In der vorliegenden Arbeit wurden neue Untersuchungsmöglichkeiten mit dem BacT/Alert 3D Mykobakterien-Testsystem erprobt. Erstens wurden Untersuchungen durchgeführt, um die Testkonzentrationen für Protionamid (PTH) und Linezolid (LIZ) für die standardmäßige Empfindlichkeitstestung von M. tuberculosis (Mtb) mit dem BacT/Alert 3D-System festzulegen. Dazu wurden die MHK-Werte für 32 Mtb-Stämme bestimmt: Referenzstamm Mtb H37Rv, sensible Patientenstämme, Patientenstämme mit verschiedenen Resistenzen (u. a. PTH-Resistenz) sowie eigens für die Arbeit isolierte LIZ-resistente Mutanten. Die PTH-MHK betrug für 20 von 21 sensiblen Mtb-Stämmen einschließlich des Referenzstammes Mtb H37Rv 0,125 - 1 mg/l (0,25 mg/l bei 11 von 21 Stämmen). Lediglich ein Stamm mit Resistenz gegenüber Isoniazid, Ethambutol und Streptomycin fiel mit einer etwas erhöhten PTH-MHK von 2 mg/l auf. Sechs PTH-resistente Stämme (z. T. mit anderen Resistenzen gegenüber Erstrang-Antituberkulotika) zeigten PTH-MHK von 4 - 16 mg/l. Die Gruppen der PTH-sensiblen und resistenten Stämme zeigten ein bimodales Verteilungsmuster, das mit einem Schwellenwert von 2 mg PTH/l gut zu differenzieren ist. Für die standardmäßige Durchführung der Empfindlichkeitstestung gegenüber PTH mit dem BacT/Alert 3D-System empfehlen wir deshalb eine PTH-Testkonzentration von 2 mg/l. Die LIZ-MHK betrug für 20 sensible Mtb-Stämme (inklusive Referenzstamm Mtb H37Rv) und sieben Stämme mit verschiedenen Resistenzen gegenüber Erstrang-Antituberkulotika 0,25 - 2 mg/l (0,5 mg/l bei 17 von 27 Stämmen). Für die vier isolierten LIZ-resistenten Mutanten betrug die LIZ-MHK 8 - 16 mg/l. Es zeigt sich auch bei der Verteilung der LIZ-MHK ein bimodales Verteilungsmuster; die Gruppen der sensiblen und resistenten Stämme sind gut zu differenzieren. Wir empfehlen für die standardmäßige Durchführung der Empfindlichkeitstestung gegenüber LIZ mit dem BacT/Alert 3D-System eine LIZ-Testkonzentration von 4 mg/l. Die festgestellten MHK-Werte von PTH und LIZ und die vorgeschlagenen Testkonzentrationen entsprechen Ergebnissen aus der Literatur, die mit ähnlichen Methoden erhoben wurden. Zweitens wurden mit dem BacT/Alert 3D-System Untersuchungen zur Kombinationstestung von Antituberkulotika bei Mtb und Stämmen des MAC-Komplexes durchgeführt, bisher liegen keine Publikationen für Untersuchungen von Wirkstoff-Kombinationen bei Mykobakterien mit diesem System vor. Es wurde geprüft, ob die MHK eines Antituberkulotikums durch die Zugabe einer subinhibitorischen Menge eines anderen Antituberkulotikums verändert wird. Bei Mtb wurden dazu folgende Kombinationen geprüft: Rifampicin (RMP) + LIZ, Moxifloxacin + LIZ, Isoniazid + PTH, RMP + PTH, PTH + LIZ. In keinem Fall konnten signifikante Effekte beobachtet werden. Ein tendenziell synergistischer Effekt der PTH-RMP-Kombination beim Stamm Mtb H37Rv (Reduktion der RMP-MHK um eine Stufe) wurde durch die Analyse der Wachstumskinetik des Stammes unterstützt. Bei zufällig ausgewählten Stämmen des MAC-Komplexes wurde die Kombination Ciprofloxacin (CIP) + Ethambutol (EMB) geprüft. Es zeigte sich bei sieben von zehn Stämmen eine Reduzierung der CIP-MHK um mindestens drei Stufen bei Zugabe einer subinhibitorischen Konzentration von EMB. Dieser synergistische Effekt wurde bereits in den 1990er Jahren mit einer ähnlichen Methode festgestellt, allerdings ohne die Stämme des MAC-Komplexes zu differenzieren (Arbeitsgruppe von S. Hoffner). Interessanterweise handelte es sich bei den von uns untersuchten Stämmen, bei denen dieser synergistische Effekt nachgewiesen wurde, um M. avium-Stämme. Diese Problematik sollte weiter verfolgt werden, da sich daraus Konsequenzen für die Therapieempfehlung ergeben könnten.

