Introdução: O gênero Acinetobacter representa um importante patógeno relacionado a infecções hospitalares, principalmente, à pneumonia associada à ventilação mecânica (PAV). O aumento de isolados de Acinetobacter baumannii resistentes aos carbapenêmicos (ABRC) representa um problema mundial, pois limita drasticamente as opções terapêuticas. A produção da carbapenamase OXA-23, uma β-lactamase da classe D de Ambler, representa o principal mecanismo responsável por esta resistência em nosso país. Objetivo: O presente estudo tem como objetivo padronizar a técnica de PCR em tempo real (qPCR) para a detecção do gene blaOXA-23 diretamente de aspirados traqueais (ATs) de pacientes com suspeita de pneumonia associada à ventilação mecânica (PAV) de unidades de tratamento intensivo (UTIs). Métodos e resultados: O DNA das amostras de ATs coletadas de pacientes em ventilação mecânica foi analisado pela técnica de qPCR, utilizando o SYBR green, para a detecção do gene blaOXA-23. Dentre os 20 ATs analisados, ABRC foi isolado em 10, A. baumannii sensível aos carbapemêmicos (ABSC) em 3 e 7 foram negativos na culura bacteriológica. O gene blaOXA-23 foi detectado tanto na colônia quanto no AT em 8 das 10 amostras de ABRC. Em uma amostra não houve detecção do gene por qPCR em nenhum dos materiais e em outra amostra houve detecção só no AT. Dos ABSC, em duas amostras não houve detecção do gene na colônia e no AT enquanto que em uma amostra o gene foi detectado somente no AT. Em nenhuma das amostras de ATs negativas na cultura foi detectado o gene. Conclusão: O estudo sugere que a técnica qPCR pode ser aplicada para a detecção do gene blaoxa-23 diretamente de amostras de AT, reduzindo assim, o tempo para o início de uma terpia antimicrobiana adequada e melhorando, consequentemente, o desfecho clínico deste pacientes. / Background: The genus Acinetobacter is an important pathogen associated with nosocomial infections mainly ventilator-associated pneumonia (VAP). The increasing of carbapenemresistant Acinetobacter baumannii (CRAB) isolates is a worldwide concern since it limits drastically the range of therapeutic alternatives. The OXA-23 producing, a carbapenemhydrolysing class D β-lactamase, is the major mechanism responsible for CRAB in our country. Objective: The study objective is to develop a real time PCR (qPCR) to detect the Acinetobacter baumannii blaOXA-23 gene directly in endotracheal aspirate (ETA) of patients under mechanical ventilation, with suspected ventilator-associated pneumonia (VAP). Methods and Results: DNA extracted from ETA samples from patients under mechanical ventilation was analyzed by qPCR, using SYBR green, for the presence of blaOXA-23 gene. Among the 20 ETAs examined, CRAB isolates were recovered in 10 quantitative cultures; carbapenem-susceptible A. baumannii (CSAB) in 3 and 7 cultures were negative to A. baumanni. Of the 10 CRAB, 8 were positive for blaOXA-23 on both the colony and ETA. In one blaOXA-23 qPCR was negative in colony and directly from ETA, while the other showed a qPCR negative result in the colony and positive in the ETA. In 3 CSAB, 2 samples showed negative results in colony and ETA and one showed a blaOXA-23 positive result only from the ETA. None of the 7 negative ETAs were positive for blaOXA-23 gene in the qPCR of the ETA. Conclusion: Our study suggests that qPCR can be applied to detect the presence of blaOXA-23 gene directly from ETAs reducing the time to an earlier initiation of appropriate therapy improving, consequently, the clinical outcomes for these patients.

Orientador: Maria Marluce dos Santos Vilela / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T20:47:23Z (GMT). No. of bitstreams: 1 Toro_AdyleiaAparecidaDalboContrera_D.pdf: 3459466 bytes, checksum: a2a86bc660c9851dab70414f47dc5420 (MD5) Previous issue date: 2008 / Resumo: INTRODUÇÃO: A infecção pelo HIV desencadeia diversificadas e extensas alterações no mecanismo de defesa do pulmão, o que se traduz em maior número de infecções por germes habituais ou oportunistas e processos imunológicos diversos como a pneumonia intersticial linfocítica (PIL), a hiperplasia linfóide (HLP) ou neoplasias. A infecção simultânea do HIV e do EBV pode aumentar o risco de HLP/ PIL. OBS.: O resumo na integra poderá ser visualizado no link ou texto completo da tese digital / Abstract: BACKGROUND: Pulmonary Lymphoid Hyperplasia (PLH) / Lymphoid Interstitial Pneumonitis (LIP) complex is common in HIV infected children. It may reflect a particular response to HIV from a developing immune system, and is also related to exposure to Epstein-Barr virus (EBV). Note: the complete abstract is avaiable with the link or full eletronic digital theses or dissertations / Doutorado / Pediatria / Doutor em Saude da Criança e do Adolescente

