Made available in DSpace on 2014-06-11T19:28:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-17Bitstream added on 2014-06-13T19:58:19Z : No. of bitstreams: 1 andrade_mc_me_arafo.pdf: 1842420 bytes, checksum: 8ab56a86c157ba82b8e80912d3966136 (MD5) / O surgimento da resistência antifúngica aos tratamentos convencionais tem exigido o desenvolvimento de novas modalidades terapêuticas para o tratamento da candidíase bucal. Nesse contexto, a Terapia Fotodinâmica (Photodynamic Therapy – PDT) tem sido sugerida como método alternativo para a inativação de microorganismos patogênicos. O sucesso da PDT depende, dentre outros fatores do tempo de pré-irradiação (TPI), que é o tempo em que o fotossensibilizador (FS) permanece em contato com os micro-organismos previamente à iluminação. O objetivo deste estudo foi avaliar a efetividade da PDT mediada pela curcumina (Cur), associando diferentes TPIs, concentrações e doses de luz, na redução da viabilidade celular de três espécies de Candida ATCC (C. albicans ATCC 90028; C. glabrata ATCC 2001; C. dubliniensis ATCC 7987), em culturas planctônicas e biofilmes. A Cur foi ativada por um aparelho de LED (luz emitida por diodo) que emite luz no espectro azul, com comprimento de onda predominante de 455 nm. Inicialmente, suspensões celulares padronizadas dos micro-organismos foram transferidas para placas de 96 orifícios, e tratadas com três diferentes concentrações de Cur (5, 10 e 20 YM), ou mantidas nos poços por 48 horas para formação dos biofilmes, seguida de tratamento com outras três concentrações de Cur (20, 30 e 40 YM). As culturas celulares foram deixadas em contato com a Cur por 1, 5, 10 ou 20 minutos, anteriormente à iluminação. Em seguida, as culturas planctônicas foram expostas à dose de luz de 5,28 J/cm2. Os biofilmes, por sua vez, foram tratados com duas doses de luz (5,28 e 10,56 J/cm2). Suspensões/biofilmes adicionais foram tratados somente com as três concentrações de Cur (pelo maior tempo usado em cada estudo) ou apenas com a maior dose de luz. Amostras controle não foram tratadas com Cur e não receberam luz. Após a PDT, as suspensões celulares... / The emergence of antifungal-resistant yeasts to conventional therapy has demanded the development of new therapy modalities against oral candidiasis. On this context, Photodynamic Therapy (PDT) has been suggested as a possible alternative treatment for inactivation of pathogenic microorganisms. PDT success depends, among other factors, on the pre-irradiation time (PIT). The aim of this study was to evaluate the effectiveness of curcumin (Cur)-mediated PDT associating different PITs, Cur concentrations and light doses, against planktonic and biofilm cultures of three different Candida species (C. albicans ATCC 90028; C. glabrata ATCC 2001; C. dubliniensis ATCC 7987). Cur was activated by a LED (light emitting diode) device, which emits light on the blue spectrum, with predominant wavelength at 455 nm. Standardized cell suspensions were treated with three different concentrations of Cur (5, 10 e 20 YM), or kept into the wells for 48 hours to allow adhesion and biofilm formation. The biofilms were treated with other three different concentrations of Cur (20, 30 e 40 YM). The cultivated fungus (planktonic and biofilms) were maintained in the dark in contact with the PS for either 1, 5, 10 or 20 minutes before irradiation. The suspensions were exposed to LED light dose of 5.28 J/cm2, while the biofilms were exposed to either 5.28, or 10.56 J/cm2. Additional samples of suspensions/biofilms were treated only with the three Cur concentrations (for the highest incubation period used in the study), without illumination, or only with the highest light dose, without Cur. Control samples had neither light nor Cur. After PDT, suspensions were plated on Sabouraud Dextrose Agar (SDA) in duplicate, while biofilm results were read using the XTT-salt reduction method. The results for the planktonic cultures were statistically analyzed by Kruskal-Wallis and Dunn, while for the biofilms, analysis of variance... (Complete abstract click electronic access below)

The typical response of cancer patients to treatment is only temporary, and is often followed by relapse. The failure of various therapeutic strategies is commonly attributed to the emergence of drug resistance. The response patterns for patients under such treatments indicate that complex dynamics regulate the response of the tumour to the therapy. The environment in which the tumour lives (the stroma) is known to be a modulator of multiple mechanisms that lead to drug resistance and seems to be a likely candidate for explaining some of this complexity. Understanding the role of stromal cells in the promotion of drug resistance is critical for the design of optimal treatment strategies, and for the development of novel therapies that selectively target both the tumour and the stroma. In this thesis we design two novel mathematical models that describe cancer growth within its environment and the evolution of drug resistance within spatially complex and temporally dynamic tumours. A compartment model captures clinically observed dynamics and allows direct comparison with experimental data, facilitating model parametrisation and the understanding of inter-tumour heterogeneity. An individual cell-based model highlights the key role of local interactions, determining heterogeneity at the tissue scale, that will eventually determine treatment outcome. A non-spatial approximation of this second model allows us to find analytic guidelines for the design of effective therapy. These tools allow the simulation of a range of treatment strategies (including combination of different drugs and variation of schedule) as well as the investigation of therapy response based on patient- or organ-specic parameters. The work developed in this dissertation is based on the paradigmatic biology of melanoma and non-small cell lung cancer. Its results are therefore applicable to a variety of cancer treatments that target similar processes, and whose therapeutic failure can be attributed to environment-mediated drug resistance.

