3 Outline Desirable qualities for POC assays Assays used for EID HIV DNAHIV RNAP24 AntigenConsiderations when selecting an EID assayKey pointsSteps to move forward
4 Desirable Qualities of a POC Test Inexpensive (< $USD 5 /test)Rapid (< 1 hour)SimpleEquipment – battery operated, few moving parts, small footprintTechnique – minimal training requiredSensitive (how sensitive? > 95%?)Specific (how specific? > 98%?)Robust - No cold chain requirementCommercially available/CE marked-FRA cleared
5 Desirable Qualities of a POC Test “Cheap, fast, or accurate. Pick 2” (Dr. Bill Rodriguez, Harvard Univ, Nov 16, 2009)
6 HIV DNA AssaysRoche AMPLICOR HIV DNA assay, v 1.5 is the gold standard – either using whole blood pellets or Dried Blood Spots (DBS)Has been successfully introduced and implemented in many countries around the world
7 POC HIV DNA AssaysCIGHT, Kelso, Northwestern Univ- LoD 5 cp/reaction (Jangam, 2010, CROI); not yet ready for field testing and on hold while work focuses on a POC p24 testMicronics – Real Time PCR (Tim Granade, CDC; CROI 2010)BioHelix – isothermal lateral flow – 2 hr TAT (Jeanne Jordan, GWU)Both Micronics and BioHelix seem to be more in the proof of concept stage and don’t yet seem ready for field testing.
8 HIV RNA Assays Have been used as alternatives to HIV DNA testing Quantitative HIV RNA assays may not be as sensitive when infants are being prophylaxed or if mothers are receiving ARVs and the child is breast-feedingQualitative Gen-Probe AptimaVery sensitive and specific (Kerr, 2009; Stevens, 2009)Works well with DBSOnly HIV RNA assay FDA approved for diagnosis (though approval is for plasma or serum, not DBS)Being used by the State of New York for EID
9 Commercially Available FDA Cleared HIV-1 RNA Assays ManufacturerAssay NameTargetHIV-1 Subgroup RecognitionRange (RNA Copies/mL)Gen-ProbeAptimaLTR and polGroup M: A-HGroup N and OQualitativeRocheAmplicor HIV Monitor v1.5Gag(underquantitates some subtypes)Standard: 400 to 750,000Ultrasensitive:50 to 100,000COBASAmpliPrep/COBASTaqMan HIV-1 Test,version 1.0, 2.0(v. 1. may under-quantitate some subtypes;improved with v.2)20-40 to 10,000,000SiemansVersant HIV-1 RNA 3.0 (bDNA)PolU.S.: ,000Ex U.S.:50-500,000AbbottRealTime HIV-1 AssayIntGroup O, N, P40 to 10,000,000bioMerieuxNucliSENS EasyQ HIV-1 v2.0 (RUO US)25 to 10,000,000cps/mLBiocentricGeneric HIV Charge ViraleLTRGroup N300 to 5,000,000CavidiCavidi ExaVir v.3RTGroup M: A-H, Group O, NHIV-2~200 to 600,000 cp equivalents/mLPerkin Elmer Life SciencesUltrasensitive p24 Ag Assayp24Group M Subtypes: A, B, C, E, AE, AGDifficult to determine from package insert
10 POC RNA AssaysIQuum – realtime PCR, LoD – ~100 cp/mL, 1 hr, 200 uL plasmaInverness – microarray, realtime detection,10 uL whole bloodHelen Lee – semiquantitative dipstick with 200 cp/mL cutoff (Lee, JID 2010)Advanced Liquid Logic - based on digital microfluidicsWave 80 – assay based on bDNA assay
11 LIAT™ Quantitative POC HIV Assay 200 uL plasma sample input (haven’t tested whole blood yet)Limit of detection - 78 copies/mL of ARNA detected in 60 minEach cartridge has an internal controlDynamic range 100 to 10 million cp/mL in 60 minDetects HIV-1 Groups M and O and HIV-2 virusesComparative data with 30 clinical specimens:Roche COBAS % correlation coefficientSiemens Versant – 92% correlation coefficientHaven’t tested whole blood yet. Limited experience with clinical samples. Can operate on a battery.
