The dog (Canis lupus familiaris) can be infected by a variety of protozoa and may develop clinical signs or remain asymptomatic. Due to the proximity of this kind with the man some protozoa present zoonotic potential, as researchers studied, including Leishmania infantum, Neospora caninum and Toxoplasma gondii. L. infantum, it is responsible for causing visceral leishmaniasis in several species, including the dog and the man with deleterious impacts on public health. The dog is considered the urban reservoir of the parasite, and it is recommended the euthanasia of seropositive, including asymptomatic. N. caninum and T. gondii are widely distributed protozoa. These agents, when affecting dogs, can cause neurological, gastrointestinal, respiratory and muscle disturbs. N. caninum is a protozoan of great economic importance and may cause reproductive problems in farm animals and the dog is the definitive host of this agent. T. gondii is an opportunistic microorganism, a zoonotic potential, with world-occurrence. The dogs as well as humans, are considered intermediate hosts, infection may be more severe in pregnant women and immunocompromised individuals. This study aimed to research anti-Leishmania infantum antibodies, Neospora caninum and Toxoplasma gondii in autopsied dogs; and to determine the occurrence of infection in animals from Agudo, Jaguari, Júlio de Castilhos, Mata, Santa Maria, Santiago, São Martinho da Serra, São Vicente do Sul and Tupanciretã, towns in the central region of Rio Grande do Sul (RS). In addition, there were correlated epidemiological data on sex, age, race, origin and occurrence of these protozoa in the seasons, with the pathological lesions. The animals were necropsied during routine at Pathology Laboratory in the Universidade Federal de Santa Maria, from November 2014 to April 2016. For detection of seropositive dogs, the Indirect Immunofluorescence Antibody Test (IFAT) was performed on stored samples in serum bank Analysis Laboratory in Veterinary Clinics. It analyzed 78 samples, where 53/78 (67.9%) were seropositive, demonstrating the presence of antibodies to one or more of the agents in the population studied. The occurrence of antibodies to L. infantum, N. caninum and T. gondii was 26/78 (33.3%) 29/78 (37.1%) and 34/78 (43.5%) respectively. Mono infections observed in 5/53 (9.4%) of L. infantum, 10/53 (18.8%) to N. caninum and 11/53 (20.7%) T. gondii, while the coinfection appeared in 27/53 (50.9%) of the dogs were detected in 4/53 (7.5%) L. infantum + N. caninum, 8/53 (15.0%) L. infantum + T. gondii, 6/53 (11.3%) N. caninum + T. gondii and 9/53 (16.9%) L. infantum + N. caninum + T. gondii, affecting animals of different ages, without predominance sex or race. There were no pathological characteristic lesions, featuring animals as asymptomatic, indicating a role for these sentinels of dogs these diseases. This study contributed to a better understanding of the epidemiology of protozoa and noted the emergence of visceral leishmaniasis in animals from areas considered harmless. In addition, this work can support the public health services in the adoption of preventive measures, avoiding possible cases of autochthonous leishmaniasis in central RS. / O cão (Canis lupus familiaris) pode ser infectado por uma variedade de protozoários, podendo desenvolver sinais clínicos ou permanecer assintomático. Devido à proximidade desta espécie com o homem e por alguns protozoários apresentarem potencial zoonótico, se realizou um estudo, incluindo Leishmania infantum, Neospora caninum e Toxoplasma gondii. L. infantum, é responsável por causar a leishmaniose visceral em diversas espécies, abrangendo o cão e o homem, com impactos deletérios na Saúde Pública. O cão é considerado o reservatório urbano deste parasita, e recomenda-se a eutanásia dos soropositivos, inclusive os assintomáticos. N. caninum e T. gondii são protozoários de ampla distribuição geográfica. Estes agentes, quando acometem os cães, podem causar distúrbios neurológicos, gastrintestinais, respiratórios e musculares. N. caninum é um protozoário de grande importância econômica, podendo ocasionar problemas reprodutivos em animais de produção e o cão é o hospedeiro definitivo deste agente. T. gondii é um micro-organismo oportunista, de potencial zoonótico, com ocorrência mundial. Os cães, assim como o homem, são considerados hospedeiros intermediários, podendo a infecção ser mais grave em indivíduos imunocomprometidos ou gestantes. Este estudo teve por objetivo pesquisar anticorpos anti-Leishmania infantum, Neospora caninum e Toxoplasma gondii em cães necropsiados; bem como determinar a ocorrência da infecção em animais procedentes de Agudo, Jaguari, Júlio de Castilhos, Mata, Santa Maria, Santiago, São Martinho da Serra, São Vicente do Sul e Tupanciretã, municípios da região central do Rio Grande do Sul (RS). Adicionalmente, correlacionou-se os dados epidemiológicos referentes a sexo, idade, raça, procedência e a ocorrência destes protozoários nas estações do ano, com as lesões anatomopatológicas. Os animais foram necropsiados durante a rotina do Laboratório de Patologia da Universidade Federal de Santa Maria, no período de novembro de 2014 a abril de 2016. Para detecção dos cães sororreagentes, foi realizada a Reação de Imunofluorescência Indireta (RIFI), nas amostras armazenadas no banco de soro do Laboratório de Análises Clínicas Veterinárias. Analisou-se 78 amostras, onde 53/78 (67,9%) apresentaram soropositividade, demonstrando a presença de anticorpos para um ou mais dos agentes na população estudada. A ocorrência de anticorpos para L. infantum, N. caninum e T. gondii foi de 26/78 (33,3%), 29/78 (37,1%) e 34/78 (43,5%) respectivamente. Observou-se mono infecções em 5/53 (9,4%) para L. infantum, 10/53 (18,8%) para N. caninum e 11/53 (20,7%) para T. gondii, enquanto que, as coinfecções apareceram em 27/53 (50,9%) dos cães, sendo detectados em 4/53 (7,5%) L. infantum + N. caninum, 8/53 (15,0%) L. infantum + T. gondii, 6/53 (11,3%) N. caninum + T. gondii e 9/53 (16,9%) L. infantum + N. caninum + T. gondii, acometendo animais de diferentes idades, sem predominância de sexo ou raça. Não verificaram-se lesões anatomopatológicas características, caracterizando os animais como assintomáticos, denotando um papel para estes cães de sentinelas destas enfermidades. Este estudo contribuiu para um maior conhecimento da epidemiologia destas protozooses e observou o surgimento da leishmaniose visceral em animais provenientes de áreas consideradas indenes. Além disso, este trabalho poderá subsidiar os serviços de Saúde Pública na adoção de medidas preventivas, evitando possíveis casos de leishmaniose autóctones na região central do RS.