Orientador: Ana Claudia Pavarina / Banca: Eunice Teresinha Giampaolo / Banca: Claudia Helena Lovato da Silva / Resumo: O surgimento da resistência antifúngica aos tratamentos convencionais tem exigido o desenvolvimento de novas modalidades terapêuticas para o tratamento da candidíase bucal. Nesse contexto, a Terapia Fotodinâmica (Photodynamic Therapy - PDT) tem sido sugerida como método alternativo para a inativação de microorganismos patogênicos. O sucesso da PDT depende, dentre outros fatores do tempo de pré-irradiação (TPI), que é o tempo em que o fotossensibilizador (FS) permanece em contato com os micro-organismos previamente à iluminação. O objetivo deste estudo foi avaliar a efetividade da PDT mediada pela curcumina (Cur), associando diferentes TPIs, concentrações e doses de luz, na redução da viabilidade celular de três espécies de Candida ATCC (C. albicans ATCC 90028; C. glabrata ATCC 2001; C. dubliniensis ATCC 7987), em culturas planctônicas e biofilmes. A Cur foi ativada por um aparelho de LED (luz emitida por diodo) que emite luz no espectro azul, com comprimento de onda predominante de 455 nm. Inicialmente, suspensões celulares padronizadas dos micro-organismos foram transferidas para placas de 96 orifícios, e tratadas com três diferentes concentrações de Cur (5, 10 e 20 YM), ou mantidas nos poços por 48 horas para formação dos biofilmes, seguida de tratamento com outras três concentrações de Cur (20, 30 e 40 YM). As culturas celulares foram deixadas em contato com a Cur por 1, 5, 10 ou 20 minutos, anteriormente à iluminação. Em seguida, as culturas planctônicas foram expostas à dose de luz de 5,28 J/cm2. Os biofilmes, por sua vez, foram tratados com duas doses de luz (5,28 e 10,56 J/cm2). Suspensões/biofilmes adicionais foram tratados somente com as três concentrações de Cur (pelo maior tempo usado em cada estudo) ou apenas com a maior dose de luz. Amostras controle não foram tratadas com Cur e não receberam luz. Após a PDT, as suspensões celulares... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The emergence of antifungal-resistant yeasts to conventional therapy has demanded the development of new therapy modalities against oral candidiasis. On this context, Photodynamic Therapy (PDT) has been suggested as a possible alternative treatment for inactivation of pathogenic microorganisms. PDT success depends, among other factors, on the pre-irradiation time (PIT). The aim of this study was to evaluate the effectiveness of curcumin (Cur)-mediated PDT associating different PITs, Cur concentrations and light doses, against planktonic and biofilm cultures of three different Candida species (C. albicans ATCC 90028; C. glabrata ATCC 2001; C. dubliniensis ATCC 7987). Cur was activated by a LED (light emitting diode) device, which emits light on the blue spectrum, with predominant wavelength at 455 nm. Standardized cell suspensions were treated with three different concentrations of Cur (5, 10 e 20 YM), or kept into the wells for 48 hours to allow adhesion and biofilm formation. The biofilms were treated with other three different concentrations of Cur (20, 30 e 40 YM). The cultivated fungus (planktonic and biofilms) were maintained in the dark in contact with the PS for either 1, 5, 10 or 20 minutes before irradiation. The suspensions were exposed to LED light dose of 5.28 J/cm2, while the biofilms were exposed to either 5.28, or 10.56 J/cm2. Additional samples of suspensions/biofilms were treated only with the three Cur concentrations (for the highest incubation period used in the study), without illumination, or only with the highest light dose, without Cur. Control samples had neither light nor Cur. After PDT, suspensions were plated on Sabouraud Dextrose Agar (SDA) in duplicate, while biofilm results were read using the XTT-salt reduction method. The results for the planktonic cultures were statistically analyzed by Kruskal-Wallis and Dunn, while for the biofilms, analysis of variance... (Complete abstract click electronic access below) / Mestre

