Ahead of Print -Independent Lineages of Highly Sulfadoxine-Resistant Plasmodium falciparum Haplotypes, Eastern Africa - Volume 20, Number 7—July 2014 - Emerging Infectious Disease journal - CDC

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Ahead of Print -Independent Lineages of Highly Sulfadoxine-Resistant Plasmodium falciparum Haplotypes, Eastern Africa - Volume 20, Number 7—July 2014 - Emerging Infectious Disease journal - CDC

Volume 20, Number 7—July 2014

Research

Independent Lineages of Highly Sulfadoxine-ResistantPlasmodium falciparum Haplotypes, Eastern Africa

Article Contents

• Methods
• Results
• Discussion
• Acknowledgments
• References
• Figure 1
• Figure 2
• Figure 3
• Figure 4
• Table
• Technical Appendix - 207 KB 
• Suggested Citation

Steve M. Taylor , Alejandro L. Antonia1, Whitney E. Harrington, Morgan M. Goheen, Victor Mwapasa, Ebbie Chaluluka, Michal Fried, Edward Kabyemela, Mwayi Madanitsa, Carole Khairallah, Linda Kalilani-Phiri, Antoinette K. Tshefu, Stephen J. Rogerson, Feiko O. ter Kuile, Patrick E. Duffy, and Steven R. Meshnick

Author affiliations: Author affiliations: Duke University Medical Center, Durham, North Carolina, USA (S.M. Taylor); University of North Carolina, Chapel Hill, North Carolina, USA (S.M. Taylor, A.L. Antonia, M.M. Goheen, S.R. Meshnick); Seattle Children’s Hospital/University of Washington School of Medicine, Seattle, Washington, USA (W.E. Harrington); College of Medicine, Blantyre, Malawi (V. Mwapasa, E. Chaluluka, M. Madanitsa, L. Kalilani-Phiri);National Institutes of Health, Bethesda, Maryland, USA (M. Fried. P.E. Duffy); Seattle Biomedical Research Institute, Seattle (E. Kabyemela); Liverpool School of Tropical Medicine, Liverpool, UK (C. Khairallah, F.O. ter Kuile); University of Kinshasa, Kinshasha, Democratic Republic of the Congo (A.K. Tshefu); University of Melbourne, Melbourne, Victoria, Australia (S.J. Rogerson);University of Amsterdam, Amsterdam, the Netherlands (F.O. ter Kuile)

Suggested citation for this article

Abstract

Sulfadoxine-resistant Plasmodium falciparum undermines malaria prevention with sulfadoxine/pyrimethamine. Parasites with a highly resistant mutant dihydropteroate synthase (dhps) haplotype have recently emerged in eastern Africa; they negated preventive benefits of sulfadoxine/pyrimethamine, and might exacerbate placental malaria. We explored emerging lineages of dhps mutant haplotypes in Malawi, the Democratic Republic of the Congo, and Tanzania by using analyses of genetic microsatellites flanking the dhps locus. In Malawi, a triple-mutant dhps SGEG (mutant amino acids are underlined) haplotype emerged in 2010 that was closely related to pre-existing double-mutant SGEA haplotypes, suggesting local origination in Malawi. When we compared mutant strains with parasites from the Democratic Republic of the Congo and Tanzania by multiple independent analyses, we found that SGEG parasites were partitioned into separate lineages by country. These findings support a model of local origination of SGEG dhps haplotypes, rather than geographic diffusion, and have implications for investigations of emergence and effects of parasite drug resistance.

Antimalarial drug resistance threatens to undermine efforts to control Plasmodium falciparummalaria. In sub-Saharan Africa, P. falciparum resistance to sulfadoxine/pyrimethamine (SP) is widespread, as shown by clinical treatment failures and the prevalence of molecular markers of drug resistance (1). Despite these findings, SP remains a major tool for malaria control when administered as a partner drug with artemisinins and as intermittent preventive therapy in infants (IPTi), children, and pregnant women (IPTp). Of these SP-based interventions, IPTi with SP is safe and effective (2), IPTi in children receiving SP and amodiaquine has shown promise in western Africa (3,4), and IPTp-SP is used widely across sub-Saharan Africa. All 3 policies are recommended by the World Health Organization for many settings in Africa (5–7). Spread of sulfadoxine-resistant parasites will compromise the effectiveness of these programs.

