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    Frequencies of Plasmodium falciparum A, dihydropteroate synthase, B, dihydrofolate reductase, and C, chloroquine resistance transporter gene haplotypes in Tanzania. Values are shown as proportions and constitute the fraction of each haplotype infection where a haplotype was dominant. The number of P. falciparum-positive samples examined in 2003, 2004, 2006, and 2007 was 107, 129, 81, and 69 in Mkokola and 69, 47, 7, and 21 in Kwamasimba, respectively.

  • 1

    Rønn AM, Msangeni HA, Mhina J, Wernsdorfer WH, Bygbjerg IC, 1996. High level of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine in children in Tanzania. Trans R Soc Trop Med Hyg 90 :179–181.

    • Search Google Scholar
    • Export Citation
  • 2

    Trigg JK, Mbwana H, Chambo O, Hills E, Watkins W, Curtis CF, 1997. Resistance to pyrimethamine/sulfadoxine in Plasmodium falciparum in 12 villages in north east Tanzania and a test of chlorproguanil/dapsone. Acta Trop 63 :185–189.

    • Search Google Scholar
    • Export Citation
  • 3

    East African Network for Monitoring Antimalarial Treatment (EANMAT), 2003. The efficacy of antimalarial monotherapies, sulphadoxine-pyrimethamine and amodiaquine in East Africa: implications for sub-regional policy. Trop Med Int Health 8 :860–867.

    • Search Google Scholar
    • Export Citation
  • 4

    Mugittu K, Ndejembi M, Malisa A, Lemnge M, Premji Z, Mwita A, Nkya W, Kataraihya J, Abdulla S, Beck HP, Mshinda H, 2004. Therapeutic efficacy of sulfadoxine-pyrimethamine and prevalence of resistance markers in Tanzania prior to revision of malaria treatment policy: Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase mutations in monitoring in vivo resistance. Am J Trop Med Hyg 71 :696–702.

    • Search Google Scholar
    • Export Citation
  • 5

    Mutabingwa TK, Anthony D, Heller A, Hallett R, Ahmed J, Drakeley C, Greenwood BM, Whitty CJ, 2005. Amodiaquine alone, amodiaquine + sulfadoxine-pyrimethamine, amodiaquine + artesunate, and artemether-lumefantrine for outpatient treatment of malaria in Tanzanian children: a four-arm randomised effectiveness trial. Lancet 365 :1474–1480.

    • Search Google Scholar
    • Export Citation
  • 6

    Lemnge M, Alifrangis M, Kafuye MY, Segeja MD, Gesase S, Minja D, Massaga JJ, Ronn AM, Bygbjerg IC, 2006. High reinfection rate and treatment failures in children treated with amodiaquine for falciparum malaria in Muheza villages, northeastern Tanzania. Am J Trop Med Hyg 75 :188–193.

    • Search Google Scholar
    • Export Citation
  • 7

    Cowman A, Morry MJ, Biggs BA, Cross GA, Foote SJ, 1988. Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proc Natl Acad Sci USA 85 :9109–9113.

    • Search Google Scholar
    • Export Citation
  • 8

    Peterson DS, Walliker D, Wellems TE, 1988. Evidence that a point mutation in dihydrofolate reductase- thymidylate synthase confers resistance to pyrimethamine in falciparum malaria. Proc Natl Acad Sci USA 85 :9114–9118.

    • Search Google Scholar
    • Export Citation
  • 9

    Brooks R, Wang P, Read M, Watkins MW, Sims PF, Hyde JE, 1994. Sequence variation of the hydroxymethyldihydropterin pyro-phosphokinase: dihydroptoate synthase gene lines of the human malaria parasite, Plasmodium falciparum, with differing resistance to sulfadoxine. J Biochem 224 :397–405.

    • Search Google Scholar
    • Export Citation
  • 10

    Triglia T, Cowman A, 1994. Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum. Proc Natl Acad Sci USA 91 :7149–7153.

    • Search Google Scholar
    • Export Citation
  • 11

    Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF, Martino LM, Mukadam RA, Rogerson SJ, Lescano AG, Molyneux ME, Winstanley PA, Chimpeni P, Taylor TE, Plowe CV, 2002. Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis 185 :380–388.

