Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-10-18T14:56:14Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CaracterizacaoEpidemiologicaGenotipica.pdf: 2006227 bytes, checksum: 29730c9982b04cd3d56cbf0c52947d27 (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-11-08T14:22:55Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CaracterizacaoEpidemiologicaGenotipica.pdf: 2006227 bytes, checksum: 29730c9982b04cd3d56cbf0c52947d27 (MD5) / Made available in DSpace on 2017-11-08T14:22:56Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CaracterizacaoEpidemiologicaGenotipica.pdf: 2006227 bytes, checksum: 29730c9982b04cd3d56cbf0c52947d27 (MD5) Previous issue date: 2012 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A criptococose é uma infecção fúngica causada por uma levedura encapsulada do gênero Cryptococcus que afeta tanto humanos quanto animais, sendo considerada como uma infecção oportunista normalmente associada à imunodepressão. Trata-se de uma das infecções fúngicas humanas de significativa morbidade e mortalidade tanto em indivíduos imunocomprometidos quanto em imunocompetentes, e manifesta-se principalmente sob a forma de meningoencefalite. O objetivo deste trabalho foi realizar a caracterização epidemiológica, genotípica e fenotípica da criptococose em uma unidade de referência no estado do Pará. A população alvo foi composta pelos pacientes atendidos no Hospital Universitário João de Barros Barreto/UFPA, de janeiro de 2010 a dezembro de 2011 com diagnóstico de criptococose confirmado laboratorialmente. Os dados pessoais, clínicos e laboratoriais foram coletados a partir da revisão dos prontuários do arquivo médico do HUJBB e os isolados foram identificados e caracterizados morfológica e bioquimicamente. O tipo sexuado e o genótipo foram identificados através de PCR utilizando iniciadores específicos. No período de janeiro de 2010 a dezembro de 2011 foram identificados 59 casos de criptococose. A maioria dos pacientes eram homens (36/59, 61%), com idade entre 5 a 70 anos, média de 30,8 anos. A faixa etária que predominou foi de 34 a 43 anos (39% dos casos). Os principais sinais e sintomas apresentados por eles foram cefaleia (85,5%), vômito (80%) e febre (76,4%). Todas as crianças (6/6) eram HIV negativas, tendo como agente causador o C. gattii (31,6%); já em adultos 71,1% das infecções eram causadas por C. neoformans, sendo a maioria HIV positivo. Os casos de óbito e de recidivas foram mais elevados entre os pacientes infectados por C. neoformans e a presença de sequelas foi mais frequente entre os pacientes infectados por C. gattii. Todos os isolados eram MAT α e houve a predominância de dois tipos moleculares: VNI (64,4%) e VGII (35,6%). Este estudo reforça que o estado do Pará é endêmico para a infecção por Cryptococcus spp., cuja forma de apresentação clinica principal, a meningoencefalite determina elevadas taxas de morbi-mortalidade. / Cryptococcosis is a fungal infection caused by an encapsulated yeast Cryptococcus genus that affects both humans and animals, and is considered as an opportunistic infection commonly associated with immunosuppression. This is one of human fungal infections cause significant morbidity and mortality in both immunocompetent and in immunocompromised individuals and appears mainly in the form of meningoencephalitis. The aim of this study was to characterize epidemiological, genotypic and phenotypic cryptococcosis in a reference unit in the state of Pará. The target population was composed of patients treated at the University Hospital João de Barros Barreto/UFPA, January 2010 to December 2011 with laboratory confirmed diagnosis of cryptococcosis. Personal, clinical and laboratory data were collected from a review of medical records file HUJBB and the isolates were identified and characterized morphologically and biochemically. The type sexed and genotype were identified by PCR using specific primers. From January 2010 to December 2011 were identified 59 cases of cryptococcosis. Most patients were men (36/59, 61%), aged 5-70 years, mean 30.8 years. The predominant age group was 34-43 years (39% of cases). The main signs and symptoms presented by them were headache (85.5%), vomiting (80%) and fever (76.4%). All children (6/6) were HIV negative, and as the causative agent C. gattii (31.6%), while in adults 71.1% of infections were caused by C. neoformans, most HIV positive. The cases of death and recurrence were higher among patients infected with C. neoformans and the presence of sequelae was more frequent among patients infected with C. gattii. All isolates were MAT α and there was a predominance of two molecular types: VNI (64.4%) and VGII (35.6%). This study reinforces that the state of Pará is endemic to infection by Cryptococcus spp., Whose main clinical presentation, meningoencephalitis determines high rates of morbidity and mortality.

