• 1.

    Falade CO, Tongo OO, Ogunkole OO, Orimadegun AE, 2010. Effects of malaria in pregnancy on newborn anthropometry. J Infect Dev Ctries 4: 448453.

  • 2.

    World Health Organization, 2017. World Malaria Report, 2017. Geneva, Switzerland: World Health Organization. Available at: http://www.who.int/malaria/publications/world-malaria-report-2017/en. Accessed August 16, 2017.

  • 3.

    Desai M 2016. Impact of sulfadoxine-pyrimethamine resistance on effectiveness of intermittent preventive therapy for malaria in pregnancy at clearing infections and preventing low birth weight. Clin Infect Dis 62: 323333.

    • Search Google Scholar
    • Export Citation
  • 4.

    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: 91149118.

    • Search Google Scholar
    • Export Citation
  • 5.

    Wang P, Read M, Sims PF, Hyde JE, 1997. Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthase and an additional factor associated with folate utilization. Mol Microbiol 23: 979986.

    • Search Google Scholar
    • Export Citation
  • 6.

    Sidhu AB, Valderramos SG, Fidock DA, 2005. Pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum. Mol Microbiol 57: 913926.

    • Search Google Scholar
    • Export Citation
  • 7.

    Malmberg M 2013. Temporal trends of molecular markers associated with artemether-lumefantrine tolerance/resistance in Bagamoyo district, Tanzania. Malar J 12: 103.

    • Search Google Scholar
    • Export Citation
  • 8.

    Djimde A 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 257263.

  • 9.

    National Malaria Control Programme, Ghana, 2015. Malaria in Pregnancy. Available at: http://www.ghanahealthservice.org/malaria/subcategory.php. Accessed October 17, 2017.

  • 10.

    Mockenhaupt FP, Eggelte TA, Till H, Bienzle U, 2001. Plasmodium falciparum Pfcrt and Pfmdr1 polymorphisms are associated with the Pfdhfr N108 pyrimethamine-resistance mutation in isolates from Ghana. Trop Med Int Health 6: 749755.

    • Search Google Scholar
    • Export Citation
  • 11.

    Mockenhaupt FP, Bedu-Addo G, Junge C, Hommerich L, Eggelte TA, Bienzle U, 2007. Markers of sulfadoxine-pyrimethamine-resistant Plasmodium falciparum in placenta and circulation of pregnant women. Antimicrob Agents Chemother 51: 332334.

    • Search Google Scholar
    • Export Citation
  • 12.

    Mockenhaupt FP, Bedu-Addo G, Eggelte TA, Homerich L, Holmberg V, von Oertzen C, Bienzle U, 2008. Rapid increase in the prevalence of sulfadoxine-pyrimethamine resistance among Plasmodium falciparum isolated from pregnant women in Ghana. J Infect Dis 198: 15451549.

    • Search Google Scholar
    • Export Citation
  • 13.

    Osarfo J, Tagbor H, Cairns M, Alifrangis M, Magnussen P, 2017. Dihydroartemisinin-piperaquine versus artesunate-amodiaquine for treatment of malaria infection in pregnancy in Ghana: an open-label, randomised, non-inferiority trial. Trop Med Int Health 22: 10431052.

    • Search Google Scholar
    • Export Citation
  • 14.

    Wooden J, Kyes S, Sibley CH, 1993. PCR and strain identification in Plasmodium falciparum. Parasitol Today 9: 303305.

  • 15.

    Alifrangis M, Enosse S, Pearce R, Drakeley C, Roper C, Khalil IF, Nkya WM, Rønn AM, Theander TG, Bygbjerg IC, 2005. Simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology. Am J Trop Med Hyg 72: 155162.

    • Search Google Scholar
    • Export Citation
  • 16.

    Thomsen TT, Ishengoma DS, Mmbando BP, Lusingu JP, Vestergaard LS, Theander TG, Lemnge MM, Bygbjerg IC, Alifrangis M, 2011. Prevalence of single nucleotide polymorphisms in the Plasmodium falciparum multidrug resistance gene (Pfmdr-1) in Korogwe District in Tanzania before and after introduction of artemisinin-based combination therapy. Am Trop Med Hyg 85: 979983.

