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Hospital Universitário Antonio Pedro / A micobacteriose pós-cirúrgica (MPC) vem emergindo nos últimos anos no Brasil e no mundo como uma infecção relacionada à assistência a saúde, representando um grave problema de saúde pública. Em 2006, vários casos foram informados à Secretaria de Estado de Saúde do Rio de Janeiro (SES/RJ). A partir de março daquele ano, teve início um trabalho conjunto dessa Secretaria e do Ministério da Saúde, que definiu diretrizes para a confirmação do surto/epidemia, para o levantamento das causas, para a identificação das espécies do patógeno envolvido e para estabelecer medidas de prevenção e controle. Este estudo teve como objetivo descrever os aspectos clínicos e sociodemográficos dos pacientes atendidos no Serviço de Infectologia do Hospital Universitário Antônio Pedro (HUAP), Universidade Federal Fluminense, com diagnóstico de MPC no período 2006-2009. Casuística e métodos: De abril de 2006 a junho de 2009 foram atendidos no Serviço de Infectologia do HUAP 125 pacientes encaminhados pela SES/RJ por serem casos suspeitos ou confirmados de micobacteriose não tuberculosa adquirida após procedimentos cirúrgicos. Os pacientes chegaram ao HUAP com a ficha própria de notificação de caso de MPC preenchida com os dados da identificação, do procedimento relacionado à infecção, da abordagem diagnóstica e da terapêutica prévia. O tratamento medicamentoso obedeceu às diretrizes estabelecidas pela SES/RJ e pela Agência Nacional de Saúde. Os dados contidos nestas fichas, bem como outras informações concernentes à evolução clínica, tratamento dispensado no HUAP e resposta terapêutica, foram inseridos em um banco de dados especialmente desenvolvido para a pesquisa. Resultados: A maior parte dos casos de MPC ocorreu em pacientes do sexo feminino (77,6%) e a colecistectomia laparoscópica foi o procedimento cirúrgico mais frequente (48,8%). A média do período de incubação foi de 41 dias e a mediana de 31 dias, O sinal clínico mais comum foi a presença de secreção (86,5%), seguida da de nodulações (65,6%). A maior parte dos casos apresentou lesões superficiais e múltiplas (44,8%). Em 45,6% dos casos foram colhidos suabes e tecido para cultura antes do início do tratamento e a positividade deste material foi de 43,5%, valor significantemente maior que o observado quando a coleta de material foi feita após o início do tratamento (16,7%). O tratamento com três fármacos (claritromicina, etambutol e terizidona) foi feito em 90,4% (113/125) dos pacientes, com duração média de 226 dias e mediana de 229 dias. Foram submetidos a pelo menos uma abordagem cirúrgica 77,6% (97/125) dos casos, principalmente aqueles que apresentavam lesões profundas (44/56). Efeitos adversos foram observados em 62,4% (78/125) dos casos, sendo boca amarga o mais frequente. Conclusão: Apesar do longo tempo de tratamento com múltiplos fármacos, a grande maioria dos pacientes aderiu ao tratamento e evoluiu para a cura sem recidivas. / Post-surgical mycobacteriosis (PSM) is emerging as a serious public health problem in Brazil and in the world. In 2006 a number of infections were reported to the Secretary of Health in the Rio de Janeiro State. Starting in March of this year, a joint effort was initiated by this Secretary and the Ministry of Health, in order to set up guidelines to confirm the outbreak/epidemic, to identify its causes, to identify the species of the pathogen involved, and to establish measures of prevention and control. The aim of this work was to describe the clinical and sociodemographic findings of the patients treated at the Infectious Diseases Service of Hospital Universitário Antônio Pedro (HUAP), Universidade Federal Fluminense, who had PSM from 2006 to 2009. Patients and Methods: From April 2006 to June 2009, 125 patients were referred by the Secretary of Health of the Rio de Janeiro State to HUAP with suspected or confirmed PSM. Each patient arrived at HUAP had a PSM case report form with data on identification, infection-related procedures, diagnostic approaches, and previous therapy. The treatment was defined by the Secretary of Health of the Rio de Janeiro State and the Health National Agency. The data from these case report forms, as well as other information associated with clinical evolution, treatment at HUAP, and therapeutic response were inserted into a database specially developed for this research. Results: Most PSM cases occurred in female patients (77.6%) and laparoscopic cholecystectomy was the most frequent surgery (48.8%). The mean incubation period was 41 days (median: 31 days). The most common presentation was drainage (86.5%) and nodules (65.6%). Most cases had multiple and superficial lesions (44.8%). Swab and tissue cultures were performed before treatment in 45.6% of the patients and their positivity was 43.5%, value significantly higher than that found when the specimens were obtained after the onset of treatment (16,7%). Most patients (90.4% - 113/125) were treated with a combined therapy using 3 drugs (clarithromycin, ethambutol and terizidone), with a mean duration of 226 days (median: 229 days). Surgical debridement was performed in 77.6% (97/125) of the cases, mainly in those with deep tissues lesions (44/56). Drug adverse effects occurred in 62.4% (78/125) of the cases, and bitter taste was the most common. Conclusion: In spite of multiple-drug and long-term treatment, most patients adhered to therapy and evolved to cure without relapses.