Cryptococcus neoformans (C. neoformans), um fungo que se encontra disseminado em várias partes do mundo, inclusive no Brasil, é o responsável pela criptococose infecção oportunista mais comum em pacientes com a síndrome da imunodeficiência adquirida (SIDA). O caráter sistêmico da criptococose pode levar esses pacientes a óbito. A finalidade de se obter um diagnóstico laboratorial rápido e acurado de C. neoformans, principalmente para o seguimento dos pacientes HIV nos levou a investigar \"seqüências iniciadoras\" (Si) A, B e C das regiões 18SrDNA, 5,8SrDNA e ITS do Cryptococcus spp. Pela Nested PCR com estas seqüências, sugerimos a melhor delas para o desenvolvimento de um diagnóstico molecular em relação aos métodos usuais. Para tal, foram avaliadas amostras de soro e líquor de 39 pacientes, que já haviam recebido tratamento clínico. Todos os casos foram selecionados em grupos, como segue:7 com criptococose (GIII), 14 HIV positivos (GIV), 18 HIV positivos associados com a criptococose (GV) em relação a 10 controles - indivíduos sadios (GI) e amostras de culturas referência (GII). Os resultados obtidos pela Nested PCR com as \"Sis\" A, B e C foram comparados àqueles obtidos pelos métodos de diagnóstico convencionais. As análises desse estudo mostraram que as \"Sis\" A, B e C detectam C. neoformans com especificidade variada, tanto no soro (SiA 91,66%, SiB-100%, SiC 75%), como no líquor (SiA 83,33%, SiB 100% e SiC 75%) mas não apresentaram falso positivo, quando esses resultados foram comparados aos obtidos das culturas heterólogas (GVI). A SiB, em líquor, apresentou sensibilidade, acurácia, valores preditivo positivo e negativo, e especificidade de 100% para a detecção de C. neoformans da mesma forma que no soro, porém neste o valor preditivo negativo foi 89%, acurácia 94% e a sensibilidade 88%. Em amostras de soro e líquor, os testes Tinta da China e Látex, mostraram resultados falso positivos para o GIV e falso negativos nos grupos GIII e GV. As análises comparativas entre as técnicas mostraram que a ordem de eficiência da sensibilidade para detecção de C. neoformans no soro foi SiB>SiA=Látex>SiC e no líquor foi SiB> SiA>tinta da China>Látex=SiC. No soro, a especificidade entre as técnicas foi SiB>SiA=Látex>SiC e no líquor foi SiB=tinta da China>Látex>SiA>SiC. De acordo com nossos dados, concluímos que, independente da doença associada (HIV) ou se o paciente for tratado, a SiB foi a melhor seqüência para a detecção de C. neoformans, tanto em amostras diretamente de soro, como de líquor para todos os grupos estudados. Tendo em vista os fatos, acreditamos que, a aplicação da técnica da Nested PCR com a \"SiB\", em amostras de líquor e soro, é um método viável e acurado para realizar o diagnóstico molecular do C. neoformans em pacientes HIV. O uso de amostras de soro, para o segmento dos pacientes com SIDA, durante o tratamento, pode ser a forma menos invasiva em relação ao líquor para a detecção do C. neoformans, com vantagens sobre os métodos utilizados / Cryptococcus neoformans (C. neoformans), a fungus that is widespread in many parts of the world, including Brazil, is responsible for cryptococcosis the most common opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). The systemic character of cryptococcosis may be fatal. In order to obtain a rapid and accurate laboratory diagnosis to follow - up of HIV-cryptococcosis patients led us to investigate the sensibility and especificity of three primers (A, B and C) of 18SrDNA, 5,8SrDNA and ITS regions of Cryptococcus spp. Using Nested PCR with those primers we suggest the best among them to be used as a method of molecular diagnosis in relation to the usual techniques. For this purpose, the serum and cerebrospinal fluid (CSF) of 39 patients, who had received medical treatment, were evaluated. All cases were separated in groups, as follows: 7 with cryptococcosis (group III), 14 HIV positive (group IV), 18 HIV positive associated with cryptococcosis (group V) were compared to, 10 healthy subjects (group I) controls, as well as to reference cultures (group II) samples. The results obtained by nested PCR with primers A and B and C were compared to those obtained by conventional diagnostic methods. The analyses of primers A, B and C detected C. neoformans both in serum (SiA 91,6%, SiB-100%, SiC 75%), and in CSF (SiA 83,3%, SiB 100% e SiC 75%). Besides, they were specific for the identification of C. neoformans and showed that there were no false positives when compared with heterologous cultures (group VI) samples. The primer B in CSF showed 100% sensitivity, 100% accuracy and 100% predictive values (positive and negative), and 100% specificity for the detection of C. neoformans, the same that occurs in serum, but in this case, with 88% in sensitivity, 89% predictive value negative and 94% accuracy. In serum and CSF samples the China Ink and the Latex tests showed false positive results for group IV and false negative for III and V groups. The comparative analysis among the techniques (Nested PCR, China Ink, Latex) indicated that the efficiency order of sensitivity for the detection of C. neoformans were PrB> PrA = Latex> PrC in serum and PrB > PrA > China Ink> Latex = PrC in CSF. For the serum and CSF specificities the same techniques were used with the following results: PrB>PrA=Latex>PrC and PrB=China Ink>Latex>PrA>PrC. According to our data we conclude that, whether the patients had been under treatment or not, the Nested PCR by PrB was the best way to detect C. neoformans both in serum and in CSF for all groups. The following up of AIDS patients, throughout the course of therapy was found to be feasible, accurate and less invasive to detect C. neoformans by using serum samples (directly)

