MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2009 / Background: RTS,S candidate malaria vaccine has been shown to be highly immunogenic in children and infants, but the protective immune mechanisms still remain to be clearly elucidated. It is believed that RTS,S elicits a strong neutralizing humoral immune response directed against surface-exposed sporozoite proteins and cell mediated immune (CMI) responses characterized by predominantly CD4+ Th1 cells. The objective of this study was to investigate humoral and cell-mediated immune responses to the RTS,S/AS02D malaria vaccine and its association with protection against infection and disease by P. falciparum. Methodology and Principal Findings: This secondary data analysis from data of a phase I/IIb randomized, double-blind, controlled trial, included 154 healthy infants living in rural Mozambique, previously immunized with RTS,S/AS02D candidate malaria vaccine or the control Engerix-B™ vaccine. Antibodies against circumsporozoite protein (CSP) and hepatitis-B surface antigen (HBsAg) were measured with a standard ELISA. Fresh blood intracellular staining assay was performed to evaluate the expression of IL-2 and IFN-γ by CD4+ and CD8+ cells in response to in vitro stimulation of specific peptides. Data was evaluated for association with the risk of malaria detected by both active and passive case detection of infection over a period of 6 months post dose 3. Anti-HBs antibody geometric mean titers declined from 10,082 mIU/mL one month post Dose 3 to 2,751 mIU/mL at 12 months post Dose 3 in the RTS,S/AS02D group; anti-HBs v geometric mean titers were 392.4 mIU/mL and 263.9 mIU/mL, respectively in the Engerix- BTM group. Anti-CSP antibody geometric mean titers declined from 199.9 EU/mL one month post Dose 3 to 7.3 EU/mL at 12 months post Dose 3 in the RTS,S/AS02D group. Median stimulation indices of HBs-specific IL-2 and IFN-γ producing CD8+ T cells was higher in the RTS,S/AS02D group than in control group (Wilcoxon rank sum p-values for IFN-γ = 0.015, for IL-2 = 0.030) at 10.5 weeks post immunization. Median stimulation indices of anti-CSP specific IFN-γ producing CD8+ T cells at the same time point was 1.13 (IQR: 0.79 - 1.67; p=0.029). For specific IL-2-producing CD4+ T cells, the median SI was 1.14 (IQR: 0.74 – 1.60, p=0.043) at 10.5 weeks post dose three. The reduction in hazards of malaria infection were 18.3 % (95% CI: -267.9 – 81.8, p=0.793) and -12.0 % (95% CI: -295 – 68.2, p=0.86) for specific IL-2 CD4+ stimulation indices; For specific CD8+ IFN-γ stimulation indices the hazards were -103.6% (95% CI: -690.9 – 47.6; p=0.305) and 48.8% (95% CI: -97.0 – 86.7; p=0.33) at four and 10.5 weeks post immunization respectively. Conclusion: The RTS,S/AS02D vaccine was immunogenic and has elicited detectable levels of CSP specific cell mediated responses. No evidence of association was found between the antibodies anti-CSP and specific cell mediated responses and the risk of malaria.

ABSTRACT INTRODUCTION : Malaria remains a serious health burden among children in sub- Saharan Africa. The Home Management of Malaria (HMM) programme was adopted by African heads of states in 2000 as a strategy to achieve high coverage of prompt and effective anti malarial treatment within 24 hours of the onset of symptoms by home caregivers in areas with poor access to facility based health care. Strategic components of the programme include communication for behavioural change, training of community based public-private health service providers and making antimalarials available in communities[1]. AIM: To determine the impact of HMM strategy in the home treatment of uncomplicated malaria in children in Kassena-Nankana District (KND) of upper-east region of Ghana. Specific objectives described the knowledge, attitude and practice and tested the association between knowledge, attitude and other factors and accurate HMM. METHOD: Secondary data from a survey on the role of health information recipients in access and utilization of treatment for malaria management in children under 5 years (U5s) conducted among 818 women in KND from 2005 to 2006 were analyzed using a cross sectional study design. A total of 708 Home caregivers (HCGs) aged 15-49 years who responded to knowledge of the treatment of uncomplicated malaria was obtained after data cleaning. Knowledge of the treatment of uncomplicated malaria was used as a proxy for accurate HMM (correct dosage and correct duration of antimalarial) in U5s because the questionnaire did not contain information on the actual treatment given by the HCGs. Data analysis was done in STATA 10 using Chi squared test for categorical variables. Logistic regression models were used to quantify the associations and adjust for potential confounders and effect modification. RESULTS: The study found that 59% of the women had good knowledge of the symptoms of uncomplicated malaria and 25% knew that only mosquitoes transmit malaria. On treatment seeking attitude (advice and autonomy), the majority (91%) of the home caregivers received various