Molecular data on

Plasmodium falciparumchloroquine and

antifolate resistance: a

public health tool

The incidence of morbidity and mortalityowing to falciparum malaria is an issue ofglobal concern. There is a pressing need toidentify appropriate and effective controlmeasures tailored to the needs of definedcommunities.

In areas where malaria transmission isintense, chemotherapy is the most practicalapproach for control. Transmissionblockers, which include insecticides andbednets, can be effective, particularly wheretransmission is less intense [1], but theseblockers have had mixed support, partlydue to fear of toxicity to the environmentand the need for additional financialcommitments by policy makers for bednetsimpregnation. Considerable effort hasbeen focused on vaccine development, butnone has been deployed yet [2].

Currently, the three main categories ofantimalarial drugs are: (1) 4-amino-quinolines and amino alcohols, which acton hemoglobin degradation and parasitefood vacuoles (e.g. chloroquine andquinine); (2) sesquiterpenes (artemisininand its derivatives); and (3) the antifolates,which are dihydrofolate reductase (DHFR)(e.g. pyrimethamine, cycloguanil and

chlorcycloguanil) and dihydropteroatesynthase (DHPS) inhibitors (e.g. sulfonamides and sulfones).Plasmodium falciparum populationsresistant to quinolines and antifolates arenow widespread throughout most malaria-endemic areas, and reports of artemisininresistance are emerging, posing challengesfor replacement with affordable andefficacious drugs [3–6]. As the mobility of apopulation increases, the risk ofintroducing resistant species into newareas grows proportionately. These factorsunderlie the necessity to increasesurveillance methods for qualitatively andquantitatively assessing resistant andsusceptible parasite populations.

Measuring drug efficacy

The WHO has outlined three ways ofmeasuring drug efficacy: (1) the clinicalresponses of patients to drug treatment (Box 1) as a standard; (2) the sensitivity ofparasites to drugs in vitro or (3) acceptedmolecular markers as complementarytools for monitoring drug resistance. Forexample, the correlation between specificmutations in the genes that encode targetsof the antifolate drugs and drug resistance,such as DHPS (targeted by sulfa drugs) andDHFR (targeted by DHFR inhibitors)genes, are well established. The correlationof particular mutations in the P. falciparumchloroquine resistance transporter gene(Pfcrt) and the P. falciparum multidrugresistance gene analog (Pfmdr1) withchloroquine resistance has also been

observed [7–9]. The procedures to determinethese drug-related parasite genotypes aresimple and well established, and are alreadyin use in many laboratories in sub-SaharanAfrica, Asia and South and Central America.These molecular data are, potentially,powerful public health tools for surveillanceof drug resistance. However, it is not yetclear how to relate the molecular data onparasite genotypes to clinical outcomes,especially in areas where a majority of thepopulation is semi-immune. For example,it has been difficult to reach a consensuson the relationship between double, tripleand quadruple mutants in DHFR andDHPS, and clinical response to antifolatetreatment. Lack of concordance betweenlaboratory clones and field trials could alsopose a problem. Despite these difficulties,the potential use of molecular data inserving as early warning signals andsurveillance tools is clear. Carefulcorrelations of clinical and molecular dataare beginning to be made [8,10,11], but theirapplication needs to be widened considerably.

Collating data

The first step in relating the clinical,parasitological and molecular data sets isthe collection and organization of theinformation available. To begin this process,we have compiled a brief summary of the published data on the moleculardefinitions of drug resistant Pfalciparum inTables 1–3. These data provide informationon the basic characteristics of parasitesthat define resistance or susceptibility to

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ParaSite – Genome Analysis

Parasitological response

S or S/R1: This is an extended test. Parasitesare defined as ‘S’ if no asexual parasites arefound by Day 6 and parasites do not reappearby Day 28. In a seven-day field test, theinfection could either be S or resistant atR1(S/R1) level if no asexual parasites arepresent on Day 7 after treatment. An S or R1response cannot be distinguished for theseven-day test because the difference betweenthe extended test and the seven day testdepends on the presence or absence ofrecrudescence between Day 8 and Day 28.RI: This is an extended test. Parasites areresistant at the R1 level if asexual parasitesdisappear by Day 7 after treatment but returnwithin 28 days and re-infection has beenexcluded.Seven-day field test: Parasites are resistant atthe R1 level if asexual parasites disappear formore than two consecutive days but theyreturn and are present on Day 7 after treatment.

