Dissertations / Theses on the topic 'Bacterial diseases – Diagnosis'

This thesis brings together 118 published studies on the microbiology of sexually transmitted diseases resulting from work performed in the University of Edinburgh Department of Medical Microbiology between 1973 and 1995. The main aim of these studies was to improve microbiological aspects of the diagnosis and management of syphilis and gonorrhoea. The earliest publication on syphilis serology was the first to recommend the use of a specific treponemal antigen test, the Treponema pallidum haemagglutination assay (TPHA) for routine screening. As a result of this study a screening schedule comprising the Venereal Diseases Research Laboratory (VDRL) and TPHA tests was introduced into routine practice in late 1973. Soon the same screening schedule was widely adopted in the United Kingdom and Europe. Appreciating the importance of computerised and automation I validated and standardised a prototype commercial enzyme immunoassay (EIA) as a single serological screening test and demonstrated that this gave a performance comparable to screening with the VDRL and TPHA tests while being suitable for automation and electronic report generation. Screening for syphilis by EIA is now becoming widespread throughout Europe. Because false positive EIA reactions may also show reactivity in the FTA-abs test, immunoblotting was evaluated as a confirmatory test. The possibility of syphilis reactivation and loss of treponemal markers in patients co-infected with HIV were also studied.

History has recorded the antiquity of serious infections in the region of the head and neck. Today, our community still experiences major life-threatening infections in these anatomical locations, which pose significant management difficulties to the oral and maxillofacial surgeon. The aim of this thesis is to review the aetiology, diagnosis and treatment of some bacterial infections involving structures of the head and neck. Such infections may spread, causing serious complications with severe morbidity and occasionally death. This theses deals only with infections of bacterial origin and does not attempt to cover viral, or fungal agents or the chronic specific diseases of tuberculosis and syphilis, and makes no attempt to address the old question of focal infection. The literature review relates especially to Ludwig’s Angina which was first described so dramatically in 1836. To this day it remains as a clinically potentially lethal disease despite the progress of modern medicine. Numerous descriptions in the literature warn of the rapid appearance of symptoms and the danger of respiratory obstruction when management of the airway is not satisfactorily undertaken. Both odontogenic and non-odontogenic causes of orofacial and neck infections are reviewed. Odontogenic problems are given special emphasis as they are now of major concern. The significance of the potential fascial spaces in the face and neck which allow the spread of dental infections is also highlighter. A thorough knowledge of these anatomical relationships is still of the utmost importance to the surgeon if he is to be successful in treatment. The principle of surgical drainage of pus is as important in 1990 as it was 150 years ago. The biological basis for the onset and progress of such fulminating infections in the head and neck region is still poorly understood. One constant need is that the bacteria, both aerobic and anaerobic, be correctly identified. Microbiological techniques are constantly improving and provide an important adjuvant investigation, which then allows the surgeon to provide the most appropriate antimicrobial therapy. Principal to the many aspects of treatment is the ability to maintain the airway of the patient and to provide the depth of anaesthesia necessary to undertake the required surgery. Major bacterial orofacial infections may have severe local and far-reaching systemic effects. Such complications are discussed in all their ramifications. It should be realised that the presentation of these patients at a late stage, when complications have already supervened, may make diagnosis difficult. There is always a necessity to ensure that the underlying cause of the disease is accurately defined and that complication are not allowed to progress further. Finally, a retrospective study of the management of 90 patients with major bacterial orofacial infections who have been treated at Westmead Hospital is presented. The outcome of this study of some major bacterial orofacial infections of the head and neck is the need to stress the importance of urgent surgical management and maintenance of the airway, together with the microbiological determination of the causative organisms and their sensitivities, so that other than empirical antibiotics can be instituted early. This must be combined with an upgrading of the patients’ medical and dental status. It was demonstrated that, in the majority of these patients, ignorance and fear combined with a lack of routine dental care resulted in major infections arising from relatively simple odontogenic causes such as dental caries, periodontal disease and pericoronal infection related to impacted teeth. Without doubt, the immediate care of these patients demanded intensive management. However, it is important to recognise that dental education forms an integral part not only of the recovery programme for the afflicted patient, but also as a community health preventive measure of profound significance.

Master of Dental Surgery
History has recorded the antiquity of serious infections in the region of the head and neck. Today, our community still experiences major life-threatening infections in these anatomical locations, which pose significant management difficulties to the oral and maxillofacial surgeon. The aim of this thesis is to review the aetiology, diagnosis and treatment of some bacterial infections involving structures of the head and neck. Such infections may spread, causing serious complications with severe morbidity and occasionally death. This theses deals only with infections of bacterial origin and does not attempt to cover viral, or fungal agents or the chronic specific diseases of tuberculosis and syphilis, and makes no attempt to address the old question of focal infection. The literature review relates especially to Ludwig’s Angina which was first described so dramatically in 1836. To this day it remains as a clinically potentially lethal disease despite the progress of modern medicine. Numerous descriptions in the literature warn of the rapid appearance of symptoms and the danger of respiratory obstruction when management of the airway is not satisfactorily undertaken. Both odontogenic and non-odontogenic causes of orofacial and neck infections are reviewed. Odontogenic problems are given special emphasis as they are now of major concern. The significance of the potential fascial spaces in the face and neck which allow the spread of dental infections is also highlighter. A thorough knowledge of these anatomical relationships is still of the utmost importance to the surgeon if he is to be successful in treatment. The principle of surgical drainage of pus is as important in 1990 as it was 150 years ago. The biological basis for the onset and progress of such fulminating infections in the head and neck region is still poorly understood. One constant need is that the bacteria, both aerobic and anaerobic, be correctly identified. Microbiological techniques are constantly improving and provide an important adjuvant investigation, which then allows the surgeon to provide the most appropriate antimicrobial therapy. Principal to the many aspects of treatment is the ability to maintain the airway of the patient and to provide the depth of anaesthesia necessary to undertake the required surgery. Major bacterial orofacial infections may have severe local and far-reaching systemic effects. Such complications are discussed in all their ramifications. It should be realised that the presentation of these patients at a late stage, when complications have already supervened, may make diagnosis difficult. There is always a necessity to ensure that the underlying cause of the disease is accurately defined and that complication are not allowed to progress further. Finally, a retrospective study of the management of 90 patients with major bacterial orofacial infections who have been treated at Westmead Hospital is presented. The outcome of this study of some major bacterial orofacial infections of the head and neck is the need to stress the importance of urgent surgical management and maintenance of the airway, together with the microbiological determination of the causative organisms and their sensitivities, so that other than empirical antibiotics can be instituted early. This must be combined with an upgrading of the patients’ medical and dental status. It was demonstrated that, in the majority of these patients, ignorance and fear combined with a lack of routine dental care resulted in major infections arising from relatively simple odontogenic causes such as dental caries, periodontal disease and pericoronal infection related to impacted teeth. Without doubt, the immediate care of these patients demanded intensive management. However, it is important to recognise that dental education forms an integral part not only of the recovery programme for the afflicted patient, but also as a community health preventive measure of profound significance.

Carrión's disease is an overlooked disease restricted to the poorest areas of the Andes, with only a few research groups working in this field around the world. Until recently, it was thought that Carrion's disease met the most relevant criteria for being eradicated, but before that happens a number of issues need to be addressed. One of the problems is the lack of well-defined effective treatment program. Both in vitro antimicrobial resistance studies and clinical trials are needed to determine the best treatment approaches. On the other hand, and perhaps the most urgent need, it is necessity to have an easy way to make the correct diagnosis. New immunological studies will help to identify new molecules with diagnostic potential. The results of this thesis have been separated into 2 different aspects of Carrión's disease; On one hand, antimicrobial resistance (Chapter I) and, on the other hand, the diagnosis and characterization of clinical samples (Chapter II). Chapter I describes a study on resistance mechanisms developed in the presence of the 4 most common antibiotics used in the treatment of Carrion's disease. The ability of Bartonella bacilliformis to become resistant to the main antibiotics used to treat Carrión's disease is evidenced both by the development of antibiotic target alterations and by the overexpression of expulsion pumps. However, total or partial reversion of acquired resistance suggests the existence of a high biological cost derived from the selection of antimicrobial resistance. These instability of the acquired antibiotic resistance may underlie the lack of antibiotic-resistant clinical isolates and the high frequency of microbiological failures during antibiotic treatments. Although studies of mutants obtained in vitro can not be transferred directly to the community, chloramphenicol appears to be the best treatment option in vitro. Chapter 2 analyzes several aspects of the diagnosis of Carrion's disease that are addressed in 3 studies. In the first study, samples obtained from patients of an suspected Oroya fever outbreak were molecularly characterized. However, we propose that this outbreak was erroneously attributed to B. bacilliformis and our results suggest that the causative agent was Sphingomonas faeni. As far as we know, this was the first reported outbreak caused by Sphingomonas spp. acquired in the community, making clear the need to develop diagnostic techniques that can be implemented in endemic areas. In a second study we evaluated the limit of detection of several PCR approaches to detect B. bacilliformis from blood. It seems that the sensitivity of these techniques could allow the diagnosis of acute cases of Carrion's disease, but its applicability to detect healthy carriers or acute cases with low bacteraemia remains unclear. In this article we propose the concomitant use of the 3 PCR approaches in combination with the clinical information. Finally, in the third study of this chapter the objective was the identification and characterization of immunogenic candidates of B. bacilliformis that may in the future be used in a rapid diagnostic tool. Four immunogenic B. bacilliformis candidates were selected: 2 proteins were identified with an anti-human IgM secondary antibody (Pap31 and SCS-α) and another 2 with anti- human IgG secondary antibody (GroEL and SCS-β). The design of treatment schemes that minimize resistance selection along with the development of diagnostic techniques that can be implemented in rural and isolated areas is essential for the control, elimination and eradication of Carrion's disease.
La enfermedad de Carrión es una enfermedad desatendida restringida a las zonas más pobres de la región Andina. Uno de los problemas es la falta de programas de tratamiento eficaces bien definidos. Por otro lado, y quizás sea la necesidad más imperiosa, es preciso tener una manera fácil de realizar el diagnóstico. Nuevos estudios inmunológicos ayudarán a identificar nuevas moléculas con potencial diagnóstico. Los resultados de esta tesis han sido separados en 2 aspectos diferentes de la enfermedad de Carrión; la resistencia a los antimicrobianos (Capítulo I) y el diagnóstico y caracterización de muestras clínicas (Capítulo II). El capítulo I describe un estudio sobre los mecanismos de resistencia desarrollados en presencia de los 4 antibióticos más comunmente utilizados en el tratamiento de la enfermedad de Carrión, evidenciándose la capacidad de Bartonella bacilliformis de volverse resistente a estos antibióticos, tanto por el desarrollo de alteraciones en sus dianas, como por la sobreexpresión de bombas de expulsión. El capítulo 2 analiza aspectos del diagnostico de la enfermedad de Carrión que se abordan en 3 estudios. En el primero se caracterizan molecularmente muestras obtenidas de pacientes de un supuesto brote de fiebre de Oroya. Sin embargo, proponemos que este brote se atribuyó erróneamente a B. bacilliformis sugieriéndose que el agente causante fue Sphingomonas faeni. En el segundo hemos evaluado el límite de detección de varios esquemas de PCR para detectar B. bacilliformis. Parece que la sensibilidad de estas técnicas podría permitir el diagnóstico de casos agudos de la enfermedad de Carrión, pero su aplicabilidad para detectar a los portadores sanos o con una baja bacteriemia sigue sin estar clara. Por fin, en el tercer estudio de este capítulo el objetivo fue la identificación y caracterización de candidatos inmunogénicos de B. bacilliformis que puedan en un futuro ser utilizados en una herramienta de diagnostico rápida. Se identificaron 4 proteínas inmunogénicas: Pap31, GroEL, SCS-α y SCS-β. El diseño de esquemas de tratamiento que minimicen la selección de resistencia junto con el desarrollo de técnicas de diagnóstico que puedan ser implementadas en zonas rurales es esencial para el control y eliminación de la enfermedad de Carrión.

