E0405681 1

WNV Confirmation in US Blood Donors 2003-2005

and Data in Support of WNV ID-NAT Triggers

Susan L. Stramer, Ph.D.

Blood Products Advisory Committee Meeting

April 27, 2007

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Confirmation of Infection among Donors Identified by Minipool and Individual Donation Nucleic Acid Amplification

Testing (NAT) for West Nile virus (WNV) RNA in the United States from 2003-2005

S.L. Stramer, J.P. Brodsky

S. G. Caglioti

D.M. Strong

representing all Roche Centers

E0405681 3

Background

Donor screening for WNV RNA by NAT began in June 2003 prior to the onset of the national epidemic for that year

During 2003-2005, all blood programs in the US performed investigational NAT for WNV in minipools (MP) or individually (ID) during epidemic periods and in epidemic locations– Conversion from MP to ID NAT was dependent on site

specific triggers; e.g., 2 positive cases and a frequency of 1:1000 positive donations

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Methods Three blood collection/testing programs contributed

data representing greater than 80% of blood collected in the US, or over 4 million donations per WNV season, and covering all geographic regions– Testing for WNV occurs throughout the year in

all US areas; however, to focus on incidence of new cases reported each year, this report covers only the epidemic periods for a given year, defined as the dates of collection between the first and last WNV confirmed-positive blood donor

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WNV Screening Tests All donations from 2003-2005 (and ongoing) were tested

for WNV RNA by the:– Gen-Probe/Chiron WNV Assay* (Transcription Mediated

Amplification; TMA) in MPs of 16 using either eSAS* (semi-automated system) or the TIGRIS (automated system)

– Sites included:

• American Red Cross (testing performed at 5 ARC National Testing Laboratories)

• United Blood Services (testing performed at 2 Blood Systems Laboratories)

• All contract collection facilities sending testing to the above

• Represents all areas within the US*Gen-Probe’s WNV Assay on the eSAS platform was FDA licensed on 12/1/05 (and on the automated TIGRIS platform on 2/20/07)

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WNV Screening Tests– Roche WNV PCR in MPs of 6 using the TaqScreen WNV Test on the

Cobas Ampliprep and Taqman platforms– Sites included (12):

• Puget Sound Blood Center (WA)• Stanford Blood Center (CA)• Gulf Coast Regional Blood Center (TX)• Community Blood Center of Greater Kansas City (MO)• Mississippi Valley Regional Blood Center (IA)• Life Source (Institute of Transfusion Medicine) (IL/PA)• Siouxland Community Blood Bank (IA)• Minneapolis Memorial Blood Center (MN)• New York Blood Center (NY)• Florida’s Blood Centers (FL)• Central Pennsylvania Alliance Laboratory (PA)• South Bend Medical Foundation (IN)• All contract collection facilities sending testing to the above

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WNV Confirmation TMA and PCR initially reactive samples considered

confirmed (+) if they met one of the following criteria– Initial test repeated as reactive in the original or modified test

format=ALT NAT (preferably from an independent sample from the index donation)

– Index donation sample tested WNV IgM/IgG positive (progam dependent)• Abbott Laboratories• Focus Diagnostics• State Public Health Laboratories

– Donor follow up samples tested reactive by• TMA and/or PCR • IgM/IgG

Quantitative PCR– National Genetics Institute

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WNV RNA Detection in US Blood Donors

2003 2004 2005 TOTALTotal Donations Screened (1st –Last Positive)

4,529,248 4,185,419 4,355,885 13,070,552

Donations Screened by ID NAT (during

epidemic period)

34,465 (0.8%)

233,908 (5.6%)

229,298 (5.3%)

497,571 (3.8%)

Date Range (1st-Last Positive)

6/25-12/1 5/3-11/29 5/26-11/30 5/3-12/1

# Confirmed Positive

721 256 352 1329

Frequency per 10,000 donations

1.59 0.61 0.81 1.02

# Presumptive Viremic Donors Reported to CDC

818 224 417 1459

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0

20

40

60

80

100

120

2003

2004

2005

Number of WNV RNA Positive Donors Reported by Week, 2003-2005

Total WNV RNA Confirmed Positive Donors2003, 2004 & 2005 ( N = 1329 )

3

34114

1

1 (DC)

26 5

1

29

16

6

5910

915

31

40

4132

5 5

13

11

12

14

288

6412

2

4

68

30

12

4

28

17

20

31

4

170

6

Legend

1 – 9 Cases

10 – 19 Cases

20 – 49 Cases

50 – 99 Cases

> 100 Cases

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WNV Confirmed Positives Sorted by Detection Method (MP or ID NAT Only)

coupled with Presence/Absence of AntibodyID

NATMP

NATAb 2003 2004 2005 Total

+ - - 14 32 21 67 (5%)

Ab Neg:

985 (74%)

+ - 534 145 239 918 (69%)

+ + 55 23 42 120 (9%)

Ab Pos:

344 (26%)+ - + 118 56 50 224

(17%)Total 721 256 352 1329

67+224=291 (22%) required ID NAT for detection

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Viral Loads WNV Confirmed Positive Donations 1013/1329 (76%) total samples submitted for quant PCR

