Antibacterial Sensitivity

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    Antimicrobial SusceptibilityAntimicrobial SusceptibilityTesting for BacteriologyTesting for Bacteriology

    LaboratoriesLaboratories

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    Objectives:Objectives:At the conclusion of this presentation participants will be able toAt the conclusion of this presentation participants will be able to

    jj Explain how to implement currentExplain how to implement current NCCLSNCCLS antimicrobialantimicrobialsusceptibility testing (AST) and reportingsusceptibility testing (AST) and reportingrecommendations.recommendations.

    jj DescribeDescribe reliable methodsreliable methods for detecting resistancefor detecting resistanceamong:among:

    EnterobacteriaceaeEnterobacteriaceae

    Pseudomonas aeruginosaPseudomonas aeruginosa

    Staphylococcus aureusStaphylococcus aureus

    Enterococcus spp.Enterococcus spp.

    Streptococcus pneumoniaeStreptococcus pneumoniae andand Streptococcus spp.Streptococcus spp.

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    At the conclusion of this presentationAt the conclusion of this presentation

    Participants will be able to(cont)Participants will be able to(cont)

    jjDescribeDescribe effective reportingeffective reporting of ASTof AST

    results.results.jj List steps that can be taken toList steps that can be taken to verifyverify

    AST resultsAST results obtained on bacteriaobtained on bacteria

    isolated from patients.isolated from patients.

    jjDiscuss NCCLSDiscuss NCCLS--recommendedrecommended QCQCproceduresprocedures and how to troubleand how to trouble--shootshoot

    outout--ofof--control QC resultscontrol QC results

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    What Does the Laboratory Need to KnowWhat Does the Laboratory Need to Know

    about Antimicrobial Susceptibility Testing (AST)about Antimicrobial Susceptibility Testing (AST)??

    jjWhichWhich organismsorganisms to test?to test?

    jjWhatWhat methodsmethods to use?to use?

    jjWhatWhat antibioticsantibiotics to test?to test?

    jjHow toHow to reportreport results?results?

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    What Does a Laboratory Need to KnowWhat Does a Laboratory Need to Know

    about AST? (cont)about AST? (cont)

    jjHow to determine theHow to determine the clinical significanceclinical significance

    of results?of results?jjHow to ensureHow to ensure accuracyaccuracy of results?of results?

    Quality control / quality assuranceQuality control / quality assurance

    jjWhen to call the MD, infection control,When to call the MD, infection control,

    public healthpublic health??

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    What Does a Laboratory Need to KnowWhat Does a Laboratory Need to Know

    about AST? (cont)about AST? (cont)

    jjWhen toWhen to ask forask for helphelp??

    jjWhere to go forWhere to go forhelphelp??

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    Brief Review of Routine ASTBrief Review of Routine AST

    MethodsMethods

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    Routine Susceptibility TestsRoutine Susceptibility Tests

    jjDisk diffusion (Kirby Bauer)Disk diffusion (Kirby Bauer)

    jjBroth microBroth micro--dilution MICdilution MIC

    NCCLS reference methodNCCLS reference method

    jjEtestEtest

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    Disk DiffusionDisk Diffusion

    TestTest

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    Select colonies

    Prepare inoculum

    suspension

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    Mix well

    Standardize inoculum

    suspension

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    Swab plate

    Remove sample

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    Add disks

    Incubate overnight

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    Measure ZonesMeasure ZonesTransmitted

    LightReflected

    Light

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    Measure ZonesMeasure ZonesTransmitted Light*Transmitted Light*

    jj

    StaphylococciStaphylococci

    oxacillin, vancomycinoxacillin, vancomycinjjEnterococciEnterococci vancomycinvancomycin

    *Measure all other drug/bug*Measure all other drug/bug

    combinations with reflected lightcombinations with reflected light

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    Zone Interpretive CriteriaZone Interpretive Criteria

    (mm)(mm)

    DrugDrug

    DiskDisk

    contentcontent

    (ug)(ug)ResRes IntInt SuscSusc

    cefazolincefazolin 3030 e 1414 1515--1717 uu 1818

    gentamicingentamicin 1010 ee 1212 1313--1414 uu 1515

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    Disk Diffusion TestDisk Diffusion Test

    jjQualitative resultsQualitative results

    SusceptibleSusceptible

    IntermediateIntermediate may respond if infectionmay respond if infectionis at body site where drug concentratesis at body site where drug concentrates(e.g. urine) or if higher than normal dose(e.g. urine) or if higher than normal dosecan be safely givencan be safely given

