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- 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.
- 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.
- Surveying genetic markers of antibiotic resistance and genomic background in Chlamydia trachomatis: insights from a multiplex NGS-based approach in clinical strains from PortugalPublication . Lodhia, Zohra; da Silva, Jorge Costa; Correia, Cristina; Cordeiro, Dora; João, Inês; Carreira, Teresa; Schäfer, Sandra; Aliyeva, Elzara; Portugal, Clara; Monge, Isabel; Gonçalves, Elsa; Matos, Susana; Dias, Ana Paula; Côrte-Real, Rita; Carpinteiro, Dina; Duarte, Sílvia; Vieira, Luís; Gomes, João Paulo; Borges, Vítor; Borrego, Maria JoséObjectives: To survey genetic markers of potential antimicrobial resistance (AMR) to macrolides and fluoroquinolones among Chlamydia trachomatis–positive samples from the collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections (STIs), and explore a multiplex PCR approach coupled with NGS to provide complementary information regarding a strain’s genomic backbone. Methods: A total of 502 C. trachomatis–positive samples, mostly anorectal exudates, were subjected to PCR and sequencing of five targets, including loci potentially driving AMR (23S rRNA, gyrA and parC) and loci potentially informative about a strain’s genomic backbone with emphasis on differentiation of lymphogranuloma venereum (LGV)/non-LGV and L2/L2b (a 9 bp insertion in pmpH, a 74 bp insertion upstream from CT105 and the polymorphic CT442). Results: No samples evidenced 23S rRNA mutations recognizably linked to macrolide resistance. Three samples harboured the Ser83Ile mutation in GyrA putatively driving fluoroquinolone resistance: two recombinant L2-L2b/D-Da (0.4%) and one L2 (0.2%). The screened regions in pmpH, upstream CT105 and CT442 were fully concordant with LGV/non-LGV differentiation. As expected, the pmpH L2b-specific genetic trait locus was detected in all L2b and recombinant L2-L2b/D-Da ompA genotypes, but also in 96.0% of L2 specimens, which also likely possess an L2b genomic backbone. The insertion upstream from CT105 exhibited full LGV specificity, constituting a promising target for the development of rapid LGV diagnostic assays. Conclusions: This study contributes to enhancing the knowledge of C. trachomatis molecular epidemiology, suggesting that the known genetic determinants of AMR are not disseminated in clinical C. trachomatis strains, and presents an exploratory approach that can be suitable for LGV/non-LGV and L2/L2b genomic background differentiation.
- Distribution of Chlamydia trachomatis ompA-genotypes over three decades in PortugalPublication . Lodhia, Zohra; Cordeiro, Dora; Correia, Cristina; João, Inês; Carreira, Teresa; Vieira, Luís; Nunes, Alexandra; Ferreira, Rita; Schäfer, Sandra; Aliyeva, Elzara; Portugal, Clara; Monge, Isabel; Pessanha, Maria Ana; Toscano, Cristina; Côrte-Real, Rita; Antunes, Marília; Gomes, Joao Paulo; Borges, Vítor; Borrego, Maria JoséObjectives: Chlamydia trachomatis is classified into 15 major genotypes, A to L3, based on the diversity of ompA gene. Here, we evaluated and characterised the distribution and diversity of ompA-genotypes over 32 years (1990–2021) in Portugal. Methods: The collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections includes 5824 C. trachomatis-positive samples that were successfully ompA-genotyped between 1990 and 2021. An in-depth analysis of ompA-genotypes distribution across the years, as well as by biological sex, age and anatomical site of infection was performed. Results: ompA-genotype E was consistently the most frequently detected across the years, with a median frequency of 34.6%, followed by D/Da (17.6%), F (14.3%) and G (10.7%). The prevalence of lymphogranuloma venereum (LGV) genotypes (mostly L2, 62.0%, followed by L2b, 32.1%) increased since 2016, reaching the highest value in 2019 (20.9%). LGV, G and Da genotypes were associated with biological sex, specifically with being male, and were the most frequent among anorectal specimens (37.7%, 19.4% and 17.7%, respectively). Notably, LGV ompA-genotypes represented 38.9% of the male anorectal specimens since 2016, and were also detected among oropharynx and urogenital samples. ompA-genotype E was the most frequently detected at the oropharynx (28.6%) and urogenital (33.9%) sites during the study period, followed by D/Da (17.4%) and F (16.0%) in the urogenital specimens, and by G (26.1%) and D/Da (25.7%) in oropharynx specimens. Our data also highlight the emergence of the recombinant L2b/D-Da strain since 2017 (representing between 2.0% and 15.5% of LGV cases per year) and the non-negligible detection of ompA-genotype B in urogenital and anorectal specimens. Conclusions: This study provides a comprehensive landscape of C. trachomatis molecular surveillance in Portugal, highlighting the continued relevance of ompA-genotyping as a complement to rapid LGV-specific detection tests. It also contributes to a deeper understanding of C. trachomatis epidemiology, diversity and pathogenicity.
