Atualmente, milhões de pessoas, por todo o globo, estão sob risco de serem infectados por um protozoário transmitido vetorialmente por pequenos insetos flebotomíneos. Este parasita é a Leishmania spp., causadora de uma patologia de amplo espectro, que varia desde a moléstia cutânea (tegumentar) até a visceral (kala-azar). A leishmaniose cutânea é a manifestação clínica de maior ocorrência (mais de 90% dos casos). A radiação ionizante, gerada em fonte de 60Co, tem sido utilizada com sucesso para promover alterações físico-químicas em diferentes protozoários, incluindo a Leishmania spp. Em trabalhos anteriores determinou-se que formas promastigotas de Leishmania amazonensis, irradiadas com diferentes doses de radiação gama, perdem sua viabilidade mantendo, porém, sua imunogenicidade. No presente trabalho, estudouse a utilização da radiação ionizante como ferramenta na seleção de formas metacíclicas do parasita em cultura axênica para a possível produção de imunógenos irradiados mais eficientes. Os resultados demonstram que culturas irradiadas com 400 Gy de radiação gama, possuem uma concentração de aproximadamente 75% de parasitas metacíclicos, capazes de produzir, in vitro, uma infecção que mimetiza a ocorrida naturalmente. Estes parasitas irradiados têm sua estrutura celular interna modificada mantendo, entretanto, seu arcabouço externo intacto. Camundongos de uma linhagem suscetível imunizados com leishmanias irradiadas com diferentes doses tiveram sua produção de imunoglobulinas aumentada, e mantiveram os títulos elevados após o desafio com parasitas não irradiados. Em outras linhagens pesquisadas este padrão se manteve, porém em títulos menores, sendo que camundongos imunodeficientes não responderam à imunização nem ao desafio. / Actually, millions of people around the globe are under the risk of infection by a protozoan transmitted by a bit of a sand fly. This parasite is a Leishmania spp. This causes a wide spectrum disease, since a coetaneous disease to a visceral one. The coetaneous form is the major clinical manifestation (above 90%). The ionizing radiation, produced in a 60Co font, had being successes used to promote physical-chemical transformations on different protozoans, including Leishmania spp. In previous work was determined that promastigotes forms of Leishmania amazonensis, irradiated with different doses of radiation, lost their viability maintaining, however, their immunogenicity. In this work, was studied the use of ionizing radiation as a tool for selection of metaciclic forms of the parasite in axenic culture, for a possible efficient irradiated immunogen production. Our results shown that cultures irradiated with 400 Gy of gamma irradiation, has 75% of metaciclic form, which are capable to produce, in vitro, an infection that is similar the natural occurrence. These irradiated parasites have their internal cellular structure modified, maintaining their external structure intact. Susceptible strain of mice immunized with leishmania irradiated with different doses had high immunoglobulin production, and maintained this production after the challenge with naive parasites. In other strains this default was similar, however in lower titles. Immunodeficient mice didnt produce immunoglobulin nor on the immunization or on the challenge.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-18T15:08:24Z No. of bitstreams: 1 sandrahelenacerratotibiriça.pdf: 1652171 bytes, checksum: 70c53fdf10ce2e1aabc9b49f47b6fd54 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-18T15:43:13Z (GMT) No. of bitstreams: 1 sandrahelenacerratotibiriça.pdf: 1652171 bytes, checksum: 70c53fdf10ce2e1aabc9b49f47b6fd54 (MD5) / Made available in DSpace on 2017-05-18T15:43:13Z (GMT). No. of bitstreams: 1 sandrahelenacerratotibiriça.pdf: 1652171 bytes, checksum: 70c53fdf10ce2e1aabc9b49f47b6fd54 (MD5) Previous issue date: 2008-09-26 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A epidemiologia da esquistossomose apresentou transformações significativas nas últimas décadas no Brasil. No Estado de Minas Gerais, a redução da morbi-mortalidade e o desenvolvimento tecnológico dos meios diagnósticos e terapêuticos foram evidentes, mas não suficientes para impedir a expansão geográfica da endemia. Clinicamente, a esquistossomose pode se manifestar com as formas agudas e as crônicas hepatoesplênicas, estas mais freqüentes nas áreas