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- Recommendations for estimating and reporting vaccine effectiveness by time since vaccination: a COVID-19 case studyPublication . Kissling, Esther; Nunes, Baltazar; Hooiveld, Mariëtte; Martínez-Baz, Iván; Monge, Susana; Robertson, Chris; Knolm, Mirjam; Sève, Noémie; Mlinarić, Ivan; Domegan, Lisa; Machado, Ausenda; Whitaker, Heather; Lazar, Mihaela; Meijer, Adam; Enkirch, Theresa; Casado, Itziar; Pérez-Gimeno, Gloria; William, Naoma; Enouf, Vincent; Kurečić Filipović, Sanja; McKenna, Adele; Rodrigues, Ana Paula; de Lusignan, Simon; Timnea, Olivia-Carmen; Latorre-Margalef, Neus; Castilla, Jesús; Pozo, Francisco; Hamilton, Mark; Masse, Shirley; Ilić, Maja; Basile, Luca; O’Donnell, Joan; Guiomar, Raquel; Riess, Maximilian; Popescu, Rodica-Manuela; M C Rose, Angela; Andrews, Nick; Bacci, Sabrina; Pastore Celentano, Lucia; Valenciano, Marta; Moren, Alain; Beutels, Philippe; Hens, Niel; I-MOVE-COVID-19 and ECDC primary care study teamsEstimating COVID-19 vaccine effectiveness (VE) by time since vaccination (TSV) is essential for understanding how protection may change over time and enables meaningful comparisons across studies. This is important for accurate comparisons of VE against different SARS-CoV-2 variants/sublineages, across age groups, during different periods post vaccination campaign, or by vaccine type/brand. We provide recommendations for case–control VE studies on estimating and reporting VE analyses by TSV, with the aim of improving quality of these estimates. Our recommendations cover study design and pre-analysis considerations, descriptive analyses, choice of categories of TSV, categorical and continuous modelling approaches, and best practices for reporting VE by TSV. Using a real-life case–control study, we apply these recommendations, and include accompanying statistical scripts in R and Stata. These recommendations will serve as a practical resource for researchers conducting VE analyses by TSV. We encourage ongoing refinement of them through input from other study groups.
- Influenza vaccine effectiveness in Europe and the birth cohort effect against influenza A(H1N1)pdm09: VEBIS primary care multicentre study, 2023/24Publication . Kissling, Esther; Maurel, Marine; Pozo, Francisco; Pérez-Gimeno, Gloria; Buda, Silke; Sève, Noémie; Domegan, Lisa; Hooiveld, Mariëtte; Oroszi, Beatrix; Martínez-Baz, Iván; Guiomar, Raquel; Latorre-Margalef, Neus; Mlinarić, Ivan; Lazar, Mihaela; Giménez Duran, Jaume; Dürrwald, Ralf; Enouf, Vincent; McKenna, Adele; de Lange, Marit; Túri, Gergő; Trobajo-Sanmartín, Camino; GOMEZ TEIXEIRA PINTO, VERÓNICA DEL PILAR; Samuelsson Hagey, Tove; Višekruna Vučina, Vesna; Cherciu, Maria Carmen; García Vazquez, Miriam; Erdwiens, Annika; Masse, Shirley; Bennett, Charlene; Meijer, Adam; Kristóf, Katalin; Castilla, Jesús; Rodrigues, Ana Paula; Kurečić Filipović, Sanja; Ivanciuc, Alina Elena; Bacci, Sabrina; Kaczmarek, MarlenaIntroduction: Influenza A(H1N1)pdm09, A(H3N2) and B/Victoria viruses circulated in Europe in 2023/24, with A(H1N1)pdm09 dominance. First influenza infections in childhood may lead to different vaccine effectiveness (VE) in subsequent years. Aim: The VEBIS primary care network estimated influenza VE in Europe using a multicentre test-negative study. Methods: Primary care practitioners collected information and specimens from patients consulting with acute respiratory infection. We estimated VE against influenza (sub)type and clade, by age group and by year of age for A(H1N1)pdm09, using logistic regression. Results: We included 29,958 patients, with 3,054, 1,053 and 311 influenza A(H1N1)pdm09, A(H3N2) and B cases, respectively. All-age VE against influenza A(H1N1)pdm09 was 52% (95% CI: 44-59). By year of age, VE was 27% (95% CI: -2 to 47) at 44 years with peaks at 72% (95% CI: 52-84) and 54% (95% CI: 41-64) among children and those 65 years and older, respectively. All-age A(H1N1)pdm09 VE against clade 5a.2a was 41% (95% CI: 24-54) and -11% (95% CI: -69 to 26) against clade 5a.2a.1. The A(H3N2) VE was 35% (95% CI: 20-48) among all ages and ranged between 34% and 40% by age group. All-age VE against clade 2a.3a.1 was 38% (95% CI: 1-62). All-age VE against B/Victoria was 83% (95% CI: 65-94), ranging between 70 and 92% by age group. Discussion: The 2023/24 VEBIS primary care VE against medically attended symptomatic influenza infection was high against influenza B/Victoria, but lower against influenza A(H1N1)pdm09 and A(H3N2). Clade- and age-specific effects may have played a role in the lower A(H1N1)pdm09 VE.