Campylobacter organisms are the most commonly reported bacterial causes of foodborne infection in the world, with Campylobacter jejuni and Campylobacter coli responsible for over 99% of reported infections. Traditionally, Campylobacter species detection is an arduous process, requiring a special incubation environment as well as specific growth media for an extended growth period. The development of a rapid and reliable diagnostic tool for the detection of Campylobacter species would be a valuable aid to the medical diagnostic decision process, especially to rule out Campylobacter infection during the enteric pre-surgical time period. Improved patient outcomes would result if this rapid assay could reduce the number of enteric surgeries. Assays performed during this dissertation project have demonstrated that both SYBR® green and hydrolysis probe assays targeting an 84 nucleotide portion of cadF, a fibronectin-binding gene of Campylobacter jejuni and Campylobacter coli, were able to detect from 101 to 108 copies of organism from stool specimens, did not detect nonspecific targets, and exhibited a coefficient of variation (CV) of 1.1% or less. Analytical validation of sensitivity, specificity and precision, successfully performed in these studies, warrants additional clinical validation of these assays.

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The objective this study produce and evaluate PPD-mallein with potential for application in the diagnosis of glanders from of the the production and purification of immunogenic proteins of B. mallei isolates from equines with glanders in Brazil. Were isolated and characterized phenotypically and by detecting the activity of proteases, polyphenol oxidases, esterases and determination of the resistance profile in vitro. Samples of B. mallei studied were susceptible to antimicrobial gentamicin, ciprofloxacin, norfloxacin, doxycycline and enrofloxacin and resistant to trimethoprim / sulfamethoxazole, amoxicillin and ampicillin. Was observed the activity of proteases, absence esterases polyphenyloxidase and bacterial growth resulted in toxic metabolites. For the production and purification of the protein partially mallein were used two strains of B. mallei Brazilian already characterized phenotypically and genotypically. Was inoculated broth samples Dorset-Henley to metabolize and get the bacterial proteins. Proteins were separated and precipitated with trichloroacetic acid and ammonium sulphate. The PPD-mallein were concentrated at 1.0 mg / mL and evaluation in guinea pigs previously sensitized with bacter were effective for identifying infection in animals truly positive and exclude the animals truly negative. Histological analysis of the application site of mallein revealed the development of Type IV hypersensitivity. Was evaluated the PPD-mallein about their power in five asinines with clinical signs, with bacteriological diagnosis and positive serology for glanders and five asinines negative in serology and bacteriology. The animals were tested according to the criteria established by paragraph IN nº24 regarding the diagnosis of glanders. After 48 hours of inoculation, there was swelling in the area of injection, presence of ocular discharge and tearing confirming the diagnosis of glanders. The other seronegative animals showed no inflammatory reaction at the site of inoculation of PPD-mallein. This immunogen produced and being tested in Brazil was effective being a new possibility for diagnosis and control of glanders in this country.
Objetivou-se nesse estudo produzir e avaliar PPD-maleína com potencial para aplicação no diagnóstico do mormo a partir da purificação de proteínas imunogênicas de Burkholderia. mallei isoladas de equídeos com mormo no Brasil. As bactérias foram isoladas e caracterizadas fenotipicamente por meio da detecção da atividade de proteases, polifenoloxidases e esterases, além da determinação do perfil de resistência à antimicrobianos in vitro. Para produção e purificação parcial da maleína foram utilizadas duas estirpes de B. mallei brasileiras fenotípica e genotipicamente caracterizadas. Inoculou-se as amostras em caldo Dorset-Henley para metabolizar e obter as proteínas bacterianas. As proteínas foram separadas e precipitadas com ácido tricloroacético e sulfato de amônia. As PPDs maleína foram concentradas em 1,0mg/mL e na avaliação realizada em cobaios previamente sensibilizados com a bactéria foram eficazes na identificação dos animais verdadeiros positivos e na exclusão dos verdadeiros negativos. Avaliou-se a PPD-maleína quanto a sua potência em cinco asininos com sinais clínicos e diagnóstico bacteriológico e sorológico positivo para o mormo e em cinco asininos negativos na sorologia e bacteriologia. As amostras de B. mallei estudadas foram sensíveis à gentamicina, ciprofloxacina, norfloxacina, doxiciclina e enrofloxacina, e resistentes a trimetoprim/sulfametoxazol, amoxacilina e ampicilina. Observou-se a atividade de proteases, ausência de esterases e polifenoloxidades e o crescimento bacteriano em meio de cultura resultou em metabólitos tóxicos. A análise histológica do local de aplicação da maleína, em cobaias, revelou o desenvolvimento de hipersensibilidade do tipo IV. Os asininos foram testados de acordo com os critérios estabelecidos pela IN nº24 do Ministério de Agricultura Pecuária e Abastecimento referente ao diagnóstico do mormo. Após 48 horas da inoculação, observou-se edema na área da injeção, presença de secreção ocular e lacrimejamento, confirmando-se o diagnóstico do mormo. Os outros animais sorologicamente negativos não apresentaram reação inflamatória no local de inoculação da PPD-maleína. Esse imunógeno produzido e em fase de teste no Brasil é uma nova alternativa para o diagnóstico e controle do mormo no país.

Une partie de ce travail de thèse a consisté à appliquer les méthodes de FISH pour l’étude de trois bactéries pathogènes intracellulaires. La viabilité de Bartonella henselae a été évaluée à partir de ganglions de patients atteints de la maladie des griffes du chat (CSD). Le faible taux d’ARN détecté par biologie moléculaire, la stérilité des cultures, l'absence de détection par analyses histologiques et FISH confirment que B. henselae n'est pas ou rarement viable dans les ganglions de patients atteints de CSD. Tropheryma whipplei, l’agent de la maladie de Whipple, a été identifié et localisé par FISH, dans les macrophages d’un ganglion et d’une biopsie pulmonaire, confirmant le diagnostic infectieux. Deux méthodes de FISH ont été testées pour détecter Coxiella burnetii dans des cas d’endocardites et d’infections vasculaires en utilisant des sondes oligonucléotidiques et des sondes PNA. Les résultats ont confirmé une meilleure efficacité des sondes PNA et démontré que les techniques de FISH sont plus sensibles que l’immunohistochimie pour le diagnostic des endocardites et des infections vasculaires à C. burnetii. Nous avons également évalué les stratégies moléculaires mises en place pour le diagnostic syndromique. Bien que la PCR conventionnelle à large spectre permette l'identification de micro-organismes fastidieux et anaérobies, la PCR spécifique en temps réel révèle une supériorité significative dans le diagnostic syndromique. En conclusion, ce travail a permis de démontrer l’efficacité et l’applicabilité de la FISH pour la détection bactérienne. Cette méthode peut être utilisée comme un outil complémentaire afin d'améliorer le diagnostic de microbiologie clinique
We applied FISH methods to the study of three intracellular pathogenic bacteria. The viability of Bartonella henselae was evaluated in a large series of lymph nodes from patients with cat scratch disease (CSD). The results obtained, associated with sterile cultures and negative histological analyzes and FISH, as well as the low level of RNA detected by molecular biology, provide evidence that B. henselae are not or are rarely viable in the lymph nodes of patients with CSD. Tropheryma whipplei has been identified by FISH in macrophages from one lymph node and for the first time in a pulmonary biopsy, confirming the diagnosis of infection. Two methods of FISH have been tested to detect Coxiella burnetii in cases of endocarditis and vascular infections using oligonucleotide and PNA probes. The results attested to the greater efficiency of PNA probes, and demonstrated that FISH were applicable for the diagnosis of C. burnetii endocarditis. We also evaluated the molecular strategies used for syndrome-driven diagnosis of infectious diseases. Although conventional broad-spectrum PCR allows for the identification of fastidious and anaerobic microorganisms, real-time specific PCR reveals a significant superiority in syndrome-driven diagnosis. The addition of specific PCRs in real time PCR would improve our molecular strategies, for example, in the case of the detection of Staphylococcus aureus for the diagnosis of lymphadenopathy. In conclusion, this work demonstrates the effectiveness and applicability of FISH for the identification of intracellular bacteria. This method can be used as an important complementary tool to the improvement of clinical microbiological diagnosis

Thesis advisor: Kenneth Williams
Due to overuse and misuse of antibiotics, the global threat of antibiotic resistance is a growing crisis. Three critical issues surrounding antibiotic resistance are the lack of rapid testing, treatment failure, and evolution of resistance. However, with new technology facilitating data collection and powerful statistical learning advances, our understanding of the bacterial stress response to antibiotics is rapidly expanding. With a recent influx of omics data, it has become possible to develop powerful computational methods that make the best use of growing systems-level datasets. In this work, I present several such approaches that address the three challenges around resistance. While this body of work was motivated by the antibiotic resistance crisis, the approaches presented here favor generalization, that is, applicability beyond just one context. First, I present ShinyOmics, a web-based application that allow visualization, sharing, exploration and comparison of systems-level data. An overview of transcriptomics data in the bacterial pathogen Streptococcus pneumoniae led to the hypothesis that stress-susceptible strains have more chaotic gene expression patterns than stress-resistant ones. This hypothesis was supported by data from multiple strains, species, antibiotics and non-antibiotic stress factors, leading to the development of a transcriptomic entropy based, general predictor for bacterial fitness. I show the potential utility of this predictor in predicting antibiotic susceptibility phenotype, and drug minimum inhibitory concentrations, which can be applied to bacterial isolates from patients in the near future. Predictors for antibiotic susceptibility are of great value when there is large phenotypic variability across isolates from the same species. Phenotypic variability is accompanied by genomic diversity harbored within a species. I address the genomic diversity by developing BFClust, a software package that for the first time enables pan-genome analysis with confidence scores. Using pan-genome level information, I then develop predictors of essential genes unique to certain strains and predictors for genes that acquire adaptive mutations under prolonged stress exposure. Genes that are essential offer attractive drug targets, and those that are essential only in certain strains would make great targets for very narrow-spectrum antibiotics, potentially leading the way to personalized therapies in infectious disease. Finally, the prediction of adaptive outcome can lead to predictions of future cross-resistance or collateral sensitivities. Overall, this body of work exemplifies how computational methods can complement the increasingly rapid data generation in the lab, and pave the way to the development of more effective antibiotic stewardship practices
Thesis (PhD) — Boston College, 2020
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