O gênero Candida é responsável pela maioria das infecções fúngicas nosocomiais. A identificação do provável foco de origem é de extrema importância para elucidar a epidemiologia desse tipo de infecção e nesse sentido, a utilização de métodos de tipagem, que avaliam características fenotípicas e moleculares dos isolados, é essencial. Assim, o objetivo desse estudo foi tipar isolados de C. albicans, antes e após armazenamento e em diferentes ocasiões de coleta, a fim de verificar a manutenção dos biotipos apresentados, e o poder de discriminação dos métodos utilizados. Foi avaliada a microbiota leveduriforme da saliva de 73 estudantes universitários (tempo 0) sendo que após 180 dias (tempo 180) foi realizada nova coleta de saliva daqueles que apresentaram isolamento de C. albicans na primeira coleta. Os isolados foram analisados por ocasião das duas coletas, quanto à produção de exoenzimas fosfolipase e proteinase, pela morfologia das colônias, pelo perfil de suscetibilidade frente à anfotericina B, fluconazol e itraconazol e pela tipagem molecular por RAPD. As leveduras, após isoladas, foram armazenadas em ágar Sabouraud dextrose (ASD) e água destilada esterilizada. Após 180 dias, foram realizadas, novamente, as provas de tipagem. Todos isolados de C. albicans foram produtores de fosfolipase, nas duas coletas, embora tenha havido oscilação de atividade enzimática entre moderada e alta, no período. O enzimotipo prevalente nos tempos 0 e 180 foi, respectivamente, 22 e 32. Com relação à proteinase, 100% das leveduras apresentaram atividade moderada da enzima no tempo 0. No tempo 180 esse percentual foi de 85%, sendo que os demais não apresentaram atividade dessa enzima. Após armazenamento em ASD e água destilada, foi detectada alteração da atividade de ambas enzimas e conseqüente mudança de enzimotipos, em 40 e 30% dos isolados, respectivamente. Foram identificados 8 morfotipos diferentes de C.albicans no tempo 0 e apenas 50% foi mantido no tempo 180. O morfotipo mais comum foi 000-0. Após armazenamento, a maioria dos isolados apresentou alteração no morfotipo. Todos isolados mostraram-se sensíveis aos antifúngicos analisados nos tempos 0 e 180, denotando apenas um antifungotipo, o 111. Somente um isolado após estocagem em ASD, teve mudança de perfil de sensível para dose dependente ao itraconazol de modo que o antifungotipo foi alterado. A tipagem molecular por RAPD, com os primers OPA-09, OPB-11 e OPE-18, mostrou 19 tipos moleculares distintos entre os isolados obtidos na primeira coleta e permitiu identificar que um isolado de C.albicans obtido no tempo 180, não era relacionado ao obtido, do mesmo indivíduo, no tempo 0. Os demais mostraram perfil de fragmentos de DNA relacionado entre as coletas. Após estocagem, por ambos métodos, todos isolados mostraram correlação genética com o padrão obtido no tempo 0. Os resultados obtidos demonstram que os métodos de conservação aplicados neste estudo não permitem a manutenção da estabilidade das características fenotípicas avaliadas. Por outro lado, além da estabilidade dos biotipos gerados, a tipagem molecular por RAPD mostrou o melhor índice discriminatório, dentre as metodologias utilizadas, ratificando sua capacidade em diferenciar isolados, de uma mesma espécie e, portanto a sua utilidade em inquéritos epidemiológicos. / The Candida genus is responsible for most nosocomial fungal infections. The identification of the probable origin focus is very important to elucidate the epidemiology of this kind of infection and so, the usage of typing methods that evaluate phenotypic and molecular characteristics are essential. Therefore, the objective of this study was to type C. albicans isolates, before and after culture storage and in two different collection occasions to verify the biotype maintenance and the discriminatory power of the utilized methods. The salivary yeast microbiota of 73 university students (time 0) was evaluated and after 180 days (time 180) a new saliva collection of those that had presented C. albicans on the first collection was made. The isolates were analysed, in the two collection occasions on the basis of exoenzymes phospholipase and proteinase production, by colonial morphology, susceptibility profile to amphotericin B, fluconazole and itraconazole and according to molecular typing by RAPD. After isolation, the yeasts were storaged on Sabouraud dextrose agar (SDA) and in sterile distilled water. After 180 days, typing tests were carried out again. All C. albicans isolates were phospholipase productors, in both collections, even though enzyme activity had oscillated between moderate and high at that period. The prevalent enzymotype at time 0 and 180 were, respectively, 22 and 32. In relation to the proteinase, 100% of the yeasts showed moderate enzyme activity at time 0. At time 180, this percentage was 85%, and the others didn`t show enzyme activity. After storage on SDA and in distilled water, it was detected activity alteration of both enzymes and consequent enzymotype change, in 40 and 30% of isolates, respectively. Eight different C. albicans morphotypes were identified at time 0 and just 50% were maintained at time 180. The most common morphotype was 000-0. After storage most isolates presented changes in the morphotype. All isolates showed susceptibility to the analysed antifungals at times 0 and 180, showing just one antifungaltype, the 111. Only one isolate, after storage on SDA had a profile change from susceptible to dose dependent susceptible to itraconazole in a way that the antifungaltype wasn`t changed. The molecular typing by RAPD, with primers OPA-09, OPB-11 and OPE-18, showed 19 distinct molecular types among first collection isolates and allowed to identify that one C. albicans isolate from time 180 wasn`t related with the isolate obtained at time 0, from the same individual. The others showed DNA fragment profiles related between collection occasions. After storage by both methods, every isolate showed genetic relatedness with the profile obtained at time 0. The obtained results showed that the preservation methods used in this study don`t allow stability maintenance of the phenotypical characteristics evaluated. On the other hand, besides biotypes generated stability, the molecular typing by RAPD showed the best discriminatory index, between methodologies used, ratifying its ability in discriminating isolates of the same specie and so, its utility in epidemiological inquiries

