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- Development of a multiplex PCR for tiled amplicon sequencing of highly variable Human Cytomegalovirus (HCMV) genomic regionsPublication . Carrasqueira, Patrícia; Ferreira, Rita; Lopo, Sílvia; Chasqueira, Maria de Jesus; Borges, Vítor; Paixão, Paulo; Gomes, João PauloHuman 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 studyPublication . 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 CommitteePurpose: 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.
- SARS-CoV-2 Seroprevalence among healthcare workers compared to the general population, 2021-22Publication . Gaio, Vânia; Amaral, Palmira; Santos, Ana João; Henriques, Camila; Guiomar, Raquel; Rodrigues, Ana Paula; Machado, Ausenda; Guiomar, RaquelHealthcare workers (HCWs) are essential as frontline responders during infectious disease health emergencies. Protecting them is crucial to ensure their health, maintain continuous patient care, and prevent transmission to patients. This study aimed to estimate the SARS-CoV-2 seroprevalence trend among HCWs from a Portuguese hospital cohort between May 2021 and June 2022. It also aimed to compare it with the seroprevalence trend in the general population aged 40-49. Additionally, the characterization of HCWs with seroconversion was performed based on their positivity for anti-nucleocapsid (Anti-N) IgG antibodies. As part of a vaccine effectiveness study, HCWs were screened for anti-RBD/Spike IgG antibodies against SARS-CoV-2 in three time points: May–July 2021, September–November 2021, and May–June 2022. At the last moment, Anti-N IgG antibodies were also assessed. To compare with data on the general population, we used results from three National Serological Surveys (NSS) phases (February–March 2021, September–November 2021, and April–June 2022) focusing on the 40–49 age group, the most representative among HCW. HCW characteristics were compared according to anti-N IgG seropositivity using the Chi-square and Mann-Whitney tests, assuming a significance level of 0.05. HCWs screening at the 3 moments included 909, 474, and 67 individuals respectively with SARS-CoV-2 seroprevalence was 86%, 90%, and 100%, respectively. These seroprevalences were similar to those found in the Portuguese general population, except for the first period (86% versus 18.8% in the general population, age group 40-49 years). At the last moment, the post-infection seroprevalence (anti-N IgG antibodies) was higher among HCWs than in the general population (41% versus 27%). A lower age and direct contact with COVID-19 patients were associated with anti-N IgG antibody positivity. The increasing trend of seroprevalence among HCWs follows the same trend in the general population. Although the time points differ, in the first moment, higher SARS-CoV-2 seroprevalence was probably linked to priority vaccine uptake. In the third moment, the higher post-infection seroprevalence among HCWs suggests a raised exposure and infection incidence in HCWs following the Omicron wave. Given the decline in COVID-19 vaccination coverage among HCWs in the post-pandemic period, ongoing monitoring of seroprevalence and COVID-19 infection rates in this group remains crucial.
- Low numbers of COVID-19 in the 2024/25 winter season: potential seasonality patternPublication . Rodrigues, Ana Paula; das Neves Pereira da Silva, SusanaBrief presentation analysing the possibility of a seasonal pattern of COVID-19 epidemics in Portugal, in the context of the VEBIS Annual Meeting.
- Nirsevimab effectiveness against hospitalised Respiratory Syncytial Virus infection in Portugal, 2024/25 seasonPublication . Gaio, Vânia; Valadas Henriques, Camila; Lança, Miguel; Machado, Ausenda; Guiomar, Raquel; Rodrigues, Ana PaulaBackground: Respiratory Syncytial Virus (RSV) is one of the leading causes of lower respiratory tract infections in younger children. In mainland Portugal, in order to reduce the risk of severe RSV post-infection complications, an immunisation strategy using Nirsevimab was implemented starting in October 2024, targeting all children aged less than 3 months, and those with high-risk conditions less than 24 months. We conducted a test-negative case-control study using the national hospital-based RSV surveillance network to estimate Nirsevimab effectiveness (NE). Methods: This multicentre study included children aged under 24 months hospitalised with severe acute respiratory infection in 14 hospitals. Cases were defined as children testing positive for RSV by RT-PCR or rapid antigen test (RAT), while controls tested negative. Immunisation status was obtained from electronic medical records. NE was estimated using logistic regression and estimated as (1 – adjusted odds ratio of immunisation) × 100, adjusting for age group, sex, month of symptom onset date, low birth weight, prematurity, and chronic conditions. Results: Between weeks 40/2024 and 12/2025, we included 111 cases and 110 controls. The median age for both groups was 2 months (IQR: 1–4). 44 (39.6%) RSV-positive and 80 (72.7%) RSV-negative infants received Nirsevimab at least 2 days before symptom onset. No significant differences were observed between cases and controls regarding sex or medical conditions. NE against RSV-associated hospitalisation was 85% (95% CI: 66–94) in the target population. Sensitivity analysis restricted to RT-PCR-confirmed cases yielded similar results. Conclusions: During the first immunisation season in mainland Portugal, Nirsevimab conferred good protection against the most severe presentation of RSV infection in young children. Our results are aligned with those reported in other countries. Given the potential for viral evolution following the introduction of universal immunisation programmes, ongoing monitoring of NE is warranted.
