The first study was designed to determine the occurrence of bacteria in dishcloths used in restaurants and bars. Coliforms were isolated from 89% of dishcloths and 70% of tabletops. Escherichia coli was isolated from 54% of dishcloths and 20% of tabletops. The numbers of heterotrophic bacteria (HPC) and coliforms were higher in bars than in restaurants. The levels of HPC found in dishcloths were 25-fold and coliforms were 60-to 120-fold lower than the levels found in dishcloths in previous home studies. The most commonly isolated genera from dishcloths in restaurants and bars differed from those in homes. The numbers of HPC on restaurant tabletops were 45-fold greater after cleaning than prior to cleaning. The mandatory use of sanitizers in restaurants and bars may therefore have reduced contamination levels and caused a shift in the microbial populations present in food service establishments. The second study was designed to determine the recovery efficiency and the survival of two respiratory viruses on produce and was compared to the survival of the enteric poliovirus 1. Adenovirus was recovered with an efficiency of 56%, 32% and 35% from lettuce, strawberries and raspberries, respectively. Coronavirus was recovered from lettuce with an efficiency of 19.6%, but could not be recovered from strawberries. Poliovirus was recovered from lettuce with an efficiency of 76.6% and 0.06% from strawberries. The survival of the viruses was observed for up to eight days. Adenovirus survived the longest on raspberries, with a log₁₀ reduction of 0.61, followed by 1.68- and 1.75-log₁₀ reductions on strawberries and lettuce, respectively. Coronavirus declined by 0.41-log₁₀ after two days and >1.34 log₁₀ by day 4 on lettuce. The enteric poliovirus 1 survived longer on produce, decreasing by only 0.37-log₁₀ on lettuce and 1.30-log₁₀ on strawberries. A microbial risk assessment was performed to assess the risk of infection from ingesting 1, 10, and 100 particles of adenovirus on lettuce. The estimated risk of infection by ingesting these numbers were 1:2000, 1:200, and 1:20, respectively and increased in a proportional way as the number of servings was increased from one to ten and 365 servings of lettuce.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / As infecÃÃes respiratÃrias agudas (IRA) sÃo importantes causas de morbidade e mortalidade em todo o mundo, acometendo principalmente crianÃas menores de cinco anos de idade. Essas infecÃÃes podem ser causadas por diferentes microrganismos, porÃm os vÃrus sÃo os mais frequentes. Esse estudo teve como objetivo descrever aspectos clÃnicos e epidemiolÃgicos de IRA de etiologia viral em crianÃas de zero a 12 anos de idade atendidas em serviÃo de emergÃncia de um hospital terciÃrio da cidade de Fortaleza-CE, no perÃodo de janeiro de 2007 a dezembro de 2008. Para tanto foram coletadas 1318 amostras de secreÃÃo de nasofaringe das crianÃas. As amostras foram submetidas à tÃcnica de imunofluorescÃncia indireta para detecÃÃo dos seguintes vÃrus respiratÃrios: vÃrus sincicial respiratÃrio (VSR), metapneumovÃrus humano (MPVh), adenovÃrus, influenza A e B e parainfluenza 1, 2 e 3. Os resultados desse estudo mostraram que pelo menos um vÃrus respiratÃrio foi detectado em 383 (29,1%) amostras. O vÃrus mais prevalente foi o VSR (44,4%), tendo o mesmo apresentado um padrÃo de sazonalidade definido, com associaÃÃo a estaÃÃo chuvosa. A co-infecÃÃo ocorreu em 12 (3,1%) amostras e o VSR foi o mais frequentemente associado. A mÃdia de idade dos pacientes foi de 23 meses e nÃo houve associaÃÃo entre o gÃnero desses pacientes e a positividade dos exames, apesar da maioria das crianÃas serem do sexo masculino. Entre os diagnÃsticos clÃnicos de etiologia viral, houve predomÃnio de infecÃÃo da via aÃrea superior (IVAS) (51,2%), e em relaÃÃo ao diagnÃstico especÃfico das infecÃÃes da via aÃrea inferior (IVAI), destacou-se a pneumonia. Portanto, os resultados desse estudo ressaltam a importÃncia dos vÃrus como causadores de IRA em crianÃas na cidade de Fortaleza, com as maiores taxas ocorrendo entre os meses de marÃo a junho, diferenciando-se da regiÃo Sul do paÃs, onde as maiores taxas sÃo encontradas nos meses de julho a outubro. / Acute respiratory infections (ARI) are important causes of morbidity and mortality worldwide, affecting mainly children under five years old. These infections can be caused by different organisms, but viruses are the most frequent. This study aimed to describe clinical and epidemiological aspects