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- Genotyping an ALAD polymorphism with real-time PCR in two populations from the Iberian PeninsulaPublication . Moreira, A.O.; Almeida, A.; Costa, S.; Laffon, B.; García-Léston, J.; Pásaro, E.; Méndez, J.; Teixeira, João PauloLead-induced toxicity varies among individuals partly because of genetic differences in their susceptibility to the metal's effects. One genetic polymorphism associated with lead toxicity is a G-to-C transversion at position 177 in the coding region of delta-aminolevulinic acid dehydratase (ALAD), originating from two codominant alleles (ALAD1 and ALAD2). We examined the distribution of this single nucleotide polymorphism in two populations from the Iberian Peninsula. Genomic DNA was extracted from whole blood, and a real-time PCR assay was designed to determine ALAD polymorphic distribution. The distribution of MspI polymorphism was similar in the two populations studied, and allelic frequencies were comparable to those obtained in other studies of Caucasians. Further studies are needed to assess fully the functional significance of this polymorphism and its influence on the toxicokinetics of lead.
- Influenza Vaccine Effectiveness in Portugal: Season 2011-12. Final ReportPublication . Nunes, Baltazar; Guiomar, Raquel; Machado, Ausenda; Pechirra, Pedro; Gonçalves, Paulo; Conde, Patrícia; Batista, Inês; João, Inês; Falcão, IsabelThe EuroEVA project is the Portuguese component of the multicentre I-MOVE study and aims to obtain estimates of the seasonal and pandemic vaccine effectiveness during and after the influenza season. Since the 2008/2009 influenza season Portugal, along with other European countries, has implemented a common protocol using a case-control study design, where influenza-like illness cases which are laboratory confirmed as influenza (ILI+) are compared to a control group consisting of ILI patients which test negative for influenza (ILI-) (Case-control Test negative design). The results presented in this report relate to the EuroEVA 2011-2012 season and aim to estimate the seasonal influenza vaccine effectiveness for the age group 60+ years and in all age groups, using two approaches: Case-control Test negative design and Screening Method. Materials and Methods Test Negative Design ILI cases were identified among patients that presented ILI symptoms to a participating EuroEVA General Medical Practitioner (GP). On a weekly basis, each GP systematically selected ILI patients (two per week with less than 60 years and all ILI patients with 60 years and more) using the EU ILI case definition. Data on potential confounding factors and effect modifiers was collected using a standardized questionnaire which included information on socio-demographic variables (age, gender, education and co-inhabitants), previous (2010-11) influenza vaccination, chronic conditions and related hospitalizations, current smoking habits, belonging to GP list and number of consultations in the previous year. An ILI patient was considered vaccinated if he/she had received one dose of the 2011/2012 trivalent influenza vaccine at least 14 days prior to onset of symptoms. VE was estimated as one minus the odds ratio of being vaccinated in cases versus controls adjusted for confounders by logistic regression. Potential confounders were investigated and included if: they changed crude OR estimate in at least 10% after adjustment by the Mantel-Haenszel method, were associated both to being a case (in the absence of the exposure factor) and to the seasonal vaccination. Screening Method ILI cases and ILI laboratory confirmed influenza cases were recruited in the context of the National I-MOVE case-control study (EuroEVA). Vaccine coverage in the population was obtained from a sample of 1074 households stratified by region (homogeneous allocation) selected from a dual sample frame: random digit dialling mobile and landline phones (ECOS sample). Relevant information was collected by CATI (Computer Assisted Telephone interview) – one respondent by household (proxy for the rest of the household members). VE was estimated by comparing the proportion of vaccinated cases to the vaccine coverage in the source population, using the Orenstein formula and the Farrington method to adjust for age group and target group for vaccination. Results In Portugal, a later beginning of the 2011/2012 influenza epidemic was observed, starting in week 4/2012 and ending at week 12/2012. In this season both influenza B and A(H3) virus types were circulating, with predominance of the later. Test negative design From the 59 GP’s that accepted to participate in the study, 35 effectively participated in the study by selecting patients (which corresponds to a 59% participation rate). After excluding 79 ILI cases (for not adhering to the inclusion criteria) the final sample consisted on 273 ILI patients. Of the 134 cases which tested positive for influenza, 98.5% were positive for influenza A(H3) and the remaining