- Distribution of the West Nile Virus vector, Culex pipiens, in mainland Portugal: A geospatial modelling studyPublication . Martinho, Júlia; Costa Osório, Hugo; Amaro, Fátima; Silva, Manuel; Marques Zé-Zé, Líbia Maria; Pereira Figueira Alves, Maria João; Nunes, Baltazar; Soares, PatriciaBackground: Culex pipiens, Portugal’s most abundant mosquito, is a vector for several pathogens including the West Nile virus. Understanding its spatial distribution can contribute to vector-borne diseases control and public health planning, given Portugal’s favourable climate. National-level data on its spatial distribution, especially in relation to climatic variables is limited. We aimed to predict the suitability of Culex pipiens distribution in mainland Portugal, considering climatic factors. Methods: A maximum entropy (Maxent) model was applied, using presence records for adult and larvae of Culex pipiens mosquitoes sampled across mainland Portugal between January 2017 and October 2023, as part of the REVIVE – Vector Surveillance Network program. Adults were sampled using CDC light traps and BG-Sentinel traps between May and October at random sites and year-round at point-of-entry sites. Larvae were sampled using dippers at breeding sites. Sampling bias was corrected by filtering presence records to one per 1 km2 cell grid. Climatic data, including temperature, precipitation and elevation, were used as predictors. Results: Out of 6,859 records, 354 unique sites were obtained after filtering and cell-duplicate removal. Suitable habitats seem to be primarily in the northern and central coastal regions. Temperature was the most important predictor. Convenience sampling bias may be present. Conclusions: Most West Nile virus case reports have come from southern Portugal, but Culex pipiens’s potential distribution covers the entire mainland territory, with seemingly higher distribution in the north. West Nile vector surveillance should be a priority in all regions to accurately assess transmission risk and implement effective control measures.
- Probe-based metagenomic pathogen detection: advancing laboratory capacity for complex diagnosisPublication . Ferreira, Rita; Coelho, Luís; Santos, João Dourado; Sobral, Daniel; Isidro, Joana; Mixão, Verónica; Pinto, Miguel; Nunes, Alexandra; Borrego, Maria José; Lopo, Sílvia; Oleastro, Mónica; Sousa, Rita; Palminha, Paula; Veríssimo, Cristina; Gargaté, Maria João; Guiomar, Raquel; Cordeiro, Rita; Macedo, Rita; Bajanca-Lavado, Paula; Paixão, Paulo; Duarte, Sílvia; Vieira, Luís; Borges, Vítor; Gomes, João PauloProbe-based pathogen enrichment, followed by NGS, is a promising tool for complex diagnosis, overcoming traditional challenges of shotgun metagenomics, namely small microbial/human genetic material ratio and demanding computational resources. Here, we assessed the combined detection performance of two Illumina probe-based panels, the Respiratory and the Urinary Pathogen ID panels (RPIP and UPIP), using 99 clinical samples of 15 different matrices (e.g., cerebrospinal fluid, plasma, serum, urine, swabs, biopsies, etc.) available from Portuguese National Reference Laboratories. This sample set involved 114 "PCR-positive hits" (Ct values range of 9.7-41.3; median of 28.4) for 52 non-redundant human pathogens. For a more detailed bioinformatics assessment, as a complement of the Illumina turnkey solution (Explify), we applied an extended version of our INSaFLU-TELEVIR(+) metagenomics pipeline. Whereas Explify analyses resulted in an initial detection frequency of 73.7% (84/114), the subsequent application of INSaFLU-TELEVIR(+), including taxonomic classification followed by confirmatory read mapping, enabled an overall detection proportion of 79.8% (91/114) of the PCR-positive hits. This translated into a detection rate increment from 54.3% (19/35) to 65.7% (23/35) for bacteria, and from 85.3% (58/68) to 89.7% (61/68) for viruses. The implemented workflow was also very satisfactory for samples with qPCR Ct values above 30, with an overall detection frequency of 71.8% (28/39) when compared with the 92.0% (46/50) observed for those with Ct ≤ 30. In summary, this study validated and established a pioneering approach at the Portuguese National Institute of Health to support clinicians in complex diagnosis, contributing to advance diagnostic capabilities toward a more informed clinical decision and potential improvement of infectious disease outcomes.