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-04-20T19:18:23Z No. of bitstreams: 2 Tese - Eliane Terezinha Afonso - 2015.pdf: 3480407 bytes, checksum: db146d8884a4aedac166a73ef268d89d (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-04-25T15:00:31Z (GMT) No. of bitstreams: 2 Tese - Eliane Terezinha Afonso - 2015.pdf: 3480407 bytes, checksum: db146d8884a4aedac166a73ef268d89d (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Made available in DSpace on 2016-04-25T15:00:31Z (GMT). No. of bitstreams: 2 Tese - Eliane Terezinha Afonso - 2015.pdf: 3480407 bytes, checksum: db146d8884a4aedac166a73ef268d89d (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) Previous issue date: 2015-08-19 / BACKGROUND: Pneumonia causes substantial morbidity and mortality in all age groups around the world. The 10-valent pneumococcal conjugate vaccine (PCV10) was introduced into the routine infant immunization in Brazil, free of charge, in March 2010. The aim of this study was to evaluate the impact PCV10 vaccination on rates of all cause pneumonia hospitalizations one year and three years after its introduction in Brazil. METHODS: We conducted two interrupted time series analysis studies. The first evaluated only the direct effect of PCV10 vaccination, in five Brazilian cities (Belo Horizonte, Curitiba, Porto Alegre, São Paulo and Recife), and was conducted one year after starting the vaccination. The second study evaluated the direct and indirect impact (individuals not vaccinated) of PCV10 vaccination in Brazil, and was conducted three years after vaccination. We used data from the Brazilian Hospitalization System from 2005-2013. The main outcome was monthly rates of all-cause pneumonia hospitalizations identified by ICD-10 codes J12-J18. We used hospitalization rates for congenital malformations and non-respiratory causes as a comparison groups. The time-series analysis was based on a generalized linear model. Pneumonia rates observed in the pre-vaccination period were used to estimate the hospitalization rates in the post-vaccination period of each study, adjusting for seasonality and secular trends. To estimate the direct (2-23 months of age) and indirect (≥5 years of age) impact of PCV10 vaccination, we calculated the percentage change in hospitalization rates, as the observed divided by the predicted rates of hospitalization in the post-intervention period minus one, with respective 95% CI and p values. The number of all-cause pneumonia hospitalizations averted by vaccination was calculated taking into account the difference between the predicted and observed number in the PCV10 post vaccination period. RESULTS: One year after introduction of PCV10 in Brazil, significant declines in hospitalizations for pneumonia in children aged 2-23 months were noted in Belo Horizonte (28.7%), Curitiba (23.3%), and Recife (27.4%). After three years of the introduction of PCV10, 461,519 pneumonia hospitalizations were averted in Brazil, and a significant decrease in rates of pneumonia hospitalization was observed in unvaccinated individuals aged 5-39 years, ranging from 14.1-17.4% (p<0.05). In contrast, an increased trend in pneumonia hospitalizations (p=0·004) was observed for elderly (≥ 65 years). CONCLUSION: Vaccination with PCV10 in Brazil was associated with reduction of pneumonia hospitalizations in vaccinated individuals. Herd effect was observed in individuals aged 5-39 years after three years of vaccination. Potential reasons for the increased trend in pneumonia hospitalization rates in the elderly should be investigated. / INTRODUÇÃO: As pneumonias contribuem com alta carga de morbimortalidades em todo mundo. No Brasil, a vacina pneumocócica conjugada 10 valente (PCV10) foi introduzida na rotina de imunização da infância em março de 2010. Este estudo teve como objetivo avaliar o impacto da vacinação nas taxas de hospitalizações por pneumonia no Brasil no curto e médio prazo do início da vacinação. METODOLOGIA: Dois estudos de séries temporais interrompidas foram conduzidos. O primeiro avaliou o efeito direto da vacinação em cinco capitais brasileiras (Belo Horizonte, Curitiba, Porto Alegre, São Paulo e Recife) e foi conduzido após um ano de introdução da PCV10 no país. O segundo estudo avaliou o impacto direto e indireto (população não vacinada) da vacinação em todo país e foi conduzido três anos após sua introdução. Os dados de hospitalizações foram obtidos do Sistema de Informações Hospitalares (SIH-SUS) de 2005 a 2013. O desfecho principal foi a taxa mensal de hospitalização por pneumonia definida pelos códigos J12-J18 da CID10. As taxas de hospitalizações por malformações congênitas e causas não respiratórias foram utilizadas como grupos de comparações. A análise de série temporal utilizou um modelo de regressão linear generalizado. As taxas de hospitalizações por pneumonia observadas no período pré-PCV10, ajustadas por tendência secular e sazonalidade, foram utilizadas para estimar as taxas no período pós-PCV10. O impacto da vacinação para cada faixa etária foi calculado como o percentual de mudança nas taxas de hospitalizações, dividindo-se as taxas observadas pelas taxas preditas do período pós PCV10, menos um. Os respectivos IC95% e os valores de p foram apresentados. O número de hospitalizações por pneumonia evitadas após três anos de vacinação foi estimado pela diferença entre os números de hospitalizações por pneumonia preditos e observados no período pós-vacinação. RESULTADOS: Após um ano de introdução da PCV10 no Brasil, observou-se significativo declínio nas taxas de hospitalizações por pneumonia em crianças de 2 a 23 meses em três das cinco capitais estudadas: Belo Horizonte (28,7%), Curitiba (23,3%), e Recife (27,4%). Após três anos da introdução da PCV10, 461.519 hospitalizações por pneumonia foram evitadas no Brasil e um significativo declínio nas taxas de pneumonia foi observado em indivíduos não vacinados de 5 a 39 anos variando de 14,1% a 17,4% (p<0,05). No entanto, observou-se um aumento significativo (9,9%, p=0,004) nas taxas de hospitalizações por pneumonia para idosos ≥65 anos. CONCLUSÕES: A vacinação com a PCV10 foi associada à significativa redução das hospitalizações por pneumonia na infância. Adicionalmente, o estudo evidenciou importante redução das hospitalizações por pneumonia em grupos etários não vacinados, sinalizando efeito indireto conferida pela vacina. A tendência de aumento das hospitalizações por pneumonias em idosos necessita de investigações para elucidação dos fatores envolvidos nesse fenômeno.