Plasmodium falciparum

artemisinin resistance:
from phenotype to genotype
Didier MENARD

SYMPOSIUM ON EMERGING INFECTIOUS DISEASES IN SEA

Phnom Penh - March 11, 2014

Didier Mnard

Malaria Molecular Epidemiology Unit

Institut Pasteur in Cambodia

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)

Malaria control tools

IRS

LLIN

Upgrading Health
care

3
Diagnostic
RDT

Artemisinin-based Combination
Therapy (ACT)

Intermittent Preventive
Treatment IPT

In 2014, the main challenges for malaria control

are
South east
Asia
Africa

Antimalarial
drugs
resistance

Malaria transmission
&
burden

Antimalarial drugs and emergence of

resistance
Introduced

First reported
resistance

Difference
(years)

Quinine

1632

1910

278

Chloroquine

1945

1957

12

Proguanil

1948

1949

Pyrimethamine

1951

1952

Sulfadoxine-Pyrimethamine

1967

1967

Mefloquine

1984

1991

Halofantrine

1989

1991

Atovaquone

1996

1996

Antimalarial drug

(Wongscrichanalai et al. Lancet ID 2002)

Antimalarial drugs and emergence of

resistance

Resistance
More drugs
used

More clinical cases

Delayed response

Recrudescent infections

Larger reservoir

Increased
transmission

Increased
gametocytes
carriage
(Talisuna et al. Lancet ID 2012)

Chloroquine: spread and evolution (1970-80)

(Mita et al. Parasitology Inter. 2009)

Sulfadoxine-Pyrimethamine: spread and

evolution (1980-90)

(Mita et al. Parasitology Inter. 2009)

Mechanisms in P. falciparum drug resistance

Mutations in or
changes in the copy
number of
Genes
relating to
the drugs
parasite
target

influx/efflux
pumps
affecting
intraparasitic
concentrations
of the drug
9

DHFR, DHPS, CytB

CRT, MDR1, MRP

P. falciparum drug resistance: impact on

mortality

10
(Murray et al. Lancet 2012)

WHO recommendation: ACTs

Artemisinin derivatives
(short half life)

&

WHO banned CQ and

artemisinin monotherapy
ACT, granted by the Global
Fund

Partner drugs
(long half life)

11

Introduction of ACTs: impact

2000

12

Introduction of ACTs: impact

2010

13

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

14

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

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)

No molecular marker
available

Infected blood from patient

isolate+ PBS
Avoids major Host factors:
Immunity
Nutrition

Resuspend in medium and serum

PATIENT ISOLATE

No in vitro phenotype

A
B
C
D
E
F
G
H

CQ
QN

Calculatee
Drug response
onse
Measure growth

DHA
MEF

Incubate at 37 C - 48 H

DRUG CONCENTRATION

16

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

17

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 stages

19

RSA0-3h 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.

20

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

21

Looking for a molecular marker of ART-R

A parasite line (ART) was selected by

culturing the ART-sensitive F32Tanzania clone under a doseescalating 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

22

Exome of F32TEM and F32ART: SNPs

23

Polymorphisms in Cambodian isolates

K13

24

Biological data: K13 polymorphisms and RSA in

Cambodian isolates

25

Epidemiological data: spread of K13 mutant-type

alleles

26

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, 2012)

28

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 RSA0-3h survival rates in vitro and parasite clearance half-lives in
vivo

29

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

30

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

31

Collaborations

Benoit Witkowski
Valentine Duru
Nimol Khim
Saorin Kim

Frderic 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 (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)

32

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Anne-Claire Andries