of viral ARI in children 0-12 years of age treated in the emergency department of a tertiary hospital in the city of Fortaleza, from January 2007 to December 2008. Therefore, we collected 1318 samples of nasopharyngeal secretions of children. The samples were subjected to indirect immunofluorescence for detection of the following respiratory viruses: respiratory syncytial virus (RSV), human metapneumovirus (hMPV), adenovirus, influenza A and B and parainfluenza 1, 2 and 3. The results of this study showed that at least one respiratory virus was detected in 383 (29.1%) samples. The most prevalent virus was RSV (44.4%), and presented the same seasonal pattern of a defined association with the rainy season. Co-infection occurred in 12 (3.1%) samples and RSV was the most frequently associated. The average age of patients was 23 months and there was no association between gender of these patients and positivity of the tests, although most children were male. Among the clinical diagnoses of viral etiology, there was predominance of upper respiratory infection diseases (URID) (51.2%), and in relation to the specific diagnosis of the lower respiratory infections diseases (LRID), stood out pneumonia. Therefore, the results of this study highlight the importance of viruses as causes of ARI in children in Fortaleza, with the highest rates occurring between the months March to June, differing from the southern region, where the highest rates are found in the months from July to October.

The introduction of the polymerase chain reaction (PCR) into the diagnostic setting has provided unprecedented opportunities in the field of respiratory medicine, not only because pathogens need no longer be cultivable for detection but also through improved sensitivity, specificity and turnaround time compared with traditional methods. The recent discovery of several novel respiratory viruses, such as human metapneumovirus (HMPV), human bocavirus and human coronaviruses (HCoVs) NL63 and HKU1 has nevertheless created significant challenges in respiratory diagnostics, as identification of which pathogens should be tested for is increasingly difficult. The recent discovery of two novel respiratory coronaviruses (HCoV-HKU1 and HCoV-NL63) presented the opportunity to undertake large scale clinical and epidemiologic study of these alongside two previously known respiratory coronaviruses, HCoV-229E and HCoV-OC43. Over 12,000 samples collected over three years were screened using a novel four-way multiplex real-time reverse transcription-PCR (RTPCR). Clinically, coronaviruses were similar to viruses currently included in routine diagnostics, with the exception of HCoV-229E which was identified as an opportunistic pathogen in immunocompromised hosts. Variability in detection frequencies of HCoVHKU1 and HCoV-OC43 was evident. The low detection frequencies of HCoVs, comparable to those of parainfluenza viruses 1 and 2 (which are included in the routine diagnostic screening panel) indicate a borderline case for inclusion of these pathogens in routine respiratory diagnostics. To investigate the epidemiology and clinical correlates of HMPV in Edinburgh, large scale retrospective screening of over 7000 respiratory samples collected over two years was conducted. Nucleotide sequencing of HMPV-positive samples was undertaken to determine phylogenetic relationships of circulating HMPV strains. HMPV comprises two genotypes, A and B. Comparisons of the clinical presentations of the two genotypes revealed little difference, with only the observation that sub-genotype B2 was more frequently associated with infection of immunocompromised patients. Detection frequencies and symptomatology associated with HMPV infections were comparable to respiratory viruses currently included in the routine diagnostic panel, mandating its inclusion in future diagnostic screening. A switch of the predominantly circulating genotype of HMPV was observed between respiratory seasons. This is a phenomenon more widely reported for the closely related respiratory syncytial virus (HRSV), which also comprises two circulating groups. To further investigate subtype (HRSV)/ genotype (HMPV) switching, evolutionary analyses of nucleotide sequence data generated from isolates collected from geographically disparate referral centres was undertaken. The fusion and attachment (G) genes were targeted, as these encode major surface proteins and are immunogenic. Analyses were by MCMC analyses using Bayesian Evolutionary