IPTp-SP has been adopted most broadly; however, its efficacy appears to be decreasing in areas with increasing parasite resistance to SP (8,9). Reduced susceptibility to sulfadoxine is conferred mainly by nonsynonymous substitutions at codons 436, 437, 540, and 581 of the P. falciparum dihydropteroate synthase (dhps) gene that encodes the enzymatic target of sulfadoxine (10). Parasites with mutant dhps haplotypes are restricted to sub-Saharan Africa, and parasites with the A437G, K540E, and A581G mutations (mutant amino acids are underlined), which are known as dhps triple mutants (haplotype SGEG across codons 436, 437, 540, and 581), have been limited to eastern Africa. In sites in Tanzania in which the SGEGhaplotype is prevalent, IPTp-SP does not appear to improve birth outcomes (9), and IPTi with SP is not effective (11).

In addition, recent evidence suggests that IPTp-SP might exacerbate placental malaria when women are infected with parasites that have the A581G mutation in dhps (12), which suggests that these parasites manifest increased pathogenicity under drug pressure. In contrast, there was no evidence of pathogenicity caused by A581G-bearing parasites in Malawi, and SP retained some efficacy in preventing illness caused by malaria during pregnancy (J. Gutman et al., unpub. data). These contrasting effects of this resistant parasite haplotype suggest that effects of the A581G mutation might vary among populations. However, if parasites from northern Tanzania dessiminate, parasites bearing the dhps A581G mutation could broadly undermine malaria control efforts in infants and pregnant women in Africa.

Because of these findings, molecular surveillance for this mutation is critical to assess the durability of SP for malaria prevention. Genetic studies have shown that mutations conferring resistance to chloroquine (13) and pyrimethamine (14) have arisen only a few times and then diffused across regions and continents. In contrast, resistance to sulfadoxine appears to have arisen independently in multiple locations (15,16), after originating only in Southeast Asia, followed by export to Africa (supported by global survey findings) (17). Efforts to prevent dissemination of the A581G mutation hinge on understanding whether the mutation arises de novo or is spread among locations.

To better understand the emergence of sulfadoxine-resistant P. falciparum in eastern Africa, we first used microsatellite genotyping to study the emergence of parasites harboring dhpshaplotypes with the A581G mutation in a longitudinal study in Malawi during 1997–2010 (8). We then compared the genetic background of these triple-mutant SGEG parasites in Malawi in a cross-sectional analysis with mutant parasite haplotypes from Tanzania and the Democratic Republic of the Congo (DRC). In these 2 investigations, we hypothesized that extant SGEA haplotypes in Malawi would share a genetic lineage with recently emerged SGEG haplotypes, and that these SGEG haplotypes from Malawi would represent a distinct lineage compared with SGEG haplotypes from other settings in eastern Africa.

Dr Taylor is an infectious disease specialist at Duke University Medical Center, Durham, North Carolina. His research interests are malaria drug resistance and pathogenesis.

Acknowledgments

We thank Jaymin Patel, Jon Juliano, Christian Parobek, Venkatachalam Udhayakumar, and Mohammed Tauqeer Alam for providing laboratory assistance and helpful discussions, and the persons who participated in the clinical studies.

