Vector-borne Diseases and Transfusion Safety

Susan L. Stramer, Ph.D.VP, Scientific Affairs

Nov 11 2016

1

Conflict of Interest DisclosureI hereby declare the following potential conflicts of interest concerning my presentation:• Research Funding: Abbott Laboratories, Hologic/Grifols,

Roche, Cerus• Honoraria: Hologic/Grifols, Roche, Cerus• Advisory: Chair, AABB Tranfusion-Transmitted Diseases

Committee; Scientific Advisory Committee, HemaQuebec

2

Vector-Borne agents that are, or have the potential to be, transfusion transmitted

Arboviruses Rickettsia Bacteria Protozoa-Yellow Fever - Dengue/Zika-Chikungunya/Ross River-St Louis Encephalitis-Colorado Tick Fever-Crimean Congo HF-Eastern EquineEncephalitis-Japanese Encephalitis-West Nile-LaCrosse-Tick-borne Encephalitis Complex (Powassan)

-Anaplasmaphagocytophilum-Ehrlichia ewingii-Erlichia chaffeensis-Orientiatsutsugamushi-Rickettsiaprowasekii-Rickettsia rickettsii

-Borrelia burgdorferi-Borrelia spp.-Yersinia pestis

-Babesia spp-Leishmania spp.-Plasmodium spp.-Trypanosoma cruzi-Trypanosoma brucei

3

BOLDED and UNDERLINED agentsare transfusion transmissible

- Western Equine Encephalitis- Severe fever with thrombocytopenia syndrome

General Patterns of Mosquito-Borne Arboviral Disease Transmission

Urban EpidemicEnzooticEnzootic

West Nile virus

Dengue virusesYellow fever virusChikungunya virusZika virus

Culex spp Aedes sppHaemogogus spp

Aedes aegypti +/-Aedes albopictus

* No known enzootic reservoir for dengue 1-4 viruses 4

Enzootic versus Urban Epidemic Spread

Enzootic transmission:WNV• Humans don’t infect

mosquitoes• Humans incidental hosts• Lifelong immunity• Human herd immunity not

relevant• Human travel irrelevant• Mosquito surveillance

important

• Humans readily infect mosquitoes

• Humans amplifying hosts• Lifelong immunity• Human herd immunity very

important• Spread by human travel• Mosquito surveillance less

important

Urban epidemic:DENV, YFV, CHIKV, ZIKV

5

West Nile virus as a Model of Success– Emerged and is endemic in the US

• 18,795 WNND cases (2002-2014)

– Transmitted by a large number of mosquitoes• 58 different mosquito species, mostly Culex spp.

– Amplified by bird hosts• 288 avian species

– > 80% asymptomatic– Transfusion/transplant transmitted

• 23 transfusion transmissions (2002)

– Intervention US/Canada = 2003 testing by MP/ID-NAT• MP-NAT “surveillance”; ID-NAT “seasonal”; 13 transmissions• 4,355 RNA confirmed-pos donors (2003-2014)

– Outside of the US = 28-day travel deferrals 6

Incidence of WNV Neuroinvasive DiseaseAnnual Incidence per 100,000 by county 1999-2011

7

Yearly statistics for WNV in the US: example of a rapidly emerging agent and a successful intervention, 2002-2014

Year (No.)

Reported

2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014

WNND(18,725)

+

2946 2866 1148 1309 1495 1227 689 386 629 486 2873 1267 1262

WNV-RNA

confirmed positive s (4,355)

N/A 714 224 417 437*

481 218 161 182 139 752 307 303

Trans-fusion cases^

(36)

23 6 1 0 2 0 2 0 1 ***

0 1 0 0

WNND = West Nile virus neuroinvasive disease.N/A = not available; prospective testing not introduced until 2003.*2003-2005 reported from CDC ArboNet; 2006-2014 reported from the AABB WNV site.^ All transfusion-transmission cases were identified from May-Oct .*** 1 WNV NAT-untested granulocyte .+ = Data available through Dec 16 2014; www.cdc.gov/westnile/StatsMaps/.

