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In this study, we analyzed blood samples obtained from 169 cadavers subjected to an autopsy from 1 October 2019 to 27 March 2020. The presence of anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies was searched by lateral flow immunochromatographic assay (LFIA) and ELISA tests and the SARS-CoV-2 RNA was tested in blood and available lung tissues by real-time PCR (RT-PCR) and droplet digital PCR (ddPCR). Five cases resulted in positives at the serological screening for anti-SARS-CoV-2. Three results were weakly positive for IgM while only one showed strong reactivity for IgG antibodies. The fifth subject (who died in December 2019) resulted positive for the ELISA test. The detection of SARS-CoV-2 RNA resulted in positive only in the blood and lung tissues of such cases. These data suggest that cadaveric blood may be a suitable substrate for the assessment of SARS-CoV-2 infection; moreover, they extend the observations of sporadic cases of SARS-CoV-2 infection in North Italy prior to the first confirmed cases.

ObjectivesTransgender people are disproportionately affected by the HIV-1 epidemic. We evaluated the origin of HIV-1 variants carried by South American transgenders living in Milan by combining accurate phylogenetic methods and epidemiological data.MethodsWe collected 156 HIV-1polsequences obtained from transgender patients engaged in sex work (TSWs) followed between 1999 and 2015 at L. Sacco Hospital, Milan, Italy. Phylogenetic analyses were conducted by HIV-TRACE, MrBayes, MacClade and Beast programs. Reference sequences were retrieved from Los Alamos and local databases. Last negative testing or proxy data from clinical records of infected individuals were used to investigate the country of infection.ResultsAmong South American TSWs, the most represented HIV-1 subtypes were B (70.5%), F1 (12.8%) and C (4.4%). Gene flow migrations of B subtype indicated significant fluxes from TSWs to Italians (21.3%) belonging to all risk groups (26.4% to heterosexuals (HEs), 18.9% to men who have sex with men (MSM), 15.1% to injecting drug users). The largest proportion of bidirectional fluxes were observed between Italians and TSWs (24.6%). For F1 subtype, bidirectional viral fluxes involved TSWs and Italians (7.1% and 14.3%), and a similar proportion of fluxes linked TSWs and Italian HEs or MSM (both 15.8%). Significant fluxes were detected from Italians to TSWs for subtype C involving both MSM (30%) and HEs (40%). Country of HIV-1 acquisition was identified for 72 subjects; overall, the largest proportion of patients with B subtype (73.5%) acquired HIV-1 infection in South America.ConclusionsOur results indicated that South American transgenders largely contribute to the heterogeneity of HIV-1 variants in our country. The high number of clusters based on all subtypes indicated numerous transmission chains in which TSWs were constantly intermixed with HEs and MSM. Our results strongly advocate interventions to facilitate prevention, diagnosis and HIV-1 care continuum among transgender people.

Nursing home residents were the most vulnerable population to be affected by Coronavirus disease 2019 (COVID-19) in Italy. The Italian vaccination strategy decided to indicate them as the target population in the first phase of the massive vaccination campaign. We carried out an analysis on an outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection which occurred in a nursing home in northern Italy (Cremona) after the administration of the complete vaccination cycle affecting most of the guests of the structure.Data relating to the outbreak were obtained through the Regional Surveillance System for Infectious Diseases of Lombardia Region.During the outbreak, among the 61 guests, 56 were vaccinated. Thirty four were found positive for COVID-19: 22 were asymptomatic, 12 were symptomatic and 4 died. The observed difference in the number of deaths between vaccinated and non-vaccinated subjects was significant. During the outbreak 104 healthcare workers (HCWs) were employed in the nursing home, only 66 were vaccinated. Eight HCWs were found COVID-19 positive, 4 of them were vaccinated and of female gender.Similarly to data reported in literature for described outbreaks, we observed that the vaccine is able to protect from the symptomatic form and a valid antibody response protect from a symptomatic disease. The low number of HCWs found positive indicates a correct use of individual protective devices.