    • Search Google Scholar
    • Export Citation
  • 12

    Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naude B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE, 2000. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6 :861–871.

    • Search Google Scholar
    • Export Citation
  • 13

    Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV, Coulibaly D, 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344 :257–263.

    • Search Google Scholar
    • Export Citation
  • 14

    Holmgren G, Gil JP, Ferreira PM, Veiga MI, Obonyo CO, Bjorkman A, 2006. Amodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of pfcrt 76T and pfmdr1 86Y. Infect Genet Evol 6 :309–314.

    • Search Google Scholar
    • Export Citation
  • 15

    Warhurst DC, 2003. Polymorphism in the Plasmodium falciparum chloroquine-resistance transporter protein links verapamil enhancement of chloroquine sensitivity with the clinical efficacy of amodiaquine. Malar J 2 :31.

    • Search Google Scholar
    • Export Citation
  • 16

    Alifrangis M, Dalgaard MB, Lusingu JP, Vestergaard LS, Staalsoe T, Jensen AT, Enevold A, Ronn AM, Khalil IF, Warhurst DC, Lemnge MM, Theander TG, Bygbjerg IC, 2006. Occurrence of the Southeast Asian/South American SVMNT haplotype of the chloroquine-resistance transporter gene in Plasmodium falciparum in Tanzania. J Infect Dis 193 :1738–1741.

    • Search Google Scholar
    • Export Citation
  • 17

    Lusingu JP, Jensen AT, Vestergaard LS, Minja DT, Dalgaard MB, Gesase S, Mmbando BP, Kitua AY, Lemnge MM, Cavanagh D, Hviid L, Theander TG, 2006. Levels of plasma immunoglobulin G with specificity against the cysteine-rich interdomain regions of a semiconserved Plasmodium falciparum erythrocyte membrane protein 1, VAR4, predict protection against malarial anemia and febrile episodes. Infect Immun 74 :2867–2875.

    • Search Google Scholar
    • Export Citation
  • 18

    Bodker R, Akida J, Shayo D, Kisinza W, Msangeni HA, Pedersen EM, Lindsay SW, 2003. Relationship between altitude and intensity of malaria transmission in the Usambara Mountains, Tanzania. J Med Entomol 40 :706–717.

    • Search Google Scholar
    • Export Citation
  • 19

    Alifrangis M, Enosse S, Pearce R, Drakeley C, Roper C, Khalil IF, Nkya WM, Ronn AM, Theander TG, Bygbjerg IC, 2005. A simple high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum cloroquine resistance transporter genes using polymerase chain reaction and enzyme linked immunosorbent assay-based technology. Am J Trop Med Hyg 72 :155–162.

    • Search Google Scholar
    • Export Citation
  • 20

    Duraisingh MT, Curtis J, Warhurst DC, 1998. Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion. Exp Parasitol 89 :1–8.

    • Search Google Scholar
    • Export Citation
  • 21

    Wang P, Lee C-S, Bayoumi R, Djimde A, Doumbo O, Swedberg G, Dao LD, Mshinda H, Tanner M, Watkins MW, Sims PF, Hyde JE, 1997. Resistance to antifolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthetase and dihydrofolate reductase alleles in a large number of field samples of diverse origins. Mol Biochem Parasitol 89 :161–177.

    • Search Google Scholar
    • Export Citation
  • 22

    Enevold A, Nkya WM, Theisen M, Vestergaard LS, Jensen AT, Staalsoe T, Theander TG, Bygbjerg IC, Alifrangis M, 2007. Potential impact of host immunity on malaria treatment outcome in Tanzanian children infected with Plasmodium falciparum. Malar J 6 :153.

    • Search Google Scholar
    • Export Citation
  • 23

    Pearce RJ, Drakeley C, Chandramohan D, Mosha F, Roper C, 2003. Molecular determination of point mutation haplotypes in the dihydrofolate reductase and dihydropteroate synthase of Plasmodium falciparum in three districts of northern Tanzania. Antimicrob Agents Chemother 47 :1347–1354.