Orientador: Fernando Rogerio Pavan / Banca: Daisy Nakamura Sato / Banca: Rodolpho Teralolli Junior / Resumo: Apesar da incidência da Tuberculose (TB) ter diminuído nos últimos anos, nota-se um expressivo aumento dos casos de TBMDR (multi-fármaco-resistente) e TBXDR (extensivamente-fármaco-resistente). A situação constitui um sério problema de saúde pública no mundo e no Brasil, com menor taxa de cura, pior prognóstico e nenhum tratamento de eficácia comprovada para contatos. A taxa estimada de cura global para TBMDR é de 52% e para TBXDR, os desfechos são ainda menos favoráveis. Nesse sentido, buscamos nesse trabalho determinar o perfil de resistência, tratamentos e evolução clínica de pacientes internados com TBMDR e TBXDR no período de 2000 a 2014 em um Hospital de Referência no Estado de São Paulo, Brasil. Os dados foram obtidos diretamente do prontuário de pacientes e do banco de dados do TBWEB, que são informações clínico-epidemiológicas, exames e de tratamento do Programa de Controle de Tuberculose do Estado de São Paulo analisados através de estatística descritiva. Foram internados 39 pacientes no período de estudo, 29 (74,3%) TBMDR e 10 (25,7%) de TBXDR. Entre os TBMDR a taxa de cura foi de 79,3% e o tempo médio de tratamento foi de 19,6 meses. E no grupo dos TBXDR, 80% evoluíram com cura e o tempo médio de tratamento foi de 25,9 meses. A taxa de cura encontrada foi maior que a descrita em outros estudos. A supervisão completa de todo o tratamento, a utilização das recomendações do MS e OMS, adicionadas a uma história minuciosa dos fármacos utilizados previamente e adequação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Although the incidence of Tuberculosis (TB) has declined in recent years, there is a significant increase in cases of MDRTBD (multi-drug resistant) and XDRTB (extensively drug-resistant). The situation is a serious public health problem in the world and in Brazil, with a lower cure rate, worse prognosis and no proven treatment for contacts. The estimated global cure rate for MDRTB is 52% and for XDRTB, outcomes are even less favorable. In this sense, we sought to determine the resistance profile, treatments and clinical evolution of patients hospitalized with MDR-TB and MDR-TB between 2000 and 2014 at a Reference Hospital in the State of São Paulo, Brazil. Data were obtained directly from the patient records and the TBWEB database, which are clinicalepidemiological information, tests and treatment of the Tuberculosis Control Program of the State of São Paulo, analyzed through descriptive statistics. Thirty-nine patients were hospitalized in the study period, 29 (74.3%) MDRTB and 10 (25.7%) MDRTB. Among the MDRTB, the cure rate was 79.3% and the mean treatment time was 19.6 months. And in the XDRTB group, 80% progressed with healing and the mean treatment time was 25.9 months. The cure rate found was higher than that reported in other studies. Complete supervision of all treatment, use of MS and WHO recommendations, added to a thorough history of the drugs previously used and adequacy of the schedules with TS results may contribute... (Complete abstract click electronic access below) / Mestre

Orientador: Ana Cláudia Pavarina / Abstract: Biofilmes formados por Candida estão relacionados a infecções bucais, como a candidíase. Embora a resistência do biofilme seja multifatorial, a proteção exercida por sua matriz extracelular (MEC) é importante para os altos níveis de resistência a drogas antifúngicas. O conhecimento dos princípios estruturais da MEC possibilita maior compreensão de como atuar para desorganizá-la e melhorar a difusão de agentes antifúngicos para que atinjam mais eficientemente o biofilme, além de, futuramente, possibilitar o desenvolvimento de terapias mais eficazes para o controle da formação de biofilmes. Sendo assim, os objetivos principais deste estudo foram: (1) verificar a influência da inativação de genes envolvidos na filamentação (EFG1 e TEC1) em características estruturais dos biofilmes e na produção de componentes da MEC; (2) verificar a influência do fluconazol (FLZ) na MEC de biofilmes de Candida albicans ATCC 90028 (susceptível a fluconazol- CaS), C. albicans ATCC 96901 (resistente a fluconazol- CaR), Candida glabrata ATCC 2001 (susceptível