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Plasmodium falciparum artemisinin resistance: from phenotype to genotype Didier MENARD Malaria Molecular Epidemiology
Plasmodium falciparum
artemisinin resistance:
from phenotype to genotype
Didier MENARD
Malaria Molecular Epidemiology Unit
Institut Pasteur in Cambodia
SYMPOSIUM ON EMERGING INFECTIOUS DISEASES IN SEA
Phnom Penh - March 11, 2014
Didier Ménard
Human population distribution falciparum malaria cases distribution 3 billion people at risk 1.1 billion high
Human population distribution
falciparum malaria cases distribution
3 billion people at
risk
1.1 billion high risk
216 million clinical
cases
655 000 deaths/y
(91% in Africa)
2
Malaria control tools IRS LLIN Upgrading Health care 3 Diagnostic Artemisinin-based Combination Therapy (ACT)
Malaria control tools
IRS
LLIN
Upgrading Health
care
3
Diagnostic
Artemisinin-based Combination
Therapy (ACT)
Intermittent Preventive
RDT
Treatment
IPT
In 2014, the main challenges for malaria control are South east Asia Antimalarial drugs Africa
In 2014, the main challenges for malaria control
are
South east
Asia
Antimalarial
drugs
Africa
resistance
Malaria transmission
&
burden
4
Antimalarial drugs and emergence of resistance First reported resistance Difference Antimalarial drug Introduced
Antimalarial drugs and emergence of
resistance
First reported
resistance
Difference
Antimalarial drug
Introduced
(years)
Quinine
1632
1910
278
Chloroquine
1945
1957
12
Proguanil
1948
1949
1
Pyrimethamine
1951
1952
1
Sulfadoxine-Pyrimethamine
1967
1967
0
Mefloquine
1984
1991
7
Halofantrine
1989
1991
3
Atovaquone
1996
1996
0
5
(Wongscrichanalai et al. Lancet ID 2002)
Antimalarial drugs and emergence of resistance Resistance More drugs used Delayed response More clinical cases
Antimalarial drugs and emergence of
resistance
Resistance
More drugs
used
Delayed response
More clinical cases
Recrudescent infections
Larger reservoir
Increased
gametocytes
carriage
Increased
6
transmission
(Talisuna et al. Lancet ID 2012)
Chloroquine: spread and evolution (1970-80) 7 (Mita et al. Parasitology Inter. 2009)
Chloroquine: spread and evolution (1970-80)
7
(Mita et al. Parasitology Inter. 2009)
Sulfadoxine-Pyrimethamine: spread and evolution (1980-90) 8 (Mita et al. Parasitology Inter. 2009)
Sulfadoxine-Pyrimethamine: spread and
evolution (1980-90)
8
(Mita et al. Parasitology Inter. 2009)
Mechanisms in P. falciparum drug resistance Mutations in or changes in the copy number of
Mechanisms in P. falciparum drug resistance
Mutations in or
changes in the copy
number of
Genes
relating to
the drug’s
parasite
target
influx/efflux
pumps
affecting
intraparasitic
concentrations
of the drug
DHFR, DHPS, CytB
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CRT, MDR1, MRP
P. falciparum drug resistance: impact on mortality 10 (Murray et al. Lancet 2012)
P. falciparum drug resistance: impact on
mortality
10
(Murray et al. Lancet 2012)
WHO recommendation: ACTs Artemisinin derivatives (short half life) & Partner drugs WHO banned CQ and
WHO recommendation: ACTs
Artemisinin derivatives
(short half life)
&
Partner drugs
WHO banned CQ and
artemisinin monotherapy
(long half life)
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ACT, granted by the Global
Fund
Introduction of ACTs: impact 2000 12
Introduction of ACTs: impact
2000
12
Introduction of ACTs: impact 2010 13
Introduction of ACTs: impact
2010
13
Emergence of artemisinin resistance in SEA (2008-2013) Noedl et al., 2008 Dondorp et al., 2009
Emergence of artemisinin resistance in SEA
(2008-2013)
Noedl et al., 2008
Dondorp et al., 2009
Phyo et al 2012
Amaratunga et al 2012
Hien et al 2012
Kyaw et al 2013
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Artemisinin resistance = clinical phenotype = increased of parasite clearance half-life Western Cambodia = ~
Artemisinin resistance = clinical phenotype =
increased of parasite clearance half-life
Western Cambodia
= ~ 6h
Thailand & Vietnam
= ~3h
15
(Dondorp et al, NEJM 2009)
Monitoring ART-R: missing tools in 2013 • No in vitro phenotype Infected blood from patient
Monitoring ART-R: missing tools in 2013
No in vitro phenotype
Infected blood from patient
isolate+ PBS
Poor correlations between
altered in vivo infection
parameters and the in vitro
drug susceptibility (standard
radioactive chemosensitivity
