Characterization of a Hepatitis B virus strain in southwestern Paraná, Brazil, presenting mutations previously associated with anti-HBs Resistance

The present study investigated if hepatitis B virus (HBV) mutants circulate in the southwestern region of the State of Paraná, Brazil, by analyzing samples from children who received immunoprophylaxis but were born to HBV carrier mothers. Samples from 25 children were screened for HBV serum markers and for HBV DNA by PCR. Only one sample was positive for HBsAg, anti-HBs and HBV DNA, although the child had been vaccinated. Analysis of the S gene sequence of this sample showed the presence of a proline at position 105, a serine at position 114, three threonines at positions 115, 116 and 140, and a glutamine at position 129. The presence of these amino acids, except for serine at position 114, has been related to monoclonal or polyclonal therapy with anti-HBs after liver transplantation, whereas the presence of threonine at position 116 has been described in immunized children from Singapore. This finding demonstrates the possible circulation of HBV strains resistant to hepatitis B immunoprophylaxis in southwestern Paraná, Brazil. The genotype of the sample was identified as genotype D, which is frequently found in the region studied. Since 36% of the children had received incomplete or no immunoprophylaxis, more extensive follow-up of children born to HBsAg-positive mothers is needed.

Experimental characterization of the out-of-plane performance of regular stone masonry walls, including test setups and axial load influence

Stone masonry is one of the oldest and most worldwide used building techniques. Nevertheless, the structural response of masonry structures is complex and the effective knowledge about their mechanical behaviour is still limited. This fact is particularly notorious when dealing with the description of their out-of-plane behaviour under horizontal loadings, as is the case of the earthquake action. In this context, this paper describes an experimental program, conducted in laboratory environment, aiming at characterizing the out-of-plane behaviour of traditional unreinforced stone masonry walls. In the scope of this campaign, six full-scale sacco stone masonry specimens were fully characterised regarding their most important mechanic, geometric and dynamic features and were tested resorting to two different loading techniques under three distinct vertical pre-compression states; three of the specimens were subjected to an out-of-plane surface load by means of a system of airbags and the remaining were subjected to an out-of-plane horizontal line-load at the top. From the experiments it was possible to observe that both test setups were able to globally mobilize the out-of-plane response of the walls, which presented substantial displacement capacity, with ratios of ultimate displacement to the wall thickness ranging between 26 and 45 %, as well as good energy dissipation capacity. Finally, very interesting results were also obtained from a simple analytical model used herein to compute a set of experimental-based ratios, namely between the maximum stability displacement and the wall thickness for which a mean value of about 60 % was found.

Comparing the performance of geostatistical models with additional information from covariates for sewage plume characterization

In this work, kriging with covariates is used to model and map the spatial distribution of salinity measurements gathered by an autonomous underwater vehicle in a sea outfall monitoring campaign aiming to distinguish the effluent plume from the receiving waters and characterize its spatial variability in the vicinity of the discharge. Four different geostatistical linear models for salinity were assumed, where the distance to diffuser, the west-east positioning, and the south-north positioning were used as covariates. Sample variograms were fitted by the Mat`ern models using weighted least squares and maximum likelihood estimation methods as a way to detect eventual discrepancies. Typically, the maximum likelihood method estimated very low ranges which have limited the kriging process. So, at least for these data sets, weighted least squares showed to be the most appropriate estimation method for variogram fitting. The kriged maps show clearly the spatial variation of salinity, and it is possible to identify the effluent plume in the area studied. The results obtained show some guidelines for sewage monitoring if a geostatistical analysis of the data is in mind. It is important to treat properly the existence of anomalous values and to adopt a sampling strategy that includes transects parallel and perpendicular to the effluent dispersion.

Ankylosing spondylitis: genomic and functional characterization of candidate genes and their repercussion in clinical practice

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.

Characterization of rabies virus isolated from a colony of Eptesicus furinalis bats in Brazil

Some bat species have adapted to the expanding human population by acquiring the ability to roost in urban buildings, increasing the exposure risk for people and domestic animals, and consequently, the likelihood of transmitting rabies. Three dead bats were found in the yard of a house in an urban area of Jundiaí city in the state of São Paulo in southeast Brazil. Two of the three bats tested positive for rabies, using Fluorescent Antibody and Mouse Inoculation techniques. A large colony of Eptesicus furinalis was found in the house's attic, and of the 119 bats captured, four more tested positive for rabies. The objectives of this study were to report the rabies diagnosis, characterize the isolated virus antigenically and genetically, and study the epidemiology of the colony.

