Supplemental Testing for HIV-1 and HCV

1
Supplemental Testing for HIV-1 and HCV
Susan L. Stramer, Ph.D. National Testing
andReference Laboratories American Red Cross
Jaye P. Brodsky Quality Analytics

• Blood Products Advisory Committee Meeting
• September 18, 2003

2
Background

• HIV-1 and HCV algorithms utilize immunoassays for
  confirmation
• HIV-1 uses Western Blot (WB) containing
  electrophoresed whole HIV lysate
• HCV uses Recombinant Immunoblot Assay (RIBA)
  containing painted recombinant HCV antigens
• Issues with supplemental assays
• Poor performance
• Unreadable or invalid
• False positivity false negativity
• High rates of indeterminate results (neither pos
  or neg) in healthy individuals

3
Background

• Issues with supplemental assays
• High costs
• Inconsistent availability (HIV)
• Is all the testing necessary, or is redundant
  testing being performed?
• Can alternatives be validated?

4
Alternative Algorithms

• HCV Alternative 1
• Use of HCV NAT in supplemental algorithms
• Already being performed as a screening test for
  all donations
• NAT results integrated into donor counseling
• Sensitivity high relative to RIBA even though NAT
  is performed in pools (16-24)
• Specificity high (false-positive donor rates less
  than 140,000)
• Already recommended for diagnostic use by CDC
• If NAT reactive, RIBA not needed
• HCV Alternative 2
• Use of S/CO to further reduce the need for RIBA
  in HCV NAT nonreactive individuals

5
Alternative Algorithms

• HIV Alternative 1
• Use of NAT and/or S/CO as proposed for HCV
• HIV Alternative 2
• Use of a second EIA where discordant EIA
  reactives (EIA-1 Rx and EIA-2 nonRx) are not
  further tested
• Feasibility for HIV versus HCV
• Anti-HCV licensed screening assays use same
  recombinant antigens, same assay format
  overlapping populations of false positives
• Anti-HIV licensed screening assays use different
  antigens (viral lysate, recombinants and
  peptides) populations of false positives are
  unique

6
Alternative Algorithms

• Issues for HIV or HCV alternatives
• Potential misclassification of donors
• Current algorithms containing RIBA or WB in
  combination with NAT (and screening S/CO value)
  provide specific counseling messages
• Kits will require labeling for intended use
  (supplemental claims to NAT assays or EIAs)
• 21 CFR 610.40 Subpart E
• You must further test each donation, including
  autologous donations, found to be reactive by a
  screening test whenever a supplemental
  (additional, most specific) test has been
  approved for such use by FDA.

