988 resultados para Dextransucrase assays
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We sought to determine the frequency of serological markers of selected infections in a population of psychiatric patients in Durango City, Mexico, and to determine whether there are any epidemiological characteristics of the subjects associated with the infections. One hundred and five inpatients of a public psychiatric hospital of Durango were examined for HBsAg, anti-HCV antibodies, anti-HIV antibodies, anti-Brucella antibodies, rapid plasma reagin and anti-Cysticercus antibodies by commercially available assays. Anti-Cysticercus antibodies were confirmed by Western blot and HBsAg by neutralization assay. Epidemiological data from each participant were also obtained. Seroprevalences of HBsAg, anti-HCV, anti-HIV, anti-Brucella, rapid plasma reagin and anti-Cysticercus antibodies found were 0.0%, 4.8%, 0.9%, 0.0%, 1.9%, and 0.9%, respectively. Overall, 9 (8.6%) inpatients showed seropositivity to any infection marker. We concluded that our psychiatric inpatients have serological evidence of a number of infections. HCV is an important pathogen among our psychiatric inpatients. Health care strategies for prevention and control of infections in Mexican psychiatric patients should be considered.
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In order to determine the role of lysozyme, an antimicrobial peptide belonging to the innate immune system, against the dimorphic fungus Paracoccidioides brasiliensis, co-cultures of the MH-S murine alveolar macrophages cell line with P. brasiliensis conidia were done; assays to evaluate the effect of physiological and inflammatory concentrations of lysozyme directly on the fungus life cycle were also undertaken. We observed that TNF-α-activated macrophages significantly inhibited the conidia to yeast transition (p = 0.0043) and exerted an important fungicidal effect (p = 0.0044), killing 27% more fungal propagules in comparison with controls. Nonetheless, after adding a selective inhibitor of lysozyme, the fungicidal effect was reverted. When P. brasiliensis propagules were exposed directly to different concentrations of lysozyme, a dual effect was observed. Physiologic concentrations of the enzyme facilitated the conidia-to-yeast transition process (p < 0.05). On the contrary, inflammatory concentrations impaired the normal temperature-dependant fungal transition (p < 0.0001). When yeast cells were exposed to lysozyme, irrespective of concentration, the multiple-budding ability was badly impaired (p < 0.0001). In addition, ultra-structural changes such as subcellular degradation, fusion of lipid vacuoles, lamellar structures and interruption of the fibrilar layer were observed in lysozyme exposed conidia. These results suggest that lysozyme appears to exert a dual role as part of the anti-P. brasiliensis defense mechanisms.
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The aim of the present study was to verify the activity of the Tri-N-Butyl Tin maleate compound against Staphylococcus aureus and Aspergillus niger, after its industrial application in 40 samples of carpets of different materials (polypropylene, polyester, polyamide and wool). The qualitative assays were performed through two methodologies: Inhibition Halo (HZ) and Inhibition of Surface (Print). The carpet with the product inhibited 100% of bacterial (Staphylococcus aureus) and fungi (Aspergillus niger) growth, under the conditions of this study. The microbial inhibition was higher in upper portion of carpets. The methodologies employed appear to be adequate to test the bactericide and fungicide activities of the Tri-N-Butyl Tin maleate. The print methodology confirmed the results obtained by the inhibition zone assay. Further studies using the same methodologies are needed to confirm our results.
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Adhesins (P-fimbriae, S-fimbriae, type 1 fimbriae and afimbrial adhesin), toxins (α-hemolysin and cytotoxic necrotizing factor type 1), iron acquisition systems (aerobactin) and host defense avoidance mechanisms (capsule or lipopolysaccharide) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. In this work, 162 Uropathogenic Escherichia coli (UPEC) strains from patients with cystitis were genotypically characterized by polymerase chain reaction (PCR) assay. We developed three multiplex PCR assays for virulence-related genes papC, papE/F, papG alleles, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, and kpsMTII, all of them previously identified in UPEC strains. The PCR assay results identified 158 fimH (97.5%), 86 kpsMTII (53.1%), 53 papC/papEF/papG (32.7%), 45 sfa (27.8%), 42 iucD (25.9%), 41 hly (25.3%), 36 usp (22.2%), 30 cnf-1(18.5%) and 10 afa (6.2%) strains. No strain was positive for cdtB. In this work, we also demonstrated that adhesins may be multiple within a single strain and that several virulence genes can occur combined in association.
