Orientador: Simone Liliane Kirszenzaft Shepherd / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T11:03:53Z (GMT). No. of bitstreams: 1 Pellegrino_AnaPaula_D.pdf: 6617098 bytes, checksum: 5e66717e28c080597cc20dc056067146 (MD5) Previous issue date: 2000 / Doutorado / Doutor em Biologia Vegetal

Publicación a texto completo no autorizada por el autor / Busca conocer la eficacia de la deshidrogenasa láctica OptiMal ® DiaMed IT con relación a la gota gruesa, en la detección del aclaramiento de la parasitemia en los días de control post tratamiento en pacientes con malaria. Se realizó un estudio de pruebas diagnósticas de pacientes identificados en el periodo marzo a diciembre del 2004. Busca evaluar las características de validez de una prueba diagnóstica (sensibilidad, especificidad, valor predictivo positivo, valor predictivo negativo) tanto de forma global y en el aclaramiento parasitario de los pacientes post tratamiento y atendidos en centros de salud de la selva oriental de Perú. Se revisó las fichas de atención de cada paciente, a quienes se les realizó la gota gruesa (prueba de referencia), se les calculó la densidad parasitaria y la prueba de OptiMal®. Se calculó la sensibilidad (S), especificidad (E), valor predictivo positivo (VPP) y valor predictivo negativo (VPN) y la concordancia de la prueba OptiMal ® con la prueba de referencia. Se incluyeron 376 casos de malaria el día 0, 310 (82.5%) casos fueron de P. vivax y 66 (17.5%) casos eran por P. falciparum. Durante el seguimiento desde el día 2 al 21 se recolectaron en total 1,570 muestras. La positividad con la prueba de gota gruesa fue de 14.6% el día 2 a 4.0% el día 14. La concordancia con la prueba Optimal® Diamed IT, fue en todos los días superior al 80%. De manera global (tanto para P. vivax o falciparum), durante el seguimiento la prueba tuvo, sin incluir el día cero, una sensibilidad S de 9.4% y especificidad E 99.5% y VPP 61.1% y VPN 93.2%. La prueba OptiMal® en este estudio demostró tener eficacia en el aclaramiento parasitario post tratamiento con relación a la gota gruesa pues tuvo alta concordancia y una buena especificidad, la baja sensibilidad se podría deber a la baja densidad parasitaria luego de iniciado el tratamiento, lo que sugiere que su aplicación sería óptima en escenarios epidemiológicos de transmisión moderada a alta. / Tesis

Les principales causes de morbidité et de mortalité en Afrique Sous-Saharienne sont les maladies infectieuses et les déficiences nutritionnelles. Plus concrètement, la carence en fer et le paludisme sont, respectivement, les principales maladies contribuant à la morbidité et la mortalité chez la femme enceinte et le nourrisson au Bénin. Pour ces raisons, une politique de supplémentation en fer et un traitement préventif intermittent (TPI) contre le paludisme ont été mis en place pendant la grossesse au Bénin. Étant donné que le TPI influence le paludisme gestationnel, qui, lui, pourrait avoir un effet sur le paludisme de l'enfant, nous avons investigué l'effet du TPI sur le paludisme de l'enfant. En parallèle, étant donné que différentes études épidémiologiques suggèrent que des niveaux de fer élevés auraient un effet délétère sur le risque de paludisme, nous avons investigué le possible risque accru de paludisme chez une cohorte de 1005 femmes enceintes et 400 enfants au Bénin. Dans des modèles multi-niveaux à intercepte aléatoire au niveau de la mère, des niveaux de fer élevés étaient associés significativement au risque d'avoir une goutte épaisse positive et à une parasitémie par P. falciparum plus élevée. Dans des modèles multi-niveaux à intercepte aléatoire au niveau de l'enfant, les niveaux de fer de élevés étaient associés significativement au risque d'avoir une goutte épaisse positive. L'extension du TPI (le nombre de jours entre les deux doses), était significativement associé à un moindre risque de paludisme.Par conséquence on devrait augmenter les doses et l'observance du TPI, et des études supplémentaires sur l'effet de différentes doses de fer, et études comparatives entre le fer donné en tant que traitement vs supplémentation systématique sont nécessaires. / In Benin malaria and nutritional deficiencies are the main diseases contributing to the disease burden. Therefore, preventive strategies targetting both diseases have been deployed for over 10 years. Pregnancy-associated malaria (PAM) is responsible for maternal anaemia, placental malaria and low birth weight (<2500g). To prevent PAM, the WHO recommends the intermittent preventive treatmentduring pregnancy (IPTp). Considering that IPTp has an effect on PAM, and thereby influences the exposure of the fœtus to the parasite, we wanted to investigate the possible effect of IPTp on malaria in infants. In parallel, iron supplements are recommended