Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: leaves 134-165.

Data compiled by the Centers for Disease Control demonstrates that African American women, in particular young people between the ages of 14-25, have an increased incidence of infection with the sexually transmitted pathogen Neisseria gonorrhoeae. Estradiol is a key regulatory hormone in female reproductive function. It has been studied extensively in the cardiovascular field, and has been linked to breast and endometrial cancers in women. However, its impact on infectious diseases is largely unknown. Given what is known about the effect of estradiol on immunologic and inflammatory disorders in women, I hypothesize that estradiol alters the infectivity of Neisseria gonorrhoeae in the female reproductive tract by altering the host inflammatory immune response. This may explain a risk factor for increased rates of infection in some populations. I sought to develop a relevant in vitro model. After screening a number of candidate cell lines, I selected the human endometrial adenocarcinoma cell line, Ishikawa. These cells express specific estrogen receptors and respond to exogenous estrogen stimulation. Estrogen treatment of Ishikawa cells did not have an impact on the invasion of N. gonorrhoeae, nor did it impact bacterial growth. However, gonococcal induced chemokine secretion was reduced by estrogen, as measured by interleukin-8 secretion. I conclude that estrogen blunts the inflammatory response to Neisseria gonorrhoeae without altering bacterial infectivity.

Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be treated with readily available antibiotics, but patients in low-resource settings often do not return to the clinic for results. As current NG diagnostic gold standards suffer from slow turnaround time to result, a rapid, sensitive molecular diagnostic would help increase appropriate treatment at the point-of-care. Here, we report on the design and development of a minimally-instrumented paper-fluidic POC diagnostic that incorporates patient swab sample lysis, isothermal nucleic acid capture, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and lateral flow visual detection. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and device clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept sensitivity and specificity study. This proof-of-principle paper-fluidic diagnostic, which can be completed within 80 minutes, selectively amplifies and detects NG in multiplex with NGIC. It has a clinically relevant limit of detection of 1000 NG cells per device. In urethral swab sample trials (N=20), the device approaches current gold standard NG diagnostic capabilities with 90% sensitivity and 100% specificity and vaginal swab sample trials (N=20) surpass the gold standard with 100% sensitivity and 100% specificity.

Made available in DSpace on 2015-12-06T23:47:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2008 / Introdução: Os profissionais de saúde estão expostos a vários riscos no desenvolvimento de suas atividades, no ambiente hospitalar, especialmente os biológicos. As doenças infectocontagiosas são as principais fontes de transmissão de microrganismos de pacientes para profissionais, através de diferentes vias. A doença meningocócica, cuja transmissão é respiratória, causa pânico na população, inclusive entre estes profissionais, devido ao seu caráter epidêmico e letalidade elevada. Objetivos: Determinar a prevalência de portadores de Neisseria meningitidis em profissionais de saúde recém-admitidos em um hospital escola e verificar a colonização da nasofaringe destes profissionais expostos ao microrganismo 12 meses após a admissão, bem como a aplicação das medidas de prevenção e uso de quimioprofilaxia preconizadas. Métodos: Estudo de painel de medidas repetidas, onde os indivíduos selecionados foram avaliados, através de coleta de cultura de nasofaringe, antes da exposição e, num segundo momento, doze meses após a exposição já definida anteriormente. Participaram da pesquisa 117 profissionais de saúde aprovados em concurso púbico e lotados no hospital entre janeiro de 2004 e dezembro de 2005. A análise descritiva das variáveis qualitativas foi apresentada em valores absolutos e relativos e as quantitativas através das medidas de tendência central e de distribuição do conjunto de medidas registradas. Foi utilizado o teste de McNemar para comparar a distribuição de duas variáveis