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Cryptococcus neoformans is the major cause of fungal meningitis, a potentially lethal mycosis. Bird excreta can be considered a significant environmental reservoir of this species in urban areas, thirty-three samples of pigeon excreta were collected within the city of Vitoria, Brazil. Cryptococcus neoformans was isolated and identified using standard biochemical assays in ten samples. PCR amplification with primer M13 and orotidine monophosphate pyrophosphorylase (URA5) gene-restriction fragment length polymorphism (RFLP) analysis discerned serotypes and genotypes within this species. All isolates were serotype A (C. neoformans var. grubii) and genotype VNI. The two alternative alleles a and α at the mating type locus were determined by PCR amplification and mating assays performed on V8 medium. All isolates were MAT α mating type but only 50% were able to mate in vitro with the opposite mating type MAT a tester strains (JEC20, KN99a and Bt63). This study adds information on the ecology and molecular characterization of C. neoformans in the Southeast region of Brazil.
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Candida glabrata is considered a major opportunistic fungal pathogen of humans. The capacity of this yeast species to cause infections is dependent on the ability to grow within the human host environment and to assimilate the carbon sources available. Previous studies have suggested that C. albicans can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources, such as carboxylic acids, contributes to the virulence of this fungus. Transcriptional studies on C. glabrata cells identified a similar response, upon nutrient deprivation. In this work, we aimed at analyzing biofilm formation, antifungal drug resistance, and phagocytosis of C. glabrata cells grown in the presence of acetic acid as an alternative carbon source. C. glabrata planktonic cells grown in media containing acetic acid were more susceptible to fluconazole and were better phagocytosed and killed by macrophages than when compared to media lacking acetic acid. Growth in acetic acid also affected the ability of C. glabrata to form biofilms. The genes ADY2a, ADY2b, FPS1, FPS2, and ATO3, encoding putative carboxylate transporters, were upregulated in C. glabrata planktonic and biofilm cells in the presence of acetic acid. Phagocytosis assays with fps1 and ady2a mutant strains suggested a potential role of FPS1 and ADY2a in the phagocytosis process. These results highlight how acidic pH niches, associated with the presence of acetic acid, can impact in the treatment of C. glabrata infections, in particular in vaginal candidiasis.
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Spleen cells from mice were examined at 5, 10, 15, 20 and 25 days post-infection (dpi) with Dermatobia hominis larva and at 5, 10, 15, 30 and 60 days post-larval emergence (dple). Cell proliferation in vitro assays were carried out with RPMI-1640 medium and larval secretory product (LSP) of D. hominis at 5, 10, 15, 20 and 25 days. When each group of mice was tested against each medium, significance was only seen for 25 dpi, with increasing order: LSP-10 d, -25 d, -5 d, -20 d, -15 d and RPMI. Significant results were also observed when each medium was tested against mice at each dpi or dple. Each dple group vs. each medium produced significant results only for 10 dple, with increasing order: LSP-5 d, -20 d, -25 d, -10 d, -15 d and RPMI. Comparative tests were also carried out between groups to refine certain observations. The LSPs were also analyzed using SDS-PAGE. The results prove that myiasis caused depletion of spleen cells, particularly under the effect of the LSP-10 and -15, but the cells tended to increase up to 60 dple. This in vitro assay may represent the real systemic immune response in the relationship LSP-D. hominis-host.
