Background The World Health Organisation currently recommends the use of artemisinin-based combination drugs for treatment of uncomplicated malaria in high malaria endemic regions. However, comprehensive understanding of factors affecting treatment of malaria with antimalarials is lacking in many rural communities in Africa. This study seeks to test the following hypothesise: 1. That socio-economic and demographic factors at the household level affect treatment of self reported malaria/fever with antimalarials in the Kilombero/Ulanga and Rufiji valleys in Tanzania 2. Distance of a household to a health facility affects treatment of malaria/fever with antimalarials in the Kilombero/Ulanga and Rufiji valleys in Tanzania. Methods: Secondary data analysis of a cross- sectional household survey on antimalarials carried out in 2005 in the Kilombero/Ulanga and Rufiji valleys in Tanzania. Georeferenced health facilities and households’ datasets from the Rufiji and Ifakara demographic surveillance systems sites were also used to estimate distance variables. Results: Out of a total of 1433 participants who reported malaria/fever, 32% (95% CI: 29.29, 34.89) obtained treatment with antimalarials. Among them, 36% obtained treatment with Sulfadoxine Pyreminthamine (SP) as a monotherapy and 44% treated malaria/fever with SP and Artesunate as a combination therapy.8% used quinine while 11 % used Amodiaquine and Artesunate. The remaining 1% used chloroquine. After adjusting for all confounding variables in a multivariate survey logistic regression model, age group, education level of the household head and district of residence were found, with statistical evidence, to be associated with treatment of reported malaria/fever with antimalarials. Background The World Health Organisation currently recommends the use of artemisinin-based combination drugs for treatment of uncomplicated malaria in high malaria endemic regions. However, comprehensive understanding of factors affecting treatment of malaria with antimalarials is lacking in many rural communities in Africa. This study seeks to test the following hypothesise: 1. That socio-economic and demographic factors at the household level affect treatment of self reported malaria/fever with antimalarials in the Kilombero/Ulanga and Rufiji valleys in Tanzania 2. Distance of a household to a health facility affects treatment of malaria/fever with antimalarials in the Kilombero/Ulanga and Rufiji valleys in Tanzania. Methods: Secondary data analysis of a cross- sectional household survey on antimalarials carried out in 2005 in the Kilombero/Ulanga and Rufiji valleys in Tanzania. Georeferenced health facilities and households’ datasets from the Rufiji and Ifakara demographic surveillance systems sites were also used to estimate distance variables. Results: Out of a total of 1433 participants who reported malaria/fever, 32% (95% CI: 29.29, 34.89) obtained treatment with antimalarials. Among them, 36% obtained treatment with Sulfadoxine Pyreminthamine (SP) as a monotherapy and 44% treated malaria/fever with SP and Artesunate as a combination therapy.8% used quinine while 11 % used Amodiaquine and Artesunate. The remaining 1% used chloroquine. After adjusting for all confounding variables in a multivariate survey logistic regression model, age group, education level of the household head and district of residence were found, with statistical evidence, to be associated with treatment of reported malaria/fever with antimalarials. Conclusion: The results suggest that participant’s age, education level of household head and location of district are important predictors of treatment of malaria with antimalarials in rural Tanzania. The implementation of any antimalarials policy in Tanzania would therefore, require a careful consideration of these factors.

