Transactions of the Royal Society of Tropical Medicine and Hygiene (2006) 100, 176—183

Increased density but not prevalence ofgametocytes following drug treatment ofPlasmodium falciparum

Eltayeb Ali a, Margaret J. Mackinnonb, Abdel-Muhsin A. Abdel-Muhsinc,Salah Ahmedd, David Wallikerb, Hamza A. Babikerb,e,∗

a Sudan Atomic Energy Commission, Khartoum, Sudanb School of Biological Sciences, University of Edinburgh, Edinburgh, UKc

Tropical Medicine Research Institute, National Institute for Research, Khartoum, Sudand Department of Biochemistry, Faculty of Medicine, University of Khartoum, Khartoum, Sudane Department of Biochemistry, Faculty of Medicine, Sultan Qaboos University, Oman

Received 10 December 2004; received in revised form 26 April 2005; accepted 29 April 2005Available online 27 October 2005

KEYWORDSPlasmodium falciparum;Chloroquine;Sulfadoxine—pyrimethamine;Drug resistance;Gametocytogenesis;RT-PCR;Sudan

Summary We monitored post-treatment Plasmodium falciparum among patientstreated with chloroquine (CQ) and sulfadoxine—pyrimethamine (SP; Fansidar®) in avillage in eastern Sudan. Parasites were examined on day 0 (pre-treatment), day 7,day 14 and day 21 (post-treatment) during the transmission season. A further samplewas taken 2 months later (day 80) at the start of the dry season. Asexual forms andgametocytes were detected by microscopy, and reverse transcriptase polymerasechain reaction (RT-PCR) was used to detect expression of gametocyte-specific pro-teins pfs25 and pfg377. Gametocyte carriage, as revealed by microscopy, increasedsignificantly following CQ and SP treatment, reaching a maximum between days 7and 14. When measured by RT-PCR, however, there was no significant difference ingametocyte rate between day 0 and days 7 or 14. RT-PCR gametocyte rates droppeddramatically by day 80 post treatment but were still 33% and 8% in the CQ- and SP-treated group at this time. Alleles associated with drug resistance of P. falciparumto chloroquine (the chloroquine resistance transporter, pfcrt, and multidrug resis-tance, pfmdr1) and to pyrimethamine (dihydrofolate reductase, dhfr) were seen ata high frequency at the beginning of treatment and increased further through timefollowing both drug treatments. Infections with drug-resistant parasites tended tohave higher gametocyte prevalence than drug-sensitive infections.© 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd.All rights reserved.

∗ Corresponding author. Present address: Department of Biochemistry, Faculty of Medicine, Sultan Qaboos University, Oman.Tel.: +968 2441 3333; fax: +968 2441 3880.

E-mail address: h.babiker@ed.ac.uk (H.A. Babiker).

0035-9203/$ — see front matter © 2005 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.trstmh.2005.04.021

Increased density of gametocytes following malaria treatment 177

1. Introduction

Drug resistance is a growing obstacle to malariamanagement and control and has a devastatingimpact on communities with poor public healthservices. Many environmental and biological fac-tors are known to influence the spread of drug-resistant malaria parasites, but drug pressure isthe primary cause of the emergence and spreadof adaptive mutant forms that survive chemother-apy (Mackinnon and Hastings, 1998). In addition,epidemiological surveys have suggested that drugpressure following use of antimalarials that haveno immediate gametocytocidal action can alsoenhance gametocyte production and transmissionof Plasmodium falciparum (Handunnetti et al.,1996; Hogh et al., 1998; Robert et al., 2000). How-ever, it is not clear whether the high gametocyteprevalence seen following antimalarials in P. falci-parum infections is due to induction of gametocyto-genesis by the drug or simply due to post-treatmentmaturation and survival of already existing game-tocytes. An association between antimalarial druguse and high gametocytaemia has been demon-strated in controlled Plasmodium chabaudi infec-tttoSrter1

suesRm1t(uataaltetc2

In this study we have monitored the prevalenceand density of pre- and post-treatment gametocy-taemias following drug treatment of acute P. falci-parum infections in a village in eastern Sudan. Themain aim of this work was to determine whetherchanges in gametocytaemia were due to induc-tion of gametocytogenesis or to release of pre-existing gametocytes from deep tissues into theperipheral circulation. We used reverse transcrip-tase polymerase chain reaction (RT-PCR) to detectRNA of gametocyte-specific protein genes (Babikeret al., 1999; Menegon et al., 2000) to reveal game-tocytes that exist at submicroscopic levels, andthen examined their dynamics following treatmentduring the dry transmission-free season of easternSudan. In addition, we typed parasites for genesdetermining resistance to CQ and pyrimethamine toexamine whether drug resistance influences game-tocyte prevalence. We discuss the implications ofthe results on the spread of drug resistance in thecontext of the marked seasonal transmission pat-tern in this area of Sudan.

