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- Auditing preanalytical phase: an autoevaluation of laboratoriesPublication . Cardoso, Ana; Correia, Helena; Faria, Ana PaulaPreanalytical phase is more prone to errors due to non-automated activities such as collection, handling, transportation and preparation of specimens. Since 2007, PNAEQ (Programa Nacional de Avaliação Externa da Qualidade) provides a specific program on preanalytical phase. In 2016, it was proposed to laboratories to audit their own preanalytical phase. The aim is to involve laboratories in evaluation and monitoring process, giving them tools for their own evaluation of this important extra-analytical phase.
- Epidemiology of Invasive Haemophilus influenzae Disease, Europe, 2007-2014Publication . Whittaker, Robert; Economopoulou, Assimoula; Dias, Joana Gomes; Bancroft, Elizabeth; Ramliden, Miriam; Celentano, Lucia Pastore; European Centre for Disease Prevention and Control Country Experts for Invasive Haemophilus influenzae Disease.We describe the epidemiology of invasive Haemophilus influenzae disease during 2007-2014 in 12 European countries and assess overall H. influenzae disease trends by serotype and patient age. Mean annual notification rate was 0.6 cases/100,000 population, with an increasing annual trend of 3.3% (95% CI 2.3% to 4.3%). The notification rate was highest for patients <1 month of age (23.4 cases/100,000 population). Nontypeable H. influenzae (NTHi) caused 78% of all cases and showed increasing trends among persons <1 month and >20 years of age. Serotype f cases showed an increasing trend among persons >60 years of age. Serotype b cases showed decreasing trends among persons 1-5 months, 1-4 years, and >40 years of age. Sustained success of routine H. influenzae serotype b vaccination is evident. Surveillance systems must adopt a broad focus for invasive H. influenzae disease. Increasing reports of NTHi, particularly among neonates, highlight the potential benefit of a vaccine against NTHi.
- Less is More: an overview on the use of RNAi as a tool to achieve Substrate Reduction in MucopolysaccharidosesPublication . Coutinho, Maria Francisca; Santos, Juliana Inês; Alves, SandraLysosomal storage diseases (LSDs) are a group of genetic disorders caused by dysfunction in enzymes responsible for the intralysosomal degradation of particular compounds. Given their complex nature and the limitations of available therapies, the shift towards the development of combination treatments to counteract more effectively the pathological burden of these disorders is in the agenda of current research viewing to improve the clinical outcome of LSD patients. We consider that treatment strategies relying on RNA interference (RNAi), as well as in other RNA-based methodologies, may be feasible and particularly promising if designed in the context of a synergistic combinatorial therapeutic approach. We are currently evaluating an RNAi-dependent strategy based upon the selective downregulation of genes involved in the biosynthesis of glycosaminoglycans (GAGs), the major substrates that accumulate in patients suffering from a subset of LSDs called mucopolysaccharidoses (MPSs). Although enzyme replacement therapy is already available for some MPSs, it has some serious drawbacks, justifying the challenge of developing additional therapies targeting this group of disorders. Our goal is to promote an effective reduction of the accumulating substrate, ultimately decreasing or delaying MPSs’ symptoms. It should be noticed that, even though some substrate reduction therapy (SRT) drugs have already been approved (miglustat for GD) or are undergoing clinical trial (genistein and/or rhodamine B for MPSs), clinical evaluation of those same drugs has unveiled a few side effects, the most well-known being those observed for miglustat, which included osmotic diarrhea and weight loss. Nevertheless, chemical drugs aren’t the only way to achieve substrate reduction. Ours is fully molecular, drug-free approach, whose major focus relies on the biosynthetic pathways giving origin to each one of the GAGs whose degradation is impaired in MPS. Taking advantage of the RNAi technology potential, we have designed and assayed specific siRNAs targeting genes on those biosynthetic cascades to decrease the levels of production of each one of the four substrates. Their efficiency is currently being evaluated in vitro. Here we present an overview of the preliminary results of this project and unveil its next steps towards a full characterization/evaluation of its potential therapeutic effect.
