College of Basic and Applied Sciences
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Item Tolerance Of Plasmodium Falciparum To Artemetherlumefantrine In The Gambia(University Of Ghana, 2020-12) Mbye, H.Antimalarial drug resistance contributes significantly to obstacles in reducing the global burden of malaria especially in sub-Saharan Africa (sSA) where the disease is most prevalent. Resistance to artemisinin-based combination therapies (ACTs), the only recommended frontline drugs for the treatment of uncomplicated malaria is now widespread in South East Asia (SEA). However, ACTs remain efficacious in sSA though in vivo delayed parasite clearance and in vitro reduced susceptibility to both components of the drug has been reported. Resistance to ACTs is therefore anticipated especially with its sustained use in endemic regions and the recent report of the emergence of de novo Pfk13 mutation that is now spreading in Rwanda. In The Gambia where artemether-lumefantrine (AL) is the first-line drug used for over 10 years, a steady increase in parasite tolerance to lumefantrine (LUM) was observed over a period of 4 years which strongly correlated with reported directional selection on a cysteine desulfarase gene (Pfnfs1). These findings are concerning and require continuous drug surveillance to track spontaneous development of AL resistant parasites and determine pathways to resistance development. This study therefore sought to investigate the prevalence and mechanisms of parasite tolerance to AL in The Gambia. A novel ex vivo drug susceptibility assay suitable to simultaneously assess parasite responses to both drugs used in AL was developed and used to assess drug susceptibility profiles of circulating parasites in western Gambia. This assay was then used to confirm identified potent compounds from the Medicines for Malaria Venture pathogen box effective against the erythrocytic stages of the parasite for future development into new antimalarial drugs. The prevalence of known drug resistance markers was assessed and novel markers that could be associated with drug resistance identified using both regression analysis and GWAS approach. Finally, CRISPR-Cas9 genome editing was used to functionally validate Pfnfs1 for its involvement in LUM tolerance using gene editing approaches. Antimalarial drug resistance contributes significantly to obstacles in reducing the global burden of malaria especially in sub-Saharan Africa (sSA) where the disease is most prevalent. Resistance to artemisinin-based combination therapies (ACTs), the only recommended frontline drugs for the treatment of uncomplicated malaria is now widespread in South East Asia (SEA). However, ACTs remain efficacious in sSA though in vivo delayed parasite clearance and in vitro reduced susceptibility to both components of the drug has been reported. Resistance to ACTs is therefore anticipated especially with its sustained use in endemic regions and the recent report of the emergence of de novo Pfk13 mutation that is now spreading in Rwanda. In The Gambia where artemether-lumefantrine (AL) is the first-line drug used for over 10 years, a steady increase in parasite tolerance to lumefantrine (LUM) was observed over a period of 4 years which strongly correlated with reported directional selection on a cysteine desulfarase gene (Pfnfs1). These findings are concerning and require continuous drug surveillance to track spontaneous development of AL resistant parasites and determine pathways to resistance development. This study therefore sought to investigate the prevalence and mechanisms of parasite tolerance to AL in The Gambia. A novel ex vivo drug susceptibility assay suitable to simultaneously assess parasite responses to both drugs used in AL was developed and used to assess drug susceptibility profiles of circulating parasites in western Gambia. This assay was then used to confirm identified potent compounds from the Medicines for Malaria Venture pathogen box effective against the erythrocytic stages of the parasite for future development into new antimalarial drugs. The prevalence of known drug resistance markers was assessed and novel markers that could be associated with drug resistance identified using both regression analysis and GWAS approach. Finally, CRISPR-Cas9 genome editing was used to functionally validate Pfnfs1 for its involvement in LUM tolerance using gene editing approaches. Keywords: Malaria, antimalarial drug resistance, ex vivo drug assays, high throughput screening genotyping, functional analysis, association studiesItem Ethnobotany, Bioactivity and Variation Studies of