Magister Public Health - MPH / HIV support groups have been widely adopted as part of care and support interventions in Zambia, yet there is very little research on the effectiveness of these groups in meeting the needs of the PLWHIV from the perspective of those who join them. This case study looks at a selected support group for People Living with HIV and AIDS (PLWHIV) facilitated by the Flying Angels, a faith based non-governmental organization established in 2007 by the Living Assemblies of God Church, in Ng‟ombe Township, Lusaka. The Support Group brings together around eighty young and old, married and single, men and women living with HIV and AIDS, to share experiences and find ways of coping with their situation. The qualitative case study sought to obtain a rich understanding of the experiences of members with a view to understanding the support they needed, the aspects of the support group that are relevant and effective to their situation, and which are not. In-depth interviews and focus group discussions methods were used to collect data. Discussion and interviews were audio-taped. Audio-recordings were translated during transcription, data organized, coded and thematically analysed.

The dynamics of T cell responses have been extensively studied during single virus infection of naïve mice. During a viral infection, viral antigen is presented in the context of MHC class I molecules on the surface of infected cells. Activated CD8 T cells that recognized viral antigens mediate clearance of virus through lysis of these infected cells. We hypothesize that the balance between the replicating speed of the virus and the efficiency at which the T cell response clears the virus is key in determining the disease outcome of the host. Lower T cell efficiency and delayed viral clearance can lead to extensive T cellmediated immunopathology and death in some circumstances. To examine how the efficiency of the immune response would impact immunopathology we studied several viral infection models where T cell responses were predicted to be less than optimal: 1. a model of co-infection with two viruses that contain a crossreactive epitope, 2. a viral infection model where a high dose infection is known to induce clonal exhaustion of the CD8 T cell response, 3. a neonatal virus infection model where the immune system is immature and 4. A model of beneficial heterologous immunity and T cell crossreactivity where mice are immunized as neonates when the T cell pool is still developing. Model 1. Simultaneous co-infections are common and can occur from mosquito bites, contaminated needle sticks, combination vaccines and the simultaneous administration of multiple vaccines. Using two distantly related arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV), we questioned if immunological T cell memory and subsequent protection would be altered following a simultaneous co-infection, where two immune responses are generated within the same host at the same time. Coinfection with these two viruses, which require CD8 T cell responses to clear, resulted in decreased immune protection and enhanced immunopathology after challenge with either virus. After primary co-infection, each virus-specific immune response impacted the other as they competed within the same host and resulted in several significant differences in the CD8 T cell responses compared to mice infected with a single virus. Co-infected mice had a dramatic decrease in the overall size of the LCMV-specific CD8 T cell response and variability in which virus-specific response dominated, along with skewing in the immunodominance hierarchies from the normal responses found in single virus infected mice. The reduction in the number of LCMV-specific CD8 memory T cells, specifically cells with an effector memory-like phenotype, was associated with higher viral loads and increased liver pathology in co-infected mice upon LCMV challenge. The variability in the immunodominance hierarchies of co-infected mice resulted in an enhanced cross-reactive response in some mice that mediated enhanced immune-mediated fat pad pathology during PICV challenge. In both viral challenge models, an ineffective memory T cell response in co-infected mice facilitated increased viral replication, possibly leading to enhanced and prolonged accumulation of secondary effector T cells in the tissues, thereby leading to increased immune pathology. Thus, the magnitude and character of memory CD8 T cell responses in simultaneous co-infections differed substantially from those induced by single immunization. This has implications for the design of combination vaccines and scheduling of simultaneous immunizations. Model 2. The balance between protective immunity and immunopathology often determines the fate of the virus-infected host. Several human viruses have been shown to induce a wide range of severity of disease. Patients with hepatitis B virus (HBV), for example, show disease progression ranging from acute resolving infection to a persistent infection and fulminant hepatitis. Certain rapidly replicating viruses have the ability to clonally exhaust the T cell response, such as HBV and hepatitis C virus (HCV) in humans and the clone 13 strain of LCMV in mice. How rapidly virus is cleared is a function of initial viral load, viral replication rate, and efficiency of antigen-specific T cells. By infecting mice with three different inocula of LCMV clone 13, we questioned how the race between virus replication and T cell responses could result in different disease outcomes. A low dose of LCMV generated efficient CD8 T effector cells, which cleared the virus with minimal lung and liver pathology. A high dose of LCMV resulted in clonal exhaustion of T cell responses, viral persistence and little immunopathology. An intermediate dose only partially exhausted the CD8 T cell responses and was associated with significant mortality, and the surviving mice developed viral persistence and massive immunopathology, including necrosis of the lungs and liver. This was a T cell-mediated disease as T cell-deficient mice had no pathology and became persistently infected like mice infected with a high dose of LCMV clone 13. This suggests that for non-cytopathic viruses like LCMV, HCV and HBV, clonal exhaustion may be a protective mechanism preventing severe immunopathology and death. Model 3. Newborns are more susceptible to infections due to their lack of immunological memory and under-developed immune systems. Passive maternal immunity helps protect neonates until their immune systems have matured. We questioned if a noncytolytic virus that produces strong T cell responses in adult mice would also induce an equally effective response in neonatal