Thesis submitted to the Faculty of Health Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2017 / Malaria remains endemic in the north-eastern areas of KwaZulu-Natal (KZN), Mpumalanga and Limpopo provinces of South Africa (SA). Anopheles arabiensis is now implicated as the main malaria vector. This vector is not completely amenable to current vector control strategies which target indoor biting and resting mosquitoes. SA is moving towards malaria elimination and there is a need for additional vector control interventions to complement existing tools. The sterile insect technique (SIT) targeting An. arabiensis was selected as a potential intervention. In a mosquito SIT programme, only sterile males should be released because females are potential disease vectors. In order to achieve male releases only, a reliable sex separation strategy is needed. Additionally, it is imperative to gather entomological baseline information on the population density, species composition, and vectorial capacity of the targeted wild population. The aim of this study was to evaluate the use of a local genetic sexing strain for SIT and to determine the population dynamics of wild An. arabiensis in northern KZN. The following objectives were initiated in this study: development of a local genetic sexing strain (GSS), evaluation of the life history and reproductive effects of irradiation on An. arabiensis, and weekly mosquito surveillance was conducted over a period of 24 months. A local GSS named GMK was established by introgressing a local wild-type population of An. arabiensis with an available GSS strain. The strain exhibited the following attributes: low egg hatch rates, fast developmental time, long adult survival and a high mating competitiveness. Dieldrin treatment of GMK eggs/larvae mainly produced males but this result remains controversial. The irradiation dose of 70 Gy induced male sterility without compromising their mating competitiveness and impacted negatively on female fitness, but not vectorial capacity. The perennial presence of An. arabiensis, the dominant anopheline species in Mamfene, was confirmed. Its population density fluctuated with season reaching a peak in summer. Clay pots were more productive than the other collection methods, collecting 16.3 mosquitoes per trap. This study recorded for the first time wild caught An. arabiensis and An. vaneedeni infected with P. falciparum in SA. An arabiensis sporozoite infection rates were 0.7% (2014) and 0.5% (2015). Anopheles vaneedeni has never been implicated as a malaria vector in nature. However, an infection rate of 1.96% was recorded (2014-2015), which implicate this species as a potential malaria vector. These results highlight the importance of intensive mosquito surveillance to establish malaria vectors responsible for low level/residual malaria transmission. The data generated provides important baseline vector surveillance information and is valuable to stakeholders and researchers to make informed decisions regarding the use of SIT against vector mosquitoes in SA. / MT 2018

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Malaria är en välkänd parasitsjukdom världen över som år 2015 ledde till 438000 dödsfall, varav 90% i den afrikanska regionen. Parasiterna förstör de humana erytrocyterna och sprids via Anopheles-myggor som är vektorer. Parasitsläktet som orsakar malaria kallas Plasmodium och består av fem arter. Orsaken bakom flest dödsfall är P. falciparum och därför går mycket diagnostik ut på att detektera just denna art. Sjukdomen uppkommer cirka en vecka från det att man blivit myggbiten. Symptomen kan vara väldigt varierande vilket gör det svårt att ställa en snabb diagnos. Effektiva läkemedel mot malariaparasiterna finns men de sätts i för många fall, in på fel sätt eller inte alls vilket beror på brister i diagnosticeringen. Kunskap och framförallt resurser saknas i endemiska områden. Idag används mikroskopi av blodutstryk (pheripheral blood smears (PBS)) som rutinmässig standard för malariadiagnostik, men för att kunna ställa tidigare och mer säkra diagnoser har nya metoder utvecklats. Det finns många kriterier som bör uppfyllas av en lyckad metod så som känslighet, snabbhet, omgivningsanpassning och en acceptabel kostnad. För att komma fram till vilken metod som passar bäst utifrån dessa aspekter samt framtidspotential jämförs i denna studie mikroskopi av tjocka och tunna blodutstryk, quantitative buffy coat (QBC), polymerase chain reaction (PCR), cell-microarray chip, loop-mediated isothermal amplification (LAMP) och rapid diagnostic tests (RDTs). Slutsatserna som kan dras är att mer arbete behöver göras innan en ny metod helt kan ersätta konventionell mikroskopi. I de endemiska områdena är kostnaden och enkelheten väldigt betydande och därför är LAMP den metod som jag efter att alla dessa metoder ha jämförts, anses ha mest potential för att uppfylla kriterierna och skulle kunna ersätta den konventionella mikroskopin om metoden vidareutvecklas. / Malaria is a parasitic disease known worldwide. In 2015 the disease caused 438000 deaths of which 90% occurred in the African region. The parasites destroy the erythrocytes in the human body and are transmitted by the Anopheles mosquitoes. There are five species of the genus Plasmodium that causes malaria. The species P. falciparum causes the most deaths and therefore most diagnostics are about detecting this species. Malaria occurs approximately a week after the infecting bite. The symptoms are often varying and nonspecific and hence complicate the diagnosis of the disease. Thick and thin blood film microscopy examination (Pheripheral blood smears) is the golden standard for malaria diagnosis. However, to be able to make quicker and more reliable diagnoses new methods need to be developed. Even though there are effective drugs against malaria, delayed diagnoses and treatment are major causes of death in many countries. Knowledge and resources are primarily lacking in the endemic areas where it is needed the most. There are many criteria for a method that should be fulfilled; such as sensitivity, rapidness, accuracy and cost-effectiveness. The method needs to be easy to use in the actual environment and it has to have potential for the future. This is a comparative study of microscopy of thick and thin blood smears (PBS), quantitative buffy coat (QBC), polymerase chain reaction (PCR), cell-microarray chip, loop-mediated isothermal amplification (LAMP) and rapid diagnostic tests (RDTs). The conclusion that can be drawn from this study is that more work needs to be done before a new method can replace conventional microscopy. In the endemic areas the aspects of cost and simplicity are important to take into consideration. Among the methods in this study, The LAMP seems to be the one with the most potential if it continues to develop in the future.