forms of advice from the older women. Such advice included: using herbs (77%), buying drugs (41%), visiting Health Clinic (24%), and visiting the Community Health Officers (19%). On receiving advice, only 15% would utilize the services of the Community Health Officers (CHOs) who are the main source of treatment information for the communities. Thirty percent (30%) of the HCGs had autonomy of health care decision-making in the households. Accurate HMM in children was 28%. Knowledge of malaria and treatment seeking attitude were not significantly associated with accurate HMM (p>0.05). In the multivariate model, the HCGs were more likely to do accurate HMM in children if they had secondary education (OR = 2.54, 95% CI 1.18 ; 5.60), were of Nankani ethnicity (OR = 3.00, 95% CI 2.08 ; 4.35) and belonged to the very poor socio-economic status (OR = 2.31, 95% CI 1.25 ; 4.30). A Chi squared analysis to further identify the differences between the women who gave drugs and those who did not showed that the women differed significantly in their ethnicity (p<0.001), occupation (p<0.001) and relationship as the biological mothers to the children (p=0.008). The major limitation of this study was that knowledge of the treatment of uncomplicated malaria was used as a proxy for accurate HMM hence the finding is not a true reflection of the actual malaria treatment practice HCGs give to U5s. Another limitation is that the study could not measure the promptness of initiating malaria treatment within 24 hours of the onset of symptoms in children because of the absence of such variable in the data. CONCLUSION: Although HCGs had good knowledge of the symptoms of uncomplicated malaria, it did not translate to accurate HMM. The study identified poor dosage of treatment with Chloroquine (the first line antimalarial at the time of the study) was responsible for inaccurate HMM. Therefore, HCGs need to receive adequate information on the dosage of the current first line Artemesinin Combination Therapy drugs which have replaced Chloroquine in the treatment of malaria. Home caregivers need to be encouraged to utilize the services of the CHOs as the main source of malaria related information in the HMM programme. Specific groups to be targeted include the older women and the HCGs at risk of inaccurate HMM. Further research on the actual treatment given to children is recommended with particular emphasis on qualitative technique to unpack culturally related ethnic beliefs associated with HMM in children.

Abstract Background In sub-Saharan Africa, malaria is the leading cause of morbidity and mortality. An estimated 15 million malaria cases and 40 000 malaria deaths were reported in Kenya. Malaria during pregnancy is associated with adverse health outcomes for both the mother as well as her foetus. The purpose of this study was to examine the relationship between socioeconomic correlates and the uptake of malaria prevention methods during pregnancy. Methodology: Data was drawn from the 2008-2009 Kenya Demographic and Health Survey. A total of 8098 women aged 15-49 were analysed. Stata version 12 was used for the management and analysis of data. Univariate, bivariate and multivariate analysis was carried out to meet the objectives of this study. Results: Forty-eight percent of women made use of Insecticide Treated Net (ITNs), 52 percent were administered with Intermittent Preventative Therapy (IPTp) and 36 percent made use of both measures during pregnancy. Multivariate results indicate that urban women were found to display slightly higher odds of ITN usage (1.13) and the combined usage of ITNs and IPTp (1.22) during pregnancy in comparison to rural women. Women with higher levels of education and women from middle income and rich households displayed higher odds of the uptake of these malaria prevention methods during pregnancy. Conclusion: This study has shown that socioeconomic indicators influence the usage of malaria prevention methods during pregnancy. It is therefore imperative that these factors be considered when designing and implementing policies aimed at improving the uptake of these measures during pregnancy.

A malária é a doença tropical com maior taxa de mortalidade global. O surgimento de resistência às terapias de primeira linha reforça a necessidade do desenvolvimento de novos candidatos a fármacos. O objetivo deste trabalho foi a descoberta de inibidores e otimização da atividade destas moléculas como candidatos a compostos líderes para o desenvolvimento de novos agentes antimaláricos. Nesse sentido, realizamos a triagem da coleção Malaria Box e identificamos 11 moléculas de diferentes classes químicas como candidatos a inibidores da enzima enolase de Plasmodium falciparum (Pfeno). Em seguida, determinamos a potência inibitória contra a enzima alvo (IC50 entre 11 &ndash; >1.000 &mu;M), atividade antiplasmodial in vitro contra a forma eritrocítica do parasito (EC503D7 entre 5,6&ndash;1.600 nM) e citotoxicidade (MCL50 > 19 &mu;M) do subconjunto de 11 compostos do Malaria Box (1&ndash;11). Ensaios de combinação