Despite over a century of research, malaria parasites (Plasmodium) still remain a major cause of mortality and morbidity worldwide. In recent years, the application of theoretical principles from ecology and evolutionary biology to the study of these parasites has started to provide insight into variety of fundamental subjects from the evolution of virulence to the facultative strategies (i.e. phenotypic plasticity) that parasites use to maximize their transmission. It is now becoming increasingly clear that to understand and predict population level patterns of virulence and transmission, the processes that occur at the between-host level must be studied in light of the interactions that happen within hosts (between parasites and between parasites and hosts). In this thesis I combine concepts from evolutionary biology and ecology with tools from molecular and cellular biology and evolutionary genetics, which allow me to study rodent malaria parasites at both evolutionary and ecological timescales. The work I present in this thesis has the following four components: 1. Phylogenetics (chapter 2): I applied recently developed phylogenetic methods to a large DNA sequence dataset that I generated, to provide a better understanding of the phylogeny of rodent malaria parasites and investigate how selection has shaped their genomes. I show that all rodent malaria subspecies can be considered species, provide the first time line for the evolution of this group of parasites and demonstrate that most loci are under purifying selection. 2. Hybridization and reproductive isolation (chapter 3): I show that hybridization between two rodent malaria parasites (P. berghei and P. yoelii) can occur, but only occurs at high levels when one of two proteins (P230 or P48/45) is absent from the surface of female gametes, which indicates that these proteins are involved in gamete recognition. I find that P230, P48/45 and P47 (a possible interaction partner) are evolving under positive selection, a feature often observed in gamete recognition proteins of other taxa. Finally, I show that the fertilization success of P. berghei is reduced in the presence of P. yoelii, but not vice-versa, which indicates asymmetric reproductive interference. 3. Sex allocation (chapter 4): I carry the first test of sex allocation’s assumption that immunity impacts on the fertility of Plasmodium male gametocytes/gametes more than on the fertility of females. I show that while the fertility of both males and females is equally affected, males are affected during gametogenesis and females are mostly affected through gamete dysfunction (i.e. gametes can mate but zygotes fail to develop), which is in agreement with the assumptions of theory. In collaboration, I incorporate these effects into sex allocation theory and predict that malaria parasites can minimize the effects of factors that kill gametocytes/gametes by adjusting their sex ratios. On the other hand sex ratio adjustment cannot compensate for gamete dysfunction or zygote death. These results have applied implications for transmission-blocking vaccines. 4. Infection dynamics of mixed-species infections (chapter 5): I develop a series of experiments to test how a focal parasite species (P. yoelii) is affected by competition with heterospecifics (P. chabaudi) and how the interaction between the two species is mediated by immunity and resource availability. I show that P. chabaudi can boost P. yoelii above its single species level (i.e. facilitation) and that this is mediated by resource availability. On the other hand, P. yoelii’s performance can also be hindered in mice that were exposed to a P. chabaudi infection. My results also reveal that host mortality is exacerbated in mixed-species infections of naïve mice, which may be due to an inability of the host to achieve the right balance between the production and the destruction of red blood cells, when dealing with a mixed-species infection. The work I present here tackles fundamental questions concerning the transmission biology and the within-host interactions of malaria parasites The results presented demonstrate the importance of interactions between hosts and parasites and between different parasite species (at the molecular and the whole organism levels) for determining the outcome of transmission, virulence and within-host parasite performance.