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Made available in DSpace on 2016-10-13T14:32:54Z (GMT). No. of bitstreams: 1 Jose Wilton Pinheiro Junior.pdf: 1486430 bytes, checksum: 24b38026485ee7cc269f6334912b551c (MD5) Previous issue date: 2008-03-07
The objective of this study was to study the production, hygienicalsanitary and reproduction aspects, and the epidemiology bacterial infectious (brucellosis and clamidofilosis) and parasitic (toxoplasmosis) diseases involved in reproduction disturbs in sheep in the state of Alagoas. Twenty seven properties distributed in twenty three municipalities in the state of Alagoas were studied. To the study of the the production, hygienical-sanitary and reproduction aspects, investigative questionnaires involving questions related to the properties were applied and analyzed. To the anti-Brucella abortus antibodies search the Tamponated Acidified Antigen, Modified Tamponated Acidified Antigen and Complement Fixation Reaction techniques were used. The agreement among the tests used in the Brucella abortus infection diagnosis was verified using the Kappa coefficient. For the study of Brucella ovis infection, the immunodiffusion technique in gelatinous agar was employed. To the anti-Chlamydophila abortus antibodies search the Complement Fixation Reaction technique with a 1:32 cutoff point was used. TheToxoplasma gondii infection diagnosis was done based on the indirect immunoflorescency technique considering the cutoff point of 1:64. To the study of risk factors associated with the Clamydophila abortus and Toxosplama gondii infection were selected variables related to properties characteristics, hygienical-sanitary management and reproduction disturbs. The analysis of the sanitary and socio-economic profile allowed to observe that the sheep creator from Alagoas is an individual with reasonable degree of education, that need information about the nutritional, reproductive and sanitary management and they still suffer the effects of the infectious and parasitic diseases occurrence, highlighting the miscarriage and reproduction disturbs. The utilization of biological techniques of reproduction is still ignorant, mainly assembles a natural in most herds studied. It was not observed positive animals to B. abortus and the agreement among the RB, RBm tests comparing to the FCR test was weak (K = 0.00; 0.00). From an amount of two hundred and seventy nine analyzed samples to B. ovis, it was observed that nine (3.2%) were positive and two hundred and seventy (96.8%) negative, distributed in six herds (37.5%) and in sixmunicipalities (46.2%). It was not possible to observe any significant statistics differences among sex, age, region, property size, creation management, significant association was observed for a historical of the reproduction disturbs (p<0,001). The results referents to the C. abortus shown that 59/274 (21.5%) were positive and 187 (68.3%) negative, noting77.7% of focus infection. The only variable that presented a significant association within the multivariate analysis statistics was the region (p<0.001; OR=3.48; I.C. 1.79 – 6.76). To the T. gondii infection, it was possible to see a significant association among the variables: age (OR=4.01; I.C. 2.03 – 7.94), property size (OR=0.48; I.C. 0.26 – 0.90), semi-intensive creation system (OR=3.17; I.C. 1.24 – 8.13), current water fount (OR=3.13; I.C. – 1.66 – 5.87), and the presence of cats (OR=1.72; I.C. 1.08 – 2.75). Based on the obtained results of the sero-epidemiologic inquiry, the conclusion is that the animals are exposed to the B. ovis, C. abortus e T. gondii infection with different epidemiological meanings in the studied population. Sanitary measures and works in health promotion should be encouraged aiming the prevention and control of the dissemination of these important agents in animal and public health.
Objetivou-se com este trabalho estudar aspectos de produção, higiênicosanitário e reprodutivo, além da epidemiologia de algumas doenças infecciosas bacterianas (brucelose e clamidofilose) e parasitária (toxoplasmose) envolvidas em distúrbios reprodutivos em ovinos no Estado de Alagoas. Foram estudadas 27 propriedades distribuídas em 23 municípios nas três mesorregiões do Estado de Alagoas. Para o estudo dos aspectos produtivos, higiênico-sanitário e reprodutivo, foram aplicados e analisados questionários investigativos, abordando perguntas objetivas relacionadas às propriedades. Para a pesquisa de anticorpos anti-Brucella abortus foram utilizadas as técnicas Antígeno Acidificado Tamponado, Antígeno Acidificado Tamponado Modificado e Reação da Fixação do Complemento. A concordância entre os testes utilizados no diagnóstico da infecção por Brucella abortus foi verificada utilizando-se o coeficiente Kappa. Para o estudo da infecção por Brucella ovis empregou-se a técnica de Imunodifusão em Gel de Agar. Para pesquisa de anticorpos anti-Chlamydophila abortus utilizou-se a técnica deReação da Fixação do Complemento com ponto de corte 1:32. O diagnóstico da infecção por Toxoplasma gondii foi realizado com base na técnica de Imunofluorescência Indireta considerado ponto de corte 1:64. Para o estudo dos fatores de risco associados às infecções por Clamydophila abortus e Toxoplasma gondii foram selecionadas variáveis relacionadas às características das propriedades, manejo higiênico-sanitário e distúrbios reprodutivos. A análise do perfil sócio-econômico e sanitário permitiu observar que o ovinocultor alagoano é um indivíduo com razoável grau de escolaridade, necessita de informações sobre práticas de manejo nutricional, reprodutivo e sanitário e ainda sofrem os efeitos da ocorrência de enfermidades infecto-contagiosas e parasitárias, destacando-se o aborto e outros distúrbios reprodutivos. A utilização das biotécnicas da reprodução ainda é insipiente, predominando a monta natural na maioria dos rebanhos estudados. Não foram observados animais positivos para B. abortus e a concordância entre os testes AAT, AATm frente a RFC foifraca (K = 0,00; 0,00). Das 279 amostras analisadas para B. ovis, observou-se que nove (3,2%) foram positivas e 270 (96,8%) negativas, distribuídas em seis rebanhos (37,5%) e em seis municípios (46,2%). Não foram observadas diferenças estatísticas significativas entre sexo, idade, região, tamanho da propriedade, sistema de criação, observou-se associação significativa para histórico de distúrbios reprodutivo(p<0,001). Os resultados referentes à infecção por C. abortus demonstraram que 59/274 (21,5%) animais forampositivos e 187 (68,3%) negativos, constatando-se 77,7% de focos da infecção. A única variável que apresentou associação significativa na análise multivariada foi a região (p<0,001; OR=3,48; I.C. 1,79 – 6,76). Para a infecção por T. gondii, observou-se prevalência geral de 32,9% e o número de focos de 100%. Na análise estatística multivariada, observou-se associação significativa para as variáveis: idade (OR=4,01; I.C. 2,03 – 7,94), tamanho da propriedade (OR=0,48; I.C. 0,26 – 0,90), sistema de criação semiintensivo (OR=3,17; I.C. 1,24 – 8,13), fonte de água corrente (OR=3,13; I.C. – 1,66 – 5,87) e presença de gatos (OR=1,72; I.C. 1,08 – 2,75). Com base nos resultados obtidos nos inquéritos soro-epidemiológicos, conclui-se que os animais estão expostos a infecção por B. ovis, C. abortus e T. gondii com significados epidemiológicos distintos na população estudada. Medidas sanitárias e trabalhos de promoção em saúde devem ser incentivados com objetivo de prevenir e controlar a disseminação destes agentes importantes na saúde animal e coletiva.

Diagnoses of tuberculosis, 'TB,' currently rely upon non-specific techniques such as X-ray exams and acid-fast microscopy. Improved diagnostics would preferably consider specific bacterial processes to provide real-time readouts of disease burden and response to chemotherapy. This dissertation presents the cell-wall incorporation of trehalose analogues (fluorescent and radioactive) by the mycobacterial antigen 85 enzymes as a novel method to label the causative bacteria of TB, Mycobacterium tuberculosis (Mtb). The trehalose mycolyltransesterase enzymes (antigens 85A, B, and C (Ag85)) serve as essential mediators of cell envelope function and biogenesis in Mtb. We show that the Ag85 enzymes display activities so broad that they allow added non-natural carbohydrate probes to be incorporated into Mtb growing in vitro and within macrophages. Design and synthesis of a library of structurally-diverse analogs of the sugar trehalose (Tre) revealed that Ag85-enzymes catalyze esterification of a wide variety of non-natural Tre structures, even stereoisomers and those appended with charged or bulky groups (Chapter 2). A novel mass-spectrometry based Ag85 enzyme assay was developed and employed to screen the library of compounds against all three isoforms of Ag85 (Chapter 3). This screen revealed that the Ag85 enzymes exhibit preference for dissacharides over monosaccharides and a broad tolerance for most modified trehalose compounds. This activity assay also afforded full kinetic analysis and the discovery of a novel, covalent inhibitor of the Ag85 enzymes. The Ag85 activity assay informed the design of a fluorescent trehalose-based compound (FITC-Tre), which is the first, non-toxic, selective, small molecule probe for mycobacterial infection. FITC-Tre was acylated with mycolyl esters by growing mycobacteria, anchoring the probe in the cell envelope resulting in fluorescent bacteria (Chapter 4). Adding FITC-Tre to Mtb-infected macrophages allowed selective, fluorescent tagging of Mtb in vivo (Chapter 5). Colocalization studies with antibodies against a variety of phagosomal associated components have hinted at the possibility of FITC-Tre as readout of cellular trafficking of bacteria. ¹⁸F-trehalose, biotin-trehalose and rhodamine-trehalose are also substrates of Ag85. ¹⁸F-trehalose shows promise as Mtb selective PET probe in an infected rabbit model of tuberculosis. Future work with these probes may allow for fluorescent tracking of the Mtb during the macrophage infection process, as well as the ability to label Mtb in infected tissue. The functional differences between the three isoforms of Ag85, A, B and C, are not well understood and may have implications for the survival and persistence of mycobacteria within humans. The differences in substrate specificity and catalytic activity between the Ag85 isoforms (discussed in Chapter 3) has been further investigated (Chapter 6). Mutation of three secondary site amino acids from Ag85C into Ag85B afforded nearly a twenty-fold gain in enzyme activity. Mutation of the equivalent Ag85B residues into Ag85C triggered nearly a twenty-fold loss in activity. Dissection of the roles of these three amino acids helps to explain the previously reported large differences in catalytic activity between Ag85A, B and C. Overexpression of Ag85A, B and C under tetracycline regulation revealed that these enzymes differentially modulate incorporation of mycolates into the cell wall. The Ag85 enzymes are not functionally redundant, and instead serve unique purposes in cell wall biosynthesis. In summary, this research has demonstrated that the broad substrate tolerance of Ag85 enzymes, coupled with their extracellular location, opens the door to probes of mycobacterial infection using many imaging modalities.