com o antimalárico artesunato e estágio de ação foram conduzidos para avaliar o potencial de associação e qual fase do ciclo eritrocítico seria mais suscetível as moléculas desse subconjunto. Os resultados obtidos indicam que alguns compostos apresentam caráter aditivo (2 e 9) ou antagônico (1, 3&ndash;8, 10 e 11) em relação ao artesunato e ação rápida (1&ndash;3, 5, 7, 9&ndash;11) ou lenta (4, 6 e 8) no desenvolvimento do parasita. Diante desses resultados, um conjunto de critérios estruturais e de atividade biológica foi estabelecido para a seleção de um candidato para estudos de relação estrutura-atividade (SAR). O derivado de hidrazinobenzimidazol (4) foi selecionado como hit inicial e 24 derivados (12&ndash;35) foram planejados, sintetizados e tiveram a atividade de inibição enzimática (IC50 entre 44 &ndash; >200 &mu;M), atividade antiplasmodial contra cepas sensível (EC503D7 entre 0,19&ndash;14 &mu;M) e resistente (EC50K1 entre 0,15&ndash;2,4 &mu;M) e citotoxicidade (MCL50 > 3,7 &mu;M) determinadas no primeiro ciclo de SAR. Os dados obtidos sugerem que a enzima Pfeno não é o alvo principal de ação dos derivados hidrazinobenzimidazol, contudo, a atividade antiplasmodial da série indicou razoável distribuição em termos de potência e variabilidade estrutural que permitiram a construção de um modelo HQSAR (q2= 0,64 e r2 = 0,93) adequado para o planejamento e predição da atividade inibitória de oito novos derivados hidrazinobenzimidazol (36&ndash;43). Os novos derivados foram sintetizados e tiveram sua atividade antiplasmodial (EC503D7 entre 0,10&ndash;2,3&mu;M), citotoxicidade (MCL50 > 3 &mu;M) e seletividade (SI > 5) determinadas. Os dados de validação prospectiva do modelo indicaram boa correlação entre a atividade real e predita para os novos derivados. Além disso, neste segundo ciclo de SAR foi descoberto o composto 41 como o mais potente (EC50 = 0,1 &mu;M) e seletivo (SI > 2.000) da série investigada neste trabalho. Nossos resultados indicam que os derivados hidrazinobenzimidazol são moléculas atrativas para a descoberta de compostos líderes para o desenvolvimento de candidatos a fármacos antimaláricos. / Malaria is the tropical disease with the highest overall mortality rate. The emergence of resistance to first-line therapies reinforces the need for the development of new drug candidates. The main goal of this work was the discovery and optimization of these molecules as lead candidates for the development of new antimalarial agents. In this sense, we screened Malaria Box collection and identified 11 molecules from different chemical classes as inhibitor candidates of Plasmodium falciparum enolase enzyme (Pfeno). Then, we determined the inhibitory potency against the target enzyme (IC50 between 11 &ndash; >1.000 &mu;M), in vitro antiplasmodial activity against the erythrocytic form of the parasite (EC503D7 between 5,6&ndash;1.600 nM) and cytotoxicity (MCL50 > 19 &mu;M) of the 11 compounds subset from Malaria Box (1&ndash;11). Combination with artesunate and stage of action assays were conducted to evaluate the potential of association and which erythrocytic stage would be more susceptible to the molecules of this subset. The results obtained indicate that some compounds showed additive (2 and 9) or antagonistic (1, 3&ndash;8, 10 and 11) effect with artesunate and fast (1&ndash;3, 5, 7, 9&ndash;11) or slow (4, 6 and 8) acting on parasite development. In view of these, a set of structural and biological activity criteria was established for the selection of a candidate for structure-activity relationship (SAR) studies. The hydrazinobenzimidazole derivative (4) was selected as hit and 24 derivatives (12&ndash;35) were designed, synthesized and had the enzyme inhibitory activity (IC50 between 44 &ndash; >200 &mu;M), antiplasmodial activity against sensitive strains (EC503D7 between 0,19&ndash;14 &mu;M) and resistant (EC50K1 between 0,15&ndash;2,4 &mu;M) and cytotoxicity (MCL50 > 3,7 &mu;M) determined in the first round of SAR. The collected data suggest that Pfeno is not the main target of the hydrazinobenzimidazole derivatives. However, the antiplasmodial activity of the series indicated a reasonable distribution in terms of potency and structural variability which allowed us the development of a HQSAR model (q2 = 0,64 and r2 = 0,93) suitable for the design and prediction of the inhibitory activity of eight new hydrazinobenzimidazole derivatives (36-43). The new derivatives were synthesized and had the antiplasmodial activity (EC503D7 between 0,10&ndash;2,3&mu;M), cytotoxicity (MCL50 > 3 &mu;M) and selectivity (SI > 5) evaluated. The prospective validation of the HQSAR model indicated good correlation between the actual and predicted activity for the new derivatives. Moreover, in the second round of SAR, compound 41 was discovered as the most potent (EC50 = 0,1 &mu;M) and selective (SI > 2,000) in these series. Our results indicate that the hydrazinobenzimidazole derivatives are attractive molecules for the discovery of new lead compounds for the development of antimalarial drug candidates.