According to the World Health Organization, nearly 3.2 billion people are at risk of malaria. The most deadliest form of human malaria is caused by the pathogen Plasmodium falciparum, which has claimed over 400,000 lives worldwide in 20151. Even when optimally treated with drug and donor blood therapies, severe malaria has a high mortality rate. The parasites target a patient’s red blood cells and convert them into paramagnetic units before eventually rupturing the host cell, further spreading the infection. Combination drug therapies using quinine and artemisinin derivatives are common but are either expensive or have associated toxicities from mis-dosing. Moreover, antimalarial drugs are becoming increasingly ineffective against the growing number of drug-resistant malaria strains. Combination drug and blood exchange therapies are often implemented to flush out malaria-infected red blood cells (iRBC) but consume a great quantity of donor blood, carry a high risk of transmitting other blood-borne diseases, and have no agreed upon advantage or disadvantage among clinicians. Due to the relative disadvantages of other treatment methods, small scale high gradient magnetic separation (HGMS) devices, used in a variety of biological applications, may be another treatment option to consider. mPharesis (“magnetic apheresis”) is a proposed low-cost, disposable magnetic blood filtration device which continually removes iRBCs from a patient’s whole blood by capitalizing on the iRBC’s unique magnetic properties. The proposed treatment-scale system will provide emergency care with parameters similar to continuous hemofiltration systems in terms of blood flow rates (up to approximately 500 mL min-1), vascular access, and treatment times (up to about 3 hours). A novel medium-scale high gradient magnetic separation device is detailed here. The device consists of a disposable photo-etched embedded wire array and acrylic layered housing on an external permanent magnet set. The magnetic force and flow field design were computationally optimized. In-vitro feasibility experiments were conducted at several flow rates and physiological hematocrits (Hct) using a blood mixture composed of healthy RBCs and a non-pathogenic paramagnetic blood analog called methemoglobin RBCs (metRBCs). The device was able to selectively remove paramagnetic RBCs without excessive loss of healthy RBCs. Simplified experiments were performed with 30% Hct with 20% metRBCs. At steady state, the concentration of metRBCs was reduced by 27.0±2.2% in a single pass at a flow rate of 77 μL min-1 as compared to 1.6±0.7% in control experiments without a magnet present. The experimental paramagnetic RBC removal rate was over 380 times greater than similar published HGMS devices. These successful results were applied to a theoretical transport model. The model was designed to compare the parasite removal and Hct level changes between combination drug and exchange transfusion (ET) therapy versus treatment-scale mPharesis-drug therapy. When the mPharesis flow rate was set to typical continuous dialysis rates, treatment times and donor blood volumes were reduced for all 10 cases. Calculated treatment times were all less than 60% of the reported ET-drug treatments, with times ranging from 47 to 71 minutes. The mPharesis-drug treatment was calculated to need between 4% and 53% less donor blood than the reported ET-drug treatments. Between 775 and 1772 mL of packed donor RBCs (3 to 6 units of whole blood) were estimated for the mPharesis-drug treatments, versus the average 5 to 20 units used during ET2. Treatment reference charts were generated to provide time and donor blood volume estimates for a range of patient sizes and disease severities. Based on the maximum flow rate of 500 mL min-1, a treatment-scale mPharesis system was estimated to be the size of three stacked briefcases, which is a feasible size for deployment in a clinical setting. Finally, the design, fabrication, and microscopic visualization of a simple, benchtop-fabricated continuous HGMS device was detailed. This proof-of-concept microfluidic device was implemented to test the effect of hematocrit and flow rate on the separation of mixtures of metRBCs (heat-treated and un-heated) and transparent ghost RBCs. An automated image processing protocol provided feasible cell concentration profiles for each flow and rheological condition with a 6.5 to 9.7% lower sum than manual counting for three samples. For the no magnet conditions, the average near-magnet concentration of paramagnetic RBCs at the outlet (within 10% of 130 μm channel height adjacent to the wire array) was between 1.3 and 2.4 times greater than the average of the rest of the flow field (degree of separation, DOS). The most effective separation was found to occur at the lowest flow rate 0.4 μL min-1 and with the 0.5% Hct metRBC sample with DOS=26. The addition of 30% ghost RBCs reduced the efficiency for all flow rates, with DOS=7.4 for best flow rate of 0.4 μL min-1. Heat treatment did not significantly affect separation with DOS=7.3, likely due to the low impact of the relatively low concentration of metRBCs (0.5%). The mesoscale fabrication and design process, clearance model, cell counting algorithm, and HGMS fabrication protocol and microscopy study described in this thesis provides a useful framework for future HGMS optimization and the further development of a clinical treatment system for severe malaria patients with often limited treatment options.