correlacionadas. Resultados: Os 117 profissionais de saúde avaliados apresentaram uma média de idade de 34 anos e 86% eram da categoria de enfermagem. Apenas 3% eram portadores de Neisseria meningitidis no exame admissional. Distribuídos nos setores do hospital, 76% foram selecionados para enfermarias, pronto atendimento e unidade semi-intensiva. Cerca de 66% dos profissionais trabalharam anteriormente em serviços de saúde e 46,1% exerciam, concomitantemente à admissão no hospital, a mesma atividade em outras unidades. Apenas 19% referiram contato recente com pacientes com doença meningocócica e 90% foram orientados a usar máscara ao prestarem assistência a pacientes com esta doença. No intervalo estudado, somente 4% destes profissionais utilizaram rifampicina como quimioprofilaxia, sendo que 68% tiveram contato com pacientes internados com doença meningocócica neste período. Apesar da orientação sobre o uso de máscara, 32% não a utilizaram. Não houve registro de nenhum resultado positivo para Neisseria meningitidis, na cultura realizada nestes profissionais, após um ano de exposição ao microrganismo, no ambiente hospitalar. Conclusões: Não houve colonização dos profissionais de saúde pela Neisseria meningitidis, o que nos permite corroborar as medidas de precaução, padronizadas universalmente, para prevenção da doença meningocócica no ambiente hospitalar / Introduction: Healthcare professionals are frequently exposed to a variety of hazards and risks whilst carrying out their activities in hospital environments, especially with regards to biological risks. The contagious diseases are the main source of transmission of infectious agents from patients to professionals through a wide and different variety of ways. The meningococcal disease, which is transmitted from respiratory secretions, causes panic in the population including these very professionals due to epidemic and lethal character. Objectives: To determine the prevalence of carriage of Neisseria meningitidis amongst the healthcare professionals whom have been recently admitted in a hospital and to verify the colonization in the nasopharynx of these very professionals exposed to the agent 12 months after their admission as well as the use of preventive measures. Methods: A panel study of repeated measurements where the selected individuals had been evaluated through the collection of cultures from the nasopharynx before exposure and, in a second instance, 12 months after the exposure previously defined. Between January 2004 and December 2005, 117 healthcare professionals, whom were successfully approved in a public admission exam and were duly assigned to a hospital, have participated in the study. The descriptive analysis of the qualitative variables was presented in absolute and relative values and the qualitative variables were presented through the measures of central tendency and distribution tendency from the set of registered measures. The McNemar Test was used in order to compare the distribution of the two correlated variables. Results: The evaluated 117 healthcare professionals presented an average age of 34 years of age and 86% were from the Nursing Category. Only 3% were carriers of N. meningitidis at the admission exam. When distributed throughout the hospital sections, 76% were selected for wards, emergency wards and semi intensive units. Approximately 66% of the professionals had previous experience in the health settings and 46.1% performed, concomitantly to the admission in the hospital, the same activity in other units. Only 19% mentioned recent contact with patients suffering from meningococcal disease and 90% were duly orientated to use a mask whenever assisting patients suffering from this disease. During the studied interval, only 4% of these professionals used rifampicin as chemoprophylaxis although 68% had actually had contact with interned patients with meningococcal disease within the said period. Independently from the orientation regarding the use of masks, 32% of them did not use it. There were no registrations of a positive result for Neisseria meningitidis in the culture carried out on these professionals after one year of exposure to the agent within the hospital environment. Conclusion: There was no colonization in the healthcare professionals by N. meningitidis thus we may corroborate the universally standardized preventive measures for the meningococcal disease in a hospital