a fluconazol- CgS) e C. glabrata ATCC 200918 (resistente a fluconazol- CgR) e (3) estudar a ação de enzimas hidrolíticas (DNase, Dextranase e β-glucanase individualmente ou em diferentes combinações) sobre a MEC de biofilmes de CaS e CaR. Biofilmes maduros (48 horas) foram analisados através de contagem de unidades formadoras de colônia (ufc/mL), peso seco total, peso seco insolúvel e proteínas insolúveis. Os componentes da MEC- polissacarídeos... (Complete abstract click electronic access below) / Resumo: Biofilms formed by Candida are related to bucal infections, such as candidiasis. Although the biofilm resistance is multifactorial, the protection exerted by its extracellular matrix (ECM) is essential for its high levels of resistance to antifungals. The knowledge of the structural principles of the ECM permits a better understanding of how to disorganize the ECM and improve the diffusion of antifungal drugs to reach the biofilm. Moreover, the study of the ECM may enable the development of more effective therapies to control biofilm formation. Thus, the main objectives of this study were: (1) to verify the influence of the inactivation of genes involved in filamentation and structural characteristics of the biofilms (EFG1 and TEC1) on the production of ECM components; (2) to verify the influence of fluconazole (FLZ) on the biofilms' ECM of Candida albicans ATCC 90028 (fluconazole-susceptible: CaS), C. albicans ATCC 96901 (fluconazole-resistant: CaR), Candida glabrata ATCC 2001 (fluconazolesusceptible: CgS) and C. glabrata ATCC 200918 (fluconazole-resistant: CgR) and (3) to study the action of hydrolytic enzymes (DNase, Dextranase and β-glucanase individually or in different combinations) on the ECM of CaS and CaR biofilms. Mature biofilms (48 hours) were analyzed by colony counting forming units (cfu/mL), total dry weight, insoluble dry-weight and total proteins. ECM components- alkali-soluble polysaccharides (ASPs), water-soluble polysaccharides (WSPs), extracellular DNA (e... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Cheminformatic approaches have been employed to optimize the bis-coumarin scaffold identified by Onywera et al. (2012) as a potential hit against the protease HIV-1 protein. The Open Babel library of commands was used to access functions that were incorporated into a markov chain recursive program that generated 17750 analogues of the bis-coumarin scaffold. The Morita-Baylis-Hillman accessible heterocycles were used to introduce structural diversity within the virtual library. In silico high through-put virtual screening using AutoDock Vina was used to rapidly screen the virtual library ligand set against 61 protease models built by Onywera et al. (2012). CheS-Mapper computed a principle component analysis of the compounds based on 13 selected chemical descriptors. The compounds were plotted against the principle component analysis within a 3 dimensional chemical space in order to inspect the diversity of the virtual library. The physicochemical properties and binding affinities were used to identify the top 3 performing ligands. ACPYPE was used to inspect the constitutional properties and eliminated virtual compounds that possessed open valences. Chromene based ligand 805 and ligand 6610 were selected as the lead candidates from the high-throughput virtual screening procedure we employed. Molecular dynamic simulations of the lead candidates performed for 5 ns allowed the stability of the ligand protein complexes with protease model 305152. The free energy of binding of the leads with protease model 305152 was computed over the first 50 ps of simulation using the molecular mechanics Poisson-Boltzmann method. Analysis structural features and energy profiles from molecular dynamic simulations of the protein–ligand complexes indicated that although ligand 805 had a weaker binding affinity in terms of docking, it outperformed ligand 6610 in terms of complex stability and free energy of binding. Medicinal chemistry approaches will be used to optimize the lead candidates before their analogues will be synthesized and assayed for in vivo protease activity.