7
Current HCV EIA 2.0 (Abbott) or 3.0 (Ortho)
Repeat Reactive
Perform RIBA 3.0 SIAN 34,656
Ind
Pos
Neg
HCV NAT
HCV NAT
HCV NAT
ReactiveIndividually
NonRxin Pool orIndividually
ReactiveIndividually
NonRxin Pool orIndividually
ReactiveIndividually
NonRxin Pool orIndividually
HCVInfected
HCV Infected?Resolved infection (20)Viral
load lt NAT cutoffFalse Pos RIBA
HCV NotInfected
HCV Infected?
17.2
38
34.5
5,967
13,198
HCV Infected?Early seroconversionFalse Rx NAT
11,963
HCV InfectedEarly seroconversion
9.6
3,319
0.5
0.15
159
50
8
Option 1 HCV EIA 2.0 (Abbott) or 3.0 (Ortho)
Repeat Reactive
Review HCV NAT ResultsN 34,656
Pool or Individual UnitNonRx(16 dtns
HIV-1/HCV TMA,24 dtns HCV PCR)
Individual UnitNAT Rx(dHCV TMA or HCV PCR)
38.7
61.3
13,407
21,249
No Further TestingHCV Infected
Perform RIBA
98.4 PPV
Ind
Pos
Neg
13,198 RIBA Pos
28.1
15.6
56.3
5,967
3,319
11,963
79.9 Sensitivity
9
Option 2 HCV EIA 2.0 (Abbott) or 3.0 (Ortho)
Repeat Reactive
Review HCV NAT ResultsN 34,656
82.9Sensitivity
Pool or Individual UnitNonRx(16 dtns
HIV-1/HCV TMA,24 dtns HCV PCR)
Individual UnitNAT Rx(dHCV TMA or HCV PCR)
38.7
61.3
21,249
13,407
Review S/CO Values of EIA
No Further TestingHCV Infected
S/CO 3.8
S/CO lt 3.8
23.2
4,936
76.8
16,313
98.4 PPV
13,198 RIBA Pos
Perform RIBA
No Further TestingHCV Infected?
S/CO 3.8
Ind
Pos
Neg
99.8 Sensitivity
55.8 PPV
31
3.5
65.5
13,177
2,753 RIBA Pos
5,055
566
10,692
10
Correlation of NAT Screening withSupplemental
HCV Serological Results(36,536 RR donations of
which 34,656 (94.9) had EIA and RIBA data)
9/8/99 to 6/30/03
RIBA Result
NATResult
Pos
Ind
Neg
Total
Rx NonRx Total
13,182(13,198)(80) 3,319 16,502(16,517)
(48)
174(159) 5,967 6,141(6,126)
50 11,963 12,013
13,407 21,249 34,656
(1.7)
140/174 (80) dHCV and PCR Rx (lt100-48,000,000
copies/mL) 16/50 (32) dHCV and PCR Rx
(lt100-24,000,000 copies/mL) 15 RIBA Ind due
to ³1 hSOD (12310 in RR samples)
11
Identification of HCV RIBA-Positive,
NAT-Nonreactive Samples
9/8/99 12/31/02
2,255HCV RIBA Pos, NAT Neg Neat
812Screened Neat,Neat Neg (36) 730
29Screened Pools,Pool Rx, Neat Neg (1) 25
1,414Screened Pools,Pool Neg (63) 1,397
Neat RetestNGI x1, orGen-Probe x2
Pos15(2.1)
Neg715
Pos1(3.7)
Neg24
Pos29(2.1)
Neg1,368
A minimum of one of two reps positive
12
Viral Loads of HCV RIBA-Positive Samples
Screened NAT Nonreactive in Pools or
Individually and that Retested NAT Reactive
Copies/mL at NGI Copies/mL at NGI Copies/mL at NGI Copies/mL at NGI
Screened in Pools (N29) 20 Quant/24 sent 100 (11)1 380 (2) 460 910 1,500 7,700 9,400 34,000 5,200,000
Screened Individually (N15) 10 Quant/12 sent 100 (5)1 200 430 2,000 7,400 12,000
Of 30 Quant, 10 (30) tested PCR positive
when diluted 116 frequency 8 of 2255 (1282)
total with viral loads gt1000 copies/mL
representing gt16 million donations screened
13
Relationship between HCV 3.0 EIA and RIBAAll