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Espécies de lactobacilos são os principais componentes da microbiota vaginal e a manutenção do predomínio lactobacilar é importante para proteção desse ambiente contra possíveis patógenos. A vaginose bacteriana é uma condição em que se observa a perda de lactobacilos e substituição desses microrganismos por espécies bacterianas, anaeróbias em sua maioria. Tal condição pode acarretar inúmeras complicações ginecológicas e obstétricas, como o aumento do risco de aquisição de infecções sexualmente transmissíveis, parto prematuro e baixo peso ao nascimento. O principal método utilizado para o diagnóstico da vaginose bacteriana é o proposto por Nugent et al. (1991) e se baseia na classificação da microbiota vaginal em flora normal, intermediária e vaginose bacteriana. Enquanto que o perfil imunológico e microbiológico da vaginose bacteriana tenha sido amplamente estudado, pouco se sabe sobre tais características na flora intermediária. Portanto, o objetivo desse estudo foi caracterizar a flora intermediária quanto aos níveis cérvico-vaginais de Interleucina (IL)1-beta, IL-6, IL-8, IL-10, fator de necrose tumoral (TNF)-alfa, antagonista de receptor de IL-1(IL-1ra), sialidases bacterianas e quanto às cargas de Gardnerella vaginalise de bactérias totais, além de verificar se o perfil geral observado na flora intermediária se assemelha ao de mulheres com flora normal ou com vaginose bacteriana. Foi realizado um estudo transversal que incluiu 526 mulheres não grávidas em idade reprodutiva. Deste total, foram constituídos os grupos de estudo de acordo com o padrão de flora vagina, segundo Nugent et al. Todos os 145 casos de vaginose bacteriana foram incluídos nas análises, bem como os 63 casos de flora intermediária e 145 das 318 mulheres que apresentaram flora normal. A determinação dos níveis cérvico-vaginais de citocinas, sialidases e a carga bacteriana foram realizados por, respectivamente, ELISA, ...

Orientador: Camila Marconi
Coorientador: Màrcia Guimarães da Silva
Banca: Andrea da Rocha Tristão
Banca: Ana Katherine da Silveira Gonçalves
Banca: Eliane Melo Brolazo
Banca: Cristina Maria Garcia de Lima Parada
Resumo: Espécies de lactobacilos são os principais componentes da microbiota vaginal e a manutenção do predomínio lactobacilar é importante para proteção desse ambiente contra possíveis patógenos. A vaginose bacteriana é uma condição em que se observa a perda de lactobacilos e substituição desses microrganismos por espécies bacterianas, anaeróbias em sua maioria. Tal condição pode acarretar inúmeras complicações ginecológicas e obstétricas, como o aumento do risco de aquisição de infecções sexualmente transmissíveis, parto prematuro e baixo peso ao nascimento. O principal método utilizado para o diagnóstico da vaginose bacteriana é o proposto por Nugent et al. (1991) e se baseia na classificação da microbiota vaginal em flora normal, intermediária e vaginose bacteriana. Enquanto que o perfil imunológico e microbiológico da vaginose bacteriana tenha sido amplamente estudado, pouco se sabe sobre tais características na flora intermediária. Portanto, o objetivo desse estudo foi caracterizar a flora intermediária quanto aos níveis cérvico-vaginais de Interleucina (IL)1-beta, IL-6, IL-8, IL-10, fator de necrose tumoral (TNF)-alfa, antagonista de receptor de IL-1(IL-1ra), sialidases bacterianas e quanto às cargas de Gardnerella vaginalise de bactérias totais, além de verificar se o perfil geral observado na flora intermediária se assemelha ao de mulheres com flora normal ou com vaginose bacteriana. Foi realizado um estudo transversal que incluiu 526 mulheres não grávidas em idade reprodutiva. Deste total, foram constituídos os grupos de estudo de acordo com o padrão de flora vagina, segundo Nugent et al. Todos os 145 casos de vaginose bacteriana foram incluídos nas análises, bem como os 63 casos de flora intermediária e 145 das 318 mulheres que apresentaram flora normal. A determinação dos níveis cérvico-vaginais de citocinas, sialidases e a carga bacteriana foram realizados por, respectivamente, ELISA, ...
Abstract: Not available
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Submitted by (edna.saturno@ufrpe.br) on 2016-10-10T15:19:58Z No. of bitstreams: 1 Giulliano Aires Anderlini.pdf: 1405471 bytes, checksum: 7d4130063d4be965f6896a3b0207d35f (MD5)
Made available in DSpace on 2016-10-10T15:19:58Z (GMT). No. of bitstreams: 1 Giulliano Aires Anderlini.pdf: 1405471 bytes, checksum: 7d4130063d4be965f6896a3b0207d35f (MD5) Previous issue date: 2009-02-10
The objective of this paper was to determine the prevalence and to identify the risks concernig to Toxoplasma gondii, Neospora caninum and Chlamidophyla abortus goats infections in the State of Alagoas. The research took place on 24 farms of goat breeding from 10 municipalities around the three different Alagoas Mesoregions. A total of 454 goat sera sample were examined for anti-Toxoplasma gondii and anti-Neospora caninum antibodies studies through out indirect immunofluorescence antibody test (IFIAT). To detect anti-Chlamidophyla abortus antibodies 255 goat serum were tested by micro method of complement fixation. The farms were analyzed by questionnaires considering their production system, nutritional, reproductive and sanitary management in order to evaluate the risks of toxoplasmosis in goats. The prevalence of anti-Toxoplasma gondii antibodies was 39% with 95,8% of farms presenting seropositive animals. Significant association was observed to mesoregion (OR = 0,23; I.C. 95% = 0,09 – 0,57), age (OR = 0,36; I.C. 95% = 0,20 – 0,64), semi-intensive herd management (OR = 8,70; I.C. 95% = 1,87 – 40,43), cats accessing to water offered to animals (OR = 3,38; I.C. 95% = 1,89 – 6,02) and cats eating placental remnants (OR = 2,73; I.C. 95% = 1,38 – 5,40). The prevalence of anti- Neospora caninum antibodies was 5,3% with 62,5% of farms presenting seropositive animals with no significant association observed to variables searched as risk factors in this study. Concerning to anti-Chlamidophyla abortus antibodies were observed general prevalence of 1,17%. We conclude that goat herd in the State of Alagoas is exposed to infections by T. gondii, N. caninum and C. abortus with events in different State Mesoregions. It is necessary to intensify and go deep on the epidemiological studies in addition to implementing orientation programs for farmers on sanitary care and goats management in order to reduce the prevalence of infection by those pathogens.
Objetivou-se com este estudo determinar a prevalência e identificar os fatores de risco associados às infecções por Toxoplasma gondii, Neospora caninum e Chlamydophila abortus em caprinos do Estado de Alagoas. A pesquisa foi realizada em 10 municípios, sendo analisadas 24 propriedades de produção de caprinos com aptidão mista situadas nas três Mesorregiões do Estado de Alagoas. Foram coletadas amostras sanguíneas de 454 animais para pesquisa de anticorpos anti-Toxoplasma gondii e anti- Neospora caninum através da prova sorológica de Imunofluorescência Indireta. Para pesquisa de anticorpos anti-Chlamydophila abortus foram utilizadas amostras sanguíneas de 255 matrizes caprinas sendo utilizada a microtécnica da Reação da Fixação do Complemento. Para o estudo dos fatores de risco foram aplicados questionários com questões referentes ao sistema de produção e manejos nutricional, reprodutivo e sanitário. A prevalência geral de anticorpos anti-Toxoplasma gondii foi de 39% com 95,8% das propriedades apresentando animais positivos. Foi observadaassociação significativa para as variáveis: Mesorregião (OR = 0,23; I.C. 95% = 0,09 –0,57), idade (OR = 0,36; I.C. 95% = 0,20 – 0,64), sistema de criação semi-intensivo(OR = 8,70; I.C. 95% = 1,87 – 40,43), acesso dos gatos à água fornecida aos animais (OR = 3,38; I.C. 95% = 1,89 – 6,02) e gatos se alimentando de restos placentários (OR = 2,73; I.C. 95% = 1,38 – 5,40). A prevalência geral de anticorpos anti-Neospora caninum foi de 5,3% com 62,5% das propriedades apresentando animais positivos não sendo observada associação significativa para os fatores analisados. Com relação a anticorpos anti- Clamydophila abortus observou-se prevalência geral de 1,17%. Conclui-se que o rebanho caprino do Estado de Alagoas está exposto às infecções por T. gondii, N. caninum e C. abortus com focos da infecção nas diferentes Mesorregiões do Estado. É necessário intensificar e aprofundar os estudos epidemiológicos além de implementar programas de orientação aos produtores sobre medidas sanitárias e de manejo dos rebanhos caprinos objetivando reduzir a prevalência de infecção por estes agentes patogênicos.