M.F. and P.E.D. were supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

References

1. Sridaran S, McClintock SK, Syphard LM, Herman KM, Barnwell JW, Udhayakumar V. Anti-folate drug resistance in Africa: meta-analysis of reported dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant genotype frequencies in AfricanPlasmodium falciparum parasite populations. Malar J. 2010;9:247 . DOIPubMed
2. Aponte JJ, Schellenberg D, Egan A, Breckenridge A, Carneiro I, Critchley J, Efficacy and safety of intermittent preventive treatment with sulfadoxine-pyrimethamine for malaria in African infants: a pooled analysis of six randomised, placebo-controlled trials. Lancet.2009;374:1533–42 . DOIPubMed
3. Dicko A, Diallo AI, Tembine I, Dicko Y, Dara N, Sidibe Y, Intermittent preventive treatment of malaria provides substantial protection against malaria in children already protected by an insecticide-treated bednet in Mali: a randomised, double-blind, placebo-controlled trial. PLoS Med. 2011;8:e1000407. DOIPubMed
4. Konaté AT, Yaro JB, Ouedraogo AZ, Diarra A, Gansane A, Soulama I, Intermittent preventive treatment of malaria provides substantial protection against malaria in children already protected by an insecticide-treated bednet in Burkina Faso: a randomised, double-blind, placebo-controlled trial. PLoS Med. 2011;8:e1000408. DOIPubMed
5. World Health Organization. WHO policy recommendation on intermittent preventive treatment during infancy with sulfadoxine-pyrimethamine (SP-IPTi) for Plasmodium falciparum malaria control in Africa 2010 [cited 2014 Apr 11].http://www.who.int/malaria/news/WHO_policy_recommendation_IPTi_032010.pdf 
6. World Health Organization. WHO policy recommendation: seasonal malaria chemoprevention (SMC) for Plasmodium falciparum malaria control in highly seasonal transmission areas of the Sahel sub-region in Africa 2012 [cited 2014 Apr 11].http://www.who.int/malaria/publications/atoz/who_smc_policy_recommendation/en/
7. World Health Organization. WHO policy brief for the implementation of intermittent preventive treatment of malaria in pregnancy using sulfadoxine-pyrimethamine (IPTp-SP)2013. [cited 2014 Apr 11].http://www.who.int/malaria/publications/atoz/Policy_brief_IPTp-SP_implementation_11april2013.pdf 
8. Feng G, Simpson JA, Chaluluka E, Molyneux ME, Rogerson SJ. Decreasing burden of malaria in pregnancy in Malawian women and its relationship to use of intermittent preventive therapy or bed nets. PLoS ONE. 2010;5:e12012. DOIPubMed
9. Harrington WE, Mutabingwa TK, Kabyemela E, Fried M, Duffy PE. Intermittent treatment to prevent pregnancy malaria does not confer benefit in an area of widespread drug resistance. Clin Infect Dis. 2011;53:224–30. DOIPubMed
10. Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF, Martino LM, Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis. 2002;185:380–8. DOIPubMed
11. Gosling RD, Gesase S, Mosha JF, Carneiro I, Hashim R, Lemnge M, Protective efficacy and safety of three antimalarial regimens for intermittent preventive treatment for malaria in infants: a randomised, double-blind, placebo-controlled trial. Lancet. 2009;374:1521–32.DOIPubMed
12. Harrington WE, Mutabingwa TK, Muehlenbachs A, Sorensen B, Bolla MC, Fried M,Competitive facilitation of drug-resistant Plasmodium falciparum malaria parasites in pregnant women who receive preventive treatment. Proc Natl Acad Sci U S A.2009;106:9027–32. DOIPubMed
13. Wootton JC, Feng X, Ferdig MT, Cooper RA, Mu J, Baruch DI, Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum. Nature. 2002;418:320–3. DOIPubMed
14. Roper C, Pearce R, Nair S, Sharp B, Nosten F, Anderson T. Intercontinental spread of pyrimethamine-resistant malaria. Science. 2004;305:1124. DOIPubMed
15. Pearce RJ, Pota H, Evehe MS. Bâ el-H, Mombo-Ngoma G, Malisa AL, et al. Multiple origins and regional dispersal of resistant dhps in African Plasmodium falciparum malaria. PLoS Med. 2009;6:e1000055.
16. Vinayak S, Alam MT, Mixson-Hayden T, McCollum AM, Sem R, Shah NK, Origin and evolution of sulfadoxine resistant Plasmodium falciparum. PLoS Pathog.2010;6:e1000830 . DOIPubMed
17. Mita T, Venkatesan M, Ohashi J, Culleton R, Takahashi N, Tsukahara T, Limited geographical origin and global spread of sulfadoxine-resistant dhps alleles inPlasmodium falciparum populations. J Infect Dis. 2011;204:1980–8. DOIPubMed
18. Taylor SM, Antonia AL, Chaluluka E, Mwapasa V, Feng G, Molyneux ME, Antenatal receipt of sulfadoxine-pyrimethamine does not exacerbate pregnancy-associated malaria despite the expansion of drug-resistant Plasmodium falciparum: clinical outcomes from the QuEERPAM study. Clin Infect Dis. 2012;55:42–50 . DOIPubMed
19. Taylor SM, Messina JP, Hand CC, Juliano JJ, Muwonga J, Tshefu AK, Molecular malaria epidemiology: mapping and burden estimates for the Democratic Republic of the Congo, 2007. PLoS ONE. 2011;6:e16420. DOIPubMed
20. Taylor SM, Antonia AL, Parobek CM, Juliano JJ, Janko M, Emch M, Plasmodium falciparumsulfadoxine resistance is geographically and genetically clustered within the DR Congo. Sci Rep. 2013;3:1165.
21. Peakall R, Smouse PE. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research–an update. Bioinformatics. 2012;28:2537–9. DOIPubMed
22. Excoffier L, Smouse PE, Quattro JM. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics. 1992;131:479–91 .PubMed
23. Nei M, Roychoudhury AK. Sampling variances of heterozygosity and genetic distance.Genetics. 1974;76:379–90 .PubMed
24. Fluxus Engineering. Phylogenetic network software [cited 2012 Oct 19].http://www.fluxus-engineering.com/sharenet.htm
25. Bandelt HJ, Forster P, Rohl A. Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol. 1999;16:37–48 . DOIPubMed
26. Hardy OJ, Vekemans X. SPAGeDi: a versatile computer program to analyse spatial genetic structure at the individual or population levels. Mol Ecol Notes. 2002;2:618–20. DOI
27. Felsenstein J. PHYLIP - Phylogeny Inference Package (version 3.2). Cladistics.1989;5:164–6.
28. Mobyle Portal. Institut Pasteur [cited 2013 Aug 27].http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms:neighbor
29. Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000;155:945–59 .PubMed
30. Hubisz MJ, Falush D, Stephens M, Pritchard JK. Inferring weak population structure with the assistance of sample group information. Mol Ecol Resour. 2009;9:1322–32.
31. Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol. 2005;14:2611–20. DOIPubMed
32. Taylor SM, Antonia A, Feng G, Mwapasa V, Chaluluka E, Molyneux M, Adaptive evolution and fixation of drug-resistant Plasmodium falciparum genotypes in pregnancy-associated malaria: 9-year results from the QuEERPAM study. Infect Genet Evol. 2012;12:282–90. DOIPubMed
33. Minja DT, Schmiegelow C, Mmbando B, Bostrom S, Oesterholt M, Magistrado P,Plasmodium falciparum mutant haplotype infection during pregnancy associated with reduced birthweight, Tanzania. Emerg Infect Dis. 2013;19:1446–54. DOIPubMed
34. Slatkin M. A measure of population subdivision based on microsatellite allele frequencies.Genetics. 1995;139:457–62 .PubMed
35. Gutman J, Mwandama D, Wiegand RE, Ali D, Mathanga DP, Skarbinski J. Effectiveness of intermittent preventive treatment with sulfadoxine-pyrimethamine during pregnancy on maternal and birth outcomes in machinga district, Malawi. J Infect Dis. 2013;208:907–16.DOIPubMed
36. McCollum AM, Poe AC, Hamel M, Huber C, Zhou Z, Shi YP, Antifolate resistance inPlasmodium falciparum: multiple origins and identification of novel dhfr alleles. J Infect Dis. 2006;194:189–97. DOIPubMed
37. Naidoo I, Roper C. Mapping ‘partially resistant’, ‘fully resistant’, and ‘super resistant’ malaria. Trends Parasitol. 2013;29:505–15. DOIPubMed

