1. 1 Susan L. Stramer, PhD and collaborators from: American Red Cross, Gaithersburg, MD American Red Cross, Charlotte National Laboratory, Charlotte, NC American Red Cross, Puerto Rico Region, San Juan, PR CDC, Dengue Branch, San Juan, PR Bio-Rad Laboratories, Redmond, WA Gen-Probe, Inc, San Diego, CA FDA, Bethesda, MD Scientific Support Office Dengue Virus Blood Donor Screening in Puerto Rico and the Florida Keys with Investigational NS1 Ag

2. 2 Outline Review background –Dengue prioritization –Repository testing in 2005 and 2007 NAT of collections in Puerto Rico –Documented transfusion transmissions Selection of NS1 Ag for investigational donation screening 2010 dengue season in Puerto Rico and S FL Results of IND screening using NS1 Ag –Reactive rates/confirmatory data to date in PR Duration of NS1 Ag/RNA reactivity –Results in the Florida Keys to date –Donor re-entry

3. 3 TRANSFUSION List and prioritize EIDs lacking a current effective intervention & posing a potential threat to transfusion safety 68 agents identified XMRV added later as 69th Highest Priority Agents: Dengue viruses Babesia vCJD http://www.aabb.org/Content/About_Blood/Emerging_Infectious_Disease_Agents/appendix2.htm

4. 4 Puerto Rico 3.95 million people 1000 mi SE of Miami Part of the US since 8198 Dengue reported in 1963; Hyperendemic Since 1998 ARC collects about half of the blood on the island (others by hospitals)

5. 5 TMA (NAT) performed on all dtns 20 Sept – 4 Dec 12 of 16,521 TMA (+) 1:1376, 5 (+) @ 1:16, 4 PCR (+) 10 3 -10 7 c/mL, DENV-2,3 detected, 3 infected mosquito cultures, 1 IgM (+) 2005 Puerto Rico Donation Repository Testing Transfusion 2008;48:1348 Number of cases Week of onset of symptoms Study Period

6. 6 Units distributed in US and PR tested (lookback study) 29 of 15,325 TMA (+) 1:529; 14 (+) @ 1:16, 12 PCR (+) 105 109c/mL, DENV-1, 2, 3 detected, 12 infected mosquito cultures, 6 IgM (+) 2007 Puerto Rico Donation Repository Testing 6 Study Period TMA positive donations

7. 7 2007 Recipient Tracing of 32 RNA Pos Units 20 distributed in Puerto Rico – all pRBCs – 4 products discarded –11 no recipient info available – 1 died shortly after transfusion – 3 no s/s reported 6 days – 2 months post tx (high titer, mod titer, low titer RNA in unit) – 1 DHF 12 distributed in US – 11 pRBCs/1 platelet –1 product discarded (platelet; high-titer RNA in unit) –4 no recipient info available –2 died shortly after transfusion –5 no s/s reported 2-4 weeks post tx (all low titer RNA in unit) 2 IgM/IgG neg when tested at CDC

8. 8 Maximum Likelihood Sequence Analysis of DENV-2 Env (1482 n’tides) DENV-2 best studied All are highly conserved, but identical sequences (D/R) recipient from an area with little reported dengue Transfusion transmission 108 copies/mL DENV-2 pRBC recipient developed DHF 4 days post transfusion Transfusion transmission 10 8 copies/mL DENV-2 pRBC recipient developed DHF 4 days post transfusion 8 0.1 Maximum Likelihood Sequence Analysis of DENV-2 Env (1482 n’tides) * * * * * Bootstrap values ≥ 70 Subclades: ISBT 2010 abstract

9. 9 Transfusion-Transmitted Dengue Singapore (NEJM 2008;359) –Donor symptomatic one day after donation –Two recipients (RBCs, FFP) developed dengue- related illness and seroconverted –Third recipient (platelet) asymptomatic but developed IgM/IgG antibodies –Donor and two symptomatic recipients positive for DENV-2 RNA Hong Kong (ProMed: Oct 11, 2002) –Donor symptomatic one day after donation –One recipient (RBC) developed dengue-related illness and seroconverted –Donor and recipient positive for DENV-1 RNA