Analyses of Sampling Trees (BEAST) software. Identification of positively selected sites was performed using Phylogenetic Analysis Maximum Likelihood (PAML). Switching of the predominantly circulating lineage does not arise for either virus due to emergence of novel strains, but through fluctuating circulation frequencies of pre-existing lineages which have been circulating for several decades, indicated by the time since the most recent common ancestor. Two HRSV-A lineages comprising genotypes undergoing turnover and replacement were identified. This finding is agreeable with serologic studies of the 1970s which reported three HRSV serogroups, two within HRSV-A and one within HRSV-B. HMPV and HRSV have similar mutation rates. Positively selected sites identified within the HRSV G gene were incongruent with those identified in a previous study, generating the hypothesis that immune evasion occurs within linear epitopes rather than at specific sites. A great deal of clinical and epidemiologic data was generated through this work, parallel studies of other respiratory viruses and through diagnostic screening results. To provide a robust indication of where resources should be diverted in terms of diagnostics, therapeutics and vaccine development, and to inform infection control measures and public health policy planning, quantification of the relative disease burden attributable to the most commonly detected respiratory viruses was calculated using the World Health Organization- endorsed Disability Adjusted Life Year (DALY) model. Relative disease burden was calculated in an age stratified manner to reflect the differences in sampling in different age groups. HRSV and influenza A were consistently one of the greatest causes of disease regardless of sampled population, although HRSV caused more disease in children under 5 than influenza A and B combined. Rhinoviruses and PIV-3 were significant pathogens in all groups except those aged 16-64 years; rhinoviruses were the leading cause of disease in the immunocompromised patient group. The potential for patient-specific diagnostic screening and guidance of interventions such as patient cohorting were clear.

jdelvall@upc.edu.pe / Acute respiratory infections are responsible for high morbi–mortality in Peruvian children. However, the etiological agents are poorly identified. This study, conducted during the pandemic outbreak of H1N1 influenza in 2009, aims to determine the main etiological agents responsible for acute respiratory infections in children from Lima, Peru. Nasopharyngeal swabs collected from 717 children with acute respiratory infections between January 2009 and December 2010 were analyzed by multiplex RT-PCR for 13 respiratory viruses: influenza A, B, and C virus; parainfluenza virus (PIV) 1, 2, 3, and 4; and human respiratory syncytial virus (RSV) A and B, among others. Samples were also tested with direct fluorescent-antibodies (DFA) for six respiratory viruses. RT-PCR and DFA detected respiratory viruses in 240 (33.5%) and 85 (11.9%) cases, respectively. The most common etiological agents were RSV-A (15.3%), followed by influenza A (4.6%), PIV-1 (3.6%), and PIV-2 (1.8%). The viruses identified by DFA corresponded to RSV (5.9%) and influenza A (1.8%). Therefore, respiratory syncytial viruses (RSV) were found to be the most common etiology of acute respiratory infections. The authors suggest that active surveillance be conducted to identify the causative agents and improve clinical management, especially in the context of possible circulation of pandemic viruses

Submitted by Erika Demachki (erikademachki@gmail.com) on 2016-08-09T20:37:17Z No. of bitstreams: 2 Dissertação - Anniely Carvalho Rebouças Oliveira - 2016.pdf: 1878998 bytes, checksum: 1afecfd5948d2abb2a5374cbeb9ff005 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2016-08-09T20:41:31Z (GMT) No. of bitstreams: 2 Dissertação - Anniely Carvalho Rebouças Oliveira - 2016.pdf: 1878998 bytes, checksum: 1afecfd5948d2abb2a5374cbeb9ff005 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-09T20:41:32Z (GMT). No. of bitstreams: 2 Dissertação - Anniely Carvalho Rebouças Oliveira - 2016.pdf: 1878998 bytes, checksum: 1afecfd5948d2abb2a5374cbeb9ff005 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The acute respiratory infections (ARIs) are an important cause of morbidity and mortality worldwide. Viruses have an important role in the etiology of the respiratory infections. Children less than five years of age have about four