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-06-21T13:42:52Z No. of bitstreams: 2 Tese - Sheila Elke Araújo Nunes - 2014.pdf: 4139252 bytes, checksum: 113368e043fc53148e1cd7b8b50b2632 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-07-10T13:36:29Z (GMT) No. of bitstreams: 2 Tese - Sheila Elke Araújo Nunes - 2014.pdf: 4139252 bytes, checksum: 113368e043fc53148e1cd7b8b50b2632 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-07-10T13:36:29Z (GMT). No. of bitstreams: 2 Tese - Sheila Elke Araújo Nunes - 2014.pdf: 4139252 bytes, checksum: 113368e043fc53148e1cd7b8b50b2632 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-12-18 / Introduction: Estimate the costs of treatment of pneumococcal diseases can aid the understanding of reduced economic burden of these after introduction of the pneumococcal conjugate vaccine (PCV), as run in Brazil, in March 2010, which introduced the PCV10 valiant in the National Program Immunization (NPI) for children between 2 and 23 months of age. Cost-effectiveness analysis (CEA) before the introduction indicated that the vaccine was cost-effective (R $ 24.930 / Daly avoided - Disability Adjusted Life Years), in the SUS perspective. Disease burden and the cost of the vaccine were identified as the main drivers of the results for sensitivity analysis. Objectives: Estimate the costs of pneumococcal disease and to evaluate the ratio of incremental cost-effectiveness (ICER) of implementing the PCV-10 brave after introduction into INP Brazil. Methods: Three steps have been performed in the SUS perspective: 1) cost of illness study: medical charts of children 28 days to 35 months of age hospitalized with clinical suspicion of bacterial pneumonia were reviewed to estimate the costs of pneumonia and to other syndromes costs were estimated by therapeutic guidelines; 2) comparison between the three methods of funding: (i) bottom-up / micro-costing by chart review; (ii) top-down / micro-costing through therapeutic guidelines; and (iii) top-down / gross-costing, through reimbursement paid by the SUS. 3) CEA: the strategy to vaccinate with PCV-10 was compared to the non-vaccination. The model used was the PneuModel. In acute otitis media from all causes, pneumococcal meningitis, pneumococcal sepsis and pneumococcal pneumonia were considered. Costs were obtained by microcusteio, epidemiological data from primary studies of population-based, dose costs and vaccination coverage in INP. The discount rate was 5%. Sensitivity analysis was conducted to test the robustness and variability of the model parameters. Results: The cost of study of hospitalized pneumonia records of 52 cases of severe pneumonia and 7 of very serious pneumonia were reviewed. Statistical analyzes of severe pneumonia data revealed that there is difference between the costing methodologies (p=0,015) and to compare the estimated costs by these methods there was no difference between the cost of compensation and the cost for therapeutic guideline (p=0,241). At ACE, annually, vaccination with PCV-10 would prevent 3,942 cases of the disease and 16,514 years of life lost in a cohort of children <1 year. The ICER was R $ 14,230 per DALY averted. In sensitivity analysis, the model was sensitive to variations in incidence and mortality of pneumonia and pneumococcal meningitis. Conclusions: The cost for therapy guideline, uncommonly used in disease cost estimates, was an alternative to funding for compensation, heavily used technique and lower accuracy. After introduction of ICER, using primary data revealed that PCV-10 is a low-cost intervention, as suggested by WHO (<1GDP / per capita - in Brazil, in 2010, US $ 10.933) and, ICER less than previous ACE. Despite uncertainties in critical parameters of the model, using secondary data, ACE can provide evidence to support decision making. After the implementation analysis can result in more accurate estimates and provide evidence to continue vaccination. / Introdução: Estimar os custos do tratamento das doenças pneumocócicas podem auxiliar no conhecimento da redução da carga econômica destas após introdução da vacina anti-pneumocócica conjugada (VPC), como corrido no Brasil, em março de 2010, que introduziu a VPC-10 valente no Programa Nacional de Imunização (PNI), para crianças entre 2 e 23 meses de idade. Análise de custo-efetividade (ACE) antes da introdução indicou que a vacina era custo-efetiva (R$ 24,930/Daly evitado – do inglês, Disability Adjusted Life Years), na perspectiva do SUS. Carga da doença e os custos da vacina foram identificados como os principais direcionadores do resultado para análise