de altas endemicidades. Outra forma incapacitante e subnotificada é a mielorradiculopatia esquistossomótica, que ocupa papel de destaque pela gravidade potencial e possibilidade de ocorrer também em áreas de baixas endemicidades, já que não depende da intensidade da infecção. Considerando a população residente, foi realizado um levantamento seccional objetivando determinar a prevalência, a intensidade da esquistossomose e a distribuição dos hospedeiros intermediários em três municípios da microrregião de Juiz de Fora. Utilizou-se estudo amostral baseado no cadastro das famílias no SIAB, com investigação de 850 famílias por meio de exames coproparasitológicos pelo método Kato-Katz e aplicação de questionário estruturado. Este estudo validou a utilização do cadastro das famílias no SIAB como base populacional para cálculo amostral na realização de pesquisas de campo. Foi pioneiro na verificação da prevalência média de 2,4% da esquistossomose mansoni nos municípios de Piau (2%), Goianá (2,1%) e Coronel Pacheco (3,1%). As características da transmissibilidade na região foram avaliadas e descritas. Os infectados predominaram na população adulta jovem, somente 10% dos escolares foram positivos e não constituíram indicadores dos domicílios com adultos doentes. Os resultados obtidos permitiram identificar o padrão de agregação, revelando os indivíduos com as maiores cargas parasitárias. As três espécies de moluscos de importância epidemiológica foram identificadas nas áreas estudadas: B. glabrata, B. straminea, e B. tenagophila. Constatou-se que, nas situações de baixa endemicidade, em municípios com população inferior a 5.000 habitantes e cobertos pela estratégia de saúde da família, a pesquisa da prevalência da esquistossomose v com base no estudo amostral a partir do cadastro das famílias no SIAB é apropriada para identificar os grupos de maior transmissão. Os indivíduos infectados foram espacialmente referenciados com os moluscos potenciais hospedeiros e indicaram os focos de maior transmissibilidade da esquistossomose nos municípios estudados. / The epidemiology of schistosomiasis has undergone significant transformation in recent decades in Brazil. In the state of Minas Gerais there has been a decline in morbi-mortality, along with advances in diagnosis and treatment, but these have not been sufficient to prevent the geographic spread of the endemic. Clinically, schistosomiasis can appear in acute and chronic hepatosplenic forms, the latter more frequent in areas of high endemicity. Another incapacitating and underreported form, schistosomotic myeloradiculopathy, deserves attention because of its potential severity and the fact it can also occur in areas with low endemicity, since it does not depend on the infection’s intensity. A cross-sectional study was carried out to determine the prevalence and intensity of schistosomiasis in the resident population and the distribution of intermediate hosts in three municipalities in the Juiz de Fora micro-region. The sample was drawn from families listed in the Basic Health Service Information System (SIAB), from which 850 families were investigated through coproparasitological tests by the Kato-Katz technique and the application of a structured questionnaire. This study was groundbreaking in verifying an average schistosomiasis mansoni prevalence of 2.4% in the municipalities of Piau (2%), Goianá (2.1%) and Coronel Pacheco (3.1%). The transmissibility characteristics in the region were evaluated and described. The infected individuals were predominantly young adults. Only 10% of the school-age subjects were positive and there were no indicators of households with sick adults. The results permitted identifying the pattern of aggregation, revealing the individuals with greater parasite loads. The three species of snail carriers were identified in the areas studied: B. glabrata, B. straminea, and B. tenagophila. Therefore, in situations of low endemicity, in regions with population under 5,000 people and covered by family preventive health programs, the study of the prevalence of schistosomiasis based on sample data from households listed in the SIAB is appropriate to identify the groups of greater transmission. The infected individuals were spatially referenced together with the host snails, indicating the probable foci of greatest transmission in the municipalities studied.