to six ARIs per year and this is a common cause of hospitalization, mainly in developing countries. In Brazil, particularly in the West-Central region, studies that evaluated the circulation of respiratory viruses in the pediatric population are scarce. Thus, this study aimed to investigate the occurrence of respiratory viruses in the pediatric population, from Goiânia-Goiás. Between May/2014 and May/2015, samples of nasal swabs were collected from children from zero to six years of age presenting or not respiratory symptoms, attended at the Materno Infantil hospital in Goiânia. For the molecular screening, 16 viruses were investiged using three Multiplex Nested-PCR protocols. It two hundred and fifty one samples were collected, of wich 90 (35,9%) were positive for at least one respiratory agent, being rhinovirus (31%), respiratory syncytial virus (27,4%) and parainfluenza virus (13,3%) the most prevalent. Children under two years of age had greater overall positive, although not statistically significant. Similar detection rate was observed among the groups symptomatic (37%) and asymptomatic (34,5%). For the symptomatic group, children whose samples were collected during the dry period (p<0,05) and those with up to two years of age were most affected (p<0,05). In the asymptomatic group no statistical difference was observed. It was observed rate of co-detection of 6,4% (16/251), mostly in samples of symptomatic patients (13/16) (p<0,05). The evaluation of the temporal profile showed a higher detection rate during the dry season, less rainfall, relative humidity and cooler temperatures. The results reinforce the importance of respiratory viruses in children and contribute to further understanding of the epidemiological and temporal factors associated with different pathogens in our region. Thus, they open the way to new studies in the state of Goiás, also providing information to assist in the construction of control measures and more effective prevention of these infections. / As infecções do trato respiratório (ITRs) representam importante causa de morbimortalidade em todo o mundo, sendo os vírus importantes agentes etiológicos. Estima-se que grande parte das crianças menores de cinco anos de idade tenha a cada ano de quatro a seis ITRs, sendo esta causa comum de hospitalização, especialmente em países em desenvolvimento. No Brasil, em particular na Região Centro-Oeste, estudos que avaliem a circulação de vírus respiratórios em população pediátrica são escassos. Neste contexto, o presente estudo teve como objetivo investigar a ocorrência de diferentes vírus respiratórios em população pediátrica de Goiânia-Goiás, apresentando ou não quadro sintomático respiratório. Durante o período de maio/2014 a maio/2015, foram coletadas amostras de swab respiratório de crianças entre zero e seis anos de idade, apresentando quadro de infecção respiratória ou assintomáticas para a mesma, atendidas no Hospital Materno Infantil, em Goiânia. As amostras foram submetidas à triagem molecular para 16 vírus respiratórios por meio de três protocolos de multiplex nested-PCR. No total, 251 amostras foram coletadas, das quais 90 (35,9%) apresentaram positividade para pelo menos um agente respiratório, sendo rinovírus (31%), vírus sincicial respiratório (27,4%) e parainfluenza (13,3%) os mais frequentes. Crianças com até dois anos de idade apresentaram maior positividade global, contudo sem diferença estatística. Índice de detecção semelhante foi observado entre os grupos sintomático (37%) e assintomático (34,5%). Para as crianças do grupo sintomático, foram mais afetadas aquelas cujas amostras foram coletadas durante período de seca (p<0,05) e aquelas com até dois anos de idade (p<0,05). No grupo assintomático, nenhuma diferença estatística foi observada. Foi observado índice de co-detecção de 6,4% (16/251), sendo a maioria de indivíduos sintomáticos (13/16) (p<0,05). A avaliação do perfil temporal demonstrou maior índice de detecção nos meses de seca, com menor precipitação pluviométrica, umidade relativa do ar e temperaturas mais frias. Os resultados obtidos no presente estudo reforçam a importância dos vírus respiratórios na população pediátrica, auxiliando na compreensão dos fatores epidemiológicos e temporais associados aos diferentes patógenos em nossa região. Desta forma, abrem caminhos para novos estudos no estado de Goiás, fornecendo ainda informações que auxiliem na construção de medidas de controle e prevenção mais eficazes contra estas infecções.

