Results of donor screening with nucleic acid amplification tests (NAT)

and implications for HIV research, diagnosis and surveillance

Michael P. Busch, MD, PhDProfessor of Laboratory Medicine, UCSF

Director, Blood Systems Research Institute

Overview of presentation

1. RNA dynamics in primary HIV infection and projected yield of NAT in low and high incidence populations

2. Design of NAT assays for blood screening

3. Yield and cost effectiveness of NAT screening of blood donors

4. Applications of donor NAT assays in research settings

HIV Viremia during early infection

HIV RNA (plasma)HIV Antibody

11

0 10 20 30 40 50 60 70 80 90 100

HIV p24 Ag

16 22

Ramp-up viremia

DT = 21.5 hrs

1st gen2nd gen

3rd gen

p24 Ag EIA -

HIV MP-NAT -

HIV ID-NAT -

Peak viremia: 106-108 gEq/mL

“blip” viremia

Viral set-point: 102 -105 gEq/mL

AIDS, 17:1871-9, 2003.

AIDS, 17:1871-9, 2003

Time Table of HIV Stage Progression based on 51 Seroconverting Plasma Donors

StageMarker Duration in Days

(95% CI)**RNA p24 Ag Ab (EIA) WB

I + - - - - 5.0 (3.1, 8.1)

II + + - - - 5.3 (3.7, 7.7)

III + + - + - 3.2 (2.1, 4.8)

IV + +/- - + IND 5.6 (3.8, 8.1)

V + +/- +/- + + * 69.5 (39.7, 121.7)

VI + +/- + + + open-ended

* without p31 band ** calculations are based on a parametric Markov model

AIDS, 17:1871-9, 2003.

Individual Rates and Linear Regression Model of HIV RNA Production during Early Infection.

LOG HIV RNA [gEq/mL]

1

2

3

4

5

6

7

8

9

-10 -5 0 5 10 15 20Day

N = 97 Samples from 44 Plasma donorsDT: 21.5 hrs (95% CI: 19.2-24.6)

Projected WP Closure and Yield of p24 Ag, MP and ID NAT Assays Relative to a Sensitive HIV-EA

Antibody Test in the Detection of WP HIV Infection

Assay Sensitivity

[gEq / mL]

WP Closure

[days]

Yield, WP HIV Infections per 1,000 Persons Tested in Various Screening Settings

[ Representative Incidence Rate / Person-Years ]

Blood Donors

[ 2 / 100,000 = 0.002% ]

STD Clinic

[ 1 / 1,000 = 0.1% ]

High Risk Clinic

[ 1 / 10 = 10% ]

p24 Ag 10,000 6 0.00033 0.016 1.6

MP NAT 1,000 9 0.00049 0.025 2.5

ID NAT 50 13 0.00071 0.036 3.6

AIDS, 17:1871-9, 2003

Proposed Laboratory Stages of Primary HIV Infection

with Semiquantitative Plot of HIV Assay Reactivities

Representative HIV Conversion Panels with Pre-Ramp-Up “Blip” Viremia

(RNA “blip” observed in 7/19 informative panels)

Days from First MP-PCR+ Test

p24 Ag HIV-1 Ab

HIV-1 RNA (copies / mL)

# Pos /

# Replicate PCR

- 21 Neg Neg < 100 1 / 10

- 19 Neg Neg < 100 7 / 8

- 14 Neg Neg < 100 0 / 8

- 11 Neg Neg < 100 0 / 8

- 7 Neg Neg < 100 0 / 8

- 4 Neg Neg < 100 3 / 8

0 Neg Neg 260 5 / 5

3 Neg Neg 27,000

7 Pos Neg 370,000

9 Pos Neg 2,800,000

16 Pos REACT 410,000

Alpha / BCP Case 1012

New Test Implementation and Declining Risk of Viral Infections from Transfusion

Busch et al. JAMA 2003; 289: 959-962

Advances in NAT systems for blood donor screening

• Fully automated platforms– Bar coded tubes to validated electronic results

• Generic extraction of RNA/DNA• Internal controls to verify amplification• Multiplex or parallel detection of mulitple viruses

(Taqman, beacon or DKA strategies)• Primers selected to amplify divergent subtypes• Rapid response to emerging pathogens

TIGRISFully Automated NAT System

Procleix Specimen ProcessingTarget Capture/Magnetic Microparticle Separation

Viral Lysis Treat specimens with heat and detergent Release nucleic acid

Nucleic Acid Capture Hybridize target sequence to capture probes Hybridize capture probe to oligomer sequence bind to magnetic particle

Removal of unwanted specimen Apply magnetic field to separate target from residual sample Remove residual specimen by washing

Magnetic Microparticle

TTTTTTTTTTTTTT

TTTTTTTTTTTTTT

TTTTTTTTTTTTTT

Ma

gn

e t

3’ AAAAAA…AAACAUCUAAC…CGU 5’

