Genetic markers associated with dihydroartemisinin–piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype–phenotype association study

Abstract

Summary Background As the prevalence of artemisinin-resistant Plasmodium falciparum malaria increases in the Greater Mekong Subregion (GMS), emerging resistance to partner drugs in artemisinin combination therapies (ACTs) seriously threatens global efforts to treat and eliminate this disease. Molecular markers for ACT failure are urgently needed to monitor the spread of partner drug resistance, and to recommend alternative treatments in Southeast Asia and beyond. Methods We performed a genome-wide association study (GWAS) 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 dihydroartemisinin-piperaquine failures. We then performed a survival analysis of 133 patients to determine whether candidate molecular markers predicted parasite recrudescence following dihydroartemisinin-piperaquine treatment. Findings Piperaquine IC50s increased significantly from 2011 to 2013 in 3 Cambodian provinces. Genome-wide analysis of SNPs identified a chromosome 13 region that associates with elevated piperaquine IC50s. A nonsynonymous 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 II and plasmepsin III genes on chromosome 14 also associate with elevated piperaquine IC50s. After adjusting for covariates, both exo-E415G and plasmepsin II-III markers significantly associate with decreased treatment efficacy (0.38 and 0.41 survival rates, respectively). Interpretation The exo-E415G SNP and plasmepsin II-III amplification are markers of piperaquine resistance and dihydroartemisinin-piperaquine failures in Cambodia, and can help monitor the spread of these phenotypes into GMS countries, 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 II-III amplification likely mediates piperaquine resistance. Funding Intramural Research Program of the US National Institute of Allergy and Infectious Diseases, National Institutes of Health; Wellcome Trust; Bill and Melinda Gates Foundation; Medical Research Council; and UK Department for International Development.

Changing Antimalarial Drug Sensitivities in Uganda

“…The A578S mutation has been described in isolates from Uganda and other African countries, and it has not been associated with artemisinin resistance (29,32,33). Recent reports have identified amplification of a gene encoding plasmepsin 2 and a SNP in an exonuclease gene (PF3D7_1362500) that encodes an E415G mutation as markers of piperaquine resistance in Southeast Asia (24,25). We assessed the prevalence of these polymorphisms in Ugandan parasites.…”

“…Of great concern is the identification of artemisinin resistance within the last decade and of piperaquine resistance very recently in Southeast Asia (22,24,25,46). Important advances from groups in Asia have identified parasitological (30,31) and molecular (21,(23)(24)(25) markers of resistance to these drugs which, when seen together, have been accompanied by high rates of treatment failure with DP (31,47).…”

“…Important advances from groups in Asia have identified parasitological (30,31) and molecular (21,(23)(24)(25) markers of resistance to these drugs which, when seen together, have been accompanied by high rates of treatment failure with DP (31,47). We utilized these new tools to characterize parasites circulating in Tororo in 2016.…”

“…pfmdr1 copy number was determined in quadruplicate using a TaqMan real-time PCR assay with 3D7 and Dd2 strain standards, as previously described (12,50). The k13-propeller-encoding domains (codons 440 to 726; PF3D7_1343700) (21) and an exonuclease gene (PF3D7_1362500) (24) were amplified and sequenced using previously described methods and primers. For the exonuclease gene, samples that failed the initial round of PCR were amplified using nested PCR with sequencing primers as previously described (24).…”

“…More recently, resistance to piperaquine also emerged in Southeast Asia, linked to an increased plasmepsin 2 (PF3D7_1408000) copy number (24,25) and, in one study, an E415G mutation in an exonuclease (encoded by PF3D7_1362500) (24). In the same region, amplification of the pfmdr1 gene has been linked to decreased sensitivity to mefloquine and lumefantrine but increased sensitivity to aminoquinolines (26,27).…”