Nucleic Acid Testing vs Infectivity

Michael P. Busch, M.D., Ph.D.Blood Centers of the Pacific

Blood Systems, Inc.

EPFA, Lisbon PortugalMay, 2001

NAT vs Infectivity Overview of stages of infections and importance

of understanding stage-specific gEq:infectivity ratios (concentration vs volume infused)

Review of NA dynamics (pre/peri/post-SC) for HIV, HCV and HBV

Review infectivity data for each stage Need for future studies

animal transmission models lookback studies (NAT/SC donors; recipient cases)

Stages of TTVIs and infectivity

• Pre-ramp-up viremia• Ramp-up viremia• Plateau phase / peak viremia• Peri-SC viremia• Post-SC viremia

– Persistent carrier (viral load set point)– “Resolved” infection?

• Immunosilent carrier• Transient Viremia w/o SC

Stages of TTVIs and infectivity

• Pre-ramp-up Viremia– Low-level, intermittent “blips” of RNA/DNA detected

prior to quantifiable ramp-up phase

– Innoculum vs focal replication that seeds dissemination vs abortive replication

– Can this occur transiently w/o subsequent ramp-up/SC?

– Is this viremia infectious?

– Kinetic, viral sequencing & infectivity studies in progress

Testing Algorithm for HCV NAT-conversion Panels

Alpha Therapeutic / NGI screening(Antibody & 512 Pool PCR)

63 HCV NAT-conversion Panels ( 774 Donations )5 Control Panels ( 54 Donations )

dHCV TMA Testing

Quantitative Viral Load( Roche Monitor )

Intermittent Pre-Ramp-Up Viremia * in 25/41 (61%) panels >3 pre-r/u units Ramp-Up & Plateau Phase

Replicate dTMA ( x 3 ) & NGI UltraQual PCR Testing

* Ramp-Up = consistent RNA detection with progressive increase in viral load.

Low-Level Intermittent HCV ViremiaPreceding Ramp-Up Phase

Assay sensitivity (6 x 102)

-60 -40 -20 0 10 20

% d

TM

A P

osit

ive

(4 r

epli

cate

s)

Days Pre/Post 1st Quantitative RNA+ Donation

x103

x102

x104

x106

x105

x108

HC

V M

onitor PC

R (gE

q/mL

)

100

50

75

25

NGI512 PCR

Neat PCR

x107

(BCP ID 10083)

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

10

100

1,000

10,000

100,000

1,000,000

-37-35-29-26-21-18-11 -8 0 3 8

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

HB

V D

NA

Loa

d (

gEq

/mL

)Representative HBV Panel with Pre-Ramp-Up Viremia Representative HBV Panel with Pre-Ramp-Up Viremia

(pre-ramp-up viremia observed in 12/23 informative panels)(pre-ramp-up viremia observed in 12/23 informative panels)H

BsA

g S/C

Day from 1st + HBsAg test

Pre-Ramp-Up Ramp-Up

+ -

Stages of TTVIs and Infectivity

• Ramp-up viremia– Persistent viremia with progressive increase in

viral load leading to peak or plateau viremia – Doubling time used to project WP differences

of ID-NAT, MP-NAT and Ag assays– Presumed infectious (chimp study in progress)

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

-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)

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)

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

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)

Window Period Viremia Parameters Derived from NAT Analysis of

Plasma Donor Panels

Virus N High-Titer Plateau Phase

Ramp-up Phase Doubling Time

pre-Ramp-up Viremia

HIV 43 N.A. 21.5 hrs (19.2-24.6) 7 / 19

HCV 63 57 days * 17.7 hrs (17.1-18.3) 25 / 41

HBV 23 N.A. 2.8 days (2.6-3.2) 12 / 23

* Based on follow-up of 30 plasma donors detected as MP-NAT-pos by Alpha and Bayer (95% CI 29-85 days)

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

Regression Model of HCV 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

dHCV R

NA

Incremental WP Closure and Projected Yield

(U.S.) of ID over MP-NAT Screening

WP Closure Yield

HCV 3.2 days 2.5 / 107

HIV 3.9 days 2.5 / 107

HBV 12.3 days 14.5 / 107

Assumes sensitivity of ID NAT = 50 gEq / mL, and MP NAT = 1000 gEq / mL Does not include detection by ID-NAT of intermittent, low-level viremic episodes preceding ramp-up viremia

Detection of HIV in antibody negative

window phase

1

10

100

1000

10000

100000

1000000

-4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12

geq/ml

Days

infectious window

NucliSens-Ampliscreen 1:48

TMA single

TMA 1:16

Relationship of Infectivity & Viral Load pre-SC Animal Models

Analyses of Plasma Stocks with Defined CID50.1. Correlate limit of detection of NAT assays to 1 CID50. 2. Characterize viral load at several CID dilutions. Calculate viral load :

CID50 ratio. 3. For HBV/HCV ~10 gEq/CID50.

