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 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 algorithms for HIV diagnostics or surveillance.

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

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)

EIAs Evaluated EIA Components 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

Rapid, NAAT, WB Tests Rapid Components Western Blot NAAT 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-1 H9/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 rLAV n=1264 96.9 98.6 Bio-Rad Genetic Systems 1/2 Peptide 98.1 Biomeriuex Vironostika 98.4 96.5 Biomerieux Vironostika Plus O 98.9 96.6 Bio-Rad Genetic Systems 1/2 Plus O 99.4 95.8 Abbott HIV1/2 rDNA 98.8 96.3 Sensitivity range 96.9-99.4 % Specificity range 95.8-98.6%

Rapid Test Sensitivity and Specificity Relative to WB OraSure OraQuick n=1264 98.0 98.9 MedMira Reveal G-1 98.4 Trinity Biotech Uni-Gold Recombigen HIV n=1197 98.5 99.4 BIO-RAD Multi-Spot n=83 97.4 97.8 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 96.7 98.7 Roche Ampliscreen n=1171 92.6 96.8 CDC In- House NAT 94.9 98.8 Sensitivity range 92.6-96.7% Specificity range 96.8-98.8%

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)

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

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% (25) 0% EIA/Rapid 1.3% (9) EIA/EIA 0.7% (5) Rapid/Rapid 1.7% (12)

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 9 Reactive Non-Reactive IND Indeterminates from 58 to 9 True answer for indeterminates???

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

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

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

Acknowledgements Industry CDC l BioMerieux Chunfu Yang BIO-RAD Wei Luo MedMira Trinity Biotech Gen-Probe Roche l 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