The main thrust of this thesis was to characterize P.falciparum genetic diversity in northern Ghana. To do this, I used simple techniques to purify P. falciparum DNA from clinical samples across a rural setting for whole-genome sequencing. The goal was to provide a framework for exploring host-parasite genetic interactions. Utilizing Illumina deep sequencing data for 277 isolates I analyzed P. falciparum genetic diversity and described within-host diversity across this area. I observed random mating (ie no population structure) in the local parasite population, and a high genetic diversity indicative of high out-crossing. Moreover, when I aggregated my data with similar published data from Burkina Faso and Mali (sites ≈500km apart), no population structure was evident. In contrast, sites sampled in Cambodia and Thailand (≈ 800km apart) were found to have greater population structure and high potential for inbreeding. This may be driven by differences in transmission intensity between the sites sampled in West Africa and southeast Asia. To demonstrate the utility of deep sequencing data, I focused on the genomic regions of pfdhfr, pfdhps and pfcrt, known to be under antimalarial drug selection. I surveyed the full diversity of point mutations already characterized in these genes and discovered previously unknown variants. However, in order to provide a means to follow up on new variants or interesting candidate regions in large clinical samples with limited parasite DNA, I assessed the Sequenom iPLEX platform for high-throughput genotyping of P. falciparum polymorphisms. This necessitated developing a method appropriate for assigning genotypes in haploid genome mixtures common in natural infections. Finally, I used this method to type host and parasite markers in a case-control sample set from this region for exploring host-parasite genetic interactions. I found that children who have the sickle-cell trait and carry parasites that have pfdhfr resistant alleles lose their protection against severe malaria as compared to children who have normal haemoglobin and are infected with parasites with these resistant alleles.

El objetivo del presente trabajo fue identificar antígenos relevantes de valor diagnóstico de aislados de P. vivax y P. falciparum provenientes del departamento de Loreto, mediante la técnica de inmunoblot. Se seleccionaron pacientes entre 3 y 64 años con diagnóstico de malaria, gota gruesa positiva, procedentes de centros de salud en el departamento de Loreto. Fueron analizadas 4 mezclas de antígenos, una de P. falciparum (PF1) y tres de P. vivax (PV1, PV4 y PV5), preparadas a partir de 36 muestras de pacientes con alta parasitemia por P. vivax (2 700 – 69 000 parásitos/μL) y P. falciparum (2 750 – 10 000 parásitos/μL). Las mezclas de antígenos fueron enfrentadas a 39 sueros (12 de P. falciparum y 27 de P. vivax) mediante ensayos de inmunoblot.

Despite the array of medical advances of our modern day society, infectious diseases still plague millions of people worldwide. Malaria, in particular, causes substantial suffering and death throughout both developed and developing countries. Aside from the socioeconomic challenges presented by the disease's prevalence in impoverished nations, one of the major difficulties scientists have encountered while attempting to eradicate the disease is the parasite's ability to become resistant to new drugs and methods of treatment. In an effort to better understand the dynamics of malaria, we analyze a mathematical model that accounts for both the treatment aspect as well as the drug resistance that accompanies it. Simulations demonstrating the effects of treatment rates and the level of resistance are studied and discussed in hopes of shedding additional light on the characteristics of this devastating epidemic.

Invasion of host erythrocytes is an essential stage in the life cycle of Plasmodium parasites and in development of the pathology of malaria. The stages of erythrocyte invasion, including initial contact, apical reorientation, junction formation, and active invagination, are directed by the coordinated release of specialised apical organelles and their parasite protein contents. Among these proteins, and central to invasion by all species, are two parasite protein families, the reticulocyte-binding protein homologue (RH) and the erythrocyte-binding like (EBL) proteins, that mediate host-parasite interactions. RH5 from Plasmodium falciparum (PfRH5) is the only member of either family demonstrated to be necessary for erythrocyte invasion in all tested strains, through its interaction with the erythrocyte surface protein basigin. Indeed, antibodies targeting either PfRH5 or basigin can block parasite invasion with high efficiency in vitro, making PfRH5 an excellent candidate for a vaccine to protect against the most deadly form of malaria. Here I present crystal structures of PfRH5 in complex with basigin and with two distinct inhibitory antibodies. This is the first structure of any RH protein, revealing a novel fold in which two three-helical bundles come together to form a kite-like architecture. The two immunoglobulin domains of basigin and the inhibitory antibodies bind to one tip of the kite. These findings provide the first structural insights into erythrocyte binding by the Plasmodium RH protein family and identify novel inhibitory epitopes to guide the design of a new generation of vaccines against the blood-stage parasite.