Orientador: Wagner Bettiol
Banca: Antonio Carlos Maringoni
Banca: Carlos Gilberto Raetano
Banca: Regina Maria Villas Boas de Campos Leite
Banca: Marcelo Augusto Boechat Morandi
Resumo: O mercado de produtos de controle biológico, principalmente os que possuem isolados de Bacillus spp. em sua formulação, tem aumentado significativamente, devido às exigências de diminuição do uso de agrotóxicos e maior sustentabilidade do segmento agrícola. Assim, o presente trabalho teve por objetivos: 1- Avaliar o potencial de bactérias do gênero Bacillus no controle de diferentes doenças de plantas; 2- Selecionar isolados de Bacillus spp. promotores de crescimento da soja e antagonistas a Sclerotinia sclerotiorium; 3- Avaliar o potencial de um produto comercial à base de B. subtilis QST 713 e outro à base de óleo de café torrado para compor um programa de manejo da ferrugem-asiática da soja (Phakopsora pachyrhizi) em aplicação sequencial ou alternada com um fungicida. Inicialmente foi avaliado o efeito de B. subtilis, B. firmus e B. amyloliquefaciens no controle dos patógenos Erysiphe diffusa, Podosphaera xanthii, Sclerotinia sclerotiorum, P. pachyrhizi, Fusarium solani, Fusarium oxysporum f. sp. phaseoli, Colletotrichum lindemuthianum e Corynespora cassiicola em condições controladas. Neste estudo, nenhum dos isolados controlou Erysiphe difusa e Podosphaera xanthii. Em folhas destacadas, todos os isolados reduziram o diâmetro da lesão de S. sclerotiorum e a severidade de P. pachyrhizi, sendo mais efetivos com aplicação preventiva. B. subtilis, B. firmus e B. amyloliquefaciens também inibiram o desenvolvimento de F. oxysporum f. sp. phaseoli, C. cassiicola e C. lindemuthia... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The market of products for biological control, especially formulations using Bacillus spp., has increased significantly, due to the requirements for the reduction of the use of agrochemicals in crop protection and sustainability of the agricultural sector. The objectives of this thesis were: 1- To evaluate the potential of Bacillus in plant disease control; 2- To select isolates of Bacillus spp. for growth promotion of soybean and antagonists to Sclerotinia sclerotiorium; 3- To evaluate the potential of a commercial product based on B. subtilis QST 713 and another product based on roasted coffee oil in order to compose a management program for asian soybean rust (Phakopsora pachyrhizi) in sequential or alternating application with a fungicide. Initially, the effect of B. subtilis, B. firmus and B. amyloliquefaciens on the control of pathogens Erysiphe diffusa, Podosphaera xanthii, Sclerotinia sclerotiorum, P. pachyrhizi, Fusarium solani, Fusarium oxysporum f. sp. phaseoli, Colletotrichum lindemuthianum and Corynespora cassiicola was evaluated under controlled conditions. In this study, Bacillus isolates did not controlled Erysiphe diffusa and Podosphaera xanthii. All isolates were effective in reducing the diameter of the S. sclerotiorum lesion and the severity of P. pachyrhizi, being more effective by preventive application in detached leaf. B. subtilis, B. firmus and B. amyloliquefaciens inhibited the mycelial growth of F. oxysporum f. sp. phaseoli, C. cassiicola and C. lindemuthianum ...
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Submitted by (edna.saturno@ufrpe.br) on 2016-11-03T12:56:44Z No. of bitstreams: 1 Romulo Menna Barreto Valenca.pdf: 688661 bytes, checksum: d80c675dd68a0dfbf5ff078ecc666613 (MD5)
Made available in DSpace on 2016-11-03T12:56:44Z (GMT). No. of bitstreams: 1 Romulo Menna Barreto Valenca.pdf: 688661 bytes, checksum: d80c675dd68a0dfbf5ff078ecc666613 (MD5) Previous issue date: 2009-10-05
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The objective of this study was to determine the prevalence and identify the risk factors associated with infection by Leptospira spp. in commercial pig farms in the state of Alagoas, Brazil. To compose the study, were used 342 pigs (312 sows and 30 boars) from at seven swine herds distributed in five districts of the state of Alagoas. The serological diagnosis of infection was done by Microscopic Agglutination Test (SAM). The risk factors analysis was performed by the application of research questionnaires, consisting of objective questions relating to the designer, the general characteristics of the property, the production management, and reproductive health. Detected a prevalence of 16.10% (55/352) of pigs positive. The risk factors associated were: The not perform quarantine (p = 0.003, OR = 5.43, CI = 1.79 -16.41) and artificial insemination utilization (p = 0.023, OR = 3.38, CI = 1.18 - 9, 66). Was foundsignificant association of sows infection with the increased number of stillborn and mummified, and with the increase frequency of estrus recurrence and increased the numbers of days from weaning to service of seropositive sows. It is concluded that infection with Leptospira spp. is disseminated in commercial pig farms in the state of Alagoas, favoring the reproductive failures and decrease of zootechnical performance. The risk factors identified in this study are facilitators in the dissemination of infection and should be adjusted to control the disease in the herds studied.
Objetivou-se com este estudo calcular a prevalência e identificar os fatores de risco associados à infecção por Leptospira spp. em suínos criados em granjas tecnificadas no Estado de Alagoas, Brasil. Para compor a amostra do estudo de prevalência foram utilizados 342 suínos, sendo 312 matrizes e 30 varrões, oriundos de sete granjas de ciclo completo e distribuídas em cinco municípios do Estado de Alagoas. O diagnóstico sorológico da infecção foi realizado pela técnica de Soroaglutinação Microscópica (SAM). A análise dos fatores de risco foi realizada por meio da aplicação de questionários constituídos por perguntas objetivas referentes ao criador, às características gerais da propriedade, ao manejo produtivo, reprodutivo e sanitário. Obteve-se uma prevalência de 16,10% (55/342) de suínos soropositivos. Os fatores associados foram: não realização de quarentena (p = 0,003; OR = 5,43; IC = 1,79 – 16,41) e a utilização da inseminação artificial (p = 0,023; OR = 3,38; IC = 1,18 – 9,66). Detectou-se associação entre a infecção dasmatrizes e o aumento do número de natimortos e mumificados, assim como com a maior freqüência de repetição de cio e aumento do intervalo desmama/estro nas matrizes soropositivas. Conclui-se que a infecção por Leptospira spp. encontra-se disseminada nas granjas suinícolas tecnificadas no Estado de Alagoas e contribui com a ocorrência de falhas reprodutivas e queda nos índices zootécnicos nessas propriedades. Os fatores de risco identificados nesse estudo são facilitadores na disseminação do agente e devem ser corrigidos para controlar a doença nos rebanhos estudados.

The overall objective of this thesis work was to develop new insights into the etiology of scleroderma, a human systemic autoimmune disease, by analyzing the autoantibodies to centrosome antigens that develop during the disease. Centrosomes are perinuclear organelles that form microtubule arrays, including mitotic spindles that ensure the faithful segregation of chromosomes during mitosis. These studies used a novel methodology to determine the prevalence of anti-centrosome autoantibodies in patients with scleroderma. Recombinant centrosome antigens were used to determine the antigenic specificity of anti-centrosome antibody subsets by immunoblotting. Centrosome marker antibodies were used in indirect immunofluorescence assays to distinguish centrosomes within the polymorphic staining pattern frequently given by scleroderma sera. We found that 43% of patients are autoreactive to centrosomes, a prevalence higher than has been reported for any other scleroderma autoantigen. Half of the centrosome-positive patients also had autoantibodies against other antigens used in scleroderma diagnosis. However, in the remaining half of these patients, anti-centrosome antibodies represented the sole class of autoantibodies that was detectable. Anti-centrosome antibodies were detected in only a small percentage of normal individuals and patients with other connective tissue diseases. These data suggest that anti-centrosome autoantibodies may represent a new diagnostic tool in scleroderma. Upon examination of anti-centrosome autoantibody development in an animal model, it appeared that this autoantibody specificity may develop in mice as a consequence of an infection. An infectious agent was isolated by plaque-formation from carrier mice. Further characterization of the infectious agent was undertaken to obtain information on its physical, morphological and cytopathological properties. The infectious agent was identified by sequence and unique antigenic properties to be homologous to the pig pathogen Mycoplasma hyorhinis. When reintroduced into naive mice, the murine mycoplasma triggered anti-centrosome autoantibody development. While anti-centrosome autoantibodies of IgM isotype are part of the repertoire of naive unimmunized mice, mycoplasma infection specifically triggered the development of anti-centrosome IgG. Moreover, centrosome autoreactivity was prevented by antibiotic treatment. The autoantibody response evolved to recruit additional specificities, having IgM isotypes, reactive to endoplasmic reticulum-associated autoantigens.

Bacillus cereus is a group of ubiquitous facultative anaerobic spore forming Gram-positive rods commonly found in soil. It has been detected and implicated in several contaminated food products and raw milk in dairy farms and it causes foodborne gastroenteritis by producing several toxins. This study is aimed at characterizing virulence determinants of B. cereus from cow‟s raw milk. A total of 400 raw milk samples were collected in Fort Hare Dairy Trust and Middledrift Dairy Farm; and cultured on Polymyxin pyruvate Egg-Yolk Mannitol Bromothymol Agar (PEMBA) for 48 hours at 37°C. DNA was isolated from the isolates and 16S rDNA was amplified and sent to Central Analytical Laboratory for sequencing. The gyrB gene of B. cereus was also used to confirm the identity of the isolates. Antibiotic susceptibility profiles of the isolates together with virulence genes were investigated. Multilocus Sequence typing was used to investigate the genetic relatedness of some selected isolates. Furthermore, spores of the isolates were produced, harvested and their concentrations determined. All (100%) of the isolates were identified as having a 96-99% similarity to B. cereus, B. thuringiensis and B. anthracis using sequencing; while gyrB gene was observed in all (100%) of the isolates. Three virulence genes nheA, nheB, nheC encoding for non haemolysin enterotoxin were amplified in all (100%) the isolates. All (100%) of the isolates were susceptible to doxycycline, gentamycin, tetracycline, ciprofloxacin, chloramphenicol and streptomycin. Resistance to rifampicin and penicillin G was predominant with equal rate of 100%, while susceptibility to erythromycin, clindamycin and doxycycline ranged from 60% to 100%. The selected isolates were related and are descendants of the same ancestor. All (100%) the isolates produced spores. The B. cereus isolates contain virulence genes, has multiple antibiotic drug resistance and produce spores, which poses a health risk to the public and cannot be used as probiotics.