The aims of this study were to characterize new SARS-CoV-2 genomes sampled all over Italy and to reconstruct the origin and the evolutionary dynamics in Italy and Europe between February and June 2020. The cluster analysis showed only small clusters including

The aim of this study was the reconstruction of SARS-CoV-2 evolutionary dynamics in time and space in Italy and Europe between February and June 2020. The cluster analysis showed that pure Italian clusters were observed mainly after the lockdown and distancing measures were adopted. Lineage B and B.1 spread between late January and early February 2020, from China to Veneto and Lombardy, respectively. Lineage B.1.1 most probably evolved within Italy and spread from central to south Italian regions, and to European countries. The lineage B.1.1.1 entered Italy only in the second half of March and remained localized in Piedmont until June 2020. In conclusion, the reconstructed ancestral scenario suggests a central role of China and Italy in the widespread diffusion of the D614G variant in Europe in the early phase of the pandemic and more dispersed exchanges involving several European countries from the second half of March 2020.

BACKGROUND: The Milan metropolitan area in Northern Italy was among the most severely hit by the SARS-CoV-2 outbreak. The aim of this study was to examine the seroprevalence trends of SARS-CoV-2 in healthy asymptomatic adults, and the risk factors and laboratory correlates of positive tests. MATERIALS AND METHODS: We conducted a cross-sectional study in a random sample of blood donors, who were asymptomatic at the time of evaluation, at the beginning of the first phase (February 24th to April 8th 2020; n=789). Presence of IgM/IgG antibodies against the SARS-CoV-2-Nucleocapsid protein was assessed by a lateral flow immunoassay. RESULTS: The test had a 100/98.3 sensitivity/specificity (n=32/120 positive/negative controls, respectively), and the IgG test was validated in a subset by an independent ELISA against the Spike protein (n=34, p45 years (p=0.002). DISCUSSION: SARS-CoV-2 infection was already circulating in Milan at the start of the outbreak. The pattern of IgM/IgG reactivity was influenced by age: IgM was more frequently detected in participants aged >45 years. By the end of April, 2.4-9.0% of healthy adults had evidence of seroconversion.

In order to reconstruct the origin and pathways of variola virus (VARV) dispersion, we analyzed 47 VARV isolates available in public databases and their SNPs. The mean substitution rate of the whole genomes was 9.41x10-6 (95%HPD:8.5-11.3x10-6) substitutions/site/year. The time of the tree root was estimated to be a mean 68 years (95%HPD:60.5–75.9). The phylogeographical analysis showed that the Far East and India were the most probable locations of the tree root and of the inner nodes, respectively, whereas for the outer nodes it corresponded to the sampling locations. The Bayesian Skyline plot showed that the effective number of infections started to grow exponentially in 1915-1920, peaked in the 1940s, and then decreased to zero. Our results suggests that the VARV major strains circulating between 1940s-1970s probably shared a common ancestor originated in the Far East; subsequently moved to India, which became the center of its dispersion to eastern and southern Africa, and then to central Africa and the Middle East, probably following the movements of people between south-eastern Asia and the other places with a common colonial history. These findings may help to explain the controversial reconstructions of the history of VARV obtained using long- and short- term calibrations.

ObjectivesHealthcare workers (HCWs) are at high risk of developing SARS-CoV-2 infection. The aim of this single-centre prospective study was to evaluate the trend of SARS-CoV-2 seroprevalence in HCWs working at the primary referral centre for infectious diseases and bioemergencies (eg, COVID-19) in Northern Italy and investigate the factors associated with seroconversion.MethodsSix hundred and seventy-nine HCW volunteers were tested for anti-SARS-CoV-2 antibodies three times between 4 March and 27 May 2020 and completed a questionnaire covering COVID-19 exposure, symptoms and personal protective equipment (PPE) training and confidence at each time.ResultsSARS-CoV-2 seroprevalence rose from 3/679 to 26/608 (adjusted prevalence: 0.5%, 95% CI 0.1 to 1.7% and 5.4%, 95% CI 3.6 to 7.9, respectively) between the first two time points and then stabilised, in line with the curve of the COVID-19 epidemic in Milan. From the first time point, 61.6% of the HCWs had received training in the use of PPE and 17 (61.5%) of those who proved to be seropositive reported symptoms compatible with SARS-CoV-2 infection. Contacts with ill relatives or friends and self-reported symptoms were independently associated with an increased likelihood of seroconversion (pConclusionThe seroprevalence of SARS-CoV-2 among the HCWs at our COVID-19 referral hospital was low at the time of the peak of the epidemic. The seroconversions were mainly attributable to extrahospital contacts, probably because the hospital readily adopted effective infection control measures. The relatively high number of asymptomatic seropositive HCWs highlights the need to promptly identify and isolate potentially infectious HCWs.