Malaria was identified as one of the first infectious diseases recognised to spread through blood transfusion. Although transfusion acquired malaria is rare, nevertheless it can be lethal if it not diagnosed or treated immediately. It is a continuous challenge for the blood services to identify and exclude asymptomatic malaria infected donors, while minimising the exclusion of uninfected donors. The diagnostic tests in current use present certain limitations which include the use of inherently antigenically variable vaccine candidate proteins that have limited sensitivity against all human malaria species. Additionally, the blood transfusion services also require alternative methods for test and reagents that may be critical to the blood supply. There is therefore a scientific and an operational requirement to use alternative strategies to develop sensitive tests to all the species of malaria. In this study, we have used immunoproteomic approach to define conserved immunogenic malaria proteins. A total of 17 target P. falciparum proteins have been identified using cohorts of malaria immune sera from adults living in endemic areas, as well as by control sera from Europeans, who have never been exposed to malaria. The identified blood stage target antigens were cloned and expressed as recombinant proteins in a suitable bacterial system. In total, 15 target proteins have been expressed with 13 of them have been successfully purified. An ELISA-based system was developed, and the antigenicity of nine target antigens were evaluated using both non-malaria and malaria sera. Single antigen testing gave overall sensitivity of 50 - 84 %, with specificity consistently over 90%. Antigens such as Alpha tubulin and 26s protease showed promising immunogenicity, while Nucleosome assembly protein achieved 100% specificity. Further development of multiple antigens in an ELISA test will be required for continued evaluation of these antigens and the humoral immune response in malaria in general.

Modified vaccinia virus Ankara (MVA) has been used extensively as a recombinant vector for delivery of antigens from diverse pathogens. Its ability to generate strong antigen specific CD8+ T cell responses in humans has been shown in clinical trials of novel vaccines against malaria, tuberculosis, HIV I AIDS, influenza and cancer. The work in this thesis describes the use of BAC recombineering technology to harness the endogenous regulatory signal (promoter) that drives the expression of non-essential open reading frames (ORFs) in MVA for immunogenic expression of a recombinant antigen. Replacement of the ORFs of four non-essential genes in MVA; C11R, F11l, A44L and B8R with an epitope tagged luciferase positioned to use the same endogenous promoter showed early transgene expression equal to or slightly higher than traditional p7.5 and short synthetic promoter (SSP) constructs. The frequency of antigen-specific CD8+ T cell induced in mice by single dose MVA or adenovirus-prime, rMVA-boost vaccination showed equivalent or slightly higher responses by the endogenous promoters compared to the traditional p7.5 and SSP constructs. Assessment of the growth rate of these viruses showed they were unimpaired and the insertions were genetically stable. Furthermore, the endogenous promoter driven insertion loci of B8R and C11R were used for the construction of a bivalent MVA expressing an epitope tagged luciferase (rLucPb9) and a Photinus pyralis (pLuc) luciferase. The frequency of antigen-specific CD8+ T cells induced in mice by bivalent MV A was equivalent to single-pLuc and single-Pb9 recombinants co-administered as a mixture, at separate sites or administered alone following single dose MV A vaccination but slightly lower for Pb9-specific CD8+ T cell following adenovirus-prime, rMVA-boost.

This study investigated the implementation of the roll back malaria (RBM) programme at household and at health post levels and examined factors that negatively impact on malaria prevention and control activities. Quantitative, descriptive, analytic crosssectional research, guided by the conceptual framework of the Health Belief Model, was conducted. Structured interviews were conducted with 857 women (for the household survey in phase 1) and 53 health extension workers (HEWs) in phase 2 of the study, in nine malaria endemic districts of Sidama Zone, southern Ethiopia. Data were analysed using SPSS version 20. The study’s findings indicate that 53.3% (n=457) of the household respondents and 24.5% (n=13) of the HEWs had low levels of overall malaria-related knowledge. Household respondents aged 25-34 years, (p<0.01); regularly received malaria-related information, (p<0.001) and the less poor women (p<0.001) had good levels of knowledge. Of the households, 38.9% (n=333) reported poor RBM practices. Wealth, knowledge, perceived threat of malaria and perceived benefits of implementing malaria preventive measures were positively associated with good RBM practices. Indoor residual spraying (63.6%; 422 out of 664), consistent use of insecticide treated bed nets (51.6%; 368 out of 713), and environmental sanitation (38.6%; 331 out of 857) were the most commonly implemented malaria prevention strategies in the study area. Out of the 252 reported malaria cases, 53.6% (n=135) occurred among children under five years of age who also comprised 50.0% (n=16) of 32 reported malaria-related deaths. The RBM practices were poorly implemented in the study area despite malaria prevention and control efforts. Slow progress in behavioural changes among household members, lack of transportation services for referring malaria patients, lack of support given to HEWs and lack of feedback and supervision from higher level health care facilities were identified as potential challenges facing RBM implementation in the study area. Future efforts need to focus on effective behavioural changes based on intervention studies and regular monitoring of the RBM programme. The workloads of the HEWs should also be reconsidered and lay health educators should be used more effectively. Health posts should always have sufficient anti-malaria drugs and other resource such as rapid diagnostic kits. / Health Studies / D. Litt. et Phil. (Health Studies)

