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Anne Piantadosi MD, PhD Massachusetts General Hospital,

Division of Infectious Diseases

Broad Institute, Laboratory of Pardis Sabeti

NEW TRICKS

FOR TICK SICKNESS:

USING GENOMICS TO DETECT AND

UNDERSTAND TICK-BORNE INFECTIONS

www.cdc.gov/ticks

GENOMICS FOR INFECTIOUS DISEASES

1) Become familiar with current techniques and

terminology in genomics.

2) Describe the current diagnostic approach for

common and emerging tick-borne infections in MA

3) Understand how cutting-edge genomic techniques

are being used to detect and study tick -borne

pathogens

OBJECTIVES

Ixodes scapularis

(Deer tick)

TICKS ARE OUT THERE!

Dermacentor variabilis

(American dog tick / wood tick)

https://www.cdc.gov/ticks/tickbornediseases/tickID.html

TICKS ARE SPREADING…

1996 2016

Eisen et al. Trends in Parasitology 2018

AND SO ARE THE DISEASES THEY CARRY:

E.G. LYME DISEASE

2001 2015

Eisen et al. Trends in Parasitology 2018

http://blog.mass.gov/publichealth/wp-content/uploads/sites/11/2017/06/Ticks.pdf

TICK-BORNE INFECTIONS ARE

ON THE RISE IN MA: “THE BIG THREE”

Lyme Disease

Anaplasmosis

Babesiosis

“THE LITTLE TWO”

“THE LITTLE TWO”

… AND MORE?

Borrelia mayonii

Heartland virus

Bourbon virus

Lyme disease

Serology to detect antibody response

TICK-BORNE INFECTIONS

CAN BE TRICKY TO DIAGNOSE

www.cdc.gov

Lyme disease


Babesiosis

“Malaria of New England”

Blood smear



https://www.cdc.gov/dpdx/babesiosis/index.html

Lyme disease


Babesiosis

Blood smear

Anaplasmosis

“Summer fever” + transaminitis, leukopenia

PCR



Lyme disease


Babesiosis

Blood smear

Anaplasmosis

PCR

Borrelia miyamotoi

Fever, similar to anaplasmosis

PCR



Lyme disease


Babesiosis

Blood smear

Anaplasmosis

PCR

Borrelia miyamotoi

PCR

Powassan virus

Encephalitis




Lyme disease


Babesiosis

Blood smear

Anaplasmosis

PCR

Borrelia miyamotoi

PCR

Powassan virus


WE HAVE DIFFERENT TESTS

FOR DIFFERENT PATHOGENS

Polymerase chain reaction

PCR IS PRETTY GOOD

Mixture of DNA or RNA

of different types

in a sample

Kanjilal et al. Seminars in Neurology 2019


PCR IS PRETTY GOOD


of different types

in a sample

Primers that match the

microbe of interest



PCR IS PRETTY GOOD


of different types

in a sample

Primers that match the

microbe of interest

Amplfication of a

small target region

Readout is detection,

+/- sequencing of the

target region


HOW CAN GENOMICS HELP US TO

DO BETTER?

Sequencing offers the ability to test for multiple

different types of pathogens at once

Genomic data and techniques allow the development

of highly sensitive, rapid, and inexpensive techniques


DO BETTER?

Better diagnostic tests for patients

Better surveillance of ticks

Pathogen genome sequencing to understand:

Transmission

Evolution

Drug resistance

WHAT IS

“NEXT GENERATION” SEQUENCING?

Massively parallel platform

Allows sequencing of millions of fragments at once

Johnsen et al. Blood 2013

GENOMICS IS VERY USEFUL IN STUDYING

INFECTIOUS DISEASE OUTBREAKS

Wohl et al. Annual Review of Virology 2016

GENOMICS IS VERY USEFUL IN STUDYING

INFECTIOUS DISEASE OUTBREAKS

Gire et al. Science 2014

Metsky et al. Nature 2017

METAGENOMIC SEQUENCING


of different types

in a sample Sequence everything Identify potential pathogen

IS THERE A NEEDLE IN THE HAYSTACK?


DO BETTER?


CASE 1: 61 YEAR OLD MAN WITH

ALTERED MENTAL STATUS

61 year old man with history of Crohn’s disease

treated with immunosuppression

Developed headache and confusion

Lumbar puncture with 430 white blood cells

Piantadosi et al. Clinical Infectious Diseases 2018



61 year old man with history of Crohn’s disease

treated with immunosuppression

Developed headache and confusion


MRI with abnormalities in multiple locations


Sample preparation – 3 days

Nucleic acid extraction

cDNA synthesis

Library construction



Sequencing – 1 day



cDNA synthesis







cDNA synthesis


Data analysis – ½ day

• Metagenomic analysis

• Virus-specific analysis






cDNA synthesis




• Virus-specific analysis Powassan virus






cDNA synthesis




• Virus-specific analysis Powassan virus

Clinical serology test positive for Powassan – 4 weeks later



No specific treatment

Patient unfortunately had

limited neurological recovery

Psychological benefits of

earlier diagnosis

Opportunity to spare

unnecessary treatment?