Philosophiae Doctor - PhD / One of the most common HIV-associated opportunistic infections is candidiasis, caused by Candida albicans or other Candida species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of Candida species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant Candida species. The mechanisms of Candida drug resistance in the African continent have also not been described. In this study, 255 Candida species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. Candida cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in Candida species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of Candida infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant Candida strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of Candida colonization mechanisms in HIV-infected African patients.

Mycobacterium bovis and other species of Mycobacterium tuberculosis complex (MTBC) can result to a zoonotic infection known as Bovine tuberculosis (bTB). MTBC has members that may contaminate an extensive range of hosts, including wildlife. Diverse wild species are known to cause disease in domestic livestock and are acknowledged as TB reservoirs. It has been a main study worldwide to deliberate on bTB risk factors as a result some studies focused on particular parts of risk factors such as wildlife and herd management. The objectives of this study were to design questionnaires from commercial farms and smallholding farms; isolate and identify MTBC from collected samples using culture and PCR assays recovered from Fort Hare, Middledrift and Seven star dairy farms; and assessing genotypic drug resistance through detection of mutations conferring resistance to INH and RMP associated with first line treatment for MTBC infection. Questionnaires were administered to thirty (30) smallholding farm owners in the two villages (kwaMasele and Qungqwala) and three (3) three commercial farms (Fort Hare dairy farm, Middledrift dairy farm and Seven-star dairy farm). Detection of M. tuberculosis complex was achieved by Polymerase Chain Reaction using primers for IS6110; whereas a genotypic drug resistance mutation was detected using Genotype MTBDRplus assays. Nine percent (9 percent) of respondents had more than 40 cows in their herd, while 60 percent reported between 10 and 20 cows in their herd. Relationship between farm size and vaccination for TB differed from forty-one percent (41 percent) being the highest to the least five percent (5 percent). The highest number of respondents who knew about relationship between TB cases and cattle location was ninety-one percent (91 percent). Approximately fifty-one percent (51 percent) of respondents had knowledge about wild life access to the farms. Relationship between import of cattle and farm size ranged from nine percent (9 percent) to thirty-five percent (35 percent). Cattle sickness in relation to farm size differed from forty-three (43 percent) being the highest to the least three percent (3 percent); while thirty-three percent (33 percent) of respondents had knowledge about health management. Respondents with knowledge about the occurrence of TB infections in farms were forty-eight percent (48 percent). The frequency of DNA isolation from samples ranged from the highest forty-five percent (45 percent) from water to the least twenty-two percent (22 percent) from soil. Fort Hare dairy farm had the highest number of positive samples forty-four percent (44 percent) from water samples; whereas Middledrift dairy farm had the lowest positive from water, seventeen percent (17 percent). Twelve (22 percent) out of 55 isolates showed resistance to INH and RMP that is, multi-drug resistance (MDR) and nine percent (9 percent) were sensitive to either INH or RMP. The mutations at rpoB gene differed from 58 percent being the highest to the least (23 percent). Fifty-seven percent (57 percent) of samples showed a S315T1 mutation while only 14 percent possessed a S531L in the katG gene. The highest inhA mutations were detected in T8A (80 percent) eighty percent and the least was observed in A16G (17 percent). The results of this study reveals that risk factors for bTB in cattle and dairy farm workers is a serious issue abound in the Eastern Cape of South Africa; with the possibility of widespread dissemination of multidrug resistant determinants in MTBC from the environment.