What happened in the US?• Unexposed population, human and avian• US strain virulent to corvids• Mosquito feeding preference shifts to humans in

summer due to the dispersal of breeding birds• Irrigation patterns, standing water (abandoned

swimming pools), tires, etc. • Movement into Caribbean, Central and S Americas

– Human disease, however, is infrequent• Why aren’t there overlapping WNV and dengue –

chikungunya epidemics? (Zika?)– Nov 2013 first confirmed cases of autochthonous

CHIKV in the Americas– March 2015 first confirmed cases of autochthonous

ZIKV in the Americas 9

10

Percent WNV confirmed-positive donors requiring ID-NAT for detection, 2002-2012, ARC

0

10

20

30

40

50

60

70

80

90

100

2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

Perc

ent o

f Con

firm

ed C

ases

ID-NAT only Identifiable

prospective

retrospective

11

050

100150200250300350400450500

<100 100-500 >500-1,000

>1,000-5,000

>5,000

MP Identifiable ID-NAT Only Identifiable

Viral Load (copies/mL)

Freq

uenc

yViral loads at index for 1508 of 1576 confirmed-positive donors

ARC, 2003-2012 Donors with PCR results at index

Mean viral load (copies/mL)

Median viral load (copies/mL)

MP-NAT Identifiable 973 24,810 3500ID-NAT Only Identifiable 535 88 5

12

0

2

4

6

8

10

12

14

16

18

20

Cas

esSeronegative (44) Seroreactive (196)

2009 2010 2011 2012

Year

Seronegative Seroreactive

Start End Start End

2009 8/10/2009 8/12/2009 7/19/2009 9/16/20092010 8/17/2010 9/27/2010 7/2/2010 10/12/20102011 8/1/2011 10/3/2011 8/1/2011 10/11/20112012 7/13/2012 11/5/2012 7/3/2012 11/8/2012

WNV confirmed-positive donors requiring ID-NAT for detection

13

14

Index viral load distributions for 635 confirmed-positive donations ARC, 2003-2012

1005-200

5,600750-28,750

1005-715

52.5-50

4000

720,000

61,000

2000

Viral loads (VL) in copies/mL (31 samples with viral loads <100 and without a final quantitative value were excluded); those listed by each marker category (ID-NAT required for identification [IDNAT P] or MP-NAT identifiable (MPNAT P] and antibody [Ab] negative [N] or positive [P]) are the medians, interquartile ranges and maximum values.Dodd et al., TMR 2015

2.3 days 6.9 days 0.9 day 10.8 days

71 213 26 325

# Tested

# TMA-

reactive

# Confirmpositive

# False

positive

% Specificity

% PPV

MP-NAT 23,910,576 1256 992 264 99.999 78.98

ID-NAT 2,883,227 1362 584 778 99.973 42.88

All NAT 26,793,803 2618 1576 1042 99.996 60.20

PVDs 26,793,803 1542 *1469 73 100.000 95.27

Performance characteristics of WNV-NAT based on the number of reactive

donations, ARC, 2003-2012

*There were 107 confirmed-positive donations that were not classified as presumed viremic donations (PVDs); thus, the overall sensitivity of the PVD designation is 93.2%. PVDs had the highest positive predictive value (PPV).

Dodd et al., TMR 201515

WNV: What did we learn?• Imported infections unpredictable and may be

overwhelming• Acute infections transmissible by transfusion• NAT offers rapid route to testing• Pooled testing may have inadequate

sensitivity• Epidemic continues to be unpredictable

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Arbovirus ComparisonCharacteristic Dengue Chikungunya Zika

Virus family Flaviviridae Togaviridae Flaviviridae

Viral genus Flavivirus Alphavirus Flavivirus

Serotypes 4 1 1

Genotypes Multiple per serotype 3-4 (ECSA) 2 (Asian)

Vectors Aedes aegyptiAedes albopictus

Aedes aegyptiAedes albopictus

Aedes aegyptiAedes albopictus

Symptoms / illness

Acute febrile illness

Acute febrileillness

Acute febrileillness

Symptomatic:Asymptomatic 25:75 75:25 25:75

Illness outcome Severe dengue –plasma leakage

Rarely severe, arthralgias

Rarely severe,microcephaly,Guillian-Barré 17

Clinical Features:ZIKV, DENV and CHIKV

Features ZIKV DENV CHIKV

Fever + +++ +++Rash +++ + ++Conjunctivitis ++ - -Arthralgia ++ + +++Myalgia + ++ +Headache + ++ ++Hemorrhage - ++ -Shock - + -