RSV is the most common cause of infant bronchiolitis, leading to morbidity and mortality in both infants and the elderly. The relationship between RSV and asthma development further highlights the need to fully understand the immune responses involved in order to develop effective vaccines and therapeutics to aid prevention and treatment of RSV infection respectively. Helminths have long been studied both as a major pathogen of humans, infecting approximately 3 billion people worldwide, and also their ability to modulate the host immune response to allow survival and chronic infection to ensue. Specifically, helminth infections are thought to modulate the host immune response through regulatory mechanisms which are not fully understood. This not only confers protection and survival of the parasites themselves, but also modulates the immune response to unrelated antigens and pathogens. In this thesis, the potential role of a strictly enteric helminth infection, with Heligmosomoides polygyrus, in the modulation of respiratory syncytial virus (RSV) infection was investigated and the associated immune mechanisms were investigated. Firstly, the effects of prior H. polygyrus infection on RSV infection and immune responses in the lung were analysed. H. polygyrus significantly reduced the number of natural killer cells, CD8+ T cells, B cells and conventional dendritic cells in the lung following RSV infection. Co-infection also reduced the production of pro-inflammatory cytokines IL-6 and TNF-α in the lungs. All of these reductions were associated with significantly lower viral titres on day 4 of RSV infection. Interestingly, this attenuation of immune responses and viral titres, correlated with reduced severity of clinical disease, as assessed by weight loss and lung function. H. polygyrus excretory secretory product (HES) was not found to be the immune-modulatory factor in this system, as HES failed to suppress viral titres and reduce immune cell responses to RSV infection. However, irradiated larvae with stunted maturation to adult worms, revealed that larval stages were sufficient to suppress viral titres. Next, the role of type 2 signalling for H. polygyrus effects on RSV infection were examined, using IL-4Rα-/- mice. H. polygyrus infection maintained the ability to attenuate RSV infection and subsequent immune responses in IL-4Rα-/- mice. Furthermore, the presence of the adaptive immune response was not required for H. polygyrus-induced attenuation of RSV infection, as demonstrated in recombinase-activating gene (RAG-/-) deficient mice. H. polygyrus induces innate type 2 immune responses indicating the release of the innate alarmin, IL-33, in the lung and consequently an accumulation of group 2 innate lymphoid cells (ILC2). Their contribution to H. polygyrus effects remain to be fully elucidated. Finally, the role of antiviral responses was explored in H. polygyrus and RSV co-infection. H. polygyrus infection alone induced expression of antiviral genes, IFN-β, OAS1A, Viperin and the antimicrobial peptide CRAMP, in both the duodenum and the lung. Expression of these genes was still higher in the lung 1 hour after RSV in H. polygyrus co-infected mice compared to controls without co-infection. The importance of type I IFN signalling pathway was demonstrated using mice deficient in the type I IFN receptor in H. polygyrus co-infection, which failed to suppress RSV titres and subsequent lung immune cell infiltration. These data highlight the ability of the strictly enteric helminth H. polygyrus to attenuate RSV infection and subsequent immune responses in the lung through the potentiation of type I IFN signalling and consequent upregulation of antiviral immune responses in the lung.

