biosafety and the 2014 ebola epidemic during the 2014 ebola outbreak... · organismic and...

Nathan Yozwiak and Danny Park Broad Institute of MIT and Harvard September 10, 2014

Harvard University!Center for Systems Biology!Organismic and Evolutionary Biology!

Biosafety and the 2014 Ebola Epidemic

Graduate-level genomics education programs

Diagnostic metagenomic studies

Genomics teaching and sequencing labs

Sample collection networks

Partnered training programs

Education Research Genomics capacity

On-site training workshops

Diagnostics core facility

BioinformaticsSustainable science careers

African Center of Excellence for Genomics of Infectious Diseases

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Centre of Excellence for

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Genomics of Infectious D

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Kenema Government

Hospital

Cheikh Anta-Diop University

University of Sierra Leone

International Partners

Senegal

University of Ibadan

Redeemer’s University

Irrua Specialist Teaching Hospital

Sierra Leone

Nigeria

Sierra Leone


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Long-standing research partnerships

3,109 suspected and confirmed cases of Ebola (Aug 31 2014)

“Six months into the worst Ebola epidemic in history, the world is losing the battle to contain it.” - Dr. Joanne Liu, MSF President to the UN, Sept 2, 2014

First outbreak in West Africa, and the biggest outbreak recorded

1000 km

GuineaSierra LeoneLiberia

DRCGabon

20142007-08

1976-77

100100 1000

RC2001-051994-96

cases

Outbreak exhibits exponential growth: 35 day doubling rate

Num

ber o

f cas

es

Month of 2014

0

500

1000

1500

2000

Apr May Jun Jul Aug

Liberia

Sierra Leone

Guinea

Exponential fit R2=0.96Doubling time = 35 days

March 2014: Pre-Outbreak Preparedness

Outbreak supplies Containment and Biosafety Ebola diagnostics

Pre-Outbreak Preparedness

3000bp

100bp

Negative Patient 1 Negative Patient 2 NC

1:1 1:10 1:100 1:1K 1:1 1:10 1:100 1:1K

EBOV seed stock spike-in dilutions

A

B

3000bp

100bp76bp

1:1 1:10 1:100 1:1KEBOV seed stock dilutions

1:1 1:10 1:100 1:1K

118bp

1:1 1:10 1:100 1:1K

Kulesh assay

419bp

FiloAB assay KGH assay

118bp

Augustine Goba, Director MoHS-KGH Laboratory

May 25, 2014: First confirmed case of Ebola in Sierra Leone

Kailahun

Ebola crosses the border from Guinea in late May

Source: Funeral of a traditional healer

Ebola spread quickly through Sierra Leone

May 25

Kenema

389 cases

1 case

For Kenema Government Hospital’s Ebola effort

1. Resupply PPE and diagnostic reagents

2. Streamline diagnostic workflow

3. Share preliminary findings with health leaders

July 2014: Outbreak Response

35

Confirmed

Suspected

Traditional

Suspected Ebola

14Confirmed Ebola

14 cases identified within first week of outbreak

Sequenced 49 confirmed and suspected cases in 10 days

Rapid sequencing and release of data

Can Trace the Origins of the Virus

13!

2007/2008 2002

1976/1977

19951994/1996

2014

Can view mutation evolution in near real-time

NSS

NC

GUINEASIERRA LEONE CLUSTER 1

SIERRA LEONE CLUSTER 2

SIERRA LEONE CLUSTER 3

NP VP35 VP40 GP VP30 VP24 L POLYMERASE

14

15

0

5

10

15

May 26 Jun 02 Jun 09 Jun 16Date in 2014

# of

new

pat

ient

s

SL1SL2SL3

15!

New mutations fall in geographic and temporal patterns

Kissi Teng

Nongowa

Kissi Tongi

Luawa

Mandu

Kpeje West

Malema

Kpeje Bongre

Jawie

Kissi Kama

Njaluahun

BoKenema

Kailahun

Kambia

Kono

SL1SL2SL3

Geneticcluster

# patients

1 32

GUINEA

LIBERIA

KissiTeng

Jawie

Mambolo

KakuaMay 31

June 15

Chi

efdo

m

?