- Source attribution of human Campylobacter 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, Vicent; Rosendal, Thomas; Clemente, Lurdes; Amaro, Ana; Aldert L. Zomer; 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 Campylobacter 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 Salmonella and Campylobacter. 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 k-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.
- Unmeasured confounding and misclassification in studies estimating vaccine effectiveness against hospitalisation and death using electronic health records (EHRs): an evaluation of a multi-country European retrospective cohort studyPublication . Humphreys, James; Nicolay, Nathalie; Braeye, Toon; Van Evercooren, Izaak; Holm Hansen, Christian; Moustsen-Helms, Ida Rask; Sacco, Chiara; Mateo-Urdiales, Alberto; Castilla, Jesús; Martínez-Baz, Iván; Machado, Ausenda; Soares, Patricia; de Gier, Brechje; Meijerink, Hinta; Monge, Susana; Bacci, Sabrina; Nunes, Baltazar; VEBIS-EHR working groupBackground: Electronic health record (EHR)-based observational studies can rapidly provide real-world data on vaccine effectiveness (VE), though EHR data may be prone to misclassification and unmeasured confounding. Methods: In VEBIS-EHR, a retrospective multi-country COVID-19 VE cohort study, we examined unmeasured confounding using a negative control outcome (death not related to COVID-19) and misclassification due to timing of data extraction. The evaluation spanned two periods (November-December 2023, January-February 2024), encompassing up to 18.7 million individuals across six EU/EEA countries. Vaccine confounding-adjusted hazard ratios (aHRs) were pooled using random-effects meta-analysis. Results: aHRs against non-COVID-19 mortality ranged from 0.35 (95% CI: 0.28-0.44) to 0.70 (0.66-0.73) when comparing vaccinated versus unvaccinated. Delaying EHR data extraction modestly increased the capture of outcome and exposure events, with some variation by vaccination status. Site-level fluctuations in aHRs did not meaningfully alter the overall pooled VE, suggesting stable estimates despite misclassification related to extraction timing. Conclusions: We observed some evidence of unmeasured confounding when using non-COVID-19 deaths as a negative outcome, though the specificity of our negative control must be considered. This result may suggest overestimation of VE, but also the need for further analysis with more specific negative control outcomes and confounding-adjustment techniques. Addressing such confounding using richer data sources and more refined approaches remains critical to ensure accurate, timely VE estimates based on retrospective cohorts constructed using registry data. Extending the delay between the end of observation and data extraction modestly improves the completeness of exposure and outcome data, with limited effect on pooled VE estimates.
- The influence of short-chain fatty acids on the survival and virulence of Arcobacter butzleriPublication . Fonseca, Inês M.; Mateus, Cristiana; Vieira, Alexandre; Domingues, Fernanda; Manageiro, Vera; Oleastro, Mónica; Ferreira, SusanaAims: Arcobacter butzleri, a widespread bacterium linked to gastrointestinal disease, can bypass host colonization resistance mechanisms; however, its response to short-chain fatty acids (SCFAs) remains poorly understood. This study investigated the impact of SCFAs on A. butzleri ’s survival and virulence. Methods and results: Eight A. butzleri isolates were assessed under varying concentrations of individual SCFAs and mixtures (m-SCFAs). Higher SCFAs concentrations inhibited bacterial growth in a strain-dependent manner. Transcript analysis of putative virulence genes revealed upregula- tion of ciaB and flaA across most m-SCFAs concentrations, while luxS expression increased at 90 mM. SCFAs generally reduced bacterial motility, with sodium propionate reducing motility but enhancing biofilm-forming ability in the model strain. Additionally, SCFAs exposure decreased the ability of A. butzleri to adhere to and invade the Caco-2 intestinal epithelial cell line. Whole-genome sequencing of the eight A. butzleri isolates revealed extensive genetic diversity, particularly in virulence- and stress-associated genes, although consistent genot ype/phenot ype correlations were not observed. Conclusions: Altogether, these findings demonstrate that SCFAs modulate A. butzleri survival and virulence, providing novel insights into their significance in shaping pathogen behaviour and host-pathogen interactions.
