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Since May 2022, a Monkeypox virus (MPXV) outbreak has been ongoing in several non-endemic countries. MPXV is usually transmitted after intimate contact, through body fluids, close contact from active lesions or through respiratory droplets. The recent outbreak occurrent in people with multiple recent sexual intercourse suggests the sexual route as the main way of transmission. However, there is no sufficient evidence to consider MPXV as a new sexually transmitted infection (STI), even though we believe that a link between MPXV and other STIs may exist with a possible facilitating action on their spreading. Herein, we illustrate the first case described during the current outbreak of a young man with both MPXV and acute HIV infection in a non-endemic country.

Covid pandemic affected donation activities worldwide, especially for living donation due to the lack of elective surgery. Moreover, the number of heart-beating and non-heart beating donors has recorded a decrease. Fondazione Banca dei Tessuti di Treviso (FBTV) is a non-profit healthcare organisation, located in Veneto Region, tasked with procurement, processing, preserving, validating and distributing human tissue for clinical use. During Covid-19 outbreak, operations in FBTV have never stopped and a great effort was required to maintain a standard trend of activity. The aim of this study was to describe the impact of Sars-CoV-2 on the activity of a multitissue bank in Italy. Moreover, we investigated the presence of the virus in tissues retrieved from two Sars-CoV-2 positive cadaver donors. Our survey demonstrated that the transplantation network of Veneto Region has positively reacted to the pandemic scenario, thanks to the effort of all personnel involved. Statistical analyses underlined that most of the activities of the tissue bank were unaffected during the Sars-CoV-2 pandemic.

In February and March 2020, two mass swab testing campaigns were conducted in Vo’, Italy. In May 2020, we tested 86% of the Vo’ population with three immuno-assays detecting antibodies against the spike and nucleocapsid antigens, a neutralisation assay and Polymerase Chain Reaction (PCR). Subjects testing positive to PCR in February/March or a serological assay in May were tested again in November. Here we report on the results of the analysis of the May and November surveys. We estimate a seroprevalence of 3.5% (95% Credible Interval (CrI): 2.8–4.3%) in May. In November, 98.8% (95% Confidence Interval (CI): 93.7–100.0%) of sera which tested positive in May still reacted against at least one antigen; 18.6% (95% CI: 11.0–28.5%) showed an increase of antibody or neutralisation reactivity from May. Analysis of the serostatus of the members of 1,118 households indicates a 26.0% (95% CrI: 17.2–36.9%) Susceptible-Infectious Transmission Probability. Contact tracing had limited impact on epidemic suppression., Vo’, Italy, is a unique setting for studying SARS-CoV-2 antibody dynamics because mass testing was conducted there early in the pandemic. Here, the authors perform two follow-up serological surveys and estimate seroprevalence, the extent of within-household transmission, and the impact of contact tracing.

Background:Generally, about half of the patients with central nervous system infections cases remain unexplained. Therefore, we aimed to describe which viruses were detected in unselected patients with a suspected central nervous system infection and the first diagnostic workflow in a university hospital laboratory.Methods:A comprehensive virus testing in cerebrospinal fluid with an in-house real-time PCR method was employed. Determining how many and which viruses to test was at the full discretion of the treating physician.Results:1462 patients were evaluated from 2011 to 2017 and 9 898 viral PCRs were made: 176 subjects (12%) had a positive result. There was great heterogeneity in the frequency of patients tested for each virus, ranging from 97.9% (1431 out of 1462) for herpes simplex virus (HSV) to 1.9% (28 out of 1462) for Parvovirus B19, positive in 1 patient. Enterovirus (EV) was the leading virus detected: the frequency was higher with respect to HSV (5.2% vs 2.4%, p=0.0004), varicella-zoster virus (VZV)(5.2% vs 2.9%, p=0.0052), human herpesvirus-6 (5.2% vs 1.7%, p=0.0014) and human herpesvirus-7 (HHV-7)(5.2% vs 2.5%, p=0.0406). Both VZV (83.5%) and HSV (97.9%) were tested significantly more than EV (68.7%, pConclusion:EV was the fifth virus frequently included in the diagnostic workflow but the most frequently detected, mostly in subjects aged less 40, as HHV-7 was. Testing these two viruses in all younger patients could reduce the number of unknown etiology.

