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Nutrientbrothforfastidiousorganismsorsterilesalinefor nonfastidiousorganisms.
2.
0.5McFarlandstandardforadjustingtheturbidityofthe inoculums.
3. 4.
5.
lotthatgivesasatisfactoryqualitycontrolresults.ThePHmust be7.2to7.4,andthedepthmustbeapproximately4mm.
6.
NonCO23537Cincubatorfornonfastidiousorganismsor 5%CO2incubationforH.infS.pneumoniaeN.meningititis QUALITY CONTROL Antibioticdiscsforsusceptibilitytestingarecheckedweekly utilizingappropriateATCCreferencestrains.Inaddition,QC testingwillbeperformedanytimewhenantibioticwithanewlot numberisusedrepeatthetesting.Documentanycorrective actionintheQClogbook.ThediscstestedforQCmustbethe samediscsusedwiththepatientspecimens.Tolerancelimitsfor antimicrobialpotencyarebasedonCLSIguidelines.Ifthezone rangelimitsareexceed,theLabDirectormustbeimmediately notifiedandnosensitivityresultswillbereported.
E coli ATCC 25922 Pseudomonas aeroginosae ATCC 27853 S. aureus ATCC 29213
ALSO
ORGANISMS
QC
STRAINS FOR
MUST BE INCLUDED
QC ORGANISMS
PROCEDURES
1.
Preparationofinoculum : a. Withasterilewireloop,touchthetopoftwotofive similarappearing,wellisolatedcoloniesonanagar platecultureaccordingtothesizeofcoloniesasfollows: largecoloniesascitrobactertouchonlythe quarterofitssize,smallcoloniesasstrepttouch fivecolonies,whilemoderatesizedcolonies touchonlytwocolonies. b. Emulsifythemin5mLofsterilephysiologicalsalineor nutrientbrothwiththehelpofvortex. c. Theturbidityoftheemulsificationisadjustedto
0.5McFarlandstandard.Turbidityismatched
againstaprintedcardorsheetofpaperinagoodlight.
3
d.
2.
Testingofantibiotics:
a.
b.
Gentlytampeachdiskdownontotheagartoprovide uniformcontact.
4
c.
INTERPRETATION
Withtheuseofaruleroratemplate,thezonesof completegrowthinhibitionaroundeachofthedisksarecarefully measuredtowithinthenearestmillimeter;Allmeasurementare madebytheunaidedeyewhileviewingthebackofthepetridish withreflectedlightagainstablack,nonreflectingbackground. Theplatesshouldbeviewedfromadirectlyverticallineofsightto avoidanyparallaxthatmayresultinmisreading. Aninterpretivecorrelate(susceptible,moderately susceptible,intermediateorresistant)isprovidedbyreferenceto publishedCLSIguidelines.
LIMITATIONS
1 Donotmoveadiskonceithascontactedtheagar,becausesomeof thedrugdiffusesalmostimmediately. 2 Susceptibilityplatespreparedwithbloodmustbeviewedfromthe agarsurfaceandmeasurementsmadewiththecoverofthePetri dishremoved.
3-
Whentestingstaphylococciagainstmethicillinoroxacillinor enterococciagainstvancomycin,incubationshouldbefor24hours.
Presenceofdistinctcolonieswithinthezoneofinhibition(2ry colonies)representeithermutantofthesamespeciesthataremore resistanttotheantimicrobialagentthanthemajorportionofthe bacterialstrainbeingtestedorthecultureisnotpureandthe separatecoloniesareofadifferentspecies.Ifitisdeterminedthat theseparatecoloniesrepresentavariantofamutantstrain,the bacterialspeciesbeingtestedmustbeconsideredresistant.Ifitis determinedtobeadifferentspecies,returntotheculturePetridish andrealizewhetheritisamissedcolonyoracontamination.If missed,doaseparateantibiogramfortheisolate.
7-
4-
GROUPU:Agentsthatshouldbetested&reportedonlyon isolatesfromurine.
