To understand transmissible human diseases, disciplines such as epidemiology and the surveillance of affected cases are as essential as the knowledge about the pathogenesis and the course of a disease. Epidemiologists categorize and estimate factors for public health risks by taking metadata into account including geographic aspects, health and social states to study a disease transmission and prevent further cases. In addition, a focus on the causative agents itself is necessary in order to understand their ecology and hence their virulence traits. The causative agents for a severe pneumonia named Legionnaires’ disease (LD) are bacteria of the genus Legionella. The putative sources of LD infection are any aerosol-generating natural or man-made fresh water systems. Due to this ubiquitous distribution of legionellae, it is difficult to find the source of infection. Therefore, it is necessary to isolate the bacterium from the suffering patients to further characterize it in the laboratory and to compare the clinical isolates with isolates obtained from probable environmental sources. The predominant species isolated from LD patients is Legionella pneumophila serogroup (Sg) 1. Intensive genotyping of L. pneumophila Sg1 isolates by using the current gold standard method, the sequence-based typing scheme (SBT), revealed limitations in the discrimination of several sequence types (ST) which could not be compensated for by additional phenotypic typing scheme. In practical terms, this means that several clones or STs are disproportional frequently found in both, patients and water systems, and cannot be distinguished by current methods. Therefore, a distorted picture of endemic and globally-spread clones is generated and current typing methods cannot add substantial information during the identification of the infectious source. The aim of this thesis is to develop and implement new typing methods for L. pneumophila isolates with a higher resolution than the gold standard methods. A DNA-DNA hybridization based microarray was designed and equipped with probes that target specifically L. pneumophila virulence factors and genes that are involved in the biosynthesis of lipopolysaccharide structures. Legionellae can be subgrouped on the basis of their lipopolysaccharide structures. Here, the usually phenotypic characterization of L. pneumophila Sg1 is successfully transmitted to a DNA-based genotypic method. Furthermore, the detailed validation of the DNA-microarray revealed a higher discriminatory power in comparison to the gold standard methods. It enables previously indistinguishable clones to be subdivided, providing valuable information about probable sources of infection. The second new tool for typing of L. pneumophila is based on the core genome of the bacteria. An extended SBT-scheme was extracted from the core genome and accordingly named core genome multilocus sequence typing (cgMLST). This genome wide gene-by-gene typing approach allows a high genomic resolution of L. pneumophila isolates by retaining epidemiological concordance. A major advantage of this genome-based method is the detection of large recombination events within the analysed genomes, which is, so far, reserved for whole genome sequencing. The population structure of legionellae is largely driven by recombination and horizontal gene transfer rather than by spontaneous mutations. Therefore, the detection of recombination events is essential for typing of L. pneumophila isolates. In addition, the cgMLST-scheme assigns a core genome sequence type to the analysed isolate and allows backwards compatibility with the current SBT-scheme. Both methods proved to be fast, reliable and robust typing methods through their application during outbreak investigations. Furthermore, both systems are particularly suited as routine molecular typing tools for the surveillance of single cases. The raw data are verified and translated into uniform portable codes, which enables the easy transfer and comparison of results. The standardized and portable quality of the results of both methods enables the establishment of a curated global database. This qualifies both methods as potential new gold standard methods for the genotyping of L. pneumophila isolates.