    • Search Google Scholar
    • Export Citation
  • 24

    Okiro EA, Hay SI, Gikandi PW, Sharif SK, Noor AM, Peshu N, Marsh K, Snow RW, 2007. The decline in paediatric malaria admissions on the coast of Kenya. Malar J 6 :151.

    • Search Google Scholar
    • Export Citation
  • 25

    Bhattarai A, Ali AS, Kachur SP, Martensson A, Abbas AK, Khatib R, Al-Mafazy AW, Ramsan M, Rotllant G, Gerstenmaier JF, Molteni F, Abdulla S, Montgomery SM, Kaneko A, Bjorkman A, 2007. Impact of artemisinin-based combination therapy and insecticide-treated nets on malaria burden in Zanzibar. PLoS Med 4 :e309.

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Five-Year Surveillance of Molecular Markers of Plasmodium falciparum Antimalarial Drug Resistance in Korogwe District, Tanzania: Accumulation of the 581G Mutation in the P. falciparum Dihydropteroate Synthase Gene

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  • 1 Centre for Medical Parasitology, Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagem, Denmark; Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Denmark; National Institute for Medical Research, Tanga Centre, Tanga, Tanzania

In January 2007, Tanzania replaced sulfadoxine-pyrimethamine (SP) with artemether-lumefantrine for treatment of uncomplicated malaria. This study examined the impact of widespread SP use on molecular markers of Plasmodium falciparum drug resistance in blood samples from persons living in two villages in Korogwe District, Tanzania, from 2003 through 2007. The prevalence of the P. falciparum dihydropteroate synthase (Pfdhps) gene 581G mutation increased from 12% in 2003 to 56% in 2007 (P < 0.001), resulting in an increase in the triple mutant Pfdhps haplotype SGEGA from 8% to 32% (P < 0.001). In contrast, the chloroquine-sensitive P. falciparum chloroquine resistance transporter (Pfcrt) CVMNK haplotype increased from 6% to 30% (P < 0.001). The dramatic increase of the triple Pfdhps mutant SGEGA haplotype may endanger the continued use of SP for intermittent presumptive treatment of pregnant women (IPTp). Further studies are needed to determine the importance of Pfdhps SGEGA haplotype parasites on the efficacy of SP for IPTp.

In Tanzania, artesunate-lumefantrine (Coartem®; Novartis, Basel, Switzerland) was officially implemented as first-line treatment against uncomplicated malaria in January 2007, replacing sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ). The change was timely after several studies in this country during the past decade showed a rapid increase of clinical failures after SP treatment, even before SP was implemented officially in 2001.14 However, SP is still being used for intermittent presumptive treatment of malaria in pregnant women (IPTp) in the country. Recently, high levels of AQ resistance has also been observed. 5,6

At molecular level, the emergence and magnitude of resistance to SP can be monitored by measuring the prevalence of single nucleotide polymorphisms (SNPs) in the P. falciparum dihydrofolate reductase (Pfdhfr) gene, responsible for pyrimethamine resistance 7,8 and the dihydropteroate synthetase (Pfdhps) gene, responsible for sulfadoxine resistance. 9,10 In Africa, the Pfdhfr triple mutation N51I + C59R + S108N with wild type at codons 50 (C50) and 164 (I164) are combined into the highly prevalent and resistant haplotype CIRNI. For Pfdhps, the double mutation, A437G + K540E, with wild type at codons 436 (S436), 581 (A581), and 613 (A613), are combined into the resistant haplotype SGEAA. The combination of the two haplotypes (CIRNI-SGEAA) has been associated with high-level of resistance to SP in vivo.11

Resistance to chloroquine (CQ) and AQ is mainly associated with a single K76T mutation in the P. falciparum chloroquine resistance transporter (Pfcrt) gene.1214 There are three main haplotypes in codons 72–76 of the Pfcrt gene, wild type CVMNK, and CQ-resistant haplotypes CVIET and SVMNT. 15 In Africa, the CVIET haplotype is the most dominant mutant haplotype; the SVMNT haplotype have only been observed sporadically. 16

In the light of two major policy changes in the choice of first-line antimalarial drugs in Tanzania involving CQ and SP in 2001 and 2007, the objective of the present study was to examine if changes in drug policy affected the distribution of the molecular markers of CQ, AQ, and SP resistance. Cross-sectional surveys were conducted annually during 2003–2007 in two villages with high and moderate malaria transmission intensity, respectively: Mkokola (300 meters above sea level) and Kwamasimba (600–700 meters above sea level) in Korogwe District in northeastern Tanzania. 17,18 The villages are approximately 15 km apart.

