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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.)

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.

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.

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.

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.)

SARS-CoV-2 serological tests are used to assess the infection seroprevalence within a population. This study aims at assessing potential biases in estimating infection prevalence amongst healthcare workers (HCWs) when different diagnostic criteria are considered. A multi-site cross-sectional study was carried out in April-September 2020 amongst 1.367 Italian HCWs. SARS-CoV-2 prevalence was assessed using three diagnostic criteria: RT-PCR on nasopharyngeal swab, point-of-care fingerprick serological test (POCT) result and COVID-19 clinical pathognomonic presentation. A logistic regression model was used to estimate the probability of POCT-positive result in relation to the time since infection (RT-PCR positivity). Among 1.367 HCWs, 69.2% were working in COVID-19 units. Statistically significant differences in age, role and gender were observed between COVID-19/non-COVID-19 units. Prevalence of SARS-CoV-2 infection varied according to the criterion considered: 6.7% for POCT, 8.1% for RT-PCR, 10.0% for either POCT or RT-PCR, 9.6% for infection pathognomonic clinical presentation and 17.6% when at least one of the previous criteria was present. The probability of POCT-positive result decreased by 1.1% every 10 days from the infection. This study highlights potential biases in estimating SARS-CoV-2 point-prevalence data according to the criteria used. Although informative on infection susceptibility and herd immunity level, POCT serological tests are not the best predictors of previous COVID-19 infections for public health monitoring programmes.

Objective: It is reported that recovery from COVID-19 chemosensory deficit generally occurs in a few weeks, although olfactory dysfunction may persist longer. Here, we provide a detailed follow-up clinical investigation in a very young female patient (17-year-old) with a long-lasting anosmia after a mild infection, with partial recovery 15 months after the onset., Methods: Neuroimaging and neurophysiologic assessments as well as olfactory mucosa swabbing for microbiological and immunocytochemical analyses were performed. Olfactory and gustatory evaluations were conducted through validated tests., Results: Chemosensory evaluations were consistent with anosmia associated with parosmia phenomena and gustatory impairment, the latter less persistent. Brain MRI (3.0 T) showed no microvascular injury in olfactory bulbs and brain albeit we cannot rule out slight structural abnormalities during the acute phase, and a high-density EEG was negative. Immunocytochemistry of olfactory mucosa swabs showed high expression of ACE2 in sustentacular cells and lower dot-like cytoplasmic positivity in neuronal-shaped cells., Discussion: The occurrence of long-term persistent olfactory deficit in spite of the absence of structural brain and olfactory bulb involvement supports the view of a possible persistent dysfunction of both sustentacular cells and olfactory neurons. The gustatory dysfunction even if less persisting for the described features could be related to a primary gustatory system involvement. Future longitudinal studies are needed to investigate the persistence of chemosensory impairment, which could have a relevant impact on the daily life., (© 2021. Fondazione Società Italiana di Neurologia.)

Objective: To investigate the presence of bacteria and fungi in bronchial aspirate (BA) samples from 43 mechanically ventilated patients with severe COVID-19 disease., Methods: Detection of SARS-CoV-2 was performed using Allplex 2019-nCoV assay kits. Isolation and characterisation of bacteria and fungi were carried out in BA specimens treated with 1X dithiothreitol 1% for 30 min at room temperature, using standard culture procedures., Results: Bacterial and/or fungal superinfection was detected in 25 out of 43 mechanically ventilated patients, generally after 7 days of hospitalisation in an intensive care unit (ICU). Microbial colonisation (colony forming units (CFU) <1000 colonies/ml) in BA samples was observed in 11 out of 43 patients, whereas only 7 patients did not show any signs of bacterial or fungal growth. Pseudomonas aeruginosa was identified in 17 patients. Interestingly, 11 out of these 17 isolates also showed carbapenem resistance. The molecular analysis demonstrated that resistance to carbapenems was primarily related to OprD mutation or deletion. Klebsiella pneumoniae was the second most isolated pathogen found in 13 samples, of which 8 were carbapenemase-producer strains., Conclusion: These data demonstrate the detection of bacterial superinfection and antimicrobial resistance in severe SARS-CoV-2-infected patients and suggest that bacteria may play an important role in COVID-19 evolution. A prospective study is needed to verify the incidence of bacterial and fungal infections and their influence on the health outcomes of COVID-19 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 © 2021 Mazzariol, Benini, Unali, Nocini, Smania, Bertoncelli, De Sanctis, Ugel, Donadello, Polati and Gibellini.)

