Cerebral malaria (CM) is a severe neurological complication of malaria infection that results from interrelated pathologies. Despite extensive research efforts, the mechanism of the disease is not completely understood. Clinical studies, postmortem analysis, and animal models have been the main research arenas in CM. In this thesis, shotgun proteomics approach was used to further understand the pathology of human and experimental CM. The mechanism by which CM turns fatal is yet to be identified. A clinical proteomics study was conducted on pooled plasma samples from children with reversible or fatal CM from the Gambia. The results show that depletion of coagulation factors and increased levels of circulating proteasomes are associated with fatal pediatric CM. This data suggests that the ongoing coagulation during CM might be a disseminated intravascular coagulation state that eventually causes depletion of the coagulation factors leading to petechial hemorrhages. In addition, the mechanism(s) by which blood transfusion benefits CM in children was investigated. To that end, the concentration and multimerization pattern of von-willebrand factor, and the concentration of haptoglobin in the plasma of children with CM who received blood transfusions were measured. In addition to clinical studies, experimental cerebral malaria (ECM) in mice has been long used as a model for the disease. A shotgun proteomics workflow was optimized to identify the proteomic signature of the brain tissue of mice with ECM.Because of the utmost importance of membrane proteins in the pathology of the disease, sample fractionation and filter aided sample preparation were used to recover them. The proteomic signature of the brains of mice infected with P. berghei ANKA that developed neurological syndrome, mice infected with P. berghei NK56 that developed severe malaria but without neurological signs, and non-infected mice, were compared to identify CM specific proteins. Among the differentially expressed proteins in mice that developed neurological signs, coagulation and acute-phase proteins were enriched. The data are in accordance with data from the clinical study. Taken together, the results support the role of coagulation and platelets adhesion in the pathogenesis of the disease.

This dissertation explores the impact of malaria on Foundation Phase learners and educators in Vhembe. Malaria is prevalent in three South African provinces, Kwazulu-Natal, Mpumalanga and Limpopo. The Limpopo province has the highest number of malaria cases. Malaria is transmitted by mosquitoes, in South Africa the An.merus and An. arabiensis, with the latter being the primary vectors. Continuous exposure to malaria infections will impact the academic performance of children. Especially in those communities where socio-economic issues, such as poverty, inadequate housing and unemployment exist, as well as weak public healthcare systems. This study was situated in an interpretive paradigm and a qualitative approach, using a case study, was followed. Data was collected by conducting interviews with principals and educators. The results indicated that principals and educators believed that parents were the most knowledgeable about malaria. There was consensus that the Department of Health promoted malaria awareness and the local clinic was the first point of contact for treatment. Schools were not involved in malaria education, except for accommodating annual healthcare visits. The empirical research findings provide evidence to show that teaching and learning continues at schools even when learners or educators are absent. Parents were responsible for the care of their children as well as for ‘catchup’ lessons. Collaboration between the Limpopo Department of Health, the Department of Basic Education officials, principals, educators, parents, and healthcare workers must be strengthened. Existing curriculum topics should be used to further advance malaria awareness. The lack of internet connectivity, efficient public transport and bad roads present a major challenge for the community in accessing healthcare services. / Dissertation (MEd)--University of Pretoria, 2017. / Early Childhood Education / MEd / Unrestricted

