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Vigilância Laboratorial da Tuberculose em Portugal: relatório 2025
Publication . Laboratório Nacional de Referência de Micobactérias
Relatório de Vigilância Laboratorial da Tuberculose em Portugal referente ao ano de 2025, elaborado pelo Laboratório Nacional de Referência de Micobactérias (LNR-TB) do Departamento de Doenças Infeciosas do INSA.
O documento apresenta a análise da vigilância molecular das estirpes do complexo Mycobacterium tuberculosis (MTC), com base em dados acumulados desde 2020, incluindo a caracterização genómica e a identificação de relações filogenéticas relevantes para a deteção de cadeias de transmissão.
Na sequência da implementação de metodologias baseadas em sequenciação do genoma total (WGS), o LNR-TB consolidou a sua capacidade para realizar, de forma sistemática, a caracterização genómica das estirpes de MTC. Esta abordagem permite a previsão do perfil de suscetibilidade aos antibacilares, incluindo de primeira linha, com elevado grau de fiabilidade, bem como a identificação precoce de eventos de transmissão. A utilização do WGS constitui, assim, uma ferramenta central para apoiar a decisão clínica e reforçar a resposta das Autoridades de Saúde, em linha com as recomendações europeias para países de baixa incidência de tuberculose.
A vigilância laboratorial das micobactérias foi ainda alargada, incluindo de forma continuada a monitorização de Mycobacterium leprae, já integrada desde 2023, e, pela primeira vez, a caracterização de casos de micobactérias não tuberculosas (MNT), refletindo a crescente relevância destas infeções no contexto clínico e epidemiológico.
Dos resultados apresentados no relatório, destacam-se os seguintes pontos:
- A confirmação bacteriológica da tuberculose mantém-se essencial para a monitorização da doença e para a determinação do perfil de suscetibilidade aos antibacilares, devendo ser sempre complementada com exames laboratoriais adequados;
- Os casos de tuberculose resistente à rifampicina ou multirresistente (TB-RR/MR) mantêm-se em vigilância, com variações anuais que justificam acompanhamento contínuo;
- A distribuição geográfica dos casos evidencia maior concentração nas regiões de Lisboa e Vale do Tejo e do Norte, à semelhança do observado em anos anteriores;
- A análise genómica permitiu identificar múltiplos clusters moleculares, incluindo cadeias de transmissão com persistência temporal, reforçando a importância da vigilância molecular contínua;
- Desde 2023, que se confirmam anualmente casos de infeção por Mycobacterium leprae, todos com enquadramento epidemiológico compatível, evidenciando a importância da capacidade diagnóstica instalada;
- A inclusão da vigilância das micobactérias não tuberculosas (MNT) constitui um avanço relevante, permitindo uma abordagem mais abrangente das infeções por micobactérias em Portugal;
- Em alinhamento com as mais recentes recomendações europeias, nomeadamente do European Reference Laboratory Network for Tuberculosis (ERLTB-Net), está em curso a transição para a utilização sistemática da sequenciação do genoma total (WGS) em todos os isolados de Mycobacterium tuberculosis, com o objetivo de substituir progressivamente os testes fenotípicos de suscetibilidade aos antibacilares de primeira linha. Esta abordagem permitirá uma resposta diagnóstica mais rápida, integrada e preditiva, reforçando a eficiência do sistema e a qualidade da informação disponibilizada a clínicos e autoridades de saúde.
Este relatório reforça o papel do INSA enquanto laboratório nacional de referência, evidenciando a importância da integração de ferramentas genómicas na vigilância laboratorial e no apoio à decisão clínica e em saúde pública.
Development of a multiplex PCR for tiled amplicon sequencing of highly variable Human Cytomegalovirus (HCMV) genomic regions
Publication . Carrasqueira, Patrícia; Ferreira, Rita; Lopo, Sílvia; Chasqueira, Maria de Jesus; Borges, Vítor; Paixão, Paulo; Gomes, João Paulo
Human cytomegalovirus (HCMV) possesses a ~236 kb genome and exhibits genetic diversity mostly concentrated in discrete hypervariable regions, such as envelope glycoprotein genes like UL55 (gB), UL73 (gN), and UL75 (gH), and recombination occurs frequently. Despite numerous efforts to associate viral polymorphisms and intra-patient population diversity with enhanced viral fitness, clinical outcomes, and latency, correlations remains inconclusive. Therefore, in this study a multiplex tiling PCR assay was developed, for the simultaneous sequencing of multiple hypervariable HCMV genomic regions, aiming for a comprehensive characterization of viral population variability and dynamics.
To take advantage of the vast potential of targeted next-generation sequencing (tNGS), a 2-pool primer scheme (51 amplicons) for multiplex tiling PCR was designed, taking into account the genetic diversity of 358 HCMV genome sequences available at NCBI Virus (https://www.ncbi.nlm.nih.gov/labs/virus). The novel multiplex targets seven independent genomic regions, encompassing 14 polymorphic loci of interest (UL33, UL55, UL73–UL75, UL100, UL115, UL128–UL131A, and UL144–UL147A). Protocol optimization and performance assays were conducted using Illumina NGS and subsequent bioinformatics analysis with the online platform INSaFLU-TELEVIR (https://insaflu.insa.pt/).
