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EID - Current tools and prospects of point of care technology Susan A. Fiscus, Ph.D. University of North Carolina at Chapel Hill.

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Presentation on theme: "EID - Current tools and prospects of point of care technology Susan A. Fiscus, Ph.D. University of North Carolina at Chapel Hill."— Presentation transcript:

1 EID - Current tools and prospects of point of care technology Susan A. Fiscus, Ph.D. University of North Carolina at Chapel Hill

2 Disclosures Honoraria: Gen-Probe, Roche, Abbott Free kits: Roche, Gen-Probe, Perkin- Elmer, Cavidi, Siemans, Abbott, Inverness, IQuum, ImmunoDiagnostics

3 Outline Desirable qualities for POC assays Assays used for EID –HIV DNA –HIV RNA –P24 Antigen Considerations when selecting an EID assay Key points Steps to move forward

4 Desirable Qualities of a POC Test Inexpensive (< $USD 5 /test) Rapid (< 1 hour) Simple Equipment – battery operated, few moving parts, small footprint Technique – minimal training required Sensitive (how sensitive? > 95%?) Specific (how specific? > 98%?) Robust - No cold chain requirement Commercially 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 Assays Roche 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 Assays CIGHT, 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 test Micronics – 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 dont 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-feeding Qualitative Gen-Probe Aptima –Very sensitive and specific (Kerr, 2009; Stevens, 2009) –Works well with DBS –Only 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 NameTarget HIV-1 Subgroup Recognition Range (RNA Copies/mL) Gen-ProbeAptimaLTR and pol Group M: A-H Group N and O Qualitative Roche Amplicor HIV Monitor v1.5 Gag Group M: A-H (underquantitates some subtypes) Standard: 400 to 750,000 Ultrasensitive: 50 to 100,000 Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, version 1.0, 2.0 Gag Group M: A-H (v. 1. may under-quantitate some subtypes; improved with v.2) to 10,000,000 Siemans Versant HIV-1 RNA 3.0 (bDNA) Pol Group M: A-H U.S.: ,000 Ex U.S.: ,000 Abbott RealTime HIV-1 Assay Int Group M: A-H Group O, N, P 40 to 10,000,000 bioMerieux NucliSENS EasyQ HIV-1 v2.0 (RUO US) Gag Group M: A-H25 to 10,000,000 cps/mL Biocentric Generic HIV Charge Virale LTR Group M: A-H Group N 300 to 5,000,000 Cavidi Cavidi ExaVir v.3 RT Group M: A-H, Group O, N HIV-2 ~200 to 600,000 cp equivalents/mL Perkin Elmer Life Sciences Ultrasensitive p24 Ag Assay p24 Group M Subtypes: A, B, C, E, AE, AG Difficult to determine from package insert

10 POC RNA Assays IQuum – realtime PCR, LoD – ~100 cp/mL, 1 hr, 200 uL plasma Inverness – microarray, realtime detection,10 uL whole blood Helen Lee – semiquantitative dipstick with 200 cp/mL cutoff (Lee, JID 2010) Advanced Liquid Logic - based on digital microfluidics Wave 80 – assay based on bDNA assay

11 LIAT Quantitative POC HIV Assay 200 uL plasma sample input (havent tested whole blood yet) Limit of detection - 78 copies/mL of ARNA detected in 60 min Each cartridge has an internal control Dynamic range 100 to 10 million cp/mL in 60 min Detects HIV-1 Groups M and O and HIV-2 viruses Comparative data with 30 clinical specimens: –Roche COBAS % correlation coefficient –Siemens Versant – 92% correlation coefficient

12 LIAT 92% correlation with Abbott m2000 with 75 clinical specimens (clades A, B, C, and D) Training took 10 minutes Easy to use Assay takes 60 minutes Fiscus, unpublished data 2010

13 IMIs CLONDIAG HIV Viral Load Point-of-Care Test Allows 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 controls The sample is applied directly onto the test cartridge The 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) versus 10 µl of Whole Blood (IMIs prototype assay) Percentage of samples with detectable viral load: COBAS (1 ml plasma) 50 % IMI VL (10 µl blood) 66 % all samples are from HIV-positive donors specificity of both assays =100% (32 HIV- negative donors) donors receiving HAART therapy naïve donors blood viral load equals plasma viral load

