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- Presence of Cryptosporidium spp. and Giardia duodenalis in drinking water samples in the north of PortugalPublication . Almeida, André; Moreira, Maria João; Soares, Sónia; Delgado, Maria de Lurdes; Figueiredo, João; Silva, Elisabete; Castro, António; Costa, José Manuel Correia daCryptosporidium and Giardia are 2 protozoan parasites responsible for waterborne diseases outbreaks worldwide. In order to assess the prevalence of these protozoans in drinking water samples in the northern part of Portugal and the risk of human infection, we have established a long term program aiming at pinpointing the sources of surface water, drinking water, and environmental contamination, working with the water-supply industry. Total 43 sources of drinking water samples were selected, and a total of 167 samples were analyzed using the Method 1623. Sensitivity assays regarding the genetic characterization by PCR and sequencing of the genes, 18S SSU rRNA, for Cryptosporidium spp. and b‚-giardin for G. duodenalis were set in the laboratory. According to the defined criteria, molecular analysis was performed over 4 samples. Environmental stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples, 8.4% (14 out of 167) with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium and 7.2% (12 out of 167) for both species. The mean concentrations were 0.1-12.7 oocysts of Cryptosporidium spp. per 10 L and 0.1-108.3 cysts of Giardia duodenalis per 10 L. Our results suggest that the efficiency in drinking water plants must be ameliorated in their efficiency in reducing the levels of contamination. We suggest the implementation of systematic monitoring programs for both protozoa. To authors’ knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in drinking water samples in the northern part of Portugal.
- Dossier ECOS - Em Casa Observamos SaúdePublication . Departamento de EpidemiologiaO Departamento de Epidemiologia [à data Observatório Nacional de Saúde (ONSA)] tem em desenvolvimento, desde 1998, um instrumento de observação que visa colher dados sobre o estado de saúde e de doença e seus determinantes, da população de Portugal Continental. A criação do “instrumento” em causa foi fundamentada na necessidade de gerar conhecimento sobre saúde, colhendo dados directamente das pessoas com a possibilidade de se produzir estimativas e disponibilizá-las com relativa celeridade. Assim, ECOS (Em Casa Observamos Saúde) nasce como um projecto que tem o objectivo de obter dados sobre saúde, com celeridade, através de entrevista telefónica utilizando uma amostra de Unidades de Alojamento de Portugal continental, com telefone da rede fixa, a Amostra ECOS, em que os indivíduos pertencentes ao agregado familiar se dispõem a ser contactadas periodicamente durante um período de tempo determinado, para responder a inquéritos sobre saúde. Considerou-se, desde da sua criação, que se trataria de um instrumento que poderia ser utilizado por outras entidades, desde que os objectivos de utilização para ele definidos não fossem desvirtuados, assim como os princípios éticos assumidos pelo DEP com as famílias participantes.
- Schistosoma haematobium and bladder cancer: what lies beneath?Publication . Botelho, Mónica Catarina; Machado, José Carlos; Costa, José Manuel Correia daSchistosoma haematobium is a parasitic flatworm that infects millions of people, mostly in the developing world, and is associated with high incidence of bladder cancer although why is not clear. But our group was able to define the mechanistic relationship for the first time between infection of S. haematobium and cancer. We used in vitro models to demonstrate the presence of informative carcinogenesis-associated phenotypes in CHO cells exposed to Sh total antigen, in which we showed increased cell proliferation, decreased apoptosis, up regulation of the anti-apoptotic molecule Bcl-2, down regulation of the tumor suppressor protein p27, and increased cell migration and invasion. We further discuss the molecular and cellular events that might be responsible for schistosomiasis-related bladder cancer.