10. 10 MAb anti-NS1 labeled with HRP (100 µL) 10 Platelia™ Dengue NS1 Ag  One step format assay  Simultaneous distribution of Diluent, Sample and Conjugate  90 min incubation time (37  1°C) ARC validation using 2007 repository samples; 3 RR/4401 screened = 1:1467; all PCR(+) 10 Incubation NS1 Capture MAb anti-NS1 + NS1 Sample (50µL) + Diluent (50µL) + TMB

11. 11 Testing began using NS1 Ag in PR on March 8, 2010 Puerto Rico has surpassed the number of dengue deaths of more than a decade (19,932 cases to Nov 11 with 31 deaths). In 1998, the virus killed 19 people and caused 17,000 illnesses. Testing began using NS1 Ag in Monroe Co, FL on Aug 16, 2010 In 2009, 28 autochthonous cases of DENV-1 were identified in Key West, Monroe County FL; 40 years since autochthonous cases had been Detected in the Keys. On Aug 13, 2010, health authorities once again confirmed the circulation of autochthonous cases (26 cases); 60 cases as of 11/27/10. Autochthonous DENV-3 was identified in a single patient on Aug 1, 2010 from Broward County FL, and in another patient (difft serotype) in Miami Dade County on Nov 11, 2010. First locally acquired case in Miami-Dade County in over 50 years.

12. 12 2010 Dengue Outbreak in PR + NS1 Ag RR Donors Reported Cases Week of onset date * * RNA pos (TMA, PCR) NS1 neut pos No. suspect dengue cases reported 19,932 (11/11) No. donations tested 47,770 (11/30) No. RR/confirmed pos 32 / 7 RR Rate 1:1493 Prevalence 1:6824 * * * * *

13. 13 Supplemental Testing Summary (Index Donation) Collect date Sero- type (CDC) NS1 Ag S/CO (ARC) c/mL (CDC) TMA S/CO (GP) % neut (Bio- Rad) IgM/IgG (CDC) C6/36 mos inf (CDC) Seq (FDA) 6/13DENV-1 2.18 3.6x10 10 30.56/ 32.24 80 negposDENV-1 gt 5 6/24DENV-4 1.24 5.1x10 7 16.95/ 4.47 85 +/- negpend 8/18DENV- 4,1 1.39 2.9x10 5 - 1x10 8 2.50/ 17.94 91 +/- pend 9/1DENV-4 4.51 2.3x10 8 30.17/ 32.31 99 -/+ pendDENV-4 gt 2 9/1neg 9.87 neg24.55/ 26.81 98 +/+ pend 10/27DENV-1 4.54 4.1x10 5 - 9.7x10 9 32.48pend -/+ pend 10/29DENV-1 11.01 4.5x10 6 - 6.3x10 10 31.08pend -/+ pend

14. 14 Dengue Seroconverter (DENV-1) SymptomOnset Day Headache Body pain 1 Fever Backache Chills 4 Rash8 * * Virus isolated/ sequenced; + in mosquito culture Dengue Seroconverter (DENV-1)

15. 15 Dengue Seroconverter (DENV-1) Duration using midpoints (+): 11 days RNA by PCR 20-21 days NS1 Ag >29 days RNA by TMA

16. 16 Why Discrepancies? Is it related to the test? –NS1 Ag capable of detection of dengue infection 10 5 -10 6 copies/mL 82% agreement with PCR in Ab-neg individuals (CDC) –Would screening with TMA (NAT) look different? Small comparative study performed (N=5135 in Sept) –Test specificity Only 7 of 35 RR donors Ag/RNA positive; PPV = 20%; specificity = 99.94%

17. 17 Why Discrepancies? Is it related to donors? –Symptomatic individuals don’t donate 53-87% of dengue cases are asymptomatic (CDC) –varies based on infecting virus, prior heterologous virus exposure and pre-existing Ab –Age distribution of those infected About 33% of symptomatic cases are in persons > 15 yrs of age (CDC) –~ 92% of donors in PR are Ab pos and have been exposed to multiple dengue serotypes (CDC) NS1 Ag sensitivity lower in secondary infection (~20% sensitive) (CDC)