- The dual action of probiotic lactobacilli in suppressing virulence and survival of Arcobacter butzleriPublication . Vieira, Alexandre; Mateus, Cristiana; Fonseca, Inês M.; Domingues, Fernanda; Oleastro, Mónica; Ferreira, SusanaArcobacter butzleri is a widely distributed foodborne and waterborne pathogen, increasingly recognized as an emerging enteropathogen. Understanding its survival mechanisms and interactions with probiotics is crucial for developing targeted intervention strategies. A. butzleri must withstand various hostile conditions to successfully colonize the gastrointestinal tract, including inhibition by probiotics, such as Limosilactobacillus reuteri, Lactobacillus acidophilus and Lactiplantibacillus plantarum. Thus, this study aimed to assess the survival of A. butzleri under acidic conditions and determine its minimum inhibitory concentration (MIC) for bile salts. Additionally, the antimicrobial potential of the lactobacilli strains was evaluated by analysing the effects of their culture-free supernatant (CFS) on A. butzleri growth, coculture interactions, and biofilm formation. The influence of lactobacilli on A. butzleri was further investigated through competition, displacement and exclusion assays using Caco-2 cell models. The results indicate that lactobacilli strains exhibit tolerance to acidic environments and physiological bile salt concentrations, whereas A. butzleri was more susceptible to acidic stress. The antagonistic effect of lactobacilli was evidenced by growth inhibition of A. butzleri in the presence of CFS or during coculture. However, CFS from certain lactobacilli strains was found to enhance biofilm formation, highlighting potential consequences. Furthermore, while lactobacilli did not demonstrate significant ntagonistic effects in competition assays, they effectively displaced and excluded A. butzleri in the Caco-2 infection model. Overall, these findings suggest that probiotic lactobacilli can inhibit A. butzleri growth, yet their impact on its virulence remains uncertain. This underscores the need for strain-specific probiotic selection to effectively target this pathogen and emphasizes that not every probiotic contribute to the prevention of A. butzleri infections.
- Uropathogenic Escherichia coli in a Diabetic Dog with Recurrent UTIs: Genomic Insights and the Impact of Glucose and Antibiotics on Biofilm FormationPublication . Rodrigues, Inês C.; Ribeiro-Almeida, Marisa; Campos, Joana; Silveira, Leonor; Leite-Martins, Liliana; Ribeiro, Jorge; da Costa, Paula Martins; Prata, Joana C.; Pista, Ângela; Costa, Paulo Martins daRecurrent urinary tract infections (UTIs) pose a significant clinical challenge in both human and veterinary medicine, due to antibiotic-resistant and biofilm-forming bacteria. We hypothesized that high glucose levels in diabetic animals enhance biofilm formation and reduce antibiotic efficacy, promoting infection persistence. This study analyzed Escherichia coli from a diabetic female Labrador Retriever with recurrent UTIs over 18 months, focusing on antimicrobial resistance, biofilm-forming capacity, and genomic characterization. Most isolates (9/11) were resistant to ampicillin and fluoroquinolones. Whole genome sequencing of six selected isolates revealed that they belonged to the multidrug-resistant ST1193 lineage, a globally emerging clone associated with persistent infections. Phylogenetic analysis revealed clonal continuity across six UTI episodes, with two distinct clones identified: one during a coinfection in the second episode and another in the last episode. High-glucose conditions significantly enhanced biofilm production and dramatically reduced antibiotic susceptibility, as evidenced by a marked increase in minimum biofilm inhibitory concentrations (MBICs), which were at least 256-fold higher than the corresponding minimum inhibitory concentration (MIC). Sulfamethoxazole-trimethoprim demonstrated the strongest antibiofilm activity, though this was attenuated in glucose-supplemented environments. This research highlights the clinical relevance of glucosuria in diabetic patients and emphasizes the need for therapeutic strategies targeting biofilm-mediated antibiotic tolerance to improve the management of recurrent UTIs.