    ResistantResistant

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    Modify methods forModify methods for

    fastidious bacteriafastidious bacteria

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    Clinical Conditions when MICs are UsefulClinical Conditions when MICs are Useful

    jj EndocarditisEndocarditis

    jjMeningitisMeningitis

    jj SepticemiaSepticemia

    jjOsteomyelitisOsteomyelitis

    jj Immunosuppressed patients (HIV, cancer, etc.)Immunosuppressed patients (HIV, cancer, etc.)

    jj Prosthetic devicesProsthetic devicesjj Patients not responding despite SPatients not responding despite S

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    MICMICjjMinimal inhibitory concentrationMinimal inhibitory concentration

    jjThe lowest concentration ofThe lowest concentration of

    antimicrobial agent thatantimicrobial agent that inhibitsinhibits thethegrowth of a bacteriumgrowth of a bacterium

    jj Interpret:Interpret:

    SusceptibleSusceptible IntermediateIntermediate

    ResistantResistant

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    Inoculum PreparationInoculum Preparation

    MIC TestingMIC Testing(NCCLS Reference Method)(NCCLS Reference Method)

    jjStandardize inoculum suspensionStandardize inoculum suspension

    jjFinal inoculum concentrationFinal inoculum concentration

    33 5 x 105 x 1055 CFU/mlCFU/ml

    (3(3 5 x 105 x 1044 CFU/well)CFU/well)

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    Microdilution MIC tray

    Prepare inoculum

    suspension

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    Dilute & mix inoculum

    suspension

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    Pour inoculum

    into reservoir and

    inoculate MIC tray

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    Inoculatepurity plate

    Incubate overnight

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    ExaminingExamining

    purity platepurity plate

    Reflected lightReflected light

    Transmitted lightTransmitted light

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    Optimal Use of Purity PlatesOptimal Use of Purity Plates

    jj SubSub final test suspensionfinal test suspension to nonto non--selective mediumselective medium

    (after inoculating MIC test)(after inoculating MIC test)jj Streak forStreak forisolationisolation (avoid several specimens per plate(avoid several specimens per plate

    -- may not reveal contaminants if no isolated colonies)may not reveal contaminants if no isolated colonies)

    jj ExamineExamine before reading MICbefore reading MIC (usually at 16(usually at 16--20 h)20 h)

    jjReRe--incubateincubate if antibiogram questionableif antibiogram questionable

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    Read MICs

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    Reporting Time: Disk Diffusion or MICReporting Time: Disk Diffusion or MIC

    jjStaphylococciStaphylococci oxacillin, vancomycin, cefoxitinoxacillin, vancomycin, cefoxitin

    jjEnterococciEnterococci vancomycinvancomycin

    jj ResistanceResistance may be reported any time growth ismay be reported any time growth isobserved after a minimum of 16 h incubationobserved after a minimum of 16 h incubation

    jjAfter minimum of 16 h, read test:After minimum of 16 h, read test:

    ififRR, report, report

    IfIfSS, re, re--incubate and read again at 24 hincubate and read again at 24 h

    NCCLS M2NCCLS M2--A8, M7A8, M7--A6A6

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    MIC on a stripMIC on a stripabbiodisk.comabbiodisk.com

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    S. pneumoniaeS. pneumoniae

    Penicillin MIC =Penicillin MIC =

    33 QQg/mlg/ml

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    MIC Interpretive Criteria (MIC Interpretive Criteria (QQg/ml)g/ml)

    DrugDrug SuscSusc IntInt ResRes

    cefazolincefazolin ee 88 1616 uu 3232

    gentamicingentamicin ee 44 88 uu 1616

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    MICs RecommendedMICs Recommended (NCCLS M100(NCCLS M100--S14)S14)

    jj ViridansViridans StreptococcusStreptococcus penicillinpenicillin

    jj

    S. pneumoniaeS. pneumoniae

    penicillin, cefotaxime/ceftriaxonepenicillin, cefotaxime/ceftriaxone(sterile sites)(sterile sites)

    jj EnterococcusEnterococcus vancomycin Int resultsvancomycin Int results

    jj StaphylococcusStaphylococcus vancomycin zonevancomycin zone ee14 mm14 mm

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    CreditsCredits

    Janet Fick Hindler, MCLS MT(ASCP)Janet Fick Hindler, MCLS MT(ASCP)UCLA Medical CenterUCLA Medical Center

    Los Angeles, CALos Angeles, CA