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CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests

Romney M. Humphries, Jane Ambler, Stephanie L. Mitchell, Mariana Castanheira, Tanis Dingle, Janet A. Hindler, Laura Koeth, Katherine Sei
Colleen Suzanne Kraft, Editor
Romney M. Humphries
aAccelerate Diagnostics, Tucson, Arizona, USA
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Jane Ambler
bWockhardt, Morton Grove Pharmaceuticals, Morton Grove, Illinois, USA
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Stephanie L. Mitchell
cClinical Microbiology Laboratory, Children's Hospital of Pittsburgh of UPMC and University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
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Mariana Castanheira
dJMI Laboratories, North Liberty, Iowa, USA
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Tanis Dingle
eProvincial Laboratory for Public Health, Edmonton, Alberta, Canada
fDepartment of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
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Janet A. Hindler
gDepartment of Pathology & Laboratory Medicine, University of California, Los Angeles, Los Angeles, California, USA
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Laura Koeth
hLaboratory Specialists, Inc., Westlake, Ohio, USA
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Katherine Sei
iBeckman-Coulter, West Sacramento, California, USA
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Colleen Suzanne Kraft
Emory University
Roles: Editor
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DOI: 10.1128/JCM.01934-17
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    FIG 1

    Cefazolin (left) and ceftriaxone (right) MIC distributions for Escherichia coli (https://mic.eucast.org/Eucast2/SearchController/search.jsp?action=performSearch&BeginIndex=0&Micdif=mic&NumberIndex=50&Antib=-1&Specium=162). Categorical agreement rates are expected to be lower for cefazolin, because the breakpoints bisect the wild-type MIC distribution. S, susceptible; I, intermediate; R, resistant.

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  • TABLE 1

    Variables to be detailed in Materials and Methods for CLSI BMD reference method

    ParameteraM07 standardData to be documented in study publication
    Test medium for nonfastidious bacteriaCA-MHB manufactured according to ISO technical standard 16782:2016 (18)Brand of CA-MHB
    Method for cation adjustment
    Any special requirements for handling CA-MHB for specific antimicrobial agents
    Test medium for fastidious bacteriaAs stipulated by M100 standard (6) or M45 guidelineBrand of alternative medium
    Sources and concentrations of supplements
    Any special requirements for handling alternative media for specific antimicrobial agents
    Antimicrobial stock solutionsAntimicrobial powder formulation designed for AST, obtained from drug manufacturer, USP, or other appropriate commercial sources; pharmacy-grade parenteral formulations should not be usedSource of antimicrobial powder and solvent and diluent used to make stock solutions
    BMD panel preparationDilution of stock solution and dispensing of 0.05 or 0.1 ml/well using pipette or dispensing deviceMethod for preparation of panels (individual or batch), volume per well, and storage conditions
    Range of concentrations tested
    Number of lot numbers of panels used in study
    Inoculum suspension preparation and panel inoculationDirect colony suspension method or growth method to achieve 0.5 McFarland standardInoculum suspension preparation and standardization method
    Dilution and inoculation of wells with pipette or inoculator device (final target of 5 × 105 CFU/ml)Method for preparation of intermediate dilutions (diluent and volumes)
    Purity plate prepared (advised) (ahWG consensus is to prepare for each isolate)
    Colony counts performed periodically
    Method and volume for panel inoculation
    Method of purity plate preparation
    Targeted final organism concentration in wells
    Procedure, acceptable range, and indications for colony counts
    Indication of whether same inoculum is used for both BMD and cAST
    Incubation procedurePrecautions taken to maintain consistent incubation temperatures and to prevent drying of panelsIncubation parameters
    Temperature, atmosphere, and time varying by species (consult M07)
    Reading of endpointsComplete inhibition of growth measured with naked eye (see exceptions to complete inhibition in M07b) (ahWG recommends ≥2 independent readers to determine MICs for each panel)Viewing device used to facilitate reading
    Number of independent readers
    Observation of any “equivocal” endpoints and how they were read (e.g., trailing) (obtaining photographs to illustrate any unique reading recommendations, if possible)
    Mechanism used to arbitrate discrepant MICs between readers
    QCAt minimum, following M07 QC recommendationsCLSI-recommended QC strains used
    Any additional QC strains used and rationale for strain selection
    • ↵a CA-MHB, cation-adjusted Mueller-Hinton broth; ISO, International Organization for Standardization; USP, U.S. Pharmacopeia; BMD, broth microdilution; cAST, commercial antimicrobial susceptibility test; ahWG, ad hoc working group; CLSI, Clinical and Laboratory Standards Institute; QC, quality control.