mice. Neonates were infected with very low doses of LCMV Armstrong and surprisingly the majority succumbed to infection between days 7-11, which is the peak of the T cell response in adult mice infected with LCMV. Death was caused by T cell-dependent pathology and not viral load as 100% of T cell deficient neonates survived with minimal lung and liver pathology. This is similar to the adult model of medium dose LCMV clone 13, but T cell responses in neonates were not partially clonal exhausted. Furthermore, surviving neonates were not persistently infected, clearing virus by day 14 post infection. In adult mice direct intracranial infection leads to LCMV replication and CD8 T cell infiltration in the central nervous system (CNS), causing CD8 T cell-mediated death. However, this does not occur in adults during LCMV intraperitoneal (ip) infections. We questioned if unlike adults LCMV could be gaining access to the CNS in neonates following ip infection. Replicating LCMV was found in the brain of neonates after day 5 post infection along with virus-specific CD8 T cells producing IFNγ at day 9 post infection. Neonates lacking perforin had complete survival when followed until day 14 post infection, suggesting perforin-mediated T cell-dependent immunopathology within the CNS of neonates was causing death after LCMV infection. Passive immunity from LCMV-immune mothers also protected 100% of pups from death by helping control viral load early in infection. We believe that the maternal antibody compensates for the immature innate immune response of neonates and controls viral replication early so the neonatal T cell response induced less immunopathology. Neonates are commonly thought to have less functional immune systems, but these results show that neonates are capable of producing strong T cell responses that contribute to increased mortality. Model 4. Due to their enhanced susceptibility to infection neonatal and infant humans receive multiple vaccines. Several non-specific effects from immunizations have been observed, for example, measles or Bacillus Calmette- Guerin (BCG) vaccines have been linked to decreased death of children from infections other than measles virus or tuberculosis. These studies mirror the concepts of beneficial heterologous immunity, where previous immunization with an unrelated pathogen can result in faster viral clearance. LCMV-immune mice challenged with vaccinia virus (VV) have lower viral loads then naïve mice and survive lethal infections, but some mice do develop fat pad immunopathology in the form of panniculitis or acute fatty necrosis (AFN). We questioned how immunological T cell memory formed during the immature neonatal period would compare to memory generated in fully mature adults during a heterologous viral challenge. Mice immunized as neonates had comparable reduction in VV load and induction of AFN, indicating that heterologous immunity is established during viral infections early in life. Interestingly, the LCMV-specific memory populations that expanded in mice immunized as neonates differed from that of mice immunized as adults. In adult mice 50% of the mice have an expansion of LCMVNP205- specific CD8 T cells while the majority of neonates expanded the LCMVGP34- specific CD8 T cell pool. This alteration in dominant crossreactivities may be due to the limited T cell receptor repertoire of neonatal mice. In naïve neonatal mice we found altered Vβ repertoires within the whole CD8 T cell pool. Furthermore, there was altered Vβ usage within virus-specific responses compared to adult mice and a wide degree of variability between individual neonates, suggesting enhanced private specificity of the TCR repertoire. Beneficial heterologous immunity is maintained in neonates, but there was altered usage of crossreactive responses. As neonatal mice were found to be so sensitive to LCMV infection we questioned if neonates could control another arena virus that did not replicate as efficiently in mice, PICV. Unlike LCMV infection, neonatal mice survived infection with PICV even with adult-like doses. However, viral clearance was protracted in neonates compared to adults, but was cleared from fat pad and kidney by day 11 post infection. The peak of the CD8 T cell response was similarly delayed. PICV infected neonates showed dose-dependent PICV-specific CD8 T cell responses, which were similar to adult responses by frequency, but not total number. As with LCMV infection there were changes in immunodominance hierarchies in neonates. Examination of the immunodominance hierarchies of PICV-infected neonates showed that there were adult-like responses to the dominant NP38- specific response, but a loss of the NP122-specific response. Six weeks post neonatal infection mice were challenged with LCMV Armstrong and there was a strong skewing of the PICV immunodominance hierarchy to the crossreactive NP205-specific response. These data further support the hypothesis that heterologous immunity and crossreactivity develop following neonatal immunization, much as occurs in adults, although TCR repertoire and crossreactive patterns may differ. Changing the balance between T cell efficiency and viral load was found to altered the severity of the developing immunopathology after viral infection.

No description available.

A doença hepática crônica causada pelo vírus da hepatite C (HCV) tornou-se, nos últimos anos, uma das principais comorbidades dos pacientes portadores do vírus da imunodeficiência humana (HIV) nos países desenvolvidos. Os pacientes coinfectados com HIV/HCV apresentam uma progressão mais rápida para a cirrose e as suas complicações que os pacientes monoinfectados com HCV. Embora os mecanismos responsáveis por esta evolução não estejam totalmente esclarecidos, a expressão da molécula de HLA-G, um HLA de classe Ib não clássico, que tem propriedades bem reconhecidas na regulação negativa da resposta imune, pode estar relacionada à progressão da doença hepática. Os objetivos deste trabalho foram analisar o perfil de expressão de HLA-G em tecido hepático de pacientes coinfectados HIV/HCV e identificar possíveis variáveis do hospedeiro, do HCV e do HIV que possam estar relacionadas com a expressão de HLA-G na biópsia hepática. Para isso, 57 amostras de biópsia hepática de pacientes coinfectados com HIV/HCV, nas quais a imuno-histoquímica para HLA-G foi realizada, foram analisadas retrospectivamente quanto à expressão desta molécula no tecido hepático. Avaliaram-se também outras características histopatológicas da biópsia como grau de fibrose, atividade inflamatória, deposição de ferro e gordura. Determinou-se o polimorfismo de inserção ou deleção de 14 pares de bases da região 3` não traduzida do exon 8 do gene do HLA-G, que está