Browsing by Author "Duarte, Sílvia"
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- Accreditation under the International Standard ISO 15189: Experience of a Genetics Laboratory in DNA SequencingPublication . Silva, Catarina; Cardoso, Ana; A Sampaio, Daniel; Carpinteiro, Dina; Mendonça, Joana; Duarte, Sílvia; Barreiro, Paula; Torgal, Helena; Isidro, Glória; Vieira, LuísIntroduction: Health care is to some extent influenced by the results of laboratory tests. In order to provide the best care for the patient, laboratories must seek to achieve high levels of quality and competence. International Standard ISO 15189 specifies these requirements and may be used by laboratories to perform accredited genetic tests of materials derived from the human body. Here we describe the procedures to establish Accreditation of DNA sequencing in our laboratory and the first Accreditation of its kind in Portugal. Methods: Our laboratory started to prepare to comply with ISO 15189 Accreditation requirements for DNA sequencing in 2010. Documents describing administrative and technical procedures of the sequencing workflow including sample registries, laboratory protocols, operation and maintenance of equipments, as well as preparation and use of reagents were produced. Regular examination of laboratory equipments by an external entity was implemented to confirm compliance with working requirements. Requisites for personnel training and demonstration of competence were also implemented. The laboratory participated regularly in the DNA sequencing scheme organized by the European Molecular Genetics Quality Network (EMQN). Results: The laboratory obtained formal recognition by Instituto Português de Acreditação (IPAC) in May 2014. A maximum genotyping score for DNA sequencing has been obtained in the external quality assessment scheme since 2010. Sequencing quality measured in terms of the quality read overlap metrics is currently of approximately 96% according to the EMQN scheme. The laboratory processes and analyzes an average of 28.750 samples per year. Discussion: Accreditation of a genetic test under ISO 15189 is a highly demanding and laborious task for a genetic laboratory. However, it is an important step in order to guarantee the highest quality and reproducibility of genetic test results.
- Avaliação do desempenho de uma core-facility de sequenciação genómica especializada em saúde públicaPublication . Vieira, Luís; Silva, Catarina; Duarte, Sílvia; Mendonça, Joana; Carpinteiro, Dina; Sampaio, Daniel A.; Ferrão, José; Santos, Daniela; Machado, Miguel; Isidro, Joana; Barreiro, Paula; Isidro, GlóriaA 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.
- Bioinformatics study of expression from genomes of epidemiologically related MRSA CC398 isolates from human and wild animal samplesPublication . Ribeiro, Miguel; Sousa, Margarida; Borges, Vítor; Gomes, João Paulo; Duarte, Sílvia; Isidro, Joana; Vieira, Luís; Torres, Carmen; Santos, Hugo; Capelo, José Luís; Poeta, Patrícia; Igrejas, GilbertoOne of the most important livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) genetic lineages is the clonal complex (CC) 398, which can cause typical S. aureus-associated infections in people. In this work, whole-genome sequencing, RNA-sequencing, and gel-based comparative proteomics were applied to study the genetic characteristics of three MRSA CC398 isolates recovered from humans (strains C5621 and C9017), and from an animal (strain OR418). Of the three strains, C9017 presented the broadest resistance genotype, including resistance to fluroquinolone, clindamycin, tiamulin, macrolide and aminoglycoside antimicrobial classes. The scn, sak, and chp genes of the immune evasion cluster system were solely detected in OR418. Pangenome analysis showed a total of 288 strain-specific genes, most of which are hypothetical or phage-related proteins. OR418 had the most pronounced genetic differences. RNAIII (δ-hemolysin) gene was clearly the most expressed gene in OR418 and C5621, but it was not detected in C9017. Significant differences in the proteome profiles were found between strains. For example, the immunoglobulin-binding protein Sbi was more abundant in OR418. Considering that Sbi is a multifunctional immune evasion factor in S. aureus, the results point to OR418 strain having high zoonotic potential. Overall, multiomics biomarker signatures can assume an important role to advance precision medicine in the years to come. SIGNIFICANCE: MRSA is one of the most representative drug-resistant pathogens and its dissemination is increasing due to MRSA capability of establishing new reservoirs. LA-MRSA is considered an emerging problem worldwide and CC398 is one of the most important genetic lineages. In this study, three MRSA CC398 isolates recovered from humans and from a wild animal were analyzed through whole-genome sequencing, RNA-sequencing, and gel-based comparative proteomics in order to gather systems-wide omics data and better understand the genetic characteristics of this lineage to identify distinctive markers and genomic features of relevance to public health.
