Page | 1 Anti-Malaria Campaign (AMC) SOP for malaria Rapid Diagnostic Test Kits SOP title: Performing malaria Rapid Diagnostic Test Kits SOP No. 01 Revision No. 0.0 Effective Date 15.03.2015 Replacement no. Dated Page no. Prepared by: Anti Malaria Campaign Date 06.03.2015 Approved: Date 10.03.2015 1. BACKGROUND: Malaria Rapid Diagnostic Test (RDT) Kits can be used as a supplementary measure to microscopy to provide rapid diagnosis of malaria. 2. PURPOSE: This Standard Operating Procedure outlines the procedure of performing a malaria Rapid Diagnostic Test. 3. SCOPE: This procedure applies to all Laboratory Technicians using malaria Rapid Diagnostic Test Kits provided by the Anti Malaria Campaign. 4. Policy: This procedure shall be reviewed whenever a new stock of malaria Rapid Diagnostic Test Kits is procured. Any deviation from the procedures outlined shall be recorded and reported. 5. REQUIREMENTS (Figure 1): 5.1 Malaria Rapid Diagnostic Test Kits (Carestart TM Malaria HRP2/pLDH (Pf/PAN) Combo) 5.2 Sterile lancets 5.3 Alcohol swab 5.4 Pipette (provided with the test kits with a marking at 5 µL level; or a micropipette to get 5 µL of blood) 5.5 Assay buffer 5.6 Puncture resistant container (sharps bin) 5.7 Infectious and Non- Infectious bins 5.8 All-purpose disinfectant 5.9 Laboratory gown/ coat 5.10 Gloves 5.11 Soap/liquid hand wash 5.12 Instruction leaflet 5.13 H/AMC/P1 form 5.14 RDT Result Report form 5.15 Pen 5.16 Timer or clock
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Anti-MalariaCampaign(AMC)
SOPformalariaRapidDiagnosticTestKits
SOPtitle: PerformingmalariaRapidDiagnosticTestKits
SOPNo. 01 RevisionNo. 0.0 EffectiveDate 15.03.2015
Replacementno. Dated Pageno.
Preparedby: AntiMalariaCampaign Date 06.03.2015
Approved: Date 10.03.2015
1. BACKGROUND:
MalariaRapidDiagnosticTest(RDT)Kitscanbeusedasasupplementarymeasuretomicroscopytoprovide
rapiddiagnosisofmalaria.
2. PURPOSE:
ThisStandardOperatingProcedureoutlinestheprocedureofperformingamalariaRapidDiagnosticTest.
3. SCOPE:
ThisprocedureappliestoallLaboratoryTechniciansusingmalariaRapidDiagnosticTestKitsprovidedbythe
AntiMalariaCampaign.
4. Policy:
ThisprocedureshallbereviewedwheneveranewstockofmalariaRapidDiagnosticTestKits isprocured.
Anydeviationfromtheproceduresoutlinedshallberecordedandreported.
5. REQUIREMENTS(Figure1):
5.1 MalariaRapidDiagnosticTestKits(CarestartTMMalariaHRP2/pLDH(Pf/PAN)Combo)
5.2 Sterilelancets
5.3 Alcoholswab
5.4 Pipette(providedwiththetestkitswithamarkingat5µL level;oramicropipettetoget5µLof
blood)
5.5 Assaybuffer
5.6 Punctureresistantcontainer(sharpsbin)
5.7 InfectiousandNon-Infectiousbins
5.8 All-purposedisinfectant
5.9 Laboratorygown/coat
5.10 Gloves
5.11 Soap/liquidhandwash
5.12 Instructionleaflet
5.13 H/AMC/P1form
5.14 RDTResultReportform
5.15 Pen
5.16 Timerorclock
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Figure1:ComponentsinaBoxofCareStartTMRDTTestKit Figure2:PartsoftheRDT
6. DescriptionoftheRDT:
CarestartTMMalariaHRP2/pLDH(Pf/PAN)CombofromACCESSBIO(CATNO.G0131)istheRDTpresently
distributedfreeofchargebytheAntiMalariaCampaignforrapiddiagnosisofmalaria.
The test kit is designed to detect 2 parasite antigens found in infected blood, namely parasite Lactate
Dehydrogenase (pLDH)present in asexual and sexual stagesofPlasmodiumvivax,P. falciparum,P.ovaleandP.malariaeandHistidineRichProtein2(HRP2)presentonlyinPlasmodiumfalciparum(trophozoites&
immature gametocytes). pLDH is an enzyme found only in live parasites and therefore is related to the
peripheralparasitedensityandisclearedfrombloodstreamsoonafterparasiteclearance(about2days).
HRP2isawatersolublepersistingantigenthatisfoundinthecirculatingperipheralblood.
7. PartsoftheRDT(Figure2)
RDTtestkitcontainsthefollowingcomponents.
