From Department of Medicine, Solna Infectious Diseases Unit Karolinska University Hospital Karolinska Institutet, Stockholm, Sweden PLASMODIUM FALCIPARUM DRUG TRANSPORTER GENES IN EMERGING MALARIA MULTIDRUG RESISTANCE

Malaria is caused by an intracellular protozoan parasite of the genus Plasmodium. The use of chemotherapy, the foremost tool available for the control of the disease, has been challenged in the last decades by the development and spread of drug resistance among malaria parasites. A clear understanding behind the mechanisms of parasite resistance is required for the improvement of treatment efficacy, policy assessment and in the development of new drugs. A common strategy used by parasites in achieving resistance involves decreasing drug accumulation inside the cell. This is typically accomplished by increasing the availability of transporter proteins that mediate the efflux of the active compound. The goal of this thesis was to better understand the involvement of drug transporter genes in the molecular mechanisms underlying drug susceptibility in Plasmodium falciparum malaria. The approaches involved clinical drug trials, clinical isolates and extensive studies of laboratory P. falciparum parasites. The contribution of pfmrp1 polymorphisms in in vivo parasite drug response was studied in P. falciparum infected patients from drug efficacy clinical trials. After Sulphadoxine-Pyrimethamine treatment, recrudescent infections selected for parasites that had a lysine at amino acid position 1466 in pfmrp1, thus providing the first indication that this transporter gene may have a role in P. falciparum antifolate drug responses in vivo. We examined the effect of the ACT partner drug, mefloquine, on the intra-erythrocytic cell cycle of P. falciparum laboratory parasites having different in vitro drug susceptibilities, while in parallel investigating the expression of four pivotal drug transporter genes: pfcrt, pfmdr1, pfmrp1 and pfmrp2. This study revealed a delay in the cell cycle of the parasite after drug pressure, accompanied by gene induction of the transporter genes studied. The genetic background of the drug transporter genes pfcrt, pfmdr1, pfmrp1 and pfmrp2 were further studied at length in field isolates collected at the Thai-Myanmar border, a historically known epicenter of resistance. The isolates were characterized in vitro for their sensitivity against a broad range of ACT relevant antimalarials. Correlation analyses revealed novel candidate markers for multidrug resistance against structurally unrelated antimalarial drugs used extensively in ACT regimens worldwide. In conclusion, these studies reinforce the concept of malaria drug resistance as a multifactorial and complex phenomenon that may involve not only the parasite’s handling of the incoming drug, but also concomitant responses of its basic physiology. LIST OF PUBLICATIONS I. Dahlström S, Veiga MI, Mårtensson A, Björkman A, Gil JP. Polymorphism in PfMRP1 (Plasmodium falciparum multidrug resistance protein 1) amino acid 1466 associated with resistance to sulfadoxine-pyrimethamine treatment. Antimicrob Agents Chemother. 2009; 53(6):2553-6. (§-shared authorship) II. Veiga MI, Ferreira PE, Schmidt BA, Ribacke U, Björkman A, Tichopad A, Gil JP. Antimalarial exposure delays Plasmodium falciparum intraerythrocytic cycle and drives drug transporter genes expression. PLoS One. 2010; 5(8). III. Veiga MI, Ferreira PE, Jörnhagen L, Malmberg M, Kone A, Schmidt BA, Petzold M, Bjorkman A, Nosten F, Gil JP. Novel polymorphisms in Plasmodium falciparum ABC transporter genes associated to major ACT antimalarial drugs. Submitted. IV. Veiga MI, Franzen O, Ferreira PE, Dahlstrom S, Lum JK, Nosten F, Gil JP. Complex polymorphism in the Plasmodium falciparum Multidrug Resistance Protein 2 gene (pfmrp2). Manuscript. Publications not included in this thesis: Ghanchi NK, Ursing J, Beg MA, Veiga MI, Jafri S, Martensson A Prevalence of resistance associated polymorphisms in Plasmodium falciparum field isolates from southern Pakistan.. Malar J. 2011;10(1):18. Dahlström S, Ferreira PE, Veiga MI, Sedighi N, Wiklund L, Mårtensson A, Färnert A, Sisowath C, Osório L, Darban H, Andersson B, Kaneko A, Conseil G, Björkman A, Gil JP. Plasmodium falciparum multidrug resistance protein 1 and artemisinin-based combination therapy in Africa. J Infect Dis. 2009;200(9):1456-64. Sisowath C, Petersen I, Veiga MI, Mårtensson A, Premji Z, Björkman A, Fidock DA, Gil JP. In vivo selection of Plasmodium falciparum parasites carrying the chloroquinesusceptible pfcrt K76 allele after treatment with artemether-lumefantrine in Africa. J Infect Dis. 2009;199(5):750-7. Veiga MI, Asimus S, Ferreira PE, Martins JP, Cavaco I, Ribeiro V, Hai TN, Petzold MG, Björkman A, Ashton M, Gil JP. Pharmacogenomics of CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and MDR1 in Vietnam. Eur J Clin Pharmacol. 2009;65(4):355-63. Dahlström S, Veiga MI, Ferreira P, Mårtensson A, Kaneko A, Andersson B, Björkman A, Gil JP. Diversity of the sarco/endoplasmic reticulum Ca(2+)-ATPase orthologue of Plasmodium falciparum (PfATP6). Infect Genet Evol. 2008;8(3):340-5. Ferreira PE, Veiga MI, Cavaco I, Martins JP, Andersson B, Mushin S, Ali AS, Bhattarai A, Ribeiro V, Björkman A, Gil JP. Polymorphism of antimalaria drug metabolizing, nuclear receptor, and drug transport genes among malaria patients in Zanzibar, East Africa. Ther Drug Monit. 2008;30(1):10-5. Eriksen J, Mwankusye S, Mduma S, Veiga MI, Kitua A, Tomson G, Petzold MG, Swedberg G, Gustafsson LL, Warsame M. Antimalarial resistance and DHFR/DHPS genotypes of Plasmodium falciparum three years after introduction of sulfadoxinepyrimethamine and amodiaquine in rural Tanzania. Trans R Soc Trop Med Hyg. 2008;102(2):137-42. Cavaco I, Asimus S, Peyrard-Janvid M, Ferreira PE, Veiga MI, Hai TN, Ribeiro V, Ashton M, Gil JP. The Vietnamese Khin population harbors particular Nacetyltransferase 2 allele frequencies. Clin Chem. 2007;53(11):1977-9. Martensson A, Ngasala B, Ursing J, Isabel Veiga M, Wiklund L, Membi C, Montgomery SM, Premji Z, Farnert A, Bjorkman A. Influence of consecutive-day blood sampling on polymerase chain reaction-adjusted parasitological cure rates in an antimalarial-drug trial conducted in Tanzania. J Infect Dis. 2007;195(4):597-601. Veiga MI, Ferreira PE, Björkman A, Gil JP. Multiplex PCR-RFLP methods for pfcrt, pfmdr1 and pfdhfr mutations in Plasmodium falciparum. Mol Cell Probes. 2006;20(2):100-4. Holmgren G, Gil JP, Ferreira PM, Veiga MI, Obonyo CO, Björkman A Amodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of pfcrt 76T and pfmdr1 86Y.. Infect Genet Evol. 2006;6(4):309-14.