Samples 9/8/99 to 6/30/03
N 34,656
NegN 12,01334.7
IndN 6,12617.7
PosN 16,51747.6
S/CO
gt5.000
4.943
4.583
3.079
2.433
1.876
1.637
1.354
1.274
95 Range
1.024 - gt5.000
1.032 - gt5.000
3.077 - gt5.000
HCV SIA (3.0) Results
14
Relationship between HCV 3.0 EIA and RIBANAT
Reactive Samples 9/8/99 to 6/30/03
N 13,407
NegN 500.4
IndN 1591.2
PosN 13,19898.4
S/CO
gt5.000
gt5.0004.9714.627
4.763
4.393
4.221
1.800
1.354
95 Range
1.026 - gt5.000
1.212 - gt5.000
4.145 - gt5.000
HCV SIA (3.0) Results
15
Relationship between HCV 3.0 EIA and RIBANAT
Nonreactive Samples 9/8/99 to 6/30/03
N 21,249
NegN 11,96356.3
IndN 5,96728.1
PosN 3,31915.6
S/CO
gt5.000
4.795
4.311
2.955
2.428
1.840
1.636
1.348
1.274
95 Range
1.024 - gt5.000
1.031 - gt5.000
1.388 - gt5.000
HCV SIA (3.0) Results
16
ARC HCV Testing 9/8/99 Through 6/30/03NAT
Reactive Specimens Only (N 13,407)
S/CO RIBANEG RIBAIND RIBAPOS Total RIBAPOS
1.0 to 1.999 27 17 8 52 15.4
2.0 to 2.999 8 6 5 19 26.3
3.0 to 3.799 1 4 8 13 61.5
3.8 to 4.999 8 70 6,954 7,032 98.9
gt 5.0 6 62 6,223 6,291 98.9
Note 13,323 of 13,407 (99.4) NAT POS specimens
had an S/CO 3.813,177/13,323 (98.9)
specimens with S/CO 3.8 confirmed POS 95 CI
98.7 - 99.1 by binomial distribution Chi-square
tests for independence were significant (plt0.0001)
17
ARC HCV Testing 9/8/99 Through 6/30/03NAT
Nonreactive Specimens Only (N 21,249)
S/CO RIBANEG RIBAIND RIBAPOS Total RIBAPOS
1.0 to 1.999 7,731 3301 198 11,230 1.8
2.0 to 2.999 2,191 1,209 181 3,581 5.1
3.0 to 3.799 770 545 187 1,502 12.5
3.8 to 4.999 953 678 1,522 3,153 48.3
gt 5.0 318 234 1,231 1,783 69.0
Note 4,936 of 21,249 (23.2) NAT NEG specimens
had an S/CO 3.82,753/4,936 (55.8) specimens
with S/CO 3.8 confirmed POS 95 CI 54.4
57.2 by binomial distribution Chi-square tests
for independence were significant (plt0.0001)
18
PPV and Sensitivity of EIA and NAT vs RIBA
RIBA RIBA RIBA
POS NEG or IND TOTAL
EIA RR, NAT Rx 13,198 209 13,407
EIA RR, NAT NR 3,319 17,930 21,249
TOTAL 16,517 18,139 34,656
PPV 13,198/13,407 98.4 Sens 13,198/16,517
79.9
19
PPV and Sensitivity of High S/CO ( 3.8) vs RIBA
RIBA
POS NEG or IND TOTAL
EIA RR, S/CO 3.8 15,930 2,329 18,259
EIA RR, S/CO lt 3.8 587 15,810 16,397
TOTAL 16,517 18,139 34,656
PPV 15,930/18,259 87.2 Sens 15,930/16,517
96.4 Note Increasing the S/CO limit to 5.0
increases the PPV to 92.3 and decreases the
sensitivity to 45.1
20
PPV and Sensitivity of High S/CO ( 3.8) vs RIBA
Where NAT is Reactive
RIBA
POS NEG or IND TOTAL
EIA RR, S/CO 3.8 13,177 146 13,323
EIA RR, S/CO lt 3.8 21 63 84
TOTAL 13,198 209 13,407
PPV 13,177/13,323 98.9 Sens 13,177/13,198
99.8
21
PPV and Sensitivity of High S/CO ( 3.8) vs RIBA
Where NAT is Nonreactive
RIBA
POS NEG or IND TOTAL
EIA RR, S/CO 3.8 2,753 2,183 4,936
EIA RR, S/CO lt 3.8 566 15,747 16,313
TOTAL 3,319 17,930 21,249
PPV 2,753/4,936 55.8 Sens 2,753/3,319
82.9
22
HCV Alternate Algorithm Summary