Introdução: O diagnóstico microbiológico da infecção por Legionella é complexo, pois a bactéria não é visualizada à coloração de Gram no escarro, e sua cultura não é realizada na maioria dos laboratórios clínicos. A imunofluorescência direta nas secreções respiratórias tem baixa sensibilidade, em torno de 40% e a técnica da “PCR” não é ainda recomendada para o diagnóstico clínico (CDC, 1997). A detecção de anticorpos no soro é a técnica mais utilizada, e o critério definitivo é a soroconversão para no mínimo 1:128, cuja sensibilidade é de 70 a 80% (Edelstein, 1993). Como critérios diagnósticos de possível pneumonia por Legionella, eram utilizados: título único de anticorpos a L pneumophila positivo na diluição 1:256, em paciente com quadro clínico compatível (CDC, 1990) e o achado de antígeno a Legionella na urina (WHO, 1990). Nos últimos anos, porém, com o uso crescente do teste de antigenúria, foram detectados casos de pneumonia por Legionella, que não eram diagnosticados por cultura ou sorologia, tornando-o método diagnóstico de certeza para o diagnóstico de pneumonia por Legionella (CDC, 1997). Por sua fácil execução, resultado imediato, e alta sensibilidade - de 86% a 98% (Kashuba & Ballow, 1986; Harrison & Doshi, 2001), tem sido recomendado para o diagnóstico das PAC que necessitam internação hospitalar (Mulazimoglu & Yu, 2001; Gupta et al., 2001; Marrie, 2001), especialmente em UTI (ATS, 2001). Vários estudos documentaram baixo valor preditivo positivo do título único positivo de 1:256, tornando-o sem valor para o diagnóstico da pneumonia por Legionella, exceto, talvez, em surtos (Plouffe et al., 1995). Outros detectaram alta prevalência de anticorpos positivos na diluição 1:256 na população, em pessoas normais (Wilkinson et al., 1983; Nichol et al., 1991). A partir de 1996, o CDC de Atlanta recomendou que não seja mais utilizado o critério de caso provável de infecção por Legionella pneumophila por título único de fase convalescente ≥1:256, por falta de especificidade(CDC, 1997). A pneumonia por Legionella é raramente diagnosticada, e sua incidência é subestimada. Em estudos de PAC, a incidência da pneumonia por Legionella nos EUA, Europa, Israel e Austrália, foi estimada entre 1% a 16% (Muder & Yu, 2000). Nos EUA, foi estimado que cerca de 8 000 a 23 000 casos de PAC por Legionella ocorrem anualmente, em pacientes que requerem hospitalização (Marston et al., 1994 e 1977). No Brasil, a incidência de PAC causadas por Legionella em pacientes hospitalizados é tema de investigação pertinente, ainda não relatado na literatura. Objetivo: detectar a incidência de pneumonias causadas por Legionella pneumophila sorogrupos 1 a 6, em pacientes que internaram no Hospital de Clínicas de Porto Alegre por PAC, por um ano. Material e Métodos: o delineamento escolhido foi um estudo de coorte (de incidência), constituída por casos consecutivos de pneumonia adquirida na comunidade que internaram no HCPA de 19 de julho de 2000 a 18 de julho de 2001. Para a identificação dos casos, foram examinados diariamente o registro computadorizado das internações hospitalares, exceto as internações da pediatria e da obstetrícia, sendo selecionados todos os pacientes internados com o diagnóstico de pneumonia e de insuficiência respiratória aguda. Foram excluídos aqueles com menos de 18 anos ou mais de 80 anos; os procedentes de instituições, HIV-positivos, gestantes, pacientes restritos ao leito; e portadores de doença estrutural pulmonar ou traqueostomias. Foram excluídos os pacientes que tivessem tido alta hospitalar nos últimos 15 dias, e aqueles já incluídos no decorrer do estudo. Os pacientes selecionados foram examinados por um pesquisador, e incluídos para estudo se apresentassem infiltrado ao RX de tórax compatível com pneumonia, associado a pelo menos um dos sintomas respiratórios maiores (temperatura axilar > 37,8ºC, tosse ou escarro; ou dois sintomas menores (pleurisia, dispnéia, alteração do estado mental, sinais de consolidação à ausculta pulmonar, mais de 12 000 leucócitos/mm3). O estudo foi previamente aprovado pela Comissão de Ética em Pesquisa do HCPA. Os pacientes eram entrevistados por um pesquisador, dando seu consentimento por escrito, e então seus dados clínicos e laboratoriais eram registrados em protocolo individual. Não houve interferência do pesquisador, durante a internação, exceto pela coleta de urina e de sangue para exame laboratoriais específicos da pesquisa. Os pacientes eram agendados, no ambulatório de pesquisa, num prazo de 4 a 12 semanas após sua inclusão no estudo, quando realizavam nova coleta de sangue, RX de tórax de controle, e outros exames que se fizessem necessários para esclarecimento diagnóstico.Todos os pacientes foram acompanhados por 1 ano, após sua inclusão no estudo.Foram utilizadas a técnica de imunofluorescência indireta para detecção de anticorpos das classes IgG, IgM e IgA a Legionella pneumophila sorogrupos 1 a 6 no soro, em duas amostras, colhidas, respectivamente, na 1ª semana de internação e depois de 4 a 12 semanas; e a técnica imunológica por teste ELISA para a detecção do antígeno de Legionella pneumophila sorogrupo 1 na urina, colhida na primeira semana de internação. As urinas eram armazenadas, imediatamente após sua coleta, em freezer a –70ºC, e depois descongeladas e processadas em grupos de cerca de 20 amostras. A imunofluorescência foi feita no laboratório de doenças Infecciosas da Universidade de Louisville (KY, EUA), em amostras de soro da fase aguda e convalescente, a partir da diluição 1:8; e a detecção do antígeno de Legionella pneumophila sorogrupo 1, nas amostras de urina, foi realizada no laboratório de pesquisa do HCPA, pelos investigadores, utilizando um kit comercial de teste ELISA fabricado por Binax (Binax Legionella Urinary Enzyme Assay, Raritan, EUA). As urinas positivas eram recongeladas novamente, para serem enviadas para confirmação no mesmo laboratório americano, ao fim do estudo. Foram adotados como critérios definitivos de infecção por Legionella pneumophila sorogrupos 1 a 6, a soroconversão (elevação de 4 vezes no título de anticorpos séricos entre o soro da fase aguda e da fase convalescente para no mínimo 1:128); ou o achado de antígeno de L pneumophila sorogrupo 1 na urina não concentrada, numa razão superior a 3, conforme instruções do fabricante e da literatura.Os pacientes foram classificados, de acordo com suas características clínicas, em 1º) portadores de doenças crônicas (doenças pulmonares, cardíacas, diabete mellitus, hepatopatias e insuficiência renal); 2º) portadores de doenças subjacentes com imunossupressão; 3º) pacientes hígidos ou com outras doenças que não determinassem insuficiência orgânica. Imunossupressão foi definida como esplenectomia, ser portador de neoplasia hematológica, portador de doença auto-imune, ou de transplante; ou uso de medicação imunossupressora nas 4 semanas anteriores ao diagnóstico (Yu et al., 2002b); ou uso de prednisolona 10 mg/dia ou equivalente nos últimos 3 meses (Lim et al., 2001). As características clínicas e laboratoriais dos pacientes que evoluíram ao óbito por pneumonia foram comparados àquelas dos pacientes que obtiveram cura. Para a análise das variáveis categóricas, utilizou-se o teste qui-quadrado de Pearson ou teste exato de Fisher. Para as variáveis numéricas contínuas, utilizou-se o teste “t“ de Student. Um valor de p< 0,05 foi considerado como resultado estatisticamente significativo (programas SPSS, versão 10). Foi calculada a freqüência de mortes por pneumonia na população estudada, adotando-se a alta hospitalar como critério de cura. Foi calculada a incidência cumulativa para pneumonia por Legionella pneumophila sorogrupos 1 a 6, em um hospital geral, no período de 1 ano. Resultados: durante um ano de estudo foram examinados 645 registros de internação, nos quais constavam, como motivo de baixa hospitalar, o diagnóstico de pneumonia ou de insuficiência respiratória aguda; a maioria desses diagnósticos iniciais não foram confirmados. Desses 645 pacientes, foram incluídos no estudo 82 pacientes, nos quais os critérios clínicos ou radiológicos de pneumonia foram confirmados pelos pesquisadores. Durante o acompanhamento desses pacientes, porém, foram excluídos 23 pacientes por apresentarem outras patologias que mimetizavam pneumonia: DPOC agudizado (5), insuficiência cardíaca (3), tuberculose pulmonar (2), colagenose (1), fibrose pulmonar idiopática (1), edema pulmonar em paciente com cirrose (1), somente infecçâo respiratória em paciente com sequelas pulmonares (4); ou por apresentarem critérios de exclusão: bronquiectasias (4), HIV positivo (1), pneumatocele prévia (1). Ao final, foram estudados 59 pacientes com pneumonia adquirida na comunidade, sendo 20 do sexo feminino e 39 do sexo masculino, com idade entre 24 e 80 anos (média de 57,6 anos e desvio padrão de ±10,6). Tivemos 36 pacientes com doenças subjacentes classificadas como “doenças crônicas”, dos quais 18 pacientes apresentavam mais de uma co-morbidade, por ordem de prevalência: doenças pulmonares, cardíacas, diabete mellitus, hepatopatias e insuficiência renal; neoplasias ocorreram em 9 pacientes, sendo sólidas em 7 pacientes e hematológicas em 2. Dos 59 pacientes, 61% eram tabagistas e 16,9%, alcoolistas. Do total, 10 pacientes apresentavam imunossupressão. Dos demais 13 pacientes, somente um era previamente hígido, enquanto os outros apresentavam tabagismo, sinusite, anemia, HAS, gota, ou arterite de Takayasu. A apresentação radiológica inicial foi broncopneumonia em 59,3% dos casos; pneumonia alveolar ocorreu em 23,7% dos casos, enquanto ambos padrões ocorreram em 15,2% dos pacientes. Pneumonia intersticial ocorreu em somente um caso, enquanto broncopneumonia obstrutiva ocorreu em 5 pacientes (8,5%). Derrame pleural ocorreu em 22% dos casos, e em 21 pacientes (35%) houve comprometimento de mais de um lobo ao RX de tórax. Foram usados beta-lactâmicos para o tratamento da maioria dos pacientes (72,9%9). A segunda classe de antibióticos mais usados foi a das fluoroquinolonas respiratórias, que foram receitadas para 23 pacientes (39,0%), e em 3º lugar, os macrolídeos, usados por 11 pacientes (18,6%). Apenas 16 pacientes não usaram beta-lactâmicos, em sua maioria recebendo quinolonas ou macrolídeos. Dos 43 pacientes que usaram beta-lactâmicos, 25 não usaram nem macrolídeos, nem quinolonas. Em 13 pacientes as fluoroquinolonas respiratórias foram as únicas drogas usadas para o tratamento da pneumonia. Do total, 8 pacientes foram a óbito por pneumonia; em outros 3 pacientes, o óbito foi atribuído a neoplasia em estágio avançado. Dos 48 pacientes que obtiveram cura, 33 (68,7%) estavam vivos após 12 meses. Os resultados da comparação realizada evidenciaram tendência a maior mortalidade no sexo masculino e em pacientes com imunossupressão, porém essa associação não alcançou significância estatística. Os pacientes que usaram somente beta-lactâmicos não apresentaram maior mortalidade do que os pacientes que usaram beta-lactâmicos associados a outras classes de antibióticos ou somente outras classes de antibióticos. Examinando-se os pacientes que utiizaram macrolídeos ou quinolonas em seu regime de tratamento, isoladamente ou combinados a outros antibióticos, observou-se que também não houve diferença dos outros pacientes, quanto à mortalidade. Os pacientes com padrão radiológico de pneumonia alveolar tiveram maior mortalidade, e essa diferença apresentou uma significância limítrofe (p= 0,05). Nossa mortalidade (11,9%) foi similar à de Fang et al. (1990), em estudo clássico de 1991 (13,7%); foi também similar à média de mortalidade das PAC internadas não em UTI (12%), relatada pela ATS, no seu último consenso para o tratamento empírico das PAC (ATS, 2001). Foram detectados 3 pacientes com pneumonia por Legionella pneumophila sorogrupo 1 na população estudada: 2 foram diagnosticados por soroconversão e por antigenúria positiva, e o 3º foi diagnosticado somente pelo critério de antigenúria positiva, tendo sorologia negativa, como alguns autores (McWhinney et al., 2000). Dois pacientes com PAC por Legionella não responderam ao tratamento inicial com beta-lactâmicos, obtendo cura com levofloxacina; o 3º paciente foi tratado somente com betalactâmicos, obtendo cura. Conclusões: A incidência anual de PAC por Legionella pneumophila sorogrupos 1 a 6, no HCPA, foi de 5,1%, que representa a incidência anual de PAC por Legionella pneumophila sorogrupos 1 a 6 em um hospital geral universitário. Comentários e Perspectivas: Há necessidade de se empregar métodos diagnósticos específicos para o diagnóstico das pneumonias por Legionella em nosso meio, como a cultura, a sorologia com detecção de todas as classes de anticorpos, e a detecção do antígeno urinário, pois somente com o uso simultâneo de técnicas complementares pode-se detectar a incidência real de pneumonias causadas tanto por Legionella pneumophila, como por outras espécies. A detecção do antígeno de Legionella na urina é o teste diagnóstico de maior rendimento, sendo recomendado seu uso em todas as PAC que necessitarem internação hospitalar (Mulazimoglu & Yu, 2001; Gupta et al., 2001); em todos os pacientes com PAC que apresentarem fatores de risco potenciais para legionelose (Marrie, 2001); e para o diagnóstico etiológico das pneumonias graves (ATS, 2001). Seu uso é indicado, com unanimidade na literatura, para a pesquisa de legionelose nosocomial e de surtos de legionelose na comunidade.
Introduction: Legionella infections are difficult to diagnose, because the bacteria is not seen at Gram stain and the sputum culture is not performed at most laboratories. Besides that, the direct fluorescent fluorescent antibody test of respiratory secretion has low sensitivity (40%) and detection by PCR techiques is still not recommended for clinical diagnosis (CDC, 1997). The most used test is antibody detection by immunofluorescence technique or by ELISA, with a demonstration of fourfold or greater rise in the reciprocal immunofluorescente antibody (IFA) titer to greater than or equal to 1:128 against Legionella pneumophila serogroup 1 between paired acute-and convalescent-phase serum specimens, which sensitivity ranges between 70 - 80% (Edelstein, 1993).Case definitions for Legionnaires´disease agreed that patients with pneumonia who have positive results in urinary antigen assays or positive results in the direct fluorescent antibody (DFA) staining of respiratory secretions, had “probable” or presumptive” disease (WHO; 1990), as well as those who have single antibody titers of ≥1:256 (CDC, 1990). The Legionella urinary antigen test have been increasingly used in the last years, showing patients with positive results despite of negative culture tests or non-diagnostic serologies. Since then, the urinary antigen test has became a valuable tool in the prompt diagnosis of Legionnaires´disease, and also a definitive criterion for the diagnosis of Legionella pneumonias (CDC, 1997). Due to its high sensitivity, in the range of 86% to 98% (Kashubba & Ballow, 1986; Harrison & Doshi, 2001), it has been recommended to the diagnosis of community-acquired pneumonia which requires hospitalization (Mulazimoglu & Yu, 2001; Gupta et al, 2001), mainly in the ICU (ATS, 2001). Concerning to the “presumptive” criterion of single antibody title of 1:256, in the absence of seroconversion, it was concluded that it shall not be used except in the outbreak setting, since it has been reported to have low predictive value (Plouffe et al, 1995); and has also low specificity (CDC, 1997), since it has been reported high prevalence of positive antibodies at 1:256 in healthy populations (Wilkinson et al, 1983; Nichol et al, 1991). Legionnaires´disease is markedly undiagnosed, either its incidence underestimated. In several studies of CAP conducted in the USA, Europe, Israel and Australia the proportion of pneumonias caused by Legionella has ranged from 1% to 16% (Muder & Yu, 2000). In USA, the incidence of Legionella CAP in patients requiring hospitalization is estimated between 8000 to 23 000 cases per year (Marston et al, 1994 ; Marston et al, 1997). Such incidence in Brazil has not yet been estimated, being an important issue to study Objective : our goal is to detect the incidence of Legionella CAP in patients requiring hospitalization for a year, at the HCPA. Material and Methods: a cohort study ( an incidence study) of adult patients with CAP who were hospitalized for one year ( from 2000-2001) at HCPA. All patients with age 18≥80 were screened for study entry except: residents in institutions, those disabled to walk, those who had been discharged from hospital in the last 15 days; either pregnant women, HIV-positives, or patients with estructural lung diseases (bronchiectasis, cistic fibrosis) or tracheostomized. Admission logs were screened daily from Monday trough Friday (including the ones who had been hospitalized in the week-end) by the researchers. Patients with an admission diagnosis either of pneumonia or acute respiratory failure were evaluated daily by the researchers, and enrolled if they had a Chest X-Ray taken within 48 hours of admission revealing a new infiltrate consistent with pneumonia and at least 1 of the following “ major criteria” : fever (axillary temperature ≥37,8ºC), cough, or sputum; or 2 of the following “minor criteria”: dyspnea, abnormal mental status, signs of consolidation by examination, pleuritic chest pain or abnormal white blood cell count (> 12.000/cm3 or band forms > 4 % ). Information about risk factors, symptoms and outcome was collected through interview and medical chart review. Urine and serum samples were collected from consenting individuals during the acute fase at the hospital. After discharge, they came to the research ambulatory to consultation 4 to 12 weeks after patient enrollment, when the research doctor asked a new Chest X-Ray and serum sample of the convalescent phase to antibody test, along with other necessary exams. All the survivors were followed for a whole year after their inclusion in the study. Acute and convalescent sera were stored at – 70ºC and sent in dry ice (in a “batch”) to the Infectious Diseases laboratory of University of Louisville ( KY, USA), where they were tested by indirect immunofluorescent assay to IgG, IgM, and IgA antibodies to L pneumophila serogroups1-6, starting at dilution of 1:8. It was used a kit test manufactured by Zeus Scientific, Inc (Raritan, NJ, USA). All the urine samples collected were immediately frozen at –70ºC to be further tested in batches, at the Research lab of HCPA, by the investigators, with a commercial EIA kit test manufactured by Binax (BINAX Legionella Urinary Enzyme Assay). The positive ones were refrozen and further sent in a “batch” to the American laboratory, to be retested by the same kit test. Patients were diagnosed as having definite infection by L pneumophila serogroups 1-6 either if they had a 4-fold rise in antibody titer to at least 1:128 or greater dilution; or if they had positive urinary antigen, performed at our lab as recommended by the manfacturer and by the literature. A comparison was made between the patients who died and the survivors, regarding his clinical and laboratory features. Testing procedures to detect significant differences between groups included the Pearson chi-squared test or Fisher exact tests for categorical variables and Student´s t-test for continuous variables. Associations were considered statistically significant if the p value was < 0,05, using a 2-tailed test (SPSS program, version 10). Death by pneumonia was definite as the patient who died primarily due to the worsening of his lung sickness; thus, was calculated the frequency of deaths in our population. Patients who improved and were discharged, were classified as “cured”. Finally, we calculated the cumulative incidence of CAP caused by Legionella pneumophila serogroups 1-6 in a general hospital, for a year. Results: during a whole year, from 645 hospital admission logs with the diagnosis of pneumonia or acute respiratory failure screened, only 82 cases of CAP were obtained. During the follow up in the hospital or ambulatory, 23 patients were excluded either because Chest X-Ray failed to show a new pulmonary infiltrate (5 patients), alternative diagnosis were made (COPD, 5 patients; heart failure, 3; tuberculosis, 2; colagenosis, 1; idiopathic pulmonary fibrosis, 1). Aditional 6 patients revealed exclusion criteria as being HIV positive (1 patient), to have bronchiectasis (4) or pneumatocele (1). Thus, 59 patients constituted the final study group, being each patient enrolled only once. The mean age was 57,6 years (ranging from 24 to 80), being 20 women and 39 men. Most of them ( 36 patients, 61%) had chronic underlying diseases; half of them had more than one disease, being more prevalent: lung diseases, heart diseases, diabete mellitus, liver diseases and renal failure. Regular cigarette smokers represented 61% of the total, and alcohhol intake, 16,9%. Cancer ocurred in 9 patients, being solid organ malignancy in 7 and haematologic malignancy in 2. From our 59 patients, 10 were classified as immunossupressed, defined as splenectomy, haematological malignancy, autoimmune disease, transplant recipient, cancer chemotherapy within 4 weeks (Yu et al, 2002), or prednisolone use ≥10 mg/day (or equivalent), for at least 3 months before admission (LIM et al, 2001). In the remaining 13 patients, only one was previously healthy, while the others had sinusitis, anemia, hypertension, or other mild diseases. At admission, Chest X-Ray showed intersticial pneumonia in only one patient; bronchopneumonia in 59,3% and airspace pneumonia in 23,7%, while both patterns ocurred concomitantly in 15,2%. Obstructive pneumonia (Fang et al, 1990) ocurred in 5 patients with lung cancer. Pleural effusion ocurred in 22%, and in 21 patients (35%) the presentation was multilobar.The antibiotic class most used were beta-lactams, in 72,9% of the patients. The remaining received at most respiratory quinolones and macrolides. From the group that used beta-lactams, 25 patients did not use either quinolones or macrolides.There were not statistic differences in mortality regarding age, sex, or treatment between the groups who received beta-lactams alone versus the group that received macrolides or respiratory quinolones. The only significant association ocurred between radiographic pattern of airspace pneumonia and greater mortality (p= 0,05). In this study 3 patients had pneumonia caused by Legionella pneumophila serogroup 1: 2 patients had seroconversion and positive antigen urinary test; the third patient had a positive urinary antigen with negative serologies, like some authors (McWHINNEY et al, 2000). The former two patients worsened with beta-lactams, prescribed before the etiological diagnosis, getting resolution of their pneumonia with levofloxacin; the third one used only beta-lactams, getting cure. There were 7 deaths for pneumonia, and 4 deaths for cancer. From 48 survivors, 33 patients (68,7%) were alive after 12 months. Our mortality rate (13,5%) is similar to the one reported in the literature (ATS, 2001). Conclusions: the incidence of hospitalized CAP by Legionella pneumophila serogroups 1-6 in our hospital in the year 2000-2001 was 5,1%, which represents the annual incidence of Legionnaires´ disease in a general hospital of South Brazil. Comments and perspectives: complementary diagnostic methods like culture, serologies to detection of all classes of immunoglobulins and urinary antigen tests shall be used to detect infections by Legionella in our country to detect the real incidence of pneumonias caused by Legionella species. At the moment, the Legionella antigen test has the greatest yeld among the available tests. It is recommended to all hospitalized PAC patients (Mulazimoglu &Yu, 2001; Gupta et al, 2001); and also to all patients who have potential risk factors for legionellosis (Marrie, 2001), as well as to the etiological diagnosis of severe pneumonias (ATS, 2001). Its use is recommended, with unanimity, to the diagnosis of community and nosocomial outbreaks.