Primary Infection Transmission Studies (Alter et al. Transfusion 1999)1 Infect 1O animals, draw samples through SC.2 Characterize serial samples by NAT, Ag, Ab assays.3 Serially transfuse eclipse and NAT(+) blood into 2O animals. 4 Correlate detection of viremia with development of infectivity.

Transfuse Human WP Samples into Test Animal1 Plasma donor SC panels.2 Pre-SC units from blood donors with documented transmission.

Infectivity Early in HCV Infection Assessed by Transmission to Chimpanzees

Phase I

(12 Weeks)

Phase II

(12 Weeks)

Phase III (?)

(12 Weeks)

Phase IV

(Control)

CHIMP I

Select 10 Donors, time “A”

50 mL each250 mL / Day X2

Same 10 Donsrs, Time

“C”Same volume

Same 10 Donors, Time

“B”Same volume

Same 10 Donsrs, Time

“D”Same volume

CHIMP II

10 Different Donors

with no “B” peak at time “C”

Same 10 donors at time

“D”

HCV RNA Level

A C

B

D3D2D

1Time

Relationship of Infectivity & Viral Load pre-SC Human Transmission Data

1. Investigate Recipients of Prior Donations by NAT / Ab SC Donors (Donor directed lookback)

Model Duration of "Infectious WP" Based on Rate of Recipient Infection Relative to ID Intervals

Petersen, et al. Transfusion 1993;33:552-7 For Cases with Documented Transmission and Available Plasma,

Characterize Viral Load and Detection by MP/SD NAT TestsSchttler, et al. Lancet 2000Robbins, et al. JAMA 2000Roth, et al. submitted

Transfuse human infectious plasma with low viral load into animals to validate sensitivity of the models

HIV Test Results for Donor, Recipients in Singapore Transmission Case

Robbins, et al. JAMA, 2000

Patient CollectionDate

HIVCopies / mL

HIVAb

p24Ag

AmplicorQuan.

NucliSensQuan.

NucliSensQual.

RT-PCRQual.

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

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

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

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

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

Reactivity (+ / Total) of Donor Screening NAT assays on a Coded Plasma Panel of Singapore transmission case and controls

Robbins, et al. JAMA, 2000

Plasma Specimen Chiron / GenProbe Roche AmpliScreen

Neg Control 0 / 3 0 / 9

Pos Control 3 / 3 9 / 9

Pos Control 1:16 3 / 3 9 / 9

Donor Undiluted 3 / 3 9 / 9

Donor 1:8

Donor 1:16

Donor 1:24

3 / 3

1 / 3

2 / 3

7 / 9

6 / 9

3 / 9

HCV Transmission by Blood Donation Negative by NAT

Schüttler CG, et al. Lancet 2000; 355:41-2

• Donation 8 weeks prior to SC donation

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

• NAT studies of FFP, incl. “enhanced input” PCR assays, negative for HCV RNA

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

HCV Transmission by Blood Donation Negative by NAT Schüttler CG, et al. Lancet 2000; 355:41-2

Follow-up study in collaboration w/ Dr. Gerlich

• Coded panel with 3 aliquots (2.5 mL) of implicated plasma and 2 positive (low-level HCV RNA ramp-up) and 1 negative controls.

• GenProbe, NGI

• Controls correctly identified by both assays

• GenProbe HCV dTMA (+) in 2 of 3 replicates; NGI Ultraqual PCR (-) on all 3 replicates

• Sequencing and chimp transmission studies in progress

Donor directed lookback studies from NAT+/SC donors

Roth et al. Lancet (submitted)

Virus # units/recips evaluated

# units/ recipients infected

HCV 22 0

HIV 11 0

HBV 13 0

Relationship of Infectivity & Viral Load pre-SC

Human Transmission Data

2. Investigate Donors to Recipients with reported post-transfusion infections (Recipient traceback)

Retest stored aliquots of donation plasma Recall implicated donors Perform sequencing/phylogenetic analysis to

confirm linkage

Analysis of Reported Post-Tx Cases in Japan Matsumoto et al. Transfusion 2001

• 141 “TA-HBV” cases reported ’97-99– 103 fully investigated: donation samples & f/u available– 15 HBV DNA(+) donations linked to 14 cases