On February 2020, the municipality of Vo’, a small town near Padua (Italy), was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. The entire population was swab tested in two sequential surveys. Here we report the analysis of the viral genomes, which revealed that the unique ancestor haplotype introduced in Vo’ belongs to lineage B and, more specifically, to the subtype found at the end of January 2020 in two Chinese tourists visiting Rome and other Italian cities, carrying mutations G11083T and G26144T. The sequences, obtained for 87 samples, allowed us to investigate viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo’. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptoms severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype, carried by an index case. The lockdown extinguished both viral spread and the emergence of new variants, confirming the efficiency of this containment strategy. The information gathered from household was used to reconstructs possible transmission events.AUTHOR SUMMARYIt is of great interest and importance to understand SARS-CoV-2 ability to mutate generating new viral strains, and to assess the impact of containment strategies on viral transmission. In this study we highlight the rapid intra-host haplotype evolution regardless of symptom severity and immune deficiencies that we observed during the first wave of the pandemic in the municipality of Vo’ in Italy. The confirmation that all the haplotypes found in this small community derive from a common ancestor haplotype, has allowed us to track the rapid emergence of new variants but lockdown and mass testing efficiently prevented their spread elsewhere.

Summary Background Medico-legal conflicts arise when it is difficult to prove the cause of nosocomial infections. Aim To report an outbreak of patient-to-patient transmission of hepatitis C virus (HCV) through the repeated use of a multi-dose saline flask during the rinsing of central venous catheters. Methods Blood samples were taken from each patient for the comparative analysis of their HCV RNA strains. No samples were available for one patient who died before the investigation started. Despite the known lability of HCV RNA, the body was exhumed four months after burial and postmortem samples were collected. HCV RNA was extracted successfully from liver and spleen samples. Genotyping of all the HCV strains was performed by sequence analysis of the 5′NC untranslated region, the E1 core conserved region and the E1/E2 hypervariable region. Findings Forensic investigators retraced the route used by two ward nurses, when saline catheter flushes were given to 14 patients with each nurse administering to seven patients. The comparative phylogenetic analysis of all case strains identified the deceased patient as the source of contamination to five patients. Conclusions This study highlights the value of sequence analysis as a tool for solving medico-legal conflicts. The High Court of Justice found that a health worker's re-use of a contaminated needle resulted in the nosocomial transmission of HCV.

We report the virological monitoring and the antiviral therapy adopted for the treatment of a patient affected by chronic B lymphocytic leukemia, who experienced a severe pneumonia with long-term shedding of influenza virus A(H1N1)pdm09, characterized by an early development of oseltamivir resistance.

SARS-CoV-2 genetic variants are emerging as a major threat to vaccination efforts worldwide as they may increase virus transmission rate and/or confer the ability to escape vaccine induced immunity with knock on effects on the level of herd immunity and vaccine efficacy respectively. These variants concern the Spike protein, which is encoded by the S gene, involved in virus entry into host cells and the major target of vaccine development. We report here that genetic variants of the N gene can impair our ability to utilize antigenic tests for both diagnosis and mass testing efforts aimed at controlling virus transmission. While conducting a large validation study on the Abbott Panbio™ COVID-19 Ag test, we noticed that some swab samples failed to generate a positive result in spite of a high viral load in Rt-PCR assays. Sequencing analysis of viruses showing discordant results in the RT-PCR and antigen assays revealed the presence of multiple disruptive amino-acid substitutions in the N antigen (the viral protein detected in the antigen test) clustered from position 229 to 374 a region known to contain an immunodominant epitope. A relevant fraction of the variants, undetected by the antigen test, contained the mutations A376T coupled to M241I. Intriguingly we found that virus sequences with this mutation were over-represented in the antigen-test-negative and PCR-positive samples and progressively increased in frequency over time in Veneto, a region of Italy that has aggressively scaled up the utilization of antigen tests, which reached nearly 68% of all the SARS-CoV-2 swab assays performed there. We speculate that mass utilization of antigen assays could create a selection pressure on the target that may favor the spread of undetectable virus variants.

