National Institute of Health Dr. Ricardo Jorge
Repositório Científico do Instituto Nacional de SaúdeNot a member yet
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Screening and in silico characterization of prophages in Helicobacter pylori clinical strains
No full textThe increase of antibiotic resistance calls for alternatives to control Helicobacter pylori, a Gram-negative bacterium associated with various gastric diseases. Bacteriophages (phages) can be highly effective in the treatment of pathogenic bacteria. Here, we developed a method to identify prophages in H. pylori genomes aiming at their future use in therapy. A polymerase chain reaction (PCR)-based technique tested five primer pairs on 74 clinical H. pylori strains. After the PCR screening, 14 strains most likely to carry prophages were fully sequenced. After that, a more holistic approach was taken by studying the complete genome of the strains. This study allowed us to identify 12 intact prophage sequences, which were then characterized concerning their morphology, virulence, and antibiotic-resistance genes. To understand the variability of prophages, a phylogenetic analysis using the sequences of all H. pylori phages reported to date was performed. Overall, we increased the efficiency of identifying complete prophages to 54.1 %. Genes with homology to potential virulence factors were identified in some new prophages. Phylogenetic analysis revealed a close relationship among H. pylori-phages, although there are phages with different geographical origins. This study provides a deeper understanding of H. pylori-phages, providing valuable insights into their potential use in therapy.Questions answered in this article: - What were the findings regarding the number of prophages identified in the H. pylori genomes? The study identified twelve new prophages in the genomes of Helicobacter pylori, with at least 10 out of the 14 sequenced strains containing at least one intact prophage, indicating a significant presence of these genetic elements in the clinical strains examined; -What percentage of H. pylori strains are compatible with the presence of prophage genes? The analysis of Helicobacter pylori genomes indicates that approximately 20% of strains are compatible with the presence of prophage genes; - What Polymerase Chain Reaction (PCR) techniques were employed to screen for prophages in H. pylori strains? The Polymerase Chain Reaction (PCR) techniques employed to screen for prophages in Helicobacter pylori strains included the use of four different primer sets targeting two regions of the integrase gene, the holin gene, and the insertion site region between specific genes. This approach aimed to enhance the detection of prophages in the clinical strains; What morphological characteristics are commonly associated with H. pylori prophages? H. pylori prophages are commonly associated with a podovirus-like morphology, as indicated by the results obtained with VIRFAM. This morphological characteristic aligns with most reported H. pylori prophages, which exhibit this specific structure; - What challenges exist in the genomic analysis and characterization of H. pylori prophages? The genomic analysis and characterization of Helicobacter pylori prophages face challenges such as the alarming rates of antibiotic resistance, which complicate treatment approaches and necessitate alternative strategies. Additionally, the variability in the genomic size and GC content among different H. pylori strains may pose difficulties in standardizing genomic analyses.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering, and Microelectromechanical Systems, LA/P/0029/2020 and Project Helicophage PTDC/SAU-PUB/29182/2017 [POCI-01-0145-FEDER-029182]. Rute Ferreira acknowledges the FCT grant (https://doi.org/10.54499/SFRH/BD/146496/2019). Luís D. R. Melo acknowledges funding from the FCT through the Scientific Employment Stimulus Program (https://doi.org/10.54499/2021.00221.CEECIND/CP1664/CT0002)
Search for Deletions/Duplications in Genes Associated with Hereditary Cancer by Digital MLPA
No full textDissertação de Mestrado em Genética Clínica Laboratorial apresentada à Faculdade de Medicina da Universidade de Coimbra, 2024Orientador João Gonçalves, Departamento de Genética Humana, INSAProjeto realizado na Unidade de Genética Molecular do Departamento
