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

14

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

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