RII: Parasites are resistant at RII level if asexualparasitemia does not clear but it is reduced to25% or less of the original pre-test level duringthe first 48 hours of treatment.RIII: Parasites are resistant at RIII level ifasexual parasitemia is reduced by <75% duringthe first 48 hours or if it continues to risefollowing treatment.

Clinical response

Adequate clinical response

This describes patients who have completedthe 14-day follow-up and meet either of twocriteria:(1) Negative smear on Day 14, irrespective ofaxillary temperature, without previouslymeeting the criteria for early treatment failure(ETF) or late treatment failure (LTF).(2) Axillary temperature of <37.5°C, irrespectiveof the presence of parasitemia, withoutpreviously meeting the criteria for ETF or LTF.

Early treatment failure

Defined by one of the following four criteria:(1) Development of danger signals or severemalaria on Day 1, 2 or 3, in the presence ofparasitemia.(2) Axillary temperature of ≥37.5°C in thepresence of parasitemia on Day 2, withparasitemia less than that counted on Day 0.(3) Axillary temperature of ≥37.5°C on Day 3 inthe presence of parasitemia.(4) Parasitemia on Day 3 is ≥25% than thatcounted on Day 0.

Late treatment failure

Defined by either of the two criteria:(1) Development of danger signs or severemalaria in the presence of parasitemia on anyday from Day 4–14, without previously meetingany of the criteria of ETF.(2) Axillary temperature of ≥37.5°C in thepresence of parasitemia on any day from Day 4–14, without previously meeting any ofthe criteria of ETF.

Box 1. WHO classification of parasitological and clinical responses to antimalarial drugs

chloroquine, DHFR inhibitors and DHPSinhibitors. Each entry shows the amino acidchanges that have been correlated withresistance to antifolates or to chloroquinein isolates globally [12–19].

To enlarge the database and to keep itupdated, we are proposing a web-baseddata bank into which the genotypes of wellcharacterized isolates of chloroquine andantifolate resistant P. falciparum can besubmitted. These data would be collatedand categorized into regional and/orgeographical forms for easy reference withcitations of the original papers or authors incases where the findings are not published.The various genotypes could then beevaluated to determine how well they canbe classified under current WHO definitionsfor adequate clinical response (ACR), early

treatment failure (ETF) and late treatmentfailure (LTF). This information could thenserve as a public health tool for determiningthe need for revising antimalarial drug use.It will also provide an easy assessment onreversions of sensitivity to drugs that hadpreviously been found to lose their efficacyin a particular region.

The entire data set available (as of May 2001) is posted at the websitehttp://depts.washington.edu/genetics/sibleylab/index.htm. A summary of thefrequency distribution of the mutations(Table 4) shows that DHFR drug-resistantphenotypes appear to be initiated with aS108N change that is followed bysubsequent changes at positions N51I and C59R in Asia, Africa and Middle East,as opposed to N51I and C50R in

South America. In both Asia and SouthAmerica, addition of an I164L change isstrongly correlated with clinicalresistance to antifolate drugs [12,13].ForDHPS, although the trend is not asobvious, it is clear that resistance isinitiated by mutations at position 437,with higher levels of resistance conferredfollowing further mutations at 436, 540and 613 in Asia and Africa, by contrastwith positions 540 and 581 in SouthAmerica. Therefore, the phenotype of drugresistance appears to be similar in Asia,Africa and Middle East, with SouthAmerica having its own unique pattern.