• Use of NAT as the first step of the supplemental
  test algorithm will reduce the amount of RIBA
  performed by approximately 40
• HCV NAT-reactive samples (even using pooled NAT
  for screening)
• Sensitivity of 79.9 (detection of RIBA confirmed
  positives)
• Those not detected by NAT will be tested by RIBA
  (as true today)
• 98.4 are RIBA pos (PPV)
• Small percent of NAT-reactive samples likely
  representing early seroconversion are RIBA ind or
  neg

23
HCV Alternate Algorithm Summary

• Use of S/CO of gt 3.8 can be applied following
  separation of RR population into NAT Rx and nonRx
• Since NAT results are available for all
  donations, and is a specific test for HCV, S/CO
  introduced for NAT nonRx to further reduce the
  dependence on RIBA
• S/CO gt 3.8 does have high sensitivity and PPV
  relative to RIBA in populations where NAT is not
  performed
• Sensitivity of 96.4
• PPV of 87.2
• However, use of a high S/CO for NAT nonRxs would
  only eliminate 23 of RIBAs performed with poor
  performance
• Sensitivity of 82.9
• PPV of 55.8

24
Cambridge BiotechHuman Immunodeficiency Virus
Type 1 (HIV -1)Western Blot Kit

• March 16, 2000 BPAC
• Any bands present but pattern does not meet
  criteria for POSITIVE INDETERMINATE
• Non-viral bands have been observed with certain
  specimens. These bands are not usually
  accompanied by any of the major viral bands of
  diagnostic significance (p24, gp41/120/160). The
  non-viral bands appear to be cell related with
  the most common in the molecular weight range of
  70K, 51-55K (possible HLA-DR) and 43K (possible
  HLA-ABC).

25
HIV-1 Blood Donor Screening/SupplementalTest
Results (ARC) March 16, 2000 BPAC
1998-1999
12.4 Million Donations Screened
11,080 Repeat Reactive (0.09)HIV-1 WB
(Cambridge Biotech)
Positive 791 (7.1)
Negative 5,128 (46.3)
Indeterminate 5,161 (46.6)
26
HIV-1 Blood Donor Screening/SupplementalTest
Results (ARC) (Cont.) March 16, 2000 BPAC
1998-1999
5,161 Indeterminate (46.6)
One Viral Band
Multiple Viral Bands
BackgroundOnly (obscures reading)
Non- ViralBands
2,752 (53.3)
589 (11.4)
724 (14)
1,050 (20.3)