de Genética Humana, INSA.A presença, na linha germinativa, de Variações do Número de Cópias (CNV) em genes de predisposição para cancro hereditário pode aumentar a suscetibilidade a esta doença. A identificação de uma CNV patogénica ou provavelmente patogénica num doente oncológico tem um impacto significativo na gestão clínica do indivíduo afetado e dos seus familiares. Tradicionalmente, a pesquisa de CNV no diagnóstico molecular de cancro hereditário é realizada apenas para alguns genes através do MLPA (Multiplex Ligation-dependent Probe Amplification) convencional. Nos últimos anos, o desenvolvimento de softwares de análise in silico de CNV com base em dados de NGS (Next-Generation Sequencing) representou um avanço significativo, ao possibilitar a pesquisa de deleções e duplicações em múltiplos genes em simultâneo. No entanto, estas ferramentas apresentam ainda limitações. Dada a relevância de uma análise abrangente que integre o maior número possível de genes relevantes no âmbito da patologia em causa, este trabalho teve como principal objetivo implementar uma nova metodologia de pesquisa de CNV em genes associados a cancro hereditário, que combina os princípios do MLPA convencional com a capacidade da NGS de analisar vários genes em simultâneo: o MLPA digital. Neste estudo, foi realizada a pesquisa de CNV por MLPA digital em amostras de doentes com história clínica e familiar de cancro, seguida de validação dos resultados utilizando outras metodologias de genética molecular e classificação das variantes identificadas segundo as recomendações da CanVIG-UK. O MLPA digital demonstrou ser eficaz na deteção de deleções e duplicações em genes associados a cancro hereditário, apresentando um desempenho adequado para a utilização em laboratórios clínicos, com sensibilidade de 100% e especificidade de 98%. A eficácia dos softwares de pesquisa in silico panelcn.MOPS e DRAGEN Enrichment foi confirmada através da concordância entre os resultados destas ferramentas e do MLPA digital. Foram identificadas cinco variantes patogénicas ou provavelmente patogénicas nos genes APC, BRCA1, BRCA2 e CHEK2, que justificam os fenótipos dos doentes. Este estudo demonstra que o MLPA digital é uma alternativa ao MLPA convencional na primeira fase de pesquisa molecular de CNV germinativas em genes associados a cancro hereditário, permitindo a análise de múltiplos genes em várias amostras em simultâneo.The presence of germline Copy Number Variations (CNV) in hereditary cancer predisposition genes may increase susceptibility to this disease. The identification of a pathogenic or likely pathogenic CNV in a cancer patient has a significant impact on the clinical management of the affected individual and their family members. Traditionally, CNV screening in the molecular diagnosis of hereditary cancer is conducted only for certain genes using conventional MLPA (Multiplex Ligation-dependent Probe Amplification). In recent years, the development of in silico CNV analysis software based on NGS (Next-Generation Sequencing) data has represented a significant advancement, enabling the screening of deletions and duplications in multiple genes simultaneously. However, these tools still have limitations. Given the relevance of a comprehensive analysis that integrates as many relevant genes as possible within the context of the pathology in question, this work aimed to implement a new methodology for CNV screening in genes associated with hereditary cancer, which combines the principles of conventional MLPA with NGS capability to analyze multiple genes simultaneously: digitalMLPA. In this study, CNV screening was performed using digitalMLPA on samples from patients with a clinical and family history of cancer, followed by validation of the results using other molecular genetics methodologies and classification of the identified variants according to CanVIG-UK recommendations. digitalMLPA proved to be effective in detecting deletions and duplications in genes associated with hereditary cancer, showing adequate performance for use in clinical laboratories, with a sensitivity of 100% and a specificity of 98%. The effectiveness of the in silico software panelcn.MOPS and DRAGEN Enrichment was confirmed through concordance between the results of these tools and digitalMLPA. Five pathogenic or likely pathogenic variants were identified in the APC, BRCA1, BRCA2, and CHEK2 genes, which account for the patients' phenotypes. This study demonstrates that digitalMLPA is an alternative to conventional MLPA in the initial phase of molecular screening of germline CNV in genes associated with hereditary cancer, allowing for the analysis of multiple genes in several samples simultaneously
