Streptococcus pyogenes (Streptocoque du Groupe A (SGA)) est un germe humain responsable d’un large éventail de pathologies invasives et non-invasives, mais aucun attribut génétique ne rend compte à lui seul de cette diversité. Notre objectif a été de rechercher des liens entre génotype, présence de gènes de virulence et caractère invasif des souches par une approche d’épidémiologie moléculaire. Une association entre génotypes et présence de certains gènes de virulence a été établie sur une collection de souches françaises de SGA responsables d’infections invasives chez des adultes. De même, la présence du locus sil, codant un système de quorum-sensing, est liée au génotype des souches, mais non à leur caractère invasif. Concernant la réponse immunitaire innée, contrairement aux souches emm1, emm4 et emm28, les souches invasives emm3 et emm89 sont plus phagocytées par les macrophages que leurs homologues non-invasives. Les souches emm89 sont plus phagocytées et survivent plus longtemps dans les macrophages que les souches des autres génotypes. Par ailleurs, les souches emm3 induisent l’apoptose des macrophages. Enfin, la cinétique de production des médiateurs pro et anti-inflammatoires est dépendante du génotype. La souche de colonisation d’un cas groupé, incluant aussi une souche invasive, présente une mutation originale dans covS (codant le senseur d’un système de régulation à deux composants). La protéine CovSY39H répond peu aux signaux de l’environnement, correspondant à une protéine CovS constitutive. Le phénotype de ce mutant, résultant de l’expression de certains gènes de virulence, est favorable à la colonisation. Sa survie dans les macrophages et sa virulence sont altérées. / Streptococcus pyogenes (Group A Streptococcus (GAS)) is a human pathogen responsible for a wide range of diseases including non-invasive and invasive infections. To date no specific GAS attribute has been associated with a type of infection although a link between genetic background and tissue tropism has been demonstrated. Our objective was to investigate the relationship between genotype, the presence of genes encoding virulence factors and invasive strains by molecular epidemiology approach. An association between genotypes and the presence of genes encoding virulence factors has been established among a collection of French strains responsible for invasive GAS infections in adults. Similarly, the presence of sil locus, encoding a quorum sensing system, is related to genotype, but not to the invasive status of the GAS strains. Regarding the innate immune response, unlike emm1, emm4 and emm28 strains, invasive emm3 and emm89 strains are more phagocytosed by macrophages than their non-invasive counterparts. The emm89 strains are phagocytosed and survive longer in macrophages than strains belonging to any other genotype. Moreover, emm3 strains induce macrophage apoptosis. Finally, the kinetics of production of pro- and anti-inflammatory mediators are genotype-dependent. A colonization strain belonging to a cluster that also includes an invasive strain, has a unique mutation in covS (encoding the sensor of a two-component system). The CovSY39H protein responds less to some environmental signals, corresponding to a constitutive CovS protein. The phenotype of the mutant, resulting in the expression of certain genes encoding virulence factors, favors a colonization state. Survival in macrophages and virulence are also altered.

La maladie pied-main-bouche (PMB) et l’herpangine sont deux maladies pédiatriques bénignes causées par les entérovirus (EV), en particulier les sérotypes de l’espèce A (EV-A). Le sérotype EV-A71 fait l’objet d’une surveillance dans les pays du Sud Est de l’Asie car il est associé à des atteintes neurologiques sévères chez les très jeunes enfants, parfois mortelles (défaillance cardio-pulmonaire). Les infections causées par les autres EV-A tel que le coxsackievirus A16 (CV-A16) provoquent rarement des atteintes sévères. En Europe, les cas de maladie PMB causés par l’EV-A71 ne font pas l’objet d’une déclaration obligatoire, car ce virus ne cause pas d’épidémies de grande ampleur. L’objectif général de la thèse était d’étudier l’épidémiologie des EV-A en Europe et nous avons utilisé une approche phylogénétique bayésienne pour analyser un échantillon de 500 souches. Nous montrons la circulation discontinue de l’EV-A71 de deux populations virales principales (sous génogroupes C1 et C2), ce qui explique la rareté des épidémies en Europe. L’épidémiologie de ce virus est aussi caractérisée par des transports de souches entre les pays Européens et sporadiquement entre l’Europe et l’Asie (sous génogroupes B5 et C4). La recombinaison génétique intertypique survient