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The effect of pulsed electromagnetic fields (PEMFs) on the proliferation and survival of matrix-induced autologous chondrocyte implantation (MACI®)-derived cells was studied to ascertain the healing potential of PEMFs. MACI-derived cells were taken from cartilage biopsies 6 months after surgery and cultured. No dedifferentiation towards the fibroblastic phenotype occurred, indicating the success of the surgical implantation. The MACI-derived cultured chondrocytes were exposed to 12 h/day (short term) or 4 h/day (long term) PEMFs exposure (magnetic field intensity, 2 mT; frequency, 75 Hz) and proliferation rate determined by flow cytometric analysis. The PEMFs exposure elicited a significant increase of cell number in the SG2M cell cycle phase. Moreover, cells isolated from MACI® scaffolds showed the presence of collagen type II, a typical marker of chondrocyte functionality. The results show that MACI® membranes represent an optimal bioengineering device to support chondrocyte growth and proliferation in surgical implants. The surgical implant of MACI® combined with physiotherapy is suggested as a promising approach for a faster and safer treatment of cartilage traumatic lesions.

The analytical performance of the new Aspergillus Galactomannan Ag VirCLIA ® mono-test (Vircell S.L.) was compared to the Platelia™ Aspergillus Ag ELISA assay (Bio-Rad). Prospective serum and bronchoalveolar lavage (BAL) samples from patients at risk of invasive aspergillosis (IA) were tested using both the Aspergillus Galactomannan Ag VirCLIA ® mono-test and the Platelia™ Aspergillus Ag ELISA assay. Concordance, sensitivity, specificity, and positive and negative predictive values were calculated using the manufacturer-recommended cutoff levels. Receiver operating characteristic (ROC) analysis and the Youden index were performed to determine the optimal cutoff. A total of 187 serum samples and 73 BAL samples were analyzed with both assays. The concordance between the Aspergillus Galactomannan Ag VirCLIA ® mono-test and the Platelia™ Aspergillus Ag ELISA assay was 87.8%, with a Cohen's kappa of 0.75. The sensitivity and specificity of the Aspergillus Galactomannan Ag VirCLIA ® mono-test were 78.6% and 96.2%, respectively, with positive and negative predictive values of 94.8% and 83.3%. The ROC curve for the Aspergillus Galactomannan Ag VirCLIA ® mono-test demonstrated an area under the curve (AUC) of 0.87, and the Youden index at the manufacturer's established cutoff was 0.73. This new Aspergillus Galactomannan Ag VirCLIA ® mono-test exhibited adequate analytical and clinical performance, showing good correlation with the Platelia™ Aspergillus Ag ELISA assay. The single-sample, semi-automated test is user-friendly, allowing small laboratories to perform the test on demand without the need for batch evaluations, providing a useful solution for timely diagnostic support for clinicians.

Carbapenemase-producing Enterobacteriales (CPE) represent an emerging threat for global public health and a serious problem for clinicians due to the limited available treatment options. The emergence of CPE has been recently described worldwide by describing different antimicrobial mechanisms. Here, we describe a CPE carrying dual-carbapenemase isolated in Italy and we provide a deep characterization of the antimicrobial resistance genes, virulence-factors and prophage regions within the genome.

Here, we characterize the complete genome sequence of Escherichia coli isolated from a newborn affected by bacterial meningitis in Italy. Genome of E. coli strain 1455 harbored a circular chromosome and two plasmids of 167.740-bp and 4.073-bp in length, respectively. E. coli 1455 belonged to the ST3, serotype O17:H18 and carried different determinants including resistance to B-lactams, tetracyclines, and quinolones. In addition, genome of E. coli strain 1455 harbored 5 integrated pro-phage regions mainly located in the chromosome, while most of the virulence factors associated to the invasiveness and clinical severity and different antimicrobial resistance determinants (bla TEM-1 , tet(A) and qnrS1) were located in the 167-Kb plasmid. Taken together, our findings suggest a possible widespread of a virulence factors-carrying plasmid worldwide and highlight the importance of genomic characterization in the diffusion of public health threats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