- Whole-genome sequencing-based surveillance system for Mycobacterium tuberculosis in PortugalPublication . Pinto, Miguel; Macedo, RitaTo improve TB surveillance and diagnosis, the Portuguese National Reference Laboratory (NRL) began implementing whole-genome sequencing (WGS) for all RR/MDR-TB cases in 2019. Since 2020, this approach has been expanded to indiscriminately include all received isolates. We describe the current WGS-based surveillance system in Portugal, framed in prospective and retrospective data (n = 1171), upgraded for antimicrobial resistance (AMR) prediction and epidemiological analysis. This system relies on three main steps: QC/QA and contamination assessment, with a novel data filtering step; genotyping and AMR prediction; and dynamic SNP-based approach, maximizing variable sites under analysis. While lineage 4 was the most prevalent (84.3 %) followed by lineage 2 (9.1 %), less common EU/EEA sub-lineages (e.g., lineages 3 and 6) showcased cross-border transmissions. Molecular clusters (n = 157) displayed distinct AMR profiles and diverse possible epidemiological contexts. Among the pipeline upgrades, we highlight: i) the novel filtering step that allowed the improvement of 123 out of 128 contaminated samples; ii) tolerating missing data per site more than doubled core variable site resolution; iii) automatic maximization of shared variable sites for in-depth cluster analysis, key for consolidating genetic links in epidemiological investigation. This study highlights the importance of sustained prospective genomic surveillance towards strengthening TB management and diagnosis in Portugal.
- Source attribution of human infection: a multi-country model in the European UnionPublication . Thystrup, Cecilie; Brinch, Maja Lykke; Henri, Clementine; Mughini-Gras, Lapo; Franz, Eelco; Wieczorek, Kinga; Gutierrez, Montserrat; Prendergast, Deirdre M.; Duffy, Geraldine; Burgess, Catherine M.; Bolton, Declan; Alvarez, Julio; Lopez-Chavarrias, Vicente; Rosendal, Thomas; Clemente, Lurdes; Amaro, Ana; Zomer, Aldert L.; Joensen, Katrine Grimstrup; Nielsen, Eva Møller; Scavia, Gaia; Skarżyńska, Magdalena; Pinto, Miguel; Oleastro, Mónica; Cha, Wonhee; Thépault, Amandine; Rivoal, Katell; Denis, Martine; Chemaly, Marianne; Hald, TineIntroduction: Infections caused by spp. represent a severe threat to public health worldwide. National action plans have included source attribution studies as a way to quantify the contribution of specific sources and understand the dynamic of transmission of foodborne pathogens like and . Such information is crucial for implementing targeted intervention. The aim of this study was to predict the sources of human campylobacteriosis cases across multiple countries using available whole-genome sequencing (WGS) data and explore the impact of data availability and sample size distribution in a multi-country source attribution model. Methods: We constructed a machine-learning model using -mer frequency patterns as input data to predict human campylobacteriosis cases per source. We then constructed a multi-country model based on data from all countries. Results using different sampling strategies were compared to assess the impact of unbalanced datasets on the prediction of the cases. Results: The results showed that the variety of sources sampled and the quantity of samples from each source impacted the performance of the model. Most cases were attributed to broilers or cattle for the individual and multi-country models. The proportion of cases that could be attributed with 70% probability to a source decreased when using the down-sampled data set (535 vs. 273 of 2627 cases). The baseline model showed a higher sensitivity compared to the down-sampled