Michael P Busch, MD, PhDBlood Systems Research Institute University of California, San Francisco

ASSURING THE SAFETY OF THE BLOOD SUPPLY

Risks of major TTVs linked to interventions, and accelerating rate of EIDs of concern to blood safety

Perkins HA, Busch MP. Transfusion-Associated Infections: 50 Years of Relentless Challenges and Remarkable Progress. Transfusion, 2010; 50(10):2080-99

ZIK

V

Recent arbovirus threats

Arboviruses

•Arthropod Borne viruses

• Descriptive, not taxonomic

• Mosquitoes, gnats, sandflies, etc

• Ticks

• Four major families

• Togaviridae, Flaviviridae, Alphaviruses, Bunyaviridae

• These are all ss-RNA enveloped viruses

•Human and sylvatic cycles

3

Simplified Patterns of Vector-Borne Disease Transmission

National Center for Emerging and Zoonotic Infectious Diseases

Division of Vector-Borne Diseases | Bacterial Diseases Branch

Scenario one:People are incidental hosts

Scenario two:People are primary hosts

Courtesy of Lyle Petersen, US CDC

Daily Airplane Flights

Evaluating an EID threat to blood safety3 basic questions need to be answered:

• Is it in the blood supply?• Necessitates a way to measure the agent in donors during epidemics

• Estimation of donor risks: prevalence, incidence, durations of detection• Estimation of blood component risks

• Temperature, preparation, storage duration effects on infectivity?• Is antibody in the infected donor or co-transfused components protective?

• Is it transfusion-transmitted and what is the risk?• Is transmission risk dependent on stage of infection or VL in the

donor/component• Do recipient antibodies from prior infection protect from TT

• If transmissible by transfusion, does it have a clinical impact in transfused recipients?• Is TT disease more or less severe than usual routes of infection

World distribution and spread of WNV

Clade 1a

Clade 1b

Clade 1c

Lignage 1

Lignage 2

U.S. WNV blood donor screening timeline

1Lanciotti, RT, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, et al. Science, 19992Pealer LN, Marfin AA, Petersen LR, Lanciotti RS, Page PL, Stramer SL, Stobierski MG et al. N Engl J Med. 20033Busch MP, Caglioti S, Robertson EF, McAuley, JD, Tobler LH, Kamel H et al. New England J Med, 2005Stramer SL, Fang CT, Foster GA, Wagner AG, Brodsky JP, Dodd RY. N Engl J Med. 2005

4Kleinman SH, Williams JD, Robertson G, Caglioti S, Williams RC, Spizman R et al. Transfusion. 20095Centers for Disease Control and Prevention. Morbidity and Mortality Weekly Report. 20096Centers for Disease Control and Prevention. Morbidity and Mortality Weekly Report. 2013

1 TTIreported4

MP NATadopted3

ID NATtriggeringadopted3

23 TTIsreported2

WNV foundin Queens, NY1

1999 2006

1 TTI reported5

20082002 2003 2004

6 TTIsreported3

1 TTI reported3

All transfusion-transmitted infections (TTIs) traced to WNV RNA(+) / Antibody(-) transfusions,

except for 2013 case in which donation had very low VL with IgM and IgG

ID NATtriggeringenhanced4

ID NATtriggeringenhanced4

2012

1 TTI reported6

101

102

103

104

105

WN

V R

NA

(g

Eq

pe

r m

L)