for type B virus. The control group, consisting of 139 ILI patients who tested negative for influenza, was statistically different (p<0.05) from the ILI+ group in the following variables: • Clinical signs and symptoms: cough (higher in cases than in controls: 95.5% vs. 89.2%, respectively) and sore throat (more frequent in controls, 89.2%, than in cases ,75.4%); • Age: controls were older than cases (median age in controls was 52 yrs vs. 39 yrs in cases); • Any chronic disease: the prevalence of at least one chronic condition relevant for influenza vaccination was higher in controls (41.7% vs 29.1%); • Seasonal vaccine in 2010-11: controls were more often vaccinated against influenza in the last season than cases (30.2% vs. 14.4%); • Co-habitants: the median number of co-habitants was higher in cases (3 vs. 2). Considering all population, vaccine coverage (VC) in controls was 27.5% statistically higher than in cases (VC=13.4%). Similar results were obtained for the sub-group target for vaccination by the National Health Authorities (VC cases =24.6% and VC controls=46.1%, p=0.010). These results indicate that crude VE estimates was 59.2% (95% CI: 21.1%-79.4%) in the general population and 61.8% (95% CI: 15.5% ;83.1%) in the target group for vaccination. After adjustment for co-inhabitants and month of onset of illness, VE adjusted estimates were 48.8% (95% CI: 0.0% ; 73.8%) and 51.6% (95% CI:-6.2%-77.9%) for the general population and for the target group, respectively. Screening Method The ECOS telephone survey was conducted during April 2012, and information was obtained from a total of 2395 individuals. According to the results, individuals were vaccinated from October 2011 through January 2012, estimating a 16.4% (95% CI: 13.6-19.6) vaccine coverage (VC) in the population. In the 60+ yrs age group, the VC was 37.3% (95% CI: 30.6-44.4) and for the individuals with chronic condition was 28.0% (95% CI: 23.0-33.7). The crude VE estimated with the Screening method for ILI+ as the outcome was 27.0% (95% CI: -19.9- 55.6) and -32.4% (95% CI: -77.7- 1.3) for ILI. Adjusted VE estimates varied from -90.4% (95% CI: -277.1- 3.9) (60+ yrs) to 6.1% (95% CI: -56.0- 43.4) (0-60 yrs) considering ILI as the outcome and from -58.6% (95% CI: -195.3- 14.8) to 56.9 (95% CI: -35.2- 86.3) for ILI positive outcomes (none were statistically significant). Conclusions Given the 3 years experience in conducting this study, logistical and implementation aspects were straightforward. The 2011-2012 season adjusted VE estimates were similar for the general population (48.8%) and for the target group (51.6%), although not statistical significant. When compared to the previous season, VE point estimate for the general population was lower (VE=58% in 2010-11), although the CI overlap. The population studied this year was older than in the last season. The time between onset of symptoms and swabbing, was also different with marginally, non significant, differences between cases and controls.
- Interlaboratory validation of an environmental monitoring method for trace analysis of endocrine disrupting compoundsPublication . Mansilha, Catarina; Rocha, Sonia; Gameiro, Paula; Carina Pinho, Carina; Ferreira, Isabel M. P. L. V. O.; Silva, Poliana; Domingues, ValentinaEnvironmental pollution continues to be an emerging study field, as there are thousands of anthropogenic compounds mixed in the environment whose possible mechanisms of toxicity and physiological outcomes are of great concern. Developing methods to access and prioritize the screening of these compounds at trace levels in order to support regulatory efforts is, therefore, very important. A methodology based on solid phase extraction followed by derivatization and gas chromatography-mass spectrometry analysis was developed for the assessment of four endocrine disrupting compounds (EDCs) in water matrices: bisphenol A, estrone, 17b-estradiol and 17a-ethinylestradiol. The study was performed, simultaneously, by two different laboratories in order to evaluate the robustness of the method and to increase the quality control over its application in routine analysis. Validation was done according to the International Conference on Harmonisation recommendations and other international guidelines with specifications for the GC-MS methodology. Matrix-induced chromatographic response enhancement was avoided by using matrix-standard calibration solutions and heteroscedasticity has been overtaken by a weighted least squares linear regression model application. Consistent evaluation of key analytical parameters such as extraction efficiency, sensitivity, specificity, linearity, limits of detection and quantification, precision, accuracy and robustness was done in accordance with standards established for acceptance. Finally, the application of the optimized method in the assessment of the selected analytes in environmental samples suggested that it is an expedite methodology for routine analysis of EDC residues in water matrices.