Flavouring Group Evaluation 80, Revision 2 (FGE.80Rev2): Consideration of alicyclic, alicyclic‐fused and aromatic‐fused ring lactones evaluated by the JECFA (61st and 82nd meetings) structurally related to an aromatic lactone evaluated in FGE.27
No full textEFSA Panel on Food Additives and Flavourings (FAF) Members: INSA - Henriqueta Louro, Departamento de Genética Humana, INSA.The EFSA Panel on Food Additives and Flavourings was requested to evaluate 14 flavouring substances assigned to the Flavouring Group Evaluation 80 (FGE.80), using the Procedure as outlined in the Commission Regulation (EC) No 1565/2000. Thirteen substances have already been considered in FGE.80 and its revision and in FGE.96 [FL‐no: 10.005, 10.024, 10.025, 10.050, 10.061, 10.069, 10.070, 10.072, 10.169, 13.009, 13.012, 13.161 and 16.055]. The remaining flavouring substance 3a,4,5,7a‐tetrahydro‐3,6‐dimethylbenzofuran‐2(3H)‐one [FL‐no: 10.057] has been cleared with respect to genotoxicity in FGE.217Rev3 and it is considered in this revision 2 of FGE.80. The substance [FL‐no: 10.057] was evaluated through a stepwise approach that integrates information on the structure–activity relationships, intake from current uses, threshold of toxicological concern (TTC) and available data on metabolism and toxicity. The Panel concluded that [FL‐no: 10.057] does not give rise to safety concerns at its levels of dietary intake, when estimated on the basis of the ‘Maximised Survey‐derived Daily Intake’ (MSDI) approach. Besides the safety assessment of the flavouring substance, the specifications for the material of commerce have also been considered and the information provided was complete for [FL‐no: 10.057]. However, for the flavouring substance [FL‐no: 10.057] in the present revision and for eight substances evaluated in previous revisions, the ‘modified Theoretical Added Maximum Daily Intakes’ (mTAMDIs) values are above the TTC for their structural class (III). For four substances previously evaluated in FGE.80Rev1 and in FGE.96, use levels are still needed to calculate the mTAMDI estimates. Therefore, in total for 13 flavouring substances, data on uses and use levels should be provided to finalise their safety evaluations. For [FL‐no: 10.050, 10.069 and 13.161], information on the composition of stereoisomeric mixtures is needed.info:eu-repo/semantics/publishedVersio
Carcinogenicity of talc and acrylonitrile
No full textIARC Monographs Working Group Members: Henriqueta Louro (Portugal), Departamento de Genética Humana, INSA.In June, 2024, a Working Group of 29 scientists from 13 countries met at the International Agency for Research on Cancer (IARC) in Lyon, France, to finalise their evaluation of the carcinogenicity of talc and acrylonitrile.International Agency for Research on Cancer, Lyon, Franceinfo:eu-repo/semantics/publishedVersio
Predominance of Genotype 5 Hepatitis Delta Virus Infection in a Portuguese Hepatology Unit
No full textThis article belongs to the Special Issue Viral Hepatitis: Prevention, Infection, and Treatment.Hepatitis delta virus (HDV) infection is the most severe form of viral hepatitis. Genotype 1 (HDV-1) is by far the most prevalent in Europe and globally, while HDV-5 predominates in Western Africa. Data about HDV seroprevalence in Portugal are scarce and genotyping studies have not been performed yet. We aimed to assess the seroprevalence and genotypes of HDV in a large cohort of HBsAg-positive patients followed in our Hepatology Unit between 2012 and 2022. The anti-HDV-positive patients were subjected to a cross-sectional analysis, including blood sample collection for HDV RNA testing and genotype determination. In the cohort of HBsAg-positive patients, 57.5% (480/835) were born in African countries and 665/835 (79.6%) had been screened for anti-HDV antibodies. The HDV seroprevalence obtained was 6.5% (43/665). Twenty-one patients (age 41.2 ± 9.9 years; 57.1% male) were included in further molecular analyses. HDV RNA was positive in 8/21 (38.0%) and classified as HDV-5 in 7 patients (6 from Guinea-Bissau and 1 from Cape Verde) and HDV-1 in 1 patient (from Ukraine). In the largest and most comprehensive study performed in Portugal regarding HDV epidemiology to date, seroprevalence and genotype distribution of HDV (with predominance of HDV-5) were strongly influenced by immigration, notably from African countries.info:eu-repo/semantics/publishedVersio