De nombreuses études ont impliqué des TLR dans le développement et la progression tumorale. Précédemment, il a été démontré que les cellules tumorales expriment TLR7, un récepteur à ARNsb, et qu’une forte expression de TLR7 par les cellules tumorales de patients atteints de cancer du poumon est associée à un mauvais pronostic. Dans un modèle murin de cancer du poumon, cet effet pro-tumoral a été reproduit lors de l’injection d’agoniste de TLR7. Mes travaux de thèse ont eu pour objectif de déterminer les mécanismes impliqués dans les effets pro-tumoraux de TLR7. La stimulation de ce récepteur au niveau des cellules tumorales induit une production plus importante de CCL2 et GM-CSF, ainsi qu’un fort recrutement de MDSC au site de la tumeur. Ces MDSC, de par leurs propriétés immunosuppressives sont responsables de l’effet pro-tumoral à la suite de la stimulation de TLR7. Nous avons également mis évidence que la stimulation de TLR7 était pro-métastatique dans un modèle murin de cancer du poumon et que les MDSC étaient également impliquées dans cet effet. Ces effets pro-métastatiques associés au TLR7 ont été confirmés chez l’homme, grâce à l’étude de transcrits de molécules associées à l’invasion, l’angiogénèse, la transition épithélio-mésenchymateuse et les métastases. Enfin nous avons démontré la présence de ligand de TLR7 chez les patients atteints de cancer du poumon et démontré que l’injection intratumorale de virus respiratoires, IAV et RSV, a un effet pro-tumoral dans notre modèle de cancer du poumon. Ces virus respiratoires pourraient donc être à l’origine de la surexpression de TLR7 et du mauvais pronostic associé à ce récepteur chez les patients atteints de cancer du poumon. Ces recherches ont donc permis de mettre en évidence de nouveaux facteurs aggravants dans le cancer du poumon, dont les virus respiratoires, et de découvrir les mécanismes impliqués. / Numerous studies have implicated some TLR in tumor development. Previously, we have demonstrated that lung tumor cells express TLR7, a receptor for ssRNA, and that high TLR7 expression confers to NSCLC patients bad clinical outcome. In mice models of lung cancer, we further demonstrated that the injection of TLR7 agonists led to a pro-tumoral effect.My thesis work has firstly demonstrated the mechanisms involved in the pro-tumoral effects of TLR7 in lung cancer: TLR7 stimulation on tumor cells induces a high production of CCL2 and GM -CSF, as well as a sharp MDSC recruitment within the tumor. These MDSC, by their immunosuppressive properties, are implicated in the pro-tumoral effect upon TLR7 stimulation. We also demonstrated that TLR7 stimulation was pro-metastatic in a mice model of lung cancer and that MDSC were also involved in this effect. These pro-metastatic effects associated with TLR7 have been confirmed in humans through the studies of transcripts and proteins involved in invasion, angiogenesis, Epithelial–mesenchymal transition and metastasis. Finally, we demonstrated that TLR7 ligands are present in tumor microenvironment of lung cancer patients and that intratumoral injection of respiratory viral infections such as IAV and RSV, have a pro-tumoral effect in lung cancer mice model. These respiratory viruses could therefore be at the origin of the overexpression of TLR7 and the poor clinical outcome associated with this receptor in lung cancer patients. This research has thus made possible to highlight new aggravating factors in lung cancer, including respiratory viruses, and to discover the mechanisms involved.

Background: Respiratory diseases are common worldwide, which are caused by various respiratory viruses. As symptoms caused by these viruses are similar, laboratory diagnosis is essential to distinguish the virus. Conventionally, respiratory viruses are isolated by cell culture with a panel of cell lines. However, handling of several cell lines is labour intensive, and the turnaround time of conventional culture is long. In previous study, the use of human colon adeno-carcinoma (Caco-2) in conventional culture was investigated. The study has proven that Caco-2 is generally susceptible to the eight common respiratory viruses, i.e. Adenovirus, Influenza A and B, Respiratory Syncytial virus, Parainfluenza virus 1, 2,3 and 4. As turnaround time of conventional culture is long; therefore, in this study, rapid shell vial culture using Caco-2 cells were evaluated. Moreover, the application of Caco-2 shell vial culture on recovering human metapneumovirus (hMPV) was also investigated. Materials and methods: This study consisted of four stages. First, recovery of viruses by conventional culture and shell vial culture of Caco-2 were compared. Specimens were added to conventional culture and shell vial simultaneously. For conventional culture, formation of CPE was examined daily and IF staining was