Polyamines play an important role in DNA, RNA and protein synthesis as well as a variety of other biological processes (cell division, differentiation and death) as outlined in Chapter 1. Assaraf and co-workers (1984) demonstrated that treatment with DFMO resulted in the inhibition of polyamine biosynthesis as well as schizogony arrest in P. falciparum. However, they did not elaborate on any other consequences that polyamine depletion could exert on the parasite. This dissertation aims to elucidate the significance of the inhibition of polyamine biosynthesis within P. falciparum by using differential transcriptome profiling. Suppression subtractive hybridisation generated transcripts which were potentially up-and down-regulated due to endogenous polyamine depletion within the human malaria parasite P. falciparum. The resulting transcripts were subjected to a restriction enzyme analysis and those with unique digestion profiles were selected and sequenced. The sequences were analysed using PlasmoDB to identify the genomic sequences to which they were best matched. To confirm that the selected transcripts were indeed differentially expressed a reverse virtual Northern dot blot was performed. Transcripts for proteins involved in protein processing, methionine and polyamine metabolism, various transporters, proteins involved in cellular differentiation and signal transduction were found to be upregulated in the absences of polyamines. This could be suggestive of a metabolic response induced by the parasite in order to overcome this deficiency. Polyamines seem to influence protein synthesis and haemoglobin degradation as well since depletion of endogenous polyamines within the parasite seems to result in increased food vacuole acidification, haemoglobin degradation, transport of proteins to the cytoplasm and protein synthesis and stabilisation. The majority of downregulated transcripts were found to be involved in cell-cell adhesion and erythrocyte invasion, protein processing and transport indicating that these processes are dependent on polyamines. Further validation of these findings by microarray as well as proteomic analysis will need to be undertaken. These results validate that polyamines do play an essential role in the cellular biology of the parasite. They also confirm that the inhibition of polyamine biosynthesis is a viable route to undertake in the search for new and improved antimalarial targets. This would be especially useful if it was combined with other antimalarials and their synergistic effects were investigated by transcriptomic, proteomic and bioinformatic analysis / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / MSc / unrestricted

Magister Pharmaceuticae - MPharm / Malaria is an infectious disease which continues to kill more than one million people every year and the African continent accounts for most of the malaria death worldwide. New classes of medicine to combat malaria are urgently needed due to the surge in resistance of the Plasmodium falciparum (the parasite that causes malaria in humans) to existing antimalarial drugs. One approach to circumvent the problem of P. falciparum resistance to antimalarial drugs could be the discovery of novel compounds with unique scaffolds and possibly new mechanisms of action. Natural products (NP) provide a wide diversity of compounds with unique scaffolds, as such, a library of virtual compounds (VC) designed from natural products with antiplasmodial activities (NAA) can be a worthy starting point.

Anemia is known to worsen treatment outcomes in malaria, but there are not many studies to identify the predictors of anemia in Nigerian children with malaria. The objective of this study is to identify some of those predictors. Socio-demographic variables are predictors of anemia among under five children in Nigeria was the hypothesis tested. This is a cross-sectional study that used the 2018 demographic health survey (DHS) data from Nigeria to explore some of the factors that determine the presence of malaria-anemia co-morbidity in Nigerian children less than five years (N= 265). The outcome variable was anemia status in children under five with malaria and the explored predictors include age, sex, residential type, region of residence, mother’s education status and family’s wealth index. The study analyzed unweighted and weighted frequencies of the variables and conducted comparison of the outcome groups based on the predictor variables using Chi-square. Univariable and multivariable logistic regression was used to explore the strength of relationship between the outcome variable and the significant predictor variables in bivariate analysis. SAS 9.4 was used for the statistical analysis. Analysis of weighted frequencies showed that 55% of the children were less than 2 years of age while the sex was almost equally distributed between males and females (50.9% vs 49.1%). Just over two-thirds lived in a rural area, 63.2% resided in the Northern part of the country, 59.1% had a rich family and majority (69.1%) had anemia. When cross-tabulated with the outcome variable of anemia status, there was a significant difference in the categories of age (P=0.0048), residential type (P=0.0031), mother’s education status (P=0.0210) and family’s wealth index (P=0.0010). Univariable logistic regression showed that children less than 2 years had over two times higher odds of developing anemia when infected with malaria compared to older children aged 3-4 years (OR:2.17, 95% CI:1.26-3.74, P=0.0052). Urban-dwelling children had 57% reduced odds of developing anemia compared to rural-dwelling children (OR:0.43, 95% CI:0.25-0.76, P=0.0034). Children of educated mothers had 50% reduced odds of developing anemia compared to children of uneducated mothers (OR:0.50, 95% CI:0.28-0.91, P=0.0222), while children in poor families had 165% increased odds of developing anemia compared to those born into rich families (OR:2.65, 95% CI 1.47-4.78, P=0.0012). Once adjusted for all significant variables in the bivariate analysis, only age remained significant as a predictor of anemia in children under five years with malaria (OR:2.29, 95% CI:1.31-4.02, P=0.0039). Younger age seems to be an important predictor of anemia in Nigerian children with malaria in real life settings given its significance on the multivariable model. This finding should inform clinicians on the need to pre-empt and treat anemia in Nigeria’s younger children with malaria for better treatment outcome.