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Klebsiella pneumoniae à um bacilo gram-negativo responsÃvel por uma parcela significativa de infecÃÃes do trato urinÃrio, respiratÃrio e corrente sanguÃnea de adultos em hospitais, alÃm de infecÃÃes em recÃm-nascidos em unidades de terapia intensiva. Sua importÃncia tem aumentado devido ao surgimento de cepas produtoras de beta-lactamases de espectro estendido (ESBL). Estas enzimas medeiam resistÃncia aos oxyimino-β-lactÃmicos. Em K. pneumoniae, a maioria das ESBL identificadas sÃo dos tipos TEM, SHV e CTX-M. Em adiÃÃo, β-lactamases que hidrolisam carbapenÃmicos dos tipos KPC e GES tem sido detectadas nestes isolados. Surtos de infecÃÃo hospitalar causados por clones de K. pneumoniae multiressistentes tem sido descritos em vÃrias regiÃes do paÃs. No entanto, este à o primeiro relato de caracterizaÃÃo genÃtica de isolados de K. pneumoniae produtores de ESBL no estado do CearÃ, Brasil. Este estudo teve como objetivo, em primeiro lugar, detectar os principais genes responsÃveis pela produÃÃo de ESBL em cepas de K. pneumoniae obtidas a partir de pacientes que desenvolveram infecÃÃes hospitalares em um hospital de cuidados terciÃrios na regiÃo norte do estado do CearÃ, de novembro de 2013 a agosto de 2014 e, em segundo lugar, analisar a similaridade genÃtica destes isolados. Trinta e seis isolados clÃnicos de K. pneumoniae produtores de ESBL foram avaliados. A detecÃÃo dos genes blaCTX-M dos grupos 1 e 2, blaSHV-like, blaTEM-like, blaKPC-like e blaGES-like foi realizada por PCR. A tipagem molecular dos isolados foi realizada por Pulsed-Field Gel Electrophoresis (PFGE). Os genes blaCTX-M dos grupos 1 ou 2 e blaSHV-like foram detectados em 100% dos isolados e os genes blaTEM-like em 55,6%. Em adiÃÃo, 55,6% dos produtores de CTX-M tambÃm produziram SHV e TEM. Nenhum gene blaKPC-like e blaGES-like foi detectado. A tipagem molecular por PFGE mostrou grande diversidade entre os isolados, contudo dois isolados coletados em diferentes clÃnicas mostraram o mesmo perfil de bandas e tinham os mesmos genes bla e, entÃo, foram considerados como pertencentes a uma Ãnica cepa. A detecÃÃo dos genes blaCTX-M em 100% dos isolados sugere que as enzimas CTX-M sÃo as principais ESBL responsÃveis pelo fenÃtipo de resistÃncia aos beta-lactÃmicos nos isolados estudados. Dados apresentados neste estudo chamam atenÃÃo para um problema de resistÃncia endÃmico causado por cepas multiclonais de K. pneumoniae multirresistentes cujo controle passa essencialmente pelo aprimoramento das polÃticas de prescriÃÃo de antimicrobianos e pela implantaÃÃo de programas de prevenÃÃo e controle da disseminaÃÃo destes patÃgenos no hospital pesquisado. / Klebsiella pneumoniae is a Gram-negative bacillus responsible for a significant portion of urinary tract infections, respiratory, and bloodstream of adults in hospitals, besides infections in neonates in intensive care units. Its importance has increased due the emergence of extended-spectrum beta-lactamase-producing strains (ESBLs). These enzymes mediate resistance to oxyimino-β-lactams. In K. pneumoniae, most of the identified ESBLs are of the TEM, SHV, and CTX-M types. In addition, carbapenem-hydrolyzing beta-lactamases of KPC and GES types has been detected in these isolates. Outbreaks of nosocomial infections caused by multidrug-resistant K. pneumoniae clones have been described in various regions of the country. However, this is the first report of the genetic characterization of ESBL-producing K. pneumoniae in the state of CearÃ, Brazil. This study aimed firstly to detect the main genes responsible for ESBL production in K. pneumoniae strains obtained from patients who developed nosocomial infections in a tertiary support hospital in the northern region of the Cearà state, from November 2013 to August 2014 and, secondly, to analyze the genetic similarity of these isolates. Thirty-six clinical isolates of ESBL-producing K. pneumoniae were evaluated. The detection of blaCTX-M groups 1 and 2, blaSHV-like, blaTEM-like, blaKPC-like, and blaGES-like genes was performed by PCR. Molecular typing of isolates was performed by pulsed-field gel electrophoresis PFGE. Groups 1 or 2 blaCTX-M and blaSHV-like genes were detected in 100% of the isolates and blaTEM-like genes in 55.6%. In addition, 55.6% of CTX-M-producers also produced SHV and TEM. No blaKPC-like and blaGES-like genes were detected. Molecular typing by PFGE showed great diversity between the isolates, although two isolates collected in different wards showed the same banding profile and had the same bla genes and so were considered to belong to a single strain. Detection of blaCTX-M genes in 100% of the isolates suggests that CTX-M enzymes are the major ESBLs responsible for the beta-lactam resistance phenotypes of the studied isolates. Data presented in this study call attention to an endemic resistance problem caused by multiclonal strains of multidrug-resistant K. pneumoniae whose control passes essentially the improvement of antimicrobial prescription policies and the implementation of prevention and control programs the spread of these pathogens in the studied hospital.