relacionada com a produção de RNA-mensageiro, em 43 destes pacientes, além do polimorfismo de IL-28B, relacionado com a resposta ao tratamento do HCV, em 44 deles. Características bioquímicas e virológicas, tanto do HIV quanto do HCV também foram avaliadas. O genótipo 1 do HCV foi o mais prevalente (87,75%), especialmente o subgenótipo 1a (60%). A expressão do HLA-G foi observada em 38 (66,7%) amostras de fígado, e foi mais frequente em estágios moderados e severos de fibrose do que em estágios mais leves (94,1% x 55%, P < 0,01). Não houve relação entre a expressão do HLA-G e os outros parâmetros estudados. Embora a progressão para a cirrose no contexto da coinfecção por HIV/ HCV seja um processo complexo, modulado por muitos factores, a associação da intensidade de fibrose com a expressão do HLA-G pode indicar que a expressão desta proteína desempenha um importante papel nos mecanismos que contribuem para a progressão da doença, por meio da regulação negativa da resposta imune contra o HCV na coinfecção pelo HIV. / Chronic liver disease induced by hepatitis C virus (HCV) infection has recently become one of the most common comorbidities in patients who are infected with the human immunodeficiency virus (HIV) in developed countries. HIV/HCV coinfected patients show faster progression to cirrhosis and its complications than the HCV monoinfected patients. Even though the responsible mechanisms for this evolution have not been entirely clarified yet, the expression of the HLA-G molecule, a HLA from the non-classic Ib class, with well-known properties of negatively regulating the immune response, may be related to the liver disease progression. The aims of the present work were to analyze the HLA-G expression profile in the liver micro ambience of HIV/HCV coinfected patients and to identify possible host factors, HIV or HCV, that may be related to the HLA-G expression on the liver biopsy. For this purpose, 57 liver biopsies of HIV/HCV coinfect patients, in which immunohistochemistry for HLA-G had been performed, were retrospectively analyzed according the HLA-G expression on the hepatic tissue. Other histopathological features in the liver biopsies, such as fibrosis degree, inflammatory activity, iron deposition and fat were also evaluated. The polymorphism of insertion or deletion in 14-base pairs of the 3`non-translated region of exon 8 of the HLA-G gene, which is related to the production of HLA-G messenger RNA, was evaluated in 43 of the patients. Also, the polymorphism of IL-28B, related to the response to HCV treatment, was evaluated in 44 of them. Biochemical and virological features of HIV and HCV were also evaluated. The HCV genotype 1 was the most prevalent (87.75%), especially the subgenotype 1a (60%). The expression of HLA-G was observed in 38 (66.7%) samples of the liver biopsies, and it was most frequent in moderate and severe stages of fibrosis than in the mild stages (94.1% x 55%, P < 0.01). There was no established relationship between HLA-G and other parameters studied. Although the progression to cirrhosis in the context of HIV/HCV coinfection is a complex process modulated by many factors, the association of HLA-G expression with the intensity of the liver fibrosis may indicate the protein expression play an important role in the mechanisms that contribute to the progression of the disease, through the negative regulation of the immune response against HCV setting of a coinfection with HIV.

A doença de Chagas é caracterizada por um amplo espectro de manifestações clínicas, que vão desde a ausência de sintomas à doença grave com comprometimento cardíaco e/ou digestivo. A influência do parasito, Trypanosoma cruzi (T. cruzi), agente etiológico da doença, nessas apresentações clínicas têm sido largamente estudada, não se tendo demonstrado o papel da diversidade genética de populações de T. cruzi na determinação das diferentes formas clínicas em humanos. Este trabalho teve como objetivos: a) geral: analisar as características moleculares de T. cruzi em pacientes com doença de Chagas com e sem imunodepressão (infecção por HIV e transplante de órgãos com e sem reativação); b) específicos: 1. Analisar comparativamente isolados do parasito quanto à distribuição em DTU; 2. Relacionar os resultados obtidos pela análise molecular do gene ND7 com a forma clínica e origem; 3. Avaliar por LSSP-PCR a variabilidade da sequência do kDNA de T. cruzi diretamente de amostras biológicas assim como em isolados de T. cruzi obtidos pelos exames de hemocultura/xenodiagnóstico; 4. Comparar os padrões polimórficos obtidos por LSSP-PCR em amostras repetidas de um mesmo paciente no mesmo sítio ou distintos sítios biológicos. Foram incluídos, após aprovação do protocolo na CAPPesq e mediante assinatura de TCLE, 106 pacientes com doença de Chagas crônica ou com imunossupressão, provenientes dos ambulatórios e enfermarias do HCFMUSP, além de 75 indivíduos controle, com provas sorológicas e moleculares negativas. Foram analisadas 187 amostras isoladas de hemocultura/xenodiagnóstico e 236 diretamente de amostras sanguíneas de pacientes. Os seguintes grupos foram constituídos: Agudo-AG, Crônico-CR, Crônico Imunodeprimido-CRI (doenças autoimunes/neoplasias), Coinfecção-CO (infecção por HIV/T.cruzi), Coinfecção-CO/RE (infecção por HIV/T.cruzi e reativação da doença de Chagas), Transplantado-TX, Transplantado-TX/RE (Transplantado com reativação da doença de Chagas). Foram identificados DTU TcI, TcV, TcVI e em maior número TcII, por ensaios de tipagem molecular do parasito, com distribuição estatisticamente significantemente de acordo com a naturalidade dos pacientes (P=0,013). Quanto ao gene ND7, observou-se que a banda de ~900 bp ocorreu em 83,0% das amostras das regiões norte, nordeste, centro-oeste e Bolívia e a de ~400pb em 54% das amostras nas regiões sul e sudeste brasileiras sendo esta diferença estatisticamente significante (P < 0,001). A comparação dos perfis observados por LSSP-PCR a partir de amostras extraídas diretamente do sangue e de isolados obtidos de hemocultura/xenodiagnóstico mostrou maior variabilidade em amostras sanguíneas, confirmada pelo dendrograma. Adicionalmente, o estudo de amostras repetidas do mesmo paciente permitiu confirmar a maior variabilidade nas amostras diretamente extraídas do sangue, com mudança dos padrões durante e após o tratamento com reaparecimento de perfis antigos não presentes no período prétratamento imediato, além de presença de perfis diferentes em distintos sítios biológicos do mesmo paciente. O encontro de DTU diferentes de TcII nos grupos CR/CRI e AG enfatiza a necessidade de atentar para a diferença em limiares de reatividade segundo DTU na análise da parasitemia por PCRq (quantitativa), conforme registrado