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- Normalization strategies for real-time expression data in Chlamydia trachomatisPublication . Borges, V.; Ferreira, R.; Nunes, A.; Nogueira, P.; Borrego, M.J.; Gomes, João PauloChlamydia trachomatis is a widespread obligate intracellular pathogen genetically non-tractable for which transcriptomics is a fundamental tool to better understand its biology. However, the suitability of endogenous controls for normalization of transcriptomic data in this bacterium still needs validation. We aimed to assess the stability of 10 genes for their potential use as endogenous controls in real-time quantitative PCR assays at both normal and stress (D-cycloserine treatment) growth conditions throughout the developmental cycle of three C. trachomatis strains with different tissue tropism. Normalization was performed by real-time absolute quantification of the bacterial genomes. We also tested the applicability of two widely used softwares (geNorm and Normfinder) to our data. For all strains, we found that 16SrRNA was the most stably expressed gene throughout the chlamydial normal developmental cycle, which indicates its potential use as endogenous control in relative expression assays. However, it was highly unstable under D-cycloserine treatment (where oppA_2 was top-ranked), suggesting prudence when using ribosomal genes in expression experiments involving stress conditions. The geNorm and Normfinder algorithms revealed contrasting results and seem inappropriate for the selected pool of genes. Considering the multiplicity of experimental conditions, there should be an in loco validation of endogenous controls, where 16SrRNA appears to be in the front line. Alternatively, normalization of expression data against genomic DNA, which is less influenced by experimental constraints that are especially relevant for intracellular organisms, likely constitutes a good option. Moreover, the number of genomes also seems to be less subject to variation than expression of endogenous controls when working under stress conditions. The present study constitutes the first evaluation of putative endogenous controls for real-time expression assays in C. trachomatis.
- Infecções e Toxinfecções alimentaresPublication . Correia, Cristina BeloInfecções e toxinfecções alimentares: mecanismos, factores determinantes,algumas manifestações adversas,transmissão de patogénicos e toxinas ao alimento, categorização de surtos, prevalência, impacto, relatórios da EFSA e dados do INSA (2004-2008).
- Seasonal and pandemic patterns of Influenza in PortugalPublication . Gonçalves, Paulo; Nunes, Baltazar; Paixão, Eleonora; Cordeiro, Rita; Pechirra, Pedro; Conde, Patrícia; Arraiolos, Ana; Furtado, Cristina; Guiomar, RaquelThe National Influenza Reference Laboratory has been collecting data on influenza activity in Portugal since 1957 through the National Influenza Surveillance Programme, including information on clinical and virological characteristics of the disease, allowing the estimation of weekly incidence rates for influenza-like illness (ILI). This information has not only been used by the National Health Authorities for the management of the disease, in its several aspects, but has also been contributing to the study of influenza by the World Health Organisation. Particularly during the past decade, the world had been preparing for a long awaited influenza pandemic, which characteristics could not been foreseen but was feared to have potentially devastating consequences. In April 2009 a new strain of influenza A(H1N1) virus of swine origin disseminated throughout the world, resulting in the first pandemic of the XXI century. To face the increasing number of diagnosis being requested, a Network of Associated Laboratories dedicated exclusively to the diagnosis of the new influenza A(H1N1) pandemic virus was activated in our country. The data on influenza collected over the past two influenza seasons, through the National Influenza Surveillance Programme and the Network of Associated Laboratories, is presented and compared. 2. Materials and methods Over 3000 ILI cases were notified to the National Influenza Reference Laboratory and to the Department of Epidemiology of the National Institute of Health, in the context of the National Influenza Surveillance Programme, from week 38/2008 through week 20/2010. The distribution by age group, gender and region, and the signs and/or symptoms present were analysed. Nasopharyngeal swabs were collected for virological characterisation of influenza viruses circulating during this period. The intensity and duration of the epidemic periods were described based on the weekly incidence rates for ILI and as a function of a defined baseline. From week 17/2009 through week 20/2010, over 53.800 ILI cases and respective biological specimens were also notified and analysed by the Network of Associated Laboratories. 3. Results Seasonal AH1, AH3 and B influenza viruses circulating in Portugal during the 2008/2009 season were replaced by the new influenza A(H1N1) pandemic virus since the beginning of the pandemic in the country on week 17/2009. When considering the data obtained though the National Influenza Surveillance Programme, lower ILI incidence rates, lower percentage of confirmed influenza cases (particularly in the population over 15 years) and fewer symptoms presented at the time of notification were observed during the 2009/2010 pandemic. The large volume of cases analysed through the Network of Associated Laboratories is currently under evaluation and will be presented at a later stage. 4. Conclusions Facing the circulation of a new virus and the threat that this could impose to the population and to the health care system, the total number of ILI cases reported and analysed in our country during the 2009/2010 winter boosted to numbers not seen in previous influenza seasons. It is a fact that the 2009/2010 pandemic has had a significant impact in Portugal in many areas, such as the adoption of health-care regulations, availability of health-care facilities, vaccination strategies, and public action/awareness. However, in terms of pattern of disease, the data collected through the National Influenza Surveillance Programme may reveal a different reality. The data analysed suggests that the 2009/2010 pandemic was milder than the previous influenza seasons. Preliminary analysis of the data collected through the Network of Associated Laboratories appears to corroborate these findings.