Standard Operating Procedure (SOP) 3: blood sampling - Partnership for the Assessment of the Risks from Chemicals
No full textLead authors: Henriqueta Louro and Maria João Silva, National Institute of Health Dr. Ricardo Jorge (INSA), Lisbon, Portugal.SOP3 Blood Sampling - presentation for PARC Training for Waste Management Survey. Aim of the presentation: Training dedicated to introduce contents of SOP for blood sampling, transport and storage, to ensure its correct implementation during the waste management occupational study. This Standard Operating Procedure (SOP) for blood sampling provides the general procedure for the collection, storage and transfer of human blood samples to be analysed within the e-waste occupational study under HBM4EU and is based on a SOP developed within a previous occupational study.Partnership for the Assessment of Risks from Chemicals (PARC) has received funding from the European Union’s Horizon Europe Research and Innovation Programme under Grant Agreement No. 101057014
Genetic Diversity of Salmonella enterica subsp. enterica Serovar Enteritidis from Human and Non-Human Sources in Portugal
No full text(This article belongs to the Special Issue Novel Insights into Pathogenesis and Antimicrobial Resistance of Salmonellae 2nd Edition)Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the leading causes of foodborne infections associated with broilers and laying hens. Portugal has had the lowest notification rates of salmonellosis in recent years, due to the vaccinations of layer and breeder flocks and strict compliance with biosecurity measures. However, data about the genetic diversity of S. Enteritidis in Portugal are scarce. In this study, 102 S. Enteritidis isolates selected from human (n = 63) and non-human sources (n = 39) were characterized by serotyping, antimicrobial susceptibility, and whole genome sequencing. The S. Enteritidis population was mainly resistant to fluoroquinolones, and a sole isolate showed resistance to extended-spectrum cephalosporins. ST11 was the most frequent sequence type, and three novel STs from human isolates (ST9236, ST4457, and ST9995) were assigned. Several Salmonella pathogenic islands (SPI) and Putative SPI were present in the genomes, namely SPI-1, 2, 3, 4, 5, 9, 10, 12, 13, and 14, C63PI, CS54_island, and 170 virulence genes were identified. The phylogenetic analysis revealed that strains from Portugal are genetically heterogeneous regarding sample type, collection date, and genetic content. This study increases the available data, essential to a better characterization of strains in a global context.This research study was carried out with the support of the ADONIS (Assessing Determinants of the Non-Decreasing Incidence of Salmonella), DISCOVER (Discovering the sources of Salmonella, Campylobacter, VTEC, and antimicrobial resistance), and OH-HARMONY CAP (One Health Harmonization of Protocols for the Detection of Foodborne Pathogens and AMR Determinants) projects, under the scope of the One Health consortium, grant agreement No 773830; and PTDC/CVTCVT/28469/2017 “CIAinVET—Food-producing animals as reservoirs of resistance to Critically Important Antibiotics” financed by the “Fundação para a Ciência e Tecnologia” (FCT). FCT for the Contrato–Programa was to A. Usié (https://doi.org/10.54499/CEECINST/00100/2021/CP2774/CT0001), and the financial support was to Research Units UIDB/05183/2020 (MED-Mediterranean Institute for Agriculture, Environment and Development) and CHANGE (LA/P/0121/2020).info:eu-repo/semantics/publishedVersio
Hepatitis B Virus Genotypes and Subgenotypes Circulating in Infected Residents in a Country with High Vaccination Rate
