Alessandro Graziani

Wastewater-based epidemiology (WBE) has emerged as a supplement to clinical investigations, facilitating the acquisition of timely and non-invasive epidemiological data. In this study, a one-year WBE study was conducted in Perugia (Umbria, Italy) to evaluate respiratory viruses using multiplex assays. Fifty-two wastewater samples were collected weekly between October 2023 and September 2024 and analyzed with the Allplex™ Respiratory Panel 2/3 (for Adenovirus (AdV), Enterovirus (EV), Metapneumovirus (MPV), Parainfluenza virus types 1/2/3/4 (PIV1–4), Bocavirus (BoV), Alphacoronavirus 229E, Alphacoronavirus NL63, Betacoronavirus OC43, and Rhinovirus (RV)) and FTD respiratory pathogens 21 (only for IFV-A and RSV). The results were descriptively compared with clinical data to explore temporal concordance between wastewater and clinical surveillance. Wastewater analysis revealed frequent detection of AdV, EV, and BoV, whereas RV was detected in 67.3% of samples. AdV, EV, and BoV were detected more frequently in wastewater than in clinical samples. RSV showed a seasonal pattern in clinical samples but was only sporadically detected in wastewater, whereas PIVs were detected more frequently in clinical samples than in wastewater. By descriptive comparison, IFV-A, MPV, and non-SARS-CoV-2 coronaviruses showed peaks occurring in similar periods in wastewater and clinical samples. These results support the potential of WBE as a complementary tool to traditional clinical surveillance and suggest that commercial multiplex assays may be useful for broad, exploratory screening of respiratory viral targets in wastewater.

HEV genotype 3 is classified into several subtypes. In Italy, 3f, 3c, 3e and, rarely, 3a subtypes are usually detected in human hepatitis E cases: the present study documents, for the first time, the detection of the 3i subtype, previously described in Italy exclusively from wild boars. Routine surveillance by short Sanger sequences highlighted five hepatitis E cases with unusual HEV subtype between 2019 and 2023 in a small geographical area in Umbria, Central Italy. Further characterization of the whole HEV genome by NGS was successful in three of them. Through phylogenetic and p-distance analysis, all the sequences could be classified as subtype 3i, and three of them proved to be highly related to some strains observed in wild boars sampled in the same geographical area. The 3i subtype had never been detected previously in humans in Italy; it is likely that increased local circulation of 3i-like strains in wild boars may have increased the chance for their transmission to humans. Investigation of the possible risk factors for HEV infection highlighted consumption of raw/undercooked products from wild boars/pigs in three cases. However, in the remaining two cases, this source seems unlikely, and transmission might have occurred indirectly from contaminated sources, such as wastewater and home-grown vegetables. Further studies are needed to investigate if these latter sources may be more common than previously thought or may play a role in relation to specific HEV subtypes and/or only in rare and exceptional circumstances.

Background: Biofilms consist of complex microbial communities embedded in an extracellular matrix which confer resistance to the most used antimicrobial agents. Chronic wounds are often associated with burns, trauma, surgery, diabetes and peripheral vascular disease. They are characterized by a marked delay in wound healing favoring the development of microbial biofilms, which in turn further delay tissue regeneration. Staphylococcus aureus, Staphylococcus epidermidis, and methicillin-resistant staphylococci biofilms are found in chronic wounds, seriously hindering wound treatment. Vitreoscilla filiformis, a Gram-negative non-pathogenic filamentous bacterium, has been shown to improve atopic dermatitis by reducing S. aureus colonization and inducing antioxidant responses in the skin. Objectives: The aim of the present study was to evaluate the antimicrobial, anti-inflammatory, and regenerative activities of the V. filiformis supernatant (VFS). Methods: The effect of VFS on bacteria growth was assessed by microbial growth kinetics and biofilm formation and dispersal. Antioxidant potential was determined by DPPH-scavenging ability and reduction in intracellular reactive oxygen species (ROS). The regenerative properties were assessed by scratch assay. Results: V. filiformis VFS holds strong anti-biofilm activity against S. aureus, S. epidermidis and methicillin-resistant S. aureus (MRSA), acting during both biofilm formation and dispersion. The decrease in biofilm mass is accompanied by a significant increase in the planktonic form compared to the untreated cells. Moreover, VFS is characterized by an interesting antioxidant activity, as demonstrated by a cell-free DPPH assay and a neutrophil-based in vitro assay. In addition, VFS can stimulate tissue regeneration in human dermal fibroblasts and keratinocytes. Conclusions: The demonstration of anti-biofilm, antioxidant and regenerative properties of V. filiformis supernatant could be exploited for the treatment of biofilm-associated wound infections.

