MOLECULAR BASIS OF

ANTIMALARIAL DRUG RESISTANCE:

Saving our last arsenal, artemisinin

D. Syafruddin
Eijkman Institute for Molecular Biology, Jakarta
Life Cycle of the malarial parasite
Plasmodium spp
101111 haploid parasites in acute
infection

Re-assortment and recombination

occur in sexual cycle to produce
new resistant genotype in each
generation

Whenever drug treatment is

required to control pathogen,
selection of drug resistance is
Inevitable (Peters, 1987)

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What is drug resistance:
“ The ability of a parasite strain to multiply or survive in the
presence of concentrations of drug that normally destroy
parasite of the same species or prevent their multiplication”
(Bruce-Chwatt et al, 1986)

Clinical classification (WHO, 2003)

Early treatment failure (ETF):

Aggravation or persistence of symptoms in the presence of
parasitaemia during the first 3 days

Late treatment failure (LTF):

Reappearance of symptoms in the presence of parasitaemia during days 4-14

Adequate clinical/Parasitological response (ACPR):

Absence of parasitaemia on the day 14 irrespective of fever, or absence
of clinical symptoms irrespective of parasitaemia, in patients not
meeting ETF or LTF criteria

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 Major achievements of WHO's
coordination and global database on
drug efficacy
77 endemic countries
changed drug policy based
on the results of their
therapeutic efficacy tests
Report on global monitoring
GFATM changed choice of
drug procurement
Detection of artesunate
resistance at Thai- Countries which need ACT policy
Cambodia border Countries which adopted ACT

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Consequences of antimalarial
drug resistance
Increased morbidity and mortality
including anaemia, low birth weight,
Increased of transmission
switch to effective drug combinations in situations of low to
moderate endemicity has always resulted in a dramatic
decrease in transmission
Economic impact
increases cost to health services (to both provider and
patient) because of returning treatment failures
Greater frequency and severity of epidemics
Modification of malaria distribution
Greater reliance on informal private sector
with the risk of using monotherapies, sub-standard and
counterfeit medicines which in turn will increase drug resistance
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History of antimalarial drug resistance

Introduced First Report Difference

of Resistance (years)

Quinine 1632 1910 278

Chloroquine 1945 1957 12
Proguanil 1948 1949 1
Sulphadoxine- 1967 1967 0
pyrimethamine
Mefloquine 1977 1982 5
Atovaquone 1996 1996 0
Artemisinin 2000 ?

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Antimalarial drugs and their predicted
target sites

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Recommended Artemisinin-based
Combination Therapy (ACTs) WHO 2010

Artemether+lumefantrine*
Artesunate+amodiaquine*
Artesunate+mefloquine
Artesunate+sulphadoxine-pyrimethamine
Dihydroartemisinin+piperaquine*

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 Chloroquine (CQ)

• 4-aminoquinoline
• Chemical structure : CQ = Amodiaquine (AQ)
= Piperaquine (PPQ)(Tarning 2007)

Piperaquine

• CQ, AQ and PPQ bind with free-haem and

inhibit hemozoin formation
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Mechanism for the antimalarial drug
resistance (Borst and Ouellette, 1995)

a. Alteration in drug transport

Quinoline antimalarials
Artemisinin (?)

b. Alteration in binding affinity to enzyme

Antifolates, Sulpha drugs,
Coenzyme Q analogues such as atovaquone
Artemisinin (?)

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Hemoglobin degradation pathways

FV Ct

FV

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Molecular basis of parasite resistance
to quinoline antimalarials
¤ Resistant-parasites accumulate less chloroquine

Resistance is reversed by Ca-channel blockers (Similar to multi-

drug resistance in cancer cells, mdr gene: ATP-dependent drug
effluxer)

¤ Identification of P-glycoprotein homologue (PfPgh1) in the

malaria parasite that is encoded pfmdr1 gene (Foote et al, 1989)

Identification of pfcrt gene and its product, pfCRT, a transporter

protein localized to food vacuole (Fidock et al, 2000)

¤ Multifactorial mechanisms?