Traditional Ghanaian Anti-Malarial Plants(University of Ghana, 2000) Asase, A.An investigation on traditional plants used in the treatment of malaria in Ghana was conducted. The study involved a collection of information from an indigenous group of people living in the Wechiau Community Hippopotamus Sanctuary Ghana about their knowledge on plant species used for the treatment of malaria. It also involved a study of the bioactivity and variations within selected plant species in the sanctuary. The investigation on the indigenous uses involved ethnobotanical interviews and field studies. Forty-one species of plants in 19 families were mentioned as used for the treatment of malaria in the sanctuary. Eight of the species of plants. namely. Afrraegle paniculata, Haemastostaphis bateri, indigofera pulchra, monathotaxis sp., Ozoroa insignis. strychnos innocua,strychnos spmosa and Xeroderris stuhlmanuii have not been previously investigated for their anti·malarial uses. The efficacy of four anti-malarial species. namely, Cassia sieberiana, Haematostaphis barteri, Mitragyna mermis and Pseudocedrela kotschyiand was determined by testing their extracts against several organisms including Plasmodium,bacteria, fungi and an insect pest. The methods of the bioassays included mic:rodilution technique for in vitro antiplasmodial assay. TLC agar-overlay diffusion for microbial bioassay and a binary choice assay for insect anti-feedant testing. The extracts were moderately active against Plasmodium and varied in their activity against the test organisms. From the stem and root barks of Haematostaphis barteri three Stilbene compounds were isolated for the first time from a member of the family Anacardiaceae.The identification of stilbenes was made through the use of NMR and APCI-MS expenmeruation. Variation study using data from a range of characters including morpholgy, anatomy of barks, ecology. distribution, Comparative phytochemistry and molecular (AFPL markers) were conducted to understand infraspecific taxonomy of tbe four species of plants and for their conservation in the sanctuary. Data obtained from the morpnomemc measurements, maceration, field ecologica1 methods, specimen computerisation, phytochemical methods such as HPLC and LC-MS and AFLP methodologjes were used for the variations analysis. The variations in the selected species were largely due to morphological and chemical variability. The variations in the species of plants were found to be due to plasticity of environmental factors and that the taxonomies of the four species are stable in the sanctuaryItem Investigating Factors Influencing Variation in P. Falciparum Invasion Phenotyping Assays(University of Ghana, 2019-07) Thiam, L.G.Malaria caused by P. falciparum, is the most life-threatening human parasitic disease, claiming over 400,000 deaths from about 200 million cases in 2017. Despite the yet unsuccessful efforts in the quest for an effective malaria vaccine, antigens expressed in the erythrocytic stage of P. falciparum represent highly promising vaccine candidates given their natural exposure to the immune system. Understanding the mechanism of erythrocyte invasion by P. falciparum mainly during active clinical infection is an important step towards developing an effective vaccine to counter the deleterious outcomes of this parasitic infection. However, comparison of pioneering studies assessing the invasion phenotypic diversity of P. falciparum is precluded by the lack of consensus in the protocols used in individual studies. As such, efforts towards the development of standard protocols necessitate the assessment of the impact of potential sources of variation in the measured outcome. Here, the effect of short-term culture adaptation on the invasion phenotypes of P. falciparum clinical isolates was assessed using freshly isolated parasites from children presenting an acute malaria illness. We also assessed the effect of short-term cryopreservation on the parasites’ invasion phenotype relative to the ex vivo invasion phenotype of the isogenic parasites. The effect of blood donor variability and some erythrocyte phenotypic features in the parasites’ invasion phenotype were also assessed here. Furthermore, we assessed the invasion phenotypic diversity of P. falciparum clinical isolates from four areas of varying transmission intensity in Ghana and compared the expression levels of the major parasites’ invasion-related ligands across isolates. Finally, we tested the invasion inhibitory activity of antibodies against two novel P. falciparum antigens and assessed the naturally-derived antibody