Finger prick or venous blood samples were obtained on filter paper from all children and adolescents less than 20 years of age. For this study, samples collected in July 2003, March 2004, May 2006, and May 2007 were available for analyses. Ethical clearance for the study was granted by the Medical Research Coordinating Committee of the National Institute for Medical Research and Ministry of Health, Tanzania. Informed consent was obtained from all participants or from their parents/guardians.

DNA extraction from bloodspots and nested polymerase chain reaction (PCR), followed by sequence-specific oligonucleotide probe (SSOP)–enzyme-linked immunosorbent assay, were performed and analyzed using SSOPs targeting SNPs/ haplotypes in Pfdhfr, Pfdhps, and Pfcrt as described. 19 To confirm the presence of the c581G SNP, the PCR–restriction fragment length polymorphism method was used. 20

During the first year (2003), the point prevalence of P. falciparum in children less than five years of age was high (79%) in Mkokola and moderate (25%) in Kwamasimba. However, during 2003–2007, the prevalence of malaria decreased to 15.5% in Mkokola and 5.2% in Kwamasimba in 2007 (Lusingu J and others, unpublished data).

The number of P. falciparum-positive samples from Kwamasimba was low in some years (n = 7 in 2006 and n = 21 in 2007). Thus, the frequency of haplotypes in Pfdhps, Pfdhfr, and Pfcrt are shown by combining data from the two villages (Figure 1).

With regard to the frequency of Pfdhps haplotypes, the triple mutant SGEGA haplotype increased from 8% in 2003 to 32% in 2007 (P < 0.001). A decrease in frequency was observed for the wildtype SAKAA haplotype and the double mutant SGEAA haplotype (Figure 1A).

The frequency of the triple mutant Pfdhfr CIRNI haplotype remained high at approximately 90% each year, and no major changes were observed (Figure 1B). By combining the Pfdhfr and Pfdhps data, the frequency of the quintuple mutant haplotype CIRNI-SGEAA decreased significantly from 75% in 2006 to 50% in 2007 (P = 0.014), and the sextuple mutant haplotype CIRNI-SGEGA increased in frequency from 17% to 32% (P = 0.118). The Pfcrt mutant haplotype SVMNT was detected in both villages in 2004, 16 but was not detected in the preceding years (Figure 1C). Interestingly, the wildtype haplotype CVMNK increased significantly in the study period (P < 0.001), most markedly during 2006–2007 from 13% to 30% (P = 0.029).

The prevalence of Pfdhps SNPs/haplotypes showed some remarkable changes during the study period (Table 1). Infections with mutants 436/437SG, 540E, and 581G increased significantly compared with pure wild types (P < 0.001 for all comparisons). Most notably, the prevalence of 581G, including mixed 581A/G infections, increased from 11.7% to 55.6% between 2003 and 2007 (P < 0.001). Some differences in prevalence were apparent between villages. Most notably, the prevalence of 436/437SG and 540E reached 100% in Kwamasimba in 2007. All of these infections were pure 436/437SG infections; only one infection contained 540K as a mixed 540K/E infection. For the high transmission village of Mkokola, Pfdhps wild types were still observed. Likewise, a trend for a higher prevalence of 581G in Kwamasimba (75.0%) than in Mkokola (50.0%) in 2007 was observed (P = 0.136).

For Pfdhfr (Table 2). the prevalence of mutant genotypes 51I, 59R, and 108N was close to 100%, with minor differences in the study period. Wild type genotypes 51N, 59C, and 108S fluctuated from year to year. For Pfdhps, the prevalence of genotypes 59R and 108N reached 100% in Kwamasimba. Wild type genotypes 59C and 108S were not found in this village, but were still found in Mkokola. For Pfcrt (Table 3). the prevalence of the wild type genotype CVMNK increased significantly from 31% in 2006 to 51% in 2007 (P = 0.021). In 2007, there was a significantly higher prevalence of CVMNK haplotype infections compared with infections without this haplotype in Mkokola (56.4%) than in Kwamasimba (33.3%), (P = 0.021).

