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 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 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 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 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 gp 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

8. 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 %
Specificity range %

9. 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 %
Specificity range %

10. 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 %
Specificity range %

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
Screening EIA
Non-Reactive
Reactive
Repeat EIA (duplicate)
Negative
+/+
+/-
-/- WB
Pos
Neg
Ind* WB
Pos
Neg
Ind*
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
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)

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 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% %
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. 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