Os receptores do tipo toll (toll-like receptors ou TLRs) são uma família de receptores na primeira linha de defesa do sistema imune inato que são capazes de reconhecer padrões moleculares associados a patógenos (PAMPs), dentre os patógenos mais importantes encontra-se Plasmodium. Variações genéticas nesses receptores estão associadas à resistência ou suscetibilidade a uma variedade de doenças infecciosas. Sabe-se que as freqüências destas mutações também podem variar de acordo com a ancestralidade biogeográfica dos indivíduos. Este trabalho avaliou a relação entre 24 polimorfismos em TLRs, ancestralidade e malária em 195 indivíduos residentes em área de Mata Atlântica do Estado de São Paulo. A ancestralidade genômica individual foi estimada usando marcadores autossômicos de inserção/deleção (INDELs) e os polimorfismos em TLR foram genotipados por PCR-RFLP, sequenciamento e eletroforese capilar. Dados prévios de malária presente ou pregressa obtidos por métodos moleculares e sorológicos foram utilizados para avaliar a relação dos polimorfismos em TLRs e malária nesses indivíduos. Os resultados mostraram que: (i) nove SNPs e um microssatélite foram polimórficos; (ii) a maior parte dos indivíduos analisados apresentou maior proporção de ancestralidade europeia; (iii) as proporções médias de ancestralidade europeia diferiram significantemente nos genótipos de TLR1 (I602S) e TLR6 (P249S); (iv) a probabilidade de ter o alelo variante G em TLR1 aumenta com o aumento da ancestralidade européia assim como aumenta a probabilidade de ter o alelo variante T no TLR6; (v) o polimorfismo em TLR9 -1237T/C foi fortemente relacionado à malária, sendo demonstrado que a presença de pelo menos um alelo C nessa posição aumentou o risco para malária 2,3 vezes; e (vi) TLR9 -1486T/C em associação com o TLR6 foi protetor, pois a associação dos alelos variantes reduziu o risco de malária em 4,4 vezes. O alelo G em TLR1 tem sido relacionado a uma resposta imune diminuída, o que poderia explicar a alta prevalência de casos de malária assintomática em áreas do sudeste brasileiro. O TLR9 tem sido relacionado com a patogênese da malária em estudos em animais e em humanos, sendo possivelmente modulado por polimorfismos na região promotora. Nossos dados também reforçam a necessidade de incluir marcadores de ancestralidade em estudos de associação genética para evitar resultados enviesados. Por fim, este é o primeiro estudo a associar polimorfismos em genes de imunidade inata e malária em amostras de Mata Atlântica. / Toll-like receptors (TLRs) are a family of receptors in the first line of defense of the innate immune system and they are able to recognize molecular patterns associated with pathogens (PAMPs), among the most important pathogens is Plasmodium. Genetic variations in these receptors are associated with resistance or susceptibility to a variety of infectious diseases. It is known that the frequencies of these mutations can also vary according to the biogeographic ancestry of the individuals. This work evaluated the relationship among 24 polymorphisms in TLRs, ancestry and malaria in 195 individuals living in the Atlantic Forest area of the São Paulo State. Individual genomic ancestry was estimated using autosomal insertion/deletion markers (INDELs) and TLR polymorphisms were genotyped by PCR-RFLP, sequencing and capillary electrophoresis. Previous data about present or previous malaria were obtained by molecular and serological methods and were used to evaluate the relationship of polymorphisms in TLRs and malaria in these individuals. The results showed that: (i) nine SNPs and one microsatellite were polymorphic; (ii) the majority of the individuals analyzed presented a greater proportion of European ancestry; (iii) the average proportions of European ancestry differed significantly in the genotypes of TLR1 (I602S) and TLR6 (P249S); (iv) the probability of having the variant G allele in TLR1 increases with increasing European ancestry as well as increases the probability of having the T allele variant in TLR6; (v) the TLR9 -1237T/C polymorphism was strongly related to malaria, and the presence of at least one C allele in this position was shown to increase the risk for malaria 2.3 times; and (vi) TLR9-1486T/C in combination with TLR6 was protective, since the association of variant alleles reduced the risk of malaria by 4.4 times. The G allele in TLR1 has been related to a decreased immune response, which could explain the high prevalence of cases of asymptomatic malaria in areas of southeastern Brazil. TLR9 has been linked to the pathogenesis of malaria in animal and human studies, possibly being modulated by polymorphisms in the promoter region. Our data also reinforce the need to include ancestral markers in genetic association studies to avoid biased results. Finally, this is the first study to associate polymorphisms in genes of innate immunity and malaria in Atlantic Forest samples.

