MOLECULAR BASIS OFANTIMALARIAL DRUG RESISTANCE:

Saving our last arsenal, artemisinin

D. SyafruddinEijkman Institute for Molecular Biology, Jakarta

Life Cycle of the malarial parasitePlasmodium spp

101111 haploid parasites in acuteinfection

Re-assortment and recombinationoccur in sexual cycle to producenew resistant genotype in eachgeneration

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Whenever drug treatment isrequired to control pathogen,selection of drug resistance isInevitable (Peters, 1987)

101111 haploid parasites in acuteinfection

Re-assortment and recombinationoccur in sexual cycle to producenew resistant genotype in eachgeneration

What is drug resistance:“ The ability of a parasite strain to multiply or survive in thepresence of concentrations of drug that normally destroyparasite 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 ofparasitaemia 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 absenceof clinical symptoms irrespective of parasitaemia, in patients notmeeting ETF or LTF criteria

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Clinical classification (WHO, 2003)

Early treatment failure (ETF):Aggravation or persistence of symptoms in the presence ofparasitaemia 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 absenceof clinical symptoms irrespective of parasitaemia, in patients notmeeting ETF or LTF criteria

Major achievements of WHO'scoordination and global database on

drug efficacy77 endemic countrieschanged drug policy basedon the results of theirtherapeutic efficacy testsReport on global monitoringGFATM changed choice ofdrug procurementDetection of artesunateresistance at Thai-Cambodia border

77 endemic countrieschanged drug policy basedon the results of theirtherapeutic efficacy testsReport on global monitoringGFATM changed choice ofdrug procurementDetection of artesunateresistance at Thai-Cambodia border

Countries which need ACT policyCountries which adopted ACT

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Consequences of antimalarialdrug resistance

Increased morbidity and mortalityincluding anaemia, low birth weight,

Increased of transmissionswitch to effective drug combinations in situations of low tomoderate endemicity has always resulted in a dramaticdecrease in transmission

Economic impactincreases cost to health services (to both provider andpatient) because of returning treatment failures

Greater frequency and severity of epidemicsModification of malaria distributionGreater reliance on informal private sector

with the risk of using monotherapies, sub-standard andcounterfeit medicines which in turn will increase drug resistance

Increased morbidity and mortalityincluding anaemia, low birth weight,

Increased of transmissionswitch to effective drug combinations in situations of low tomoderate endemicity has always resulted in a dramaticdecrease in transmission

Economic impactincreases cost to health services (to both provider andpatient) because of returning treatment failures

Greater frequency and severity of epidemicsModification of malaria distributionGreater reliance on informal private sector

with the risk of using monotherapies, sub-standard andcounterfeit medicines which in turn will increase drug resistance

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Introduced First Report Differenceof Resistance (years)

Quinine 1632 1910 278Chloroquine 1945 1957 12Proguanil 1948 1949 1Sulphadoxine- 1967 1967 0pyrimethamineMefloquine 1977 1982 5Atovaquone 1996 1996 0Artemisinin 2000 ?

History of antimalarial drug resistance

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Introduced First Report Differenceof Resistance (years)

Quinine 1632 1910 278Chloroquine 1945 1957 12Proguanil 1948 1949 1Sulphadoxine- 1967 1967 0pyrimethamineMefloquine 1977 1982 5Atovaquone 1996 1996 0Artemisinin 2000 ?

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Antimalarial drugs and their predictedtarget sites

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Recommended Artemisinin-basedCombination Therapy (ACTs) WHO 2010

Artemether+lumefantrine*Artesunate+amodiaquine*Artesunate+mefloquineArtesunate+sulphadoxine-pyrimethamineDihydroartemisinin+piperaquine*

Artemether+lumefantrine*Artesunate+amodiaquine*Artesunate+mefloquineArtesunate+sulphadoxine-pyrimethamineDihydroartemisinin+piperaquine*

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Chloroquine (CQ)

• 4-aminoquinoline

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

• CQ, AQ and PPQ bind with free-haem andinhibit hemozoin formation

• 4-aminoquinoline

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

• CQ, AQ and PPQ bind with free-haem andinhibit hemozoin formation

Piperaquine

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Mechanism for the antimalarial drugresistance (Borst and Ouellette, 1995)

a. Alteration in drug transportQuinoline antimalarialsArtemisinin (?)

b. Alteration in binding affinity to enzymeAntifolates, Sulpha drugs,Coenzyme Q analogues such as atovaquoneArtemisinin (?)