Orientadores: Manzélio Cavazzana Júnior, Regina Maura Bueno Franco / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T19:07:23Z (GMT). No. of bitstreams: 1 Malta_RobertoCarlosGrassi_D.pdf: 3672169 bytes, checksum: 25ac1445db6b62e9892dee0a7eea948b (MD5) Previous issue date: 2011 / Resumo: As infecções parasitárias são um dos principais problemas de saúde pública, apresentando-se de forma endêmica em diversas áreas do Brasil. Podem apresentar estreita relação com fatores sócio-demográficos e ambientais, tais como: precárias condições socioeconômicas, consumo de água contaminada, deficiente estado nutricional dos indivíduos e outros, sendo frequentemente a população infantil a mais atingida. Com o objetivo de investigar a prevalência de parasitas intestinais em populações indígenas e populações carentes e os fatores-chave envolvidos na epidemiologia de enteroparasitoses, foi realizado levantamento enteroparasitológico em moradores de 02 reservas indígenas Reserva Bororó/MS e Reserva Xingu/MT (tribos Kayabí e Juruna) -, e também em moradores de 02 cidades - Pontes e Lacerda/MT e Ibateguara/AL. A coleta de dados foi realizada de 2002 a 2009. Foram analisadas 2754 amostras de fezes pelos métodos de Faust, Hoffman, Kato-Katz, Rugai, Direto e Ziehl-Neelsen modificado. Foram obtidos dados pessoais e parâmetros socioeconômicos. Observou-se a presença de 73% de enteroparasitas na reserva indígena de Dourados, 62,77% na reserva indígena do Xingu, 52,61% no município de Pontes e Lacerda/MS e 67,42% no município de Ibateguara. As espécies de maior prevalência no sexo masculino foram Entamoeba coli (22,5%), Giardia duodenalis (11,6%), Entamoeba histolytica (13,9%) e Ascaris lumbricoides (13,6%). No sexo feminino foram Entamoeba coli (24,1%), Giardia duodenalis (8,8%), Entamoeba histolytica/díspar (13,8%) e Ascaris lumbricoides (13,3%). A prevalência de protozoários (42,6%) foi maior que de helmintos (31,1%). Para a maioria dos grupos analisados não houve diferença entre o quadro clínico de diarreia e o tipo e número de enteroparasita. O poliparasitismo foi detectado em 12,8% das amostras e o monoparasitismo em 46,5%. Os grupos etários de menor idade apresentaram predomínio de infecções por protozoários / Abstract: The parasitic infections are the major public health problems, presenting an endemic form in several areas of Brazil. They may present narrow relationship with social-demographical and environmental factors, such as: social-economical precarious conditions, consumption contaminated water, deficient nutritional condition of individuals and others, being frequently the infant population the most affected. In order to investigate the prevalence of intestinal parasites in indigenous and deprived populations and the key factors involved in the epidemiology of intestinal parasites, it was realized intestinal parasitological survey in residents of two Indian reservations: Bororó Reservation/MS and Xingu Reservation/MT (Kayabí and Juruna tribes); and also in residents of two cities: Pontes e Lacerda/MT and Ibateguara/Al. The data collection was conducted from 2002 to 2009. 2,754 faeces samples were analyzed by the methods of Faust, Hoffman, Kato-Katz, Rugai, Direct and modified Ziehl-Neelsen. The study obtained personal data and social-economical parameters. It was observed the presence of 73% of intestinal parasites in the Bororó Reservation, 62.77% in the Xingu Reservation, 52.61% in the cities of Pontes e Lacerda/MS and 67.42% in the Ibateguara city. The species of most prevalence in male individuals were Entamoeba coli (22.5%), Giardia duodenalis (11.6%), Entamoeba histolytica (13.9%) and Ascaris lumbricoides (13.6%). In female individuals were Entamoeba coli (24.1%), Giardia duodenalis (8.8%), Entamoeba histolytica/ E. díspar (13.8%) and Ascaris lumbricoides (13.3%). The prevalence of protozoan (42.6%) was higher than helminths (31.1%). For most analyzed groups there was no difference between the diarrhea clinical situation and the intestinal parasite type and number. The multiple intestinal parasite was detected in 12.8% of samples and monoparasitism in 46.5%. The minor age individuals presented the preponderance of protozoan infections / Doutorado / Parasitologia / Doutor em Parasitologia