    • Search Google Scholar
    • Export Citation
  • 17.

    Afoakwa R, Boampong JN, Egyir-Yawson A, Nwaefuna EK, Verner ON, Asare KK, 2014. High prevalence of PfCRT K76T mutation in Plasmodium falciparum isolates in Ghana. Acta Trop 136: 3236.

    • Search Google Scholar
    • Export Citation
  • 18.

    Sisowath C, Petersen I, Veiga MI, Mårtensson A, Premji Z, Björkman A, Fidock DA, Gil JP, 2009. In vivo selection of Plasmodium falciparum parasites carrying the chloroquine-susceptible Pfcrt K76 allele after treatment with artemether-lumefantrine in Africa. J Infect Dis 199: 750755.

    • Search Google Scholar
    • Export Citation
  • 19.

    Venkatesan M 2014. Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine. Am J Trop Med Hyg 91: 833843.

    • Search Google Scholar
    • Export Citation
  • 20.

    Mousiliou A, De Tove YS, Doritchamou J, Luty AJF, Massougbodji A, Alifrangis M, Deloron P, Ndam NT, 2013. High rates of parasite recrudescence following intermittent preventive treatment with sulphadoxine-pyrimethamine during pregnancy in Benin. Malar J 12: 195.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

Molecular Markers of Plasmodium falciparum Drug Resistance in Parasitemic Pregnant Women in the Middle Forest Belt of Ghana

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  • 1 Ghana Health Service, Effiduase District Hospital, Effiduase, Ashanti Region, Ghana;
  • 2 School of Medicine, University of Health and Allied Sciences, Ho, Ghana;
  • 3 Centre for Medical Parasitology, Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark;
  • 4 Department of Veterinary and Aquatic Sciences, Section for Parasitology and Aquatic Diseases, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark;
  • 5 Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark

Data on prevalence of antimalarial molecular resistance markers in pregnant women in Ghana is scarce. Prevalence of single nucleotide polymorphisms/haplotypes in the Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes was assessed in a cross-sectional study involving 200 pregnant women. Almost 90% of infections were wild type at the Pfcrt gene whereas the Pfmdr1 NFD mutant haplotype occurred in 43% of samples. Prevalence of Pfdhfr/Pfdhps quadruple mutation was 92.6% whereas Pfdfr/Pfdhps quintuple mutation with K540E was not observed. The study provides important updates of antimalarial resistance markers in Ghanaian pregnant women and suggests increased tolerance to one of the first-line treatment options in Ghana: artemether–lumefantrine. The data support the view that sulfadoxine–pyrimethamine is still efficacious for intermittent preventive treatment in Ghana, but the impact of increased doses on selection of mutations needs to be assessed. Continuing the surveillance of resistance markers is important to inform changes in antimalarial drug policy in pregnancy.

Pregnancy-associated malaria (PAM) leads to maternal and newborn morbidity and mortality.1 Control measures include intermittent preventive treatment with sulfadoxine–pyrimethamine (IPTp-SP); regular and correct use of long-lasting insecticide-treated nets; and prompt diagnosis and treatment with efficacious antimalarials, including artemisinin-based combination therapies (ACTs). However, parasite resistance to antimalarial drugs and mosquito resistance to insecticides mitigate these measures.2

Particularly, Plasmodium falciparum resistance to SP has resulted in the declining protective efficacy of IPTp-SP mainly in eastern Africa.3 Stepwise mutations in mainly codons 108, 51, and 59 of the Pfdhfr gene and codons 436, 437, 540, 581, and 613 in the Pfdhps gene lead to resistance in vitro and in vivo.4,5 The Pfmdr1 single nucleotide polymorphisms (SNPs) are linked to altered sensitivity to various antimalarials including monotherapies lumefantrine, amodiaquine (AQ), quinine, and artemisinins and the ACTs as well.6,7 Altered parasite sensitivity to AQ is also linked to similar mechanisms for chloroquine (CQ) resistance, which in turn is associated with mutations at codons 72–76 of the Pfcrt gene, particularly at codon 76 (K76T).8 Wild-type Pfcrt variants have been reportedly selected by artemether–lumefantrine (AL).7

In Ghana, PAM accounts for 17.6% of outpatient department attendance, 13.7% of admissions among pregnant women, and 3.4% of maternal deaths.9 Quinine is used to treat uncomplicated malaria in the first trimester whereas since 2005 and 2008, artesunate–amodiaquine and AL, respectively, are used in the second and third trimesters. Sulfadoxine–pyrimethamine has, since 2005, been used for IPTp alone.