Background: In February and Mach 2020, two mass swab testing campaigns conducted in Vo’, Italy demonstrated the extent of asymptomatic SARS-CoV-2 infection and the feasibility of epidemic suppression. Methods: We tested 86% of the Vo’ population (2,602 subjects) in May with three immuno-assays detecting antibodies against the spike (S) and nucleocapsid (N) antigens, a neutralisation assay and Polymerase Chain Reaction (PCR). Subjects testing positive to PCR in February/March or a serological assay in May were tested again in November. Findings: Combining the results obtained with the three assays, we estimate a seroprevalence of 3.5% (95% Credible Interval (CrI) 2.8%-4.3%) in May. In November, all assays showed a reduction in antibody titres, though 98.8% (95% Confidence Interval (CI) 93.7%-100.0%) of sera still reacted against at least one antigen. Conversely, 18.6% (95% CI 11.0%-28.5%) showed a marked increase of antibody or viral neutralisation reactivity between May and November, linked to documented or likely re-exposures. We found significant differences in the magnitude and persistence of the antibody response by age group but not by symptom occurrence, hospitalisation, or sex. Analysis of the serostatus of 1,118 households indicated a 27.3% (95% CrI 19.2%-34.6%) probability of SARS-CoV-2 transmission among household members and that 81.8% (95% CrI 55.9%-95.2%) of transmission could be attributed to 20% of infections. Contact tracing correctly identified 44% of the infected subjects and had limited impact on the epidemic. Interpretation: We find evidence of antibody persistence up to nine months post infection. Different assays provided significantly different seroprevalence estimates, making it challenging to compare seroprevalence estimates globally. Due to the high population susceptibility and the limited impact of contact tracing, rigorous testing and improvements in contact tracing are essential to control SARS-CoV-2. Funding: Veneto Region, Medical Research Council, Wellcome Trust, Royal Society, University of Padua, UK National Institute for Health Research. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The first and second serosurveys of the Vo' population were approved by the Ethics Committee for Clinical Research of the province of Padova.

Lactoferrin (Lf), a multifunctional cationic glycoprotein synthesized by exocrine glands and neutrophils, possesses an in vitro antiviral activity against SARS-CoV-2. Thus, we conducted an in vivo preliminary study to investigate the antiviral effect of oral and intranasal liposomal bovine Lf (bLf) in asymptomatic and mild-to-moderate COVID-19 patients. From April 2020 to June 2020, a total of 92 mild-to-moderate (67/92) and asymptomatic (25/92) COVID-19 patients were recruited and divided into three groups. Thirty-two patients (14 hospitalized and 18 in home-based isolation) received only oral and intranasal liposomal bLf, 32 hospitalized patients were treated only with standard of care (SOC) treatment, and 28, in home-based isolation, did not take any medication. Furthermore, 32 COVID-19 negative, untreated, healthy subjects were added for ancillary analysis. Liposomal bLf-treated COVID-19 patients obtained an earlier and significant (p &lt, 0.0001) SARS-CoV-2 RNA negative conversion compared to the SOC-treated and untreated COVID-19 patients (14.25 vs. 27.13 vs. 32.61 days, respectively). Liposomal bLf-treated COVID-19 patients showed fast clinical symptoms recovery compared to the SOC-treated COVID-19 patients. In bLf-treated patients, a significant decrease in serum ferritin, IL-6, and D-dimers levels was observed. No adverse events were reported. These observations led us to speculate a potential role of bLf in the management of mild-to-moderate and asymptomatic COVID-19 patients.

Purpose We report on the first identified cluster of the B.1.1.7 variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in the northeast of Italy. Methods The cluster was recognized in January 2021 with an epidemiological started from the hospitalization of a 68-year-old man suffering from coronavirus disease 2019 (COVID-19) related pneumonia and we surprisingly found three families involved in the same cluster. Results We retrospectively rebuilt the pathway of infection and performed a virological analysis. Conclusion This allow us to make clear the very high attack rate and the great infective capacity of this B.1.1.7 variant of SARS-CoV-2.

In viral diseases T cells exert a prominent role in orchestrating the adaptive immune response and yet a comprehensive assessment of the T-cell repertoire, compared and contrasted with antibody response, after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is currently lacking. A prior population-scale study of the municipality of Vo’, Italy, conducted after the initial SARS-CoV-2 outbreak uncovered a high frequency of asymptomatic infected individuals and their role in transmission in this town. Two months later, we sampled the same population’s T-cell receptor repertoire structure in terms of both diversity (breadth) and frequency (depth) to SARS-CoV-2 antigens to identify associations with both humoral response and protection. For this purpose, we analyzed T-cell receptor and antibody signatures from over 2,200 individuals, including 76 PCR-confirmed SARS-CoV-2 cases (25 asymptomatic, 42 symptomatic, 9 hospitalized). We found that 97.4% (74/76) of PCR confirmed cases had elevated levels of T-cell receptors specific for SARS-CoV-2 antigens. The depth and breadth of the T-cell receptor repertoire were both positively associated with neutralizing antibody titers; helper CD4+ T cells directed towards viral antigens from spike protein were a primary factor in this correlation. Higher clonal depth of the T-cell response to the virus was also significantly associated with more severe disease course. A total of 40 additional suspected infections were identified based on T-cell response from the subjects without confirmatory PCR tests, mostly among those reporting symptoms or having household exposure to a PCR-confirmed infection. Taken together, these results establish that T cells are a sensitive, reliable and persistent measure of past SARS-CoV-2 infection that are differentially activated depending on disease morbidity.