15 SAMBA HIV-1 POC Test Lee, et al JID 201 Suppl 1:S65-72

16 SAMBA HIV-1 POC Test From Lee, et al JID 201 Suppl 1:S65-72 FeatureAbbott RealTime NucliSensRoche COBAS SAMBA HIV-1 Cold storage <10 C o 2-8 C o Not required Sample volume 200 uL1.0 mL200 uL240 uL Sensitivity150 cp/mL176 cp/mL400 cp/mL200 cp/mL SubtypesM: A-H, N, O, PM: A-H M: A-K, N, O Comparison of the Simple Amplification-Based Assay (SAMBA) HIV-1 Test with Commercially Available HIV-1 Nucleic Acid Tests

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% specific Abbott is developing a total nucleic acid assay as well Both require expensive new instrumentation – Roche Taqman or Abbott m2000 –Limited information about the performance of these assays in more resource constrained settings –Probably suitable for large centralized labs

18 HIV-1 p24 Antigen Tests The ultrasensitive, heat dissociated p24 antigen assay has been shown to work well for EID –With both plasma sensitivity % specificity % N= 2314 samples from 9 publications –And DBS Sensitivity – % Specificity – 100% N=1328 from 3 publications

19 Point of Care p24 Antigen Tests Inverness Determine Combination Ab/p24 Ag ImmunoDiagnostics mBio Diagnostics Northwestern –Abbott partnership - David Kelso

20 p24 Antigen Rapid Test for Diagnosis of Acute Pediatric HIV Infection Plasma volume: 25 L Immune Disruption: 90 o C heat shock Assay Steps: 1. Add 25 L plasma to 75 L 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/mL p24 Rapid Test Strip

21 Results from pre-clinical trials in Cape Town 394 infant samples tested at NHLS Virology Lab, Groote Schuur Hospital, Cape Town, South Africa 86% of samples were from infants under 6 months of age, 53% from infants under 2 months of age Reference 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 results

22 Point-Of-Care p24 Antigen Rapid Test Under Development 1. Separate plasma 2. Pretreat sample in processor Whole blood volume: 80 L Immune Disruption: Heat shock Total Assay Duration: 35 min. Consumables: Plasma separator, reaction tube, reaction buffer, rapid test strip Processor: Battery operated Cost per Assay: $1-2 per test Ready early 2012? 3. Insert rapid test strip and read results Assay Procedure

23 Cheap, fast, or accurate. Pick 2 Cheap: (< $ 5 USD) Fast: < 60 min Accurate: Sensitivity: > 95% Specificity: > 98% IQuum?????? Inverness????????????? SAMBA??????< 2 hrs CIGHT p24

24 Factors to be Considered When Selecting an EID Assay Performance characteristics –Sensitivity and specificity –Specimen type and volume –HIV subtype(s) in the population Technical and support issues –Volume and throughput – 1-2 or 1000/day –Equipment footprint –Printable results –Training requirements Acceptance by MOH and clinicians External and internal quality assurance

25 Centralized vs POC Testing Centralized Testing using DBS Can be implemented now Better control on training, supply logistics, internal and external QA Potential for high through- put Huge backlog of DBS in some countries with long turn around times Delays and problems in returning results Point of Care Results ready in an hr or less Possibly fewer problems with mislabeling Able to confirm positive test results immediately Potential problems with training, competency, logistics Not yet ready for prime time

26 Key Points POC assays should be inexpensive, rapid, simple, sensitive, specific, and robust Promising POC assays today include: IQuums LIAT, SAMBA, CIGHTs p24, and possibly Clondiags Timeline for field testing and implementation: –CIGHT p24 – Late 2011-early 2012 –IQuum - ???? –Inverness - ???? –SAMBA - ?????

27 Steps to move forward Continue lab validation of new POC tests Field test new assays under controlled conditions Expand 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


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