Evaluation of a single round polymerase chain reaction assay using dried blood spots for diagnosis of HIV-1 infection in infants in an African setting.docVIP

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Evaluation of a single round polymerase chain reaction assay using dried blood spots for diagnosis of HIV-1 infection in infants in an African setting.doc

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Evaluation of a single round polymerase chain reaction assay using dried blood spots for diagnosis of HIV-1 infection in infants in an African setting

Chohanetal.BMCPediatrics2011,11:18 /1471-2431/11/18 TECHNICAL ADVANCE OpenAccess Evaluationofasingleroundpolymerasechain reactionassayusingdriedbloodspotsfor diagnosisofHIV-1infectionininfantsinan Africansetting BhavnaHChohan1,2?,SandraEmery3?,DaltonWamalwa4,GraceJohn-Stewart5,MaxwelMajiwa4,MusaNg’ayo6, SteveFroggett3,JulieOverbaugh3* Abstract Background:Theaimofthisstudywastodevelopaneconomical‘in-house’singleroundpolymerasechain reaction(PCR)assayusingfilterpaper-driedbloodspots(FP-DBS)forearlyinfantHIV-1diagnosisandtoevaluateits performanceinanAfricansetting. Methods:An‘in-house’singleroundPCRassaythattargetsconservedregionsintheHIV-1polymerase(pol )gene wasvalidatedforusewithFP-DBS;firstwevalidatedthisassayusingFP-DBSspikedwithcellstandardsofknown HIV-1copynumbers.Next,wevalidatedtheassaybytestingthearchivedFP-DBS(N=115)frominfantsofknown HIV-1infectionstatus.Subsequentlythis‘in-house’HIV-1polPCRFP-DBSassaywasthenestablishedinNairobi, KenyaforfurtherevaluationonfreshlycollectedFP-DBS(N=186)frominfants,andcomparedwithfindingsfrom areferencelaboratoryusingtheRocheAmplicor? HIV-1DNATest,version1.5assay. Results:TheHIV-1polPCRFP-DBSassaycoulddetectoneHIV-1proviralcopyin38.7%oftests,2copiesin46.9% oftests,5copiesin72.5%oftestsand10copiesin98.1%oftestsperformedwithspikedsamples.Usingthe archivedFP-DBSsamplesfrominfantsofknowninfectionstatus,thisassaywas92.8%sensitiveand98.3%specific forHIV-1infantdiagnosis.Using186FP-DBScollectedfrominfantsrecentlydefinedasHIV-1positiveusingthe commerciallyavailableRocheAmplicorv1.5assay,178FP-DBStestedpositivebythis‘in-house’single-roundHIV-1 polPCRFP-DBSPCRassay.Uponsubsequentretesting,the8infantFP-DBSsamplesthatwerediscordantwere confirmedasHIV-1negativebybothassaysusingasecondbloodsample. Conclusions:HIV-1wasdetectedwithhighsensitivityandspecificityusingbotharchivedandmorerecently collectedsamples.Thissuggeststhatthis‘in-house’HIV-1polFP-DBSPCRassaycanprovideanalternativecost- effective,reliableandrapidmethodforearlydetectio