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Dengue Incidence is Rapidly Increasing in the Americas

0

500000

1000000

1500000

2000000

250000019

80

1982

1984

1986

1988

1990

1992

1994

1996

1998

2000

2002

2004

2006

2008

2010

2012

Num

ber C

ases

Year

Source: Pan American Health Organization (PAHO) 19

Dengue viruses• Mosquito-borne flavivirus ; 4 closely related “types”• Most important arbovirus• Rapidly expanding global footprint; >2.5 billion people (~1/3 world’s

population) live in areas of risk; endemic in >100 countries– Asia/Latin America – leading cause of hospitalization in children

• Humans are the amplifying host• No vaccine or specific treatment; vector control is the only effective

intervention• Immunity to a given type is lifelong but cross reactivity between types is

short lived and increases risk for severe dengue • 50-80% asymptomatic• Transfusion transmission reported• Kidney, BM transplant, need-stick and lab infections• No FDA-licensed test• Testing under IND occurred in Puerto Rico; overall yield comparable to

WNV 20

TT-DENV: seven cases/clusters by year• Hong Kong, 2002: one case with

PCR and serologic, no sequence confirmation

• Singapore, 2007: cluster of 3 cases from single donation, confirmed by envelope sequencing

• Puerto Rico, 2007: single case confirmed by envelope sequencing

• Brazil, 2012: viremic donors transmit to 6 recipients with minimal disease, retro study; no sequence confirmation

• Puerto Rico, 2011-12: 2 (3?) transmissions from Ag negative/TMA positive donors, retro study; no sequence confirmation

• Brazil, 2014: single case from regular platelet donor without sequence comparison

• Singapore, 2014: single case with sequence identity with donor

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2007 Puerto Rico Donation Retrospective StudyStramer et al. Transfusion 2012;52:1657

• 29 of 15,325 TMA (+) 1:529; 12 PCR (+) 105-109copies/mL, DENV-1, 2, 3 • 12 infected mosquito cultures, 6 IgM (+)

Study Period

TMA positive donations

week of onset of symptoms22

TMA = Transcription Mediated Amplification

230.1

Mara4 VenezuelaJamaica 2008Dominican Rep. 2003

Colombia 2007Mexico 2002

Brazil 2006NGC

Thai 2006Thai 2003

Thai 2001

16681

India 2006 Sri Lanka 1989

P7-863 Malaysia 1969Australia 1993

Burkina Faso 1983

PR159

IQT2133 PeruVen2 Venezuela

West Africa 1981

Guinea 1981

PR 2008PR 2008

PR 2007

PR3PR3-1

2007

PR 2009

Asian American

SE Asian

Indian Pacific

South American

Sylvatic

Maximum Likelihood Sequence Analysis of DENV-2 Env(1482 nucleotides)

*

*

*

*

* Bootstrap values ≥ 70

DENV-2 best studiedAll are highly conserved, butidentical sequences (D/R)

Subclades:

Transfusion transmission108 copies/mL DENV-2pRBC recipient developedDHF 3 days post transfusion

Stramer et al. Transfusion 2012;52:1657 23

Comparison to estimated prevalence of viremic donations - PR, 1995-2010; Petersen et al. Transfusion 2012 (Aug)

Dashed lines are simultaneous 95th percentile-t confidence bands;light lines depict 100-sample realizations of the 500 used to compute the

average prevalence of dengue viremiaboxed% = RNA (TMA) donor prevalence from all years of testing

20050.07%

20070.19%

20100.31%

20110.03%

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25

No. Donations

Tested;N=270,049

No. Reactive;

N=386

No. (%) Confirmed Positive;

N=173

Rate of Confirmed Positives

No. False Positive;

N=213

Rate of False

Positives

NS1 Ag 2010-2012 181,232 117 10 (9) 1:18,123 107 1:1,693Retrospective TMA 2010-2012 53,449 98 8 (8) 1:6,681 90* 1:594