At the beginning of the COVID-19 pandemic, an ad hoc organisational framework was established between academic, local government and community partners to implement the "Sentinella - Identify, Trace and Prevent" screening programme in Verona, north-east Italy. Between September 2020 and May 2021, key populations not covered by any screening policies at the local and national level were screened for SARS-CoV-2. Target populations were: older adult residents (males >65 years and females >75 years), bus and taxi drivers, social workers, supermarket employees, hospital cleaning and catering staff, researchers working in the local hospitals, students, and people experiencing homelessness (PEH). Five dedicated swab clinics, home testing facilities, and one mobile clinic were activated to collect nasopharyngeal swabs. Molecular analysis was performed for all the subjects; an antigen-rapid diagnostic test (Ag-RDT) was also implemented as a point-of-care test for PEH. Medical follow-up, psychological support, and quarantine facilities were organised for subjects who tested positive for SARS-CoV-2. Overall, 2075 subjects participated in the surveillance programme. Amongst these, 1,572 were residents/workers, whilst 503 were PEH. A total of 127 (6.2%) participants tested positive for SARS-CoV-2. Sixty-nine were residents, 58 PEH. The incidence rate was 4 per 10.000 person/day (95% CI 3.1-5.0). The highest prevalence and incidence rates were found amongst supermarket employees (9.7% and 8.5 per 10.000 person/day, 95% CI 3.81-18.86, respectively), followed by hospital cleaning staff (8.1%, 7.6 per 10.000 person/day, CI 95% 4.9-11.7). Regarding PEH, the prevalence of SARS-CoV-2 was 11.5%. All PEH identified as positive were isolated in dedicated shelter facilities. Amongst the 69 residents/workers who were quarantined, 53 were reached for initial psychological support for assessing the presence of any psychological distress or psychiatric pathology. Amongst the subjects evaluated, 10 (18.9%) presented clinically significant psychological discomfort and accessed the stepped-care psychological intervention. The community partnerships played a pivotal role in optimising early case detection. Promotion of testing helped to prevent and contain more efficiently potential clusters through strategic planning, especially for PEH. Insights from the study highlight the importance of community partnerships in public health emergencies, particularly in the context of highly transmissible diseases pathways., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Tebon, Davis, Savoldi, Soriolo, Walters, Nosè, Barbui, Turrini, Danese, Lippi, Cecchetto, Mazzariol, Gibellini, Caliskan, Marchetti, Verlato, Princivalle, Porru, Tacconelli and De Nardo.)

We evaluated the activity of piperacillin in relation to INCREASING TAZOBACTAM CONCENTRATION against ESBL-producing Enterobacterales collected from patients with bacteraemia. Increasing tazobactam concentration (4, 12 or 24 mg/L) exerted a reduction of piperacillin MICs under the clinical breakpoint in a concentration-dependent manner (0%, 60% and 90% of clinical isolates). Also, activity of piperacillin/tazobactam based at higher achievable serum concentrations (123/14 mg/L) is needed to reduce the bacterial growth in 92% of ESBL-producers. CHANGES IN THE PIPERACILLIN MIC IN RELATION TO INCREASING TAZOBACTAM SUGGEST THAT REALTIME TDM COULD BE USED FOR DRIVEN ANTIMICROBIAL THERAPY WITH PIPERACILLIN/TAZOBACTAM IN BSI DUE TO ESBL STRAINS., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We investigated the activity of cefiderocol/β-lactamase inhibitor combinations against clinical strains with different susceptibility profiles to cefiderocol to explore the potentiality of antibiotic combinations as a strategy to contain the major public health problem of multidrug-resistant (MDR) pathogens. Specifically, we evaluated the synergistic activity of cefiderocol with avibactam, sulbactam, or tazobactam on three of the most "Critical Priority" group of MDR bacteria (carbapenem-resistant Enterobacterales , Pseudomonas aeruginosa , and Acinetobacter baumannii ). Clinical isolates were genomically characterized by Illumina iSeq 100. The synergy test was conducted with time-kill curve assays. Specifically, cefiderocol/avibactam, /sulbactam, or /tazobactam combinations were analyzed. Synergism was assigned if bacterial grow reduction reached 2 log 10 CFU/mL. We reported the high antimicrobial activity of the cefiderocol/sulbactam combination against carbapenem-resistant Enterobacterales , P. aeruginosa , and A. baumannii ; of the cefiderocol/avibactam combination against carbapenem-resistant Enterobacterales ; and of the cefiderocol/tazobactam combination against carbapenem-resistant Enterobacterales and P. aeruginosa. Our results demonstrate that all β-lactamase inhibitors (BLIs) tested are able to enhance cefiderocol antimicrobial activity, also against cefiderocol-resistant isolates. The cefiderocol/sulbactam combination emerges as the most promising combination, proving to highly enhance cefiderocol activity in all the analyzed carbapenem-resistant Gram-negative isolates, whereas the Cefiderocol/tazobactam combination resulted in being active only against carbapenem-resistant Enterobacterales and P. aeruginosa, and cefiderocol/avibactam was only active against carbapenem-resistant Enterobacterales.