Resumo: De acordo com a OMS, a tuberculose mata 5000 pessoas por dia, e se não controlada, são estimados 35 milhões de novos casos nos próximos vinte anos. A alta susceptibilidade dos infectados por HIV para a tuberculose, bem como a proliferação da tuberculose multidroga-resistente (MDR), tem criado um grande interesse mundial de expansão nos programas atuais de pesquisas sobre a tuberculose. A Purina Nucleosídeo Fosforilase do Mycobacterium tuberculosis (MtPNP) é um potencial alvo para novas drogas anti-tuberculose visto que é responsável pela síntese de novo de ribonucleotídeos de purina. A Inibição específica da PNP poderia potencialmente levar o M. tuberculosis ao estado latente. O objetivo desde trabalho é resolver a estrutura da MtPNP complexada com adenina, e analisar as interações com os ligantes. A MtPNP foi cristalizada utilizando condições experimentais descritas posteriormente, e o ligante (adenina) foi adicionado por soaking. Os dados de difração de raios X foram coletados no detector CCD utilizando fonte de radiação síncotron (Laboratório Nacional de Luz Síncrotron, LNLS, Campinas, Brazil). O programa MOSFLM foi utilizado para o processamento, e os dados foram escalonados através do programa SCALA, apresentando o grupo espacial ortorrômico P21212 (a=118,96Å, b=134,65 Å, c=44,42 Å). O cristal foi determinado utilizando o método de substituição...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In according to the WHO, the tuberculosis kills 5000 people every day, if does not controlled, are esteemed 35 millions of new cases in next 20 years. The high susceptibility of human immunodeficiency virus infected persons to the disease and the proliferation of multidrug-resistant (MDR) strains have created a worldwide interest in expanding current programs in tuberculosis research. The Purine Nucleoside Phosphorylase from Mycobacterum tuberculosis (MtPNP) is a potential target for new drug anti-tuberculosis, whereas is responsible for de novo synthesis of purine ribonucleotides. The specific inhibition of M. tuberculosis PNP could potentially lead to the latent state of M. tuberculosis. The objective of this work is to solve the structure from MtPNP complexed with adenine, and to analyze their interactions with the ligand. MtPNP was crystallized using the experimental conditions described elsewhere, and the ligand (adenine) was added by soaking. The X-ray diffraction data were collected on a CCD detector using synchrotron radiation source (Laboratório Nacional de Luz Síncrotron, LNLS, Campinas, Brazil). It was used the MOSFLM program to processing, and the data were scaled through the program SCALA, presenting orthorhombic spatial group P21212 (a=118,96Å, b=134,65 Å, c=44,42 Å). The crystal was determined by molecular replacement methods using the program AMoRe. The final model has Rfree and Rfactor of 23.87% and 17.51% respectively, and maximum resolution of 1.86Å. It was observed a large...(Complete abstract click electronic access below) / Orientador: Fernanda Canduri / Coorientador: Walter Filgueira de Azevedo Júnior / Banca: José Roberto Ruggiero / Banca: Flávio Augusto Vicente Seixas / Mestre

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis that has a damaging economic and welfare impact on the livestock industry. Johne's disease in cattle is known to reduce milk yield and carcass value, making it of economic concern to both dairy and beef farmers. In addition, there is cause for concern regarding zoonotic transmission, as there is an unconfirmed but potential relationship between MAP infection and human Crohn's disease, which presents similar clinical symptoms. MAP is most often contracted by neonates through the faecal-oral route, but can also be spread through contact with contaminated milk and colostrum, as well as in utero. Once the host receives an oral dose, the bacteria traverse the gut epithelium and are phagocytosed by gut macrophages residing in the lamina propria and