Prospective TMA 2012-2013 88,817 171 155 (91) + 1:573

(0.17%)16 1:5,551

Dengue Blood Donation Screening under INDin Puerto Rico (2010-2013)

* NS1 Ag positive control cross contamination+ 20 (13%) NS1 Ag positive at index

Transfusion 2015

0,0E+00

5,0E+09

1,0E+10

1,5E+10

2,0E+10

2,5E+10

3,0E+10

3,5E+10

4,0E+10

4,5E+10

5,0E+10

0

5

10

15

20

25

30

35

40

-10 0 12 19 54

PCR

Vira

l Loa

d (c

opie

s/m

L)

Rel

ativ

e N

umbe

r

Days Post Donation

054V58459 Donor #9NS1 Ag S/CO Mean TMA S/CO IgM P/N IgG Titer Quant PCR

NS1 Ag Screening 2010Dengue Seroconverter (DENV-1)

Duration (+):

<12 days RNA by PCR

<19 days NS1 Ag

~36 days RNA by TMA (mid-point)

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Dengue Donor Follow-up• Follow donors to development of symptoms, determine

viral and immune dynamics• 13 symptoms occurred at significantly higher rates in RNA-

pos donors (cases) vs false pos (controls)– Secondary (n=72) >> Primary (n=6); of those at p< 0.01

• Fever, backache, headache, chills, sore throat, body pain, joint pain

• Rash, backache, chills, fever• Viral median decline from first detection => 7.5 days • IgM median appearance => 4 days => maximum at 21.5

days => decline by day 50

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0

5

10

15

20

25

30

35

40

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

TMA

S/CO

Days28

0

5

10

15

20

25

30

35

40

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

TMA

S/CO

Days

Observed period of RNA detection from time of first detection 51/80 followed donors provided follow-up within 60 days of index34/80 followed donors provided follow-up within 21 days of index

N Median 95% CI

34 7.5 6.66-11.75

IgM neg 19 7.5 6.13-14.66

IgM pos 15 7.5 5.23-10.17

p=0.7172

0

1

2

3

4

5

6

7

8

9

100 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96 100

104

108

112

116

120

124

IgM

S/C

O *

21.5 days to peak IgM

29* - MAC ELISA and InBIOS ELISA

Appearance of IgM8 IgM NR donors with complete profiles including

2 positive samples or 1 positive with an S/CO > 2 within 30 days of index

Days Post Index

IgM Reactive Donors at Index Donors 40Samples 197

0

1

2

3

4

5

6

7

8

9

10

0 50 100 150 200 250 300 350 400

IgM

S/C

O *

Days post Index* - MAC ELISA and InBIOS ELISA 30

(N= 8/17) (N= 35/155)

Copi

es/m

L

1.3x10^10Primary

p= 0.00037.7x10^72nd

(N= 25) (N= 19)

Copi

es/m

L 7.0x10^8DENV-1

7.1x10^7DENV-4p=0.0382

(N= 35/81) (N= 9/87)

Copi

es/m

L 5.3x10^8IgM Neg

4.5x10^7IgM Posp=0.0325

Comparisons of DENV viral loads

(N= 8/30)(N= 18/48)

Copi

es/m

L4.6x10^7Asympt

1.0x10^9Sympt

p=0.2665

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Why are there only 7 clusters of TT dengue despite high levels of viremia in donors?

• Unknown why the number of TT-dengue cases is so low in the face of massive outbreaks• Lack of effective hemovigilance

• Cannot distinguish mosquito from blood-borne transmission

• Lack of recognition of dengue nonspecific symptoms in severely ill patients

• Transfusion of Ab-pos units in endemic areas• Recipients in an endemic area have antibody

• Heterologous type => severe dengue

• Different outcomes dependent on the route of transmission: mosquito vs transfusion

• Promotors in mosquito saliva 32

DENV-4 in Brazilian Donors

37.5% (6/16) infected

vs. 0.93% of control recipients

16 to 16 susceptible recipients

42 DENV RNA + units into 35 recipients

0.80% confirmed RNA positive in Recife

0.51% confirmed RNA positive in Rio

39,134 donors consented

Record review finds no significant differences between cases and controls re: morbidity or mortality