O desenvolvimento de imunidade naturalmente adquirida na malária é lento e depende de fatores como o número de malárias prévias, intervalo entre cada malária, exposição a variantes antigênicas múltiplas e idade do indivíduo. Mecanismos imunes efetores dependentes de anticorpos são importantes no desenvolvimento dessa imunidade. Vários estudos têm mostrado que as subclasses IgG1 e IgG3 anti-P. falciparum, conhecidas por sua ação citofílica, são anticorpos protetores, enquanto anticorpos não citofílicos como IgG4 reconhecendo os mesmos epítopos seriam bloqueadores dos mecanismos protetores. Dados recentes sugerem que sob determinadas condições, IgG2 também pode ter ação citoíflica e participar da proteção na malária. Neste trabalho, nós estudamos pela primeira vez, a evolução das subclasses de IgG contra formas eritrocitárias de P. falciparum de pacientes com malária falciparum não complicada internados em hospital por até 42 dias, sob tratamento com mefloquina. As subclasses de IgG foram avaliadas por ELISA em 48 pacientes (7 amostras de soro de cada um colhidas nos tempos 0h, 48h, 7, 14, 28, 35 e 42 dias), quanto à quantidade (concentração, ug/ml; índices de reatividade, IR; ou em freqüência, %) e quanto à avidez dos anticorpos (índice de avidez, IA). Amostras de soro de 14 pacientes (tempos: 0, 48h, 7, 21 e 28 dias) também foram avaliadas quanto à especificidade e a avidez de reconhecimento das diferentes bandas protéicas do antígeno de P. falciparum por Immunoblotting. As subclasses IgG1, IgG2, IgG3 e IgG4 anti-P. falciparum, na maioria de alta avidez, estavam presentes no início do tratamento, respectivamente, em 100%, 39,5%, 80,6% e 28,4% dos pacientes e com concentrações médias de 20, 2; 3,8; 1,5 e 0,05 ?g/mL. As concentrações máximas das subclasses de IgG foram alcançadas no 7o dia, e os IAs máximos de IgG1 e IgG3 foram alcançados no 7o dia, e os de IgG2 no 14o dia e os de IgG4 no 2o dia. A concentração inicial dos anticorpos IgG3 anti-P. falciparum apresentou correlação negativa com o tempo de clareamento parasitário (TCP) e a relação das somas dos anticorpos IgG1, IgG2 e IgG3 pelos níveis de IgG4 se correlacionaram negativamente com a parasitemia inicial. No Immunoblotting, foram identificadas frações protéicas que podem estar relacionadas com o reconhecimento imune protetor, por serem reconhecidas pelas subclasses IgG1, IgG2 e IgG3 e não reconhecidas ou reconhecidas tardiamente por IgG4: 125, 96, 86, 75, 55 e 47 kDa. A resposta predominante das subclasses IgG1, IgG2 e IgG3 observada nestes pacientes, todos com malária não complicada, pode indicar que esses anticorpos estão cooperando para o controle de formas graves da doença e refletirem um certo grau de desenvolvimento de imunidade adquirida / The development of naturally acquired immunity to malaria is slow and depends of several factors as number of previous malaria, interval between each malaria attack, exposure to parasite multiple antigen variant and ageassociated maturation of the immune system. Antibody-dependent effector immune mechanisms are believed to be important to the protective immunity. A number of studies have showed that anti-P. falciparum IgG1 and IG3, named cytophilic antibodies, are protective, whereas the noncytophilic, IgG4, that recognize the same epitopes may block the protective mechanisms. Recent data have suggested that in certain situations, IgG2 can also act as cytophilic and to cooperate in protection. In this work, we have studied, for the first time, the evolution of the IgG subclasses against P. falciparum blood stages in uncomplicated falciparum malaria patients taken into hospital upon mefloquine treatment and followed up 42 days. These antibodies were determined by ELISA in 48 patients (7 serum samples from each patient collected in different times: 0h, 48h, 7, 14, 28, 35 and 42 days). The results were expressed in concentration (ug/ml), index of reactivity (IR) or frequency (%) and the avidity were expressed as index of avidity (IA). Serum samples 14 patients (time of collection: 0, 48h, 7, 21 and 42 days) were also evaluated by Immunoblotting as their specificity and avidity against different proteins of the P. falciparum blood stages The subclasses Anti-P. falciparum IgG1, IgG2, IgG3 and IgG4, high avidity predominantly, were present since the beginning of the treatment, respectively, in 100%, 39,5%, 80,6% and 28,4% of the patients with the following concentrations: 20, 2; 3,8; 1,5 and 0,05 ?g/mL. The highest concentrations were reached at day 7, and IgG1 and the highest IgG3 IAs were reached at day 7, and the highest IgG2 IAs at day 14 and the highest IgG4 IAs at day 2. The initial concentration of anti-P. falciparum IgG3 showed a negative correlation with the parasitemia clearance time (PCT) and the ratio between the sum of IgG1, IgG2 and IgG3 levels to IgG4 levels was negatively correlated with the initial parasitemia. Six protein fractions were identified by the Immunoblotting that can be related to protective immune recognition, because they were recognized by IgG1, IgG2 and IgG3 antibodies and not or only later recognized by IgG4 antibodies: 125, 96, 86, 75, 55 and 47 kDa. The predominant IgG1, IgG2 and IgG3 responses observed in these uncomplicated malaria patients may suggest that these antibodies are cooperating to the control of severe disease and reflecting a certain development of protective immunity

