SARS-CoV-2 has been circulating in northern Italy since december 2019: evidence from environmental monitoring

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease COVID-19, a public health emergency worldwide, and Italy is among the most severely affected countries. The first autochthonous Italian case of COVID-19 was documented on February 21, 2020. We investigated the possibility that SARS-CoV-2 emerged in Italy earlier than that date, by analysing 40 composite influent wastewater samples collected - in the framework of other wastewater-based epidemiology projects - between October 2019 and February 2020 from five wastewater treatment plants (WWTPs) in three cities and regions in northern Italy (Milan/Lombardy, Turin/Piedmont and Bologna/Emilia Romagna). Twenty-four additional samples collected in the same WWTPs between September 2018 and June 2019 (i.e. long before the onset of the epidemic) were included as ‘blank’ samples. Viral concentration was performed according to the standard World Health Organization procedure for poliovirus sewage surveillance, with modifications. Molecular analysis was undertaken with both nested RT-PCR and real-rime RT-PCR assays. A total of 15 positive samples were confirmed by both methods. The earliest dates back to 18 December 2019 in Milan and Turin and 29 January 2020 in Bologna. Virus concentration in the samples ranged from below the limit of detection (LOD) to 5.6 × 104 genome copies (g.c.)/L, and most of the samples (23 out of 26) were below the limit of quantification of PCR. Our results demonstrate that SARS-CoV-2 was already circulating in northern Italy at the end of 2019. Moreover, it was circulating in different geographic regions simultaneously, which changes our previous understanding of the geographical circulation of the virus in Italy. Our study highlights the importance of environmental surveillance as an early warning system, to monitor the levels of virus circulating in the population and identify outbreaks even before cases are notified to the healthcare system

First detection of SARS-CoV-2 in untreated wastewaters in Italy

Several studies have demonstrated the advantages of environmental surveillance through the monitoring of sewage for the assessment of viruses circulating in a given community (wastewater-based epidemiology, WBE). During the COVID-19 public health emergency, many reports have described the presence of SARS-CoV-2 RNA in stools from COVID-19 patients, and a few studies reported the occurrence of SARS-CoV-2 in wastewaters worldwide. Italy is among the world's worst-affected countries in the COVID-19 pandemic, but so far there are no studies assessing the presence of SARS-CoV-2 in Italian wastewaters. To this aim, twelve influent sewage samples, collected between February and April 2020 from Wastewater Treatment Plants in Milan and Rome, were tested adapting, for concentration, the standard WHO procedure for Poliovirus surveillance. Molecular analysis was undertaken with three nested protocols, including a newly designed SARS-CoV-2 specific primer set. SARS-CoV-2 RNA detection was accomplished in volumes of 250 ml of wastewaters collected in areas of high (Milan) and low (Rome) epidemic circulation, according to clinical data. Overall, 6 out of 12 samples were positive. One of the positive results was obtained in a Milan wastewater sample collected a few days after the first notified Italian case of autochthonous SARS-CoV-2. The study confirms that WBE has the potential to be applied to SARS-CoV-2 as a sensitive tool to study spatial and temporal trends of virus circulation in the population

Pepper mild mottle virus in different water matrices

Abstract Background Pepper mild mottle virus (PMMoV), a plant virus belonging to Virgoviridae, has recently been suggested as a potential viral indicator for faecal pollution in aquatic environments, since it has been found to be abundantly excreted from healthy human subjects. Methods The occurrence, amount and diversity of PMMoV was investigated in water environments by nested RT-PCR and TaqMan based quantitative PCR. During 2017-2019, 251 water samples (92 urban wastewaters, 32 treated effluents, 16 surface water samples, 9 estuarine samples, 20 seawater samples, 67 groundwater samples, and 15 drinking waters) were analysed. Results PMMoV was detected in 73/92 (79%) wastewater samples, 22/32 (69%) treated sewages, 11/16 (69%) river samples, 6/9 (67%) estuarine samples, 5/20 (25%) bathing waters, and 9/67 (13%) groundwaters, whilst drinking water samples tested always negative. Mean viral concentrations (genome copies/L) were: raw sewage 4.2 × 106, treated sewage 7.4 × 105, river 3.2 × 103, estuarine waters 9.6 × 102, seawaters 3.0 × 102, groundwaters 7.7 × 101. Conclusions This study highlights the significant occurrence of PMMoV in aquatic environment in Italy, and a clear gradient of viral prevalence and concentrations from polluted to clean waters (wastewaters to drinking waters). Key messages PMMoV is ubiquitous throughout the water cycle in Italy with different concentrations. Studies are needed to evaluate the suitability of PMMoV as a viral indicator for human fecal pollution in waters

Chronic obstructive pulmonary disease and neutrophil infiltration: role of cigarette smoke and cyclooxygenase products