2. Materials and methods

2

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ions in mice (Buckling et al., 1997). In additiono the effects of drugs on gametocyte produc-ion in general, there is the question of the rolef resistant parasites in maintaining transmission.ome in vivo studies have indicated that drug-esistant malaria parasites produce more game-ocytes and can be more infectious (Sutherlandt al., 2002), although no association with drugesistance was noted in vitro (Buckling et al.,999).

In central and eastern Sudan, malaria transmis-ion is distinctly seasonal and antimalarial drugsage prevails in the short rainy season. How-ver, during the lengthy dry and malaria-free sea-on in this area, antimalarial drug usage is scanty.esistance to chloroquine (CQ) (the first-line anti-alarial drug) is well established (Bayoumi et al.,

989); clinical failure of the drug fluctuates withhe amount of annual rains and can reach up to 85%Babiker et al., 2005). This has led to a switch to these of sulfadoxine—pyrimethamine (SP; Fansidar®)s an alternative antimalarial. Subsequently, resis-ance to pyrimethamine was reported in easternnd central Sudan (Babiker et al., 1991; Ibrahim etl., 1991). Clinical failure of Fansidar® in Sudan isimited; for example, clinical failure of Fansidar® inhe Khartoum area is approximately 2.7% (Alifrangist al., 2003). However, the frequency of resis-ance to pyrimethamine has increased signifi-antly in eastern Sudan (Abdel-Muhsin et al.,004).

.1. Study areas

he study was carried out in Asar village, Gedareftate, eastern Sudan, where malaria transmissions short and distinctly seasonal following thennual rains (July to November), reaching a peakn October. The number of malaria cases dropsubstantially by January. Entomological surveysave shown no evidence of transmission during theong dry season (Hamad et al., 2002). Plasmodiumalciparum is the predominant malaria parasite,ccounting for more than 90% of all malarianfections in this area. Anopheles arabiensis ishe main mosquito vector. The entomologicalnoculation rate is less than one bite per personuring the transmission season (Babiker et al.,997).

.2. Patients

uring October 1999, 106 patients with acute P.alciparum infections were recruited and ran-omly allocated to either CQ or SP treatment.ixty-one patients, aged 6—50 years (average7.5 ± 1.33 years), with a mean parasite density of0 110.57 ± 4933 parasites/�l blood were given CQ25 mg/kg over 3 d). Forty-five patients, aged 6—67ears (average 20.53 ± 1.93 years), with a meanarasitaemia of 6024.67 ± 2903.51 parasites/�l

178 E. Ali et al.

blood were given SP (25 mg/kg sulfadoxine and0.25 mg/kg pyrimethamine in a single dose).Finger-prick blood samples were taken on day0 (pre-treatment), day 7, day 14 and day 21(post-treatment). A further sample was taken 2months later (day 80). At the same time, thickand thin blood films were prepared and examinedfor asexual parasites and gametocytes. A film wasdeclared negative after examination of 100 thickfields. The blood samples were processed andstored at −70 ◦C for RNA isolation (Babiker et al.,1999). At the same time, 20 �l blood was spottedonto filter paper for DNA isolation.

The study aims and protocol were explainedto all patients, and blood samples were collectedwith the informed consent of all patients and theguardians of children involved. The study was givenethical clearance by the Ministry of Health, Sudan.

2.3. RNA extraction and detection ofsubmicroscopic gametocytes using RT-PCR

RT-PCR was used to amplify RNA of the gametocyte-specific protein genes, pfs25 and pfg377 to detectgametocytes in blood samples with low and

sequence of allele pfmdr1-86N but not pfmdr1-86Y.

2.4.2. Pyrimethamine resistance gene dhfrDihydrofolate reductase dhfr alleles at position108 were analysed using allele-specific oligonu-cleotide probes specific for alleles dhfr-108S, dhfr-108N and dhfr-108T (Adbel-Muhsin et al., 2002).PCR fragments of dhfr containing codon 108 wereamplified using primers described elsewhere (Ploweet al., 1995), blotted onto a nylon membraneand hybridised with labelled allele-specific probes(Adbel-Muhsin et al., 2002).