Sabino EC et al.JID. 2015

1:200 1:125

33

Cases 1 and 2 were considered presumptive DENV-transfusion transmissions based on medical record review.Matos et al., Transfusion 2015

Fever 2 weeks post transfusion; n=13

Died ≤ 24 hrs after transfusion; n=3

Discharged ≤ 48 hrs after transfusion; n=1

Lived ≥ 24 hrs after transfusion; n=42

Recipients of DENV TMA-reactive blood transfusionsAll recipients of DENV RNA-reactive blood transfusions; n=46

No Fever >2 weeks of transfusion; n=29

New fever; n=6

Continuous fever;n=7

New or other dengue-like symptoms;

n=12

No new or other dengue-like symptoms;

n=17

New or other dengue-like

symptoms; n=2

No new or otherdengue-like

symptoms; n=4

1. 57452-RNA pos (qPCR) 7x10^7 c/mL

2. 627533. 65936(IgM pos)

4. 42418** 4. 40126

(TMA RR) prior units transfused

34

35

36

Days1-3

Days 4-8

Days >8

NAT + + -

IgM - + +

IgG (PRNT) - - +

Chikungunya Virus Response• 2005-2007 Reunion and islands of the Indian Ocean, >300,000

cases; >40% of population infected with 75% symptomatic– Mutation viral env protein associated with increased viremia– ECSA strain

• 2006 N Italy 337 cases (spread via Ae. albopictus, ECSA strain imported from India); risk of a viremic donation est’d @ 1.05/100,000 donations

• Interventions:– Suspend/stop blood collection in areas with “risk” above a

certain threshold (e.g., > that of HBV transfusion risk)– Implemented platelet pathogen inactivation– CHIKV NAT– Donor deferral if lives in or traveled to an epidemic area

• Public concern undoubtedly demanded an intervention37

Petersen, Stramer, Powers. Transfusion Med Reviews 2010;24

Brouard et al., 2008 Transfusion 48

Distribution of CHIKV symptomatic infections per weekReunion Island

Overall risk = 132 per 100,000 dtns (1:758)Peak risk = 1500 per 100,000 dtns (1:67)

38

39

27,084 suspected cases

French West Indes1 Dec 2013 – 24 Apr 2015

Guadeloupe: 81,350 cases

Puerto Rico

Martinique: 72,520 cases

Puerto Rico: 31,433 cases

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Puerto Rico, Passive Dengue Surveillance System

199917

Oct 28,

French Measures in the CaribbeanGallian P et al, Blood 2014;123:3679

• Strategy– INTERCEPT PC in place since 2010– Surveillance via French HV system– 72h quarantine for blood products that are not pathogen inact’d

waiting for post-donation information (PDI)– PCR of all samples in batches post transfusion (Marseilles)

• Real-Star QPCR by Altona; 63% LOD = 140 copies/mL• Results

– 4 RNA pos/2149 screened Martinique (1:250)– 104 - 2x108 copies/mL 10 - 2x105 pfu/mL– 2 symptomatic (61-66 yo)– 2 asymptomatic (41-43 yo)– No CHIKV or any transfusion transmissions (TTI) reported

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42

Prospective detection of CHIKV in blood donors, Caribbean 2014

Gallian et al.,

Martinique Feb 24-Apr 94 RNA pos/2149 screened104 - 2x108 copies/mL 2 symptomatic (61-66 years old)2 asymptomatic (41-43 years old)

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Blood transfusion during arbovirus outbreaksFrench Polynesia: global strategy

Blood safety

Blood productquarantine => rash and fever

PathogenInactivation(Intercept)

NAT

Courtesy of Didier Musso

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Intercept and Arbovirus Inactivation(infectivity models)

Platelet(log10 reduction)

Plasma(log10 reduction)

WNV > 6 > 6.8CHIKV > 6.4 > 7.6DENV > 5ZIKV > 5.2-6.2 > 6.6

Musso et al., Transfusion; 2014Aubrey et al., Transfusion; 2016Santa Maria et al., AABB; 2016

