We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    et al. The threat of artemisinin-resistant malaria. N. Engl. J. Med. 365, 1073–1075 (2011).

  2. 2.

    et al. Artemisinin resistance in Plasmodium falciparum malaria. N. Engl. J. Med. 361, 455–467 (2009).

  3. 3.

    et al. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet 379, 1960–1966 (2012).

  4. 4.

    et al. In vivo susceptibility of Plasmodium falciparum to artesunate in Binh Phuoc Province, Vietnam. Malar. J. 11, 355 (2012).

  5. 5.

    et al. Reduced susceptibility of Plasmodium falciparum to artesunate in southern Myanmar. PLoS ONE 8, e57689 (2013).

  6. 6.

    et al. Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study. Lancet Infect. Dis. 12, 851–858 (2012).

  7. 7.

    Tanabe, K. & Kita, K. Spread and evolution of Plasmodium falciparum drug resistance. Parasitol. Int. 58, 201–209 (2009).

  8. 8.

    et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505, 50–55 (2014).

  9. 9.

    et al. Spread of artemisinin resistance in Plasmodium falciparum malaria. N. Engl. J. Med. 371, 411–423 (2014).

  10. 10.

    et al. Using CF11 cellulose columns to inexpensively and effectively remove human DNA from Plasmodium falciparum–infected whole blood samples. Malar. J. 11, 41 (2012).

  11. 11.

    et al. Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing. Nature 487, 375–379 (2012).

  12. 12.

    et al. Improved linear mixed models for genome-wide association studies. Nat. Methods 9, 525–526 (2012).

  13. 13.

    et al. Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia. Proc. Natl. Acad. Sci. USA 110, 240–245 (2013).

  14. 14.

    et al. A major genome region underlying artemisinin resistance in malaria. Science 336, 79–82 (2012).

  15. 15.

    et al. Spread of artemisinin resistance in Plasmodium falciparum malaria. N. Engl. J. Med. 371, 411–423 (2014).

  16. 16.

    et al. Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia. Nat. Genet. 45, 648–655 (2013).

  17. 17.

    et al. Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia. J. Infect. Dis. (1 September 2014).

  18. 18.

    & Apicoplast translation, transcription and genome replication: targets for antimalarial antibiotics. Trends Parasitol. 24, 279–284 (2008).

  19. 19.

    , , , & Molecular interaction of ferredoxin and ferredoxin–NADP+ reductase from human malaria parasite. J. Biochem. 142, 715–720 (2007).

  20. 20.

    & Hydroxylated metabolites of the antimalarial drug primaquine. Oxidation and redox cycling. J. Biol. Chem. 267, 6848–6854 (1992).

  21. 21.

    et al. Identification and functional validation of the novel antimalarial resistance locus PF10_0355 in Plasmodium falciparum. PLoS Genet. 7, e1001383 (2011).

  22. 22.

    et al. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol. Cell 6, 861–871 (2000).

  23. 23.

    , , & Characterization of the pfmdr2 gene for Plasmodium falciparum. Mol. Biochem. Parasitol. 62, 83–92 (1993).

  24. 24.

    , , & Whole genome re-sequencing identifies a mutation in an ABC transporter (mdr2) in a Plasmodium chabaudi clone with altered susceptibility to antifolate drugs. Int. J. Parasitol. 41, 165–171 (2011).

  25. 25.

    et al. The F423Y mutation in the pfmdr2 gene and mutations N51I, C59R, and S108N in the pfdhfr gene are independently associated with pyrimethamine resistance in Plasmodium falciparum isolates. Antimicrob. Agents Chemother. 56, 2750–2752 (2012).

  26. 26.

    et al. pfmdr2 confers heavy metal resistance to Plasmodium falciparum. J. Biol. Chem. 281, 27039–27045 (2006).

  27. 27.

    et al. Polymorphism in Plasmodium falciparum drug transporter proteins and reversal of in vitro chloroquine resistance by a 9,10-dihydroethanoanthracene derivative. Antimicrob. Agents Chemother. 48, 4869–4872 (2004).