Electrochemical characterization of Dps, a DNA-protecting protein

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100% of isolates, the tdh gene was identified in two (10.5%) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.

Characterization of the molluscicidal activity of Bauhinia variegata and Mimusops elengi plant extracts against the fasciola vector lymnaea acuminata

The molluscicidal activity of Bauhinia variegata leaf and Mimusops elengi bark was studied against vector snail Lymnaea acuminata. The toxicity of both plants was time and concentration-dependent. Among organic extracts, ethanol extracts of both plants were more toxic. Toxicity of B. variegata leaf ethanolic extract (96h LC50- 14.4 mg/L) was more pronounced than M. elengi bark ethanolic extract (96h LC50-15.0 mg/L). The 24h LC50 of column purified fraction of B. variegata and M. elengi bark were 20.3 mg/L and 18.3 mg/L, respectively. Saponin and quercetin were characterized and identified as active molluscicidal component. Co-migration of saponin (Rf 0.48) and quercetin (Rf 0.52) with column purified bark of M. elengi and leaf of B. variegata on thin layer chromatography demonstrate same Rf value i.e. 0.48 and 0.52, respectively. The present study clearly indicates the possibility of using M. elengi and/or B. variegata as potent molluscicide.

Molecular characterization of Aeromonas spp. and Vibrio cholerae O1 isolated during a diarrhea outbreak

This work aimed to assess pathogenic potential and clonal relatedness of Aeromonas sp. and Vibrio cholerae isolates recovered during a diarrhea outbreak in Brazil. Clinical and environmental isolates were investigated for the presence of known pathogenic genes and clonal relatedness was assessed by intergenic spacer region (ISR) 16S-23S amplification. Four Aeromonas genes (lip, exu, gcat, flaA/B) were found at high overall frequency in both clinical and environmental isolates although the lip gene was specifically absent from selected species. A fifth gene, aerA, was rarely found in A. caviae, the most abundant species. The ISR profile revealed high heterogeneity among the Aeromonas isolates and no correlation with species identification. In contrast, in all the V. cholerae isolates the four genes investigated (ctxA, tcpA, zot and ace) were amplified and revealed homogeneous ISR and RAPD profiles. Although Aeromonas isolates were the major enteric pathogen recovered, their ISR profiles are not compatible with a unique cause for the diarrhea events, while the clonal relationship clearly implicates V. cholerae in those cases from which it was isolated. These results reinforce the need for a better definition of the role of aeromonads in diarrhea and whether they benefit from co-infection with V. cholerae.

Prevalence and genetic characterization of Cryptosporidium spp. and Cystoisospora belli in HIV-infected patients

Cryptosporidium spp. and Cystoisospora belli are monoxenic protozoa that have been recognized as the causative agents of chronic diarrhea in immunocompromised individuals, especially HIV-infected subjects. The objective of this study was to evaluate the frequency of these intestinal protozoa in HIV-positive patients in the Triângulo Mineiro region of Brazil and to correlate the presence of these infections with clinical, epidemiological and laboratory data of the patients. Oocysts were detected in stool samples of 10 (16.9%) of the 59 patients studied, while Cryptosporidium spp. were present in 10.1% (6/59) and C. belli in 6.7% (4/59). The frequency of these parasites was higher among patients with diarrheic syndrome and CD4+ T lymphocyte counts < 200 cells/mm 3 , demonstrating the opportunistic characteristic of these infections. A significant association was observed between the lack of adherence to antiretroviral therapy and the presence of Cryptosporidium spp. and/or C. belli. Parasitism with Cryptosporidium spp. was more frequent in February and April, the months following the period of high rainfall. The same was not observed for C. belli. Genetic characterization of two isolates led to the identification of Cryptosporidium parvum, one of the main species associated with the zoonotic transmission of cryptosporidiosis.