- Attributable sources of the five most prevalent non-typhoidal Salmonella serovars across ten European countriesPublication . Teunis, Gijs; Dallman, Timothy J.; Zajac, Magdalena; Skarżyńska, Magdalena; Petrovska, Liljana; Pista, Ângela; Silveira, Leonor; Clemente, Lurdes; Thépault, Amandine; Bonifait, Laetitia; Kerouanton, Annaelle; Chemaly, Marianne; Alvarez, Julio; Soderlund, Robert; Nielsen, Eva Moller; Chattaway, Marie; Burgess, Kaye; Byrne, William; Zomer, Aldert L.; van den Beld, Maaike; Hendrickx, Antoni P.A.; Franz, Eelco; Pires, Sara; Hald, Tine; Mughini-Gras, LapoNon-typhoidal Salmonella is the second most frequently reported zoonotic pathogen in the European Union and European Economic Area. Most human infections are caused by serovars Enteritidis and Typhimurium. Genomic characterisation of Salmonella isolates from humans and animals has become a routine public health surveillance tool in many countries. In this study, the relative contributions of several potential sources of human infection of the five frequently reported Salmonella serovars were estimated using machine-learning methods based on a large, cross-sectional collection of genomes from human cases, and animal and environmental sources, across ten European countries. To define the population structure, core-genome Multilocus Sequence Typing was performed. A supervised machine-learning approach was applied for source attribution in the form of a Random Forest classifier. The source and country attribution models achieved moderate accuracy (F1=0.6–0.9), which is lower than in previous studies using machine-learning on Whole Genome Sequencing data. However, attributions of human clinical isolates to different sources were generally in line with previous findings for these five serovars. While the lack of clonality in some sources hindered their prediction, it is also likely that certain sources (e.g., pets) do not serve as major contributors to human infection. Therefore, in most cases attributing these sources to the livestock species they are typically associated with, is likely appropriate. Country attributions showed that substantial human cases are attributable to countries other than their own, indicating geographical interrelatedness of sources. This highlights the value of internationally harmonised Salmonella-control policies in the food production chain.
- COVID-19 Vaccine Effectiveness Against Medically Attended Symptomatic SARS-CoV-2 Infection Among Target Groups in Europe, October 2024-January 2025, VEBIS Primary Care NetworkPublication . Delaunay, Charlotte Laniece; Verdasca, Nuno; Monge, Susana; Domegan, Lisa; Sève, Noémie; Buda, Silke; Meijer, Adam; Lucaccioni, Héloïse; Torrijos, Miriam López; McKenna, Adele; Enouf, Vincent; Dürrwald, Ralf; In't Velt, Eline; Laiglesia, Mª Ángel de Valcárcel; Bennett, Charlene; Masse, Shirley; Erdwiens, Annika; Hooiveld, Mariette; Mlinarić, Ivan; Túri, Gergő; Rodrigues, Ana Paula; Martínez-Baz, Iván; Lazar, Mihaela; Latorre-Margalef, Neus; Borges, Vitor; Kaczmarek, Marlena; Bacci, Sabrina; Kissling, Esther; European primary care VE groupWe estimated the effectiveness of 2024/25 COVID-19 vaccination against medically attended SARS-CoV-2 infection in Europe, among target groups. We included 3204 patients (8/139 cases vaccinated: 6%; 517/3065 controls vaccinated: 17%) from a multicentre, test-negative design study at primary care level. Vaccine effectiveness was 66% (95% CI: 34-85) overall, 73% (95% CI: 21-94) and 54% (95% CI: -3 to 83) in the first and second months post-vaccination, respectively. Overall vaccine effectiveness was 67% (95% CI: 33-86) among older adults (≥ 60 or ≥ 65 years). This relatively high COVID-19 VE (compared with previous seasons), as well as trends by time since vaccination, should be confirmed with additional data, as sample size was low.
- Genomic Analysis of Antibiotic Resistance and Virulence Profiles in Escherichia coli Linked to Sternal Bursitis in Chickens: A One Health PerspectivePublication . Ribeiro, Jessica; Silva, Vanessa; Freitas, Catarina; Pinto, Pedro; Vieira-Pinto, Madalena; Batista, Rita; Nunes, Alexandra; Gomes, João Paulo; Pereira, José Eduardo; Igrejas, Gilberto; Barros, Lillian; Heleno, Sandrina A.; Reis, Filipa S.; Poeta, PatríciaAbstract: Sternal bursitis is an underexplored lesion in poultry, often overlooked in microbiological diagnostics. In this study, we characterized 36 Escherichia coli isolates recovered from sternal bursitis in broiler chickens, combining phenotypic antimicrobial susceptibility testing, PCRbased screening, and whole genome sequencing (WGS). The genetic analysis revealed a diverse population spanning 15 sequence types, including ST155, ST201, and ST58. Resistance to tetracycline and ciprofloxacin was common, and several isolates carried genes encoding β-lactamases, including blaTEM-1B. Chromosomal mutations associated with quinolone and fosfomycin resistance (e.g., gyrA p.S83L, glpT_E448K) were also identified. WGS revealed a high number of virulence-associated genes per isolate (58–96), notably those linked to adhesion (fim, ecp clusters), secretion systems (T6SS), and iron acquisition (ent, fep, fes), suggesting strong pathogenic potential. Many isolates harbored virulence markers typical of ExPEC/APEC, such as iss, ompT, and traT, even in the absence of multidrug resistance. Our findings suggest that E. coli from sternal bursitis may act as reservoirs of resistance and virulence traits relevant to animal and public health. This highlights the need for including such lesions in genomic surveillance programs and reinforces the importance of integrated One Health approaches.