na literatura. Os dados observados por LSSP-PCR acrescentam informações adicionais não revelados por tipagem molecular, representando novos desafios para o entendimento da relação hospedeiro-parasito em pacientes com doença de Chagas sem e com imunossupressão, ao lado de fatores como nível de parasitemia e pressão seletiva de medicamentos antiparasitários e imunossupressores / Chagas\' disease is characterized by a broad spectrum of clinical manifestations, ranging from asymptomatic cases to severe cardiovascular and/or gastrointestinal involvement. The clinical presentations are thought to be determined primarily by genetic diversity of populations of Trypanosoma cruzi (T. cruzi), but no correlation was clearly demonstrated yet. This study aimed to: a) general: to analyze the molecular characteristics of T. cruzi in patients with Chagas\' disease with and without immunosuppression (HIV infection and organ transplantation with or without reactivation); b) specifics: 1.To analyze comparatively isolates from the parasite as for the distribution in DTU; 2. To describe the results obtained by molecular analysis of gene ND7 in relationship with the clinical form and origin; 3. To assess by LSSP-PCR the variability of the sequence of the T. cruzi kDNA directly from biological samples, as well as in T. cruzi isolates obtained by examination of blood culture/xenodiagnosis; 4. To compare the polymorphic patterns obtained by LSSP-PCR in repeated samples of the same patient on the same site or different biological sites. After approval of the protocol in CAPPesq and by signing an informed consent, 106 patients with chronic Chagas disease or immunosuppression, from the HCFMUSP\'s clinics and wards, and 75 control subjects with negative serological and molecular tests were included. They were analyzed 187 isolated samples from blood culture/xenodiagnosis and 236 directly from blood samples of patients. The following groups were formed: Acute-AC, Chronic-CR, Chronic immunocompromised-CRI (autoimmune diseases/ neoplasms), Coinfection-CO (HIV/T. cruzi infection), Coinfection-CO/RE (HIV/T. cruzi and reactivation of Chagas disease), Transplantation-TX, Transplantation-TX/RE (Transplantation/ with reactivation of Chagas\' disease). DTU TcI, TcV, TcVI and higher TcII number were identified for molecular typing assays of the parasite and the distribution of DTU was statistically significant according to patient´s naturality (P=0.013). As for ND7 gene, was observed that the band of ~ 900 bp was prevalent in 83% of the samples in the North, Northeast, Midwest regions and Bolivia and the band of ~400bp occurred in 54% of the samples of Brazilian\' South and Southeast regions, this distribution was statistically significant (P < 0.001). The comparison between the profiles observed by LSSP-PCR from samples taken directly from blood and isolates obtained from blood culture/xenodiagnosis showed greater variability in blood samples, confirmed by dendrogram. Additionally, the study of repeated samples from the same patient allowed to confirm the greater variability in blood samples taken directly, with changing patterns during and after the treatment with reappearance of old profiles not present in the immediate pre-treatment period, and the presence of different profiles at different biological sites in the same patient. The presence of other DTUs than TcII in chronic and chronic immunosuppressed patients and AC groups emphasizes the need to pay attention to the different reactivity thresholds for the various DTU in the analysis of parasitaemia by PCRq (quantitative), according to the data registered in the literature. The results observed by LSSP-PCR add further information not revealed by molecular typing, representing new challenges for the understanding of the host-parasite relationship in patients with Chagas\' disease with and without immunosuppression, alongside factors such as level of parasitaemia and selective pressure of antiparasitic drugs and immunosuppressive

Nos últimos anos o tratamento da infecção crônica pelo HCV passou por importantes mudanças. Recentes avanços em biologia molecular proporcionaram o melhor conhecimento sobre a estrutura molecular do HCV e permitiram o desenvolvimento de moléculas que tem como alvo proteínas específicas integrantes do ciclo replicativo do vírus, denominados agentes antivirais de ação direta (DAAs). No Brasil, atualmente, os DAAs aprovados para o tratamento da Hepatite C são: Simeprevir (2ª geração de inibidor de protease), Daclatasvir (inibidor de NS5A) e o Sofosbuvir (inibidor análogo nucleotídeo de polimerase). A combinação dessas diferentes classes de DAAs permite maior eficácia no tratamento do HCV, reduz a duração do tratamento e o risco da emergência de resistência. Ao mesmo tempo em que o desenvolvimento de DAAs promete melhorar a chance de sucesso do tratamento dos pacientes crônicos infectados pelo HCV, a emergência de variantes associadas à resistência representa um grande desafio ao sucesso da terapia antiviral atualmente proposta e informações sobre a presença dessas mutações ainda são escassas. Este estudo tem como objetivo o mapeamento de variantes associadas à resistência (RAVs) primárias aos inibidores da polimerase (NS5B) do HCV em pacientes monoinfectados (HCV) e em pacientes coinfectados (HCV/HIV). Para tal, o rastreamento de substituições de aminoácidos foi realizado entre as posições 159 e 495 da proteína NS5B do HCV nas sequências de 244 pacientes infectados pelo HCV-1: 133 monoinfectados [1b (n=93); 1a (n=40)] e 111 coinfectados [1a (n=93); 1b (n=18)]. A ocorrência natural de RAVs nos resíduos S282, L320 e P495 não foi observada nas sequências analisadas neste estudo. As RAVs encontradas no grupo de monoinfectados foram: L159F (16,1% - 1b), C316N (16,3% - 1b) e A421V (21,4% - 1a; 3,2 - 1b) ; e no grupo de coinfectados foram: C316N (7,1% - 1b), V321A (1,6% - 1a), M414V (1,3% - 1a); A421V (23,7% - 1a; 6,3% - 1b), A421G (1,3% - 1a); Y448H (1,3% - 1a). Entre os pacientes monoinfectados, a região NS5B do HCV-1a apresentou menor número de variantes associadas à resistência (RAVs) quando comparada ao subtipo 1b, ao contrário do observado nos coinfectados. A variante C316N foi a única que ocorreu em combinação com outras variantes. Houve a ocorrência concomitante das variantes L159F e C316N em 8 pacientes monoinfectados pelo HCV-1b (8/56; 14,3%). Entre os coinfectados, foi observada a ocorrência concomitante das variantes C316N e A421V em apenas 1 paciente infectado pelo HCV-1b (1/14; 7,1%). A presença de RAVs foi detectada nas duas populações estudadas neste estudo. Contudo, outros estudos são necessários para que se possa avaliar o real impacto dessas mutacões na resposta ao tratamento / The treatment