- SARS-CoV-2 Seroprevalence among hospital healthcare workers in comparison with the general population, 2021-2022Publication . Gaio, Vânia; Amaral, Palmira; Santos, Ana João; Henriques, Camila; Valadas Henriques, Camila; Guiomar, Raquel; Rodrigues, Ana Paula; Machado, AusendaBackground/Objectives: Healthcare workers (HCWs) are essential as frontline responders during infectious disease health emergencies. Protecting them is crucial to ensure their health, maintain continuous patient care, and prevent transmission to patients. This study aimed to estimate the SARS-CoV-2 seroprevalence trend among HCWs from a Portuguese hospital cohort between May 2021 and June 2022. It also aimed to compare it with the seroprevalence trend in the general population aged 40-49. Additionally, the characterization of HCWs with seroconversion was performed based on their positivity for anti-nucleocapsid (Anti-N) IgG antibodies. Methods: As part of a vaccine effectiveness study, HCWs were screened for anti-RBD/Spike IgG antibodies against SARS-CoV-2 in three time points: May–July 2021, September-November 2021, and May–June 2022. At the last moment, Anti-N IgG antibodies were also assessed. To compare with data on the general population, we used results from three National Serological Surveys (NSS) phases (February–March 2021, September–November 2021, and April–June 2022) focusing on the 40–49 age group, the most representative among HCW. HCW characteristics were compared according to anti-N IgG seropositivity using the Chi-square and Mann-Whitney tests, assuming a significance level of 0.05. Results: HCWs screening at the 3 moments included 909, 474, and 67 individuals respectively with SARS-CoV-2 seroprevalence was 86%, 90%, and 100%, respectively. These seroprevalences were similar to those found in the Portuguese general population, except for the first period (86% versus 18.8% in the general population, age group 40-49 years). At the last moment, the post-infection seroprevalence (anti-N IgG antibodies) was higher among HCWs than in the general population (41% versus 27%). A lower age and direct contact with COVID-19 patients were associated with anti-N IgG antibody positivity. Conclusions/Recommendations: The increasing trend of seroprevalence among HCWs follows the same trend in the general population. Although the time points differ, in the first moment, higher SARS-CoV-2 seroprevalence was probably linked to priority vaccine uptake. In the third moment, the higher post-infection seroprevalence among HCWs suggests a raised exposure and infection incidence in HCWs following the Omicron wave. Given the decline in COVID-19 vaccination coverage among HCWs in the post-pandemic period, ongoing monitoring of seroprevalence and COVID-19 infection rates in this group remains crucial.
