The Technology and Innovation Unit of the National Institute of Health: A sequencing and bioinformatics core facility specializing in public health genomics

The National Institute of Health (INSA) has a long tradition in investigating the molecular etiology of genetic and complex diseases. These activities greatly benefit from centralized sequencing services provided by the Technology and Innovation Unit (UTI). Its mission is to perform sequencing and genotyping assays in the framework of research, diagnosis and epidemiological surveillance, as well as to implement data analysis pipelines for the study of human gene variants. The equipment portfolio includes a NextSeq 550, a MiSeq, two 3500 AB Genetic Analyzers, a Fragment Analyzer and a 7500 Real-time PCR system. UTI provides results for average of 36.000 sequencing/fragment samples per year. The team has already performed >300 small genome, amplicon, gene panel (including clinical exome), 16S rRNA gene and RNA/microRNA next-generation sequencing assays for INSA and for several Universities in the scope of scientific collaborations. Technical procedures are conducted under a quality control system that includes external quality assessment for next-generation sequencing/Sanger sequencing and ISO 15189 accreditation for Sanger sequencing. UTI plays a key role in public health genomics, providing state-of-the-art equipment, centralized resources, technical expertise and short response times.This work was supported by Centre for Toxicogenomics and Human Health - UID/BIM/00009/2019 - and GenomePT project – POCI-01-0145-FEDER-022184info:eu-repo/semantics/publishedVersio

Clostridium difficile: diversidade genética e perfis de suscetibilidade aos antimicrobianos

Este trabalho teve como objetivo estudar a variabilidade genética e o perfil de suscetibilidade aos antimicrobianos de estirpes de C. difficile recebidas no Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA) entre julho de 2012 e dezembro de 2014

Portuguese Diaspora: Life Stories / Documentaries

The return is a change that is configured at various levels - relationships , routines , roles - and can interfere with the self - concept and the concept that individuals have about the other . (Schlossberg , Waters & Goodman , 1995).The mobility in this case the return is a transition and / or a crisis in that the individual initiates an adaptation process . This process is influenced by several variables , including the subjective perception regarding the event and the different characteristics of the contexts of exit and reception and the characteristics of the actors in this event. (Pine and Ferreira , 2002; Evans & Forney , Guido DiBrito , 1998). If migrations occur regularly , this process can not interfere with the return movement of the Portuguese diaspora since decolonization that constituted for the Portuguese a transition not expected and , but configuring contexts breakthrough.The intention is to understand the contexts of life of the people involved in the process ( rupture, integration , opportunities , etc. ) through the documentaries made for the purpose

Overview of Clostridium difficile Infection: Life Cycle, Epidemiology, Antimicrobial Resistance and Treatment

The use of antimicrobial agents and acquired resistances explains in part the emergence and spreading of epidemic strains of Clostridium difficile. Continued use of antimicrobial therapy still represents an acute danger in triggering the emergence and spreading of new resistant and multiresistant strains including against first-line antibiotics. We examine the pathway of peptidoglycan synthesis in this organism and associated resistances, as well as resistance to other classes of antibiotics. The life cycle of C. difficile involves growth, spore formation and germination. Spores endow the organism with a formidable capacity of persistence in the environment and in the host, resistance, dissemination and infectious potential. Highly resistant spores produced by antibiotic-resistant/multiresistant strains may be one of the most serious challenges we face in what concerns the containment of C. difficile. Finally, we review recent developments in the treatment and prevention of C. difficile infection

Acesso aos Cuidados de Saúde A Saúde como Pilar de Desenvolvimento Regional NUTE III Alto Alentejo

