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Evaluation of Commercially Available HIV Assays to Address Alternative Screening/Diagnostic Algorithms S. Michele Owen, 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

Background • Why Evaluate Tests and Consider Alternative Algorithms? – Abundance: Multiple new FDA 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)

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

Methods- Samples • 1002 specimens from the U. S. (Boston Biomedica Inc. ) Ø Methods- Samples • 1002 specimens from the U. S. (Boston Biomedica Inc. ) Ø Plasma centers or blood banks. • 62 non-U. S. samples (Boston Biomedica Inc) Ø World Wide Performance Panels • 205 non-U. S. samples (Cameroon Blood Bank Study) Ø 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

Methods-Testing • Plasma samples: Ø randomized and blinded Ø tested by 6 EIAs, 4 Methods-Testing • Plasma samples: Ø randomized and blinded Ø 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: Ø Positive if the Western Blot was positive (current “gold standard”) Ø 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 Ampli. Screen NAT testing was done by individuals certified by the company to run the test)

EIAs Evaluated EIA Bio. Merieux Vironostika HIV-1 (2 nd) Components HIV-1 viral lysate Bio. EIAs Evaluated EIA Bio. Merieux Vironostika HIV-1 (2 nd) Components HIV-1 viral lysate Bio. Merieux Vironostika HIV-1 Plus O (2 nd) HIV-1 viral lysate, purified viral env proteins, and synthetic peptide from transmembrane epitope of HIV-1 Group O BIO-RAD Genetic Systems r. LAV (2 nd) LAV lysate and recombinant gp 41 BIO-RAD Genetic Systems 1/ 2 Peptide (2 nd) Synthetic peptides from env and pol regions of both HIV-1 and HIV– 2 BIO-RAD Genetic Systems HIV 1/2 Plus (3 rd) HIV-1 recombinant gp 160 and p 24, HIVgp 36 synthetic peptide, and HIV-1 group O synthetic oligopeptide Abbott HIVAB HIV-1/HIV-2 (r. DNA) (3 rd) Recombinant HIV-1 core and env proteins and HIV-2 env protein

Rapid, NAAT, WB Tests Rapid Components Med. Mira Reveal conserved immunodominant peptides Ora. Sure Rapid, NAAT, WB Tests Rapid Components Med. Mira Reveal conserved immunodominant peptides Ora. Sure Ora. Quick peptides, gp 41, gp 36 Trinity Biotech Uni-Gold Recombigen recombinant immunodominant proteins BIO-RAD Multispot HIV-2 gp 36 peptide, HIV-1 gp 41 peptide, recombinant gp 41 Western Blot Calypte Biomedical Cambridge Biotech HIV-1 H 9/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

EIA Sensitivity and Specificity Relative to WB Test Sensitivity Specificity Bio-Rad Genetic Systems r. EIA Sensitivity and Specificity Relative to WB Test Sensitivity Specificity Bio-Rad Genetic Systems r. LAV n=1264 96. 9 98. 6 Bio-Rad Genetic Systems 1/2 Peptide n=1264 98. 1 98. 6 Biomeriuex Vironostika n=1264 98. 4 96. 5 Biomerieux Vironostika Plus O n=1264 98. 9 96. 6 Bio-Rad Genetic Systems 1/2 Plus O n=1264 99. 4 95. 8 Abbott HIV 1/2 r. DNA n=1264 98. 8 96. 3 Sensitivity range 96. 9 -99. 4 % Specificity range 95. 8 -98. 6%

Rapid Test Sensitivity and Specificity Relative to WB Test Sensitivity Specificity Ora. Sure Ora. Rapid Test Sensitivity and Specificity Relative to WB Test Sensitivity Specificity Ora. Sure Ora. Quick n=1264 98. 0 98. 9 Med. Mira Reveal G-1 n=1264 98. 4 Trinity Biotech 98. 5 Uni-Gold Recombigen HIV n=1197 99. 4 BIO-RAD Multi-Spot n=83 97. 8 97. 4 Sensitivity range 97. 4 -98. 5% Specificity range 97. 8 -99. 4%

NAT Sensitivity and Specificity Relative to WB Test Sensitivity Specificity Gen-Probe Procleix n=1264 96. NAT Sensitivity and Specificity Relative to WB Test Sensitivity Specificity Gen-Probe Procleix n=1264 96. 7 98. 7 Roche Ampliscreen n=1171 92. 6 96. 8 CDC In- House NAT n=1264 94. 9 98. 8 Sensitivity range 92. 6 -96. 7% Specificity range 96. 8 -98. 8%

Indeterminate Characteristics • 58 indeterminate samples Ø 5 U. S. plasma donors Ø 52 Indeterminate Characteristics • 58 indeterminate samples Ø 5 U. S. plasma donors Ø 52 Cameroon samples Ø 1 BBI non-U. S. performance panel sample • Most had 3 or fewer EIA/Rapid positive results Ø low S/CO values on EIA Ø One or few bands on WB § p 24 >> p 66 > p 55 • 4 samples positive on multiple EIAs Ø 2/4 positive by NAT Ø 1/4 almost complete WB pattern (known O from Spain) Ø 3/4 p 24 Ag positive (BBI)

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

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

Summary Potential Algorithms Relative to Current EIA/WB False Negative False Positive EIA/NAAT 3. 3% Summary Potential Algorithms Relative to Current EIA/WB False Negative False Positive EIA/NAAT 3. 3% (25) 0% EIA/Rapid 1. 3% (9) 0% EIA/EIA 0. 7% (5) 0% Rapid/Rapid 1. 7% (12) 0%

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

Important Caveats • No follow-up samples available for discordant samples (true answer unknown) • 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. Ø 1 freeze/thaw of specimen prior to NAT testing Ø 1 -2 freeze/thaws prior to Serological testing

Summary • Range of sensitivity observed for all tests was 92. 1% - 99. 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 NATbased 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

Conclusions • All FDA approved HIV detection assays have comparable Sensitivity and Specificity Ø Conclusions • All FDA approved HIV detection assays have comparable Sensitivity and Specificity Ø 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

Acknowledgements CDC Chunfu Yang Wei Luo Chou Pau Nick Delatorre Chin-Yih Ou Tom Spira 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 Mc. Dougal Industry Bio. Merieux BIO-RAD Med. Mira Trinity Biotech Gen-Probe Roche l