Peyer's patches. MAP are able to evade the macrophage response by resisting intracellular degradation within phagosomes. Infected macrophages respond to the infection by secreting several pro-inflammatory cytokines that drive the downstream immune response and granuloma formation. This work aimed to elucidate key early responses of bovine monocyte derived macrophages (MDM) to MAP infection, and determine the reliability of using the reference strain, K10 (which is likely to have undergone lab adaptation) to model the infection in vitro, by comparing the MDM response to K10 with the response to a recent clinical isolate, C49. At a multiplicity of infection of 5 (MOI 5), there was a significant decrease in K10 intracellular survival (~90%), compared to C49 intracellular survival, over a 24 hour infection time-course. This suggests that K10 may have lost some virulence mechanism through lab adaptation. Understanding the mechanisms of how MDM respond to these two strains could be informative for the design of targeted vaccines When further investigating the MDM response to both strains, it was found that, at MOI 5, MDM infected with K10 secreted higher levels of IL-1β and IL-10, compared to MDM infected with C49. Both cytokines are associated with mycobacterial infection and could perhaps indicate that MDM are more responsive to the K10 strain at early time-points. In addition, MDM infected with K10 produced significantly higher levels of reactive nitrogen species (RNS). RNS are antimicrobial products that can destroy invading pathogens, and have been shown to have bactericidal effects on MAP. The production of RNS could, therefore be a potential mechanism by which MDM are able to kill K10 more efficiently than C49. An additional aim of this project was to understand the importance of the route of phagocytosis in determining the outcome of MAP infection. MDM express several phagocytic receptors, including Fc receptors (FcRs), complement receptors (CR), Ctype lectin receptors and scavenger receptors. This project mainly focused on the role of the mannose receptor (MR) on bacterial uptake and downstream immune responses, as past studies have suggested that other species of mycobacteria such as M. tuberculosis, target the mannose receptor in order to regulate macrophage immune responses. Blocking the MR reduced intracellular survival for both strains of MAP; however, the mechanism by which the MR influences intracellular survival remains poorly understood The effect of opsonisation on MAP prior to uptake by phagocytic cells was also investigated, as presence of opsonins, such a complement proteins and antibody, can change the mechanism by which pathogens are phagocytosed. MAP were incubated in serum from either MAP- negative or MAP- positive cattle, prior to infection and the percentage uptake and survival assessed by performing colony counts. Opsonisation in serum from Johne's negative cattle resulted in marked increase in MAP uptake but not intracellular survival, whereas opsonisation in serum from Johne's positive cattle did not increase uptake but decreased the intracellular survival rate by 24 HPI. This finding highlights a potential protective role of antibody early in the infection process, and could significantly impact how the infection is modelled in future, as anti-MAP antibody may be present in contaminated milk at the point of infection. Taken together, the data presented in this thesis show that bacterial strain has a significant impact on MDM response to MAP infection, which may have important implications for the interpretation of previous studies and the design of future studies investigating host-pathogen interactions in the context of paratuberculosis. Additionally, this work has shown that RNS production and the mechanism of uptake can affect intracellular survival rates, and although this needs further investigation, the findings could have implications for the design of future vaccines.