Cryptococcus neoformans e C. gattii são as principais espécies do gênero que causam infecção no homem, C. albidus e C. laurentii são espécies menos envolvidas. O presente trabalho teve por objetivos avaliar a patogenicidade in vivo, os fatores e os genes relacionados à virulência, e verificar o perfil de suscetibilidade ao fluconazol de 10 isolados ambientais de cada uma das espécies: C. neoformans, C. albidus e C. laurentii, antes e após a inoculação em camundongos BALB/c imunocompetentes; pesquisar os sorotipos, mating types e realizar a tipagem molecular. Proteinase, fosfolipase, urease, produção de melanina e crescimento à 37ºC foram pesquisados utilizando metodologias clássicas, e a pesquisa dos genes e determinação dos sorotipos e mating types foram feitas por PCR. A tipagem molecular foi realizada por PCR-fingerprinting, com os iniciadores (GACA)4 e M13. A determinação da CIM do fluconazol foi realizada pelo método da microdiluição em caldo. Todos os isolados de C. neoformans foram sorotipos A e MAT-alfa. A inoculação em animais mostrou que 9 isolados de C. neoformans mataram 100% deles em até 33 dias, e 1 levou os animais à morte num período entre 40 e 82 dias; 9 isolados foram recuperados dos pulmões e cérebro dos animais em 7 e 14 dias, e um deles levou todos os animais à morte em 12 dias, sendo possível recuperá-lo somente no 7º dia. Os animais inoculados com C. albidus e C. laurentii permaneceram vivos até negativação das culturas dos órgãos avaliados. C. albidus foi isolado principalmente do fígado e dos pulmões até 10 dias após a inoculação, C. laurentii dos pulmões e do cérebro até 120 dias. Todos os isolados das 3 espécies produziram cápsula antes e após a inoculação. Todos C. neoformans, 6 C. albidus e 6 C. laurentii cresceram à 37ºC antes e depois da inoculação. Melanina foi produzida por todos os isolados de C. neoformans e nenhum C. albidus nas duas ocasiões; e por 6 e 9 isolados de C. laurentii, antes e depois da inoculação, respectivamente. Seis isolados de C. neoformans e 1 de C. laurentii produziram proteinase nas duas ocasiões. Sete isolados de C. albidus produziram proteinase antes e todos depois da inoculação. Fosfolipase foi produzida por todos C. neoformans e C. albidus, e por 6 C. laurentii nas duas ocasiões. A avaliação da atividade da urease realizada em meio líquido foi positiva em 24 a 48 horas pelos isolados de C. neoformans e C. laurentii, e em 24 a 96 horas por C. albidus. A CIM de fluconazol variou de 2 a 8 ug/mL para C. neoformans, de 8 a >= 64 ug/mL para C. albidus, e de 1 a 64 ug/mL para C. laurentii, nas duas ocasiões. Todos os isolados de C. neoformans apresentaram os genes lacase (Lac1), fosfolipase (PLB1), proteinase (cnap1), calcineurina (CNA1), urease (URE1), e ERG11, com os oligonucleotídeos utilizados. A PCR com ERG11 mostrou uma banda no gel de agarose para todos C. albidus, porém nenhum dos outros genes pesquisados foram amplificados em C. albidus e C. laurentii. A tipagem molecular por PCR-fingerprinting dos isolados de C. neoformans revelou 2 tipos moleculares: VNI (7 isolados) e VNII (3 isolados). A maioria dos isolados de C. albidus apresentou homogeneidade nos padrões de bandas gerados, e C. laurentii foi a espécie que demonstrou maior diversidade genética por esta metodologia. Concluímos que a passagem dos isolados pelos animais não alterou os fenótipos estudados e nenhuma alteração foi detectada pela análise molecular. No entanto, verificamos a grande heterogeneidade molecular dos isolados de C. laurentii estudados. / Species of Cryptococcus neoformans and C. gattii are the main ones in the genus causing infection in man while C. albidus and C. laurentii are less involved. This study evaluated the in vivo pathogenicity, factors and genes related to virulence and the susceptibility to fluconazole before and after inoculation in immunocompetent BALB/c mice of ten environmental isolates of C. neoformans, C. albidus and C. laurentii. Serotypes, mating types and molecular typing were also determined to complete the evaluation. Enzymes like proteinases, phospholipase, urease, production of melanin and growth at 37oC were investigated by classical methods, but gene characterization and determination of serotypes and mating types were investigated by PCR. Molecular typing was done by PCR-fingerprinting with primers (GACA)4 and M13. The microdilution method was used to determine the minimum inhibitory concentration (MIC) of flucozanole. All C. neoformans isolates were serotype A and MAT-alfa and 9 of them when inoculated in animals killed 100% in up to 33 days. One isolate inoculated killed the animals in 40 to 82 days. Nine isolates were recovered from the animal lungs and brain in 7 and 14 days and the one which killed all animals in 12 days was only recovered on the 7th day. Animals inoculated with C. albidus and C. laurentii were alive until the tissue cultures of evaluated organs were negative. C. albidus was isolated mainly from the liver and lungs in up to 10 days after inoculation and strains of C. laurentii from the lungs