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The objective of the present study was to evaluate the serum viral load in chronically infected Hepatitis B virus (HBV) patients and to investigate the distribution of HBV genotypes in São Paulo city. Quantitative HBV-DNA assays and HBV genotyping have gained importance for predicting HBV disease progression, have been employed for assessing infectivity, for treatment monitoring and for detecting the emergence of drug resistance. Twenty-nine Brazilian patients with suspected chronic hepatitis B were studied, using real time PCR for viral load determination and direct DNA sequencing for the genotyping. The serology revealed chronic HBV infection in 22 samples. The HBV-DNA was positive in 68% samples (15/22). The phylogenetic analysis disclosed that eleven patients were infected with HBV genotype A, two with genotype F and two with genotype D. Thus, the genotype A was the most prevalent in our study.
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Toxoplasma gondii causes severe fetal disease during acute infection in pregnant women, thus demanding early diagnosis for effective treatment and fetus preservation. Fetal tests are inefficient and risky, and diagnosis is based on maternal IgM serology, which had weak screening ability due to increased sensitivity, with alternative IgG avidity tests. Here, we performed ELISA and avidity assays using a recombinant T. gondii antigen, rROP2, in samples from 160 pregnant women screened from a large public hospital who were referred due to positive IgM assays. IgG serology and avidity assays were compared using whole T. gondii extract or rROP2. ELISA IgG detection with rROP2 showed good agreement with assays performed with T. gondii extract, but rROP2 IgG avidity assays were unrelated to whole extract antigen IgG avidity, regardless of the chaotrope used. These data show that avidity maturation is specific to individual antigen prevalence and immune response during infection. ELISA rROP2 IgG assays may be an alternative serological test for the diagnosis of toxoplasmosis during pregnancy, although our data do not support their use in avidity assays.
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In view of the high circulation of migratory birds and the environmental and climatic conditions which favor the proliferation of arthropods, the Brazilian Pantanal is susceptible to circulation of arboviruses. However, the amount of data concerning arbovirus vectors in this area is scarce; therefore the aim of this study was to conduct a preliminary investigation of Culicidae species in the Nhecolândia Sub-region of South Pantanal, Brazil and their potential importance in the arbovirus transmission. A total of 3684 specimens of mosquitoes were captured, 1689 of which caught in the rainy season of 2007, were divided into 78 pools and submitted to viral isolation, Semi-Nested RT-PCR and Nested RT-PCR, with a view to identifying the most important arboviruses in Brazil. Simultaneously, 70 specimens of ticks found blood-feeding on horses were also submitted to the same virological assays. No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative. Nevertheless, a total of 22 Culicidae species were identified, ten of which had previously been reported as vectors of important arboviruses. The diversity of species found blood-feeding on human and horse hosts together with the arboviruses circulation previously reported suggest that the Nhecolândia Sub-region of South Pantanal is an important area for arbovirus surveillance in Brazil.
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RESUMO: O efluxo de compostos antimicrobianos é um mecanismo importante na multirresistência em bactérias. Bombas de efluxo codificadas em plasmídeos, como a QacA e a Smr, estão implicadas na susceptibilidade reduzida a biocidas, geralmente utilizados na prevenção e controlo de infecções nosocomiais, incluindo as causadas por estirpes de Staphylococcus aureus resistentes à meticilina (MRSA). Neste trabalho pretendeu-se avaliar a relevância de QacA e Smr no perfil de susceptibilidade dos isolados clínicos MRSA SM39 e SM52, que transportam os plasmídeos pSM39 e pSM52 com os determinantes qacA e smr, respectivamente. A actividade de efluxo das estirpes SM39 e SM39 curada (sem pSM39) e das estirpes SM52 e RN4220:pSM52 (estirpe susceptível RN4220 transformada com pSM52) foi caracterizada por: (1) determinação da concentração mínima inibitória (CMI) de biocidas, corantes e antibióticos, na ausência e presença dos inibidores de efluxo tioridazina, clorpromazina, verapamil e reserpina; e (2) fluorometria em tempo-real. A determinação de CMIs demonstrou que a actividade