Days following infectious mosquito bite

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

RNA IgM

IgG

MP-NAT

ID-NAT

3.4

9.5

5.8

IgM SC

IgG SC

6.1

6.9 days

13.2

3.9

7.7

1x ID-TMA

6x ID-TMA

MP-TMA

detection

Window Periods for acute WNV infection parameters,

and need for tragetted ID-NAT

Busch et al, JID, 2008

2003230

2004125

2005123

2006112

2007162

200879

200980

No WNV positive cases during winter months

201076

WNV Positive Donations 2003-2012, CTS 201148

0

61

130

36

3000000

811

39

46

14

52000000

4

21

51

42

50000000

3

28

53

24

40000000

9

28

75

37

10

21000011

17

31

21

7

1000000

7

2428

19

200000002

13

35

24

500000002

15

35

27

10000000

16

83

118

48

31

40

0

20

40

60

80

100

120

140

Jun

e

Sep

t

De

c

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rch

Jun

e

Sep

t

De

c

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Jun

e

Sep

t

De

c

Ma

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Jun

e

Sep

t

De

c

Ma

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Jun

e

Sep

t

De

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Jun

e

Sep

t

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Ma

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Jun

e

Sep

t

De

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Jun

e

Sep

t

De

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Sep

tem

ber

De

cem

ber

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Jun

e

Sep

tem

ber

De

cem

ber

2012353

2010 2011 2012

Estimated DENV prevalence in PR blood donors from 1995 to 20102

2006 2007

Seroprevalence in PR blood donors1

DENV RNA in blood donors and TTI in PR3

DENV RNA in Brazilian blood donors4

2002

TTI in Hong-Kong

TTI in Singapore

2008

IND NS1Ag screening implemented by ARC in PR & S Florida

NS1 Ag + rapid retrospective TMA testing

IND prospective TMA screening implemented

Dengue virus blood screening timeline

1 Mohammed H., Tomashek K.M., Stramer S.L., Hunsperger E., Transfusion August 20122 Petersen L.R, Tomashek K.M., Biggerstaff B.J., Transfusion August 2013 Stramer S.L., Linnen J.M., Carrick J.M., et, Transfusion August 20124 Dias L.L., Amarilla A.A., Poloni T.R., Covas D.T., Aquino V.H., Figueiredo L.T.M., Transfusion August 2012

REDS-III Study of donor viremia and TT in Brazil

TTI in PR

Estimated and observed RNA donor viremiain Puerto Rico

2005

0.07%

2007

0.19%

2010

0.31%

2011

0.03%

TMA: 2012-2013 = 0.18%

Petersen, Biggerstaff, Stramer

REDS-III Dengue Study Sites and Epidemic ActivityRecife and Rio de Janeiro

0

100

200

300

400

500

600

700

800

900

1 3 5 7 9 1113151719212325272931333537

Nu

mb

er

of

case

s

Weeks

Dengue Epidemic in Recife

2011

2012

Dengue Epidemics in Recife

(2011-2012)N

um

ber

of

case

s p

er

wee

k

Weeks

Dengue Epidemics in Rio de Janeiro (2008-2012)

Sabino et al, JID 2016Busch et al, JID 2016

Subject accrual & results of DENV RNA testing

Sabino et al, JID 2016

2%

9%

IgM Rate6.2%

NAT

9.1 days (95%CI: 4.4-13.9 days) period of detectable RNA by ID-NAT

Busch et al, JID 2016

1 case of clinical dengue diagnosed for every 3 infections

853 cases of reported clinical disease per NAT yield donation

DENV symptoms for case and control patients during the 45-day chart review period

Sabino et al, JID 2016

Chikungunya VirusMakonde language: “to dry up or be contorted”

Alphavirus

3 genotypes

• West African

• East/Central/Southern/East African (ESCA)

• Asian

Like dengue

• Man-mosquito-man transmission cycle

• Aedes aegypti is the traditional urban vector

• Also spread by Aedes albopictus

Chikungunya Virus Disease

Acute onset of fever and severe pain in multiple joints

Mortality low but extremely debilitating; symptoms often last months to years

Most infected persons become symptomatic

No antiviral therapy; treatment is symptomatic

Lifelong immunity following infection

No licensed vaccines; prevention relies on avoidance of mosquito bites & mosquito control

Reported Chikungunya cases and Number of Countries/Territories

with Local Transmission in the Americas, Dec 2013–Mar 2015

Cases reported to PAHO

to April 24 (week 16 2015)

50 countries/territories in the Americas

Susp’d Lab conf’d Imprt’d Deaths

US 0 11 2574 0

PR 31,433 4349 31 21

All 1,367,343 30,580 3637 191

Real-time TMA Assays on the Panther

Target amplification by “biphasic” real-time TMA

Amplification reaction is divided into 2 steps (linear/exponential)

Allows higher level of multiplexing

Detection occurs during the amplification step using “molecular torches” or beacons (paired fluorophore/quencher)

Quantitative or qualitative assays

Up to 6 real time fluorometer modules could be mounted under the incubator

• The real-time TMA technology is currently under development and is not available for commercial sale; the performance characteristics of this product in development have not been established.