During the last decades, newly developed medical devices often came along with unappropriate designs, increasing the likelihood of misuse through the operator. Part of the root cause was that no sufficient measures were applied to assimilate user needs. Consequently, usability engineering approaches are now stronger emphasized to ensure that new devices are not only safe to use but are also designed for users’ needs. Besides, testing processes in clinical microbiology laboratories are currently reshaped due to new generations of rapid testing methods. Hence, it is particularly important to apply usability engineering frameworks during the development phase to make sure devices address users’ needs and also fit into the new work- and communication flows. Based on that, this research project applies a usability engineering approach to the design process of a new rapid antibiotic susceptibility testing system of Astrego Diagnostic AB that is supposed to be used in clinical microbiology laboratories in the near future. The research questions focus on how this device can be designed to enable integration into clinical laboratories. -       How can a rapid AST testing system be integrated into the workflow of clinical microbiology laboratories? -       What are the remaining uncertainties for integrating a rapid AST system into the workflow of a clinical microbiology laboratory on the example of Astrego’s AST system? Several methods were used to address these questions, which include literature research, a competitive audit, subject matter interviews and semi-structured interviews, and observations of targeted users. The findings show that a rapid antibiotic susceptibility testing system may be used in several different ways, which also impacts its design. Process-wise, it could be used after Gram staining and bacterial identification has been conducted and, more realistically, simultaneously bacterial identification to pave the way for additional time savings further. However, uncertainties remain regarding the design of the new testing system. Depending on the number of devices that targeted laboratories need to implement to accommodate their testing volume, it makes sense to design a built-in user interface or an external one that can be accessed through a tablet or desktop. Thus, it is uncertain to what extent manual input of bacteria ID is relevant as the dRAST system fully enables manual input of Gram type and bacteria IDs while it might also be possible to avoid manual interaction by receiving this information through software interfaces.

La découverte récente du premier gène de résistance plasmidique à la colistine mcr-1 a mis en évidence le besoin urgent d'améliorer la détection des isolats résistant à la colistine dans les laboratoires de microbiologie clinique afin de pouvoir rapidement isoler les patients porteurs. L’objectif de ma thèse a ainsi été de répondre à cette problématique par le développement et l’évaluation d’outils spécifiques. Une revue a été rédigée visant à résumer les nouveaux outils développés pour détecter la résistance aux polymyxines, y compris la présentation des méthodes actuelles phénotypiques, antibiogrammes et génotypiques. Le milieu de culture LBJMR a été développé pour détecter toutes les bactéries à Gram négatif résistantes à la colistine et également les souches d’entérocoques résistants à la vancomycine qui représentent un autre problème majeur de santé publique. LBJMR est polyvalent et a permis de dépister 3 bactéries d'intérêt clinique à partir d’un même échantillon: E. coli et K. pneumoniae résistantes à la colistine, et E. faecium résistant à la vancomycine. Un brevet a été déposé. Le kit UMIC Colistine est un dispositif commercial de détermination de la CMI basé sur la méthode de référence. Évalué conformément à la norme ISO 20776, sa sensibilité était excellente pour la détection des souches porteuses du gène mcr-1. Le kit UMIC Colistine constitue ainsi une méthode rapide et fiable pour évaluer la CMI des isolats cliniques dans les laboratoires de microbiologie clinique. Enfin, mes travaux de thèse ont compris l’analyse d’une souche de K. pneumoniae hétérorésistante à la colistine et la description d’une nouvelle espèce bactérienne : Pseudomonas massiliensis. / The recent discovery of mcr-1, the first plasmid-mediated colistin resistance gene, highlighted the urgent need to implement the detection of colistin-resistant isolates in clinical microbiology laboratories, in order to isolate carrier patients. My thesis aimed to address this issue by developing and evaluating specific tools. A review was redacted to summarize the new tools developed to detect polymyxin resistance including the current phenotyping, susceptibility testing and genotyping methods. The LBJMR culture medium has been developed to detect all colistin-resistant Gram-negative bacteria and also the vancomycin-resistant enterococci which represent another major problem of public health. The LBJMR medium allowed the detection of different bacteria of interest from the same clinical sample: colistin-resistant E. coli and K. pneumoniae and also a vancomycin-resistant E. faecium. This project led to a patent filing. The UMIC Colistine kit is a ready-to-use device based on the reference method to determine the polymyxin MIC. Evaluated in accordance with ISO 20776 standard, its sensitivity was excellent for the detection of colistin-resistant strains harboring the mcr-1 gene. The UMIC Colistin system is a rapid and reliable method to determine colistin MIC of clinical isolates in clinical microbiology laboratories. My thesis project also included the analysis of a colistin-heteroresistant Klebsiella pneumoniae strain, and the description of a new bacteril species, Pseudomonas massiliensis. These studies highlight the need to improve the detection of colistin-resistant strains by using reliable methods, suitable for diagnosis in clinical microbiology laboratories.