and brain in up to 120 days. All isolates in the 3 species were capsule producers before and after inoculation. All strains of C. neoformans, 6 C. albidus and 6 C. laurentii grew at 37oC both before and after inoculation. All C. neoformans produced melanin and 6 C. laurentii produced it before inoculation and nine after. None was produced by C. albidus. Six isolates of C. neoformans and one of C. laurentii produced proteinases in both situations, before and after inoculation. Seven C. albidus isolates produced the protein hydrolyzing enzyme before inoculation and all after. Phospholipase enzyme was produced by all C. neoformans, and C. albidus and by 6 C. laurentii in both conditions, before and after inoculation. Urease activity was detected between 24 and 48 hours after incubation in a liquid medium for C. neoformans and C. laurentii cultures and after 24 to 96 hours for C. albidus. Fluconazole MICs ranged from 2 to 8 ug/ mL for C. neoformans isolates, from 8 to >= 64 ug/mL for C. albidus and from 1 to 64 ug/mL for C. laurentii in both conditions. Genes laccase (Lac1), phopholipase (PLB1) proteinase (cnap1), calcineurine (CNA1), urease (URE1) and ERG11, detected with the primers used were present in all C. neoformans. With exception of ERG11, which showed a band in agarose electrophoresis by all C. albidus, the other genes were not amplified in C. albidus and C. laurentii. Molecular typing by PCR-fingerprinting showed two molecular types in C. neoformans: VNI in 7 isolates and VNII in 3 isolates. Most C. albidus showed homogenous patterns in the bands generated and C. laurentii was the species with the higher genetic diversity by this methodology. It is concluded that isolate inoculations in animals does not alter phenotypes and no alteration is detected by molecular analysis. However, the high molecular heterogeneity of C. laurentii was detected.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Salmonella infection is a relevant problem in the public health, being one of the most important causes of the world´s enteric pathologies, with 1,3 million ill people, resulting in 600 deaths/year. The Salmonella spp. transmission occurs especially through water consumption and contaminated food. The diagnosis is realized using biochemical, serologic, and molecular tests. The RAPD (Randomly Amplified Polymorphic DNA) test is able to detect the genetic variation present in the isolated Salmonella spp. samples, allowing a molecular typing. This research aimed at achieving molecular typing from Salmonella spp. samples isolated from various resources (oyster, chicken, potable water, blood, and human feces) utilizing the RAPD technique. 33 Salmonella spp. samples, that came from bacteria located at the Laboratório de Virologia Comparada-DMO/CCBS/UFS, and two standard samples were utilized. Six randomized primers were used from the Ready-To-Go System RAPD; the products amplified were submitted to an electrophoretic run in a 5% polyacrilamid gel, and silver dyed. The band standard observed was analyzed by the NTSYS program. After a computational analysis it was possible to discriminate the 35 Salmonella spp. samples, resulting in 35 RAPD individual and distinct patterns, showing that the samples are genetically diversed and that there is an ample genetic biodiversity in the circulating samples in Aracaju-SE. To the grouping the Salmonella spp. samples was observed the epidemiological relationship between human samples and chicken. / A infecção por Salmonella é um relevante problema de saúde pública, sendo uma das mais importantes causas de patologias entéricas do mundo, com 1,3 milhões de doentes, resultando em aproximadamente 16000 hospitalizações e 600 mortes/ano. A transmissão da Salmonella spp. ocorre principalmente através do consumo de água e alimentos contaminados. O diagnóstico é realizado através de testes bioquímicos, sorológicos e moleculares. A técnica de RAPD (Randomly Amplified Polymorfhic DNA) é capaz de detectar as variações genéticas presente nos isolados, permitindo a tipagem molecular. Este estudo teve como objetivo realizar a tipagem molecular das amostras de Salmonella spp. isoladas de diversas fontes (ostra, frango, água residual, sangue e fezes humanas) utilizando a técnica de RAPD. Foram utilizadas 33 amostras de Salmonella spp. da bacterioteca do Laboratório de Virologia Comparada DMO/CCBS/UFS e duas amostras padrões. Foram utilizados seis primers randômicos do Sistema Read-To-Go RAPD, os produtos amplificados foram submetidos à corrida eletroforética em gel de poliacrilamida 5% e corado pela prata. O padrão de bandas observadas foi analisado pelo programa NTSYS. Após análise computacional foi possível discriminar as 35 amostras de Salmonella spp., resultando em 35 padrões de RAPD individuais e distintos, mostrando que as amostras são geneticamente diversas e que existe uma ampla biodiversidade genética nas amostras circulantes em Aracaju-SE. Ao grupar as amostras de Salmonella spp. observou-se o relacionamento epidemiológico entre as amostras humanas e de frango.