environment. / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2015-07-08T12:28:14Z (GMT). No. of bitstreams: 2 17.pdf: 1549058 bytes, checksum: d1d338fb698a95bb0d0ea23528f8cf74 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A doença meningocócica permanece como um problema de saúde pública, estando relacionada a altas taxas de morbidade e mortalidade, principalmente em crianças e lactentes. A rápida evolução da doença requer uma intervenção terapêutica específica e precoce, e enfatiza a necessidade de disponibilizar vacinas que possam ser utilizadas na prevenção dessa doença. Estratégias utilizando proteínas de membrana externa de Neisseria meningitidis como antígeno estão sendo desenvolvidas para a produção de uma vacina universal contra o sorogrupo B, que pode, inclusive, conferir proteção contra outros sorogrupos. Duas vacinas se encontram atualmente em ensaios clínicos. A vacina recombinante rfHbp possui duas variantes da proteína fHbp (V1 e V2) como antígeno, e a vacina 4CMenB é uma vacina multicomponente que inclui três proteínas (fHbp, NHBA e NadA) combinadas com OMVs da cepa epidêmica da Nova Zelândia. Embora estes antígenos sejam relativamente conservados, os mesmos já demonstraram variabilidade em diferentes regiões. O objetivo deste estudo foi avaliar a prevalência e diversidade genética destes antígenos em isolados clínicos de N. meningitidis sorogrupos B e C circulantes no Brasil. A caracterização dos isolados pelo MLST revelou seis complexos clonais principais (CCS ST-41/44, ST-1136, ST-32/ET-15, ST-8/A4, ST-11/ET-37 E ST 103). Todos os isolados apresentaram o gene de fHbp, onde a variante 1 foi predominante para sorogrupo B e a variante 2 para o sorogrupo C. O gene NHBA também foi identificado em todos os isolados. Apenas 29,73% dos isolados apresentaram o gene nadA. Nenhuma das cepas analisadas apresentou o mesmo subtipo de PorA presente na OMV que compõe a vacina, não sendo interessante a inclusão desta vesícula em uma possível vacina brasileira. Apenas três variantes de VR3 de PorA foram identificadas, demonstrando a baixa variabilidade desta região da proteína e a possível aplicação em uma vacina. / Meningococcal disease remains a public health problem causing high case-fatality rates worldwide, especially among young children and young adults. The rapid evolution of the disease requires specific and fast therapeutic actions and the use of prophylaxis through vaccination is the best choice to halt the spread of the disease. New vaccine approaches using surface exposed proteins from Neisseria meningitidis have been developed for the design of a universal vaccine against serogroup B, which could also elicit protection against other serogroups. Two vaccines using this strategy are currently through clinical trials. The recombinant vaccine rfHbp with two fHbp antigenic variants (V1 and V2) and the multicomponent vaccine 4CMenB including three proteins (fHbp, NHBA and NadA) combined with an OMV from the New Zealand vaccine strain. Although these are highly conserved antigens, some genetic variation has been already observed within their genes. The main objective of this study was to evaluate the prevalence and genetic diversity of these antigens among N. meningitidis serogroup B and C isolates circulating in Brazil. The characterization of such isolates by MLST analysis, revealed the presence of six main clonal complexes (CCS ST-41/44, ST-1136, ST-32/ET-15, ST-8/A4, ST-11/ET-37 E ST 103). The gene fHbp was amplified from all isolates and Variant 1 was predominant among serogroup B and Variant 2 among serogroup C. Gene NHBA was also amplified from all isolates. Gene nadA was only amplified form 29.73% of all isolates. We didn´t find any strain matching the same PorA subtype of the OMV used in the 4CMenB vaccine, thus the inclusion of such bacterial structure in a future vaccine to be used in Brazil would be useless. Only three PorA VR3 variants were detected showing the low diversity of this protein region and the possible inclusion of this structure on a future vaccine. We have detected a possible indication of capsule “switching” among some isolates which shows the need of a wide surveillance of meningococcal strains that can escape the protection of serogroup C conjugate vaccines. The rfHbp vaccine suggested that it could show a higher efficiency when compared with 4CMenB, but it is important to cover possible scape mechanisms showed by several strains.