The emergence of antimicrobial resistance represents a serious threat to public health and for infections due to multidrug-resistant (MDR) microorganisms, representing one of the most important causes of death worldwide. The renewal of old antimicrobials, such as colistin, has been proposed as a valuable therapeutic alternative to the emergence of the MDR microorganisms. Although colistin is well known to present several adverse toxic effects, its usage in clinical practice has been reconsidered due to its broad spectrum of activity against Gram-negative (GN) bacteria and its important role of "last resort" agent against MDR-GN. Despite the revolutionary perspective of treatment with this old antimicrobial molecule, many questions remain open regarding the emergence of novel phenotypic traits of resistance and the optimal usage of the colistin in clinical practice. In last years, several forward steps have been made in the understanding of the resistance determinants, clinical usage, and pharmacological dosage of this molecule; however, different points regarding the role of colistin in clinical practice and the optimal pharmacokinetic/pharmacodynamic targets are not yet well defined. In this review, we summarize the mode of action, the emerging resistance determinants, and its optimal administration in the treatment of infections that are difficult to treat due to MDR Gram-negative bacteria.

We compared SARS-CoV-2 positivity between the foreign-born adult working population and Italians living in the Verona area to investigate whether being a foreign-born adult could confer an increased risk of infection or lead to a diagnostic delay. The present study included 105,774 subjects, aged 18-65 years, tested for SARS-CoV-2 by nasopharyngeal swabs and analyzed at the University Hospital of Verona between January 2020 and September 2022. A logistic regression model was used, controlling for gender, age, time of sampling, and source of referral. A higher proportion of SARS-CoV-2 positivity in Italian (30.09%) than in foreign-born (25.61%) adults was reported, with a higher proportion of SARS-CoV-2 positivity in men than women in both cohorts analyzed. The difference in swab positivity among Italian and foreign-born adults was the highest in people aged 18-29 years (31.5% vs. 23.3%) and tended to disappear thereafter. Swab positivity became comparable between Italian and foreign-born adults during the vaccination campaign. Multivariable analysis confirmed the lower risk of swab positivity among foreign-born adults (OR = 0.85, 95% CI 0.82-0.89). In the Verona area, foreign-born adults showed a lower rate of SARS-CoV-2 positivity than the native population, likely because of underdiagnosis. Hence, public health should increase attention toward these particularly vulnerable populations., Competing Interests: The authors declare no conflicts of interest.

The availability of new technologies for deep sequencing, including next-generation sequencing (NGS), allows for the detection of viral genome variations. The epidemiological determination of SARS-CoV-2 viral genome changes during the pandemic waves displayed the genome evolution and subsequent onset of variants over time. These variants were often associated with a different impact on viral transmission and disease severity. We investigated, in a retrospective study, the trend of SARS-CoV-2-positive samples collected from the start of the Italian pandemic (January 2020) to June 2023. In addition, viral RNAs extracted from 938 nasopharyngeal swab samples were analyzed using NGS between February 2022 and June 2023. Sequences were analyzed with bioinformatic tools to identify lineages and mutations and for phylogenetic studies. Six pandemic waves were detected. In our samples, we predominantly detected BA.2, BQ.1, BA.5.1, BA.5.2, and, more recently, XBB.1 and its subvariants. The data describe the SARS-CoV-2 genome evolution involved in viral interactions with the host and the dynamics of specific genome mutations and deletions.

Flaviviruses cause numerous pathologies in humans across a broad clinical spectrum with potentially severe clinical manifestations, including hemorrhagic and neurological disorders. Among human flaviviruses, some viral proteins show high conservation and are good candidates as targets for drug design. From an epidemiological point of view, flaviviruses cause more than 400 million cases of infection worldwide each year. In particular, the Yellow Fever, dengue, West Nile, and Zika viruses have high morbidity and mortality-about an estimated 20,000 deaths per year. As they depend on human vectors, they have expanded their geographical range in recent years due to altered climatic and social conditions. Despite these epidemiological and clinical premises, there are limited antiviral treatments for these infections. In this review, we describe the major compounds that are currently under evaluation for the treatment of flavivirus infections and the challenges faced during clinical trials, outlining their mechanisms of action in order to present an overview of ongoing studies. According to our review, the absence of approved antivirals for flaviviruses led to in vitro and in vivo experiments aimed at identifying compounds that can interfere with one or more viral cycle steps. Still, the currently unavailability of approved antivirals poses a significant public health issue.

Next-generation sequencing (NGS) from SARS-CoV-2-positive swabs collected during the last months of 2022 revealed a large deletion spanning ORF7b and ORF8 (426 nt) in six patients infected with the BA.5.1 Omicron variant. This extensive genome loss removed a large part of these two genes, maintaining in frame the first 22 aminoacids of ORF7b and the last three aminoacids of ORF8. Interestingly, the deleted region was flanked by two small repeats, which were likely involved in the formation of a hairpin structure. Similar rearrangements, comparable in size and location to the deletion, were also identified in 15 sequences in the NCBI database. In this group, seven out of 15 cases from the USA and Switzerland presented both the BA.5.1 variant and the same 426 nucleotides deletion. It is noteworthy that three out of six cases were detected in patients with immunodeficiency, and it is conceivable that this clinical condition could promote the replication and selection of these mutations.