Resistance to artemisinin combination therapies (ACTs) has emerged in southeast Asia threatening the most widely used treatment against antimalarial-resistant Plasmodium falciparum worldwide. Artemisinin resistance has been associated with a reduced rate of parasite clearance following treatment with an ACT and is attributed to increased survival of ring-stage parasites. Single nucleotide polymorphisms (SNPs) in kelch gene (K13) has been associated with delayed in vivo clearance half-life of artemisinin-resistant P. falciparum and is the only known molecular marker of resistance. The absence of reliable in vitro phenotypes for artemisinin resistance has limited our understanding of the resistance mechanism(s) and fitness costs, therefore we have culture adapted and cloned patient isolates from Thailand and Cambodia that had clinical resistant phenotypes. Stable reduced susceptibility to artemisinin derivatives and mefloquine was observed using a modified [3H]hypoxanthine drug susceptibility assay. In addition we devised an in vitro phenotype assay of artemisinin resistance, known as the delayed clearance assay (DCA), that was positively correlated with the in vivo delayed clearance and presence of K13 SNPs of artemisinin resistance. Remarkably we discovered for the first time altered patterns of intraerythrocytic development in artemisinin-resistant P. falciparum. In the absence of drug pressure most artemisinin-resistant clones have a prolonged ring phenotype with temporally compressed trophozoite stage, yet with the normal overall asexual life cycle period. Parasites remain in ring-stage up to 14 hours longer than wild-type, whereas time spent in trophozoite-stage is dramatically reduced. One parasite, PL08-09 (5C), exhibited an accelerated 36-hour life cycle in the absence of drug pressure and progressed through asexual development in equal time spent at each intraerythrocytic stage. These data support our hypothesis that parasite resistance to artemisinin results from reduced exposure to drug at the most susceptible stage of development (trophozoite). Interestingly, the most prevalent K13 mutation C580Y is associated with both cell cycle phenotypes. Another cell cycle phenotype, ring-stage dormancy, has been associated with artemisinin resistance in vitro reducing the dormant period of artemisinin-resistant parasites following dihydroartemisinin exposure allowing resistant parasite cultures recrudesce before wild-type. However, sensitive parasites have the ability to enter ring-stage dormancy causing recrudescence in vitro. It is possible that multiple cell cycle phenotypes enhance the survival and fitness of the resistant parasite population as a whole in the face of antimalarial exposure. We have demonstrated that there is a fitness cost associated with artemisinin resistance and remains an important component in the spread of genetic mutations associated with artemisinin resistance. Resistant parasites outcompeted sensitive in vitro only when exposed to dihydroartemisinin. Two mutations associated with artemisinin resistance, including a mutation in K13, were lost in artemisinin resistant parasite by the end of the study. Conversely, parasite cultures maintained artemisinin resistance phenotypes in vitro only if exposed to artemisinin drug pressure every 21-42 days. The mechanism of artemisinin resistance remains elusive and how the parasites alter their intraerythrocytic development is unknown. Therefore. we transfected green fluorescent protein (GFP) or luciferase into artemisinin-resistant P. falciparum clones from Thailand and Cambodia to aid the study the cell cycle phenotypes associated with artemisinin resistance. Artemisinin resistance phenotypes were maintained in stably transfected clones. Increased growth of artemisinin-resistant clones was observed following exposure to ACT partner drug. Low concentrations of antimalarials synchronized the luciferase expression of artemisinin-resistant parasites having different cell cycle phenotypes in the absence of drug pressure. Ring-stage dormancy was observed with many antimalarial drugs and contributes to recrudescence observed by antimalarials other than artemisinin. Our results show evidence that current ACT treatments are selecting multidrug resistant parasites in the field that are better able to tolerate all antimalarials through regulatory cell cycle mechanisms. These cell cycle phenotypes associated with artemisinin resistance contribute to reducing the fitness cost associated with genetic mutations causing artemisinin resistance. This leads to the survival of the most fit population of parasites that survive combination drug treatments, thus demonstrating the importance of discovering novel drugs to target ACT-resistant P. falciparum.