Casos de malária no Brasil atingiu seu nível mais baixo em 35 anos e o Plasmodium vivax é responsável por 85% dos casos em todo o país. A vigilância epidemilógica da transmissão residual da malária persistente na Amazônia é o próximo grande desafio para os esforços vigentes de eliminação. Esta situação nos levou a avaliar uma estratégia para detecção de portadores do parasito (sintomáticos ou não) em áreas que se aproximam a eliminação da malária. Nossa hipótese é que o monitoramento sistemático de moradores de domicílios com um ou mais casos de malária vivax clínica confirmados pela vigilância passiva de rotina (referido como casos índices) e seus vizinhos tem um melhor custo-benecífio na identificação de novas infecções em relação aos inquéritos em massa da população em geral. Para testar essa hipótese, foram recrutados 41 casos índices (24 indivíduos classificados como autóctones, 11 como possíveis recaídas e, seis como casos importados), 163 moradores dos domicílios índices (indivíduos morando na mesma residência do caso índice), 878 moradores vizinhos (moradores de domicílios a um raio de < 3 km) e 841 controles (moradores da mesma localidade, porém a > 5 km de distância do domicílio índice) entre os meses de fevereiro a julho de 2013. Participantes residiam em comunidades rurais pertencentes a Acrelândia, onde o P. vivax é a única espécie implicada na transmissão da malária humana. Todos os participantes foram convidados a fornecerem amostra de sangue capilar para realização do diagnóstico para malária no momento de identificação do caso índice (dia 0), 30, 60 e 180 dias após a primeira visita. Em geral, 6028 análises por microscopia revelaram um aumento na prevalência de malária nos domicílios índices (6,1%; odds ratio [OR] = 36,3, P < 0,001) e vizinhos (2,6%; OR = 13,6, P < 0,001) comparados aos controles (0,1%). Não houve casos positivos para P. falciparum. Moradores dos domicílios índices e vizinhos foram associados com uma maior probabilidade de infecção pelo P. vivax em comparação com os indivíduos controles, após análise ajustada por potenciais confundidores (modelos de regressão logística para efeitos mistos), além desses participantes albergarem > 90% da biomassa parasitária circulante. Nos quatro seguimentos da RCD a microscopia identificou somente 49,5% das infecções diagnosticadas por qPCR, porém 76,8% do total da carga parasitária circulante nas proximidades do domicílio índice. Embora, moradores dos domicílios controles foram responsáveis por 27,6% das amostras positivas por qPCR, 92,6% desses indivíduos eram portadores assintomáticos da infecção, que provavelmente não seriam alvos da RCD. Tipagem molecular dos parasitos utilizando três marcadores polimórficos - msp1F3, MS16 e pv3.27 revelaram alta diversidade de P. vivax, consistente com a complexidade das vias de transmissão e múltiplas fontes de infecção dentro dos aglomerados, potenciais complicações para os programas de eliminação de malária. / Malaria burden in Brazil has reached its lowest levels in 35 years and Plasmodium vivax now accounts for 85% of cases countrywide. The epidemiological surveillance residual malaria transmission entrenched in the Amazon is the next major challenge for ongoing elimination efforts. This situation prompted us to evaluate a strategy for targeted detection of parasite carriers (either symptomatic or asymptomatic) in areas approaching malaria elimination. We hypothesize that repeated screening of households (HHs) with one or more slide-confirmed clinical vivax malaria cases diagnosed by routine passive surveillance (referred to as index cases) and their neighbors is more cost-effective for finding new malaria infections than population-wide mass blood surveys. To test this hypothesis, we recruited 41 index cases (24 subjects classified with indigenous, 11 possible relapsing and 6 cases imported) 163 index household members (subjects sharing the household with index cases), 878 neighbors (subjects living within a radius of up to 3 km from index cases) and 841 controls (subjects living in the same locality, but > 5 km from the index case) between January and July 2013. Study participants lived in rural communities surrounding Acrelândia town, where P. vivax is the only malaria parasite diagnosed in humans. They were invited to contribute finger-prick blood samples, for laboratory diagnosis of malaria, at the time of index case diagnosis (day 0) and 30, 60, and 180 days later. Overall, 6,028 microscopical analysis revealed an increased prevalence of infection in index households (6.1%; odds ratio [OR] = 36.3, P < 0.001) and neighbors (2.6%; OR = 13.6, P < 0.001) compared to controls (0.1%). There were no positive cases for P. falciparum. Subjects in index and neighbor households were significantly more likely to be parasitemic than control households members, after adjusting for potential confounders (mixed-effects logistic regression models), and together harbored > 90% of the P. vivax biomass in study subjects. Four rounds of microscopy-based RCD would identify only 49.5% of the infections diagnosed by qPCR, but 76.8% of the total parasite biomass circulating in the proximity of index HHs. However, control HHs accounted for 27.6% of qPCR-positive samples, 92.6% of them from asymptomatic carriers who were beyond the reach of RCD. Molecular genotyping analysis of parasites with three polymorphic molecular markers (msp1F3, MS16, and Pv3.27) revealed high P. vivax diversity, consistent with complex transmission networks and multiple sources of infection within clusters, potentially complicating malaria elimination efforts.