Despite the concerted efforts of researchers, policy makers and public health workers worldwide, malaria persists as a significant disease threat for nearly half the world’s population. Recent advances in vector control measures, diagnostics and antimalarial drug therapies have contributed greatly to reducing the incidence of clinical disease, and by extension, the number of deaths attributable to malaria in the past two decades; however, the latter remains high—over 400,000 people die each year from malaria, the vast majority of these being children under the age of five. Our ability to rapidly and effectively treat malaria has been a cornerstone of efforts to control and eradicate this devastating disease. Nonetheless, the constant evolution and spread of drug-resistant forms of the Plasmodium parasites that cause malaria—particularly the most virulent of these, Plasmodium falciparum—have historically greatly hindered these efforts, compromising the efficacy of every previous first-line treatment. Today, treatment of P. falciparum malaria relies on artemisinin derivatives, an exquisitely potent and fast-acting class of antimalarials that are deployed ubiquitously in artemisinin-based combination therapies, or ACTs. Now, emerging resistance to ACTs threatens to once again reverse the hard-fought advances made in the global fight against malaria. Resistance to artemisinin itself was first documented in western Cambodia and northwest Thailand in 2009 and has continued to spread throughout Southeast Asia at alarming rates. Reports of resistance to ACTs followed soon thereafter. Artemisinin resistance has also emerged de novo in other parts of the world. The major concern is that it will spread to Africa, where the disease burden is highest. Previous studies have provided compelling evidence that resistance to artemisinin results primarily from specific point mutations in the C-terminal Kelch propeller domain of the P. falciparum protein K13. Here, we have addressed two central aims regarding the role of this protein in mediating resistance to artemisinin. The first was to genetically dissect the contribution of a panel of K13 polymorphisms to artemisinin resistance and parasite fitness as assessed in vitro, with the latter being a key factor impacting the spread of resistance-conferring alleles in high-transmission settings. These experiments were conducted by CRISPR-Cas9-mediated gene editing, which allowed us to successfully engineer K13 mutations into a variety of strain backgrounds, including, for the first time, recently culture-adapted African parasites. These experiments clearly show that there is no genetic obstacle to the acquisition of artemisinin resistance in African parasites; however, they also suggest that fitness costs associated with these mutations may counter-select against the spread of resistance. The second aim relating to K13 was to investigate the underlying biology of this protein. To this end, we raised monoclonal antibodies to recombinant K13 and generated transgenic lines expressing tagged versions of the protein. Using these tools, we describe the subcellular localization of K13 in wild-type and mutant parasites in the presence and absence of drug pressure, and identify potential K13-associated proteins. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13, suggesting that mutations result in a loss of protein function. In order to overcome K13-mediated artemisinin resistance, novel therapeutics with distinct modes of action will be required. In our last aim, we characterize inhibitors of a particularly promising new antimalarial drug target, the proteasome. We report that these covalent peptide vinyl sulfone inhibitors are highly potent against genetically diverse parasites, including K13-mutant, artemisinin-resistant lines. Moreover, we observe that parasites do not readily acquire resistance to these compounds, nor do related compounds select for cross-resistance to one another. We also observe strong synergy between artemisinin and related compounds with these inhibitors in both K13 mutant and wild-type parasites. These results highlight the potential for targeting the Plasmodium proteasome as a means of overcoming artemisinin-resistant malaria.

Indiana University-Purdue University Indianapolis (IUPUI) / Severe malaria, primarily caused by Plasmodium falciparum infection, is among the leading causes of childhood mortality globally. A key virulence factor and source of antigenic variation and immune evasion during infection is P. falciparum erythrocyte membrane protein 1 (PfEMP1). Encoded for by approximately 60 var genes, this complex protein mediates cytoadherence of infected erythrocytes to the host endothelium and is a prominent immune target for the anti-malarial immune response in children. During severe malaria, specific domains of PfEMP1 that bind to endothelial protein C receptor (EPCR) and intercellular adhesion molecule-1 (ICAM-1) on host endothelial cells, are more prevalently expressed. The interaction of these proteins and infected erythrocytes mediates the sequestration of infected erythrocytes and plays a role in severe malaria pathogenesis. Antibodies to these domains develop over time with exposure to the parasite and are thought to contribute to immunity against severe malaria in children. In this study, whole blood samples from children with different forms of severe malaria, enrolled in two observational prospective cohort studies were used to quantify the expression of PfEMP1 domains using RT-qPCR and to measure the antibody response to PfEMP1 domains via a bead-based multiplex immunoassay. Using these samples, we demonstrated that although the expression of var transcripts encoding PfEMP1 domains was generally similar across children with different forms of severe malaria, the expression of variants encoding specific EPCR-binding domains was associated with thrombocytopenia and severe anemia. The antibody response to PfEMP1 domains in children with severe malaria was highest in children with SMA and children with asymptomatic parasitemia, but not associated with decreased risk of additional malaria episodes. Overall, the results of this study suggest that PfEMP1 is acting similarly across different forms of severe malaria but that it can be related to pathogenesis and severe malaria immunity.

BACKGROUND: Malaria adversely affects pregnant women and their fetuses or neonates. Estimates of the malaria burden in pregnant women based on health facilities often do not present a true picture of the problem due to the low proportion of women delivering at these facilities in malaria-endemic regions. METHODS: Data for this study were obtained from the Healthy Beginning Initiative using community-based sampling. Self-identified pregnant women between the ages of 17-45 years were recruited from churches in Enugu State, Nigeria. Malaria parasitaemia was classified as high and low based on the malaria plus system. RESULTS: Of the 2069 pregnant women for whom malaria parasitaemia levels were recorded, over 99 % tested positive for malaria parasitaemia, 62 % showed low parasitaemia and 38 % high parasitaemia. After controlling for confounding variables, odds for high parasitaemia were lower among those who had more people in the household (for every one person increase in a household, OR = 0.94, 95 % CI 0.89-0.99). CONCLUSION: Results of this study are consistent with hospital-based estimates of malaria during pregnancy in southeastern Nigeria. Based on the high prevalence of malaria parasitaemia in this sample, education on best practices to prevent malaria during pregnancy, and resources in support of these practices are urgently needed.

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