- Sequence analysis of resistance markers in pandemic influenza A (H1N1) 2009 viruses isolated in PortugalPublication . Pechirra, Pedro; Arraiolos, Ana; Conde, Patrícia; Gonçalves, Paulo; Cordeiro, Rita; Guiomar, RaquelBackround: In April 2009, a new influenza A (H1N1) virus (AH1N1pdm) was detected circulating in humans. Its rapid widespread transmission led to the declaration by the WHO of an influenza pandemic. This virus had origin in a reassortment between a North American swine lineage (already a triple reassortant, circulating in pigs since the late 1990’s) and a Eurasian swine lineage (which contributed to the AH1N1pdm with the neuraminidase (NA) and matrix (MP) gene segments). The NA inhibition assay is the base test to determine the susceptibility to NA inhibitors (NAI), however it requires virus isolation, and the consequent detection of a resistant viral strain requires confirmation by sequencing of the NA gene segment present in the original biological product. Material and Methods: In Portugal, during the 2009 influenza pandemic, about 16500 clinical samples were tested by the National Influenza Reference Laboratory for the presence of influenza AH1N1pdm virus. From near 8000 AH1N1pdm-positive samples, 153 were isolated in MDCK cells and of these 53 isolates were taken for sequence analysis of the NA and MP gene segments. The NA inhibition assays were performed by the WHO collaborating centre (London). Results: As all AH1N1pdm viruses, sequenced Portuguese isolates carried the S31N change in the M2 protein, which confers resistance to the adamantanes. The most common mutation in N1 neuraminidase associated with resistance to oseltamivir is the H275Y change. From our 53 sequenced isolates, only one carried this mutation. This was from an 8-years-old child with chronic diseases such as a DiGeorge syndrome diagnosed in 2004 and Evans Syndrome with auto-immune haemolytic anaemia diagnosed in 2006. Initially, this child was treated with an underdosing of oseltamivir, however, as the influenza-like syndrome persisted with a suspicion of viral pneumonia, led to the antiviral treatment dose review. The NA inhibition assay confirmed that this virus was resistant to oseltamivir. The NA inhibition assays performed in our viral isolates and accordingly to our sequence data, revealed that the majority of Portuguese AH1N1pdm viruses were sensitive to NAI. All neuraminidases presented the N248D substitution, except for A/Lisboa/35/2009, a viral strain from an early clade (at least, previous to clade 4) as it lacks also V106I amino acid change. Other amino acid substitutions, such as E119V, I223V, Q136K, known to reduce the susceptibility to NAI, were not found in our isolates. Conclusions: The use of conventional sequence analysis for monitoring the molecular markers of antiviral resistance in influenza A virus provides a valuable tool for an early detection of antiviral resistant strains.
