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- 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.
- Coordinated implementation of a conventional PCR assay to detect all Ebola and Marburg virus species in a European laboratory networkPublication . Heimsch, K.C.; Bleicker, T.; Best, T.D.; Presser, L.D.; Molenkamp, R.; Jääskeläinen, A.J.; Milewska, A.; Smahelová, J.; Baronti, C.; Pappa, S.; Tabain, I.; Cordeiro, Rita; Marsili, G.; Huik, K.; dos Reis, V. Pinho; Barzon, L.; Maes, P.; Drosten, C.; Corman, V.M.Background: Filoviruses, including Ebola and Marburg viruses, cause severe hemorrhagic fever in humans and primates. These viruses pose significant threats to public health, making rapid and sensitive detection critical for controlling outbreaks. We developed and validated a hemi-nested generic PanFilo assay to detect all Ebola virus species, Marburg viruses, and recently discovered bat filoviruses. This assay was deployed to 15 European laboratories and evaluated through testing of eight non-infectious samples. Objectives: Laboratories were asked to determine the detection limit of positive controls and test all samples using the assay provided. The deployed assay enables direct Nanopore sequencing of PCR products, by using tagged primers during the second round of PCR. Sequencing of the samples was carried out on a voluntary basis. Results: Multicenter validation revealed a 95 % limit of detection of 5309 RNA copies/μL for Ebola, 10,273 copies/μL for Marburg, and 2145 copies/μL for Mengla virus. In an implementation quality assessment, 93.3 % (84/90) of samples containing filovirus RNA were correctly identified and 100 % (30/30) of filovirus-negative samples were correctly identified. Thirteen laboratories sequenced PCR products, with nine identifying all positive samples correctly. Conclusion: The assay enables rapid and reliable detection of filoviruses, with sequencing capabilities for identifying both known and novel variants. This assay might be used for detection during the initial phase of an emerging filovirus outbreak, before a specific assay has been developed. However, our distribution across 15.
- Multi-country and intersectoral assessment of cluster congruence between pipelines for genomics surveillance of foodborne pathogensPublication . Mixão, Verónica; Pinto, Miguel; Brendebach, Holger; Sobral, Daniel; Santos, João Dourado; Radomski, Nicolas; Uldall, Anne Sophie Majgaard; Bomba, Arkadiusz; Pietsch, Michael; Bucciacchio, Andrea; de Ruvo, Andrea; Castelli, Pierluigi; Iwan, Ewelina; Simon, Sandra; Coipan, Claudia E.; Linde, Jörg; Petrovska, Liljana; Kaas, Rolf Sommer; Joensen, Katrine Grimstrup; Nielsen, Sofie Holtsmark; Kiil, Kristoffer; Lagesen, Karin; Di Pasquale, Adriano; Gomes, João Paulo; Deneke, Carlus; Tausch, Simon H.; Borges, VítorDifferent laboratories employ different Whole-Genome Sequencing (WGS) pipelines for Food and Waterborne disease (FWD) surveillance, casting doubt on the comparability of their results and hindering optimal communication at intersectoral and international levels. Through a collaborative effort involving eleven European institutes spanning the food, animal, and human health sectors, we aimed to assess the inter-pipeline clustering congruence across all resolution levels and perform an in-depth comparative analysis of cluster composition at outbreak level for four important foodborne pathogens: Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Campylobacter jejuni. We found a general concordance between allele-based pipelines for all species, except for C. jejuni, where the different resolution power of allele-based schemas led to marked discrepancies. Still, we identified non-negligible differences in outbreak detection and demonstrated how a threshold flexibilization favors the detection of similar outbreak signals by different laboratories. These results, together with the observation that different traditional typing groups (e.g., serotypes) exhibit a remarkably different genetic diversity, represent valuable information for future outbreak case-definitions and WGS-based nomenclature design. This study reinforces the need, while demonstrating the feasibility, of conducting continuous pipeline comparability assessments, and opens good perspectives for a smoother international and intersectoral cooperation towards an efficient One Health FWD surveillance.
