E011375A (03-13-01) 1

Update on the U.S. NAT Experience for Update on the U.S. NAT Experience for HIV, HCV, HBV and WNV:HIV, HCV, HBV and WNV:

Successes and Near SuccessesSuccesses and Near Successes

Susan L. Stramer, Ph.D.Susan L. Stramer, Ph.D.Executive Scientific OfficerExecutive Scientific Officer

American Red CrossAmerican Red Crossfor,for,

Recent Advances in Transfusion Medicine and Recent Advances in Transfusion Medicine and Annual General Meeting of HKABTHAnnual General Meeting of HKABTH

22 November 200322 November 2003

E011375A (03-13-01) 2

New Test Implementation and Declining Risk of Viral Infections from Transfusion

Updated from AuBuchon, Birkmeyer, Busch. Ann Intern Med 1997;127:904-9.

E011375A (03-13-01) 3

Current Safety Estimates:Current Safety Estimates:Risk of Infection Risk of Infection

HBVHBV 1:205,0001:205,000--488,000 488,000

HCVHCV 1:1,935,0001:1,935,000

–– PrePre--NATNAT 1:276,0001:276,000

HIVHIV 1:2,135,0001:2,135,000

–– PrePre--NATNAT 1:1,468,0001:1,468,000

HTLVHTLV 1:514,0001:514,000--2,993,0002,993,000

Based on data from ARC repeat donations, 2000-2001,representing 4.2 million person-years of observation;Dodd et al., Transfusion, August 2002

HIV-1/HCV NAT Confirmed Positivesby Site 3/3/99 to 9/30/03

San Diego3/3/99

Detroit3/23/00

Charlotte3/26/01

St. Louis2/13/01

Philadelphia5/14/01

Total

# UnitsScreened

# HIV-1 POS

# HCV POS

# False POS

12,429,452 7,814,247 3,091,5443,555,426 3,271,580 30,162,249

4 2 40 0 101:3,016,225

46 19 2319 22 1291:233,816

3791:37,597

1761:44,399

1021:30,309

471:75,647

331:99,139

7371:40,926

E011375A (03-13-01) 5

HIVHIV--1/HCV NAT Results1/HCV NAT ResultsPools of 16 Pools of 16

* One p24 Ag confirmed positive* One p24 Ag confirmed positive

9/8/99 to 9/30/039/8/99 to 9/30/03

869 NAT Reactive, Seronegative Donations (0.0031%)869 NAT Reactive, Seronegative Donations (0.0031%)

308 HCV NAT Rx308 HCV NAT Rx(Disc. Rx)(Disc. Rx)

504 Disc.504 Disc.NonNon--RxRx

(1:55,206)(1:55,206)

122 HCV Yield 122 HCV Yield SamplesSamples

(1:228,065)(1:228,065)

31 Disc. QNS31 Disc. QNS 26 HIV NAT Rx26 HIV NAT Rx

10 HIV Yield*10 HIV Yield*Samples Samples

(1:2,782,394)(1:2,782,394)

27,823,945 Donations 27,823,945 Donations

E011375A (03-13-01) 6

0 10 20 30 40 50 60 70 80 90 100

Number of Days

T N71

T N161

T N119

T N123

T N110

T N165

P C221

T N33

P C225

P C226

T N143

CP 8

T N69

T N218

T N95

T N120

T N173

DN 0005

T N80

T N211

T N 181

T N83

T N224

T N91

T N192

T N43

T N142

T N158

T N242

CP 2

T N221

T N179

T N27

T N29

Seroreactive, NAT Reactive

Seronegative, NAT Reactive

Seroreactive, NAT Negative

Seronegative, NAT Negative

5-day tail added to indicate the NAT & seroreactivity of the last donation

HCV NAT Reactive Donations Confirmed with Follow Up Sample(s) N = 34 Follow up days ≤ 92

E011375A (03-13-01) 7

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450

Number of Days

CP 9

T N78

T N113

T N168

T N104

P C184

T N200

T N167

T N207

T N199

T N136

T N183

P C232

T N144

CP 3

T N189

T N 185

T N146

T N175

T N202

T N195

T N107

P C231

T N49

P C209

T N232

T N184

T N31

T N196

T N171

T N160

T N90

T N150

T N148

Seroreactive, NAT Reactive

Seronegative, NAT Reactive

Seroreactive, NAT Negative

Seronegative, NAT Negative

5-day tail added to indicate the NAT & seroreactivity of the last donation

HCV NAT Reactive Donations Confirmed with Follow Up Sample(s) N = 34 Follow up days ≥ 97

E011375A (03-13-01) 8

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

PCR

5 Days5 Days

E001372C 8

Individual Rates and Linear Regression Model of Individual Rates and Linear Regression Model of

HIV RNA Production during Early InfectionHIV 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)

Representative HIV Conversion Panels withRepresentative HIV Conversion Panels withPrePre--RampRamp--Up “Blip” ViremiaUp “Blip” Viremia