Surveillance of molecular markers allows early detection of changing antimalarial drug susceptibility and may inform changes in drug policy. However, data on current prevalence of antimalarial resistance markers in Ghanaian pregnant women, compared with the general population, are scarce. Prevalences of 69% for Pfcrt K76T, 66% for Pfmdr1 N86Y, 80% for Pfdhfr N108S, and 36–73% for Pfdhfr triple mutations were reported in studies conducted from 1998 to 2006.1012 With increasing coverage and doses of IPTp-SP and wider ACT utilization, it is appropriate to evaluate how these changes have influenced the prevalence of antimalarial resistance markers in this population.

We assessed the prevalence of SNPs/haplotypes in the Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes in a cross-sectional study of asymptomatic pregnant women of all gravidity enrolled in a drug trial from antenatal clinics at St. Michael’s and Bekwai Government hospitals in the Bosomtwe and Bekwai districts, respectively, in Ghana.13 The geometric mean parasite density was 224/μL (95% confidence interval; 193, 289).13 Some women were likely exposed to IPTp-SP in previous pregnancies whereas ACT use cannot be ruled out given that second and third trimester women were enrolled.

Filter paper spots from 200 women, picked by simple random selection, were collected at recruitment from July 2011 to October 2012. The study protocol was approved by the Committee for Human Research and Publication Ethics, Kwame Nkrumah University of Science and Technology, Ghana (CHRPE 190/10 and CHRPE/AP/236/12). Written informed consent was obtained from each participant.

Parasite DNA extraction was by the Chelex-100 method.14 Polymerase chain reaction amplification of segments of the Pfcrt, Pfdhfr, Pfdhps, and Pfmdr1 genes and analyses of SNPs at these genes were as described earlier.15,16 Laboratory parasite strains 3D7, HB3, FCR3, and DD2 and blood samples obtained from non-malaria–exposed Danish citizens served as controls. Data were entered in Microsoft Excel 2007 and analyzed using proportions for prevalence and frequency of SNPs and haplotypes. Prevalence measures included mixed infections whereas frequency excluded them.

Data analysis included 199 samples. Successful Pfcrt haplotyping of codons 72–76 was achieved in 83.9% of samples (167/199). Infections with the CVMNK wild-type haplotype were predominant with a prevalence of 88.6% (148/167). At the Pfmdr1 gene, wild-type SNPs N86 and D1246 were dominant with a prevalence of 87.8% and 98.7%, respectively (see Table 1). The single mutant N86-184F-D1246 (NFD) and the wild-type N86-Y184-D1246 haplotypes were equally frequent at 43.2%. YYD and NFY haplotypes also occurred in equal frequencies of 1.6% whereas YFD and YFY had frequencies of 10.4% and 0.8%, respectively. The triple mutant 86Y-184Y-1246Y haplotype was not observed.

Table 1

Prevalence and frequency of Pfmdr1 SNPs at codons 86, 184, and 1,246

Pfmdr1 SNPsPrevalenceFrequency*
Evaluable samples (N)Number with specified SNP (n)% (n/N × 100)Evaluable samples (N)Number with specified SNP (n)% (n/N × 100)
N8616414487.815813887.9
86Y1642515.21581912.1
184F1498859.11377655.5
Y1841464846.61376144.5
D124615515398.715515398.7
1246Y14421.414421.4

SNPs = single nucleotide polymorphisms.

Frequency excludes mixed infections whereas prevalence includes mixed infections.

Mutations at Pfdhfr codons 51, 59, and 108 were predominant with frequencies of 75.7% (103/136), 85% (119/140), and 87.8% (122/139), respectively. Neither the S108T nor the I164L mutations were observed (see Table 2).