Im Rahmen der Zusammenarbeit des Missionsärztlichen Instituts in Würzburg mit dem Sacred Heart Hospital (Nigeria) wurden vor Ort im Hinblick auf das Problem der Arzneimittelfälschungen in Nigeria und dem Auftreten von einzelnen Resistenzen gegen Artemisinin-Derivate Untersuchungen bezüglich der aktuellen Situation im Kampf gegen Malaria im Großraum Abeokuta durchgeführt. Der Kenntnisstand über Malaria und das Gesundheitsverhalten einer für die nigerianische Bevölkerung möglichst repräsentativen Probandengruppe (n=100) wurden mithilfe eines Fragebogens erfasst. Ebenfalls mithilfe eines Fragebogens wurden die Therapiestrategien der einheimischen Ärzte (n=34) gegen Malaria untersucht und die Verfügbarkeit und Qualität von Artemisinin- Derivaten im Untersuchungsgebiet durch den Erwerb von Medikamenten-Samples (n=29) und anschließende Labortests überprüft. Die Befragung der Bevölkerung ergab, dass Wissen bezüglich der Ursachen, Symptome und Prävention der Malaria durchaus vorhanden ist, wobei große Unterschiede abhängig vom Bildungsstand bestanden. Vor allem ältere Menschen verfügten über wesentlich geringere Schulbildung und verließen sich deshalb sehr viel mehr auf die traditionelle Medizin. Darüber hinaus war eine oftmalige Bagatellisierung der Malaria auffällig, weshalb viele Probanden (53%) sich im Krankheitsfall gegen das Aufsuchen eines Krankenhauses entschieden. Die Befragung bezüglich der Therapiestrategien der einheimischen Ärzte zeigte, dass die Richtlinien der WHO bezüglich der Verwendung von ACT offensichtlich optimal angenommen und angewandt werden. Als mögliches Problem stellte sich die von 76,7% der Ärzte nur selten angewandte Labordiagnostik dar, eine Tatsache, die Fehldiagnosen begünstigt. Bei der Testung der Medikamente erwiesen sich 14,3% der Proben als minderwertig oder sogar gefälscht, was offiziellen Angaben entspricht. Zudem handelte es sich bei 37,9% der Arzneimittelproben um Monopräparate, was im Hinblick auf Resistenzbildung mehr als bedenklich ist. Diese Resultate weisen darauf hin, dass im Südwesten Nigerias die Malaria-Problematik noch immer nicht adäquat gelöst ist. Immer noch erhalten viel zu wenige Menschen eine optimale Therapie, was zu einem großen Teil an fehlendem Wissen und damit verbundenem falschem Gesundheitsverhalten, an dem großen Einfluss der traditionellen Medizin und an der Präsenz von gefälschten, wirkungslosen Arzneimitteln auf dem Markt liegt. / Considering the problem of counterfeit drugs in Nigeria and the occurrence of resistances against artemisinin-derivates, investigations regarding the current situation in Abeokuta and its surrounding areas had to be carried out. Knowledge about malaria and healthcare-seeking behaviour of the local population (n=100) was investigated by means of a questionnaire, as well as antimalarial treatment strategies of local doctors (n=34). Furthermore availability and quality of artemisinin-derivates were checked by purchasing drug samples (n=29) and subjecting them to laboratory tests. Questioning the local population showed that knowledge about malaria (causes, symptoms, prevention) is definitely existing, although major differences depending on the level of education could be observed. Especially the elderly had a very low level of education and, therefore, relied a lot more on traditional medicine. Moreover it became obvious that malaria is often trivialized, so that many people didn’t choose to go to the hospital in case of an infection. Questioning the local doctors regarding their treatment strategies showed that WHO treatment guidelines were obviously widely accepted and applied. A possible problem could be that 76,7 % of the interviewed doctors stated that they rarely use laboratory test for diagnostics. A fact, which favours misdiagnosis. By testing the drug samples, 14,3% proved to be substandard or even counterfeit. Moreover, 37,9% of the samples were monotherapies, which is more than alarming regarding the development of resistances. These results indicate that the problem of malaria is still not adequately solved in south-west Nigeria. Too few people receive an optimal therapy due to lack of knowledge, wrong healthcare-seeking behaviour, the big influence of traditional medicine and the presence of ineffective counterfeit drugs on the market.