Resistance to antimalarial drugs continues to be a major obstacle in controlling and eradicating malaria. The identification of genetic markers of resistance is vital for disease management but they can be difficult to predict before resistance arises in the field. This thesis describes an alternative approach to gene identification, combining an in vivo experimental evolution model, Linkage Group Selection (LGS) and Solexa genome re-sequencing. Here this model was used to resolve the genetic basis of chloroquine and artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi. AS-30CQ is a parasite with high resistance to chloroquine and resistance to artemisinin. It was crossed with the genetically different drug-sensitive strain AJ. The resulting progeny were selected with drugs and backcrossed to the sensitive parent. Both crosses were treated with increasing concentrations of chloroquine and artemisinin. The frequency of markers from the sensitive parasite were analysed in order to characterize the signatures of drug selection. Three loci involved progressively in chloroquine resistance were identified on chromosomes 11, 3 and 2. One main locus on chromosome 2 was identified with artemisinin selection. The Solexa platform was used to re-sequence the genomes of both AS-30CQ and its sensitive progenitor, AS-sens. The differences between the two genomes were integrated with the LGS data to identify: 1) a strong candidate for the main CQresistance determinant - a putative amino acid transporter on chromosome 11 (aat1) 2) two candidates for high level chloroquine resistance on chromosome 3. and 3) a mutation in ubp1 gene on chromosome 2 that is likely to contribute to the highest level of chloroquine resistance and be main determinant of the artemisinin resistance phenotype. In addition the last section of this thesis describes two otherwise isogenic clones showing low- and high levels of chloroquine resistance were grown competitively to evaluate the effect of these mutations on parasite fitness. The highly resistant strain demonstrated a loss of fitness in relation to its more sensitive progenitor and was outcompeted in untreated and low-treated infections.

Artemisinin-resistant Plasmodium falciparum parasites have been reported in the Greater Mekong Subregion since 2007. Artemisinin combination therapy (ACT) is the mainstay of antimalarial treatment and is responsible for decreases in malaria-related morbidity and mortality over the past fifteen years. The slowed parasite clearance rates following ACT indicates resistance to artemisinin derivatives. This resistance places increasing selective pressure for variants or traits that confer resistance to the partner drug used in combination and has led to the rapid failure of several partner drugs. While a single nucleotide polymorphism (SNP) in kelch13 has been shown to mediate some resistance phenotypes, the complete mechanism of artemisinin resistance is poorly understood. The known mechanisms of resistance hint at a connection to autophagy, an intracellular pathway that cells use to degrade waste molecules or organelles in response to stress and starvation, which is poorly characterized in Plasmodium. In this doctoral thesis project, I investigated the role of an autophagy-like mechanism in P. falciparum in the mechanism of artemisinin resistance. I found a SNP in autophagy-related gene 18 (atg18) that was associated with clinical delayed parasite clearance half-life following ACT. This gene encodes PfAtg18, a protein that I characterized as being similar to mammalian/yeast homologues in terms of structure, binding abilities, and ability to form puncta in response to stress. In order to investigate the contribution of the mutation in this protein, I edited the atg18 gene using CRISPR/Cas9 and screened the mutant and parent parasites against a drug library of over 6000 unique compounds. I discovered that while the SNP did not change the mutant parasite's susceptibility to any of the antimalarial compounds using a 72-hour drug pulse, it did alter the susceptibility to 227 other compounds. Further, I found that the SNP offers parasites a fitness advantage by allowing them to grow better in nutrient-limited settings. Finally, I determined that neither this atg18 SNP nor several polymorphisms in kelch13 modulate a dormancy phenotype that appears to be involved in the artemisinin-resistance mechanism.

