Abstract
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 × 10-8 and 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.
Highlights
Artemisinin combination therapy (ACT) – the use of a short-acting artemisinin derivative and a long-acting partner drug – is recommended worldwide for the treatment of Plasmodium falciparum malaria.[1]
Artemisinin resistance, which has evolved across the Greater Mekong Subregion (GMS),[7,8,13] threatens to compromise the efficacy of dihydroartemisinin-piperaquine and other ACTs in the global treatment and elimination of malaria.[14]
These findings, and the discovery that piperaquine-resistant parasites are sensitive to the former ACT partner drug mefloquine,[4] have recently led Cambodia’s national malaria control program and the World Health Organisation to recommend artesunate-mefloquine as the first-line ACT in 10 Cambodian provinces, including Pursat and Preah Vihear
Summary
Artemisinin combination therapy (ACT) – the use of a short-acting artemisinin derivative and a long-acting partner drug – is recommended worldwide for the treatment of Plasmodium falciparum malaria.[1] Dihydroartemisinin-piperaquine, a current frontline ACT in Cambodia, Vietnam, Thailand, Myanmar, China, and Indonesia is failing in Cambodian provinces where artemisinin resistance[2] has emerged and spread.[3,4,5,6,7,8,9,10,11,12] This situation likely arose because the survival of parasites after artemisinin exposure increases the chance that they develop spontaneous genetic resistance to piperaquine, survive declining piperaquine plasma concentrations, and propagate via mosquitoes to other humans. Molecular markers are urgently needed for large-scale surveillance programs to predict dihydroartemisinin-piperaquine failures in Cambodia and other GMS countries, and investigate the molecular mechanism of piperaquine resistance
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