• 1,925 (70)p24 GAG only

• 464 (79) GAG reactivity with or without
  other banding

• 569 (79)p70

• 16 (35)ENV only

65.7 Viral Banding
27
Calypte Biotech HIV-1 Western Blot
gp160
gp120
p66
p55/51
gp41
p31
p24
p17
28
Calypte HIV-1 Western Blot
gp160
gp120
p66
p55/51
gp41
p31
p24
p17
29
HIV-1 RNA Concentrations During SC Based on NGIs
PCR of 28 Plasma Donor Panels (N221)
Categories
E001372A 29
30
Comparison of HIV-1 p24 Ag PositiveWindow-Case
U.S. Blood Donors
Sample Collection(days)
HIV-1/HIV-2 Ab S/CO
RNA Copies/mL
p24 Ag S/CO
PercentNeut.
SC
WB
1 Index 6 x 105 14.0 95 0.1 12 3 x
104 1.2 95 2.3 POS minus p31 22 1 x 105 0.3
5.6 POS all bands 2 Index 2 x
106 28.5 94 0.8 20 1 x 105 0.7 1.6 POS
minus p31 43 1 x 104 0.1 5.3 POS all
bands 3 Index 1 x 105 8.1 95 0.1 13 5 x
105 26.5 90 8.8 POS minus p31 4 Index 1 x
105 2.4 95 0.0 13 1 x 106 17.5 89 2.7 IND
p66 75 5 x 104 0.1 17.4 POS all
bands 5 Index 3 x 105 18.6 99 0.3 10 4 x
105 0.2 4.3 POS minus p31 17 1 x 105 0.9
12.5 POS minus p31 24 3 x 104 0.7 8.5 POS
minus p31 31 2 x 104 0.1 13.1 POS minus p31
31
Current HIV-1/HIV-2 EIA(Abbott rDNA or GSC pEIA)
Perform WBN 17,090
Ind
Pos
Neg
HIV NAT
HIV NAT
HIV NAT
ReactiveIndividually
NonRxin Pool orIndividually
ReactiveIndividually
NonRxin Pool orIndividually
ReactiveIndividually
NonRxin Pool orIndividually
HIVInfected
HIV Infected?Viral load lt NAT cutoffFalse Pos WB
HIV NotInfected
HIV NotInfected?
4.4
51
44
0.3
759
HIV Infected?Early seroconversionFalse Rx NAT
8,654
7,525
HIV InfectedEarly seroconversion
59
0.3
0.2
56 (6)
37 (0)
32
Option 1 HIV-1/HIV-2 EIA(Abbott rDNA or GSC
pEIA)
Review HIV-1 NAT ResultsN 17,090
Pool or Individual UnitNonRx(16 dtns
HIV-1/HCV TMA,24 dtns HIV PCR)
Individual UnitNAT Rx(dHIV TMA or HIV PCR)
5
95
852 (765)
16,238
No Further TestingHIV Infected
Perform WB
89.1 PPV(99.2)
Ind
Pos
Neg
53.3
0.4
46.3
759 WB Pos
8,654
59
7,525
92.8 Sensitivity
33
Option 2 HIV-1/HIV-2 EIA(Abbott rDNA or GSC
pEIA)
Review HIV-1 NAT ResultsN 17,090
33.9Sensitivity
Pool or Individual UnitNonRx(16 dtns
HIV-1/HCV TMA,24 dtns HIV PCR)
Individual UnitNAT Rx(dHIV TMA or HIV PCR)
5
95
16,238
852 (765)
Review S/CO Values of EIA
No Further TestingHCV Infected
S/CO 15
S/CO lt 15
89.1 PPV(99.2)
1.6
261
98.4
15,977
Perform WB
759 WB Pos
No Further TestingHIV Infected?
S/CO gt 15
Ind
Pos
Neg
7.7 PPV
99.6 Sensitivity
53.3
0.2
46.5
20 WB Pos
8,510
39
7,428
694
34
Correlation of NAT withSupplemental HIV
Serological Data(17,791 RR donations of which
17,090 (96.1) had EIA and WB data)
9/8/99 to 6/30/03
Western Blot Result
NATResult
Pos
Ind
Neg
Total
Rx NonRx Total
759(89.1) 59(0.4) 818(4.8)
56 8,654 8,710
37 7,525 7,562
(10.9)
852 16,238 17,090
6/56 (11) dHIV and PCR Rx (9,500-800,000
copies/mL) 0/37 (0) dHIV and PCR Rx
35
Characteristics of HIV-1 WB Pos/TMA Nonreactive
Samples
9/8/99 8/31/00
Sample Pool Neat HIV-1/HIV-2 S/CO WB HIV
PCR 1 NR 1.43 41, 120, 160 Neg 2 NR 1.03 41,
160 Neg 3 NR 1.62 41, 160 Neg 4 NR 1.11 41,
55, 160 Neg 5 NR 2.50 24, 41, 51, 61,
160 Neg 6 NR 20.18 all bands Neg 7 NR 1.22 24,
41, 160 Neg 8 NR 1.11 17, 41, 120,
160 Neg 9 NR 1.84 41, 160 Neg 10 NR 1.40 24,
41, 160 Neg 11 NR 1.70 17, 24, 41, 51,
160 Neg 12 NR 20.00 all bands Pos (200
copies/mL) 13 NR 17.84 all bands Neg
All samples p24 Ag negative
36
Characteristics of HIV-1 WB Pos/TMA Nonreactive
Samples
9/1/00 12/29/01
Sample Pool Neat HIV-1/HIV-2 S/CO WB HIV
PCR 14 NR 8.73 17, 24, 160 Neg 15 NR 10.56 24
, 55, 120, 160 Neg 16 NR 19.30 all