5’ GUAGAUUG…GCA 3’

Capture Oligo

Target RNA orDNA

TT

TT

TT

TT

TT

TT

TT

TT

TT

TT

TT

TT

TT

TT

Procleix Detection Dual Kinetics Analysis (DKA)

Used to differentiate Internal Control (IC) signal from target signal

Utilizes Acridinium Ester (AE) labeled probes with differential kinetics of light-off

Ortho Fluoro Acridinium Ester labeled probe = flasher probe, hybridizes to IC

2’Methyl Acridinium Ester labeled probes = Glower probes, hybridize to West Nile virus amplicon

ETF Algorithm deconvolutes light-off and calculates each signal

0

2000

4000

6000

8000

10000

12000

14000

16000

1 4 7 10

13

16

19

22

25

28

31

34

37

40

43

46

49

Interval

RLU

Flasher

Background

Glower

Analytical Sensitivity, HIV-1Chiron Procleix™ HIV-1/HCV Assay and HIV-1 Discriminatory Assay

02 04 06 08 0

1 0 01 2 0

0 1 3 1 0 3 0 1 0 0 3 0 0

HIV/HCV, Lot AHIV Discr, Lot AHIV/HCV, Lot BHIV Discr, Lot B

50% detection at 8 Copies/ml

Copies/ml

Pos

itivi

ty r

ate

Roche Fully Automated TaqScreen™ System

COBAS AmpliPrep

COBAS TaqMan

Hamilton Pipettor

TaqMan™ 5' Nuclease AssayFluorescence Energy Transfer Probe

Q

Qh

R

R

R

Primer ProbeR Fluorescence Emission

Quenched

R = ReporterQ = Quencher

R Fluorescence EmissionDetected

h Q

Testing Algorithm for Plasma Pools usingChiron/G-P Procleix™ Assays

Plasma PoolsPlasma Pools

Non-Reactive PoolsNon-Reactive PoolsReactive PoolsReactive Pools

Non-Reactive SamplesNon-Reactive SamplesReactive SamplesReactive Samples

Discriminated SamplesDiscriminated Samples Non-Discriminated SamplesNon-Discriminated Samples

HIV-1 and HCV Discriminatory ProbesHIV-1 and HCV Discriminatory Probes

Chiron Procleix HIV-1/HCV AssayChiron Procleix HIV-1/HCV Assay

Chiron Procleix HIV-1/HCV AssayChiron Procleix HIV-1/HCV Assay

Constituent SamplesConstituent Samples

Results of NAT Screening in U.S.

Virus Dates Units tested NAT+/Ab-

HCV 4-10/99 to 12/04

53.3 million 230 (1/230,000)

HIV 4/99-12/00 to 12/04

50.3 million 18(1/3.1 million)

HBV 8/02 to 12/04

1.7 million 5(1/330,000)

WNV 7/03 to 11/04

4.8 million 968(1/5,000)

Stramer, Glynn, Kleinman, Caglioti, Strong, Busch . NEJM, 2004Gandhi, Strong, Kleinman et al. Blood102 (11):192A, 2003

Morb Mortal Wkly Rep 52:1160

Correlation of routine HIV NAT screening with supplemental HIV serological data

5,972 HIV-EIA RR of 7.6 million donations at BSL, 04/99 - 07/03

NAT Result

Pos

Ind

Neg

Total

Pos

(dHIV R)

267

(93%)

2**

(0.1%)

0

269

Neg (mTMA on

MPs)

18*

286

2216

2218

3468

3468

5702

Western Blot Result

* 9/18 autologous donations, 6/9 from 3 donors. 1 false positive WB, f/u EIA R, WB(-), NAT(-). ** 1 w/ 1+ p24 band-only; 1 w/ +/- p24, p55, gp120 & 2+ gp160 bands.