- Harmonization of nucleic acid testing for Zika virus: development of the 1st World Health Organization International StandardPublication . Baylis, S.A.; Hanschmann, K-M.O.; Schnierle, D.S.; Trösemeier, J-H.; Blümel, J.; Zika Virus Collaborative Study GroupBACKGROUND: With the ongoing public health emergency due to Zika virus (ZIKV), nucleic acid testing (NAT) is essential for clinical diagnosis and screening of blood donors. However, NAT for ZIKV has not been standardized, and this study was performed to establish a World Health Organization (WHO) International Standard (IS) for ZIKV RNA; WHO ISs have been used to improve detection and quantification of blood-borne viruses. STUDY DESIGN AND METHODS: The candidate IS (cIS), code number 11468/16, was prepared by heat inactivation and lyophilization of a ZIKV strain isolated from a patient in French Polynesia in 2013. The cIS was evaluated together with other reference materials, including both Asian and African ZIKV lineages as well as a panel of clinical samples and in vitro-transcribed RNAs. The samples for evaluation were distributed to 24 laboratories from 11 countries. The assays used consisted of a mixture of in-house developed and commercial assays (available or in development). RESULTS: The potencies of the standards were determined by quantitative and qualitative assays. In total, 37 sets of data were analyzed: 19 from quantitative assays and 18 from qualitative assays. Data demonstrated wide variations in reported potencies of the cIS and the other study samples. CONCLUSIONS: Assay variability was substantially reduced when ZIKV RNA concentrations from the biological reference materials and clinical samples were expressed relative to the cIS. Thus, the WHO has established 11468/16 as the 1st IS for ZIKV RNA, with a unitage of 50,000,000 IU/mL.
- Characterization of the molecular pathogenesis of a malformation syndrome associated with a complex double chromosome translocationPublication . Marques, Mariana; David, Dezso; Dias, Deodália Maria AntunesCongenital anomalies are devastating conditions responsible for high neonatal mortality, as well as high morbidity of the surviving individuals. Chromosomal rearrangements are a leading cause of severe congenital malformations and are associated with about 25% of perinatal deaths due to congenital anomalies. The aim of this study is the identification of candidate genes responsible for the phenotype characterized by intrauterine growth retardation, severe developmental delay, brain malformations and refractory epilepsy identified in an individual with an apparently balanced de novo double chromosomal translocation t(2;7)(q23;q32),t(5;6)(q23;q26)dn. Identification and mapping of the structural chromosomal aberrations were performed by whole-genome array analysis, array painting with genomic amplicons of the derivative chromosomes and by whole genome sequencing of large-insert jumping libraries (liWGS). Subsequently all junction fragments were amplified and the breakpoints were identified at nucleotide resolution by Sanger sequencing. Genome array analysis identified a 651.76 kb deletion at 14q24.3 (g.76,673,181-77,324,937 [GRCh37/hg19]). Transforming growth factor beta 3 (TGFB3), a gene associated with autosomal dominant arrythmogenic right ventricular dysplasia and Loeys-Dietz syndrome (OMIM #107970 and #615582), is situated 224 kb upstream from the proximal deletion breakpoint.. Translocation breakpoints were identified both by array painting and liWGS. The 2q23.3 breakpoint of the t(2;7)(q23.3;q32.1), disrupts IVS5 of pre-mRNA processing factor 40 homolog A (PRPF40A), a protein coding gene related to Huntington’s disease (OMIM#143100). The calcium channel, voltage-dependent, beta-4 subunit (CACNB4) gene, localized 600 kb upstream of this breakpoint, is associated with three epilepsy related autosomal dominant disorders (OMIM #613855, 607682 and 607682). The Staphylococcal nuclease and tudor domain containing 1 (SND1) gene disrupted by the 7q32.1 breakpoint, is not presently associated with any known phenotype. However, the RNA binding motif protein 28 coding gene (RBM28), situated 300 kb downstream of the 7q32.1 breakpoint, has been associated with progressive neurological defects (OMIM #612079). Concerning the t(5;6)(q23.2;q26) translocation, the 5q23.2 breakpoint is situated in an intergenic region whereas the 6q26 breakpoint disrupts IVS3 of PARK2 co-regulated gene (PACRG). This gene shares a bidirectional promoter with parkin RBR E3 ubiquitin protein ligase (PARK2), which is associated with early onset Parkinson disease. About 300kb downstream of this breakpoint is the homolog of quaking mouse (QKI) gene that also plays a role in brain development. The application of liWGS unveiled the presence of two additional cryptic alterations on der(6), an excision/insertion and an inversion. The cryptic excision at 6q22.33 disrupts protein tyrosine phosphatase receptor type K (PTPRK), a gene from the protein tyrosine phosphatase family which is associated with tumor suppression. As a result of the