Tübingen, Univ., Diss., 2007.

Zugl.: Heidelberg, Univ., Diss., 2007

Mestre -- Escola Nacional de Saude Publica, Rio de Janeiro, 2003.

Despite progress through expanded use of bed nets and anti-malarial drugs, Plasmodium falciparum (Pf) malaria caused about 200 million cases and 500,000 deaths in 2015. An ideal vaccine would reduce the burden of disease and interrupt transmission. Despite decades of effort, there is no vaccine that can adequately address the global burden of malaria. This thesis focuses on two potential weaknesses in the parasite life-cycle. First, I investigate two vaccination strategies aimed at improving the antibody response to RH5, an essential and conserved protein for erythrocyte invasion. Due to instability of the resultant recombinant vaccine constructs, these efforts have required re-engineering of the vaccine platform, which remains an ongoing effort. Second, the immunogenicity and mechanism of protection of a live-attenuated whole sporozoite vaccine, PfSPZ Vaccine, was assessed. In a study that examined PfSPZ Vaccine at intravenous (IV) doses between 1.35 &tiles; 10⁵ to 4.5 &tiles; 10⁵ PfSPZ, I demonstrate that PfSPZ antibody responses correlated with durable sterile protection against controlled human malaria infection (CHMI). Surprisingly, the pre-vaccine frequency of Vγ9⁺Vδ2⁺ T cells, an innate T cell that recognizes conserved Plasmodium phosphoantigens, also correlated with durable sterile protection. Regarding the mechanism of protection, PfSPZ-specific antibodies as well as CD8 and CD4 T cells in the blood decreased substantially over time, yet sterile protection was maintained. In non-human primates, the CD8 T cell response in the liver at a memory time point was measured to be about 100-fold higher than found in the blood. Collectively, these data suggest that PfSPZ Vaccine confers durable protection in humans by long-lived, tissue-resident CD8 T cells. These findings were extended with a study using 9.0 x 10⁵ PfSPZ, wherein I demonstrate that T cell responses peaked immediately after the first vaccination with minimal T cell activation despite additional immunizations. This suggests that anti-PfSPZ immunity may be limiting the effectiveness of subsequent immunizations. Finally, I examined the T cell response to PfSPZ attenuated by chloroquine (termed PfSPZ-CVac). T cell responses were substantially higher than achieved with comparable PfSPZ Vaccine doses. Additionally, a significantly higher proportion of PfSPZ-specific CD4 T cells were polyfunctional, simultaneously expressing IFN-γ, IL-2, and TNF-α, in subjects that were protected from CHMI. In sum, these studies provide insight into the immunobiology of a protective immune response that may guide future malaria vaccine development efforts.

Malaria is one of the most prevalent diseases in Africa and the Plasmodium falciparum species is widely accepted as the most virulent, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials there has been little progress towards a proven vaccine. Pending a long term solution, endemic countries rely heavily on the development of innovative drugs with acute efficacy coupled with rapids mode of action. Until recently the rate of drug action has been measured by light microscopic examination of parasite morphology using blood slides of drug treated parasite cultures at regular time intervals. This technique is tedious and, most importantly, subject to interpretation with regards to distinguishing between viable and comprised parasite cells, thus making it impossible to objectively quantitate the rate of drug action. This study aimed to develop a series of bioassays using the calcein-acetoxymethyl and propidium iodide vitality probes which would allow the rate of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other. A novel bioassay using these fluorescent vitality probes coupled with fluorescence microscopy was developed and optimized and allowed the rate of drug action on malaria parasites to be assessed i) rapidly (in relation to current assay techniques) and ii) in a semi-quantitative manner. Extrapolation to flow cytometry for improved quantification provided favourable rankings of drug killing rates in the pilot study, however, requires further development to increase throughput and approach the ultimate goal of producing a medium-throughput assay for rapidly assessing the rate of action of antimalarial drugs. Attempts to adapt the assay for use in a multiwell plate reader, as well as using ATP measurements as an indication of parasite vitality after drug treatment, was met with erratic results. The viability probes assay as it stands represents an improvement on other assay formats in terms of rapidity and quantification of live/compromised parasites in cultures.