A steep increase in the frequency of Pfdhps SGEGA haplotypes was observed in the study period. Except for a study published in 1997 in Ifakara, Tanzania, where a prevalence of 27.8% was observed (n = 18), 21 the 581G mutation has, to our knowledge, until now been absent in Tanzania. 4,22,23 In addition to the present study, a recent study in the Hale, Tanga region of Tanzania detected a frequency of the 581G mutation of > 50% (Gesase S, unpublished data). Presumably, the intense use of SP in the east African region the past 5–6 years has resulted in additional accumulation of mutations conferring sulfadoxine resistance. Whether SGEGA parasites are more sulfadoxine resistant in vivo than double mutant SGEAA-parasites is unknown.

The frequency of the wild type CVMNK haplotype increased from 6% in 2003 to 30% in 2007. This finding may reflect decreasing drug pressure of 4-amino-quinolines on parasite populations in the area. Amodiaquine was discontinued as a second-line drug in January 2007. Thus, selection pressure of AQ on Pfcrt mutants is most likely of less importance compared with that of CQ. The trends in distribution of Pfdhps and Pfcrt haplotypes observed did not seem to be affected by the time of sampling because sampling in March 2004 was during the low transmission season and sampling in 2003, 2006 and 2007 was during the high transmission season.

There were noteworthy differences in prevalence of polymorphisms between the two villages. For Pfdhps and Pfdhfr, the prevalence of mutants 436/437SG and 540E in Pfdhps and 59R and 108N in Pfdhfr reached 100% in 2007 in the low transmission village of Kwamasimba. The difference in transmission intensity between the two villages may constitute the main reason for a higher frequency of SP- and CQ/ AQ-resistant mutants in Kwamasimba because a lower degree of transmission appears to favor less multi-clonality and thus a smaller reservoir of diverse drug-sensitive/resistant parasites. Because parasite prevalence decreased significantly in the study period, as in other parts of east Africa 24,25 this finding may cause persistence of Pfdhps/Pfdhfr mutants even after SP drug pressure has ceased.

In the study area, the main source of antimalarial drugs is community resource persons, who stopped prescribing SP in February 2007. However, a continued antifolate drug pressure is persisting beyond the formal policy change because SP is still prescribed for IPTp and other sulfonamide-antifolate drugs, such as sulfamethoxazole-trimethoprim, are increasingly being used as chemoprophylaxis against opportunistic infections in person infected with human immunodeficiency virus. Further studies are needed to determine whether continued accumulation of mutations in Pfdhps may endanger the efficacy of SP for IPTp.

Table 1

Prevalence of single nucleotide polymorphism (SNP) haplotypes in the Plasmodium falciparum dihydropteroate synthase (Pfdhps) gene, Tanzania, 2003–2007*

Table 1
Table 2

Prevalence of single nucleotide polymorphism (SNP) haplotypes in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene, Tanzania, 2003–2007*

Table 2
Table 3

Prevalence of single nucleotide polymorphism (SNP) haplotypes in codon 72–76 of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene, Tanzania, 2003–2007*

Table 3
Figure 1.
Figure 1.

Frequencies of Plasmodium falciparum A, dihydropteroate synthase, B, dihydrofolate reductase, and C, chloroquine resistance transporter gene haplotypes in Tanzania. Values are shown as proportions and constitute the fraction of each haplotype infection where a haplotype was dominant. The number of P. falciparum-positive samples examined in 2003, 2004, 2006, and 2007 was 107, 129, 81, and 69 in Mkokola and 69, 47, 7, and 21 in Kwamasimba, respectively.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 80, 4; 10.4269/ajtmh.2009.80.523

*

Address correspondence to Michael Alifrangis, Centre for Medical Parasitology, Institute for International Health, Immunology and Microbiology, CSS, Øster Farimagsgade 5, Building 22+23, PO Box 2099, 1014 Copenhagen K, Denmark. E-mail: micali@sund.ku.dk