SARS-CoV-2, the etiological cause of the COVID-19 pandemic, can cause severe illness in certain at-risk populations, including people with cystic fibrosis (pwCF). Nevertheless, several studies indicated that pwCF do not have higher risks of SARS-CoV-2 infection nor do they demonstrate worse clinical outcomes than those of the general population. Recent in vitro studies indicate cellular and molecular processes to be significant drivers in pwCF lower infection rates and milder symptoms than expected in cases of SARS-CoV-2 infection. These range from cytokine releases to biochemical alterations leading to morphological rearrangements inside the cells associated with CFTR impairment. Based on available data, the reported low incidence of SARS-CoV-2 infection among pwCF is likely a result of several variables linked to CFTR dysfunction, such as thick mucus, IL-6 reduction, altered ACE2 and TMPRSS2 processing and/or functioning, defective anions exchange, and autophagosome formation. An extensive analysis of the relation between SARS-CoV-2 infection and pwCF is essential to elucidate the mechanisms involved in this lower-than-expected infection impact and to possibly suggest potential new antiviral strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lotti, Lagni, Diani, Sorio and Gibellini.)

SARS-CoV-2 infection is mainly detected by multiplex real-time RT-PCR from upper respiratory specimens, which is considered the gold-standard technique for SARS-CoV-2 infection diagnosis. A nasopharyngeal (NP) swab represents the clinical sample of choice, but NP swabbing can be uncomfortable to the patients, especially for pediatric-age participants, requires trained healthcare personnel, and may generate an aerosol, increasing the intrinsic exposure risk of healthcare workers. The objective of this study was to compare paired NP and saliva samples (SS) collected from pediatric patients to evaluate whether the saliva collection procedure may be considered a valuable alternative to the classical NP swab (NPS) sampling in children. In this study, we describe a SARS-CoV-2 multiplex real-time RT-PCR protocol for SS, comparing the results with the paired NPS specimens from 256 pediatric patients (mean age 4.24 ± 4.40 years) admitted to the hospital emergency room of Azienda Ospedaliera Universitaria Integrata (AOUI), Verona, and randomly enrolled between September 2020 and December 2020. The saliva sampling demonstrated consistent results when compared to NPS use. The SARS-CoV-2 genome was detected in 16 out of 256 (6.25%) NP samples, among which 13 (5.07%) were positive even when paired SS were analyzed. Moreover, SARS-CoV-2-negative NPS and SS were consistent, and the overall concordances between NPS and SS were detected in 253 out of 256 samples (98.83%). Our results suggest that saliva samples may be considered a valuable alternative to NPS for SARS-CoV-2 direct diagnosis with multiplex real-time RT-PCR in pediatric patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Diani, Silvagni, Lotti, Lagni, Baggio, Medaina, Biban and Gibellini.)

A total of 866 anaerobic strains isolated from clinical samples were tested by E-TEST for antimicrobial susceptibility. The most frequent antimicrobial resistance among the isolated genera, both Gram-positive and Gram-negative, was observed for clindamycin, and therefore, it cannot be considered as an empirical treatment. The antimicrobial resistance to benzylpenicillin was predominant among the Gram-negative bacteria, in particular the Bacteroides spp. The resistance percentages to meropenem and metronidazole are still low. However, metronidazole showed a considerable resistance in Finegoldia magna isolates, alone or in combination with other antibiotics. These data provide novel and useful epidemiological information on infections promoted by anaerobic bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: Detection strategies in vulnerable populations such as people experiencing homelessness (PEH) need to be explored to promptly recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks. This study investigated the diagnostic accuracy of a rapid SARS-CoV-2 Ag test in PEH during two pandemic waves compared with gold standard real-time multiplex reverse transcription polymerase chain reaction (rtRT-PCR)., Methods: All PEH ≥ 18 years requesting residence at the available shelters in Verona, Italy, across two cold-weather emergency periods (November 2020-May 2021 and December 2021-April 2022) were prospectively screened for SARS-CoV-2 infection by means of a naso-pharyingeal swab. A lateral flow immunochromatographic assay (Biocredit ® COVID-19 Ag) was used as antigen-detecting rapid diagnostic test (Ag-RDT). The rtRT-PCR was performed with Allplex™ SARS-CoV-2 assay kit (Seegene). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated as measures for diagnostic accuracy., Results: Overall, 503 participants were enrolled during the two intervention periods for a total of 732 paired swabs collected: 541 swabs in the first period and 191 in the second. No significant differences in demographic and infection-related characteristics were observed in tested subjects in the study periods, except for the rate of previous infection (0.8% versus 8%; p < 0.001) and vaccination (6% versus 73%; p < 0.001). The prevalence of SARS-CoV-2 in the cohort was 8% (58/732 swabs positive with rtRT-PCR). Seventeen swabs were collected from symptomatic patients (7%). Among them, the concordance between rtRT-PCR and Ag-RDT was 100%, 7 (41.2%) positive and 10 negative pairs. The overall sensitivity of Ag-RDT was 63.8% (95% CI 60.3-67.3) and specificity was 99.8% (95% CI 99.6-100). PPV and NPV were 97.5% and 96.8%, respectively. Sensitivity and specificity did not change substantially across the two periods (65.1% and 99.8% in 2020-2021 vs. 60% and 100% in 2021-2022)., Conclusions: A periodic Ag-RDT-based screening approach for PEH at point of care could guide preventive measures, including prompt isolation, without referral to hospital-based laboratories for molecular test confirmation in case of positive detection even in individuals asymptomatic for COVID-19. This could help reduce the risk of outbreaks in shelter facilities., (© 2023. The Author(s).)