Em todo o mundo, menos de 5% dos doentes com tuberculose (TB), sejam casos novos ou previamente tratados, tem a avaliação dos isolados quanto ao perfil de sensibilidade aos antibioticos. No Rio Grande do Sul, estado localizado no sul do Brasil, cerca de 4700 casos novos de TB são registrados a cada ano, com uma taxa de cura de 68,9%, e uma taxa de abandono de 7,5%. A identificação rápida da resistência às drogas, em isolados clínicos de M. tuberculosis é importante para o estabelecimento de uma quimioterapia eficaz bem como para evitar a propagação de cepas resistentes. Os objetivos deste estudo foram caracterizar os pacientes de TB com maior risco de possuir TB-­‐MDR, analisando o perfil de resistência às drogas dos isolados e o perfil epidemiológico desses pacientes. Além disso utilizou-­‐se as amostras clínicas para avaliar o teste comercial (GenoType® MTBDRplus) e para desenvolver e padronizar um novo teste (Detect-­‐TBMR) para detectar as mutações mais frequentes associadas a resistência à INH e RIF. Uma proporção significativamente maior (75% versus 20%, p = 0,009) de pacientes do gênero masculino foi encontrada entre os casos resistentes às drogas do que entre os casos suscetíveis. 43,8% dos pacientes demoraram mais de 30 dias para procurar assistência médica e no grupo TB MDR, 25% dos casos não tinha sido submetido a qualquer tratamento prévio anti-­‐TB. Em nossas amostras, encontramos uma proporção de 48,3% de TB-­‐ MDR. A família T foi a família de spoligotipo mais frequente. Comparado com o método da proporções, a sensibilidade e especificidade do ensaio MTBDRplus foram 82% e 94% para a resistência à RIF, 60% e 94% para resistência à INH. Comparado com sequenciamento, a sensibilidade e especificidade do ensaio MTBDRplus foi 92% e 97% para a resistência à RIF e 100% e 100% para a resistência à INH, respectivamente. Para detectar resistência à RIF e INH, o ensaio Detect-­‐TBMDR mostrou sensibilidade e especificidade de 79,3% e 77,0% e 100% e 65%, respectivamente, em comparação com o método da proporções. Comparado com o sequenciamento, a sensibilidade e especificidade do ensaio Detect-­‐TBMDR foi de 81,2% e 94,7% e 100% e 96,2%, para detectar e resistência à RIF e INH, respectivamente. Ainda existem discordâncias entre o método das proporções e a abordagem molecular, particularmente em relação a resistência à INH. Contudo, estes métodos são muito importantes para o manejo mais rápido e correto dos pacientes, auxiliando na escolha do melhor esquema terapêutico. / In most parts of the world, less than 5% of new and previously treated tuberculosis (TB) patients are tested for multidrug resistance (MDR) TB. In Rio Grande do Sul state, the southern most Brazilian state; approximately 4700 new cases of TB are recorded each year, with a cure rate of 68.9%, and a noncompliance rate of 7.5%. Rapid identification of drug resistance in clinical isolates of Mycobacterium tuberculosis is important to facilitate rapid and adequate chemotherapy of TB, and to prevent the spread of resistant strains. The aim of this study was to characterize TB patients at higher risk of having MDR-TB, to analyze the drug resistance and epidemiological profile of these patients. Use the clinical samples to assess the commercial test (GenoType® MTBDRplus) and develop and standardize a new test (Detect-MDRTB) for detecting the most frequent mutations associated with resistance to INH and RIF. A significantly higher proportion (75% versus 20%, p = 0.009) of males were found among drug-resistant cases than drug susceptible cases. 43.8% of patients took longer than 30 days to seek medical care and in the MDR group 25% of the cases did not undergo any previous anti-TB treatment. In our samples we found a proportion of 48.3% of MDR-TB. The T family was the most frequent spoligotype family. Compared with the proportion method, the sensitivity and specificity of the MTBDRplus assay were 82% and 94% for RIF-resistance, 60% and 94% for INH resistance. Compared with sequencing, the sensitivity and specificity of the MTBDRplus assay were 92% and 97% for RIF-resistance, 100% and 100% for INHresistance. To detect RIF and INH-resistance, the Detect-TBMDR assay showed a sensitivity and specificity of 79.3% and 77.0%, and 100% and 65%, respectively, compared to proportion method. When compared with sequencing, Detect-TBMDR assay, to detect RIF and INH-resistance, showed a sensitivity and specificity of 81.2% and 94.7% and to 100% and 96.2%, respectively. Discordances still exist between the proportion method and molecular approach, particularly regarding INH-resistance. However, these methods are very important for the management faster and correct patient, helping to choose the best treatment regimen.