  28. 28.

    , , & Epidemiology of drug-resistant malaria. Lancet Infect. Dis. 2, 209–218 (2002).

  29. 29.

    Did medicated salt hasten the spread of chloroquine resistance in Plasmodium falciparum? Parasitol.Today 4, 112–115 (1988).

  30. 30.

    , , , & Mind the gaps—the epidemiology of poor-quality anti-malarials in the malarious world—analysis of the WorldWide Antimalarial Resistance Network database. Malar. J. 13, 139 (2014).

  31. 31.

    , & Variations in frequencies of drug resistance in Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 94, 9389–9393 (1997).

  32. 32.

    , , & Genetic predisposition favors the acquisition of stable artemisinin resistance in malaria parasites. Antimicrob. Agents Chemother. 55, 50–55 (2011).

  33. 33.

    & QTL analysis for discovery of genes involved in drug responses. Curr. Drug Targets Infect. Disord. 4, 53–63 (2004).

  34. 34.

    et al. A new world malaria map: Plasmodium falciparum endemicity in 2010. Malar. J. 10, 378 (2011).

  35. 35.

    , , & Standardizing the measurement of parasite clearance in falciparum malaria: the parasite clearance estimator. Malar. J. 10, 339 (2011).

  36. 36.

    et al. An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing. PLoS ONE 6, e22213 (2011).

  37. 37.

    et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008).

  38. 38.

    & Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

  39. 39.

    & SNP-o-matic. Bioinformatics 25, 2434–2435 (2009).

  40. 40.

    Some distance properties of latent root and vector methods used in multivariate analysis. Biometrika 53, 325–328 (1966).

  41. 41.

    & The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425 (1987).

  42. 42.

    , & Fast model-based estimation of ancestry in unrelated individuals. Genome Res. 19, 1655–1664 (2009).

  43. 43.

    , & Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14, 2611–2620 (2005).

Download references


We thank the following colleagues for their efforts in support of this work: P. Vauterin, G. Band and Q.S. Le; J. Anderson, D. Dek, S. Duong, R. Gwadz, S. Mao, V. Ou, B. Sam, C. Sopha, V. Try and T. Wellems; the personnel at Phuoc Long Hospital, Bu Gia Map Health Station, Malaria Control Center of Binh Phuoc Province, Vietnam; M. Phommasansack, B. Phimphalat and C. Vilayhong; and A.K. Tshefu. Special thanks are given to V. Cornelius and K. Johnson for their continual support of the analysis group. The sequencing for this study was funded by the Wellcome Trust through core funding of the Wellcome Trust Sanger Institute (098051). The Wellcome Trust also supports the Wellcome Trust Centre for Human Genetics (090532/Z/09/Z), the Resource Centre for Genomic Epidemiology of Malaria (090770/Z/09/Z) and the Wellcome Trust Mahidol University Oxford Tropical Medicine Research Programme. The Centre for Genomics and Global Health is supported by the UK Medical Research Council (G0600718). This work was funded in part by the Bill and Melinda Gates Foundation (OPP1040463), the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, US National Institutes of Health and the Department for International Development (PO5408). P.R. is a staff member of the World Health Organization. The views expressed in this publication are those of the authors and do not necessarily reflect the positions, decisions, policies or views of their employers or of the funding organizations.

Author information

Author notes

    • Olivo Miotto
    •  & Roberto Amato

    These authors contributed equally to this work.


  1. Wellcome Trust Sanger Institute, Hinxton, UK.

    • Olivo Miotto
    • , Roberto Amato
    • , Bronwyn MacInnis
    • , Jacob Almagro-Garcia
    • , Daniel Mead
    • , Samuel O Oyola
    • , Magnus Manske
    • , Jim Stalker
    • , Eleanor Drury
    • , Susana Campino
    •  & Dominic P Kwiatkowski
  2. Medical Research Council (MRC) Centre for Genomics and Global Health, University of Oxford, Oxford, UK.