No full textThis article belongs to the Special Issue Elimination of Viral Hepatitis: Improving Diagnosis, Treatment and Surveillance, 2nd Edition.Despite the availability of a vaccine against hepatitis B virus (HBV), this infection still causes public health problems, particularly in susceptible populations. In Portugal, universal free vaccination started in 1994, and most HBV infections are diagnosed in immigrants from high-prevalence countries. Our aim was to assess the pattern of HBV genotypes/subgenotypes in samples collected between 2017 and 2021 from a convenience sample of 70 infected residents in Portugal. The HBV pol/HBsAg region was amplified and sequenced, allowing the analysis of RT sequences submitted to phylogenetic analysis and mutations assessment. A total of 37.1% of samples were from native Portuguese, aged 25–53 years (mean: 36.7 years), and the remaining samples were from individuals born outside of Portugal. A high diversity of HBV was identified: subgenotypes A1–A3 in 41.0% (16/39); D1, D3, and D4 in 30.7% (12/39); E in 23.1% (9/39); and F4 in 2.6% (1/39). Besides genotypes A and D, Portuguese were also infected with genotypes E and F, which are prevalent in Africa and South America, respectively. Resistance mutations in RT sequences were not found. The findings provide valuable insights for updating the HBV molecular epidemiology in Portugal. However, successful strategies to prevent and control the infection are still needed in the country, especially among susceptible and vulnerable populations.info:eu-repo/semantics/publishedVersio
16.ª Reunião Anual PortFIR - Sistemas Alimentares, Solo, Composição de Alimentos e Saúde: Resumo da reunião
No full textResumo da 16.ª Reunião Anual PortFIR subordinada ao tema “Sistemas Alimentares, Solo, Composição de Alimentos e Saúde”. A publicação apresenta as comunicações e abstracts/posters submetidos, bem como os resultados da avaliação ao grau de satisfação dos participantes no evento.info:eu-repo/semantics/draf
Mucopolysaccharidosis in the pediatric population: results of an early diagnosis approach in the scope of the FIND project
Get PDFAs Mucopolissacaridoses (MPSs), constituem um subgrupo das Doenças Lisossomais de Sobrecarga, causadas por deficiências em enzimas lisos somais, que catalisam a degradação dos glicosaminoglicanos. As MPSs têm apresentação multissistémica, heterogénea e consequentemente de diagnóstico difícil. O projeto FIND tem como objetivo alertar os clínicos para sinais e sintomas de risco ao mesmo tempo que disponibiliza uma ferramenta de diagnóstico.
Este estudo pretende descrever uma abordagem desenvolvida no âmbito do projeto FIND para identificar doentes com mucopolissacaridoses (MPSs) em idade pediátrica, antes do aparecimento dos sintomas mais graves. A identificação atempada permitirá uma intervenção terapêutica precoce, assim como oferecer aconselhamento genético às famílias afetadas.
O projeto FIND permitiu a identificação de 12 doentes de MPSs e a sua refe renciação para os respetivos centros de tratamento.
Este estudo, para além da sua vertente educativa, coloca à disposição dos clínicos um ótimo meio para a identificação e caraterização de casos sinto máticos de MPS em idade pediátrica.Mucopolysaccharidoses are a subgroup of Lysosomal Storage Disorders
characterized by the accumulation of glycosaminoglycans in cells, tissues,
and organs due to specific enzyme deficiencies. In early disease stages,
diagnosis can be postponed for years or even missed in late onset forms.
This study aims to describe an approach developed within the scope of
the FIND project to identify patients with mucopolysaccharidoses (MPSs)
a in pediatric age, before the appearance of the most severe symptoms, allowing early therapeutic intervention, as well as offering genetic
counseling to affected families.
The FIND project was designed to claim awareness to the red flags of MPS
at pediatric age and to provide a tool for physicians to diagnose these
pathologies, since most of them are amenable to enzyme replacement
therapy. The clinical suspicion was addressed by performing seven distinct
enzymatic assays in dried blood spots. In the cases of a deficiency, the
quantification and identification of the glycosaminoglycans were performed
and the genetic study carried out.
In the first eight years of the project, we have identified twelve patients
among the 385 samples received. FIND project allows an early identifica tion and characterization of symptomatic MPSs patient, as well as other related pathologies at pediatric age.info:eu-repo/semantics/publishedVersio