rarement parmi les populations d’EV-A71 en circulation et ne contribue pas significativement à leur diversité génétique. Cependant, ce mécanisme génétique est relié à l’émergence d’un sous génogroupe CV-A16 qui circule en France depuis 2011. Comparés à l’EV-A71, les sérotypes CV-A2, CV-A4, CV-A6 sont plus fréquemment sujets à des événements de recombinaison intertypiques. L’analyse de la sélection à l’échelle moléculaire indique que la fixation des mutations dans les protéines de capside de l’EV-A71 est lente, probablement à cause des contraintes structurales et fonctionnelles. La surveillance des infections à EV-A71 en Europe devrait être renforcée à cause de la neurovirulence de ce virus, de l’introduction récente et répétée de souches variantes « asiatiques » et de l’existence d’une grande diversité de génogroupes en Afrique et en Inde encore peu explorée. / Hand-Foot and Mouth Disease (HFMD) and Herpangina are two benign pediatric diseases caused by Enteroviruses (EV), especially enterovirus A species serotypes (EV-A). Infections caused by the EV-A71 serotype are monitored in countries of South East Asia because they are associated with severe neurological symptoms in young children and may be fatal (cardiopulmonary failure). Infections caused by the other EV-A serotypes, e.g. coxsackievirus A16 (CV-A16), rarely induce severe symptoms. In Europe, EV-A71 HFMD cases are not notifiable because this virus does not cause large-scale epidemics. The overall objective of this thesis was to study the EV-A epidemiology in Europe and we used a Bayesian phylogenetic approach to analyze 500 viral strains. We show a discontinued circulation of two EV-A71 populations (C1 and C2 subgenogroups), which explains the rare outbreaks in Europe. The epidemiology of this virus is characterized by transportation events of viral strains between European countries and sporadically between Europe and Asia (C4 and B5 subgenogroups). Intertypic genetic recombination occur rarely among circulating EV-A71 populations and does not contribute significantly to their genetic diversity. We found that genetic mechanism was related to the emergence of a new CV-A16 subgenogroup, which is circulating in France since 2011. In comparison with EV-A71, a number of serotypes (CV-A2, CV-A4, and CV-A6) are more frequently involved in intertypic recombination events. The structural and functional constraints are possible factors involved in the slow mutation fixation in the EV-A71 capsid proteins as determined by analyses of molecular selection. Neurovirulence, the recent and repeated introductions of variants “Asian” strains, and the diversity of genogroups in Africa and India call for strengthened surveillance of EV-A71 infections among European countries.

O presente estudo teve por objetivos caracterizar os vírus Influenza circulantes na cidade de São Paulo e verificar a variabilidade genética do gene da neuraminidase (NA) dos Influenzavírus A. Um total de 3009 amostras de crianças internadas no Hospital Universitário da USP, no período de 1995 a 2006, foram submetidas à duplex RT-PCR para detecção dos Influenzavírus A e B (IA e IB). As amostras positivas para IA foram submetidas à subtipagem pela multiplex RT-PCR. Cento e trinta e três amostras (4,4%) foram positivas, sendo 88,0% (117/133) IA e 12,0% (16/133) IB. Entre as amostras IA, 94 eram H3N2, 7 H1N1 e 16 não foram subtipadas pela multiplex. Um total de 74 amostras (71 H3N2 e 3 H1N1) tiveram o gene da neuraminidase sequenciado (total ou parcialmente). As sequências obtidas foram submetidas à análise filogenética, sendo verificado o agrupamento das cepas circulantes em clusters por ano de isolamento, demonstrando sua evolução temporal. Quando comparadas com as cepas vacinais utilizadas no período, foi verificada uma boa similaridade. / The aim of this study was to characterize Influenza virus circulating in São Paulo city and the neuraminidase (NA) gene sequence of Influenzavirus A in 3009 samples from children hospitalized at USP University Hospital, from 1995 to 2006. Samples underwent duplex RT-PCR for detection and typing of Influenza virus A and B (IA and IB) and also were submitted to multiplex PCR for subtyping of IA. Among 3009 samples, 4.4% were Influenza virus, being 88.0% IA (117/133) and 12.0% IB (16/133). Among samples of IA, 94 were characterized as H3N2, 7 H1N1 and 16 were not subtyped. A total of 74 samples (71 H3N2 and 3 H1N1) had the NA gene sequenced (total or partially). Sequences obtained were compared to sequences of the world and sequences of vaccine strains. Brazilian samples were grouped in clusters with samples isolated in the same year. It was also found a good similarity between circulating strains and vaccine strains.