Orientador: Carlos Emilio Levy / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T23:13:59Z (GMT). No. of bitstreams: 1 Fiolo_Kateli_M.pdf: 2870883 bytes, checksum: 6e6dd0df208caa395482319a31dda8b1 (MD5) Previous issue date: 2011 / Resumo: Streptococcus agalactiae, conhecido como Estreptococo beta-hemolítico do grupo B (EGB), é classificado por diferenças capsulares que podem variar em dez sorotipos, alguns responsáveis por infecções materno-infantis sérias e debilitantes ou podendo ainda levar ao óbito. O EGB pode ocasionar também, infecções graves em adultos e idosos. OBJETIVO: Descrever e analisar o perfil epidemiológico dos sorotipos prevalentes de Streptococcus agalactiae, provenientes de infecção em recém-nascidos (RN), do Centro de Atenção Integral á Saúde da Mulher (CAISM /UNICAMP) e casos de infecção por EGB de diversos materiais do Hospital de Clínicas da Unicamp (HC UNICAMP) e de sete laboratórios que prestam serviços a outros hospitais maternidade na cidade de Campinas SP e região. MÉTODOS: Estudo transversal laboratorial realizado, no período de janeiro de 2007 a dezembro de 2010. As cepas de EGB foram triadas por provas laboratoriais manuais padronizadas, ou por automação microbiológica, Vitek®2 (BioMeriéux). A seguir foram tipadas por PCR, utilizando sucessivamente primers específicos para espécie e para nove sorotipos de Streptococcus agalactiae. RESULTADOS: Durante os anos de 2007 e 2008 o programa de triagem materna do CAISM coletou 2.022 amostras de secreção retovaginal com média de 20,5% de positividade. Entre janeiro de 2007 a dezembro de 2010, foram selecionadas 120 amostras de EGB, isoladas de pacientes do HC UNICAMP, de diferentes materiais: urina (72,5%), sangue (hemocultura) (15,8%), secreção de feridas e abscessos (4,1%), líquor (2,5%), secreção ferida cirúrgica (1,6%), outras secreções (3,3%). Foram também selecionados, entre setembro de 2008 a setembro de 2009, 383 amostras de EGB isolados por laboratórios que prestam serviço a hospitaismaternidade de Campinas e região em: urina (54,3%), secreção retovaginal (37,8%), esperma (3,4%), sangue (2,3%), secreções gerais (1,8%) e líquor (0,2%). Foram avaliados, por análise molecular os sorotipos de 70 destas amostras, sendo 22 isoladas de sangue, 5 de líquor e 43 de outros materiais clínicos, escolhidos aleatoriamente, revelando a predominância do sorotipo tipo V (61,4%), seguido pelo tipo Ia (24,3%), tipo III (10,0%), tipo Ib (2,8%) e o tipo IV (1,4%). Dentre as amostras analisadas apenas seis eram provenientes de processos infecciosos em RNs do CAISM, sendo 1, 2,1 e 2 casos, respectivamente para cada ano, de 2007 até 2010, estimando-se para este período uma incidência média 0,55 casos de EGB por 1.000 nascidos vivos. Apenas mais um caso de RN foi isolado no Hospital Estadual de Sumaré no ano de 2009. Entre esses sete casos de RN, em dois foram encontradas amostras pareadas de mãe-filho. Nas amostras de RNs houve predominância do sorotipo V com 42,8%, seguido pelo tipo III e Ia com 28,5% cada um, nas amostras das duas mães foram encontrados os mesmos sorotipos de seus recém-nascidos. CONCLUSÕES: O número de amostras obtidas de recém nascidos foi abaixo do esperado, possivelmente em conseqüência da eficiência do programa de triagem e profilaxia materna do EGB, não podendo ser excluída a possibilidade de limitações dos recursos laboratoriais utilizados. Os sorotipos encontrados são os mais prevalentes na literatura mundial e associados à maior virulência. A técnica de PCR revelou ser muito útil para estudos epidemiológicos e de elevada especificidade / Abstract: Streptococcus agalactiae, also known as beta-hemolytic streptococcus group B (GBS), is classified by capsular differences that can vary in ten serotypes, some responsible for maternal and infant debilitating or serious infections and can even lead to death. The GBS can also cause, serious infections in adults and elderly. OBJECTIVE: To describe and analyze the epidemiology of prevalent serotypes of Streptococcus agalactiae, isolated from newborns (NB), from the Center for Integral Attention to Women's Health (CAISM / UNICAMP) and cases of GBS infection of various materials from Hospital de Clinicas, Unicamp (HC UNICAMP) and seven laboratories that provide services to other maternity hospitals in Campinas, São Paulo, Brazil and region. Methods. It was a cross-sectional laboratory study conducted in the period from January 2007 to December 2010. GBS strains were screened by standard manual microbiological laboratory tests, or by automation by Vitek ® 2 (bioMérieux). Following, they were typed by PCR, using specific primers for species and nine serotypes of Streptococcus agalactiae. RESULTS: During the years of 2007 and 2008 the CAISM maternal screening program collected 2,022 rectovaginal secretion samples with an average of 20.5% positivity. From January 2007 to December 2010, were selected a total of 120 GBS strains isolated at the HC UNICAMP as follows: urine (72.5%) blood (15.8%), secretion