Lactoferrin, a multifunctional cationic glycoprotein, secreted by exocrine glands and neutrophils, possesses an antiviral activity extendable to SARS-CoV-2.We performed in vitro assays proving lactoferrin antiviral activity through direct attachment to both virus and cell surface components. This activity varied according to concentration (100/500μg/ml), multiplicity of infection (0.1/0.01) and cell type (Vero E6/Caco-2 cells).Interestingly, the in silico results strongly supported the hypothesis of a direct recognition between the lactoferrin and the Spike S glycoprotein, thus hindering the viral entry into the cells.Hence, we conducted a clinical trial to investigate effect and tolerability of a liposomal lactoferrin formulation as a supplementary nutraceutical agent in mild-to-moderate and asymptomatic COVID-19 patients.A total of 92 mild-to-moderate (67/92) and asymptomatic (25/92) COVID-19 patients were recruited and divided in 3 groups according to the administered regimen. Thirty-two patients, 14 hospitalised and 18 in home-based insolation received oral and intranasal liposomal bovine lactoferrin (bLf), 32 hospitalised patients were treated with standard of care treatment (hydroxychloroquine, azitromicin and lopinavir/darunavir), and 28, in home-based isolation, did not take any medication. Furthermore, 32 COVID-19 negative, not-treated, healthy subjects were added as a control group for ancillary analysis.bLf-supplemented COVID-19 patients obtained an earlier and significant (p < 0,0001.) median rRT-PCR SARS-COV-2 RNA negative conversion than standard of care-treated and non-treated COVID-19 patients (14.25 vs 27.13 vs 32.61 days, respectively).In addition, bLf-supplemented COVID-19 patients showed significant fast clinical symptoms recovery than standard of care-treated and non-treated COVID-19 patients. Moreover, in bLf-supplemented patients, a significant decrease of either serum ferritin or IL-6 levels or host iron overload, all parameters characterizing inflammatory processes, were observed. Serum D-dimers was also found significantly decreased following bLf supplement. No adverse events were reported.These in vitro and in vivo observations led us to speculate a potential and safe supplementary role of Blf in the management of mild-to-moderate and asymptomatic COVID-19 patients.

Since February 21, 2020, SARS-CoV-2 has spread exponentially worldwide. Neonatal patients needing intensive care are considered a vulnerable population. To report the results of a policy based on multi-timepoint surveillance for SARS-CoV-2 of all neonates admitted to the neonatal intensive care unit (NICU), their parents, and all healthcare providers in a part of Italy with a high prevalence of the infection. Observational study conducted from 21 February to 21 April 2020. Intervention consisted of (a) parental triage on arrival at the neonatal ward; (b) universal testing with nasopharyngeal swabs and blood testing for SARS-CoV-2 IgM and IgG antibodies; (c) use of continuous personal protective equipment at the NICU by parents and staff. A total of 6726 triage procedures were performed on 114 parents, and 954 nasopharyngeal swabs were collected from 226 individuals. Five (2.2%) asymptomatic individuals (2 parents and 3 healthcare providers) tested positive on nasopharyngeal swabs and were kept isolated for 14 days. Of 75 admitted newborn, no one tested positive on nasopharyngeal swabs or antibody tests. Three parents presented with fever or flu-like symptoms at triage; they tested negative on swabs.Conclusion: With universal screening of neonates, parents, and staff, there were no cases of SARS-CoV-2 infection among the neonates admitted to a NICU in an area with a high incidence of SARS-CoV-2. Our experience could be usefully compared with other strategies with a view to developing future evidence-based guidelines for managing high-risk neonates in case of new epidemics. What is Known: • The novel coronavirus named SARS-CoV-2 has since spread worldwide at a remarkable rate, with more than 2.5 million confirmed cases. • Pediatric population may be less affected from COVID-19 than adult population but infants and newborn babies seem to be more vulnerable to SARS-CoV-2 infection. What is New: • Using an approach based on triage; testing with nasopharyngeal swabs and serology; and use of personal protective equipment, there were no cases of SARS-CoV-2 infection among neonates in a NICU in a high incidence of SARS-CoV-2 area. • Positive and asymptomatic individuals were identified and isolated early allowing the containment of infection's spread among healthcare providers and parents.