• 9 WP units - donors SC to anti-HBc/anti-HBc on f/u– 5 tested HBsAg EIA+, all w/ >4,000 gEq/mL

• 2 units anti-HBc+ at donation and f/u• 3 low-level HBV DNA(+) donations w/ HBV(-) donors on f/u

– 1 case w/ 43 HBV DNA- donations, 1 donor SCd on f/u

• 92 reported “TA-HCV” cases: all donations NAT(-) and all donors (-) on f/u

• 0 cases of “TA-HIV” reported

Stages of TTVIs and infectivity

• Plateau phase (HCV)– Prolonged, stable high-titer viremia preceding SC

– Minor fluctuations in viral load may reflect host cell capacity to support viral replication

• Peak viremia (HIV & HBV)– Rapid rise then fall in viral load corresponding to

immune control and clearance of virus

– Results in clearance or post-SC steady state viremia

Stages of TTVIs and infectivity

• Peri-SC phase– cellular and humoral immune response results

in down-modulation of viral production and, in some cases, clearance

– Smooth decline in viral load to clearance or steady state viremia in most cases

– Some cases evidence marked fluctuation in viremia including intermittent neg-pos ID NAT results

Virologic/Serologic Profile – HCV Panel 6211

Virologic/Serologic Profile – HCV Panel 6211

DaysDaysDaysDays

S/COS/COS/COS/CO

HC

V P

CR

Qu

anti

fica

tio

nH

CV

PC

R Q

uan

tifi

cati

on

HC

V P

CR

Qu

anti

fica

tio

nH

CV

PC

R Q

uan

tifi

cati

on

46 Days46 Days

100

1,000

10,000

100,000

1,000,000

10,000,000

00 2020 4040 6060 8080 100100 120120 140140 160160 180180 200200

1122334455667788991010

PCR

Time to Detection of Transfusion-Transmitted HCV Infection by Test

RNA

0

.2

.4

.6

.8

1

Cum

. S

urvi

val

0 20 40 60 80 100 120 140 160Days post-transfusion

EIA 3.0

ALT

Time to Mean 95% CIRNA 12.6 1.5-23.7ALT 51.5 18.5-84.5EIA3.0 70.7 32.8-128.6

HEPATOLOGY Vol. 29, No. 3, 1999

HCV NAT-Positive Donor HCV NAT-Positive Donor with fluctuating viremia during plateau-SC phase

0

250

500

750

1,000

1,250

1,500

0 30 60 90 120

ALT

0

5

10

15

20

25

30

(S/CO)

-147

102

103

104

105

106

-365

Viral load

RIBA – – – – – – Ind Ind + +

ALT

TMA

EIA

Viralload

Stages of TTVIs and infectivity

• Immunosilent carriers– Persistent viremia in absence of detectable SC

• Case reports for HIV, HCV and HBV• Recipient infection documented for HCV

– Durand et al. Ann Intern Med 2000– Peoples et al. Transfusion 2000

• Transient Viremia without SC– Confirmed by alternate sample and f/u with serotyping– Frequency in HR populations detectable by high

sensitivity NAT is now under study.

0 25 50 75 100

125

150

175

200

225

250

275

300

325

350

375

400

425

450

475

500

525

550

575

600

CP9TN78

TN104CP3

TN107TN49

TN113TN31TN90TN71

TN119TN110TN33CP8

TN69TN123TN95

TN120TN80TN83TN91TN43CP2

TN136TN27TN29

Seroreactive

Seronegative

Seroreactive, NAT Negative

Seronegative, NAT Negative

ARC HCV NAT Reactive Donations Confirmed with Follow up Sample(s) N = 25

Number of Days

Stages of TTVIs and infectivity

• Post-SC– Chronic seropositive carrier state

• Set-point viral load (NA vs Ag levels)• Variable infectivity to NA/Ag ratio

– Defective viral particles– Excess Ag production– Neutralizing Ab– Impact of unit storage and recipient susceptibility

– “Resolved” infection • Sensitivity of NAT vs infectivity in blood?• Persistence of infections virus in liver or other tissues• Waning Ab titers w/ persistent cellular immune responses

Rate of HCV Viremia (dHCV TMA) among Seropositive Subjects with Divergent Risk

Factors and Immune Status

Study Category Sero+

No.

