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Abstract

Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (‘K13-propeller’) with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

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Acknowledgements

We thank the patients and field staff involved in clinical trials and sample collections. We are grateful to the provincial health department directors and other staff of the Cambodian Ministry of Health. Clinical trials and sample collections were supported in part by the Global Fund Grant Malaria Program Rounds 6 (CAM-607-G10M-CNM3) and 9 (CAM-S10-G14-M), the Bill and Melinda Gates Foundation and USAID (through the World Health Organization), the US DOD Global Epidemic Information System, and the Intramural Research Program, NIAID, NIH. Laboratory work was supported by grants from Banque Natixis (to O.M.-P. and D.M.) and Laboratoire d’Excellence IBEID (Agence Nationale de la Recherche, France) and Institut Pasteur, Division International (ACIP A-10-2010). B.W. was supported by a postdoctoral fellowship from Institut Pasteur, Division International; J.B. by an Institut Pasteur Paris Master-Pro fellowship; and D.M. by the French Ministry of Foreign Affairs. We are grateful to the Wellcome Trust Sanger Institute and the MalariaGEN resource centre for sequencing, genotyping and population structure analysis of some Cambodian clinical samples, funded by the Wellcome Trust (098051; 090770/Z/09/Z) and the MRC (G0600718). We thank the Rotary Club-Versailles for funding computer equipment. P.R., D.M.B. and W.O.R. are staff members of the World Health Organization and the US Navy, respectively. They alone are responsible for the views expressed in this publication, and they do not necessarily represent the decisions, policy or views of the World Health Organization or the US Navy.

Author information

Author notes

    • Frédéric Ariey
    • , Johann Beghain
    • , Sandie Ménard
    • , Antoine Berry
    •  & Jean-Christophe Barale

    Present addresses: Institut Pasteur, Genetics and Genomics of Insect Vectors Unit, 75724 Paris Cedex 15, France (F.A.); Institut Pasteur, Functional Genetics of Infectious Diseases Unit, 75724 Paris Cedex 15, France (J.B.); Centre de Physiopathologie de Toulouse-Purpan, Institut National de la Santé et de la Recherche Médicale UMR1043, Centre National de la Recherche Scientifique UMR5282, Université Toulouse III, 31024 Toulouse Cedex 3, France(S.M. & A.B.); Institut Pasteur, Unité de Biologie et Génétique du Paludisme, Team Malaria Targets and Drug Development, 75724 Paris Cedex 15, France (J.-C.B.).

    • Rick M. Fairhurst
    • , Françoise Benoit-Vical
    • , Odile Mercereau-Puijalon
    •  & Didier Ménard

    These authors contributed equally to this work.

Affiliations

  1. Institut Pasteur, Parasite Molecular Immunology Unit, 75724 Paris Cedex 15, France

    • Frédéric Ariey
    • , Johann Beghain
    • , Anne-Claire Langlois
    • , Thierry Fandeur
    • , Jean-Christophe Barale
    •  & Odile Mercereau-Puijalon
  2. Centre National de la Recherche Scientifique, Unité de Recherche Associée 2581, 75724 Paris Cedex 15, France

    • Frédéric Ariey
    • , Johann Beghain
    • , Anne-Claire Langlois
    • , Jean-Christophe Barale
    •  & Odile Mercereau-Puijalon
  3. Institut Pasteur du Cambodge, Malaria Molecular Epidemiology Unit, Phnom Penh, Cambodia

    • Benoit Witkowski
    • , Nimol Khim
    • , Saorin Kim
    • , Valentine Duru
    • , Pharath Lim
    • , Thierry Fandeur
    •  & Didier Ménard
  4. Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Chanaki Amaratunga
    • , Pharath Lim
    •  & Rick M. Fairhurst
  5. Institut Pasteur, Plate-forme Génomique, Département Génomes et Génétique, 75724 Paris Cedex 15, France

    • Christiane Bouchier
    •  & Laurence Ma
  6. National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia

    • Pharath Lim
    • , Rithea Leang
    • , Socheat Duong
    • , Sokunthea Sreng
    • , Seila Suon
    •  & Char Meng Chuor
  7. SSA WHO, Drug Monitoring in Cambodia, National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia

    • Denis Mey Bout
  8. Service de Parasitologie et Mycologie, Centre Hospitalier Universitaire de Toulouse, 31059 Toulouse Cedex 9, France

    • Sandie Ménard
    •  & Antoine Berry
  9. Naval Medical Research Unit #2 Detachment, Phnom Penh, Cambodia

    • William O. Rogers
  10. Swiss Tropical and Public Health Institute, 4051 Basel, Switzerland

    • Blaise Genton
  11. MRC Centre for Genomics and Global Health, University of Oxford, Oxford OX3 7BN, UK

    • Olivo Miotto
  12. Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok 10400, Thailand

    • Olivo Miotto
  13. Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK

    • Olivo Miotto
  14. Global Malaria Program, World Health Organization, 1211 Geneva, Switzerland

    • Pascal Ringwald
  15. Centre National de Référence du Paludisme, CHU Bichat-Claude Bernard, APHP, PRES Sorbonne Paris Cité, 75018 Paris, France

    • Jacques Le Bras
  16. Centre National de la Recherche Scientifique, Laboratoire de Chimie de Coordination UPR8241, 31077 Toulouse Cedex 4, France

    • Françoise Benoit-Vical
  17. Université de Toulouse, UPS, Institut National Polytechnique de Toulouse, 31077 Toulouse Cedex 4, France

    • Françoise Benoit-Vical

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Contributions

B.W., S.M., A.B. and F.B.-V. produced the F32-ART5 and F32-TEM clonal lines and analysed their survival rates. F.A. and J.B. developed computational components of the whole-genome sequence analysis. C.B. and L.M. performed whole-genome sequencing. F.A., C.A., S.K., V.D., P.L., R.L., S.D., Se.S., So.S., C.M.C., D.M.B., W.O.R., B.G., T.F., P.R., J.L.B., R.M.F. and D.M. conducted clinical studies and collected parasite isolates. A.-C.L., N.K., S.K., V.D., S.M. and A.B. performed PCR and sequencing analyses. B.W., F.B.-V., V.D. and D.M. performed in vitro assays (RSA0–3 h). O.M. provided genotyping and population structure data for Cambodian parasite isolates. J.-C.B. and O.M.-P. performed three-dimensional structure modelling. F.A., R.M.F., F.B.-V., O.M.-P. and D.M. conceived of the study, supervised the project, processed the data and wrote the manuscript with contributions from B.W., C.A., A.B. and J.-C.B.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Frédéric Ariey or Rick M. Fairhurst or Françoise Benoit-Vical or Odile Mercereau-Puijalon or Didier Ménard.

The following reagents have been deposited to the MR4/BEI by D.M.: MRA-1236 (Plasmodium falciparum IPC 3445 Pailin Cambodia 2010), MRA-1237 (Plasmodium falciparum IPC 3663 Pailin Cambodia 2010), MRA-1238 (Plasmodium falciparum IPC 4884 Pursat Cambodia 2011), MRA-1239 (Plasmodium falciparum IPC 5188 Ratanakiri Cambodia 2011), MRA-1240 (Plasmodium falciparum IPC 5202 Battambang Cambodia 2011) and MRA-1241 (Plasmodium falciparum IPC 4912 Mondulkiri Cambodia 2011).

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https://doi.org/10.1038/nature12876

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