assay, which monitors
parasite multiplication in the
presence of drugs)
Avoids major Host factors:
Immunity
Nutrition
Calculate
e
Drug response
nse
Resuspend in medium and serum
B A CQ
Measure growth
C D QN
F E DHA
H G MEF
Incubate at 37 C - 48 H
DRUG CONCENTRATION
No molecular marker
available
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PATIENT ISOLATE
Definition of the in vitro phenotype of ART-R • Drug exposure to DHA similar to
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- NS NS 18
Pailin (W) vs Ratanakiri (E) in vitro testing
Standard isotopic 48-
NS
NS
18
Pailin (W) vs Ratanakiri (E) in vitro testing Ring-stage survival assay : significant early ring
Pailin (W) vs Ratanakiri (E) in vitro testing
Ring-stage survival assay : significant early
ring stages
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RSA 0-3h and parasite clearance time 13 patients with fast-clearing infections (filled circles) and 13
RSA 0-3h and parasite clearance time
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) Ex-vivo ring-stage survival assays (RSAs) were done
Ex vivo RSA and parasite clearance time (r=0.74)
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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Looking for a molecular marker of ART-R A parasite line (ART) was selected by culturing
Looking for a molecular marker of ART-R
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”)
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Whole-genome sequences were
obtained at different time point
Exome of F32TEM and F32ART: SNPs 23
Exome of F32TEM and F32ART: SNPs
23
Polymorphisms in Cambodian isolates K13 24
Polymorphisms in Cambodian isolates
K13
24
Biological data: K13 polymorphisms and RSA in Cambodian isolates 25
Biological data: K13 polymorphisms and RSA in
Cambodian isolates
25
Epidemiological data: spread of K13 mutant-type alleles 26
Epidemiological data: spread of K13 mutant-type
alleles
26
Clinical data: K13 SNPs are associated with delayed parasite clearance 27
Clinical data: K13 SNPs are associated with delayed
parasite clearance
27
K13 SNPs predict delayed parasite clearance more accurately than founder population membership (Miotto et al,
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 resistance 1. Progressive loss
K13-propeller polymorphism fulfills the definition of
a molecular marker of ART resistance
1. 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. 14 as
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. correlation with RSA 0-3h survival rates in vitro and parasite clearance half-lives in
vivo
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What next? Define the role of the Kelch protein in ART-R? Regulating cytoprotective and protein
What next? Define the role of the Kelch protein in
ART-R?
Regulating cytoprotective
and protein degradation
responses to external
stress
Homology with human KLHL12 and KLHL2,
involved in ubiquitin-based protein
degradation, and KEAP1, involved in cell
adaptation to oxidative stress
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What next? • Validate specific SNP(s) as predictive of artemisinin resistance outside Cambodia in the
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 (20,000
samples collected after 2012 - 38 countries in Asia, Africa and South
America
• Explore the conditions of emergence and spread of K13 mutants and
the parasites gene flow
• Identify additional genetic loci involved in ART resistance and partners
drugs/associated to clinical treatment failures
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• RSA: Screening new drugs effective on ART-R parasites (Sanofi/MMV)
Collaborations Benoit Witkowski Valentine Duru Nimol Khim Saorin Kim Fréderic Ariey Odile Mercereau-Puijalon
Collaborations
Benoit Witkowski
Valentine Duru
Nimol Khim
Saorin Kim
Fréderic Ariey
Odile Mercereau-Puijalon
Johann Beghain
Anne-Claire Langlois
Jean Christophe Barale
Christiane Bouchier
National Center for
Parasitology , Entomology
and Malaria Control (CNM)
Rick Fairhurst
Chanaki Amaratunga
Pharath Lim
CHU Toulouse Sc Parasitologie/INSERM/CNRS (Françoise 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)
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34 Anne-Claire Andries
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Anne-Claire Andries