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

2,800,0002,800,000NegNegPosPos99

370,000370,000NegNegPosPos77

27,00027,000NegNegNegNeg33

5 / 55 / 5260260NegNegNegNeg00

3 / 83 / 8< 100< 100NegNegNegNeg-- 44

0 / 80 / 8< 100< 100NegNegNegNeg-- 77

410,000410,000REACTREACTPosPos1616

0 / 80 / 8< 100< 100NegNegNegNeg-- 1111

0 / 80 / 8< 100< 100NegNegNegNeg-- 1414

7 / 87 / 8< 100< 100NegNegNegNeg-- 1919

1 / 101 / 10< 100< 100NegNegNegNeg-- 2121

# Pos /# Pos /

# Replicate PCR# Replicate PCR

HIVHIV--1 RNA 1 RNA (copies / mL)(copies / mL)

HIVHIV--1 1 AbAb

p24 Agp24 AgDays from First Days from First MPMP--PCR+ TestPCR+ Test

Alpha / BCP Case 1012

HIV Test Results for Donor, Recipients inHIV Test Results for Donor, Recipients inSingapore Transmission CaseSingapore Transmission Case

Robbins, et al. JAMA, 2000Robbins, et al. JAMA, 2000

Patient Collection Date

HIV Copies / mL

HIV Ab

p24 Ag

Amplicor Quan.

NucliSens Quan.

NucliSens Qual.

RT-PCR Qual.

BD 6 / 97 5 - 39 - - - - + +

BD 10 / 31 / 97 16,000 + nd nd + nd +

PR 10 / 28 /97 2,800 + nd nd + nd +

RBCR 10 / 3 / 97 13,000 + nd nd + nd +

BD = Blood Donor; PR = Platelet Recipient; RBCR = Red Blood Cell Recipient

1/00 1/01 1/02

Pre-seroconversionDonation(rpt donor4 in 2000)

Day 5Transfusion;51 yo male CABG(9 units RBCs; Implicated unit180 copies/mL;FFP @ NGI)

Seropositive donation Plasma used for viral genetic analysis (T)

Transfused patientidentified as seropositive

Transfused patientsampled for genetic analysis (F)

Time Course of Donor and Recipient Events in HIV Transmission Case #1

Delwart, Kalman, Busch et al., submitted

E011375A (03-13-01) 13

Linkage between NAT+/Ab- donor and recipient

Envelope Gag p17

E011375A (03-13-01) 14

Case Report #2Case Report #222ndnd case: case: seroconversionseroconversion in a repeat donorin a repeat donor–– Donations 9/12/01, 11/12/01, 1/10/02, 3/12/02 Donations 9/12/01, 11/12/01, 1/10/02, 3/12/02

(transmitting), 5/7/02 ((transmitting), 5/7/02 (seroconvertedseroconverted))

–– All All interdonationinterdonation intervals 56intervals 56--60 days60 days

–– No retention sample for further testingNo retention sample for further testing

3/12/02 donation3/12/02 donation

–– S/CO = 0.23 HIV S/CO = 0.23 HIV AbAb

–– S/CO = 0.14 p24 AgS/CO = 0.14 p24 Ag

–– S/CO = 0.28 NAT (pool of 16; GenS/CO = 0.28 NAT (pool of 16; Gen--Probe)Probe)

–– Transmitted HIV to 2 recipientsTransmitted HIV to 2 recipients

•• red cell and FFP; platelets outdatedred cell and FFP; platelets outdated

E011375A (03-13-01) 15

Case Report #2Case Report #2

5/7/02 donation5/7/02 donation–– S/CO = 7.57S/CO = 7.57--7.75 HIV 7.75 HIV AbAb

–– S/CO = 21.85 (pooled NAT), 17.92 (resolution), S/CO = 21.85 (pooled NAT), 17.92 (resolution), 14.71 (14.71 (dHIVdHIV))

Female donor (17 Female donor (17 yoyo) heterosexual ) heterosexual contact with the infected source within 7 contact with the infected source within 7 days of donationdays of donation

Genetic analysis at FDA and CDC confirms Genetic analysis at FDA and CDC confirms donor/recipient HIV relatednessdonor/recipient HIV relatedness

E011375A (03-13-01) 16

Case Report #3Case Report #35/30/02 female FT donor (33 5/30/02 female FT donor (33 yoyo))–– All testing All testing nonreactivenonreactive including HIV including HIV AbAb, ,

p24 Ag and NATp24 Ag and NAT–– Red cells and FFP distributed to hospitals on Red cells and FFP distributed to hospitals on

6/3/02 and 6/8/036/3/02 and 6/8/03–– No retention sample availableNo retention sample available

10/30/02 donor returns for 210/30/02 donor returns for 2ndnd donationdonation–– Donor uses CUE to not use her donationDonor uses CUE to not use her donation–– AntiAnti--HIV RR (Abbott), WB confirmed pos HIV RR (Abbott), WB confirmed pos

((CalypteCalypte) and NAT reactive (pool of 16, ) and NAT reactive (pool of 16, GenGen--Probe); p24 Ag Probe); p24 Ag nonreactivenonreactive

–– Retention sample availableRetention sample available

E011375A (03-13-01) 17

Case Report #3Case Report #3

Donor acknowledged unprotected Donor acknowledged unprotected heterosexual contact with partner who heterosexual contact with partner who died in summer of 2002 of kidney died in summer of 2002 of kidney failure (donor was unable to be reached failure (donor was unable to be reached until 1/28/03)until 1/28/03)–– Donor knew of her HIV infection Donor knew of her HIV infection

and initiated antiand initiated anti--retroviral therapy retroviral therapy July 02 (prior to her 10/30/02 July 02 (prior to her 10/30/02 donation on which she donation on which she CUE’dCUE’d))