M. Tech. (Department of Chemistry, Faculty of Applied and Computer Sciences), Vaal University of Technology / The present study includes the use of a green synthetic method to prepare copper and silver nanoparticles using chitosan, aqueous extracts of Camellia sinensis, Combretum molle and Melia azedarach linn leaves. This study aims to investigate the influence of capping and precursor concentration on the properties of silver nanoparticles with emphasis on the medicinal plants chosen. The effect of capping agent on the properties of copper nanoparticles is also investigated. The phytochemical properties of plant extracts and the antimicrobial activity of the synthesized particles were also studied; this was achieved by using microdilution bioassay. Decoction method was used to extract secondary metabolites from plant leaves. Preliminary phytochemical screening carried out on the aqueous extracts of the plant leaves showed the presence of tannins, proteins, flavonoids, phenols, and carbohydrates. The total phenolic and flavonoids content of the aqueous extract was determined using spectroscopic methods. The highest phenolic content was found in the aqueous extract of Combretum molle (135 mg/g), and the highest flavonoid content was found in the aqueous extract of Camellia sinensis (0.4 mg/g). Characterization was done by a combination of spectroscopic, microscopy and XRD techniques. Both the size and shape of the synthesized silver nanoparticles were dependent on the identity of the capping molecule, precursor and capping agent concentration as depicted from their TEM and XRD results. Silver nanoparticles were found to be predominantly spherical. The capping agent concentration was also found to influence the degree of agglomeration, with an increase in capping agent concentration giving lesser agglomeration. FTIR spectral analysis showed that silver nanoparticles interact with bioactive compounds found in the plants through the hydroxyl functional group. Other shapes including diamond were observed for the effect of precursor concentration. The XRD micrographs revealed a face-centered cubic geometry and the phase remained the same with an increase in precursor concentration. The synthesized silver nanoparticles were all blue shifted compared to the bulk material. The TEM results revealed that copper nanoparticles with different sizes and shapes were successfully synthesized. All the prepared copper and silver nanoparticles showed satisfactory antifungal and antibacterial activity against Candida albicans, Cryptococcus neoformans, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumonia and Pseudomonas aeruginosa. The capping molecules used in this study also showed some antibacterial and antifungal activity against the selected strains. However nanoparticles performed better than these capping molecules. Both silver and copper nanoparticles were found to be more active against gram-negative bacteria compared to gram-positive bacteria. Amongst all the prepared silver nanoparticles Combretum molle capped nanoparticles were found to be the most active nanoparticles. Also with copper nanoparticles, it was found that Combretum molle capped nanoparticles were the most active nanoparticles. Between the two metal nanoparticles, silver nanoparticles showed high antibacterial and antifungal activity compared to copper nanoparticles. The antioxidant activity of silver nanoparticles was assessed using 2.2-diphenyl-1-picrylhydrazyl. Silver nanoparticles were found to have some antioxidant activity. However, the capping molecules were found to be more active than the synthesized nanoparticles. This observation is attributed to the presence of some bioactive compounds in the plant extracts.

<p>Systemic fungal infections brought about by <em>Cryptococcus</em> species are associated with some of the highest mortality rates of any infectious disease. Alarmingly these pathogens have overtaken tuberculosis as the second greatest killer among Sub-Saharan AIDS patients and are an emerging disease among immunocompetent populations on the Pacific Coast of North America. This clinical threat has been exacerbated by our inability to discover novel compounds that specifically target fungal cellular architecture at the genus level. To confront this challenge, we have made a concerted effort to biologically prospect the vast chemical potential of Actinomycete bacteria isolated from diverse and underexplored niches around the world. A novel phenotypic screen was developed whereby bacterial small molecule producers were co-cultured on agar plates in an intimate setting with evolutionary distant fungal pathogens <em>Candida albicans</em> and <em>Cryptococcus neoformans</em>. Diffusible small molecules released by the organisms created a signaling environment that stimulated profound phenotypic changes both in the Actinomycetes and the pathogens. We were able to discern a unique relationship whereby the growth of <em>C. neoformans</em> was specifically inhibited by Nigerian soil Actinomycete isolate curated as WAC 2288. Further bioactivity guided purification and chemical analysis lead to the identification of ibomycin, a previously undescribed 34 membered macrolactone decorated with seven sugar moieties. A draft genome of WAC 2288 revealed a 140kb gene cluster containing 12 type I PKS modules and downstream capacity to generate rare sugars are responsible for ibomycin biosynthesis. Purification of ibomycin analogs has revealed that the terminal vancosamine on the molecule is dispensable for bioactivity, establishing a chemical antecedent for target identification through affinity chromatography. Throughout these studies the unprecedented anticryptococcal activity of ibomycin is consistently recapitulated. Future work on the molecule may validate ibomycin as an effective antifungal therapy.</p> / Master of Science (MSc)