Objectives The direct identification of SARS-CoV-2 RNA in nasopharyngeal swabs is recommended for diagnosing the novel COVID-19 disease. Pre-analytical determinants, such as sampling procedures, time and temperature storage conditions, might impact on the end result. Our aim was to evaluate the effects of sampling procedures, time and temperature of the primary nasopharyngeal swabs storage on real-time reverse-transcription polymerase chain reaction (rRT-PCR) results. Methods Each nasopharyngeal swab obtained from 10 hospitalized patients for COVID-19 was subdivided in 15 aliquots: five were kept at room temperature; five were refrigerated (+4 °C); five were immediately mixed with the extraction buffer and refrigerated at +4 °C. Every day and for 5 days, one aliquot per condition was analyzed (rRT-PCR) for SARS-CoV-2 gene E and RNaseP and threshold cycles (Ct) compared. To evaluate manual sampling, 70 nasopharyngeal swabs were sampled twice by two different operators and analyzed separately one from the other. Results A total of 6/10 swabs were SARS-CoV-2 positive. No significant time or storage-dependent variations were observed in SARS-CoV-2 Ct. Re-sampling of swabs with SARS-CoV-2 Ct lower than 33 resulted in highly reproducible results (CV=2.9%), while a high variability was observed when Ct values were higher than 33 (CV=10.3%). Conclusions This study demonstrates that time and temperature of nasopharyngeal swabs storage do not significantly impact on results reproducibility. However, swabs sampling is a critical step, and especially in case of low viral load, might be a potential source of diagnostic errors.

On the 21st of February 2020 a resident of the municipality of Vo’, a small town near Padua, died of pneumonia due to SARS-CoV-2 infection1. This was the first COVID-19 death detected in Italy since the emergence of SARS-CoV-2 in the Chinese city of Wuhan, Hubei province2. In response, the regional authorities imposed the lockdown of the whole municipality for 14 days3. We collected information on the demography, clinical presentation, hospitalization, contact network and presence of SARS-CoV-2 infection in nasopharyngeal swabs for 85.9% and 71.5% of the population of Vo’ at two consecutive time points. On the first survey, which was conducted around the time the town lockdown started, we found a prevalence of infection of 2.6% (95% confidence interval (CI) 2.1-3.3%). On the second survey, which was conducted at the end of the lockdown, we found a prevalence of 1.2% (95% CI 0.8-1.8%). Notably, 43.2% (95% CI 32.2-54.7%) of the confirmed SARS-CoV-2 infections detected across the two surveys were asymptomatic. The mean serial interval was 6.9 days (95% CI 2.6-13.4). We found no statistically significant difference in the viral load (as measured by genome equivalents inferred from cycle threshold data) of symptomatic versus asymptomatic infections (p-values 0.6 and 0.2 for E and RdRp genes, respectively, Exact Wilcoxon-Mann-Whitney test). Contact tracing of the newly infected cases and transmission chain reconstruction revealed that most new infections in the second survey were infected in the community before the lockdown or from asymptomatic infections living in the same household. This study sheds new light on the frequency of asymptomatic SARS-CoV-2 infection and their infectivity (as measured by the viral load) and provides new insights into its transmission dynamics, the duration of viral load detectability and the efficacy of the implemented control measures.

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.

West Nile virus (WNV) lineage 2 is expanding and causing large outbreaks in Europe. In this study, we analyzed the epidemiological, clinical, and virological features of WNV lineage 2 infection during the large outbreak that occurred in northern Italy in 2018. The study population included 86 patients with neuroinvasive disease (WNND), 307 with fever (WNF), and 34 blood donors. Phylogenetic analysis of WNV full genome sequences from patients&rsquo, samples showed that the virus belonged to the widespread central/southern European clade of WNV lineage 2 and was circulating in the area at least since 2014. The incidence of WNND and WNF progressively increased with age and was higher in males than in females. Among WNND patients, the case fatality rate was 22%. About 70% of blood donors reported symptoms during follow-up. Within the first week after symptom onset, WNV RNA was detectable in the blood or urine of 80% of patients, while 20% and 40% of WNND and WNF patients, respectively, were WNV IgM-seronegative. In CSF samples of WNND patients, WNV RNA was typically detectable when WNV IgM antibodies were absent. Blunted or no WNV IgM response and high WNV IgG levels were observed in seven patients with previous flavivirus immunity.