Of course, such a database is rapidlyoutdated. We hope that scientists workingon various aspects of drug resistance willfind a way to keep the database current.Even more important, exchanges of opinionand data will allow us all to define usefulrelationships of parasite genotypes withclinical responses (ACR, ETF and LTF)and parasitological responses (RI, RII andRIII) to both antifolates and quinolines.Moreover, surveillance of the changes inprevalence of these alleles within apopulation could signal the need foralternate choice of drugs, providingvaluable tools for public health decisions.Perhaps a forum, as has been initiated bythe UNDP/World Bank/WHO SpecialProgramme for Research and Training inTropical Disease (TDR), including scientistsinvolved in this area of research, couldspeed greatly the progress of this effort.

Acknowledgements

The authors are grateful to Pascal Ringwald for critically reading themanuscript and for his contributions.

References

1 Baird, K.J. (2000) Resurgent malaria at themillennium: control strategies in crisis. Drugs 59,719–743

2 Miller, L.H. and Hoffman, S.L. (1998) Researchtowards vaccines against malaria. Nat. Med. 4,520–524

3 Foote, S.J. and Cowman, A.F. (1994) The mode of action and mechanism of

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185Forum

Table 2. Allelic changes associated with antifolate resistance

Dihydrofolate reductase residues Dihydropteroate synthase

residues

Position number 16 50 51 59 108 140 164 436 437 540 581 613Susceptible A C N C S V I S or A A K or L A AResistant V R or I I R N or T L L F G E G S or T

Table 3. Dihydrofolate reductase combinations observed in antifolate resistant malariaa

Plasmodium falciparum Dihydrofolate reductase Reference strain designation Refs

Wild type S108 3D7 [22]Single mutant S108N HB3 [23]Double mutants C59R + S108N K1 [24]

N51I + S108N 7G8 [25]A16V +S108T FCR3 [26]

Triple mutants N51I + C59R + S108N W2 [27]Quadruple mutants N51I + C59R + S108N + I164L V1S [28]

aThese variants could combine with different mutants of dihydropteroate synthase at positions 437, 540 , 436 and 613.

Table 4. Global variations in the distribution of dihydrofolate reductase and dihydropteroate synthase alleles associated with antifolate

drug resistance in Plasmodium falciparuma

Dihydrofolate reductase Dihydropteroate synthase

Amino acid position 16 50 51 59 108 140 164 436 437 540 581 613South America 0 7 9 11 6 0 1 1 6 6 6 0Southeast Asia 2 0 20 43 43 2 6 4 9 0 7 2Africa 1 0 22 27 34 0 0 10 17 7 0 1Middle East 0 0 1 4 5 0 0 0 0 0 0 0

aEach data point is a literature report of a parasite isolate found within a particular locality. These data reflect reports from up to May 2001 and references can be found at thewebsite http://depts.washington.edu/genetics/sibleylab/index.htm

Table 1. Allelic changes associated with chloroquine resistancea

Amino acid position

Pfcrt Pfmdr1

72 74 75 76b 97 220 271 326 356 371 86c

Susceptible S M N K H A E N I R NResistant C I E T Q S Q S or D T or L I or T Y

aRecent studies show that factors other than allelic changes in the gene encoding Pfcrt are paramount when allparasites carry the resistant allele (T76) [20,21]. bThis mutation has the most consistent correlation with chloroquine resistance from clones and field isolates ofPlasmodium falciparum.cMutation at position 86 appears not to confer chloroquine resistance, which is independent of the resistant alleleof Pfcrt.

resistance to antimalarial drugs. Acta Trop. 56, 157–171

4 Phillips, R.S. (2001) Current strategies of malariaand potential for control. Clin. Microbiol. Rev. 14,208–226

5 White, N.J. (1992) Antimalarial drug resistance:the pace quickens. J. Antimicrob. Chemother. 30,S71–S78

6 Das, B. et al. (2000) Emerging resistance ofP falciparum to artemisinine and relatedcompounds. J. Assoc. Physicians India 48, 443–444