of chronic HCV infection has undergone important changes recently. Advances in molecular biology provided a better knowledge of the HCV molecular structure and allowed the development of molecules which target specific proteins that have important roles in the viral replicative cycle, known as direct action antiviral agents (DAAs). In Brazil, DAAs approved for treating hepatitis C are Simeprevir (2nd generation protease inhibitor), Daclatasvir (NS5A inhibitor) and sofosbuvir (nucleotide analogue NS5B polymerase inhibitor). The combination of these different DAAs classes leads to a greater efficacy in the treatment of HCV, reducing its duration and the risk of resistance associated variants (RAVs) emergence. DAAs increase the chance of successful treatment of HCV chronic infected patients. RAVs emergence represents a major challenge to the success of antiviral therapy. Nevertheless, data about the presence of these mutations are still scarce. The aim of this study was to verify the presence of primary RAVs associated to HCV polymerase inhibitors primary resistance in monoinfected (HCV) and coinfected (HCV / HIV) patients. For this purpose, amino acid substitutions identification was conducted between positions 159 and 495 of the HCV NS5B protein in the sequences of 244 patients with HCV-1: 133 monoinfected [1a (n=40); 1b (n=93)] and 111 coinfected [1a (n=93); 1b (n=18)]. Naturally occurring RAV in S282, L320 and P495 residues were not observed among the sequences analyzed in this study. RAVs found in monoinfected patients were L159F (16.1% - 1b), C316N (16.3% - 1b) and A421V (21.4% - 1a; 3.2% - 1b); while in co-infected group, the following RAVs were identified: C316N (7.1% - 1b), V321A (1.6% - 1a), M414V (1.3% - 1a); A421V (23.7% - 1a; 6.3% - 1b), A421G (1.3% - 1a); Y448H (1.3% - 1a). Among monoinfected patients, HCV-1a NS5B region showed fewer RAVs when compared to HCV-1b, conversely to what was observed in HIV coinfected patients. Variant C316N occurred in combination with other ones: there was the simultaneous occurrence of L159F and C316N variants 8/56 (14.3%) 8 HCV-1b monoinfected patients Among the coinfected patients, it was observed concomitant occurrence of C316N and A421V variant in only 1/14 (7.1%) HCV-1b infected patient. RAVs were detected in both HCV and HCV/HIV infected populations in this study. Further studies are required to assess the real impact of these changes in response to treatment

A epidemia HIV/AIDS é um grave problema de saúde enfrentado no Brasil e no mundo. Desde o surgimento do vírus, na década de 80, muitos esforços foram realizados para esclarecer o curso da infecção que resulta no comprometimento do sistema imune em indivíduos sem tratamento. A ativação imune crônica pode levar a um status de imunossenescência exacerbada, morte celular, alteração da resposta imune e uma imunodeficiência generalizada. Percebe-se que diversos fatores do hospedeiro interferem na progressão para Aids, como deleção de 32 pares de base do gene CCR5 (CCR5delta32), perfis de HLA desfavoráveis (*B35) e coinfecções, principalmente citomegalovírus, tuberculose e hepatites B e C. Estudos recentes com o GBV-C, pertencente à família Flaviviridae, gênero Pegivirus, possibilitaram uma nova perspectiva no entendimento do curso da infecção causada pelo HIV, uma vez que nenhuma doença foi relacionada à presença do vírus GB tipo C, além de promover um atraso na progressão para a Aids e aumento da sobrevida dos pacientes portadores do vírus. Assim, o objetivo desse estudo foi avaliar o perfil de ativação, senescência e exaustão celular em indivíduos recém-infectados pelo HIV-1 e coinfectados pelo GBVC. Foram investigados a contagem de linfócitos T CD4+ e CD8+, razão CD4/CD8, presença do GBV-C e HIV-1, além da análise da expressão de marcadores de ativação (CCR5, CD38 e HLA-DR), senescência e exaustão celular (PD-1, CD95, CD57 e CD28). Diante dos critérios de inclusão do estudo, foram selecionados nove pacientes com infecção persistente com o vírus GBV-C para o grupo 1 (HIV-1+/GBV-C+), e oito pacientes sem viremia para GBV-C foram incluídos no grupo 2 (HIV-1+/GBV-C-), sendo a média de idade dos pacientes selecionados de 31,6 e 31,7 anos, respectivamente, sexo masculino e homens que fazem sexo com homens (HSH). Na visita de inclusão no estudo (V1) nenhum dos dados analisados (células T CD4+ e CD8+, carga viral e razão CD4/CD8) apresentou diferença estatística, assim como os marcadores de ativação, senescência e exaustão celular. Na análise longitudinal da diferença (deltaVn-V1), percebeu-se uma diminuição dos marcadores de ativação e senescência no grupo HIV-1+/GBV-C +, sem significância estatística entre esses dados. Foi observado, contudo, que houve uma diminuição de células T CD4+ e CD8+ naïve no grupo HIV-1+/GBV-C+, também notou-se redução na subpopulação de células T CD8+ naïve e memória central expressando CD28, houve uma diminuição das subpopulações de memória intermediária e efetora terminal, assim como na subpopulação efetora terminal expressando HLA-DR+, no grupo HIV-1+/GBV-C+. Os resultados demonstraram que a infecção pelo GBV-C reflete na diminuição da estimulação imune, ativação celular e também na redução de marcadores de senescência e exaustão celular nas subpopulações de células T, sugerindo um envolvimento na modulação da progressão do HIV / The HIV/AIDS epidemic is a serious health problem in Brazil and in the world. Since its emergence in the 1980s, many efforts have been made to understand this infection, resulting in a compromised immune system if left untreated. Chronic immune activation may lead to exacerbated immunosenescence, cell death, altered immune response, and a generalized immunodeficiency. Several host factors play an important role in the progression to AIDS, such as the 32 base pairs deletion in the CCR5 gene (CCR5delta32), unfavorable HLA molecules (*B35), and coinfections, mainly cytomegalovirus, tuberculosis, and hepatitis B and C. Recent studies with the GBV-C (Flaviviridae family, genus Pegivirus) have provided a new perspective in the understanding of the HIV infection natural history. GBV-C coinfection delays progression to Aids and increases patient survival. In addition, no symptoms have been associated to its occurrence. The aim of this study was to evaluate the profile of cellular activation, senescence, and exhaustion in recently HIV-infected individuals coinfected with GBVC. Patients were selected from a prospective cohort diagnosed with recent HIV-1 infection with known results for levels of CD4+ and CD8+ T lymphocytes, CD4/CD8 ratio, GBV-C plasma levels, HIV-1 plasma viremia, and markers for cellular activation (CCR5, CD38, and HLA-DR) and senescence and exhaustion (PD-1, CD95, CD28, and CD57). Nine