during pregnancy to prevent maternal anemia. As there is some epidemiological evidence that iron might enhance malaria episodes and their severity we wanted to analyse the association of iron levels with malaria in pregnancy and infancy. Therefore, we conducted a cohort study of 1005 pregnant women and 400 infants from 2010 to 2012 in Allada (Benin). We showed that IPTp duration (number of days between doses) was inversely correlated with malaria risk and P. falciparum parasitemia, possibly due to the reduction of the exposure of the fœtus to the parasite in utero. We also found that iron levels during pregnancy and infancy were associated to increased malaria risk and P. falciparum parasitemia, with a possible dose effect. In a context of growing resistance to SP, IPTp doses and observance should increase. In addition, complementary interventional data are needed to determine the benefits and risks of differently dosed iron supplements, in order to ascertain their impact on infant health in malaria-endemic regions.

The discovery of new β-haematin inhibitors has become one focus for researches in response to the resistance of P. falciparum malaria parasites that emerged towards well-known antimalarials. While hundreds of new β-haematin inhibitors have been discovered using detergent mediated high-throughput screening methods, a crucial aspect is understanding exactly how these β-haematin inhibitors behave in the malaria parasite and inhibit the formation of haemozoin. What is known, is that well-known β-haematin inhibitors like chloroquine cause increased amounts of exchangeable haem in the parasite digestive vacuole and form a Fe(III)PPIX-inhibitor complex by accumulating at high concentrations which consequently inhibits parasite growth. Another important focus is on understanding the digestion of haemoglobin and its role in haemozoin formation. This research investigates the in vitro modelling of cellular haemozoin and inhibition by various β-haematin inhibitors across different scaffolds and the role of haemoglobin degradation in P. falciparum malaria parasites. The investigated β-haematin inhibitors resulted in micromolar IC50 (NF54) values and decreased parasite growth with increases in concentration. Using a pyridine-based parasite haem fractionation plate method, these β-haematin inhibitors were shown to target haemozoin formation by causing increased amounts of exchangeable haem that corresponded to decreasing amounts of haemozoin in chloroquine-sensitive parasites. The amounts of exchangeable haem were shown to be inversely proportional to the percentage of parasite growth in the presence of these β-haematin inhibitors. It was apparent that there was a tendency for parasite growth inhibition activity to decrease as the amount of exchangeable haem present in chloroquine sensitive parasites increased, although, the trend was not statistically significant. Moreover, it was observed that experimental cellular accumulation ratio values were low in comparison to chloroquine and amodiaquine. Based on the experimental cellular accumulation ratio values, it was deduced that the accumulation of these β-haematin inhibitors was not primarily due to pH trapping and more complex than previously proposed. Further investigations into the exchangeable haem amounts as a function of intracellular test compound amounts at the IC50 values of these β-haematin inhibitors highlighted that there was an apparent 1:1 relationship with the amount of intracellular exchangeable haem, indicative of complex formation. Transmission electron microscopy images were obtained for untreated parasites that showed intact parasites inside red blood cells with clearly visible haemozoin crystals dispersed throughout the parasite digestive vacuole, whilst, treated parasites showed less defined haemozoin crystals as a result of inhibition. Moreover, electron energy-loss spectroscopy revealed that untreated parasites exhibited a strong iron signal which was associated with haemozoin in the parasite digestive vacuole with a weaker signal attributed to the red blood cell cytoplasm. Similarly, a strong iron signal was shown in the digestive vacuole of treated parasites which was associated with less defined haemozoin crystals. A halo around these haemozoin crystals was observed and was suggested to be indicative of the build-up of exchangeable haem. Additionally, a strong bromine signal attributed to a bromine-containing β-haematin inhibitor, test compound 1, was also observed in the same region as the haemozoin crystals. Overlaid signal distribution maps for iron and bromine showed direct evidence of Fe(III)PPIX and test compound 1, suggesting complexation. High-quality Raman spectra were obtained for the Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetically prepared samples for the Fe(III)PPIX porphyrin dominated spectral region of 1700-500 cm-1 at an excitation wavelength of 532 nm. From the spectra, a putative Fe(III)PPIX-test compound 1 complex was identified and shown to be similar to the synthetically prepared counterpart, haematin-test compound 1 mixture. It was highlighted that a unique peak at 1080 cm-1 indicated π- π interactions between the pyrrole-imidazole ring and thus confirming that the formation of this putative Fe(III)PPIX-inhibitor complex occurs. The confocal Raman true mapping technique proved to be efficient and reliable for imaging the signal distribution of haemozoin at the Raman peak of 754 cm-1 and 1080 cm-1 for the Fe(III)PPIX-test compound 1 complex which co-localized in the digestive vacuole of chloroquine sensitive parasites. Moreover, oxy- and deoxy-haemoglobin was observed to be localized to the red blood cell, where, deoxy-haemoglobin was located on the outer parts of the parasite. Principle component analysis, based on the Raman peak positions, exhibited significant differences in the spectra for Fe(III)PPIX species in red blood cells, chloroquine sensitive parasites and synthetic samples where clusters were observed to separate mainly along principle component 1. These data proved that the spectra of the Fe(III)PPIX-test compound 1 complex was the same as its synthetically prepared counterpart but different from the remaining Fe(III)PPIX species. In comparison to the Fe(III)PPIX-test compound 1 complex, the cluster separations were observed to be significant, where, no significant separation was observed for the Fe(III)PPIX-test compound 1 complex and the haematin-test compound 1 mixture. Based on this, it was evident that a Fe(III)PPIX-test compound 1 complex existed in the digestive vacuole of treated chloroquine sensitive parasites. To fully understand the inhibition of haemozoin, the development of a haem pathway model is necessary, but, requires certain prerequisites. Bioinformatics data from PAXdb and ExPASy revealed that chloroquine resistance (Dd2) parasites, containing 1337 previously identified proteins with an average abundance-weighted molecular weight of 40,483 ± 77 g/mol. With this, the protein mass per cell for red blood cells, chloroquine-sensitive and - resistant parasites were consistent across three protein quantification methods was measured and revealed that chloroquine resistant parasites had a significantly higher protein mass per cell than chloroquine sensitive parasites and in turn a higher total number of protein molecules per cell. Aspartic proteases are 4-fold higher in concentration than cysteine proteases with histo-aspartic protease having the highest concentration in chloroquine resistant parasites. Along with these data, a time point quantification for chloroquine sensitive parasites throughout the blood-stage showed that the amount of haemoglobin decreased in a sigmoidal manner and corresponded to a linear increase in the amount of haemozoin and relatively constant exchangeable haem amount. This was consistent with Giemsa smears that showed that for early time points, large initial decreases in the amount of haemoglobin were observed between the early trophozoite to late trophozoite stage.

Malaria is a severe public health concern in certain regions, causing 445,000 deaths and over 200 million cases in 2016 (Ashley et al., 2018). The vast majority of these cases and deaths are located in warm climates where the Anopheles mosquito is present, especially in sub-Saharan Africa and southeast Asia. Current efforts aim to prevent the disease through vaccination, which has proven to be challenging. Plasmodium falciparum, the pathogen responsible for malaria, is transmitted between humans via the female vector Anopheles mosquito. This parasite has a complex life cycle that is not fully understood, making it difficult to treat the infection and even more difficult to inoculate a population through vaccination. P. falciparum is also capable of polymorphism, changing structure once a host antibody has identified a pathogenic antigen. For this reason, it is very technically challenging to develop a vaccine that is able to confer a high enough immune response through sufficient host antibody production. This thesis will begin with a review on the prevalence and severity of malaria and an overview of the principles of immunology and vaccine development. We will then discuss the parasitic life cycle and how it results in the pathogenesis of the disease. Current antimalarial treatments and resistance to those treatments will be analyzed. This thesis will conclude with an in-depth analysis of the current prophylactic vaccines against P. falciparum, focusing on their mechanism, efficacy, and probability of success.