18. 18 NS1 Ag neg samples from Sept 2010 tested by TMA Frequency S/CO Donations tested5135 Initially reactive22 (0.43%) Repeat reactive16 (0.31%) 2 pos with NS1 Ag (1:2909) vs 18 RRs with TMA (1:285); TMA 10-fold higher clinical sensitivity 14/16 RR x 3; 10/13 reactive using alt TMA

19. 19 NS1 Ag Testing (as of 11/30) Total No. donations tested 49,884 No. RR/confirmed pos 35 / 7 RR Rate 1:1425 Prevalence N/A Puerto Rico No. donations tested 47,770 No. RR/confirmed pos 32 / 7 RR Rate 1:1493 Prevalence 1:6824 CBCF Florida Keys No. donations tested 2114 No. RR/confirmed pos 3 / 0 RR Rate 1:704 Prevalence N/A

20. 20 Dengue NS1 Ag Reentry Criteria Requires a donor follow-up sample collected at any time following an NS1 Ag-reactive donation to test –nonreactive for NS1 Ag and viral RNA Once above has been met, donor may be reinstated at 120 days post index No donor has been reentered to date –Unconfirmed = 3/28 eligible (to 11/30) –Confirmed = 2/7 eligible (to 11/30) Used WNV as a model; why 120 days? –1/148 donors TMA reactive at 104 days (1/5 reps); 5 others reactive at 40-83 days (1-2/5 reps); all IgM/IgG pos (BSRI) –1/186 donors TMA reactive at 51 days (ARC) 20.5 day median (range 6.5-56.4 days); all IgM/IgG pos 99% RNA clearance at 50 days

21. 21 Dengue NS1 Ag Persistence in Reactive Donors (20 unconf’d > 1 follow up, 3 eligible for reentry as of 11/30) S/CO Time (Days)

22. 22 Dengue NS1 Ag Persistence in Reactive Donors (20 unconf’d > 1 follow up, 3 eligible for reentry as of 11/30) S/CO Time (Days)

23. 23 Dengue NS1 Ag Persistence in Reactive Donors (5 conf’d pos > 1 follow up, 2 eligible for reentry as of 11/30) S/CO Time (Days)

24. 24 Dengue NS1 Ag Persistence in Reactive Donors (5 conf’d pos > 1 follow up, 2 eligible for reentry as of 11/30) S/CO Time (Days)

25. 25 Dengue NS1 Ag Persistence in Reactive Donors (days post index with first case removed) Days from first to last detectable Total No. donors25 Min6 days Max> 89 days Mean> 42 days Unconfirmed No. donors20 Min6 days Max> 89 days Mean> 43 days Confirmed No. donors5 Min12 days Max19 days Mean16 days

26. 26 Summary  DENV is endemic/epidemic in Puerto Rico with rates of donor RNA of:  1:1376 (2005 RNA by TMA)  1:529 (2007 RNA by TMA)  1:1467 (2007 NS1 Ag; conf’d by RNA); 3-fold lower than TMA  NS1 Ag is not as sensitive as RNA for blood donor screening, but does detect high RNA titers  1:6824 (2010-NS1 Ag Puerto Rico overall)  Testing over the same time period (Sept):  1:285 RNA by TMA  1:2909 for NS1 Ag (conf’d by RNA); 10-fold lower than TMA  Specificity comparable to other blood donor screening tests; neutralization test needed

27. 27 Summary  Duration of RNA 11 => 29 days/20-21 days NS1 Ag  NS1 Ag false positivity is persistent (> 89 days), but both NS1 Ag/RNA confirmed and unconfirmed donors should be eligible for reentry using the criteria defined

28. 28 Support Federal earmark 1H75CK 000159-01 –For blood donation screening ARRA GO Grant 5RC2HL 101632-02 – Follow up study including: Virology/serology Cellular immunology: 42 cytokines/chemokines; testing at BSRI (no results to date) Creation of a plasma/PBMC NHLBI-sponsored repository