- Kalirin: a novel genetic risk factor for ischemic strokePublication . Krug, T.; Manso, H.; Gouveia, L.; Sobral, J.; Xavier, J.M.; Albergaria, I.; Gaspar, G.; Correia, M.; Viana-Baptista, M.; Simões, R.M.; Pinto, A.N.; Taipa, R.; Ferreira, C.; Fontes, J.R.; Silva, M.R.; Gabriel, J.P.; Matos, I.; Lopes, G.; Ferro, J.M.; Vicente, A.M.; Oliveira, S.A.Cerebrovascular and cardiovascular diseases are the leading causes of death and disability worldwide. They are complex disorders resulting from the interplay of genetic and environmental factors, and may share several susceptibility genes. Several recent studies have implicated variants of the Kalirin (KALRN) gene with susceptibility to cardiovascular and metabolic phenotypes, but no studies have yet been performed in stroke patients. KALRN is involved, among others, in the inhibition of inducible nitric oxide synthase, in the regulation of ischemic signal transduction, and in neuronal morphogenesis, plasticity, and stability. The goal of the present study was to determine whether SNPs in the KALRN region on 3q13, which includes the Ropporin gene (ROPN1), predispose to ischemic stroke (IS) in a cohort of Portuguese patients and controls. We genotyped 34 tagging SNPs in the KALRN and ROPN1 chromosomal region on 565 IS patients and 517 unrelated controls, and performed genotype imputation for 405 markers on chromosome 3. We tested the single-marker association of these SNPs with IS. One SNP (rs4499545) in the ROPN1-KALRN intergenic region and two SNPs in KALRN (rs17286604 and rs11712619) showed significant (P < 0.05) allelic and genotypic (unadjusted and adjusted for hypertension, diabetes, and ever smoking) association with IS risk. Thirty-two imputed SNPs also showed an association at P < 0.05, and actual genotyping of three of these polymorphisms (rs7620580, rs6438833, and rs11712039) validated their association. Furthermore, rs11712039 was associated with IS (0.001 < P < 0.01) in a recent well-powered genomewide association study (Ikram et al. 2009). These studies suggest that variants in the KALRN gene region constitute risk factors for stroke and that KALRN may represent a common risk factor for vascular diseases.
- Development of a PCR-RFLP marker to genetically distinguish Prosorhynchus crucibulum and Prosorhynchus aculeatusPublication . Francisco, Claire Juliana; Almeida, André; Castro, António Manuel; Santos, Maria JoãoThe cercariae stages of Prosorhynchus crucibulum and Prosorhynchus aculeatus are morphologically indistinguishable. However, the differentiation of these two species is crucial to understand the transmission dynamics between these primary hosts (mussels) and the secondary hosts (fish). In this way, the objective of this study is to develop an accurate molecular identification tool to differentiate the cercariae stage of P. crucibulum and P. aculeatus. We targeted the 18S nuclear ribosomal DNA region by PCR amplification and sequenced this amplicon. By generating these sequences, we developed a RFLP tool with the use of the enzymes HincII and FokI that produced different restriction profiles between P. crucibulum and P. aculeatus. Each enzyme generated different-sized fragments specific to the species examined and no cross-reaction between the species was detected in their restriction pattern. By sequencing, no intraspecific-polymorphism was detected since there is 100% homology among P. aculeatus or P. crucibulum. These results indicate that PCR-linked restriction analysis of the 18S rDNA region provided us with rapid and reliable molecular tools for distinction of the cercariae of these species. The sequences generated were deposited in GenBank accession numbers for P. crucibulum cercariae (FJ463407, FJ463408 and FJ463409) and adult worm (FJ429096, FJ429097), and for P. aculeatus adult (FJ429094 and FJ429095).
- Occurrence of Aflatoxins and Ochratoxin A in Baby FoodsPublication . Alvito, Paula; Sizoo, Eric A.; Almeida, Cristina M. M.; Egmond, Hans P.Infants have a more restricted diet and they generally consume more food on a body weight basis than adults. Therefore, the significance and potential health risk of any contaminant in foods consumed by infants is increased and diligent attention must be paid to this particular area. The present study aims to determine the occurrence of aflatoxin M1 (AFM1), aflatoxin B1 (AFB1) and ochratoxin A (OTA) in processed cereal-based foods (flours) and infant formulae (milk powder) available in the Portuguese market, both sold as conventional and organic origin. Mycotoxin determination was carried out using a method previously applied to duplicate diet samples. This method employed chloroform extraction, liquid–liquid extraction, immunoaffinity column (IAC) cleanup and HPLC analysis with fluorescence detection after postcolumn derivatisation. Quantification limits were 0.014, 0.004 and 0.028 μg kg− 1 for AFM1, AFB1 and OTA, respectively. These toxins could only be quantified in 12 of 27 analysed samples (15 positive results): two samples with AFM1, two samples with AFM1 and OTA, one sample with AFB1 and OTA and seven samples with OTA. Positive results concerned four for AFM1 (26%), one for AFB1 (7%) and ten for OTA (67%). For these samples, contents ranged between 0.017–0.041 μg AFM1 kg−1, 0.034–0.212 μg OTA kg−1, and one sample had a value of 0.009 μg AFB1 kg−1. Considering the presented results, we could provisionally conclude that the presence of these mycotoxins in baby foods does not constitute a public health problem. These are the first results concerning the occurrence of mycotoxins in marketed baby foods in Portugal and this is the first study using the HPLC method, proposed for duplicate diets, in baby food sample analysis.
