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- The Sensitivity and Resistance of Yeasts Isolated from Women with Vulvovaginal Candidiasis to Common Antifungal drugs Using Disc DiffusionPublication . Moallaie, Hossein; Verissimo, Cristina; Brandão, João; Rosado, LauraBackground and Purpose: Due to the ever-increasing use of antifungal drugs especially those of azole group, the prevalence of recurrent forms of vaginal infections and the number of drug-resistant yeasts are on the rise. Therefore, the rpesent study is conducted to investigate the sensitivity and resistance of yeasts isolated from vaginal infections to antifungal drugs. Methods and Materials: This cross-sectional descriptive analytical study was conducted on 118 yeasts isolated from 436 suspects of vulvovaginal candidiasis; their sensitivity and resisitance to drugs belonging to imidazoles group including clotrimazole (CTR), fluconazole (FCA), ketoconazole (KET), miconazole (MCZ) and econazole (EC) as well as nystatine (NY) belonging to polyene group using the standard disc diffusion technique. To determine their relationship clinical symptoms and the raltion of resistance to one drug with resistance to other drugs, relevant tests were used including chi-square, kappa and linear regression coefficient in SPSS 11. Results: The results showed no resistance to nystatine from polyene group and econazole from azole group; however, 53 cases (%44.9) were resistant to fluconazole, 26 cases (%22) to miconazole, 10 cases (%8.5) to clotrimazole and 2 cases (%1.7) to ketoconazole. Conclusion: The results of linear correlation showed a negative correlation between the sensitivity of yeasts to CTR and KET and clinical symptoms.
- Novel method for picking up large heterozygous deletions with semiquantitative PCR in patients with mucolipidosis III alpha/betaPublication . Coutinho, Maria Francisca; Encarnação, Marisa; Lacerda, Lúcia; Ribeiro, Helena; Prata, Maria João; Alves, SandraMucolipidosis II (ML II alpha/beta) or I-cell disease is a rare genetic disease in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent. GlcNAc-phosphotransferase is a multimeric enzyme encoded by two genes: GNPTAB and GNPTG. Although a wide spectrum of mutations in GNPTAB has recently been reported to cause ML II alpha/beta, there are some patients that were previously reported as possessing only one heterozygous mutation while the second one was missing. Here we support the idea that some of these cases may be attributable to the methodological problem of direct sequencing since large heterozygous deletions are undetectable through this method. Methods: We developed a semiquantitative approach by multiplex PCR and capillary electrophoresis. In order to do so, we co-amplified two exon fragments in each PCR reaction, including the one corresponding to GNPTAB exons and an internal fragment that would work as a control (primer pairs for LIPA exons, the gene that codes for acid lipase, another lysosomal enzyme). Results: After testing this technique in the two Portuguese patients that present heterozygous missense mutations but with the second mutation missing, we found evidences that support the idea that, in both patient, the last exons (20 and 21) of the GNPTAB gene are missing in one of the alleles. Conclusion: Here we present the first molecular evidence of the existence of large deletions in the GNPTAB, underlying Mucolipidosis type III as well as a novel method for detecting these types of alterations when present in heterozygous patients. This semiquantitative PCR approach is a valuable tool that allows the screening of large deletions in the GNPTAB gene. In conclusion, ML III cases with only one heterozygous mutation already detected trhpugh direct sequencing methods should be further screened for the possible presence of a large heterozygous deletion mutation.
- Registo Nacional de Anomalias Congénitas: relatório de 2002-2007Publication . Departamento de EpidemiologiaO Registo Nacional de Anomalias Congénitas (RENAC) é um registo epidemiológico de base populacional, destinado a permitir a observação e a vigilância epidemiológica da ocorrência de casos de anomalias congénitas em Portugal. O RENAC recebe notificações de diversas origens, principalmente dos Serviços de Obstetrícia e de Neonatologia dos hospitais portugueses, sendo registados todos os casos com pelo menos uma anomalia major. O Registo cobre todo os recém-nascidos vivos, cujas anomalias sejam detectadas até ao final do período neo-natal, assim como os abortos espontâneos, os fetos mortos e as interrupções médicas de gravidez, com pelo menos uma anomalia congénita. O presente relatório abrange um período de 6 anos, compreendido entre 2002 e 2007, em que a população sob vigilância variou entre o número máximo de 80840 nascimentos em 2002 e o número mínimo de 57088 nascimentos, em 2006, representando, respectivamente, 76,8% e 57,2% de todos os nascimentos ocorridos em Portugal. A cobertura média no período sob vigilância foi de 66,6% do número total de nascimentos ocorridos nos hospitais que notificaram para o RENAC, de acordo com as estatísticas oficiais. O número total de casos notificados neste período foi de 5815, com 8643 anomalias congénitas. O número máximo de casos verificou-se em 2002, 1306 casos e o valor mínimo, 684 casos, no ano 2006. O número máximo de anomalias registadas foi de 2062 anomalias, isoladas ou em associação, no ano 2002 e de 1009 anomalias, no ano 2006. A prevalência máxima observada foi de 233,6/10000 no ano 2002 e a mínima de 165,5/10000 no ano 2006. De entre todas as anomalias registadas, o grupo mais frequente foi o das anomalias do aparelho circulatório (25,5%) a que correspondeu a prevalência de 50,5 por cada 10000 nascimentos. Seguiram-se as anomalias do sistema osteomuscular (20,7%; 40,9 casos por cada 10000 nascimentos) e do aparelho urinário (14,5%; 28,7 casos por cada 10000 nascimentos).