Introduction: The radiopacity is a required property in all dental materials. This characteristic allows this materials be able to be distinguished from the tooth surfaces through the radiography. Portland cement (PC) presents physico-chemical and biological properties similar to mineral trioxide aggregate (MTA), except for radiopacifier. Objective: To evaluate the radiopacity, antimicrobial activity and pH of the MTA, pure PC and PC added different radiopacifiers. Materials and Methods: The radiopacity of MTA, pure PC and PC added the radiopacifying: iodoform, lead oxide, zirconium oxide, bismuth subnitrate, barium sulfate and bismuth oxide in the proportions 15%, 20% and 30% were analyzed. The radiopacity was carried out using a semi-direct digital radiography system (Instrumentarium Kavo EXPRESS®). The PC and cements selected with adequate radiopacity (ANSI/ADA No. 57) were evaluated for their antimicrobial activity and pH. Antimicrobial activity was analyzed by microdilution tests and agar diffusion. The microorganism tested were Enterococcus faecalis, as a standard strain (ATCC 29212) and six clinical strains (LMA 26, LMA 27, LMA 28, LMA 29, LMA 31 and LMA 34). The pH was measured using a digital pH meter (pH Meter Pocket-sized). Statistical analyzes were performed using the Shapiro-Wilk normality test, ANOVA and Tukey with significance level of 5%. Results: Pure PC had the lowest radiopacity. The MTA, PC + iodoform 20% (3.59 mmEq.Al), PC + bismuth oxide 20% (4.79 mmEq.Al), PC + bismuth subnitrate 20% (3.12 mmEq.Al), PC + 20% lead oxide (3.18 mmEq.Al) and PC + 30% zirconium oxide (4.28 mmEq.Al). Regarding the agar diffusion test, the LMA 26 strain was inhibited by PC, PC + Iodoform 20%, PC + 30% Zirconium Oxide, PC + Bismuth subnitrate 20%, and PC + Bismuth Oxide 20%. The LMA 31 strain was inhibited by PC + zirconium oxide 30% and PC + bismuth subnitrate 20% and LMA 27 strain was inhibited by PC + bismuth subnitrate 20%. The other strains were not inhibited. In the microdilution test, most strains was inhibited by tested materials, except the ATCC 29212 strain which was not inhibited by PC + lead oxide 20%. LMA 28 29, 31 and 34 strains were not inhibited by PC + lead oxide 20%and PC + zirconium oxide 30%, in which LMA 28 and 29 strains were not inhibited by PC + bismuth oxide20%. All cements tested showed alkalinity in all experimental periods, in which MTA had the lowest pH. Conclusions: MTA and PC added the radiopacifiers subnitrate bismuth and iodoform in concentrations of 20% by weight, provided satisfactory radiopacity, alkaline medium and promoted bactericidal and bacteriostatic activities against strains of E .faecalis tested. / Introdução: A radiopacidade é uma propriedade obrigatória em todos os materiais odontológicos. Esta característica permite este material seja distinguido das superfícies dentárias através da radiografia. O cimento Portland (CP) apresenta propriedades físicoquímicas e biológicas semelhantes ao Agregado Trióxido Mineral (MTA), exceto pela partícula radiopacificadora. Objetivo: Avaliar a radiopacidade, atividade antimicrobiana e pH do MTA, CP puro e CP adicionado de diferentes radiopacificadores. Materiais e Métodos: Foi analisada a radiopacidade do MTA, CP e CP adicionado dos radiopacificadores: iodofórmio, óxido de chumbo, óxido de zircônio, subnitrato de bismuto, sulfato de bário e óxido de bismuto nas proporções 15%, 20% e 30%. A radiopacidade foi avaliada por meio do sistema de radiografia digital semi-direto (Instrumentarium Kavo EXPRESS®). O CP e os cimentos selecionados com radiopacidade adequada (ANSI/ADA nº 57) foram submetidos à avaliação da atividade antimicrobiana e do pH. A atividade antimicrobiana foi analisada pelos testes de microdiluição e difusão em ágar. O micro-organismo testado foi o Enterococcus faecalis, sendo uma cepa padrão (ATCC 29212) e seis cepas clínicas (LMA 26, LMA 27, LMA 28, LMA 29, LMA 31 e LMA 34). O pH foi aferido com pHmetro digital (pH Meter Pocket-sized). Os testes estatísticos utilizados foram os testes Shapiro-Wilk de normalidade, análise de variância ANOVA e Tukey a nível de significância de 5%. Resultados: O CP puro apresentou o menor valor de radiopacidade, O MTA, CP + iodofórmio 20% (3,59 mmEq.Al), CP + óxido de bismuto 20% (4,79 mmEq.Al), CP + subnitrato de bismuto 20% (3,12 mmEq.Al), CP + óxido de chumbo 20% (3,18 mmEq.Al) e CP + óxido de zircônio 30% (4,28 mmEq.Al). No teste de difusão em ágar, a cepa LMA 26 foi inibida pelo CP, CP + Iodofórmio 20%, CP + Óxido de Zircônio 30%, CP + Subnitrato de Bismuto 20% e CP + Óxido de Bismuto 20%. A cepa LMA 31 foi inibida pelas associações CP + óxido de zircônio 30% e CP + subnitrato de bismuto 20% e a cepa LMA 27 foi inibida pelo CP + subnitrato de bismuto 20%. As demais cepas não foram inibidas. No teste de microdiluição, a maioria das cepas foi inibida pelos materiais-testes, exceto a cepa ATCC 29212 que não foi inibida pelo CP + óxido de chumbo 20%, as cepas LMA 28, 29, 31 e 34 não foram inibidas pelas associações CP + óxido de chumbo 20% e CP + óxido de zircônio 30%, sendo que as cepas LMA 28 e 29 também não foram inibidas pelo CP + óxido de bismuto 20%. Todos os cimentos testados apresentaram alcalinidade em todos os períodos experimentais, sendo que o MTA apresentou os menores valores de pH. Conclusões: O MTA e as concentrações de CP adicionado de 20% do agente radiopacificador subnitrato de bismuto e CP adicionado do agente radiopacificador iodofórmio, 20% em peso, proporcionaram radiopacidade satisfatória, alcalinizaram o meio e promoveram atividades bactericida e bacteriostática contra as cepas de E. faecalis testadas.