performed when CPE was indicated; meanwhile, shell vial culture were incubated for seven days and stained with IF to detect infected cells. In stage two, the effect of incubating shell vial culture in rolling drum was investigated. Shell vials inoculated with the same specimen in duplicate were incubated in rolling drum and without rolling drum simultaneously. IF staining was performed in day 2, and results were obtained. For those which are IF negative in day 2, second shell vial was further incubated to seven days before harvest. In the next stage, a large batch of samples was used to evaluate on the use of Caco-2 shell vial culture in day 2 and day 7. Lastly, Caco-2 shell vial and conventional culture and LLC-MK2 conventional culture were tested for isolation of hMPV. Results: Compared to Caco-2 conventional culture, recovery rate of shell vial culture was elevated slightly. When experimenting on the effect of incubation in rolling drum, results showed that recovery rate was raised in shell vial with rolling drum in day 2, moreover, the percentage of positive cells were increased significantly (p value < 0.05). Furthermore, in the evaluation of Caco-2 shell vial in day 2 and day 7, 75% of samples were isolated in day 2 while 85% were recovered in day 7. Lastly, in the investigation on recovery of hMPV, 53%, 42% and 17% hMPV positive cases were isolated by Caco-2 shell vial, Caco-2 conventional culture and LLC-MK2 conventional culture respectively. Conclusion: First, although recovery rate by shell vial and conventional culture were similar, turnaround time was reduced from a week to a few days by shell vial culture. Therefore, Caco-2 shell vial culture is a more efficient than Caco-2 conventional culture in isolating respiratory viruses. The study also showed that incubation of shell vial in rolling drum able to increase the number of positive cells. Furthermore, in this study, Caco-2 cells were also shown to be more efficient in isolating hMPV when compare to LLC-MK2 cells. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-14T08:11:41Z No. of bitstreams: 1 Dissertação - ítalo de Araújo Castro - 2015.pdf: 2281966 bytes, checksum: fa56d97881ce8b52bd310d3446c61552 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-14T08:16:03Z (GMT) No. of bitstreams: 1 Dissertação - ítalo de Araújo Castro - 2015.pdf: 2281966 bytes, checksum: fa56d97881ce8b52bd310d3446c61552 (MD5) / Made available in DSpace on 2015-12-14T08:16:03Z (GMT). No. of bitstreams: 1 Dissertação - ítalo de Araújo Castro - 2015.pdf: 2281966 bytes, checksum: fa56d97881ce8b52bd310d3446c61552 (MD5) Previous issue date: 2015-04-23 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Acute respiratory infection (ARI) is a major cause of morbity and mortality worldwide, particularly among children, and most of these infections are caused by viruses. Respiratory viral infections can cause symptoms ranging cough, coryza, sneezing, fever and airflow obstruction. Furthermore, the infection poses as an important trigger of asthma exacerbation, frequent clinical condition in children, and its prevalence has been rising in the last years. There are few epidemiologic studies analyzing the relationship between ARIs and asthma in Brazil. Based on this background, the aim of the study was investigate the occurrence of viral respiratory infections in pediatric patients with and without asthma in Goiânia – Goiás. Between august, 2012 and august, 2013 225 nasal aspirates and/or nasal swab samples were obtained from children with four to 14 years old. The samples were screened by Multiplex Nested-PCR for detection of 16 common respiratory viruses. From 225 samples, 42 had at least one virus detected. Samples from four different patients had more than one virus detected. The viral detection rate in ARI patients (25%), exacerbated asthma (16.3%) and stable asthma (14.8%) showed no significant difference. The most frequent viruses detected were Rhinovirus (28.6%), FLUA (11.9%), Adenovirus (11.9%), HBoV (11.9%) and RSVA (9.5%). The monthly detection rate was higher during the rainy season, period marked by great rainfall and high relative air humidity. Among the positive samples, RSV were detected during the great rainfall months and high air humidity, while the FLU and HBoV were detected during the winter months, period with low air humidity in the Mid-West region. The seasonal profile from the other viruses was unclear. The obtained results reinforces the importance of the viral pathogens in the pediatric population. Although the viral detection rate was not statistically significant, the presence of these pathogens in children is an important matter for consideration, especially to delineate control and prevention measures concerning ARIs and its impact on preexistent asthma. Hence, this study is the first of the kind