2.5. Statistical analysis

The overall objectives of the statistical analysiswere to examine the effect of antimalarial drugson gametocyte prevalence and on the frequency ofresistant parasites during the weeks following drugtreatment. Logistic regression analysis was carriedout to determine the effects of day post treat-ment, drug (CQ or SP) and resistance genotype onasexual and gametocyte prevalence (binomial vari-ables). Age and sex of the host were also fitted inteoicou(

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subpatent parasitaemias (Babiker et al., 1999;Menegon et al., 2000). RT-PCR was carried out forboth genes as there is slight variation in the sen-sitivity of detection of the two genes. Total RNAwas first isolated using a High Pure RNA Isolation kit(Roche, Lewes, UK). Reverse transcription and sub-sequent amplification of cDNA were carried out in asingle tube as previously described (Babiker et al.,1999; Menegon et al., 2000). Gametocyte-infectedblood obtained from an in vitro culture of the P. fal-ciparum 3D7 clone was used as a positive control.Conventional PCR was run using the same RT-PCRpre-mix to exclude possible contamination of RNAwith co-extracted DNA.

2.4. DNA extraction and PCR analysis ofdrug resistance genes

2.4.1. CQ resistance genes, pfcrt and pfmdr1Plasmodium falciparum DNA was prepared from fil-ter papers as described by Plowe et al. (1995). DNAwas amplified using primers flanking residue 76 ofthe pfcrt gene in two rounds of PCR. The 145 bpnested PCR product was then digested with restric-tion enzyme ApoI that cuts pfcrt-76K but not pfcrt-76T (Djimde et al., 2001).

Detection of pfmdr1 alleles was carried out asdescribed by Duraisingh et al. (2000). Following tworounds of PCR, pfmdr1 alleles at codon 86 wereidentified by ApoI digestion, which cuts the coding

he model in preliminary analyses, with a randomffect for person to allow for repeated measuresn the same individual, but these terms were notncluded in the final model as they had no signifi-ant effects. Similar analyses were also carried outn log-transformed gametocyte density (a contin-ous variable) using multi-way analysis of varianceANOVA).

Data on the frequency of resistance alleles werenalysed as a binary trait using logistic regressiontting the effects of day, which was fitted eithers a categorical variable or as a linear covariate.eparate analyses were performed for each drugreatment group first, and then on all data com-ined.

Owing to drug failure in some cases, both drugsere taken by the patient: in these cases, dataere excluded from the analyses.

. Results

.1. Parasitaemia following treatment withQ and SP

f 61 P. falciparum infections treated withQ, approximately 20% showed parasitaemiaetectable by microscopy on days 7, 14, 21 and0 (Figure 1a). However, using PCR, a significantlyigher proportion of patients were found to be

Increased density of gametocytes following malaria treatment 179

Figure 1 (a, b) Prevalence of asexual parasites in Plasmodium falciparum infections as detected by microscopy (BF)and PCR following chloroquine (a) and sulfadoxine—pyrimethamine (b) treatment. (c, d) Prevalence of gametocytes asdetected by microscopy (BF) and RT-PCR, and geometric mean gametocyte density, following treatment with chloro-quine (c) and sulfadoxine—pyrimethamine (d).

parasitaemic until day 21 (P < 0.001). By day 80there was a significant drop in parasite preva-lence when measured by PCR (P < 0.001; Figure 1a),although 33% of patients remained positive at thisstage.

Higher sensitivity to SP compared with CQ wasseen among 45 P. falciparum patients treated withSP (Figure 1b). Less than 8% of patients had par-asitaemia detectable by microscopy post treat-ment. However, similar to CQ treatment, mostpatients had PCR-detectable parasitaemias untilday 21, after which parasites were cleared frommost patients.

3.2. Gametocyte prevalence amongpatients treated with CQ and SP

Using microscopy, a significantly higher gametocyteprevalence was detected on days 7 and 14 amongthe 61 P. falciparum patients treated with CQcompared with pre treatment on day 0 (P < 0.001)(Figure 1c). A similar pattern was seen among the45 patients treated with SP (P < 0.05) (Figure 1d).Only minor changes between days 7 and 14 wereobserved, but after that, gametocyte prevalencedP

l

treated with CQ and SP (Figure 1c and d). There wasno significant change in gametocyte rate (game-tocyte carriers) from days 0 to 21. However, itdropped significantly between days 21 and 80 forboth drugs (P < 0.001 for the CQ group; P > 0.10 forSP; P < 0.05 combined) (Figure 1c and d). By day 80,11% and 5% of patients in the CQ and SP groups,respectively, had gametocytes detectable by RT-PCR.