Made available in DSpace on 2014-07-29T15:30:40Z (GMT). No. of bitstreams: 1 Dissertacao CrystianeAraujo.pdf: 881195 bytes, checksum: da026b75ed78f3c38d78f44f6a15271e (MD5) Previous issue date: 2007-05-31 / Dermatophytes are keratinophilic fungi that colonize and invade the stratum corneum of the skin, hair and nails causing the dermatophytosis. An increasing number of antifungal agents has become available for the treatment of dermatophytosis, however not all species have the same susceptibility pattern and may occur relative or absolute resistance of some dermatophytes. The document M38-A developed by Clinical and Laboratory Standards Institute (CLSI) for determining the minimal inhibitory concentration (MIC) of different antifungal agents against filamentous fungi, has not included the dermatophytes. The broth microdilution method has been evaluated by various researchers, and some parameters as inoculum size, temperature and duration of incubation and endpoint determination has been investigated. In this study, the in vitro activity of fluconazole, itraconazole, ketoconazole, griseofulvin and terbinafine against 60 dermatophyte isolates, belong to three species, using the broth microdilution technique, with modifications at temperature and incubation time was used. Additionally, the MIC values obtained by broth microdilution method were compared with those obtained by the agar dilution technique. The results obtained by broth microdilution method showed that all isolates produced clearly detectable growth at 28oC and the MIC values could be determined after 4 days of incubation for the isolates of Trichophyton mentagrophytes and 5 days for T. rubrum and Microsporum canis isolates. Itraconazole, ketoconazole and terbinafine had the lowest MIC values (0.03 μg/ml) for 33.3%, 31.6% and 15% of the isolates, respectively. A good concordance was observed between the agar dilution and broth microdilution methods. The levels of agreement were 91.6% with ketoconazole and griseofulvin, 83.3% with itraconazole, 81.6% with terbinafine and 73.3% with fluconazole for all the tested isolates. In summary, the results of this study suggest that an incubation time of 5 days and temperature at 28oC used in broth microdilution and agar dilution methods can contribute to define and to better interpret the MIC values. Beside, until a reference method for testing the susceptibilities of dermatophytes is standardized, the similar results with broth microdilution method become the agar dilution useful for testing the susceptibility of these fungi. / Os dermatófitos são fungos filamentosos queratinofílicos capazes de colonizar e invadir o extrato córneo da pele, pêlo e unhas produzindo as dermatofitoses. Há vários agentes antifúngicos disponíveis para o tratamento das dermatofitoses, entretanto as espécies de dermatófitos possuem perfis de suscetibilidade diferentes, podendo ocorrer resistência relativa ou absoluta. O documento M38-A desenvolvido pelo Clinical and Laboratory Standards Institute (CLSI) para determinar a concentração inibitória mínima (CIM) de diferentes agentes antifúngicos para fungos filamentosos, não inclui os dermatófitos. O método de microdiluição em caldo tem sido avaliado por vários pesquisadores, e alguns parâmetros como tamanho do inóculo, temperatura e tempo de incubação e determinação das leituras de CIM são investigados. Neste trabalho, foi avaliada a atividade in vitro de fluconazol, itraconazol, cetoconazol, griseofulvina e terbinafina para 60 isolados de dermatófitos, pertencentes a três espécies, através da técnica de microdiluição em caldo, com modificações na temperatura e tempo de incubação. Adicionalmente, os valores de CIM obtidos pelo método de microdiluição em caldo foram comparados com aqueles obtidos pela técnica de diluição em ágar. Os resultados encontrados através do método de microdiluição em caldo mostraram que todos os isolados produziram crescimento claramente detectável a temperatura de 28oC e os valores de CIMs foram determinados após 4 dias de incubação para os isolados de Trichophyton mentagrophytes e 5 dias para os isolados de T. rubrum e Microsporum canis. Itraconazol, cetoconazol e terbinafina mostraram os menores valores de CIM (0,03 μg/ml) para 33,3%, 31,6% e 15% dos isolados, respectivamente. Uma boa concordância foi observada entre os métodos de diluição em ágar e microdiluição em caldo. A concordância entre os dois métodos foi de 91,6% para cetoconazol e griseofulvina, 83,3% para itraconazol, 81,6% para terbinafina e 73,3% para fluconazol para todos os isolados analisados. Em conclusão, os resultados deste estudo sugerem que uma incubação de 5 dias e temperatura de 28oC, utilizados no método de microdiluição em caldo e diluição em ágar, podem contribuir para definir e melhor interpretar os valores de CIM. Além disso, até que um método de referência de teste de suscetibilidade para dermatófitos seja padronizado, os resultados similares entre os métodos de microdiluição em caldo e diluição em ágar tornam este último útil para testar a suscetibilidade in vitro destes fungos.