Dissertação de Mestrado em Gestão e Políticas PúblicasO Alto Alentejo é uma região caracterizada pelas altas taxas de desemprego, fraco desenvolvimento regional, entre outros. O setor da saúde assume-se particularmente como um sector mais frágil e pouco desenvolvido e, portanto, mais ‘’sensível’’ de analisar. É neste sentido que surge a presente investigação ‘’Acesso aos Cuidados de Saúde: Saúde como pilar de Desenvolvimento Regional NUTE III Alto Alentejo’’. Urge então refletir mais profundamente e detalhadamente acerca deste sector, na medida em que o cenário atual não é o que melhor responde às necessidades da população local, além dos recursos humanos de que dispõem serem bastante reduzidos, o que limita qualidade dos serviços bem como o desenvolvimento regional e vice-versa. Foram múltiplas as políticas implementadas por forma a promover o acesso aos cuidados de saúde e consecutivamente o desenvolvimento regional. Desde à menção do artigo 64º da Constituição da República Portuguesa, criação de Lei de Bases da Saúde, criação do SNS, bem como das políticas que criaram entidades regionais como a ARSA e ULNSA, E.P.E. para aproximar os serviços de cuidados de saúde da população. Contudo, não foram suficientes para diminuir as desigualdades de acesso aos cuidados de saúde nem para atenuar as diferenças relativas ao desenvolvimento regional. Esta investigação pretende assim contribuir, através de uma discussão aprofundada, a implementação de medidas no setor. A metodologia adotada é essencialmente qualitativa, fundamentada em documentos secundários, bases de dados nacionais e entrevistas semiestruturadas. O presente trabalho tornou-se um caso de estudo da Região do Alto Alentejo. Diversos autores, como adiante veremos, afirmam que são várias as barreiras ao acesso aos serviços de saúde e ao desenvolvimento regional, não só os recursos financeiros como também a educação, distâncias geográficas e a oferta de serviços muitas vezes não correspondente às necessidades dos cidadãos.Alto Alentejo is characterized by its unemployment, difficult access to entities that provide services to citizens, by its weak regional development, among others. In this sense, it arises within the scope of “Access to Health Care; Health as a pillar of Regional Development NUTE III Alto Alentejo ”. The health sector is a “sensitive” sector to explore and it is necessary to do so as the current scenario is not the one that best meets the needs of the local population, the human resources available to do so are reduced, limiting the quality of services as well as regional development and vice versa. There were multiple policies implemented in order to promote access to health care and consecutively regional development. Since the mention of article 64 of the Constitution of the Portuguese Republic, creation of the Basic Health Law, creation of the NHS, as well as the policies that created regional entities such as ARSA and ULNSA, E.P.E. to bring health care services closer to the population. However, they were not enough to reduce inequalities in access to health care or to mitigate differences in regional development, as we shall see. This investigation thus intends to contribute to the decision when implementing measures in the sector. The adopted methodology is essentially qualitative based on secondary documents, national databases and semi-structured interviews. The present work has become a case study in the Alto Alentejo Region. Several authors, as we will see later, affirm that there are several barriers to access to health services and regional development, not only financial resources but also education, geographical distances and the offer of services often not corresponding to the needs of citizens.N/

Portuguese Diaspora: Life Stories / Documentaries

Abstract The return is a change that is configured at various levels -relationships , routines , roles -and can interfere with the selfconcept and the concept that individuals have about the other . (Schlossberg , Waters & Goodman , 1995).The mobility in this case the return is a transition and / or a crisis in that the individual initiates an adaptation process . This process is influenced by several variables , including the subjective perception regarding the event and the different characteristics of the contexts of exit and reception and the characteristics of the actors in this event. (Pine and through the documentaries made for the purpose

Genomic epidemiology and phenotyping reveal on-farm persistence and cold adaptation of raw milk outbreak-associated Yersinia pseudotuberculosis