Los genotipos de Plasmodium vivax, junto con la densidad parasitaria pueden estar relacionados al grado de severidad de la malaria. El conocimiento de esta relación puede ayudar a un mejor manejo de la enfermedad, tratamiento con drogas y elaboración de posibles vacunas. El objetivo del trabajo fue determinar el número de genotipos de Plasmodium vivax presentes en la Amazonía Peruana. Para la genotipificación se usó el gen que codifica la proteína de superficie del merozoito (MSP3 alfa) y el polimorfismo generado por secuencias repetitivas de nucleótidos (TR) encontrado en un segmento de 100 Kilobases (Kb) de Plamodium vivax “sinténico” al cromosoma 3 de Plasmodium falciparum. Se trabajó con 302 muestras de sangre de pacientes, a todas ellas se les realizó el examen de la gota gruesa y frotis para el diagnóstico y conocimiento de la densidad parasitaria, dicho diagnóstico fue confirmado por Nested PCR. A las muestras confirmadas se les realizó la genotipificación. Con el marcador MSP3 alfa se identificaron 9 genotipos, de los cuales uno de ellos se encontró asociado a severidad de la enfermedad (P7) (P<0.05, OR>1), y otro relacionado con infecciones mixtas (P9), 1/9 (11%). Para los TR se seleccionaron 9 pares de “primers” de un total de 33. Encontrándose 102 genotipos diferentes de los cuales 24/102 (24%) fueron infecciones por genotipos mixtos. El hecho que exista una inserción en el tamaño de su ADN originalmente reportado, aumenta la posibilidad que se dé la enfermedad en forma más severa. Estos resultados indicarían que poblaciones de Plasmodium vivax son altamente diversos y que infecciones por múltiples clonas se darían en la región hipoendémica de la Amazonía Peruana, las cuales podrían representar un desafío para evaluación posterior de drogas y vacunas. / Genotypes of Plasmodium vivax along with parasite density may be associated with the severity level of malaria, and knowledge of this relation can help to better understand the disease, drug treatments and the development of new vaccines. The object of this project was to determine the number of Plasmodium vivax genotypes present in the Peruvian Amazon. This genotyping utilized the gene encoding a merozoite surface protein (MSP3 alpha) and the polymorphism generated by a sequence of nucleotide repeats (TR) found in a 100 kilobases (Kb) de Plasmodium vivax syntenic chromosome 3 of Plasmodium falciparum. In this project we used 302 blood samples from patients. A blood droplet and droplet smear were obtained for the diagnostic and observation of parasite density; these results were later confirmed through Nested PCR. All samples were submitted for genotyping. Using the marker MSP3 alpha, 9 genotypes were identified, one was found associated with disease severity (P7) (P<0.05, OR>1) and other with mixed infections (P9) 1/9 (11%). For the TR, 9 pairs of primers were selected from a pool of 33 describing 102 different genotypes of which 24 (24 %) were mixed infections. Observation supported by the existence of a DNA insertion that increases the original size of the sequence, increasing the possibility that the disease become more severe. These results would indicate that populations of Plasmodium vivax are highly diverse and can result in multiple infections by different clones in hypoendemic regions such as the Peruvian Amazon, making later evaluation of drugs and vaccines more challenging. / Tesis