E011375A (03-13-01) 18

Case Report #3Case Report #3FFP transfused 7/30/02 to a 60FFP transfused 7/30/02 to a 60--yo female ER patientyo female ER patient–– Hospital tested recipient following LB notification from Hospital tested recipient following LB notification from

ARC; recipient tested antiARC; recipient tested anti--HIV pos by independent lab HIV pos by independent lab (12/20/02; 5 months following transfusion)(12/20/02; 5 months following transfusion)

–– No samples are made available to ARC for HIV genetic No samples are made available to ARC for HIV genetic analysisanalysis

RBCsRBCs transfused at 6 days (6/5/02) to an 89transfused at 6 days (6/5/02) to an 89--yo male yo male multiple multiple myelomamyeloma patient patient –– 3/14/03 (8 months following transfusion) recipient tests 3/14/03 (8 months following transfusion) recipient tests

antianti--HIV, p24 Ag and RNA (TMA and PCR) HIV, p24 Ag and RNA (TMA and PCR) nonreactivenonreactive (3/2/03 had tested anti(3/2/03 had tested anti--HIV HIV nonreactivenonreactive by by hospital)hospital)

–– RBC plasma volume approx. 20mL versus 292mL FFPRBC plasma volume approx. 20mL versus 292mL FFP

0.29>15.94>15.49(x2)*,**

14.45(450

copies/mL)

12.898.1110/30/02(5 months)

0.190.26NANA0.895/30/02

p24 Ag(Abbott)

S/CO

Anti-HIV-1/2(Abbott)

S/CO

dHIVIndividualPool of 16

Date of Donation

NAT (TMA S/CO)

* all bands present on licensed HIV-1 Western Blot (Calypte)

** donor tested nonreactive on less-sensitive EIA (OTC) indicating recent infection

HIV Lookback Case #3

0.340.340.300.30<5<50.090.093/14/033/14/03RBC RBC RecipientRecipient

(8 months)(8 months)

0.280.28>16.42>16.42>17.05(x2)*,**>17.05(x2)*,**

<5<52.29 2.29 (MP)(MP)

12.2912.29((dHIVdHIV))

1/28/031/28/03DonorDonor

(8 months)(8 months)

p24 Agp24 Ag

(Abbott)(Abbott)

S/COS/CO

AntiAnti--HIVHIV--1/21/2(Abbott)(Abbott)

S/COS/CO

PCR PCR

(NGI)(NGI)

copies/copies/mLmL

TMATMA

IDTIDT

S/COS/CO

DateDateFollowFollow--Up Up

SamplesSamples

*all bands present on licensed HIV-1 Western Blot (Calypte)

** donor tested nonreactive on less-sensitive EIA (OTC) indicating recent infection

HIV Lookback Case #3

Effect of RNA Load & Storage on HIV Transmission

0 10 30 50

1

2

3

4

5

6RBC (+)

RBC (–)

Platelets (+)

FFP (+)

FFP (–)

Days to Administration HIV

-1 R

NA

Loa

d (L

og10

) C

opie

s/m

L

Busch et al. JID 1994

E011375A (03-13-01) 22

87.5%87.5%

33.3%33.3%

67.5%67.5%

100%100%

SummarySummary

Effect of Dilution on Detection of Effect of Dilution on Detection of HIVHIV--WP Plasma Transmitting HIVWP Plasma Transmitting HIV

5/95/96/96/93/33/31/31/31:161:16

8/98/97/97/93/33/33/33/31:81:8

2/92/9

1/11/1

TXTX

3/93/9

9/99/9

SingaporeSingapore

RocheRoche

1/31/32/32/31:241:24

1/11/13/33/3UndiluteUndilute

TXTXSingaporeSingapore

ChironChiron

Singapore: Singapore: JAMAJAMA 2000. 284(2);2102000. 284(2);210--214. 214. TX: Unpublished (TX: Unpublished (DelwartDelwart, et al.), et al.)

E011375A (03-13-01) 23

HCV Panel 6211 HCV Panel 6211 ––Virologic/Serologic ProfileVirologic/Serologic Profile

46 Days46 Days

Neat and 1:128Neat and 1:128

PCR

DaysDays

S/C

OS

/CO

HC

V P

CR

Qua

ntita

tion

HC

V P

CR

Qua

ntita

tion

100

1,000

10,000

100,000

1,000,000

10,000,000

0 20 40 60 80 100 120 140 160 180 200

0

1

2

3

4

5

6

7

8

9

10

Antibody Multiplex Neat Multiplex 1:128

E001372C 23

LowLow--Level Intermittent HCV ViremiaLevel Intermittent HCV ViremiaPreceding RampPreceding Ramp--Up PhaseUp Phase

Assay sensitivity (6 x 102)

-60 -40 -20 0 10 20

% d

TM

AP

osit

ive

(4 r

epli

cate

s)

Days Pre/Post 1st Quantitative RNA+ Donation

x103

x102

x104

x106

x105

x108

HC

V M

oni tor PC

R (gE

q/mL

)

100

50

75

25

NGI512 PCR

Neat PCR

x107

(BCP ID 10083)