3.3. Gametocyte density

There was a significant increase in gametocyte den-sity for both groups between days 0 and 7 (P < 0.001;Figure 1c and d). Gametocyte density peaked ondays 7 and 14 among the CQ- and SP-treated groups,respectively. No differences in asexual forms andgametocyte densities between the two groups werefound after accounting for drug resistance effects.However, there were very few data from sensitiveparasites and therefore resistance effects on game-tocyte density cannot be ruled out.

3.4. Frequency of pfcrt, pfmdr1 and dhfralleles following CQ and SP treatment

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ecreased significantly (P < 0.05 across both groups;> 0.05 each drug separately).RT-PCR revealed a 40% higher gametocyte preva-

ence compared with microscopy among patients

he frequency of the resistance allele pfcrt-76T,hich was approximately 85% at the start of treat-ent, very slightly increased over time both in

180 E. Ali et al.

Figure 2 Frequencies of the mutant alleles of pfcrt,pfmdr1 and dhfr genes in a cohort of Plasmodiumfalciparum-infected patients in Asar village followingtreatment with (a) chloroquine or (b) sulfadoxine—pyri-methamine.

the CQ-treated group (P = 0.16; Figure 2a) and theSP-treated group (P = 0.08; Figure 2b), and thisincrease was significant when the data were com-bined (P = 0.03), and was similar in the two groups(P > 0.10 for drug by day interaction). Similarly,there was a slight increase in the frequency of thepfmdr1-86Y allele across days in the two groups(P = 0.12 for CQ; P = 0.14 for SP; P = 0.05 for com-bined data; interaction P > 0.10). The frequency ofthe dhfr-108N allele increased significantly acrossdays in both groups (Figure 2a and b; P < 0.001 forCQ; P = 0.06 for SP; P < 0.001 for combined; interac-tion P > 0.10). There was no significant differencebetween the two drug groups in the frequency ofany of the alleles (P > 0.05 across all days).

3.5. Drug resistance and gametocyteprevalence

There was a tendency towards higher gametocyteprevalence among resistant compared with sensi-tive P. falciparum infections for both drugs (whenmeasured by RT-PCR: 66% for resistant vs. 56% forsensitive infections in the CQ group, 76% vs. 61% inthe SP group; when measured by microscopy: 24%

4. Discussion

In this study we examined the post-treatment per-sistence of parasites and gametocyte productionamong 106 P. falciparum acute malaria patientstreated with either CQ or SP. Significant increasesin gametocyte densities, detected by microscopy,were observed for both drugs during the first 2weeks following treatment, although there was norise in RT-PCR-detectable gametocyte prevalenceamong the patients examined. Drug-resistant par-asites increased in frequency over time in bothtreatment groups, and there was some evidence ofincreased gametocyte prevalence among patientsinfected with drug-resistant parasites comparedwith those with sensitive P. falciparum infections.

Gametocyte rates, as detected by microscopy,peaked among both treatment groups between days7 and 14, in agreement with findings of recent stud-ies in The Gambia (Targett et al., 2001), Senegal(Robert et al., 2000) and Nigeria (Sowunmi andFateye, 2003). It takes 10—12 d for P. falciparumgametocytes to mature and appear in the circu-lation. This long duration is possibly an adaptivemechanism to evade the deleterious effect of acutecftird2

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for resistant vs. 0% for sensitive infections in the CQgroup, 25% vs. 15% in the SP group). These differ-ences were significant when data were pooled forthe two drug groups together (P = 0.06 for RT-PCR;P = 0.03 for microscopy).

linical paroxysms (Naotunne et al., 1991). There-ore, gametocytes that appear shortly after drugreatment (by day 7) can be attributed to theirncreased density, possibly owing to drug-inducedelease from sites of sequestration, rather thanrug-triggered gametocytogenesis (Targett et al.,001).

In the present study, in addition to microscopye have used PCR and RT-PCR to obtain a higher

esolution of detection of P. falciparum and itsametocytes, respectively. The RT-PCR techniqueas proven to be specific, sensitive and robust, andas been tested for possible transcription leakageBabiker et al., 1999; Menegon et al., 2000). Weherefore believe that RT-PCR is ideal for analysisf the epidemiology of gametocytes as they con-istently exist in lower densities compared withsexual forms (Taylor and Read, 1997). In addition,ature gametocytyes have a short half-life and lim-

ted longevity (Hawking et al., 1971); therefore,stimates of the prevalence of asexual forms byonventional PCR are unlikely to be confounded byo-extracted gametocyte DNA.