TiO 2 -Ag doped nanoparticulate (TiO 2 -Ag-NP) adhesive photocatalytic films were used to assess the ability in dropping down the burden of indoor microbial particles. The application of an easy-to use photocatalytic adhesive film to cleanse indoor living spaces from microbial pollution, represents a novelty in the field of photocatalytic devices. Reduction was attained by photocatalysis in selected spaces, usually with overcrowding (≥ 3 individuals) in the common working daily hours, and upon indoor microclimate monitoring. TiO 2 -Ag doped nanoparticulate (TiO 2 -Ag-NP) adhesive photocatalytic films were applied within five types of living spaces, including schools and job places. The microbial pollution was assessed at time 0 (far from routine clean, ≥ 9 h) and throughout 2-4 weeks following the photocatalyst application by relative light unit (RLU) luminometry and microbial indirect assessment (colony forming units per cubic meter, CFU/m 3 ). TiO 2 -Ag-NP photocatalyst reduced RLU and CFU/m 3 by rates higher than 70% leading to RLU ≤ 20 and microbial presence ≤ 35 CFU/m 3 . The described TiO 2 -Ag-NP is able to reduce microbial pollution to the lowest RLU threshold (≤ 20) within 60 min in open daylight in a standardized test room of 100 m 2 . The correlation between RLU and CFU/m 3 was positive (r = 0.5545, p < 0.05), assessing that the microbial reduction of indoor areas by the TiO 2 -Ag-NP adhesive film was real. Titania photocatalysts represent promising tools to ensure air cleaning and sanitization in living indoor microclimates with a low cost, feasible and straightforward approach. This approach represents an easy to handle, cost effective, feasible and efficacious approach to reduce microbial pollution in indoor spaces, by simply attaching a TiO 2 -Ag-NP adhesive film on the wall., (© 2023. The Author(s).)

Several reports have indicated that SARS-CoV-2 infection displays unexpected mild clinical manifestations in people with cystic fibrosis (pwCF), suggesting that CFTR expression and function may be involved in the SARS-CoV-2 life cycle. To evaluate the possible association of CFTR activity with SARS-CoV-2 replication, we tested the antiviral activity of two well-known CFTR inhibitors (IOWH-032 and PPQ-102) in wild type (WT)-CFTR bronchial cells. SARS-CoV-2 replication was inhibited by IOWH-032 treatment, with an IC 50 of 4.52 μM, and by PPQ-102, with an IC 50 of 15.92 μM. We confirmed this antiviral effect on primary cells (MucilAir TM wt-CFTR) using 10 μM IOWH-032. According to our results, CFTR inhibition can effectively tackle SARS-CoV-2 infection, suggesting that CFTR expression and function might play an important role in SARS-CoV-2 replication, revealing new perspectives on the mechanisms governing SARS-CoV-2 infection in both normal and CF individuals, as well as leading to potential novel treatments.

COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of proinflammatory genes, similar to that promoted by R848. Furthermore, by using CU-CPT9a, a TLR8-specific inhibitor, we found that SCV2-RNA2 stimulates neutrophils exclusively via TLR8-dependent pathways. In sum, our study proves that single-strand RNAs from the SARS-CoV-2 genome potently activate human neutrophils via TLR8, thus uncovering a potential mechanism whereby neutrophils may contribute to the pathogenesis of severe COVID-19 disease.

