PfK13-Independent determinants of susceptibility of African Plasmodium falciparum to artemisinin and partner drugs in vitro

Artemisinin-based combination therapy (ACT) is currently the most effective treatment strategy against Plasmodium falciparum malaria. There have been reports from Southeast Asia of parasite resistance to artemisinin and its derivatives, and there are fears that resistance to this class of drugs will spread to Africa. Artemisinin resistance is recognised by a relatively slow parasite clearance rate in patients receiving an ACT or artemisinin. In-vitro or ex-vivo methods are conventionally used to determine the EC50 value – the effective concentration of drug that inhibits parasite growth by 50% for parasite lines in the laboratory. To date, there have been no consistent correlations between the estimated in-vivo half-life of P. falciparum treated with artemisinin in-vivo and in-vitro EC50 estimates from standard 48hr artemisinin susceptibility assays. This study investigates alternative methods of assaying artemisinin resistance by in vitro exposure to dihydroartemisin (DHA) - the major metabolite of all artemisinins. DHA has a short half-life (1-2hr) in-vivo and so short drug pulses were applied to parasites in an effort to better mimic in-vivo conditions. A 6hr drug pulse assay and a standard 48hr assay were used to observe any differences that may exist in EC50 values among field isolates from Kenya (HL1204), Ghana (HL1210) and Nigeria (HL1212). In all three isolates tested, the mean DHA EC50 values for the 6hr pulse assays were higher than estimates from standard 48hr assays. This trend was seen as an elevated EC50 value in the dose response curve, allowing discrimination of DHA EC50 6hr values among the isolates. Field isolates from Ghana (6 fold higher than 3D7 lab strain) and Nigeria (3.4 fold higher) showed elevated mean DHA EC50 6hr value compared to that from Kenya (1.7 fold higher than 3D7). When this assay was applied to parasites from Cambodia with known PfK13- determined artemisinin resistance, an elevated EC50 value in the 6hr pulse assay was again observed, but discrimination between Cambodian resistant and sensitive isolates was not achieved. We present results from a new DHA in vitro susceptibility assay format we have devised to overcome this problem. Experiments suggest this format can identify both PfK13- dependent Asian phenotypes and PfK13-independent phenotypes in African parasites. 6 Susceptibility to partner drugs lumefantrine and piperaquine was assessed with standard 48hr assays for all isolates. The genetic basis of the susceptibility phenotypes observed was explored by whole genome sequencing for each African isolate. Mutations present in 11 genes of interests for drug resistance loci were identified. As these isolates are multiclonal, all haplotypes present in selected genes of interests for every isolate were described. Implications of this work for continuing studies of parasite susceptibility of ACT in Africa are considered. This includes a new method that can be used to monitor and control the spread of drug resistance on the African continent.