Introdução: Resistência aos antirretrovirais pode limitar opções de tratamento, principalmente em pacientes com acúmulo de falhas terapêuticas, o que pode comprometer resultados clínicos. Objetivos: caracterizar o perfil de mutações na transcriptase reversa e protease do HIV-1 de pacientes em falha ao tratamento. Secundariamente, avaliar associação entre mutações e número de falhas terapêuticas, associação entre mutações e subtipos do HIV-1 e apresentar a evolução temporal da prevalência dos subtipos do HIV-1, no estado do Pará, ao Norte do Brasil. Método: Estudo transversal, no qual se avaliam genotipagens, entre janeiro de 2004 a dezembro de 2013, com dados obtidos de formulário de solicitação do exame padronizado pela RENAGENO e de impressos dos resultados, ambos arquivados em quatro serviços de atendimento especializados. Foram incluídos os testes realizados por laboratório da RENAGENO, em maiores de 18 anos, e o primeiro exame daqueles que o realizaram em mais de um momento, totalizando 377 amostras. As mutações são descritas de acordo com o banco de dados de resistência do HIV da Universidade de Stanford (http://hivdb.stanford.edu), estimam-se suas prevalências e avaliam-se mutações de resistência de acordo com o número de falhas no momento da genotipagem, bem como diferenças de mutações entre subtipos B e não-B do HIV-1. Resultados: A mutação M184V foi a mais prevalente (80,1%), seguida da K130N (40,6%) e TAM. Em pacientes multiexperimentados previamente à genotipagem, resistência a ZDV, d4T e TDF foi associada às mutações M41L, D67N, V118I, L210W, K219Q e T69D; bem como resistência a todos os IP/r associou-se às mutações principais M46I, V82A, L90M, I54V, I84V, M46L e L76V. O subtipo B é o predominante no Pará (90,7%) e diferenças de prevalência de mutações entre subtipos ocorreram entre as mutações L63P e A71T versus subtipo B, enquanto as mutações L76V, M36I, K20R, L10V, L89M e F53L associaram-se ao subtipo não-B. Conclusão: A seleção de mutações de resistência do HIV-1 relacionada aos antirretrovirais é similar ao descrito em literatura. O acúmulo de falhas ao tratamento favorece a emergência de mutações, o que reforça o monitoramento de falha virológica, seguida de genotipagem para minimizar o impacto de resistência. Estudos adicionais de epidemiologia molecular são necessários para avaliar melhor a questão da prevalência de subtipos de HIV-1 no estado e possíveis associações com mutações de resistência do HIV-1. / Introduction: Resistance to antiretroviral treatment can limit treatment options, especially in patients with accumulation of therapeutic failures, which may compromise clinical outcomes. Objectives: characterizing the profile of mutations in the protease and reverse transcriptase of HIV-1 patients in the treatment failure. Secondarily to evaluate the association between mutations and the number of treatment failures, association between mutations and subtypes of HIV-1 and present the temporal evolution of the prevalence of subtypes of HIV-1 in the state of Pará in northern Brazil. Method: cross-sectional study in which genotyping is evaluated between January, 2004 and December, 2013 with data obtained from the standardized application form for the examination RENAGENO and printed the results, both filed in four specialty care services. We included those by laboratory RENAGENO in 18 years and the first examination in those who underwent more than one time, totaling 377 samples. Mutations are described according to the database of HIV resistance at Stanford University (http://hivdb.stanford.edu), estimated their prevalence and resistance is evaluated according to the number of failures at the time of genotyping as well as differences between mutations and subtype B and non-B HIV-1. Results: The M184V mutation was the most prevalent (80.1%), followed by K130N (40.6%) and TAM. In patients who received at least three treatments prior to genotyping, resistance to ZDV, d4T and TDF was associated with mutations M41L, D67N, V118I, L210W, K219Q and T69D; well as resistance to all PI / r was associated with the major mutations M46I, V82A, L90M, I54V, I84V M46L and L76V. HIV-1 subtype B was the most prevalent (90.7%) and there were differences between subtypes B versus mutations: L63P and A71T were more frequent in the subtype B, whereas mutations L76V, K20R, L10V, L89M and F53L were in non-B subtypes. Conclusion: The selection of resistance mutations in HIV-1 related to antiretroviral is similar to that described in the literature. The accumulation of failures to treatment favors the emergence of mutations, reinforcing the monitoring and evaluation of virologic failure by genotyping to minimize the impact resistance. Additional molecular epidemiological studies are needed to better assess the issue of prevalence of subtypes of HIV-1 in the state and possible associations with resistance mutations in HIV-1.

