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Dengue Virus Blood Donor Screening in Puerto Rico and the Florida Keys with Investigational NS1 Ag

Dengue Virus Blood Donor Screening in Puerto Rico and the Florida Keys with Investigational NS1 Ag. Susan L. Stramer, PhD and collaborators from: American Red Cross, Gaithersburg, MD American Red Cross, Charlotte National Laboratory, Charlotte, NC

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Dengue Virus Blood Donor Screening in Puerto Rico and the Florida Keys with Investigational NS1 Ag

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  1. Dengue Virus Blood Donor Screening in Puerto Rico and the Florida Keys with Investigational NS1 Ag 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

  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. 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. 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. 2005 Puerto Rico Donation Repository TestingTransfusion 2008;48:1348 • TMA (NAT) performed on all dtns 20 Sept – 4 Dec • 12 of 16,521 TMA (+) 1:1376, 5 (+) @ 1:16, 4 PCR (+) 103-107 c/mL, DENV-2,3 detected, 3 infected mosquito cultures, 1 IgM (+) Study Period Number of cases Week of onset of symptoms

  6. 2007 Puerto Rico Donation Repository Testing • 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 (+) Study Period TMA positive donations 6

  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. Maximum Likelihood Sequence Analysis of DENV-2 Env (1482 n’tides) PR 2009 PR 2008 PR3 2007 PR 2008 PR3-1 PR 2007 Asian American Mara4 Venezuela Jamaica 2008 Dominican Rep. 2003 Colombia 2007 Mexico 2002 Brazil 2006 NGC Thai 2006 SE Asian Thai 2003 Thai 2001 16681 India 2006 Sri Lanka 1989 Indian Pacific P7-863 Malaysia 1969 Australia 1993 Burkina Faso 1983 PR159 South American IQT2133 Peru Ven2 Venezuela West Africa 1981 Sylvatic Guinea 1981 0.1 Maximum Likelihood Sequence Analysis of DENV-2 Env (1482 n’tides) Subclades: 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 108 copies/mL DENV-2 pRBC recipient developed DHF 4 days post transfusion * * * * 8 ISBT 2010 abstract * Bootstrap values ≥ 70

  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. + Incubation TMB NS1 Sample (50µL) + Diluent (50µL) NS1 + Capture MAb anti-NS1 MAb anti-NS1 labeled with HRP (100 µL) 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 10

  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. 2010 Dengue Outbreak in PR + NS1 Ag RR Donors 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 Reported Cases *RNA pos (TMA, PCR) NS1 neut pos * * * * * * * Week of onset date

  13. Supplemental Testing Summary (Index Donation)

  14. Dengue Seroconverter (DENV-1) Dengue Seroconverter (DENV-1) * Virus isolated/ sequenced; + in mosquito culture *

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

  16. Why Discrepancies? • Is it related to the test? • NS1 Ag capable of detection of dengue infection • 105-106 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. 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. NS1 Ag neg samples from Sept 2010 tested by TMA 14/16 RR x 3; 10/13 reactive using alt TMA 2 pos with NS1 Ag (1:2909) vs 18 RRs with TMA (1:285); TMA 10-fold higher clinical sensitivity Frequency S/CO

  19. NS1 Ag Testing (as of 11/30)

  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. 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. 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. 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. 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. Dengue NS1 Ag Persistence in Reactive Donors (days post index with first case removed) Days from first to last detectable

  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. 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. 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

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