Preliminary results of the application of the novel multiplex tNGS assay to clinical samples (n = 11) from different matrices (urine, BALF, plasma, placenta) collected between 2020 and 2024 demonstrated its high potential, with successful amplification and sequencing of all amplicons in all samples with a Ct value ≤25. The assay also proved effective in sequencing highly polymorphic regions and detecting mixed infections. We anticipate that this innovative approach will open new avenues for characterizing circulating HCMV diversity and investigating whether viral population diversity and specific genetic profiles correlate with clinical outcomes, ultimately supporting earlier interventions and improved management of HCMV infections (e.g. congenital infections).
Impact of a revised late HIV diagnosis definition on late HIV estimates in Europe: A multi-country pilot study
Publication . Kirwan, P.; Stengaard, A.; Brännström, J.; Van Beckhoven, D.; van Sighem, A.; Op de Coul, E.; Bartmeyer, B.; Koppe, U.; C. Martins, H.; Maly, M.; Wessman, M.; Tsiara, C.; Ferentinos, G.; Suligoi, B.; Grabar, S.; Sullivan, A.K.; Reyes, J.; Pharris, A.; Kuchukhidze, G.; Kirk, O.; Croxford, S.; Delpech, V.; Raben, D.; EuroTEST Steering Committee
Purpose: Late HIV diagnosis has been defined as a CD4 count <350 cells/mL or AIDS-defining event. With improvements in HIV tests and testing frequency, more people in Europe are diagnosed during the acute/seroconversion phase, when their CD4 count can be temporarily low. A revised consensus definition of late HIV diagnosis* enables better distinction between people diagnosed late and those diagnosed during the acute/seroconversion phase. We aimed to pilot this revised definition with European countries.
Method: Pseudo-anonymised HIV diagnosis records for 2022-2023 were collected from nine countries. Records included markers of recent HIV acquisition from laboratory evidence (RITA, p24), testing history (negative HIV test within 12 months), or clinical evidence (e.g. seroconversion illness). We applied the revised definition to reclassify those with recently acquired HIV as ‘not-late’. Late diagnosis correction factors were calculated as: (number reclassified)/(number with
CD4<350 or AIDS-defining event) and evaluated by demographic factor.
Results: Availability of recent acquisition evidence varied by country and individual marker. Of 10,241 diagnoses with CD4 counts reported, 56% (5,696/10,241) had a CD4<350 or AIDS-defining event, i.e. were initially classified as late. Of these, 563 had evidence of recent HIV acquisition: 168 had laboratory evidence, 238 testing history evidence, and 260 clinical evidence (could have multiple). After reclassification the late diagnosis rate was reduced from 56% to 50%,with an overall correction factor of 10% (563/5,696), ranging between 3-25% across countries . The correction factor was higher for younger individuals compared to older, and for MSM compared to other transmission routes .
Conclusions: Without reclassification, late HIV diagnosis rates are overestimated, by up to 25% in young MSM. This correction addresses a lack of progress in
reducing the percentage of people diagnosed late. For countries to undertake this correction, improved collection of recent acquisition markers at clinic and national
levels is needed.
Multiomics profiling of Red Blood Cells (RBCs) combined with Deep Machine Learning analysis – reveals potential Diagnostic Biomarkers for Acute Venous Thromboembolism
Publication . Febra, Cláudia; Saraiva, Joana; AlKhnbashi, Omer; Vaz, Fátima; Macedo, João; Uddin, Mohammed J.; Penque, Deborah; Soares, Nelson C.
Background and aims: Venous Thromboembolism (VTE) is a leading cause of cardiovascular mortality. The diagnosis of acute VTE is still based on complex imaging exams due to the lack of biomarkers. However, studies assessing the diagnostic capacity of novel metabolomics biomarkers in VTE are scarce. Our aim was to determine whether patients with acute VTE exhibit differences in proteomic and metabolomic profiles.
Multiomics screening of red blood cells (RBCs) reveals new candidate diagnostic biomarkers for acute venous thrombosis
Publication . Febra, Cláudia; Saraiva, Joana; AlKhnbashi, Omer; Pinto, Frederico G.; Vaz, Fátima; Macedo, João; Uddin, Mohammed J.; Goswami, Nandu; Penque, Deborah; Soares, Nelson C.
Venous Thromboembolism (VTE) is a leading cause of cardiovascular mortality. The diagnosis of acute VTE is still based on complex imaging exams due to the lack of biomarkers. However, studies assessing the diagnostic capacity of novel biomarkers in VTE are scarce. We aimed to determine if patients with acute VTE have differences in the proteomics/metabolomic profile. This observational trial included 62 patients with clinical suspicion of acute deep vein thrombosis (DVT) or pulmonary embolism (PE) admitted to the emergency room (ER).