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Replacement of HIV-1 p24 Antigen Replacement of HIV-1 p24 Antigen Screening with HIV-1 RNA Nucleic Acid Screening with HIV-1 RNA Nucleic Acid

Testing (NAT) for Whole Blood DonationsTesting (NAT) for Whole Blood Donations

S.L. Stramer, R.A. Porter, J.P. Brodsky, S.L. Stramer, R.A. Porter, J.P. Brodsky, K.J. Waldman, R.Y. Dodd, T.A. ArmstrongK.J. Waldman, R.Y. Dodd, T.A. Armstrong

and G.D. Griffinand G.D. Griffin

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BackgroundBackground

Since the implementation of p24 HIV-1 antigen Since the implementation of p24 HIV-1 antigen (Ag) screening in March, 1996, only 5 Ag positive (Ag) screening in March, 1996, only 5 Ag positive window-case donations have been identifiedwindow-case donations have been identified

Due to low Ag yield and the improved sensitivity Due to low Ag yield and the improved sensitivity of NAT screening for HIV-1, replacement of Ag of NAT screening for HIV-1, replacement of Ag with HIV-1 NAT should be possiblewith HIV-1 NAT should be possible

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HIV-1 p24 Antigen Testing ExperienceHIV-1 p24 Antigen Testing Experience

During 2 years of testing 14 million donations (ARC), During 2 years of testing 14 million donations (ARC), 136 samples were HIV-1 p24 Ag confirmed positive136 samples were HIV-1 p24 Ag confirmed positive 58 were false positive (based on RNA negativity, 58 were false positive (based on RNA negativity,

lack of seroconversion, and nonreproducible Ag lack of seroconversion, and nonreproducible Ag neutralization) = 1:241,379neutralization) = 1:241,379

74 were HIV Ab positive74 were HIV Ab positive 4 were recently infected seroconverting donors4 were recently infected seroconverting donors As of November 1999, 5 seroconverting donors As of November 1999, 5 seroconverting donors

identified in 23 million donationsidentified in 23 million donations– 1:4.6 million (ARC)1:4.6 million (ARC)– 1:9.2 million (US)1:9.2 million (US)

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Samples for TestingSamples for Testing

p24 Ag and HIV NAT reactive yield samples (N=4)p24 Ag and HIV NAT reactive yield samples (N=4)– Tested undiluted and diluted 1:128 in RNA-negative Tested undiluted and diluted 1:128 in RNA-negative

defibrinated plasmadefibrinated plasma

Commercial seroconversion samples (N=25)Commercial seroconversion samples (N=25)– Tested undiluted and diluted 1:128Tested undiluted and diluted 1:128

Diluted p24 Ag “External Control” sample that is Diluted p24 Ag “External Control” sample that is tested on all NAT runs (N=437)tested on all NAT runs (N=437)– 3 Ag+/Ab– units (S/CO 1-2 Coulter) pooled3 Ag+/Ab– units (S/CO 1-2 Coulter) pooled

– Pool diluted 1:16Pool diluted 1:16

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Comparison of HIV-1 p24 Ag Positive Comparison of HIV-1 p24 Ag Positive Window-Case U.S. Blood DonorsWindow-Case U.S. Blood Donors

11 IndexIndex 6 x 106 x 1055** 14.0*14.0* 9595 0.10.11212 3 x 103 x 1044 1.21.2 9595 2.32.3 POS – minus p31POS – minus p312222 1 x 101 x 1055 0.30.3 5.65.6 POS – all bandsPOS – all bands

22 IndexIndex 2 x 102 x 1066** 28.5*28.5* 9494 0.80.82020 1 x 101 x 1055 0.70.7 1.61.6 POS – minus p17, p31POS – minus p17, p314343 1 x 101 x 1044 0.10.1 5.35.3 POS – all bandsPOS – all bands

33 IndexIndex 1 x 101 x 1055 8.18.1 9595 0.10.11313 5 x 105 x 1055** 26.5*26.5* 9090 8.88.8 POS – minus p31 POS – minus p31

44 IndexIndex 1 x 101 x 1055 2.42.4 9595 0.00.01313 1 x 101 x 1066** 17.5*17.5* 8989 2.72.7 IND – p66IND – p667575 5 x 105 x 1044 0.10.1 17.417.4 POS – all bandsPOS – all bands

55 IndexIndex 3 x 103 x 1055 18.6*18.6* 9999 0.30.31010 4 x 104 x 1055** 0.20.2 4.34.3 POS – minus p31POS – minus p311717 1 x 101 x 1055 0.90.9 12.512.5 POS – minus p31POS – minus p312424 3 x 103 x 1044 0.70.7 8.58.5 POS – minus p31POS – minus p313131 2 x 102 x 1044 0.10.1 13.113.1 POS – minus p31POS – minus p31

SCSC

Sample Sample CollectionCollection

(days)(days) WBWBRNA RNA

Copies/mLCopies/mLp24 Ag p24 Ag S/COS/CO

PercentPercentNeut.Neut.