O gênero Candida é responsável pela maioria das infecções fúngicas nosocomiais. A identificação do provável foco de origem é de extrema importância para elucidar a epidemiologia desse tipo de infecção e nesse sentido, a utilização de métodos de tipagem, que avaliam características fenotípicas e moleculares dos isolados, é essencial. Assim, o objetivo desse estudo foi tipar isolados de C. albicans, antes e após armazenamento e em diferentes ocasiões de coleta, a fim de verificar a manutenção dos biotipos apresentados, e o poder de discriminação dos métodos utilizados. Foi avaliada a microbiota leveduriforme da saliva de 73 estudantes universitários (tempo 0) sendo que após 180 dias (tempo 180) foi realizada nova coleta de saliva daqueles que apresentaram isolamento de C. albicans na primeira coleta. Os isolados foram analisados por ocasião das duas coletas, quanto à produção de exoenzimas fosfolipase e proteinase, pela morfologia das colônias, pelo perfil de suscetibilidade frente à anfotericina B, fluconazol e itraconazol e pela tipagem molecular por RAPD. As leveduras, após isoladas, foram armazenadas em ágar Sabouraud dextrose (ASD) e água destilada esterilizada. Após 180 dias, foram realizadas, novamente, as provas de tipagem. Todos isolados de C. albicans foram produtores de fosfolipase, nas duas coletas, embora tenha havido oscilação de atividade enzimática entre moderada e alta, no período. O enzimotipo prevalente nos tempos 0 e 180 foi, respectivamente, 22 e 32. Com relação à proteinase, 100% das leveduras apresentaram atividade moderada da enzima no tempo 0. No tempo 180 esse percentual foi de 85%, sendo que os demais não apresentaram atividade dessa enzima. Após armazenamento em ASD e água destilada, foi detectada alteração da atividade de ambas enzimas e conseqüente mudança de enzimotipos, em 40 e 30% dos isolados, respectivamente. Foram identificados 8 morfotipos diferentes de C.albicans no tempo 0 e apenas 50% foi mantido no tempo 180. O morfotipo mais comum foi 000-0. Após armazenamento, a maioria dos isolados apresentou alteração no morfotipo. Todos isolados mostraram-se sensíveis aos antifúngicos analisados nos tempos 0 e 180, denotando apenas um antifungotipo, o 111. Somente um isolado após estocagem em ASD, teve mudança de perfil de sensível para dose dependente ao itraconazol de modo que o antifungotipo foi alterado. A tipagem molecular por RAPD, com os primers OPA-09, OPB-11 e OPE-18, mostrou 19 tipos moleculares distintos entre os isolados obtidos na primeira coleta e permitiu identificar que um isolado de C.albicans obtido no tempo 180, não era relacionado ao obtido, do mesmo indivíduo, no tempo 0. Os demais mostraram perfil de fragmentos de DNA relacionado entre as coletas. Após estocagem, por ambos métodos, todos isolados mostraram correlação genética com o padrão obtido no tempo 0. Os resultados obtidos demonstram que os métodos de conservação aplicados neste estudo não permitem a manutenção da estabilidade das características fenotípicas avaliadas. Por outro lado, além da estabilidade dos biotipos gerados, a tipagem molecular por RAPD mostrou o melhor índice discriminatório, dentre as metodologias utilizadas, ratificando sua capacidade em diferenciar isolados, de uma mesma espécie e, portanto a sua utilidade em inquéritos epidemiológicos. / The Candida genus is responsible for most nosocomial fungal infections. The identification of the probable origin focus is very important to elucidate the epidemiology of this kind of infection and so, the usage of typing methods that evaluate phenotypic and molecular characteristics are essential. Therefore, the objective of this study was to type C. albicans isolates, before and after culture storage and in two different collection occasions to verify the biotype maintenance and the discriminatory power of the utilized methods. The salivary yeast microbiota of 73 university students (time 0) was evaluated and after 180 days (time 180) a new saliva collection of those that had presented C. albicans on the first collection was made. The isolates were analysed, in the two collection occasions on the basis of exoenzymes phospholipase and proteinase production, by colonial morphology, susceptibility profile to amphotericin B, fluconazole and itraconazole and according to molecular typing by RAPD. After isolation, the yeasts were storaged on Sabouraud dextrose agar (SDA) and in sterile distilled water. After 180 days, typing tests were carried out again. All C. albicans isolates were phospholipase productors, in both collections, even though enzyme activity had oscillated between moderate and high at that period. The prevalent enzymotype at time 0 and 180 were, respectively, 22 and 32. In relation to the