Malaria, a disease caused by parasites of the Plasmodium genus, infects over 220 million people annually, resulting in over 400,000 deaths. Most of these deaths occur in Africa in children < 5 years of age. Artemisia annua L., an ancient Chinese medicinal herb, is known for its foremost phytochemical constituent, artemisinin (AN). Semisynthetic derivatives of AN form the primary component of artemisinin combination therapies (ACTs), the frontline treatment for malaria worldwide. However, ACTs have several drawbacks including cost and availability. Thus, cheaper, more readily available antimalarials are needed. Recent clinical data suggested dried leaves of A. annua (DLA) administered orally as a tea infusion may be as efficacious as ACTs despite a significantly lower AN dose delivered. In mice, AN plasma concentration was improved when administered as DLA compared to pure AN. I therefore hypothesized that phytochemicals within DLA enhanced the oral bioavailability of AN. To investigate this hypothesis, here I examined the effects of DLA on the underlying mechanisms that govern oral bioavailability. Using an in vitro human digestion model, I showed that AN solubility was greater when delivered as DLA, largely due to essential oil in the plant. Furthermore, AN intestinal permeability was enhanced in a Caco-2 cell model of the intestinal epithelium. Extracts, teas, and phytochemicals produced by Artemisia also inhibited the activity of CYP2B6 and CYP3A4, the enzymes responsible for first-pass AN metabolism in the liver. Additionally, AN tissue distribution was improved when delivered as DLA and AN accumulation in tissues was higher in female vs. male rats. Finally, I showed that DLA was a more efficacious anti-inflammatory than pure AN in rats, potentially due to enhanced AN bioavailability. Taken together, these results shed light on the mechanisms behind enhanced oral bioavailability afforded by DLA and demonstrate the potential for DLA to be used as a therapeutic for malaria and other diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / Plasmodium vivax malaria is geographically widespread and remains a significant public health burden in the Americas, Southeast Asia, and the western Pacific. In order to achieve the end goal of malaria eradication, a highly effective vaccine targeting P. vivax is urgently needed. Unlike pre-erythrocytic vaccines that aim to confer sterile immunity that prevents malaria infection altogether, Plasmodium vivax blood-stage vaccines aim to confer clinical immunity that protects against malarial disease by controlling parasitemia and mitigating the symptomatic manifestations of malaria after infection. To design an effective P. vivax blood-stage vaccine, it is essential to understand the acquisition and longevity of natural humoral immune responses against promising P. vivax blood-stage vaccine candidate antigens. We hypothesize that acute vivax malaria induces differential humoral immune responses against P. vivax antigens that exhibit antigen-specific kinetic and compositional profiles, which can be used to identify vaccine candidates that elicit durable humoral responses. Therefore, we compared the kinetic profiles and half-lives of naturally acquired IgG antibodies reactive against nine promising P. vivax blood-stage vaccine candidate antigens up to 180 days post-infection in Brazilians with acute vivax malaria. Naturally acquired IgG antibodies against these antigens have previously been associated with a reduced risk of vivax malaria. Among the P. vivax antigens evaluated, the merozoite antigen Pv12 elicited the most durable IgG antibodies, whereas the DBP-FL elicited the most short-lived responses. Neither patient age nor prior malaria exposure significantly correlated with the magnitude and durability of IgG responses to any P. vivax antigen. Seropositivity, against Pv12, was generally maintained for at least 30 days after acute vivax malaria. These findings suggest that a blood-stage vaccine targeting Pv12 may benefit from boosting IgG antibodies against this antigen after natural vivax “breakthrough” infections. Further studies will be needed to determine the Pv12-specific memory B cell response as well as the functional role for naturally acquired Pv12-specific antibodies in reducing parasitemia and/or clinical disease. In summary, the current study has provided insight into the longevity of IgG antibody responses to important P. vivax antigens after an acute malaria episode.