in the region, and the data provided tried to fill the knowledge gaps about seasonality and circulation of these pathogens. / Infecções agudas do trato respiratório (ITR) representam importante causa de morbidade e mortalidade entre crianças e os vírus constituem os principais agentes etiológicos responsáveis por essas infecções. Além da capacidade de causar doenças com sintomas comuns desde a tosse, coriza, espirros, febre e obstrução nasal, estão frequentemente associados à exacerbação de quadros de asma, condição clínica crônica mais frequente em crianças cuja prevalência vem aumentando nos últimos anos. Considerando a população de asmáticos, os dados epidemiológicos relacionando infecções respiratórias virais e asma no Brasil são escassos. Neste contexto, o presente estudo objetivou investigar a ocorrência de infecções por vírus respiratórios em população pediátrica asmática e não asmática da cidade de Goiânia – Goiás. Para isso, entre agosto de 2012 e agosto de 2013 foram coletadas 225 amostras de aspirado nasofaríngeo e/ou swab nasal de crianças entre quatro e 14 anos de idade, as quais foram submetidas à triagem molecular para detecção de 16 vírus respiratórios, por meio de três protocolos de Multiplex Nested-PCR. Das 225 amostras, 42 apresentaram positividade para pelo menos um vírus. Amostras de quatro pacientes apresentaram mais de um vírus detectado. Os índices de detecção viral encontrados para pacientes apresentando ITR (25%), asma exacerbada (16,3%) e asma estável (14,8%) não apresentaram diferenças significativas. Os vírus mais frequentemente detectados foram os Rinovírus (28,6%), Influenza A (FLUA) (11,9%), Adenovírus (11,9%), Bocavírus Humanos (HBoV) (11,9%) e Vírus Sincicial Respiratório A (RSV) (9,5%). Maiores índices de detecção viral foram observados durante a estação chuvosa da região, período de maior precipitação pluviométrica e umidade relativa do ar (UR). Dentre as amostras positivas, os RSV foram detectados nos meses de maior precipitação pluviométrica e UR, enquanto os FLU e HBoV foram detectados durante os meses de inverno coincidindo com a estação seca. Os demais vírus não apresentaram perfil sazonal definido. Os dados obtidos ressaltam a importância dos agentes virais na população pediátrica. Embora o índice de detecção viral não tenha sido significativo entre os pacientes apresentando asma exacerbada e asma estável, a presença desses agentes infecciosos em crianças constitui um fator a ser considerado ao traçar-se estratégias de prevenção e controle dos quadros de ITR, e seu impacto em quadros preexistentes de asma. Além disso, os resultados agregam conhecimento sobre a circulação e sazonalidade desses patógenos, uma vez que este é o primeiro estudo visando a detecção molecular de vírus respiratórios em crianças na região Centro-Oeste.

Ce travail vise à estimer l’impact de co-infections sur le terrain et à mieux comprendre le synergisme possible entre agents pathogènes en conditions expérimentales. Nous nous sommes intéressés à E. coli (O78) et au virus influenza faiblement pathogène (LPAIV, H6N1) dans le modèle dinde. Les oiseaux ont été infectés par voie aérosole pour reproduire l’infection respiratoire. L’excrétion virale ainsi que les lésions ont été plus importantes lors de la co-infection, ce qui suggère une patho-génicité accrue. Ces résultats montrent que E. coli et LPAIV ont un effet additif sur la maladie respiratoire lors qu’ils ont été inoculés soit simultanément soit en différé (à 3 jours d’intervalle) à des dindes naïves. En parallèle, nous avons étudié les virus respiratoires en circulation dans les élevages pakistanais. Des co-infections avec le LPAIV H9 (lignage G1) et le virus de la maladie de Newcastle (génotype VII) ont été fréquemment observées. / The purpose of this study was to assess the burden of co-infections in the field and to better understand the possible synergism between pathogens in a laboratory setting. We focussed on E. coli (O78) and low pathogenic avian influenza virus (LPAIV, H6N1) in turkey model and infected the birds via the aerosol route to reproduce respiratory disease. Viral shedding and lesions were more severe and persisted longer during coinfection indicating possible enhancement of pathogenesis for LPAIV by E. coli and vice versa. These findings all endorse our conclusions that E. coli and LPAIV exercise an additive pathogenic effect in the reproduction of respiratory disease if given simultaneously or spaced by three days between the viral and the bacterial challenges to susceptible turkeys. In parallel, we studied avian respiratory agents circulating in the field in Pakistani farms. There, we focussed on co-infections as well, targeting viruses only as a first study. We observed frequent LPAIV H9 (G1 lineage) and Newcastle disease virus (genotype VII) coinfections in the field.