bands Neg 17 NR 19.47 all bands Pos (200
copies/mL) 18 NR 1.04 41, 66, 120,
160 Neg 19 NR 4.72 41, 160 Neg 20 NR 1.50 24,
41, 160 Neg 21 NR 1.53 24, 41,
160 Neg 22 NR 1.60 24, 160 Neg 23 NR 17.60 all
bands Neg 24 NR 19.30 all bands Neg
All samples p24 Ag negative
37
Characteristics of HIV-1 WB Pos/TMA Nonreactive
Samples
9/1/00 12/29/01
Sample Pool Neat HIV-1/HIV-2 S/CO WB HIV
PCR 25 NR 1.00 24, 120, 160 Neg 26 NR 1.19 41,
120, 160 Neg 27 NR 17.43 all
bands Neg 28 NR 1.37 41, 160 Neg 29 NR 1.40 24
, 41, 120, 160 Neg 30 NR 1.43 24,
160 Neg 31 NR 15.20 17, 24, 120,
160 Neg 32 NR 18.80 all bands Neg 33 NR 15.93
all bands Neg 34 NR 1.34 41, 160 Neg
All samples p24 Ag negative
38
Relationship between HIV-1/HIV-2 EIA andWestern
Blot-All Samples 9/8/99 to 6/30/03
N 17,090
NegN 7,56244.2
IndN 8,71051.0
PosN 8184.8
19.178
18.030
16.008
EIAS/CO
2.600
2.672
1.602
1.598
1.225
1.231
95 Range
1.018 11.370
1.018 12.329
1.513 20.952
Western Blot Results
39
Relationship between HIV-1/HIV-2 EIA and Western
BlotNAT Nonreactive Samples 9/8/99 to 6/30/03
N 16,238(95 of total)
NegN 7,52546.3
IndN 8,65453.5
PosN 590.4
16.268
EIAS/CO
2.602
2.672
2.390
1.602
1.599
1.400
1.225
1.230
95 Range
1.018 11.368
1.018 12.258
1.013 20.092
Western Blot Results
40
Relationship between HIV-1/HIV-2 EIA and Western
BlotNAT Reactive Samples 9/8/99 to 6/30/03
N 852(5 of total)
NegN 374.3
IndN 566.6
PosN 75989.1
19.298
18.182
16.296
EIAS/CO
2.545
2.560
1.578
1.581
1.314
1.299
95 Range
1.076 12.398
1.012 18.385
8.130 20.952
Western Blot Results
41
ARC HIV Testing 9/8/99 Through 6/30/03NAT
Reactive Specimens Only (N 852)
S/CO WBNEG WBIND WBPOS Total WBPOS
1.0 to 4.999 35 48 5 88 5.7
5.0 to 9.999 1 2 23 26 88.5
10.0 to 14.999 1 3 37 41 90.2
15.0 to 19.999 0 3 591 594 99.5
gt 20.0 0 0 103 103 100.0
Note 697 of 852 (81.8) NAT POS specimens had
an S/CO 15.0694/697 (99.6) specimens with
S/CO 15.0 confirmed POS 95 CI 98.8 - 99.9
by binomial distribution Chi-square tests for
independence were significant (plt0.0001)
42
ARC HIV Testing 9/8/99 Through 6/30/03NAT
Nonreactive Specimens Only (N 16,238)
S/CO WBNEG WBIND WBPOS Total WBPOS
1.0 to 4.999 6,769 7,749 35 14,553 0.2
5.0 to 9.999 515 609 3 1,127 0.3
10.0 to 14.999 144 152 1 297 0.3
15.0 to 19.999 86 133 18 237 7.6
gt 20.0 11 11 2 24 8.3
Note 261 of 16,238 (1.6) NAT NEG specimens had
an S/CO 15.020/261 (7.7) specimens with s/co
15.0 confirmed POS 95 CI 4.7 11.6 by
binomial distribution Chi-square tests for
independence were significant (plt0.0001)
43
PPV and Sensitivity of HIV-1/HIV-2 EIA and NAT vs
Western Blot
Western Blot
POS NEG or IND TOTAL
EIA RR, NAT Rx 759 93 (6) 852 (765)
EIA RR, NAT NR 59 16,179 16,238
TOTAL 818 16,272 17,090
PPV 759/852 89.1 (759/765 99.2) Sens
759/818 92.8
44
PPV and Sensitivity of High S/CO ( 15.0) vs
Western Blot
Western Blot
POS NEG or IND TOTAL
EIA RR, S/CO 15.0 714 244 958
EIA RR, S/CO lt 15.0 104 16,028 16,132
TOTAL 818 16,272 17,090
PPV 714/958 74.5 Sens 714/818 87.3
45
PPV and Sensitivity of High S/CO ( 15.0) vs WB
Where NAT is Reactive
Western Blot
POS NEG or IND TOTAL
EIA RR, S/CO 15.0 694 3 697
EIA RR, S/CO lt 15.0 65 90 155
TOTAL 759 93 (6) 852 (765)
PPV 694/697 99.6 Sens 694/759 91.4
46
PPV and Sensitivity of High S/CO ( 15.0) vs WB
Where NAT is Nonreactive
Western Blot
POS NEG or IND TOTAL
EIA RR, S/CO 15.0 20 241 261
EIA RR, S/CO lt 15.0 39 15,938 15,977
TOTAL 59 16,179 16,238
PPV 20/261 7.7 Sens 20/59 33.9
47
HIV Alternate Algorithm Summary