- Candida auris in Intensive Care Setting: The First Case Reported in PortugalPublication . Henriques, João; Mixão, Verónica; Cabrita, Joana; Duarte, Tiago Isidoro; Sequeira, Tânia; Cardoso, Sofia; Germano, Nuno; Dias, Liliana; Bento, Luís; Duarte, Sílvia; Veríssimo, Cristina; Gomes, João Paulo; Sabino, RaquelCandida auris is an opportunistic human pathogen that has rapidly spread to multiple countries and continents and has been associated with a high number of nosocomial outbreaks. Herein, we report the first case of C. auris in Portugal, which was associated with a patient transferred from Angola to an ICU in Portugal for liver transplantation after a SARS-CoV-2 infection. C. auris was isolated during the course of bronchoalveolar lavage, and it was subjected to antifungal susceptibility testing and whole-genome sequence analysis. This isolate presents low susceptibility to azoles and belongs to the genetic clade III with a phylogenetic placement close to African isolates. Although clade III has already been reported in Europe, taking into account the patient’s clinical history, we cannot discard the possibility that the patient’s colonization/infection occurred in Angola, prior to admission in the Portuguese hospital. Considering that C. auris is a fungal pathogen referenced by WHO as a critical priority, this case reinforces the need for continuous surveillance in a hospital setting
- Caraterização virológica dos vírus da gripe que circularam em Portugal na época 2014/2015Publication . Pechirra, Pedro; Costa, Inês; Cristóvão, Paula; Roque, Carla; Barreiro, Paula; Duarte, Sílvia; Machado, Ausenda; Rodrigues, Ana Paula; Nunes, Baltazar; Guiomar, RaquelIntrodução e objetivo: A monitorização contínua das propriedades antigénicas e genéticas dos vírus da gripe é essencial, quer para a seleção anual das estirpes virais a incluir na vacina, quer para identificar novas linhas de orientação da terapêutica antiviral. O presente estudo descreve as caraterísticas antigénicas e genéticas dos vírus da gripe identificados em Portugal no inverno de 2014/2015.
- Cephalosporin-resistant Neisseria gonorrhoeae isolated in Portugal, 2019Publication . Pinto, Miguel; Matias, Rui; Rodrigues, João Carlos; Duarte, Sílvia; Vieira, Luís; Gonçalves, Inês; Gonçalves, Maria João; Ramos, Maria Helena; Gomes, João Paulo; Borrego, Maria JoséWe report a multidrug resistant Neisseria gonorrhoeae exhibiting resistance to ceftriaxone and cefixime, isolated in Portugal in 2019. Whole-genome sequencing was performed for typing and identification of genetic determinants of antimicrobial resistance. Due to its antimicrobial susceptibility profile, awareness should be raised for the circulation of this strain.
- Comparative Effectiveness of COVID-19 Vaccines in Preventing Infections and Disease Progression from SARS-CoV-2 Omicron BA.5 and BA.2, PortugalPublication . Kislaya, Irina; Casaca, Pedro; Borges, Vítor; Sousa, Carlos; Ferreira, Bibiana I.; Fonte, Ana; Fernandes, Eugénia; Dias, Carlos Matias; Duarte, Sílvia; Almeida, José Pedro; Grenho, Inês; Coelho, Luís; Ferreira, Rita; Ferreira, Patrícia Pita; Borges, Cláudia Medeiros; Isidro, Joana; Pinto, Miguel; Menezes, Luís; Sobral, Daniel; Nunes, Alexandra; Santos, Daniela; Gonçalves, António Maia; Vieira, Luís; Gomes, João Paulo; Leite, Pedro Pinto; Nunes, Baltazar; Machado, Ausenda; Peralta-Santos, AndréWe estimated comparative primary and booster vaccine effectiveness (VE) of SARS-CoV-2 Omicron BA.5 and BA.2 lineages against infection and disease progression. During April-June 2022, we implemented a case-case and cohort study and classified lineages using whole-genome sequencing or spike gene target failure. For the case-case study, we estimated the adjusted odds ratios (aORs) of vaccination using a logistic regression. For the cohort study, we estimated VE against disease progression using a penalized logistic regression. We observed no reduced VE for primary (aOR 1.07 [95% CI 0.93-1.23]) or booster (aOR 0.96 [95% CI 0.84-1.09]) vaccination against BA.5 infection. Among BA.5 case-patients, booster VE against progression to hospitalization was lower than that among BA.2 case-patients (VE 77% [95% CI 49%-90%] vs. VE 93% [95% CI 86%-97%]). Although booster vaccination is less effective against BA.5 than against BA.2, it offers substantial protection against progression from BA.5 infection to severe disease.