7 Fidock, D.A. et al. (2000) Mutations in theP. falciparum digestive vacoule transmembraneprotein PfCRT and evidence for their role inchloroquine resistance. Mol. Cell 6, 861–871

8 Djimde, A. et al. (2001) A molecular marker forchloroquine-resistant falciparum malaria. New Engl. J. Med. 344, 257–263

9 Warhurst, D.C. (2001) A molecular marker forchloroquine-resistant falciparum malaria. New Engl. J. Med. 344, 299–301

10 Djimde, A. et al. (2001) Application of a molecularmarker for surveillance of chloroquine-resistantfalciparum malaria. Lancet 358, 890–891

11 Kublin, J.G. et al. (2002) Molecular markers fortreatment failure of sulfadoxine-pyrimethamineand chlorproguanil–dapsone for falciparummalaria and a model for practical application inAfrica J. Infect. Dis. 185, 380–388

12 Cowman, A.F. et al. (1988) Amino acid changeslinked to pyrimethamine resistance in thedihydrofolate reductase-thymidylate synthasegene of Plasmodium falciparum. Proc. Natl. Acad.Sci. U. S. A. 85, 9109–9113

13 Vasconcelos, K.F. et al. (2000) Mutations inPlasmodium falciparum dihydrofolate reductaseand dihydropteroate synthase of isolates from theAmazon region of Brazil. Mem. Inst. OswaldoCruz 95, 721–728

14 Peterson, D.S. et al. (1990) Molecular basis ofdifferential resistance to cycloguanil andpyrimethamine in Plasmodium falciparummalaria. Proc. Natl. Acad. Sci. U. S. A. 87,3018–3022

15 Nzila, A.M. et al. (2000) Towards an understandingof the mechanism of pyrimethamine-sulfadoxineresistance in Plasmodium falciparum: genotypingof dihydrofolate reductase and dihydropteroatesynthase of Kenyan parasites. Antimicrob. AgentsChemother. 44, 991–996

16 Wang, P. et al. (1997) Resistance to antifolates inPlasmodium falciparum monitored by sequenceanalysis of dihydropteroate synthetase anddihydrofolate reductase alleles in a large numberof field samples of diverse origins. Mol. Biochem.Parasitol. 89, 161–177

17 Basco, L.K. and Ringwald, P. (2001) Analysis ofthe key pfcrt point mutation and in vitro and in vivo response to chloroquine in Yaounde,Cameroon. J. Infect. Dis. 183, 1828–1831

18 Dorsey, G. et al. (2001) Polymorphisms in thePlasmodium falciparum pfcrt and pfmdr-1 genesand clinical response to chloroquine in Uganda.J. Infect. Dis. 183, 1417–1420

19 Plowe, C.V. et al. (1997) Mutations in Plasmodiumfalciparum dihydrofolate reductase anddihydropteroate synthase and epidemiologicpatterns of pyrimethamine–sulfadoxine use andresistance. J. Infect. Dis. 176, 1590–1596

20 Mayor, A.G. et al. (2001) Prevalence of the K76Tmutation in the putative Plasmodium falciparumchloroquine resistance transporter (pfcrt) geneand its relation to chloroquine resistance inMozambique. J. Infect. Dis. 183, 1413–1416

21 Babiker, H.A. et al. (2001) High-level chloroquineresistance in Sudan isolates of P. falciparum isassociated with mutations in the chloroquineresistance transporter gene pfcrt and multidrug

resistance gene pfmdr-1. J. Infect. Dis. 183,1535–1538

22 Walliker, D. et al. (1987) Genetic analysis of thehuman malaria parasite Plasmodiumfalciparum. Science 236, 1661–1666

23 Bhasin, V.K. and Trager, W. (1984) Gametocyte-forming and non-gametocyte-forming clones ofPlasmodium falciparum. Am. J. Trop. Med. Hyg.33, 534–537