proteinase, 100% of the yeasts showed moderate enzyme activity at time 0. At time 180, this percentage was 85%, and the others didn`t show enzyme activity. After storage on SDA and in distilled water, it was detected activity alteration of both enzymes and consequent enzymotype change, in 40 and 30% of isolates, respectively. Eight different C. albicans morphotypes were identified at time 0 and just 50% were maintained at time 180. The most common morphotype was 000-0. After storage most isolates presented changes in the morphotype. All isolates showed susceptibility to the analysed antifungals at times 0 and 180, showing just one antifungaltype, the 111. Only one isolate, after storage on SDA had a profile change from susceptible to dose dependent susceptible to itraconazole in a way that the antifungaltype wasn`t changed. The molecular typing by RAPD, with primers OPA-09, OPB-11 and OPE-18, showed 19 distinct molecular types among first collection isolates and allowed to identify that one C. albicans isolate from time 180 wasn`t related with the isolate obtained at time 0, from the same individual. The others showed DNA fragment profiles related between collection occasions. After storage by both methods, every isolate showed genetic relatedness with the profile obtained at time 0. The obtained results showed that the preservation methods used in this study don`t allow stability maintenance of the phenotypical characteristics evaluated. On the other hand, besides biotypes generated stability, the molecular typing by RAPD showed the best discriminatory index, between methodologies used, ratifying its ability in discriminating isolates of the same specie and so, its utility in epidemiological inquiries

Resistensbestämningar som genererar ett kvantitativt MIC-värde är värdefullt för att kunna välja adekvat antibiotikabehandling. Referensmetoden för MIC-bestämning är mikrobuljongspädning där kommersiella antibiotikapaneler underlättar handhavandet av metoden på kliniska laboratorier. Syftet med studien var att verifiera mikrobuljongspädning med Sensititre™-panelerna SEMSE7 och SEMST7 avsedda för grampositiva kocker, genom att jämföra erhållet resultat med tidigare uppmätta referensvärden av MIC samt SIR-kategorier. Mikrobuljongspädning utfördes med SEMSE7-panel och MH-buljong för Staphylococcus spp. (n=21) och Enterococcus spp. (n=13) samt med SEMST7-panel och MH-F buljong för Streptococcus pneumoniae (n=20). Avläsning av MIC utfördes samt tolkades enligt SIR-systemet. Resultaten jämfördes mot kända referensvärden tidigare uppmätta med mikrobuljongspädning. Isolat analyserade med SEMSE7-panel och SEMST7-panel erhöll en överensstämmelse av MIC på 88,2% respektive 96,7%. Motsvarande kategorisk överensstämmelse av SIR var 92,5% respektive 92,6%. Resultatet visar att Sensititre™-panelen SEMST7 är godkänd i verifieringen av mikrobuljongspädning med god överensstämmelse med referensvärden av MIC samt SIR-kategorier. Vidare bedöms att fortsatt verifiering krävs av SEMSE7-panelen eftersom ej tillfredställande överenstämmelse med referensvärden av MIC erhölls, trots god överenstämmelse av SIR-kategorier. Studien visar att SEMST7-panelen kan implementeras för Streptococcus pneumoniae i den kliniska verksamheten på mikrobiologilaboratoriet, Jönköping. / Antimicrobial susceptibility testing methods generating a quantitative MIC are valuable for choosing adequate antibiotic treatment. Broth microdilution is the reference method for MIC determination and commercial panels with antibiotics facilitate the procedure in clinical laboratories. The aim of the study was to verify broth microdilution with the Sensititre™ panels SEMSE7 and SEMST7 designed for gram-positive cocci, by comparing results with previously measured reference values of MIC and SIR. Broth microdilution was performed using the SEMSE7 panel with MH broth for Staphylococcus spp. (n=21) and Enterococcus spp. (n=13), and using the SEMST7 panel with MH-F broth for Streptococcus pneumoniae (n=20). Reading MIC endpoints was performed and interpreted according to SIR system. Isolates analysed with the SEMSE7 and SEMST7 panel obtained essential agreement of 88,2% and 96,7%, and categorical agreement of 92,5% and 92,6% respectively. In conclusion, the Sensititre™ panel SEMST7 is approved in the verification of broth microdilution with satisfactory essential agreement and categorical agreement. Furthermore, it is considered that further verification is required for the SEMSE7 panel as unsatisfactory essential agreement was obtained, despite satisfactory categorical agreement. The study shows that the SEMST7 panel can be implemented for Streptococcus pneumoniae in the clinical practice at the microbiology laboratory, Jönköping.

