1. Early Infant Diagnosis: 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. Acknowledgments Dr. Shuqi Chen, IQuum Dr. Robert Coombs, Univ of Washington Dr. John Gerdes, Micronics Dr. Jeanne Jordan, George Washington Univ Dr. David Kelso, Northwestern Univ Dr. Helen Lee, Univ of Cambridge Dr. Laura Mazzola, WAVE 80 Dr. Christopher Myatt, mBio Diagnostics Dr. Avi Pelossof, Inverness Dr. Michael Pollack, Advanced Liquid Logic

4. Desirable Qualities of a POC Diagnostic Test  Rapid (< 1 hour)  Sensitive (how sensitive? > 95%)  Specific (how specific? > 98%)  Inexpensive (< $5/test)  Simple  Equipment – battery operated, few moving parts, small footprint  Technique – minimal training required  Robust - No cold chain requirement  Commercially available  CE marked/FDA cleared

5. Desirable Qualities of a POC Diagnostic Test  “Cheap, fast, or accurate. Pick 2” (Dr. Bill Rodriguez, Harvard Univ, Nov 16, 2009)

6. HIV DNA and Total Nucleic Acid Assays Roche AMPLICOR HIV DNA assay, v 1.5 is the gold standard –Has been successfully introduced and implemented in many countries around the world –Can use whole blood pellets or Dried Blood Spots (DBS) Roche Qualitative Total Nucleic Acid Assay has been introduced (Stevens, et al, JCM 2008) –Works on whole blood and DBS –100% sensitive and 99.7% specific Abbott also developing a DNA assay Both Roche and Abbott assays require large, expensive new equipment –Probably suitable for large centralized labs

7. POC HIV DNA Assays CIGHT, Dr D 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; HIV Diagnostics Mtg, 2010) 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 Qualitative Gen-Probe Aptima –Only HIV RNA assay FDA approved for diagnosis (though approval is for plasma or serum, not DBS) –Works well with DBS –Very sensitive and specific (Kerr, 2009; Stevens, 2009) –Being used by the State of New York for EID 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

9. Commercially Available HIV-1 Viral Load Assays ManufacturerAssay Name RocheAmplicor HIV Monitor v1.5, COBAS RocheTaqMan HIV-1, version 1.0, 2.0 SiemansVersant HIV-1 RNA 3.0 (bDNA) AbbottRealTime HIV-1 Assay bioMerieuxNucliSENS EasyQ HIV-1 v2.0 (RUO US) BiocentricGeneric HIV Charge Virale CavidiCavidi ExaVir v.3

10. POC RNA Assays  Univ of Cambridge & Diagnostics for the Real World – isothermal amplification with visual detection by dipstick, LoD 75 cp/mL using 250uL plasma, <90 min  IQuum – realtime PCR, LoD – ~100 cp/mL, 1 hr, 200 uL plasma  Inverness – microarray, realtime detection,10 uL whole blood Advanced Liquid Logic - based on digital microfluidics Wave 80 – assay based on bDNA assay

11. SAMBA HIV-1 POC Test Lee, et al 2010. JID 201 Suppl 1:S65-72

12. SAMBA Semi-quantitative VL assay with cut-off of 1,000 cp/mL Qualitative assay appropriate for EID 250 uL plasma - limit of detection 75 cp/mL 100 uL whole blood - limit of detection 400 cp/mL Robust – no cold chain required, can be battery operated Simplified sample preparation chemistry Sample prep and amplification/detection equipment not linked currently Little training required Field testing in resource-limited settings in September 2010 Clinical trial for regulatory approval in 2011

13. LIAT™ Quantitative POC HIV Assay 200 uL plasma sample input (haven’t tested whole blood yet) Realtime PCR 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 Can be battery operated Very simple training and operation Add 200 uL plasma to cartridge, cap, and insert in instrument

14. LIAT  92% correlation with Abbott m2000 with 75 clinical specimens (clades A, B, C, and D)  Training took 5 minutes  Easy to use  Assay takes 60 minutes Fiscus, unpublished data 2010

15. IMI’s CLONDIAG HIV Viral Load Point-of-Care Test  Can use 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

16. IMI CLONDIAG HIV Viral Load Test 1 ml of EDTA Plasma (COBAS Ampliprep/Taqman) versus 10 µl of Whole Blood (IMI’s 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 ——— N=258

17. HIV-1 p24 Antigen Tests The ultrasensitive, heat dissociated p24 antigen assay has been shown to work well for EID –With both plasma sensitivity - 91-100% specificity - 95-100% N= 2314 samples from 9 publications –And DBS Sensitivity – 98-100% Specificity – 100% N=1328 from 3 publications

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

19. p24 Antigen Rapid Test for Diagnosis of Acute Pediatric HIV Infection Assay Steps: 1. Add 25  L plasma to 75  L buffer 2. Heat in water bath at 90 o C for 4 min 3. Insert test strip & read after 20 min. Assay Sensitivity: 50pg/mL or 40,000 RNA copies/mL p24 Rapid Test Strip

20. 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 babies < 6 months of age 53% from infants < 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 98- 100%) 5 samples gelled (1.3%) giving invalid results

21. 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

22. “Cheap, fast, or accurate. Pick 2” Cheap: (< $ 5 USD) Fast: < 60 min Accurate: Sensitivity: > 95% Specificity: > 98% Whole blood Robust (battery operated and no cold chain) IQuum?????In develop- ment Needs cold chain Inverness????? SAMBA$10- 20? < 90 min CIGHT p24

23. Centralized vs POC Testing Centralized Testing using DBS Can be implemented now Better control on training, supply logistics, internal and external QA 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

24. Key Points POC assays should be inexpensive, rapid, simple, sensitive, specific, and robust Promising POC assays today include: IQuum’s LIAT, SAMBA, CIGHT’s p24, and possibly Clondiag’s VL assay

25. 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  Continuous QA of POC facilities  Confirmation of all positives at a central/reference lab

26. IQuumInvernessSAMBACIGHT p24 Cheap: (≤ $ 5 USD)??? $10-20? Fast: < 60 min ≤ 90 min Limit of detection <100 cp/mL ???75 cp/mL 40,000 cp/mL Accurate: Sensitivity: ≥ 95% Specificity: ≥ 98% ??? Quantitative Semi No Whole blood * Robust (battery operated and no cold chain) Recommend 4°C, but >1 month at 37°C “Cheap, fast, or accurate. Pick 2” * Plan to evaluate