Objectives: The Milan metropolitan area in Northern Italy was among the most severely hit by the SARS-CoV-2 outbreak. The epidemiological trends of mild COVID-19 are however still unknown. The aim of this study was to examine the seroprevalence of SARS-CoV-2 infection in healthy asymptomatic adults, the risk factors, and laboratory correlates. Design: We conducted a cross-sectional study during the outbreak. Presence of anti-SARS-CoV-2 IgM/IgG antibodies against the Nucleocapsid protein was assessed by a lateral flow immunoassay. Setting: Blood center at a leading academic hospital serving as COVID-19 referral center. Participants: We considered a random sample of blood donors since the start of the outbreak (February 24th to April 8th 2020, n=789). Main outcome measures: The main outcome was the prevalence of IgG/IgM anti-SARS-CoV-2 antibodies . Results: The test had a 98.3% specificity and 100% sensitivity, and for IgG was validated in a subset by an independent ELISA against the Spike protein (N=34, P

This report describes the isolation, molecular characterization, and phylogenetic analysis of the first three complete genomes of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) isolated from three patients involved in the first outbreak of COVID‐19 in Lombardy, Italy. Early molecular epidemiological tracing suggests that SARS‐CoV‐2 was present in Italy weeks before the first reported cases of infection.

This report describes the isolation, the molecular characterization and the phylogenetic analysis of the first three complete genomes of SARS-CoV-2 isolated from three patients involved in the first outbreak of COVID-19 in Lombardy, Italy. Early molecular epidemiological tracing suggests that SARS-CoV-2 was present in Italy weeks before the first reported cases of infection.

To reconstruct the evolutionary dynamics of the 2019 novel‐coronavirus recently causing an outbreak in Wuhan, China, 52 SARS‐CoV‐2 genomes available on 4 February 2020 at Global Initiative on Sharing All Influenza Data were analyzed. The two models used to estimate the reproduction number (coalescent‐based exponential growth and a birth‐death skyline method) indicated an estimated mean evolutionary rate of 7.8 × 10−4 subs/site/year (range, 1.1 × 10−4‐15 × 10−4) and a mean tMRCA of the tree root of 73 days. The estimated R value was 2.6 (range, 2.1‐5.1), and increased from 0.8 to 2.4 in December 2019. The estimated mean doubling time of the epidemic was between 3.6 and 4.1 days. This study proves the usefulness of phylogeny in supporting the surveillance of emerging new infections even as the epidemic is growing., Highlights The aim of this study was to investigate the temporal origin, rate of viral evolution and population dynamics of SARS‐CoV‐2.The Bayesian approach used indicated a most probable origin of the epidemic between October and November 2019.The estimated effective reproductive number increased from 0.8 to 2.4 in December 2019 when the mean doubling time was about 4 days.This study proves the usefulness of phylogeny in supporting the surveillance of emerging new infections.

To reconstruct the evolutionary dynamics of the 2019 novel coronavirus, 52 2019-nCOV genomes available on 04 February 2020 at GISAID were analysed.The two models used to estimate the reproduction number (coalescent-based exponential growth and a birth-death skyline method) indicated an estimated mean evolutionary rate of 7.8 × 10−4 subs/site/year (range 1.1×10−4–15×10−4).The estimated R value was 2.6 (range 2.1-5.1), and increased from 0.8 to 2.4 in December 2019. The estimated mean doubling time of the epidemic was between 3.6 and 4.1 days.This study proves the usefulness of phylogeny in supporting the surveillance of emerging new infections even as the epidemic is growing.

