Browsing by Author "Sobral, Daniel"
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- 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.
- 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.
- Development of an amplicon-based sequencing approach in response to the global emergence of mpoxPublication . Chen, Nicholas F.G.; Chaguza, Chrispin; Gagne, Luc; Doucette, Matthew; Smole, Sandra; Buzby, Erika; Hall, Joshua; Ash, Stephanie; Harrington, Rachel; Cofsky, Seana; Clancy, Selina; Kapsak, Curtis J.; Sevinsky, Joel; Libuit, Kevin; Park, Daniel J.; Hemarajata, Peera; Garrigues, Jacob M.; Green, Nicole M.; Sierra-Patev, Sean; Carpenter-Azevedo, Kristin; Huard, Richard C.; Pearson, Claire; Incekara, Kutluhan; Nishimura, Christina; Huang, Jian Ping; Gagnon, Emily; Reever, Ethan; Razeq, Jafar; Muyombwe, Anthony; Borges, Vítor; Ferreira, Rita; Sobral, Daniel; Duarte, Silvia; Santos, Daniela; Vieira, Luís; Gomes, João Paulo; Aquino, Carly; Savino, Isabella M.; Felton, Karinda; Bajwa, Moneeb; Hayward, Nyjil; Miller, Holly; Naumann, Allison; Allman, Ria; Greer, Neel; Fall, Amary; Mostafa, Heba H.; McHugh, Martin P.; Maloney, Daniel M.; Dewar, Rebecca; Kenicer, Juliet; Parker, Abby; Mathers, Katharine; Wild, Jonathan; Cotton, Seb; Templeton, Kate E.; Churchwell, George; Lee, Philip A.; Pedrosa, Maria; McGruder, Brenna; Schmedes, Sarah; Plumb, Matthew R.; Wang, Xiong; Barcellos, Regina Bones; Godinho, Fernanda M.S.; Salvato, Richard Steiner; Ceniseros, Aimee; Breban, Mallery I.; Grubaugh, Nathan D.; Gallagher, Glen R.; Vogels, Chantal B.F.The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.
- Distribution and Clinical Significance of HPV16 Variants in Head and Neck Squamous Cell Carcinomas: Data from a Portuguese Cohort and Systematic ReviewPublication . Cochicho, Daniela; Nunes, Alexandra; Sobral, Daniel; Gomes, João P.; Esteves, Susana; Mendonça, Joana; Vieira, Luis; Martins, Luís; Cunha, Mario; Montalvão, Pedro; Magalhães, Miguel; Gil da Costa, Rui M.; Félix, AnaIntroduction: Genomic variants of the human papillomavirus type 16 (HPV16) are thought to play differential roles in the susceptibility to head and neck squamous cell carcinomas (HNSCC) and its biological behaviour. This study aimed to establish the prevalence of HPV16 variants in an HNSCC cohort and associate them with clinical pathological characteristics and patient survival. Methods: We retrieved samples and clinical data from 68 HNSCC patients. DNA samples were available from tumour biopsy at the time of the primary diagnosis. Targeted next-generation sequencing was used to obtain whole-genome sequences, and variants were established based on phylogenetic classification. Results: 74% of samples clustered in lineage A, 5.7% in lineage B, 2.9% in lineage C, and 17.1% in lineage D. Comparative genome analysis revealed 243 single nucleotide variations. Of these, one hundred were previously reported, according to our systematic review. No significant associations with clinical pathological variables or patient survival were observed. The E6 amino acid variations E31G, L83V, and D25E and E7 N29S, associated with cervical cancer, were not observed, except for N29S in a single patient. Conclusion: These results provide a comprehensive genomic map of HPV16 in HSNCC, highlighting tissue-specific characteristics which will help design tailored therapies for cancer patients.