Table 2

Prevalence and frequency of SNPs at the Pfdfhr and Pfdhps genes

CodonSNPsPrevalenceFrequency
Evaluable sample (N)Number with specific SNP (n)% (n/N × 100)Evaluable sample (N)Number with specific SNP (n)% (n/N × 100)
Pfdhfr
 50/51CN1423625.51363324.3
CI14210675.513610375.7
 59C1462416.41402115.0
R14612283.614011985.0
 108S1391712.21391712.2
N13912287.813912287.8
T00
 164I132132100.0132132100.0
L00
Pfdhps
 436/437AA14732.014132.1
AG9564.69567.4
SA32.010.7
SG4530.64129.1
FA10.710.7
 540K14314299.3141141100.0
E10.70
 581A15214796.715014697.3
G53.342.7
 613A14813893.214613793.8
S106.896.2
Pfdhfr and Pfdhps haplotypes*
Pfdhfr (N = 126)
  CIRNI9777
  CNCSI97.1
Pfhdps (N = 130)
  AGKAA8061.5
  SGKAA3930
  AGKAS64.6
  AGKGS10.8
  AAKAA32.3
  SAKAA10.8
Pfdhfr/ (N = 81)
  CIRNI/AGKAA (quadruple mutation)4251.9
  CIRNI/SGKAA (quadruple mutation)3340.7
  CIRNI/AGKAS (quintuple mutation)56.2
  CIRNI/AGKGS (sextuple mutation)11.2

SNPs = single nucleotide polymorphisms.

Only frequencies are reported for haplotypes and N is evaluable samples whereas n is the number of haplotypes.

The Pfdhfr double mutations (CNRNI + CICNI + CIRSI) collectively constituted 15.9%.

At the Pfdhps gene, the combined frequency of the 437G mutation (as either 436/437AG or 436/437SG) was 96.5%. Only one sample that was a mixed infection showed the K540E mutation (see Table 2). The frequencies of A581G and A613S mutations were 2.7% and 6.2%, respectively.

Combining Pfdhfr and Pfdhps SNPs into haplotypes showed that only 7.1% of isolates (9/126) were pure Pfdhfr wild type (CNCSI) whereas 77.0% (97/126) carried the triple mutation (CIRNI). For Pfdhps, 3.1% (4/130) were pure wild type (three AAKAA and one SAKAA haplotypes). Single Pfdhps mutated haplotypes AGKAA and SGKAA were present in 91.5% (119/130) of samples whereas only one isolate exhibited the triple mutated haplotype AGKGS (see Table 2).

The Pfdhfr/Pfdhps quadruple mutation (CIRNI-437G) was present in 92.6% (75/81) of isolates. The quintuple mutation (51I + 59R + 108N/437G + 613S) was found in 6.2% (5/81) whereas the sextuple mutation (51I + 59R + 108N/437G + 581G + 613S) was present in 1.2% (1/81) of isolates. The quintuple mutation with Pfdhps K540E (51I + 59R + 108N/437G + 540E) was not observed.

Close to 90% of P. falciparum infections exhibited the CVMNK wild-type haplotype at codons 72–76 of the Pfcrt gene. The prevalence of the CVIET mutant haplotype (Pfcrt K76T) is likely the lowest reported in Ghana since changing from CQ treatment of uncomplicated malaria in 2005 and contrasts sharply with a recent report in nonpregnant populations in Ghana.17 Apart from repopulation of wild-type P. falciparum parasites following CQ withdrawal, another possible reason for the predominance of the wild-type parasites could be selection by AL.18 The findings suggest a prospect for reintroducing CQ for malaria treatment in pregnancy. However, CQ should be combined with another drug to combat early reemergence of resistance and restricted to those with laboratory-confirmed parasitamia.

The frequency of the Pfmdr1 NFD was 43.2% and is the first such report in Ghanaian pregnant women. Increasing levels of this haplotype and the N86 SNP are reported to underlie ACT tolerance.19 A higher frequency than observed was expected following the introduction of AL use in pregnant women since 2008, but it is possible that low drug pressure in pregnant women has kept the selection of NFD low.

