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- The role of pfmdr1 in Plasmodium falciparum tolerance to artemether-lumefantrine in AfricaPublication . Sisowath, Christin; Ferreira, Pedro E.; Bustamante, Leyla Y.; Dahlstrom, Sabina; Martensson, Andreas; Bjorkman, Anders; Krishna, Sanjeev; Gil, José PedroObjective Artemether-lumefantrine (AL), presently the most favoured combination therapy against uncomplicated Plasmodium falciparum malaria in Africa, has recently shown to select for the pfmdr1 86N allele. The objective of this study was to search for the selection of other mutations potentially involved in artemether-lumefantrine tolerance and/or resistance, i.e. pfmdr1 gene amplification, pfmdr1 Y184F, S1034C, N1042D, D1246Y, pfcrt S163R and PfATP6 S769N. Methods The above mentioned SNPs were analysed by PCR-restriction fragment length polymorphism and pfmdr1 gene amplification by real-time PCR based protocols in parasites from 200 children treated with AL for uncomplicated P. falciparum malaria in Zanzibar. Results A statistically significant selection of pfmdr1 184F mostly in combination with 86N was seen in reinfections after treatment. No pfmdr1 gene amplification was found. Conclusion The results suggest that different pfmdr1 alleles are involved in the development of tolerance/resistance to lumefantrine.
- Plasmodium falciparum drug resistance phenotype as assessed by patient antimalarial drug levels and Its association With pfmdr1 polymorphismsPublication . Malmberg, Maja; Ferreira, Pedro; Tarning, Joel; Ursing, Johan; Ngasala, Billy; Bjorkman, Anders; Martensson, Andreas; Gil, José PedroBackground. Multidrug-resistant Plasmodium falciparum is a major threat to global malaria control. Parasites develop resistance by gradually acquiring genetic polymorphisms that decrease drug susceptibility. The aim of this study was to investigate the extent to which parasites with different genetic characteristics are able to withstand individual drug blood concentrations. Methods. We analyzed 2 clinical trials that assessed the efficacy and effectiveness of artemether-lumefantrine. As a proof of concept, we used measured day 7 lumefantrine concentrations to estimate the concentrations at which reinfections multiplied. P. falciparum multidrug resistance gene 1 (pfmdr1) genotypes of these parasites were then correlated to drug susceptibility. Results. Reinfecting parasites with the pfmdr1 N86/184F/D1246 haplotype were able to withstand lumefantrine blood concentrations 15-fold higher than those with the 86Y/Y184/1246Y haplotype. Conclusions. By estimating drug concentrations, we were able to quantify the contribution of pfmdr1 single-nucleotide polymorphisms to reduced lumefantrine susceptibility. The method can be applied to all long-half-life antimalarial drugs, enables early detection of P. falciparum with reduced drug susceptibility in vivo, and represents a novel way for unveiling molecular markers of antimalarial drug resistance.
- Assessing the cost-benefit effect of a plasmodium falciparum drug resistance mutation on parasite growth In vitroPublication . Froberg, Gabrielle; PE, Ferreira; Martensson, Andreas; Ali, Abdullah; Bjorkman, Anders; Gil, J. P.Plasmodium falciparum mutations associated with antimalarial resistance may be beneficial for parasites under drug pressure, although they may also cause a fitness cost. We herein present an in vitro model showing how this combined effect on parasite growth varies with the drug concentration and suggest a calculated drug-specific cost-benefit index, indicating the possible advantage for mutated parasites. We specifically studied the D-to-Y change at position 1246 encoded by the pfmdr1 gene (pfmdr1 D1246Y) in relation to amodiaquine resistance. Susceptibilities to amodiaquine, desethylamodiaquine, and chloroquine, as well as relative fitness, were determined for two modified isogenic P. falciparum clones differing only in the pfmdr1 1246 position. Data were used to create a new comparative graph of relative growth in relation to the drug concentration and to calculate the ratio between the benefit of resistance and the fitness cost. Results were related to an in vivo allele selection analysis after amodiaquine or artesunate-amodiaquine treatment. pfmdr1 1246Y was associated with decreased susceptibility to amodiaquine and desethylamodiaquine but at a growth fitness cost of 11%. Mutated parasites grew less in low drug concentrations due to a predominating fitness cost, but beyond a breakpoint concentration they grew more due to a predominating benefit of increased resistance. The cost-benefit indexes indicated that pfmdr1 1246Y was most advantageous for amodiaquine-exposed parasites. In vivo, a first drug selection of mutant parasites followed by a fitness selection of wild-type parasites supported the in vitro data. This cost-benefit model may predict the risk for selection of drug resistance mutations in different malaria transmission settings.