La tuberculose MDR/XDR (multi et ultrarésistante aux antituberculeux) causée par Mycobacterium tuberculosis constitue un problème de santé publique mondial. L’étude et l’identification des mutations responsables de la résistance sont des facteurs clés pour le contrôle et la surveillance de la tuberculose MDR/XDR. L’expansion de lignée L2/Beijing, une famille de souches originaire du Sud-Est de la Chine (Guangxi) potentiellement plus virulente, complique la maitrise de cette maladie. Dans ce contexte, nous avons développé le TB-EFI et le TB IS-NTF/RINT, deux méthodes moléculaires rapides, multiplexées et haut débit (développées sur le système Luminex xMap), prêtes à utilisation. Nous avons initié le développement d’une méthode moléculaire par la sélection de marqueurs moléculaires pertinents en vue de la discrimination des souches Beijing par la technique MLPA-Beijing. Le TB-EFI est un test qui permet d’identifier les mutations fréquentes (polymorphismes de nucléotides simples) dans les gènes associés à la résistance des souches de Mycobacterium tuberculosis aux antituberculeux de deuxième ligne dont la Fluoroquinolone, les Injectables, et à l’antituberculeux de première ligne, l’Ethambutol. Le TB-EFI pourrait être un test utilisable dans les études rétrospectives en vue du suivi de la résistance d’une population. Le test IS-NTF/RINT est un test spécifique aux souches Beijing qui type la séquence d’insertion IS6110 au sein du locus NTF (Ancien/moderne) et détecte les mutations responsables de la résistance de ces souches à la Rifampicine et l’Isoniazide (les deux antibiotiques principaux de première ligne). Ce test est d’une importance capitale pour l’identification et le contrôle des souches épidémiques, mais aussi pour une vision sur l’évolution du phénomène de résistance dans le temps et l’espace. Il est peu discriminant pour la différenciation des souches Beijing. En vue d’une discrimination complète et précise des souches Beijing, nous avons proposé un lot de SNP qui serviront pour la technique MLPA-Beijing. Par ailleurs, ces méthodes ainsi que le spoligotypage sur microbille, nous ont permis d’effectuer des études d’épidémiologie moléculaire de la tuberculose au Kazakhstan, en Nouvelle Guinée Papouasie, en Italie, au Mozambique, au Pérou. Les techniques développées dans cette thèse pourraient contribuer de manière significative au contrôle de la tuberculose XDR dans les zones « hot-spot », et à la surveillance mondiale de l’évolution des souches Beijing spécialement des souches MDR épidémiques. / MDR / XDR (multidrug and extensively resistant to tuberculosis) TB caused by Mycobacterium tuberculosis is still a global public health problem. The study and identification of mutations responsible for resistance are important factors for the control and surveillance of MDR / XDR TB. The expansion of the L2 / Beijing lineage, a family of strains originating from South-East of China (Guangxi) and potentially more virulent, complicates the control of this disease. In this context, we have developed TB-EFI and TB IS-NTF / RINT, two high-speed, multiplexed and high-throughput molecular methods ready to use (developed on the Luminex xMap system). We initiated the development of a molecular method by the selection of relevant molecular markers for the discrimination of Beijing strains by the MLPA technique. TB-EFI is a test that identifies frequent mutations (single nucleotide polymorphisms) in the genes associated with the resistance of Mycobacterium tuberculosis strains to second-line anti-TB drugs including Fluoroquinolone, Injectable, and first-line antituberculosis drug, Ethambutol. TB-EFI may be used in retrospective studies to monitor resistance in a population. The IS-NTF / RINT test is a test specific to Beijing strains that types the IS6110 insertion sequence within the NTF locus (Ancient / Modern) and detects the mutations responsible for the resistance of these strains to Rifampicin and Isoniazid (the two leading primary antibiotics). This test is of paramount importance for the identification and control of epidemic strains, but also for a vision on the evolution of the phenomenon of resistance in time and space. It is not very discriminating among Beijing strains. In view of complete and precise discrimination of the Beijing strains, we have proposed a set of SNPs that will be used for a technique that will be called MLPA-Beijing. In addition, these methods as well as spoligotyping on microbeads allowed us to carry out molecular epidemiological studies of tuberculosis in Kazakhstan, Papua New Guinea, Italy, Mozambique and Peru. The techniques developed in this thesis could contribute significantly to the control of XDR tuberculosis in hot-spot areas, and to the global monitoring of the evolution of Beijing strains especially epidemic MDR strains.

No description available.

