There are an estimated 216 million cases of malaria worldwide resulting in 655,000 deaths. Plasmodium falciparum is the intracellular parasite responsible for the 91% of the world’s malaria morbidity and mortality burden in humans. Efforts to control and eradicate malaria have been hampered by the accelerated evolution of drug resistance in the parasite. To date, the parasite has developed resistance to all major antimalarial drugs, raising concerns about the spread of drug-resistant parasites and the ability to effectively treat malaria. Understanding the genetics and genomics of P. falciparum allows for greater biological insights into drug resistance and virulence of the parasite, allowing for more informed control decisions.
The development of molecular tools to assay genomic variation in P. falciparum is crucial. A novel microarray (the CNV-SNP Array) with an optimized probe design for copy number variation and SNP genotyping in the P. falciparum genome is presented that demonstrates variable length and isothermal probes are superior to static length probes. Sample preparation and hybridization conditions were optimized to mitigate the effects of host DNA contamination in field samples and can identify copy number variants (CNVs) and SNPs with 95% accuracy in a single hybridization.
The advent of in vitro culturing of P. falciparum revolutionized malaria research by allowing for controlled parasite propagation, preservation, and experimentation, but the effect of culture adaptation on the parasite genome has not been comprehensively or experimentally assessed. Using comparative genomic hybridization (CGH), copy number fluctuation of parasite isolates adapted to in vitro culture was assessed and show little copy number fluctuation during the course of adaptation. The few copy number changes seen during culture adaptation, do not lessen the power of using culture-adapted parasites in the study of P. falciparum, but advocate caution when using culture-adapted parasites to make inferences on population diversity.
The role of DNA rearrangements has been largely ignored in malaria biology despite large variation in the Plasmodium karyotype, known rearrangements in laboratory lines, and the known influence of copy number variation on drug resistance. The extent and functionality of genome rearrangements in P. falciparum, as well as their origins and evolutionary dynamics, were assessed in 148 parasite samples from Southeast Asia, (Cambodia, Laos, and Thailand) and Africa (Malawi and The Gambia) using the CNV-SNP array. CNV size, gene content, and frequency within and between parasite populations were determined and the roles of drift and selection (positive and purifying) in shaping the CNV distributions evaluated. Geographical variation in CNVs were determined and used to better understand the evolution of genome rearrangements in P. falciparum.
Understanding the genomic components responsible for parasite phenotypes like drug resistance, while vital, is only one aspect of malaria control. Economic, social, and political components also play key roles in combating malaria. The difficulties facing national malaria control programs in Uganda was assessed in a rural region of Uganda and the presence of substandard antimalarials in the private drug market confirmed.
There are many aspects to consider in the fight against malaria and requires a broad and comprehensive view to solve. A few tools and findings are outlined in this dissertation with the hopes that it may be of some value in the fight against this deadly scourge.