The molecular basis of Plasmodium falciparum resistance to the antimalarial lumefantrine

Malaria control is compromised by the development and spread of Plasmodium falciparum resistance to antimalarial drugs, which has caused an increase in malaria related morbidity and mortality. Artemisinin based combination therapy (ACT) has been implemented in almost all malaria endemic areas in an attempt to suppress drug resistance. The ACT artemether-lumefantrine (Coartem®; Novartis) is one of the most important and most widely used ACTs, especially in Africa. The aim of this thesis was to investigate a possible molecular basis of P. falciparum resistance to the antimalarial drug lumefantrine, especially the role of the P. falciparum chloroquine resistance transporter gene (pfcrt) and the P. falciparum multidrug resistance gene 1 (pfmdr1). The results showed that treatment with artemether-lumefantrine selects parasites with pfmdr1 N86, 184F, D1246 and pfcrt K76 alleles in high transmission Africa. The observed selection occurred in newly acquired infections after treatment (reinfections). Therefore these alleles were interpreted as markers of tolerance rather than resistance. Selection of pfmdr1 N86 and pfcrt K76 carrying parasites was not observed after treatment with sulfadoxine-pyrimethamine (SP), indicating a drug specific selection. The involvement of pfcrt K76 in lumefantrine response was confirmed in vitro in isogenic pfcrt-modified parasite lines. Pfmdr1 gene amplification has been suggested to increase the risk of artemetherlumefantrine treatment failure. In an in vitro study on field samples from Thailand, we observed that pfmdr1 amplification was associated with a decreased susceptibility to lumefantrine as well as to its metabolite desbutylbenflumetol (DBB) and to artemisinin. Pfmdr1 amplification was not detected in any of the African study sites in Zanzibar, Tanzania and Uganda, but may potentially represent a further development towards resistance to lumefantrine. Overall, the results indicate that pfmdr1 and pfcrt may be involved in the mechanism of resistance to lumefantrine. Pfmdr1 N86 and pfcrt K76 as markers for tolerance may be of practical use, functioning as an early warning of emerging resistance. Pfcrt 76T, the molecular marker for chloroquine resistance, incurs lumefantrine sensitivity and may increase susceptibility to artemisinin. This suggests that artemether-lumefantrine is ideal in areas of chloroquine resistance. However, as some alleles are associated to both lumefantrine and artemisinin susceptibility, there is a risk for lumefantrine to accelerate the development of resistance to artemisinin derivatives. Although artemisinins may not select for these alleles themselves due to their fast elimination, lumefantrine pressure may provide the main selective force and drive a decrease in susceptibility to both drugs. This shows that an understanding of the molecular basis of drug resistance is crucial. LIST OF PUBLICATIONS I. Christin Sisowath, Johan Stromberg, Andreas Martensson, Mwinyi Msellem, Christine Obondo, Anders Bjorkman, Jose Pedro Gil. In vivo selection of Plasmodium falciparum pfmdr1 86N coding alleles by artemetherlumefantrine (Coartem®). J Infect Dis. 2005 Mar 15;191(6):1014-7. II. Christin Sisowath, Pedro E Ferreira, Leyla Y Bustamante, Sabina Dahlstrom, Andreas Martensson, Anders Bjorkman, Sanjeev Krishna, Jose Pedro Gil. The role of pfmdr1 in Plasmodium falciparum tolerance to artemether-lumefantrine in Africa. Trop Med Int Health. 2007 Jun;12(6):736-42. III. Christin Sisowath, Ines Petersen, M Isabel Veiga, Andreas Martensson, Zul Premji, Anders Bjorkman, David A Fidock, Jose Pedro Gil. In vivo selection of Plasmodium falciparum parasites carrying the chloroquine-susceptible pfcrt K76 allele after treatment with artemether-lumefantrine in Africa. J Infect Dis. 2009 Mar 1;199(5):750-757. IV. Christin Sisowath*, Sabina Dahlstrom*, Walther Wernsdorfer and Jose Pedro Gil. Association between pfmdr1, pfcrt, pfmrp1 and PfATP6 mutations and in vitro susceptibility of fresh Plasmodium falciparum isolates to antimalarial drugs. Manuscript.