FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Dentre as arboviroses, a dengue apresenta-se como a mais importante, constituindo um grave problema de saÃde pÃblica a nÃvel mundial. As regiÃes tropicais e subtropicais sÃo as mais afetadas, ocorrendo aproximadamente 100 milhÃes de infecÃÃes por ano. Os principais sintomas que caracterizam a infecÃÃo sÃo febre, cefalÃia, dor retro-orbitÃria, mialgia, artralgia, prostraÃÃo e exantema. No entanto, outras infecÃÃes apresentam sintomas bastante semelhantes com a dengue, como a leptospirose. Ambas, dengue e leptospirose, podem causar surtos durante as estaÃÃes chuvosas, visto que à o melhor perÃodo para a disseminaÃÃo do mosquito Aedes aegypti e a contaminaÃÃo de Ãreas alagadas pela bactÃria Leptospira sp.. Dessa forma, este estudo teve como objetivo empregar diferentes mÃtodos para o diagnÃstico laboratorial de dengue e leptospirose em pacientes com sÃndrome febril. Oitenta e seis amostras de 86 pacientes com suspeita de dengue foram analisadas por meio do teste imunocromatogrÃfico NS1, ELISA-NS1, teste imunocromatogrÃfico de captura de IgM e IgG, ELISA-IgM e RT-PCR. Dentre os 86 pacientes avaliados, 48 (55,8%) foram positivos para pelo menos um dos testes diagnÃsticos realizados. ApÃs testados para dengue, 68 pacientes foram avaliados pelo teste imunocromatogrÃfico de captura de IgM e pelo teste de ELISA-IgM, ambos para identificaÃÃo de anticorpos anti-Leptospira. Cinco (7,3%) foram positivos para esta infecÃÃo. à de grande importÃncia pesquisar outras causas infeciosas, como a leptospirose, que tambÃm à uma doenÃa febril, em pacientes com suspeita clÃnica de dengue, pois o controle das doenÃas mais prevalentes sà se torna possÃvel quando conhecemos os agentes infecciosos responsÃveis por elas. Portanto, faz-se necessÃrio o uso de diagnÃsticos especÃficos para cada infecÃÃo, permitindo com que os programas de controle epidemiolÃgicos sejam realizados. / Among the arboviruses, dengue is the most important, constituting a serious public health problem worldwide. The tropical and subtropical regions are most affected, occurring approximately 100 million infections per year. The main symptoms that characterize the infection are fever, headache, retro-orbital pain, myalgia, arthralgia, prostration and rash. However, other infections have very similar symptoms to dengue, such as leptospirosis. Both dengue and leptospirosis, can cause outbreaks during the rainy seasons, since it is the best period for the dissemination of Aedes aegypti and contamination of wetlands by Leptospira sp.. This study aimed to employ different methods for the laboratory diagnosis of dengue and leptospirosis in patients with febrile syndrome. Eighty-six samples of 86 patients suspected of being infected with dengue were analyzed by NS1 immunoassay, NS1-ELISA, immunoassay IgM and IgG capture, IgM-ELISA and RT-PCR. Among the 86 patients evaluated, 48 (55.8%) were positive for at least one of diagnostic tests. After tested for dengue, 68 patients were evaluated by immunoassay IgM capture and by ELISA-IgM, both for identification of anti-Leptospira. Five (7.3%) were positive for this infection. It is very important to search for other infectious causes, such as leptospirosis, which is also a febrile illness, in patients with clinical suspicion of dengue, since the control of diseases prevalent only becomes possible when we know the infectious agents responsible for them. Therefore, it is necessary to use specific diagnoses for each infection, allowing epidemiological control programs can be carried out.