responses against both antigens in children presenting at the hospital with symptomatic malaria. We showed that short-term cryopreservation has a minimal effect on the parasites’ invasion phenotype while short-term culture adaptation was found to affect the genotypic diversity and, to a lesser extent, the phenotypic diversity of P. falciparum clinical isolates. Blood donor variability was also found to affect P. falciparum invasion efficiency. However, this variation was associated with none of the erythrocyte phenotypic features tested here (e.g. blood group, hemoglobin genotype, receptor density). The erythrocyte invasion phenotyping experiments showed the predominance of sialic acid independent invasion pathway in Ghanaian clinical isolates and a high dependency on the CR1/PfRh4 mediated invasion pathway across all isolates. Moreover, invasion into trypsin-treated erythrocytes was found to be strongly correlated with the invasion into chymotrypsin-treated erythrocytes. The assessment of the transcript levels of invasion-related genes showed significantly higher expression levels of PfEBA family genes relative to the PfRh family genes, while the pattern of expression was similar across sites. Further analysis revealed a strong negative correlation between PfEBA175 and all other genes except for PfEBA140. Furthermore, the growth inhibition assays showed different ranges of dose-dependent inhibitory activities of individual antibodies against the target antigens used in his study. However, the combination of antibodies against different antigens showed no synergistic invasion inhibitory activities across all isolates. Finally, the level of antibody responses against the different antigens were strongly associated with transmission intensity. Taken together, our findings showed that differences in protocols may account for most of the variations in previously reported invasion phenotyping data across different countries. Therefore, this work has provided preliminary data upon which more rigorous experimental design should be based on for the optimization and establishment of standardized invasion phenotyping assays, which is critical for large-scale studies.Item Targets and Patterns of Erythrocyte Invasion Inhibitory Antibodies in Malaria(University of Ghana, 2018-07) Mensah-Brown, H.E.Background As malaria transmission continues to decline, a better understanding of how transmission intensity and patterns influence clinical presentation of disease and the acquisition of immunity to malaria is important for our understanding of the pathophysiology of malaria and the changes in immune responses. Erythrocyte invasion is a crucial step in the life cycle of Plasmodium falciparum. However, the targets of naturally acquired invasion inhibitory antibodies remain unclear. In this study, the differences in clinical and haematological presentation of disease, the predictors of anaemia severity in children with malaria, living in three ecologically distinct areas of Ghana, with different transmission intensities was determined. Patterns of invasion inhibitory antibodies were also assessed to explore the impact of transmission intensity on these parameters. Additionally, the relationship between antibody levels and functionality of antibodies to key invasion ligands were evaluated in adults living in a holoendemic area of Ghana. Methods Blood samples were taken from children between the ages of 2 and 14 years with confirmed malaria in hospitals in three areas with different transmission intensities (Kintampo>Navrongo>Accra). Whole blood was used to perform comprehensive analysis of parasitological, clinical and haematological variables, whilst socio-economic details were collected using a questionnaire. Plasma samples separated from the whole blood collected were tested by enzyme linked immunosorbent assays for antibodies to P. falciparum invasion antigens, including erythrocyte binding antigens (EBA) 175, EBA140, EBA181, and reticulocyte binding-like homologue (Rh) 2, Rh4 and Rh5. To identify the targets of invasion inhibitory antibodies, plasma samples were collected from 50 male adults living in a high transmission area with no recent history of clinical malaria. Antibodies against EBA and Rh proteins were detected using enzyme linked immunosorbent assays (ELISA). Immunoglobulin (IgG) fractions were purified from the plasma and used in erythrocyte invasion assays. Results Severity of malarial anaemia was more pronounced in children living in areas of high malaria transmission compared to children living in low endemic areas. Sickle cell trait was protective against