The frequency of the Pfdhfr triple mutation and Pfdhfr/Pfdhps quadruple mutation (CIRNI-437G) is comparable with previous reports in west African pregnant women.20 The Pfdhfr CIRNI triple mutation appears to have reached saturation within the pregnant population in Ghana, having recorded 73% prevalence in 200612 and 77% in the present study. Furthermore, the K540E mutation was present as a mixed infection in only one sample (1/142), consistent with reports of its rarity in west Africa along with the A581G and A613S mutations.20 The low prevalence of these SNPs suggests that IPTp-SP still retains good efficacy in Ghana. The study did not include an assessment of the novel Pfdhps mutation I431V which may be related to SP resistance. In addition, not all samples were evaluable, thus reducing the numbers included in data analysis. This may be because of long storage durations of the filter paper spots (≥ 10 months) at possibly suboptimal temperatures/humidity with some DNA degradation. The study findings are, however, consistent with previous reports.20

The study provides an updated picture of antimalarial resistance markers in pregnant women in Ghana. With its presence in more than a third of the samples, the Pfmdr1 NFD haplotype may suggest an increasing tolerance to AL. The National Malaria Control Program must strengthen education on targeted treatment with ACTs to help reduce drug pressure and delay resistance development. In line with World Health Organization recommendations, Ghana increased the number of IPTp-SP doses from 3 to ≥ 3 in 2013. It is important to monitor how this will impact the selection of Pfdhfr/Pfdhps mutations, especially the I431V and its distribution, and whether this may compromise the continuing protective efficacy of IPTp.

Acknowledgments:

We are grateful to Ulla Abildtrup, Centre for Medical Parasitology, University of Copenhagen for her invaluable contribution to the genetic analysis. We also thank the district health management teams of Bosomtwe and Bekwai districts, the study participants, and the staff.

REFERENCES

  • 1.

    Falade CO, Tongo OO, Ogunkole OO, Orimadegun AE, 2010. Effects of malaria in pregnancy on newborn anthropometry. J Infect Dev Ctries 4: 448453.

  • 2.

    World Health Organization, 2017. World Malaria Report, 2017. Geneva, Switzerland: World Health Organization. Available at: http://www.who.int/malaria/publications/world-malaria-report-2017/en. Accessed August 16, 2017.

  • 3.

    Desai M 2016. Impact of sulfadoxine-pyrimethamine resistance on effectiveness of intermittent preventive therapy for malaria in pregnancy at clearing infections and preventing low birth weight. Clin Infect Dis 62: 323333.

    • Search Google Scholar
    • Export Citation
  • 4.

    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: 91149118.

    • Search Google Scholar
    • Export Citation
  • 5.

    Wang P, Read M, Sims PF, Hyde JE, 1997. Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthase and an additional factor associated with folate utilization. Mol Microbiol 23: 979986.

    • Search Google Scholar
    • Export Citation
  • 6.

    Sidhu AB, Valderramos SG, Fidock DA, 2005. Pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum. Mol Microbiol 57: 913926.

    • Search Google Scholar
    • Export Citation
  • 7.

    Malmberg M 2013. Temporal trends of molecular markers associated with artemether-lumefantrine tolerance/resistance in Bagamoyo district, Tanzania. Malar J 12: 103.

    • Search Google Scholar
    • Export Citation
  • 8.

    Djimde A 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 257263.

  • 9.

    National Malaria Control Programme, Ghana, 2015. Malaria in Pregnancy. Available at: http://www.ghanahealthservice.org/malaria/subcategory.php. Accessed October 17, 2017.

  • 10.

    Mockenhaupt FP, Eggelte TA, Till H, Bienzle U, 2001. Plasmodium falciparum Pfcrt and Pfmdr1 polymorphisms are associated with the Pfdhfr N108 pyrimethamine-resistance mutation in isolates from Ghana. Trop Med Int Health 6: 749755.

    • Search Google Scholar
    • Export Citation
  • 11.

    Mockenhaupt FP, Bedu-Addo G, Junge C, Hommerich L, Eggelte TA, Bienzle U, 2007. Markers of sulfadoxine-pyrimethamine-resistant Plasmodium falciparum in placenta and circulation of pregnant women. Antimicrob Agents Chemother 51: 332334.

    • Search Google Scholar
    • Export Citation
  • 12.