Jun

Jul

Aug

Sep

tO

ctNov

Dec Ja

nFe

bM

ar0.0

0.5

1.0

1.5

0

5

10

15

20

25

% N

AT

reactive

Calculated ID rate IgG%

Sero

reactiv

e

Simmons et al, EID 2016

High Incidence of Chikungunya Virus and Frequency of Viremic Blood Donations during Epidemic, Puerto Rico, 2014

Sep

tOct

Nov

0

1

2

3

% N

AT

reactive

ID NAT only

MP detectable

NAT Serology #

ID.only. Seronegative 2

ID.only. IgM.and/or.IgG 30

MP.detectable Seronegative 11

MP.detectable IgM.and/or.IgG 13

NAT Serology #

ID-NAT+ only no antibody 2

1/16 diln (1/2 tests positive) no antibody 1

1/16 diln (2/2 tests positive) no antibody 10

1/16 diln IgM+ 6

1/16 diln IgM+ & IgG+ 7

ID-NAT+ only IgM+ & IgG+ 30

Detection of CHIKV RNA+ donations by HologicCHIKV/DENV assay applied to 3008 individual donations

Simmons et al, EID 2016

Dynamics of CHIKV viremia in blood donations

ID onlySeronegative

(n=2)

MP+veSeronegative

(n=11)

MP+veIgM positive

(n=6)

MP+veIgM and IgG

positive

(n=4)

ID onlyIgM and/or IgG

positive

(n=33)

100

102

104

106

108

CH

IKV

vir

al lo

ad

(c

/ml)

Eclipseperiod

ID-NATonly

MP-NATID-NAT &

seropositive

Indeterminate 0.5 days±0.8 5.1 days±1.0 8.0 days±5.3

Simmons et al, EID 2016

24

Zika virus (ZIKV)

• Arbovirus surveillance (Dick et al. 1952):

• Isolated from the blood of a sentinel rhesus monkey (placed

in the forest in 1947)

• Isolated from a pool of Aedes africanus mosquitoes in Ziika

forest, Uganda (1952)

• Humans:

• Serosurveillance: detection of neutralizing antibodies in

human sera collected from East Africa (Smithburn et al.

1952)

• Isolated from a human in Nigeria in 1954

• First isolation of the virus itself from ZIKV-infected human

(Simpson et al. 1964)

25

ZIKV is a Flavivirus related to DENV

Classification:

• Flaviviridae family

• Flavivirus genus

• West Nile virus

• Dengue

• Japanese encephalitis

• Yellow fever

• Spondweni virus clade

Structure:

• 50 nm in diameter

• Enveloped: cell membrane

derived

• Capsid: icosahedral

symmetry

• + single stranded RNA

~11kb

Asian strains

East Africa

West Africa

26

Transmission Cycle of ZIKV

Transmission cycle of ZIKV

aegypti, albopictus, stegomyia, polynesiensis, africanus, apicoargenteus, furcifer, hensilli, luteocephalus, and vitattus , etc.

Vector: Aedes mosquitoesSylvaticUrban Different Aedes mosquitoes can transmit

ZIKV including:

Aedes aegypti

• Nervous mosquito• Feeds on Humans meal • Year round

Aedes albopictus

• Calm• Feeds on multiple animals• Diapause in winter

Figure 2. Distribution of ZIKV mosquito vector

WHO cumulative detection of mosquito-borne ZIKV infections, Jan 2013 to April 2017

77 countries since 2007- 70 first outbreak since 2015- 13 countries reporting sexual transmission- 29 countries reporting congenital abnormalities- 22 countries reporting increases in Guillain-Barré syndrome

Why ZIKV Transfusion Transmission Concerns?

