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Background: Information on vaccine effectiveness in a context of novel variants of concern (VOC) emergence is of key importance to inform public health policies. This study aimed to estimate a measure of comparative vaccine effectiveness between Omicron (BA.1) and Delta (B.1.617.2 and sub-lineages) VOC according to vaccination exposure (primary or booster). Methods: We developed a case–case study using data on RT-PCR SARS-CoV2-positive cases notified in Portugal during Weeks 49–51, 2021. To obtain measure of comparative vaccine effectiveness, we compared the odds of vaccination in Omicron cases versus Delta using logistic regression adjusted for age group, sex, region, week of diagnosis, and laboratory of origin. Results: Higher odds of vaccination were observed in cases infected by Omicron VOC compared with Delta VOC cases for both complete primary vaccination (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.8 to 2.4) and booster dose (OR = 5.2; 95% CI: 3.1 to 8.8), equivalent to reduction of vaccine effectiveness from 44.7% and 92.8%, observed against infection with Delta, to 6.0% (95% CI: 29.2% to 12.7%) and 62.7% (95% CI: 35.7% to 77.9%), observed against infection with Omicron, for complete primary vaccination and booster dose, respectively. Conclusion: Consistent reduction in vaccine-induced protection against infection with Omicron was observed. Complete primary vaccination may not be protective against SARS-CoV-2 infection in regions where Omicron variant is dominant. Grant no. 2021/PHF/23776; POCI-01-0145-FEDER-022184; Project ALG-D2-2021-06 info:eu-repo/semantics/publishedVersion

We propose a novel, non-discriminatory classification of monkeypox virus. Together with the World Health Organization, we named three clades (I, IIa and IIb) in order of detection. Within IIb, the cause the current global outbreak, we identified multiple lineages (A.1, A.2, A.1.1 and B.1) to support real-time genomics surveillance.

A Unidade de Tecnologia e Inovação (UTI) do Departamento de Genética Humana foi criada em 2009 pelo despacho normativo n.º 15/2009. Apesar de estar integrada num departamento técnico científico, esta unidade constituiu-se desde logo como core-facility de sequenciação genómica do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA). Este papel envolve uma gestão contínua de prioridades dos serviços a prestar aos utilizadores, no âmbito da resposta a diferentes problemas de saúde pública, aliada a uma preocupação permanente com a qualidade dos resultados e os tempos de resposta. Neste trabalho, apresentamos os resultados da avaliação do desempenho da UTI, desde a introdução da tecnologia de Next-Generation Sequencing (NGS) em 2013, em termos de: (i) métricas de produção da Unidade, (ii) impacto dos resultados publicados no âmbito de colaborações científicas com os grupos de investigação do INSA ou de entidades externas e de (iii) avaliação dos serviços através de um inquérito dirigido aos utilizadores. Até final de 2021, o número de ensaios de NGS e de citações dos trabalhos publicados cresceram, por ano, 39% e 61%, respetivamente. Os utilizadores avaliaram de forma muito positiva os serviços prestados pela UTI em 2021. Globalmente, estes resultados demonstram que o modelo de trabalho de "core- -facility" exercido pela UTI é uma mais-valia na resposta aos problemas da saúde pública em Portugal. The Technology and Innovation Unit (UTI) of the Department of Human Genetics was established in 2009 by the normative order no. 15/2009. Despite being integrated in a technical and scientific department, this unit was from the beginning constituted as the genomic sequencing core-facility of the National Institute of Health Doutor Ricardo Jorge (INSA). This role involves continuous management of service priorities to be provided to users, within the scope of responding to different public health problems, combined with a permanent concern with the quality of data and response time. In this work, we present the results of the UTI performance evaluation since the introduction of the Next-Generation Sequencing (NGS) technology in 2013, in terms of: (i) Unit production metrics, (ii) impact of the results published in the scope of scientific collaborations with INSA research groups or external entities, and (iii) evaluation of services through a survey aimed at users. By the end of 2021, the number of NGS assays and citations of published works grew by 39% and 61% annually, respectively. Users evaluated very positively the services provided by the UTI in 2021. Overall, these results demonstrate that the "core-facility" work model exercised by the UTI is an asset in responding to public health problems in Portugal. Este trabalho foi desenvolvido no âmbito do projeto GenomePT (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 Fundação para a Ciência e a Tecnologia (FCT). Este trabalho foi também suportado por Fundos FEDER através do Programa Operacional Fatores de Competitividade – COMPETE e por Fundos Nacionais através da Fundação para a Ciência e a Tecnologia no âmbito do projeto UID/BIM/00009/2019 (Centro de Toxicogenómica e Saúde Humana – ToxOmics) info:eu-repo/semantics/publishedVersion