- Do COVID-19 and Influenza vaccines influence susceptibility to other respiratory viruses? A population based studyPublication . Almeida Santos, João; Gomez, Verónica; Guiomar, Raquel; Verdasca, Nuno; Gomes, Licínia; Machado, Ausenda; Rodrigues, Ana PaulaIntroduction: Studies have raised concerns that Influenza and COVID-19 vaccination may influence susceptibility to other respiratory viruses (ORV), potentially increasing the risk of non-target infections. This challenges a key assumption of test-negative design studies—that vaccines do not affect the risk of other infections within the same clinical syndrome. Nevertheless, current evidence remains inconclusive. This study aimed to evaluate the association between COVID-19 and influenza vaccination and the risk of non-influenza/COVID-19 respiratory virus infections. Methods: Test-negative design (TND) study using Portuguese data from a primary care vaccine effectiveness study (VEBIS Primary Care study) between October/2022-April/2025. Data on influenza/COVID-19 vaccination status, age, sex and chronic conditions were collected. Samples were tested by RT-PCR for influenza, SARS-CoV-2 and ORV. Patients with laboratory-confirmed influenza/COVID-19 infection were excluded. Logistic regression estimated adjusted odds ratios (aOR) of being vaccinated among cases (ORV positive) and controls (pan-negative). Results: Of the 1096 patients included, 4.5% received the COVID-19 vaccine, 5.9% the influenza vaccine, 13.3% both, and 76.3% neither. Human Rhinovirus (44.2%), human Coronavirus (14.6%) and Respiratory Syncytial Virus (14.1%) were the viruses more frequently identified. Individuals 65+ exhibited significant lower odds of infection with ORV (OR=0.45, 95%CI:0.25-0.81) compared to younger age group (<18yo). Vaccination status, including influenza only (aOR=1.01, 95%CI:0.59‐1.72), COVID only (aOR=0.88, 95%CI:0.48‐1.59), and both vaccines (aOR=1.38, 95%CI:0.92‐2.06), were not associated to ORV infection risk. Conclusions: Our results suggest that vaccination status—whether for influenza, COVID-19, or both—was not significantly associated with the risk of ORV infections. This supports the use of test-negative controls for influenza/COVID-19 within the same clinical syndrome, as it upholds a key TND assumption of no association between vaccination and risk of non-target infections. While mechanisms such as reduced cross-protection from natural infection or potential vaccine-induced cross-immunity have been proposed, our findings reinforce the validity of the primary methodological assumption rather than suggesting evidence for these alternative effects.
- Vigilância da infeção por vírus sincicial respiratório durante a primeira campanha de imunizaçãoPublication . Gaio, Vânia; Henriques, Camila; Lança, Miguel; Guiomar, Raquel; Rodrigues, Ana PaulaResumo: O vírus sincicial respiratório (VSR) é um dos principais agentes etiológicos de infeções do trato respiratório inferior em crianças, especialmente em bebés e crianças pequenas (< 24 meses). Em 2024 foi estabelecida a imunização com o Nirsevimab em Portugal para as crianças menores de 3 meses e crianças de risco acrescido de infeção grave por VSR. Este trabalho tem como objetivos: 1) caraterizar a epidemia de VSR em Portugal em 2024/25; 2) caraterizar as crianças internadas com infeção respiratória durante a primeira campanha de imunização com Nirsevimab em Portugal, segundo toma de nirsevimab, idade, prematuridade, baixo peso ao nascer e doenças crónicas. Material e Métodos: Portugal tem um sistema de vigilância sentinela hospitalar, que integra 15 hospitais públicos e privados, os quais notificam os internamentos por infeção respiratória aguda de crianças menores de 24 meses. Para cada um dos doentes foi reportada informação clínica, epidemiológica, antecedentes vacinais e resultado de teste laboratorial para RSV. Entre 30 de setembro de 2024 e 23 de março de 2025 foi estimada a incidência semanal de internamento por VSR, a proporção de doentes internados em Unidades de Cuidados Intensivos(UCI) e caraterizados os doentes imunizados, positivos para VSR [VSR(+)] e negativos para VSR [VSR(-)], de acordo com as suas caraterísticas demográficas e clínicas. Resultados: A taxa de incidência máxima ocorreu na semana 51/2024 (89,6 /105), 21,0 % das crianças tinham idade inferior a 3 meses de idade, 16,3 % eram prematuras e 5,2 % foram internadas em UCI ou necessitaram de ventilação mecânica. 211 crianças eram elegíveis para imunização [111 doentes VSR(+) e 110 doentes VSR(-)], das quais 124 (56,1 %) foram imunizadas com Nirsevimab (39,6 % dos doentes VSR(+) e 72,7 % dos doentes VSR(-)). Não se observaram diferenças significativas entre imunizados e não imunizados em relação às doenças crónicas, prematuridade e baixo peso à nascença. Apenas a idade (1,5 meses) foi inferior nos doentes VSR(+) imunizados, relativamente aos doentes VSR(+) não imunizados. Conclusões: Na primeira época com imunização com Nirsevimab, a incidência de internamento por VSR em menores de 24 meses foi inferior ao observado em épocas anteriores. Ainda assim, apenas 56,1 % dos doentes elegíveis para vacinação, que participaram neste estudo, tinham tomado Nirsevimab, não sendo evidentes diferenças entre doentes imunizados e não imunizados no que se refere a doenças crónicas, baixo peso à nascença e prematuridade. Estes resultados mostram a necessidade de estudar os fatores associados à toma de Nirsevimab de modo a contribuir para melhorar futuras campanhas de imunização.