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a. Alteration in drug transportQuinoline antimalarialsArtemisinin (?)

b. Alteration in binding affinity to enzymeAntifolates, Sulpha drugs,Coenzyme Q analogues such as atovaquoneArtemisinin (?)

CtFV

Hemoglobin degradation pathways

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n

FV

Molecular basis of parasite resistanceto 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 drugeffluxer)

¤ Identification of P-glycoprotein homologue (PfPgh1) in themalaria parasite that is encoded pfmdr1 gene (Foote et al, 1989)

Identification of pfcrt gene and its product, pfCRT, a transporterprotein localized to food vacuole (Fidock et al, 2000)

¤ Multifactorial mechanisms?

¤ Resistant-parasites accumulate less chloroquine

Resistance is reversed by Ca-channel blockers (Similar to multi-drug resistance in cancer cells, mdr gene: ATP-dependent drugeffluxer)

¤ Identification of P-glycoprotein homologue (PfPgh1) in themalaria parasite that is encoded pfmdr1 gene (Foote et al, 1989)

Identification of pfcrt gene and its product, pfCRT, a transporterprotein localized to food vacuole (Fidock et al, 2000)

¤ Multifactorial mechanisms?

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Topology and function of the Plasmodiumfalciparum chloroquine resistance transporter

(PfCRT)

Sanchez et al, 2010

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K76T is used as molecular markers for chloroquine resistance in P. falciparum fieldisolates. The 76T allelic distribution declines significantly in Malawi whenchloroquine was replaced with SP in 1993Their roles in P. vivax and P. chabaudi were not proven. Other modulatory factor(s)?

Topology and function of thePlasmodium falciparum Multi drug resistance 1

(PfMDR1)

Sanchez et al, 2010

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Allelic replacement experiment (Reed et al, 2000) of 184F,1034C, 102D and 1246Y confers resistance to CQ andmodulate sensitivity to QN, MF, HF and ART

Topology and function of the Plasmodium falciparumMulti drug resistance-associated Protein 1 (PfMRP1)

Sanchez et al, 2010

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Polymorphisms in the pvCRTand its corresponding sites at pvCRT-o

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Polymorphisms in the pvMDR1and its corresponding sites at pfMDR1-o

Orjuela-Sanches et al. AAC 2009

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Located on chromosome 8

Map of pppk-dhps geneof the malaria parasite

nt1784pppk

dhpsPfF3717 PfR186

PfJR82 PfRJR83

PfR199

PfFJR84 PfRJR85

PfF153 Pf165R

dhps

RFLP analysisMspA1I for S436F/AAva II for A437GFok I for K540EBstU1I and Bsl1 for A581GMwoI for A613S/T

L L’

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437G is the most common allele

Chromosome 4

DHFR Junction TS

1875 bp

Map of the dhfr-ts gene of the malarialparasite

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108N is the most common allele108T+16V confers resistance to Cycloguanilbut retains sensitivity to Pyrimethamine

DHFR Junction

JR78 JR79JR77JR76

Restriction enzymesNlaIII for 16ValTai I and Mae II for 50ArgTsp5091 for 51IleXmnI for 59ArgBsrI for 108Asn, ScrfI for 108ThrAlu I for Ser108

CO I

CO III

Cytb

6kb

Extrachromosomal genomesin the malarial parasites

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Predicted Secondary Structure of the putativeapocytochrome b of Plasmodium berghei

Cytoplasmic

H HGVFL

AS

RY

SP

EIS

YA YY S I Q H I LRE

LWS

GW

CF R

YMA T F

I GYVLPWGQ

MS

YW

GA

TV

I

TN

LL

SG

I

PS

LV

IW L C

GG

YT

VSDPT

IKR

FFVF G V I PL

SH

PD

NA

IV

VN

TY

V TP

LQ I V P

EWY

FLPFYAML

P

KT

I

SKNAGL VI IG C Q L

P Q DIFI

L YG

QoQo

L

50

250

M133I

L144S

V284F

G131, N245, F264 MucidinI141, T142 StigmatellinF123, G131, G137, MyxothiazolY268N

Y268C M133, L144, I258, F267,Y268, L271, K272, P275,G280, L283, V284AtovaquoneH H