    • ↵b Exceptions include the evaluation of trimethoprim-sulfamethoxazole, where 80% growth inhibition is read as the endpoint.

  • TABLE 2

    Variables to be detailed in Materials and Methods for the cAST under evaluation

    ParameteraTesting considerationsData to be documented in study publication (if applicable)
    Regulatory status of cASTcASTs that have not undergone regulatory approval may not be optimizedRegulatory status of cAST (RUO, IUO, or IVD)
    Version/edition of cAST hardware/softwareNewer approved versions/editions are often designed to enhance performanceVersion/edition of hardware/software
    Range of antimicrobial agent concentrations testedSome concentration ranges may differ from those in BMD panelsRange of concentrations tested and description of how MICs are evaluated if concentrations in cAST panel differ from those in BMD panel
    Lot numbersPerformance variability among lot numbers of cAST panels or gradient diffusion strips may be encounteredNumber of lot numbers evaluated
    Inoculum suspension preparation and panel inoculationSingle inoculum for reference BMD and cAST?Indication of whether same inoculum was used for both BMD and cAST
    QCAcceptable ranges for CLSI QC strains for cASTs may be slightly different from those published in M100Manufacturer's QC instructions followed precisely
    • ↵a cAST, commercial antimicrobial susceptibility test; RUO, research use only; IUO, investigational use only; IVD, in vitro diagnostic; QC, quality control.

  • TABLE 3

    Additional variables to be detailed when cAST method is gradient strip or disk diffusion method

    ParameteraTesting considerationsData to be documented in study publication (if applicable)
    MHA or supplemented MHAPerformance may vary across brands and lot numbers of MHABrand of MHA
    Disks and gradient strips are typically developed with a single brand of MHANumber of lot numbers evaluated
    Troubleshooting of MHA source if performance issues are noted (determining whether brand used is different from that used by gradient strip manufacturer)
    Method used to determine MIC endpoints and management of colonies within zoneSome use a magnifying glass or digital camera/software for endpoint measurementMethod used to determine MIC endpoints, including managing any colonies within zone or equivocal endpoints
    MIC values may differ depending on how colonies in the zone of growth inhibition are evaluatedAny procedure for rounding between 2-fold dilutions
    Interpretation of endpoints may be challenging; follow manufacturer's instructions precisely (obtain photographs for guidance, if possible)
    • ↵a MHA, Mueller-Hinton agar.

Additional Files

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  • Supplemental material

    • Supplemental file 1 -

      Tables S1 (Calculation of method comparison agreement and error categories and acceptable criteria), S2 (Acceptance performance rates for cASTs, by the error rate bound method, for antimicrobials with an intermediate category), S3 (Acceptance performance rates for ASTs, by the error rate bound method, when no intermediate category exists), and S4 (Hypothetical examples of arbitrating discrepancies between cAST and BMD)

      PDF, 304K

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CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests
Romney M. Humphries, Jane Ambler, Stephanie L. Mitchell, Mariana Castanheira, Tanis Dingle, Janet A. Hindler, Laura Koeth, Katherine Sei on behalf of the CLSI Methods Development and Standardization Working Group of the Subcommittee on Antimicrobial Susceptibility Testing
Journal of Clinical Microbiology Mar 2018, 56 (4) e01934-17; DOI: 10.1128/JCM.01934-17

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CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests
Romney M. Humphries, Jane Ambler, Stephanie L. Mitchell, Mariana Castanheira, Tanis Dingle, Janet A. Hindler, Laura Koeth, Katherine Sei on behalf of the CLSI Methods Development and Standardization Working Group of the Subcommittee on Antimicrobial Susceptibility Testing
Journal of Clinical Microbiology Mar 2018, 56 (4) e01934-17; DOI: 10.1128/JCM.01934-17
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  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • STUDY DESIGN
    • EVALUATION OF RESULTS
    • QUALITY CONTROL
    • SUMMARY
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

antimicrobial susceptibility testing
CLSI
evaluations

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