JIP06-COVID19-COVRIN-WP2-T2.1 Genome analyses JIP06-COVID19-COVRIN-WP2-ST2.1.2 Harmonisation sampling and methods NGS DeliverableD-COVRIN.2.1.2 - Report on the comparisons of variants and reference sample exchange ______________________________________________________________________ T2.1 leader: Vítor Borges (INSA, 36) ST2.1.2 leader: Gabriele Vaccari (ISS, 27) Task participants: All partners Partners participating in Bioinformatics Ring Trial:INSA (36), ISS (27), ANSES (01), APHA (21), FLI (10), INIAV (35), IZSAM (28), Sciensano (04) _______________________________________________________________________ This deliverable describes the activities and outcomes of the COVRIN WP2-T2.1 sub-task ST2.1.2 (Harmonisation sampling and methods NGS), in which aBioinformatics Ring Trial was carried out in order to assess the reproducibility of bioinformatics pipelines (i.e., from raw reads to consensus sequences, using reference-based mapping) currently being applied for genome analyses of SARS-CoV-2 (and other coronaviruses - CoV) in the partners institutes.

Funding: This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. This work is also a result of 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), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). This work was also supported by Fundos FEDER through the ProgramaOperacionalFactores de Competitividade–COMPETE and by FundosNacionais through the Fundação para a Ciência e a Tecnologia within the scope of the project UID/BIM/00009/2019 (Centre for Toxicogenomics and Human Health -ToxOmics). Dissemination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in healthcare institutions affects both patients and health-care workers (HCW), as well as the institutional capacity to provide essential health services. Here, we investigated an outbreak of SARS-CoV-2 in a "non-COVID-19" hospital ward unveiled by massive testing, which challenged the reconstruction of transmission chains. The contacts network during the 15-day period before the screening was investigated, and positive SARS-CoV-2 RNA samples were subjected to virus genome sequencing. Of the 245 tested individuals, 48 (21 patients and 27 HCWs) tested positive for SARS-CoV-2. HCWs were mostly asymptomatic, but the mortality among patients reached 57.1% (12/21). Phylogenetic reconstruction revealed that all cases were part of the same transmission chain. By combining contact tracing and genomic data, including analysis of emerging minor variants, we unveiled a scenario of silent SARS-CoV-2 dissemination, mostly driven by the close contact within the HCWs group and between HCWs and patients. This investigation triggered enhanced prevention and control measures, leading to more timely detection and containment of novel outbreaks. This study shows the benefit of combining genomic and epidemiological data for disclosing complex nosocomial outbreaks, and provides valuable data to prevent transmission of COVID-19 in healthcare facilities. publishersversion published

