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Transcript of 1 Susan L. Stramer, PhD and collaborators from: American Red Cross, Gaithersburg, MD American Red...
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
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 Rico3.95 million people
1000 mi SE ofMiami
Part of the US since8198
Dengue reported in1963; HyperendemicSince 1998
ARC collects abouthalf of the blood onthe island (othersby hospitals)
5
• 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 (+)
2005 Puerto Rico Donation Repository TestingTransfusion 2008;48:1348
0
50
100
150
200
250
300
350
400
450
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52
Suspected
Confirmed
Nu
mb
er o
f ca
ses
Week of onset of symptoms
Study Period
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
0
100
200
300
400
500
600
700
800
900
1000
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51
Not processed
Indeterminate
Laboratory-negative
Laboratory-positive
Study Period
TMA positive donations
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)
DENV-2 best studied All are highly conserved, but• identical sequences (D/R)• recipient from an areawith little reported dengue
Transfusion transmission 108 copies/mL DENV-2 pRBC recipient developed DHF 4 days post transfusion
Transfusion transmission108 copies/mL DENV-2pRBC recipient developedDHF 4 days post transfusion
80.1
Mara4 Venezuela
Jamaica 2008
Dominican Rep. 2003
Colombia 2007
Mexico 2002
Brazil 2006NGC
Thai 2006
Thai 2003Thai 2001
16681
India 2006 Sri Lanka 1989
P7-863 Malaysia 1969
Australia 1993
Burkina Faso 1983
PR159
IQT2133 Peru
Ven2 VenezuelaWest Africa 1981
Guinea 1981
PR 2008PR 2008
PR 2007
PR3
PR3-1 2007
PR 2009
Asian American
SE Asian
Indian Pacific
South American
Sylvatic
Maximum Likelihood Sequence Analysis of DENV-2 Env (1482 n’tides)
*
*
*
*
* Bootstrap values ≥ 70
Subclades:
ISBT 2010 abstract
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
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 (371°C)
ARC validation using 2007 repository samples; 3 RR/4401 screened = 1:1467; all PCR(+)
10
Incubation
NS1NS1
Capture MAb anti-NS1
+
NS1NS1 Sample (50µL)
+
Diluent (50µL)
+
TMB
11
Testing began using NS1 Ag in PR on March 8, 2010
Puerto Rico has surpassed the number of dengue deaths of more than adecade (19,932 cases to Nov 11 with 31 deaths). In 1998, the virus killed19 people and caused 17,000 illnesses.
Testing began using NS1 Ag in Monroe Co, FL onAug 16, 2010
In 2009, 28 autochthonous cases of DENV-1 were identified in Key West,Monroe County FL; 40 years since autochthonous cases had beenDetected in the Keys. On Aug 13, 2010, health authorities once againconfirmed the circulation of autochthonous cases (26 cases); 60 casesas of 11/27/10.
Autochthonous DENV-3 was identified in a single patient on Aug 1, 2010from Broward County FL, and in another patient (difft serotype) in MiamiDade County on Nov 11, 2010. First locally acquired case in Miami-DadeCounty in over 50 years.
12
2010 Dengue Outbreak in PR + NS1 Ag RR Donors
0
100
200
300
400
500
600
700
800
900
1000
1 6 11 16 21 26 31 36 41 46 51
0
1
2
3
4
5
6
7
8
9
10
NS1 Ag Reactives Mean Threshold 2010
Rep
ort
ed C
ases
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
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)
6/13 DENV-1 2.18 3.6x1010 30.56/ 32.24
80 neg pos DENV-1 gt 5
6/24 DENV-4 1.24 5.1x107 16.95/ 4.47
85 +/- neg pend
8/18 DENV-4,1
1.39 2.9x105-1x108
2.50/ 17.94
91 +/- pend pend
9/1 DENV-4 4.51 2.3x108 30.17/ 32.31
99 -/+ pend DENV-4 gt 2
9/1 neg 9.87 neg 24.55/ 26.81
98 +/+ pend pend
10/27 DENV-1 4.54 4.1x105-9.7x109
32.48 pend -/+ pend pend
10/29 DENV-1 11.01 4.5x106-6.3x1010
31.08 pend -/+ pend pend
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Dengue Seroconverter (DENV-1)054V58459 Donor #9
-5
0
5
10
15
20
25
30
35
40
-10 0 12 19 54
Days Post Donation
Rel
ativ
e N
umbe
r
-1.E+11
0.E+00
1.E+11
2.E+11
3.E+11
4.E+11
5.E+11
6.E+11
7.E+11
8.E+11
PC
R V
iral L
oad
(Bill
ions
)
NS1 Ag S/CO Mean IgM P/N IgG Titer Quant PCR
Symptom Onset Day
Headache Body pain
1
Fever Backache
Chills
4
Rash 8*
* Virus isolated/sequenced; + in mosquito culture
Dengue Seroconverter (DENV-1)
15
Dengue Seroconverter (DENV-1)054V58459 Donor #9
-5
0
5
10
15
20
25
30
35
40
-10 0 12 19 54
Days Post Donation
Rel
ativ
e N
umbe
r
-1.E+11
0.E+00
1.E+11
2.E+11
3.E+11
4.E+11
5.E+11
6.E+11
7.E+11
8.E+11
PC
R V
iral L
oad
(Bill
ions
)
NS1 Ag S/CO Mean TMA S/CO IgM P/N IgG Titer Quant PCR
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
0
1
2
3
4
5
6
7
8
9
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Initial Reactive (all) Repeat Reactive Initial Reactive only
Fre
qu
ency
S/CO
Donations tested 5135
Initially reactive 22 (0.43%)
Repeat reactive 16 (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
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
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/C
O
Time (Days)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
0 20 40 60 80 100 120 140 160 180
22
Dengue NS1 Ag Persistence in Reactive Donors(20 unconf’d > 1 follow up, 3 eligible for reentry as of 11/30)
S/C
O
Time (Days)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
0 20 40 60 80 100 120 140 160 180
23
Dengue NS1 Ag Persistence in Reactive Donors(5 conf’d pos > 1 follow up, 2 eligible for reentry as of 11/30)
S/C
O
Time (Days)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
0 20 40 60 80 100 120 140 160 180
24
Dengue NS1 Ag Persistence in Reactive Donors(5 conf’d pos > 1 follow up, 2 eligible for reentry as of 11/30)
S/C
O
Time (Days)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
0 20 40 60 80 100 120 140 160 180
25
Dengue NS1 Ag Persistence in Reactive Donors (days post index with first case removed)
Days from first to last detectable
Total
No. donors 25
Min 6 days
Max > 89 days
Mean > 42 days
Unconfirmed
No. donors 20
Min 6 days
Max > 89 days
Mean > 43 days
Confirmed
No. donors 5
Min 12 days
Max 19 days
Mean 16 days
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