- South European Kales: a Cross Country, Cross Cultural ResearchPublication . D’Antuono, L.F.; Manco, M.A.; Costa, H.S.; Sanches-Silva, A.; Albuquerque, T.G.; Koçaoglu, B.; Hayran, O.BaSeFood (Sustainable exploitation of bioactive components from the Black Sea Area traditional foods) is a EU funded program in which local crops are being considered and investigated, comparing their uses, composition and related traditional foods, in a cross-country perspective. Within the BaSeFood partner countries, kales are present along the Black sea coast of Turkey, in the northern Apennines of Italy and in the north of Portugal. The available literature on the kale populations of these areas indicates glucobrassicin as a characteristic component of their glucosinolate profile. A research was planned to comparatively investigate several aspects of kale distribution, present and potential uses, traditional foods and bioactive composition. In the three countries considered, kales are extensively used and locally valued, but are lesser known as major vegetables. Although differences in the present knowledge of kale traditional uses do exist, common patterns, due to the peculiar ecological character¬istic of kales, in combination with the social conditions of some growing areas, are clearly identifiable. Local recipes are being collected, and examined also in the perspective of scaling-up production. Winter soups are the main form of utilisation, corresponding to the need of combining vegetables with carbohydrate supplying ingredients. Seed samples were collected from representative populations and are being used to plant a comparative field trial, aimed at investigating glucosinolate and phenolic profiles in a common environment. Finally, some targeted experiments about the retention of bioactive components during the preparation of a common dish in the three countries (kale soup) are being planned.
- Selective pressure acting on influenza virus neuraminidase protein and relation with development of resistance to antiviral drugsPublication . Correia, V.; Santos, L.A.; Giria, M.; Rebelo-de-Andrade, H.Neuraminidase (NA) protein of influenza viruses has the particularity of being under antibody and antiviral drug selective pressure, as it is one of the main surface antigens and the target of neuraminidase inhibitors(NAIs). The aim of this study is to investigate the selective pressure(SP) acting on the NA of seasonal and pandemic influenza viruses. It comprises two objectives: (a)to evaluate the contribution of positive SP for the emergence of NAIs resistant viruses; and (b)to determine the impact of NAIs introduction into clinic and its wide use during pandemic on the SP acting on NA. For the 1st objective it will be analysed the SP acting on the sites associated with NAIs resistance or reduction in susceptibility. The 2nd objective implies a differential evolutionary pressure analysis according to time, with 3 sub-datasets of NA sequences being considered: (1)before worldwide introduction of NAIs into clinic(1999); (2)before wide use of oseltamivir during A(H1N1)2009 pandemic(2009); and (3)from 2009 to date. A large dataset of full-length NA coding sequences will be used for each (sub)type/variant, comprising sequences obtained at national level(since 2000/2001) and sequences available at GISAID and NCBI. A(H1N1)seasonal dataset was already created, including a total of 1523 sequences, from which 94 belong to 1st sub-dataset, 1094 to 2nd and 335 to 3rd. All SP analysis will be performed using the expertise acquired with this workshop. This study may contribute for understanding the role of antiviral drug selective pressure in NAIs resistance, patterns of emergency of resistant viruses and NA evolutio
- Risk factors for human papillomavirus infection among women in Portugal: The CLEOPATRE Portugal StudyPublication . Pista, Angela; Oliveira, Carlos; Cunha, Maria João; Paixão, Teresa; Real, Odete; CLEOPATRE Portugal Study GroupObjective: To investigate demographic, socioeconomic, lifestyle, and medical factors that might predispose women to cervical human papillomavirus (HPV) infection. Method: A cross-sectional population-based study was performed. Women aged 18–64 years who attended selected obstetrics and gynecology or sexually transmitted disease (STD) clinics in mainland Portugal between February 2008 and March 2009 were recruited, according to an age-stratified sampling strategy. Liquid-based cytology samples were analyzed centrally for HPV genotype and for cytologic features. Univariate and multivariate logistic regression analyses identified risk factors for HPV infection. Results: Among the 2326 women evaluated, the crude prevalence of HPV infection was 19.4%. Lifetime number of sexual partners was a strong predictor of HPV infection (odds ratio 5.44 for 5–10 partners versus 1 partner; Pb0.001). Other risk factors were young age (particularly among women aged 20–24 years; Pb0.001); country of birth other than mainland Portugal (P=0.002); education up to secondary school level (P=0.010); smoking history (≤10 years; P=0.004); and any STD in the past 12 months (P=0.052). Conclusion: Data from the present study may aid identification of women at increased risk of HPV infection and target prevention strategies.