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A dengue à a arbovirose mais importante no mundo, causando mais de 100 milhÃes de casos de dengue clÃssico (DC) e mais de 250 mil casos de febre hemorrÃgica da dengue (FHD), anualmente. A infecÃÃo com o vÃrus dengue (DENV), famÃlia Flaviviridae, causa um amplo espectro de manifestaÃÃes clÃnicas que variam desde formas assintomÃticas a quadros graves, potencialmente fatais, como os casos hemorrÃgicos e/ou de choque hipovolÃmico. Na maioria das vezes, a doenÃa se apresenta com sintomas inespecÃficos. Dessa forma, torna-se difÃcil diferenciar a dengue de outros casos febris de natureza infecciosa como leptospirose, febre amarela e outras arboviroses, apenas com base nas manifestaÃÃes clÃnicas iniciais. Diante disso, o objetivo deste estudo foi identificar a etiologia de 82 pacientes com quadro clÃnico semelhante ao de dengue e com resultado negativo no isolamento viral (IV) para o DENV. O IV foi realizado pelo LaboratÃrio Central de SaÃde PÃblica do Cearà (LACEN-CE), em 2008. Neste estudo, as amostras desses pacientes foram avaliadas para dengue por meio da detecÃÃo de anticorpos especÃficos contra o vÃrus pela tÃcnica de IgM-ELISA (PanBio DiagnosticsÂ) e pela reaÃÃo em cadeia da polimerase apÃs transcriÃÃo reversa (RT-PCR). As amostras negativas para dengue foram testadas para a detecÃÃo de anticorpos IgM especÃficos contra bactÃrias do gÃnero Leptospira por ELISA (PanBio DiagnosticsÂ). Foram testadas tambÃm amostras de 73 pacientes quanto a infecÃÃo por hantavirus, atravÃs da detecÃÃo de anticorpos especÃficos (IgM e IgG) contra antÃgenos de hantavÃrus e RT-PCR. Trinta e cinco pacientes (35/82; 42,68%) foram positivos para dengue, sendo que destes, todos foram positivos no IgM-ELISA e 4 foram positivos tambÃm no RT-PCR. Das 47 amostras dengue-negativas, apenas 43 foram testadas para infecÃÃo por Leptospira devido ao volume insuficiente das amostras. Seis pacientes (6/82; 7,32%) foram positivos IgM-ELISA para leptospirose. TrÃs pacientes foram positivos para hantavÃrus, entretanto apenas 1 (1/82; 1,22%) foi positivo no IgM-ELISA e 2, no IgG-ELISA. A infecÃÃo dos 35 (42,68%) pacientes negativos em todos os testes de detecÃÃo de infecÃÃo aguda foi classficada como sÃndrome febril indiferenciada (SFI). Esta à a primeira evidÃncia de infecÃÃo por hantavÃrus no Estado do CearÃ. Essas doenÃas podem causar infecÃÃo clinicamente indistinguÃvel da dengue e, portanto, deveriam ser incluÃdas no diagnÃstico diferencial no contexto dessas sÃndromes febris. / Dengue is the most important arborvirosis in the world, causing approximately 100 millions cases of classical dengue fever (DF) and more than 250.000 of dengue hemorrhagic fever (DHF), annually. The dengue virus (DENV) belongs to the Flaviviridae family and its infection causes a wide clinical spectrum ranging from assymptomatic forms to severe manifestations, potentially fatal, as in hemorrhagic forms or dengue shock syndrome (DSS). Most of times the disease presents inespecific symptoms. Thus, DF is difficult to distinguish from other acute febrile illnesses, including arboviral ones and leptospirosis, based only on clinical criteria. Given this, the aim of this study was to identify the etiology of 82 patients with clinical picture of dengue-like illness, negative in DENV isolation. The virus isolation was done in Laboratorio Central de SaÃde PÃblica do Cearà (LACEN-CE), Brazil, in 2008. In the present study, the serum samples from these patients were evaluated for dengue infection by IgM-ELISA (PanBio DiagnosticsÂ) and RT-PCR, following Lanciotti et alli protocol (1992). Negative samples to dengue infection were tested to leptospirosis by IgM-ELISA (PanBio DiagnosticsÂ). Seventy-three patients were also tested for hantavirus infection by IgM and IgG by ELISA and RT-PCR. Dengue infection was diagnosed in 35 patients (35/82; 42.68%) of which all were positive in IgM-ELISA, and 4 were also positive in RT-PCR. Of 47 (47/82; 57.32%) DENV-negative samples, only 43 were tested to evaluate lesptospiral infection because of insufficient sample volume. Six patients (6/82; 7,32%) were positive to leptospirosis in the IgM-ELISA. Three patients were positives to hantavirus infection, but only 1 (1/82; 1,22%) was positive in the IgM-ELISA and the two others, in the IgG-ELISA. Thirty and five patients (35/82; 42,68%) remained negative em all tests. They were classified as having other febril illness (OFI). To the best of our knowledge, this is the first evidence of hantavirus infection in humans in the state of CearÃ, Brazil. These diseases, including leptospirosis, may cause infection clinically indistinguishable from DF and therefore should be included in the differential diagnosis of febrile illnesses in this setting.