CHARACTERIZATION OF MOLLUSCICIDAL COMPONENT OF Moringa oleifera LEAF AND Momordica charantia FRUITS AND THEIR MODES OF ACTION IN SNAIL Lymnaea acuminata

SUMMARY The molluscicidal activity of the leaf powder of Moringa oleifera and lyophilized fruit powder of Momordica charantia against the snail Lymnaea acuminata was time and concentration dependent. M. oleifera leaf powder (96 h LC50: 197.59 ppm) was more toxic than M. charantia lyophilized fruit powder (96 h LC50: 318.29 ppm). The ethanolic extracts of M. oleifera leaf powder and Momordica charantia lyophilized fruit powder were more toxic than other organic solvent extracts. The 96 h LC50 of the column purified fraction of M. oleifera leaf powder was 22.52 ppm, while that of M. charantia lyophilized fruit powder was 6.21 ppm. Column, thin layer and high performance liquid chromatography analysis show that the active molluscicidal components in M. oleifera leaf powder and lyophilized fruit of M. charantia are benzylamine (96 h LC50: 2.3 ppm) and momordicine (96 h LC50: 1.2 ppm), respectively. Benzylamine and momordicine significantly inhibited, in vivo and in vitro, the acetylcholinesterase (AChE), acid and alkaline phosphatase (ACP/ALP) activities in the nervous tissues of L. acuminata. Inhibition of AChE, ACP and ALP activity in the nervous tissues of L. acuminata by benzylamine and momordicine may be responsible for the molluscicidal activity of M. oleifera and M. charantia fruits, respectively.

MOLECULAR CHARACTERIZATION OF INFLUENZA B VIRUS OUTBREAK ON A CRUISE SHIP IN BRAZIL 2012

In February 2012, an outbreak of respiratory illness occurred on the cruise ship MSC Armonia in Brazil. A 31-year-old female crew member was hospitalized with respiratory failure and subsequently died. To study the etiology of the respiratory illness, tissue taken at necropsy from the deceased woman and respiratory specimens from thirteen passengers and crew members with respiratory symptoms were analyzed. Influenza real-time RT-PCR assays were performed, and the full-length hemagglutinin (HA) gene of influenza-positive samples was sequenced. Influenza B virus was detected in samples from seven of the individuals, suggesting that it was the cause of this respiratory illness outbreak. The sequence analysis of the HA gene indicated that the virus was closely related to the B/Brisbane/60/2008-like virus, Victoria lineage, a virus contained in the 2011-12 influenza vaccine for the Southern Hemisphere. Since the recommended composition of the influenza vaccine for use during the 2013 season changed, an intensive surveillance of viruses circulating worldwide is crucial. Molecular analysis is an important tool to characterize the pathogen responsible for an outbreak such as this. In addition, laboratory disease surveillance contributes to the control measures for vaccine-preventable influenza.

HIV AND HCV COINFECTION: PREVALENCE, ASSOCIATED FACTORS AND GENOTYPE CHARACTERIZATION IN THE MIDWEST REGION OF BRAZIL

A cross-sectional study on prevalence, associated factors and genotype distribution of HCV infection was conducted among 848 HIV-infected patients recruited at reference centers in the Midwest Region of Brazil. The prevalence rate of HIV-HCV coinfection was 6.9% (95% CI: 5.2 to 8.6). In multivariable analysis, increasing age, use of illicit drugs (injection and non-injection), a history of blood transfusion before 1994, and the absence of a steady partnership were significant independent associated factors for HIV-HCV coinfection. The phylogenetic analysis based on the NS5B region revealed the presence of two major circulating genotypes of HCV: genotypes 1 (58.3%) and 3 (41.7%). The prevalence of HIV-HCV coinfection was lower than those reported in studies conducted with HIV-infected patients in different regions of Brazil, due to the fact that illicit drug use is not a frequent mode of HIV transmission in this region of Brazil. Serologic screening of HIV-patients for HCV before initiating antiretroviral treatment, a comprehensive identification of associated factors, and the implementation of effective harm reduction programs are highly recommended to provide useful information for treatment and to prevent HCV coinfection in these patients.

Algorithm for the Parkinson’s disease behavioural models characterization using a biosensor

Dissertação para obtenção do Grau de Mestre em Engenharia Biomédica