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.

The repeated emergence of drug resistance in Plasmodium falciparum underscores the importance of understanding the genetic architecture of current resistance pathways, as well as any associated fitness costs. Why resistance emerges in particular regions of the world has been linked to particular genetic backgrounds that better tolerate resistance-associated polymorphisms; this is likely to play a key role in driving the epidemiology of drug resistance, however is infrequently studied at a large scale in a laboratory setting. The first results chapter establishes a barcoding approach for P. falciparum with the aim of tracking parasite growth in vitro. The strategy used was adapted for P. falciparum by using a pseudogene (PfRh3) as a safe harbour to insert unique molecular barcodes. These libraries of barcoded P. falciparum vectors were also used as a readout of transfection efficiency. The second chapter establishes a proof of principle for phenotyping by barcode sequencing, using a panel of barcoded parasites generated in different genetic backgrounds that comprise sufficient genetic diversity to pilot the method. These were grown in the presence and absence of antimalarial compounds, and growth phenotypes were measured in parallel using BarSeq. The third results chapter studies the contribution of mutations in Pfkelch13, a molecular marker of artemisinin resistance, to parasite fitness. Combining CRISPR/Cas9-based genome editing and high throughput sequencing, the impact of Pfkelch13 alleles on fitness in the context of particular strain backgrounds is revealed. In particular, the impact of genetic background in the emergence and spread of drug-resistant lineages (referred to as KEL1) in Southeast Asia carrying a Y580 Pfkelch13 allele. Overall, given the current pace of genome sequencing of pathogenic organisms such as P. falciparum, it will be important to increase the scale of experimental genetics, in order to tackle in real-time natural variation that might be under constant selection from drugs, thus anticipating the emergence of drug resistance in changing parasite populations. Through this work, tools were developed to facilitate parallel phenotyping by measuring in vitro growth using high-throughput sequencing. The work also develops novel approaches to address the importance of genetic background and a potential role for positive epistasis in a lineage responsible for the recent outbreak of drug-resistant malaria in Southeast Asia.

Introduction : Bien que les données officielles fassent état d’une diminution globale du nombre de cas de paludisme en Guyane, les orpailleurs travaillant sur les sites illégaux au cœur de la forêt amazonienne semblent très touchés par cette pathologie. L’objectif principal de cette étude était de déterminer la prévalence du paludisme dans cette population. Les objectifs secondaires évaluaient la proportion des différentes espèces plasmodiales et leur distribution géographique, le niveau de résistance des parasites aux dérivés de l’artémisinine, les connaissances attitudes et pratiques vis à vis de cette pathologie, et des données de santé de cette population. Matériel et méthodes : Les inclusions ont eu lieu sur les sites de repli des orpailleurs le long du fleuve Maroni. Après recueil du consentement éclairé, un test de diagnostic rapide du paludisme était effectué, ainsi qu’un questionnaire, un examen clinique, et un prélèvement de sang pour microscopie, PCR et tests de résistance (RSA et génotypage du gène pfK13 pour les PCR positives à Plasmodium falciparum). Résultats : De janvier à juin 2015, 421 orpailleurs ont été inclus, majoritairement des hommes (70,6%) brésiliens (93,8%), de médiane d’âge de 37 ans. La prévalence du portage de plasmodies déterminée par PCR était de 22,3% (IC95% : 18,3-26,3) à 84% asymptomatiques. Les espèces identifiées étaient principalement P. falciparum (47,9%) puis P. vivax (37,2%) avec 10,6% de coinfections. Lors du dernier accès palustre, 52,4% des orpailleurs avaient eu recours à l’automédication, majoritairement avec des dérivés de l’artémisinine (93,8%) avec une mauvaise observance (37,8%). Le fait d’être en Guyane était fortement associé à l’automédication (AOR=22,1). Le test RSA montrait un taux de survie supérieur à 1% pour un échantillon mais l’analyse du gène pfK13 ne mettait pas en évidence de mutations associées à la résistance à P. falciparum Discussion: La prévalence élevée de porteurs asymptomatiques de paludisme constitue un réservoir important pourla transmission du paludisme dans la région. L’utilisation massive de dérivés de l’artémisinine associée à une mauvaise observance sont des facteurs de risque d’émergence de résistance, ce qui entraînerait des conséquences sanitaires et économiques importantes. Avec une volonté politique, des actions sont possibles pour limiter ce risque, comme la distribution de kits d’autodiagnostic et d’auto-traitement avec une formation au niveau des sites de repli. / Introduction: Although official data show a global decrease of malaria in French Guiana, this disease often affects illegal gold miners working in the deep Amazonian forest. The main objective of this study was to evaluate the malaria prevalence in this population. The secondary objectives were to evaluate and map the proportion of Plasmodium species, to assess behavior, attitudes and practices regarding malaria in this population, to measure the artemisinin resistance level in parasites and to evaluate their general health. Material and methods: Inclusions took place at the gold miners’ resting sites, spread along the Maroni river. After recording their informed consent, a malaria rapid test was performed, as well as a questionnaire, a clinical exam and a blood sample for microscopy, PCR and resistance test (RSA and PfK13 genotyping for P. falciparum positive samples). Results: From January to June 2015, 421 gold miners were included, mainly men (sex ratio 2.4), Brazilian nationals (93.8%), with a median age of 37 years. Plasmodium prevalence using PCR was 22.3% (CI95%: 18.3 - 26.3), of whom 84% were asymptomatic. During the last malaria attack, 52.4% selfmedicated with artemisinin derivatives (93.8%) and a poor treatment adherence (37.8%). Being in French Guiana when the malaria attack occurred was strongly associated with selfmedication (AOR=22.1). One sample showed a survival rate higher than 1% in RSA test but PfK13 genotyping did not reveal any mutation in P. falciparum. Discussion: This high prevalence of asymptomatic carriers constitutes a huge reservoir for malaria transmission in the region. Massive use of artemisinin derivatives associated with poor treatment adherence are factors that may contribute to the emergence of artemisinin resistance. That would have huge sanitary and economical consequences. With political will, actions are possible to limit this risk, as the distribution of kits for self diagnosis and self-treatment with training on resting sites.