The SIS-NET ICU study aimed to describe the epidemiology of severe community-acquired pneumonia (CAP) among patients admitted to Italian intensive care uni

Background/Objectives: Chronic skin wounds are characterized by inflammation, persistent infections, and tissue necrosis. The presence of bacterial biofilms prolongs the inflammatory response and delays healing. Ozone is a potent antimicrobial molecule, and many formulations have been used in the advanced therapeutic treatment of chronic wounds. The aim of this work was to determine the antimicrobial, anti-inflammatory, and regenerative activity of a stable ozone-gel formulation over time. Methods: The antimicrobial property was assessed by measuring the minimal inhibitory concentration and the antibiofilm activity. The anti-inflammatory effect was evaluated by TNF-α determination, and the regenerative effect was measured by scratch assay. Results: The ozone gel demonstrated antimicrobial and antibiofilm activity in all ATCC microorganisms examined and on most clinical isolates. Higher concentrations of the ozone gel were also useful in the dispersion of preformed biofilm. The ozone gel also showed anti-inflammatory activity by reducing the production of TNF-α and regenerative activity in human fibroblasts and keratinocytes. Conclusions: Given all these antimicrobial, anti-inflammatory, and regenerative characteristics, the ozone gel could be, in this formulation, used in the treatment of wounds. The ozone-gel formulation described here retains stability for over 30 months, which facilitates its use compared to formulations that lose efficacy quickly.

Hepatitis E virus (HEV) is a global health problem, causing an estimated 20 million infections annually. Thus, the management of HEV requires special consideration. In developed countries, hepatitis E is mainly recognized as a foodborne disease (mainly transmitted via undercooked meat consumption) that is generally caused by genotype 3 and 4 circulating in various animals, including pigs and wild boars. The current absence of officially recognized protocols for the analysis of HEV in foods and the lack of awareness of this disease among healthcare workers, together with the high percentage of asymptomatic cases, make HEV infection highly underestimated. Most HEV-3 infections in immunocompetent individuals are self-limited. Nevertheless, the possibility of serious forms of liver disease, especially in patients with co-morbidities, should be considered because it can lead to a fatal outcome. Here, we report a case of fatal hepatitis related to HEV-3 infection in a 67-year-old male patient with underlying chronic liver disease (CLD) and living in a region where a high prevalence and genetic heterogeneity of HEV-3 in wild boar has been recently demonstrated. Our case report describes the interdisciplinary approach used (from the diagnosis to the virus phylogenetic characterization) in order to improve epidemiologic HEV surveillance in central Italy.

The emergence of SARS-CoV-2 and the non-pharmacological interventions adopted to counter its spread appear to have led to changes in the normal circulation and seasonality of respiratory viruses. Our study aims to investigate changes related to the circulation of respiratory viruses, not SARS-CoV-2, among hospitalized patients in Perugia, Central Italy, between 2019 and 2023. The samples were collected from individuals who went to the emergency room (ER) or were hospitalized and analyzed using a molecular multiplex test. The results underline that non-pharmaceutical interventions altered the typical seasonal circulation patterns of different respiratory viruses. Those mostly affected were enveloped viruses like influenza viruses that disappeared in 2021; the least impact was recorded for Rhinovirus, which was detected during the pandemic period, maintaining the same seasonality observed in the pre-pandemic period although with a reduction in the number of positive samples. Our data underline the importance of the continuous monitoring of these viruses, especially to understand the timing with which prevention measures, not only non-pharmacological interventions but also the equipment of vaccine doses and monoclonal antibodies, should be adopted to reduce their circulation, particularly in the population at risk of developing severe forms of lower respiratory tract infection.