Realizou-se estudo comparando o uso da reação em cadeia da polimerase à evolução clínica e painel de exames tradicionais para diagnóstico em pacientes com suspeita clínica de tuberculose cutânea e micobacteriose atípica. Observou-se sensibilidade da reação em cadeia da polimerase de 88%, especificidade de 83%, valor preditivo positivo de 82%, valor preditivo negativo de 88% e acurácia de 85% com concordância pelo teste de McNemar (p= 0,655). Os exames do painel de maior acurácia, após a reação em cadeia da polimerase, foram o teste tuberculínico com acurácia de 79% e a presença de dermatite crônica granulomatosa com reação em cadeia da polimerase positiva com acurácia também de 79%, ambos com concordância pelo teste de McNemar (p= 0,179 e p= 0,655, respectivamente) / A study was performed comparing the polymerase chain reaction and the traditional panel of exams for the diagnosis in patients with a clinical suspicion of cutaneous tuberculosis and atypical mycobacteria infection to the clinical evaluation. It was observed that the sensitivity of the PCR was 88%, the specificity was 83%, the positive predictive value was 82%, the negative predictive value was 88% and the accuracy was 85% in agreement with the McNemar test (p=0.655). The panel exams of second highest accuracy, were the tuberculin test with an accuracy of 79% and the chronic granulomatous dermatitis with positive PCR, also with an accuracy of 79%, both in agreement with the McNemar test (p=0.179 and p=0.655, respectively)

Thesis (M.Sc. (Medical Microbiology)) --University Limpopo, 2013 / Genotyping of TB is essential to investigate and confirm transmission of the multi-drug resistant tuberculosis and of great value in optimizing strategies for the determination of strains causing the increased mortality rates of TB outbreaks. Sputum samples (207) were collected from National Health Laboratory Services in Polokwane laboratory for determining mutations and genotypes of the Mycobacterium tuberculosis strains using GenoType®MTBDRplus (Hain LifeScience, Germany) and Real-Time PCR (Roche, South Africa) techniques. Of the 207 samples, 28 (13.5%) exhibited drug resistance. Thirteen of the 28 (46%) MDR-TB strains belonged to the non-Beijing family, with mutations at codons rpoB 516 and rpoB 526 for RIF and katG 315 and inhA 15 for INH resistance. The Non-Beijing strains 75% (21/28) were monoresistant to RIF 14% (3/21) at codons 516, 526, 531 of rpoB gene and INH 19% (4/21) at codon 315 of katG and codon 15 of inhA 5% (1/21). Of the eight Beijing strains, 3(8%) were INH- resistant at codon 315 for katG and codon 15 for inhA and 3(8%) were RIF-resistant with mutations at codons 516 and 526. Two samples were typed as MDR for the Beijing strains with codon 315 for INH and codons 526 and 531 for RIF. The sample with a co-infection for Beijing and non-Beijing was an MDR-TB strain with mutations in rpoB codons 526, 531, katG 315 and inhA 8, 15 and16. The study showed a high rate of drug resistance with the non-Beijing compared to Beijing strains and mutations in specific codons for RIF and INH are variable for the TB families.

Trotz weltweiter kostenlos zur Verfügung stehender multi drug therapy und Eradikationsbemühungen der WHO liegt die Leprainzidenz seit fünf Jahren bei etwa 250 000 Neuerkrankungen pro Jahr. Der massive Abfall der Prävalenz seit 1985 ist zum einen rechnerisch und durch die Art der Datenerhebung bedingt (s. 5.1.1), zum anderen lässt sie keine Rückschlüsse auf die Transmissionsereignisse, also die Aktivität der Krankheit zu. Dazu dient die Inzidenz, die nur unverhältnismäßig gesunken ist (s. 2.4.1). Für eine effektive Bekämpfung der Lepra muss jedoch eine Reduktion der Neuerkrankungen erreicht werden. Bekannt ist, dass nur ein Bruchteil der mit M.leprae exponierten Individuen eine manifeste Krankheit entwickelt. Die Suche nach Wirtsfaktoren für eine Lepraerkrankung ist für gezielte Präventions- und Prophylaxe-Maßnahmen, die Neuerkrankungen verhindern sollen, demnach von großer Bedeutung. In der vorliegenden Arbeit werden die Ergebnisse einer Literaturrecherche zum Thema "Talglipide als Wirtsfaktoren für M.leprae" präsentiert und eine Methode zur Talganalyse entwickelt. Anhand wissenschaftlicher Publikationen wurde der aktuelle Stand der Forschung in den verschiedenen Bereichen dieser Hypothese reviewartig dargestellt. Der aktuelle Erkenntnisstand hinsichtlich der Lepraübertragung ist vereinbar mit mehreren Übertragungswegen, wobei die Tröpfchen- und die Hautübertragung