Parasitic diseases remain a health burden affecting more than 500 million people worldwide with malaria having the highest mortality rate. The parasites can be transferred to the human bodies either through the mouth by ingestion of contaminated food and water or through the skin by bug bites or direct contact to environments harbouring them. Epidemiological control seems to be impossible since there is failure to control the insect vectors as well as practice of hygiene. Therefore, this has led to the development of a number of vaccines, chemotherapy and disease control programs. However, parasites have increasingly developed resistance to traditionally used anti-parasitic drugs and due to that fact there is need for alternative medication for parasitic diseases. Heat shock protein 90 (Hsp90) facilitates the folding of proteins in all living cells and their role is more important to parasites because of their environmental changes, from vector to host. Hsp90s play a major role; therefore this justifies the need for a deeper analysis of the parasitic Hsp90s. Recent studies have revealed that, the Plasmodium sp. Hsp90 has an extended linker region which increases the protein’s affinity for ATP and its inhibitors. Therefore we hypothesize that there are also significant features in other parasitic Hsp90s which would lead to Hsp90 being defined as potential drug targets. In the present study an attempt was made to gain more insight into the differences in primary structure of human and parasitic Hsp90s. The sequences were retrieved from the NCBI database and analysis was done in three groups basing on the localization of the Hsp90. The physicochemical properties were calculated and in every group, the protozoan Hsp90s showed significant differences when compared to the human orthologs. Multiple sequence alignments (MSA) showed that endoplasmic reticulum Hsp90s have an extended region in the middle domain indicating their ability to bind to a unique subset of client proteins. Sequence identities between the human and parasites showed that the protozoan Hsp90s are less related to the human Hsp90s as compared to the other parasites. Likewise, motif analysis showed the trypanosomatids and apicomplexan groups have their own unique set of motifs and they were grouped together in the phylogenetic analysis. Phylogenetic analysis also showed that, the protozoan Hsp90s forms their own clades in each group while the helminths did not form in endoplasmic reticulum group. In this study, we concluded that, Hsp90 can be a potential drug target for the protozoan species and more specifically those from the apicomplexan and trypanosomatids groups.

Orientador: Ana Maria Aparecida Guaraldo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T08:35:35Z (GMT). No. of bitstreams: 1 Ferreira_GlaucoRogerio_D.pdf: 2964649 bytes, checksum: 7665afd2ae49a0524acb8e13d66e8985 (MD5) Previous issue date: 2013 / Resumo: A Organização das Nações Unidas (ONU) no ano 2000 definiu oito Objetivos do Milênio; dentre eles pode-se destacar o sexto que é Combater o HIV/AIDS, a malária e outras doenças. As doenças parasitárias constituem ainda um sério problema de saúde pública. A erradicação ou controle desses parasitas requer melhorias das condições sócio-econômicas, do saneamento básico e educação. O presente trabalho teve como objetivo avaliar o Ensino a Distância como ferramenta de ensino para agentes de saúde, sobre as temáticas Doenças parasitárias; Criar conteúdo e disseminar conhecimento para capacitar agentes municipais de saúde em medidas profiláticas, transmissão e prevenção de parasitoses intestinais, através do preparo e aplicação de curso a distância (EAD); Analisar a Plataforma TelEduc para difundir essa capacitação a outros agentes. O curso foi montado e realizado em Plataforma TeldEduc, hospedada no Instituto IPES (Instituto de Projetos Especiais), com duração de 180 horas, sendo 148 horas por EAD e 32 presenciais. A maioria das aulas presenciais foi realizada na Faculdade Municipal "Professor Franco Montoro", no município de Mogi Guaçu-SP. O público alvo foi constituído pelos agentes de saúde e profissionais da saúde; 158 alunos se inscreveram no curso e a taxa de evasão foi de 24,8%. As aulas presenciais eram constituídas por aulas práticas, visitas técnicas ou revisão de conteúdo. Foi aplicado um questionário no início e outro no término para avaliar os conhecimentos pré existentes sobre a temática e os adquiridos pelo curso. Foi empregado o teste Qui-Quadrado para avaliar o nível de significância entre as respostas dos questionários. Concluiu-se que o EAD pode e deve ser utilizado, como uma alternativa de educação em doenças parasitárias, como mais uma ferramenta útil para acesso e ampliação do conhecimento / Abstract: The Organization of the United Nations (ONU) in 2000 set eight Millennium Development Goals; among them we can highlight which is the sixth: Combat HIV / AIDS, malaria and other diseases. Parasitic diseases are still a serious public health problem. The eradication or control these parasites requires improvements in socioeconomic conditions, sanitation and education. This study aimed to evaluate the Distance Learning as a teaching tool for health workers, concerning parasitic diseases. Create content and disseminate knowledge to empower local health agents in prophylactic