- Qualidade do Ar InteriorPublication . Proença, Maria do Carmo; Cano, ManuelaUma vez que 80-90% da nossa vida é passada no interior, nas últimas décadas tem-se verificado uma crescente preocupação da comunidade científica com a Qualidade do Ar Interior (QAI), devido aos efeitos que a mesma pode exercer sobre a saúde dos seres humanos. Com o intuito de melhorar a eficiência energética, os edifícios actuais são mais estanques favorecendo a acumulação de contaminantes gerados no interior quando a ventilação é insuficiente. Em Portugal, na sequência da Directiva 2002/91/CE foi revista a legislação e, em 2006, foi publicado o RSECE - eficiência energética de edifícios com sistemas de AVAC e que, entre outros, estabelece requisitos para a Qualidade do Ar Interior. Este artigo refere os principais contaminantes do ar interior, suas principais fontes e efeitos na saúde. Descreve a metodologia de avaliação da QAI bem como os valores de referência adoptados. São descritos alguns casos de estudo, suas principais conclusões e recomendações.
- BaSeFood: Sustainable exploitation of bioactive components from the Black Sea Area traditional foodsPublication . D'Antuono, F.; Sanches-Silva, A.; Costa, H.S.The Sustainable exploitation of bioactive components from the Black Sea Area traditional foods (BaSeFood) is a 3-year collaborative research programme, funded by the 7th Framework Programme, launched on the 1st of April 2009. The project, which is coordinated by Dr Filippo D’Antuono (University of Bologna), consists of a research consortium of 13 partners, namely Italy (two), the United Kingdom, Greece, Portugal, Serbia and six Black Sea area countries: Russian Federation, Ukraine (two), Romania, Bulgaria, Turkey and Georgia. BaSeFood will contribute scientifically by studying the bioactive compounds within traditional foods of the Black Sea area using rigorous analytical and biological assays. The vast array of characteristics of traditional foods will be considered, as well as any associated consumer-perceived benefits, related to health claims, so that they can be properly understood by the consumer and exploited by food processors to produce more healthy traditional foods.
- Escherichia coli-cloned CFTR loci relevant for human artificial chromosome therapyPublication . Rocchi, Lucia; Braz, Carla; Cattani, Sonja; Ramalho, Anabela; Christan, Sulith; Edlinger, Marlene; Ascenzioni, Fiorentina; Laner, Andreas; Kraner, Simone; Amaral, Margarida; Schindelhauer, DirkClassical gene therapy for cystic fibrosis has had limited success because of immune response against viral vectors and short-term expression of cDNA-based transgenes. These limitations could be overcome by delivering the complete genomic CFTR gene on nonintegrating human artificial chromosomes (HACs). Here, we report reconstruction of the genomic CFTR locus and analyze incorporation into HACs of three P1 phage-based and F factor bacteria-based artificial chromosomes (PACs/BACs) of various sizes: (1) 5A, a large, nonselectable BAC containing the entire wild-type CFTR locus extending into both adjacent genes (296.8-kb insert, from kb -58.4 to +51.4) containing all regulators; (2) CGT21, a small, selectable, telomerized PAC (134.7 kb, from kb -60.7 to + 2) containing a synthetic last exon joining exon 10, EGFP, exon 24, and the 3' untranslated region; and (3) CF225, a midsized, nonselectable PAC (225.3 kb, from kb -60.7 to +9.8) ligated from two PACs with optimized codons and a silent XmaI restriction variant to discriminate transgene from endogenous expression. Cotransfection with telomerized, blasticidin-S-selectable, centromere-proficient α-satellite constructs into HT1080 cells revealed a workable HAC formation rate of 1 per ∼25 lines when using CGT21 or 5A. CF225 was not incorporated into a de novo HAC in 122 lines analyzed, but integrants were expressed. Stability analyses suggest the feasibility of prefabricating a large, tagged CFTR transgene that stably replicates in the proximity of a functional centromere. Although definite conclusions about HAC-proficient construct configurations cannot be drawn at this stage, important transfer resources were generated and characterized, demonstrating the promise of de novo HACs as potentially ideal gene therapy vector systems.