favorisiert werden (s. 2.5.1). Die Interpretation der Genomanalysen von M.leprae hat in Kombination mit früheren biochemischen Erkenntnissen Aufschluss über die metabolischen Fähigkeiten von M.leprae gegeben. M.leprae besitzt ein reduktives "Minimalgenom", das sich an die intrazelluläre Nische, in der es lebt, außerordentlich adaptiert hat. Der Verlust wichtiger Kohlenstoffquellen und eine stark eingeschränkte Atmungskette stehen im Gegensatz zu den fast vollständig erhaltenen anabolen und katabolen Stoffwechselwegen der Lipide. Sowohl für die Zellwandsynthese als auch zur Energieproduktion ist M.leprae auf wirtsbezogene Lipide angewiesen. Freie Fettsäuren werden dabei veresterten Fettsäuren vorgezogen (s. 5.2.1.1). Epidemiologische Erkenntnisse stehen ebenfalls in Einklang mit unserer Lipidhypothese. Als Ursache für das Clustering von Leprafällen in Haushalten kommen sowohl genetische als auch zufällig verteilte Faktoren sowie ein auf den gesamten Haushalt wirkender "Haushaltsfaktor" in Frage. Als genetischer Faktor, der die Talglipidzusammensetzung beeinflusst, wären Enzymaktivitäten für die Squalen- bzw. Sapiensäuresynthese denkbar (vgl. 5.2.1.3). Einen in der Bevölkerung zufällig verteilten Suszeptibilitätsfaktor könnte die residente Keimflora der Haut darstellen, die für die Menge der im Talg vorkommenden freien Fettsäuren verantwortlich ist (s. 5.2.1.2). Als möglicher Haushaltsfaktor kommt "Armut" in Betracht, der nachgewiesenermaßen mit schlechter Körper- und Kleidungshygiene einhergeht (s. 5.2.2.3). In Kombination mit einer vermutlich ebenfalls mit Armut assoziierten häufigeren kutanen Verletzung durch Parasiten und Mücken könnte dies eine kutane Schmierinfektion mit M.leprae begünstigen. Für eine Schmierinfektion über kontaminierte Gegenstände spricht auch die gute extrakorporale Überlebensfähigkeit des Erregers und der Nachweis von M.leprae in Bodenproben (s. 5.2.2.2). Die epidemiologischen Daten zu Geschlechterverhältnis und Infektionszeitpunkt lassen ebenfalls eine Rolle der Talglipide als Wirtsfaktoren für eine Lepraerkrankung vermuten (s. 5.2.1.2). Auf Basis der Erkenntnis, dass Lipide und insbesondere Talglipide als Wirtsfaktoren für M.leprae in Betracht kommen, wurde zu diesbezüglich relevanten Talgkomponenten recherchiert. Als trophischer Wirtsfakor kommen die individuell in unterschiedlichen Mengen vorliegenden freien Fettsäuren des Sebums in Betracht. Die Menge an Squalen und Sapiensäure im Talg hat nachgewiesenermaßen erheblichen Einfluss auf die Barrierefunktion der Haut gegen bakterielle Erreger (s. 5.2.1.3). Es wurde folglich eine auf Dünnschicht- und Gaschromatografie beruhende Methode zur Quantifizierung dieser Talgbestandteile entwickelt (s. 4.6). Die Probenentnahme erfolgt mittels Sebutape, was eine bewährte, reproduzierbare und auch unter tropischen Bedingungen leicht anwendbare Methode darstellt. / Despite worldwide free available multi drug therapy and eradication efforts of the WHO, the leprosy incidence is for five years at about 250 000 new cases per year. The massive drop in prevalence since 1985 is on one hand mathematically and due to the way of data collection (see 5.1.1), on the other hand it does not allow conclusions about the transmission events, ie the activity of the disease. An appropriate marker of the disease activity would be the incidence, which is only fallen disproportionately (see 2.4.1). For an effective fight against leprosy, however, a reduction of new cases must be achieved. It is known that only a small part of individuals exposed to M.leprae develop a manifest disease. The search for host factors for leprosy is therefore of great importance for well-directed preventive and prophylactic measures. In the present work the results of a literature review on the subject "Sebum lipids as host factors for M. leprae" and the development of a method for sebum analysis are presented. Based on scientific publications the current state of research in the various areas of this hypothesis was presented review-like . The current state of knowledge regarding leprosy transmission is compatible with multiple transmission paths, wherein the droplets and the skin transmission are favored (see 2.5.1). The interpretation of genome analysis of M. leprae together with previous biochemical findings shed light on the metabolic capabilities of M. leprae. M. leprae has a reductive " minimal genome ", which has become extremely adapted to the intracellular niche in which it lives. The loss of important