Consumption of raw or undercooked wild boar (WB) meat is considered an important risk factor for hepatitis E virus (HEV) infection in humans. The possibility of HEV contamination during the slaughtering practices may pose an additional risk. Based on these assumptions, we evaluated HEV contamination of WB meat hunted in Umbria (central Italy) during the 2022–2023 hunting season by real-time RT-PCR. Herein, we show that 10.8% of livers from slaughtered WB were positive for HEV RNA, thus providing an estimate of HEV infection in WB in the Umbria region. Then, by evaluating paired liver–muscle samples from both HEV-positive and HEV-negative animals, we found evidence of muscle HEV contamination in 33% and 14% of cases, respectively. This is the first report on the detection of HEV in WB meat in Umbria, an Italian region with diffuse WB hunting and consumption. The evidence of contamination provided by our study underscores the importance of adopting good hygienic practices in the processing stages of hunted WB carcasses to significantly reduce meat contamination and the risk posed for the final consumer.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 6 million deaths worldwide, and the spread of new variants over time increased the ability of this virus to cause infection. The Omicron variant was detected for the first time in Umbria, a region of central Italy, in November 2021 and it induced an unprecedented increase in the number of infection cases. Here, we analysed 3300 SARS-CoV-2 positive samples collected in Umbria between April 2022 and December 2023. We traced the molecular evolution of SARS-CoV-2 variants over time through the Next-Generation Sequencing (NGS) approach. We assessed correlation between SARS-CoV-2 infection and patients’ health status. In total, 17.3% of our samples came from patients hospitalised as a consequence of COVID-19 infection even though 81.4% of them received at least three vaccine doses. We identified only Omicron variants, and the BA.5 lineage was detected in the majority of our samples (49.2%). Omicron variants outcompeted each other through the acquisition of mutations especially in Spike glycoprotein that are fingerprints of each variant. Viral antigenic evolution confers higher immunological escape and makes a continuous improvement of vaccine formulation necessary. The continuous update of international genomic databases with sequencing results obtained by emergent pathogens is essential to manage a possible future pandemic.

The pathogenesis of coronavirus disease 2019 (COVID-19) is associated with a hyperinflammatory response. The mechanisms of SARS-CoV-2-induced inflammation are scantly known. Methylglyoxal (MG) is a glycolysis-derived byproduct endowed with a potent glycating action, leading to the formation of advanced glycation end products (AGEs), the main one being MG-H1. MG-H1 exerts strong pro-inflammatory effects, frequently mediated by the receptor for AGEs (RAGE). Here, we investigated the involvement of the MG-H1/RAGE axis as a potential novel mechanism in SARS-CoV-2-induced inflammation by resorting to human bronchial BEAS-2B and alveolar A549 epithelial cells, expressing different levels of the ACE2 receptor (R), exposed to SARS-CoV-2 spike protein 1 (S1). Interestingly, we found in BEAS-2B cells that do not express ACE2-R that S1 exerted a pro-inflammatory action through a novel MG-H1/RAGE-based pathway. MG-H1 levels, RAGE and IL-1β expression levels in nasopharyngeal swabs from SARS-CoV-2-positive and -negative individuals, as well as glyoxalase 1 expression, the major scavenging enzyme of MG, seem to support the results obtained in vitro. Altogether, our findings reveal a novel mechanism involved in the inflammation triggered by S1, paving the way for the study of the MG-H1/RAGE inflammatory axis in SARS-CoV-2 infection as a potential therapeutic target to mitigate COVID-19-associated pathogenic inflammation.