H

N

MN

YNSINLVKTHLIN

YPC

PL N I NF LWN

YG

FL LGI I

F FIQ ILT

GVFL M

A TGA SLVF FLT YLHI

LRGLN

YS Y

LYL

PL

SW I S GL I IF A L FI V TA F

I GY

L I F

AV FP

I C L

I F V

F F I

LH

LH

SGT

NP

L G Y D T A L K I P F YP

NL

LS

LD

VK

GF

NN IL I LF L I QS IFG V IL GL VIV IASL QLL FLL

A

EQ

RN

LT

TI

IQ

F KMVF

SA

RE

YS

FPI I

W FMC SF Y A LL W IG C Q L YGR LFII LFF SSGL FSL

VQ

KK T H YD

YSS

QANI

MatrixQi

QiNH2

COO-

L

N27, G33, L217 Antimycin AI14, L215 Diuron

100

200

300

M133, L144, I258, F267,Y268, L271, K272, P275,G280, L283, V284Atovaquone

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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.

Genotyping of malaria parasites isolatesin 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

AllelicCombination

Prevalence (%)

Wet Season Dry Season

Genotypic Profile of P. falciparum isolates inWest Sumba District and its association to

antimalarial drug resistance

CQ Resistance 76T + 86 Y 23.6(41/174*)

24.73(24/174)

76T + 1042D 65.4(125/191)

24.1 (42/174)

SP Resistance 108N/T + 51I+ 59R

0 (99) 0 (73)

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

Source: Asih et al, 2009, Malaria J

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)

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

Source: Asih et al, 2009, Malaria J

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Asih et al, IJP, 2010

Haplotypes of the P. vivax isolates insevere malaria cases in Papua

Haplotype Gene FrequencyPercentage

(%) (n= 94)Pvdhfr Pvdhps

A I13/57L/58R/61M/117T 383G/A553 48 51.1B I13/57L/58R/61M/117T A383/A553 30 31.9C I13/F57/58R/61M/117T 383G/A553 5 5.3C I13/F57/58R/61M/117T 383G/A553 5 5.3D I13/F57/58R/61M/117T A383/A553 1 1.1E I13/57L/58R/61M/117N 383G/A553 3 3.2F I13/57L/58R/61M/117N A383/A553 2 2.1G I13/F57/58R/61M/117N 383G/A553 4 4.3H I13/F57/58R/61M/117N A383/A553 5 5.3I I13/57L/58R/T61/117T 383G/A553 1 1.1J I13/57L/S58/61M/117T A383/A553 1 1.1K I13/57L/58R/T61/117N 383G/A553 2 2.1L I13/57L/58R/61M/117N 383G/A553 2 2.1M I13/57L/58R/61M/117S 383G/A553 4 4.3N I13/57L/58R/T61/117S 383G/A553 4 4.3

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Does Artemisinin-resistant parasite emergein Indonesia?

Does Artemisinin-resistant parasite emergein Indonesia?

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Source: Asih et al, 2009, Am J Trop MedEIJKMAN INSTITUTE

Parasite clearance day prolonged after treatment withArtemisinin-based Combination Therapy (ACT)

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

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

Conclusions1. Molecular assays indicated that multi drug-resistant

strains of malarial parasites has been emerging inIndonesia

2. ACTs showed high efficacy againsts uncomplicatedmalaria in Indonesia but early sign of reduced efficacy invivo has been detected

3. Appropriate use of ACTs and regular monitoring usingdrug sensitivity test in vivo and molecular markers shouldbe performed to contain or delay the spread of artemisinin-resistant parasites

Conclusions1. Molecular assays indicated that multi drug-resistant

strains of malarial parasites has been emerging inIndonesia

2. ACTs showed high efficacy againsts uncomplicatedmalaria in Indonesia but early sign of reduced efficacy invivo has been detected

3. Appropriate use of ACTs and regular monitoring usingdrug sensitivity test in vivo and molecular markers shouldbe performed to contain or delay the spread of artemisinin-resistant parasites

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National Institute of Health, Research and Development,The Ministry of Health (LITBANGKES)/United States Naval MedicalResearch Unit 2 (NAMRU-2), JakartaSekartutiRita MarletaWilliam O Rogers

UMCN, Nijmegen, The NetherlandsRW SauerweinAJAM van der Ven

Collaborating InstitutionsEijkman Institute for Molecular BiologyDin SyafruddinFarah CoutrierPuji BS AsihSangkot MarzukiJosephine E Siregar

National Institute of Health, Research and Development,The Ministry of Health (LITBANGKES)/United States Naval MedicalResearch Unit 2 (NAMRU-2), JakartaSekartutiRita MarletaWilliam O Rogers

UMCN, Nijmegen, The NetherlandsRW SauerweinAJAM van der Ven

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Thank you

Funding Sources

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Thank you