Three protein targets are used in malaria rapid diagnostic tests (RDTs). These are Plasmodium falciparum histidine rich protein 2, Plasmodium lactate dehydrogenase and aldolase. A thrust of research in RDTs is to improve on their specificity and sensitivity. In this study the current diagnostic target, P. falciparum lactate dehydrogenase (PƒLDH) was compared to a new target glyceraldehyde-3-phosphate dehydrogenase (PƒGAPDH) that was identified based on transcriptional data. These proteins are conserved amongst all Plasmodium species, with minor amino acid sequence variations which were evaluated as possible species-specific peptide epitopes for PƒLDH: LISDAELEAIFDRC and PƒGAPDH: CADGFLLIGEKKVSVFA; CAEKDPSQIPWGKCQV, where common peptides were identified as pan-malarial epitopes for pLDH: APGKSDKEWNRDDLC and pGAPDH: CKDDTPIYVMGINH. The chosen peptides were located on the surface of their predicted 3D crystal structure models. Antibodies were raised against these peptides in chickens (IgY) and affinity purified. PƒLDH and PƒGAPDH were recombinantly expressed in E. coli BL21(DE3) cells and their coding inserts confirmed by sequencing. The recombinant proteins were detected in Western blots with specific anti-His₆ tag antibodies at approximately 35 kD (PƒLDH ~ 36 kD and PƒGAPDH ~ 39 kD) which compared with their expected values. Both recombinant proteins were found to form tetramers in solution and were used to raise IgY antibodies for comparison of Pheroids™ and Freund’s adjuvants. Pheroids™, like Freund’s appeared to exhibit a depot effect, however Freund’s adjuvant gave higher affinity purified IgY yields. The anti-recombinant and anti-peptide IgY specifically detected their respective recombinant and native antigens and did not cross-react with other human blood proteins. Immunoprecipitation detected higher levels of PƒGAPDH to PƒLDH in P. falciparum culture lysates. A double antibody sandwich ELISA detected 17.3 ng/ml PƒLDH and 138.5 ng/ml PƒGAPDH at 1% parasitemia in in vitro cultures, however this needs to be further evaluated. These findings suggest PƒGAPDH to be at least as good a protein target as PƒLDH for malaria diagnosis and further trials using it as a target in an RDT format should be considered. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Malaria affects 200 million people annually, resulting in 584,000 - 1,238,000 deaths. The majority of these deaths occur in children, less than 5 years of age, in sub-Saharan Africa and are due to cerebral malaria (CM), a neuropathology induced primarily by the species Plasmodium (P.) falciparum. The pathogenesis of CM remains poorly understood and the mechanisms involved in acquired protection against the syndrome in malaria-endemic regions are undefined. Utilising the well characterised P. berghei ANKA experimental infection model of cerebral malaria (ECM), results presented in this thesis show that the development of ECM is associated with the accumulation and arrest of pathogenic CD8+ T cells within the perivascular spaces of the brain. Accumulation of activated CD8+ T cells, without arrest, was observed in the perivascular spaces of the brains of mice infected with the non-ECM causing P. berghei NK65 strain. These data show that the behaviour of intracerebral CD8+ T cells specifies their pathogenic function during malaria infection. The development of ECM was associated with extensive disruption to the BBB, which developed in the absence of extensive CD8+ T cell-dependent endothelial cell apoptosis. We modified the ECM model, establishing an infection-drug cure strategy, to investigate the immunological basis of parasite exposure-induced resistance to ECM development. Three rounds of infection-drug cure promoted resistance to ECM, which was associated with reduced intracerebral expression of genes involved in defence response, regulation of apoptosis, chemotaxis, CTL activity, antigen processing and presentation and cell adhesion, compared with ECM susceptible mice. Additionally, CD8+ T cell activation was suppressed in exposure-induced resistant mice and was associated with the antibody dependent expansion of a splenic plasmacytoid DC population, with a regulatory phenotype. The infection-induced protection against ECM was critically dependent upon secreted antibody production. A long standing problem in studying the immune response to malaria infection has been the inability to track parasite-specific CD4+ T cell responses. To address this, we generated and validated new transgenic P. berghei parasites expressing the model antigen, ovalbumin (OVA), either in the parasite cytoplasm or on the parasitophorous vacuole membrane (PVM). We found that cellular location and expression level of the antigen influence the induction and magnitude of parasite-specific T-cell responses. These parasites thus provide knowledge on the factors that influence the recognition of parasite antigens by the immune system and represent useful tools to study the development and function of antigen-specific T-cell responses during malaria infection. The results in this thesis improve our understanding of the events that lead to the development of CM, and the host immune responses that develop following parasite exposure to protect against it. The results should contribute towards the rational development of adjunctive therapies and effective vaccines for human CM.