12 LIAT92% correlation with Abbott m2000 with 75 clinical specimens (clades A, B, C, and D)Training took 10 minutesEasy to useAssay takes 60 minutesFiscus, unpublished data 2010
13 IMI’s CLONDIAG HIV Viral Load Point-of-Care TestAllows determination of HIV load in fingerstick, whole blood, or plasma.Multiple HIV-1 and HIV-2 targets are detected simultaneously by a proprietary microarray real time detection method.The test includes internal controlsThe sample is applied directly onto the test cartridgeThe cartridge is processed by a compact, battery driven instrument
14 IMI CLONDIAG HIV VL Test Data generated on 1 ml of EDTA Plasma (COBAS Ampliprep/Taqman) versus10 µl of Whole Blood (IMI’s prototype assay)IMInegativepositiveCOBASplasma73 (28 %)56 (22 %)<40 cp/ml8 (3 %)21( 8 %)6 (2 %)94 (36%)Percentage of samples with detectable viral load:COBAS (1 ml plasma) 50 %IMI VL (10 µl blood) 66 %all samples are from HIV-positive donorsspecificity of both assays =100% (32 HIV-negative donors)258 clinical samples from HIV infected individuals of unknown subtypes were tested in both the Clondiag POC test using 10 ul whole blood and Roche Taqman using 1.0 ml of plasma. I’m not sure whether this was version 1.0 or 2.0 of Taqman.donors receiving HAARTtherapy naïve donorsblood viral load equals plasma viral load———
15 SAMBA HIV-1 POC TestLee, et al JID 201 Suppl 1:S65-72
16 SAMBA HIV-1 POC TestComparison of the Simple Amplification-Based Assay (SAMBA) HIV-1 Test with Commercially Available HIV-1 Nucleic Acid TestsFeatureAbbott RealTimeNucliSensRoche COBASSAMBA HIV-1Cold storage<10 Co2-8 CoNot requiredSample volume200 uL1.0 mL240 uLSensitivity150 cp/mL176 cp/mL400 cp/mL200 cp/mLSubtypesM: A-H, N, O, PM: A-HM: A-K, N, OFrom Lee, et al JID 201 Suppl 1:S65-72
17 Total Nucleic Acid Assays Roche Taqman - CAP/CTM HIV-1 Qual is being introduced (Stevens, et al, JCM 2008)Works on whole blood and DBS – 100% sensitive/99.7% specificAbbott is developing a total nucleic acid assay as wellBoth require expensive new instrumentation – Roche Taqman or Abbott m2000Limited information about the performance of these assays in more resource constrained settingsProbably suitable for large centralized labs
18 HIV-1 p24 Antigen TestsThe ultrasensitive, heat dissociated p24 antigen assay has been shown to work well for EIDWith both plasmasensitivity %specificity %N= 2314 samples from 9 publicationsAnd DBSSensitivity – %Specificity – 100%N=1328 from 3 publications
19 Point of Care p24 Antigen Tests Inverness Determine Combination Ab/p24 AgImmunoDiagnosticsmBio DiagnosticsNorthwestern –Abbott partnership - David Kelso
20 p24 Antigen Rapid Test for Diagnosis of Acute Pediatric HIV Infection Plasma volume: 25mLImmune Disruption: 90oC heat shockAssay Steps: 1. Add 25mL plasma to 75mL buffer 2. Heat in water bath for 4min 3. Insert test strip & read after 20 min.Assay Sensitivity: 50pg/mL or 40,000 RNA copies/mLp24 Rapid Test Strip20
21 Results from pre-clinical trials in Cape Town 394 infant samples tested at NHLS Virology Lab, Groote Schuur Hospital, Cape Town, South Africa86% of samples were from infants under 6 months of age, 53% from infants under 2 months of ageReference Assay: Total Nucleic Acid PCR (Roche Ampliprep/COBAS Taqman HIV-1)p24 Assay Sensitivity: 23/24 = 95.8% (95% CI 80-99%)p24 Assay Specificity: 363/365 = 99.4% (95% CI %)5 samples gelled (1.3%) giving invalid results21
22 Point-Of-Care p24 Antigen Rapid Test Under Development Assay Procedure1. Separate plasmaWhole blood volume: 80mLImmune Disruption: Heat shockTotal Assay Duration: 35 min.Consumables: Plasma separator, reaction tube, reaction buffer, rapid test stripProcessor: Battery operatedCost per Assay: $1-2 per testReady early 2012?2. Pretreat sample in processor3. Insert rapid test strip and read results22
24 Factors to be Considered When Selecting an EID Assay Performance characteristicsSensitivity and specificitySpecimen type and volumeHIV subtype(s) in the populationTechnical and support issuesVolume and throughput – 1-2 or 1000/dayEquipment footprintPrintable resultsTraining requirementsAcceptance by MOH and cliniciansExternal and internal quality assurance
25 Centralized vs POC Testing Centralized Testing using DBSCan be implemented nowBetter control on training, supply logistics, internal and external QAPotential for high through-putHuge backlog of DBS in some countries with long turn around timesDelays and problems in returning resultsPoint of CareResults ready in an hr or lessPossibly fewer problems with mislabelingAble to confirm positive test results immediatelyPotential problems with training, competency, logisticsNot yet ready for prime time
26 Key PointsPOC assays should be inexpensive, rapid, simple, sensitive, specific, and robustPromising POC assays today include: IQuum’s LIAT, SAMBA, CIGHT’s p24, and possibly Clondiag’sTimeline for field testing and implementation:CIGHT p24 – Late 2011-early 2012IQuum - ????Inverness - ????SAMBA - ?????
27 Steps to move forward Continue lab validation of new POC tests Field test new assays under controlled conditionsExpand usage and evaluate the effects of POC on key operational parameters:% of infants tested% of infants who receive their results% of infected infants who access care% of infected infants who die or are hospitalized before age 2 years