de sensibilidade. Objetivos: Estimar os custos da doença pneumocócica e avaliar a razão de custo-efetividade incremental (RCEI) da implementação da VPC-10 valente após introdução no PNI do Brasil. Métodos: Três etapas foram executadas, aplicadas a perspectiva do SUS: 1º) estudo de custo de doenças: prontuários de crianças com 28 dias a 35 meses de idade internadas por suspeita clínica de pneumonia bacteriana foram revisados para estimar os custos da pneumonia e para demais síndromes os custos foram estimados por diretrizes terapêuticas; 2º) comparação entre as três metodologias de custeio: (i) bottom-up/micro-costing através da revisão de prontuários; (ii) top-down/micro-costing através de diretriz terapêutica; e (iii) top-down/gross-costing através de ressarcimento pago pelo SUS. 3º) ACE: a estratégia de vacinar com a VPC-10 foi comparada com a não vacinação. O modelo empregado foi o PneuModel. Neste, otite média aguda por todas as causas, meningite pneumocócica, sepse pneumocócica e pneumonia pneumocócica foram consideradas. Os custos foram obtidos por microcusteio, dados epidemiológicos a partir de estudos primários de base populacional, custos da dose e de cobertura vacinal no PNI. A taxa de desconto aplicada foi de 5%. Análise de sensibilidade foi conduzida para testar a robustez e variabilidade de parâmetros do modelo. Resultados: No estudo de custo da pneumonia hospitalizada prontuários de 52 casos de pneumonias graves e 7 de pneumonias muito graves foram revisados. Análises estatísticas dos dados de pneumonias graves revelaram que há diferença entre as metodologias de custeio (p=0,015) e ao comparar os custos estimados por estas metodologias não houve diferença entre o custeio por ressarcimento e o custeio por diretriz terapêutica (p=0,241). Na ACE, anualmente, a vacinação com VPC-10 evitaria 3.942 casos da doença e 16.514 anos de vida perdidos em uma coorte de crianças <1 ano. A RCEI foi de R$ 14.230 por DALY evitado. Na análise de sensibilidade, o modelo foi sensível às variações de incidência e letalidade de pneumonia e meningite pneumocócica. Conclusões: O custeio por diretriz terapêutica, pouco empregado nas estimativas de custo de doença, se mostrou uma alternativa ao custeio por ressarcimento, técnica muito utilizada e de menor acurácia. A RCEI pós introdução, com dados primários, revelou que a VPC-10 é uma intervenção de baixo custo, como sugerido pela OMS (<1PIB/per capita – no Brasil, em 2010, R$ 10,933) e, com menor RCEI que ACE anterior. Mesmo com incertezas em parâmetros críticos do modelo, usando dados secundários, ACE podem fornecer evidências para apoiar tomadas de decisões. Analise pós-introdução pode resultar em estimativas mais precisas e fornecer evidências para continuar a vacinação.

Made available in DSpace on 2014-08-20T13:32:49Z (GMT). No. of bitstreams: 1 dissertacao_andressa_fisch.pdf: 708004 bytes, checksum: 48d7e7a2a394dafd06e3209289b60dde (MD5) Previous issue date: 2014-02-19 / Enzootic Pneumoniae (EP), caused by Mycoplasma hyopneumoniae, generates significant economic losses to the pig industry worldwide. The currently available vaccines confer partial protection against the EP and the development of new vaccine strategies have proven necessary. In this paper, humoral immune response in mice of six comercial bacterins against sexteen recombinant antigens previously characterized of M. hyopneumoniae was evaluated by indirect ELISA. Seroconversion Seroconversion was used to determine biologically significant reactivity. The antigen MHP_0067 was recognized by sera from one inoculated group. The antigens MHP_0513 and MHP_0580 were recognized by sera from four inoculated groups each. For other antigens, not seroconversion was detected. Identify potentially protective antigens underexpressed in these vaccines and associate them with conventional vaccination may promote increased levels of protection. These antigens are potential targets for the composition of a recombinant subunit vaccine associated with the commercial vaccine. / A Pneumonia Enzoótica Suína (PES), causada pela bactéria Mycoplasma hyopneumoniae, gera importantes perdas econômicas para a indústria suinícola em todo o mundo. As vacinas atualmente disponíveis diminuem as perdas produtivas, mas não impedem a infecção dos rebanhos. O desenvolvimento de novas estratégias vacinais têm se mostrado necessário. Neste trabalho, avalIou-se a resposta imune humoral induzida pela inoculação, em camundongos, de seis bacterinas comerciais contra quinze fatores de patogenicidade de M. hyopneumoniae previamente caracterizados por nosso grupo. A detecção de anticorpos foi realizada através de ELISA indireto. Soroconversão foi utilizada para determinar reatividade biologicamente significativa. O antígeno MHP_0067 foi reconhecido pelo soro de um único grupo inoculado. Os antígenos MHP_0513 e MHP_0580 foram reconhecidos pelos soros de quatro grupos inoculados cada. Para os demais antígenos não houve soroconversão pelos grupos inoculados com as bacterinas comerciais. Identificar antígenos potencialmente protetores ausentes nestas vacinas e associá-los à vacinação convencional pode promover o aumento dos níveis de proteção. Estes antígenos são potenciais alvos para a composição de uma vacina de subunidade recombinante associada à vacina comercial.