- Metabolite composition of chestnut (Castanea sativa Mill.) upon cooking: Proximate analysis, fibre, organic acids and phenolicsPublication . Gonçalves, Berta; Borges, Olga; Costa, H.S.; Bennett, Richard; Santos, Mariana; Silva, Ana PaulaThe aim of this research was to study the processing effects (roasting and boiling) on primary and secondary metabolite composition of fruits from the following chestnut (Castanea sativa Mill.) cultivars (cvs.) of three Protected Designation of Origin (PDO) areas in the Trás-os-Montes e Alto Douro region (Portugal): PDO Terra Fria (cvs. Aveleira, Boaventura, Côta, Lamela and Trigueira), PDO Padrela (cvs. Judia, Lada, Longal and Negra) and PDO Soutos da Lapa (cvs. Longal and Martaínha). The cooking processes significantly (p < 0.0001) affected primary and secondary metabolite composition of the chestnuts. Roasted chestnuts had higher protein contents, insoluble and total dietary fibre and lower fat contents whilst boiled chestnuts had lower protein, but higher fat contents. Cooking increased citric acid contents, especially in roasted chestnuts. On the other hand, raw chestnuts had higher malic acid contents than cooked chestnuts. Moreover, roasted chestnuts had significantly higher gallic acid and total phenolics contents, and boiled chestnuts had higher gallic and ellagic acids contents, when compared to raw chestnuts. The present data confirms that cooked chestnuts are a good source of organic acids and phenolics and have low fat contents, properties that are associated with positive health benefits.
- Antigenic and genetic analysis of pandemic influenza A(H1N1) 2009 viruses from PortugalPublication . Pechirra, Pedro; Arraiolos, Ana; Conde, Patrícia; Gonçalves, Paulo; Cordeiro, Rita; Guiomar, RaquelBackround: The influenza AH1N1 2009 pandemic virus (AH1N1pdm) was first detected in Portugal in May 4th 2009. This virus had origin in a reassortment between a North American swine lineage (already a triple reassortant, circulating in pigs since the late 1990’s) and a Eurasian swine lineage. As the HA (North American swine lineage) continues to circulate in the human population, its antigenic sites will continue to be targeted by antibody-mediated selection pressure. Therefore it is important from a public health perspective, continue to characterise the HA and to monitoring the antigenic and genetic properties of the AH1N1pdm viruses in order to detect any changes and thus any necessity for selecting further vaccine candidates or changes in antiviral recommendations. In this study, is presented a genetic and antigenic characterisation of influenza AH1N1pdm viruses, isolated in Portugal over the 2009 influenza pandemic. Material and Methods: In Portugal, during the 2009 influenza pandemic, about 16500 clinical samples were tested by the National Influenza Reference Laboratory for the presence of influenza AH1N1pdm virus. From near 8000 AH1N1pdm-positive samples, 147 were isolated in MDCK-SIAT1 cell cultures and characterised antigenically by hemagglutination-inhibition assays (HI). Of these, 56 isolates were taken for sequence analysis of the HA1 gene segment. Results: Antigenically, the AH1N1pdm viruses are homogeneous, being similar to A/California/7/2009 and the later pandemic H1N1 viruses A/Bayern/69/2009 and A/Lviv/N6/2009. However, 12 of the isolated viruses show 4-fold or greater reductions in the HI titre against most of the HI sera panel. They react better with sera raised against the A/Bayern/69/2009 and A/Lviv/N6/2009. Three of these isolated viruses presented amino acid substitutions at hemagglutinin antigenic sites (G155E in epitope B; R205K in epitope D and E258D in epitope E of the HA1 subunit) and in one strain was observed the amino acid substitution V199I in the vicinity of epitope D. Changes in positions 153-157 of the HA have been highly associated with reduced HI titers with ferret antisera to the A/California/7/2009 vaccine virus. These changes usually emerge after virus propagation in cell cultures. Sequenced hemagglutinins of portuguese isolates show that these viruses, with two exceptions, belong to the clade 7, already described in the literature. As known, viruses from this clade have a S203T mutation. One of our strains, A/Lisboa/31/2009, belongs to clade 6, as presents the Q293H amino acid change. This viral strain was isolated from a patient that arrived from the USA (Boston, New York) in June 2009. Another viral strain, A/Lisboa/35/2009, belongs to an earlier clade (at least, previous to clade 4) because it don’t presents the S203T neither the Q293H in its hemagglutinin and it lacks also the V106I and N248D amino acid changes in its neuraminidase. Additionally, mutations P83S in HA present in all the portuguese isolated viruses and I321V in the majority of them have been observed in all the non-clade 1 isolates. Conclusions: The great majority of influenza AH1N1pdm viruses isolated in Portugal were similar to the vaccine strain A/California/7/2009. They were representative of the clade 7, except two strains with foreign travel history. Most of the observed amino acid changes in the HA were located at antigenic sites or in their vicinity.