Cigarette smoke is the main cause of chronic obstructive pulmonary disease (COPD), where it can contribute to the observed airway inflammation. PGE(2) is produced within human airways, and both pro- and anti-inflammatory activities have been reported. We quantitated PGE(2) concentrations in induced sputum supernatants from different groups of subjects and correlated the obtained values to neutrophil infiltration as well as to the expression of cyclooxygenase-2 (COX-2). Cigarette smoke extract (CSE) was used to evaluate the effect of smoking on COX-2 and PGE(2) receptor expression as well as on PGE(2) release in neutrophils and alveolar macrophages (AM) obtained from normal donors. The effects of PGE(2) and of PGE receptor agonists and antagonists were evaluated on the adhesion of neutrophil to a human bronchial epithelial cell line (16HBE). PGE(2) levels, COX-2 expression, and neutrophil infiltration were significantly higher in normal smokers and COPD smokers (P < 0.0001) compared with controls and COPD former smokers. Induced sputum supernatant caused neutrophil adhesion to 16HBE that was significantly reduced, in COPD smokers only, by PGE(2) immunoprecipitation. In vitro experiments confirmed that CSE increased PGE(2) release and COX-2 and PGE(2) receptor expression in neutrophils and AM; PGE(2) enhanced the adhesion of neutrophils to 16HBE, and a specific E-prostanoid 4 (EP(4)) receptor antagonist blunted its effect. These results suggest that CSE promote the induction of COX-2 and contributes to the proinflammatory effects of PGE(2) in the airways of COPD subjects.Cigarette smoke is the main cause of chronic obstructive pulmonary disease (COPD), where it can contribute to the observed airway inflammation. PGE(2) is produced within human airways, and both pro- and anti-inflammatory activities have been reported. We quantitated PGE(2) concentrations in induced sputum supernatants from different groups of subjects and correlated the obtained values to neutrophil infiltration as well as to the expression of cyclooxygenase-2 (COX-2). Cigarette smoke extract (CSE) was used to evaluate the effect of smoking on COX-2 and PGE(2) receptor expression as well as on PGE(2) release in neutrophils and alveolar macrophages (AM) obtained from normal donors. The effects of PGE(2) and of PGE receptor agonists and antagonists were evaluated on the adhesion of neutrophil to a human bronchial epithelial cell line (16HBE). PGE(2) levels, COX-2 expression, and neutrophil infiltration were significantly higher in normal smokers and COPD smokers (P < 0.0001) compared with controls and COPD former smokers. Induced sputum supernatant caused neutrophil adhesion to 16HBE that was significantly reduced, in COPD smokers only, by PGE(2) immunoprecipitation. In vitro experiments confirmed that CSE increased PGE(2) release and COX-2 and PGE(2) receptor expression in neutrophils and AM; PGE(2) enhanced the adhesion of neutrophils to 16HBE, and a specific E-prostanoid 4 (EP(4)) receptor antagonist blunted its effect. These results suggest that CSE promote the induction of COX-2 and contributes to the proinflammatory effects of PGE(2) in the airways of COPD subjects

Magnetic field confinement by meridional flow and the solar tachocline

We show that the MHD theory that explains the solar tachocline by an effect of the magnetic field can work with the decay modes of a fossil field in the solar interior if the meridional flow of the convection zone penetrates slightly the radiative zone beneath. An equatorward flow of about 10 m/s penetrating to a maximum depth of 1000 km below the convection zone is able to generate almost horizontal field lines in the tachocline region so that the internal field is almost totally confined to the radiative zone. The theory of differential solar rotation indeed provides meridional flows of about 10 m/s and a penetration depth of < 1000 km for viscosity values that are characteristic of a stable tachocline.Comment: 5 pages, 6 figures, submitted to A&

Wastewater surveillance of SARS-CoV-2 variants in October–November 2022 in Italy: detection of XBB.1, BA.2.75 and rapid spread of the BQ.1 lineage

: This study adds insight regarding the occurrence and spread of SARS-CoV-2 Variants of Concern (VOCs) and Variants of Interest (VOIs) in Italy in October and November 2022, by testing urban wastewater collected throughout the country. A total of 332 wastewater samples were collected from 20 Italian Regions/Autonomous Provinces (APs) within the framework of national SARS-CoV-2 environmental surveillance. Of these, 164 were collected in the first week of October and 168 in the first week of November. A ∼1600 bp fragment of the spike protein was sequenced by Sanger (for individual samples) and long-read nanopore sequencing (for pooled Region/AP samples). In October, mutations characteristic of Omicron BA.4/BA.5 were detected in the vast majority (91 %) of the samples amplified by Sanger sequencing. A fraction of these sequences (9 %) also displayed the R346T mutation. Despite the low prevalence documented in clinical cases at the time of sampling, amino acid substitutions characteristic of sublineages BQ.1 or BQ.1.1 were detected in 5 % of sequenced samples from four Regions/APs. A significantly higher variability of sequences and variants was documented in November 2022, when the rate of sequences harbouring mutations of lineages BQ.1 and BQ1.1 increased to 43 %, and the number of Regions/APs positive for the new Omicron subvariant more than tripled (n = 13) compared to October. Moreover, an increase in the number of sequences with the mutation package BA.4/BA.5 + R346T (18 %), as well as the detection of variants never observed before in wastewater in Italy, such as BA.2.75 and XBB.1 (the latter in a Region where no clinical cases associated with this variant had ever been documented) was recorded. The results suggest that, as predicted by the ECDC, BQ.1/BQ.1.1 is rapidly becoming dominant in late 2022. Environmental surveillance proves to be a powerful tool for tracking the spread of SARS-CoV-2 variants/subvariants in the population