Variola virus (VARV), the causative agent of smallpox, is an exclusively human virus belonging to the genus Orthopoxvirus, which includes many other viral species covering a wide range of mammal hosts, such as vaccinia, cowpox, camelpox, taterapox, ectromelia, and monkeypox virus. The tempo and mode of evolution of Orthopoxviruses were reconstructed using a Bayesian phylodynamic framework by analysing 80 hemagglutinin sequences retrieved from public databases. Bayesian phylogeography was used to estimate their putative ancestral hosts. In order to estimate the substitution rate, the tree including all of the available Orthopoxviruses was calibrated using historical references dating the South American variola minor clade (alastrim) to between the XVI and XIX century. The mean substitution rate determined by the analysis was 6.5 × 10-6 substitutions/site/year. Based on this evolutionary estimate, the time of the most recent common ancestor of the genus Orthopoxvirus was placed at about 10 000 years before the present. Cowpox virus was the species closest to the root of the phylogenetic tree. The root of VARV circulating in the XX century was estimated to be about 700 years ago, corresponding to about 1300 AD. The divergence between West African and South American VARV went back about 500 years ago (falling approximately in the XVI century). A rodent species is the most probable ancestral host from which the ancestors of all the known Orthopoxviruses were transmitted to the other mammal host species, and each of these species represented a dead-end for each new poxvirus species, without any further inter-specific spread.

Objectives The aim of this study was to evaluate acquired drug resistance in Italy in the 2009–2018 period. Methods We analysed 3094 patients from the Italian ARCA database who had failed antiretroviral treatment and who had received a genotypic test after 6 months of treatment. Drug resistance mutations were identified using International AIDS Society (IAS)-USA tables and the Stanford HIVdb algorithm. The global burden of acquired resistance was calculated among all subjects with antiretroviral failure. Time trends and correlates of resistance were analysed using standard statistical tests. Results Patients of non-European origin and non-B subtypes increased significantly from 11.5% (103/896) to 19.2% (33/172) and from 13.1% (141/1079) to 23.8% (53/223), respectively, over time. Overall, 14.5% (448/3094), 12.1% (374/3094) and 37.8% (1169/3094) of patients failed first, second and later lines, respectively. According to both IAS and HIVdb, in the study period resistance to any class, nucleoside reverse inhibitor, non-nucleoside reverse inhibitor, and protease inhibitors (PIs) declined significantly. Integrase strand transfer inhibitor (INSTI) resistance declined significantly from 31% (36/116) to 20.8% (41/197) according to HIVdb but not to IAS. Divergent data were highlighted regarding the proportion of non-European patients carrying any, PI and INSTI resistance using IAS tables compared with the Stanford HIVdb algorithm, as the former failed to detect a decrease in resistance while the latter indicates a reduction of 1.6-, 5- and 1.8-fold resistance for such drug classes. In the multivariate analysis, the risk of resistance increased in patients with a larger number of treatment lines and higher viraemia and decreased in those starting therapy in the last biennium of the study. Discussion A marked reduction in drug resistance was observed over 10 years, compatible with higher genetic barrier and potency of new antiretrovirals. Nonetheless, concerns remain for subjects with non-B subtypes when using mutation lists instead of interpretation systems because of the extensive polymorphism of the protease region.

Because of the high variability of Hepatitis C virus (HCV), it might be important to characterize in vivo the evolution of resistance-associated mutations (RAVs) to direct-acting antivirals (DAAs) in different genotypes. NS3-, NS5A- and NS5B-HCV substitutions were studied by next generation sequencing (NGS) on 74 HCVinfected patients who started a DAA regimen. RAVs with frequencies of 1% and 15% were analyzed. Globally, 43, 15, 12 and 4 patients were infected with subtype 1a, 1b, genotype 4 and subtype 3a, respectively. The majority of patients (64.8%) had cirrhosis, 70.3% were HIV-coinfected and 14.9% were DAA-experienced. Overall baseline prevalence of RAVs was 74.3%, 52.2%, 45.9% and 36.8% to any NS3, NS5B and NS5A inhibitors available at that time, respectively, and dropped to 39.2%, 26.1%, 22.8% and 16.2%, respectively, when only mutations associated with the ongoing regimen were considered. The highest proportion of mutations was detected in subtype 1a (81.4%, p=.026), particularly in NS3 region (76.9%, p