Mon projet de doctorat était de créer un pipeline pour taxono-génomique pour la comparaison de plusieurs génomes bactériens. Deuxièmement, je automatisé le processus d'assemblage (NGS) et annotation à l'aide de divers logiciels open source ainsi que la création de scripts de maison pour le laboratoire. Enfin, nous avons intégré le pipeline dans la description de plusieurs espèces bactériennes de laboratoire sur. Cette thèse est divisée principalement en Taxono- génomique et Microbiogenomics. Les avis de la section taxono-génomique, décrit sur les avancées technologiques en génomique et métagénomique pertinentes dans le domaine de la microbiologie médicale et décrit la stratégie taxono-génomique en détail et comment la stratégie polyphasique avec des approches génomiques sont reformatage de la définition de la taxonomie bactérienne. Les articles décrivent les bactéries cliniquement importantes, leur séquençage complet du génome et les études génomiques comparatives, génomiques et taxono-génomique de ces bactéries. Dans cette thèse, j'ai inclus les articles décrivant ces organismes: Megasphaera massiliensis, Corynebacterium ihumii, Collinsella massiliensis, Clostridium dakarense. Bacillus dielmoensis, jeddahense, Occidentia Massiliensis, Necropsobacter rosorum et Pantoea septica. Oceanobacillus
My PhD project was to create a pipeline for taxono-genomics for the comparison of multiple bacterial genomes. Secondly I automated the process of assembly (NGS) and annotation using various open source softwares as well as creating in house scripts for the lab. Finally we incorporated the pipeline in describing several bacterial species from out lab. This thesis is subdivided mainly into Taxono-genomics and Microbiogenomics. The reviews in taxono-genomics section, describes about the technological advances in genomics and metagenomics relevant to the field of medical microbiology and describes the strategy taxono-genomics in detail and how polyphasic strategy along with genomic approaches are reformatting the definition of bacterial taxonomy. The articles describes clinically important bacteria, their whole genome sequencing and the genomic, comparative genomic and taxono-genomic studies of these bacteria