Pattern of “Blip” Viremia in 6 HCV Pattern of “Blip” Viremia in 6 HCV PanelsPanels

0

0.2

0.4

0.6

0.8

1

1.2

-60 -50 -40 -30 -20 -10 0Days before first minipool reactive

% o

f 4

dH

CV

test

s re

acti

ve

10011 10029 1005010083 1008510034

HCV RNA Production Rate and Linear HCV RNA Production Rate and Linear Regression Model during RampRegression Model during Ramp--up up

ViremiaViremia

-2

0

2

4

6

8

10

12

-20 -15 -10 -5 0 5 10 15 20

Day from first HCV RNA positive sample

N = 101 samples from 37 donorsLog HCV RNA[gEq/mL] DT = 14.9 hrs (95% CI: 12.9-17.8)

Projections of HCV Window Period Reductions by MP / Projections of HCV Window Period Reductions by MP / ID NAT Using the Linear Regression Model ofID NAT Using the Linear Regression Model of

HCV ProductionHCV Production

-2

0

2

4

6

8

10

12

-20 -15 -10 -5 0 5 10 15 20

Day from first HCV RNA positive sample

Log HCV RNA[gEq/mL]

MP NAT[1,000 gEq/mL]

ID NAT[50 gEq/mL]

a

bc

d

HCV RNA

Chimp 325

2.5

3.5

4.5

5.5

6.5

7.5

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108

Weeks Post-challenge

Vir

al lo

ad (

log

10

cop

ies/

ml)

0.1

1

10

100A

nti

-HC

V A

bs (

un

its/m

l)

Viral Load (log 10 copies/ml) Anti-HCV Ab (units/mL)

1 RNA(+)

Virion

10 RNA(+)

Virions

100 RNA(+)

Virions

Cutoff anti-HCV

HCV Transmission by BloodHCV Transmission by Blood DonationDonationNegative by NATNegative by NAT

SchüttlerSchüttler CG, et al. Lancet 2000; 355:41CG, et al. Lancet 2000; 355:41--22

Donation 8 weeks prior to SC donationDonation 8 weeks prior to SC donation

HCV transmission by platelet concentrate HCV transmission by platelet concentrate (~50mL plasma) but not RBC (~5mL plasma)(~50mL plasma) but not RBC (~5mL plasma)

NAT studies of FFP, including “enhancedNAT studies of FFP, including “enhancedinput” PCR assays, negative for HCV RNAinput” PCR assays, negative for HCV RNA

Conclusion: Conclusion: “Even a negative NAT test “Even a negative NAT test cannot completely prevent transmission of cannot completely prevent transmission of HCV”HCV”

HCV Transmission by Blood DonationHCV Transmission by Blood DonationNegative by NATNegative by NAT

SchüttlerSchüttler CG, et al. Lancet 2000; 355:41CG, et al. Lancet 2000; 355:41--22

FollowFollow--up study in collaboration w/ Dr. up study in collaboration w/ Dr. GerlichGerlichCoded panel with 3 aliquots (2.5 mL) of Coded panel with 3 aliquots (2.5 mL) of implicated plasma and 2 positive (lowimplicated plasma and 2 positive (low--level HCV level HCV RNA rampRNA ramp--up) and 1 negative controls. up) and 1 negative controls. GenProbe, NGIGenProbe, NGIControls correctly identified by both assaysControls correctly identified by both assaysGenProbe HCV GenProbe HCV dTMAdTMA (+) in 2 of 3 replicates; (+) in 2 of 3 replicates; NGI NGI UltraqualUltraqual PCR (PCR (--) on all 3 replicates) on all 3 replicates

Relation of HCV RNA Level in AntiRelation of HCV RNA Level in Anti--HCV+ Units HCV+ Units and Transmission to Recipientsand Transmission to Recipients

OperskalskiOperskalski et al. Transfusion, Oct. 2003et al. Transfusion, Oct. 2003

63 (98%)63 (98%)6464>> 101044

20 (95%)20 (95%)212110102 2 -- 10103.93.9

10 (100%)10 (100%)1010PCR(PCR(--)/TMA(+))/TMA(+)

1 (8%)1 (8%)1212PCR(PCR(--)/TMA()/TMA(--))

SeroconversionSeroconversion

(94(94 with 13 w/o SCwith 13 w/o SC))

Total ExposedTotal Exposed

(107)(107)

Transmission to RecipientTransmission to Recipient

Donor RNA Donor RNA StatusStatus

Donor HCV RNA Levels and Seroconversion in Recipients (N=107);where R=TMA reactive/quant. PCR nonreactive, and

NR=TMA and quant. PCR nonreactive

E011375A (03-13-01) 33

HBV DNA Levels Relative toHBV DNA Levels Relative toHBsAg(HBsAg(––) Window Periods) Window Periods

0

500

1500

2500

3500

Presented at 1998 AABB: Detection of Hepatitis B Seroconversion Presented at 1998 AABB: Detection of Hepatitis B Seroconversion by Highly Sensitive Assays by Highly Sensitive Assays for Surface Antigen and HBV DNA. M.C. Kuhns, A.L. McNamara, B. Pfor Surface Antigen and HBV DNA. M.C. Kuhns, A.L. McNamara, B. Peterson, A. DiMarco, eterson, A. DiMarco, G.A. Satten. 1998. G.A. Satten. 1998. TransfusionTransfusion. Vol. 38 Supplement: 91S. Abstract S342.. Vol. 38 Supplement: 91S. Abstract S342.