Background Usutu virus (USUV) is a mosquito-borne flavivirus, which shares its transmission cycle with the phylogenetically related West Nile virus (WNV). USUV circulates in several European countries and its activity has increased over the last 5 years. Aim To describe human cases of USUV infection identified by surveillance for WNV and USUV infection in the Veneto Region of northern Italy in 2018. Methods From 1 June to 30 November 2018, all cases of suspected autochthonous arbovirus infection and blood donors who had a reactive WNV nucleic acid test were investigated for both WNV and USUV infection by in-house molecular methods. Anti-WNV and anti-USUV IgM and IgG antibodies were detected by ELISA and in-house immunofluorescence assay, respectively; positive serum samples were further tested by WNV and USUV neutralisation assays run in parallel. Results Eight cases of USUV infection (one with neuroinvasive disease, six with fever and one viraemic blood donor who developed arthralgia and myalgia) and 427 cases of WNV infection were identified. A remarkable finding of this study was the persistence of USUV RNA in the blood and urine of three patients during follow-up. USUV genome sequences from two patients shared over 99% nt identity with USUV sequences detected in mosquito pools from the same area and clustered within lineage Europe 2. Conclusions Clinical presentation and laboratory findings in patients with USUV infection were similar to those found in patients with WNV infection. Cross-reactivity of serology and molecular tests challenged the differential diagnosis.

Background In Italy the pandemic of COVID-19 infection has placed an enormous burden on health authorities: contact precautions are required to avoid viral transmission and people should be subjected to standard infection control procedures. This is crucial in a country experiencing a high number of confirmed cases of COVID-19 infection in Europe and where multidrug-resistant Gram-negative bacteria (MDR-GN) are endemic.The aim of this study was to compare the prevalence of MDR-GN in surveillance rectal swabs (SRS) and in clinical samples (CS) in the period March 1,2020-April, 24 2020 with respect to the previous 2-month period and to the previous year. Methods The first SRS and the first CS with a MDR-GN isolate detected from 01/01/2019 to 24/04/2020 were included. Analysis was made by comparing three different study periods in 2019 and 2020 (Jan-Dec 2019, Jan-Feb 2020, and Mar-Apr 2020), for medical department, surgical department and intensive care department. Results Overall, 612 MDR-GN organisms were identified (399 SRS and 213 CS): carbapenemase-producing Klebsiella pneumoniae and Acinetobacter baumanii (CPAB) were the most frequently detected (Figure 1). We observed an increased relative frequency of patients with MDR-GN detected in CS respect to those found in SRS (32.7% vs 44.5% vs 70.6%, p=0.0005): 5/12 CS detected in the last period were isolated from the respiratory tract (Figure 2). Nine patients with COVID-19 pneumonia had MDR-GN. All but two patients had a previous negative SRS performed 4 days before (median value) and the median interval between COVID-19 positivity and MDR-GN positivity was 7 days.The six patients with CPAB isolation were all hospitalized in the same ward, with partially overlapping hospital stays during the study period. In 5 of them, CPAB was detected in the respiratory tract (Figure 3). Conclusion The first detection of MDR-GN in CS and the nosocomial MDR-GN acquisition despite cohorting due to COVID-19 infection underline the need to reinforce infection control measures in a high prevalence country during COVID-19 pandemia. A correct antimicrobial policy urged because, according to published data, most patients with COVID-19 infection received antimicrobial therapy: furthermore MDR-GN infection could play a role in the negative outcome of these patients. Disclosures All Authors: No reported disclosures

Despite efforts to improve surveillance and vaccination coverage, measles virus (MeV) continues to cause outbreaks also in high-income countries. As the reference laboratory of the Veneto Region, Italy, we analyzed changes in population immunity, described measles outbreaks, investigated MeV genetic diversity, and evaluated cross-protection of measles vaccination against MeV epidemic strains. Like most European areas, the Veneto Region has suboptimal measles vaccination coverage and is facing a growing public mistrust of vaccination. A progressive decline of measles vaccine uptake was observed during the last decade in the Veneto Region, leading to immunity gaps in children and young adults. Measles outbreaks were caused by the same MeV genotype B3, D4, and D8 strains that were circulating in other European countries. Eleven cases of measles were observed in immunized subjects. These cases were not associated with particular MeV genotypes nor with mutations in epitopes recognized by neutralizing antibodies. Accordingly, sera from fully vaccinated subjects cross-neutralized epidemic MeV strains, including the genotypes B3, D4, and D8, with the same high efficiency demonstrated against the vaccine strain. In fully vaccinated subjects, high MeV IgG antibody titers persisted up to 30 years following vaccination. These results support the use of the current measles-containing vaccines and strategies to strengthen vaccination.