- Plasmodium falciparum multidrug resistance protein 1 and artemisinin-based combination therapy in AfricaPublication . Dahlstrom, Sabina; Ferreira, Pedro; Veiga, Maria Isabel; Sedighi, Nazli; Wiklund, Lisa; Martensson, Andreas; Farnert, Anna; Sisowath, Christin; Osorio, Lyda; Darban, Hamid; Andersson, Bjoern; Kaneko, Akira; Conseil, Gwanaelle; Bjorkman, Anders; Gil, José PedroPlasmodium falciparum response mechanisms to the major artemisinin-based combination therapies (ACTs) are largely unknown. Multidrug-resistance protein (MRP)-like adenosine triphosphate (ATP)-binding cassette transporters are known to be related to multidrug resistance in many organisms. Therefore, we hypothesized that sequence variation in pfmrp1 can contribute to decreased parasite sensitivity to ACT. Through sequencing of the pfmrp1 open reading frame for 103 geographically diverse P. falciparum infections, we identified 27 single-nucleotide polymorphisms (SNPs), of which 21 were nonsynonymous and 6 synonymous. Analyses of clinical efficacy trials with artesunate-amodiaquine and artemether-lumefantrine detected a specific selection of the globally prevalent I876V SNP in recurrent infections after artemether-lumefantrine treatment. Additional in silico studies suggested an influence of variation in amino acid 876 on the ATP hydrolysis cycle of pfMRP1 with potential impact on protein functionality. Our data suggest for the first time, to our knowledge, the involvement of pfMRP1 in P. falciparum in vivo response to ACT.
- Antimalarial Exposure Delays Plasmodium falciparum Intra-Erythrocytic Cycle and Drives Drug Transporter Genes ExpressionPublication . Isabel Veiga, Maria; Ferreira, Pedro; Schmidt, Berit Aydin; Ribacke, Ulf; Bjorkman, Anders; Tichopad, Ales; Gil, José PedroBackground: Multi-drug resistant Plasmodium falciparum is a major obstacle to malaria control and is emerging as a complex phenomenon. Mechanisms of drug evasion based on the intracellular extrusion of the drug and/or modification of target proteins have been described. However, cellular mechanisms related with metabolic activity have also been seen in eukaryotic systems, e. g. cancer cells. Recent observations suggest that such mechanism may occur in P. falciparum. Methodology/Principal Findings: We therefore investigated the effect of mefloquine exposure on the cell cycle of three P. falciparum clones (3D7, FCB, W2) with different drug susceptibilities, while investigating in parallel the expression of four genes coding for confirmed and putative drug transporters (pfcrt, pfmdr1, pfmrp1 and pfmrp2). Mefloquine induced a previously not described dose and clone dependent delay in the intra-erythrocytic cycle of the parasite. Drug impact on cell cycle progression and gene expression was then merged using a non-linear regression model to determine specific drug driven expression. This revealed a mild, but significant, mefloquine driven gene induction up to 1.5 fold. Conclusions/Significance: Both cell cycle delay and induced gene expression represent potentially important mechanisms for parasites to escape the effect of the antimalarial drug.