    • Olivo Miotto
    • , Roberto Amato
    • , Bronwyn MacInnis
    • , Jacob Almagro-Garcia
    • , Magnus Manske
    • , Jim Stalker
    • , Susana Campino
    • , Lucas Amenga-Etego
    • , Chris C A Spencer
    • , Gilean McVean
    •  & Dominic P Kwiatkowski
  3. Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

    • Olivo Miotto
    • , Elizabeth A Ashley
    • , Charles Woodrow
    • , Francois Nosten
    • , Aung Pyae Phyo
    • , Caterina I Fanello
    • , Nicholas P Day
    • , Arjen M Dondorp
    •  & Nicholas J White
  4. Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

    • Roberto Amato
    • , Jacob Almagro-Garcia
    • , Chris C A Spencer
    • , Gilean McVean
    •  & Dominic P Kwiatkowski
  5. Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

    • Elizabeth A Ashley
    • , Mehul Dhorda
    • , Charles Woodrow
    • , Hien Tinh Tran
    • , Francois Nosten
    • , Aung Pyae Phyo
    • , Paul N Newton
    • , Mayfong Mayxay
    • , Caterina I Fanello
    • , Marie Onyamboko
    • , Nicholas P Day
    • , Arjen M Dondorp
    •  & Nicholas J White
  6. Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA.

    • Chanaki Amaratunga
    • , Pharath Lim
    •  & Rick M Fairhurst
  7. National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia.

    • Pharath Lim
    • , Char Meng Chuor
    • , Chea Nguon
    • , Seila Suon
    •  & Sokunthea Sreng
  8. Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, USA.

    • Mehul Dhorda
    • , Christopher G Jacob
    • , Shannon Takala-Harrison
    •  & Christopher V Plowe
  9. WorldWide Antimalarial Resistance Network (WWARN), Asia Regional Centre, Mahidol University, Bangkok, Thailand.

    • Mehul Dhorda
  10. Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

    • Mallika Imwong
    • , Sasithon Pukrittayakamee
    •  & Kesinee Chotivanich
  11. Navrongo Health Research Centre, Navrongo, Ghana.

    • Lucas Amenga-Etego
  12. Oxford University Clinical Research Unit, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam.

    • Thuy-Nhien Nguyen Thanh
    •  & Hien Tinh Tran
  13. Global Malaria Programme, World Health Organization, Geneva, Switzerland.

    • Pascal Ringwald
  14. Department of Immunology and Medicine, US Army Medical Component, Armed Forces Research Institute of Medical Sciences (USAMC-AFRIMS), Bangkok, Thailand.

    • Delia Bethell
  15. Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand.

    • Francois Nosten
    •  & Aung Pyae Phyo
  16. Lao Oxford Mahosot Wellcome Trust Research Unit (LOMWRU), Mahosot Hospital, Vientiane, Laos.

    • Paul N Newton
    •  & Mayfong Mayxay
  17. Faculty of Postgraduate Studies, University of Health Sciences, Vientiane, Laos.

    • Mayfong Mayxay
  18. Center of Malariology, Parasitology and Entomology, Ministry of Health, Vientiane, Laos.

    • Maniphone Khanthavong
    •  & Bouasy Hongvanthong
  19. Department of Medical Research, Lower Myanmar, Yangon, Myanmar.

    • Ye Htut
    • , Kay Thwe Han
    •  & Myat Phone Kyaw
  20. Malaria Research Group and Dev Care Foundation, Dhaka, Bangladesh.

    • Md Abul Faiz
  21. Kinshasa School of Public Health, Kinshasa, Democratic Republic of the Congo.

    • Marie Onyamboko
  22. Department of Paediatrics and Child Health, University of Ilorin, Ilorin, Nigeria.

    • Olugbenga A Mokuolu
  23. Howard Hughes Medical Institute, University of Maryland School of Medicine, Baltimore, Maryland, USA.

    • Christopher V Plowe
  24. Department of Statistics, University of Oxford, Oxford, UK.

    • Gilean McVean


  1. Search for Olivo Miotto in:

  2. Search for Roberto Amato in:

  3. Search for Elizabeth A Ashley in:

  4. Search for Bronwyn MacInnis in:

  5. Search for Jacob Almagro-Garcia in:

  6. Search for Chanaki Amaratunga in:

  7. Search for Pharath Lim in:

  8. Search for Daniel Mead in:

  9. Search for Samuel O Oyola in:

  10. Search for Mehul Dhorda in:

  11. Search for Mallika Imwong in:

  12. Search for Charles Woodrow in:

  13. Search for Magnus Manske in:

  14. Search for Jim Stalker in:

  15. Search for Eleanor Drury in:

  16. Search for Susana Campino in:

  17. Search for Lucas Amenga-Etego in:

  18. Search for Thuy-Nhien Nguyen Thanh in:

  19. Search for Hien Tinh Tran in:

  20. Search for Pascal Ringwald in:

  21. Search for Delia Bethell in:

  22. Search for Francois Nosten in:

  23. Search for Aung Pyae Phyo in:

  24. Search for Sasithon Pukrittayakamee in:

  25. Search for Kesinee Chotivanich in:

  26. Search for Char Meng Chuor in:

  27. Search for Chea Nguon in:

  28. Search for Seila Suon in:

  29. Search for Sokunthea Sreng in:

  30. Search for Paul N Newton in:

  31. Search for Mayfong Mayxay in:

  32. Search for Maniphone Khanthavong in:

  33. Search for Bouasy Hongvanthong in:

  34. Search for Ye Htut in:

  35. Search for Kay Thwe Han in:

  36. Search for Myat Phone Kyaw in:

  37. Search for Md Abul Faiz in:

  38. Search for Caterina I Fanello in:

  39. Search for Marie Onyamboko in:

  40. Search for Olugbenga A Mokuolu in:

  41. Search for Christopher G Jacob in:

  42. Search for Shannon Takala-Harrison in:

  43. Search for Christopher V Plowe in:

  44. Search for Nicholas P Day in:

  45. Search for Arjen M Dondorp in:

  46. Search for Chris C A Spencer in:

  47. Search for Gilean McVean in:

  48. Search for Rick M Fairhurst in:

  49. Search for Nicholas J White in:

  50. Search for Dominic P Kwiatkowski in:


C.A., P.L., M.D., M.I., L.A.-E., T.-N.N.T., H.T.T., D.B., F.N., A.P.P., S.P., K.C., C.M.C., C.N., S. Suon, S. Sreng, P.N.N., M. Mayxay, M.K., B.H., Y.H., K.T.H., M.P.K., M.A.F., C.I.F., M.O. and O.A.M. carried out field and laboratory work to obtain P. falciparum samples for sequencing. E.A.A., C.A., P.L., T.-N.N.T., H.T.T., D.B., F.N., A.P.P., S.P., K.C., C.M.C., C.N., P.N.N., M. Mayxay, M.K., B.H., Y.H., K.T.H., M.P.K., M.A.F., C.I.F., M.O. and O.A.M. carried out clinical studies to obtain parasite clearance data. D.M., S.O.O., E.D., S.C. and B.M. developed and implemented methods for sample processing and sequencing library preparation. J.S. and M. Manske managed data production pipelines. E.A.A., C.W., P.R., C.G.J., S.T.-H., C.V.P., N.P.D., A.M.D., R.M.F., N.J.W., O.M., B.M. and D.P.K. contributed to study design and management. O.M., R.A., J.A.-G., C.C.A.S., G.M. and D.P.K. performed data analyses. O.M., R.A., J.A.-G., C.C.A.S., G.M. and D.P.K. performed data analyses. O.M., R.A. and D.P.K. drafted the manuscript, which was reviewed by all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Dominic P Kwiatkowski.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–14, Supplementary Tables 1–15 and Supplementary Note.

About this article

Publication history






Further reading