750 (74%) were Ab neg

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0-99 100-999 1000-9999 10000-99999 100000+

AB Pos

AB Neg

N = 202 48 10 3 0 = 263

N = 46 195 206 248 55 = 750

Copies/mL

0

50

100

150

200

250

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36

Gen-Probe TMA S/CO Values of Confirmed Positive (CP) and False Positive (FP) WNV Reactive Blood Donors at Index

N S/CO Median

FP MP 168 2.2

FP ID 477 2.0

CP MP 856 32.4

CP ID 281 21.9

S/CO

Num

ber 1005 of 1137 (88%) confirmed pos have S/CO > 17

>

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Total False Positives Reported During Relevant Date Ranges

2003 2004 2005 Total

Total False Positives

448 154 120 722*

False Positives

during ID NAT

285 (64%)

148 (96%)

67 (56%)

500 (69%)

False Positives

per 10,000 donations

0.99 0.37 0.28 0.55

*540 donors of 722 had follow up samples testing both RNA and Ab nonreactive

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Results of WNV Confirmatory TestingN=1329

Seroconversion at follow up

(f/u)

Ab Pos at Index (IgM/IgG)

Repeat reactivity (same or alt NAT)

Number (%)

No sample available or data not collected

if sample obtained- + 200 (15.2)

190 no f/u; 6 RNA+ at f/u; 4 f/u samples

not tested for Ab

Follow up samples collected and conf’d Ab pos (N=83 / 83)

+ - 128 (9.7)

Follow up samples collected and conf’d Ab pos (N=156 /156)

+ + 217 (16.4)

+ - - 10 (0.8)

+ - + 764 (57.9)

+ NA NA 10 (omitted)

Total (%) 345 (26.2) 1181 (89.5) 1319

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1559 IR with index and f/u testing

0conf’d (+) index

but (-) f/u

10conf’d (+)on f/u only

1009conf’d (+)

at index and f/u

540false (+)

920conf’d (+)

based on repeat NAT (+)and Ab (+) at f/u

239conf’d (+)

based on Ab (+) at indexand Ab (+) at f/u

PPV screening = 1019/1559 = 65.4% NPV screening = 540/550 = 98.2% PPV index confirmation = 1009/1009 = 100% Sens index confirmation = 1009/1019 = 99.0% Sens repeat NAT = 920/1019 = 90.3% Sens Ab index = 239/1019 = 23.5%

Flow Diagram of WNV NAT Reactiveswith Complete Index and Follow-up Data

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Same vs Alternate NAT Reactivity at Index1196 of 1309 Total Tested by Both Methods

1094 (91.5%) Reactive by at Least One Method

Primary (same)

Total+ -

Alternate

+ 989 49 6 Ab neg at

index

1038 (86.8%)

- 56 13 Ab neg at

index

102 158

Total 1045 (87.4%)

151 1196

Primary and Alternate NAT have equivalent sensitivity

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Conclusions - PPV The PPV of the screening algorithm (65%)

indicates the need for confirmatory testing– 69% of false positives driving the lower PPV

obtained during periods of ID NAT

The PPV of index donation confirmatory algorithm is 100% using follow-up testing results as the gold standard– All donors who are confirmed positive based on

index donation results have been accurately classified as WNV infected (i.e., no false positives observed)

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Conclusions - Sensitivity

Sensitivity of the confirmatory algorithm based on index sample testing approximates that based on follow-up sampling, indicating very little additional value is obtained by follow-up testing – 99.0% based on repeat NAT (+) and Ab (+) at index

(including those Ab (+) at index and conf’d by f/u)• 90.3% by repeat NAT (+)• 23.5% by index Ab (+)

A confirmatory algorithm requiring follow-up testing will never have 100% sensitivity in practice because not all donors will participate in follow up

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Conclusions - NPV

The few true positive donors who would not be classified as confirmed positive based on index testing would already have been counseled for possible WNV infection, been deferred for 120 days, and components from their donations would have been quarantined; thus there is no adverse impact on blood safety by eliminating follow-up testing

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Triggering Need to trigger (convert from MP=>ID NAT) during epidemic periods

– based on low viral loads of WNV compared to HIV or HCV– 22% of WNV NAT (+) samples detected required ID NAT– 26% of detected samples were Ab positive of which majority (81%)

required ID NAT Most systems have implemented some type of trigger

– not standardized– no method exists for site to site communication

Triggering has been successful; however, 2 WNV breakthrough cases occurred in 2006 (MMWR 2007)

No. of/year (CDC) 2002 2003 2004 2005 20069902 WNND cases 2946 2866 1148 1309 1491

1820 WNV (+) donors n/a 818 224 417 36133 Transf-Transmissions 23 6 1 0 2

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Triggering Based on need for improvement, AABB WNV

Task Force representing blood industry developed Assn Bulletin #07-02 (4/3/07)– Received input from CDC and FDA