- Dengue and Oropouche virus co-infection in a traveller from Cuba to PortugalPublication . Zé-Zé, Líbia; Laranjinha, Joana; Borges, Vítor; Graça, Ana Luísa; Sobral, Daniel; Santos, João Dourado; Carvalho, Ana Cláudia; Faria, Nuno R.; Gomes, João Paulo; Alves, Maria JoãoIn 2024, unprecedented outbreaks of dengue and Oropouche were reported in the Americas. We describe a documented co-infection with dengue and Oropouche viruses in a 35-year-old traveller from Cuba detected in Portugal. RT-PCR and next-generation sequencing confirmed both viruses. Our findings highlight the need for multiplex arboviral diagnostics in travellers from regions with concurrent outbreaks.
- Sustained importance of Streptococcus pneumoniae among pediatric complicated Pneumonia in Portugal (2019-22)Publication . Gomes-Silva, J.; Silva-Costa, Catarina; Pinho, Marcos; Friães, Ana; Ramirez, Mário; Melo-Cristino, José; Portuguese Group for the Study of Streptococcal Infections; Portuguese Study Group of Pediatric Invasive Streptococcal DiseaseBackgrounds: To improve the etiological diagnosis of culture-negative pediatric complicated pneumonia (PCP), we expanded our real-time PCR assay to include other bacterial agents and evaluate potential changes in etiology after 7 years of near universal use of 13-valent conjugate pneumococcal vaccine (PCV13). Methods: We collected 156 culture-negative pleural fluid and empyema samples from children (<18 years), in 62 hospitals in Portugal, from January 2019 to December 2022. Our assay included Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Mycoplasma pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis and Streptococcus agalactiae. For S. pneumoniae cases we performed molecular serotyping. Results: Overall, 78 samples were negative for all bacteria tested (50.0%). Among the remaining 78 samples, the majority was positive for S. pneumoniae (n=64, 41.0%). S. pyogenes was found in 7 samples (4.49%), S. aureus in 5 samples (3.20%), H. influenzae in 2 samples (1.28%), and M. pneumoniae in 1 sample (0.64%). We did not detect M. tuberculosis nor S. agalactiae. In 2 samples, we detected the presence of DNA from both S. pneumoniae and another species: S. aureus (n=1) and H. influenzae (n=1). Among the pneumococcal samples, 44 were serotype 3 (56.4%), 5 were serotype 8 (6.41%), 2 were serotype 14 (2.56%) and serotypes 15A, 16F, 19A, 19F and 6C/6D were detected in 1 sample each (1.28% each). The remaining were negative for all serotypes tested (4.49%). Conclusions/Learning Points: After two decades of pneumococcal conjugate vaccine use, S. pneumoniae is still responsible for most culture-negative PCPs, with PCV13 serotype 3 responsible for most cases. Expanding the molecular diagnostic panel to other species allowed the identification of the etiology of only an additional 9.62% of cases, suggesting that bacteria other than S. pneumoniae remain infrequent despite PCV13 use.
- Biofilm Formation by ST17 and ST19 Strains of Streptococcus agalactiaePublication . Silvestre, Inês; Borrego, Maria José; Jordão, LuísaBacterial biofilms are an important virulence factor with a vital role in evasion from the host immune system, colonization and infection. The aim of the present study was to evaluate in vitro the effects of three environmental factors (H+ , glucose and human plasma) in biofilm formation, by carrier and invasive S. agalactiae strains of ST17 and ST19 sequence types, including DNase producers and nonproducers. Bacteria ability to assemble biofilms was classified based on crystal violet assay. Biofilm formation was also monitored by scanning electron microscopy. Depending on the growth medium used, each bacterial isolate could fit in different biofilm production categories. Our data showed that optimal conditions for S. agalactiae biofilm assembly were reached after 48 h incubation at pH 7.6 in the presence of glucose and inactivated human plasma. In the presence of inactivated human plasma, the biofilm biomass of ST19 strains experienced a higher increase than ST17 strains. The composition of the extracellular polymeric matrix of the three strongest biofilm producers (all from ST17) was accessed by enzymatic digestion of mature biofilms and proteins were shown to be the predominant component. The detailed identification of the extracellular protein components should contribute to the development of new therapeutic strategies to fight S. agalactiae infections.