- Dissecting whole-genome sequencing-based online tools for predicting resistance in Mycobacterium tuberculosis: can we use them for clinical decision guidance?Publication . Macedo, Rita; Nunes, Alexandra; Portugal, Isabel; Duarte, Sílvia; Vieira, Luís; Gomes, João PauloWhole-genome sequencing (WGS)-based bioinformatics platforms for the rapid prediction of resistance will soon be implemented in the Tuberculosis (TB) laboratory, but their accuracy assessment still needs to be strengthened. Here, we fully-sequenced a total of 54 multidrug-resistant (MDR) and five susceptible TB strains and performed, for the first time, a simultaneous evaluation of the major four free online platforms (TB Profiler, PhyResSE, Mykrobe Predictor and TGS-TB). Overall, the sensitivity of resistance prediction ranged from 84.3% using Mykrobe predictor to 95.2% using TB profiler, while specificity was higher and homogeneous among platforms. TB profiler revealed the best performance robustness (sensitivity, specificity, PPV and NPV above 95%), followed by TGS-TB (all parameters above 90%). We also observed a few discrepancies between phenotype and genotype, where, in some cases, it was possible to pin-point some "candidate" mutations (e.g., in the rpsL promoter region) highlighting the need for their confirmation through mutagenesis assays and potential review of the anti-TB genetic databases. The rampant development of the bioinformatics algorithms and the tremendously reduced time-frame until the clinician may decide for a definitive and most effective treatment will certainly trigger the technological transition where WGS-based bioinformatics platforms could replace phenotypic drug susceptibility testing for TB.
- Draft Genome Sequence of an Escherichia coli Strain Isolated from a Gallus gallus Broiler Producing the Novel CTX-M-166 VariantPublication . Manageiro, Vera; Clemente, Lurdes; Duarte, Sílvia; Vieira, Luís; Caniça, ManuelaWe report here the draft genome sequence of the CTX-M-166-harboring O6:H16 sequence type 48 (ST48)-fimH34 Escherichia coli strain recovered from a Gallus gallus broiler. Sequence analyses revealed the presence of an IncI1/ST103-ISEcp1-blaCTX-M-166-orf477 plasmid region and of diverse antibiotic resistance and virulence-acquired genes.
- Epidemiology and genetic diversity of invasive Neisseria meningitidis strains circulating in Portugal from 2003 to 2020Publication . Bettencourt, Célia; Nunes, Alexandra; Nogueira, Paulo; Duarte, Sílvia; Silva, Catarina; Gomes, João Paulo; Simões, Maria JoãoInvasive meningococcal disease (IMD) continues to be a public health problem due to its epidemic potential, affecting mostly children. We aimed to present a detailed description of the epidemiology of IMD in Portugal, including insights into the genetic diversity of Neisseria meningitidis strains. Epidemiological analysis included data from the Portuguese National Reference Laboratory of Neisseria meningitidis during 2003 to 2020. Since 2012, N. meningitidis isolates have also been assessed for their susceptibility to antibiotics and were characterized by whole genome sequencing. During 2003–2020, 1392 confirmed cases of IMD were analyzed. A decrease in the annual incidence rate was observed, ranging from 1.99 (2003) to 0.39 (2020), with an average case fatality rate of 7.1%. Serogroup B was the most frequent (69.7%), followed by serogroups C (9.7%), Y (5.7%), and W (2.6%). Genomic characterization of 329 isolates identified 20 clonal complexes (cc), with the most prevalent belonging to serogroup B cc41/44 (26.3%) and cc213 (16.3%). Isolates belonging to cc11 were predominantly from serogroups W (77.3%) and C (76.5%), whereas cc23 was dominant from serogroup Y (65.7%). Over the past 4 years (2017–2020), we observed an increasing trend of cases assigned to cc213, cc32, and cc11. Regarding antimicrobial susceptibility, all isolates were susceptible to ceftriaxone and 61.8% were penicillin-nonsusceptible, whereas 1.4% and 1.0% were resistant to ciprofloxacin and rifampicin. This is the first detailed study on the epidemiology and genomics of invasive N. meningitidis infections in Portugal, providing relevant data to public health policy makers for a more effective control of this disease.