Os autores deste artigo dirigem um especial agradecimento ao consórcio Portuguese network for SARS-CoV-2 genomics, o qual engloba todos os laboratórios a nível nacional que enviaram amostras para caracterização genética do SARS-CoV-2, a equipa do INSA envolvida no diagnóstico laboratorial da COVID-19, a equipa da Unidade de Genómica do Instituto de Gulbenkian Ciência e colaboradores no campo da bioinformática (Hugo Martiniano e Miguel Pinheiro). Agradece-se, ainda, de forma particular, às autoridades de saúde nacionais, regionais e locais pela partilha de dados demográficos e epidemiológicos. Os primeiros casos de COVID-19 em Portugal foram reportados no início de março, verificando-se rapidamente uma subida exponencial dos mesmos até ao início de abril. Neste âmbito, o Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA) iniciou o estudo da variabilidade genética do SARS-CoV-2 no país, criando um consórcio constituído por mais de 60 laboratórios, os quais possibilitaram a recolha de cerca de 3000 amostras positivas, de 159 concelhos. Nesta fase sequenciaram-se cerca de 1800 genomas de SARS-CoV-2, colocando Portugal como um dos países a nível mundial com maior empenho na caracterização das variantes genéticas do novo coronavírus em circulação. Os resultados obtidos possibilitaram a caracterização do início da epidemia em Portugal, permitindo, de alguma forma, avaliar a adequação de medidas de saúde pública que foram tomadas. O presente artigo debruça-se sobre os principais resultados obtidos neste âmbito, enumerando também uma série de estudos genéticos mais específicos que foram sendo realizados pelo INSA e com relevância em saúde pública. The first cases of COVID-19 in Portugal were reported at the beginning of March, with an exponential increase rapidly occurring until the beginning of April. In this context, the Portuguese National Institute of Health (INSA) initiated the study of SARS-CoV-2 genetic variability in the country, creating a consortium of more than 60 laboratories, which made it possible to collect about 3000 positive samples from 159 counties. Currently, around 1800 SARS-CoV-2 genomes have been sequenced, placing Portugal as one of the countries worldwide with the greatest commitment to characterize the genetic variants of the new coronavirus in circulation. The results obtained enabled the characterization of the epidemic start in Portugal, and assess the adequacy of some public health measures. Although this article essentially focuses on results obtained pursuing this objective, it also lists a series of more specific genetic studies with Public Health relevance that have been carried out by INSA. Este estudo é cofinanciado pela Fundação para a Ciência e Tecnologia e pela Agência de Investigação Clínica e Inovação Biomédica (234_596874175) no âmbito da call RESEARCH 4 COVID-19. info:eu-repo/semantics/publishedVersion

Chlamydia trachomatis is the most prevalent sexually transmitted bacterium worldwide and the causative agent of trachoma. Its strains are classified according to their ompA genotypes, which are strongly linked to differential tissue tropism and disease outcomes [ocular disease, urogenital disease and lymphogranuloma venereum (LGV)]. While the genome-based species phylogenetic tree presents four main clades correlating with tropism/prevalence, namely ocular, LGV, urogenital T1 (more prevalent genotypes) and urogenital T2 (less prevalent genotypes), inter-clade exchange of ompA is considered a rare phenomenon probably mediating marked tropism alterations. An LGV epidemic, associated with the clonal expansion of the L2b genotype, has emerged in the last few decades, raising concerns particularly due to its atypical clinical presentation (ulcerative proctitis) and circulation among men who have sex with men (MSM). N/A

The National Institute of Health (INSA) is the state laboratory in the health sector. INSA has a long tradition in investigating the molecular etiology of genetic and complex diseases and in the identification of pathogenic organisms responsible for disease outbreaks and environmental imbalances. These activities benefit greatly from the existing centralized sequencing services provided by the Technology and Innovation Unit (UTI). Its mission includes performing sequencing and genotyping assays in the framework of research, diagnosis and epidemiological surveillance, as well as implementing data analysis pipelines for the study of variation in human genes. The equipment portfolio includes two next-generation sequencers and two capillary electrophoresis instruments for Sanger sequencing/fragment analysis, that altogether process an average of 36.000 samples/year. The team performed over 300 next-generation sequencing workflows for small genomes, amplicons, gene panels, clinical exome, 16S rRNA gene and RNA/microRNAs. Standard of operation procedures are conducted by trained technicians under a quality control system that includes external quality assessment and ISO 15189 accreditation. UTI plays a key role in public health genomics, providing state-of-the-art equipment, centralized resources, technical expertise and short response times for public health problems. This work was supported by Centre for Toxicogenomics and Human Health - UID/BIM/00009/2019 - and GenomePT project – POCI-01-0145-FEDER-022184. info:eu-repo/semantics/publishedVersion