Current malaria treatments are ineffective in sub-Saharan Africa due to problems beyond the disease. Approximately 90% of malaria mortalities occur in sub-Saharan Africa, and 77% percent of these are children under the age of five. At the same time, sub-Saharan Africa is also the recipient of 80% of international aid. With international malaria funding increasing in recent years, there must be an analysis on the practicability of funded interventions as malaria continues to be a tremendous burden in the region. This review highlights the complexity of malaria pathology and its association with poverty that makes treatments ineffective. Available, frontline antimalarial drugs and insecticides have shown increased resistance that has spread throughout many malaria endemic regions. This resistance aggravates the disease as the parasite and the vector evolve, resulting in increased transmission, increased severity of symptoms, and a high risk of mortality. In addition, the heavily funded malaria vaccine under development by GlaxioSmithKline and PATH shows partial efficacy that languishes over time, putting to question the practicability of such heavily funded interventions. The limitations of available treatments necessitates a holistic approach that responds to the economic state of endemic regions in order to effectively alleviate the burden of disease. An example of a holistic approach is the Multisectoral Action Framework for Malaria. This approach considers the socioeconomic development and fragile markets of endemic nations to encourage partnerships between governments and healthcare sectors in eradicating malaria. Although it will take years to demonstrate results, the burden of malaria calls for sustained efforts to alleviate the burden of the disease along with the poverty that perpetuates it.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Saúde Pública. / Made available in DSpace on 2012-10-21T01:28:20Z (GMT). No. of bitstreams: 1 191406.pdf: 766588 bytes, checksum: c3e2812fa7c4fca064d6f3a7df70142c (MD5) / A história da malária em Santa Catarina foi abordada sob três principais enfoques: a evolução da ocorrência e da magnitude da doença no Estado, os estudos referentes aos fatores de transmissão e os métodos de combate ao longo do tempo. No que se refere à ocorrência e à magnitude, o período abrangido estende-se de 1877 a 1994, com inúmeras lacunas de maior ou menor amplitude e caracterizadas pela quase ausência de informações disponíveis. Em 1877, um relatório faz referência à ocorrência freqüente de malária no litoral catarinense. Em 1947, o número de casos registrados da doença no Estado era de 26724. Esse número caiu no decorrer dos anos, de tal maneira que, em 1958, foram detectados 1384 casos. Quanto aos fatores de transmissão, procura-se descrever as pesquisas que levaram à descoberta, no início da década de 1940, no litoral do Brasil e, particularmente, no litoral de Santa Catarina, do chamado complexo bromélia-malária, caracterizado pela existência, nas florestas, das bromélias, como criadouros dos mosquitos do sub-gênero Kerteszia, únicos vetores da malária na região. A área correspondente ao complexo bromélia-malária, em Santa Catarina, estende-se, no sentido norte-sul, da divisa com o Paraná à divisa com o Rio Grande do Sul, numa faixa compreendida entre as serras Geral e do Mar ao oceano. No que diz respeito aos métodos de combate utilizados, eles foram diferentes, em função do período histórico considerado.. Até a descoberta do complexo bromélia-malária, os métodos consistiam em obras de drenagem e saneamento de coleções hídricas e no tratamento dos pacientes. Com a descoberta do complexo bromélia-malária, os métodos passaram a ser radicalmente diferentes e consistiam na destruição das bromélias, no combate às larvas de Kerteszia, no tratamento de pacientes com antimaláricos e no combate aos mosquitos alados com inseticidas de efeito residual. O objetivo era manter a doença sob controle. Essa metodologia variada de combate persistiu até 1962, quando foi instituída a campanha de erradicação. A partir daquele ano, o método principal de combate era a dedetização intradomiciliar, complementada com o tratamento radical dos casos detectados. O objetivo era erradicar a malária do Estado. A interrupção da transmissão foi conseguida em 1986, quando ocorreram os últimos casos autóctones da doença.

Orientador : Prof. Dr. Alan Guilherme Gonçalves / Coorientador : Prof. Dr. Diogo R. B. Ducatti / Coorientador : Prof. Dr. Roberto Pantarolo / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas. Defesa: Curitiba, 25/02/2016 / Inclui referências : f. [121-139] / Área de concentração: Insumos, medicamentos e correlatos / Resumo: Uma doença de longo histórico existencial, a malária é uma parasitose que ainda coloca diversas regiões do mundo em risco, ocasionando milhares de mortes. A Organização Mundial de Saúde (OMS) recomenda a terapia combinada com artemisininas (ACT) como a primeira linha de tratamento medicamentoso para a doença, frequentemente em associações com o cloridrato de mefloquina. De modo similar a outras doenças multifatoriais, diversos estudos vêm sendo realizados acerca do tratamento da malária com o uso de compostos multifuncionais (Designed Multiple Ligands, DMLs), os quais apresentam potencial de agir simultaneamente sobre diferentes fatores patológicos. Uma das estratégias para a síntese de DMLs consiste na ligação molecular de fármacos distintos utilizando grupos conectores (linkers). As propriedades físico-químicas dos monossacarídeos e seus derivados os tornam um bom alvo para uso como grupos conectores para a síntese de antimaláricos multifuncionais. Até o momento, não foram descritos na literatura o uso de derivados do ácido tartárico na associação molecular entre a mefloquina e outros antimaláricos. O presente trabalho teve como foco a avaliação de um monossacarídeo (D-glucose) e um ácido aldárico (ácido L-(+)-tartárico) como grupos conectores em duas estratégias distintas para a formação de DMLs de artemisinina e mefloquina. Modificações na estrutura do ácido tartárico geraram um linker isopropilideno derivado, o qual foi ligado ao nitrogênio piperidínico do cloridrato de mefloquina por meio de uma reação de Steglich na presença de DCC, com a formação de um grupo amida. O padrão de desdobramento de sinais da mistura diastereoisomérica formada sugere a formação predominante de um dos confôrmeros distintos para um dos dois diastereoisômeros formados. Esses produtos apresentaram indícios de ligação à dihidroartemisinina frente a análises espectrométricas, para a formação de DMLs. Os resultados apresentados constituem um importante ponto de partida na aplicação de fármacos associados a carboidratos e derivados no tratamento da malária e, consequentemente, demais doenças multifatoriais. Palavras-chave: Malária, Artemisinina, Mefloquina, DML, monossacarídeos. / Abstract: A long-existing disease, malaria still is a parasitosis endangering many regions around worldwide, causing thousands of deaths. World Health Organization (WHO) recommends artemisinin combination therapy (ACT) as the fisrt-line medical treatment for malaria, often associating it with mefloquine hydrochloride. In the same way to other multifactorial diseases, several studies have been conducted on using designed multiple ligands (DMLs) on malaria treatment. This class of compounds demonstrates potential on acting simultaneously over multiple targets of the pathologies. One of the strategies for DML synthesis consists on linking distinct drugs covalently through connecting groups (linkers). The physiochemical properties of monosaccharides and their derivatives make them an interesting study subject as antimalarial DMLs connecting groups. ). Recent literature does not describe the use of tartaric acid derivatives as connecting groups between mefloquine and other antimalarials. This study focused on evaluating a monosaccharide (D-glucose) and an aldaric acid (L-(+)-tartaric acid) as linkers in two different strategies for artemisinin-mefloquine DML synthesis. Modifications in tartaric acid structure generated an isopropylidene linker, which reacted with mefloquine hydrochloride's piperidine nitrogen by Steglich amidation in the presence of catalyst DCC. NMR signal patterns of the diastereoisomerical mixture suggest the predominant formation of one of the two obtained conformers. This diastereoisomerical mixture showed spetrometrical evidence on DML formation through dihydroartemisinin conjugation. The present results can be considered an important starting point for the use of carbohydrate-associated drugs for malaria and other multifactorial diseases treatment. Keywords: Malaria, Artemisinin, Mefloquine, DML, monosaccharides.