- Multiplex PCR for detection of microcystins-producing cyanobacteria from freshwater samplesPublication . Valério, Elisabete; Chambel, Lélia; Paulino, Sérgio; Faria, Natália; Pereira, Paulo; Tenreiro, RogérioThe aim of this study was to develop a PCR-based method of gene-directed multiplex PCR to rapidly identify microcystins producing cyanobacteria, regardless of their taxa, that could be applied in routine freshwater monitoring. Instead of using the amplification of only one or two mcy gene fragments, a multiplex PCR that simultaneously amplifies mcyA-cd, mcyAB, and mcyB fragments of the microcystin gene cluster was validated with DNA from 124 cyanobacterial isolates and applied in 37 environmental samples. The toxicological status of the isolates was assessed by high-performance liquid chromatography also used as the "gold standard" for the evaluation of multiplex mcy genes-based PCR, where a sensitivity of 92.3% and a specificity of 100% have been obtained. For the environmental samples, a rapid protocol for their direct use in the PCR reaction has been developed and, by using ELISA results as "gold standard" for the presence of microcystins in these samples, a sensitivity of 80% and a specificity of 100% were achieved, showing that this multiplex PCR test is a rapid, reliable, and economical way of assessing the microcystin-producing potential of cyanobacteria in freshwaters, regardless of their taxa or microcystins variant produced.
- Best Practice Guidelines on molecular diagnostics in Duchenne/Becker muscular dystrophiesPublication . Abbs, S.; Tuffery-Giraud, S.; Bakker, E.; Ferlini, A.; Sejersen, T.; Mueller, C.R.Introduction: A meeting of 29 senior scientists from Europe, the USA, India and Australia, was held in Naarden, The Netherlands on November 14–16, 2008, to establish consensus Best Practice Guidelines for molecular diagnosis of Duchenne and Becker muscular dystrophy (DMD/BMD). New therapeutic trials for DMD demand accurate diagnosis of the disorder, especially where the therapy is targeted towards specific mutations. These guidelines aim to help diagnostic laboratories attain that accuracy by describing the minimum standards for acceptable molecular diagnostic testing of DMD. For the different types of clinical referral received by a molecular diagnostic laboratory, the guidelines recommend the appropriate tests to be carried out, interpretation of the results and how those results should be reported.
- Molecular Characterization of the Portuguese Patients with defects in the GLB1 gene: evidences of a strong genotype-phenotype correlation.Publication . Coutinho, Maria Francisca; Lacerda, Lúcia; Ribeiro, Helena; Prata, Maria João; Alves, Sandra
- Molecular characterization of a new isolate of Borrelia lusitaniae derived from Apodemus sylvaticus in PortugalPublication . Carvalho, Isabel Lopes de; Zeidner, Nordin; Ullmann, Amy; Hojgaard, Andrias; Amaro, Fátima; Zé-Zé, Líbia; Alves, M.J.; Sousa, Rita de; Piesman, Joseph; Núncio, Maria SofiaA total of 196 small mammals were collected in Portugal and tested for Borrelia burgdorferi sensu lato. Tissue samples were taken from each animal and cultured in Barbour-Stoenner-Kelly (BSK)-II medium. The single strain of spirochete isolated was confirmed as Borrelia lusitaniae by genetic analyses. This is the first report of B. lusitaniae isolated from Apodemus sylvaticus.