The present study reports the first complete genome sequence analysis of West Nile virus (WNV) strains isolated from brain samples from raptors. The results prove the circulation of closely related WNV lineage II strains in central Europe and genetic analysis revealed seven amino acid substitutions in structural (PrM3, E159 and E231) and in non-structural (NS1109, NS5259, NS5310 and NS5600) proteins. Observed amino acid substitutions Phe3 and Ser231 were common only within the lineage VII Koutango strain isolated from Rhipicephalus guilhoni tick in Senegal. Further research could reveal whether these substitutions influence the biological properties of WNV, including virulence and neuroinvasiveness.

In 2018, there was a large West Nile virus (WNV) outbreak in northern Italy. We observed five atypical cases of WNV infection that were characterised by the presence of WNV RNA and WNV IgG at the time of diagnosis, but no IgM response during follow-up. Neutralisation assays demonstrated pre-existing Usutu virus immunity in all patients. Besides challenging diagnosis, the immunological crosstalk between the two viruses warrants further investigation on possible cross-protection or infection enhancement effects.

Background This longitudinal study described Cytomegalovirus (CMV) DNA, Epstein-Barr (EBV) DNA and human herpesvirus 8 (HHV-8) DNA asymptomatic salivary shedding in HIV-positive men who have sex with men (MSM). We aimed to 1-analyze frequency and persistence of herpesvirus shedding, 2-correlate herpesvirus positivity and HIV viroimmunological parameters and 3-assess the association between HIV-RNA suppression and herpesvirus replication. Methods Herpesvirus DNA was tested with an in-house real-time PCR in 2 salivary samples obtained at T0 and T1 (24 months after T0). HIV-RNA was evaluated in the 24 months prior to T0 and in the 24 months prior to T1; MSM were classified as successfully suppressed patients (SSPs), viremic patients (VPs) and partially suppressed patients (PSPs). EBV DNA load was classified as low viral load (EBV-LVL, value ≤10,000 copies/ml) and as high viral load (EBV-HVL,> 10,000 copies/ml). Mann-Whitney U test tested the difference of the median between groups of patients. Chi-squared test and Fisher’s exact test compared categorical variables according to the frequencies. Kruskal-Wallis test compared continuous data distributions between levels of categorical variables. Results Ninety-two patients (median CD4+ count 575 cells/mm3, median nadir 330 CD4+ cells/mm3) were included: 40 SSPs,33 VPs and 19 PSPs. The more frequently single virus detected was EBV, both at T0 and at T1 (in 67.5 and 70% of SSPs, in 84.8 and 81.8% of VPs and in 68.4 and 73.7% of SPSs) and the most frequently multiple positivity detected was EBV + HHV-8. At T1, the percentage of CMV positivity was higher in VPs than in SSPs (36.4% vs 5%, p

The aim of the study was to evaluate cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNA salivary shedding in HIV-positive men who have sex with men (MSM) and to determine whether viro-immunological parameters and long-term (24 months) plasma HIV RNA (pHIV) detection may predict herpesviruses replication. A total of 193 HIV-positive MSM were consecutively recruited (mean CD4+ cell count 607 cells/mm(3) and mean nadir value 333 cells/mm(3) ); pHIV was analyzed for 24 months prior to saliva sampling: patients were categorized as successfully suppressed (SS) and not suppressed (NS). The EBV viral load was categorized as high viral load (HVL), intermediate (IVL), or low (LVL), CMV DNA as positive or negative. NS patients experienced both herpesviruses detectability more frequently respect to SS patients (P = 0.034); conversely, no salivary shedding was more frequent in SS patients (P = 0.014). HVL EBV was more frequent in NS patients than in SS subjects (P = 0.038 for isolated EBV detection and P = 0.001 when CMV shedding was associated). NS subjects with HVL EBV had a median pHIV of 43,820 copies/ml, significantly higher respect to IVL and LVL patients (P = 0.027 and P = 0.0005, respectively). CMV shedding was mostly associated to EBV shedding. NS patients showed a significantly higher frequency of saliva HVL EBV detection compared to SS patients; moreover, NS patients with HVL EBV had a higher pHIV respect to those with IVL and LVL shedding. Our results suggest that a successful pHIV suppression could reduce the burden of salivary EBV replication and likely the risk of herpesviruses-related cancers.