• Use of NAT as the first step of the supplemental
  test algorithm will reduce the amount of HIV WB
  performed by only 5
• HIV NAT-reactive samples (even using pooled NAT
  for screening)
• Sensitivity of 92.8 (detection of WB confirmed
  positives)
• Those not detected will be tested by WB only
  0.4 of positives remain and majority are false
  positive on WB (as indicated by repeat NAT
  nonRx, low EIA S/CO and weak WB banding patterns)

48
HIV Alternate Algorithm Summary

• Use of S/CO gt 15 following separation of RR
  population into NAT Rx and nonRx is not practical
• Vast majority of HIV RR samples are not from
  individuals with HIV infection and consequently
  are NAT nonRx and have low S/CO values
• Of those with an S/CO gt 15, the PPV is 7.7
  (20/261)
• Of those with an S/CO lt 15, it is likely that all
  WB pos are false pos
• Other options
• Dual EIA Algorithm

49
Dual EIA Algorithm

• Feasibility based on the concept
• If two assays with comparable sensitivity are
  composed of differing rare reagents and have a
  different format, the false positive populations
  should have limited cross over the more unique
  the tests, the greater the separation of false
  positive populations
• Used successfully for HTLV and HBsAg to eliminate
  gt60 of repeat reactives requiring further
  testing by expensive, complicated, error prone
  and unavailable/unlicensed tests (e.g., HTLV)

50
ARC HTLV Supplemental Results
Number

State of California Testing Initiated
Month (2000-2002)
51
HIV Dual EIA Algorithm Qualification

• Qualified in both directions based on the two FDA
  licensed HIV-1/HIV-2 EIAs
• Abbott (EIA-1) Genetic Systems (EIA-2) (ARC)
• Genetic Systems (EIA-1) Abbott (EIA-2) (BSL)
• All HIV EIA repeat reactive samples from
  1/1/00-3/31/02 having adequate volume for
  additional testing were evaluated
• Western Blot and NAT (TMA pools of 16)
  test-of-record data were used for analysis
• All 2nd EIA testing was performed centrally at BSL