Made available in DSpace on 2014-08-20T13:32:53Z (GMT). No. of bitstreams: 1 dissertacao_silvana_marchioro.pdf: 699240 bytes, checksum: 32b6c311c57b68941cfdd0ee832643a1 (MD5) Previous issue date: 2008-11-27 / Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), one the most common respiratory diseases of pigs worldwide. The disease is characterized by retarded growth rate in animals of mid-finishing to slaughter age, causing significant economic losses to swine production. Vaccination is the most cost-effective strategy for the control of this disease. However, the vaccines against the disease available on the market have a high production cost and only offer partial protection, not preventing the establishment from infection and the presence of pigs carriers. This issue highlights the need for the use of new strategies aiming at circumventing this problem. The recombinant DNA technology is an important tool for the development of vaccines. The publication of the sequence of three genomes of M. hyopneumoniae, two pathogenic strains (232 and 7448) and a non-pathogenic (J) is helping the researches, and accelerating the process of identification of new antigenic proteins. From the sequence data and proteomic analysis, it was possible to identify coding sequences (CDS) of transmembrane proteins. These antigens were cloned and expressed using E. coli as an expression system. A total of seven recombinant proteins were purified and evaluated regarding immunogenicity in mice and antigenicity with sera from animals with EP. All the antigens evaluated were able to induce a significant immune response in mice. The evaluation of antigenicity properties revealed the proteins reacted with sera from pigs infected with M. hyopneumoniae. The recombinant proteins evaluated in this study may be of value to use in the development of vaccines and tests for immunodiagnosis of swine enzootic pneumonia. / Mycoplasma hyopneumoniae é o agente etiológico da pneumonia enzoótica suína (PES), uma das doenças respiratórias de suíno mais comum em todo o mundo. Ela é caracterizada por retardo no desenvolvimento em animais na fase de crescimento e terminação, causando perdas econômicas consideráveis na indústria suinícola. A vacinação parece ser a forma mais efetiva de controlar a PES, entretanto, as vacinas contra a doença disponíveis no mercado apresentam um elevado custo de produção e conferem uma proteção parcial, não impedindo o estabelecimento da infecção e nem a presença de suínos portadores. Desta forma, surge à necessidade da utilização de novas estratégias visando contornar este problema. A tecnologia do DNA recombinante constitui uma ferramenta importante para o desenvolvimento de vacinas. As publicações das seqüências de três genomas de M. hyopneumoniae, duas cepas patogênicas (232 e 7448) e uma não patogênica (J) estão contribuindo com pesquisas e, acelerando o processo de identificação de novas proteínas imunogênicas. A partir dos dados gerados e da análise proteômica complementar, foi possível a identificação de seqüências codificadoras (CDS) de proteínas transmembrana. Estas seqüências foram clonadas e expressas em E. coli. Um total de sete proteínas recombinantes foram purificadas e avaliadas quanto à imunogenicidade em camundongos e à antigenicidade frente a soros de suínos com PES. Todas as proteínas avaliadas foram capazes de induzir uma resposta imune significativa em camundongos. O estudo das propriedades antigênicas revelou que estas são reconhecidas pelo soro de suínos infectados com M. hyopneumoniae. As proteínas recombinantes avaliadas neste estudo poderão ser de valia para utilização como vacinas de subunidade e em testes de imunodiagnóstico da PES.

La grippe porcine est une infection enzootique touchant 50% du cheptel français. Elle passe parfois inaperçue, mais peut également induire une forte morbidité au sein des lots d’animaux touchés, entraînant une baisse des performances zootechniques et des pertes économiques importantes. La sévérité de l’infection à virus influenza A chez le porc peut dépendre de divers facteurs, comme les virus eux-mêmes, les pratiques d’élevage, le statut immunitaire des animaux, les infections concomitantes par d’autres pathogènes respiratoires, etc. De la même manière, diverses formes épidémiologiques de la grippe existent en élevage. Ainsi, des infections peuvent se répéter à un âge déterminé, sur toutes les bandes successives d’un élevage, notamment chez des jeunes présentant une immunité passive. Afin de mieux comprendre cette diversité clinique et épidémiologique de la grippe porcine, et aider à l’élaboration de stratégies d’intervention adéquates pour le contrôle de la maladie, nous avons cherché à apporter de nouvelles connaissances quant à certains facteurs pouvant favoriser l’exacerbation du syndrome grippal et/ou son caractère récurrent, et plus généralement aux mécanismes sous-jacents à la pathogenèse des virus influenza A chez le porc, en relation avec les réponses de l’hôte infecté. Nous avons d’abord comparé, suite à inoculations expérimentales de porcs exempts d’organismes pathogènes spécifiés, la pathogénicité des deux virus influenza porcins les plus fréquemment rencontrés chez le porc en France, l’un du lignage européen « avian-like swine H1N1 » (H1avN1), l’autre du lignage européen « human-like reassortant swine H1N2 » (H1huN2), seuls ou en association avec Mycoplasma hyopneumoniae (Mhp), autre pathogène respiratoire répandu en élevage. Nous avons montré que l’infection H1huN2 induit une pathologie plus marquée que l’infection H1avN1, et que la pré-infection des porcs par Mhp induit