June 17June 7

unknown

HCW-ADriver

HCW-B

GUINEA

LIBERIA

Kissi Teng

Jawie

Mambolo

Kakua

KGH

Kissi Teng

Nongowa

Kissi Tongi

Luawa

Mandu

Kpeje West

Malema

Kpeje Bongre

Jawie

Kissi Kama

Njaluahun

BoKenema

Kailahun

Kambia

Kono

SL1SL2SL3

Geneticcluster

# patients

1 32

GUINEA

LIBERIA

Intrahost variation elaborates transmission epidemiology

New mutation is rapid and random during outbreak

2014 outbreak

All of EBOV

Po

ste

rio

r p

rob

ab

ility

Subs. rate (site-1xyear -1

x10-3)

0 1 2 3 4

0.0

0.1

0.2

0.3

0.4

Faster rate of change

0

20

40

60

80

May 26 Jun 02 Jun 09 Jun 16

Cum

ulat

ive

tota

l

Patients

Date in 2014

Unique mutationsUnique lineages

Continually increasing viral diversity

NSS

136

559

55

79

75

110

p = 3.0e-07p = 1.2e-8

Betwee

n outb

reaks

With

in ou

tbrea

ks

With

in ho

sts

1.00

0.75

0.50

0.25Fr

actio

n

Enriched for non-synonymous SNPs

Largest, deepest dataset from an Ebola outbreak

Complete, reliable genomes

HIV-1

Human enterovirus A

Aphid lethal paralysis virus

GB virus C

Lassa virus

Plasmodium

Suspected EVD Patients

A

EBOV conf.

10

20

% m

alar

ia in

fect

ion

B

EBOV susp

.

Find any and all human pathogens

CommunityHospital/ Lab

Internationalcollaborators

Patient samples

Increases accuracyof diagnosis

Sample collection

Local diagnosis

Refined diagnostics:cheaper/field deployable

High throughput genomicanalysis and serology

The impact of genomic surveillance



1. Lab practices 2. Lab design 3. Researcher training

BL3

BL2+

BL1

Safety consideration!(for both field and advanced labs sites)

Blood

Inactivated plasma

Nucleic acid



Lab Practices

• PPE additions: Coveralls and face-shields are required at all times, in addition to the usual PPE of double gloves, booties, gowns and N95 face masks.

• All absorbent paper has been removed - except what is needed for the ELISAs.

• All surfaces are sprayed with bleach and ethanol every evening and again in the morning.

• All material is discarded into bleach – including all tips, tubes and blood collection tubes.

• A disposable glove box has been installed; confirmed (or highly suspected) EBOV samples can thus be handled in complete isolation.

• A third biosafety cabinet has been installed in the main lab so all ELISA plate washing and reading equipment can be contained.

• Standard protocols have been posted in the lab, and personnel have been trained to help maintain biosafety.

Enhanced safety strategies:



Kenema Government Hospital Laboratory

Ebola ward, Kenema, Sierra Leone

Lab Design



Irrua Specialist Teaching Hospital Laboratory, Nigeria

RNA Extraction&

PCR set-up

Mater Mixset-up

Sample Processing&

Inactivation

Research&

Sample Storage

Work Room

Storage Room

PCR RoomBL3 lab space

Office

Patient Sampling

Room

StaffRoom

Restroom

Restroom

Gel-electrophoresis&

Imaging Room

Converted tostorage

Gel

imag

ing

Sink Fridge

Gel running stationGel prep area

PCR machinesqPCR

AnteRoom

Sinkbenchtop

clean hoodnot vented

Frid

ge

blood chemistrymachine

buck

etce

ntrif

uge

Lab coats

SinkExtraction Area

Freezer

Vacufuge Centri-fuge

Thermomixer

Centrif.Chemicalstorage

under counter

A/C

Freezer

Mas

ter m

ix a

dditi

on Fridge

Freezer

Sam

ple

colle

ctio

n ki

t as

sem

bly

and

pick

-up

Diss

ican

t bo

x

Storage

Sink

Sink

Fridge Freezer

Freezer

LN2Freezer

Comp-uter

SampleLabel printer

Mic

rosc

ope

Mal

aria

Dia

g.

Freezer

FreezerHallway

LN2Freezer

Freezer

AnteRoomExit

ELIS

A st

atio

n

Mobile Glovebox

Incu

bato

r

Fire Exit

BL3 Sample Storage

RNA Extraction&

PCR set-up

Mater Mixset-up

Restroom

Sample Processing&

Inactivation

Research&

Sample Storage

Work Room

Storage Room

PCR RoomELISA

Office

Restroom

Patient Sampling

Room

StaffRoom

Restroom

Gel-electrophoresis&

Imaging Room

Converted tostorage

Gel

imag

ing

Sink Fridge

Gel running stationGel prep area

PCR machinesqPCRCloset

AnteRoomcoat and shoe

area

Autoclave

Sinkbenchtop

clean hoodnot vented

Biosafety HoodFr

idge

Incubator

ELISA station

blood chemistrymachine

buck

etce

ntrif

uge

Lab coats

SinkExtraction Area

Freezer

Vacufuge Centri-fuge

Thermomixer

Centrif.Chemicalstorage

under counter

A/C

Freezer

Mas

ter m

ix a

dditi

on Fridge

Freezer

Sam

ple

colle

ctio

n ki

t as

sem

bly

and

pick

-up

Diss

ican

t bo

x

Storage

Sink

Sink

Fridge Freezer

Freezer

LN2Freezer

Comp-uter

SampleLabel printer

Mic

rosc

ope

Mal

aria

Dia

g.