    Mockenhaupt FP, Bedu-Addo G, Eggelte TA, Homerich L, Holmberg V, von Oertzen C, Bienzle U, 2008. Rapid increase in the prevalence of sulfadoxine-pyrimethamine resistance among Plasmodium falciparum isolated from pregnant women in Ghana. J Infect Dis 198: 15451549.

    • Search Google Scholar
    • Export Citation
  • 13.

    Osarfo J, Tagbor H, Cairns M, Alifrangis M, Magnussen P, 2017. Dihydroartemisinin-piperaquine versus artesunate-amodiaquine for treatment of malaria infection in pregnancy in Ghana: an open-label, randomised, non-inferiority trial. Trop Med Int Health 22: 10431052.

    • Search Google Scholar
    • Export Citation
  • 14.

    Wooden J, Kyes S, Sibley CH, 1993. PCR and strain identification in Plasmodium falciparum. Parasitol Today 9: 303305.

  • 15.

    Alifrangis M, Enosse S, Pearce R, Drakeley C, Roper C, Khalil IF, Nkya WM, Rønn AM, Theander TG, Bygbjerg IC, 2005. Simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology. Am J Trop Med Hyg 72: 155162.

    • Search Google Scholar
    • Export Citation
  • 16.

    Thomsen TT, Ishengoma DS, Mmbando BP, Lusingu JP, Vestergaard LS, Theander TG, Lemnge MM, Bygbjerg IC, Alifrangis M, 2011. Prevalence of single nucleotide polymorphisms in the Plasmodium falciparum multidrug resistance gene (Pfmdr-1) in Korogwe District in Tanzania before and after introduction of artemisinin-based combination therapy. Am Trop Med Hyg 85: 979983.

    • Search Google Scholar
    • Export Citation
  • 17.

    Afoakwa R, Boampong JN, Egyir-Yawson A, Nwaefuna EK, Verner ON, Asare KK, 2014. High prevalence of PfCRT K76T mutation in Plasmodium falciparum isolates in Ghana. Acta Trop 136: 3236.

    • Search Google Scholar
    • Export Citation
  • 18.

    Sisowath C, Petersen I, Veiga MI, Mårtensson A, Premji Z, Björkman A, Fidock DA, Gil JP, 2009. In vivo selection of Plasmodium falciparum parasites carrying the chloroquine-susceptible Pfcrt K76 allele after treatment with artemether-lumefantrine in Africa. J Infect Dis 199: 750755.

    • Search Google Scholar
    • Export Citation
  • 19.

    Venkatesan M 2014. Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine. Am J Trop Med Hyg 91: 833843.

    • Search Google Scholar
    • Export Citation
  • 20.

    Mousiliou A, De Tove YS, Doritchamou J, Luty AJF, Massougbodji A, Alifrangis M, Deloron P, Ndam NT, 2013. High rates of parasite recrudescence following intermittent preventive treatment with sulphadoxine-pyrimethamine during pregnancy in Benin. Malar J 12: 195.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Joseph Osarfo, Ghana Health Service, Effiduase District Hospital, Effiduase, Ashanti Region, Ghana. E-mail: josarfo@yahoo.co.uk

Financial support: This forms part of J.O.’s doctoral research work which was funded by the Malaria Capacity Development Consortium (MCDC). MCDC is in turn funded by the Wellcome Trust Grant WT084289MA and Bill & Melinda Gates Foundation Grant 51941 (http://www.mcdconsortium.org).

Authors’ addresses: Joseph Osarfo, Ghana Health Service, Effiduase District Hospital, Effiduase, Ashanti Region, Ghana, E-mail: josarfo@yahoo.co.uk. Harry Tagbor, School of Medicine, University of Health and Allied Sciences, Ho, Ghana, E-mail: htagbor@uhas.edu.gh. Pascal Magnussen, Centre for Medical Parasitology, Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark, and Department of Veterinary and Aquatic Sciences, Section for Parasitology and Aquatic Diseases, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, E-mail: pma@sund.ku.dk. Michael Alifrangis, Centre for Medical Parasitology, Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark, and Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark, E-mail: micali@sund.ku.dk.

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