didier-menard 1 art
TRANSCRIPT
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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
vivo29
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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