excision/insertion, the excised 48 kb fragment containing PTPRK exon 7 and flanking intronic sequences is inserted 36 Mb further distal at 6q26. Located 70kb from the PTPRK gene, the laminin 2 (LAMA2) gene was reported has being involved in brain malformations, including polymicrogyria. The inversion breakpoint at 5q23.2 is located within an intergenic region. In conclusion, these findings suggest that disruption of PRPF40A and PACRG genes, in association with misregulation of CACNB4, RBM28, PARK2, QKI and LAMA2 genes from the breakpoint regions are the most likely candidate genes responsible for this complex malformation phenotype. Additionally the modulating effect of TGFB3 gene cannot be excluded. Comparative analysis of this complex chromosome rearrangement by array painting and liWGS demonstrates that currently only liWGS is able to identify the full spectrum of balanced, otherwise cryptic, structural alterations. In this way, liWGS allows high-throughput delineation of chromosomal rearrangements, allowing a better phenotype-genotype association. A major drawback of studying chromosome anomalies is the unavailability of relevant human biological material or of data from such samples. Theoretically, to overcome this issue, animal or induced pluripotent stem cells models can be used. During this study, the obtainment of a proband-specific iPSC model was attempted. Unfortunately, the complexity of the pluripotency induction process, the associated costs and the requisites of using non-viral vectors hinder the development of such cellular models for the study of the molecular pathogenesis of congenital anomalies. Proband derived lymphoblastoid cell line (LCL), non-integrative episomal plasmids containing the four Yamanaka factors – OCT3/4, c-MYC, SOX2 and KLF4 and an electroporation platform were used for the pluripotency induction experiments. Electroporated cells were maintained on a human foreskin fibroblasts (HFF) feeder-layer. While performing the reprogramming experiments, several technical difficulties were identified. A major difficulty is achieving high transfection efficiency of LCL with episomal plasmids without high cell mortality rates. Although no LCL derived iPSC colonies were obtained, the identification of the critical steps in the induction protocol of LCL derived cells will certainly contribute for further development of such cellular models. Furthermore, the availability of individual-derived iPSCs will definitely lead to a robust cellular model for the study of the molecular pathogenesis of chromosome rearrangements associated with congenital anomalies.
- Lysossomal acid lipase activity in dried blood spots - preliminar resultsPublication . Gaspar, Paulo; Alves, Sandra; Vilarinho, LauraLysosomal storage diseases (LSDs) are a group of heterogeneous and multisystemic disorders caused by defects in enzymes responsible for the intralysosomal degradation of particular compounds. One of them is Lysosomal Acid Lipase Deficiency (LALD) that is caused by the deficiency of the enzyme Lysosomal Acid Lipase (LAL), which is responsible for the hydrolysis of cholesterol esters and triglycerides in the lysosome.
- Lysosomal acid lipase deficiency: a hidden disease among cohorts of familial hypercholesterolemia?Publication . Chora, J.R.; Alves, A.C.; Medeiros, A.M.; Mariano, C.; Lobarinhas, G.; Guerra, A.; Mansilha, H.; Cortez-Pinto, H.; Bourbon, M.Highlights: - Dyslipidemia phenotype of patients with familial hypercholesterolemia and lysosomal acid lipase deficiency (LALD) can overlap; - Familial hypercholesterolemia negative patients should be investigated to identify possible LALD patients; - Correct identification of LALD patients is important for patient prognosis. Background: Lysosomal acid lipase deficiency (LALD) is an autosomal recessive disorder and an unrecognized cause of dyslipidemia. Patients usually present with dyslipidemia and altered liver function and mutations in LIPA gene are the underlying cause of LALD. Objective: The aim of this study was to investigate LALD in individuals with severe dyslipidemia and/or liver steatosis. Methods: Coding, splice regions, and promoter region of LIPA were sequenced by Sanger sequencing in a cohort of mutation-negative familial hypercholesterolemia (FH) patients (n = 492) and in a population sample comprising individuals with several types of dyslipidemia and/or liver steatosis (n = 258). Results: This study led to the identification of LALD in 4 children referred to the Portuguese FH Study, all with a clinical diagnosis of FH. Mild liver dysfunction was present at the age of FH diagnosis; however, a diagnosis of LALD was not considered. No adults at the time of referral have been identified with LALD. Conclusion: LALD is a life-threatening disorder, and early identification is crucial for the implementation of specific treatment to avoid premature mortality. FH cohorts should be investigated to identify possible LALD patients, who will need appropriate treatment. These results highlight the importance of correctly identifying the etiology of the dyslipidemia.