Plasmodium vivax agente etiológico de la malaria, exhibe una gran variabilidad genética durante episodios recurrentes de la enfermedad. Esta recurrencia es informada como de baja prevalencia asociada con la malaria asintomática. Así mismo los episodios recurrentes (reinfecciones o relapsos) a menudo pueden ser confundidos por resistencia a fármacos como la cloroquina. Por lo tanto el objetivo principal de este estudio fue relacionar los patrones de recurrencia y la resistencia con la variabilidad genética de P. vivax. En este estudio se evaluaron las muestras secuenciales de individuos provenientes de una región endémica del Perú (Mazán-Iquitos), diagnosticados previamente con malaria, por microscopía, durante seguimientos activos y sometidos a un régimen de tratamiento estándar con cloroquina. La genotipificación realizada en base al gen pvmsp3-α, utilizando el Nested PCR y la digestión enzimática, permitió identificar una alta variabilidad genética de P. vivax, a partir de la cual, se identificaron los patrones de recurrencia, establecidos como relapsos, a partir de estadios latentes o hipnozoitos homólogos (con haplotipos idénticos) y reinfecciones (con haplotipos diferentes). Los rangos de tiempo permitieron una identificación más precisa, observándose mayores frecuencias de relapsos por hipnozoitos homólogos antes de los 90 días post-primera evaluación y mayores frecuencias de reinfecciones después de este periodo. Así mismo las recurrencias en el primer periodo de tiempo, por haplotipos diferentes, pueden deberse también a hipnozoitos heterólogos. Complementando el estudio, el análisis de secuenciamiento del gen pvmdr1, permitió identificar SNPs, codificantes de mutaciones no sinónimas, relacionadas con resistencia a cloroquina. Estos SNPs, a través del software U-Melt (análisis in sílico), presentaron variaciones en las temperaturas de fusión. Finalmente los resultados de cuantificación relativa con qPCR Real Time no mostraron diferencias significativas en el número de copias del gen pvmdr1. Palabras clave: Cloroquina, Genotipificación, Haplotipos, Hipnozoito, Malaria Asintomática, Recurrencia, Variabilidad. / --- Plasmodium vivax etiologic agent of malaria has a large genetic variability during recurrent episodes of the disease. This recurrence is reported as low prevalence associated with asymptomatic malaria. Also recurrent episodes (reinfection or relapse) can often be mistaken for drug resistance as chloroquine. Therefore the main objective of this study was to correlate the patterns of recurrence and resistance to the genetic variability of P. vivax. In this study, we evaluated the sequential samples of individuals from an endemic region of Peru (Mazán-Iquitos), previously diagnosed with malaria microscopy during active follows and subjected to a standard treatment regimen with chloroquine. Genotyping based on the pvmsp3-α gene, using Nested PCR and enzymatic digestion, identified high genetic variability of P. vivax, from which were identified recurrence patterns established as relapse, from latent stages or homologous hypnozoites (with identical haplotypes) and reinfections (with different haplotypes). The time ranges allow more accurate identification, with higher frequency of relapses by homologous hypnozoites before 90 days post-first evaluation and higher frequencies of reinfection after this period. Also recurrences in the first period of time, for different haplotypes may also be due to heterologous hypnozoites. Complementing the study, the sequencing analysis of the gene pvmdr1, identified SNPs, encoding nonsynonymous mutations related to resistance to chloroquine. These SNPs, through U-Melt software (in sílico analysis), showed variations in the melting temperatures. Finally the results of relative cuantification with Real Time qPCR no showed significant differences in copy number of the pvmdr1 gene.} Keywords: Chloroquine, Genotyping, Haplotypes, Hipnozoite, Recurrence, Asymptomatic Malaria, Variability. / Tesis