Made available in DSpace on 2015-07-08T12:28:20Z (GMT). No. of bitstreams: 2 14.pdf: 2053160 bytes, checksum: 6a9f2af0a7daaa1f0f06673bc00622fc (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A doença meningocócica (DM) é uma enfermidade aguda, grave, de evolução rápida com taxas de mortalidade de 15 a 20% e que atinge principalmente as faixas etárias mais jovens, considerada um grave problema de saúde pública mesmo em países desenvolvidos. A DM está associada à colonização da nasofaringe de humanos, único hospedeiro, por uma bactéria Gram negativa, Neisseria meningitidis. O método considerado padrão ouro para a tipificação molecular da Neisseria meningitidis é o MLST, que consiste no sequenciamento de 7 genes Housekeeping , o MLST é uma técnica complexa que exige uma demanda de custo e tempo, tornando-se inviável sua implementação em laboratórios públicos que recebem amostras de Nm, uma estratégia que poderá contornar este obstáculo seria a PCR em tempo real que com poucas reações e em poucos dias fornecerá o perfil de MLST de isolados com alta porcentagem de confiabilidade. O objetivo do estudo é detectar os complexos clonais do MLST causadores dos grandes surtos de DM através de sondas específicas pela PCR em Tempo Real Qualitativa. A análise pelo MLST de 98 isolados estudados caracterizou todos os 15 isolados do sorogrupo B como integrantes do complexo clonal ST-32/ET-5 além de outras 11 cepas do sorogrupo C. O ST-103 foi o complexo clonal predominante entre as cepas do sorogrupo C com 43 isolados, outros STs foram encontrados para o sorogrupo C,ST-11 (n=11), ST-41/44 (n=2), ST-8/4A (n=9), ST-35 (n=1), ST-269 (n=1) e outros 5 isolados não foram associados a nenhum complexo clonal. A análise pela PCR em Tempo Real forneceu dados importantes como a caracterização de grande número dos isolados com 100% de certeza e de outros isolados com probabilidade acima de 75%. A PCR em Tempo Real qualitativa mostrou ser uma ferramenta rápida e sensível no auxílio a estudos epidemiológicos para controle de surtos regionais quando há a necessidade de ações rápidas por parte das autoridades de saúde pública. / Meningococcal disease (MD) is an acute illness, severe, rapidly evolving with mortality rates of 15 to 20% and that primarily affects younger age groups, considered a serious public health problem in developed countries. DM is associated with colonization of the nasopharynx of human host by Gram negative bacteria, Neisseria meningitidis (Nm). The method considered the "gold standard" for molecular typing of Neisseria meningitidis is the MLST, which consists in sequencing of 7 genes "Housekeeping", the MLST is a complex technique that requires a time and cost consuming, making it impractical implementation in public laboratories that receive Nm samples, a strategy that could circumvent this obstacle would be the real-time PCR with few reactions in a few days coud provide the MLST profile of isolates with a high percentage of reliability. The objective in this study is to detect the MLST clonal complexes causing major outbreaks of DM through specific probes for Real Time PCR Qualitative method. The analysis by MLST of 98 isolates studied featured all 15 isolates of serogroup B as clonal complex ST-32/ET-5 plus 11 other strains of serogroup C. The ST-103 was the predominant clonal complex among strains of serogroup C isolates with 43. Others STs were found for serogroup C, ST-11 (n=11), ST-41/44 (n=2), ST-8/4A (n=9), ST-35 (n=1), ST-269 (n=1) and other five isolates were not associated with any clonal complex. The analysis by Real Time PCR provided important data such as the characterization of large numbers of isolates with 100% certainty and other isolates with probability above 75%. The Real-Time PCR using the qualitative method proved to be a highly useful tool to aid in rapid and epidemiological studies for outbreak control is necessary when regional rapid actions of public health authorities.