Background: Recent in-vitro data have shown that the activity of monoclonal antibodies (mAbs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) varies according to the variant of concern (VOC). No studies have compared the clinical efficacy of different mAbs against Omicron VOC., Methods: The MANTICO trial is a non-inferiority randomised controlled trial comparing the clinical efficacy of early treatments with bamlanivimab/etesevimab, casirivimab/imdevimab, and sotrovimab in outpatients aged 50 or older with mild-to-moderate SARS-CoV-2 infection. As the patient enrolment was interrupted for possible futility after the onset of the Omicron wave, the analysis was performed according to the SARS-CoV-2 VOC. The primary outcome was coronavirus disease 2019 (COVID-19) progression (hospitalisation, need of supplemental oxygen therapy, or death through day 14). Secondary outcomes included the time to symptom resolution, assessed using the product-limit method. Kaplan-Meier estimator and Cox proportional hazard model were used to assess the association with predictors. Log rank test was used to compare survival functions., Results: Overall, 319 patients were included. Among 141 patients infected with Delta, no COVID-19 progression was recorded, and the time to symptom resolution did not differ significantly between treatment groups (Log-rank Chi-square 0.22, p 0.90). Among 170 patients infected with Omicron (80.6% BA.1 and 19.4% BA.1.1), two COVID-19 progressions were recorded, both in the bamlanivimab/etesevimab group, and the median time to symptom resolution was 5 days shorter in the sotrovimab group compared with the bamlanivimab/etesevimab and casirivimab/imdevimab groups (HR 0.53 and HR 0.45, 95% CI 0.36-0.77 and 95% CI 0.30-0.67, p<0.01)., Conclusions: Our data suggest that, among adult outpatients with mild-to-moderate SARS-CoV-2 infection due to Omicron BA.1 and BA.1.1, early treatment with sotrovimab reduces the time to recovery compared with casirivimab/imdevimab and bamlanivimab/etesevimab. In the same population, early treatment with casirivimab/imdevimab may maintain a role in preventing COVID-19 progression. The generalisability of trial results is substantially limited by the early discontinuation of the trial and firm conclusions cannot be drawn., Funding: This trial was funded by the Italian Medicines Agency (Agenzia Italiana del Farmaco, AIFA). The VOC identification was funded by the ORCHESTRA (Connecting European Cohorts to Increase Common and Effective Response to SARS-CoV-2 Pandemic) project, which has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement number 101016167., Clinical Trial Number: NCT05205759., Competing Interests: FM, MM, AS, PD, MM, MT, MA, GD, LC, DD, ES, ED, DG, IM, GS, NM, ET No competing interests declared, LS Lolita Sasset has served as a paid consultant to Abbvie, Janssen, MSD, Gilead Sciences, Janssen, MSD and ViiV Healthcare; she does not have any financial competing interests with this study, AC Annamaria Cattelan has served as a paid consultant to Abbvie, Janssen, MSD, and received research fundings from Gilead Sciences, Janssen, MSD and ViiV Healthcare; she does not have any financial competing interests with this study, CT Carlo Tascini has received grants from Correvio, Biotest, Biomerieux, Gilead, Angelini, MSD, Pfizer, Thermofisher, Zambon, Shionogi, Avir Pharma and Hikma outside the submitted work in the last two years, (© 2022, Mazzaferri et al.)

Background: We described a SARS-CoV-2 thrice-infected case series in health workers (HW) to evaluate patient and virus variants and lineages and collect information on variables associated with multiple infections., Methods: A retrospective analysis of clinical and laboratory characteristics of SARS-CoV-2 thrice-infected individuals was carried out in Verona University Hospital, concurrent with the ORCHESTRA project. Variant analysis was conducted on a subset of available specimens., Results: Twelve HW out of 7368 were thrice infected (0.16%). Symptomatic infections were reported in 63.6%, 54.5% and 72.7% of the first, second and third infections, respectively. Nine subjects were fully vaccinated at the time of the third infection, and five had an additional booster dose. The mean time to second infection was 349.6 days (95% CI, 138-443); the mean interval between the second and third infection was 223.5 days (95% CI, 108-530) ( p = 0.032). In three cases, the second and third infections were caused by the Omicron variant, but different lineages were detected when the second vs third infections were sequenced., Conclusions: This case series confirms evidence of multiple reinfections with SARS-CoV-2, even from the same variant, in vaccinated HW. These results reinforce the need for continued infection-specific prevention measures in previously infected and reinfected HW.

Oral microbiome is a complex population of micro-organisms, which by cross-talking with the local immune system, plays a major role in the immune homeostasis of the oral cavity, further contributing in the physiology of the gastro-intestinal microbiota. Understanding their involvement in the onset and pathogenesis of oropharyngeal cancers is paramount, despite very few reports deal with the fundamental role exerted by oral microbiota disorders, such as dysbiosis and impairment in the oral microbiome composition as causative factors in the development of oropharyngeal tumors. Current research, via metabolomic or meta-transcriptomic analyses, is wondering how this complex microbial population regulates the immune homeostasis in oral and pharyngeal mucosa and whether changes in bacterial composition may give insights on the role of oral microbiome in the development of oropharyngeal tumors, so to prevent their occurrence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nocini, Muzio, Gibellini, Malerba, Milella, Chirumbolo and Zerman.)