56-
GROUP C ( GROUP 2) for H.Infleuanza & Enterobacteriacae & antimicrobial combinations will be done . IN THE THRID DAY Another combination will be tested in multi resistant strains
Antimicrobial agents with FDA clinical indication that should be considered for routine testing
Acinetobacter Fortum(CAZ) Tienam(IPM)orMeronam(MEM) Unasyn(SAM) Ciprofloxacin(CIP)orLevofloxacin(LEV)
H.Influenza Ampicillin(AMP) Sutrim(SXT) Unasyn(SAM) Cefuroxime(CXM) Cefotaxime(CTX) Rocephine(CRO) Fortum(CAZ) Azithromycin(AZM) Augmentin(AMC) Cefopodoxime(CPD) Ciprofloxacin(CIP) orLevofloxacinLEV Tienamormeronam
NisseriaGonorrhea
NB : Testing of B lactamase is mandatory for both isolates using either penicillin disc or nitrocefin sticks .
Sutrim(SXT)
Burkholderia
Stenotrophomonas
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.Haemolyticstrept(pyogens)
Ampicillin(AMP) Pencillin(P) Erythromycin(E)/AZM Clindamycin(Cd) Maxipime(FEP)
S.pneumoniae
Erythromycin(E)/AZM Oxacillin(ox)testing penicillin Sutrim(SXT) Clindamycin(Cd) Levofloxacin(LEV) Ofloxacian(OFX) Tetracycline(TE) Vancomycin(VA) OptochineOP
Rocephine(CRO)
Ofloxacin(OFX) BacitracinBC
N.B If oxacillin sensitive S.pneumoniae report blindly all penicillins & cephalosporins sensitive but if resistant MIC for 3rd generation cephalosoprins is mandatory
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Staph(nonurine)
Sutrim(SXT)
Tetracycline(TE)
Erythromycin(E)
CIPorOFXorLEVinMSSA DOorTE
Gentamycin(CN) Caphalothin(CF)
Furadantin(F) Sutrim(SXT)
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ESBLconfirmationinKlebsiella,Ecoli&Proteus
(bloodisolatesonly):
DISCAPPROXIMATIONTEST
AMCbetterorSAM(atthecenteroftheplate) CPDaloneorCAZ&CTXtogether(2.5cmaroundAMC fromcentertocenter) FOX30g(besidecephalosporin) TZPORSCF FEP(mandatory) IPMORMEM(besidecephalosporin) OFXORLEV/NORinurineonly CNORAK Inurineaddfuradantin
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IPM OR MEM
TZP or SCF
CHART FOR
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Pseudomonas Fortum(CAZ)
Augmentin(AMC) Cefepime(FEP)
cefotrioxne(CRO)or Cefobid(CFP)
Cefotaxime(CTX) Sulperazone(SCF)
Ciprofloxacinofloxacinor Tienam(IPM)or
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GramnegativebacilliotherthanEcoli&Klebsiellain urine:
Norfloxacin(NOR) OFX/LEV/CIP Furadantin(F) Lomefloxacin Sutrim(SXT) Cefobid(CFP) Augmentin(AMC) Gentamycin(CN) Carbencillin Tazocin FEP SCF
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CLSIrecommendations:
1. Thefollowingantimicrobialagentmaybeappearactivein vitrobutarenoteffectiveclinicallyandshouldnotreportedas susceptible:
Salmonellaand Shigella MRSA 1stand2ndgenerationcephalosporinsand aminoglycoside All.Lactam,carpenams,
cephalosporins,clindamycin,SXT Yersinia Listeria ESBL Ampicillin resistant enteroccoci .Lactam Cephalospornis Penicillin,cephalosporins&azactam Penicillin,Blactamaseinhibitor(AMC, SAM)&carbapenemsthemechanismis alteredPBPs
2.
Warning(CSF):Thefollowingantimicrobialagentshould notberoutinelyreported: a.
b.
c.
d.
e. f.
3.