The malaria-causing parasite Plasmodium falciparum 1s dependent on tightly regulated gene expression for its progression through the intra-erythrocytic life cycle, pathogenesis and establishment of persistent infection by evasion of the human host's immune system. Evidence points towards P. falciparum being unusually dependent on nuclear architecture and genomic organisation for the control of gene expression. Spatially defined nuclear regions of transcriptional activity have been detected and the spatial positioning of loci may determine their transcriptional potential. Additionally, a number of epigenetic markers have been shown to occupy spatially distinct subcompartments of the nuclear volume. Limitations of microscopic assays used until now have left us with a stereotyped and incomplete image of the organisation of the parasite nucleus and the transcriptional and epigenetic factors involved in the regulation of parasite gene expression, and the possible dynamics thereof. This work focused on the use of high-content and super-resolution fluorescent microscopy for the study and graphical representation of the spatial organisation of various nuclear factors involved in transcriptional regulation in P. falciparum parasites. The first objective (chapter 2) establishes P. falciparum parasite sample preparation and fluorescent labeling techniques for microscopy. Immunofluorescent labeling of var gene associated transcription repressive and permissive histone modifications, H3K9me3 and H3K9ac, respectively, as well as serine 2- phosphorylated RNA polymerase II and the putative transcription and splicing factor PfMyb2, was optimised. DNA fluorescent in situ hybridisation was also optimised for labeling of var gene exons. In the second objective (chapter 3), the assays established in the previous chapter are used for high-content combinatorial labeling in thousands of nuclei, followed by analysis using a bespoke computational algorithm for the detection and classification of different labeling patterns. This approach revealed a high level of diversity in the nuclear distributions of each assayed target. Superresolution stochastic optical reconstruction microscopy was used to further study the sub-diffraction organisation of selected labeling patterns. The data presented in this dissertation reveal that the complex spatial organisation of certain nuclear factors is subject to greater diversity within the nucleus of P. falciparum parasites than previously thought. / Dissertation (MSc)--University of Pretoria, 2012. / gm2013 / Biochemistry / unrestricted

Background: Malaria is a major world health issue and its continued burden is due, in part, to difficulties in the diagnosis of the illness. The World Health Organization recommends confirmatory testing using microscopy-based techniques or rapid diagnostic tests (RDT) for all cases of suspected malaria. In regions where Plasmodium species are indigenous, there are multiple etiologies of fever leading to misdiagnoses, especially in populations where HIV is prevalent and children. To determine the frequency of malaria infection in febrile patients over an 8-month period at the Regional Hospital in Bamenda, Cameroon, we evaluated the clinical efficacy of the Flourescence and Staining Technology (FAST) Malaria stain and ParaLens AdvanceTM microscopy system (FM) and compared it with conventional bright field microscopy and Giemsa stain (GS). Methods: Peripheral blood samples from 522 patients with a clinical diagnosis of "suspected malaria" were evaluated using GS and FM methods. A nested PCR assay was the gold standard to compare the two methods. PCR positivity, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined. Results: Four hundred ninety nine samples were included in the final analysis. Of these, 30 were positive via PCR (6.01%) with a mean PPV of 19.62% and 27.99% for GS and FM, respectively. The mean NPV was 95.01% and 95.28% for GS and FM, respectively. Sensitivity was 26.67% in both groups and specificity was 92.78% and 96.21% for GS and FM, respectively. An increased level of diagnostic discrepancy was observed between technicians based upon skill level using GS, which was not seen with FM. Conclusions: The frequency of malarial infections confirmed via PCR among patients presenting with fever and other symptoms of malaria was dramatically lower than that anticipated based upon physicians' clinical suspicions. A correlation between technician skill and accuracy of malaria diagnosis using GS was observed that was less pronounced using FM. Additionally, FM increased the specificity and improved the PPV, suggesting this relatively low cost approach could be useful in resource-limited environments. Anecdotally, physicians were reluctant to not treat all patients symptomatically before results were known and in spite of a negative microscopic diagnosis, highlighting the need for further physician education to avoid this practice of overtreatment. A larger study in an area with a known high prevalence is being planned to compare the two microscopy methods against available RDTs.