- Avian malaria infections in western European mosquitoesPublication . Ventim, R.; Ramos, J.A.; Osório, H.; Lopes, R.J.; Pérez-Tris, J.; Mendes, L.Avian malaria infections in western European mosquitoes. Ventim R, Ramos JA, Osório H, Lopes RJ, Pérez-Tris J, Mendes L. SourceInstitute of Marine Research (IMAR/CMA), Department of Life Sciences, University of Coimbra, Apartado 3046, 3001-401 Coimbra, Portugal. ritaventim@gmail.com Abstract In the complex life cycle of avian malaria parasites (Plasmodium sp.), we still have a poor understanding on the vector-parasite relationships. This study described the community of potential avian malaria vectors in four Portuguese reedbeds. We tested if their geographical distribution differed, and investigated on their Plasmodium infections. The mosquitoes' feeding preferences were evaluated using CO(2), mice, and birds as baits. The most abundant species were Culex pipiens, Culex theileri, and Ochlerotatus caspius (and, in one site, Coquillettidia richiardii). Plasmodium lineages SGS1 and SYAT05 were found in unengorged Cx. pipiens and Cx. theileri, respectively, suggesting that these mosquitoes were competent vectors of those lineages. The species' abundance was significantly different among sites, which may help to explain the observed differences in the prevalence of SGS1. At the study sites, SGS1 was detected in the most abundant mosquito species and reached a high prevalence in the most abundant passerine species. Probably, this parasite needs abundant hosts in all phases of its cycle to keep a good reservoir of infection in all its stages. Cq. richiardii showed an opportunistic feeding behavior, while Cx. pipiens appeared to be more mammophilic than previously described, perhaps because the used avian bait was not its preferential target. In one of the study sites, mosquitoes seem to be attracted to the Spotless Starling Sturnus unicolor, an abundant bird species that may be an important local reservoir of avian malaria infections. To our knowledge, this is the first report of detection of avian Plasmodium DNA from European mosquitoes.
- Genotoxic effects of occupational exposure to lead and influence of polymorphisms in genes involved in lead toxicokinetics and in DNA repairPublication . García-Lestón, Julia; Roma-Torres, Joana; Vilares, Maria; Pinto, Rui; Prista, João; Teixeira, João Paulo; Mayan, Olga; Conde, Joao; Pingarilho, Marte; Gaspar, Jorge Francisco; Pásaro, Eduardo; Méndez, Josefina; Laffon, BlancaLead is still widely used in many industrial processes and is very persistent in the environment. Although toxic effects caused by occupational exposure to lead have been extensively studied, there are still conflicting results regarding its genotoxicity. In a previous pilot study we observed some genotoxic effects in a population of lead exposed workers. Thus, we extended our study analysing a larger population, increasing the number of genotoxicity endpoints, and including a set of 20 genetic polymorphisms related to lead toxicokinetics and DNA repair as susceptibility biomarkers. Our population comprised 148 workers from two Portuguese factories and 107 controls. The parameters analysed were: blood lead levels (BLL) and δ-aminolevulinic acid dehydratase (ALAD) activity as exposure biomarkers, and T-cell receptor (TCR) mutation assay, micronucleus (MN) test, comet assay and OGG1-modified comet assay as genotoxicity biomarkers. Lead exposed workers showed markedly higher BLL and lower ALAD activity than the controls, and significant increases of TCR mutation frequency (TCR-Mf), MN rate and DNA damage. Oxidative damage did not experience any significant alteration in the exposed population. Besides, significant influence was observed for VDR rs1544410 polymorphism on BLL; APE1 rs1130409 and LIG4 rs1805388 polymorphisms on TCR-Mf; MUTYH rs3219489, XRCC4 rs28360135 and LIG4 rs1805388 polymorphisms on comet assay parameter; and OGG1 rs1052133 and XRCC4 rs28360135 polymorphisms on oxidative damage. Our results showed genotoxic effects related to occupational lead exposure to levels under the Portuguese regulation limit of 70 μg/dl. Moreover, a significant influence of polymorphisms in genes involved in DNA repair on genotoxicity biomarkers was observed.