measures, transmission and prevention of intestinal parasites, through the preparation and application of distance learning course (DLC); Analyze Platform TelEduc to disseminate this training to other agents. The course was set up and held in TeldEduc Platform, hosted at the Institute IPES, lasting 180 hours, with 148 hours of classroom and 32 DLC. Most regular classes were held in the Faculty Franco Montoro. The course was facing health workers and health professionals, 158 people enrolled in the course and 115 completed, which generated a dropout rate of 24,8%. The classroom consisted of practical sessions, technical visits or content review. A questionnaire was applied at the beginning and another at the end to assess the pre-existing knowledge on the subject and acquired by the course. It was employed a chi-square test to assess the level of significance between the answers of questionnaire. It was concluded that the DLC can and should be used as an alternative education on parasitic diseases and means a very useful tool to improve the access and knowledge / Doutorado / Relações Antrópicas, Meio Ambiente e Parasitologia / Doutor em Biologia Animal

Este trabalho teve como objetivo caracterizar e padronizar métodos para a tipagem molecular de marcadores genéticos, para futuro uso em estudos de genética populacional de Plasmodium vivax na Amazônia brasileira. As amostras sanguíneas utilizadas neste estudo foram colhidas no Brasil, Camboja, Sri Lanka e Estados Unidos. Foram selecionados polimorfismos de única base (SNPs) distribuídos ao longo de cromossomos distintos de P. vivax; para estes polimorfismos, sete ensaios de tipagem de SNPs foram padronizados com sucesso. Com a tipagem molecular, foi possível definir haplótipos que caracterizam cada amostra, assim como identificar infecções geneticamente mistas (co-ocorrência de clones distintos na mesma amostra). No entanto, com este conjunto de marcadores não foi possível agrupar amostras de acordo com sua localização geográfica, por estes marcadores não serem suficientemente informativos do ponto de vista genético. Os sete SNPs avaliados, quando comparados a 13 marcadores de DNA microssatélite, revelaram menor proporção de infecções geneticamente mistas e menor diversidade genética. / This study aimed to characterize and standardize methods for molecular typing of genetic markers to be used in the future in studies of population genetics of Plasmodium vivax population in the Brazilian Amazon. Blood samples used in this study were collected in Brazil, Cambodia, Sri Lanka and United States. Single nucleotide polymorphisms (SNPs), distributed over different P. vivax chromosomes were chosen and seven typing assays were sucessfully standardized. Molecular typing of polymorphisms allowed to define haplotypes that characterize each sample, as well as to identify genetically mixed infections (co-occurrence of different clones in the same sample). However, with this markers set, it was not possible to group samples according to their geographical location, because probably they are not sufficiently genetically informative. Compared to 13 microsatellite markers, these seven SNPs revealed a lower proportion of mixed-clone infections and lower genetic diversity.

Casos de malária importada, contraídos em região endêmica, mas diagnosticados em um país não-endêmico, são um evento raro mas com desfecho potencialmente fatal. Nosso objetivo foi investigar se a análise de genomas mitocondriais permite inferir a origem geográfica de casos importados de malária vivax diagnosticados nos EUA, comparando os resultados com aqueles obtidos por análise de DNA microssatélite. Foi sequenciado o genoma mitocondrial completo de 63 amostras de P. vivax provenientes de infecções importadas dos EUA, além de 7 amostras do Brasil e 6 do Panamá. A rede de haplótipos com DNA mitocondrial foi construída com 412 sequências e foi possível classificar com precisão a origem geográfica presumida dos isolados da América do Sul, Coréia, Sudeste Asiático e Melanésia, porém os isolados do Sul da Ásia, América Central e África não puderam ser classificados geograficamente. A análise bayesiana realizada com a tipagem de marcadores de microssatélites não apresentou sucesso quanto à classificação geográfica dos isolados de P. vivax. / Cases of imported malaria, infection acquired in an endemic region, but diagnosed in a non-endemic country, are rare but can lead to a fatal outcome. Our objectives were investigate whether the analysis of mitochondrial genomes allows inferring the geographic origin of isolates of P. vivax derived from cases of imported malaria diagnosed in the USA, and compare the performance of mitochondrial genome and DNA microsatellite analysis. We sequenced full mitochondrial genomes from 63 P. vivax isolates collected at the USA from imported infections, and 7 samples from Brazil and 6 Panama. A network of mitochondrial DNA haplotypes was built with 412 genomic sequences and were able to classify accurately isolates from South America, Korea, Southeast Asia and Melanesia according to their presumed geographic origin, but failed to do so with samples from South Asia, Central America and Africa. The Bayesian analysis performed by typing microsatellite markers showed no success on the classification of geographical isolates of P. vivax.

