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Molecular & Biochemical Parasitology 177 (2011) 77–82

Contents lists available at ScienceDirect

Molecular & Biochemical Parasitology

eview

pdate on genetic markers of quinine resistance in Plasmodium falciparum

ohn Okomboa, Eric Ohumaa, Stephane Picotb, Alexis Nzilaa,∗

Kenya Medical Research Institute (KEMRI)/Wellcome Trust Collaborative Research Program, PO Box 230, 80108 Kilifi, KenyaMalaria Research Unit (MRU), University Lyon 1, Faculty of Medicine, 8 Avenue Rockefeller, 69373 Lyon, France

r t i c l e i n f o

rticle history:eceived 28 October 2010eceived in revised form 13 January 2011ccepted 19 January 2011vailable online 1 February 2011

a b s t r a c t

The emergence and spread of antimalarial resistance remain burgeoning issues. Any strategy to slowdown or overcome these problems requires an understanding of the genetic changes underlying thisresistance. Quinine, the first antimalarial, has been central in the treatment of severe malaria, and hasbeen proposed as second line treatment for uncomplicated malaria in many African countries. Somereports have indicated the emergence of quinine resistance in South East Asia and in Africa, however

eywords:alariarug resistanceuininefnhefcrt

doubts have been raised about this quinine resistance in Africa. New and interesting data are emergingon the mechanism of quinine reduced susceptibility. In this report, we have reviewed work on the in vivoefficacy and in vitro activity of quinine, and discussed recent data on genetic markers of resistance to thisdrug. Overall, quinine still remains efficacious in Africa, and pfnhe, the sodium hydrogen exchanger, maybe one of the genetic markers underlying quinine in vitro resistance.

© 2011 Elsevier B.V. All rights reserved.

fmdr1icrosatellite

ontents

1. Historical perspective and background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772. Clinical use and pharmacological properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783. How widespread is in vivo quinine resistance? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784. In vitro activity and pharmacokinetics of quinine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785. Genetic markers of resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

5.1. Role of Pfcrt and Pfmdr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795.2. Phnhe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

5.2.1. Pfnhe biochemistry and physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795.2.2. Pfnhe and QN resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

5.3. Pfnhe in association with Pfmdr1 and Pfcrt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81Appendix A. Supplementary data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

. Historical perspective and background

Quinine (QN) (Fig. 1) is the oldest antimalarial agent and haseen used for four centuries for the treatment of malaria. Theecorded historical account of this drug dates from the 1630s, when

∗ Corresponding author. Current address: Departments of Chemistry and Clinicalharmacology, University of Cape Town Rondebosch, 7701 Cape Town, South Africa.el.: +27 21 650 2553; fax: +27 21 650 5195.

E-mail addresses: alex.nzila@uct.ac.za, alexisnzila@yahoo.co.uk (A. Nzila).

166-6851/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.molbiopara.2011.01.012

Jesuits in Peru used the bark of the cinchona tree to treat fevers.However, this antipyretic effect was also known by native localIndians as cinchona tree quina-quina (barks of barks). In the 1630s,Jesuits used quina bark to cure the Countess of Cinchón, wife of theViceroy of Peru, of her fevers, and both the Countess and the Jesuitsthen brought this Peruvian bark to Europe (Spain). In the mid-

1600s, evidence was produced that fevers responsive to cinchonabark were specifically intermittent fevers, characteristic of malaria,making it the first antimalarial agent [1]. In 1820, Pierre-JosephPelletier and Joseph Bienaimé Caventou isolated an alkaloid from

78 J. Okombo et al. / Molecular & Biochemic

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inchona (or quina) bark and named it quinine. The purified com-ound began to be used instead of powdered bark to treat malaria,nd in the mid-1800s, the structure of quinine was elucidated1].