Authors’ addresses: Michael Alifrangis, Michael B. Dalgaard, Lasse S. Vestergaard, Insaf F. Khalil, Thor G. Theander, and Ib C. Bygbjerg, Centre for Medical Parasitology, Institute for International Health, Immunology and Microbiology, CSS, Øster Farimagsgade 5, Building 22+23, PO Box 2099, 1014 Copenhagen K, Denmark, E-mails: micali@sund.ku.dk, michael.dalgaard@cmp.dk, l.vestergaard@cmp.dk, insafk@sund.ku.dk, thor@sund.ku.dk, and iby@sund.ku.dk. John P. Lusingu, Bruno Mmbando, Deus Ishengoma, and Martha M. Lemnge, National Institute for Medical Research, Tanga Medical Research Centre, PO Box 5004, Tanga, Tanzania, E-mails: jlusingu@tanga.mimcom.net, b.mmbando@biostat.ku.dk, dishengoma@tanga.mimcom.net, and lemnge@tanga.mimcom.net.

Acknowledgments: All study participants, including their parents or guardians, are kindly acknowledged. We thank Ulla Abildtrup and Charles Brown (Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana) for excellent technical assistance. The study was conducted under the auspices of the Joint Malaria Programme, a collaborative research initiative between Centre for Medical Parasitology at the University of Copenhagen and Copenhagen University Hospital, Kilimanjaro Christian Medical College, London School of Hygiene and Tropical Medicine and the Tanzania National Institute for Medical Research.

Financial support: The field study was supported by the Tanzania-Denmark ENRECA programme (104.Dan.8.L.312) and project 91106 by the Danish International Development Agency.

REFERENCES

  • 1

    Rønn AM, Msangeni HA, Mhina J, Wernsdorfer WH, Bygbjerg IC, 1996. High level of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine in children in Tanzania. Trans R Soc Trop Med Hyg 90 :179–181.

    • Search Google Scholar
    • Export Citation
  • 2

    Trigg JK, Mbwana H, Chambo O, Hills E, Watkins W, Curtis CF, 1997. Resistance to pyrimethamine/sulfadoxine in Plasmodium falciparum in 12 villages in north east Tanzania and a test of chlorproguanil/dapsone. Acta Trop 63 :185–189.

    • Search Google Scholar
    • Export Citation
  • 3

    East African Network for Monitoring Antimalarial Treatment (EANMAT), 2003. The efficacy of antimalarial monotherapies, sulphadoxine-pyrimethamine and amodiaquine in East Africa: implications for sub-regional policy. Trop Med Int Health 8 :860–867.

    • Search Google Scholar
    • Export Citation
  • 4

    Mugittu K, Ndejembi M, Malisa A, Lemnge M, Premji Z, Mwita A, Nkya W, Kataraihya J, Abdulla S, Beck HP, Mshinda H, 2004. Therapeutic efficacy of sulfadoxine-pyrimethamine and prevalence of resistance markers in Tanzania prior to revision of malaria treatment policy: Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase mutations in monitoring in vivo resistance. Am J Trop Med Hyg 71 :696–702.

    • Search Google Scholar
    • Export Citation
  • 5

    Mutabingwa TK, Anthony D, Heller A, Hallett R, Ahmed J, Drakeley C, Greenwood BM, Whitty CJ, 2005. Amodiaquine alone, amodiaquine + sulfadoxine-pyrimethamine, amodiaquine + artesunate, and artemether-lumefantrine for outpatient treatment of malaria in Tanzanian children: a four-arm randomised effectiveness trial. Lancet 365 :1474–1480.

    • Search Google Scholar
    • Export Citation
  • 6

    Lemnge M, Alifrangis M, Kafuye MY, Segeja MD, Gesase S, Minja D, Massaga JJ, Ronn AM, Bygbjerg IC, 2006. High reinfection rate and treatment failures in children treated with amodiaquine for falciparum malaria in Muheza villages, northeastern Tanzania. Am J Trop Med Hyg 75 :188–193.