une exacerbation de l’infection H1avN1, mais pas H1huN2. Nous avons utilisé le modèle de co-infection Mhp/H1avN1 pour évaluer deux approches alternatives qui permettraient de diminuer l’impact des infections grippales et de leurs complications : l’apport de composés aux propriétés antioxydantes via l’alimentation ; et la restriction alimentaire de courte durée. Dans ces deux cas nous avons montré des effets bénéfiques sur les paramètres zootechniques pendant les jours suivant l’infection grippale. Ce travail a également apporté de nouvelles connaissances quant aux modifications des marqueurs plasmatiques de stress oxydant, ainsi que sur les modifications métaboliques faisant suite à la co-infection Mhp/H1avN1. La sévérité des manifestations cliniques de la grippe étant liée à la qualité de la réponse immunitaire mise en place chez l’hôte infecté, nous avons entrepris d‘étudier les réponses immunitaires du porc touché par la grippe et d’évaluer l’impact de facteurs tels que la présence de Mhp ou d’anticorps d’origine maternelle sur ces réponses. Nous avons ainsi montré que l’infection virale induit une inflammation et une réponse interféron. La pré-infection par Mhp exercerait un effet additif sur cette inflammation et pourrait favoriser la persistance du virus dans le poumon. Nous avons également montré que la présence d’immunité passive protège cliniquement le porcelet mais n’empêche pas l’excrétion du virus, retarde la réponse lymphocytaire T et inhibe la réponse humorale post-infectieuse. Malgré la réponse immunitaire humorale défaillante, les animaux étaient totalement protégés d’une seconde infection homologue lorsque les anticorps maternels avaient disparus. Ces travaux ont permis d’apporter de nouvelles connaissances sur les facteurs influençant l’infection grippale en élevage porcin ainsi que sur les mécanismes sous-jacents, ce qui est une prérequis pour l’amélioration des mesures de lutte et de maitrise de la maladie. Ils permettent, d’envisager d’améliorer la santé des animaux en agissant sur leur régime alimentaire. / Swine influenza is an enzootic infection affecting 50% of the French livestock. The infection can be unnoticed but can also induce high morbidity among batches of affected animals, resulting in lower production performance and significant economic losses. The severity of influenza A virus in pig is influenced by many factors such as the virus strain, husbandry practices, the immune status of animals, concomitant infections with other respiratory pathogens, etc. In the same way, various epidemiological forms of influenza exist in farms. Thus, infections can be repeated in all successive batches within a farm, especially among young animals with passive immunity. In order to better understand the clinical and epidemiological diversity of the swine flu, and help develop appropriate strategies to control the disease, we tried to bring new knowledge about factors that promote the exacerbation of the flu syndrome and/or its recurrence, and more generally to give new information about the mechanisms underlying the pathogenesis of influenza viruses in pigs, in relation to the response of the infected host. Firstly, we compared, through experimental infections of specific pathogen free pigs, the pathogenicity of the two swine influenza viruses mostly detected in pigs in France, i.e. one from the European “avian-like swine H1N1” lineage (H1avN1) and the other one from the European “human-like reassortant swine H1N2” lineage (H1huN2), each one alone or in co-infection with Mycoplasma hyopneumoniae (Mhp), another respiratory pathogen widespread in French farms. We showed that the H1huN2 infection induced a more marked pathology than the H1avN1 infection, and that Mhp pre-infection induced the exacerbation of the H1avN1, but not the H1huN2, infection. Then, we used the Mhp/H1avN1 co-infection model to evaluate alternative approaches that could reduce the impact of influenza infections and their complications: firstly, a supply of compounds with antioxidant properties in food; and secondly, a feed restriction of short duration. In both cases, we showed beneficial effects on zootechnical parameters the days following influenza infection. This work has also brought new knowledge on modulation of oxidative stress markers in plasma, as well as metabolic changes following the co-infection with Mhp and H1avN1 in pigs. The severity of flu clinical manifestations being related, among other, to the quality of the immune responses developed by the infected host, we studied these responses in pigs experimentally infected by H1avN1 and assessed the impact of factors such as the presence of Mhp or maternal derived antibodies on these responses. We showed that the viral infection induced inflammation and interferon response. The Mhp pre-infection exerted an additive effect on inflammation of lung tissue and may promote the virus persistence in the lung. Finally, we have shown that the presence of maternally-derived immunity protected the piglets clinically but did not prevent viral shedding, delayed the T cell response and strongly inhibited the post-infectious humoral response. However, despite the failed humoral immune response, animals were completely protected from a second infection occurring when maternal antibodies had disappeared. Therefore, this work have brought new knowledge on factors influencing influenza infection in pig as well as the underlying mechanisms, which is a prerequisite for improving disease control. They allow, between-other, to consider improving the health and welfare of animals by acting on their diet.