HIV-1/HIV-1/HIV-2 Ab HIV-2 Ab

S/COS/CO

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Detection of HIV-1 p24 Ag and NAT Yield Detection of HIV-1 p24 Ag and NAT Yield Samples by HIV-1 NAT (1:128 Dilution)Samples by HIV-1 NAT (1:128 Dilution)

ARC 2ARC 2 AgAg 6x106x1055 ++ (16.08)(16.08)

ARC 3ARC 3 AgAg 1x101x1055 ++ (16.06)(16.06)

ARC 5ARC 5 AgAg 3x103x1055 ++ (29.59)(29.59)

MilwaukeeMilwaukee NATNAT 2x102x1044 ++ (7.86)(7.86)

CaseCaseIndexIndex

Detected ByDetected By

Index RNAIndex RNAConc. UndilutedConc. Undiluted

(copies/mL)(copies/mL)

MultiplexMultiplex1:128*1:128*(S/CO)(S/CO)

* Diluted in RNA screened negative defibrinated plasma* Diluted in RNA screened negative defibrinated plasma

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HIV Panel 6240 – HIV Panel 6240 – Virologic/Serologic ProfileVirologic/Serologic Profile

DaysDays

S/C

OS

/CO

HIV

PC

R Q

uant

itatio

nH

IV P

CR

Qua

ntita

tion

PCR

100

1,000

10,000

100,000

1,000,000

0 10 20 30 40 50 60

0

5

10

15

20

25

30

35

HIV-1 p24 Antigen HIV 1/2 Antibody

5 5 DaysDays

2 2 DaysDays

100

1,000

10,000

100,000

1,000,000

0 10 20 30 40 50 60

0

5

10

15

20

25

30

35

HIV-1 p24 Antigen HIV 1/2 Antibody Multiplex Neat Multiplex 1:128

HIV Panel 6240 – HIV Panel 6240 – Virologic/Serologic ProfileVirologic/Serologic Profile

DaysDays

S/C

OS

/CO

HIV

PC

R Q

uant

itatio

nH

IV P

CR

Qua

ntita

tion

2 Days 2 Days 1:1281:128

7 Days 7 Days

NeatNeat

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PCR

5 Days5 Days

PCR

Days

S/C

OH

IV P

CR

Qua

ntita

tion

100

1,000

10,000

100,000

1,000,000

0 20 40 60 80 100 120 140 160 180 200

0

10

20

30

HIV-1 p24 Antigen HIV 1/2 Antibody

HIV Panel PRB932 –HIV Panel PRB932 –Virologic/Serologic ProfileVirologic/Serologic Profile

Cutoff

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PCR

Days

S/C

OH

IV P

CR

Qua

ntita

tion

100

1,000

10,000

100,000

1,000,000

0 20 40 60 80 100 120 140 160 180 200

0

10

20

30

HIV-1 p24 Antigen HIV 1/2 Antibody Multiplex Neat Multiplex 1:128

HIV Panel PRB932 –HIV Panel PRB932 –Virologic/Serologic ProfileVirologic/Serologic Profile

Cutoff

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14 Days 14 Days

HIV Panel PRB943 –HIV Panel PRB943 –Virologic/Serologic ProfileVirologic/Serologic Profile

Days

S/C

OH

IV P

CR

Qua

ntita

tion

100

1,000

10,000

100,000

1,000,000

0 5 10 15 20 25

0

10

20

30

40

50

HIV-1 p24 Antigen HIV 1/2 AntibodyPCR

Cutoff

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12 Days 12 Days 2 Days 2 Days

PCR

HIV Panel PRB943 –HIV Panel PRB943 –Virologic/Serologic ProfileVirologic/Serologic Profile

Days

S/C

OH

IV P

CR

Qua

ntita

tion

100

1,000

10,000

100,000

1,000,000

0 5 10 15 20 25

0

10

20

30

40

50

HIV-1 p24 Antigen HIV 1/2 Antibody Multiplex Neat Multiplex 1:128

Cutoff

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5 Days 5 Days 7 Days 7 Days 2 Days 2 Days

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Clinical Sensitivity, Clinical Sensitivity, HIV Panels, Gen-ProbeHIV Panels, Gen-Probe

0

5

10

15

20

25

30

0 5 10 15 20 25 30

Multiplex Assay S/CO (Neat)

p24

Ant

igen

S/C

O

Cutoff

Lot 809355Lot 809355HIV Seronegative BleedsHIV Seronegative Bleeds

(92 samples from(92 samples from25 individuals)25 individuals)