MSc Applied Mathematics) / Department of Mathematics and Applied Mathematics / See the attached abstract below

MSc (Biochemistry) / Department of Biochemistry / Plasmodium falciparum is a unicellular protozoan parasite that causes malaria in humans. The parasite is passed to humans through mosquito bites and migrates to the liver before it infects host erythrocytes. It is at the erythrocytic stage of development that the parasite causes malaria pathology. Malaria is characterized by the modification of host erythrocytes making them cytoadherent. This is as a result of formation of protein complexes (knobs) on the surface of the erythrocyte. The knobs that develop on the surface of the erythrocyte are constituted by proteins of host origin as well as some proteins that the parasite ‘exports’ to the host cell surface. Nearly 550 parasite proteins are thought to be exported to the infected erythrocyte. Amongst the exported proteins is P. falciparum heat shock protein 70-x (PfHsp70-x). Hsp70 proteins are known to maintain protein homeostasis. Thus, the export of PfHsp70-x may be important for maintaining protein homeostasis in the host cell. PfHsp70-x is not essential for parasite survival although is implicated in the development of parasite virulence. This is possibly through its role in facilitating the trafficking of parasite proteins to the erythrocyte as well as supporting the formation of protein complexes that constitute the knobs that develop on the surface of the infected erythrocyte. The main objective of the current study was to investigate protein interaction partners of PfHsp70-x. It is generally believed that PfHsp70-x interacts with various proteins of human and parasite origin. Potential candidate interactors include its protein substrates, Hsp70 co-chaperones such as Hsp40 members, and human Hsp70-Hsp90 organizing protein (hHop). The establishment of the PfHsp70-x interactome would highlight the possible role of PfHsp70-x in the development of malaria pathogenicity. Based on bioinformatics analysis, PfHsp70-x was predicted to interact with some exported P. falciparum Hsp40s, hHop and human Hsp90 (hHsp90). Recombinant forms of PfHsp70-x (full length and a truncated form that lacks the C-terminal EEVN motif implicated in co-chaperone binding) were expressed in E. coli BL21 Star (DE3) cells. Recombinant hHop and hHsp70 were expressed in E. coli JM109 (DE3) cells. The proteins were successfully purified using nickel affinity chromatography. Co-affinity chromatography using recombinant PfHsp70-x and immuno-affinity chromatography using PfHsp70-x specific antibody did not confirm the direct interaction of PfHsp70-x with human Hop. However, the direct interaction of hHop and PfHsp70-x has previously been validated in vitro and the current bioinformatics data support ii the existence of such a complex. PfHsp70-x was not stable in the cell lysate that was prepared and this could explain why its interaction with hHop could not be ascertained. However, taken together the evidence from a previous independent study, and the predicted interaction of PfHsp70-x with human chaperones suggests cooperation of chaperone systems which possibly facilitates the folding and function of parasite proteins that are exported to the infected erythrocyte. / NRF

Polyamines are ubiquitous components of all living cells and their depletion usually causes growth arrest or cytostasis, a strategy employed for treatment of West-African trypanosomiasis. In the malaria parasite, Plasmodium falciparum, polyamine biosynthesis is regulated by the uniquely bifunctional protein, Sadenosylmethionine decarboxylase/ornithine decarboxylase (PfAdoMetDC/ODC). The unique nature of this protein could provide a selective mechanism for antimalarial treatment. To validate polyamine depletion and specifically PfAdoMetDC/ODC, as drug target for antimalarial therapeutic intervention, polyamine biosynthesis was completely restrained via the inhibition of both catalytic sites of PfAdoMetDC/ODC with DFMO and MDL73811. The physiological effects during the resulting cytostasis were studied with a comprehensive functional genomics approach. The study was preceded by various assays to determine the treatment dosage that would result in complete cytostasis, without non-specific chemical cytotoxicity. The results obtained revealed that the cytostatic mechanism with growth arrest of the treated parasites and normal progression of the untreated controls require special