The worldwide increasing number of persons affected by largely preventable diseases like diabetes demands better prevention and treatment. Insulin is required for effective utilisation of circulating nutrients. Impaired responsiveness to insulin (insulin resistance, IR) is a hallmark of type 2 diabetes and independently raises the risk of heart attack and stroke. The pathophysiology of IR is incompletely understood. High-throughput measurement of large numbers of circulating biomarkers may provide new insights beyond established risk factors. The aims of this thesis were to (i) use proteomics, metabolomics and genomics methods in large community samples to identify biomarkers of IR; (ii) assess biomarkers for risk prediction and insights into aetiology and consequences of IR; and (iii) use Mendelian randomisation analysis to assess causality. In Study I, analysis of 80 circulating proteins in 70-to-77-year-old Swedes identified cathepsin D as a biomarker for IR and highlighted a tentative causal effect of IR on raised plasma tissue plasminogen activator levels. In Study II, nontargeted fasting plasma metabolomics was used to discover 52 metabolites associated with glycaemic traits in non-diabetic 70-year-old men. Replication in independent samples of several thousand persons provided evidence for a causal effect of IR on reduced plasma oleic acid and palmitoleic acid levels. In Study III, nontargeted metabolomics in plasma samples obtained at three time points during an oral glucose challenge in 70-year-old men identified associations between a physiologic measure of IR and concentration changes in medium-chain acylcarnitines, monounsaturated fatty acids, bile acids and lysophosphatidylethanolamines. Study IV provided evidence in two large longitudinal cohorts for causal effects of type 2 diabetes and impaired insulin secretion on raised coronary artery disease risk. In conclusion, the Studies in this thesis provide new insights into the pathophysiology and adverse health consequences of IR and illustrate the value of combining traditional epidemiologic designs with recent molecular techniques and bioinformatics methods. The results provide limited evidence for the role of circulating proteins and small molecules in IR and require replication in separate studies and validation in experimental designs.

Abstract Metabolic syndrome is a constellation of metabolic abnormalities predisposing to cardiovascular diseases (CVD), type 2 diabetes, and increased mortality. Due to the high prevalence and severe co-morbidities, metabolic syndrome constitutes a major burden for both public health and the global economy. Improved understanding of the detailed molecular mechanisms could provide novel strategies for the treatment and preferably prevention of the metabolic syndrome-related health issues. Recent advancements in ‘omics’ technologies have facilitated the development of novel tools to examine the links between genetic variation and human health. The new techniques allow determination of millions of genotypes or quantification of hundreds of metabolic measures from a single blood sample. In this thesis, genomics and metabolomics approaches are coupled to improve our understanding of the metabolic syndrome-related health issues. More precisely, my projects evaluate the metabolic effects of two lipid-lowering therapies and non-alcoholic fatty liver, as well as assess genetic determinants of chronic inflammation. The present results indicate generally consistent metabolic effects of statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) genetic inhibition. The subtle discrepancies observed could potentially contribute to differences in the efficacy to lower CVD risk between statins and PCSK9 inhibitors. The dissimilar metabolic effects of the four genetic variants that increase the risk of non-alcoholic fatty liver disease (NAFLD) highlight the heterogeneity of the molecular mechanisms involved in NAFLD pathogenesis. The results further suggest that fatty liver by itself might not promote unfavourable metabolic aberrations associated with fatty liver on a population level. The newly identified loci associating with inflammatory phenotypes elucidate the genetic mechanisms contributing to the inflammatory load. In particular, the present results suggest the important role of the locus determining the ABO blood types in the regulation of the soluble adhesion molecule levels. To conclude, this thesis successfully complements the