Introduction: The anti-spike (S) IgG assay is the most widely used method to assess the immunological response to COVID-19 vaccination. Several studies showed that subjects with perivaccination infection have higher anti-S IgG titers. However, a cut-off has not yet been identified so far for distinguishing infected subjects after vaccination. This study thus evaluates the performance of the anti-S IgG assay in identifying subjects with breakthrough infections (BIs) and its potential usefulness for screening healthcare workers (HCWs). Methods: Out of 6400 HCWs of the University Hospital of Verona vaccinated with two doses of BNT162b2, 4462 never infected before subjects who had completed primary vaccination were tested for IgG anti-S 6 to 9 months after the second dose. Of these, 59 (1.3%) had a BI. The discriminant power of IgG anti-S in detecting previous breakthrough infection was tested by constructing receiver operating characteristic (ROC) curves. Results: The discriminant power for BI was rather good (area under the curve (AUC), 0.78) and increased with decreasing time elapsed between antibody titer assessment and previous SARS-CoV-2 infection. Accuracy (AUC) sensitivity increased from 0.78 (95% CI 0.70−0.85) for BI in the previous six months to 0.83 (95% CI 0.67−0.99) for those in the previous two months, and from 0.68 to 0.80, respectively. The specificity (0.86) and optimal cut-off (935 BAU/mL) remained unchanged. However, BI were rather rare (1.3%), so the positive predictive value (PPV) was low. Only 40 of the 664 HCWs with antibody titer > 935 BAU/mL had previously confirmed BI, yielding a PPV of only 6.0%. When adopting as cut-off the 90th percentile (1180 BAU/mL), PPV increased to 7.9% (35/441). Conclusions: The anti-S IgG assay displayed good sensitivity and specificity in discriminating subjects with BI, especially in recent periods. However, BIs were rare among HCWs, so that the anti-S IgG assay may have low PPV in this setting, thus limiting the usefulness of this test as a screening tool for HCWs. Further studies are needed to identify more effective markers of a previous infection in vaccinated subjects.

SARS-CoV-2 replicates in host cell cytoplasm. People with cystic fibrosis, considered at risk of developing severe symptoms of COVID-19, instead, tend to show mild symptoms. We, thus, analyzed at the ultrastructural level the morphological effects of SARS-CoV-2 infection on wild-type (WT) and F508del (ΔF) CFTR-expressing CFBE41o- cells at early and late time points post infection. We also investigated ACE2 expression through immune-electron microscopy. At early times of infection, WT cells exhibited double-membrane vesicles, representing typical replicative structures, with granular and vesicular content, while at late time points, they contained vesicles with viral particles. ∆F cells exhibited double-membrane vesicles with an irregular shape and degenerative changes and at late time of infection, showed vesicles containing viruses lacking a regular structure and a well-organized distribution. ACE2 was expressed at the plasma membrane and present in the cytoplasm only at early times in WT, while it persisted even at late times of infection in ΔF cells. The autophagosome content also differed between the cells: in WT cells, it comprised vesicles associated with virus-containing structures, while in ΔF cells, it comprised ingested material for lysosomal digestion. Our data suggest that CFTR-modified cells infected with SARS-CoV-2 have impaired organization of normo-conformed replicative structures.

Background: The research aimed to investigate the incidence of SARS-CoV-2 breakthrough infections and their determinants in a large European cohort of more than 60,000 health workers., Methods: A multicentric retrospective cohort study, involving 12 European centers, was carried out within the ORCHESTRA project, collecting data up to 18 November 2021 on fully vaccinated health workers. The cumulative incidence of SARS-CoV-2 breakthrough infections was investigated with its association with occupational and social-demographic characteristics (age, sex, job title, previous SARS-CoV-2 infection, antibody titer levels, and time from the vaccination course completion)., Results: Among 64,172 health workers from 12 European health centers, 797 breakthrough infections were observed (cumulative incidence of 1.2%). The primary analysis using individual data on 8 out of 12 centers showed that age and previous infection significantly modified breakthrough infection rates. In the meta-analysis of aggregated data from all centers, previous SARS-CoV-2 infection and the standardized antibody titer were inversely related to the risk of breakthrough infection ( p = 0.008 and p = 0.007, respectively)., Conclusion: The inverse correlation of antibody titer with the risk of breakthrough infection supports the evidence that vaccination plays a primary role in infection prevention, especially in health workers. Cellular immunity, previous clinical conditions, and vaccination timing should be further investigated.