anaemia severity in Kintampo (P=0.016), although this association was not statistically significant in Accra (P=0.379) and) and Navrongo (P=0.529). Parasitaemia was not a significant predictor of haemoglobin level, after controlling for age and gender. In addition, antibodies against invasion antigens were negatively correlated with parasitaemia, and increased in an age-dependent manner. Regression analysis showed that breadth of antibody reactivity was exposure and age-dependent. Levels of antibodies to EBA and RH antigens generally correlated with invasion inhibitory activity and breadth of antibody reactivity was predictive of inhibitory activity of purified IgG (ρ= 0.437, P= 0.009). Conclusions Our findings demonstrate significant differences in the haematological presentation and severity of malaria among children residing in areas with different transmission intensities. Patterns of antibody responses against invasion proteins are both antigen and exposure dependent. Growth inhibitory activity was significantly associated with antibody levels and the number of different antigens recognized.Item Understanding the Acquisition and Kinetics of Naturally Acquired Immunity to Pfrh5 Complex Proteins in Malaria(University of Ghana, 2018-03) Partey, F.D.Plasmodium falciparum causes the severe form of malaria affecting mainly children below the age of five years and pregnant women. In the last decade, there has been a global decline in the reported malaria case incidence. Despite the reduction in malaria-associated morbidity and mortality, the disease is still prevalent in many developing countries in the tropics. With emergence of insecticide resistance and drug resistance to the limited range of effective drugs currently available, malaria vaccines remain a critical component of any strategic plan to eliminate and eventually eradicate malaria. Individuals living in endemic regions do develop immunity to malaria after repeated exposure to the parasite and never achieve sterile immunity. Developing an effective malaria vaccine has been difficult mainly due to the complex parasite life cycle, antigenic variation and polymorphism of parasite ligands. Much of the vaccine studies have focused on the asexual blood stage of the parasite life cycle as the clinical symptoms associated with malaria are due to this stage. In spite of this, there is no blood stage vaccine. P. falciparum reticulocyte binding-like protein homolog 5 (PfRH5) and its interacting proteins P. falciparum cysteine-rich protective antigen (PfCyRPA) and P. falciparum protein 113 (Pf113) have emerged as promising blood stage vaccine candidates due to the essential role of PfRH5 in merozoite invasion, its less polymorphic nature and the strain-transcending inhibitory effect of antibodies induced against the PfRH5 complex proteins. Nevertheless, not much is known about the role of PfRH5, PfCyRPA and Pf113 in naturally acquired immunity. In the present studies, the immunogenicity of PfRH5 complex proteins and the kinetics of antibodies induced against the PfRH5 complex proteins in natural infections were examined. In total, 206 Ghanaian children between the ages of 1-12 years who were either symptomatic, asymptomatic or non-parasitemic and healthy were recruited at the Hohoe municipal hospital. Plasma levels of antigen-specific IgG antibodies among acute malaria patients on day of admission showed seroprevalence of PfRH5, PfCyRPA and Pf113 was low compared to other well-studied merozoite antigens. The predominant IgG subclass response to the PfRH5 complex proteins was IgG1 and IgG3. Plasma IgG levels were found not to correlate with protection from malaria, severity of disease, or parasitemia on day of admission. Following treatment, antibodies to the studied antigens rapidly declined suggesting plasma IgG levels to the PfRH5 complex proteins were markers of recent parasite exposure. To better understand the mechanism of action of anti-PfRH5 and anti-PfCyRPA antibodies in naturally acquired immunity, human monoclonal antibodies were isolated from semi-immune Ghanaian adults and tested for functionality. The antibodies showed poor reactivity to PfRH5 and PfCyRPA recombinant proteins and did not exhibit inhibition against P. falciparum in vitro. In contrast, a panel of human monoclonal antibodies isolated from individuals vaccinated with viral vectored PfRH5 elicited strain-transcending inhibitory effects in vitro. Thus, knowledge gained from the functional and structural characterization of the human anti-PfRH5 monoclonal antibodies would greatly advance the rational design of PfRH5 based vaccines.