- Preliminary spectrum of genetic variants in familial hypercholesterolemia in ArgentinaPublication . Bañares, V.G.; Corral, P.; Medeiros, A.M.; Araujo, M.B.; Lozada, A.; Bustamante, J.; Cerretini, R.; López, G.; Bourbon, M.; Schreier, L.E.Highlights: - First description of familial hypercholesterolemia mutations in Argentina; - Identification of 7 patients with severe familial hypercholesterolemia; - Wide genetic heterogeneity with 1 relatively common allele, the Lebanese mutation; Description and deep bioinformatics characterization of 4 novel genetic variants; - Studying the exon 14 in a first step could be a low-cost approach for this population. Background: Familial hypercholesterolemia (FH) is a genetic disorder characterized by elevated low-density lipoprotein cholesterol and early cardiovascular disease. As cardiovascular disease is a leading cause of mortality in Argentina, early identification of patients with FH is of great public health importance. Objective: The aim of our study was to identify families with FH and to approximate to the characterization of the genetic spectrum mutations of FH in Argentina. Methods: Thirty-three not related index cases were selected with clinical diagnosis of FH. Genetic analysis was performed by sequencing, multiplex ligation-dependent probe amplification, and bioinformatics tools. Results: Twenty genetic variants were identified among 24 cases (73%), 95% on the low-density lipoprotein receptor gene. The only variant on APOB was the R3527Q. Four were novel variants: c.-135C>A, c.170A>C p.(Asp57Ala), c.684G>C p.(Glu228Asp), and c.1895A>T p.(Asn632Ile); the bioinformatics’ analysis revealed clear destabilizing effects for 2 of them. The exon 14 presented the highest number of variants (32%). Four variants were observed in more than 1 case and the c.2043C>A p.(Cys681*) was carried by 18% of index cases. Two true homozygotes, 3 compound heterozygotes, and 1 double heterozygote were identified. Conclusion: This study characterizes for the first time in Argentina genetic variants associated with FH and suggest that the allelic heterogeneity of the FH in the country could have 1 relative common low-density lipoprotein receptor mutation. This knowledge is important for the genotype–phenotype correlation and for optimizing both cholesterol-lowering therapies and mutational analysis protocols. In addition, these data contribute to the understanding of the molecular basis of FH in Argentina.
- Programa Nacional de Avaliação Externa da Qualidade (PNAEQ) - Fases Extra-Analíticas: folheto de divulgação 2017Publication . Cardoso, Ana; Correia, Helena; Faria, Ana PaulaObjetivo: A implementação de um programa de AEQ nacional e específico para as Fases Extra-Analíticas tem como objetivo melhorar a qualidade do serviço prestado pelos laboratórios participantes. Ações: - Distribuição de diferentes tipos de ensaios AEQ, incluindo folhas de registo, checklists e outras ferramentas - Recolha de informações através de questionários para levantamento de dados - Reuniões com fornecedores de material de colheitas - Reuniões com os participantes para esclarecimento de dúvidas - Criação de um Grupo de Trabalho para seleção de indicadores, avaliação dos resultados, divulgação de dados e formação
- Inquérito Alimentar Nacional e de Atividade Física, IAN-AF 2015-2016: relatório metodológicoPublication . Lopes, Carla; Torres, Duarte; Oliveira, Andreia; Severo, Milton; Alarcão, Violeta; Guiomar, Sofia; Mota, Jorge; Teixeira, Pedro; Ramos, Elisabete; Rodrigues, Sara; Vilela, Sofia; Oliveira, Luísa; Nicola, Paulo; Soares, Simão; Frost Andersen, Lene; Consórcio IAN-AFO Inquérito Alimentar Nacional e de Atividade Física (IAN-AF) 2015-2016 teve como objetivo primário recolher informação de representatividade nacional e regional (dos 3 meses aos 84 anos de idade) sobre o consumo alimentar (incluindo a ingestão e suplementação nutricionais, a segurança dos alimentos e a insegurança alimentar) e sobre a atividade física (incluindo os comportamentos sedentários, as atividades desportivas/de lazer e as escolhas