For centuries, quinine has been used to treat malaria, and itemains the drug of choice in the treatment of severe malaria,nd has been chosen as a second line treatment (in combinationith antibiotics) after artemisinin combination therapy (ACT) in

he treatment of uncomplicated malaria [2].Quinine resistance has been reported both in Africa and South

ast Asia [3–7], and this has led to the investigation of thertemisinin derivative artesunate as a possible alternative for thereatment of severe malaria [8–10], and intravenous artesunateas recently been recommended as the drug choice in severealaria [11]. However, this strategy could be compromised with

he emergence of artemisinin resistance [12]. Investigations haveeen carried out to unravel the mechanisms of quinine resistance,owever, important information such as the correlation between

n vivo and in vitro resistance, and the mechanism of resistances still awaiting elucidation. In this review, we present the cur-ent knowledge on the in vivo efficacy and in vitro activity of thisrug, and discuss genetic markers that can be used to monitoresistance.

. Clinical use and pharmacological properties

In the treatment of uncomplicated malaria, QN is administeredrally at a dose of 10 mg salt/kg, 8 hourly, for 7 days. The drug iselatively well absorbed. In severe malaria, QN therapy starts with areloading dose of around 7 mg (salt)/kg infused at a constant rate,y intramuscular (im) or intraperitoneal (ip) route, over 30 min,ollowed by 10 mg/kg over 4 h. Alternatively, 20 mg/kg salt can benfused over 4 h [13]. After preloading, quinine is administered atdose of 10 mg/kg salt every 8 h up to 7 days [13].

At these doses, the maximum concentration (Cmax) of QN of0–46.2 �M (10–15 mg/L) can be reached in vivo. This drug has ahort half-life of around 8–12 h, thus the necessary “3 times peray” dosing. Though the in vivo effective concentration of QN isigh, however the bulk of it is bound to protein (87%), leading tomax of pharmacologically active QN to be in the range of 3.9–6 �M13].

. How widespread is in vivo quinine resistance?

Several reports have indicated the emergence of quinine resis-ance in vivo, however, there is no evidence of widespreadesistance worldwide despite the fact that this drug has been in use

or more than 400 years. For instance, a review of clinical trials of itsntimalarial effect showed that, over a period of 30 years, from the970s up to early 2000, the efficacy of QN has not changed in mostndemic areas, yet during the same period, the efficacy of chloro-uine, pyrimethamine and mefloquine had substantially decreased

al Parasitology 177 (2011) 77–82

[14]. Only recently, in the last 5 years, cases of bona fide in vivo resis-tance have been reported in South Asia [6,7], and this resistance isdefined as the failure to clear parasite following the 7-day QN treat-ment. In Africa, a reduced cure-rate of 80–60% has been reported,indicating the emergence of resistance [3,5,15]. However, detailedobservation of these data indicates that the in vivo resistance inAfrica may not be as high as reported.

According to WHO (WHO/CDS/CSR/DRS/2001.4), antimalarialdrug resistance is defined as the “ability of a parasite strain to sur-vive and/or multiply despite the administration and absorption ofa drug given in doses equal to or higher than those usually recom-mended but within tolerance of the subject”. This definition impliesthat the drug is used at recommended doses and in the right regi-men. As discussed earlier, QN is administered for 7 days, every 8 h,for a total 21 doses, posing a real problem of compliance. In addition,most side effects attributable to quinine are experienced duringthe second half of the treatment course, due to reduced plasma“quinine binding proteins” in already convalescent, asymptomaticpatients [16]. This development of side effects in the absence ofclinical symptoms coupled with the bitter taste of quinine limitadherence to full treatment. In support of this, rates up to 30%non-compliance have been reported [17]. Thus, the full 7-day treat-ment has to be complied with before concluding on in vivo reducedefficacy or resistance.

A detailed observation of recent publications reporting on invivo QN resistance in Africa shows that these studies were pri-marily effectiveness trials [3,5,15]. Therefore, since full treatmentadherence was not established, it is difficult to separate reduceddrug efficacy or resistance due to inadequate drug intake from bonafide resistance. It is interesting to note that in all these reports, theauthors themselves were cautious about the overestimation of fail-ure cases due to poor compliance. In support of this, most studiesthat have attempted to assess QN efficacy by lowering the numberof treatments (so as to reduce the lengthy treatment time), showedsubstantially reduced drug efficacy [18,19]. So, in vivo QN resistancestill needs to be established in Africa, while in South East Asia, in vivoresistance has been established based on efficacy studies, thus thereis bona-fide resistance [6,7].