Mea

n H

BV

DN

AC

opie

s/m

L

DaysInfection

HBsAg EIA (+)

PRISM (+)

HBV DNA (+)

6.8 da 12.6 da

N=25 Panels

E011375A (03-13-01) 34

00

1010

2020

3030

4040HBV DNA Copies/mLHBV DNA Copies/mL

DaysDays

S/COS/CO

00 1010 2020 3030 4040 5050 6060 7070 8080 9090 100100 110110 120120 130130100100

1,0001,000

10,00010,000

100,000100,000

1,000,0001,000,000

Representative Results from Representative Results from SC Panels Based on NGI’s PCRSC Panels Based on NGI’s PCR

1386713867

HBV DNAHBV DNA HBsAgHBsAg AntiAnti--HBcHBcAntiAnti--HBsHBs

E001372B 34

E011375A (03-13-01) 35

PCR+/HBsAgPCR+/HBsAg––HBcAbHBcAb––/HBsAb/HBsAb––

(n=13)(n=13)

PCR+/HBsAg+PCR+/HBsAg+HBcAbHBcAb––/HBsAb/HBsAb––

(n=14)(n=14)

PCR+/HBsAg+PCR+/HBsAg+HBcAb+/HBsAbHBcAb+/HBsAb––

(n=5)(n=5)

PCR+/HBsAg+PCR+/HBsAg+HBcAb+/HBsAb+HBcAb+/HBsAb+

(n=0)(n=0)

PCRPCR––/HBsAg/HBsAg––HBcAb+/HBsAb+HBcAb+/HBsAb+

(n=3)(n=3)

CategoriesCategories

99Median DaysMedian Days

100100

1,0001,000

10,00010,000

100,000100,000

1,000,0001,000,000

10,000,00010,000,000

3535 44

HBV DNA Concentrations During SC Based on NGI’s PCRHBV DNA Concentrations During SC Based on NGI’s PCRof 13 Plasma Donor Panels (N=181)of 13 Plasma Donor Panels (N=181)

HB

V R

NA

Cop

ies/

mL

HB

V R

NA

Cop

ies/

mL

E001372B 35

Individual Rates and Linear Regression Model Individual Rates and Linear Regression Model of HBV DNA Production during Early HBV of HBV DNA Production during Early HBV

InfectionInfection

0

1

2

3

4

5

6

7

8

-60 -50 -40 -30 -20 -10 0 10 20 30

Day from first HBsAg positive sample

Log HBV DNA[gEq/mL]

N = 70 samples from 21donorsDT: 2.6 days (95% CI: 2.6-3.2)

E011375A (03-13-01) 37

00

PC

R Q

uan

t.P

CR

Qu

ant.

100100

1,0001,000

10,00010,000

100,000100,000

1,000,0001,000,000

10,000,00010,000,000

1010

21.3121.31 27.2327.23 33.5333.53 37.4737.47 38.7638.76

Days from First Qual. PCR PosDays from First Qual. PCR Pos

First PCRFirst PCR Last PCRLast PCR PRISMPRISMAbbottAbbott

CC

Ortho Ortho BB

GSCGSC2.0 2.0

StaticStatic

Summary of First Detected Summary of First Detected HBV Pos SamplesHBV Pos Samples

E0201627A 37

E011375A (03-13-01) 38

Estimated WP differences between 7 HBsAg assays (A-G) and Pooled (P-S) vs Single-Sample (S-S) NAT

Biswas et al. FDA/REDS HBV Study

E011375A (03-13-01) 39

Composite data: HBsAg Negative,Composite data: HBsAg Negative,AntiAnti--HBc Reactive UnitsHBc Reactive Units

ParameterParameter REDSREDS ARCARC

Sample dates Sample dates 19911991--9595 20012001

# tested # tested 395395 30003000

# DNA pos# DNA pos 4 194 19

Rate: Rate: calculated directcalculated direct

per HBc (+) per HBc (+) 0.24%0.24% 0.63%0.63%

per otherwise per otherwise txtx unit 1:49,000 1:37,000unit 1:49,000 1:37,000

Copies/Copies/mLmL all all << 100 68% 100 68% << 100100

E011375A (03-13-01) 40

US HBV Clinical Trials (Roche pools 24)US HBV Clinical Trials (Roche pools 24)N=778,792 Donations TestedN=778,792 Donations Tested

2 2 (1 anti(1 anti--HBsHBs Rx; Rx; 1 1 negneg at F/U)at F/U)

--++--1 (pool)1 (pool)++--++

21 (21 (3 yield; 3 yield; 18 false positive18 false positive))

++----44++++--

4056 (14 IDT Rx)4056 (14 IDT Rx)----++17 (11 IDT Rx)17 (11 IDT Rx)--++++

102102++++++777,579777,579------

No.No.DNADNAHBsAgHBsAgAntiAnti--HBcHBc

E011375A (03-13-01) 41

Estimates for HBV Risk fromEstimates for HBV Risk fromIncidence/Window Period ModelIncidence/Window Period Model

US HBV Clinical Trials (Roche pools 24)US HBV Clinical Trials (Roche pools 24)