• Up to 80% of ZIKV infections asymptomatic/illness is generally mild • public health surveillance based on clinical case reporting is insensitive

• ZIKV can result in severe congenital syndromes & Guillain-Barré syndrome

• ZIKV RNA detected in asymptomatic blood donors

• ZIKV transfusion transmission reported – 4 cases (Brazil); 3 donors

• ZIKV may persist in whole blood longer than plasma

• 1-3 months vs weeks (diagnostic assays)

• Unknown impact of travel/sexual contact

• ZIKV interventions are available

• 2 investigational screening assays on newly available platforms

• Licensed pathogen inactivation for plasma/platelets; investigational methods in development (red cells/whole blood)

• FDA classification as a Relevant Transfusion-Transmitted Infection

ZIKV disease cases reported to CDC by US state or territory

(Jan 1 2016-March 1 2017)

http://www.cdc.gov/zika/geo/united-states.html

US Local TravelContinental 221 (215 FL) 4,779

(6 TX)Territories 38,161 145

2014 2015 2016

ECDCrecommenda on

FDAguidelines

20132007

HighseroprevalenceinYappopula on

ZIKVTTIsinBrazil

FDArevisedguidelines

WHOrecommenda ons

Poten alforZIKVTTI

CDCEUADiagnos cassay

WHOInterna onalStandard

FDAapprovedINTERCEPTTM(Cerus)

1stNATforbloodscreening(RocheIND)

2ndNATforbloodscreening(HologicIND)

YapIsland2007

FrenchPolynesia10/2013–02/2014

Brazil/La nAmerica03/2015–present

CaribbeanIslandsincludingPuertoRico01/2016–present

ZIKVoutbreaks:

Con guousU.S.includingFLandTX08/2016–present

SouthEastAsia08/2016–present

Distribu onofZIKVassayanaly cperformancepanelstoaddi onaldiagnos candresearchlabs.

Returnofresults.03/2016-06/2016Produc on/characteriza on

ofZIKVviralstocks12/2015–03/2016

Discussionofdonorscreeningop onwithCDC,FDA,andbloodscreeningcompanies

12/2015-04/2016

Distribu onofZIKVassayanaly cperformancepanelstobloodscreeningcompanies,CDC,andFDAlabs.

02/2016

2015

AccesstoZIKVisolates10/2015

2016

Compila onofresultsandanalysesoftestperformance.07/2016-09/2016AccesstoBrazilZIKVTTIRNA+plasma02/2016

31

Nucleic acid detection assays for blood screening (IND)

• Roche cobas® 6800/8800 System

– cobas® ZIKV test

• Hologic/Grifols Procleix Panther system

– Procleix Zika virus assay

Weekly Detection Rate of ZIKV RNA in Blood Donated in Puerto Rico

This project has been funded in wholeor in part with Federal funds from theBiomedical Advanced Research and Development Authority, Office of theAssistant Secretary for Preparednessand Response, Office of the Secretary,Department of Health and HumanServices, under Contract #HHSO100201600010C.

365 (0.53%)68.380 tested

ZIKV Confirmed Positive Blood Donations in US StatesN=47 (Aug 14 2016 - Mar 18 2017)

6.25 million donations screened47 confirmed positive/210 reactive

Conf’d based on repeat 1° NAT, alt NAT, Ab (IgM)1:133,000 conf pos; 22.4% PPV; 99.9974% spec

Of 47, state of residence:25 (53%) FL, 6 CA, 4 NY, 5 TX, 1 NV, 1 AZ, 1 MA, 1 WV, 1 TN, 1 PA, 1 IA

Majority have remote travel to an active area

ID-N

AT

only

IgM

neg

(n=1

4)

MP-N

AT

reac

tive

IgM

neg

ativ

e

(n=1

92)

MP-N

AT

reac

tive

IgM

pos

itive

(n=2

6) ID-N

AT

only

IgM

pos

itive

(n=9

3)

102

104

106

108

1010

RN

A c

op

ies/m

l

Black symbols = Peurto Rico (n=306)Red symbols = Continental US (n=19)