Packaged raw milk contaminated with Yersinia pseudotuberculosis mediated a large yersiniosis outbreak in southern Finland in 2014. The outbreak was traced back to a single dairy farm in southern Finland. Here we explore risk factors leading to the outbreak through epidemiologic investigation of the outbreak farm and through genomic and phenotypic characterization of the farm’s outbreak and non-outbreak associated Y. pseudotuberculosis strains. We show that the outbreak strain persisted on the farm throughout the 7-month study, whereas the non-outbreak strains occurred sporadically. Phylogenomic analysis illustrated that the outbreak strain was related to previously published genomes of wild animal isolates from Finland, implying that wild animals were a potential source of the outbreak strain to the farm. We observed allelic differences between the farm’s outbreak and non-outbreak strains in several genes associated with virulence, stress response and biofilm formation, and found that the outbreak strain formed biofilm in vitro and maintained better growth fitness during cold stress than the non-outbreak strains. Finally, we demonstrate the rapid growth of the outbreak strain in packaged raw milk during refrigerated storage. This study provides insight of the risk factors leading to the Y. pseudotuberculosis outbreak, highlights the importance of pest control to avoid the spread of pathogens from wild to domestic animals, and demonstrates that the cold chain is insufficient as the sole risk management strategy to control Y. pseudotuberculosis risk associated with raw drinking milk

New era on the surveillance of multidrug resistant tuberculosis in Portugal: whole genome sequencing

A tuberculose multirresistente continua a ser um dos principais desafios no controlo da Tuberculose em Portugal. Os métodos de diagnóstico e vigilância clássicos são trabalhosos, muito demorados e nem sempre fáceis de interpretar. A introdução de metodologias moleculares permitiu ultrapassar alguns destes obstáculos, mas, sendo limitada no número de alvos genéticos que analisa, não permite uma análise completa das características das estirpes de M. tuberculosis isoladas. Para ultrapassar esta questão, tivemos como objectivo a implementação de uma metodologia baseada na sequenciação total do genoma que, para além de permitir um screening de todas as mutações conhecidas associadas a resistência, permite fazer uma vigilância molecular das estirpes com uma sensibilidade muito elevada, possibilitando a inter venção das Autoridades de Saúde de forma atempada e otimizada. O Laboratório Nacional de Referência de Micobactérias/Tuberculose do Instituto Nacional de Doutor Ricardo Jorge está, neste momento, capacitado para responder eficazmente a estas necessidades garantindo um dignóstico e vigilância em “tempo real” de todas as estirpes de M. tuberculosis multirresistentes.Multidrug-resistant tuberculosis continues to be one of the main challenges for Tuberculosis´ control in Por tugal. Classical diagnostic and sur veillance methods are laborious, time-consuming and not always easy to interpret and per form. The introduction of molecular methodologies overcame some of these obstacles, but, as they are limited in the number of genetic targets analy zed, they do not allow a complete analysis of the characteristics of the isolated M. tuberculosis strains. As such, we aimed to implement a methodology based on whole genome sequencing that, in addition to enable the screening of all known mutations associated with resistance, it makes it possible to carr y out molecular sur veillance of the strains with a ver y high sensitivity, allowing a timely and optimized intervention of the Health Authorities. The National Reference Laborator y of Mycobacteria / Tuberculosis is now capable of responding ef fectively to these needs, ensuring a "real time" diagnosis and sur veillance of all multidrug resistant M. tuberculosis strains.Este trabalho foi financiado pela Fundação para a Ciência e a Tecnologia (FCT) no âmbito do projeto Centro de Toxicogenómica e Saúde Humana (UID/BIM/0009/2016) e desenvolvido no âmbito do projeto GenomaPT (POCI-01-0145-FEDER-022184), cofinanciado pelo Programa Operacional Competitividade e Internacionalização (Compete2020), Programa Operacional Regional de Lisboa (Lisboa 2020) e Programa Operacional Regional do Algarve (CRESC Algarve2020), através do Portugal 2020 e do Fundo Europeu de Desenvolvimento Regional (FEDER), e pela FCT.info:eu-repo/semantics/publishedVersio

Genomic Epidemiology and Phenotyping Reveal on-Farm Persistence and Cold Adaptation of Raw Milk Outbreak-Associated Yersinia pseudotuberculosis