Alguns estudos caracterizaram a microbiota de canais radiculares de dentes decíduos com necrose pulpar como polimicrobiana, com predomínio de microrganismos anaeróbios. No entanto, nenhum estudo até o presente momento investigou a presença de genes de resistência a antimicrobianos em diferentes ecossistemas da cavidade oral de crianças. Este estudo tem por objetivo determinar a presença de espécies de Prevotella e do gene cfxa/cfxa2 associados à resistência à beta-lactâmicos, em diferentes ecossistemas orais de crianças com necrose pulpar. Vinte e sete crianças, que estavam sob cuidados odontológicos no Ambulatório de Clínica Infanto-Juvenil da Faculdade de Odontologia da UFRGS, e que apresentavam, pelo menos, um dente decíduo com necrose pulpar foram selecionados para este estudo. Foram coletadas amostras de saliva, biofilme supragengival, biofilme da câmara pulpar e do canal radicular de 32 dentes (27 posteriores e 5 anteriores). Após isolamento do DNA microbiano, a presença das bactérias Prevotella intermedia, Prevotella nigrescens, Prevotella tannerae e do gene cfxA/cfxA2 foi avaliada através do método de PCR. A amostra foi composta de pacientes com idade média de 5,5 anos (± 1,76). A taxa total de espécies de Prevotella foi de 29,1%, 25%, 21,8% e 32,29% em amostras de saliva, biofilme, câmara pulpar e canal radicular, respectivamente. As três espécies juntas não foram detectadas em todos os micro-ambientes do mesmo paciente, mas estavam presentes em 3,1% (n=1) de amostras do canal radicular. Prevotella nigrescens foi a bactéria mais frequentemente encontrada em todos os ecossistemas estudados. Foram observadas diferenças estatisticamente significativas em relação à presença de P. nigrescens em pelo menos um local e idade do paciente (teste t, p = 0,04). Também foi encontrada associação entre a presença desta bactéria, em pelo menos um local e uso de antimicrobianos (teste exato de Fisher, p = 0,014). A presença do gene de resistência à beta-lactâmicos, cfxA/cfxA2, foi testada em 12 pacientes da amostra, nos quatro micro-ambientes orais. Entre esses pacientes, 55,6% eram meninas, com idade média de 6 anos (± 2,5). Não foi detectada a presença deste gene em nenhuma amostra investigada. Os dados sugerem que a cavidade bucal de crianças com necrose pulpar apresenta presença diversificada de espécies de Prevotella em diferentes micro-ambientes orais. A ausência do gene cfxA/cfxA2 foi observada em todas as amostras investigadas. Estudos futuros, testando a presença de outros genes de resistência à beta-lactâmicos, são importantes para uma investigação abrangente. / Some studies characterized the microbiota of root canals of primary teeth with pulp necrosis as polymicrobial, with a predominance of anaerobic microorganisms. However, no study to date has investigated the presence of antimicrobial resistance genes in different ecosystems of the oral cavity of children. This study aims to determine the presence of Prevotella species and genes associated with resistance to beta-lactams in different oral enviroments of children with pulp necrosis. Twenty-seven children who were under dental care at the Children and Youth Clinic (Dental School, UFRGS, Porto Alegre, Brazil), and who had at least one primary tooth with pulp necrosis were selected for this study. Saliva, supragingival biofilm, pulp chamber biofilm and root canal biofilm were collected of 32 teeth (27 posterior and 5 anterior). After isolation of microbial DNA, the presence of Prevotella intermedia, Prevotella nigrescens, Prevotella tannerae and cfxA/cfxA2 gene were evaluated using the PCR. The sample consisted of patients with a mean age of 5.5 years (± 1.76). The total rate of Prevotella species was 29.1%, 25%, 21.8% and 32.29% in saliva samples, biofilm, pulp chamber and root canal, respectively. The three strains were not detected in all enviroments of the same patient, but were present at 3.1% (n = 1) of the root canal samples. Prevotella nigrescens was the most common bacteria in all oral enviroments. Statistical significant differences were observed for the presence of P. nigrescens at least one oral enviroment and age of the patient (t-test, p = 0.04). Also an association was observed, among the presence of these bacteria in at least one enviroment and use of antimicrobials (Fisher's exact test, p = 0.014). The presence of the resistance gene to beta-lactams, cfxA/cfxA2, was tested on 12 patients of the sample at all four oral enviroments. Among these patients, 55.6% were girls with a mean age of 6 years (± 2.5). Absence of this gene in the sample investigated was detected. The absence of cfxA/cfxA2 gene was observed in all the investigated samples. Future studies testing the presence of other resistance genes to beta-lactams, are important for a comprehensive investigation.