Water distillationmachine

Freezer

Freezer

-80 Freezer

Fire Exit

Hallway

A/C

Current Laboratory Space Proposed changes to Laboratory Space

BL3 designated spaceBL1 designated space

AnteRoomEnter

-80 Freezer

A/C A/CA/C

A/C

A/C

Biosafety Hood

RestroomRestroom

Chimney

Fire Exit

Freezer Room/Storage



Autoclave

Irrua, Nigeria

Lab Design



Genomics Laboratory, Redeemer’s University, Nigeria

CDC Resource Needs Assessment

Lab Design



Training ACEGID 2014 Summer Genomics Training - Harvard/Broad

7-week training program Focus:• Microbiology theory • Genomic diagnostic tools

• PCR, deep sequencing • Bioinformatics • Teaching pedagogy • Biosafety



ACEGID 2014 Summer Genomics Training - Harvard/BroadTraining

Training-of-Trainers Courses Developed:

• Foundations of Genomic Technology • Lassa Diagnostics: Clinical

applications of molecular technology • Foundations of PCR • Intro to Molecular Cloning

Biosafety training



ACEGID 2014 Summer Genomics Training - Harvard/Broad

• General lab safety at Harvard • BL3 field site training at Broad • Lecture: Ara Tahmassian, Chief Research

Compliance Officer • Glovebox tutorial • NIEDL BL-4 lab tour

Training

August 2014: Resupplying before departure

Expanded scientific capabilities to enable sustainable, collaborative and independent health research

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The Goals of ACEGID

Kenema Government Hospital and the University of Sierra Leone

Tulane University and the VHFC

Harvard University and the Broad Institute

In memoriam…22

Supplemental material

Thanks to our partners

- Genomic Center for Infectious Diseases - Sequencing Platform - Technology Labs- Center for Systems Biology

- Department of Organismic and Evolutionary Biology

- Kenema Government Hospital - University of Sierra Leone - Sierra Leone Ministry of Health and Sanitation


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Gaps and Challenges Remaining* Potential Research Contributions

Coordination of the outbreak Single or multiple outbreaks

Financing Donations, Sustainable testing

Communication Open-access data release

Cross border collaboration Pathogen origin location

Logistics Diagnostic-Centered Response

Case management Pathogen origin date

Infection control Containment decisions

Surveillance Strain-specific diagnostics

Contact tracing Transmission chains

Community participation Improved public trust

… and Research

* As identified at the Emergency Ministerial meeting on Ebola Virus Disease Accra 2-3 Jul 2014 http://www.afro.who.int/en/media-centre/pressreleases/item/6695-health-ministers-agree-on-priority-actions-to-end-ebola-outbreak-in-west-africa.html

The Importance of Viral Diagnostic Research in Outbreak Settings

http://www.afro.who.int/en/media-centre/pressreleases/item/6695-health-ministers-agree-on-priority-actions-to-end-ebola-outbreak-in-west-africa.html

Using genomic surveillance to refine current diagnostics

Project Goals (1) Develop African research capacity in genomics by building a critical mass of well-trained scientists;

(2) Empower African researchers to utilize genomics-based tools towards the control and elimination of infectious diseases;

(3) Create genomics curricula to support and promote cutting-edge genomics-based research; and

(4) Engage communities in prevention efforts and public health education.

!

World Bank funded Project


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Foundational training 2-month training for ACEGID members at Harvard/Broad Institute Training program planned for June-August 2014 in Cambridge, MA, USA !

On-site Workshops Annual 2-week trainings at each of the 4 ACEGID sites for larger scientific

community !

Advanced training Annual multi-week advanced training for PIs held at Broad Institute

ACEGID Action Plan for Learning Excellence

Northwest Labs, Harvard Cambridge, Massachusetts, USA

Education

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Training%focus:%•  Laboratory%procedures%•  Molecular%biology%theory%•  Lab%Safety%•  Science%communicaAon%

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1%

Education

Year 2-4 Design and Construction of new Genomics Center at Redeemer’s 1. Genomics Teaching Labs 2. Genomic Diagnostic Research Labs 3. Core scientific production facility 4. Administration and guesthouse

Design Group

GHESKIO Tuberculosis Hospital, Port-Au-Prince, Haiti

Umubano Primary School, Kigali, Rwanda

ACEGID Infrastructure

biosafety and the 2014 ebola epidemic during the 2014 ebola outbreak... · organismic and...

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