Figures

• Figure 1. Genetic relatedness of Plasmodium falciparum dihydropteroate synthase (dhps) haplotypes from Malawi over time based on median-joining network of microsatellite profilesMedian joining network was calculated based on microsatellite profiles for...
• Figure 2. Principal coordinates analyses of wild-type (SAKA) and mutant (SGEA and SGEG)Plasmodium falciparum dihydropteroate synthase (dhps) halotypes from eastern Africa based on analysis of variance (R_ST)Pairwise R_ST values were...
• Figure 3. Neighbor-joining network of Plasmodium falciparum wild-type (SAKA) and mutant (SGEA and SGEG) dihydropteroate synthase (dhps) haplotypes, eastern AfricaPairwise linear genetic distances among 193 parasite isolates were...
• Figure 4. Predicted population structure of Plasmodium falciparum parasites from eastern Africa among those with A) double-mutant (SGEA) or triple-mutant (SGEG) dihydropteroate synthase (dhps) haplotypes and B) those with only the...

Table

• Table. Pairwise Φ_PT values and Nei genetic distances among major Plasmodium falciparum dhpshaplotypes by year, Malawi

Technical Appendix

• Technical Appendix. Additional population genetic indices and fragment lengths of microsatellite loci for Independent lineages of highly sulfadoxine-resistant Plasmodium falciparum haplotypes, eastern Africa.  207 KB 

Suggested citation for this article: Taylor SM, Antonia AL, Harrington WE, Goheen MM, Mwapasa V, Chaluluka E, et al. Independent lineages of highly sulfadoxine-resistant Plasmodium falciparumhaplotypes, eastern Africa. Emerg Infect Dis [Internet]. 2014 Jul [date cited].http://dx.doi.org/10.3201/eid2007.131720

DOI: 10.3201/eid2007.131720

1Current affiliation: Duke University School of Medicine, Durham, North Carolina, USA.