knowledge of the molecular mechanisms involved in metabolic syndrome-related traits and provides examples of how to couple omics technologies in the study of complex traits or in the evaluation of drug effects. / Tiivistelmä Metabolinen oireyhtymä on tila, jossa useiden aineenvaihdunnallisten riskitekijöiden kasautuminen suurentaa riskiä sairastua tyypin 2 diabetekseen ja sydän- ja verisuonitauteihin sekä lisää kokonaiskuolleisuutta. Vakavista liitännäissairauksista ja suuresta esiintyvyydestä johtuen metabolinen oireyhtymä kuormittaa merkittävästi sekä terveydenhuoltoa että kansantaloutta. Jotta metabolisen oireyhtymän hoitoon ja ennaltaehkäisyyn voitaisiin kehittää uusia keinoja, on tärkeää ymmärtää paremmin oireyhtymän syntyyn vaikuttavat täsmälliset molekyylimekanismit. Niin sanottujen ’omiikka-tekniikoiden’ viimeaikainen kehitys tarjoaa uusia mahdollisuuksia tutkia geenimuutosten vaikutuksia terveyteen. Uusien tekniikoiden avulla voidaan määrittää miljoonia genotyyppejä tai satoja aineenvaihdunnan merkkiaineita yhdestä verinäytteestä. Tässä väitöskirjatyössä yhdistetään genomiikan ja metabolomiikan menetelmiä metaboliseen oireyhtymään liittyvien terveysongelmien tutkimiseksi. Väitöskirjani osatöissä arvioin kahden lipidilääkkeen sekä ei-alkoholiperäisen rasvamaksan aineenvaihdunnallisia vaikutuksia sekä pyrin tunnistamaan krooniseen tulehdukseen vaikuttavia geneettisiä tekijöitä. Tulosten mukaan statiinien ja PCSK9:n (engl. proprotein convertase subtilisin/kexin type 9) geneettisen eston aineenvaihduntavaikutukset ovat hyvin samankaltaiset. Kuitenkin havaitut pienet poikkeavuudet tietyissä merkkiaineissa voivat vaikuttaa eroavaisuuksiin siinä, kuinka tehokkaasti lääkeaineet alentavat sydäntautiriskiä. Suuret erot rasvamaksan riskiä lisäävien geenimuutosten vaikutuksissa aineenvaihduntaan korostavat rasvamaksaan liittyvien molekyylimekanismien monimuotoisuutta. Tulosten perusteella vaikuttaa siltä, että rasvan kertyminen maksaan ei luultavasti itsessään aiheuta suuria muutoksia verenkierron aineenvaihduntatuotteiden pitoisuuksiin. Tulehdusmerkkiaineisiin assosioituvat uudet geenialueet täydentävät tulehduksen molekyylimekanismeihin liittyvää tietoa. Tulokset korostavat ABO-veriryhmän määräävän geenin vaikutusta liukoisten adheesiomolekyylien pitoisuuksiin. Kaiken kaikkiaan väitöskirjan osatyöt tuovat uutta tietoa metaboliseen oireyhtymään liittyvien terveysongelmien molekyylimekanismeihin. Projektit havainnollistavat, miten omiikka-tekniikoita voidaan hyödyntää monitekijäisten fenotyyppien tutkimuksessa sekä lääkeaineiden aineenvaihduntavaikutusten arvioinnissa.

Made available in DSpace on 2014-07-29T15:26:22Z (GMT). No. of bitstreams: 1 TeseMariaAparecidaSVieira2010.pdf: 800058 bytes, checksum: 8dafcb160a1972a06f2d836a5f52d813 (MD5) Previous issue date: 2010-07-05 / Nasal carriage of Staphylococcus aureus methicillin-resistant (MRSA) is known to be a risk for subsequent infection. The MRSA carriers are an emergent and hidden reservoir in community and in the health-care environment. The aim of this investigation were to assess the prevalence and risk factors for MRSA nasal carriage in children attending emergency departments (ED) and Neonatal Intensive Care Units (NICU), and to describe the molecular features of such isolates. Methods: Nasal swabs were obtained from children less than 60 months of age attending ED, and from newborns of the four NICUs of Goiânia city, central Brazil, in 2007 and 2008. The definition of MRSA followed the CLSI criteria. Exposure variables to S. aureus and MRSA carriers were gathered through in-person interviews with mothers and hospital records. Univariate and multivariate logistic regression were performed to identify risk factors for S. aureus and MRSA carriage. Molecular typing was evaluated by pulsed field gel electrophoresis, staphylococcal cassette chromosome mec (SCCmec) typing, and multilocus sequence type (MLST). Results: A total of 2,735 children were enrolled. At the ED (n=2.034), the prevalence respectively of nasal carriages for S. aureus and MRSA were 20% (n=408) and 0.2% (n=4). Among NICUs (total of infants = 701), the prevalence of nasal carriage ranged from 0.03% to 15.7% for S. aureus and, from 0.0% to 2.0% for MRSA. At the ED, MRSA carriage was independently associated with child-care attendance in the previous 6 months (OR=10.6; p=0.045) and congenital malformation (OR=26.8; p=0.002). All nasal carriers at NICUs were from private hospitals. Only length of hospitalization