Memoria para optar al Título Profesional de Médico Veterinario / El objetivo de este estudio fue indagar la exposición frente a Brucella canis y Leptospira spp. a través de la detección de anticuerpos en cánidos silvestres y domésticos en la Isla Grande de Tierra del Fuego. Se analizó un total de 56 sueros, 15 de zorros culpeo fueguino, 12 de zorros chilla y 29 de perros domésticos. Dos técnicas serológicas fueron utilizadas para la detección de anticuerpos: contrainmunoelectroforesis (CIEF) y aglutinación microscópica (MAT) para brucelosis canina y leptospirosis, respectivamente. Los resultados mostraron la ausencia de anticuerpos contra B. canis y la presencia de anticuerpos contra Leptospira spp., con prevalencias de un 20% para zorros culpeo, 8,3% para zorros chilla y 3,4% para perros domésticos. Estos resultados se pueden explicar por factores tales como las características intrínsecas del agente infeccioso y/o ubicación de los animales analizados. Así, una comprensión más profunda sobre la interacción de los cánidos silvestres y domésticos con otras especies animales en un medio ambiente en común, es una herramienta esencial hacia la conservación de las especies endémicas en peligro de extinción de la Isla Grande de Tierra del Fuego como el zorro fueguino. / The objective of this study was to explore exposure against Brucella canis and Leptospira spp. through the presence of antibodies in wild and domestic canids in Isla Grande de Tierra del Fuego. A total of 56 sera, 15 from Fuegian culpeo foxes, 12 from grey foxes and 29 from domestic dogs, were analyzed. Two serological techniques were used for antibody detection: counterimmunoelectrophoresis (CIEF) and microscopic agglutination (MAT) for canine brucellosis and leptospirosis, respectively. The results showed the absence of antibodies against B. canis and the presence of antibodies against Leptospira spp., with prevalence’s of 20% for culpeo foxes, 8.3% for grey foxes and 3.4% for domestic dogs. These results could be explained by factors such as intrinsic characteristics of the infectious agent and/or location of the test animals. Thus, a deeper understanding of the interaction of wild and domestic canids with other animal species in an environment in common, is an essential tool towards the conservation of the endangered endemic species from the Isla Grande de Tierra del Fuego like the Fuegian fox.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Objective: This study was carried out to determine the prevalence of leptospirosis among febrile patients with a suspicious clinical diagnosis of dengue fever in northern Peru. Results: A total of 276 serum samples from patients with acute febrile illness (AFI) and suspected diagnosis for dengue virus (DENV) were analyzed. We identified an etiological agent in 121 (47.5%) patients, DENV was detected in 30.4% of the cases, leptospirosis in 11.2% and co-infection by both pathogens was observed in 5.9% of the patients. In this study the most common clinical symptoms reported by the patients were: headache 89.1%, myalgias 86.9% and arthralgias 82.9%. No differences in symptomatology was observed among the different study groups. / National Research Foundation of Korea / Revisión por pares

A utilização de vacinas destaca-se como medida preventiva contra a leptospirose. Sete potenciais antígenos vacinais de L. interrogans sorovar Copenhageni foram estudados: LipL32, LipL23 e LipL22 (lipoproteínas), SphH e Sph4 (hemolisinas), AnkB (domínio anquirina) e OmpA76 (domínio OmpA). Os genes foram amplificados, clonados e as proteínas expressas em E.coli e Salmonella enterica para ensaios de imunização e desafio. As sete proteínas foram purificadas. No primeiro desafio, hamsters foram imunizados com a salmonela recombinante para expressar a OmpA76 e desafiados com sorovar Pomona. Sobreviveu 30% do grupo salmonela recombinante, 100% da vacina comercial e 10% dos controles. No segundo desafio, hamsters foram imunizados com pool das sete proteínas e desafiados com sorovar Copenhageni. Sobreviveram 30% do grupo pool de proteínas, 90% da vacina comercial, 100% da bacterina e 0% do grupo PBS. A LipL32, OmpA76, LipL23 e LipL22 reagiram com soros de hamsters infectados. Extratos de leptospiras foram reconhecidos pelos soros específicos contra LipL32, OmpA76 e LipL23. / Preventive measures as vaccines are the best strategies to combat leptospirosis. Seven potential vaccine candidates from L. iInterrogans serovar Copenhageni were studied: LipL32, LipL23 and LipL22 (lipoproteins), SphH e Sph4 (hemolysins), AnkB (ankyrin domain) and OmpA76 (OmpA domain). The genes were amplified, cloned and the proteins expressed in E. Coli e Salmonella enteric for challenge assays. In the first assay, hamsters were immunized with the recombinant salmonella to express OmpA76 in vivo, and challenged with serovar Pomona. The survival was 30% of the recombinant salmonella group, 100% of the commercial vaccine and 10% of the control groups. In the second assay, hamsters were immunized with a pool of the purified proteins and challenged with serovar Copenhageni. The survival was 30% of the group pool of proteins, 90% of the commercial vaccine, 100% of the bacterin and 0% of the PBS group. LipL32, OmpA76, LipL23 and LipL22 reacted with hamsters infected sera. Leptospiral extracts were recognized by specific sera against LipL32, OmpA76 and LipL23.