presented persistent GBV-C infection and were selected for group 1 (HIV- 1/GBV-C+), mean age of 31.6 years. Another set of eight patients without GBV-C viremia were selected as controls and included in group 2 (HIV-1/GBV-C-), mean age of 31.7 years. All participants were male, in most cases men who have sex with men (MSM). At baseline visit (V1), no variable (levels of CD4+ and CD8+ lymphocytes, viral load, CD4/CD8 ratio, and cellular activation, senescence, and exhaustion markers) presented no statistical significant differences, suggesting that all selected patients shared similar characteristics. Longitudinal analysis (delta, Vn-V1) revealed a nonsignificant decrease in activation and senescence markers for both groups. However, it was observed a decrease in naïve CD4+ and CD8+ T cells in group 1, and also a reduction in the subpopulations of naïve and central memory (CD28+) CD8+ T cells. The HIV+/GBV-C+ group also presented diminished intermediate memory and terminal effector subpopulations, as well a decrease in HLA-DR+ terminal effector cells. The data demonstrate that GBV-C infection results in reduced immune stimulation, cellular senescence, and cell exhaustion, suggesting an involvement in the modulation of HIV progression

Analisar a ocorrência de coinfecção por TB/HIV/aids, os desfechos internação e mortalidade e seus fatores de risco nos casos notificados por TB/HIV/aids, no município de Poro Alegre. Foram estudados casos de coinfecção no período de 2009 a 2013. Realizou-se um estudo de coorte retrospectivo, utilizando-se como fonte de informação as bases de dados do Sistema Nacional de Agravos de Notificação, Sistema de Informação Hospitalar e Sistema de Informação sobre Mortalidade. Foram analisadas as seguintes variáveis ano, idade, escolaridade, raça/cor, Gerência Distrital, situação de entrada e encerramento, indicação e realização de tratamento supervisionado e agravos que poderiam estar associadas aos desfechos internação e mortalidade por coinfecção TB/HIV/aids. Utilizou-se análise de regressão logística uni e multivariada para estimar as medidas de associação, considerando-se o nível de significância de 5%. Modelo de regressão de Poisson foi utilizado como variável resposta para o desfecho por número de internações e número de mortes, considerando o ano como variável explicativa. Foram encontrados 2419 casos de coinfecção por TB/HIV/aids com taxa média de prevalência, mortalidade e internação de 34,10/100.000, 9,36/100.000, 53,83/100.000 habitantes, respectivamente. Entre as gerências distritais, a Partenon e Lomba do Pinheiro é a que concentra as taxas mais elevadas de prevalência, internação e mortalidade. Os casos de coinfecção eram na maioria do sexo masculino, com baixa escolaridade e predomínio na população não branca. Permaneceram no modelo multivariável para o desfecho internação as variáveis: escolaridade (p<0,001), cuja menor escolaridade apresentou o maior risco; entrada (p= 0,021) cujo reingresso após abandono apresentou 1,30 vezes mais risco de internação; encerramento (p<0,001), cujas categorias abandono, óbito e Tuberculose Multirresistente apresentaram associação positiva com o desfecho, juntamente com a variável gerência distrital (p<0,001) com maior risco para duas gerências. Permaneceram no modelo multivariável para o desfecho mortalidade as variáveis: idade (p<0,001), indicação Tratamento Supervisionado (p<0,001), escolaridade (p=0,001), cuja categoria até 7 anos apresentou um risco de 2,80 de ocorrência do óbito; e entrada (p= 0,010), cuja categoria reingresso após abandono apresentou um risco de 1,41 vezes maior do que a categoria cura para mortalidade. A regressão de Poisson demonstrou tendência no aumento da ocorrência de internação de 11,9% a cada ano. A identificação de fatores preditores à internação e óbito pode aumentar o nível de alerta das equipes ao tratarem indivíduos coinfectados por TB/HIV/aids, visando diminuir a ocorrência destes desfechos. A taxa de internação também parece ser um indicador para acompanhar a morbimortalidade e direcionar as ações dos profissionais e serviços de saúde, visando melhorar a adesão ao tratamento, evitando assim a ocorrência da tanto de internação quanto do óbito. / To analyze the occurrence of co-infection TB/HIV/aids the outcomes of admission and mortality and their risk factors in the notified TB/HIV/aids cases in the municipality of Porto Alegre. The study comprised co-infection cases dated from 2009 to 2013. A study of retrospective cohort was made by utilizing as information source the National System of Notifiable Diseases, Hospital Information System and Mortality Information System data bases. The following variables have been analyzed: year, age, education, race/color, District Management, entry and closing situation, Supervised Treatment indication and performance and damages that could be associated to the outcomes of admission and mortality due to TB/HIV/aids coinfection. The univariate and multivariate logistic regression analysis have been applied in order to estimate the association measures, by considering the significance level of 5%. The model of Poisson regression has been utilized as the variable response for the outcome per number of admissions and number of deaths, by considering the year as an explicative variable. A total of 2,419 TB/HIV/aids cases were found with mean rate of prevalence, mortality, admission of 34,10/100.000, 9,36/100.000, 53,83/100.000 inhabitants respectively. Among the district, the Partenon e Lomba do Pinheiro is the one that concentrates the highest rates of prevalence, admission and mortality. The coinfection cases were mostly of masculine sex with low education level and predominance within the non-white population. The following variables remained in the multivariable model for the admission outcome: education (p<0,001) whose lowest school level presented the highest risk; entry (p= 0,021) whose reentrance after abandonment presented 1.30 times higher risk of admission; closing (p<0,001) whose categories abandonment, death and Multiresistant Tuberculosis showed positive connection with the outcome together with the district variable (p<0,001) with higher risk for two managements. The following variables remained in the multivariable model for the mortality outcome: age (p<0,001), Supervised Treatment indication (p<0,001), education level (p=0,001) whose category up to 7 years showed a risk of 2.80 occurrence of death; and, entry (p= 0,010) whose category re-entrance after abandonment showed a risk 1.41 times higher than the category cure for mortality. The regression of Poisson demonstrated a trend in the increase of the admission occurrence at the rate of 11.9% each year. The identification of factors