Indiana University-Purdue University Indianapolis (IUPUI) / Artemisinin resistance was first observed in Southeast Asia (SEA) and could pose a threat to malaria treatment all over the world. Recently mutations in the propeller region of Pfkelch13 gene have been used as a genetic marker for resistance observed in SEA. We investigated the presence of mutations in the Pfkelch gene in children in Kampala, Uganda with severe malaria (SM) treated with intravenous quinine, or with asymptomatic P.falciparum infection (AP) treated with artemether-lumefantrine. We sequenced the Pfkelch gene (2178bp) in 157 children with SM and 49 children with AP infection. In children with SM and AP we identified 106 (60.8%) and 27 (55.1%) parasites with mutations upstream of the Pfkelch13 propeller region. The two most prevalent mutations were 142NN (26.1% in SM, 33% in AP) and K189T (16.5% in SM, 12.2% in AP). In SM, only a single infection had a mutation in the propeller region (A578S), while in AP, mutations in the propeller region included A578S (n=1) and S522C (n=1). In children with SM, parasites with 142NN insertion compared to 3D7 Pfkelch13 parasites had lower parasite density (p=0.02) and lower parasite biomass (p=0.03). Children with SM who either had 142NN or K189T mutation cleared parasites after quinine treatment faster than those with the 3D7 Pfkelch13 genotype (P<0.001 for both mutations compared to 3D7). In this cohort mutations, upstream of the Pfkelch13 propeller region were common. Future studies will assess the presence of Pfcrt and Pfmdr mutations in this cohort, and how these relate to the Pfkelch13 mutations and to parasite clearance.

Artemisinin-based Combination Therapies (ACTs) are currently the frontline treatment against Plasmodium falciparum malaria, but parasite resistance to artemisinin (ART) and its derivatives, core components of ACTs, is spreading in the Mekong countries. In this study, we report the synthesis of several novel artemisinin derivatives and evaluate their in vitro and in silico capacity to counteract Plasmodium falciparum artemisinin resistance. Furthermore, recognizing that the malaria parasite devotes considerable resources to minimizing the oxidative stress that it creates during its rapid consumption of hemoglobin and the release of heme, we sought to explore whether further augmentation of this oxidative toxicity might constitute an important addition to artemisinins. The present report demonstrates, in vitro, that FM-AZ, a newly synthesized artemisinin derivative, has a lower IC50 than artemisinin in P. falciparum and a rapid action in killing the parasites. The docking studies for important parasite protein targets, PfATP6 and PfHDP, complemented the in vitro results, explaining the superior IC50 values of FM-AZ in comparison with ART obtained for the ART-resistant strain. However, cross-resistance between FM-AZ and artemisinins was evidenced in vitro

Introduction: Antimalarial resistance, particularly artemisinin resistance, is a major threat to P. falciparum malaria elimination efforts worldwide. Urgent intervention is required to tackle artemisinin resistance but field data on which to base planning of strategies are limited. The aims were to collect available field data and develop population level mathematical models of P. falciparum malaria treatment and artemisinin resistance in order to determine the optimal strategies for elimination of artemisinin resistant malaria in Cambodia and treatment of pre-hospital and severe malaria in Cambodia and Bangladesh. Methods: Malaria incidence and parasite clearance data from Cambodia and Bangladesh were collected and analysed and modelling parameters derived. Population dynamic mathematical models of P. falciparum malaria were produced. Results: The modelling demonstrated that elimination of artemisinin resistant P. falciparum malaria would be achievable in Cambodia in the context of artemisinin resistance using high coverages with ACT treatment, ideally combined with LLITNs and adjunctive single dose primaquine. Sustained efforts would be necessary to achieve elimination and effective surveillance is essential, both to identify the baseline malaria burden and to monitor parasite prevalence as interventions are implemented. A modelled policy change to rectal and intravenous artesunate in the context of pre-existing artemisinin resistance would not compromise the efficacy of ACT for malaria elimination. Conclusions: By being developed rapidly in response to specific questions the models presented here are helping to inform planning efforts to combat artemisinin resistance. As further field data become available, their planned on-going development will produce increasingly realistic and informative models which can be expected to play a central role in planning efforts for years to come.