• Samplewell(denotedbySinthetestkit)• Bufferwell(denotedbyAinthetestkit)• Nitrocellulosestrip(showingtestandcontrollines)
8. TestingProcedure
8.1. Record informationofthepatientdata inaH/AMC/P1form(SerialNo.,date,name,age,sex,addressandhistoryoffever)
8.2. Collectalltheitemsrequiredtoperformthetest.IftheRDTshavebeenrefrigerated,itisbettertogettheRDTtotheroomtemperature.
8.3. Takeoutthetestkitfromthesachetandkeepitonaflatleveledsurface.
8.4. Writeonthetestkitwithamarkerpen,penorapencil,thenameofthepatient,date.
8.5. Collectbloodforthetest.8.5.1. Iffingerprickbloodisused
8.5.1.1. Holdingpatient'slefthandpalmupwards,selectthethirdfingerfromthethumb.(Thebigtoecanbeusedwithinfants).
8.5.1.2. Clean the finger with a piece of cotton soaked with 70% alcohol, using firm strokes toremovegreaseanddirt from theballof the finger.Puncture theballof the fingerwithasterilelancet,usingaquickrollingaction.
Samplewell
Bufferwell
Nitrocellulose
strip
stripSample
well
1.assaybuffer2.bloodlancet3.alcoholpad4.pipette5. test kit (inaluminumpouch)
13
5
4
2
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8.5.1.3. Applygentlepressuretothefingertoexpressthefirstdropofbloodandwipeitawaywithadrypieceofcottonwool.(Makesurethatnostrandsofcottonremainonthefingertobelatermixedwiththeblood.
8.5.1.4. Takea samplepipetteandwhilegently squeezing the tube immerse theopenend in theblooddropandthengentlyreleasethepressuretodrawbloodintothesamplepipetteuptotheblackline.
8.5.2. Ifanticoagulatedbloodisused,collect5µlofbloodusingthesamplepipetteprovided(uptotheblackline)orwithamicropipette.
8.6. Add5µlofbloodtothesamplewell(indicatedbyS).
8.7. Addtwodropsof(60µl)ofassaybufferintothebufferwell(indicatedbyA)andwritedownthetimeofaddingthebuffertothebufferwell(preferablyontheRDT).
8.8. Readtheresultin20minutes.(Sincenonspecificbindingcanoccurafter20minutes,careshouldbetakentoexaminethetestkitin20minutes).
8.9. WritetheresultasinterpretedaccordingtothestepsgiveninInterpretationsection(#11).
8.10. KeeptheRDTforfurtherreference.
9. STORAGEandTRANSPORT
9.1. StoreandtransporttheRDTswithinthetemperaturerangeof1o-40oC(34o-104oF).RDTscanbestoredwithinthemaincomponentoftherefrigerator,Itshouldneverbekeptinfreezercompartment.
9.2. Ifthetestkitisstoredinarefrigerator,thedeviceneedtobeequilibratetoroomtemperaturepriortouse.
10. SAFETYRULES
10.1. Readprovidedinstructionsforusebeforeusingthetestkit.10.2. Donotusecomponentsfromother lots.However,buffersolutionsandother itemscanbeusedwith
theRDTsofthesamelotwhichareprovidedlater.
10.3. DonotusetheRDTifthepackageisdamaged.
10.4. Use the test device and optional components (lancet/alcohol pad) immediately after opening itspackage.
10.5. Usedisposableprotectivegloveswhileperformingthetest.
10.6. Keepoutofreachofchildren.
11. INTERPRETATION
RDTshouldbeexaminedunderclearlightforinterpretation,sincesometimesfaintcontrolandtestlinesmaybevisible.Interpretationofresultsshouldbedonein20minutesasfollows(figure3).
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Invalid Negative PANPositive P.falciparumPositive PositiveP.falciparummonoinfectionormixedinfection
Testshouldbeconsidered
invalidifthelineinthecontrol
area(C)doesnotappear.Other
linesmayormaynotbepresent.
Thetestshouldberepeated
Onlythelineinthecontrolarea
(C)ispresent.
Thelineinthecontrolarea(C)
andthePanspecificline(line2)
arepresent.Thisindicatesa
positiveresultforP.vivax,P.falciparum,P.ovaleorP.malariae.
Thelineinthecontrolarea(line
C)andtheHRPspecificline(line
1)arepresent.
Controlline(C)andthetwotest
lines(line1and2)arepresent.
Abloodsmearhastobe
examinedforspecies
confirmation
Ifmalariaishighlysuspected,
- Repeatthetest(asverylowP.vivaxandP.falciparum
parasitaemiamaygiveanegative
result)- Performmicroscopy(asP.malariaeandPlasmodiumovalemayfailtogiveapositiveresult)
GenerallyifP.falciparumispresentHRP2line(line1)isalsovisible,
howevercertainP.falciparumstrainsdonothavetheHRP2
antigen.Suchstrainswillshowonly
thepLDHline.
AlsoinP.falciparumhyperparasitaemicconditions,dueto
prozoneeffect,theHRP2linemay
notappearinitially.Insuch
instancesitmayappearinfollowup
tests.
SinceHRP2antigenisawater
soulbleantigensecretedbythe
parasiteandfoundincirculating
peripheralblood,thislinewill
appearevenwhenparasitesare
sequestered.(Inthisinstancea
bloodsmearcouldgiveanegative
result)HRP2maypersistupto3weeks
afterparasiteclearanceandhence
couldgiveafalsepositiveresult
evenaftercompleteparasite
clearance.
Figure3-InterpretationoftheCareStartTMRapidDiagnosticTest
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