> 5.7

Puerto Rico Executive Order Summary• In response to the CHIKV epidemic declared in Puerto Rico,

Governor issued an Executive Order in July 2014 requiring additional pre-donation screening questions and deferral• Targeted symptoms => past 7 days• Exposure to someone with DENV or CHIKV => past 7 days

• 28-day deferral if a “yes” response• 3-day “required” platelet hold and call-back of donors for

evidence of symptoms (RBCs 7-day passive hold)• Products discarded if symptoms are reported or if contact was not

established• The Red Cross elected to suspend platelet collections in

Puerto Rico in August 2014, following by the introduction of PI in March 2015• Platelets to meet patient need supplied from the US mainland

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CHIKV in PR Donors

• 557 collections Apr 4-Aug 14 2014

• Neg for ID markers including DENV RNA by TMA

• Screened by CHIKV TMA95% detection 16 copies/mLSinglet => dilutions to 10^8

• No donor reported PDI up to 12 days post donation

• 3 (0.54%, 1:186) TMA RR and confirmed by orthogonalmethods

(3 pos and 3 neg controls)1/3 reported PDI from call back

Chiu et al., EID 2015:21(8). Genomic assays forIdentification of CHIKV in Puerto Rico 2014

copies/mL: 2.9x105 – 9.1-107

46

3 CHIKV-pos donors were in the Caribbean clade- Molecular clock analysis showed this clade was an offshoot of the Asian strain predicted to have emerged in the Western Hemisphere in early 2013- PR isolates diverged from the other Caribbean strains 1.7 years prior to the study

47

First probable case of TT-Ross River Virus

•

Hoad VC, Speers DJ, Keller AJ, Seed CR et al.: First reported case of transfusion-transmitted Ross River virus infection. Med J Aust. 2015;202: p. 267-270.

• RBC recipient - symptoms consistent with RRV IgM detected Haemagglutination inhibition (HI) positive Archive sample tests RRV PCR positive

50

Estimated donor deferral rate (%) in the US from a14- or 28-day deferral by geographic region

comparing 2 surveys

Area of travelin the past:

Summer Survey Fixed sites only; paper survey;

Americas only primarily

Winter SurveyAll sites; electronic survey;

Worldwide

14-day 28-day 14-day 28-dayMexico 0.19 0.52 0.40 0.92

Caribbean 0.16 0.48 0.48 1.16Central and

South America0.05 0.15 0.20 0.46

All above* 0.39 1.17 0.96 2.23All travel (global)*

N/A 2.64 1.35 4.02

51

*Individual areas and sums are not the same due to incomplete reporting of traveldestination and travel to multiple places.

Spencer et al., AABB 2015; Plenary

DENV CHIKV ZIKV

Year Local Import Local Import Local Import

2014 7 (FL) 650 12 (FL) 2,799 0 0

2015 1 (FL) 748 1 (TX) 895 0 0

2016 (Nov 8)

2 (FL) 573 0 119 219 (FL) 3,989

Comparison of US-reported cases of DENV, CHIKV and ZIKV to the CDC, 2014-2016

52

• Mayaro• Adapting to urban Aedes aegypti & albopictus?• Chikungunya-like illness• Recent cases in Haiti, far from former regions

• Rift Valley fever• Expanding range out of Africa• Increasing virulence (i.e., more “hemorrhagic fever”)• 19 potential mosquito vectors in North America• Wild/domestic epizootics with increased human contact

• Crimean-Congo hemorrhagic fever• Tick transmission by old world Hyalomma sp. present in Germany & U.K.

Importation of vectors to North America reported• High fatality rate • Spread from Africa to Asia, two cases in Spain in 2016

• Usutu• Mosquito-borne by Culex pipiens (Aedes albopictus?)• Avian amplification & reservoirs with massive bird die offs in EU• 6% seroprevalence around Modena, Italy• Encephalitic syndrome in immunocompromised

Summary

• Mosquito-borne viruses are an increasingly recognized threat to transfusion safety

• Interventions are not widely available, have long development times and are costly

• Processes for determining when decisions to “do more” are needed

55