24 Thaithong, S. and Beale, G.H. (1981) Resistanceof ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitrotests. Trans. R. Soc. Trop. Med. Hyg. 75, 271–273

25 Zolg, J.W. et al. (1989) Point mutations in thedihydrofolate reductase-thymidylate synthasegene as the molecular basis for pyrimethamineresistance in Plasmodium falciparum. Mol.Biochem. Parasitol. 36, 253–262

26 Trager, W. et al. (1981) Clones of the malariaparasite Plasmodium falciparum obtained bymicroscopic selection: their characterization withregard to knobs, chloroquine sensitivity, andformation of gametocytes. Proc. Natl. Acad. Sci.U. S. A. 78, 6527–6530

27 Oduola, A.M. et al. (1988) Plasmodiumfalciparum: induction of resistance to mefloquinein cloned strains by continuous drug exposurein vitro. Exp. Parasitol. 67, 354–360

28 Udeinya, I.J. et al. (1983) Plasmodium falciparum:effect of time in continuous culture on binding tohuman endothelial cells and amelanoticmelanoma cells. Exp. Parasitol. 56, 207–214

Isaac Quaye

Carol Hopkins Sibley*

Dept of Genome Sciences, University ofWashington, Seattle, WA 98195-7730, USA.*e-mail: sibley@genetics.washington.edu

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186 Forum

ParaSite

Eaten alive on the Net

General parasitology

(bionet-parasitology@net.bio.net)

Confusing…This list has baffled bystanders. Itacquired a moderator and then went dead[ParaSite (2001) Trends Parasitol. 17, 299].Furthermore, in December 2001, anannouncement appeared that it was to beremoved from Usenet altogether. ‘Yet’,said James Mahaffy (Dordt College, IA,USA), ‘I find some posts on here. Does thismean that bionet is still alive? I hope so.’This was followed by: ‘The moderator is asconfused as everyone else. As long asmessages appear, they will be posted. YourHumble Moderator.’Then some life creptback. A variety of questions were asked.‘Phil’, a vet (Swiss Federal Institute ofTechnology) enquired about treating miceinfected with Chilomastix bettencourti(a lumen-dwelling flagellate) with

metronidazole: could a high enoughconcentration be obtained in the gutlumen? An anonymous correspondentwanted to know how to increase yields ofdigested genomic DNA and, Faith Russell,who has adopted a son from Russia,wanted to know how to deal with his many infections (Helicobacter pylori,Giardia, Blastocystis hominis andDientamoeba fragilis). ‘But answer camethere none.’

Toxoplasma gondii and human behaviour

Then in January, an excited V.Z. Nuri,stimulated by reading the book Parasite Rex by Carl Zimmer (http://www.carlzimmer.com/parasite_1.html), posted along, enthusiastic discourse on howToxoplasma gondii might be manipulatinghuman behaviour. The parasite has beenshown to alter the behaviour of rats, sothat they are less fearful of cats, itsdefinitive host, their reaction time isslower, making them easier prey, and

they ‘even “seek out” cat smells’. [SeeBerdoy, M. et al. (2000) Fatal attraction inToxoplasma-infected rats: a case ofparasite manipulation of its mammalianhost. Proc. R. Soc. London Ser. B 267,1492–1594]. Another paper suggests thatbehavioural changes of infected micecould be due to nonspecific by-products ofinfection, rather than specific manipulationby the parasite [Hrda, S. et al. (2000)Transient nature of Toxoplasma gondii-induced behavioral changes in mice.J. Parasitol. 86, 657–663.] Work byJaroslav Flegr was quoted as findingpsychological effects in people infected withT. gondii, women becoming more sociable(outgoing and warm-hearted) and menbecoming less moral (more jealous andsuspicious) [see Flegr, J. et al., (1996)Induction of changes in human behaviour by the parasitic protozoanToxoplasma gondii. Parasitology 113,49–54; Flegr, J. et al. (2000) Correlation ofduration of latent Toxoplasma gondii