de efluxo mediada por QacA e Smr está envolvida na susceptibilidade reduzida aos biocidas e corantes testados, que incluíram o brometo de hexadeciltrimetilamónio, a cetrimida, o cloreto de benzalcónio, a berberina, o cloreto de dequalínio, a pentamidina e o brometo de etídeo. Os ensaios fluorométricos confirmaram a elevada actividade de efluxo presente nas estirpes com os genes qacA ou smr. A determinação de CMIs para antibióticos β-lactâmicos em conjunto com o teste da nitrocefina revelou a presença simultânea do gene qacA e de uma β-lactamase no plasmídeo pSM39. Este trabalho evidencia a importância das bombas de efluxo QacA e Smr na resistência a biocidas em estirpes MRSA e na sobrevivência destas estirpes em ambiente hospitalar e na comunidade, para além de destacar a questão da potencial co-resistência entre biocidas e antibióticos.--------------- ABSTRACT: Drug efflux has become an important cause of multidrug resistance (MDR) in bacteria. Plasmid-encoded MDR efflux pumps, such as QacA and Smr, are implicated in reduced susceptibility to biocides, generally used in the prevention and control of nosocomial infections, including the ones caused by methicillin-resistant Staphylococcus aureus (MRSA). In this work, we aimed to evaluate the relevance of QacA and Smr to the susceptibility profile of the clinical MRSA isolates SM39 and SM52, which harbor the plasmids pSM39 and pSM52 that carry the determinants qacA and smr, respectively. Efflux activity of strain SM39 and its plasmid-free counterpart, SM39 cured, SM52 and RN4220:pSM52 (susceptible strain RN4220 transformed with pSM52) was characterized by: (1) determination of minimum inhibitory concentration (MIC) of biocides, dyes and antibiotics, in the absence and presence of the efflux inhibitors thioridazine, chlorpromazine, verapamil and reserpine; and (2) real-time fluorometry. MIC determination showed that QacA and Smr mediated efflux was involved in the reduced susceptibility profile to the biocides and dyes tested, which included hexadecyltrymethylammonium bromide, cetrimide, benzalkonium chloride, berberine, dequalinium chloride, pentamidine and ethidium bromide. Fluorometric assays confirmed the higher efflux activity present in strains harboring qacA or smr genes. Moreover, MIC determination for β-lactam antibiotics together with the nitrocefin test confirmed the presence of a β-lactamase in the plasmid carried by SM39 strain, pSM39. This work highlights the relevance of QacA and Smr to the biocide resistance in MRSA strains, and consequently to their survival and maintenance in the hospital environment and in the community. Furthermore, the presence of a β-lactamase and qacA determinants in the the same plasmid reinforces the question of the potencial biocide/antibiotic co-resistance in MRSA strains.
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RESUMO: Introdução: A espondilite anquilosante (EA) é uma doença inflamatória crónica caracterizada pela inflamação das articulações sacroilíacas e da coluna. A anquilose progressiva motiva uma deterioração gradual da função física e da qualidade de vida. O diagnóstico e o tratamento precoces podem contribuir para um melhor prognóstico. Neste contexto, a identificação de biomarcadores, assume-se como sendo muito útil para a prática clínica e representa hoje um grande desafio para a comunidade científica. Objetivos: Este estudo teve como objetivos: 1 - caracterizar a EA em Portugal; 2 - investigar possíveis associações entre genes, MHC e não-MHC, com a suscetibilidade e as características fenotípicas da EA; 3 - identificar genes candidatos associados a EA através da tecnologia de microarray. Material e Métodos: Foram recrutados doentes com EA, de acordo com os critérios modificados de Nova Iorque, nas consultas de Reumatologia dos diferentes hospitais participantes. Colecionaram-se dados demográficos, clínicos e radiológicos e colhidas amostras de sangue periférico. Selecionaram-se de forma aleatória, doentes HLA-B27 positivos, os quais foram tipados em termos de HLA classe I e II por PCR-rSSOP. Os haplótipos HLA estendidos foram estimados pelo algoritmo Expectation Maximization com recurso ao software Arlequin v3.11. As variantes alélicas dos genes IL23R, ERAP1 e ANKH foram estudadas através de ensaios de discriminação alélica TaqMan. A análise de associação foi realizada utilizando testes da Cochrane-Armitage e de regressão linear, tal como implementado pelo PLINK, para variáveis qualitativas e quantitativas, respetivamente. O estudo de expressão génica foi realizado por Illumina HT-12 Whole-Genome Expression BeadChips. Os genes candidatos foram validados usando qPCR-based TaqMan Low Density Arrays (TLDAs). Resultados: Foram incluídos 369 doentes (62,3% do sexo masculino, com idade média de 45,4 ± 13,2 anos, duração média da doença de 11,4 ± 10,5 anos). No momento da avaliação, 49,9% tinham doença axial, 2,4% periférica, 40,9% mista e 7,1% entesopática. A uveíte anterior aguda (33,6%) foi a manifestação extra-articular mais comum. Foram positivos para o HLA-B27, 80,3% dos doentes. Os haplótipo A*02/B*27/Cw*02/DRB1*01/DQB1*05 parece conferir suscetibilidade para a EA, e o A*02/B*27/Cw*01/DRB1*08/DQB1*04 parece conferir proteção em termos de atividade, repercussão funcional e radiológica da doença. Três variantes (2 para IL23R e 1 para ERAP1) mostraram significativa associação com a doença, confirmando a associação destes genes com a EA na população Portuguesa. O mesmo não se verificou com as variantes estudadas do ANKH. Não se verificou associação entre as variantes génicas não-MHC e as manifestações clínicas da EA. Foi identificado um perfil de expressão génica para a EA, tendo sido validados catorze genes - alguns têm um papel bem documentado em termos de inflamação, outros no metabolismo da cartilagem e do osso. Conclusões: Foi estabelecido um perfil demográfico e clínico dos doentes com EA em Portugal. A identificação de variantes génicas e de um perfil de expressão contribuem para uma melhor compreensão da sua fisiopatologia e podem ser úteis para estabelecer modelos com relevância em termos de diagnóstico, prognóstico e orientação terapêutica dos doentes. -----------ABSTRACT: Background: Ankylosing Spondylitis (AS) is a chronic inflammatory disorder characterized by inflammation in the spine and sacroiliac joints leading to progressive joint ankylosis and in progressive deterioration of physical function and quality of life. An early diagnosis and early therapy may contribute to a better prognosis. The identification of biomarkers would be helpful and represents a great challenge for the scientific community. Objectives: The present study had the following aims: 1- to characterize the pattern of AS in Portuguese patients; 2- to investigate MHC and non-MHC gene associations with susceptibility and phenotypic features of AS and; 3- to identify candidate genes associated with AS by means of whole-genome microarray. Material and Methods: AS was defined in accordance to the modified New York criteria and AS cases were recruited from hospital outcares patient clinics. Demographic and clinical data were recorded and blood samples collected. A random group of HLA-B27 positive patients and controls were selected and typed for HLA class I and II by PCR-rSSOP. The extended HLA haplotypes were estimated by Expectation Maximization Algorithm using Arlequin v3.11 software. Genotyping of IL23R, ERAP1 and ANKH allelic variants was carried out with TaqMan allelic discrimination assays. Association analysis was performed using the Cochrane-Armitage and linear regression tests as implemented in PLINK, for dichotomous and quantitative variables, respectively. Gene expression profile was carried out using Illumina HT-12 Whole-Genome Expression BeadChips and candidate genes were validated using qPCR-based TaqMan Low Density Arrays (TLDAs). Results: A total of 369 patients (62.3% male; mean age 45.4±13.2 years; mean disease duration 11.4±10.5 years), were included. Regarding clinical disease pattern, at the time of assessment, 49.9% had axial disease, 2.4% peripheral disease, 40.9% mixed disease and 7.1% isolated enthesopathic disease. Acute anterior uveitis (33.6%) was the most common extra-articular manifestation. 80.3% of AS patients were HLA-B27 positive. The haplotype A*02/B*27/Cw*02/DRB1*01/DQB1*05 seems to confer susceptibility to AS, whereas A*02/B*27/Cw*01/DRB1*08/DQB1*04 seems to provide protection in terms of disease activity, functional and radiological repercussion. Three markers (two for IL23R and one for ERAP1) showed significant single-locus disease associations. Association of these genes with AS in the Portuguese population was confirmed, whereas ANKH markers studied did not show an association with AS. No association was seen between non-MHC genes and clinical manifestations of AS. A gene expression signature for AS was established; among the fourteen validated genes, a number of them have a well-documented inflammatory role or in modulation of cartilage and bone metabolism. Conclusions: A demographic and clinical profile of patients with AS in Portugal was established. Identification of genetic variants of target genes as well as gene expression signatures could provide a better understanding of AS pathophysiology and could be useful to establish models with relevance in terms of susceptibility, prognosis, and potential therapeutic guidance.
