WNV Confirmation in US Blood Donors 2003 -2005

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 E 0405681 1

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 E 0405681 2

Background u Donor screening for WNV RNA by NAT began in June 2003 prior to the onset of the national epidemic for that year u 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 E 0405681 3

Methods u 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 E 0405681 4

WNV Screening Tests u 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 e. SAS* (semiautomated 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 e. SAS platform was FDA licensed on 12/1/05 (and on the automated TIGRIS platform on 2/20/07) E 0405681 5

WNV Screening Tests – Roche WNV PCR in MPs of 6 using the Taq. Screen 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 E 0405681 6

WNV Confirmation u 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 Ig. M/Ig. G positive (progam dependent) • Abbott Laboratories • Focus Diagnostics • State Public Health Laboratories – Donor follow up samples tested reactive by • TMA and/or PCR • Ig. M/Ig. G u Quantitative PCR – National Genetics Institute E 0405681 7

WNV RNA Detection in US Blood Donors Total Donations Screened (1 st – Last Positive) Donations Screened by ID NAT (during epidemic period) Date Range (1 st. Last Positive) # Confirmed Positive Frequency per 10, 000 donations # Presumptive Viremic Donors Reported to CDC E 0405681 8 2003 2004 4, 529, 248 4, 185, 419 34, 465 (0. 8%) 6/25 -12/1 233, 908 (5. 6%) 2005 4, 355, 885 TOTAL 13, 070, 552 229, 298 (5. 3%) 497, 571 (3. 8%) 5/3 -11/29 5/26 -11/30 5/3 -12/1 721 256 352 1329 1. 59 0. 61 0. 81 1. 02 818 224 417 1459

Number of WNV RNA Positive Donors Reported by Week, 2003 -2005 E 0405681 9

Total WNV RNA Confirmed Positive Donors 2003, 2004 & 2005 ( N = 1329 ) 12 4 64 1 12 31 68 4 17 13 12 288 6 29 26 34 114 132 59 5 30 5 15 20 28 1 3 4 2 5 10 4 11 170 Legend 1– 9 Cases 10 – 19 Cases 20 – 49 Cases 50 – 99 Cases > 100 Cases 3 16 40 6 1 14 9 1 (DC)

WNV Confirmed Positives Sorted by Detection Method (MP or ID NAT Only) coupled with Presence/Absence of Antibody ID NAT MP NAT + + Total Ab 2003 2004 2005 + + 14 32 21 534 145 239 55 23 42 118 56 50 721 256 352 Total 67 Ab Neg: (5%) 985 918 (74%) (69%) 120 Ab Pos: (9%) 344 224 (26%) (17%) 1329 67+224=291 (22%) required ID NAT for detection E 0405681 11

Viral Loads WNV Confirmed Positive Donations 1013/1329 (76%) total samples submitted for quant PCR 750 (74%) were Ab neg N = 202 N = 46 E 0405681 12 48 10 3 0 195 206 248 55 Copies/m. L = 263 = 750

Gen-Probe TMA S/CO Values of Confirmed Positive (CP) and False Positive (FP) WNV Reactive Blood Donors at Index S/CO Median FP MP 168 2. 2 FP ID 477 2. 0 CP MP 856 32. 4 CP ID Number N 281 21. 9 1005 of 1137 (88%) confirmed pos have S/CO > 17 > S/CO

Total False Positives Reported During Relevant Date Ranges 2003 2004 2005 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 E 0405681 14

Results of WNV Confirmatory Testing N=1329 Seroconversion at follow up (f/u) Ab Pos at Index (Ig. M/Ig. G) Repeat reactivity (same or alt NAT) Number (%) No sample available or data not collected if sample obtained - + 200 (15. 2) 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) + NA NA 10 (omitted) Total (%) 345 (26. 2) 1181 (89. 5) 1319 E 0405681 15 190 no f/u; 6 RNA+ at f/u; 4 f/u samples not tested for Ab 764 (57. 9)

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Same vs Alternate NAT Reactivity at Index 1196 of 1309 Total Tested by Both Methods 1094 (91. 5%) Reactive by at Least One Method Primary (same) Total + - + 989 49 1038 (86. 8%) - 56 102 158 1045 (87. 4%) 151 1196 Alternate Total 13 Ab neg at index 6 Ab neg at index Primary and Alternate NAT have equivalent sensitivity E 0405681 17

Conclusions - PPV u 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 u 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) E 0405681 18

Conclusions - Sensitivity u 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 (+) u A confirmatory algorithm requiring follow-up testing will never have 100% sensitivity in practice because not all donors will participate in follow up E 0405681 19

Conclusions - NPV u 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 E 0405681 20

Triggering u 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 u Most systems have implemented some type of trigger – not standardized – no method exists for site to site communication u Triggering has been successful; however, 2 WNV breakthrough cases occurred in 2006 (MMWR 2007) No. of/year (CDC) 9902 WNND cases 1820 WNV (+) donors 33 Transf-Transmissions E 0405681 21 2002 2946 n/a 23 2003 2866 818 6 2004 1148 224 1 2005 1309 417 0 2006 1491 361 2

Triggering u 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 u 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) E 0405681 22

AABB AB #07 -02 u 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) u Reversion back to MP NAT following 7 days without a repeatable or Ab (+) ID NAT reactive u 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 u 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 E 0405681 23

AABB AB #07 -02 u 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 E 0405681 24

Minimum Criteria u u 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 1 st and 2 nd reactive that may lead to false negative MP NAT results u 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 1 st-2 nd is 7 days (rolling period) • >5000/week => interval between 1 st-2 nd is 3 days • Monitor number of donations between 1 st-2 nd ; trigger if <2000 collections during interval E 0405681 25

Cumulative 2006 Data as of 3 am, Jan 03, 2007* National Cumulative Human Disease Cases: 4180 These 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 Disease Incidence 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 Network U. S. /Canada Map: Suspected Cases by Postal Code 439 CP; 64 FP; 4 cannot conclude

ARC Distribution of WNV Confirmed Positive Donors 2006 (One indicator per county N = 212) 1 6 4 1 7 63 6 1 49 3 2 7 1 3 2 11 6 1 1 2 3 2 1 2 6 1 9 3 3 1 1 2 1

Logistics u WNV ID NAT is a balance between sensitivity and capacity u Largest labs may have capacity for 1000 samples/day or 1200 samples/automated instrument/day u 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 E 0405681 33

2006 WNV ID NAT Calendar (as reported by regions)

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

Course of Infection in Platelet Recipient Leiby et al, NEJM, 1999; 341(16): 1237 -1239 Signal/Cutoff (s/c) 3 2 1 0 PCR & HC positive 0 20 40 60 80 100 120 140 160 Days Posttransfusion E 011375 A (03 -13 -01) 36