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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