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Dissertação para obtenção do Grau de Mestre em Biotecnologia
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A existência de contribuições significativas de águas de infiltração e de águas pluviais nas redes de drenagem de águas residuais urbanas, motivaram a realização deste trabalho. Este estudo foi realizado no sistema de saneamento de Gondomar, utilizando os registos de caudais e qualidade das águas residuais em quatro estações de tratamento de águas residuais, durante quatro anos. O principal objectivo deste trabalho foi estimar as afluências de infiltração e precipitação ao sistema de saneamento e a identificação dos subsistemas críticos. Concluiu-se que a percentagem de volume excedente é cerca de 30% nos anos com maior ocorrência de precipitação. O propósito deste estudo foi também apresentar soluções para este problema, incluindo técnicas de inspecção e reparação dos elementos de drenagem, as quais foram definidas para os subsistemas identificados como críticos, Freixo e Rio Tinto. No Freixo mostrou-se importante a inspecção dos colectores, ramais e câmaras de visita, uma vez que o caudal afluente a este subsistema é muito superior ao esperado e a água residual apresenta-se diluída. Em Rio Tinto evidenciou-se como prioritária a detecção e eliminação de ligações abusivas, dado que são frequentes as ocorrências de exfiltração, com danos significativos. Para além das técnicas de detecção apresentadas considera-se que é necessário manter a monitorização de caudais e recipitação nos subsistemas, e se possível com registo contínuo ao longo do dia. Para a quantificação de precipitação é sugerida a utilização de três udómetros, de forma a permitir estabelecer com maior exactidão, a relação entre as contribuições pluviais e o caudal afluente. São apresentadas propostas, que incluem além de inspecções, ensaios e reparações, como um ponto de partida para a recolha de informação para actualização do adastro, com vista a uma futura aplicação da modelação matemática de previsão do comportamento destes subsistemas.
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The present work evaluated the diagnostic accuracy of detection of Dengue NS1 antigen employing two NS1 assays, an immunochromatographic assay and ELISA, in the diagnostic routine of Public Health laboratories. The results obtained with NS1 assay were compared with virus isolation and, in a subpopulation of cases, they were compared with the IgM-ELISA results obtained with convalescent samples. A total of 2,321 sera samples were analyzed by one of two NS1 techniques from March to October 2009. The samples were divided into five groups: groups I, II and III included samples tested by NS1 and virus isolation, and groups IV and V included patients with a first sample tested by NS1 and a second sample tested by IgM-ELISA. Sensitivity, specificity, positive and negative predictive values, Kappa Index and Kappa Concordance were calculated. The results showed that NS1 testing in groups I, II and III had high sensitivity (98.0%, 99.5% and 99.3%), and predictive values and Kappa index between 0.9 - 1.0. Groups IV and V only had Kappa Concordance calculated, since the samples were analyzed according to the presence of NS1 antigen or IgM antibody. Concordance of 92.1% was observed when comparing the results of NS1-negative samples with IgM-ELISA. Based on the findings, it is possible to suggest that the tests for NS1 detection may be important tools for monitoring the introduction and spread of Dengue serotypes.