In the last 6 years, Italy accounted for 36 % of the total autochthonous European West Nile virus (WNV) cases reported to ECDC. Since 2001, the country put in place a multi-species national surveillance plan. The plan was enhanced in 2020 by adopting a fully integrated “One Health” approach, including human, wild bird, equine, and mosquito surveillance for the early detection of WNV. In this context, the systematic acquisition of whole viral genetic information from human patients and animals is fundamental to obtain an in-depth knowledge on the patterns of virus evolution and transmission and to gain insights on the role virus genetics in morbidity and mortality, The purpose of this pilot study was thus to design a One-Health surveillance framework based on the genomic surveillance of WNV circulating at the vector-human-animal interface, in the endemic territory of Romagna (North-Eastern Italy) during the 2023 transmission season. Whole genome sequencing (WGS) analyses confirmed the circulation of WNV lineage 2 showing high nucleotide and amino acid identity of 99.82 % and 99.92 % respectively among viral sequences from human patients, vectors and birds. All the sequences clustered with other Italian strains in the Central and Southern European clade with robust bootstrap support and BLASTn identity exceeding 99.7 %. The highest nucleotide identity was observed with sequences from Emilia-Romagna and Veneto regions (Italy), confirming a local virus circulation and overwintering of WNV lineage 2 with a confined virus spread and no (or limited) external introduction of viral strains. Our results, support the adoption of a One Health approach to WNV surveillance, based on WGS and integrating the clinical diagnosis, epidemiology, and genomic characterisation, to create a suitable operational process for the characterisation of autochthonous and imported Arboviruses circulating in Romagna to effectively integrate the already established surveillance plan.

Developing low-cost vaccine production strategies is crucial to achieving global health equity and mitigating the spread and impact of disease outbreaks. High hydrostatic pressure (HHP) technology is a widely used technology employed in the food industry for long-term preservation. This project aims at validating HHP as a cost-effective method for the production of highly immunogenic thermal stable whole-virus SARS-CoV-2 vaccines. Structural studies on HHP-inactivated viruses demonstrated pressure-dependent effects, with higher pressures (500–600 MPa) destabilizing viral morphology. Immunogenicity assessments, in animal models, revealed that 500 MPa treatment elicited the most robust humoral and cellular immune responses, outperforming heat inactivation. Additionally, HHP-inactivated viral preparation retained thermostability for 30 days at 4 °C, reducing cold-chain dependencies and enabling vaccine distribution also in low-resource settings. With its rapid, cost-effective, and scalable production process, HHP presents a transformative, equitable solution for global vaccine development, particularly for emerging pathogens.

Since the first SARS-CoV-2 outbreak, mutations such as single nucleotide polymorphisms (SNPs) and insertion/deletions (INDELs) have changed and characterized the viral genome sequence, structure and protein folding leading to the onset of new variants. The presence of those alterations challenges not only the clinical field but also the diagnostic demand due to failures in gene detection or incompleteness of polymerase chain reaction (PCR) results. In particular, the analysis of understudied genes such as N and the investigation through whole-genome next generation sequencing (WG-NGS) of regions more prone to mutate can help in the identification of new or reacquired mutations, with the aim of designing robust and long-lasting primers. In 48 samples of SARS-CoV-2 (including Alpha, Delta and Omicron variants), a lack of N gene amplification was observed in the genomes analyzed through WG-NGS. Three gene regions were detected hosting the highest number of SNPs and INDELs. In several cases, the latter can interfere deeply with both the sensitivity of diagnostic methodologies and the final protein folding. The monitoring over time of the viral evolution and the reacquisition among different variants of the same mutations or different alterations within the same genomic positions can be relevant to avoid unnecessary consumption of resources.

Toscana virus (TOSV), a Phlebovirus transmitted by sandflies, is a leading cause of aseptic meningitis in the Mediterranean region. Despite its clinical significance, underreporting and limited availability of complete genomic data hinder a thorough understanding of its genetic diversity and evolution. This study presents a novel amplicon-based whole-genome sequencing (WGS) method using Illumina library preparation kits and proprietary software to optimize workflows and enhance bioinformatic analyses. Primers targeting TOSV lineage A genomes were designed with PrimalScheme to generate 400 bp amplicons, incorporating degenerate bases to improve coverage. Library preparation utilized Illumina Microbial Amplicon Prep (iMAP) kits, followed by de novo assembly using BaseSpace DRAGEN Targeted Microbial software. The method’s sensitivity was tested on viral propagates at various RNA concentrations (10⁴ to 10 copies/μL), demonstrating robust performance at concentrations above 10² copies/μL. Validation on high-titre viral propagates (n = 7), low-titre clinical samples (n = 15), and phlebotomine pools (n = 5) confirmed its reproducibility and ability to comprehensively cover coding regions. Cerebrospinal fluid samples yielded the most consistent results compared to urine and sandfly pools. This innovative WGS approach represents a significant advancement in TOSV genomic surveillance, enabling large-scale studies of its genetic diversity and evolutionary dynamics, which are critical for improving diagnostics and public health strategies.

Sexually transmitted infections (STIs) have seen a considerable increase in the last years and given the health burden they may represent from both a personal and community perspective, they require surveillance and prevention programmes based on a timely and decentralized diagnosis. In this context, user-friendly rapid molecular tests may represent a good trade-off between diagnostic accuracy, accessibility and affordability. In this study we evaluated the diagnostic performance of a new real-time LAMP (Loop Mediated Isothermal Amplification) method for the rapid detection and differentiation of 7 major sexually transmissible pathogens by analysing real clinical samples (genital and extra-genital matrices) from individuals with suspected STIs. The assay showed good overall diagnostic performances in terms of sensitivity, specificity and concordance with a gold-standard PCR-based molecular method. This assay, not requiring specialised laboratory technicians or expensive instrumentation, but nonetheless capable of guaranteeing accurate results, is within the reach of outpatient settings, obstetrics, and gynaecology clinic, hence ensuring on-field access to early diagnosis.

Acute infectious gastroenteritis (AIG) is caused by diverse bacterial and viral enteric pathogens with overlapping clinical presentations, limiting conventional pathogen-directed workflows. This retrospective before–after study evaluated the diagnostic, microbiological, operational, and economic impact of multiplex molecular syndromic testing for AIG within a regional hub-and-spoke laboratory network in Italy. Two 19-month periods were compared: a pre-implementation period based on conventional diagnostics and a post-implementation period using multiplex syndromic panels as first-line tests. Diagnostic investigations totalled 25,574 before and 24,509 after implementation. Overall test positivity increased from 5.43% to 11.59% (p < 0.001), with significant increases for bacterial and viral targets. Pathogen detection events increased from 1388 to 2839, including broader detection of Campylobacter spp., Shigella spp./enteroinvasive Escherichia coli (EIEC), Yersinia enterocolitica, Aeromonas spp., Astrovirus, Sapovirus, and Norovirus genogroups. Mean turnaround time decreased from 63 to 47 h from sample collection and from 56 to 41 h from laboratory check-in. Although direct diagnostic costs increased moderately, total laboratory costs decreased when personnel costs were included, and cost per diagnostic detection declined from €233.80 to €120.86. Multiplex syndromic testing improved microbiological detection, diagnostic efficiency, turnaround time, and laboratory organisation in routine AIG diagnosis.