was associated with MRSA nasal carriage at NICUs (p=0.023). Among four MRSA nasal carrier at ED, one harbored SCCmec type III, and three SCCmec type IV. Among four children from at the NICUs two infants harbored SCCmec type III, and two SCCmec type IV. All MRSASCCmec type III were multidrug-resistants. Strains related to Pediatric/USA800 and Brazilian MRSA clones were detected in both, ED and NICUs. One MRSA cluster related to Western Australia/USA400 was detected in ED. Conclusions: Children visiting ED, especially those reporting day-care attendance, and neonates from NICUs may play a role in spreading MRSA in healthcare settings. The study suggests cross transmission of MRSA type III and type IV between ED and hospital environments. / Portador nasal de Methicillin-resistant Staphylococcus aureus (MRSA) é um preditor de infecção subseqüente. Portadores de MRSA são um reservatório oculto, emergente na comunidade e nos serviços de saúde. Os objetivos deste estudo foram avaliar a prevalência e fatores de risco do portador nasal por S. aureus e MRSA em crianças atendidas em Pronto Atendimento (PA) e admitidas em Unidades de Cuidado Intensivo Neonatal (UCINs) e descrever as características moleculares de tais isolados. Método: Swabs nasais foram obtidos de crianças menores de 60 meses atendidas em PA e de neonatos de quatro UCINs do município de Goiânia, Brasil, em 2007 e 2008. A definição de MRSA seguiu critérios definidos pelo CLSI. Variáveis de exposição para portadores de S. aureus e MRSA foram obtidas por meio de entrevistas com mães e registros hospitalares. Regressão logística multivariada foram realizadas para identificar fatores de risco. A tipagem molecular foi feita por meio de staphylococcal cassette chromosome mec (SCCmec) typing, Pulsed Field Gel Electrophoresis (PFGE) e seqüenciamento de multilocus enzimáticos (MLST). Resultados: Um total de 2.735 crianças foi recrutado. No PA (n=2.034), as prevalências de portador nasal para S. aureus e MRSA foram de 20,1% (n=408) e de 0,2% (n=4), respectivamente. Nas UCINs (n= 701), a prevalência de portador nasal variou de 0,03% a 15,7% para S. aureus (n=64) e, de 0,0% a 2,0% para MRSA (n=4). No PA, o portador de MRSA foi independentemente associado à frequência de creche nos últimos 6 meses (OR=10,6; p=0,045) e malformação congênita (OR=26,8; p=0,002). Todos os portadores nasais de MRSA nas UCINs eram crianças internadas em hospitais privados e a única variável associada ao portador MRSA foi tempo de internação (p=0,023). Das quatro crianças portadores de MRSA no PA, uma portava SCCmec tipo III e, três, SCCmec tipo IV. Nas UCINs, duas crianças eram SCCmec tipo III e duas, SCCmec tipo IV. Todas as cepas SCCmec tipo III eram multidrogarresistentes (CLSI). Cepas MRSA relacionadas ao clones pediátrico/USA800 e epidêmico brasileiro foram detectados no PA e nas UCINs. Um cluster MRSA relacionado ao clone Western Australia/WA-1/ USA400 foi encontrado no PA. Conclusão: Crianças atendidas no pronto atendimento, especialmente aquelas que freqüentaram creche, e neonatos de UCINs apresentam potencial para disseminação de MRSA nos serviços de saúde. O estudo sugere transmissão cruzada de MRSA SCCmec tipo III e tipo IV entre serviços de emergência e hospitais.

Made available in DSpace on 2014-07-29T15:26:24Z (GMT). No. of bitstreams: 1 tese caritas medicina tropical.pdf: 1784866 bytes, checksum: b85c7ab5508fd90ff809159f179430cc (MD5) Previous issue date: 2009-02-17 / Objectives: (i) to identify risk factors for S. pneumoniae penicillin nonsusceptible isolates (PNSp) in children attending day-care centers (DCCs) in Goiânia, Brazil and to assess the genetic patterns of pneumococcal isolates; (ii) to estimate the coverage for carriage serotypes for the 7-valente (PCV7) pneumococcal conjugate vaccine, and for the investigational 10 (PCV10) and 13-valent (PCV13) vaccines; (iii) to assess the genetic relatedeness between isolates expressing capsular type 14 and those non(sero)- typeable isolates (NTPn); (iv) to investigate if carriage isolates match genetically to any international pneumococcal clone (PMEN network). Methods: A cross-sectional survey of carriage PNSp was conducted among 1.192 children, 2 months to 5 years of age, attending 62 DCCs in Central Brazil. Capsular typing was performed in PNSp isolates (CLSI, 2007) and in a sample of isolates susceptible to penicillin (PSSp) matched to PNSp and DCCs whenever possible. Serotyping was performed by Quellung reactions and confirmed