ativas na rotina diária) e a sua relação com determinantes em saúde, nomeadamente os socioeconómicos. O projeto foi desenvolvido por um Consórcio, envolvendo investigadores da Universidade do Porto (Promotor), da Universidade de Lisboa, do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), da Universidade de Oslo e da empresa SilicoLife. Os participantes foram selecionados aleatoriamente por um processo de amostragem bietápica, a partir do Registo Nacional de Utentes do Serviço Nacional de Saúde: i) seleção aleatória de Unidades Funcionais de Saúde em cada Unidade Territorial para Fins Estatísticos (NUTS II), ponderada para o número de inscritos; ii) seleção aleatória de indivíduos registados em cada Unidade Funcional de Saúde, com um número fixo de elementos por sexo e grupo etário. Avaliaram-se 6553 participantes através de uma entrevista presencial e destes, 5811 completaram as duas entrevistas previstas, intervaladas no tempo entre 8 a 15 dias e distribuídas ao longo de 12 meses (outubro de 2015 a setembro de 2016), incluindo as quatro estações do ano e todos os dias da semana, de forma a ajustar para a variabilidade intra-individual e para a sazonalidade dos comportamentos alimentares e de atividade física. A metodologia utilizada incluiu ferramentas e protocolos harmonizados no contexto Europeu, de acordo com o definido no inquérito pan-Europeu EU-MENU, promovido pela Autoridade Europeia para a Segurança dos Alimentos (EFSA), integrados numa plataforma eletrónica assistida por computador, especificamente desenvolvida (Plataforma “You eAT&Move”). O Módulo “You” permite a recolha de informação sociodemográfica, de saúde geral, de antropometria, de propensão alimentar e de insegurança alimentar. Os parâmetros antropométricos (peso, estatura, perímetros do braço, cintura e anca) foram objetivamente medidos de acordo com procedimentos padronizados. A insegurança alimentar foi avaliada através de uma adaptação do questionário desenvolvido por Cornell/Radimer (1990), que fornece estimativas a nível nacional de insegurança alimentar, para as famílias, com e sem menores de 18 anos, recolhendo informação sobre quatro dimensões subjacentes à experiência de insegurança alimentar: disponibilidade, acesso, utilização e estabilidade/resiliência. O módulo “eAT24” permite a recolha de informação alimentar através de dois questionários às 24 horas anteriores (ou diários alimentares de dois dias nas crianças com idade <10 anos), sincronizada com dados de composição nutricional dos alimentos e receitas da Tabela da Composição de Alimentos Portuguesa (INSA), adaptada. A quantificação de porções alimentares incluiu um manual fotográfico especificamente desenvolvido para o efeito (1006 fotos de alimentos e receitas e 11 fotos de medidas caseiras). A classificação e descrição dos alimentos foram realizadas com base no sistema FoodEx2. Esta informação permite caracterizar as dimensões de consumo alimentar e nutricional e de segurança dos alimentos. O módulo “Move” permite a recolha de informação de atividade física e inclui os sub-módulos International Physical Activity Questionnaire (IPAQ), Activity Choice Index (ACI) (≥ 15 anos) e diários de atividade física de 4 dias (2 dias consecutivos e 2 dias de fim de semana) em crianças e adolescentes (6-14 anos), sincronizados com os dados de equivalentes metabólicos associados aos diferentes tipos de atividades. Esta informação permite caracterizar as dimensões de comportamentos sedentários, atividades desportivas e escolhas ativas na rotina diária. A criação de evidência nacional, desagregada por áreas geográficas, para diferentes grupos populacionais (crianças, adolescentes, adultos, idosos), constitui uma base descritiva essencial para o planeamento em saúde. O conhecimento produzido pelo IAN-AF 2015- 2016 permite dar resposta a prioridades estratégicas em saúde, nacionais e internacionais, e constitui uma base sólida para o desenvolvimento de políticas de educação alimentar e de atividade física, bem como de políticas de segurança alimentar, em Portugal e na União Europeia.