A malária é uma doença infecciosa causada pelos parasitas do gênero Plasmodium e transmitida pelo mosquito Anopheles spp. Devido ao surgimento de casos de resistência aos fármacos disponíveis novos alvos e candidatos a fármacos são necessários. Recentemente, a enzima N-miristoiltransferase (NMT) foi confirmada como essencial para o parasita e validada como alvo terapêutico para o desenvolvimento de candidatos a fármacos antimaláricos. O objetivo desse trabalho foi identificar os determinantes moleculares responsáveis pela atividade inibitória de uma série de derivados benzotiofênicos frente à NMT. Nesse sentido, estudos de relação quantitativa estrutura-atividade (QSAR) 2D e 3D foram desenvolvidos para dois conjuntos de dados de derivados benzotiofênicos como inibidores da enzima do parasita (PfNMT) e a homóloga humana (HsNMT). Além disso, estudos de modelagem por homologia da PfNMT foram conduzidos. Os estudos de QSAR 2D foram desenvolvidos pelo método de Holograma QSAR (HQSAR). O modelo estrutural de PfNMT foi aplicado na construção dos modelos QSAR 3D CoMFA (Comparative Molecular Field Analysis) e CoMSIA (Comparative Molecular Similarity Index Analysis). Os estudos de QSAR 3D foram conduzidos com diferentes métodos de cálculo de carga parcial atômica (Gasteiger-Hückel, MMFF94 e AM1-BCC, CHELPG e Mulliken) e de alinhamento molecular (Máxima Subestrutura Comum, alinhamento flexível e baseada no alvo molecular). Os melhores modelos construídos pelos métodos de QSAR 2D e 3D foram robustos, internamente consistentes e com elevada capacidade de predição da atividade de novos compostos contra a PfNMT. Os mapas de contribuição e de contorno geraram informações importantes sobre a relação estrutura-atividade dos compostos. Os resultados permitiram a identificação das bases moleculares responsáveis pela atividade dos inibidores benzotiofênicos e são úteis para o planejamento de novos inibidores mais potentes e seletivos para a enzima do parasita. / Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium and transmitted by Anopheles spp. mosquitos. Due to the emerging resistance to current available drugs, great efforts for new molecular target and drugs are required. Recently, N-myristoyltransferase (NMT) was confirmed as an essential enzyme to malaria parasites and validated as a chemically tractable target for the development of new drug candidates against malaria. This work aimed to shed light on the molecular requirements underlying the inhibitory activity of benzothiophene derivatives against NMT. Therefore, 2D and 3D quantitative structure-activity relationship (QSAR) studies were developed for two datasets of benzothiophene derivatives as P. falciparum NMT (PfNMT) and the human homologue (HsNMT) inhibitors. Also, homology modeling studies for PfNMT were developed. The 2D QSAR studies were developed by the Hologram QSAR (HQSAR) method. The PfNMT structural model was applied in the construction of 3D QSAR models CoMFA (Comparative Molecular Field Analysis) and CoMSIA (Comparative Molecular Similarity Index Analysis). Different molecular alignment (maximum common substructure, flexible alignment and structure based) and atomic partial charge calculation (Gasteiger-Hückel, MMFF94, AM1-BCC, CHELPG and Mulliken) methods were used to build the 3D QSAR models. The best models showed internal consistency and high predictive ability of biological activity against PfNMT. The contribution and contour maps gave important information about compounds structure-activity relationship. The results allowed the identification of the molecular requirements underlying the inhibitory activity and should be useful for the design of novel potent and selective PfNMT inhibitors as antimalarial drug candidates.

A malária é a doença parasitária mais comum no mundo e é causada por protozoários do gênero Plasmodium spp., sendo transmitida por dípteros do gênero Anopheles spp. para hospedeiros vertebrados. Ambos, parasitas e vetores, têm desenvolvido resistência aos tratamentos e medidas profiláticas, respectivamente, indicando a necessidade de novas formas de controle. Chaperonas moleculares e co-chaperonas são interessantes alvos de estudo para desenvolvimento de terapias mais eficazes, uma vez que estas biomoléculas desempenham papel importante no processo de adaptação e sobrevivência do protozoário. As chaperonas da família Hsp90 participam de vários processos fisiológicos, que não somente auxiliar o enovelamento de proteínas clientes. Cada protômero da Hsp90 é composto por três domínios denominados N, M e C, e a proteína se organiza na forma de homodímeros flexíveis. As co-chaperonas são proteínas auxiliares, essenciais para modulação do ciclo funcional da Hsp90. A co-chaperona Aha1 (do inglês Activator of the Hsp90-ATPase activity 1) estabiliza a Hsp90 em um estado conformacional intermediário e estimula a atividade ATPásica da mesma. Neste contexto, é usual a busca por inibidores potenciais diretos e indiretos para Hsp90 e por respostas sobre seu mecanismo de inibição. O objetivo deste trabalho foi a obtenção e caracterização bioquímica e biofísica da proteína Hsp90 recombinante de Plasmodium falciparum (PfHsp90) e construções PfHsp90NM e PfHsp90M, além do mapeamento da interação com a co-chaperona Aha4 de P. falciparum (PfAha4). Os experimentos de caracterização estrutural revelam que o domínio N é menos estável termicamente do que o domínio M e também é o mais rico em estrutura secundária do tipo hélice &alpha;. A PfHsp90 comporta-se majoritariamente como homodímero alongado e flexível em solução, sendo o domínio C essencial para a dimerização, todavia as construções PfHsp90NM e