- Molecular diagnosis of familial hypercholesterolemia: an important tool for cardiovascular risk stratificationPublication . Alves, A.C.; Medeiros, A.M.; Francisco, V.; Gaspar, I.M.; Rato, Q.; Bourbon, M.Familial hypercholesterolemia (FH) is associated with an increased risk of premature coronary heart disease. Molecular identification of these patients can reduce the burden of mortality from cardiovascular disorders simply by the correct identification of the disease early in life, followed by counseling and appropriate lifestyle modifications, and therapeutic measures when required. Recent studies show that, in Portugal, this disease is severely under-diagnosed. After more than 10 years of research through the Portuguese FH Study, it is now possible to translate the original research results into clinical application. AIMS: The main aims of the present work were to determine whether clinical characterization is sufficient to identify these individuals at high risk of developing CHD and to evaluate the clinical applicability of molecular diagnosis for FH. METHODS: All patients described in this study were recruited for the Portuguese FH Study. The diagnostic criteria used to select the index patients were adapted from the Simon Broome Heart Research Trust. To analyze the usefulness of the molecular diagnosis, graphs of total and LDL cholesterol values by age were constructed for 622 possible FH patients. The lipid profile of patients genetically identified as having FH, before and under medication, were analyzed to assess whether these patients were receiving appropriate treatment. The data are shown separately for children and adults and for female and male propositi (index cases and hypercholesterolemic relatives), both with and without a detectable mutation in the LDLR gene. RESULTS: The Portuguese FH Study has already genetically identified 404 individuals (171 index patients and 233 relatives) among more than one thousand individuals sent for study. A total of 78 different mutations in the LDLR gene were found in 171 index patients, 2 different mutations were found in the apoB gene of 4 patients and 2 patients had a unique PCSK9 mutation. Statistical analysis revealed that there are significant differences between total cholesterol (p < 0.001) and apoB (p = 0.026) values in the group of children (male and female) with and without a mutation in LDLR. For female children LDL values were also significantly different (p < 0.001) between subgroups but for male children this difference did not reach statistical significance. In adult women there is a statistically significant difference for total cholesterol (p = 0.049), LDL cholesterol (p = 0.031) and apoB (p = 0.003) values in the subgroups with and without a LDLR mutation. In adult males there is a statistical difference for total cholesterol (p = 0.002). LDL cholesterol (p = 0.003) and apoB (p = 0.0023) in subgroups with and without an LDLR mutation. Nevertheless there was considerable dispersion of values and individually it is not possible to distinguish between patients with and without a mutation in the LDLR gene, based only on lipid profile. CONCLUSIONS: By analysis of the clinical data of 696 possible FH patients, the present report shows evidence that clinical characterization is not sufficient to distinguish between patients with genetic or environmental dyslipidemia, and so molecular diagnosis is useful in clinical practice, allowing correct identification of FH patients and their relatives, and the early implementation of therapeutic measures to reduce the elevated cardiovascular risk of these patients. In general, molecular diagnosis of FH is feasible and could be obtained in 1-2 months if the technology is available. In Portugal the test will be offered to the population by our Institute at a cost of about 500 euros, like many other genetic tests or exams such as nuclear magnetic resonance.
- Biological and genetic characterization of Cryptosporidium spp. and Giardia duodenalis isolates from five hydrographical basins in northern PortugalPublication . Almeida, André; Moreira, Maria João; Soares, Sónia; Delgado, Maria de Lurdes; Figueiredo, João; Silva, Elisabete; Castro, António; Costa, Alexandra Viana da; Costa, José Manuel Correia daTo understand the situation of water contamination with Cryptosporidium spp. and Giardia spp. in the northern region of Portugal, we have established a long-term program aimed at pinpointing the sources of surface water and environmental contamination, working with the water-supply industry. Here, we describe the results obtained with raw water samples collected in rivers of the 5 hydrographical basins. A total of 283 samples were analyzed using the Method 1623 EPA, USA. Genetic characterization was performed by PCR and sequencing of genes 18S rRNA of Cryptosporidium spp. and b-giardin of Giardia spp. Infectious stages of the protozoa were detected in 72.8% (206 of 283) of the water samples, with 15.2% (43 of 283) positive for Giardia duodenalis cysts, 9.5% (27 of 283) positive for Cryptosporidium spp. oocysts, and 48.1% (136 of 283) samples positive for both parasites. The most common zoonotic species found were G. duodenalis assemblages A-I, A-II, B, and E genotypes, and Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis, and Cryptosporidium muris. These results suggest that cryptosporidiosis and giardiasis are important public health issues in northern Portugal. To the authors’ knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in raw water samples in the northern region of Portugal.
- Case control study for measuring influenza vaccine effectiveness in Portugal - Final report Season 2009-10Publication . Nunes, Baltazar; Guiomar, Raquel; Machado, Ausenda; Falcão, Isabel; Gonçalves, Paulo; Conde, Patrícia; Batista, Inês; Dias, Carlos Matias; Marinho Falcão, JoséO Departamento de Epidemiologia em conjunto com o Departamento de Doenças Infecciosas do INSA realizaram um estudo, na época de 2009-2010, que teve por objectivo estimar a efectividade da vacina sazonal (em indivíduos com 65 e mais anos) e pandémica (todas as idades). O estudo contou com a participação de: Portugal, Espanha, Irlanda, França, Itália, Hungria e Roménia. Este estudo surge enquadrado no projecto europeu multicêntrico - I-MOVE- Monitoring influenza vaccine efectiveness during influenza seasons and pandemics in the European Union, coordenado pela EpiConcept SARL e financiado pelo ECDC, que pretende estimar a efectividade da vacina sazonal e pandémica durante e após a época de gripe.