Genetic markers associated with dihydroartemisinin-piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype-phenotype association study.
As the prevalence of artemisinin-resistant Plasmodium falciparum malaria increases in the Greater Mekong subregion, emerging resistance to partner drugs in artemisinin combination therapies seriously threatens global efforts to treat and eliminate this disease. Molecular markers that predict failure of artemisinin combination therapy are urgently needed to monitor the spread of partner drug resistance, and to recommend alternative treatments in southeast Asia and beyond.



We did a genome-wide association study of 297 P falciparum isolates from Cambodia to investigate the relationship of 11 630 exonic single-nucleotide polymorphisms (SNPs) and 43 copy number variations (CNVs) with in-vitro piperaquine 50% inhibitory concentrations (IC50s), and tested whether these genetic variants are markers of treatment failure with dihydroartemisinin-piperaquine. We then did a survival analysis of 133 patients to determine whether candidate molecular markers predicted parasite recrudescence following dihydroartemisinin-piperaquine treatment.



Piperaquine IC50s increased significantly from 2011 to 2013 in three Cambodian provinces (2011 vs 2013 median IC50s: 20·0 nmol/L [IQR 13·7-29·0] vs 39·2 nmol/L [32·8-48·1] for Ratanakiri, 19·3 nmol/L [15·1-26·2] vs 66·2 nmol/L [49·9-83·0] for Preah Vihear, and 19·6 nmol/L [11·9-33·9] vs 81·1 nmol/L [61·3-113·1] for Pursat; all p≤10-3; Kruskal-Wallis test). Genome-wide analysis of SNPs identified a chromosome 13 region that associates with raised piperaquine IC50s. A non-synonymous SNP (encoding a Glu415Gly substitution) in this region, within a gene encoding an exonuclease, associates with parasite recrudescence following dihydroartemisinin-piperaquine treatment. Genome-wide analysis of CNVs revealed that a single copy of the mdr1 gene on chromosome 5 and a novel amplification of the plasmepsin 2 and plasmepsin 3 genes on chromosome 14 also associate with raised piperaquine IC50s. After adjusting for covariates, both exo-E415G and plasmepsin 2-3 markers significantly associate (p=3·0 × 10-8and p=1·7 × 10-7, respectively) with decreased treatment efficacy (survival rates 0·38 [95% CI 0·25-0·51] and 0·41 [0·28-0·53], respectively).



The exo-E415G SNP and plasmepsin 2-3 amplification are markers of piperaquine resistance and dihydroartemisinin-piperaquine failures in Cambodia, and can help monitor the spread of these phenotypes into other countries of the Greater Mekong subregion, and elucidate the mechanism of piperaquine resistance. Since plasmepsins are involved in the parasite's haemoglobin-to-haemozoin conversion pathway, targeted by related antimalarials, plasmepsin 2-3 amplification probably mediates piperaquine resistance.



Intramural Research Program of the US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, and UK Department for International Development.
Wellcome Trust Sanger Institute, Hinxton, UK; Centre for Genomics and Global Health, Wellcome Trust Centre for Human Genetics, Oxford, UK. Electronic address: ra4@sanger.ac.uk.
Roberto
R
Amato
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA; National Center for Parasitology, Entomology, and Malaria Control, Phnom Penh, Cambodia.
Pharath
P
Lim
Wellcome Trust Sanger Institute, Hinxton, UK; Centre for Genomics and Global Health, Wellcome Trust Centre for Human Genetics, Oxford, UK; Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand.
Olivo
O
Miotto
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.
Chanaki
C
Amaratunga
National Center for Parasitology, Entomology, and Malaria Control, Phnom Penh, Cambodia.
Dalin
D
Dek
Wellcome Trust Sanger Institute, Hinxton, UK; Centre for Genomics and Global Health, Wellcome Trust Centre for Human Genetics, Oxford, UK.
Richard D
RD
Pearson
Wellcome Trust Sanger Institute, Hinxton, UK; Centre for Genomics and Global Health, Wellcome Trust Centre for Human Genetics, Oxford, UK.
Jacob
J
Almagro-Garcia
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.
Aaron T
AT
Neal
National Center for Parasitology, Entomology, and Malaria Control, Phnom Penh, Cambodia.
Sokunthea
S
Sreng
National Center for Parasitology, Entomology, and Malaria Control, Phnom Penh, Cambodia.
Seila
S
Suon
Wellcome Trust Sanger Institute, Hinxton, UK.
Eleanor
E
Drury
Wellcome Trust Sanger Institute, Hinxton, UK.
Dushyanth
D
Jyothi
Wellcome Trust Sanger Institute, Hinxton, UK.
Jim
J
Stalker
Wellcome Trust Sanger Institute, Hinxton, UK; Centre for Genomics and Global Health, Wellcome Trust Centre for Human Genetics, Oxford, UK.
Dominic P
DP
Kwiatkowski
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA. Electronic address: rfairhurst@niaid.nih.gov.
Rick M
RM
Fairhurst
D000962:Antimalarials / Q000494:pharmacology / Q000627:therapeutic use; D037621:Artemisinins / Q000627:therapeutic use*; D002160:Cambodia / Q000453:epidemiology; D004351:Drug Resistance; D004359:Drug Therapy, Combination; D056726:Genetic Association Studies; D005819:Genetic Markers; D055106:Genome-Wide Association Study; D006801:Humans; D020128:Inhibitory Concentration 50; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000453:epidemiology*; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines / Q000627:therapeutic use*; D017211:Treatment Failure; D010879:Piperazines
D000962:Antimalarials; D037621:Artemisinins; D005819:Genetic Markers; D011804:Quinolines; C039060:artenimol; C034759:piperaquine; D010879:Piperazines