consideration for basic comparisons of response in terms of the assay methodology used and data analysis. This is particularly important when studying a multistage organism such as P. falciparum, which constantly develops and change during the intraerythrocytic developmental cycle, such that growth arrest compared to normal progression would result in significant differences merely due to stage. This critical principle was kept in mind throughout the investigation and was applied to the relative quantification of RNA, proteins and metabolites via a relative time zero approach as opposed to the standard parallel time point comparison. Three independent functional genomics investigations, namely transcriptomics, proteomics and metabolomics were conducted, in which highly synchronised 3D7 parasite cultures were treated during the schizont stage and parasites were sampled during a time course at three time points (just before and during cytostasis). Transcriptome analysis revealed the occurrence of a generalised transcriptional arrest just prior to the growth arrest. To our knowledge this is the first time that transcriptional arrest as the preceding mechanism of cytostasis due to polyamine depletion, was demonstrated. However, despite the transcriptional arrest, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses: the increased abundance of the transcripts for lysine decarboxylase and ornithine aminotransferase (OAT) and the decreased abundance of that for S-adenosylmethionine synthetase (AdoMet synthetase). Pearson correlations indicated more subtle effects of the perturbation on the proteome and even more so on the metabolome where homeostasis was generally maintained, except downstream to the enzymatic blockade at PfAdoMetDC/ODC. The perturbation-specific compensatory roles of OAT in the regulation of ornithine and AdoMet synthetase in the regulation of AdoMet were confirmed on both the protein and metabolite levels, confirming their biological relevance. The results provide evidence that P. falciparum respond to alleviate the detrimental effects of polyamine depletion via the regulation of its transcriptome and subsequently the proteome and metabolome, which supports a role for transcriptional control in the regulation of polyamine and methionine metabolism within the parasite. The study concludes that polyamines are essential molecules for parasite survival and that PfAdoMetDC/ODC is a valid target for antimalarial drug development. / Thesis (PhD)--University of Pretoria, 2008. / Biochemistry / unrestricted

Malaria is one of the deadliest parasitic diseases worldwide, causing 219 million infections and 435 thousand deaths per year. As such, this mosquito-borne illness is a major target for global eradication efforts. One critical arm of the eradication strategy is chemotherapy. For a therapeutic to advance the eradication agenda, it must cure the patient of infection and eliminate transmission stage parasites (called gametocytes) from the blood, thereby breaking the cycle of transmission. Currently, first-line treatments against malaria infection consist of an artemisinin derivative in combination with another antimalarial drug from a different drug class. Although artemisinin and its derivatives are highly efficacious at curing malaria, these drugs are ineffective at preventing disease transmission. However, recent in vivo studies have suggested that whole plant Artemisia annua (the botanical source of artemisinin) delivered as tea can cure patients of infection and eliminate transmission stage parasites from the bloodstream. To validate these in vivo results in vitro, experiments were performed to measure the killing efficacy of A. annua and A. afra tea infusions against three different stages of the parasite life cycle— one stage of the asexual cycle, immature gametocytes, and mature gametocytes. Killing effects were observed using light microscopy and gametocyte gene-specific RT-qPCR analyses. Results suggested that A. annua tea was nearly as effective as artemisinin at killing all three tested stages of the parasite. A. afra tea, which contains low levels of artemisinin, showed comparable killing efficacy against late stage gametocytes, but not against the other two tested stages. These results supported the notion that A. annua tea is an effective antimalarial and also provides evidence that both A. annua and A. afra teas may be a viable therapeutic option for eliminating gametocytes during human infection.