Total

EIA signal/cutoff and WB band data for 18 BSL donor samples that tested WB-pos /

NAT-negNAT HIV EIA HIV S/CO HIV S/CO HIV S/CO GS_BLOT E18 E24 E31 GS40 E41 E51 E55 E65 E120 E160

NR R POS + + +/- + + + + + + +NR R 2.246 1.874 1.781 POS - + - - - - - - - +NR R POS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++NR R 7.813 6.281 5.905 POS +/- ++ ++ ++ ++ ++ ++ ++ ++ ++NR R 8.031 6.678 7.331 POS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++NR R 7.592 7.197 6.945 POS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++NR R 7.577 7.278 7.281 POS +/- + ++ ++ ++ + ++ ++ + ++NR R POS - ++ + ++ + + ++ ++ + ++NR R 6.722 6.878 6.879 POS + ++ ++ ++ ++ ++ ++ ++ ++ ++NR R 7.624 6.28 6.089 POS ++ ++ ++ ++ ++ + ++ ++ ++ ++NR R 7.93 8.062 7.843 POS ++ ++ ++ ++ ++ + ++ + ++ ++NR R 7.519 7.888 7.473 POS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++NR R 6.994 7.003 7.385 POS + ++ ++ ++ ++ + ++ ++ ++ ++NR R 7.331 6.898 7.427 POS +/- ++ ++ ++ + ++ ++ + ++ ++NR R 7.314 6.691 6.965 POS +/- ++ +/- ++ + +/- ++ ++ ++ ++NR R 6.985 6.196 7.042 POS +/- ++ ++ ++ ++ +/- ++ ++ ++ ++NR R 7.492 6.574 6.716 POS + ++ ++ ++ ++ + ++ + ++ ++NR R 7.577 7.105 6.563 POS ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

17 infected donors with probable very low-level viremia 1 donor with false positive WB

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

4 0 0

4 5 0

J u l y A u g S e p O c t

M P - N A T I D - N A T

Yield of WNV NAT screening of 4,585,573 donations from July-October, 2003

ARC & ABC (~95% of U.S. collections)

944 confirmed viremic donors.: - 770 detectable by MP-NAT - 174 ID-NAT-only (BSI and ARC screened 36,269 donations)

Incremental WP Closure and Projected Yield (U.S.) of ID over MP-NAT Screening

Assumes 20 member pools: 20-fold differential sensitivity of ID-NAT and MP-NAT (e.g., 5 gEq/mL vs. 100 gEq/mL)

Does not include detection by ID-NAT of intermittent, low-level viremic episodes preceding ramp-up viremia

WP Closure (days)

Yieldper 107

HCV 3.2 2.5 (1:4,000,000)

HIV 3.9 2.5 (1:4,000,000)

HBV 12.3 14.5 (1:690,000)

Risk (per unit) of transmission of major viruses calculated using I x WP model

HIVHIV 1 : 1,200,0001 : 1,200,000 1 : 1,800,0001 : 1,800,000 1 : 2,800,000 1 : 2,800,000

HCVHCV 1 : 230,0001 : 230,000 1 : 1,600,0001 : 1,600,000 1 : 2,300,0001 : 2,300,000

HBVHBV 1 : 220,0001 : 220,000 1 : 260,0001 : 260,000 1 : 500,0001 : 500,000

pre-NATpre-NAT MP-NATMP-NAT ID-NATID-NAT

Yield and Cost-Effectiveness of Enhanced Blood Screening Procedures

$ / Test Yield / 107 Units

$ / QALY

HIV Ab $ 2 1,000 3,600

HCV Ab $ 3 2,400 - 94,400

HIV p24 Ag $ 4 5 (1) > 1,880,000

HIV/HCV MP-NAT $ 15 3 / 45 4,300,000

HIV/HCV ID-NAT $ 30 6 / 48 7,300,000

Incremental CE of ID-NAT over MP-NAT: $15 million / QALY

Jackson, Busch, Stramer, AuBuchon. Transfusion, 2003.

Application of donor NAT screening assays in HIV research, diagnostic and public health settings

• Pathogenesis and treatment studies (AIEDRP)– Mechanisms of viral clearance/control– Immune and viral evolution during primary infection– Response of early vs delayed therapy– Antiviral vs immune enhancement strategies

• Vaccine trials– Establish incidence in enrolled populations– Discriminate vaccine response from breakthrough Tx– Precisely define infection dates for assessment of

efficacy• PEP trial

– Real-time NAT sensitivity and specificity

Yield of RNA and LS-EIA screening for enrollment

into AIEDRP primary HIV infection study (SF Options Project 6/96 to 12/04)

HIV RNA -309

HIV RNA +71

Antibody -/Ind380

Antibody -0-6 months

91

LS-EIA NR28

LS-EIA R31

Antibody -7-12 months

59

Doc. HIV -< 1year ago

150

LS EIA NR263

LS-EIA R194

No Doc. HIV Test457

Antibody +607

Screened987

71 of 486 cases of primary infection detected in viremic pre-SC stages

40 EIA-, 31 WBind

Use of NAT Testing to Determine Time of Infection in Two Phase III Trials of an HIV Vaccine (VAX004/005)

• 30 of 711 infections (1.8%) detected by SC occurred in volunteers who were already viremic at baseline - would have been misclassified as vaccine failures

– Based on 15-day (+/-5) NAT+/Ab- WP, baseline annualized HIV incidence estimated at 5.4% (1.2%-13.7%, combined 95%CI).

• Additional 34% of infections had their date of infection changed based on NAT - important in the time-to-event analyses of efficacy

• No vaccine effect on rate of detection of RNA+/Ab- samples – no evidence for accelerated or retarded seroconversion