Estimated at 1:63,000 (95% CI:31,000Estimated at 1:63,000 (95% CI:31,000--147,000) 147,000) from 1991from 1991--1993 REDS data using adjusted IR 1993 REDS data using adjusted IR of 9.54/10of 9.54/1055 pypy and 59and 59--day day wpwp

1:58,0001:58,000--1:149,000 REDS and 1:269,000 ARC1:149,000 REDS and 1:269,000 ARC

WP reduced by an estimated 11 days by Roche WP reduced by an estimated 11 days by Roche PCR relative to PCR relative to HBsAgHBsAg assays used in the assays used in the clinical trial; this is a 19% reduction (11/59 clinical trial; this is a 19% reduction (11/59 days); therefore, predicted yield based on initial days); therefore, predicted yield based on initial REDS IR estimate is 1:331,000 REDS IR estimate is 1:331,000

Yield from trial was 1:269,000 Yield from trial was 1:269,000 –– 4 yield cases to date; viral loads 2004 yield cases to date; viral loads 200--61,000 61,000

copies/copies/mLmL

Overall Incidence Rates in Repeat DonorsOverall Incidence Rates in Repeat Donors(Per 100,000 person(Per 100,000 person--years)years)

19911991--19931993 19961996--20002000 pp--valuevalue

HIV 3.3 1.7 HIV 3.3 1.7 (1.55)(1.55) 0.030.03

HBV 13.0 6.4 HBV 13.0 6.4 (1.27)(1.27) 0.010.01

HCV HCV ---------- 3.1 3.1 (1.89)(1.89) ----------

ARC IR for 2000ARC IR for 2000--2001; HBV is unadjusted (no anti2001; HBV is unadjusted (no anti--HBcHBc correction factor)correction factor)

Window Periods from Theoretical Infectivity Window Periods from Theoretical Infectivity (1 copy/20 (1 copy/20 mLmL) to Detection by) to Detection by Current Current

MarkersMarkers

Detected Doubling Time Detected Doubling Time WP daysWP days

(copies/(copies/mLmL) (95% CI) (95% CI)) (95% CI) (95% CI)

HIV 80 20.5 h (18.2HIV 80 20.5 h (18.2--23.4) 9.1 (8.123.4) 9.1 (8.1--10.4)10.4)

HCV 192 14.9 h (12.9HCV 192 14.9 h (12.9--17.7) 7.4 (6.417.7) 7.4 (6.4--8.8)8.8)

HBV 2200 2.9 HBV 2200 2.9 dada (2.2(2.2--4.3) 45.0 (34.14.3) 45.0 (34.1--66.1)66.1)

E011375A (03-13-01) 44

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

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

Does not include detection by IDDoes not include detection by ID--NAT of intermittent, lowNAT of intermittent, low--level level viremicviremic episodes preceding rampepisodes preceding ramp--up up viremiaviremia

14.5 (1:690,000)12.3HBV

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

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

Yieldper 107

WP Closure (days)

Lost Lost QALYsQALYs from HIVfrom HIV--infected Blood Transfusions in US infected Blood Transfusions in US Relative to Changes in Incidence and Screening AssaysRelative to Changes in Incidence and Screening Assays

500

250

1984No Test

(0.1% prev)

1996p24 Ag EIA(16 d. WP)

19872nd-gen EIA(33 d. WP)

19923rd-gen EIA(22 d. WP)

19851st-gen EIA(56 d. WP)

1999MP NAT

(11 d. WP)

?ID NAT

(7d. WP)

92,000

481

284

2865

44

189

IR = 3.4 per 100,000 py IR = 1.6 per 100,000 py

QALYslost/year

~90,000 QALYs

~20 QALYs

~200 QALYs

E011375A (03-13-01) 47

WNV BackgroundWNV BackgroundEpidemic proportion in US in 2002Epidemic proportion in US in 2002–– all except 4 states had human and/or animal casesall except 4 states had human and/or animal cases

•• largest WNME outbreak ever documentedlargest WNME outbreak ever documented•• largest largest arbovirusarbovirus meningoencephalitismeningoencephalitis outbreak of any cause outbreak of any cause

in N. Americain N. America–– 4156 human cases in 39 states and DC (May 2003) 4156 human cases in 39 states and DC (May 2003) –– 284 deaths284 deaths–– Transfusion investigation of 61 individualsTransfusion investigation of 61 individuals

•• 23 confirmed 23 confirmed

Expectation was that 2003 will be equal or higher than Expectation was that 2003 will be equal or higher than 20022002–– 8470 human cases in 44 states and DC (Nov.19); 29% WNME8470 human cases in 44 states and DC (Nov.19); 29% WNME–– 189 deaths189 deaths–– Transfusion investigation of >14 individualsTransfusion investigation of >14 individuals

•• 2 confirmed; 2 probable2 confirmed; 2 probable

E011375A (03-13-01) 48

WNV Final Guidance for IndustryWNV Final Guidance for IndustryFDA May 1 2003FDA May 1 2003

Our current thinking is that we would Our current thinking is that we would recommend routine and appropriate use of recommend routine and appropriate use of licensed donor screening tests to detect licensed donor screening tests to detect acute donor infections with WNV once acute donor infections with WNV once such tests are available. We also such tests are available. We also anticipate that appropriate tests could be anticipate that appropriate tests could be used in widespread investigational studies used in widespread investigational studies conducted under FDA regulationsconducted under FDA regulations

