Plasmodium falciparum resistance to amodiaquine in monotherapy and in combination therapy with artesunate

In response to the increasing resistance to antimalarial monotherapies, artemisinin basedcombination therapy (ACT) is now recommended as first line therapy against uncomplicatedPlasmodium falciparum malaria. However, the choice of partner drug to artemisinin (ART) iscritical for ACT efficacy to endure. One main partner drug option for ACT is amodiaquine(AQ), with its active long half-life metabolite desethyl-amodiaquine (DEAQ). AQ is related tochloroquine (CQ) and has been widely used in Africa for decades, but despite widespread CQresistance it has remained relatively effective. CQ resistance has been associated with mutationsin the P. falciparum CQ resistance transporter (pfcrt) gene and the P. falciparum multiple drugresistance 1 (pfmdr1) gene. Possible mutations associated with AQ/DEAQ resistance haveremained unclear. In this thesis we explore whether mutations, especially in the pfcrt andpfmdr1 genes, are associated with tolerance/resistance to AQ/DEAQ in monotherapy and inACT, as well as a possible associated parasite fitness cost. The thesis is based on (a) in vivo clinical trials in East-Africa with AQ monotherapy or ARTplus AQ (ASAQ) combination therapy and (b) in vitro studies on isolates from Colombia and reference clones in which the pfmdr1 gene has been modified by allelic exchange. Mutationanalyses were done by PCR followed by either RFLP and/or DNA pyrosequencing or fullsequencing and gene amplification analysis was done by TaqMan probe based Real-Time PCR.The genetic results were related to drug susceptibilities determined by an HRP2-ELISA assayand parasite growth in competition experiments in vitro and/or the clinical outcome in vivo. The treatment failure rate after AQ therapy was relatively high (20%), while after ASAQtherapy it fulfilled the efficacy criteria of WHO (