Staging of ZIKV NAT yield cases with valid IgM results

◊ Participate in development of INDs to enable testing of the US blood supply

◊ Study viral and immune parameters and clinical outcomes in ZIKV+ donors

◊ Characterize viral, serological and cellular immune markers of early stages of asymptomatic vs symptomatic ZIKV infection (with and w/o prior DENV Abs)

◊ Investigate compartmentalization of Zika virus in blood components

◊ Identify and store index blood components and follow-up samples that can be further studied to characterize the performance of existing and future assays,

◊ Characterization of transfusion-transmission of ZIKV in macaques (collaboration between REDS-III with the UC Davis Primate Center)

◊ ZIKV/CHIKV/DENV NAT testing surveillance of minipools from the 4 REDS-III Brazil Hemocenters

◊ ZIKV/CHIKV/DENV transfusion-transmission study being launched at the largest hospital in Latin America (Hospital das Clinicas, Sao Paulo)

◊ ZIKV/CHIKV transfusion-transmission study in chronically transfused SCD patients

NHLBI-funded REDS-III Studies of ZIKV

37

Zika RNA positive donor enrollment and follow-up activities

Month 9 Month 126

Extended Follow-up• Characterization of humoral and cellular immunity• Discriminate recent vs remote infections• Detect ZIKV reinfections

WNV RNA compartmentalization in blood

• Rios et al. Clinical Infectious Diseases. 2007

• 1 log higher WNV viral load in whole blood than in plasma

• Lai et al. Transfusion. 2012

• Longitudinal cohort of 10 WNV+ blood donors

• Higher WNV viral load in whole blood than in plasma after seroconversion

• WNV RNA cleared in plasma within 15 days post-index in 9/10 donors

• Persisted in whole blood up to 90 days post-index in 5/10 donors

• Confirmed in cohort of 56 WNV+ blood donors, Lanteri et al. Transfusion,

• Re-testing plasma PBMC and whole blood WNV persists in the RBC fraction

• WNV RNA is more persistent in symptomatic WNV+ blood donors pathogenesis

• How does WNV stick to the RBCs?

6 symptomatic travelersSerum + for 3 days; WB + for 2 months

5 Asymptomatic donorsPlasma - 10 days (range 7–37)WB -22 days (range 14–100)VL higher in whole blood

Higher levels of ZIKV RNA in red cells vs plasma in index donation samples after IgM seroconversion

Plasma RBC unit0

1

2

3

4

5

6

7

8Pre-IgM (n=65)

Lo

g1

0Z

IKV

RN

AIU

/mL

Plasma RBC unit0

1

2

3

4

5

6

7

8Post-IgM (n=45)

Lo

g1

0Z

IKV

RN

AIU

/mL

Longer persistence of ZIKV RNA in whole blood and RBC blood compartments than in plasma and body fluids

0 1 2 3 4 5 6100

101

102

103

104

105

106

107

Visit

ZIK

V IU

/mL

Plasma

0 1 2 3 4 5 6100

101

102

103

104

105

106

107

Visit

ZIK

V IU

/mL

PBMC

0 1 2 3 4 5 6100

101

102

103

104

105

106

107

Visit

ZIK

V IU

/mL

Red blood cells

0 1 2 3 4 5 6100

101

102

103

104

105

106

107

Visit

ZIK

V IU

/mL

Urine

0 1 2 3 4 5 6100

101

102

103

104

105

106

107

Visit

ZIK

V IU

/mL

Whole Blood

0 1 2 3 4 5 6100

101

102

103

104

105

106

107

Visit

ZIK

V IU

/mL

Saliva

1. Testing whole blood extends detection period for diagnosis of clinical cases and monitoring pregnant women and travelers.

2. Impact on donation policy: to extend deferral period or consider NAT testing whole blood.3. Considerations for testing for solid organ, tissue and semen donations 4. IS RBC-ASSOCIATED VIRUS INFECTIOUS?