Background: To report the baseline phase of the SIEROEPID study on SARS-CoV-2 infection seroprevalence among health workers at the University Hospital of Verona, Italy, between spring and fall 2020; to compare performances of several laboratory tests for SARS-CoV-2 antibody detection., Methods: 5299 voluntary health workers were enrolled from 28 April 2020 to 28 July 2020 to assess immunological response to SARS-CoV-2 infection throughout IgM, IgG and IgA serum levels titration by four laboratory tests. Association of antibody titre with several demographic variables, swab tests and performance tests (sensitivity, specificity, and agreement) were statistically analyzed., Results: The overall seroprevalence was 6%, considering either IgG and IgM, and 4.8% considering IgG. Working in COVID-19 Units was not associated with a statistically significant increase in the number of infected workers. Cohen's kappa of agreement between MaglumiTM and VivaDiagTM was quite good when considering IgG only (Cohen's kappa = 78.1%, 95% CI 74.0-82.0%), but was lower considering IgM (Cohen's kappa = 13.3%, 95% CI 7.8-18.7%)., Conclusion: The large sample size with high participation (84.7%), the biobank and the longitudinal design were significant achievements, offering a baseline dataset as the benchmark for risk assessment, health surveillance and management of SARS-CoV-2 infection for the hospital workforce, especially considering the ongoing vaccination campaign. Study results support the national regulator guidelines on using swabs for SARS-CoV-2 screening with health workers and using the serological tests to contribute to the epidemiological assessment of the spread of the virus.

Coronavirus disease 2019 (COVID-19) may result in a life-threatening condition due to a hyperactive immune reaction to severe acute respiratory syndrome-coronavirus-2 infection, for which no effective treatment is available. Based on the potent immunomodulatory properties of mesenchymal stromal cells (MSCs), a growing number of trials are ongoing. This prompted us to carry out a thorough immunological study in a patient treated with umbilical cord-derived MSCs and admitted to the Intensive Care Unit for COVID-19-related pneumonia. The exploratory analyses were assessed on both peripheral blood and bronchoalveolar fluid lavage samples at baseline and after cellular infusion by means of single-cell RNA sequencing, flow cytometry, ELISA, and functional assays. Remarkably, a normalization of circulating T lymphocytes count paralleled by a reduction of inflammatory myeloid cells, and a decrease in serum levels of pro-inflammatory cytokines, mostly of interleukin-6 and tumor necrosis factor-α, were observed. In addition, a drop of plasma levels of those chemokines essential for neutrophil recruitment became evident that paralleled the decrease of lung-infiltrating inflammatory neutrophils. Finally, circulating monocytes and low-density gradient neutrophils acquired immunosuppressive function. This scenario was accompanied by an amelioration of respiratory, renal, inflammatory, and pro-thrombotic indexes. Our results provide the first immunological data possibly related to the use of umbilical cord-derived MSCs in severe COVID-19 context.

Since the beginning of the SARS-CoV-2 pandemic, COVID-19 appeared as a unique disease with unconventional tissue and systemic immune features. Here we show a COVID-19 immune signature associated with severity by integrating single-cell RNA-seq analysis from blood samples and broncho-alveolar lavage fluids with clinical, immunological and functional ex vivo data. This signature is characterized by lung accumulation of naïve lymphoid cells associated with a systemic expansion and activation of myeloid cells. Myeloid-driven immune suppression is a hallmark of COVID-19 evolution, highlighting arginase-1 expression with immune regulatory features of monocytes. Monocyte-dependent and neutrophil-dependent immune suppression loss is associated with fatal clinical outcome in severe patients. Additionally, our analysis shows a lung CXCR6 + effector memory T cell subset is associated with better prognosis in patients with severe COVID-19. In summary, COVID-19-induced myeloid dysregulation and lymphoid impairment establish a condition of 'immune silence' in patients with critical COVID-19.