Nov. 4,5 2002 FDA workshopNov. 4,5 2002 FDA workshop

March 13, 2003 BPACMarch 13, 2003 BPAC

E011375A (03-13-01) 49

WNV Blood Screening as a Public WNV Blood Screening as a Public Health Surveillance Tool Health Surveillance Tool

325 (1.14%)3035528,411DOD/GPC

4,933,349

1,396,282

1,271,802

2,236,854

DonationsTested

11 (0.001%)103114ABC/Roche

993 (0.02%)972*1,963Total

717

777

Initial TestReactive

452

385

Positives

Second test result

Nucleic Acid Testing

265 (0.02%)

392 (0.02%)

Negatives

ABC/GPC

ARC/GPC

System TestFormat

The following data are submitted from the blood collection facilities (as of 10/26/03)

ARC – American Red Cross; ABC – America’s Blood Centers; GPC – GenProbe/Chiron* Includes MD Anderson of Houston, TX report of 2 positive donors

E011375A (03-13-01) 50

WNV Presumed Positive Blood DonorsWNV Presumed Positive Blood DonorsARC and ABC ARC and ABC -- as of October 26, 2003as of October 26, 2003

Pattern indicates blood donors

Transfusion associated WNV human case(s)

RI

NJ

WA

ID

MT(10)

WY(11)

ND(41)

SD(45)

NE(187)

CO(182)CA*

(3)

NM(8)

TX(86)

OK(62)

KS(96)

MN(21)

IA(7)

MO(9)

AR(4)

LA(17)

WI(8)

IL(6)

IN(7)

MI(8)

KY(1)

TN (5)

MS(2)

AL(3)

GA(10)

FL(21)

SC

NC(1)

OH(10)

VA (3)

PA (16)

NY(12)

VT ME

NHMA

CT

DEMDDC

AK

HI

OR*(1)

NV(1) UT

(2)

AZ*(3)

(7)WV(1)

(6)

N = Donors4.9M Tested972 Positives(14 in Canada)

(1)

* Travel Related

Viral loads in Confirmed Positive WNV Hot CasesN=364 confirmed pos of 372 total (8 quants pending)/385 hot cases

Median Viral Load = 1,900 copies/mLMean Viral Load = 28,273 (SD 69,995) copies/mL

N = 52Median = 99

N = 113Median = 200

N = 81Median = 4500

N = 93Median = 39000

N = 25Median = 200000

WN

V c

op

ies/

mL

1

10

100

1000

10000

100000

1000000

0-99 100 - 999 1000 - 9999 10000 - 99999 >99999

WNV copies/mL

N = 52Median = 99

N = 113Median = 200

N = 81Median = 4500

N = 93Median = 39000

N = 25Median = 200000

48 IgM Pos; 31<100 copies/mL; 17 with 100-970 copies/mL

Cases by week, starting from 7/7/03

0

5

10

15

20

25

30

35

40

7/7-7/13 7/14-7/20 7/21-7/27 7/28-8/3 8/4-8/10 8/11-8/17 8/18-8/24 8/25-8/31 9/1-9/7 9/8-9/14 9/15-9/21 9/22-9/28

Nu

mb

er o

f N

AT

po

siti

ves

Nebraska

Kansas

6207124Total

518427*216

IDT Rx; Pool NonRx

11015*26

IDT Rx; Pool Rx Discordant

0138295

PCR Pending

PCR NegPCR PosIDT Rx; Pool Rx

*42 not repeat reactive in pools and PCR positive

Results of Pool and IDT WNV NAT

For 2 Regions; N=337

(of 349 reactives/30,501 screened IDT)

RNA/IgM Reactivity Over Time

12.1Total RNA+IgM(days)

7.5-8.5Total RNA only (days)

2.85.32.41.66.51-2

Window

Period (days)

+ IR only

+++

+ neat

+++

++ 1:2

++

++ 1:2

+

+++ 1:16

--

+ neat

--

Category

RNA:

IgM:

71364160No. Detected (N=46)

WNV Viremic/Serologic Profile from ARC DonorsUsing a Single Donor with Increasing Viral Load as an

“Anchor”

1

10

100

1,000

10,000

100,000

1,000,000

0

10

20

30

40

50

60

70

4 days Arbitrary Days

Vir

al L

oad

(co

pie

s/m

L)

IgMS

/N

1 5 10 15 20 25 30 35

Anchor Viral Load (copies/mL) IgM S/N

E011375A (03-13-01) 56

Infectivity of WNV Screened BloodInfectivity of WNV Screened Blood

11,240 donations from areas with high WNV 11,240 donations from areas with high WNV prevalence were screened retrospectively by prevalence were screened retrospectively by WNV IDT (after testing WNV IDT (after testing nonRxnonRx in a pool of 16 in a pool of 16 and product release); Blood Systems studyand product release); Blood Systems study–– 43 IR total (33 IR only + 10 RR)43 IR total (33 IR only + 10 RR)

•• 33 IR only 33 IR only --> 6 infected donors F/U> 6 infected donors F/U•• 10 RR 10 RR --> 10 infected donors F/U> 10 infected donors F/U