that predict admission and death may increase the alert level of the staff upon taking care of subjects coinfected by TB/HIV/aids aiming at diminishing the occurrence of these outcomes. The admission rate also seems to be an indicator to follow up morbimortality and to guide staff actions and health services with the purpose of improving the adhesion to the treatment and thus avoiding the occurrence of admission and death as well. / Analizar la ocurrencia de coinfección por TB/HIV/aids, los desenlaces internación y mortalidad y sus factores de riesgo en los casos notificados por TB/HIV/aids, en la municipalidad de Porto Alegre. Se han estudiado casos de coinfección en el período de 2009 a 2013. Se realizó un estudio de cohorte retrospectivo, utilizándose como fuente de información las bases de datos del Sistema Nacional de enfermedades de declaración obligatoria, sistema de información hospitalaria y Sistema de Información de Mortalidad. Se analizaron las siguientes variables: año, edad, escolaridad, raza/color, distrito, situación de entrada y cierre, indicación y realización de Tratamiento Supervisado y agravios que podrían estar asociadas a los desenlaces internación y mortalidad por coinfección TB/HIV/aids. Se utilizó el análisis de regresión logística uni y multivariante para estimar las medidas de asociación, considerándose el nivel de significancia de 5%. El modelo de regresión de Poisson se ha utilizado como variable de respuesta para el desenlace por número de internaciones y número de muertes, considerando el año como variable explicativa. Se han encontrado 2419 casos de coinfección por TB/HIV/aids con tasa media de prevalencia, mortalidad e internación de 34.10/100.000, 9.36/100.000, 53.83/100.000 habitantes, respectivamente. Entre los distrito, la Partenon e Lomba do Pinheiro es la que concentra las tasas más altas de prevalencia, internación y mortalidad. Los casos de coinfección eran en su mayoría del sexo masculino, con baja escolaridad y predominio en la población no blanca. Permanecieron en el modelo multivariante para el desenlace internación las variables: escolaridad (p<0,001), cuya menor escolaridad presentó el mayor riesgo; entrada (p= 0,021) cuyo reingreso tras abandono presentó 1.30 veces más riesgo de internación; cierre (p<0,001), cuyas categorías abandono, óbito y Tuberculosis Multirresistente presentaron asociación positiva con el desenlace, juntamente con la variable distrito (p<0,001) con mayor riesgo para dos gerencias. Permanecieron en el modelo multivariante para el desenlace mortalidad, las variables: edad (p<0,001), indicación Tratamiento Supervisado (p<0,001), escolaridad (p=0,001), cuya categoría hasta 7 años presentó un riesgo de 2.80 de ocurrencia de óbito; y, entrada (p= 0,010), cuya categoría reingreso tras abandono presentó un riesgo de 1.41 veces mayor que la categoría cura para mortalidad. La regresión de Poisson demostró tendencia en el aumento de la ocurrencia de internación de 11.9% a cada año. La identificación de factores predictores de internación y óbito puede aumentar el nivel de alerta de los equipos al tratar a individuos coinfectados por TB/HIV/aids, buscando disminuir la ocurrencia de estos desenlaces. La tasa de internación también parece ser un indicador para acompañar la morbimortalidad y direccionar las acciones de los profesionales y servicios de salud, con vistas a mejorar la adhesión al tratamiento y, así, evitar la ocurrencia tanto de internación cuanto de óbito.

L'infection chronique ou occulte par le virus de l'hépatite B (HBV) est l'un des facteurs de risque les plusimportants pour le développement de carcinome hépatocellulaire viro-induit. Malgré la petite taille dugénome et les contraintes imposées par l'organisation de ce génome, le HBV présente une grandevariabilité génétique qui est le résultat des erreurs générées lors de l’étape de reverse transcription maisaussi, lors des processus de sélection et d’adaptation. Durant l'infection, une relation étroite hôte-viruss'établit. La population virale est caractérisée par la présence de quasi-espèces pouvant influencerl'évolution de la maladie hépatique et favoriser les phénomènes d'échappement. Le but de notre travail aété 1) d'étudier la variabilité virale associée aux infections occultes et l'impact de certaines modificationsde l'AgHBs (sY100C) sur la reconnaissance des tests de détection enzymatiques, et l'impact des mutations(sK122R, sD144E) sur la reconnaissance des anticorps anti-HBs d'un patient 2) de développer un nouveloutil d'amplification de génomes entiers de HBV que nous avons appliqué à l'étude préliminaire de quasiespècesd'un mutant Core. Cet outil d'amplification est plus sensible que les techniques existantes, et ilpermet l'étude des propriétés moléculaires et fonctionnelles des isolats virales. En conclusion, nous avonspu montrer que les processus d'échappement sont complexes, et font intervenir des facteurs du virus et del'hôte. Ce travail contribue à l'amélioration de la compréhension de la biologie de l'infection par le HBV,et illustre la complexité des interactions hôte-virus. / Pas de résumé en anglais / A infecção crônica e a infecção oculta pelo vírus da hepatite B (HBV) representam fatores de riscoconhecidos para o desenvolvimento de carcinoma hepatocelular. Apesar do pequeno tamanho do genomae as restrições impostas pela organização do genoma, o HBV tem uma alta variabilidade genética que é oresultado dos erros gerados durante a etapa de transcrição reversa e também durante o processo de seleçãoe adaptação do vírus. Durante a infecção uma relação de proximidade se estabelece entre o vírus e ohospedeiro. A população viral é caracterizada pela presença de quasispecies que poderiam influenciar aevolução da doença hepática, e também promover a seleção de variantes de escape imunológico. Osobjetivos do nosso estudo foram 1) estudar a variabilidade do HBV associada às infecções ocultas e oimpacto de certas modificações do HBsAg (sY100C) no reconhecimento de testes de detecçãoenzimáticos. Estudar também o impacto das mutações (sK122R, sD144E) sobre o reconhecimento deanticorpos anti-HBs de um paciente, 2) desenvolver um novo método para amplificar o genoma completodo HBV que foi aplicado a um estudo para identificação de quasispecies de um mutante da região do coreviral. Este método de amplificação é mais sensível do que as técnicas existentes e permite o estudo depropriedades moleculares e funcionais dos virus. Em conclusão, mostramos que os processos de escapeviral são complexas e envolvem fatores relacionados tanto com o vírus como com o hospedeiro. Estetrabalho contribui de maneira significativa para a compreensão da biologia da infecção pelo HBV eilustra a complexidade das interações vírus-hospedeiro.