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Made available in DSpace on 2014-07-29T15:04:30Z (GMT). No. of bitstreams: 1 dissertacao marleneandrade.pdf: 776220 bytes, checksum: 89f7e969fbef687591092b3ded9dd40a (MD5) Previous issue date: 2008-04-30 / Assessing patients with chronic wounds poses a challenge to professionals and doubts still persist concerning characterization of the wound infection status. We believe that the Basic Health Units are in places of reference for the public to submit chronic wounds and should have full attendance and resolution were objectives of this study: characterizing patients with chronic wounds attended as spontaneous demand in the bandage room; characterizing patients chronic wounds regarding the presence of classical signs and additional infection criteria; isolating and identifying aerobic bacteria and fungi in the samples of chronic wounds clinically signaling to infection; verifying strains susceptibility when isolated before antimicrobial agents often used in praxis as well as new antibiotics and analyzing the relationship of local factors with infection status. This is a cross-sectional study, carried out in ambulatory bandage rooms in basic health units with emergency service in the municipality of Goiânia. Data was collected from June to July, in 2007. Data was obtained through the use of structured interviews containing questions about characterization of patients and a check list for the assessment of chronic wounds through the signs and clinical symptoms indicating infection and analysis of wound samples by using swabs in accordance with Levine s technique. After consent and approval of the ethics committee, 46 patients were evaluated and 60 wound samples were assessed. Data bank was organized in Excel table and statistics analysis in SPSS- 15.0. The average age was 55 years, 37 (80.4%) male, 20 (43.5%) belonging to E social class, 23 (50%) retired people having basic sanitation items. 28 (61%) presented venous ulcerated lower limbs, 31 (67.4%) performed the bandage both in the BHU and at home. 45 (75%) out of 60 wounds were infected and 15 (25%) noninfected. Through bivariate analysis we verified an association of the infection status with the following characteristics: width depth of tissular damage, necrotic tissue and exudate amount. Among signs and symptoms, classical ones occurred in a frequency higher than 65% both in the infected and noninfected group. Regarding the additional ones, we verified variance in occurrence in both wound groups. Staphylococcus aureus was predominat in 65% of cases and was sensitive to most antibiotics tested. Among Gram-negative bacteria the most frequent were: Pseudomonas aeruginosa (23%), resistant to amoxylin +clavulanic acid, cefalexin and cefotaxim; Proteus mirabilis (16.6%) and Proteus vulgaris (15.0%), all sensitive to gentamicin, aztreonam, ciprofloxacin, and amicacin. These results indicate the need to structure an integrated net to treat patients with chronic wounds and evidence additional criteria to be employed in the elaboration of service protocols pursuing improvement of quality of assistance given in basic health units. / A avaliação de pacientes com feridas crônicas representa um desafio para os profissionais, e dúvidas, ainda persistem em torno da caracterização do status de infecção, nessas lesões. As Unidades Básicas de Saúde constituem-se locais de referência para a população portadora de feridas crônicas, onde deveriam ter atendimento integral e resolutividade. Objetivos deste estudo: caracterizar os pacientes com feridas crônicas, atendidos como demanda espontânea, na sala de curativos; caracterizar as feridas crônicas dos pacientes atendidos em relação à presença de sinais clássicos e critérios adicionais de infecção; isolar e identificar bactérias aeróbias e fungos das amostras de feridas crônicas, com indícios clínicos de infecção; verificar a susceptibilidade das cepas isoladas, frente aos antimicrobianos freqüentemente utilizados na prática e novos antibióticos, e analisar a relação de fatores locais, com o status de infecção. Trata-se de estudo transversal, realizado em Salas de Curativos Ambulatoriais de Unidades Básicas de Saúde (UBS), com atendimento de urgência do Município de Goiânia. Os dados foram coletados no período de junho a julho de 2007 e obtidos por meio de entrevista com roteiro estruturado, contendo questões de caracterização dos pacientes e de um check-list, para a avaliação das feridas crônicas, por meio dos sinais e sintomas clínicos indicativos de infecção, e análise de amostras de feridas utilizando o swab, mediante técnica de Levine. Após aprovação pelo Comitê de Ética e Consentimento, foram avaliados 46 pacientes, e analisadas amostras de 60 feridas. Os programas computacionais utilizados para organizar e analisar estatisticamente os dados foram: o Excel e o SPSS (versão 15.0), respectivamente. A média de idade foi de 55 anos, 37 (80,4%) eram do sexo masculino, 20 (43.5%) pertenciam à classe social E, 23 (50%) eram aposentados e possuíam itens de saneamento básico, 28 (61%) apresentaram úlceras venosas, localizadas nos membros inferiores, 31 (67,4%) realizavam o curativo nas UBS ou em domicílios. Das 60 lesões, 45 (75%) estavam infectadas e 15 (25%) não infectadas. Mediante análise bivariada, verificaram-se associação com o status de infecção as seguintes características: profundidade da extensão do dano tissular, quantidade de tecido necrótico e exsudato. Dentre os sinais e sintomas, os clássicos ocorreram numa freqüência superior a 65%, tanto no grupo de infectadas quanto não infectadas, e nos adicionais verificou-se variação na ocorrência, em ambos os grupos de feridas. Houve predominância do Staphylococcus aureus em 65% dos casos e a maioria sensível aos antibióticos testados. Entre as bactérias Gram-negativas, as mais freqüentes foram: Pseudomonas aeruginosa (23,3%), resistentes à amoxicilina+ácido clavulânico, cefalexina e cefotaxima; Proteus mirabilis (16,6%) e Proteus vulgaris (15,0%) sensíveis a gentamicina, aztreonam, ciprofloxacino e amicacina. Estes resultados indicam a necessidade de se estruturar uma rede integrada, para o atendimento dos pacientes com feridas crônicas. Também evidenciam critérios adicionais que podem ser empregados na elaboração do protocolo de atendimento, buscando a melhoria da qualidade da assistência prestada nas unidades básicas de saúde.