ZIKV IgM non-reactive (P/N 3.0) at index

ZIKV IgM

0 50 100 150 2000

5

10

15

20

Day(s) from Index

P/N

ZIKV IgG

0 50 100 150 2000

5

10

15

20

Day(s) from Index

P/N

ZIKV IgM Equivocal (P/N 2.0 to

Characterization of transfusion-transmission of ZIKV in macaques (collaboration between REDS-III Central Lab (BSRI) and the UC Davis Primate Center)

Aims:

Dynamics of acute ZIKV TT infection in a macaque model

Characterization of minimal infectious dose for ZIKV in pre and post-Ab SC stages of infection

Characterization of the effect of pathogen-reduction on transmission—specifically at high viral loads

FDA and HRSA leveraging this study by extending monitoring of ZIKV infected macaques to investigate distributions/persistence in tissues and organs of interest

Characterization of blood transfusion-transmission of

Zika virus in macaques

Acute ZIKV Infection Dynamics in Blood in Macaques

Coffey et al., PlosOne, 2017

Coffey et al., PlosOne 2017

ZIKV RNA in organs/tissues 14 days post-infection in Macaques

Characterization of blood transfusion-transmission of Zika virus in macaques

Aim 2A. Minimal infectious dose in ramp-up phase

Plasma from ZIKV

RNA+ IgM- blood donor

Serial dilutions in

macaque plasmaSerial follow-up

For ZIKV infection

Aim 2B Minimal infectious dose in the presence of ZIKV antibodies

Plasma from ZIKV

RNA+/IgM+/IgG blood donor

Serial dilutions

Serial follow-up

For ZIKV

infection

Intravenous

infection

of macaques

Aim 3. Analysis of efficacy pathogen reduction technologiesSerial follow-up

For ZIKV

infection

PRT of plasmaPlasma from ZIKV RNA+

blood donors

Re-challenge of

uninfected animals

with non-PRT

plasma

Dilution in

macaque

plasma

Viral load

(IU/ml)

In vitro infectivity

(PFU/ml)

Mouse

infectivity

adjusted to be equivalent of

1ml

Macaque

infectivity

using 1 ml

“100” 228500 648 Not Tested Not Tested

“30” 68500 396 Not Tested Not Tested

“10” 27900 270 Not Tested Not Tested “3” 8900 72 - 1/1 infected

“1” 3325 24 - 1/2 infected

“0.3” 813 4 6/6 infected 0/2 infected

“0.1” 423 Below LOD 6/6 infected 0/2 infected

“0.3” 130 Below LOD 4/6 infected 0/1 infected “0.01” 49 Below LOD (~0.2) 6/6 infected 0/1 infected

“0.03” ND (~13) Below LOD (~0.04) 1/6 infected ND

“0.001” ND (~5) Below LOD 0/6 infected ND

Infectivity of Brazilian plasma implicated in TT

Macaque infection occurred between 3,000 – 9,000 RNA copies and 24 – 72 pfu.

• REDS-III Central Laboratory, Blood Systems Research Institute

• Brian Custer

• Marion Lanteri

• Thema Gonzalez

• Sonia Bakkoor

• Tzong-Hae Lee

• Graham Simmons

• Mars Stone

• REDS-III Brazil Program

• Ester Cerdeira Sabino, the Fundaçao Faculdade de Medicina and Hospital das Clinicas of the Medical School of the University of São Paulo with participation of 4 blood centers located in: Bello Horizonte - Minas Gerais(Fundaçao Hemominas), Recife - Pernambuco (FundaçaoHemope), Rio de Janeiro (Fundaçao Hemorio), and São Paolo (Fundaçao Pro-Sangue).

• REDS-III Data Coordinating center, RTI International

• Don Brambilla

• Marian Sullivan

Acknowledgements

• UC Davis

• Koen Van Rompay

• Lark Coffee

• REDS-III Chair

• Steve Kleinman

• REDS-III ZIKV Oversight Committee

• Jay Epstein, FDA

• Hira Nakhasi, FDA

• Matt Kuehnert, CDC

• Lyle Peterson, CDC

• Brad Biggerstaff, CDC

• NHLBI

• Simone Glynn

• Allison Cristman

• Kelli Malkin

• Shimian Zou