- SARS-CoV-2 seroprevalence studies: the Portuguese experience in last 2 years and next stepsPublication . Rodrigues, Ana Paula; on behalf of the ISN COVID-19 groupPortuguese experience in SARS-CoV-2 seroprevalence studies
- Epidemiology and molecular characterization of invasive disease in children twenty years after the implementation of Haemophilus influenzae serotype b vaccine in Portuguese Immunization ProgrammePublication . Bajanca-Lavado, Maria Paula; Bettencout, Célia; Cunha, Florbela; Gonçalo-Marques, José; Study Group of invasive Haemophilus influenzae disease in of the Pediatric Infection Disease SocietyBackground: Haemophilus influenzae is an important human pathogen responsible for severe childhood invasive disease, despite the implementation of the vaccine against serotype b isolates (Hib), in our National Immunization Programme (NIP), in June 2000. The use of the vaccine lead to a reduction in Hib invasive disease, together with the emergence of non-encapsulated (NTHi), and capsulated non-b-type isolates. This study aims to characterize H. influenzae invasive disease in children, twenty years after the introduction of the Hib vaccine in NIP. Methods Hundred-twenty invasive H. influenzae isolates collected from children in 33 Hospitals, between January 2010 and December 2020, were characterized at the National Reference Laboratory for Haemophilus influenzae. Antibiotic susceptibility was assessed by a microdilution assay. Capsular status was identified by PCR as previously described. MLST was performed as described in the literature. Sequences were analysed and submitted to the MLST website (https://pubmlst.org/hinfluenzae/) for assignment of the sequence type (ST). goeBURST analysis was performed using the PHYLOViZ platform. Results Childhood invasive disease was mainly due to NTHi (55.8%; 67/120), although Hib still in circulation (29.2%; 35/120). Twenty-two cases of vaccine failures were responsible for 62.9% of Hib disease, with 59% of cases occurring in last four years. Non-b capsular types isolates were distributed as follow: 9.2% serotype a (11/120), 1.6% serotype e (2/120) and 4.2% serotype f (5/120). Most isolates were susceptible to all antibiotics studied, with 8.3% (10/120) being ampicillin resistant by β-lactamase producing. MLST revealed, as expected, high genetic variability (77.1%), with 37 different STs among 48 NTHi isolates. In opposition, encapsulated isolates were clonal with Hia assigned to CC23 (ST23-n=6; ST1511-n=1), Hib to CC6 (ST6-n=27, ST190, ST1149 and ST1231 with one isolate each), Hie to CC18 (ST18-n=2) and Hif to CC124 (ST124-n=2, ST1188-n=1). Conclusions Our data suggests that after vaccine implementation, invasive disease among Portuguese children is mainly due to highly genetically diverse, susceptible NTHi isolates. Nevertheless, we are concerned about Hib disease (~30%) despite the higher vaccine coverage observed in our country. Ongoing surveillance should be continued, in order to monitor the burden of the disease, especially Hib, and develop additional public health prevention strategies.
- Molecular identification of fungi from tissue samplesPublication . Sabino, RaquelTissue samples from patients with suspected invasive fungal disease (IFD) should be examined not only by mycological culture but also by microscopy and specific fungal stains should be included. For histopathological diagnosis of fungal infection is required deep knowledge about fungal morphology in tissue and also about the various reactions of the tissue in response to that infection. Highly experienced histopathologists are therefore essential to detect fungal structures and also to recognize tissue reactions associated with IFD, distinguishing them from staining artifacts. This presentation discusses different molecular methods applied to the diagnosis of invasive fungal infections. These methods may help to circunvent some constraints of histology and cultural methods.