–– 33 IR only 33 IR only --> 7 transfused> 7 transfused•• 6 6 IgM/IgGIgM/IgG negneg at indexat index•• 1 1 IgMIgM neg/IgGneg/IgG pos at indexpos at index

–– 10 RR 10 RR --> 4 transfused> 4 transfused•• 3 3 IgM/IgGIgM/IgG negneg at indexat index•• 1 1 IgM/IgGIgM/IgG pos at indexpos at index

E011375A (03-13-01) 57

Infectivity of WNV Screened BloodInfectivity of WNV Screened Blood33 33 IRsIRs --> 7 transfused > 7 transfused --> 2 cases investigated; 5 donors not > 2 cases investigated; 5 donors not investigated lack of investigated lack of seroconversionseroconversion on F/U; other 2 on F/U; other 2 subsequently no subsequently no seroconversionseroconversion on F/U; 1 recipient deceased; on F/U; 1 recipient deceased; neither had any indication of WNV infectionneither had any indication of WNV infection

10 10 RRsRRs --> 4 transfused > 4 transfused --> 4 cases investigated> 4 cases investigated

10.34 10.34 (Ab (Ab negneg at at index; F/U index; F/U IgM/IgGIgM/IgG+)+)

2.5 2.5 ((AbAb negneg at at index; F/U index; F/U IgM/IgGIgM/IgG+)+)

32.1232.12((IgM/IgGIgM/IgGpos at pos at index and index and F/U)**F/U)**

34.75 34.75 (Ab (Ab negneg at at index; F/U index; F/U IgM/IgGIgM/IgG+)+)

2.032.031.591.59IDTIDT

0.570.57

TX*TX*

0.220.22

SDSD

0.250.25

AZAZ

0.920.92

TXTX

0.030.03

SDSD

0.120.12

TXTX

PoolPool

RRRRRRRRRRRRRRRRIRIRIRIRS/COS/CO

*Case 2; MMWR 52 (38):916-919; **no recipient symptoms

E011375A (03-13-01) 58

Infectivity of WNV Screened BloodInfectivity of WNV Screened Blood

2 cases reported 2 cases reported –– 1st case1st case–– Donor TX Donor TX -- 48 48 yoyo male donor; NAT pool of male donor; NAT pool of

16 (Gen16 (Gen--Probe) Probe) nonRxnonRx, IDT Rx, IDT Rx

•• NAT NAT nonRxnonRx and and IgM/IgGIgM/IgG pos at 30 dayspos at 30 days

–– Recipient TX Recipient TX –– 71 71 yoyo male aortic graft male aortic graft surgery surgery --> 5 units > 5 units RBCsRBCs

•• Day 3 fever and encephalitisDay 3 fever and encephalitis

•• Day 11 WNV Day 11 WNV IgMIgM and and neutneut AbAb pospos

•• Days 2, 7 and 11 samples NAT RxDays 2, 7 and 11 samples NAT Rx

•• Patient recoveringPatient recovering

E011375A (03-13-01) 59

Infectivity of WNV Screened BloodInfectivity of WNV Screened Blood2 cases reported 2 cases reported –– 22ndnd casecase–– Recipient NE Recipient NE –– 80 80 yoyo male cardiac surgery male cardiac surgery --> 27 units > 27 units

from 26 donors: 8 from 26 donors: 8 RBCsRBCs, 12 platelets, 6 FFP, 12 platelets, 6 FFP•• Day 10 patient dischargedDay 10 patient discharged•• Day 13 fever and encephalitis; WNV Day 13 fever and encephalitis; WNV IgMIgM pospos•• Patient recoveringPatient recovering

–– Donors SE NE Donors SE NE –– tested pool NAT tested pool NAT nonRxnonRx (pool of 6; (pool of 6; Roche)Roche)•• 20/26 donors donated late July20/26 donors donated late July--early August 03; 6 early August 03; 6

donated in Feb 02 donated in Feb 02 •• 20 donors tested individually20 donors tested individually

–– Linkage to one Linkage to one seroconvertingseroconverting donor who tested:donor who tested:•• CDC equivocal CDC equivocal --> pos (0.1 > pos (0.1 pfu/mLpfu/mL=40 =40 c/mLc/mL))•• Rx commercial labRx commercial lab•• Rx IDT and Rx IDT and nonRxnonRx pool 16 (smaller pools +/pool 16 (smaller pools +/--))•• NonRxNonRx IDT and pool of 6 IDT and pool of 6

E011375A (03-13-01) 60

SummarySummary

Serologic testing has been the norm for the Serologic testing has been the norm for the detection of most infectious disease agents since detection of most infectious disease agents since the introduction of the introduction of HBsAgHBsAg in 1971in 1971Additional safety measures obtained by NAT Additional safety measures obtained by NAT screening have decreased the risk of viral screening have decreased the risk of viral transmission through blood components to transmission through blood components to exceedingly rare events!exceedingly rare events!NAT platform provides the ability to add new NAT platform provides the ability to add new tests in remarkably short time framestests in remarkably short time framesHowever, because donations may be infectious However, because donations may be infectious below the cutoffs of tests in place, pressure below the cutoffs of tests in place, pressure exists to do moreexists to do more–– Single unit NATSingle unit NAT–– Pathogen reduction?Pathogen reduction?