from wounds and abscesses (4.1%), cerebrospinal fluid (2.5%), wound secretion (1.6%) and other secretions (3.3%). From September 2008 to September 2009, were also selected 383 samples of GBS isolated by laboratories that provide service for maternity hospitals of Campinas region as follows: urine (54.3%), rectovaginal secretion (37.8%), sperm (3.4%), blood (2.3%), general secretions (1.8%) and cerebrospinal fluid (0.2%). Of these samples 70 strains were evaluated by molecular typing analysis, 22 isolated from blood, 5 from cerebrospinal fluid and 43 randomly selected isolates from other clinical materials, revealing the predominance of serotype V (61.4%), followed by serotype Ia (24.3%), serotype III (10.0%), serotype Ib (2.8%) and serotype IV(1.4%). Among the 70 samples, six were from newborns of CAISM with infectious processes, with 1, 2, 1 and 2 cases occurred respectively in each year from 2007 to 2010. For this period were estimated an average incidence of 0.55 cases of GBS for 1,000 born alive. Only one additional case of NB infection was isolated in the Hospital Estadual de Sumaré in 2009. Among these seven cases of NB infections, for only two were found paired EGB isolates from mother and newborn. In the NB samples was found predominantly the serotype V ( 42.8%), followed by type Ia and III with 28.5% for each, and in two samples of mothers were found the same serotype of their newborns. CONCLUSIONS: The number of samples of newborns was lower than expected, possibly due to the efficiency of the maternal GBS screening program and prophylaxis, but can not be excluded the limitations of laboratory resources used. The founded serotypes are the most prevalent in the literature and associated with increased virulence. The PCR technique has proved to be very useful for epidemiological studies and a have a high specificity / Mestrado / Saude da Criança e do Adolescente / Mestre em Ciências

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-18T16:42:00Z No. of bitstreams: 1 Fleming, Maria Emília de Castro Kling [Dissertação, 2011].pdf: 3850457 bytes, checksum: 76e7a64373d2898f956190ecd2aa26c3 (MD5) / Made available in DSpace on 2017-04-18T16:42:00Z (GMT). No. of bitstreams: 1 Fleming, Maria Emília de Castro Kling [Dissertação, 2011].pdf: 3850457 bytes, checksum: 76e7a64373d2898f956190ecd2aa26c3 (MD5) / Introdução: As infecções de corrente sanguínea estão entre as mais graves infecções, principalmente se causadas por microrganismos resistentes a antimicrobianos. O objetivo deste estudo foi avaliar a prevalência e o perfil de resistência de microrganismos isolados em hemoculturas em um hospital público e outro privado localizados em Niterói, Rio de Janeiro, Brasil. MÉTODOS: O estudo foi conduzido em um hospital geral de natureza pública com 227 leitos e um hospital geral, privado contendo 123 leitos, entre Agosto de 2009 a Agosto de 2010. Todas as amostras provenientes de pacientes maiores de 18 anos foram consecutivamente coletadas após identificação por métodos de rotina de cada laboratório de microbiologia. As cepas de E. coli, K. pneumoniae, K. oxytoca e P. mirabilis foram testadas quanto a produção de ESBL (Extended Spectrum Betalactamase) de acordo com as recomendações do CLSI. As enterobactérias foram testadas quanto a produção de carbapenemases pelo teste modificado de Hodge (CLSI). As amostras de P. aeruginosa foram testadas para a produção de MBLs pelo teste fenotípico de disco combinado. A reação em cadeia da polimerase (PCR) foi utilizada para a detecção dos genes (blaIMP, blaVIM, blaSPM), relacionados com a produção de metalo-beta-lactamases (MBLs). A similaridade genética entre as cepas de P. aeruginosa foi avaliada pela técnica de eletroforese em gel de campo pulsado (PFGE). RESULTADOS: Foram coletadas 195 amostras de microrganismos isolados em hemoculturas no hospital público e 123 amostras no hospital privado. Os nãofermentadores foram a maior causa de bacteremias na unidade de terapia intensiva (UTI) da instituição pública. As enterobactérias foram os microrganismos mais prevalentes nas enfermarias da unidade privada. No hospital público foram detectadas amostras produtoras de ESBL, enquanto no hospital privado foram identificadas cepas produtoras de carbapenemases. Dentre as cepas de P. aeruginosa, 40 amostras foram testadas para a produção de MBLs. Treze cepas (32,5%) foram positivas no teste fenotípico e no PCR, todas positivas para o gene blaSPM-1, sendo que apenas uma foi proveniente da instituição pública e 12 do hospital particular. Nenhuma amostra carreadora dos genes blaIMP-1 e blaVIM-2 foram detectadas. Os resultados de PFGE mostraram que todas as cepas carreadoras do gene blaSPM-1, isoladas no hospital privado, foram geneticamente relacionadas (Pulsotipo A), o que pode indicar uma transmissão cruzada entre os pacientes e profissionais de saúde. CONCLUSÕES: As características dos microrganismos isolados em hemoculturas variou entre as unidades de internação e entre os hospitais, demonstrando