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/publishedVersion

A infeção por Clostridium difficile é a principal causa de diarreia infeciosa associada aos cuidados de saúde. Neste estudo, caracterizámos um conjunto de estirpes clínicas de Clostridium difficile, provenientes de diversos hospitais portugueses, com o objetivo de estudar a resistência aos carbapenemos neste agente patogénico. Um total de 191 estirpes clínicas, isoladas entre 2012 e 2015 de 15 hospitais em Portugal, foram incluídas no estudo; a suscetibilidade ao imipenemo foi determinada por um método de gradiente de difusão em agar. Foram selecionadas estirpes sensíveis e resistentes ao imipenemo, para estudos fenotípicos adicionais e para contributo da sequenciação do genoma completo. A resistência ao imipenemo foi detetada em 24 (12,6%) das estirpes, 22 das quais pertencentes ao ribotipo (RT) 017 (apenas toxina B positivo), todas provenientes do mesmo hospital, durante o período em estudo, e com perfil de multiresistência. Pela análise dos dados de sequenciação dos genomas, foram identificadas duas substituições de aminoácidos (Ala555Thr e Tyr721Ser) nos domínios funcionais de duas enzimas envolvidas na síntese do peptidoglicano (penicillin-binding proteins - PBP). Uma PBP adicional foi também identificada nas estirpes RT017. Este estudo descreve pela primeira vez alterações em PBPs como base genética provável da resistência ao imipenemo em C. difficile. Clostridium dif ficile is a major cause of healthcare-associated infections. Here, we characterized C. dif ficile strains isolated in Por tuguese hospitals, in order to search for impenem resistance and the underlying genetic determinants. Imipenem susceptibility testing by agar gradient dif fusion was per formed on 191 C. dif ficile strains, isolated from 15 portuguese hospitals, between 2012-2015. Some of the imipenem-resistant and imipenem-susceptible strains were selected for downstream phenotypic analyses and for whole genome sequencing (WGS). Resistance to imipenem was detected in 24 (12.6 %) strains, 22 of which were ribotype (RT) 017 strains, only positive for toxin B, isolated in the same hospital, and presenting resistance to several other antibiotics. Through analysis of WGS data, two amino acid changes (Ala555Thr and Tyr721Ser) targeting the transpeptidase domain of two penicillin-binding proteins (PBP) were identified. An additional PBP was also identified in this ribotype. We describe, for the first time, mutations in PBP-encoding genes as the probable genetic basis for C. dif ficile imipenem resistance. Este trabalho foi financiado pelo INSA (projeto 2016DDI1284) e pela Fundação para a Ciência e Tecnologia (bolsa de investigação no âmbito do projeto Pest-C/EQB/LA0006/2011; programa IF IF/00268/2013/CP1173/CT0006, a MS; bolsa de doutoramento PD/BD/105738/2014, a ALM). info:eu-repo/semantics/publishedVersion

Resumen del trabajo presentado a la 9th International Conference on Tularemia, celebrada en Montréal (Canada) del 16 al 19 de octubre de 2018., [Background and aim]: Tularemia is caused by the gram-negative intracellular pathogen Francisella tularensis. In Europe, the region of Castilla y León, Northwest Spain, is a major hotspot for tularemia, where the largest outbreaks of the disease have been recently reported. While rodents and lagomorphs are recognised as the main mammalian hosts in Europe, the common voles (Microtus arvalis) are documented key agents for human tularemia in Northwestern Spain, as evidenced by a spatial and temporal coincidence between human cases and increases in vole abundance. This study aimed to perform comparative genomics of F. tularensis isolates from tissue samples of F. tularensis positive voles with 11 human isolates from 2014, when an increased number of human cases was observed in the same area of Spain. [Material and Methods]: For this study, we selected thirty-four trapped voles, sampled in 80 km2 of farmland in Palencia Province, Spain (42°1′N, 4°42′W), that tested Ft-positive, by conventional PCR and hybridization by reverse line blotting (targeting lpnA), and the multi-target TaqMan PCR, tul4 and ISFtu2 assays. Tissues (liver, spleen and lung) from each animal were minced, inoculated in chocolate agar PolyViteXTM at 37°C in 5% CO2, and observed at 24, 48 and 72h post-inoculation. So far, after DNA extraction, four isolates, showing typical Ft growth, were subjected to paired-end whole-genome sequencing in an Illumina MiSeq apparatus, followed by genome assembly and bioinformatics analysis. [Results and conclusions]: All trapped voles tested positive for F. tularensis subsp. holarctica. A preliminary core-genome SNPs-based analysis, representing >99% of the genome, showed that all isolates (N=15) were distinguishable by 33 single nucleotide variant sites, representing two clear phylogenetic clusters. Noteworthy, four newly sequenced vole-isolated strains segregated together with five human-associated strains from 2014, suggesting the potential persistence of this strain in this region.