Made available in DSpace on 2015-05-14T16:53:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-04-15Bitstream added on 2015-05-14T16:59:11Z : No. of bitstreams: 1 000828911.pdf: 473053 bytes, checksum: 63407242b7f0b46a1ef661b3ea907e82 (MD5) / Introdução: O envolvimento do sistema cardiovascular em pacientes com malária por Plasmodium vivax tem sido pouco estudado. Neste trabalho, avaliamos as estruturas cardíacas, a função ventricular e marcadores sistêmicos de lesão cardiovascular em pacientes com a forma não-grave da malária por P. vivax, em Manaus, estado do Amazonas, Brasil. Métodos: Foram avaliados, prospectivamente, 26 pacientes adultos com malária por P. vivax em tratamento ambulatorial, no período de janeiro de 2012 a março de 2013. Os resultados foram comparados com grupo controle de 25 indivíduos saudáveis, pareados por gênero e idade. Avaliação clínica, exames laboratoriais e ecocardiografia transtorácica foram realizados na primeira avaliação após o diagnóstico de malária (dia zero, D0) e sete dias após (D7) início do tratamento. Resultados: Os Casos apresentaram maiores valores do diâmetro sistólico do ventrículo esquerdo (VE; 28,8±2,82 vs 30,9±4,03 mm; p=0,037) e do volume diastólico do VE (82,4±12,3 vs 93,8±25,9 ml; p=0,05) e menor fração de ejeção do VE (método de Teicholz: 73,2±6,59 vs 68,4±4,87; p=0,004) que os Controles. A fração de variação da área do ventrículo direito (VD) foi menor (54,7±5,11 vs 50,5±6,71 %; p=0,014) e o índice de performance miocárdica do VD (0,21±0,71 vs 0,33±0,19; p=0,007), a área diastólica do VD (13,0±3,19 vs 15,3±2,96 cm2; p=0,009 ), a área de sistólica do VD (6,41±1,27 vs 7,45±1,46 cm2; p=0,009) e a resistência vascular pulmonar (1,13±0,25 vs 1,32±0,26 unidades Woods; p=0,012) foram maiores nos Casos que nos Controles. A fração de variação da área do VD foi também menor e a resistência vascular pulmonar maior nos Casos no D0 que no D7. No D0, os Casos apresentaram maior concentração sérica de bilirrubina indireta, da molécula de adesão celular vascular solúvel tipo 1, do fragmento N-terminal do pró-peptídeo natriurético cerebral e da troponina T, e menor ... / Purpose: Malaria is an endemic disease in the Amazon Region. In Brazil it has been largely attributed to Plasmodium vivax infection since the 1990’s. Cardiovascular system involvement in patients with P. vivax malaria has been poorly addressed. The aim of this study was to evaluate cardiac structures and function and serum markers of cardiovascular injury in patients with the nonsevere form of P. vivax malaria in Manaus, Amazonas state, Brazil. Methods: In an observational study, we prospectively evaluated 26 patients with P. vivax malaria in an outpatient referral hospital from January 2012 to March 2013 and compared results with a control group of 25 gender- and age-matched healthy individuals. Patients underwent clinical evaluation, laboratory tests, and transthoracic echocardiography at first evaluation after malaria diagnosis (day zero, D0) and seven days after starting malaria treatment (day seven, D7). Results: Echocardiography showed higher left ventricular (LV) systolic diameter (28.8±2.82 vs 30.9±4.03 mm; p=0.037) and LV diastolic volume (82.4±12.3 vs 93.8±25.9 ml; p=0.05), and lower LV ejection fraction (Teicholz method: 73.2±6.59 vs 68.4±4.87; p=0.004) values in patients than controls. Right ventricle (RV) fractional area change (54.7±5.11 vs 50.5±6.71%; p=0.014) was lower, and RV myocardial performance index (0.21±0.71 vs 0.33±0.19; p=0.007), RV diastolic area (13.0±3.19 vs 15.3±2.96 cm2; p=0.009) and systolic area (6.41±1.27 vs 7.45±1.46 cm2; p=0.009), and pulmonary vascular resistance (1.13±0.25 vs 1.32±0.26 Woods unit; p=0.012) were higher in patients than controls. RV fractional area change was lower and pulmonary vascular resistance higher in patients in D0 than D7. In D0, patients presented higher serum levels of indirect bilirubin, soluble vascular cell adhesion molecule–1 (sVCAM-1), Nterminal prohormone brain natriuretic peptide, and troponin T, and lower levels of nitric oxide than D7 and controls