Recommendations involve use of a minimum trigger that has been shown to be feasible and has relatively high effectiveness (81%; Custer et al., 2004)– First validation was 2002 retrospective study based on

frequency of WNV clinical disease (1:1000) and observation of 1 MP-neg unit/4 MP-pos units (Stramer et al., NEJM, 2005)

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AABB AB #07-02 Minimum criteria based on initial reactive donations and

rapid time to respond (within 24 h) due to the short duration of the ID-NAT-only window period (2-7 days)

Reversion back to MP NAT following 7 days without a repeatable or Ab (+) ID NAT reactive

Communication plan based on the existing testing sites that have entered data into the AABB web site and therefore have had communication plans in place for their institutions and their customers

Missing link then is communication between facilities; contact information and states for which collected donations are tested are provided as an attachment to the AB

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AABB AB #07-02 Sites for which collections occur in adjacent/overlapping

areas should be communicating!– Tools for tracking activity

• Site specific maps, etc.

• AABB WNV NAT-reactive donor website for which new cases should be added as quickly as feasible

– Donors entered by residential zip code

• Maps provided by CDC/USGS for human WNV activity

• CDC reports of avian/mosquito activity

– These tools can be used as part of planning activities within facilities and between facilities; can be sent by email on a weekly basis to trigger communication

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Minimum Criteria Feasible, or it wont be done! Real time 2 WNV NAT reactives Rate of >1:1000

– If fewer than 1000 collections/week, use weekly collections; should combine with adjacent/overlapping facilities• May have long intervals between 1st and 2nd reactive that may lead to

false negative MP NAT results Defined geographic area to which the above two criteria are applied

– Most feasible/standardized method is based on number of collections• <1000/week cannot segment your facility• <5000/week => interval between 1st-2nd is 7 days (rolling period)• >5000/week => interval between 1st-2nd is 3 days• Monitor number of donations between 1st-2nd ; trigger if <2000

collections during interval

 Cumulative 2006 Data as of 3 am, Jan 03, 2007* National Cumulative Human Disease Cases:4180These data are provisional and may be revised or adjusted in the future. * States shown in yellow are those in which virus activity has been reported historically, but all counties in these states have not.

2006 West Nile Virus Activity in the United States(Reported to CDC as of January 3, 2007) N= 4,180

2006 West Nile Virus Human Neuroinvasive DiseaseIncidence in the United States

(Reported to CDC as of January 3, 2007)

2006 West Nile Virus Viremic Blood Donor Activity in the United States(Reported to CDC as of January 3, 2007) N= 340

West Nile Virus Biovigilance NetworkU.S./Canada Map: Suspected Cases byPostal Code

439 CP; 64 FP; 4 cannot conclude

ARC Distribution of WNV Confirmed Positive Donors 2006(One indicator per county N = 212)

2

2

49

63

1

4

7

2

1

1

2

3

2

9

11

3

6

1

6

61

7

2

3

11

3

3

6

1

1

1

1

E0405681 33

Logistics WNV ID NAT is a balance between

sensitivity and capacity

Largest labs may have capacity for 1000 samples/day or 1200 samples/automated instrument/day

Reagent performance issues or other sources of false positivity may cause sites to artificially trigger early or extend the time of ID NAT– Repeat NAT can be used to eliminate

probable low-level false positives

2006 WNV ID NAT Calendar(as reported by regions)

ARC Region 1 2 3 4 1 2 3 4 5 1 2 3 4 1 2

Midwest 2,995 2,345 1,315 2,658 3,186 2,757 3,686 3,206 2,622 3,442 3,177 3,093 2,935 2,887N. Central 1,500 4,425 1,919Lewis & Clark 2,870 4,708 4,619 4,507 4,644 4,735 3,595 4,822 4,532 4,526 1,761Central Plains 1,850 2,351 1,719 370 1,956 2,242 618 2,123 2,616Alabama & Gulf Coast 659 1,914Badger Hawkeye 493 3,424 3,432 10Grtr Ozarks & Arkansas 811 1,646Indiana-Ohio 1,313 665Northern Ohio 2,457 711Western Lake Erie 365 1,258Mid Atlantic 1,050 2,555Arizona 534 1,246 956 304

Weekly Totals 4,495 6,770 6,104 7,366 10,314 12,833 15,119 11,743 13,368 16,229 9,573 10,698 7,616 2,887

July August September October

2006 WNV ID NAT Total Donations Tested(as reported by regions)

RegionLab 009 017 020 002 026 032 055 038 042 050 029 007 Totals

Charlotte 2,573 1,361 3,934Detroit 7,359 1,978 3,168 1,623 14,128St. Louis 40,304 7,844 15,845 2,457 2,244 68,694Portland 45,319 3,040 48,359

Totals 40,304 7,844 45,319 15,845 2,573 7,359 2,457 1,978 3,168 1,623 3,605 3,040 135,115

E011375A (03-13-01) 36

Days Posttransfusion

Sig

nal

/Cu

toff

(s/

c)

0 20 40 60 80 100 120 140 160

0

1

2

3

PCR & HC positive

Course of Infection in Platelet Recipient

Leiby et al, NEJM, 1999; 341(16):1237-1239