Background: In many forensic cases, the medical records of the deceased are not available at the time of the autopsy; therefore, no information about the deceased's state of health, including any infectious diseases contracted during life, is accessible. The detection of some of the principal viral infections, such as hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1), could contribute to determining causes of death and interesting applications could be found in medico-legal practice, such as occupational risk assessment. To date, accurate and sensitive serological and molecular assays capable of detecting these viruses have been validated on biological samples taken from living beings, while their efficiency on forensic post-mortem biological samples has yet to be thoroughly assessed. To further this aim, this study evaluated whether the nucleic acid amplification techniques (NAATs) for the detection of viral genomes that are applied in clinical settings can be used, with the same success rate, for these latter samples., Methods: Manual viral nucleic acid extraction processes and fully-automated amplification-based detection techniques developed in-house were evaluated on blood samples taken during the routine autopsies of 21 cadavers performed 2 to 9 days after death. Information on HBV, HCV, and HIV-1 seropositive status was previously known for only four of these cadavers., Results: Using automated quantitative real-time PCR (qPCR) and qualitative PCR (end-point) analyses, it was possible to confirm the presence of viral genomes in the four post-mortem whole blood samples with previously reported specific serological positivity. In addition, the genomes of HCV and/or HIV-1 genomes were detected in three other blood samples with unknown serological status at the time of autopsy., Conclusions: Therefore, our findings suggest that molecular assays may detect the presence of viral genomes in forensic post-mortem blood samples up to five days after death. This provides an additional means of investigation that can contribute to the determination of the deceased's cause of death., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s). Published by IMR Press.)

People with cystic fibrosis should be considered at increased risk of developing severe symptoms of COVID-19. Strikingly, a broad array of evidence shows reduced spread of SARS-CoV-2 in these subjects, suggesting a potential role for CFTR in the regulation of SARS-CoV-2 infection/replication. Here, we analyzed SARS-CoV-2 replication in wild-type and CFTR-modified human bronchial epithelial cell lines and primary cells to investigate SARS-CoV-2 infection in people with cystic fibrosis. Both immortalized and primary human bronchial epithelial cells expressing wt or F508del-CFTR along with CRISPR/Cas9 CFTR-ablated clones were infected with SARS-CoV-2 and samples were harvested before and from 24 to 72 h post-infection. CFTR function was also inhibited in wt-CFTR cells with the CFTR-specific inhibitor IOWH-032 and partially restored in F508del-CFTR cells with a combination of CFTR modulators (VX-661+VX-445). Viral load was evaluated by real-time RT-PCR in both supernatant and cell extracts, and ACE-2 expression was analyzed by both western blotting and flow cytometry. SARS-CoV-2 replication was reduced in CFTR-modified bronchial cells compared with wild-type cell lines. No major difference in ACE-2 expression was detected before infection between wild-type and CFTR-modified cells, while a higher expression in wild-type compared to CFTR-modified cells was detectable at 72 h post-infection. Furthermore, inhibition of CFTR channel function elicited significant inhibition of viral replication in cells with wt-CFTR, and correction of CFTR function in F508del-CFTR cells increased the release of SARS-CoV-2 viral particles. Our study provides evidence that CFTR expression/function is involved in the regulation of SARS-CoV-2 replication, thus providing novel insights into the role of CFTR in SARS-CoV-2 infection and the development of therapeutic strategies for COVID-19.

Background: Residual symptoms can be detected for several months after COVID-19. To better understand the predictors and impact of symptom persistence we analyzed a prospective cohort of COVID-19 patients., Methods: Patients were followed for 9 months after COVID-19 onset. Duration and predictors of persistence of symptoms, physical health and psychological distress were assessed., Results: 465 patients (54% males, 51% hospitalized) were included; 37% presented with at least 4 symptoms and 42% complained of symptom lasting more than 28 days. At month 9, 20% of patients were still symptomatic, showing mainly fatigue (11%) and breathlessness (8%). Hospitalization and ICU stay vs. non-hospitalized status increased the median duration of fatigue of 8 weeks. Age > 50 years (OR 2.50), ICU stay (OR 2.35), and presentation with 4 or more symptoms (OR 2.04) were independent predictors of persistence of symptoms at month 9. A total of 18% of patients did not return to optimal pre-COVID physical health, while 19% showed psychological distress at month 9. Hospital admission (OR 2.28) and persistence of symptoms at day 28 (OR 2.21) and month 9 (OR 5.16) were independent predictors of suboptimal physical health, while female gender (OR 5.27) and persistence of symptoms at day 28 (OR 2.42) and month 9 (OR 2.48) were risk factors for psychological distress., Conclusions: Patients with advanced age, ICU stay and multiple symptoms at onset were more likely to suffer from long-term symptoms, which had a negative impact on both physical and mental wellbeing. This study contributes to identify the target populations and Long COVID consequences for planning long-term recovery interventions., Competing Interests: Declaration of Competing Interest The authors have no competing interests, (Copyright © 2022. Published by Elsevier Ltd.)