by multibead assay. NTPn isolates and serotype 14 were tested by PCR for capsule genes. Odds ratio for PNSp carriage and respective 95% confidence interval (95%CI) were assessed by logistic regression. Pulsed field gel electrophoresis (PFGE) was applied to assess the genetic similarity between PNSp serotype 14 and NTPn isolates. PCR was performed for the presence of pneumococcal capsule gene locus. For comparison purpose we also evaluated the genetic profile of PNSp serotype 14 invasive strains derived from the current pneumococcal invasive disease surveillance for the same pediatric population. Isolates were epidemiologically related if they shared &#8805;80% similarity on the dendrogram (Dice coefficient). A cluster was defined as three or more related isolates. Results: A total of 686 pneumococci were isolated for a colonization rate of 57.6% and 178 (25.8%) were PNSp. Among the PNSp isolates the usual common types were found: 14 (53%), 23F (10.2%), 6B (6%), 19F (4.8%) and 19A (4.2%). PSSp isolates displayed 30 different serotypes although serotype 14 was the most common. Overall a high prevalence of NTPn (11.1%) was observed with 62.9% PNSp. Serotypes coverage xvi for the PCV7, PCV10 and PCV13 vaccines were 55.2%, 55.9% and 65.1%, respectively. Being less than 24 months of age (OR=1.79; p=0.006), hospitalization in the previous three months (OR=2.19; p=0.025), and recurrent acute otitis media (OR=2.89; p=0.013) were independently associated with PNSp in a multivariate model. Among the 123 PNSp submitted to PFGE (106/carriage and 17/ invasive isolates) a major group of 34 serotype 14 strains (8 invasive and 26 carriage) was identified and found to be genetically related to the global pneumococcal clone Spain 9V-3 (82.7% similarity). All NTPn presented capsule gene locus and 10 (45.4%) of them presented capsule gene locus to type 14. Conclusions: (i) DCC attendees with history of recurrent AOM could significantly contribute to the spread of nasopharyngeal PNSp strains into the community; (ii) epidemiologic and molecular evidences support the findings that pneumococcal nonypeable carriage isolates are genetically similar to carriage and invasive isolates expressing capsular type 14; (iii) carriage and invasive isolates circulating in Goiânia belong to a serotype 14 variant of the Spain 9V -3 clone and play a critical role in the spread of PNSp strains to the entire pediatric community of Goiânia / Objetivo: (i) identificar fatores associados à colonização nasofaríngea por S. pneumoniae não suscetíveis à penicilina em crianças que frequentam creches no município de Goiânia-GO e caracterizar geneticamente as cepas não suscetíveis; (ii) determinar a cobertura das vacinas conjugadas pneumocócicas 7, 10 e 13 valente; (iii) avaliar o relacionamento genético entre cepas do sorotipo 14 e pneumococos não tipáveis (PnNT); (iv) identificar a presença de cepas colonizadoras relacionadas geneticamente aos clones internacionais de S. pneumoniae. Metodologia: Um estudo de prevalência de portador de pneumococo não suscetível à penicilina (SpNP) foi conduzido de agosto a dezembro de 2005, em 1192 crianças de dois a 59 meses de idade, atendidas em 62 creches em Goiânia. Os testes de suscetibilidade antimicrobiana seguiram as recomendações do CLSI de 2007 e a sorotipagem foi realizada pela reação de Quellung e confirmada por ensaio multibead. Isolados PnNT e do sorotipo 14 foram analisados por reação de PCR. Odds ratio para portador de SpNP e respectivos intervalos de 95% de confiança foram estimados por regressão logística. Para avaliar a similaridade genética entre os isolados de portador (sorotipo 14 e PnNT) e isolados invasivos (sorotipo 14) obtidos de crianças de Goiânia utilizou-se amostras de isolados invasivos de um estudo maior de vigilância populacional que vem sendo conduzido desde 2007. Assim, eletroforese em campo pulsado (PFGE) foi utilizada para a tipagem molecular. Definiu-se como linhagem a presença de três ou mais cepas resistentes com similaridade genética &#8805; 80%. Resultados: S. pneumoniae foi isolado de 686 (57,6%) crianças das creches e 178 (25,9%) dessas eram portadoras de SpNS. Sorotipo 14 (53%), 23F (10,2%), 6B (6%), 19F (4,8%) e 19A (4,2%) estavam presentes em 78,2% dos PnNS. Detectou-se alta prevalência (11,1%) de isolados não tipáveis, dos quais 62.9% eram resistentes à penicilina. A cobertura dos sorotipos colonizadores para as vacinas 7-valente, 10- valente e 13-valente foi respectivamente 55,2%, 