En infectiologie, comme en cancérologie, la médecine personnalisée se développe. Ainsi, les traitements antibiotiques probabilistes cèdent leur place à des traitements adaptés aux pathologies et aux patients. En plus des risques d’échecs liés à une thérapie non adaptée, le traitement probabiliste d’une infection est associé à l’augmentation des résistances acquises chez les bactéries. Cependant, cette orientation nécessite de disposer de tests compagnons, c’est-à-dire des tests diagnostiques sensibles et spécifiques pouvant précocement identifier les bactéries et les marqueurs de résistance à partir des liquides biologiques. A côté des méthodes moléculaires largement développées mais ayant des limites de multiplexage, les techniques protéomiques ont récemment été intégrées dans le diagnostic en infectiologie. Ce travail de thèse a consisté à développer des méthodes de spéctrométrie de masse et à les appliquer à la détection de pathogènes et de marqueurs de résistance. Ce travail s’est focalisé sur trois applications : 1) l’identification, la caractérisation et la quantification précoce de micro-organismes dans des échantillons primaires (aspirats endotrachéaux (AET)) de patients atteints de pneumopathies acquises sous ventilation mécanique (PAVM), 2) la détection d’éléments génétiques de la résistance aux antibiotiques : les intégrons, 3) la détection de phénotypes de résistance aux antibiotiques chez Staphylococcus aureus. / Personalized medicine for infectious diseases or cancer becomes more and more important in modern therapy. Furthermore, probabilistic treatment has been associated with the development of resistant bacteria causing infectious diseases. As a result, probabilistic treatments are replaced by adapted treatment for pathologies and patients. However, this new approach needs available companion diagnosis tests that are sensitive but also specific tests able to provide rapid pathogen and resistance markers identification in biological fluids. Beside molecular methods, widely developed but with multiplex limits, proteomic technics have recently joined the infectious diagnosis. This work consisted in developing mass spectrometry technics for bacteria and resistance marker identifications. This work focused on 3 applications: 1) identification and quantitation of microorganisms in crude samples (endotracheal aspirates (ETA)) from patient suffering of ventilator associated pneumonia (VAP), 2) detection of genetic elements involved in antibiotic resistance : the integrons, 3) detection of antibiotic resistance phenotypes in S. aureus.

Les infections graves entraînent une dysrégulation de la réaction inflammatoire associée à une immunodépression complexe associée à la survenue d’infections nosocomiales. Les mécanismes cellulaires et moléculaires qui régulent ces phénomènes demeurent largement incompris. A l’interface entre système immunitaire inné et adaptatif, les cellules dendritiques et les lymphocytes innés pourraient être impliqués dans l’immunodépression post-infectieuse. Par ailleurs, les récepteurs de type Toll (TLR) déterminent l’amplitude de la réponse inflammatoire initiale, mais leur contribution dans le développement de l’immunodépression post-infectieuse n’a pas été établie. Les objectifs de ce projet de recherche étaient d’investiguer le rôle des cellules dendritiques, des lymphocytes de type innés et des voies de signalisation dépendantes des TLRs dans l’immunodépression induite par le sepsis. Nous avons mené ce programme de recherche en combinant une double approche translationnelle et expérimentale. Nous avons étudié la cinétique des cellules dendritiques circulantes chez le patient septique et montré que leur déplétion était associée à la survenue d’infections nosocomiales. L’analyse des trois souspopulations de lymphocytes T innés (lymphocytes γδ, NKT et MAIT) chez le patient septique a montré que seuls les lymphocytes MAIT présentaient une déplétion associée au sepsis sévère, dont la persistance était également corrélée à la survenue d’infections nosocomiales. Enfin, à l’aide de souris knockout nous avons étudié le rôle de TLR2, TLR4 et TLR5 sur la réponse anti-bactérienne dans un modèle murin de pneumonie secondaire à P. aeruginosa à distance d’un sepsis polymicrobien sublétal. Nous avons montré que les souris déficientes pour TLR2 étaient protégées de l’infection secondaire grâce à une meilleure clairance bactérienne. Ce travail introduit des perspectives nouvelles dans la physiopathologie de l’immunodépression post-infectieuse et suggère des applications thérapeutiques potentielles. / Severe sepsis leads to a dysregulated inflammatory response followed by a complex immunosuppressive state that can favor the emergence of nosocomial infections. The cellular and molecular mechanisms that drive the post-infective immunosuppression remain poorly understood. They may involve immune cells that link innate and adaptive immunity such as dendritic cells or innate-like lymphocytes. Furthermore, Toll-like receptors (TLR) are critical determinants of the inflammatory response but their role to the development of sepsis-induced immune dysfunction are unknown. The aim of this research project was to investigate the role of dendritic cells, innatelike T cells and TLR-dependent signalling pathways in the sepsis-induced immunosuppression process. For this purpose, we combined a translational and experimental approach. We assessed dendritic cells blood count in septic patients and showed that the depletion of dendritic cells was associated with the advent of nosocomial infections. We studied 3 populations of innate-like T-cells (γδ lymphocytes, NKT- and MAIT-cells) in septic patients and demonstrated that only the MAIT-cells presented a significant depletion following severe sepsis, the persistence of which was correlated with the advent of nosocomial infection. Last, using knockout mice, we analyzed the relative contribution of TLR2, TLR4 and TLR5 to the host response in a model of late-onset secondary Pseudomonas aeruginosa pneumonia following a sublethal polymicrobial sepsis. We observed that TLR2 deficient mice were specifically protected against the secondary pneumonia through a better bacterial clearance. Our results provide new insights in the pathophysiology of post-infective immunosuppression and suggest potential therapeutic applications.

