S. Michele Owen, Ph.D. Laboratory Branch Division of HIV AIDS Prevention

1. Evaluation of Commercially Available HIV Assays to Address Alternative Screening/Diagnostic Algorithms S. Michele Owen, Ph.D. Laboratory Branch Division of HIV AIDS Prevention Centers for Disease Control and Prevention

2. Background • Why Evaluate Tests and Consider Alternative Algorithms? • Abundance: Multiple new FDA approved testing methods- NAT, Rapid Tests, New EIAs • Ambiguity: reduce or eliminate WB indeterminate results • Cost and Efficiency: sequential EIAs have been effectively used internationally (WHO/UNAIDS)

3. Objective Compare performance of commercially available HIV tests as a basis for evaluating alternative algorithms for HIV diagnostics or surveillance.

4. Methods- Samples • 1002 specimens from the U.S. (Boston Biomedica Inc.) • Plasma centers or blood banks. • 62 non-U.S. samples (Boston Biomedica Inc) • World Wide Performance Panels • 205 non-U.S. samples (Cameroon Blood Bank Study) • Units that were reactive in one or more screening tests for HIV, HBV, HCV or Syphilis • All samples were collected, processed and stored using standard diagnostic protocols

5. Methods-Testing • Plasma samples: • randomized and blinded • tested by 6 EIAs, 4 rapid tests and 3* NAT- based tests. • Any sample found to be reactive by any of the above tests was subjected to Western Blot • Sample was considered to be: • Positive if the Western Blot was positive (current “gold standard”) • Negative if any sample was either • negative by all 13 tests described above or • Western Blot negative. • All tests were performed by trained laboratory personnel (Gen-Probe and AmpliScreen NAT testing was done by individuals certified by the company to run the test)

6. EIAs Evaluated EIAComponents BioMerieux Vironostika HIV-1 (2nd)HIV-1 viral lysate BioMerieux Vironostika HIV-1 Plus O (2nd) HIV-1 viral lysate, purified viral env proteins, and synthetic peptide from transmembrane epitope of HIV-1 Group O BIO-RAD Genetic Systems rLAV (2nd) LAV lysate and recombinant gp41 BIO-RAD Genetic Systems 1/ 2 Peptide (2nd) Synthetic peptides from env and pol regions of both HIV-1 and HIV–2 BIO-RAD Genetic Systems HIV 1/2 Plus (3rd) HIV-1 recombinant gp160 and p24, HIVgp36 synthetic peptide, and HIV-1 group O synthetic oligopeptide Abbott HIVAB HIV-1/HIV-2 (rDNA) (3rd) Recombinant HIV-1 core and env proteins and HIV-2 env protein

7. Rapid, NAAT, WB Tests Rapid Components MedMira Reveal conserved immunodominant peptides OraSure OraQuick peptides, gp41,gp36 Trinity Biotech Uni-Gold Recombigen recombinant immunodominant proteins BIO-RAD Multispot HIV-2 gp36 peptide, HIV-1 gp41 peptide, recombinant gp41 Western Blot Calypte Biomedical Cambridge Biotech HIV-1H9/HTLV-IIIB Lysate BIO-RAD Genetic Systems HIV-1 CEM/HIV LAV Lysate NAAT Gen-Probe Procleix LTR and Pol Roche Ampliscreen Gag In house LTR

8. EIA Sensitivity and SpecificityRelative to WB Sensitivity range 96.9-99.4 % Specificity range 95.8-98.6%

9. Rapid Test Sensitivity and SpecificityRelative to WB Sensitivity range 97.4-98.5% Specificity range 97.8-99.4%

10. NAT Sensitivity and SpecificityRelative to WB Sensitivity range 92.6-96.7% Specificity range 96.8-98.8%

11. Indeterminate Characteristics • 58 indeterminate samples • 5 U.S. plasma donors • 52 Cameroon samples • 1 BBI non-U.S. performance panel sample • Most had 3 or fewer EIA/Rapid positive results • low S/CO values on EIA • One or few bands on WB • p24 >> p66 > p55 • 4 samples positive on multiple EIAs • 2/4 positive by NAT • 1/4 almost complete WB pattern (known O from Spain) • 3/4 p24 Ag positive (BBI)

12. Current Diagnostic Algorithm WB WB Pos Pos Neg Neg Ind* Ind* Screening EIA Non-Reactive Reactive Repeat EIA (duplicate) Negative +/- +/+ -/- Negative *follow-up sample, HIV-2

13. Potential Simple Algorithms • EIA Screen /NAAT Confirmation • EIA Screen/Rapid Confirmation • EIA Screen/Alternate EIA Confirmation • Rapid Screen/Alternate Rapid Confirmation

14. Summary Potential Algorithms Relative to Current EIA/WB

15. Proposed Blood Bank Algorithm HIV EIA Repeat Reactive (BIO-RAD Plus) 713 Gen-Probe NAT 38 675 Reactive Non-Reactive No WB or Alternate EIA Required (optional) Alternate EIA Vironostika Plus O 26 12 Non-reactive Reactive 675 HIV-1 Positive 12 Negative 2/12 False Negative WB 17 0 9 Reactive Non-Reactive IND Indeterminates from 58 to 9 True answer for indeterminates???

16. Important Caveats • No follow-up samples available for discordant samples (true answer unknown) • Limited demographic or epidemiological data available • Collection, processing, and storage of samples was conducted using routine diagnostic procedures. • 1 freeze/thaw of specimen prior to NAT testing • 1-2 freeze/thaws prior to Serological testing

17. Summary • Range of sensitivity observed for all tests was 92.1% - 99.4% • Range of specificity observed for all tests was 95.8% - 98.8% • Discordant results between serological and NAT-based tests were observed. • True answer unknown • Most indeterminate samples • Non-U.S. • Few WB Bands • Low EIA S/CO values • 4 Indeterminate samples likely or known positive

18. Conclusions • All FDA approved HIV detection assays have comparable Sensitivity and Specificity • Lower values may be due to the stringent testing methods employed in the study • NAAT alone can not replace WB for confirmation • EIA/EIA, EIA/Rapid or Rapid/Rapid algorithms yielded better sensitivity than EIA combined with NAT • Proposed Blood Bank algorithm would likely reduce indeterminate WB results • Much work left to do to establish “best algorithm” • Seroconversion samples • Discordant/Indeterminate samples with follow-up

19. Industry BioMerieux BIO-RAD MedMira Trinity Biotech Gen-Probe Roche l Acknowledgements CDC Chunfu Yang Wei Luo Chou Pau Nick Delatorre Chin-Yih Ou Tom Spira Bharat Parekh Faye Cowart Susan Kennedy Debbie Kuehl Debra Candal Donna Rudolph Tammy Barnett Silvina Masciotra Marcia Kalish Steve McDougal