- Invasive Haemophilus influenzae Disease, Europe, 1996-2006Publication . Ladhani, S.; Slack, M.P.; Heath, P.T.; von Gottberg, A.; Chandra, M.; Ramsay, M.E.; European Union Invasive Bacterial Infection Surveillance participantsAn international collaboration was established in 1996 to monitor the impact of routine Haemophilus influenzae type b (Hib) vaccination on invasive H. influenzae disease; 14 countries routinely serotype all clinical isolates. Of the 10,081 invasive H. influenzae infections reported during 1996-2006, 4,466 (44%, incidence 0.28 infections/100,000 population) were due to noncapsulated H. influenzae (ncHi); 2,836 (28%, 0.15/100,000), to Hib; and 690 (7%, 0.036/100,000), to non-b encapsulated H. influenzae. Invasive ncHi infections occurred in older persons more often than Hib (median age 58 years vs. 5 years, p<0.0001) and were associated with higher case-fatality ratios (12% vs. 4%, p<0.0001), particularly in infants (17% vs. 3%, p<0.0001). Among non-b encapsulated H. influenzae, types f (72%) and e (21%) were responsible for almost all cases; the overall case-fatality rate was 9%. Thus, the incidence of invasive non-type b H. influenzae is now higher than that of Hib and is associated with higher case fatality.
- Genotyping of Giardia duodenalis cysts by new real-time PCR assays for detection of mixed infections in human samplesPublication . Almeida, André; Pozio, Edoardo; Cacciò, Simone M.Of the seven genetic groups, or assemblages, currently recognized in the Giardia duodenalis species complex, only assemblages A and B are associated with human infection, but they also infect other mammals. Recent investigations have suggested the occurrence of genetic exchanges among isolates of G. duodenalis, and the application of assemblage-specific PCR has shown both assemblages A and B in a significant number of human infections. In this work, three real-time quantitative (qPCR) assays were developed to target the G. duodenalis triose phosphate isomerase, glutamate dehydrogenase, and open reading frame C4 sequences. Primers were designed to allow the specific amplification of the DNA of assemblage A or B and to generate products distinguishable by their melting curves or, after qPCR, by their sequences, sizes, or restriction patterns. The assays showed full specificity and detected DNA from a single trophozoite (4 to 8 target copies). We applied these assays, as well as a TaqMan assay that targets the beta-giardin gene, to genomic DNA extracted from 30 human stools and to Giardia cysts purified by immunomagnetic capture from the same samples. Simultaneous detection of both assemblages was observed in a large number of DNAs extracted from stools, and experiments on the cysts purified from the same samples showed that this was essentially attributable to mixed infections, as only one assemblage was detected when dilutions of cysts were tested. In a few cases, detection of both assemblages was observed even when single cysts were tested. This result, which suggests the presence of recombinants, needs to be confirmed using more accurate methods for cyst separation and enumeration. The assays described in this study can be used to detect Giardia cysts infectious to humans in samples from animals and in water and food.
- Alterações do estado de saúde associadas à alimentação: contaminação microbiológica dos alimentosPublication . Viegas, Silvia Judite
- Variation in the measurement of DNA damage by comet assay measured by the ECVAG† inter-laboratory validation trialPublication . Forchhammer, Lykke; Johansson, Clara; Loft, Steffen; Möller, Lennart; Godschalk, Roger, W. L.; Langie, Sabine A. S.; Jones, George D. D.; Kwok, Rachel W. L.; Collins, Andrew R.; Azqueta, Amaya; Phillips, David H.; Sozeri, Osman; Stepnik, Maciej; Palus, Jadwiga; Vogel, Ulla; Wallin, Hakan; Routledge, Michael N.; Handforth, Catherine; Allione, Alessandra; Matullo, Giuseppe; Teixeira, João Paulo; Costa, Solange; Riso, Patrizia; Porrini, Marisa; Møller, PeterThe comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0–10 Gy) and used to construct a calibration curve to calculate the number of lesions per 106 base pair. All laboratories detected dose–response relationships in the coded samples irradiated with ionizing radiation (1.5–7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levene's test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay.