MYA-4439"’
`Tinea rubr‘un': MRI. 666 A'I‘CC® MYA-4438'”
`
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`ix
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`

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`Volume 28
`
`M33-/\2
`
`Reference Method for Broth Dilution Antifungal Susceptibility Testing of
`Filamentous Fungi; Approved Standard—Second Edition
`
`1
`
`Scope
`
`This document describes a method for testing the susceptibility of filamentous fungi (moulds) that cause
`invasive (A.rperg:'Hzrs spp., Fusarium spp., Rhfzopus oryzae [R. arrhizus], P.rezrdaHe.scheria boydii
`[Scedosporiimi apiospermum], Sporothrix schenckif, and other pathogenic moulds) and cutaneous (the
`dermatophytes Trichophyron, MfCI‘0Sp0rur?I, and Epiderinriphyron spp.) fungal
`infections to antifungal
`agents.3'5‘1° Addressed in this document are testing conditions including inoculuin preparation and
`inoculum size,
`incubation time
`and temperature, medium formulation,
`and criteria end~point
`determination.?‘9 Quality control (QC) reference ranges are also provided.6’”
`
`This standard focuses on the fully defined synthetic medium RPMI-1640 for testing of moulds because of
`the suitability ofthis test medium for antifiingal susceptibility testing of yeasts.2‘3’”"2
`
`Refer to CLSI document M2?” for drug stock solution preparation and dilution procedures.
`
`2
`
`Introduction
`
`The method described in this document is intended for testing common filamentous fungi or moulds,
`including the dermatophytes, which cause invasive and cutaneous infections, respectively. These moulds
`encompass Aspergfffus spp., Fusarium spp., Rhizopns spp., P. boydif (S. apio.rpemnnn_), S. pt-olfficans, the
`mycelial form of S. schenckii, other Zygomycetes and opportunistic monilaceous and dematiaceous
`niouldsfm as well as the dermatophyte Trichophyton, Mr'crr)sporiun, and Epidermophyron spp. [0 Caution
`should be used when interpreting the minimal
`inhibitory concentration (MIC) and minimal effective
`concentration (MEC) results for any mouldfdrug combination. The method has not been used in studies of
`the yeast or mould form of dimorphic fungi, such as Btasromyces derniariridis, C0ccidi0ide.s irnmiris,
`Coccfdiriides pomdasif, Hfsropfasma capsrrfarrtrir variety cap.su:'atuni, Peniciflinni marneffei, or S.
`schenckfi. The method also has not been used in studies of dermatophytes with the echinocandins or
`nonderniatophyte moulds with ciclopirox, griseofulvin, or terbinafine.
`
`This document is a “reference” standard developed through a consensus process to facilitate agreement
`among laboratories in measuring the susceptibility of moulds to antifungal agents. It is emphasized that
`the relationship between in virro vs in vivo data has only been evaluated in animal models.” An important
`use of a reference method is to provide a standard basis from which other methods can be developed,
`which also will result in interlaboratory agreement within specified ranges. Such methods might have
`particular advantages, such as case of performance, economy, or 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~A2.
`
`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 infectious agents and thus are
`more comprehensive than universal precautions, which are intended to apply only to transmission of
`blood-borne pathogens. Standard and universal precaution guidelines are available from the US Centers
`for Disease Control and Prevention.” For specific precautions for preventing the laboratory transmission
`
`ii"(':'iJI:‘:rtrI and LaImr'um;_1' .‘\'rin:rdr.':'tir I::.m'mrt». AH rigJ't.'.s' r'e.\‘t’n'c:('_
`
`]
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`Number 16
`
`M3 8«A2
`
`infectious agents from laboratory instruments and materials and for recommendations for the
`of all
`management of exposure to all infectious disease, refer to CLSI document M29.”
`
`.4
`
`‘v.;\
`
`4 Definitions
`
`antibiogram — overall profile of antimicrobial susceptibility results of a microbial species to a battery of
`antimicrobial agents.
`
`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
`causes a specified reduction in visible growth of a microorganism in an agar or broth dilution
`susceptibility test.
`
`quality control (QC) — the operational techniques that are used to ensure accuracy and reproducibility.
`
`5 Antifungal Agents
`
`5.1
`
`Source
`
`reference powders can be obtained commercially, directly from the
`standards or
`Antifungai
`drug manufacturer. 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 [pg]
`or International Units per mg of powder), and its expiration date. Store the powders as recommended by
`the manufacturers, or at -20 °C or below (never in a self-defrosting freezer), in a desiccator, preferably in
`a vacuum. When the desiccator is removed from the freezer, allow it to come to room temperature before
`opening (to avoid condensation of water).
`
`5.2 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. Thus, a laboratory rnust standardize its
`antifungal solutions based on assays ofthe lots ofantifungal powders used.
`
`"Use either of the following formulas to determine the amount of powder or diluent needed for a standard
`solution:
`
`W ' lt
`mg] (mg)
`
`=
`
`Volume (mL) ' Concentration (pg:’mL)
`Potency (uo mg)
`
`or
`
`V01ume(mL)_
`
`'
`I
`W "Uh
`otenc) (ug mg)
`erg ttmg)
`Concentration {pg/rnL)
`
`- P
`
`1
`
`)
`
`l
`
`(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 that required and to calculate the volume of diluent needed to
`obtain the concentration desired.
`
`Ix)
`
`C(_.‘z"I'J'i|'.f't.'Cl'r" and I.ubr:."a!a:j_v SImiri'w'uls' i’m‘rim.'c. AH rigi't.“.i' r:?.v¢’r‘t-‘ml.
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`Volume 28
`
`M3 8-A2
`
`Example: To prepare 100 mL of a stock solution containing 1280 ttg of antifungal agent per mL with
`antifungal powder that has a potency of 750 ttg/mg, use the first formula to establish the weight of
`powder needed:
`
`1230 tigfmL
`1001111.
`(Desired Cone.) = 1 TKO‘? mg
`Weight : (Target Vol.)
`(ms)
`?50 us/ms
`
`(Potency)
`
`(3)
`
`Because it is advisable to weigh a portion ofthe powder in excess of that required, deposit powder on the
`balance until approximately 180 mg is reached. With that amount of powder weighed, use formula (2)
`above to determine the amount of diluent to be measured:
`
`182.6 mg
`?50 pg/mg
`Volume m (Powder Weight) ' (Potency)
`(mt) _
`1280 ttg/mL
`
`=10'i".0mL
`
`(Desired Concentration)
`
`(-'1)
`
`Therefore, dissolve the 182.6 mg ofthe antifunga] powder in lU7'.0 mL of diluent.
`
`5.3 Preparing Stock Solutions
`
`least 1280 ttgfrnls or 10 times the highest
`Prepare antifungal stock solutions at concentrations of at
`concentration tested, whichever is greater. Some antifungal agents of limited solubility, however, require
`lower concentrations. In all cases, information provided by the drug manufacturer should be considered as
`part of determining solubility.
`
`5.3.1
`
`Use of Solvents Other Than Water
`
`Somc drugs must be dissolved in solvents other than water (see Table 1). Information on the solubility of
`an antifungal compound should be included with the drug. Such drugs should be dissolved at
`concentrations at least 100 times higher than the highest desired test concentration. Commonly used
`agents include analytical grade dimethyl sulfoxide (DMSO), ethyl alcohol, polyethylene glycol, and
`carboxy methyl cellulose. When such solvents are used, a .s'err'es of dilirrions 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 Table 1).
`This procedure avoids dilution artifacts that result from precipitation of compounds with low solubility in
`aqueous media.
`
`For example, to prepare for a broth microdilution test series containing a water-insoluble drug that can be
`dissolved in DMSO, for which the highest desired test concentration is 16 ttg/1nL, first weigh 4.8 mg
`(assuming 100% potency) of the antifiingal powder and dissolve it in 3.0 mL DMSO. This will provide a
`stock solution at 1600 tig/mL. Then prepare further dilutions of this stock solution in DMSO (see Table
`2). Diiute the solutions in DMSO 1:50 in test medium (see Section 6.2), and a further 2x (twofold)
`dilution will occur when inoculated (see Section 6.4), 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 Section 5.2 should be applied.
`
`it 't‘i.'ir1‘urI amt’ £w'mmrurj1' .S'.=‘a:rdam“.\' Iii.s‘rr'r.-He. AH r:'ghr.~' I'e.vr'r't‘cd.
`
`'3
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`Number 16
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`5.3.2
`
`Filtration
`
`M3 8-A2
`
`Normally, stock solutions do not support contaminating microorganisms and they can be assumed to be
`sterile. If additional assurance of sterility is desired, filter them through a membrane filter. Do not use
`paper, asbestos, or sintered glass filters, which may adsorb appreciable amounts of certain antifungal
`agents. Whenever filtration is used,
`it is important to document the absence of adsorption by results of
`appropriate assay procedures.
`
`5.3.3
`
`Storage
`
`Dispense small volumes of the sterile stock solutions into sterile polypropylene or polyethylene vials,
`carefillly seal, and store (preferably at —60 °C or below, but never at a temperature greater than -20 °C).
`Remove vials as needed and use the same day. Discard any unused