    • Search Google Scholar
    • Export Citation
  • 7

    Cowman A, Morry MJ, Biggs BA, Cross GA, Foote SJ, 1988. Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. Proc Natl Acad Sci USA 85 :9109–9113.

    • Search Google Scholar
    • Export Citation
  • 8

    Peterson DS, Walliker D, Wellems TE, 1988. Evidence that a point mutation in dihydrofolate reductase- thymidylate synthase confers resistance to pyrimethamine in falciparum malaria. Proc Natl Acad Sci USA 85 :9114–9118.

    • Search Google Scholar
    • Export Citation
  • 9

    Brooks R, Wang P, Read M, Watkins MW, Sims PF, Hyde JE, 1994. Sequence variation of the hydroxymethyldihydropterin pyro-phosphokinase: dihydroptoate synthase gene lines of the human malaria parasite, Plasmodium falciparum, with differing resistance to sulfadoxine. J Biochem 224 :397–405.

    • Search Google Scholar
    • Export Citation
  • 10

    Triglia T, Cowman A, 1994. Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum. Proc Natl Acad Sci USA 91 :7149–7153.

    • Search Google Scholar
    • Export Citation
  • 11

    Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF, Martino LM, Mukadam RA, Rogerson SJ, Lescano AG, Molyneux ME, Winstanley PA, Chimpeni P, Taylor TE, Plowe CV, 2002. Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis 185 :380–388.

    • Search Google Scholar
    • Export Citation
  • 12

    Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LM, Sidhu AB, Naude B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE, 2000. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 6 :861–871.

    • Search Google Scholar
    • Export Citation
  • 13

    Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV, Coulibaly D, 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344 :257–263.

    • Search Google Scholar
    • Export Citation
  • 14

    Holmgren G, Gil JP, Ferreira PM, Veiga MI, Obonyo CO, Bjorkman A, 2006. Amodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of pfcrt 76T and pfmdr1 86Y. Infect Genet Evol 6 :309–314.

    • Search Google Scholar
    • Export Citation
  • 15

    Warhurst DC, 2003. Polymorphism in the Plasmodium falciparum chloroquine-resistance transporter protein links verapamil enhancement of chloroquine sensitivity with the clinical efficacy of amodiaquine. Malar J 2 :31.

    • Search Google Scholar
    • Export Citation
  • 16

    Alifrangis M, Dalgaard MB, Lusingu JP, Vestergaard LS, Staalsoe T, Jensen AT, Enevold A, Ronn AM, Khalil IF, Warhurst DC, Lemnge MM, Theander TG, Bygbjerg IC, 2006. Occurrence of the Southeast Asian/South American SVMNT haplotype of the chloroquine-resistance transporter gene in Plasmodium falciparum in Tanzania. J Infect Dis 193 :1738–1741.

    • Search Google Scholar
    • Export Citation
  • 17

    Lusingu JP, Jensen AT, Vestergaard LS, Minja DT, Dalgaard MB, Gesase S, Mmbando BP, Kitua AY, Lemnge MM, Cavanagh D, Hviid L, Theander TG, 2006. Levels of plasma immunoglobulin G with specificity against the cysteine-rich interdomain regions of a semiconserved Plasmodium falciparum erythrocyte membrane protein 1, VAR4, predict protection against malarial anemia and febrile episodes. Infect Immun 74 :2867–2875.

    • Search Google Scholar
    • Export Citation
  • 18

    Bodker R, Akida J, Shayo D, Kisinza W, Msangeni HA, Pedersen EM, Lindsay SW, 2003. Relationship between altitude and intensity of malaria transmission in the Usambara Mountains, Tanzania. J Med Entomol 40 :706–717.

    • Search Google Scholar
    • Export Citation
  • 19

    Alifrangis M, Enosse S, Pearce R, Drakeley C, Roper C, Khalil IF, Nkya WM, Ronn AM, Theander TG, Bygbjerg IC, 2005. A simple high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum cloroquine resistance transporter genes using polymerase chain reaction and enzyme linked immunosorbent assay-based technology. Am J Trop Med Hyg 72 :155–162.

    • Search Google Scholar
    • Export Citation
  • 20

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