RecommendationP/AMP/cefazolin/clindamycin/ erythromycin. 4. P.mirabilisshouldbeaddedtoE.coliandK.pneumoniaein screeningforESBLinbacterimicisolatesonly(blood)because reportsofESBLinnonbacterimicisolateshavebeen relativelyrareduetolowfrequencyofplasmidconjugate. 5. Levofloxacinshouldbeusedforstenotrophomonaswith SXT. 6. Susceptibilitytestingisnotrecommendedfor S.saprophyticusinurinaryisolatesNOV(R)<16mm. 7. LabshouldidentifyS.lugdunensis(anuncommon)butone causeofendocarditis:(PYRtest+veandornithinede carboxylase+ve).
8.
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9.
Fororganisms(campy,corynebacterium,bacillusspp.) consultationwithaninfectiousdiseasespecialistis recommendedforguidanceindeterminingtheneedfor susceptibilitytestingandintheinterpretationofresults, publishedreportsinmedicalliteratureandcurrentconsensus recommendationsfortherapyofuncommonisolates,may obviatetheneedfortesting.Ifnecessaryadilutionmethod usuallywillbethemostappropriatetestingmethodandthis mayrequiresubmittingtheorganismtoareferencelab. NB:
CephalothinCF/CF/CListhedisc
representativeforthe1st,2ndgenercephaland cefopodoxime
Tetracycline:isrepresentativeforDo,
minocyclin.
Erythromycine:isrepresentativefor
macrohide.
10.
Oxacillinscreeningdiskdiffusionusedtodetecthighrate ofpenicillinresistanceinS.pneumoniae(>20mm
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susceptible,<19mm(doMICtesting)andcorrelatewiththe bodysiteitiscollectedfrom.
11.
12.
13.
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15.AsregardsESBLdetection:
TestingofcephamycinsisrecommendedinESBL producingisolates.
TheseenzymescanbeinducedbycertainAbs,AAs,or bodyfluids.
MeronamisthemostactivewithMICsgenerallylower thanthoseofIPM(0.030.12mg/mlvs0.060.5mg/ml).
AlsoESBLactivityisinhibitedbyclavulonicacid,the onlyinfectionsthatcanbetreatedsafelywith
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ANTIMICROBIAL
Diskapproximationtest :
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Principle: Thismethodhasbeenexploredtoassessprimarilyina qualitativefashiontheinteractionofantimicrobialsasthey diffusethroughagarplatesseededwithatestorganism. Advantages: Simple. Theuseofreadilyavailablematerials(discsand MullerHintonagar). Disadvantages: Qualitativemethodonly. Lowsensitivityandspecificitycomparedtodilution methods. i.e.: Theresultsofthistestmaydifferfromresultsobtainedwhen thesameagentsandorganismaretestedinliquidmedia.
Procedure:
25
Afterovernightincubation(1618hrs)at37Ctheplate arereadyforexamination.
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Example of antibiotic combinations used for multi resistant organisms "by Dilution methods":
1. Pseudomonas: Bactericidal: Ciprofloxacinandtienam(CIPandIPM). CiprofloxacinandAmikin(CIPandAK). CiprofloxacinandAzactam(CIPandATM). CiprofloxacinandFortum(CIPandCAZ).
LevofloxacinandMaxipime(LEVandFEP).
CiprofloxacinandMaxipime(CIPandFEP).
LevofloxacinandMeronam(LEVandMEM).
CiprofloxacinandTazocin(CIPandTZP).
LevofloxacinandTazocin(LEVandTZP). LevofloxacinandGentamycin(LEVandCN). TazocinandGentamycin(TZPandCN).important 27
TazocinandTienam(TZPandIPM). Bacteriostatic:
AugmentineandAmpicillin(AMCandAMP).notused VanocomycinandCarbencillin(VAandPip).notused
AzactamandMaxipime(ATMandFEP).
2.
3. o
o o o o o o
4.
5.
CiproflxocinandAmpicllin(CIPandAMP).
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