A proteína de ligação em Duffy (PvDBP) dos merozoítos de P. vivax se liga a glicoproteínas de membrana, chamadas de grupo sanguíneo Duffy, conhecidas como DARC. Indivíduos DARC negativos são normalmente resistentes a infecção estágio sanguínea por P. vivax; portanto, PvDBP é um forte antígeno canditato a vacina. Aqui, investigamos respostas de anticorpos contra três proteínas derivadas de PvDBP e MSP119, em 343 indivíduos de uma região Amazônica brasileira. Anticorpos contra Sal III-His, OII-His, Sal III-IV-GST e MSP119-GST foram encontrados em 43,7%, 39,0%, 14,3% e 38,8% dos indivíduos. Os indivíduos FY*BESFY*BES foram os que menos apresentaram anticorpos contra PvDBP, porém encontramos proporções similares de respondedores entre indivíduos com outros genótipos. A análise de sequências revelou muitas variantes da região II de PvDBP em 41 isolados. A mais comum (encontrada em 28,8% dos isolados), difere de Sal I e PNG- O em seis codóns de aminoácidos. Nenhuma variante tipo Sal I, cujo protótipo de vacina está em teste clínico, foi encontrada nos parasitas locais. / The Duffy binding protein (PvDBP) of the P. vivax merozoites, which binds to a erythrocyte membrane glycoprotein known as Duffy blood group antigen for chemokines (DARC). DARC-negative individuals are usually resistant to blood-stage infection with P. vivax; therefore, PvDBP is a major vaccine candidate antigen. Here, we investigated antibody responses to three proteins derived from PvDBP and MSP119, in 343 subjects in the Amazon of Brazil. Antibodies to Sal III-His, OII-His, Sal III-IV-GST and MSP119-GST were found in 43,7 %, 39,0%, 14,3% and 38,8% of the subjects. FY*BESFY*BES individuals were less likely to have antibodies to PvDBP, but we found similar proportions of seropositive subjects with other genotypes. Sequence analysis revealed several region II PvDBP variants in 41 local P. vivax isolates. The most common of them (found in 28,8% isolates) differs from Sal I and PNG-O alleles in six amino acid codons. No Sal I-type variant, from which a PvDBP-based vaccine prototype currently under clinical testing has been derived, was found in local parasites.

The rapid development of DNA sequencing technologies has opened up new avenues of research, including the investigation of population structure within infectious diseases (both within patient and between populations). In order to take advantage of these advances in technologies and the generation of new types of data, novel bioinformatics tools are needed that won’t succumb to artifacts introduced by the data generation, and thus provide accurate and precise results. To achieve this goal I have create several tools. First, SeekDeep, a pipeline for analyzing targeted amplicon sequencing datasets from various technologies, is able to achieve 1-base resolution even at low frequencies and read depths allowing for accurate comparison between samples and the detection of important SNPs. Next, PathWeaver, a local haplotype assembler designed for complex infections and highly variable genomic regions with poor reference mapping. PathWeaver is able to create highly accurate haplotypes without generating chimeric assemblies. PathWeaver was used on the key protein in pregnancy-associated malaria Plasmodium falciparum VAR2CSA which revealed population sub-structuring within the key binding domain of the protein observed to be present globally along with confirming copy number variation. Finally, the program Carmen is able to utilize PathWeaver to augment the results from targeted amplicon approaches by reporting where and when local haplotypes have been found previously. These rigorously tested tools allow the analysis of local haplotype data from various technologies and approaches to provide accurate, precise and easily accessible results.