55,9% e 65,1%. Crianças menores de 24 meses de idade (OR=1,79; p=0,006), hospitalização nos últimos três meses (OR=2,19; p=0,025), e otite média aguda recorrente (OR=2,89; p=0,013) foram fatores xiv independentemente associados com SpNS na análise multivariada. Entre os 123 isolados submetidos à PFGE, 106 eram de nasofaringe de crianças das creches, dos quais 84 expressavam a cápsula tipo 14 e 22 eram isolados PnNT. Todas as cepas invasivas eram sorotipo 14. A maior linhagem agrupou 34 pneumococos do sorotipo 14, com 82,7% de similaridade, os quais foram geneticamente relacionados ao clone Spain 9V-3. Todas as cepas PnNT apresentaram locus para o gene da cápsula para o tipo 14. Houve uma diferença estatisticamente significante entre os valores da CIM para a penicilina entre as três principais linhagens (Krukal-Wallis, p<0,001). Conclusões: (i) crianças com otite média recorrente podem exercer papel importante na disseminação de pneumococos resistentes para a comunidade; (ii) Evidências genéticas apóiam os achados de que cepas de pneumococo não tipáveis assemelham-se ao genótipo das cepas do sorotipo 14; (iii) isolados de portadores e invasivos que circulam em Goiânia pertencem a um sorotipo 14 variante do clone Spain9V-3, responsável pela disseminação da resistência do pneumococo na população pediátrica de Goiânia

In der vorliegenden Arbeit wurde die Sensitivität des Nachweises von Leishmanien in Giemsa-gefärbten Bioptaten aus Hautulzerationen mittels direkter Mikroskopie mit der Sensitivität der ITS1-PCR verglichen. Bei der ITS1-PCR wurde eine Sensitivität von 87 % mit einem positiven predictive value von 100 %, sowie eine Spezifität von 100 % mit einem negativen predictive value von 85 % nachgewiesen. Weiterhin wurden vier verschiedene Nachweismethoden miteinander verglichen: die in vitro Kultivierung in NNN Medium, die direkte Mikroskopie von Giemsa gefärbten Hautbioptaten, die PCR Amplifizierung der ITS1 Region aus auf Filterpapier aufgetragenen Hautbioptaten (FP) sowie die ITS1-PCR von ungefärbten Hautbioptaten (US). Die PCR der US erwies sich als die sensitivste Methode. Die Verbreitung von Leishmanien Arten in Jericho wurde mittels molekularer Epidemiologie untersucht. Die räumliche (Spatial) Analyse zeigte drei statistisch relevante Cluster innerhalb der kutanen Leishmaniose (CL): ein Cluster mit L. major und zwei L. tropica Cluster. Bei der Raum-Zeit–Analyse wurden vier Cluster von Kutanen Leishmaniose, zwei L. major und drei L. tropica Cluster nachgewiesen. Insgesamt 106 Stämme, die aus verschiedenen endemischen Regionen in Zentralasien, im Nahen Osten und Afrika stammen, wurden mit 10 Mikrosatellitenmarkern untersucht. Die Auswertung erfolgte über zwei Analysemethoden: die Distanz-basierte und die Modell-basierte Methode. Anhand der L. major Genomsequenz wurden PCR-Primer zur Amplifizierung von Mikrosatellitenloci von L. major entwickelt, die auf den Chromosomen 1, 3, 5, 21 und 35 liegen. Sieben unterschiedliche L. major Populationen einschließlich zweier genetisch isolierter Populationen im Nahen Osten wurden mit diesen Markern nachgewiesen. / In this study we compared the sensitivity of the diagnosis of Giemsa-stained skin scrapings by standardized graded direct microscopy with that of ITS1-PCR. ITS1-PCR showed a sensitivity of 87% with positive predictive value of 100% and a specificity of 100% with negative predictive value of 85%. In-vitro cultivation using NNN medium and direct smear microscopy of Giemsa-stained slides, PCR amplifying region 1 of internal transcribed spacer (ITS1) using skin scrapings spotted on filter papers (FP) and unstained tissue smears (US) were compared. PCR using US was more sensitive than all other methods Molecular epidemiology was used to study the distribution of Leishmania species in Jericho. Spatial analysis showed three statistically significant clusters of CL, one cluster for L. major and two clusters for L. tropica. In the case of space-time, four clusters for CL, two for L. major and three for L. tropica were detected. A total of 106 strains isolated in different endemic regions of Central Asia, Middle East and Africa were analysed using 10 pairs of microsatellite markers under two cluster methods: distance and model-based. Markers were designed to amplify microsatellite loci identified in the genome sequence of L. major on chromosomes 1, 3, 5, 21 and 35. Seven discrete populations of L. major including two genetically isolated populations in the Middle East were revealed.