Similar to phosphorylation, transient conjugation of ubiquitin to target proteins (ubiquitination) mediated by the concerted action of ubiquitin ligases and de-ubiquitinating enzymes (DUBs) can affect substrate function. As obligate intracellular parasites, viruses rely on different cellular pathways for their own replication and the well conserved ubiquitin conjugating/de-conjugating system is not an exception. Viruses not only usurp the host proteins involved in the ubiquitination/de-ubiquitination process, but they also encode their own ubiquitin ligases and DUBs. Here we report that an N-terminal variant of the herpes simplex virus (HSV) type-1 large tegument protein VP1/2 (VP1/2(1-767)), encompassing an active DUB domain (herpesvirus tegument ubiquitin specific protease, htUSP), and TSG101, a component of the endosomal sorting complex required for transport (ESCRT)-I, functionally interact. In particular, VP1/2(1-767) modulates TSG101 ubiquitination and influences its intracellular distribution. Given the role played by the ESCRT machinery in crucial steps of both cellular pathways and viral life cycle, the identification of TSG101 as a cellular target for the HSV-1 specific de-ubiquitinating enzyme contributes to the clarification of the still under debate function of viral encoded DUBs highly conserved throughout the Herpesviridae family.

Summary Objectives Very few data exist on viral meningitis and encephalitis in elderly patients (>65 years old). Methods This study investigated the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), human herpes virus 6 (HHV-6), HHV-7, HHV-8, cytomegalovirus (CMV), Epstein–Barr virus (EBV), enterovirus (EV), human adenovirus (HAdV), human parechoviruses (HPeVs), and tick-borne encephalitis virus (TBEV) through real-time PCR (RT-PCR) in patients >65 years old who had cerebrospinal fluid (CSF) tested for a suspected central nervous system infection. Results A total of 2868 RT-PCRs were performed on 502 CSF samples. Overall, 65 positive RT-PCRs were found: 23 for HSV (35.4% of positives), 15 for EV (23.1% of positives), 14 for EBV (21.5% of positives), 12 for VZV (18.5% of positives), and one for CMV (1.5% of positives). A positive RT-PCR in CSF was detected in 24 (17.4%) patients aged ≥80 years and in 35 (9.6%) patients aged 65–79 years ( p =0.02). VZV was more frequently detected in the oldest subjects (5.9% vs. 1.6%, p =0.03). Conclusions HSV was the most common viral aetiology identified in the study, with VZV infection being recognized more frequently in those patients aged ≥80 years.

Objectives We described Klebsiella pneumoniae producing carbapenemase (CPKP) spread from 01/01/2015 to 13/09/16 in a tertiary level hospital. Methods The first positive surveillance rectal swab (SRS) or clinical sample (CS) collected in the medical department (MD), surgical department (SD) and intensive care department (ICD) were included in the study. A validated in-house Real-Time PCR method was used to detect carbapenemases; multilocus sequence typing (MLST) was used for further characterization of the strains. Results 21535 patients were included: 213 CPKP strains from surveillance rectal swab (SRS) and 98 from clinical samples (CS) were collected. The percentage of CPKP detected in SRS with respect to CS increased in the medical MD from 2015 to 2016 (p=0.01) and in ICD from 2012 to 2015 (p=0.0001), while it decreased in SD from 2014 to 2016 (p=0.003); 68.5% of the positive SRS had a previous negative SRS; CPKP was more frequently identified in CS than in SRS in MD. Twelve strains harboured more than one carbapenemase gene. Many other species harbouring a carbapenemase gene were collected. Conclusions MDs need more inclusive surveillance criteria. The late detection of positive SRS underlined the risk of colonization during hospitalization.

During 2008–2009, several human cases of WNV disease caused by an endemic lineage 1a strain were reported in areas surrounding the Po river in north-eastern Italy. Since 2010, cases have been recorded in nearby northern areas, where, in 2011, both lineage 1a and 2 were detected. We describe here two new WNV complete genome sequences from human cases of WNV infection occurring in 2011 in the Veneto Region. Phylogenetic analysis showed that both genome sequences belonged to lineage 1a and were related to WNV strains of the Western Mediterranean subtype. The novel WNV genomes had high nucleotide and amino acid sequence divergence from each other and from the WNV strain circulating in Italy in 2008–2009. The presence of different WNV strains in a relatively small geographical area is a novel finding with unpredictable impact on human disease that requires further investigation.

We describe the dynamics of Zika virus (ZIKV) infection in a man in his early 40s who developed fever and rash after returning from Haiti to Italy, in January 2016. Follow-up laboratory testing demonstrated detectable ZIKV RNA in plasma up to day 9 after symptom onset and in urine and saliva up to days 15 and 47, respectively. Notably, persistent shedding of ZIKV RNA was demonstrated in semen, still detectable at 181 days after onset.