que os dados locais podem orientar a terapia antimicrobiana e as medidas de controle e prevenção das infecções. Devido ao impacto das infecções de corrente sanguínea e a presença de microrganismos resistentes no ambiente hospitalar, estudos adicionais e medidas de vigilância são necessárias / Introduction: Bloodstream infections are one of the most serious bacterial infections, especially if caused by resistant microorganisms. The purpose of this study was to assess the prevalence and resistance profile of pathogens isolated from blood cultures in a public and a private hospital of Niterói, Rio de Janeiro, Brazil. METHODS: A case-series of patients with blood stream infection was conducted at a 227-bed public general hospital and at a 123-bed private general hospital from August 2009 to August 2010. All isolates were consecutively detected from patients minimum age of 18 years and identified by the routine methodology used at each laboratory. Every E. coli, K. pneumoniae, K. oxytoca and P. mirabilis isolates were tested for ESBL (Extended Spectrum Beta-lactamase) production using the CLSI guidelines. The Enterobacteriaceae was tested for carbapenemase production using the Modified Hodge test (CLSI). Every P. aeruginosa were tested for metallo-betalactamases (MBLs) producing by the phenotypic method of combined disk. The polymerase chain reaction (PCR) was used to detect the MβLs genes (blaIMP, blaVIM, blaSPM). The genetic similarity between the strains was evaluated in samples which were positive for MBLs using the pulsed field gel electrophoresis technique (PFGE). RESULTS: Were collected 195 samples of microorganisms isolated in blood cultures in the public hospital and 123 samples in the private hospital. The non-fermentatives were the major cause of bacteremia in the ICU of public hospital. The Enterobacteriaceae were the most prevalent in the the wards of private hospital. In the public hospital, we found strains producing ESBL and in the private hospital, strains producing carbapenemase. Forty samples of P. aeruginosa were tested for MBL producing. Thirteen strains (32,5%) were positive in phenotypic test and in PCR, every sample were positive for blaSPM-1, and only one was from the public institution and 12 of the particular hospital. No blaIMP-1 and blaVIM gene were detected. The PFGE analysis showed that all blaSPM-1 gene-carrying strains isolated in private hospital were genetically related (Pulsetype A), suggesting a cross transmission between patients and health professionals. CONCLUSIONS: The characteristics of the microorganisms isolated from blood culture varied from hospital to hospital and between inpatient units, showing that local data can help with therapeutic choices and with the prevention and control of infection. Due to the impact of bloodstream infections and the presence of resistant microorganisms in the hospitals, additional studies and monitoring measures are necessary

Blind people cannot enter text on touch devices using common input methods. They use special input methods that have lower performance (i.e. lower entry rate and higher error rate). Most blind people have muscle memory from using classic physical keyboards, but the potential of using this memory is not utilized by existing input methods. The goal of the project is to take advantage of this muscle memory to improve the typing performance of blind people on wide touch panels. To this end, four input methods are designed, and a prototype for each one is developed. These input methods are compared with each other and with a standard input method. The results of the comparison show that using input methods designed in this report improves typing performance. The most promising and the least promising approaches are specified.

Dynamically Typed Assembly Language (D-TAL) is not only a lightweight and effective solution to the gap generated by the drop in security produced by the translation of high-level language instructions to low-level language instructions, but it considerably eases up the burden generated by the level of complexity required to implement typed assembly languages statically. Although there are tradeoffs between the static and dynamic approaches, focusing on a dynamic approach leads to simpler, easier to reason about, and more feasible ways to understand deployment of types over monomorphically-typed or untyped intermediate languages. On this occasion, DISM, a simple but powerful and mature untyped assembly language, is extended by the addition of type annotations (on memory and registers) to produce an instance of D-TAL. Strong-DISM, the resulting language, statically, lends itself to simpler analysis about type access and security as the correlation between datatypes and instructions with their respective memory and registers becomes simpler to observe; while dynamically, it disallows operations and further eliminates conditions that from high level languages could be used to violate/circumvent security.