- Molecular characterization of Neisseria meningitidis strains causing non-invasive disease in Portugal from 2012-2024Publication . Bettencourt, Célia; Camões, InêsIntroduction: Non-invasive meningococcal disease (NIMD) is not notifiable, and the prevalence of serogroups and antimicrobial resistance (AMR) are unknown. This study aims to investigate the genetic diversity of non-invasive isolates identified in Portugal (2012-2024), assess their genomic relationships with Portuguese invasive isolates and identify AMR profiles. Material and Methods: All non-invasive N. meningitidis isolates were characterized by whole genome sequencing and the sequences submitted to the PubMLST/Neisseria.database. For antimicrobial susceptibility testing, antibiotic gradient strip diffusion (Etest) was used. Results: A total of 141 non-invasive isolates were characterized by WGS with 88% identified from respiratory secretions. Serogroup B was the most prevalent (40.4%), followed by serogroups Y (10.6%), C and E (3.5% each), W, X and Z (1.4% each). Capsule null (cnl) isolates accounted 33.3%. In silico analysis revealed the main clonal complexes (cc): B-cc41/44 (21%) and cc162 (14%), Y-cc23 and cc103 (33.3% each) and cnl-cc53 (38.3%). Isolates belonging to cc11 were predominantly serogroup C (40%) and only 1 isolate was identified as serogroup W. All isolates were sensitive to ceftriaxone and 67.9% of the isolates were penicillin-nonsusceptible, while 2.9% and 3.9% were resistant to ciprofloxacin and rifampicin, respectively. Conclusions: In this study, we identified non-invasive populations with similar genetic diversity when compared to invasive populations in previous studies[1]. In contrast, NIM isolates showed increased levels of resistance to penicillin and several isolates showed resistance to antibiotics used in IMD prophylaxis. These results emphasise the need for more studies on AMR among meningococci in order to ensure the effective use of antibiotics in the treatment of meningococcal disease.
- Surveillance of invasive meningococcal disease in Portugal, from 2020 to 2024Publication . Bettencourt, Célia; Nunes, A.; VigLab-DM – Network for the Laboratory Surveillance of Meningococcal Disease; Bajanca-Lavado, M.P.; .Introduction: Since 2002, laboratory surveillance of Invasive Meningococcal Disease (IMD) has been carried out by the National Reference Laboratory for Neisseria meningitidis, at the National Institute of Health Doutor Ricardo Jorge, Portugal. This study aims to analyse the epidemiology of IMD and the genetic diversity of Neisseria meningitidis strains from 2020 to 2024. Material and Methods: Suspected IMD cases and N. meningitidis isolates were sent to the reference laboratory for confirmation and strain characterization. Invasive isolates were characterized by WGS (Illumina) and sequences were submitted to the PubMLST/Neisseria database. Results: Between 2020 to 2024, 125 IMD cases were confirmed. Annual incidence rate ranged from 0.36 cases/100,000 inhabitants in 2020 to 0.32 in 2023 [1, 2]. Serogroup B was the most prevalent (49.6%), followed by serogroups Y (14.4%), W (13.6%) and C (5.6%). Serogroup W mainly affected those over 45 years old (58.8%). In silico analysis of 89 (71.2%) isolates identified major clonal complexes (cc): B-cc213 (22%) and cc41/44 (18%), Y-cc23 (80%), W-cc11 (66.7%), and C-cc11/cc103 (33.3% each). Conclusions: Compared to previous studies (2003-2020), the incidence of IMD in Portugal has decreased [1-3]. However, serogroup B remains the leading cause of IMD, raising concerns, particularly due to cases in children and emerging clusters with low vaccination coverage (e.g. serogroup B cc213) [4]. In contrast, serogroup W cases have increased, especially among adults [2, 3]. This study highlights the importance of laboratory surveillance for understanding IMD epidemiology and monitoring long-term trends.