Thesis submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirement for the degree of Doctor of Philosophy 2019 School of Public Health University of the Witwatersrand, South Africa / Background: Spatial targeting of interventions is increasingly recognized as essential for malaria control, particularly in areas aiming for elimination. The associations between house characteristics and malaria transmission is known, but gaps remain on whether transmission is also influenced by factors such as distances between households or the degree to which houses are clustered. It is also important to identify new interventions that can be implemented by targeting critical points in the biology of major malaria vectors and common characteristics of human settlements. Such efforts will be essential to complement current major tools. Aims: This study had the following aims; (i) understand how households densities and their distances influence malaria transmission the study area , (ii) identify housing-related factors affecting residual transmission risks in the area (iii) assess opinions of residents on how house characteristics, settlement patterns and other environmental factors influencing malaria transmission, and (iv) identify effective complementary approaches that could be used to improve malaria control in these residual transmission area with high coverage of basic interventions such as long lasting treated nets. Methodology: A mixed methods approach was used, which included: (i) indoor and outdoor mosquito collections from fixed as well as randomly selected households over 12 months, (ii) characterization of physical and microclimatic conditions of the houses and surrounding environmental variables, (iii) assessing effects of spatial clustering of households on malaria transmission risks, and (iv) assessing community concerns regarding household and environmental factors influencing transmission. During these studies, swarms of Anopheles mosquitoes were discovered for the first time in this area. Therefore, additional quantitative and qualitative observations were made to characterize the swarms and assess how they could be targeted to complement malaria control efforts. Key findings: Despite the expansive use of LLINs in the area, factors associated with housing characteristics and settlement patterns as well as people’s perceptions contribute to persistent malaria transmission, and will need to be addressed for eventual elimination to be reached. The main findings of this study were as follows: (i) household occupancy influenced indoor mosquito density (ii) high house densities increased Anopheles biting risk but mosquito density declined as distances between houses increased beyond 50m; (iii) abundance of the vector Anopheles arabiensis peaked during high rainfall months (February-May), but An. funestus densities remained stable into the dry season (May-August); (iv) there were higher indoor densities of An. arabiensis and An. funestus in houses with mud walls compared to plastered or brick walls, open eaves compared to closed eaves and unscreened windows compared to screened windows; (v) most respondents were aware of associations between their house characteristics and malaria risks but carrying out improvements was constrained by financial costs and other household priorities; (vi) in all our surveys, An. funestus mosquitoes contributed approximately 85% of ongoing malaria transmission in the areas, even when occurring in far smaller densities than An. arabiensis; and (vii) with regards to complementary interventions, small scale studies demonstrated that mating swarms of both An. arabiensis and An. funestus could be readily identified and characterized (in this case by volunteer community members), and that targeting the swarms using aerosol spraying could possibly reduce overall biting risk in the communities. Conclusions: This study yielded evidence that malaria transmission risks are significantly lower when the distances between houses are high. Variation in malaria transmission risk within a village might be important for planning and implementing spatially targeted interventions. With limited resources, malaria control efforts must be prioritized in locations with significantly higher risk in order to obtain maximum benefit. Thus, the distances between houses should be considered when planning malaria vector control strategies. Furthermore, the overall clustering of indoor densities of major disease-transmitting mosquitoes is significantly associated with household occupancy and its spatial distribution within the villages. Further research is needed to assess the potential of using this information for predicting, identifying and targeting the most intense foci of mosquito-borne disease transmission based on household occupancy. Understanding the factors influencing malaria transmission at a small scale is crucial for planning malaria control interventions. Malaria transmission heterogeneity does not only exist on a large scale, but also at small scales, which may be influenced by small variations of environmental features, such as seasonality. These variations should be considered when improving malaria control interventions. While An. funestus showed variation in density during the year, numbers of mosquitoes remained high throughout the dry season, whereas An. arabiensis showed a definite peak in the rainy season with a subsequent drop during the dry season. Further studies are required to investigate the survival strategies of major malaria vectors during the dry season. Understanding this particular feature of An. funestus may lead to the design of new control strategies or improve existing interventions so as to reduce the malaria burden in such rural areas. This study also documented high mosquito densities in mud-walled houses compared to houses with brick or plastered walls. These findings further indicate the necessity of considering house improvement as a malaria control strategy. While the community members had fairly high awareness that improved houses were associated with reduced mosquito nuisance, many families in rural areas are living in poorly constructed houses. It is therefore necessary to consider inter-sectoral collaborations to integrate housing into health policies in Tanzania. Further studies are also needed to provide epidemiological evidence on how housing design affects malaria transmission. Furthermore, community-based house improvement programs should be promoted, including simple, scalable and affordable house improvements for the populations living in high malaria endemic areas in rural communities. Generally, this study has generated information which sets the basis for further studies on the relationship between the exact house location and malaria transmission risk in rural malaria endemic countries. / MT 2020