4. In vitro activity and pharmacokinetics of quinine

In vitro resistance is defined by a right shift of the sigmoidrepresenting the drug response curve (cell growth inhibition orinhibition cell growth as a function of drug concentration), and thisright shift reflects the increase in inhibitory concentration that kills50%, 90% or 99% of parasitaemia (IC50, IC90 or IC99) [20]. However,this increase in IC50 does not necessarily translate to in vivo thera-peutic failure. The correlation between drug resistance in vitro andin vivo leads to establish in vitro cut-off points or thresholds, whichpermit to discriminate drug sensitive and resistant parasite popu-lations. Different cut-off points of 800, 500, or 250 nM have beenproposed for quinine [21,22]. However, in the absence of bona-fidein vivo resistance (at least in Africa), and of well-defined geneticmarkers of drug resistance, these cut-off points remain arbitrary.In the view of obtaining a better insight into the range of in vitroactivity of QN, we have reviewed major works on in vitro QN activ-ity published in the last 30 years. In total, we have reviewed 71publications (40 from Africa and 31 from South East Asia), using atotal of more than 6000 Plasmodium falciparum isolates. Detailedinformation on the selection criteria and sources of parasite are

summarized in Table 1S, Supplementary data in the supplementalmaterial.

Fig. 2 summarises the median or means of the IC50 values overthe period 1980–2010 in Africa and South East Asia. African iso-lates have lower IC50values, ranging from 20 to 600 nM, while in

J. Okombo et al. / Molecular & Biochemic

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1980–2010). Each dot is a geometric or arithmetic mean, or median of 6–295solates, representing a total of more than 6000 isolates.

outh East Asia, IC50 can reach values above 1200 nM (Fig. 2). Thus,arasites from South East Asia are less susceptible to QN, and noturprisingly, this is an area where bona-fide quinine resistance haseen reported [6,7].

. Genetic markers of resistance

.1. Role of Pfcrt and Pfmdr1

Early reports demonstrated the existence of in vitro cross resis-ance between QN and QN structural analogues such as chloroquineCQ), lumefantrine (LM), mefloquine (MFQ) and halofantrine (HLF)23–26], leading to the hypothesis that these drugs may have someommon mechanisms of resistance [27]. This led to the explorationf the role of the transporter protein, pfcrt and pfmdr1 in relationith QN in vitro activity. Pfcrt and Pfmdr1 genes encode a 45 and

62-kDa putative transporters, respectively, and they both localizeo the digestive vacuole membrane. Pfmdr1 consists of two homol-gous halves, each with six predicted transmembrane domains andconserved nucleotide binding domain (ATB binding domain), andfcrt has 10 transmembrane domains, and belongs to the “drugetabolite transporter family”.Conflicting results were observed on the association of a Pfcrt

utation at codon 76 (Pfcrt K76T) and QN reduced susceptibility.orrelations were found in some studies, but not all [28–33], whileransfection studies indicated an increase in QN susceptibility intrains with Pfcrt K76T mutants [34,35]. In South East Asia, QNn vitro activity tend to be inversely related to CQ activity, while,

orldwide, their activities appear to be positively correlated [33].hese conflicting observations explain the multi-factorial nature ofN resistance.

Concerning Pfmdr1, transfection studies showed that poly-orphisms, mainly at codons N1042D, S1034C or D1246Y, were

ssociated with increased resistance to QN [34,36–38], and thisas been supported in field isolates [28,30], though other studieshowed no association [39–41]. Increased Pfmdr1 number reduceshe susceptibility to QN, and to other aryl-amino quinolines suchs mefloquine and lumefantrine [29,39–43]. Thus, Pfmdr1 has aearing on the activity of QN.