Dissertação (professor titular) - Universidade Federal do Parana, Setor de Ciencias da Saude / Resumo: Os objetivos do presente estudo foram pesquisar a prevalência da bacteria Neisseria gonorrhoeae endocervical em mulheres assintomáticas, usuárias de dispositivo intra-uterino TCu 380A (grupo de estudo - grupo 1) comparando com as usuárias de método anticoncepcional natural ou laqueadura tubária (grupo de controle - grupo 2) e avaliar a percepção da usuária do DIU em relação à informação recebida no serviço. O trabalho foi realizado no Setor de Anticoncepção - Disciplina de Reprodução Humana - Departamento de Tocoginecologia - Universidade Federal do Paraná, no periodo compreendido entre dezembro de 1994 até fevereiro de 1996. A pesquisa constou da avaliação de duzentas pacientes - divididas igualmente nos dois grupos - selecionados aleatoriamente durante o atendimento de rotina nos ambulatórios. O resultado, demonstrou prevalência nula da bactéria Neisseria gonorrhoeae em mulheres assintomáticas de ambos os grupos. A presença de temor de uso do DIU, desconhecimento do método e de seus fatores de risco revelou que a informação recebida foi pouca ou insuficiente para neutralizar as informações que a paciente tinha sobre o método. Sugere-se a necessidade de melhoria do relacionamento médico - paciente para modificar a visão da usuária. / Abstract: The aim of this study is to research the predominance of the endocervical bacteria Neisseria gonorrhoeae in asymptomatic women who use IUDs TCu 380A (group of study - group 1) in comparison with women using natural contraceptive method or tubal ligature (control group - group 2) and to evaluate the perception of the IUD user regarding information received at the clinic. The research was carried out at the Contraception Sector - Human Reproduction Discipline - Tocogynecology Department - Federal University of Paraná, between December of 1994 and February of 1996. The research comprised the evaluation of two hundred patients - divided equally into two groups - selected randomly during the daily routine in the outpatient care. The results showed a null predominance of the bacteria Neisseria gonorrhoeae in asymptomatic women in both groups. The fear of using IUD, the unfamiliarity with the method and its risks revealed that the information received was poor or unsatisfactory for neutralizing information the patients already had about the method. Improvements in the physician-patient relationship are needed to modify the user's point of view.

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No description available.

Die zunehmende Antibiotikaresistenz vieler Krankheitserreger ist ein weltweites Problem, welches zu einem klinischen Bedarf an neuen antimikrobiellen Substanzen führt. Sphingolipide einschließlich Ceramide stellen eine vielfältige Gruppe strukturverwandter Lipide dar und bestehen aus einem Sphingosin-Grundgerüst, welches mit einer Fettsäure verbunden ist. Sowohl das Sphingosin-Grundgerüst allein als auch Sphingolipide zeigen eine antibakterielle Wirkung gegenüber einer Vielzahl pathogener Mikroorganismen. Die Intensität der Hemmung hängt von der Sphingolipidstruktur und dem Mikroorganismus ab. Neuere Studien konnten zeigen, dass Sphingosin, Ceramide und Ceramid-Analoga in N. meningitidis aufgenommen werden und eine bakteriostatische oder bakterizide Wirkung zeigen. Jedoch ist die antibakterielle Wirkungsweise noch nicht genau bekannt. Um mehr über den Wirkmechanismus zu erfahren haben wir die ultrastrukturellen Veränderungen von N. meningitidis nach Inkubation mit azido-funktionalisierten Sphingolipiden mit elektronenmikroskopischen Verfahren (transmissionselektronenmikroskopische und rasterelektronenmikroskopische Aufnahmen) untersucht. Mittels korrelativer Licht- und Elektronenmikroskopie (CLEM) konnten wir die azido-funktionalisierten Sphingolipide nach Aufnahme in N. meningitidis lokalisieren. Zum Anfärben der funktionalisierten Sphingolipide wurde die kupferfreie Azid-Alkin-Cyccloaddition verwendet. / The increasing antibiotic resistance of many pathogens is a worldwide problem that leads to a clinical need of new anti-microbial compounds. Sphingolipids, including ceramides, represent a diverse group of structurally related lipids and are composed of a sphingosine backbone coupled to a fatty acid. Solely the sphingosine backbone as well as the sphingolipids show antibacterial activity against a wide range of pathogenic microorganisms. The rate of inhibition depends on the sphingolipid structure and the microbial strain. Recent studies revealed the uptake of sphingosine, ceramides and ceramide analogues by N. meningitidis and a bacteriostatic or bactericidal effect. However, the mechanism of the bactericidal effect is still unknown. To elucidate the antibacterial mechanism, we studied the ultrastructural changes after incubation of N. meningitidis with azido-modified sphingolipids by using electron microscopy techniques (transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Due to correlative light-electron microscopy (CLEM) we were able to localize the azido-modified sphingolipids after incorporation in N. meningitidis. Copper-free azide-alkyne cycloaddition was used to stain the azido-modified sphingolipids.