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 EvaluatedEIA ComponentsBioMerieux 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 ComponentsMedMira 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 BlotCalypte 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

Rapid, NAAT, WB Tests

EIA Sensitivity and SpecificityRelative to WB

Test Sensitivity Specificity

Bio-Rad Genetic Systems rLAV

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 HIV1/2 rDNA

n=1264

98.8 96.3

Sensitivity range 96.9-99.4 % Specificity range 95.8-98.6%

Rapid Test Sensitivity and SpecificityRelative to WB

Test Sensitivity Specificity

OraSure OraQuick

n=1264

98.0 98.9

MedMira Reveal G-1

n=1264

98.4 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 SpecificityRelative 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

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-ReactiveReactive

Repeat EIA (duplicate) Negative

+/+ +/- -/-

NegativeWB

Pos Neg Ind*

WB

Pos Neg Ind* * 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) 0%

EIA/EIA 0.7% (5) 0%

Rapid/Rapid 1.7% (12) 0%

Proposed Blood Bank Algorithm HIV EIA Repeat Reactive

No WB or Alternate EIARequired(optional)

(BIO-RAD Plus)

NAT

Non-ReactiveReactive

Alternate EIA

Reactive Non-reactive

713

67538

Vironostika Plus O

Gen-Probe

1226

675 HIV-1 Positive WB

Reactive Non-Reactive

17 0

IND9

Indeterminates from 58 to 9

12 Negative2/12 False Negative

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 testing1-2 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

AcknowledgementsIndustryBioMerieuxBIO-RADMedMiraTrinity BiotechGen-ProbeRoche

l

CDCChunfu Yang

Wei Luo

Chou Pau

Nick Delatorre

Chin-Yih OuTom SpiraBharat ParekhFaye CowartSusan KennedyDebbie KuehlDebra CandalDonna RudolphTammy BarnettSilvina MasciotraMarcia KalishSteve McDougal