52
HIV Dual EIA AlgorithmGenetic Systems pEIA
(EIA-1) at BSL
N 1,657 RR Samples (1/1/00 3/31/02)
Tested by Western Blot (Bio-Rad)
Ind544 (32.8)
Pos80 (4.8)
Neg1,033 (62.4)
Abbott
EIA-2
Abbott
EIA-2
Abbott
EIA-2
RR 80
NR 0
RR 31
NR 513
RR 49
NR 984
NAT
NAT
NAT
79
1
0
31
0
513
0
49
0
984
(all WB bandshigh S/COboth EIAs)
53
HIV Dual EIA AlgorithmAbbott rDNA EIA (EIA-1) at
ARC
N 6,227 RR Samples (1/1/00 3/31/02)
Tested by Western Blot (Calypte)
Ind2,890 (46.4)
Pos266 (4.3)
Neg3,071 (49.3)
GSC
EIA-2
GSC
EIA-2
GSC
EIA-2
RR 250
NR 16
RR 16
NR 2,874
RR 13
NR 3,058
NAT
NAT
NAT
237
13
1
15
0
2,874
0
13
0
3,058
0
16
(/- 24, 160)
54
N 13, Abbott RR, WB Pos, GSC RR, NAT NR
WB Banding Pattern Abbott S/CO GSC S/CO
1 24, 31, 41, 51, 66, 120, 160 17.43 5.80
2 17, 24, 120, 160 15.52 2.91
3 all bands 15.93 9.30
4 all bands 17.19 8.62
5 all bands 19.30 8.87
6 17, 24, 61, 160 2.40 7.06
7 24, 31, 41, 51, 66, 120, 160 20.95 4.39
8 24, 41, 120, 160 1.40 1.33
9 all bands 19.30 8.07
10 41, 66, 120, 160 1.04 9.38
11 all bands 17.60 8.59
12 all bands 17.84 8.93
13 all bands 18.80 11.12
55
N 16, Abbott RR, WB Pos, GSC NR, NAT NR
WB Banding Pattern Abbott S/CO GSC S/CO
1 17, 41, 160 1.53 0.17
2 17, 41, 160 1.50 0.20
3 41, 160 1.34 0.30
4 41, 120, 160 4.90 0.25
5 41, 160 4.72 0.31
6 24, 120 1.52 0.29
7 41, 120, 160 4.92 0.25
8 24, 41, 160 1.00 0.38
9 41, 120, 160 1.19 0.29
10 24, 160 1.43 0.22
11 24, 160 1.60 0.32
12 17, 24, 41, 120, 160 2.36 0.33
13 17, 41, 120, 160 1.11 0.17
14 24, 55, 120, 160 10.56 0.57
15 17, 24, 160 8.73 0.62
16 41, 160 1.37 0.28
Both samples PCR and repeat TMA (undilute) NR
one of two reported participation in an HIV
vaccine trial
56
HIV Dual EIA Summary
GSC BSL N 1,657 No. Abbott RR No. NAT Rx
No. WB Pos 80 (5) 80 (100) 79
(Bio-Rad) Ind 544 (33) 31 (6) 0
Neg 1,033 (62) 49 (5) 0
Abbott ARC N 6,227 No. GSC RR No. NAT Rx
No. WB Pos 266 (4) 250 (94) 237
(Calypte) Ind 2,890 (46) 16 (0.6) 1
Neg 3,071 (49) 13 (0.4) 0
57
HIV Dual EIA Algorithm Findings

• Using NAT as the gold standard
• Sensitivity 100 (317/317)
• 95 CI 98.84-100
• Specificity 98.4 (7,447/7,567)
• 95 CI 98.11-98.68
• No. WBs eliminated 98.4 (7,764/7,884)
• No. indeterminate interpretations eliminated
  98.6 (3,387/3,434)