RNA -pos

No. ( % )

ARC Donors 4,565 3,653 (80%)

BSI Donors 1,514 1,193 (79%)

UHS IVDU 300 255 (85%)

VATS HIV+ Patients

112 93 (83%)

Systematic Review of Role of Polymerase Chain Reaction in Defining Infectiousness

among People Infected with Hepatitis C VirusDore GJ, Kaldor JM, McCaughan GW

Br Med J 1997; 315:333-7

•2022 people exposed to anti-HCV(+) sources•1148 exposed to PCR(+) sources: 148

transmissions•874 exposed to sources (-) for HCV: 0

transmissions•Transmission rates after:

•Perinatal exposure 6.2%

•Needlestick 6.1%

•Solid organ / bone marrow transplantation 78%

•Blood components 88%

Results of SD-NAT Re-testing ofHCV-RIBA-3-positive Specimens that Tested Negative by MP vs SD NAT

Original NAT Screening

# RIBA+ / NAT- Specimens Tested

# (%) pos on SD-NAT (+)

Minipool

Single Donation

60

15

15 (25%) *

2 (13%)

* Mean SD-NAT s/co = 5.0 (range 1.05 – 9.2)

HCV viral load in anti-HCV+ donors and outcome in recipients

Operskalski et al. AABB 2001

Donation HCV

RNA level

# recipients

exposed

# (%) recipients

infected

Neg (<102.2) 15 6 (40%)

102.2 – 103.9 18 15 (83%)

104.0 – 106.3 53 49 (92%)

Correlation of HIV NAT withSupplemental HIV Serological Data

ARC 09/08/99 – 08/31/0009/08/99 – 08/31/00

Western Blot ResultNATResult

Pos

Neg

Total

Pos Ind Neg

213(94%)

13

226(5.6%)

38

1,901

1,939

21

1,856

1,877

(1.5%)

Total

272

3,770

4,042

Characteristics of WB Pos/TMA Neg Samples identified by ARC NAT Screening

All samples p24 Ag negativeAll samples p24 Ag negative

Sample Pool Neat HIV-1/HIV-2 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

09/08/99 – 08/31/0009/08/99 – 08/31/00

HIV-1 Transmission vs RNA Load and Storage

0 10

30

50

1

2

3

4

5

6 RBC (+)

RBC (–)

Platelets (+)

FFP (+)

FFP (–)

Days to Administration HIV

-1 R

NA

Load (

Log

10)

Cop

ies/

mL

Busch et al. JID 1994

HBV DNA Detection in anti-HBc+HBsAg- US blood donations

Kleinman et al. AABB 2001• 5121 anti-HBc-only donations from 5 REDS sites

tested• 1231 anti-HBc+, anti-HBs <100 IU/mL by Prism• 395 rep samples screened by PCR (<10 IU/mL) w/

confirmation by 2 PCR assays on sep aliquot• 4 HBV DNA+ detected (4/107 anti-HBs-neg)

– HBV DNA load 10-100 copies/mL

• Project 1 HBV DNA+ per 48,955 Tx units, similar to projected yield of HBV DNA+ pre-HBsAgWP units

Infectivity of HBV DNA PCR+, HBsAg-, anti-HBc+ blood

Prince et al. Transfusion 2001

• 3 patients sampled 25-30 years post acute HBV

• HBsAg-/anti-HBc+/anti-HBs+• HBV DNA+ w/ 200-1300 copies/mL• 1.35 mL serum + 2-4 x 107 PBMC

inoculated• Chimps monitored 15 months • All chimps neg for HBsAg, HBV DNA, etc.

Infectivity of HBsAg (-) Hepatic Allografts with Antibodies to HBV

Dodson, et al. Transplantation 1997;64:1582-4

Donor Recipient N InfectedN (%)

anti-HBc

anti-HBs

anti-HBc

anti-HBs

- + - - 25 0

+ - - - 25 18* (72%)

+ - - + 7 0

+ - + - 15 2* 4/18 transmitting donors had low-titer anti-HBs

Conclusions

Relationship between infectious units and RNA/DNA levels varies by stage of infection

During primary (pre-SC) infection, 1-10 gEq/mL plasma appears able to transmit infection

Ab+/NAT- donations must be presumed infectious (can’t d/c serology or reenter donors)

Need for future studies at international level1. animal transmission model systems2. donor lookback studies (prior units from NAT+ and Ab

SC donors)3. investigation of donors in TA-recipient infections