PfHsp90M comportam-se como monômeros. Ensaios de fluorescência revelaram que as construções exibem diferenças na estabilidade química e que possuem estrutura terciária local com seus triptofanos parcialmente expostos ao solvente. A atividade ATPásica da PfHsp90 foi estimulada por PfAha4, e a interação entre elas foi resolvida com estequiometria de duas moléculas de PfAha4 por dímero de PfHsp90. Tal interação foi entalpicamente dirigida, ocorrendo liberação de moléculas de água, com interação mediada principalmente por contatos hidrofóbicos. O mapeamento das regiões de contato sugere que o cerne da interação ocorra entre a PfAha4 e o domínio M da PfHsp90. As diferenças exibidas pela PfHsp90 com relação as propriedades de proteínas ortólogas podem ter relação com os resíduos de aminoácidos que conectam os domínios N e M em sua estrutura, devido a sua flexibilidade, tamanho e composição. / Malaria is the most common parasitic disease in the world and is caused by protozoa of the genus Plasmodium spp., and transmitted by dipterans of the genus Anopheles spp. for vertebrate hosts. Both parasites and vectors have developed resistance to treatments and prophylactic measures, respectively, indicating the need for new forms of control. Molecular chaperones and co-chaperones are interesting targets for the development of more effective therapies, since these biomolecules play an important role in the process of adaptation and survival of the protozoan. The chaperones of the Hsp90 family participate in several physiological processes, which not only aid in the folding of client proteins. Each Hsp90 protomer have three domains called N, M and C, and the protein is organized as flexible homodimers. Co-chaperones are assistant proteins, they are essential for modulating the functional cycle of Hsp90. The Aha1 (Activator of the Hsp90-ATPase activity 1) co-chaperone stabilizes Hsp90 in an intermediate conformational state and stimulates the ATPase activity thereof. In this context, it is usual the search for direct and indirect potential inhibitors for Hsp90 and for responses about its inhibition mechanism. The objective of this work was the biochemical and biophysical characterization of the Hsp90 recombinant protein of Plasmodium falciparum (PfHsp90) and PfHsp90NM and PfHsp90M constructions, as well as to map interactions with the Aha4 co-chaperone of P falciparum (PfAha4). Structural characterization experiments show that the N domain is less thermally stable than the M domain and is also the richest in &alpha;-helix secondary structure. PfHsp90 behaves mostly as elongated and flexible homodimer in solution, domain C is essential for dimerization, on the other hand the constructs PfHsp90NM and PfHsp90M behave as monomers. Fluorescence assays revealed that the constructs exhibit differences in chemical stability and that have local tertiary structure with their tryptophans partially exposed to the solvent. The ATPase activity of PfHsp90 was stimulated by PfAha4, and the interaction between them was resolved with stoichiometry of two molecules of PfAha4 by PfHsp90 dimer. Such interaction was enthalpically driven, releasing of water molecules, with interaction mediated mainly by hydrophobic contacts. The mapping of contact regions suggests that the core of the interaction occurs between PfAha4 and the M domain of PfHsp90. The differences exhibited by PfHsp90 concerning the properties of orthologous proteins, may be related to the amino acid residues that connect the N and M domains in its structure, due to its flexibility, size and composition.

O sistema imune exerce uma contribuição fundamental na proteção contra a infecção pelo plasmódio, o agente causador da malária. Entre os mecanismos envolvidos no controle dos parasitas do sangue destacam-se aqueles mediados por anticorpos. Entretanto, outros mecanismos do sistema imune também parecem exercer papéis fundamentais, e ao mesmo tempo foram pouco estudados. O presente trabalho teve como objetivo descrever alguns dos mecanismos efetores importantes na resposta imune à malária experimental. O projeto subdivide-se em duas partes: a) verificar a relação entre o priming por IFN-g e a imunidade ao parasita durante a fase crônica da infecção sanguínea pelo Plasmodium; e b) descrever separadamente o papel de células dendríticas e macrófagos do baço na fagocitose de eritrócitos infectados durante a infecção em modelo experimental, e o subsequente papel destas células na resposta imune durante esse período. Os resultados obtidos neste projeto deverão contribuir para a compreensão dos mecanismos envolvidos na imunidade contra a malária. / The immune system is considered crucial for the protection against Plasmodium infection. Among the mechanisms involved in the control of blood-stage parasites, the ones mediated by antibodies are widely recognized. However, other mechanisms of immune system can also exert fundamental roles, being less studied. This work had the objective of describing some of the effector mechanisms important in the immune response to blood-stage experimental malaria. This work was subdivided in two main branches: a) to verify the relation between the IFN-g-induced priming and the protective immunity to the parasite during the chronic phase of blood-stage infection; and b) to describe separately the role of dendritic cells and splenic macrophages as phagocytic cells during acute Plasmodium chabaudi infection, and the subsequent role of these cells in the immune responses during this period. The results obtained in this project may contribute for the comprehension of the mechanisms involved in the immunity against malaria.