The purpose of the study was to compare the occupational aptitude scores of students enrolled in beginning and advanced clerical courses in the School of Business Administration at North Texas State College, Denton, Texas, with the academic grades of these same students to determine the relationship between the academic grades received and the occupational aptitude score indicated by the General Aptitude Test Battery.

Au sein des mycoplasmes pathogènes pour l’homme, il existe des mycoplasmes à tropisme respiratoire, parmi lesquels M. pneumoniae, et d’autres dont le tropisme est la sphère urogénitale, comme M. genitalium. M. genitalium est un agent émergent dont l’épidémiologie est encore mal connue. Il est responsable d’infections sexuellement transmissibles, urétrites chez l’homme et cervicites chez la femme. M. pneumoniae est responsable d’infections respiratoires aiguës chez l’enfant et l’adulte jeune. M. genitalium est une espèce extrêmement fastidieuse dont la culture est exceptionnelle à partir de prélèvements de patients. Dans le but d'enrichir notre collection de prélèvements positifs pour M. genitalium un protocole de recherche clinique (FeminIST) proposant un dépistage systématique par PCR du portage de M. genitalium chez les femmes infectées par le VIH de la cohorte Aquitaine a été mis en place. Les méthodes génotypiques ont été largement appliquées pour le typage moléculaire des Mollicutes, mais peu de méthodes simples, automatisées et discriminantes l’ont été à M. genitalium et M. pneumoniae. La MLVA (« Multi-Locus Variable-Number of Tandem-Repeats Analysis ») est une méthode qui analyse le polymorphisme associé aux répétitions en tandem, présentant de nombreux avantages, comme un pouvoir discriminant élevé et la possibilité d’être réalisée directement à partir des prélèvements. Cette technique a été appliquée à M. genitalium et M. pneumoniae et comparée aux méthodes de typage déjà disponibles. Six et cinq VNTR ont été respectivement identifiés comme discriminants pour M. genitalium et M. pneumoniae. Les PCR ont été multiplexées et les amorces marquées pour faciliter et automatiser l’analyse réalisée par électrophorèse capillaire. La méthode a été réalisée sur notre collection de 265 souches cliniques de M. pneumoniae et directement à partir de 123 prélèvements positifs de M. genitalium. La MLVA a permis de typer 89,4 % des prélèvements positifs pour M. genitalium et toutes les souches de M. pneumoniae. Elle s’est révélée plus discriminante que les autres méthodes pour les deux espèces. Les représentations hiérarchiques des résultats confirment l’hétérogénéité de l’espèce M. genitalium et, en revanche, l’homogénéité de l’espèce M. pneumoniae. En résumé, la MLVA s’avère être un outil de typage moléculaire performant pour M. genitalium et M. pneumoniae donnant des résultats facilement échangeables entre laboratoires. / Human pathogenic mycoplasmas include respiratory tract species, such as M. pneumoniae and urogenital species, such as M. genitalium. M. genitalium is an emerging agent for which epidemiology is unclear. It is involved in sexually transmitted infections, mainly urethritis in men and cervicitis in women. M. pneumoniae is responsible for acute respiratory infections especially in children. M. genitalium is a fastidious species for which culture remains extremely difficult. In order to extend our collection of samples positive for M. genitalium, a clinical research study (FeminIST) was conducted. It consisted in a PCR screening for M. genitalium in the urogenital tract of HIV-infected women of the Aquitaine cohort. Genotyping methods have been widely applied to Mollicutes, but few simple and automatized methods have been developed for M. genitalium and M. pneumoniae. The MLVA (Multi-Locus Variable-Number Tandem-Repeats Analysis) method analyzes the genome polymorphism associated with tandem repeats. Its advantages are a high discriminatory power and the possibility of being used directly from clinical samples. This technique was applied to M. genitalium and M. pneumoniae and compared with other available genotyping methods. Six and five VNTR were selected for M. genitalium and M. pneumoniae, respectively. The use of multiplex PCR and capillary electrophoresis enabled a high-throughput analysis and allowed an easy interpretation of the results. The method was applied to our collection of 265 M. pneumoniae clinical strains and used directly from 123 clinical samples positive for M. genitalium. 89.4% of M. genitalium PCR-positive samples and all the M. pneumoniae isolates were amplified and typed. We showed a higher discriminatory power for our MLVA than for other genotyping methods, without the need of a fastidious sequencing step. The hierarchical representation of results confirms the M. genitalium species heterogeneity and the M. pneumoniae species homogeneity. MLVA appears to be a good tool for molecular typing of these two mycoplasma species, allowing an easy exchange of data between laboratories.