Abstract Professional practice in critical care settings is characterized by the application of relevant theories, research and evidence-based guidelines to clinical practice. However, critical care nurses’ knowledge and skills in adhering to evidence-based protocols and guidelines for avoiding ventilator-associated pneumonia are inadequate. The aim of the study was to evaluate critical care nurses’ knowledge and skills in adhering to best-practice endotracheal suctioning recommendations and ventilator bundles, to develop and validate instruments to evaluate the care of mechanically ventilated patients, and to evaluate the effectiveness of continuing education on critical care nurses’ knowledge and skills, with a focus on ventilator bundles. In the first study, a descriptive and cross-sectional correlation study was conducted to evaluate critical care nurses’ (n&#160;=&#160;40) endotracheal suctioning practices in relation to current best-practice recommendations. In the second study, a descriptive design with a literature review was conducted to assess the effectiveness of educational programmes in preventing ventilator-associated pneumonia. In the third study, an instruments validation study was conducted to develop and test the psychometric properties of the Ventilator Bundle Questionnaire (VBQ) and Observation Schedule (VBOS). In the fourth study, the effectiveness of human patient simulation education was evaluated among thirty (n&#160;=&#160;30) critical care nurses who were randomly allocated to intervention and control groups (n&#160;=&#160;15 each). Critical care nurses’ knowledge and skills in adhering to best-practice endotracheal suctioning recommendations and ventilator bundles continues to be inadequate. However, educational programmes were linked to significant improvements in learning and clinical outcomes. The VBQ and VBOS were developed and shown to have acceptable psychometric properties (CVI 0.99–1.0, ICC 0.93–1.0). After human patient simulation education, the mean skill scores of the intervention group increased significantly (pt*g&#160;=&#160;0.02). Educational programmes may have a significant impact on clinical outcomes and thus, patients’ safety and quality of care, through improvements in nurses’ knowledge and skills in adhering to evidence-based guidelines in critical care settings. The VBQ and VBOS can provide an objective method measuring whether evidence-based guidelines are being used in clinical practice. In addition, there was a significant transfer of learned skills to clinical practice following human patient simulation education. / Tiivistelmä Teho-osastoilla ammatillinen erityisosaaminen edellyttää tutkitun tiedon, teorioiden sekä näyttöön perustuvien hoitosuositusten soveltamista kliiniseen käytäntöön. Kuitenkin tehohoitajien tiedot ja taidot noudattaa näyttöön perustuvia hoitokäytäntöjä ja suosituksia hengityslaitehoitoon liittyvän keuhkokuumeen ehkäisyksi ovat olleet puutteellisia. Tutkimuksen tarkoituksena oli arvioida tehohoitajien tietoa ja taitoa noudattaa hyväksi havaittuja hengitysteiden imukäytäntöjä sekä hengityslaitehoitoon liittyviä hoitosarjakäytäntöjä, kehittää ja validoida mittareita hengityslaitehoitoa saavien potilaiden hoidon laadun arvioimiseksi sekä arvioida täydennyskoulutuksen vaikuttavuutta tehohoitajien tietoihin ja taitoihin noudattaa hengityslaitehoitoon liittyviä hoitosarjakäytäntöjä. Ensimmäisessä osatyössä arvioitiin kuvailevan ja korrelatiivisen tutkimusasetelman avulla tehohoitajien (n = 40) alahengitysteiden imukäytäntöjä suhteessa hyväksi havaittuihin toimintakäytäntöihin. Toisessa osatyössä arvioitiin kuvailevan kirjallisuuskatsauksen avulla koulutusinterventioiden vaikuttavuutta hengityslaitehoitoon liittyvän keuhkokuumeen ehkäisyssä. Kolmannessa osatyössä kehitettiin ja testattiin hengityslaitehoitoon liittyvä hoitosarjakysely (VBQ) sekä havainnointimittari (VBOS). Neljännessä osatyössä arvioitiin simulaatiokoulutuksen vaikuttavuutta satunnaistetussa koeasetelmassa interventio- (n&#160;=&#160;15) ja kontrolliryhmän (n&#160;=&#160;15) välillä. Tehohoitajien tiedot ja taidot noudattaa hyväksi havaittuja hengitysteiden imukäytäntöjä sekä hengityslaitehoitoon liittyviä hoitosarjakäytäntöjä olivat edelleen puutteellisia. Kuitenkin koulutusinterventioiden vaikuttavuus kliinisiin hoitotuloksiin sekä oppimistuloksiin oli merkittävä. VBQ- ja VBOS-mittareiden psykometriset ominaisuudet osoittautuivat hyväksyttäviksi (CVI 0,99–1,0, ICC 0,93–1,0).Simulaatiokoulutuksen jälkeen interventioryhmän taidot noudattaa hoitosuosituksia lisääntyivät merkittävästi (pt*g&#160;=&#160;0,02). Koulutusinterventioiden kliininen vaikuttavuus potilasturvallisuuden ja hoidon laadun kehittämisessä voi olla merkittävää, kun hoitajien tietoa ja taitoa noudattaa näyttöön perustuvia hoitosuosituksia lisätään kliinisessä tehohoitotyössä. VBQ- ja VBOS-mittarit voivat tarjota objektiivisen tavan arvioida tutkitun tiedon siirtymistä kliiniseen käytäntöön. Simulaatiokoulutuksen jälkeen opittujen taitojen siirtovaikutus kliiniseen käytäntöön oli merkittävä.