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Procleix® WNV Assay: A TMA-based Assay for Screening Blood

Donations for West Nile Virus RNA

Jeff Linnen, Ph.D.Research and Development

Gen-Probe Incorporated,San Diego, CA USA

SoGATJuly 3, 2003

Procleix® is a registered trademark of Chiron Corporation

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Procleix WNV Assay

• Based on Transcription Mediated Amplification (TMA)

• Uses same instrument platform as Gen-Probe’s licensed NAT blood screening assay– Procleix Semi-automated System currently used with

Procleix HIV-1/HCV Assay

TECAN Target Capture System (TCS) Luminometer

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Specificity of the Procleix WNV Assay

• 1,680 normal blood donations were tested at Gen-Probe– 99.8% initial specificity; 100% resolved specificity– over 40,000 archived samples from 2002 high risk populations have been

tested by American Red Cross (S. Stramer)

• No cross reactivity to other blood borne viruses – Testing included HTLV, HIV-1/-2, HCV, HBV, HGV, Rubella, HAV, CMV,

EBV, HCV, Parvo B19

• Assay designed to specifically detect West Nile virus:– No cross reactivity to other flaviviruses: Dengue (1-4), Yellow Fever Virus,

and St. Louis Encephalitis virus; weak cross reactivity to Murray Valley Encephalitis virus

– Detects Kunjin virus (Australian subtype of West Nile virus)

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Analytical Sensitivity

Percent Positive

Copy Level BBI Lineage 1Virus* (N = 10)

BBI Lineage 2Virus* (N = 20)

300 100% 100%

100 100% 100%

30 100% 100%

10 100% 100%

3 50% 65%

1 10% 5%

0.3 0% 10%

0 0% 0%*virus quantified by BBI TaqMan Assay (BBI Diagnostics, West Bridgewater, MA).

Based on data from Procleix WNV Assay kit lot manufactured at 2 million test scale

Percent Positive

Copy LevelImpath BCPLineage 1**

(N =30)50 100%

25 100%

5 100%

1 100%

0 0%**Quantitation for this panel is probably not accurate

Probit Analysis (results from BBI panels):Lineage 1: 95% detection at 4 to 14 copies/mLLineage 2: 95% detection at 4 to 8 copies/mL

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Samples from CDC WNV Transfusion Transmission Case Investigations (2002)

Quantitation Procleix WNV AssayCase

IDCDC PCRResults

NGIPCR

Copies/mL

ARCTaqManPFU/mL

Neat 1:8 1:16 IgMSero-conversion

1 POS 7,700 16.4 ++ +++ +++ +

2 POS 3,300 13.5 ++ +++ +++ NA3 POS 3,900 7.1 ++ +++ +++ NA4 POS 1,200 2.3 ++ +++ +++ NA5 POS 31,000 1,412 ++ +++ +++ NA6 POS 6,600 13.3 ++ +++ +++ +7 POS 37,000 1,225 ++ +++ +++ +8 POS 39,000 327 ++ +++ +++ +9 POS 9,000 7.9 ++ +++ +++ +

10 NEG/POS(Hi input) 630 0.9 ++ +++ +++ +

11 NEG/POS(Hi input) 480 0.06 ++ +++ +++ +

12 NEG NEG 0.013 ++ + - - - - - +++ - - - + (EQV at index)

13 NEG POS;< 100 NEG - - - - + NT NT +

• Positive Results from testing 383 blinded samples sent to Gen-Probe from the American Red Cross• All donations implicated in transfusion transmission were detected at 1:16 dilution with TMA

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Nationwide WNV Blood Screeningin the United States, 2003

• Testing using the Procleix WNV Assay started on June 19– Implemented nationwide on July 1– Test development which normally takes 2 to 3 years was condensed into

less than 9 months.

– Procleix WNV Assay is being used to test over 80% of the US blood supply

• Most donations are being tested in pools of 16 (some sites are testing individual donations)– reactive pools will be resolved to individual donation

• Testing will reduce the risk of WNV transfusion transmission– will provide real time surveillance of human WNV activity in North America

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Acknowledgements• Gen-Probe WNV Assay Development Team

– (Front to back, left to right) Mike Shih, Josh Cary, Geoffrey Dennis, Janice Cline, Martha Alden, Wen Wu, Mackenzie Lewis, Michelle Cass, Amy Broulik, Jeff Linnen, Stephanie Miller

• National Heart, Lung, and Blood Institute (NHLBI) for partial funding• Susan Stramer, American Red Cross• Chiron Corporation