Further investigations have shown that other transporters mod-late QN activity, and the most important one is the Na+/H+

xchanger transporter (Pfnhe) [36,44].

al Parasitology 177 (2011) 77–82 79

5.2. Phnhe

5.2.1. Pfnhe biochemistry and physiologyUsing quantitative trait loci (QTL) on the genetic cross of HB3

and Dd2 strains and in vitro activity of QN, Ferdig et al. identifiedcandidate genes on segments of chromosomes (Chr) 13, 7 and 5associated with QN reduced susceptibility. The mapped segmentsof Chr 7 and 5 contain Pfcrt and Pfmdr1, respectively, while thesegment of Chr 13 was narrowed down to Ppfnhe gene, encoding aputative Na+/H+ exchanger [36].

Pfnhe is a 226 kDa protein of 1920 amino acids, and localizedon the parasite plasma membrane. Fig. 3 shows the structureof the protein. It contains 12 transmembrane domains, and 3microsatellite regions, msR1, ms3580 and ms4760. msR1, consist-ing of TCDNNNMPNNNMSNNN, is an asparagine (N) rich sequence,and is repeated 1–3 times. ms3580 is rich in N, isoleucine (I) andhistidine (H), and this microsatellite is known as NIH [36,45,38].

The microsatellite ms4760 is the most variable region ofthe gene. It is produced by the insertion or deletion of 5blocks: DNND (block I), DNNND (block II), NHND (block III),DKNNKND (block IV), DDNNNDNHNDD (block VI). The numberof repeats of each block determines the ms4760 sequence, andso far, over 33 different sequences have been reported (Fig. 3)[28,36,45,38]. The Block II repeats (DNNND) vary between 1 and4, though less than 3% of isolates analysed so far have 4 repeats(6/226).

Several investigations have been dedicated in determining thephysiological role of Pfnhe protein. Anaerobic glycolysis, an impor-tant biochemical pathway that generates ATPs in malaria parasite,is associated with an increase in cytosolic pH as the result of thegeneration of lactic acid. The parasite actively pumps H+ out of thecell through the Pfnhe, thus maintaining the parasite pH at around7.3 [46,47]. However these results have been questioned, on thebasis that the parasite could use V-type H+ ATPase to reduce itsacid burden [48–50]. Thus, role of Pfnhe in the parasite still awaitsfull elucidation [50–52].

5.2.2. Pfnhe and QN resistanceFerdig et al. [36] demonstrated that, using 71 P. falciparum

strains from several parts of the endemic area, parasites withmore than 2 DNNND repeats had a significantly reduced QN activ-ity compared with those with only 1 copy number [36]. Thiswork provided the first evidence of the involvement of DNNNDin reduced QN susceptibility. Since this seminal work, 5 otherpublications have addressed the relationship between ms4760variation and QN in vitro activity. Fig. 4 summarizes data of 5for the 6 publications. These studies (except two [28,53]), con-firmed that the increase in DNNND repeats, from 1 to 2 or more,was associated with a significant decrease in QN activity. Detailedobservation of the data revealed other features. In two studies,the presence of 1 versus 3 and 2 versus 3 repeats was asso-ciated with the restoration of QN susceptibility (Fig. 4) [36,38].The same observation was made in Andriantsoanirina et al. andBaliraine et al. [28,53], though the difference was not signifi-cant. However, in two other studies, the opposite was observed,with a trend towards decreased QN activity in parasites with 3repeats when compared with parasites with 1 or 2 repeats (Fig. 4)[45,54].

The analysis of DDNNNDNHNDD did not yield clear-cut result.Indeed, two studies showed a decrease in QN activity when 1repeats was compared to 2 or 3, and 2 other studies showed an

were less susceptible to QN than those with 1 repeat, and thosewith 3 repeats were the most susceptible, however these differ-ences were not significant [53]. Using the published work, we

80 J. Okombo et al. / Molecular & Biochemical Parasitology 177 (2011) 77–82

94, 95

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Fig. 3. (a) Diagram of Pfnhe-1 gene. Asterisks (*) indicate polymorphic codons (8

ave also investigated the impact of the association repeats inNNND/DDNNNDNHNDD with QN activity, but no relationshipas found.