- Using clinical criteria on seasonal and pandemic influenza: what to look for?Publication . Gonçalves, Paulo; Paixão, Eleonora; Machado, Ausenda; Cordeiro, Rita; Nunes, Baltazar; Pechirra, Pedro; Guiomar, RaquelThe reliability of clinical criteria on the diagnosis of influenza has been a question which has generated much debate in the scientific community. In Portugal, Influenza-like illness (ILI) cases have been notified by two sentinel networks, one of general practitioners (“Médicos-Sentinela”) and another of emergency rooms of hospitals and health centres, to the National Influenza Reference Laboratory in the context of the National Influenza Surveillance Programme, using clinical criteria adapted from the International Classification of Health Problems in Primary Care. With the emergence of a novel influenza A(H1N1) virus circulating in humans, the clinical definition of an influenza case was reformulated to accommodate the clinical features observed at that time. But how clinically different was the new variant A(H1N1) infection when compared to the seasonal influenza? In this study we propose to evaluate the signs and symptoms present in influenza cases diagnosed in Portugal during a seasonal influenza winter with those reported during the recent A(H1N1) pandemic. Also, we intend to evaluate the clinical criteria used for the notification of influenza cases during these two periods. 2. Materials and methods ILI cases were diagnosed for influenza at the National Influenza Reference Laboratory in the context of the National Influenza Surveillance Programme during the 2008/2009 influenza winter season and 2009/2010 pandemic period, from week 38/2008 through week 20/2010. Clinical information on those cases included the presence of signs and symptoms related with ILI as defined by the International Classification on Health Problems in Primary Care and on the case definition introduced by the European Commission Decision 202/253/EC (signs/symptoms analysed: sudden onset of symptoms, presence of fever, weakness, headache, myalgia, cough, sore throat, respiratory difficulty, chills and contact with an influenza patient). The odds ratio (OR) of being positive for influenza for each sign/symptom was calculated, independently and using multivariable logistic regression with all the signs/symptoms. Results were compared with the clinical definition of influenza used during both periods. 3. Results During the 2008/2009 winter, when seasonal influenza A(H3) viruses were dominant, all signs/symptoms analysed revealed an OR associated with a risk of being positive for influenza. Those with a higher risk were cough (OR 9.5, CI95% 4.7-19.3), fever (OR 4.5, CI95% 2.8-7.1), chills (OR 2.3, CI95% 1.5-3.5), myalgia (OR 1.7, CI95% 1.1-2-7) and contact with another ILI patient (OR 1.5, CI95% 1.1-2.0). Adjusting for all the signs/symptoms, the multivariable logistic regression reveals cough (OR 10.7, CI95% 5.2-22.0), fever (OR 5.1, CI95% 3.2-8.2) and contact with another patient (OR 1.4, CI95% 1.0-1.9) to be statistically significant. For the pandemic season 2009/2010, the signs/symptoms associated with a higher risk of being positive for influenza were fever (OR 4.5, CI95% 2.5-8.3), cough (OR 3.2, CI95% 2.1-4.8) and contact with another patient (OR 2.0, CI95% 1.4-2.8). Multivariable logistic regression also indicates these signs/symptoms to be statistically significant, after adjustment. 4. Conclusions Although initial evidences that the new influenza pandemic variant A(H1N1) could cause a clinically different infection, and fears that the disease would present a more severe profile than seasonal influenza, data collected in our country through the National Influenza surveillance Programme indicates that both situations were clinically similar in their presentation. This fact has been supported by studies from other countries.