29 NAT +/p24 Ag –29 NAT +/p24 Ag –

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Cutoff

Clinical Sensitivity, Clinical Sensitivity, HIV Panels, Gen-ProbeHIV Panels, Gen-Probe

Lot 809355Lot 809355HIV Seronegative BleedsHIV Seronegative Bleeds

(92 samples from(92 samples from25 individuals)25 individuals)

Multiplex Assay S/CO (1:128)

p24

Ant

igen

S/C

O

0

5

10

15

20

25

30

0 5 10 15 20 25 30

21 NAT +/p24 Ag – 1:12821 NAT +/p24 Ag – 1:128

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Effect of Dilution on NAT Reactivity Effect of Dilution on NAT Reactivity (Gen-Probe HIV-1/HCV Assay)(Gen-Probe HIV-1/HCV Assay)

Lot 355Lot 355

HIV-1 (N=25 Panels)HIV-1 (N=25 Panels)

Lot 356Lot 356

HCV (N=22 Panels)HCV (N=22 Panels)

Lot 355Lot 355 Lot 356Lot 356

UndilutedUndiluted 162162 167167 162162 160160

Diluted 1:128Diluted 1:128 145 (90%)145 (90%) 148 (89%)148 (89%) 151 (93%) 151 (93%) 151 (94%)151 (94%)

p24 Agp24 Ag 82 (57%)82 (57%) 82 (55%)82 (55%) —— ——

ALT (ALT (60)60) —— —— 50 (44%)*50 (44%)* 49 (43%)**49 (43%)**

* Of 114 tested;* Of 114 tested;** Of 113 tested** Of 113 tested

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Gen-Probe Linked Study – Phase I Gen-Probe Linked Study – Phase I External Control PanelExternal Control Panel

MemberMember

HIV-1 300 ± 150

HCV 300 ± 150

NEG —

Weak HIV-1 p24 Ag(S/CO 1-2 Coulterdiluted 1:16) 6,800

TargetTargetCopies/mLCopies/mL

Gen-Probe Linked Study – Phase IGen-Probe Linked Study – Phase IExternal Control DistributionsExternal Control Distributions

Fre

quen

cy

S/CO

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

50

100

150

200Control N Mean S/CO Range

HIV-1 435 18.66* 8.9 – 37.6

HCV 438 8.27* 2.1 – 10.4

NEG 436 0.28 0.1 – 0.9

HIV-1/p24 Ag 437 20.06* 12.1 – 25.2

*p<0.05 (437 runs total)

0

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ConclusionsConclusions

High RNA titers (High RNA titers (101055 copies/mL) correspond copies/mL) correspond with p24 Ag positivitywith p24 Ag positivity

No p24 Ag reactive sample was found NAT No p24 Ag reactive sample was found NAT nonreactive even using a pool size of 128nonreactive even using a pool size of 128

NAT, even using pools of 128, is more sensitive NAT, even using pools of 128, is more sensitive than screening with the currently licensed tests than screening with the currently licensed tests for HIV-1 p24 Ag. According to these data, the for HIV-1 p24 Ag. According to these data, the Ag test could be replaced by the use of licensed Ag test could be replaced by the use of licensed pooled NATpooled NAT

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1

2

3

4

5

6

7

8Lo

g R

NA

Lev

el [

copi

es/m

L]

-2 -1.5 -1 -.5 0 .5 1 1.5 2

Log p24 S/CO

N = 146R2 = 0.78

Linear Relationship Between HIV RNA Linear Relationship Between HIV RNA Levels and p24 S/CO RatiosLevels and p24 S/CO Ratios

4.03 log (596-190,108 copies/mL) = 1

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-2

-1

0

1

2

3

4

5

6

7

8

9Lo

g H

IV R

NA

Lev

el [

copi

es/m

L]

-2 -1.5 -1 -.5 0 .5 1 1.5 2Log p24 S/CO

N = 146

95% Prediction Limits for Relationship of 95% Prediction Limits for Relationship of HIV RNA Levels and p24 S/CO RatiosHIV RNA Levels and p24 S/CO Ratios

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1

2

3

4

5

6

7

8Lo

g H

IV R

NA

Lev

el [

copi

es/m

L]

RNA Only (N=61) p24 Pos (N=85)

P = <0.0001*

* Mann-Whitney Test

HIV-1 RNA Levels During “RNA Only” HIV-1 RNA Levels During “RNA Only” and “p24 Positive” Window Periodsand “p24 Positive” Window Periods

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1

2

3

4

5

6

7

8Lo

g R

NA

Lev

el [

copi

es/m

L]

-2 -1.5 -1 -.5 0 .5 1 1.5 2

Log p24 S/CO

Linear Relationship Between HIV-1 RNA Levels Linear Relationship Between HIV-1 RNA Levels and p24 S/CO Ratios in 32 Casesand p24 S/CO Ratios in 32 Cases