carbon sources and a severely impaired respiratory chain are in contrast to the almost perfectly preserved anabolic and catabolic pathways of lipids. For both the cell wall synthesis and for energy production M.leprae is dependent on host related lipids. Free fatty acids are thereby preferred to esterified fatty acids (see 5.2.1.1 ). Epidemiological findings are also in line with our lipid hypothesis. Concerning the causes for the clustering of leprosy cases in households, genetic factors as well as randomly distributed factors as well as a “household factor” must be considered. Enzyme activities for squalene- or sapienic acid-synthesis are conceivable as a genetic factor that influences the sebum lipid composition (see 5.2.1.3 ). A randomly distributed susceptibility factor could be the resident bacterial flora of the skin, which is responsible for the amount of free fatty acids occurring in the sebum (see 5.2.1.2 ). The household factor might be "poverty" which has been shown to correlate with poor body and clothing hygiene (see 5.2.2.3 ) . In combination with poverty-related frequent cutaneous injury caused by parasites and mosquitoes, this could favor a cutaneous infection with M. leprae. A smear infection via contaminated objects would be in accord with the good extracorporeal survival of the pathogen and the detection of M. leprae in soil samples (see 5.2.2.2 ) . The epidemiological data on sex ratio and age of infection also point to the fact that sebum lipids might be host factors for leprosy (see 5.2.1.2 ). Based on the recognition that lipids, and in particular sebum lipids must be taken into consideration as host factors for M.leprae, research has been done to identify possibly relevant sebum components. Individually different amounts of free fatty acids in the sebum must be considered as a trophic host factor. The amount of squalene and sapienic acid in the sebum has been shown to have significant influence on the barrier function of the skin against bacterial pathogens (see 5.2.1.3 ). We thus developed a method based on thin-layer and gas chromatography to quantifiy these sebum components (see 4.6). The sampling is done with “Sebutape”, which is a proven, reproducible and easily applicable method, even under tropical conditions.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Mycobacterium vaccae JOB5 and Graphium sp. were studied to evaluate their ability to cometabolize methyl tert-butyl ether (MTBE) and its metabolites after growth on two different alkanes, propane and iso-pentane. Both cultures were capable of cometabolizing MTBE and the metabolites, tert-butyl formate (TBF) and tert-butyl alcohol (TBA). MTBE, TBF, and TBA did not support growth of either microbe. Higher degradation rates were obtained in the bacterial system when the cultures were grown on iso-pentane. Nonlinear least squares regression and direct linear plot methods were used to estimate kinetic coefficients and provided comparable results. The enzymes from Mycobacterium vaccae JOB5 and Graphium sp. that promote the cometabolism of MTBE and its metabolites exhibited similar kinetics and substrate inhibition. The presence of the substrate decreased the degradation rate of MTBE and TBA suggesting competitive inhibition and preference for the substrate. Blockage experiment with acetylene suggested the presence of an alkane monooxygenase for the metabolism of MTBE and TBA, and a hydrolytic enzyme for the degradation of TBF. The presence of a hydrolase enzyme was supported by the fact that TBF was degraded to TBA under either aerobic or anaerobic conditions and was not inhibited by the presence of acetylene, propane, or isopentane. Measured rates of abiotic hydrolysis of TBF were significantly less than biodegradation rates. Acetylene acted as a reversible inhibitor for both cultures when tested in the presence of the growth media and as an inactivator when tested in the presence of a phosphate solution for the bacterial system. Growth-batch reactor experiments were conducted to compare the degradation of iso-pentane and MTBE with the predicted degradation rates based upon kinetic constants determined from single and dual-compound experiments. Experimental data was modeled with Monod kinetics and STELLA�� software. Reasonable predictions of reactor performance were achieved when Monod maximum utilization rates were increased compared to single and dual-compound experiments. / Graduation date: 2002

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.