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 Topology and function of the Plasmodium
falciparum chloroquine resistance transporter
(PfCRT)

Sanchez et al, 2010

K76T is used as molecular markers for chloroquine resistance in P. falciparum field

isolates. The 76T allelic distribution declines significantly in Malawi when
chloroquine was replaced with SP in 1993
Their roles in P. vivax and P. chabaudi were not proven. Other modulatory factor(s)?

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 Topology and function of the
Plasmodium falciparum Multi drug resistance 1
(PfMDR1)

Sanchez et al, 2010

Allelic replacement experiment (Reed et al, 2000) of 184F,

1034C, 102D and 1246Y confers resistance to CQ and
modulate sensitivity to QN, MF, HF and ART EIJKMAN INSTITUTE
Topology and function of the Plasmodium falciparum
Multi drug resistance-associated Protein 1 (PfMRP1)

Sanchez et al, 2010

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 Polymorphisms in the pvCRT
and its corresponding sites at pvCRT-o

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 Polymorphisms in the pvMDR1
and its corresponding sites at pfMDR1-o

Orjuela-Sanches et al. AAC 2009

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 Map of pppk-dhps gene
of the malaria parasite
Located on chromosome 8

nt1784
pppk

dhps
PfF3717 PfR199 PfR186

PfJR82 PfRJR83

PfFJR84 PfRJR85

PfF153 Pf165R
RFLP analysis
MspA1I for S436F/A L L’
Ava II for A437G
Fok I for K540E
BstU1I and Bsl1 for A581G
MwoI for A613S/T

437G is the most common allele

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Map of the dhfr-ts gene of the malarial
parasite

Chromosome 4

1875 bp

DHFR Junction TS

JR76 JR77
JR78 JR79 Restriction enzymes
NlaIII for 16Val
Tai I and Mae II for 50Arg
Tsp5091 for 51Ile
XmnI for 59Arg
BsrI for 108Asn, ScrfI for 108Thr
Alu I for Ser108

108N is the most common allele

108T+16V confers resistance to Cycloguanil
but retains sensitivity to Pyrimethamine

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Extrachromosomal genomes
in the malarial parasites
CO I Cytb

6kb
CO III

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Predicted Secondary Structure of the putative
apocytochrome b of Plasmodium berghei

QoL144S Q I VP
WLC L E Cytoplasmic
Qo
I
A L V P
I Q H G I L G T W
I
YS L W V N GI
L G V Y
Y Y
A Y T T S PS Y F
S
Y R
S T N
T
FP
L G131, N245, F264 Mucidin
E V V 250 AY
I L M
S V M I141, T142 Stigmatellin
E S
W Q TPD I KL Y268N
P G M133I G
I A T F123, G131, G137, Myxothiazol
S W K N I
P
Y268C M133, L144, I258, F267,
50 Y W D
C R
R
S
F
R
P
L
F
H
P V284F K
S
P
QD
I
Y268, L271, K272, P275,
F
A S N F G280, L283, V284
L V H M Y YV G L
V L I P GA C QL I
F G A
GT AF T
I H F GV F
L V G I
L
G Y Atovaquone
T
LQ I A LS
I
L FP S I Q V I I LW L R FL
A
N27, G33, L217 Antimycin AI F F F V F I L F A V L V
I L SLQ Y S
A I I L
I L A C F L L F FF S
I14, L215 Diuron I L
L F
G T
L
Y F
L I I I
F V L I
I
L
L F M F
C
S
H G
HI F I NN
L W LG F
G I IS A I LS
Y L W F F I
N R S L G E P V
W L Q Q
N FL G
P
H K
R
F
S N
I L V
LN L L H D N Y K
P
NH N
Y
Y S L 300 L E
R
KT
H YD

Qi
C
SYL
Matrix
2 S T
P M G L T A Y
1 T L I S
Y N S

Qi COO
N 0 N N I F
I Y - S
L HT K V L N I S N 0 P P Q V
LG F Y
F KM I N AQ
2 YD T A LK I P
0
0

1. Budimulja et al, 1997. Mol. Biochem. Parasitol, 84:137-141.

2. Syafruddin et al 1999. Mol Biochem. Parasitol, 104:185-194.
3. Siregar et al, 2008. 2008. Parasitol Int, 57:229-232.

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Genotyping of malaria parasites isolates
in Indonesia using drug resistant alleles