A leptospirose é uma zoonose mundial causada por bactéria do gênero Leptospira. O desenvolvimento de uma vacina de subunidade eficaz seria de grande interesse em saúde pública. Propusemos investigar 4 antigenos de Leptospira interrogans srv Copenhageni, genes LIC12659, LIC12660, LIC12631 e LIC10868, os quais foram amplificados e clonados nos vetores de expressão pAE e pAEsox. As proteínas VapB, VapC, VapBC e Sph2 foram expressas em E. coli e em salmonela SL3261 e purificadas por cromatografia de afinidade a metal. As atividades hemolítica de Sph2 e ribonucleásica de VapC não foram evidenciadas, possivelmente devido a diferenças estruturais entre as proteínas recombinantes e as nativas. Os clones de E. coli com os genes do módulo toxina-antitoxina vapBC sofreram os efeitos de inibição (vapC) e recuperação (vapBC) de crescimento, como descrito. As proteínas recombinantes, VapB purificada ou em salmonlea e Sph2, mostraram-se imunogênicas em camundongos e em hamsters. A imunização com Salmonela-VapB determinou a melhor proteção no teste de desafio. / Leptospirosis is a zoonosis worldwide distributed, caused by bacteria from the genus Leptospira. The development of an efficient vaccine of sub-unities would be of major interest for public health. We proposed to investigate 4 antigens from Leptospira interrogans srv Copenhageni, genes LIC12659, LIC12660, LIC12631 and LIC10868, which were amplified and cloned into expression vectors pAE and pAEsox. The proteins VapB, VapC, VapBC and Sph2 were expressed in E. coli and in salmonella SL3261, and purified by metal-affinity chromatography. The activities hemolytic of Sph2 and ribonuclease of VapC were not confirmed, possibly due to differences between native and recombinant proteins. The clones of E. coli with genes of the toxin-antitoxin module vapBC suffered inhibition (vapC) and recovery (vapBC) of growth rate as described. The recombinant proteins VapB purified or in salmonella and purified Sph2, showed to be immunogenic in mice and in hamsters. The immunization with Salmonella-VapB determined better protection on challenge assay.

La Leptospirosis es una enfermedad que puede pasar desapercibida para muchos médicos, debido a que los signos y síntomas se pueden mimetizar con otras patologías comunes dificultando su diagnóstico definitivo. En este trabajo se calculó el valor de los Costos Directos, generados por la atención clinica del paciente con Leptospirosis en una fase precoz y otra tardía; y los Costos Indirectos generados por los pacientes que al estar hospitalizados dejan de ir a trabajar. Para el cálculo de los costos directos en una fase precoz se tomó el Protocolo de atención de la Leptospirosis y se sacó el valor del paradiagnóstico y tratamiento. Para el cálculo de los Costos Directos en una fase tardía de la Leptospirosis, en este caso UTI; se tomaron 16 pacientes positivos a Leptospirosis complicadas sacando el valor de cada item utilizado durante su estadía. Para los Costos Indirectos se tomaron estos 16 pacientes internados en la UTI, se calculó el valor del día no trabajado según el salario mínimo legal vigente del año en que estuvieron hospitalizados.