The pre-treatment gametocyte rates revealedy RT-PCR were very high (59—65%) comparedith only 6—7% detected by microscopy, andere not different from those seen on days 7 and4 post treatment. However, microscopy rates,hich reflect high gametocyte densities, increased

ignificantly by day 7 both following CQ and SPreatment. It is therefore likely that the subpatent

Increased density of gametocytes following malaria treatment 181

gametocytes already existing pre-treatment hadsimply increased in density in the blood follow-ing drug administration. CQ has been found todissociate antigen—antibody complexes, and redblood cells treated with chloroquine diphosphatehave demonstrated some dissociation of thecoating immunoglobulin (Edwards et al., 1982).However, the concentration of CQ used in thatwork (20 mg/ml) was much higher than the ther-apeutic level. Thus, antimalarial-induced releaseof sequestered gametocytes is probably the causeof the observed post-treatment gametocytaemia.However, we cannot rule out self-medication priorto recruitment.

In this study we have used the main determinantgenes, pfcrt-76T and dhfr-108N, as markers for CQand pyrimethamine resistance, respectively (Fidocket al., 2000; Peterson et al., 1988). A large numberof laboratory and field studies have demonstratedthat the pfcrt-K76T mutation is the major cause ofCQ resistance (Wellems and Plowe, 2001). However,the consensus is that CQ resistance is controlledby multiple loci (Babiker and Walliker, 2002), withpfcrt being the major determinant. Transfectionwork has demonstrated the role of pfmdr1 in alter-iWgdaltC

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(Abdel-Muhsin et al., 2004). The increase infrequency of the mutant dhfr allele among theCQ-treated group is unexpected: one possibleexplanation is that because CQ resistance is wellestablished in the village (Babiker et al., 1991),people who do not clear their symptoms soon afterCQ treatment often self-treat with SP.

The increase in post-treatment drug-resistantparasites was coupled with a slight increase ingametocyte prevalence among patients infectedwith drug-resistant parasites. Previous surveys havesuggested that drug-resistant parasites producemore gametocytes than sensitive ones, whichresults in high rates of mosquito infectivity (e.g.Hogh et al., 1998; Mendez et al., 2002; Sutherlandet al., 2002). This probably reflects the survival ofresistant parasites following drug selection ratherthan an intrinsic characteristic. In a controlled invitro study, no association was found between drug-resistant P. falciparum phenotype and gametocyto-genesis in some laboratory clones (Buckling et al.,1999).

We have previously noted increases in frequencyof drug-resistant P. falciparum parasites at the startof the dry season, which could be explained by thedthdadoadsudetosaoad

phgs1mdmAa

ng the level of CQ resistance (Reed et al., 2000).ith regard to SP, we have examined the main

enetic determinant of pyrimethamine resistance,hfr-108N (Peterson et al., 1988). Nevertheless,dditional mutations in this gene can augment theevel of resistance (Kublin et al., 2002). We haveherefore used the above markers for detection ofQ- and pyrimethamine-resistant parasites.

Resistant alleles for both genes increased overime following treatment. These findings are ingreement with previous reports that demon-trated selection of these genes following anti-alarial treatment (Curtis et al., 1998; Sutherland

t al., 2002). The appearance of parasites withhe sensitive pfcrt allele following CQ treatments probably owing to persistence of gametocytes ofrug-sensitive clones. With regard to SP resistance,arasites with the wild-type dhfr allele were alsoetected post treatment. This may be due to thelow effect of SP compared with CQ, since by days8 and 80 no parasites with the wild-type dhfr alleleere seen among the SP-treated group (Figure 2b).Recent work in Asar has shown an increase

n drug resistance genes during the dry seasonompared with the start of the transmission seasonAbdel-Muhsin et al., 2004). This increase mighte due to selection following treatment of acutealaria cases during the wet season, as shown

n the present study. The subsequent decrease inrequency of these alleles during the dry season haseen attributed to a fitness cost of drug resistance

rug selection that prevails towards the end of theransmission season (Abdel-Muhsin et al., 2004). Weave recently shown that parasites that survive thery season as asymptomatic subpatent infectionsre capable of producing low-density gametocytesetectable by RT-PCR (Nassir et al., 2005). More-ver, we have noted that sensitive parasites appearnd increase in frequency towards the end of thery season and at the beginning of the transmis-ion season (Abdel-Muhsin et al., 2004; H. Babikernpublished data). This pattern of fluctuation ofrug sensitive and resistant parasites in this area ofxtreme seasonal transmission has been attributedo a fitness cost of drug resistance in the absencef drug selection during the dry season and at thetart of the transmission season (Abdel-Muhsin etl., 2004). Thus, parasites that cause the first wavef clinical cases and produce high gametocytaemiasre expected to be more transmissible than the low-ensity parasites that persist during the dry season.