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/31156582/Packaged raw milk contaminated with Yersinia pseudotuberculosis mediated a large yersiniosis outbreak in southern Finland in 2014. The outbreak was traced back to a single dairy farm in southern Finland. Here we explore risk factors leading to the outbreak through epidemiologic investigation of the outbreak farm and through genomic and phenotypic characterization of the farm's outbreak and non-outbreak associated Y. pseudotuberculosis strains. We show that the outbreak strain persisted on the farm throughout the 7-month study, whereas the non-outbreak strains occurred sporadically. Phylogenomic analysis illustrated that the outbreak strain was related to previously published genomes of wild animal isolates from Finland, implying that wild animals were a potential source of the outbreak strain to the farm. We observed allelic differences between the farm's outbreak and non-outbreak strains in several genes associated with virulence, stress response and biofilm formation, and found that the outbreak strain formed biofilm in vitro and maintained better growth fitness during cold stress than the non-outbreak strains. Finally, we demonstrate the rapid growth of the outbreak strain in packaged raw milk during refrigerated storage. This study provides insight of the risk factors leading to the Y. pseudotuberculosis outbreak, highlights the importance of pest control to avoid the spread of pathogens from wild to domestic animals, and demonstrates that the cold chain is insufficient as the sole risk management strategy to control Y. pseudotuberculosis risk associated with raw drinking milk.This research was supported by the Finnish Ministry of Agriculture and Forestry grant Dnro: 1395/311/214, the Walter Ehrström Foundation, and the University of Helsinki Doctoral Program in Food Chain and Health. The sequencing of the strains and the salary of AJ and JI were supported by the INNUENDO project (https://sites.google.com/site/theinnuendoproject/) which received funding from the European Food Safety Authority, grant agreement GP/EFSA/AFSCO/2015/01/CT2 (“New approaches in identifying and characterizing microbial and chemical hazards”).info:eu-repo/semantics/publishedVersio

Multidrug-Resistant Salmonella enterica Serovar Rissen Clusters Detected in Azores Archipelago, Portugal

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/31950026/Gastrointestinal infections caused by nontyphoidal Salmonella (NTS) remain one of the main causes of foodborne illness worldwide. Within the multiple existing Salmonella enterica serovars, the serovar Rissen is rarely reported, particularly as a cause of human salmonellosis. Between 2015 and 2017, the Portuguese National Reference Laboratory of Gastrointestinal Infections observed an increase in the number of clinical cases caused by multidrug-resistant (MDR) S. enterica serovar Rissen, particularly from the Azores archipelago. In the present study, we analyzed by whole genome sequencing (WGS) all clinical, animal, food, and environmental isolates received up to 2017 in the Portuguese Reference Laboratories. As such, through a wgMLST-based gene-by-gene analysis, we aimed to identify potential epidemiological clusters linking clinical and samples from multiple sources, while gaining insight into the genetic diversity of S. enterica serovar Rissen. We also investigated the genetic basis driving the observed multidrug resistance. By integrating 60 novel genomes with all publicly available serovar Rissen genomes, we observed a low degree of genetic diversity within this serovar. Nevertheless, the majority of Portuguese isolates showed high degree of genetic relatedness and a potential link to pork production. An in-depth analysis of these isolates revealed the existence of two major clusters from the Azores archipelago composed of MDR isolates, most of which were resistant to at least five antimicrobials. Considering the well-known spread of MDR between gastrointestinal bacteria, the identification of MDR circulating clones should constitute an alert to public health authorities. Finally, this study constitutes the starting point for the implementation of the "One Health" approach for Salmonella surveillance in Portugal.This work was partially funded by the GenomePT project (POCI-01-0145- FEDER-022184), supported by COMPETE 2020–Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), and Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and by the Fundação para a Ciência e a Tecnologia (FCT)info:eu-repo/semantics/publishedVersio