From the above observations, DNNND modulates QN suscepti-ility, and repeat variation from 1 to 2 or more is strongly associated

ig. 4. Relationship between quinine (QN) in vitro activity and repeat variation of DNNNDhat kills 50%/90% of parasitaemia (IC50), and values in parentheses are isolate numbers44], values are represented as IC90, NHNDNHNNDDD repeat was not associated with QNs IC50.

0 and 1437). (b) Some of the reported ms4760 microsatellite sequence profiles.

with decreased QN susceptibility. However, more data are neededto establish the contribution of DDNNNDNHNDD repeats in themodulation of QN activity.

A recent analysis using allelic exchange (of truncated Pfnhegene) and transfection in parasite with different genetic back-

and NHNDNHNDDD. Y-axis and labels on each column represent QN concentration. Stars (*) indicate differences that are statistically significant (p < 0.05). In Study 1activity and data were not available. In the other 4 studies, values are represented

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round (CQ sensitive and CQ resistance) has confirmed the rolef Pfnhe in conferring in QN resistance, however this was depen-ant upon strains. Indeed CQ truncated Pfnhe gene in CQ sensitivetrains did not alter their susceptibility to QN, while the oppo-ite was observed with CQ resistant strain [48], a clear indicationhat Pfcrt (and Pfmdr1, as discussed earlier) could be contributingactors.

.3. Pfnhe in association with Pfmdr1 and Pfcrt

As discussed earlier, Pfcrt and Pfmdr1 can modulate QN in vitroctivity. Thus, it is important to establish the impact of these mark-rs in relation with Pfnhe and QN in vitro activity. However, thisas not been fully explored yet. Indeed, so far, 2 studies [38,53]ave investigated Pfcrt and Pfmdr1 genotypes in relation to Pfnheolymorphisms and QN activity. This first study has shown thatarasites with 2 DNNND repeats with a background of wild-typefmdr1-86 had QN IC50 of 92 nM, while this value rose almost-fold in Pfmdr1-86 mutant parasites (QN IC50 of 249 nM). In con-ract, no difference was found with regard to pfcrt-76 (211 nMersus 234 nM) [38]. In the second study, pfmdr1 mutations atodons 86 and 1246 were associated with an increase in QNC50 in parasite harbouring 2 DNNND repeat (median 151 nM),ompared to pfmdr1 wild type with 2 repeats (median 111 nM),owever the difference was not significant [53]. These prelimi-ary results tend to indicate that the association of DNNND repeatsnd pfmdr1 genotype may be a better marker for QN in vitroeduced activity. However, more studies are needed to confirm thisbservation.

. Concluding remarks

QN is the oldest antimalarial agent, however, it is still activelysed and it remains the drug of choice in the treatment of severealaria, and in some instances, of uncomplicated malaria. In South

ast Asia, the emergence of in vivo resistance is well established,nd some reports indicate that the same may be happening infrica. However, a detailed observation of clinical studies indicates

hat in vivo resistance still needs to be established in Africa. Indeed,he reported in vivo QN resistance in Africa may be due to poorompliance of the 7-day QN treatment.

However, the emergence of parasites with decreased suscep-ibility in vitro is now common in Africa, and most of them have

copies of DNNND repeats in the ms4067 microsatellite of thefnhe gene, and the presence of Pfmdr1 may contribute in furtherecreasing QN activity. Though QN is still efficacious in Africa, it

s on the background of in vitro reduced susceptibility parasiteshat in vivo QN resistance will emerge. Thus, polymorphism of

icrosatellite 4760 (in combination with Pfmdr1 mutations) coulde used to monitor the selection and spread of QN reduced in vitroctivity.

cknowledgments

We thank the director of the Kenya Medical Research Instituteor permission to publish this manuscript. This study was supportedy European & Developing Countries Clinical Trials PartnershipEDCPT), through the Award of the best African scientist of the year009 (to AN).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.molbiopara.2011.01.012.

[

al Parasitology 177 (2011) 77–82 81

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