- Three-way translocation (X;20;16)(p11;q13;q23) in essential thrombocythemia implicates NFATC2 in dysregulation of CSF2 expression and megakaryocyte proliferationPublication . Vieira, Luís; Vaz, Andreia; Matos, Paulo; Ambrósio, Ana Paula; Nogueira, Manel; Marques, Bárbara; Pereira, AM; Jordan, Peter; da Silva, Maria GomesEssential thrombocythemia (ET) is a myeloproliferative neoplasm essentially characterized by excessive production of platelets. Molecular pathogenesis of ET is linked in approximately half of the patients to intracellular cytokine signaling dysregulation as a result of thrombopoietin receptor or Janus kinase 2 (JAK2) mutations. However, genetic defects underlying cytokine transcription have not been associated with ET. Using molecular cytogenetics and whole-genome array analyses, we uncovered a submicroscopic deletion at 20q13.2 in a JAK2V617F-positive ET patient with an acquired complex chromosome translocation. The deletion encompassed the nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 (NFATC2) gene that encodes a transcription factor involved in the regulation of hematopoietic cytokines. RNA interference-mediated suppression of NFATC2 mRNA or pharmacological inhibition of NFATC2 protein with 11R-VIVIT in cultured JAK2V617F-positive SET-2 megakaryocytes increased colony stimulating factor 2 (granulocyte-macrophage) (CSF2) mRNA and promoted cell proliferation. Moreover, impairment of NFATC2-calcineurin interaction with 11R-VIVIT further reduced the transcription of the NFATC2 gene. Antibody-mediated neutralization of CSF2 cytokine in inhibitor-treated cells prevented 11R-VIVIT-induced cell proliferation, indicating that impairment of NFATC2-calcineurin interaction promotes megakaryocyte proliferation through up-regulation of CSF2 transcription. Our results suggest a model in which haplo-insufficiency of NFATC2 cooperates with activation of the JAK-STAT signaling pathway in the pathogenesis of JAK2V617F-positive ET with del(20q). These results further indicate that pathogenesis of ET may be linked to genetic defects of other transcription factor genes involved in the regulation of cytokine expression.
- Vegetables production in NW Portugal, agriculture practices and groundwater qualityPublication . Aguiar, Ana; Melo, Armindo; Mansilha, Catarina; Ferreira, IsabelIn NW of Portugal, the region between Esposende and Vila do Conde was established into a unique system called masseiras; masseiras are small fields that were created at the beginning of XX century by removing sand from dunes. Since the 80’s sand between closed masseiras has been removed giving way for larger fields, the “enlarged masseira with greenhouses”. This region is part of vulnerable zone nº1 (VZ1). Nowadays, the masseiras have almost disappeared; they had been replaced by “enlarged masseiras with plastic greenhouses” exploited by a new generation of growers. As water quality is an indicator of agriculture practices, 8 wells were selected and water samples were collected on four dates from September 2009 to May 2010. Laboratorial analysis was performed on nitrates and 28 pesticides. The results confirm that water in VZ1 is contaminated with nitrates (as it was expected) and also with pesticides. The water was contaminated in both dug wells and drilled wells all year round. Masseiras’ wells have more nitrates than greenhouses’ wells, probably due to poor agricultural practices used by growers who still employ high quantities of fertilizers and water. Pesticides detected in water samples include some that have been sprayed in the field and others that have not. Some forbidden substances were found. Our results confirm that, in VZ1, there is a diffusion contamination, which origin is unknown. In this communication, we put in picture the history of masseiras fields: how masseiras fields have been created and how they have disappeared; what trends exist in pest management strategies and the use of fertilizers, pesticides and water resources. These results reinforce the need to change the agricultural system in this region of traditional vegetable production.