Specific high-sensitivity enzymatic reporter unlocking

SHERLOCK IS A SENSITIVE, TARGETED

CRISPR-BASED TECHNIQUE

Myhrvold et al. Science 2018

Specific high-sensitivity enzymatic reporter unlocking

Targeted (like PCR)

Can be performed directly from clinical sample in <2h

SHERLOCK IS A SENSITIVE, TARGETED

CRISPR-BASED TECHNIQUE


79 year old healthy male gardener and physician

Developed dizziness, slurred speech, confusion, fever


MRI with abnormalities in multiple locations





Sample preparation – ~24 hours


cDNA synthesis

SHERLOCK for Powassan virus Positive

Confirmed by sequencing over the next 2 days

Patient unfortunately passed away

Family committed to raising awareness about

Powassan virus

SHERLOCK CAN DETECT OTHER


Slide courtesy of Gordon Adams and Jacob Lemieux MD, DPhil


DO BETTER?



http://blog.mass.gov/publichealth/wp-content/uploads/sites/11/2017/06/Ticks.pdf

Targeted

Can be performed directly from primary sample in <2h

Lateral flow assay

FIELD-DEPLOYABLE SHERLOCK


METAGENOMIC SEQUENCING FOR

SURVEILLANCE AND DISCOVERY

33 viruses, 24 of which were novel

Caveat: this technique will find many things, most of

which are unlikely pathogenic to humans

Tokarz et al. mSphere 2018


DO BETTER?



Pathogen genome sequencing to understand:

Transmission

Evolution

Drug resistance

GENOMICS ALLOWS US TO ASK:

Is the pathogen population size growing?

Are there mutations associated with increasing

human infection or pathogenesis?

POWASSAN VIRUS IS UNDER-STUDIED

• 22 complete virus genomes previously available

– 17 from ticks

– 5 from humans

(from Russia and Canada)

0.02

MG647780.1

HM440563.1

KT224350.1

HM440559.1

HQ231414.1

KT224351.1

MG647781.1

HM440561.1

EU770575.1

KJ746872.2

KU886216.1

NC_003687.1

AF311056.1

MG647782.1

EU543649.1

EU670438.1

HM440558.1

MG647779.1

HM440562.1

HQ231415.1

MG647783.1

HM440560.1

Lineage II

Lineage I

Northeast

U.S.

Midwest

U.S.

ONGOING EFFORTS TO UNDERSTAND

POWASSAN VIRUS DIVERSITY

0.03

ME_2018_PF3.019_1

HM440560.1

HQ231414.1

EU770575.1

MA_2018_CSF_B156

MG647782.1

MA_2016_brain_B22IS

EU543649.1

MG647781.1

DTV_SPO_M1V2B2

KT224351.1

HQ231415.1

IPS001

FB_DV_1

ME_2018_PF3.057_1

AF311056.1

HM440558.1

DTV_MB5_12_73_B2

FA_12_40_B2

DTV_FB5_13_1_B2

EU670438.1

HM440563.1

MG647779.1MG647780.1

HM440562.1

ME_2018_PF3.017_1

ME_2018_PF3.020_1

HM440561.1

KJ746872.2

HM440559.1

MA_2016_brain_M30

MG647783.1

KT224350.1

ME_2016_brain_ST001

KU886216.1

NC_003687.1

Lineage II

Lineage I

Northeast

U.S.

Midwest

U.S.

• 14 new Powassan virus genomes

– 4 from humans

– 10 from ticks

BABESIA MICROTI IN NEW ENGLAND

CT, RI,

MA, NH

Nantucket

Reference

Group

WI, MN

177 years

[51-499]

220 years

[73-583]

1293 years

[327 – 3933]

5043 years

[1341-14708]

●●●●●●●●●●●●●●

●

●●●●●●●●

●●●

●●

●

Lemieux et al. Nature Microbiology 2016

BABESIA POPULATION GROWTH

IN NEW ENGLAND


GENOMICS ALLOWS US TO ASK:

Is the pathogen population size growing?

Are there mutations associated with increasing

human infection or pathogenesis?

Are there mutations associated with drug resistance?

Patient with relapsing babesiosis, despite treatment

Mutations associated with drug resistance

RELAPSING BABESIOSIS


CAN WE TREAT VIRAL INFECTIONS USING

NEW GENOMIC TECHNIQUES?

Slide courtesy of Catherine Freije

~2/3 of viruses that infect humans are RNA viruses

(Woolhouse ME et. al . Emerg. Infect. Disease . 2016.)

Many have limited antiviral treatment options

(De Clercq E & Li G. Clin. Microbiol . Review . 2016.)




SHERLOCK as an antiviral:

Virus-infected cell

Cas13-crRNA therapy




SHERLOCK as an antiviral:

Before:

• DEET, picaridin

• Permethrin

• Long, light clothing

PREVENTING TICK-BORNE INFECTION

After:

• Shower

• Tick check

• Heat your clothes

Tick-borne infections are increasing in MA:

Lyme disease, anaplasmosis, babesiosis

Powassan virus, borrelia miyamotoi

Others?

Cutting-edge genomic techniques including

SHERLOCK, metagenomic sequencing, and pathogen

genome sequencing will allow us to improve:

Diagnostics for patients

Surveillance of ticks

Understanding pathogen evolution and pathogenesis

SUMMARY

Single-cell sequencing

Human cell atlas

Evolution of cancer cells

CRISPR for genomic screens and gene editing

EXCITING DIRECTIONS IN GENOMICS

Pardis Sabeti MD, DPhil

Jacob Lemieux MD, DPhil

Shibani Mukerji MD, PhD

Sam Telford ScD

Gordon Adams

Lisa Freimark

Erica Normandin

Siavash Zamirpour

KL2 TR001100

K08 AI139348

Broadnext10

ACKNOWLEDGEMENTS

new tricks for tick sickness - maphn.org - home...erica normandin siavash zamirpour kl2 tr001100 k08...

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