model, where samples per source were more evenly distributed. The proportion of cases attributed to non-domestic source was higher but varied depending on the sampling strategy. Both models showed that most cases could be attributed to domestic sources in each country (baseline: 248/273 cases, 91%; down-sampled: 361/535 cases, 67%;). Discussion: The sample sizes per source and the variety of sources included in the model influence the accuracy of the model and consequently the uncertainty of the predicted estimates. The attribution estimates for sources with a high number of samples available tend to be overestimated, whereas the estimates for source with only a few samples tend to be underestimated. Reccomendations for future sampling strategies include to aim for a more balanced sample distribution to improve the overall accuracy and utility of source attribution efforts.
- Signature cytokine-associated transcriptome analysis of effector γδ T cells identifies subset-specific regulators of peripheral activationPublication . Inácio, Daniel; Amado, Tiago; Pamplona, Ana; Sobral, Daniel; Cunha, Carolina; Santos, Rita F.; Oliveira, Liliana; Rouquié, Nelly; Carmo, Alexandre M.; Lesourne, Renaud; Gomes, Anita Q.; Silva-Santos, Brunoγδ T cells producing either interleukin-17A (γδ cells) or interferon-γ (γδ cells) are generated in the mouse thymus, but the molecular regulators of their peripheral functions are not fully characterized. Here we established an Il17a-GFP:Ifng-YFP double-reporter mouse strain to analyze at unprecedented depth the transcriptomes of pure γδ cell versus γδ cell populations from peripheral lymph nodes. Within a very high fraction of differentially expressed genes, we identify a panel of 20 new signature genes in steady-state γδ cells versus γδ cells, which we further validate in models of experimental autoimmune encephalomyelitis and cerebral malaria, respectively. Among the signature genes, we show that the co-receptor CD6 and the signaling protein Themis promote the activation and proliferation of peripheral γδ cells in response to T cell antigen receptor stimulation in vitro and to Plasmodium infection in vivo. This resource can help to understand the distinct activities of effector γδ T cell subsets in pathophysiology.
- Wolbachia Screening in Aedes aegypti and Culex pipiens Mosquitoes from Madeira Island, PortugalPublication . Fernandes, Rita; Melo, Tiago; Marques Zé-Zé, Líbia Maria; Campos Freitas, Inês; Silva, Manuel; Dias, Eva; Santos, Nuno C.; Gouveia, Bruna R.; Seixas, Gonçalo; Costa Osório, HugoSimple Summary: Mosquitoes can spread serious diseases like dengue and West Nile virus. On Madeira Island, two mosquito species—Aedes aegypti and Culex pipiens—are present and may pose a risk to public health. Scientists are exploring new ways to control these mosquitoes using a natural bacterium called Wolbachia, which can reduce a mosquito’s ability to transmit viruses and even lower mosquito populations. However, for these methods to work, it is important to know first if the mosquitoes in the area already carry this bacterium. In this study, we tested Ae. aegypti and Cx. pipiens from Madeira for Wolbachia. Wolbachia was absent in all 100 Ae. aegypti tested but present in all 40 Cx. pipiens. We also found that the Wolbachia in Cx. pipiens belonged to a group commonly seen in other parts of the world. These results are important because they help us understand which mosquito control strategies might work in Madeira. Specifically, if scientists want to use Wolbachia to control Ae. aegypti on the island, they would need to introduce it artificially. This information can help improve public health efforts and reduce the risk of mosquito-borne diseases in the region.