Source: Syafruddin et al, 2005

Genotypic patterns of P. falciparum isolates in Western Indonesia

Source: Syafruddin et al, 2005

Genotypic patterns of P. falciparum isolates in Eastern Indonesia

Source: Syafruddin et al, 2005

Genotypic Profile of P. falciparum isolates in
West Sumba District and its association to
antimalarial drug resistance

Allelic Prevalence (%)

Combination
Wet Season Dry Season

CQ Resistance 76T + 86 Y 23.6 24.73

(41/174*) (24/174)
76T + 1042D 65.4 24.1 (42/174)
(125/191)

SP Resistance 108N/T + 51I 0 (99) 0 (73)

+ 59R
437G + 540E 0 (92) 0 (92)

Source: Asih et al, 2009, Malaria J

Allelic frequency of genes associated with chloroquine
resistance among P. falciparum isolates in West Sumba

Gene Allele Prevalence (%)

Wet Season Dry Season

Pfcrt 76T 92.9 (198/213*) 84.9 (147/174)

Pfmdr1 copy number ≥ 1 - 24.1 (43/178) 42.6 (23/54)

Pfmdr1 86Y 44.3 (51/115) 40,32 (25/62)

1034C 0 (0/191) 0 (0/191)
1042D 72.8 (139/191) 53.3 (96/180)
1246Y 0 (0/191) 0 (0/191)

Source: Asih et al, 2009, Malaria J

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Asih et al, IJP, 2010
 Haplotypes of the P. vivax isolates in
severe malaria cases in Papua

Percentage
Haplotype Gene Frequency (%) (n= 94)
Pvdhfr Pvdhps
A I13/57L/58R/61M/117T 383G/A553 48 51.1
B I13/57L/58R/61M/117T A383/A553 30 31.9
C I13/F57/58R/61M/117T 383G/A553 5 5.3
D I13/F57/58R/61M/117T A383/A553 1 1.1
E I13/57L/58R/61M/117N 383G/A553 3 3.2
F I13/57L/58R/61M/117N A383/A553 2 2.1
G I13/F57/58R/61M/117N 383G/A553 4 4.3
H I13/F57/58R/61M/117N A383/A553 5 5.3
I I13/57L/58R/T61/117T 383G/A553 1 1.1
J I13/57L/S58/61M/117T A383/A553 1 1.1
K I13/57L/58R/T61/117N 383G/A553 2 2.1
L I13/57L/58R/61M/117N 383G/A553 2 2.1
M I13/57L/58R/61M/117S 383G/A553 4 4.3
N I13/57L/58R/T61/117S 383G/A553 4 4.3
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Does Artemisinin-resistant parasite emerge
in Indonesia?

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Source: Asih et al, 2009, Am J Trop Med
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Parasite clearance day prolonged after treatment with
Artemisinin-based Combination Therapy (ACT)

Source: Asih et al, 2009, Am J Trop Med

AS+AQ or DHP is given in three days (D0, D1 and D2)

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Conclusions
1. Molecular assays indicated that multi drug-resistant
strains of malarial parasites has been emerging in
Indonesia

2. ACTs showed high efficacy againsts uncomplicated

malaria in Indonesia but early sign of reduced efficacy in
vivo has been detected

3. Appropriate use of ACTs and regular monitoring using

drug sensitivity test in vivo and molecular markers should
be performed to contain or delay the spread of artemisinin-
resistant parasites

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Collaborating Institutions
Eijkman Institute for Molecular Biology
Din Syafruddin
Farah Coutrier
Puji BS Asih
Sangkot Marzuki
Josephine E Siregar
National Institute of Health, Research and Development,
The Ministry of Health (LITBANGKES)/United States Naval Medical
Research Unit 2 (NAMRU-2), Jakarta
Sekartuti
Rita Marleta
William O Rogers

UMCN, Nijmegen, The Netherlands

RW Sauerwein
AJAM van der Ven

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Funding Sources

Thank you

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