Regardless of the biological basis, the increase inost-treatment gametocyte density in P. falciparumas significant epidemiological consequences. Highametocyte densities have been associated withuccess of mosquito infection (Tchuinkam et al.,993), which leads to an increase in secondaryalaria cases from resistant parasites selected byrug treatment. Increased mosquito infectivity andalaria incidence in areas of high transmission infrica have been attributed to drug-resistant par-sites (Lines et al., 1991; Trape, 2001). Similarly,

182 E. Ali et al.

in areas with low and seasonal transmission, drug-resistant malaria epidemics have been associatedwith years of heavy rains and floods (Bayoumi etal., 1989; Novell et al., 1988). Laboratory andfield studies suggest that drug pressure enhancesmalaria parasite gametocytogenesis and transmis-sion (Buckling et al., 1999; Sutherland et al., 2002).Therefore, the use of antimalarial combinationsthat cure as well as reduce transmission may limitmalaria incidence and the spread of drug resistance(Targett et al., 2001). The use of such combina-tions in eastern Sudan could greatly limit the annualburden of malaria caused by the short annual trans-mission in this region.

Conflicts of interest statementThe authors have no conflicts of interest concerningthe work reported in this paper.

Acknowledgements

We would like to thank the inhabitants of Asarfor their co-operation and help. We are grate-ful for the help of the Malaria Research Group ofthe Biochemistry Department, Faculty of Medicine,

village in eastern Sudan. 2. Drug resistance, molecular kary-otypes and the mdr1 genotype in recent isolates. Trans. R.Soc. Trop. Med. Hyg. 85, 578—583.

Babiker, H.A., Lines, J., Hill, W.G., Walliker, D., 1997. Populationstructure of Plasmodium falciparum in villages with differentmalaria endemicity in east Africa. Am. J. Trop. Med. Hyg. 56,141—147.

Babiker, H.A., Abdel-Wahab, A., Ahmed, S., Suleiman, S.,Ranford-Cartwright, L.C., Carter, R., Walliker, D., 1999.Detection of low level Plasmodium falciparum gametocytesusing reverse transcriptase polymerase chain reaction. Mol.Biochem. Parasitol. 99, 143—148.

Babiker, H.A., Satti, G., Ferguson, H.M., Bayoumi, R., Walliker,D., 2005. Drug resistant Plasmodium falciparum in an areaof seasonal transmission. Acta Trop. 94, 260—268.

Bayoumi, R.A., Babiker, H.A., Ibrahim, S.M., Ghalib, H.W.,Saeed, B.O., Khider, S., Elwasila, M., Karim, E.A., 1989.Chloroquine-resistant Plasmodium falciparum in easternSudan. Acta Trop. 46, 157—165.

Buckling, A.G., Taylor, L.H., Carlton, J.M., Read, A.F., 1997.Adaptive changes in Plasmodium falciparum transmissionstrategies following chloroquine chemotherapy. Proc. Biol.Sci. 264, 553—559.

Buckling, A., Ranford-Cartwright, L.C., Miles, A., Read, A.F.,1999. Chloroquine increases Plasmodium falciparum game-tocytogenesis in vitro. Parasitology 118, 339—346.

Curtis, J., Duraisingh, M.T., Warhurst, D.C., 1998. In vivo selec-tion for specific genotype of dihydropteroate synthase ofPlasmodium falciparum by pyrimethamine—sulfadoxine butnot chloroproguanil—dapsone treatment. J. Infect. Dis. 177,

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Khartoum University, the Malaria Administrationof the Sudanese Ministry of Health, and the staffof Gedaref Hospital, Sudan. Financial support wasreceived from the Medical Research Council, UK,the Wellcome Trust, UK, the Gordon Memorial Col-lege Trust Fund, UK, the University of Edinburgh,UK, the Royal Society of London, UK, and the Inter-national Atomic Energy Agency (IAEA).

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