A leptospirose é uma zoonose mundial causada por bactéria do gênero Leptospira. O desenvolvimento de uma vacina de subunidade eficaz seria de grande interesse em saúde pública. Propusemos investigar 4 antigenos de Leptospira interrogans srv Copenhageni, genes LIC12659, LIC12660, LIC12631 e LIC10868, os quais foram amplificados e clonados nos vetores de expressão pAE e pAEsox. As proteínas VapB, VapC, VapBC e Sph2 foram expressas em E. coli e em salmonela SL3261 e purificadas por cromatografia de afinidade a metal. As atividades hemolítica de Sph2 e ribonucleásica de VapC não foram evidenciadas, possivelmente devido a diferenças estruturais entre as proteínas recombinantes e as nativas. Os clones de E. coli com os genes do módulo toxina-antitoxina vapBC sofreram os efeitos de inibição (vapC) e recuperação (vapBC) de crescimento, como descrito. As proteínas recombinantes, VapB purificada ou em salmonlea e Sph2, mostraram-se imunogênicas em camundongos e em hamsters. A imunização com Salmonela-VapB determinou a melhor proteção no teste de desafio. / Leptospirosis is a zoonosis worldwide distributed, caused by bacteria from the genus Leptospira. The development of an efficient vaccine of sub-unities would be of major interest for public health. We proposed to investigate 4 antigens from Leptospira interrogans srv Copenhageni, genes LIC12659, LIC12660, LIC12631 and LIC10868, which were amplified and cloned into expression vectors pAE and pAEsox. The proteins VapB, VapC, VapBC and Sph2 were expressed in E. coli and in salmonella SL3261, and purified by metal-affinity chromatography. The activities hemolytic of Sph2 and ribonuclease of VapC were not confirmed, possibly due to differences between native and recombinant proteins. The clones of E. coli with genes of the toxin-antitoxin module vapBC suffered inhibition (vapC) and recovery (vapBC) of growth rate as described. The recombinant proteins VapB purified or in salmonella and purified Sph2, showed to be immunogenic in mice and in hamsters. The immunization with Salmonella-VapB determined better protection on challenge assay.

A utilização de vacinas destaca-se como medida preventiva contra a leptospirose. Sete potenciais antígenos vacinais de L. interrogans sorovar Copenhageni foram estudados: LipL32, LipL23 e LipL22 (lipoproteínas), SphH e Sph4 (hemolisinas), AnkB (domínio anquirina) e OmpA76 (domínio OmpA). Os genes foram amplificados, clonados e as proteínas expressas em E.coli e Salmonella enterica para ensaios de imunização e desafio. As sete proteínas foram purificadas. No primeiro desafio, hamsters foram imunizados com a salmonela recombinante para expressar a OmpA76 e desafiados com sorovar Pomona. Sobreviveu 30% do grupo salmonela recombinante, 100% da vacina comercial e 10% dos controles. No segundo desafio, hamsters foram imunizados com pool das sete proteínas e desafiados com sorovar Copenhageni. Sobreviveram 30% do grupo pool de proteínas, 90% da vacina comercial, 100% da bacterina e 0% do grupo PBS. A LipL32, OmpA76, LipL23 e LipL22 reagiram com soros de hamsters infectados. Extratos de leptospiras foram reconhecidos pelos soros específicos contra LipL32, OmpA76 e LipL23. / Preventive measures as vaccines are the best strategies to combat leptospirosis. Seven potential vaccine candidates from L. iInterrogans serovar Copenhageni were studied: LipL32, LipL23 and LipL22 (lipoproteins), SphH e Sph4 (hemolysins), AnkB (ankyrin domain) and OmpA76 (OmpA domain). The genes were amplified, cloned and the proteins expressed in E. Coli e Salmonella enteric for challenge assays. In the first assay, hamsters were immunized with the recombinant salmonella to express OmpA76 in vivo, and challenged with serovar Pomona. The survival was 30% of the recombinant salmonella group, 100% of the commercial vaccine and 10% of the control groups. In the second assay, hamsters were immunized with a pool of the purified proteins and challenged with serovar Copenhageni. The survival was 30% of the group pool of proteins, 90% of the commercial vaccine, 100% of the bacterin and 0% of the PBS group. LipL32, OmpA76, LipL23 and LipL22 reacted with hamsters infected sera. Leptospiral extracts were recognized by specific sera against LipL32, OmpA76 and LipL23.

Tests on hamsters reveal that there is a decided protective index from the soluble antigen prepared by the acid-heat extraction of living cultures of Leptospira pomona. They also reveal that the antigen must be given in quantities sufficiently small to be amenable to the system of the test animal or a condition of ”immunogenic paralysis“ results. Due to the number of hamsters in any one group whose pooled sera was tested, a fair evaluation could not be made between various groups in the agglutination-lysis test. Tests on the stability of the immunogenic agent used show that it is completely stable for at least a period of three months when stored in a refrigerator at 5 °C. / Master of Science