- An endoribonuclease of the YicC-like family delays sporulation via sRNA degradation in Clostridioides difficilePublication . Martins, Diogo; Salgueiro, Bruno; Sobral, Daniel; Gragera, Marcos; Hensel, Zach; Henriques, Adriano O.; Romão, Célia V.; Serrano, MónicaClostridioides difficile CD25890 is a YicC-like endoribonuclease involved in regulating sporulation initiation, a process critical for the host-host transmission of this anaerobic pathogen. Using comparative transcriptomics we identified a small RNA, SQ528, that accumulates at higher levels in a CD25890 deletion mutant and we show that purified CD25890 cleaves SQ528 in a metal-dependent manner. Moreover, the overexpression of SQ528 increases sporulation under certain nutritional conditions phenocopying a CD25890 deletion mutant. CD25890 is an hexamer in solution and in vivo. An N-terminal domain, which self-interacts as assessed by size exclusion chromatography and a two hybrid assay, is essential for oligomerization of CD25890. A C-terminal domain harbours residues H230, E254, and E258, conserved among orthologues, important for catalysis. AlphaFold2 modelling and cryo-EM suggest an elongated barrel-like structure with an internal cavity lined with basic residues that may aid in RNA binding. We show that CD25890 forms a complex with polynucleotide phosphorylase which combines the endoribonuclease activity of the first with the exonucleolytic activity of the latter and leads to the complete degradation of SQ528. This study identifies a native substrate for the YicC-family of ribonucleases and advances our understanding of the role of CD25890 in sporulation initiation in C. difficile.
- European pilot interlaboratory comparison study on Mpox virus whole genome sequencingPublication . Fuchs, Jonas; Bertelli, Claire; Pillonel, Trestan; Cordeiro, Rita; Izopet, Jacques; Pasquier, Christophe; Lewandowski, Kuiama; Maks, Anastasija; Michel, Janine; Rodriguez-Sanchez, Belen; Sanches-Seco, Maria Paz; Ledesma, Juan; Sobral, Daniel; Vercauteren, Koen; de Block, Tessa; Rezende, Antonio Mauro; Brinkmann, Annika; Nitsche, Andreas; Greub, Gilbert; Panning, MarcusObjectives: Since 2022, distinct Mpox virus (MPXV) clades have been spreading across different geographic regions, causing a challenging epidemiological situation. Whole genome sequencing (WGS) proved to be instrumental for patient management and global public health. We report a pilot interlaboratory comparison study for MPXV WGS. Methods: We distributed noninfectious DNA samples, including the main MPXV clades I and II, to eight European laboratories. We included one cowpox (CPXV) sample as a specificity control. Participants were free to choose their WGS pipeline of choice to mimic a real-world scenario and were asked to report on the sequencing pipeline used, average genome coverage, and MPXV species, clade, and subclade assignments. Results: Seven of the eight invited laboratories reported results back. All participants largely identified the MPXV clades and reported high-quality genomes with minimal variations, specifically for MPXV clade IIb 2022 outbreak strains. However, reconstructed genomes showed high variability for nonclade IIb MPXV strains. The CPXV sample was correctly identified by three laboratories. Conclusions: Although results for MPXV clade IIb 2022 outbreak strains are reassuring, the inclusion of MPXV clade I and IIa strains highlights pitfalls for targeted sequencing approaches and subsequent bioinformatic analyses. Our findings underscore the need for standardized external quality assessment studies.
- INSaFLU-TELEVIR: an open web-based bioinformatics suite for viral metagenomic detection and routine genomic surveillancePublication . Santos, João Dourado; Sobral, Daniel; Pinheiro, Miguel; Isidro, Joana; Bogaardt, Carlijn; Pinto, Miguel; Eusébio, Rodrigo; Santos, André; Mamede, Rafael; Horton, Daniel L; Gomes, João Paulo; TELEVIR Consortium; Borges, VítorBackground: Implementation of clinical metagenomics and pathogen genomic surveillance can be particularly challenging due to the lack of bioinformatics tools and/or expertise. In order to face this challenge, we have previously developed INSaFLU, a free web-based bioinformatics platform for virus next-generation sequencing data analysis. Here, we considerably expanded its genomic surveillance component and developed a new module (TELEVIR) for metagenomic virus identification. Results: The routine genomic surveillance component was strengthened with new workflows and functionalities, including (i) a reference-based genome assembly pipeline for Oxford Nanopore technologies (ONT) data; (ii) automated SARS-CoV-2 lineage classification; (iii) Nextclade analysis; (iv) Nextstrain