La bacteriose a e. Chrysanthemi pv. Dianthicola (echr) facteur limitant de la production du dahlia est transmise par la multiplication vegetative. Afin de proposer une methode de diagnostic plus precise que la detection visuelle, les methodes immunoenzymatiques ont ete etudiees et adaptees pour la detection d'echr dans les tissus du dahlia. La methode das-elisa (double antibody sandwich) est evaluee par rapport aux methodes de diagnostic de reference (isolement et immunofluorescence). Son utilisation, pour l'analyse sanitaire du materiel de propagation vis-a-vis d'echr seul et associe eventuellemnt a la mosaique du dahlia (damy), est etudiee en vue d'une selection sanitaire. Les etudes effectuees pour optimiser les reactifs, pour determiner les parametres pouvant modifier la reaction antigene-anticorps

Thesis (Ph. D.)--University of Wisconsin--Madison, 1985.
Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Probiotics are live microorganisms that can confer health benefits to the host. They have long been consumed through fermented foods. While the specific mechanisms of probiotics are largely unclear, there is evidence that their beneficial effects may be attributed to the microbes’ ability to modify the gastrointestinal (GI) environment, to modulate host immune response, or to produce natural products that directly inhibit pathogens in the gut. With the increasing awareness of the important functions that the gut microbiota plays in affecting host heath, probiotics may no longer just stay as simple dietary supplements, but become a promising approach to disease management. With recent advances in synthetic biology, novel functions can be introduced into these “good” microbes to provide additional benefits. Genetically engineered bacteria have been developed to specifically target pathogens or effectively deliver therapeutics to the GI tract. However, there are significant limitations to the existing systems developed. For example, the engineered pathogen sensors largely rely on the similarity between the host and the pathogens, the therapeutics delivery systems are usually constrained by the molecular structures, and the majority of the works have been limited to laboratory settings. In this dissertation, I present a system we have developed based on a food-grade probiotic, Lactococcus lactis, and demonstrate a synthetic biology methodology that could be applied to build biosensors of other pathogens or environmental signals, as well as a generalizable peptide delivery vehicle to the GI tract. I will present my work in three parts. (1) The discovery of an effective antagonistic effect of L. lactis against the infectious diarrheal disease cholera, and elucidation of the mechanism with an infant mouse model. (2) The development of a diagnostic circuit in L. lacits that enables in situ detection of the pathogen and easy readout through fecal sample analysis. (3) The design of a generalizable therapeutic peptide delivery system utilizing the endogenous secretion pathway of L. lacits. Overall, my work exploits the natural and engineered benefits of the probiotic L. lactis and demonstrates its use in the intestinal disease diagnosis and therapy.
2019-02-20T00:00:00Z

Surface-Enhanced Raman Scattering (SERS) has the potential to be a rapid disease diagnostic platform. SERS is a well-known ultrasensitive, label-free method for the detection and identification of molecules at low concentrations. The Raman cross-sections are primarily enhanced by plasmonic effects for molecules close to (< 5 nm) the surface of nanostructured metal substrates. Due to the unique Raman vibration features that provide molecular signatures, we have shown that SERS can provide a rapid (< one hour), label-free, sensitive and specific diagnosis for a number of diseases. This work demonstrates the capability of SERS to be an effective optical diagnostic approach, in particular, for bacterial infectious diseases such as urinary tract infections (UTI) and sexually transmitted diseases (STD), and cancer cell identification. More specifically, this work demonstrates the ability of SERS to distinguish different vegetative bacterial cells with species and strain specificity based on their intrinsic SERS molecular signatures. With the exception of C. trachomatis - the causative agent of chlamydia - whose SERS molecular signatures are found to be aggregated proteins on the cell membrane, all bacterial SERS molecular signatures are due to purine molecules resulting from nucleic acid metabolism as part of the rapid onset of the starvation response of these pathogens. The differences in relative contribution of different purine metabolites for each bacterium gives rise to the SERS strain and species specificity. The ability of SERS to distinguish cancer and normal cells grown in vitro based on changes of SERS spectral feature as a function of time after sample processing is also demonstrated. Furthermore, the difference of spectral features on the gold and silver SERS substrate of the same bacteria can be used as additional attribute for identification. This work demonstrate the potential of SERS platform to provide antibiotic-specific diagnostics in clinical settings within one hour when combined with a portable Raman microscopy instrument, an effective enrichment procedure, multivariate data analysis and an expendable SERS reference library with drug-susceptibility profile for each bacterial strain determined a priori, as well as the ability of SERS platform as a powerful bioanalytical probe for learning about near cell membrane biochemical processes.

碩士
國立交通大學
分子醫學與生物工程研究所
107
Chronic Kidney Disease (CKD) affects 11.9% of adult population in Taiwan. CKD not only affects people's health but also costs large amount of money to provide CKD patient medical resources from the national government. Other complications are problems that can happen with CKD patient such as diabetes, hypertension, and cardiovascular diseases, etc. These complications can lead to a rapid deterioration of the disease and the depletion of existing medical resources. Therefore, early diagnosis and prevention of CKD become more important. In recent years, studies have pointed out that the microbial flora of the intestine is closely related to many diseases. The microbial flora of the intestine is not only co-evolved with humans, but also can affect the health of humans, synthesis of the body's metabolites, inhibition of intestinal pathogens, and reduce the toxicity of toxic substances on the human body has established a good immune system. But not all bacteria are beneficial to humans. Some anaerobic bacteria break down the metabolites of protein synthesis in the intestine but are toxic to humans. Previous studies have confirmed that derivatives of bacterial metabolites, "indoxyl sulfate" are highly accumulated in the blood of patients with chronic kidney disease, and are toxic to vascular cells, accelerating the deterioration of renal function. Therefore, it is more important to evaluate the amount of Indole. By searching for the bacteria that produce Indoxyl-sulfate to design a detection kit, develop a method to evaluate the ability of bacteria to produce Indole to detect the condition of end-stage chronic kidney patients, hoping to be in CKD patients found representative bacteria. It is expected that by changing the intestinal flora and significantly slow down the development of chronic kidney disease and effectively improve the health status of patients.

Yes
The majority of Mycobacterium tuberculosis isolates resistant to isoniazid harbour a mutation in katG. Since these mutations cause a wide range of minimum inhibitory concentrations (MICs), largely below the serum level reached with higher dosing (15 mg/L upon 15–20 mg/kg), the drug might still remain partly active in presence of a katG mutation. We therefore investigated which genetic mutations predict the level of phenotypic isoniazid resistance in clinical M. tuberculosis isolates. To this end, the association between known and unknown isoniazid resistance-conferring mutations in whole genome sequences, and the isoniazid MICs of 176 isolates was examined. We found mostly moderate-level resistance characterized by a mode of 6.4 mg/L for the very common katG Ser315Thr mutation, and always very high MICs (≥19.2 mg/L) for the combination of katG Ser315Thr and inhA c-15t. Contrary to common belief, isolates harbouring inhA c-15t alone, partly also showed moderate-level resistance, particularly when combined with inhA Ser94Ala. No overt association between low-confidence or unknown mutations, except in katG, and isoniazid resistance (level) was found. Except for the rare katG deletion, line probe assay is thus not sufficiently accurate to predict the level of isoniazid resistance for a single mutation in katG or inhA.
European Research Council (Starting Grant INTERRUPTB 311725 to CM, LR and BdJ), The Damien Foundation

My placement was with the Zoonoses, Foodborne and Emerging Infectious Diseases section in the Office of Health Protection at the Australian Government Department of Health during 2014-2015. I focused on the following five projects. I described the epidemiology of bacterial toxin mediated foodborne outbreaks in Australia and identified risk factors and risk groups, from an analysis of outbreak data recorded from 2001 to 2013. The main risk factor for bacterial toxin mediated foodborne outbreaks is temperature abuse (storage between 4C and 60 C) of solid masses of foods, such as lasagna. Residents of aged care facilities were the group at highest risk of illness and death from bacterial toxin mediated foodborne outbreaks. Few outbreaks were identified with food prepared at home. As part of the national response to the large outbreak of Ebola virus disease (EVD) in West Africa (2014-2015), a national surveillance system was established to enable reporting and to provide information to jurisdictions to assist monitoring of travellers at risk of contracting EVD. I conducted an evaluation of this system. The system was considered useful and achieved its aims; however a coordinated central, online database would improve reporting and ease of use. Culture independent diagnostic testing (CIDT) for bacterial causes of gastroenteritis is becoming commonly used in pathology laboratories in Australia. These tests are rapid, cheap and require less technical expertise than traditional culture based laboratory tests. However, these tests do not provide the subtyping information required for public health surveillance, including outbreak detection. For the time being, laboratories are continuing culture in addition to CIDT. My project documents this transition in Queensland. In May 2014, three cases of bacteraemia caused by Ralstonia species were diagnosed in South Australia. The only common exposure was propofol, a sedative. An investigation into the cause of this cluster of cases was led by the Therapeutic Goods Administration (TGA) as the agency responsible for the regulation of propofol. Additional cases were identified from Queensland (four cases), and Victoria (one case). I was part of the epidemiology team assisting and advising the TGA. The investigation incorporated evidence from the case series, molecular analysis of isolates (including whole genome sequencing), assessment of causal association and expert consultation through the Delphi method. The cases of Ralstonia bacteraemia were determined to be associated with at least two separate exposures. Cases in Queensland were linked to contaminated bottled water, while cases in South Australia and Victoria were associated with propofol. The mechanism of contamination of the propofol was unable to be determined. During a multi-jurisdictional investigation into an outbreak of hepatitis A associated with consumption of frozen mixed berries in 2015, a national case control study was conducted. I assisted OzFoodNet Queensland with interviewing controls and conducted a sub-analysis of cases and controls from Queensland. Frozen mixed berries were the only food exposure with a statistically significant association with illness, supporting the implication of frozen mixed berries as the source of the outbreak, as indicated by traceback and microbiological evidence.

No
Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.
European Research Council grant (INTERRUPTB; no. 311725), European Research Council grant (TB-ACCELERATE; no. 638553), Foundation for Innovative New Diagnostics, German Center for Infection Research (DZIF), Deutsche Forschungsgemeinschaft (German Research Foundation) under Germany’s Excellence Strategy (EXC 22167–390884018), FWO Odysseus G0F8316N, US National Institutes of Health BD2K K01 (MRF ES026835), Agence Nationale de la Recherche (ANR-16-CD35-0009)