Tracing the footprints of SARS-CoV-2 in oceanic waters

The detection of SARS-CoV-2 in water environments has predominantly focused on wastewater, neglecting its presence in oceanic waters. This study aimed to fill this knowledge gap by investigating the occurrence of SARS-CoV-2 in remote sea and oceanic waters, at large distances from the coastline. Forty-three 500-liter samples were collected between May 2022 and January 2023 from the Atlantic Ocean, the Mediterranean Sea, the Arctic region, the Persian Gulf and the Red Sea. Using molecular detection methods including real-time RT-qPCR and nested PCR followed by sequencing, we successfully detected SARS-CoV-2 RNA in 7 of the 43 marine water samples (16.3&nbsp;%), and specifically in samples taken from the Atlantic Ocean and the Mediterranean Sea. The estimated concentrations of SARS-CoV-2&nbsp;genome copies in the positive samples ranged from 6 to 470 per 100&nbsp;l. The presence of mutations characteristic of the Omicron variant was identified in these samples by amplicon sequencing. These findings provide evidence of the unforeseen presence of SARS-CoV-2 in marine waters even at distances of miles from the coastline and in open ocean waters. It is important to consider that these findings only display the occurrence of SARS-CoV-2 RNA, and further investigations are required to assess if infectious virus can be present in the marine environment

Global quantification and distribution of antibiotic resistance genes in oceans and seas: Anthropogenic impacts and regional variability

The global spread of antibiotic resistance genes (ARGs) in the marine environment poses a significant threat to public health and natural ecosystems. This study quantified and analysed the distribution and co-occurrence patterns of ARGs in a wide range of oceans and high seas, including the Atlantic, Arctic and Indian Ocean, the Mediterranean Sea and the Persian Gulf. Focusing on beta-lactamases (blaOXA-48, blaCTX-M-1 group, and blaTEM), sulfonamides (sul1) and tetracycline (tetA), our results showed that sul1 was ubiquitous, indicating widespread dissemination. Notably, the Mediterranean Sea exhibited higher levels of multiple ARGs in single samples, suggesting significant anthropogenic impact. Interestingly, the Arctic Ocean, particularly around the Svalbard Islands, also showed the presence of multiple ARGs, highlighting the pervasive occurrence of antibiotic resistance in remote areas. We employed two clustering approaches to explore ARG patterns, primarily focusing on identifying geographic trends and differences in ARG abundance. Additionally, we investigated potential sources of contamination, including proximity to wastewater treatment plants, ports, marine traffic, and currents. These findings clearly demonstrate that antibiotic resistance gene contamination is widespread across diverse marine environments, with significant regional variations. This underscores the urgent need for tailored intervention strategies and global collaboration to mitigate the spread of ARGs and manage their complex dynamics in marine ecosystems

Wastewater surveillance of SARS-CoV-2 variants in October-November 2022 in Italy: detection of XBB.1, BA.2.75 and rapid spread of the BQ.1 lineage

This study adds insight regarding the occurrence and spread of SARS-CoV-2 Variants of Concern (VOCs) and Var-iants of Interest (VOIs) in Italy in October and November 2022, by testing urban wastewater collected through-out the country. A total of 332 wastewater samples were collected from 20 Italian Regions/Autonomous Provinces (APs) within the framework of national SARS-CoV-2 environmental surveillance. Of these, 164 were collected in the first week of October and 168 in the first week of November. A similar to 1600 bp fragment of the spike protein was sequenced by Sanger (for individual samples) and long-read nanopore sequencing (for pooled Region/AP samples).In October, mutations characteristic of Omicron BA.4/BA.5 were detected in the vast majority (91 %) of the samples amplified by Sanger sequencing. A fraction of these sequences (9 %) also displayed the R346T mutation. Despite the low prevalence documented in clinical cases at the time of sampling, amino acid substitutions characteristic of sublineages BQ.1 or BQ.1.1 were detected in 5 % of sequenced samples from four Regions/APs. A significantly higher variability of sequences and variants was documented in November 2022, when the rate of se-quences harbouring mutations of lineages BQ.1 and BQ1.1 increased to 43 %, and the number of Regions/APs positive for the new Omicron subvariant more than tripled (n = 13) compared to October. Moreover, an increase in the number of sequences with the mutation package BA.4/BA.5 + R346T (18 %), as well as the detection of variants never observed before in wastewater in Italy, such as BA.2.75 and XBB.1 (the latter in a Region where no clinical cases asso-ciated with this variant had ever been documented) was recorded.The results suggest that, as predicted by the ECDC, BQ.1/BQ.1.1 is rapidly becoming dominant in late 2022. Environ-mental surveillance proves to be a powerful tool for tracking the spread of SARS-CoV-2 variants/subvariants in the population