phylogeographic and temporal analysis (SARS-CoV-2, human and avian influenza, monkeypox, respiratory syncytial virus (RSV A/B), as well as a "generic" build for other viruses); and (v) algn2pheno for screening mutations of interest. Both INSaFLU pipelines for reference-based consensus generation (Illumina and ONT) were benchmarked against commonly used command line bioinformatics workflows for SARS-CoV-2, and an INSaFLU snakemake version was released. In parallel, a new module (TELEVIR) for virus detection was developed, after extensive benchmarking of state-of-the-art metagenomics software and following up-to-date recommendations and practices in the field. TELEVIR allows running complex workflows, covering several combinations of steps (e.g., with/without viral enrichment or host depletion), classification software (e.g., Kaiju, Kraken2, Centrifuge, FastViromeExplorer), and databases (RefSeq viral genome, Virosaurus, etc.), while culminating in user- and diagnosis-oriented reports. Finally, to potentiate real-time virus detection during ONT runs, we developed findONTime, a tool aimed at reducing costs and the time between sample reception and diagnosis. Conclusions: The accessibility, versatility, and functionality of INSaFLU-TELEVIR are expected to supply public and animal health laboratories and researchers with a user-oriented and pan-viral bioinformatics framework that promotes a strengthened and timely viral metagenomic detection and routine genomics surveillance. INSaFLU-TELEVIR is compatible with Illumina, Ion Torrent, and ONT data and is freely available at https://insaflu.insa.pt/ (online tool) and https://github.com/INSaFLU (code).
- 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.
- Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virusPublication . Isidro, Joana; Borges, Vítor; Pinto, Miguel; Sobral, Daniel; Santos, João Dourado; Nunes, Alexandra; Mixão, Verónica; Ferreira, Rita; Santos, Daniela; Duarte, Silvia; Vieira, Luís; Borrego, Maria José; Núncio, Sofia; de Carvalho, Isabel Lopes; Pelerito, Ana; Cordeiro, Rita; Gomes, João PauloThe largest monkeypox virus (MPXV) outbreak described so far in non-endemic countries was identified in May 2022 (refs. 1-6). In this study, shotgun metagenomics allowed the rapid reconstruction and phylogenomic characterization of the first MPXV outbreak genome sequences, showing that this MPXV belongs to clade 3 and that the outbreak most likely has a single origin. Although 2022 MPXV (lineage B.1) clustered with 2018-2019 cases linked to an endemic country, it segregates in a divergent phylogenetic branch, likely reflecting continuous accelerated evolution. An in-depth mutational analysis suggests the action of host APOBEC3 in viral evolution as well as signs of potential MPXV human adaptation in ongoing microevolution. Our findings also indicate that genome sequencing may provide resolution to track the spread and transmission of this presumably slow-evolving double-stranded DNA virus.
- Preclinical assessment of an antibiotic-free cationic surfactant-based cellulose hydrogel for sexually and perinatally transmitted infectionsPublication . Calado, Rita D.A.; Mendes, Bárbara B.; Conniot, João; Ravasco, João M.J.M.; Sobral, Daniel; Ferreira, Carolina; Ferreira, Rita; Rodrigues, João Carlos; Santos, Daniela; Duarte, Sílvia; Vieira, Luís; Inácio, Ângela S.; Carrêlo, Henrique; Vaz, Winchil L.C.; Gomes, João Paulo; Nunes, Alexandra; Conde, João; Vieira, Otilia V.Sexually transmitted infections and urogenital-perinatal infections are significant health challenges owing to their asymptomatic nature, multidrug-resistant pathogens, and lack of effective vaccines. Surfactants are under investigation as potential antimicrobial agents and alternatives to traditional antibiotics. Here, we discovered that N-dodecylpyridinium bromide (C12PB), a cationic quaternary ammonium surfactant, has very low potential to induce antimicrobial resistance with no antibiotic cross-resistance or inflammation in vitro. Therefore, we developed a preclinical antibiotic-free cationic surfactant-based cellulose hydrogel for treating sexually transmitted infections. The C12PB-hydrogels provided sustained surfactant release, enhancing their biocompatibility and antibacterial activity without inflammation or epithelial disruption of the vaginal tract. In a preclinical model of Neisseria gonorrhoeae infection, a single application of the C12PB-hydrogel showed a 2- to 3-fold reduction in infection. This lays the foundation for the future development of C12PB-hydrogels for sexually transmitted infections, demonstrating potent antibacterial activity and minimal risk of antimicrobial resistance or inflammation.