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Didier MENARDMalaria Molecular Epidemiology Unit

Institut Pasteur in Cambodia

SYMPOSIUM ON EMERGING INFECTIOUS DISEASES IN SEA

Phnom Penh - March 11, 2014

Plasmodium falciparumartemisinin resistance:

from phenotype to genotype

Didie

rMnard


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Human population distribution

falciparummalaria cases distribution3 billion people at

risk

1.1 billion high risk

216 million clinical

cases

655 000 deaths/y

(91% in Africa)


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Malaria control tools

IRS LLIN Upgrading Health

care

Diagnostic Artemisinin-based Combination Intermittent Preventive

RDT Therapy (ACT) Treatment IPT

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In 2014, the main challenges for malaria control

are

Africa

Malaria transmission&

burden

South east

AsiaAntimalarialdrugs

resistance


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Resistance

Delayed response

Recrudescent infections

Increasedgametocytes

carriageIncreased

transmission

Larger reservoir

More clinical cases

More drugsused

(Talisuna et al. Lancet ID 2012)

Antimalarial drugs and emergence of

resistance

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Chloroquine: spread and evolution (1970-80)

(Mita et al. Parasitology Inter. 2009)

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Sulfadoxine-Pyrimethamine: spread and

evolution (1980-90)

(Mita et al. Parasitology Inter. 2009)

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Mechanisms in P. falciparum drug resistance

Genes

relating to

the drugs

parasite

target

influx/efflux

pumps

affecting

intraparasitic

concentrationsof the drug

Mutations in or

changes in the copy

number of

DHFR, DHPS, CytB

CRT, MDR1, MRP

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P. falciparum drug resistance: impact on

mortality

(Murray et al. Lancet 2012)

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WHO recommendation: ACTs

Artemisinin derivatives(short half life)

&

Partner drugs(long half life)

WHO banned CQ andartemisinin monotherapy

ACT, granted by the Global

Fund

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Introduction of ACTs: impact

200012


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Introduction of ACTs: impact

201013


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Emergence of artemisinin resistance in SEA

(2008-2013)Noedl et al., 2008

Dondorp et al., 2009

Phyo et al 2012

Amaratunga et al 2012Hien et al 2012Kyaw et al 2013

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Artemisinin resistance = clinical phenotype =

increased of parasite clearance half-life

(Dondorp et al, NEJM 2009)

Western Cambodia

= ~ 6 h

Thailand & Vietnam

= ~3h

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No in vitro phenotypePoor correlations between

altered in vivo infectionparameters and the in vitro

drug susceptibility (standard

radioactive chemosensitivity

assay, which monitorsparasite multiplication in the

presence of drugs)

No molecular marker

available

Monitoring ART-R: missing tools in 2013

Infected blood from patientisolate+ PBS

Resuspend in medium and serum

Incubate at 37 C - 48 H

Measure gro wth

DRUG CONCENTRATION

A

B

C

DE

F

G

HPATIENTISOLATE

CalculateDrug response

Avoids major Host factors:

ImmunityNutrition

CQ

QN

DHA

MEF

nse

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Definition of the in vitro phenotype of ART-R

Drug exposure to DHA similar to physiological

exposure = 6h - 700 nM DHA compared to 48h -

0.1 to 64 nM DHA Readout: survival rates compared to parasite

growth

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Pailin (W) vs Ratanakiri (E) in vitro testing

Standard isotopic 48-

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NS NS


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Pailin (W) vs Ratanakiri (E) in vitro testing

Ring-stage survival assay : significant early

ring stages

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RSA0-3hand parasite clearance time

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13 patients with

fast-clearing

infections (filled

circles) and

13 patients with

slow-clearing

infections (open

circles)

in Pursat in 2010.


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Ex vivo RSA and parasite clearance time (r=0.74)

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Ex-vivo ring-stage

survival assays (RSAs)

were done on

parasite isolates

obtained directly

from patients with

malaria in Pursat

(red), Preah Vihear

(blue), and Ratanakiri

(green) in 2012


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A parasite line (ART) was selected by

culturing the ART-sensitive F32-

Tanzania clone under a dose-

escalating regimen of artemisinin

(F32-ART)

F32-TEM is its sibling clone cultured

without artemisinin (fast runner)

Whole-genome sequences were

obtained at different time point

Looking for a molecular marker of ART-R

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Exome of F32TEM and F32ART: SNPs

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Polymorphisms in Cambodian isolates

K13

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Biological data: K13 polymorphisms and RSA in

Cambodian isolates

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Epidemiological data: spread of K13 mutant-type

alleles

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Clinical data: K13 SNPs are associated with delayed

parasite clearance

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K13 SNPs predict delayed parasite clearance more

accurately than founder population membership(Miotto et al, 2012)

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K13-propeller polymorphism fulfills the definition of

a molecular marker of ART resistance1. Progressive loss of wild-type parasites in Western Cambodia during the decade of

emerging ART resistance in this region;

2. Mutant parasites cluster in Cambodian provinces where ART resistance is well

established and are less prevalent where ART resistance is uncommon;

3. located 5.9 kb upstream of the 35-kb locus identified by Cheeseman et al.14as

being under recent positive selection, and within the region of top-ranked

signatures of selection outlined by Takala-Harrison et al.16;

4. multiple mutations, all non-synonymous, are present in the K13-propeller,

reflecting positive selection rather than a hitchhiking effect or genetic drift;

5. mutations occur in a domain that is highly conserved in P. falciparum

6. correlationwith RSA0-3hsurvival rates in vitroand parasite clearance half-lives in

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Regulating cytoprotectiveand protein degradation

responses to external

stressHomology with human KLHL12 and KLHL2,

involved in ubiquitin-based protein

degradation, and KEAP1, involved in cell

adaptation to oxidative stress

What next? Define the role of the Kelch protein in

ART-R?

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What next?

Validate specific SNP(s) as predictive of artemisinin

resistance outside Cambodia in the GMR (20 known SNPs)

Validate by cross genetic studies (collaboration with

D. Fidock, Columbia University, NY) Tool for mapping ART-R :

In the GMR - Mapping parasite migration patterns to identify areas

at risk of resistance: Redefine Tier 2

Worldwide mapping: KARMA project leads by RIIP/IPP (20,000samples collected after 2012 - 38 countries in Asia, Africa and South

America

Explore the conditions of emergence and spread of K13mutants and

the parasites gene flow Identify additional genetic lociinvolved in ART resistance and partners

drugs/associated to clinical treatment failures

RSA: Screening new drugs effective on ART-R parasites (Sanofi/MMV)


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Frderic Ariey

Odile Mercereau-Puijalon

Johann Beghain

Anne-Claire LangloisJean Christophe Barale

Christiane Bouchier

Rick Fairhurst

Chanaki Amaratunga

Pharath Lim

National Center forParasitology , Entomologyand Malaria Control (CNM)

CHU Toulouse Sc Parasitologie/INSERM/CNRS (Franoise Benoit Vical, Antoine Berry)

Wellcome Trust Sanger Institute - MORU (Olivo Miotto)

Swiss TPH (Blaise Genton)

WHO (Pascal Ringwald)

NAMRU-2 (William Rogers)

CNRP France (Jacques Le Bras)

Collaborations

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Benoit Witkowski

Valentine Duru

Nimol Khim

Saorin Kim


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Anne-Claire

Andries

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