Magister Pharmaceuticae - MPharm / Malaria remains one of the top mortality causes in the sub-Saharan African region, especially among pregnant women and infants. Despite several measures being implemented within the affected areas such as the use of treated mosquito nets, sulfadoxine and pyrimethamine (SULPYR) as an intermittent preventive treatment (IPTp-SP) is still considered the standard prophylactic regimen for pregnant women. Recently, the WHO increased the regimen of IPTp- SP from three to four doses on a monthly interval, this recommendation poses a potential risk of toxicity and resistance to the drugs. An improvement towards this challenge is under clinical trial and consists of the inclusion of azithromycin (AZI), a macrolide antibiotic, to the current IPTp-SP treatment regimen. This will not only aid in the prophylaxis of malaria in pregnant women but will also assist in other pregnancy related infections. All three these drugs exhibit poor aqueous solubility; requiring high concentrations for oral administration to achieve therapeutic plasma concentrations. / 2024

Malaria causes high morbidity and mortality, especially among the most vulnerable populations, including pregnant women. Malaria in pregnancy (MiP) can be prevented by compliance with the 3 core measures: sleeping under insecticide-treated nets (ITNs), 3 doses of sulfadoxine-pyrimethamine as intermittent preventive treatment (IPTp-SP), and effective case management of malaria and anemia. The purpose of this cross-sectional household survey was to examine the composite risk behaviors that enhance the transmission of MiP. Stratified and multistage sampling methods were used to select a sample of 300 pregnant women in Abuja, Nigeria. Bivariate and multivariate analysis were conducted. According to study findings, participants' mean age was 28.6 years, many (117 or 68.0% of the participants) used an ITN the night before the survey, and some (113 or 38.0%) had used SP for IPTp. Many participants (183 or 61.0%) were of high malaria risk status (MRS). The predictors of MRS were knowledge, OR 3.282, 95% CI [1.091, 9.873], p=0.03; number of pregnancy, OR=5.589, 95% CI [1.465, 21.316], p=0.01; attendance at antenatal clinic, OR= 3.777, 95% CI [1.119, 12.746], p=0.03; level of education, OR=0.050, 95 CI [0.013, 0.197], p=0.000; perceived barriers of ITN, OR=0.308, 95% CI [0.165, 0.575], p=0.000; and perceived benefits of SP for IPTp, OR=3.156, 95% CI [1.879, 5.301], p=0.000 . Perceived seriousness, perceived severity of malaria, age, and religion were not significantly related to MRS. This study leads to positive social change by providing information for policy makers to review MiP-related policies and programs to ensure quality messages, providers, and products are accessible and affordable across rural and urban settings where the target population live and work.

There is lack of evidence on which of the two highly recommended malaria prevention methods, insecticide treated bednets and indoor residual spraying, is more effective than the other. There is also limited peer reviewed literature that compares the characteristics of people who use the two malaria prevention methods. Based on the Health Belief Model, the research questions tested whether there is any relationship between the use of mosquito bednet or the use of indoor residual spraying and contracting malaria, and whether there is any relationship between sociodemographic and socioeconomic factors and the use of malaria prevention methods. Using a quantitative research design, secondary data from the 2011 Angola malaria indicator survey were analyzed. Chi-square for association, logistic regression, and multinomial logistic regression tests were used. There was no statistically significant association between the use of mosquito bednet and having malaria. However, the use of indoor residual spraying significantly reduced the likelihood of getting malaria. There was also a statistically significant association between place of residence, wealth index, level of education, and number of household members and using mosquito bednet and between wealth index and using indoor residual spraying. In conclusion, the malaria prevention programs should focus on indoor residual spraying. It is recommended that all households in southern Africa malaria prone areas should be regularly sprayed. The findings of this study contribute to positive social change in the sense that by using more effective malaria prevention method, individuals will be able to function normally on daily basis, save on expenses related to employment loses or treatment and care of the sick, as well as loss of life and improve own economic status.